Tobacco Documents Online

. Applications for supplements . Resubmitted application (modified . New applications from present or recent grantees e. New applications

SCIENTIFIC ADVISORY BOARD MEE'TI'NG ~.f'.f;... ... ' ..~ . ', .. .. . . . ' . Administrative actions taken since May 20, 1967 . Staff Reports on matters referred by the Board Considerationof applications a. Renewal applications b. Supplements requested . Resubmitted (modified) application d. New applications from present or recent grantees e. New applications Program planning Special session for staff reperts on recent developments in certain projects and policy discussions of methods for effective follow-up.

4 af .~~ . , ,~. y IGTIAL REPORT CONFIDE SCIENTIl'IC ADVISORY BOARD MEETING ;;,. . . New York, New York ' - . S, May 20-21, 1967 . y..~..: i ,Y( Dr. Kenneth M. Lynch,`Chairman ~' Dr. C. C. Little ~ Scientific Director, CTR ~tiz . Dr. Richard J. Bing K Dr. McKeen Cattell ~_ Dr. Leon 0. Jacobson - 4WW', ' Dr. Clayton G. Loosli'~ -Dr. Stanley P'. Reimann f Dr. Sheldon C. Sommers ~ N A Dr. John W. Fertig (5/20/67) Columbia University Mr. W. T. Hoyt Executive Director, CTR X Dr. Robert C. Hockett Associate Scientific Director, C'iR Dr. J. Morrison Brady Associate Scientific Director, CTR Dr. John H. Kreisher Associate Scientific Director, CTR Dr. Vincent F. Lisanti Scientific Associate, CTR . The report of actions taken at the December meeting was approved by the Board. Administrative Actions ~ #390R2 -Peter H. Knapp, Ph.D. A balance of $252.36 from the completed project was received and deposited. w. ~ Cr: y _: #~+08R1 Benjamin Burrows, M.D. A balance of $1,500.00 from the completed project was received and deposited. #466R1 E. C. Hoff, Ph.D., M.D. The grantee was authorized to use the une~Tended'balance of approximately $4,000.00 for the needs of the ! project after the termination date of May 31, 1967. #510R1 Donald J. Massaro, Mi.D. This grant will be transferred to Duke University Medical Center following receipt of financial report and refund from Georgetown University. #592 Donald J. Massaro, Mi.D. $7,820.00 Transferred to Duke University Medical Center. 1003546613 #528R1 Sue Buckingham, M.D. After staff negotiation, the final sl:.m approved for this year's grant is $15,155.00 with the understanding-that the money still remaining from her previous grants be used also to defray , the costs of this project to a total of $31,624.00, plus $2,000.00 4 - from carryover for contingencies. ` , Q)LJ\(ai. FOr 7'c>z;AccO PrsrAI Z crr-U.S.A. LV .'.,t.y.~sa.t.:v:~.r....i.+:...... _..iaw.i.h~::..~...t,:-.. :.c.:.uaa..raa..ss.+.i-t~.. .. . , .... - -..,..:a.a...~..y. .. _...~~...~..._.._.~.2s..~L..e.aa+...

from the personnel category to travel ($450) and supplies & ` Ulrich Schaeppi, M.D. $10,175.00 Ulrich 3chaeppi,-M.D. $17,260.00 The request of a transfer of both grants from The Worcester - Foundation for Exrperimental Biology to Ma,son Research Institute, also of Worcester, Massachusetts, approved. The question of a contract or grant should be clarified and decided before the letter of approval is sent. . , . -,.: .. - .: ,.n.~''.::.~~,.~-. . .. ~:~:. , '.., ., .... .... . _..'. .. .....,... .:.__.... . ..-. .. a1.Mww......:.1' .ff7'lie '1?. Timoth.y Crocker, M.D. 'Permissi=was granted~ to*transfer $1,000 ; services . ~.r.. been answered'and the grant will proceed. #579 Naiter M. Chopra, Ph.D. -$23,955.00 Abproved. All questions have r~r585 project funds to travel expenses. #607 Samuel Bellet, M.D. Permission was granted to transfer $800 of the agreed upon, pending Dr. Larson's discussions with publishers. grants is to be returned~to The Council. Approved. The balance of $13,879.67 remaining from previous George W. Smetters, M.D. (Thomas C. Laipply, M.D.) $11,963.00 #591 Paul S. Larson, Ph.D. The final amount for this grant has not been #610 Caroline Bedell Thomas, M.D. $29,865.00 A terminal one-year grant - for activation as of July 1, 1967. University has been received and the grant has been authorized #609 Donald B. Louria, M.D. Proper official approval from Cornell application on the agenda for the September meeting, was aAproved in order to summarize work status and to allow transfer of such information to Mr. Kurt Enslein for evaluation. These monies a.<re to provide for salaries to keep Dr. Thomas'' group together and are not for the purpose of continuing the project " indefinitely. The Board agreed that Mr. Enslein should be ; remuneraLea ror tnis service in accord with past procedures. #611 John R. Rowlands, Ph.D. The committee recommended placing this . Supplements #516R1S Donald M. Pace, Ph.D. $2,003.60 Approved. #5585 G. H. Friedell, M.D. $5,z +03'.00~ Approved. #566s Kenneth M. Lynch, M.D. and Forde A. McIver, M.D. $1,200.00 Anrroved.

#547R1 #548R1 #554R1 'Rel Apliti enwapcaons . . ... ~,~. Bertram Eichel, D.D.S. $29,594.00 Approved for the terminal yeax ~ tf" <of a three-year p"roe am at the previous rate of $17,950.00. Rose Marie Pangborn, M.S. $22,433.00 Approved. ,..:,:.;_. ._ . .. . .. Thomas C. Westfall, Ph. D. $11, 500~.00 Approved for the terminal year of a three-year program. . . ' r. ~" `lc ~ James E. P. Toman, Ph.D. $15,225.00 Approved for the first year of a three-year program. Marcus N. Carroll Jr., Ph.D. $19,620X0 Approved for one year at the requested amount contingent upon assurance from the institution :that his salary will be secure for the entire period of the grant. Stephen M. Ayres, M.D. $22,890.00 Approved for the terminal year of a three-year program. Walter M. Booker, Ph.D. $19,568.00 Approved for the terminal year of a three-year project at the previous rate of $17,705.00. Hyman Engelberg, M.D. $24,966.00 Approved for the terminal year of a two-year program. Dr. Loosli will request that Dr. Samuel Rappaport review this research program before the next meeting. Cesare Biancifiori, M.D.' $10,100.00 Approved for the second ye ar of a program originally submitted as a three-year plan. It was suggested that urethane be used as a control to produce carcinoma of the lung. The staff will review this program for the next meeting and a visit by a Board member is contemplated. q -. , . Roger K. Larson, M.D. $17,424.00 Approved for the terminal year of a two-year program. #534R1. Paul Goldhaber, D.D.S. $23,396•75 Approved for the second year of a -three-year program -with the understanding that any residual funds remaining from the previous year be applied against this grant total. Sheldon C. Sommers, M.D. $14,536.00 ;°: a three-year plan. Ines Mandl, Ph.D. $25,969.00 Approved for the second year of a three- year plan. Gustave A. Laurenzi, M.D. $12,229.00 Approved. A future conference should be developed around this program. S. N. Pradhan, M.D., Ph.D. $9,928.00 Approved for the second year of a three-year plan. Broda 0. Barnes, Ph.D., M.D. $,23,000.00 Denied. Dr. Loosli will contact WHO, NCI; etc., to see if an overall effort for an ongoing survey is possible. 100354661s Ph, Gene M. Smith, Z Q.D. $14,184.00 Approved as a terminal grant.

~Harry S. Bernton, X.D. $21,460.00 Denied. Jay D. Coffman, M:D. $5,192.00 Approved. R. Ernest Clark, Ph.D. $23,328.80 Denied. Maurice S. Segal, M.D. $85,650.00 Approved in the amount of 430,000.00 plus overhead. Final arrangements are to be made by `` staff. r ._the Helsinki Declaration. . ; . . Board thought this was an excellent research proposal but not n Lhe grant applicatz= forms to the effect that the investigators should abide by .withinthe scope of The Council's program. A. Weinstock, Ph.D. $40,036.00 Approved as a one-year program with review by the staff at the end of nine months. . . ,,.,. . . .. ,;,..:--w . i '- T#615 Charles E. Tobin, Ph.D. $7,634.85 Denied. #616 .. Robert A.,Kuhn, M.D. $30,072.50 Denied. T1617 Luben G. Angeloff $12,805.00 Denied. i619 H. A. I. hTewman, Ph.D. $17,675.00 Denied without prejudice. The year of a two-year program. #618 Edmond Antony Murphy, M.D., Sc.D. $8,430.00 Approved for the first #620 #621 Ole A. Holtermann, M.D. $12,978.00 Denied. . The staff was requested to look into the forms used by other granting agencies concerning research carried out on humans, and possibly incorporate a statement - 7. The staff will send to Dr. Ratzenhofer a copy of the Scientific Director's - ill emphasize the non-prejudicial attitude toward foreign applications for Report and a letter of exvlanation of the mission of The Council. This letter research grantW. ti. The next meeting of the Scientific Advisory Board will be held in New York City on September 23-24, 1967.

~~~.. ' ,.... i ® ~ ~. Sa `? yr ~ °° ,A AIB~NISTRATIVE' ACTIONS a ~ Reports torScientific Advisory Board .No Action Required _ _ #534R1 Paul Goldhaber, D.D.S. The final sum approved for the renewal grant is $22,938.75 which, with an unexpended balance of $458.00 from the previous grant totals $23,396•75, the amount originally requested . . , #614 Maurice S. Segal, M.D. The final sum authorized for the new grant is $34,500,00; representing,$30,000.00 plus 15% overhead in the amount of $4,500.00. °-S Refunds Received : #385R2 William F. McNary Jr., Ph6D. Refund of $3,107.27 received and deposited. #4$9 E. T. Angelakos, M.D., Ph,D. A refund of $247.84 received and deposited. #506 Roger K. Larson, M.D. A refund of $486.57 received and deposited. , ; #585 George W. Smetters, M.D. The balance of $13,879.67 (grants #363, F, ~ 450A and 450B) was returned as requested and deposited. ~ ., a.. W~. r. _.. . . . . , Fori Toa_~cco Rrsz;nlzcll U S.A: Followup , . ,._ . . , r. ~. . , a : #,477MR2 Marcus N. Carroll Jr., Ph.D. Word has been received from the institutionaL authorities that Dr. Carroll's salary has been guarant'eed until June 30, 1968. His grant has, therefore, been activated effective July 1, 1967 in the amount of $'19,620.00. ' 4 v. #510R11 Donald J. Massaro, M.D. A refund of $4,105.83:was received from ~' Georgetown University and deposited. A new check in this amount has been issued to Duke University for continuation of Dr. Massaro's grant. ~bRe.,_...;r _ .'.'. . ,..:.,........ : ~ .. ~ports to Scientific Advisory Board Action Required September23-2k, 1 . 1ts s., l PZ 7_ -Q, Time Extensions #415R1 Duane G. Wenzel, Ph.D. A time extension was authorized for one year, without additional funds, to May 31, 1968. #k48Ri John S. Waugh, Ph.D. A time extension to June 30, 1968 and use of uncommitted balance of $11,495.42 was authorized for the purposes of the project. #495 Walter Redischy M6D. Permission granted to extend the grant from June 30 to September 30, 1967, and to use the uncommitt'ed funds for the purposes of the project during that interval. A financial report was requested. 0 ~

Budget Reallocations authorized-to cover travel expenses in the amount of $229.00. --. #493N4, Barbara K. Watson, Ph.D. Reallocation of the granc budget w , as #599 Domingo M. Aviado, M.D. 'Permission was granted to realloca.te the . . . _ . _ . .. . . . ,. . . . : l . . , a. .:. , , r'~T.q{}~~ #409R2 Frederik B. .Bang, M.D. Permission requested to carry over'balance of $5,184.67 from the 1966 grant to the current year. ~,' ~ #493m1 , ,,r . Barbara K. Watson, Ph.D. Permission requested to carry over balance of $5,072.88 from previous grant. budget to provide $750.00 for traT~el expenses. „~~~~`~ 'Carryover of Grant Funds excess funds. of Nebraska requesting a financial statement and return of the: ~ personnel and equipment. A letter has been sent to the University to the University of the Pacific to be used for additional balance of $1,000+ be transferred from the University of Nebraska #516 D. M. Pace, Ph.D. The grantee has requested that an estimated #559R1 Sheldon C. Sommers, M.D. Permission requested to carry over balance of $1.,359•29 from previous grant. ... . ~~~: A supplement in the amount of $3,300.00 Walter M. Booker, Ph.D. The May minutes record the sum approved `7';:j as $17,205.00 whereas the correct amount is $17,705.00.

~PPLICAtIOA'S TO BE CONSIDERED Amount Anticipated 'Last Requested Duration Grant Category Renewa]; Applications 223R8 McKennis .361R4 Gey 3 years 52'1598 58,400 3 years 43,434 CR'`'~e (16 months) cuit;we 30,6og :18,500 32,428 409R3 . Bang 455R1 Rose 523R1 Jones 558MF1 Friedell 561R1 Brinkrnan 566R1. Lynch 567R1 Severi 569R1 Moser 573R1 Loosli . 31,050 12,109 322570 15,000 15,980 51,576 3 years 21, 835 1 year 3-5 years 13,300. 10,000 Cattell Jacobsori , , Bing ~ Little, Lynch x Reimann, Loosli Sommers Reimann, Sommers Jacobson Epidemiol- Sommers, Cattell~ 1 year 362453 2 years 15,653 1 year 27,121 2 years 16,000 2 years 15,137 Carcinogen- Bing, Sommers esis Reimann Carcinogen- Reimann, Jacobson esis Loosli, Sommers Carcinogen- Little, Sommers esis Reimann, Loosli -Cardiovas- Cattell, Bing ~ cular Jacobson, 2 y a ears 53,755 J cobson, Lynch ~R N c~~3 Little Jacobson, Bing~ '';=i'. Lynch, Loosli Reimarin, Jacobson Patho±o tC Lynch Reimanny Sommers Supplemental Applications 456R2 Homburger 6,000 1 year .1+67R2 Westfall ~ 7,500 Modified Application 613M Clark 23,328 New Applications from 338-A Homburger 626 Redi,sch,:- 631 Domino 632 Strong 7Q,000 Kaasfa.y • Little, Jacobson ~ Sommers, Reimann 11,500 Pharmacol- Bing, Cattell ogy Jacobson, Lynch 1 year Little, Loosli aati-e-= Cattell 1003546621 Present or Recent Grantees 55,617 16,795 28,565 20,516 5 years 59 ,096 Sommers Jacobson . ~ , 1 year 12,075 Cardiovas- Loosli, Reimann Bing, Cattell 3 years 33,483 cular Pharmacol- Jacobson Cattell, Jacobson 3 years 17,531 ogy Cardiovas- Little Reimann, Bing cular Cattell, SoLmcrs

jIPPLICATIO\ 3 TO BE C0INSIDuRM , Grant Principal . Amount Anticipated 'Last .io. Investigator Requested D;:ra;.ion Grant 611 Rowlands 46,058 3 years ~~:5~ e Jacobson, Cattell . . . . - . _ ~ ' . . ~ . .. ~ . .. ~ ~ 622 Geller - .. 29,442 3 years e a2 c_o J_ Cattell, Little :; t Bing 627 Beller 26,391 3 years Cardiovas- Bing'r Cattell : cular Somers 625 Brown~ 12,548 .2 years ~ Cardiovas- Cattell, Bing ~ Reimann 624 Noakes 30,900 3 years r:Scsxl"wu `~'~ Jacobson, Loosl •sna tre Jacobe on r=4 ~,~~ 623 Essman 13,570 2 years ~ - g Little, Catt~ ell cular Jacobson Lynch 628 Finley 26,105 3 years (~`~r^dN~ Sommers, Loosli ~^~ ~~~ Reimann, Bing-' 629 Erickson chosomatic ogy - Psy-_ Jacobson • 93350 1 year . . Pharmacol- Cattell, Little 630 Parshley 20,169.50 2 years .rk) ^%flN"Tri.pstre- Loosli, Sommers ogy Jacobson Reim2•nn, Little 633 Boyarsky 61)950 3 years -.Pharmacol- Cattell, Bing 4

PLJRRET+1T GREiIVTS '!BY SUBJECT

Carcinogenesis 413 C. Leuchtenberger 4/1/67 55,150 451 M. S. Protzel 2/1/67 31,353 558' G. H. Friedell 11/1/66 36,453 521 C. Biancifiori 3/1/67 10,100 603' S. L. Kauffman 9/1/67 18,439 606 A. Furst 4/1/67 33 314 ~ ~ 564 J. U. Schlegel 1/1~67 23',000 566 K. M. Lynch & F. A. McIver 10/1/66 27,121 567 L. Severi 10/1/66 16,ooo 561 - G. L. Bririlanan 11/1/66 15,653 23b, 5 83 $445,740 3% 6--

310 R. J. Bing 467 T. C. Westfall 472 J. E. P. Toman 477 M. N. Carroll Jr. 513 H. Engelberg 569 K. M. Moser 607 S. Bellet 612 J. D. Coffman 618 E. A. Murphy Pharmacology Activated Amount 7/1/67 11,261 9/1/67 11,500 7~1%67 19,620 7/1/67 24,966 10/15/66 15,137 7/1/67 17,000 7/1/67 8~430 1 206 E. F. Domino 1/1/67 491 J. P. Long 6/1/67 492 W. M. Booker 7/1/67 543 U. Schaeppi 6/1/67 548 S. N. Pr adhan $ 588 B. Bhagat %1%67 598 U. Schaeppi 6/1/67 Chemistry 223 H. S. McKennis Jr. 7/1/66 486 A. A. Albanese 1/1/67 534 P. Goldhaber 7/1/67 579 N. N. Chopra 6/1/67 609 D. B. Louria 7/1/67 620 A. Weinstock 6/1/67 33,483 8,550 17,705 17,260 9,695 100 ~75 , 52,598 132869 23,396 (?) - 23,955 28,092 ~40 03~6 lti1, 946 12% $4'17, 073 ~ 28% W Clt .~ . Q? Q? N ~ CARDIOV'ASCULAR, -PHARMACOLOGY & CHEMISTRY

EPIDEMIOLOGY, PSYCHOSOMATIC & STATISTICS • Activated ' Amount 455 C. L. Rose 1/1/67 510 D. J. Massaro .2/1/67 576 J. H. Rogers (18 mos.) 2/1/67 800 9,315 15,000 25,115 Psychosomatic 517 A. Damon 2/1/67 527 N. W. Heimstra (to 12/31/67) 6/1/66 555 G. M. Smith 7/1/67 610 C. B. Thomas 6/1/67 ^ LUNG PHYSIOLOGY 478 S. M. Ayres 497 A. A. Liebow 519 L. A. Soloff 528 S. Buckingham 546 I. Mandl 547 G. A. Laurenzi 565 L. S. Tsai 592 D. J. Massaro 599 D. K. Aviado 604 R. H..Earle PATHOLOGIC DIAGNOSTIC 423 526 559 585 614 B. Eichel R. K. Larson S. C. Sommers G. W. Smetters W'. F. McNary Jr. M. S. Segal 13,282 9,487 14,184 29 865 $91,933 7/1/67 22,890 10/1/66 9,499 2/1/67 33,600 7/1/67 15,155 7/1/67 25,969 7/1/67 12,229 7/1/67 18,000 5/1/67 7,820 4/1/67 24,497 7/1/67 14,970 8/1/67 17,950 8/1/67 17,424 4 1%1%67 11,963. W 6/1/67 16,906 7/1/67 34 500 , ~ ~ $113,279 N

24 P. S. Larson 80 Fellowship Program 591 P. S. Larson Activated Amount 1o/1/66 21,835 4/1/67 24,845 10/1/66 10,000 1/1/67 16,835 lo/1/66 53,755 $123,270 7/1/67 22,433 4/1/66 7,430 $293 7/1/67 23,921 6/1/67 56,000 $79,921 $1,485,708 F. B. Bang B. K. Watson 0. R.. Jones J. E. Craighead C. G. Loosli ORAL CAVITY 451 R. M. Pangborn~ 529 J. H. Manhold (18 mos.) MI'SCELLANE0LIS

R}ti;~+:d-.

® ® r 1 '-' ~, ~ 206 E F Doanino ~ ~~ ~ ~`` 455 C L Rose ,~~'~+` F. Hamburger A. A. Albanese 550 J. E. Craighead M1 Ift 0 _xr F Hamburger ~r~ ~ f'- ' 451 M S Protzel j ~~ V ~ ~ :510D J Massaro xr,C .t7516 D M Pace ~ ~ 517 A Damon 519 L A. `Soloff f i o. Grantee' 486 564 J. U.'Schlegel 585 G. W. Smetters a t :± 588 B. Bhaga . . 338' 572 T. T. Crocker 576 J. H. Rogers (18 mos. 521 C. Biancifiori r CURRENT GRANTS ' (By Date of Activation) Date 1966 2/1 3/1 1 7 33,483 70,000 .13,869. 16,835 23,000 11,963 9,695 33,131 15,000 10,100 $401,452 11 ~ - -- - _ ~a-- '~.-TFIE COUNCIL FOR TOBACCO RESEARCH-U.B.A.

0 . Grantee 413 C. Leuchtenberger .493 B. K. Watson 529 J. H. Manhold (18 mos.) 599 D. M. Aviado 606 A. Furst 592 80 491 527 543 579 598 ~ 6o8 D. J. Massaro Fellowship Program J. P. Long N. W. Heimstra (to U. Schaeppi N. M. Chopra U. Schaeppi W. F. McNary Jr. 12/31/57) 610 C. B. Thomas 618 E. A. Murphy 620 A. Weinstock ._.~--- ------_-_,. 4/1 55,150 " 20,845 I, m m 5/1 7,430 7,820 6/1 56,000 of 8,550 n n Q m m ,t m n 9,487 17,260 23,955 10,175 16,906 29,865 8,430 4o, 036 $16,917 $352,803 ~ O O W ~ ~ ~ ~ C.~ O

• H. S. McKennis Jr. 310 R. J. Bing 461 R. M. Pangborn .472 J. E. P. Toman 477 M. N. Carroll Jr. 478 S.' M. Ayres 492 W. M. Booker 513 H. Engelberg 528' S. Buckingham 534 P. Goldhaber 546 I. Mandl 547 G. A. Laurenzi 555 G. M. Smith 559 S. C. Sommers 565 L. S. Tsai 604 R. H. Earle 607 S. Bellet 609 D. B. Louria 612 J. D. Coffman 614 M. S. Segal 423 B. Eichel 526 R. K. Larson 54$ S. N. Pradhan Date 7/1 m m m 22,433 n 15,225 m 19,620 ,, 22,890 ir 17,705 of 24,966 n 15,155 „ n ti m m ® ® 25,969 12,229 14,184 14,536 18,000 14,970 17,000 ~ 28,092 5,192 34,500 ~ O p 17,950 Ca CA 17,424 ~ 9,928 W N ® i, 8/1 ®

No. Grantee 467 T. C. Westfall 591 P. S. Larson 3 S. L. Kauffman Date 9/1 $52,598 $456,485

. 409 497 523 566 567 573 569 558 561 F. B. Bang 10/1 21,835 A. A. Liebow " 9,499 rr 0. R. Jones 10,000 K. M. Iynch & F. A. McIver " 27,121 L. Severi " 16,000 C. G. Loosli " 53,755 K.. M. Moser 10/15 15,137 G. H. Friedell 11/1 G. L. Brinlcnan "' 36245315,651 $205,453

t

Tm~ CUUtcIL FOR TOBACCO RESEARCIi-U.S.r'1. TO: -The committee comprising Dr. Cattell, Chm., Dr. Jacobson and Dr. Bing l '.~v . , FROM: Robert C. Hockett SUBJECT: Renewal application from Herbert McKennis, Jr., Ph.D, - No. 223-R8. "f,"t recent years. year study, with support at approximately the same rate as we provided in basis, but it should be noted that the present proposal anticipates a three- letter whs.ch describes the change in emphasis to biological and enzymatic studies, is included. 'In the past the grant has been renewed on an annual Jr., of the Medi.cal College of Virginia, Richmond, Virginia. His covering We enclose herewith a renewal application from Dr. Herbert McKennis$ Council has supported for the past eleven years. This has been a very worthwhile and productive program, which the we would need to scale down appropriations for work in this area. indefinitely (a point also raised the year before), and it was indicated that _ When the grant was renewed last year it was emphasized that we would not be able to maintain support for studies on nicotine metabolites R. C. H.

DEPARTMENT.OFPHARMACOLOGY. RICHMOND.. VIRGINIA.23219 ..July 12, 1967 Dr. Robert C. Hockett Associate Scientific Director The Council for Tobacco Research - U.S.A. 633 Third Avenue New York, N. Y. 10017 Dear Bob: I am enclosing an application that describes in a brief way research thoughts in~the general area of nicotine metabolism. We are very tardy in submitting this application. The reason, in part, has been our desire to run through some preliminary or range-finding experiments and to establish a feeling that we are on the right course and had developed sufficiently workable techniques so that contemplated work could proceed without delay. We now feel that this is the case and hope that the Council will see fit to consider our application, which in many ways is a continuation, with more of an emphasis on biological and enzymatic studies -- in contrast to much of the earlier work in which we concentrated on the problems of structure and syntheses of many of the nicotine metabolites. To bring you up-to-date in an informal way on some of the more recent happenings that have not yet been reduced to manuscript or report form a few comments will be made. ~ Preliminary experiments were conducted on the effect of age on the ~ ability of rat-microsomal preparations to metabolize nicotine. Using our Q younger rats (150-200g) as a standard it appeared quite consistently that Cj equivalent weights of the liver fractions from the older animals (400-500g) CA showed a lesser ability to metabolize nicotine. Studies to relate this ~ apparent deficit to the composition of the liver fraction and comparative ~ studies on the ability of animals of varying ages to metabolize labelled ~ nicotine are contemplated. Isotopic material is already on~hand for these ~ studies. At earlier dates we talked about the progress made on the electron-capture studics with pentafluropropionylmetanicotine that had been derived from nicotine and pentafluropropionic acid anhydri~de as a basis for the quantitative determination of nicotine (with support from the kMA Education and Research Foundation). The major drawbacks to the analytical method, in our goal to reach 1 x 10-12 grams included a broad peak which could not be sharpened by .. . . . . . - . _. . -._. .. . -._. .. .J:..r._._. . _ «~-....a......... .. ._.«~......-.r...

Dr. Hockett Page Two • July 12, 1967 increasing column temperature because of the backgroundor trash that appeared in biological material. This problem seems to have been overcome in part by modification of the injection part of the apparatus (a standard procedure). Additionally, the basic chemistry of the reaction has been extended to nornicotine. The analytical sample of pentafluropropionylmetanicotine (by analysis) shows a retention time different from that of the corresponding nicotine derivative. In consequence we feel more and more secure about the method. In the midst of other things we are setting up to run kinetic studies on the conversion of y-3-pyridyl-y-methylaminobutyric acidito cotinine. At least at physiological pH ranges this should be relatively simple to accomplish polarimetrically in view of the marked differences in the specific rotations of the two compounds. The data should help persuade people of the care needed in isolating cotinine. We shall be grateful as always for any considerationigiven to our application. A copy with the signature of the college business officer will follow. Please do not hesitate to write or phone in the event of any questions. We are grateful for the generous and helpful support of the past and' close with our best personal wishes. Sincerely, Herbert McKennis, Jr. HMcK/bb

Date. . Nameoflnvestigotbr(s)e(inciudeTitleandDegrees) Herbert McKennis, Jr., Ph.D., Professor of Pharmacology z Uutttutton & • Address: i`ledical College of Virginia, Richmond, Virginia 23219 Factors Controlling the Biological Disposition of Pyridine .;Coupounds of Tobacco Smoke {~. . . _ :• I. ProposedStartingDate: July 1, 1967' =` S. AnGcipoted Duration ofithis Specific Study: 1967-197D (3 years) 'it has been possible to•describe the broad outlines of many of the routes involved in the 6. Brief Descripton of Objectives or Specific Aims: From a series of experiments with support of the Couneid for Tobacco Researeh-U.S.A., .:~r -__c:;, i, ..c;.•oammalian metabolism of nicotine, nornicotine, and 3-acetylpyridine -- three of the many ; pyridine compounds that occur in tobacco and tobacco smoke. [The general nature of the 4igiRf. FI previous studies are given in the publications which are enumerated in an attached 1ist.) Through. isolation or total synthesis it has been possible to supply many of the mammalian .::pharmacologica3 properties of many nicotine metabolites. •' metabolites to biological investigators and. to assist them in a variety of work on the +=" , It is now desirable for us and interesting to us to expand our knowledge of the factors ~.='~'''which determine the disposition of pyrid3ne compounds arising from tobacco smoke and eaptore oiocnemicai1 inieraccions OeLWCeID Yml1AC WNpvucuw. rLC16 a~s aireauy in tne puo- lished literature some data that suggests that desensitization or tachyphylaxis to nicotine arises in part through a stimulation of the enzymes that are concerned with, the : aetabolism-of nicotine. It has been suggested that this stimulation of metabolism is ; produced by nicotine and:that other substances have a similar capacity to enhance the metabolism-of nicotine. Direct studies on nicotine metabolism-are however very limited ., in their scope. "determine the nature of stimulation or inhibition of metabolism at almost all- of the key C points that are involved in mammalian systems. Similarly, it should be possible to determine individual differences in the capacity to metabollize nicotine and related pyridine compounds. (continued on sheet la attached). 7. Give a Brief Statement of your,Working Hypothesis: Current data on the effect of various smoke components on the metabolism and transfer of nicotine and other alkaloids are limited or apparently contradictory. A study of these factors should be helpful to an- understanding of the.biological effects of tobacco smoking. have already described,, and through synthetic routes which make possible the labelLing of nicotine and related in almost any desired position, we believe that it is now possible to Through the current availability of synthetic routes to nicotine metabolites, which we [o-`mm a- CiAW 49MM ~ ~

Brief Description of Objectives or Specific Aims: (continued) ~.,_ . ~ . .. . . -~~. ..',. -.. ,. , . . .-. In the case of some of the simpler metabolites of nicotine, such as methylamine, 'it is "i relatively easy to obtain a broad picture of the nature of compounft which interfere with metabolic events. Work with 1`'C-methylamine in our own laboratory (The Pharmacologist, Fall 1967, in press) and others illustrates that interference with oxidation of rz ;}` T methylamine maY be produced by some types of monoamine-oxidase inhibitors. The data " suggest, however, that the inhibition may be referrable to-a so-called methylamine : oxidase which has not been fully characterized. Liver preparations obtained from rats after administration of monoamine-oxidase inhibitors have thus far been shown to be as fully capable as control livers in tests which, involve a spectrophotometric measurement of the disappearance of•nicotine. From another series of experiments, the data now suggests that the oxidation of nicotine can be impeded by the presence of large quantities of cotinine. It would, be desirable to study possible effects of other pyridine compounds 'T of smoke on the metabolism of nicotine and related substances. The.need for a biologically. , oriented study appears to be re-emphasized by apparently conflicting chemical data on the _ oxidation of nicotine. For instance, the pyrolidine-N-oxide of nicotine has been considered; (by J. C. Craig) to be the logical key intermediate in the biological.formation of cotinine, while Linnell has reported that the same oxide in vitro inhibits the oxidation ;..;: ~f nicotine to cotinine. 0

1 8. Details offiExperimentat Design and Procedures: (Attach Separate Pages) With the aid of isotopically labelled.nicotine and its metabolites it is feasible to study by a number of different methods factors influencing the disposition of nicotine. It is anticipated that during the current studies both in vivo and!in vitro methods will be employed. _ Initial experiments to determine the suitability of the rat as an experimental animal for initial studies have already beem conducted. Liver microsomal preparations from both :young and old rats of the >:,'?Ystar strain have been examined for their capacity to metabolize . ;;;,y nicotine in vitro. The general picture currently presented is that equivalent fractions " by weight of liver that are obtained from older animals (250-550g body weight) do not ~: metabolize nicotine as effectively as do the respective fractions from younger animals (150-175 g body weight). It is not currently:known whether or not the observed deficiency •in the older animals can be attributed to a lesser amount of metabolizing cellular •'•.fractions per unit of liver weight, or an overa•11 lessening of metabolic ability that exists throughout the animal. It would-be desirable to settle this point and to determine ; whether or not the elimination of unchanged nicotine by the older animals suffers any ` impairment. The additional matter of elimination can.be settled without modification of the experimental design of the metabolic experiments. Questions of this type can be .•"answered by comparing the ability of groups of young and old'animals to'metabolize (continued--on sheet 2a attached) • . 9. • Physical Facilties Available (Where Other than Administering Organization Indicate Geographical Location) Laboratories and animal facilities are.located on the fourth and fifth floors of McGuire Hall at the Medical College of Virginia. Gas chromatography and isotope counting facilities are available in areas convenient to the working laboratories but separate from these. The equipment list includes a preparative ultracentrifuge, polarimeter, Wa bu a lratus, hydrogenation apparatus, and!the general equipment of chemical, and 1~ A~citiortequirements: biological laboratories. , Additional requirements, if any, would depend!upon the outcome of the research. J • 11. Biographical sketches of all:principal and professional personnel (append) 12. List of publications: (Five most recent as pertinent) (append) -~~ _..,...,.....- .._.KS,.r. ~ . . • ... .. ... ._ . ..- . _.._ .. .._ .~.._....._._,.. .__ .. ...._.,_..,...- .~.~.,.~,.._,..~ ......_

• 2a. 8. Details of Experimental Design,and Procedures: (continued) 14C-labelled nicotine and to eliminate unchanged alkaloid. For all of this isotopic material is now available.as a result of our synthetic work and previous :; nutriculture: nicotineC (random), nicotine-1'C-methyl, and nicotine-2-14C. -"`'In additiom, six of the mammalian metabolites of (-)-nicotine are available in both• _:tF ' isotopic and'non-isotopic form so that quantitative data can be obtained. 0

R: REDACTED MATERIAL •- E. Consvmabie Supplies (iist by catepories) --Chemi.cal Reagents .Isotopes Glass ware : Animals and feed Year 2 Year 3 C. Other: Expenses Qtemize) Reprints and page charges •Travel (Presentation of reports and conferences) E.. Overhead (] 5% of A+ B+ C) EstimatedFuture Requirements: Salaries Consumable Suppl. It is understood thotthe applica in applying fona grant have re the Council's "Statement oi. Pot and?erms Under Which Project Sub Totai I OtherExpenses PermanentEquiR. 250 500 Ovencead Total S,S00 _ 1,000 1,000 6,675 52,175 6,000_ 1,000 1,000 7,050 55,050 // ~ Siqnature 1'..~- ~ t. / / /y~.( r t and institutionai officen 644-9851 xt. 8153 ~ ad and found accept661t - cy Containing Conditions Signature Telephone Grants Are Made-`° wu ~aoflk..ohhtlmGru6°n ~•1NJE. ~L [;~: ;S VICE Te4ephone n i ML VJ ~a );h W 'L•-•- "~ +~ at meetings Ar=

i , List financial support fea research from oPl sources, tndudinp own instituNon, for this and/or related research projects. . Quantitative Methods for the'Detesmination*of the Distribution of Nicotine and Its Congeners in Biological Systems Institution supplies salary of principal-investigator and all secretarial help currently obtained through other funds None other than this application for the Council for Tobacco Research - U.S.A. VD99PSC00t

R: REDACTED MATERIAL B'iographical'Inyormation on HERBERT I•icKEtJi+tll S , JR. '9-.. ~- _ _ ~ Attended elementary schools in Scarsdale, N.Y. and Loomis School, Windsor, Conn. , Harvard S~.II . , ~ and Cornel•-l. Fh..D-. Chemist at Nuodex Products Co., 1938-1939, and Ciba Pharmaceutical Products Co,, 1940-1942, Appointed Assistant in Biochemistry, Cornell University Medical College, 1942; Assistant Professor of Chemistry, Medical College of Virginia, 1945; Instructor in Physio- logical.Chemistry, the Johns H'opkins-University, 19:46; Associate Professor of-Biochemistry, Medical College of Virginia, 1948; fie•ad, Basic Sciences Res'earch Department, Naval C. E. Laboratory, 1949; Associate-Professor-, 1953, Professor of Pharmacology, Medical College-of Virginia, 1955. Biochemist; specialist in intermed!iary metabolism, anti- biotics, alkaloids, polymeric compoundis. U..S. and-foreign patents: soil stabilization and antispasmodic. Has done a wide variety of scientific-advisory and technical consulting work for industrial firms, government agencies,' scientific organizations, and universities. Visiting Professor•, University of Chile, 1960; Hono•ra•ry member-, Faculty of Medicine, University of Chile, Sociedad de Biologia, de Santiago. Sigma Xi. Phi Lambda Upsilon. American-Chemical Society, American Society of Biological Chemists, The Society for Experimental Biology and Medicine, International Oceanographic R'oundation, The New York Academy of Sciences, American Society for Pharmacology and Experi- mental Therapeutics, American Associ!_&ti:orn for the Advancement of Science, Society of American, Mil-itary Engineers, American Institute of Chemists, Virginia Academy of Science, Society of Toxicology.

s- Edward R. Bowman Born: West Virginia 1927 Medical C.>llege of Virginia Citizen U.S.A. Richmond, Virginia Education: -Concord College B.S. 1952 (Biology - Chemistry). West Virginia University 1953 (Physi:ology). Duke University 1955-56• (Graduate Student-in Physiology). %=. Medical College of Virginia PheD. 1963 (Pharmacology). Experience: 1961 - present • 19'56 - 1958 1955 - 1956 1954 - 1955 ~ 1952 - 1953 1952 195 0 - 1951 •.19'47 - 1950 •1944 - 1946 Research Associate Department of Pharmacology Medical College of Virginia Richmond, Virginia Graduate Student, Major - Pharmacology Minor - Physiology & Biochemistry Medical College of Virginia, Richmond., Virginia Research Assistant Department of Pharmacology Medical College of Virginia Richmond, Virginia Graduate Student, Major - Physiology Minor - Anatomy Duke University Durham, North Carolina Bacteriologist State Department of Health Richmond, Virginia Graduate Student, Physiology West Virginia University Morgantown, West Virginia Student, Biology & Chemistry Concord College N Athens West Vir inia O , g O U.S. Army ~JI EA Student, Biology & Chemistry Concord College ~ Athens , West Vir inia g Q? U.S. Arm.y

R: REDACTED MATERIAL ACTED 0

PUBLICATIONS OF PROJECT WORK SUPPORTED BY THE COUNCIL FOR TOBACCO RESEARCH - U.S.A. 1. Synthesis and properties of pyridylalanines. Herbert McKennis, Jr., and Edward R. Bowman. Virginia Journal of Science, 8, 314 (1957). 2. Metabolism of y-(3-pyridyl)-Y-oxobutyric acid. Lennox B. Turnbull, Edward R. Bowman, and Herbert McKennis, Jr. Federation Proceedings, 17, 325 (1958). 3. Y-(3-Pyrid)rl)-Y-methylaminobutyric acid as a urinary metabolite "of nicotine. Herbert McKennis, Jr., Leiinox B. Turnbull, and Edward R. Bowman. Journal of the American Chemical Society, 79, 6342 (1957). 4. Metabolites of nicotine and a synthesis of nornicotine. Herbert McKennis, Jr., Lennox B. Turnbull, Harvey N. Wingfield, Jr., and Lovell J. Dewey. Journal of the American Chemical Society, 80, 1634 (1958). 5. The role of cotinine in nicotine metabolism Herbert McKennis, Jr., Lennox B•. Turnbull, and Edward R. Bowman. Abstracts of Communications, IV. International Congress of Biochemistry, Vienna, Sept. 1-6, 1958. 6. 7. 8. A constant rate infusion apparatus. Quentin S. McKennis, Edward R. Bowman, and Herbert McKennis, J Toxicology and Applied Pharmacology, 1, 61 (1959). Metabolism of nicotine to (+)-Y-(3-pyridyl)-y-methylaminobutyric acid. Herbert McKennis, Jr., Lennox B. Turnbull,*and Edward R. Bowman. Journal of the American Chemical Society, 80, 6597 (1958). Metabolism of nicotine in the human and excretion of pyridine compounds by smokers. Edward R. Bowman, Lennox B. Turnbull, and Herbert McKennis, Jr. The Journal of Pharmacology and Experimental Therapeutics, 127, 92 (1959). Demethylation of cotinine in vivo.. N O ~ Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, and Einosuke P+ada. Journal of the American Chemical Society, 81, 3951 (1959). GJ CA ~,. Oxidation of cotinine in vivo. ~ Edward R. Bowman, Lennox B. Turnbull, and Herbert McKennis, Jr. ~ ~ Federation Proceedings, 18, 371 (1959).

-2- 11. Depressor activity from nicotine metabolites. Edward R. Bowman, H. B. Kennedy, Jr., Einosuke Wada, and Herbert McKennis, Jr. - Abstracts of Communications, XXI. International Congress of Physiological Sciences., 41, Buenos Aires, August 9-15, 1959. The isolation andistructure of a ketoamide formed in the metabolism of nicotine. Herbert McKennis, Jr., Edward R. Bowman, and Lennox B. Turnbull. Journal of the American Chemical Society, 82, 3974 (1960). 13. Methylation-in the.metabolism of (-)-nicotine. •Lennox B. Turnbull, Edward R. Bowman, and Herber•t'McKennis, Jr. Federation Proceedings, 19, 268 (1960). 14. The excretion and metabolism of nicotine. Herbert MeKennis, Jr. Annals of the New York Academy of Sciences, 90, 36 (1960). 15. Norcotinine (desmethylcotinine) as a urinary metabolite of nornicotine. Einosuke Wada, Edward R. Bowman, Lennox B. Turnbull, and Herbert McKennis, Jr. Journal of Medicinal and:Pharmaceutical Chemistry, 4, 21 (1961). 16. Depressor effects arising from (-)-cotinine. Joseph F. Borzelleca, Edward R. Bowman, and Herbert McKennis, Jr. The Pharmacologist, 2, 72 (1960). 17. Oxidation of nicotine-C-14 and nicotine-methyl-C-14 in vivo. Lennox B. Turnbull, Einosuke Wada, Edward R. Bowman, and Herbert McKennis, Jr. Federation Proceedings, 20, 172 (1961). 18. Metabolism,of nicotine in the human. Edward!R. Bowman, Lennox B. Turnbull, and-Herbert McKennis, Jr. Virginia Journal of Science, 9, 438 (1948). 19. Degradation of (-)-cotinine in the human. Herbert McKennis, Jr., and Edward R. Bowman. Abstracts of Communications, V. International Congress of Biochemistry, Moscow, August 10-16, 1961, p. 393. 20. The mammalian degradation of (-)-nicotine to 3-pyridylacetic acid and other compounds. Herbert McKennis, Jr., Edward R. Bowman, and Lennox B. Turnbull. Proceedings of the Society for Experimental Biology and Medicine, 107, 145 (1961). 21. Metabolism of (-)-cotinine in the rat. Is Sorell L. Schwartz, Edward R. Bowman, and Herbert McKennis, Jr. Virginia Journal of Science, 12, 196 (1961).

-3- 22. Demethylation in the metabolism of (-)-nicotine. Herbert McKennis, Jr., Lennox B. Turnbull., Sorell L. Schwartz, Einosuke Tamaki. and Edward R. Bowman. The Journal of Biological Chemistry, 237, 541 (1962). .23. Metabolism of (-)-cotinine to a keto acid. . Sorell L. Schwartz, and Herbert McKennis, Jr. -Federation Proceedings, 21, 183 (1962). 24. .Studies on the respiratory and cardiovascular effects of (-)-cotinine. Joseph F. Borzelleca, Edward R. Bowman., and Herbert McKennis, Jr. The Journal of Pharmacology and Experimental Therapeutics, 137, 313 (1962). 25. Studies on the metabolism of (-)-cotinine in the human. Edward R. Bowman, and Herbert McKennis, Jr. The Journal of Pharmacology and Experimental Therapeutics, 135, 306 (1962). _ 26. Routes in the mammalian metabolism of (-)-nicotine to 3-pyridylacetic acid. Sorell L. Schwartz, Edward R. Bowman, and,Herbert McKennis, Jr. Abstracts of Papers, 16th Tobacco Chemists' Research Conference, Richmond, Virginia, Sept. 26-28, 1962, p. 7. 27. Studies on the metabolic fate of 3-acetylpyridine. Lennox B. Turnbull, Edward R'. Bowman, and Herbert McKennis, Jr. Abstracts of Papers, 16th Tobacco Chemists' Research Conference, Richmond, Virginia, Sept. 26-28, 1962, p. 6. 28. The corrected structure of a ketoamide arising from the metabolism of (-)-nicotine. Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, and Sorell L. Schwartz. Journal of the American Chemical Society, 84, 4598 (1962). 29. N-Methylation of nicotine and cotinine in vivo.' Herbert McKennis, Jr.,•Lennox B. Turnbull, and Edward R. Bowman. The Journal of Biological Chemistry, 238, 719 (1963). 30. Studies on-the degradation of the pyrrolidine ring of (-)-nicotine in vivo. Formation of Y-(3-pyri,dyl)-Y,-oxobutyric acid. Sorell L. Schwartz, and Herbert A•fcf:ennis, Jr. The Journal of Biological Chemistry, 238, 1807 (1963). 31. The synthesis of hydroxycotinine and studies on its structure. Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, and Einosuke Tamaki. Journal of Organic Chemistry, 28, 383 (1963). 32. (-)-Cotinine. Edward R. Bowman, and Herbert McKennis, Jr. Biochemical Preparations, 10, 36 (1963).

33. Conjugate formation in the metabolisni of 3-acetylpyridine..' Lennox B. Turnbull, Edward R. Bowman, and Herbert McKennis, Jr. Abstracts of Papers, 17th-Tobacco Chemists' Research Conference, Montreal, Quebec, Canada, Sept. 22-25, 1963, p. 11. A comparative study of the metabolic release of methyl groups from a series of N-methylpyridinium compounds. Herbert McKennis, Jr., Edward'R. Bowman, Antonio Horvath, and John P. Bederka, Jr. ~ Nature, 202, 699 (1964). i • 35. Mammalian degradation of (-)-demethylcotinine. Sorell L. Schwartz, and Herbert McKennis, Jr. Nature, 202, 594 (1964). 36. Disposition and fate of (-)-cotinine-H3 in the mouse. Edward R: Bowman, Eskil Hansson, Lennox B. Turnbull, Herbert McKennis, Jr., and Carl G_ Schmiterlt5w. The Journal of Pharmacology and!Experimental Therapeutics, 143, 301 (1964). 40. Alternate routes in the metabolic degradation of the pyrrolidine ring of nicotine. Herbert McKennis, Jr., Sorell L. Schwartz, and-Edward R. Bowman. The Journal of Biological Chemistry,, 239, 3990 (1964). Herbert MeF:ennis, Jr., Sorell L. Schwartz, Lennox B. Turnbull, Einosuke Tamaki, and Edward R. Bowman. The Journal of Biological Chemistry, 239, 3981 (1964). 37. Additional routes in the metabolism:of 3-acetylpyridine. Herbert McKennis, Jr., Lennox B. Turnbull, and Edward R. Bowman. The Journal of Biological Chemistry, 239, 121S (1964). 38. Effect of cotinine and other nicotine metabolites in-yitro on duod'enum-and;ileum segments. K. S. Kim, Joseph F. Borzelleca, Edward R. Bowman, and Herbert McKennis, Jr. Federation Proceedings, 23, 330. (1964). 39. The metabolic formation of y-(3-pyridyl)-y-hydroxybutyric acid and its possible intermediary role in the mammalian metabolism of nicotine. 41. Metabolism of 3-acetylpyridine to an analog of mandelic acid. Lennox B. Turnbull, C. N1. Lukhard,, and Herbert McKennis, Jr. Toxicology and Applied Pharmacology, 6, 362 (1964). 42. Disposition and fate of nicotine in animals. Herbert McKennis, Jr. Tobacco Alkaloids and Related Compounds, U. S. Von Euler, editor, Pergamon Press, Oxford, 1965, p. 53.

43. Urinary excretion of conjugate forms.of 1-(3-pyridyl)ethanol aftei administration of 3-acetylpyridine. Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman., and C. Norman Lukhard. The Journal of Biological Chemistry, 241, 1878 (1966). 44. The structure of dibromoticonine, a bromination product of nicotine. • New York, N. Y., Sept. 12-16, 1966. Abstracts of Papers, 152nd Meeting, American Chemical Society, Herbert McKennis, Jr., Edward R. Bowman, L. D. Quin, and R. C. Denney: 45. Studies on the synthesis and metabolism*of 4-(3-pyridyl)-4- methylaminobutyric acid-4-14C. Paolo L. Morselli, Edward R. Bowman, Helen H. 0ng, and! Herbert McKennis, Jr. Virginia Journal of Science, 17, 345 (1966). 46. The fate and distribution of 1-(3-pyridyl)ethanol methiodide in relation to the toxicity.of 1-(3-pyridyl)ethanol and 3-acetylpyridine. John P. Bederka, Jr., Eskil Hansson, Edward R. Bowman, and Herbert McKennis, Jr. Biochemical Pharmacology, 16, 1 (1967). 47. Studies on the separation of acidic metabolites of nicotine by gas chromatography. Herbert McKennis, Jr., Edward R. Bowman, and Mohammad Saeed Dar. Virginia Journal of Science, 18, 13 (1967). 48. Pharmacological action and intermediary role of 5-(3- -pyridyl)tetrahydrofuranone-2. Edward R. Bowman, Pavol lirdina and; Herbert McKennis, Jr. Federation Proceedings, 26, 616 (1967). 49. Metabolism of (')-Cotinine-2-Z 4C in the rat. Paolo L. Morselli, Helen H. Ong, Edward R. Bowman, and Herbert McKennis, Jr. Journal of Medicinal Chemistry, in press. 50. 1`1ethylamine metabolism in normal and, mao-inhibitor-treated Paolo L. biorselli, Edward R. Bowman,, and Herbert McKennis, The Pllarmacologist, in press. rats. Jr.

-6- 51. On•the congeners of whiskey. Herbert McKennis, Jr.,:-and;Harvey B. Haag. Journal of the American Geriatrics Society, 7, 848 (1959). 52. ,Depressor effects arising from (-)-cotinine. Joseph F. Borzelleca, Edwar.d R. Bowman, and Herbert McKennis, Jr. •The Pharmacologist, 2, 72 (1960). 53. The excretion and metabolism of nicotine. Herbert McKennis, Jr. Annals of the New York Academy of Sciences, 90, 36 (1960). 54. The isolation and structure of a ketoamide formed in the metabolism of nicotine. Herbert McKennis, Jr., Edward R. Bowman, and: Lennox B. Turnbull. . Journal of the American Chemical Society, 82, 3974 (1960). 5S. Methylation in the metabolism of (-)-nicotine. Lennox B. Turnbull, Edward R. Bowman, and Herbert McKennis, Jr. Federation Proceedings, 19, 268' (1960). 56. L-y-Glutamylhydrazine and the metabolism of hydrazine. Herbert McKennis, Jr., Allan S. Yard, Elizabeth J. Adair, and J. H. Weatherby. The Journal of Pharmacology and Experimental Therapeutics, 131, 152 (1961). ` 57. Demethylation in the metabolism,of (-)-nicotine in vivo. Herbert McKennis, Jr., Einosuke Wada, Edward R. Bowman, and Lennox B. Turnbull. Nature, 190, 910 (1961). 58. Norcotinine (Desmethylcotinine) as a urinary metabolite of nornicotine. Einosuke R'ada, Edward R. Bowman, Lennox B. Turnbull, and Herbert McKennis, Jr. Journal of Medicinal and Pharmaceutical Chemistry, 4, 21 (1961). 59. The isolation of 3-pyridylacetic acidi, a urinary metabolite of (-)-cotinine. Edward R. Bowman,, Lennox B. Turnbull, and Herbert McKennis, Jr. Abstracts of Papers, 14th Tobacco Chemists'- Research Conference, Oct. 13-14, 1960, Winston-Salem, North-Carolina. 60. Oxidation,of nicotine-C14 and nicotine-methyl-C14 in viyo. Lennox B. Turnbull, Einosuke iiada-, Edward R. Bowman, and Herbert McKennis, Jr. " Federation Proceedings, 20, 172 (1961). 61. Inhibition by thyroxine of y-aminobutyrate-a-ketoglutarate transaminasc. Antonio Horva:th, Fernando Orrego, and Herbert McKennis, Jr. Federation Proceedings, 20, S (1961).

62. Factors controlling the metabolism of Y-aminobutyric acid. Antonio Horvath, Fernando Orrego, and Herbert McKennis, Jr. The Journal of Pharmacologyy and Experimental Therapeutics, 134, ,222 (1961). 63. Mammalian degradation of (-)-nicotine to 3-pyridylacetic acid and other compounds. . Herbert McKennis, Jr., Edward R. Bowman, and Lennox B: Turnbull. Proceedings of the Society for Experimental Biology and Medicine, 107, 145 (1961). 64. Selective toxicity. (Book review). Herbert McKennis, Jr. Revista Medica de Chile, 88, 864 (1960). 65. Los oxoesteroides, el uso de-hidrazidos fen6licos para su- detecci6n, caracterizaci6n y medici6n,. (Book review). Herbert McKennis, Jr. Revista Medica de Chile, 88, 626 (1960). 66. Metabolism of nicotine in the human. Edward R. Bowman, Lennox B. Turnbull, and Herbert McKennis, Jr. Virginia Journal of Science, 9, 438 (1958). 67. Metabolism of (-)-cotinine in the rat. Sorell L. Schwartz, Edward:R. Bowman, andiHerbert McKennis, Jr. Virginia Journal of Science, 12, 196 (1961). 68. Degradation of (-)!-cotinine in the human.. Herbert McKennis, Jr., and Edward,R. Bowman. Abstracts of Communications, V. International Congress of Biochemistry, Moscow, August 10-16, 1961, p. 393. 69. Aspects of the metabolism of isoniazid and acetylisoniazid in the human and the dog. Allan S. Yard, and Herbert McKennis, Jr. Journal of Medicinal and Pharmaceutical Chemistry, 5, 196 (1962). 70. Demethylation in the metabolism of (-)-nicotine. , Herbert McKennis, Jr., Lennox B. Turnbull, Sorell L. Schwartz, Einosuke Tamaki, and Edward R. Bowman.. The Journal of Biol~ogical Chemistry, 237, 541 (1962). Inhibition of the catabolism of aspartic-4-C14 acid by thyroxine in vivo. Antonio Horvath, and•Herbert McKennis, Jr. Enzymologia 24 91 (1962) N 0 , , . Studies on-the metabolism of (-)-cotinine in the human. G.? C11 .~ Edward R. Bowman, and Herbert McKennis, Jr. ~ The Journal of Pharmacology and Experimental Therapeutics, ~ 135, 306 (1962). ~

73. Metabolism.of (-)-cotinine to a keto acid. Sorell L. Schwartz, and Herbert McKennis, Jr. Federation Proceedings, 21, 183 (1962). 74. The_excretion and metabolism of triethylene glycol. Herbert McKennis, Jr., Robert A. Turner, Lennox B: Trunbull, .and Edward R. Bowman. W. W. Muelder, M. P. Neidhardt, and"Carl L. Hake. - Richard Henderson, Herbert G. Nadaeu, and Samuel Spencer. Toxicology and Applied Pharmacology, 4, 411 (1962)1. 75. Studies on the respiratory and cardiovascular effects of (-)-cotinine. Joseph F. Borzelleca, Edward R. Bowman, and Herbert McKennis, Jr. The Journal of Pharmacology and-Experimental Therapeutics, 137, 313 (1962). 76. Routes in the mammalian metabolism of (-)-nicotine to 3-pyridylacetic acid. Sorell L. Schwartz, Edward R. Bowman, and Herbert hlcKennis, Jr. Abstracts of Papers, 16th. Tobacco Chemists' Research Conference, Sept. 26-28, 1962, Richmond, Virginia, p. 7. 77. Studies on the metabolic fate of 3-acetylpyridine. Lennox B. Turnbull, Edward R. Bowman, and Herbert McKennis, Jr. Abstracts of Papers, 16th Tobacco Chemists' Research Conference, Sept. 26-28, 1962, Richmond, Virginia, p. 6. 78. Acetylhydrazine as an intermediate in•the metabolism of aroylhydrazines. Lennox B. Turnbull, Allan S. Yard, and Herbert McKennis, Jr. Journal of Medicinal and Pharmaceutical Chemistry, 5, 1327 (1962). 79. N-Methylation of nicotine and cotinine in vivo. Herbert McKennis, Jr., Lennox B. Turnbull~, aE-d Edward R. Bowman. The Journal of Biological Chemistry, 238, 719.(1963). > 80. The synthesis of hydroxycotinine and:studies on its structure. Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, and Einosuke Tamaki. Journal of Organic Chemistry, 28, 383 (1963). 81. The corrected structure of a ketoamide arising from the metabolism of (-)-nicotine. Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, 82. and Sorell L. Schwartz. Journal of the American Chemical Society, 84~, 4598 (1962). (-)-Cotinine. Edward R. Bowman, and Herbert McKennis, Jr. Biochemical Preparations, 10., 36 (1963). ti

83.. Studies on the degradation of the pyrrolidine ring of (-)-nicotine in vivo. Formationm of.7-(3-pyri•dyl)-Y-oxobutyric acid. Sorell L., Schwartz, and Hkerbert McKennis, Jr. The Journal of Biological Chemistry, 238, 1807 (1963). 84. Conjugate formation in the metabolism of 3-acetylpyridine. Lennox R. Turnbull, Edward R. Bowman, and Herbert McKennis, Jr. Abstracts of Papers, 17th Tobacco Chemists', Research Conference, Sept. 22-25, 1963, Montreal, Quebec, p. 11. 85. Metabolic release of methyl groups from a series of N-methylpyridinium compounds. Herbert McKennis, Jr., Edward R. Bowman, Antonio Horvath, and John P. Bederka, Jr. Nature, 202, 699 (1964). 86. Mammalian degradation•bf (-)-demethylcotinine. Sorell L. Schwartz, and Herbert McKennis,, Jr. Nature, 202, 594 (1964). 87. Disposition and fate of (-)-cotinine-H3'in the mouse. Edward R. Bowman, Eskil Hansson, Lennox B. Turnbull, Herbert McKennis, Jr.; and Carl G. Schmiterlbw. The Journal of Pharmacology and:Experimental Therapeutics, 143, 301 (1964). 88. Additional routes in the metabolism of 3-acetylpyridine. Herbert McKennis, Jr., Lennox B. Turnbull, and Edtaard R. Bowman. The Journal of Biological Chemistry, 239, 1215 (1964). 89. The metabolic formation of Y-(3-pyridyl)-y-hydroxybutyric acid and its possible intermediary role in the mammalian metabolism of nicotine. Herbert McKennis, Jr., Sorell L. Schwartz, Lennox B. Turnbull, Einosuke Tamaki, and Edward R. Bowman. The Journal of Biological Chemistry, 239, 3981 (1964). 90. Alternate routes in the metabolic degradation of the pyrrolidine ring of nicotine. Herbert McKennis, Jr., Sorell L. Schwartz, and Edward R. Bowman. The Journal of Biological Chemistry, 239, 3990 (1964). 91. Effect of cotinine and other nicotine nmetabolites in vitro on duodenum and ileum segments. K. S. Kim, Joseph F. Borzelleca, Edward R. Bowman, and Herbert McKennis, Jr. Federation Proceedings, 23, 330 (1964). 92. Metabolism of 3-acetylpyridine to an, analog of mandelic acid. Lennox B. Turnbull, C. N_ Lukhard, and Herbert McKennis, Jr. Toxicology and Applied°Pharmacology, 6, 362 (1964). ..~,.~._.;~::....,..d.~~...~.».-.: ~~w...

.-10- + 93. Disposition and fate of nicotine irn animals. Herbert McKennis, Jr. ' Tobacco Alkaloids and Related Compounds, U. S. Von Euler, editor, Perganon Press, Oxford, 1965, p.'53. 94. Viewpoints of the study of drug toxicity. (Book review). Herbert McKennis, Jr. American Journal of Pharmaceutical Education, 28,469 (1964). 95. Urinary excretion of conjugate forms of 1-(3-pyridyl)ethanol after administration of 3-acetylpyrid2ne. Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman, and C. Norman Lukhard. The Journal of Biological Chemistry, 241,.1878 (1966). 96. The structure of dibromoticonine, a bromination product of nicotine. Herbert McKennis, Jr., Edward R. Bowman, L. D. Quin, and R. C. Denney.

Papers Published During the Period of This Report i 1. Urinary excretion of conjugate forms of 1•-(3-pyridyl)ethanol after administration of 3-acetylpyridine. Herbert McKennis, Jr., Lennox B. Turnbull, Edward R. Bowman,, and C. Norman Lukhard. The Journal of Biological Chemistry, 241, 1878 (1966). 2. The structure of dibromoticonine, a bromination.product of nicotine. Herbert McKennis, Jr., Edward R. Bowman, L. D. Quin, and R. C. Denney. Abstracts of Papers, 152nd Meeting, American Chemical Society, New York, N. Y., Sept. 12-16, 1966. 3. Studies on the synthesis and metabolism of 4-(3-pyridy.l)-4- methylam%nobutyric acid-4~I`'C. Paolo L. Morselli, Edward R. Bowman, Helen H. Ong, and Herbert McKennis, Jr. , Virginia Journal of Science, 17, 345 (1966). 4. The fate and distribution, of I-(3-pyridyl)ethanol methiodide im relation to the toxicity of 1-(3-pyridyl)ethanol and 3-acetylpyridine. John P. Bederka, Jr., Eskil Hansson, Edward R. Bowman, and Herbert McKennis, Jr. Biochemical Pharmacology, 16, 1 (1967). 5. Studies on the separation of acidic metabolites of nicotine by gas chromatography. ` Herbert bicKemtis, Jr., Edward R. Bowman, and Mohammad Saeed Dar. Virginia Journal of Science., 18, 13 (1967). 6. Pharmacological action and intermediary role of 5-(3- pyrid'y1)tetrahydrofuranone-2. - Edward R. Bowman, Pavol lirdina and Herbert McKennis, Jr. Federation Proceedings, 26, 616 (1967). 7. b1etabolism• of (=)-cotinine-2-14C in the rat. Paolo L. Morselli, Helen H. Ong, Edward R. Bowman, and Herbert McKennis, Jr. Journal of Medicinal Chemistry, in press. 8. Methylamine metabolism in normal and mao-inhibitor-treated rats. F.1 Paolo L. Morselli, Edi,rard~ R. Bowman, and Herbert McKennis, Jr. O The Pharmacologist, in press. CO CA 4 0? M ~ ~ . . o~

Talks Given During the Period of this Report 1. Studies on the synthesis and metabolism,o.f 4-(3-pyridyl)-4- methyiaminobutyric acid-4-14C. Paolo L. Morselli, Edward R. Bowman,•'tlelen`II: Ong, and Herbert McKennis, Jr. Annual Meeting 'of the Virginia Academy of Science, Harrisonburg, Virginia, May 4-6, 1966.- 2. The structure of dYbromoticonine, a bromination produce of nicotine. Herbert McKennis, Jr., Edward R. Bowman, L. D. Quin, and R. C. Denne'y:--i52nd-Meeting, American Chemical Society, New York, N. Y., Sept: 12-16, 1966. 3. Some new-synrthetic routes to metabolites of nicotine. IIerbert.. McKennis, Jr., Biological Seminar, Medical College of Virginia, Richmond, Virginia, Sept. 28', 1966. 4. Quantitative methods for the determination of the distribution of nicotine and its congeners in biological systems. Iierbert McKennis, Jr., American Medical Association 14orkshop,, Colorado Springs, Colorado, Nov. 1-3, 1966. 5. Studies on 1''C-labell-ed nicotine metabolites. Edward R. Bowman and Herbert McKennis, Jr., American Medical Association Workshop, Colorado,Springs, Colorado, Nov. 1-3, 1966. ~ a... r. n.. . Pharmacological action and intermediary role of 5-(3-pyridyl)tetrahydrofuranone- Edward R. Bowman and Pavel Itrdina. S1st Annual Meeting of the Federation of American Societies for Experimental Biology, ;-Chicago, Illinois, April 16-21, 1967. . Pharmacological effects of some nicotine metabolites and,related r-compounds. --K.--S. Kim and J. F. Borzelldca, S1st Annual Meeting of the Fed'eration of American Societies for Experimental Biology, Chicago, Illinois, April 16-21, 1967. 8._ PentafluropropionyLation in•the determination of nicotine. "`llerbert McKennis, Jr., S. C. Srivastava, and Edward R. Bowman. Annual Meeting of the Virginia Academy of Science, Roanoke, Virginia, May 6, 1967.

---

'r.8'_.1T=.~ . T C HF OU\CIL FOR TOBACi;O RE-T .SLAI2CFIT,S,A, ( ' ~ MEr ')RAr]DUM . . . ~. ... .. l ~--~ 4 _ . . '. ~ . _ :_~}i . \~ . .1 }. , l 4 The committee comprising Dr. Little 1. r.«. v Y.. •. i.: u FROM: ~ Dr. Loosli and Dr. Sommers. -` '• ° ~Robert C. Hockett 1 WAS w Au :J . ..~ k y ! st 16, 1967 •' .. I 4~ .h!T. Chm., Dr. Lynch, Dr. Reimann, . _~- r.~ from George 0. Gey, M.D. SUBJECT: Application for renewal and extension No. 361-R4. - :: _ ,.. ... ' We enclose herewith a renewal and extension application from Dr G 0 . , ;Following earlier grants, a five-year plan of research was ap :. •proved by the Board and activated as of September 1, 1962. It was renewed ; th t eorge ..Gey of the Johns Hopkzns Medical Institutions of Baltimore, Maryland, together with a comprehensive Progress Report . . left a final six months for the five~-year plan. t a .. . . .. - . w g of September 17-18, 1966) to keep the prbject operating while comprehensive a~_w progress reports were prepared. This six month extension to March 1, 1967.;'` i ree mes as of the annl.versary date. However, as of September 1, 1966, ; the renewal was made for six months only at Dr. Gey's request (SAB'meetin ~ o e ouncil. t" 1Y ~S7 r _f This comprehensive report of progress has now been received It contains six parts. Of these, parts four and five seem to the undersigned'` ~~~ especially relevant to the current special interests f th C gran , ence e present application is for sixteen months. •The rate h ~t The report is accompanied by an application for continued support ~~ for the last six months of the five-year plan, retroactive in effect to x~,~~ March 1, 1967 and also requests a new grant for the period'September 1, 1967 ,'" . to June 30, 1968 in order to conform the anniversary to that of his other major t H th . J. This new grant application forecasts two more years at a comparable While the application proper does not give detail of plans and Council support to'Dr. Gey has been long-continued and substantial. We regard this as justifying appointment of large subcommittee for careful evaluation of the_.rather lengthy reports. per mont is, however, unchanged. ~ rate. ;objectives, these are set forth in context in the six-part progress report. 4~t,;. h'y r :.I ..I~ ~'.:SN1T ~ 1 y.

Renewed: 9/1/66 (6 ma. to 3/1/67) cr. #L34 Activated: 7/3/56 Renewed annua].I,y to 9/L/6i Date: August 4-, 1967 Geor•e 0. Ge M.D. Assoc. Prof. :`_3 ameof (mestigotor(s): (includeTUe ond Degrees) PRINCIPLE INVESTIGATOR: 6E g y~ ~ Asst. Prof. Surg. S Phys. Chem.; 2)Mary Reed, Ph.D., Res. Assoc.; 3)Roland Pattillo, M.D., Fellow S 4 C h r Armstron ne at e Emer. Surg. & Dir. Finney-Howell Can, Res. Lab. CO-INVESTIGATORS: 1)Chiun, T. Ling, Dt:D., Scd., i g urg., ) d antibody. Department of Surgery ••5)Darwin-Chee, Grad. Student '' The Johns Hopkins Medical-Institutions .6)Tom Depner, Med. Student eells,maylbe observed to grow and function with the elaboration after challenge of specific circumstances under which immunologically competent cells, such as lymphocytes and plasma (Ling f, Gey). One prime objective is the evaluation of the nutritional requirements and This J - For the 16-montfi period given in item 4 with two additional years required. & Brief Descripton.of Objecluves or5pecificAims: 1) Studies on nutrition and. environmental, factors affecting the growth of cells in culture request to June 30, 1968. 5. Anticipoted Durotion of this Specific Study: C-• OF FELLO{4SHIPS. 4. Propoted5tartinD-Date: March 1, 1967 (Period March 1-Aug. 31 previously committed). cell to virus (Chee f,, Gey). 4) Evaluation of cellular transformation in vitro--Role of tumor DNA (teznikoff & Gey). 5) Evaluation of HeLa cell antigens in a study of genetic exchange of information from host o , c arac erization o c orionic gona r p t ate, Address: Finney-Howe1L Cancer Research Laboratory Reznskoff, Ph•D. cands ., Baltimore, Maryland 21205 yr. IV, Res. Assoc. 3. ShortTideofBrojed: CONTINUATION OF STIIDLES ON CHARACTERISTICS OF NORMAL CELL. GROhTH IN RELATION TO INVASIVE CARCINOMA, EMBRYONAL TO ADULT FORMS. SUPPORT Isolation and studies of normal and malignant Iung cells--cytology, karyology, low temper- ature storage properties, mucous secretion and surfactant production (Reed & Gey). ) Factors contributing to functional growth of malignant trophoblast. Che>aical and biological t o i derived in vitro (Pattillo f Ge ) f h d h 6) Search for immunoglobulinS.in cultured lymphoblasts of the MBILI (deBruyn-Gey) line (Depner & Gey). , on known histogenic cell types. 17 We have already created a number of in vitro cell isolates in attempts to evaluate in vivo cell systems especially in a study of neoplastic processes. Cells "in culture C.i•Dermit fund'amental studies of growth, differentiation and mallignant transformation. tsikich- can also be learned of the functional capacity of normal, and maLignant cells and. may, indeed, provide the best systems for the evaluation. of a vari-ety of specific stresses 7. Give a Brief Statement of your. Working Hypothesis: .r domm `-

A. Physical Facilfies Available (Where Other 1hanAdministering Organization Indicate Geographical Location) ~. Biographical sketches of all principal and professional personnel (append). SEE ATTACHED:SNEETS'. 12. list of publications: (Fi've most recent as pertinent) (append) SEE ATTACHED SHEET. cy- 0 ~WR V~I y~I W =

R: REDACTED MATERIAL A. Sa(aries (Penonnel by names) Professional '7,George 0. Gey, M.D., Assoc. Prof. Emer'. Surg. i::',`,thiun T. Ling, M.D., Sc.D., Nut_i. Biochem. F, Asst. t.:"Surg. (also Asst. Prof. Physiol•. Chem.) I•; Mary V. Reed, Ph.D., Research Associate : ;;: _ . . . . . , - •niml " • I,;.Hedda Linden, Ti~ssue Culture Tech. `.hfary Jo Koryto, Secretary " "'Charles Lane, Laboratory Assastant Ph A t l Pf if L b F Mi h oratory. . ae e er, a otog. ss ~ c r _ Franklyn Greene, Laboratory Asst. E. Consumablo Supplies Qist bycotegories). ': j `'sGlassware "Chemicals (including photographic) .Biologicals (serum, extracts and procurement) •Cost of animals and care. . 'Photographic film, plates and prints SubJotot" -- C. Other Expenses (itemize) Prof. 100%' 50% 100 : 100 :' Sub1QoQa9 (6k): Welfare Benefits 2,b12.T8 $764.00 600.00 600.00 300.00 40000 . Sub Tota! $7i2;615.12 .S2 0,( 6.10, Contingency Cephone Rental avel D.,Permanent Equipment (t.mize) ~ This amount to cover part of $3,332.00 purchase of Technicon.Sequential Amino Acid Analyzer ($12,340.00). Balance from American. Cancer Society and other sources E. Orerhead (l5'/0 of A+B+C) Estimated Future Requirementf: Salaries Consumable Suppl. Other Expenses Totall Permanent Equip. 7,182.'f8 $58,L00.00 i wu kcat-' 19 G}I ~~ /', - ,_Overhea Total d Year2 _We hope to.stress pathogenesis of early tumors such as choriocarcinoma (3 mo.(?) Year3 ;,induction period) in comparative normal versus carcinoma cell siudies. More travel expe ses needed to retrieve specimens. Total cost pre~t basis. It is undentood that the applicant and institutional offiten In apphling for a grant have reod and found acceptable the Councif's "Statement of,Policy Containing Conditions and Terms Under Which Project Grants Are Made: " similar to "1~..~. Signature ~ r / r' ~ Sig:iatu.e_ .rue... arr... et t,. ie,rsw .n h T l ep one e Telephone

Ust f•mancial support for r.s.arch from all sourea, Ineludinp own Institution, for this and/or r.lated research aroiects. I -------- --- - "Evaluating Factors Contributinr; to Nornal and Malignant Cell Growth" •F-273-1 t, ! Personnel support in collaborative studies with Dr. Milton Edgertonts group on systemotio wave leng-th dependent laser studies Sourc. Ameriean Cancer Society ,, Amount I Duration 7/1/67 - 6/3o/Eg ' 4 years , .

I'ACI LI'f ICS AVaI LIIBLE Tiie F_innev-(-fht:ell Cancer Research Laboratory of the•Department of Surgery has its fa:cilities established in the Johns 1,Ibp3cins 1?bspital. Its facilities include: -. (a) An integrated and cor~plete tissue culture laboratory including, cubicles ' accommodating our regular group and pre and post-doctu2a,l cancer trainees; also, diusti.lled water autoclave, refrigerator centrifuges, low-temperature refri.€erai:ors, speci:ad incubators and speciall ob:,crtiation booths for tissue cu].ture studf•. (b) A Cryo',ii.ology storage faci Pity includina nit.rogern refrigerators and pxogr.am!;tinb eCl;uipIlent. (c) A biochemical laboratory for cell nutriltion studies and other cheriical studies on che~:iicali1y-clefined media, serum proteins, and tissie-c culture products. (d) A motion picture laboratory including: 1) A shock-prcof time lapse motion pi.cturey asscn:blj• and a special ti.me lapse data• recording microscope ('Rei,mcr and Gcy) fone cell pc,•nula~tion stud7.es. .. _. ~ 2) A 35 mm. antomatic fil:m: processor. - 3) Dark 'rocm f-aci.].ities. (e) Electr•on microscope laboratory (Siemens-1bl•a].si.e unit) con~pl.etc with dark rooms and thin sectioning equipment. - (f) Adequate animal housing quarters. (g) Adequate animal cperating rooms.• - Some of our hrojects ane carriedi out in collaboration r:ith others as ~ indicated iii the statement regarding interdepartmental, collFboration. Of W -. particular importance are the- opportunities for collaboration with the Department of PalWobiology (Dr. F. B. Bang) on viruses, the Department of f~ Gynecology and Obstetrics in cooperati~on with Dr. 1-lug5 Davis and: others on ~ studies on the developmentt of invasive carcinoma o.f the ccr.•ix. Of speciall ~ importance is our access to operative specimens from tlaiis. and reany other ~ hospitals and, the excellent esprit de corps here:and in many. remote places• i•n• the i•:orUd allor:ing, rare specimens to be obtained by our f oa•:'.rer fel lows or other frienchs.. The frec{:u^nt nccd for choriocarcinoma and' other raire specir-men-, requires that the facilities of others be availahle ar.d in aKldition. to the T=issuc; i'rocuTei^ent D'ivisions'of the \.C..I. which are aTso: bei,ig u_.ed. Ourr personnel and speciai facilities have aJ.vayss bcen at-a:ilable. to othe-s in thiss and other institutic-.is for conferences and especially for their benefit in allor:'ing pPOCUremerat of coatinuous culltures of nuuny different cell types.

R: REDACTED MATERIAL Present Address: Finney-Howell Cancer Research Laboratory., 13th Floorj RU #1 -'. ' SCIENTIr"IC ArJI} xC1iDE`tIC POSITIONS : 1921 - Carnegie Research Assistant, Cold Springs Harbor,, L.I., N.Y.. 1921-22 - Instructor in Zoology, University of Pittsburgh. 1922-23: - Guest Research Student, Dept. of E,-nbryoloey, Carnegie Inst., - Baltir.,ore j }id.. 1923-29 - Cancer Research Fellorr, Colunbia--.Hospital, !1ilrraukee, ~ffisc.. 1929-33 - Director, Tissue Culture Laboratorg, Dept, ol" Suro ery, and Student Assistant in Sur;ertr, Johns Hopkins ',-edical School. 1929-39'-- Guest Research Associate, Dept. of Embryology, Carnegie Inst, of Washington, Balt:Lmore, "d.. 1933-38 - Assistant in Surgery.. Johns Hopkins ?-tedical School. 1933-36 - Surgeon (R), U.S.P,H~.S., National Cancer Institute. Director, Cancer Research - CorOperative.Studies, i7.S.P.N•.S, and Johns Hopkins Medical School, Baltimore, ?1d.. 1940-41 - Finney-Hov+ell Cancer Research FeTlow, 1941-45 - Comr.iander, Emergency ;ledical Services, Office of Civilian• Defense, Eastern. District, Baltimore, Md.. 193$-46 - Instructor in Surgery, Johns Hopkins Medical School. 1940-Ct6 - Assistant. Director, Division of Ce11.Physiol:ogy j Dept. of Surgery, Johns Hopkins Medical Schbol. y- 194•7- - - Assistant Professor of Surgery, Johns Hopkins -tedical School. 190- - - Director, vinney-Ho•rr.ell Cancer Rescarch Laboratory, DepartrLent of Sur~ery, The Johns• Hopkins 'redieGl School and Hospital. -195h, - Lectr. Pathobiolo p~/-, School of xiygiene and Public Health, The Johns I3opkins "edical School. 100354656'7 ~ 1958 - Associate Prof. Surg. The Johns Hopkins Medical School , . 1966 - Associate Prof. Surg. (Emeritus): Active Duty, The Johns Hopkins bledical School.

George 0. (with Lewis, IT.H. ): Clasnoatocytes and Tumor Cells in Cultures of ;; 0 Mouse Sarcoma, Bull. Johns Hoj~lcins Hosp, p L:369, 1923. . and Thalhimer, W. 1924, Observations on the Effects of Insulin Introduced into the Medium of Tissue Cultures, . J.A,?1,A. ~, 82':1609. with Cron, R.S. 1927. The Viability of t'ne Cast.-Off Menstrual Endometrium: Am, J, Obs, & Gyn. 13:61i5. Studies on the Cultivation of Human Tissue Outside.ttie Body. 1929'. - bifZsconsln J. J, 28:11. An Improved Technic for Massive Tissue Cultures, 1933. Am. J. Cancer 17:752. with Stone, H.E.,~and O:vingsy J.C.__ 1933. LLiving Grafts of Endo- crine Glands, CaZifornia and'%Jestern t:tedicine~ 38: No, 6; 39: Nos. 1 and 2, June, July and AuJust, 1933. r ~ Glands, Am. Jour. Surgery, rTejv Series, 21 :1934. wtith. Stone., H.R., and O:vings:, J.C., Living Grafts of Eiadocrine with, Stone, H,B..* and 0•ringsj J.C. -1931x. Transplantation of Living, Grafts of Thyroid and:Parathyroid Glands. Annals of Surg. 262, with, Stone, H.B., and Owings, J,C. Feb. 1M, Living Grafts of Thyroid and Parathyroid!Glands~. Surg. Gyn. Obs.. ar.d,M,K, Gey. The Haintenance of Human Normal Cells and Human Tumor Cells in Continuous Culture. .1. Preliminary Report. Cultivation of i,iesoblastic Tumors and Normal Tissue and; Notes on Methods. 'of Cult1vation,. Am. J. Cancer 27:45. May 1936. ° with Seegar., G.E., and Hellman, L.ri, The Production of a ~ Gonadotrophic Substance (Prolan,) by Placental Cells im Tissue Culture. Sci. 88:306 1938. ; 'Assembly, Memphis, Tenn. Feb, 16, 1938, with Stone, H.B'., and Gtivirigs, J.C, 1938. Further Reports on Grafting of Endocrine. Glands, AFiis-South. Post.-Graduate Medical and• Bana,, F.B,. Eacperimental Studies on the Culttvcal Behavior and the Infectiv'ity, of Lymphopathia. Venerea. Virus 11taintained i,n Tissue Culture, Bull. Johns Hopkins Hosp., 45: No.s$ Nov. 1939, with Gemmill, C.L., and' Austrian, a. The ;.:etabolism of Tissue Cultures of Walker Rat Sarcoma! 319~ Bull, Johns Hopkins Hosp., 66: 167-18l1., 191'10. - - _ ~ 1003546668

PUBLTC~;r:TIONS (CONTtD) Gey, George 0.s Cytological and Cultural Observatioras'on Transplantable Rat Sarco- -tained in Continuous Culture: Cancer Research, 1:737, 1941. mata Produced by the Innoculation of Altered Normal Cells 1'.'ain- Hopl:ins Hosp. 72:26. by Placental Cells 1-4aintained in Continuous C1z7.ture: Bull. Johns."; with Seager Jones t G.E~, and Gey, M.K. 1943. Hormone Production. and F3.ror,. i~~:: ~. 191t5. Observations on the Conversion of Nbrmal Into Malignant Cells. 'Annals of Sur.= 121. Yiith Gey., M.K., Inui, F. and Ved•der, H. 1944. Penicillin Action, on Strains of Normal and Cancer Cells. A,A.A.S'. Res. Conf. on Cancer, 321. _ Gey,. M.?C., Inuii, -=F.,, and Vedder, H. The: Effects of Crude and Purified Penicillin on Continuous Cultures of Normal and ?11alignant cells. Bull. Johns Hopkins Hosp. 77:116-131•, 19450 with Firor, Phase Contrast ?ficroscop,y of Living Cells. Annals - •of Sur., 125:604. _ Hanks, J.H., and Barrett, Rachel. 1948. Retardation of Growth ~ and Ttetabolism of Normal and Malignant Cells During Continuous Cultivation. . Gcowth 12:69D with Bang, F.B. 1948. A Fibrillar Structure in Rat Fibroblasts as seen by Electron iTicroscopy. Proc. Soc. Exptl. Biol. '!ed;. .• 69:86. with Bang, F.B. 1949. Electron 1licroscopy of Tissue Cultures Infected tirith t~ze Virus of Eastern Equine Encephalomyelitis. Proc. Soc. Expt1.. Biol. ,'•fed. 71:78. Gey., ?a.K., Firor, and~ Selif, V%0. 1949. Cultural and Cytologic Studies on Autologous Normal and Malignant Cells of Specific in vitro•Origin. A,cta. Un. Int. Con. •Cancrum. 6:706. and;Bang, F.B. 19,5•1. Viruses and Cells. A Study in Tissue :._ C!Lilture Applications. I. Cells Involved-Availability and Sus- ceptibility. Trans. N.Y. Acad. Sci. 14:15. with Bang, F.B. 1951. Viruses and Cells. A Study in Tissue Culture Applications. II. Effect of Several Viruses on Cell TSrpes and the 1'.mount of Virus Produced. Trans. N.Y. Acad. Sci. 13 :324. • with Bang., F.B, and Levy, E. 1951. Some Observations on Host- ' Cell Virus Relationships in: Forl Pox. I. Gr~ o:vth~ in Tissue Culture, IT. The Inclusion Produced by•the Virius on the Chick Chorio- P;llantoic 1•2embrane. J. Im.•mzm. 66; 329. 10U3S46r69

' PIMLIGATIoM (coNrM) C2yt George 0,., with Bangs F,B, ~'1952. Comparative Susceptibility of Cultured Cell Strains to the Virus of Eastern Equine Encephalomye]:itis, Bull. Johns Honkins Hosp, 91:427. Cancer Inst, 13:1099. 1952. Proceedings of the Research:Sessi,on on Endocrinologic Aspects of Aging, Edited by V, Korenchevsky. J. Gerontology°, 7, 1953.' Discussion in "Problems of Lging" edited by Nathan'•V•. Siiocks from 15th ?facy Conference, with Ehrmann, R.L. 1953. The Use of Coll Colonies on Glass for Evaluating Nutrition and Growth in Roller-Tube Cultures. J. Natl, with Shooter~ R.A. 1952. Studies on the ,iineral Requirements of Mammalian Cells, Brit, J. aptl. Path,. 33;:98. with Glinosj Andre D.* 1952. HiLmoral Factors. Involved in the Induction of Liver Regeneration in'the Rat. Proc. Soc, Exptl. Biol, Med. 80:421. with Scherer, '7.F., and Syvflrton? J.T. 1953. Studies on the Propagation in vitro of Poliomyelitis Viruses. IV. Viral 'Mu1ti- plication-in a Stable Strain of Human Malignant Epithelial Cells (strain hela):derived from an Epidermoid;Carcinoma of the Cervix. J. E~cptl. Ked. 9'T: 695 « 1954-55, Some Aspects of the Constitution and,Behavior of Normal and ~ialignar,t Cells Maintained in Continuous Culture, The Harvey Lectures, Series L. Academic Press Inc,) N.Y. 154. . ngitated:, Fluid t4ediumy Ann,, N.Y. Acad. Sci. s58:1039-1055. Bangs F'.B., and Gey, 19.K. 1954. Responses of a Variety of Normal and Malignant Cells to Continuous Cultivat:.cn and Some Practical -. -Applications of these Responses to Problems in the Biology Disease. Ann. N.Y. l.cad. Sci. 58:976, with 0rzens., O.v.H.s and Geys M.K. 1954. Growth of Cells in Shaprass. P.,,,. and Boryskos E, 1954, Activities and Responses of Living Cells and their Components as Recorded by Cinephase f'licro- scopy and Electron Microscopy. Ann. N,Y. Acad, Sci:. 58s1089s M.K, 1954, An, Evaluation of'Some Compara- &3nga F.B., and Geys I tive Studies on, Cultured Strains of Normal and rfalignant Cells of Animals and ?.ian4 Texas Reports:. Biol, 'I'[ed. 12:805, 1003546670

PITBLICATIONS (CONTID) Gey, George 0., vaith I:o, 1%H:.Y., Bang, F.B: 'and Shapras a P. 1955. • Rous Sarcoma Z`issue Cultures. "Bul.l. Johns Hopkins I•Iosp. 97:227. genic Effect of the Rous Sarcoma Virus ontChickeniFibroblasts in, Virus Infections in the Chick Enbryo I and II and The Cytiopatho-» .: ti .,... Shapras, P.3 Bang, F.B. and Gey, .1955, Some Relations of Inclusion. Drc-olets (tiinocvtosis-»Levis) and Hitochondrial Behavior :in, Normal and malignant Cel1s. Symposium on Fine Structure of ::_.~gf6j9;;. Ce11s E P Noordhoff Ltd Gron inger~ 38 with- R. L. Ehrmann, 1956. The Grotixth of Cells on a Transparen:t Gel of Reconstituted Rat Taih Collagen, J. IJatl. Cancer Inst. 16:1375. .. Discussion, tissue Culture in the Study of Animal and Human Ttimors. 1957. 19:78lt--785y d. Nat. Cancer Inst. •. Factors Influencing Proliferation of Viruses in Normal and Malignant Cells. 1957. Texas Reports. No. 3• 534r-539. C Nith F. B. Bang, 14. Ford and D. ?r.iranegan, 195•7. Chronic Infections Pr•oduced in Cultured Cell Strains by the Virus•of Eastern Equine Encephalomyelitis. Icad. Press Inc., Virolog,y, 4, No. 3. with H. N. Kent, 1960. Selective Uptake of Serum Globulins and Glycoproteins by'Cells Growing,in Vi,tro. Science, 131:666-68. with M. V. Reed, 1962. Cultivation of Normal and Malignant Human Lung Tissue. Lab. Investigations, 11:8, pp.. 638-52. Paper presented at the Tissue Culture Association Meeting, Washington, _ D.C., May. 31, 1962. ~ trated Corodice for Continuous Cell Culture (7 C's Solution). with J. K. Frost, 1962. Symposium: Cell Modulations, Maturation, and bleoplastic-Transformation,. Acta Cytologica., 6:399-402. : with,C. T. Ling and V. Richters. Chemically Characteri_zed•Concen- with• R. A. I4filcli and M. A. Naughton, 1965, Snbmit Patterns of Rapidly Proliferating Connective Tissues. Biochem. Biophys. Acta 1001:623-26. - C with D'f.. K. Gey, M. Svotelis, and H. Linden. A Motion Picture Sequence Illustrating the Production of Giant Cells from Hypo- diploid Rat Strains NSAT-14pH and -14pL. Presented at the 16th Annual Meeting of the Tissue Culture Association., May 31-June 3, 1965. 1oo3s4ss'~1 with C. T. Ling: The Role and Interaction of Trace Elements in Cell Nutrition. Presented at the 17th Annual Meeting of the Tissue Culture Association, May 31-June 3, 1966. with C. T. Ling. Further Studies on the Role and Interaction of TYace E1emeats in Cell Nutrition. Presented at the 18th Annual Meeting-of :.,ze'Tissue Culture Association, June 4-7, 1967.

R: REDACTED MATERIAL ~ •. .... ::; .. ! SCIEr1TIFIC 0%rdIn,71IQNS ROACTED . PRQFESSId':TAL. CLUBS INUT~.STIG.1TOR .,ND GR'.rFTEE The International Cancer Research Foundation 1932 37. National Cancer Institute 1948. ' Frances P. Garvan Cancer Fund., Chem.ical.Foundation 1925t-31. American Cancer Society 19h9'. ' ?4edical Consultant and Gnantees Committee on Hemorrhagic Fever), tumed Epidemiology Board 1953. National Foundation for Infaintile Paralysis 1953-55. AWARDS ....~.r.. Judd Arard: Cancer Research 19541, Wein Award Cancer Cytology 19568 t • Forces ~ O O Gj Ul ~ ~ 09

SCII1=1-IC RE-3:::1?HCII 1+'Ih;:s PET,r,ttS;Ms • Gojr) G. 00a and he-wis, l-l.N.~ THE CUT;iI~IATIOI:~ 0T I:Jf~r1II `'LifOIcS Iii t~STPLO~ Garvan C.,•ncer Research. Founda-i:ioil., Cizen. I`ouudation ~"s'ic., N.Y., 1931. FF'r'w :1'S Ur, IR,~',~U?1l,lI0PI 0?4 TISSu 1 CUi,Ttlf?ES' Eas•(;esiY Conference of Radl.olozis ts 1936. GeYy Ii.K,s S'cones H.E., ancd 04ru'i,'s,, d.C.S TISSI3E C;;L`ilTi-a,;i Ule L111JiiC1L1i#L - O.11tG-_"`.1S. Amer.i.can Association of 1lnatomists, 1938. Letr.i s' If. s.~ Geyi CiJ7m2IIZrArSGI-I Ox BT~~00 Ch,iJ, aT,.-LOFui OrF irlIE, 2ItJLSE. Amer's cart Associat;ion for Caz.cer Research., 193u", t1-1ii: b`axors tT,,•i,y OFrSERVATIUId 0:I RAT WN0t: Ci jIS I'iA.Y PIO 1-ceV;:G, CRILS Iir VITFtO. hrlericam Cancer SocYety., IT.Y.j 3.945. IrIth. >~i.ror., ssT.I•I.y and SelX9 1y,0.., CYTOI:OJICAL I3ITROFIO.Z 0T J1U•i07.A~MJS IIOfu•SAh !i';7t:;Ull~'G,iAI:F CT;hL.S.* A Siud:y vri'+h pltase cor.:f;ras~ microaCO~~ ?ni:. Cancer Co .^-.sss 1 9 ~6, _ _ .. _ _ _ ... .. _ _ .,. d .. _ ,_ ~' Sh<?pr2s,, P.,, and F~anC~ F.13.~ P'0_Zi.2L 11ID IIAI~GIIAi.T CE.L~S~ (a) STi.fUI'''S h7i IALL AhD :;AhI,f.'FmYsT 13P_T CE;LS 0?~' I50i;EJIC Or.I(ixli't Pa?ase atx2 13-eetron ,:icrosconc Oi,se:cvatl.ons. Y r w r r. w N r`.r w-- r W !O .r _-N (b) OLtaJRYA`I!l:vNS 0'I Iil;a•iAid Ch,::C2.':C=:.I~T~a,, S::rai~.ns D1 Re~ A. I'i; 4n:~ ~i•r~~- 7 zj•• ~ CIi:~~T` ~ AG;.IV'ITY a . i; t(,l, i'+ fOC ~Cv t~ r•. .~l.ullas 5 Ca%~t~•.~s.~rd•~:~ HeT?l, ul~s~Sc, !'~•II~..J~Cktcv f I. ABD 3-UTOC:I-7N:JIUAh FEI;&ITIO:i9. 1L^..er:.cun Cancer Sociei.yr 19t;5 19525. Sha.pras, P.., 13m=g, F.33:j, cncl Pazcne --., h., ACTIVITY 04IF I4ITOIlIOi:DZSd AIh^3 P7u'l`:~I.A i)a^."a-tBRANE Ti•I I:4 LLilf;r, 1u:I3 ilfJ':Iii.`IAi;F CET,LS 1~'T AND .IIt1i>LAPh Eliaezd.cu.n Cance'r Socictys 1'9I;G z95Y o ._ Gey, M. K., Svotelis, M., and Linden, H'., A Motion Picture Sequence Illustrating the Production of Gianti Cells from Hypodiploid Rat Strains NSAT-14pH and -14ph. Presented at the 1Gth Annual Meeting of the Tissue Culture Association, May 31-June 3, 1965.

R: REDACTED MATERIAL CURRICULUM VITAii NA~titE: Chiun T. Ling .~.-_..____.....-. a___. i P= LANGUAGES: English, German, French, Japanese, Chinese. Tung Iti'en College, Fukien., China (Premedical), 1919--21 Nippon Medical,College, Tokyo, Japan (Premedical), 1921-22 Nippon Medical Col lege., Tokyo., Japan (Medical), 1922-26 STUDIES AND DEGREES. Degree: M.D-., 1926 Johns Hopkins University, Baltimore, Maryland, 1945=46 Degree: M.P.H;:, 1946 VOKIM Harvard•University, Boston, Massachusetts, 1946-4.7 (Research in Nutrition) - Johns Hopkins University, Baltimore, Maryland, 1948-52 Degree: Sc.D:. (Biochemistry), 1952 Internship: University Hospital, Tokyo, Japan, 1926-27- Resident in surgery: Red Cross Hospital, Tokyo, Japan, 1929,-30 ACADEMIC APPOINTMENTS: Assistant Resident: University Hospital, Tokyo, Japan, 1927-29 Research Associate in Biochemistry, Johns Hopkins U15iversity, 1952-53 Assistant Professor of Biochemistry, Jefferson Medical College, 1953-57 Assistant Professor of Biological Chemistry, University of Michigan, Medical School, 1957•-59-' -Research Associate, Simpson Memorial~ Institute, Uni~versity of Michigan, 1957-59 Instructorr in Siurgery, Johns Hopkins University, School of Medicine, 19S9~-62 Instructor in Physiological Chemistry, Johns Hopkins. University, SchooL' of Medicine, 1959-6.2 Asst. Prof. Surgery; Asst. Prof. Physiological Chemistry, Johns Hopkins University School of r4ediclne:, 1962-to date EXPERIENCE: Over 15 years of experience in biochemica•1 and nutritional research. The following outlines the nature of probliems studied and the techniques used.

Curriculwn Vitae - Dr. Chiun T. Ling Animal experimentation and in vitro studies with tissue materials, on the functions of vitamins and their effects on carbohydrate, protein, and lipid metabolism. Quantitative micro techniques for the analyses of lipids.and nucleic acids, coenzyme assays, etc. Chromatographic and electrophoxetic analyses. Radioisotope.work with Co60, Fe59, andiCl4. Isolation of leucocytes and determination of thymidine contents in normal and leukemic cells. Isolation of erythrocyte stromata and;studying their chemical composition-in normal and pathologic cells.• ~ Five.years of experience in teaching medical biochemistry, consisting o a) Formal illustrated lectures 17 - 18 hours each,year b) Supervision of group experiments by medical students, c) Supervision of laboratory work, group~conferences and students counseling d) Supervision of graduate students PREVIOU&RESEARCH ACTIVITIES: Studies on tlze formation of lipid components of blood cells in bone marrow; incorporation of labeled precursors into components of globoside, phospho- lipids, and, sterol of the cells in yitro, with bone marrow from normal, anemic and,leukemic aninals; changes in enzyme and coenzyme activities; effects of _ vitamin antagonists in these systems. t 1.. Studies on the Nutrition of hormal and Malignant Cells in,Culture. PRESENT RESEARCH ACTIVITIES: A concentrated chemicallyy characterized medium has been developed..which maintains MBIII lymphoblasts and Walker Rat Carcino-sarcoma-256- cells in continuous culture. Investigations•in progress include: a) The influence of intracellular amino. acid concentration on the forma- tion of cell proteins and its. dependence on amino acid levels in culture media.' b) Studies. on the quantitative evaluation of cell growth in vitro; inter- relationships. betweerL cell volume, cell count, cell protein and nucleic acid contents (DNA/RNA• ratio) . • c) Effect of the nature:of glass walls off culture vessels and, their chemical treatment on cell survivali, growth and behavior.

Curriculum Vitae - Dr. Chiun T. Ling 2. Lipid Metabolism of Cells in Culture 3. • l 's0.~-. an.. .. ..., Investigations,on the Metabolic Interaction between Cholesterol, Phospholipids and;Fatty. Acids. Other Research Interests Nutritional requirements of normal bone marrow cells in factors affecting cell differentiati.on,. Culture requirement of leukemic cells Immunochemical reactions between tissue cells in vitro TEACHING ACTIVITIES: culture and Teaching and condUcting experiments with,-studentis in nutritional biochemistry as part of the biochemistry course g1ven,duri,ng one quarter only to second year medical students by the Department of Physiological Chemistry. Students' work includes studies.of changes in tissue enzymic activities in nutritional deficiency. Reference - Laboratory Manual for Biochemistry, Section VIII,. Dept. of Physiological Chemistry. i

LIST OF PUBLICATI:ONS AND REPORTS.- Dr. Chiun T. Ling Ling, C. T., Hegsted, D. M., and Stare., F. J.: The Effect of -Pyridoain Defi- ~' f t` R ts h ane Trans oima ion ciency on the hiaczn~Tryptop J. Biol. Chem., 174:803, (1948). Chow, B. Arch. of Biochem,. & Biophys., 34:151, (11951),. F., Bazrows, L., and L',ing, C. T.: The Distribution of Radioactivity in the Organs of the Fetus or of the Young Rats Born of Mothers Injected with B12 containiaig Co60 Ling, C. T., and Chow., B. F.: The Effect of V•itamin,B12 on the Body Composition of Rats. J. Bioli. Chem., 198:439, (1952). • Ling, C. T., and Chow, B, F.: The Effect of Vitamin B12 on the Levels of Solu- ble Sulfhydryl Compounds in,Blood. J. Biol. Chem., 202:445, (1953). Ling, C. T., and Chow, B. F.: The Influence of Vitamin B12 on Carbohydrate and Lipidi Metabolism. J. Biol. Chem., 206:797, (1954). Ling, C. T., and Chow, B. F.: The Effect of Vitamin B1,2,on Ribose Formation by Erythrocytes. Federation Proc., 13:253, (1954)'. Ling, C. T., and Chow, B. F.: Vitamin BZi2, and Carbohydrate Metabolism in First _ European Symposium on Vitamin B'12. and Intrinsic Factor. Ferdinand Enke Verlag, Stuttgart, Germany (1956),. Ling, C. T.: Vitamin B1,q and Lipid Metabo.lism:. Abstracts of Papers, Am. Chem_ Soc. 134th National Meetings, 57C, (Ii958) . Ling, Chiun T.: Role_of Vitamin B12 in Lipid Metabolism. Federation Proc. 18 :489•, (1959). . Ling, Chiun T., Gey, G. 0., and Richters., V..: Seven Cs Solutions: Chemically " Characterized Concentrated Corodice for Continuous.Ce11 Culture. Submitted for presentation to,the 53r.d Annual Meeting of the American Association for Cancer Research, April, 1962. Ling, C. T.,- and;Gey, G. 0.: The Role and Interaction of Trace Elements in Cell Nutrition. Submitted for presentation to the 17th Annual Meeting of the Tissue Culture!Association, May 31-Jtme 3, 1966. Ling, C. T., andiGey, G. 0.: Further Studies on the Role and Interaction of Trace Elements in Cell Nutrition. Submitted for presentation to the 18th-Annual Meeting of the Tissue Culture Association, June 4-7, 1967. • Copy-of paper submitted with application. To be~ published soon,.

R: REDACTED MATERIAL Mary V. Reed (Business)i 601 N. Broadway,°Baltimorc, Ma-ryland 21205 Education: Goucher College Smith,College Johns Hopkins University School of Hygiene F:Public Health Dates Degree Date of Degree A. B. June, 1931 A.M. June, 1933 Specid l Student University of Maryland Ph,lg, Januaiy, 1954 (Medical School) Master's Thesis - The effect of certain triihenylmethane dyes on Staphylococcus aureus and Escherichia coli communior Doctoral dissertation - A comparative study of the-metabolism of a ch1_oramphenicol-sensitive strain and a chloramphenicol-resistant strain of Escherichia coli Professional Society Memberships / ACTED ACTED }~] .. Academic Honors - Sigma Xi - Johns Hopkins University Teaching Positions Graduate Assistant in Bacteriology, Smith College:, Northampton, Massachusetts, ~ 1931-1933. Assistant in Bacteriology, Smith College,.1933-1i934. Instructor in-Science and Mathematics, The Knox School, Cooperstown, New York, 1934-39, 1945-46. ~ Instructor In Science, Mount.Vprnorn Seminary >a Junior College, Washington, D.C., 1939-45. Assistant Professor in Chemistry and• Nutrition, University of Tennessee, Knoxvillc, Tennessee, 1946-48.

Curriculum Vitae - Dr. Mary V. Reed Instructor in Physiology and Bacterioliogy, Goucher College, Baltimore, Maryland, 1949-1954, Assistant Professor, 1954-55_ • Postdoctoral Fellow., The Johns Hopkins Medical School, Baltimore, Maryland, 1955-1965. Research Associate, The Johns Hopkins Medical School, Baltimore, Maryland, 1965-to date. Publications Reed, M. V., and Genung, E. F.: The Effect of Certain Triphenylmethane dyes on Staphylococcus aureus and Escherichia coli communior. J. Bact., 27, 29, (1934). =_ Reed, M. V., and;Genung, E. F.: Titration of Dyes for the•ir Bacteriostatic Action. Stain. Tech., 9, 117-128, (1~934);. Reed, M,.V'., and Gey, G. 0.: Cultivation of Normal and Malignant Human Lung Tissue. 1. The establishment of three adenocaz•cinoma,cell: strains. Lab. Investigations, 11, 638-652, (1962),.

R: REDACTED MATERIAL CURRICULUM VITAE ROLAND ANTHONY PATTILLO Address: . ~ACTED PMA~'iTED (Business) 601 N. Broadway, Baltimore, Maryland Education: Blessed Sacrament High School, Beaumont, Texas. 1951 ~. B.S. Xavier University, New Orleans, Louisiaha 1955, M.D. St. Louis University School of Medicine 1959. Inter.nship: Milwaukee County Ceneral Hospital Milwaukee, Wisconsin kesidency: Obstetrics $ Gynecology 1959-1960i Under Dr. Richard F. Mattingly, Professor f, Chairman, and Dr. Eleanor Delfs, Professor of Obstetrics, Department of Gynecology $ Obstetrics Marquette University School of Aiedicine Inclus.ive of Exchange Resident rotations: Boston,Lying.-In Hospital, under Dr. Duncan.Reid,, Professor & Chairman, Department of Obstetrics $, Gynecology, Harvard University, Boston,, Massachusetts General Surgery & Urology, under Dr. Edwin Elllison'•s direction, Professor and Chairman:, Department of Surgery, Marquette University School of Medicine General, Gy.necolob2c F Obstetric Pathology, under Dr. Paul Kimmelstiel, Research ProfessTz-"of Pathology, Marquette University School of Medie%ne Academic: Instructor, Department of Gynecology $.Obstetrics 1964 - Marquette University School of Medicine Research Fellow, National Institute of hlealtlr 1964 - Fellow in Surgery, The Johns Hopkins University 1966 - Hospital A}?pointments : ~ Staff: St. Joseph's Hospital; Mt. Sinai Hospital St. Michael FIos.p:ital, Milwaukee, 11'isconsin, Attending Staff: Mi1•,•:a~2kee County General Hospital

R: REDACTED MATERIAL t . -2- ~ Curriculum Vitae - Dr. Roland A. Pattillo Family: Professional Associations: 0 ACTO ACTED RWAOTED Bibliography: Pattillo, R. A., Foliey, D. V., aiid Mattingly, R. F.: _Internal Pelvic Ilymphography. American Journal of Obstetrics & Gynecology, 88:110, ~2964-). Pattillo, R. A. and San Felippa, P. M.: Carcinoma of the Vulva. Marquette Medical Review, 31:121, (May., 1965). Pattillo, R-. A., Smith,.T. E., Delfs., E.,. and'Mattingly, R. F.: Cytologic and Hormonal Activity of the Trophoblast in Explant Culture: American Journal of Obs.tetrics. and Gynecology, 96:337, (1966). Patt-illo, R. A.: The Human Trophoba ast--Its Endocrine and Neoplastic Properties. Marquette-N9edical Review-, 32, No. 4, (1966). Pattiilo, R. A., Mattingly, R. F. and Messinger, P.. S,: Visicocervicorectal Fistula: A New- Gynecologic Entity. Obstetrics &• Gynecology, 27:432, (1966). ~ - Continuing Investigation,: Carcinogenic Stimulation of the Rat Placenta - Attempts are being-made to create:an animal model for choriocarcinoma. The carcincgen 7,12 dimethylbenzanthracene is d'eposited, at the gestational site in early and mid pregnancy in Hooded rats. Hormonal support in the form of estrogen:`and progesterone is supplied exogenouslyin order tp maintain:placental viab•ility: Evidence for neoplastic changes is being sought. Rea•ctions• of the Human 'Prophoblast as Ezplanrt andi Transplant- Human trophoblastic tissue obtained stenilely from ecto.pic gestations and sterile mature pLacentas:from:Cesarean sectionss are propagated inlruller tube, T-Flask and organ cultures. Studies are directed toward identifying the ce-11 of origin of cho•rionic gonadotropim and steroid hormones. Comparisons. of the:differences in behavior of hydatiform mole and chorio--• carcinoma as to functional and cytologic chaiTar-teristics are being sought. Immunologic factors in normal trophoblast and their'variants and similarities in trophoblastic ncnp.lasms arc being•evaluated by transplantation to thymectomized nconatal r?ts. .

ugust 23, 1967 2O: The committ ee m D 3 •, . ers an comprlsi.ng Dr. Reimann~ C'hm Dr Sdm d . r. Jacobson. Renewal application from Frederik B. Bang, M.D. - No. 409-R3. We enclose herewith a renewal ap licatio f D p n rom r Frederick B .. Bang of the John H , s opkins Unversit Blti iy,amore, Maryland. This request represents the fourth ear f i y o an or ginal five year plan. At the time of the last renewal prior consideration was promised for a further two vears ;n tho -1,,. .,4- ; _r it should be noted that the anticipatedvdurationjofuthelstud• isonower, given as three years which w ld ou extend the totl dti auraon to six years. Tr1r-~ CouzTcm FOR ToBAcco RrsEAl`c-n _ U rA "

E s :'-~,,.~~.~r~--.;:~.fi,r~ r YIRUS, PREVIOUS INFECTIONS Tme Courrcu, FOR TaE &eca RESr.Atzc$ - U.S.A. 633 1BIItD A4F.NLTE `,(,"QMT'j'F;E : NESV YORII. N: Y. 10017 Dr. Reimann, Chm4 Dr. Sotmers Dr. Jacobson Application For Research Grant #R3 Activated: 10 1 6k Renewed: 10/1/65 Renewed: 10/1/66 ` i. Name of Investigator(s): (include Titie and Degrees), :'<-Frederik B. Bang, M.D., Professor of Pathobiology :..The Johns Hopkins University School of Hygiene and Public Health 615 North Wolfe Street, Baltimore, Maryland -._ 21205 3. ShortTitle of Project: Pathogenesis of virus infections of respiratory mucosae 4. ProposedStarting Date: - To be continuous with present grant- 3. Anticipated Duration of this Specific Study: Three years r S. Brief Descripton of Objectives or Specific Aimse " l 1) Continue to study the effects of drugs and of internal and'ex- ternal environmental factors on susceptibility to viral infections, with emphasis on the initiation of infection. 2) Initiate a sequential study of regeneration and re-organization of mucosal cells following acute desquamation;"for this study, nu- clei will be labelled with tritiated thymidine, a method used exten- sively in studies of intestinal mucosae. 3) Study the effects of avitaminosis A on the susceptibility of chickens to the viruses of Newcastle disease and laryngotracheitis; we have found in~preliminary studies that avitaminosis A (known to affect mucous epithelia) shows the first apparent effects histolo- gically on the inner surface of the scroll of the maxillary concha, an area which we have found emiriently vulnerable to virus infection. 4) 1'iith the collaboration of Dr. Donald F. Proctor, we propose to determine the effects of acute upper respiratory infections on the rate of nasal mucus,clearance before, during, and after the acute clinical phase of infection; this will be a study on human volun- teers, using a method which has proved reliable in a recent study which involved several hundred tests. (Items 1, 3, and 4 will extend studies which have been primarily supported by the Council for Tobacco Research, and which have been published or are in press, as noted under item 12 below) 7. Give a Brief Statement of your Working Hypothesis: Items 7, 11; 12 are on a separate attached page `-~

X* 8. Details of Experimental Design and Procedures. (Attach Separate Pages) t'1: Drugs are administered to chicks intramuscularly or locall,r; NDV is inocu- ';labelled eells.deternined at successive time intervals. j: Chickens fed for `'bine labelling will be done in pulses, and the progress and persistence of ~fected cells is then determined by plaque techniques. 2s Tritiated thymi- >,-"cised, extensively washed, scraped, and the cells trypsini~zed; the number of. -,~rated intranasally. Chicks are killed at intervals, the maxillary conchae ex- ..;-Llnning it simultaneously with~a radioactive tracer in a series of volunteers. ;,before, during, and after spontaneous acute upper tract infection. This is ,`,~~done by injecting a chemically inert tracer dye (Direct Sky Blue, Wyeth) onto "_a particular site on the anterior mucociliated part of the nasal fossa and -timing its appearance in the nasopharynx, a method pre-tested for accuracy by -.Clearance of the upper respiratory tracts of human volunteers will be-recorded ,will be followe&by titrations and by histology. 4: The rate of mucocili'~ary lated with the two viruses and the spread of virus in the respiratory tract- :3 NeeKs arter natching on a commercial vitamin A-deprived diet-•will be inocu- r:s., ... '%°given in Item 6) ,~}(The numbers above are consistent with those of the experimental objectives 9. Physical Facilties Available (Where Other than Adlninistering Organization Indicate Geographical Location). Fully equipped virus and histological laboratories. 10. Additional Reqpirements: l) A full time histological technical trainee for routine histo- logical sectioning; the present technical assistant, Mr. Raoul Spicker, to train this apprentice and to devote most-of his own~ efforts toward autoradiography. 2) Half-time support for an additional animal man, to cover the increased use of animals requiring reliable supervision. C 11. Biographical sketches of all principal and professionat personnel (append) (appended) 12. List of publications: (Five most recent as pertinent) (append) ( app end ed') k (

. Working hypothesis 1) Alterations in the mucous-cil.iated epithelium which are induced by changes in the external and/or internal environment of '~the host will affect viral entry into mucosal cells. ~ 2} The progress of infection from cell to cell, and the re- covery of the mucous-ciliated epithelium following infection, are affected by mucous secretion. Avitaminosis A, known to induce ";conversion of mucous epithelium.to squamous epithelium, should irus infection l y myxov alter the progress of infection, particular Van-nevl vani ~_ A.B. Johns HoDkins University 1935: M.D. Johns Hop- Biogra hical sketches Frederik B. Bang, M.D., -` b. November 5, 1916 in Philadelphia x. - h ns kins School of 14iedicine 1939. Associate Professor of b7edicine, Jo :Hopkins School of Medicine 1949-1953; Professor of Pathobiology, Johns Hopkins School of Hygiene and Public Health 1953-1967. Special 'interests: virus pathogenesis; invertebrate immunology. Betsy G. Bang, A.B., - b. July 9, 1912 in Lancaster, South Carolina. A.B. George Washington University 1933; Certification in "Art as Applied to Medicine, Johns Hopxins School of Medicine 1937. Illustrator in Comparative Anatomy, American Museum Natural History 1937 - 1940; Research Associate in Pathobiology, Johns Hopkins School --'of Hygiene and Public Health 1962-1967. Specific interest: compara- tive functional anatomy of the upper respiratory tract. • Prantika Som, D.V.M., - b. August 31, 1942, Silchar, India. ll i i f 1 60 vers ; ege, Un Calcutta 9 ty o Inter-Science, Lady Bradbourne Co B.V.Sc. and A.H., and D.V.1f2. Bengal Veterinary College, University __ of Calcutta 1965. Candidate for Sc.D. degree in Department of Patho- biology, Johns Hopkins University School of Hygiene and Public Health. 12. Recent pertinent publications (1) Bang, F. B., and Foard, hi. : Interaction of respiratory epithe- lium of the chick and Newcastle disease virus. Amer. J. Hygiene 1964 79:260 - (2) Huang, J., and Bang, F.B.: The susceptibility of chick embryo skin organ cultures to influenza virus following excess vitamin A J. Exp. Med. 1964 120:129-148 (3) Bang, F. B., Bang, B. G., and Foard, M. : Responses of upper f„A respiratory mucosae to drugs and' viral infections. Amer. Review of p Resp. Dis. 1966 93:142-149 O (4) Bang, B.G., and Bang, F.B.: haryngotracheitis virus of ~ chickens: a model for acute desquamating rhinitis. J. Bxp hTed . . • 1967 125 : 4i09-4 28 (9 } Bann B G 14iukher j e e A L an(I Ban F B • Human nasal 1-1 . 0 • 1 P 0 • f .`Z) 1 . . . . . , mucus flow rates. (In press: Johns Hopkins Medical Journal) (2n press: simultaneous abstract in J. Amer. Med. Assoc.)

TECHNICAL Raoul Spicker r To be named , ieehnicaFHenry Johnson Juhan Labidas Linda Endemann Gwendolyn Futcher Research Asst. in Pathobiology Histologist Histological Tranee Animal Attendant Animal Attendant Glassware Washer Secretary . Consumabfe Supplies (list by categories) Animal Food Histological Material Publications Sub-Total C. Other Expenses Qtemize) Travel-Domestic-to attend clinics Requisition Payroll-to cover temporary summer help, termination payroll and special services .Fringe Benefits-6X--salaries and wages E. Orerhead (15%af A+$+C) Estimated Future Requirements: L.l Salaries Consumable Suppl. OtherExpenses Year 2 $21318.00 $260000 2610,00'_ Year 3 52304S.00 52600:00 2 1'.00' It is understood that the applicant and institutional officers In applying for a grant have read and found acceptable the Council's "Statement oF'Policy Containing Conditions' and?erms Uhder Whiah Project Grants Are Made." 50% $3625.00 507. 3000.00 100% ' 5000;00 88% ' ." 3060.00 507' 1500,00 1007 3645.00 12% 752.00 Sub-Total $20582.00 Sub-Total 1000.00 1000.00 600.00 800.00 Permanent'Equip. Overhead Total $1000:,00: $3980.00 $31508.00 Q $1000.00 S4254 00 $33612.00 Q Signature y l Dknaur of v,coj.ce " Telephone ~ ~~ N+ Signature tso3tnus Otfic.r of th. t.:rr.r o. Telephone VI' ~ `=

r* 'S i List financtol support for research from all sources, including own Institution, for this and/or related research projecfs. ~ I This grant to B,G, Bang finances a study of the comparative functional anatomy of the nasal folfa, t~899~SE00~ I 0 ~

TFm CouNcir. FoR To13Aeco Rr,,SrAi:Cr-r- V. S. A. Bell, M.D. - #455R1. On April 1, 1965, the Scientific Advisory Board awarded a $10,000 "piggy-back" grant to Mr. Charles Rose and Dr. Benjamin Bell of the Boston V.A. Outpatient Clinic for a.ssisting a retrospective longevity study. On August 1, 1967', ~vrith the approval of the committee indicated', an additional $3,300 was given as a supplement to carry personnel through to October 1, 1967. This new proposal will carry forward the "seeding" concept initiated by this group. . The unusual husbanding of funds along eighteen months beyond'the The committee comprising Dr. Sommers, Chairman, Dr. Cattell an I1r. Little. SU&TECT: A renewal application from Charles L. Rose, A.M. and Benjamin original termination date is one of the minor recommendations for this award. The major recommendations evolve around the fact that these data plus the new Normative Aging Studies, prospective, serve as the keystone to the staff project being carried out by Kurt Enslein~dealing with the develop- mqnt of computer methods for analyzing multivariate data. He has successfully developed methods to handle in excess of 100 variables simultaneously. of other characteristics and does not bear a primary relationship to longevity. It has already become evident that smoking is a result of a number As indicated in the enclosed progress reports, your support to this group is reaping the benefit of $154,00aper year of V.A. funding and at least an equal amount of other funding and effort fromiother sources. The impact of C.T.R. support is being appreciated and felt by the Veterans Administration, the U.S. Public Health S'ervice and the National .Institute of Child Health and Human Development, the Retina Foundation, Massachusetts Eye and'Ear Hospital, Massachusetts General Hospital, Harvard School of Public Health, Harvard'University Department of Anthropology, Boston University Department of S'ocioloE^y and Anthropology,, Harvard School of Dental Medicine, Tufts University School of Dental Medicine, Boston V.A. Hospital Special Laboratory, Veterans Administration Central Office, U.S.P.H.S. Hospital, Baltimore, and National Institute of Dental Research. An additional indirect benefit may be mentioned. As a result of these informally coordinated effortsp monitored by the CTR staff, a number of our grantees are serving on advisory c a-nmittees which appear to be influencing programs underway or contemplated by other agencies. 1003546630

It should also be noted tlat the methods developed by Mr. Enslein are being used to evaluate data related to smoking habits supplied by Dr. C. B. Thomas of the Johns Hopkins University Medical School, which had not been fully evaluated before. A small poriion of the data obtained by Drs. Sanford Chodosh and Maurice Sega1l is also now being evaluated by these new techniques. Reference: Progress Report The committee has received Progress Report No. 2, which covers the period from July 1, 1964' through June 30, 1967. In this rather long document the part marked "Report 3.1 Accomplish- ments, F.Y. 1967" describes the new approaches being developed and used. In particula.r, the section identified as Longevity Study by Charles Rose is relevant to CTR interests. It is the staff's opinion that the history of this project shows results beyond our expectations: The concept of using small sums to "seed" possible new approaches that may later be incorporated into a larger ongoing investigation is very appealing. The assurances of good judgment underline this group's past activities and projection.5 for the future. Though the application is for a twelve-month period we expect, from performance, that these funds will be used sparingly and judiciously. The budget understandably cannot be explicitly spelled out in the usual format, but the general target areas are outlined under Item 8'and appended Item 13. This project had the sympathy of Dr. E. B. Wilson, Chairman of the original subcommittee, as a case in which a small "piggy-back" grant might provide opportunity to correlate smoking habits with a large body of other data collected at the expense of other agencies. His prophetic judgment appears to have been justified. Vincent F. Lisanti, D.M.D. Robert C. Hockett, Ph.D.

p P% I F i'X A`L No, 455R1 Activated: 1• 5 THE COUNCIL FOR TOBACCo RESEARCH - U.S.A. " . . . ess TIIII2D SvENLrs . ) STATISTICS (Methodolo - r7EPIDEMLOIOtiY a:~ Addresss a ; 2 Institution & 43 E:'F~ry,.c:°s °` t = ``~`• Director and Assistant Director respectively of Normative Aging Study. Date•. August 25, 1967 - 1. Name of Investigator(s): QncludeTitle and Degrees) :_~Co-principal Investigators: Benjamim Bell, N.D. and Charles L. Rose, A.M~.: Veterans Administration Outpatient Clinic . gy - ~ Dr. Little Application For Research Grant ~ ... _ .. . •Dr. Cattell `_ Dr. Sommers, Chm. ~ COMMITTEE: N$w Yoas. N. z 1ooiT i - 17 Court Street, Boston, Massachusetts 0210B; r.....~.;..,,..:}.~„~~ .~.:...: . . 9. Shorf Title of Projed: ~~ -;15 lt Aite to the Normative Aging andLongevity Study ::emenasssanc-. ',..~>_ uPP =Y ~ ; /. Proposed Starting Data October 1, 1967 : S. Anticipated Duration oF,lhis Specific 51udy: -Twelve months Q@rsef Descripton of Objectives or Specific Aimsa Lti~dinal Study of Healthy Aging!',, and reprints are attached. Briefly, this is a s g erans m n e e t e: er the t nserted into the literature un ;:;..The objectives and specific aims of the Normative Aging Study have alreadybeen tration Lon i- V Ad i i i Th t d l " ~~,,large scale longitudinal aging study sponsored by the Veterans Administration with , ",j°.formative stages, and is expected to grow and enlarge particularly over the next five "a budget in excess of $154,000 pe'r year. The supplementary assistance we are re- questing is primarily for "seed money" purposes. The Study is still in its beginning ,": Fdti ihhllilhtt t th Std Th Retina ounaonn optamoogca researc a negligible cosoeuy.e buted by A. He are also stretching our research dollar through our policy of cross- which modest funds from funding. source A (e.g. The Council for Tobacco Research) are added to the larger funds already appropriated by funding source B (e.g. The Veterans Administration) so that the net result is many times the augmentation factor contri- as an ongoing orogram also makes possible the "piggy backing" principle, through y ng Stu ::possi~ble subsequent budgetary develoonent many times over, the Normative Ag i~velopment. In addition to the "seeding" concept through. which:small grants make d i :;years. "Seed maiey" at this early stage will assist us in making possible this de- :•linking with, otherstudies. We utilize the sophisticated resources of the Retina A Foundation benefits by having our study nonulation made available to them including administrative services of scheduling and record keeping as well as other data which: bave been collected on.our Subjects. The same sort of arrangement has been worked out with the Psychiatric Department of the Mass. General Hospital in,the field of Information Processing of the Central Nervous System. Most recently we have linked up with the.LongirGudinal Studies of the Gerontology Branch, of NICHD in Baltimore through the,establishment of a Joint Scientific Committee so.that these two major studies mi~ght coordinate their procedures and- produce findings on combined data. For the rationale and, accomn3ishments of the Normative Aging Study, the reader is aQ i f~rrn o h a ta '+e3 reorint: "The Veterans Administration Longitudinal . 7. Givca ne So ement o ny$qr~ _Cm~ yp y, stt:,, Study e~ Hea~t~iv Aging ~~ddionaL material on the development and accomplishments of the Study are contained in the official reports of the last threeyears, and copies i C

of these are also attached A l p g a stica l tools for multivarzate an .alysis, and devel'opment of the .. 4.. ..i.,.a _. . ~ speeia aspect of the lFormative Aging Study is the prob- =~•~C ~t lem of deve l in st ti ~ o •=.--"y unknown area or multivariate treatment of time series (longxtudinall data. The solutions to these problems will be apnlicable to all complex aging and other biomed" 1 d' ica stu ies whether- they take place at one point in time or stuitiple points in time. 8. Details of Experimental Design and Procedures: (AttacTi Se arat B ) p e ages _.Funds requested to carry out the,proposed procedures are described in attachement sheet. The emnhasis of r df t eques e unding is in thf f .e exploration oeasibility and development of inethodology. 9: Physical Fadlties Available (Where Other thon Administering Organization Indicate Geographical tocation); The physical facilities of the Boston VA Outpatient Clinic and those of collaborating ipvestigators in nearby hospitals and universities•are available. 10. Additiona(Requirementse C The continuing relationship of the Council for Tobacco Research with. Kurt Enslein, an. internationally known specialist in biomedical anolicatioris of computers, is indis- pensable to the development of our data acquisition and comoutational program. The ' statistical, programming, and comouter resources offered by Mr. Enslein is at the top -of the•"State of the Art" in computer technology. For examole, Mr. Ensleints data link with the computer of the Smithsonian- Astrophysical Laboratory. in Cambridge pro- vides the best computational services of its kind available anywhere. -- C 11. 6ioflraphiiol sketches of all principal andproiessional personnel (append) Biographical sketches of the princinal co-investigat.ors are apnended, 12. lisloof publications: (Five most recent as pertinen.)'.(npgend). .. List of five most recent relevant oub).ications. is appended., on attachment sheet.

C. Other. Expenses (Itemize) 0. Permanent Equipment (Itemize) E. Overhead (15% of A-F B-I- C) Esf'unated Future Requirements: Total Salaries Consumable Suppf. Other•Expenses Pormanant Equip. Yeor 2 Year 3 It is understood that the applicant and institutional'officen In applj•ing for a grant have read and found acceptable the Covncil's "Statement of Poliey Containing Conditions and Terms Under Which Project Grants Are Made " i Sub•Total Sub-Totall Overhead ' Total ~ - ~ Signature'i~/•., /.n Li.7-}3;_J :4r ~ Sianafure .! I / -- - • r4L x• - --T TelepHone 'Ca los7neir ofRcs of 1h. tr.dim4.n Telephone C, ~011 ~ W~J

. S69gPSE:00Z ' Ust ffnanelal support for rs.arch from all rourc.., Inctudlnp own Initltotion, for thfs and/orralaNd ra.orah prol.eN.

ATTACHMENT SHEET' charge from the brain evoked by a stimulus. he processing o e involves a special purpose computer which carries out on line calculations ra c study consists o e e ro-encep a oIo ; • T f th data ' ~ phic recording of electri,cal dis- t h 1 a f 1 which have never been carried out longitudinally. The evoked potential "` Hospital. The Study involves evoked potential and'EEG investigations `.~ by available equipment and investigators at the Massachusetts General .total costs of the Study. The remainder of the costs would. be absorbed at the h4assachusetts General Hospital which would amount to 100 of the : (1) The funding off a part time technician for neuroohysiologic studies' so that directions and amplitudes of the stimulus might be varied. r,ama:inAAr_ Tha RPtina -Foundation is world famous for its develonment of less than 10oof the total cost with the Retina Foundatiom picking up the fied image of the nerioheral retina and choroid. The fundin.g would invoive k (2) The funding of develonment of instrummentation to ohotoaraoh a magni- ).~~~ :~,~:~ well equinped laboratories and w.orkshons. The obj:ective of this proposal instrumentation and has available sophisticatedd biomedical engineers and peripheral retinal vasculature and choriocapillar•ies. •~-<<s"~r~ is to develov means for objective renresentation and measurement of the , V (4) Funding of services bv Dart time yhvsicians to accomplish unforseen jobs that come up in research development, such as reading EKG's, inter- preting medical records to generate coded quantifiedidata, and X-ray read- ing. . . able at the Surgical-Vascular Laboratory of the Peter Bent Brigham Hospital. The sophisticated apparatus to carry out this procedure is already avail- sion,. The nroblem here is to fund the services of a part time physician. calculation of systolic slope, as a predictor of peripheral vascular occlu-.,{,; ,; . (3) The fundinff of a snecial mPthod of pulse reaistration with automated (5) Funding of technical asnects of manuscrint preoaration such as photo- 2'RI®R ~ graphs, drac•rings, graphs, slides, bibliographic materials, etc. Also, fund- ing-costs of obtaining official documents such as birth and death certi- 0 r ficates and hospital records. CJ , ~... (6) FundinF of trave]: for professional meetings and local travel for searching hospital or other official records. ~ ~ ~ Item 12: Publications ~ (1) B. Bell, C.L. Rose and A. Damon "The VA Longitudinal Study of Healthy Aging.." Gerontologist 6:179-184, Dec• 1966. ~ (2) C.L. Rose, R. Nuttall and K. Enslein "ttethodolog•ic Problems irr Predic- tion of Lonaevity" Proc 7th Znt. Congress of Gerontology 7:255-258 Vienna, 1966. 1 Item 8: PROPOSEDPPROCEIDURES

Item 12: Publications_ (Continued) Item in Lonc_itudinal Studies of Agin¢" J.Amer. Geriat. Soc. .(3) r-.L. Rose and B. Bell "Selection of Geographically Stable Subjects " 4.:~ . 12:143-151, 1965 • (4) C.L. Rose "Representativeness of Volunteer Subjects in a Longitudinal Study of Aging'lHum,.Dev. 8:152-156, 1965. . . _..,~.__ . W. use as described in item. 8, it would be difficult a:priori to specify each item in.the budget, so that a general budget is offered instead: In view of the need for flexibility and the principles of supplementary fund, A total of $18,500, is. requested broken down as follows: A, Personnel - - - - - - - - - - - - - - - - - - 20 0 B. Consu.mable Suoplies - - - - - - - - - - - - - 30 % C. Other expenses (Services, travel, etc.) - - - 50% D. Equioment - - - - - - - - - - - - - - - - - - 0 0 E. Overhead - - - - - - - - - - - - - - - - - - 0% • .

R: REDACTED MATERIAL Biograpttical Sketch ~ BELL,.Benjs%in DATES 1924 to 1928 1936 to 1932 1938 to da te 1939 to 1940 1940 to 1942 154•2 to 1946 1946 to 1947 1946 to date 1960 to date 1960 to 1962 1962 to date. Ze.r12 to elcte 1963 tc uaie PLACE Boston Latin Sc`nool. Harvard Co3Zege Jefferson Medical Co11ege Philadelphia Jefferson Medical Colie~e E?osni'tal, Philadelphia . !4BSSdChusettS lVedicc21. Society; Norfolk District itgdical Societq; American Med ica.l Assoc i a-tion.. ?3essUchusztts Ge::eral E:osnital, Boston Boston. Disnensary, Eos-ton RhinoDlasty course (Fornan)< Mi].itar•y Hospitals, Arr^y of United States Boston City 'r"osui-~a;. Ai:ra1 Servi.ce Veterans AdmInis°tration Ourpati.eni Clinic, Boston Veterans Administration Qutr.atiert Clinic, Boston Clinical Associate in Otolarvn'oiogy, Boston Gnisre^sity School of 'Sediyine BOStoZ• U':i Yersi*y'Scihoo3, of ?iedicine Fv,,tion Grnit ansity School of H•_dicir.c Veterzins -",d:a:r ictrz:ic<i Cutoatien; CLi.nic, 5ostcn PCSITiON OR DEGREE A.B,, cum laude, in Biochemical Scfences lf.D. House D~H icer, (Surp,,ical)' :`iP_Sn}Je2' Graduate Assistant Gr adtate Assi srant ( Lt;2 ) ChIeE of EE;iT, Basic Science Course Chier, EEc?T Service S_a=f Chief of ,: '. ~w:~x Assr:. C1i.nicah °:•of~~ssar ol` 0i.o1ar ;.':7z~-)1'o?Y LC.^.iar C» on .r.+E3h gt;-i.U7.(71or y Chief, t:orr^ativ-a r?S•i.o-a S t l•..; y 100354669$ J

PUBLTCATIOt?S' .• • +• y IL~/• ; .. . .v 4C ~ ' T , _ ~ ~ ' '-• :~L i 'J . . . u.r1 Pertyoff, V,A.; and Bell, $,,: Urj Histeriut;e Des Prenlzres Recherches Bell, D,,: Office OnhthalTColoay -?lornal Fundi, ?tass,. G. Practice t1-Vrs, . •; IV, 3:6-F7 (t:aS'1 J.95t). • ~'~::.'~ . Be11, B. : Office Onh•thalrro2o„.I - Abr.orial Fcandi, }iass, G Practice ;dei~s, ;~ ) I956 Y ( e , 4:3-5 July I :.hA;; Bell, B.: Hill, G,E,; and Wyeth, JaH,: tiabilization of Staoes Ooerati:on for Deafness due to Clinical Otosclerosis, Medical Research in the Veterans Admini•stration, 2:735•737• ('?ay) 1125d. Bell, B.: and-Coc ell, • LoLo: Effect of Svstenic Cort:costeroid Tharany on.Ir.traocular Pressure, Medical Research in the Veterans Aciminis- trztio'o, 2:736 (May) 1959. Bell, B,; Harrison, R.; Trotter, :t,R•,; and tiolft, Ea: f+quecur Dynamics in. 107 Spanisn-i,:1~2rican "War Veterans, presented at Dlational Veterans Administra:.ion Research Coni'evence in Cir.cinr,ati December 1951; Abstract, Journal of Gerontology, October 1962„ Harrison, P.•,E,, Holf, E,,, and Bell, B,,: Ocular s'tudies with• sneciall reference to glaucotr:a in: a Drour+ of 107 eld~er1y males, Aail. J. Ophthalmology 57:235-•240, 1954, Rose, C,:,.y, and Bell, B, : Seie4t ion cf GeognaT}hical'! V Stable Subjects in the f5oiigi•L`1',diiicll Study of tging,. J's or the Anio Ga^,r_c?tY'lc Society, February 1`Jo5 a PR^PFSSIG'dAL C0:'~4:3'iiTY RELATIONS rnvited Lcctau'er to Clinical Asser,b].>es (1935, 1960 £ 1961) of American Acader.ry of Genera7. Practice (IiasJ.n Cbapter), tifass:, Speech and tiearinr; Society (1950), i'.ehnbil'ztati.on Sci:iinar at Boston College (1951}, F{edi:cal TV (ycu and Your Sea?t'h, 1954), and; RaZio I'ro;;raMs (Ask the Doctor, and Medical Foram of the Air, 1953: to 19E1), Task Force on AginE of iiass:, Mental Health Planning Pro;.ect-, 19o3•-1584s

R: REDACTED MATERIAL C~ ~r,~t,Zss v~ ~~: ~ ~~•. . . _. r1_re t e;~~lc~ L. 8oc= A. tiv, L~rtSve.aitl ©~ tiac^~~o, 1~~:5, G'ac:aate Sc~aol.o~ SvcT~i :ervicc s::a'y,uiat_a_tr~a Pagt Pcofesslonal Ex~rriar.c::: SccL`n' ~ , ` 1 s~ .. ,~'zSr-^::^.'.S a. t'L'D~'.iG' iiiiYare, ' ; nn (S;.r.cc I'ove--,?er 1960) InaYructca, ;;ort',r_natern Ilnive_•s?~Lys uafver; ity Co? 1 ,;;a, zoatctn, Hza.^,aeLwsctta (Since 1962) Edsca; ion: _ A. Do , I.crvard, 1937- - P=yc ;clc~,.: Eaiy::a!tqr :I:~d:crds 2la~ucchLa~tta (Sic:cc April i:~_40) ReseBZch S3cia1 ;'o?•iti2r, YA. C1ztpctier.t Clinic, 17 Co-r?: Et,, Bczeoa, 2•If:ar.ac.asz;etcu -_C-zrreat Poaitio•a: Ee.aarCh jocfS)i.ie3: a:l it Ye9ZjaaSE~=~ :.Mt::4e9 of flr- ~ iaat iicr Sup~^rted si,~ "- ~'. "'CZ6 S , ~t~~7-2952 x 4a.. ~~Y 19i;5-194?; SuFervic0"s 'i~ ~ V~a iit9.i•a1 ` , C:z_c~ ~3, 29s~~1;.,~2; Cafc~.-:ca Ca~ s eelcr I1liaciU St^`C~ - r ~' r ' ir.a~ni.co ,,c ' zafl't lcr Lo"~a _ f Z9~2a1~~:5; Scciai tici':ar, i7s~' Ilos~ital - liaZiQP.^,1 i•:avCCifZ?vA Cf a~.0Cic"il i+0:.LCsr$ ;;, E„ Socicf~y Fe~: F'n:ecx c`; in PFy chintr.i Boston Soziety fci Gero>ztClagic pa,•chicLry }.'~'~TZ;:. I ril'1 10: i a lo "Czrracttve :3ya{c~l ,esL:r,bi?j.taticn ~ t.n Ezfcctiye AnpYa:chfl 1 i K ? Sy-j ~~ • 33 : 3 2;~I 35, 19hS I* Ci IYC^>~ 7:rE? Gb:1E'-%rci1ce in r.e~ahi 1 itat•ia~'r : 3e rr~ ~'j,n ZiT. ai:~c L'Z~^~ Ae ;~_z-3: ..l•L%aliV, i'jl !n Et:l Cti?6i7 of rln_?:.,'A,BiG^S tJ E Y: ztal 1:v^p-!taVr o rL'.;yt2;aG 1952 35. Gc~,t u a;Itu to th..ti- C.-- fs ~ .,.~~.v>?± y ls7XCb 1J,53 '

._.Ctzazles, L. Rose: (^ontiruod ku6lira4ioa3) 60 `~"Groip P.Eam-arch by Stu:'ent So•'wial 1:Ork-:;a", 1~ Fo ,c.•.iav l~Sqc~ t:'k,. 3;:nuaxy 1954 n "SOCiL11 .'a:Zvi.CC Fir;67 Cit?rr9ctivC il]°Ld19w" Ry,~hr.bo, 9:uS°,138, 1955 , "Ftelatives' AttitusJes End 2~:entaZ Nospitalizaticn", ~!,r.tZl f1M, April 1958 90 "Attituv-loa of RolAti-7c~ of ,:nr-tal. Pu^ti2cta", read at Mnos~ Coafe.re :cL of Sacial t'crti, zoocon, ?,3-'ss„ , IinW_''Gt3,er 5, 1959 •-10. nlntcrcorstiation of r adicaZ, Anr ;repr.•"^a:rtc s:d aocial? Indicc3 in OcCvBC.~a; ~±a",. raad at i)'ier:•2aiirn:3l CLIi,-'rE'SB of Catontolobye Cope.raagen, Au;-tat 1963 11., ; rl~Lcr of Soas' t<'ani~;. IZln,~3~ o,: Olc2z _T'~: ~~ t rfbad SC GZrV:,ColC*;ici:'}, Society A:Z:'3u+1 'sZ.azCisZ$,: Ro :tca, F;asa, , t•Iovezbwr 19-53 12. nS0,:i:21 rAC€oVa I:1 1,01%i.11OVity'r, Ti.o 4:27-1?, 1964 • 13. ".oL`i82. Co.::al':1tiL'•s of M1IvVj.ty";~ Ctle 5 l.rc n ,,,,y ~ ~y •+ h0 N~.4`~~~1'+L:~..z~' (GU) t Spti:%(?2', Uo Z'o , 11.1)C4 14. uS; lrcticzt of G=rSrrak .ically SyaSle Subj::cts in the Iot'; it}3d2.:3sI ,°,+:G;dy of n, _..:...;.._.,~..,.. _. ~-.,.. ~ ....,, ~., .s

0 TFIL COUNCII. FOR TOB ACCO RESE AI:CIi-ZT. S.A. MF,NIORANDUM TO: The co:mmittee comprising Dr. Jacobson, Chm., Dr. Bing, Dr. Lynch and~ Dr. Loosli. FROM: Robert C. Hockett SUBJECT: Renewal grant application from Oswald Jones, M.D. - No. 523R1 l level of support (of the order of that requested in the original applica- tion). Dr. Jones is now working in collaboration with~another group at Ithaca who have obtaiined~similar results from the effect of viruses in from Dr. Oswald~R. Jones of St. Luke's Hospital, New York, New York. This represents the second year of the two years for which a grant was approved~as a contribution to the overall project at the rate of $10,000 per year. It should be noted that the present request is for support for a period of from three to five years at a much higher We enclose herewith a renewal application with covering letter dogs. R. C. H. .

DR.OSWALD R.JONES 71 EAST SEVENTY-FIRST STREET NEW'YORK.21, N.lt August 2, 1967 ..I}r. Robert C. Hockett Council for Tobacco Research 633 3rd Avenue New York City Dear Doctor Hockett: I am enclosing my application for research grant from the Council. I hope it is properly filled out. If not let me know. As you may remember, we are starting on the second part of a two year grant of $10,000. per annum. As the study has continually grown in size and scope so have the expenses. Would it be poqsible for the Council to consider starting this fall the research grant for which this anplication has been submitted? ::in most of these was the virus, type not determined. During the past year our study has completedthe bulmonary tests on thirty-one children ranging in ages seven to fourteen. '!`vnanty of these were controls, healthy children. The remaining eleven were children who had had a period of pulmonary infection previously, which took the form -.-of'bronchitis, bronchiolitis, pneumonia or asthma. The etiological factor findings and shall continue this study. This coming,year, due to the establishment of a diagnostic viral laboratory at St. Luke's HosDital, we l will be able to identify the type of virus or other organism~which has caused the lung damage. paper, a copy of which will be sent to you. We are most encouraged in our :` The results • of this study are now being put together for our preliminary . The controls shovred'normal pulmonary tests, including diffusion studies, while the others all showed definite signs of lung damage. I am enclosing three copies of a rebort which show canine distemper virus in the respiratory tract of infected dogs. We think it is an amazing piece of work from our Ithaca study on the e=fects of canine distemper on the resnira.tor,y tract. As distemaer in the dog is practically the same as measles in the child, we can safely conclude that the resbirator:)T tract of children probably is injured by measles and similar o~.her viruses. Our original thouRhts held that pulmonary emohysema probably vras caused by childhoo dviral infections, such as measles, chicken pox and resniratory syncytial viruses. Our animal and human studies are beginning to show a definite trend in that direction.

DR.OSWALD R.JONES 71 EAST SEVENTY-FIRST STREET NEW YORK 21, N:.Y. I do hope that the Council will affirm this request for a research grant which will make it possible to continue our present well oriented studies and to augment them through virological studies. Oswald R. Jones, M.D.

~t A t1 1967 a e. ugus , . Name of Investigator(s): (incfud. Title and Degrees) Oswald R. Jones, M.B. - Director of Research Ptoject 2. InslBution & Addresse gt, Luke rs, Hospital -Irest 113th Street, New lork City ,_ =; 3 Short Tide of Project; Cabbon monozide uptake and diCfusion capacity in children. This• test together with others will be used to evaluate puLnonary function in children starting at the earliest age of cooperation. This evaluation of 4. Proposed5tartingDote: pulmonary function will be applied to normal children, those with asthma bronehitis, or chronic pneumonia which conditions were subse7tuent to S Mti ' t~Qgr~tign etthjp~egfjhS,yt j}•~az.{d~arlq those caused by knovrn viral agents. Octotier 1, 967• - 3-5 years. 6. Erief Descripton of Objedives or Specific Aims:. Measurements of carbon monoxide (CO), uptake and diffusion capacity (i) have been modified to obviate painful arterial puncture by the use of end tidal PC0 at reat (2, 3, jtr S) and during graded hyperventilation (6). Application o8 these methods in the dog, and in. normal humans an& patients :with chronic chest diseases have established the reJ.ationship between~CO uptake and diffusion as graded. hyperventilation- in, normal and diseased ; a~dults (7, 10 'supporting 8,9) `'•. Normal children will be grouped according to age and size and normal values for CO uptake and. diffusion established as controls. Children with asthma bronchitis and post viral pneumonitis will be studied and the results. compared. ~ 7. Givea Brief Statemenlofyovr Working Hypothesis: The objectives of the study are: (1) to establ'ish normal values:for CO untake ¢Dd diffusion capacitv at rest and during hynerventilation in children. (2). To study the relationship of asthrcay bronchitis and viral pneumonitis on diffusion capacitv 3n d}rtdron as a background for the development of chronic pulnonary insuff iei-enc;; 4-- desm `=

9. Physical Facilties Available (Where Other than Administering Organization4ndicate Geographical tocation) The cardiopulnonary laboratory of St. Lukets Hosoital is a recognized research- • laboratory in pulmonar,-T diseases with four full time physicians, three blood gas technicians and all equipment and facilities to perform the studies outlined. New CO measurement and recording equipment is requested for . specific application to small children and;infants. r>>~t#d~nxa7s ~ . The pedicatric department of St. Luke's Hospital is headed by a full time director and operates an active inpatient service of fifty-four beds with ~ 1500'patients per one:year, as well as an outnatient depart-nent with more than, 30000 vi.sits per one year. A full time pediatric carcli ologist will devote part time ( 1/3) to this project. Antibody titre for viruses. F7.uorescent antibody search in tissues obtained by biopsy or at post•mortem, also cultures of the above tissues for oresence of bacteria and viruses. As mentioned CO nptslce and diffusion capacity techniques have been developed in this laboratory which,have been validated; in adults and which are specifically a)bplicable in children as.described. 11. Biographical sketches of all principal and professional personnell(append)' 12. List of pubGcmions: (Five most recent as pertinent) (oppend),

1 -liaterial and Method Z~+renty healthy boys, age 9 to 14 years,. from, the Cathedral School,, • . . . • . . . ~_r=~ u and that multiple results on- one person were obtazned using graded hyperventilation. - modified in such a way that correction for CO back pressure was introduced ;" helium-dilntion method, diffusion studies using carbon monoxide method of extensive spirometric measurements, lung.volume evaluation using and 11 patients from the Pediatric Department of St. Luke's Hospital of a similar age group were studied, for their lung function. Studies consisted .. tt.t _i~__~ _ .3 _1 : _1 ~ . . . . . " ~:~.: ..: L3151~Uj'y CSLLCL Ci.L1111(:cl.L CAGL111L1d"Vi1 nGI-G J.JGi'1:V.WlllGll ULL -L1+3. 110u1.4L1y UVya• clinical impressions, and the results were then compared. patients were categorized; as 1) normal~ 2) those with•min5.mal lung damage, if any~ 3) those with mild.lung damage,$ and A) those with moderate lung damage. Evaluation of the physiologic studies was done independently, without knowledge of the A1l hospital patients were examined and evaluated by a pediatrician. Hospital 'Observations Normal boys: Spirmnetric and volumetric studies have shown increase in lung volun es related to the growthr of the person studies, as expected. The ratios of the subdivisions of the lung volumes did not show any significant difference in this particular span of years. Diffusion studies have shown an lanexpected decline in efficiency of the alveolocapillary membrane when. exnressed per unit of lung,volume for more : mature boys. The findings indicate that the size of alveoli chanees with. age, so that actual alveolocapillary membrane reduces per•unit of volume. This, •' can be explained by increase of the size of the alveoli. This is, to our knowledge, the first time that this has been sho:m physiologically, although t.his. was to be expected because the growth of the lungS by prol!3f eration of the alveolar units stops. approximate.Ly at tne age or tien ana arter tinati tine lung volume increases essentially by expansion of the alveolar space. Sick persons: It vras found that the majority of the sick individuals have shown a distorted ratio of lung subdivisions (residual volume:total lung volume), although a discrimination toward normal persons "qas impossible because of the wide span of.normal values. Diffusion studies were able to differentiate sick persons from their healthy age-mates. Even mildly sick persons had. their diffusion values at the lower limit of normal or below that limit. In conclusion$ it is possible to showv that we have reliable tools for measuring lunj functions. The means are so sensitive that they can show the physiological changes related to the body growth in this age group. In addition, the results can discrininately show pathologic reduction in functions caused by chronic lung disease in:children.. 3fore studies will be needed to establish definite soan of normal values for each, age and size group, as well as to evaluate damage induced by different noxious agents. 1003546708 Added to our investigative facilities, will be a new diagnostic viral laboratory at St. Lukets ti•rere we can- identify the causative agent of pulmonary .damage in childhood. ..:.s.a:.,..,.::: ._,:_ ... •. : _.. .. ...v .. . ~ . .. _. ,.. ....... . .~

R: REDACTED MATERIAL 1- - Sub-Total r 2, 800>.00 2,900.00 28,9~i2.00 7 r$(>.30 Totd Salaries Consumable Suppl. Other Expenses Permanent Equip. Itis understood;thatthe applicant and institutional officers In applying for a, grant have read and found acceptable the Council's "Statement of Policy Containing Conditions and Terms Under Whieh Project Grants Are Made" 2~.h . .30 Overhea& Total None = Same 26 728 i0 26,728 30 ' /. Signatur// oK d f.n.i.N ~•jelephone ' W~inar Offfoe..f thL Ihnirvfi°e Telephone dc~ am=

tJrt finandol wpport for r.soarch from all roure+r, includiny own institution, for this and/or rolat.d r.s.arch projmctr. Sourca Stqny Yro1d Corporation Amount $90000 1 ear Duration \

0. R: REDACTED MATERIAL CI~; RICUT,tT.'.t VITAP: OS:'iALD R. JO'i :S3, LA.D. 1?- 9, •,";aterbury' Conn-. B.fi.p 1919 Yale College, V.D. COI:t1T'1yJiII 1923- St. ?.ul;© ts I'd~ital., N.Y. med. 192j-?_5, Be1.Iqvue . IiosII.j, 2I.Y. chest oerv. 1925-26, asslt Dath. St. I,ukels flosp.' 1926 27. Inst. in '.'•ed. Col. Univ. 1927 32. Assoc. in ?'ed. 1932 37. Asst. Clin. - Prof. of Med. 1937-62. F.A,C.P. Dipl. Tnt. `zed., Ac. t'ed.t Dir. .Car~a.o Respiratory Lab. St. Lukels. Cons. nhy. St. Lukols., Cons. Phy. _ Chest dis. S out.~:aepton Hosp., Cons. nhy. chest serviea- B©Ilevue ITosp. "Rc~ferences: 1. Jones, 0.q.s ?3easles - A Cause of Emhyse^.as An Hyoot'l:esis Ccncerning Chronic ~b.lr.onary ?:z?hysena, a Poss3.ble Cause end its Prevmtion, Am. Pzv. P.esF. I'rls. S'ol. $7# Lcr A.pr..1963 . Jones, 0.a., Platt, "7.D., and Amill, L.A. -'jiliary ":ht•erculosis Caused b~ Intravenous Self-In3ection of ;'u'-erc1Q- Pa:ci111., Treated .`alccessfulZ;l'•Tiah Strente*.xycin 'i'hcrauy.p ~n. PevR of T.II., Vol. 60, tlo. h Oct. 19h9 ' _ 3. Jonas, O.R. nnd Cournand A. - The Shsmikea FuLmns.r3* I:o1ae •rriih Chronic Bronciiiecvasis., Am. I?ev. of T.B. Vol. 2% ho. 3 Sept. 1933 Mler, J.A.# s.nd Jonos, 0.R.. - Primary Carcinosa of the Lung. Am. Rev. of T.B. Vol. 21, Flo. 1, Jan. 1930 Jones, O.p. and Burfor*3, G.R. - has;rive Atelect<isis following Cyclcp:ropane I!nEsth"sia - Jour. A: t.A. vo11 11% Apr. 2, i93~-.

R: REDACTED MATERIAL ~ LUCY Fi. S:711F T U.A . CttP'?.ICT1W1 "ITTAF. _ ~ P, ~ ritev York Cityp Barnarfl C.allegep tlerr York City .191t9' A.B.p Co11c .~;a of Ptysicians & SurCeons, Coluztbia ttn•iversit;r, N.Y,C. '. 11.D. 1953. Uary I=Ccne hassett Foseital, Coopersto;rn, N.Y. 1953 (Ju].y-Dec.) ;;..St. Luke'o i?osp. r~.i.C., 196C-62, clin, fe21o~ pediatric cardiolo;~ 1962- j-.•1961s, Preabyterian f?osp. I3anies F?osp. N.Y.C.,v Fes. lssoc- phem. 19514-1955 • =, 1?iv. F3p. Che:r,ollierapyp S1oan-9-otterin; rnst. 1T.Y.--:ad. Lic. ,1.Y.S. 1962p Am. Bd. of P©d. L'ipl. 1965„ Instr. Pediatrics Colu;:bia -Lni.v. 196?c-j, Asst. Att. Ped.(Cardio) St. Luko4s tTosp. 1964-, Ass&-.-Ped. Co1umbiF= res. L'.ed. Center, 4.Y.C. 1954 - 1!ssoc. Visitinv Ped., -I?zrl.cm I'osp. N.Y. 3.965 j. 1IEvd. Soc. of Co:u.tty of 21.Y.* t1.Y.State Led. Soc. Amer. ?'ed. !!ssoc.. References 1. S4iftj, L.it.$ and Giiffiths, S.P.: Cardiac 1!rrhytimias in the First Year of Life; ihe. Heart Bnllstin- 14: ]13-116, 1965 (1rov. I?©c.) I •

`.. Veljko Joseph Krstulovic, -M. D. Chronological Listing of'AII Activities Since Graduation From Professional School• I TO . Month Year Month Year Type of Activity, including Name and Address of Employer, Beginning with Date of Graduation from Professional School 1936 Baccalaureafie from Second State`s Boys Gymnasium, Zagreb. Oct. 1941 Graduation from Medical School of the University of Zagreb. Nov. 1941 May 1942 Internship in Pediatrics, Dermatology & Gynecology at ~ May 1942 Feb. 1943 The University of Zagreb. Continuation of intemship in the Chief Army Hospitaj. Zagreb. Postdocforai Training, ~ Aug. 1947 Oct. 1947 Course in Avia; ion Medicine, Zemun, Yugoslavia July 1950 Nov. 1951 While in the army, attended daily medical conf~rences, ward I ec. 951 I ec. 954 rounds, cardiac conferences and EKG reading in Medical Department of University Hospital in Zagreb. Chief, Dr. lvan Botteri, Professor of Clinical, Medicine. Residency in Internal Medicine at University"Hospital. Chief, Jan. 1955 Dec. 1956 Dr. Ivan Botteri, Professor of Clinical Medicine, succeeded by Dr. Arpad. Hahn, Professor of Clinical Medicine. Residency in Cardiology at University Hospital'. Dr. Radovcn July ' 1957 Ivancic, Associate Professor in Medicine. Board of Intemal Medicine. Croafii-a-Yugoslavia. Professional Appointments Feb. 1943 I Medical Officer in Yugoslav Army. April 1945 July 1947 Medical Officer in Air Force batta(!ion (equivalent. to flight surgeon in U,. S. Air Force). 1003546713 Jul y 1947 i Nov. 1951 Chief of Clinical Medicine in Air Force Division. IN Dec. + 1951: Jan. 1957 Insfiructor in Medicine at Medical School, University of •Zagreb. Chief, Dr. Ivan Botteri and; Dr. Arpod Hahn, Professor in, CFinical Medicine.

Veljko Joseph :;rstulovic, M. D. . ~ ~an, May FROM 1961 1965 TO Monfih ~ Year .1 ' June 1960 4 .I ' June 1960 . 9 June 196 1 'Dec. 1964 present 1966 present Type of Activity, incl uding Name and Address of Employer,'Beg inning with Date of Graduation from Professionai School Chief of Cardiopulmonary Section of Internal Medicine Polyclinic at Medical School. in Zagreb. Chief, Dr. 'k`t Vinko Vuletic, Professor of Clinical Medicine. Associate Supervisor in Charge of Physical Diagnosis Course at the Medical School in Zagreb. Chief, Dr. Vinko Vuletic, Professor of Clinical Medicine. Research Fellow at Cardiovascular Lab at Cornell Medical School. Chief, Dr. Irving S. Wright, Professor of Clinical Medicine Cornell Medical School , . Research Fellow in Cardiopulmonary Laboratory at St. Luke's Hospital Center, New York City-. Chief, Dr. A. L. Loomis Bell, Jr. '. Research Associate in Cardiopulmonary Laboratory at St. Luke's Hospital Center. - Consultant for Heart an& Lung. Diseases, Harlem Hospital.

Ve1 ~I:e ,!csc; ?~ Kr~: u1cv:Cr 1V s.~'. tiSf of P~s~Sic,c;ic: ~s c;ic:~ ss ir Y~ _ .:yi..::-!v...c:.~ ~'.U C~ vlc~ .ZCO cs G.:.c~i r ~ C C....i~~....~. 9' 1- 73 1 _ _._..~.._ ....1.. .. ,C^' . m^~SC•' 4 .' ° . ~ it ~i:iF.: t v0:!> >9.7~. ~ ..7~J~... S V.^iV::.CT jCta Vii ..... . . •~:. • . • KC:-.`uIGViCr •p ~ ~ f.1^Ga7c:-."; I:~ ~ C;..GIG7JSi:}Cac:~f .: Cs~:C:~i C ~~ ~• c+15CC~CS. i ubC:CufCSiS. 10: i~? {`3.r,'~;. ~ . l;;i ~i.:.0:2., ry `+ t . . , ' 0! 1C::lOGCFv , l . j S'Ei :T.,r G'J.7.>_r="+ta . ,,..,..._ o ......vViC,, V CG~IOVC •I Da'iirC:C:'CI.:C C.^..~,1JS:: :S3 L~tr7 .. 5) C..f i~o tcc, N., IIt..~-:4cvvicr N; • CSLiIC C S •J ~F5 iJf,^r'l2C~ V.: CGitCL'.~•Q CaJiC^ . (C•'- t ..n~ : l,c~ ~..:1 ia f :~ Scr~ ci:ILi1 -t. J . ti 1, ... ~..,.!? ^^-•btv ~ f • ~ " fl r i;t ~ ~+ -~ ' Cc- ZoC' I Ji.av..(~.. ~ ^ l` IGL:1960. .7) X-L:LiSGViC, V.: VC.^.0'v"'i ie^ . C:7 ...r .. G3 a CiY:Ci.~.:l~i.:i fr2C,^ya.•~:: iC i::CC. ~iSl .,n ~. r~...^ , I G . v1 . J1. ... jJ.Vw/. f".s.ufovlc V• rV' -~ • , , Gvc ,,:iGvlcf V.,, ~ ~~,.....~.:.: C:: C; i ' ~: c.. ,. t C:::C. t'Jli'i: t5~?:CliO L~Q:~Ml (. "••iC- ~a, r. . t~ tt • ` , ~ M ~': . . •J V.V /ilY .I. ..~.i3~...J'~'ltri~ 1 ~~~ ~ ~-...t ,C..~.C c.:.: 1-3 D';~'^a^ xi .SZa7i:.Jvdl:'$. ~ ~•.. Iv..lo t1

Veljko Joseph Krstulovic, M. D. Lisfi of Publications in United States of America T. t1) Shimomura, Seiichi, Pierson, Richard N., Jr., Krstulovic, Veljko, and Beli, A. L. L., Jr.: .:Primary and secondary pulmonary vasopressor responses to acefiylcholine demonstrated by fihe :,.wedged catheter perfusion technique. (Abstract), Bull. N. Y. Acad. Med., 38: 839, 1962. 2) -Bell, A. L. L., Jr., Kighfiling.er, Benjamin, Shimomura; Seiichi, and Krstulovic, Veljko: Postvalvular pulmonary artery stenosis. Hemodynamic and radiologic definition . Circ. 26: 685, 1962. (Abstract). 3) Shimomura, Seiichi, Krstulovic, Veljko, Pierson, Richard N., Jr., and Bell., A. L. L., Jr.: Separation of the primary vasoconstrictor effect of acetylcholine in the pulmonary vascular bed from systemically induced response utilizing the wedged perfused segment in the intact dog. Circ.. 26: 785, 1962. (Abstract); - 4) Kightbinger, Benjamin N., Shimomura, Seiichi, Krstulovic, Vetjko, Kittred'ge, Richard D., and Bell, A. L. Loomis, Jr.: Posfivalvular pulmonary artery stenosis. Hemodynomic and fadiologic definition (Abstract), Bull. N. Y. Acad. Med. 39: 58, 1963. • 5) Haynes, W. F., Jr., Krstulovic, V. J., Bell, A. L. L. Jr.: Smoking laabit.ond incidence of t i ti f d l i f i l R D A 93 3 9 h R n on ratory trac ec n group o a o ~ resp escenfi ma es. m. ev. esp. is. : 7 0, 1 6 . ., , Krstulovic, V. J., Haynes, W. F., jr., and-Bell, A. L. L. Jr.: Spirometry in adolescent .;.'tnales. l.. Normal values. Submitted for publication. 7) Krstulovic, V. J., Haynes, W. F., Jr., and Bell, A. L. L. Jr.: Spirometry in adolescent males. II. Comparison of spirometric data,. Submitted for publication. 8). Krstulov'rc, Veljko: Graded hyperventilation for eval,u~ofiion of dynamics of diffusion. In preparat i on . 9) Krstulovic, Veljko J., Felton, Charles, and Bell-, A. L. L. Jr.: Effect of respiratory rafie on lung diffusion using graded hyperventilation. In preparation. 10) Krstulovic, Veljko J., Jones, Oswald R., Bell;, A. L. L. Jr.: Age related changes in the efficiency of the alveolar capillary membrane in children obtained by hyperventilation diffusion study. In preparation. . lv O

.

THL CouNkoiL Foz. Torhkcco RrsEArcrr-U.S.A. August 3, 11967 MEMORANDUM .The committee comprising Dr. Reimann, Chm., Dr. Jacobson and Dr. Lynch. Robert C. Hockett SUBJECT: Renewal application from Gilbert H. Friedell, M.D. - No. 558'-M-Rl. We enclose herewith a renewal application from Dr. Gilbert H. Friedell of the New England Deaconess Hospital, Boston, Massachusetts. This application is accompanied by a progress report. The present request is for the second year of the program, for which prior consideration in the allocation of funds was promised. It should be noted that the U.S. Public Health service has provided funds for the continuation of the study and Dr. Friedell does not contemplate asking the Council for additional support. Robert C. Hockett -' .:.,.. .. _ .-..~_ -,...:.:. .,....., , ,..... _ _......_ .w..,,....~ ._z..,,~~..:..~~.:~.. _

NO. 558-M-Rl :. Activated: 11 1 TirII: COUNCIL FOR TOBACCO REsEARCIi . U.s.A. C©MMLTTEE : Dr. Reimann, Chm. Dr. Jacobson Dr. Lynch 633 TY!1RD AVEtiUZ NEW YORK, N. Y. 10017 Application For Renewal of Research Grant First IX Second Q Date: July 25, 1967 1. Name of Investigator(s): (include title and degrees) ~ Gilbert H. Friedell, M.D., Senior Research Associate 2. Institution & New England Deaconess Hospital Address: Cancer Research Institute 185 Pilgrim Road Boston, Massachusetts 02215 3. Short Title of Project: The Pathogenesis of Human Bladder Cancer 4. Proposed Renewa) Starting Date: (Anniversary or other) November 1, 1967 5. Discuss any Important Changes or Additions to Objectives or Specific Aims: In this project we propose to study the pathogenesis of cancer of the urinary bladder in human patients. We propose to investigate the role, if _ ar.y, of cigarette smoking in this disease, but only as part of a broader investigation of etiologic factors. For this reason our Detailed Plan of Procedure is divided into the following sections: Introduction Specific Aims Methods of Procedure Previous Work in this Field Selected Personal Publications 6. Give a Brief Statement of your Working Hypothesis if altered or modified: Not modified, see #5 above.

7. Changes or Additions to Experimental Design and Procedures: (Attach Separate Pages) None 8. Additional Requirements: 9. Changes in Personnel with Biographical Sketches of new Personnel (append) No changes 10 A. Publications or Papers in Press resulting from the Project or closely related' work None at this time.

f wa. a © 11. Budget (for coming year) Pathologist, part-time Technical Interviewer Research Assistant Laboratory Technician , D. Permanent Equipment (itemize) B. Consumable Supplies (list by categories) A. Salaries (Personnel'by names or category) Professional Laboratory & office supplies 1,000 1,000 a- C. Other Expenses (itemize) Shipment of urine samples-local Shipment of urine samples-Madison Travel expenses for interviewer Epidemiological and statistical consultations It is understood that the applicant and institutional officers in applying for a grant have read and found acceptable the Council's "Statement of Policy Containing Conditions and Terms Under Which i Project Grants Are Mode." Sub-Totbl 4,500 Total 4 ,050 31,050~ ~ ~ Signatore`/ `.r' aGrector oi IProjsct Telephone Signature bwinnr Otfirm.ot th. Imfiiu7iun Telephone Q ~

List financial support for research from all sources, Including own institution, for this and/or related research projects. Source Amount $149,188 (D.c.) Duration u/1/67- 3/31/71

Tuu, 0ouNcir, For Z`om~,cco P.Eisr"a'~`~i,cr z',crz' CTR GRANT NO. 558' G. H. FRIEIDELL, M.D~. Cancer Research Institute r New England Deaconess Hospital 194' Pilgrim Road Boston, Massachusetts 02215 ` PROGRESS REPORT NO. 1 THE PATHOGLI`ESIS OF HUt4A.N BLADDER CANCER The specific aims of this project were to study the relationship between: 1. Detailed smoking, occupational, residential and family histories;. I will briefly discuss ou-r procgress with regard to each of these specific afms. 2. Levels of urinary excretion of urinary metabolites; and 3. The pathology ar.d clinical course of patients with bladder cancer. - Interview Studies • We have interviewed141 patients withbladder cancer, 106 males and 35 females. A majority of interviews were patients originally treated at either the New F,nglar.d Deaconess or New England Baptist Hospitals, but recently we have N ~ ~.. Well. We are pleased at gaining access to this latter group of patients for we CJ begun to inte.rvicw patients treated in the Boston Veteran's Administration Hospital as CA feel it represents a different population than we had previously been studying. *4: For example, in 4 of the first 16 cases seen at the V. A. Hospital a history of employment in the rubber industry was found. We have interviewed 176 control subjects, 90 male and 86 female. The selection of an appropriate control population for such an interview study has been soMewhat difficult, however, and we have enlisted the assistance of epidemiologists at the Harvard~School of Public Health in making this selection. They have beemof oxeat help to us, but in addition, and perhaps of greater sionific2.nce, I believe members ~ of that department have become sufficiently interested in exploring the epidemiology of

bladder cancer to collaborate in a more extensive interview study. This study . would cover the greater Boston area and should more accurately reflect the pos- sible influence of environmental factors in the pathogenesis of this disease. Thus far 340 of the females and ?5°,; of the male bl.tdder cancer patients Tryptophan Metabolites With regard to tryptophan metabolite excretion we have carried out 24, 4~C or 96 • who have been interviewed have smoked. This is approximately the same ratio as in our control population, but at present we are attaching no significance to tnese '!, i'igures. 11`o sionificant trend is yet apparent in our data concerning occupational or residential history but 48% of the female and 430 of the male population with bladder cancer have a family history of cancer. Again I am reluctant to comment about the sionificance of these numbers until we have reviewed our data with our Council For Tobacco Research-USA we had obtained the part-time services of Dr. Carl of a second trained interviewer and with the additional funds furnished by Th By the end of the period covered in this report we had obtained the services statistical consultants. Cornil, a trained~ urologist. hour studies on 63 patients and 32 controls. t,ot all of the urine samples have been assayed but thus far it would appear that approximately one third of the patients with bladder tumors have abnormal excretion of tryptophan n,etabolites. If one excludes patients with papillomas from the group of tu~aor cases, approximately one half of the remaining patients have abnormal values. This latter figure is similar to the one originally published by Price and his co-workers for the 41 Madison patients with bladder tu^:ors. 1QQ3S4s i 24 In~our material it would appear that the histolo~ical grade and extent of the tumors are related to abnormal urinary metabolite concentrations. A review of the original patholo3ical material from Dr. Price's group of 41 cases suggests that in

this group also the extent of the tumor was related~ to the presence or absence of abnormal urinary metabolite values. This review was conducted by Dr. Safouh Atassi and me in Madison within the past few months. r.. Pathology and Clinical Course . Spencer Burney and I, in Boston, have almost completed our review of the patholo~y from all of the bladder tumor patients whom we have thus far interviewed. sections were prepared. These latter studies should be suitable for publication In~addition we are almost through with a review of 25 cases in which giant histologic within the next few months. 1'; ,; 7 A great deal of time and eneray has been spent followirg u the h-storie of 11 '' patients patients treated for bladder cancer at the New E'ngland Deaconess and~ A*ew Enoland Baptist Hospitals. From this review the fact has emerged that the majority of patients survi- ving their primary therapy will be found in the first 5 years after treatment. will shortly have fairly complete information about 5, 10 and 15 year survival in. • bladder cancer patients treated at these two institutions. Such information has heretofore not been available. Financial Support Finally, it should be noted~that we have been awarded a researchigrant from the • U. S. Public Health Service to continue our bladder cancer studies for a 4 year period. This grant will supplement funas from The Council For Tobacco Research-USA for the first 2 years and then will furnish entire support for the last 2 years of our pro:osed research project. The grant year for the ?ublic Health Service award began April 1, 1967. The sums awarded for each of the 4 grant years are $57,185.00, and $13,701.00. t31,307.00, $45a935.CO, At this time we hope that The Council For Tobacco Research-USA will see fit to renew its aNard to us for a second year beginning November 1, • 1967. Vle do not anticipate asking The Council for additional support for this project beyotid the second grant year.

---

THE COU\CIL FOR TOBACCO RESEARCFI-U.S.A, August 1, 1967 MEMORANDUM TO: The committee comprising Dr. Bing, Chm., Dr. Sommers and Dr. Reimann,. FROM: Robert C. Hockett SUBJECT: A renewal application from Geoffrey L. Brinkman,--M.D. - No. 561R1. We enclose herewith a first renewal application from Geoffrey L. Brinkman, M.D. of Wayne State University School of Medicine, Detroit, Michigan. = Dr. Brinkman's observatiomof intracellular structures in~the human bronchial mucosa are very exciting and an effort which warrants continued support. Also the opportunity to obtain bronchial biopsies from children undergoing lung resection might provide needed information of normal bronchial structures and their variations, This application, with a reduced budget, includes a progress report and covers the second of a three-year program for which prior con- sideration in the allocation of funds was promised. Robert C. Hockett John H. Kreisher

BIOASSAY- CARCINOGENFSIS & TISSUE CULTURE THL COUNCIL FOR TOBACCO RESLARCIi - U.S.A. 633 TIIl[2D AVBNIIE NEW YOIiK, N. Y. 10017 CO'iMITTF.,E : Dr. Bing, Chm. Dr. Sommers Dr. Reimann Application For Renewal of Research Grant First Z] Second Q 1. Name of lnvestigator(s): (include title and degrees) Geoffrey L. Brinkman, M.D., Associate Professor of Medicine Wayne State University School of Medicine 1400 Chrysler Freeway Detroit, Michigan 48207 - : 3. Short Title of Projects The Effect of Cigarette Smoking on the Ultramicroscopic Structure of the Bronchial Mucosa 4. Proposed Renewal Starting Date: (Anniversary or other) December 1, 1967 5. Discuss any Important Changes or Additions to Objectives or Specific Aims: • Recently in the bronchial mucosa from an adult, non-smoking patient, some -structures have been seen which have the typicaL morphological appearance of tumor whether these virus-like bodies can be identified in other individuals, both in the bronchial mucosa and in lung tumors. For this reason, I plan to broaden the scope `of the study to include study of the ultramicroscopic structure of lung cancers. If ,neoplasms cannot be.decided at this time. However, further study is indicated to see ; particular biopsy. Whether they are oncogenic or in any way related to bronchogenic virus (Figures 1 & 2). These virus-like bodies were seen in several cells from this '-~..:-these virus-like bodies can be recognized in lung tumors, then further study will be ,justified to determine whether they have oncogenic properties. Representative photomicrographs of some recent biopsies are attached (Figures 3-10). Up until now it has been impossible to obtain bronchial biopsies in normal individuals and we have had to rely on patients who are undergoing lung resection and in whom there was no clinical~or pathological evidence of bronchial mucosal ab- normality. I have now been able to make arrangements with Children's Hospital of Michigan to obtain bronchial biopsies from children undergoing lung resection for removal of congenital pulmonary abnormalities, and as a result we will be able to ~A obtain bronchial tissue which probably can be regarded as normal. C 0 C031 Ul ~ ~ ~ ~ J 6. Give a Brief Statement of your Working Hypothesis if altered or modifieda ~. ..~.~:.A........,~..:.:~.. .__. ~_.._.._ ~. ._._.,..~ :,:,

. Changes or Additions to Experimental Design and Procedures: (Attach Separate Pages). No basic change in experimental design or procedure is planned, but we have ;`enable us to determine what is the best procedure for processing human bronchiaL these variations of technique are proceeding and ultimately will, I am sure, embedding material (Vestophal and Maraglass). Experience with comparison of -experimented during the year with different strength fixatives and different mucosa. 9 9. Changes in Personnel with Biographical Sketches of new Personnel (append)'. My original technician is now working on a part-time basis because of domestic problems, but as well, I have another part-time technician who is an undergraduate student in Biology at Wayne State University. Between the two technicians I have the equivalent of a full-time technician. *0. Publications or Papers in Press resulting from the Project or closely related work

71 rA W rJL PrI {9 t: Technical ,Mary Ann Perczak Social Security C. Other Expenses (itemize) .Service Contract D. Permanent Equipment (itemize) Viewing binoculars E. Overhead (15% of A+ B-FC) 6 w : -.., ~ , . , , .. .. .. . _ . . . . _.. .. } A. Salaries (Personnel by names or category) Professional Geoffrey L. Brinkman B. Consumable Supplies(list by categories) :Photographic Chemicals, embedding materials Glassware }1. Budget (for coming year) t~SL ~ E - ^-4_~`• Total Sub-Total. Sub-Total I Sub-Total 100 w%C W*J dd .. _. . . ; ... . .. . ~ ~ ' . ~,.k ., , % time Amount 33-1/3 Nil 7,000. 290. 7,290. 750'. 500. 100. 1,350. 1,400. 1,400. 563.' 1,506, 12.109. 5Y It is understood that the applicant and institutional officers in applying for a grant have read and found acceptable the Councills "Statement of Policy Containing Conditions and Terms Under Which Projecf Grants Are Made." / Signature ~"/. Dive o~ of~Pruj..ct .~, . -7% /Telephone Signature Of/i<.r of tM. InstltuNon.

w ® List finaeial support for research fro m all sources, including own institutioa for this and/or related research projects. \ I :, . I c ~= Other Sources of Financial Support Rand Foundation Source Detroit TB & Health Society American Cancer Society II Q Amount $39,000. $20,795. $11,847. '~F' ~ ~~.J. lw}I Duration LB; , w d a 3 2 , , Current Gift to purchase electron microscope'and to equip darkroom. Effect of Cigarette Smoking on Ultramicroscop Structure of the Human Bronchial Mucosa f

2T49DSCOOt Cr = cilia P = pellicular structures = lumen = "whiskers" Dg = degenerating cell = cell membrane = microvilli = intercellular spaces = vacuoles = mitochondria = cisternae Tf = tonofibrils D = nucleus = desmosomes R = ribrosomes Sc = scrolls G = granule

M49DSE001 FIGURE 1. A cell in the bronchial mucosa showing large cisternae which probably represent Golgi organ. In the surrounding cytoplasm are numerous vacuoles, some of which contain virus-like bodies consisting i n of a central core with a surrounding two or three-layered concentric wall. These are better, seen the next figure.

FIGURE 3. This photomicrograph was obtained from a man who was a heavy smoker and shows the luminal surface of a ciliated cell with several cilia which appear normal and a great deal of pellicular material between the cilia. This pellicular material is a branching form of particularly large microvilli and greatly increases the specific surface area of the cells. On the right hand side there is a dark cell which is an exhausted goblet cell. The microvilli on this cell are covered with "whiskers", which are seen in greater detail in Figure 4.

( FIGURE 4. Shows the microvilli with "whisker" formation. This has been noted frequently on cells which appear to be exhausted and about to be shed into the bronchial lumen. On the left, the pellicular material is seen in greater detail. .

i FIGURE 5. This is a cross-section of the tracheal mucosa and shows cells in the process of being shed into the tracheal lumen. The cell nearest the lumen on the right has a light cytoplasm and very little intracellular structure. The intercellular processes which are normally interlocking are well seen.

FIGURE 6. This was also obtained from the tracheal mucosa, but shows a cell in the process of being expelled into the lumen.

FIGLTRE 7. Shows two cells adjacent to the bronchial lumen. The one on the rijht is a normal ciliated cell with~~~~eil-formed cilia and pellicular material containin~ laroe numbers of mitochondria which extend up close to the basal body of the cilia. • The cell on the left has a nude appearance due to the loss of the luminal structures. The mitochondria in this cell are much sparser, giving the cell an empty appearance. This cell is probably in~a state of exhaustion and about to be shed into the lumen.

FIGURE 8. Shows tracheal mucosal cells which were obtained adjacent to a squamous cell carcinoma of the tracliea. This figure shows well-developed desmosomes, while in the cells themselves there are numerous tonofibrils which may indicate that this cell is reverting to a more primitive state. sM9VSEOOZ

FICURF 9. The next four photomicrographs were obtained from grossly normal bronchial mucosa in a paticnt witli squamous cell carcinoma. This figure shows a large cell containing numerous vacuoles with varyin~; types o' substructure within. As well, large cisternae are noted. The cell on the right of this figure shows well-developed tonofibrils.

FIGURI: 10. Shows the granules seen in the previous figure two and three-layered membranes are forming, some of which of the virus-like bodies seen in Figures 1 and 2. in greater detaii. Within these granules are concentric and begin to have the appearance

FIGURE 11. Shows these granules at greater magnification with these unusual scroll-like forming within the granules. zV4stscooi substructures

FIGURF 12. Further demonstration of these scroll-like substructures, some of which bear close re- semhlance to the virus-like bodies. The possibility that this cell is the site of either multiplication or destruction of viruses must be entertained. EV49VSEOOZ

4p

THE C.''OUxCIL FbR TOBACCO RESEARCH - U.S.A. The committee comprising Dr. Reimann, Chm., Dr. Jacobson, Dr. Loosli and Dr. Sommers. SUBJECT: A renewal application from Kenneth M. Lynch, M.D., and Forde A. - .McIver, M.D. - No. 566-R1. We enclose herewith a grant renewal request from Dr. Kenneth M. Lynch and Forde A. McIver of the Medical College of Sfluth~Carolina, Charleston, South Carolina. This represents support for the final year of an~original two- year request for which prior consideration was promised in the allocation of available funds. Also enclosed is a five-month~progress report through July 15, 1967 and a copy of a M.S. Thesis by Martha R. McGee, entitled "Asbestos as a Possible Co-Carcinogen" representing effort on the current and pre- vious project. Robert C. Hockett John H. Kreisher .. ,..,,. . ~ ....+~>.,._.

BIOASSAY, CARCINOGENESIS and TISSUE CULTURE TII~ COUNCIL FOR TOBACCO RESEARCIi - U.S.A. tObIlITTEE Dr. Reimann, Chm. Dr. Jacobson Dr. Loosli Dr. Sommers 633 Z7:IHD AVF.'VUE ATEAY~ YaRI{. N. Y~.~ 10017~ Application For Renewal of Research Grant First IN Second D I ,.. ;.~.i+.",. r ;. -- No. 566-Ri '' Activated: 10 1,66 Cf. #276 Activated: 6/1/60 Renewed annually as of June l, 1961- 1965 inclusive. Supplement: 6/1/66 1. Name of Investigator(s): (include title and' degrees) Kenneth M. Lynch, M.D., Eneritus Professor of Pathology or e. McIver, M.D., Associate Professor of Pathology 2. Institution & Address: Medical College of South Carolina 80 Barre Street Charlest on, South Carolina 29401 3. Short Title of Project: Environmental Factors and Pulmonary Carcinogenicity 1. Asbestos Dust II. Asbestos and Co-carcinogens, Viral and Chemical 4. Proposed Renewal Starting Date: (Anniversary or other) 1 October 1967 5. Discuss any Important Changes or Additions to Objectives or Specific Aims: No significant changes or additions to objectives or specific aims. ' 6. Give a Brief Statement of your Working Hypothesis if altered or modified: No alteration in working hypothesis. .:._. ., _:.. __ ,... ~. :.::... .<,.,.__ . ._._. .. . . . : . . . .. . . _._ . _.. _ ._. _ _ ._ ~-.u:~ ~ ~.- ' . . .~:. , . ,..__.

7. Changes or Additions to Experimental Design and' Procedures: (Attach Separate Pages) No major changes in experimental design,.but this feature is restated and brought up to date in the Progress Report. 8. Additional Requirements: Except for the continued advice and assistance of the Staff of the Oak Ridge National Laboratories, no additional requirements are anticipated. The desire to report portions of this work at the Second International Conference on the Biological Effects of .,Asbestos in Dresden, Germany in April 1968 is reflected in the . budget request. O G7 U't EA 0? ~ . ~ 9. Changes in Personnel with Biographical Sketches of new Personnel (append) No changes in professional personnel. Mr. Grimsley, who served as Project Assistant last year, has entered graduate school and Mr. Cummings has taken over both posi- tions. Heyward Wesley has replaced Ben Simmons as Animal Caretaker. Paul Gillette and James Powers, medical students, have been employed to take care of animals and supervise the project on week-ends and holidays. 10. Publications or Papers in Press resulting from the Project or closely related work •Manuscript on exposure of dogs to asbestos dust in preparation for presentation at Second International Conference on the Biological Effects of Asbestos. Manuscript (thesis) on Asbestos as a Possible Co-carcinogen completed by Martha R. McGee (see attached copy). i , _ .. . _ ...,. _~ __.. _. . . . ._.~.:._:.~:.r,...,r.b::- . .,.- , ' ~ - ,. ~:.~ :'. .~ ~;. .:. -.. . . . . ,. . . .:,.. . , . . .. . _. a,~- , ... . . ~ ........_. .. >. .a~...._.,...

~ ~ : . t! M1 ~.. }':?rt ',1 t. Budget (for coming year) .r Technical > A. Salaries (Personnel by names or category) Professional Kenneth M Lynch, M.D. ,:.Forde A. McIver, M.D. (See attached page 1) B. Consumable Supplies (list bycategories) C. Other Expenses Ctemize) D. Permanent Equipment (itemize) None E. Overhead (15% of A+ B-HC) See attached page 1) (See attached page 2) C Sub-Total Sub-Total' Sub-Total % time Amount 0.00 0.00 14,340.00 1,395.00 4,248.00 Total 32,570.00 i C• h is undentood that the applicant and institutional officers in applying for a grant have read and found acceptable the Council's "Statement of Policy Containing Conditions Ond Terms Under Which Project Grants Are Made." 0,00 Signature~_ ' tea Fo7rde A. Pf1¢fve-k;'_v , 723-9411 Telephone Signatui 4~lC . ~~ !us neu O licer of the Institution 723=94'11 John E, Wise, T 'e bone Vice President for Business A~f~'irs ~ `_

1 Attached Page 1 11. Budget (for coming year) ,Mrs. Patricia Dumas 100 Michael Cummings 100 Heyward Wesley 100 Ben Martin 2 Paul Gillette* 10 James Powers* 10 4,800.00 3,600.00 3,300.00 ' 240.00 1,200.00 1,200.00 $14,340.00 *Paid at high rate per hour for week-end~and~holiday supervision and care of animals. Animal Care Committee maintenance charge for mice without caretaker @ 0.01 per animal/day Animal Care Committee maintenance charge for 20 dogs without caretaker @ 0.15 per animal/day Animal Care Committee maintenance charge for 70 guinea pigs with animal caretaker @ $0.08 per animal/day Laboratory supplies for light and electron microscopy Photographic supplies Asbestos and dusting supplies 7,020.00 1,026.00 2,016.00 1,350.00 375.00 O 0 450 00 w . 91 ~ 350.00 ~ ~ $12,587.00 ~ *Please note that new Medical College policy has resulted~in a sharp increase in the cost of animal care. Nevertheless, these charges are 25% to 40% below those of neighboring medical colleges. . Animal care supplies - gloves, shoe covers, coveralls, disinfectants, etc. C. Other Expenses (itemize) Travel to report on previous studies at the Second International Conference on the Biological Effects of Asbestos and for advice and consultations at Oak Ridge National Laboratories 875.00 ~~9'~

Attached Page 2 Services by Surgery Department for broncho- Sub-total $1,395.00 Permanent equipment (itemize) None Sub-total 0.00 4,248.00 T otal $32,570.00

Current list financial support for research from all sources, including own institution, for this and/or related research projects. Title of Project No other funded research at this time. .i,: Pending No other research applications pending. ZS49VSEOOZ

Timi, CovxciL ror. Tonltieco RESLARCr1- U. S'. A. . , . ,;a~,. The committee comprising Dr. Little, Chm., Dr. Sommers, Dr. Reimann and Dr. Loosli. Robert C. Hockett SUBJECT: Renewal application from Lucio Severi, M.D. - No. 567-Ri. We enclose herewith a renewal application from Professor Lucio Severi, Professor in the Division of Cancer Research, University of Perugia, Perugia, Italy. It represents the first renewal of an original three- year request for which prior consideration was promised in the allocation of available funds. The amount requested'is $1,000 less than was granted for the current year. This study seems to be going forward according to plan. Under item #10 of the.application, three papers are listed. The first Progress Report (October 1, 1966 - April 1, 1967) was distributed to the Scientific Advisory Board on June 23, 1967.

~ BIqASSAY, CAROINUGl;Nk;615 and T1S5Uh• CULT'UHE: NO. 567R1 Activated: 10 1/6b : Txr, Couxcu, roR ToBicco RicsmRcu - U~S.A. ' ~ ---- ' T - • 633l1atD AvEXUE . . . . COl4.ffTTEE: . NEW 7ocu:: rr: r. 20017 Dr. Little, Chm. • Sommers )?" ;;' Dr. R0im8[1n - App(ication For Renewal of Research Grant -D L 'r.oosli . _ _ F•sst 9 Second [] : Datrr, 19th July, 1967 1. NameafInrestigator(:)o(includetitleandde9rees) PTOf". I)uei0 Severi, iZ`.D. Principal Investigator; '.Cesare Biancifiori, M.D. Go-Principal Investigator; ~ RqdQlro Ribacchi, a.D. Assistant; Emilio Buceiarelli, rii.D. Assistant; `~ 2 Huhtutian :Gaetano Giraldo, M.D•. Assistant ~;"'r`'Adtlress k ;z; -- ~~ .M:-. .-. . C-':'~'`:i•£... ; Division of Cancer Research, University of Perugia 0. Box 327 ' :r;•. 'FERUGIA ZTAT~Y Fn ~1 U~:x~::;iC%"ti~~~c~ ~ ,~ r3. ShortTitleof Proiect: -, :5"6n Approach to the Study of Internal Factors in Dung Carcinogenesis. `-Influence'oi.Hormones. d. ProposedRen.wafStadingDates(Anniversaryorother) OetOber 1St, 1907 3 - . s" s. Discuuseny,important Changes or Additioni to objectlre: or Specific Atms: There have been no basic changes ~W"';.made to the ob3ectives proposed•in the Application of 2nd August, 1966 and the Semiannual Progreas Report no. 1 forwarded in April, 1967 to The +c: Council for Tobacco Research. ' .~. cta '? The observation of adranallimage in carcinogenesis b ;oreal dy hydrazine/isonicotinic acid' hydi•azide (IP)H) seems a profitable line of .; invest3gation.:":~In lurg cancer patients changes in endocrine glands have been described. ''.This is still the starting point for study on ~~~ ~yrhether internal factor exists in iungcaneer. -~ an1' Y .3'~*~~The aims of this ;research' ther•efor•e remain chiefly, o study whether the adrenals are involved in spontaneous .i and INH; and hydrazine sulphate (h.s.)..induced pulman.arg _ .tunours in mice and To study whether the thyroid•is involvedin-spontaneous .• 'and INH and h.a. induced pulmonary tumours in mice. '~q. . Q

7. Changes or Additions to Experimental Design and Procedures: (Attach Separate Pages) (SEE ATTAC_TiED PAGES) 8. Additional Requirements: Nil 9. Changes in Personnel with Biographical Sketches of new Personnel (append), New personnel: Dr. Gaetano Giraldo (See biographical sketch attached) •10. Publications or Papers in Press resulting from the Project or closely related work (SEE ATTACIiED LIST )

Changes or Additions to Experimental' Design and Procedures: A. Spontaneous and 1NFI and h.s. induced pulmonary tumours in. . the mouse in relation to adrenal hormones. : in susceptible and resistant mice. - .. Incidence of spontaneous and induced pulmonary tumours .There have been no changes made to these experiments, which have been described in the Application and in Progress Report no. 1. Incidence of spontaneous pul.nonary tumours in susceptible adrenalectomized mice into which, the adrena7.s of resistant mice have been grafted, and vice versa. At the beginning of this-dxperiment (see Application and Progress Report no. 1) A/Lae/Se and C57BL/Cb/Se mice, respectively susceptible and resistant to lung tumour induetion, were used. To assure that the adrenals take in the host it is necessary to .treat the test mice at birth with lymphocytes of the donor mouse. .:, The administration (intravenous or intraperitoneal) of lyaphocytes . between these two substrains, however, is badly tolerated: none of the C57BZ,/Cb/Se mice treated with A/Lac/Se lymphocytes lived beyond a few ~ days; the same thing happened when A/Lac/Se nice were the tests and the C57/Cb/Se the donors. None of the technical procedures used was effective in preventing the death of the test mice. For this reason, the C57BL/Cb/Se mice were replaced by CBA/Cb/Se. The tolerance ~betweeri CBA/Cb/Se and A/Lac/Se mice has proved good provided the test , ,.,.. mice are treated within a few hours of birth. CBA/Cb/Se mice are considered resistant to lung tumour induction and therefore may replace the 057BL/Cb/Se. Several newborn mice of the two substrains have already been `treated, and survival is good; at 8 weeks they will be adrenalectomized, and have the adrenals of the lymphocyte donor grafted into them; they will be examined at natural death. This has already been described in the Application and in Progress Rcport no. 1. c. Pulnronary tu~~origene sis by IlNH and h.s. in susceptible adrenalect-ouized. mice into which the,adrenals of resistant mice have beengrafted, and vice versa. These experiments will be carried out as described in the Application, with CBA/Cb/5e mice replacing the G57BI,/Cb/Se. 11 100354G'756

incidence and the susceptibility to lung tumour induction are different. If these characteristics in the adrenalectomized hybrid test mice are the same as in the parent donor of the adrenals, the influence of these glands in lung carcinogenesis could be hypothesized. 1 . Incidence of spontane ous pulraonary tumours in intact and . ~:. in,adrenalectomized (C5?BZ/Cb/Se x A/Lac/Se)Fl hybrid mice. No:change has been made to the experiment as described in the :,.:Application but, as mentioned in Progress Report no. 1, : fi t ~ ;(BALB/c/Cb/Se• x C57BL/Cb/Se)F' hybrid' mice will also be used. ''Incidenc.e of sp ont ane ous pulmonary tumours in adrenale ct o- "; `;': ;: • niized (C57BL/Cb/Se x A/Lac/Se)F mice into which the e n grafted. : adrenals of the parents have be • No changes: the experiments will be carried out as described e x ~e y w s u . _.Q, ; in the Application. In these experiments as well (BALB/c/Cb/Se ' ill al o be sed C57BL/Cb/~ )F h brid mic Pu]:monary tumorigenesis by INH and h.s. in intact and in ! adrenalectomized (C57Bh/Cb/Se x A/Lac%Se)F1 hybrid mice. (as shown in Progress Report no. 1). (BALB/c/Cb~Se x C57B1,/Cb/Se)Fi hybrid mice willl be used Also in these experiments, in addition to the (C57BL/Cb/Se x A/Lac/Se)F hybrid nice (as shown in the Applicat'ion), g. Pulnonary tuinorigenesis by INR, and h.s. in adrenalectomized 05?BL/Cb/Se x A/hao/ae)Fi hybrid mice into which the adrenals of the parents have been grafted. - These•experiments will be carried out as previously described in the Application; (BALB/c/Cb/Se x.C57BL/Cb/Se)Fl hybrid e m.ice will be added. If it seems of use, (CBA/Cb/Se x A/hac/Se)F1 hybrid mice will be ".'added to the experiments described'in paragraphs d,e,f,g. These different types of hybrids will be used because they are the product of substrains of mice in each of which the spontaneoug B•. Spontaneous and J21H and h.s. induced pulmonary tun¢ours in the mouse in relation to thyroid hormones. No changes made to the experiments, as described in a,b,e,d- of the Application. 1003546'7S7

0. Additional experiments. In addition to the original experimental design, investigations ;` are be irig carried out, on: Mitotic cell activity in the mouse lung and Immunologzcal {:. .approaches of INH and h. s. induced lung tumours in mice. . a. Preliminary studies on the mitotic cell turnover in the mouse lung. This experiment is being carried out tirith special fixation and staining techniques in lung of lung.tumour induction susceptible (BALB/c/Cb/Se) and~ resistant (CBA/Cb/Se and' C5?BL/Cb/Se) mice, treated and non treated and in different hormonal states. This should allow us to observe the early cell changes and the cells•chiefly involved in the regenerative process. Differences between the three groups of mice - treated, non-treaiied and in different hormonal states - could be of significant interest. b. T.mmunological approaches to chemically induced lung tuzaours in nice. (i) Attempts to show whether lung tumours obtained with INH and h.s. are merely accelerated spontaneous tumours or whether they are indtzced. -:It has been shown that chemically induced tumours develop specific antigens, whereas spontaneous tumours do not. Moreover, whereas virus in.duced tumours usually carry antigens specific to the virus and therefore cor_L-aon to every tumour induced by that virus, the tumour specific antigens of chemically induced tumours differ from tumour to tumour. The application of this approach to INFi and h,.s. induced lung tumours could proceed along the following lines: part of a large lung adenoma could be transplanted into mice of the same strain; when it grew out, a part of it could be excised an•d fragments iMplanted into other mice, and removed when they started to grow out. The ability of further fragments of the same tunour, or of measured numbers of cells from it, to be rejected by the immunized mice would • de:aonstrat e the ant igeni city of the se tumours. 100354r.7sg

R: REDACTED MATERIAL This experiment will have to be repeated several tiMes. shoul.d, however, be confirmed also by other experiments, with the ,a,iA of showing that the participation. of a virus in the development .of the pulmonary tumour can be excluded, but this will not show that : a hormonal factor is not involved. The latter is•an internal component _ which probably does not act by itself but, in favourable conditions, might allow the action of 7Iv'H, or h.s. or of any other careinogen. , (ii) Attempts to influence the growth of urethane induced - primary lung tumours in BAhB/c mice by stimulation of imTaunocompetent cells of the host. Previous work done in this laboratoxy (1965, 1966) has shownn that (the depression of in~.'nunoco-apetent. cells obtained by) neonatal : y thynmectomy poten.tiates the oneogenic effect of a single •dose of ~ urethane in BALB/c mice. In fact, in thym-ectomized mice lung• tursours develop earlier, are more nu.*ierous, grow to"a.-greater size, metastasize more frequently than in controls treated' with the sane dose of urethane, and they only rarely show foci of necrosis and/or fibrosis. Theoretically, a stimulation of immunocompetent cells in mice treated with a single dose of urethane could reduce the oncogenic effect of the chemical compound. Following this approach, BAhB/c mice inoculated with urethane will be treated with incomplete and complete Friend's adjuvant, with urethane-induced secondary BAZB/c lung tum•our grafts, and with a cell-free homogenate of the same tu.*~our. - _ 8. (See printed form) I 9. Biographical Sketch of Dr. Gaetan~o Giraldo. Title: Assistant scientist, Division of Cancer Research, -Universi:ty of Peru gia a _ Place of Birth: Naples, Italy Pre-sent Citizenship: Italian Sex; Male Education and Experience: Dr. Giraldo graduated M.I?. from the University of Naples in. 1962. H:e is a staff member of the Institute of Pathological Anatomy. and Division of Cancer Research, University of Perug•ia, from 19b3- Dr. Girald6 has:published 17 papers on-various subjects. ~ _ 1oo3i4s'7s9 -

-Publications or Papers in Pre.ss resulting.from the Project or closely related work. Ribaechi, R. and Girald•o, Cr.: Sui fattori intrinseci della cancerogenesi polraonare del topo: effetti della tinectomia '; ~ .r.r ' neonatale. hav. Anat. Pat. Peruga a 26, 127-136, 1966. Severi, Z. and Biancifiori, C.: Hepatic carcinogenesis in CBA/Cb/Se mice and Cb/Se rats by hydraza.ne sulphate. "~ 29th-30th October, 1966. (in:press) International Conference on Hepatic Hegeneration, Montecatini., 3. Severi, Z. and Biancifiori, C.: Hepatic carcinogenesis in CBE1 mice and Cb rats by isonicotinic acid hyd-rGzide and hydrazine sulphate. Symposium on Hepatomas, Philadelphia, 19th-20th May, 1967. (The paper will be forwarded to the Journal of the National Cancer Institute for publication) 0

R: REDACTED MATERIAL -- 'c,....-. . .. • ~ R Consumable Supplies (list by categories). ? Animal food and sawdust ~~,Chem1cal drnizs 5taining nateriala ,•_,:Sttrgical instruraents 01,000 500 . 250 150 100 Record oards, registers, labels :Glassware C. Olher Expenses (itemize) 300 -1,700 1,700 15:000 L it is understood•that the applicant and institutional officers in appljring for a grant have read and found accepttble the Counc3l's "Statement of;Policy Containing Conditions and:Terms Under Which Project Grants Are Made." Sub-Tatal 300 Tatal $11,600 Signature Ls-,+% ofrsctar ot h.ieet Signature~ ~+4nps Ollibi at'Iha ImGwltow Telephone `

Oth.r sourca of Financial Support ! iF . Ulttinanciat,upport for ret.arch from ali .ourc.s, Including own InNttution, for this and/or r.iat.d rtswrch pro ~ ?! Hormonal i'actor(s)'in pulmonary oaroi.ndgeneeis'. (by hydrazine). (Severi, L. and Bianoifiori, 0.) Lung tumorigenesis by Isoniazid (INH), its' i netabolite hvdrazine su].nhate and derivativesi of hydrazine. (8iancifiori, 0.) , • V . The Counoil for Tbbacoo ;. i4i - Research, 633 Third Avenue,t~ ' New York N.Y. 10017

Trrr CovzTCIL For ToBAeco RLS1 CIAr.CH -U. S.A. MEMORANDUMi r August 3, 1967 TO: The committee comprising Dr. Cattell, Chm., Dr. Bing and Dr. Jacobson. FROM: Robert C. Hockett SUBJECT: A renewal application from Kenneth M. Moser, M.D. - NO. 569-Ri. We enclose herewith a renewal application from Dr. Kenneth M. Moser, of Georgetown University Medical Center, Washington, D.C. The research program~seems to be going forward according to plan, and since this request is for the second year of an original two- year plan, it is essentially routine. Robert C. Hockett I*

CARDIOVASCULAR, PHARMACOLOGY and CHEMISTRY Activated: 10/15/63 C010ITTEE: 633 TIIIItD AVE.UE - NEW YORK. N. Y. idoi~ Renewed t 10/15/64 Dr. Cattell, Chm. Renewed: 10/15/65 Tiar,, CoU;r•CiL FOR To13Acco Frf:SL11RCH - U.S.A. Cf. #3 - Activated: 10.15 No. 56 -R1 Dr. Jacobson Dr. Bing :. ~`.. Application For Renewal of Research Grant First (5 1. Name of Investigator(s): (include title and degrees Second 0; Kenneth M. Moser, M.D. Associate Professor of Medic' .2. tnstitutiond~ Georgetown University Medical School, Washi Address: 3. ShortT,tleofProject: Smoker- Non-Smoker Differences in Activation of Fibrinolytic-Coagulation Systems. d. Proposed Renewal Starting Date:(Anniversory or other) E November, 1967. 5. Discuss any Important Changes or Additions to Objectives or Specific Aims: No ma jor changes have occurred in objectives or specific aims from those stated in the 1966 application. The goal is still to ~ define smoker-non-smoker differences in coagulation-fibrinolytic behavior which might bear upon the statistic relationsh,ip•6etween heart disease (especially coronary thrombosis) and cigarette smoking. • 6. Give a Brief Statement of your Working Hypothesis if altered or modified: The basi c hypothesi s remai ns unaltered; namely, that smoking may enhance thromboatherogenesis via the coagulation-fibrinolytic system.

I 2. =.::..7. Changes or Additions to Experimental Design and Procedures: (Attach Separate Pages) At present there have , , ~, been no majo, changes in experimental design. However, if current trends in data are substantiated ::. three additiot,s to the protocol may be instituted: 1) studyof the changes in "platelefi adhesiveness" associated with smoking (in smokers) or h erventilation (non-smokers), 2) limited studies f yp ; o • fibrinogen turnover rates in age-sex matched smokers and non-smokers; 3) addition of an exercise ~~ test to the nicotinicacid stimulus; i.e., to determine whether exercise produces the some type of k- k d'ff s e d t' ' erences rxc mo r non smo er t as oes ntco iaci . :_~"~;..,,,~ The rationale for these otenfial additio s lies i the data e ded (Tabfe f-'A W7~ n n n s .,.:.: p app , e ave recenfily subjected a considerable amount of our data in this study to computer statistical analysis 'mean; standard deviation, standard error, correlation and "t" tests) . This analysis has revealed, '~ry :`several instructive smoker-non-smoker differences. In basel:fne-only studies (Table I) smokers have a statistically significant (P <•05) higher level of plasminogen. While other differences exist , they are not statistically significant. However, in this analysis there is a significant positive : eorrelation. between! age and fibrinogen concentration in non-smokers - not in smokers. Thus ;•smokers do not apper to have the expected age-related' rise in fibrinogen, although the mean :: fibrinogen is higher in smokers. (Continued - pages 2-A thru 2-E) 8. Additional Requirements: • We currently have no major additional requirements. 9. Changes in Personnel with. Biographical Sketches of new Personnel (append)' Dr. Paul G: Harsanyi joined our research staff this year. (see attached bibliographic sketch,.page 2-F) • •io. Publications or Papers in Press resulting from the Projecl or closely related work is currently being prepared for publication. The datQ presented i n Ta6f es 1-3

-Page2-A 7. Changes or Additions, cont. On the acute smoking (or hyperventilation) studies, other interesting data appear. (Table 2) . Again, there is a positive age-fibrinogen correlation in non-smokers only, with fibrinogen levels again higher (but not quite significant at P < .05) in smokers. The plasminogen levels are again significantly higher in the smokers. In both broups, there is a higFitiy significant rise in plasminogen (P < .001) with smoking (or hyperventilation).. Partial tiiromboplastin time behavior is also interesting. In this study,hasdine PTT was longer in smokers (not significantl.y) but fell, with smoking and after smoking. In non-smokers, PTT increased slightly with and following hyperventilation. , In the nicotinicacid study (Table 3) to date, instructive data have been generated. The most striking is the fact that nicotiryc acid provokes a much greater (and apparently more sustained) enhancement of activator activity in the non-smokers than in the smokers. Significant differences (as measured by both ELT and A-P plates) exist, between smokers and non-smokers at all intervals. Thus, the non-smoker seems to release activator more readily than the smoker. If this is true under all circumstances, itwould; suggest thrombolysis may be impaired in smokers in terms of its response to a-given stress. f As fihe data are exteded fthtlill bidt (A ; •n,urer compuer anayses we carre ou. program h b dld fh) aseeneveopeor tis specific purpose . Analysis of smoker-non-smoker differences between different age and sex-matched groups are contemplated. These may reveal differences obscured by combining all data, as has been done in the analysis reported in Tables 1-3. I ~ N I ~ , ~ . . O O W i ~ ~ ~ ~ ~ ;

Page 2-B Smokers (60) Non-Smokers (50) Pack-Years 18.0 f 15.4 Pro. T. (Sec) 17.9 ~ 3.1 P.T.T. (Sec) 89.3 ~' 30.7 ELT (Min) 234.0 t 74.0 Fibrinogen (mgm%) 451.0 ± 147.0 A-P (mm2) . 192.0 f 121.0 P`gen (mm2) . 329.0 ± 70.0 C. P'gen (CRU) 5.6 ± 4.5 3.7 ± 3.3 Table One: Smokers versus non-smokers. Fasting morning values. .. u _ _ . n~.. ..., . ~ .~. . . .. r...al+. .. - . ...a.. ....J .a<. +.+

Pack-Years .. . 19.8 P.T. 17.2 } 2.8 17.2 ± P.T.T. ELT A-P P'gen C. P'gen N'on-Smokers (40) Age 41.5 f 21.2 6.0 P. T. 16.6 ~ 2.1 17.0 ± 2.4 16.9 } 2.3 s...., . 2.8 17.2 ~'. 33.8 85.9 f 75.0 242.0 ± 151.0 443.0 ~' 123.0 191.0 ~' 81.0 307.0 ~ 5.4 7.5 ± Pack-Years 0 -- -- 96.3 38.1 88.8 t 241.0 ~ 76.0 229.0 t 451.0 t 156.0 451.0 ± 196.0 ~ 126.0 206.0 t 307.0 t 73.0 314.0 ± 5.5 t 5.0 13.4 ± 82.9 t 21.7 92.6 ± 46.7 85.7 f 49.9 243.0 ± 64.0 250.0 t 60.0 240.0 ± 69.0 411.0 ~' 140.0 404.0 ~" 139.0 402.0 ~ 149.0 A-P 172.0 } 74.0 183.0 f 88.0 196.0 f 85..0 h-h . O P'gen 288.0 ± 60.0 285.0 ± 69.0 290.0 ± 60.0 W Table Two: Smokers and non-smokers studied before, at completion and 60 minutes (O after completion of a 30 minute smoking or mild hyperventilation period. C. P'gen 3.8 ~ 3.9 11.9 +' 3.4 4.6 t. 3.6 ~ ~ VI

Age • w . ~.~ Pack-Years P. T. P.T.T. ELT Fibrinogen A-P P'gen C. P'gen ~A) nicotinic acid given intravenously. Pagei (Page 1. of Table Three Smokers 0 10 20 40 60 37.8 * 12.8 . . : ' . , 16.3 t 9.8 _ -- ° -" _ 16.0 + 1.9 16.4 t 2.4 16.4 1- 2.2 16.3 t 1.8 16.1 ± 1.8 . 90.3 * 35.7 97.8 t 42.7 102.0 t 45.0 ~98.9 ± 44.0 86.1. '~ 55.9 . 0 t 60.0 263 151.0 ± 112.0 147.0 f 111.0 178.0 ± 96.0 ` 199.0 ± 90.0 ~ • . . . ~ 419.0 t 126.0 408.0 t 156.0 272.0 f 77.0 419.0 ± 138.0 ~ 439.0 f 145.0 ' 135.0 ± 142.0 345.0 ± 277.0 354.0 ± 279.0 271.0 ± 220.0 233.0 ~ ,180:0 262.0 ± 79.0 266.0 t 77.0 265.0 * 73.0 • 274.0 ± 61.0 . 275.0 f '75.0 3.8 ± 2.4 4.4 ± 2.6 ' 4.4 ± 2.8 4.2 ± 6.6 • 3.9 ± 2.2 Table Three: Smokers (38) and non-smokers (30) studied before and at 10, 20, 40, and 60 minutes after 100 mgm 0449MOOZ

Age Pack-Years P. T. P.T.T. ELT Fibrinogen A-P P'gen ;: C. P'gen 16.4 87.6 223.0 355.0 167.0 280.0 1.9 74.0 •` 1 85.0 274.0 - * , 159.0 508.0 ± 288.0 264.0 f 78.0 4.2 ~ ~ 2.6 ± 85.2 . ~ . _ . . ~ - - i 124.0 ~' 120.0 102.0 94.0 .: ; .; ~"143.0 316.0 f T 98.0 314.0 153.0 '~. ;' ', 360.0 , 265.0 * 3.3 f 2.3 73.0:~ Smokers (38) and rion-smokers (30) studied before and at 10, 20; 40, and 60 minutes after 100 mgm nicotinic acid given intravenously. 2.0 + + t 73.0 * 126.0 f 109.0 83.0

R: REDACTED MATERIAL 2- F . Page 2, Item 9: Personnel with Biographical S::etches: Dr. Paul G. Harsanyi. . , • Date of birth: Place of birth: Budapest, Hungary Marital status: ~ Q- M.D.: University of Budapest, 1962. Internship: _',: • University of Budapest Medical Center, 1962-62. Residency: °..'University of Pecs Medical Center, 1963-66. • Visiting Research „ i - : Scientist: • University of Vienna: Medical School, I966. : . RPSenrch AssncintPr - •, .... Georgetown University Medical School, 1966 to present. (NOTE: Dr. Harsanyi; who joined our Program in 1966, : has made a considerable contribution to our. research efforts. His departure from. Hungary interrupte.d a promising research career there. We are del-ighted he has been able to resume it with us.) . r 0

tl7s understood that'theappl7cant and institutionol'officen in applying fona grant Hare read and found acceptable the Counc+!'s "Statement of Policy Containing Conditions and Terms Under Which Project Grants Are Made." 500.00 600.00 Sub-Total 1,100.00 300.00 200.00 300:00 400.00 sub-Total. -- 1,200.00 20b-.00 200.00 I 400.00 / 2,032.00 Total 15,980.00 Signature a;r.ww af.Mai.ct Si natu e r g otca.,r a. tew~w .. GCMM `1= R: REDACTED MATERIAL A. Salaries (Persannel by names orcateqory) : Professionaf - Kenneth M. Moser, M. D. George C. Hojjar, M,. D, Paul G. Harsanyi, M. D. : Tedin'icol _Charles Brunswick, B.S. _ :: Dorothy Brown, lohnnie Burbank "' :Fringe Benefits (7,12?k) : L ConsumableSupplies (list by categories) Reagents, solutions .. Glassware, needles, miscellaneous C. Other Expenses Gtemi:e) Equipment maintenance and repair Preparation of slides, graphs Data analysis (computer time) Phyments to volunteers 1...... ~ . .. .. .. - O. Permanent Equipment (itemize) " Double Vortex Mixer Hand Calculator

i 11 Lisl Hnandotsupport fa ra+orch from all aource., lncludinp own InilituFlon, forthG ond%r reloted r.s.arch prol.eh. ~ r CurreM Title of pralect ' Role of Fibrlnolysis In Pulmonary Embolism t Pending Control Mechanisms oF the Fibrinolytic System In Man ~~~9~SEOOti

dgz= .. . ~`~s~. ~ .-

TFrr Cot7-NLciL For ToBAcco RESEARCrn - U. S. A. • MEMORANDUM . The committee comprising Dr. Jacobson, Chm.; Dr. Lynch, Dr. Reimann '- and Dr. Sommers. FROM: Robert C. Hockett SUBJECT: First renewal application from Clayton G. Loosli, M.D. - No. 573R1. We enclose herewith the first renewal application of Dr. Clayton G. Loosli of the University of Southern California School of Medicine. second and third years. - This represents the second year of a three-year program that was activated on October 1, 1967 and promised priority in consideration for the attached). - A detailed Progress Report accompanies the application (copy R. C. H. Z1. •'.a-.i.'.:~i~:_Tw:~{L~.~_.-.'..~-an-..'~i.~.. ..•~... . .......~. . .. .~ - „-.'. . .... .... a. _.... .. .. ..-.. . . .. ...............,~.r.~~_.. .r.. ..Y.~.':../-~ ....

NO. 573R1. . .. . :. Activated: 10 66 'IiE COUNCIL FOR TCBACCO RESEARCH - U.S.A. NEW YORK. N.. Y. 10017 Application For Renewal of Research Grant :Hastings Professor of Medicine and Pathology, USC School of Medicine 1. NameofInvestigator(s):Gndudetitleanddegrees) Clayton G. Loosli, Ph.D., M.D. 2025 Zonal Avenue, Los Angeles, California 90033 ' The Attenc}ing Staff Association Los Angeles County General Hospital 1200 North State Street ° Los Angeles, California 90033 LUNG TISSUE REACTIONS TO AIRBORNE CHEMICAL AND BIOLOGICAL AGENTS 4. Proposed Renewal Starting Datet (Anniversary or other) Oc tober 1, 1967' after which the mice began to die. The rapidity of development of lung lesions is directly related to the concentration of N02 in the air. In order to observe what the lung c anges were iTce in the final stages*before death, the higher concentration was chosen. This coming year, with the availability of several separate exposure chambers, comparative studies will be made of the lungs of animals exposed to on animals exposed to 38 to 42 ppm over periods from 1 day to 8 weeks,, The objectives are the same. This past year the pathogenesis of lung changes in mice exposed'to atmospheres of N02 in concentrations of 10, 20, 40 ppm have been made. Most of our studies have been made S. Discuss any Importanti Changes or Additions to Objectives or Specific Aims: the three concentrations (10, 20, 40 ppm of NOZ) by histological, histo- chemical, biochemical and electronmicroscopic procedures. -.Studies of the lungs and other organs (liver, spleen, kidney, L adrenal, brain, heart) of mice exposed to different concentrations of So2 will be continued. Studies of the pathogenesis of PR8 influenza A virus infections in N02 and S02 exposed animals will be continued. An attempt will be made to elucidate the mechanism(s) of increased resistance of N02 exposed mice to influenza A infections. The patho- genesis of PR8 inflvenza A airborne virus infections in S02 mice will be studied to determine if they, as do N02 exposed mice, develop a resistance. The susceptibility of N02 and S02 exposed mice to airborne staphy- lococcus aureus will be continued and, in addition, the pneumococcus and hemophilus influenza organisms will be employed. 6. Givea 8rief Statement ofyourHMorking Hypothesis if altered or modified: There is no change in our working hypothesis. Our aim is to define the lesions due to NO2 and SO exposure, the 1'esions ue to bactc>ria and influenza A virus, an `t e e ec of super imposition of these lesions in the same animal. N 0 0 w CA ~ C deem `_ .

7. Changes or Additions to Experimental Design and Procedures: (Attach Separate Pages) ~,"F~>~'M14 zThere is no basic change in the experimental design and procedures. - A full complement of skilled and experienced technical personnel have been procured in histology, biochemistry, histochemistry, bacteriology, d 1 ' f t I b l h ronmicroscopy. viro ogy an e ec e ieve t e great importance o :our study rests in the simultaneous study of the histological, biochem- 'ical and histochemical changes in the lungs and other organs of mice `subjected to N02 and 802 exposure alone and in combination with .exposure to the above named viruses and bacteria. This past year oxygen consumption, alkaline and acid phosphatase and lactic dehydrogenase measurements have been made in N02 exposed animals. This coming year glycolysis, lipid metabolism and anti- trypsin activity will be measured in the lungs of N02 exposed animals, as well as urinary steroids and transaminase in the serum. Generally, all the facilities to carry on the above studies are chambers in which to expose mice to different concentrations of NO2 available. The output can be greatly increased by having several - and S02 for different periods of time. This will allow us to make short term observations while allowing other mice to be observed estimated that very satisfactory chambers can be constructed for $500 each, including fans, flexible exhaust piping, etc. :for longer periods of time at low levels of NO2 and S02. It is Also, a drying oven is needed in the biochemistry section to facilitate the analysis of tissues. 9. Changes in Penonnel'w•rth BiographicaliSketches o6new Personnel (append) There are no changes in personnel. Ramon D. Buckley, Ph.D., Assistant Professor of Biochemistry, directs the biochemical studies, and is co-director with salary support from the NIH grant. Between the two grants he devotes 80 percent of his time to the studies. The technical personnel are all experienced. 10. Publicotions or Papers in Press resulting from the Projqct or closely related'work No papers have been published or are in press as yet from studiess supported by this grant.

1, 6udgat(ior comint; yaar) r A. Salarics (P rsonnol Iby names or category) Prof~ssipnot . ,.''-Clayton G. Loosli, M.D., Ph.D. 5U• _:*Ramon D. Buckley, Ph.D. 75. - Technical *SyIvia Moss, M.S., Histol-Biochemist . 100 *Elizabeth Vinceguerra, B.S., Biochemist 100 *Johnnie Bilbrew, Jr., Animal Caretaker 100 . Marv Bresnahan. B.S.. Viroloerist 100 • Edna M. Stone, M.S., Histology-Histochem. John Hardy, B.S., Electronmi~croscopist 100 .Ernestine ?ruiet, B.S., Bacteriologist 100 Technician 100 S- Consumob'a Sunp!1as (iist b•/ cosegoriesj *Paid'' fro:n NIH grant (see page 4) u- o.o Fringe Benefits, 8% S bT I Glassware......... .................. ... 500 Gen.&spec.chem.reagents,enzymes,cofactors 1,000 Supplies, histol., bacteriol. & virol.... 2,500 Nlice and rabbits........... ........... 6..$ 2,000 Travel for Principal Investigator and Dr. Buckley ..........................$ 750 Service, Spinco, spectrophotometers, etc. 1,000 Repairs on equipment, office supplies, publication costs, misc, supplies ....... 1,250 Dry ice, water de-ionizers, etc.......... 1,000 C. Other ~XC3:1Sa. C:eTlza}' chambers•Wlt1 motors, fans, etc. None None None $7,560 7,560 7,119 7,000' 7,000 -. Sub-Total 3,000 D. Permenent:c,uipmeat ite:nixe) Construction of three exposure 'c. Gver:aad(15%of:,=3=C) 15% of $43,544 Total undea:acd ti:a: tnenpplkcctand insatu:ionei aficers fcr a g:or.t hove recc end'found eccep:cble tsa Cc-cii: "S:o:ernent of ?oiity, Cor.:oining Conci:ions end Tt:rms Under Which 2re;ec: C.ran:s Are N.eda:" I.-:)-O0 Signature 6,532 51,576 3L15 225- leas.orawef,h.inaYudce Gbtlt Ext. 3826 - Ji S . •Business Manager Te(cp4onc

list financial support for research from all sources, including own institution, for this and/or reiated research projects. Source I Duration Jan-Dec 1967 1968 1969

Tm;,, C.ou.NCiL For ToBAcco RE, S EAr,Cr-1-ZT. S.A. TO: The committee comprising Drs. Little, Chm., Dr. Jacobson, Dr. Sommers and Dr. Reimann. - FROM: Robert C. H:ckett SUBJECT: Request for a supplementary grant from Freddy Homburger, M.D. No. 456R2. We enclose herewith a letter from Dr. Freddy Homburger of Bio- Research Consultants, Inc., requesting additional funds. Payment for the additional smoking machines and the cost of the patent, mentioned in the first two paragraphs of the letter, have already been approved. The request for a consultant fee of $6,000.00 for Mr. Walton beginning April 1, 1967 is at the same rate as we provided for the previous year. R. C. H.

BICIASSAY. CARCINOGENESIS and TISSUE CULTURE No. l+ 6R2--•`''• ~ Activated: 1 11 /65 THE COUNCIL FOR TOBACCO RESEARCH - U.S.A. Renewed: 1/1/66 ; CON1MITTEE : Dr. Little, Chm. Dr. Jacobson 633 T.IIII2D AP".CViJLr NEW YOR&, N. Y. 10017 Name of lnvestigator(s): Gnclud'e Title and Degrees) FRIDDY HOMBURGER, M.D. President and Director BIO-RESEARCH CONSULTANTS, INC. 9 Commercial Avenue Cambridge 41, Massachusetts I machines prior to shipping them, at your direction, to other investigators. Renewed: l/1/67 In reply to your letter of June 16th, I wish to thank you for formalizing the contract to purchase from Mir. Richard Walton six additional machines at $ 1,495. each, and we shall be prepared to inspect and' test these or its designee. r that assignment of the patent will be made to The Council for Tobacco Research i A ~ . ~ ~K' .. i the patent for the'Walton machine' approximately $ 500. with the understanding • We also have passed on to Mir. Walton your decision to pay for which will be paid to Mr. Walton quarterly for his participation in our develop- ment of a''Walton smoking machine" for the preparation of cigarette smoke con- densates and for his continued consultation in our adaptation of the present 'Walton smoking machine' to studies on other species, and in the adaptation of the 'Walton smoking machine' to the exposure of organs other than the respira- tory tract. Further to follow up on your letter, this is to request formally .that the Scientific Advisory Board of The Council approve an additional con'- sultant fee for Mr. Walton of $ 6, 000. for one year beginning April 1st, 1967, another year from that date to be helpful' to us in the matters referred to above. W We are submitting; this request to you retroactive to April 1st, © 1967 because Mr. Walton has continued since that date and will continue for Q We appreciate your most helpful cooperation. r . .- ~ .~ !'..9.1 ^'.AYi....i. .. . Yours sincerely, President an irector eddy Hombu ger, M. D.

I

0 TFII7 COUNCIL FOI: T013_1CC0 IRESEARCFI-U. S'.A. August 28, 1967 MEMORANDUM . . ..__ .~~. and Dr. Lynch. TO: The committee comprising Dr. Bing, Chm., Dr. Catte]1, Dr. Jacobson FROM: Robert C. .Hockett M.S., Ph.D. - No. 467R2-S. SUBJECT: Request for Supplementary grant from Thomas C. Westfall, A.B., We enclose herewith a request for a supplement grant from Dr. Thomas C. Westfall of the University of Virginia School of Medicine. On June lst he requested permission to reallocate $3,000 from the present year's budget toward purchase of a liquid scintillation spectrometer and was given staff permission to do so, subject to ratification by the Board at their next meeting. The Board has already approved (May 20, 1967) a grant of $11,500.00 effective on September 1, 1967 to complete the three-year research plan that Dr. Westfall undertook beginning September 1, 1965. coming meeting. The present application for an additional appropriation is quite detailed and is distributed for consideration on its merits at the forth- R. C. H.

1 , Renewed: , ..- 9/1/66 .THE COUNCIL FOR TO:;ACCO Rip.,SizARCII - U.S.A. Renewed: ". 9/i/67_ N3.W YORS. 11t. Y. 10017 633 THIItD APErP[SE ` ' PEE COMVII;T • -Chm:- _~. Dr. Birig,-• -Dr. •Cattell Dr. Jacobson Application For Research Grant Dr. Lynch 1. Name of InvesiigatoE(s): (include Title and Degrees) 2. Institution & Address: . .. CARDIOVASCUTAR; PHARI~ACOLOGY and CI~ISTRY N0. 467-R2-SUB,'~ - Activated: 9/1/65 Thomas C. Westfall, A.B., M.S., Ph.D. Department of Pharmacology University of Virginia,School of Medicine Charlottesville, Virginia 22901 3. ShortTitleofProject: Supplemental Grant Request " Funds to aid in the purchase of a Scintillation Spectrometer (to supplement grant entitled, "Action of Nicotine on Subcellular Distribution of Catecholarnines in Brain and-Heart" ) 4. Proposed Starting Date: . October 1, 1967 5. Anticipated Duration of this SpecifirStudy: on separate sheet •6. Brief Descripton of Objectives or Specific Aims: on separate sheet 7. Give a Brief Statement of your Working Hypothesis: on separate sheet v

C.1. Eiographical sketches of all prinGpal and professional personnel (append). on separate sheet 9. Physical Facilties Available (Where Other than.Administering Organization Indicate Geographical Location) `_ see append'ed curriculum,vitae l 12. List of publications: (Five most recent as pertinent) (append) . - see appended curriculum vitae

To aid in the purchase of a Packard Automatic Tri Carb Scintillation Spectrometer Sub-Total 7,500 Total 7,500 Estimated Future Requirements: Salaries Consumable Suppl. Other Expenses PermanenbEquip. Overhead - Total Year 3 his understood Ihat,the appficant and institutionaf officers in applying for a grant have read and found acceptablh the Council's "Statement,of Policy Containing Conditions ond Terms Under Which Projed Grants Are Madm" Signature -0 Dir.r.tarol Pcoi.d y~/ Telephone Signature a72Y W un.u 0117- of th. luNluKaw Telep.hane ~ Q qw=

List flnandol support for research from oil sourca, insludln9 own inslitution, }o+thfs ond/or nlat.d m.orch pro/se1s. • i Catecholamine Metabolism Following Cigarette Smoking and Nicotine Administration The American Medical: i Association Education and - Research Foundation Committee foT Research on Tobacco and Health

Duration of Specific Study " ~.~•~~. 4; This equipment would be used to tielp•complete the project . listed above which is currently being sponsored.by The Council. .'In addition, it is anticipated that this instrument will be used in many future projects under contemplation by the principle investigator. `Imgortance of a Scintillation Spectrometer to the Specific Aims and Obiectives of the Principle Investigator. The purpose of this supplemental grant application is to request funds which will aid in the purchase of a scintillation .spectrometer. This instrument is greatly needed in achieving the following overall specific aims. 1) To ascertain.the importance of certain biogenic amines in helping to explain the central and;peripheral actions of nicotine. 2) To specifically determine if •there is an adrenergic mechanism involved in the central action of nicotine. 3) To specifically determine whether or not nicotine is exerting; any of its peripheral action by the release of norepinephrine directly from,adrenergic nerve termina:ls. CENTRAL ADRENERGIC ACTIONS OF NICOTINE Attempts at determining whether or not there is an adrenergic mechanism involved in the central action of nicotine are currently being:carried out in this laboratory by studying the effect of nicotine on the subcellular distribution of certain amines,in specific-brain regions where these amines are present in large }y concentrations. Studies have been made on brain areas containing O large concentrations in an attempt to prevent a masking of a O possible effect that might be taking place due to tissue which co has very little or no amine.content. Our results, to date, ~ indicate that there are certain changes in the level of these amines in various regionz of the central nervous system ~ (Westfall, et al., 1967). These studies are limited., however, j~ to measurement of changes in the• static storage levels of ~ catecholamines and other amines in the brain,. In addition,• they have not included areas such as the cerebellum which contains very little endogen.ous norepinephrine but may be very important because of the active metabolism of amines recently observed to be taking place there. Although these above mentioned studies can produce important results and can give us some ideas of.a release of amines by nicotine, they tell us nothing about the dynamic aspects of metabolism of amines in the brain. _

We are becoming.more and more aware that the storage-levels of norepinephrine, dopamine and serotonin are not static but"r'•eflect dynamic.equilibria between the rates df formation and the rates of utilization of the amines (see reviews by Glowinski and . Baldessarini, 1966; Hornykiewicz, 1966).. In other words, there is a constant amount of. norepinephrine in nerve endings because,_._ a continuous synthesis or input is balanced by a continuous • efflux onto receptors or metabolizing enzymes. The level of norepinephrine will be altered, therefore, if one of these rates h bhd N iti hhid tht asseen cange.umerousnvesga+orsave empaszea ..syn'Chesls, storage release anCt metabolism are not unrelatec! phenorn.ena and should.be considered together in interpreting ~drug action (Neff; et al., 1965; Costa,.et al., 1966). Therefore, it may be thats changes in the turnover rate of these amines are .a better measur•e of drug action than mere changes in the concentrations of the amines which could remain constant or even ' decline despite an increased rate of syntHe-sis. ., •' Recent studies by Iversen and Glowinski (1966), and Glowinski and Iversen (1966a!,b.) have shown that different turnover rates of norepinephrine occur in several diff erent regions, of the rat brain. These investigators observed that the brain regions seemed.to fall into three classes. The cerebellum appears•to have the fastest turnover of norepinephrin;e, with a half-life of about 2 hours. Although this brain region has such a fast turnover, it is quite interesting that this area has a! very low endogenous norepinephrin!e content.. This would seem to indicate that the small stores of norepinephrine are in an unusually active metabolic state. This.rapid turnover may even be correlated in some way with its ability to form adenosine 3',5'-phosphate. Along with the low endogenous norepinephrinScon~tent, the cerebellum seems to `- accumulate very little H-norepinephrins. On the other hand, the bellu r a e s to ine hrine c c l t b lit f H t m pp ar e a cumu a norep p e a es o e more -me o when compared to brain regions with slower turnover times. Following the cerebellum, the brain regions with the next fastest turnover rates seem-to be the cortex and hippocampus with ~ half-lives of about 3 hours. The regions with the slowest rates W- : of turnover of norepinephrine are the hypothalamus and the medulla j~ oblong,ata with half-lives of 4 hours. Interestingly, these same rA regions have the highest concentration of endoger~ous norepinephrine ~ and seem'to accumulate thg greatest amounts of H-norepinephrine ~ and the least amount of H-metabolites. These experiments point out the important fact that an inverse relationship between•turnover and, N endogenous concentrations of norepinephrine in various parts of the brain seem to exist. .This proba•bly indicates that areas with small endogenous levels actually h,ave a faster active metabolism of catecholamines than regions containing higher levels of norepinephrine. These observations emphasize, therefore, the importance of studying turnover rates rather than only measuring steady state levels of catecholamines in various brain regions following drug administration. . At the present time there appears to be three available methods for measuring the rate of turnover of norepinephrine in the brain. Two of these methods involve the use of labelled techniques. The first method measures catecholamine turnover by introducing

0 removal of an,equivalent amount of labelled and unlabelled amine. -:.turning over of the endogenous norepinephrine stores at a -con stant rate, by the continual synthesis of norepinephrine and •..decline of H3-norepinephrine that occurs is interpreted as'the labelled norepinephrine into the lateral ventricle and follows the changes in specific radioactivity with time. The'exponential `:-as tyrosine, dopa or dopamine and measurina-the amount of formed ~,~norepinephrine. The second is by using;labelled precursors such turnover rate and;therefore, the rate of synthesis of endogenous ~,The slope of the exponential decline is a reflection of the , .':; amine stores. :: central stores.gives a measure of the rate of utilization of i y disappearance of endogenous catecholamines from~peripheral and ;inhibiting catecholamine biosynthesis with such,drugs as aC-methyl-p--tyrosine When s nthesi s is blocked~ the rate of .norepinephrine from these precursors. The final method is by . Of these three methods of measuring catecholamine turnover in ''• the brain, the use of exogenous tritiated n,orepinephrine appears to have distinct advantages. The use of A-methyl-p-tyrosine, for •°-';instance, is very limited by the difficuity of measurina very low other precursors is an improvement over the previous method'but may ,r : q es ai a e, e use o - opamine or ' concentrations of endogenous norepine.phrine in discre~e brain areas ~ ~' with the chemical techni u av ' 1 bl Th f H d complicated by the possibility that a prolonged synthesis of ,-to suggest that this fact does not occur to any important extent ":`with H3-d`opamine (Iversen and Glowinski, 1966)1. :.-of the labelled precursor. The available evidence, however, seems H3-norepinephrine may occur in the presence of persistant amounts -It can be concluded, therefore, that equipment necessary to ''which has been demonstrated to have a rapid;metabolism but which has 'This seems particularly important for regions such as the cerebellum -;would give a direct measurement of the influence of nicotine on the r;dynamic metabolism of catecholamines in the central nervous system. ;of catecholamines in the brain in response to nicotine.• These studies measure labelled substances is required to adequately measure turnover : t b d t di f _;s;;~.'...•_. ;.,no een s even u e or changes in static catecholamine levels .because ot the technical problems in measuring the low levels 1003545793 •.(see IMestfall, et al., 1967) present there. PERIPHERAIL.ADRENERGIC ACTIONS OF NICOTINE Another important use of this instrument will be its application in studies aimed;at further evaluating the role of nicotine on peripheral amine stores in adrenergic nerve terminals. As in the central nervous system, there is likewise a dynamic equilibria between the rates of formation and rates of utilization of catecholamines in peripheral sympathetic nervous structures. Therefore, it is equally • important to study the influence of nicotine on the turnover of norepinephrine in adrenergically innervated:organs such,as the heart and spleen. Similar to different brain, regions,'there are differences in the turnover rate of norepinephrine in various organs as well ' .(Burack and Draskoczy, 1964).

Previou,s publications from this laboratory have pointed out the inconsistencies and controversies that exist in the literature concerning the ability of nicotine to release norepinephrine from storage sites in adrenergically innervated organs (Westfall, 1965a,b;'Westfall, et al., 1967). One of the main reasons f or these discrepancies is probably a result of the difficulty in measuring and detecting,the'small changes in endogenous amine -that are taking place following the admin-istration of nicotine.. A more convenient method for studying cardiac nor:epine hrine in the rat has been used by Herttin and: co-workers (1961~ and by Daly, et al., in the mouse (19663. In both cases they have shown that following tracer doses of labelled norepinephrine the amines are taken up into the heart, equilibrate with,endogernous amine and most important are affected in a similar manner by agents that cause or inhibit norepinephrine release. This method;has been described as being rapid, simple, economical and quite reliable. Other methods have been described:using a perfused heart preparation (Nash, et al., 1967). It is anticipated that similar methods would be very valuabl!e and useful in studying; the influence of nicotine on norepinephrine stores. These methods appear even, more valuable when it is realized that d,rugs can-act by releasing norepinephrine from,nerve endings without arn appreciable change in the endogenous level as measured by the currently available biochemical methods. Because of the dynamic equilibria that exists between formation and utilization, any release of amine may be quickly replaced by synthesis and/or reuptake into nerve terminals. With the use of labelled amines described above, such a release can be much more easily detected. - Another potential use of the scintillation spectrometer would be to study the influence of nicotine on the uptake of norepinephrine o into adrenergic nerves. Such a ossible effect is suggested by the data.of Nedergaard, et al. (1966). These investigators have reported that nicotine can potentiate the response of vascular smooth muscle to nerve stimulation by up to 60%. The same doses of nicotine occasionally produced small increases in resting tone. Similar types of potentiation of sympathetic effects exist for a large number of drugs such as cocaine or imipramine which are well known to block uptake or reuptake of norepinephrine into adrenergic nerve termin,als (Carlsson, 1966; Trend;elenburg, 1966). Therefore, it is q:uite possible that nicotine also possesses some ability to antagonize the uptake of norepinephrine into adrenergic nerve terminals. Such a possibility certainly deserves investigation. 7.. Workinq Hypothe si s. The synthesis, storage, release and metabolism of biogenic amines are not unrelated phenomenon and should be considered:tog,ether in interpreting the action of nicotine in the central and peripheral nervous system. Changes im the turnover rate of amines such as norepinephrine will give a much clearer picture of the effect of nicotine on the dynamic metabolism,of amines in brain and,peripheral organs.

. Details of'Experiments Utilizing a Scintillation Spectrometer. Ky" . . • " . ... •S TURNOVER OF CATrCHOLANEINES IN TNE CENTRAL NERVOUS SYSTEM BEFORE AND AFTER NICOTINE ADMINISTRATION— in specific activity of exogenously administered tritiated .:.norepinephrine. • offer the most advantages is the one which follows the chang,es As mentioned above, there are three available methods for x=•-studying the turnover of catecholarnines in then brain at the `, present time. Of these three methods, the one which seems to doses will be administered;prior to or following the intraventricular The labelled,amine will be injected into the lateral ventricle of rats anesthetized lightly with-pentobarbital, using the recently described method of Nobel, et al. (1967). Nicotine in various be dissected:out on an ice cooled glass plate including the following. -and brains quickly removed, blotted and chilled. Seven regions will decapitation at various tumes--after the injecti.on of norepinephrine : injection ot DL-H-3-norepin-ephrine. T11e rats will be Kilied by cerebellum, medulla oblongata hypothalamus d) midbrain e) striatum f) hippocampus g) cortex . Glowinski and Iversen (1966). In addition, the plates in,"Craigie's Complete details of these dissections are given in the report by :'Neuroanatomy of the Rat" (Zeman and Innes, 1963) will be closely ~i;''-.f ol lowed . ... . ... ~.- . The tissues will be quickly weighed and homogenized in 10 to 15 ml of cold 0.4 N perchloric acid in a Ultra-turrax homogenizer. The homogenates will then be centrifuged at 10,000 x g for 10 min. After the addition of 0:1 ml of 1% disodium ethylenediamine- ~,& tetraacetate (EDTA) and 0!.1 ml of a freshly prepared 1% solution 0 of ascorbic acid, the pH will be adjusted to 8.3 - 8.5, with N 0 normal RaOH. The sample will then be passed through a column GJ containing aluminum oxide-to remove the free catecholamines while Q1 the combined effluent and:washing! will be used for the estimation ~ of the o-methylated metabolite, n-ormetanephrine. Elution of the ~ catecholamines fromf the alumina column, will be carried out• by the addition of 0.2 N HC1. An• aliquot of this eluate will be used ~ to determine the radioactivity of the free norepinephrine, while the tritiated d!eaminated metabolites will be extracted from the alumina-eluates according to the method of Kopin, et al. (1961). Briefly, this involves taking an.aliquot, acidifying it with 6 N HC1 and saturating the solution with NaCI. The nonamine catechols are then extracted into ethylacetate. After centrifugation, an aliquot of the organic phase will be evaporated in a current of air

::':in a glass vial and the radioactivity determined-. The o-methylated metabalite,=normetanephrine will be--:assayed by the method described by Iversen, et al. (1966). Briefljr;., the effluent and. washing:s from the alumina column used to extract -' the catecholamines will be adjiusted. to pH 6.5•and passed through _ a column (6 x 20 mm) of Dowex 50W-X4 in the sodium form. The ;.:resin will then be washed with glass distilled water, and the ._ normetanephrine eluted with a mixture of equal quarts of 6 N hydrochloric acid and ethanol. Aliquots of this eluate will then':44: -.The free catecholamines, normetanephrine and the deaminated metabolites will be assayed in a scintillation spectrometer after the addition of 4 ml ethanol and 10 quantities of 0.4% 2,5-diphenyloxa:zole and 0.005% 1,4-di(2-5-phenyloxazole)benzene in toluene. Tritiated o-methylated, deaminated metabolites can be estimated by the difference be~ween total radioactivity of tissue extracts and the sum of (H ) norepinephrine and other ~ :~;-;'~; metabolites. A flow chart, enciosed in this report, gives a quick overall picture of ths-extraction procedures. In some experiments, various subcellular constituents of the various brain regions will be assayed for their f ree catecholamine and metabolite content. In these experiments, the tissues will be initially homog:enized in 0-.3 M sucrose. On,e-fourth of the sample will be taken for assay of total radioactivity and the remalning;extract spun at a low speed (10,000 x g;- 10 min) to,remove the coarse fraction. The low speed supernatant will then be spun at 100,000 x g for 40 min, yielding a• supernatant and particulate fraction. The low speed sediment,high speed supern-atant, high speed particulate and.totai tissue samples will then be extracted in 0.4 N perchloric acid and extracted according:to the procedure described above for the differentiation of free, deaminated, o-methylated and deaminated ~ ~"o-methylated amines. . .~ _., ~- . . ., . - . yY STUDY OF THE RELEASE OF NOREPINEPHRINE FROM ADRENERGIC NERVES BY NICOTINE As ha;s been mentioned in the previous section, controversies still exist as to whether or not nicotine releases appreciable quantities of norepinephrine from adrenergic nerves. Most of this controversy exists as a result of discrepancies in the literature because of the difficulty of available chemical methods for determining the small amounts of norepinephrine that might be released. _ In the present studies, the influence of nicotine on the . release of norepinephrine will be studied by measuring the amount of labelled morepineph,rin,e remaining in the heart at a g;iven period of time, following;the prelabeiling of the norepinephrine stores. 4 p Y • ~~' be eva orated to dr ness in vials -~-~, .;.,

These studies can be carried out in both,mice and rats. assayed according to the method of von Euler and Lisha•jko (1961). centrifuged (10,000 x g for 10 min) and the supernatants taken for assay in a:scintiilation spectrometer, similar to the method described above. Endogen-ous norepinephrine will be simultaneously •turrax apparatus. Following homogenization, the extract will be after 3 hours by a blow on the heads and the hearts quickly removed. The hearts will be immediately exsanguinated:and , homogenized in 10 vol of 0.4 N perchloric acid:in an Ultra- norepinephrine will be injected into the tail vein of male white mice. Nicotine in various doses will then be administered subcutaneously one hour later. The mice will then be sacrificed 50 mg of heparin/1) of 1.0i'mµ mole (5µ curies) of tritiated For mice, a 0.1 ml solution (isoton-ic sodium chloride,'con.taining {.• except that about 10,µC of tritiated norepinephrine will be administered by a tail vein_ and the animals sacrificed by decapitation. In the studies on rats a similar procedure will be followed distribution of the labelled norepinephrine will be studied. In In some experiments the influence of nicotine on the subcellular • ultracentrifuge. All fraction-s resulting from the differential centrifugation will then be taken for assay in a scintillation . and extracted•in 10% trichloroacetic acid for assay of total heart. The remaining homogenate will be spun in a refrigerated centrifuge at a slow,speed (10,000 x g, for-10 min). The slow speed supernatant will then be spun at 100,000 x g for 45 min in a these experiments, the tissues will be homogenized in ice cold potassium phosphate buffer, pH 7.5. An aliquot will be taken counter or adsorbed on- alumina, for endogenous norepinepttrine determination following,extractions in 10% trichloroacetic acid. INFLUENCE OF NICOTINE ON THE UPTAKE OF NOREPIN'EPHRINE The influence of nicotine on the uptake of norepinephrirne can be conveniently studied by administering the nicotine prior to the intravenou.s injection of tritiated norepinephrine as described above. Similar experiments can be carried out on the perfused guinea pig heart where, in addition.to net uptake, the arterial-venous difference can also be studied. This.can be accomplirshed-by measuring,the perfusate content as well as the tissue content of N norepinephrine. ~ CW ~ . ~ . ~

EXTRACTION, SEPARATION AN D ANALYSIS OF CATECHOLAMINES AND METABOLITES Animals injected, sacrificed (decapitation), tissues dissected out and weighed. - W " Homogenization, (0.4 N perchloric acid, if Ul'tra-turrax apparatus) Centrifugation (10,000-x g - 10 mm) W Ascorbic acid, EDTA added pH adjusted to 8.3-8.5 (NaOH!) W Alumina Columns washed with water y Effluent + washings .(o-methylated metabolites) I Dowex column (50W-X4 ), washed with water eluted with 6 N H.C1+Ethan,ol if added Ethylacetate ~ Contrifugati tY Assayed" for Assayed in Assayed in Normetanephine in Scintillation Counter Scintillation Coun Scintillation Counter Ad j usted to-Ph• 6. 5 Aliquot (taken for total radioactivity determination) Catecholamintes e.luted with 0.2 N' MC1 A1:iquot (assay Aliquot(dearninate of norepinephrine) metabolites), I - 6 N HC1, NaC 1003546798 Ira

Burack, W. R. and P. R. Draskoczy. The turnover of endogenously labelled catecholamines in several regions of the sympathetic nervous system. J. Pharmacol. exp. Ther., 144: 66-, 1964. •Carlsson~ A. Pharmacological depletion of catecholamine stores. : Pharmacol. Rev., 18: 541, 1966.. Costa, E., Bouilin,, D. J., Hammer, W., Vogel, W. and B'. B'. Brodie. Interactions of drugs with adrenergic neurons. Pharmacol. Rev., 18: 577, 1966. f°' Daly, J. W., Greveling, C. R. and. B. Witkop. The chemorelease o noreninephrine from mouse hearts. Structure-activity relationships. I. Sympathomimetic amines and relatediamines. J. Med. Chem., 9: 273, 1966. ~ Daly, J. W.,_Creveling, C. R. and B. Witkop. The chemorelease of norepinephrine from mouse hearts. Structure-activity relationships.' II. Drugs affecting!the sympathetic and central nervous system. J. Med. Chem,., 9: 280, 1966. • Glowinski, J. and R. J. Balidessar•ini. Metabolism of n-orepinephrine in the central nervous system. Pharmacol. Rev., 18: 1201, 1966. Glowinski, J. and L. L. Iversen. Regional studies of catecholamines in the rat brain. I. The disposition of (H~) norepinephrine, ~:....(H3) dopamine and (H3) dopa in various regions of the brain. J. Neurochem., 13': 655, 1966. .Glowinski, J. and L. L. Iversen. Regional studies of catecholamin-es ...in the rat brain. III. Subcellular distribution of endogenous and exog;enous,catecholamines in various brain regions. Biochem. : Pharmacol., 15: 977, 1966. Hertting•, G., Axelrod, J. and R. W. Pa~trick Actions of cocaine and ~ -norepinephrine in the N tyramine on the uptake and release of H .. heart. Biochem. Pharmacol., 8: 246, 1961. CO Hornykiewicz, 0,. Dopamine (3-hydroxy tryptamin,e) and brain functions. Pharmacol. Rev., 18: 925; 1966. Iversen, L. L. and J. GLowinski, 0,. Regional studies of catecholamines in the rat brain- II. Rate of turnover of catecholamin.es in various brain region,s. J. Neurochem., 13: 671, 1966. Kopin,, I. J., Axelrod J. and E. Gordon:. The metabolic fate of H3- epinephrine and Ci4-metanephrine irn the rat. J. Biol. Chem., 236: 2109, 1961. Montanari, R., Costa, E., Beaven, M. A. and;B. B. Brodie. Turnover rates of norepinephrine in hearts of intact mice, rats and guinea•pig-s. using tritiated noTepinephrine. Life Sci., 2: 232, 1963'.

• Nash, C. W.,,Wolff, S. A. and B. A. Ferguson. The action of from isolated perfused: rat hearts. Fed. Proc., 26: 570, 1967. svnpathomimetic amines on the release of noradrenaline ~• , •. 7 Neff, N. H., Tozer, T. N., Hammer, W. and B. B. Brodie. -Kinetics of release of norepinephrine by tyramine. Life Sci.,.4: 1869, 1965. . • . Noble, E. P., Wurtmann, R. J. and J. Axelrod. A simple and rapid method for injecting H3-n,orepinephrine into the lateral ventricle of the rat brain. Life Sci., 6: 281, 1967. Irend'elenburg, U. Mechanisms of supersensitivity and subsensitivity ..to sympathomimetic amines. Pharmacol. Rev., 18: 629, 1966. Westfall, T. C. Effect of nicotine and nicotine analogues on tissue •• and urinary catecholamines in the rat. Acta physiol. Scand., 63: 77, 1965. .Westfall, T. C. Tobacco alkaloidis and the release of catecholamines, in tobacco. alkaloids and related compounds. Ed. by U. S. von Euler, Pergamon Press, 4: 179, 1965. • Westfall, T. C.,.Fleming, R. M., Fudger, U. K. and W. G. Clark. Effect of nicotine and related substances on amine levels in the brain. Ann. N. Y. Acad. Sci., 14 2: 83, 1967. Zeman, W. and J. R. M.. Innes. Craigie's Neuroanatomy of the Rat. Academic Press, New York, 1963.

FUNDS FOR THE PURCHASE OF A LIQUID SCINTILLATION SPECTROMETER Present Reques Additional Funds: Second year budget of a grant entitled, Action of Nicotine on Subcellular Distribution of Catecholacnines,in Brain and Heart, Council for Tobacco Research -'-U.S.A. . . . . . Research and Development Fund University of Virginia School of Medicine . . . . . Total. . $15,500.00 Cost of Instrument. . $15,500.00- 0

A we171 equipped Department of Pharmacology with adequate ;`differen.tial estimation of epinephrine and norepinephrine, the ' ~;•:.fluorometer with the necessary f ilter combinations for for this project includes: Farrand Model A photoelectric .laboratory and office space is available. Equipment available. room as well'as rooms for animals within the Department and located in the Department of Biochemistry on the floorahove is also available. Our Department has a well equipped shop and dark ~ultra-centrifuge. The use of a Spinco preparative ultracentrifuge -'analytical balances, Grass polygraphs, and'Type 40 rotor for :Medical Electronics fractionator, Beckman spectrophotometers, .-homog;enizer, International refrigerated centrifuge, Gilson .Beckman expandomatic pH meter, chromatographic,column•s for :`.alumina absorption, ultra-turrax high speed tissue floor below) an Aminco-Bowman spectrophotofluorometer, ~°=use of (on loan from Cancer Research Laboratory, located on . ' ~rrecc + fl.n C.-hnnl n~s Medicine. - :} ~- ---- _.._...-- -~------- ---... _.. _..---- -- Staff. ` 1. Thomas'C. Westfall, Ph.D. Principle Investigator 2. Hirofumi Osada, M.D. Research Fellow 3. Leslie Frank .Research Assistant ~ •-'4. William Moore Graduate Student

R: REDACTED MATERIAL CURRICULUM VITAE • N ame • Thomas C. Westfall Date of Birth: ~- Place of Birth: I:atrobe, Penn,sylVania Marital Status: ; R- .. Education : - - in Pharmacology. 1963-1964. Naticsnal Heart Institute Postdoctoral 1959 A.B., Biology and Chemistry;:West Virginia University 1961 M.S., Pharmacology., West Virginia University 1962 Ph.D., Pharmacology, Dr. Daniel T. Watts, supervisor, West Virginia-University Education,al Awards: ~ - 1959-1962. National. Institutes of Health Predoctoral Traineeship Academic Positions Held•: Award. 1962-1963'. Instructor in Pharmacology, West Virginia University Medical. Center. 1965- Assistant Professor of Pharmacolooy, University of Virginia School of ?J:edicin,e.- Sweden, Professor U. S. von Euler, supervisor. 1964-1965. Assistant Professor of Pharmacblogy, West Virginia : University Medical Center. 1963-1964. Research Fellow of National Heart In,stitute, Department of Physiology, Karolinska Irnstitute, Stockholm; Teaching Experience: I 1962 thru 1965. Gave approximately 20 lectures per year to medical, nursing and graduete students at West Virginia University Medical Center. This in,volved participation in the laboratory course of instruction to the same groups. 1966 to present. Gave about one-fourth of-he lectures'in Pharmacology to the sophomore medical class at- the University of Virginia. This also-involved active participation in all = laborator.y, exerci ses. • Active teaching at the grad-uate. 1eve1 and supervision of graduate students was also carried out. Committee Assionments. (University of Virginia. School of Medicine): 1965-1966. Research Society, School of Med_c"ne. 1966-1967. Search Committee for Pharmacology Chairman.. • YI"RO KERO' • • ~XERO~ yr~'O ~./...~ _-

R: REDACTED MATERIAL Karolinska Institute, Stockholm, Sweden. Philadelphia General Hospital, Cardiology Division, Philadelphia, Duke ;University School of Medicine, Durham., N. C. Medical College of Virginia, Richmond,'Va. SYmposia Participation (by invitation).: • 1.966-196.7. 1967- . 1967- . ; Search Committee for Psychiatry Chairman.. AD,HOC Curriculum Committee of Basic Sciendest,-,~.AD HOC Committee, Cell Biology course for first ~~._ .year medical students. ;Society Memberships: REDACTED Professional Lectures (by invitation): REDACTED University of Texas School of Medicine, Galve.stsdn, Texas. .Turku Uni,versity School of :lhedicine, Turku,--Finland. and the Release of Catecholamines. Fourth International Wenner-Gren Center Symposium.. Tobacco Alkaloid•s and Related Compounds.. Held in:Stockholm, Sweden, February, 1964. Paper presen-ted: Tobacco Alkaloids New York Academy of Sciences Symposium on The Effects of Nicotine ..•, and Smoking on the Central N'ervous System. Held, in New. York City, April 7-9, 1966. Paper pre-sented: Effect of N icotine and Related Substances on Amine Levels.in the Brain. Central and peripheral autonomic effects of nicotine. Influence of drugs on uptake,storage, release and inactivation of biocenic amines. ;XEnp: ' X:aq ., gQa. . , ~ r. . American Medical Association Workshop on•Tobacco and Health. Held' at Broadmoor Hotel, Colorado Spring.s., Colorado, November 1-3, .1966. 'Paper presented: Influence of Nicotine on Catecholamine Metabolism. N[ajor Fields of Investigation : _ XCnJ XCno •ci-•r

BIBI:IOGP.APxY ~ of . Thoma s C. Westf all: Malor Publication•s • Westfall, T. C. and Watts, D. T. The on t?-.c cardiovascular respon-se to h siol., 17: 471, 1962'. . Westfall, T. C. and Watts, D. T. The effect of•cigarette Exp. Biol. Med;., 112: 843, 1963. :_ smoke on epinephrine secretion in the dog. _roc. Soc. 3.- Westfall, T. C. and Watts, D. T. Catecholamine bxcretion in smokers and nonsmokers. J. Aool. Physiol., 19: 40, 1964. , . • • +i+L... M. . Westfall, T. C. and Watts, D. T. The effect of nicotine on brain and urinary amines in the rat. J. Neurochem., 11: 397, 1964. - - 5. Westfall,' T. C. Tobacco alkaloid-s and. the release of catecholamines ir, Tobacco Alkaloid:s and.Related• Compounds, Ed. by U. S. von,Euler, Pergarnon Press, 4: 179, 1965. 6. Westfall, T. C. Effect of nicotine and nicotine analogues •.• on tissue and urinary'catecholamin,es in the rat. Acta: physiol. Scand., 63: 77, 1965. 7. Westfall, T. C. Uptake and exchange of catecholamines in rat tissues after d and 1-adrena~line. Acta ohysiol. Scand., 63: 336, 1965. ... • .. . . .:~ . . 8. Westfall, T. C. and Peach, M. J. Action of angiotensin on myocardial and renal catecholamines in the rabbit. Biochem. Pharmacol., 14: 1916, 1965. - 9. Westfall, T. C., Cippoloni, B. and Edtnundowicz, A. Influence of propranolol on the hemodynamic changes and pla•sma catecholamine levels following cigarette , smoking;and nicotine. Proc. Soc. Exo. Biol. Med, 123: 174, 1966. 10. Westfall; T. C., Fleming, R. M., Fudg;er,. M. K. and. Clark, W. G. Effect of nico•tine and related substances on amine levels in the brain. Ann. N. Y. Acad. Sci., 142: 83, 1967. ;xr_rro' . • ixF.PO• .xr.wp ~...

• • -.-- Westfall, T. C. Accumulation of norepinephrine in rat ' _ tissue following treatment- with three bet&adr'energic • 67. antagonists. Arch. int. Pharmacodvn., 167:`69,:1.q . .' . .12. Westfall, :. C. and Anderson, G. P. Influence of nicotine on catecholamine metabolism in the~rat. Arch. int. Pharmacodyn., (in press) 1967. Westfall, T. C. Effect of beta adrenergic blockers on the-.:;; -.noradrenaline content of rat heart and spleen before : and after noradrenaline infusion. B•rit. J. Pharmac. Chomoi;ner., (in press) 1967. storage in the perfused guinea pig;heart. Influence of Beta adrenergic antagonists on norepinephrine Action of nicotine-on subcellular amine levels in brain • and.heart. stores following depletion with various pharmacological ~agents. . 7 Influence of adrenahectomy on repletion of catecholamine 20: 89, 1961. Westfall, T. C.* and V`latts, D. T. .hhe effect of reserpine on the cardiovascular response to smoking. Fed. Proc., 21: 193, 1962. Westfall, T. C.* and Watts, D. T. The effect of cigarette smoke on epinephrine secretion in the dog. Fed. Proc:, Westfall, T. C.* and Watts, D. T. Catecholamine excretion in' smokers and nonsmokers. Fed. Proc., 22: 1509, 1963'. . Peach, M. J. and Westfall, T. C. Action of angiotensin on 3. myocardial catecholamines in the rabbit. Fed. Proc., 24: 488, 1965. 5. Westfiall, T. C.* Influence of pron.ethalol, propran,olol and iproveratril on u^Lake and storage of norepinephrine. Fed. Proc., 25 : 260-, 1-966.. 6. Westfall, T. C.*, Fleming, R. P4-., Fudger, M. K. and Clark, W. G.. Effect of nicotin-e and related substances on aminee levels in the brain. Svmoo.sium abstract. Syrnao.sium on The Effects of Nicotine and Smoking on the Central.Nervo.us Sys;:em. N. Y. Acad. Sci., April, 1966. 7. Westfall, T. C.* Uptake and. storage of norepinephrine following the administi-ation, of three beta adrenergic an tag;omi st s. Va. J. Sci., 17 : 354, 1966•. XF_RO 4f•Y. . r. . .. . ~.:- ,. .'.itt:..a ~ . .. .. - ..

Westfall, T. C.* Influence of nicotine on catecholamine . metabolism. Symposium a: .tract_. Tobacco and Health. °, Amer. Med. Assn., November, 1966. ,--,- - Westfall, T. C.* Influence of beta adrenergic antagoni•sts on the norepinephrine content in,rat heart before and after NE infusion. Fed. Proc., 26: 569, 1967. - Westfall, T. C.* Influence of beta adrenergic blockers on norepinephrine storage in the perfu-sed guinea pig heart. Va. J. Sci., 00: 000,, 1967. lyestfall, T. C.* The effect of beta adrenergic blocking, drugs on the norepinephrine level in the perfused guinea pig heart following•NE infusion. The Pharmacologist, 9: 249, 1967. III. Texts Contributor to: _ Medicinal Chemistry, AlfredlBurger, Editor, third edition, John Wiley and Sons, Inc., Interscience Publishers, New-York. " Introduction to Neuropharmacology (co-author, with E. D. Brand). is XEPO •XERO `:°RO • XI'R.l cunr, 'C^c~r-' ~C.,

R: REDACTED MATERIAL CURRICULUM, VITAE Name: Hirofumi 0sad'a ~- .~:::~_~:~•--- . • P1'ace of Birth: Kawasaki, Japan Education: University of Tokyo, Doctorate of Medical Science (Internal Medicine), March 1964. University of Tokyo Hospital, Internship, April 1959 - Ma-rch 1960. Yokohoma Municipal Un-iversity, M.D., 'Ma.rch 1959.. Academic Positions: 1964-1966. Instructor in Internal Medicine, University of Tokyo. 1966-1967. Research Fellow, Division of -CardUology, Philadelphia General Hospital, Philadelphia, Pa. 1967- . Research Fellow, Depart:nent of Pharmacology, University of Virginia School of Medicine, • rrrrp ^t++.v Charlottesville, Val. Society Membershios ACTED AMED I ;xrwa •3w•. ,. .. ••..~.~t

Publications : _ . .... . ~ . . .:~'. • . , . _ • ... . .. . . .. • drug (Nialamide). In Japanese. J. Therap., 43: 1961, 1961. s2rotonin and catecholamine metabolism •of new anti-anginal - ' Shimizu, K. and Osada, H. Clinical experience and effects on : Kobayashi, T., Ito, Y., Kobayashi, M., Kajihara, T.; Takeuchi, M. 2. and Aoki, T. Acute cardiac death-of unknown etiology. In English. Jap. Heart J, 3: 4!42, 1962. ~.~;.;,` ~_ ti:i . . s In Japanese. Diaonosis and Therapy, 50: 925, 1962. , y- . . , . , f g > > > , , > > Makai N. Kono, R - Yo~shimura S Su ai M Komizo U -,; .{~... 3. Kobayashi, T., Ito, Y., Kajihara, T.', Kishii, T., Osada, H., f .:.: .:....... .4. KobaYalshi> T., Ito, Y.> Kajihara> T:> Shimizu> K.e Kishii,T. Osada, H. and Komizo, U. . Clinical experience and effects of (J,-methyl dopa). In Japanese. J1. Geriatrics, 7: 330, 1963. Ej,ima,'M., Osada, H. and Tkeda, T. Paragang:lioma. In Japanese. 5. Kobayashi, T., Ito, Y., Kajih,ara, T., Shimizu, K., Kishii, T., Osada, H. and;Komizo, U. A new anti-hypertensive drug : . ao. , , e J. Yoshimura, S., Kobayashi., T. and 0sada, R. Acute cardiac death. • d-methvl dopa on serotoni,n and catecholamine. In Japanese. '"'?Rrx`° 44 1962 1888 Th r Endocrinoloay and Metabol? sm, 4: 99, 1963. . Yoshimura, S., Kobayashi, T. and Osada, H. Acute cardiac death. .In Japanese. J. Jao. Med. Assn., 49: 1015, 1963. 8. Kobayashi, T., Osada, H. and Komizo, U. c{-methyi dopa as a,anti- hypertensive drug. In Japanese. Respiration and Circulation, 11: 507, 1963. 9. Osada, H., Komizo, U., Kondo, K., Kishii, T., Kajihera, T., KobaJyashi, T. and Okano, S. Experimental and clinica~l trial of H.-catecholamine, esp.ecialLy as tos:-methyl dopa. In Japanese. Saishin-loaku, 19: 198, 1964. 10. Kobayashi, T.,,Kajihara., T. and Osada, H. Some con•siderations on the causative diseases of acute cardiac death, In Japanese. J. Chest. Dis., 8: 465, 1964. 1]:. Yoshimura, S., Kobayashi, T., Kajihara;, T. and Osada, H. Acute cardiac death of un'•:no:;~n, etiology. In Japanese. J. -Chest.. _ Dis., 8: 529•, 1964. s...a'_..~... ~. . xcr~o~ XCpo 1003546809

, -.. _ . . i: .. '. 12. Osad_:a, H. The effects of reserpine, .(-methyl dopa, nialamide and dexamethazone on the uptake and-metabolism-of •H3-catecholamine:r' , In English. Jap. Heart J., 5: 224, 1964 Progress on research ='•s:rf: H and Osada Kajihara T 13 Koba ashi T , . , .: . y , ^.:.~.. ., t erapy o ~-~- n in the world in 1964. Etiology, diagnosls an x hypertension. In Japanese. Jan. Med. J., 2129': 3, 1965. . 14.- Kobayashi T. and Osada, H. Anti-hypertensive effects of reserpine. In Japanese. Jan. Med'. J., 2147: 139, i965• 15. Kobayashi, T. and Osada, H. Basa;l and'casual blood pressure. In Japanese. Jao. Med. J., 2140: 130-, 1965. 16•. Kobayashi, T., Osada, H., Kondo, K. and Tawara, I. Anti- anginal and anti-anrhythmic effects of propranolol. In Japanese. J. Therao., 48: 775, 1966.. 17. Kobayashi, T., Kajihara, T., Osada, H., Komizo, U. and Ishii, K. b lism t i t h l i H l • . a o am ne me -ca ec o ycer ne on The effect of nitfog In English. Jaot. Heart_J-., 7: 430, 1966: 18.__Kobayashi, T., Kajihara, T., Osada, H. and Okada, T. The effects of ethacrynic acid'. In Japanese. J. Therap., 1966 (under contribution). %. 19. Osada, H. Review and Practice. The rnethod's of ineasuiements of catecholamine•in urine, blood and tissues. In Japanese-. Librar for Chemical P.nal,sis, Vol. 6, Jap. Chemical Analystics Assn, in Tokyo under contribution). Y6fi0 eXYlt'. ~'COPY 'r,t.l'-:,

.Tr-nm, Couti.ciL For, Tomwco l.LSl,nrcr-.U. S.A. September 5, 1967 .The committee comprising Dr. Little, Chm., Dr. Loosli and' Dr. Cattell. We enclose herewith a modified proposal from Drs. R. Ernest Clark and Donald B. Giddon of Tufts University School of Dental Medicine, Boston, Nlassachusetts. Their original proposal, #613, considered by the Board in~ May, 1967, did not receive approval. Four questions in particular were .raised in the discussion. These questions were relayed with Dr. Clark by the staff and have been taken into consideration in the revised pro- posal which he now asks the Board to review. In order to maintain the continuity of the proposal a copy of the original proposal is enclosed along with a copy of the original memo. The budget appears to be reasonable in light of the fact that' the principal investigator intends to devote a good deal of time to this proposed investigation. You will note that we'are not competing in the bud~et for graduate students, and possibly questionable quality that may result from the use of departmental graduate students. They are asking for one year's support only and intend to submit a new proposal based on the results and data obtained from this exploratory effort if it looks promising. Vincent F. Lisanti Robert C. Hockett Robert C. Hockett A modified proposal from R. Ernest Clark, Ph.D. and Donald B. Giddon, D.M.D., Ph.D. - No. 613-bi.

Tur, CoUCrr. Folt, ToBACCO Rr'srAIZC1I - U.S.A. Address: • - _ z iJ,@VA~xd B. Giddon, D.M.D. Ph.D. Professor and Chairman, Dept. of Social Tufts University School of Dental Medicine Dentistry Date:, Augus t 21, 1967 .R. Ernest Clark, Ph.D. Associate Professor of Psychology (effective 9/1/67) First J] Second "` 1: Nhme ofJnvestigator(s)c (include title and degrees) , :~-.--,- .OJJ llllliL a..L.\ub COh4fifiLTTEE. NEW Yotac. N. Y. 10017 Dr. Little, Chm. I Dr. Loosli, ';`-Dr. Cattel1 Application For Renewal of Research Grant' Boston, Massachusetts 136 Harrison Avenue 3. Sho.tritieofrraject, A Psychophysiol'ogical Study of the Act of Reaching for a , Cigarette. 4. Proposed Renewal Starting Date: (Anniversary onoth'er) 5. Discuss any Importe~itGhanbg i dr d8i}ions to Objectives or Specific Aims: Re recommendations of the Council: teristics). nonsmokers, Psychol. Bull, 1960, 57, 493-513, Table 6 - Differentiating Charac- Matarazzo, and 3aslow (Psychological and'related characteristics of smokers and etc), but more comprehensibly within the framework of the reported'findings of' 1. It has been the intention of the investigators to consider the "factors as potential modifiers of habits" indicated (i.e., financial security, 2. Because glucose taken orally can block ttie usual cardiac effects of smoking a cigarette, and because of the high positive correlation between smo ing and coffee drinking (usually with sugar), it remains reasonable to determine the smokers joint usage of cigarettes and coffee (especially with sugar) tor'regulate"'his bodily processes. 3. It is certainly of future interest to evaluate psychophysiological differences among smokers associated with differences iu background'character- ist'ics as to why they started to smoke and why they continued,, but such questions are beyond the immediate scope of this project. 4. A small group of nonsmokers is necessary to evaluate normal (non-smoker related) variation in psychophysiological measurement during the 2 hr experimental sessions. It is certainly true, however, that the investigators should' attempt to select non-smokers to be homogenlous as to their reasons for being non-smokers. ' 6. Give a Brief Statement of your Working Hypothesis if allered or modified: Same as previous application. 46= dc~ ON=

7. Changes or Additions to Experimental Design and. Procedures: (Attach Separate Pages) ^ 47 The original proposal indicated that questionnaires would be given to about 600 students i th n e principal intit' id vesgaorsntrouctor h l y psyc o ogy classes at Northeastern University. This can no longer be done. These classes are now ~ taithin . .,L ..c _I _ . oiwulu a granae oe received for the proposed •~ research, other subjects will have_ to be sou ht. As man as g y possible will be ~ rt~,~~ ~Y. tested, but the actual nu ' d m J er an sources arek n. % ~ u Wh nown.en potential subject samples' (students in various colleges and universities in the Boston region) have been identified uesti i , q onna res will bediitdb amnsere to otain information're ardin g g use of tobacco products coff l ee a cohol sugarll if ,,,, as we asnormation to differentiate among the smoker and nonsmoker groups. Consistent with the recommendations of the Tobacco~Council, the two groups will be selected to maximize intra-groups homogene~ty. 8. Additional Requirements: None. 9. Changes in Personnel with Biographical Sketches of new Personnel (append), None. ~0. Publications or Papers in Press resulting from the Project or closely related work Same as previous application.

according to the findings of the exploratory study. Publications and fees $200 2350 4L= C. Other Expenses (itemize) .Electronic maintenance & consultation $900 Travel (Psychophysiological meeting, . San Diego) $500 Payment to Subjects $750 sWb-Totai D. Permanent Eqyipment (itemize) ~:l Beckman 2-Channel offner polygraph and accessories Total New proposal will be submitted at the end of the first year It is understood that the applicant and institutional officers in applying for a grant have read and'#ound acceptable the Council's "Statement of Policy Containing Conditions and Terms Under Which Project Grants Are Made:" ' businss Off3der of 7heInstitution Telephone 1011111=

ep% ^ 4. Other Sources'of Finanolal Support . CI a Current Pending 4tB9VSE00Z f List financial support for research from all sources, including own Institution, for this and/or related research pro(ecta. Same as previous application I , Title of fho(ect Source

year's grant in the amount of $2,572. Reasons for the request are stated briefly in~ his letter. ..of Harvard's Department of Pathology for a supplement to his current quite relevant to the main-stream interests of the Council. some account of the work going on and this appears to be both promising and project is a young one, but a progress report dated January 30, 1967 gave be settled at the forthcoming meeting, it can be added to the agenda. The We enclose herewith a request from Dr. John E. Craighead This request reached us after mailing of the meeting book for September 23-24. If the committee feels'that the matter can nevertheless n

VIRUS, Previous Infection DEPARTMENT OF PATHOLOGY HARVARD MED1Cr*_ SCHOOL s w a...+r....+r e+er.. . ^ Mr. W. T. Hoyt, Executive Director September 6, 1967 -. BOBTON. MASS: 02115 ................................... The Council for Tobacco Research - U.S.A. 633 Third Avenue, New York, N. Y: 10017 .Dear Mr. Hoyt: I am writing pursuant to my telephone conversation and with a high degree of efficiency. .certain of our investigations. I believe a modest increase in ,our budget will make it possible to conduct our studies productively the grant entitled "'Biology and Cytopathic Effects of Respi.ratory and Oncogenic*Viruses in Organ Cultures of Human Respiratory Tract Tissue." As indicated in my report of June 30, 1967, the work supported by this grant has progressed most satisfactorily although costs have been greater than initially anticipated. This is in part due to the success of our initial studies but more because increases in supply costs and'salaries have been substantial during 1967. In addition we have found it necessary to utilize commercially supplied cell cultures in order to conduct with your staff member regarding a revision of the budget for Enclosed please find a revised budget for the 3 years h of grant support. You will note that the budget for the present first year has been increased by $2,572. This will compensate for a wage adjustment for non-professional personnel made by the University on July 1, 1967, increased demands on our part- time histologist, and the use of commercially supplied primary human cell cultures. Proportional increases have been made in the revised budget for the second and third year. These changes anticipate the growing scope of our work and increased costs. I trust the Progress Report'submitted to your office on January 30, 1967 will provide detailed information on our work. Should a more comprehensive up-to-date evaluation of these studies be required, please feel free to contact me. I would be happy to provide specific details on matters of salaries and supply costs. Sincerely yours, „ f 1 0 ~ © GJ ~ ,cfohn E. Craighead, M!!(D ~ssistant Professor t

REVISED BUDGET Biology and cytopathic Effects of Respiratory and Oncogenic Viruses in Organ Cultutes of Humin Respiratory Tract Tissue A Principal Investigator: J. E. Craighead Principal Investigat br. Retirement, Social Security, - etc. 16% Technician (full time Histologist (shared) Secretary, bookkeeper, dishwasher, (shared) 9 1/4% Supplies Media, serum, chemical Commercial-supplied cell cultures Glassware Disposable plastics Histology supplies Paper, secretarial supplies Miscellaneous Equipment Replacement parts and service Year 1 2790 446 5175 1500 1600 765 5800 2000 1800 888 1200 1250 1300 1500 2000 2200 500 500 500 500 550 600 200 200 200 100 100 100 300 300 ~ 300

i

The committee comprising Dr. Sommers, and Dr. Reimann. New research proposal from Freddy Hoaaburger - No. 338-A. We enclose herewith a new research proposal from Freddy Homburger of the Bio-Research Institute, Inc., of Cambridge, Massachusetts, which is intended to succeed the present -#338'-R4. K,..., As he points out in his application, CTR Grant -#338 - was ac- tivated on October 1, 1962 as a five-year plan which will terminate on •: October 1, 1967. This was the so-called "basic research" grant whichihas been considered quite separate from the concurrent grant -#456 for smoke -inha3ation studies. The latter is considered by Homburger to be in the nature of a contract. He now submits a summary review of progress during these five years, which is enclosed with_a manuscript: The present application proposes a group of studies to follow-up and build upon the findings of project - #338 with practical objectives in view. Because of its relation~to -#338y we have designated it as No. 338-A to maintain continuity of labelling yet recall that it is a new proposal • insofar as commitments are concerned. This proposal was generated without any instigation from the . staff and is transmitted for consideration on its merits. R. C. H. ; p.S. The manuscript mentioned in the enclosed progress report - "Acceleration of Growth of Chemically Induced Tumors by Use of Transplan- tation Technic", F. Homburger and A. Treger, Cancer Res. 27: 1205-1213, 1967)., is forthcoming and will be forwarded when received. Cigarette Smoke Condensates and Carcinogen - Related Substances". F. Homburger, A. Treger and E. Boyer. To be published in the Journal of the National Cancer Institute. "Experimentali Studies on the Inhibition of Carcinogenesis by R.C'.H. C

1 I I BIOASSAY, CARCINOGENESIS and TISSUE CULTURE Dr. Reimann Date: August 18th, 1967 DDr. Loosli Application For Researc6 Grant five year plan. Dr. Jacobson #456 is concurrent on a OP 1 1 N Dr. Sommers, Chm. Ig'M' ; 0 7 #224 - 1955 - 1902 mc1. SucCFSSOR TO THE and.•enewed annually thryu _ TOBACCO IIVDUSTRY RESEARCH CO~VthfIYTEE October 1; 1966. '` yv'- . ~~qh,• If! ~ COMt+II]TTEE: 03s irulzv avr:~~u~: Preceded by #29B, #2].2 & E V N Y 0 / 1. Name of Investigator: Freddy Homburger, M. D. President and Director Bio-Research Institute, Inc. 9 Commercial Avenue Cambridge, Massachusetts 02141 4. Project or Subject: BIOASSAY OF TOBACCO SMOKE CONDENSATES AND RELATED PROBLEMS 5. Detailed Plan of Procedure (Use additional pages if more space is required.) INTR ODUCTION It is realized that our current grant from CTR has been made for one year without further commitment in order to enable us to conclude a five-year program initiated late in 1962. The present application is to request the Council to consider support for another five-year program, based upon our past performance sum- marized in the progress report. The three major projects for which we seek long-term support are the following: 1. Acceleration of growth of chemically induced tumors for the purpose of developing rapid carcinogen testing methods. 2. Systematic study of inhibitors of chemical carcinogenesis with the aim to neutralize alleged carcinogens contained in cigarette smoke condensates. '~ ' # . . , Cf 33 THB COUNCIL FOR TOBACCO RESEARCH - U.S. . Activated on 1011/62 .:..i.::: ~~.. ..: .

Bioassay of Tobacco Smoke Condensates and Related Problems conditions the decline of carcinogenicity and of co-carcinogenicity for mouse: • "skin that appears to have occurred since 19'60 in cigarette smoke condensate. entific knowledge and will contribute to the technology necessary to formulate ``;~ . ,•,~ 3. Skin-painting studies in mice to measure under cornparable '' Each of these projects is based on many years of experience in the particular field and clearly promises to yield valuable fundamental sci- cigarettes that will produce smoke condensates incapable of producing cancers when paintea on the slein oi mice. 1) Acceleration of Carcinogen Testing Th ff d i l ere are e orts un erway n many aboratories to develop rapid screening procedures for the detection of carcinogenic substances. These range from in vitro tissue culture work to the use_ of neonatal mice, newts and~ other `~`~ff'Kr tion sites represent the first significant step in this direction. We believe that it will be possible to obtain even. shorter times of' latency than so far possible by extracting from the initial carcinogen-injection or -application sites large numbers of transformed cells for transfer into fresh hosts, time to make it practical to detect even weak cancer-causing chernicals. Our studies on the transfer of multiple pooled carcinogen injec- light for paramecia and other biological phenomena have been, correlated with carcinogenic potency. However, the most reliable carcinogenesis test would still be the production of tumors in, a mammalian species in a sufficiently short species. The destruction of sebaceous glands and the increased lethality of U. V. Initial experiments on this subject are. already und'erway. The subcutaneous injection sites of C57BL/6 mice are excised after 3 to 5 weeks of contact with carcinogen (benzo[rst]pentaphene is being used as standard car- cinogen) and after mechanical dispersion by means of Snell's cytosieve, the cell suspensions are centrifugated in Ficoll®, a neutral high molecular dextran-like polysaccharide of low osmotic pressure. The cells are thereby separated ac- cording to their specific gravity and the various cell layers, some of which will contain concentrated amounts of malignant cells, are injected' into fresh hosts. By this method, it is possible to inject into a single mouse many times the number of transformed cells coming from numerous induction sites. -Based on the studies of several authors using transplanted tumors and confirmed by our own work, the larger the number of transferred' malignant cells, the more rapid the growth of tumors. It is believed that it may be possible to get a 100% tumor yield 2 weeks after transfer into new hosts or 5 to 7 weeks after the car- cinogen was first injected, 1003546825

.Bio-Research Institute, Inc. Bioassay.of Tobacco Smoke Condensates and Related Problems in carcinogen injection sites in the subcutaneous tissue five weeks after in ~' .4. jection of carcinogen. By transfer of pooled multiple injection sites, we were ~observing changes in from 2 to 3 weeks after carcinogen injection but not earlier. Thus, the shortest theoretically possible test period would be 4 weeks In the mouse, the first cytologically malignant cells are found In hamsters, on the other hand, Nettleship and Smith (Proc. Soc. Exp. Biol. Med. 74:800-802, 1950) showed morphologically transformed' fibro- possible test period wouldi be 2 to 3 weeks. I transfers could be made 1 or 2 days after injection of carcinogen and the shortest trates, will produce palpable tumors in a shorter period than in mice. Such that Nettleship's observations can be confirmed) that transfer of multiple injec- tion sites in hamsters and even more so, transfer of transformed cell concenl-. cutaneously induced tumors -is the same as in mice. It is likely (assuming blasts 24 hours after injection of methylcholanthrene. Hence, it would appear that the subcutaneous tissue of hamsters would lend itself even better than that of the mouse to carcinogen testing. The times of latency in hamsters for sub- and tumor cells transferred fronn, females into males could be identified as be- longing to the original female host and hence as induced by the carcinogen in- jected into the first (female) host. In addition, chromosome studies are readily feasible in hamsters Institute of Technology, and to carry out the above described studies. Dr. Janis Gabliks, currently Associate Professor of Cell Biology at Massachusetts culture laboratory under the direction (either part time or later on full time) of . We propose to establish during the next 2 to 3 years a tissue .transfer large numbers of cells exposed to carcinogens in vitro back into ham- ster cheek pouches. In this way, it may be possible to obtain tumor growth even more rapidly than is possible with in vivo systems alone. ' In addition, it will then become possible to extend the work of Berwald and Sachs on transformation of hamster fibroblasts in vitro and to serial transplantation. We are quite confident that these techniques applied to subcutaneous tissue, epidermis and lung tissue of mice and hamsters will make such pro- cedures as mouse-skin painting obsolete and replace them by carcinogen tests lasting less than two months and having as endpoints histologically demonstrable malignant tumors, the truly neoplastic nature of which can be ascertained by 1003546826 • While these studies will initially be done wit'h, chemical carcino- gens (strong, weak and intermediate), we shall soon be able to apply them as

Bio-Research Institute, Inc. C Bioassay of Tobacco Smoke Condensates, and Related Problems tion later this summer and condensate will be available in adequate amounts. ~ Bio-Research Consultants, originally scheduled for this spring, will be in opera- well to tobacco srnoke condensates, since the condensate producing machine of found that oxidative derivatives also have this effect. 2) Systematic Study of Inhibitors of Chemical Carcinogenesis . While Falk and Kotin showed that reduction derivatives of poly- cyclic hydrocarbons inhibit the carcinogenic effect of these hydrocarbons, we the di-quinone should be more active. corresponding to the quinones which, if the peroxide hypothesis were correct, would not possess the chemotherapeutic activity of the quinones. Conversely, they possess chemotherapeutic (in contrast to merely carcinogenesis inhibiting) effects. The hypothesis that hydrogen peroxide, which may form from quinones, is the cytotoxic agent is susceptible to test by synthetizing the aza derivatives The quinones appear to be a group of special interest because neutralize the carcinogenic effects (as tested by our own new rapid methods and by skin painting) of cigarette smoke condensates. It is the purpose of a systematic study of derivatives of benzo [rst]pentaphene, of other polycyclic hydrocarbons and of terpenes (such as li- monene) and their derivatives to find those compounds that are most active in counteracting the carcinogenic effects of polycyclic hydrocarbons and that are themselves least carcinogenic. Such compounds could be used eventually to which are known to be precursors of the cytotoxic quinones in vivo. test the trifluoroacetyl derivatives of some hydroquinones and their glycyl esters, In order to render these compounds more volatile and better suited for use as adjuvants in the tobacco blends, it is suggested to prepare and IV -_... I ...._- -1- ,a :v~ . These compounds are represented by the following examples: ~A . 0 1. 9, 10-Phenanthrene hydroquinone di~(trifluoroacetate), IV © 2. 9, 10-Phenanthrene hydroquinone-bis-trifluoroacetylglycyl `~ 3. ester, V 3, 4, 9, 10-dibenzpyrene-5, 8-di(trifluoroacetoxy), VI y~ V VI

Bio-Research Institute, Inc. Bioassay of Tobacco Smoke Condensates arnd Related Problems for the volatilization of amino acids (see, for example, Cruickshank and Sheehan easily, and the Trifluoroaceti~c acid~ and its derivatives are completely non-Jtoxic~ Anal. Chem. 36:1191, 1964). The trifluoroacetate group is known to split ver- and physiologically inert (see "Toxic Aliphatic Fluorine Compounds" by F. L; Pattison, Elsevier Pub. Co. , 1959', pp. 20;27;62). Skin-Painting Studies in Mice genicity of smoke condensates, and Wynderts early work, for lack of a better method, as a measure of carcino-' Skin painting in mice has been used since Croninger, Graham control for a comparison between mouse carcinogenicity of condensates of 1960 in 1960 of cigar tobacco which have been preserved and which could serve as a", earlier (more carcinogenic) experiments. We have some 20, 000 cigarettes made prepare at the time of his latest study condensates from cigarettes used in his W'ynder and by Bock. However, neither of these authors was in a position to duced carcinogenicity of cigarette smoke condensates have been published by originally studied by Wynder, Kensler and ourselves. Such observations on re= . ;.,,~...;,..~ This approach is based on methods used in gas •chrornatography There are indications that present day cigarettes may be less car-~. cmogensc an ess co-carcinogenic in terms of mouse skin response than those ,, zf{ and' of 1967. The evidence which suggests such an experiment is shown in Table I, summarizing our own mouse-skin painting experiments using conden- sates from various unfiltered cigarettes done in 1960, 1963, 64, 65 and 66. •..yR There is a strong suggestion here that the mouse-skin carcinoizenicity " has declined and, even more striking, that co-carcinogenicity has practically disappeared6 While the cigarettes used were the same brand in most of these studies, the source of the condensates, the machines used for smoking and the handling of the condensates were different. For these reasons, the results shown here are only suggestive and not conclusive. We are convinced, however, that a repetition of our earlier study sponsored by CTR in 1960 to 61 would yield similar and conclusive evi- dence if indeed the composition of present day cigarettes has changed. We are 1003546828 Condensates could be prepared by Bio-Research Consultants in an identical mariner for all cigarettes smoked. Condensates would be diluted with equal parts of acetone as in our previous studies, planning the experiment summarized in Table II.

Bioassay of Tobacco Smoke Condensates and R elated~ Problems By use of the ATC cigarettes (which we obtained from Dr. • 40 experiment), we could obtain a check on the reproducibility of' the first (19'60) ;. ~. Hockett and understand to be made of the cigar tobacco used in our original 1960 experiment and thereby determine that CAF1 mice respond today in a manner. , similar to 1960. We could by this device obtain a comparison with the Millerton 7 mice used by Wynder in most of his published studYes. Inclusion of the two most " ..:.:~ widely smoked non-filter cigarettes of today is logical, condensates. croton oil as a promotor yields early information on the carcinogenicity of the . The use of primed animals having received 400y of benzpyrene as initiator measures the co-carcinogenicity of the condensates, and the use of ment. It will provide conclusive evidence not obtainable by any other means and This is an elaborate, complex and protracted (two years) experi- carcinogenicity of present day cigarettes, might well demonstrate the absence of co-carcinogenicity and greatly attenuated come may be predicted! as likely. In view of our latest skin-painting studies (Table I) such an out-

. _ able I SUMMARY OF SOME RECENT ' MOUSE SKIN PAINTING EXPERIMENTS 1 ~ Date of beginning of project 1961 -62 . Jan. 1963 May 1964 Mice 0 CAF-1/Jax Millerton Charles River CAF-1/Jax Millerton , 92 Swiss yg Swiss 29 ~~ Swiss Q$ Millerton~) Swiss eg ~ N Primed with benzo[a]pyrenet - - + + A 9 "5 ~rll Cigarette condensate applied• 0 ' j Per cent tar 50% 50% 50% 18. 6% ' 50% ' 50% ` 18, f9Jo 50oJo 18.01 % 5 ^ t ~ 1 Per cent water -, - 15.4% - 15•4oJo - . ~ ~ No, of mice per group at start 100 200 45 200 150 100 50 50 50 50 00 y c;60 Papillomas after: . _ . . i . . Y .~~ . 30 weeks " 56%' 0. 5% 14% ~ 40 42 " 1% 10% ~' 58% ~ 8% 0% 0. 2% 0: ~s '~v 57 " 9% 1% a 66 " 20% 39% 6% 0. 7% 76 " 32% r 60% 12% 21% 7% ~sc f Published J. Nat. Cancer Inst. 31:1445, 1963. 4001Ag per mouse. .~ ocsftscoo-r , :;. . July 1964 ,. April 1965 Dec. ` 1965 Feb.' 1966' . P.uguet;-196E ~ . ta+:".'t~~ai; ~rr . Q

ME ® Group No. 10 11 12 Total 950 :rx+-V" ~ Application Th Council for Tobacco esearch-U.S.A, s. Bio-Research Institute, Inc. Table II ~.. • s/ . ! . . . . . r,J ~ +~ . t PROPOSED EXPERIMENT FOR CTR MOUSE SKIN PAINTING Strain of Mice CAF/1 0 No. Mice per Group 100 , " 100 Millerton Sw~ss 100 50 50 50 50 Primer (mg per mouse) 0.4 0.4' 0.4 0.4 Treatment of Mice Cigarette Smoke Condensate Derived from ATCt Brand A Br:~ne g none~ ATCt ATCt Brana 13 none # Croton Oil 0.75 d 0. 75 ~ 4 * Benzo[a]pyrene #* All cigarettes of regular length (70 mm) t All-tobacco cigarettes # Acetone control

rt Y5 ~ "6. Budget Plan: Salaries Expendable Supplies Other Expenses Permanent Equipment "" " Overhead (15% of a, b, c) 4 2/1/68-1/31/69 :~ Anticipated Duration of Work: Five years f Based on current level of endeavor._: ~ , t,... £;~8 Facilities•and Staff' Available: The same personnel as in the past will remain active in '~ "this work," In addition, a new group is being developed for studies on tissue , ~ 3 ~E ~~~`cultures" and hamster oncology in our new Cummington Street annex, It is ~ anticipated that with~ addition of increased staff in the new division for work with hamsters, some of the mouse work will be reduced so that during each of _~'X the next five years, the budget for this project will remain approximately the . ~ . _ . . . ~ .. _•;. . ~ : ~:-: : •. ~.., : ..~.; ' . . . .. ...... .. . ..'.:~ .. ..• -,:r- ~'."~F;i! .~ -. . . , _ . . , , . . .v. ..-. .. . . _ ~ . .. ~: - . .~- : ... + . . . . 2. .. . .. ~ . . n Additional Requirements: Condensates for the carcinogen inhibition and carcinogen ~. acceleration studies will be provided by Bio-Research Consultants free of charge for labor costs only because their smoking machine was developed under GTR contract. In the case of skin-painting studies where large amounts of con-" * .~ densate are required, this will have to be purchased by CTR at the cost incurred ~" A: by necessary addition of technical personnel for the production of these large ~~ '`amounts of condensate. However, it is anticipated' that the new condensate ma-~ -: ~10 Additional Information (Including relation of work to other projects and other sources of support) chine will be so ~~ -~i.r .. . .:. . . .. . . - .,,. ,.,'•. the past, it has been possible to share some personnel with much more. efficien ' ~` . W , r 'carcinogenesis studies carried out under National - Institutes than existing:, s_ ~,mok ~ri ou 'ng ~of Health, National Cancer Institute Research Grant No. CA machines that this cos , ~~04869. Since this grant has been discontinued, this is no longer will be small. ~~ possible. :.Thus,` support for these studies by CTR is assuming added importance.~ ' for us. `°Even with it (at the requested rate), we must reduce our total effort iri the carcinogenesis field. Without CTR support, we should have drastically to ,.~reduce our efforts in this field in which we have worked since 1948 and for which 34, 760. 5,300, 3,085_ 6, 000_ 6,472, Total 55, 617. the Insti)ution ' . ~3. aYr irl.+T l ® 0 Y N m we have developed a uniquely competent team. Signature Director of Project ll, .~ ^ Business Officer of .w

THr,i CoUNCiL FOR TOBACCO RrsEARCri-U.S.A. The committee comprising Dr. Bing, Chm., Dr. Jacobson. New application from Walter Redisch, M.D. - No. 626. We enclose herewith an application for a research grant from Dr. Walter Redisch of New York University Medical Center, New York, N.Y. We have supported Dr. Redisch's program for a number of years, as shown on the first page,„but since the latest was designated a ter- minal grant, a new number has been assigned to the present application. It includes a comprehensive summary of work done under previous grants, which we requested.

I 1 3. _f, li IJ: ;' 7. Gir* a 6rtef Statement of your Workinp Hypothesis: tao. 626 - • 'cF. .9~ U.S.A. Activated: 7/1/66 CF. #3l+4 - 1962-164 COtdPARE: 3955 - 1957 #i6o - 1957 #213 -1959 - 1961 #301 -1961 - 1962 •r deiined vithout

t`d) A d ( d' B: : con PPen e : F' 9 P6Ys+ml Focilties Available (Where Other Shan Administering Organization Indiwte GeograP hicn! location ) ~ ew York University Researeh Laboratories, Goldwater Memorial Hospital,. Welfare Islanrl, N.Y. ... --Vascular•Research, Laboratory, New York University Medical Qenter, 550 First Ave., N.Y. l."b~c.-..:._a.::.....' - • , .. . . .- ;.~ _ ~ "L~'irY-^,~. •.a. ~~ ~:'~ ~ . •r ~'. Detoib of Ex erimenlal Des~ ( and Procedures ~P° Attach rate Po ts ~"' `~ r. . .• ~,v {N. v n: The method to be used in this study is di°_ferential venous occlusion Ple thYsmoBraPhY. ar.^4:.s: . ' .. ¢ ~, Plethysmographlc measurements of the foot and^ca.lf are taken, those of the foot being -Far_iepresentative of skin blood flow while measurementa•of the calt are representative of e cle blood flow. The calf and foot are placed in the appropriate plethysmographic L"f'* ; ~*l~nusucite chambers. The chamEers are then made airtight with vaseline and foam'rubber. Volume chenges of the ldmb are caloulated from the effect of a kaowa volume, namely ;; { 5 nl air ia~ected into the chamber, has oa the deflectioa of the recorder. On the =`°= =~recording a time-scale permits measuret>~at of tlie distaace equivalent to 10 seconds ti . fi~;on the base line. At this point a perpendicuLar is erected. Hlood Yiow in ml/mfnute n''in 100 cc of tissue is calculated from the Pormula: _ ., . . ~9~,. .:.. . ... .. . . .. _- _ .. .- . _ .. . • _. k€:= 600 x. L_ Blood Flow c t. °~ tt ~y K z V r<ml~ per minute per 100 mll of tissuej: the perpendicular rise of the slope in >ffi, during 10 seconds. K is the call- ; bratioa constant of the recording apparatus in >an deflection per ml of change in ~alume. V is the volume of the enclosed ca1P or foot in ml which is deterratned by ~ iNi: `. ,.r- ~ the water displacement overflnw.method. ;. • wyZ: Y: ,, ._~- .,, ..n. . . . _ .. . . ,. .. .. .... ... . ' ' _.. ~. 11. biopraphical sketches of all principal and professional personnel (append) K t' t Appended D !' ., . .. 12 L9st of publications: (Five most recent as pertinent) (append) Appended 0 ® T `_

R: REDACTED MATERIAL . Estimated Fulu re Requirements: , , vr...: Salaries Consumable Suppl. Other Expenses Permanent Equip. Overhead Total Y.ar 2 _ Year3 -. : Siqnature ' <<- 7 / ~- ~'%lSl It is understood that the applicant and instilutional officers le applying for a grant liare read and found;acceptable tbe•Council's "Statement•af Policy Containing Conditions and Terms Under Which,Preject Grants Are Made:' otr.d-.r.,a.6 688-3500 Ext 50T % r , Telephone Signature' .`, ~ twinns o(f~.. of iM t.»~~wio. ) ,Telephone l

THE COUNCIL FOR TOBACCO RESEARCH - U.S.A. Application For Research Grant • • . Give a Brief Statement of your Working Hypothesis: the effects of cigarette smoking on the cardio-vascular system show• some essential similarities to the pharmacologic effects of nicotine. have the same effects on~various physiologic parameters. There is like- wise no definite proof that these effects aree dlze to nicotine. However, There is no proof that cigarette smoking and tobacco smoking in general The pharmacologic action of nicotine consists.of a primary transient stimulation and secondary more persistent depression of all sympathetic and parasympathetic ganglia. This includ'es the adrenal medulla and epinephrine is thereby discharged. Nicotine and tobacco smoking have also been shown to exert an antidiuretic action This effect is thought to be the result of stimulation and of the supraoptical-hypophyseal• system iaith the subsequent release of posterior pituitary anti-diuretic hormones. Nicotine also markedly stimulates the central nervous system which is particularly evident for the respiratory and vasomotor centres of the medazlla. Due to the multiple sites of action one may encounter initially a slowing of the heart rate due to,stiYmulation of the central vagal nuclei and cardiac.vagal ganglia. Later owing to the stimulation of sympathetic ganglia, and the central vasomotor centre, tachycardia: and a peripheral vasoconstriction ma.y become prominent. may dil"a.te. .After the stage of sympathetic ganglionic stimulation is eucceeded by paralysis, smaller vessels of the periphery, especially those of the skin, These responses shbuld be studied to ascertain-the presence or absence of an increased catecholamine activity in the systemic blood vessels of patients with hypertension and'the presence or absence of a basic abnormality in catecholamine metabolism in primary hypertension. Such findings in untreated patients with primary hypertension should be compared to findings in patients•iireated with various drugs to lower blood pressure. Also the effect of cigarettee smoking on the treatment of hypertension with diuretics should be studied. 4r

TFID+:COUNCIG.FOR TOBACCO RESEARCH - U.S.A. Application For Research Grant :Dr. Walter Redisch `'New York University School of Medicine New York University Research Service ' 8. Details of Experimental Design and Procedures: (cont'd) When measuring blood flow through the calf (i.e. muscle vascular bed) two pressure cuffs are applied to the extremity to be measured. The distal tocclusion) cuff is inflated to 270 mm Hg, the one proximl to the plastic case (collecting cia•ff )• to the individual'sc diastolic blood pressure minus 10, mm Hg. When measuring blood flow to the foot (i.e skin vascular bed), only the collecting cuff is used which is inflated to the individU•al's diastolic blood pressure minus 10 mm Hg. Following sudden• occlusion, the linear portion of the rising slope of the recorded curve is used to calculate the rate of flow according to the formula mentioned above. All experiments of course will be d'one in a constant temperature laboratory, .t at temperatures of 20 and 28°C . Humidity will be held constanti at 50- In order to avoid environmental influences, patients izb basal state will be brought to the constant temperature laboratory where they will rest on a bed while quasi-continuously the skin temperature on both big toes and the .right middle finger are recorded using.a six ehannel Speedomax. A patient is considered to be adjusted to-the environment of the room when the skin temperature of his toes and fingers has come down to constant room~ temper- ature and stayed there for thirty minutes. In,patients whose skin temperature does not come down•to room temperature despite prolonged exposure, maintenance of a constant skin temperature of toes and fingers for 30 minutes is con- sidered.to represent adaptation. For smoking, our standard proceduxe will be used, subjects will be instructed and supervised to smoke at the rate of one inhalation every thirty seconds for six minutes (total of twelve inhalations). • •

, THE COUNCIL FOR TOBACCO RESEARCH - U.S.A. Application For Research Grant Studies.in Tbbacco Hypersensitivity. Fontana, V.J., Reddsch, W, Nemir, R.,. Smith, M.K.,. DeCrinis, K. and Sulzberger, M6B. J. of Allergy, 30: 241, 1959. _ Tobacco;Hypersensitivity: Peripheral Circulatory Implications. Walter Redisch, M.D. - Presented at the New York Acader{y of Sciences,, March 25, 1960. Annals, N.Y. Acad. of Sciences go,: Art._ l, 142-l44, Sept. 27, 1960. • -Vascular Responses. to Smoking Tobacco Compared with Responses to Skin Testing of Tobacco Extracts. DeCrinis, K., Redischy W., Fontana, V., Lewis, A., Sulzberger, M.B. and Steele, J.M. Annals Int. Med. 52: No. 5, 1960. Charles. C. Thomas, Publisher, Springfield, Illinois, pp. 352, 1962. Studies on Effects of Catecholamines Upon Ektremity. Blood Flow in Man. Redisch, W. Metabolismus Parietis Vasorum!- Praha Diebus - 4- 9 September 1961. Tobacco Allergy and Vascular Responses. Redischy W. Reprinted from BOOK - TOBACCO AND HF.I4LTH, James and Rosenthal, et al.. Evaluation of Vascular Responses to Cigarette Smoking. Redischy W., Messina,. E.J., Terry, E.N., Rouen, L.R. and Steele, J.M6 Submitted-for publication, 1967, Angiology. (Accepted 7/27/67) A Manifestation of Diabetic Microangiopathy in Nailfold Capillaries. Terry, E.N., Messina, E.J., Schwartz, M.S., Redisch, W. and Steele, J.M~ Diabetes, in press. ~ Blood Flow-Measurements in Response to Bamethane Sulfate in Man. O• Terry, E.N., Messina,, E.J., Redisch, W. and Steele, J.M. w Angiology- 18: 161-173, March 1.96.7 ,.,. W1 EP ff3 ~ r. Walter'Redisch New York University School of Medicine :;.New York University Research Service a

R: REDACTED MATERIAL CURRIv'ULUM VITAE • -:.Marital Status: . ' Former Appointments: Present Appointments: Walter Redisch, M.D., F:A.C.P., P.R.S.A . ~ Demonstrator, Dept. of Physiol., German University of Prague Fellowship, Heart Station,, Vier.na University Fellowship, Dept. of Internal Medicine, Franz 1921-33 1927 Joseph's Hospital, Vienna 1929 Fellowship, Dept. of Internal Medicine, University of Tuebingen, Tubingen, Germany 1924 University iss't., Dept. of Cen._and E:~. Path. and Clin:. Propedeutics, GerLlan Uriiversity Med. School of Prague and•Univ. Hospital Associate Professor of Clinical Medicine, New York University School of Medicine, New York, t~TY Research Associate, New York University Research Service, Goldwater Memorial Hospital, New York, NY (Director, Dr. J. Murray Steele) Visiting Physician, New York University Medical . Research Division, Goldwater Memorial Hospital, New York, NY : Associate Visiting Physician, . New• York, NY ., Bellevue Hospital, 1923-32 Associate Attending Physician, University Hospital, New Yor::, NY Physician-in-charge, Vascular Section, New York University Cardiovascular Clirtic; Bellevue Hospital. Physician-in-charge, Medical-Surgical Vascular Group; New York University Medical Center (Surgeon-in-charge, Dr. Roy Clauss)• Member, Advisory Board', Council on,Circulation, A.H.r.. A C 0 Consultant, Vascular Disease, St. Michael's Hospital, Newar::, New Jersey. 0) ~ ~

R: REDACTED MATERIAL - CurricUuiu Vitae W~Llter Redisch, M.I}. / -2- Participe.ted in a project for the United States Navy of the Office of Scientific Research and Development under the directicn of Dr. Steele Major, USA Medical Corps 1°irst.t on- New Guinea and then,Chief of Medical Service, Regional. Hospital, Ca:ap Shelby., Mississippi ~ .During 37 months of imr service on official leave of absence frozi the Universit f. ACTED ACTED l i 1943-46.

PRESENT APPOIN2MENTS : MD!BER OF: R: REDACTED MATERIAL CURRl_'.ULUM VI'rAE Erwin N. Terry, M.D. L R- Vienna, Austxia Tel-Aviv High-Schooi of Cotmnerce; Palestine 1940 No. 7 Formation College - 1945-46 Medical Faculty of the University of the Saar 1951-56 Major, British Army and Israel Defense Forces 1941-46 Moniteur, University of Saar, Department of Psychiatry -1954-56 • Civilian Contract Physician, P.A.C. Hospital,, U.S. Air Force in Germany 1956-61 Associate Medical Director, C.H. Boehringer Sohn, Ingelheim/Rhein, Germany .1961.-63• Medical Director, Pharma Research Canad;a;, Ltd., Pointe Clair, Qitebec 1963- - Research Fellow, New York University Research Service, Goldwater Memorial Hospital, Welfare Island, NY, NY 1964- ACTED PMACOTED1 , • !

Comprehensive Report : Walter Redisch, H.D. - . • , , . Go].dwater Memorial Hospital New York University Research Service ;'Research Associate . ;Associate Professor of Clinical Medicine, New York University School of Medicine. BLOOD FLOW STUDIES ]N HYPERTIlNSIVE SMOKERS ,.,,•.,:, . ,:: •.,, .:. `;This laboratory has studied vascular responses to cigarette smoking in•man sInce Z955• There have been 4 phases of the investigation so far. 1955 - 1959 In cooperation with Drs. Ma.rion Sulzberger and'Vincent Fontana it, was first ascertained that in healthy volunteers the percentage of subjects with strongly tobacco• extract had:, no decrease in peripheral blood flow• in response to smoking•. : on significant correlation,: 90%• of those who did not react to skin testing with ;~extremity measured by plethysmography were then ascertained in 80 healthy subjects and correlated to other hemodynamic parameters as well as to the results of testing- ,:for skin sensitivity to tobacco. extracts. Comparison with• skin, testing revealed ,, -` to cigarette smoking. The effects of cigarette smoking•on blood flow in the lower the percentage of subjects who showed a significant•decrease in blood flow response positive reactions to skin testing with tobacco extract was about, the same as 1~9 - 1962 .,'5ince b]•ood flow responses to smoking in the lower extremity seemed erratic, that some test subjects showed unequivocal increase in total flow, it, soon ;'~'::became evident that it was necessary to employ some acceptable means of separately • most obvious errors. . following permits,a rough estimation of skin and muscle flow by correcting for the : ence between skin-mass/muscle mass•ratio!in the foot and in the leg. The formula ,.;'.estima.ting skin flow and muscle flow. A method of differential plethysmography 'has been developed in our laboratory on the basis of the rather constant differ- Pf - Pi Ut a = 1-Cf-cl ~ F= Flow Rate V= Volume ~ P = Perfusiion, Rate ~ L= Leg F' = Foot S= Skin M= Muscle C= Correction Factor - Fl + Ff = F c.c./min.) V1+Vf =V ~c.cj p = v (c.c./100, c.c./min•.~ }.~ O Ps = Pf + Cf,& Cf = o'. 20 Q Pm=P]1-ClA C1=026 ~ Of course, all experiments are done in the Constant Temperature Laboratory; . the ones reported here were done at 25°C and 55% humn•di'try.

In order to be reasonably sure that the differences be meaningful, we • • considered as significai.it only changes of more than 1 1/2 ml. per 100 m-l. of . aissue~min. - . ,4,, At the present time 34 subjects have been.tested:,with this method. There '~ were 25,males and nine females.. Their ages ranged from 24 to S7 years with an •arteriosclerosis, while the rest had no demonstrable vascullar disease. Nine of the group were significantly sensitive to one or more specific tobacco extracts. Skin and muscle flow responses•of these 9 subjects were tested for the smoking of tobacco types to.wYiich their skin was sensitive. :.. average of 56.3. Seven of the 34 subjects exhsbited evi.dence of obliteratz.ve Skin I+'1ow. Mu.scle Flow ase No Change Increase Decrease No Change Increase Total Allergic J 1 0 2 3 4 No OAS 8 7, 1 0 2 2 ~+ .OAS 1 - 0 0 0 1 0 - In response to smoking,. in eight of these nine tobaeco-sensitive persons a significant decrease in skin blood flow was observed. One•he.d a decrease not significant according to our criteria and was therefore listed as showing "no change't; none had an increase in skin flow. Skin• Flow - Nfuscle Flow Decrease No hange Increase Decrease No Chan e Increase Total Non- ' A1Ll.er ic 2 2 1 8 2 12 11 No OAS l9 1 10 8 2 9 8 OAS 6 1 0 0 3 3 In contrasty of the 25 subjects who•were skin negative, eight showed an increase in skin flow•, 15 had no change and in only two a significant decrease in skin flow was observed. There was no, conclusive trend in mw.scle flow changes i,n, either group-. The df,fiierences in skin flow responses are-h2ghlyy significant. 3) 1962 - 1964 ' The study now turned to patients with,non-gangerous occlusive athero- sclerosis. sclerosis. 22 patients were sttudied'in more than 100 experiments. The results

The Council for Tobacco Research -' "-were by and large identicali with those in healthy subjects. The last phase of the project was devoted to a critical re-evalnzation.-'~.; .The following conclusions were reached: pletnysmography "opthalmic artery pulsensor", capillaroscopy and'photography and skin sensitivity testing for tobacco extracts, the following conclusions seem justified: : On the basis of studies carried out using segmental venous occlusion .1) Vascular response to•cigarette smoking in the lower extremity differs in skin and muscl:e. A decrease in skin perfusion-is generalhyy accom- panied. by an increase in muscle perfusion and vice versa. 2) Changes in relationship between,skin and muscle perfusion depend ever muscle perfusion is increased in response to cigarette smoking at 20 C, it will decrease at 25°C inresponse to-the same stimulus. Whenever skin perftasion is decreased at 20°C following cigarette smoking, it will show an increase in response to cigarette smoking 25 C. o en- ,.responses to cigarette smoking are quantitatively at their lowest. ',;quantitatively and qualitatively on environmental temperature. 3) At "neutral" temperatures between 22.5°C to 23.5°C., vascular 5) There is almost constant relationship between skintand muscle • perfusion in vascular responses to smoking in.the lower extremity. As a rule, increase in one is accompanied by a decrease in•the other and vice versa. of the rapid rate, at whichrthey occur. 6) Microcirculatory changes in response to tobacco-smoking are dynamic in nature. The anal,ysis of these changes require microcineamatography because 7) s`Opthalmic artery pulsensor" studies do not seem to be of value in assessing the response to smoking. 8) Skin sensitivity reaction to tobacco extracts may be dependent to, a very large extent on preparation and composition of the extract. ~ 0 ~ ~ ~ + = ~

. Total Essay of Proposal BLOOD FLOW ST7DIES IN HYPERTENSIVE; SMOKERS No-reference could be found to a studyy of effects of cigarette smoking on blood:- The effects of cigarette smoking on the peripheral circulation have been •studied extensively in healthy subjects and in patients with arteriosclerosis. ;:flow in patients with hypertension, untreated or under treatment with variouS substances. recently it was thought that cardiac output and stroke volume in primary hyper- tension are within normal limits. There was little or no evidence for over- activity of the sympathetic nervous system, vasoconstrictive reflexes remaining at normal levels. In the majority of cases catecholamine excretion, as a measure of sympathetic activity, was found to be within normal limits. be due to an increased peripheral resistance in the circulatory system. Until .. In primary hypertension, the elevation of blood pressure is considered to In the early labile stage of primary hypertension, some moderate elevati,on of catecholamine excretion ha•s beenishown in some cases. This elevation was particularly evident during peak elevations of blood pressure. The whole concept of peripheral vascular resistance is ill defined. It, has been shown that circulation in various organs or vascular beds may respor_d differently to a different stimulus. It has also been demonstrated that the ststus of a particular vascular bed, prior to stimulation, will influence the subsequent response. Observing vascular responses in one vascular bed does not necessarily permit the prediction of similar responses in other areas This concept of variability of vascular response in differing vascular beds would appear to contradi,ct the concept of increase in total peripheral vascular resistance in essential hypertension. amine metabolism. Though it would appear that overall synthesis of catecholamines and their release from storage by neurone discharge as judged by urinaryy catechol- amine excretions is normal, it has been shown that systemic blood vessels of subjects with hypertension are more reactive to catecholamines suchas Nore- pinephrine than, those of normals. Mis then,, would suggest that the Norepine- phrine released in response to nerve discharge is modulated dYfferently. It has also been suggested that a defect of the storage mechanism of Nbrepi,nephrine, in primary hypertension one is basically dealing with an abnormality in catechol- A number of drugs currently in use to•lower blood pressure affect catechol- 'aaiine function in one way or another. Thus the question has been, raised whether being smaller in hypertension compared to the normal, would lead to a greater response to infused or endogenous Norepinephrine as well will be absorbed, leaving more available for vasoconstriction. A third possibility to explain the increased vascular reactivity in primary hypertension has been suggested, • namely insufficient tissue binding of exogenous or end•ogenous~ norepinephrine C thus leading to~an exaggerated vasoconstrictor response. Q W The pharmacologic action of nicotine consists of a primary transient• ~ stimulation and secondary more persistent depression of all sympathetic and ~ _ parasympathetic ganglia. This includes the adrenal medulla and epinephrine ~ a.

s Blood ?low Studies in Hypertensive Smokers ;is thereby discharged Nicotine and tobacco smoking have also-been shown to release of posterior pituitary anti-diuretic hormones. Nicotine also markedly stimulates the. central nervou's, system which is exert an•antidiuretic action. This effect is thought to be the result of stimulation of the supraoptical-hypophyseal system with the subsequent .: become prominent. central vasomotor centre, tachycardia and a peripheral vasoconstriction may vagal ganglia. Ia.ter owing to stimulation of sympathetic ganglia, and the .the heart rate due to stimulation of the central vagal nuclei and cardiac ;_-Due to-the multiple sites of action one may encounter initially a slowing of = particul:arly evid'ent for the respiratory and. vasomotor centres 'of the medulla. . 'After the stage of sympathetic ganglionic stimu]lation is succeeded by may dilate. . paralysis, smaller vessels of the periphery, especially those of the skin, ,..pressure. Also the effect of smoking on the treatment of hypertension with diuretics shoul.d be studied. Such findings in untreated patients with primary hypertension shoul(lbe com- pared to findings in patients treated with various drugs to lower blood .'blood vessels of patients with hypertension and the presence or absence of : a basic abnormality in catecholamine metabolism iniprimary hypertension. These combined effects of nicotine should be studied:,to ascertain the presence or absence of an increased catecholamine activity in the systemic It is suggested that the effect of cigarette smoking be studied an.dd compared in the following groups: 1) patients with primary hypertension who have not been treated before and are not being treated while the study is in progress 2) patients with primary hypertension treated. with reserpine 3) patients-with primary hypertension tkeated with thiazide ~~ patients with a primary hypertension treated with a reserpine thiazide combination 5) patients with!primary hypertension treated with guanethedin 6) normotensive subjects • a Es.ch group to consist,of 5 subjects Essential hypertension for the purposes of this study Ls.defined as a sustained diastolic pressure of 100: mm Hg.or higher without detectable underlying cause.

_ ,`,.. . . . . Blood ~'low Studies in Hypertensive Smokers y The method used in this study is differential venous occlusion plethys- ,:~.... mography. _ Plethysmographic measuTements.of the fot.ad ca73 are taken, those of the- , • PPro- • - . :,.... . reDresentative of muscle blood flow. The calf and foot are placed in the a foot being representative of skin blood flow while measurements of the calf are ~~ ', with vaseline and foam rubber. Volutne changes of the limb are callculated from ''..priate plethysmographic lucite chambers. The chambers are then made airtight :•.• tissue is calculated from the formula: • measurement of the distance equivalent to 10 seconds on the base line. At : this point a perpendicular is erected. Blood flovin ml/minute i,n 100 cc of on the deflectionlof the recorder. On the recording a time-scale permits the effect of a known volume, namely 5 ml air injected into the chamber, has ' (00 x L _ Blood Flow K x V (ml, per minute per 100 ml of tissue) L is the perpendicular rise of the slope in mmy during.10 seconds. K is the calibration constant of the^recording apparatus in mm deflection per ml of change in volume. V is the volume of the enclosed calf or foot in ml which-is determined by the water displacement overflow method. When measuring blood flow through the calf (i e. muscle vascular bed) two pressure cuffs are applied toathe extremity to be measured. The distal (occlusion) cuff is- infliated to 270 mm Hg, the one proximal to the plastic case (collecting cuff) to the indi;vidtzal's diastolic blood pressure minus 10;mm Hg. When measuring blood' flow to the foot (i e. skin vascular bed) only the collecting cuff is used which is inflated to the individual's diastolic blood pressure minus 10 mm Hg. Following.sudden occlusion,, the linear portion of the rising slope of the recorded curve is used to calculate the rate of flow according to the formula mentioned above. All experiments.will of course be done in a constant temperature roomy at temperatures of 20 and 28oC. Humidity to•be held constant at 55%, A In order to. avoid environmental influences°, patients will be brought to the constant temperature laboratory where they will rest on a bed while quasi- continuously the skin temperature on both big toes andithe right middle finger are recorded using a six channel Speedomax. A patient is considered to be adjusted to the environment of the room when the skintemperature of his toes and:,fingers has come down to constant roomitemperature and. stayed there for thirty minutes. Inpatients whose skin temperature does not come down to. room temperature despite prolonged exposure;. maintenance of a constant skin temperature of toes and fingers for 30 minutes is considered to represent. adaptation. Walter ReB:isch, M.D. New York University School of Medicine.

C0Mi1TTEE• Dr. Cattell, Chm. Dr. Jacobson, Dr•. Little 1. Name of Investigator(s): rindud.jitte and Degrees) $dward- F. Domino, M.S., M~D., Professor of Pharmacology 4. dutitvtion b. z*'c'r 5-r+. ~, •. .'i:'.'. _ . . .: Addtess: University of Michigan Department of Pharmacology 6440 Medical Science Bldg. ":- Ann Arbor, Michigan 48104 year_ 3. Short Title of Projocf: Effects of Tobacco Smoking and Nicotine on the Central Nervous System• I. Proposed Starting Date: January 1, 1968 : 5.Antiupated;DorattonofthisSpecificSrody: Three years. Phase "a" should be completed within I 6. Brief Descripton of Objeqives or Specific Aims: 7. Givea6:iefStatementofyourWorkingHypotHesis: People smoke tobacco for the psychopharmacological, effects of nicotine. These effects are identifiable and. measurable us ing objective a. To determine effects of inhaling tobacco smoke of high, and low nicotine con- . To compare these effects with i.v. nicotine injections. . To further delineate the CNS actions of nicotine. ~ :,taining cigarettes on skeletal orsscle tone in animals and man. endpoints of central nervous system action. 0

_.'J_ ....-.._.-~... . r _i.,. 6 D.tailsofExperimentalDesignondProcedureso(AttachSeparatePages) f•ur research approach has been oriented to the question of why people smoke tobacco. Tb date this research has dealt with the behavioral arousal effects of r.nicotine and tobaceo smoking. These effects are very mild and are accompanied by ;;4;41s..-...r previously by others as described in Tobaecot Experimental' and CLinical Studies" a unique relaxing efFect. -This unique muscle relaxant effect has been. observed by Larsony flaag and Silvette. Many. or the previous studies have used large toxic doses•of nicotine so that the relationship of data so obtained to tobacco smoking is unclear. Furthermore, it is our hypothesis that the pharmacologically active substance in tobacco, i.e., nicot•ine, acts mostly on the central nervous system in causing a reduction in skeletal muscle tone. Most of the known peripheril effects of small doses of nicotine are to increase skeletal muscle tone. The proposed research will be conducted in two phases. 1. Effects of tobacco smoking of high and, low nicotine content on skeletal -'•muscle tone. Effects of nicotine given intravenously on skeletal musale tone. 9. Physical Facitties Available (Where Other:than Administering Organization Indicate Geographical Location) (Cont 'd•. p. 2a) . :,':~`" nieotine and tobacco smoking. These have been published in detail by the principal Standard techniques will be used to evaluate the'skeletal muscle effects:of i:,>;::..:..'.n•::..:....;;r. ..... . .:a - Department of Pharmacology 6440 MedicaL Science Building Cigarettes of known tar and nicotine content are desireds These must look identical and hopefully taste somewhat similar. It is estimated about two cartons of each-type would be required. C 11. Biographical sketches of cll principal and professional personnel (append) : See appended vita of Drs. Domino, Miyasakarand Nakai 12. List of publications: (Five most recent as pertinent).(append) .,

2a invest.gator (see N. Y. Acad. Sci. 64: 705-729, 1956 and more recently, Centrally Acting Skeletal Muscle Relaxants, Chapt. 15, 313-324 in Evaluatioff of Drug Acti-:' vities: Pharmacometrics, Vol. 1~ D.R. Lawrence and A. L. Bacharach, Eds. Academic Press, Lond, 1964). In man the-EMG and patellar reflex will be measured in res=t' ponse to fixed muscle tensions. This will be done before, during, and after . y _ . .- ,tobaceo smoking of cigarettes of known nicotine content as phase I of this pro= Ject. A total of 10 smokers and 10 former or occasional smokers.will be studied: Each will be given 2 cigarettes to smoke at each recording session. Each M, cigarette will be smoked over a fixed-period of 3 minute$ during which muscle activity will be recorded. At weekly intervals the subjects will be given a cigarette of..t high, low, zero nicotine content and a sham-smoke in a Latin square design. During phase II the study will switch to the use of intravenous nicotine over-'1WA a 5 minute period to simulate the amount inhaled•from.the high nicotine content cigarette. A maximal dose of 10-20 microgm/kg will be given depending upon tolerance, etc. Similar studies will be conducted in the chioralose anesthetized cat using this as a model system to determine more precisely the site of action of nicotine. ';,=' r~r4 In addition our previous studies (seeProgress Report #9) on the effects of nicotine on the visual sensory input and its modulation will be continued as described previously. The correlation of nicotine content in the brain w~th its neurological ,., .: and behavioral effects will be studied as soon as a supply of 1'+C-labelled nicotine becomes available to us through Dr. Armitage of England. :

R: REDACTED MATERIAL Estimated Future Requirements: Total `L Consvmable 5upplies Qist by catepories) Animal Surgical Chemical Glassware _:,'PHotographia Polygraph recording paper Sub-Total . C Other Expenses (itemize) . 12%-Staff benefits for personnel Student Volunteers Travel to yearly Pharmacology Meetings D. Nrmanent Equipment ('rkmize) Sub-Total. ' -' ' Infusion pump '• ' Strain gauge, •amplifier' and coupler Respirator _' SMG amplifier and integrator Year 2• Year 3, In applying for a grant have read and found acceptable TelepHone It is understood that the applicant and institutional officers DW~r.f~t qe~t Salaries 9'. Consumable.Suppt. Olher Expenses Permanent Equip. 1800 1000 300 3100 400 400 500 700 2000' 5250 3200 2000 -3307 • 29,507 5512 3300 2000 3472 30.821 Signature /• .•+~".- • f _-7 -.0,i i r• :~t. Qverhead the Council's "Stotement.of Policy Containing Conditions Signaturc and Terms Under Which Project Grants Are Model" IuJ..u olru r.t 1h. bueti.e.. Telephone 40M 611~

list flnonttal support for research from all:ouress, including own InstituNon, for this andlor related stsarch'projacts." : The above grant will terminate 8-31-67. A.competing renewal grant is being submitted with a May 1, 1968 start- ing date.. However, even if approved this grant only is in- directly related to the tobacco and nicotine project as it involves central cholinergic mechanisms since nicotine is not a neurotransmitter substance. 9SS9PSEOOZ JPII

R: REDACTED MATERIAL Name• Title- ~ - Matue Miyasaka • 40 Placee o,f Birth: "Postdoctoral Fellow Present Nationality: Sex: -Sucaa City, Nagano Prefecture Japanese ~ Japan EDUCATION Birthdate•.. . P_ Male Institution: De ree: Year: Tokyo Medical and Dental University M.D. 1955 Tokyo Medical and Dental University D.Med.Sc. 1960 Maior Research Interest: Relationship to Pro•'tect: Postdoctoral Fellow Research and Professional Experience: Rotating internship, First National Hospital of Tokyo 1955-1956 Postgraduate Course, Department of Neuro-psychiatry 1956-1960 Official staff, Neuropsychiatric Department, Tokyo University 1960-1965 Postdoctoral Fellow, Department of Pharmacology, , University of Michigan 1966-present Publications: Twenty-seven major publications.have been published in various medical and scientific journals.

R: REDACTED MATERIAL Edward,F. Domino Place of Birth: Professor of Pharmacology "Present Nationality: ,Chicago, Illinois :. U S Citizen EDUCATION • • Instiiu tion• University of Illinois, Urbana, Illinois University of I1linois, Chicago, Illinois University of Illinois, Chicago, Illinois De ree- B.S. 1948 ri.s. (Pharmacology)1951 M.-D. _(With, honors) 1951 Ma:ior Research Interest: Experimental and Clinical Neuropsychopharmacology Relationship to Pro;ect: - Principal Investigator Research and Professional Experience: Professor of Pharmacology, The University of Michigan Associate Professor of Pharmacology, The University of Mich. Assistant Professor of Pharmacology, The University of Mich. Instructor, Pharmacology, The University of Michigari Instructor, Pharmacology, University of Illinois Rotating Internship, Presbyterian Hospital, Chicago Publications: 1962 to present 1958-1962 1954-1958. 1953-1954 1952-1953 1951-1952 Over 160 full-length manuscripts and abstracts on neuro- and psychopharmacology have been published in various:scientific journals. t ~..~._._.. .

R: REDACTED MATERIAL Name: ~ Yoshihisa Nakai Place of Birth: BIOGRAPHICAL SKETCHES.-= -- - Title • . .. Postdoctoral Fellow Present Nationality: Sex: Institution: Japanese EDUCATION De -ree: Male Year: Kyoto University, Kyoto, Japan B.S. 1955 Kyoto University, Kyoto, Japan M.D. 1959 Kyoto University, Kyoto, Japan D.Med.Sc. 1965 Major Research Interest: Sensory evoked responses Relationship to Project: Postdoctoral Fellow Research and Professional Experience: Internship at Kyoto University Hospital 1959-1960 Residency at Kyoto University Hospital 1960-1962 Postgraduate course at the Department of Pharmacolagy, Faculty of Medicine at Kyoto University 1961-1965 Postdoctoral Fellow, Department of Pharmacology, University of Michigan Publications: 1966-present Seven major publications have been pu~lished'covering various aspects of the neuropharmacology of psychoactive drugs. ~

The committee comprising Dr. Reimanny Chm.,'Dr. Bing, Dr. Cattell and Dr. Soamers. FROM: ' Robert C. Hockett SUBJECT: New grant application from Jack P. Strong, M.D. -#632. . We enclose herewith a new grant application from Dr. Jack P. Strong , Yeteran members of the Board will recognize this as a proposal to continue, vithout essential change, the autopsy study of the degree of sclerosis of certain arteries as it may be correlated with a few selected life-history factors on which information is collected retrospectively from~"next'-of-ki'ln" after decease. These factors include smoking history. : of the Louisiana State University Medical Center ici New Orleans, Louisiana. situation, without priorities. with notification that this completed commitments of the Council and any fur- ther application would be considered "de novo"'in the light of the current : ance with this, a seventh renewal was approved, effective on February 1, 1965, 1964 with one additional year of priority consideration promised. In accord- started originally under Dr. R.L. Holman ifl 1958'and continued, first under Dr. H.C. McGill and then under Dr. J.P. Strong for a total of eight years. In line with our policy of trying to limit implied commitments to some realistic time basis, we askediDr. Strong in 1963 to make an estimate as to how much longer the study would need to be continued to make possible statis- tically significant conclusions. His estimate was that two more years would be needed from February 1, 1964. Our budget projections were made on that basis, and a sixth renewal grant was approved to be..effeetive on February 1, The project, in an earlier and somewhat different phase, was sufficient'for the collection of the estimated minimu number [ry underscorinejof cases needed'in most of the major sub-elassiiica~ions. ?art of an ad- ditional year will be required for processing the intervieu data, completing the evaluation of the arterial specimens, andifor conducting the definitive analysis of the data." application. At the present rate of case complet'ion, another year should be ,'the study another year in accordance with the plan outlined in our lsst In this last application Dr. Strong stated, "we wish to continue The SAB authorized an extension of the project, without additional funds, from February 1, 1966 to August 1, 1966 for some of this data.processing. The project then terminated technically as of the latter date and a final comprehensive progress report and financial accounting are due. (The last financial accounting was received on April' 9, 19o'1t,covering the period from February 1, 1963 to January 31, 1964). A"final" progress report will no doubt depend on the completion of the interim data analysis mentioned in the appli- cation on page 2-a. Meanwhile addition of cases has continued. Q

Tnr•. CouNciL I+`ol: `1`oBAcco PE sLrkl:cr1-U.S.A. 'A first public report on the study was made at the October 19-20, • (See attached memo of J.M.B. and my memorand=to the Board dated July-l8, smoking per se was responsibile for speeding the atherosclerotic process £_ the degree of atherosclerosis could not be interpreted as showing that the ' the platform that the association he observed between smoking history and '` sentation. Persons present at the meeting reported that he did state on ~' copy of the release by the American Heart Associati= on the day of pre- ~~ ,letter of comment I wrote to Dr. Strong on receipt of this abstract and a-~' paper presented at that time is attached. Also attached is a copy of the 1956 Annual Meeting, of the American Heart Association. An abstract of the 1961)'. and pathologically very similar to human ones and can be scored for severity ; cholesterol and coconut oil, arterial lesions appear that are anatomically 114~ and on a diet of what they call their basic "monkey chow" with added butter, the wild state they show no lesions, but in captivity with limited activity monkeys may turnout to be a good animal model for atherosclerosis work. In~, and learn more about his animal studies. He told rne about their comparative atherosclerosis studies in baboons, rhesus monkeys and squirrel monkeys both in the wild state and in captivity. They believe that the small squirrel , by Drs. McGill and Strong on the induction of atherosclerosis in primates. Following preliminary correspondence on this subject (copies enclosed) I made a visit to Dr. Strong on January 4, 1967 to reviewthe project work ` Meanwhile there were newspaper reports of studies being conducted thyroid for induction of lesions. autopsy material. The monkeys, unlike dogs, do not have to be made hypo- by methods very similar to thQse in use by Strong's group for scoring human inite results and this study probably ought to be rounded out by somebody. ~ argue that if further cases are to be added, this should be done now while ~ the personnel and procedures are ready for the task. It does seem that if iPb any job is worth doing at all, it is worth doing well enouoh to yield def- CT~ The tr,ain item in our grants has been provision for her salary. They will ally good at this and Dr. Strong evidently wants to keep her onithe job. days of Dr. Holman. He and his successors all think that she is exception- (Mrs. Richards has been the interrogator in this project since the we have received the proposal for extension of the autopsy study. whether chronic nicotine absorption superimposed upon a tested atherogenic regimen would'influence the degree of atherosclerosis resulting. Instead ceived would suggest controlled experiments with small primates to deter,~ine discussed with Strong. I had rather expected that the next proposal we re-'` Yq letter of May 9, 1967 comments further on some of the topics I the strenoth of the correlation. If the association vanished, definite con- cases mmiEbt confirm the association more definitely or conceivably diminish PERSONAZ ~ CU^.EP;T: ~ I cannot judge fromiany data I have seen as to how signi- ficant Strong's reported association between smoking history and severity of atherosclerotic lesions really is. An extension of the study to more r n . i persisted, we would remain where we are - clusions could be d * If t aFl ' ' - T

, i Triv. Cou.Ncil, Foiz 'hom%-cc. o RLSEaAr.cri - U. S. A. I do contribute causally to the progression of arteriosclerosis. Controlled in a state of uncertainty as to whether nicotine and/or smoking actually animal experiments with chronic nicotine or smoking exposure and studies istically, might provide better clues toward a solution of this key question of how other characteristics and life practises cluster with smoking stat= and thus be a better investment. R. C. H. ._.,~.:,... <~..:,~.._......_~..

#17k - February 1, 1958 - February 1, 1959' #174R1 - February 1, 1959 - February 1, 1960 1174R2 - February 1, 1960 - February 1, 1961 #174R3 - February r #174R4 - February #174R5 - February February 1, 1962 February 1, 1963 February 1, 1964 February 1, 1965 February 1, 1966 TOTAL $7,705.00 7,705.00 7,705.00 (+ 3,500.00 SUP 14,292.00 14,989.o0 15,822.00 15,775.00 17,531.00 MEW dml~

Rrae Suhca_3ttea on Project F'?74(Strci:-, sr3 2'^an), Dra. Catte] 1 L3 . n 3 • ~ F .rt £wxFcS:CL'. r C7•^~•~i to ^:.^.CCrt3~•TI t`:c ir:"~ratiCn,F3 Of tl{A' 'no•+v '- trin °•~ 1°e _:, r•.oj, i:xntil Fe'rtsry l, 1; %?, . ~ been s ' x 3tt d . , C. cn 7iy:,ch4 Pohst C. Ha iett An k-giicatian for cor Li 3 . nuo teh ~,- has rc,t, -, Dr. I'An.r;; C ~-eGi?1 altha.r , y '•i u.~z t`~.~ q-d w ,a 'r.ia co1le u~~, p.. tren~ w a*Mr beea r. ed as p, i Sr~1 Yrtvc si ir tcr. 7U3 ~t3:e cc--Xee c° ath:•xoscl.c.^otic cis^~e as deter"-ny:i at r•.;Lf c-ay, ~.'i L i1 aa Iy o~ ecci 3hn t v~.ctlr:a,l:a, for c~e3s•~ioa rah l:abits o: 3i: e, Fr-1 ei'~i311g cza,3 A1t,.-,•,,~;h tY.r an„itc^ A + t x . ~ J arould be Iie:1 in an efPOrt toy > i,. x., ~*i~ y,, c,~ 3,,'r~ t a ci~i~yar.t:;r`ro: c., c,_t: st;~tr,.1~n;; beer L:~~ o.~• futura n].aaIn. In rcrc:ri4z the ~ rt 3 t~ ~ G .3 Zw x.~2•~'7Ls•c_ia`.:Lr?~~7 'L~;~ ~Ckark ~`-C7"'•.~J:BC;i C: ~•y o!:e }~ ~- ~5 O" r. r- Ya t.a cerc__~ry s's:o x?:; ha ~?; in t3:r r.t,,.~,._ ir.. ;4d • s- aL^a •Ci- ~_. va..culjs: ~ ~ ~ . ~ o_ . ,.e.::3M: F~o_~1e. (3) „aetih:: tlr•~ ?esicn;, '•.n otl=er ~~ •ts of' ^{,~ (l) :ze r.cci72cy k~.th a~.icr ct::~:'osc3erotic lcsio_s cc : be Ci'e:tcct. (2)' 1:e re3iaW:.Lity ol i:;~cr Mtic*,I tcan tra o3t~~Aned on the life L~Mfi- ^ ~~ l,:cOc auc;ctiora insi.u3c-j: (4) 4' ~ "ee'x:::.. ot --- m ,ear'_ „~ i3 ,TC_i,3 ~re C^ iri L32'ii st7 tl`>^>e, mr.ny' :.C :1C3 of c..:_;••-Y`lsUc C?'t,.^. CC•1:'Ci Dc ol;'~._^.irn-i by ccc. :!t-aUt!;n 5 Fr• , e'.:1~ () at l:i n:i., of coaeiuJ.foas ci,ouIci L•:± po:,aib?e ir t} e da*.:a could be coliectca c.:ic-I the eurrel . to ,~ . :ere rry rav;.- bcen ot:,eru. T:.t c:Pcrer:ce on ~,r_ro 3c:c~i ~ t• leN~ {, ~. r, ~ t c~. 4 u., l_.•._r.. 0 1 ttc o• rire thin': .J ~~~) ~~ 1i ~.c.~.liS.i ~. ,•'J~ 1G:. PiF~. ~.,y I E!`1F.(a, 7 ~t :i: 2'A J~n1, C l.`.:3 L•. =. C~fI~~ ~. ~.:, Od :~'.:C j~ ~.LJ. ~iC i'~~3~~ . IC..~• ~ J ' 3 _i _':~ J. C14.e ..-.. ~ i .. of , . .O_ 1i11Z:.; £ )J`r 3y ;:•1 t su*'•c tY:oc r•lvice r~s ,~i ;1 1 , ! i :a z 1 . >_ta ~ ; , *c c_ rsa t on taia ' ' L t~.: 2 tL'3'' CUJjC,.i: h•C^ CjC3~~~ C-3 ~. ~ k_. C=n c'o e:;'a^,1 r 4;? 1_. ,ctt ` ~ v r , c cnk. a.: al....• •~l c. , .~ ioa a-1 ~ Prc4'eat. M-)t, of the of t::3 L'x.LC_'L'~:_~L` 1.1Cik`~`LCLen.x:'C!] C,^J.?JS a t:a re, rati ;•, ia ts,,at L{a n L%.aa for £L ~i 't i i:: t~i:'? C 1•,. r e `_

`, he rccomizes diet.~ry in..'ormtioa a3 equalJ.y or c::xe Lrz*rt..nt, he sees this as r,•zch r.ore d.ifficult ar_d does not ittclv.de it in hi:a l.+r^.e.ent plan, thou3ir the r^.attAr 13 u_Ider cor.tinuirz study by hin colle:zs-ueo at tne University of Louisianx. "dith respect to other parts of the vacct+lar s;;atc.ey he hva a coroTar,r arteries. r.ot included in his proposaS to us, r;nica is corair._d; to study of the ': hopwa to develop a co11,ateral study of t'ais sys tem. Rhis, 7;:rever, is coJ.le^Z:e v:o is int.:: estrd prticulnrly in the cereb-resl circvJ aion cn3 The ccn£creaco d3d not reveal e-no#hrr studies nou urzv.emmy with use of tecrniques stu'ficiently siniJ.a.r to s-a;;,3est that any* poolin;; of data would be poa3ible. The subeo-ittee xsat, therefore, reach nju?~o:•.ent as to tt eooprrative Froject or by other rradificc.tiorr. investm^nt or whctiier it c.rabe rade a gocd ir,vests.ent by ektan ,ion into vhether the exger4it'ure rcqueatccl for the stated purpose3 fri11 be a roc1 With renueet to t'.le kin33 of corclu,ioia tYxzt be drr-^r tra-n thL~ vcr?: if it cnsr be ca.*ried cut uucccsoiWlly, th;.-r e seem to be three po:, :ibi].ities: (1) There ray be a m~it,i_re co_-relation between t•a••± de-zzce of ecror-q:y CrLcm o3elerosis a: J. t..e e=.~"t of c.. :.:in;. 2) .ncl e ray be a co •relatzvn, or ~3) incre rra.y be r~ cozr it:tion. If.there s1hauld be a pasitive correlation, tre Een:xrl chseo of getv'ir., some real perspective. other life habits co:L1d be st:.35o1 E:Lmul'tanrousJ.y °,,.?:ere wo-ald 'cc a bett^r corcltiwioa uas 3ti..c'ci.ficd or not, OS' ccrc.tie, if a suffici.nt mr-'aer of as e`other evidence that tobccc:o use is to he<zlt'sr, urethrs• this indict tobe.cco F:ou13' urcloubtrl7:y seize urm any such rt.:ult rnd e~,gloy it up the lvo~ess, or v'r.ether the asso,;iation rras inaree'~ oth_r interv --~dinte factors aal r.ot actually cc.uaa1. .~osz tir3?o are ea ;e•r to laca+r uhcif.r^ the smoisirl; Sa usA3 t1:e Ereater d<jToc of ciseTsz by s=;eZ-diri; sitLUtien v~s? 3 be rach as it i3 =a.lreac:.y. lf:..`t i:r, tie tro;Iid r;.t re•:11}• If t.':c-re should be a negutive correlation, tho d_;Tee of this would be one pu:;sibLe kT~otacsis. co <C1U=1:r, tyYt to'i•deco u3e .^a>_o.a*".a CiOF71 ti.:e i:i::?:^YO3eZG'i*UtiC D:^C~--L:3 iat:?VUJI conclu;i•rersss would be si:-~~i?ar. t;e wo1:].d not re<:J.ly be Ju,;tifi::d in If ther.; s1_7-A1d be r.o cxrcis.tien, t? c rssults would be r. ara conclussive, sir_ce if t'r.e precision of t: e c'atn ti:erc: sufficient to ^_:_' : it cer t i.2 t7mt t::c•re wa s e: ea1 lncZ c: c i- ~] .ticn En:.i r_Dt __ df a neti:cdolo&ic:+1 ind~ter:L:acy, Sfe could tia;2 corcl2a^ t. :;, tol:.acco uti_ aetl s:Liy c:cea not acceierate uthe;.•oacleroJls oll t)'c: coro::.Z` C•2'i.Cricc. ~ Ii L7 2•7?GG7i12_ 13 co*_'i.^;;t, the st•l:.dy tpj:::n••3 a3 er.^ble in w:icli "'nea3s we win bu'u t ils •r.e co .:i; J:oa, fcr C' tJr-I ( of i.e., sclc l;?Yie^11v trou-,h <<c na ' ~ ht in t::_ rc.:,lrz,r,_» 1 r hc: ,l..nea cr:-1 e:~c:: at t:_e ~ ~k3~ o ciu lJ 8•o~ps"). Y ~ i Cn dmg~ ~ ~ `=

Is ti:er epny b"is fo.r GuiiJJinZ. u:~t will be found? In a so^.e;:h.:.t €iMS.]-:r study., Snain fm::d po correlation between smosr.in;; c,r. the mou.nt of di'sCaJc in the crn•sno-anf arterzes. At our conf e-rencey ~'noL:.~s Iauber rexmrted t}mi; the Frmi.n^,iL~xa .. stu,i;,r found no co?°L elotinn teIn'reen s4,oar ; e~.~a the ir.c ider_ce of s,n~?irs '~ ractori:>. ?hey have done reL.i:i•reiy little .:rauo:,.ic or ;;'s-tGroZ.,,;ical vroe1-,. , tTOr"2Yi.._"j* A.f CnO WuEi',:..e: V^.,M. E•.^vIi'..as 3.3 'c.' Ci 1r©h,aC lr'amfC'ti~:~.~J.C~ Cti~:~.~J.C~ .~J.C~ Of gCll'..ra1i4eaat:Z:VJ\•1eY Vs73.4i of ti-v 4.4iQ~i.-~..' wYy V1-4c11.t:.:7.0 the ClbJ~,.'.~„,R:iI-ti~o i:i~oi: +;{ cuZ.,-,ea•t that rrohui:ly no coz:Te3atioz wo 't1d be f oUnd bel`rw:;i n s*.:a?tins erZ the re;c:^olo;.;.ic=.1 condition fou.:d on c"torpsy. She k L-~~.i._ group diC2 ij.:;J 8a positive CCr'1`C'~...t7oIA bniou{3e:1 Et:.':::-UTis t'ma r~roca;.^C~ac?.l 1]3:Ci::'CtioYl• The two Cb3-_~xra''ioSls can be re.^.onC3Z.ei1 by flrsStm3.^..Z tl3;.t. FimLltiin!; Ijui y dil:ec.ly or irad':.:: 'c3C'.f:ly. COlits'].:bLii,e to "'i.}:C u.;''c^.c-I;)itE:t]:on of &Y7 2..^.ui;e epi~.soo-u: ui?,~n t;ae ur.derlfi.r. ; ca. ~lai nrs ~..r ara c;~. 1ucive. uz; s is a h;t_r.onmtica.l conce_r,'c, er.d I do not know of w.~y r: wcl::'~aisms that have been clezcMxi ed or j even _r.rc os le-Y `~ ~.'. ~..~.^a..,..• ~ ^ n`.-^` ^" ~.~~ y eC~ir uA.. .~3 '~, .e rL~i~i vc.."~ 1J'~V Cl'at:._v vl' a...Uit~.,.'''_g on clottfn ; ti-me or r.c :>atcari t is cCnsidvre-J. to : c suca. To detcr:aine s:}°.ct2:, r c:-ia'.ir,3 doeu or do:;a not -nfffect the .I'ate of C.'`4?:':?:.ozclaroSi ° by diZ'GC t CL` ii.::'ly a3ct, r:':mm S.TOU}.Ct' S:;= 7,r br° G~::le af C.Lm L'.:iaf'. ].m?Jo.,'t!?.a i: r:a;'rs'i+i G'xmy?'ovle:'=S. If tie CaS3Ta^3'4 t,::'L' by ..v .:._ ~ n • • ~ t ~ ~ - 1 ~l t~si s~Ct~.~~ of 2.u: x~'cv..wi~.1~ ~2~p ci:ae~' c.l~.ci?r:~,..i.ve•4.ci?r:~,..i.ve ~:n ir ~ ~.~,::.1. Our E,°f fortt3 .^zlGil.~; th ."..:re Z].i:^.;.' v:t.'''L.''-.~Y Dr. ZiCT'izel lF.'Gr'.?- not t`i:3.'y Fl03JE'VCT'~ in Qr 2'ECE::1" i.Co>liCiE:me kTiVl3 rrz'. s'T: ir'ZL'iL u`?`_E.'., he ~~I ?~ ei::res3e:~ tae cpL~iaon that en'3~TM:zl e-:i~c=i':cnts co~.].~' t~e ~=~~~ c~e, ~ c . i;_ y.~'?a 1 ~''~_~.-~ ~~"- ~'',~~-2~~° r ~.ci . '» ~:..ch~ci.. .; _,°1.7 ._ s c d9:eL~:,.°y r;.~..T..~..,5..T..~.., ci...;_, in ara I2.';:1 GLI°~' cl.ei:t1; j`•'' L'?i ~ ' i.. ' „ i ` ~ ~ f!TU:t Si,»:17.G°. ..G~k.A dGs![,I".F'il to Bi12 5r i«.T"'~ro'.'~..C Ji.JbE=o t}=£e inih:.:1:Stj o22 L`.y an G'a bu iiC cl'C•t3: ro- s~.`a.L;"t.'oL`o,ge>3ic rc,'-1w::n in' h~.' a G=::2'i different 5;~:;C~f;:>> 'li;GlL::,''u'~::::g ^ ei'fec,,..a,,.. ' , of ~2:~ n:is ~~ s •~1'.&CCrE'. fu:1 1 ~h ~.~:3 '' ~.ecrer on 1 `~e ' 7re r•o blcm my be fF:_part~...nt eno_+.;,h to justZi'y toth r~.~n r.:3 aniT.al s laurL es. X131.c.n to r.zr :iu-io d'z scusuionz z"ur'Wiie.^ with :~tnw'e e~se3. Lim ('rG.'."`e 0.]' f,:^.-~, 'E..~'1iF:^ work. h'.ActrXM2e fi.C L::iAJt deter~~i,e how far ve mnL to ~r-ib?e on McGi31's nplrc;ach'. Fi. L' e R. RC"i;se:: •

1 CARDIOVASCULAR, PHARNtACOLOGY & CKRff STRY THE. COUNCIL rOR TOBACCO Itn, SnAnC}3 - U_,q_A_ Activatedt.,2f ^ ''- • • --- 833 TlriltD A4ENUE COhMCTTEE Dr. Reitnarin.;•• Cfim;: """ Dr. Bing - Dr. Catitell. •:- Dr. Sommers NMti YORIC. N. Y. 1007.7 Application For Research Grant 1. Name of fnvestigatoF(s): (include?itte and•Degrees) lteneweat Annua on 2j3- 1959- Inclusive . .... .~::z• Jack P. Strong, M. D. - Myra L. Richards, A. M. 2. Institution & Address: Louisiana State.Uhiversity, Baton Rouge, La. 3. Short Title of Project: -; Relationship of Smoking and Other Environmental Factors to Atherosclerotic Lesions A. Proposed Starding Date: February 1, 1968 .5. Anticipated Duration of this Specific Study: 3 years 6. Brief Descripton of Objectives or Specific Aims: ,;i~ With reference to studies already in progress and, to others being considered in the Department of Pathology at the Louisiana State University Medical Center, we wish to request reinstatement of support for continuation of our present autopsy study .;j . of the relationship of smoking and other environmental factors to atherosclerotic ~.°. lesions. Support toward this retrospective study of a cumulative sample of autopsied .. : , .s male deaths in New Orlean& was provided by the Tobacco Industry Research Committee ,._. . throughout the major developmental and testing phases begining February 1, 1958, and ' on through the first 3 years of the definitive phase. The last grant year ended on - January3T; I~'6S;1,vlth ari extension to July 31, 19b6, for the use of unexpended funds. • .::,•~~:`~ ~3ri: The purpose (and plan) of our study continues. to be to investigate selected environ- mental factors, inclkiding lifetime history of cigarette usage, occupation and occupational h sical activit (l t 5 di h b p y y years), as etary practices, a itual level of salt intake, and educational level attained, in relation to various measurements of type and extent of atherosclerotic lesions in the aorta and coronary arteries. 7. Give a Brief Statement of your Working Hypothesis; For human male s, holding age, rac e, and ca us e of death classification constant, amount of cigarette smoking is positively associated - with atherosclerotic involment of both the aorta and the coronary arteries.

t Details of Exoarimentol Desian andProcedures: (Attach Separate Poaes) rno aample ox cases to oe 3nvestigatea is comprised of white and Negro resident r e . ia e v sua , an ra iograp ic met ods, previously developed : C autopsiedi at the Charity Hospital of Louisiana at New Orleans or the laboratories of f 1' 1.11 i I d ~1' h h •the Parish Corone R ; males, agedi 20 through 64 years at death, who die in' Orleans Parish and who are . in our laboratories in connection with the International Atherosclerosis Proiect_ fnT to the specimens which are dissected at autopsy by our technicians, processed for quantitation of various aspects of lesion development are applied by the pathologists ;' I ' prior to the evaluationk the pathologists have no information t+ertainine to the identi- preservation, preservation, and stored:for reevaluation. The specimens are blind-coded'y and ' sacauon oz• rne suo_ieci or to zne ciinicai or autoosv aata. -_ '•data for transfer to IBM punch cards by the Department of Biometry, LSU6M, where " view of surviving family members or other close associates,and prepare the basic viewers, *adequately trained and; experienced in social wor14 obtain the data by inter- The collection and preparation of the pathologicali data and that oE the environ- mental data are two distinct and; separate operations. Research associa•te inter- "`~ % it is programmed for computer analysis. Prior to the analysis stage of the study, : ~;<v° the interviewers have no knowledge of the artery findings. (see attached sheet for con- 9. -~' 9. Physical FadGies Available (Where Other than Administering Organization Indicate Geographical Location) Anuation) There is a large research team in the Department of Patholbgy engaged: in long term research on atherosclerosis. The head of•-the department (principal investigator)i co-investigator, and chief of the biophysics section are directly concerned with providing professional supervision, interviewing, (see attached sheet for continuation) 10. Additional Requirements C C 1. biographical sketthes of all principol and professionol personnel (oppend) Jack P. Strong, M. D. - Myra L. Richards, A. M. 14. List of publicotions: (Rve most recennas pertinent) (oppend) See attaehed sheet. . .~ -

,v.,.. as,t•,..,....:.~'~.+r.. - 'f..:....- . . i~ . .. t.. ; M ~ z !' ,~, ,~ - ~~t ~ a4(7 i ' ~ '~ ' 4x ~ 1 ',scecognizmg mai a sampie ot necrops:ea suojects is a setectea group ana tnere- `••2~-•:••~ ~ ' • s ~ . - . . . ..,:.a ''' = fore not representative of the living pppulation, it is necessary that co;nparative - •' study of the values derived by measurement be made within appropriate race-age ~ groups, subdivided further according to major cause-of-death classification. In -~;: this connection, preliminarY tabulations were -made in late 1966 of the firstr645 . ;' cases completed, and in Table I(at•tached) is presented the race-age distribution of ::the sample according to the classification of "basal", without evidence of Coronary ' Heart Disease (CHD), and "non-basal", with evidence of CHD. ":;:~::... .. . . , • .. . . » The sample is 54%sNegro and 46%white. It is of interest that 208 (60%4 of .the 347 Negro cases fall, into the basal[ group, while the 298 white cases break about evenly between basal and non-basal; 51 %.and 49 %a, respectively. It follows that, since 45 % of alb Negro cases fall in the younger age groups (20 through 44 : years), a large proportion of the Negro cases would be basal, --that is, free of evidence of CHD or disease thought to be related to CHD or to smoking (hypertension, stroke, diabetes, lung cancer, emphysema). .: We are preparing for an interim analysis of the present total of 756 cases in which both the pathological and the environmental data have been transferred to punch cards. This interim sample represent•s approximately the half-way point in reachin our revi d L 1 500- f L h l R i i f g se goa o a case samp ev ,. e ( s on o t e origina goal of 500 cases was necessary in order to ama§s a greater number and a more nearly equal distribution of the cases•among the sub-groups, thereby permitting more meanin ful anal ses ) g y . '..,.. . - Envisonmental histories have been obtained at interview of quali£ied' family members or other close associat'es in another 200 cases. These cases now await coding and the visual and; radiographic measurements of the lesions by the pathologists and by the staff of the Biophysics Section, Department of Pathology. Another 40-odd new cases were assigned for interview in,April, and upon com- pletion of these and others awaiting assignment, the total cumulated sample should exceed 1, 000 cases by September first. This cumulation, of cases• will represent the obtainable environmental data: on all white and Negro male necropsied deaths, begin- ning January 1, 1963', whose artery specimens were collected by our technicians at necropsy and who have met the criteria for the study sample as defined in our Stand- ard Operating Protocol. (The autopsies of the 70 male cases of the pilot study, now - included in the study sample, were performed between June I, 1961, and May 31, 196Z. );.( ' At our present rate of completing• the cases, the collection of the last 500 will require approximately 2 years, and the analysis of the data for the total 1500-case sample, one additional year. Continuation to question 9, second page • or obtaining and evaluating•anatomie materiabfor this specific project. A medical research technologi,st and typist-clerk are engaged in the project and there is a posi- tion open for a• social worker interviewer. Ample autopsy material from Charity Hosp- ital of Louisiana at New Orleans and the Office of the Coroner, Orleans Parishi is available. The Department, of Biometry LS'U School of Medicine has supervised the de- velopment and, testing of the methods of collecting information on smoking habits and is collaborating in analyzing the data. Qr Q • `=

^ o Relationship of Environmentax Factors to Athoreclerosis Table 1. Distribution of the Cumulated Sample bf 645 Cases (N) for Preliminary Analysiso of the Basal Grou I Number , . .., • of WHITE • NEGRO ' ' Cases Jc AGE - •in ~Ion-Ba®al* ~ All Non=Basa1# ~ Sam le p All Basal Group' Group Basal Group' Group . (N) :~ ~._ ~. (white) -.~•,.: . .....-........r...~..~..> (the remainc',er) ~.~....~.e..,_..._ (Negro) (ttn iecnainder 20-g,} 38 ? 4 13 1 24 21 3 25-3.4 89 29 .. Z7 . 2. .. 60 . 52 8. , ~ . 35~4 ,.4 lzz 51 31 , . 20 7 1 47 24 . - . 45-54 191 93 I 43 50 98 .46 52 ... r':'. 55-64 ~ 205 ~ 111 38 73 94 42 52 ' . TOTAL 645 298 152 146 347 208 139 (46% of sa-3e) (51% of white ) (49% of wh4te ) ,(54% of aunp]e) (60% ct NqTro) (40~10 of Nagro) of 360 Casoo. and of the Non-Basal Group (the Remainder) of 285 Casce, by Race and by Ago. "Basal" = thoee subjects without evidence of CHD or discase related to CI•ID or to smoking ~e "Non-T3asaP" = those subjects with it to it It •11 11 11 " " " " ~ UBWSE00T I

R: REDACTED MATERIAL Technical :; Mariz Longoria; Typist-Clerk IIh 50 :'Tunius Solomon, Medical Research Techno- 50 logi sG. , . . _ . . . - . G.t:_T..~..I L Consurnable SuppGes (list by categories). ,Computer time Interview forms and supplies C. Other Expenses Gtemize) D. Pernwnent•Equipment (temize) _ Travel to professional meetings; one trip to England(London School of Hygiene)for consultation on analyses and results(May not be utilized until following year). • E. Overhead (15°.U•of.A+af C) Estimated Future Requirementsc Year 2 Year3 Solaries Consumable Suppl. ~ 1,500 i~. 1,500, Sub-Total I 500 1,000. 2,675 17,841 Toto!' 2 ©, 516 OtherExpanses PermanentEquip, Overhead * Total Travel, 5001 ~ 2,586 19,826 Travel 500 2, 64fi 20, 283 SignatOre. It is understood -that the applicant and institulional,afficers in applying for a grant hove read and found acceptalile the Council's "Statement of Policy Containing Conditions and Terms Under Which Project Grants Are Made." O~nen.nr9,utear /1 r/ . ~ JI Telehone ' J <j. "p /1 5ignotureL W.;n.u OUiu,r of t'w Icf IituFon TelephonE di= 4=5K

' -~ lht finondal.upport far nteorch from all wurcet,lncludtng own tniHtvtton, for thls and/a..nlat.d m.arch proj.dt: Organized Research Budgeteupporting Athero®- clerosis reeearch.

R: REDACTED MATERIAL CURRICULUM VITAE-" Tack Perry Strong, M.1). av . . ~ " - l ' ..` - . . . - . . . , . ' . . . i _ .. Secondary Education: Phillips High School - 1941- - 1945, College: B. S. University of Alabama, 1948 M. D. Louisiana State University'School of Medicine, 1951 i Honors: Phi Beta Kappa - 1947 Alpha Omega Alpha - 195 0' Phi Kappa Phi. - 1967 Academic and; Professional Appointments: . . ,: `' Internship (rotating), Jefferson Hillman Hospital, Birmingham, Alabama, 1951 - 1952 Assistant, Department of Pathology, Louisiana State University School of Medicine, 1952 - 1953- Medical Officer and Pathologist, Ui. S. A. F. , 1953 - 1955 Consultant in Pathology, Southwest Foundation for Research an Education, 1954 - 1955 Instructor in Pathology, Louisiana State University School of Medicine, 1955 - 1957 , - - Assistant Professor of Pathology, Louisiana State Uhiversity School of Medicine, 1957 - 1960 Associate Professor of Pathology, Louisiana State University School of Medicine, 1960 - 1964 -- Assigned to Professor J. N. Morris, Social Medicine Research Unit of the Medical Researeh-Council, London, England September 1962 - September 1963 %~ Combined Course in Medical Statistics by Professors P. Armitage and D. D. Reid, London School of Hybiene and Tropical~ . Medicine, Jianuary - Ap-ril', 1963• Professor of Pathology, Louisiana State University School'of Medicine, 1964 to date Head, Department of Pathology, Louisiana State University School of Medicine, 1966 to date . ~ ....,~ .. _ . . . . . . ~ ~ . ~ • . . . _ •,~.< . :a.=~ •

t t i men s: n ~Hospital Appo 5 p ar y o s , at o; Assisting Visiting :_. ital of Louisiana Hos Ch it i E h l P ` 2 - 1953; 1955 - 1958 - at New Orleans, 19 ' .. Visiting,Pathologist, CharibyHospital of Louisiana at New ' Orleans, 1958 to 1966 : Division, 1966 ta date :•Senior Visiting Pathologist and Pathologist-in-Chief, LSU .`. Louisiana Hearb Association Research Committee, 1960 to date 71. 1965 to date • World Health Organization Atherosclerosi•s Project, 1962-1963 Pathology A Study Section, United States Public Health Service, Fellowships ; Senior Research Fellow, United States Public Health Service, 1957 - 1962 American Association of Pathologists and Bacteriologists; . ' Assistant Secretary, I959=196Z American Society for Experimental Pathology International Academy, of Pathology American Society of Clinical Pathologists; Councilor for Louisiana, 1966 American Society for the Study of Arteriosclerosis Research Career Development A*ard, United States Public Health Service, 1962-1964 Profes sional Organizations: College of American Pathologists ResearchInteresYs: Epidemiology, geographic pathology and pathogenesis of atherpsclerosis; atherosclerosis in primates Current Grants: Natural and Experimental Atherosclerosis (HE-08974) Pathology Training (GM-01202) Frost Foundation Comnuuiity, Activities,: :. Anatomy, 1957; Clinical Pathology, 1958 Diplomate of American Board of Pathology - Pathologic President, Bissonct Elcincntary Parent Teachers Association, 1961• Viee.President, Jefferson. Conueaittec for Better Schools, 1-964 Mrn.hnr nf G)fficial llnardL Mnnholland' Meihodist Church. L96S

R: REDACTED MATERIAL I Curriculuwn yitae :.Place of tirth: , - Marital status: ; ~Education ~. Secondary: Liberty High School :College: `Graduate: L1_- Tulane University: Completion,of the 2-year curriculum Medical-SociW, 1930-1932. Myra L. Richards SchoQl of Social lYork* Specialty, - ROACTED Graduate School____------------------ ___1930-1932...A.A'I.deeree-7~.~ Experience - Bocial Service Dept., Charity Hospital La. State Univ. School of Medicine Public Health Dept., Night V-D Clinic OSocial Service Dept. Charit_y- Hospital Social Research,Inc. (of Chicago) La. State University Medical Center Dep`t. of Pathology -(see page 2 for positions 1932-1942 held in- the Dept. ) - Field Supervisor of 6 1933--1934 senior medical students in "Social Aspects of bledicine" - biec:'.,i:al-Socia1 Worker ....... 1934-1935 (approx. '5 - Medical-Social SYorker... 1955 (Sept.-Nov.) part-time Eye clinics and:wards. - Survey interviewer. . . - 1957, 1958 part time , N.O.Dir., Dr.Rob't.Stone, Tulane: (1) "alcoholic beverages" ; (2) "attitudes tow•ard the dr•, ft (parents of draft-age boys, prior to induction). F-~ - Research Assistant(Clinical).. ~ pa:rt-time ... _ 1956-1958 - Research Associate (Clin.ical) . . tjt part-time.... - 1958-1960 ~ - Associate (Research)......... 1960-1966 ~ -,Instructor ................... 1966_to date ~ *G-raduate School: Tuition Award (library) .............................. (1 yr.) °5ocial Service Dept. - Medical-Social SYorker........ 193].-32 (half-day) Emerg. Relief A•dmin...certification interviewer. .. . .1931 (3 months) Charity Hospital

I Social Serv . July, 1931 Dec.1935- ~ Oct..1937 (aPProx. ) ~ June-Dec. 1941 Dec.1941- July 1942 Sept. -Nev. . , . . 1955 -.Formulated operational policies and procedures for a: _ _ joint W.P.A. - Charity Hospital medical certi~ieation `' project, (staffed by ~I~PA physician and registered nurse for screening applicants; served as liaison worker in the Charity clinics for applicants who required further examination and evalt~ation for work ' ~ . . . , . . . . . . . . . . .. ..:~i: ;9 clinics and wards. Medical-Social Case Worker --_-9 general and special ivhite Organized and supervised the Dept's. Intake Service; ` supervised clerical staff of 15 and an N.Y.A. clerical project. ` Accident Rooms. Set up a•nd super.vised the first program of inedical- social services:in Charity Hospital Admitting and Designed and supervised the hospital's first program of establishing eligibility by standardized interview procedure; trained the interviewers. Case Supervisor of 7 medical-social woxkers,in clinics .and Admitting Room. Director, Social Service Dept. (acting)---professional of 16. (Unable to accept permanent appointment as husband was to be commissioned in War II). 'staff of 35 medical-social w•orkers and clerical staff Supervisor: Charity Hospital A'dmissions Unit interviewers and 7 medical-social workers in,clinics and Adm. Dept. (Resigned to join husband at air base --ZYar II). (S•ee-preceding page). Professional Organizations Publicat.i ons N O O C..W Grl Co-author -"Relatioiiship Between Cigarette Smoking Habits and ~p Coronary Atherosclerosis in Autopsied JIales." ~ Circulation Suppl No III - 0ctober 1966 , , . , , V01. XXXIV, No. 4, p. 31. Abs-iract. ACTED ACTED .ru....~iL...L.'.r ., .S':... .z~.i...sa. a.._ ..-~.. .. w.' ~ .. . .~. .. ... . .... _.. . ... ....... .-. ., , .~......._ ...........f::...,~,..~-....-,.... ~...._.«,t.:::--__.>.-<'...

PUBLICATIONS .,::= Strong, J. P., and.H. C. McGill, Jr. '1962. The natural history of `' coronary atherosclerosis. Amer. J. Path. 40(1);37-49 aortic atherosclerosis: relationship to race, sex, and coronary lesions in New Orleans. Exp. Molec. Path., Suppl. T;15-27. -Strongy J. P., and H. C. McGill, Jr. 1963. The natural history of to 72. 7n The Milbank Memorial Fund Quarterly, Comparability in international epidemiology. Milbank Memorial Fund, New York. Strong, J. P., and D. A. Eggen. 1965. Atherosclerotic lesions, p. 57 and race, sex, and topographic distribution,. Circulation 32•948-955. Eggen, D. A. , J. P. Strongy and H. C. McGill, Jr. 1965. Coronary :' Calcification: relationship to clinically significant coronary lesions 1966. Relationship between cigarette smoking, habits and coronary atherosclerosis in autopsied males. Circulation 34 (Suppl. 3):3 1. (Abstr. Strong, J. P., H. C. McGill, Jr., M. L. Richards, and D. A. Eggen. ~ C. . s .. .. ..._

T` .+` -'.M1.>...._~.-4 GtuuuUVE1Sty1,Ax. rllAltt•1ACOL0i,Y analcfED1LSTRY COt1,\CiL ON A(;iEQfOSCL[ttOSIS - Ah1ERICaN SOCI[TY FOR THE S7Up'Y OF A'2'C . . Cowaittce R^turn to. ~ . . ~*"{ng"""A6SCitICAN' hEART ASSOCIATION, 44 East 23+d Strcot, Now York, Niy.r 10010 Dr ~ ttell . - .: „ .- ' ' Abstruct of paper to bc considcrod for presenlotlon ot Iho _ - 20th Annual Mcclin57r Octo6cr 19-20, 1966 Now York, Nc w York D'cADLINC for submtss;onr postmarkcd MAY 16, 1966 TY?; ALL INFO2'dATIONI DO NOT USE INK FOR CGi.+2:CiiO\SI SU>YI'61T IN DUPLICATEI #174R7 Autivated': 2 15 ,, Renewed annually on • Dfl2tltiT FtLL, Us159 - '6 Incl. Numbor A. Roc"d REL4TIONSFQP BETiv'EEN CICAREPfE 51:OYSNG' HABITS 11Im CORONARY ATHEROSCLEP.OSI'S IN ~.UTOPSIEU MkLES Eggen, Ph.D. Ner Orleans, Louisiana _ _" L-i order co toot the association of cigarette smoking with coronary' o.tbcroscloroate; w•e- coli,:ct;.d coror.:ry artorioa irom 645 autopsicd maloa, ZO-64 yeara. of azo. Tntcrvicwc.-•a o5tai;.od` estimatca of ci;,.'. cttc omo?:f ~- habits of thes© deceased porsor,a fron aurvivic:;; relativca. The intcrviowera used a~.._..:aos that had been tested for valiaitg and reliabii : pais u of livinC persona from the same houaehold. Independently, we estimatad vic::a;l•; t'c.c ca:.c:t of each type of atherosclerotic lesion and rrscastue& tho extent of ealci:icatio.: .:7.d >r e: : coronary wall thiclcneas by optical-electronic acannin3 of radioo:•aphs. For the princi;.» ~ _ - ..r:.lyzcs wo c•.:cluded patients having diseases thought to be asoociatcd with smokin5 (c=a'.•:y aoma, lun- cancor, ctc. ) or with coronary heart disease (myocardial infarctidn, diabetes, strol;e, ete. f. The rnoan percent of the coronary intimzl atiuf4ca Sz; pc.tcd by mII grant liLi Otinf and by a grant fromm the Tobacco Research Cot:ncii._ . .• _ Council on Tobacco Rcscarc'r.-::. = 49WE `= occ::picc by raiacd at..eroaclerotie leaions (fibrous plaques, complicated lesions, or calcificu lecio :~} was a;, rc._i:nntcly twice aa groat in heavy cmoheru (•> 25 cigarettes per day) and in light smokero (< 25 cii;ar cttea per day) than in noa-rsrrokerc. Calci__..~ Inaio :s .^_::4 rncan coronary wall thicknoao measured radiograp'aically --vere, oa tuc svcr; •:, SiiLI nct in heavy omolcora and loweat in iton-nmokcra. Dilforencoa amonS thccc ccto;;o_ico.vero genorally arcatost at yowgor ngoa. Cccupational physical activi:7 .;d cduc:.:ional level ncl:icvcd could not account for obaervcd difforcnccs in c:ctcnt of leL"i.:.::z. Strong, M.D. " New Orleans, Louisiana ` ~.~ `McCill, Jr.,, M.D;; ., Nhw Orleans, Louisiana Richards, M.A. New Orleans, Louisiana

Jack P. Stronr, M.D. ProfecEor of P`itholoM.y School of 1'edicine Iaui:siana State University 1542 rimlane Avenue P.1ew Orleans, Louisiana 70112 Dear Doctor Stror:g: - === Mr. Hoyt has turned over to rce the abstract you prepared for the sic?erah7~r influence tr.e fut±rre di.rection anl cr:p':.ir~la in our reccarcn pro4Jrer~, long-te~1:, study rat:.ar irupatientl.y in the c::ia~^cta~;ion that they would con- , rcad F.ith F;reat interest. Indeed we have t,ee;~ ati~ aiti~rS the resul'~s of this r~ m,ericaii Ifedrt Associt:tion r^: etin3 in Vesr York on Cct:,ber 20th. It has been, '; . T'his prasises to be the czse. c,evi:.e some e_•:perim-Vntal aphrocchery to this p.ro'bl::r3, in the expectation that ""Mt t~Lri fhtui'e ir.tcrest in cardiovascular diccase vould J•ie in this area. fai)y to occru: waclc .,uc.h _r•~,,1ox~J are ~ery F~uvz?ncc~~. r~n cil~rt ~aas M.;~a~ to frE~,uently occur: ~1 hwre there iw r`l,.tiit~aly Iittl:e at:_eLosL3.zro:~i a:ia and aftei, + .. ~ . ather:,.^,cl erosiz tends in Ceneral to prosi.sPone to int'arct:lon, yet the 1...~tt;c~r infox-L-al conference on thi:, latter subjc:ct S:;?3 h?2d in our ofi'ice83 on Jture 10, " ' ~cilit "' h.~, atterued by about a dozen :; e a a y. lhey t:ec:.,ed' to a;;~~ea that t:~oa~;n tion in : ub;;ects vho have dcvcd opcd a prc.+dic,posi_r: ; do~;rer of se'! ea•oai::. An ania:^s that precipitate acute clinical events sacli as thr--:a'cosi:. arcl/os, ir_x^r-c-~ ~ i:e could then have focussed our attention chiefZy upon the a;i.11-::~ascurc mech :'4 eleroti.c precaaceU and could virtually have vritten t:+_is off a^ a po::azbili'ty, w„ of any other ci&arette cuo::e inSre'ient sy ;;i2if i c.~:ili;i_y~ tl?y? f:tf:2r'3:j- mi;;1it have been warranted. We cculd have asst;r:e3, rather dc.?i ~zitr•1;~, that neither chronic nicotine absorption at cusico7i:-ix*y levels, nor the absorption s:?O~Sii'1n and the der%e..^ hc^.rJ scler osi. so~ r~:~t} i:r G.ef yni vi3 c7i:^~.'•1;. J;72'9 `~ ~ ~~ n ~f at _^, .~' a .. , tA If you had found a definite lack of correlr.ti o:r bet,,.een ci,;:ar.ette tion. 'L'he rensoz;n for this exaactation were Pa fello .+s : We h,a rather e::pccted that you would fir•d sucP+_ a lack of co: rcla- 1. Gur own cni*-3l gtu:dics involving t:.uperpaditaon of chroni c nic- otii:c dosaSt; up:,n an athero;;enic dii;t sao::cd no consiytent difi"ercr.ces bctweLn the ni:ii;.zlr, receivi.n; tl:c aLha~].oid ard t21n.;e on the die t aloizc. S 2. The sivila2• pasii- ortcm p':tliolo;:,y ctudies of ;f3let:s i1n;l Pl;_.ir and of Spwin, which F-t:ocatia little r,:;coci ation. 3. Tiie Fr. ry3n~;'ae~ :~.11? Zr,y n ti.~lice sho,: i:n~ that anZii;a pectoris no oft:c»er or carlicr thar: no>>-ai~."j.-.:a;:cra. 1003546880

The fact that cigar and pipe s:.:okcrs e:,pcricnce little greater:' 7 mortality frou•I iucli4raic heart diseascs than no:I-cml<,ers althoujj~ they probably absorb as much nicotine as ciryarettry sz.zo'iera. = (This rer:•:.~ins to be de:~onstratetl definitely). ~ 1•~r re2aoning throu;hout the eourse of your atuety has been that the ' deronstration of a non-corre2ation would pclmjt definite scientil'ic concZus- ion3. On the other hand, the findin.Z of a positive correlation would leave us, froT:1 the standpoint of lo-ic, very nearly where we were beforc. That is, the o^cibility that ni cot ine (or soms other sI~ohe inyr_edient) ray contribute to the Ati.era3c'lerotic process would reiairj open, but the exiotcnce of a cor- relation would IIo-t be sufficiel,t to d:.monstr.ate t:°_xt nicoti.I.e or othcr in,;,rcd- ient actual.l,y d;res so. ihe -Y'canon, of course, is tliat cne subjects a:ere not ranctv.~Iized before the irp:sitioli oi nicotil,L do30-e (or :,moxing) s4. would be the case in or sni"ml cai:cri:+._ont, but were we17 -avlc'c4cd. If sm•~''a.rg is a respora;e to constitutio.nal ia.ct'oru of oersana.ity, ability to copL with stres3 and alL,ieLy etc. ai.d ic t.z-3,refore c.caoci..,tod 1"'it'ri a v}.o?.e ~•stylr of ].ii°e "°e" Cunt is different fro,i i;'_at of eVLl0 r.cea sces to in3icc.te, then thc;~c ft.ctoz•~: or liic: h:b_i.ts (e,:crcize, sl.ce_), use oif aQplri.n, coffee drilir-inZ, alcohol coI.::t1:ip'U*_;,n, diet etc.) ::Z_,;Zt be tiae cau:,cs otf thIe dii'.icz•Cnc-_s in dC~i'::c of arcerinl only be rez 1_.ectc~d in the s~lo:,ing pr~icLis°.,. Your fin(ZiI?£;F3 !'ill;,gefit tvlat we f2?~v.3 t::) U:hie i`t li:e i~t= 1.1 Si1! 2' contro]_lc;l £I13L:1l e_q:ericanis, hopc;,1,tlly u:.an2y~ beU:r mdlals Fc::a dc:t the previouy one:s, to i.ro]ai;c a:A evaluate [illy speci.ii.c n ic'~Lit.e ei 'ei:t t:::at mly exist. They E.'ZfJJ €Liv"~~ Cil: that.ve I:'.uy I;~i.'.:;:Z to l?J`:tenGi "1:u:r thl":1' our S3'uvt:iJ:c7Li of perZor.a2ity an3: "styi.e of life" it7 smo-:::r:, as cc+~~~:zr:~d' to non-Uz=~:.er,. Sti.l), furthcr, they su;;ost tlint we r:ust ,:o,rtihun, a&a intai:aiz'y ouu-.- effovts to prri"ect bioche:aica1 I-:etl:ods of caaparir.~, the aci+T•A n:.cocire abcor•.p;a.an by p1.pe, ci, ar. and ci.Laz'e tte s-n?.ol:e)'s under their il?'bi;tu.al co.;r?i 4io r,N of tobacco us4. I an afraid that uhen tliis paper is pre::cni;cfl publicl.y, 1-re rzgy he called upon for theu ,ii w•e t;entral.a:y di.;J:i~r:e to do :;o in the pu.oZic pres:.. If co_:.~c?s.c;i to do oo, the i:i.iz:Is o' cJ;-ncat~ ~ra would be inci:inc;d to Ls~I:e are i~'p3.'Lect in thc,e ot~.t?incd above. It wot+?d be quitL un i~ri~i.r:at~c, I thin::, if t'hL.r.^ ttpp;.ar.2d to be any d'iv-parity bnt.;een Yaui• vic.:;; ans :air.: on the intcrpreiation or ic:nli.c::tions of tlr;;:,e re~ulc::, ",,'!-mvt th•are :hc,te?.,1 be ^,.^ sriu c any actual das,`y to I~e ~-cry un1:.::ely, but re,,ortAr.:_,ortAr.: li-, to el~~cc^.te the Ay)_)cai.•a2:ce oi a contrJversy. 11'}2i$ could prc~bably be c.vclcled if you sce the i<sn2icclticns as ;,•;; s.^:: tner.i nI:j di:a?~:j the pres:: by inc1u '1ir.~; in your prc, cr.i;, tion a caution A-si_nat ovcrin'ccrorei;ution. Ti-,.n, if we wa•re forced to ce:_:_ent, we cculd sirply E~p:~2oi~e our a~;ree.a~lt with you. ~ 1003546881

i If you fe41 that thia mitter needs discuNsion I can probably arrange to atoh ofof in I'cw Or).eana on wy fort-hcomin- trip to Teaa:y. Robert C. Hoc':r:tt, Pi.D. ,,.:;~, ~ Associat•c Scientific Director yjY , . ~. < P.S. If f1-,11 co~iev of the varici:i- p,t)crs you are - k4scr:~,at~~ at t?le Cccti rg could b: r.cdc Z-vcilaule to tzs in advarce, it trvuld be very hclpf xLL.

LOUISIANA STATE UNIVERSITY MEDICAL CENTER School of Medicine 1542 TULANE AVENUE . NEW ORLEANS • LOUISIANA • 70112 DEPARTMENT OF PATHOLOGT~ October 13, 1966 ~ . Dr. Robert C. Hockett The Council for Tobacco Research ` 633 Third Avenue New York, New York 10017 I appreciate the comments included in your letter to me additional grant support for animals, cages, and some personnel for more expanded studies. I would like to make application to The Council for Tobacco Research to support sorne of this work on atherosclerosis. We are now at the stage where we need on pilot studies for using primates -to study the effect of smoking . particularly apropos to our situation since we have been carrying concerning the abstract on Smoking Habits and Atherosclerotic Lesions. Your comment concerning future experiments was and would appreciate it if you would send me the necessary ~ application forms. Many thanks for your suiDport in the past. 9 JPS :lml i ~ Sincerely, ~ /Jack P. Strong, M. D , ~ '-15'rofessor and' Head -'

- S':I LC_'. r e 39th Scien4iiic Session' of the Awrica.. ii,=t Associa =o:,, ?;sw ., York C=ty, October _1-2 ; lc,ao. Also the ?neric•a_. Sxi'e•'cf'cr the .- Study of Srteriosclaros:s and the Co•.Lncil on ''sterioscl'e-ros`_s of t: e ~ A:^erican Heart Association, October 19-20, 19S5. .. TA2 '-p^2: e='Oy CTR G2'£ nte•Jjsei Stro n.~,. nZelati On3^i"n deu4l°E : was not £c8.i_'9e o'2ther03e1Erosis '"i.Lct that sLo'iin„,_J1U5sC-2 1:n:-:nC?Z'_ Et.CrJ•SCi'E='J&'_.. in t3e;?e£vy S:Ilo:;er. Dr. .StrCnZ .eOrciudc^dt_^.:t.Sm i^_; --_ sa , p_'.:er :Oi_-t-._- out the '8ssociatio.^.t, of c_G8r;.tt.- o:'7Cin%• and -_r_c;.^:c-ce o' stiory of the ..ee vin;; gic-:ec up by T'ne Piew a.:r.f ^i =2s on Fr_cay, Y= e" e'' s,,. -.aritS and Co'_'o_.S•ry:$:Rerosclerosis i n. ::3to'3sied '~1E3; u wc's the on~ 7 e;EYO`s__ ir- t':e s:noaer. Dr. E^<ge13~.~.:g 2.s~:ed _- the nu-::oer of _:tic:: t.: s' _:ae-r- ?acto:" cJ'.:'.d lie, or ~.-rall ;.ay be, the cause of inc-rEasad incide_~ ce of Et~cros- a hiFfi:..v_:21._..:on Oet-wee_ poor nutrition'c-.,., a~Cierosclr;r....is.. S,erest_1-'•_ KOrz;." ever this material 8^a~ il:dicated t.^_=' tREr2 <^-.^._ =a:'e:. :.Gbeo -the i n-'luenc_ of n:e..n ition in this study. :+~yMz Ric'_:azrds re•„_ ted .___z t__y • t'-.at St_ onL := d st: t:1 a di_ ec : causal relat:cnshio_ .:o ^ention was made of ~ d3->'e _ke_.t20_.3'±ly 51.~r'ili'iC;?lt.n irf.2rE::O: in T'Pc:v6r'_i__...E3 _c:__ _ - Ve_e s t-,:;.=3t=cClly Si „ni_'-02.^_u. Stro:"'r5 rc71y:... "i :e' ._ ^0ers £=8 :2_ _y .._•a-- c °r'd y_C:t:.°.. ii= .. -aari'il has been~ B'o-_Ci_ teu to -,Pest2_" of the :.e::._..~_..~_ sc: oo_ .C1e_+Js'_s,:=''^- inter,-a..ior2i. ..tady o'17Co1•Ynt._i•es sLi__ in _':osrcSs. -- _~ a-)De£rs that +.=.Cse spc.C_.-_„Pls .r., co11zC t,•,_ I'-_^o_^= t~'12' 10'+.'e5 t of the .._c- -.._ co'.~_,.ri'es . A L?r--rtt2:1 _ pO_'t isdl:.. _., te C_--+5 Dr. R. r:c0i11 regor~irz on the "GEC _ ical ?at= o-oar o' : . ' - ur-^-,.r co :S ~. .... ~ioA'_T-' San n-^.ton. i0, r'e:Fcs . .- .'}...9.~°.~ 0;1 "Coronary Art„r-y Disease in M=~ato:ySteel;_eVd _- ...~° S,-as wall re-zu'_tir_~ accelcrat-.d ad;encortical activity. any ici-_i:n ,-_ blood strea_... 3L,fcr interest is the r:e~~;ane_~~io- ^-;ane_~~io-^- of t_e e. ~e_-~- to sa? „..aaer her is no sex dil°erence. The sc1r.:J n a`s no ii: or =_t of dur_ -,..-e li:'e span - the art: _al c^anSes ccM71,^..ely reverse after :`resh Mater. ra::ever, the steellead -ay rett:r : to' salt :wat`c_^ once or _.._ fish a_°'7er The steel:ead trout has the salia cPa Zes occur aa__ {n of Se3-:..`1 .:2Ll1'cLio-^_. This e_ 3-^.Se in the saImQ?lre sil' ts°_n 3e3t^ _ 'Z--.- ii1eS2' c_~?:>,2s. 2re'i.hou'%e t to be dL:et0 :yUe cd"En.,Co°_'tici8.:..8s _I: -;; -^3 p- o'+':°c'l _^'-i3.3con ~ai.^i^,~r'., intiL''2l tissue -.--,- fraY ented:..e;'n al e_ :s t_,- re£::l by Dr. Van ('.iti:er soI' Seattle. ^_:1e ?Ja}`JTOEy Co-.'-slsts o_ ._ti2_..-.i '-- Dr. _S_~t~rstain of Da1 las, presc_ tac * e.. --.a',:a-s.a3 on the ttac:-..-.- of ._ e._..c::•Fn t rc:.__~^e of the caj)i11'wry. sli ..a2:Lar_cs s::o•.: t-_is ..-_ La,, th. for : e study were a1- __en by r-eedle I:z'ocsy the & cs.._....- n2-_- 'Tais c:?nZe ,.anotec in' ail ciabcl._c3 and _- zdd_ use.:;lr-ess o_°;,his teca nicuc in c'ie~aoa_'s, t'.ac 7ener __.. `_n to a n,_._„Z_ of o'c-_,._ _=-De_s involvir- va_=a-3 in cas,.-__Y~~_,,,. -.-etaco_is-: as a basic to d2` -. .. tive diz,-ase. If :a3 c^"'_1• . -` . -. _ _ .=:'y c: ;•,:- 'g t:..;n .C:':IL''yStt':e vaso vLJJr_ o_'t.-.- ara;at . Vesse_5..._c tze :o: __ :nn,:w of deZe.e_ wtive di seasc of the a... .,u. J. O ~ _ • =No ~ ~I ~' ~/ ^~ ' 00

i S Herx~,{ C. ;•:cuil:l, Jr., ?•i,D. . i~C'ial!~ aeajDenartmer.t- of ?athDloE,~,r Louisiana 3tai.;e University School of I•:edicine lyt:2 `_'ulane Avenue Ue:r Crleal:s 12, Louisiana Dear Doc'sor 1:cGill: WC.' have rCC'GI=f,-1.y noted SOr e Dubl1C]'j~- ~ i.:. i.:'1'G h'^,.c1'^ CJT:Ce].'i:iI?~ a 1~>_•^v~jC'Cit tu te^Ci: }Jab00I:S to S;1G:a? Cc'arc'-..13'C:.`. ie S'7GL1i~C'i G~^ _=,t F3:CS~GC~ 1Ti j.E.' 3_^i?1T2v a little L:17.'e about t'ia°_Se utl:cti eS 4_r:;•r+I:Li rely p. „ z- ou.`.' oSA1 s:aoi:e ir]halato:1, C-L'~:.'~.ies so :.' ha i1 r7Z'l r~ C:i1 r a1=CC1 rats, i"i `r•:"_St'r.°,1's and Qoa:.-.. 1h°- "w1 a ° ol Tirle .r.SCS_:.'J,.l., Jvrli-°iC __--lC b:~ ,~.~.-• tion ro;atc e involves :-~a.:"~ procle . ~ . i le s • .., • d .. ..C- 1.? ~7I~e~ttt ..v 1 i". Jr s•n icra ~. ~C.,._.~, 'r.eepir.Z7 ttie r e ~ ~, C:. '.LiL~:1Ji.S of CJ 3a:.~ t:_O_4 C~.G„ i. yl_,7-.^ L, . .:?'.. hw_ans ~ ' _ 1?i T7:7"~.l The S m7:i3 f1;?S i.o .`.: Q'i,1Lwt?:1., pJ_ t'o:1 of air caI_l ti:elia've17aQ to t>?•C 1:uT2,'- SL:_'iU:cCS ?3r; v[`_A' :4gi_', 1:1 ^C^,- l oTadS or i r: C'GiGi:3 i:.~'.v'reof, io1~:! -Dt7iT•^ ~'ErtCr",t ~''' ~' t%ti° Q.._ t'Z "~'.'rar: ~'e1 i le .L L . __ c; ps ei~:s rLl`'.-'1t, ^ be yi..pl' fiel ~ in r: •_ '.a case o°;1. ~ M , rcr'~ ~ ~i. Z . ~ ti~~~_ ~ o ~'C..`J_.J...,...o. I~ i2'1 the ?•1a11ii.t,°T' C,= ~11°:.`^C.n i7 tr bl i: 7f (~C,::z 3t? oth^Z' D_%'~rv ^ •~.~ ...^ a ^ . $Sl~i' : :a o: u1:iT:-.als, hot,s_r.r1,, coCts etc. ].zrall~~~a;.-y or explorat;owy or pari; of 2.nz c:ec,ensive p1.:;1?. A SD~C? S?~-?~C~ . ..L'J J.. =~~a.l1y ou~r't r:ot to be alloue-3 to pa'ss 71 t's,e n^~a.l ~ -~ : ~ ~. 3,.3 . LiC._ f - 1.il ~. ~ ter out a uu::.^.';n~Gi : c^.1 T.J"J'37? i iiD:'i J_' v:E -. 1__ ~- ~..:~, it ~. ' 'J1.=L1C: 1'~. ~ ~' wS f'.'- ,lLzr-,; at :1].. .~:ost o= ::ork t':;.t1 e: So in J~T ~ cr' na;:wl i:. ;ala•tior:. Still f:not'rerr ir:nor'•^..nt problem that inc1i: -I-~~s r_^..Ii : n:: t;:'.;o-,r,- 1S to GleYi3e %3. ?:E-'t tlot' . of i1P^.&L'Z'1.`:,T : j Vi.3 c'vL:~'.1 _ c'{pOSu.rc o= h r.. c ^ ln.~^ to . - total S 1 J,iA. l~'ie Tll~.~:•~i~_ ' •.~.~:I, •~ up iT_ ~ CI Ci" l L't S~ 1'~t:~i°~~ t a S?.v }f a (as ~:L'LiaC:Cl fTa". :i) is :7; a V^';' ~QOi' O"' 1LI :..~7S..`•L of waS'ic.~.'1.:DI:;i i2: :; Q . ~ 7 of llt' ~'a b'i.1!'JI7, , if : r;n. r ''o~t ~' R",C 'on, tr~'ti~ ~ sr.- '~~n d~. .' ~•l7~~ ~< ,.:_° ~: lo~~ Pti~y"S1o11 );-l.C•_^.1 of "7T lLtC~ ].:1T J L• CSi,i . a ~i' ' t.1v .~ Wia'c 14~ .r u 1003546885 The ri7rii, so +v..}.,.. ~ r"' a.C:{: of ~ ^ 'l ' L, :1 far is I1.3L l . 17:! CLn i- t77T"i I:1o'."J',.1C?. T'C~i:: is ., 7 .~ I in a yT'L''11 L'u: ., f:.c^.._'"1:'% C Jna 3!:+. t:C•Jn in .^.V_.a..2&.?v' _. t a' r • ~ry ` t t?t ia 11 L L1_ II..CJ_ U.. . 1Z`Ol~" 1 .^.., ''JUt i i .'.:V._~.i~.j ..:.I .:.° i' ~..?.7:::;:2 ~1:. ~ ~Y^ 11 1r .::=,- bi•C,;.I;eS. lil',ce nU1 "''' l~ ; ,~^'. CV(:. Ca, 1T -:~le or I:o.. :, Ui_ ~:.- . e 'LlJ ~..:,:Jr~ ,. at :,~ . . ed, ur-~ h:.•; e tJ_,. ~ rr ala .. ' ..,~tii...a2.:xz?n'.. tt.e r. ..` ~ to +, ~ ~oz~s~ t_ e, lll~.i:e( ~.i:e(~ 7raCeilU'L'eH. t1l~i`rOLI`'~1 carJ7n L:oT:o::iC~e is T?Ve1:i;`.:.?.1.1;J Ch?tefid fb'nI:

It ~. the Z7lrJod aL;n=-n, t'hic; pro:.''•sS, tli:l.'.j::e e :i:et%?r1.7l1.•.".mi of Ilic?-l UIIE3, ]s Sl:id @nola~a' SO t~at ar_al~'~1Ca1 *rlor ti cari ue cC1'r]:ed ollt CJ'?i 3: i;C Jl?~ *.Ii thoU•t unuue Canti,er of cor_fiusion in the resalts attra'aatable to c1allij~es d,:1?•iII, the t'--:e het7-recn cxpoUure and sa:a~lir_~. After aufficie:it s•.e hope t; a;, v,e can v.se blood of +~ ^ l ,~n , to aro:SG 2,<2" 7 if C.I'.;;T Cfi7X"t to oba:'? C " i ;,3 1.'~ d?', -2 ( ~~)*JI ma'tl ~,~,t 'tl ~, [ ."i. .itC"rt.c ?I_L~r ~:~.ulYl. SoVi.C'r o--". l-n.tie-!'. -J U100.L' be used !,r1vil' i.n,_- .'t. Ij..'.ii r .~I~ _ n.~ , ~ /~ .~ ^ could V(~.'i C.'vi- _n .... ~,Y•.. C^.•..L-~1 ./~' Nr".l ~.l~ o.. t•1e ZJ .)J+O,',;.:'_Zy nwal l• VK .'i.i . - , ~ ~ n1~ r ! : ',.. Y .. ra_~44 s . o.-_ ~.-..~ o~~:._:~:,t, ,. _cv.~_ _ G7'~ tiT.~', ..e_i o_^ I?1C'.:]tll:a i.$ ^u' or;;°n. ?r: if e::Y~ .~_., . , . ..-..~_.~...... ) J - -•- G .. l~r.vl~~.I 1ii y.vil ValY::10 WLi:Y'(J'111 aryoilox.Lde, hG,...~"1 C:14~-'' ~. f..•.L - ' .v _C.. !:.:.rCe 4- 0'•'1 i:.ir il- 'n~- tr'Jr of" 1'.3I"i". 3'tir fc?CC C _ -3 ~ ..0 ~,~jy 4J V c :.' ^ , LT ~~ L be ~ Lt.IJ:~G•.fA ~'1lwSV . L.iW. .\e .:.^ J~.J Ql C•:r,-n .._.~! J.. L.1:~J\l~I~I:L lu"~'Jt:•. aJ triA TLI3=J~ :~u ~:..-v.i~.. V~~ iLamol _:4'v7 nearly CV:1Jt.211b in i.lzi.11,7• `.Li; ;:<^.,jnoiy ba 101,e e .._-}y` •~~. G4]LL 7.L Ur'.,''..Va`~. . t'^J~VV.'e . 1_V:Je 1.- pa c~ "te lz~~-<Zf , ~e~~17;:c _.1 tllec? L:•1i,a1:) J if ti:?y b1'a: e _~ t!':° _.^`, s$ in J r"' -,~ - w v_ c, tr'-e J* ^- r.•Ei. : .. ~:S eJ u... .-c .J ~_.c ve ~:i~:'_e in Le 'CtW_:'7:nti L1~~I ~ .. , • iQ1 1JE? i °i1J:I'l y J.fL l•.i.1S i:nu:,e z,..2.•iD ) i. C 1n Tl~ ni .,. k1T::L of -'?r3Jr1 ii C:"? one *.D11o Z.i :It ; rCl~: ~ L`I'~~_' fe': b;L , u J t '_".:c^.'i~:,/ In:'_uICi S I"i~y be Ci~.].u£.'Yu£.' C21.fiE3ie:1uYu f1'^., ,~` i.Jt_ 1'-~-;=•I1".~Ci, u c d i...,q r,:e C..^J.1tJ.t ~ '+'t. J .a. u :.sl~.t,ns~.Ib jec-;;.; can o_^dira-ril;;l jl:d~:e very ~~:e?1 ~7 z^ I.:;,aer t':c;~ or all aside f._Ja'' ~LI'y c~no'Llol:wll faCto_S that r?1`f, 7':i:ji"t1iC£,' ~•'_'t?i. i;_~'~' S2'r t:'.~.;~' 'o. I think '~:na u i_t3 !'li..:?.^, Ca,bci1 :~:1J:~i:~~r 13" -'JC 3 F, ]"~ 'i bet~"~.^.-,^.?n ni_nz:bcr.; 12J11V ~lL ~irl,l ~a...aVu_•r'.*S Gf ~'-~ ~L , 1 l~ ._• V i ..:.,,.te a. ~La_~.f ir_~ c.~' l::a r be c,tit-i~e j.I1."ii:;'t„.. V i'l~ ^'+7.t_ 2.t_2 reSp.,,Gli. to 7??r Y7'3 1'aOlLa, for ti '-::,,1Q2 ::"10 1 GiiiS a laI`~;C? r~L':;ltir of c].La''rCti:e^a but takes fe?~T' real 7i11fS I`w f bC' C~'LI']i:CG' a iiil.iCrCllt , ~Ler ~.)w:r::.ed) .)c~, ec^.y. DiscrepaIlcies samm}.irn x.ore or le:s routinely of ,, ca a::_et?:o l ax ~'act Cil:eSt? CI'raire ` t: l: 'o;;recs •r"^o-: t o:_ or.e o^ t: e `i,-n :] •~ •- f~^ • n i. Jr for of -ic^._. C1':ie':L as SL'C;:1 a`l.`Ol . all U:aol.. ...,, . O • Si21Cc2?C11f "pLL•.., W CA YA C. L 00 JC.J:`JIl.:~»,.~ ~C

Dr. Robert C. Hockett Associate Scientific Director The Council for Tobacco Research-U. S. A. 633 Third Avenue New York, New York 10017 Thank you for your informative. letter of August 31 and' the . had expected at this stage. followed our work here quite closely, went a little farther than we a report in a local paper by one of the feature writers who has enclosed reprints. Publicity on this project, which started with The project is entirely preliminary and is an attempt to cigarettes, we will be able to assign baboons randomly to the addition, it is our hope that if other animals can be taught to smoke to the experimental work with, smaller laboratory animals. In eliminate some of the objections that you describe in your letter experimental group. So far all efforts have been directed toward developing smoke with the mouth and lips just as does man. the passage of smoke through the nasal passages since the animals whole smoke. At any rate, we will certainly be able to eli rninate monitoring experiments to test exposure of the lung surface to procedures for teaching the animals and we have carried~ out no Any further information that you develop or that you come across relating to how one can determine actual exposure of the lung surface to smoke will be greatly appre ciated. Sincerely, HCM:lml cc Dr. Jack P. Strong Henry C. McGill, Jr.., M. D. Professor and Chairman Department of Patholoby

1 w Jack P. Stror,;, bS.D. Iiead Professor and Departmen"t of Fatholoa Iou.i.siz~na State University .'•s`edicaa. Cez;ter 1Y42 T\c._,:e Avcltue 2yew ©rleen,, Iouisi sna 70112 Decemb2r 5, 1566 Derr hoctor St_orz: 1 In acco,: ~ance yrnth your rel~1L''3 Y, ne sent you a}~plicat: o t for•~s o:~ Octo3er 2'st, a..dassk~,c that t:e s~oo1d : c rect~ivy~ a for Fsz a;~r~zl S{il?~ jj on i:eff ~,C "~ J~' .^i ~lo~C1IIr ?_1~ Y'_"! CC v7 i2c: = c JG?? :: ..".Cl^._`'O.^, :S . 1 :i:2oiLlCi ~ .aa:~ J. a :1Cyt ::~}, ~1 ?.e:.1 t: E'.r:'iio:1T•i poila out ~ :.ir :: .:~~ sze ravisj.nG oas• fo~ ~Zn J E.r .d ;~ y ' be E'~i^:?~..~~)._ i, _i in ^ fL;i Wc^.c''.~iS ..~iS. i:v^ iT'~11 :.'.'.Cl' ~'.7L:. C0y)1.c3 of ±~.'sC3C~ 2I1~ ' '~i_ri:'.y I7.°.y reach J ou in t_j1,.e for use in outlinir. ; y:.:]r pl a",~Jc: e3 i~t r_si~~a4~ c_z. ~ :'1,n.e haIl. •"ie::1Z Li'3.vc.'•'lc.`~.e r"l?ferC:1CC'j' to 4:: 1C _~~ t. '_lr ;lJL' ai.'it''' been J~3 O. by ' ia C'Jl'.A. J c u1 `JiZ S l l'l :~~'a3 :~Olt .i.f:.~.s i+ ~~"1 T:23'1. t.U dClI { ~ b!'..~.^,.ooa."i. 'I'Z?l3 pt7.2'1pClC.' of us1n`i b£:Loo::S i.1 cTr.'cd~n'I?.scul.ox c^,.S Y L.i1 C. ~i:?11~. '' ~ ~F~M:^ siJr".ZJ 'th:V ~ ~• ..a•' ClO:c^7,)~' i~2~^~.:i-^:1 s.:i-^:1s to ~ii~:^ hv?1G1•? 5~.~ is J '~'Y.Z~1L J"~i~ 1.°..:.° ~ F211I.. )(,L':\ ~O ~ 1G .. ,v ' • J\ J. disease. l;lso, since it is : c' •~ :~u .': ; cn.t' c~: ,~d ;_L^os~ any kil,,± of ea^c_. .a~n~a1 die;, ti•.ct .~cf Ue of iii{;^rat in to c_;ii;o- ~. ~..,.~1..... ,.. vt?.vCa'LIL.' Ci1.°C::~C. On th^ o~'1.1a°r h?_`•.id:, Yl.',.•a c'~:J~`i of i~„ a.:......,.~_.1'IZ3 bCbooi3:S rCLi?Cr 1'.i'ic;ii.Z?IiS I:C 's:?.^.?l I ti:•i?£\t T:Ltst be i?]vOhi::Ci. l:c°9.2' nol::al JL.CfC sr,~t.n JJ w..\~..JJ.I. t'n c1~L'it.. 1~~7•1-JJU.S2~l ` •: ~•J 4:L'~C..t lC~.'j, lss ol ii0:.a1.la.l~ bt~ + uC. ~.t J - - alveCasaly. l+.Yl.^JS.'h..n.' reason for vZ33 .".Cltscti v-1 of tJeJCo:1S, Cw~'CO.'lL.lll~~j. b0 :Jo.'i+C that they cc~ be t._t;,.1~ ~,:*t J to c~ •ro~'-.,. ci~~-arctt.cs ,arcttcs as ~no:~ ~~e t•i>^,,.:..~ reports, is t.]o iit ~ls . `i'his fac'c, if it is true, su<;Gcstc t:Za.t t'c,,eir r.::c in' tc'swvic.:cl .tLr'.±'es i.light be Si?-t~S "~ On tl '.c o'° Yr.~r .~_ e' ~ta ^1, o- e C~<~.,~ p: ..o~le.1, eV~n ~.Il h....s:1 sti_?i?-t~ •~_ qua:Y_ of s3to~~:r;, is -it?e dii'ficuJ.-cs of relctir_-; zctuc_L c]~.o: x desage to euny biolo-ical or h;,+-~Yoi;hctical consequence of s::,o):i;1<;. L•?e ha-re been conluctir,-, sro?:^ inh•:1ntton e::p°ri.-:c]Its with sne11. IJ eaZ~iT:~sls fo.^ ::.h^,_:';: ten y~rrs. T?,^ origi*ta1 *_c°et'~ods ti~e_~e pret~y ccude bttt, little O ~ ~. ,~L,___;, l:~ve bc ..i so that .o r ~.e fcel that ti,L i2to1~ O by ~..~ ~tle, ,. „ c,~ q es ?_ p o: ed ~ Motr to crsrf~ o~.t ~rto':^ inhal~:tio~i exi~:Jsure ssures t;r: cr coiidi{~;rci:, clo:.:~~ : es;.=~~lin~ '~ ~"ti`~'~.. ' enrQ~`3i'CC~` r~Cl in ii!C' .n'.n tT~.th o~Js- "ci.iV3 of ac~.ui!1 L'.aQo.^~U.Y'b' to ~'i^..th ~` v_A., 1'ih2 P%'.i t1cU-1__t3 %111 ea sp'2a:cs of {:h,-smoliC r W,°?C p ~.c^:.\'I' to C:1'l.E'.:".:1 th^,se svU^1e.. J.n ~ an effort to ~c ~~o~~3 dczc resi o:ac r:c~ ,~rc' ei?ia in tc~: ts of pal.^i~onaxnf Qp m : ~ In thirJ f_tell -L•l:~-re a_c great aclrranJGar-es iIi the usc of 1=iec silace 00 I ~cll-St<~~ ~.~Y'c?L~^4 ii::-leC ~leC~ 3J.rui:1,5 are rCliy~liCli ntti)er3 Oi eni'm rls cc;i' Ue u.sed s'or li`_'etil:-n e, c~^:~L :o~n;a . Th _ pro5]~e~:^p o" n swa tze ~: c~ela:^.iI .f1 -, • t dif.i c~:1 4. I~Iice I.~~ c~el for Lt~;ies of , -'.,a.xs " ~o be s' ;:~la r ~tl' ,:rosclc;•osi.s y ~7~ _a _ Y

•Jack P. Strong, M.D. . t b i ' ~ no L verf sat sfactory for such ~ror r.. We sponso:ed a few studies several years aZo in w.ach -rabbits and hens, fed high cholesterol die ts, 'Wer^ used as. the nodel. This techrdqi.e vas not eltogcther satisfactory ti:e, thought, culd WL batie Yioped that a better raodel miGht now be available for a reixsti tion of this kind of work. W.e have been told for several yea.rr that a coloq* of 'Mall primates was to be set up in Boston in connection with the Hasvard School of Public Hea].th.'s ~ r _.. Depart.ent ox Ilutz•itzoi. This ha,s now boen done and sev~:rald varieties of small primates are being bred in captivity and stanei.<ti-diced strains developed. Since dietary factors az•e regarded as havin ; a relatioln to atherosclerosis, this colony has se::m_d to us to be a possibly prosising locale for 'atheroge7esis studies i•Ahere sta_nda±-dized diets could be caazain=d wi;:h eitner chz°oaLc nicotine dos4se or actual sno'r_e e}rosure. We have not yet enctered into any actuall neUotiatio.ris with that group, az:d would certainly like to ka:o1;* r..ore abov.t the bwckgrous_d work and pilot studies tih-st- have been carr ied out by you and your associates. I think it Slouldd be a very good thing if we couiLd' get toge tner in the very near futw•c for a leisurely discuss.z on of tlti^ whole p2c~ale;~ and the vaxious trar^s in tinUch it znight be anproa.chvd.. A.rour.d th;; ttLrr_ of the yL,a,r I F;:, pla-ra.iir.; to rse0ke a trip to Houston and proba.bl,}r Sun ts.Lonio oa other r1a.LtA:.Ts. Ferh:~:as vre could a1so have a conference in t.ic course of that triP, and if there is wok k on athLrosc1Grozis in San Lntonio that I shol`.~,.d sEe I niCtl'i~ I'~...aj.4 orGe vi sit sex re' sevc;ral p112'p: J;;e s . I = enclosing copies of t,:o publications Ll~at I haiJr,a,~c:d to ruwa iTito . recently a-al which nay be of srs:n.,, interest to you. With kind re;;r~~ds, RCH: ek SincFrc7y, Robert C. Hoc::;.•tit, 1-"n.D. Associate Scien;;ir.ic Director encl.a. 1. A. S. Milton. The effect of nicotine on blood glucose levels and plasma non-esterified fatty acid levels in the intact and adrenalectomized ca.t. Brit. J. Pharmacol., 26/1:256-263 (1966). 2. Robert E. CGrrol].. The relationship of cadmiura in the air to cardiovascular disease death rates. JI.A.M.A., 198/3:267-269, Oct. 17, i;b6. 40

LOUISIANA STATE UNIVERSITY MEDICAL CENTER 1542 TULANE AVENUE • NEW ORLEANS • LOUISIANA • 701f2 • .Robert C. Hockett, Ph.D. Associate Scientific Director The Council for Tobacco Research - U,S,A, 633 Third Avenue New York, N. Y. 10017 Several months ago I spoke with you concerning a smoke substance "acetonitrile" which identifies smokers f rom non- smokers when isolated in the urine. We are most anxious to learn more about this test as we would like to employ it in a study of deaths of 25-44 ,year old males in New Orleans. We plan to take smoking histories from members of the family who can be located but would like to have this objective type measure if possible. It would also be very helpful'in those for whom we cannot find a respondent. - We would greatly appreci-ate any information which you are able to provide to us on this test. Sincerely, JPS: tn Professor and Head Department of Pathology ack P. Strong, M.D.

Jack P. Strong, M.D. Professor and Head Departrent of Patholo~r Lotiisiana State University Medical Center 151+2 T.i? ar.e Avenue r1ew Orleans, Louisiana 70112 Dear Doctor Strong: ~ I have been planning to write to you ever since our visit last January, about the several items in my notes. Your letter now pr_ ,rides the stim,zlus to tuke ection. Acetonitr-, ic 2r.ost of what has been done on tRi s' is no'it yet publisi:e3 and ve thina that corsiderably more chemical: croa•: is sti?l n:.•eded to validate the L:ethoe?olo,>y and espe•ciuhy~ to solve the probler^:.~ ol" kirC.'f:~ '7.cs in' £tbsJrp''vion and excretion. S'G4diL.s ' :.iT the subject w:.3'e i.nit-iac'cd at the Suuth:.7est Research Insti tute in San Antonio un de:c government contrect-s and then continued under a ~sant fro-i this Council. Before the vor1: iiay completed, the mn *;ino ;-;ns r':,o::t corce.-nea v*ith it L'"x^.dC a move. We have just received a "I'].n°.l" r epor t btit this 1.e-.-': as many lo7:,e ends. Under sE.rm-rate cover, ty:' are sending -%'%-.,u c:)pies o2' the brief notes that Y•,ave been pticlishra and excarpt, fmm t}ze repor'L.s we 'hk^.tTe. Vnese are to be considcre:i confid4ntia:1l and, as ycai wil?' see, do not addd tip to a validated method ready for r:.}atil:e aj al icztion. Tf you sris,z to (,;Pt into thi.s subject, it m:.3..]l reduiie so_.e constriicti•ze thin':.- ing and further re:,earch. The mterial iw ir,t; n~;3 to s<ve time and as; ist in research into applicahility rather than as a da:cription of a teyf:e•d r:e th:.eIo7.oZy. Inhalation St-ndiew I thin?; you have received a copy of our Arlnu^1 Y.epo,.t for 7.955-l5~~. On pa„es 11-13 yau fri_ll ftir.d a report of our conference on tecilnini:es for exaosure of anjr:;ls to ci;3rE:ttc s,no'.ic illial- ation, incli:clir., a statcr.ent of the conditions that we feel r:ust be net to 1=::'.-:L ..^;Llch ralaLy Li.^•f'fZ,L1: in dE.'.`.'cribing dose-response Until there is so:-_c satisfactoi f I_etho:l for controlli:~~ and es>r~:,ttir~ the aCtu.~~ ]_t'.i - sL1I'f c ce e2.`?37211~ C3 to {;h~ ?32rticllL'Ite ai, dgas plu: vapor p:1.;lses 1Jf smol-.e, attellpts to relut` specific h1s l oil,'~n,tlloLogicz1 cl_an;;es to do::W;;e will reL:ain im"ther vaZ~Lely q!aali tative. t1e are concentrutir.i-3 on, these probl.mr_s at present and have r:~cte pr~o,-;ress ir.sofar as si!al1 €tniLials are cor,cerne•d. .~,`zmn].l r.;dents, however, have r.ot been use•d very 1:'i'.Ich for studies of the carili o:rC4.JcLlJ__ir. S,r.i tCM , Lilal, so far as I know, there is little bacnZic-:r,d on the proce'-ses of Ertez•iW sclerosis in such zni;::aLs.

. lines. They will surel,y preyent a more difficult problcm because of We are just now undertalcing an inl:a].ation stuJy with dogs to vorI: out better method.oloa.r. Wo rlve not tacklicd primiteU along these their ffarni•a7l de.Yterity. to physio7.o~ice,1, p atholo-ica,h orUbioch ::aicall ones. . For tslese rc:Nso:Is, I am sure our Board mc,bers feel that spon- ttlneous s~olcin~ by baboons cat present, contribute in a ~;eaninGf7z1 n:anner rather to stud;,• of pSyd'~l_of-ieal and r:':otivations.~l p`aenoz~~er.a than pila'.s or by injection) at uev~?2c-A levels for oC.wcrvation oi any di2fe?rt:ncvs induced by the a]%m].oi'd. The pocsibility of using prir,atec, especially s~ller onpN, as a Lodel for atherosclerosis studies see.aG r<o.:a prc•misin~ for tiie i~Ii~leai~at eate future. If conditions can be established for i,rof?ucin- lesions closely rese,mblinn the Ir;~:an ones, in a c3.r ~ ~~.~~_ s ii t~•nt aazd renrocl~~cibtn "^i.,~•, it ~ , nig_zt be pousible to' suPorir•:posc chronic .ic~%c I~:~ s:;er, food, cr~Zn~~.r-~; heter3~;er.eous aerosol. thi,^a scCr:LS not y,~"C- to be at hand. L`.';:1 f1'Jre in'1:c-2'E'..Stin~--n,, perii':75, would be supe=•i^~p:,sEd in.hc:l.ation of tac Gas p:L:se of s?~ohe or of cc1;;_~zIc cor.- stituen4s, eSp<:ci:.'..?:y car'con' zloT.oni'de (see ilsurizp). The last ::i;o-irld. be far eZtier e:~erimentclJ:y since it would not inwolve t'sie h--r.d,'!in~ of a rapidly _It 4rould actua.ll . be much Ltore interesting to s:tp-a•rir:n;,ue uhole sraohe inh??ntin:a upon the at-hero~;e.zic rc.;;i,:_c:I, but t.Yie r~eth,3oloa for To riake any such studier practical, we would nced a basic a4i:ox- o~4nic regu.n giving rathc?• unifo3.-a ca°fectn sa tha.t we Ffo',u.d i;ot have to ;tlye >andiL?y large nur.?bers of anii:.al.s. ~ • utJfia;'l s ~~?u..~y At2ong wl'L the subjects t;e discussed, it seen-3 Loat a_r.r:,nos to provide for continuation off youz p,esent autopsy si,uc!.y until a tatall of 1500 cases has bi Cil acctLT:'1'.LZ1tGd, a2'sd to Colltinuc9 ana~.]ysiN of d•'-l,t,.~'. ArtCricl of ••o;; L"T I::^I1 2'Ace rehltior. ,. ...... -~....-..~ . ~..,~_..._...J_ Thi:: lcihd of t'clir.~ ur.:~oubtc;c'>f ou~ht to be donc• but I n~I tun ceT•t,in as to h:=u directly reZev,:r.L to our purpoCec; shch stuciies c:iL be co ~.ic'crel by our i?.card. Lur•~ lz11 i_c?s Since seveval. studies orI tiie effects of e:uonic inflt.ences on lung st:rfactant arc r_c,;r under way unc?er our sponsol:iri:p c)r trnt of the A.~:I.A.---'. car:::ot ca:'+i1J' JLI::~~? Slhet2'i-r oilz' .^U3a1'd :7'il1_ fCe.l tl?<^t L"J~1"e is n~'.'.L.C:.~i ..L.C:.~i. It r1~;ht depend on 01lct?Ier >7oi~e pra;s~isin- r.I^ttiocis or bci;ter c ntel desij~I;s can-bc cu.`zestcd.

Jack P. Strong, M.D. «e are enclosing a copy of the recent paper by Torii3orn Lundmn It presents very co3ent evidence of the Senetic influence in coronary ;. heart disease as cor:ipared to enviror^ientwl ones. Yeanwhile proaress is being mde in the study of ho,•r other habits and practises tend to °cluster"' t::ith ci,3rette smokinS. Mr. Kurt Ehs3.ein who is aaplyinS rmltivaria.te analysis to large bodies of descriptive data fron. epi«emiolo~i cal studies erpres; es this by sayin~ that "s::o:;inS and drinking appear to be stu?-:arizin-g other and p_rh aps un M4asured variables". In termr of such variabi.es, he is attermptin- to "describe" the smoker es compared to the non-smoker. We hope that the co.riJin`1t7.on of data from such tiore sopnisticated epidt-mio]:o~;,ical studies and expcrit-iental one;; will produce some defir.ite ans1'ieis ln~ time. With kind regards, Sincerely yours, Robert C. Yo.^::?i:t, Tit.D. Associate Scientific Director P.S. We enclose three sets of our revised apul.i catiot, for;:1. ilte• next logical tai•Get date wi]1 be the Se,Aedber r:.cet~.nS of our Scicntific Advisozy raM rd. The dcad].ine is AuC-;ust first but c_ v1_er receipt is an advu.nta ;e since it spreads out the Boe.rd''s wor k load.

. ~:aiQ~k~'~.a`+:t+~.~L::'r.r~s +'~ . `~Y~:~.~: ~, a~~ ~..

THE CoUNCrL FOR TOBACCO RESEARCH-U.S.A. TO: The committee comprising Dr. Jacobson, Chm., Dr. Cattell and Dr. Bing. FROM: . Robert C. Hockett SUBJECT: A new grant application from John R. Rowlands, Ph~.D. - No. 611.. Enclosed herewith is a new project proposal with supporting material from John R. Rowlands, Ph.D. of Southwest Research Institute, San Antonio, Texas. ~ The preliminary investigations have shown an electron paramag- netic resonance (EPR) pattern believed to be produced by the interactions of free radicals and/or reactive diamagnetic molecules with a metal com- plex, presumably hemoglobin, present inthe cells of rabbit lung tissues subjected to smoke. The exact nature of ~his interaction or bonding is not clear, but presumably entails covalen bonding to the sixth coordin- ation site on the iron of hemoglobin - the same point at which oxygen is bound. A study by Dr. John Waugh at M.I.T. has shown the EPR spectrum of interacted free radicals in the smoke condensate. He has not worked with lung tissue. He suggested the broad absorption may be due to a "two- electron triplet state" or to compounds and complexes of transition metals. These studies are primarily definitive chemistry in approach. Further work wil]l be necessary to define the interaction and suggest a means of separating the complex mixture undoubtedly involved. The application has been under study for over a year and was distributed to the committee on March 29, 1967, but has not yet been con- sidered by the S.A.B. Robert C. Hockett John H. Kreisher ~..e: .~-.~..~_._.,~.:...: a.....,.:. .

CFENLISTRY COWLTTEE • THE COUNCIL FOR TOBACCO RESEARCH - TT.S.A. SUCCESSOR TO THE TOBACCO INDUSTRY RESEARCH COMMITTEE Dr. Jacobson, Chm. Dr. Cattell Dr. Bing 1. Name of Investigator: John R. Rowlands . Date: May 27,. 1966 Senior Research Scientist . 3. Institution & Southwest Research Institut'e Address: 8500 Culebra Road San Antonio, Texas 78206 / C" 4. Project or Subject: A detailed investigation of the nature of the reaction between biological materials and atmospheric contaminants using Electron Paramagnetic Resonances and optical spectroscopic techniques is proposed. Such a program could lead to the development of experi- mental methods for assessing the relative importance of various atmospheric contaminants as biological hazards. In recent years a great amount of statistical information has leld to the belief that there exists a close relationship between cigarette smoking .and lung cancer. In addition, studies of this sort have also indicated a significantly higher incidence of lung cancer in areas of high atmospheric polution. - It has so far proven to be very difficult to establish in an objective way the nature of the effects of air polution~ and smoking because of the lack of a convenient experimental method. The advent of Electron Spin Resonance spectroscopy has provided ~ the most powerful experimental technique that is currently available for Q the detection:and identification of both free radicals and compounds con- Q taining pararnagnet'ic metals. The technique has been used with~ great CJ success in this last decade in such diverse areas as radiation biology, ~ chemical kinetics and providing important experimental parameters for ~ improving ligand field theory. Inst'ead of discussing in detail here the ~ experimental and theoretical aspects of electron paramagnetic resonance ~ we have included as appendices two reprints which indicate in some detail " the potential of the method. •.:..,_... ~:.,~:..... .. .. Detailed Plan of Procedure (Use additional pages if more space is required.) ' 633 T_IIIRD AVENIIE NE~y YOR.S. N. Y. 10017 Application For Research Grant I

In the course of studies sponsored' by.NCI involving the- chemical nature and properties of the constituents of tobacco smoke we have demonstrated that the reactive species found in the smoke interact quite dramatically with lung tissue to produce a complex elec-tron paramagnetic resonance signal. The cigarettes were smoked directly into freshly excised rabbit lung. Details of our experimental procedure and results will be published shortly. The most interesting feature of the results is that the resonance signal we observe is not just that of a free radical signal; but consists of both~ a free radical signal and a resonance which may be attributed to at least one type of' paramagnetic metal complex. No electron paramagnetic resonance signal was detected in control lungs under the same conditions used for the smoked lungs. The paramagnetic metal resonance signal does not exist in cigarette smoke itself and has obviously been produced~ by the reaction between cigarette smoke and lung tissue. We have not as yet identified the species giving rise to the signal but work is currently in progress in an attempt to make this identification. We have included as Figures 1, 2 and 3 the spin resonance signals of trapped cigarette smoke, a rabbit lung which has smoked six cigarettes and~ a rabbit lung which had smoked! no cigarettes. In a brief series of experiments we have also shown similar reactions of lung tissue with common atmospheric pollutant gases suggesting that a thorough investigation of the reaction between such contaminants and freshly excised lungs in addition to certain model biochemical systems such as metal-porphyrin complexes, has great promise in the study of the biological effect of atmospheric contaminants. It is suggested that the Council for Tobacco Research-U. S. A. will find in this experimental procedure a method which has the potential of allowing quantitative estimates of the biochemical (biological) effects of pollutants and further, of allowing a study of the combined biological effects off pollutants and tobacco smoke. •

See addenda for detailed breakdown for first year cost a. Salaries b. Expendable Suppljes c. Other Expenses d. Permanent Equipment e. Overhead (15% of a, b, c) 7. Anticipated Duration of Work: 3 years 8. Facilities and Staff Available: Total! In addition to the senior staff listed on the cost estimate and the few small items of equipment requested in the budget all the remaining facilities and personnel are available and~ will be assigned as requested. 9. Additional Requirements: 10. Additional Information (Including relation of work to other projects and other sources of support)! The proposed program is designed' to be complimentary to the existent NCI program entitled "Physical & Chemical Properties of Free Radicals and Alkylating Agents in Tobacco Smoke, " which deals explicitly with the effect of tobacco smoke alone. It is anticipated that in the latter phases of the proposed prograrn~ effortss will be devoted to the study of'the synergsstic effects of tobacco smoke coupled with atmospheric pollutants. Together these two programs should provide an excellent experimental approach to the vexing program to the connection between the breathing of contaminants of one form or another and the onset of lung cancers. In order to indicate the scope of our action in~ the approach of electron spin resonance to a variety of chemical and biochemical problems we have appended a partial listing of sponsored programs both completed and in progress. Signature / Directpr of Project Business Officer of the Institution W to

COST ESTIMATE J. R Rowlands, Principle Investigator Research Scientist and Tech. Assistants Material and Supplies and glassware 1500 2,000 . Travel Expenses - Based on 2 Man Trips to New York Transportation 500 Subsistence - Based on 5 Man Days 100 Outside Consultant (Mr. Arthur Gross, 1250 Pico Laboratories) 10 days at $125/day Photography 250 Telephone Tolls and Telegraph 100 Animals and animal supplies 500 Miscellaneous chemicals ` D. Surcharge 11076 Salaries Special Equipment 2,200 20, 878 Miscellaneous optical components for spectroscopic equipment 2, 000 Total Estimated Cost $46, 058 Signature Director of Project 1003546900 Direct Labor Cost First Year C. Other Expenses Business Officer of the Institution

PREVIOUS AND PRESENT' PROGRAMS 4. Sponsor Rome Air Development Center 5: San Antonio R&D Pro- curement, Brooks AFB 6. 7. Corporation "Free Radicals in Paper by Electron Spin Resonance, '' 4-7-58 to 1-7-59. ` "Detection of Radiation Induced Free Radicals by Paramagnetic Resonance, Contract No. DA 19-129-QM-387, 5-18-55 to 10-18-57. "Detection of Radiation Induced Free Radicals by Paramagnetic Resonance, Contract No. 19-129-QM-10b0,. 11-14-57 to 7-13-59. "Investigation of Applications of Magnetic Resonance Absorption Spectroscopy to the Study of the Effects of Microwaves in Biologicali Materials, " Contract No. AF 30-(602)-1843, 5-1-58 to 5-1-59. "The Study of Radiation Induced Free Radicals in Chemical and Biological Systems, " Contract No. AF 41(657)-246, 8-1-58 to 10-29-63. Southwest Research "Basic Research-Free Radicals 'in, Institute Biological Materials," 8-28-57 to 1-31-58. - Thiokol Chemical "Devel~opment of Rapid! Methods and 8. Thiokol Chemical' Corporation Techniques for Analysis of Solid Propellants Based on Nuclear and Electron Paramagnetic Resonance, "' 10-31-57 to 12-8-58. "Electron Spin and Nuclear Spin Resonance Studies of PBAA Binder Systems„" 12-5-58 to 3-20-60. 9. Commercial Sponsor "Application of Magnetic Resonance Methods to the Study of Reaction Mechanisms Involving Free RadYcals, "' 9-1-59 to 12-31-61. 10. Quartermaster Corps "Study of Radiation Induced Reactions in Food Constituents by Resonance Methods," Contract No. DA 19-129-QM-1740, j 12-30-60 to 12-29-62. 1003546901

• San Antonio R&D Pro- curement Office, Brooks Air Force Base, Texas 12. Commercial Sponsor 13. Comrnerical Sponsor 14. Southwest Research Institute 15. Department of Health, Education & W elfare, Public Health Service 16. Brooks Air Force Base, Texas 17. Brooks Air Force Base, Texas 18. Food~ and Drug Administration 19. U. S. Army Dugway Proving Ground 20. Wright-Patterson AFB, Ohio Program "Thq Study of Radiation Induced Free Radicals in Chemical and Biological Systems," Contract No. AF 41(657)- 407, 8-f-51 to 11-30-63. "Application of Magnetic Resonance Methods to the Study of Energy Con- • version Processes of Photosynthesis," 9-1-59 to 12-31-62. "The Mechanisms and Inhibition of Enzyme Action by Electron Spin Resonance," 5-14-62 to 5-13-63. "An Electron Spin Resonance Study of Negative Ions of Porphyrins, 8-24-62 to 12-1-65. "Study of Radiation~ Damage Mechanisms to Biological Materialis,';' 10-1-65 to 9-30-68. "Determination of Free Radical Content in Chemical Systems, " Contract No. AF 41'(609)-2771, 2-15-65 to 3-11-66.. "The Effects of Ionizing Radiation on Oxidation States of Biological Systems," Contract No. AF 41(609)-2816,. 7-1-65 to 7-31-66. "'Investigation of Chemical-Eiectrical Sensing Methods for the Determination of Organ Phosphate Insecticides," Contract No. I?RO-C_. =63--105 (Neg. ) 6-3-63 to 6-2-64. "Design and Development of Electron Capture and Flame Ionization~ Techniques for Tracing Biological Aerosols in the Fie1d, " Contract No. DA 42-00'7-AMC-82(R), 1-29-64 to 7-1-65. "Analysis of Amino Acids by Gas Chroma- tography," Contract No. AF 33(6T5)-1823, 6-10-64 to 6-15-65. 1003546302

22. Southwest Research Institute 6-23-64 to 6-22-66. • 23. Ag riculture U. S. Department of "A Study of the Basic Methods of Action "The Correlation of Acetonitrile in Body Fluids to Tobacco Usage," 4-1-64 to 3-31-65. "Separation and Detection of Nitrogen Compounds of Biological Interest," - of' Several Fungicides Using Radio Frequency Absorption Methods, " Grant No. 12-14-100-802(34), 6-28-65 to 12-28-67. 24. Public Health~ Service "Radiation Damage Mechanisms in = Biological Materials, "' Grant No. RH GM 00384-01, 1-1-65 to 9-30-68. 25. National Institutes of "Physical & Chemical Properties of Health Free Radicals and Alkylating Agents in Tobacco Smoke," PH43-65-100, 6-23-65 to 6-22-66.

i 1003546905

B500 CULEBRA ROAD 5AN ANTONIO,TEXAS 7B2pB Dr. Robert C. Hockett Associate Scientific Director. The Council for Tobacco Research - U. S. A. • 633 Third Avenue New York, New York, 10017 tained in my proposal in which the total smoke condensate was allowed to react with the hemoglobin solution. These results strongly support the viewpoint I have already expressed in my proposal , that a detailed program involving the reactions of the constituents of the gaseous phase with selected enzyme systems be undertaken. I look forward' to hear- .Here are, as promised, the electron spi~n~ resonance spectra of a hemoglobin solution which has been treated with~ the gaseous fraction of cigarette smoke. The spectra are quite similar to the ones con- Physical and Biological Sciences JOHN R. ROW L.ANDS Staff Scientist, Department of enclosures <

.,\\ C I COa1zSE-rTS, SMo; r,aQTc c vL~ iJ1=t c.T ~f A~ ~ ,'z.

, @ - !°WC. S cAO w wTI4 2So &AvSS F~Gcs~~ 2- ESR, or Fic.TErZca c iGAgE; TIF Sszoj~E lZr--AcTP-D 1dlTN ~~I6~Uf31F.3 / D1'~50. FiCST TF-Sr

J SMo N_C-. P-EAeTED . W1Ti-1 h'ceNtGLoE3Jm/Dlqs©.

• ctcbuC~E 4 - ~52 ~t2. f1U~t cr~r~teil /Dt-tso &.TtOL.

30 3S i PIGOPES OcD%r-- I(o; ES(Z SCAA QR Fc LTC-.aED (3iGOSI, S. SA~`,.aCF- P-r-4CTC--D u.)IT'b{ USntGLo3ir-3/brASO.

1003546912

Dr. J. Morrison Brady Associated Scientific Director The Council for Tobacco Research-U. S. A. 633 Third Avenue , New York, New York 10017 Several weeks ago I had a telephone conversation with one of your ~ colleagues whose name I cannot recall. However the result of our :t point which I have overlooked~ or not explained sufficiently lucidly, enclosed document covers the essential points we discussed and that it will meet with your review board's approval. If there is any informa#ion concerning my grant request No. 5-4727. I hope the conversation was that I was asked to submit some supplementary please do not hesitate to write or telephone me for the extra information. Yours sincerely, John R. Rowlands Senior Research Scientist Department of Physical and Biological Sciences N 0 0 cj CA ~ ~ w

ESR STUDIES OF BIOCHEMICAL EFFECTS OF ATMOSPHERIC POLLUTANTS TIM COUNCIL FOR TOBACCO RESEARCH, U. S. A. 633 Third Avenue New York, New York Attention: Dr. J. Morrison Brady Associated Scientific Director

lung into which had been smoked six popular brand cigasettes. A short pre- paramagnetic resonance signal from an.homogcnate of a freshly excised rabbit print of a publication, Appendix A, which outlines these results is included i with this proposal supplement. In our proposal 05-47Z7, I indicated that I had observed an electron In a telephone conversation I was asked if I could give some additional information to strengthen my grant application. cussion will supply the necessary information. I believe the following dis- As indicated in Appendix A, I believe the observed electron para- magnetic resonance pattern is produced by the interaction of free radicals and,(or ~.;.! . reactive diarnaonetic molecules with a metal complex present in the rabbit lungs. To be more specific I believe the bulk of the observed sional is due to the reaction of these free radicals and/or reactive molecules with the haer.zoglobin molecules present in the red blood cells. Such an interaction is represented In the haemoglobin molecule the iron is present as a ferric ionic ~,e3+. The paramagnetic resonance spectra of Fe3~ ionic complexcsA have been investigated by many workers. In particular however, detailed electron spin resonance studies have been performed on four derivatives of the haemoGlobin molecule, in order to discover the detailed form of bonding between the central iron atom and the particular group on the sixth coordination point, the point at which oxygen attachment occurs. 1.003545915 . The tcntativc: explanation of the reported ESR signal from smoked rabbit lung is the attachment of an active molecule (s) from tobacco smoke to the sel£sarne sixth coordination point. On the basis of this hypothesis several

Is this the transport mechanism to the liver which leads 0 the formation of acetonitrile by the liver? . Why does the lung membrane pass these molecules so readily? . What molecules or radicals are thus passed and picked up 'and are these identical to those in smoke? . Is thert a connection between these modified ha emoglobin molecules and the observed vessel constriction in smokers? Since none of these questions-lie within the scope of our present NCI program (primarily because of the unexpected nature of this discovery), TRC support there are several rather direct questions which must first be dealt with; for example: .attempt an exploration of the more complex biological questions raised above, s essential to allow the investigation of any or all of these. Before we . What types of molecules and/or radicals will react to form .haemoglobin complexes such as we have observed? 2. What is the stability of these complexes at physiological conditions ? 3. Are such complexes formed by other atmospheric contaminents;i. e. , smog, automobile exhaust fumes, etc. ?

THEORETICAL CONSIDERATIONS The structure of the central portion of the haemoglobin molecule is represented in Fig. 1 where the haem-porphyrin plane is shown perpen- dicular to the axis joining the iron atom to the globin molecule and the ligand is the relative ease with which the ligand of the sixth coordination point can be changed which makes it 'feasible to study a whole series of derivatives . at the sixth coordination point. The main feature of the hacmoglobin molecule formed by substituting different ligands into this position. Single crystal stud'ies have been made on the acid mathaemoglobin, acid metn-jyoglobin, metmyoglobin fluoride andl metmyoglobin azide. -The extreme g factors for each derivative are listed in Table I. TABLE I , Derivative Group attached gJ` ~r,in Bohr magnetons Acid methaemoglobin H20 - 2.0 6. 0 ' 5.84 Acid metmyoglobin HZO 2.0 6.0 5.85 Metmyoglobin fluoride .F 2.0 6. 0 5.92 Metmyoglobin azic'e N3 2.8 1.70 2.84 The parallel suffix refers to directions parallel to the axis through the iron atom normal to the haem plane and the perpunclicular suffix to directions at right angles to this axis (i. e. , in the hacni planeX It can be seen from Table I- that the four derivatives so far studied are di vided into two classes by both 1003546917

the resonance and susceptibility measurements. As a result of the suscep#i- bility data these two groups were clasbified' as "essentially ionic'"' and '!essentially covalent" corresponding to the cases of a spin quantum number = 5/2 or 1/2. Thus, if ionic binding is assumed, the central positively charged ferric ion will be left with five 3d electrons. These will line up with their spins parallel according to Hund's rule and so half fill each of the five vacant 3d orbitals. A total electronic spin moment of 5/2 is thus formed which will itself be quantized' in any applied field. On the other hand, if covalent bonding takes place six pairs of electrons are required to form with the surrounding ligands. These electrons donated by the surrounding atoms will occupy certain orbitals of the ferric iron, anu it can be shown that the most stable configuration for octahedral bonding is the one in which two 3d orbitals and the one 4s orbital and three 4p orbitals are employed. The five non bonding 3d electrons of the ferric iron are therfore, left with only three 3d orbitals to occupy and hence four are forced to pair leaving one unpaired and a resultant S = 1/2. If this simple interpretation were correct the resonance spectrum obtainc:d from the essentially ionic conzpounds would be expected' to have the same genoral features as that of the other ionic ferric salts. Thus, the total spin and associated magnetic moment corresponding to the configuration with S- 5/2 would take up six different orientations varying from +5/2 to -5/2. Transitions can then be induced between each of these levels according to the selection rulep,S;±li and thus five electronic absorption lines would be expected centered on a g factor of 2. 0. However, the work on single cryEtals of 1003546918

'' acid mEthaemoglobin, metmyoglobin and metmyoglobin fluoride showed that the g factor was in fact, anisotropic varying from a g// of 2. 0 to a g-L of 6. 0, and that there was only one electronic transition observable. . A theoretical explanation of the observed anisotropic values has been provided by assuming that very strong asymmetrical crystalline fields This highly anisotrdpic g factor of the "essentially ionic" haemo- globin derivatives makes the detection of the pararr,agnetic resonance signal are active on the iron atoms so that the energy difference between the six different orientations of the total spin vector of 5/'2 are much larger than of amorphous samples difficult, since the resonance line extends from approximately 1300 gauss.to 3250 gauss. This of course, accounts for the each of any apparent signal from the sample of untreated rabbit lungs. In contrast to the large variation from g = 2 to g = 6 observed for the ionic derivatives the azide derivative which is "essentially covalent" has g, factor spread across the free spin value, indicating an S of 1/2 with considerable orbital interactions. The signal we observe from rabbit lungs which have been treated with tobacco smoke is als o located at approximately the free spin value. Consequently, we have tentatively attributed at least part of our observed signal to "essentially covalent" derivatives of haen.o- globin being formed by reaction of the normal hatrro;lobin with constituents 0 of the tobacco smoke. 1003546919 So far the above discu9aion o the nature of the resonance is hypothesis based upon what is known about iron in other complexes and ha:

_ , . ...:. :. . ,..,,,,;. to be verified.experimentally. F'owever, assuming the hypothesis is correct, such a complex provides a vehicle for carrying the chemicals present in cigarette smoke to the liver'for the production of for example, acetonitrile.

resonance signal is in fact as we have outlined. 1. To establish that the origin of our observed electron spin • PROPOSED PROGRA1v1 . To determine the types of molecules and free radicals that will react with haemoglobin to produce the type of complex we have ob- . served from the reaction of tobacco smoke with haemoglobin. In this in tobacco smoke, smog, and automobile exhaust.- Since all three systems study we will restrict ourselves to compounds which are normally found contain many conUtituents in common, at this stage we will restrict our- selves to.the investigation of representative examples of these common species. is the complex once formed stable or is the decomposition of the complex to reform undamaged haemoglobin:a probable reaction? 4. To determine what effect the formation o. such a comp,ex has 3. Having established the necessary requirements for the pro- duction of such a complex, to investigate the stability of the complex, i. e. , on the general reactivity of the haemoglobin molecule. Since the bonding of the iron atom to both the porphyrin ring and~ the globin molecule changes upon formation of such a complex this is accompanied by a change in electron distribution throughout the whole molecule. Such a change in electron distribution will necessarily cause changes different areas of this complex molecule. ~ 0 in the chemical reactivities of CA FL~ ~ ~ ~"~

SUPNISSIO I OF REPORTS A bi-annual report will be submitted to the Tobacco Research Council throughout the course of this program, with a final report summarizing the entire program. It is anticipated that much of this work will be appropriate for publication in the open Scientific Literature, but prior to publication the Sponsor requested to review the manuscript and give his consent to the publication.

REFERENCES Ingram, Free Radicals as Studied by Electron Spin Resonance, 0 Academic Press, (1958). Swartz, et al, Biochem. and Biophysical Res. Commun., 21:61 (1965).

.Original aigned by prank C, Whitmore Frank C. Whitmore Deputy Director Department of Physical and Biological Sciences Approved: Z%t"1 . A. C. Hulen, Treasurer hn R. Rowland's Senior Research Scientist

1003546925

v THE EFFECTS OF TOBACCO SMOKE ON LUNG TISSUE AS MEASURED BY ELECTRON SPIN RESONANCE John R. Rowlands, David G. Cad'ena, Jr. and Arthur L. Gross* Southwest Research Institute . .. 8500 Culebra Road San Antonio, Texas This work was supported by the National Cancer Institute, National In:,titutes'of Health under Contract No. PI-I43-65-100. *Present Address Pico- Laboratories, Incorporated 8222 Broadway San Antonio, Texas 0

I. INTRODUCTION The presence of free radicals in tobacco smoke has been previously reported. During the course of a program concerned with free radicals ; and alkylating agents in tobacco smoke it was observed that the concen- tration oi free radicals in tobacco smoke condensate varied considerably as a function of methods used for collecting and subsequent treatment with solvents. In order to determine the biological effects of free radicals it became apparent that a method was needed whereby smoke could be directly applied to lunb tissue without first colltcting and processing the condensate. The method reported herein enabled lung tissue to be directly exposed to cigarettc smoke in a manner similar to normal smoking and resulted in the observation of distinct changes in the lung tissue as measured by electron spin resonance. •

II. EXPERIMEN'TAL Large adult rabbits of unkown strain and obtained locally were used. Each animal was sacrificed by cervical dislocation and the lungs and the trachea were excised intact as rapidly as possible. This was accomplished within ten minutes after the animals were sacrificed. The trachea was then attached to the apparatus shown in Figure 1 using thin twine to hold it in place. The smoking apparatus (Figure 1) consists of a bell jar, valving, and a Phipps and Bird smoking machine that was modified to partially exhaust the bell jar, hold it in the exhausted state, and then admit atmospheric air into the jar. The timing of the cycle is controlled by the cam and motor arrangement contained in the smoking machine. The smoking parameters used were: puff frequency ---------------------30 seconds puff duration----------------------- 2 seconds inhalation time------------------- - 4 seconds butt length-- ---- ----------------- 15 mm The puff volume varied with the capacity of the lungs during each experiment. The volume was adjusted so that the lungs would fully inflate without rupturing. During the initial phases of this work several lungs did rupture thereby allowing smoke to enter the bell jar. This was later prevented by - first using the mininial puff volume that would inflate the lungs and step-wise increasing the voiur.-±e until the lungs would inflate with a turgid appearance. King size, non-filtered cigarettes, manu actured in the United States were 10Q3546928

~ ~ CIGARET,T:E: ;~ b h1ORMAL-~ ::: I I ::) NIO~-M?.LLY ~ LY i CLOSE.. ~ ..._.__~t=V E CLOSED c i VALUE _11 ~ i I L , .~~ _ FIGURE 1. SMOKING APPA tiATUS

used. The cigarettes were not preconditioned prior to use.. They were inserted into a holder composed of rubber tubing and were ignited by means of a lit cigarette of the same brand. A total of six cigarettes were smoked into the lungs for each experiment. As soon as the smoking was completed the lungs were removed from the apparatus and homogenized at room temperature for thirty seconds in an ©mnimixer. A cylindrical sample of the homogenate was then prepared and placed into a Varian liquid nitrogen dewar. Electron spin resonance spectra . of the sample were then recorded at 77 0 K using a Varian V 4500 X band spectrometer using 100 mc/sec modulation. A typical spectrum is shown in Figure 2 together with the spectrum obtained from an unsmoked lung using the same instrument parameters, and sample size. The observed spectrum was reasonably reproducible although small changes in the relative magnitudes of the three line pattern superimposed~ on the broad resonance P line were noted from sample to sample indicating that the observed pattern is composite. A considerable amount of additional data is required before • we can hope to arrive at any definite conclusions about the species giving rise to the signal. However, the similar pattern that Swartzob-erved in irradiated blood suggests that the observed spectrum may be produced by the interaction of the free radicals in tobacco smoke with the red blood Q . O cells. This is being further investigated on further lung experiments and ~ on a series of model systerns including metal porphyrin complexes. In ~ Fi;ure 3 we have included the spectrum obtained from freeze dried smoke ~ exposed lung tissue. Figure 3A represents the spectrum obtained at 77 °i{,

B FIGURE 2. (A) ESR'OF LUNG WHICH HAS SMO1'11D SIX CIGARETTES (B) EPR OF CONTROL

and Figure 3B the same sample run at room temperature. The room - temperature signal consists of a single narrow line which is typical of the electron spin resonance signal obtained from tobacco sinoke condensate. On refreezinb to T?°K the electron spin resonance signal observed reverts measurement at low temperature is consistent with it being due to a paramagnetic metal complex resonance, which becomes saturated and to that shown in Figure 3A. The reappearance of the broad signal upon hence not detected at room temperature. Conclusions ~ It is. apparent from this work that the smoking system that is reported has proven to be a reliable method for exposing intact lung tissue to fresh cigarette smoke comparab2e to the manner of' human exposure. The results are not biased by changes in the smoke that occur as a result of trapping and processing. We cannot at this time be certain of the origin of our observed electron spin resonance signal. However, due to the similarity of it to that ob ! served by Swartz upon irradiation of red blood cells we anticipate that both his and our observations may be explained by free radical attack of the haernoglobin molecule at the sixth coordination position leading to a covalent hexacoordinated complex. N ~ ~ W L1"1 ~ ~ • ~ ~ _:.~ ,.:.....: .._ _.~.::.::.,.,4.:..,

FIGURE 3A. EPR OF VACUIUVM L~TF %~-UO_~=JL L"u NG AT 770X . . .. .~.a=.....j . .. . . ..:.. _ . _. . . .. f.S:! .~ .._ . ~ _

[j~Viy~ .,:\ o FIGURF, 3B. EPR OF VACUUM L`RTED LUZG AT ROOM ' Ai'URE .

APPENDIX A. 0

PROPOSED PROGRAM 1. To establish that the origin of our observed electron spin resonance signal' is in fact as we have outlined. selves to the investigation of representative examples of these common contain many constituents in common, at this s.tage we will restrict our- study we will restrict ourselves to compounds which are normally found in tobacco smoke, smog, and automobile exhaust. Since all three systems served from the reaction of tobacco smoke with haemoglobin. In this . To determine the types of molecules and free radicals that will react with haemoglobin to produce the type of complex we have ob- 3. Having established the necessary requirements for the pro- duction of such a complex, to investigate the stability of the complex, i. e. , is the complex once formed stable or is the decomposition of the complex to reform undamaged haemoglobin a probable reaction? 4. To determine what effect the formation of such a complex has on the general reactivity of the haemoglobin molecule. Since the bonding of the iron atom to both the porphyrin ring and the globin molecule changes upon formatio.n of such a complex this is accompanied by a change in electron distribution throughout the whole molecule. Such a change in electron O 0 distribution, will necessarily cause changes in the chemical reactivities of W ~ different areas of this complex molecule. ~ ~ W ~

lung into which had been smoked six popular bra.r,d cigarettes. A short pre- paramagnetic resonance signal from anhomogenate of a freshly excised rabbit • print of a publication,.Appendix A, which outlines these results is included with this proposal supplement. In our proposal #5-4727, I indicated that I had observcd an electron In a telephone conversation I was asked if I could give some additional information to strengthen~ my grant application. I believe the foilowing dis- cussion will supply the necessary information. As indicated in Appendix A, I believe the observed electron para- magnetic resonance pattern is-produced by the interaction of free radicals and/or reactive diamagnetic molecules with a metal complex present in the rabbit lungs. To be more specific I believe the bulk of the observed signal is due to the reaction of these free radicals and/or reactive molecules with the haemoglobin molecules present in the red blood cells. Such an, interaction is represented in Figure 1. In the haemoglobin molecule the iron is present as a ferric ionic 3+ complex i. e. Fe . The paramag.netic resonance spectra of Fe3+ ionic complexes have been investigated by many workers. In particular however, detailed electron spin resonance studies have been performed on four derivatives of the haemoglobin molecule, in order to discover the detailed form of bonding between the central iron atom and the particular group on the sixth coordination point, the point at which oxygen attachment occurs. The tentative explanation of the reported ESR signal from smoked rabbit lung is the attachment of an active molecule (s) from tobacco smoke to the selfsame sixth coordination point. On the basis of this hypothesis several soo3s4ss37

• I %

, extremely important questions arise: 2. Why does the lung membrane pass these molecules so readily? 3. What molecules or radicals are thus passed and picked up 4. Is there a connection between these modified ha emoglobin . molecules and the observed vessel constriction in smokers? Since none of these questions lie within the scope of our present NCI program (primarily because of the unexpected nature of this discovery), TRC support is essential to allow the investigation of any or all of these. Before we attempt an exploration of the more complex biological questions raised above, there are several rather direct questions which must first be dealt with; for example: 1. What types of mol'ecules and/or radicals will react to form haemoglobin complexes such as we have observed'? 2. What is the stability of these complexes at physiological conditions? 3. Are such complexes formed by other atmospheric contaminents;i. e. , smog, automobile exhaust fumes, etc. ? P

dicular to the axis joining the iron atom to the globin molecule and the ligand is represented in Fig. 1 where the haem-porphyrin plane is shown perpen- at the sixth coordinatio.n point. The main feature of the haemoglobin molecule is the relative ease with which the ligand of the sixth~ coordination point can be changed which makes it feasible to study a whole s ries of derivatives formed by substituting different ligands into this position. THEORETICAL CONSIDERATIONS The structure of the central portion of the haemoglobin molecule Single crystal studies-have been made on the acid methaemoglobin, acid metmyoglobin, metmyoglobin fluoride and metmyoglobin azide. The extreme g factors for each derivative are listed.in Table I. TABLE I • Derivative Group attached . gCl µ . in Bohr magnetons Acid methaemoglobin . H2O 2.0 6. 0 5.84 - _ ~ Acid metmyoglobin H20 2.0 6. 0 5.85 O Metmyoglobin fluoride F 2.0 6. 0 5.92 CJ ~ Metmyoglobin azide N 2.8 1.70 2.84 ~ 3 ~ ~ The parallel suffix refers to directions parallel to the axis through the iron atorn~ normal to the haem plane and the perpendicular suffix to directions at right angles to this axis (i. e. , in the haem plane). It can be seen from Table I that the four derivatives so far studied are divided into two classes by both

the resonance a.nd susceptibility,measureme.nts. As a result of the suscepti- . bility data these two groups were classified as "essentially ionic" and "essentially covalent" corresponding to the cases of a spin quantum number = 5/2 or 1/2. Thus, _if ionic binding is assumed, the central positively charged ferric ion will be left with five 3d electrons. These will line up with their spins parallel according*to Hund's rule and so half fill each of the five vacant 3d orbitals. A total electronic spi.n moment of 5/2 is thus formed which will itself be quantized in any applied field. On the other hand, if covalent bonding takes place six pairs of electrons are required to form with the surrounding ligands. These electrons donated by the surrounding atoms will occupy certain orbitals of the ferric iron, and it can be shown that the most stable configuration for octahedral bonding is the one in which two 3d orbitals and the one 4s orbital and three 4p orbitals are employed. The five non bonding 3d electrons of the ferric iron are therfore, left with only three 3d orbitals to occupy and hence four are forced to pair leavi.ng one unpaired and a resultant S= 1/2. If this simple interpretation were correct the resonance spectrum obtained from the essentially ionic compounds woul& be expected to have the same general features as that of the other ionic ferric salts. Thus, the total spin and associated magnetic moment corresponding to the configuration with S = 5/2 would~ take up six different orientations varying from +5/2 to -5/2. Transitions can then be induced between each of these levels according to the selection rule NS=±1 and thus five electronic absorption lines would be expected centered on a g factor of 2. 0. However, the work on single crystals of 1003546941

acid methaemoglobin, metmyoglobin and metrnyoglobin fluoride showed that the g factor was in fact, anisotropic varying from a gf/ of 2. 0 to a - g j. of 6. 0, and that there was only one electronic transition observable. A theoretical explanation of the observed anisotropic values has been : provided by assuming that very strong asymmetrical crystalline fields different orientations of the total spin vector of 5/2 are much larger than are active on the iron atoms so that the energy difference between the six the microwave quanta. This highly anisotropic g factor of the "essentially ionic'"' haemo- 0 globin derivatives xnake s the detection of the paramagnetic resonance signal of amorphous samples difficult, since the resonance line extends from approximately 1300 gauss to 3250 gauss. This of course, accounts for the each of any apparent signal from the sample of untreated rabbit lu.ngs. In contrast to the large variation from g = 2 to g = 6 observed for the ionic derivatives the azide derivative which is "essentially covalent'T' has a g factor spread across the free spin value, indicating an S of 1/2 with considerable orbital interactions. The signal we observe from rabbit lungs which have been treated~ with tobacco smoke is also located~ at approximately the free spin value. Consequently, we have tentatively attributed at least part of our observed signal to "essentially covalent"' derivatives of haemo- globin being formed by reactio.n of the normal haemoglobin with constituents of the tobacco smoke. So far the above discussion of the nature of the resonance is hypothesis based upon what is known about iron in other complexes and has 1003546942

to be verified experimentally. However, assuming the hypothesis is correct, such a complex provides a vehicle for carrying the chemicals present in heW ~ crn~OJ'n3s. cigarette smoke to the liver for the production of fo-r_e-ce:ntpl , le. •

SUBMISSION! OF REPORTS A bi-annual report will be submitted' to the Tobacco Research Council throughout the course of this program, with a final report summarizing the entire program. It is anticipated that much of this work will be appropriate for publication in the open Scientific Literature, but prior to publication the Sponsor requested to review the manuscript and give his consent to the publication. 41

REFERENCES Ingram, Free Radicals as Studied by Electron Spin Resonance, Academic Press, (1958). Swartz, et a1 , Biochem. and Biophysical Res. Commun. 21:61 (1965).

rank C. Whitmore Deputy Director Department of Physical and Biological Sciences e Approved: Approved: A. C. Hulen, Treasurer ._ J6hn R. Rowla.nds Senior Research Scientist

THE ci Cov.NLciL FOR ToBACCO RESr',ARCR - U. S. A. The committee comprising Dr. Cattell, Chm., Dr. Little and Dr. Bing. Robert C. Hockett SUBJECT: New grant application from Irving Geller, F'h.D. - No. 622. We enclose herewith a new grant application from Irving Geller, Ph.D. of Southwest Foundation for Research and Education, San Antonio, Texas. The first portion has been already answered by Bonet et al (ref. to Morrison and Armitage, Effects of Nicotine on Behavior, N.Y. Acad. Sci. 142, 268 (1967) who observed that nicotine in moderate dosage facilitated the learning of the task and, under certain conditions, improved the performance of the rats. The second section of this proposal would test the tranquilizing effect of nicotine, and whether nicotine dosage can be shown to allow an animal to over- come a negative (shock) stimulous in order to obtain food. Comparison should be made to the application of Essman, #623. Robert C. Hockett John H. Kreisher ::~.~.... ,..._

• sysot pau6!sso 041 jo uol;!s!nboo 4t!M pa;otoosso /4a!xuo ~0 6uuoMOl o o; pa{nq!uto aq A!q0ntaouoo plnoo au!;o?!u aapun 6u!ujoal p!doj ajoW •slow!uo AJoaaJoqoi y1lMIuol;ont!s 6u!uJoal ow! sisay;odA y s!y1 }sa; o; s! qojoasaj 's!y+ f o;ua;u!ayj ;;(,l)1aa~ia6u!xo!ai;puo6ulz!i!nbuoj;'ti!JUOU!wopaia~ eu:oa,ouawaos{augoP~g'L 6u!4ows ;oy} aq W sioaddo sjoytno wapow fiuowo snsuasuoo y„ 4 H I M,nodd ~ (;aays payae;}o uo uo!}onu!;uo:)) •aauowio}jad auyi-asoq o{ wnlai .Goiodwaa o 6u!MOlioj puo•uo!}oalu! 6njp ay} ja!jo sAop aaJya pannmoo s{oajja paAoiap paounouajd-{nA 'XI}46!Is Jo!noyaq 6u!w!} ay pa;drus!p au!;oo!u jo suo!;odlul •{Jodo spuooas,ZZ ol OZ paaods sasuodsaj ianal asoy; poo) y;!en;6u!pjoMaj .(q.Aias!oajd aw!; o} paula14 ajam s}oi Aj6uny '{uawuadxa s!yy ul •a,{oj;jo}!q au,y _oo!u f o suo!;oaiu! I!ows Aq paoRpaed -1o!noyafl uo;oajia paAolap o pa;uodailoyn+ (6) san6oalloo,puo sqto6!;sanu! s!N; Lq uoyoo!Iqnd snoinajd o u! pa;{!!FJwaxa s! 1o!noya5{ uoi;ou!w;los!p,{o ad.(t y -sloW!uo .(q saIlo;JadaJ !oJo!noyaq pJop ' s~oa~ aaiy j 4p"!S?BpadS qyy;a uo!{ojnd patodo!tuy •q a ~ } ! los walqo~d jo w6!po~od iD1uaWiadxa uo ol w j j0 6u!woai '6u!n ~ yn ns {sa; oj (l,) slow!uo puo~uow u! i}oaJia 6u!z;l!ribuo~} .CI{uou!wopaid o}jaxa ,(ow au!a ;® o V {oy; uo!aou aE{+,wwj swa}s losodosd yoJcasaj s!y1 ~o} ssoq ayj swryo!}uadgnsan!wtqp}ouotduxa au ; ({toodoo wnw!xow 40 wioij ad uoo ws!uu Jo s l • I d ~ssaoau o{snw auo 'stsay;odAy ! _aui;ooiu }oyt ;sa66ns }lo!ym o}op ~ou!wllai awos p I lonsin ogo 6u!woal ay; a}oi;snil! II{!M losodo~d s!yt u! paquosap aq o} s;uawuadxa ayj ~~(}!I!qo Pt'o-,;_ : pazyltnbuoa;„ ay} ajayrn uo!}on}!s awos 6 notxuo ssa Jo -ou!w!vos!p aqa JO uo!{!s!nboo p!do' ajow aauay p -- _1aMOI o}oalia, qyB!w sainsa~ yong •slow!uo ia1}uoo uo4} 34}}aq nso1 944 uioal ~(ow sIow!uo pa;oalu; _- d uo s owluo .(jo;ojofloi .Gj6uny ut ua;oulwuos!p uo so{ 6utwoal ayI yt/m peIo!Dosso /4a!xuo jo 6ui ay} JO Ma!naJ an!suayajdwo:) y'(9 9 q E Z) i • jos}oaj3 a ay{ }i1ln~ 6u!!oap y7joasaj •jo ~!onod o sloana~ ain{oja}!1 ay{ )o ena!nas an!sua;xa vy ` . •uo!; ,. a L p opojolo7 u! 6u!{aaw pasoio'o 40 pa{uasald'1}lo al fo s, V 1 1 4 1 y owwo UI suo+}on}ts ssa~;s j0 6ujujoai uo au!}oolu 4oyM puo pa~oo!sano aq 04 pua; au!}oo(u ~ ~ u! aouowiofsad uo au!;oo!u jo s;oa3}a a4j y4!^'~ }I°ap 47!4"` suo!{o6!;sanu!'n~a) o papnpu! (9) s6u!jds :' aysilqnd uaaq soy sa!pr4s 6ulls!xa I Jas -,( d ) _uojs 30 aouowiojjad ay; uo s{oa{pjoi s{uoInwi;s ua+ou'11y3!M aui;oo!u ajodwoo :CI!onsn sa!prys yons Ut panlonu! sJo{D6!{sanul •au!}ooiu JO sat{iadoid;uolnw!;s ay} 1o uo!}oj}suowap o st s}lnsa~ ~(Ilonsn o suot{oo apqns 'satpn{s Nons ul 'SIDWIUD I~1Q}O1O9DI L961 jaqwa}das L:otoQ bulpetS pasodoid Y' 90Z8L soxaj 'o!uo}uy uos : . (}aaty$ aojawwo:) }saM 00001) 96ZZ xo8 • O• d . uo!}oonP3 puo ya~oasaa joj uo!}qpunoj }saMy{noS • ,nwppy • ....F;.;.••~.~:...: = (saa,apnpue):(t)Joto6asaAU!;oowoNl 6aQPuoe!4~ . , ,.. eJ;6u!n~l •d•ya r~aiia _ _ _ .. . _.. .. __ ....... .. _ _..- . .._ ... _~i'i'L•f'-~•. ~~ slow!uy,Go;oJoqo1 u! 6u!uwoal UO au!}oo!N 3 os}aaJi3 :palojd~ap!1Po4S 6 ^ puo .(6olooowioyd Ijo {uaw}jodaQ tuow;!oyj `' .. ' . xo ' , j oo C6o ~ ! ~ l

• Objectives and, Specific Aims (Continued) It was conjectured that these delayed changes in behavior following nicotine injections could .; -conceivably be related to epinephrine output. Si.l'vette et al., in their extensive review (1), found ;~ ''':'::it".difficult to ascribe the above reported effects to any pharmacolocrical action of a sina(e iniection of nicotine three days previously. A more likely conclusion would seem to be that the animals werey ~ ::effected by some other part of the experimental procedure rather than the nicotine per se." :=1t is of interest to note that a later study (10) reported'an increase in excretion of urinary epinephrine produced by chronic injections of nicotine. The excretion reached a peak after three ;.,.days of treatment. . The intent of the proposed research is to measure rate of acquisition of discrimination behavior '_.i'n laboratory animals who are administered nicotine chronically during acquisition training. 0 ~ OO W CJ1 ~ CA 0

s ~ @t I y ~ 9t 4 ~t Ae =:' tt R ^l' f:l 8f L 1,L SL • s t ~ -~- - - t M - - - - - - - - - - - - fi -- - - - - - - - - - - - - a - ;_ _ - - _ -- - ~ ---- -- --- -- -- _ - -- - - - r - - ' - - - - - - - - - - - - - -- - - - - - - -- - _ ~ - _ -- - - - -- - ~ - -- -- ~ - " - i. , - - - - - - _ ~ I ' - - F _ ~- - - -~ -~ ~ - - -" - - -- - - -- - - - I 'Y'@'fl NI 30VW . OOSL•L 03 '@ 'A'N 'SOY@ N11Wl0100 40011 : HONI % IHl OL 0 r @ ~ , .

food rewards, the expertmental condt trons wi 11 be as fol ows: ran om presentahons O tg t s tmu F', =tion of nicotine on the learning of an auditory discrimination 8ased;on punishmenti("conflict"). ~ ,.,. s'iVi- .% ~ PrnCrdurP A_ Hunarv animals wilN feam,to aress a lever in order to obtoin a Iiauid or solid the general tQcEltt/eS at tne ~outnwest tounaarlon ror tcesearcn ano cuucanon ure ovunuuie I arthis project. These include adequate office, laboratoryy and onimaf room space. C] 1. Biographical sketches of all principal and professional peraonnel (append). L ~,food reward'. Once the animals learn that depressing the lever is associated with the delivery at I d fl'h t' Ii ype}c61~4^: . '.v.,c:.:~:~.::: ~ ~{, _ p.xpenmenta(Des:gncndProcedures:(Attach5eparatePages) The subjectswill be white rats, squirrel ij.ADetailsof "-monkeys and cynomologous monkeys. They wil1i be gradually reduced to 80a/o of tl.eir body weight x and maintained at this weight by limited feedings throughout the course of the experiment. The r, apparatus will consist of two operant conditioning chambers fon rats, conditioning chambers for • A.;squirrel.monkeys and two Foringer monkey chairs. '. ' . . Sg*`; y~s:-Two experimental procedures wilt be employed. Procedure A will involve the effects ot ~."t.zchro nic administration of nicotine on the learning of a visuahor auditory discrimination based:on ~}.•positive reinforcement (food reward). Procedure B will involVe the effects of chronic administra- the .. erimental chambers will serve as a signol that a lever response will be incorrect and will the ex p a tone stimulus occom- If howevea cond eriod of darkness for the animals nished b a 30 s `u'p~'~b . , p y e pu e •-3~;r panies the presentation of the tight stimulus, a lever response wi!l be correct' and the hungry animal will be reinforced with- food, For some animals light alone wi(b indicate an incorrect response, F••while light and'tone will indicate a correct response. For another group ot animals tone alone will ...:.: tndtcate an tncorrect response, wHtte tone pius•Itght will tndtcate a correct response. ( ee ttac ment) 9 Physical Focilties Available (Where.Other fNan Adminut0ring Organizotion Jndicate Geographical Location) 12. Listofpubllcations:(Fivemostrocentaspertinent){append) Attached Attached

.shown efficiency or how well the animal . is* performing. TheRse points are determined by taking"' -AIl experimental animals will receive chronic administrations of nicotine during acquisition :,'training, while control animals wil(: receive saline. ~.. . I the ratio of right responses divided by right and wrong or -RTy- = efficiency. bf the animal is making an equal number of right and wrong responses, he is not discriminating and the point would 2C •~~ - :. fall at 0.50. If the animal's wrong responses exceed the right, responses, the point would fall below_ :;T 0.50. Points lying above .50 indicate that right responses exceed wrong responses and: thal" the ~_~a, .:: animal is acquiring the discrimination. The solid red line in the figure representing data for the li i l d d i f h h l h h : ne an ma an t e sa iscr mination aster t - . is anima acquire t e nicotine rat shows that t ; When the performance of the discrimination began to drop, on the 20 to 23 day, the nicotine dose .~'n` . Experimental, Design and Procedures (Continued) • : Treatment of Data: In Figure 1 are preliminary data for two of four experimental rats which illustrate nicotine effects on the acquisition of such a. discrimination. On the ordinate'is ' m orm e n e g n was mcrease rom . o. mg . g an e per e i p ~_'S a ' airn b came su erior to ~" al a a c of th d f 0 T t 25 /k d th f ; that of the sal ine rat. d d' i i i l i h di i i i h h i on more ne an ma o acqu m nat e propose exper ment, t e n cot s re t e scr If, in t it may be of interest to determine whafi effect the discontinuance rapidly than the saline controls , of nicotine injections will have on the performance of the discrimination. Possible Results: T. Nicotine treated animals will acquire the discrimination more rapidly than saline controls. 2. Saline controls will acquire the behavior more rapidly than nicotine animals. . There will be no significant differences bet~ • en nicotine and saline animals in the rate of acquisition of the discrimination. • Procedure B: This procedure, developed by this- investigator and colleagues in 1957, is ..currently used extensively in the pharmaceutical industry for the pre-clinical evaluation of the minor tranquilizers (anti-anxiety agents). The behavior involves the learning of an auditory dis- crimi'nation (or visual) based on punishment. The auditory discrimination is condilioned in hungry laboratory animals by simultaneously rewarding! with food and punishing wi.th- mild electric shock all lever responses made in the presence of a discriminative stimulus (tone or light). Appropriate setting of the shock intensity results in suppression of ongoing lever pressing, in the presence of the discriminative stimulus. The intent of this experiment is to investigate the rate of acquisi- tion of such a discrimination in animals administered nicotine chronically. The hungry laboratory animals first learrn to press a lever irrorder to obtain food rewards which are obtainable on the average of once every two, minutes (2 minute VI). When lever pressing rates have stabilized a tone or light stimulus of 3 minute duration is introduced at regular intervals during the lever-pressing session. This stimulus serves as a signal for the animal that all lever responses will be reinforced with food. The discriminative stimulus signals a change from a reLatively undesirable schedule ofi reinforcement (2 minute VI) to a schedule with a higher '"pay-off" of reinforcements (continuous reinforcement). When the behavior has stabilized, a. " 1003546953

~8. Experimental Design and Procedures (Continued) punishment contingency is added so• that, during• the discriminative stimulus,lever pressing is 's:"~; t~.= .d rewarded with food but also produces mild shock to the feet of the animal . The hungry animal must balance the positive aspects.of obtaining food against the negative aspects of accepting ;~r;~electric shocks in order to obtain the food. After a period of time these experimental contin- • s ' ~;;gencies result in a suppression of lever-pressing behavior during stimulus periods. Anti-anxiety raining and•will later be compared with saline controls. . tion in laboratory animals. Animals will be administered nicotine chronically during acquisition .~ • This investigation wil.lt test the effects of nicotine on the acquisition of such a discrimina- agents will reinstate lever pressing behavior that has been suppressed by punishment (11,12,13). ` Possible Results: . Nicotine animals will acquire the punishment discrimination•more rapidly than saline controls. • Saline animabs will acquire the punishment discrimination more rapidly than nicotine animals. • There will be no significant difference between saline and;nicotine animals in acquisition of the punishment discrimination. :....The data from these experiments will show. whether nicotine treated animals will acquire - a discrimination more rapidly than saline controls. They will also show if the findings are similar (d inf-mation nishment riminati ns ba d n ositi d a ll as will fo dis Th i ' p pu . y e r c o se o ve rewar s we ey . - with regard to reproducibiliity of the phenomenon between species. Findings may or may not be :_i_qualitatively similar for rats, squirrel monkeys, and old world monkeys.

REFERENCES. Med. Monthly, 85, 472-484, 1958. Silvette, H., Larson, P. S. and Haag, H. B.: Medical uses of tobacco past and presen Smith, C. S., Rosenfeld, S. and'Sacks, L. J.: Study of the effect of nicotinism in the ` ~ albino rat J Pharmacol . 55 274-287 1935. Kuschinsky, G. and Hotovy, R.: Uber die zentral erregendeWirkung des nicotins. Klin Wschr. 22, 649-650, 1943. . Heimstra, H. W., Grodsky, M. A. and Davis, R. T.: Sanddigging behavior of rats. Proc. S. Dak. Acad. Sci. 34, 96-102, 1955. Cervalo, B. A. and Cheskey, C.: Effects of nicotine sulfate injections on sand digging behavior of rats. Proc. S. Dak. Acad~. Sci. 36, 222-225, 1957. . Bonta, I.L.; Delver, A., Simons, L. and De Vos, C. J.: A newly developed motility apparatus and its applicability in two pharmacological designs. Arch. tnt. Pharmacodyn.. ,:129, 381-394, 1960. central nervous system functions, Pharm. Rev. 14, 137-173, 1962. . Silvette, H., Hoff, E. C., Larson, P. S. and Haag, H. B.: The actions of nicotine on Research Conference and Workshop on Nicotine. Sponsored by AMA and held in Colorado Springs, November 1-3, 1966. . Geller, I., De Marco, A. 0.. and Seifter, J.: Delayed effects of nicotine on timing -behavior in the rat. Science. 131: 1960 ., 10. Westfall, T. C. and Watts, D. T.: The effect.of nicotine on amines of brain and urine in 'the rat. J. of Neurochem. 11, 397-402, 1964. 11. Geller, I. and Seifter, J.: Effects of ineprobamate, barbiturates, d-amphetamine an.d promazine on experimentally induced conflict in the rat. Psychopharmacologia. I, 1960. 12. Geller, I. and Seifter, J.: The effects of monourethanes, diurethanes and barbiturates on a punishment; discrimination. J. Pharmacol . Exptl. Therapeutics. 136, No. 3, 1962. 13. Gell.er, I.: Use of approach avoidance ESehavior (conflict) for evaluating depressant drugs. Chapter 33, in Nodine (ed.), Psychosomatic Medicine, The First Hahnemann Symposium, Lea and Febiger: Philadelphia; 1962. Is 1UU3546955

R: REDACTED MATERIAL . Date. of Birth: •~` Educa"tion: Institution CURRICULUM V'ITAE Irving Geller, Ph. D. : George Washington Univ. B. A. 1949 Pre-med. ... George Washington Univ. M. A. _ 1951 Psychology American University Ph. D. 1957 Psychology ;.Present Position (s): '.Chairrnan- of the Department of Pharmacology, Division of Biological Growth and ~Developrnent, Southwest Foundation for Research and Education. August 1966 -. revious Positions: iation Psychologist - Naval Res. Lab. , Washington, D. C. ; Forbes Air Force Base, : Topeka, Kansas, March 1951-November 1951. 'Research Psychol'ogist - VIWalter Reed Army Inst. of Research, Washington, D. C. .<.::1952-1957. =Ser.ior Research Scientist - Wyeth Labs. Inc. , Radnor, Penna. 1 957'-1964. aAssociate Research Professor of Pharmacology - New York Med;ical College, 1964 - - 1 966. ~:. `_.•=Professiona.l Societies: :: American Psychologi-ca=1 Association "'' h~ew York A'cademy of Science . .r_: ... ~ -:.:Psychonomic Society -'Sigma Xi " American Society for Pharmacology and EXperi•mental Therapeutics.

PUBLICATIONS lrving Geller, Ph. D. ;I . Geller, Irving, M. Sidman and J.:.V. Brady, "The Effects of Electroconvulsive Shock on a Conditioned. EmotionaG Response: A Control for Acquisition, R'ecency, ° J. Comparative and:Physiol. Psychol., Vol,. 48, Nb. 2, 130-131 (1955). . Geller', Irving, and J. Seifter, "The Effects of Promazine and Phenergan on Mulltipfe ' Sched'ule Reinforcement Performance in the Albino Rat," Fed. Proc., Vol. 18, (1959). ,• 3. Geller, lrvin.g, A. O. DeMarco and.J. Seifter, "Delayed Effects of Nicotine on TWing Behavior in the Raty" Science, Vol. 131, No. 3402, 735-737(1960)'. 4. Geller, Irving, and Joseph Seifter, "The Effects of Meprobamate, Barbiturates, d-Amphetamine and Promazine on Experimentally Induced Conflict in the Rat," Psycho- . pharmacologia, 1, 482=492, (1960). --5. Geller, Irving, "The Acquisition tand Extinction of Conditioned Suppression as a Function ; : of the Base-Line Reinforcer," J. Expmntl. Anal. Behavior, Vol. 3, N'o. 3, 235-240 (1960)'. 6. -Geller, Irving, "Behavioral Procedures Used' in Evaluation of the Psychopharmacollogical! Effects of Carphenazine," Diseases of the Nervous System, Supplement, Vol. XXII, No. 2 7. Geiler, Irving, and•J . V. Brady, "Effect of Electroconvulsive Shock on an Extinguished ."Fear" Response," Science, Vol.. 133, No. 3458, 1080-1081 (1961):. 8. Ge ller, Irving, "Use of Approach Avoidance Behavior (Conflict) for Evaluating Depressant Drugs," 1st Hahnemann,Symp. on, Psychosomatic Medicine, Chap. 33, 267-274 (1962). 9. Geller, Irving, J. T. Kulak, Jr. and J. Seifter, "The Effects of Chlordiazepoxide and 7:Chlorpromazine on a Punishment Discrimination," Psychopharmacologia, 3, 374-385, (1962). '10. Geller, Irving, "Experimentally (nduced Conflict for Evaluation of Psychopharmacologic Agents" (A Scientific Exhibit~) (1962)•. 1•1 . Geller, Irving, E. Bachman and J. Seifter, "Effects of Reser.pine and'Morphine on Behavior Suppressed by Punishment," Life Sciences, No. 4, 226-2311 (1963). 12. Geller, Irving and'J. Seifter, "The Effects of Mono-Urethans, Di-Urethans and B'arbiturates on a Punishment Discrimination," J. Pharm!. and Exptl . Therapeutics, Vol. 136, No. 3, 284-288 (1962) (one copy only). 13. 'GeII'er, Irving, "Conriitioned "Anxiety" and: Punishment EfEects on Operant Behavior of Goldf ish (Carassius auratus)." Science, 141, 3578, 351-353 (1.963),. 0 1003 546957.

.0 )Ji •? . ~ . ... .. .. _ „ '...'_. . .cl~r.:'~_. a:r. " ^4h yi 14. DelYlarco, A. O. and (. Geller, "Effects of Acceleration Forces on Timin( Bzhavior in the White Ratt," Aerospace Medicine, Vol. 35, No. 1, 30-32 (1964). .. ~ ~,: , . . . . ' 15. Geiler, Irving, "Relotive Potencies of Benzodiazepines as Measured by Their Effects on ConflIctBehavior, Arch. cnt. Phanmacodyn, 149 , No. 1-2, 243-247(1964). . . . . .,..:. ::~ ;;:16. Geller, Irving, "Conditioned Suppressiora im Goldfish as a Function of Shoclc-Reinforcemen z~ Schedul+eJ. Experimental Analysis of Behavior, Vol . 7, No. 5, 345-349 (1964). y: . Geller, Irving, "Psychopharmacology of tybamate," Journal of Psychopharmocology, ~~ ~ VoL. 1, No. 2; 47-55.(1966). ;. ...:..:*i..a~.-...~:...d..~.~ ...w....~......... -.~...,...........~.s... .._ _.,...~a._..-..... ~ .w n ... _..,. .._ _.n... _a... ....L•,~.+.~c:.-,..~na~J'.r.+c'-`- :.' -- - '~ ,~y,~-SL°-- .....

R: REDACTED MATERIAL Operant conditioning chambers for squirrel monkeys (we have) .:.~` . _ . R. Consumable Supplies (Ust by categories) 100% 100%• Sub-Total . 2. Recorder paper, ink, pens, electrical supplies and : • Animals - Rots and Monkeys . Animal food - wood shavings - -• syringes - vitamins - food reinforcers. replacement parts C. Other Expenses (itemize) Travel - visit to scientific meetings Publicatiomcosts - illustrations, slides, etc. D. Pertnanent Equipment (itemlze) . Cages for. housing squirrel monkeys and'rats - includes devices for watering and feeding. Sub•Totaf Operant• conditioning chambers for• rats • - Behavioral Control equipment and record'ers (see attachment) E. Overhead (15% of A-fi 6-H C). Salaries Consumable Suppl. $2000.00_ $2300.00 Other Expenses PermonentEqbip. Overhead Totai $1200.00 $1500.00 $2438.00 $20,189.00 $1000.00 $1000.00 0 $2535.00 P, 435.00 It is understood that the applicant and institutienaf officors in applying for a grant have read and found acceptable the Council's "Stotement of Policy Containing Conditions Sipnatur and Terms Under Which Project Grants Are Made." Iwi..u o/Ik.r a! n.. lnstilwion EDWARD F. FEITH TAEASURER elhphone i .c.c('o~cStL cr,y.~yro Telephone `_

Behavioral ControL Equipment and: Recorders 3 6 6 1 6 6 4 3 4 6 6 6 3 11 9 3 0 Cumulative Recorders 4027 JM Reg.. Power Supply 4005 J Timer 4005 JM, Timer 4013 J Pulse Generator 4069 J Dual Pulse Generators 4001 J and Gate 4035 J Dual and Gate 4043 J Dual Exc. OR Gate 4011 J Diode Panels 4024 J Flip Flop R/S Binary 4072 J Basic Timer 4020 J Probability Generator 4023 J Delay Generator - 4018 J Cradle Relay 4066 J Binary Relay (Alt.) 4068 J Session Timers 4028 J Pre Deter Counters 4010'J Reed Logic Relays 4025 J Noise Generator , GRAN.D TOTAL ~ 450.00 203.50 156.25 193•.75 51.25 60.00 43.75 43.75 44.45 30.00 56.25 101.25 124.85 58.75 31.00 65.00 64.50 248.00 25.00 139.50 ~ 1350.00 610.50 • '468.75 581.25 ' . . 307.50 180.00 262.50 262.50 44.45 180.00 337.50 405.00 374.55 235.00 186.00 390.00 387.00 744.00 275.00 418.50 $ 8000.00

I Ust1(nanaial svpporf for rsoon:h from al1 sourea, Including own In1lFatlon, for this and/or related resmarch proiecb, • 1, Titl* of Project Experimentai Studies in Psychophannacology ~9~9i~SC00~ ~

4~% I , : yh ~ ~ ~.Srl ~'V~Y~ ~h ~`J ~y~ ~ti~.~~' ,.. \... 1 ,~ ' _ _ ..`1~~.~ ~'¢~.••~• ~~ ~tiii~R4 +;S7f S'

HE CpUNCIL FOR TOBACCO RESEARCFI U S A . . , . ,1

Studies of Nicotine Aetion upon Memory Consolidation 01.1 •.' A. propo:ed 5tarting Dbte~ Oatober l, lyb (;•=,,: _. ~, .- :. b. Mticipated Duration of this Specific Study: 11ia years. C 6. Brief Desuipton of ObjectivesorSpecificAimu ?.` The rpajor objective of this proposed research is to assess the possible role of =`-•' - aicotine in determining the tenporal character of memory conso lidation in experimaita7. '; animalls. The proposed e-Veriments represent an attempt to relate the effects of nicotine :=centrnl avents_ _ SnracifSeallv_ the effect of nicotine on lvain aminP coneentration and ->+ "Yn the central nervous system to the behavioral processes which are dependent upon sueh " s Y P Y s~,_. P s of msno consolidation (the rocess b tumover -Ul 5e' which the r5~ zelated, Yo the m ce r basis upon-whieh memory consolidation,may be facilitated. The parameters to be con- - ;idgred in th~,~ronosed studies are nicotine d~sage and the intensity of the amnesic event which, for the present experiments, will be electroconvulsive shock. A one-trial conditioning technique will be utilized to establish a passive avoidance response, the ,retention of which will be tested, based upon the interaction of the effects of nicotine : on the central nervous system and the effects of electnoconvulsive shock both on the -.,central nervous system as well as an 2rcnesic event specific to the behavior conrerned ~,..grop.osed- studies. : ~rax~ amzne levels, specifi.cally aerotonin, the studies proposed herein are based upon t?*~tf~e+assqmptian that nicotine-induced changes in brain anine levels will eonsti.tute the z~r~~a~y~~Eiais.a~ay be shortened or lengthenedy dependirg upon.changes brought about ia ; " "' previous work in this laboratory has demonstrated that the tenporal course for mmtory , fined in terms of tha time interval following learning within which. memory for an acquired :..„;.~ c.-event may be dLsnuuted by ttie introduction of external agents attd/or events) . Since the t. {, ~~menory trace• oecomes zixatea~ in tne central nervous systemj tnls may De emp2ri.caliy ae- -~10-= 7 Giveo6riefSlatementofyourWorking,Nypomhesis: Memory cons,lidation, as deflned• by the post-conditioning time int'erval, within which retention of a conditioned response may be, disrupted by electro- convulsive shock will be facilitated through. the prior administration of nicotine. Such facilitation wiS1 be defined by a reduction in the time interval fbllowing learning, wterein. eltctroconvulsive shock results in a subsequent retrograde annesia for the corziiti.oned behavior. _

C ~1. 6ioprophical sketches of all prineipa[ and professional personnel(append) See attached page 10. 12. List of puhlications: (Five most recent as pertinent) (append) See attached page 11,

R: REDACTED MATERIAL ~ _ $2s50© $500 $7.,800 $13,80o Y.ar3 Slgnature.cl Itisunderstoodthat theapplicant.andinstitutionol officers otr.aorof Proi.cr in applying!for a grant hove read'andYoand acceptable Telephone the Councilk'"Stetement of Poliry Containing Conditions Signature r. . Mj 6-(966 andTerms Under Which Rrajpct Grants Are Made." wv..r. otr<a.tah. t.utiwb. ! - I Telephone FiL 5-7500 Ext.202 .

Detaf 1s of Experimental Design and Procedures: ' i ;Y ay { ._a* J _' ~"`All experiments will be based upon the use of a reliable technique which pro-~ vides for the establishment of a condit3•oned passive awidance response in mice, E;utilizing a single training trial (Essman & Alpern' 1965). The apparatus basically r•-' •~;;~consi sted of a small transparent lucite chamber f5 xed to a hole opening into a:,:;~ ~`arger.opaque chamber. The floor of the op.e ue chamber consisted of stainless sfi~eel aq -'~'•~grids which were wired in series through a cam-operated grid scrambler to a 95 volt °:power supply. Completion of the circuit by ~ anuaal stepping •firom,.the outer ch~amber~ ~ ~ :into the inner one, through any 2 adjacent grids? ~ovided for a 3.2 ma, shock '::,; 'i flelivered' thro u~ the pa~rs of the ani.mal for 1 sec. The interval between placement ~. . ~~: of the animal in the outer chanber and its entry into the larger ad3acent chambe r ~ ~ : was ne asured' (response latency}'-_ : w : .. ; r.. . .. ..,r:~!ii!• . . ..- . . ..,.; - - ..... . ...-_.... - .. :. __. .- _ ._.. . --. -•. .. _. - . . . -.. . .. - _ All animals utilized in the proposed exQeriments will be male, CF-i strain mice Nius Musculus ), to be obtained from a commerc3~a1l vendor (Carworth Farms, Inc .) at ; ; ; wening 2 days) ,, and fblloSSing adaptation to conditions of laboratory housing ~~ '` (1Q anim~ls t~er caaewill be utilized as experimental subjects at approximatnely- ~:' f~~~ ~:30 d~{~s of a ~'e ~jb. `.V The specific experiments will be concerned kd:th: 1: Dltihi fotf ose-response reaonspsr nicotine sulfate and rae o memory ;; consolidation; the latter will be determined from the incidence of ; reduced retrograde amnesia induced by electroconvulsive shock (ECS). . The temporal gradient for ECS-induced retrograde amnesia in animals pre-;: treated with nico tine sa! fate. ., The effect of nicotine sulfate on thr incidence of conditioned response acquisition as a funrtion of con3itioning stimulus intensity. The effect of nicotine sulfate on the incidence of conditioned response retention as a function of the intensity of thec stimulus (ECS) - - amnesi. ~; W ".- In the first series of experiments nicatine salfate will be administered i.p. mice in dose ranges of 0.25 mg/kg to 2.00 mg/kg (larger doses would not be indicated in view of neurotoxic effects). Control groups will be given an equivalent i.p* volume of 0,~ saline. Each group will be given a single training trial one hr: ,~ l :post-in3ection (stable CvS stimulant effect reached) in the apparatus described above, =- utilizing a 3.0 mA, 3 sec. training shock (yielding approximately 100% conditioned e'~ ,±~~response acquisition)~, followed either iimnediately, 5, 10, 15, 20, 30, !~5, or 60 min ~•~~by a 10 mA electroconvulsi.v eve shock (ECS), applied transcorneall.y for 200 msc. (sufficient to produce approximately 100% retrograde amnesia in control mice, when applied immediately po st-trai,ning). A testing ~ trial. -will be given to all groups of mice 2!~ hrs. following the training trial. Conditioned response retention will be evaluated on the basis of response latencies in the testing trial (L y 30 sec.) and , retrograde amnesia wy~1l also be defined in terms of the absence of conditioned response as determined from latency measures (L ~ 5 sec.), Control groups, both, drug and. saline-treated:, as described above, will be given no conditioning shock followed at the appropriate intervals by ECS, conditioning shock with subsequent sham-ECS (corneall electrodes applied, but no current passed)~ and no, conditioning shock and sham-ECS. The outcome of the control group treatments is expected' to be _ very similar to that obtaired in pilot studies reported in the ~eliminary supporting data appended to this application. The data resulting from the proposed experiments :. wi11 allow for oonclusi•ons regardir~g the dose-response relationship for nicotine .,= • sulfate and the rate of memory co nsolidation, determn.ned from the incidence of :' reduced retrograde amnesia induced by ECS. Alro the tem oral , p gradients for ECS- ' ~~;... induced retrograde amnesia will be determined for nicotine sulfate. Since this ,~~:~ ~%~}» +u~ a.`. '~`:.ttn .'t~ ...f~•

compound, within the proposed dose range, does s, not alter the susceptibi,lity of mice =;to electroshock-induced convulsiont one may therefore assume that• ariy change in the„,~ P'.amnesic effect of electroshock is due to a central event rather than a threshold °= ,, haige. The usual tenporal gradient for electroshock-irduced amnesia has in our ., ~~~ : _ r~,~s experience, fallen witrii~ 1-hr. post-training, with; a reduced ircidence of ar~nesia ='~4ui ftif th tiilth occrrn as auncon oeranng eecrosoc ~ k time interval. From psel >!ra na: 4>,: data it appears as though the.gradient is appreciably shortened when• animals have : been cotinetdieshould "; ~':~~ ivle~n lusively establishlthe temporalboundaries of the amnesic ~fectSOf~S•~y `•-..A The second series of proposed experiments will concern the effects of nicotine 'slft t btilid i th d uaeoe uzeneose ran e ecified ab oveu the acauisition and Pon t;ak, , g' sp s r retention of conditioned avoidance behavior as a function of (a) conditionin Yg stimulus intensity (i.Q.. 2.0, or 3.0 mA) and (b) post-training ECS intensity (5.0, : ,~:10.0, or 20.0 mA). The incidence of oonditioned response acquisition and the dura- :-tion of stability of this response has been shown to increase as the conditioning• ushock intensity is increasedy and the incidence of electroshock induced amnesia is increased as the ECS intensity is increased. In the first experiment of this series animal.s will be conditioned., utilizing¢~ ,;_,the technique described above and will be given either a foot shock of 1.0s 2.0, or :-;3.0 mA, applied through the grid floor upon, successful completion of the response: .", ::.Control animals in this situation will be given no conditioning shock. Fbr each of ` these conditions a post-training electroconvulsive shock will be given at 5.0., 10.0, : o 200ih 6 r. mA, eter immediately following the training trial or at 10, 20. 30, or0z : minutes followi,ng the training trial All animals wi11 be tested for acquisition .. !`f thditid ftif thtii dfiti oe conone response as auncon oe pre-ranngosage o ncone sulfate (0.25 mg/kg to 2.00- mg/kg), 24 hrs. follojing the training trial where the conditioning shock was varied. These data, for the effects of the drug upon re- ~~ tention as a fumtion of the conditioning shock intensity, will be available from :Y ~;~, ..... the non-convulsed control animals for which a saline-injected group will serve as a :oontrol for the drug dosage. Data relevant to the issue of post-trainingECS , ~-~y intenslty and the incidence of retrograde alnnesia as a flxraction thereof will be ._ determined from nicotine sulfate and saline-treated groups given these injections :=; one hour prior to the training trial. The testing trial will again be given 24 hours "• folloh2ng training, and on the basis of the incidence of retrognade amnesia obtained `y~ statements with respect to the temporal gradient far retrograde amnesia as a function of an intensity of ttae amnesic event, electroconvulsive shock, may be made. On the basis of lot studies it is antici ated that nicotine sulfate will reduce the Pi p temporal gradient for the amnesie effect of electroconvulsive shock, and that such :,a reduction will be a function of decreased ECS intensity as well. . --,, ..•,..:...: . . . ., . Another series of experiments will be concerned'with the biochemical effects of nicotine sulfate in the dose range which proves to be effective in facilitating memory cmnsolidation, as determined from the experiments outlined above. Those specific biochemical, changes with. which' the experiments will be concerned will deal primarily with, the biogenic amines serotonin (5-hydroxytryptamine) and norepinephrine, as we11 as the substrates to which thiltd Silii ese amnes are reae.nce premnary data has suggested that in the strain of mouse used • there are no appreciable changes over titae in norepinephrirE, this observation will again be verified experim_entally and the majority of the chemical determinations: to be carried out will concern themselves with serotonin metabolism; in this connectiony 5-hydro.Vtryptoghane, the serotonin precursor and 5-hydroxyindoleacetic acid, the serotonin metabolite, will be assayed, - ~'/ ,•.0® j, 3545969 f.7,`,~ = y . .:..kf 'Z.

1 :x. ~d.~C ,`~y `.. i ~{~ ,4 utili.zing spectrofluorometric procedures which have bren well standardized in this a ~'i laboratory. Through these means serotonin concentration can be determined and !.~.A ~ -_`t+ reliable estimates of serotonin turnover may be obtained. These chemical deteri+iina=.' .tions tiull be carried out under conditions of both, experimental as well as control ; `.:.C' . drug treatment and under conditions wherein electroconvulsive shock is administered`~ or controlled.for by sham treatment. •It is anticipated that as a result of electro=~ ~ `~~convulsive shock whole brain serotonin concentration in control animals will 'be : ••:.. ; z':.elevated. ~.:This hypothesis is based upon previous observations in this (Essman,B::- M. W~:~x Chan ges in memory consolidation with alterations in neural RNA. -Proc . Coll. ` Int: `~ Neuzo psychophaim., 1967, 108-113) and other laboratories (Kato, h., et al. . Histam epinephrine and norepinephrine in the rat brain, following convulsions.`~ ~~ ~° Int. J. Neurops3'c h.'1967, 3, l~6-51). It is further hYP.o-t,hesized that the de ee":' ---~ ~' rto wfuch eleetroshock elevates brain serotonin will be limited under conditions `~ .where nico tine sulfate results in decreased brain' serotonin levels as a fZnction . of dosage and post-injection time, and that under these conditions either seroton3ii =..~> ~,... ,. concentration or sero•to nin turnover may be statistically correlated with the be- ~. . .. "=?>.havioral data indicating the incidence under these conditions of conditioned respo~ ~,~.. . retention; i.e., facilitated memory consolidation,, inferred from nicotine sulfate- ~~ uiduced attenuation of electroshock-induced retrograde amnesia, will be Positivel :~ - Y :"~ correlated at, thos•e dosages and post-injection times where maxiimall changes in brain', ' serotonin concentration and/or turnover are observed. The proposed experiments may be summarized as an attempt to explore the relationship.between the effects of nicotine sulfate on amine metabolism 3n the .~' : ~.:. ~ .;. central nervous system and tihe process of iner• ory consolidation based upori the ~...;~~.; hypothesis that nicotine-induced alterati.ons in brain amine levels may account ~4. for a nicotine-induced facilitation of inemory consolidatiOn • .. , . . . . ... ..e .,_.. ra1.,.: .. . .~T" . . .. . . . - - . 11 7 Y~~ K ,. W'. 1 i y

~ ~- -. , _:. ....<• , .,. .,- . .: _ Several experiments coinpleted in this laboratory have indicated the feasibility :; :of the experi-ments propos id. The first of these studies was a series concerned with ;~ :the acquisition by mice of a simple maze response requiring a choice di.scrimination~;£ -, 3n order to escape from a water maze. Gmups of an3ma3 s were given either 0.9% salane, ~ 2?;;, 0.25 mg/kg~ 0.50 mgf kg, or 1.00 m gf kg of nico tine sulfate, intraperitoneal?'y. ,t 15 min . K•following injection all animals were given 4 training trials spaced at 15-min. 3nter=; ~va1s in a water maze. Their escape'times and incidence of errorless performance in:~ ~ Vthe acquisition of the escape task were recorded. When the esc~e timA s for all 4.r, , ±-~ = groups o£ animals were linearly transformed across all trials in order to derive a ~~--y ;,decreraent score indicative of the rate of maze acquisition, the only group reachin stati.stical significance was that comprised of those mice which were given 1.00 m ; of nicotine sulfate 15 min. prior to the first training, trial (t - 1.92; E-C.05) : A co,riparimn between groups. for thee incidence of errorless performance throughout , ~ ;' .the acquisition of the escape response indicated that there was a significant differ-".j Swtence between drou s in errorless erformance and that this sionificance was ]ar el b P P o g y:: %..'accounted for by the high incidence of errorless performance among those animals "`;ii 100/kfitilft ('2 195 1 ~02) Th~ -recevr.g. mgg o ncone ssae -1., df = , E..ere was a ~ incidence of errorless performance a~nong animals treated with 1.00 mg/kg of : . nicotine sulfate, as compared with an averabe of 28% errorless performance among ~ `th i thitd Tt/e e other groupsns suy.hese findings suggesed that 1.00 mgkg of nicotin " sulfate exerted a significant effect in facilitating the acquisition of a simple escape response by mice. - i:- - ~ s:~:r:' • - .. . . .. .. . . _ . . . . =' Subsequent studies were concerned withe the effect of a 1.00 mg/kg dose of nieotine sulfate in relation to the memory consolidation process. In these studies animals were treated with either nicotine s~.ilfate or physiological saline 60 min. :1 ~=" prior to being given a single training trial to establish a conditioned avoidance ; ='~,response. The training trial was followed immediately by a 10 mA electroconvulsive ~~ shock administered transcorneally for 200 msec.; 24 hrs. following the training . trial all animals were tested for the incidence of retention and/or retrograde ~";. arnnasia. Saline-treated, animals showed between 90% and 100% incidence of retrograde ' amnesia, as defined by the absence of any conditioned avoidance behavior, whereas Dapproximately 66% of the nicotine sulfate-treated 2nimals showed retention of the r conditioned avoidanae response, with the remaining 34% showing the same retrograde ~ ;: annesia for the conditioned avoidance behavior, as was shozr2 by almost, all of the ~1 saline-treated co ntrol animals. This finding suggested that the amnesic effect of -.electroconvulsive shock was either attenuated by nicotine sulfate treatment or that : the time interval required for fixation of the memory trace was appreciably shortened through the effects of nicotine sulfate and was thereby less vulnerable to the• amnesic °. effects characteristically exerted by post-learning electroconvulsive shock. The first alternative suggested by these data does not seem to appear dependent upon any alteration in the threshold to con,.m]sion produced by electroshock since, for the dosage used, independent experiments established that for electroshock, varied from 5 through 20 mA, there was no alteration, in the incidence of a full clonic-tonic : convulsion following such electroshock administration at intervals from 15 mi.m. ta 60 min after injection of 100 mg/kg oflft .. nicotine suae. 1003546971 In another study where the interval between administration of 1.00• mg/kg of nicotine sulfate and the training trial, i*nmediatelyy folloUred by electroshock, was - varied from 15 min. thruuph 60 tmi.n., the data indicated that only when the co und ` - ... 7 was ad~inistered 60 min. prior to co nditioning trial did a si~ificant (n t.01~ ,:.~..: : _. . =;°tinddenee of conditiored response retention oecur. These data were consistent with "those:{those obtained in the previous study in that comparable results emerged, further ,~ . _ ` .. . .-- ~ - ,~~.

;41,. ~_~, ?::=..,- >- .. .. .. - . ;. . .~ ... _ . _..., . : ... ~ .- . ...,. _ . . ..: _ .. , the observation that nicotine s.ilfate exerts a peak effect upon menory ns~ lidation when its central action occurs ona hr. follo-A ng, administration of •.supportiflg c o This ohservation is supported by some biochemical data in which~ to mice /k 100 m : . g g ; . :; icotine sulfate at ` f /k 00 1 g o n m g v for saline-injected 2nimals or• mice treated with i . . who7:~e brain serotonin and the renoval of brain tissue ecedin l t i ' , g s pr erva n various i ~1o waarr~ c7Ct:rM[i aSSdVed snectrofluorometrically. Contrary to a c~~~~a.~ -.--r----r------- ~ ct of nicvtine and related substaxices ;,:~ Eff 1 t T C ~ e . a . ., e ecent report (Westfall,, 83-100) no changes ]I~2 d Sci 1967 i NY A . , , ca An n the brain ine levels 9:~.....9 upon an 6 i 0 m n. in norepinephrine were observed, as fLintion of pst-in3ection time up to • ~ a trend to~rard tl biti didllhow tion whereas whoeran seroonn generay s ' s ec o ~~.:p J I ~ These data and the bazavioral data previously : n of time ti f t ~ ' . o unr as a decremen ~ . ~ ~rreferred to are summarized in Table 1. ~ " Mean Whole Brain, Serotonin Concen-hration. and Per Cent Incidence of Conditioned Response Retention after Electro- ~'~}~~~~'?=~f~ shock for Mice given 0.9% Saline or 1.00 mdkg of Nicotine - StLl.fate at. Varyinfz Intervals Prior to Cond3,tioning. Per Cent Incidence ' Mpan Whole of cK xetention Brain, Seroto nd.n ~' after ECS• .,[.ev.. - . _._.....- : _ - ,. - . -.. kT. , an ges • w°~These data are consistent with a previous suggestion Essman `~~P , ~ :: ?~~F~~~mPmnrv ns~lidation with alterations in neural RNA. Proc. Coll. Int. Neuro- m :..,..: .. s•ti~• ~ 1= sP ychopharm., 1967, 108-113) that a reduction in brain serotonin level or pharnaco -:" ` - ~ d aA: ~~ ; ~'illid liittin rte of serotonin turnover in the brain ten oecay-.mposemaons o thea- ~~ 'limit the degree to which amnesic agents or events become eriectizve zn zmposa.ng 3, ' a retso grade amnesia, and~ that these observations may be interpreted as a facilita- time interval fo llos~ ng learning th r b h lid ti i '` , e y e e on p rocess w tion of the menory co ns~ a :. ;: -; .r <? r = ; :, tive effects of knocai di t th ll l bl t , ~ srup o e :I, y vu nera e race is norma ~. wherein tt~e msnory amnesic agents or events, is appreciably shortened by those conditions which tend : to lead to a reduc tion in or limitation on the activity of bra3,n s erotonin. It is• `:; 1i_.• felt• that nicotine sulfate is one such agent which, at the dosage uZisIzea, proviaes for a wide margin of safety insofar as neurotoxic effects are concerned.

Ph.D. - University of North Dakota, 1957 A. - University of North Dakota, 1955 B. A. - New York University, 1954 eriencel _ . .. . . . . , _~•.,,.,. _ ~,. . .. < : . . . .. . . ,Instructor, Dept. of Neuropsychiatry,'U.S. Army Medical Service School, 1958-59.-' ,, Director, Psychophysiological Research, U.S. Army Surgical Research Unit, '?~;~~,, j. , 1958-59 Senior Post-Doctoral Fellow, Neurophysiology, Albert Einstein College of . Medicine, 1959"61 Research Associate, Dept. of Physiolog5*, Albert Einstein Co].lzge of Medicine~`° . . . . . : ', . ~ . -_ . ~ . . .: _. . .. .... , . . ' :'^~•.'... '' c'a ' ee.! 1961-62. Assistant Professor,' Queens College, 1962-64. Associate Professor, Queens College, 1965-66. :'. Professor, Queens College, 1967 - Present. ;: Research Fellowy Laboratory of Neurochemistry, Nt. Sinai Ho spitali, New York,y .~ 1964-66.. .Research, Associate, Laboratory of Neurochemistry,,Mt. Sinai Hospits7, :; New'York, 1966 - Present. - Lecturer (Neurochemistry), Coltunbi•a University, 1964 - Present. Areas of Major Research Interest: ' 1. Biochemistry of the central nervous system. 2. - Neurochemieal correlates of behavior. 3. Memory processes. Member•ships: :Amera.can, Psychological Association. Physiological Society : American Society of Zoologists New York Academy of Sciences American Association for the Advzncement of Science Psi Chi • " • Phi Delta Kappa Alpha Pi Zeta Sigma Xi .: „

x r 6 M -1 .S. v. ;P~ a 0 -c: k

® © 0 FROM: = Robert C. Hockett , ~ T0 .The committee comprising Dr. 3acobson, Chm6, Dr. Loosli and lr:oa • i~ ~~. .. , . Dr. Reimann. SUBJECT: New grant application from John E. Noakes, Ph.D. - No. 624. n 0 ..~ . ....:.. ... . .. , , . . L'y :. "' We enclose 'herewith a new grant applicatiori- from Dr:~ John E. : Universities, Oak Ridge, Tennessee 37830. Noakes of the Oak Ridge Associated '.': .., . . : . r . : . . . , . . . Dr. Noakes made two or three visits to this office to discuss his past research and new plans, before sub _ . , . ~tting the application here enclosed. _ . . t _ .. ,.~ r a a a ~ . ~y~l.'~."',~5!F..n:.~~:'~"r.~`~t~`'.:_.... • _. .. •.. ]:PoU~CIL FOR TOBACCn RESLARCFi-U.S.A. In talking with him I had in mind the general attitude of the Board as expressed when the subject of Polonium-210 in tobacco first arose. The consensus, as I recall it, was that any'realistic effort to define the possible health hazard'due to polonium in tobacco must be part of the general problem of dose-response relationships in exposure to any alpha- 'emitting radioactive material. Since the Atomic Energy Commission is very deeply and extensively involved in suchistudies it seemed inadvisable for the Council to dabble with that nroblem on a small scale. - ,- } The applications from Werthamer (#`"j95 and #596) were submitted to the Board in March 1967 to relieve the staff of responsibility in dis- .,position. It was denied, (1) because of doubts expressed by Dr. Hasterlick -about the proposed methodology, (2) because it proposed to study differences .~in lung burden of polonium in smokers vs non-smokers without any kind of provision for determining "how much is too much" in terms of'hazards and (3) on the general grounds mentioned above. ,.. . ~. , The Noakes proposal is of quite a different character. The man impressed me as knowing what he was talking about and his institutional ; connection suggest that this should be the case. The proposed worY, is aimed at following up clues as to the origin of polonium-210 in tobacco with a possibility of demonstrating relatively simple and inexpensive means of re- ducing the content materially. The same study may also be pertinent to many other crop plants. Whether or not polonium-210 is presently a hazard in tobacco, or whether this can easily be shown, it seemed to me that the industry, mig7ht be interested in such means of mini.mizing this contaminationy as a matter of public policy. I know that the companies have sponsored some work on polonium but do not know whether this has included anything substantially ,equivalent to what Noakes proposes here. Hence, I am,sending a copy of this application to a member of the industry technical group for comment, and memorandum.

" ' R Ou\'CIL FO TOBACCO RESEARCI3 - U. S. A. im. ; MEMORANDUM - CONT' D' ' y y r • g p q p . ~~ S abe ance at present We have been tr in to develo bette e ui ment and ,.polonium-210 grown-in" without full consultation concerning methods of exposure and of systematic biochemical and pathological examination. He merely indicates that he would be glad to cooperate in setting up such studies and in providing the materials if this kind of follow-up should-be wanted after the first phase. I think this questi=must be left in Noakes included parts 2 and 3 of the proposal in a tenta ve manner. ti `'He is not a biologist and would not undertake systematic studies of animal -' exposures to smokes from tobaccos containing different known levels of n ~ / ` ~ r, a~y .w.'1h. ..~' direct carcinogenic effect of cigarette smoke on the lungs. Hence, we cannot judge at present how worthwhile it would be to try comparing the actions of three smokes with different polonium levels. Further experience gained the next year or so may help decide this question. Similar questions`r~ pertain to such biological test systems as that which Dr. Furst has been~ ±.~ using, the Heston intravenous administration methodithat Dr. Homburger has w~ been trying out, or to the mouse-skin painting method. - -- . , , . : . .. - _methoas ror exposing small animals chronacally to whole, fresh, "normal" cigarette smoke and for measuring the actual exposures achieved. Such exposures have not so far given any clear or reproducible evidence of a ~ R. C. H. .4 ! ._..--.,. . . , ,... . ,, ... .. .. . ... .. . . Y;kM~~ P.S. I am enclosing a copy of a k~ paper by C. W. Francis and G. Chesters of , the University of Wisconsin entitled Radioactive Ingrowth of Polonium- 210 in Tobacco Plants, which may anticipate Noakes to some degree. . R L. A copy has been sent to Noakes for his comment. .._ .. .. . _:` i a~a

.Name ' of Investigator (s ) John E. Hoakes, Senior Scientist, ,: , . .. . : Institution & Address: Post Office Box 317 Oak Ridge Associated Universities Oak Ridge, Tennessee 37830 . - .;-~.. . .. . . .... -..`.: Proposed Starting Uate: September 1, 1967 yr . . ,,. ' ~ .. . ~ ~ ~ . C . .. ,. ' ~'~ .. .. Short Title of Project: ':"Po-210. in Tobacco" Physical Facilities Available (Where Other ;"'Indicate Geographical Location) tate full-time uninterrupted use. In addition to the above enuinment. one pH •would be made of this instrument in the proposed tobacco research would necessi- c='...but teaching activities take priority. It is felt that 4he extensive use that ':'is available at the Special Training Division-for shorti-term,research projects. : metric d'etermination of Ra-226. Pb-210 and Po-210. This instrumentation • .'.A multichannel analyzer is needed for pulse height analysis in the radio- ~"l~s ~'~ ~~ ld h x b ~ t l t sui Se °~' ~ z ~ d n n ~rou ave to e n e arged and heat and light nhouse nstalle gr i the •-~- -

R: REDACTED MATERIAL Q 2,000 7-, 5ao Zt is understoo& that the anniicnnm ,, ,.a g a n ve read and found acceptable ~ i nstltutional offi'cers in applyiog for ~_o:jeca Di:ec-or ra ±'~'' t h the Council's State~ent of Policy Con- •,Signature~,~~ ~ YA?nfM~ C!nn.il+a....e _,_ _-_ ._ . Fxecutive Director

Current .~; .. **.Mechanisms of Radiation Injury Studies related to treatment of radiation'•injury. :. Title of Project Environmental Radiation Studies *Metals_ metabolism and medical radioisotope develop- '•: ment, Therapy with radiation., Radioisotopes in diagnosis) Biologically important radioisotopic ma,roeriais : NnsorpLion~ wnoie boay xetention ana Deposition in Specific Organs; NASA Study on. ~ : radiation effects~ Instrument development and ~= • : methodology * Research by Special Traintng Division ." .. . *)~Research by Aedical Division - while not'direetly '- related to the first phase of the proposedd projectj, : . later work in this project wi_ 11 involve the Medical ,''•: " Divis3.on. •.

R: REDACTED MATERIAL _.;_-M. S. - Texas A & M University - 1959 Ph. D. - Texas A & M Universit y - ~62 ' ... . -. . ..{~- ..,. - --. _:~:' .... --Professional :' American. Chemistry Honorary SiBr.a X! - .: ._ .. ._~;.... Experience: -1962-Present: -;-Sen_:or Scient3,st, 'Special Training Division, =-: Oak Ridge Associated Universities, sen ior _ = lecturer with research in. geochronology of ; msrine sed•iments. and, environ_*•:ental studies. .'.Assistant Professor (Research), Iastitute of ' Marine Science' University of Alaska; worx concerned with establishment of a marine -=`laboratory and research in clay minerology. it • Research Scientist II, Texas A & M U y; nivers =_.. responsible for. setting up and operatin; the carbon-24 dating laboratory using the liquid scintillation method. - -~ _.-_.. . _ .. . . _. _. . _ . . . ":; Rese arch Chem3.st, Texas A& M University; ~-IGY Progran, "Mahole" Investigation, and course instructor. Research and teaching assistant and work in '`deve].opment of an analytical method for boron eva7:uation in marine Wa.ters; =d investigation, •. of long chain unsaturated fatty acids. i955-six months Graduate Assistant at Syracuse University : ~.,._ . . = - ::Tndustrial Ecperience: - - _ 1959162 -9 Served as.a consultant for toxicity work applied to industrial application.. Research Chemist and:, JLnio_ Ri:ecutive position•, Cla:.k-Cleveland Company. Work in preduc-. devzl- opWeat in the field of plastics. Personsl contact with Battelle Ne~orial and Evans Research Centers with laboratory eval- -.uation of their subrsitted data. Soils Rngineering - New York S'Cate, Soil Anal,yst 1965-Pz~-616:.F-- Consu]tant - Picker Instrumeat Co:y-many

how Temperature Conversioa of Acetylene to PPare Benzcne #+248G'+ . "The Distribution of Boron aad. Boric Acid Cotaplexes in the Sea," Master Thesis, Texas A& M liniversity, 1559 :"Boron-Boric'Acid Complexes inSea 4Fater," presented at the Internationa2 11nio n of Geodesy and Geopqysics, Helsinssi, ': FinZand, 1960 and Noakes, J. E. and D W. Hood, Jour. of .Deep Sea Researcb, 8, 121-129, 1951. .. : . .. ...., .. • "Ci4/C~ Ratios of the Organic and Incr;anic Carbon Frac- ;:tion of' Waters of the Caribbean, and Gulf of A4exico," -: Final Report A & M) Project 235, N. S:', Grant-610232, :.:.. 15 February 1961. - • . "Benzene Synthesis Acids Cl4 'Dating," C"nemica7l and Zhgineeri, g Nesr , October 9, 19b1. ;"Carbon Dating by Liquid Scintil.ation," Noakes, J. E. and D. W. Hood, presented at the•Ouk Ridge Special Caurse, "Nuclesr ,L-thods as Applied to Oceanography, "' November, 1961. "'Conversion of Carbon Diox.ide to Beazene for Liquid Scfntil- :lation CcWUting," Noakes, J. E., A. S. Isbel.l, and D. W. Hood, ••'Trans.. Aaer. Geophys. Union, 42, No. 2, 226, 1961. "I7niv. of 2exas R_diocarbon Dates I, ° Stipp, J. J., E. M. Davis, J. E. Noases, and T. E. Hoover, Amer. Jour. of Sci. Radio- carboa, Skrppl. ~ 1962: "Natural Radiocarbon Measurements by Liquid Scintillation Counting," Dissertation, Texas A & M University, 1962. "Low Teap Benzene Synthesis for Carbon-14 Dating," Noakes, `= J.•E., A. F. Isbe11, J. J. Stiop and D. W. Hood., Geochemica - et Cosmochim.ica Acta, 2' No. 7) 797-804., 1963• "Texas A & M University Radiocarbon Dates I," Noakes, J. E., J. J. Stipp,.and D. W. xood, Amer. Jour. of Sci. Radio- carbon Sitppl., 6 189-193, 1964. "Geochronology- of the Gulf of h:.xico, Pa•.~-t I," Rona, E., L. K. Akers, J. E. Noakes, I. R. Supernaw, Prog*;ress in 0ceanography., Vol. 3, Perganon Press,, 1965.

"A Cherdca7. Studyy of an, j'L-,biEnt Tempcratu.re Catay,ytic Benzene Synthesis Used in Radioc=bon Datingy Nonlces, ; ,. J E M Kim G' Thd L K .., S..,. A.omas, a.n.. Al:ers , "' ; 'A "ORL~1S Publication 4,4b, Novemb er, 1964. "Cobaltt f.- Molybdate Catalysor Ambient ioient Ten;perat TMe S f~ F:thesis of Benzene for Liquid Scintillation Radiocarbon .` . Dati ng, " Noakes, J. E., S. M. Kim and L. K. Axers, ;; ti:' ORTwS-50,. s,prils 1965• "Chemieal and Counting.Advances in Liquid Scintillatyoa ;Counting," J. E. Noakes, S. M6 Kim and J. J. Stippi Pullman Conf June 711 1965 PtC erence --,:}aper presene 'and published in- proceedings. U.S.A.E.C. Ganf,-650652 "Electrodeposition of Actinides and L:antr.anides," .Kim,S, M.~ John E. ~it~alces, L. K. Akers, W. "v7. l~Llle_,. : ORIrISZi$, 15 1965 : - December.-. : aElectrodeposition IM--thoci- for Counting Alp: a. and Beta Eaitters," Kim, S. M:, J. E. Noakes, and W. rl. MilZer, Nucleonics, 24• #3, March, 1966, "Anoma.lies in the Th230fTh232 Activity Ratio in Some Mississippi River Sediments, Noakes, J. E„ and I. R. Supernaz•, (presented at AGU N-meting in April!J 1965. Jaarnal Geophysical Research- (in press) "Recent Improvements in Benzene Chemistry for Radio- _ carbon Dating," J.E. Noakes, S. M. Kim and• L. K. Akers; Peochiaica and Cosmochimica Acta (in press) .: . "Oek. Ridge Institute of Nuclear Studies Radioearbon Dates'~ . I," Noakes, J. E.I S. M. Kim and L. K. Akers, Jour, Of ~;Sci Radiocarbon Suppl (i) ..n press "The Mass of a Neut"ron: A Student Exercise", H. E. Banta and J. E. Hoakes, American Journal of Physics (aecepuez for publication) "Uranium Content of G•,zlf of Mexico Sea Water", J. E. veakes, S M Kira and L K Ak Ptd A ....ers,resene at-aeri can Geophysica]. UnioL) Apr.il,:'19&7;.(to be ptiolished) • "A New i~'ethd' f Thi A' loororumnalysis from Marine Seci ent" , ,- S M J Ek P ~ .. Kim,.. Noaes,resented:,at Americ an Geophysical •Union, April, 1967 (to be published) ~

- APPLICATION FOR RE3?CH, G;Aw, Objectives of Pronosed Po-210 Toba.cco Re;,.curch ' The first phase of the proGram would be a Zeochcmicai study of the sources of'Po-210 which mig7ht be available to tobacco plzntc snd- :'the gross mechanism by which Po-210 could eutcr thc.plant system. : The second pha,e would be a medical study to deter:u:ne if and at what concentration levels Po-210 in biological systems could be 'conszdered to b e carcinoaenic. ' Specific Aims on a Yearly Basis "(a) Id'entification of parent radioactive source ;r.aterial .;.ost responsible for the occurrence of Po-210 in tobacco (~a-226, Rn-222~ Po-210). Mechanism by which Po-210 parent finds its way into ihe plant -.system;(air•-plant interface, soil-root intcr•~ace, eLc.)' c) 'r'.stablish conditions for decreasing Po-210 content in tobacco . plants grown in a natural environment (phosphate fertilizer, soil pH control, etc.). . ) Verify control!led;;ponditions for growin<; tobacco with ]:ow, and high coneentrati.ons of Po-210,for medical studies.. .), Bulk amounts of tobacco grown with high and ultra low Po-210 :: content under conditions established from ~first year stUdJ. ) Bulk tobacco of high and low Po-210 content analyzed for trace metal content, tars, nicotine, etc., to be used as standardized reference tobacco sources-for biological studies. c) Consultation and preliminary evali:ation or most suitable bio- logical specimens to be used in medical studies. I _Perfection of inethods.and equipment for introducing tobacco smoke into.biological specimens and analytical methods to be eMpioyad in the evaluation of results. (c and d= Gurrent tobacco research projects to be evaluated.) Third Year (a)- Medical studies of biological sp eci:nens' sub jected to hi ;h a:d low concentratio n of Po-2101 tobacco smo:ce. (b) Pathologica7: and: che:^ical ana ly$is to be carried out on suc- rificed. animals to determine ?o-210 content in various or,=ans any evid'ence of alteration in cell development. . .._.,.:

:Fourth Year - - - y;. ) Continuation of inec2ical investigation looking into spec„fic aspects of third year study which indicate:, further invclsti- :gation is warranted. ) 'slorkinr;, H-ypothesis of tobaceo-smoking individuals. The radiation dose rate in bronchial ;conttif P210fd ih cenra.on oo-'ounn te respirntory and pulmonary syste:rs ``•: Radfor.d and Hunt (Science 1964) were first,`to ~~i ~ ' publish the fi..a~n„ of ci!T ` d calculated to. be as high, as 200 RD1. This value far exceeds L'c,e m:x: m pituelium for long duration smokers (2 packs a day for 25 yearo) wa.: should be a prime suspect as a cancer forming agent. etc., of tobacco-snolcing people wr.icht has led to supposition tha-t it recent studies have reported Po-210 found in the liver, lzidneys, blood, important source for initiating bronchial cancer im smokers. '-:ore -was sufficient to .cause alteration in cell development and. could be :n Council for Radiation Protection (ICRP). They concluded that the dose dose of 1.1 n.:~til to an entire lung as recommended by the Intercatioaa.i.. organs, no mathematical model can,be constructively applied. biological half-life of Po-210, and its alpha rad i ationt damagc to various,', of the rather limited experimental and analytical data available for the =~=formulated mathematical models to substantiate their statement. Bccause Skrable (Science 1964) and others have taken c:ceepLiotm to th~:s work ,nc:, _:; 1-0.5 pc/g of tobacco. . °: iT. S. tobaccos rank among the highest in Po-210 content with values of tobacco obtained from all parts of the world show a wide variation. n en n r th Stdi b G(S6) e plant.uesyregory-cience 195 on the Po-210 co t t i the source and mechanism by which this radionuclide finds its way into The occurrence of Po-210 in tobacco has also raised the question as to interface. Atmospheric Rn-222 concentration is between,50-200 pc%u~ and resulting fron~the .assimilation of Rn-222 of gas at the piant-atu,osnaer e Berger (Science 1965) has attriliuted the Po-210 content of tobacco as . ... .. .. ... - .:--. ..-.. .'. . . .. . - ~ in tobacco occurs after.harvest and during the curing period. -"has concentrated on showing, that the major portion of the Po-220 uptake originates fron,the decay of Ra-226.in ground materials. Berger's wo=k ~ Tso (Science 1966) has attempted to• refute this work by showing. that a more plausible explanation for Po-210 in tobacco is in plant root uptake of Pb-210 from the soil. He demonstrated in his studies that tobacco plants subjected to high-concentrations of Ph-210 for a short growth period can contain Po-210 as high as 150 pc/g of tobacco.

Po-210 contcnt of tobacco has been of ON.U, sin_ff interest si;:cc t::a first reports appeared in tFie literature. Spceulo,tion• vras that o::e 1.-.source of Po-210 in tobaeco- could be the Florida phosphate rcci. used as a phosphorous source for essentially all fertilizers irn taa U:;itcd ::'States. _ ahis phosphate rock is rcported to contain o.01-0.05io u_a- niurr, and would be expected; to have in secul :r ec,uilibriun;, e ; wdio- active daug,hter Po-210: Ra.diometric analysis of the•,~,ajor constitucnts of co:;Tercial tobaeco fertilizers have revealed that Po-210 occarG in - thc phospnat~_o rock fraction in concentrations of 20-25 Pc1g• Ti:e ,ycarly-app.lication of high•phosphate fertilizer to American tob,,.cco growing soils may well be the prime reason for the high Po-210 found• :in U. S. tobaccos. rrirther :st.ucr'ie5 have been carried out at ORI:iJ to determine i= Po-210 free tobacco could be grown in a controlled enviror.rr,ent. Tobacco :.-pla.nts grown hydroponically in nutrient solution free of Po-210 or ,_~its precursors have been.found to contain Po-210 in concentrations of .<.01 pe/g of tol~acco. These studies and those of Tso, clearly show `.that Po-210 content in tobacco can be regulated to desired amounts ". under controlled experimental Concli.tions. These studies also• i_.dicate thai similar controlled experiments could be devised to dcte=:nine tYte °sour•ce and mechanism for uptake of Po-210 into tobacco plants. ::,: The ability to produce tobacco,with regulated amounts of Po-210 also ;;offers potential application to biological studies. Introduction of .controlled amounts of Po-210 in smoke to biological specimens wo•ald ;=facilitate quantitative evaluation of such parameters as Po-210 upt`:_e, body distribution and retention times. It would-also enable comparison f biological 'specimens possessing low and. high Po-210 content for carcinogenic- evaluation. :.;~:: .. . .. ~ ~ ~ Experimental AesiAn ...~ _ -- . - . _- . ~-The experimentali procedures proposed: for accomplishing the fir st year 's Y: ~;=~; ; goals set forth in sect~on 6 of this proposal can best be described as ~-_ ..... =~-~r _;~--e)e-sets-o Y--experiments trhich will be concurrently carried out. . Radioactive materials responsible for Po-210 in tobacco plants and. : gross mechanism of plant uptake. ~~ Pe`~groups of tobaceo plants will be greenhouse raised. One G--oi.p of plants willl be grown in quartz sand free of Po-210 or any parent radioactive material (photo 1). The second plant group w:_I11 be grown in typical virgin Tennessee tobacco soil. The thl'rd- group of plants will be grown in similar soil, but wi11 have p'r.osphate fcrti- lizer added' to them which eontains appreciable a.;.ounts of Po-210. :'. All plant groups will be nourished with nutrient solution, --ade with reagent grade chemicals free of any Po-210 or parent radioa.:tive material.

'A3,1 tobacco plants from the three eroup s,•ti 'l be harvesteci• at during time of growing and curing. The tobacco Po-210. content --uptake of parent Rn-222 at the atmospi.e-rc-plant inter nhase rtaturity. The. quartz sandgrownf:obacco plants should s:.ow. '-the lowest Pb-210 content of all toba.ccos. and reflec t only the ~.from atmospheric Rn-222 co:atributio7 during curing will be evaluated by preferential curing methods. :. • : . . - . :. of the Po-210 contribution- from Rn-222. add'dillflt thiltkdth R222titi •e w reece so upae an"en- conrouoa. The Po-2iQ,furniched by the soil can be calculated by sub:acti-on " Thc Po-210 content of tobacco plantsgrosnm soil with nutr:ent " soil and Rn-222 contribution will be known, the fertilizer Po-210 soil contribution and fertilizer eontribucion. Sin ce the Po-210 Ti;e Po-210 content of tobacco plants grot•m in soil tirit'rh p'_.ospc,ate ~ertilizcr added will be a composite value of 3n-222 uptake,, plus '.contribu4ion to the tobacco can be calculated. - tobacco plant at the time of harvest can be composite value of the It should be pointedi out. that the Po-210 soil contribution to t he : harvest and a'c later times will perr,d.t calculation of the decay o_ -direet non-supported Po-210 uptake from the soil und . parent -,row_ .. in. Ra-225y Pb-2]:•J and-Po-210 analysis of the nature tobacco at .the non-supported Po-210 and the grow in of zhe Po-210 parent con- tribution. These analyses will also enable identification of the ` sponsible for the •Fo-210 content in aged.smoking tobaccos. : ' Po-210,parent which is entering the plant-root system and soley re- e greenhouse conditions. Each plant will receive a known arwun: of .= .Three groups of tobacco plants will be grown in Tennessee soil ur.der Conditions .or decreasing Po-210 content in soil grown tooacco plu,nts: radioactive Po-210,in equilibrium with its daughter Po-210 and •- 7nutrient solution of controlLed pH. • . . ...,. _... : „ _. ,.. _.. .._ _ .. ... .. _ . _- _.... _. . . i: . ~' ~ ~ • "-=- . The third group will have nutrient solution of pfl7 with added s•1faze pH5. The second group will be raised with nutrient solution- of ph?. The first group of plants will be raised t•rith, nutrient solution of ions should' indicate the degree of fo.=atiorn of insoluole Pb-210 the third group under pH7 soil conditions ;-~ith excecs av'ailci~le s~;lfate uptake under acid soil conditions which shouid reflect leas ten;iency of the soil to hold the available Pb-210 and Pb-210. The second plant grouD • will represent uptake of t o-23A and Po-210 from the added radioactive spike and the norr.•,al soil contribution. Plants grova in Plants will be grown to r•,aturity. and araly Led at harvest time for Fo-210 and Po-210- content. The first group of plants will represent ions. sulfate as compared to available Po-2l0 for plant uptake. ~,

The presence of high concentrations of sulphate ions in co,=ner•cial , ----fertilizers.is due to the sulfuric acid treatment of phosphate ~ tion of insoluble PoS01+ in soil at raised.pl'i should therefore be - rock during fertilizer manufacturing, The possibility of the forma- considered: , _~ Soi1 pH is known to be a prime factor in affecting plant trace metal 'uptake. If the tobacco uptake of Pb-210 and Po-210 is found to be : altered by so11 pH, it may be one explanation for'the wide variations It may also reveal advantages to be gained by incorporation of addi- --tives to fertilizer or soils to better control soil pH. -.• -In Po-210 content found in tobaccos growain all parts of the world. 111. rp-'~1:u anaa ro-eIU todaceo uptatie stuaies to estaelisri conditions x•or growing tobaccos of high Po-2].0 content. the third group 100x,. where x = p curie levels. - administered to the first group i-rill be lx, the second group.lOx and Pb-210 in equilibrium with .its dau;;hter. Po-210. Radioactivity greenhouse conditions. All plants will be fed nutrient solution • for their entire growth period. i,2.ch group of plants, early in its development izill be subjected to a designated amount of radioactive Three groups of tobacco•pl.antts will be grown in quarta sand under A11 tobacco plants will be raised to maturity and analyzed at time of harvest for Fb-210 and Pb-220 content. These results.will establish studies. - growino tobacco of desired Po-210 content to be used in the medical and Po-210, will be used to,establish predictable conditions for A plot of the Po-210 tobacco, uptake with regard'to available Pti-210 Low level Po-210, tobacco data will be supplied from seri es I experiments. the upper levels of Po-210t content which can be obtained in tobacco. ' AHALYTIGr'1L METHODS . Tobacco trace metals., tar and nicotine content Tobacco grown under experimental conditions (photo 2) will be analyzed for trace metals,, tar and nicotine content. Similar analyses of comercial tobaccos will be conducted for co:ipara- tive pruposes. Trace metal analysis will be carr:ted oult using non-d:iestructive slow~ and-fast.nautron ac tivation analysis. The tar and nicotine analyses will be conducted by various methods. 0

Sink with hot and; cold runnirgwate-r 'Distilied'water Bench top, 3'xl0' . . . Fume hood with elec- -~;trical outlets and water ., . 'StoraJe cabinets . " and shelves _ "" . Desk ...i..i: . . ' Greenhouse. aAproximatelv, : :108 square feet .-=Liquid Scintillation .,,Counter, prop. 78798 . '-Ton,Chamber prop`. -ihl49- facilit y --

I

210P0 ALPHA SPECTRUM 22 - h®ur c®lwi . I }/Sbb9~5500~ Ol - I I I 1 I I 78 82 86 90 94 98 102 106 108 Ch-I ANR! E L RI UMD C R

0 0 ci C1t ~ ~ co

TAE GUU\TCIL FOR TQB_xCCO RESEARCFI-U.S.A. We enclose herewith a new application from Dr. Harry Darrow Brown of the University of Texas, Galveston, Texas. The application includes a supplement on "Nicotine Effects Upon Cardiac Membranes" and is accompanied by six reprints (one an abridgement) of studies by the author on ATPase as listed below. coplasmic Reticulum ATPase On a Solid Support. Biochem. Biophys. Res. Comm. 25, 304, (1966). Harry Darrow Brown. Azasteroids and Heart Adenosine Tri- phosphatase. Biochem. Pharmacal., 15, 2007, (1966). Harry Darrow Brosra, Swaraj K. Chattopadhyay, Anil B. Patel.' Properties of Butanol-Extracted and Detergent-Solubilized Membrane ATPase. Arch. Biochem. Biophysics, 120, 222, (1967). H. D. Brown, S.K. Chattopadhyay, A. Patel and R. H. Rigdon. Glycoside Effect Upon Membrane Enzymes of Erythrocytes and boscle in Duck AIyopathy. Experientia. Harry Darrow Brown. A characterization of the ouabain sensitivity of heart microsomal ATPase. Biochem. Biophys. Acta, 120, 162, (1966). . Harry Darrow Brown, S.K. Chattopadhyay, and A. Patel. Sar- H, D. Brown (?) (Authors not shown). Abridgement of the Manuscript .'Erythrocyte Abnormality in Human Wopathy."

CARDIOVASCU7AR! PHAMCOLOGY and CHENIISTRY ~'Y' :~w,:: ~ -': Na•625 ..~ #~:,'' Nicotine effect upon cardiac membrane enzymes '*} ~ 4. Proposed Starting Date: October 1st, 1967 R . Anticipated Duration of this Specific Study: 2 years 6. Brief Descripton of Objectives or Specific Aims: ,_ . _ _ . . , .. . . . ~ • . . in experiunental am.mals. We propose here to extend this observation to a study of the primary :.~r....,., . _:.It has been reported that the administration of nicotine elevates serum cholesterol level cardiac tissues. :: and secondary effects of nicotine upon enzymes associated with subcellular membranes of A ~-' ;=:The specific aim of the proiect is to test the thesis that nicotine in chan--inQ• the •nature : muscle. ,... of those enzymes which. are bound; in the native state, to membrane portions of cardiac ~ of the lipid pool, from which cellular membranes are synthesized, may alter the function :.. ._ Give a brief statement of your working hypothesis. It is known that nicotine intervenes in, the biosynthesis of lipids and this: in turn may tration affects the ion transport system and so is an event preliminary to cardiomyopathy. ; systems which are part of the membrane structural network. It is primarily the ion transport, membrane adenosine triphosphatase and the hormonally -controlled adenyl cyclase systems which we propose to study. The thesis of membrane change will• be further examined by a consideration of serum enzymes whose levels in serum, are relateable to cellular changes. We hypothesize that the reported change in lipid composition which follows nicotine adminis- lipid composition of the membrane system will result in a changed function of those enzyme ., sarcoplasmic reticular and plasma membranes. From, this, we may predict that the changed change the nature of the lipid pool available for and ultimately incorporated into heart muscle

T-- 1:.'IILP....-:.,s ' , , . . . . .- __ . - • . ~ . --'8 DetailsofExperimento(DesignandProceduresr(AttachSeparatePages) Rodents and rabbits will be used as experimentab animals. Continued ..F{~i~ . .. . ., . ~ . ~irV A. Biological matenal ...t Injection of nicotine following a dose rate based upon, Kershbaum, et al. (Am, Hearo Jour. B. Observation of serum lipids. • - :: During the course of administration of nicotine, the level of serum lipids will assayed by thin-layer chromatography using silicic acid. Additional experimentaL pro- : differenbes observed in consequence of nicotine administration may be intensified in another =_;' cedures will be designed,upon the basis of the changed pattern of serum lipids so that the .,:Aw~... , ' . [ . ...•. • . . 206, 1965) will be undertaken. `" the prime'experimental group. 4• group of experimental animals by the inlection of hpids in ratios relative to those foundiin s ea o re e It h be ur a t find th t th d trati f 20 25 dia h 1 t I s e m o a zac o es ero in As In natural myopathic states, erythrocytes and muscle tissue are altered . ;.^L'_ _._ l•rls+'in1•s•. p:4 None C 11. @iographical sketches of alliprindpal andprofessional personnel'(append). t• cells and muscle membranes. . to experimental animals greatly affects-the activity pattern of transport A'TPase of red blood a a , n g a uus on (continued page 2A) 9 Physical Facilties Available (Where Other than Administering Organization indicale Geographical Lbcation) 10. AdditionalRequirementse 12. List obpuhlications: (Five most recent as pertinent) (append) . (see page 2A) (see page 2B) (see page 2C)

by the administration of diazacholesterol. This phenomenon we attribute to a change in r;=r'the lipid components of the membrane s to which the protein enzyme is bound. The changed ,.r= ; ~~__... .{. 'k°~~~ ~: ctivity pattern• ( response to metals and to the cardiac steroid membrane ) represents an ; extremely sensitive indicator of membrane subcellular organization. : : . ,_.. .:... _. . .. .., _ - . A'Sw..4~. . . . ' . - - . .. . . Enzyrne assay . -_,' systems chosen for primary study are known to be associated with Yriembrane structures of the cell. The transport ATPase is probably the best documented ,.~.. . model for transport phenomena and its vectorial role in cell function closely relates to -,~~; u membrane structure The adenyl cyclase system appears to be part of a structure-related . _ complex of catalysts acting upon ATP as substrate. The pivotal position of adenyl cyclase, -:: whose catalysis is facilitated by epinephrine and whose product in turn controls the phos- .. .,t, s cataolism and its ycolysis may be a key to interpretation of ener.getic .~ `> phorolysi step of gi ., ~.. ' biological control. These two membrane related enzymes will serve as indicators of drug nieotine ) induced changes. . ~', . . _ Aldolase and creatine phosphokinase elevation in serum are widely used indicators of tissue degeneration. These will be assayed and correlated with the hypothesize .cardiomyopathy. - . • . : Blood will be collected in a haemolyzing solution. The blbod haemolyzate will be centrifuged at 20, 000 x g for 20 minutes. The supernatant discarded and the pellet washed and recentrifuged in the Tris buffer. The final pellet obtained will, be collected and ;; stored as the erythrocyte ghost preparation, and used as the source of ATPase activity. ,~; rt{ Muscles will be macerated and, sarcoplasmic reticulttm isolated by a differential centrifugation: ~ procedure in sucrose. Heart muscle membrane preparations prepared in, much the same ;~ way will be assayed for adenyl cyclase activity by ultra violet analysis following ion exchange" ~ F... chromatography. These methods and those for the assay of aldolase and creatine phospho- kinase activity in serum are presently in use in our laboratory. The ATPase will be assayed ;` by measurement of inorganic phosphate; aldolase by the Sibley-Leihninger method and creatin.e ~ phosphokinase by measurement of phosphoryl group transfer. -• '`- D. Analysis of data, r + Findings will be interpreted in terms of the effect of nicotine administration upon changes in membrane enzyme and in serum enzyme levels (presumably related to linkage from cells ). This deviation will be correlated with induced changes in the metabolic lipid pool and all factors considered as a function of possible cardiomyopathy. Facilities available. -Three laboratories- are in use by the principal'investigator. Many major analytic tools are available. Specific holdings directly available to this project are: Beckman, B flame spectrophotometer, International HR i refrigerated centrifuge, Beckman L ultra-centrifuge, RSCo fraction collector, ISCO fraction collector, deionizing column, water baths, Zeromatic pH meter, Cahn electro-balance, Roller-Smith balance, Beckman DB recording spectrophotometer, refrigerators, freezers, necessary minor laboratory devices and ware. Held by a colleague in an adjoining laboratory, and available to the project are F and M gas chromatographs, Cary 14 spectrophotometer, and P-E infrared spectrophotometer. 1003547000

R: REDACTED MATERIAL ~ ~ Additional common facilities held by the Department in another building, available to this project, include a 2000 square. foot laboratory, animal rooms, refrigeration equip= ment, Mettler balance, free electrophoresis unit r analytic ultracentrifuge and recording .. , . . • spectrophotometer. Also the resources of the Institution include electron microscope <. ;._. r, . . •.. facilities, infrared spectrophotometer, Beckman amino acid analyzer, scintillation '_counter, and•generally, most of the instrumentation and facflities•appurtaining to the -laboratory investigation. A unique dual differential microcalorimeter is in use in this 1a13o.ratozy and appropriate to aspects of the proposed study: . . Biographical sketches of all principal and professiona•1 personnel. Harry Darrow Brown m., 3 c. Ph.D. A.M. B.S. 1964- t 1964- 1963 -1964' ' 1961-1963 1957-1961 1954-1956 Occasional Anil B. Patel M.S * M.Sc. B .Sc . 1966- 1963 -1965 ` 1962-1963 91 1957 Columbia. University. Faculty of Pure Science 1952 same 1950 Long Island University - Assistant Professor, Department of Biochemistry and Nutrition, University of Texas Medical Branch; Galveston, Texas. Collaborator, United States Dept. of Agriculture. Chemist ( Biochemist ), USDA, New Orlea.ns. Associate Professor, Southern Illinois University, Edwardsville. Assistant Professor, Loyola University, New Orleans. Lecturer, Hunter College, New York. Editor: Macmillan, Acad. Press, Schol. Lib. University of Cincinnati 1961 University of Bombay 1953 University of Bombay Research Associate, Department of Biochemistry, The University of Texas Medical Branch; Galveston, Texas. Research assistant in orgaze.i.c chemistry, University of Cincinnati. Teaching assistant in chemistry, University of Cincinnati. * Residence requirements complete; thesis in preparation.

List of five recent publiLations pertinent to the proposed research. .. .. . . .. . _.., . .. . . . ..:,, ,~ . ..,.: . ......, . ~ .,. ... . .., • ~ . ~. .: . ... - _ .. . . mahty ui human myopathy. Ms, submitted. `Brown, H.D,, Chattopadhyay,S.K., andPatel, A.B., (1967) Erythrocyteabnor- •..Glycoside effect upon membrane enzymes of erythrocytes: and muscle in duck : Brown, H. D., Chattopadhyay, S. K., Patel, A., and Rigdon, R, H., (1967) - 'myopatny. Experientia, in press. '~ ' Brown, H. D., Chattopadhyay, S. K., and Patel, A. (1966) SarcoDlasmic- reucuium pi-trase upon a solid support. Bioch. Bfoph. Res;. Comm. 25 (3): : Brown, H. D., (1966 ) A characterization of the ouabain sensitivity of heart micro- Pharmacol., 15: 2007-2011. Brown, H. D., (1966) Azasteroids. and heart adenosine triphosphatase. Biochem . somal ATPase. Bioch. Bioph. Acta 120: 162-165.

R: REDACTED MATERIAL -; " -11. Salaries (Personnel by names) :, Professionai Principal investig-4tor ' Research associate,, Anili Patel Technical . Secretarial services TPrhniral aSSistant o&Sr, wct ~ I!. Consumable Supplies (fistiby categories) •. Chetnicals 380~ :•- t-:nart paper, stanonery aW. Hardware, small tools 75. Glassware 160'. Silica curvettes (41 80: C. Other Expenses (itemize) Craftsman's services 70. Communications, xerox 80. T`tavel (to consult with-colleagues and to present results ) 450. Publication costs 70.. Animalsand animal care 800. D. Permanent Equipmentfi(itemize)' •.Tanks and- accessories for thin-Iayer chromatography 350. E. Overhead (7 5% oF A+ B+ C) aao-wrar Sub-Tota1 I 1591. 15911. Total Estimoted Future.Requirements: 12548, Salaries Consumable Suppl. Other Expenses Permanent Equip. Overhead Total Yean2 ~ 745. 1470. none I654•. 12681. i l Year 3 It is understood that the applicant and instituiional officers In applying for a,grani have read and found acceptable the Council's "Statement of Policy Containing Conditions and Terms Under WHith Project Grants Are Mbde." " Signature~ ~ _ Dir.amraf Fr~.e 713'SO 5 - 4="K `_

List finoncial support for reswrch from oll sourc.s, lncluding own Institution, for this and/or ralated research projeds. No support for this p Source ~oject is available from other sources. ~%d Amount Duration ~

R: REDACTED MATERIAL M.; 3 c. 1557 Columbia: University Ph• D Faculty of Pure Science . , , . A.M. 1952 same :,;. ...:........:~ 1950 Long Island University ~:~ - - ~.-. . _.. . . . . . . .. .- ... . _ ... _ : - ;.1964- Assistant Professnr, Department of Biochemistry and Nutrition,•' University of Tcxa•s Medical BrAnclr Galveston, Texas: , ::~w: =~'~ ~ - • = - r= ~ • . . . _. _ .. . ~ i ;:u;. - - . . . . .. . .. .. . . " _ •'.:~.'-~i 'r: . :d!§-.-. r;•.~• 1964- Collaborator United States Depty'of A riculture 1963-1964 Chemist (Biochemist), USDA, New Orleans. 1961-1963 Associate Professor, Southern Illinois University, Edwardsville. 1957-1961 Assistant Professor, Lolrola-University, New Orleans. 1954-1956 Lecturer, Hunter College, New York. 1950-1953 Teaching Assistant, Columbia University. . Occasional Editor: Macmillan, Acad. Press, Schol. Lib. #I Pre~ ent areas of scholarship: The enzymes of vectorial ( transport ) metabolism, relationship of subcellular structure to enzyme function, microcalorimetry applied to biochemical events. ~ .. , -•~.. _ Manuscripts in preparation: . , ~ . Membrane ATPase and muscle disease; enzyme studies in the -' myopathic mouse; routine calorimetric medica•1 analysis; a book 'Biochemical Microcalorimetry' being written together with a group of distinguished collaborators, . . ..~ "; "•~ under my editorship, will be published by Academic Press. ~ . ~ ..~; . . . . , - • - . . ~ . - .~ ~<+r-,`,•`a Recent bibliography: i Brown, H. D., Chattopadhyay, and. Patel, A. B. ('1967) Erythrocyte abnormality in human myopathy. Ms. submitted. . Patel, A. B., Brown, • H. D. (1967) On the mechanism of trypsin esterase activity. Ms. submitted. Brown, H. D., Rigdon, R. H., Chattopadhyay, S. K., and Patel, A. (1967) O Enzyme studies in the myopathic duck. r~:s, submitted. C ' • • . • • ~ ~ . . _ _. . . _:..

; Brown, H. D-., Chattopadhyay, S. K., and. Patel, A. B, (1967) Erythrocyte ghost ATPase. Ms. submitted. . Patel, A. B., and Brown, H. D'. (1967) Selective deamination of.nucleosides by 2, 4 dinitrophcnyl hydrazine, Nature, 214: 402. Brown, H. D., Chattopadhyay,'S. K., and Patel, A. (1967) Properties of butanol- extracted and detergent-solubilized membrane ATPase. Arch. Biochem. Biophys. . 120: 222. Schuster, C. F., Shannon, G. R., and Brown, H. D. (1967) Temperature control of collected. fractions. Ms, subn;ittcd. ~:;; adh a S K and Patel A (1967 ) The a arent multi- Brown H D Chatto pp y ; p y .,,,••,,• >^r{ plicity, of membrane ATPa se activities. Biophys. J. Supp , VII, Abst. VYA8. y' Chattopadhyay, S, K., Patel, A., Rigdon, R. H., and Browns H: D. (1967 ) ~' _;'~~ s~ Transport enzyme abnormality in myopathy. , Proc. 7th Intl. Biochem. Congress (abst,) in press, ,- . Brown, H. D., Chattopadhyay, S. K., Patel, A., and Rigdon, R. H. (1967 ) ; ;- Glycoside effect upon membrane enzymes of erythrocytes and muscle in duck myopathy. • 'Experientia, In rress, =, Brown, Ii.D„ Chattopadhyay, S. K., and Patel, A. (1966) Sarcoplasmic-reticulum ATPase upon a solid support. Bioch. Bioph. Res. Comm. 25 (3 ): 304-308. Brown, H. D., Chattopadhyay, S. K., and Patel, A. (1966) Restructuring, by =tY' linkage to insoluble matrices, of enzymes solubilized from cell particles. J. Cell Biol., 31 (2): 17A (abst.). - Brown, H• D•, Chattopadhyay, S. K., and Patel, A, (1966 ) Characteristics of an ATPase in membrane particles, solubilized, and linked to a cellulose matrix. i E l i .,.. nzymo og a n press ,. Brown, H. D. (1966) Simultaneous 3-element microcalorimetry of experimental .' and control enzyme reactions. Proc. Second Intl, Biophysics Congress. #6 (abst. 92), Brown, H. D., Patel, A., and Chattopadhyay, S. K. (1967) Cellulose-matrix supported biological substances. Ms. submitted. ' Brown, H. D. (1966) Azasteroids and heart adenosine triphosphatase. Biochem.- Pharma.col., 15: 2007-2011. Brown, H. D., Jackson, R. T., and Wai.tzman, M. B. (1967) Ciliary process ATPase: azasteroid and erythrophleum alkaloid inhibition. . Life Sciences, in press.; ' Chattopadhyay, S. K., and Brown, H. D. (1966) A new detergent useable in enzyme solubilization. Texas Jour. Sci,, 18 (3): 324. Brown, H. D•, Patel, A., and Chattopadhyay, S. K. (1966) The properties of 1 : apyrase upon a solid support. Plant Physiol,, 41 (supp.) Txvi (abst.}, . • ~. ~.~. Hutfinan, Gary and Brown, H. D. (1966 ) Heart membrane ATPase-model system ~ for inotroPic activity Texas Reports of Biol and Med 24 ( 3 ): 512 (abst ) , , . • • Brown, H. D. (1966) Sampler for automated chemical• analysis. Chemist-Analyst. 55 (3): 88-89. Brown, H. D. (1966) A characterization of the ouabain sensitivity of heart micro- somalATPase, Bioch. Bioph. Acta 120: 162-165. Brown, H. D. (1966 ) Membrane preparation and steroid-azasteroid sensitivity of transport ATPase. In, Sneil, F., Membranes and transport phenomena. Biophy. Soc., St. Louis. Brown, H. D. (1966 ) Membrane. preparation and steroid azasteroid• sensitivity of transport ATPase. Biophys. Jour, VI Supp.: 135 (abst.). Brown, H. D•, Neucere, N. J,, Altschul, A. M., and Evans; W. J. (1965) Activity patterns of purified ATPase from Arachis. Life Sciences 4: 1439•1447. i ~ 100354'7006 • =

R: REDACTED MATERIAL = calorimetry: schema for the continuous observation of the movement of Brown, H. D., Evans, W..J., and Altschul,, A. M. (1965 ) Applications of glucose across a biological rrtembrane. Bioch,. Biophys. Acta 94 (1): 302-304. ': Altschul, A. M., Evans, W. J., Carney, W'. B., TVicCourtney, E. J., and Brown, H. D. :: (1964•) Some aspects of preparative electrophoresis on polyacrylamide gel: ' application to bovine serum albumin. Life Sciences 3: 611-615. . Brown, H. D., and Altschul, A. M. (1964) Glycoside-sensitive ATPase from -'fl,rachis hypogaea.--Bioch. Bioph. Res. Comm. 15: 479-483. N. J. (1964 ) Char- Altschul A. M., Evans and Neucere - Brown H. D. W. J. , ,• , , , , , acterizaUion of a purified ATPase from. Arachis hypogaea with special reference to the relationship-of'activity to conformation. Plant Physiol. 39 ( supp ) lxi-Jxi.i " (abst.). t3rown, H. U., tvans, W. J., antl Altsctlul, A. M. ( lyb4 ) Study oL A"1'Pase by differential calorimetry.. Fed. Proc. 23 (2): 175 ( abst .) . Brown, H. D., Jackson, R. T., and DuPuy, H. J. (1964) Transport of sugar i:n ' Allium: Effects of inhibitors and ethylene. Nature 202 ( 4933 ): 722-723. 0 calorimetry of ATPase activity in potato apyrase and red-blood-cell ghosts. - A Brown, H. D., Evans, W. J., and Altschul, A. M. (1964) Analysis by differential -`s- (1964) Properties of a preparative polyac .rytamide gel electropharesis column as •'Life Sciences 3: 1487-1492. ;: Altschul, A. M., McCourtney, C. J., Evans, W. J., Carney, W. B., and Brown, H "; they affect yield and column stability. ACS al?st. Chicago, Sept. 1964. Brown, H. D. (Ed.) (1963 ) Cell Interface Reactions. Schol. Lib., N~. Y. epidermis. J. Cell. and Comp. Physiol•. 61 (3): 215-222.. • Jackson; R. T., and Brown, H. IJ. (1963 )~ Cation and glucose transport in onion ~ Memberships: I

Application for Research Grant NICOTINE EFFECT UPON CARDIAC' MEMBRANE ENZYMES Harry Darrow Brown The University of Texas Medical Branch at Galveston Galveston, Texas 77550 713 - SO 5 - 1107

Diazacholesterol effect upon membrne ATPase HARRY DARROW BROWN, SWARAJ K. CHATTOPADHYAY and ANIL B. PATEL' :Galveston Texas Conditions resembling myopathy have been induced in experimental animals by the administration of steroids. Response of animals to these compounds has been reported as characteristically a moderate to severe muscular weakness bearing a after contraction, indistinguisha?;le from naturally occurring rrnyotonia. ~ and in the goat reported that the drug induced a delayed muscle fiber relaxation similarity to myotonia.la 2 Winer, et al.3 using 20, 25 diazacholesteroli in man We have found in man and in ;he duck4t 5 that certain, myopathic states can be correlated with an altered respor.se to the cardiac glycoside, ouabain, of rnembrane- skeletal muscle. In the present study we undertook a proionged' administration of bound ATPase from erythrocyte ghosts and! from the sarcoplasmic reticulum of 20, 25 diazacholesterol to rabbits while monitoring the response of isolated erythro- • ATPase preparations was assayed at the termination of the treatment period. cyte-ghosts ATPase to ouabain. Effect of the drug course upon muscle membrane Fifteen male rabbits (6-7 lbs.) were used in three groups. Each animal from Group I was treated with a 5 mg daily dose of 20, 25 diazacholesterol in 1 ml of water daily. The control Group I1'_. was maintained upon a daily administration of of the drug which were progressively increased from 10 mg to 50 mg in 1 mii of water intramuscularly. Similarly, animals from Group II were given higher doses 1 ml of distilled water. From the Biochemistry Departmezt, The University of Texas Medical Branch at Galveston, Texas.

Nine to ten ml of blood was collected in• a glass tube containing 10 ml haemo- lyzing solution ( Tris buffer, 0.002'M, pI-: 7.4, with 0.005 M Na2EDTA ) from each animal. Blood samples were co;ic ::od before the treatment and~ after 7, 8, 1'.' and 13 days of administration. Ske1, <: muscle samples were collected after 16 days of drug therapy. Each time, trc :'•'Iod' haemolyzate was centrifuged at 20, 000 x g for 20 minutes,` The supernatant .:.s discarded and the pellet washed and recen- , : r ..~ trifuged 4 to 5 times in the same ',is buffer, and 2mM NaC1. Before each wash p waer theellets hornognized in a I;•c.xer driven Teflon-pestle homogenizer fo 5 minutes to ensure complete disi,i ion of blood cells. The reddish-white pellet obtained in final centrifugation wa_- collected and stored at -10°C as the erythro- cyte ghost preparation, and used as the source of ATPase activity. Muscles were macerated~ in 10 volumes of 0.1 M cold Tris buffer, pH 7.2, with 0.25 M sucrose preliminary to separation of the membrane fractions by centri- ' fiigation. The slurry was first cerLtrifuged 600 x g for 20 minutes. The pellet was rejected and~ the supernatant dialyzed against the same Tris-sucrose buffer with~ 5 mM Na2EDTA for 4 hours. Afte-=ward the dialysates were centrifuged 10, 000 x g and 20, 000 x g for 30 minutes, and'i each time the pellets were discarded. This supernatant was then centrifuged in 10 ml tubes at 80, 000 x g for 30 minutes and again 100, 000 x g for 70 minutes. The 100, 000 x g pellet was resuspended in 2 ml- Tris-sucrose buffer, pH 7.2, as the enzymatically active fraction ( sarcoplasmic reticulum ) . ATPase activity was measured as inorganic phosphate evolved in reaction mixtures containing 0.1 ml of enzyane preparation (ghost or membrane fraction 0.8 ml substrate ( 0.1 M Tris-sucrose buffer, pH 7.2, with~ Na2ATP r0.3 mg/ml of reaction mixture~ together with 0.001 M MgC12, 0.0021VI KCl, 0.001 M NaCl ), 0.1 ml water or of inhibitor in water. Multiple reactions were run so that they might be taken as samples in sequence to allow consideration of the extent of reaction as a function of time. Reaction mixtures were incubated at 42°. After incubation, i 0.1 ml of 50170 trichloroacetic acid was added and the mixture was centrifuged at ~ 600 x g for 6 minutes. The supernatant was assayed~ for inorganic phosphate. C Protein was determined in sample aliquots of the enzyme preparation. 0 C4 ~ ~ ~

Blood was drawn from each of thc- animals before the initiation of the admin- istration of diazacholesterol .' At this time erythrocyte -ghost ATPase activity tvas inhibited by 10-4.IVf ouabain in the incubation mixture, without exception. ' In the first experimental group, animals receiving 5 mg daily doses, the erythro- cyte adenosine triphosphatase activity was stimulated by ouabain, rather than 'inhibited as were the control preparations. This was true also of preparations from blood of rabbits in experimental Group II, which had received larger doses. These data are presented in Table I. Muscular weakness began the 4th day of drug administrati= and became pro- gressively severe. This was accompanied by an apparent muscle atrophy. Figure 1 A is a graphic representation of the result of a series of experiments in which the catalytic activity was inhibited by 10-41VI' ouabain. Identical experi- in which ATPase activity of ghosts was plotted as a function of time and compared~ (dotted line ) with a similar series, using ghosts prepared' from the same animal, reticulum ) ATPase was similar to that apon the red-blood-cell ATPase; enzyme The results of the drug course upon tInc t00, 000 x g muscle fraction~ ( sarcoplasrnicc effect of ouabain here was to stimLlatc: rather than to inhibit the ATPase activity. ministration of 20, 25 diazacholesterol LLre presented by Figures 1 B and 1 C. The ments using preparations from anirnals which had been carried on courses of ad- stimulated. Data obtained in representative series of experiments is presented in activity of'control animals was inhibited while that of the experimental animals was Figure 2. The characteristic response of membrane ATPase systems to ouabain is an inhibition of the rate of catalytic hydrolysis of ATP. Enzymatic activity of ghost preparations and a muscle merrnbrane fraction from untreated rabbits was inhibited by ouabain. Sustained treatment of animals with 20,25 dYazacholesterol changed the character of the ATPase activity such that a stimulation of the catalytic rate was effected by the presence of ouabain in the incubation mixture. We have

suggestedb, I that the ATPase respons:, to ouabain is a reflection of the conformation of the enzyme which it derives in part from, its association withi the membrane. Myopathic changes in skeletal,muscle following the administration of cortico- steroids has been discussed by Awad, ct al.1- In their experimental work the possibility_of neural induction of the observed myopathy was eliminated and they concluded that steroid myopathy is a primary muscular disorder. .The use of 20,25 diazacholesteroi as a cholesterol lowering agent was discon- tinued in medical, practice when, it became known that the site of action was at the _point of coiiv~rsion of desmosterol to cholesterol resulting in the accumulation of desmosterol. The present results wL'.:.?z show an effect upon membrane related! ion ,transport enzyme (Na+ + K+ -ATPase: may provide an element of support to the : thesis that diazacholesterol causes myatonia by affecting the muscle membrane system. Such an effect could conceiva:)iy follow a change in the lipid pool from which the membrane elements in turn, are synthesized. It is known that the ATPase interrelationship of lipid pool to functional membrane is a matter which requires, activity is affected by changes in the lipid components of the membrane 8 The and is subject to, direct experimental test. Rabbits in the experimental groups visibly showed indications of changes in muscle tone which may relate to the observations of Winer, et al 3 that the diaza- - cholesterol is capable of inducing myotonia. The transport ATPase response to ouabain which we have found in erythrocytes from myotonic patients4 is paralleled port phenomena and appears to indicate that diazacholesterol adrninistration directly affects the membrane transport system. f4id'i.ngs a-ccord _with the common supposition that myotonia involves aberrant trans- by the present experimental results in nhe diazacholesterol-treated rabbit. These 5

Table I'. Effects of ouabain upon a:"' Psse activity# ( µmoles Pi/mg Protein/min ) _ Group II progressively increasing c;oses 10-50 mg/day; Group III, controls.) of erythrocyte ghosts .( Group I a~: :~als received 5 mg diazacholesteroL/day• 6 140 mg _10 Group .roup III. 360 mg 140 mg 360 mg 180 mg 36&mg 360 mg - 310 mg 360 mg Activity Control Ouabain 10-4 M % stimulation inhibition 0.20 0.27 +35 0.22 0.26 +18 0.16 0.17 + 6 0.19 0.21 +10 0.48 0.47 - 2. 0.26 0.39 +50 0.19 0.19 + 0 0.40 0.59 +47 0.35 +52 0.21 0.25 0.39 0.36 0.13 0.15' +15 0.32 0.36 +12 • 0.29 0.46 0.28 0.55 0.24 0.49 +58 +96 + 104 0.15 0.24 +60 0.21 0.32 +52 0.14 0.11 -21 0.16 6.11 -.25 0.14 0.06 -57 0.15 0.12 -20 0.44 ' 0.28 - 36 * Upon the basis of 40 minute incubation.

REFERENCES . Faludi, Georgina, Mills, Lewis C., and Chayes, Zev W.: Effect of steroids on muscle, Acta Endocrinologica 45: 68-78, 1964. ;•_.; ir.'. . ._ .. . ... . Awad, Essam A., Swaiman~ Kenneth F., and Kittke, Frederic J.: Changes in the structure, innervation, electromyographic patterns and enzymes of skeletal muscle resultind from experimental treatment with triamcinolone, Arch. Physical Med. and''Rehabilitation 46: 297, .1965 . Winer, Nathaniel, Martt, Jack M., Somers, John E., Wolcott, Lester, Dale, Homer E., and Burns, Thomas W.: Induced myotonia in man aad goat, J. of Laboratory and Clin. Med. 66: 758-769, 1965. . Brown, H. D., Chattopadhyay, S. K., and Patel, A. B. Erythrocyte ab- normality in human myopatl-: yT . Ms. submitted,. . Brown, H. D., Chattopadr yay. S. K., Patel, A., and Rigdon, R. H.: Glycoside effect upon n-.er_z:;:-:ne enzymes of erythrocytes and muscle in duck myopathy. Expcric_ _ia, in press. • Brown, H. D.: A chasacterizarion of the ouabain sensitivity of heart microsomal ATPase. Bioch. Bioph. Acta 120: 162-165, 1966. . Brown, H. D., Neucere, N.J., Altschul, A.M., and Evans, W. J.: Activity patterns of purified ATPase from Arachis, Life Sciences 4: 1439-1447, 1965. . Schatzmann, H.,J.: Lipoprotein nature of red cell adenosine triphosphatase, Nature 196: 677, 1962. a 4 . ~: ~:•4. 4 yY. +~°'S•i 1:r #Z>ti :.i 'A,,' ~~ L' ;h,

Y:n! r 1s , s. t (Abstzact ) . . . .. _ .. . .. . .. . . - . _ . '. . ..v.. ATPase activity of erythrocytee ghost prer.arations and of a muscle membrane fraction (sarcoplasmic reticulum )-frornl untreated r~.bbits was inhibi'ted by the cardiac glycoside, ouabain.. Sustained treatment of animals with 20,25 diaza- ~. ,_ . . .~., cholesterol, reported by Winer e`t al.,( this Journal, 66: 758, 1965) to in-, " R. ,<duce myotonia, changed the character of the ATPase activity of identical . .. . . ' . . . . :Y: preparations such that a stimulation of the catalytic rate was effected by the . presence of ouabain in the incubation mixture. We have earlier suggested that the ATPase response to ouabain is a reflection of the conformation of the enzyme which it derives in part from its association with the membrane. The 'present findings accord with the common supposition that myotonia involves .`aberrant transport phenomena and~ appears to indicate that` diazacholesterol administration directly affects the membrane transport systemi. • ;sq vr. X! fu .,, w! s s. r x . ... , ._. .., . _ . ., , .. , _ .. - . _ . .

ARCHIVE VARIANCE SHEET THE NUMBER ( RANGE ) I O QJ'J'g' rI ©17 I S ( ) MISSING ( ) MISSING IN ALL SETS MISSING IN CLOSED AND REVIEW SETS ( ) DELETED ( ) CHANGEDTO ( x) DELETED AND CHANGED TO ~Oo~JJ4 LQaG~S A ( ) NOT USED ( ) OTHER DATE OF CHANGE CLOSED SET REVIEW SET WAREHOUSE SET

III: IDOUXCIL FOR TOBACC0 RESEARCFI - U. S. A. -r MEMORANDUM = z ''D ! Y rYi ' S ~ ~ t ..L+r w : 'h Au u t i8, 1 6 g s 9 7 TO: Tie committee comprising Dr. Bing, Chm., Dr. Cattell aid Dr. Jacobson. FROM: -': Robert C. Hockett SUBJECT: New grant application from Fritz K. Beller, MD., Sc.D. - No. 627. We enclose herewith a new grant application from Dr. Fritz K. ` Beller, New York University School of Medicine, New York, New York. The author has developed a method for producing disseminated '''intrava.scular coagulation in rabbits by the infusion of endotoxin. Using this model he proposed to determine the effects of various factors includ- ',:, ing tobacco combustion products. Details of the technique for administer- ing ing the combustion products of tobacco are not revealed. R. C. H. In support of this application we enclose seven~reprints: a4 ,i s ., Sw 1. Beller, F.K. and. Graeff, H.: Deposition of Glomerular Fibrin in the Rabbit after Infusion with Endotoxin. Nature, in ,,. :., press. (July 15-copY) .; ., _. .. . , r s ?x~~ . . K Y ~F; :a' r 5. . Beller, F.K., Mitchell, P. and Gorstein, F.: Fibrin Deposition in the Rabbit Kidney Produced by Protease Inhibitors. Tbrombos }t,~ , ~~ Diathes. Haemorrh. 17:~+27,(1967) < exper. Med. 118;245,(1963) of the Lethal Effect of Endotoxin by Heterologous Plasma. J. Beller, F.K., Debrovner, Ch.H. and Douglas, G.W.,: Potentiation Enzyme System in Newborn. Amer. J. Obstetr. Gynec. 96:977, (1966) Beller, F.K., Douglas, G.W. and Epstein, M©.: The Fibrinolytic Beller, F.K. and Porges, R.: Blood Coagulation and Fibrinolyti~c Enzyme Studies during Cyclical and Continuous Application of Progestational Agents. Amer. J. Obstetr. Gynec. 97:j+~48,(1967) 6. Maki, M. and F.K.Beller: Comparative Studies of Fibrinolytic Obstetr. Gynec. 20 117, (1962 ). 1Q03C,~,•M $ Inhibitors in Vitro. Thrombos. Diathes. Haemorrh. 16:668,(1966) Beller, F.K., Goessner, W. and Herrschlein, Hans J.: Tissue Activator of the Fibrinolytic System in Placental Tissue. .:. . _ - _ : ,.- ,.. . .. , . _ - . . , .. . , . '.. ..~~. . . . Y~ .lL. Y a.w .M _...•... ,:LM1t.64'-

THt: COUNcIL FOR TOBACCO RE SEARCH - U.S.A sss TrMin bFF1v'crE COPOffTTEE: NEw YORK. rr. Y: 10017 . Dr. Bing Dr. Cattell Dr. Jacobson Dr. Lynch _... _... . •: ....._ _. ~~~ ._.., .. .. .._ . ... - ..: D Med li M it K B F ,, . . z e er, r . ::~ ~. ; ' ` 2. Institution & New York University School of Medicine :, 550 First Avenue, New York, N.Y. 10016 4. Proposed Starting Date: pcCober,1, .1967 ~ :° 5. Anticipated Duration of this Specific Study: 3 years • 6. Brief Descripton of Objectives or Specific Aims: •'. The inference of tobacco combustion products, including nicotine and carbon Address: _.which intravascular coagulation is initiated under carefully controlled monoxide on blood coagulation will be studied in an experimental model in - condit..ons. : =• intravenous infusion of a sublethal dose of endotoxin in rabbits produces : It has been shown recently'in this laboratory that a single continuous _ glomerular fibrin deposition and renal cortical necrosis. Although the .histopathology of fibrin deposition is identical to that of the generalized ` . =: Shwartzman reaction, this experimental model differs from that of the generalized Shwartzman reaction where two injections of a sublethal dose of endotoxin are administered 24- hours apart. The difficulty of interpreting the overlapping effects of tcvo doses of endotoxin is avoided by the continuous intravenous infusion, which is also able to initiate fibrin deposition as a dose related phenomenon. The pathophysiology of disseminated fibrin deposition has not been fully elucidated. However, endotoxin has several distinct, direct and indirect effects on blood coagulation. Platelets .•. fall in a linear fashion, followed;by a subsequent decrease of the "consumable" coagulation factors including fibrinogen. Leukocytes decrease initially but increase after 4-5 hours during the course of the infusion. We have observed that the complement titer falls in concert with hemolysis and .• fibrin N. deposition. The thrombi in-the lung, liver and heart developed in an O irregular pattern in the early phases, that is 2-3 hours following the C initiation of the infusion lomer ot seen until fib l i d iti i ; g ~ u ar n on s n r epos approximately 6 hours after th n R l t t d t ti f , on. a e e s ar o ra en otoxin adminis WDA h td i l d ocvn • s u s re ate to glomerular fibrin deposition. The development of glomerular capillary necrosis is apparently dependent on the following: C5 (con tinued on next page) .: 7. Give a Brief Statement of your Vi/orking Hypothesis: Our initial observations indicate that the infusion of endotoxin in rabbits .' produces a reliable and reproducible method for disseminated intravascular (continued on next page)

. Question 6, continued; .2. the reactivity of the kidney andits ability to lyse fibrin 1. the amount of glomerular involved 1. the gaseous combustion product of tobacco in its entirety The study proposed is designed to examine the effect of: 3. the size of the dead vessels involved 2. nicotine . carbon monoxide ..- i... In both acute ad chronic experiments utilizing animals n : pretreated with a continuous intravenous infusion of doto i ._, : en x n. .coagulation as well as the initiation of the fibrinolytic system. =*:~ :.Such a model permits the investigation of a variety of substances ~ •` which might either protect the animal on the one hand or d) any combination of these effects. c) the failure of lysis of fibrin or delay of lysis of fibrin_ -b) the earlier initiation of coagulation or fibrin deposition. . } .. .:~ • . fibrin deposition ,~r? a) in the lowering of the total dose of endotoxin required for. e es n severa Y man was: Y th if t i l the combustion products of tobacco effect the coagulation system per:~g, =^x?. en o ox n on e o er I . potentiate the dilatarious effects of d t i th th f W`In addition, an effect not obvious in the untreated animal may `become manifest. .

E. DeloitsofExparimentalDesianandProced~res:.fAtfach5en.,..,t.v.....a While the basis of the investigation will be the infusion o$ rabbits with a con.tinuous dose of endot 2 o 0 8 1 = Plan 1: In the study of the acute effects of tobacco combustion products, animals will be infused with endotoxin and ex o d' t xin ( - 0 mg/Kg/hour) for periods of 6-14'hours, several distinct experimental plans will b f 1 ; o nicotine, carbon l p se t monoxide and tobacco smoke Either si . mu aneously o r immediately prior to ~ -= -ttie-infusion of endbtoxin animals will be t di s u ed with rest t : sia:; peco: ~;Wa) the dose range of endotoxin in controls as comparedto treatd 4 e 3 l ., an ma s « tt; b) the time lag,required for lomerular fibrin deposition, and -c) ariy change in the g ~~;f. parameters to be studied by serial blood sampling. ,~'.*:~ In the second plan endot ox l in wi l be infused for a period of 10-14 G'h i CfiQuK$3nfold•owing which, one kidney will be surgically removed (control) . _--_ The_animals.will then be exposed to tobacco combustion products for an additional 10-14 hours and sacrificed. The kidney so obtained will be --oompared•to that of the control kidney with respect to-the depos3stion of - ;. fibrin•in glomerular capillaries. In plan 3 the animals will be exposed to-tobacco combustion products over extended periods of time and subsequently infused with•endotoxin. The ''do'se"rarige and- parameters of coagulation will be studied in, comparison with that of the controls. (contindt) ue on nex page 9. Physical Faci(ties Available (Where Other 1han,Adminiatering-Organization Indicate Geograpfiicaf location)' -'A fu11y equipped laboratory for the study of blood eoagu3ation and,the fibrinoiytic enzyme system,with~standardized techniques as well as histological and histopathological techniques ' i l b f C• 10. Adddwnal$equirements:. Smoking machine ( at proposal of Dr. Kuschner, Department of Pathology): `: C~11. Biographicol4ketcNes of all principal and professionol personnel (append) check appended:pages 12: List ofpublications: (Five most recentos pertinent) (append) check appended pages ` s avai a le. Thas.laboratory is located in Bellevue Hospitali as part of the Department of Obstetrics.and;Gynecology.

Methods: Among the methods emp-loyed will be frozen and permanent tissue =1ung, heart, pituitary as well as kidney. Immunofluorescent methods demonstration of fibrin. Among the organs studied will be the liver, sections utilizing conventional histochemical techniques for the .to substantiate the specificity of fibrin deposition. Histochemical :employing anti rabbit fibrinogen.produced= in the rat will be utilized ':; identification.of tissue activator will be studied utilizing the . The coagulation system will be studied from the following points: . ,fibrin films and,suitable tissue sections (Todd). c) urinary output, d) body temperature, e) blood pRand pC02, f) leucocytes., g) hemolysis. (Johnson et al.) fibrin plates heated and unheated (Astrup). : Biological parameters: a) arterial pressure, b) osmolarity, :4:.:..'Study of the fibrinolytic enzyme system: a) euglobulin lysis ° time, b)plasminogen, c) plasmin inhibitor and kinase inhibitor factor VIII levels (TGT) : 3- thromboelastogram (Hartert) . 1- platelet enumeration (phase contrast) .2- fibrinogen (Ratnaff and Menzie), factor V (on phase method) and Question 11: ' D. 1955). Assistant and Associate Professor, University of Tuebingen.1:956- ;i Research Council 1954},Dozent Obs & Gyn, University of Giessen (Med. Sci.::':; Internship and residency in Obstetrics & Gynecology, University of Giesseml948-1954. Max Plank Institute of Radiobiology (Trainee German -Principle Investigator: Fritz K. Beller, M.D., age 43. MD Marburg 1948, -1961. Professor of Obstetrics & Gynecology, Tuebingen 1961.- Visiting Associate Professor, NYU School of Medicine, Dept. Ob-Gyn, ..1961-1963. Associate Professor, Dept. of Ob-Gyn 1963-1967. Professor .of Ob-Gyn, NYU School of Medicine, September 1, 1967. Career Scientist •,!~1.i; .•:- German Specialty Board, 1957. Diplomate American Board or Obstetrics or the Health Research Council of the City of New York 1961 to present. .,;04, : and Gynecology 1967. State Board New York 1963. NYU Chapter XS. U.S. Citizenship; 1966. . ., . ., ., g ..Amer Soc Exp Pathol Intern Colle e Pathol New York Acad. Sci . =.~,,.. .f,. Societies: FACOG, FRSM (London) Soc. Gynecol. Investigation, Co-Investigator: Fred Gorstein, M.D „ age 37, MD NYU School of Med icine 1955. `` Intern Bellevue Hospital 1956-1957. Assistant Resident Pathology, Bellevue Hospital 1957-1960. Fellowships: PHS Training Fellow Pathology 1957-19b0, PIiS Post doctoral Fellow Pathology 1960-1961. Career Scientist of the Health Research Council of the City of New'York 1963 - present. Instructor Pathology, NYU School of Medicine 1960-1963. Assistant Professor 1963-1967. Associate Professor, Sept. 1,1967. Diplomate American Board;of Pathology 1961,1967. Societies: N.Y. Acad. Sci., Harvey Soc., Citizen; U.S. - 100354T022

, . . . '*Senior technician: Peter Mitchell, age 25, B.S. Cornell University 1963 Worked in this laboratory since 1963. Continued school at NYU at night. uestion 11: continued: M.S. expected, 1968. Citizen; .f~~ • ;-"•Technieian: Helge Bornhausen; age 33 German Gymnasium Sehool for f.Medical Technicians Mainz 1955-1957. Joined this laboratory in 1966. 4 'C; .-itizen•Germany. :Immigrant Visa. uestion 12: ; 1) Beller,F.K, and Graeff,H.: Deposition of glomerular fibrin in the .. _.3) Beller, F.K., Debrovner,Ch.H. and Douglas,G.W,: Potentiation of -,rabbit after infusion with endotoxin. Nature, in press. il 2) Beller,F.K., Mitchell,P. and Gorstein,F.: Fibrin deposition in the Haemorrh. 17:427, 1967. ~ :-rabbit kidney produced by protease inhibitors. Thrombos. Diathes. the lethal effect of endotoxin by heter•ologous plasma. J. exper. system in newbornes. Amer. J. Obstetr. Gynec. 96:977, 1966. Beller,F.K., Douglas,G.W. and Epstein,M.D,: The fibrinolytic enzyme .Med. 118:245, 1963. . 5) Beller,F.K. and Porges,R.: Blood coagulation and fibrinolytic enzyme studies during cyclical and continuous application of progestational agents. Amer. J. Obstetr. Gynec. 97•448, 1967.

R: REDACTED MATERIAL C A. Solaries (P,ersonnel by.names): Professional Fritz K. Beller, M.D. Fred Gorstein, M.D. (co-investigator) Fringe Benefits 'Technical 1 technician 1 senior technician Fringe Benefits L Consumable Supplies (list by.categories) Glassware Laboratory reagents (chemicals iimnune sera, endotoxins, etc.) C. Other Expenses (itemize) Laboratory animals D. Permanent Equipment (itemize) Thromboelastogram Water bath - Deep freeze unit 2 ovens: slide file cabinet E. Overhead (15% of A + 6fC)1 2601._ 26391 Estimated Future Requirementsc =• Salaries Consumable Suppl. Other Expenses Permanent Equip Overhead Totbil Year2• -.f61GF'- 1500 3000 Year3 r16600i=' 1500 3000. 1/ is understood lhotltHe opplicant'and institutionaliolficern in applying for. a grant have read and found acceptable the Council's "Statement.oF Policy Contaihing Conditions and Terms Under WhicH Pro,~ectlGranfs Are Alade." 500 i.3100• 242_67_ 500`); 3195 .'• 249,95! Signature DK.~..ar.rrol"0R9-3200 ext. 2732 -Telbphone Signature 4 016 i Ih -• i Jr .. ,iU.w ~ e ~. t w . Arms Norg ' rong, M.D. D ixec to r Telephone 0 ~ 4_ `_

i a r~ n OtfMr Sources of Finanetal5upport I Lhtlinanetal support for ns~arch from all sources, lncluding own (nstitution, forthit and/orrnlatod retwrch pro)ec1h, 5Z04,PSE00T i B I Source

. .'tr..~t.. •. •t . . - . . . . • . , . , .. . . -_-- ~ , .. Additional information to Grant reqL-:!st; Dr.Beller. . - L . ~~;'- . . . ... ~ . . .. . . The principle parameter for the evaluation of smoke andismoke substances are primarily not paramiters of the coagulation and fibrinolytic enzyme -system in peripheral blood. They will be assayed but are of secondary i.nterest, The primary interest will be focused on biological parameters in 33CtW--X organ systems, especially the kidney after disseminated coagulation is produced by endotoxin infusion. The experimental design is best appreciated, from the following scheme. ... .:~., _ Removal of Removal of 1 kidney 2nd;kidney ."I. Pretreatment phase:Smoke or smoke substances will be applied to animals ;:'acute or ehronically before endotoxin infusion is induced. •t.Parameters: 1) Assaying the concentration of endotoxin needed to produce fibrin deposition with and without smoke substances. 2) Measuring the time 9 of phase II until fibrin deposition occurs. Assays and methods: 1) histologic sections and 2) histochemical sec#ions of the kidney, p'ituitary, liver and .luno. The kidney removed in different time lags in between 4-14 hours is Urine particularly suitable. 3) Renal excretion study. 4) ?0M osmolarity. 5) Venous and-artea.•ial pH. 6) Leucocytes. 7) Coagulation-assays. 1003547026 II. Infusion phase: Smoke and smoke substances will be applied' duririo phase II together with-the endotoxin infusion. Parameters and assays as under I. III. Postinfusion phase: Smoke and'smoke substances will be applied in phase III after terminating the endotoxin infusion.. 1) Histolooical sections and Development of fibrin deposition Reactive-thrombolysis .Endotoxin infusion for 14.hours Recovery phase

:.histochtmical sections in the second•kidney will be compared to the kid~ie ~ . . - . . - =_ . .. . . . . . . . . . .t,~ _.:~:;~, ; h~: ~ on us (con ro ) This experiment will b d .eone after termination of the inf i ' t l .. . . . . •. •:•.' . .. :,f;e~.tW in order to evaluate inh3bition or activation of the reactive thrombolytic =Y~ ::recoveryy p ase. Additional assays as under 1. h ; ssay 1) Histologicall sectionin thekidney: 100 glomeruli will be counte . ...... d s 'A ~ ~ n-each section and the number of glomerula involved with fibrin depositioli I am-enclosing a detailed procedure of the method. See also, Beller et al.,1961 •: expressed in /. 2) Fibrin layer according to Todd (J. Bacteriol..,78:281.) . rges ,.,.. : and 1967. The coagulation assays are described in Beller and Po l967 $ ;. 3) Activator assay on tissue extracts measured on fibrin plates (Astrup and; ',_~ . ... . .... Muellertz, Lit, in Albrechtsen,O.K.: Fibrinolysis in the org'anism... Acta .. - • , .•.

Technique: (Mitchell, Weiss z ` ~ Prepartif fibi fil .aon ornm: :'bufftr is added to a te--t tube containing 10 ml of fibrinogen solution. . , . .- . ..' , . . ... _ , Bavine fibrinogen highly contaminated 14 ftithaelis Veronal buffer' (pH a 7.35) to a concentration of 300 mg to ~ ~ •tTg per 10 cc. 20 units of Parke.Davis Topical thrornbin in 1 cc of atb i itell euesnvered gnt:y to mix the soutions and is then poured uniforml 1.• . =:.onto an 8 cm x 14 cm piece of wettable cellophane (dialysis paper) wh.ich ;has been saturated with the Veronal buffer and placed ori a perfectly level :"~.surface. The solution is permitted to clot at room temperature and is then placed in a refrigerated (4°C) wet chamber for at least one half hour. 2 x 3 cm pieces of the film-may then be cut fro,n.the 8 x-14 cm piece using a sfliconized surgical scissor. These sections are inverted on clean micro- '' seo slidi tht bbbl f :~pees usng care soa nouesorm under the film. The cellophane is then peeled off leavin; a 2 mm fibrin film on th lid Th e s e . ese ~ films are stable, wheorefrigerated, for three days. Activator inhibitors ~-=may be incorporated into the films prior to the addition of thrombin. If .:.-::. =...:. _- ': ... .: . . . . . . 1the film# ~ to be heated to destroy the plasminogen (S6°C,/3(1 min.), ~.., . . . . : : ., . `~` steps should be carried out in a wet chamber after the ` = ' `' ` • •....:. .. :- .. . . ;> .,._..... ~ o thelid n se. ': ,: Tissue activator assay: film slides with a precooled forceps. If many filras are to be run, they are placed in. the refrigerated wet eli;3mber and t1h,eir incul:atior.s all comnense V • 1003547028 The tissue used in this.assay must be received fresh, and immediately quick frozen onto a cryostat block. In this ste-te.the tissue is stable for several days if it is sealed in parafilm to prevent drying. The tissue sections are then cut 8 microns thick and placed on the fibrin . , .~;

incubation is carried out in a moist 37°C incubator of time. A control slide (0 min=••incubation) is prepared for and to aid in interpretation of themsults. Incubations of 1) rinse slides in tap water bath 2) place in acid hematoxylin 90 sec. (or until red-blue color results 5) rinse in water • 6) 50% ethyl alcohol x 15 min. 7) 70•/G ethyl alcohol x 15 min. 8) 95% ethyl alcohol x 30 min. 9)10(/, ethyl alcohol x 45 min. 10) eosin Y x 20 sec. 11) wash in 100°; ethyl alcohol three times 12) 50% ethyl alcohol - 50% xylol 13) 100% xylol x 20 min. x 20 min. 14) Trim excess fibrim film from the slides with sharp knife and cover with synthetic mounting medium and a cover slip. The entire area under the cover slip must be filled with mountir.g meditnn to prevent drlrinh of the film. 'T};e sVes are •icw CoCi'JIIP.Int; S T-a 6 fe ) QuJ &_ILC~y 6,Z SyoreY; ~r_ot- The slides are best observecd witlh, a lm: pnwer stercoscopic micruscope. Lysed zones will first appear as depressedi ar.eps under the tissue section. 3) rinse in water 4) develop in dilute ammonia water until blue -*.Sei.vely inactive tissues have been carried :~r~ods of up to 24 hours. The reaction - 4 LCl% €ormaline-saline solution. : .~_ . ~: ..• . • .~. . ~ .' .' Staining procedure: 9

. . . . . . .:'~:'~J; may be made with reference to the time of incubation necessary : ~Semi-qeiontitative comparisons of activator activity in different ; ~ Sli~.fi iubated fc~r tonger pe : ~ds will have holes through the entire ri . . ..._ . . . .. . .. .. . . . "• -- • . .x, ..} fibrin film at areas under the tissue that correspond to the earlier, . . • #• - .. . _ . • ... . <; n ftresaions.-.Lysi:i an hea'ted_•films or those containing activator ,~ :~inhibitors ( e.q. K_x 10'~'i~AEA) is indicative of proteolytic activi in the tissc.e not due to plasminogen activator. ,.to procl~sce a certain observed amount of lysis. However, these comparisons mwat be made only between slides which are prepared with the same batch ". of fibrinogen on one film and with simultaneous incubations. It is preferable to use one batch of fibrinogen in which the plaminogen contamination is known (Ear assay in the true sense of the definition since the plasmin spreads. The Gh3~e~,=`-~xk~-: The method is not a histochemical localization is therefore dependent on a proper incubation time. Excessive-incubation of the slides results in autolysis of the entire tissue seetion:'';~:` 1

We enclose herewith a new grant application from Drs. Theodore N. , • _ . , . . . . , .SUBJECT: New grant application from Theodore N. Finley, M.D. and Aaron Ladman, Ph.D. - No. 628. Finley and Aaron J. Ladman of the University of New Mexico School of Medicine, Albuquerque, New Mexico. The application is supported by reprints of four recent papers on related subjects by the applicants: The Anatomical Record 157, 559 (1967). Comparison of the Composition and"Surface Activity of "Alveolar" and Whole Lung Lipids in the Dog. T.E. Morgan, T.N. Finley and : H. Fralkow. Biochem. Biophys. Acta 106, 403.(1965). Alterations in Pulmonary Surface Active Lipids during Exposure to Increased OxygenTension. T.E. Morgan, T.N. Finley, G.L. Huber and H. Fralkow. J. Clin. Invest. 44, 1737 (1965). Bronchopulmonary Lavage in Normal Subjects and Patients with Ob- :.structive Lung Disease. T.N. Finley, E.W. Swenson, W.S. Currany .,G.L. Huber and A.J. Ladman. Ann. Internal Med. 66, 651 (1967). The Fine Structure of the Ductuli Efferents. A.J. Ladman. General evaluation of the scientific merits of the proposal committee. Staff questions are: To what extent would this study duplicate or supplement others now in progress under CTR or AMA sponsorship? 2. If cigarette smoke inhalation by dogs is an essential feature of the project, do we have suitable techniques for monitored and con- trolled administration of whole, fresh, "normal" smoke? Are the applications willing to accept the best suggestions and guidance that the CTR staff canprovide in this area from accumulated ex- `r,~ i'SY4 '';i{3° "y ugust± 17s '1967 perience? 100354'7032 3. In their experimental design will they distinguish between chronic differences between smokers and non-smokers and short-term effects of recent smoke exposure?

:.: rPHYSTOI,OGY OF THE RESPIRATORY SYSTEhI: THB COUNCIL FOR TOBACCO RESriARCH - U. S.A. 633 TIICFtI7 .dVENLTE NMSr YOR%, N. Y. 10017 Date: ? August • .' Name of Inve3figatar(sj: (iric(ude Title and Degrees) • :..; Theodore K. F i n 1 ejr;-'M,D. , Assoc i ate Profes sor of Med i c i ne -'Aaron J. Ladman, Ph.D., Professor and Cha•irman, Department of Anatomy. 2. Institution & : Address: : r e 2'ic' : ' The Undvers_ity_ o• N6r Flex i co School of Med i ci ne -915 Stanford Avenue N.E. Albuquerque, New Mexico 87106 3: Short Title of Project:: .. . . -Effect of cigarette smoking on lipids and morphology of alveolar lining material AS and macrophages. Proposed Starting Date: ~ ~' January 1, 1968 :: 5. Anticipated Duration of this Specific Study; ,. 3 years to December 31, 1970 '' 6 Brief Descripton of. Objectives or Specific Aims: . Four studies will be undertaken: :' d a d r h li l "' f ff th i h f i ki i D ~ ~ s n mo o n on e o ono e e ect o c nat t eterm 9arette smo (a) 9 P P 9Y:w;~ . non-smoking humans. of alveolar lining material and macrophages recovered by saline lavage of smoking and K. ~ lo `' '`Determinetion of acute an b d chronic effects of smoking orn liPi. ds and morPho { ),._._..._ 9Y of dog alveolar"lining material and macrophages in situ as well as on fractions recovPred ; a 1i i ne lMage. circulating whi!te cells with radioactive lipids and amino acids and monitoring!the transport of these labelled'cells in:to the alveoli of the lung by radioautographic microscopy. ..(c) Determination of the effect of cigarette smoking on the incorporation of radioactive lipids into alveolar lining material and macrophages of the dog. (d)"~ Determination of the origin of the alveolar macrophage by labelling ,7. Give a Brief Statement of your Working Hypothesis: -.. . . ,- - . . . (See attached sheet) _ ~_.~,. . .....-.~........ ......... ~w+ir:y.~,,.._. ......:..,a::u.,...~:.........~.w~...~..1.~..:...~..

Detaili of Experimentol Design and Procedures: (Attach Separate P4ges) 9. Physical FacSlties Available (Where Other than Administering Organization Indicate Geographical!tacalion) See Attached Pages ( 10. Additional Aequiremenfs: See Attached Pages C 11. Blographical sleetches of all princlpal and professional personne( (qppend) See AttacHed Pagt?s I 12. List of publications: (Five most recent as pertinent) (append) tSee Attached Pages 17"ft. -NUM.- E'-"-PMNWM Eft"ft-=,F-

;; Preliminary experiments have shown morphologic differences in* the alveolar macrophages recovered by saline lavage of smokers when compared to non-smokers.'~.;" The alveolar macrophages of smokers contain an increased number of inclusion " bodies of various types. In addition, the fatty acid distributi-on patterns.of the phospholipids extracted from both the cellular and non-cel,lular fractions of the lavage differ in smokers and non-smokers. There appear to be more unsaturated long chain fatty acids in the phospholipids of smokers. More studies. are necessary to confirm these.observations. . _ : . . • . .. . . ,.;: The incorporation of labelled lipids and thei!r precursors in alveolar` macrophages of control dogs and; dogs exposed to cigarette smoke will be studied to determine aitered: l,ipid pathways. We feel the above studies are necessary before more detailed studies of alterations in lung morphology and lipid metabol- EXPERIMENTAL EXPERIMENTAL DESIGN AND PROCEDURES: Surface activity of the acellular alveolar tining, material will be measured on a modified WilheTmy balance. ism in patients with various lung diseases are undertaken. In addition, studies to determine the origin of the alveolar macrophage will be attempted because smoking may alter not only the quality but also the quantity of these cells. Lipids will be extracted from the cellular and acellular alveolar lining material by several Folch washes using CHC13•:Me0H:H2O. The various classes of _ --••lipids (neutral and phosphol i pilds) will be separated by thin layer chromatography using silica gel H and CHC1 :MeOH:acetic acid for phospholi.pids and petroleum .ether:diethyl ether:acetic Kiid for the neutral lipids. The lipids will be local'ized on the thin. layer plates with 2,7 dich°lorofliuorescein, and removed by ; scraping. Fatty acid methyl-esters will be formed,by refluxing!in Me0F1 and H SO and will then be separated by gas liquid chromatography at 210° using a butanedtio ': succinate column. : The lavage sediment containing alveollar lining!material and cells will be centrifuged at 10,000 x G at 0° C for 20 minutes, fixed in cold,2.5% glutaralde- hyde buffered with 0.15 M Na cacodylate to pH 7.2 for two hours, washed:several times in•buffer, fixed in cold phospha.te buffered 1% 0s04 for two hours, rap-idly dehydrated in ethanol and then embedded in Epon 812. Sections for light and electron microscopy will be made from the eporn blocks. In attempts.So isolate relatively pure monocytes from circulating blood, buffy coats from dog bliood obtained by exsanguinati:on will be processed by an ~ electronic separator so that monocytes can be separated. They w-H-l be labelled with rad.ioactilve:substances and injected into dogs. Their course into the alveolar spaces will be followedd by appropriate radioautographic microscopy. ~ The exper i,men-ta l des iign w i 1 l emp 1 oy the procedures ou t l i ned i n the f i rs t three paragraphs above, on humans who are smokers; non-smokers will serve as controls. (The appended reprints contain further information about our methods ~.:•. t..: and observations and are included as an aide for the reviewer)

9. PHYSICAL FACILITIES AVAILABLE `Tr has approximately 4,500.square feet of research~.` The Department of Anatomy. . ,_. ..,space which is available for use_in support of the outlined program. This includes`~ •three electron microscopes, four ultrami-crotomes, two fully equipped photographic' , darkrooms with the supporting equipment to do excellent photomicrography.' Suitable 'supporting technical and secretarial personnel are currently employed ~ :.to assstn tempemenaton•o te program~ ..: ;.. • .. - .. . . . '- . . •_ _ . ", .. . .~• _ , . . ,- . , .. . ..A ' outlined. has the equipment and facilities to accomplish the biochemical procedures as '. r, medical complex. Dr. Finley's laboratory ds composed of 1000 square feet and,:;~= Members of the Department of Medicine have laboratory areas within'the :f Sufficient animal quarters are present to provide adequate space to house and ma i nta i n the needed suppl i es of dogs. catheter. . hospitals, to perform the lung lavagos on human subjects. X-ray and fluoros- copic equipment are at hand to monitor the positionirig of the intraalveolar Albuquerque Veteran's Administration Hospital, the Universitiy's teaching Rooms are available at the Bernaiil'llo-County-Indian Hns.pital; and 10. ADDITIONAL REQUIREMENTS that a base-line for the non-smoker and the smoker can be establi!shed. So far, ';` We are attempting to survey a sufficiently large number of humans so ,,., $25.00 to each individual who has volunteered for the lung lavage procedure. y g n e av g p p ,, v a ne n v der aic! er fift subjects have u on ast we ha s li a e l the e o ] d a iil rt k the th d h I t t ti ld th it l h e e e even an un ow reac a w un a n r on s ou occur, osp r e ma i ntenance and care of the pat i ent. Add i t i ona 1 funds to cover such added costs should be budgeted so that effective patient-care can be provided; . . .. :*,: . i

t CURRICULUM VITAE P-1 ' ,.Seattle, Washington Assistant Resident in Medicine University of Ca!liifornia Medical Center, 1955-1956 San.Francisco County Hospi,tat, 1956-1957- r POSITIONS: Assistant Resident in Medicine Research Fellow, 1957-1958 University of Buffalo Department of Physiology CI i n ica I Instructor i n Med ic i ne, 958-196.f Cardiovascular Research Institut+e= Unavers it~y-of Ca I' i forn i a Med;ica II Center Associate Staff Member, 1960-1961 Cardiovascu;lar Research .Institute University of California bedical, Center ~ • SOCIETIES: R: REDACTED MATERIAL University of Washingtori;: IS44-1950! Degree: B. S.. (Chemistry) - Johns Hopkiins Medicai Schoo-l-, 19504954 Degree: M. D. San Francisco County HosRital, 1954-1955 American Trudeau Society Research Fellow, 1958-1961 Associiate Professor, July 1964 and B'iiophysics; Director of Anesthesiology Research University of Washington School of Medicine 1961; Assistant Professor of Anesthesiology, Physi:o,logy Assoc iiate Prof essor of M_ed.Ic i ne, Un ivers i ty of uew-Mexico School of Medicine., September 1964 to• present. American Physiological Society We.stern Society for Clinicali Research American Society of Clini.cail Investigation Amer ican Fed'erat ion for CI i ndca I Research

B1BL10GRAPHY - T. N. Firi:ley, M. D. Nbrgan, Thomas E., T. N. Finley, Gary L. Huber, and Helen F-ialkow. -Alt+erations in Pulmonary Surface Active Lipids duciing Exposure to • Acute effects of saline lavage on pulmonary mechanics and morphology. J. Clin. Invest. (irn Press) Lung Lipids in the dog. Bioch!im. Biophys. Acta, t06-:403-413,1965. 3.A Huber, G. L., Edmunds, L. H. and Finley, T. N. of the Composition and Surface Activity of "AJveolar11 and Whole 2. Morgan, Thomas E., Theodore N;. Finley and Helen Fialkow. Comparison Increased Oxygen Tension. J. Clin. Invest. 44:-1737,II965. . Finley, T. N., Swenson, E. W., Curran, W. S., Hu,ber, G. L. and Ladman•, A. J. Bronchopulmonary lavage in normal subjects and patitents with obstrucfiive lung disease. - Ann. I n-tt . Med. 6b : 65 l, 1967 . 5. Finley, T. N., Ladman, A. J., Brewer, L., andi MrKay, ML B. A morphological and' 1 ipid ana,lysis of the alveolar I ining, rreterial in the dog. J. Lip-id Res. Un Press)

R: REDACTED MATERIAL CURRICULUM V'fTAE i MAJOAt' RESEARCHI .:1NTEREST: -Aaron J: Ladmany Ph. D. P-1 :" Jama ica, New• York, U. S. R: New York Univers•ity, New Yor:k, N. Y. Indiana University, Bloomington, Ind. American Cancer Soc., Postdoctoral Research Fellow 1952-1955; Special Research Fellow ofs U.S.P.H.S. 1955-1957; Career Development Award 1962-64; Visiti.ng.Summer Investi.gator, Jackson Laboratory, Bar Harbor, Maine 1949-1951, 1953•, 1954, t956; - 1957, 1959; Fellow•of American Associ-ation for the Advancement of Sc ience, 1966.. Funct iona I Cytology-aind Cytocfiemi stry RESEARCH AND PROFESSIONAL ..EXPERIENCE: Research Assistant, Jackson Laboratory, Bar Harbor Maine, 1947-1948; Teac h i ng!Fe I I ow im-Anatomy, Un i vers i ty of C i nc i-nnat i, Depa rtment of Anatomy, C i nc i ranat i, Oh i o, 1949-1949; Teaching Fellow i:n Anatomy, I.ndiana Uni.versity Department of Anatomy, Bloomi-ragton, Indiana, 1949-(952; Research Fel low im, Anatomy,' Harvard Medical School, Department of Anatomy, Boston, Vass., 1952-1955; Associate-in Anatomy., Harvard Medical School, Department of Anatomy, Boston, Ma,ss., L955-196D; 1964 to presen-h. ; e ca ennessee - n ts Memph i s ,.,, Professor of Anatomy, Chairman, Department of Anatomy, The Un ivers i ty- of New Mex ico Schoo I cf Med;i c i.ne, y, 1961 I 964 d i I U T M i Un ivers.ity of Tennessee" Assoc iate Professor of Anatom

. ~ B1BL:OGRAPHY - A. J. LADMAN, Ph.D. .: Ladman, A.J. and F i n iey, '1'. N. E1 ectron m1croscop-i c observat i ons o pulmonary surfactant and the cells which produce it. 2. Finl•ey, T.N., Swenson, E.W,., Curran, W.S., Huber, G.L. and Ladman, A..J. Bronchopulmonary Lavage in Normal Subjects and Patients with Obstructive Anat. Rec. 154:372,1966. (Abstract) .Lung-Disease. Ann. Int. Med. 66:651,1967. Ladman, A.J. The fine structure of the ductuli efferentes of the opossum. Exfoliative cytology of the lung alveolus: Preliminary electron microscopic observations on cells obtained i_n,vivo from human lungs. Elec. Micr. Soc. Am. 1967. (tn Press) Anat. Rec. 157:559,1,967. Finley, T.N•., Conley, G.R., Huber, G.L. and Ladman, A.J. of human alveolar lung,mate'ri!al and cells. Clin. Res. 14:365,1966. ,(Abstract) Ladman, A.J., Pratt, S.A., and Finley, T.N.

R: REDACTED MATERIAL Chemicals Glassware Reagents EM,supplies L Consumable Supplies (list by cntegories) C C. Other Expenses (itemize) Dr. Susan A. Pratt - electron microscopist Technical Laboratory Technician: Secretarial assistance Animals, maintenance & procurement 1/2 service contract for EM• Consullting fee to• Dr. Swenson Patient; reimbursements 100. 100 25• Sub-Total -- .. : - --7711 Amount 500- .. 500 --- 500 1,000 Sub-Total $,2,500 1,000 700 500 3,000 Sub-rotalt D. Permanent Equipment (temize) I E. Overhead (15% of A+B+C) Estimoted huture Requiremenls: Year 2 Year 3 s,200- $3,405 Total $26, f05 Salaries Consumable Suppl`. Other Expenses Permanent;Equip- _ Overhead ~ Total P- 2,500 4,200 P-. - 2,500 4,200 11 is understood thotAhe opplicant and institutional officers fn•applying for a grant have read and found acceptable the Council's "Stotement ofi Policy Containing Conditions andTerms Under Which Project Grants AreMade."' 3,368 25,818: /T 4 26 ~ Sgnature Signoture Telephone ~-r-i ewn.,, orr ..r tn. t.,rk.ttoe Telephone C GC!M

I 4. Other Sources of Financial Support List financial support for re.earch from all cources, including own in.titution, for this and/or related research projmcts. Current Titie of Project Fine structure of tissues during transport behavior ibid. Respiratory Distress - Role of Lung 8 Blood Lipids National Sourc. Institutes of Health Amount $38,435 9= Durotion 1-66- Grant #1 R01 GM14435-01 8-31-67 ibid. Grant #1 RO1 GM14435-02 $28,676 9- 1-67- 8-31-68 National Institutes of Health $28,500 9- 1-67- Grant #5 ROl HE09491-04 8-31-68 S t w

.. '.~s. , ... FII: CUII\CIL FvP TOB_1CCOQ RESEARCFI-ZT.S,A, The committee comprising Dr. Cattell, Chm., Dr. Little and. Dr. Jacobson. SUBJECT: New grant application from Carlton K. Erickson, Ph.D. - N Kansas. It is supported by reprints of two studies as follows: We enclose herewith a new grant application from Dr. Carlton K. Erickson of the School of Pharmacy, The University of Kansas, Lawrence, 70~ (1966)• meeting. (See application Nos. 622 and 623). We are already supporting Avoidance Respondingin Rats. Arch. Int. Pharmacodyn, 163, Involvement in Cholinergic-Induced Blockade of Discriminated 1. C.K. Erickson and R.K.Chalmers. Hippocampal Theta Rhythm 2. Carlton K. Erickson. Facilitated Responding in a Discriminated . Lever Press Avoidance Situation. Psychom. Sci., 8, 37, (1967). This is the third new application for the support of studies con- cerning the effects of nicotine on learning, on the agenda for our September s al t th i l ever pro~ec s n e same genera area. It is interesting to note the number of different experimental approaches which have been designed for the study of the influence of plan which looks good. 1-: nicotine on learning. This proposal describes a well controlled research R.C.H.

I. CARDIOVASCtfIAR, PIll1MfRCOLOGY and CHEbffSTRY II. EFIDSMOLOGY, STATISTICS• and;PSYCHOSObfATIC N0: 6- 1. Name of Inrestigator(s): (indude Title and Degrees). _ - - ' B.S., MyS,, Ph.D. 2. lnstitution & ~ddrEik-< = Sehoo•l-of Pharmacy Date: August 7, 1967 Carlton K. Erickson, Assistant Professor of Pharmacology and Toxicology, The University of Kansas Lawrence, Kansas. 66044! •,,;-ed alertness andilearning. Recent research, however, has demonstrated sorption on the subject's alertness and learning ability. Obviously, itg.`cattile `wott13•'be considered socially unacceptable if it seriously impair- COtIIdLTTEE: Dr. Cattell, Chm. Litbl e _. qpp~icatfon For Research Grant ts D J r. aco son For obvious social and medical reasons, nicotine is one of the common drugs which has been studied for its effect on learning and performance. In humans who smoke regularly and inhale a significant amount of nicotine, :°obvious questions arise concerning the effects of prolonged nicotine ab- C ; Mechanism of Learning Facilitation by Nicotine ; 1. Proposed Sthrting Data. November 1, 1967 S. Anticipated Duration of this Specific Studja 12 months 6. Brief Descripton of Objectives or Specifio Aims: that while large doses of nicotine depress learning, small doses can ; enhance perforr.tance in rats and mice (1,3)': Unfortunately, there has not been a concerted effort to study the true mechanism of nicotine's observed facilitatio7_pf.learning. McGaugh and Petrinovich. (9) have cautioned ~gain'st- assurting that changes in performance reflect actual cban;es in ' effic,iency of the neural processes involved in learning (i.e., the so- called formation of the memory traee). It is of special practical impor- tance with nicotine to differentiate a transient change in motivation or ---a2ertrcess fr'afn`t?ie relatively permanent alteration of neural processes involved in learnino. Er.perience has shown that in order to convincingly demonstrate that nicotine (or any centrally-active drug) is actuaLly affecting neural learnimg necnanisms, 3 important criteria must be fulfilled;: 1 The nhenn7eann of "drua disso^:.~.tion" r'>>:t t:r, rrle•_ out; ilcGauy~h `~aYih.Fetrinovic:i (9) have stated that "subsequent stLdies oE nicotine `on learnin.-) should ta.'.:e into account the possibility that dissociation 77ay occur with this eo :7-pound", Dru;; dissociation refers to• the-situation in which habits learned by subjects in a drugged state do not transfer to ( t the normal s tate, but can be evol;ed again when ever the sub ject is dru;-,~,_^,ed . ` In other e•+ords, nicotine may act as a secondary stimultRs dur ng, learnin~; 7. G"rveoariefSiatementofyourWarkingNkpothesis: tto pa"e la) hicotine enbances central nervous processes involved in Iearning, Er.d does not demonstrate deleterious ef.fects on, beaaviorai perform,ance.in doses comparable to those s_nvolved in snto'.:ir.g, 4N==K `_

ectives and Specific Aims (continued): ih: of a response •or other information; when the drug is not pr.,sent, the ..;,,'• :~,.learn•ed material may no-t be recalled. Peripheral actions of the drug must be ruled out. It is well knoc~n RP that changes in peripheral functiioning such as autonomic and ganglionic ~ ='transmission changes, sight and hearing changes, etc, can influence :~.learning, These must be shown to be inoperative in any suspected drug . ; .'- ; =;,effects on the learning process. The contribution of central "side effects" must be determined, . ; 1 , •1~• d t th th ff f t ti d b d t t ll i i t it or ve- er e e ec a cen ra y-ac rug e p npo n o s a o e °action on central-learning processes, other central effects such as changes ~in motivation (hunger, thirst); and excitability level (anxiety, confusion,, ':hyperesthesia), must be ruled, out. Contribution of these effects to :}"observed performance would not be considered part of true learning altera=-• :-':tion. since they are temporary and do not directly inf luence ermanent: p • ~memory. :This project, -then, is designed to fulfill the preceding criteria for •.:nicotine by aa series of approprilate pha.rmacologic and neurophysiolo~ic experiments, Since the hippocampus, a limbic system structure, has been ;.' imp licated by many workers as a ma j or bra in, area invo lved in lea1 Liino, .;--.thi,s as well as other suspected. structures (cortex, reticular activating system) will be conce.ntrated upon. It is interesting that low doses of ,:.~.nicotine which. enhance learned performance in animals also produce a :.characteristic theta rhythm (4-7 cps slow, wave activity) in the hippocampal , ELG (8). Conversely, high doses of nicotine which block learned behavior can elicit seizure activity in the hippocampus. Although these observations have never been corre.lated in the same animal, they provide an•interes•tino theoretical basis for studying the relations between nicotine, learning, : and the hippocanpus and other related brain structures, ~ A unique.technique to be used in this study concerns the "chronic" r administration of small doses of nicotine during a relatively lengthy avoidance training session•. Because of the short duration of action of nicotine, rats will be fittedi with:chronically-indwelling intravenous catneters-for repeated nicotine administration. It is felt that this will : also clo$e1y parall•al the effects of a chain smoker performing a• relatively ' l :co:apex_ mental or physical task.

, .. • "S. fletaNSs oE Expedmental Desi9n and Procedures: (Attach Separate Pages). Animals - Rats will be used throughout the study, in order to satis•fy' `:•,.eurrent housing requirements and to allow data accumulation on a large ;~eu h 'number of inexpensive subjects; ' Trainin- procedure - Since most of the published behavioral studies •':7::`:- avoidance response, it seems best to expand upon these results with with rats and,nicotine have utilized either the shuttlebox or pole-climb .similar behavioral situation. This worker is most familiar with the ;,' '4discriminated. lever-press avoidance 'resPonse and its interP retations , . ..;'_.: ; " ;-detail in an acqompanying reprint (Psychonoiri. Sci,, 1967), The pr.ocedure ~-;~ ~so this training situation will be used. The procedure is.described in ;~ is especiatiy aavantageous tor Learning studies because acquisition of :of changes in response rate due to drug treatment, °'the response is relatively slow, thereby allowing accurate observation• -.•Dru7 administration method - Since the purpose of this project is to, during training sessions only. That is, doses will be administered up to the time when a preset criterion is reached (either 85% avoidance averaoe study the effects of nicotine on learning rather than on previously- learned behavior, nicotine and saline (control) wi11 be administered -for 3 consecutive sessions, or 15 sessions, iehichever is reached first). • •Although doses, onsets, and durations will have to be determined for our The applicant has at his di.sposal for• tha.s project the following facilities and equipment: . : particular test conditions and animals, the literature does provide 9. Physica[ Faciities Availabl'e (Where Other than Administering Organization Indicate Geographical Location) ( t0 page 2a ) 1-Staff office (air conditioned) 1-Laboratory-office combination containing desks for 5 students • and animal surgery area (air conditioned) (to page 2,0 • 0. Additional Requirements: - - ; For completion of pro~ramming equipment in available test chambers: 2-Grason Stadler shocker-scrarb lers, Model E1064GS 1-Heavy-duty power supply, 28VDC (Grason, Stadler), Model E783DA 2-Lehigh Valley basic interval timer, Model 1309 1-Lehigh Valley session timer, Model 1350C 1-Lehigh Valley digital counters, Model 1425-10 Z- • 11. Biographical sketches of all principal and professional personnel (append) See page 2c? , .. • • = 12. List of puLrlications: (Five most recent as pertinent) (append) See page 2e. ._:~•,. . .- . . ~. .:~ .._ . _ .. : .:.: .. _: _._ _ ... -. .•_~_.......__-,... .. . ...:_

Experimental Desien•and Procedures (continuedi)s ;.; . . 'information which can be used to design the dose schedule. Domino (3) has shown that nicotine in relatively small doses reaches its peak after minutes and has a duratioin of approximately 30 minutes. Since the ' avoidance training method to be used requires 4-hour sessions every other ;;'day, it is obvious that nicotine will have to. be given more than once in ;;f;`order to provide prolonged drug.action for an antire session. Thus animals wi11 be fitted with chronic-indwelling catheters vial the jugular vein according to a method described by Weeks and Davis (11L) and modified by us (5). The cannula will be attached•to a swivel fluid-lock assembly y mounted in the top of the test chamber which will allow the animal' -to ~:move freely without twisting the cannula. Injections will then be made . ;at desired intervals without physically interrupting the animalts ,training session. 4hi;.oDoses to be used - According to Johnston (6), 1/50, gr. of intravenous nicotine produces, in a habitual smoker, the same psychic effects as smoking one cigarette. Assuming this effect occurs in a 70 kg. man, the :!Ocigarette equivalent" of nicotine received is calculated to be 18.6 ug/kg. ~~ of body weight. Since our experimental design will utilize intravenous nicotine administration, the nicotine "cigarette equivaLent", as well as j~other,simi:lar small doses, will be thoroughly investigated. Along these Alines, it is interesting that the doses used by Domino (3) to facilitate avoidance behavior (40-80 ug/kg, subcutaneously) and by Broc•m (2) to. evoke hippocar.ipal responses (30-50 ug/kg. subcutaneously) are similar to !Four calculated cigarette equivalenr. It further seems very possible that i'; ur 186 ug/kg dose given intravenously may produce the same effects as ,, 0rthe Domino and Brown subcutaneous,doses, Experimental pLan, - The 3 criteria discussed•under Objectives will be `investigated'using nicotine as followss U~' ~~1.- Drug dissociation - In order to determine the presence or absence of ~Znicotine dissociation of learning, groups of rats will be trained in the conditioned avoidance situation under the effects of nicotine and saline. Rate of response acquisition to the preset criterion will be recorded to determine enhancement of response rate with nicotine. Later sessions will `' consist of performance measurement of both groups.treated with saline. Drug dissociation will be ruled out if the nicotine-trained rats continue to perform at the pre-saline level . Central action of nicotine - The usual method of determining whether g drug is acting centrally is to demonstrate a behavioral action with the s.•=k 'drug followed by loss of the action with a quaternary derivative which -- , _ - . . ll not pass the blood-brain barrier. In this re~ard, Domino (4) has ;reported that congeners such as nicotine methiodide are relatively ineffec- 4tive incontrast to nicotine in blocking conditioned, avoidance behavior _ ~kin rats. Nicotine methiodide will be used to confirm these results in our conditions, and to control for central specificity in other parts of the study. . . .- . . . • - .... Another method to test for specificity of drug effects in the brain is simpLy to place the:drug into various structures of interest by means of ~''`a cannula. This provides a. means of determining significant differences %';in drug effect from parenteral administration. Using apparatus similar to that described above for intravenous injections, rats with chronically- idllil ih biil bdihll dfi ,nweng cannuasn teran wle teste wt smaoses o nco- tine injected into the cortex, hippocampus, and reticular foi Vation; Intracerebral quantities of nicotine will apptoximate I x 10' moles, and C"solution volumes will be not more than 2 uL/injection. A modification of •the nylon-stainless steel assemblies.of Valenstein et a_1. (10) will be used for cannula construction, and nicotine will be administered• intra- cerebraLly at times comparable to parenteral administration. c Q C

LD, Conficmation of cannula placements•will be performed histologically. by this drug. each session wouZd•strongly indicate facilitation of memory trace formation nhancement of conditioned avoidance learning when nicotine is given•after Dom ino, E.F., "Some behavioral actions ofnicotine!', in Tobacco (1967). W ' .- Browm, B'.B, , "Relationship between evoked response changes and behavior ~ following,small doses of nicotine", Ann. Y•.Y. Acad; Sci: 142r19•0-200 , a~~ y .avoidance conditioning of inbred:strains of mice", Psychopharmr. 10t : 5 (1966) References : Bovet, D,,, Bovet-Nitti, F:, and Oliverio, A•,,'"Effects of nicotine on : ReLated Comnoarzds, ed. Von EuZer, The i4aci•i211an• Company, .":e~. Yark 965), ;''kUts optical isomer•, and• related: compounds", in Tbhacco AZ'_:aloidts and omino, S.F,, "Some comparative pharmacological actions of (-)-nicouine, '= .. . . .. . . ; . . • . ., .. _ ... .. . ;r., ,_. .. . ... ... . ' . . - . .. .; ' : . .. .. ... . . . . . : , , New York (1965), pp • 4 -Q . - .. . : _ . _ Alklidd RLd CM C,x aos aneateomnounds The b;acillanorapany r, ed. Von Euler • 3 = 3. ~ .:::;.;,~ ' Frozen sk:ctid•ns of 50 micron;thickness will be cut, placed on slides, and stainediwithtcres,vlechtvioletl and methyLene blue according to the :•',storage effects by ruling out motivational changes and excitability factors. fi ~;•~;"•r••adopted by a r.tunber of investigators as a means o assessng memory ;j method' of i:luver and Barrera•(7)•. 3i Direct action on neural learning processes - The procedure of ;:;:admnistering drugs after trainino,rather• than'before training•has been . " --~;and the stronger the incoming stimulus, the'more deeply the memory trace " '.ing the CNS'initiate reverberating neural pathways'which•must continue for `_'A current popular theory'of learning and memory states that stimuli enter- fixed time to foum a memory trace; The longer the pathway reverberates i ld: Thidiil (:lhki Usanteus any overrng stmuuse,g, eectrosoc or certan ` =:.drug~ tahich, disrupts the reverberating neural pathway (assuming the path- . way is'eLectrical-chemical in nature) will inhibit learning and trace in this manner (9) On the other handl stilats suh as ~~., centramunc ~:-rmphetasnine and strychnine given after training trials in aninals have a c e~ q g pro "" '= ?~formation: Post-trial eLectroshocl: and various dru $ such as barbiturates g f,,, :"=y"', and tran uilizers have indeed been shoxm to fife rnin cess t the l ~ { y u a e ve w or rugs are max ma y e ect n m nutes an c t :~g 's<which ~re metabolized Prior• to retention tests, as is ni.cotine, Thus ~ a "been shown to enhance the learning process (9). This technique is especi- s 11 it bl f d ll ff d whi h i i i hi i C ; Johnston, L,it,, "Tobacco smol:ing and nicotine", Lancet 243:742 (1942). y. ... . . . . -- ' Erickson, C,'_{,, uapublished data on chronic intravenous alcohol infusion in rats: . - . : . - . . ' . . . . ~ .. . ... . . . •7: Kluver, I;. and Barrera, E.A., "A method for the combined study of cells• and fibers in the nervous system~~, J. I'europzthol. exp. heuroi. 12s •:•400-3 (1953), • 8. Lon_-o, V.G., r3vnta, F., and Scotti de Carolis, A., "Effects of nicotine on the electroencephalo--ram of, the rabbit", Y?:.Y. Acad. Sci. 142:159-69• (1967), , Q C

~ c, ~ t•1cu^augh, J.L•: 'and Petrinovich, L.F., "'Effects of drugs menory", Intern: Pev; Neurobiol. 8s 139-96 (1965) ; k'Gn... . ~ . . _._ . : Valenstein, E.S., Hados, jV:, and Stein, L,;, "A simplified electrode- ~h1assembly for implanting chronic electrodes in the brains of small animalsrt Am. J. Psychol. 7<<:125-5 (1961). ; Weeks, J.i<.. and Davis, J.D,., "Chror:ic intravenous AppL. Physiol. 19:540-1 (1964); Physical Facilities Available (continued): _1-Behavioral laboratory (air conditioned) containing 9 rat test .be available for this project; so*:e prooranmin(-n equipment is chambers and relay progranr.ling equipment (3 test chambers will required, to complete the char,~bers - see Addi tior_ai Recuirements) work), with Microto~:~e equipped for carbon dioxide and paraffin 1-Histoloo-i.cal laboratory ( for confirmation of cannula placer.:ent sectioning -

1 Z ::4i' , ...,._ . . :°~'~ • ti: _ halmers, R.K. and Eric'..tson, C.K., "Central choliner~ic blockade of the••"`~~"~~. ~~~"~~.~ ~ ;jt ~;. ~, conditioned avoidance response in rats'~. Psychopharmacoloooia 6t 31-+1 ;`_~~:-•; s~a..F:,t t, r ~:,c t'FE faC~ » ~~ ~Erickson, C.IL, and, Chalmers. P.,K,, 11l~ippocampal theta rhythrt•involvement hZ.,kin cholinergic-induced blocicade ot discriininated avoidance responding ` Ran rats", Arch. int, Pharm.acodyn; 163: 70-8• (1966), ~Ericnson, C.v., "Facilitated responding in a discriminated lever press avoidance situation"~ Psychonom. Sci, 8s37-8 (19b7). :•'Erickson, C,K,,. "A reliable lever-press avoidance training method in artthe rat", Fed, Proc. 25:738 (1967). :- ---• ~ ; ., •. ... atel, J: and Erickson, C.K., "Enhanced avoidance acquisition by loio- ;intensity hippocarnpal stimuli", Pharmacologist 9:200 (1967). .........x:......=...:.._r..V.. ~~.' d5.'•=-~awr.•:w:..«.~...-.~.::.+.+s.....,.:......

R: REDACTED MATERIAL A. Salades (Personnel by names) _. % time Professional • -. ~ Carlton,I:. Erickson, Prin, Invest. 9 months academic year 207. 3 months st.trmter 1007. Fringe benefits Jitendra B-. Patel, Graduate Student 12 months calendar year 50% None Sub•Total Sab-Total D. Permanent Equipment Qtemize) Amount 400 100 100 50 2-Grason Stadler Shockers with Grid Scrambler, Model E10G4GS ? $ 285.00 $ 570 1-Grasorn Stadler Heavy-duty Power Supply, 281JDC, Model E783DA 175 2-Lehigh- Valley Basic Interval Timers, Model 1309 0 ; ~o ~ $ 95. 0: 3 _ - - a) (to page .. Total-$ 1,214: E. Orerhead(16%•ofA-}s6+C)' $ 1.0m Total q 5n $ Q Estimated Future RequlPements: NO:+E s Salories Consumable SuppU Other Expenses Permanent Equip. Overhead` ' Total C Animals and feed Cannula materials Dru--s: and••histologlcal chemicals Recorder paper, notebooks C. Other Expenses (temize) Yean2 Year 3 Signature t•tiTx1~C C1Y,_ K C[/La.GI': So-r~ It is undlntood thotlhe applicant and institutional iofficers oi-+...f r..i«t 913 UN4-4004 in,applying for a gronthavs read and found acceptable , n Q Te[ephone & _ ~/36 the Council's "Statement of Paliry Conlaining Conditionz Signaturt ~~LL !~T_16L t i( f f!~ L~ t/ 1 14 and.Terms Under Which Projed Grants Are Made." 4.4- Oflkrr ef,the InitAUkon LIYFCH-{'vfFC'e_ a(~Telephone . r=att 1 lnrAx,cr:j

... • `? -- s ... .a Permanent Equipment ( continued•) 1-Lehigh Valley Digital Counters, Model 14•:25-10 1-i.ehigr Valley Session Tiner, Model 1350C

i a ^ List finandal support for research from all wynp, indudlnp own Instttutlon, for this ond/ort.lated nsearch projects. Thle of Project "Addictive Ability of Self-injected Intravenous Alcohol in P,a:s" ~. "Behavioral Hyperexcitability Induced by Alcohol ?lithdravra1 in P.ats" "Enhancement of Learning by Limbic System Stimulation" VSO4tScOOI fi i A

Tx~ CoUNciL FOR Toal%-cco PES1E1]AxcFr-U.S.A. .The committee comprising Dr. Loosli, Chm., Dr. Sommers, and Dr. Little. Robert C. Hockett SUBJECT: New grant application from Mary Stearns Parshley, Ph.D. - No. 630. reprints, from Dr. Mary Stearns Parshley of Columbia University College o We enclose herewith~a new grant application, supported by two Physicians and Surgeons, New York, New York. humanirespiratory epithelium in vitro. of the effects of the constituents of tobacco smoke on normal and malignant A well documented application for the support of a broad study R. C. H. Reprints: of Malignant Cells in vitro by a Component of Normal Adult Connective Tissue. Nature, 208, 5012 (1965). . Mary Stearns Parshley and Ines Mandl. - Inhibition of Growth 2. Effect of Inhibitors from Adult Connective Tissue on Growth of a Series of Human Tumors in vitro. Mary Stearns Parshley. Cancer Research, 25, 3 (1965).

*'• - .. - .. ~ - `C :; t~~-;BICASSAY, CARCINOGENESIS and TISSUE CULTURE 1i~''~. . ,. ...-... . _._.__..~ .. _ . . -T= CoUlvca FOR T©sAccG Rrsr.nRCS . U.S.A. Ess TIIIFtD 19EAFUE NEH' IoIiH. N. T. 10017 - Dr. Loos13, Chm6 '_ Di. Sommers Apprcation For Researeh Grant -DY : •'Reimann Dr. Little 1. Nameoflnvestipator(s)d(i,ncfudeTitleandDeyrees): Mary Stearns PARSNTE3C, Ph.D. . Assistant Professor of Anatosy in Obstetrics & Gynecolo gy 4 Institur~on ~ Columbia University Address: College of Physicians & Surgeons 630 West 168th Street Nev York, N. Y. 10032 3. 5horf Title of Projech EFFECT OF CONSTITIIENIS OF TOBACCO SMOKE ON! NORMAL AND MAIZGNANT HUMAN RESPIRATORY EPITHELIDM IN VITRO. 1. Proposed Starting Date Novetnber 1, 1967 S.Anticupated-DuranonofthisSpecificStudy: Although some meaningful results should be obtained by the end of the first year, it is expected that an additional period; will be required. C• 6. Brief Descriplon of O6jectives or5pecific Aims: There is an,increasing emphasis on tobaeco smoke as a contributory factor to the development of lung cancer. Tobacco products, the polycyclie aromatic hydro- " carbons in particular, have been shown to induce or enhance neoplastic transfor- taat~oA in laboratory animals (4,9,15). On the other hand soma constituents, namely the oibnoearboxylic acids, have been. reported to have an anti-tumor effect (12);. There is little precise knovledge of the qualitative and quantitative effects on norma1'respiratory epithelium of the nearly 300 organic and inorganic compounds rpported as constituents of tobacco smoke (4,8). These include netal~s, hydro- carbons, alkaloids chieflynicotine, nitrogen products, and volatile elements. Either similar factors.found in polluted air or still other factors must be respon- -r----sib3e-forthe•-occurrence of lung.eancer in non-smokers, and the higher incidence of this disease among male smokers. A great deal of study has been devoted; to the biological activity of tobacco tars or condensates with. emphasis on the pol,ycyclic hydrocarbons which are present only in traces (1,2,5,6,9,15). Not only has it been shown. that these hydrocarbons are not specifically ea'rc•iftogcnia for epithelium (6,9), but the fact that fractions of smoke condensate not containing hydrocarbons also K7~vpToduae neoplastic changes in Iung•epithelium has been demonstrated (6,9). Most animal experiments deal with the carcinogenic effect on skin of smoke condensates and their fractions (4,7,8615). Studies in which animals actually inhaled smoke gave inconsistent results (7). The skin is quite different from the mucus-coated respiratory epithelium and apparently more sensitive to the polycyclic hydrocarbons. Although nicotine content has been considered to be detrimental to the cleansing activity of the respiratory cilia (3), very little is established as to the local biological effect of nicotine (4). Littlo attention has been paid to,the volatile / substances such as carbon monoxide, hydrogen~cyanid'e, and'nitrogen.oxides, present `!• in-many times higher concentration- than that considered to be "safes' in industry (8;1f+). It is possible that neoplastia changes in normal respiratory epithelium 7. G'rveaBriefStatemento.ryourWorkingHypothcsis: (cont. on p.la)- If the development.of malignancy in normal human•respiratory epitheliun is the result of an interference with, the oxygen supply and normal respiration, a simulation , (cont. on p. 1a) G= ~ `_

~ may be caused by an interference on the part of some of these volatile constituents of tobacco and polluted air with the oxygen supply to these cells necessary for aerobic respiration. This anoxia could result in a mutation toward anaerobic glycolysis as a source of energy characteristic of bron,hogenic carcinoma (1k). `,'z e:F!. ~ ~.'::i::;_ ~ The technique of tissue culture makes possible the direct observation of human ,:;...tissues under high magnifications. The biological effect of whole tobacco smoke ;f ; as well as some of its volatile constituents on these cells can be studied microsco_` pically, and the metabolic byproducts of the cells under different conditions can be analyzed. Cell lines provide a source of uniform. test material. In addition test : materials can. be added in a concentration not possible in the intact animal and under .conditions of prolonged contact permitting the equilibrium of poorly soluble or slowly diffusing substances between the medium and the cells. A number of materials possibly related to a neoplastic activity of either tobacco smoke or polluted air have been studied by this method-. Hyperplasia and metaplasia considered preneoplastic - in cultures of human foetal hl.ng treated with polycyclic hydrocarbons and tobacco smoke condensate and its fractions was reported by I,aznitski (5,6). Crocker and coworkers havecarried on similar studies with rat and hamster trachea in tissue culture (1,2,9). These workers reported a nonspecific effect of these carcinogens, and also that analogs of these hydrocarbons not known to be carcinogenic produced similar abnormalities in cultures of respiratory epithelium,. Toxic effects of organic constituents of tobacco smoke and polluted air, acetaldehyde, nitrogen dioxide, , and monocarboxylie acids, on lines of human and animal cells in tissue culture were observed by Pace and his coworkers. (10,11,12). Rounds also observed that nitrogen dioxide introduced as sodium nitrite into the medium of cultures of trypsinized and - suspended cells from the lungs of mice and rats caused nuclear and cytoplasmic changes • • which accompanied a reversible inhibition of respiratory activity (13). See continuation sheet lb for Pertinent Literature Ref, cited above, ~ . . .. . . . .. ... •;::..'.•.;';`n~~:"1' . . It is proposed to study the effect of some of the gaseous constituents of tobacco smoke and polluted air on established lines of normal and malignant human respira_ tory epithelium from. the pharynx, larynx, and lung and to compare their charaeteris- ties as evidenced in tissue culture under these experimental conditions. Effect on rate of proliferation, cell movement and contact relationship, nuclear and cyto- plasmic structure and chromosome content will be correlated with differences in metabolic activity and related to neoplastic capability. A clear understanding of the morphological and metabolic differences between- normal and malignant human respiratory "` epithelium should contribute to an understanding of the neoplastic process itself., 7.,cont. of these conditions in tissue cultures of these cells by a manipulation of the gaseous atmosphere of the culture. should demonstrate this. Similar experimentation with the gaseous phase over respiratory cancer cells in tissue culture might lead to changes in the morphologic and metabolic characteristics of these altered cells which could give a clue to the causes of carcinogenesis. These studies have a direct bearing on neoplastic development and the biological and biochemical mechanisms responsible. ' 1003547058

continuation sheet lb cont. Pertinent Literature References :"l, Crocker, T. T., Nielson, B,:I., and Laznits.ki, I, Carcinogenic hydrocarbons , Arch. of Environ• Health, 10:2:240. 1965 ~ ~ . ..~. . .,.;.; :~~~~:;. ~:: ~• • . . ... • . . . .: . .,. . .- . .. .., . . ,. ,, ., .... . ...,-~ . • ,. . : . .,;~ :,.. '2, --------------, and Nielson, B. I. Effects of carcinogenic and non-carcinogen drocarbons on respiratory epithelium in orhan culture. Tissue Culture Assn. Proc,' Miami June 1965 56 , . r p• • i: ..} . . ' .L 3,Dawson, F. W. Effects of nicotine and influenza on human ciliary activity•in vitro• Fed. Am, Soc, Exp. Biol. and Med. Proc., 24: part I, #260, 1965. 4. Kensler, C. J. The pharmacology of tobacco smoke effects of chronic exposure,. in "Tobacco and Healthil, ed. by G. James and T. Rosenthal, Chap. 1, Charles Thomas;?" Springfield, I11. 1962, p, 5. 5•Iaznitski, I. Effect of 3,4-Benzpyrene on human foetal lung grown in vitro. Brit. J. Cancer, 10:510, 1956, 6, ---------- Observations on the effects of condensates from cigarette smoke on human foetal lung in vitro. Brit. J. Cancer, 12:547. 1958. 7. Leuchtenberger, C., and Leuchtenberger, R. A correlated histological, cyto-"~ logical, and cytochemical study of the major bronchi from mice exposed to cigarette .; smoke. in "Tobacco and Health", ed, by G. James and T. Rosenthal, Chap, 8, Charles Thomas, Springfield, Ill. 1962, p. 81. •- 8. Lindsey, A. J. Some observations upon the chemistry of tobacco smoke. "Tobacco and Health11, ed. by G. James and T. Rosenthal, Chap. 2, Charles Thomas, -~.Springfield, I11., 1962, p. 21. 9, Nielson, B. I., and Crocker, T. T. Epithelial and mesenchymal interactions', :+ in hamster respiratory mucosa exposed. to polycyclic hydrocarbons in organ culture,F Tissue Culture Assn. Proc., Philadelphia, June, 1967, p, 15. • 10. Pace, D. M., and Elliott, A. Studies on the effect of acetaldehyde on R`"r tissue cells cultivated in vitro. Cancer Res,, 20:868, 1960. 11, -----------, Thompson, J. R., Aftonomos, B., and Holck, G, 0. Effects of N02 : and salts of NOZ on established cell lines. Canad, J. Biochem,"and Phys., 39:1247, 1961:- :,.., . 12, -----------, Aftonomos, B., Elliott, A., and Sommer, S. ObserQations on some effects of the sodium salts of certain monocarboxylic acids on established cell lines• Canad. J. Biochem., 45:81, 1967. 13. Rounds, D. E„ and Bils, R. F. Effects of air pollution on cells in culture. Arch. Environ. Health., 10:251, 1965. 14. Stevens, K. M. Lung cancer, an evolutionary approach. Austral. J. Exp. Biol, and Med. Sci,, 43,:421, 1965. 15, Wynder, E, L„ Hoffmann, D., and Auerbach, 0. The role of particulate and volatile components in tobacco carcinogenesis. Am. Assn. Cancer Res., 6:69, 1965. 1003547059

t . Details of Experimental Design and Procedures: (Attach Separate Pages) . It is planned to maintain the following lines of normal and mali•mant human•ies '" ~ ` . piratory epithelium as monolayers of, cells on glass in milk dilution bottles in mix_ ~"'tures of serum (human placental,•horse, and calf) in synthetic media, Parker's :'° Medium 199 or Eagle's minimal essential medium (r1Er1) under an atmosphere of 5% COI~3 i air. :.-. ..a. Line KB, human epidermoid carcinoma of the pharynx ' Line Fd.Ep.42 human epidermoid carcinoma of the larynx carried in our ]aborato- , , i132l h fl l ne L-, normaumanetaung epithelium ,:.We also intend to set up•other lines of freshly explanted normal and malignant "` of Dr. Sheldon C. Sommers, Professor of Pathology at Delafield Hospital. 'These lines`'~ iuman epithelium from biopsy material which will be obtained through the cooperation ~ tissue as opposed to the older lines. will be used to compare the effect of the experimental conditions on freshly explanted' ExpEriments to determine the effect of tobacco smoke and some of its volatile : components Will be carried out as follows. At intervals•series of tubes with and without coverslips will be seeded with a known number of cells. After the cells have '-`s"`r -become confluent the medium will be renewed and the cultures will be gassed by means ` of an apparatus used routinely in our laboratory for adjusting the gaseous phase of the culture with mixtures of 0 and N02 for short periods. In other experi- CO N2 2, . 2, , 9:'i:~y3i2ai Facilties Available (Where Other than Administering Organization Indicate Geographical Location)• (eont, on p.'~2a large well-equipped tissue culture laboratory with glass enclosed sterile room, incubators, refrigerators, deep-freezes, large and small centrifuges, Mettler micro- analytical balances, torsion balances, Leitz Ortholux research microscope with equip- `:1-V~44 ~ ment for bright light and phase contrast microsco py, Zeiss inverted researeh- microscope with, some light and phase contrast equipment, Leitz binocular microscope with Leitz : •th ',, ... • 10I'i~2l~iRiaUi~qSid~6T#t3~ent for photography with lightmeter, camera back, and other equipment f ~.,~ for microphotography and microscopy, sterilizers, stills, special glassware and equip.. „r~;v ment for Carrel flask, Ylaximow slide, Leighton tube, and bottle culture, equipment for histolo ical and histochemi a2 studies g , „• c electrophorator. Dr. Mandl is Asst. Professor of Biochemistry in Obstetrics & Gynecology, phoretic equipment, Spinco I2 ultracentrifuge with swinging bucket, and Elphor FF separation, including cold room, deep freezes, refrigerators, freezer-dryers, analytical balances, homogenizers, two high speed ultracentrifuges, chromatographic and electro- Mandl who has several rooms in Delafield Hospital equipped for biochemical analysis an The chemical investigation will be carried out in collaboration with Dr. Ines r •11. Biographical sketches of ali principal and professional personnel (append) See attached curriculum vitae. ~ 12. List of publications: (Five most recent as pertinent) (append) See attached continuation sheet 2b.

continuation sheet 2a `:. ments the salts, NaSCN; and NaN02, will be added directly to the cult~ure medium or to balanced salt solution in serial dilut2on and allowed to remain in cont t a . c with the cells for.short periods of time. Ci.iltures of freshly explanted normal ';:'~ hu:nan respiratory epithelium will be exposed directly to diluted fresh tobacco ,~~~ •~ ~ smoke by the same apparatus. .,. . .. .... ' . ~ F_S'11y/•I. ia'4 . .. .. , . . . . . , _ .. ~ i• . .. ... , .. .,S.r:.`.~: ~ ~. ~=H.i••~",i iy-.••The effect of these substances on the cells will be determined by the followin 1. Growth rate studies. Effect on rate of growth will be evaluated by dye' exclh bld usion cell counts wit trypanue, an mitotic c OuntS. 2. Mor hp ological Studies. Studies of differences in cell morphology and irC'ty ;number and c aracter of chromosomes will be made as well as histochemical prepara-~`' ..tions to demonstrate different enzyme content. Parallel cultures on coverslips in : Leighton tubes will be fixed and stained w3,th°H. and E., Giemsa, Feulgen, Methyl r Green and Pyronin•Y to observe effect on mitosis and nucleic acid content. Enzyme'coit :tent 'of the cells 'will be observed from preparations in which=the cells have been " reaeted; for sites of alkaline phosphatases, beta_glucuronidase, succinic dehydro-• 4' genase, and leucyl beta-napthylamidase activity. For chromosome studies, cultures will be treated with colchicine to halt mitosis in the metaphase, fixed, spread, stained with acetic-orcein and observed under the oil immersion objective of the :~ microscope and photographed. r 3. Biochemical Studies. The cells and culture medium will be analyzed to evaluate the differences in the metabolic by-products of the eell lines, both treated and-untreated, • a. The effect on cellular respiration and g1YcolYsis will be observed usin g standard l larbur earance and laetic acid ' techni ues In lucose disa articular ! p pp tr.y; r g q g production in the used culture medium will be estimated by the methods of Trevelyan _,~j and Harrison (Biochem. J., 50:299, 1952) and Hullin and Noble (Biochem. J., 55.289. 1953)• . .•.° ._ . . ... ~ •: . b. The cells will be fractionated in a Spinco IZ ultracentrifuge and the mitochondrial fraction. extracted and studied, in the Warburg apparatus by the method of Umbreit (Manometric Techniques and Tissue Metabolism, Chap. I) and the --ratios of oxygen uptake to phosphorus utilization will be estimated and compared. ~~ c.Differences in protein patterns of the metabolic products of the cells will be established by acrylamide gel electrophoresis. .• n ~ d. The enzymatic profiles of both cells and utilized culture medium will be .studied with special reference to proteolytic enzyme activity using chromagenic : substrates and modifications of the Bratton-I:arshall method (Goldberg, Pineda, and Rutenburg, Am. J. Clin. Path., 32:571, 1959; Blackiaood and riandl, Anal. Biochem6, 2: • 3go, 1961). 0 :,;

continuation sheet 2b A ~~ Supporting Data: i . , ~ • .. . , . ... • _ -- • ~ - . .. • .• ~ ' .. . ... , S • A. Previous work done in this or related fields. The principal investigator .'" ~.'.....has carrie on e enszve s u ies of t e e avior of normal and malignant cells in •,rabbit's ear chambers and in tissue cultures, In col.laborationn with H. S. Simms :.,,;j several hundred biological substances and organic and inorganic chemicals were . More recentlv Prowth stimulatine and erowth inhibitin~ materials have been extracted'; .tested for their effect on growth of normal epithelium and connective tissue cells,"-~ ;~Trom normal adult connective tissue and, tested for effect on growtn•ol numan tpmor :~ biopsies, Rous sarcoma, and many lines of normal and malignant human and animal ;~ materials were also tested against animal tumors in vivo, Sarcoma-180 in the mouse cells initiated in this laboratory and maintained in conrinuous cultivation. These:a and Flexner-Jobling carcinoma in the rat and against heterologously transplanted :t9? ~ human ovarian carcinoma in eonditioned rats. The inhibitory material reduced the on Sephadex-G200 and Biogel-300. At the present time biochemical analysis is in specific growth stimulating and growth inhibiting materials have been separated from crude extracts by ion-exchange DEAE-cellulose chrom3tography and•by gel filtration ,.. . in animals when transplanted heterologously into conditioned weanling rats. Active the same line of highly malignant ovarian carcinoma cells in tissue culture and also' effective against malignant epithelium in another. The latter factors inhibited :,0- for malignant fibroblasts in vitro and; in vivo in one case, and materials primarily ~ growth inhibitory properties. We have obtained substances with specific inhibition ".using different methods of fractionation we have separated materials with different showed that these factors inhibited the growthof animal tumors also. Recently by growth of malignant cells in tissue culture from- ?5-100~, Collateral studies progress to determine the exact chemical nature of these inhibitors. • B. Six most pertinent recent references. papers included, 1959. "Transplantation of Tfissuest', ed. by L. Peer. Williams & Wilkins & Co. Baltimore parshley, M. S. Tissue Culture of Adult Tissue. Chap. 17 in Vol. II of the Simms, H. S., and parshley, M. S. The effect of proteins and other substances on , ;,t the growth of adult tissue in vitro. Chap. 7 in "Protein and Amino Acid Nutrition't,:; ed, by A. Albanese. Academic Press. New York, 1959. parshley, M. S., and Mand1, I. Separation and study of a connective tissue consti- for Cancer Assoc Am cell strains m f t hibit t th th i it t i , . , ..-.rSV u or n v ro o ory e g,row o tuen n Res. Proc., 4:1:50, 1963. • parshley, M. S. Effect of inhibitors from•adult connective tissue on•growth of a series of human tumors in vitro. Cancer Research, 25:387, 1965. Parshley, M. S., and•Mandl, I. Inhibition of malignant cells in vitro by a compo- nent of normal adult connective tissue. Nature, 208':800, 1965. Parshley, Ni. S., Mandl, I., and I;anahan, J. Separation and in vitro study of ~ Q tumor cell inhibitors. Tissue Culture Assn, Proc., San Francisco, June, 1966. . W C!1 ~

R: REDACTED MATERIAL C " Reserirch Assistant 100 Diener Fringe Benefits (10) 50, Sub-Total d. Consumable Supplies (list by cotegories) _ Special gUssware and equipment for tissue culture ?ledia, chemica•ls, and stains Sub-Total C.. Other Expenses (itemize) $ 2,000 Travel to scientific meetings $ 500 Sub-Total D. Permanent Equipment (itemize) $ 500 Zeiss Phototaicroscope $ 5,820 Hark Scholander Respirometer ` 97$ E. Overhead (T59'o of lt+Si• C) Estimated Future Requirements: Salaries Consumable Supp{. OtherExpenses Permanent Equip. Overhead ' Totol C Year 2 {'~ Yeor3 ~ ~ .t 2,000 t 500 t 18)5 $14. 070- $ 2,000 $ 500 $ 1 ~4 ~$14 , 440 ItisunderstaodthattheapplIcantandinstitutionalofficen D(r.dw•otProj.ce / l ~ in applying for a grant have read and found acceptable Telephone. . the Council's "Statement of Policy Containing! Conditions Signature and •Terms Uhd'tr Which Project Grants Are Made.:' tu,Lreu Otltc.r oi rF.'. t~rGlmie~ ylCE•FRnS!DENT II'1 DHAKtit Telephone ~ OF MEDICAL AFFAIRS I 111111100=7 ~;

a ~ Olh.r Sourmof Ffnanctal Supporl Ust flnancial support for mwrch from afl sourea, tnciuding own imtitutlon, fortMr and/or..loNd r.raarch proj.cft, ' Tille of Projec Study of Naturally Occurring Growth Inhibitors Growth Inhibition of Malignant Cells in vitro and in vivo - Sourca National Cancer Institute Damon Runyan Memorial Fund Effect of Vinca alkaloids on Human Neoplastic Cells in tissue culture Leukemia Society, Inc. Scholarship

R: REDACTED MATERIAL CURRICUhUM VITAE -630 West 168th Street New York New York 10032 -• College of physicians and Surgeons, Columbia University ' ' ' ;.Gs . P~- _ 1- - .Zfarried: Thomas Fredricson parshley, New York City, April 15, 1938 Mary Stearns Parshley Assistant Professor of Anatonjy in Obstetrics and Gynecology Child: Elisabeth, Stearns Parshley,.born June 5,--1947 ,.:~;:Academic Background: . ~ . Smith College, A,B.. with High Honors 1933 (Zoology) University of Pennsylvania, M,A, 1935 (Anatomy, Medical Sciences University of Pennsylvania, Ph-,D. 1938 (Anatomy, Medical Sciences Travelling Fellow•from Smith College at the University of Penna 1934_1938 (Wilder,. Nicolson, and Fine fellowships). University Scholar, University of Pennsyl'vania, 1935-1936. Bennett Fellow, University of Pennsylvania, 1936-1937. . .. . . .. ~- ,= Professional Experience: 1, Laboratory assistant, Willard Parker Hospital, New York City, • 1933-1934. 2. Fellow in Anatomy, University of Pennsylvania, 1938-1939. Finney-Howell postdoctoral research Fellow from the Johns Hopkins University, 193$-I939• __ 3. Research Assistant in Pathology, Columbia University, 1940_1945, ',' Research Associate in pathology, Columbia University,' 1945-1949,..` 4. Research Associate in Surgersr, Columbia University, 1949-i963, Assistant Professor of Anatomy in Surgery, Columbia University,- ' 1963-1965. , 5. Lecturer in Biology, Hunter College Graduate School, 1957-1963. 6. Assistant Professor of Anatony in Obstetrics and Gynecology, Columbia University, 1965- _ vessels, tissue transplantation, atherosclerosis, cancer research. World War II: Office of Scientific Research and Development, 1942_1945. (Certificate, O,S.R.D., bronze plaque, Columbia University). Special Fields of'Interest: Microscopic Anatomy and Cell Physiology. The study of living cells and'tissues: by the Clark-Sandison rabbitIs ear chamber method, and-by a variety of tissue culture techniques. Wound healing, regeneratiomof connective tissue, skin and blood Summer: 1. Smith Scholar, Marine Biological Laboratory, Woods-Hole, Mass. 1932- 2, Laboratory Assistant, St. John's Hospital, Brooklyn-, New York, 1934. Societies and Committees: Phi Beta Kappa, Sigma Xi, AAAS, Am. Assn. Anat., physiol, Soc., Phila., 1issue,Culture Assn., Am. Heart Assn., Am. Assn. Study of Arteriosclerosis,.Am. Soc. Cell Biol„ Am. Assn, Cancer Research. Corresponding Sec., Tissue Culture Assn., 1946-1952, Member Course Cozrmiittee and Registrar Tissue Culture Assn. Summer Course, 1946-1964, c

PUBLICATIONS - MARY STEARNS PARSHIEY . Stearns, Mary L. the rabbit's ear. I. Am. J. Anat.Z6:° 133_176, 1940. Parshley, M.S., and Si.mms, H.S. tive,tissue in transparent chambers in-; ' the rabbit's ear. II. Am. d. Anat, 67: 55-97. 1940. and Parshley, M. S. Simms, H. S., Parshley, Nf. S., . . J. Geront. 2:3:205_216, 1947. Growth,and differentiation of con- ~ ~ nective tissue as observed micro-`~ scopically in the living rabbit.`Am.`~ J. Med. Sci: 198:144_145, 1939. Proc~ ;:Phys, Soc., Phila., 14:20_21, 1.938_1939 Growth-and differentiation of con-Y ..,,~ -nective tissue as observed microsco-.'- pically in the living rabbit. Anat, ~ .. Rec. 73:3. 1939 Suppi. 2, p.49 (abstract : .. , ' ... .:."s.r:~Y l1~ Studies on the development of connec-,~zl tive tissue in,transparent chambers iri': Studies on the development of connec-:'M. Conditions favoring the growth of adult skin epithelium in vitro. Anat. Ree. 94:3:486 1946 (abstract) , . '.h`~' y Studies on the fat depositing mechaniste' Proc. Am, Soc. Study Arteriosclerosis;A Am. Heart J. 35:860_861, 1947. Fat deposition in vitro caused by lip' and Pitt, R. B. . fanogens and opposed by antilipfanogen.' 8. Simms, H. S., Parshley, M. S., Pitt, R. B., and Fulton, J. B. Further studies on the fat depositing mechanism in vitro. Am. Heart J. 36: •_ 469, 1948 (abstract). (abstractl. YRhi , . , . . , ,. ., Simms, H. S. sue in vitro. Anat. Rec. 103_453, 1949 ~ 9. Fulton J Parshley B . M Growth of blood vessels from adult tis. ;k- ; S 11. Parshley, M,. S'., and Deterling, Cultivation in vitro of fresh and;frozen R. A., Jr., and Coleman, C. C.,Jr.segments of the abdominal aorta of the ': dog. Anat. Rec. 106:64, 1950, (abstract).- 10. Parshley, Nr. S.., and Simms, H, S. Cultivation of adult skin,epithelial cells, chicken, and human, in vitro. Am. J. Anat. 86:163-190, 1950.

0 18. 21. Parshley, M, S.. Deterling, R. A., Jr., C. C. - Colemarn, Jr., and Parshley, M. S. Deterling, R. A., Jr., C. C. Coleman, Jr., and Parshley, Coleman, C. C., Jr., R. A. Deterling, Jr., and 14, S. Parshley 17. Pate, J. W., P. Sawyer, R. A. Deterling, Jr., J. W. Blunt and N. S. Parshley M. S. 20. Parshley, M S R A Deterlino A preliminary report on experimental ,r studies of the frozen homologous aor- ;.,-tic graft. N. Y. Med. 6:19-22, 19S0, . . ' .}i'Lt rTn`,~ Experimental studies of the frozen homologous aortic graft. Surgery 29:419_440, March 1951, Growth in vitro of blood vessels frota - the bone marrow of adult chickens,~ • Aia; « J Anat 89:321-3~ 1951 ..,. _.. Experimental studies of preserved aor~ tic homografts. Ann. Surg. 134:868 1951. 16, Hui, K.K.L., E.B.C. Keefer, R. A. Early results of experimental studies '; °~ Deterling, Jr., J. W. Blunt and of the action of high intensity elec=' M. S. Parshley • trons on aortic homografts. Surgical Forum 2:255-260, 1951. W. B. Saunders Co. Early results in experimental use of freeze-dried arterial grafts. Surgi- cal Forum 3:147-152, 1952. W. B. Saunders Co., Phila. , .. . . . .. ~.1.~~~y~l Deterling, R. A., Jr., and M. S. A critical study of present criteria Parshley and J. W. Blunt ' governing selection of blood vessel :~ : grafts. Surgery 33:213-232, Feb. i953; 19. Parshley, M. S., and R. A. Deterling, The effect of storage at low tempera-:~: ture on growth in vitro of adult dog ` Jr, aorta. Anat. Rec. 115:2:357, Feb. 1953 '' (abstract). ~,. Tissue culture studies of blood vesse ., , o,l :~ Jr., and C. C. Coleman, Jr. grafts. I. The cultivation in vitro of Y~. fresh normal adult aorta (dog, cat, rab- bit, ' bit, goat, monkey, and human). Am. J. Anat. 93:2:221-272, Sept. 1953. The-development of tubular structures in cultures of epithelium from the cor- ~~. tex of adult dog lddney. Anat. Rec. 118:2:452, Feb. 1954• (abstract). 1003547067. •

PIIBLICATIONS - continued Coleman, C. C, , Jr. , R. A. - Deterling, Jr., and M. S. 1ParshleY : ~. , . . r Parshley, M. S. and R. A. Deterling, Jr. 25. Parshley, M. S. 26. Parshley, M. S. 1 •27, Si.mms, H. S., and M: S, Parshley 28'. Parshley, M. S. 29. Parshley, M. S. 30, parshley, M.*S „ and I. Frandl 31, Parshley, M. S. ., Some long-term observations on aortic•~' _ homografts. Surgery, 37:64-79, Jan, 1955. The effect of storage at low temperatures on growth of adult dog aorta.• A. J.'Anat; 97:3:359-394, Nov. 1955. • ~~ Factors affecting the character of the, outgrowth of tissue culture from the "" ~ bone marrow of adult human rib. Anat. :' Rec. 124:429, 1956 (abstract). The effect of serum from patients with the nephrotic syndrome on tissue cul- •„L " tures of human-embryonic kidney. -Anat, Rec. 127:439, 1957 (abstract). •s; The tissue culture of adult tissues. Chap.17 in the "Transplantation of Tis- sues", Vol•.II, Iyndon Peer, ed., Will- UR iams and Wilkins Co., Baltimore, pp.593- "`t ; The effect of proteins and amino acids on the growth of adult tissue in vitro.- Chap.7 in "Protein and Amino Acid Nutri- tiont', A. Albanese, ed,, Academic Press,. 143 k N Y 195 1959 - ew or , pp, . , Isolation of a growth inhibitor from adult connective tissue. Excerpta Afed,,•--'>'•=:'•~ Vo1.15, No.7, Sec.I, p.662, 1961 (abstract). ~ Effect of growth inhibitors from adult :s connective tissue on growth in vitro of a series of human tumors. Excerpta Med „ Vo1.16, No.9, p.814, 1962. Separation and study of a connective tis- sue constituent inhibitory to the growth in-vYtro of tumor cell strains. Proc. Am. Soc. Cancer Res „ 4:1:50, 1963. Effect of inhibitors from adtilt connective tissue on growth-of a series of hunan tu- mors in vitro. Cancer Res. 25:387-399. 1965 1oo3S47o9S ~ ...:,~

0 .. ' 33. Parshley, M. S., Blackwood, 38. C. E., and Mandl, I. Parshley, M. S., and Mandl, I. Parshley, M. S., Mandl, I., and Manahan, J. Levi, M., YAndl, I., Parshley, M. S., and Swartz, D. P. Levi, M., Parshley, M-., and Mandl, I. Martorelli, Braz, Jr., Parshley, M. S., and Moore, J. G. ix~:sx;r Inhibition-of malignant cells in vitro '; by a component of normal adult connec-X"a~~ " tive tissue:-'Nature, 208:800, Nov. -1965'' 5eparat2.on ana i.n vitro szuay oi tumor cell inhibitors. Proc. Tissue Culture Assn., San Francisco, June, 1966. In Vitro, 2:124. 1966. , r stadenocarci Antibod illar c t a - y g y o p p noma of the ovary. proc. Pan American Cancer Gytology Congress, May 1967, New York City. Antigenicity of ovarian carcinoma cells : grown-in tissue culture. Proc. Tissue Culture Assn., June 1967, Philadelphia. Effect of chemotherapeutic agents on-a ~ ' " line of human breast carcinoma cells in +_i cciiP m~l i.iTr~z _ Urne _ Ti ssue f:ul t tarc~ d h =:} 6 ia, elp 7, Phi.la Assn,, May 19

: :Tur Cou`'CIL FoR TOB11CC0 ~.'ESLARCH-U. S.A,. .The committee comprising Dr, Cattell, Chm., Dr. Bing and Dr. Jacobson. Equipment grant application from Saul Boyarsky, M.D. of Duke Universit Medical School - N0. 633. We enclose herewith an application for a grant to purchase an item-o major equipment from Dr. Saul Boyarsky of Duke University Medical School in Durham, North Carolina. whether this fact influenced the S.A.B. decision). Health on July first of the same year. (btr meeting notes do not show for certain . from the American Medical Association's Committee for Research on Tobacco and consideration may have been the fact that he received a grant for the same purpose motility. This application was deniedby the Board in September, 1966. One study the effect of nicotine and other neuropharmacologic agents on ureteral In July of 1966, Dr. Boyarsky applied for a grant of $16,907.00 to The present application, requesting a great deal more money for a• of the normal and abnormal ureter and increases the force of contraction". "have demonstrated conclusively that nicotine accelerates the peristaltic rate cinefluorographic unit"'also includes progress reports on the nicotine work which -.'.'Hydradjust Table and nine inch image intensifier Vidicon with 500 MA generator ';the same committee with Boyarsky's earlier application, they are being kept on well as bibliographies of their publications. Since these were distributed to Boyarsky furnishes lengthy curricula vitae of the investigators as file in the office for reference by the committee chairman. R. C. H.

THl; COUNCIL FOR TOBACCO R~SEARCH - U.S.A • 88s TIUMD AV'ENIIE =4+I1TTEE: . NE{V YORK. N. Y. 10017 Dr. Catte~, Chm6 Dr. Bing Dr. Jacobson Application FonResearch Grant Date3rAugust, 1967 of Urology; Peregrina Labay, M.D. , Research~Associate in Urology 2. Institution & Address: Division of Urology, Department of Surgery Duke University Medical Center Durham, North Carolina 27706 3. Short file of Project: Nicotine Effect on Ureter in Man: A Possible Therapy for Hydronephrosis. 1. MlameofInvestigator(s):(includeTitteandDegrees) Saul Boyarsky, M.D.,Prof. of Urology, Director, Urological Research; James F. Glenn, M. D. ,Prof. of Urology, Chairman Division 4. Proposed Starting Date: December 1, 1967 S.AnticipatedDurationofthisSpecificStudy: Three years C Q Brief Descripton of Objectives or Specific Aims: In recent animal experiments we have demonstrated conclusively that nicotine accelerates the peristaltic rate of the normal and abnormal ureter and increases the force of contraction. The renal pelvis and ureter are thereby emptied super- normally by nicotine action. This nicotine effect' has been demonstrated in our laboratory by several in vivo techniques, cinefluorography, peristaltic pressures, surgical observation, as well as in vitro and in abnormal ureter such as diverted'y ligated' and pharmacologically- blocked ureters. Nicotine proditces these effects whether administered by the intravenous, intiraarterial, pulmonary or topical routes. The drug releases catecholamines from sympathetic nerve endings and the adrenal gland in addition to its cholinergic action, and its release of antidiuretic hormone. Hence, cigarette smoking may be a "natural" for the treatment of hydronephrosis which is characterized by depressed motility of the renal' pelvis and ureter, and excessive collection of urine in the renali pelvis. The human uretea resembles the dog ureter pharmacologicalUy in every respect we have had an opportunity to study. Our assays of surgical specimens have shown that the dog and human ureters have a similar catecholamine content. The humann ureter responds in vitro to the same spectrum of drugs as the dog ureter. 7. GiveaBriefStatementofyourWorkingNypothesii: Since nicotine accelerates ureteral'peristalisis in dogs, and the human and dog ureter are similar pharmacologically, the effect of nicotine on the normal and hydronephrotic human ureter would be eXpected' to stimulate peristalsis and to restore tone. C~

8. Details of!Experimental Design and Procedures: (Attach Separate Pages) It is proposed to study normali voluntee: s and urologic patients by cinefluorography to determine the effect of nicotine and tobacco smoke upon ureteral peristalsis in . ~ health and disease. laboratories in related fields provides the background for this study in a university Duke University Medical Center with its library, faculty and plenitude of research passed also. 9. Physical Facilties Available (Where Other than Administering Organization Indicate Geographical location)', metric pressure. studies of peristalsis will' be performed whenever catheters are catecholamine assays of surgically excised ureters and renal pelves; and c) mano- -in terms of rate, rhythm and character of bolus progression. contrast medium willi allow us to describe ureteral peristalsis and pelvic peristalsis ' 1) Observation of the ureter during a slow intravenous infusion of concentrated 2) The effect of nicotine administration and'tobacco smoking upon this control rate will be determined. 3) Patients with ureteral obstruction, hydronephrosis, dilated' renal pelves and surgical lesions of the pelvis will then be studied t'o note the effect of nicotine upon ureteral and pelvic function in these patients. 4) Ancillary studies will include a) in vitro motility studies; b) fluorozrnetric C medical center. See addendum Page 2a, 10i Additional Requirements: This proposal is actually for a part of our total needs. The requested unit' will be an integral part of several larger projects mentioned above and already in progress. _ Salaries, supplies and staff are available through these projects. The annual budget is actually between 2 to 3 times the amount of the present request. Our present need' is for this equipment, not salaries or supplies. The salaries needed to use the equipment are being funded from other sources, so that the budgetary deletions would represent a "savings" in one sense to the Council for Tobacco Research. ( 11, Biographical sketches of oil principal and professional personnel (append) ' 12. list of publications: (Five most recent as pertinent) (append)

Council for Tobacco Research Saul Boyarsky, M. D. Addendum to No. 9, Physical Facilities Available ,.: Three hospital services, the Duke Medical Center, Durharn Veterans Administration Hospital and the affiliated Watts community Hospital provides some 80-100 urologic beds. :;.The research laboratories include 2200 sq. ft. in~ the new Clinical Research II Building and some 1200 sq. ft. in the Bell Building animal laboratories and the ~ Yeterans Administration Hospital adjacent to the Duke campus. `; At present these laboratories are complete for animal cinefluorography (obsolete for patient use), animal surgery, electrornanometry, chemical, histo= '. logic and physiologic studies. However, we have not yet fund'ed the cinefluorographic unit for which adequate space has been provided in our new laboratories. These laboratories will pass from the contractor to Duke University within the next 6-8 weeks. ~. The research staff is one of the largest in any urology department in the country We have a full-time research professor who is the principal investigator of thils . project, a full-time research associate, a pha"rmacologist and a bioengineer in the ` Division of Urology, a half-time radiologist as well as a collaborating biochernist, ;r (Dr. Norman Kirshner of catecholamine fame) and a physiatrist, Dr. Robert Gregg .' A USPHS academic urology training program supports three pre-residency ~ research fellows and two post-residency research fellow s at present engaged in r'? research (three full-time an6 two half-time). We have five research technicians, ~; two research secretaries, and two research nurses in the urology research laboratory "As will be d'etailed, below under the additional research support section and in the presentations section, active research programs are in progress in surgical .physiology of the ureter, in the area of catecholamine biochemistry, in ureteral neurology, in human diagnostic cinefluorography of the ureter, ureteral reconstruction and pyelonephriitis. Funds are needed for the cinefluorographic unit so that patients can be studied in the urologic facility properly, rather than when an opening on a unit located in the Radiology Department becomes available. Backing from Dr. Richard Lester, Professor and Chairman of Radiology, is enthusiastic as is evidenced' in part by his assignment of Dr. William Barry, Associate Professor of Radiology to Uroradiology.

4~ : ` Council for Tobacco Research Saul Boyarsky, M. D. ' Addendum to No. 12, List of Publications ,Boyarsky, S. , Labay, P. , Kirshner, N., and Gerber, C. : Does the Ureter Have Nervous Control?, J of Urol., 97:627, 1967 (April). The Neurogenic Bladder, Saul Boyarsky, M. D. (Ed. ) The Williams and . Boyarsky, S., Kirshner, N. and Catacutan-Labay, P. : Catecholamine Monographs of.the Surgical Sciences, Vol. I, pp. 173-2:1i3, June, 1964. No. 2 -Wilkins Co., Baltimore, Maryland, March, 1967. Boyarsky, S'. , Labay, P., and Kirshner, N. : Accelexation~ of Ureteral Peristalsis by Adrenal Compression, Science,, 154:669, 1966. Content of the Normal Dog Ureter, Invest. Urol. , 4:97, 1966. . Boyarsky, S. : Surgical Physiology of the Renal Pelvis and Ureter,

r A. Salaries (Personnel by names) Professional B. Consumable Suppiies(list by categories) i % time Amount ' Sub-Total Sub-Total :Saul Boyarsky, M. D. 'Duke Utiiv Med Ctr D. Permanent Equipment (itemize) $61, 950 61 50 Hydradjust Table 9'r image intensifier Vidicon with 500 MA generator cinefluorographic unit C Saiaries Consumable SupplJ Other Expenses Permanent Equip. C . 13. Budget: (lst year) It is understood that the applicant and'institutional officers in applying for a grant have read'and found acceptable the Council's "Statement of Po6icyLontaining Conditions and Terms Under Which Project Grants Are hSode." Signature ttAJ _ 2 681 b D~ <rlorosvroi«t , 919) rJ ~ , _~ Teiephone ~ ~ ~/ Signature BuePnns Obflkecof th<.IhstiluNon S.; C. Harward ComtrolLer Telephone 111111CM1111111111111 `_

4. Other Sources of Financial Support List financial support for research from all sources, including own institution, for this and/or related research projects. Current ritle of Projed Pathophysiology of the Scarred Ureter in Dogs Pathophysiology of Ureteral Obstruction in Man The Ureter in Pyelonephritis Workshop on the Optimal Care of the Neurogenic Bladder Patient In Vitro Studies of the Ureter Ureteral Pharmacology Toxicology and Pharmacology of Ureteropelvic Motility Source NIH NIH NIH NIH Vocational Rehabilitation n Administration NIH NII-I' Army Amount Duration 17, 500 7/1/67 - 6/30/68 17,000 7/1/67- 12/31/67 14,565 7/1/67- 6/30/68 20, 410 8/1/67- 7/31/68 14,966 8/1/67- 7/31/68 36,828 7/1/67- 6/31/70 20,614 7 /1/ 67- 6/30/73 19,248 7/1/67- 6/30/68

• Saul Boyarsky, M. D, Duke University Med Ctr Progress Report: lnclude in sufficient detail, for, purposes of evaluation, accomplishments related -`; to the original goals of this project, and information concerning any new , lines of investigation which have been followed. (Tables and figures may be included.) Attach additional pages as required. ;;Effect of Nicotine: r The effect of nicotine upon the function of the renal pelvis and ureter has een established. Ureteral peristaltic frequency is accelerated by a factor of 2 to 5 under optimal doses. This has been demonstrated by the methods of peristaltie` ~.~.- . pressures, cinefluorography, and direct' observation at laparotomy in the dog. Our studies show that nicotine reaches ureteral receptors whether administered topically intravenously, intraaortically, or by inhalatirn . The classical biphasic effect of r~ ° nicotine is observed on the ureter as well as elsewhere, that of preliminary excit- afion followed by a longer lasting depression of activity. Dose-response curves have not been established completely and definitely yet; work is underway to gather this `~ data. r z s`. Localization to Ureter: Yl.~ Critical experiments have demonstrated that the nicotine effect is entirely. ;4separate from that of any antidiuretic effect which may be also introduced. In vitro °.~tudies have demonstrated inconstant, unpredictable and weak effect of the drug ~ acting d'irectl'y on the srnooth muscle of the ureter. These studies are continuing. Nicotine Analogues and Metabolites: With, the cooperation of Dr. Charles H. Jarboe, Assistant Dean and Associate Professor of Pharmacology, University of. Louisville, School of Medicine, we have ' instituted pilot studies on the ef2•c cts of nicot:n-_ moaon-lethiod'ide and other analogues. here does seem to be ureteral activity inherent in, these compounds. , , ; .. a; ~ ~~.:. .. . ..: ~ . . ~ r;%. . . . . .. . . . Studies with a batch of cotlinine, kindly supplied by Dr. Herbert McKennis, Professor of Pharmacology, the Medical College of Virginia, has shown that these breakdown products of nicotine are inactive. Mechanisin of Action: The rrech3nism, of action of nicotine on the ureter is under investigation. Studies on the adrenallectornizcd dog and reserpinized dog, are continuing in order to demonstrate the rolie of intrinsic c vechoian-Lines, vis-a-vis the role of systemic catecholartiines secre'ed by the adrenal gland. Correlati•,n \srith Re:nal F,ffects: ~The long series of experinler.t:i oni tht:~ different:all effect of topical and nicotinic • nerve stimulation has shown that thore are overlapping effects between ureteral perist'alsis and renal secretion of uri:1e. 1003547078

AMA Progress Report `° Effect of Nicotine, on, the Urtnary Trae-t ~ Ureteral Peristalsis Sau1 Boyarsky, M.D. :;. . 'At present problems remaining to be investigated include: e74effect of adrenal cortical hormones in supporting muscular contraction role of the cholinergic system in ureteral peristalsis.. ontimuing-call-c-cti~on-of data to fill out dose-response curves ~,r;:<; Elucidation of the renal pelvis and ureter as a delicate neurornuscular .mechanisrn -rather than an autonomous muscular tube represents a fundamental advance in Urology and in U'seteral Physiology which should have far reaching s `..diagnostic and therapeutic effects. The rapy: Therapeutic uses are suggested by present research and should be welcome. One of the objects of our research is to develop drugs which can be used in the . reatment of ureteral colic and similar disorders. =Other Benefits of Research: .~ _ he Tesult of this project on our laboratory has been most beneficial. Our g1'oup ha`s`grown-to embrace bioengineer, pharmacologist, and radiologist in addition '.to the Urolo y research fellows. The additional equipment has increased the efficiency of.our efforts, particularly the tape recorder which has allowed immediate analysis ;~,.of cinefluorographic experiments and longer periods of observation, at lesser expense than was previously possible with cinefluorographic film alone. Does the Ureter Have Nervous Control?, Physiologic, Pharmacologic and Biochemical _ Evidence for Sympathetic Innervation, Peregrina Labay, American Physiological Society, Houston, Texas, August29-Sept 2, 1966. ftecent Advances in Ureteral Physiology, Saul Boyarsky, Pan Pacific Surgical Association Annual Meeting, Honolulu, Hawaii, Sept 20-28, 1966. ~: Symposium on Urologic Education, Saul Boyarsky, Landmark Inn, Duke University Sponsor, Div. of Urology, Durham, N. C. August 29-Sept. 2, 1966.

~~.Ureteral Pharmacology and Toxicology, Saul Boyarsky, Edgewood Army Arsenal, Edgewood Arsenal, Maryland (March 24, 1967)~. re Ureteral Nerves Functional?, N. Kirshner, S. Boyarsky, P Labay, C ,,Gerber .,~ and J.. F._ Glenn, American Heart Association, New York, New York -' Oct•ober 21-23, 1966. urther Ureteral Neurophysiology Explorations -- Evidence for Nerve Influence 11 on Ureteral Peristalsis, Saul Boyarsky, Spinal Cord Injury Conference, West Roxbury, Massachusetts, Nov. 7-9, 1966. Ureteral Peristalsis from Conduit to Control Mechanism (Taylor Lecture) Saul `' : Boyarsky, University of Oklahoma, Oklahoma City, Okla., Nov. 17-19, 1966. >, -Is There a Physiologic Basis for Ureteral Pharmacology?, Saul Boyarsky, Physiology Pharmacology Seminar, University of Oklahoma, Oklahoma City, Okia. Nov. 17-19, 1966. EXHIBIT: Physiologic Diagnosis Through Urodynamics, R. D. Ensor, S. Boyarsky,.*; 1 and J. F. Glenn, Southeastern Section of American Urological Association, y; Hollywood Beach, Florida, April 9-13, 1967. 'reteral Peristaltic Acceleration by Renal Nerve Stimulation, Peregrina Labay and- .;: }, Saul Boyarsky, American Physiological Society, FASEB, Chicago, Illinois, April18-19, 1967. More Evidence for Ureteral Nerve Function and Its Therapeutic Implications, Saul Boyarsky, Peregrina Labay and J. F. Glenn, Annual Meeting of the Society of Genito-Urinary Surgeons, Rye, New York, May, 1967. EXHIBIT- Physiologic Diagnosis Through Urodynamics, R. Dale Ensor, Saul Boyarsky, and James F. Glenn, American Urological Association, New York May 1967, lst Prize. In Vitro Studies of Ureteral Smooth Muscle, J. Malin, S. Boyarsky, and P. Labay, American Urological Association, New York, May 1967. The Effect of Topical Nicotine on Ureteral Peristalsis, Peregrina Labay, and Saul Boyarsky, American Medical Association 116th Annual Convention, Atlantic City, New Jersey, June 18-22, 1967.

~ ..A.~ ...,V-~. . r ~.• - ~ ... ~ ~`~ A i L ma , n ` G. June Yiautz, Yerzgrina Labay anr! Saul 13oyarsky MOVIE - 'The Effect of Nicotine on Ureteral Peristalsis - The Experirnental. presented at the following meetings: . Symposium on Urologic Education, Landmark Inn, Duke Univ. Division o Urology, Sponsor, Durham, N. C. , Sept. 1966. American Urological Association, Annual Meeting, New York, May, 1967 .116th Annual Convention of the American Medical Association, Atlantic City, New Jersey, June 18-22, 1967 n Does the Ureter Have Nervous Control?, Saul Boyarsky, Peregrina Labay, Norman Kirshner, and Carl Gerber, J. of Urol. , 97:627, 1967 (april) AMA Prot rens Report Effect of Nicotine on the Urinary Tract: ~~Ureteral Peristalsis Saul Boyarsky,-M: D. _ The Effect of Topical Nicotine on Ureteral Peristalsis, Peregrina Labay . and Saul Boyarsky, JAMA, 200:209, 1967.

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---