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N1M012013'/ ( CJum. •8tol. (ntensationa. : 3(:97 a 1 31"-331 ® Flaoner Saantilic Pudliaaing Cumpony. Amator,tan - Printed :n The NetbeNands S1tiDIES ON PULbiONaRY AILYL HYDROCARBON , HYDROXYLASE ACTIVITIP 4V I,YBRED STIiAL'4S OF .NUCE RICHARD B. KOURL THOM.6J RUDE, PA(JL E.'IH064A.S.° and CdRIitH L WHI7a'lIRE . Depertrnant of 8loeheneiea!-0nroto~r. .Nierobio/ogieal Aaaoeiatae, i7?3 Bathesde dewws, 8ethetda. lfd 20014. and ' Dep•artmant of Afoel+emiutry and Drng Mataboilan4 hfolhnan•LaRoche l,m.. Nntlop. N.J. 07Zl0 (U.S..LI (Reeetved July 16eb, 19751 (ReTiawn nxea.ed and aceapted Dacmober 5th. 1975) SUMMARY PuLaonary and hepatiG leveis of aryl hydrocmcbon hydroltyiase (AHH) were studied in inbred strains of mice following inctatracheal (i. :.) inscillation of 3-methylcttolanttirene (MCA). Lt. tnstillaaon of 188 Feg MCA in stsrile 02% gelatin in saline resulted in pt'eferetltial induction of pulmonaey AHH. After treatroent vath this dose of MCd, the pulmonary ?,tiH levels of stsAUts C5 7BLl6Cum. C37BL(6i, BALB/a.Mai. C3EIJi14[ai. and C57LJJ wete ob- served to be induced within 24 h after ueatment. Strains DBAl2C+>;al, a KRld. SJLJJ. DBA/2d and RF/d expresseri ao such incresse. At a dose of 500 ;ag ViCA., the pulmonary time of DBA/2 mice did express a 4-fold :ntsease. This increase in AHH was determined to be quita different from the increase observed in C878L/6 mice by: (1) specific activity of the enzymes, (2) gmietic reguiatton. (3) snsceptibi3lty to inhibition by 7,11-benaotlavoae. and (4) spectrrl properties of the associated cytoch.romea. It was of major impor- ;ance that induction of pulmonary AiiIt was observed to be regulated by a single dominant gene in crosses iavolvi" the CS78W6Cuo and DBAi2Cum suauls of miae. Rewlts were discussed with the view in mind that theae genetically tegulated levels of .1FiE3 may play a role in suaceptibility to cancets induced by polycydic, aromatic hydrocarbon csrcinogens.. Abbrenauoni: Al{Ei. xpl hydroaarbon hydrosytase; 3P. benae(alpyrene; DMSO, dnteehyltul[axide: MCA, 3-mathylcholaothsrna; TCI1D, 2,3,7,9-eettachloeodibansaLp)- dS07Xin. 31'

HT00120138 \ ( wTtiODCCTIOa AFIFl is the name given to one oi Wn multicomlmnent niixcd•Cuiletion oxi- clases which convert a variery of lipid•soluble compounds to water-soluble forms, usually for subsequent elimination from the body (see refs. 1.2). The presence or uiducibdity of AHH is associated wir,h the cytotoxic action of certain chemical carcinogens in vivo (3] and in vitro [4.51 and the detosifi- cation [6-8] or activation (9-131 of polycyclic aromatic hydrocarbons in chemical cardnogenesis. Thus. one is confronted with the paradoxtcal atua- tion .that the same eazysne• syst8m may both detoxify and acuwate the sarae chemical aarcinogens. The AHH enzyme in inbred strains of mice posaesses two properties which make it particulaly amenable for studying its role in chemically induced cancers, tlsac is, the system is inducible °[14,13], and this iaductbility is genetically controlled. Dependiog on the strains used, hepatic AHH inducibility mn be nVlated by & single autosoaial donsinant gene (14,16], a single autoaomal codominant gone [17,181 or non-ospon- siveness can be the result of a dominant allele [181. Using these genetic sys- terns, recent information suggesca that this enzyme system plays a major role in the susceptibility to IMCa (19-21 ]- and BP (Z".1 -induced carcinogenesis. The lungs and shin of mice seem to be under a different kind of control because, these orgaas seem to be slightly inducible in those mouse strains in which the liver is completely non-inducible [23-261. 11us apparent dichot- omy is very important to understand because it suggests that the genetics of AHH inducibility is orgap-dependenti rather than strain-dependent as origin- ally conceived (14,16] . In this paper. we show that there is both a qualita4ive and quantitative difference between the increased levels of ASH observed in the AHH "non•inducible" mice and the increase observed ui the AHH "inducible'• mice. Moreover, in crosses in•,olvug the C5'Bi.l6Cuas and DB,A/2Cum stzains of mice, pu4nonary AHH reaponsiveness to intrztracheally odminWet+ed MCA is regulated by a single autosomal dominant gene. SXPSRS'DfENTAL PR'nC8DU88 .lfaterrals The polycyclic hydrocarbons 3P and ylCA were purclaaseid from Sigma Wteisdcsls (St. Louis, Mo.) and poroed by nectysrallization from benzene. 7.8-Benzoflavoete was purchased from alclrich Chemicals (Cedar Kaoils, Y.J.). Metaphane was purchased from Pftman-Moore (Washfngton Czossing. N.J.). The sources of mice ~rere either Cumberland View Farats (Clinton, Tenn.), The Jackson Laboiatory (Bar Harbor, Maine). or Microbiological Associates ($ethesda, Md.). For inUW=2denl inatillations, a Bausch and Lomb stsreo- ° Tho wrnt "inducibility" as used in arie papar. dQnota a relacive increase in raws of de novo syrnehoafa or ai aeeivation of eazyms aeuviey from pre-exiaUna moiaumL or in rar® of both, w6es compared to rate of bwnkdown. Vo parcicular mechamsm u implied. 313 I

0 H TE-01201 39 ( microscope equipped with fiber optic illuminacion and Hamilton syringes wich 22 ga oy 38 mm (with 1.5 mm bails) feeder needles were used. Methods Care and feeding of mice were as stated previously (271. Animais were always treated betweern the times of 9:00 and 10:00 a.m. to avoid diunaal, variations. The i.t. iastillation technique was similar to that described recent• ly by Ho and Fuesc (28J. Aniaials were anesthesized by inhalation of ineta- phaae and were placed oa special boards designed to hold their mouths open and at the correct angle for instillation. After positioning the animals, 0.02 ml of solution was given i.t. directly into the lung. The solution consisted of MCA suspended in 0.2% gelatin in sterile saWte [29] or MCA dissolved in trioctanoiin. At various times post-treatment, the lungs and livers were excised and frozen at 70°C until assayed. Microsomes were prepared from lung or liver tissues according to the methods of Nebert and Gielen (301 and were stored at 70°C for up to 72 h before being assayed. Samples were diluted with 0.1 N Tris-HCL buffer (pH 7.4) to a Enai ratio of 1.0 ml of rniaosomal suspension per gram wet weight tissue. The assay for aEiEi was basically that of Neben and Gelboin (151 as modified by Nebert and Gielen (30) :and Thomas at al. (14j. Tissues were cuefully weighed and homogenized in 0.05 X Tris: 0.25 M sucrose buffer (pH 7.,*) at a 1: 10 (wlv) dilution for lunes and 1: 20 (w/v) dilution for livess. 0.2 ml of homagenate was added to 0.79 ml buffer containing 100 gmoles Tris, 0.90 µmoles vADP11, 0.78 µmoles VADf•l. 6 µ:noles VLgCh and 200 feg bovine serum albamus (Cohn Fraction V). 80 nmoles BP were added in 0.01 ml acetone to initiate the :eaction, and cubes were incubated with shaking at 3?°C for 20 min. The assay was stopped by :be addition of cold acetone-hexane (1 : 3.3) and the polarmerabolitea in the hexane phase were bac4:-extracted with 2.0 ml of 1 N NaOH. The amount of fluorescence sssociated with the presence of 3-OH BP was deterauned with e.ccitation at 398 nm and emission at,522 nNn in an aminco-Boavmaa spectrophototluoro- meter. 1ilfcrasomal preparations were assayed for 4HFi activity in a similar raanner, however, the amount of microsomal protein was determir,ed by the ;ttethod of Oyama and Eagle (311. A unit of AHH activtty is deHned to be that amount of enzyme producing the fluorescent equivalent of 1.0 amoles 3-0H BP per min at 37°C. For hepatic and pulmonary tissttes, this is given in terms of units/g wet weight times, and for microeomal preparations in tezras of units/µg protein. 7,8•BenzoOavone inhibition of AFIIi in microsomal preparations was determined according to the procedures of Goujon et al. (321 and Nlebel et al. [24,33}. Concentrations of 7,8-benzotlavone were dissolved in DMSO and 0.01 ml was added to tubes contaiaing all factors for the AHH assay. occluding BP. The tubes were incubated at 37°C for 1 min, and then $P was added. For the spec=rl studies. =crosomal pellets were resu.6panded by homo- 319

0 HYV120140 I E c Gcnization in 0.1 N3 I:•PO.,, pH 7.4. Spcctral studies were prrformed on an .\minco-Chance DW•'' V'Vlvis spcctroj,lu,wmctcr in uic split bcam mode. Cytochrome P-4S0 was deterrnined after bubbling CO. adding 0.5 mINt NADIi. dividing the sample between both cuvettes. correctinc tbe baseline and then recording the spectra a[tcr adding sodium ditltiionil.c tu Uhe sample cuvette (34). This procedure cancels out the contribucion from cytoclaPome b, and hemoglobin. BESULZ9 .4FIhf levels follorufrtq i.t. admtrsiscrarioR of MCA Effect of uehicle. Tables t and II demonstrate the pulmonary and hepatic responses of B6D2F,Cum mice 24 h after treatment with various doses of M.+~CA pven in either 0.2mo gelatin or trioctanoin. MCA suspended in the gelatin solution seemed preferendaily to induce pulmonary AHH at dose levels e 188 µg. MCA ln trioctanoin induced both pulmonary and hepatic AHH at every dose level tested. All subsequent studies were done with MCA suspended in 0.2%, gelatin so thati pulmonary AHH could be specifically studied. Effect of aarental strains. The hepatic and pulmonary responses of CS i BL/ 6Cum (86) and DBA/2Cum (D2) are shown in Table fII. The 86 strain re• spom9ed to MCA much like the B6D2F, hybrid (Table I): however, ac doses > 1881q, MCA induced both pulmonary andhepatic AHH of 86 and BBD2F, mice. Pulmonary AHH levels in 02 mice were induced Ilightly by treatmenc TAS[.8 I EFFECTS OF 114TRATit.aC8S.1f. IIdST1I,,L.ITION OF vAg10C5 31CA DOSGS iN T8i0CPANOIId ON PVLYIONARY AND HEPATIC At1I? • IN 3603F;Carn MICE jtg MCA Lung AUi Liaer AEW Uatraawd '14ioo MCA• Trioe Ind. ° Unrreatad Trioe MCA- 'rd= Ind. 0 0.32 o.ZO - - 12.6 9.8 - - 25 1.0 9.0 19.6 2.0 SO L' 3.3 19.6 2.0 100 L6 8.0 38.2 3.9 200 L9 9.5 39.2 4.0 400 LG 8.0 . 62.7 6.i 500 L2 8.0 44.1 4.3 •,lM aaeivity givea in terres of unrta per g vrel aei91:.t ttewe. A unit ie that amount of onaymo caueinq the lluoresoant equivalent of 1 nmolo uf 3-0E{ UP per rnia ar 37°C. Neao of at laast three anmalr emyed in duplicetU; valuee for individud mice aera usuolly within 10% of each other. d Ind.. IndudbiLity: the relative inereasa of 1ICa-ereat.d tiesue wer vehiole-aaated Conaoi tissua. 320

NT00120141 l l TABLE II EFFECTS OF INTMa T ZAC:IEAL IINSTILLATION OF VARIOUS VICA DOSFS IN J.:.°a GELATIN ON PUt.4ON.1RY AND HEPATIC Aall + IN 88D2F,Cum MICC u81r1C.1 Luna AHH Liree ,iHH 0 5 11 23. 47 94 I88 375 500• Untreawd Gal NICA: Ind. Unrsearsd Gal MCA: [nd. . Gal God 0.36 0.28- - 1:.6 10.0 - - 0.56 2.210 7.81 0.78 0.76 "-90 L0.30 L03 Ld0 5.40 : 10.110 L10 1.30 5.00 14.40 L40 L80 6.90 LI.,I0 L10 2.00 7.70 18.90 1.90 :.i0 9.20 24.00 2.40 '.a80 9.60 28.30 Z70 Soe Table I [ar [ootaoet~ TABLE DI EFFECTS OF INTRATRACHEAL INSTILLATION OF VARIOUS IMCA D0SE8 IN 0.:°6 GELATD+I ON PULMONABY AND HEPATIC AFIH ` IN CS78Ld6Cusm AND D8A/2Cwm MICE Mg YICA Lung .1HH Liver AHH Uactsnted Gal %1Ca Gal [ndJ d Uncewsad Gal MCA Gal Iad. .1. C9TBL! BCum 0 0.40 0.32 - - 17.8 14.5 - - 5 0."3 2.30 8.3 0.57 11 0.89 280 13.3 0.92 22 1.30 4.70 13.1 0.90 47 1.40 i.i0 14.5 LOiJ 94 1.70 5.30 24.7 1.70 188 :.40 7.50 32.3 2.Z0 375 2.80 1.30 35.0 2.,I0 S00 2,40 7.30 3Z.3 Z10 B. DBAJ 3Cum 0 0.210 0.18 - - 9.80 ' 9.80 - - 188 0.1': 1.00 10.1 1.10 500 0.58 3.10 10.2 1.10 See Tabio I lae foomocoir. ' 321

NT60120142 ( 111 with 500 gg N1CA but not by 188 rg 1dCa. winc:eas hepatic ?IEtH was un- affected at either dose. The average inducibility :or pulmonary tissue of ten D2 mice following MCA txeatment was 3.1 : 0.6 (s.e.) In teaas of specific activity, the D2 strnin 71as very little pulmonary AHH aetiviry: the highest induced activity was about the 9aate as the constitutive (or control) activity of the other two e*ramQ- Both sexes oa the two inbred strauns and their 60 30 20 0 1 I 2 0 0 A t 0 0 o--4b 02 G-s 86 a--r 8602Ft 10 11 12 13 • OArS F(q. L. Kioetlas of pulenonaay and hepetie AliEi f0((owEng i.tL adntinimatlon of MCA. (a) 11mo•dmpendsat iname in pulmbnsry AHH foUowine l.tL trentment arittt 138 µ8 MCA into 02 36 io--Q1 and 88D2Fi (a---o). (b) Timedependene increaee in pu(monary AHFI fofloona8 i.t raeatmant with 500 AB MCA into 02 (d~~l. 06 ie,--®i and OsD2F, (O-~~:) miee. (c)'PimrdepfndOnc mcreae in hepatic ~1FiH fotloanpg i. a traatmoeu Wesla 500 µg 78CA into Q2 (.r--s ). 36 ~a------0 ) aad 86D2F L (O~o) mict. 32.:

TABLE IV KFAECTS OP INTRATflACiiliAl. INb"PiL4AT1ON OP 180 NB MCA IN 0.2% QB4ATIN ON 1'ULaIONAIiY ANU IIl{P.1T11: A1111 • 1?: VABInUS BTRAINS OP MICB 99mue b ({AI.NlcL1d C31UIElai C67WJ C67U1./t3J AKII/J SJLIJ DO Al3J Iti•'1J -- ' l.ung ' Conlrol 0.71 0.33 0.64 0.28 0.28 0.10 0.20 0.41 1NG:A 8.1(4.4) 2.6(7.2) S.4(b.3) 2.4(8.OI 0.46(1.6/ 0.28(1.6) 0.26(1.d/ 0.5111.3) Liver Coalnrl 10.2 7.9 138 16.1 11.6 10.8 B.9 13 b i1l:A a2.2(1.2) 7.310.9) 27.442.11 32.0(2.0) lb.s(0.9' 11.711.11 B.slo.0) 136110) ° A1111 aetlaity gl.en In lerms of unils per g wel wuigbt lisaue. A unit is tbad amount of ansyme csusing Ilre fluurescent aquir°lenr uf 1.0 nurule of 3•O11 11P per min at 37°l!. The r°I°tire inducibility Is Qivsn in p°reatbea°o. b Tirssues wero remosJ 24 16 after 1.8. Irn°Inuenl witb ei/bur gelslin s+.llno or 188 pg 61CA. .

d.JC0120144 ( ayurid were assayed for :hesr pulmonary and •nepatlc :\Flll responses tailow- in¢ 1IC.1 artd no differencos were obscrved (data not eho«•n I. Ctangcg in pultaonary AI*I with t;mc following i.t. instillation in cclaun of 188 p¢ NiCa into 36, D2 and 86D?JF, mice are shown in Fig. la. The induction time course was similar for both .86 and 66D2F, mice: maximum inauction was obseved by 24 h and remained constant for up ta 96 !1. The maximum observed incresse for each strain and hy brid was similar; about 6-foid. Tlie responses of pulmonary and hepatic AHH following i.tL administrationr of 500 µg MCA are shown in ?*.g. lb and Fig. ic. Pulmanary AHH levels re- rssined meximelly induced .or at least 7 days. Hepatic levels deGeased by day two and were almost.at bsckground level by the third day.'A subsequent treatment on the seventh day resulted in levels of AHH activity similar to that obseroed for the Gxat treatment. I Ef fects on uorfous straUs of ntft Table IV demonstrates the hepatic -attd ~ pulmonary AHH levels follovQiag i.tL irstilletion of 188 jig MCA. The AHH ~ levels in pulmonary tissues of smins BAL8/c Mai, C3Hlf Vlat, CS7L/J and ~ C57BL16d were induced by MCA while scrains AKRlJ, SJL1J, DBA/2J and ! RF/J showed little or no increase. Hepatic responses were low for all stravis ~ except perhaps for the C57BL/6J and Ca7L/J, which did show a 100% ~ increase. Genetic regulation of pulrnwtary AHH induction. Induction of pulmonary ~( AHII by i.t. instilled HC.1 segregated as a single autosomal dominant gene :n ; rrosses between the 86 and 02 strains of mice (Table V). Backcross antl F, ' animals were cloasiQed as inducible or non-inducible if, afler i.c. troatmr.nt ' with 188 ug MCA. pulmonary A1H8 levels were 2.5 (:0.5) ur.its/R tissue (inducible) or 0.3 (c0.03) units/g tissue (non•inducible). Among 90 bzck- crosg animals tested, 47 were inducible (52%) and among 50 Fa animals tessed, 36 were inducible (7?.yal. These numben were not statistically 3ifier- I TaBL$ V CMtSTIC SEGREGATION OF PULYIGNARY ,iKH IiY CROSSFS INVOLVING THE CSTBL/BCum AND DBA/2Cam ST8AR4S OF 31ICB • Sts®ia ' Yamber teeated Numb.e induced % iadtuxion 36 30 s0 100 D2 30 0 0 86D7F, I00 100 100 88D2F,X 86 52 52 100 88DaF, X 02 92 4' 82 S8D2F3 • s0 36 7: ' atlee wale troated with 188 !lq a+1CA10.02 ml d.2:5 gelatin aoluunn i.t. and :1 h;ater. the pulmoaary AHH waa awyed. A mouae aae conaiderod inuucihle if. aiter \1C.1 Lrcu• moat, pulmonary AHH wae 2.5 (aO.S) uninlQ tiswe and considered nontnducililc if auintonary AHH was 0.3 (s0.08) urtihr6 t4stte. The sea of tlte prepay pkiyad no rose in sagre0,ttion pattarrt. . 324 ,

HT90120145 ( aa r,s-bma Eig.:. Lo vitro eWata of 7.846onsef(aeane on AHF[ activity of pulmonaey rni¢eoaomal prepaeattoas trom coatrol and MCa•treeced (m--®) 86 miee. and contsol (a~d) ae.d MC.&-trsatad (~^s) 02 miee. Spedita aacivitles of the miaoeomn6 pnpaeatioas +.as 5.0. 84.0. L.T and 38 unies .t88 ancEvity aaslj8 protain, cespactlre(y. ant (P < 0.01, using cbi square analysis) from the expw--d 50% and 75% r.tdoa ttsara would be expected if a single autmmal dominsnt gene waa reg(a- lating this indueibility. AMR lenels in rniicrosoniad prepanations.llse effect of 7,"em:oQavoae. s known compett&e inhibitor oi the 'snduced, or P4i8-medieted enzyme, on the in vitro metabolism of BP by microsomea derived from pulmonary tissues of that 36 and D2 mice is shown in F[g. 2. lt.mults.-4vith the untreatad (no 7,8- benzoDavoae) preperations gerteraily agreed with the neiults shown in Tables III arsd IV; that te, 02 pulmonary t'sasue eapeeased much Iess enzyme aativity . than 66 tissue, MCA tteatmenc of D2 mice resulted in a small increase in pulmonaty aFfK levels to about tlo,obsasved in contro186 mice, and J4CA- tteataaent of B5 mice resulted ia a much higlzer level of pulmonary AHFi acalvLty (about L4fobd in tlmae mierosomes). 7,8-8ensoAavoo.e inhibited AHH activity ftotn control and induced D2 tissue and control 86 tfsnte in a aimilar manner, while the AEIH activity from induced 30 tiasue was much more sensitive. Foreaaaaple, at concentraeions of 7,8-bmtoflavone equimolar to the BP concentration, the enzymes from the D2 tissue and Zrom control B6 tissue were inhibited by about 40%, while the enzymes doas induced 36 cia6ues were inhibited by greater than 80%. Speetro/ studies in nsicroso.nad preperations. Spectral determinations of P• 450 in lung microsomes 8+om both control and MCA inducecd mice wese attempted (Tabl4 V`Il. These studies were compltcatad by a large absorbance I , a 325

N T ®01 2Q 146 k ( T.1BLE VT SPEL"M.1L STtlD[Cq ON ?IJi.StONARY MICROSOMES DERIVF.D FROM 02 A1VD BB vllCE Straina TteatmAnt " Spntral peak (nn) nmotee cyrocfuatne P-4501 me protoin 02 Gel 450 ~I 0.1.98 02 %1CA 448-409.5 0.093 86 Gef 430 0.101 Be MCA 448 0.093 a Twopty mieQ nt aaalf ssraia w&ta treatod i.t. arltlt eitker 300 a8 MCA af 0.2% gelatiu- =lioe rolutiom ned. saselLled 48 h laeac i.uaa microeomm wem pevpmtvd au gives in a9ATERaAIeS AND dWTHODS& in the 420-424 am n3gioa of the CO difference spectra which equalled or ecceaded the abaorbance in the region of 450 nm. This "P-t20-like" materia, was present even thouga the CO difference spectra were recorded under con- ditons designed to blank out cytoctlrome b: and hemoglob(n, aand regardless of whether the microsomes were tresh or frozen. We do not lotow to what eat,ent this "P-i20-Ilks" material might intertere with the P450 determina- ti+ons. Even under tttese conditions. a shift in the CO ditference spectra from 450 rms to 448 nm in C37BL16af lung 2 days after adminisrraeion of MCA was observed. DB&V2J mice analyzed at the same time showed at most a 1«.m shift Pxom 450 nm to 449 nm after MCA induction. Thom resulLs are very similar to those ra.ently described for mivrosomal, preparations of rat lung (351. Follovving tteatment with MCA, the content of P450 in putmon- arp tissue of D2 aQica actually decreased (see Table Vi). Total P-d50 content of pulmomaty ttssae fracn B8 mice was not attered by MCA txeattnent. The natuie of " decreaso is ineaplicable at this time. GISCUSSION In tl»s report we describe some of the major pantcneters regulating the conatitutive ared MCA-induced Levels of pulmonary AfiN utRvity in inbred sttainm of mice. The intiateaoheai route was used so as to att~mpt to limit the eazymatic response to pulmonary tiasue. Utilizing the iabud strains C378Li6 and DBAJ2„ whoae hepatic ARE responses have been e:ttansively studt ed [i4-18] . we sww in this teport that: (a) MCa given in either trioctanoin or a 0.2% gelattn solution induces puttrtonary AHH, and t[tis induction is doae- dependeat (Tables I, II, III, IV7; (b) a dose of 188 Ag xCA given in 0.2°'0 gelatin induces pulmonary AHH. but has very limited effect on hepatic MK levels (?ablea II aM III); (c) pulmonary t1I1H can be indutxd in Da mice by Lt. administration oP MCA. but hepatic aElH levels are never induced (Tible III: F(g.1): (d) although pulmonary AU3 :s it=duced in D2 miee. the levels 326