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-~~ ~. iAl N 00925013 Effect of Different Doss of Tobacco Smoke on Ciilary Ac4ivity /n Cat. Variations In Amount of TebaGbp Smo::e, interval Between Ciga- rettes, CoInteat of "Tar," Nicotine, and Phenol TonE DeLBa3ta, 11l.Ti., lratitute of Hygiene, garolinsx•¢ lnstitutet, Stockholm, Sweden i i INTEREST in the multitudinous effects of to- bacco smoke on the biological organism increases. This attention chiefly concerns the reactions in the airwa y-s of mammals-particularly, mucosal changes (1, Q), changes in pulmonary clearance (3), and ciliotoaic and mucotosic effects. The experiments described in this paper summarize our results from earlier work and unpublished studies concerning the ciliotosic effects of cigarette smoke as outlined below. The latter will soon be published in greater detail (.¢). ETperiments on the ejf'cct of cig,trette em.oke on c:7iarT activity Various factora A. Components in smoke: B. Interva.l between cigarettes: 1. Total particulate phase 0, 2.5, 5, 10, and 15 minutes Total gas phase Filterless 2. Particle phase ("tnr" 0-19 Cellulose acetate filter mg/cigarette) C. Amount of smoke 05,1, 5. 10, 8. Nicotine 0.7 and 1.8 and 20 ml mg/eib rette 11lterless 4. Pheno12.7 and 18.8 mg/100 Cambridge filter cigarettes METHODS Two techniques were mainly used in the study of exposure to smoke. The first was fully mechanized, the c1t being exposed ereq third breath to 1 ml of fresh tobacco smmke taken from a 35 ml pufl. Aftcr25 puffs, the 79

i t i ! so n.u.naMv HK1C92501 4 animal Neas allowed to rest for 5 minutes before the exposure was relmated (5). Because it proved impossible to avoid a certain amount of l'eakage.rith this technique (so that I tn] fresh smoke corresponded to only about half this amount, i.e., 0.5 nil), subsequent esperiments were performed manually. One ml n•as taken from 35 ml fresh smoke at I puff a minute and the cigarette was smoked until a°_S mm stub remained. There was no 1eakatge. These two series are thus not entirely comparable, but compari- sous can be ma de bet wccn the esperiment s in each series. 17ie methods for ob=erring ciliary activitc have been described in detail el-eo•liere. Ciliary activity in the trachea was observed after tracheotomy with an Oiropak microscope (6). RESULTS Components in Smoke 1. Total particulate phase, total yas phase: These experiments (7) con- cern the effect of cariations in the gaseous and/or the particulate pbases (table 1), by use of the method described eLsewhere (b). The results showed that an average of 91 puffs of unfil~tered smoke was required to elicit ciliostasis. "Mhen the gaseous phase was reduced to only a small fraction of its original value and the particulate phase was left largely intact, the ciliostatic effect diminished from 91->t i 0 puffs. Reduoo- tion of the particulate phase and maintenance of the gaseous phase gave a figure of 194 puffs.lS']te.n both components were ~educed at the same time, the ciliostatic effect of the smoke was gtr.atly diminished to 512 puffs. However, the ciliostatic effect of the smoke could also be substantially diminished by complete elimination of only the particulate phase. More than 600 puffs were then required to elicit ciliostasis. The correlation ecefficients for the relationship between the particulate phase ("tar" in- cluding nicotine and phenol) and ciliotosicity were ltigh throughout (0.79-0.G9). High correl9timis were aleo found for certain components in the gaseous rhase, for instance for toluene (0.56). In view of the relevance of the particulate phase in the present context, subsequent experiments were con rined to varying the measured componerits of this phase, that is, the "tar;" nicotine, or phenol, and keepina the other two components more orless constant. 2. Fartku7ate phase: Experiments (8) were conducted by the same techniqnes as described in Section 1. Fi. e series of 5 amimals each were studied, the"tar" content of the smoke var; inr from 6.2-19.2 mg/cigarette. The number of puffs before ciliostasis, recorded as in previous experiments, is gieen in text-figure 1. These eaperiments again showed n strong correla- tion between tar content and ciliotocicits. Control experiments with expo. xAT1oJ:at. CANCER La71TUTa atp.ocnaPH iCO. 28

e ~ ~ Teats 1.-(Iliioatatte effect of tobacco smoke of varying eonopoaition (identioal tobaoeo--8lters vsried)' ~ r n 0 ~ ea Numbex pv Cigarette otfor(ta -11loetrsfa (menn Of 5 IInltnala) Tar - Nieotine 247.3 Phenol 195.5 Toluene 111 Acetonitrile 82 Methyl etbyl Kotone 70.0- Acetone 56.5- Isopyrene 34 Acetaldahydo 21 1 (i1 20.3 1.3.3 B.2 26- 31 37 57 5U - 94 2 170- 1?~:4 1.27 6.4 1 1- 1 R 9 27 3 194 1'l.8 0.87 1:0 20 20 28 41) 52 8!t 4 512 14.0 0.99 2.6 1 1 1 8 8 31 5 0U0 0.8 0.02 0.4 U 10 25 iU 40 89 Correlation aoe/Hcient -0. 74 -0. 73 -0. 69 -0. s0 -0. 30 -0. 27 -0. 16 -0. 28 'Tv ana woottne etpceaed ag tag/¢t:sette, otliae eompaands n tenttn otyeklgssette. Numadont adpes nnda enmpounda indtmte bonlnq potnt tn eentlgndo O ogrem I bw O ii

nAiaia~c.v 82 vurvs 0 "Di U . . " 0 aons s +e n „ TAR TErr-riovas 1. Egect of ditter ent tar content of cigarette emol:e on eliiary aMivityM N 14CQ925A16 CWoTOStc t l njl from a 35 mW ~o' was made c~ . d.G7 and 1.78 mSl t i,tuctically constant (l ~elt-frgure 2. Ciliostae , ontent after 75 pufTs. :. bi8h nicotine con : i'nifieant ~ Phenoti: Finall. nicotine and r" c' 11 Rere studied by effect. The concent r: n,8/100 cigarettes) (ca. 7 mg/100 cib^ar induced by a mc''n sure to air alone Arere conducted in this, as well as in a11 the other studies, Wond+ mdTCat'ng oenol in the concc' without displaying any effect on the eiliary actiaity. ttie tintent of nicotine onl rar t y y a ext step was F. Nacotine: The n These esperitnents were conducted by the tnanual technique described; the cigarette was smoked down to a 28 mm stub at a rate of 1 puff a minute and Puils 050-1 0 0 e e 0 0 1W 0 M e • 0 e 6 i ~ b0 e 8 0 I 0 ~ "?ar" 13.2 13.6 Nicotine 0.67 1.79 TESrrtotTee 2.-8tfect of ntcotlne on ciliary activtty. o=tndtrsdual vatnce; e=mean valne. 1 40 NATIONAL OAI: CEn IN671TOTE ~LON00>tA~'~ ~ro' TanIs 2.- phen0l en tmUJ1o0 Tu stuas tll" and tlie lengib from 0 mv11lc cigarettes vccr. Eeri ~~{e A cacdi interval ' Text•86^'rc ducted bs the and the Sltet the smoko a, the iuterval+ in other Wp1 cant diffem, . alvcar Ten" 6lterlesa Tmv+nn A 2.u .+,

, es, I CII.IOTOaIC EFFECTS OF TOaSVCe 8]IO7iE IN THE CAT N00925017 sa I nil from'n 35 nil sniohe puU was manually injected into the trachea. The study..as niade with 2 seriv:; of 0 aniulals eache l71e nicotine contents were 0.67 and 1.79 mg/cigaret(e, respectit•eli•, the "tar" content being kept practically constant (13 .2 and 13.6 nir'ci,arette). The results are shown in t.eat-figtu•e 2. Ciliostasis was elicited 1,y thc cigarettes with a low nicotine content after 73 puffs, while 67 puffi weli required with the cigarettes with a high nicotine content. The difference betmeell these values is not significant. 4. Phenoi': Finally, the phe,iol content was varied while the contents of nicotine and "tar" were kept largely constant. Ttr:o series of 5 an iuaa.ls each were studied by the mainual technique used for thc tm•estigation of nicotine effect. The concentration of phenol was low and rery high (2.7 and 18.8 mg/100 cigarettes) compared to the concentratSon in not•mal cia rettes (ca. 7 mb/100 cigarettes). The results are given in table 2. Ciliostasis was induced by a mean of 135 pufl; in the first series and by 72 puffs in the second, indicating a clear correlation bet w'een ciliostasis and the amount of plienol in the conceartrations st tidied. TABLE 2.-L@'ect of different pbenol oontent on ciliarl• activity Phenol concentration Puffs to "Tar" and nicotine (mg/100 cigarettes) ciUostasis (mg/cigarette) 2 7 135 19 18. 8 72 1.2 Interval Between Cigarettes To study the relationship between the ciliostatic effect of cigarette smoke and the length of the interval between cigarettes, this interval was raried from 0 minutes to 2.5, 5, 10, and 15 minutes. In addition, two types of cigarettes were used, one without a filter and one with a filter of cellulose acetate.ll series of 5 animals was studied for each type of cigarette and each interval (0). The cigalrt tes were commercial brands. Teat-figttre 3 shows the results of these experiments, which were con- ducted by the fully mechanical technique. For both the filterless cigarettes and the filter.tipped, there is a clear correlation between the toxicity of the smoke and the length of the interval between cigarettes: the longer the interval, the greater the number of puU's before the onset of ciliosta:.is- in other words, a clear dose-response relationship. There is also a signifi- cant difference between the two commercial ci,..arettes, the one tt ith a filter always requiring a greater number of puffs to elicit ciliostasis than the filterless. Townnn A i.rs.c nAtntl'l'L CIG.\nF:TrE 28 2qa-o1D- r..-_s

NK@o925o1s 84 DALH9111r NUMBER OF PUFFS FOR CILIOSTASIS i 2:0 700 tS0 1* a.] 0 ~---•--- S 10 1S 2A MINUTES BETWEEN CIGARETTES TEITFICIISE $.--Ct1tOSt&tIC effect Of smoke from filter-Upped (BQearea) and EUteriess (trtangtca) ciga- rettes, with rarying time intervals between cigarettes. The means from groaps of Sve anlmals are plotted. i I 1 1 -Amount of Smoke Sevpral authors have discussed the effect of cigarette smoke on the air nass in terms of its ciliotoxic influence as well as its effect on the transport of mucus and pulmonary clearance (1t9-IS). 17Ie experimental methods used for exposure to cigarette smoke have varied considerably, ranging from the use of mussels and similar animals through in z;tro studies on frogs and mammals to :n vi:;o investigations of mamnials. Various tech- niques have also been used for producing the smoke. The esperiments described below concern the effect of different amounts of smoke (4). The experiments were made in two series, one with unfiltered smoke and the other with smoke filtered through a Cambridge filter. The amounts of smoke administcred once a minute tmtil the time that a 28 mm stub re- mained were 0.5, 1.0, 5,10, and 20 ml. Each group comprised 5-10 animals. Tltelrsultsareshown inteet-figure4. A clear dose-response relationship was found for both types of cigarette smoke. Moreover, nt all dose levels studied, the unfiltered smoke proved more toxic than the same amount of filtered smoke, though it is interesting that. this difference appears to be less for t1le larger amounts of smoke ad- ministered. When 20 inl puffs -scere used, the difference benceen the number of puffs o; filtered and unfiltered smoke required to elicit ciliostasis was about 20, whereas for I Inl puffs t1le difference was no less than 150. t tt el ca ca nt at, rcl pa thi foi in pal ant 7 (ox of `' pla; Co1U edn the NATIOI:AL C.SNCIiR INSTITUTL XfO\OOItAPII ItO. 28

~ HKO0925019 , CII.IOT031C EFFECTS OF TOBACCO BMOHE L\ THE CAT 85 o' 0.5 ; S ~0 7Cmvo." TesrfzccuE 4.-Cillotoxielty of whole smoke (Laccr curre) and Cambridge filtered smoL-e (upecr curve), at varyirrs dose levela CO1 CLUSI O\ S i ~port I tods ling ; on ech- lents Iand s of re- tala Vtte wed ang ad- iber was . 28 The experiments presented concern the effect on ciliary actirity in the trachea of the cat of short-term exposure to tobacco smoke. It should be emphasized that from such experiments one naturally cannot draw any conclusions about the genesis of variotis respiratory diseases in man in connection with cigarette sntolking. \'o detailed investigations have been made of the correlation between ciliotoxic and mucotoxic agents of the above type attd respitatoty diseases, though the probability of such a relationship exists. To judge from results of the experiments, both the particulate and the e scous phases possess ciliotoxic properties. This ro ises the question of which indiridual component or components are responsible for this effect. Several diilerent substances are probably biologically active in this respect and tLet~e are no doubt s3•netgistic effects as well. The particulate phase is ~uerally regarded as consisting of "tar," nicotine, and phenol, though some phenol is also foutul in the gascotts phRse. The present investigations started in an attempt to establish the cilio- t.oaicity of the various cbmponents of the particulate phase. The content of utur" (which is itself it mixture of more than 500 compounas) thus dis• playod a strong correlation to the ciliary activity,.rhereas nicotine, in the concentrations studied, had no detectnble effect. The phenol esperiment admittedly showed a elear correlation, but it must be borne in mind that the higher concentration (1S.S mg;100 ci;arettes) is much motr thau one TOW.utU .1 LESS lLttiMFFI. CtQ.IIiE't`r4: i

NK609250211 ( 86 D.aLtiAISN tinds in a normal cigarette. Smaller rariatioils in the content of pllenol have produced contradictoiT results. Individual compounds in the gaseous phase were not investigated, but lienslva•and Batti;tl (13) reportthat, with their technique,both hydrogen cyanitle and acrolein are essential in the ciliotosic effect of the gaseous phase. ~ Concerning the length of' he interval between cibarette-smoke exposures, ~~ a clear dose-rc: ponm irlat ionship.ras found to ciliotosicits. Finally, it is quite clear that the smaller the dose of smoke, the stnaller the actue toxic etiect. This applies to the unfiltered smoke as well as to rl t;:e smnlce p;isscd through a Cambridge filter. The difference between these two types of smoke is Dot constant, however; it diminished with increasing (1 doses. This trlationship should be borrte in mind when deciding on the technique of exposure; other-wise it is easy to orerest imate or underestimate the toxicity bf the total smoke or of its individual components. (t SUGGESTIOI'S To study the ciliotoxic and muemtosic effect of cigarette smoke in a meaningful manner, it seems necessary to design more experiments with conditions that agree as cloself as possible with the conditions obtaining when a person smokes. It has been pointed out that the tecimique used for exposure to smoke is important in the interpretation of the results obtained. The technique and the biological system involved in the obserration of the ciliary activity appear equally important. These problems thus warrant re-examination, without Rbich it will be difficult to make further proa ess in the study of cil iotosic effeets of cigaret te smoke. REFERENCES (7) Gtnce:c. G. dt.. and CeaoLrx, D.: The depressant effeet of cigarette smoke In the In vitro auttbacterial ni4lrit,c of alreolar macroPhages. lesr Eng J Med 276: 4221427, 19Ri. (2) Lsocarsaasecsa, C., LECCHTECBE6GLa, R.. Roca, F., Tsuaette, K., and Tssase, T.: C,.-tological and cctocbemical alterations in the respiratory tract of mice after esposure to cigarette smoke, SnBuenza .irus, and both. Cancer Res 28: LSSvst9, 19C3. (3) Rns~oEe. R. : Pulmonary defense meehnnisms to airborne bacteria. Acta Pbys. iol :•cand Snppl300: 1-59,1963. (4) DAI.HA:1.v, T., and RrLaNDCa. R: Cillatoafclt,r of cigarette Rmoke and its vola- tile components. Submitted for publication In Amer Rer Resp Dia (5) DsLasu<, T., cor Esssr, fi.. SAaiA~D, A., and R1'LA.rara, R.: A machine for Introducing a regulated amount of tobacco smoke into an animal. Air R'ater Poitut 7:611-i15, 1t188. ~ . T'ATIOIi AL CANCER 1N81TTOTE 1tONO0RAP17 NO. 28 .roRr

L'.-.._ ., ._.,.. .. .. .. .... .... . . . . . . . .. - '.?idA.'--: .:_ . . '.X;,_.u. .. . CIL101'OXI1i EFFECfB OY TOB.ICCO SMOKE IN Tllln CAT oI yltCnol ibmated, but h hydrogen I the gaseous ~ a eaposures, the BnAtllle1' 4wo astA tween t}leBe 1 iIICl'eAstll.b iing on the derestimate smoke in a iments with as obtaining HKU0925021 81 (6) Dat.u,.M.v, T.: The deternoinatiou In v:vo of the rate of cilinry beat in the tra- e6ea. Aeta Pbyslol Seand 49 : 243-280, 1960. (7) Dwr.neus, T., and Rruxqrs, R.: Cigarette svnoke a>q.d c)itastasis. Effect of vsry- ing composition of smol:e. Arch Environ FlealtU (Chicago) 19:47-a0, 196G. (8) -: Tar content and cAlatosicity of cigarette smoke. Acta Pharnwcol Tosicol 25: S6`J472, 1967. (9) : Cillastatic action of cigarette smoke: Varying eaposure Utues. Arch Otolarsag (Chicabro) $1: 379-.1Z,196J. (10) CAasoa, S.. Got.arneuea, R., and Caarsrrss, Ii.: Restwases of ciliated epitbelium to Irritants. Jlueus transport in the respiratory tract. Amer Rev Reap Die 03: 8(-92, 1966. (11) FAr.x, FI. L., Tat:u>ra. II. 1I., and Sortx, P.: Effect of cig:Zrette smoke and Its constituents on ciliated mucus•secreting epitbelhnm. J idt Cancer Inst 23: 9911`1012, 1959. (12) G^^* =wt, 13., BAUat, R., and Ficsox, B.: Effets inldblteurs de la fum8e de tabac sur I'acUvitd ellialre de I'EpitbSlium respiratoire, et nature des composants responsables. Buil Aced Nat Med (Paris) 145: 416-123, 1961. (18) Sexsi.ca, C. J.. and BArrrsrA, S. P.: Components of cigarette smoke with dliary deDre.¢sant activity. NeR Eng 3 11ed 200: 1161-1166',1963. ,riv smoko 1 le l..afiique iary activity sxaminntion, the study of t smoke in the ngJ>Sfed 278: , and TAtaASe. r tract of mice 7antrr Res 23: dn. Acta Phys- :e and 1ta voln• )1s. 3.: A machine ati animal. Air ; iP.Ap13 NO. 28 'roWann A L'ESS IlAnaiFGt. CIOJREITE (