[CANCER RESEARCH 28, 2357-2362, November 1968]

Tobacco Carcinogenesis1

B. L. Van Duuren

Laboratory of Organic Chemistry and Carcinogenesis, Institute of Environmental Medicine, New York University Medical Center,New York, New York 10016

Summary

A brief review is presented on tobacco Carcinogenesis withparticular emphasis on problems that are currently beingpursued in various laboratories as well as approaches that areworthy of further study. The presence of tumor-initiatingagents which are weakly carcinogenic or noncarcinogenic andof tumor-promoting agents in tobacco leaf and smoke isdiscussed.

Introduction

Although this symposium is to deal with natural productcarcinogens, it is imperative that a discussion of tobacco dealalso with its all-important thermal degradation and distillationproduct-tobacco smoke.

During the 1950's intensive studies in a number of labo

ratories led to the isolation, characterization, and quantitationof a number of aromatic hydrocarbons from cigarette smokecondensate (21), and for a short period of time it was felt thatthese compounds were the main tumorigenic agents in thecondensate. However, a closer examination of the biologicresults and further chemical and biologic experimentationsoon revealed that other factors must be involved (13).Phenolic fractions from cigarette tars showed tumor-promoting activity (18, 30). Phenol, isolated from cigarettetar (5), has long been known to possess tumor-promotingactivity in initiation-promotion experiments (4). Quantitativeanalysis of the known aromatic carcinogens and phenols andtheir known biologic response on mouse skin made it evidentthat these compounds alone do not account for the carcinogenic activity of cigarette tars (15).

Research efforts in various laboratories continued in a variety of directions in the search for carcinogens, tumor-promoting agents, etc. in tobacco smoke. The complexity ofthis problem is evident if one considers the large number ofcomponents of tobacco leaf. A recent review (21) lists over1,200 known organic compounds, and there remain many yetto be characterized. A substantial number of these components are volatilized during burning, including some high molecular weight components; and still other compounds, including the aromatic hydrocarbons, are formed during combustion of the leaf components.

'This work was supported by contract PH-43-64-938 from the National Cancer Institute and ES-00260 from the NIH.

This report will give a brief review of the known tumorigenicagents of tobacco leaf and smoke, some new areas of research,and some of the problems related to bioassay and chemicalanalysis of tobacco and smoke components.

Bioassay

For Carcinogenicity. The most commonly used bioassay hasbeen, and still is, mouse skin application. These tests, in orderto be meaningful, must be carried out for at least one year or,preferably, for the lifetime of the test group. It is frequentlyoverlooked that tobacco smoke condensate is a weak carcinogen, and this fact adds to the problems of bioassay. Highdoses, 25 mg or more per application applied 3 times weekly,on mouse skin are required and even then do not result intumors until after one year (29).

It is also desirable to include other routes of administration,and this has been done by subcutaneous injection in rats (7),resulting in sarcomas. In tests carried out in this laboratory bysubcutaneous injection in 20 female Sprague-Dawley rats, 3mg of cigarette tar in 0.1 ml dimethylsulfoxide, once weekly,for 64 weeks did not result in any local tumors. These resultsunderline the fact that tobacco tar is indeed weakly carcinogenic. The subcutaneous route as a bioassay procedure hasbeen criticized by various groups (8). However, in studiescarried out in this laboratory on the Carcinogenicity of anextensive series of epoxides, lactones, and related compoundsby skin application on mice, subcutaneous injection in miceand rats and feeding in rats led us to conclude thatsubcutaneous injection in rats is as reliable as any of the otherroutes of administration (25).

A comprehensive study on the tumorigenicity of tobaccotars is that of Rockey et al. (16). These workers applied tarsdirectly to the bronchus of dogs by using a trachéalfenestra-tion procedure. They observed at various stages squamousmetaplasia, carcinoma in situ, and invasive carcinoma. Theseresults were obtained after prolonged application.

Various short-term bioassays have been attempted, but thesehave usually resulted in unreliable results.

Although cell culture experiments for bioassay of wholecarcinogens would appear to be useful in short-term studies,they have been largely disappointing. During recent years,however, several reports have appeared on successful neoplas-tic transformation of cells grown in vitro with benzo(a)pyreneand other carcinogens (2, 10). This aspect has been exploredalso in our laboratories (20). In these experiments theoccurrence of transformation in embryonic hamster cells wasfound to be a rare event. Seven different experiments were

NOVEMBER 1968 2357

Research. on October 7, 2020. © 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from

B. L. Van Duuren

carried out with primary or secondary hamster embryocultures. Only one of these experiments resulted in a line ofcells that induced tumors when injected subcutaneously intonewborn hamsters or into cheek pouches of cortisonizedyoung adult hamsters. The carcinogen used was benzo(a)-pyrene at a concentration of 0.1 /ug/ml in the growth medium,and the cultures were carried for 34 generations beforeinoculated cells resulted in tumors (Table 1). In the other sixexperiments, the main effect observed was toxicity due tothe carcinogens benzo(a)pyrene and 7,12-dimethylbenz(a)-

anthracene (DMBA). Attempts to induce malignant transformation in mouse embryo cultures were unsuccessful. Recentwork (11) suggests that not all cells of a population aresusceptible to the induction of a transformation by benzo-

(a)pyrene. Furthermore, morphologic changes observed in cellculture during transformation, such as loss of contact inhibition, are not reliable markers for malignant transformation.Nevertheless, further studies in cell culture are clearly indicated, in order to develop rapid and, hopefully, reliableshort-term biologic indicators of carcinogenicity. An additional benefit of cell culture systems is the prospect ofexperimentation with cells of human origin.

Table 1

Generationsattimeoftest7722346262Route(0.5

to 1.0 X IO6cellsinoculated)S.C.,

newbornS.C.,adultS.C.,adults.o.,newbornCheek

pouch,adult0Cheekpouch, adult"Tumor

yield0005/75/5

cloned3/4unclonedTime

tofirsttumor(days)1402252

Tumorigenicity of benzo(a)pyrene-treated hamster embryo fibroblasts Results at 8 months, except where noted (20).

"Results at 2 months.

One major problem concerning bioassay of cigarette smokeis that of extrapolating from mouse skin and other laboratoryexperiments to human lung. In order to overcome thisproblem partially, it would be desirable to carry out bioassaysof fresh smoke in avian or mammalian lung which will meanlong-term testing and the accompanying expense.

In summary, then, while skin application in mice andsubcutaneous injection in mice and rats are still the mostcommonly used for carcinogenicity studies, in vitro cellculture systems and long-term test of fresh smoke in lungs

should be encouraged.For Tumor-Promoting Activity. Tumor-promoting activity

can be established by a protocol involving a single applicationof a subcarcinogenic dose of a carcinogen on mouse skin,usually DMBA or benzo(a)pyrene, 20 to 150 /;g, followed byrepeated application of the tumor-promoting agent. In thisway, tumor-promoting activity has been established for tobacco leaf extracts (3, 29), and for cigarette tars (9, 29).Another procedure, which may be more appropriate intobacco carcinogenesis studies, is the simultaneous and repeated application of a subcarcinogenic dose of benzo-(a)pyrene and the promoting agent to be tested. This test

procedure was used by Berenblum in his original studies on thecocarcinogenicity of croton oil (1).

In a recent study (27) we have also explored systemicinitiation followed by skin application of the promoting agent.The initiators used were DMBA, benzo(a)pyrene, and urethangiven as single doses by subcutaneous or intraperitonealinjection followed by repeated skin application of the promoting agent. DMBA was shown to be a potent systemic initiatorat doses as low as 5 ¿ig,while benzo(a)pyrene, even at 500 Mg,was ineffective as an initiator for skin tumors.

The permanent nature of such systemic initiation was shownby experiments in which skin tumors were induced whenpromoting treatment was delayed for 246 days. In theseexperiments there was no diminution in tumor yield, and thelatent period was shorter when compared to experiments inwhich the interval between initiation and promotion was 14days. Both whole tobacco extract and whole cigarette tar wereactive tumor-promoting agents on mouse skin when theinitiating agent was administered systemically. The latentperiod was longer and tumor yields lower than in corresponding experiments in which croton resin was used as a promoter.From these experiments it was concluded that skin applicationof the initiator is preferable to systemic initiation. Theseresults are summarized in Table 2 together with results inwhich the initiator was applied topically.

In the previous presentation, the effect of tumor-promotingagents in cell culture was briefly described. It is possible thatthese mixed culture experiments may yet be useful for rapidbioassay of tumor-promoting agents (19).

Carcinogens in Tobacco Leaf and Smoke Condensate

It is surprising that, in spite of efforts in several laboratories,no new carcinogens have been isolated from tobacco or smokecondensate since the appearance of the U.S. Surgeon General's

Report on Smoking and Health (15). In that report 7 aromatichydrocarbons are listed as known carcinogens. These compounds are: benzo(a)pyrene, dibenz(a,i)pyrene, dibenz-

(a,h)anthracene, benzo(c)phenanthrene, dibenz(aj)acridine,dibenz(a,h)acridine, and 7H-dibenzo(c,g)carbazole. A recentreview (31) also lists chrysene, benz(a)anthracene, benzo(e)-pyrene, benzo(b)fluoranthene, benzo(j)fluoranthene, and in-deno(l,2,3-cd)pyrene as known carcinogenic hydrocarbonsfrom cigarette smoke. The carcinogenicity of the first 3compounds included in the latter listing is questionable on thebasis of published carcinogenicity assays, although 2 of themmay be important tumor-initiating agents as will be describedbelow. However, it is clear that even with the increased lengthof this list, summarized in Table 3, some other carcinogensand/or tumor-initiating and -promoting agents must be presentin tobacco smoke.

Several conflicting reports have appeared recently on thepresence of nitrosamines in cigarette smoke condensate (21).It is, at present, not clear whether these compounds occur inthe fresh smoke or whether they are indeed formed duringcollection and storage of cigarette smoke condensate, and thisaspect merits further study.

The role of free radicals, which has been shown by electronspin resonance studies in cigarette tars (12), may be of

2358 CANCER RESEARCH VOL. 28

Research. on October 7, 2020. © 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from

Tobacco Carcinogenesis

Table 2

RouteSkinSubcutaneousIntraperitonealPrimarytreatment,and

doseDMBA,

150pgDMBA,150MgNoneNoneDMBA,

50¿IgUrethan,20mgDMBA,

500¿lgBenzo(a)pyrene,500 HgSecondary

treatment(25mg in 0.1 ml

solvent 3timesweekly)WCTWTEWCTWTEWTEWCTWCTWCTNo.

of micewithpapillomasTotal

No. miceat timeoffirst

tumor11/205/20001/173/184/122/18Daystofirst

tumor78133161195137202

Comparison of various routes of administration of initiating agent. 20 9 ICR/Ha Swiss mice per eroup. Results at 365days (27, 29).

WCT, whole cigarette tar; WTE, reconstituted tobacco extract; DMBA, 7,12-dimethylbenz(a)anthracene.

Table 3

Benzo(a)pyreneDibenz(a,h)anthraceneBenzo(b)fluorantheneBenzo(j)fluoranthcneDibenzo(a,i)pyrene

Indeno(1.2,3— ed) py reneDibenz(a,j)acridineDibenz(a,h)acridinc7H-Dibenzo(c,g)carbazole

Carcinogenic aromatics in cigarette tars.

importance and requires further study. The contribution madeby traces of polonium-210 and other radionuclides in tobaccocarcinogenesis remains questionable (14).

It is worthwhile to consider the possibility of other as yetunidentified carcinogens in cigarette tars. Certainly the searchfor nitrogen oxides and nitroso compounds in fresh condensâtesshould be continued. Over the past 6 years an intensivestudy on the carcinogenicity of epoxides, ß-and 7-lactones,hydroperoxides, peroxides, and halo-ethers have been carriedout in our laboratory (22, 23). A substantial number of thesecompounds have shown moderate to weak carcinogenicactivity and a representative series of these types of carcinogens are listed in Table 4. Many of these compounds are highlyreactive alkylating agents, and their detection in cigarettesmoke condensate will be most difficult because of their rapidrate of reaction with nucleophilic compounds, of which thereare many in cigarette smoke.

The a, /3-unsaturated -y-lactone, 0-angelicalactone, earlier

reported to be carcinogenic has been shown by extensive testsin our laboratory (24) to be inactive. Nevertheless, otherlactones isolated from tobacco leaf, the levantenolides I (a-and ß-epimers)and polyalcohols such as the duvanols, e.g.,duvanol.II, should be tested for carcinogenic and promotingactivity respectively. These compounds occur in small amountsin tobacco leaf, and this presents a problem in terms of theirbioassay for carcinogenicity since they are not availablesynthetically.

Tumor-promoting Agents in Tobacco Leaf and Smoke

It is important to isolate and characterize tumor-promotingagents in tobacco leaf and smoke in order to provide guidelines

Me Me

Table 4

(3-Propiolactone(J-ButyrolactoneGlycidaldehyde1,2,4,5-Diepoxybutane1,2,4,5-Diepoxypentane

1,2,5,6-Diepoxyhexane1,2,6,7-Diepoxyheptane1,2,7,8-DiepoxyoctaneVinylcyclohexene hydroperoxideBis(chloroemthyl)ether

Carcinogenic epoxides, lactones, peroxy compounds, and halo-ethers (22,

23).

for the analysis of cigarette tars for harmful agents. At themoment very little is known about the nature of thesematerials, except that both tobacco leaf extracts and thewhole condensate possess tumor-promoting activity in initiation-promotion experiments.

There are two possible approaches that can be used in thesearch for tumor-promoting agents in cigarette smoke and/ortobacco leaf. One is to test for tumor-promoting agents,known or suspected components of leaf or smoke of which thestructures are suggestive of possessing promoting activities.These structural features include lipophilic-hydrophilichydroxy-esters, phenols and polyphenols, etc. A substantialnumber of compounds in this class have been tested but havenot shown tumor-promoting activity (28). The only outstanding compound in this series is phenol, which has been shownby Boutwell and Bosch (4) to be tumor promoting, and this

NOVEMBER 1968 2359

Research. on October 7, 2020. © 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from

B. L. Van DuurenTable 5

Treatment

Primary (single dose) Secondary, dose per animal (tobacco leaf extracts)Cumulative No. of mice

with papillomaDays to first papilloma

(from initiation)

DMBA, 150 /ig in 0.1 ml acetone

None

DMBA, 150/Jgin 0.1 ml acetone

None

DMBA, 150 Mg¡n0.1 ml acetone

None

DMBA, 150 fJg in 0.1 ml acetone

None

Tobacco ether extract, 25 mg in 0.1 ml benzene.

Tobacco ether extract, 25 mg in 0.1 ml benzene.

Tobacco chloroform extract, 1 mg in 0.1 ml acetone.

Tobacco chloroform extract, 1 mg in 0.1 ml acetone.

Tobacco methyl alcohol extract,25 mg in 0.1 ml water.

Tobacco methyl alcohol extract,25 mg in 0.1 ml water.

Reconstituted tobacco extract, 25 mg in 0.1 mlacetone-water (1:1).

Reconstituted tobacco extract, 25 mg in 0.1 mlacetone-water (1:1).

146

168

216

133

Initiation-promotion: skin tumors in mice (29). 20 9 Swiss mice per group, promoter applied 3 times weekly; results indicated are at one year.

activity has been subsequently confirmed (29). The otherphenols tested by us have been inactive or have exhibitedmarginal activity, e.g., eugenol (29).

The second approach, which is no less laborious and timeconsuming, is continued fractionation and bioassay for tumor-promoting activity of tobacco leaf and smoke condensate. Weare currently employing this approach also. In this study, ofwhich a preliminary report has appeared (29), powderedflue-cured cigarette tobacco was extracted successively withether, chloroform, and methyl alcohol. These extracts weretested separately and they were also recombined in theproportions in which they were obtained as extracts and testedas a reconstituted tobacco extract. The results of these testsare summarized in Table 5. In order to avoid destruction ofbiologically active materials during fractionation, the procedures used in our isolation of the active principles of croton oil(26) were used in this work also, and that is solvent distribution, extensive countercurrent distribution, and thin-layerchromatography. Biologic activity, i.e., tumor-promotingactivity, has been established for some of these fractions, butthe results are not sufficiently advanced to warrant furtherdiscussion at this point.

Cigarette Tar Aromatic Hydrocarbons as Initiating Agents

It has been shown in earlier studies that weak carcinogenicagents such as epoxides and ß-lactonescan function as initiating agents by a single application of the agent on mouse skinfollowed by repeated application of a promoting agent, such asthe mixture of phorbol esters obtained from croton oil (23). Ithas also been reported earlier that benz(a)anthracene, a borderline carcinogen, is a relatively potent initiating agent (17).It was, therefore, of interest to test some of the otherborderline and noncarcinogenic hydrocarbons, most of whichare known to occur in cigarette tars for their initiating activity.The results, published in detail elsewhere (28, 29), are sum-

Table6

CompoundTumor-initiating

activityCarcinogenic

activity

Dibenz(a,c)anthracene, V6-Methylanthanthrene, VIChrysene, IVBenz(a)anthracene, IIIBenzo(e)pyrene, IX1-Methylpyrene, VIIBenz(m,n,o)fluoranthene, VIIIAnthanthrenc. XCoronene, XI

Tumor-initiating agents.

marized in Table 6. All of these compounds have beenreported in cigarette smoke condensate (21). Three borderlinecarcinogens and 1 noncarcinogen showed notable tumor-initiating activity, whereas 5 other noncarcinogens were notinitiating agents.

It has been pointed out by numerous workers that carcinogenic aromatic hydrocarbons alone do not account for thetumorigenicity of tobacco tars, and Roe has shown this ininitiation-promotion experiments (18). The present resultshelp to account for tumorigenic activity of cigarette tars, inaddition to that which may be ascribed to known carcinogensand tumor-initiating and -promoting agents.

These results raise the distinct possibility that, in tobaccocarcinogenesis, one is probably dealing with a host of carcinogens, initiating and promoting agents acting in concert.Furthermore, the above findings underline the complexity ofthe problem of chemical indicators for a less hazardouscigarette. It does suggest that a reduction of total aromatichydrocarbon content is desirable but that other agents, particularly alkylating agents, may also be present and act aswhole carcinogens and/or tumor-initiating agents.

2360 CANCER RESEARCH VOL. 28

Research. on October 7, 2020. © 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from

Tobacco Carcinogenesis

ni IV

VI

ACTIVE INITIATING AGENTS

CH,

INACTIVE AS INITIATING AGENTS

In conclusion, it should be pointed out that the problems inthe chemical examination of tobacco smoke center aroundmethodology, i.e., the chemical analysis of fresh cigarettesmoke as it emerges from the burning cigarette. There is noquestion that many chemical reactions occur during trapping,subsequent handling, and storage of cigarette smoke condensate, and in these processes compounds that are vitally important from the biologic point of view may be destroyed orundergo detoxifying chemical reactions.

ACKNOWLEDGMENTS

The author is indebted to his coworkers C. Katz, S. Melchionne, Dr.A. Segal, and Dr. A. Sivak, whose names appear in the references citedon work from this laboratory.

REFERENCES1. Berenblum, I. The Cocarcinogenic Action of Croton Resin. Cancer

Res. 1: 44-48, 1941.2. Berwald, Y., and Sachs, L. In Vitro Transformation of Normal

Cells to Tumor Cells by Carcinogenic Hydrocarbons. J. Nati.Cancer Inst, 35: 641-661, 1965.

3. Bock, F., Moore, G. E., and Crouch, S. K. Tumor-promotingActivity of Extracts of Unburned Tobacco. Science, 145:831-833,1964.

4. Boutwell, R. K., and Bosch, D. The Tumor-promoting Action ofPhenol and Related Compounds for Mouse Skin. Cancer Res., 19:413-424,1959.

5. Clemo, G. R. Some Aspects of the Chemistry of Cigarette Smoke.Tetrahedron,.?: 168-174, 1958.

6. Dickens, F., and Jones, H. E. H. Further Studies on theCarcinogenic Action of Certain Lactones and Related Substances inthe Rat and Mouse. Brit. J.Cancer 19: 392-403, 1965.

7. Druckrey, H., and Schildbach, A. Quantitative Untersuchungen zurBedeutung des Benzpyrens fürdie Carcinogene Wirkung vonTabakrauch. Z. Krebsforsch., 05: 465-470, 1963.

8. Food Protection Committee. Problems in the Evaluation ofCarcinogenic Hazard from Use of Food Additives, p. 27. Washington, D.C.: Nati. Acad. Sci.-Natl. Res. Council, Pubi. 749, 1960.

9. Gellhorn, A. The Cocarcinogenic Activity of Cigarette TobaccoTar. Cancer Res., 18: 510-517, 1958.

10. Heidelberger, C., and lype, P. T. Malignant Transformation in vitroby Carcinogenic Hydrocarbons. Science, 755: 214-217, 1967.

11. Huberman, E., and Sachs, L. Cell Susceptibility to Transformationand Cytotoxicity by the Carcinogenic Hydrocarbon Benzo(a)py-rene. Proc. Nati. Acad. Sei. U. S., 56: 1123-1129, 1966.

12. Lyons, M. J. Assay of Possible Carcinogenic Hydrocarbons fromCigarette Smoke. Nature, 777: 630-631, 1956.

13. Orris, L., Van Duuren, B. L., Kosak, A. I., Nelson, N., and Schmitt,F. L. The Carcinogenicity for Mouse Skin and the AromaticHydrocarbon Content of Cigarette-smoke Condensâtes. J. Nati.Cancer Inst., 21: 557-561, 1958.

14. Radford, E. P., and Hunt, V. R. Polonium-210: A VolatileRadioelement in Cigarettes. Science, 143: 247-249, 1964.

15. Report of the Advisory Committee to the Surgeon General of thePublic Health Service, Smoking and Health, p. 58, U.S. Departmentof Health, Education and Welfare, Publication No. 1103, Washington, D.C.: U.S. Government Printing Office, 1964.

16. Rockey, E. E., Speer, F. D., Ahn, K. J., Thompson, S.A., andHirose, T. The Effect of Cigarette Smoke Condensate on theBronchial Mucosa of Dogs. Cancer, 15: 1100-1116, 1962.

17. Roe, F. J. C., and Salaman, M. H. Further Studies on IncompleteCarcinogenesis: Triethylene melamine (T.E.M.), 1,2-benzanthra-cene and (J-propiolactone as Initiators of Skin Tumor Formation inthe Mouse. Brit. J. Cancer, 9: 177-203, 1955.

18. Roe, F. J. C, Salaman, M. H., Cohen, J., and Burgan, J. G.Incomplete Carcinogens in Cigarette Smoke Condensate. TumorPromotion by a Phenolic Fraction. Brit. J. Cancer, 13: 623-633,1959.

19. Sivak, A., and Van Duuren, B. L. Phenotypic Expression ofTransformation: Induction in Cell Culture by a Phorbol Ester.Science, 157: 1443-1444, 1967.

20. Sivak, A., and Van Duuren, B. L. Studies with Carcinogens andTumor-promoting Agents in Cell Culture. Exptl. Cell Res., 49:572-583, 1968.

21. Stedman, R. L. The Chemical Composition of Tobacco andTobacco Smoke. Chem. Rev., 68: 153-207, 1968.

22. Van Duuren, B. L., Goldschmidt, B. M., Katz, C, Langseth, L.,Mercado, C., and Sivak, A. a-Halo Ethers: A New Type ofAlkylating Carcinogen. Am. Med. Assoc. Arch. Environ. Health, 16:472-476, 1968.

NOVEMBER 1968 2361

Research. on October 7, 2020. © 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from

B. L. Van Duuren

23. Van Duuren, B. L., Langseth, L., Goldschmidt, B. M., and Orris, L.Carcinogenicity of Epoxides, Lactones and Peroxy Compounds,VI. Structure and Carcinogenic Activity. J. Nati. Cancer Inst, 39:1217-1228, 1967, and earlier papers cited therein.

24. Van Duuren, B. L., Langseth, L, Orris, L., Baden, M., andKuschner, M. Carcinogenicity of Epoxides, Lactones, and PeroxyCompounds, V. Subcutaneous Injection in Rats. J. Nati. CancerInst., 39: 1213-1216,1967.

25. Van Duuren, B. L, Langseth, L, Orris, L., Teebor, G., Nelson, N.,and Kuschner, M. Carcinogenicity of Epoxides, Lactones andPeroxy Compounds, IV. Tumor Response in Epithelial and Connective Tissue in Mice and Rats. J. Nati. Cancer Inst., 37:825-838, 1966.

26. Van Duuren, B. L., and Orris, L. The Tumor-enhancing Principlesof Croton tiglium L. Cancer Res., 25: 1871-1875, 1965.

27. Van Duuren, B. L., Sivak, A., and Langseth, L. The Tumor-promoting Activity of Tobacco Leaf Extract and Whole CigaretteTar. Brit. J. Cancer, 21: 460-463, 1967.

28. Van Duuren, B. L., Sivak, A., Langseth, L Goldschmidt, B. M., andSegal, A. Initiators and Promoters in Tobacco Carcinogenesis. J.Nati. Cancer Inst., Monograph No. 28, 173-180, 1968.

29. Van Duuren, B. L, Sivak, A., Segal, A., Orris, L., and Langseth, L.The Tumor-promoting Agents in Tobacco Leaf and TobaccoSmoke Condensate. J. Nati. Cancer Inst., 37: 519-526, 1966.

30. Wynder, E. L., and Hoffman, D. A Study of Tobacco CarcinogensNo. 8. Role of Acidic Fractions as Promoters. Cancer, 14:1306-1315,1961.

31. Wynder, E. L., and Hoffman, D. Tobacco and Tobacco Smoke.Studies in Experimental Carcinogenesis, pp. 338-339. New York:Academic Press, 1967.

2362 CANCER RESEARCH VOL. 28

Research. on October 7, 2020. © 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from

1968;28:2357-2362. Cancer Res   B. L. Van Duuren  Tobacco Carcinogenesis

  Updated version

  http://cancerres.aacrjournals.org/content/28/11/2357

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/28/11/2357To request permission to re-use all or part of this article, use this link

Research. on October 7, 2020. © 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from