Anthracene Related to Carcinogenicity1

[CANCER RESEARCH 29,506â€”509,March1969]

Molecular Site of Substituents of Benz(a)anthracene Related to Carcinogenicity1

JohnPatakiandCharlesHuggins The Ben May Laboratory for Cancer Research, The University ofChicago, Chicago, Illinois 60637

SUMMARY The tumor-producing effect of dibenz(a,h)anthracene led Cook (5) to undertake experiments showing that 9-isopropyl Benz(a)anthracene did not induce tumors in rats, but its BA is a (weak) carcinogenand Fieseret aL (9) synthesizedthe derivatives were highly and equally carcinogenic when they stronger cancer-producing substances 7-methyl-BA and possessed two or three methyl groups in any combination at 7,8-DMBA. Considerably later it was found that 7,12-DMBA the following special sites: positions 6, 7, 8, or 12. Derivatives (1), 7,8,12-TMBA (1), and 6,7,8-TMBA (21) are very powerful of benz(a)anthracene with pairs of methyl groups at sites 1 carcinogens. It was evident that substitution of BA at positions and 12 or 3 and 9 were not carcinogenic. 7,12-Dimethylbenz 7 and 8 to form cholanthrene or 7,8-DMBA was more effective (a)anthracene and 7,8,12-trimethylbenz(a)anthracene are in promoting carcinogenic potency than substitution of BA at equivalent in their ability to elicit neoplasms in rats and far positions 8 and 9 was, irrespective of whether a new ring was exceed 3-methylcholanthrene and benzo(a)pyrene in this formed or the substituents remained as alkyl groups. regard. MATERIALSANDMEThODS INTRODUCTION Chemicals. Of 26 compounds which were tested, four were In this paper it will be demonstrated that the addition of new. These were synthesized in our laboratories. The new one, two, or three small alkyl substituents at certain positions compounds (Table 2) are: 7-methoxy-12-methyl-BA (XVIII); in benz(a)anthracene is highly advantageous in transforming 7-ethoxy-12-methyl-BA (XIX); 6,7,12-TMBA (XXIV), and this molecule to an active carcinogen, whereas the introduc 6,8,12-TMBA (XXV). Satisfactory elemental analyses were ob tion of pairs of methyl groups into other sites was ineffective tamed for each compound. Their nuclear magnetic resonance in this regard. (NMR) spectra are in complete agreement with the assigned Kennaway and Cook made the basic discoveries in the sys structures. In addition, thin-layer chromatography (12) with tematic correlation of cancer-producing activity with chemical migration as a single spot was the minimum requirement for constitution. Benz(a)anthracene (7), benz(a)acridine (3) and purity of the compounds. benz(c)acridine (19) do not elicit cancer, but the ring system Lipid emulsions for i.v. injection were prepared by the of each is potentially carcinogenic, and cancer-producing prop method of Schurr (22). The hydrocarbon-concentration (w/w) erties are developed by substitution in suitable positions. The of our emulsions (Table 1) was: BA, BP, 3-MC, 0.25%; basic structures are activated to carcinogenic status by the 7,12-DMBA, 7,8,12-TMBA, 0.5%. addition of a supplementary ring or by the introduction of one Adrenal Apoplexy. Female rats of Sprague-Dawley strain or more small alkyl groups into the molecule. were injected i.v. with massive doses of each emulsion at 50 The strong 3-banded fluorescence spectrum of the carci days of age (Day 0); necropsy was performed on Day 3, and nogenic tars derived from isoprene and acetylene led to the the hemoglobin content of the adrenals was determined (16). identification of dibenz(a,h)anthracene (7) as the first pure Induction of Mammary Cancer. Normal female rats of aromatic carcinogen. It was found soon thereafter that 3- Sprague-Dawley strain weighing 140â€”160 gm were given a methylcholanthrene (24) and benzo(a)pyrene (6) greatly cx commercial ration (Rockland Mouse/Rat Diet, Tekiad, Inc., ceeded DBA2 in carcinogenic potency in the sense that these Monmouth, Illinois) and water ad libitum and kept in an air compounds gave rise to tumors more rapidly than DBA did. conditioned room at 25 Â±1Â°C.Vaginal smears were examined daily. The rats remained healthy in the sense that (a) estrus cycles were regular and (b) there was no pulmonary disease. There were 8â€”10 animals in each group. â€˜This investigation was supported by grants from American Cancer The animals were injected i.v. on 3 occasions with the lipid Society, Jane Coffin Childs Memorial Fund for Medical Research, and Walter Schwimmer. emulsion containing 2 mg of the hydrocarbon (approx. 13 2Abbreviations used: BA, benz(a)anthracene ; BP, benzo(a)pyrene; mg/kg) at 50 (Day 0), 53, and 56 days of age. The time of DBA, dibenz(a,h)anthracene;DMBA, dimethylbenz(a)anthracene; 3.MC, appearance of palpable mammary cancers was determined. The 3-methylcholanthrene ; TMBA, trimethylbenz(a)anthracene. animals were sacrificed on Day 98; active centers are mam Received September 3, 1968; accepted October 30, 1968. mary cancers found at necropsy.

506 CANCER RESEARCH VOL.29

Table 1Appearance

ofpalpablecancers, days. ActiveNo. withMeancentersHydrocarbonratscancerRangeofRats S.D.(mean)Benz(a)anthracene280Benzo(a)pyrene30957â€”95 Â±

1813-MC20744â€”98 71 Â± 68Â±151.27,12-DMBA747428â€”58 35Â±76.97,8,12-TMBA202029â€”62 39 Â±107.1

Mammary cancer evoked by pulse-doses of hydrocarbons. Female Sprague-Dawley rats, approx. 150 gm, on 3 occasions were given an intravenous injection of hydrocarbon respec tively at 50, 53, and 56 days of age respectively. Each dose of hydrocarbon was 2 mg (13 mg/kg). Active centers are cancers found at necropsy. See Footnote 2 for abbreviations.

Induction of Sarcoma. Solutions of hydrocarbons (Table 2) of rats injected with 7-ethyl-BA (IV), 7-methoxy-BA (V), or in sesame oil, 0.5% w/v, were freshly prepared. The recipients 12-ethyl-BA (VIII). 7-Formyl-BA (VI) did not evoke tumors. were males, 25 days old (Day 0), Long-Evans strain; each was The following compounds (Table 2) with two alkyl groups injected i.m. in a hind leg with 0.5 ml of oil containing 2.5 mg were strong carcinogens, and sarcoma became evident in all of a compound. There were 8 animals in each group. The groups at about the same time: 6,7-DMBA (XI), 6,8-DMBA experiment was terminated on Day 270 when necropsy was (XII), 6,12-DMBA (XIII), 7,8-DMBA (XIV), 7,12-DMBA performed. (XV), 8,1 2-DMBA (XVI). 7-Methoxy-12-methyl-BA (XVIII), By our definition, a strong carcinogen induced sarcoma with 7-ethoxy-12-methyl-BA (XIX), and 7-formyl-1 2-methyl-BA in 6 months in every animal in a group of 8 rats; a weak (XXII)werealsostrongcarcinogens.Tworelatedcompounds, carcinogen elicited sarcoma more slowly and only in a propor 7,12-dimethoxy-BA (XX) and 7-hydroxymethyl-12-methyl-BA tion of the members of the group; a compound is inactive (XXI),wereweakcarcinogens. when no member of the group developed sarcoma in 9 Two derivatives of BA (Table 2) possessing pairs of methyl months. groups failed to cause sarcoma. The inactive compounds were 1,12-DMBA (IX) and 3,9-DMBA (X). 9,10-DMBA (XVII) was RESULTS a feeble carcinogen. Each of four derivatives of BA possessing three methyl Adrenal Effects. In the adult rat a single large dose of groups (Table 2) was a very strong carcinogen and equivalent 7,12-DMBA (16) and closely related compounds causes selec in the speed with which sarcoma became evident: 6,7,8-TMBA tive necrosis of the adrenal cortex with adrenal apoplexy. (XXIII), 6,7,12-TMBA (XXIV), 6,8,12-TMBA (XXV), and We found that an i.v. injection of a lipid emulsion of 7,8,12-TMBA (XXVI). 7,8,12-TMBA causes the same sort of adrenocorticolysis but less efficiently than 7,12-DMBA does. In female Sprague Dawley rats, 50 days old, the minimum quantity of hydrocar DISCUSSION bon to cause adrenal apoplexy in every member of a group of 4 animals was: 7,12-DMBA, 18 mg/kg; 7,8,12-TMBA, 90 The carcinogenicity of polycycic aromatic hydrocarbons for mg/kg. the rat was tested by 2 methods; each of these has advantages Adrenal hemorrhage was not observed in rats injected with peculiar to itself. emulsions of BA, BP, or 3-MC; in each case the dose of hydro The classical method (4) of intramuscular injection of solu carbon was 60 mg/kg. tions of hydrocarbons in oil is advantageous when the amount Mammary Cancer. A set of pulse-doses of 7,12-DMBA and of compound available is small; a pool of injected oil remains 7,8,12-TMBA evoked mammary cancer rapidly and in every for more than 1 year as a marker of the site of injection. But member of the group (Table 1). The number of active centers the development of sarcoma is slow and, as an additional dis was large and equal in rats injected with emulsions of each advantage, a pool of sesame oil in muscle can serve as a local compound (Table 1). Similar sets of pulse-doses of BP or 3-MC izing agent (14) for circulating carcinogens. The appearance of evoked mammary cancer respectively in 30 and 35% of the sarcoma in a small proportion of injected animals is not proof rats; the appearance of palpable tumors was delayed and the of carcinogenicity of the injected compound. yield of active centers was 1. Our emulsion of BA did not The use of injectable emulsions (15) of hydrocarbons is induce cancer. highly useful. Intravenous injection in rodents is extremely Sarcoma. Spontaneously occurring sarcoma has never been simple; anesthesia is not required. At a given point of time, the observed in rats in our colonies. The solvent, sesame oil, and entire dose of compound is deposited in the blood. In suscep BA (II) did not elicit sarcoma (Table 2), but BA was activated tible rats mammary cancers appear rapidly and are easily de by monoalkyl substitution at the C7 and C12 positions. 7- tected. Both the time of appearance of mammary cancers and Methyl-BA (III) was a strong carcinogen and 1 2-methyl-BA the number of active centers at necropsy are useful indicators (VII) wasweak.Sarcomaoccurredinaverysmallproportion of carcinogenicity.

MARCH 1969 507

Table 2Detection sarcomaNo. of of with No. S.D.I Compound ratsNo. sarcomadaysRangeMean Â±

240II Sesame oil, controls 160a. Benz(a)anthracene Monoalkylbenz(a)anthraceneIII and related derivatives of 21IV 7-Methyl-BA 282875â€”147108 Â± 241188V 7-Ethyl-BA 161216VI 7-Methoxy-BA 160VII 7-Formyl-BA 42VIII 12-Methyl-BA 1611142â€”247197 Â± 241246b. 12-Ethyl-BA Dialkylbenz(a)anthraceneIx and related derivativesof 80x 1,12-DMBA 80XI 3,9-DMBA 16XII 6,7-DMBA 8870â€”130104 Â± 8895â€”123111Â±11XIII6,8-DMBA 18XIV 6,12-DMBA 8843â€”11379Â± 18XV 7,8-DMBA 8882â€”137104 Â± 20XVI 7,12-DMBA 202067â€”11687Â± 8885â€”10991Â±8XVII8,12-DMBA 81180XVIII 9,10-DMBA 21XIX 7-Methoxy-12-methyl-BA 151537â€”10186Â± 7xx 7-Ethoxy-12-methyl-BA 8881â€”10190 Â± 63XXI 7,12-Dimethoxy-BA 86108â€”261161 Â± 164147â€”178162XXII7-Hydroxymethyl-12-methyl-BA 20C. 7-Formyl-12-methyl-BA 151581â€”140102 Â± benz(a)anthraceneXXIIITrialkyl derivatives of 23XXIV 6,7,8-TMBA 7754â€”13096 Â± 18XXV 6,7,12-TMBA 88102â€”134116Â± 14XXVI 6,8,12-TMBA 8869â€”11292Â± 7,8,12-TMBA 8843â€”9883 Â±18 Spindle-cell sarcoma evoked by alkyl derivatives of benz(a)anthracene. Male Long-Evans rats, 25 days old, were injected i.m. with a solution ofsesame oil, 0.5 ml, containing each compound, 2.5 mg. The experiment was terminated at 9 months. See Footnote 2 for abbreviations.

Mammary glands of healthy young adult female rats of the In the present study it was found that all derivatives of BA Sprague-Dawley strain are extremely susceptible to induction were strongly carcinogenic when two or three methyl groups of cancers by polycyclic aromatic hydrocarbons and also by were present in any combination at positions 6, 7, 8, or 12; total-body irradiation (11, 13, 23). the high yields of neoplasms in rats injected with derivatives In the present experiments it was evident that a methyl sub of this sort are impressive. In contrast, 1,12-DMBA and stituent is of high significance in rendering benz(a)anthracene 3,9-DMBA did not elicit sarcoma, and 9,10-DMBA was a carcinogenic, and its site in the molecule is decisive in this feeble carcinogen. regard. Derivatives of BA with a single ethyl or methoxy group The advantageous sites in the benz(a)anthracene molecule were feeble carcinogens. The twelve monomethyl derivatives for methyl activation to produce carcinogenic compounds of benz(a)anthracene have been tested for carcinogenicity in form a geometric figure, the triangle of carcinogenicity. The earlier studies. Badger et al. (2) found in mice that only 3 of presence of an alkyl group in the advantageous area is not these derivatives were strong carcinogens: 7-methyl-BA, 8- obligatory for carcinogenesis but the fact remains that methyl methyl-BA, and 12-methyl-BA. Dunning and Curtis (8) in substitution at sites other than this triangle results in corn jected into rats solutions of each of the monomethyl deriva pounds with weak carcinogenicity or in inactive compounds. tives of benz(a)anthracene in hot paraffin: 4 of the derivatives The influence of the fluorine atom upon carcinogenicity of were strong carcinogens (6-, 7-, 8-, and 12-methyl-BA); 5 were polynuclear aromatics has been studied by Miller and Miller weak carcinogens (1-, 4-, 5-, 9-, and 10-methyl-BA); 3 com (20).4-Fluoro-benz(a)anthraceneisa strongandtoxiccarci pounds did not elicit sarcoma (2-, 3-, and 11-methyl-BA). nogen, whereas 4-methyl-BA is an inactive carcinogen. Miller We have found (17) that all 12 of the monomethyl deriva and Miller (20) also found that 7-methyl-BA and 6-F-7-methyl tives of BA can have intimate association with cell constitu BA were strong carcinogens, whereas 5-F-7-methyl-BA and ents. Among the large biochemical events which they initiate is 9-F-7-methyl-BA did not elicit cancer. the powerful induction of protein synthesis. When a big dose Lacassagne et al. (19) studied the carcinogenic activity of was fed to a group of rats, each of the 12 compounds greatly alkyl groups in angular benzacridines. The benz(c)acridine stimulated in vivo synthesis of menadione reductase in liver. series is richer in carcinogenic compounds than the benz(a) But only 4 of the compounds elicited sarcoma after injection acridine series is. In both series, all carcinogenic members with of an oil solution in muscle; the carcinogens were 6-methyl-, out any exception have a methyl substituent in the meso 7-methyl-, 8-methyl-, and 12-methyl-BA. anthracenic carbon (Chart 1). Monomethyl derivatives of

508 CANCER RESEARCH VOL.29

J@3 4. Burrows, H., Hieger, I., and Kennaway, E. L. The Experimental * I II Production of Tumours of Connective Tissue. Am. J. Cancer, 16: I0â€”@ 57â€”67, 1932. 5. Cook, J. W. The Production ofCancer by Pure Hydrocarbonsâ€”Part 8' 7 6* II. Proc. Roy. Soc. (London) 5cr. B, 111: 485â€”496,1932. 6. Cook, J. W., Hewett, C. L., and Hieger, I. The Isolation ofa Cancer BENZ(Â°.)ANTHRAC ENE Producing Hydrocarbon From Coal Tar. Parts I, II & III. J. Chem. Soc., 395â€”405, 1933. 7. Cook, J. W., Hieger, I., Kennaway, E. L., and Mayneord, W. V. The Production of Cancer by Pure Hydrocarbonsâ€”Part I. Proc. Roy. Soc. (London) SeriesB, 111: 455â€”484, 1932. 8. Dunning, W. F., and Curtis, M. R. Relative Carcinogenic Activity of Monomethyl Derivatives of Benz(a)anthracene in Fischer Line 344 o@9 Rats. J. Nati. Cancer Inst., 25: 387â€”391, 1960. BENZ(o.)ACRIDINE BENZ(c)ACRIDINE 9. Fieser, L. F., Fieser, M., Hershberg, E. B., Newman, M. S., Selig man, A. M., and Shear, M. J. Carcinogenic Activity of the Cho Chart 1. Introduction of one or more small alkyl groups at special lanthrenes and of Other 1: 2-Benzanthracene Derivatives. Am. J. (*) sitestransformstheinactiveparentmoleculetoacarcinogen. Cancer, 29: 260â€”268, 1937. 10. Ford E., and Huggins, C. Selective Destruction in Testis Induced by 7,12-Dimethylbenz(a)anthracene. J. Exptl. Med., 118: 27â€”40, benz(c)acridine were studied for carcinogenicity. Whereas 1963. 7-methylbenz(c)acridine was carcinogenic, its 8-methyl-, 11. Hamilton, J. G., Durbin, P. W., and Parrott, M. The Accumulation 9-methyl-, and 10-methyl- analogs were inactive. and Destructive Action of tin21 (eka-Iodine) in the Thyroid It has been found that a pulse-dose of 7,12-DMBA causes Gland of Rats and Monkeys. J. Clin. Endocrinol. Metab., 14: selective destruction (10) in the tubules of rat's testis. The 1161â€”1178, 1954. damage is exclusively in spermatogonia and in resting sperma 12. Harvey, R. G., and Halonen, M. Charge-Transfer Chromatography tocytes; these are the only cells in germinal epithelium which of Aromatic Hydrocarbons on Thin Layers and Columns. J. Chromatogr.25:294â€”302,1966. synthesize deoxyribose nucleic acids. Cells which multiply by 13. Huggins, C., and Fukunishi, R. Cancer in the Rat after Single Ex meiosis (they do not synthesize DNA) are spared from injury. posures to Irradiation or Hydrocarbons. Radiation Res. , 20: The evidence demonstrates that there are 3 molecular parame 493â€”503, 1963. ters (17, 18) of critical significance in determining carci 14. Huggins, C., and Grand, L. C. Sarcoma Induced Remotely in Rats nogenicity in polynuclear aromatic hydrocarbons: (a) the elec Fed 3-Methylcholanthrene. Cancer Res., 23: 477â€”480, 1963. tron transfer factor; (b) the geometric factor; (c) molecular 15. Huggins, C., Grand, L., and Fukunishi, R. 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MARCH 1969 509

John Pataki and Charles Huggins

Cancer Res 1969;29:506-509.

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