Highly selective bioactivation of 1- and 2-hydroxy-3-methylcholanthrene to mutagens by individual human and other mammalian sulphotransferases expressed in Salmonella typhimurium.

The benzylic alcohols 1- and 2-hydroxy-3-methylcholanthrene (OH-MC) are major primary metabolites of the carcinogen 3-methylcholanthrene (MC). We investigated them for mutagenicity in TA1538-derived Salmonella typhimurium strains expressing mammalian sulphotransferases (SULTs). 1-OH-MC was efficiently activated by human (h) SULT1B1 (2400 revertants/nmol), weakly activated by hSULT1C3 and hSULT2A1 (2-9 revertants/nmol), but not activated by the other hSULTs studied (1A2, 1A3, 1C2 and 1E1). Mouse, rat and dog SULT1B1 activated 1-OH-MC (8-100 revertants/nmol) with much lower efficiency than their human orthologue. The other isomer, 2-OH-MC, was activated to a potent mutagen by hSULT1A1 (4000-5400 revertants/nmol), weakly activated by hSULT1A2 or hSULT2A1 (1-12 revertants/nmol), but not activated by the other hSULTs. In contrast to their human orthologue, mouse, rat and dog SULT1A1 did not appreciably activate 2-OH-MC (<1 to 6 revertants/nmol), either. Instead, mouse and rat SULT1B1, unlike their human and canine orthologues, demonstrated some activation of 2-OH-MC (15-100 revertants/nmol). Docking analyses indicated that 1- and 2-OH-MC might bind to the active site of hSULT1A1 and hSULT1B1, but only for (S)-2-OH-MC/hSULT1A1 and (R)-1-OH-MC/hSULT1B1 with an orientation suitable for catalysis. Indeed, 1- and 2-OH-MC were potent inhibitors of the hSULT1A1-mediated sulphation of acetaminophen [concentration inhibiting the enzyme activity by 50% (IC50) 15 and 13nM, respectively]. This inhibition was weak with mouse, rat and dog SULT1A1 (IC50 ≥ 4 µM). Inhibition of the SULT1B1 enzymes was moderate, strongest for 1-OH-MC/hSULT1B1. In conclusion, this study provides examples for high selectivity of bioactivation of promutagens by an individual form of human SULT and for pronounced differences in activation capacity between orthologous SULTs from different mammalian species. These characteristics make the detection and evaluation of such mutagens extremely difficult, in particular as the critical form may even differ for positional isomers, such as 1- and 2-OH-MC. Moreover, the species-dependent differences will complicate the verification of in vitro results in animal studies.

Early morphologic alterations in mouse skin after topical application of 3-methylcholanthrene and its metabolites.

The effects of topical administration of 3-methylcholanthrene (MCA) or its metabolites on BALB/cKi mice were reported on inflammatory skin reactions, the alterations in epidermal thickness, the number of nucleated cells, pyknotic nuclei and/or nuclear fragments, and mitotic figures in the interfollicular epidermis (IFE). In the two-stage carcinogenesis system, MCA, the powerful complete carcinogen, induced an ordered sequence of cell changes strikingly similar to those caused by tumor-promoting agents such as the phorbol esters. These changes were absent after application of the "K-region" oxide of MCA. Other MCA metabolites also failed to induce notable inflammation, epidermal hyperplasia, and/or hypertrophy. Several MCA derivatives, however, caused a thinning of IFE paralleled by an increase in the relative number of pyknotic nuclei and a decrease in the total number of epithelial cells. The inhibitor of polycyclic hydrocarbon metabolism alpha-naphthoflavone did not prevent MCA-mediated skin reactions but, under suitable conditions, apparently potentiated the hyperplastic effects of MCA. The findings indicate that important events in the promotion phase of MCA-mediated skin carcinogenesis might be associated with the parent compound rather than with one of its metabolites.

Breakage of human cell DNA after exposure to 3-methylcholanthrene-11,12-oxide.

Damage to and repair of DNA isolated from human neonatal and fetal skin cells were measured by alkaline sucrose gradient analysis. 3-Methylcholanthrene did not induce single-strand breaks in DNA of the cells in culture, whereas the 11,12-oxide of 3-methylcholanthrene was very effective in this regard. The cis-1,2-dihydroxy, trans-11,12-dihydroxy, and cis-11,12-dihydroxy derivatives of 3-methylcholanthrene exerted little effect. The breaks in DNA caused by 3-methylcholanthrene oxide occurred during a 60-min incubation period and were repaired during the following 60 min. Methylmethane sulfonate also induced breaks in the DNA within 60 min.

Binding of K- and non-K-region arene oxides and phenols of polycyclic hydrocarbons to polyguanylic acid.

Arene oxide derivatives of carcinogenic polycyclic hydrocarbons have been postulated as the reactive intermediates responsible for the in vivo binding of the parent hydrocarbon to cellular nucleic acids. In this study the reaction of 12 different K- and non-K-region arene oxides and 7 benzo(a)pyrene phenols with polyguanylic acid in aqueous acetone solutions has been investigated. The extent of binding of the polycyclic hydrocarbon was monitored by changes in the ultraviolet absorption and fluorescence spectra of the reisolated polyguanylic acid. The most reactive compound was the K-region arene oxide of 7,12-dimethylbenz(a)anthracene. A lower but significant level of binding was detected with the K-region arene oxides of benz(a)anthracene, benzo(a)pyrene, and 3-methylcholanthrene. Very low or negligible binding was detected with the K-region arene oxides of pyrene and phenanthrene; the non-K-region arene oxides of benzo(a)pyrene, phenanthrene, and naphthalene; and all of the benzo(a)pyrene phenols. Significant differences in the fluorescence spectra of polyguanylic acid modified with three different benzo(a)-pyrene arene oxides were observed.

Morphological transformation and DNA adduct formation by benz[j]aceanthrylene and its metabolites in C3H10T1/2CL8 cells: evidence for both cyclopenta-ring and bay-region metabolic activation pathways.

Benz[j]aceanthrylene (B[j]A), a cyclopenta-fused polycyclic aromatic hydrocarbon related to 3-methylcholanthrene, has been studied to identify the major routes of metabolic activation in transformable C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts in culture. Previous studies have reported that the major (55% of total) B[j]A metabolite formed by C3H10T1/2 cells was (+/-)-trans-9,10-dihydro-9,10-dihydroxy-B[j]A (B[j]A-9,10-diol), the dihydrodiol in the bay-region ring, with moderate amounts (14% of total) of (+/-)-trans-1,2-dihydro-1,2-dihydroxy-B[j]A (B[j]A-1,2-diol), the cyclopenta-ring dihydrodiol. The morphological transforming activities of three potential intermediates formed by metabolism of B[j]A by C3H10T1/2 cells, (+/-)-anti-trans-9,10-dihydro-9,10-dihydroxy-B[j]A-7,8-oxide (B[j]A-diol-epoxide), B[j]A-9,10-oxide, and B[j]A-1,2-oxide as well as the two B[j]A-dihydrodiols were examined. B[j]A, B[j]A-diol-epoxide, B[j]A-1,2-oxide, and B[j]A-9,10-diol were found to have moderate to strong activities with B[j]A-diol-epoxide the most active compared to B[j]A, while B[j]A-1,2-diol was inactive. B[j]A-9,10-oxide was found to be a weak transforming agent. At 0.5 microgram/ml, the following percentage of dishes with type II or III foci were observed: B[j]A, 59%; B[j]A-diol-epoxide, 75%; B[j]A-1,2-oxide, 25%; and B[j]A-9,10-diol, 17%. DNA adducts of B[j]A, B[j]A-9,10-diol, B[j]A-diol-epoxide, B[j]A-9,10-oxide, and B[j]A-1,2-oxide in C3H10T1/2 cells were isolated, separated, identified, and quantitated using the 32P-postlabeling method. B[j]A forms two major groups of adducts: one group of adducts is the result of the interaction of B[j]A-1,2-oxide with 2'-deoxyguanosine and 2'-deoxyadenosine; the second group of adducts is a result of the interaction of B[j]A-diol-epoxide with 2'-deoxyguanosine and 2'-deoxyadenosine. Qualitative and quantitative analysis of the postlabeling data suggests that B[j]A is metabolically activated by two distinct routes, the bay-region diol-epoxide route and the cyclopenta-ring oxide route, the former being the most significant.

Metabolism of [3H]-methylcholanthrene in the perfused rat liver.

The metabolism of [3H]-3-methylcholanthrene (3-MC) was studied in the isolated, perfused rat liver. Following addition of 250 mug to the perfusion fluid, 3-MC disappeared rapidly. After 2 hr, approximately 34% of the radioactivity was excreted in the bile, 6% remained in the perfusate, and 60% was found in the liver. Of the liver radioactivity, 80% was unchanged 3-MC, 11% was conjugated metabolites, 4% was free hydroxymetabolites, and 4% was nonextractable, presumably bound to macromolecules. Of the perfusate radioactivity, 82% was conjugated metabolites, 3% was free hydroxymetabolites, and 15% was unchanged 3-MG. A similar distribution was observed in intact, bile-cannulated rats, but biliary excretion was about one-fourth as high with double the i.v.-injected dose. Biliary excretion in perfused livers rose rapidly during the first 30 to 40 min, then decreased steadily. It was nearly twice as high in male as in female rat livers. Pretreatment of rats with 3-MC more than doubled the biliary excretion rate over the first 20 to 30 min in livers of both sexes and raised that of the female to that of the male rat liver. Neither retinol acetate nor 7,8-benzoflavone had any appreciable effect on biliary excretion of 3-MC metabolites. 2-Diethylaminoethyl-2,2-diphenylvalerate, a well-known microsomal oxygenase inhibitor, lowered excretion by 80 to 90% and lengthened the lag period, and dibutyryl-3',5'-cyclic adenosine monophosphate markedly increased the rate of excretion of 3-MC metabolites. Fractionation of bile by chromatography on Sephadex LH-20 revealed six well-defined peaks of radioactivity. In contrast, bile of intact rats given 3-MC gave a pattern on Sephadex chromatography consisting of only three peaks. Preliminary data suggest that these consist of conjugates of dihydroxymetabolites as well as more highly hydroxylate derivatives. The data obtained indicate that the perfused liver is an appropriate experimental model for studies on the hepatobiliary metabolism of carcinogens.

Mutagenicity of cyclopenta-fused isomers of benz(a)anthracene in bacterial and rodent cells and identification of the major rat liver microsomal metabolites.

The microsomal metabolites and mutagenic activity of four cyclopenta-fused benz(a)anthracenes, benz(j)aceanthrylene [B(j)A], benz(e)aceanthrylene [B(e)A], benz(l)aceanthrylene [B(l)A], and benz(k)acephenanthrylene [B(k)A], have been studied. Aroclor 1254-induced rat liver microsomes metabolized B(j)A to B(j)A-1,2-dihydrodiol, B(j)A-9,10-dihydrodiol, B(j)A-11,12-dihydrodiol, and 10-hydroxy-B(j)A; B(e)A-1,2-dihydrodiol, B(e)A-3,4-dihydrodiol, and B(e)A-5,6-dihydrodiol; B(l)A to B(l)A-1,2-dihydrodiol, B(l)A-4,5-dihydrodiol, and B(l)A-7,8-dihydrodiol; and B(k)A to B(k)A-4,5-dihydrodiol and B(k)A-8,9-dihydrodiol. With each polycyclic aromatic hydrocarbon, metabolism occurred on the cyclopenta ring. All four isomers were active as gene mutagens in Salmonella typhimurium and in Chinese hamster V79 cells. In the S. typhimurium mutation studies, using Aroclor 1254-induced rat liver S9, B(j)A, B(e)A, and B(l)A required significantly less microsomal protein for maximal mutation response than B(k)A and B(a)P, suggesting a one-step activation mechanism, presumably on the cyclopenta-fused ring. B(j)A, B(e)A, and B(l)A were significantly more mutagenic than B(k)A and B(a)P in S. typhimurium. In the Aroclor 1254-induced rat liver S9-mediated V79 mutagenesis system, all four isomers were active, with B(l)A the most active. When Syrian hamster embryo cells were used as the metabolic activation component for V79 cells, only B(l)A produced a significant response and was equivalent in activity to B(a)P. A helical configuration for B(l)A is inferred from the identification of two trans-B(l)A-1,2-dihydrodiols, syn and anti, which have been synthesized, separated, and characterized. The metabolically formed dihydrodiol is anti-trans-B(l)A-1,2-dihydrodiol, and experimental evidence suggests that the metabolically formed B(l)A-1,2-oxide is the anti-isomer. Synthetic B(l)A-1,2-oxide was found to be a direct-acting mutagen in S. typhimurium and Chinese hamster V79 cells and is estimated to account for up to 40% of the mutagenic activity of the parent hydrocarbon. Therefore, certain cyclopenta-ring fusions on benz(a)anthracene appear to markedly increase its genotoxic and carcinogenic activities.

Comparison of the skin tumor initiating activity of 3-methylcholanthrene and 3,11-dimethylcholanthrene in mice.

The abilities of 3-methylcholanthrene (3-MC) and 3,11-dimethylcholanthrene (3,11-DMC) to initiate skin tumors in Sencar mice were determined by using a 2-stage system of tumorigenesis. 3,11-DMC was found to have very weak skin tumor initiating activity when compared to the potent activity of 3-MC. The only difference between 3-MC and 3,11-DMC is the substitution of a methyl group in position 11 which is part of the 'K-region' or the 'peri' position. From these results, we suggest that an unhindered peri position adjacent to an angular benzene ring is necessary for carcinogenic activity of 3-MC.

Tumor-initiating activity of the 9,10-dihydrodiol- and 9,10-dihydrodiol-7,8-epoxide of 3-methylcholanthrene in SENCAR mice.

The skin tumor-initiating activities of several bay-region metabolites of 3-methylcholanthrene (3-MCA) were determined in SENCAR mice. 3-MCA-anti-9,10-diol-7,8-epoxide possessed weak tumor-initiating activity when tested at 100 and 200 nmol/mouse doses (0.27 and 0.67 papillomas per mouse after 18 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA)). 3-MCA-trans-9,10-diol at initiating doses of 50 and 100 nmol/mouse was as active as 3-MCA. 3-MCA-trans-9,10-diol was also tested for mutagenic activity toward V79 cells in cell-mediated assays and found to be approximately 2-times more potent than 3-MCA. The data suggest that 3-MCA-trans-9,10-diol is a proximate carcinogen for mouse skin.

Mouse skin tumor-initiating activity of benz[j]aceanthrylene in SENCAR mice.

Benz[j]aceanthrylene (B[j]A), a cyclopenta-fused derivative of benz[a]anthracene, has been reported to be an active bacterial cell and mammalian cell gene mutagen, a morphological transforming agent in C3H10T1/2CL8 mouse embryo fibroblasts and a mouse lung tumorigen in strain A/J mice. B[j]A was evaluated as a skin tumor initiator in female SENCAR mice and was found to induce papilloma formation in the range of 40-400 micrograms/mouse. B[j]A was found to be extremely active, inducing 8.7 papillomas/mouse after an initiating dose of 40 micrograms/mouse. At this dose, 100% of the mice bore tumors. Comparison with four other cyclopenta-fused polycyclic aromatic hydrocarbons suggests that B[j]A is extremely potent.

The effect of steric strain in the bay-region of polycyclic aromatic hydrocarbons: tumorigenicity of alkyl-substituted benz[a]anthracenes.

3,6-Dimethylcholanthrene (3,6-DMC) and 7,11,12-trimethylbenz[a]anthrene (7,11,12-TMBA) were tested for tumor-initiating activity on mouse skin as an approach to evaluate the potential role of steric strain in the bay-region on tumorigenic potency. Methyl-substitution at the 6-position of 3-methylcholanthrene (3-MC) increases steric strain in the bay-region of the hydrocarbon as it does at the 12-position of benz[a]anthracene (BA) causing both hydrocarbons to become non-planar. 3,6-DMC had at least 2- to 3-fold higher tumor-initiating activity than did 3-MC. Introduction of an 11-methyl group in 7,12-dimethylbenz[a]anthracene (7,12-DMBA) results in the formation of a more highly hindered (buttressing effect) hydrocarbon. 7,11,12-TMBA had 5% or less of the tumor-initiating activity of 7,12-DMBA, although the hydrocarbon still had relatively high tumorigenic activity on mouse skin. The results obtained with 3,6-DMC and studies reported previously with other methyl-substituted hydrocarbons, show that hydrocarbons possessing steric strain in the bay-region of the molecule can have enhanced tumorigenic activity. The basis of this steric effect remains unclear, however, as a result of the decreased tumorigenic activity of the 11-methyl-substituted derivative of 7,12-DMBA. The weak tumor-initiating activity of BA was enhanced at least 4- to 8-fold as a result of methyl-substitution at the 6- and 8-positions (6,8-dimethylbenz[a]anthracene). The higher tumorigenic activity of 6,8-dimethylbenz[a]anthracene compared to BA is consistent with a presumed decrease in metabolic detoxification of the dimethyl-substituted derivative at the 5,6- and 8,9-double bonds.

The metabolism of 1-hydroxy- and 2-hydroxy-3-methylcholanthrene by liver microsomes: effect of enzyme inducing agents.

The metabolism of both 1-hydroxy-3-methylcholanthrene (1-OH-3-MC) and 2-hydroxy-3-methylcholanthrene (2-OH-3-MC) by rat liver microsomes was investigated. Metabolites were separated by high pressure liquid chromatography (HPLC). Both compounds were shown to be substrates for further metabolism. Metabolism of 1-OH-3-MC gave rise to at least 13 products while 2-OH-3-MC showed the formation of at least 10 metabolites. At least 3 products of the 1-OH-3-MC metabolism are diol derivatives of the substrate. Using 2-OH-3-MC as substrate generated one major diol derivative that represented between 20% and 50% of total metabolite formation. Pretreatment of animals with known microsomal enzyme inducing agents did not result in the expected induction patterns. In some instances, the microsomes from induced animals were less efficient at metabolizing substrate than those from control. The discovery of a major peak from 2-OH-3-MC metabolism is of extreme importance considering the potency of the parent hydroxy compound in tumor initiation studies.

The binding of polycyclic aromatic hydrocarbon diol-epoxides to DNA.

Diol-epoxide derivatives of polycyclic aromatic hydrocarbons are the ultimate carcinogenic forms of these compounds encountered in vivo. Using a novel electro-fluorescence apparatus, we report that, like the (+/-)-anti-diol-epoxide of benzo[a]pyrene (BP), the similar diol-epoxides of benzo[a]anthracene (BA) and 3-methylcholanthrene (3MC) also bind to the DNA helix at an inclination of approximately 50 degrees. This compares with the essentially perpendicular, intercalative-type binding generally found with saturated aromatic compounds, such as the native hydrocarbons, and may indicate a systematic behavior in the molecular associations precursive to carcinogenesis.

Further investigations into the effect of non-benzo ring bay-region methyl substituents on tumor-initiating activity of polycyclic hydrocarbons.

The effect of a methyl substituent at the non-benzo ring bay-region position of benzo[e]pyrene (B[e]P), cholanthrene (CA) and dibenz[a,j]anthracene (DB[a,j]A) on skin tumor-initiating activity was examined. A methyl substituent at the 1-carbon of B[e]P enhanced tumor-initiating activity of the parent compound (0.18 vs. 2.4 papillomas/mouse for B[a]P vs. 1-methyl-B[e]P, respectively, with an 800 nmol initiating dose). A methyl substituent at the 7- and 14-carbons of DB[a,j]A increased the activity of this weak initiator more than 13 times. The introduction of a methyl substituent at the non-benzo ring bay-region position of CA (i.e. carbon atom 6) dramatically increased tumor-initiating activity in SENCAR mice. 6-Methyl-CA was found to be a more potent skin tumor-initiator than 3-methyl-CA, and nearly as potent as 7,12-dimethylbenz[a]anthracene, one of the most potent initiators yet tested in the 2-stage initiation-promotion mouse skin model. When taken together with previous results from our laboratories, the data further support the generality of the enhancing effect of a non-benzo ring bay-region methyl substituent on polycyclic hydrocarbon tumor-initiation.

Metabolism of benz[j]aceanthrylene (cholanthrylene) and benz[l]aceanthrylene by induced rat liver S9.

The metabolites of benz[j]aceanthrylene (B[j]A) produced by incubation with liver S9 proteins from rats induced with Aroclor-1254 and phenobarbital have been identified as: trans-B[j]A-1,2-dihydrodiol, B[j]A-9,10-dihydrodiol, B[j]A-11,12-dihydrodiol, and 10-hydroxy-B[j]A. The major metabolite formed (58-60%) by both induced S9 preparations was trans-B[j]A-1,2-dihydrodiol, the cyclopenta-ring dihydrodiol while oxidation at the k-region or the proximal-bay region was minor. There were no statistical differences in individual or total B[j]A metabolite rates between the 2 induced S9 preparations. B[l]A was metabolized by Aroclor-1254 and phenobarbital induced rat liver S9 preparations to trans-B[l]A-1,2-dihydrodiol, B[l]A-7,8-dihydrodiol, and B[l]A-4,5-dihydrodiol. The major B[l]A metabolite formed (28-40%) by both induced S9 preparations was B[l]A-7,8-dihydrodiol, the k-region dihydrodiol. Cyclopenta-ring oxidation to trans-B[l]A-1,2-dihydrodiol was approximately 50% of that observed for k-region oxidation. Both induced S9s produced similar rates of B[l]A metabolites except for B[l]A-7,8-dihydrodiol formation which was higher for Aroclor-1254-induced S9.

The induction of sister chromatid exchanges by dihydrodiols derived from 7,12-dimethylbenz[a]anthracene and 3-methylcholanthrene.

The in vitro induction of sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells by the polycyclic hydrocarbons, 7,12-dimethylbena[a]anthracene and 3-methylcholanthrene and some of the related dihydrodiols was investigated. Increased numbers of SCEs were seen in the chromosomes of cells exposed to non-K-region dihydrodiols. The most active compounds were the 3,4-dihydrodiol of 7,12-dimethylbenza[a]anthracene and the 7,8- and 9,10-dihydrodiols of 3-methylcholanthrene: the parent hydrocarbons and their corresponding K-region dihydrodiols were relatively less active. The results are consistent with others that suggest that the metabolic activation of both hydrocarbons proceeds through the conversion of non-K-region dihydrodiols into vicinal diol-epoxides.

Morphological transformation of C3H10T1/2CL8 cells by cyclopenta-fused derivatives of benzo[a]pyrene and benzo[e]pyrene.

Cyclopenta-fused homologs of polycyclic aromatic hydrocarbons (PAH) have proven to be more genotoxic and tumorigenic than their parent PAHs. In an effort to uncover their mechanisms of metabolic activation, the morphological transforming activities of dibenzo[k,mno]acephenanthrylene (CP(3,4)B[a]P), dibenzo[j,mno]acephenanthrylene (CP(1,12)B[a]P) and naphtho[1,2,3,4-mno]acephenanthrylene (CPB[e]P) were studied in C3H10T1/2CL8 mouse embryo fibroblasts. CP(3,4)B[a]P, a PAH with a blocked K region and unblocked bay region, was highly active inducing an average of 1.1 Type II and III foci/dish at 5 micrograms/ml with an average of 67% of the dishes containing foci. This activity was similar to that of benzo[a]pyrene. CP(1,12)B[a]P and CPB[e]P were inactive. The relative positions of the cyclopenta-ring and bay region may play an essential role in the metabolic activation of these PAHs and their biological activities.

Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of fluorinated derivatives of benzo[a]pyrene and 3-methylcholanthrene.

Comparative studies of tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland were conducted with benzo[a]pyrene (BP) and 3-methylcholanthrene (MC) derivatives. SENCAR mice were initiated with BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-methylBP, 7-FBP, 8-FBP, 9-FBP, 10-FBP, or 10-azaBP and promoted with tetradecanoyl phorbol acetate. The same compounds plus BP 7,8-dihydrodiol were tested by intramammillary injection in female Sprague-Dawley rats. Tumor-initiating activity in mice and/or carcinogenicity in rats were observed for BP, 6-methylBP, 6-, 7-, 8-, and 10-FBP, whereas 9-FBP was inactive in both experiments and 10-azaBP was only marginally active in the mammary gland. BP 7,8-dihydrodiol was carcinogenic in rat mammary gland, although it was less potent than BP. MC, 8-FMC, 10-FMC, and 3-methylcholanthrylene were also tested in Sprague-Dawley rats by intramammillary injection. All compounds were carcinogenic, with MC displaying the most potent activity. The less potent carcinogenic activity of BP 7,8-dihydrodiol in the mammary gland, compared with BP, and the moderate-to-weak tumor-initiating and/or carcinogenic activity of 7-, 8-, and 10-FBP suggest that the bay-region diol-epoxide pathway does not play a significant role in the activation of BP in these two target tissues. Similarly, the carcinogenic activity of 8-FMC and 10-FMC, in which the bay-region diol-epoxide pathway is blocked, suggests that this mechanism of activation is not important in the carcinogenicity of MC in rat mammary gland.

Carcinogenicity and metabolic profiles of 3-methylcholanthrene oxygenated derivatives at the 1 and 2 positions.

Trapping of 3-methylcholanthrene (MC) radical cation by nucleophilic compounds occurs specifically at the 1-carbon atom. With the purpose of providing more evidence for the hypothesis that the critical mechanism of activation of MC is one-electron oxidation, the carcinogenicity of MC was compared to that of 1-hydroxy-3-methylcholanthrene (MC-1-OH), 3-methylcholanthrene-1-one (MC-1-one), 2-hydroxy-3-methylcholanthrene (MC-2-OH), 3-methylcholanthrene-2-one (MC-2-one) and 3-methylcholanthrylene (MCL) by repeated application on mouse skin. Seven-week-old female Swiss mice in 6 groups of 30 were treated on the back with 0.2 mumol of compound in acetone twice weekly for 20 weeks. In addition, the metabolism of MC and its derivatives was studied using mouse skin homogenates. The compounds tested were classified according to carcinogenicity in 4 groups: MC and MC-2-OH, the strongest carcinogens; MC-2-one and MCL, weaker than MC and MC-2-OH; MC-1-OH, the weakest carcinogen; and MC-1-one, non-carcinogenic. These results support the hypothesis that one-electron oxidation for MC, MC-2-OH and MC-1-one might be the critical mechanism of carcinogenic activation, with C-1 the binding site to cellular nucleophiles. The carcinogenic effect of MC-1-OH is speculated to be the formation of an ester bearing a good leaving group, which might be the ultimate alkylating compound in the in vivo reaction. The lack of carcinogenic activity for MC-1-one may be attributed to absence of nucleophilic trapping at C-1 via the radical cation pathway as well as the inability of mouse skin to reduce MC-1-one to the carcinogenic MC-1-OH.

Metabolism and activation of cyclopenta polycyclic aromatic hydrocarbons in isolated human lymphocytes, HL-60 cells and exposed rats.

The metabolism of radiolabelled benz(j)aceanthrylene (B(j)A) was studied in suspensions of isolated human peripheral mononuclear blood cells (lymphocytes), using high performance liquid chromatography (HPLC). The only known metabolite found after 24 h exposure to 30 microg/ml (120 microM) B(j)A, was B(j)A-1,2-dihydrodiol, representing approximately 35% of the total metabolites formed. B(j)A, benz(l)aceanthrylene (B(l)A) and benzo(a)pyrene (B(a)P) all caused DNA adducts in human lymphocytes, as well as in the human promyelocytic cell line HL-60 cells, as measured by the 32P-postlabelling technique (30 microg/ml, 24 h). The total DNA adduct levels in human lymphocytes exposed to B(j)A, B(l)A or B(a)P were 0.13 +/- 0.03, 1.10 +/- 0.62 and 0.37 +/- 0.10 fmol/microg DNA, respectively, and similar levels were obtained in HL-60 cells (0.18 +/- 0.14, 0.97 +/- 0.35 and 0.29 +/- 0.17 fmol/microg DNA, respectively). For each compound, the human lymphocytes and HL-60 cells in addition showed similar DNA adduct patterns. Cells exposed to B(j)A revealed only one DNA adduct, which, judged by its TLC properties, resulted from B(j)A-1,2-epoxide. As measured by the alkaline filter elution technique, only low levels of single strand DNA breaks (SSB) were observed in both human lymphocytes and HL-60 cells after exposure to B(j)A, B(l)A or B(a)P (24 h, 30 microg/ml). By adding cytosine-1-beta-D-arabinofuranoside (Are C) and hydroxyurea (HU) 1 h prior to analysis to prevent strand break rejoining, some increase in SSB (2-3 times) was observed in the lymphocytes. Co-incubation of human lymphocytes with liver microsomes from PCB-treated rats for 1 h and exposure to B(j)A or B(l)A, increased the DNA adduct levels in the lymphocytes to 12.3 and 37.8 fmol/microg DNA, respectively. A large increase in SSB was also observed, whereas no such increase was observed after co-incubation with human liver microsomes. In vivo exposure of rats to 30 mg/kg B(j)A (i.p.) for 24 h revealed one major DNA adduct in lymphocytes and lung tissue (only one of three rats showed an adduct in liver tissue), apparently resulting from B(j)A-1,2-epoxide. The total DNA adduct level in the lymphocytes was 0.09 fmol/microg DNA, and in lung tissue between 0.10 and 0.62 fmol/microg DNA. Overall, the present data suggests that oxidation at the cyclopenta-ring is an important activation pathway for B(j)A in rats as well as in humans.