Benzo(a)pyrene-7,8-dihydrodiol 9,10-oxide adenosine and deoxyadenosine adducts: structure and stereochemistry.

The structure and absolute stereoconfigurations of four adenosine adducts with (+/-)-7 alpha,8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) and their deoxyadenosine analogs have been determined. They result from both cis and trans addition of the N6 amino group of ademine to the 10 position of both enantiomers of BDPE. This was determined from studies of the nuclear magnetic resonance spectra, mass spectra, and circular dichroism spectra, as well as from their pKa values and chemical reactivities.

Benzo(a)pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo.

Evidence has been obtained that a specific isomer of a diol epoxide derivative of benzo(a)pyrene, (+/-)-7 beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, is an intermediate in the binding of benzo(a)pyrene to RNA in cultured bovine bronchial mucosa. An adduct is formed between position 10 of this derivative and the 2-amino group of guanine.

Comparison of the cellular DNA-bound products of benzo(alpha)pyrene with the products formed by the reaction of benzo(alpha)pyrene-4,5-oxide with DNA.

DNA isolated from mouse embryo cell cultures that had been treated with [3H]benzo(alpha)pyrene was degraded with enzymes to deoxyribonucleosides, and the hydrocarbon-deoxyribonucleoside products were isolated by chromatography on a Sephadex LH20 column eluted with a water: methanol gradient. The hydrocarbon-deoxyribonucleoside products were not identical to those found in similar chromatograms of enzyme digests of DNA that had been reacted with benzo(alpha)pyrene-4,5-oxide in aqueous ethanol solution. This finding suggests that the metabolic activation of benzo(alpha)pyrene that results in this hydrocarbon becoming covalently bound to DNA in mouse embryo cells in culture may be more complex than simply formation of a K-region epoxide and reaction of that compound with the cellular DNA.

Oxidation of the trans-3,4-dihydrodiol metabolites of the potent carcinogen 7,12-dimethylbenz(a)anthracene and other benz(a)anthracene derivatives by 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase: effects of methyl substitution on velocity and stereochemical course of trans-dihydrodiol oxidation.

The homogeneous 3 alpha-hydroxysteroid-dihydrodiol dehydrogenase of rat liver cytosol catalyzes the NADP-dependent oxidation of a wide variety of polycyclic aromatic trans-dihydrodiols and has been implicated in their detoxification (T.E. Smithgall, R.G. Harvey, and T.M. Penning, J. Biol. Chem., 261:6184-6191,1986). This study examined the influence of methyl groups on the velocity and stereochemical course of enzymatic benz(a)anthracene (BA) trans-dihydrodiol oxidation. The racemic trans-3,4-dihydrodiols of BA and 7-methylbenz(a)anthracene (7-MBA) were completely consumed by the purified dehydrogenase, indicating that both stereoisomers are substrates. However, 50% of the ( +/- )-trans-3,4-dihydrodiols of 12-methylbenz(a)anthracene (12-MBA) and 7,12-dimethylbenz(a)-anthracene (DMBA) were oxidized, suggesting that only one stereoisomer is utilized in each case. At low substrate concentrations, enzymatic oxidation of the trans-3,4-dihydrodiols of BA, 12-MBA, and DMBA followed simple first-order kinetics. By contrast, oxidation of the 3,4-dihydrodiol of 7-MBA was of higher order, due to differences in the rate of oxidation of each stereoisomer. Rate constants estimated for each reaction indicate that the non-bay-region methyl group at position 7 has a greater enhancing effect on the rate of oxidation than the bay-region methyl group at position 12 (10- versus 4-fold, respectively). The 3,4-dihydrodiol of DMBA, which possesses both non-bay- and bay-region methyl groups, is oxidized more than 30 times faster than the unmethylated parent hydrocarbon. The absolute stereochemistry of the preferentially oxidized dihydrodiols was assigned by circular dichroism spectrometry. For the 3,4-dihydrodiols of DMBA and 12-MBA, the stereoisomer oxidized has the 3S,4S configuration. A large negative Cotton effect was observed in the circular dichroism spectrum of the 7-MBA 3,4-dihydrodiol which remained at the end of the rapid phase of oxidation of this racemic substrate, indicating that the dehydrogenase displays partial stereochemical preference for the 3S,4S enantiomer. These results suggest that methylation of BA at C-7 greatly enhances the oxidation of the 3S,4S-dihydrodiol, while the presence of a bay-region methyl group at C-12 completely blocks the oxidation of the 3R,4R-stereoisomer. Rapid, stereoselective oxidation of methylated polycyclic aromatic trans-dihydrodiols by this route in vivo may significantly influence their carcinogenicity.

Molecular structure of the K-region cis-dihydrodiol of 7,12-dimethylbenz[a]anthracene.

The molecular structure and conformation of the cis-5,6-dihydrodiol of 7,12-dimethylbenz[a]anthracene has been determined by an X-ray crystallographic analysis. The compound crystallizes in the space group P21/a with cell dimensions a equals 17.799(6), b equals 33.211(8), c equals 5.241(1) A, beta equals 91.88(2)degrees. There are two molecules, designated A and B in the asymmetrical unit, that are not related to each other by crystallographic symmetry. Their conformations are almost identical, and there are no significant differences in their bond lengths or angles. In both molecules the 5-hydroxyl group is equatorial while the 6-hydroxyl group is axial. This conformation is probably forced by steric hindrance between the hydroxyl group, 0-6, and the hydrogen atoms of the 7-methyl group. The molecules pack in the crystal by forming hydrogen bonds between the hydroxyl groups of adjacent molecules, A with A, B, with B, and A with B. The ring system of the cis-5,6-dihydrodiol is much more buckled than is that in 7,12-dimethylbenz[a]anthracene itself. The angle between the two outermost rings is 36 degrees, the deviation from planarity being primarily a consequence of the partial saturation in the ring containing the two hydroxyl groups. Extrapolation of these results to other dihydrodiol derivatives of carcinogenic hydrocarbons permits some predictions of preferred molecular geometry. Thus, the 8,9-dihydrodiol-10,11-epoxide of 7,12-dimethylbenz]a[anthracene, analogous to the biologically active 7,8-dihydrodiol-9,10-epoxide of benzo]a[pyrene, a mutagen that is believed to be an active intermediate in carcinogenesis by benzo]a[pyrene, should probably exist preferentially in a conformation bearing the8-hydroxyl group in the axial orientation.

Enhancing effect of a bay region methyl group on tumorigenicity in newborn mice and mouse skin of enantiomeric bay region diol epoxides formed stereoselectively from methylchrysenes in mouse epidermis.

The stereochemistry of diol epoxide formation in mouse epidermis upon topical application of [3H]-1R,2R-dihydroxy-1,2-dihydro-5-methylchrysene ([3H]-5-MeC-1R,2R-diol) and [3H]-6-MeC-1R,2R-diol, and the tumorigenicity in mouse skin and in newborn mice of the R,S,S,R and S,R,R,S enantiomers of 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (5-MeC-1,2-diol-3,4-epoxide), 5-MeC-7,8-diol-9,10-epoxide, and 6-MeC-1,2-diol-3,4-epoxide were examined. Analysis of tetraols and their derived tetraacetates present in mouse epidermis, 2 h after application of [3H]-5-MeC-1R,2R-diol or [3H]-6-MeC-1R,2R-diol, demonstrated greater than 90% stereoselectivity in formation of 5-MeC-1R,2S-diol-3S,4R-epoxide and 6-MeC-1R,2S-diol-3S,4R-epoxide. Taken together with previous data, these results demonstrate that there is a high degree of stereoselectivity for formation of R,S,S,R enantiomers of 5-MeC- and 6-MeC-1,2-diol-3,4-epoxides in mouse skin. The results of the tumorigenicity studies in mouse skin and in newborn mice clearly demonstrated that 5-MeC-1R,2S-diol-3S,4R-epoxide was the most tumorigenic of the diol epoxide enantiomers tested; 6-MeC-1R,2S-diol-3S,4R-epoxide was inactive. The results of this study show that the high tumorigenicity of 5-MeC compared to 6-MeC is due to the remarkable tumorigenic activity of 5-MeC-1R,2S-diol-3S,4R-epoxide which, in contrast to 6-MeC-1R,2S-diol-3S,4R-epoxide, has a methyl group in the same bay region as the epoxide ring. We propose that such methyl bay region diol epoxides of other carcinogenic methylated polynuclear aromatic hydrocarbons will also show unique tumorigenic properties.

Identification of the major adducts formed by reaction of 5-methylchrysene anti-dihydrodiol-epoxides with DNA in vitro.

5-Methylchrysene is metabolically converted to the bay-region dihydrodiol-epoxides, trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I), in which the methyl group and the epoxide ring are in the same bay region, and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene (DE-II). Previous studies have indicated that DE-I is more important in 5-methylchrysene carcinogenesis than is DE-II. Both DE-I and DE-II were individually reacted with calf thymus DNA in vitro. The DNA was enzymatically hydrolyzed to deoxyribonucleosides, and the modified deoxyribonucleosides were separated by chromatography on Sephadex LH-20 and analyzed by high-performance liquid chromatography. One major adduct and seven minor adducts were formed from each dihydrodiol-epoxide. The major adduct was, in each case, characterized by its pH-dependent partition coefficient, stability to base, mass spectrum, ultraviolet spectrum, and nuclear magnetic resonance spectrum as a deoxyguanosine derivative resulting from addition of the exocyclic amino group of deoxyguanosine to the benzylic carbon of the epoxide ring of the dihydrodiol-epoxide. The results of this study show that the major DNA adducts formed from 5-methylchrysene via DE-I and DE-II are structurally similar.

Molecular structure of benzo(a)pyrene 4,5-oxide.

An X-ray crystallographic study of benzo(a)pyrene 4,5-oxide, a metabolite of the carcinogen benzo(a)pyrene (BP), as given information on the geometry of this molecule. The carbon skeleton of BP itself has been shown by others to be early planar; the planarity of the carbon skeleton has been shown by this work to be perturbed very little by epoxidation of the 4,5-double bond. Epoxidation has, however, increased the double bond character of C-11--C-12, C-9--C-10, and C-7--C-8. The hydrogen atom on C-3 points directly toward the oxygen atom of another molecule. This C--H... O interaction, although weak, suggests that C-3 might be slightly acidic. An analysis of the experimentally determined bond lengths indicates that, after the highly reactive epoxide ring, the most reactive positions are at C-1, C-6, C-7, C-11, and C-12. The oxide ring of BP, unlike that for the K-region oxide of 7,12-dimethylbenz(a)anthracene, is symmetrical (with C--O distances equivalent within experimental error). The C--O distances are longer than those found in most oxides, including those in 7,12-dimethylbenz(a)anthracene-5,6-oxide. Thus it has been shown that the oxide rings of the K-region oxides of the two potent carcinogens BP and 7,12-dimethylbenz(a)anthracene are not similar in dimensions.

Age-, tissue-, and Ah genotype-dependent differences in the binding of 3-methylcholanthrene and its metabolite(s) to mouse DNA.

The induction of transplacental carcinogenesis by 3-methylcholanthrene (MC) in mice is determined, in part, by the genotype at the Ah locus. The relationship of Ah genotype and MC-induced DNA adducts was tested by comparing the response of pregnant and fetal C57BL/6 mice (Ahb Ahb; responsive to the induction of MC metabolism) and DBA/2mice (Ahd Ahd; nonresponsive). On day 17 of gestation (day 1 = presence of vaginal plug), C57BL/6 mice were treated i.p. with 100 mg/kg MC and DBA/2 mice with 30 mg/kg. Mice were sacrificed 24 h later and the tissues were analyzed for the presence of DNA adducts using the P1 nuclease version of the 32P-postlabeling method. With a 3.3-fold difference in administered dose, the total adduct levels in fetal DNA were (a) similar in both strains with the exception of liver, for which C57BL/6 mice had more adducts; (b) higher in the lung than skin, liver, or thymus; and (c) only 1/4 to 1/14 of the adult levels. Maternal DBA/2DNA contained more adducts in the thoracic lymph nodes and liver but fewer in the placenta and lung, compared to maternal C57BL/6 DNA. More adducts were detected in lung DNA than liver DNA in C57BL/6 mice. In contrast, these levels were similar in DBA/2 mice. When the difference in dose administered was considered in conjunction with this, less MC bound to DNA of C57BL/6 than DBA/2 mice overall. To identify adducts, oxidized metabolites of MC, 1-hydroxy-, 2-hydroxy-, 9,10-dihydrodiol-, or 3-methoxymethyl-MC, were topically applied to the dorsal skin of both strains. All of these metabolites produced adducts. Approximately 14 different adduct spots were detected. The two most abundant adducts were produced by 1-hydroxy-, 2-hydroxy-, and 9,10-dihydrodiol-MC. One of these also contained a 3-hydroxymethyl group. Several adducts did not contain the 9,10-dihydroxy group. The adducts derived from 3-methoxymethyl-MC were consistently found in greater abundance in DNA from C57BL/6 tissues, compared with DBA/2. Thus, oxidation of the 3-methyl group may be enhanced by Ah-dependent induction of MC metabolism. Together, these results suggest that the individual and total adduct levels are influenced by the genotype at the Ah locus, the route of administration, and the metabolite(s) with tissue and age specificity.

Metabolic activation of benzo[g]chrysene in the human mammary carcinoma cell line MCF-7.

Benzo[g]chrysene (BgC) is an environmental pollutant, and recent studies have demonstrated that anti- BgC-11,12-dihydrodiol 13,14-epoxide (anti-BgCDE) is a potent mammary carcinogen in rats. To determine whether BgC can be metabolically activated to anti-BgCDE in human cells, the human mammary carcinoma cell line MCF-7 was treated with BgC and with the racemic trans-3,4- and 11,12-dihydrodiols. The DNA adducts formed in these experiments were examined using 32P-postlabeling, and specific adducts were identified through comparisons with adducts obtained by the reaction of the racemic syn- and anti-BgCDEs with calf thymus DNA and with purine deoxyribonucleoside-3'-phosphates in vitro. It was found that BgC is metabolically activated in MCF-7 cells to form major DNA adducts through both the syn- and anti-11,12-dihydrodiol 13,14-epoxide metabolites. BgC is therefore a potential environmental risk to humans. The major BgC-DNA adducts formed from both the dihydrodiol-epoxide diastereomers were deoxyadenosine adducts. Thus, BgC has DNA-binding properties that are very similar to those of the potent mammary carcinogens 7,12-dimethylbenz[a]anthracene and dibenzo[a,l]pyrene.

Potent tumor-initiating activity of the 3,4-dihydrodiol of 7,12-dimethylbenz(a)anthracene in mouse skin.

The abilities of the racemic trans-3,4-, 5,6-, and 8,9-dihydrodiols of 7,12-dimethylbenz(a)anthracene to initiate skin tumors in mice were determined by using a two-stage system of tumorigenesis. The 7,12-dimethylbenz(a)anthracene trans-3,4-dihydrodiol was found to be much more active as a tumor initiator than the parent hydrocarbon. The 7,12-dimethylbenz(a)anthracene trans-5,6- and 8,9-dihydrodiols were essentially inactive as skin tumor initiators. Our results suggest that the 3,4-dihydrodiol of 7,12-dimethylbenz(a)anthracene is a proximal carcinogen and that the "bay region" diol-epoxide may be the ultimate carcinogenic form of DMBA.

Influence of the alkyl substituent on mutagenicity and covalent DNA binding of bay region diol-epoxides of 7-methyl- and 7-ethylbenz(a)anthracene in Salmonella and V79 Chinese hamster cells.

The anti-isomers of the bay region diol-epoxides of the strong carcinogen 7-methylbenz(a)anthracene and of the weak carcinogen 7-ethylbenz(a)anthracene were investigated for mutagenicity in Salmonella typhimurium (reversion of the his - strains TA98 and TA100 to prototrophy) and V79 Chinese hamster cells (acquisition of resistance to 6-thioguanine and ouabain; formation of micronuclei). In addition, in the V79 cells, the levels of the DNA adducts formed were determined by 32P-postlabeling analysis. In terms of mutations per nmol compound administered, the methyl derivative was four to 10 times more potent, depending on the genetic endpoint, than its ethyl congener. However, when the results were expressed as mutations per adduct, the difference between the two diol-epoxides was small. Therefore, a higher level of DNA modification appears to be the major reason for the stronger mutagenicity of the methyl derivative. However, both diol-epoxides had similar half-lives (about 9 min) in physiological buffer, as determined from the decline in mutagenic activity after preincubation of the test compound. These results suggest that the effect of the 7-alkyl group on the extent of reaction with DNA is more a result of steric factors than of a change in the intrinsic chemical reactivity of the diol-epoxides.

Reactivity with DNA bases and mutagenicity toward Salmonella typhimurium of methylchrysene diol epoxide enantiomers.

The reactions with DNA and mutagenic activities toward Salmonella typhimurium TA 100 of the R,S,S,R and S,R,R,S enantiomers of anti-1,2,-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (anti-5-MeC-1,2-diol-3,4-epoxide), anti-5-MeC-7,8-diol-9,10-epoxide, and anti-6-MeC-1,2-diol-3,4-epoxide were compared because among these compounds only the R,S,S,R enantiomer of anti-5-MeC-1,2-diol-3,4-epoxide is highly tumorigenic. The major products formed in the reaction of each racemic diol epoxide with DNA were two pairs of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts; one product in each pair was formed from the R,S,S,R enantiomer and the other from the S,R,R,S enantiomer of each racemic diol epoxide. Formation of products from R,S,S,R enantiomers exceeded formation of those from S,R,R,S enantiomers in each case. Among the R,S,S,R enantiomers, 5-MeC-1,2-diol-3,4-epoxide, which has a methyl group in the same bay region as the epoxide ring, was most reactive toward DNA, and in particular toward dGuo. The dGuo/dAdo adduct ratios were greater for the products formed from the R,S,S,R enantiomer compared to the S,R,R,S enantiomer of each diol epoxide. The dGuo/dAdo adduct ratios were also greater for the enantiomers of anti-5-MeC-1,2-diol-3,4-epoxide than for the enantiomers of either anti-5-MeC-7,8-diol-9,10-epoxide or anti-6-MeC-1,2-diol-3,4-epoxide. In S. typhimurium TA 100, the R,S,S,R enantiomer of anti-5-MeC-1,2-diol-3,4-epoxide was the most mutagenic compound (6700 revertants/nmol), followed by the R,S,S,R enantiomer of anti-5-MeC-7,8-diol-9,10-epoxide (1500 revertants/nmol). The other diol epoxide enantiomers were weakly active or inactive at the doses tested. The results of this study demonstrate that both the absolute configuration of a diol epoxide and the position of the methyl group have major effects on its reactivity with DNA. The greatest reactivity is seen in an R,S,S,R enantiomer with the methyl group and epoxide ring in the same bay region, e.g., the highly tumorigenic and mutagenic 5-MeC-1R,2S-diol-3S,4R-epoxide. Comparison of the dGuo/dAdo adduct ratios of the various diol epoxides with their tumorigenic and mutagenic activities suggests that dGuo adducts are important in the expression of biological activity of methylchrysene diol epoxides.

Carcinogenicity and mutagenicity of benz(a)anthracene diols and diol-epoxides.

Benz(a)anthracene (BA) and its five possible trans-dihydrodiols were evaluated for determination of their skin tumor-initiating activity and their mutagenic activity in Chinese hamster V79 cells. In addition, the skin tumor-initiating abilities of five diol-epoxides of BA were tested. Results showed (+/-)-trans-3,4-dihydroxy-3,4-dihydrobenz(a)anthracene (BA 3,4-dihydrodiol) to be approximately 10 times more mutagenic than was BA and about 20 times more mutagenic than were the other possible dihydrodiols in the V79 cells cocultivated with irradiated hamster embryo cells. As a skin tumor initiator, BA 3,4-dihydrodiol was approximately 5 times more active than BA, whereas the other BA dihydrodiols were all less active tumor initiators. (+/-)-trans-3alpha,4beta-Dihydroxy-1alpha,2alpha-epoxy-1,2,3,4-tetrahydrobenz(a)anthracene was found to be approximately 20% more active as a tumor initiator than was BA 3,4-dihydrodiol, whereas the other diol-epoxides of BA were less active than BA itself. The results suggest that the bay-region diol-epoxide of BA may be the ultimate carcinogen and mutagenic form of BA.

High stereoselectivity in mouse skin metabolic activation of methylchrysenes to tumorigenic dihydrodiols.

The stereoselectivity of mouse skin metabolic activation to dihydrodiols of the strong carcinogen 5-methylchrysene (5-MeC) and the weak carcinogen 6-methylchrysene (6-MeC) was investigated. Synthetic 1,2-dihydro-1,2-dihydroxy-5-methylchrysene (5-MeC-1,2-diol), 5-MeC-7,8-diol, and 6-MeC-1,2-diol were resolved into their R,R- and S,S-enantiomers by chiral stationary phase high performance liquid chromatography. The absolute configurations of the enantiomers were assigned by their circular dichroism spectra. Using these enantiomers as standards, the metabolism of 5-MeC and 6-MeC in vitro in rat and mouse liver and in vivo in mouse epidermis was investigated. Only the R,R-enantiomers of each dihydrodiol predominated (greater than 90%). The dihydrodiol enantiomers were tested for tumor initiating activity on mouse skin. In each case, the R,R-dihydrodiol enantiomer was significantly more tumorigenic than the S,S-enantiomer. The most tumorigenic compound was 5-MeC-1R,2R-diol; it was significantly more active than either 5-MeC-7R,8R-diol or 6-MeC-1R,2R-diol. The results of this study demonstrate that there is a high degree of stereoselectivity in the metabolic activation of 5-MeC and 6-MeC to proximate tumorigenic dihydrodiols in mouse skin. The bay region methyl group has no effect on the stereoselectivity of activation to 1,2-dihydrodiol metabolites in the chrysene system.

Tumorigenicity of 5-methylchrysene dihydrodiols and dihydrodiol epoxides in newborn mice and on mouse skin.

5-Methylchrysene, (+/-)-trans-1,2-dihydro-1,2-dihydroxy-5-methylchrysene, (+/-)-trans-7,8-dihydro-7,8-dihydroxy-5-methylchrysene, (+/-)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (anti-DE-I), (+/-)-trans-1,2-dihydroxy-syn-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (syn-DE-I), and (+/-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene (anti-DE-II) were tested for tumorigenicity in newborn mice and for tumor-initiating activity on mouse skin. In newborn mice, a total dose of 56 nmol of anti-DE-I induced 4.6 lung tumors/mouse and 1.2 liver tumors/mouse. These incidences were significantly higher than observed for any of the other metabolites, tested at equimolar doses. The results indicate that anti-DE-I, but not syn-DE-I or anti-DE-II, is a major ultimate carcinogen of 5-methylchrysene in the newborn mouse. Anti-DE-I was also more tumorigenic than anti-DE-II on mouse skin, inducing 4.4 tumors/mouse after an initiating dose of 100 nmol, compared to zero tumors per mouse induced by anti-DE-II. However, anti-DE-I was less tumorigenic on mouse skin than was its metabolic precursor, trans-1,2-dihydro-1,2-dihydroxy-5-methylchrysene or its parent hydrocarbon, 5-methylchrysene.

Rates of hydrolysis and extents of DNA binding of 5-methylchrysene dihydrodiol epoxides.

The rates of hydrolysis in the absence and presence of native and denatured DNA, and the extents of DNA binding of five dihydrodiol epoxides derived from 5-methylchrysene (5-MeC) and chrysene have been determined. The compounds studied were: trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-MeC; trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-Mec; trans-1,2-dihydroxy-syn-3,4-epoxy-1,2,3,4-tetrahydro-5-MeC; trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydro-5-MeC; and trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene. In the absence of DNA, at pH 7 and 37 degrees C half-lives of trans-1,2-dihydroxy-syn-3,4-epoxy-1,2,3,4-tetrahydro-5-MeC and trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-MeC were similar, t 1/2 = 62 and 59 min, while trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydro-5-MeC hydrolyzed faster than trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-MeC, t 1/2 = 5.4 versus 17.5 min; trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene had the slowest rate of hydrolysis, t 1/2 = 104 min. Studies of the effects of native and denatured DNA on the rates of hydrolysis of the dihydrodiol epoxides indicated that native DNA remarkably accelerated these rates for all dihydrodiol epoxides, but the degree of acceleration varied for the different dihydrodiol epoxides. The acceleration of hydrolytic rates by native DNA relative to that by denatured DNA was correlated with the covalent binding of these dihydrodiol epoxides with DNA in vitro. The catalytic effect of DNA in enhancing the rates of hydrolysis of dihydrodiol epoxides and the relative extents of covalent binding of the dihydrodiol epoxides to DNA were in the following order: trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-MeC greater than trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-MeC greater than trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene greater than trans-1,2-dihydroxy-syn-3,4-epoxy-1,2,3,4-tetrahydro-5-MeC greater than trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydro-5-MeC. The results of this study suggest that physical interactions with DNA are important in determining the relative extents of binding of these dihydrodiol epoxides to DNA in vitro.

Both (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides initiate tumors in mouse skin that possess -CAA- to -CTA- mutations at Codon 61 of c-H-ras.

We have determined the tumor-initiating activity of (+/-)syn- and (+/-)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (syn- and anti-DMBADE), the two metabolically formed bay-region diol epoxides of DMBA, and we have also analyzed mutations in the H-ras gene from tumors induced by these compounds. Using a two-stage, initiation-promotion protocol for tumorigenesis in mouse skin, we have found that both syn- and anti-DMBADE are active tumor initiators, and that the occurrence of papillomas is carcinogen dose dependent. All of the papillomas induced by syn-DMBADE (a total of 40 mice), 96% of those induced by anti-DMBADE (a total of 25 mice), and 94% of those induced by DMBA (a total of 16 mice) possessed a -CAA- to -CTA- mutation at codon 61 of H-ras. No mutations in codons 12 or 13 were detected in any tumor. Topical application of syn- and anti-DMBADE produced stable adducts in mouse epidermal DNA, most of which comigrated with stable DNA adducts formed after topical application of DMBA. Further analysis of the data showed that levels of the major syn- and anti-DMBADE-deoxyadenosine adducts formed after topical application of DMBA are sufficient to account for the tumor-initiating activity of this carcinogen on mouse skin. Previously, we showed that both the syn- and anti-DMBADE bind to the adenine (A182) at codon 61 of H-ras. Collectively, these results indicate that the adenine adducts induced by both bay-region diol epoxides of DMBA lead to the mutation at codon 61 of H-ras and, consequently, initiate tumorigenesis in mouse skin.

Synthesis of the tumorigenic 3,4-dihydrodiol metabolites of dibenz[a,j]anthracene and 7,14-dimethyldibenz[a,j]anthracene.

Syntheses are described of the trans-3,4-dihydrodiol derivatives (2a and 2b) of dibenz[a,j]anthracene and 7,14-dimethyldibenz[a,j]anthracene (1a and 1b), implicated as their proximate carcinogenic metabolites. Conversion of 2a to the bay region anti-diol epoxide derivative 3a, its putative ultimate carcinogenic metabolite, is also reported. The related diol epoxide derivative of 2b could not be prepared due to its chemical instability. Tumorigenicity assays confirm that 1b and 2b are potent carcinogens on mouse skin, while 1a and 2a are only relatively weakly active. The diol epoxide 3a exhibited significantly higher tumorigenicity than its dihydrodiol precursor 2a. These findings are consistent with the hypothesis that the bay region diol epoxide metabolites are the active carcinogenic forms of these hydrocarbons. They also support the generalization that methyl substitution in bay regions enhances the carcinogenic activity of polycyclic aromatic hydrocarbons.

Biologically active dihydrodiol metabolites of polycyclic aromatic hydrocarbons structurally related to the potent carcinogenic hydrocarbon 7,12-dimethylbenz[a]anthracene.

Syntheses of the trans-dihydrodiol derivatives implicated as the proximate carcinogenic metabolites of the polycyclic hydrocarbons cholanthrene, 6-methylcholanthrene, benz[a]anthracene, and 7- and 12-methylbenz[a]anthracene are described. These compounds are useful models for research to determine the molecular basis of the strong enhancement of carcinogenicity consequent upon methyl substitution in nonbenzo bay molecular sites and meso regions of polycyclic hydrocarbons. Synthesis of the bay region anti-diol epoxide derivative of cholanthrene, its putative ultimate carcinogenic metabolite, is also described. Tumorigenicity assays indicate that the 9,10-dihydrodiol derivatives of cholanthrene and its 3- and 6-methyl derivatives are all potent tumor initiators on mouse skin. The most active member of the series is the dihydrodiol derivative of 6-methylcholanthrene, which contains a bay region methyl group. The ability of the dihydrodiols 3a-c and the trans-3,4-dihydrodiol of 7,12-dimethylbenz[a]anthracene (3d) to induce chromosomal aberrations in rat bone marrow cells was also examined. The observed order of activity was 3d greater than 3c greater than 3b greater than 3a. These findings are consistent with the hypothesis that the diol epoxide metabolites of these dihydrodiols are the active carcinogenic forms of the parent hydrocarbons.