Bay Region Dihydrodiol Epoxide Research Articles

Microsomal Biotransformation of Benzo[ghi]perylene, a Mutagenic Polycyclic Aromatic Hydrocarbon without a “Classic” Bay Region

Carcinogenic polycyclic aromatic hydrocarbons (PAH), e.g., benzo[a]pyrene (BaP), possess a bay region comprising an ortho-fused benzene ring. Benzo[ghi]perylene (BghiP) represents the group of PAHs lacking such a "classic" bay region and hence cannot be metabolically converted like BaP to bay region dihydrodiol epoxides considered as ultimate mutagenic and carcinogenic metabolites of PAH. BghiP exhibits bacterial mutagenicity in strains TA98 (1.3 his(+)-revertant colonies/nmol) and TA100 (4.3 his(+)-revertant colonies/nmol) of Salmonella typhimurium after metabolic activation by the postmitochondrial hepatic fraction of CD rats treated with 3-methylcholanthrene. Inhibition of microsomal epoxide hydrolase (mEH) with 1,1,1-trichloro-2-propene oxide raised the bacterial mutagenicity of BghiP in TA98 almost 4-fold indicating arene oxides as ultimate mutagens. To confirm this assumption, the biotransformation of BghiP was elucidated. Incubation of BghiP with liver microsomes of CD rats treated with Aroclor 1254 yielded 17 ethyl acetate extractable metabolic products. Twelve metabolites were identified by a combination of chromatographic, spectroscopic, and biochemical methods. The microsomal biotransformation of BghiP proceeds by two pathways: Pathway I starts with the monooxygenase attack at the 7-position leading to the 7-phenol, which is transformed to the 7,8- and 7,10-diphenols followed by oxidation to the 7,8- and 7,10-quinones. On pathway II, the K regions of BghiP are successively converted to arene oxides yielding the indirectly identified 3,4-oxide and the 3,4,11,12-bisoxides. Enzymatic hydrolysis of the 3,4-oxide leads to the trans-3,4-dihydrodiol, which is oxidized to the 3,4-quinone. Similarly, the trans-3,4-trans-11,12-bisdihydrodiols and the trans-3,4-dihydrodiol 11,12-quinone are generated from the 3,4,11,12-bisoxides. The trans-3,4-dihydrodiol and the trans-3,4-trans-11,12-bisdihydrodiols are preferentially formed as R,R and R,R,R,R enantiomers, respectively. The intrinsic bacterial mutagenicity of the 3,4,11,12-bisoxides is rather low and hardly explains the strong increase in bacterial mutagenicity of BghiP after inhibition of mEH. Thus, we believe that the 3,4-oxide plays a more important role as the ultimate mutagenic metabolite of BghiP.

Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: A review

Polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic compounds (HACs) constitute a major class of chemical carcinogens present in the environment. These compounds require activation to electrophilic metabolites to exert their mutagenic or carcinogenic effects. There are three principal pathways currently proposed for metabolic activation of PAH and HAC: the pathway via bay region dihydrodiol epoxide by cytochrome P450 enzymes (CYPs), the pathway via radical cation by one-electron oxidation, and the ortho-quinone pathway by dihydrodiol dehydrogenase (DD). In addition to these major pathways, a brief description of a minor metabolic activation pathway, sulfonation, for PAHs that contain a primary benzylic alcoholic group or secondary hydroxyl group(s) is included in this review. The DNA damages caused through the reactive metabolites of PAH/HAC are described involving the DNA covalent binding to form stable or depurinating adducts, the formation of apurinic sites, and the oxidative damage. The review emphasizes the chemical/biochemical reactions involved in the metabolic processes and the chemical structures of metabolites and DNA adducts.

Mutational spectra for polycyclic aromatic hydrocarbons in the supF target gene

An SV40-based shuttle vector system was used to identify the types of mutational changes and the sites of mutation within the supF DNA sequence generated by the four stereoisomers of benzo[ c]phenanthrene 3,4-dihydrodiol 1,2-epoxide (B[ c]PhDE), by racemic mixtures of bay or fjord region dihydrodiol epoxides (DE) of 5-methylchrysene, of 5,6-dimethylchrysene, of benzo[ g]chrysene and of 7-methylbenz[ a]anthracene and by two direct acting polycyclic aromatic hydrocarbon carcinogens, 7-bromomethylbenz[ a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[ a]anthracene (7-BrMe-12-MeBA). The results of these studies demonstrated that the predominant type of mutation induced by these compounds is the base substitution. The chemical preference for reaction at deoxyadenosine (dAdo) or deoxyguanosine (dGuo) residues in DNA, which is in general correlated with the spatial structure (planar or non-planar) of the reactive polycyclic aromatic hydrocarbon, is reflected in the preference for mutation at A⋅T or G⋅C pairs. In addition, if the ability to react with DNA in vivo is taken into account, the relative mutagenic potencies of the B[ c]PhDE stereoisomers are consistent with the higher tumorigenic activity associated with non-planar polycyclic aromatic hydrocarbons and their extensive reaction with dAdo residues in DNA. Comparison of the types of mutations generated by polycyclic aromatic hydrocarbons and other bulky carcinogens in this shuttle vector system suggests that all bulky lesions may be processed by a similar mechanism related to that involved in replication past apurinic sites. However, inspection of the distribution of mutations over the target gene induced by the different compounds demonstrated that individual polycyclic aromatic hydrocarbons induce unique patterns of mutational hotspots within the target gene. A polymerase arrest assay was used to determine the sequence specificity of the interaction of reactive polycyclic aromatic hydrocarbons with the shuttle vector DNA. The results of these assays revealed a divergence between mutational hotspots and polymerase arrest sites for all compounds investigated, i.e., sites of mutational hotspots do not correspond to sites where high levels of adduct formation occur, and suggested that some association between specific adducts and sequence context may be required to constitute a premutagenic lesion. A site-specific mutagenesis system employing a single-stranded vector (M13mp7L2) was used to investigate the mutational events a single benzo[ a]pyrene or benzo[ c]phenanthrene dihydrodiol epoxide–DNA adduct elicits within specific sequence contexts. These studies showed that sequence context can cause striking differences in mutagenic frequencies for given adducts. In addition, these sequence context effects do not originate only from nucleotides immediately adjacent to the adduct, but are also modulated by more distal nucleotides. The implications of these results for mechanisms of polycyclic aromatic hydrocarbon-induced mutagenesis and carcinogenesis are discussed.

Metabolic Activation of 4H-Cyclopenta[def]chrysene in Human Mammary Carcinoma MCF-7 Cell Cultures

The tumor initiating activities of 4H-cyclopenta[def]chrysene (C[def]C) and its two putative reactive metabolites, trans-1, 2-dihydroxy-anti-3,3a-epoxy-1,2,3, 3a-tetrahydro-4H-cyclopenta[def]chrysene (C[def]C-3,3a-DE) and trans-6,7-dihydroxy-anti-8,9-epoxy-6,7,8, 9-tetrahydro-4H-cyclopenta[def]chrysene (C[def]C-8,9-DE), were evaluated previously in mice [Amin, S., et al. (1995) Carcinogenesis 16, 2813-2817]. C[def]C-3,3a-DE was the more active inducer of lung tumors and elicited twice as many tumors as C[def]C-8,9-DE. In this study, the route of metabolism of C[def]C to DNA-reactive metabolites in the human mammary carcinoma cell line (MCF-7) was investigated using the 32P-postlabeling assay. The results show that metabolic activation to DNA-binding species proceeds through the formation of both trans-1,2-dihydrodiol and trans-6,7-dihydrodiol metabolites of C[def]C. At a 1 microM dose, adducts from the methylene-bridged (C[def]C-3,3a-DE) and bay region (C[def]C-8,9-DE) dihydrodiol epoxides were detected in comparable amounts. In contrast, the majority of the postlabeled adducts recovered from cells exposed to a 10 microM dose were derived from the bay region dihydrodiol epoxide, C[def]C-8,9-DE. Using markers from reactions of the dihydrodiol epoxides with deoxyguanosine 3'-phosphate and deoxyadenosine 3'-phosphate, it was shown that the major radioactive spots formed with both anti-C[def]C-3,3a-DE and anti-C[def]C-8,9-DE chromatographed with deoxyguanosine adduct markers. Thus, the human cells used in these studies can activate C[def]C to carcinogenic metabolites.

Cytochrome P450-mediated activation of phenanthrene in genetically engineered V79 Chinese hamster cells

V79 Chinese hamster cells genetically engineered for rat cytochromes P450 1A1, 1A2, 2B1 and human cytochromes P450 1A1, 1A2, 2A6, 2E1, and 3A4 are being applied in metabolism studies on polycyclic aromatic hydrocarbons. This study presents the results on phenanthrene as the prototypic polycyclic aromatic hydrocarbon possessing a bay region. Phenanthrene is of less importance regarding cytotoxicity and carcinogenicity as compared to e.g. benzo[ a]pyrene or 7,12-dimethylbenz[ a]anthracene. However, phenanthrene is more readily converted to metabolites which are exreted in higher amounts than those from any other polycyclic aromatic hydrocarbon. Therefore, its metabolites are of diagnostic value in epidemiological and occupational exposure studies. For this reason, it is worthwhile to understand the metabolism of phenanthrene in detail, e.g. allocating metabolites and cytochromes P450s. In accordance to previous observations cytochromes P450 1A1 and 1A2 were the most active forms towards phenanthrene. However, metabolite profiles differed between rat and human homologues of cytochromes P450, in particular for cytochrome P450 1A2. The predominant metabolite formed by rat cytochrome P450 1A2 was the K region trans-9,10-dihydrodiol, whereas human cytochrome P450 1A2 produced similar amounts of the trans-1,2-, trans-3,4- and trans-9,10-dihydrodiol. High amounts of trans-1,2-dihydrodiol, the metabolic precursor of the bay-region dihydrodiol epoxide, were also formed by human cytochrome P450 1A1 compared to its rat homologue. Unexpectedly, human cytochrome P450 2E1 showed a remarkable catalytic activity to metabolize phenanthrene to its trans-9,10-dihydrodiol. Utilizing recombinant CYPs in live V79 cells appears to be a valuable too yielding results important for the evaluation of exposure data and risk assessment for humans.

Polycyclic Aromatic Hydrocarbons: Chemistry of DNA Adduct Formation

Polycyclic aromatic hydrocarbon carcinogens are usually activated for DNA binding by the metabolic formation of bay region dihydrodiol epoxides with R,S,S,R stereochemistry. Such metabolites from planar hydrocarbons reacted preferentially with the amino groups of deoxyguanosine residues, whereas those from nonplanar hydrocarbons were more efficiently trapped by DNA and reacted preferentially with the amino groups of deoxyadenosine residues, in some cases. Molecular modeling and NMR measurements indicated that the conformations of DNA adducts depend upon the hydrocarbon involved and the cis or trans opening of the epoxide ring during adduct formation. The structural characterization of carcinogen-DNA adducts and investigations of relationships between specific adducts and biological effects represent an important background that can be valuable in molecular epidemiological approaches.

Detection of the major DNA adducts of benzo[b]fluoranthene in mouse skin: role of phenolic dihydrodiols.

The primary mechanism of activation of benzo[b]fluoranthene (B[b]F) to a genotoxic agent does not involve the bay region dihydrodiol epoxide trans-9,10-dihydroxy-anti- 11,12-epoxy-9,10,11,12-tetrahydrobenzo[b]fluoranthene (B[b]F-9,10-DE). Previous results have suggested that the major DNA adduct(s) formed in vivo with B[b]F are more polar than those formed from B[b]F-9,10-DE. Employing newly-developed synthetic methods for the formation of B[b]F derivatives, trans-9,10-dihydro-5,9,10-trihydroxybenzo[b]fluoranthene (5-OH-B[b]F-9,10-diol) and trans-9,10-dihydro-6,9,10-trihydroxybenzo[b]fluoranthene (6-OH-B[b]F-9,10-diol) were synthesized and evaluated for tumor-initiating activity at initiation doses of 0.04, 0.12, and 0.4 mumol per mouse. In contrast to previous bioassay results, both of these phenolic dihydrodiols exhibited tumor-initiating activity. 5-OH-B[b]F-9,10-diol was the more potent tumor initiator, inducing a 90% incidence of tumor-bearing mice with an average of 7.50 tumors per mouse at a total initiator dose of 0.4 mumol. 6-OH-B[b]F-9,10-diol was significantly less active as a tumor initiator in this bioassay, inducing a 50% incidence of tumor-bearing mice with an average of 0.60 tumor per mouse at a total dose of 0.4 mumol. 32P-Postlabeling analysis was employed to evaluate the DNA adducts formed in vivo in mouse skin from both 5- and 6-OH-B[b]F-9,10-diol. HPLC analysis of B[b]F-DNA adducts formed in mouse skin demonstrated the presence of a single major adduct together with four additional minor adducts. The DNA adducts formed with 5-OH-B[b]F-9,10-diol had identical HPLC retention to the major and one of the minor adducts observed for B[b]F. These two DNA adducts account for 58% of the modified nucleotides detected in mouse skin following the topical application of B[b]F. Methods for the preparation of trans-9,10-dihydroxy-anti-11,12-epoxy-5-hydroxy-9,10,11,12- tetrahydrobenzo[b]fluoranthene (5-OH-B[b]F-9,10-DE) were developed. The DNA adducts formed with 5-OH-B[b]F-9,10-DE were similar to the adducts formed with 5-OH-B[b]F-9,10-diol. The results of the DNA binding studies along with the tumor-initiating data indicate that 5-OH-B[b]F-9,10-diol is the major proximate tumorigenic metabolite of B[b]F on mouse skin. Studies performed with deoxyguanosine 3'-phosphate suggest that the principal adducts formed with anti-5-OH-B[b]F-9,10-DE are the result of the interaction with deoxyguanosine.

Mutagenic activity of the 4,5‐ and 9,10‐dihydrodiols of benzo[J]fluoranthene and their syn‐ and anti‐dihydrodiol epoxides in salmonella typhimurium

The objective of this study was to determine the relative mutagenic activities of the major dihydrodiol metabolites of benzo[j]fluoranthene (B[j]F) and their corresponding syn- and anti-dihydrodiol epoxides. Salmonella typhimurium tester strains TA97a, TA98, and TA100 were used to evaluate the mutagenic potencies of the parent hydrocarbon and these suspect proximate and ultimate mutagenic metabolites. B[j]F and the trans-dihydrodiol metabolites were active only in the presence of an external metabolic activation system (S9) with the exception of the B[j]F-4,5-diol, which was weakly active in TA98 and TA100 in the absence of S9. The B[j]F-4,5-diol was more mutagenic than the B[j]F-9,10-diol in tester strains TA98 and TA100, whereas the opposite effect was observed in TA97a. In the absence of S9, the anti-B[j]F-4,5-diol epoxide was more mutagenic than the syn-B[j]F-4,5-diol epoxide and the syn- and anti-B[j]F-9,10-diol epoxides in tester strains TA97a and TA100. The exceptional mutagenic potency of the anti-B[j]F-4,5-diol epoxide in TA100 resembles that observed by epoxides located within a fjord, or by the anti-diol epoxides of bay region methylated polycyclic aromatic hydrocarbons. In contrast, the mutagenicity of the pseudo bay region dihydrodiol epoxides arising from the B[j]F-9,10-diol more closely resembles that observed with the classical bay region dihydrodiol epoxides of chrysene. In summary, both dihydrodiol metabolites of B[j]F are mutagenic in S. typhimurium, and the relative potency varies among the tester strains. The highest mutagenic response was achieved in tester strain TA100, which detects base-pair substitutions. The most potent direct-acting dihydrodiol epoxide in this tester strain was the anti-B[j]F-4,5-diol epoxide, which agrees with the results of mouse skin painting studies that indicate that the B[j]F-4,5-diol is more tumorigenic that the parent hydrocarbon or the B[j]F-9,10-diol. A covalent DNA adduct formed between the anti-B[j]F-4,5-diol epoxide and deoxyguanosine was the major species of DNA adduct formed in S. typhimurium. This adduct corresponds to the major DNA adduct formed in mouse skin following application B[j]F.

Thermodynamic Properties of the Arene Epoxides and the Relative Carcinogenicities of Benzo[a]pyrene and Benzo[e]pyrene

Abstract The thermodynamic stabilities of all possible arene oxides of benzo[a]pyrene and benzo[e]pyrene, and of the two parent compounds, have been obtained using semiempirical AM1 and molecular mechanics calculations. The calculations predict that all K-region epoxides are highly stabilized compared to other epoxides for both PAHs. The terminal ring epoxides include proximal and distal types, either close to or remote from the bay-region, respectively. The distal epoxides correspond to the necessary first intermediates in the enzymatic activation process leading to the ultimate bay-region dihydrodiol epoxide carcinogens. There are marked differences in the calculated heats of formation of the distal benzo[a]pyrene-7,8-epoxide and benzo[e]pyrene-9,10-epoxide (compared to either the respective parent PAHs or the K-region epoxides) favoring the epoxide from benzo[a]pyrene. One can propose that these thermodynamic differences may be a principal factor responsible for facile initiation of the activating meta...

7-Suffooxymethyl-12-methylbenz[a]anthracene is an electrophilic mutagen, but does not appear to play a role in carcinogenesis by 7,12-dimethylbenz[a]anthracene or 7-hydroxymethyl-12-methylbenz[a]anthracene

Although a bay-region dihydrodiolepoxide metabolite has been considered as a principal ultimate electrophilic and carcinogenic form of 7,12-dimethylbenz[a]anthracene (DMBA), other reactive metabolites might also play a role in the activation of this hydrocarbon in vivo. Earlier studies suggested the hydroxylation of a meso-anthracenic methyl group with subsequent formation of a benzylic ester bearing a good leaving group (e.g. sulfate) as a metabolic activation pathway for DMBA. In support of this hypothesis, the formation of an electrophilic and mutagenic sulfuric acid ester of 7-hydroxymethyl-12-methylbenz[a]anthracene (HMBA) by rat liver cytosolic sulfotransferase activity has previously been demonstrated, but no data have been reported on the carcinogenicity of this reactive ester. In the present study, we compared the carcinogenicity of chemically synthesized sodium 7-sulfooxymethyl-12-methylbenz[a]anthracene (SMBA) with that of the parent methyl and hydroxymethyl hydrocarbons. For this purpose, tests were made in several animal tumor models: induction of hepatomas in male B6C3F1 mice, lung adenoma induction in A/J mice, initiation of mouse skin tumors, development of sarcomas in rats at the injection sites, and initiation of preneoplastic enzyme-altered foci in rat liver. Data from all of these studies indicate that SMBA is not more carcinogenic than DMBA or HMBA. In addition, the carcinogenic activity of HMBA was not altered by dehydroepiandrosterone, a strong inhibitor of sulfotransferase activity for HMBA. DMBA produced only a low level of hepatic benzylic DNA adducts in rats when a relatively high dose was administered. These adducts constitute less than 5% of total DMBA residues bound to hepatic DNA. The rest of the adducts appear to be associated with other electrophilic intermediates including the dihydrodiol epoxide metabolites. Based on the results of our present study, it is unlikely that DMBA exerts its carcinogenic activity via metabolic activation to SMBA.

Formation and removal of dibenzo[a,e]fluoranthene-DNA adducts in mouse embryo fibroblasts

In vivo binding of dibenzo[a,e]fluoranthene (DBF) to mouse embryo fibroblast DNA was compared with that observed previously in vitro on calf thymus DNA incubated with mouse liver microsomes. The h.p.l.c. elution patterns of the adducts formed by DBF metabolites with DNA and obtained in vivo at the optimal exposure time of 42-48 h were qualitatively very similar to the patterns obtained in vitro, but their amplitude was quantitatively reduced. There are two striking differences between the in vivo and in vitro results. Firstly, the most polar peak A, very abundant in vitro, was absent in vivo. Secondly, the reactivity of the two major proximate metabolites of DBF, the bay and pseudo-bay region dihydrodiols, was very different in intact cells compared with the results in vitro. When incubated in vitro, pseudo-bay region dihydrodiol DBF was twice as reactive as bay region dihydrodiol DBF. The opposite reactivities were observed in vivo. The major DBF-DNA adducts formed in vivo were collected in the peaks E, B and C. The predominant peak E contained DNA adducts of both bay and pseudo-bay region dihydrodiolepoxides which are the major ultimate metabolites of DBF in vivo and in vitro. The other two prominent peaks B and C contained DNA adducts of 3-hydroxy DBF pseudo-bay region dihydrodiolepoxide and the 7-hydroxy DBF bay region dihydrodiolepoxide, respectively. After adduct formation, post incubation of fibroblasts for a further 48 h, in the absence of DBF, eliminated half the amount of adducts present. Peak B adducts were repaired more efficiently than those of peaks E, C D and F. The carcinogenic initiating activity of DBF appears to be a complex process in which several DNA adducts play a role.

7,12-Dimethylbenz[ a]anthracene-DNA adducts in cultured cells from mouse fetuses of different gestational ages

Primary cell cultures prepared from individual litters of NIH Swiss mouse fetuses of different gestational ages were incubated with 7,12-[ 3H]dimethylbenz[ a]anthracene (DMBA) for 24 h. Levels of binding of DMBA to DNA and the distribution of individual DMBA-deoxyribonucleoside adducts were similar in all cultures derived from fetuses of 13–15 days of gestation. However, in cells from fetuses at 17–19 days changes in DMBA-DNA binding were noted. In particular the syn bay region dihydrodiol epoxide of DMBA was responsible for a significantly greater fraction of the total DMBA-DNA binding in the cultures from the more mature fetuses.

Effects of butylated hydroxyanisole and butylated hydroxytoluene on 7,12-dimethylbenz[ a]anthracene—DNA adduct formation in mouse embryo cell cultures

Neither butylated hydroxyanisole (BHA) nor butylated hydroxytoluene (BHT) significantly reduced overall 7,12-dimethylbenz[ a]anthracene (DMBA)—DNA adduct formation in mouse embryo cell cultures. However, analysis of DMBA—DNA adducts by Servacel DHB chromatography and high-pressure liquid chromatography showed that treatment of cells with BHA, but not with BHT, resulted in a decreased contribution from the syn bay region dihydrodiol epoxide to overall binding.

5-Methylchrysene metabolism in mouse epidermis in vivo, diol epoxide—DNA adduct persistence, and diol epoxide reactivity with DNA as potential factors influencing the predominance of 5-methylchrysene-1,2-diol-3,4-epoxide—DNA adducts in mouse epidermis

5-Methylchrysene (5-MeC) can form two bay region dihydrodiol epoxides: 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I) which has the methyl group and the epoxide ring in the same bay region, and 7,8-dihydroxy-9,10-epoxy-7,8,10-tetrahydro-5-methylchrysene (DE-II). In a previous study, we observed that the ratio of DE-I:DNA adducts to DE-II:DNA adducts in mouse epidermis, 24 h after application of [3H]5-MeC metabolites was 2.7 to 1. To investigate the basis for this observation we have now studies: (i) the formation of [3H]5-MeC in mouse epidermis in vivo at various time intervals from 0.33 to 24 h; (ii) the persistence of DE-I:DNA adducts and DE-II:DNA adducts in mouse epidermis at 4-48 h after application of [3H]5-MeC; and (iii) the reactions of DE-I and DE-II with calf thymus DNA in vitro. In contrast to results obtained with mouse liver 9000 grams supernatant, the dihydrodiol precursors of DE-I and DE-II were present in equivalent quantities in mouse epidermis in vivo at every time point studied. The ratio of DE-I:DNA adducts to DE-II:DNA adducts in mouse epidermis was constant throughout the time period studied. However, the extent of formation of DE-I:DNA adducts was greater than that of DE-II:DNA adducts upon reaction of DE-I or DE-II with calf thymus DNA in vitro. These results suggest that differences in reactivity with DNA of DE-I and DE-II may bw responsible for the higher levels in mouse epidermis of DE-I:DNA adducts compared with DE-II:DNA adducts and provide a possible basis for the observed enhancing effect of a bay region methyl group on the carcinogenicity of polynuclear aromatic hydrocarbons.

Tumour initiating activity of dihydrodiols of benzo[b]fluoranthene, benzo[j]fluoranthene, and benzo[k]fluoranthene.

The tumor initiating activities on mouse skin of benzo[b]fluoranthene, (B[b]F), benzo[j]fluoranthene (B[j]F), benzo[k]fluoranthene (B[k]F] and three of their dihydrodiols, 9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene (B[b]F-9,10-diol), 9,10-dihydro-9,10-dihydroxybenzo[j]fluoranthene (B[j]F-9,10-diol), and 8,9-dihydro-8,9-dihydroxybenzo[k]fluoranthene (B[k]F-8,9-diol) were evaluated. Among the parent hydrocarbons, B[b]F was the most potent tumor initiator, with activity greater than that of B[j]F but less than that of benzo[a]pyrene. B[k]F also showed tumor initiating activity, in contrast to its lack of complete carcinogenic activity on mouse skin. B[b]F-9,10-diol, which can form a bay region dihydrodiol epoxide, was as active as B[b]F. B[j]F-9,10-diol, which would form its dihydrodiol epoxide in a four sided pseudo-bay region, was less active than B[j]F. B[k]F-8,9-diol was inactive. These results, together with parallel metabolic studies, suggest that the formation of bay region dihydrodiol epoxides may not be the major activation mechanism in benzofluoranthene tumorigenesis.

Identification of metabolites of benzo[b]fluoranthene.

The metabolism by rat liver 9000 x g supernatant of the environmental carcinogen benzo[b]fluoranthene was investigated. The major metabolites were identified, by comparison to synthetic samples, as 5- and 6-hydroxybenzo[b]fluoranthene and 4- and 7-hydroxybenzo[b]fluoranthene. The principal dihydrodiol metabolite formed under these conditions was trans-11,12-dihydro-11,12-dihydroxybenzo[b]fluoranthene, which was identified by comparison to the synthetic compound. 1,2-Dihydro-1,2-dihydroxybenzo[b]fluoranthene was identified, by its mass spectrum and by comparison if its u.v. spectrum to that of a synthetic model compound, 11-ethylidene-11H-benzo[b]fluorene. No evidence was obtained for the formation of 7b,8-dihydro-7,8-dihydroxybenzo[b]fluoranthene or trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene. The latter would be the precursor to a bay region dihydrodiol epoxide of benzo[b]fluoranthene.