Oxidation of the carcinogens benzo [a] pyrene and benzo [a] anthracene to dihydrodiols by a bacterium.

A mutant strain of Beijerinckia, after growth with succinate plus biphenyl, contains an enzyme system that oxidizes benzo [a] pyrene and benzo [a] anthracene to mixtures of vicinal dihydrodiols. The major dihydrodiol formed from benzo [a] pyrene was identified as cis-9, 10-dihydroxy-9, 10-dihydrobenzo [a] pyrene by comparison with a synthetic sample. Benzo [a] anthracene was metabolized to four dihydrodiols, the major isomer being cis-1, 2-dihydroxy-1, 2-dihydroxy-1, 2-dihydrobenzo [a] anthracene.

Binding of benzo[a]pyrene 7,8-diol-9,10-epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity.

The formation, stereostructure, and cellular reactions of the 7,8-diol-9,10-epoxide metabolites of the carcinogen benzo[a]pyrene have been examined after topical application of benzo[a]pyrene to the skin of mice. In this known target tissue, polymer adducts from diastereomeric diol epoxides, (+)-(7S, 8R, 9R, 10R) and (+)-(7R, 8S, 9R, 10R), were formed stereospecifically from their corresponding 7,8-dihydrodiols. Both diol epoxides bind with proteins, RNA, and DNA in vivo. For the nucleic acids, binding occurs preferentially at the 2-amino group of guanine in cellular RNA and DNA in vivo. Methods for establishing the structure of the cellular adducts as well as the possible biological implications of their formation are discussed.

Neoplastic effects of oral administration of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and their inhibition by butylated hydroxyanisole.

The neoplastic effects of administration of benzo[a]pyrene (BP) and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP 7,8-dihydrodiol) by oral intubation to noninbred female Ha:ICR mice have been determined. Under the experimental conditions, BP induced papillomas of the forestomach. BP 7,8-dihydrodiol also induced papillomas of the forestomach and was more potent than BP. In addition, administration of BP 7,8-dihydrodiol caused a large number of pulmonary adenomas and lymphomas. Butylated hydroxyanisole (BHA) added to the diet at a concentration of 5 mg/g inhibited BP-induced neoplasia of the forestomach. BHA also inhibited neoplasia of the forestomach, lungs, and lymphoid tissues that was caused by administration of BP 7,8-dihydrodiol. These data suggest that the inhibitory effect of BHA on BP carcinogenesis may entail events that occur subsequent to the formation of BP 7,8-dihydrodiol.

Tumorigenicity of the diastereomeric benz[a]anthracene 3,4-diol-1,2-epoxides and the (+)- and (-)-enantiomers of benz[a]anthracene 3,4-dihydrodiol in newborn mice.

The tumorigenic activity of benz[a]anthracene (BA), the (+)- and (-)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene (BA 3,4-dihydrodiol), and the racemic diastereomers of the BA 3,4-diol-1,2-epoxides [i.e., either or both of the diastereomeric 1,2-epoxides derived from BA 3,4-dihydrodiol in which the epoxide oxygen is cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 4-hydroxyl group) was examined in newborn Swiss-Webster mice. The mice were administered ip a total dose of 280 nmoles of compound in divided doses consisting of 40 nmoles within 24 hours of birth, 80 nmoles at 8 days of age, and 160 nmoles at 15 days of age. The experiment was terminated when the animals were 26 weeks of age. BA 3,4-diol-1,2-epoxide-2 was the most potent compound tested. All animals treated with BA 3,4-diol-1,2-epoxide-2 developed pulmonary tumors with an average of 13.3 tumors per mouse. BA 3,4-diol-1,2-epoxide-1 produced pulmonary tumors in 42% of the mice with an average of only 0.56 tumors per mouse. The (-)-enantiomer of BA 3,4-dihydrodiol with [3R,4R] absolute stereochemistry was the second most tumorigenic derivative of BA tested; it produced pulmonary tumors in 71% of the mice with an average of 1.88 tumors per mouse. BA and the (+)-enantiomer of BA 3,4-dihydrodiol had little or no tumorigenic activity at the dose tested. A comparison of the average number of pulmonary tumors per mouse revealed that BA 3,4-diol-1,2-epoxide-2 was about 30-fold more tumorigenic than was BA 3,4-diol-1,2-epoxide-1, 8-fold more tumorigenic than was (-)-BA 3,4-dihydrodiol, and greater than 85-fold more tumorigenic than was BA. These data indicate that in newborn mice BA 3,4-dihydrodiol and a BA 3,4-diol-1,2-epoxide are proximate and ultimate carcinogenic metabolites of BA, respectively.

Mouse skin tumor-initiating activity of 5-, 7-, and 12-methyl- and fluorine-substituted benz[a]anthracenes.

As an approach for elucidation of structure activity relationships that underlie the exceptionally large difference in carcinogenic activity between benz[a]anthracene and 7,12-dimethylbenz[a]anthracene (7,12-DMBA), 11 methyl- and/or fluorine-substituted benz[a]anthracenes were evaluated for tumor-initiating activity on mouse skin. Outbred CD-1 and outbred Sencar mice received a single topical application of the hydrocarbons followed by twice weekly applications of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate for 16-26 weeks. 7,12-DMBA was almost two orders of magnitude more active as a tumor-initiator than 7- and 12-methylbenz[a]anthracene. Methyl substitution at the 7- and 7,12-positions of benz[a]anthracene was significantly more effective in the enhancement of tumorigenic activity than fluorine substitution at these positions. Although 7-fluorobenz[a]anthracene, 12-fluorobenz[a]anthracene, and 7,12-difluorobenz[a]anthracene had only 0.15, 0.26, and less than 0.005 times the tumor-initiating activity of their respective methyl-substituted derivatives, they were severalfold more active than benz[a]anthracene. 7-Fluorobenz[a]anthracene was slightly less active than 12-fluorobenz[a]anthracene, whereas 7-methylbenz[a]anthracene was about twofold more than 12-methylbenz[a]anthracene. For 7,12-di-substituted benz[a]anthracenes, 7-methyl-12-fluorobenz[a]anthracene was more than twice as tumorigenic as 7-fluoro-12-methylbenz[a]anthracene, but each was individually more active than 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene, respectively. Both fluorinated compounds were much less active than 7,12-DMBA. Substitution of fluorine or methyl at the 5-position of 7-methylbenz[a]anthracene and substitution of fluorine at the 5-position of 12-methylbenz[a]anthracene dramatically reduced their tumorigenic activity.

Fluorine substitution as a probe for the role of the 6-position of benzo[a]pyrene in carcinogenesis.

The tumorigenic activities of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-F-BP) were compared to determine whether an unsubstituted 6-position is important for the carcinogenic effect of BP. Highly purified samples of 6-F-BP and BP had similar activities for the induction of lung adenomas in Swiss Webster mice treated before weaning. The 6-fluoro derivative, however, had about one-half as much activity as BP for the initiation of skin papillomas in CD-1 mice. Similarly, 6-F-BP (approximately equal to 90% purity) had about one-half the activity of BP for the induction of skin tumors in C57BL/6J mice given repetitive treatments of the hydrocarbons and for the induction of sarcomas in C3H/fCum mice given a single sc injection. 6-F-BP (approximately equal to 90% purity) had activity similar to that of BP for induction of sarcomas at the sc injection site in Fischer 344 rats. These results and related data indicate the need for detailed metabolic studies whenever fluorine substitution is used as a probe to assess the role of the unsubstituted position in the carcinogenicity of the parent compound.

Sequence specificity in the interaction of the four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides with the supF gene.

The shuttle vector pS189 was treated with each of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide, and the modified DNA was used as a template in a polymerase arrest assay examining the supF gene. Sites at which polymerase (Sequenase, version 2.0) progress along the template was blocked were presumed to be at or near sites of adduct formation. The polymerase arrest sites were compared with recently reported mutation hotspots induced by these agents in this gene (Bigger et al., Proc. Natl. Acad. Sci. USA, 89: 368-372, 1992). For 31 of 32 mutation hotspots, a polymerase arrest band was present at or 1 or 2 nucleotides 3'- to that site, indicating that adduct formation tended to be associated with mutation hotspots. However, the arrest bands near mutation hotspots were not particularly prominent in all cases, and there were many sites of substantial polymerase arrest that were not in the vicinity of mutation hotspots. Thus, factors in addition to chemical selectivity must play key roles in determining sites of mutation.

Lack of carcinogenicity of 4-, 5-, 6-, 7-, 8-, 9-, and 10-hydroxybenzo(a)pyrene on mouse skin.

Seven phenols of benzo(a)pyrene (4-, 5-, 6-, 7-, 8-, 9-, and 10-hydroxybenzo(a)pyrene) were tested for carcinogenicity on mouse skin by application of 0.4 mumole of compound once every two weeks for 56 weeks. None of the seven phenols tested was carcinogenic to mouse skin, while treatment with the same dose of benzo(a)pyrene produced tumors in 92% of the treated animals. The lack of carcinogenicity of 7- and 8-hydroxybenzo(a)pyrene indicates that the strong carcinogenic activity previously reported for benzo(a)pyrene 7,8-oxide was not due to either phenolic isomerization product of this arene oxide.

Murine strain differences in ovotoxicity following intraovarian injection with benzo(a)pyrene, (+)-(7R,8S)-oxide, (-)-(7R,8R)-dihydrodiol, or (+)-(7R,8S)-diol-(9S,10R)-epoxide-2.

Murine ovarian tumors produced by polycyclic aromatic hydrocarbons like benzo(a)pyrene (BP) require small oocyte destruction. Small oocyte destruction was evaluated in C57BL/6N (B6), DBA/2N (D2), and C57BL/6J X DBA/ 2JF1 (B6D2F1) mice following intraovarian injection with BP, (+)-( 7R ,8S)-oxide, (-)-( 7R , 8R )-dihydrodiol [(-)-DHD], or (+)-( 7R ,8S)-diol-(9S, 10R )-epoxide-2 [(+)- DE2 ] at doses ranging from 0.01 to 30 micrograms/ovary. BP, (-)-DHD, and (+)- DE2 produced small oocyte destruction in a dose-dependent fashion. The (+)-( 7R ,8S)-oxide did not destroy small oocytes at the highest dose tested (10 micrograms/ovary). The rank orders of the calculated doses which resulted in the destruction of 50% of the small oocytes (ED50S) for small oocyte destruction were BP approximately equal to (-)-DHD greater than (+)- DE2 in all three groups of mice. However, the ED50S for BP and (-)-DHD differed considerably among B6, D2, B6D2F1 mice; ED50S were smallest in B6 mice and largest in D2 mice. The ED50S for oocyte destruction in B6D2F1 mice were intermediate or similar to ED50S for B6 mice, depending on the method used for calculation. In spite of large strain differences in ED50S for BP and (-)-DHD, the ED50S for (+)- DE2 were similar in B6, D2, and B6D2F1 mice. The similar ED50 for (+)- DE2 suggests that it is an ultimate ovotoxin and ovarian carcinogen and that the target molecule(s) and mechanism(s) of detoxification are similar in B6, D2, and B6D2F1 mice.

Tumorigenicity studies with diol-epoxides of benzo(a)pyrene which indicate that (+/-)-trans-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene is an ultimate carcinogen in newborn mice.

The tumorigenic activities of benzo(a)pyrene(BP), (+/-)-trans-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxide 1), (+/-)-trans-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxide 2), (+/-)-trans-7,8,-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP 7,8-dihydrodiol), and the tetraols derived from the hydrolysis of diol-epoxide 2 were evaluated in newborn mice. The mice were given injections sequentially of 4, 8, and 16 nmoles of each compound on the first, eighth, and fifteenth days of life, and the animals were killed when they were 28 weeks old. Diol-epoxide 1 was highly toxic in newborn mice, and most of the animals treated with this compound died before weaning. Diol-epoxide 2 and BP 7,8-dihydrodiol were, respectively, about 40- and 15-fold more active than BP in causing pulmonary adenomas. Vehicle-treated control animals had an average of 0.13 lung adenoma/mouse, whereas animals treated with BP, BP 7,8-dihydrodiol, or diol-epoxide 2 had, respectively, 0.24, 1.77 and 4.42 pulmonary adenomas/mouse. Diol-epoxide 1 and the tetraols derived from diol-epoxide 2 did not induce pulmonary adenomas. The inactivity of diol-epoxide 1 under the conditions of our study should be interpreted with caution because of the high toxicity of this compound. The results of our study provide evidence that BP 7,8-dihydrodiol is a proximate carcinogenic metabolite and that diol-epoxide 2 is an ultimate carcinogenic metabolite of BP in the newborn mouse.

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.

Stereochemical specificity in the metabolic activation of benzo(c)phenanthrene to metabolites that covalently bind to DNA in rodent embryo cell cultures.

Benzo(c)phenanthrene (BcPh) has only weak carcinogenic activity in rodent bioassays. However, bay-region diol-epoxides of BcPh have the highest tumor-initiating activities of all hydrocarbon diol-epoxides tested to date. To determine whether BcPh is metabolically activated to bay-region diol-epoxides that bind to DNA in cells, Sencar mouse, Syrian hamster, and Wistar rat embryo cell cultures were exposed to [5-3H]-BcPh, and the BcPh-deoxyribonucleoside adducts formed were analyzed by immobilized boronate chromatography and reverse-phase high-performance liquid chromatography. Greater than 74% of the BcPh-deoxyribonucleoside adducts formed in all 3 species resulted from reaction of (4R,3S)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-BcPh [(-)-BcPhDE-2] with DNA to yield deoxyadenosine and deoxyguanosine adducts in a ratio of 3:1. A much smaller proportion of BcPh-deoxyribonucleoside adducts were formed by reaction of (4S,3R)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-BcPh [(+)-BcPhDE-1] with deoxyadenosine. No BcPh-deoxyribonucleoside adducts arising from either (+)-BcPhDE-2 or (-)-BcPhDE-1 were detected. The absence of adducts from these isomers of BcPhDE was not due to failure of these isomers to react with DNA in cells, for reaction of (+/-)-BcPhDE-1 or (+/-)-BcPhDE-2 with DNA in solution or in hamster embryo cell cultures resulted in the formation of DNA adducts from both the (+)- and (-)-enantiomers of each BcPhDE. These results indicate that both the (+)- and (-)-3,4-dihydrodiols of BcPh are formed and that their metabolic activation to diol-epoxides occurs with high stereospecificity in cells from all 3 species of rodents. The finding that the major DNA-binding metabolite is (-)-BcPhDE-2, the diol-epoxide with the (R,S)-diol-(S,R)-epoxide absolute configuration that is associated with high carcinogenic activity of diol-epoxides of other hydrocarbons, demonstrates that these cells are able to activate BcPh to an ultimate carcinogenic metabolite. The fact that a high proportion of the BcPh-DNA adducts are deoxyadenosine adducts suggests that BcPh has DNA-binding properties similar to those of the potent carcinogen 7,12-dimethylbenz(a)anthracene. The stereospecificity observed in the metabolic activation of BcPh to DNA-binding metabolites and the reaction of these metabolites with both deoxyguanosine and deoxyadenosine suggest that studies of the interactions of BcPh with DNA in vivo may be a valuable approach for establishing the role of specific activation pathways and DNA adducts in tumor induction.

Dose-dependent differences in the profile of mutations induced by (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in Chinese hamster V-79 cells.

Chinese hamster V-79 cells were exposed to a high dose (0.30-0.48 microM; 32% cell survival), an intermediate dose (0.04-0.10 microM; 100% cell survival) or a low dose (0.01-0.02 microM; 97% cell survival) of (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+)-BPDE] which is the ultimate carcinogenic metabolite of benzo(a)pyrene. The mutation frequency for cells treated with dimethyl sulfoxide vehicle or with low, intermediate or high dose of (+)-BPDE were 1, 10, 52 or 514 8-azaguanine-resistant colonies/10(5) survivors, respectively. Independent 8-azaguanine-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene was amplified by the polymerase chain reaction and sequenced. Altogether, 368 (+)-BPDE-induced mutant clones were examined. At all doses, base substitutions were the most prevalent mutations observed (about 72% of the mutant clones), followed by exon deletions (about 26% of the mutant clones) and frame-shift mutations (about 6% of the mutant clones). At the high cytotoxic dose, 7 of 120 base substitutions occurred at AT base pairs (6%) and 113 at GC base pairs (94%). At the intermediate noncytotoxic dose, 20 of 82 base substitutions occurred at AT base pairs (24%) and 62 at GC base pairs (76%). At the low noncytotoxic dose, 27 of 76 base substitutions were at AT base pairs (36%) and 49 were at GC base pairs (64%). The results indicated that decreasing the dose of (+)-BPDE decreased the proportion of mutations at GC base pairs and increased the proportion of mutations at AT base pairs. At the dose of (+)-BPDE was decreased, there was a dose-dependent decrease in the proportion of GC-->TA transversions (from 69% to 42% of the base substitutions) and a dose-dependent increase in the proportion of AT-->CG transversions (from 1% to 25% of the base substitutions). The data also indicated dose-dependent differences in (+)-BPDE-induced exon deletions and hot spots for base substitutions at GC and AT base pairs. Although more than 99% of the (+)-BPDE-induced mutations at guanine occurred on the nontranscribed strand of DNA, (+)-BPDE-induced mutations at adenine occurred on both the transcribed and nontranscribed strands. The ratio of mutations at adenine on the transcribed strand to mutations at adenine on the nontranscribed strand was 35:19 in (+)-BPDE-treated V-79 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Tumorigenic activity of benzo(e)pyrene derivatives on mouse skin and in newborn mice.

The tumorigenic activities of benzo(e)pyrene and several of its derivatives were determined in two mouse tumor models. Newborn Swiss-Webster mice were given i.p. injections of 0.4, 0.8, and 1.6 mumol of compound on the first, eighth, and 15th day of life, respectively. When the mice were 62 to 66 weeks old, the experiment was terminated by killing the animals. Benzo(e)pyrene, trans-4,5-dihydroxy-4,5-dihydrobenzo(e)pyrene, and trans-9,10-dihydroxy-9,10-dihydrobenzo(e)pyrene had little or no tumorigenic activity in lung tissue, although trans-9,10-dihydroxy-9,10-dihydrobenzo(e) pyrene did induce a significant number of hepatic tumors. The tumor-initiating activities of benzo(e)pyrene and several of its derivatives were determined on the skin of female CD-1 mice. A single topical application of 1.0 to 6.0 mumol of the test compound was followed 7 days later by twice-weekly applications of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate for 35 weeks. Control mice and mice treated with 6.0 mumol of benzo(e)pyrene, trans-4,5-dihydroxy-4,5-dihydrobenzo(e)pyrene, trans 9,10-dihydroxy-9,10-dihydrobenzo(e)pyrene, and trans-9,10-dihydroxy-9,10,11,12-tetrahydrobenzo(e)pyrene had a tumor incidence of less than 20% and had less than or equal to 0.25 papillomas/mouse. 9,10-Dihydrobenzo(e)pyrene was the only derivative tested that had significant tumor-initiating activity on mouse skin; an initiating dose of 2.5 mumol gave a 67% tumor incidence and 1.43 papillomas/mouse.

Carcinogenicity of 2-hydroxybenzo(a)pyrene and 6-hydroxybenzo(a)pyrene in newborn mice.

Benzo(a)pyrene (BP), 2-hydroxybenzo(a)pyrene (2-HOBP), and 6-hydroxybenzo(a)pyrene (6-HOBP) were tested for tumorigenicity by i.p. injection into newborn mice. The mice were treated sequentially with 200, 400, and 800 nmol of compound on the first, eighth and fifteenth day of life, and the animals were killed at 24 weeks of age. Treatment with 2-HOBP caused about 4-fold more pulmonary tumors than BP, while 6-HOBP had little or no tumorigenic activity. Newborn mice treated with 2-HOBP, BP, and 6-HOBP had a 98, 81, and 11% incidence of pulmonary adenomas with an average of 24, 6.4, and 0.11 adenomas per mouse, respectively. In the control group, 7.5% of the animals had pulmonary adenomas with an average of 0.08 adenoma per mouse. When 25, 50, or 100 nmol of BP or 2-HOBP was applied to mouse skin once every 2 weeks for 60 weeks, both compounds had about the same carcinogenic activity. These results demonstrate the importance of evaluating the carcinogenic potential of chemicals in more than one tumor system. BP and 2-HOBP were tested for mutagenicity towards two strains of Salmonella typhimurium and towards Chinese hamster V79 cells in the presence of hepatic microsomes from rats pretreated with Aroclor 1254. The products formed during the metabolism of 2-HOBP or BP by liver microsomes had significant mutagenic activity.

Bacterial and mammalian cell mutagenicity of four optically active bay-region 3,4-diol-1,2-epoxides and other derivatives of the nitrogen heterocycle dibenz[c,h]acridine.

The mutagenic activities of the enantiomers of the diastereomeric pair of bay-region 3,4-diol-1,2-epoxides of the nitrogen heterocycle, dibenz[c,h]acridine, have been evaluated in histidine-dependent strains of Salmonella typhimurium and in an 8-azaguanine-sensitive line of Chinese hamster cells. In strains TA 98 and TA 100 of S. typhimurium the pair of enantiomers with [1R,2S,3S,4R] and [1S,2R,3R,4S] absolute configuration and the benzylic hydroxyl group trans to the epoxide oxygen are 2 to 4 times more mutagenic than the [1S,2R,3S,4R] and [1R,2S,3R,4S] isomers in which the benzylic hydroxyl and epoxide oxygen are cis. In both strains of bacteria there is very little difference in mutagenic activity between the enantiomers of each diastereomer. In contrast to these results in bacteria, the bay-region 3,4-diol-1,2-epoxide isomer with [1R,2S,3S,4R] absolute configuration is 5 to 7 times more mutagenic to Chinese hamster V79 cells than are the other 3 isomers. The enantiomeric pair of bay-region tetrahydro-1,2-epoxides of dibenz[c,h]acridine are at least 7 times more mutagenic than the diol-epoxides in the Salmonella assay, and no difference in mutagenic activity is observed between enantiomers. In the Chinese hamster V79 cell system, however, the tetrahydro-1,2-epoxide with [1R,2S] absolute configuration is 2- to 3-fold more mutagenic than its enantiomer with [1S,2R] absolute configuration. Homogeneous rat liver epoxide hydrolase does not catalyze the hydration of the diol-epoxide isomers to nonmutagenic products, although the tetrahydroepoxides, especially the tetrahydro-3,4-epoxide, are metabolized by the enzyme. Results of metabolic activation experiments with the bacterial mutagenesis system and microsomes from Aroclor 1254-treated rats are consistent with the mutagenicity data described above and support the concept that dibenz[c,h]acridine is metabolically activated to a bay-region diol-epoxide. Notably: (a) 3,4-dihydrodibenz[c,h]acridine, the expected precursor of a bay-region tetrahydroepoxide, is metabolized to a potent mutagen; (b) racemic dibenz[c,h]acridine 3,4-dihydrodiol is metabolized to products which are several-fold more mutagenic than are products of the metabolism of dibenz[c,h]acridine or its 1,2- or 5,6-dihydrodiols; and (c) the tetrahydro-3,4-diol, which lacks the isolated bay-region double bond, is not metabolically activated to a bacterial mutagen.

Inhibitory effect of 3-hydroxybenzo(a)pyrene on the mutagenicity and tumorigenicity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.

The 12 isomeric phenols of benzo(a)pyrene were tested for their ability to inhibit the mutagenic activity of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [B(a)P 7,8-diol-9,10-epoxide-2], an ultimate mutagenic and carcinogenic metabolite of benzo(a)pyrene. 3-Hydroxybenzo(a)pyrene [3-HO-B(a)P], a major metabolite of benzo(a)pyrene, was the most potent antagonist tested. Approximately 3 nmol of 3-HO-B(a)P, 14 nmol of 10-HO-B(a)P, and 5-8 nmol of 1-, 2-, 4-, 5-, 6-, 7-, 8-, 9-, 11-, and 12-HO-B(a)P inhibited the mutagenic activity of 0.05 nmol of B(a)P 7,8-diol-9,10-epoxide-2 by 50% in Salmonella typhimurium strain TA 100. The importance of the phenolic group for antimutagenic activity was indicated by the lack of antimutagenic activity of benzo(a)pyrene itself. 3-HO-B(a)P also inhibited the mutagenic activity resulting from the metabolic activation of benzo(a)pyrene and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene by rat liver microsomes. This inhibition may have resulted from an effect of 3-HO-B(a)P on the metabolic activation of these carcinogens and/or from a direct effect on the action of B(a)P 7,8-diol-9,10-epoxide-2. In a mammalian cell culture system utilizing Chinese hamster V79 cells, 3-HO-B(a)P (8 microM) inhibited the mutagenicity of B(a)P 7,8-diol-9,10-epoxide-2 (0.2 microM) by 50%. Although 3-HO-B(a)P was a potent inhibitor of the mutagenic activity of bay-region diol epoxides of benzo(a)pyrene, dibenzo(a,h)pyrene, and dibenzo(a,i)pyrene in S. typhimurium strain TA 100, higher concentrations of 3-HO-B(a)P were needed to inhibit the mutagenicity of the chemically less reactive benzo(a)pyrene 4,5-oxide and the bay-region diol epoxides of benz(a)anthracene, chrysene, and benzo(c)phenanthrene. Both 3-HO-B(a)P and 10-HO-B(a)P accelerated the disappearance of B(a)P 7,8-diol-9,10-epoxide-2 from 1:9 dioxane-water solutions at pH 7 and 25 degrees C. 3-HO-B(a)P, the most effective antimutagen of the B(a)P phenols tested, was much more reactive with the diol epoxide than 10-HO-B(a)P, the least effective antimutagen. The rate constant for the reaction of 3-HO-B(a)P with the diol epoxide exhibited a nonlinear (greater than first-order) dependence on the concentration of the phenol. Evidence was obtained for covalent adduct formation between the diol epoxide and each of the two phenols.(ABSTRACT TRUNCATED AT 400 WORDS)

High tumorigenicity of the 3,4-dihydrodiol of 7-methylbenz[c]acridine on mouse skin and in newborn mice.

The tumorigenicities of 7-methylbenz[c]acridine (7MB[c]ACR) and its five metabolically possible trans-dihydrodiols were determined in two mouse tumor models. In initiation-promotion studies on mouse skin, a single topical application of 0.15 to 0.75 mumol of compound was followed 9 days later by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate for 20 wk. Comparison of the average number of skin tumors per mouse indicated that 7MB[c]ACR 3,4-dihydrodiol, the metabolic precursor of a bay-region diol-epoxide, was 4- to 6-fold more active than the parent compound as a tumor initiator. The 1,2-, 5,6-, 8,9-, and 10,11-dihydrodiols of 7MB[c]ACR had no significant tumor-initiating activity at the doses tested. In newborn mice, a total dose of 0.35 mumol of compound was administered i.p. during the first 15 days of life, and tumorigenic activity was determined when the mice were 32 to 36 wk old. 7MB[c]ACR 3,4-dihydrodiol induced about 8-fold more pulmonary tumors per mouse and 9-fold more hepatic tumors per male mouse than the parent aza-substituted hydrocarbon. The other four dihydrodiols of 7MB[c]ACR had no significant tumorigenic activity. The high tumorigenic activity of 7MB[c]ACR 3,4-dihydrodiol in both tumor models suggests that a bay-region 3,4-diol-1,2-epoxide may be an ultimate carcinogenic metabolite of 7MB[c]ACR. 7MB[c]ACR was at least 5-fold more active as a tumor initiator on mouse skin than was the unsubstituted aza-aromatic compound, benz[c]acridine. This latter result indicates that substitution of a methyl group at position 7 of benz[c]acridine leads to enhanced tumor-initiating activity, as has been previously demonstrated for benz[a]anthracene and its 7-methyl derivative.

Tumorigenicity of optical isomers of the diastereomeric bay-region 3,4-diol-1,2-epoxides of benzo(c)phenanthrene in murine tumor models.

Tumorigenic activities of the (+)- and (-)-enantiomers of the diastereomeric, bay-region benzo(c)phenanthrene 3,4-diol-1,2-epoxides were evaluated in two mouse tumor models. In an initiation-promotion experiment on mouse skin, a single topical application of 10, 25, or 75 nmol of the compounds was followed by 20 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate. Of the four optical isomers of the bay-region diol epoxides, (-)-(R,2S,3S,4R)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrogenzo(c )phenanthrene [(-)-diol epoxide-2] and (+)-(1R,2S,3R,4S)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo(c) -phenanthrene [(+)-diol epoxide-1] had equally high tumor-initiating activity while (+)-[1S,2R,3R,4S]-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo (c)phenanthrene [(+)-diol epoxide-2] had less than one-half of the activity of (-)-diol epoxide-2 and (+)-diol epoxide-1. (-)-(1S,2R,3S,4R)-3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo(c) -phenanthrene [(-)-diol epoxide-1] was inactive at the doses tested. In newborn mice, (-)-diol epoxide-2 was almost 10-fold more active in producing lung tumors (average number of lung tumors/mouse) than the next most active compound, (+)-diol epoxide-2, at a total dose of 10 nmol. The enantiomers of diol epoxide-1 were inactive at this dose. When the total dose of each optical isomer was increased to 50 nmol, (-)-diol epoxide-1 was still inactive, and (+)-diol epoxide-1 produced a significant number of lung tumors (0.9 lung tumor/mouse), but this isomer still had less than 10% of the activity of the (+)- and (-)-diol epoxide-2 isomers. (-)-Diol epoxide-2, but none of the other optical isomers, also produced a significant incidence of hepatic tumors at the higher dose, and this compound was found to be the most tumorigenic bay-region diol epoxide ever tested in newborn mice. Racemic diol epoxide-1 had approximately 1% of the tumorigenic activity of racemic diol epoxide-2 in newborn mice, but both racemates had equal tumor-initiating activity on mouse skin. These results dramatically illustrate the complexities involved in ranking the relative tumorigenic activities of compounds in different tumor models.

Dose-dependent differences in the mutational profiles of (-)-(1R,2S,3S,4R)-3,4-dihydroxy-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene and its less carcinogenic enantiomer.

Chinese hamster V-79 cells were treated with high cytotoxic or low noncytotoxic concentrations of the highly carcinogenic and mutagenic (-)-(1R,2S,3S,4R)-3,4-dihydroxy-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene [(-)-B[c]PhDE; fjord-region diol epoxide] or its biologically less active (+)-(1S,2R,3R,4S) enantiomer [(+)-B[c]PhDE]. The benzylic 4-hydroxyl group and the epoxide oxygen are trans in both enantiomers. Independent 8-azaguanine-resistant clones were isolated. The coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene was amplified by reverse transcription-PCR and sequenced. For (-)-B[c]PhDE, mutation frequencies were 10- or 356-fold above background for the low (0.01-0.1 microM; 97% cell survival) or high (1.0-1.25 microM; 26% cell survival) doses, respectively. For the high dose group, 20 of 64 base substitutions occurred at GC base pairs (31%) and 44 at AT base pairs (69%). For the low-dose group, 6 of 55 base substitutions were at GC base pairs (11%), and 49 were at AT base pairs (89%). For the less active (+)-B[c]PhDE, mutation frequencies were 17- or 372-fold above background for the low (0.12-0.5 microM; 95% cell survival) or high (2.0-3.0 microM; 31% cell survival) doses, respectively. In contrast to the results with the (-)-B[c]PhDE, both the high- and the low-dose groups for (+)-B[c]PhDE gave a 50:50 distribution of base substitution at GC versus AT base pairs. Our data indicate that: (a) transversions were the predominant base substitutions observed for both the (+)- and (-)-enantiomers of B[c]PhDE; (b) (-)-B[c]PhDE showed high selectivity for causing AT --> TA transversions, whereas considerably less selectivity was observed for (+)-B[c]PhDE; (c) (-)-B[c]PhDE had a different hot spot profile for base substitutions than did (+)-B[c]PhDE, but some common hot spots were observed for both compounds; and (d) decreasing the dose of (-)-B[c]PhDE increased the proportion of mutations at AT base pairs and decreased those at GC base pairs, but this was not observed for (+)-B[c]PhDE.