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.

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.

The carcinogenic and electrophilic activities of N-benzoyloxy derivatives of N-methyl-4-aminoazobenzene and related dyes.

The N-benzoyloxy derivatives of N-methyl-4-aminoazobenzene, its 4'-methyl and 4'-ethyl derivatives, and N-ethyl-4-aminoazobenzene were synthesized for comparison of their carcinogenic activities and their reactivities with nucleophilic reagents. Each of the 4 esters had a similar degree of nonenzymatic reactivity with methionine and guanosine at neutral pH. Each of the dyes induced sarcomas at the site of s.c. injection in rats, but N-benzoyloxy-N-methyl-4-aminoazobenzene was considerably more carcinogenic than were any of the other dyes. N-benzoyloxy-N-methyl-4-aminoazobenzene was also more stable in neutral lipid, and this stability may have contributed to its greater carcinogenicactivity. Neither the electrophilic reactivities nor the s.c. carcinogenicities of these dyes paralleled the hepatocarinogenic activities of the parent dyes.

The metabolic activation of the carcinogen 1'-hydroxysafrole in vivo and in vitro and the electrophilic reactivities of possible ultimate carcinogens.

Administration of [2',3'-3H]-1'-hydroxysafrole to rats or mice resulted in the formation of hepatic DNA-, ribosomal RNA-, and protein-bound 3H derivatives. Alkaline digestion of the 3H-protein released 0.1 to 0.3% of the 3H as a derivative that was identified as 3'-methylmercaptoisosafrole by its cochromatography in five solvent systems with the synthetic compound. 1'-Hydroxysafrole was metabolized at a low rate by rat and mouse liver cytosols in a 3'-phosphoadenosine 5'-phosphosulfate-dependent reaction to a derivative (presumably the sulfuric acid ester) that was captured by its reaction with RNA. Likewise, 1'-hydroxysafrole was oxidized at a low rate by rat and mouse liver microsomes to 1'-hydroxysafrole-2',3'-oxide in a reduced nicotinamide adenine dinucleotide phosphate-dependent reaction. Both of these electrophilic metabolites are candidate ultimate carcinogenic derivatives of 1'-hydroxysafrole. The electrophilic reactivities of various safrole derivatives with nucleosides were determined to be in the order of 1'-oxosafrole greater than 1'-acetoxysafrole greater than 1'-acetoxysafrole-2',3'-oxide greater than 1'-hydroxysafrole-2',3'-oxide greater than safrole-2',3'-oxide greater than or equal to 1'-oxosafrole-2',3'-oxide. The major reactions were generally observed with guanosine. A major reaction product of 1'-acetoxysafrole and guanosine 5'-monophosphate yielded 3'-hydroxyisosafrole under very mild acidic conditions. These data further substantiate the previous characterization of this reaction product as O-6-(isosafrol-3'-yl)guanylic acid. The syntheses of 1'-oxosafrole, 2',3'-dehydrosafrole, [2',3'-3H]-1'-hydroxysafrole, and the 2',3'-oxed.

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.

Mutagenicity and cytotoxicity of benzo(a)pyrene arene oxides, phenols, quinones, and dihydrodiols in bacterial and mammalian cells.

Twenty-nine benzo(a)pyrene derivatives were tested for mutagenic acitivity without metabolic activation in Salmonella typhimurium strains TA98, TA100, and TA1538 and in Chinese hamster V79 cells. The compounds studied included 4 arene oxides, all 12 isomeric phenols, 5 quinones, and 8 dihydrodiols. Benzo(a)pyrene 4,5-oxide was the most mutagenic of the compounds tested in both the bacterial and mammalian systems. The other arene oxides [benzo(a)pyrene 7,8-, 9,10-, and 11,12-oxides] were only weakly mutagenic in the S. typhimurium strains. However, in Chinese hamster V79 cells benzo(a)pyrene 11,12-oxide. Among the phenols, 6-hydroxybenzo(a)pyrene and 12-hydroxybenzo(a)pyrene were moderately mutagenic in strain TA98 of S. typhimurium, and 6-hydroxybenzo(a)pyrene was moderately mutagenic in V79 cells. The other 10 phenols, 5 quinones [benzo(a)pyrene 1,6-, 3,6-, 4,5-, 6, 12-, and 11,12-quinones] and 8 dihydrodiols [benzo(a)pyrene cis-4,5,trans-4,5-, cis-7,8-, trans-7,8-, cis-9,10-, trans-9,10-, cis-11,12-, and trans-11, 12-dihydrodiols] were eitherinactive or only weekly mutagenic. 1-Hydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene were weakly mutagenic in strain TA98 of S. typhimurium, and benzo(a)pyrene 7,8-dihydrodiol was weakly mutagenic in V79 cells. Benzo(a)pyrene 11,12-quinone was extremely cytotoxic to the V79 cells but had no observable toxicity in the bacterial strains.

Mutagenicity and cytotoxicity of benzo(a)pyrene benzo-ring epoxides.

Four benzo-ring epoxides of the environmental carcinogen benzo(a)pyrene (BP) were tested for mutagenic and cytotoxic activity in 3 strains of Salmonella typhimurium (TA1538, TA98, and TA100) and in Chinese hamster V79 cells. Although very unstable in aqueous solution, 7beta,8alpha-dihydroxy-0beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol epoxide 1), with the 7-hydroxyl group on the same face of the molecule as the epoxide oxygen, was 1.5 to 4 times as mutagenic in the bacterial strains as was its more stable stereoisomer 7beta,8alpha-dihydroxy-9alpha,10beta-epoxy-7,8,9.10-tetrahydrobenzo(a)pyrene (diol epoxide 2). In V79 cells, diol epoxide 1 had one-third the mutagenic activity of diol epoxide 2 but was at least 10 times more labile than diol epoxide 2 in the tissue culture medium. The half-life of diol epoxide 1 in tissue culture medium was about 30 sec, whereas the half-life of diol epoxide 2 was between 6 and 12 min. 9,10-Epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, which is saturated in the benzo ring, is also very unstable and has mutagenic activity equal to or greater than diol epoxide 1 in the bacterial and mammalian cells. 7,8-Epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was more stable in aqueous solution than any of the 9,10-epoxides of BP but was much less mutagenic in both the bacterial and mammalian cells. In v79 cells, diol epoxides 1 and 2 and 9,10-opoxy-7,8,9,10-tetrahydrobenzo(a)pyrene were more than 40 times more cytotoxic than 7,8-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. The mutagenicity of the 2 tetrahydro epoxides toward strain TA98 of S. typhimurium was readily abolished by purified epoxide hydrase, whereas the mutagenic activity of the 2 diol epoxides was relatively unaffected by coincubation with the enzyme.

Exceptional carcinogenic activity of benz[a]anthracene 3,4-dihydrodiol in the newborn mouse and the bay region theory.

Benz[a]anthracene and the five metabolically possible trans-dihydrodiols of benz[a]anthracene were tested for carcinogenicity in newborn Swiss-Webster mice. Four hundred, 800, and 1600 nmoles hydrocarbon i.p. were sequentially injected on Days 1, 8, and 15 of life. The mice were killed at 22 weeks of age. Of the mice treated with trans-3,4-dihydroxy-3, 4-dihydrobenz[a]anthracene, 24% developed malignant lymphoma, whereas 4% of the animals treated with benz[a]anthracene had malignant lymphoma. None of the animals treated with the trans-1,2-dihydroxy-1,2-dihydrobenz[a]anthracene, trans-5,6-dihydroxy-5,6-dihydrobenz[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenz[a]anthracene, or trans-10,11-dihydroxy-10,11-dihydrobenz[a]anthracene had malignant lymphoma. trans-3,4-Dihydroxy-3,4-dihydrobenz[a]anthracene caused about 35-fold more pulmonary adenomas than did benz[a]anthracene, whereas the trans-1,2-dihydroxy-1,2-dihydrobenz[a]anthracene, trans-5,6-dihydroxy-5,6-dihydrobenz[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenz[a]anthracene, and trans-10, 11-dihydroxy-10,11-dihydrobenz[a]anthracene had little or no activity. The exceptionally high carcinogenicity of trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene is consistent with the metabolism of this compound to either or both of the diastereomeric bay region 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracenes, and the data support the bay region theory of polycyclic hydrocarbon carcinogenesis.

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.

Marked differences in the carcinogenic activity of optically pure (+)- and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in newborn mice.

Optically pure (+)- and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrenes [(+)- and (-)-BP 7,8-dihydrodiol] were tested for carcinogenicity by giving newborn mice i.p. injections of 20, 40, and 80 nmol of compound on Days 1, 8, and 15 of life. The animals were killed at 17 weeks of age. Control mice had 0.10 pulmonary adenoma per mouse, whereas animals treated with (+)-BP 7,8-dihydrodiol and (-)-BP 7,8-dihydrodiol had 0.16 and 9.28 pulmonary adenomas per mouse, respectively. When a 5-fold higher dose was administered according to the above dosage schedule, (+)-dihydrodiol caused 2.34 pulmonary adenomas per mouse and (-)-BP 7,8-dihydrodiol caused 32.2 pulmonary adenomas per mouse. When 200, 400, and 800 nmol of benzo(a)pyrene or (+)-BP 7,8-dihydrodiol were administered sequentially on Days 1, 8, and 15 of life, 4.13 and 18.5 pulmonary adenomas per mouse, respectively, were observed when the mice were 17 weeks of age. This high dose of (-)-BP 7,8-dihydrodiol killed most of the mice. Administration of (-)-BP 7,8-dihydrodiol caused a high incidence of malignant lymphomas, whereas (+)-BP 7,8-dihydrodiol and benzo(a)pyrene had little or no ability to cause malignant lymphomas.

Carcinogenicity of benzo-ring derivatives of benzo(a)pyrene on mouse skin.

Benzo(a)pyrene (BP) and several benzo-ring derivatives of BP were tested for carcinogenic activity in mice by topical application of each compound once every 2 weeks for 60 weeks. Chronic treatment of C57BL/6J mice with (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (0.025 to 0.10 micronmole/application) indicated that the dihydrodiol was slightly more active as a complete carcinogen than the parent hydrocarbon BP. 7,8-Dihydroxy-7,8,9,10-tetrahydro-benzo(a)pyrene, a compound related to (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene but which lacks the double bond at position 9,10, was inactive as a carcinogen on mouse skin. These results indicate the importance of the double bond at position 9,10 for the carcinogenic activity of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene. Chronic treatment of mice with -.4 micronmole of the highly mutagenic (+/-)-7,beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, (+/-)-7beta,8alpha-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, or 9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene every 2 weeks for 60 weeks resulted in tumor incidences of 0, 8, and 4%, respectively, whereas BP at this dose caused a 100% tumor incidence. The high reactivity of the three epoxides may account for their inactivity or their weak carcinogenic activity on mouse skin.

Tumorigenicity of benzo[alpha]pyrene 4,5-,7,8-,9,10- and 11,12-oxides in newborn mice.

Benzo[alpha]pyrene (BP) and its 4,5-,7,8-,9,10 and 11,12-oxides were tested for carcinogenicity by intraperitoneal injection into newborn mice. The mice were treated sequentially with 200, 400 and 800 nmol of hydrocarbon on days 1,8 and 15 of life, and the animals were killed at 24 weeks of age. Mice treated with BP, BP7,8-oxide and BP 11,12-oxide had a 93%, 72% and 20% incidence of pulmonary adenomas, respectively, with an average of 10.0, 2.1 and 0.32 adenomas/mouse, respectively. Eight percent of the control animals had pulmonary adenomas with an average of 0.08 adenomas per mouse. The moderate tumorigenic activity of the relatively unstable BP 7,8-oxide, together with earlier data on the strong carcinogenic activity of BP, 7,8-dihydrodiol, supports the idea that BP 7,8-oxide is a proximate carcinogenic metabolite of BP in the newborn mouse.

Drug residue formation from ronidazole, a 5-nitroimidazole. VIII. Identification of the 2-methylene position as a site of protein alkylation.

Ronidazole protein-bound adducts were generated by the in vitro anaerobic incubation of [2-methylene-14C]ronidazole with microsomes from the livers of male rats. Acid hydrolysis of the protein adducts yielded an imidazole ring fragment bearing the radiolabel and an amino acid residue derived from the proteins. This fragment has been identified as carboxymethylcysteine by co-chromatography of the amino acid and its dansyl derivative with known standards under a variety of conditions. The carboxymethylcysteine was estimated to represent at least 15% of the radioactivity derived from the protein-bound adducts and provides unequivocal evidence that nucleophilic attack by protein cysteine thiols occurred at the 2-methylene position of ronidazole.

Drug residue formation from ronidazole, a 5-nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro.

Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methytlviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxyethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively.

Drug residue formation from ronidazole, a 5-nitroimidazole. V. Cysteine adducts formed upon reduction of ronidazole by dithionite or rat liver enzymes in the presence of cysteine.

When ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reduced by either dithionite or rat liver microsomal enzymes in the presence of cysteine, ronidazole-cysteine adducts can be isolated. Upon reduction with dithionite ronidazole can react with either one or two molecules of cysteine to yield either a monosubstituted ronidazole-cysteine adduct substituted at the 4-position or a disubstituted ronidazole-cysteine adduct substituted at both the 4-position and the 2-methylene position. In both products the carbamoyl group of ronidazole has been lost. The use of rat liver microsomes to reduce ronidazole led to the formation of the disubstituted ronidazole-cysteine adduct. These data indicate that upon the reduction of ronidazole one or more reactive species can be formed which can bind covalently to cysteine. The proposed reactive intermediates formed under these conditions may account for the observed binding of ronidazole to microsomal protein and the presence of intractable drug residues in the tissues of animals treated with this compound. They may also account for the mutagenicity of this compound in bacteria.

Drug residue formation from ronidazole, a 5-nitroimidazole. VI. Lack of mutagenic activity of reduced metabolites and derivatives of ronidazole.

The potential toxicity of ronidazole residues present in the tissues of food-producing animals was assessed using the Ames mutagenicity test. Since ronidazole is activated by reduction, reduced derivatives of ronidazole and metabolites formed by enzymatic reduction of ronidazole were tested for mutagenicity. When tested at levels several orders of magnitude higher than that at which ronidazole was mutagenic, 5-amino-4-S-cysteinyl-1,2- dimethylimidazole , a product of the dithionite reduction of ronidazole in the presence of cysteine, the 5-N-acetylamino derivative of ronidazole and 5-amino-1,2- dimethylimidazole all lacked mutagenic activity in Ames strain TA100. The metabolites of ronidazole formed by the incubation of ronidazole with microsomes under anaerobic conditions were also not mutagenic. These data demonstrate that although ronidazole is a potent mutagen, residues from it which may be present in the tissues of food-producing animals lack any mutagenic activity.

Mutagenicity of benzylic acetates, sulfates and bromides of polycyclic aromatic hydrocarbons.

Studies were performed to determine the direct mutagenicity of the acetates and some bromides and sulfates of hydroxymethyl polycyclic aromatic hydrocarbons in S. typhimurium strains TA98 and TA100. Benzylic acetates, bromides and sulfates were synthesized and characterized. The compounds tested were benzyl alcohol, 5-hydroxymethylchrysene, 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, 6-(2-hydroxyethyl)benzo[a]pyrene, 6-hydroxymethylanthanthrene, 9-hydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, 7-hydroxymethylbenz[a]anthracene, 7-(2-hydroxyethyl)benz[a]anthracene, 12-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, 12-hydroxymethyl-7-methylbenz[a]anthracene, 1-hydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene, 3-hydroxy-3, 4-dihydrocyclopental[cd]pyrene and 4-hydroxy-3, 4-dihydrocyclopental[cd]pyrene. The benzylic sulfate esters of 6-hydroxymethylbenzo[a]pyrene and 7-hydroxymethylbenz[a]anthracene were the most mutagenic compounds, whereas the aliphatic sulfate ester of 7-hydroxyethylbenz[a]anthracene did not cause an increase in mutations above background. All meso-anthracenic benzylic acetate esters were mutagenic in both strains with various degrees of activity, whereas the corresponding non-benzylic esters were inactive, as expected. Of the non-meso-benzylic acetate esters, only the 3-acetoxy-3, 4-dihydrocyclopenta[cd]pyrene was mutagenic. In the benzylic bromide series, only the eight mesoanthracenic were mutagenic, whereas benzyl bromide and 5-bromomethylchrysene were inactive. The aliphatic bromides, 6-(2-bromoethyl)benzo[a]pyrene and 7-(2-bromoethyl)benz[a]anthracene did not display significant activity. The potencies of the acetate esters more accurately reflect the mutagenicity because the rate of solvolysis did not compete with the reactivity of the esters with bacterial DNA. In the case of benzylic sulfates and bromides, the rate of solvolysis was very rapid and could have diminished the level of mutagenicity, depending on the assay conditions. These results demonstrate that meso-anthracenic benzylic acetates, sulfates and bromides are mutagenic, whereas benzylic acetate esters attached to other carbon atoms are inactive.

Drug residue formation from ronidazole, a 5-nitroimidazole. I. Characterization of in vitro protein alkylation.

The metabolic activation of [14C]ronidazole by rat liver enzymes to metabolite(s) bound to macromolecules was investigated. The alkylation of protein by [14C]ronidazole metabolite(s) was catalyzed most efficiently by rat liver microsomes, in the absence of oxygen utilizing NADPH as a source of reducing equivalents. Based on a comparison of total ronidazole metabolized versus the amount bound to microsomal protein, approximately one molecule alkylates microsomal protein for every 20 molecules of ronidazole metabolized. Protein alkylation was strongly inhibited by sulfhydryl-containing compounds such as cysteine and glutathione whereas methionine had no effect. Based on HPLC analysis of ronidazole, cysteine was found not to inhibit microsomal metabolism of ronidazole ruling out a decrease in the rate of production of the reactive metabolite(s) as the mechanism of cysteine inhibition.

Drug residue formation from ronidazole, a 5-nitro-imidazole. IV. The role of the microflora.

When radioactive 1-methyl-5-nitroimidazole-2-methanol carbamate, ronidazole, labeled at the 4,5-ring positions was administered orally to germ-free and conventional rats, a much larger fraction of the radioactivity was excreted in the feces of the conventional animals. Determination of the total radioactive residues present in the carcass, blood, plasma, liver, fat and kidney 5 days after dosing indicated that the carcass of the germ-free animals contained a greater quantity of residue than that of conventional rats. On the other hand, the blood of the conventional animals contained a much higher level of radioactivity than that of the germ-free animals. These results show that while the microflora influence the distribution of the drug their presence is not obligating for the formation of persistent tissue residues in rats dosed with ronidazole.

Identification of in vitro (rat liver postmitochondrial S9 fraction) metabolites of the antiprotozoal agent 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole .

Twelve in vitro oxygenated metabolites of 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole (MK-0436) were produced by incubation of this antiprotozoal agent with the postmitochondrial supernatant (S9) fraction isolated from the livers of rats treated with phenobarbital. Metabolite structure elucidation was achieved using NMR and mass spectrometry. Seven monohydroxy and two dihydroxy metabolites were fully characterized; two other metabolites were partially characterized as dihydroxy derivatives of the drug. The major in vitro metabolite is the 5 axial hydroxy compound, and a minor metabolite is the corresponding ketone. In all cases metabolite formation involved biotransformation on the hexahydrobenzisoxazole ring.