Toxicological evaluation of brominated vegetable oil in Sprague Dawley rats.

Brominated vegetable oil (BVO) has been approved by the US Food and Drug Administration on an interim basis as a food additive. Past studies have raised concerns about potential toxicities from consuming BVO. To investigate further these toxicities, we conducted a 90-day dietary exposure study in Sprague Dawley rats and analyzed tissue distribution of the main metabolites. Six-week-old male and female rats were fed diets containing 0 (control), 0.002%, 0.02%, 0.1%, or 0.5% BVO by weight. Statistically significant increases were observed in the serum bromide in the high-dose group of both sexes and in the incidence of thyroid follicular cell hypertrophy in the two highest dose groups of males and the high-dose group of females. An increase in serum TSH was observed in the high-dose group for both sexes, as well as a decrease in serum T4 in the high-dose males. A clear dose-response was observed in di- and tetra-bromostearic acid levels in the heart, liver, and inguinal fat. These data expand upon previous observations in rats and pigs that oral exposure to BVO is associated with increased tissue levels of inorganic and organic bromine, and that the thyroid is a potential target organ of toxicity.

Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats.

Furan is a food processing contaminant found in many common cooked foods that induces liver toxicity and liver cancer in animal models treated with sufficient doses. The metabolism of furan occurs primarily in the liver where CYP 2E1 produces a highly reactive bis-electrophile, cis-2-butene-1,4-dial (BDA). BDA reacts with nucleophilic groups in amino acids and DNA in vitro to form covalent adducts. Evidence for BDA-nucleoside adduct formation in vivo is limited but important for assessing the carcinogenic hazard of dietary furan. This study used controlled dosing with furan in Fischer 344 rats to measure serum and liver toxicokinetics and the possible formation of BDA-nucleoside adducts in vivo. After gavage exposure, furan concentrations in the liver were consistently higher than those in whole blood (∼6-fold), which is consistent with portal vein delivery of a lipophilic compound into the liver. Formation of BDA-2'-deoxycytidine in furan-treated rat liver DNA was not observed using LC/MS/MS after single doses as high as 9.2 mg/kg bw or repeated dosing for up to 360 days above a consistent background level (1-2 adducts per 10(8) nucleotides). This absence of BDA-nucleoside adduct formation is consistent with the general lack of evidence for genotoxicity of furan in vivo.

Comparative carcinogenicity of 4-aminobiphenyl and the food pyrolysates, Glu-P-1, IQ, PhIP, and MeIQx in the neonatal B6C3F1 male mouse.

The tumorigenic activities of four representative heterocyclic amine food pyrolysates, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), were assessed in the neonatal male B6C3F1 mouse and were compared with that of the potent human carcinogen, 4-amino-biphenyl (4-ABP). These aromatic amines were administered by i.p. injection at two dose levels on days 1, 8, and 15 after birth; and the incidence of tumors was examined at 8 and 12 months. Glu-P-1, IQ, PhIP, MeIQx, and 4-ABP each induced a significant incidence of hepatic adenomas, as compared to the solvent-treated (DMSO) control. Hepatocellular carcinomas were also observed with 4-ABP, SO, and MeIQx. Overall tumorigenicity was in the order: 4-ABP greater than Glu-P-1 greater than IQ approximately PhIP greater than MeIQx greater than DMSO. In the neonatal B6C3F1 mouse, these heterocyclic aromatic amines showed potent tumorigenicity after 8 and 12 months at total doses that were 5-10,000-fold less than those employed in standard chronic bioassays.

Inhibitory effect of caloric restriction on tumorigenicity induced by 4-aminobiphenyl and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) in the CD1 newborn mouse bioassay.

The tumorigenicity of 4-aminobiphenyl (4-ABP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were studied in combination with caloric restriction in the male neonatal CD1 mouse bioassay. 4-ABP and PhIP exhibited moderate and weak tumorigenicity, respectively, in ad libitum fed mice; however, none of the caloric restricted mice developed tumors. These results indicate that caloric restriction, even when begun 3 months after the conclusion of compound treatment, markedly inhibited 4-ABP- and PhIP-induced tumors in the CD1 mouse.

Tumorigenicity and liver tumor ras-protooncogene mutations in CD-1 mice treated neonatally with 1- and 3-nitrobenzo[a]pyrene and their trans-7,8-dihydrodiol and aminobenzo[a]pyrene metabolites.

The environmental pollutants 1- and 3-nitrobenzo[a]pyrene (1- and 3-NBaP) are metabolized by mammalian microsomes through ring oxidation to 1-NBaP trans-7,8-dihydrodiol and 3-NBaP trans-7,8-dihydrodiol, and by nitroreduction to 1- and 3-aminobenzo[a]pyrene. To determine if these compounds are tumorigenic, 1- and 3-NBaP, along with several of their metabolites and the parent benzo[a]pyrene (BaP) and its trans-7,8-dihydrodiol metabolite, were tested in the neonatal CD-1 mouse bioassay. Male mice were administered i.p. injections at a total dose of 100 or 400 nmol per mouse on 1, 8 and 15 days after birth. While the liver tumor incidences for BaP, BaP trans-7,8-dihydrodiol, and the positive control 6-nitrochrysene (6-NC) were significantly higher than in the solvent control animals, all the other tested compounds exhibited no tumorigenicity. The frequency of Ha- and Ki-ras mutations in liver tumors of mice treated with BaP, BaP trans-7,8-dihydrodiol, and 6-NC were higher than in the few liver tumors isolated from control mice or mice treated with the NBaPs or their metabolites. Since 1- and 3-NBaP and their metabolites are potent mutagens in the Salmonella assay and moderate mutagens in the Chinese hamster ovary (CHO) mammalian mutagenicity assay, our results indicate that the in vitro mutagenicity of these compounds does not correlate with their tumorigenicity.

Potent tumorigenicity of 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene in the neonatal B6C3F (1) male mouse.

The tumorigenicity of 7-chlorobenz[a]anthracene (7-Cl-BA, and environmental contaminant, and 7 bromobenz[a]anthracene (7-Br-BA) was determines in the male B6C3F(1) newborn mouse. Mice receiving 7-Cl-BA and 7-Br-BA by i.p. injections at a dose of 1600 nmol per mouse on 1, 8, and 15 days after birth developed 92 and 96% hepatocellular adenomas, and 100 and 83% hepatocellular carcinoma, respectively. Metabolism by liver microsomes of 15-day-old mice each produced the corresponding trans-3,4-dihydrodiol. Analysis by (32)P-postlabeling/HPLC indicated the presence of DNA adducts derived from 7-Cl-BA trans-3,4-dihydrodiol and 7-Br-BA trans-3,4-dihydrodiol. Our results indicate that both 7-Cl-BA and 7-Br-BA are potent carcinogens and that bay-region diol epoxides are the ultimate metabolites that lead to DNA adduct formation and tumor initiation.

Liver tumors induced in B6C3F1 mice by 7-chlorobenz[a]anthracene and 7-bromobenz[a]anthracene contain K-ras protooncogene mutations.

We previously examined the tumorigenicity of 7-chlorobenz[a]anthracene (7-Cl-BA) and 7-bromobenz[a]anthracene (7-Br-BA) in the neonatal mouse bioassay and found that 7-Cl-BA and 7-Br-BA induced hepatocellular adenoma in 92 and 96% of the mice and hepatocellular carcinoma in 100 and 83% of the mice, respectively. In the present study, mRNA was isolated from each of the liver tumors induced by the two compounds and reverse-transcribed to cDNA. Portions of the K- and H-ras oncogene coding sequences were then amplified and analyzed for DNA sequence alterations. Eighty-three percent (20/24) of 7-Cl-BA-induced and 91% (20/22) of 7-Br-BA-induced liver tumors had activated ras protooncogenes. In contrast to the general finding of H-ras mutations in B6C3F1 mouse liver tumors, both compounds had 95% (19/20) of the mutations located at the first base of K-ras codon 13, resulting in a pattern of GGC --> CGC. Thus, our results demonstrate that 7-Cl-BA and 7-Br-BA induce a unique type of ras (K-ras) oncogene activation in liver tumors of B6C3F1 mice.

Tumorigenicity of nitropolycyclic aromatic hydrocarbons in the neonatal B6C3F1 mouse bioassay and characterization of ras mutations in liver tumors from treated mice.

The nitropolycyclic aromatic hydrocarbons (nitro-PAHs) 1-, 2-, and 3-nitrobenzo[a]pyrene, 1- and 3-nitrobenzo[e]pyrene, 2- and 3-nitrofluoranthene, 9-nitrodibenz[a,c]anthracene, and two of the parent PAHs fluoranthene and dibenz[a,c]anthracene were tested for tumorigenicity in the neonatal male B6C3F1 mouse. 6-Nitrochrysene was used as a positive control. Mice were administered three intraperitoneal injections of test agent (400 nmol total) on 1, 8, and 15 days after birth and evaluated for liver and lung tumors at 12 months of age. 2-Nitrobenzo[a]pyrene and 6-nitrochrysene induced a high incidence of liver tumors (91-100%), while the remaining test compounds did not induce tumors at a rate significantly higher than the solvent control. 6-Nitrochrysene was the only test agent to produce a significant increase in the frequency of lung tumors. K- and H-ras mutations were analyzed in liver tumors of treated mice and mainly occurred at the first base of K-ras codon 13, resulting in GGC --> CGC transversion. Since most of the tested nitro-PAHs are mutagens in vitro, the results of this study indicate that the in vitro mutagenicity of these compounds does not correlate with their tumorigenicity in the neonatal B6C3F1 mouse bioassay. Also, the results indicate that liver tumors from mice treated with nitro-PAHs possess ras mutations typical of PAHs and their derivatives.

Induction of rat hepatic cytochromes P-450 by environmental nitropolycyclic aromatic hydrocarbons.

Nitrated polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that result from various incomplete combustion processes. We have examined the activity of hepatic microsomal enzymes in rats pretreated with a series of environmentally occurring nitrated PAHs including: 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 6-nitrochrysene, 7-nitrobenz[a]anthracene, 3-nitrofluoranthene, and 1-, 3-, and 6-nitrobenzo[a]pyrene. None of the compounds increased the cytochrome P-450 content more than 2-fold. 1,8-Dinitropyrene, 6-nitrochrysene, and 1- and 3-nitrobenzo[a]pyrene significantly increased arylhydrocarbon hydroxylase activity 2- to 8-fold higher than solvent-treated controls. The induction of 7-ethoxycoumarin O-deethylase activity paralleled that found with arylhydrocarbon hydroxylase. The maximum induction of aminopyrine N-demethylase was only 1.5-fold, and none of the nitrated PAHs caused significant increases in epoxide hydrase or NADPH-cytochrome c reductase. 1-Nitropyrene reductase activity was induced by each of the compounds with the exception of 6-nitrobenzo[a]pyrene. The greatest increase was caused by 1-nitrobenzo[a]pyrene followed by 1,3-dinitropyrene, 3-nitrobenzo[a]pyrene and 6-nitrochrysene. These data suggest that nitrated PAHs may potentiate the effects of subsequent exposures to various chemical carcinogens.

DNA adducts and carcinogenicity of nitro-polycyclic aromatic hydrocarbons.

We have been interested in the structure-activity relationships of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs), and have focused on the correlation of structural and electronic features with biological activities, including mutagenicity and tumorigenicity. In our studies, we have emphasized 1-, 2-, 3-, and 6-nitrobenzo[a]pyrenes (nitro-B[a]Ps) and related compounds, all of which are derived from the potent carcinogen benzo[a]pyrene. While 1-, 2-, and 3-nitro-B[a]P are potent mutagens in Salmonella, 6-nitro-B[a]P is a weak mutagen. In vitro metabolism of 1- and 3-nitro-B[a]P has been found to generate multiple pathways for mutagenic activation. The formation of the corresponding trans-7,8-dihydrodiols and 7,8,9,10-tetrahydrotetrols suggests that 1- and 3-nitro-B[a]P trans-7,8-diol-9,10-epoxides are ultimate metabolites of the parent nitro-B[a]Ps. We have isolated a DNA adduct from the reaction between 3-nitro-B[a]P trans-7,8-diol-anti9,10-epoxide and calf thymus DNA, and identified it as 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-ni tro-B[a]P . The same adduct was identified from in vitro metabolism of [3H]3-nitro-B[a]P by rat liver microsomes in the presence of calf thymus DNA. A DNA adduct of 3-nitro-B[a]P formed from reaction of N-hydroxy-3-amino-B[a]P, prepared in situ with calf thymus DNA was also isolated. This adduct was identified as 6-(deoxyguanosin-N2-yl)-3-amino-B[a]P. The same adduct was obtained from incubating DNA with 3-nitro-B[a]P in the presence of the mammalian nitroeductase, xanthine oxidase, and hypoxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of isomeric nitrobenzo[a]pyrenes leading to DNA adducts and mutagenesis.

We have been interested in determining the structural and electronic features that may be useful in predicting the mutagenic activity of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs). We have previously found that a correlation between structural and electronic features and direct-acting mutagenicity in Salmonella typhimurium cannot be made using nitro-PAHs with different molecular size. In this study, a series of structurally related nitro-PAHs, the environmental contaminants 1-, 3-, and 6-nitrobenzo[alpha]pyrene (NBaP) and their derivatives, was used to determine structure-activity relationships. It was found that isomeric NBaPs are activated to DNA damaging and mutagenic derivatives by nitroreduction, ring-oxidation, or by a combination of these two pathways. A general finding was that NBaPs and derivatives with their nitro substituent oriented perpendicular to the aromatic system exhibit either very weak or no direct-acting mutagenicity in S. typhimurium strains TA98 and TA100. In this paper, we also discuss the effect of the location of the nitro group on the metabolism and the mutagenicity of NBaPs and the effect of oxygen-containing functional groups on the mutagenicity of NBaP derivatives. These findings provide a useful molecular basis for interpreting and predicting the direct-acting mutagenicity of nitro-PAHs.

Comparative direct-acting mutagenicity of 1- and 2-nitropyrene: evidence for 2-nitropyrene mutagenesis by both guanine and adenine adducts.

The mutations and DNA adducts produced by the environmental pollutant 2-nitropyrene were examined in Salmonella typhimurium tester strains. 2-Nitropyrene was a stronger mutagen than its extensively studied structural isomer 1-nitropyrene in strains TA96, TA97, TA98, TA100, TA102, TA104 and TA1538. Both 1- and 2-nitropyrene were essentially inactive in TA1535. The mutagenicity of 1- and 2-nitropyrene in TA98 was much higher than in TA98NR and the activity of these compounds in TA100 was much higher than in TA100NR. While 1-nitropyrene exhibited similar mutagenicity in strains TA98 and TA98/1,8-DNP6, the mutagenicity of 2-nitropyrene in TA98/1,8-DNP6 was much lower than in TA98. Analysis of DNA from TA96 and TA104 incubated with 2-nitropyrene indicated the presence of two adducts, N-(deoxyguanosin-8-yl)-2-aminopyrene and N-(deoxyadenosin-8-yl)-2-aminopyrene. The results suggest that 2-nitropyrene is metabolized by bacterial nitroreductase(s) to N-hydroxy-2-aminopyrene, and possibly by activation to a highly mutagenic O-acetoxy ester. DNA adduct formation with deoxyguanosine and deoxyadenosine correlates with the mutagenicity of 2-nitropyrene in tester strains possessing both G:C and A:T mutational targets.

Characterization of DNA adducts in Chinese hamster ovary cells treated with mutagenic doses of 1- and 3-nitrosobenzo[a]pyrene and the trans-7,8-diol-anti-9,10-epoxides of 1- and 3-nitrobenzo[a]pyrene.

The environmental contaminants 1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are mutagens in Chinese hamster ovary (CHO) cells with exogenous metabolic activation. Previous studies demonstrated the potent direct-acting mutagenicity of the oxidized metabolites, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-1-nitrobenzo[a] pyrene (1-NBaPDE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydro-3-nitrobenzo[a]pyrene (3-NBaPDE), and the partially nitroreduced metabolites, 1- and 3-nitrosobenzo[a]pyrene (1- and 3-NO-BaP). In this study, we have identified the major adduct formed by incubation of calf thymus DNA with 1-NBaPDE and used this standard in conjunction with other adduct standards to characterize the 32P-postlabeled DNA adducts produced by 1- and 3-nitro-BaP metabolites in CHO cultures. The major adduct from 1-NBaPDE exposure was 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-1- nitrobenzo[a]pyrene; from 3-NBaPDE, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3- nitrobenzo[a]pyrene; from 1-NO-BaP, 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene; and from 3-NO-BaP, 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene. For comparison, the adducts formed by trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the related nitroreduced derivative 6-nitrosobenzo[a]pyrene were also examined. The nitrobenzo[a]pyrene DNA adducts described in this study are proposed to be involved in the mutagenicity of 1- and 3-nitro-BaP upon either oxidative or reductive metabolism.

Immunochemical, 32P-postlabeling, and GC/MS detection of 4-aminobiphenyl-DNA adducts in human peripheral lung in relation to metabolic activation pathways involving pulmonary N-oxidation, conjugation, and peroxidation.

4-Aminobiphenyl (ABP) is a recognized human bladder carcinogen, whose presence in cigarette smoke results in DNA adduct formation in the human urothelium. Since preliminary studies indicated that even higher levels of ABP-DNA adducts may be present in human peripheral lung, we utilized a sensitive immunochemical assay, in combination with 32P-postlabeling, to quantify the major 4-aminobiphenyl (ABP)-DNA adduct, N-(guan-8-yl)-ABP, in surgical samples of peripheral lung tissue from smokers and ex-smokers. No differences in adduct levels were detected between smokers and ex-smokers by immunoassay. In contrast, the 32P-postlabeling method showed statistically significant differences between adduct levels in smokers and ex-smokers; however, a relatively high background of smoking-related adducts chromatograph near the major ABP adducts and may compromise estimation of the level of ABP-DNA adducts in smokers. Furthermore, the levels measured by 32P-postlabeling were 20- to 60-fold lower than that measured by immunoassay. Since 32P-postlabeling may underestimate and immunochemical assays may overestimate adduct levels in the lung, selected samples were also evaluated by GC/MS. The immunochemical and GC/MS data were concordant, leading us to conclude that N-(guan-8-yl)-ABP adducts were not related to smoking status. Since ABP-DNA adduct levels in human lung did not correlate with smoking status as measured by immunoassay and GC/MS, the metabolic activation capacity of human lung microsomes and cytosols was examined to determine if another exposure (e.g., 4-nitrobiphenyl) might be responsible for the adduct. The rates of microsomal ABP N-oxidation were below the limit of detection, which was consistent with a lack of detectable cytochrome P4501A2 in human lung. N-Hydroxy-ABP O-acetyltransferase (but not sulfotransferase) activity was detected in cytosols and comparative measurements of N-acetyltransferase (NAT) using p-aminobenzoic acid and sulfamethazine indicated that NAT1 and NAT2 contributed to this activity. 4-Nitrobiphenyl reductase activity was found in lung microsomes and cytosols, with the reaction yielding ABP and N-hydroxy-ABP. Lung microsomes also demonstrated high peroxidative activation of ABP, benzidine, 4,4'-methylene-bis(2-chloroaniline), 2-aminofluorene, and 2-naphthylamine. The preferred co-oxidant was hydrogen peroxide and the reaction was strongly inhibited by sodium azide but not by indomethacin or eicosatetraynoic acid, which suggested the primary involvement of myeloperoxidase rather than prostaglandin H synthase or lipoxygenase. This was confirmed by immunoinhibition and immunoprecipitation studies using solubilized human lung microsomes and antisera specific for myeloperoxidase. These data suggest that ABP-DNA adducts in human lung result from some environmental exposure to 4-nitrobiphenyl. The bioactivation pathways appear to involve: (1) metabolic reduction to N-hydroxy-ABP and subsequent O-acetylation by NAT1 and/or NAT2; and (2) metabolic reduction to ABP and subsequent peroxidation by myeloperoxidase. The myeloperoxidase activity appears to be the highest peroxidase activity measured in mammalian tissue and is consistent with the presence of neutrophils and polymorphonuclear leukocytes surrounding particulate matter derived from cigarette smoking.

High-performance liquid chromatographic separation of ring-oxidized metabolites of nitro-polycyclic aromatic hydrocarbons.

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are widespread genotoxic environmental pollutants, which require metabolic activation to exert their biological activities. Metabolism of nitro-PAHs generates ring-oxidized metabolites including epoxides, phenols, dihydrodiols and tetrahydrotetrols. Separation of the oxidized metabolites and related compounds of a series of isomeric nitro compounds derived from anthracene, benz[a]anthracene, benzo[a]pyrene and benzo[e]pyrene was studied by high-performance liquid chromatography (HPLC) of different types of columns (monomeric and polymeric; reversed-phase and normal-phase). In the reversed-phase HPLC system, the general elution order of these compounds is: parent nitro-PAHs greater than phenolic derivatives greater than epoxides greater than dihydrodiols greater than tetrahydrotetrols. Among the geometric isomers, trans-dihydrodiols with both hydroxyl groups at the quasiaxial positions were eluted earlier than those with the hydroxyl groups at the quasiequatorial positions. Orientation of the nitro substituent has also been found to be an important structural feature for determining the relative retention order. Among the geometric isomers of nitro-PAHs and trans-dihydrodiols, the isomers with their nitro groups perpendicular or nearly perpendicular to the aromatic rings were eluted faster than the analogues with their nitro groups parallel or nearly parallel to the aromatic rings. Normal-phase HPLC gave opposite retention order, but with different separability among some of the compounds. Therefore, combination of both reversed- and normal-phase HPLC provides efficient separation of the ring-oxidized derivatives of nitro-PAHs. Results are also presented to compare the separation efficiency among different types of columns used. The results suggest that the polarity of solutes is the principal factor for determining their HPLC retention time.

Stereoselective metabolism of 9-methyl-, 9-hydroxymethyl- and 9,10-dimethylanthracenes: absolute configurations and optical purities of trans-dihydrodiol metabolites.

Stereoselective metabolism of 9-methylanthracene (9-M-A), 9-hydroxymethylanthracene (9-OHM-A) and 9,10-dimethylanthracene (9,10-DM-A) by liver microsomes from untreated rats, and from rats pretreated with either 3-methylcholanthrene or phenobarbital was studied. The metabolites were separated by h.p.l.c. and characterized by analysis of the u.v.-visible, mass and n.m.r. spectral data and compared to published spectral data. The identified trans-dihydrodiol metabolites were 9-M-A trans-1,2- and 3,4-dihydrodiols, 9-OHM-A trans-1,2- and 3,4-dihydrodiols, and 9,10-DM-A trans-1,2-dihydrodiol. The absolute configuration and optical (enantiomeric) purity of the trans-dihydrodiol metabolites were determined. In order to assist in determining the absolute configuration of these trans-dihydrodiols, metabolism of 9-bromoanthracene by liver microsomes of rats pretreated with 3-methylcholanthrene was performed and its trans-1,2- and 3,4-dihydrodiol metabolites were determined to both possess an R,R absolute configuration. Absolute configurations of the trans-dihydrodiol metabolites of 9-methylanthracene, 9-hydroxymethylanthracene and 9,10-dimethylanthracene were then determined by comparison of their CD spectra with those of anthracene 1R,2R-dihydrodiol or 9-bromoanthracene 1R,2R-dihydrodiol. The optical purities of the trans-dihydrodiol metabolites were determined by analysis of the chiral stationary phase h.p.l.c. profiles of the dihydrodiols or their corresponding tetrahydrodiol derivatives. The results indicated that the R,R enantiomers were the predominant products and that liver microsomes of rats pretreated with 3-methylcholanthrene exhibited much higher stereoselectivity than the liver microsomes of untreated rats or rats pretreated with phenobarbital in the formation of trans-dihydrodiols from anthracene, 9-methylanthracene, 9-hydroxymethylanthracene and 9,10-dimethylanthracene. The methyl and hydroxymethyl substituents slightly decreased the stereoselectivity of the trans-dihydrodiol formation.

Stereoselective metabolism of 7-chlorobenz(a)anthracene by rat liver microsomes. Absolute configurations and optical purities of trans-dihydrodiol metabolites.

Metabolism of 7-chlorobenz(a)anthracene (7-Cl-BA) by liver microsomes from untreated rats, and from rats pretreated with either 3-methylcholanthrene or phenobarbital was studied. The metabolites were isolated by HPLC and characterized by UV-visible, mass, and proton NMR spectral analyses and identified as 7-Cl-BA trans-3,4-, 5,6-, and 8,9-dihydrodiols, 3-hydroxy-7-Cl-BA, and 4-hydroxy-7-Cl-BA. Proton NMR spectral analysis indicated that 7-Cl-BA trans-3,4-dihydrodiol preferentially adopted a quasidiequatorial conformation while trans-5,6- and 8,9-dihydrodiols preferentially adopted a quasidiaxial conformation. Comparison of circular dichroism spectra with those of 7-bromobenz(a)anthracene trans-dihydrodiol metabolites of known absolute stereochemistry indicated that the major enantiomeric 7-Cl-BA trans-3,4-, 5,6-, and 8,9-dihydrodiols had R,R absolute configuration. Application of chiral stationary phase HPLC for direct resolution of the trans-dihydrodiols and their hydrogenated and dechlorinated derivatives enabled determination of the optical purity of each dihydrodiol metabolite obtained from the three microsomal systems. In vitro incubation of 7-Cl-BA under molecular oxygen-18 produced 7-Cl-BA trans-3,4-, 5,6-, and 8,9-dihydrodiols, each containing one oxygen-18 atom. Mass spectral analysis of the dehydration products of the oxygen-18-containing trans-dihydrodiol metabolites indicated that 7-Cl-BA 3S,4R-epoxide and 7-Cl-BA 8R,9S-epoxide were the predominant enantiomeric intermediates.

Metabolism of 9-nitroanthracene by rat liver microsomes: identification and mutagenicity of metabolites.

Aerobic metabolism of 9-nitroanthracene by uninduced rat liver microsomes produced four metabolites identified as trans-1,2- and 3,4-dihydrodiols, 1,2,3,4-tetrahydrotetrol of 9-nitroanthracene, and anthraquinone. Further metabolism of the predominant metabolite, 9-nitroanthracene trans-3,4-dihyrodiol, yielded a 1,2,3,4-tetrahydrotetrol with a trans-cis-trans configuration, indicating that a trans-dihydrodiol anti-epoxide is formed as an intermediate. The mutagenic activities of both dihydrodiols and 9-nitroanthracene in strains TA98 and TA100, both in the presence and in the absence of S9 enzymes, were very low. When 9-nitroanthracene was metabolized under anaerobic conditions, nitroreduction did not occur. The results thus explain the weak mutagenic activity of 9-nitroanthracene.