Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus).

1. The in vitro metabolism of [3H]benzo[a]pyrene (BP) and [14C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo. 2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of of BP-7,8-diol (predominantly the [-] enantiomer) and smaller amounts of BP-4,5-diol. 3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides. 4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates. 5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25-30 days. The major adduct was anti-BPDE-deoxyguanosine.     

Effect of glutathione and uridine 5'-diphosphoglucuronic acid on mutagenesis by benzo[a]pyrene and aflatoxin B1 in the Salmonella/microsome assay

In the Salmonella/microsome plate or liquid assay, the addition of glutathione (GSH) and uridine 5'-diphosphoglucuronic acid (UDPGA), both cofactors for GSH-S-transferases or UDPGA-transferases, altered the rat-liver microsome-mediated mutagenesis of benzo[a]pyrene (BP) and aflatoxin B1 (AFB). With either BP or AFB, an increased, unchanged or decreased number of revertant colonies of S. typhimurium was observed, depending on the substrate concentration, the source of rat-liver 9000 X g supernatant (S9), the time of incubation and the type of mutagenicity test (liquid or plate assay). Several factors responsible for quantitative changes in the pattern of BP and AFB metabolites under various assay conditions in vitro, which alter the overall mutagenic activity of the parent compound, are discussed.     

Selenium modified mutagenicity and metabolism of benzo[a]pyrene in an S9-dependent system

Selenium added to the incubation mix containing rat-liver S9 modified both the metabolism and mutagenicity of benzo[a]pyrene (BaP) and several of its metabolites. Selenium (Na2SeO3) inhibited the S9-dependent mutagenic effects of BaP on Salmonella typhimurium strain TA100 as indicated by the number of histidine-dependent revertants counted. This inhibition was concentration-dependent over a range of 12.5 to 100 ppm. When used as the substrate the BaP metabolites 7,8-dihydrodiol, 9,10-dihydrodiol and 3-hydroxy also produced significantly fewer revertants in TA100 when selenium was included in the incubation mix. High-performance liquid chromatographic analysis of metabolites from S9-dependent metabolism of BaP indicated that selenium inhibited the formation of 3-hydroxy-BaP, 9,10-dihydrodiol, 7,8-dihydrodiol, 1,3- and 3,6-quinone. Eluting samples on an alumina column to isolate the conjugated metabolites showed that selenium caused 12% less binding to glucuronides, no significant differences in binding to sulfate esters or glutathione but the amount of unmetabolized BaP and unconjugated metabolites was increased by 48%. These results suggest that selenium inhibits S9-dependent BaP metabolism therefore reducing the mutagenic effects of this compound.     

Inhibition of the mutagenicity of benzo[a]pyrene in the V79/HGPRT system by bioantioxidants

In the presence of metabolic activation (S9 microsomal fraction of mouse-liver homogenate) the mutagenicity of benzo[a]pyrene (BP) in Chinese hamster V79 cells was inhibited by the phenolic bioantioxidants (BA) Dibunol (2,6-di-tert-butyl-4-methylphenol-D) and 5-methylresorcine(5-MR). The mixture BP + D and BP + 5-MR at molar ratios of 1:1 and 1:85 respectively showed no mutagenic activity compared to the control. One can assume that D and 5-MR inhibited BP-induced mutagenesis by binding the free radicals of BP metabolites with the formation of less active phenolic derivatives and also by linkage with cytochrome P-450, which prevents further metabolic activation of BP.     

Host-mediated mutagenicity experiments with benzo[a]pyrene and two of its metabolites

Benzo[a]pyrene (BP) and two of its major metabolites, the ultimate mutagen BP-4,5-oxide and the proximate mutagen trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) were investigated for mutagenicity in Salmonella typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-mediated assay. BP and BP-4,5-oxide were not mutagenic under any experimental conditions. BP-7,8-diol was inactive with the strain TA1538 but was mutagenic with the strains TA98 and TA100. The effect was potentiated by pretreatment of the host mice with the cytochrome P-450 inducer 5,6-benzoflavone. We conclude: (i) one of the reasons for the observed insensitivity of the intrasanguineous host-mediated assay towards BP is that BP-4,5-oxide, which contributes to the microsome-mediated mutagenicity of BP, is inactive in the host-mediated assay; (ii) the finding that BP-7,8-diol is mutagenic in the host-mediated assay demonstrates that the lack of mutagenicity of BP is not intrinsic; (iii) the potentiated mutagenicity after treatment of the hosts with 5,6-benzoflavone suggests that cytochrome P-450 is more important in the activation of BP-7,8-diol in this system than other enzymes (e.g. prostaglandin synthase) that can also activate this compound in vitro.     

Effect of vitamins A, C and glutathione on the mutagenicity of benzo[a]pyrene mediated by S9 from vitamin A-deficient rats

Vitamin A deficiency has been shown to enhance the mutagenicity of benzo[a]pyrene (Narbonne et al., 1985). Here we report that this is not a result of increased benzo[a]pyrene metabolism but might be a consequence of either a lack of vitamin A or a decreased level of scavengers (ascorbic acid and glutathione) in the liver. However, the addition of vitamin A in vitro in the form of retinyl palmitate strongly inhibits the benzo[a]pyrene mutagenicity. An enhancing effect on the mutagenicity of benzo[a]pyrene is observed with addition of ascorbic acid when incubated with high amounts of the precarcinogen. In vivo addition of high levels of glutathione also reduces the mutagenicity of benzo[a]pyrene.     

Mutagenicity testing of seminal fluid: seminal fluid increases the mutagenicity of the precursor mutagen benzo[a]pyrene in the presence of S9 mix

Small amounts of seminal fluid strongly enhanced the mutagenicity of the precursor mutagen benzo[a]pyrene (BP) in the Salmonella/microsome test. This previously unreported effect was found only in the presence of S9 mix for metabolic activation. The increase far exceeded the additive effect expected from experiments where seminal fluid and BP were tested separately with S9 mix. Testing of the direct-acting mutagen 4-nitro-o-phenylene-diamine (NPD) together with seminal fluid resulted in a lower mutagenic activity than that of NPD alone. Seminal fluid had a bactericidal effect on the Salmonella bacteria, thus only volumes up to 40 microliter could be used per plate. The mutagenic effect of only seminal fluid and S9 mix was slightly increased over controls in a standard Ames test, but was equal to the spontaneous mutation rate with a preincubation test modified according to Kado and coworkers. There were no significant differences between seminal plasma from smokers and non-smokers in any experimental series. Seminal fluid concentrated 20-fold by extraction with the mutagen-removing adsorbant Mutasorb did not have any enhancing effect on the mutagenicity of BP, nor did it exhibit any mutagenic activity in itself with or without S9 mix.     

Carcinogenicity and metabolic profiles of 6-substituted benzo[a]pyrene derivatives on mouse skin

The ability was tested of appropriate substituents of benzo[a]pyrene (BP) at C-6 to decrease or suppress the carcinogenic activity for these BP derivatives relative to the parent compound. 8-week-old female Swiss mice in 9 groups of 30 were treated on the back with 0.2 mumol of compound in acetone 4 times weekly for 20 weeks. The following compounds were administered: BP, 6-methylbenzo[a]pyrene (BP-6-CH3), 6-hydroxymethylbenzo[a]pyrene (BP-6-CH2OH), benzo[a]pyrene-6-carboxaldehyde (BP-6-CHO), benzo[a]pyrene-6-carboxylic acid, 6-methoxybenzo[a]pyrene, 6-acetoxybenzo[a]pyrene, 6-bromobenzo[a]pyrene, and 6-iodobenzo[a]pyrene. Two additional groups received BP or BP-6-CH3 twice weekly for 20 weeks at a total dose 25% of that above. In addition, the metabolism of selected 6-substituted BP derivatives was studied, using mouse skin homogenates in vitro and mouse skin in vivo. Only four compounds were carcinogenic; the order of potency was BP greater than BP-6-CH3 greater than BP-6-CH2OH and BP-6-CHO. The difference in carcinogenicity between BP-6-CH2OH and BP-6-CHO could not be assessed by this experiment. In a further tumorigenesis experiment the carcinogenicity of BP-6-CH2OH was compared to that of BP-6 CHO, BP-6-CH3 and 6-hydroxymethylbenzo[a]pyrere sulfate ester (BP-6-CH2OSO3Na) on mouse skin. 9-week-old female Swiss mice in groups of 28 were treated at three dose levels with 0.8, 0.2 and 0.05 mumol of compounds in dioxane--dimethyl sulfoxide (75 : 25) twice weekly for 40 weeks. After 40 experimental weeks BP-6-CH2OSO3Na proved to be a more potent carcinogen than BP-6-CH2OH, which, in turn was more active than BP-6-CHO. The greater carcinogenicity of BP-6-CH3 relative to BP-6-CH2OH and BP-6-CHO is confirmed, suggesting that BP-6-CH2OH is not a proximate carcinogenic metabolite for BP-6-CH3. Since BP-6-CHO is a weaker carcinogen than BP-6-CH2OH and is efficiently reduced metabolically to BP-6-CH2OH, the latter compound may be a common proximal carcinogenic metabolite. The stronger potency of BP-6-CH2OSO3Na, compared to its alcohol, suggests that an ester of BP-6-CH2OH might be the ultimate alkylating compound reacting with cellular nucleophiles.     

Mutagenicity of some methylated benzo[a]pyrene derivatives

The mutagenicity of benzo[a]pyrene (BP) and a number of methylated derivatives towards Salmonella typhimurium has been tested. The most mutagenic derivative tested was 6-methylbenzo[a]pyrene which produced about twice the number of revertants as did BP, 11-Methylbenzo[a]pyrene was slightly more mutagenic than BP. All the other compounds tested (7-, 8-, 9- and 10-methylbenzo[a]pyrene and 7,8- and 7,10-dimethylbenzo[a]pyrene) were significantly less active than benzo[a]pyrene. With the exception of 6-methylbenzo[a]pyrene, these results closely parallel the known carcinogenicity of the methylated benzo[a]pyrenes, and support the view that metabolic activation of BP may involve the 7-10 positions which are blocked in the methylated compounds.     

The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[alpha]pyrene in Salmonella typhimurium strains

The comutagenic and desmutagenic effect of quercetin on the mutagenicity of typical mutagens e.g. 2-acetylaminofluorene (AAF), 4-nitroquinoline-1-oxide (4NQO) and benzo[alpha]pyrene (B[a]P), in Salmonella typhimurium TA98, TA100 and TA98/1,8 DNP6 were examined. In the mixed application of AAF with quercetin in the presence of mammalian metabolic activation system (S9 mix), the numbers of revertants in TA98 increased by as much 2.2-5.0-fold compared with the sum of those in the separate applications of AAF and quercetin. A 1.4-2.7-fold increase was observed in TA100. Quercetin did not affect the mutagenicity of 4NQO, and depressed that of B[a]P. Dose-response curves for mutagenicity of quercetin with or without AAF (5 micrograms/plate) were examined. The results suggest that quercetin, present in a molarity of up to 1.5 times that of AAF, is apparently effective in enhancing the mutagenicity of AAF, because a linear dose-response curve was observed in the range of 0-5 micrograms/plate quercetin with AAF although quercetin alone was not mutagenic in the same range. Dose-response curves for mutagenicity of quercetin with or without 5 micrograms/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was reduced to about 60% of the sum of separate application at doses ranging from 25 to 100 micrograms/plate of quercetin. Since enhancement and depression of mutagenicity by quercetin were observed for indirect mutagens, AAF and B[a]P, respectively, in the presence of S9 mix, quercetin may affect the metabolic pathway of these mutagens.     

Effect of induction by DDT on metabolism and mutagenicity of benzo[a]pyrene

Liver microsomal enzymes are essential for the detection of benzo[a]pyrene (B[a]P)-mediated mutagenesis in the Salmonella/mammalian microsome mutagenicity test and, furthermore, this mutagenicity is considerably enhanced by induction of hepatic enzymes involved with drug metabolism. Although Aroclor 1254 is most commonly used for induction of S9 enzymes, DDT is also capable of this induction. This paper reports a comparison of liver S9 fraction induced by the two agents: there is a marked difference in their concentration optima for metabolism of B[a]P; greater numbers of revertant colonies are seen with Aroclor-induced S9, which is optimal at a concentration of 10% (v/v), whereas DDT-induced S9 is optimal at 2.5% (v/v); Aroclor induces aryl hydrocarbon hydroxylase (AHH), cytochrome P-450 and epoxide hydrase while DDT induces only AHH, to about half the level detected in the Aroclor-induced S9 fraction. A comparison of metabolite distribution for Aroclor- and DDT-induced hepatic microsomes reveals quantitative differences only. DDT-induced microsomes yield a greater proportion of B[a]P-4,5-oxide and its metabolic product B[a]P-4,5-dihydrodiol than do Aroclor-induced microsomes. Time course studies on the mutagen half-life measured on the agar plate provides good evidence that metabolites responsible for mutagenicity were different for each inducer.     

Mutagenicity of nitro-azabenzo[a]pyrene and its related compounds.

The mutagenicity of nitrated benzo[a]pyrene (BP) and the related compounds, 1- and 3-nitrobenzo[a]pyrene (NBP), 1- and 3-nitro-6-cyanobenzo[a]pyrene (N-6-CBP), 1- and 3-nitro-6-azabenzo[a]-pyrene (N-6-ABP), 1- and 3-nitro-6-azabenzo[a]-pyrene-N-oxide (N-6-ABPO) and 1,6- and 3,6-dinitrobenzo[a]-pyrene (DNBP), was investigated. The mutagenic activities of 3-N-6-CBP and 3-N-6-ABP were 117 and 76 times, respectively, that of 3-NBP. In addition, 3,6-DNBP was more mutagenic than 1,6-DNBP. It is suggested that the mutagenic activation differs with the position of NO2 substitution in the chemical structure. A nitro derivative with NO2 substitution at the 3 position of the aromatic ring of BP was more mutagenic than that with the substitution at the 1 or 6 position. The reducibility of DNBPs was then determined by detecting 1- or 3-amino-6-nitrobenzo[a]pyrene (A-6-NBP), a metabolite of DNBP; 3,6- and 1,6-DNBP were reduced to 3- and 1-A-6-NBP at frequencies of 958 +/- 26 and 79 +/- 8, respectively, pmole per mg of protein, when the compound was incubated anaerobically with rat liver S9 mix at 37 degrees C for 15 min. NO2 substituted at the 3 position of the aromatic ring of BP was readily reduced by a microsome enzyme to form an amino derivative. The result suggests that these compounds have a structure-activity relationship between mutagenicity and NO2 substitution of BP.     

Vitamin K1 amplification of benzo(a)pyrene metabolism in chick embryos

Seventeen-day-old chick embryos were used as a test system to assess the effect of vitamin K1(K1) on benzo(a)pyrene (BP) metabolism as measured by the induction of arylhydrocarbon hydroxylase (AHH) and cytochrome P-450 and the levels of glutathione (GSH) and glutathione S-transferase (GST) in liver. Twenty-four hours after injection of BP into the air sac there was a sharp rise in AHH and P-450 and a drop in GSH. When K1 was injected 24 hr prior to BP there was a decrease in GST activity as compared with the control plus an augmented increase in AHH induction. This augmentation in BP metabolism (Phase I) together with a concomitant decrease in at least one mechanism of Phase II conjugation is in keeping with other evidence that K1 can play an adjuvant role in BP induced mutagenicity and carcinogenicity. Ubiquinone has a much lesser effect on BP metabolism than does K1 in equimolar concentration.     

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

Induction and repair of DNA strand breaks in cultured human fibroblasts exposed to various phenols and dihydrodiols of benzo[a]pyrene

Cultured human fibroblasts from healthy donors were incubated for 30 min with nine different benzo[a]pyrene (BP) derivatives in the presence or absence of liver microsomes from 3-methylcholanthrene treated rats. The induction and repair of DNA strand breaks were analysed by alkaline unwinding and separation of double and single stranded DNA (SS-DNA) by hydroxylapatite chromatography immediately after the incubation or at various times after the treatment. In the absence of microsomes DNA stand breaks were detected in fibroblasts exposed to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP) and the three BP dihydrodiols (BP-4,5-, BP-7,8- or BP-9,10-dihydrodiol). After removal of the BP derivatives from the medium the DNA strand breaks disappeared within 24 h. alpha-Naphthoflavone (alpha-NF) caused a decrease in the induction of strand breaks by 1-, 3- and 9-OH-BP but did not affect the induction of strand breaks in cells exposed to BP-7,8-dihydrodiol. In the presence of microsomes DNA strand breaks were found after exposure to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP), as well as BP-7,8- and 9,10-dihydrodiol. In contrast BP-4,5-dihydrodiol did not induce strand breaks under these conditions. The induction of strand breaks by BP-7,8-dihydrodiol was enhanced in the presence of cytosine-1-beta-D-arabinofuranoside (AraC). In all cases the DNA strand breaks had disappeared 24 h after removal of the BP derivatives and microsomes except after treatment with BP-7,8-dihydrodiol.     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus)

• 1.1. The in vitro metabolism of [³H]benzo[a]pyrene (BP) and [¹⁴C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo.
• 2.2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [−] enantiomer) and smaller amounts of BP-4,5-diol.
• 3.3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides.
• 4.4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates.
• 5.5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25–30 days. The major adduct was anti-BPDE-deoxyguanosine.

     

Double base lesions of DNA by a metabolite of carcinogenic benzo[a]pyrene.

Carcinogenic benzo[a]pyrene (BP) is generally considered to show genotoxicity by forming DNA adducts of its metabolite, BP-7,8-diol-9,10-epoxide. We investigated oxidative DNA damage and its sequence specificity induced by BP-7,8-dione, another metabolite of BP, using (32)P-5'-end-labeled DNA. Formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at G residues of 5'-TG-3' sequence and at poly(C) sequences, in DNA incubated with BP-7,8-dione in the presence of NADH and Cu(II), whereas piperidine treatment induced cleavage sites at T mainly of 5'-TG-3'. BP-7,8-dione strongly damaged the G and C of the ACG sequence complementary to codon 273 of the p53 gene. Catalase and a Cu(I)-specific chelator attenuated the DNA damage, indicating the involvement of H(2)O(2) and Cu(I). BP-7,8-dione with NADH and Cu(II) also increased 8-oxo-7,8-dihydro-2'-deoxyguanosine formation. We conclude that oxidative DNA damage, especially double base lesions, may participate in the expression of carcinogenicity of BP in addition to DNA adduct formation.     

Characteristics of microsomal reduction of benzo[a]pyrene 4,5-oxide

NADPH-reduction of benzo[a]pyrene 4,5-oxide (BP-4,5-oxide) to BP required four components from rat liver: cytochrome P-450, NADPH cytochrome P-450 reductase, phosphatidylcholine and a soluble, heat-sensitive factor which was present in 105 000 × g supernatant and was also released from microsomes by sonication. The requirement for this factor contrasts with recently reported results from Sugiura et al. (Cancer Res., 40 (1980) 2910). Oxide-reduction was 40 times faster under anaerobic conditions, but oxygen did not affect the stimulation factor. This stimulation was highest (× 15) at low concentrations of microsomal protein (<0.1 mg/ml) and was almost absent at high concentrations of microsomal protein (>1 mg/ml). Oxide-reduction activity was proportional to microsomal protein concentration in the presence of added 105 000 × g supernatant, but for microsomes alone (>0.1 mg/ml) exhibited a parallel plot with an intercept at 0.08 mg/ml microsomal protein. Stimulation was highest at high concentrations of BP-4,5-oxide and a linear plot of V⁻¹ vs. [BP-4,5-oxide]⁻¹ was only obtained in the presence of 105 000 × g supernatant (K_m = 3 μM, V_max = 3.3 nmol/mg/min). Microsomal hydration of BP-4,5-oxide (inhibited in reductase assays) was unaffected by 105 000 × g supernatant, suggesting that stimulation of oxide-reduction did not derive from solubilization of BP-4,5-oxide. Stimulation was observed in the initial rate of reaction and was independent of incubation time. Inhibition of lipid peroxidation, removal of peroxides and deoxygenation were all excluded as explanations of the stimulatory effect.     

Effects of the hepatic S9 fraction from aroclor-1254-treated rats on the mutagenicity of benzo[alpha]pyrene and 2-aminoanthracene in the Salmonella/microsome assay.

The mutagenicity of benzo[alpha]pyrene and 2-aminoanthracene for Salmonella typhimurium TA98 in the plate-incorporation test was studied using liver S9 from untreated and aroclor-1254-treated rats. The induction of liver S9 protein, arylhydrocarbon hydroxylase (AHH), and cytochrome P448/450 was followed with time. There was no change in protein concentrations with induction; AHH and cytochrome levels were increased at 1, 3, 5 and 7 days post Aroclor treatment. Benzo[alpha]pyrene mutagenicity was enhanced with Aroclor treatment while 2-aminoanthracene mutagenicity was depressed. The benzo[alpha]pyrene mutagenicity showed a positive correlation with the levels of AHH and cytochrome on the plate; 2-aminoanthracene showed a negative correlation with activity in induced samples.     

Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100

Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella typhimurium strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver S9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of S9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60% of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without S9. BP, 3MC and 3MCE were not mutagenic in either strain without S9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the S9 preparation revealed decreased AHH activity with increase in Se concentration. The decreased mutagenicity and AHH activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as Na2SO3). Inhibition of AHH activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. typhimurium TA98 and TA100.