Nitrogen-substitution effect on in vivo mutagenicity of chrysene

We have previously reported the in vivo mutagenicity of aza-polycyclic aromatic hydrocarbons (azaPAHs), such as quinoline, benzo[f]quinoline, benzo[h]quinoline, 1,7-phenanthroline and 10-azabenzo[a]pyrene. The 1,10-diazachrysene (1,10-DAC) and 4,10-DAC, nitrogen-substituted analogs of chrysene, were shown to exhibit mutagenicity in Salmonella typhimurium TA100 in the presence of rat liver S9 and human liver microsomes in our previous report, although DACs could not be converted to a bay-region diol epoxide, the ultimate active form of chrysene, because of their nitrogen atoms. In the present study, we tested in vivo mutagenicity of DACs compared with chrysene using the lacZ transgenic mouse (Mutatrade markMouse) to evaluate the effect of the nitrogen substitution. DACs- and chrysene-induced mutation in all of the six organs examined (liver, spleen, lung, kidney, bone marrow and colon). The mutant frequencies obtained with chrysene showed only small differences between the organs examined and ranged from 1.5 to 3 times the spontaneous frequency. The 4,10-DAC was more mutagenic than chrysene in all the organs tested. The highest lacZ mutation frequency was observed in the lung of 4,10-DAC-treated mice and it was 19 and 6 times the spontaneous frequency and the frequency induced by chrysene, respectively. The 1,10-DAC induced lacZ mutation in the lung with a frequency 4.3- and 1.5-fold higher than in the control and chrysene-treated mice, respectively, although the mutant frequencies in the other organs of 1,10-DAC-treated mice were almost equivalent to those of chrysene-treated mice. Not only chrysene but also DACs depressed the G:C to A:T transition and increased the G:C to T:A transversion in the liver and lung. These results suggest that the two types of nitrogen substitutions in the chrysene structure may enhance mutagenicity in the mouse lung, although they showed no difference in the target-organ specificity and the mutation spectrum.     

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (MutaMouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.     

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.     

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (Muta™Mouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.     

Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse)

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.     

The 3,4-oxide is responsible for the DNA binding of benzo[ghi]perylene, a polycyclic aromatic hydrocarbon without a "classic" bay-region

The polycyclic aromatic hydrocarbon (PAH) benzo[ghi]perylene (BghiP) lacks a "classic" bay-region and is therefore unable to form vicinal dihydrodiol epoxides thought to be responsible for the genotoxicity of carcinogenic PAHs like benzo[a]pyrene. The bacterial mutagenicity of BghiP increases considerably after inhibition of the microsomal epoxide hydrolase (mEH) indicating arene oxides as genotoxic metabolites. Two K-region epoxides of BghiP, 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) and 3,4,11,12-bisepoxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisoxide) identified in microsomal incubations of BghiP are weak bacterial mutagens in strain TA98 of Salmonella typhimurium with 5.5 and 1.5 his+-revertant colonies/nmol, respectively. After microsomal activation of BghiP in the presence of calf thymus DNA three DNA adducts were detected using 32P-postlabeling. The total DNA binding of 2.1 fmol/microg DNA, representing 7 adducts in 10(7) nucleotides, was raised 3.6-fold when mEH was inhibited indicating arene oxides as DNA binding metabolites. Co-chromatography revealed the identity between the main adduct of metabolically activated BghiP and the main adduct of the 3,4-oxide. DNA adducts of BghiP originating from the 3,4,11,12-bisoxide were not found. Therefore, a K-region epoxide is proposed to be responsible for the genotoxicity of BghiP and possibly of other PAHs without a "classic" bay-region.     

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.     

A comparison of mutational specificity of mutations induced by S9-activated B[a]P and benzo[a]pyrene-7,8-diol-9,10-epoxide at the endogenous aprt gene in CHO cells

We have determined the mutational specificity of S9-activated benzo[a]pyrene (B[a]P) at the endogenous aprt locus in a hemizygous Chinese hamster ovary cell line. The aprt gene of recovered mutants was amplified using the polymerase chain reaction (PCR) and directly sequenced. This spectrum was then compared to mutations recovered following treatment with the B[a]P metabolite, benzo[a]pyrene diol-epoxide (BPDE). No significant difference between the two spectra in the types of mutations produced, or their distribution was observed. This observation supports the hypothesis that BPDE is the reactive metabolite of B[a]P, responsible for the significant biological effects caused by this ubiquitous polycyclic aromatic hydrocarbon. The major mutation recovered was the G:C-->T:A transversion, and mutations were primarily localized within runs of guanines. We also confirmed our previous finding that mutation by B[a]P is non-random, targeting events in runs of guanines flanked by adenine residues. This same target hotspot region is found in codon 61 of the human c-Ha-ras1 proto-oncogene. This may help explain the selective activation of this codon by BPDE.     

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.     

The presence of the mutagenic polycyclic aromatic hydrocarbons benzo[a]pyrene and benz[a]anthracene in creosote P1

Several fractions of creosote P1 separated by TLC showed mutagenicity towards Salmonella typhimurium TA98. Thus mutagenicity is probably caused by the presence of mutagenic aromatic hydrocarbons. The mutagenic polycyclic aromatic hydrocarbons, benzo[a]pyrene and benz[a]anthracene, were detected in concentrations of 0.18 and 1.1% respectively. Because these compounds are probably not essential for the wood-preserving properties of creosote , a more selective composition of the product should be considered.     

Factors determining mutagenic potential for individual cis and trans opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts

Four adducts that would result from trans opening at C-1 of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (B[c]PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B[c]PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC [context I(A)] and 5'-CAGATTTAGAGTCTGC [context II(A)]. After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B[c]PhDE-dAdo adducts were determined. These findings were compared with data [Pontén et al. (1999) Biochemistry 38, 1144-1152] for cis opened B[c]PhDE-2-dAdo adducts in the same sequence contexts. In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%). For adducts derived from B[c]PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A). For adducts derived from B[c]PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context. Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration. However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity.     

Importance of the route of administration for genetic differences in benzo[a]pyrene-induced in utero toxicity and teratogenicity

C57BL/6N (Ahb/Ahb) mice have a high-affinity Ah receptor in tissues, whereas AKR/J and DBA/2N (Ahd/Ahd) mice have a poor-affinity Ah receptor. The cytochrome P1-450 induction response (enhanced benzo[a]pyrene metabolism) occurs much more readily in Ahb/Ahb and Ahb/Ahd than in Ahd/Ahd mice, at any given dose of the inducer benzo[a]pyrene. Embryos from the AKR/J X (C57BL/6N)(AKR/J)F1 and the reciprocal backcross were studied during benzo[a]pyrene feeding of the pregnant females. Oral benzo[a]pyrene (120 mg/kg/day) given to pregnant Ahd/Ahd mice between gestational day 2 and 10 produces more intrauterine toxicity and malformations in Ahd/Ahd than Ahb/Ahd embryos. This striking allelic difference is not seen in pregnant Ahb/Ahd mice receiving oral benzo[a]pyrene. Pharmacokinetics studies with [3H]benzo[a]pyrene in the diet and high-performance liquid chromatographic analysis of benzo[a]pyrene metabolism in vitro by the maternal intestine, liver, and ovary and the embryos of control and oral benzo[a]pyrene-treated pregnant females are consistent with "first-pass elimination" kinetics and differences in benzo[a]pyrene metabolism by the embryos and/or placentas versus maternal tissues. In the pregnant Ahd/Ahd mouse receiving oral benzo[a]pyrene, little induction of benzo[a]pyrene metabolism occurs in her intestine and liver; this leads to much larger amounts of benzo[a]pyrene reaching her embryos, and genetic differences in toxicity and teratogenesis are manifest. In the pregnant Ahb/Ahd mouse receiving oral benzo[a]pyrene, benzo[a]pyrene metabolism is greatly enhanced in her intestine and liver; this leads to less benzo[a]pyrene reaching her embryos, much less intrauterine toxicity and malformations, and no genetic differences are manifest. More toxic metabolites (especially benzo[a]pyrene 1,6- and 3,6-quinones) are shown to occur in Ahd/Ahd embryos than in Ahb/Ahd embryos. In additional studies, no prenatal or neonatal "imprinting" effect in C57BL/6N mice by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 on benzo[a]pyrene metabolism later in life was detectable. These genetic differences in intrauterine toxicity and teratogenicity induced by oral benzo[a]pyrene are just opposite those induced by intraperitoneal benzo[a]pyrene [Shum et al., '79; Hoshino et al., '81). The data in the present report emphasize the importance of the route of administration when the teratogen induces its own metabolism.     

Depurination of benzo[a]pyrene-diolepoxide treated DNA

Rat liver DNA was treated in vitro with benzo[a]pyrene-diolepoxide (BPDE), the ultimate carcinogenic metabolite derived from the polycyclic hydrocarbon benzo[a]pyrene. On incubation of the reacted DNA, apurinic sites developed which gave rise to strand breakage in alkaline solution. The reduction in molecular weight produced by these breaks was measured by analytical ultracentrifugation. In the case of anti-BPDE this depurination was shown to occur in two stages. The first was mainly due to attack at the 7-position of guanine, to yield an adduct which was lost from the DNA within a few hours. The second stage was due to much slower loss of the major N2-guanine adduct. The separated enantiomers, (+)- and (-)-anti-BPDE, and syn-BPDE all caused depurination to various extents. It is argued that although these processes are important in a study of the action of BPDE on DNA in vitro, their contribution to the biological activity of BPDE is probably negligible.     

Isolation and characterization of rat liver cytosolic aldehyde dehydrogenases induced by phenanthrene or benzo[a]pyrene

The cytosolic aldehyde dehydrogenase was isolated from the liver of Wistar rats treated with phenanthrene (non-carcinogenic) or benzo[a]pyrene (carcinogenic polycyclic aromatic hydrocarbon). The benzo[a]pyrene-induced enzyme has higher Km values for small aliphatic aldehydes and a lower molecular weight than the phenanthrene-induced enzyme. It is more resistant to changes of pH and to inhibition by disulfiram, but more sensitive to heat denaturation than the phenanthrene-induced enzyme. The phenanthrene-induced aldehyde dehydrogenase is very similar to the normal uninduced aldehyde dehydrogenase, whereas the benzo[a]pyrene-induced aldehyde dehydrogenase has common properties with the TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced enzyme and the hepatoma-specific enzyme.     

Photolysis primes biodegradation of benzo[a]pyrene

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

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.     

The role of repair protein Rad51 in synergistic cytotoxicity and mutagenicity induced by epidermal growth factor receptor inhibitor (Gefitinib, IressaR) and benzo[a]pyrene in human lung cancer

Rad51 protein is essential for homologous recombination repair of DNA damage, and is over-expressed in chemo- or radioresistant carcinomas. The polycyclic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) affects MAPKs transduction pathways. Gefitinib (IressaR, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and ERK1/2 activation. We hypothesized that gefitinib enhances B[a]P-mediated cytotoxicity by decreasing ERK1/2 activation. Exposure of human lung cancer cells to gefitinib decreased B[a]P-elicited ERK1/2 activation and induced Rad51 protein expression. Gefitinib and B[a]P co-treatment decreased Rad51 protein stability by triggering degradation via a 26S proteasome-dependent pathway. Expression of constitutive active MKK1/2 vectors (MKK1/2-CA) rescues the decreased ERK1/2 activity, and restores Rad51 protein level and stability under gefitinib and B[a]P co-treatment. Gefitinib enhances B[a]P-induced growth inhibition, cytotoxicity and mutagenicity. Co-treatment with gefitinib and B[a]P can further inhibit cell growth significantly after depletion of endogenous Rad51 by siRad51 RNA transfection. Enhancement of ERK1/2 activation by MKK1-CA expression decrease B[a]P- and gefitinib-induced cytotoxicity, and B[a]P-induced mutagenicity. Rad51 protein protects lung cancer cells from synergistic cytotoxic and mutagenic effects induced by gefitinib and B[a]P. Suppression of Rad51 protein expression may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib.     

Comparative metabolism of benzo[f]quinoline by liver microsomes from brown bullheads and rats

1. Liver microsomes from rats were considerably more active in metabolizing benzo[f]quinoline (B f Q) than those from brown bullheads (Ictalurus nebulosus). 2. The main B f Q metabolites formed by both rat and brown bullhead liver microsomes were qualitatively similar and included B f Q-7,8-dihydrodiol, B f Q-9,10-dihydrodiol, B f Q-N-oxide, 7-hydroxy B f Q, and 9-hydroxy B f Q. 3. The liver microsomes from control brown bullheads and rats metabolized B f Q primarily at the 7,8-and 9,10-positions, respectively, whereas in the case of microsomes from 3-methylcholanthrene (3-MC)-treated rats or brown bullheads, the major site of metabolic attack was the 7,8-position. 4. A 3-MC-type of cytochrome P-450 appears to be primarily responsible for the oxidation of B f Q by control brown bullhead liver microsomes, whereas a phenobarbital-inducible type of cytochrome P-450 seems to be involved in the metabolism of B f Q by control rat liver microsomes.     

Inhibition of benzo[a]pyrene-induced mutagenesis in Chinese hamster V79 cells by hemin and related compounds

The inhibitory effects of hemin and related compounds on the mutagenicity of benzo[a]pyrene (BP) were investigated in Chinese hamster V79 cells co-cultivated with X-irradiated hamster embryo cells. Mutant V79 cells were selected by their resistance to ouabain. The mutation frequency induced by BP was substantially inhibited dose dependently by hemin. The mutagenicity of BP (1 microgram/ml) on V79 cells was reduced to 6.5% by hemin, 52% by biliverdin, 73% by protoporphyrin and 85% by chlorophyllin at the highest concentration of the compounds tested (15 microM).     

The effect of norharman on the metabolism of benzo[alpha]pyrene by rat-liver microsomes in vitro in relation to its enhancement of the mutagenicity of benzo[alpha]pyrene.

The effect of norharman on the metabolism of benzo[alpha]pyrene by rat-liver microsomes was studied. Separation of the metabolites into hydrophilic and hydrophobic fractions showed that norharman inhibited the conversion of hydrophobic metabolites to hydrophilic ones. Analysis of the hydrophobic metabolites by high-pressure liquid chromatography showed that norharman also inhibited the disappearance of benzo[alpha]pyrene itself. However, large amounts of hydrophobic metabolites, such as phenol, quinones and diols, were formed in the presence of norharman, and formation of the strong mutagen 7,8-dihydroxybenzo[alpha]pyrene was increased 10-fold by norharman. The increase in formation of this compound may be one of the chief reasons why norharman enhances the mutagenicity of benzo[alpha]pyrene on Salmonella typhimurium.