2-(4'-CHLOROPHENYL)-1,4-BENZOQUINONE INCREASES THE FREQUENCY OF MICRONUCLEI AND SHORTENS TELOMERES

The toxicity of polychlorinated biphenyls (PCBs) has been attributed widely to receptor-mediated effects, buttressed by the popularity of the Toxic Equivalency Factor. We propose that a crucial toxic mechanism of lower-chlorinated PCBs is their enzymatic biotransformation to electrophiles, including quinoid metabolites, that bind intracellular sulfhydryl groups, such as those found in microtubulin and enzymes like telomerase. To test this hypothesis, we have examined micronuclei induction, cell cycle, and telomere shortening in cells in culture. Our findings show a large increase in micronuclei frequency and cell cycle perturbation in V79 cells, and a marked decrease in telomere length in HaCaT cells exposed to 2-(4'-chlorophenyl)-1,4-benzoquinone (PCB3pQ).     

An experimental approach to identifying the genotoxic risk from cooked meat mutagens

In order to define the toxicological risk to the human population from the chemical compounds formed during the process of cooking animal meat, which have been described as possessing mutagenic, genotoxic and carcinogenic activities, an extensive study was undertaken of cooked meat extract and two cooked meat mutagens, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) and 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx). The study involved toxicokinetics and mouse-tissue distribution studies of the two chemicals, in vitro and in vivo mutagenicity/genotoxicity analyses (i.e. the detection of gene mutations, chromosome aberrations and micronuclei in mouse bone marrow cells, and mouse urine and faeces mutagenicity tests), as well as in vivo protein and DNA binding assays. IQ and MeIQx were found to be positive for the induction of gene mutations in Salmonella typhimurium TA98, but not in Chinese hamster V79 cells; IQ only was found to be positive for the induction of chromosome aberrations in Chinese hamster ovary cells and cultured human lymphocytes. IQ and MeIQx were negative for the induction of micronuclei in mice treated with 40 mg chemical/kg body weight; the lowest effective dose administered to the mice that produced mutagenic urine was 0.4 mg IQ/kg body weight and 0.04 mg MeIQx/kg. A dose of 40 mg IQ/kg, given orally by gavage to mice, produced an excretion of 1-4% of the applied dose in the urine and 0.1-2% of the applied dose in the faeces, when evaluated chemically or mutagenically. The number of DNA adducts in the liver correlated with the dose of IQ or MeIQx administered to the mice. All the data have been used for defining a possible risk estimate to the human population as a consequence of a cooked meat diet.     

Mutagenicity of 3-methylcholanthrene, pcb3, and 4-oh-pcb3 in the lung of transgenic bigblue rats

Recent findings of high levels of predominantly lower chlorinated biphenyls in indoor and outdoor air open the question of possible health consequences. Lower chlorinated biphenyls are more readily metabolized to reactive and potentially harmful intermediates, acting as mutagens and cancer initiators. The goal of this study was to assess the mutagenicity of PCB3 in the lungs of rats. Male BigBlue? 334 Fisher transgenic rats, which carry the bacterial lacI gene as a target of mutagenicity, were given intraperitoneal injections of corn oil, 3-methylcholanthrene (3-MC, positive control), 4-monochlorobiphenyl (PCB3) or its metabolite 4-hydroxy-PCB3 (4-OH-PCB3) weekly for 4 weeks. Lungs tissue was harvested to determine mutant frequencies, mutation spectra, and pathological changes. 3-MC caused a 15-fold increase in mutant frequency and an increase in transversion type mutations; a very early occurrence of this type of mutation in lung tissue was previously identified in Ki-ras oncogenes of lung tumors from 3-MC exposed mice. The 2-fold increase in the mutant frequency after treatment with PCB3 and 4-OH-PCB3 was not statistically significant, but a shift in the mutation spectra, especially with PCB3, and an increase in mutations outside of the hotspot region for spontaneous mutations (bp 1-400), suggest that PCB3 and possibly 4-OH-PCB3 are mutagenic in the rat lung.     

Mutagenic activity of major mammalian metabolites of cyclophosphamide toward several genes of Escherichia coli.

Representative intermediates of the major mammalian metabolic pathway of cyclophosphamide, a drug that is not mutagenic as such unless it is metabolically activated, were assayed for their direct mutagenic activity toward a bacterial indicator strain, Escherichia coli 343/113. The compounds tested were 4-hydroperoxycyclophosphamide and the two urinary metabolites, carboxyphosphamide and 4-ketocyclophosphamide. Further, the degradation products of 4-hydroxycyclophosphamide, phosphoramide mustard, acrolein, and nornitrogen mustard, were also tested. The mutagenicity test systems were those used previously to demonstrate the liver enzyme-mediated mutagenic activity of cyclophosphamide in E. coli 343/113: stationary cell suspensions were treated for 180 min at 37 degrees C with different concentrations of the compound under test; the induction of forward mutations from 5-methyltryptophan sensitivity to resistance (MTR) and from galRs18 to gal+ as well as back mutations from arg56 to arg+ was measured by plating aliquots of the treated bacterial population on different selective mutation media. Except for acrolein, all cyclophosphamide metabolites tested are directly mutagenic toward E. coli 343/113. With all substances the highest induced mutation frequency is that of arg+ mutations, followed by gal+ and MTR mutations; this indicates that mostly base-pair substitution type mutations are induced. The mutagenic potential, however, differs greatly between compounds at concentrations between 0.1 and 20mM. The results show that the first step in the mammalian biodegradation of cyclophosphamide gives rise to compounds that are directly mutagenic, and that this mutagenicity is retained and even enhanced through all further metabolic steps to produce the compound of highest mutagenicity, nornitrogen mustard.     

Mutagenicity of the mycotoxin alternariol in cultured mammalian cells

The mycotoxin alternariol (AOH) is an important contaminant of fruit and cereal products. Concern about exposure to low levels of AOH was raised after the disclosure that contamination of food with the AOH-producing species Alternaria alternata is associated with oesophagal cancer. Previously we have reported that AOH induces kinetochore-negative micronuclei in cultured Chinese hamster V79 cells. The present study investigates the mutagenicity of AOH at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus in V79 cells and at the thymidine kinase (TK) gene locus in mouse lymphoma L5178Y tk(+/-) cells (MLC). Concentrations of 10 microM AOH and more gave rise to a significant and concentration-dependent induction of HPRT and TK mutations in V79 cells and in MLC, respectively. The mutagenic potency of AOH was about 50-fold lower than that of the established mutagen 4-nitroquinoline-N-oxide in both cell lines. Discrimination between small and large colonies in the TK assay revealed the predominant induction of small colonies, which are indicative for extensive chromosomal deletions and which correlated with the induction of micronuclei in MLC. The mutagenicity of AOH may have a bearing on the carcinogenicity of this mycotoxin.     

Effects of vitamin C and PCB metabolites on the inhibition of aryl hydrocarbon hydroxylase and suppression of mutagenicity of benzo[a]pyrene

The reverse mutations of Salmonella typhimurium TA98 and TA100 induced by benzo[a]pyrene (BP) were suppressed by about 90% by vitamin C (1500 μg/plate). Of 11 polychlorinated biphenyl (PCB) metabolites tested (150 μg/plate), 3-methylsulfonyl-3′,4,4′,5-tetrachlorobiphenyl (3-MSF-3′,4,4′,5-tetraCB), 3-MSF-3′,4,4′,5,5′-pentaCB and 4-MSF-3,3′,4′,5,5′-pentaCB also strongly suppressed the mutagenicity of BP. In addition, vitamin C reduced the activity of aryl hydrocarbon hydroxylase (AHH) in microsomes from aromatic hydrocarbon-responsive (Ah-responsive) and Ah-non-responsive strains of mice that had been pretreated with olive oil (vehicle), 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Vitamin C affected the AHH activities in the same way in both strains of mice, but the extent of inhibition was somewhat smaller in the Ah-responsive strain than in the Ah-non-responsive strain. In contrast, 3-MSF-3′,4,4′,5-tetraCB, one of the most potent AHH inhibitors among 11 PCB metabolites showed various inhibitory effects depending upon the origin of the microsomes. It is suggested that vitamin C is directly bound to the substrate BP, to cytochrome P-450 itself or to the substrate-enzyme complex, thus lowering the rate of AHH reaction, and that the mechanisms of inhibition of AHH and suppression of mutagenicity by vitamin C are different from those of PCB metabolites.     

Initiating activity of 4-chlorobiphenyl metabolites in the resistant hepatocyte model.

We recently reported that several mono- to tetrachlorinated biphenyls have initiating activity in the livers of Fischer 344 rats. In the present study, we investigated the metabolic activation of one of those compounds, 4-chlorobiphenyl (PCB 3). Monohydroxy (400 micromol/kg), dihydroxy (200 micromol/kg), and quinone (100 micromol/kg) metabolites of PCB 3 were evaluated for their initiating activity. Fischer 344 male rats were fasted for 4 days; 24 h after feeding again, they were injected (ip) with metabolites, vehicle, or diethylnitrosamine (DEN, 20 or 40 mg/kg). All animals were treated with selection agents as follows: three daily p.o. doses of 2-acetylaminofluorene (2-AAF, 30 mg/kg), followed by a single p.o. dose of carbon tetrachloride (2 ml/kg) and three additional daily treatments of 2-AAF. Rats were killed 2 weeks after the last 2-AAF intubation. Livers were evaluated for changes in morphology, and the number and volume of gamma-glutamyl transpeptidase-positive foci were measured. Of the metabolites tested, only one monohydroxy and one quinoid metabolite showed initiating activity. The metabolic activation of PCB 3, therefore, proceeds via parahydroxylation and oxidation to the ortho 3,4-quinone, the ultimate carcinogen. This is the first report to demonstrate that specific PCB metabolites possess initiating activity in the rodent liver in vivo. The results support the conclusion that 4-OH PCB 3 and 3,4-BQ PCB 3 act as proximate and ultimate carcinogenic metabolites resulting from the bioactivation of PCB 3 in rat liver.     

Mutagenic potential of binary mixtures of nitro-polychlorinated dibenzo-p-dioxins and related compounds

The mutagenic potential of binary mixtures of nitro-polychlorinated dibenzo-p-dioxins and other environmentally related compounds was determined using Salmonella typhimurium strain TA98 in the standard plate incorporation assay. Samples tested included binary mixtures of 4-nitro-4'-chlorobiphenyl with 6-nitro-4,2',3',4',5'-pentachlorobiphenyl, 4-nitrobenzo-p-dioxin with 4-nitro-2,3,8-trichlorodibenzo-p-dioxin, and benzo[a]pyrene with either nitropentachlorobiphenyl or nitrotrichlorodibenzo-p-dioxin. Inhibition was the primary interaction observed for the mixtures of polyhalogenated dioxins or biphenyls with the direct-acting mutagens nitrodibenzo-p-dioxin or nitrochlorobiphenyl. At the highest dose tested, nitrotrichlorodibenzo-p-dioxin inhibited the bacterial mutagenicity of nitrodibenzo-p-dioxin by almost 50%, while pentachlorobiphenyl inhibited the mutagenicity of nitrobiphenyl by 34%. Conversely, synergism was the primary interaction observed for mixtures of halogenated aromatics with the promutagen benzo[a]pyrene. The addition of nitrotrichlorodioxin to benzo[a]pyrene enhanced the mutagenicity of the latter compound by as much as 70%, while the mutagenic potential of a mixture of benzo[a]pyrene and nitropentachlorobiphenyl was approximately 50% greater than the mutagenicity of benzo[a]pyrene alone. In summary, mixtures of nonmutagenic nitropolyhalogenated biphenyls or dibenzo-p-dioxins appear to inhibit the mutagenicity of direct-acting mutagens, while these same compounds seem to enhance the mutagenic potential of the promutagen benzo[a]pyrene.     

Measurement of mutant frequency in T-cell receptor (TCR) gene by flow cytometry after X-irradiation on EL-4 mice lymphoma cells

It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency (MF) on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytometry. In this study, we developed an in vitro system using murine EL-4 lymphoma cells and observed frequency of cells defective in TCR gene expression after exposure to ionizing irradiation. EL-4 cells were stained with fluorescein-labeled anti-CD4 and phycoerythrin-labeled anti-CD3 antibodies. They were analyzed with a flow cytometer to detect mutant EL-4 cells lacking surface expression of TCR/CD3 complexes which showed CD3-, CD4+ due to a somatic mutation at the TCR genes. Mutant cells could be observed at 2 days after 3 Gy irradiation. MF of EL-4 cells was 6.7x10(-4) for 0 Gy and the value increased to the maximum level of 39x10(-4) between 4 and 8 days after 3 Gy irradiation and these data were found to be best fitted by a linear-quadratic dose-response model. After the peak value the TCR MF gradually decreased with a half-life of approximately 3.2 days. We also examined the hprt mutant frequencies at seven days after irradiation and the cytokinesis-blocked micronucleus frequency at 20 hrs after irradiation. The frequencies of hprt mutation and micronuclei were found to be best fitted by a linear-quadratic dose-response model and a linear dose-response model, respectively. The method to detect mutation on TCR gene is quick and easy in comparison with other methods and is considered useful for the mutagenicity test.     

Dihydroxylated metabolites of cannabinol formed by rat liver in vitro.

Cannabinol (CBN) was metabolized in vitro by a 10,000 g supernatant from rat liver. After removal of unchanged CBN and its monohydroxylated metabolites four dihydroxylated metabolites were isolated. By nuclear magnetic resonance and mass spectrometry the compounds were identified as 1',7-dihydroxy-CBN. Side chain hydroxylation occurred predominantly at C-4' anc C-3'.     

Mutagenesis and DNA adduct formation by 1-nitropyrene in Chinese hamster ovary cells without exogenous metabolic activation.

The direct-acting mutagenicity of 1-nitropyrene (1-NP), a tumorigenic environmental pollutant and model nitropolycyclic aromatic hydrocarbon, was studied in Chinese hamster ovary (CHO) cells. Previous reports indicated that 1-NP, a potent direct-acting mutagen in Salmonella typhimurium, was mutagenic in CHO cells only in the presence of an exogenous activation system. In this study, a DNA-repair-deficient CHO cell line, CHO-UV5, and the repair-proficient CHO-K1-BH4 cell line were used to measure the direct-acting mutagenicity of highly purified samples of 1-NP. Exposure of CHO-K1-BH4 cells for 5, 12, and 24 hr did not increase mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (p greater than 0.1; paired t test; n = 5-6), while treatment of CHO-UV5 cells for 24 hr produced a statistically significant induction of mutants (22 +/- 6 mutants per 10(6) clonable cells vs a solvent control value of 9 +/- 3 per 10(6) cells; p less than 0.01; n = 6). 32P-Postlabeling analysis of the DNA adducts formed in CHO-UV5 cells following 1-NP exposure revealed the presence of a single major adduct. Based on its chromatographic properties, its sensitivity to nuclease P1 digestion, and its resistance to hydrazine treatment, the adduct appeared to be N-(deoxyguanosin-8-yl)-1-aminopyrene, which was presumably produced by nitroreduction of 1-NP to N-hydroxy-1-aminopyrene. These results suggest that the use of extended treatment times and DNA-repair-deficient cells may be required to assess adequately the mutagenicity of nitropolycyclic aromatic hydrocarbons in CHO cells.     

Non-coplanar 2,2′,3,3′,4,4′,5,5′,6,6′-decachlorobiphenyl (PCB 209) did not induce cytochrome P450 enzyme activities in primary cultured rat hepatocytes,was not genotoxic,and did not exhibit endocrine-modulating activities

2,2′,3,3′,4,4′,5,5′,6,6′-Decachlorobiphenyl (PCB 209) is a fully chlorinated, non-coplanar biphenyl. To demonstrate that PCB 209 is not likely to exhibit human health hazards common to coplanar PCBs it was tested for cytochrome P450 (P450) enzyme induction potentials, genetic toxicity, and endocrine-modulating activity. PCB 209 (dose from 0.005 to 5000 ng/mL) did not significantly induce P450 CYP1A, 2A, 2B, 3A, or 4A enzyme activities in primary cultured rat hepatocytes. In contrast, Aroclor 1260, a PCB mixture that contains approximately 60% chlorine by weight, showed significant induction of P450 CYP1A, 2A, 2B, and 3A within the same dose range. PCB 209 (dose from 100 to 5000 μg/plate) was negative in the bacterial mutagenicity (Ames) test in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 or in Eschericia coli strain WP2uvrA. PCB 209 (dose from 25 to 150 μg/mL) was also negative for forward mutations at the thymidine kinase (TK^+/−) locus of L5178Y mouse lymphoma cells. The Ames and the mouse lymphoma assays were both conducted in the absence and presence of rat liver S9 fraction. PCB 209 (dose from 500 to 2000 mg/kg by single dose oral gavage) did not induce an increase in the frequency of micronuclei in polychromatic erythrocytes in mouse bone marrow in vivo. PCB 209 did not induce estrogenic effects when administered by gavage to ovariectomized adult female rats at 500 and 1000 mg/kg for 4 days, nor did it produce alterations consistent with endocrine-modulating activity in adult intact male rats when administered by gavage at 500 and 1000 mg/kg for 15 consecutive days.     

Induction of cytochrome P450 1B1 in MDA-MB-231 human breast cancer cells by non-ortho-substituted polychlorinated biphenyls

The effects of 12 non-ortho-substituted polychlorinated biphenyl (PCB) congeners on the induction of human cytochrome P450 1B1 (CYP1B1), an estradiol 4-hydroxylase, were investigated in MDA-MB-231 breast cancer cells. Three independent quantitative assays were used, in which the rates of estrogen metabolism, the levels of the CYP1B1 and CYP1A1 mRNAs, and luciferase activities under the control of the CYP1B1 promoter were measured. Of the congeners investigated, 3,4,4',5-tetrachlorobiphenyl (PCB 81), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 3,4',5-trichlorobiphenyl (PCB 39) and 3,3',4,5-tetrachlorobiphenyl (PCB 78) were the most potent in each assay, causing four to 10-fold increases in response. Exposure to 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) resulted in elevated CYP1B1 mRNA and increased CYP1B1-promoter driven luciferase activity, but caused depressed rather than elevated rates of E(2) metabolism due to inhibition of CYP1B1. The relative magnitudes of CYP1B1 induction by the PCB congeners, as determined by the three assays, were in close agreement, with the exception noted for PCB 169. These results indicate that PCB structure-activity relationships for the induction of human CYP1B1 are similar to those observed for human CYP1A1, but differ somewhat from what has been reported for induction of rat CYP1A1.     

Activating and inactivating reactions controlling 2-naphthylamine mutagenicity

Factors controlling 2-naphthylamine mutagenicity were studied using the Ames test. 1) Both rat liver microsomes and cytosolic proteins were required for generation of mutagenic metabolites. 2) 1-Hydroxy-2-naphthylamine, the major metabolite of 2-naphthylamine, was not mutagenic but cytotoxic to bacteria. 3) Ascorbic acid, reduced glutathione and conjugation reactions, such as glucuronidation, were strongly inhibiting 2-naphthylamine mutagenicity. 4) When isolated hepatocytes were used as the activating system mutagenic metabolites could not be detected. However cytotoxicity was detectable at doses of 2-naphthylamine greater than 0.2 mol/10⁶ cells.The results suggest that the formation of genotoxic metabolites of 2-naphthylamine is largely prevented in the intact, non-dividing rat hepatocyte.     

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is enantioselectively oxidized to hydroxylated metabolites by rat liver microsomes

Developmental exposure to multiple ortho-substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB)-, dexamethasone (DEX)- and corn oil (VEH)-treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzyme. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer did not affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of PCBs and their metabolites in wildlife and humans are due to metabolism by P450 enzymes but also suggest that the enantioselective formation of neurotoxic PCB 136 metabolites, such as 4-OH-PCB 136, may play a role in the developmental neurotoxicity of PCBs.     

Genotoxicity of pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) are common constituents of many plant species around the world. PA‐containing plants are probably the most common poisonous plants affecting livestock and wildlife. They can inflict harm to humans through contaminated food sources, herbal medicines and dietary supplements. Half of the identified PAs are genotoxic and many of them are tumorigenic. The mutagenicity of PAs has been extensively studied in different biological systems. Upon metabolic activation, PAs produce DNA adducts, DNA cross‐linking, DNA breaks, sister chromatid exchange, micronuclei, chromosomal aberrations, gene mutations and chromosome mutations in vivo and in vitro. PAs induced mutations in the cII gene of rat liver and in the p53 and K‐ras genes of mouse liver tumors. It has been suggested that all PAs produce a set of (±)‐6,7‐dihydro‐7‐hydroxy‐1‐hydroxymethyl‐5H‐pyrrolizine‐derived DNA adducts and similar types of gene mutations. The signature types of mutations are G : C → T : A transversion and tandem base substitutions. Overall, PAs are mutagenic in vivo and in vitro and their mutagenicity appears to be responsible for the carcinogenesis of PAs. Published in 2010 by John Wiley & Sons, Ltd.     

Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl with liver microsomes of phenobarbital-treated dog; the possible formation of PCB 2,3-arene oxide intermediate.

1. Metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) was investigated in vitro using liver microsomes of one male beagle dog after phenobarbital treatment. 2. Three major metabolites were isolated and identified as 3-hydroxy-2,4,5,2',4',5'-HCB, 2-hydroxy-4,5,2',4',5'-pentachlorobiphenyl (PenCB), and 2-hydroxy-3,4,5,2',4',5'-HCB, by comparison of g.l.c.-mass spectrometry and 1H-n.m.r. data with those of authentic samples. 3. 2-Hydroxy-3,4,5,2',4',5'-HCB was found as a metabolite of 2,4,5,2',4',5'-HCB for the first time using dog liver microsomes. Present result indicate that this metabolite and the dechlorinated PenCB are derived from a metabolic intermediate, namely, 2,3-epoxy-2,4,5,2',4',5'-HCB. 2,3-Epoxide formation is a new metabolic pathway of PCB.     

Mutation analyses of a series of TNT-related compounds using the CHO-hprt assay

Trinitrotoluene (TNT) and related compounds were tested for induction of mutation in the CHO-hprt mutation assay. The parent compound, TNT, was consistently found to be mutagenic at concentrations above 40 microg ml(-1), whether or not S9 activating enzymes were added. Five TNT metabolites gave statistically significant but small increases in mutation frequency over solvent controls: 4-amino-2,6 dinitrotoluene, 2,4',6,6'-tetranitro-2',4-azoxytoluene, 2,2',6,6'-tetranitro-4,4'-azoxytoluene, 2',4,6,6'-tetranitro-2,4'-azoxytoluene and triaminotoluene. Clear dose-response relationships could not be established for the mutagenic response of these compounds. They are considered as very weak mutagens in this mammalian test system. Five compounds did not produce statistically significant mutation frequencies at the levels tested: 2-amino-4,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene, 1,3,5-trinitrobenzene, 2,6-diamino-4-nitrotoluene and 4,4',6,6'-tetranitro-2,2'-azoxytoluene. The results indicate that none of the TNT metabolites tested pose a significant mutational health risk, at least as judged by the CHO-hprt assay.     

Structure-dependent induction of CYP2B1/2 by 3-methylsulfonyl metabolites of polychlorinated biphenyl congeners in rats

The effects of eleven 3-methylsulfonyl (3-MeSO(2))-metabolites of polychlorinated biphenyl (PCB) congeners (which were reported to remain in Swedish mother's milk and Japanese Yusho patient's tissues) and their two structurally similar 3-MeSO(2)-PCBs on the hepatic drug-metabolizing enzyme activities were compared with those of phenobarbital (PB) and 3-methylcholanthrene (3-MC).The induction profile of the drug-metabolizing enzymes, CYP2B1 and CYP2B2 in the hepatic microsomes of rats treated with nine 3-MeSO(2) derivatives, namely 3-MeSO(2)-2,4',5-trichlorobiphenyl, 3-MeSO(2)-2,2',4',5-tetrachlorobiphenyl (3-MeSO(2)-2,2',4',5-tetraCB), 3-MeSO(2)-2,2',5,5'-tetraCB, 3-MeSO(2)-2,3',4',5-tetraCB, 3-MeSO(2)-2,2',3',4',5-pentachlorobiphenyl (3-MeSO(2)-2,2',3',4',5-pentaCB), 3-MeSO(2)-2,2',4',5,5'-pentaCB, 3-MeSO(2)-2,2',3',4',5,5'-hexachlorobiphenyl (3-MeSO(2)-2,2',3',4',5,5'-hexaCB), 3-MeSO(2)-2,2',3',4',5,6-hexaCB and 3-MeSO(2)-2,2',4',5,5',6-hexaCB, was similar to that of rats treated with PB, but was different from that of rats treated with 3-MC. These findings indicate that 3-MeSO(2) metabolites derived from nine PCBs are PB-type inducers of microsomal drug-metabolizing enzymes. The relative inducing potencies of 3-MeSO(2) derivatives on the hepatic drug-metabolizing enzyme activities differed with the extent of chlorination and the positions of chlorine substituent on the phenyl rings. The results of present study show that the structure-CYP2B1/2 induction relationship exists for the 3-MeSO(2) derivatives studied. The inducing abilities of 3-MeSO(2)-2,2',4',5-tetraCB and 3-MeSO(2)-2,2',4',5,5'-pentaCB (2 μmol/kg) on the content of cytochrome P450 were higher than those of 2,3',4,4',5-pentaCB (mono-ortho-substituted PCB) (80 μmol/kg), 3,3',4,4'-tetraCB (coplanar PCB) (80 μmol/kg) and 3,3',4,4',5-pentaCB (coplanar PCB) (0.5 μmol/kg). The inducing effects of the administration of 3-MeSO(2)-2,2',4',5-tetraCB and 3-MeSO(2)-2,2',4',5,5'-pentaCB at 2 μmol/kg on the contents of total cytochrome P450, CYP2B1 and CYP2B2 corresponded to those of PB at 431 μmol/kg twice at a 24 h interval. It is noticeable that 3-MeSO(2)-2,2',4',5-tetraCB and 3-MeSO(2)-2,2',4',5,5'-pentaCB have highly potent PB-type inducing activity on drug-metabolizing enzyme systems.     

Detection and mechanism of formation of the potent direct-acting mutagen 2-bromoacrolein from 1,2-dibromo-3-chloropropane

The nematocide 1,2-dibromo-3-chloropropane (DBCP) was converted to products which are mutagenic for Salmonella typhimurium TA 100 in the presence of rat liver microsomes, NADPH, and oxygen. Typical in vivo and in vitro inhibitors of cytochrome P-450 decreased DBCP mutagenicity in the presence of microsomes. Addition of glutathione to cytosolic preparations failed to bioactivate DBCP to mutagenic metabolites. Mutagenicity studies with selectively deuterated analogs showed that substitution of deuterium for hydrogen at C-1 or C-3 of DBCP modestly decreased mutagenicity, but that deuteration at both C-1 and C-3 markedly decreased mutagenicity. The formation rates of the potent direct-acting mutagen, 2-bromoacrolein (2-BA), in incubations of DBCP and its deuterated analogs with rat liver microsomes, correlated with the isotope effects on mutagenicity. Characterization of 2-BA was accomplished by gas chromatography-mass spectrometry using positive-ion chemical ionization. Mass spectral analysis of 2-BA formed from specifically deuterated analogs of DBCP indicated that initial oxidative dehalogenation at C-1 followed by a spontaneous beta-elimination reaction was the preferred pathway in the formation of 2-BA from DBCP. These results demonstrate that mutagenic metabolites of DBCP are formed by cytochrome P-450-mediated oxidative metabolism, and that 2-BA is a major mutagen formed.