Yoshimitsu Oda

Specificity and sensitivity of Salmonella typhimurium YG1041 and YG1042 strains possessing elevated levels of both nitroreductase and acetyltransferase activity

Acetyltransferase and nitroreductase are enzymes involved in the intracellular metabolic activation of nitroarenes and/or aromatic amines in Salmonella typhimurium. The plasmid carrying both the acetyltransferase and nitroreductase genes was introduced into S. typhimurium TA98 and TA100. The resulting strains, YG1041 and YG1042, respectively, showed high levels of both enzyme activities and were more sensitive to the mutagenic action of some nitro-aromatic compounds such as 2-nitrofluorene, 1-nitropyrene and p-nitrophenetole than did the sensitive strains previously established in this laboratory or the conventional strains. These results indicate that the new strains permit the very efficient detection of the mutagenicity of nitroarenes in the environment.

7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes

7-Ethoxycoumarin O-deethylation has been used widely as a marker activity for assessing substrate specificities of cytochromes P450 (P450) in liver microsomes of mammals, and extensive studies have shown that in rats and mice the major catalysts are P450 1A1, 1A2, and 2B enzymes. In contrast to findings in experimental animal models, P450 2E1 has been reported to be a principal enzyme involved in 7-ethoxy-coumarin O-deethylation in human livers. In this study, we further examined the roles of individual forms of human P450 involved in 7-ethoxycoumarin O-deethylation using microsomes from different human liver samples and from human lymphoblastoid cells expressing human P450 enzymes and purified P450 enzymes isolated from the membrane of Escherichia coli expressing modified P450 proteins. Kinetic analysis showed that there were at least two different enzymes involved in 7-ethoxycoumarin O-deethylation in different human samples. Samples that contained high amounts of P450 2E1 in liver microsomes showed biphasic curves for O-deethylation with relatively high turnover numbers, whereas P450 1A2-rich samples tended to have low Km values with low Vmax values. Anti-human P450 2E1 antibodies inhibited markedly (P < 0.05) the 7-ethoxycoumarin O-deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 microM). However, we also found that anti-P450 1A2 antibodies suppressed O-deethylation activities only at a low substrate concentration (10 microM). Recombinant human P450 1A2 was found to have a low Km value for 7-ethoxycoumarin O-deethylation, whereas P450 2E1 showed a high Km value. Of the P450 enzymes examined, P450 1A1 gave the highest O-deethylation activities with a low Km value, although this enzyme is reported to be expressed extrahepatically in humans. Other human P450 enzymes, including P450 2A6, 2C10, 2D6, 3A4, and 3A5, did not show significant O-deethylation activities except that P450 2B6, a minor P450 component in human livers, was found to have a Vmax value similar to that of P450 1A2 and a Km value similar to that of P450 2E1. These results suggest that P450 1A2 is a low Km enzyme for 7-ethoxycoumarin O-deethylation in human liver microsome, although it has a low Vmax value than P450 2E1.

Inhibitory effect of curcumin on SOS functions induced by UV irradiation

The antigenotoxic effects of curcumin, including the inhibition of SOS induction and mutagenesis by UV light, were investigated in Salmonella typhimurium TA1535/pSK1002 and Escherichia coli K-12 strains. Induction of the SOS gene (umuC) expression was assayed by measuring accumulated beta-galactosidase activity. We found that curcumin blocked umuC induction promoted by UV irradiation in a dose-dependent manner. Also, with another SOS response, Weigle reactivation, we observed that curcumin effectively inhibited phage reactivation by UV irradiation. Furthermore, we tested the effect of curcumin on UV mutagenesis. We showed that mutagenesis induced by UV irradiation was suppressed by the addition of curcumin. Together these results indicate that curcumin acts as an inhibitor of SOS functions including UV mutagenesis.

Identification of a carcinogenic heterocyclic amine in river water

Abstract We investigated the existence of carcinogenic heterocyclic amines in water samples from a river near Kyoto, where several sewage-treatment plants discharge their effluents and water purification plants downstream use this polluted water as their source. Trp-P-2 is one of the carcinogenic heterocyclic amines included in charred meat and is often excreted in human waste in genotoxic fractions. We analysed Trp-P-2 by reverse-phase HPLC using an ODS column and the UV spectrum. Genotoxic potent activities for blue-rayon extracts of river water were detected in the samples at sampling locations. We identified Trp-P-2 in a genotoxic fraction by MS analysis. The concentrations of Trp-P-2 were calculated in the order of 10−6 mg l−1. These results indicated the existence of carcinogenic heterocyclic amines in the river water. The authors concluded that these carcinogens derive from the effluent of sewage treatment plants including human waste.

A loss of uvA function decreases the induction of the SOS functions recA and umuC by mitomycin C in Escherichia coli

We have studied the levels of recA and umuC protein synthesis in Escherichia coli as a probe for regulatory and mechanistic events involved in mitomycin C mutagenesis. Both RecA and UmuC protein induction were greatly stimulated by mitomycin C in the wild-type strain, reached a peak at about 60 min for the recA gene, and at 90 min for the umuC gene, respectively, and maintained a plateau. The induction was blocked by recA and lexA(Ind-) mutations that conferred no mutagenesis on the cell. Mutation affecting uvrA protein markedly decreased induction of the recA gene as well as the umuC gene by mitomycin C. The results established that UvrA protein is involved in the induction of recA and umuC, and account, at least in part, for the mitomycin C nonmutability of uvrA mutants.

Protective effects of baicalein and wogonin against benzo[a]pyrene- and aflatoxin B1-induced genotoxicities

To evaluate the protective effects of baicalein and wogonin against benzo[a]pyrene- and aflatoxin (AF) B(1)-induced toxicities, the effects of these flavonoids on the genotoxicities and oxidation of benzo[a]pyrene and AFB(1) were studied in C57BL/6J mice. Baicalein and wogonin reduced benzo[a]pyrene and AFB(1) genotoxicities as monitored by the umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. Baicalein added in vitro decreased liver microsomal benzo[a]pyrene hydroxylation (AHH) activity with an ic(50) of 33.9 +/- 1.4 microM at 100 microM benzo[a]pyrene. Baicalein also inhibited AFQ(1) and AFB(1)-epoxide formation from AFB(1) (50 microM) oxidation (AFO) with ic(50) values of 22.8 +/- 1.4 and 5.3 +/- 0.8 microM, respectively. However, the in vitro inhibitory effects of wogonin on AHH and AFO activities in liver microsomes were less than those of baicalein as inhibition by 500 microM wogonin was only about 51-65%. Treatment of mice with liquid diets containing 5 mM baicalein and wogonin resulted in 22 and 49% decreases in hepatic AHH activities, respectively. Baicalein treatment resulted in 39 and 32% decreases in AFQ(1) and AFB(1)-epoxide formation from liver microsomal AFO, respectively. Wogonin treatment resulted in 39 and 47% decreases in AFQ(1) and AFB(1)-epoxide formation, respectively. A 1-week pretreatment with wogonin significantly decreased hepatic DNA adduct formation in mice treated with 200 mg/kg of benzo[a]pyrene via gastrogavage. These in vitro and in vivo effects suggested that baicalein and wogonin might have beneficial effects against benzo[a]pyrene- and AFB(1)-induced hepatic toxicities and that wogonin had a stronger protective effect in vivo.

Genotoxicity of 3,6-dinitrobenzo(e)pyrene, a novel mutagen in ambient air and surface soil, in mammalian cells in vitro and in vivo

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP), newly identified in airborne particles and surface soil, is a potent mutagen in Salmonella typhimurium. The present study investigated the genotoxic potency of 3,6-DNBeP in vitro and in vivo using mammalian cell strains (Chinese hamster CHL/IU and human HepG2) and ICR mice, respectively. In the hprt gene mutation assay using HepG2 cells, the spontaneous mutant frequency was 61.1 per 10(5) clonable cells, which increased to 229 per 10(5) clonable cells after treatment with 1.0 microg/ml (3 microM) 3,6-DNBeP. Notably, in HepG2 cells with increased N-acetyltransferase 2 activity, the mutant frequency increased to 648 per 10(5) clonable cells by treatment of 1.0 microg/ml (3 microM) 3,6-DNBeP. The sister chromatid exchange frequency increased approximately three times the control level in HepG2 cells treated with 3,6-DNBeP at a concentration of 1.0 microg/ml (3 microM). In HepG2 and CHL/IU cells, the frequency of the cells with micronuclei was 0.9 and 1.2%, and the frequencies increased to 2.3 and 7.6% after 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment, respectively. The H2AX phosphorylation level increased 8-fold compared with the background level with 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment in HepG2 cells. Moreover, the comet assay showed that 3,6-DNBeP produced DNA damage in the cells of liver, kidney, lung and bone marrow in ICR mice 3 h after intraperitoneal injection at 40 mg/kg (0.12 mmol/kg) body weight. These data indicate that 3,6-DNBeP is genotoxic to mammalian cells in vitro and in vivo.

Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/umu assay

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.

Mutagenicity of TCDD in Big Blue® transgenic rats

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a significant environmental contaminant resulting from such industrial processes as pulp and paper production. TCDD is a suspected human carcinogen and its ability to induce cancer in laboratory rodents is well documented. Its mechanism of tumor initiation, however, is not well understood and in vitro mutagenicity studies have yielded inconsistent results. In this study, Big Blue lacI transgenic rats were used to assess the mutagenicity of TCDD in both male and female animals. After 6 weeks of exposure to 2 microg/kg TCDD neither an increase in mutation frequency nor any change in mutation spectrum was observed in either male or female animals.

Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay.

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)–catalyzed N‐hydroxylation and subsequent esterification by O‐acetyltransferase (O‐AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH‐P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113–120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW″/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O‐AT gene in the pOA101‐expressing umuC″lacZ gene) in S. typhimurium TA1535. In addition, as an O‐AT–deficient strain, we developed the OY1003/1A2 strain by introducing pCW″/1A2:hNPR and pOA101 into O‐AT–deficient S. typhimurium TA1535/1,8‐DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and ≧0 nmol isoniazid/min/mg protein as the O‐AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2‐amino‐1,4‐dimethylimidazo[4,5‐f]quinoline (MeIQ), 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx), 2‐aminoanthracene, 2‐amino‐6‐methyldipyrido[1,2‐a::3,2′‐d]imidazole, 3‐amino‐1,4‐dimethyl‐5H‐pyrido[4,3‐b]indole, and 3‐amino‐1‐methyl‐5H‐pyrido[4,3‐a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O‐acetyltransferase. Environ. Mol. Mutagen. 36:121–126, 2000.