Toshiko Tanaka-Kagawa

Functional characterization of two variant human GSTO 1-1s (Ala140Asp and Thr217Asn).

Glutathione-S-transferase class Omega (GSTO 1-1) belongs to a new subfamily of GSTs, which is identical with human monomethylarsonic acid (MMA(V)) reductase, the rate limiting enzyme for biotransformation of inorganic arsenic, environmental carcinogen. Recombinant GSTO 1-1 variants (Ala140Asp and Thr217Asn) were functionally characterized using representative substrates. No significant difference was observed in GST activity towards 1-chloro-2,4-dinitrobenzene, whereas thioltransferase activity was decreased to 75% (Ala140Asp) and 40% (Thr217Asn) of the wild-type GSTO 1-1. For MMA(V) reductase activity, the Ala140Asp variant exhibited similar kinetics to wild type, while the Thr217Asn variant had lower V(max) (56%) and K(m) (64%) values than the wild-type enzyme. The different activities of the enzyme variants may influence both the intracellular thiol status and arsenic biotransformation. This can help explain the variation between individuals in their susceptibility to oxidative stress and inorganic arsenic.

Determination of UDP-glucuronosyltransferase UGT1A6 activity in human and rat liver microsomes by HPLC with UV detection

A simple and sensitive method for the determination of UDP-glucuronosyltransferase UGT1A6 activity using 4-methylumbelliferone (4-MU) and 4-nitrophenol (4-NP) as substrates in human and rat liver microsomes by high-performance liquid chromatography (HPLC) with uv detection is reported. The method was validated for the determination of 4-methylumbelliferyl beta-D-glucuronide (4-MUG) and 4-nitrophenyl beta-D-glucuronide (4-NPG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. There was no interference from matrix and non-enzymatic reactions. Calibration curves for 4-MUG and 4-NPG are linear from 0.5 to 500 microM. Average recoveries ranged from 98 to 100% in spiked liver microsomes samples. 4-MUG and 4-NPG were stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was found to be more sensitive than previous methods using a spectrophotometer, a spectrofluorometer and HPLC. The detection limit for 4-MUG and 4-NPG (signal-to-noise ratio of 3) was 14 and 23 nM, respectively. The intra- and inter-day precision (relative S.D. (RSD)) and accuracy (relative mean error (RME)) was <5 and 9%, respectively. The intra- and inter-day reproducibility (RSD) of UGT1A6 enzyme assay in liver microsomes was <6%. With this improved sensitivity, the kinetics of UGT activities toward 4-MU and 4-NP in human and rat liver microsomes could be determined more precisely. In addition, the method could determine the non-inducible, and 3-methylcholanthrene- and phenobarbital-inducible activities of UGT1A6 in rat liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with UGT1A6 isoform in mammals using small amounts of biological samples.

Overexpression of manganese-superoxide dismutase prevents methylmercury tox1city in hela cells

HeLa cells were stably transformed with plasmid constructs that allowed constitutive expression of antioxidant enzymes such as catalase, glutathione peroxidase (GSH-Px), Cu,Zn-superoxide dismutase (Cu,Zn-SOD) or Mn-superoxide dismutase (Mn-SOD) to examine the involvement of reactive oxygen generation in methylmercury toxicity. Overexpression of catalase, GSH-Px or Cu,Zn-SOD did not affect the sensitivity of HeLa cells against methylmercury. However, the sensitivity of HeLa cells against methylmercury was decreased by overexpression of Mn-SOD, an enzyme localized in matrix of mitochondria and which decomposes superoxide anions. These results suggest that formation of superoxide anions in the mitochondria might be involved in the mechanism of the cytotoxicity of methylmercury.

Interaction of bisphenol A with rat hepatic cytochrome P450 enzymes

The effect of bisphenol A (BPA) on the kinetics of cytochrome P450 (P450)-dependent monooxygenases in rat liver microsomes was studied. Testosterone 16beta-hydroxylase (TS16BH) and testosterone 2alpha-hydroxylase (TS2AH) activities were extensively inhibited by BPA at 100 microM (69% and 74%, respectively). The inhibition type was mixed for both P450-dependent monooxyganases. The Ki of TS16BH and TS2AH from Lineweaver-Burk plots were 25.9 and 24.9 microM, respectively. The activities of acetanilide 4-hydroxylase (AA4H), 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1-hydroxylase (BF1H), chlorzoxazone 6-hydroxylase (CZ6H) and testosterone 6beta-hydroxylase (TS6BH) were also effectively inhibited by BPA at 100 microM (43-52%). The inhibition type of these P450-dependent monooxygenases was mixed or uncompetitive, and the K(i)s (50.5-88.5 microM) were higher than those of TS16BH and TS2AH. By contrast, the values of IC50 and Ki of testosterone 7alpha-hydroxylase (TS7AH) and lauric acid omega-hydroxylase (LAOH) for BPA were >1000 microM. These results suggest that BPA interacts with rat hepatic CYP1A2, CYP2A2, CYP2B2, CYP2C11, CYP2D1, CYP2E1 and CYP3A2 in vitro.

Initial Response and Cellular Protection through the Keap1/Nrf2 System during the Exposure of Primary Mouse Hepatocytes to 1,2-Naphthoquinone

Quinones are reactive chemical species that cause cellular damage by modifying protein thiols and/or catalyzing the reduction of oxygen to reactive oxygen species, thereby promoting oxidative stress. Transcription factor Nrf2 plays a crucial role in cellular defense against electrophilic modification and oxidative stress. In studies using 1,2-naphthoquinone (1,2-NQ) as a model quinone, we found that Keap1, the negative regulator of Nrf2, was readily arylated at its reactive thiols by 1,2-NQ. Exposure of primary mouse hepatocytes to 1,2-NQ resulted in the activation of Nrf2 and the upregulation of some of Nrf2s downstream genes. This interaction was further investigated in hepatocytes from Nrf2 knockout mice in which the proteins responsible for the metabolism and excretion of 1,2-NQ are minimally expressed. The chemical modification of cellular proteins by 1,2-NQ was enhanced by Nrf2 deletion, resulting in increased toxicity. However, deletion of the negative regulatory protein, Keap1, drastically reduced the covalent binding by 1,2-NQ and its cellular toxicity. Experiments with chemicals that inhibit the biotransformation and extracellular excretion of 1,2-NQ suggest that 1,2-NQ undergoes detoxification and excretion into the extracellular space predominantly by two-electron reduction and subsequent glucuronidation by NAD(P)H:quinone oxidoreductase 1 and uridine 5-diphosphate-glucuronosyltransferases, followed by multidrug resistance-associated protein-dependent excretion. These findings suggest that the Keap1/Nrf2 system is essential for the prevention of cell damage resulting from exposure to 1,2-NQ.

Functional analysis of three CYP1A2 variants found in a Japanese population.

Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previously reported three naturally occurring genetic polymorphisms (125C>G, Pro42Arg, CYP1A2*15; 1130G>A, Arg377Gln, *16; and 1367G>A, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation revealed that Vmax and Vmax/Km of all three variants were less than 3 and 1% of the WT, respectively, although the Km value was significantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins produced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggesting marked reductions in their hemoprotein formation. These results suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoenzyme.

In vitro biotransformation of atrazine by rat liver microsomal cytochrome P450 enzymes

We studied atrazine (ATZ) metabolism in male and female rat liver microsomes in vitro, and the major metabolite was deisopropylatrazine (DeiPr-ATZ) with deethylatrazine (DeEt-ATZ) and 1-hydroxyisopropylatrazine (iPrOH-ATZ) as minor metabolites in both sexes. The enzyme kinetics of ATZ biotransformation were examined by means of Eadie-Hofstee analyses. Although no remarkable sex difference of Michaelis Menten values for each pathway was observed, Cl(int)S (Vmax/Km) for DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ were slightly higher in female than in male rats. The formation of DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ from ATZ was substantially inhibited by SKF-525A, metyrapone, diallyl sulfide, 7-ethoxycoumarin, benzphetamine, nicotine, testosterone and lauric acid in both sexes. Cimetidine effectively inhibited the formation of all metabolites in male rats. On the other hand, the inhibition rates of the formation of DeiPr-ATZ and iPrOH-ATZ by cimetidine in female rats were lower than those in male rats, and DeEt-ATZ was hardly affected by the chemicals. In contrast with the results for cimetidine, the inhibition of ATZ biotransformation by bufuralol was more effective in female than in male rats. Anti-rat CYP2B1 and CYP2E1 antibodies effectively inhibited DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ formations in both sexes. Anti-rat CYP2C11 antibody also inhibited the three metabolites in both sexes, with the inhibition rates higher in male than in female rats, similar to cimetidine. In the case of anti-rat CYP2D1 antibody, the inhibitory effect on ATZ biotransformation in male rats was less than that in female rats. On the other hand, anti-rat CYP1A2, CYP3A2 and CYP4A1 antibodies did not affect the ATZ biotransformation in either sex. There was no significant correlation between the formation rate of ATZ metabolites and P450 isoform levels in either sex. These results may mean that CYP2B2, CYP2C11, CYP2D1 (only iPrOH-ATZ formation) and CYP2E1 in male rats, and CYP2B2, CYP2D1 and CYP2E1 in female rats are involved ATZ metabolism in liver, and that the substrate specificity of P450 isoforms for ATZ is broad.

Interaction of bisphenol a with human UDP‐glucuronosyltransferase 1A6 enzyme

The effects of bisphenol A (BPA) on UDP‐glucuronosyltransferase 1A6 (UGT1A6) activities in microsomes from human livers and yeast cells expressing human UGT1A6 (humUGT1A6) were investigated. Serotonin (5‐HT) and 4‐methylumbelliferone (4‐MU) were used as the substrates for UGT1A6. BPA dose‐dependently inhibited 5‐HT and 4‐MU glucuronidation activities in both enzyme sources. The IC50 values of BPA for 5‐HT and 4‐MU glucuronidation activities were 156 and 163 μM for liver microsomes, and 84.6 and 80.3 μM for yeast cell microsomes expressing humUGT1A6, respectively. The inhibitory pattern of BPA for 5‐HT and 4‐MU glucuronidation activities in human liver microsomes exhibited a mixture of competitive and noncompetitive components, with Ki values of 84.9 and 72.3 μM, respectively. In yeast cell microsomes expressing humUGT1A6, 5‐HT glucuronidation activities were noncompetitively inhibited by BPA (Ki value, 65.5 μM), whereas the inhibition of 4‐MU glucuronidation activities by BPA exhibited the mixed type (Ki value, 42.5 μM). These results suggest that BPA interacts with human UGT1A6 enzyme, and that the interaction may contribute to the toxicity, such as hormone disruption and reproductive effects, of BPA.

Haplotypes and a Novel Defective Allele of CES2 Found in a Japanese Population

Human carboxylesterase 2 (hCE-2) is a member of the serine esterase superfamily and is responsible for hydrolysis of a wide variety of xenobiotic and endogenous esters. hCE-2 also activates an anticancer drug, irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin, CPT-11), into its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38). In this study, a comprehensive haplotype analysis of the CES2 gene, which encodes hCE-2, in a Japanese population was conducted. Using 21 single nucleotide polymorphisms (SNPs), including 4 nonsynonymous SNPs, 100C>T (Arg34Trp, *2), 424G>A (Val142Met, *3), 1A>T (Met1Leu, *5), and 617G>A (Arg206His, *6), and a SNP at the splice acceptor site of intron 8 (IVS8-2A>G, *4), 20 haplotypes were identified in 262 Japanese subjects. In 176 Japanese cancer patients who received irinotecan, associations of CES2 haplotypes and changes in a pharmacokinetic parameter, (SN-38 + SN-38G)/CPT-11 area under the plasma concentration curve (AUC) ratio, were analyzed. No significant association was found among the major haplotypes of the *1 group lacking nonsynonymous or defective SNPs. However, patients with nonsynonymous SNPs, 100C>T (Arg34Trp) or 1A>T (Met1Leu), showed substantially reduced AUC ratios. In vitro functional characterization of the SNPs was conducted and showed that the 1A>T SNP affected translational but not transcriptional efficiency. These findings are useful for further pharmacogenetic studies on CES2-activated prodrugs.

Detection of 34 plasticizers and 25 flame retardants in indoor air from houses in Sapporo, Japan.

Various plasticizers and flame retardants are contained in building materials and furniture produced for indoor environments. However, some of these material inclusions have been reported to cause endocrine-disrupting and mucosa-irritating effects. Because of the local climate, buildings in Sapporo are better insulated against cold weather than those in many other areas in Japan. In this study, we measured 59 compounds, including plasticizers (phthalates, adipates, and others) and flame retardants (organo-phosphates and brominated compounds), from indoor air samples from six houses in Sapporo. These compounds were measured separately in the gas phase and the particle phase using a two-stage cartridge equipped with a quartz fiber filter (1 μm mesh) and C18 solid-phase extraction disk for sampling and analyzed by GC/MS and LC/MS/MS (for the detection of brominated flame retardants). Among the 59 compounds measured in this study, 34 compounds were detected from the indoor air of the six houses. The highest concentration among the 34 compounds found in a newly built house was 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate (TXIB) at 20.8 μg/m(3). Di(2-ethyl-1-hexyl)terephthalate (DEHT), which has been used in recent years as an alternative to di(2-ethyl-1-hexyl)phthalate (DEHP), was found in all six houses, although at low concentrations ranging from 0.005 to 0.027 μg/m(3). To our knowledge, this is the first report of DEHT in indoor air in Japan. Among the compounds detected in this study, those with lower molecular weights tended to be captured in the C18 solid-phase extraction disk rather than in the quartz fiber filter. These results suggest that compounds with higher volatility exist preferentially in the gas phase, whereas compounds with lower volatility exist preferentially in the particulate phase in indoor air.