Hideto Jinno

UGT1A1 Haplotypes Associated with Reduced Glucuronidation and Increased Serum Bilirubin in Irinotecan‐administered Japanese Patients with Cancer

A comprehensive haplotype analysis of UGT1A1 in the Japanese population was conducted, and the effects of these haplotypes were investigated with respect to UGT1A1‐related phenotypic parameters in patients with cancer who received irinotecan.

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.

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver

Abstract We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg. Among the P450-dependent monooxygenase activities, testosterone 2α-hydroxylase (T2AH) and testosterone 6β-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA. The levels of the control activities were 13 and 50%, respectively. Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C11/6 and CYP3A2/1 protein levels in rat liver microsomes. In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19–73%). The Km values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats. The Vmax for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg. On the other hand, lauric acid ω-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (1.5- and 1.7-fold, respectively). Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression. However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7α-hydroxylase (T7AH) were not affected by BPA at any dose. These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA.

Haplotype structures of the UGT1A gene complex in a Japanese population.

Genetic polymorphisms of UDP-glucuronosyltransferases (UGTs) are involved in individual and ethnic differences in drug metabolism. To reveal co-occurrence of the UGT1A polymorphisms, we first analyzed haplotype structures of the entire UGT1A gene complex using the polymorphisms from 196 Japanese subjects. Based on strong linkage disequilibrium between UGT1A8 and 1A10, among 1A9, 1A7, and 1A6, and between 1A3 and 1A1, the complex was divided into five blocks, Block 8/10, Block 9/6, Block 4, Block 3/1, and Block C, and the haplotypes for each block were subsequently determined/inferred. Second, using pyrosequencing or direct sequencing, additional 105 subjects were genotyped for 41 functionally tagged polymorphisms. The data from 301 subjects confirmed the robustness of block partitioning, but several linkages among the haplotypes with functional changes were found across the blocks. Thus, important haplotypes and their linkages were identified among the UGT1A gene blocks (and segments), which should be considered in pharmacogenetic studies.

Comprehensive UGT1A1 Genotyping in a Japanese Population by Pyrosequencing

Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), is important in the detoxification and enhanced elimination of a large number of endogenous and exogenous substrates. The human UGT1A gene complex contains at least nine variations of exon 1, common exons 2–5, and a single exon 1 splices to exons 2–5 (1). Of the UGT1A isoforms, UGT1A1 is primarily responsible for the glucuronidation of bilirubin in the human liver and can also conjugate phenols, anthraquinones, flavonoids, and a variety of therapeutic drugs and their metabolites (e.g., SN-38, an active irinotecan metabolite) (2)(3). Several functional polymorphisms in UGT1A1 are associated with reduced bilirubin glucuronidation activity and cause hyperbilirubinemia (Gilbert and Crigler–Najjar syndromes). UGT1A1 TATA box variants [A(TA)6TAA>A(TA)5/7/8TAA] are associated with enhanced [(TA)5] or reduced [(TA)7/8] UGT1A1 transcription (4). Among them, the (TA)6 and (TA)7 repeats have been reported in Asians. The variant (TA)7 is associated with reduced glucuronidation of SN-38 and bilirubin, as well as the pathogenesis of Gilbert syndrome (5). In addition, a T-to-G substitution at nucleotide −3279 (A of the translational start codon in GenBank accession no. AF297093.1 is nucleotide number 1) in the UGT1A1 phenobarbital-responsive enhancer module reduces transcriptional activity (6). The most common nonsynonymous single-nucleotide polymorphism (SNP) (211G>A) that causes an amino acid alteration (glycine to arginine at codon 71) is found in Asian populations at frequencies of 13–23% (7)(8). The 686C>A (P229Q) variation in the Taiwanese has a frequency of 2.8% (8). Also associated with Gilbert syndrome are 211G>A (G71R) and 686C>A (P229Q). Rare in Japanese and Taiwanese patients is 1456T>G (Y486D), which is associated with the more severe type II Crigler–Najjar syndrome (8)(9). Our previous study demonstrated that …

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.

Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) in liver microsomes from human, monkey, rat and mouse by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than previous methods using a spectrofluorimeter and fluorescence plate reader. The detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol/assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver microsomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in liver microsomes from all animal species exhibited a monophasic kinetic pattern, whereas the pattern of EROD activities in rat and mouse liver microsomes was biphasic. In addition, the method could determine the non-inducible and 3-methylcholanthrene-inducible activities of EROD and MROD in rat and mouse 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 cytochrome CYP1A isoforms in mammals.

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.

Resolution of enantiomers of alcohols and amines by high-performance liquid chromatography after derivatization with a novel fluorescent chiral reagent

4-(2-Chloroformylpyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole [R( + )-NBD-Pro-COCl and S( − )-NBD-Pro-COCl], optically active tagging reagents, have been synthesized for resolution of enantiomers of amines and alcohols by high-performance liquid chromatography. The reagents react with amino and hydroxyl functional groups in the presence of pyridine to produce the corresponding diastereomers. The optimum excitation and emission wavelengths for the diastereomers in water-acetonitrile (1:1) were approximately 485 nm and 530 nm, respectively. The excitation and emission wavelengths were independent of the amine or alcohol portion of the diastereomer. The resulting diastereomers can usually be efficiently resolved by normal-phase chromatography with n-hexane-ethyl acetate as the eluent. When R( + )-NBD-Pro-COCl was used as the derivatization reagent, the diastereomers corresponding to the R-configurations of amines and alcohols were eluted faster than those from the S-configuration. The elution order was reversed when the diastereomers were prepared with S( − )-NBD-Pro-COCl. The Rs values of the diastereomers derived from amines and alcohols by normal-phase chromatography are in the range of 3.23–4.32 and 2.99–4.10, respectively. After derivatization with NBD-Pro-COCls the alcohol enantiomers were also separated adequately by a reversed-phase column with a water-acetonitrile mixture.