Biomedical subjects

Hideto Jinno

Publications and source records attributed to Hideto Jinno.

At least 19 recordsLinked to original sources

PubMed2006

Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A6 enzymes.

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major isoform in the human liver that glucuronidates numerous drugs, environmental chemicals and endogenous substrates. In this study, human and cynomolgus monkey UGT1A6 cDNAs (humUGT1A6 and monUGT1A6, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate UGT1A6s. The enzymatic properties of UGT1A6 proteins were characterized by the kinetic analysis of serotonin (5-hydroxytryptamine, 5-HT) and 4-methylumbelliferone (4-MU) glucuronidation. humUGT1A6 and monUGT1A6 showed 96% identity in their nucleotide and amino acid sequences. Immunoblotting analysis using an antibody raised against human UGT1A6 showed that protein staining intensities were different between human and cynomolgus monkey UGT1A6 enzymes in microsomal fractions from livers and yeast cells, although both enzymes were detectable. The apparent K(m) value (15 mM) for 5-HT glucuronidation of cynomolgus monkey liver microsomes was significantly higher than that (8.6mM) of human liver microsomes, whereas V(max) values were lower in cynomolgus monkeys (2.8 nmol/min/mg protein) than in humans (8.6 nmol/min/mg protein). No significant species difference was observed in K(m) (approximately 90 microM) or V(max) (approximately 25 nmol/min/mg protein) values for liver microsomal 4-MU glucuronidation. In yeast cell microsomes, K(m) values (approximately 6mM) for 5-HT glucuronidation by recombinant UGT1A6s were similar, while a V(max) value (0.1nmol/min/mg protein) of monUGT1A6 was significantly lower than that (0.7 nmol/min/mg protein) of humUGT1A6. In 4-MU glucuronidation, both K(m) (210 microM) and V(max) (3.5 nmol/min/mg protein) values of monUGT1A6 were significantly higher than those of humUGT1A6 (K(m), 110 microM; V(max), 1.5nmol/min/mg protein). These findings suggest that the enzymatic properties of UGT1A6 were extensively different between humans and cynomolgus monkeys, although humUGT1A6 and monUGT1A6 showed high homology at the amino acid level. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.

Amino Acid Sequence↗

PubMed2006

Inducibility of UDP-glucuronosyltransferase 1As by beta-naphthoflavone in HepG2 cells.

UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms (UGT1A1, UGT1A6 and UGT1A9), mainly expressed in the human liver, and examined the inducibility of UGT1As by beta-naphthoflavone (BNF) in human hepatoma HepG2 cells. The cells were pretreated for 72 h with BNF at concentrations of 25, 50 and 100 microM. 7-Ethyl-10-hydroxycamptothecin (SN-38) glucuronidation, used as a probe for UGT1A1, showed sigmoidal kinetics with a Hill coefficient (n) of 1.2-1.3 in control and BNF-pretreated HepG2 cells. The Vmax values were significantly increased 3.6- to 4.3-fold by BNF, whereas there was no significant change in the S50 values by BNF at any concentration examined. On the other hand, 4-methylumbelliferone (4-MU) glucuronidation as a probe for UGT1A6 and UGT1A9 in the control and BNF-pretreated HepG2 cells exhibited a biphasic kinetic pattern. Although Km1 values for the low-Km phase were similar between the control and BNF-pretreated HepG2 cells, Km2 values for the high-Km phase of BNF-pretreated HepG2 cells were reduced to 54-69% of control HepG2 cells. The values of Vmax1 and Vmax2 for the low- and high-Km phases, respectively, were significantly increased 1.9- to 2.6-fold by BNF at 25 and/or 50 microM but not 100 microM. With respect to Vmax (Vmax1 and Vmax2) and Vmax/Km (Vmax1/Km1 and Vmax2/Km2), the values were significantly increased 2.0- to 3.2-fold by BNF at all concentrations examined. Furthermore, real-time reverse transcription polymerase chain reaction using TaqMan probes demonstrated that BNF concentration-dependently induced mRNA levels of UGT1A1 but not UGT1A6 or UGT1A9 in HepG2 cells (1.3- to 6.0-fold). These results suggest that the inducibility of UGT1A isoforms in HepG2 cells by BNF is different from other aryl hydrocarbon receptor agonists previously reported, and should provide useful information for the prediction of drug-drug interactions and toxicological assessment of environmental chemicals.

Cell Line, Tumor↗

PubMed2005

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 V(max) and V(max)/K(m) of all three variants were less than 3 and 1% of the WT, respectively, although the K(m) 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.

Animals↗

PubMed2005

Functional analysis of four naturally occurring variants of human constitutive androstane receptor.

The human constitutive androstane receptor (CAR, NR1I3) is a member of the orphan nuclear receptor superfamily that plays an important role in the control of drug metabolism and disposition. In this study, we sequenced all the coding exons of the NR1I3 gene for 334 Japanese subjects. We identified three novel single nucleotide polymorphisms (SNPs) that induce non-synonymous alterations of amino acids (His246Arg, Leu308Pro, and Asn323Ser) residing in the ligand-binding domain of CAR, in addition to the Val133Gly variant, which was another CAR variant identified in our previous study. We performed functional analysis of these four naturally occurring CAR variants in COS-7 cells using a CYP3A4 promoter/enhancer reporter gene that includes the CAR responsive elements. The His246Arg variant caused marked reductions in both transactivation of the reporter gene and in the response to 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), which is a human CAR-specific agonist. The transactivation ability of the Leu308Pro variant was also significantly decreased, but its responsiveness to CITCO was not abrogated. The transactivation ability and CITCO response of the Val133Gly and Asn323Ser variants did not change as compared to the wild-type CAR. These data suggest that the His246Arg and Leu308Pro variants, especially His246Arg, may influence the expression of drug-metabolizing enzymes and transporters that are transactivated by CAR.

Animals↗

PubMed2005

Functional analysis of six human aryl hydrocarbon receptor variants in a Japanese population.

Aryl hydrocarbon receptor (AhR) is an important transcriptional regulator involved in the induction of CYP1A1, CYP1A2, CYP1B1, UGT1A1, and UGT1A6. In this study, functional properties of four novel naturally occurring human AhR variants (K401R, N487D, I514T, and K17T/R554K) were examined along with the single variants K17T and R554K. The luciferase reporter assay using the CYP1A1 promoter reporter in HeLa cells treated with beta-naphthoflavone or 3-methylcholanthrene, which are known as typical agonists for AhR, showed that reporter activities of the K401R and N487D variants were reduced to 40 to 58% of those of wild-type (WT) but not of the other variants. Similarly, the K401R and N487D variants also reduced the omeprazole-induced reporter activities to approximately 56 and 74% of those of the WT, respectively. The reduced activities of the two variants were probably caused by the reduced protein expression levels, since the protein levels of the K401R and N487D variants were approximately 52 and 47% of the WT, respectively, without any changes in their mRNA levels. The reduced protein levels were recovered by treatment with a proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal], suggesting that the reduced protein levels were caused by the accelerated proteasomal degradation by a proteasome. Together, the current data demonstrate that the K401R and N487D variants reduce their apparent transcriptional activities, both ligand-induced and omeprazole-induced activation, probably through reduced protein expression. Thus, these two variants may influence drug metabolism through reduced induction of CYP1A1 and other target enzymes.

Genes, Reporter↗

PubMed2005

Functional characterization of five novel CYP2C8 variants, G171S, R186X, R186G, K247R, and K383N, found in a Japanese population.

Cytochrome P450 2C8 is one of the primary enzymes responsible for the metabolism of a wide range of drugs such as paclitaxel, cerivastatin, and amiodarone. We have sequenced the CYP2C8 gene from 201 Japanese subjects and found five novel nonsynonymous single nucleotide polymorphisms (SNPs): 511G>A (G171S), 556C>T (R186X; X represents the translational stop codon), 556C>G (R186G), 740A>G (K247R), and 1149G>T (K383N), with the allele frequency of 0.0025. The CYP2C8 variants were heterologously expressed in COS-1 cells and functionally characterized in terms of expression level, paclitaxel 6alpha-hydroxylase activity, and intracellular localization. The prematurely terminated R186X variant was undetectable by Western blotting and inactive toward paclitaxel 6alpha-hydroxylation. The G171S, K247R, and K383N variants exhibited properties similar to those of the wild-type CYP2C8. Paclitaxel 6alpha-hydroxylase activity of the R186G transfectant was only 10 to 20% that of wild-type CYP2C8. Furthermore, the R186G variant displayed a lower level of protein expression in comparison to the wild type, which was restored by the addition of a proteasome inhibitor (MG-132; Z-Leu-Leu-Leu-aldehyde). The reduced CO-difference spectral analysis using recombinant proteins from an insect cell/baculovirus system revealed that the R186G variant has a minor peak at 420 nm in addition to the characteristic Soret peak at 450 nm, suggesting the existence of improperly folded protein. These results indicate that the novel CYP2C8 SNPs, 556C>T (R186X) and 556C>G (R186G), could influence the metabolism of CYP2C8 substrates such as paclitaxel and cerivastatin.

Animals↗

PubMed2005

Inorganic arsenic compounds and methylated metabolites induce morphological transformation in two-stage BALB/c 3T3 cell assay and inhibit metabolic cooperation in V79 cell assay.

We have performed two-stage transformation assay using BALB/c 3T3 cells to determine initiating and promoting activities of disodium arsenate, sodium arsenite, monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA). Treatment with these arsenic compounds at the initiating stage induced significant numbers of transformed foci when cells were post-treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). Disodium arsenate was active at the concentrations of 15-30 microM, sodium arsenite 5-20 microM, and DMAA 1-2 mM. MMAA required 10 mM to induce cell transformation. The concentrations of these compounds (except DMAA) that induced transformation were highly growth-inhibitory (more than 50%). DMAA induced transformation foci at growth inhibition levels of 66 to 84%. In experiments on promoting activity, cells pretreated with a sub-threshold dose of 20-methylcholanthrene (MCA, 0.2 microg/ml) or sodium arsenite (10 microM) were used. Transformation was enhanced by post-treatment with disodium arsenate (1-10 microM), sodium arsenite (0.5-2 microM), and MMAA (200-1000 microM), but not with DMAA. Studies of gap junctional intercellular communication using the V79 cell metabolic cooperation assay showed that the arsenic compounds (except DMAA) exhibited inhibitory activity. Thus, most arsenicals were shown to have not only initiating activity, but also promoting activity. In addition, inorganic arsenicals, especially trivalent sodium arsenite, were more active than organic ones and exhibited promoting activity at one-order of magnitude lower than initiating activity. These results suggest that from the viewpoint of human hazard, more attention should be paid to the tumor promoting activity of inorganic arsenic compounds.

Animals↗

PubMed2005

Transfection assays with allele-specific constructs: functional analysis of UDP-glucuronosyltransferase variants.

Adverse drug reactions (ADRs) are a major clinical problem. A rapidly growing body of evidence suggests that genetic factors, at least in part, determine individual susceptibility to ADRs. A large number of pharmacogenetic studies have identified a number of polymorphisms as predictors of drug efficacy and/or adverse events. These candidate markers should be investigated further to ascertain the underlying mechanism of action, for example, changes in the kinetic parameters of an enzyme, or transcriptional activity of a promoter region. In this chapter, we describe a transient transfection assay for the functional characterization of naturally occurring variants of UDP-glucuronosyltransferase (UGT) 1A1. This phase II drug metabolizing enzyme is involved in the glucuronidation of SN-38, an active metabolite of the anti-cancer drug irinotecan. Single-nucleotide polymorphisms of the UGT1A1 gene have been correlated to irinotecan-induced ADRs. Variant UGT1A1s are heterologously expressed in COS-1 cells and characterized in terms of the level of protein expression and enzyme kinetics.

Adult↗

PubMed2005

Genetic variations and haplotypes of UGT1A4 in a Japanese population.

Nineteen genetic variations, including 11 novel ones, were found in exon 1 and its flanking region of the UDP-glucuronosyltransferase (UGT) 1A4 gene from 256 Japanese subjects, consisting of 60 healthy volunteers, 88 cancer patients and 108 arrhythmic patients. These variations include -217T>G and -36G>A in the 5'-flanking region, 30G>A (P10P), 127delA (43fsX22; frame-shift from codon 43 resulting in the termination at the 22nd codon, codon 65), 175delG (59fsX6), 271C>T (R91C), 325A>G (R109G), and 357T>C (N119N) in exon 1, and IVS1+1G>T, IVS1+98A>G and IVS1+101G>T in the following intron. Among them, 127delA and 175delG can confer early termination of translation, resulting in an immature protein that probably lacks enzymatic activity. Variation IVS1+1G>T is located at a splice donor site and thus may lead to aberrant splicing. Since we did not find any significant differences in the frequencies of all the variations among the three subject groups, the data were analyzed as one group. The allele frequencies of the novel variations were 0.006 for IVS1+101G>T, 0.004 for 30G>A (P10P) and 357T>C (N119N), and 0.002 for the 8 other variations. In addition, the two known nonsynonymous single nucleotide polymorphisms (SNPs), 31C>T (R11W) and 142T>G (L48V), were found at 0.012 and 0.129 frequencies, respectively. The SNP 70C>A (P24T), mostly linked with 142T>G (L48V) in German Caucasians, was not detected in this study. Sixteen haplotypes were identified or inferred, and some haplotypes were confirmed by cloning and sequencing. It was shown that most of 142T>G (L48V) was linked with -219C>T, -163G>A, 448T>C (L150L), 804G>A (P268P), and IVS1+43C>T, comprising haplotype *3a; haplotype *4a harbors 31C>T (R11W); 127delA (43fsX22) and 142T>G (L48V) were linked (haplotype *5a); 175delG (59fsX6) was linked with 325A>G (R109G) (*6a haplotype); and -219C>T, -163G>A, 142T>G (L48V), 271C>T (R91C), 448T>C (L150L), 804G>A (P268P), and IVS1+43C>T comprised haplotype *7a. Our results provide fundamental and useful information for genotyping UGT1A4 in the Japanese and probably Asian populations.

Arrhythmias, Cardiac↗

PubMed2005

Genetic polymorphisms of UGT1A6 in a Japanese population.

Thirteen single nucleotide polymorphisms (SNPs), including 6 novel ones, were found in exon 1 and its flanking region of UDP-glucuronosyltransferase (UGT) 1A6 from 195 Japanese subjects. Several novel SNPs were identified, including 269G>A (R90H), 279A>G (S93S), and 308C>A (S103X) in exon 1, and IVS1+109C>T, IVS1+120A>G, and IVS1+142C>T in the intron downstream of exon 1. Among these SNPs, 308C>A confers termination of translation at codon 103, resulting in the production of an immature protein that probably lacks enzymatic activity. The allele frequencies were 0.003 for all the 6 SNPs. In addition, the 3 known nonsynonymous SNPs were detected: 19T>G (S7A), 541A>G (T181A), and 552A>C (R184S) with frequencies of 0.226, 0.218, and 0.226, respectively. High linkage disequilibrium was observed among 19T>G (S7A), 315A>G (L105L), 541A>G (T181A), 552A>C (R184S), and IVS1+130G>T, as reported in Caucasian and African-American populations. Then, 11 haplotypes in UGT1A6 were estimated. The novel nonsynonymous variant, 269A or 308A, was shown to be located on the same DNA strand together with 19G, 315G, 541G, 552C, and IVS1+130T. Our results provide fundamental and useful information for genotyping UGT1A6 in the Japanese, and probably Asian populations.

Asian People↗

PubMed2005

Survey of volatile organic compounds found in indoor and outdoor air samples from Japan.

Indoor air quality is currently a growing concern, mainly due to the incidence of sick building syndrome and building related illness. To better understand indoor air quality in Japan, both indoor and outdoor air samples were collected from 50 residences in Iwate, Yamanashi, Shiga, Hyogo, Kochi and Fukuoka Prefectures. More than 100 volatile organic compounds (VOCs) were analyzed by thermal desorption-gas chromatography/mass spectrometry method. The most abundant class of compounds present in the indoor air samples were identified (i.e. alkanes, alkylbenzenes and terpenes). For 30% of the indoor air samples, the sum of each VOC exceeded the current provisional guideline value for total VOC (TVOC, 400 microg/m3). The major component of these samples included linear and branched-chain alkanes (possibly derived from fossil fuels), 1,4-dichlorobenzene (a moth repellent), alpha-pinene (emission from woody building materials) and limonene (probably derived from aroma products). As an unexpected result, one residence was polluted with an extremely high concentration of 1,1,1,2-tetrafluoroethane (720 microg/m3), suggesting accidental leakage from a household appliance such as a refrigerator. The results presented in this paper are important in establishing the Japanese target compound list for TVOC analysis, as well as defining the current status of indoor air quality in Japan.

Air↗

PubMed2004

Racial variability in haplotype frequencies of UGT1A1 and glucuronidation activity of a novel single nucleotide polymorphism 686C> T (P229L) found in an African-American.

Ethnic differences in genetic polymorphisms in UDP-glucuronosyltransferase 1A1 (UGT1A1) were investigated among African-Americans, Caucasians, and Japanese using samples obtained from 150 individuals for each population. Genotyping of -3279T>G in the phenobarbital-responsive enhancer module, TA repeats in the TATA box, 211G>A (G71R) and 686C>A (P229Q) in exon 1, and three single nucleotide polymorphisms (SNPs) (1813C> T, 1941C>G, and 2042C>G) in the 3'-untranslated region in exon 5 was performed. Eight haplotypes of block 1 (exon 1 and its 5'-flanking region) harboring the first four variations were assigned to each individual. The dominant haplotype for African-Americans was *28b (-3279G;TA(7); 211G;686C) (0.446), whereas that for the Japanese was *1a (-3279T; TA(6);211G;686C) (0.610). Frequencies of the two haplotypes *1a and *28b were comparable in Caucasians. Haplotype *6a (-3279T;TA(6); 211A;686C) was characteristic of the Japanese, whereas haplotypes *36b and *37b (-3279T;TA(5) and TA(8);211G;686C) were found mostly in African-Americans. Although the three SNPs in block 2 (exons 2-5) were in complete linkage in the Japanese, they were not completely linked in African-Americans or Caucasians. These differences in haplotype distribution patterns among the three populations suggest the possibility of ethnic differences in toxicity profiles of drugs detoxicated by UGT1A1. A novel SNP, 686C>T (P229L), was found in an African-American. The intrinsic clearance of 7-ethyl-10-hydroxycamptothecin (SN-38) by P229L UGT1A1 expressed in COS-1 cells was about 3% of the wild type. The results of Western blotting and real-time reverse transcription-polymerase chain reaction suggest that the low glucuronidation activity of the variant was partly due to its low stability. The variation 686C>T may cause high toxicity during 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) therapy or hyperbilirubinemia in patients.

Black or African American↗

PubMed2004

UGT1A1 haplotypes associated with reduced glucuronidation and increased serum bilirubin in irinotecan-administered Japanese patients with cancer.

PURPOSE: 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. METHODS: The UGT1A1 gene, including the enhancer, the promoter, and all 5 exons and their flanking regions, was sequenced from 195 Japanese subjects. The gene was divided into 2 blocks, and the haplotypes of each block were assigned. The association of these haplotypes with area under the concentration-time curve (AUC) ratios (7-ethyl-10-hydroxycamptothecin glucuronide [SN-38G]/7-ethyl-10-hydroxycamptothecin [SN-38]) and pretreatment levels of serum total bilirubin was investigated in 85 cancer patients who received irinotecan. RESULTS: Four haplotype groups (*1, *60, *28, and *6) were assigned in block 1, and 2 haplotype groups (*IA and *IB) were in block 2. The majority of the *IB haplotypes in block 2 were linked to either the *1 or the *60 haplotype but not to *28 in block 1. Highly significant associations were obtained between the *28 haplotypes and both a reduced AUC ratio (P =.0014, Jonckheere-Terpstra [JT] test) and an increased total bilirubin level (P =.0007, JT test). Increased total bilirubin levels in the *60 (P =.0048, JT test) and *IB groups (P =.0224, JT test) were also observed. The reduction in the AUC ratio by the *6 group was moderate (P =.0372, JT test) but was remarkable in combination with *60 (*6/*60) or *28 (*6/*28) as compared with the *1 group (*1/*1) (P =.049 and P =.0071, respectively; nonparametric Dunnett test). CONCLUSION: This study identified several UGT1A1 haplotypes significantly associated with the reduced AUC ratio (*28 and *6) and with the increased total bilirubin level (*28, *60, and *IB) and suggested that the novel haplotype *IB might be functionally important. These findings will be useful for further pharmacogenetic studies on adverse reactions to irinotecan.

Aged↗

PubMed2004

Involvement of human hepatic UGT1A1, UGT2B4, and UGT2B7 in the glucuronidation of carvedilol.

Carvedilol ((+/-)-1-carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol) is metabolized primarily into glucuronide conjugates. In the present study, we identified the human UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of carvedilol by thin-layer chromatography using microsomes from human liver or insect cells expressing recombinant UGT isoforms. We observed two forms of carvedilol glucuronides, namely G1 and G2, in hepatic microsomes. The glucuronidation of carvedilol was catalyzed by at least three recombinant UGT isoforms: UGT1A1, UGT2B4, and UGT2B7. UGT2B4 formed both G1 and G2, whereas UGT1A1 and UGT2B7 were responsible for the formation of glucuronide G2 and G1, respectively. The enzyme kinetics for carvedilol glucuronidation by UGT1A1, UGT2B4, and UGT2B7 in addition to human liver microsomes were examined by Lineweaver-Burk analysis. The values of Km and Vmax for human liver microsomes were 26.6 microM and 106 pmol/min/mg protein for G1, and 46.0 microM and 44.5 pmol/min/mg protein for G2, respectively. The Km values for UGT1A1, UGT2B4, and UGT2B7 for G1 and G2 (22.1-55.1 microM) were comparable to those of the liver microsomes, whereas the Vmax values were in the range of 3.33 to 7.88 pmol/min/mg protein. The Km and Vmax/Km values for UGT2B4 and UGT2B7 for G1 were similar, whereas UGT2B4 had lower Km and higher Vmax/Km values for G2 compared with those of UGT1A1. These results suggest that G1 formation is catalyzed by UGT2B4 and UGT2B7, whereas G2 is formed by UGT2B4 and UGT1A1. These three hepatic UGT isoforms may have important roles in carvedilol metabolism.

Adolescent↗

PubMed2004

Identification of novel alternative splice variants of human constitutive androstane receptor and characterization of their expression in the liver.

Human constitutive androstane (or active) receptor (hCAR), a member of the nuclear receptor superfamily NR1I3, regulates the expression of several genes that are mainly involved in the metabolism of endogenous and xenobiotic compounds (e.g., CYP2B6, CYP3A4, and UGT1A1). We found four novel splice variants in the ligand-binding domain (LBD) of hCAR (NCBI reference sequence, NM_005122; designated SV0 herein). The variants designated SV1 and SV2 contained in-frame 12- and 15-base pair (bp) insertions, respectively. SV3 carried both of the insertions, and SV4 contained an in-frame 117-bp deletion. The insertion site of SV1 is located in the alpha6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices alpha8 and alpha9. SYBR Green real-time reverse transcription-polymerase chain reaction analysis of each splice variant revealed that the hepatic expression of SV2 was almost comparable with that of SV0 (approximately 40%), whereas other variants accounted for 6 to 10% of the total hCAR transcripts. In the reporter gene assays employing the phenobarbital-responsible enhancer module (PBREM) from CYP2B6 and UGT1A1 genes, the splice variants, except for SV1, were inactive, whereas SV1 transactivated the CYP2B6 PBREM but not the UGT1A1 PBREM reporter. A nuclear translocation assay in rat hepatocytes revealed that all the splice variants lack the responsiveness toward phenobarbital and 6-(4-chloropheny-l)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in terms of the ligand-dependent nuclear translocation. Further characterization, such as the identification of specific ligands, will help elucidate physiological implication of these hCAR splice variants.

Alternative Splicing↗

PubMed2004

Single nucleotide polymorphisms and haplotype frequencies of UGT2B4 and UGT2B7 in a Japanese population.

Both UDP-glucuronosyltransferase 2B4 (UGT2B4) and UGT2B7 are expressed mainly in the human liver and have several overlapping substrates; e.g., catechol estrogens, bile acids, codeine, and carvedilol. To identify novel single nucleotide polymorphisms (SNPs) and haplotypes in a Japanese population, the enhancer/promoter regions, all the exons, and the surrounding intronic regions of UGT2B4 and UGT2B7 were sequenced from 136 Japanese individuals. We found 16 and 21 polymorphisms, including 10 and 4 novel ones in UGT2B4 and UGT2B7, respectively. The novel nonsynonymous SNPs were 1364A>G (K455R) and 1531T>C (C511R) in UGT2B4 and 1192G> A (D398N) in UGT2B7. From linkage disequilibrium analysis, several SNPs in UGT2B7 were found to be highly linked with each other. No close linkage between the SNPs in UGT2B4 and UGT2B7 was observed, indicating that each gene is located within an independent haplotype block. Thus, haplotype analysis was separately performed for the two genes. In UGT2B4, we unambiguously determined 8 haplotypes and inferred an additional 12 haplotypes using an expectation-maximization-based program. In UGT2B7, five haplotypes were unambiguously assigned and an additional eight haplotypes were inferred. The haplotype structure of UGT2B7 was more diverse than that of UGT2B4 in terms of the number of frequent SNPs. In addition, ethnic differences in the UGT2B4(*)2 and UGT2B7(*)2 haplotypes between the Japanese and the Caucasian and/or African populations were found. Our findings provide fundamental and useful information for genotyping UGT2B4 and UGT2B7 in the Japanese, and probably other populations.

Asian People↗

PubMed2003

Functional characterization of human UDP-glucuronosyltransferase 1A9 variant, D256N, found in Japanese cancer patients.

SN-38 (7-ethyl-10-hydroxycamptothecin), an active metabolite of the antitumor prodrug irinotecan, is conjugated and detoxified to SN-38 10-O-beta-d-glucuronide by hepatic UDP-glucuronosyltransferase (UGT) 1A1. Recent studies have revealed that other UGT1A isoforms, UGT1A7 and UGT1A9, also participate in SN-38 glucuronidation. Although several genetic polymorphisms are reported for UGT1A1 and UGT1A7 that affect the SN-38 glucuronidation activities, no such polymorphisms have been identified for UGT1A9. In the present study, UGT1A9 exon 1 and its flanking regions were sequenced from 61 Japanese cancer patients who were all treated with irinotecan. A novel nonsynonymous single nucleotide polymorphism was identified in UGT1A9 exon 1, heterozygous 766G>A resulting in the amino acid substitution of D256N. The wild-type and D256N UGT1A9s were transiently expressed at similar protein levels in COS-1 cells, and their membrane fractions were characterized in vitro for the glucuronidation activities toward SN-38. The apparent Km values were 19.3 and 44.4 microM, and the Vmax values were 2.94 and 0.24 pmol/min/mg of membrane protein for the wild-type and D256N variant, respectively. The SN-38 glucuronidation efficiency (normalized Vmax/Km) of D256N was less than 5% that of wild-type UGT1A9. These results clearly indicate that the D256N variant is essentially nonfunctional with regard to SN-38 glucuronidation. These findings highlight the importance of further studies into the potential influence of UGT1A9 D256N variant to irinotecan metabolism in vivo.

Amino Acid Substitution↗

PubMed2003

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.

Arsenicals↗