ABSTRACT:Cytochrome P450 (P450) 2B6 is a hepatic enzyme of potential importance for the metabolism of clinically used drugs and environmental or abused toxicants. Genetic polymorphisms of CYP2B6 (CYP2B6*2, CYP2B6*3, CYP2B6*4, CYP2B6*5, CYP2B6*6 and CYP2B6*7; wild-type, CYP2B6*1) were found previously in white and Japanese populations. In the present study, the goal was to investigate the effects of amino acid substitutions on CYP2B6 function. Wild-type (CYP2B6.1) and all of the known variants of CYP2B6 (CYP2B6.2, CYP2B6.3, CYP2B6.4, CYP2B6.5, CYP2B6.6, and CYP2B6.7) were transiently expressed in COS-1 cells, and their 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activities were determined. The levels of the variant CYP2B6 proteins were relatively low compared with that of CYP2B6.1, although the differences were not significant. The activities of 7-ethoxy-4-trifluoromethylcoumarin O-deethylation on the basis of the CYP2B6 protein level at low (0.5 M) and high (50 M) substrate concentrations varied among wild-type and variant CYP2B6 proteins. All CYP2B6 enzymes showed typical Michaelis-Menten kinetics. The K m value of CYP2B6.6 was significantly higher than that of CYP2B6.1. Those CYP2B6 variants having a Lys262Arg substitution (CYP2B6.4, CYP2B6.6, and CYP2B6.7) showed increased values for V max and V max /K m , whereas the kinetic parameters of CYP2B6.2 and CYP2B6.3 were not affected by the corresponding amino acid substitution. These results may mean that Lys262 in combination with other amino acid residues such as Gln172 and Arg487 is associated with the CYP2B6 function and that the genetic polymorphism of CYP2B6 leads to interindividual differences in xenobiotic metabolism.

ABSTRACT:Ethnic differences in genetic polymorphisms in UDP-glucuronosyltransferase 1A1 (UGT1A1) were investigated among AfricanAmericans, 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.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:7-Ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of antitumor agent irinotecan (CPT-11), is conjugated and detoxified to SN-38-glucuronide by UDP-glucuronosyltransferase (UGT) 1A1. Genetic polymorphisms in UGT1A1 are thought to contribute to severe diarrhea and/or leukopenia caused by CPT-11. In this regard, it has been reported that polymorphisms in the promoter region could affect the CPT-11 pharmacokinetics and interindividual variation of toxicity. However, little information is available on the influence of UGT1A1 polymorphisms in the coding region on the SN-38 glucuronidation activity. In the present study, wild-type (WT) and three variant (G71R, P229Q, and Y486D) cDNAs of human UGT1A1s were transiently expressed in COS-1 cells, and the kinetic parameters of these UGT1A1s were determined for SN-38 glucuronidation. A partially reduced UGT1A1 protein expression was observed in COS-1 cells for G71R and Y486D. WT UGT1A1 catalyzed SN-38 glucuronidation with an apparent K m value of 11.5 M, whereas those of G71R, P229Q, and Y486D were 14.0, 18.0, and 63.5 M, respectively. The SN-38 glucuronidation efficiency ratio (V max /K m ) normalized for the level of expression was 1.4, 0.66 (47% of WT), 0.73 (52%), and 0.07 (5%) l/min/mg of protein for WT, G71R, P229Q, and Y486D, respectively. Thus, the SN-38 glucuronidation activity of Y486D was drastically reduced, whereas the reduction in the G71R and P229Q activities was fractional. The decreased SN-38 glucuronidation efficiency ratio of G71R and P229Q could be critical in combination with other polymorphisms in the UGT1A1 gene.

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 ␣6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices ␣8 and ␣9. 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-chlorophenyl)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.

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 Nrf2's 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.

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, an active metabolite of the antitumor prodrug irinotecan, is conjugated and detoxified to SN-38 10-O-␤-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 766GϾA 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 K m values were 19.3 and 44.4 M, and the V max 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 V max /K m ) 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.Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; Fig. 1) is an antitumor prodrug of a potent topoisomerase I inhibitor, SN-38 (7-ethyl-10-hydroxycamptothecin), which is now applied clinically with or without other chemotherapeutic agents such as 5-fluorouracil and cisplatin to a variety of malignant tumors including gastrointestinal and lung cancers (Rothenberg, 2001). Irinotecan is hydrolyzed by carboxylesterases in the human liver, intestinal mucosa, and plasma to liberate SN-38 (Rivory et al., 1996;Ahmed et al., 1999;Kehrer et al., 2000), which undergoes subsequent glucuronidation by UDP-glucuronosyltransferases (UGTs) into an inactive 10-O-␤-D-glucuronide (SN-38G; Fig. 1) and biliary excretion (Gupta et al., 1994;Iyer et al., 1998). Although the precise mechanism remains to be elucidated, the hepatic SN-38 glucuronidation capacity is thought to be an important determinant factor of severe diarrhea, the major dose-limiting side effect of irinotecan (Ratain, 2002). Therefore, genetic polymorphisms in UGT1A could influence the incidence of the irinotecan-induced diarrhea.The UGT1A family is known to include 9 functional isoforms (UGT1A1, UGT1A3 to UGT1A10). UGT1A2P, UGT1A11P, UGT1A12P, and UGT1A13P are thought to be pseudogenes based on their frame-shift mutations. The UGT1A family members have common carboxyl terminal sequences (245 amino acids in length) derived from four This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences...

ABSTRACT: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 6␣-hydroxylase activity, and intracellular localization. The prematurely terminated R186X variant was undetectable by Western blotting and inactive toward paclitaxel 6␣-hydroxylation. The G171S, K247R, and K383N variants exhibited properties similar to those of the wild-type CYP2C8. Paclitaxel 6␣-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.