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We reported previously that subjects homozygous for the cytochrome P450 2A6 (CYP2A6) (*)4 have a lower risk of lung cancer. The purpose of this study was to clarify whether or not the alterations of smoking behavior and risk for lung cancer could be found in subjects possessing novel CYP2A6 variants discovered recently. An epidemiological study was performed with 1094 cases and 611 controls in male Japanese smokers. It was found that the amounts of daily cigarette consumption in subjects who harbored CYP2A6(*)4/(*)7, (*)4/(*)10, (*)7/(*)7, (*)7/(*)9 and (*)4/(*)4 genotypes were significantly less than those in subjects carrying the (*)1/(*)1 genotype (P < 0.01). Even after adjustment with cigarette consumption, the adjusted odds ratios (ORs) for lung cancer were significantly lower in subjects who harbored CYP2A6(*)1/(*)4, (*)1/(*)7, (*)1/(*)9, (*)1/(*)10, (*)4/(*)4, (*)4/(*)7, (*)4/(*)9, (*)7/(*)7 and (*)7/(*)9 genotypes than those who possessed the (*)1/(*)1 genotype (P < 0.05). When participants were classified into four groups according to the CYP2A6 genotypes, group 1 ((*)1/(*)1), group 2 (heterozygotes for the (*)1 and a variant allele), group 3 (heterozygotes and homozygotes for variant alleles except for (*)4/(*)4) and group 4 ((*)4/(*)4), lung cancer risk was found to be less in subjects with the variant of CYP2A6 alleles [group 2, OR of 0.59 [95% confidence interval (CI), 0.44-0.79]; group 3, OR of 0.52 (95% CI, 0.37-0.72); group 4, OR of 0.30 (95% CI, 0.16-0.57)]. The reduced risk for lung cancer was seen more clearly in heavy smokers than in light smokers. Additional stratification analysis showed that the ORs for squamous cell carcinoma (OR of 0.07) and small cell carcinoma (OR of 0.10) were lower than that of adenocarcinoma (OR of 0.39) in group 4. These results suggest that the CYP2A6 is one of the principal determinants affecting not only smoking behavior but also susceptibility to tobacco-related lung cancer.

A genetic polymorphism was identified in the 5'-flanking region of human CYP1A2 gene, and its effect on the transcriptional activation of the CYP1A2 gene was investigated. Nucleotide sequence analysis revealed the existence of a point mutation from guanine (wild type) to adenine (mutated type) at position -2964 in the gene. This point mutation was detected by a polymerase chain reaction-restriction fragment length polymorphism method using DdeI or BslI restriction enzyme, and was proven to be genetically inherited. Allele frequency in 116 Japanese subjects showed 0.77 and 0.23 for the wild and mutated types of allele, respectively. The point mutation caused a significant decrease of CYP1A2 activity measured by the rate of caffeine 3-demethylation in Japanese smokers (p<0.05). Gel retardation analysis showed the existence of protein bound to the polymorphic locus. These results suggest that this polymorphism is a causal factor of decreased CYP1A2 inducibility.

Monkeys are widely used as a primate model to study drug metabolism because they generally show a metabolic pattern similar to humans. However, the paucity of information on cytochrome P450 (P450) genes has hampered a deep understanding of drug metabolism in the monkey. In this study, we report identification of the CYP2C76 cDNA newly identified in cynomolgus monkey and characterization of this CYP2C along with cynomolgus CYP2C20, CYP2C43, and CYP2C75. The CYP2C76 cDNA contains the open reading frame encoding a protein of 489 amino acids that are only approximately 80% identical to any human or monkey P450 cDNAs. Gene and protein expression of CYP2C76 was confirmed in the liver of cynomolgus and rhesus monkeys but not in humans or the great apes. Moreover, CYP2C76 is located at the end of the CYP2C gene cluster in the monkey genome, the region of which corresponds to the intergenic region adjacent to the CYP2C cluster in the human genome, strongly indicating that this gene does not have the ortholog in humans. Among the four CYP2C genes expressing predominantly in the liver, the expression level of CYP2C76 was the greatest, suggesting that CYP2C76 is a major CYP2C in the monkey liver. Assays for the capacity of CYP2C76 to metabolize drugs using several substrates typical for human CYP2Cs revealed that CYP2C76 showed unique metabolic activity. These results suggest that CYP2C76 contributes to overall drug-metabolizing activity in the monkey liver and might account for species difference occasionally seen in drug metabolism between monkeys and humans.Cytochrome P450s (P450s) are one of the most important drug-metabolizing enzymes and form a superfamily consisting of a large number of subfamilies (Nelson et al., 1996(Nelson et al., , 2004. The cDNA sequences encoding P450s have been reported for many species of not only mammals but also birds, insects, plants, bacteria, and others (see http://drnelson. utmem.edu/CytochromeP450.html). In humans, 57 functional genes have been identified to date (Nelson et al., 2004). The human CYP2C subfamily, comprising CYP2C8, CYP2C9, CYP2C18, and CYP2C19, is essential in metabolizing approximately 20% of all prescribed drugs, including tolbutamide, phenytoin, warfarin, and ibuprofen (Goldstein, 2001). The CYP2C subfamily consists of multiple members in each mammalian species, including 15 in mice, 12 in rats, and 9 in rabbits (for the latest information, see http://drnelson.utmem.edu/CytochromeP450.html). Between humans and rodents, the number of the subfamily members is different, and none of the CYP2Cs seems to show a clear orthologous relationship between the two species, suggesting that the data from rodents must be cautiously interpreted and extrapolated to humans (Nelson et al., 2004).For monkeys, which generally mean Old or New World monkeys, three CYP2C cDNAs have been identified in the macaque and cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) monkeys. Two sequences have been published including cynomolgus CYP2C20 (Komori et al., 1992) and rhesus CYP...

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.

Biochemistry 29, . The sequences of three genomic clones for CYP3A4 were analyzed for all exons, exon-intron junctions and the 5'-flanking region from the major transcription site to nucleotide position -1105, and compared with those of the CYP3A7 gene, a fetal-specific form of cytochrome P450 in humans. The results showed that the identity of 5'-flanking sequences between CYP3A4 and CYP3A7 genes was 91 %, and that each 5'-flanking region had characteristic sequences termed as NFSE (P450N,-specific element) and HFLaSE (P450HFLa specific element), respectively. A basic transcription element (BTE) also lay in the 5'-flanking region of the CYP3A4 gene as seen in many CYP genes [Yanagida, A., Sogawa, K., Yasumoto, K. & Fujii-Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470-14751. The BTE binding factor (BTEB) was present in both adult and fetal human livers.To examine the transcriptional activity of the CYP3A4 gene, DNA fragments in the 5'-flanking region of the gene were inserted in front of the simian virus 40 promoter and the chloramphenicol acetyltransferase structural gene, and the constructs were transfected in HepG2 cells. The analysis of the chloramphenicol acetyltransferase activity indicated that (a) suecific element(s) which could bind with a fa&or(s) in livers was present in the the transcriptional activity.Cytochrome P450 is a heme-containing enzyme which plays central roles in the oxidative and reductive metabolism of a variety of endogenous as well as exogenous compounds. It is generally known that there are numbers of forms of cytochrome P450, which are classified into families according to the homology of their nucleotide sequences 111.Among families of cytochrome P450, the CYP2C and CYP3A families are unique in that there are many forms in each family, and that these enzyme proteins are present in larger amounts in human liver microsomes, compared with other forms of cytochrome P450 121. The CYP3A, CYP3A4 (P450N,.) and CYP3 A7 (P450HFLa) proteins have been purified from human adult and fetal livers, respectively [3-51.Correspondence to T. Kamataki, Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, N12W6, Kitaku, Sapporo, Hokkaido, 060 JapanAhhreviations. BTE, basic transcription element ; BTEB, BTE binding factor; CAT, chloramphenicol acetyltransferase; CUP, cytochrome P450; DMEM, Dulbecco's modified Eagles medium; ER, estrogen receptor; ERE, estrogen response element; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HFLaSE, P450HFLa-specific element; HNF-4, hepatic nuclear factor-4 ; HNF-5, hepatic nuclear factor-5 ; NFSE, P450,,-specific element; PR, progesterone receptor; PRE, progesterone response element; SV40, simian virus 40.Enzymes. Cytochrome P450 (EC 1.14.14.1 .); chloramphenicol acetyltransferase (EC 2.3.1.28.).Note. The 5'-flanking sequence data of CYP3A4 gene published here have been deposited with the DDBJ/EMBL/GenBank and are available under the accession number D11131. ~, I5'-flanking region of the CYP3A4 gene to show In addition, f...

The purpose of the study was to elucidate human intestinal cytochrome P450 isoform(s) involved in the metabolism of an antihistamine, ebastine, having two major pathways of hydroxylation and N-dealkylation. The ebastine dealkylase in human intestinal microsomes was CYP3A4, based on the inhibition studies with antibodies against CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A isoforms and their selective inhibitors. However, ebastine hydroxylase could not be identified. We then examined the inhibitory effects of anti-CYP4F antibody and 17-octadecynoic acid, an inhibitor of the CYP4 family, on ebastine hydroxylation in intestinal microsomes, since CYP4F was recently found to be the predominant ebastine hydroxylase in monkey intestine; and a novel CYP4F isoform (CYP4F12), also capable of hydroxylating ebastine, was found to exist in human intestine. However, the inhibitory effects were only partial (about 20%) and thus it was thought that, although human CYP4F was involved in ebastine hydroxylation, another predominant enzyme exists. Further screening showed that the hydroxylation was inhibited by arachidonic acid. CYP2J2 was selected as a candidate expressed in the intestine and closely related to arachidonic acid metabolism. The catalytic activity of recombinant CYP2J2 was much higher than that of CYP4F12. Anti-CYP2J antibody inhibited the hydroxylation to about 70% in human intestinal microsomes. These results demonstrate that CYP2J2 is the predominant ebastine hydroxylase in human intestinal microsomes. Thus, the present paper for the first time indicates that, in human intestinal microsomes, both CYP2J and CYP4F subfamilies not only metabolize endogenous substrates but also are involved in the drug metabolism. Ebastine is a potent nonsedative H 1 -receptor antagonist (Fig. 1), and after oral administration to experimental animals and humans, the agent is almost completely metabolized to the pharmacologically active principle, the carboxylated metabolite (carebastine), and other inactive metabolites Matsuda et al., 1994;Yamaguchi et al., 1994). Carebastine alone was the major metabolite detectable in the blood. Our previous in situ studies using rats indicated that the small intestine extensively converted the orally given ebastine to carebastine via hydroxylated ebastine and the dealkylated metabolite (Fujii et al., 1997). Therefore, it seemed that small intestine plays an important role in the first-pass metabolism of this drug, and the enzymes responsible for its metabolism exist there.We reported that ebastine was primarily metabolized by human liver microsomes to two metabolites, hydroxy-and desalkyl-ebastine (Hashizume et al., 1998). N-Dealkylation to desalkyl-ebastine was mediated by CYP3A4, whereas hydroxylation to hydroxy-ebastine, the most important intermediate metabolite yielding carebastine, was mediated by unidentified P450(s) other than CYP3A4. Our recent studies revealed that two novel CYP4F isoforms (P450 MI-2 and CYP4F12) obtained from monkey and human small intestine, respectively, were ...

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