摘要:

7-Ethoxy-4-trifluoromethyIcoumarin (7-EFC) was examined as a substrate for cytochrome P450 (P450) in microsomes from human livers and expressed in B-lymphoblastoid cells. The Odeethylation of 7-EFC to 7-hydroxy-4-trifluoromethyIcoumarin (7-HFC) varied over a liver bank (n=19) by a factor of 13 (40-507 pmol min-'mg-' protein). When compared with the ability of the bank of human liver samples to metabolize form-selective substrates of the P450, 7-HFC formation correlated strongly with the formation of the S-mephenytoin metabolite, nirvanol (r2=0.86, p<0.0001). a-Napthoflavone (ANF), diethyldithiocarbamate (DDC) and chloramphenicol (CAP) inhibited the O-deethylation of 7-EFC by microsomes from human livers by greater than 60%. Orphenadrine (ORP), a reported specific CYP2B6 inhibitor, was a less potent inhibitor of 7-HFC formation by microsomes from human liver than DDC or ANF. Using microsomes from B-lymphoblastoid cells expressing specific P450s, CYP2B6 and CYP1A2 were found to produce substantial levels of 7-HFC whereas CYP2E1 and CYP2C19 produced detectable amounts of this metabolite. ORP inhibited expressed CYP2E1 and CYP2B6 mediated 7-HFC formation to a greater extent than the inhibition observed for CYP1A2. Methoxychlor and S-mephenytoin inhibited expressed CYP2B6 but not CYP1A2 mediated 7-EFC O-deethylation. Livers (n=5) with high relative rates of 7-HFC formation displayed biphasic enzyme kinetics with the lowmsite (averagem=3.3 HM) demonstrating allosteric activation. Five livers with low relative rates of 7-HFC formation also exhibited biphasic kinetics but lacked evidence of an allosteric mechanism being involved in the lowmcomponent (averagem=2.4 JUM). Furthermore, expressed CYP2B6 and CYP2E1 converted 7-EFC to 7-HFC with allosteric activation indicated, while CYP1A2 mediated metabolism of 7-EFC to 7-HFC best fit the classic Michaelis-Menten model. A commercially available antibody to rat CYP2B, suggested to be specific for CYP2B6, was found to cross react with all members of the CYP2 family examined including CYP2C19, which possessed a nearly identical electrophoretic mobility to that of CYP2B6 in the system examined. In total, the evidence presented indicates that multiple P450s are involved in the formation of 7-HFC from 7-EFC, therefore this does not appear to be a useful or a selective probe of CYP2B6 catalytic activity. Furthermore, the specificity of both antibody and chemical inhibitor (ORP) probes previously suggested to be specific for CYP2B6 is also questioned.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Dihydralazine-induced hepatitis is characterized by the presence of anti-liver microsomal (anti- LM) autoantibodies in the sera of patients. Cytochrome P450 1A2 (CYP1A2), involved in the metabolism of dihydralazine, was shown to be a target for autoantibodies. In order to investigate further the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, and since the specificity of anti-LM autoantibodies towards CYP1A2 has been determined, the antigenic site was further localized. By constructing fragments derived from CYP1A2 cDNA and probing the corresponding proteins with several anti-LM sera, we were able to define a region (amino acid 335-471) which was immunoreactive with 100% of sera. An internal deletion in this region led to the loss of recognition by anti-LM autoantibodies, confirming that the epitope was conformational. Epitope mapping studies had previously been performed for CYP2D6, CYP17, CYP21A2, and recently for CYP3A1 and CYP2C9. Those data were compared with results obtained in the present study for CYP1A2.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The polymorphic cytochrome P450 2D6 (CYP2D6)‡ causing poor, extensive or ultrarapid metabolism of several clinically important drugs exhibits pronounced interethnic variation. Ultrarapid metabolism is caused by multiple copies of active CYP2D6 genes and recently 29% of an Ethiopian population has been shown to carry duplicated or multiduplicated CYP2D6 genes, whereas the corresponding frequency in other black, Oriental and European populations investigated is 1—2%. In order to characterize the distribution of alleles with multiple CYP2D6 copies in a neighbouring population and to characterize the CYP2D locus in general among Saudi Arabians, the CYP2D6 genotype of a Saudi Arabian population was examined using restriction fragment length polymorphism (RFLP) analysis and allele-specific polymerase chain reaction (PCR) amplification. Of 101 Saudi Arabians studied, 21 subjects had an EcoRI fragment indicative of CYP2D6 gene duplication. In contrast, only two individuals were heterozygous for a deletion of the whole gene (CFP2D6*5). The allele frequency of CYP2D6*4, the most common defective allele among Caucasians, was only 3.5% in the Saudi population. Two other alleles, CYP2D6*10 and *17', common in certain populations and which cause diminished enzyme activity, were found only at low allele frequencies of 3.0% each. These findings are in agreement with earlier Saudi Arabian phenotyping studies which reported a low frequency (1-2%) of poor metabolizers for CYP2D6 probe drugs. In conclusion, the Saudi Arabian population studied exhibited very few defective alleles and a large number of subjects carried duplicated CYP2D6 genes, implying a high conservation on functional CYP2D6 genes possibly due to dietary reasons and reveal the Saudi Arabians as an unique population in comparison with others examined.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

A, the 1795 Tdel and the 2637 Adel accounted for 65.8%, 6.2% and 4.8% respectively, within the poor metabolizer subgroup. Fifty-three different alleles were characterized from the mutation pattern and by allele-specific sequencing. They are derived from three major alleles, namely the wild-type CYP2D6*1A, the functional CYP2D6*2 and the null CYP2D6*4A. Five allelic variants (CYP2D6*1A, *2, *2B, *4A and *5) account for about 87% of all alleles, while the remaining alleles occur with a frequency of 0.1%-2.7%. These data provide a solid basis for future epidemiological, clinical as well as interethnic studies of the CYP2D6 polymorphism and highlight that the described single strand conformation polymorphism method can be successfully used in designing such studies.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

We have examined the kinetics of substrate metabolism by cDNA-expressed human CYP2C9 and the R144C variant. Both enzymes exhibited similar apparentmvalues for (S)-warfarin 7- hydroxylation, diclofenac 4'-hydroxylation and lauric acid 11 -hydroxylation. In contrast, the R144C variant (relative to CYP2C9) had slower rates of metabolism for all three substrates. The difference was most pronounced for (S)-warfarin. Surprisingly, the magnitude of the difference was found to be dependent on the cytochrome P450 to NADPH-cytochrome P450 reductase (OR) ratio in the system (the difference being more pronounced at higher OR to P450 ratios) implying that the R144C change affects interaction of the P450 with OR. The rates of (S)-warfarin 7- hydroxylation by CYP2C9 and the R144C variant also exhibited differential dependence on salt concentration which further supported a difference in interaction with OR. When OR was bypassed and the hydroxylation was supported by cumene hydroperoxide, no difference in the rates of diclofenac 4'-hydroxylation was observed for CYP2C9 and the R144C variant regardless of OR to P450 ratio. However, for (S)-warfarin 7-hydroxyIation, some OR-dependence was maintained even when the reaction was supported by cumene hydroperoxide. Finally, we compared CYP2C9 activity and CYP2C9 protein levels for human Iymphoblast expressed (high OR to P450 ratio) to human liver microsomes using immunoblotting and enzyme selective substrates. Human liver microsomal CYP2C9 and human lymphoblast-expressed CYP2C9 showed comparable amounts of activity per unit enzyme. This final observation indicates that the high OR to P450 ratio is the preferred model and predicts that the R144C change in human liver microsomal CYP2C9 should markedly reduce the rates of substrate metabolism. The implications of these observations for the interpretation of results with cDNA-expressed enzymes is discussed.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Cyclophosphamide and ifosfamide are alkylating agent prodrugs that require activation by cytochrome P450 (CYP) to manifest their cancer chemotherapeutic activity. The present study investigates the activity of four individual human CYP2C enzymes and their allelic variants in cyclophosphamide and ifosfamide activation as an initial attempt to gain insight into the underlying basis for the large interpatient differences in the clinical pharmacokinetics and metabolism of these anticancer drugs. Recombinant CYP2C8, CYP2C19, two allelic variants of CYP2C18, and six variants of CYP2C9 expressed in a yeast cDNA expression system were each enzymatically active, as judged by the ability of the isolated microsomes to catalyse 7-ethoxycoumarin Odeethylation after reconstitution with purified NADPH-cytochrome P450 reductase and cytochrome b5. With cyclophosphamide as substrate, CYP2C19 had the lowest apparent Km, followed by CYP2C9, CYP2C18 and CYP2C8, whereas in the case of ifosfamide, the rank order was:KmCYP2C19<CYP2C18<CYP2C9<CYP2C8. CYP2C18 had the highest in vitro intrinsic clearance/ catalytic efficiency (apparent Vmax/Km) in cyclophosphamide and ifosfamide activation, followed by 2C19>2C9 ~2C8. Examination of a panel of CYP2C allelic variants revealed that CYP2C18-Thr3S5 had both a higher Vmaxand a higher apparent Kmtoward cyclophosphamide than CYP2C18-Met385with no difference in catalytic efficiency, whereas with ifosfamide the Thr385 allele exhibited a strikingly lower apparent Kmresulting in a six-fold higher catalytic efficiency. In the case of CYP2C9, a Ile35- J to Leu mutation associated with poor metabolism of the hypoglycemic drug tolbutamide decreased catalytic efficiency toward cyclophosphamide by increasing the apparent Kin, whereas the same mutation reduced the efficiency of this P450 toward ifosfamide by decreasing the Vmax. Substitution of CYP2C9-Gly417by Asp resulted in a two-fold lower catalytic efficiency for cyclophosphamide metabolism but a three-fold higher efficiency for ifosfamide metabolism. A His276to Gly substitution resulted in an increase in both Vmax and apparent Kin with no net change in catalytic efficiency for either oxazaphosphorine. Mutations at CYP2C9 residues 144 and 358 had little or no effect. Thus (a) wild type CYP2C19 and CYP2C9 are relatively low Kmcatalysts of cyclophosphamide and ifosfamide activation, and (b) all four human CYP2C enzymes activate these two anticancer prodrugs with varying efficiencies and with striking differences among naturally occuring allelic variants in the case of CYP2C9 and CYP2C18.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Genetic factors are known to influence the preference for drinking alcohol - in humans as well as certain inbred strains of laboratory animals. Here we examined the possible role of the aromatic hydrocarbon receptor (AHR) in alcohol-preferring C57BL/6I (B6, high-affinity AHR) and alcoholavoiding DBA/2J (D2, low-affinity AHR) inbred mouse strains, and in the two congenic lines B6.D2-Ahrd(>99% B6 genome with the D2 low-affinity AHR) and D2.B6-Ahrb-1(> 99% D2 genome with the B6 high-affinity AHR). This laboratory had previously shown an association between resistance to intraperitoneal ethanol-induced toxicity and the high-affinity AHR. Offering the choice between drinking water and 10% ethanol, we found that alcohol preference is three- to four-fold greater in B6 than D2 mice, as well as three- to four-fold greater in B6.D2- Ahrdthan D2.B6-Ahrb-1mice - indicating that alcohol preference is AHR-independent. The prototype AHR agonist 2,3,7,8-tetrachIorodibenzo-p-dioxin (TCDD; dioxin) did not affect the rates of chronic alcohol consumption in B6 or D2 mice, suggesting that dioxin-inducible metabolism does not play a major role in alcohol drinking preference. In B6 mice, we found that oral treatment with the aldehyde dehydrogenase (ALDH) inhibitor disulfiram decreased alcohol preference by 50%, whereas oral treatment of the catalase inhibitor 3-amino-l,2,4-triazole increased alcohol drinking preference by 15-20%. Although liver and brain ALDH activities were both significantly higher in D2 than B6, these activities were not related to alcohol consumption. Hepatic and brain catalase activities, on the other hand, were two- to three-fold higher in D2 and D2.B6-Ahrb-1mice, compared with that in B6 and B6.D2-Ahrd. Furthermore, brain acetaldehyde levels were inversely related to the quantity of alcohol voluntarily consumed. We conclude that the alcohol drinking preference between the B6 and D2 inbred mouse strains is independent of the Ah receptor - but is genetically determined, in part, by the level of brain catalase activity which, in turn, regulates brain acetaldehyde concentrations.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The NQO1 locus on chromosome 16q2.2 encodes NAD(P)H:quinone oxidoreductase, an enzyme implicated in detoxication and protection against redox cycling. Two alleles have been identified in the human population, the rarer one, termed the null-allele, coding for a nonfunctional enzyme. Since lack of NQOR activity has been suggested to increase susceptibility to certain cancers, the distribution of the two alleles was determined by polymerase chain reaction-restriction fragment length polymorphism analysis in patients with renal cell carcinoma (n=131) and urothelial carcinoma (n=99) compared with a normal population (n=260). Allele distribution in the normal population followed a Hardy-Weinberg distribution with frequencies of 0.867 for the major allele and 0.133 for the null-allele. Increased frequencies of the null-allele were found in the tumour patient groups (0.191 and 0.182, respectively) due to an increased number of both homo- and heterozygotes. The odds ratios for homozygous null-allele vs. wild-type genotypes were 1.7 and 3.6 for renal cell carcinoma and urothelial carcinoma, respectively. These data are compatible with the assumption that diminished activity of NQOR in some individuals increases susceptibility to certain cancers.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Twenty-six healthy Caucasian subjects were evaluated for polymorphic N-acetyltransferase (NAT2) metabolic activity in vivo by sulfamethazine phenotyping and for their respective NAT2 genotype. Application of discriminant analysis allowed the separation of the rapid and slow acetylators solely on the base of their respective mutation pattern with identical results as achieved by the classical method of discrimination according to the phenotyping results. Multiple linear regression analysis was used to obtain a quantitative relationship between allelic pattern and the phenotypic outcome. It is shown that the computation methods produce relationships enabling the influence of particular mutations and/or allelic configurations on the metabolic activity in vivo to be estimated. This may be important in cases of discordant or overlapping phenotype and genotype results as well as in investigating the NAT2 polymorphism as a risk factor for cancer and other disease in epidemiological studies.

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID

ISSN:0960-314X    

出版商:OVID    

年代:1997

数据来源: OVID