Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes

Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the K _m values by these three P450 enzymes were determined to be 11.0, 105, and 132 μM, respectively, and the V _max values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 μM) substrate concentration. CYP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 μM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 μM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 μM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti-CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 μM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 μM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes. Received: 27 October 1998 / Accepted: 11 January 1999     

A novel CYP2A6 allele, CYP2A6*23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent

OBJECTIVES: CYP2A6 is the main enzyme involved in nicotine metabolism in humans. We have identified a novel allele, CYP2A6*23 (2161C>T, R203C), in individuals of Black-African descent and investigated its impact on enzyme activity and association with smoking status. METHODS: Wild-type and variant enzymes containing amino acid changes R203C (CYP2A6*23), R203S (CYP2A6*16) and V365M (CYP2A6*17) were expressed in Escherichia coli. The effect of CYP2A6*23 in vivo was examined in individuals of Black-African descent given 4 mg oral nicotine. RESULTS: CYP2A6*23 occurred at an allele frequency of 2.0% in individuals of Black-African descent (N=560 alleles, 95% confidence interval, 0.8-3.1%) and was not detected in Caucasians (N=334 alleles), Chinese (N=288 alleles) or Japanese (N=104 alleles). In vitro, CYP2A6.23 had greatly reduced activity toward nicotine C-oxidation similar to CYP2A6.17, as well as reduced coumarin 7-hydroxylation. Conversely, CYP2A6.16 did not differ in activity compared with the wild-type enzyme. The trans-3'-hydroxycotinine to cotinine ratio, a phenotypic measure of CYP2A6 activity in vivo, was lower in CYP2A6*1/*23 and CYP2A6*23/*23 individuals (mean adjusted ratio of 0.60, n=5) compared with CYP2A6*1/*1 individuals (mean adjusted ratio of 1.21, n=150) (P<0.04). CYP2A6*23 trended toward a higher allele frequency in nonsmokers (3.1%, N=9/286 alleles) compared with smokers (0.7%, N=2/274 alleles) (P=0.06). CONCLUSION: These results suggest the novel CYP2A6*23 allele impairs enzyme function in vitro and in vivo and trends toward an association with lower risk of smoking.     

Ethnic variation in CYP2A6*7, CYP2A6*8 and CYP2A6*10 as assessed with a novel haplotyping method

Cytochrome P450 2A6 is the main human nicotine metabolizing enzyme coded for by a highly polymorphic gene, CYP2A6. CYP2A6*7, CYP2A6*8 and CYP2A6*10 are variant alleles common to Asian ethnicities. The CYP2A6*7 and CYP2A6*8 alleles each contain a non-synonymous single nucleotide polymorphism (SNP) 6558T>C and 6600G>T, respectively, whereas the CYP2A6*10 haplotype allele contains both. We have developed the first haplotyping assay; it can unambiguously distinguish the CYP2A6*7, CYP2A6*8 and CYP2A6*10 alleles. The allele frequencies of these three variants were assessed using the novel haplotyping assay in Chinese-Canadian (n=112), Chinese-American (n=221), Taiwanese (n=319), Korean-American (n=207) and Japanese-Canadian (n=64) populations, as well as in Caucasian (n=110) and African-Canadian (n=113) populations. Our new method demonstrated higher frequencies of CYP2A6*7 and CYP2A6*10, and a lower frequency of CYP2A6*8 in Asian populations, but no significant change of allele frequencies in Caucasian or African-Canadian populations.     

CYP2A6 genetic polymorphisms and liver microsomal coumarin and nicotine oxidation activities in Japanese and Caucasians

Genotypes of CYP2A6, namely CYP2A6(*)1 (wild-type), CYP2A6(*)2, and CYP2A6(*)3, were examined in liver DNA of 39 Japanese and 43 Caucasians using two-step polymerase chain reaction (PCR) methods. We first amplified a DNA fragment (1725 bp) located between near middle of exon 1 and end of exon 4 of the CYP2A6 gene and further amplified using a forward primer 't' or 'mut' (middle of exon 3) and a reverse primer 'E3R' (middle of intron 3) for the detection of CYP2A6(*)2-genetic polymorphism. The 1725 bp fragment was also used for the amplification between exon 3 and near middle of intron 3 of the CYP2A6 gene and the fragment thus obtained digested with XcmI or DdeI to detect and confirm the CYP2A6(*)2- and CYP2A6(*)3-types, respectively. Only one DNA sample from a Japanese origin (J18) was not amplified by CYP2A6-specific primers; liver microsomes from this individual had very low activity of coumarin 7-hydroxylation and were devoid of protein(s) immunoreactive to anti-CYP2A6 antibody. Thus, this individual was suggested to be due to the gene deletion in CYP2A6. By analyzing the remaining 38 Japanese and 43 Caucasians, we found that there were no cases of CYP2A6(*)3-type polymorphism in the samples examined in this study, and no cases of CYP2A6(*)2-type polymorphism in the Japanese samples. Of Caucasians studied two individuals were classified into heterozygous CYP2A6(*)1/(*)2-type. Liver microsomal coumarin 7-hydroxylation activities in these two Caucasians were found to be lower than those of the other 41 Caucasians. Kinetic analysis showed that two CYP2A6(*)1/(*)2 individuals had a very low ratio of V(max) to K(m) for nicotine C-oxidation as well as coumarin 7-hydroxylation in liver microsomes, compared with those of homozygous CYP2A6(*)1-type. These results suggest that among 39 Japanese and 43 Caucasians examined one Japanese is classified to be CYP2A6 gene deletion and two Caucasians are heterozygous CYP2A6(*)1/(*)2-genotype. Thus the race-related differences in the occurrence of CYP2A6 genetic polymorphisms were supported.     

Gene-gene interactions between CYP2B6 and CYP2A6 in nicotine metabolism

OBJECTIVES: CYP2A6 is the major enzyme involved in nicotine metabolism, yet large interindividual variations in the rate of nicotine metabolism exist within groups of individuals having the same CYP2A6 genotype. We investigated the influence of genetic variation in another potential nicotine-metabolizing enzyme, CYP2B6, and its interaction with CYP2A6, on the metabolism of nicotine. METHODS: Two hundred and twelve healthy Caucasian adult twin volunteers underwent an intravenous infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, for characterization of pharmacokinetic and metabolism phenotypes. Five CYP2B6 genetic polymorphisms causing amino acid substitutions (R22C, Q172 H, S259R, K262R, and R487C) were analyzed. RESULTS: We observed that the CYP2B6*6 haplotype (defined as having both Q172 H and K262R variants) was associated with faster nicotine and cotinine clearance, and that such associations were more prominent among individuals having decreased-activity CYP2A6 genotypes. Statistically significant interactions between CYP2B6 and CYP2A6 genotypes were observed (P<0.003 for nicotine clearance and P<0.002 for cotinine clearance). CONCLUSIONS: Our results indicate that CYP2B6 genetic variation is associated with the metabolism of nicotine and cotinine among individuals with decreased CYP2A6 activity. Further investigation of the roles of CYP2B6 and the interaction between CYP2B6 and CYP2A6 genotypes in mediating nicotine dependence and tobacco-related diseases is merited.     

The molecular and enzyme kinetic basis for the diminished activity of the cytochrome P450 2D6.17 (CYP2D6.17) variant. Potential implications for CYP2D6 phenotyping studies and the clinical use of CYP2D6 substrate drugs in some African populations

In this study, the basis for the diminished capacity of CYP2D6.17 to metabolise CYP2D6 substrate drugs and the possible implications this might have for CYP2D6 phenotyping studies and clinical use of substrate drugs were investigated in vitro. Enzyme kinetic analyses were performed with recombinant CYP2D6.1, CYP2D6.2, CYP2D6.17 and CYP2D6.T107I using bufuralol, debrisoquine, metoprolol and dextromethorphan as substrates. In addition, the intrinsic clearance of 10 CYP2D6 substrate drugs by CYP2D6.1 and CYP2D6.17 was determined by monitoring substrate disappearance. CYP2D6.17 exhibited generally higher K(m) values compared to CYP2D6.1. The V(max) values were generally not different except for metoprolol alpha-hydroxylation with the V(max) value for CYP2D6.17 being half that of CYP2D6.1. CYP2D6.1 and CYP2D6.2 displayed similar kinetics with all probe drugs except for dextromethorphan O-demethylation with the intrinsic clearance value of CYP2D6.2 being half that of CYP2D6.1. CYP2D6.17 exhibited substrate-dependent reduced clearances for the 10 substrates studied. In a clinical setting, the clearance of some drugs could be affected more than others in individuals with the CYP2D6(*)17 variant. The CYP2D6(*)17 allele might, therefore, contribute towards the poor correlation of phenotyping results when using different probe drugs in African populations. To investigate effects of CYP2D6(*)17 mutations on the structure of the enzyme, a homology model of CYP2D6 was built using the CYP2C5 crystal structure as a template. The results suggest an alteration in position of active-site residues in CYP2D6.17 as a possible explanation for the reduced activity of the enzyme.     

Genetic polymorphisms in human CYP2A6 gene causing impaired nicotine metabolism

AIMS: Previously, we determined the phenotyping of in vivo nicotine metabolism and the genotyping of the CYP2A6 gene (CYP2A6*1 A, CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4 and CYP2A6*5 ) in 92 Japanese and 209 Koreans. In the study, we found one Korean and four Japanese subjects genotyped as CYP2A6*1B/CYP2A6*4 who revealed impaired nicotine metabolism, although other many heterozygotes of CYP2A6*4 demonstrated normal nicotine metabolism (CYP2A6*4 is a whole deletion type). After our previous report, several CYP2A6 alleles, CYP2A6*6 (R128Q), CYP2A6*7 (I471T), and CYP2A6*8 (R485L), have been reported. The purpose of the present study was to clarify whether the impaired nicotine metabolism can be ascribed to these CYP2A6 alleles. Furthermore, we also determined whether the subjects possessing CYP2A6*1x2 (duplication) reveal higher nicotine metabolism. METHODS: Genotyping of CYP2A6 alleles, CYP2A6*6, CYP2A6*7, CYP2A6*8, and CYP2A6*1x2 was determined by PCR. RESULTS: The five poor metabolizers were re-genotyped as CYP2A6*7/CYP2A6*4, suggesting that a single nucleotide polymorphism (SNP) causing I471T decreases nicotine metabolism in vivo. Furthermore, we found that two subjects out of five with a lower potency of nicotine metabolism possessed SNPs of CYP2A6*7 and CYP2A6*8 simultaneously. The novel allele was termed CYP2A6*10. In the 92 Japanese and 209 Koreans, the CYP2A6*6 allele was not found. The allele frequencies of CYP2A6*7, CYP2A6*8, and CYP2A6*10 were 6.5%, 2.2%, and 1.1%, respectively, in Japanese, and 3.6%, 1.4%, and 0.5%, respectively, in Koreans. The CYP2A6*1x2 allele was found in only one Korean subject (0.5%) whose nicotine metabolic potency was not very high. CONCLUSIONS: It was clarified that the impaired in vivo nicotine metabolism was caused by CYP2A6*7 and CYP2A6*10 alleles.     

Two novel haplotypes of CYP2D6 gene in a Japanese population

Two novel haplotypes of CYP2D6 were found in Japanese subjects. One haplotype of the human CYP2D6 gene, newly designated as CYP2D6(*)44 allele, had both a novel single nucleotide polymorphism (SNP) of 2950G>C in intron 6 donor splice junction and a known SNP (82CG, -1235A>G, -740C>T, -678G>A, and a gene conversion with CYP2D7 gene in intron 1 associated with CYP2D6(*)21. Both CYP2D6(*)44 and CYP2D6(*)21B alleles would cause a splicing error or a frameshift with impaired drug metabolizing function mediated by CYP2D6.     

Isoflavones inhibit nicotine C-oxidation catalyzed by human CYP2A6

The inhibitory effects of isoflavones (daidzein, genistein, and glycitein) on human cytochrome P450 (CYP) 2A6 activities were investigated. Daidzein, genistein, and glycitein uncompetitively inhibited nicotine C-oxidation catalyzed by recombinant CYP2A6 expressed in baculovirus-infected insect cells with Ki values of 1.3 +/- 0.3 microM, 0.7 +/- 0.2 microM, and 5.2 +/- 0.8 microM, respectively, but not coumarin 7-hydroxylation. Effects of the intake of soy isoflavones on in vivo nicotine metabolism were investigated with 7 healthy Japanese homozygotes of CYP2A6*1. The cotinine/nicotine ratio of the plasma concentrations 2 hours after chewing 1 piece of nicotine gum under the basal condition (after abstaining from soy foods for 1 week) was 8.8 +/- 2.6 (4.4-11.4). The ratio was significantly (P < .05) reduced to 6.7 +/- 1.6 (4.0-8.2) after consumption of a soy isoflavone supplement (60 mg of total isoflavones/d) for 5 days. The authors found that isoflavone contained in soy products significantly decreased nicotine metabolism.     

Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.     

Characterization of the novel CYP2A6*21 allele using in vivo nicotine kinetics

Objective The impact of CYP2A6*21 (K476R) on in vivo nicotine metabolism and disposition was investigated.Methods A two-step allele-specific PCR assay was developed to detect the 6573A>G single nucleotide polymorphism (SNP) in CYP2A6*21. Nicotine metabolism phenotypes from a previously described intravenous labeled nicotine and cotinine infusion study [1] was used to assess the impact of CYP2A6*21. Genomic DNA samples from 222 (111 monozygotic and dizygotic twin pairs) Caucasian subjects were genotyped for CYP2A6 alleles (CYP2A6*1X2, -*1B, -*2, -*4, -*7, -*9, -*10, -*12, and -*21). The pharmacokinetic parameters were compared between individuals with no detected CYP2A6 variants (CYP2A6*1/*1, n=163) and individuals heterozygous for the CYP2A6*21 allele (CYP2A6*1/*21, n=9).Results The frequency of the CYP2A6*21 allele was found to be 2.3% in Caucasians (n=5/222 alleles, evaluated in one twin from each twin pair). In vivo pharmacokinetic parameters, such as nicotine clearance (1.32±0.37 vs. 1.18±0.20 L/min), fractional clearance of nicotine to cotinine (1.02±0.36 vs. 0.99±0.23 L/min), nicotine half-life (111±37 vs. 116±29 min), and the trans-3′-hydroxycotinine to cotinine ratio (1.92±1.0 vs. 1.55±0.58) indicated no substantial differences in nicotine metabolism between those without the variant (CYP2A6*1/*1, n=163) and those with the variant (CYP2A6*1/*21, n=9), respectively.Conclusions CYP2A6*21 does not have a detectable impact on nicotine metabolism in vivo. Our data suggest that CYP2A6*21 may not be important for future studies of nicotine metabolism and the resulting impacts on smoking behaviors.Nael Al Koudsi and Jill C. Mwenifumbo contributed equally to this work.     

Catalytic roles of CYP2C9 and its variants (CYP2C9*2 and CYP2C9*3) in lornoxicam 5'-hydroxylation.

The effects of allelic variants of CYP2C9 (CYP2C9*2 and CYP2C9*3) on lornoxicam 5'-hydroxylation were studied using the corresponding variant protein expressed in baculovirus-infected insect cells and human liver microsomes of known genotypes of CYP2C9. The results of the baculovirus expression system showed that CYP2C9.3 gives higher K(m) and lower V(max) values for lornoxicam 5'-hydroxylation than does CYP2C9.1. In contrast, K(m) and V(max) values of CYP2C9.1 and CYP2C9.2 for the reaction were comparable. Lornoxicam 5'-hydroxylation was also determined in liver microsomes of 12 humans genotyped for the CYP2C9 gene (*1/*1, n = 7; *1/*2, n = 2; *1/*3, n = 2; *3/*3, n = 1). A sample genotyped as *3/*3 exhibited 8- to 50-fold lower intrinsic clearance for lornoxicam 5'-hydroxylation than did samples genotyped as *1/*1. However, the values for intrinsic clearance for *1/*3 were within the range of values exhibited by samples of *1/*1. In addition, no appreciable differences were observed in kinetic parameters for lornoxicam 5'-hydroxylation between *1/*1 and *1/*2. In conclusion, this study showed that lornoxicam 5'-hydroxylation via CYP2C9 was markedly decreased by the substitution of Ile359Leu (CYP2C9.3), whereas the effect of the substitution of Arg144Cys (CYP2C9.2) was nonexistent or negligible. Additional in vivo studies are required to confirm that individuals with homologous CYP2C9*3 allele exhibit impaired lornoxicam clearance.     

Functional characterization of allelic variants of polymorphic human cytochrome P450 2A6 (CYP2A6*5, *7, *8, *18, *19, and *35)

Cytochrome P450 2A6 (CYP2A6) catalyzes important metabolic reactions of many xenobiotic compounds, including coumarin, nicotine, cotinine, and clinical drugs. Genetic polymorphisms of CYP2A6 can influence its metabolic activities. This study analyzed the functional activities of six CYP2A6 allelic variants (CYP2A6*5, *7, *8, *18, *19, and *35) containing nonsynonymous single-nucleotide polymorphisms. Recombinant variant enzymes of CYP2A6*7, *8, *18, *19, and *35 were successfully expressed in Escherichia coli and purified. However, a P450 holoenzyme spectrum was not detected for the CYP2A6*5 allelic variant (G479V). Structural analysis shows that the G479V mutation may alter the interaction between the A helix and the F-G helices. Enzyme kinetic analyses indicated that the effects of mutations in CYP2A6 allelic variants on drug metabolism are dependent on the substrates. In the case of coumarin 7-hydroxylation, CYP2A6*8 and *35 displayed increased K(m) values whereas CYP2A6*18 and *19 showed decreased k(cat) values, which resulted in lower catalytic efficiencies (k(cat)/K(m)). In the case of nicotine 5-oxidation, the CYP2A6*19 variant exhibited an increased K(m) value, whereas CYP2A6*18 and *35 showed much greater decreases in k(cat) values. These results suggest that individuals carrying these allelic variants are likely to have different metabolisms for different CYP2A6 substrates. Functional characterization of these allelic variants of CYP2A6 can help determine the importance of CYP2A6 polymorphisms in the metabolism of many clinical drugs.     

CYP2A6 AND CYP2B6 are involved in nornicotine formation from nicotine in humans: interindividual differences in these contributions.

Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent K(m) = 173 +/- 70 microM, V(max) = 57 +/- 17 pmol/min/mg) and low-affinity (apparent K(m) = 619 +/- 68 microM, V(max) = 137 +/- 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent K(m) values of CYP2A6 (49 +/- 12 microM) and CYP2B6 (550 +/- 46 microM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 microM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 microM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.     

Hepatic CYP2B6 is altered by genetic,physiologic, and environmental factors but plays little role in nicotine metabolism

1. Human cytochrome P4502B6 (CYP2B6) is predominantly expressed in the liver and it plays a major role in the metabolism of several therapeutically important drugs and environmental toxicants.

2. The objective was twofold: (1) to determine the role of genetic, physiological, and environmental factors in predicting hepatic CYP2B6 protein expression; and (2) to investigate the role of CYP2B6 in nicotine C-oxidation.

3. Human livers (n?=?40) were assessed for CYP2B6 protein and genotype.

4. Linear regression analyses indicated that CYP2B6 genotype (10%), gender (14%), and exposure to inducers (21%), but not age, were predictors of CYP2B6 protein amounts. Livers with at least one CYP2B6*5 or *6 allele were associated with lower CYP2B6. Female livers and livers exposed to inducers (phenobarbital and/or dexamethasone) were associated with higher CYP2B6.

5. A weak correlation between CYP2B6 and nicotine C-oxidation activity was observed, which was abrogated when controlling for CYP2A6 protein levels. CYP2B6*6 was not associated with different nicotine kinetics.

6. In summary, CYP2B6 protein expression was associated with genotype, gender, and exposure to inducers, but not with nicotine C-oxidation activity.

     

Characterization of cytochrome P450 2D6.1 (CYP2D6.1), CYP2D6.2, and CYP2D6.17 activities toward model CYP2D6 substrates dextromethorphan, bufuralol, and debrisoquine.

Over 50 allelic variants of cytochrome P450 2D6 (CYP2D6) encoding fully functional, reduced-activity, or nonfunctional proteins have been described. Compared with Caucasians, studies in black populations demonstrate a tendency toward slower CYP2D6 activity, attributed in part to the presence of a variant allele associated with reduced activity, the CYP2D6*17 allele. To investigate the kinetic characteristics of this variant protein, expression constructs coding for CYP2D6.1, CYP2D6.2, and CYP2D6.17 gene products were prepared and transfected into mammalian COS-7 and insect (Trichoplusia ni) cells for expression. Microsomal fractions containing the expressed proteins were used to determine the kinetic parameters K(m), V(max), and intrinsic clearance (Cl(int)) for the model substrates dextromethorphan, bufuralol, and debrisoquine. Relative to the wild-type CYP2D6.1 protein expressed in COS-7 cells, CYP2D6.17 exhibited a 2-fold higher K(m) and a 50% reduction in V(max) using dextromethorphan as the substrate. In contrast, no appreciable change in bufuralol K(m) was observed with CYP2D6.17 whereas V(max) was decreased by 50%. When expressed in the baculovirus expression system, CYP2D6.17 exhibited a 6-fold increase in K(m) but no change in V(max) with dextromethorphan as the substrate, a 2-fold higher K(m) and 50% reduction in V(max) with bufuralol, and a 3-fold increase in K(m) and no change in V(max) with debrisoquine relative to CYP2D6.1. These data indicate that CYP2D6.17 exhibits reduced metabolic activity toward all three commonly used CYP2D6 substrates, although specific effects on substrate affinity and turnover demonstrate some substrate dependence.     

Down-regulation of hepatic nicotine metabolism and a CYP2A6-like enzyme in African green monkeys after long-term nicotine administration

Nicotine metabolism is decreased in smokers compared with nonsmokers, but the mechanism(s) responsible for the slower metabolism are unknown. Nicotine is inactivated to cotinine by CYP2A6 in human liver [nicotine C-oxidation (NCO)]. CYP2B6 also metabolizes nicotine to cotinine but with lower affinity than CYP2A6. To evaluate the effects of long-term nicotine treatment on hepatic levels of CYP2A6 and CYP2B6, and nicotine metabolism, an African green monkey (AGM) model was developed. As in humans, approximately 80 to 90% of in vitro hepatic NCO is mediated by a CYP2A6-like protein (CYP2A6agm) in this species, as determined by inhibition studies. Male AGM (n = 6 per group) were treated for 3 weeks with nicotine (s.c., 0.3 mg/kg, b.i.d.), phenobarbital (oral, 20 mg/kg, as a positive control for P450 induction), and/or saline (s.c., b.i.d.). Immunoblotting demonstrated a 59% decrease (p < 0.05) in hepatic CYP2A6agm protein in nicotine-treated animals. A CYP2B6-like protein (CYP2B6agm) was modestly and insignificantly decreased (14%, p = 0.11). In vitro NCO was decreased by 41% in the nicotine-treated group (p < 0.05), mediated by a decrease in CYP2A6agm, as demonstrated using inhibitory antibodies. CYP2A6agm mRNA (33%, P < or = 0.05) and CYP2B6agm (35%, p < 0.01) mRNA were also significantly decreased in the nicotine-treated group. Phenobarbital-treated animals demonstrated an increase in CYP2B6agm (650%, p < 0.001), but not CYP2A6agm (20%, p = 0.49). NCO was increased in the phenobarbital-treated group (55%, p < 0.05) by an increase in CYP2B6agm-mediated NCO. Consistent with the slower nicotine metabolism observed in smokers, nicotine may decrease its own metabolism in primates by decreasing the expression of the primary nicotine-metabolizing enzyme CYP2A6.     

A novel duplication type of CYP2A6 gene in African-American population.

Human CYP2A6 is responsible for the metabolism of nicotine and its genetic polymorphisms affect smoking behavior and risk of lung cancer. In the present study, we identified a novel type of CYP2A6 gene duplication that is created through an unequal crossover event with the CYP2A7 gene at 5.2 to 5.6 kilobases downstream from the stop codon. The novel duplication type of CYP2A6 was found in African Americans (n = 176) at an allele frequency of 1.7%, but was not found in European-American (n = 187), Korean (n = 209), or Japanese (n = 184) populations. The plasma cotinine/nicotine ratio in subjects possessing the novel CYP2A6 gene duplication with the CYP2A6*1 allele (10.8 +/- 7.0, n = 4) was 1.4-fold higher than that in homozygotes of the wild type (8.0 +/- 5.0, n = 87), although the difference was not statistically significant. The findings in the present study suggested that the novel duplicated CYP2A6 allele, which is specific for African Americans, would increase nicotine metabolism and may affect smoking behavior.