Determination of cytochrome P450 2E1 activity in microsomes by thin-layer chromatography using [2-14C]chlorzoxazone

A thin-layer chromatographic assay was developed as an alternative method for the determination of cytochrome P450 2E1 (CYP2E1) in microsomes using [2-¹⁴C]chlorzoxazone. After incubation of microsomes with 0.125 μCi/mmol chlorzoxazone, chlorzoxazone and its single metabolite, 6-hydroxychlorzoxazone, were extracted using chloroform-2-propanol (85:15, v/v) and chromatographed on silica gel 60 F254 plates with acetone-hexane (45:55, v/v) as solvent. The plates were then exposed to X-ray film for 2 days to localize the radiolabelled chlorzoxazone and 6-hydroxychlorzoxazone. The metabolite and substrate regions were scraped and counted in a liquid scintillation analyzer. This method is sensitive enough to determine constitutive and induced CYP2E1 activities in liver or kidney microsomes. The precision of the method was similar to that of the HPLC method. The correlation coefficient between both methods was found to be 0.97 (n=21). Therefore, the TLC method constitutes a valuable tool for the determination of chlorzoxazone metabolism in microsomes.     

Expression of CYP2E1 in human nasopharynx and its metabolic effect in vitro

It was evident that nitrosamines can act directly on target tissue and result in carcinogenesis. As has been shown, the carcinogenic activity of nitrosamines relied on its bioactivation by Cytochrome P450 2E1 (CYP2E1). In this study, we investigated the expression of CYP2E1 in Nasopharyngeal carcinoma (NPC) cells, embryonic nasopharyngeal epithelial tissue (ENET) specimens, and NPC biopsies by RT-PCR analysis. CYP2E1 was expressed in all NPC cell lines (6/6, including 7429) and ENET (6/6), and 80% of NPC biopsie (8/10). The fact that Human nasopharynx expresses CYP2E1 suggests that CYP2E1 may play an important role in the course of NPC by indirect carcinogens nitrosamines. To further evaluate the function of CYP2E1, the CYP2E1 was stably expressed in the cell line NIH 3T3/rtTA under a tetracycline-controlled transactivator. The expression of CYP2E1 was tightly regulated in a dose-dependent manner by Doxycycline (Dox) When the catalytic activity of CYP2E1 was assayed, the result showed that the generation of 6-hydroxychlorzoxazone (6-OH-CZ) from chlorzoxazone (CZ) was dose- and time-dependent on Dox addition to the medium. In the presence of 1 μg/ml Dox, the CZ 6-hydroxylase activity of the cell line was found to be 0.986 ± 0.034 nmol/10⁶ cells/h. The metabolic activation of Tet/3T3/2E1-6 cells was also assayed by N,N′-dinitrosopiperazine (DNP) cytotoxicity, and the viability of Tet/3T3/2E1-6 cells treated with Dox was lower than that of untreated cells with a significant difference between them in 80 and 160 μg/ml DNP (P ( 0.05, t test. This cell line will be useful not only to assess the metabolic characteristics of CYP2E1, but also will be useful to investigate the role of CYP2E1 in metabolic activation of carcinogenic nitrosamines in vitro.     

Polysaccharide peptides from Coriolus versicolor competitively inhibit model cytochrome P450 enzyme probe substrates metabolism in human liver microsomes

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy or health supplement in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited rat CYP2C11-mediated tolbutamide 4-hydroxylation and in human CYP2C9. In this study, the effects of the water extractable fraction of PSP on the metabolism of model CYP1A2, CYP2D6, CYP2E1 and CYP3A4 probe substrates were investigated in pooled human liver microsomes. PSP (1.25-20μM) dose-dependently decreased CYP1A2-mediated metabolism of phenacetin to paracetamol (IC(50) 19.7μM) and CYP3A4-mediated metabolism of testosterone to 6β-hydroxytestosterone (IC(20) 7.06μM). Enzyme kinetics studies showed the inhibition of CYP1A2 activity was competitive and concentration-dependent (K(i)=18.4μM). Inhibition of testosterone to 6β-hydroxytestosterone was also competitive and concentration-dependent (K(i)=31.8μM). Metabolism of dextromethorphan to dextrorphan (CYP2D6-mediated) and chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1-mediated) was only minimally inhibited by PSP, with IC(20) values at 15.6μM and 11.9μM, respectively. This study demonstrated that PSP competitively inhibited the CYP1A2- and CYP3A4-mediated metabolism of model probe substrates in human liver microsomes in vitro. The relatively high K(i) values for CYP1A2 and CYP3A4 would suggest a low potential for PSP to cause herb-drug interaction related to these CYP isoforms.     

Monolithic silica rod liquid chromatography with ultraviolet or fluorescence detection for metabolite analysis of cytochrome P450 marker reactions

In vitro cytochrome P450 assays are used in metabolism studies in support of early phases of drug discovery to investigate, e.g., metabolic stability, enzyme inhibition and induction by new chemical entities. LC-UV and LC-fluorescence are traditional analytical tools in support of such studies. However, these tools typically comprise different methods of relatively low throughput for the various metabolites of probe reactions. In recent years, LC-MS methods have been developed to increase throughput. Increased throughput can also be achieved by means of modern chromatographic tools in combination with UV and fluorescence detection. This approach is especially suitable when cytochrome P450 isoforms are investigated by means of single probe incubations. Here, an LC-UV/fluorescence system based on a monolithic porous silica column is described for the analysis of metabolites of nine cytochrome P450 marker reactions [phenacetin to paracetamol (CYP1A2), coumarin to 7-hydroxycoumarin (CYP2A6), paclitaxel to 6alpha-hydroxypaclitaxel (CYP2C8), diclofenac to 4-hydroxydiclofenac (CYP2C9), mephenytoin to 4-hydroxymephenytoin (CYP2C19), bufuralol to 1-hydroxybufuralol (CYP2D6), chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1), midazolam to 1-hydroxymidazolam (CYP3A4), and testosteron to 6beta-hydroxytestosteron (CYP3A4)]. While offering sensitivities and linear ranges comparable to previously reported methods, the set-up described here provides ease of use and increased throughput with maximum cycle times of 4.5 min.     

Effects of glucose supplementation on the pharmacokinetics of intravenous chlorzoxazone in rats with water deprivation for 72 h

It was reported that in rats with water deprivation for 72 h with food (dehydration rat model), the expression of CYP2E1 was 3-fold induced with an increase in mRNA level and glucose supplementation instead of food during 72-h water deprivation (dehydration rat model with glucose supplementation) inhibited the CYP2E1 induction in dehydration rat model. It was also reported that chlorzoxazone (CZX) is metabolized to 6-hydroxychlorzoxazone (OH-CZX) mainly via CYP2E1 in rats. Hence, the effects of glucose supplementation on the pharmacokinetics of CZX and OH-CZX were investigated after intravenous administration of CZX at a dose of 25 mg/kg to control male Sprague-Dawley rats and dehydration rat model and dehydration rat model with glucose supplementation. Based on the above mentioned results of CYP2E1, it could be expected that increased formation of OH-CZX in dehydration rat model could decrease in dehydration rat model with glucose supplementation. This was proven by the following results. In dehydration rat model with glucose supplementation, the AUC of OH-CZX was significantly smaller (1900 versus 1050 microg min/ml), AUC(OH-CZX)/AUC(CZX) ratio was considerably smaller (105 versus 34.3%), C(max) was significantly lower (20.6 versus 8.08 microg/ml), total amount excreted in 24-h urine as unchanged OH-CZX was significantly smaller (62.3 versus 42.7% of intravenous dose of CZX), and in vitro V(max) (2.18 versus 1.20 nmol/min/mg protein) and CL(int) (0.0285 versus 0.0171 ml/min/mg protein) were significantly slower than those in dehydration rat model.     

Evaluation of Tetrahydropalmatine Enantiomers on the Activity of Five Cytochrome P450 Isozymes in Rats Using a Liquid Chromatography / Mass Spectrometric Method and a Cocktail Approach

The aim was to evaluate the effects of tetrahydropalmatine (THP) enantiomers on the activity of five cytochrome P450 (CYP450) isozymes in vivo. A liquid chromatography / mass spectrometric (LC‐MS) method was developed for simultaneous determination of five specific probe substrates including metoprolol (2D6), caffeine (1A2), dapsone (3A4), chlorzoxazone (2E1), and tolbutamide (2C9) in rat plasma. Analytes were separated with the mobile phase consisting of 0.1% acetic acid aqueous solution and acetonitrile in a gradient elution. The mass spectrometric detection via selected ion monitoring (SIM) was operated in both positive ion mode (for metoprolol m/z 268, caffeine m/z 195, and dapsone m/z 249) and negative ion mode (for chlorzoxazone m/z 168 and tolbutamide m/z 269) in the same run. Linear correlation was obtained (r² > 0.99) over the concentration range of 0.050–25.0 µg/mL for caffeine and dapsone, 0.025–10.0 µg/mL for metoprolol, 0.050–50.0 µg/mL for chlorzoxazone, and 0.25–100.0 µg/mL for tolbutamide. Intra‐ and interday precision were less than 12.09%. The matrix effect ranged from 87.50% to 109.25% and the absolute recoveries were greater than 70%. The method was successfully applied to evaluate the effect of THP enantiomers on the activity of CYP450 isozymes by a cocktail approach. The pharmacokinetic results of five probe drugs indicated that there were stereoselective differences between the two THP enantiomers, i.e., d‐THP had the potential to inhibit the activities of CYP2D6 and CYP1A2 isozymes, while l‐THP inhibited CYP1A2 isozyme and induced CYP3A4 and CYP2C9 isozymes. Chirality 27:551–556, 2015. © 2015 Wiley Periodicals, Inc.     

Determination of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma and urine by high-performance liquid chromatography

A sensitive and reproducible method is described for the determination of the cytochrome P450 enzyme 2E1 substrate chlorzoxazone and its primary metabolite 6-hydroxychlorzoxazone in human plasma and urine. Plasma or diluted urine were acidified, incubated with β-glucuronidase and then were extracted with diethyl ether. Separation of the analytes was achieved on a C₁₈ column with UV detection set at 283 nm. Excellent linearity was observed over the concentration ranges of 100–3000 ng/ml and 4–400 μg/ml in plasma and urine, respectively. The intra-assay variability was 5.1% and the inter-assay variability was 8.2% for each compound in each matrix. The method presented is applicable to pharmacokinetic and pharmacogenetic studies utilizing chlorzoxazone.     

Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes

Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3% identity in their amino acid sequences. The functional CYP content in yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7% of that of humCYP2E1, although no significant differences were statistically observed. The K(m) values of microsomes from human livers and yeast cells expressing humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 microM for chlorzoxazone 6-hydroxylation, and 422 and 514 microM for 4-nitrophenol 2-hydroxylation, respectively. The K(m) values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not significantly different from those of humans in any enzyme source. V(max) and V(max)/K(m) values of human liver microsomes for CYP2E1-dependent oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and 2990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were comparable to or lower than those of human liver microsomes (49.5-102%). In yeast cell microsomes expressing humCYP2E1, V(max) and V(max)/K(m) values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.     

Heterologous expression of mouse cytochrome P450 2e1 in V79 cells: construction and characterisation of the cell line and comparison with V79 cell lines stably expressing rat P450 2E1 and human P450 2E1

A V79 Chinese hamster cell line was constructed for stable expression of mouse cytochrome P450 2e1 (Cyp2e1), as an addition to the existing cell battery consisting of cell lines stably expressing rat CYP2E1 and human CYP2E1 (V79 Cell Battery). The aim was to establish a cell battery that offers the in vitro possibility of investigating species-specific differences in the toxicity and metabolism of chemicals representing substrates for CYP2E1. The newly established cell line (V79m2E1) effectively expressed Cyp2e1 in the catalytically active form. The expression of catalytically active CYP2E1 in V79m2E1 cells was maintained over several months in culture, as demonstrated by Western Blotting and chlorzoxazone (CLX) 6-hydroxylase activity. The cells exhibited CLX 6-hydroxylase activity with a Km of 27.8 microM/l and Vmax of 40 pmol/mg protein/minute, compared with a Km of 28.2/28.6 microM/l and a Vmax of 130/60 pmol/mg protein/minute from V79r2E1/V79h2E1 cells. Furthermore, the CYP2E1-dependent mutagenicity of N-nitrosodimethylamine could be demonstrated in the V79m2E1 cells. Therefore, the new cell battery permits the interspecies comparison of CYP2E1-dependent toxicity and of metabolism of chemicals between humans and the two major rodent species--the rat and the mouse--that are usually used in classical toxicity studies.     

Tet调控CYP2E1基因表达细胞系的代谢能力分析

应用Tet-On基因表达系统,调控CYP2E1基因在NIH 3T3细胞中的表达水平,探讨CYP2E1在化学致癌物二甲基亚硝胺(N-nitrosodimethylamine, NDMA)代谢中的作用．先后将 Tet-on基因表达系统的调控质粒pRevTet-on和反应质粒pRevTRE-2E1转染NIH 3T3细胞,分别用G418和潮霉素筛选,并通过RT-PCR和Western印迹检测,成功获得了3个具有良好诱导性Tet调控的CYP2E1基因表达细胞系（Tet/3T3-2E1）．应用不同浓度强力霉素(doxycycline, Dox)处理Tet/3T3-2E1细胞诱导CYP2E1表达,HPLC分析细胞对CYP2E1特异性药物探针氯唑沙腙的原位代谢能力及MTT法分析CYP2E1介导的NDMA细胞毒性作用．结果显示,Tet/3T3-2E1细胞CYP2E1的表达及其代谢能力有明显的Dox浓度依赖性,而且NDMA对Dox诱导组细胞有明显浓度依赖性细胞毒性作用,其IC₅₀值为12.06 μmol/L,无Dox诱导组未见明显的NDMA细胞毒性作用．该细胞模型的成功建立对于开展与CYP2E1相关的毒物、致癌物代谢研究,筛选抗毒物、抗癌物等具有重要价值．     

Cocktail探针药物法评价傣药"雅解沙把" 对大鼠肝药酶P450亚型 CYP1A2、CYP2C19、CYP2E1、CYP3A4 活性的影响

目的:采用cocktail探针药物法研究傣药"雅解沙把"对肝细胞色素P450亚型CYP1A2、CYP2C19、CYP2E1、CYP3A4的影响。方法:将SD大鼠随机分为空白对照组、苯巴比妥钠组(10.8 mg/kg)、"雅解沙把"低剂量组(0.27 g生药/kg)和"雅解沙把"高剂量组(2.43 g生药/kg),按上述剂量灌胃给药,空白对照组灌胃蒸馏水。连续灌胃7天后处死动物,取肝脏制备肝微粒体,以甲硝唑为内标,建立HPLC方法检测Cocktail探针药物奥美拉唑、氯唑沙宗、咖啡因、氨苯砜的代谢情况。结果:与空白对照组比较,"雅解沙把"低剂量组和高剂量组氯唑沙宗的代谢明显升高,氯唑沙宗的含量显著降低(P0.01),"雅解沙把"高剂量组奥美拉唑和氨苯砜的代谢明显升高,奥美拉唑和氨苯砜的含量明显降低(P0.05)。"雅解沙把"低剂量组和高剂量组虽咖啡因代谢较与空白对照组有上升的趋势,但差异无统计学意义(P0.05)。结论:傣药"雅解沙把"能促进肝药酶CYP3A4、CYP2C19、CYP2E1的活性,加速药物代谢,这可能是其解药物毒的作用机制之一。     

Rapid and sensitive high-performance liquid chromatographic assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomes

A high-performance liquid chromatographic assay was developed for the quantitation of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. These compounds along with phenacetin, the internal standard, were extracted from incubation mixtures using ether extraction. The extracts were analyzed on a Brownlee Spheri-5 C₈ column with a mobile-phase of acetonitrile–0.5% phosphoric acid (30:70, v/v). The assay utilized UV detection at 287 nm which provided sensitivity and specificity to simultaneously quantify chlorzoxazone and 6-hydroxychlorzoxazone from liver microsomal samples at amounts of 10 ng and greater. The mean correlation coefficient of the standard curves for 6-hydroxychlorzoxazone and chlorzoxazone was 0.998 and 0.993, respectively, over the range of 25–400 ng, and the regression curves were found to be linear at least through 1600 ng. All components eluted within 7 min, resulting in a total analysis time of 8 min. The inter-day and intra-day coefficients of variation were <7 and <3%, respectively. This method provides a rapid, sensitive and cost-effective assay for 6-hydroxychlorzoxazone and chlorzoxazone in liver microsomal incubations.     

Presence of cytochrome P450 enzymes in human CD34+ haematopoietic progenitor cells

Human CD34+ cells (haematopoietic stem and progenitor cells separated from peripheral blood) were shown to express CYP2E1 protein by Western blotting. For the first time, the specific CYP2E1 activity (chlorzoxazone 6-hydroxylation) was also detected. No CYP3A4 protein neither the CYP3A-specific nifedipine oxidase activity were detectable. The results obtained indicate differences in content of active CYP proteins in populations of stem and progenitor cells from different species as the CYP3A2 (a rat form similar to human CYP3A4) was shown to be expressed in bone marrow derived cells by RT-PCR (Avital et al. 2001).     

Expression of constitutive and inducible cytochrome P450 2E1 in rat brain

Studies initiated to investigate the expression of cytochrome P450 2E1 (CYP2E1) in rat brain demonstrated low but detectable protein and mRNA expression in control rat brain. Though mRNA and protein expression of CYP2E1 in brain was several fold lower as compared to liver, relatively high activity of N-nitrosodimethylamine demethylase (NDMA-d) was observed in control rat brain microsomes. Like liver, pretreatment with CYP2E1 inducers such as ethanol or pyrazole or acetone significantly increased the activity of brain microsomal NDMA-d. Kinetic studies also showed an increase in the Vmax and affinity (Km) of the substrate towards the brain enzyme due to increased expression of CYP2E1 in microsomes of brain isolated from ethanol pretreated rats. In vitrostudies using organic inhibitors, specific for CYP2E1 and anti-CYP2E1 significantly inhibited the brain NDMA-d activity indicating that like liver, NDMA-d activity in rat brain is catalyzed by CYP2E1. Olfactory lobes exhibited the highest CYP2E1 expression and catalytic activity in control rats. Furthermore, several fold increase in the mRNA expression and activity of CYP2E1 in cerebellum and hippocampus while a relatively small increase in the olfactory lobes and no significant change in other brain regions following ethanol pretreatment have indicated that CYP2E1 induction maybe involved in selective sensitivity of these brain areas to ethanol induced free radical damage and neuronal degeneration.     

Determination of 6-hydroxychlorzoxazone and chlorzoxazone in porcine microsome samples

A simple, accurate and sensitive HPLC method for the in vitro determination of 6-hydroxychlorzoxazone and chlorzoxazone in porcine microsome samples is described. Chromatography was performed on a YMC-Pack ODS-AQ column using a mobile phase of 0.05% phosphoric acid pH 3-methanol (60:40, v/v). UV detection was carried out at 287 nm. The detector response was linear over the concentration range 25-2000 ng/ml. This assay produced quick, accurate, and repeatable results.     

Upregulation of CYP2e1 and CYP3a activities in histamine-deficient histidine decarboxylase gene targeted mice

Histamine is a biogenic amine with multiple physiological functions. Its importance in allergic inflammation is well characterized; moreover, it plays a role in the regulation of gastric acid production, various hypothalamic functions, such as food uptake, and enhancing TH2 balance during immune responses. Using histidine decarboxylase gene targeted (HDC(-/-)) BALB/c mice, we studied the effect of the absence of histamine on four cytochrome p450 enzyme activities. Their selective substrates were measured: ethoxyresorufin O-dealkylase activity of CYP1A, pentoxyresorufin O-dealkylase activity of CYP2B, chlorzoxazone 6-hydroxylase activity of CYP2E1 and ethylmorphine N-demethylase activity of CYP3A.The results indicate a significant elevation of CYP2E1 and CYP3A activities, however, no change in CYP1A and CYP2B activities was seen in HDC targeted mice compared to wild type controls with identical genetic backgrounds.     

Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms

The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.     

Expression of cytochrome P450 2A6 in Escherichia coli: purification, spectral and catalytic characterization, and preparation of polyclonal antibodies.

Cytochrome P450 (CYP) 2A6 is the principal human enzyme catalyzing coumarin 7-hydroxylation and is known to be involved in the metabolism of halothane, nicotine, and metabolic activation of butadiene and nitrosamines. In this paper expression of CYP2A6 in Escherichia coli is reported. In order to achieve expression, the N-terminus of protein was modified by PCR mutagenesis. The N-terminal variant with only a single amino acid change showed expression of 210 nmol of CYP2A6/liter of culture. Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized. Absolute spectra were typical for CYP proteins and indicated low spin characteristics of isolated protein. Due to a hydrophobic segment the N-terminal amino acid sequence of recombinant CYP2A6 was blocked. The N-terminal formylmethionine block was removed by mild acid treatment. Purified CYP2A6 had good catalytic activity toward marker substrate coumarin in a reconstituted system (K(m) = 1.48 +/- 0.37 microM, V(max) = 3.36 +/- 0.18 nmol product/min/nmol CYP). Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione. CYP2A6 was shown to metabolize chlorzoxazone in the reconstituted system with activity of 0.32 nmol of product/min/nmol of CYP, and thus caution should be taken when interpretation of CYP2E1 in vivo phenotyping data is performed. Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immunoblotting and immunoinhibition studies. Availability of this expression system and specific antibodies should facilitate characterization of the role of CYP2A6 in the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of this enzyme.