Prenatal and neonatal exposure to bisphenol-A enhances the central dopamine D1 receptor-mediated action in mice: enhancement of the methamphetamine-induced abuse state.

Bisphenol-A (BPA), one of the most common environmental endocrine disrupters, has been extensively evaluated for toxicity in a variety of tests in rodents, including developmental and reproductive toxicity, and carcinogenicity. However, little is known about its action on the CNS. In this report, we show that prenatal and neonatal exposure to BPA in mice leads to the enhancement of the dopamine D1 receptor-dependent rewarding effect induced by a psychostimulant methamphetamine. Furthermore, this treatment with BPA markedly enhanced hyperlocomotion and its sensitization induced by methamphetamine, which reflects extensive abuse associated with sociological and psychiatric problems. We also demonstrated that chronic exposure to BPA produced an up-regulation of dopamine D1 receptor function to activate G-protein in the mouse limbic forebrain, which is thought to be a critical site for the expression of rewarding effects by abuse drugs. Additionally, chronic BPA exposure produced a significant increase in levels of the dopamine D1 receptor mRNA in the whole brain. In contrast, no change in protein levels of methamphetamine-targeted proteins, dopamine transporter or the type 2 vesicle monoamine transporter in the brain was observed by prenatal and neonatal exposure to BPA. The present data provide the first evidence that prenatal and neonatal exposure to BPA can potentiate the central dopamine D1 receptor-dependent neurotransmission, resulting in supersensitivity of methamphetamine-induced pharmacological actions related to psychological dependence on psychostimulants.

Escape from tolerance of organic nitrate by induction of cytochrome P450.

The mechanism of organic nitrate tolerance is poorly defined. We studied the rat P450-catalyzed conversion of organic nitrate to nitric oxide (NO) by purified P450 isoforms relationship between P450 expression and nitrate tolerance following continuous infusion of organic nitrates in rats. The hypotensive effect of an nitroglycerin (NTG) bolus injection was abolished in rats that had been previously provided a continuous 48 h infusion of NTG. This effect was accompanied by a gradual but marked decrease in plasma and urinary nitrate levels following a peak at 18-24 h. Nitrate tolerance was reversible; the decline in the hypotensive effect and P450 levels observed after 2 d of continuous infusion was followed by restoration to control levels 2 d after cessation of the infusion. Similarly, the hypotensive action disappeared in P450-depleted, and -inhibited rats. At 48 h after infusion, NTG-induced NO generation of the vessels increased in acetone (a P450 inducer) -pretreated rats. The appearance and disappearance of P450 paralleled the conversion of organic nitrates to NO. Our observations indicate that nitrate tolerance is in large part the result of decreased P450 expression and activity. Interventions that maintain or increase P450 activity may be a strategy to provide relief from ischemic conditions in humans.

Functional evaluation of cytochrome P450 2D6 with Gly42Arg substitution expressed in Saccharomyces cerevisiae.

A single amino acid-substituted mutant protein, CYP2D6 (G42R) was expressed in Saccharomyces cerevisiae and its enzymatic properties were compared with those of other single (P34S, R296C and S486T) and double amino acid-substituted mutant proteins (P34S/S486T and R296C/S486T) expressed in yeast cells, all of which were known to occur in the CYP2D6 gene as single nucleotide polymorphisms. The protein levels of G42R, P34S and P34S/S486T in microsomal fractions and their oxidation capacities towards debrisoquine as a prototypic substrate and bunitrolol as a chiral substrate were different from those of wild-type CYP2D6, while the R296C, S486T and R296C/S486T behaved similarly to the wild-type in these indices. The CYP contents both in yeast microsomal and in whole cell fractions indicated that some part of G42R protein was localized in the endoplasmic reticulum membrane fraction, whereas most of G42R protein was in some subcellular fractions other than endoplasmic reticulum. In kinetic analysis, the G42R substitution increased apparent Km and decreased Vmax for debrisoquine 4-hydroxylation, while it increased both Km and Vmax for bunitrolol 4-hydroxylation. The P34S substitution did not drastically change Km but decreased Vmax for debrisoquine 4-hydroxylation, whereas Km was increased and Vmax unchanged or decreased for bunitrolol 4-hydroxylation by P34S substitution. These results suggest that the G42R substitution causes a change in the CYP2D6 conformation, which may be different from the change produced by the P34S substitution.

Comparison of the levels of enzymes involved in drug metabolism between transgenic or gene-knockout and the parental mice.

Drug-metabolizing enzymes are involved in the metabolic activation or detoxification of carcinogens. To evaluate animals developed as models for alternative carcinogenicity testing, we investigated whether or not a gene manipulation including the transgene of ras and the knocking out of a tumor suppressor gene such as p53 or XPA could alter the expression of representative drug-metabolizing enzymes directly or indirectly. Expression of several isoforms of cytochrome P450 (CYP) in the liver of rasH2, p53 (+/-), Tg.AC, and XPA (-/-) mice with or without treatment of prototype inducer. phenobarbital or 3-methylcholanthrene, was analyzed by Western immunoblotting in comparison with their parental strains of mice. In addition, the activities of 3 major phase II enzymes, UDP-glucronosyltransferase, sulfotransferase, and glutathione S-transferase, were compared between the gene-manipulated and the corresponding parental strains of mice. Results demonstrate that XPA gene knockout appeared to increase constitutive expression of CYP2B and CYP3A isoforms. Overexpression of human c-Ha-ras gene or p53 gene knockout appeared to increase constitutive UGT activity toward 4-nitrophenol. The content or activities of almost all other enzymes examined in the present study do not appear to be affected by the gene manipulation.

Progesterone oxidation by cytochrome P450 2D isoforms in the brain.

The existence of cytochrome P450 2D isoforms in the brain has been demonstrated, although their physiological functions remain to be elucidated. In this study we demonstrated that recombinant rat cytochrome P450 2D1 and 2D4 and human cytochrome P450 2D6 possess progesterone 6 beta- and 16 alpha- hydroxylation activities; 2 beta- and 21-hydroxylation activities; and 2 beta-, 6 beta-, 16 alpha- and 21-hydroxylation activities, respectively. Cytochrome P450 2D4 had the lowest K(m) value and the highest maximum velocity value toward these activities. Progesterone 2 beta- and 21-hydroxylation activities were also detected in rat brain microsomes, and these activities were completely inhibited by anticytochrome P450 2D antibodies. The presence of endogenous 2 beta- and 21-hydroxyprogesterones in rat brain tissues was also demonstrated. The mRNAs of cytochrome P450 2D4, CYP11A, and 3 beta-hydroxysteroid dehydrogenase were detected in the rat brain, suggesting that progesterone was generated from cholesterol by CYP11A and 3 beta-hydroxysteroid dehydrogenase and then underwent hydroxylation to hydroxyprogesterones by cytochrome P450 2D4 in rat brain. Collectively, our findings support the idea that cytochrome P450 2D may be involved in the regulation (metabolism and/or synthesis) of endogenous neuroactive steroids, such as progesterone and its derivatives, in brain tissues.

Kinetics of testosterone 6beta-hydroxylation in the reconstituted system with similar ratios of purified CYP3A4, NADPH-cytochrome p450 oxidoreductase and cytochrome B5 to human liver microsomes.

Kinetics of testosterone 6beta-hydroxylation were determined using a reconstituted system that consisted of CYP3A4, cytochrome b5 and NADPH-cytochrome P450 oxidoreductase (OR) with similar ratios as those seen in human liver microsomes and compared with those determined using human liver microsomes. Two reconstituted systems were constructed in accordance with two human liver microsomal samples that showed extremely high and low ratios of OR/CYP3A4. The Km values of testosterone 6beta-hydroxylation obtained from the reconstituted systems with high and low OR/CYP3A4 ratios were 29.3 and 35.2 microM, respectively, which were similar to that of the corresponding human liver microsomal samples (23.2 and 40.0 microM, respectively). However, Vmax values obtained from the reconstituted systems (3.7 and 0.8 pmol/min/pmol CYP3A4) were much lower than those from the human liver microsomes (44.2 and 31.1 pmol/min/pmol CYP3A4). The results suggest that the interaction between substrate and CYP3A4 in the reconstituted systems appear to be similar to human liver microsomes but that the velocity of the substrate metabolism in the reconstituted systems is different from that in human liver microsomes. In conclusion, our reconstituted systems could be used for the determination of affinity but not for the determination of the maximum velocity of substrate metabolism. Further studies on the protein-protein interactions between CYP3A4, OR, cytochrome b5 and/or a specific lipid environment are required to establish a reconstituted system showing similar kinetic properties to those of human liver microsomes.

Study of cytochrome P4502E1 mRNA level of mononuclear cells in patients with alcoholic liver disease.

BACKGROUND: Cytochrome P-4502E1 (CYP2E1) is an important enzyme because of its unique ability to convert many substrates to cytotoxins. The increased production of reactive intermediates by elevated enzyme concentrations leads to various pathological conditions. Therefore, it is important to detect induced CYP2E1 levels in alcoholic individuals to avoid xenobiotic-promoted liver injury. In the present investigation, we detected CYP2E1 mRNA levels of mononuclear cells obtained from 10 ml of blood by using competitive polymerase chain reaction (PCR) method. METHODS: Mononuclear cells were obtained from healthy individuals who did and did not drink habitually and patients with alcoholic liver disease (ALD). Complementary DNA synthesis was performed with RNA obtained from mononuclear cells by reverse transcription-PCR. Competitive PCR of CYP2E1 was performed with the sense (5'-CTGCAACGTCATA-GCCGACA-3') and antisense (5'-TCCATTTCCACGAGCAGGCA-3') primer and competitor DNA. Competitive PCR of beta-actin also was performed. Electrophoresis was scanned, and each band was digitized. The concentration of CYP2E1 and beta-actin mRNA was calculated from the ratio of competitor DNA. RESULTS: In healthy individuals who did and did not drink habitually, CYP2E1 mRNA levels were 103.3 copies/microl RNA and 101.7 copies/microl RNA, respectively. In actively drinking patients with ALD, CYP2E1 mRNA levels were 103.5 copies/microl RNA, but those levels decreased to 101.7 copies/microl RNA after 4 days of abstinence. No significant difference was observed in CYP2E1 mRNA levels between alcoholic fibrosis and cirrhosis. As control, we measured beta-actin mRNA levels in mononuclear cells in all samples. The mean value of beta-actin mRNA was 104.3 copies/microl RNA in all cases, which included patients with ALD. CONCLUSIONS: The results demonstrated that it is possible to measure the CYP2E1 mRNA levels of mononuclear cells in a 10 ml blood sample. The CYP2E1 mRNA level in mononuclear cells increases during drinking and decreases in abstinence for a short period of 3 to 4 days. It is concluded that CYP2E1 mRNA level may be used as an effective marker for alcoholic intake.

A transgenic mouse expressing human CYP4B1 in the liver.

The human CYP4B1 protein was expressed in the liver of a transgenic mouse line under the control of the promoter of the human apolipoprotein E (apo E) gene. Hepatic microsomes of transgenic mice catalyzed omega-hydroxylation of lauric acid and also activated 2-aminofluorene (2-AF), which is a typical substrate for CYP4B1, to mutagenic compounds detected by an umu gene expression assay. These activities observed in transgenic mouse were efficiently inhibited by CYP4B1 antibody. However, such inhibition was not observed in control mice. This is the first report to indicate catalytic activities of human CYP4B1. For further characterization of human CYP4B1, a fusion protein of CYP4B1 and NADPH-P450 reductase was expressed in yeast cells. It was able to activate 2-AF and was also able to catalyze omega-hydroxylation of lauric acid. This transgenic mouse line and the recombinant fusion protein provide a useful tool to study human CYP4B1 and its relation to chemical toxicity and carcinogenesis.

A novel cytochrome P450 enzyme responsible for the metabolism of ebastine in monkey small intestine.

Small intestinal microsomes of cynomolgus monkeys were found to catalyze hydroxylation and dealkylation of an H(1)-antihistamine prodrug, ebastine. To identify the main enzyme responsible for ebastine hydroxylation, which has been hitherto unknown, we purified two cytochrome P450 isoforms, named P450 MI-2 and P450 MI-3, from the intestinal microsomes on the basis of the hydroxylation activity. P450 MI-2 and P450 MI-3 showed the respective apparent molecular weights of 56,000 and 53,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The internal amino acid sequence of P450 MI-2 had high similarity with those of human CYP4F2, CYP4F3, and CYP4F8. The first 27 amino acid residues of P450 MI-3 were highly homologous with those of monkey CYP3A8 and human CYP3A4/5/7. Furthermore, P450 MI-2 and P450 MI-3 were recognized by anti-CYP4F and anti-CYP3A antibodies, respectively, in immunoblot analysis and catalyzed leukotriene B(4) omega-hydroxylation and testosterone 6beta-hydroxylation, which are known to be mediated by CYP4F and CYP3A, respectively. Although both enzymes had ebastine hydroxylation activity, the V(max) value of P450 MI-2 was much higher than that of P450 MI-3 (37.0 versus 0.406 nmol/min/nmol of P450), and the former K(M) (5.1 microM) was smaller than the latter K(M) (10 microM). Anti-CYP4F antibody inhibited the hydroxylation in small intestinal microsomes strongly (70%), but anti-CYP3A antibody did not. These results indicate that P450 MI-2 belongs to the CYP4F subfamily and is mainly responsible for hydroxylation of ebastine in monkey small intestinal microsomes. This suggests that the small intestinal CYP4F enzyme, P450 MI-2, can play an important role in the metabolism of drugs given orally.

Androgen regulation of CYP4B1 responsible for mutagenic activation of bladder carcinogens in the rat bladder: detection of CYP4B1 mRNA by competitive reverse transcription-polymerase chain reaction.

Significant sex differences exist among cases of bladder cancer in humans as well as in experimental animals such as rats. Aromatic amines such as benzidine and 2-naphthylamine are known to induce bladder cancer. These carcinogenic amines are activated to genotoxic substances by cytochrome P 450 CYP4B1, which is present in bladder mucosa. In this study, regulation of CYP4B1 was investigated to elucidate sex difference in bladder carcinogenesis. Competitive reverse transcription-polymerase chain reaction was used to investigate the expression of rat CYP4B1 mRNA occurring in small amounts of tissue such as bladder tissue. Expression of CYP4B1 in the bladder of male rats increased with development but not in that of female rats. Moreover, mature male rats exhibited higher expression of CYP4B1 in the bladder than did mature female rats. Castration of male rats decreased CYP4B1 levels and treatment with testosterone led to a partial recovery of CYP4B1 levels. These results indicate that CYP4B1 levels in the rat bladder are partly regulated by androgens. Furthermore, the present findings suggest that the sex difference observed in bladder carcinogenesis was due to sex-different expression of CYP4B1 in bladder tissue.

Involvement of human liver cytochrome P4502B6 in the metabolism of propofol.

AIMS: To determine the cytochrome P450 (CYP) isoforms involved in the oxidation of propofol by human liver microsomes. METHODS: The rate constant calculated from the disappearance of propofol in an incubation mixture with human liver microsomes and recombinant human CYP isoforms was used as a measure of the rate of metabolism of propofol. The correlation of these rate constants with rates of metabolism of CYP isoform-selective substrates by liver microsomes, the effect of CYP isoform-selective chemical inhibitors and monoclonal antibodies on propofol metabolism by liver microsomes, and its metabolism by recombinant human CYP isoforms were examined. RESULTS: The mean rate constant of propofol metabolism by liver microsomes obtained from six individuals was 4.2 (95% confidence intervals 2.7, 5.7) nmol min(-1) mg(-1) protein. The rate constants of propofol by microsomes were significantly correlated with S-mephenytoin N-demethylation, a marker of CYP2B6 (r = 0.93, P < 0.0001), but not with the metabolic activities of other CYP isoform-selective substrates. Of the chemical inhibitors of CYP isoforms tested, orphenadrine, a CYP2B6 inhibitor, reduced the rate constant of propofol by liver microsomes by 38% (P < 0.05), while other CYP isoform-selective inhibitors had no effects. Of the recombinant CYP isoforms screened, CYP2B6 produced the highest rate constant for propofol metabolism (197 nmol min-1 nmol P450-1). An antibody against CYP2B6 inhibited the disappearance of propofol in liver microsomes by 74%. Antibodies raised against other CYP isoforms had no effect on the metabolism of propofol. CONCLUSIONS: CYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes.

Presence of a no-observed effect level for enhancing effects of development of the alpha-isomer of benzene hexachloride (alpha-BHC) on diethylnitrosamine-initiated hepatic foci in rats.

The dose dependence of the promoting effects of the alpha-isomer of benzene hexachloride (alpha-BHC) on hepatocarcinogenesis was investigated in a medium-term rat liver bioassay (Ito test). A total of 195 F344 male rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at the start of the experiment and subjected to two-thirds partial hepatectomy at week 3. Two weeks after the administration of DEN, alpha-BHC were fed to rats at doses of 0, 0.01, 0.1, 0.5, 1, 2, 4, 7.5, 15, 30, 60, 125 and 500 ppm in diet for 6 weeks. All surviving animals were killed at week 8, and their livers were examined immunohistochemically for detection of glutathione S-transferase placental form (GST-P)-positive foci, surrogate preneoplastic lesions. Quantitative values for numbers and areas were dose-dependently increased in rats given alpha-BHC at 0.5-500 ppm. However, those for groups treated with 0.01 and 0.1 ppm were decreased, albeit not significantly in comparison to the controls. Cytochrome P450 3A2 (CYP3A2) protein levels and activities showed a good correlation to the number and area of GST-P-positive foci. These results support evidence of hormesis and indicate a no-observed effect level for alpha-BHC promoting potentials may exist regarding rat liver carcinogenesis, which correlates with expression of CYP3A2 in the liver.

cDNA cloning and expression of a novel cytochrome p450 (cyp4f12) from human small intestine.

A cDNA encoding a novel human CYP4F enzyme (designated CYP4F12) was cloned by PCR from a human small intestine cDNA library. RT-PCR analysis demonstrated that CYP4F12 is expressed in human small intestine and liver. This cDNA contains an entire coding region of a 524-amino-acid protein that is 81.7, 78.3, and 78.2% identical to CYP4F2, CYP4F3, and CYP4F8, respectively. When expressed in Saccharomyces cerevisiae, the P450 catalyzes leukotriene B(4) omega-hydroxylation and arachidonic acid omega-hydroxylation, typical reactions of CYP4F isoforms. Their activity levels are, however, much lower than those of CYP4F2. Interestingly, CYP4F12 catalyzes the hydroxylation of the antihistamine ebastine with significantly higher catalytic activity relative to CYP4F2 (385 vs 5 pmol/min/nmol P450). These results indicate that CYP4F12 has a different profile of substrate specificity from other CYP4F isoforms, enzymes responsible for metabolizing endogenous autacoids, therefore suggesting that it may play an important role in xenobiotic biotransformation in the human small intestine.

CYP2C9*3 influences the metabolism and the drug-interaction of candesartan in vitro.

Candesartan cilexetil is an angiotensin II receptor antagonist, and candesartan, its active metabolite, is metabolized by CYP2C9. However, the effect of CYP2C9*3 on candesartan metabolism is not established. We characterized the kinetics of candesartan by CYP2C9*1/*1 and CYP2C9*1/*3 in human liver microsomes. The difference between the two was not significant. Subsequently, CYP2C9*1 and CYP2C9*3 (Leu359) were expressed in yeast, and the kinetics of candesartan were determined. The wild-type showed the lower Km (345 vs 439 microM; 3/4) and higher Vmax/Km (1/3) than the Leu359 variant. Also, we investigated potential interaction between candesartan and warfarin with both the wild-type and the Leu359 variant. Candesartan had no effect on S-warfarin 7-hydroxylation. In contrast, S-warfarin inhibited candesartan metabolism by the wild-type (K = 17microM) greater than by the Leu359 variant (Ki = 36 microM). These findings suggest that CYP2C9*3 may change not only the metabolic activity but also the inhibitory susceptibility compared with CYP2C9*1.

CYP4B1 is a possible risk factor for bladder cancer in humans.

In experimental animals such as rats and rabbits, CYP4B1 has an important role in mutagenic activation of procarcinogens in bladders. In human bladders, it is not clear whether CYP4B1 has such role or not. In the present study, human bladder microsomes activated 2-aminofluorene which is a typical substrate for CYP4B1 and is a bladder carcinogen. CYP4B1 was detected in the human bladder microsomes by immunoblotting. Furthermore, we developed a microassay for CYP4B1 mRNA by performing real-time RT-PCR. Using this method, CYP4B1 mRNA levels were assayed in transurethal resection samples from the bladders of patients with bladder tumors. The bladder-tumor patients had a significantly higher expression of CYP4B1 than the nonbladder tumor patients. These findings suggest that a high expression of CYP4B1 increases the risk of bladder tumor by activation of carcinogenic aromatic amines. This approach could be an important tool in the assessment of human bladder cancer risk.

Chemopreventive effects of coumaperine from pepper on the initiation stage of chemical hepatocarcinogenesis in the rat.

This study was designed to investigate the chemopreventive action of three natural products, coumaperine, aurapten and an extract from rosemary, against the initiation stage of rat hepato-carcinogenesis. Coumaperine has been isolated from white pepper as a naturally occurring antioxidative agent, but its potential modifying effects on carcinogenesis remain unclear. In experiment 1, a modification of the model developed by Tsuda et al. was applied, with assessment of numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci in male F344 rats. Coumaperine, aurapten and the extract from rosemary were administered i.g. at 100 mg / kg / day once daily for 5 days with initiation by diethylnitrosamine (DEN) on day 4 (20 mg / kg, i.p.). Numbers and areas of GST-P-positive foci in each group given test chemicals tended to be decreased as compared to the vehicle control group values, significance being achieved for number with coumaperine. Experiment 2 was planned to investigate the mechanism of the inhibitory effects of coumaperine. Livers at 8 h after initiation by DEN were examined with coumaperine administered at 100 mg / kg / day once daily for 3 days. Proliferating cell nuclear antigen (PCNA)-positive cells tended to be decreased as compared to the vehicle control, but no effects on apoptosis or cytochrome P-450 (CYP) 2E1 expression were apparent. Our results suggest that coumaperine provides protection against initiation of hepatocarcinogenesis, and that this is related to inhibition of cell proliferation.

The decreased in vivo clearance of CYP2D6 substrates by CYP2D6*10 might be caused not only by the low-expression but also by low affinity of CYP2D6.

CYP2D6 exhibits genetic polymorphism with interindividual differences in metabolic activity. We have found a significant influence on the pharmacokinetics of venlafaxine by the CYP2D6*10 allele in a Japanese population. CYP2D6.10, which is translated from CYP2D6*10, has two amino acid substitutions: Pro34 --> Ser and Ser486 --> Thr. In this study, CYP2D6.10 was expressed in Saccharomyces cerevisiae and its catalytic activity for CYP2D6 substrates was investigated. The CYP2D6*10B- and *10C-associated cDNA were isolated from human lymphocyte genotyped as CYP2D6*10. In addition, three forms of CYP2D6, Pro34/Thr486 (PT), Ser34/Ser486 (SS), and Pro34/Ser486 (wild type, CYP2D6.1), were constructed by PCR-site mutagenesis to clarify the effects of the two amino-acid substitutions. The expression of CYP2D6 protein was confirmed by immunoblotting using CYP2D antibody. The absorbance at 450 nm was measured by CO-reduced difference spectra from five all microsome preparations. The CYP2D6 forms with Pro34 --> Ser amino acid substitution were at a lower expression than CYP2D6.1 from the findings of immunoblotting and spectral analysis. The apparent K(m) values of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 1.7, 8.5, and 49.7 microM, respectively, for bufuralol 1'-hydroxylation, and 9.0, 51.9, and 117.4 microM, respectively, for venlafaxine O-demethylation, respectively. The V(max) values were not significantly different among the three variants. These findings suggest that the decreased in vivo clearance by CYP2D6*10 was caused not only by low expression of but also the increased K(m) value of CYP2D6.

Area under the plasma concentration-time curve of inorganic fluoride following sevoflurane anesthesia correlates with CYP2E1 mRNA level in mononuclear cells.

BACKGROUND: Because the amount of inorganic fluoride released after anesthesia with sevoflurane depends on the dose of administered sevoflurane and cytochrome P450 (CYP) 2E1 activity in the liver, a reliable and noninvasive probe for CYP2E1 would be useful for predicting plasma inorganic fluoride levels after anesthesia. In this study, the authors evaluated the relation between plasma concentration of inorganic fluoride after sevoflurane anesthesia and CYP2E1 mRNA level in mononuclear cells. METHODS: Twenty patients (American Society of Anesthesiologists physical status I), aged 20-68 yr undergoing body surface surgery with general anesthesia with sevoflurane were enrolled. One milliliter of blood was obtained before administration of sevoflurane and mononuclear cells were obtained. Levels of CYP2E1 mRNA in mononuclear cells were measured by competitive reverse transcription polymerase chain reaction with a specific primer and competitor for CYP2E1 mRNA. RESULTS: There was a significant correlation between level of CYP2E1 mRNA in mononuclear cells and the area under the plasma concentration-time curve of plasma inorganic fluoride from the beginning of sevoflurane administration to infinity in uninduced and uninhibited patients (r2 = 0.56; P < 0.01). CONCLUSIONS: Area under the plasma concentration-time curve of inorganic fluoride after sevoflurane anesthesia correlates with CYP2E1 mRNA in mononuclear cells in peripheral blood.