Omeprazole limited sampling strategies to predict area under the concentration-time curve ratios: implications for cytochrome P450 2C19 and 3A phenotyping.

PURPOSE: To develop a limited sampling strategy (LSS) to predict area under the concentration-time curve (AUC) ratios of omeprazole (AUC(OPZ)) to its metabolites 5-hydroxyomeprazole (AUC(5OH)) and omeprazole sulfone (AUC(SUL)) as phenotyping parameters for cytochrome P450 (CYP) 2C19 and 3A. METHODS: Data were obtained from 37 (4 women) Caucasian, Chinese, and Korean healthy adults from three published studies. The AUC(OPZ), AUC(5OH), and AUC(SUL) were calculated via noncompartmental analysis. Observed AUC(OPZ, OBS)/AUC(5OH, OBS) and AUC(OPZ, OBS)/AUC(SUL, OBS) were determined. Plasma concentrations of omeprazole, 5-hydroxyomeprazole, and omeprazole sulfone at 1, 1.5, 2, 3, 4, 6, and 8 h post-dose were used to generate limited sampling strategy (LSS) models to predict AUC(OPZ,PRE)/AUC(5OH,PRE) and AUC(OPZ,PRE/)AUC(SUL,PRE). Bias and precision were assessed via percentage mean prediction error (%MPE) and percentage mean absolute error (%MAE), with acceptable limits being <15%. RESULTS: For CYP2C19, the AUC(OPZ,OBS)/AUC(5OH,OBS) was [mean ± standard deviation (SD)] 2.10 ± 1.63. Five LSS models of AUC(OPZ,PRE)/AUC(5OH,PRE) were generated, but none met the bias or precision criteria. Upon stratification by CYP2C19 genotype and ethnicity, a three-timepoint (at 1, 2, and 4 h) LSS model accurately predicted AUC(OPZ)/AUC(5OH) in Caucasian CYP2C19*1/*1 subjects. For CYP3A, AUC(OPZ,OBS)/AUC(SUL,OBS) (mean ± SD) was 1.79 ± 0.67. All LSS models had unacceptable %MAE, even when stratified by CYP2C19 genotype and ethnicity. CONCLUSIONS: A LSS model to predict AUC(OPZ)/AUC(5OH), and thus CYP2C19 activity, was generated for Caucasian CYP2C19*1/*1 subjects. However, additional model validation is needed prior to general use. LSS models to predict AUC(OPZ)/AUC(SUL), and thus CYP3A activity, were not possible, even upon stratification by CYP2C19 genotype and ethnicity.

Pharmacokinetics of Ampreloxetine, a Norepinephrine Reuptake Inhibitor, in Healthy Subjects and Adults with Attention-Deficit/Hyperactive Disorder or Fibromyalgia Pain.

BACKGROUND AND OBJECTIVE: Ampreloxetine is a novel norepinephrine reuptake inhibitor in development for the treatment of symptomatic neurogenic orthostatic hypotension. The objectives of this analysis were to define the pharmacokinetics of once-daily oral ampreloxetine and provide dose recommendations for clinical development. METHODS: We fitted a population pharmacokinetic model to ampreloxetine plasma concentrations from single- and multiple-ascending dose trials in healthy subjects and two phase II studies in adult subjects with attention-deficit/hyperactive disorder or fibromyalgia at doses of 2-50 mg. RESULTS: Ampreloxetine pharmacokinetics was best described by a two-compartment model with first-order absorption and elimination. The terminal half-life was 30-40 h, resulting in sustained drug concentrations for the entire 24-h dosing interval at steady state. Covariates of age, weight, or renal impairment did not impact ampreloxetine exposure. Cytochrome P450 2D6 phenotype had no influence on ampreloxetine exposure. Sex and smoking status were identified as statistically significant covariates, suggesting a role for cytochrome P450 1A2 in the elimination of ampreloxetine. Despite statistical significance, differences in ampreloxetine exposure in male vs female subjects and smokers vs non-smokers were not clinically meaningful at the recommended dose. At the 10-mg dose, > 75% norepinephrine transporter inhibition and < 50% serotonin transporter inhibition are anticipated for adult subjects. CONCLUSIONS: The population pharmacokinetic model effectively described the plasma concentration-time profile of ampreloxetine after single and multiple doses. Population pharmacokinetic/pharmacodynamic analysis justified using a fixed dosing regimen with no dose adjustments across a broad population and can be used to inform dosing strategies in future clinical studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier numbers NCT01693692 (fibromyalgia); NCT01458340 (attention-deficit/hyperactive disorder).

Safety, Pharmacokinetics, and Pharmacodynamics of TD-0714, a Novel Potent Neprilysin Inhibitor in Healthy Adult and Elderly Subjects.

TD-0714 is an orally active, potent, and selective inhibitor of human neprilysin (NEP) in development for the treatment of chronic heart failure. Oral administration of TD-0714 in rats resulted in dose-dependent and sustained increases in plasma cyclic guanosine monophosphate (cGMP) over 24 hours consistent with NEP target engagement. Randomized, double-blind, placebo controlled, single ascending dose (50-600 mg TD-0714) and multiple ascending dose (10-200 mg TD-0714 q.d. for 14 days) studies were conducted in healthy volunteers. TD-0714 was generally well-tolerated and no serious adverse events or clinically significant effects on vital signs or electrocardiogram parameters were observed. TD-0714 exhibited dose-proportional pharmacokinetics (PKs) with high oral bioavailability, minimal accumulation after once daily dosing, and negligible renal elimination. Pharmacodynamic (PD) responses were observed at all dose levels studied, as reflected by statistically significant increases in plasma cGMP concentrations. The increases in cGMP were significantly above the baseline (~ 50-100%) on day 14 for the entire 24-hour interval indicating that sustained cGMP elevations are achieved at steady-state. Maximal steady-state cGMP response was observed in plasma and urine at doses ≥ 50 mg. The TD-0714 PK-PD relationship and safety profile were similar in elderly vs. younger adult subjects. The TD-0714 PK and PD profiles support further clinical development of TD-0714 and suggest the potential for once-daily administration and predictable exposure in patients with cardiorenal diseases regardless of their renal function.

Regulation of human CYP2C18 and CYP2C19 in transgenic mice: influence of castration, testosterone, and growth hormone.

The hormonal regulation of human CYP2C18 and CYP2C19, which are expressed in a male-specific manner in liver and kidney in a mouse transgenic model, was examined. The influence of prepubertal castration in male mice and testosterone treatment of female mice was investigated, as was the effect of continuous administration of growth hormone (GH) to transgenic males. Prepubertal castration of transgenic male mice suppressed the expression of CYP2C18 and CYP2C19 in liver and kidney to female levels, whereas expression was increased for the endogenous female-specific mouse hepatic genes Cyp2c37, Cyp2c38, Cyp2c39, and Cyp2c40. Testosterone treatment of female mice increased CYP2C18 and CYP2C19 expression in kidney, and to a lesser extent in liver, but was without effect in brain or small intestine, where gene expression was not gender-dependent. Continuous GH treatment of transgenic males for 7 days suppressed hepatic expression of CYP2C19 (>90% decrease) and CYP2C18 ( approximately 50% decrease) but had minimal effect on the expression of these genes in kidney, brain, or small intestine. Under these conditions, continuous GH induced all four female-specific mouse liver Cyp2c genes in males to normal female levels. These studies indicate that the human CYP2C18 and CYP2C19 genes contain regulatory elements that respond to the endogenous mouse hormonal profiles, with androgen being the primary regulator of male-specific expression in kidney, whereas the androgen-dependent pituitary GH secretory pattern is the primary regulator of male-specific expression in liver in a manner that is similar to the regulation of the endogenous gender-specific hepatic genes.

Generation of mice transgenic for human CYP2C18 and CYP2C19: characterization of the sexually dimorphic gene and enzyme expression.

CYP2C19 is an important enzyme for human drug metabolism, and it also participates in the metabolism of endogenous substrates, whereas the CYP2C18 enzyme is not expressed in human liver despite high mRNA expression. Mice transgenic for the human CYP2C18 and CYP2C19 genes were generated. Quantitative mRNA analysis showed CYP2C18 and CYP2C19 transcripts in liver, kidneys, and heart to be expressed in a sexually dimorphic manner, with male mice having 2- to 100-fold higher levels. Transcript levels in the small intestine were somewhat higher than liver but were similar in both sexes. Transgene mRNA expression was much lower in lung and brain and least in the heart. Immunoblotting using an antipeptide antiserum, reactive with human CYP2Cs and mouse CYP2C70, revealed increased immunoreactive protein in liver microsomes from heterozygous transgenic male mice and a concomitant increase in 5'-hydroxylation of R-omeprazole and S-mephenytoin intrinsic clearance, consistent with CYP2C19 overexpression. A CYP2C18-specific antiserum showed that this enzyme was not expressed in livers or kidneys from heterozygous transgenic mice, but the antiserum had high affinity for recombinant CYP2C18 expressed in COS-7 cells. It is concluded that 1) both the CYP2C18 and CYP2C19 genes are subject to sexually dimorphic regulation in murine liver, kidney, and heart; 2) the CYP2C18 protein is not expressed in murine liver or kidney despite high levels of the corresponding mRNA; and 3) this transgenic model may be suitable for studying sex-dependent regulation of the human CYP2C genes and possibly serve as an in vivo model for CYP2C19-dependent drug metabolism.

Increased omeprazole metabolism in carriers of the CYP2C19*17 allele; a pharmacokinetic study in healthy volunteers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: The only existing study of CYP2C19*17-associated alterations in drug pharmacokinetics was retrospective and compared probe drug metabolic ratios. The CYP2C19*17 allele had been associated with a two- and fourfold decrease in omeprazole and S/R-mephenytoin metabolic ratios. WHAT THIS STUDY ADDS: This study characterized the single-dose pharmacokinetics of omeprazole, along with the 5-hydroxy and sulphone metabolites, in CYP2C19*17/*17 and CYP2C19*1/*1 subjects. The observed differences in omeprazole AUC(infinity) suggest that the CYP2C19*17 allele is an important explanatory factor behind individual cases of therapeutic failure. AIMS To investigate the influence of the CYP2C19*17 allele on the pharmacokinetics of omeprazole, a commonly used CYP2C19 probe drug, in healthy volunteers. METHODS: In a single-dose pharmacokinetic study, 17 healthy White volunteers genotyped as either CYP2C19*17/*17 or CYP2C19*1/*1 received an oral dose of 40 mg of omeprazole. Plasma was sampled for up to 10 h postdose, followed by quantification of omeprazole, 5-hydroxy omeprazole and omeprazole sulphone by high-performance liquid chromatography. RESULTS: The mean omeprazole AUC(infinity) of 1973 h nmol l(-1) in CYP2C19*17/*17 subjects was 2.1-fold lower [95% confidence interval (CI) 1.1, 3.3] than in CYP2C19*1/*1 subjects (4151 h nmol l(-1), P = 0.04). A similar trend was observed for the sulphone metabolite with the CYP2C19*17/*17 group having a mean AUC(infinity) of 1083 h nmol l(-1), 3.1-fold lower (95% CI 1.2, 5.5) than the CYP2C19*1/*1 group (3343 h nmol l(-1), P = 0.03). A pronounced correlation (r(2) = 0.95, P < 0.0001) was seen in the intraindividual omeprazole AUC(infinity) and omeprazole sulphone AUC(infinity) values. CONCLUSIONS: The pharmacokinetics of omeprazole and omeprazole sulphone differ significantly between homozygous CYP2C19*17 and CYP2C19*1 subjects. For clinically important drugs that are metabolized predominantly by CYP2C19, the CYP2C19*17 allele might be associated with subtherapeutic drug exposure.

Kinetics of omeprazole and escitalopram in relation to the CYP2C19*17 allele in healthy subjects.

PURPOSE: Ultrarapid drug metabolism of antidepressants has been associated with therapeutic failures. The CYP2C19*17 allele has been associated with higher levels of CYP2C19 gene transcription and increased rates of omeprazole and mephenytoin metabolism. The aim of this study was to compare the impact of the CYP2C19*17 allele on omeprazole single-dose kinetics with escitalopram exposure at steady state in volunteers genotyped as either CYP2C19*17/*17 or CYP2C19*1/*1. METHODS: Sixteen healthy volunteers participated in both study parts, five homozygous for CYP2C19*17 and 11 homozygous for CYP2C19*1. Individual pharmacokinetic parameters were determined after single-dose omeprazole of 40 mg and after 1 week on escitalopram 5 mg b.i.d. RESULTS: Escitalopram area under the concentration time curve from zero to 12 h (AUC(0-12h)) was 21% lower in homozygous carriers of CYP2C19*17 compared with CYP2C19*1 (p = 0.08). There was a significant correlation between escitalopram exposure at steady state and the single-dose kinetics of omeprazole (Spearman correlation coefficient of 0.67; p = 0.006). CONCLUSION: Based on our investigation using two different CYP2C19 substrates, we concluded that a clinically significant difference in escitalopram or omeprazole kinetics between the genotypes appears unlikely.

A genome-wide association study identifies novel loci for paclitaxel-induced sensory peripheral neuropathy in CALGB 40101.

PURPOSE: Sensory peripheral neuropathy is a common and sometimes debilitating toxicity associated with paclitaxel therapy. This study aims to identify genetic risk factors for the development of this toxicity. EXPERIMENTAL DESIGN: A prospective pharmacogenetic analysis of patients with primary breast cancer, randomized to the paclitaxel arm of CALGB 40101, was used to identify genetic predictors of the onset and severity of sensory peripheral neuropathy. A genome-wide association study in 855 subjects of European ancestry was conducted and findings were replicated in additional European (n = 154) and African American (n = 117) subjects. RESULTS: A single nucleotide polymorphism in FGD4 was associated with the onset of sensory peripheral neuropathy in the discovery cohort [rs10771973; HR, 1.57; 95% confidence interval (CI), 1.30-1.91; P = 2.6 × 10(-6)] and in a European (HR, 1.72; 95% CI, 1.06-2.80; P = 0.013) and African American (HR, 1.93; 95% CI, 1.13-3.28; P = 6.7 × 10(-3)) replication cohort. There is also evidence that markers in additional genes, including EPHA5 (rs7349683) and FZD3 (rs10771973), were associated with the onset or severity of paclitaxel-induced sensory peripheral neuropathy. CONCLUSIONS: A genome-wide association study has identified novel genetic markers of paclitaxel-induced sensory peripheral neuropathy, including a common polymorphism in FGD4, a congenital peripheral neuropathy gene. These findings suggest that genetic variation may contribute to variation in development of this toxicity. Validation of these findings may allow for the identification of patients at increased risk of peripheral neuropathy and inform the use of an alternative to paclitaxel and/or the clinical management of this toxicity.

Toxicity and metabolism of methylnaphthalenes: comparison with naphthalene and 1-nitronaphthalene.

Naphthalene and close structural analogues have been shown to cause necrosis of bronchiolar epithelial cells in mice by both inhalation exposure and by systemic administration. Cancer bioassays of naphthalene in mice have demonstrated a slight increase in bronchiolar/alveolar adenomas in female mice, and in inflammation and metaplasia of the olfactory epithelium in the nasal cavity. Similar work in rats demonstrated a significant, and concentration-dependent increase in the incidence of respiratory epithelial adenomas and neuroblastomas in the nasal epithelium of both male and female rats. Although the studies on the acute toxicity of the methylnaphthalene derivatives are more limited, it appears that the species selective toxicity associated with naphthalene administration also is observed with methylnaphthalenes. Chronic administration of the methylnaphthalenes, however, failed to demonstrate the same oncogenic potential as that observed with naphthalene. The information available on the isopropylnaphthalene derivatives suggests that they are not cytotoxic. Like the methylnaphthalenes, 1-nitronaphthalene causes lesions in both Clara and ciliated cells. However, the species selective lung toxicity observed in the mouse with both naphthalene and the methylnaphthalenes is not seen with 1-nitronaphthalene. With 1-nitronaphthalene, the rat is far more susceptible to parenteral administration of the compound than mice. The wide-spread distribution of these compounds in the environment and the high potential for low level exposure to humans supports a need for further work on the mechanisms of toxicity in animal models with attention to whether these processes are applicable to humans. Although it is tempting to suppose that the toxicity and mechanisms of toxicity of the alkylnaphthalenes and nitronaphthalenes are similar to naphthalene, there is sufficient published literature to suggest that this may not be the case. Certainly the enzymes involved in the metabolic activation of each of these substrates are likely to differ. The available data showing extensive oxidation of the aromatic nucleus of naphthalene, nitronaphthalene and the methylnaphthalenes (with some oxidation of the methyl group) contrast with the isopropylnaphthalene derivatives, where the major metabolites involve side chain oxidation. Overall, these data support the view that ring epoxidation is a key step in the process involved in cytotoxicity. Whether the epoxide itself or a downstream metabolite mediates the toxic effects is still not clear even with naphthalene, the best studied of this group of compounds. Additional work is needed in several areas to further assess the potential human health consequences of exposure to these agents. These studies should involve the definition of the extent and severity of methylnaphthalene toxicity after single dose exposures with attention to both the nasal and respiratory epithelia. The cytochromes P450 responsible for the initial activation of these agents in rodents with subsequent complimentary studies in primate models should help determine whether key metabolic processes responsible for toxicity occur also in primates. Finally, the precise involvement of reactive metabolite formation and adduction of cellular proteins in toxicity will be important in not only assessing the potential for human toxicity, but also in developing an understanding of the genetic and environmental factors which could alter the toxicity of these agents.

Bioactivation of the pulmonary toxicants naphthalene and 1-nitronaphthalene by rat CYP2F4.

Naphthalene, a ubiquitous environmental contaminant, produces cytotoxicity in nonciliated bronchiolar epithelial (Clara) cells in mice; rats are refractory to lung cytotoxicity from naphthalene. In contrast, 1-nitronaphthalene is a potent toxicant in both species. Naphthalene is metabolized by CYP2F to a 1,2-epoxide, the first and obligate step in events leading to cytotoxicity. 1-Nitronaphthalene is metabolized to both the 5,6- and the 7,8-epoxides with the 7,8-epoxide predominating in lung. Previous studies have demonstrated recombinant CYP2F2 (mouse) to efficiently metabolize both naphthalene and 1-nitronaphthalene. To better understand the mechanism for the unique toxicity profiles for both compounds, a CYP2F ortholog (CYP2F4) was isolated from rat lung and expressed using a baculovirus system. Recombinant CYP2F4 efficiently generates 1R,2S-naphthalene oxide (K(m) = 3 microM, V(max) = 107 min(-1)) and the 5,6- and 7,8-epoxides of 1-nitronaphthalene (K(m) = 18 microM, V(max) = 25 min(-1) based on total generated glutathione conjugates). Kinetics and regio/stereoselectivity of rat CYP2F4 were indistinguishable from mouse CYP2F2. These results, combined with our recent immunomapping studies demonstrating minimal pulmonary CYP2F expression in rats, indicate that CYP2F expression is the factor most clearly associated with susceptibility to naphthalene-induced pneumotoxicity. CYP2F4 failed to display an enhanced ability to bioactivate 1-nitronaphthalene, an ability that could have potentially compensated for the lower CYP2F pulmonary expression levels in the rat, yet equal species susceptibilities. These results suggest the importance of other P450 enzymes in the epoxidation/bioactivation of 1-nitronaphthalene. Expression of recombinant CYP2F1 (human) yielded an immunoreactive protein with no detectable CO-difference spectrum suggesting inadequate heme incorporation.

Pulmonary bioactivation of trichloroethylene to chloral hydrate: relative contributions of CYP2E1, CYP2F, and CYP2B1.

Pulmonary cytotoxicity induced by trichloroethylene (TCE) is associated with cytochrome P450-dependent bioactivation to reactive metabolites. In this investigation, studies were undertaken to test the hypothesis that TCE metabolism to chloral hydrate (CH) is mediated by cytochrome P450 enzymes, including CYP2E1, CYP2F, and CYP2B1. Recombinant rat CYP2E1 catalyzed TCE metabolism to CH with greater affinity than did the recombinant P450 enzymes, rat CYP2F4, mouse CYP2F2, rat CYP2B1, and human CYP2E1. The catalytic efficiencies of recombinant rat CYP2E1 (V(max)/K(m) = 0.79) for generating CH was greater than those of recombinant CYP2F4 (V(max)/K(m) = 0.27), recombinant mouse CYP2F2 (V(max)/K(m) = 0.11), recombinant rat CYP2B1 (V(max)/K(m) = 0.07), or recombinant human CYP2E1 (V(max)/K(m) = 0.02). Decreases in lung microsomal immunoreactive CYP2E1, CYP2F2, and CYP2B1 were manifested at varying time points after TCE treatment. The loss of immunoreactive CYP2F2 occurred before the loss of immunoreactive CYP2E1 and CYP2B1. These protein decreases coincided with marked reduction of lung microsomal p-nitrophenol hydroxylation and pentoxyresorufin O-dealkylation. Rates of CH formation in the microsomal incubations were time-dependent and were incremental from 5 to 45 min. The production of CH was also determined in human lung microsomal incubations. The rates were low and were detected in only three of eight subjects. These results showed that, although CYP2E1, CYP2F, and CYP2B1 are all capable of generating CH, TCE metabolism is mediated with greater affinity by recombinant rat CYP2E1 than by recombinant CYP2F, CYP2B1, or human CYP2E1. Moreover, the rates of CH production were substantially higher in murine than in human lung.

Comparison of pulmonary/nasal CYP2F expression levels in rodents and rhesus macaque.

Naphthalene is a ubiquitous environmental contaminant that results in dose-dependent and tissue-, species-, and cell-selective necrosis of murine Clara cells upon exposure. Naphthalene is metabolized by CYP2F to a 1,2-epoxide, the first and obligate step in events leading to cytotoxicity. The studies reported here examine the relationship between levels of transcript (mRNA) and CYP2F protein in the respiratory tract of rodents with tissue susceptibility to injury. In both mice and rats, the lung contains more CYP2F transcript than liver; levels in kidney were undetectable. Mice expressed 4- and 8-fold greater CYP2F transcript in lung and liver tissue, respectively, than rats. Quantitative immunoblot blot analysis of CYP2F in airway subcompartments revealed mice to have 30- (minor daughters/terminal bronchioles), 20- (major daughter), 40- (trachea), and 6- (parenchyma) fold higher levels of CYP2F protein than rats. Within the lungs of both rodent species, the highest CYP2F expression was found in the distal airways. The kidney contained undetectable amounts of CYP2F; multiple immunoreactive bands in liver precluded quantification. The olfactory epithelium contains the greatest amount of cytochrome P450 protein of all tissues studied in the rat, consistent with the observed pattern of in vivo injury. Overall, these studies in rodents demonstrate a strong association between CYP2F expression levels and susceptibility to naphthalene-induced cytotoxicity. Of all primate tissues studied, only the nasal ethmoturbinates contain quantifiable amounts of CYP2F, roughly 10- and 20-fold less than the corresponding tissues in rats and mice, respectively. These results suggest that rhesus macaques may be refractory to naphthalene-induced pulmonary injury.

Bioactivation of 1,1-dichloroethylene to its epoxide by CYP2E1 and CYP2F enzymes.

1,1-Dichloroethylene (DCE) exposure to mice elicits lung toxicity that selectively targets bronchiolar Clara cells. The toxicity is mediated by DCE metabolites formed via cytochrome P450 metabolism. The primary metabolites formed are DCE epoxide, 2,2-dichloroacetaldehyde, and 2-chloroacetyl chloride. The major metabolite detected is 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. In this investigation, studies were undertaken to test the hypothesis that CYP2E1 and CYP2F2 are involved in bioactivation of DCE to the epoxide in murine lung. We have developed a method using liquid chromatography/mass spectrometry (LC/MS) to evaluate the kinetics of the rates of production of conjugate [C] by recombinant CYP2E1 and CYP2F enzymes and lung microsomes. Concentration-dependent formation of conjugate [C] was found in incubations of DCE with recombinant CYP2E1 and CYP2F enzymes and lung microsomes from CD-1, wild-type (mixed 129/Sv and C57BL), and CYP2E1-null mice. Recombinant rat CYP2E1 exhibited greater affinity and catalytic efficiency for DCE metabolism than did recombinant human CYP2E1, mouse CYP2F2, goat CYP2F3 or rat CYP2F4. In the lung microsomal incubations, the rates of conjugate [C] production were higher in CD-1 mice than in either wild-type or CYP2E1-null mice; the level of [C] in CYP2E1-null mice was about 66% of that in wild-type mice. These results demonstrated that LC/MS analysis is a suitable method for detection and quantitation of conjugate [C], and that CYP2E1 and CYP2F2 catalyze the bioactivation of DCE to the epoxide in murine lung. The results also demonstrated that CYP2E1 is the high-affinity enzyme involved in DCE bioactivation.