Physiological Significance and Expression of P450s in the Developing Eye

Expression of 10 CYP orthologs (Families 1–3) in developing mouse conceptus is constitutive. These forms have specific temporal and spatial expression. Studies on CYP1B1 indicate its requirement for normal eye development, both in human and mouse. The distribution of the enzyme in the mouse eye is in three regions, which may reflect three different, perhaps equally important, functions in this organ. Its presence in the inner ciliary and lens epithelia appears to be necessary for normal development of the trabecular meshwork and its function in regulating intraocular pressure. Its expression in the retinal ganglion and inner nuclear layers may reflect a role in maintenance of the visual cycle. Its expression in the corneal epithelium may indicate a function in metabolism of environmental xenobiotics.     

Physiological significance and expression of P450s in the developing eye

Expression of 10 CYP orthologs (Families 1-3) in developing mouse conceptus is constitutive. These forms have specific temporal and spatial expression. Studies on CYP1B1 indicate its requirement for normal eye development, both in human and mouse. The distribution of the enzyme in the mouse eye is in three regions, which may reflect three different, perhaps equally important, functions in this organ. Its presence in the inner ciliary and lens epithelia appears to be necessary for normal development of the trabecular meshwork and its function in regulating intraocular pressure. Its expression in the retinal ganglion and inner nuclear layers may reflect a role in maintenance of the visual cycle. Its expression in the corneal epithelium may indicate a function in metabolism of environmental xenobiotics.     

Metabolism of retinoids and arachidonic acid by human and mouse cytochrome P450 1b1.

The cytochrome P450 family 1 (CYP1) is considered to be one of the xenobiotic-metabolizing enzyme families and is responsible for oxidative metabolism of polycyclic aromatic hydrocarbons. For example, mouse Cyp1b1 was originally identified as the enzyme responsible for oxidative metabolism of 7,12-dimethylbenz(alpha)anthracene (DMBA). A comparison of the kinetics of this metabolism by mouse and human CYP1B1 orthologs revealed the mouse enzyme to have a more favorable metabolism of DMBA, with a catalytic efficiency ratio (CER) of 0.23. However, CYP1 enzymes are also capable of metabolism of endobiotics, and in the present study, the metabolism of retinoids and lipid endobiotics by human CYP1B1 and mouse Cyp1b1 orthologs was compared. Both hemoproteins oxidized retinol to retinal and retinal to retinoate, but did not oxidize retinoate. The CYP1B1 to Cyp1b1 CERs were 13 and 26 for the two steps, respectively; the Cyp1b1 K(m(app)) values for retinoids were 20-fold higher. Human family 1 cytochromes P450 had unique regional specificities for arachidonate oxidation: the major metabolites of CYP1A1, CYP1A2, and CYP1B1 were 75% terminal hydroxyeicosatetraenoic fatty acids (HETEs), 52% epoxyeicosatrienoic fatty acids (EETs), and 54% mid-chain HETEs, respectively. CYP1A1 and CYP1B1 K(m(app)) values for arachidonate were about 30 microM, whereas CYP1A2 K(m(app)) was 95 microM. The major metabolites of arachidonic acid by Cyp1b1 were EETs (50%) and midchain HETEs (37%). The mouse ortholog had a CER for metabolite production of 64 due to a K(m(app)) of 0.5 mM for arachidonate.     

A dual function of the furanocoumarin chalepensin in inhibiting Cyp2a and inducing Cyp2b in mice: the protein stabilization and receptor-mediated activation

Chalepensin, a furanocoumarin, is present in several medicinal Rutaceae plants and causes a mechanism-based inhibition of human and mouse cytochrome P450 (P450, CYP) 2A in vitro. To address the in vivo effect, we investigated the effects of chalepensin on multiple hepatic P450 enzymes in C57BL/6JNarl mice. Oral administration of 10?mg/kg chalepensin to mice for 7?days significantly decreased hepatic coumarin 7-hydroxylation (Cyp2a) and increased 7-pentoxyresorufin O-dealkylation (Cyp2b) activities, whereas activities of Cyp1a, Cyp2c, Cyp2e1, and Cyp3a were not affected. Without affecting its mRNA level, the decreased Cyp2a activity was accompanied by an increase in the immunodetected Cyp2a5 protein level. In chalepensin-treated mice, microsomal Cyp2a5 was less susceptible to ATP-fortified cytosolic degradation than that in control mice, resulting in the elevated protein level. The in vitro inactivation through NADPH-fortified pre-incubation with chalepensin also protected microsomal Cyp2a5 against protein degradation. Using cell-based reporter systems, chalepensin at a concentration near unbound plasma concentration activated mouse constitutive androstane receptor (CAR), in agreement with the observed induction of Cyp2b. These findings revealed that suicidal inhibition of Cyp2a5 and the CAR-mediated Cyp2b9/10 induction concurrently occurred in chalepensin-treated mice.     

Role of the aryl hydrocarbon receptor and Cyp1b1 in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin

The role of aryl hydrocarbon receptor (AhR) and cytochrome P450 (Cyp) 1 family in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in vivo. Immature (21 days old) AhR, Cyp1a2, or Cyp1b1 knockout (–/–) mice were treated intraperitoneally with estradiol (E2, 20 ng/mouse per day, for 14 consecutive days) and/or TCDD (200 ng/mouse per day, on days 7, 9, 11, and 13). Uterine wet weight and uterine peroxidase activity (UPA) were measured as markers of estrogen responsiveness. UPA was a better marker of estrogen responsiveness than the uterine wet weight. In AhR wild-type (+/+) mice, UPA (208.1±81.6 units/g tissue) was increased by the administration of E2 (to 297.2±178.7 units/g). The administration of TCDD significantly (p<0.01) decreased the UPA (10.5±3.4 units/g) compared with that in the control mice. Co-administration of TCDD with E2 also significantly (p<0.05) decreased the UPA (18.8±19.9 units/g) compared with that in E2-treated mice. In AhR(–/–) mice, UPA (162.9±146.7 units/g) was significantly (p<0.01) increased by the administration of E2 (486.8±108.2 units/g). In contrast to the results in AhR(+/+) mice, UPA was not affected by the administration of TCDD (51.8±70.6 units/g) compared with control, and co-administration of TCDD with E2 (545.8±189.4 units/g) compared with that in E2-treated mice. In Cyp1a2/1b1(+/+) mice, UPA was significantly (p<0.05) increased by the administration of E2 (70.0±36.4 units/g). Co-administration of TCDD with E2 significantly (p<0.05) decreased the UPA (29.6±22.2 units/g) compared with that in E2-treated mice. In Cyp1a2(–/–) mice, co-administration of TCDD with E2 significantly (p<0.01) decreased the UPA (6.8±5.1 units/g) compared with that in E2-treated mice. In Cyp1b1(–/–) mice, UPA (5.5±8.1 units/g) was significantly (p<0.05) increased by the administration of E2 (56.6±34.1 units/g). In contrast to the results in Cyp1a2/1b1(+/+) mice or Cyp1a2(–/–) mice, UPA was not affected by the co-administration of TCDD and E2 (52.6±30.1 units/g) compared with that in E2-treated mice. This is the first demonstration that Cyp1b1 as well as AhR is involved in the antiestrogenic effects of TCDD.     

Role of P450 1A1 and P450 1A2 in bioactivation versus detoxication of the renal carcinogen aristolochic acid I: studies in Cyp1a1-/-, Cyp1a2-/-, and Cyp1a1/1a2-/- mice

Exposure to aristolochic acid I (AAI) is associated with aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial cancer. Individual differences in xenobiotic-metabolizing enzyme activities are likely to be a reason for interindividual susceptibility to AA-induced disease. We evaluated the reductive activation and oxidative detoxication of AAI by cytochrome P450 (P450) 1A1 and 1A2 using the Cyp1a1(-/-) and Cyp1a2(-/-) single-knockout and Cyp1a1/1a2(-/-) double-knockout mouse lines. Incubations with hepatic microsomes were also carried out in vitro. P450 1A1 and 1A2 were found to (i) activate AAI to form DNA adducts and (ii) detoxicate it to 8-hydroxyaristolochic acid I (AAIa). AAI-DNA adduct formation was significantly higher in all tissues of Cyp1a1/1a2(-/-) than Cyp1a(+/+) wild-type (WT) mice. AAI-DNA adduct levels were elevated only in selected tissues from Cyp1a1(-/-) versus Cyp1a2(-/-) mice, compared with those in WT mice. In hepatic microsomes, those from WT as well as Cyp1a1(-/-) and Cyp1a2(-/-) mice were able to detoxicate AAI to AAIa, whereas Cyp1a1/1a2(-/-) microsomes were less effective in catalyzing this reaction, confirming that both mouse P450 1A1 and 1A2 are both involved in AAI detoxication. Under hypoxic conditions, mouse P450 1A1 and 1A2 were capable of reducing AAI to form DNA adducts in hepatic microsomes; the major roles of P450 1A1 and 1A2 in AAI-DNA adduct formation were further confirmed using selective inhibitors. Our results suggest that, in addition to P450 1A1 and 1A2 expression levels in liver, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.     

Phenotype of the Cyp1a1/1a2/1b1-/- triple-knockout mouse

Crossing the Cyp1a1/1a2(-/-) double-knockout mouse with the Cyp1b1(-/-) single-knockout mouse, we generated the Cyp1a1/1a2/1b1(-/-) triple-knockout mouse. In this triple-knockout mouse, statistically significant phenotypes (with incomplete penetrance) included slower weight gain and greater risk of embryolethality before gestational day 11, hydrocephalus, hermaphroditism, and cystic ovaries. Oral benzo[a]pyrene (BaP) daily for 18 days in the Cyp1a1/1a2(-/-) produced the same degree of marked immunosuppression as seen in the Cyp1a1(-/-) mouse; we believe this reflects the absence of intestinal CYP1A1. Oral BaP-treated Cyp1a1/1a2/1b1(-/-) mice showed the same "rescued" response as that seen in the Cyp1a1/1b1(-/-) mouse; we believe this reflects the absence of CYP1B1 in immune tissues. Urinary metabolite profiles were dramatically different between untreated triple-knockout and wild-type; principal components analysis showed that the shifts in urinary metabolite patterns in oral BaP-treated triple-knockout and wild-type mice were also strikingly different. Liver microarray cDNA differential expression (comparing triple-knockout with wild-type) revealed at least 89 genes up- and 62 genes down-regulated (P-value < or = 0.00086). Gene Ontology "classes of genes" most perturbed in the untreated triple-knockout (compared with wild-type) include lipid, steroid, and cholesterol biosynthesis and metabolism; nucleosome and chromatin assembly; carboxylic and organic acid metabolism; metal-ion binding; and ion homeostasis. In the triple-knockout compared with the wild-type mice, response to zymosan-induced peritonitis was strikingly exaggerated, which may well reflect down-regulation of Socs2 expression. If a single common molecular pathway is responsible for all of these phenotypes, we suggest that functional effects of the loss of all three Cyp1 genes could be explained by perturbations in CYP1-mediated eicosanoid production, catabolism and activities.     

Monitoring Cyp2b10 mRNA expression at cessation of 2-year carcinogenesis bioassay in mouse liver provides evidence for a carcinogenic mechanism devoid of human relevance: the dalcetrapib experience

Introduction

Dalcetrapib is a cholesteryl ester transfer protein (CETP) modulator in clinical assessment for cardiovascular outcome benefits. In compliance with regulatory requirements, dalcetrapib was evaluated in rodent 2-year carcinogenesis bioassays. In the mouse bioassay, male mice demonstrated increased liver weight and statistically increased incidences of hepatocellular adenoma/carcinoma. Hepatic cytochrome p450 (Cyp) 2b10 mRNA induction and increased Cyp2b10 enzyme activity signify activation of hepatic nuclear receptor constitutive androstane receptor (CAR), a widely established promoter of rodent-specific hepatic tumors. We therefore monitored hepatic Cyp2b10 mRNA and its enzyme activity in a subset of dalcetrapib-treated male mice from the bioassay.

Methods

Liver samples were obtained from ~ 1/3 of male mice from each dose group including vehicle-controls (mean and earliest study day of death 678 and 459 respectively). Quantitative real time PCR (qRT-PCR) was performed to determine Cyp2b10 mRNA expression and Cyp1a-, Cyp2b10- and Cyp3a-selective activities were monitored.

Results

Cyp2b10 mRNA was strongly induced by dalcetrapib with an expected wide inter-individual variation (5-1421-fold). Group average fold-induction versus vehicle-controls showed a dose-related increase from 48-fold (250 mg/kg/day) to 160-fold (750 mg/kg/day), which declined slightly at 2000 mg/kg/day (97-fold). Cyp enzyme activities showed approximate doubling of total Cyp P450 content per milligram protein and a 9-fold increase in Cyp2b10-selective pentoxyresorufin O-dealkylase activity (750 mg/kg/day).

Discussion

These data from hepatic Cyp2b10 monitoring are strongly suggestive of CAR activation by dalcetrapib, a mechanism devoid of relevance towards hepatocarcinogenesis in humans; results show feasibility of Cyp2b10 as a surrogate marker for this mechanism at cessation of a carcinogenesis bioassay.     

Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro

The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E₂) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition, sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E₂ metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E₂, testosterone, androstenedione, and progesterone (P₄) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17β-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), 3β hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels.     

The cyp2e1-humanized transgenic mouse: role of cyp2e1 in acetaminophen hepatotoxicity.

The cytochrome P450 (P450) CYP2E1 enzyme metabolizes and activates a wide array of toxicological substrates, including alcohols, the widely used analgesic acetaminophen, acetone, benzene, halothane, and carcinogens such as azoxymethane and dimethylhydrazine. Most studies on the biochemical and pharmacological actions of CYP2E1 are derived from studies with rodents, rabbits, and cultured hepatocytes; therefore, extrapolation of the results to humans can be difficult. Creating "humanized" mice by introducing the human CYP2E1 gene into Cyp2e1-null mice can circumvent this disadvantage. A transgenic mouse line expressing the human CYP2E1 gene was established. Western blot and high-performance liquid chromatography/mass spectrometry analyses revealed human CYP2E1 protein expression and enzymatic activity in the liver of CYP2E1-humanized mice. Treatment of mice with the CYP2E1 inducer acetone demonstrated that human CYP2E1 was inducible in this transgenic model. The response to the CYP2E1 substrate acetaminophen was explored in the CYP2E1-humanized mice. Hepatotoxicity, resulting from the CYP2E1-mediated activation of acetaminophen, was demonstrated in the livers of CYP2E1-humanized mice by elevated serum alanine aminotransferase levels, increased hepatocyte necrosis, and decreased P450 levels. These data establish that in this humanized mouse model, human CYP2E1 is functional and can metabolize and activate different CYP2E1 substrates such as chlorzoxazone, p-nitrophenol, acetaminophen, and acetone. CYP2E1-humanized mice will be of great value for delineating the role of human CYP2E1 in ethanol-induced oxidative stress and alcoholic liver damage. They will also function as an important in vivo tool for predicting drug metabolism and disposition and drug-drug interactions of chemicals that are substrates for human CYP2E1.     

Dose-response relationship of cytochrome P4501b1 mRNA induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin in livers of C57BL/6J and DBA/2J mice

 The dose-response relationship of cytochrome P4501b1 (Cyp1b1) and Cyp1a1 induction in livers of TCDD-treated female C57BL/6J and DBA/2J mice are described. The animals were treated i.p. with 0.001, 0.01, 0.1, 1, 10 and 50 μg TCDD/kg for 24 h, and Cyp1b1 and Cyp1a1 mRNA expression was analyzed by RT-PCR. In the livers of both mouse strains, the Cyp1b1 and Cyp1a1 mRNA content was increased after TCDD exposure in a dose-dependent manner. These effects were more pronounced in TCDD-responsive C57BL/6J mice than in the less responsive DBA/2J mice, although Cyp1a1 was more responsive to TCDD than Cyp1b1 in both strains. The calculated ED₅₀ values for Cyp1b1 and Cyp1a1 induction in livers of TCDD-treated C57BL/6J mice were 1.3 and 0.08 μg TCDD/kg, respectively. The corresponding values for half-maximal induction response in livers of DBA/2J mice were 3.4 μg TCDD/kg for Cyp1b1 and 1.5 μg TCDD/kg for Cyp1a1. These results show that Cyp1b1 mRNA expression is less inducible by TCDD than Cyp1a1. Both genes are highly inducible in TCDD-responsive C57BL/6J mice expressing the high affinity arylhydrocarbon receptor (Ah receptor), suggesting that Cyp1b1, like Cyp1a1, is a potential Ah receptor-regulated gene. Received: 8 December 1995/Accepted: 6 February 1996     

Role of mouse CYP2E1 in the O-hydroxylation of p-nitrophenol: comparison of activities in hepatic microsomes from Cyp2e1(-/-) and wild-type mice.

Enzymatic activities are routinely used to identify the contribution of individual forms of cytochrome P450 in a particular biotransformation. p-Nitrophenol O-hydroxylation (PNPH) has been widely used as a measure of CYP2E1 catalytic activity. However, rat and human forms of CYP3A have also been shown to catalyze this activity. In mice, the contributions of CYP3A and CYP2E1 to PNPH activity are not known. Here we used hepatic microsomes from Cyp2e1(-/-) and wild-type mice to investigate the contributions of constitutively expressed and alcohol-induced murine CYP2E1 and CYP3A to PNPH activity. In liver microsomes from untreated mice, PNPH activity was much greater in wild-type mice compared with Cyp2e1(-/-) mice, suggesting a major role for CYP2E1 in catalyzing PNPH activity. Hepatic PNPH activities were not significantly different in microsomes from male and female mice, although the microsomes from females have dramatically higher levels of CYP3A. Treatment with a combination of ethanol and isopentanol resulted in induction of CYP3A proteins in wild-type and Cyp2e1(-/-) mice, as well as CYP2E1 protein in wild-type mice. The alcohol treatment increased PNPH activities in hepatic microsomes from wild-type mice but not from Cyp2e1(-/-) mice. Our findings suggest that in untreated and alcohol-treated mice, PNPH activity may be used as a specific probe for CYP2E1 and that constitutively expressed and alcohol-induced forms of mouse CYP3A have little to no role in catalyzing PNPH activity.     

Lentiviral-mediated RNAi knockdown yields a novel mouse model for studying Cyp2b function

There are few in vivo knockout models available to study the function of Cyp2 members involved in the metabolism of endogenous and exogenous chemicals. These models may help provide insight into the cytochrome P450s (CYPs) responsible for the detoxification and activation of drugs, environmental toxicants, and endobiotics. The aim of this work is to produce a potent Cyp2b-knockdown (KD) mouse for subsequent study of Cyp2b function. We made a quintuple Cyp2b-KD mouse using lentiviral-promoted short hairpin RNA (shRNA) homologous to all five murine Cyp2b subfamily members (Cyp2b9, 2b10, 2b13, 2b19, and 2b23). The Cyp2b-KD mice are viable, fertile, and without obvious gross abnormalities except for an increase in liver weight. Expression of the three hepatic Cyp2b members, 2b9, 2b10, and 2b13, is significantly repressed as demonstrated by quantitative real-time PCR and Western blotting. The constitutive androstane receptor activator, 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), was used to determine if shRNA-mediated Cyp2b10 repression could be outcompeted by Cyp2b10 induction. TCPOBOP-treated Cyp2b-KD mice show 80-90% less Cyp2b protein expression than TCPOBOP-treated wild-type (WT) mice, demonstrating that Cyp induction does not outcompete the repressive function of the shRNA. Untreated and TCPOBOP-treated Cyp2b-KD mice are poor metabolizers of parathion compared with WT mice. Furthermore, Cyp2b-KD mice are sensitive to parathion, an organophosphate insecticide primarily metabolized by Cyp2b enzymes, when compared with WT mice. In summary, we designed an shRNA construct that repressed the expression and activity of multiple Cyp2b enzymes. We foresee that this novel Cyp2b-KD mouse model will significantly improve our understanding of the role of Cyp2b enzymes in chemical sensitivity and drug metabolism.     

Effects of aqueous extract of Ruta graveolens and its ingredients on cytochrome P450, uridine diphosphate (UDP)-glucuronosyltransferase,and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-quinone oxidoreductase in mice

Ruta graveolens (the common rue) has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes, effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP), uridine diphosphate (UDP)-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H):quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg) induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(P)H:quinone oxidoreductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (F_mix) increased Cyp2b activities in males. The mixture of rutin and F_mix increased Cyp1a and Cyp2b activities. These results revealed that rutin and F_mix contributed at least in part to the P450 induction by rue.     

Functional Cyp2e1 is required for substantial in vivo formation of 2,5-hexanedione from n-hexane in the mouse

Neurotoxicity of n-hexane is mediated by its metabolite 2,5-hexanedione (2,5-HD). Cytochrome P4502E1 (CYP2E1) has been suggested but not shown to be involved in the formation of the metabolite. An objective of the current study was to assess the essentiality of CYP2E1 for in vivo 2,5-HD formation from n-hexane. This was accomplished by comparing urinary levels of the gamma-diketone in n-hexane-treated mice in which the Cyp2e1 gene has been deleted (Cyp2e1-/-) with that in n-hexane-treated wild-type (Cyp2e1+/+) mice. 2,5-HD was detectable not as the free compound but as further metabolites, at levels that were comparable in both strains of mice, following a daily 200 mg/kg i.p. dose of the alkane for 10 days. Continued daily n-hexane treatment resulted in increased urinary levels of 2,5-HD metabolites in Cyp2e1+/+ but not in Cyp2e1-/- mice. Only in Cyp2e1+/+ mice and only on day 21 of n-hexane treatment was a trace level of unchanged 2,5-HD detected. 3-Hexanol was the only other n-hexane metabolite detected in the mice but its concentration was higher in Cyp2e1-/- than in Cyp2e1+/+ mice. In n-hexane-treated rats, in contrast to mice, multiple metabolites of the alkane, including unchanged 2,5-HD, were detected. The results indicate that substantial in vivo formation of 2,5-HD from n-hexane in the mouse requires CYP2E1, and suggest that further detoxification of the metabolite may be very efficient in this species.