Role of CYP3A and CYP2E1 in alcohol-mediated increases in acetaminophen hepatotoxicity: comparison of wild-type and Cyp2e1(-/-) mice.

CYP2E1 is widely accepted as the sole form of cytochrome P450 responsible for alcohol-mediated increases in acetaminophen (APAP) hepatotoxicity. However, we previously found that alcohol [ethanol and isopentanol (EIP)] causes increases in APAP hepatotoxicity in Cyp2e1(-/-) mice, indicating that CYP2E1 is not essential. Here, using wild-type and Cyp2e1(-/-) mice, we investigated the relative roles of CYP2E1 and CYP3A in EIP-mediated increases in APAP hepatotoxicity. We found that EIP-mediated increases in APAP hepatotoxicity occurred at lower APAP doses in wild-type mice (300 mg/kg) than in Cyp2e1(-/-) mice (600 mg/kg). Although this result suggests that CYP2E1 has a role in the different susceptibilities of these mouse lines, our findings that EIP-mediated increases in CYP3A activities were greater in wild-type mice compared with Cyp2e1(-/-) mice raises the possibility that differential increases in CYP3A may also contribute to the greater APAP sensitivity in EIP-pretreated wild-type mice. At the time of APAP administration, which followed an 11 h withdrawal from the alcohols, alcohol-induced levels of CYP3A were sustained in both mouse lines, whereas CYP2E1 was decreased to constitutive levels in wild-type mice. The CYP3A inhibitor triacetyloleandomycin (TAO) decreased APAP hepatotoxicity in EIP-pretreated wild-type and Cyp2e1(-/-) mice. TAO treatment in vivo resulted in inhibition of microsomal CYP3A-catalyzed activity, measured in vitro, with no inhibition of CYP1A2 and CYP2E1 activities. In conclusion, these findings suggest that both CYP3A and CYP2E1 contribute to APAP hepatotoxicity in alcohol-treated mice.     

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.     

Role of cytochrome P450 1A2 in bilirubin degradation Studies in Cyp1a2 (-/-) mutant mice

In congenital jaundice, which is due to defects of bilirubin gluruconidation, bilirubin is degraded by an alternative pathway into unidentified products. Previously, it was shown that plasma bilirubin levels can be decreased in rats with this defect by inducers of CYP1A enzymes. Here, liver microsomes from rats or mice treated with beta-naphthoflavone (BNF) or 3-methylcholanthrene (3 MC) had increased activity for bilirubin degradation. The activity was further stimulated by addition of the coplanar molecule 3,4,3',4'-tetrachlorobiphenyl (TCB). There was more stimulation of bilirubin degradation by TCB in microsomes from BNF-treated rats than in microsomes from BNF-treated mice. CYP1A1 to CYP1A2 ratios were greater in rats treated with BNF. In Cyp1a2 (-/-) mutant mice, 3-MC treatment did not increase the rate of bilirubin degradation, but TCB increased this degradation severalfold. Between SWR and C57BL/6 inbred mouse strains that have a 2-fold difference in hepatic constitutive CYP1A2 levels, there was also a 2-fold difference in bilirubin degradation; TCB did not stimulate in either strain. We conclude that CYP1A2 is responsible for microsomal bilirubin degradation in the absence of TCB. TCB was required for bilirubin degradation by CYP1A1. Manipulation of CYP1A2 may be of therapeutic benefit in patients with these diseases of bilirubin conjugation.     

Role of CYP1A2 and CYP2E1 in the pentoxifylline ciprofloxacin drug interaction

In this study the drug interaction between ciprofloxacin (CIPRO) and pentoxifylline (PTX) was investigated and the role of CYP1A2 in the drug interaction was determined with the aid of a selective CYP1A2 inhibitor, furafylline (FURA), and the Cyp1A2 knockout mouse. Serum concentrations of PTX (83.4+/-1 micromol/l) and metabolite-1 (M-1) (13.7+/-2.8 micromol/l) following a single injection of PTX (100 mg/kg i.p.) were significantly higher (P<0.05) in mice treated with CIPRO (25 mg/kg i.p. 9 days) compared to serum concentrations of PTX (46.3+/-0.5 micromol/l) and M-1 (6.4+/-1.1 micromol/l) in mice administered saline. Murine hepatic microsomes were incubated with PTX alone or the combination of PTX and CIPRO. The metabolism of PTX in the murine hepatic microsomes containing both CIPRO and PTX was significantly decreased compared to microsomes incubated with PTX alone, suggesting that CIPRO may inhibit the metabolism of PTX. To further clarify the role of CYP1A2 in the metabolism of PTX in mice, the effect of a selective CYP1A2 mechanism based inhibitor, FURA, on the metabolism of PTX was investigated and our results indicate that FURA inhibited metabolism of PTX. We then investigated PTX elimination in the Cyp1A2 knockout mouse. Blood levels of PTX were assessed at 2 and 20 min following a single injection of PTX (32 mg/kg i.v). Serum concentration of PTX was determined in Cyp1A2 knockout mice compared to Cyp1A2 wild type control mice. The serum concentration of PTX in Cyp1A2 wild type mice (n=9) was 22.2+/-3.2 micromol/l at 20 min following injection of PTX. The serum concentration of PTX in Cyp1A2 knockout mice (n=11) was significantly elevated at 20 min following injection of PTX compared to Cyp1A2 wild type mice. These results clearly indicate that inhibition of CYP1A2 catalytic activity that occurs in the Cyp1A2 knockout mice is sufficient to alter metabolism of PTX and result in markedly elevated levels in serum of Cyp1A2 knockout mice. The results of Western analysis in murine microsomes suggest that CYP1A2 protein levels were not altered by CIPRO indicating that CIPRO did not downregulate Cyp1A2. The results of Western analysis also indicated that CIPRO treatment increased CYP2E1 in mouse microsomes and the implications of these will be discussed.     

CYP1A2 is essential in murine uroporphyria caused by hexachlorobenzene and iron

Using Cyp1a2(-/-) mice we previously showed that CYP1A2 is absolutely required for hepatic uroporphyrin accumulation caused by iron and 5-aminolevulinate (ALA) treatment, both in the presence and absence of an inducer of CYP1A2. In this study we have used these mice to investigate whether CYP1A2 has an obligatory role in hepatic uroporphyria caused by hexachlorobenzene (HCBZ), an inducer of CYP2B and CYP3A, as well as CYP1A2. Here we treated mice with HCBZ and iron, with and without the porphyrin precursor, ALA, in the drinking water. In iron-loaded wild-type mice given a single dose of HCBZ and ALA, hepatic uroporphyrin (URO) accumulated to 300 nmol/g liver after 37 days, whereas in Cyp1a2(-/-) mice, there was no hepatic URO, even after an additional dose of HCBZ, and a further 29 days of ALA treatment. A similar requirement for CYP1A2 was found in uroporphyria produced in HCBZ and iron-treated mice in the absence of ALA. As detected by Western immunoblotting, HCBZ induced small increases in CYP2B and CYP3A in the livers of all animals. In the wild-type animals, HCBZ also induced CYP1A2 and associated enzyme activities, including uroporphyrinogen oxidation, by about 2-3-fold. In the Cyp1a2(-/-) mice, HCBZ did not increase hepatic microsomal uroporphyrinogen oxidation. These results indicate that, in mice, CYP1A2 is essential in the process leading to HCBZ-induced uroporphyria. Contributions by other CYP forms induced by HCBZ appear to be minimal.     

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.     

Role of CYP2E1 in thioacetamide-induced mouse hepatotoxicity

Previous experiments showed that treatment of mice and rats with thioacetamide (TAA) induced liver cell damage, fibrosis and/or cirrhosis, associated with increased oxidative stress and activation of hepatic stellate cells. Some experiments suggest that CYP2E1 may be involved in the metabolic activation of TAA. However, there is no direct evidence on the role of CYP2E1 in TAA-mediated hepatotoxicity. To clarify this, TAA-induced hepatotoxicity was investigated using Cyp2e1-null mice. Male wild-type and Cyp2e1-null mice were treated with TAA (200 mg/kg of body weight, single, i.p.) at 6 weeks of age, and hepatotoxicity examined 24 and 48 h after TAA treatment. Relative liver weights of Cyp2e1-null mice were significantly different at 24 h compared to wild-type mice (p<0.01). Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in Cyp2e1-null mice were significantly different at both time points compared to wild-type mice (p<0.01). Histopathological examination showed Cyp2e1-null mice represented no hepatototoxic lesions, in clear contrast to severe centriobular necrosis, inflammation and hemorrhage at both time points in wild-type mice. Marked lipid peroxidation was also only limited to wild-type mice (p<0.01). Similarly, TNF-alpha, IL-6 and glutathione peroxidase mRNA expression in Cyp2e1-null mice did not significantly differ from the control levels, contrasting with the marked alteration in wild-type mice (p<0.01). Western blot analysis further revealed no increase in iNOS expression in Cyp2e1-null mice. These results reveal that CYP2E1 mediates TAA-induced hepatotoxicity in wild-type mice as a result of increased oxidative stress.     

Short-term treatment with alcohols causes hepatic steatosis and enhances acetaminophen hepatotoxicity in Cyp2e1(-/-) mice

CYP2E1 has been reported to have an essential role in alcohol-mediated increases in hepatic steatosis and acetaminophen hepatotoxicity. We found that pretreatment of Cyp2e1(-/-) mice with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, for 7 days resulted in micro- and macrovesicular steatosis in the livers of all mice, as well as a dramatic increase in acetaminophen hepatotoxicity. In Cyp2e1(-/-) mice administered up to 600 mg acetaminophen/kg alone and euthanized 7 h later, there was no increase in serum levels of ALT. In Cyp2e1(-/-) mice pretreated with ethanol and isopentanol, subsequent exposure to 400 or 600 mg acetaminophen/kg resulted in centrilobular necrosis in all mice with maximal elevation in serum levels of ALT. Acetaminophen-mediated liver damage was similar in males and females. Hepatic microsomal levels of APAP activation in untreated females were similar to those in males treated with the alcohols. However, the females, like the males, required pretreatment with the alcohols in order to increase APAP hepatotoxicity. These findings suggest that, in the Cyp2e1(-/-) mice, the alcohol-mediated increase in acetaminophen hepatotoxicity involves the contribution of other factors, in addition to induction of CYP(s) that activate acetaminophen. Alternatively, CYP-mediated activation of acetaminophen measured in vitro may not reflect the actual activity in vivo. Our findings that a 7-day treatment with ethanol and isopentanol causes extensive hepatic steatosis and increases acetaminophen hepatotoxicity in Cyp2e(-/-) mice indicate that CYP2E1 is not essential for either response.     

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.     

Inhibition of urethane-induced carcinogenicity in cyp2e1-/- in comparison to cyp2e1+/+ mice.

Urethane is an established animal carcinogen and has been classified as "reasonably anticipated to be a human carcinogen." Until recently, urethane metabolism via esterase was considered the main metabolic pathway of this chemical. However, recent studies in this laboratory showed that CYP2E1, and not esterase, is the primary enzyme responsible for urethane oxidation. Subsequent studies demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in Cyp2e1-/- compared to Cyp2e1+/+ mice. Using Cyp2e1-/- mice, current studies were undertaken to assess the relationships between urethane metabolism and carcinogenicity. Urethane was administered via gavage at 1, 10, or 100 mg/kg/day, 5 days/week, for 6 weeks. Animals were kept without chemical administration for 7 months after which they were euthanized, and urethane carcinogenicity was assessed. Microscopic examination showed a significant reduction in the incidences of liver hemangiomas and hemangiosarcomas in Cyp2e1-/- compared to Cyp2e+/+ mice. Lung nodules increased in a dose-dependent manner and were less prevalent in Cyp2e1-/- compared to Cyp2e+/+ mice. Microscopic alterations included bronchoalveolar adenomas, and in one Cyp2e1+/+ mouse treated with 100 mg/kg urethane, a bronchoalveolar carcinoma was diagnosed. Significant reduction in the incidence of adenomas and the number of adenomas/lung were observed in Cyp2e1-/- compared to Cyp2e1+/+ mice. In the Harderian gland, the incidences of hyperplasia and adenomas were significantly lower in Cyp2e1-/- compared to Cyp2e+/+ mice at the 10 mg/kg dose, with no significant differences observed at the high or low doses. In conclusion, this work demonstrated a significant reduction of urethane-induced carcinogenicity in Cyp2e1-/- compared to Cyp2e1+/+ mice and proved that CYP2E1-mediated oxidation plays an essential role in urethane-induced carcinogenicity.     

Midazolam metabolism in cytochrome P450 3A knockout mice can be attributed to up-regulated CYP2C enzymes

The cytochrome P450 3A (CYP3A) enzymes represent one of the most important drug-metabolizing systems in humans. Recently, our group has generated cytochrome P450 3A knockout mice to study this drug-handling system in vivo. In the present study, we have characterized the Cyp3a knockout mice by studying the metabolism of midazolam, one of the most widely used probes to assess CYP3A activity. We expected that the midazolam metabolism would be severely reduced in the absence of CYP3A enzymes. We used hepatic and intestinal microsomal preparations from Cyp3a knockout and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo metabolite formation was determined after intravenous administration of midazolam. We were surprised to find that our results demonstrated that there is still marked midazolam metabolism in hepatic (but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found comparable amounts of midazolam as well as its major metabolites in plasma after intravenous administration in Cyp3a knockout mice compared with wild-type mice. These data suggested that other hepatic cytochrome P450 enzymes could take over the midazolam metabolism in Cyp3a knockout mice. We provide evidence that CYP2C enzymes, which were found to be up-regulated in Cyp3a knockout mice, are primarily responsible for this metabolism and that several but not all murine CYP2C enzymes are capable of metabolizing midazolam to its 1'-OH and/or 4-OH derivatives. These data illustrate interesting compensatory changes that may occur in Cyp3a knockout mice. Such flexible compensatory interplay between functionally related detoxifying systems is probably essential to their biological role in xenobiotic protection.     

Decrease in 4-aminobiphenyl-induced methemoglobinemia in Cyp1a2(-/-) knockout mice

Methemoglobin formation, as well as hemoglobin or DNA adducts, are useful biomarkers of occupational exposure to certain arylamines. It has been suggested that, in liver from animals not treated with a cytochrome P450 (CYP) inducer, hepatic CYP1A2 is the major P450 involved in N-hydroxylation. This is the first step in the metabolic activation of many arylamines, such as the human urinary bladder carcinogen 4-aminobiphenyl (ABP). The product of this catalytic step, N-hydroxy-4-ABP, reacts in the blood with oxyhemoglobin to form methemoglobin and nitrosobiphenyl. We therefore examined the role of CYP1A2 in causing methemoglobinemia in ABP-treated Cyp1a2(-/-) knockout mice. Application of ABP (100 micromol/kg body wt) to the skin resulted in a marked depletion in the levels of the hepatic thiols (reduced glutathione and cysteine) after 2 h, which rebounded to basal levels 24 h later, and we found no differences between the Cyp1a2(-/-) and wild-type Cyp1a2(+/+) animals. Unexpectedly, the methemoglobin levels were significantly (p < 0.05) higher in Cyp1a2(-/-) than Cyp1a2(+/+) mice at 2, 7, and 24 h following topical ABP. Treatment with dioxin, 24 h prior to ABP, decreased methemoglobin levels by about half at each of the time points in both the Cyp1a2(-/-) and Cyp1a2(+/+) mice. These data suggest that CYP1A2 does not play a positive role in methemoglobin formation via the activation of ABP; rather, the absence of CYP1A2 enhances ABP-induced methemoglobinemia. Because liver CYP1A2 levels are known to vary more than 60-fold between humans, our findings may be relevant to patients who are exposed to arylamines in the workplace.     

Prediction of human liver microsomal oxidations of 7-ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome P-450 enzymes.

Different roles of individual forms of human cytochrome P-450 (CYP) in the oxidation of 7-ethoxycoumarin and chlorzoxazone were investigated in liver microsomes of different human samples, and the microsomal activities thus obtained were predicted with kinetic parameters obtained from cDNA-derived recombinant CYP enzymes in microsomes of Trichoplusia ni cells. Of 14 forms of recombinant CYP examined, CYP1A1 had the highest activities (V(max)/K(m) ratio) in catalyzing 7-ethoxycoumarin O-deethylation followed by CYP1A2, 2E1, 2A6, and 2B6, although CYP1A1 has been shown to be an extrahepatic enzyme. With these kinetic parameters (excluding CYP1A1) we found that CYP1A2 and 2E1 were the major enzymes catalyzing 7-ethoxycoumarin; the contributions of these two forms were dependent on the contents of these CYPs in liver microsomes of different humans. Similarly, chlorzoxazone 6-hydroxylation activities of liver microsomes were predicted with kinetic parameters of recombinant human CYP enzymes and it was found that CYP3A4 as well as CYP1A2 and 2E1 were involved in chlorzoxazone hydroxylation, depending on the contents of these CYP forms in the livers. Recombinant CYP2A6 and 2B6 and CYP2D6 had considerable roles (V(max)/K(m) ratio) for 7-ethoxycoumarin O-deethylation and chlorzoxazone 6-hydroxylation, respectively; however, these CYP forms had relatively minor roles in the reactions, probably due to low expression in human livers. These results support the view that the roles of individual CYP enzymes in the oxidation of xenobiotic chemicals in human liver microsomes could be predicted by kinetic parameters of individual CYP enzymes and by the levels of each of the CYP enzymes in liver microsomes of human samples.     

Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the liver of C57BL/6J mice is a model for clinical sporadic porphyria cutanea tarda (PCT). There is massive uroporphyria, inhibition of uroporphyrinogen decarboxylase (UROD) activity, and hepatocellular damage. A variety of evidence implicates the CYP1A2 enzyme as necessary for mouse uroporphyria. Here we report that, 5 weeks after a single oral dose of TCDD (75 microg/kg), Cyp1a2(+/+) wild-type mice showed severe uroporphyria and greater than 90% decreases in UROD activity; in contrast, despite exposure to this potent agent Cyp1a2(-/-) knockout mice displayed absolutely no increases in hepatic porphyrin levels, even after prior iron overload, and no detectable inhibition of UROD activity. Plasma levels of alanine-aminotransferase (ALT) and aspartate aminotransferase (AST)-although elevated in both genotypes after TCDD exposure-were significantly less in Cyp1a2(-/-) than in Cyp1a2(+/+) mice, suggesting that the absence of CYP1A2 also affords partial protection against TCDD-induced liver toxicity. Histological examination confirmed a decrease in hepatocellular damage in TCDD-treated Cyp1a2(-/-) mice; in particular, there was no bile duct damage or proliferation that in the Cyp1a2(+/+) mice might be caused by uroporphyrin. We conclude that CYP1A2 is both necessary and essential for the potent uroporphyrinogenic effects of TCDD in mice, and that CYP1A2 also plays a role in contributing to TCDD-induced hepatocellular injury. This study has implications for both the toxicity assessment of TCDD and the hepatic injury seen in PCT patients.     

淫羊藿总黄酮对大鼠肝微粒体CYP1A2、CYP3A4和CYP2E1活性的影响

评估淫羊藿总黄酮对大鼠肝细胞色素P450及其主要亚型活性的潜在影响。淫羊藿总黄酮以300mg／kg／d的剂量对SD大鼠进行连续灌胃处理15天，测定肝微粒体中cYP450含量与CYP1A2、CYP3A4和CYP2E1亚型活性，观察淫羊藿总黄酮的效应。CYP1A2的活性用荧光比色法进行测定，CYP3A4和CYP2E1的活性用紫外可见分光光度法测定。淫羊藿总黄酮处理后的大鼠肝脏CYP450含量及CYP1A2、CYP3A4和CYP2E1亚型活性均明显增高，其中CYP1A2和CYP2E1活性升高显著（P〈0．01）。淫羊藿总黄酮对大鼠肝脏CYP450及主要亚型CYP1A2、CYP3A4和CYP2E1活性均有诱导效应。     

Roles of cytochromes P450 1A2, 2A6, and 2C8 in 5-fluorouracil formation from tegafur, an anticancer prodrug, in human liver microsomes.

Tegafur, an anticancer prodrug, is bioactivated to 5-fluorouracil (5-FU) mainly by cytochrome P450 (P450) enzymes. The conversion from tegafur into 5-FU catalyzed by human liver microsomal P450 enzymes was investigated. In fourteen cDNA-expressed human P450 enzymes having measurable activities, CYP1A2, CYP2A6, CYP2E1, and CYP3A5 were highly active in catalyzing 5-FU formation at a tegafur concentration of 100 microM. Kinetic analysis revealed that CYP1A2 had the highest V(max)/K(m) value and that the V(max) value of CYP2A6 was high in 5-FU formation. In human liver microsomes, the activities of 5-FU formation from 10 microM, 100 microM, and 1 mM tegafur were significantly correlated with both coumarin 7-hydroxylation (r = 0.83, 0.86, and 0.74) and paclitaxel 6 alpha-hydroxylation (r = 0.77, 0.62, and 0.85) activities, respectively. Coumarin efficiently inhibited the 5-FU formation activities from 100 microM and 1 mM tegafur catalyzed by human liver microsomes that had high coumarin 7-hydroxylation activity. On the other hand, furafylline, fluvoxamine, and quercetin, as well as coumarin, showed inhibitory effects in liver microsomes that had high catalytic activities of 5-FU formation. The other P450 inhibitors examined showed weak or no inhibition in human liver microsomes. Polyclonal anti-CYP1A2 antibody, monoclonal anti-CYP2A6, and anti-CYP2C8 antibodies inhibited 5-FU formation activities to different extents in those two microsomal samples. These results suggest that CYP1A2, CYP2A6, and CYP2C8 have important roles in human liver microsomal 5-FU formation and that the involvement of these three P450 forms differs among individual humans.     

Role of cytochrome P450 2E1 in protein nitration and ubiquitin-mediated degradation during acetaminophen toxicity

It is well established that following a toxic dose of acetaminophen (APAP), nitrotyrosine protein adducts (3-NT), a hallmark of peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where cytochrome P450 2E1 (CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in APAP-mediated toxicity. This study was aimed at investigating the relationship between CYP2E1 and nitration (3-NT formation) followed by ubiquitin-mediated degradation of proteins in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400 mg/kg) for 4 and 24 h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of nitric oxide synthase (NOS), was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible than the native proteins for ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity.     

Activities of cytochrome p450 enzymes in liver and kidney microsomes from systemic carnitine deficiency mice with a gene mutation of carnitine/organic cation transporter

Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-2 degrees strain, exibit a systemic carnitine deficiency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine/organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the effects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6beta-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs/jvs-type mice compared to jvs/wt- or wt/wt-type mice. Coumarin 7-hydroxylation activities (marker for Cyp2a) were decreased to 0.7-fold in the male jvs/jvs-type mice. The activities of lauric acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver microsomes were increased 1.4- to 1.9-fold in female jvs/jvs-type mice. Genotoxic activation of 2-aminofluorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not affected by the SCD phenotype. These results demonstrated that the SCD phenotype affected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.