Cytochrome P450 2E1 Inducers Research Articles

The induction of cytochrome P450 2E1 by ethanol leads to the loss of synaptic proteins via PPARα down-regulation.

Ethanol, one of the most commonly abused substances throughout history, is a substrate and potent inducer of cytochrome P450 2E1 (CYP2E1). Our previous study showed that brain CYP2E1 was induced by chronic ethanol treatment and was associated with ethanol-induced neurotoxicity in rats. We therefore investigated the possible mechanism of brain CYP2E1 involvement in ethanol-induced neurodegeneration. Compared with the controls, chronic ethanol treatment (3.0g/kg, i.g., 160days) significantly increased CYP2E1 mRNA levels in the rat cortex, but the mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and the pre- and post-synaptic proteins (synaptophysin, SYP, and drebrin1, DBN1) were decreased. Ethanol treatment dose-dependently induced CYP2E1 mRNA expression, and CYP2E1 overexpression exacerbated the ethanol-induced neurotoxicity. Pretreatment with p38 inhibitor (SB202190) and ERK1/2 inhibitor (U0126) attenuated the induction of CYP2E1 mRNA and protein levels by ethanol; however, no change was observed with JNK inhibitor pretreatment. Ethanol exposure or CYP2E1 overexpression significantly decreased PPARα, SYP, and DBN1 expression as indicated by the data from real-time RT-PCR, Western blotting and immunocytochemistry. The activation of PPARα by WY14643 increased the activity of the SYP and DBN1 promoters and attenuated the inhibition of these genes by ethanol. The specific siRNA for CYP2E1 significantly attenuated the ethanol-induced inhibition of PPARα, SYP and DBN1 mRNA levels. These results suggest that the induction of CYP2E1 by ethanol may be mediated via the p38 and ERK1/2 signaling pathways in neurons but not via the JNK pathway. The CYP2E1-PPARα axis may play a role in ethanol-induced neurotoxicity via the alteration of the genes related with synaptic function.

Relationship between oxidative stress and hepatic glutathione levels in ethanol-mediated apoptosis of polarized hepatic cells

To investigate the role of reactive oxygen species (ROS) in ethanol-mediated cell death of polarized hepatic (WIF-B) cells. In this work, WIF-B cultures were treated with pyrazole (inducer of cytochrome P4502E1, CYP2E1) and/or L-buthionine sulfoximine (BSO), a known inhibitor of hepatic glutathione (GSH), followed by evaluation of ROS production, antioxidant levels, and measures of cell injury (apoptosis and necrosis). The results revealed that ethanol treatment alone caused a significant two-fold increase in the activation of caspase-3 as well as a similar doubling in ROS. When the activity of the CYP2E1 was increased by pyrazole pretreatment, an additional two-fold elevation in ROS was detected. However, the CYP2E1-related ROS elevation was not accompanied with a correlative increase in apoptotic cell injury, but rather was found to be associated with an increase in necrotic cell death. Interestingly, when the thiol status of the cells was manipulated using BSO, the ethanol-induced activation of caspase-3 was abrogated. Additionally, ethanol-treated cells displayed enhanced susceptibility to Fas-mediated apoptosis that was blocked by GSH depletion as a result of diminished caspase-8 activity. Apoptotic cell death induced as a consequence of ethanol metabolism is not completely dependent upon ROS status but is dependent on sustained GSH levels.

Effects of Pyridine Exposure upon Structural Lipid Metabolism in Swiss Webster Mice

Pyridine is a prototypical inducer of cytochrome P450 (CYP) 2E1, an enzyme associated with cellular oxidative stress and membrane damage. To better understand the effect of this treatment on cellular lipids, the influence of pyridine exposure (100 mg/kg/day i.p. for 5 days) on fatty acids, fatty esters, and fatty alcohol ethers in brain, heart, liver, and adipose tissue from male Swiss Webster mice was investigated. Lipid levels in cholesterol esters, triglycerides, free fatty acids, cardiolipin, sphingomyelin, and glycerylphospholipids were quantified. Pyridine altered the level and composition of lipids involved in membrane structure (i.e., sphingomyelin, phosphatidylethanolamines, and plasmalogens), energy metabolism (i.e., free fatty acids), and long-chain fatty acid transport (i.e., cholesterol esters) in a tissue-specific manner. Subtle changes in cholesterol esters were observed in all tissues. Sphingomyelin in the brain and heart were depleted in monounsaturated fatty acids (1.4- and 1.5-fold, respectively), while the liver sphingomyelin concentrations increased (1.5-fold). Pyridine exposure also increased heart free fatty acids by 1.3-fold, enriched cardiac phosphatidylethanolamine in long-chain polyunsaturated fatty acids by 1.3-fold, and depleted cardiolipin-associated plasmalogens by 3.8-fold. Phosphatidylethanolamines in the brain were also enriched in both saturated fatty acids (1.2-fold) and polyunsaturated fatty acids (1.3-fold) but were depleted in plasmalogens (2.9-fold). In particular, the levels of phosphatidylethanolamine-associated arachidonic (AA) and docosahexaenoic acid (DHA) in both brain and cardiac tissues significantly decreased following pyridine exposure. Considering the hypothetical role of plasmalogens as membrane-bound reactive oxygen scavengers, the current findings suggest that the brain and heart should be the focus of future studies on the toxicity of pyridine, as well as other CYP 2E1 inducers.

Negundoside, an irridiod glycoside from leaves of Vitex negundo , protects human liver cells against calcium-mediated toxicity induced by carbon tetrachloride

To evaluate the protective effect of 2'-p-hydroxybenzoylmussaenosidic acid [negundoside (NG), against carbon tetrachloride (CCl(4))-induced toxicity in HuH-7 cells. CCl(4) is a well characterized hepatotoxin, and inducer of cytochrome P450 2E1 (CYP2E1)-mediated oxidative stress. In addition, lipid peroxidation and accumulation of intracellular calcium are important steps in the pathway involved in CCl(4) toxicity. Liver cells (HuH-7) were treated with CCl(4), and the mechanism of the cytoprotective effect of NG was assessed. Silymarin, a known hepatoprotective drug, was used as control. NG protected HuH-7 cells against CCl(4) toxicity and loss of viability without modulating CYP2E1 activity. Prevention of CCl(4) toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species (ROS), a decrease in lipid peroxidation and accumulation of intracellular Ca(2+) levels and maintenance of intracellular glutathione homeostasis. Decreased mitochondrial membrane potential (MMP), induction of caspases mediated DNA fragmentation and cell cycle arrest, as a result of CCl(4) treatment, were also blocked by NG. The protection afforded by NG seemed to be mediated by activation of cyclic adenosine monophosphate (cAMP) synthesis and inhibition of phospholipases (cPLA2). NG exerts a protective effect on CYP2E1-dependent CCl(4) toxicity via inhibition of lipid peroxidation, followed by an improved intracellular calcium homeostasis and inhibition of Ca(2+)-dependent proteases.

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

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

Ethanol increases mitochondrial cytochrome P450 2E1 in mouse liver and rat hepatocytes

Enhanced hepatic levels of cytochrome P450 2E1 (CYP2E1) may play a key role in the pathogenesis of some liver diseases because CYP2E1 represents a significant source of reactive oxygen species. Although a large fraction of CYP2E1 is located in the endoplasmic reticulum, CYP2E1 is also present in mitochondria. In this study, we asked whether ethanol, a known inducer of microsomal CYP2E1, could also increase CYP2E1 within mitochondria. Our findings indicated that ethanol increased microsomal and mitochondrial CYP2E1 in cultured rat hepatocytes and in the liver of lean mice. This was associated with decreased levels of glutathione, possibly reflecting increased oxidative stress. In contrast, in leptin-deficient obese mice, ethanol administration did not increase mitochondrial CYP2E1, nor it depleted mitochondrial glutathione, suggesting that leptin deficiency hampers mitochondrial targeting of CYP2E1. Thus, ethanol intoxication increases CYP2E1 not only in the endoplasmic reticulum but also in mitochondria, thus favouring oxidative stress in these compartments.

Lipopolysaccharide-induced liver injury in rats treated with the CYP2E1 inducer pyrazole

Elevated LPS and elevated cytochrome P-450 2E1 (CYP2E1) in liver are two major independent risk factors in alcoholic liver disease. We investigated possible synergistic effects of the two risk factors in causing oxidative stress and liver injury. Sprague-Dawley rats were injected intraperitoneally with pyrazole (inducer of CYP2E1) for 2 days, and then LPS was injected via tail vein. Other rats were treated with pyrazole alone or LPS alone or saline. Eight hours later, blood was collected and livers were excised. Pathological evaluation showed severe inflammatory responses and necroses only in liver sections from rats in the pyrazole plus LPS group; blood transaminase levels were significantly elevated only in the combination group. Activities of caspase-3 and -9 and positive terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were highest in the LPS alone and the LPS plus pyrazole group, with no significant difference between the two groups. Lipid peroxidation and protein carbonyls in liver homogenate as well as in situ superoxide production were maximally elevated in the LPS plus pyrazole group. Levels of nitrite plus nitrate and inducible nitric oxide (NO) synthase (iNOS) content were comparably elevated in LPS alone and the LPS plus pyrazole group; however, 3-nitrotyrosine adducts were elevated in the combined group but not the LPS group. It is likely that LPS induction of iNOS, which produces NO, coupled to pyrazole induction of CYP2E1 which produces superoxide, sets up conditions for maximal peroxynitrite formation and production of 3-nitrotyrosine adducts. CYP2E1 activity and content were elevated in the pyrazole and the LPS plus pyrazole groups. Immunohistochemical staining indicated that distribution of CYP2E1 was in agreement with that of necrosis and production of superoxide. These results show that pyrazole treatment enhanced LPS-induced necrosis, not apoptosis. The enhanced liver necrosis appears to involve an increase in oxidative and nitrosative stress generated by the combination of LPS plus elevated CYP2E1 levels.

Determinação das substâncias reativas ao ácido tiobarbitúrico como indicador da peroxidação lipídica em ratos tratados com sevoflurano

Sevoflurane is a fluorinated ether with low blood solubility and biotransformed by an oxidative enzymatic liver system involving cytochrome P450 2E1. Lipid peroxidation occurs during ethers biotransformation process under action of cytochrome P450, a possible mechanism for liver and kidney toxicity promoted by such compounds. This study aimed at determining the levels of substances reactive to thiobarbituric acid (TBARS), as an index for lipid peroxidation in sevoflurane-treated rats, previously treated or not with isoniazid, enzymatic inducer of cytochrome P450 2E1.Forty two male Wistar rats were randomly distributed in 4 groups receiving respectively: G1 - 1 L.min-1/60 minutes of 100% oxygen for 5 consecutive days; G2 - 4% sevoflurane in 1 L.min-1/60 minutes of 100% oxygen for 5 consecutive days; G3 - intraperitoneal isoniazid (50 mg.kg-1/day) for 4 consecutive days and then treated as G1; G4 - intraperitoneal isoniazid (50 mg.kg-1/day) for 4 consecutive days and then treated as G2. Animals were sacrificed 12 hours after the last treatment, plasma was collected for TBARS analysis and the liver left lobe and both kidneys were removed for histological evaluation.Results have shown increased TBARS levels in G3 and G4, with mild increase in G2. Histological evaluation has revealed focal liver necrosis in rats pretreated with isoniazid (G3).Sevoflurane has promoted lipid peroxidation only when associated to isoniazid.

P-Nitrophenol and glutathione response in medaka ( Oryzias latipes) exposed to MX, a drinking water carcinogen

When chlorine is introduced into public drinking water for disinfection, it can react with organic compounds in surface waters to form toxic by-products such as 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5 H]-furanone (MX). We investigated the effect of exposure to MX on cytochrome P450 2E1 (CYP2E1)-like activity and total glutathione (GSH) in the liver of the small fish model, medaka ( Oryzias latipes). The multi-site carcinogen methylazoxymethanol acetate (MAMAc) was the positive control compound. Both medaka liver microsome preparations and S-9 fractions catalyzed the hydroxylation of p-nitrophenol (PNP), suggesting CYP2E1-like activity in the medaka. Male medaka exposed for 96 h to the CYP2E1 inducers ethanol and acetone under fasted conditions showed significant increases in PNP-hydroxylation activity. Furthermore, total reduced hepatic GSH was reduced in fish fasted for 96 h, indicating that normal feeding is a factor in maintaining xenobiotic defenses. Exposure to MX and MAMAc induced significant increases in hepatic CYP2E1-like activity, however MX exposure did not alter hepatic GSH levels. These data strengthen the role of the medaka as a suitable species for examining cytochrome P450 and GSH detoxification processes and the role these systems play in chemical carcinogenesis.

Genetic Repeat Polymorphism in the Regulating Region of CYP2E1: Frequency and Relationship With Enzymatic Activity in Alcoholics

Background: Differences in the regulatory region of the CYP2E1 gene could be responsible for the interindividual variation in the cytochrome P-450 2E1 (CYP2E1) involved in ethanol oxidation. Recently, a polymorphic repeat sequence in the human gene was described between −2178 and −1945 base pairs. Its frequency seemed to vary among different ethnic populations, and it was suspected to be related to an increased inducibility to further ethanol intake. In the study reported here, the frequency of this polymorphism was investigated in a white French population. Its relationship with the previously described Pst I/Rsa I or Dra I CYP2E1 polymorphisms, alcoholism, alcoholic liver disease, and inducibility of CYP2E1 by ethanol was examined. Methods: The polymorphic region was characterized by polymerase chain reaction in 103 controls, 148 alcoholic subjects without liver diseases, and 98 others with liver cirrhosis. By using in vivo chlorzoxazone (CHZ) metabolism, CYP2E1 phenotype was assessed in 36 non–ethanol-induced subjects (17 controls and 19 withdrawn alcoholics) and in 14 ethanol-induced subjects (10 controls after ingestion of 0.8 g/kg ethanol and four alcoholics with 100 g of daily intake). This phenotype was expressed as the 6-hydroxy CHZ/CHZ ratio. Results: The rare allele frequency was found to be 1.58% in whites (n= 349). Neither significant association with alcoholism or alcoholic liver diseases, nor relationship with the Pst I/Rsa I polymorphism, was observed. But the Dra I polymorphism was more frequent among the heterozygous subjects when compared with wild-type homozygous ones (p < 0.05). The CYP2E1 phenotype was similar in wild-type homozygotes and in heterozygotes at the constitutive level, as well as after induction with ethanol. Conclusions: Our data suggest that CYP2E1 repeat polymorphism does not seem to constitute a major factor for interindividual differences in CYP2E1 expression and susceptibility to alcohol-related disorders in whites.

Genetic Repeat Polymorphism in the Regulating Region of CYP2E1: Frequency and Relationship With Enzymatic Activity in Alcoholics

Differences in the regulatory region of the CYP2E1 gene could be responsible for the interindividual variation in the cytochrome P-450 2E1 (CYP2E1) involved in ethanol oxidation. Recently, a polymorphic repeat sequence in the human gene was described between -2178 and -1945 base pairs. Its frequency seemed to vary among different ethnic populations, and it was suspected to be related to an increased inducibility to further ethanol intake. In the study reported here, the frequency of this polymorphism was investigated in a white French population. Its relationship with the previously described PstI/RsaI or DraI CYP2E1 polymorphisms, alcoholism, alcoholic liver disease, and inducibility of CYP2E1 by ethanol was examined. The polymorphic region was characterized by polymerase chain reaction in 103 controls, 148 alcoholic subjects without liver diseases, and 98 others with liver cirrhosis. By using in vivo chlorzoxazone (CHZ) metabolism, CYP2E1 phenotype was assessed in 36 non-ethanol-induced subjects (17 controls and 19 withdrawn alcoholics) and in 14 ethanol-induced subjects (10 controls after ingestion of 0.8 g/kg ethanol and four alcoholics with 100 g of daily intake). This phenotype was expressed as the 6-hydroxy CHZ/CHZ ratio. The rare allele frequency was found to be 1.58% in whites (n = 349). Neither significant association with alcoholism or alcoholic liver diseases, nor relationship with the PstI/RsaI polymorphism, was observed. But the DraI polymorphism was more frequent among the heterozygous subjects when compared with wild-type homozygous ones (p < 0.05). The CYP2E1 phenotype was similar in wild-type homozygotes and in heterozygotes at the constitutive level, as well as after induction with ethanol. Our data suggest that CYP2E1 repeat polymorphism does not seem to constitute a major factor for interindividual differences in CYP2E1 expression and susceptibility to alcohol-related disorders in whites.

Carbon Monoxide Generation from Trihalomethane Metabolism in Rats

Carboxyhaemoglobin (COHb) formation in rats after oral administration of trichloromethane (TCM), bromodi chloromethane (BDCM), chlorodibromomethane (CDBM) and tribromomethane (TBM) increased in the order TCM &lt; BDCM &lt; CDBM &lt; TBM. The trihalomethanes are sub strates for cytochrome P450 2E1 (CYP2E1) and cyto chromes P450 2B1/2 (CYP2B1/2) as shown by: (a) the inhibi tion of COHb formation due to simultaneous administra tion of CDBM or TBM and diethyldithiocarbamate, an inhibitor of CYP2E1, and (b) the initially higher levels of COHb after gavage of CDBM or TBM in rats pretreated with isoniazid, an inducer of CYP2E1, or pretreated with phenobarbital, an inducer of CYP281/2. There is an en hancement of the COHb level after daily administration of CDBM or TBM for 7 days in comparison to a single gav age, but the COHb levels were not higher due to chronic intake of CDBM with the drinking water (2.4 μmol.l-1) in comparison to the basic levels, measured during 26 weeks.

Expression of cytochrome P4502E1 in human liver: assessment by mRNA, genotype and phenotype.

Cytochrome P4502E1 (CYP2E1) is constitutively expressed in human liver and is responsible for the metabolic bioactivation of a wide variety of xenobiotics, including a number of protoxins and procarcinogens. CYP2E1 expression is regulated at several levels including pre-transcriptional, transcriptional and post-transcriptional levels, and any variation in enzyme concentration and hence activity may represent increased risk of toxicity or carcinogenicity. We have investigated variability in the levels of CYP2E1 mRNA, protein and functional activity in a human liver bank, and attempted to relate these parameters to the RsaI restriction fragment length polymorphism in the 5'-flanking region. Variation in CYP2E1 mRNA (18-fold) was greater than the variation seen in CYP2E1 protein (twofold) and functional activity (fourfold) determined using two probe substrates, chlorzoxazone and p-nitrophenol. Although protein and functional activity showed a significant correlation (r = 0.93 and r = 0.83 for chlorzoxazone and p-nitrophenol, respectively), there was no correlation between any of these parameters and mRNA levels. Also, the variation in CYP2E1 activity could not be directly accounted for by the RsaI polymorphism in our samples. In conclusion, our results are consistent with a complex regulation of CYP2E1 and the fact that it is highly conserved in the human population. The absence of a relationship between the RsaI polymorphism and CYP2E1 activity is consistent with other studies performed in Caucasians, but does not exclude an effect of this polymorphism on inducibility of CYP2E1.

The Detection of Cytochrome P450 2E1 and Its Catalytic Activity in Rat Testis

Cytochrome P450 2E1 participates in the bioactivation of a wide variety of environmental and occupational pollutants. Such reactions may lead to the production of active carcinogenic metabolites. The presence of P450 2E1 in the testis and prostate has not yet been reported. In the present study, cytochrome P450 2E1 mRNA has been identified in the rat prostate and testis by reverse transcription PCR, southern blotting, and DNA sequencing. P450 2E1 protein from rat testis could be detected with immunoblot analysis, but was not detected in the prostate. The hydroxylation ofp-nitrophenol, known to be mediated by P450 2E1, was demonstrated by HPLC measurement of product formation in microsomal fractions from the rat testis, but again not from prostate. Exposure of rats to pyridine resulted in a 2.9-fold increase ofp-nitrophenol hydroxylation by testicular microsomes. Diethyldithiocarbamate, a selective mechanism-based inhibitor of P450 2E1, or a P450 2E1 monoclonal antibody, caused marked inhibition of testicular microsomalp-nitrophenol hydroxylase activity. These results indicate that cytochrome P450 2E1 is present in the rat testis, and that it is elevated by treatment of the animals with pyridine. Thus, the presence and inducibility of cytochrome P450 2E1 in the testis may be of significance in the bioactivation of environmental chemicals to genotoxic metabolites.

Chlorodibromomethane metabolism to bromide and carbon monoxide in rats

The chlorodibromomethane (CDBM) metabolites bromide and CO were analysed as bromide level in plasma and carboxyhaemoglobin (COHb) level in blood of rats, respectively. The mean basic levels of bromide in plasma of rats receiving vehicle were 0.075 +/- 0.036 mmol/l (n = 27). After administration of CDBM at 0.4, 0.8, 1.6, and 3.1 mmol/kg p.o., the mean bromide levels rose to maximal values that were higher by a factor 27, 48, 69, and 135, respectively. Bromide elimination was slow and the plasma level was significantly increased following repeated administration in comparison to a single administration of CDBM. The CDBM concentrations in blood and in fat tissue 6 h after the last of 7 administrations of 0.8 mmol CDBM/ kg p.o., once a day for 7 consecutive days, were significantly lower than 6 h after a single gavage of this CDBM dose. The mean normal level of 0.45 +/- 0.32% COHb in rats (n = 30) was significantly increased following oral CDBM uptake. Initially higher COHb levels were measured after 7 consecutive applications of 0.8 mmol/kg CDBM. After a single administration of CDBM the level of glutathione disulphide in the liver was significantly increased; this effect was reversible. The oxidative CDBM metabolism was influenced by the glutathione (GSH) concentration in the liver. The rate of COHb and bromide formation was decreased after GSH depletion due to pretreatment of rats with buthionine sulphoximine (BSO) and increased following enhancement of the GSH concentration due to pretreatment of the animals with butylated hydroxyanisole (BHA). CDBM is a substrate for cytochrome P-450 2E1 (CYP2E1), as demonstrated by the inhibition of bromide and COHb formation due to simultaneous administration of CDBM and the CYP2E1 inhibitor diethyldithiocarbamate (DDTC); also by the initially higher levels of bromide in plasma and COHb in blood after gavage of CDBM pretreated with isoniazid (INH), an inducer of CYP2E1. The increase of bromide formation after CDBM administration in phenobarbital (PB)-pretreated rats indicated that cytochrome P-450 2B1 and 2B2 (CYP2B1 and CYP2B2) play a role as catalysts of the CDBM biotransformation. It is shown that m-xylene pretreatment, which activates CYP2E1 as well as CYP2Bs, leads to a higher bromide level after CDBM administration than the INH or PB pretreatment. In liver microsomes of rats treated with CDBM (0.8 mmol/kg p.o., seven daily applications), the p-nitrophenol hydroxylase (p-NPH) activity, a market of CYP2E1, was increased. It is concluded that CDBM may be an inducer of CYP2E1. These results combined with literature data demonstrate that the oxidation of CDBM was catalysed mainly by CYP2E1 and CYP2Bs and that there may be a risk of bromide accumulation following repeated uptake of the trihalomethane.

Expression, catalytic activity, and inducibility of cytochrome P450 2E1 (CYP2E1) in the rat central nervous system.

Cytochrome P450 2E1 (CYP2E1) metabolizes several neuroactive substrates, including exogenous compounds such as anesthetics, organic solvents, and muscle relaxants as well as endogenous substrates such as arachidonic acid. CYP2E1 and its mRNA were found to be expressed in the rat hippocampus, where the enzyme was localized mainly to the microsomal fraction. Chlorzoxazone (CZN), a CYP2E1 substrate, was 6-hydroxylated in hippocampal homogenates with a K(m) of 25.5 microM and a Vmax of 0.22 pmol/mg/min. CYP2E1 was also expressed in vitro in cortical glial cultures, where CYP2E1 mRNA levels were found to be 1,000-fold lower than in rat liver. Exposure of cortical glial cultures to 25 or 100 mM ethanol for 24 h caused a fourfold and sixfold increase, respectively, in the rate of CYP2E1-dependent 6-hydroxylation of CZN. After a continuous exposure to 100 mM ethanol for 48 or 72 h, however, the hydroxylation rate was down-regulated. Chlormethiazole, a potent inhibitor of hepatic CYP2E1 transcription, inhibited the ethanol-dependent induction of CYP2E1 by 50%. In vivo, acute ethanol treatment of rats (24 h, 3 g/kg) resulted in a 1.8-fold increase in the rate of CZN 6-hydroxylation in hippocampal homogenates. It is concluded that CYP2E1 is expressed and catalytically active in the rat CNS, and that CYP2E1 can be induced by a relatively low concentration of ethanol in cortical glial cultures. It is suggested that CYP2E1 substrates may be metabolically activated in situ in the CNS.

12] Chlorzoxazone: An in vitro and in vivo substrate probe for liver CYP2E1

Publisher Summary Cytochrome P4502E1 (CYP2E1) is involved in the oxidation of ethanol and a diverse group of suspect carcinogens, including nitrosamines, benzene, phenol, chloroform, trichloroethylene, ethylene dihalides, methylene dihalides, styrene, butadiene, vinyl halides, and urethane. The same chemical probe would have had to be convenient for in vitro studies, including the determination of CYP2E1 catalytic activities in microsomal preparations or in cell cultures. Chlorzoxazone, a potent muscle relaxant used in the treatment of painful muscle spasms, has been shown to be an ideal chemical probe specific for CYP2E1 in both in vitro and in vivo studies. In vitro studies comparing the abilities of several purified recombinant human P450 enzymes have demonstrated that CYP2E1 is the major catalyst of chlorzoxazone 6-hydroxylation in human liver. Data obtained from in vivo studies in alcoholic patients, before or after ethanol withdrawal, or in patients treated with either CYP2E1 inducers (isoniazid) or inhibitors (disulfiram) are in agreement with data obtained from in vitro liver microsomal preparations. Therefore, chlorzoxazone should be a suitable in vitro and in vivo probe for human liver CYP2E1.

Acetylsalicylic acid--inducer of cytochrome P-450 2E1?

Pretreatment of rats with acetylsalicylic acid or sodium salicylate stimulates the metabolism of dichloromethane to carbon monoxide as measured by the carboxyhemoglobin level in blood. Simultaneous administration of dichloromethane and acetylsalicylic acid or sodium salicylate, respectively, was accompanied by reduced carboxyhemoglobin formation. In liver microsomes of rats pretreated with acetylsalicylic acid the p-nitrophenol hydroxylase activity was increased. It is concluded that (i) cytochrome P-450 2E1 is involved in the metabolic conversion of both dichloromethane and salicylic acid, and (ii) salicylic acid may be an inducer of cytochrome P-450 2E1.

Investigation of the mechanistic basis of N,N-dimethylformamide toxicity. Metabolism of N,N-dimethylformamide and its deuterated isotopomers by cytochrome P450 2E1.

Dimethylformamide (DMF) is an industrial solvent with hepatotoxic properties. The toxicity of DMF has been associated with its metabolism to S-(N-methylcarbamoyl)glutathione (SMG). The major urinary metabolite of DMF is N-(hydroxymethyl)-N-methylformamide (HMMF). HMMF undergoes oxidation in the formyl moiety, possibly via the intermediacy of its hydrolysis product N-methylformamide (NMF), and the reactive intermediate thus generated reacts with glutathione to yield SMG. Experiments were conducted to elucidate enzymatic details of the metabolism of DMF. Generation of HMMF from DMF in microsomes from rats which had received acetone, an inducer of cytochrome P450 2E1, was increased by 175% over that observed in control microsomes. In liver microsomes from 4 humans the metabolism of DMF to HMMF was inhibited by a monospecific antibody against rat liver P450 2E1, and the metabolic rates were correlated with those of NMF to SMG, a process known to be mediated via P450 2E1. DMF was also metabolized by purified rat liver P450 2E1. The kinetic parameters which characterize the metabolism of DMF or its deuterated isotopomers to the respective HMMF isotopomers, of HMMF to SMG and of NMF to SMG in liver microsomes, were computed from Eadie-Hofstee plots. The affinity of DMF for the metabolizing enzyme in rat liver microsomes is considerably higher (apparent Km = 0.20 mM) than that of NMF (Km = 4.28 mM) or of HMMF (Km = 2.52 mM). The respective values observed with human microsomes are very similar. The apparent Km values for the N-methyl oxidation of N,N-dimethyldeuterioformamide ([2H1]DMF) and N,N-bis(trideuteriomethyl)formamide ([2H6]DMF) in rat microsomes are 0.14 and 0.21 mM, respectively. The apparent Vmax for the oxidation of [2H1]DMF is similar to that computed for DMF, and the Vmax for [2H6]DMF is less than half of that computed for DMF. The kinetic deuterium isotope effect (KDIE) on DMF metabolism was determined in incubations with rat microsomes in three ways: (i) the noncompetitive intermolecular KDIE by the ratio of Vmax/Km for DMF to Vmax/Km for [2H6]DMF, (ii) the competitive intermolecular KDIE as the quotient of metabolic products HMMF to N-(hydroxydideuteriomethyl)-N-(trideuteriomethyl)formamide in incubations of DMF together with [2H6]DMF, and (iii) the intramolecular KDIE as the quotient of the ratio of N-(hydroxymethyl)-N-(trideuteriomethyl)formamide to N-(hydroxydideuteriomethyl)-N-methylformamide generated from N-(trideuteriomethyl)-N-methylformamide ([2H3]DMF). The respective values were found to be (i) 2.4, (ii) 5.0, and (iii) 5.2. DMF inhibited the oxidation of NMF or HMMF to SMG.(ABSTRACT TRUNCATED AT 400 WORDS)