Jean-François Ménez

Assessment of cytochrome P4502E1 induction in alcoholic patients by chlorzoxazone pharmacokinetics

Chlorzoxazone is mainly metabolized to 6-hydroxychloroxazone (6-OHchlorzoxazone) by the ethanol-inducible cytochrome P450 2E1 (CYP2E1). To evaluate the impact of ethanol consumption on the enzyme induction, the pharmacokinetics of chlorozoxazone and 6-OHchlorzoxazone were studied in alcoholic and control subjects. Fifteen alcoholic male inpatients (all smokers, daily intake 333 +/- 191 g of absolute ethanol) and 20 healthy male volunteers (10 smokers and 10 non-smokers, weekly intake < 100 g of absolute ethanol) participated in this study. Following a 12 hr fasting period, each subject was orally administered 500 mg of chlorzoxazone. Venous blood and urine samples were collected over a 10 hr period. Areas under the curve of plasma concentration versus time (AUC) of chlorzoxazone and 6-OHchlorzoxazone was calculated. The total plasma clearance of chlorzoxazone was measured as the dose/AUC ratio. The mean total plasma clearance was not different between smoker and non-smoker controls but it was enhanced by 73% in alcoholic patients. These results indicate a negligible and non-significant effect of cigarette smoking in controls but an increased metabolism of chlorzoxazone in alcoholic patients (P < 0.05). This increase was corroborated by the 2-fold enhancement of the 6-OHchlorzoxazone/chlorzoxazone AUC ratio, compared to controls. A good correlation was found between this AUC ratio and the 6-OHchlorzoxazone/chlorzoxazone concentration ratio at t = 2 hr in patients and in controls (r = 0.88 and 0.85, respectively, P < 0.01). The concentration ratio increased by 150% in alcoholic patients and decreased by 65% in the seven alcoholics tested after 7 days of alcohol abstinence. It is therefore concluded that the 6-OHchlorzoxazone/chlorzoxazone concentration ratio at t = 2 hr could constitute a simple and non-traumatic marker of CYP2E1 induction.

Ethanol-induced cell death in cultured rat astroglia

Because of the important role of glial cells in brain maturation and reports on their delayed proliferation following ethanol exposure, it was considered of interest to investigate the mechanism of ethanol action on these cells. Biochemical parameters related to the apoptotic and necrotic processes in astroglial cells exposed for 1 week to 50 and 100 mM ethanol were examined. Ethanol increased intracellular calcium levels without changing transglutaminase activity and nitrite levels. Moreover, DNA fragmentation was noted with flow cytometry and with the random oligonucleotide primed synthesis assay but neither following agar gel electrophoresis nor in UV microscopy of cell nuclei. The DNA patterns obtained were different from these seen in programmed cell death. Additionally, immunocytochemical analysis showed greater fragility of astrocytes than oligodendrocytes to ethanol. These results support the hypothesis that astroglial cells in vitro exposed to ethanol die due to necrotic but not apoptotic mechanisms.

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.

High-performance liquid chromatographic determination of chlorzoxazone and 6-hydroxychlorzoxazone in serum: a tool for indirect evaluation of cytochrome P4502E1 activity in humans.

Chronic alcohol consumption is known to induce the enzyme cytochrome P4502E1 (CYP2E1), which is involved in the toxicity and carcinogenicity of a number of solvents and xenobiotics. It was recently suggested that in vivo chlorzoxazone metabolism could be a potential tool as a non-invasive probe for measuring CYP2E1 activity in humans. Therefore, a simple and sensitive method was developed for the determination of chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone in both serum and urine. Biological samples were hydrolysed by Helix pomatia juice, deproteinized with perchloric acid, and then extracted using ethyl acetate. The compounds were separated by high-performance liquid chromatography on an octadecylsilane column with a mobile phase of acetonitrile-0.5% acetic acid in water (30:70, v/v) and detected at 287 nm. The linearity of the method was tested in the concentration range 0.5-20 micrograms/ml, and the limit of detection in biological samples was found to be 0.5 microgram/ml. Within- and between-run precision was below 5% and 10%, respectively, for both compounds at three concentrations (0.5, 10 and 20 micrograms/ml). The accuracy of the procedure was in the range 0.3-6%. Serum levels and urinary excretion of chlorzoxazone and its metabolite were studied in five healthy controls and five alcoholic patients, following oral administration of 500 mg of chlorzoxazone. The concentration ratio 6-hydroxychlorzoxazone/chlorzoxazone in blood was shown to be a valuable tool for the evaluation of CYP2E1 activity in humans.

Cytochrome P450 4A and 2E1 expression in human kidney microsomes.

Laurate and arachidonate omega and (omega-1)-hydroxylase activities, cytochrome P450 2E1 (CYP2E1), and CYP4A content were measured in 18 human kidney microsomal samples. The rates of laurate and arachidonate were found to be very different from those measured in human liver samples, with a laurate omega/omega-1 ratio of approximately 22 in human kidney vs 0.75 in human liver. Immunoblot analysis of the 18 human kidney microsomal samples identified 1 CYP4A electrophoretic band, but CYP2E1 was not detectable in human kidney, contrary to liver. Laurate and arachidonate omega-hydroxylase activities were significantly correlated with CYP4A content (r = 0.86 and 0.75, respectively). Polyclonal antirat CYP2E1 antibody did not affect omega-hydroxylase activity, whereas the polyclonal antirat CYP4A1 antibody inhibited it by 60%. These results suggest that, in contrast to other species, human kidney microsomes do not contain significant amounts of CYP2E1, but possess CYP4A and fatty acid omega-hydroxylase activity.

Induction of liver and kidney CYP1A1/1A2 by caffeine in rat☆

Caffeine metabolism by hepatic microsomal P450 enzymes is well documented in experimental animals and humans. However, its induction effect on P450 enzymes has not been thoroughly studied. In a preliminary experiment, the time-dependent incubation of 1 mM caffeine with rat hepatocyte culture resulted in an increase of its own metabolic rate. The dose-dependent expression of rat hepatic and renal cytochromes (CYP) 1A1/1A2 was then investigated after per os administration of caffeine. P450 expression was monitored by using specific enzymatic activities and Northern blot analysis. Caffeine caused a dose-dependent elevation of hepatic CYP1A1/1A2 activities in microsomal preparations, which ranged from 1.7- to 6-fold for ethoxyresorufin O-deethylase and 3- to 8.9-fold for methoxy-resorufin O-demethylase according to the dose regimen of 50 and 150 mg caffeine/kg/day for 3 days, respectively. Northern blot analysis demonstrated that caffeine treatment increased liver CYP1A1 and CYP1A2 mRNA levels over the dose regimen of 50-150 mg caffeine/kg/day for 3 days, respectively. The result of this study demonstrates that caffeine increases its own metabolism in a dose-dependent manner and induces CYP1A1/1A2 expression through either transcriptional activation or mRNA stabilization.

Validation of the (ω-l)-hydroxylation of lauric acid as an in vitro substrate probe for human liver CYP2E1

Abstract The (ω-1)-hydroxylation of lauric acid (11-OH-LA), a model substrate of fatty acids, was previously shown to be due to CYP2E1 in rat liver microsomes. The present study examined changes in hepatic CYP2E1 content and 11-OH-LA in a panel of 29 human liver microsomes. The 11-OH-LA activity was strongly correlated with the CYP2E1 content, quantitated by immunoblot ( r = 0.75) and with four monooxygenase activities known to be mediated by CYP2E1: chlorzoxazone-6-hydroxylation ( r = 0.73), 4-nitrophenol hydroxylation ( r = 0.84), N -nitrosodimethylamine demethylation ( r = 0.79) and n-butanol oxidation ( r = 0.73). The (ω-1)-hydroxylation of lauric acid was inhibited by ethanol ( K i = 3.5 mM), acetone (IC 50 = 10 mM), dimethylsulfoxide, chlorzoxazone (competitive inhibitors of CYP2E1), diethyldithiocarbamate, and diallylsulfide (both selective mechanism-based inactivators of CYP2E1). The weak value of ethanol K i on the (ω-1)-hydroxylation of lauric acid suggested that low levels of alcohol could modify fatty acid metabolism in the liver. Furafylline and gestodene, suicide substrates of CYP1A and CYP3A4, respectively, did not modify the 11-hydroxylation of lauric acid. Polyclonal antibody directed against rat CYP2E1 inhibited the formation of 11-OH-LA without affecting 12-OH-LA activity. Taken together, these results suggest that CYP2E1 is involved in the (ω-1)-hydroxylation of lauric acid in human liver microsomes, and ω-hydroxylation is mediated by another enzyme. Finally, the use of yeasts and mammalian cells genetically engineered for expression of 9 human P450s demonstrated that CYP2E1 was the one enzyme involved in the (ω-1)-hydroxylation of lauric acid.

Acetaminophen metabolism and cytotoxicity in PC12 cells transfected with cytochrome P4502E1

Abstract Although a number of studies confirm the important role of metabolites in the cytotoxicity of acetaminophen, its precise mechanisms remain unknown. Acetaminophen is metabolized by microsomal enzymes. Cytochrome P4502E1 (CYP2E1) mediated N-hydroxylation results in the formation of N-acetyl-benzo-quinoneimine, a highly reactive intermediate. We examined biochemical parameters related to necrotic and apoptotic processes in acetaminophen-exposed PC12 cells is and in a PC12 cell line genetically engineered to express human CYP2E1. Both the [3H]thymidine incorporation test and the protein assay uniformly showed dose- and time-related significant growth retardation in both cell lines exposed to the drug. This was more evident in CYP2E1-transfected cells. Moreover, the cytotoxic effect of acetaminophen was increased as evidenced by lactate dehydrogenase activity in the culture medium. Both random oligonucleotide primed synthesis assay and enzyme-linked immunosorbent assay revealed significant DNA fragmentation in both cell lines, which was greater in transfected cells, reaching about 11% of total cellular DNA. These results were confirmed by flow cytometry and microscopic examination of cell nuclei. Intracellular calcium levels were increased only in transfected cells, approximately threefold when 5 mM acetaminophen was administered for 48 h. These results indicate the cytotoxic effects of acetaminophen via apoptosis, necrosis, and growth retardation. While the precise mechanism remains obscure, it seems that DNA fragmentation and apoptotic cascade represent a preliminary biochemical event in acute cell death, and that acetaminophen biotransformation by CYP2E1 stimulates this pathway.

Caffeine increases its own metabolism through cytochrome P4501A induction in rats

Caffeine is one of the most widely used - and maybe abused - xenobiotic compounds in the world. If numerous pharmacological properties of caffeine have been reported, the effects of caffeine treatment on the hepatic drug-metabolizing enzyme system have been scarcely studied. Pretreatment of rats for 3 days with 150 mg/kg/day of caffeine dramatically increased P4501A and P4502B dependent catalytic activities determined in vitro. Furthermore, N-demethylations and C-8 oxidation of caffeine were increased by about 2 fold by caffeine treatment. Immunoblot analysis demonstrated that the liver contents of P4501A2 and P4502B1/2B2, known to be involved in these monooxygenase activities, increased also by about 2 fold. Cytochrome P4503A1 and 2E1 were not modified. Taken together, there data suggest that caffeine increases its own metabolism through P4501A induction.

Simultaneous radiometric and fluorimetric detection of lauric acid metabolites using high-performance liquid chromatography following esterification with 4-bromomethyl-6,7-dimethoxycoumarin in human and rat liver microsomes

The formation of (omega-1)-hydroxylauric acid from lauric acid (LA) can be used as an indicator of the activity of cytochrome P450 2E1 (CYP2E1) in rat and human liver microsomes. A high-performance liquid chromatographic (HPLC) method that is capable of identifying and measuring the two main metabolites of lauric acid, (omega-1)- and omega-OH-LA, has been developed and used in the study of rat and human liver microsomes. Measurement of the enzymatic activities, based on the esterification of the metabolites and substrate with the fluorescent agent, 4-bromomethyl-6, 7-dimethoxycoumarin, is described using both radiometric and fluorimetric detection methods. Extraction efficiencies of metabolites and residual substrate were calculated using radioactivity and were greater than 85%. The assay is accurate and reproducible and has a detection limit of 75 pg (0.37 pmol). Additionally, a strong correlation between the two techniques was found in both human (r = 0.945, n = 15, p < 0.01) and rat (r = 0.949, n = 18, p < 0.01) livers, for the (omega-1)-hydroxylauric acid.