2E1 Substrates Research Articles

Mutagenicity of cytochrome P450 2E1 substrates in the Ames test with the metabolic competent S. typhimurium strain YG7108pin3ERb 5

Poor metabolic competence of in vitro systems was proposed to be one of their major shortcomings accounting for false negative results in genotoxicity testing. For several “low molecular weight cancer suspects” this was specifically attributed to the lack of cytochrome P450 2E1 (CYP2E1) in conventional in vitro metabolising systems. One promising attempt to overcome this problem is the transfection of “methyltransferase-deficient” S. typhimurium strains with the plasmid pin3ERb 5. This plasmid contains DNA encoding for a complete electron transport chain, comprising P450 reductase, cytochrome b 5 and cytochrome P450 2E1. In order to answer the question if CYP2E1 substrates that yield negative or inconclusive results in the Ames test can be activated by metabolic competent bacterial strains, we used YG7108pin3ERb 5 to investigate the following compounds: acetamide, acrylamide, acrylonitrile, allyl chloride, ethyl acrylate, ethyl carbamate, methyl-methacrylate, vinyl acetate, N-nitrosopyrrolidine, trichloroethylene and tetrachloroethylene. N-Nitrosodiethylamine served as a positive control. In addition to these known or proposed CYP2E1 substrates, we investigated the polycyclic aromatic hydrocarbon benzo[α]pyrene and the heterocyclic aromatic amines 2-aminofluorene and 2-aminoanthracene. Results The extensive metabolic competence of the transformed strain is underlined by results showing strong mutagenicity between 10 and 500 μg N-nitrosopyrrolidine per plate. Unexpectedly, 2-aminoanthracene was mutagenic at a concentration range between 25 and 250 μg per plate using YG7108pin3ERb 5. Moreover, we demonstrate for the first time a clear response of sufficiently characterised allyl chloride in the Ames test at a reasonably low concentration range between 300 and 1500 μg per plate. We achieved similar results in the parent strain YG7108 with conventional metabolic activation. Without metabolic activation less pronounced mutagenicity occurred, suggesting a contribution of a direct alkylating effect. Propylene oxide is usually contained in allyl chloride as stabilizer at amounts up to 0.09%. Though YG7108 revealed to be very sensitive towards propylene oxide, allyl chloride dissolved in water was not mutagenic, showing that no water soluble compounds contribute to its mutagenicity. None of the remaining compounds showed mutagenic effects using YG7108pin3ERb 5. Conclusion YG7108pin3ERb 5 and its parent strain YG7108 are sensitive for compounds which are negative in conventional tester strains including N-nitrosodiethylamine, N-nitrosopyrrolidine, propylene oxide and allyl chloride.

Conformational Modulation of Human Cytochrome P450 2E1 by Ethanol and Other Substrates: A CO Flash Photolysis Study

The alcohol-inducible cytochrome P450 2E1 is a major human hepatic P450 which metabolizes a broad array of endogenous and exogenous compounds, including ethanol, low-molecular weight toxins, and fatty acids. Several substrates are known to stabilize this P450 and inhibit its cellular degradation. Furthermore, ethanol is a known modulator of P450 2E1 substrate metabolism. We examined the CO binding kinetics of P450 2E1 after laser flash photolysis of the heme-CO bond, to probe the effects of ethanol and other substrates on protein conformation and dynamics. Ethanol had an effect on the two kinetic parameters that describe CO binding: it decreased the rate of CO binding, suggesting a decrease in the protein's conformational flexibility, and increased the photosensitivity, which indicates a local effect in the active site region such as strengthening of the heme-CO bond. Other substrates decreased the CO binding rate to varying degrees. Of particular interest is the effect of arachidonic acid, which abolished photodissociation in the absence of ethanol but had no effect in the presence of ethanol. These results are consistent with a model of P450 2E1 whereby arachidonic acid binds along a long hydrophobic binding pocket and blocks exit of CO from the heme region.

Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides.

A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.

Effect of pyrazole and dexamethasone administration on cytochrome P450 2E1 and 3A isoforms in rat liver and kidney: lack of specificity of p-nitrophenol as a substrate of P450 2E1.

The induction effects of pyrazole and dexamethasone (known to be specific to P450 2E1 and 3A enzymes, respectively), given alone or simultaneously, were studied in rat liver and kidney microsomes. Pyrazole treatment induced the catalytic activity and the amount of P450 2E1 enzyme in both organs. Immunoreactive P450 2E1 and 4-nitrophenol 2-hydroxylation increased 8- and 13-fold, respectively (versus control), in the kidney, but only 2.4- and 2.7-fold (versus control) in the liver after pyrazole treatment. As assessed by nifedipine oxidation activity, dexamethasone treatment increased the P450 3A catalytic activity approximately 4-fold (versus control) in the liver, but not in the kidney, suggesting that P450 3A was not inducible in the kidney. Pyrazole decreased P450 3A activity in the liver but did not modify it in the kidney. A combination of both chemicals induced both enzymes, but to a lesser extent than treatment with each single chemical compound. Furthermore, the 2-hydroxylation of p-nitrophenol, considered one of the most specific substrates for monitoring the level of P450 2E1, was mediated also by P450 3A, at least in dexamethasone-treated rats. Finally, this experimental work demonstrated that P450 3A induction is organ-specific, and it also demonstrated the lack of specificity of p-nitrophenol as a P450 2E1 substrate.

Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1: RATE-LIMITING PRODUCT RELEASE ACCOUNTS FOR EFFECTS OF ISOTOPIC HYDROGEN SUBSTITUTION AND CYTOCHROME b5 ON STEADY-STATE KINETICS

A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of approximately 5 on Km (DK = DKm/HKm), but not on kcat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observed DKm values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k >1000 min-1) even in the absence of substrate. These results indicate that the basis for the increase in Km is in the latter portion of the catalytic cycle. The intrinsic isotope effect (Dk) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min-1), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which the kcat approximates the rate of product release, and Km is an expression in which the denominator is dominated by the rate of C-H bond breaking.

Determination of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma and urine by high-performance liquid chromatography.

A sensitive and reproducible method is described for the determination of the cytochrome P450 enzyme 2E1 substrate chlorzoxazone and its primary metabolite 6-hydroxychlorzoxazone in human plasma and urine. Plasma or diluted urine were acidified, incubated with beta-glucuronidase and then were extracted with diethyl ether. Separation of the analytes was achieved on a C18 column with UV detection set at 283 nm. Excellent linearity was observed over the concentration ranges of 100-3000 ng/ml and 4-400 micrograms/ml in plasma and urine, respectively. The intra-assay variability was < or = 5.1% and the inter-assay variability was < or = 8.2% for each compound in each matrix. The method presented is applicable to pharmacokinetic and pharmacogenetic studies utilizing chlorzoxazone.

Kinetic Analysis of the Activation of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone by Heterologously Expressed Human P450 Enzymes and the Effect of P450- Specific Chemical Inhibitors on This Activation in Human Liver Microsomes

The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the α-methyl and α-methylene groups, leading to the formation of reactive species which can pyridyloxobutylate and methylate DNA, respectively. The present study examined the kinetic parameters of NNK-derived keto alcohol (α-methyl hydroxylation), and keto aldehyde (α-methylene hydroxylation) formation catalyzed by human P450s heterologously expressed by either the baculovirus–insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane preparations of the expressed P450s catalyzed the α-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol. Human P450 2A6 showed the lowestKM(118 μm) for the formation of keto aldehyde. A similarKMwas observed for keto alcohol formation by expressed P450 2A6, but thekcatwas lower than the value obtained for keto aldehyde formation. The addition of exogenous b5increased the expressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both α-hydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capacity for keto alcohol formation; however, theirKMvalues for this reaction were in the millimolar range. Expressed human P450 3A4 oxidized NNK to keto aldehyde also with a highKM. Ten human liver microsomal samples were each shown to activate NNK to keto aldehyde and keto alcohol. A positive correlation coefficient of 0.74 was found between keto aldehyde formation and both coumarin 7-hydroxylation (P450 2A6) and 6β-testosterone hydroxylation (3A4) activity in characterized human liver microsomes. Keto alcohol formation showed a significant correlation with ethoxyresorufin O-dealkylation (P450 1A2) in human liver microsomes. Both coumarin and troleandomycin, specific inhibitors of P450 2A6 and 3A4, respectively, inhibited the formation of keto aldehyde, but inhibited the formation of keto alcohol only slightly in human liver microsomes. Both furafylline, a P450 1A2 inhibitor, andN-nitrosodimethylamine, a P450 2E1 substrate, inhibited the formation of keto alcohol but not keto aldehyde in human liver microsomes. Quinidine, a specific inhibitor of P450 2D6, was not an effective inhibitor of NNK metabolism. These results demonstrate that P450s 2A6 and 3A4 may be important P450s for the activation of NNK to a DNA-methylating agent and keto aldehyde via the α-methylene hydroxylation pathway. P450s 1A2, 2E1, and 2D6 are shown to be selective for α-methyl hydroxylation of NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent.

Chloroethylene mixtures: pharmacokinetic modeling and in vitro metabolism of vinyl chloride, trichloroethylene, and trans-1,2-dichloroethylene in rat.

Environmental and occupational exposures are typically to mixtures of chemicals, although most toxicity information is for individual compounds. Interactions between chemicals may involve pharmacokinetic and/or pharmacodynamic effects resulting in modulation of toxicity. Therefore, physiologically based pharmacokinetic modeling has been used to analyze data describing the metabolism of vinyl chloride (VC) and trichloroethylene (TCE) mixtures in rats. A single saturable pathway was modeled, representing cytochrome P450 2E1. This was partially validated using preexposure to trans-1,2-dichloroethylene (tDCE) which virtually eliminated in vivo metabolism of both VC and TCE at low concentrations. Microsomes from tDCE-exposed animals showed inhibition of metabolism of P450 2E1 substrates (chlorzoxazone, p-nitrophenol, and TCE) and no effect on 7-ethoxycoumarin deethylation. Studies with liver microsomes from VC-exposed animals found that neither suicide inhibition nor induction occurred during 6-hr exposures to high concentrations. Therefore, these effects were not modeled. Modeling of mixtures of VC and TCE was successful only using competitive inhibition, as might be predicted for cytochrome P450 2E1 substrates, and not uncompetitive or noncompetitive inhibition. These results were further confirmed by determining the depletion of glutathione due to VC metabolism. The validation of a detailed model for the inhibition kinetics of metabolism of these two compounds permits better understanding of the implications of coexposures for toxicity. It is notable that competitive inhibition only becomes significant at relatively high concentrations (tens of ppm), while at typical low environmental concentrations (ppb), absorption is perfusion limited and enzyme is in excess so that the chemicals will be metabolized independently.

Probing the Active Sites of Rat and Human Cytochrome-P450 2E1 with Alcohols and Carboxylic Acids

Cytochrome P450 2E1 (P450 2E1) catalyzes the biotransformation of many low-molecular-weight compounds including industrial solvents, indoor pollutants, alcohol, and drugs. In order to understand the nature of the P450 2E1 active site, we studied the competitive inhibition of the P450 2E1-catalyzed N-nitrosodimethylamine demethylation by alcohols and carboxylic acids with different alkyl chain lengths. Using microsomes from acetone-treated rats as the enzyme source of P450 2E1, the K i value for each compound was measured. With primary alcohols and secondary alcohols, the K i decreased with the increase in the carbon chain length until the carbon number reached 6 or 7; the free energy increment of binding was 0.28 kcal/mol CH 2 group. Similar inhibitory effects were also observed with human P450 2E1 heterologously expressed in Hep G2 cells. These results suggest that both rat and human P450 2E1 contain a pocket with hydrophobicity that serves as a binding site for low-molecular-weight substrates. Among a series of carboxylic acids and ω-hydroxycarboxylic acids investigated, dodecanoic acid was the strongest inhibitor ( K i = 22 μM), and 12-hydroxydodecanoic acid had the lowest K i (320 μM) within the series of ω-hydroxycarboxylic acids. The free energy increment of binding for carboxylic acids was 0.35 kcal/mol CH2 group. 1,10-Decanedicarboxylic acid, which contains a carboxylic group at each end, showed a K i > 20 mM. We suggest that for optimal interaction of a carboxylic acid moiety with the active site of P450 2E1, the hydrocarbon end of the molecule binds to the substrate binding site, leaving the carboxylic acid group outside of a proposed substrate access channel. The length of optimal substrates such as dodecanoic acid may reflect the length of the substrate access channel and the size of the active site. We estimate that the distance from the opening of the access channel to the oxygenation site is about 15 Å. Based on the structural features of P450 2E1 substrates and competitive inhibitors, we propose a conceptual model to illustrate the binding of these molecules in the active site of P450 2E1.

Hepatoprotective mechanism of silymarin: No evidence for involvement of cytochrome P450 2E1

The involvement of the alcohol-inducible cytochrome P450 2E1 in the hepatoprotective mechanism of the plant flavonoid extract silymarin, and its main active component silybin, was investigated in isolated hepatocytes. Allyl alcohol toxicity, associated lipid peroxidation and GSH depletion was efficiently counteracted by silymarin (0.01–0.5 mM), and at higher concentrations by silybin. Cell damage by t-butyl hydroperoxide was also prevented by silymarin and silybin, but less efficiently. However, the covalent binding of the acetaminophen intermediate, formed via P450 2E1, was unaffected by addition of the flavonoids. Silybin did not influence microsomal 2E1-catalyzed demethylation of N-nitrosodimethylamine. Neither did demethylation of N-nitrosodimethylamine or aminopyrine by isolated microsomes affect the in vivo administration of silybin. Addition of silymarin or silybin to primary cultures of isolated hepatocytes did not prevent cell damage induced by exposure to the P450 2E1 substrate CCl 4. In contrast, the mere presence of low concentrations (25–50 μM) of these compounds was found to inhibit cell attachment to the matrix and eventually resulted in cell damage. We conclude that contrary to earlier reports we found no evidence for an interaction of silymarin or silybin with cytochrome P450 2E1. This suggests that the antioxidant and free radical scavenging properties may account for most of the therapeutic effect of these compounds. The untoward effect of silymarin on cultured cells may have consequences when considering long-term prescription of this therapeutic agent.

Exposure to various benzene derivatives differently induces cytochromes P450 2B1 and P450 2E1 in rat liver.

Benzene (B), toluene (T), ethylbenzene (EB), styrene (S) and xylene isomers (oX, mX, pX) are important environmental pollutants and B is a proved human carcinogen. Their inhalation by male Wistar rats (4 mg/l, 20 h/day, 4 days) caused cytochrome P450 (P450) induction. The degree of P450 2B1 induction increased and that of 2E1 decreased in the series B, T, EB, S, oX, mX and pX, as estimated by Western blots, while neither solvent was as effective for 2B1 induction as phenobarbital and B was more effective for 2E1 than ethanol. The levels of several other P450s decreased after exposure to these solvents, B being most effective. Exposure to these solvents increased in vitro hepatic microsomal oxidation of B and aniline (AN) (2E1 substrates) 3 to 6-fold, indicating induction of this P450. T oxidation was increased 2 to 4-fold and chlorobenzene (ClB) oxidation 3-fold. Sodium phenobarbital (PB, 80 mg/kg/day, 4 days, i.p.) did not increase ethylmorphine (EM) and benzphetamine (BZP) demethylation (2B1 substrates), neither of the B derivatives did so, and oX decreased it; however, pentoxyresorufin O-dealkylation was well related to the immunochemically detected 2B1 levels in control, PB and B microsomes. PB did not increase B, but increased T and ClB oxidation 2-4 and 3-fold, respectively, indicating possible 2B1 role in their oxidation. B oxidation after various inducers was related to immunochemical 2E1 levels, T and ClB oxidation to both 2B1 and 2E1 and AN oxidation to 2E1 and 1A2 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b 5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent K m and V max values for NDMA demethylation were 22 μ m and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK 143 cells, which do not contain b 5, displayed K m and V max values of 31 μ m and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b 5 into TK −143 microsomes increased the V max 2.2-fold and decreased the K m to 22 μ m. Addition of b 5 to Hep G2 microsomes resulted in a 1.6-fold increase in V max, but showed no effect on the K m . P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a K m of 47 μ m and V max of 213 pmol/min/mg microsomal protein. Addition of b 5 lowered the K m to 27 μ m, but had no effect on V max. These results demonstrate conclusively that P450 2E1 is responsible for the low K m forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b 5.

Inhibition of cytochrome P-450 2E1 by diallyl sulfide and its metabolites

Diallyl sulfide, a major flavor ingredient from garlic, was previously shown to inhibit chemically induced carcinogenesis and cytotoxicity in animal model systems. It modulated cytochrome P-450 compositions by inactivating P-450 2E1 and inducing P-450 2B1. The present studies examined the inhibition of P-450 2E1 mediated p-nitrophenol hydroxylase activity by diallyl sulfide and its putative metabolites diallyl sulfoxide and diallyl sulfone (DASO2). Each compound displayed competitive inhibition of p-nitrophenol hydroxylase activity in incubations using liver microsomes from acetone-pretreated male Sprague-Dawley rats. Preincubation of the microsomes with DASO2 inactivated p-nitrophenol hydroxylase activity in a process that was time- and NADPH-dependent and saturable, exhibited pseudo-first-order kinetics, was protected by alternate substrate, was accompanied by a loss of microsomal P-450-CO binding spectrum, and was unaffected by exogenous nucleophile. The Ki value for DASO2 was 188 microM and the maximal rate of inactivation was 0.32 min-1. DASO2 was ineffective in the inactivation of ethoxyresorufin dealkylase, pentoxyresorufin dealkylase, or benzphetamine demethylase activity. Purified P-450 2E1 in a reconstituted system was inactivated in a time- and NADPH-dependent manner by DASO2. The metabolic conversion of diallyl sulfide to the sulfoxide and sulfone was observed in vivo and in vitro. The results suggest that diallyl sulfide inhibits the metabolism of P-450 2E1 substrates by competitive inhibition mechanisms and by inactivating P-450 2E1 via a suicide-inhibitory action of DASO2.