Human P450 2E1 Research Articles

Roles of Cytochrome P450 in Metabolism of Ethanol and Carcinogens.

Cytochrome P450 (P450) enzymes are involved in the metabolism of carcinogens, as well as drugs, steroids, vitamins, and other classes of chemicals. P450s also oxidize ethanol, in particular P450 2E1. P450 2E1 oxidizes ethanol to acetaldehyde and then to acetic acid, roles also played by alcohol and aldehyde dehydrogenases. The role of P450 2E1 in cancer is complex in that P450 2E1 is also induced by ethanol, P450 2E1 is involved in the bioactivation and detoxication of a number of chemical carcinogens, and ethanol is an inhibitor of P450 2E1. Contrary to some literature, P450 2E1 expression and induction itself does not cause global oxidative stress in vivo, as demonstrated in studies using isoniazid treatment and gene deletion studies with rats and mice. However, a major fraction of P450 2E1 is localized in liver mitochondria instead of the endoplasmic reticulum, and studies with site-directed rat P450 2E1 mutants and natural human P450 2E1 N-terminal variants have shown that P450 2E1 localized in mitochondria is catalytically active and more proficient in producing reactive oxygen species and damage. The role of the mitochondrial oxidative stress in ethanol toxicity is still under investigation, as is the mechanism of altered electron transport to P450s that localize inside mitochondria instead of their typical endoplasmic reticulum environment.

Roles of Human CYP2A6 and Monkey CYP2A24 and 2A26 Cytochrome P450 Enzymes in the Oxidation of 2,5,2',5'-Tetrachlorobiphenyl.

2,5,2',5'-Tetrachlorobiphenyl (TCB) induced type I binding spectra with cytochrome P450 (P450) 2A6 and 2A13, with Ks values of 9.4 and 0.51 µM, respectively. However, CYP2A6 oxidized 2,5,2',5'-TCB to form 4-hydroxylated products at a much higher rate (∼1.0 minute-1) than CYP2A13 (∼0.02 minute-1) based on analysis by liquid chromatography-tandem mass spectrometry. Formation of 4-hydroxy-2,5,2',5'-TCB by CYP2A6 was greater than that of 3-hydroxy-2,5,2',5'-TCB and three other hydroxylated products. Several human P450 enzymes, including CYP1A1, 1A2, 1B1, 2B6, 2D6, 2E1, 2C9, and 3A4, did not show any detectable activities in oxidizing 2,5,2',5'-TCB. Cynomolgus monkey CYP2A24, which shows 95% amino acid identity to human CYP2A6, catalyzed 4-hydroxylation of 2,5,2',5'-TCB at a higher rate (∼0.3 minute-1) than CYP2A26 (93% identity to CYP2A6, ∼0.13 minute-1) and CYP2A23 (94% identity to CYP2A13, ∼0.008 minute-1). None of these human and monkey CYP2A enzymes were catalytically active in oxidizing other TCB congeners, such as 2,4,3',4'-, 3,4,3',4'-, and 3,5,3',5'-TCB. Molecular docking analysis suggested that there are different orientations of interaction of 2,5,2',5'-TCB with the active sites (over the heme) of human and monkey CYP2A enzymes, and that ligand interaction energies (U values) of bound protein-ligand complexes show structural relationships of interaction of TCBs and other ligands with active sites of CYP2A enzymes. Catalytic differences in human and monkey CYP2A enzymes in the oxidation of 2,5,2',5'-TCB are suggested to be due to amino acid changes at substrate recognition sites, i.e., V110L, I209S, I300F, V365M, S369G, and R372H, based on the comparison of primary sequences.

Enhanced phytoremediation of mixed heavy metal (mercury)–organic pollutants (trichloroethylene) with transgenic alfalfa co-expressing glutathione S-transferase and human P450 2E1

Soil contamination is a global environmental problem and many efforts have been made to find efficient remediation methods over the last decade. Moreover, remediation of mixed contaminated soils are more difficult. In the present study, transgenic alfalfa plants pKHCG co-expressing glutathione S-transferase (GST) and human P450 2E1 (CYP2E1) genes were used for phytoremediation of mixed mercury (Hg)–trichloroethylene (TCE) contaminants. Simultaneous expression of GST andCYP2E1 may produce a significant synergistic effect, and leads to improved resistance and accumulation to heavy metal–organic complex contaminants. Based on the tolerance and accumulation assays, pKHCG transgenic plants were more resistant to Hg/TCE complex pollutants and many folds higher in Hg/TCE-accumulation than the non-transgenic control plants in mixed contaminated soil. It is confirmed that GST and CYP2E1 co-expression may be a useful strategy to help achieve mixed heavy metal–organic pollutants phytoremediation.

Oxidation of Methyl and Ethyl Nitrosamines by Cytochrome P450 2E1 and 2B1

Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine [N,N-dimethylnitrosamine (DMN)], a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥8. The KIE was not attenuated in noncompetitive intermolecular experiments with rat liver microsomes {(D)V = 12.5; (D)(V/K) = 10.9 [nomenclature of Northrop, D. B. (1982) Methods Enzymol. 87, 607-625]} but was with purified human P450 2E1 [(D)V = 3.3; (D)(V/K) = 3.7], indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine [N,N-diethylnitrosamine (DEN)], the intrinsic KIE was slightly lower and was not expressed [e.g., (D)(V/K) = 1.2] in noncompetitive intermolecular experiments. The same general pattern of KIEs was also seen in the (D)(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH(3)NH(2), CH(3)CH(2)NH(2), and NO(2)(-)). Experiments with deuterated N-nitroso-N-methyl-N-ethylamine demonstrated that the lower KIEs associated with ethyl versus methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 [Chowdhury, G., et al. (2010) J. Biol. Chem. 285, 8031-8044]. These same features (no lag phase for HCO(2)H formation and a lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has a lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkyl nitrosamine oxidation appears to be shared by a number of P450s.

Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

The metabolism of α,β-unsaturated aldehydes, e.g., 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently, we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O(2), and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 and rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice a diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of the reduction of α,β-unsaturated aldehydes in the liver.

Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors.

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. "Toxic hits" uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as "nanoreactors" to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD 50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC-MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.

Identification of covalent modifications in P450 2E1 by 1,2-epoxy-3-butene in vitro

1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.

Novel Metabolic Pathways of p-n-Nonylphenol Catalyzed by Cytochrome P450 and Estrogen Receptor Binding Activity of New Metabolites

Nonylphenol, which is used industrially as a surfactant, is an endocrine-disrupting chemical (EDC) which has estrogenic activity. The novel biotransformation of nonylphenol was investigated, based on our previously reported ipso-metabolism of para-substituted phenols by cytochrome P450 (P450). Three novel metabolites of nonylphenol, i.e., nonylquinol, 4'-hydroxynonanophenone (CO-NP) as benzyl-oxidized nonylphenol, and hydroquinone, were detected in a rat liver microsome reaction mixture. On the other hand, production of 1-(4'-hydroxyphenyl)nonan-1-ol (OH-NP), namely benzyl-hydroxylated nonylphenol, was detected in a human liver microsome reaction mixture. The formation of all these metabolites was suppressed by the addition of P450 inhibitor. This showed that all nonylphenol metabolism was catalyzed by P450. To identify which P450 isoenzyme is involved in each reaction, fourteen human P450 (CYP) isozymes, CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, and CYP4A11, were examined. CYP1A1, 1A2, and CYP2B6 effectively catalyzed the production of nonylquinol. CYP2B6 also catalyzed the benzyl-hydroxylation to give OH-NP. Hydroquinone was formed mainly from OH-NP, not via CO-NP. We examined the estrogenic activity of these new metabolites by estrogen receptor (ER)-binding reporter gene assay. Nonylquinol, OH-NP and hydroquinone have no ER-binding activity. However, CO-NP showed the same level of estrogen receptor binding activity as nonylphenol. Moreover, the amount of CO-NP formed was small. Therefore, the novel metabolic pathways led overall to metabolic inactivation, as concerns the estrogenic activity of nonylphenol through the ER.

Rapid Conformational Dynamics of Cytochrome P450 2E1 in a Natural Biological Membrane Environment

Among the members of the cytochrome P450 superfamily, P450 2E1 is most often associated with the production of reactive oxygen species and subsequent cellular toxicity. We sought to identify a structural basis for this distinguishing feature of P450 2E1 by examining its carbon monoxide binding kinetics as a probe of conformation/dynamics. We employed liver microsomes from wild-type and P450 2E1 knockout mice in order to characterize this P450 in a natural membrane environment. The CO binding kinetics of the P450s of wild-type microsomes had a rapid component that was absent in the knockout microsomes. Data analysis using the maximum entropy method (MEM) correspondingly identified two distinct kinetic components in the wild-type microsomes and only one component in the knockout microsomes. The rapid kinetic component in wild-type microsomes was attributed to endogenous P450 2E1, while the slower component was derived from the remaining P450s. In addition, rapid binding kinetics and a single component were also observed for human P450 2E1 in a baculovirus expression system, in the absence of other P450s. Binding kinetics of both mouse and human P450 2E1 were slowed in the presence of ethanol, a modulator of this P450. The unusually rapid CO binding kinetics of P450 2E1 indicate that it is more dynamically mobile than other P450s and thus able to more readily interconvert among alternate conformations. This suggests that conformational switching during the catalytic cycle may promote substrate release from a short-lived binding site, allowing activated oxygen to attack other targets with toxic consequences.

Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego

The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84+/-0.09 mM and kcat of 2.98+/-0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65+/-0.08 mM and kcat of 0.95+/-0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

GLUTATHIONE TRAPPING TO MEASURE MICROSOMAL OXIDATION OF FURAN TOCIS-2-BUTENE-1,4-DIAL

Furan is a liver carcinogen and toxicant. Furan is oxidized to the reactive dialdehyde, cis-2-butene-1,4-dial, by microsomal enzymes. This reactive metabolite readily reacts with glutathione nonenzymatically to form conjugates. A high-performance liquid chromatography-electrochemical method for the detection of cis-2-butene-1,4-dial-glutathione (GSH) conjugates in microsomal preparations was developed to measure the extent of furan metabolism to cis-2-butene-1,4-dial in vitro. Previously unobserved mono-GSH reaction products of cis-2-butene-1,4-dial were detected in addition to the already characterized bis-GSH conjugates. Chemical characterization of these compounds indicated that the alpha-amino group of glutathione had reacted with cis-2-butene-1,4-dial to form a thiol-substituted pyrrole adduct. The analytical method was used to estimate the extent of furan oxidation in rat liver microsomes from untreated or acetone-pretreated F344 rats as well as in human P450 2E1 Supersomes. Our results confirm that cytochrome P450 2E1 can catalyze the oxidation of furan to cis-2-butene-1,4-dial. However, the data are also consistent with the involvement of other P450 enzymes in the oxidation of furan in untreated animals. This assay will be a valuable tool to explore tissue and species differences in rates of furan oxidation.

Direct electrochemistry of immobilized human cytochrome P450 2E1.

This communication reports the first electrochemical study of the human P450 2E1 either absorbed or covalently linked to different electrode surfaces. Glassy-carbon and gold electrodes gave reversible electrochemical signals of an active P450 2E1. Molecular modeling of the enzyme helped to rationalize the results. A monolayer coverage was obtained on gold modified with cystamine/maleimide that covalently linked surface accessible cysteines of P450 2E1. The midpoint potential measured for the oriented P450 2E1 was -177 +/- 5 mV comparable to that of the FeIII/FeII of other P450 enzymes. The observed electron-transfer rate for this electrode was 10 s-1. The turnover of the active enzyme was measured with the P450 2E1 specific substrate p-nitrophenol, resulting in a KM of 130 +/- 3 muM and the formation of 2.2 muM of the p-nitrocatechol product upon application of a -300 mV bias.

Heterologous Expression of Mouse Cytochrome P450 2e1 in V79 Cells: Construction and Characterisation of the Cell Line and Comparison with V79 Cell Lines Stably Expressing Rat P450 2E1 and Human P450 2E1

A V79 Chinese hamster cell line was constructed for stable expression of mouse cytochrome P450 2e1 (Cyp2e1), as an addition to the existing cell battery consisting of cell lines stably expressing rat CYP2E1 and human CYP2E1 (V79 Cell Battery). The aim was to establish a cell battery that offers the in vitro possibility of investigating species-specific differences in the toxicity and metabolism of chemicals representing substrates for CYP2E1. The newly established cell line (V79m2E1) effectively expressed Cyp2e1 in the catalytically active form. The expression of catalytically active CYP2E1 in V79m2E1 cells was maintained over several months in culture, as demonstrated by Western Blotting and chlorzoxazone (CLX) 6-hydroxylase activity. The cells exhibited CLX 6-hydroxylase activity with a Km of 27.8 microM/l and Vmax of 40 pmol/mg protein/minute, compared with a Km of 28.2/28.6 microM/l and a Vmax of 130/60 pmol/mg protein/minute from V79r2E1/V79h2E1 cells. Furthermore, the CYP2E1-dependent mutagenicity of N-nitrosodimethylamine could be demonstrated in the V79m2E1 cells. Therefore, the new cell battery permits the interspecies comparison of CYP2E1-dependent toxicity and of metabolism of chemicals between humans and the two major rodent species--the rat and the mouse--that are usually used in classical toxicity studies.

Rate-limiting steps in cytochrome P450 catalysis.

Cytochrome P450 (P450) reactions are of interest because of their relevance to the oxidative metabolism of drugs, steroids, carcinogens, and other chemicals. One of the considerations about functional characterization is which steps of the catalytic cycle are rate-limiting. Detailed analysis indicates that several different steps can be rate-limiting with individual P450 reactions. N-Dealkylation of para-substituted N,N-dimethylanilines is a function of the electron withdrawing/donating properties of the substituent and the oxidation-reduction potential of the substrate, supporting a role in rate-limiting electron transfer from substrate to the high valent P450. In the oxidations of ethanol and acetaldehyde by human P450 2E1, a step following product formation must be the slow step (but not product release per se). Several oxidations catalyzed by human P450s 1A2 and 2D6 show slow C-H bond breaking, and apparent high-valent iron complexes accumulate in the reaction steady-state. Kinetic simulations were used to test the suitability of potential schemes and to probe the effects of changes in individual reaction steps.

Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology

This paper reports on the application of the molecular Lego approach to P450 enzymes. Protein domains are used as catalytic (P450 BM3 haem domain and human P450 2E1) or electron transfer (flavodoxin and P450 BM3 reductase) modules. The objectives are to build assemblies with improved electrochemical properties, to construct soluble human P450 enzymes, and to generate libraries of new P450 catalytic modules based on P450 BM3. A rationally designed, gene-fused assembly (BMP–FLD) was obtained from the soluble haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP) and flavodoxin from Desulfovibrio vulgaris (FLD). The assembly was expressed successfully and characterised in its active form, displaying improved electrochemical properties. Solubilisation of the human, membrane-bound P450 2E1 (2E1) was achieved by fusing key elements of the 2E1 enzyme with selected parts of P450 BM3. An assembly containing the first 54 residues of P450 BM3, the whole sequence of P450 2E1 from residue 81 and the reductase domain of P450 BM3 was constructed. The 2E1–BM3 assembly was successfully expressed in the cytosol of Escherichia coli. The soluble form of 2E1–BM3 was reduced in carbon monoxide atmosphere and displayed the typical absorption peak at 450 nm, characteristic of a folded and active P450 enzyme. Finally, the alkali method previously developed in this laboratory was used to screen for P450 activity within a library of random mutants of P450 BM3. A number of variants active towards non-physiological substrates, such as pesticides and polyaromatic hydrocarbons were identified, providing new P450 catalytic modules. The combination of these three areas of research provide interesting tools for exploitation in nanobiotechnology.

Oxidation of 2,6-dimethylaniline by recombinant human cytochrome P450s and human liver microsomes.

2,6-Dimethylaniline (2,6-DMA) is classified as a rodent nasal cavity carcinogen and a possible human carcinogen. The major metabolite of 2,6-DMA in rats and dogs is 4-amino-3,5-dimethylphenol (DMAP) but oxidization of the amino group to produce metabolites such as N-(2,6-dimethylphenyl)hydroxylamine (DMHA) is also indicated by the occurrence of hemoglobin adducts of 2,6-DMA in human and rats. Previous studies have shown a large interindividual variability in human 2,6-DMA hemoglobin adduct levels. In the present study, 2,6-DMA oxidation in vitro by human liver microsomes and recombinant human P450 enzymes was investigated to assess whether the hemoglobin adduct variability could be attributed to metabolic differences. At micromolar concentrations, the only product detectable (UV) was DMAP, while at 10 nM, DMHA was a substantial product. 2E1 and 2A6 were identified as the major P450s in human liver microsomes responsible for the production of DMAP by using P450-specific chemical inhibitors and mouse monoclonal antibodies that selectively inhibit human P450 2E1 and 2A6. 2A6 was identified as the major P450 responsible for the N-hydroxylation. Native P450 2E1 and human liver microsomes catalyzed the rearrangement of DMHA to DMAP independent of NADPH. Consistent with a mechanism involving oxygen rebound to the heme iron center, labeled oxygen was not incorporated into DMAP from either 18O2 gas or H2 18O in this rearrangement. Results presented here suggest much of the observed interindividual variability of 2,6-DMA hemoglobin adduct levels could be due to differences in the relative amounts of hepatic 2E1 and 2A6.

Reaction of trichloroethylene and trichloroethylene oxide with cytochrome P450 enzymes: inactivation and sites of modification.

Trichloroethylene (TCE) has been shown to be toxic to experimental animals and humans. TCE oxide is a reactive electrophile formed during TCE oxidation and rearranges to acylating intermediates [Cai, H., and Guengerich, F. P. (1999) J. Am. Chem. Soc. 121, 11656-11663], which may be related to the toxicity. Mice treated with TCE have been reported to contain N(6)-dichloroacetylLys residues in P450 2E1, as detected by immunochemical methods. TCE can be oxidized by both P450 2E1 and (rat) 2B1. In this work, direct reaction of TCE oxide with either human P450 2E1, P450 2B1, or NADPH-P450 reductase was shown to lead to enzyme inactivation, and no recovery of the activity of either enzyme occurred, consistent with the view of inactivation reactions with Lys groups and not hydroxyls or Cys. Furthermore, Lys adducts were detected in the reaction of TCE oxide with both P450 2E1 and NADPH-P450 reductase, with a larger amount of N(6)-formylLys observed compared to N(6)-dichloroacetylLys in both cases. Inactivation of P450 2E1 during NADPH-dependent TCE oxidation was not observed, compared to control experiments. However, inactivation of P450 2B1 during NADPH-dependent TCE oxidation was detected. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of tryptic peptides indicated that the direct reaction of TCE oxide with human P450 2E1 resulted in the modification of peptides containing Lys87 (AVKEALLDYK), Lys251 (VKEHHQSLDPNCPR), and Lys487 (YKLCVIPR), with either a formyl or dichloroacetyl group attached. Lys87 and Lys487 of human P450 2E1 appear to be modified during the oxidation of TCE, using the same approach. The results are considered in the context of comparison of species and P450s.

Metabolism of the beta-oxidized intermediates of N-nitrosodi-n-propylamine: N-nitroso-beta-hydroxypropylpropylamine and N-nitroso-beta-oxopropylpropylamine.

The rat liver carcinogen N-nitrosodi-n-propylamine (NDPA) is metabolized to a propylating and methylating species in vivo. Metabolism to a methylating species is believed to require an initial hydroxylation by cytochrome P450s (P450s) to N-nitroso-beta-hydroxypropylpropylamine (NHPPA), which is oxidized to N-nitroso-beta-oxopropylpropylamine (NOPPA), followed by a P450-mediated depropylation to beta-oxopropyldiazotate, which non-enzymatically breaks down to the methylating agent. Purified rat liver P450 2B1 and rabbit liver 2E1 in the reconstituted system and liver microsomes from phenobarbital (PB) and pyridine (Pyr) treated rats readily metabolized NOPPA to a methylating species as determined by the in vitro formation of 7-methylguanine (m7Gua) in DNA. Exposure of cells derived from the human liver epithelium transfected with human 2E1 (T5-2E1) to NOPPA resulted in the formation of m7Gua DNA adducts and a dose dependent toxicity. In vitro incubation of NHPPA with microsomes from PB, Pyr and non-treated (NT) rats and a human microsomal sample also resulted in m7Gua formation. P450s 2B1 and 2E1 oxidized NHPPA to NOPPA, forming 16.5 +/- 3.1 and 20.0 +/- 4.4 pmol NOPPA/pmol P450 in 1 h, respectively. Rat liver cytosol, in the presence of NAD+, oxidized NHPPA to NOPPA at a rate of 13.7 +/- 3.0 pmol/min/mg protein while microsomes from NT rats catalyzed this reaction at 95.6 +/- 16.5 pmol/min/mg protein. Cells derived from hamster lung tissue (V79 control) and T5-neo cells oxidized NHPPA to NOPPA. This oxidation was about 15 fold higher in T5-2E1 or V79 cells transfected with human 2E1 or rat 2B1, respectively. The results are consistent with the putative sequential oxidation pathway and suggest that, at the concentrations tested, oxidation of NHPPA to NOPPA may be predominantly mediated by cytochrome P450s. In addition, it appears that rabbit, rat and human P450 2E1 can catalyze both oxidations.

Metabolism of Ochratoxin A: Absence of Formation of Genotoxic Derivatives by Human and Rat Enzymes

Ochratoxin A (OTA) is a potent renal carcinogen in male rats, although its mode of carcinogenicity is not known. The metabolism and covalent binding of OTA to DNA were investigated in vitro with cytochromes P450, glutathione S-transferases, prostaglandin H-synthase, and horseradish peroxidase. Incubation of OTA with rat or human liver microsomes fortified with NADPH resulted in formation of 4-(R)-hydroxyochratoxin A at low rates [10-25 pmol min(-1) (mg of protein)(-1)]. There was no evidence of OTA metabolism and glutathione conjugate formation with rat, mouse, or human kidney microsomes or postmitochondrial supernatants (S-9) [<5 pmol min(-1) (mg of protein)(-1)]. Recombinant human cytochromes P450 (P450) 1A1 and 3A4 formed 4-(R)-hydroxyochratoxin A at low rates [0.08 and 0.06 pmol min(-1) (pmol of P450)(-1), respectively]; no oxidation products of OTA were detected with recombinant human P450 1A2 or 2E1 or rat P450 1A2 or 2C11 [<0.02 pmol min(-1) (pmol of P450)(-1)]. Prostaglandin H-synthase produced small amounts of an apolar product [33 pmol min(-1) (mg of protein)(-1)], and OTA products were not formed with horseradish peroxidase. There was no evidence of DNA adduct formation when [(3)H]OTA was incubated with these enzyme systems in the presence of calf thymus DNA (<20 adducts/10(9) DNA bases); however, these enzymes catalyzed DNA adduct formation with the genotoxins aflatoxin B(1), 2-amino-3-methylimidazo[4,5-f]quinoline, benzo[a]pyrene, and pentachlorophenol. There was also no detectable [(3)H]OTA bound in vivo to kidney DNA of male Fischer-344 rats treated orally with [(3)H]OTA (1 mg/kg, 100 mCi/mmol, 24 h exposure, <2.7 adducts/10(9) DNA bases), based upon liquid scintillation counting. However, (32)P-postlabeling experiments did show evidence of DNA lesions with total adduct levels ranging from 31 to 71 adducts/10(9) DNA bases, while adducts in untreated rat kidney ranged from 6 to 24 adducts/10(9) DNA bases. These results do not support the premise that OTA or metabolically activated species covalently bind to DNA and suggest that the (32)P-postlabeled lesions are due to products derived from OTA-mediated cytotoxicity.

Acylation of protein lysines by trichloroethylene oxide.

Stable lysine adducts were formed in proteins following reaction with trichloroethylene (TCE) oxide, the major reactive compound generated by the metabolism of TCE. The order of formation of these adducts is N(6)-formyllysine > N(6)-(dichloroacetyl)lysine >> N(6)-glyoxyllysine, with the ratio being influenced by the particular protein. Protein lysine adducts were also analyzed following the enzymatic oxidation of TCE with several different cytochrome P450 (P450) enzyme systems. The ratio of formyl/dichloroacetyl lysine adducts was influenced by the enzyme system that was used. Chloral and TCE oxide formation was more extensive with rat liver microsomes isolated from phenobarbital-treated rats than with rat microsomes in which P450 2E1 was induced by treatment with isoniazid or in human P450 2E1 systems. Glutathione (GSH) and GSH transferase had inhibitory effects on the reaction of TCE oxide with albumin, with formylation being atteunated much more than the formation of dichloroacetyllysine. GSH is likely to react with the reactive acyl chloride intermediates formed from TCE oxide hydrolysis, instead of direct reaction with TCE oxide, as judged by the lack of an effect of GSH on the rate of decomposition of TCE oxide. Studies with the model enzymes aldolase and glucose-6-phosphate dehydrogenase, both known to have sensitive lysine groups, indicate that TCE oxide has effects similar to known acylating agents that form the same adducts; concentrations of TCE oxide (or the model acylating agents) in the low-millimolar range were needed for inhibition. The characterization of TCE-derived protein adducts can be used as a basis for consideration of the exposure and risk of TCE to humans. Human P450 2E1 was less likely to oxidize TCE to form TCE oxide and protein lysine adducts than rat P450 2B1, and the difference is rationalized in terms of the influence of the protein on chloride migration in an enzyme reaction intermediate.