Human Cytochrome P450 2E1 Research Articles

Glutathione metabolism modeling: A mechanism for liver drug-robustness and a new biomarker strategy

BackgroundGlutathione metabolism can determine an individual's ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to simulate perturbations observed when human liver derived THLE cells, transfected with human cytochrome P452E1 (THLE-2E1 cells), were exposed to paracetamol (acetaminophen). MethodsHuman liver derived cells containing extra human cytochrome P4502E1 were treated with paracetamol at various levels of methionine and in the presence and absence of an inhibitor of glutamyl-cysteine synthetase (GCS). GCS activity was also measured in extracts. Intracellular and extracellular concentrations of substances involved in glutathione metabolism were measured as was damage to mitochondria and proteins. A bottom up mathematical model was made of the metabolic pathways around and including glutathione. ResultsOur initial model described some, but not all the metabolite-concentration and flux data obtained when THLE-2E1 cells were exposed to paracetamol at concentrations high enough to affect glutathione metabolism. We hypothesized that the lack of correspondence could be due to upregulation of expression of glutamyl cysteine synthetase, one of the enzymes controlling glutathione synthesis, and confirmed this experimentally. A modified model which incorporated this adaptive response adequately described the observed changes in the glutathione pathway. Use of the adaptive model to analyze the functioning of the glutathione network revealed that a threshold input concentration of methionine may be required for effective detoxification of reactive metabolites by glutathione conjugation. The analysis also provided evidence that 5-oxoproline and ophthalmic acid are more useful biomarkers of glutathione status when analyzed together than when analyzed in isolation, especially in a new, model-assisted integrated biomarker strategy. ConclusionA robust mathematical model of the dynamics of cellular changes in glutathione homeostasis in cells has been developed and tested in vitro. General significanceMathematical models of the glutathione pathway that help examine mechanisms of cellular protection against xenobiotic toxicity and the monitoring thereof, can now be made.

Inhibitory effects of sanguinarine on human liver cytochrome P450 enzymes

Sanguinarine (SAG) has been recognized as an anticancer drug candidate. However, the drug-drug interactions (DDI) potential for SAG via the inhibition against human cytochrome P450 (CYP) enzymes remains unclear. In the present study, the inhibitory effects of SAG on seven major human CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C8, 2C9 and 3A4 were investigated with human liver microsomes (HLM). The results showed that SAG was a potent noncompetitive inhibitor of CYP2C8 activity (Ki=8.9 μM), and competitive inhibitor of CYP1A2, CYP2C9 and CYP3A4 activities (Ki=2.7, 3.8 and 2.0 μM, respectively). Furthermore, SAG exhibited time- and NADPH-dependent inhibition towards CYP1A2 and CYP3A4 with KI/kinact values of 13.3/0.087 and 5.58/0.029 min(-1) μM(-1), respectively. Weak inhibition of SAG against CYP2E1, CYP2D6 and CYP2A6 was also observed. In vitro-in vivo extrapolation (IV-IVE) from HLM data showed that more than 35.9% of CYP1A2, CYP2C9, CYP2C8 and CYP3A4 activities in vivo could be inhibited by SAG, suggesting that harmful DDIs could occur when SAG or its medical preparations are co-administered with drugs primarily cleared by these CYP isoforms. Further in vivo studies are needed to evaluate the clinical significance of the data presented herein.

Functional PstI/RsaI Polymorphisms in the CYP2E1 Gene among South Indian Populations

Human cytochrome P4502E1 (CYP2E1) is a well-conserved xenobiotic-metabolizing enzyme expressed in liver, kidney, nasal mucosa, brain, lung, and other tissues. CYP2E1 is inducible by ethanol, acetone, and other low-molecular weight substrates and may mediate development of chemically-mediated cancers. CYP2E1 polymorphisms alter the transcriptional activity of the gene. This study was conducted in order to investigate the allele frequency variation in different populations of Andhra Pradesh. Two hundred and twelve subjects belonging to six populations were studied. Genotype and allele frequency were assessed through TaqMan allelic discrimination (rs6413419) and polymerase chain reaction-sequencing (-1295G>C and -1055C>T) after DNA isolation from peripheral leukocytes. The data were compared with other available world populations. The SNP rs6413419 is monomorphic in the present study, -1295G>C and -1055C>T are less polymorphic and followed Hardy-Weinberg equilibrium in all the populations studied. The -1295G>C and -1055C>T frequencies were similar and acted as surrogates in all the populations. Analysis of HapMap populations data revealed no significant LD between these markers in all the populations. Low frequency of CYP2E1 c2 could be useful in the understanding of south Indian population gene composition, alcohol metabolism, and alcoholic liver disease development. However, screening of additional populations and further association studies are necessary. The heterogeneity of Indian population as evidenced by the different distribution of CYP2E1 c2 may help in understanding the population genetic and evolutionary aspects of this gene.

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.

Negatively Cooperative Binding Properties of Human Cytochrome P450 2E1 with Monocyclic Substrates

Human CYP2E1 accounts for almost 2% of total CYP enzymes in the liver cells, and plays a crucial role in the metabolism of small molecular weight compounds. This enzyme is associated with the nearly 6% metabolisms of the currently clinical drugs. However, it is found that CYP2E1 has a non-hyperbolic kinetic profile that can not be explained by the common Michaelis-Menten mechanism. Further studies show that the non-hyperbolic kinetic behaviors are associated with multiple substrate binding, which is also known as the cooperative binding properties. However, the detailed mechanism for the cooperative binding is not clear by now. In this paper, we summarized the experimental and theoretical studies on the cooperative binding mechanism. Based on the structural analysis, a second substrate binding site is confirmed in human CYP2E1, which is located neither in the region near Leu103, Leu210 and Phe478, nor far from the active site. Additionally, two important residues Thr303 and Phe478 are also identified to be the key factors in the cooperative binding on the short-range and long-range effects, respectively. The former plays a crucial role in the positioning of substrates and in proton delivery to the active site; the latter is located between the substrate access channel and the active site, and exhibits directly effects on substrate access or on substrate positioning in the active site. All these points can provide useful information for the cooperative binding in human CYP2E1, revealing the detailed mechanism for the non-hyperbolic kinetic behaviors.

Investigation of Indazole Unbinding Pathways in CYP2E1 by Molecular Dynamics Simulations

Human microsomal cytochrome P450 2E1 (CYP2E1) can oxidize not only low molecular weight xenobiotic compounds such as ethanol, but also many endogenous fatty acids. The crystal structure of CYP2E1 in complex with indazole reveals that the active site is deeply buried into the protein center. Thus, the unbinding pathways and associated unbinding mechanisms remain elusive. In this study, random acceleration molecular dynamics simulations combined with steered molecular dynamics and potential of mean force calculations were performed to identify the possible unbinding pathways in CYP2E1. The results show that channel 2c and 2a are most likely the unbinding channels of CYP2E1. The former channel is located between helices G and I and the B-C loop, and the latter resides between the region formed by the F-G loop, the B-C loop and the β1 sheet. Phe298 and Phe478 act as the gate keeper during indazole unbinding along channel 2c and 2a, respectively. Previous site-directed mutagenesis experiments also supported these findings.

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.

A Negative Cooperativity Mechanism of Human CYP2E1 Inferred from Molecular Dynamics Simulations and Free Energy Calculations

Human cytochrome P450 2E1 (CYP2E1) participates in the metabolism of over 2% of all the oral drugs. A hallmark peculiar feature of this enzyme is that it exhibits a pronounced negative cooperativity in substrate binding. However the mechanism by which the negative cooperativity occurs is unclear. Here, we performed molecular dynamics simulations and free energy calculations on human CYP2E1 to examine the structural differences between the substrate-free and the enzymes with one and two aniline molecules bound. Our results indicate that although the effector substrate does not bind in the active site cavity, it still can directly interact with the active site residues of human CYP2E1. The interaction of the effector substrate with the active site leads to a reorientation of active site residues, which thereby weakens the interactions of the active substrate with this site. We also identify a conserved residue T303 that plays a crucial role in the negative cooperative binding on the short-range effects. This residue is a key factor in the positioning of substrates and in proton delivery to the active site. Additionally, a long-range effect of the effector substrate is identified in which F478 is proposed to play a key role. As located in the interface between the active and effector sites, this residue structurally links the active and effector sites and is found to play a significant role in affecting substrate access and ligand positioning within the active site. In the negative cooperative binding, this residue can decrease the interactions of the active substrate with the active site by π-π stacking which then lowers the hydroxylation activity for the active substrate. These findings are in agreement with previous experimental observations and thus provide detailed atomistic insight into the poorly understood mechanism of the negative cooperativity in human CYP2E1.

Alcohol Disrupts Endoplasmic Reticulum Function and Protein Secretion in Hepatocytes

Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo. HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP). VL-17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced. Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD.

Pathways of liver injury in alcoholic liver disease

The development of ethanol-induced liver injury including liver cirrhosis and severe alcoholic steatohepatitis (ASH) is a complex process involving various liver cell types and mainly factors released under the control of the innate immune system. Chronic ethanol consumption induces oxidative stress and production of reactive oxygen species (ROS), cytokine release, mitochondrial dysfunction, endoplasmic reticulum stress, and others. ROS initiate lipid peroxidation that directly damages plasma and intracellular membranes and leads to the production of reactive aldehydes with potent pro-inflammatory and pro-fibrotic properties. Oxidative stress and ROS are predominantly generated through the induction of cytochrome P450-2E1 (CYP2E1). A key role for this enzyme in ethanol-induced liver injury has been demonstrated by its inhibition through chlormethiazole and by the finding that CYP2E1 knock-out (KO) mice do not show evidence of ethanol-induced liver disease [1]. Furthermore, transgenic overexpression of human CYP2E1 in a mouse model results in more severe liver disease. Both the hydroxyethyl radical and acetaldehyde, the first products of ethanol metabolism, can bind glutathione (GSH), a tripeptide that acts as a direct free radical scavenger. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protects cells against xenobiotic and oxidative stress. Nrf2 KO mice exhibit a dramatically increased mortality after ethanol feeding, highlighting the important role of oxidative stress in ethanol-induced injury [1] (Fig. 1).

Role of CYP2E1 in Ethanol-Induced Oxidant Stress, Fatty Liver and Hepatotoxicity

Background/Aims: Several pathways contribute to mechanisms by which ethanol induces oxidant stress. While some studies support a role for cytochrome P450 2E1 (CYP2E1), others do not. There is a need to develop oral models of significant ethanol-induced liver injury and to evaluate the possible role of CYP2E1 in ethanol actions in such models. Methods: We evaluated chronic ethanol-induced liver injury, steatosis and oxidant stress in wild-type (WT) mice, CYP2E1 knockout (KO) mice and in humanized CYP2E1 knockin (KI) mice, where the human 2E1 was added back to mice deficient in the mouse 2E1. WT mice and CYP2E1 KO and KI mice (both provided by Dr. F. Gonzalez, NCI) were fed a high-fat Lieber-DeCarli liquid diet for 3 weeks; pair-fed controls received dextrose. Results: Ethanol produced fatty liver and oxidant stress in WT mice, but liver injury (transaminases, histopathology) was minimal. Ethanol-induced steatosis and oxidant stress were blunted in the KO mice (no liver injury) but restored in the KI mice. Significant liver injury was produced in the ethanol-fed KI mice with elevated transaminases and necrosis. This liver injury in the KI mice was associated with elevated oxidant stress and elevated levels of the human CYP2E1 compared to levels of the mouse 2E1 in WT mice. Activation of JNK was observed in the ethanol-fed KI mice compared to the other groups. Fatty liver in WT and KI mice was associated with lower levels of lipolytic PPAR-α. No such changes were found in the ethanol-fed KO mice. Conclusions: These results show that CYP2E1 plays a major role in ethanol-induced fatty liver and oxidant stress. Restoring CYP2E1 in the CYP2E1 KO mice restores ethanol-induced fatty liver and oxidant stress.

Expression of a human cytochrome P4502E1 in Nicotiana tabacum enhances tolerance and remediation of γ-hexachlorocyclohexane

Lindane (γ-hexachlorocyclohexane), a persistent organo-chlorine insecticide widely used in developing countries, has a negative effect as a polluting agent of soil and surface waters. Plants can be used for remediation of organic pollutants and their efficiency can be enhanced by introduction of heterologous genes. Mammalian cytochrome P4502E1 (CYP2E1), an important monooxygenase is involved in the degradation of a wide range of xenobiotics including environmental pollutants/herbicides and pesticides. Here, we report the development of transgenic tobacco plants expressing human CYP2E1 and the efficacy of plants for remediation of lindane. Transgenic tobacco plants with CYP2E1 showed enhanced tolerance to lindane when grown in hydroponic medium and soil compared to control plants. Remediation of (14)C-labeled lindane from hydroponic medium was higher in transgenic plants compared to that of control plants, with the best performing line showing 25% higher removal of lindane from solution than control plants. Similar results were seen in plants grown in soil spiked with lindane. The present study has shown that transgenic plants expressing CYP2E1 gene have potential use for remediation of lindane from contaminated solutions and soil.

Comparative analysis of binding affinities between styrene and mammalian CYP2E1 by bioinformatics approaches

Cytochrome P450 2E1 (CYP2E1) is a cytochrome P450 enzyme involved in styrene metabolism. This study compared the binding affinities between styrene and 11 mammalian CYP2E1 systems using bioinformatics methods. Firstly, amino acid sequences of CYP2E1s were obtained from the Swiss-Prot database. Then, taking the crystal structure of human CYP2E1 as a template, 3D models of the CYP2E1s of other mammals were constructed using the SWISS-MODEL program. Finally, the generated homology models were applied to calculate their docking capacities against styrene and polystyrene using the Surflex-Dock program, which could automatically dock ligands into a receptor's ligand binding site using a protomol based approach and assess the affinity by an empirically derived scoring function. Docking experiments showed that the studied mammalian CYP2E1s had high binding affinities with styrene. For polystyrene, the dimmer of styrene has high binding affinities with CYP2E1s, however, trimer and other high polymers were found hard to be docked into the CYP2E1s. The results of this study indicated that bioinformatics approaches might be useful tools to predict styrene and polystyrene affinities with mammalian CYP2E1s.

In Vitro Cytochrome P450 2E1 and 2A Activities in the Presence of Testicular Steroids

Cytochrome P450 2E1 (CYP2E1) and 2A (CYP2A) are the main enzymes involved in the metabolism of skatole in pigs. In this study, physiological concentrations of androstenone, 17β-oestradiol and testosterone were tested for their ability to regulate CYP2E1 and CYP2A activity in liver microsomes isolated from entire male and female pigs as well as in microsomes from Saccharomyces cerevisiae expressing either human recombinant CYP2E1 or CYP2A6. We found that physiological concentrations of androstenone and oestradiol had the ability to inhibit CYP2E1 activity. The magnitude of this inhibition (approximately 30%) was similar in recombinant human CYP2E1 and microsomes from entire male pigs. This inhibition was only seen when adding the steroid to the assay 15 min before the substrate. Interestingly, CYP2E1 activity in the microsomes from female pigs was not affected. None of the investigated steroids modified the activity of recombinant human CYP2A6. However, CYP2A activity was slightly increased in the microsomes from female pigs in the presence of oestradiol, but the magnitude of this increase was very low (below 10%) and probably irrelevant. Overall, these results indicate that physiological concentrations of androstenone and oestradiol have a potential to inhibit CYP2E1 activities in vitro, and that this inhibition is gender-specific. Further studies are needed to investigate the biochemical mechanisms underlying those differences between the genders.

Genotoxicity of acrylamide in vitro: Acrylamide is not metabolically activated in standard in vitro systems

The recent finding that acrylamide (AA), a genotoxic rodent carcinogen, is formed during the frying or baking of a variety of foods raises human health concerns. AA is known to be metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA), which is responsible for AA's in vivo genotoxicity and probable carcinogenicity. In in-vitro mammalian cell tests, however, AA genotoxicity is not enhanced by rat liver S9 or a human liver microsomal fraction. In an attempt to demonstrate the in vitro expression of AA genotoxicity, we employed Salmonella strains and human cell lines that overexpress human CYP2E1. In the umu test, however, AA was not genotoxic in the CYP2E1-expressing Salmonella strain or its parental strain. Moreover, a transgenic human lymphoblastoid cell line overexpressing CYP2E1 (h2E1v2) and its parental cell line (AHH-1) both showed equally weak cytotoxic and genotoxic responses to high (>1 mM) AA concentrations. The DNA adduct N7-GA-Gua, which is detected in liver following AA treatment in vivo, was not substantially formed in the in vitro system. These results indicate that AA was not metabolically activated to GA in vitro. Thus, AA is not relevantly genotoxic in vitro, although its in vivo genotoxicity was clearly demonstrated.

IL-4-mediated transcriptional regulation of human CYP2E1 by two independent signaling pathways

Cytochrome P450 2E1 (CYP2E1), the alcohol-inducible member of the cytochrome P450 super family, plays an important role in both physiological and pathophysiological processes. The present study focused on the induction of human CYP2E1 transcription by the anti-inflammatory cytokine interleukin-4 (IL-4) in human hepatoma B16A2 cells and revealed that this regulation is mediated by two independent pathways. RNA interference and overexpression of STAT6, indicated that the JAK–STAT signaling pathway is involved in IL-4-dependent induction and mutagenesis revealed the presence of a STAT6 binding site in CYP2E1 proximal promoter region (−583/−574-bp). However, inhibition of the JAK–STAT6 pathway using JAK1 siRNA constructs could only partially inhibit the induction of CYP2E1 promoter constructs indicating the presence of a second IL-4 responsive element. Indeed by using a series of truncated CYP2E1 promoter constructs a second more distal IL-4 responsive element (−1604/−1428-bp) was identified, which was further shown to involve the activation of IRS1/2. This induction was dependent on the transcription factor NFATc1 as IL-4-induced CYP2E1 expression was altered by silencing or overexpressing NFATc1. A NFATc1 binding site was identified in the second distal IL-4 responsive element (−1551/−1545-bp) by chromatin immunoprecipitation (ChIP) analysis. Finally simultaneous siRNA-mediated down-regulation of both STAT6 and NFATc1 or mutation of both STAT6 and NFATc1 binding sites abolished the IL-4-dependent transcriptional induction of CYP2E1, demonstrating that both pathways are required for maximal activation. In conclusion, the present study indicates that the induction of CYP2E1 transcription by IL-4 is mediated through two independent parallel pathways, involving JAK–STAT6 and IRS1/2 and NFATc1.

Chronic alcohol-induced liver injury and oxidant stress are decreased in cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice

A major pathway for chronic ethanol-induced liver injury is ethanol-induced oxidant stress. Several pathways contribute to mechanisms by which ethanol induces oxidant stress. Although some studies support a role for cytochrome P450 2E1 (CYP2E1), others do not. Most previous studies were conducted in the intragastric infusion model of ethanol administration. There is a need to develop oral models of significant liver injury and to evaluate the possible role of CYP2E1 in ethanol actions in such models. We evaluated chronic ethanol-induced liver injury, steatosis, and oxidant stress in wild-type (WT) mice, CYP2E1 knock out (KO) mice, and humanized CYP2E1 knock-in (KI) mice, in which the human 2E1 was added back to mice deficient in the mouse 2E1. WT mice and the CYP2E1 KO and KI mice (both provided by Dr. F. Gonzalez, National Cancer Institute) were fed a high-fat Lieber–DeCarli ethanol liquid diet for 3 weeks; pair-fed controls received dextrose. Ethanol produced fatty liver and oxidant stress in WT mice but liver injury (transaminases, histopathology) was minimal. Ethanol-induced steatosis and oxidant stress were blunted in the KO mice (no liver injury) but restored in the KI mice. Significant liver injury was produced in the ethanol-fed KI mice, with elevated transaminases, necrosis, and increased levels of collagen type 1 and smooth muscle actin. This liver injury in the KI mice was associated with elevated oxidant stress and elevated levels of the human CYP2E1 compared to levels of the mouse 2E1 in WT mice. Activation of JNK and decreased levels of Bcl-2 and Bcl-XL were observed in the ethanol-fed KI mice compared to the other groups. Fatty liver in the WT and the KI mice was associated with lower levels of PPARα and acyl-CoA oxidase. No such changes were found in the ethanol-fed KO mice. These results show that CYP2E1 plays a major role in ethanol-induced fatty liver and oxidant stress. It is the absence of CYP2E1 in the KO mice that is responsible for the blunting of steatosis and oxidant stress because restoring the CYP2E1 restores the fatty liver and oxidant stress. Moreover, it is the human CYP2E1 that restores these effects of ethanol, which suggests that results for fatty liver and oxidant stress from rodent models of ethanol intake and mouse CYP2E1 can be extrapolated to human models of ethanol intake and to human CYP2E1.

Human Cytochrome P450 2E1 Structures with Fatty Acid Analogs Reveal a Previously Unobserved Binding Mode

Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9-C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with omega-imidazolyl-octanoic fatty acid, omega-imidazolyl-decanoic fatty acid, and omega-imidazolyl-dodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the omega-1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe(298) side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B' helices at successive distances from the active site.

Breaking self-tolerance toward cytochrome P4502E1 (CYP2E1) in chronic hepatitis C: Possible role for molecular mimicry

Circulating auto-antibodies targeting conformational antigens on cytochrome P4502E1 (CYP2E1) are detectable in patients with chronic hepatitis C (CHC) and are associated with more severe necro-inflammation. This study investigated the antigen specificity and the possible origin of these auto-antibodies. CYP2E1 site-directed mutagenesis and molecular simulation were used to characterize the epitope specificity of CHC-associated anti-CYP2E1 auto-antibodies. Immunoprecipitation experiments using differently mutated human CYP2E1s revealed that conformational anti-CYP2E1 antibodies targeted two epitopes located on the molecule surface in an area between Lys(324)-Glu(346) at J-K'' helices overlapping. Such epitopes were not recognized by the sera targeting linear CYP2E1 antigens. The CYP2E1(324-346) peptide showed good homology with two sequences (NS5b(438-449) and NS5b(456-465)) within the NS5b protein of hepatitis C virus (HCV). Consistently, conformational anti-CYP2E1 IgG bind to GST-conjugated NS5b(438-449) and NS5b(456-465) more efficiently than those recognizing CYP2E1 linear antigens. Competition experiments confirmed the cross-reactivity of conformational anti-CYP2E1 IgG with both NS5b(438-449) and NS5b(456-465). Moreover, mice immunized with GST-conjugated NS5b(438-449) or NS5b(456-465) peptides developed antibodies recognizing human CYP2E1. In CHC patients cross-reactivity between CYP2E1 and specific sequences in HCV-NS5b protein can promote the development of auto-antibodies targeting conformational epitopes on the CYP2E1 surface that might contribute to hepatic injury.

Control of Human Cytochrome P450 2E1 Electrocatalytic Response as a Result of Unique Orientation on Gold Electrodes

Oriented immobilization of human cytochrome P450 2E1 and its catalytic activity by direct electrochemistry was achieved by engineering two multisite mutants of P450 2E1: MUT261 (C268S-C480S-C488S) and MUT268 (C261S-C480S-C488S). Here, all the exposed cysteines are mutated into serines, with the exception of one (C261 for MUT261 and C268 for MUT268) that is able to link covalently to a modified gold electrode. The P450 2E1 wild type, as well as the two mutants, were immobilized onto gold electrodes using dithio-bismaleimidoethane as a self-assembled monolayer. The catalytic activity of the wild type and of the two cysteine mutants were determined using p-nitrophenol as a substrate, and the amount of the electrocatalysis product (p-nitrocatechol) was determined spectrophotometrically. The amounts of product formed by the mutants on the electrodes were 2-fold to 3-fold higher than those of the wild type. Control experiments performed in solution using the cytochrome P450 reductase as the electron donor show no significant differences in the level of product formed. The higher level of product formation of the two mutants on the electrode is ascribed to the controlled immobilization on the gold surface: the heme electron transfer proximal side is linked to the electrode, while the substrate binding distal side is exposed to the bulk solution. This is the first evidence that the control over the orientation of the human cytochromes P450 is key to maximize the electrocatalytic efficiency of these enzymes.