CYP2E1 Protein Research Articles

Protective effects of tiopronin against high fat diet-induced non-alcoholic steatohepatitis in rats

To study the protective effects of tiopronin against high fat diet-induced non-alcoholic steatohepatitis in rats. Male Sprague-Dawley rats were given a high-fat diet for 10 weeks. The rats were administered tiopronin (20 mg/kg) or a positive control drug ursodeoxycholic acid (UDCA, 15 mg/kg) via gavage daily from week 5 to week 10. After the rats were sacrificed, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), free fatty acids (FFA), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C), and liver homogenate FFA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured using commercial analysis kits. The expression levels of CYP2E1 mRNA and protein were determined using RT-PCR and immunoblot assays, respectively. Tiopronin significantly lowered both the serum ALT and AST levels, while only the serum ALT level was lowered by UDCA. Tiopronin significantly decreased the serum and liver levels of TG, TC and FFA as well as the serum LDL-C level, and increased the serum HDL-C level, while UDCA decreased the serum and liver TC levels as well as the serum LDL-C level, but did not change the serum levels of TG, FFA and HDL-C. Tiopronin apparently ameliorated the hepatocyte degeneration and the infiltration of inflammatory cells in the livers, but UDCA did not affect the pathological features of the livers. Both tiopronin and UDCA ameliorated the mitochondrial abnormality in the livers. The benefits of tiopronin were associated with increased SOD and GSH-Px activities, and with decreased MDA activity and CYP2E1 expression in the livers. Tiopronin exerts protective effects against non-alcoholic steatohepatitis in rats, which may be associated with its antioxidant properties and regulation of lipid metabolism.

Analysis of ethanol metabolic enzymes in primary human mammary epithelial cells and MCF‐10A cells: possible participation of CYP2E1 in ethanol‐induced oxidative stress and epidermal growth factor receptor (EGFR) activation

Ethanol (EtOH) consumption is a well‐established risk factor for breast cancer in women. However, the mechanism by which EtOH exerts its carcinogenic activity in breast tissue remains known. In the present work, we analyze the ethanol metabolic enzymes in primary human mammary epithelial cells (HMEC) and MCF10A cells and to study the role of those enzymes in ethanol‐ induced oxidative stress and EGFR activation. Our results show that EtOH decreased CYP2E1 protein expression at 10, 30 and 100 mM when cells were treated every 12 h for 3 days. We also found that 30 and 100 mM EtOH increased ROS levels after 2 h treatment in CYP2E1over‐expressing 4.1.2E1 cells. Additionally, we found that in CYP2E1over‐expressing 4.1.2E1 cells increased the level of pY1086 EGFR phosphorylation after 18 h ethanol treatment. These studies suggest that ethanol can transactivate EGFR and activate downstream cell signaling in MCF10A cells, mediated by oxidative stress. These studies were supported in part by NIH RO1 ES‐07259 and by CONACYT ‐Mexico a Fellowship to Angel León‐Buitimea number 212849.

Expression of the hepatic skatole- and androstenone-metabolising enzymes in entire male pigs of two live weights

The aim of the present study was to investigate an in vivo relationship between increased levels of testicular steroids and protein expression of the key enzymes involved in hepatic metabolism of skatole (cytochrome P4502E1 and CYP2E1 and CYP2A) and androstenone (3β-hydroxysteroid dehydrogenase — 3β-HSD, and sulfotransferase SULT2B1). For this purpose, we compared the protein expression in the liver from high weight (115kg) and low weight (90kg) pigs and related it to the level of the testicular steroids testosterone, oestrone sulphate and androstenone. We demonstrated that protein expressions of both skatole and androstenone-metabolising enzymes were affected by live weight of pigs. Expressions of CYP2E1 and 2A proteins were significantly lower (P=0.05 and 0.001, respectively) in the liver microsomes of high weight pigs compared to that of low weight pigs. The expression of androstenone-metabolising 3β-HSD was also lower (P=0.003) in high weight pigs whilst the expression of SULT2B1 was higher in high weight pigs (P=0.036). We concluded that the age-related decrease in activities of cytochrome P4502E1 and 2A and thus increased fat skatole levels are likely due to decreased protein expression of those enzymes in older (heavier) pigs with higher levels of testicular steroids. The age-related increase in androstenone levels can be at least partly due to decreased protein expression of 3β-hydroxysteroid dehydrogenase, but not SULT2B1.

Hepatocellular hypertrophy and cell proliferation in Sprague–Dawley rats from dietary exposure to potassium perfluorooctanesulfonate results from increased expression of xenosensor nuclear receptors PPARα and CAR/PXR

The present study investigated the potential role for activation of PPARα and CAR/PXR by potassium PFOS (K+PFOS) with respect to the etiology of hepatic hypertrophy and hepatocellular adenoma in rats. Male Sprague–Dawley rats were fed K+PFOS (20 or 100ppm) for either 1, 7, or 28 days. Wyeth 14,643 (Wy 14,643, 50ppm) and phenobarbital (PB, 500ppm) were the controls for PPARα and CAR/PXR activation, respectively. Measurements included: plasma ALT, AST, cholesterol, triglycerides, and glucose; liver protein and DNA content; liver activities of palmitoyl CoA oxidase (ACOX), Cyp4A, CYP2B, and CYP3A; induction of liver CYP4A1, CYP2E1, CYP2B1/2, and CYP3A1 proteins (SDS-PAGE and Western blots); liver and thyroid microscopic histopathology, apoptotic index, and cell proliferation index. Terminal body weight was decreased by K+PFOS (100ppm) and Wy 14,643. All test-compound treatments increased liver weight. Plasma lipids were decreased by both PFOS and Wy 14,643. After treatment for 1 day, K+PFOS (100ppm), PB, and Wy 14,643 increased mean hepatic DNA concentration and total hepatic DNA, and total DNA remained elevated after treatment for 7 days and 28 days (PB and Wy 14,643 only). Hepatic P450 concentration was elevated after 7 and 28 days by K+PFOS and by PB. K+PFOS and Wy 14,643 increased liver activities of ACOX and CYP4A as well as increased liver CYP4A1 protein. By 28 days of treatment, K+PFOS and PB increased liver activities of CYP2B and CYP3A as well as increased liver CYP2B1/2 and CYP3A1 proteins, and Wy 14,643 increased CYP2B enzyme activity to a slight extent. All test compounds increased the liver cell proliferative index and decreased the liver apoptotic index. No histological changes of the thyroid were noted; however, PB and WY increased thyroid follicular cell proliferation index (seven-day treatment only), while K+PFOS did not. The thyroid follicular cell apoptotic index did not differ between groups. The hepatomegaly and hepatocellular adenoma observed after dietary exposure of Sprague–Dawley rats to K+PFOS likely are due to the increased expression of xenosensor nuclear receptors PPARα and CAR/PXR. Given the markedly lower or absent response of human hepatocytes to the proliferative stimulus from activation of PPARα and CAR/PXR, the hepatocellular proliferative response from activation of these receptors by PFOS observed in rats is not expected to be of human relevance.

Effects of dietary inulin, statin, and their co-treatment on hyperlipidemia, hepatic steatosis and changes in drug-metabolizing enzymes in rats fed a high-fat and high-sucrose diet

BackgroundRats fed a high-fat and high-sucrose (HF) diet develop hepatic steatosis and hyperlipidemia. There are several reports that a change in nutritional status affects hepatic levels of drug-metabolizing enzymes. Synthetic inulin is a dietary component that completely evades glucide digestion. Supplementing a HF diet with inulin ameliorates hypertriglycemia and hepatic steatosis, but not hypercholesterolemia. This study aimed at distinguishing the effects of synthetic inulin and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin), which inhibit cholesterol biosynthesis.MethodsWe examined effects of co-treatment with synthetic inulin (5%) and fluvastatin (0, 4, and 8 mg/kg, per os) on body weight, epidydimal white adipose tissue weight, serum and hepatic lipid profiles, and hepatic cytochrome P450 (CYP) mRNA and protein profiles in rats fed a standard diet or a HF diet for 3 weeks.ResultsTreatment with the synthetic inulin (5%) or fluvastatin at 4 mg/kg (lethal dose in rats fed the HF diet, 8 mg/kg) ameliorated the elevation in hepatic triacylglycerol and total cholesterol levels in rats fed the HF diet. Whereas co-treatment with the inulin (5%) and fluvastatin (4 mg/kg) had a tendency to more strongly suppress the elevation in serum levels of very low density lipoprotein triacylglycerol than either treatment alone, no additive or synergistic effect was found in decrease in hepatic lipid levels. Hepatic levels of CYP1A1/2 and CYP2E1 mRNA and protein and methoxyresorufin O-demethylase and ethoxyresorufin O-deethylase activities were reduced in rats fed the HF diet. The synthetic inulin alleviated the reduction in hepatic levels of CYP1A1/2 and CYP2E1 mRNA and protein more strongly than fluvastatin, and no synergistic effects were observed on co-treatment. Furthermore, hepatic levels of aryl hydrocarbon receptor mRNA were decreased in rats fed the HF diet and recovered to near normal values with the intake of dietary inulin, which correlated with change in CYP1A1/2.ConclusionsDietary inulin alone was effective to prevent the development of hepatic steatosis, ameliorate nutritional effects, and alleviate the hepatic change in the expression of CYP1A1/2 and CYP2E1, while co-treatment with statin did not have additive or synergistic effects and statin may cause adverse effects in rats fed the HF diet.

Furocoumarins affect hepatic cytochrome P450 and renal organic ion transporters in mice

Furocoumarins are a group of natural products with many biological activities. Clinical evidences have demonstrated the important contribution of furocoumarins to the toxicity of some foods and herbs. In order to assess liver and kidney toxicity of furocoumarins, male mice were orally administrated with psoralen, isopsoralen, imperatorin, isoimperatorin and xanthotoxin at 20 and 40 mg/kg once daily for 28 days, respectively. No changes of food or water intake were observed in furocoumarins-treated mice. Only 40 mg/kg isopsoralen reduced body weight. 40 mg/kg furocoumarins altered serum activities of alanine transaminase, aspartate aminotransferase, alkaline phosphatase, and/or levels of albumin, showing hepatotoxicity. Furthermore, furocoumarins increased activity and protein expression of hepatic microsomal cytochrome P450 (CYP450) 3A11. CYP 2E1 activity and protein expression were suppressed by psoralen and isopsoralen and increased by xanthotoxin. Renal protein levels of organic cation/carnitine transporters (OCT1, OCT2 and OCTN2) and organic anion transporter 3 were increased by most furocoumarins. Renal urate transporter 1, glucose transporter 9 and multidrug resistance protein 4 were influenced by furocoumarins. These findings suggest that furocoumarins may interfere in metabolism, excretion and bioavailability of endogenous and exogenous compounds to impair liver and kidney functions mediated by affecting hepatic CYP450 and renal organic ion transport system.

Effect of mosquito mats (pyrethroid-based) vapor inhalation on rat brain cytochrome P450s

The effect of transfluthrin (TF) or D-allethrin (DA) pyrethroid (PYR) vapors, often contained as main ingredients in two commercially available mosquito repellent mats, on cytochrome P450 (CYP) enzymes of rat brain and liver was assessed. Immunodetection of CYP2E1 and CYP3A2 proteins revealed their induction in cerebrum and cerebellum, but not in liver microsomes of rats exposed by inhalation to TF or DA. This overexpression of proteins correlated with an increase of their catalytic activities. The specifically increased expression of CYP isoenzymes, due to PYR exposure in the rat brain, could perturb the normal metabolism of endogenous and xenobiotic compounds and leads to increased risks of neurotoxicity by bioactivation, lipid peroxidation and DNA damage.

Depression by a Green Tea Extract of Alcohol-Induced Oxidative Stress and Lipogenesis in Rat Liver

We determined the effects of a green tea extract with 36% alcohol on the blood alcohol content, oxidative stress, lipogenesis, inflammation and liver function of female Wistar rats. Tea alcohol significantly decreased the O₂⁻, H₂O₂ and HOCl amounts via catechins and not caffeine. Thirty days of alcohol gavage improved the level of reactive oxygen species (ROS) in the liver, bile and blood, increased the 4-hydroxynonenal-protein adducts, Kupffer cell infiltration and lipid accumulation in the liver, and elevated the plasma alanine aminotransferase level. A western blot analysis showed reduced expression of the oxidative enzymes (CYP2E1 and NADPH oxidase p47phox protein) and lipogenic enzymes (SREBP-1c and fatty acid synthase) in the alcohol-treated liver. Tea alcohol significantly attenuated these elevated parameters. We conclude that the green tea extract in alcohol efficiently reduced the amounts of O₂⁻, H₂O₂ and HOCl primarily due to the catechin content, and not caffeine. The developed tea liquor attenuated alcohol-induced oxidative injury and lipogenesis in the liver by the synergetic action of catechins and caffeine.

Independent and Combined Effects of Ethanol Self-Administration and Nicotine Treatment on Hepatic CYP2E1 in African Green Monkeys

Cytochrome P450 2E1 metabolizes ethanol and also bioactivates many toxins and procarcinogens. Elevated levels of hepatic CYP2E1 are associated with an increased susceptibility to chemical toxicity and carcinogenesis. This study investigated the induction of hepatic CYP2E1 by ethanol and nicotine, alone and in combination, in a nonhuman primate model. Monkeys that self-administered ethanol and that received subcutaneous injections of nicotine (0.5 mg/kg b.i.d.), alone and in combination, were compared with control animals (four groups, n = 10/group). Chlorzoxazone (CZN) was used as a probe drug to phenotype in vivo CYP2E1 activity before and after chronic ethanol and/or nicotine exposure. CYP2E1 protein levels and in vitro chlorzoxazone metabolism were assessed in liver microsomes. Average daily ethanol consumption was ≈3.0 g/kg (blood ethanol levels ≈24 mM) and was unaffected by nicotine treatment. Ethanol self-administration and nicotine treatment, alone and in combination, significantly increased in vivo CZN disposition compared with that in control animals. The effect of ethanol was only observed at higher levels of intake. Ethanol and nicotine increased CYP2E1 protein levels and in vitro CZN metabolism, with combined exposure to both drugs resulting in the greatest increase. The effect of ethanol was also dependent on level of intake. Chronic exposure to ethanol and nicotine induced hepatic CYP2E1 activity and protein levels, particularly when both drugs were used in combination and when ethanol intake was high. These results have important implications for public health, given the association between elevated CYP2E1 and disease, and the large proportion of individuals who are exposed to ethanol and nicotine, often in combination.

Chronic alcohol intake-induced oxidative stress and apoptosis: role of CYP2E1 and calpain-1 in alcoholic cardiomyopathy

Cytochrome P-450 2E1 CYP2E1 induction has been linked to oxidative stress in a number of experimental models. The aim of this study was to investigate the relationship between CYP2E1 activity and markers of oxidative stress and cardiac cell apoptosis during the development of alcoholic cardiomyopathy (ACM). Changes in left ventricular morphology were evaluated in 4 groups of chronically instrumented dogs (control; alcohol-receiving; and alcohol-receiving plus treatment with either valsartan or carnitine) after 6 months of treatment. CYP2E1 and calpain-1 protein expression were determined by Western blotting, and apoptosis evaluated by TUNEL and immunohistochemistry. Malonyl dialdehyde levels were assessed as a marker of oxidative stress, while superoxide dismutase and glutathione peroxidase levels were evaluated as markers of antioxidant defense mechanisms. Expression of CYP2E1 was increased in the alcohol-receiving group compared with controls (P<0.05) and was associated with oxidative stress. Similarly, expression of Bad and calpain-1 protein was increased after chronic alcohol exposure, while Bcl-xL protein expression remained at a low level. Bad and calpain-1 protein expressions were significantly inhibited by treatment with valsartan or carnitine, while expression of Bcl-xL protein was increased (P<0.05). Collectively, our results indicate a possibly significant role for CYP2E1 in the oxidative stress associated with chronic alcoholism. The resulting increase in oxidative stress is accompanied by cellular apoptosis and may ultimately contribute to tissue remodeling and ACM. Importantly, these alcohol-induced effects may be abrogated by means such as angiotensin 1 receptor blockade or carnitine supplementation.

Role of Kupffer cells in pathogenesis of sepsis‐induced drug metabolizing dysfunction

The present study aimed to determine the role of Kupffer cells (KCs) in cytochrome P450 (CYP) isozyme activity and the expression of its gene during polymicrobial sepsis. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl(3)) at 48 and 24 h before cecal ligation and puncture (CLP). The depletion of KCs was confirmed by measuring the mRNA level of the KC marker gene CD163. Serum aminotransferase levels and lipid peroxidation showed an increase and hepatic glutathione content showed a decrease at 24 h after CLP. These changes were prevented by GdCl(3) pretreatment. Catalytic activities of CYP1A1, 1A2 and 2E1 showed a significant reduction at 24 h after CLP but were prevented by GdCl(3). A reduction in the levels of CYP2E1 protein and CYP2B1 and CYP2E1 mRNA expression was prevented by GdCl(3). Phosphorylation of CYP1A1/1A2 markedly increased 24 h after CLP, which was prevented by GdCl(3). The increased serum level of high mobility group box 1, hepatic level of Toll-like receptors 2 and 4, and inducible nitric oxide synthase protein expression were prevented by GdCl(3). In addition, elevated serum concentrations of tumor necrosis factor-α and interleukin-6, and increased hepatic mRNA levels of tumor necrosis factor-α and interleukin-6 were decreased by depletion of KCs. Our findings suggest that ablation of KCs protects against hepatic drug-metabolizing dysfunction by modulation of the inflammatory response.

Secretory phospholipase A 2-mediated progression of hepatotoxicity initiated by acetaminophen is exacerbated in the absence of hepatic COX-2

We have previously reported that among the other death proteins, hepatic secretory phospholipase A 2 (sPLA 2) is a leading mediator of progression of liver injury initiated by CCl 4 in rats. The aim of our present study was to test the hypothesis that increased hepatic sPLA 2 released after acetaminophen (APAP) challenge mediates progression of liver injury in wild type (WT) and COX-2 knockout (KO) mice. COX-2 WT and KO mice were administered a normally non lethal dose (400 mg/kg) of acetaminophen. The COX-2 KO mice suffered 60% mortality compared to 100% survival of the WT mice, suggesting higher susceptibility of COX-2 KO mice to sPLA 2-mediated progression of acetaminophen hepatotoxicity. Liver injury was significantly higher at later time points in the KO mice compared to the WT mice indicating that the abatement of progression of injury requires the presence of COX-2. This difference in hepatotoxicity was not due to increased bioactivation of acetaminophen as indicated by unchanged cyp2E1 protein and covalently bound 14C-APAP in the livers of KO mice. Hepatic sPLA 2 activity and plasma TNF-α were significantly higher after APAP administration in the KO mice. This was accompanied by a corresponding fall in hepatic PGE 2 and lower compensatory liver regeneration and repair ( 3H-thymidine incorporation) in the KO mice. These results suggest that hindered compensatory tissue repair and poor resolution of inflammation for want of beneficial prostaglandins render the liver very vulnerable to sPLA 2-mediated progression of liver injury. These findings are consistent with the destructive role of sPLA 2 in the progression and expansion of tissue injury as a result of continued hydrolytic breakdown of plasma membrane phospholipids of perinecrotic hepatocytes unless mitigated by sufficient co-induction of COX-2.

Transgenic plants of Petunia hybrida harboring the CYP2E1 gene efficiently remove benzene and toluene pollutants and improve resistance to formaldehyde

The CYP2E1 protein belongs to the P450 enzymes family and plays an important role in the metabolism of small molecular and organic pollutants. In this study we generated CYP2E1 transgenic plants of Petunia using Agrobacterium rhizogenes K599. PCR analysis confirmed that the regenerated plants contained the CYP2E1 transgene and the rolB gene of the Ri plasmid. Southern blotting revealed the presence of multiple copies of CYP2E1 in the genome of transgenic plants. Fluorescent quantitative PCR revealed exogenous CYP2E1 gene expression in CYP2E1 transgenic plants at various levels, whereas no like expression was detected in either GUS transgenic plants or wild-types. The absorption of benzene and toluene by transgenic plants was analyzed through quantitative gas chromatography. Transgenic plants with high CYP2E1 expression showed a significant increase in absorption capacity of environmental benzene and toluene, compared to control GUS transgenic and wild type plants. Furthermore, these plants also presented obvious improved resistance to formaldehyde. This study, besides being the first to reveal that the CYP2E1 gene enhances plant resistance to formaldehyde, also furnishes a new method for reducing pollutants, such as benzene, toluene and formaldehyde, by using transgenic flowering horticultural plants.

Acetaminophen Induces Apoptosis in Rat Cortical Neurons

BackgroundAcetaminophen (AAP) is widely prescribed for treatment of mild pain and fever in western countries. It is generally considered a safe drug and the most frequently reported adverse effect associated with acetaminophen is hepatotoxicity, which generally occurs after acute overdose. During AAP overdose, encephalopathy might develop and contribute to morbidity and mortality. Our hypothesis is that AAP causes direct neuronal toxicity contributing to the general AAP toxicity syndrome.Methodology/Principal FindingsWe report that AAP causes direct toxicity on rat cortical neurons both in vitro and in vivo as measured by LDH release. We have found that AAP causes concentration-dependent neuronal death in vitro at concentrations (1 and 2 mM) that are reached in human plasma during AAP overdose, and that are also reached in the cerebrospinal fluid of rats for 3 hours following i.p injection of AAP doses (250 and 500 mg/Kg) that are below those required to induce acute hepatic failure in rats. AAP also increases both neuronal cytochrome P450 isoform CYP2E1 enzymatic activity and protein levels as determined by Western blot, leading to neuronal death through mitochondrial–mediated mechanisms that involve cytochrome c release and caspase 3 activation. In addition, in vivo experiments show that i.p. AAP (250 and 500 mg/Kg) injection induces neuronal death in the rat cortex as measured by TUNEL, validating the in vitro data.Conclusions/SignificanceThe data presented here establish, for the first time, a direct neurotoxic action by AAP both in vivo and in vitro in rats at doses below those required to produce hepatotoxicity and suggest that this neurotoxicity might be involved in the general toxic syndrome observed during patient APP overdose and, possibly, also when AAP doses in the upper dosing schedule are used, especially if other risk factors (moderate drinking, fasting, nutritional impairment) are present.

Ethanol Induction of CYP2A5: Permissive Role for CYP2E1

CYP2A5 metabolizes xenobiotics and activates hepatocarcinogens, and induction occurs in response to hepatic damage and cellular stress. We evaluated whether ethanol can elevate CYP2A5 and whether CYP2E1 plays a role in the ethanol induction of CYP2A5. Wild-type (WT), CYP2E1 knockout (KO), and CYP2E1 knockin (KI) mice were fed ethanol for 3 weeks. Ethanol increased CYP2E1 and CYP2A5 protein and activity in WT mice but not in the KO mice. Induction of CYP2A5 (and CYP2E1) was restored in the KI mice. Ethanol induction of CYP2A5 occurred only after CYP2E1 was first induced. Immunohistochemical staining revealed that CYP2E1 and CYP2A5 colocalize to the same zones in the liver. Ethanol also elevated CYP2A5 mRNA levels in WT and KI mice but not in KO mice. Induction of CYP2A5 by cadmium was partially decreased in KO mice compared with WT or KI mice. Ethanol elevated CYP2A4 mRNA levels in all mice although the extent of induction was lowest in the KO mice. In summary, ethanol elevated mouse hepatic CYP2A5 levels, which may be of toxicological significance because CYP2A5 metabolizes nicotine and other drugs and activates hepatocarcinogens. Induction of CYP2A5 by ethanol is potentiated by the induction of CYP2E1. We speculate that ethanol induction of CYP2E1 followed by increases in reactive oxygen species and activation of Nrf2 are important steps in the mechanism by which ethanol induces CYP2A5. The possibility that induction of CYP2E1 is permissive for the induction of CYP2A5 may reflect a new contribution by CYP2E1 to the actions of ethanol.

Effect of Alcohol on Drug Efflux Protein and Drug Metabolic Enzymes in U937 Macrophages

ATP-binding cassette (ABC) proteins and cytochrome P450 (CYP) enzymes regulate the bioavailability of HIV-1 antiretroviral therapeutic drugs, non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs). They are also involved in regulating, and responding to, oxidative stress in various tissues and organs including liver. This study is designed to assess the effect of alcohol on the ABCC1 and CYP enzymes involved in the metabolism of NNRTIs and PIs (CYP2B6, CYP2D6, and CYP3A4) and oxidative stress (CYP1A1, CYP2A6, and CYP2E1) in U937 macrophages. The U937 cell line has been utilized as an in vitro model of human macrophages. The expression levels of the ABCC1 and CYP enzymes in U937 macrophages were characterized in terms of mRNA quantification, protein analysis, and assays for functional activity. In addition, oxidative stress was monitored by measuring the activities of oxidative stress marker enzymes and production of reactive oxygen species (ROS). The order of mRNA expression in U937 macrophages was ABCC1 ∼ CYP2A6 > CYP3A4 ∼ CYP2E1 ∼ CYP1A1 > CYP2D6 > CYP2B6. Alcohol (100 mM) increased the mRNA levels of ABCC1 and CYP2A6 (200%), CYP2B6 and CYP3A4 (150%), and CYP2E1 (400%) compared with the control. Alcohol caused significant upregulation of ABCC1, CYP2A6, CYP2E1, and CYP3A4 proteins (50 to 85%) and showed >50% increase in the specific activity of CYP2A6 and CYP3A4 in U937 macrophages. Furthermore, alcohol increased the production of ROS and significantly enhanced the activity of oxidative stress marker enzymes, superoxide dismutase, and catalase in U937 macrophages. Our study showed that alcohol causes increases in the genetic and functional expressions of ABCC1 and CYP enzymes in U937 macrophages. This study has clinical implications in alcoholic HIV-1 individuals, because alcohol consumption is reported to reduce the therapeutic efficacy of NNRTIs and PIs and increases oxidative stress.

Сравнительный анализ генов ортологичных белков цитохрома Р450 2Е1 человека и млекопитающих

Aim. To carry out a comparison analysis of nucleotide sequences for the cytochrome P450 2E1 protein ortologs genes and reestablish the connection of gene evolution with nucleotide content. Methods. In silico: BLAST, ClustalW, MEGA4, PHP. Results. A general phylogeny of CYP2E1 genes is described. The most affinity is found for human translated sequences with Pan troglodytes. The transition N > T is the most often, it occurs in introns by 2.6 times more than in exons. The correlation of keto-amino skew of CYP2E1 genes and evolution age of species is stated. Conclusions. The analysis carried out in the paper allows us to assume that a common ancestor of the CYP2E1 protein isoform lived before the divergence between rodent and Primates orders, i.e. 70 million years ago. The single nucleotide substitution is accumulated in introns during evolution.

Bariatric Surgery-Induced Weight Loss Reduces Hepatic Lipid Peroxidation Levels and Affects Hepatic Cytochrome P-450 Protein Content

To evaluate the effects of surgical weight loss on hepatic lipid peroxidation levels and cytochrome P-450 protein expression in patients with nonalcoholic fatty liver disease (NAFLD). NAFLD and nonalcoholic steatohepatitis (NASH) affect hepatic cytochrome P-450 (CYP) protein expression and activity, and CYP2E1 may play a role in the pathogenesis of NAFLD and NASH through induction of oxidative stress and lipid peroxidation. NAFLD and NASH are associated with increased systemic lipid peroxidation levels and elevated hepatic CYP2E1 activity, but hepatic CYP3A4/5 activity is decreased. Liver biopsies from 20 patients with NAFLD who underwent bariatric surgery were obtained intraoperatively and at 15 +/- 7 months following surgery. Hepatic malondialdehyde (MDA) levels (a marker of lipid peroxidation), CYP2E1 and CYP3A4/5 protein expression, and steatosis, as a percent of total area, were measured by immunohistochemistry followed by digital image quantitation. Following weight loss, as reflected by reduced BMI (54 +/- 9 vs. 37 +/- 9 kg/m2; P < 0.001), features of the metabolic syndrome, grade and stage of liver disease, and liver histology were all significantly improved (P < 0.01). Hepatic MDA staining (35 +/- 18% vs. 23 +/- 14%; P = 0.02), CYP2E1 protein content (68 +/- 9% vs. 56 +/- 11%; P < 0.001), and steatosis (17 +/- 7% vs. 2 +/- 3%; P < 0.001) were significantly reduced following weight loss. CYP3A4/5 protein content was unchanged (57 +/- 13% vs. 55 +/- 13%; P = 0.433). The reduction in lipid peroxidation was independently associated with changes in CYP2E1 protein expression after bariatric surgery (r = 0.477; P = 0.033). Elevations in hepatic lipid peroxidation and CYP2E1 expression that are seen in NAFLD improve significantly with weight loss induced by bariatric surgery.

Cytochrome 3A and 2E1 in human liver tissue: Individual variations among normal Japanese subjects

Aims The metabolism of drugs, xenobiotic compounds, and other endogenous/exogenous substrates generally begins with their oxidation through cytochrome P450 (CYP). The results of recent pharmacogenetic analyses have demonstrated CYP's polymorphisms to be related to individual differences in metabolism, but only a limited number of CYP3A4 and CYP2E1 variant alleles influence enzymatic activities. Therefore, CYP gene expression profiling of both normal and pathological human livers should provide critical information for an evaluation of the biological significance of CYPs. Main methods In our present study, we first characterized the individual differences in CYP3A4 and CYP2E1 expression levels among Japanese normal or non-pathological liver tissue obtained from autopsy or surgery using immunohistochemistry and quantitative RT-PCR array of phase I metabolic enzymes with combined laser capture microscopy and qPCR analysis. Key findings Both CYP3A4 and CYP2E1 mRNA and proteins were predominantly detected in hepatocytes surrounding central veins in normal liver, but there were marked individual differences in both CYP3A4 and CYP2E1 mRNA and proteins among the 23 Japanese subjects examined. Individual differences in CYP3A and CYP2E1 subtypes were also detected in the livers obtained from monozygotic neonatal Japanese female twins with different survival periods. CYP3A and CYP2E1-positive cells were decreased in number in non-pathological hepatocytes of diseased livers compared to those in disease-free livers from autopsy. Significance The above results suggest that individual differences in CYP3A4 and CYP2E1 exist among normal human liver tissues and in non-pathological hepatocytes between diseased and normal liver, and these differences may be important in evaluating the pharmacodynamics of various substances.

Ethanol Enhances Hepatitis C Virus Replication through Lipid Metabolism and Elevated NADH/NAD+

Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD(+) ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD(+). Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting beta-oxidation with beta-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. Therefore, lipid metabolism and alteration of cellular NADH/NAD(+) ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.