Overexpression Of Cytochrome P450 2E1 Research Articles

Identification of Cytochrome P450 2E1 as a Novel Target in Glioma and Development of Its Inhibitor as an Anti-Tumor Agent.

Glioblastoma (GBM) is a devastating inflammation-related cancer for which novel therapeutic targets are urgently required. Previous studies of the authors indicate Cytochrome P450 2E1 (CYP2E1) as a novel inflammatory target and develop a specific inhibitor Q11. Here it is demonstrated that CYP2E1 overexpression is closely related to higher malignancy in GBM patients. CYP2E1 activity is positively correlated with tumor weight in GBM rats. Significantly higher CYP2E1 expression accompanied by increased inflammation is detected in a mouse GBM model. Q11, 1-(4-methyl-5-thialzolyl) ethenone, a newly developed specific inhibitor of CYP2E1 here remarkably attenuates tumor growth and prolongs survival in vivo. Q11 does not directly affect tumor cells but blocks the tumor-promoting effect of microglia/macrophage (M/Mφ) in the tumor microenvironment through PPARγ-mediated activation of the STAT-1 and NF-κB pathways and inhibition of the STAT-3 and STAT-6 pathways. The effectiveness and safety of targeting CYP2E1 in GBM are further supported by studies with Cyp2e1 knockout rodents. In conclusion, a pro-GBM mechanism in which CYP2E1-PPARγ-STAT-1/NF-κB/STAT-3/STAT-6 axis fueled tumorigenesis by reprogramming M/Mφ and Q11 as a promising anti-inflammatory agent for GBM treatment is uncovered.

Evaluation on Hepatoprotection of Dihydromyricetin in Acetaminophen-Induced Hepatotoxicity Based on Analysis of Inflammation and Apoptosis Mediated by PI3K/AKT Pathway

Purpose: We aimed to investigate whether dihydromyricetin (DHM) could alleviate acetaminophen (APAP)-induced liver damage in mice, and to verify whether the process is associated with the PI3K/AKT signaling pathway. Methods: The contents of DHM in serum and related physiological indicators in blood and liver tissue were measured, respectively. We used haematoxylin and eosin (H&E), TUNEL, Hoechst 33,258, immunofluorescence assay and western blot methods to comprehensively assess the protective mechanism and therapeutic effect of DHM on liver damage induced by APAP (250 mg/kg) in mice. Results: APAP (250 mg/kg) could increase the expression of alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β) and cause 4-hydroxy-2-nonenal (4-HNE) and Cytochrome P450 2E1 (CYP2E1) overexpression and stress response in the PI3K/AKT pathway. DHM was also detected in the serum of mice about five minutes after administration. DHM pretreatment could reverse GSH depletion and CYP2E1 overexpression, reduce the expression of ALT, AST, malondialdehyde, 4-HNE, TNF-α, and IL-1β, meanwhile it could reverse the abnormal expression of PI3K/AKT signaling pathway-related proteins which were induced by APAP. DHM pretreatment significantly reduced APAP-induced liver tissue apoptosis, necrosis, and inflammatory infiltration. Conclusion: DHM had a hepatoprotective effect on hepatotoxicity induced by APAP, which was shown by inhibiting oxidative stress and inflammatory responses, and reducing hepatocyte apoptosis by activating the PI3K/AKT signaling pathway.

High-Risk HPV with Multiple Infections Promotes CYP2E1, Lipoperoxidation and Pro-Inflammatory Cytokines in Semen of Asymptomatic Infertile Men

High-risk human papillomavirus (HR-HPV) infection, followed by chronic inflammation and oxidative stress, is a major risk factor of male infertility. In this study, we explored the potential impact of high-risk (HR) HPV genotypes in single infection (SI) and multiple infections (MI) that promote CYP2E1 expression, oxidative damage and pro-inflammatory cytokines, possibly contributing to sperm damage and male infertility. Semen samples from 101 infertile military men were studied. We analyzed seminal parameters, namely, HPV genotyping, cytochrome P450 2E1 (CYP2E1), oxidative stress biomarkers (total antioxidant capacity (TAC), catalase (CAT) and superoxide dismutase (SOD)), lipid peroxidation (LPO), 8-hydroxiguanosine (8-OHdG) and pro-inflammatory cytokines (IFN-γ, IL-1β, IL-4, IL-6 and IL-8). Eighty-one men (80.2%, 81/101) were positive for HPV infection, and MI-HR-HPV was higher than SI-HR-HPV (63% vs. 37%). HPV-52 was the most frequently detected type (18.5%), followed by HPV-33 (11.1%), and the most frequent combination of genotypes detected was HPV-33,52 (11.1%), followed by HPV-18,31 (6.2%). The group with infected samples presented lower normal morphology and antioxidant levels compared to non-infected samples. In concordance, the infected group showed high levels of LPO, IFN-γ, IL-1β, IL-4 and IL-6 and downregulation of CAT and SOD enzymes. Interestingly, changes in motility B, low levels of TAC, overexpression of CYP2E1, LPO and IL-8 levels were higher in MI-HR-HPV than SI-HR-HPV, suggesting that HPV infection promotes a chronic inflammatory process and a toxic and oxidative microenvironment, which increases with MI-HPV infections.

Downregulation of mitogen-activated protein kinases (MAPKs) in chronic ethanol-induced fatty liver

Mitogen-activated protein kinases (MAPKs) are versatile proteins that have been suggested to be involved in the regulation of lipid metabolism. This study was designed to investigate the responses of MAPK signaling to chronic ethanol exposure in vivo and in vitro, and try to explore its role in the pathogenesis of alcoholic fatty liver (AFL). Mice were fed with Lieber-Decarli liquid diet (5% ethanol, w/v) for 4 weeks to induce fatty liver, and the chronological changes of MAPK phosphorylation were measured using western blotting. We found that chronic ethanol feeding led to accumulation of triglyceride (TG), decreased phosphorylation of MAPKs, decreased protein level of peroxisomal proliferator activation receptor α (PPARα), and increased protein expression of cytochrome P4502E1 (CYP2E1) in mice liver. In vitro study showed that overexpression of CYP2E1 blunted the response of MAPKs to ethanol, and MAPK phosphatase 1 (MKP-1) knockdown by siRNA led to upregulation of PPARα protein level. Lastly, epidermal growth factor (EGF), a well-known MAPK activator, significantly suppressed chronic ethanol-induced hepatic fat accumulation and decline of PPARα expression in mice liver. Collectively, MAPK suppression, possibly due to the activation of hepatic CYP2E1, may be involved in chronic ethanol-induced hepatic steatosis.

Linking Dysregulated AMPK Signaling and ER Stress in Ethanol-Induced Liver Injury in Hepatic Alcohol Dehydrogenase Deficient Deer Mice.

Ethanol (EtOH) metabolism itself can be a predisposing factor for initiation of alcoholic liver disease (ALD). Therefore, a dose dependent study to evaluate liver injury was conducted in hepatic alcohol dehydrogenase (ADH) deficient (ADH−) and ADH normal (ADH+) deer mice fed 1%, 2% or 3.5% EtOH in the liquid diet daily for 2 months. Blood alcohol concentration (BAC), liver injury marker (alanine amino transferase (ALT)), hepatic lipids and cytochrome P450 2E1 (CYP2E1) activity were measured. Liver histology, endoplasmic reticulum (ER) stress, AMP-activated protein kinase (AMPK) signaling and cell death proteins were evaluated. Significantly increased BAC, plasma ALT, hepatic lipids and steatosis were found only in ADH− deer mice fed 3.5% EtOH. Further, a significant ER stress and increased un-spliced X-box binding protein 1 were evident only in ADH− deer mice fed 3.5% EtOH. Both strains fed 3.5% EtOH showed deactivation of AMPK, but increased acetyl Co-A carboxylase 1 and decreased carnitine palmitoyltransferase 1A favoring lipogenesis were found only in ADH− deer mice fed 3.5% EtOH. Therefore, irrespective of CYP2E1 overexpression; EtOH dose and hepatic ADH deficiency contribute to EtOH-induced steatosis and liver injury, suggesting a linkage between ER stress, dysregulated hepatic lipid metabolism and AMPK signaling.

Upregulation of CYP2E1 expression causes oxidative damage induced by 2-chloroethanol in primary cultured rat astrocytes

Brain edema caused by subacute poisoning with 1,2-dichloroethane (1,2-DCE) has gained much attention during recent years, but its underlying mechanism is poorly understood. As an intermediate metabolite of 1,2-DCE in vivo, 2-chloroethanol (2-CE) can be transformed into chloroacetaldehyde and reactive oxygen species (ROS) through cytochrome P450 2E1 (CYP2E1) mediated metabolism. In previous studies, it was found that CYP2E1 expression is enhanced in the brain of mice treated with 1,2-DCE. This study was designed to verify the roles of CYP2E1 overexpression in 2-CE induced cytotoxicity in rat astrocytes, and the contribution of specific signaling molecules to the upregulation of CYP2E1 expression caused by 2-CE. The results of this study demonstrate that treatment with 2-CE can enhance CYP2E1 protein and mRNA levels, cause an increase in ROS and MDA levels, and higher percentages of apoptotic cells in rat astrocytes. Pretreatment with either diallyl sulfide or vitamin C, the inhibitor of CYP2E1 or scavenger of ROS, respectively, can suppress the levels of CYP2E1 expression, ROS and MDA, ameliorate cell apoptosis, and attenuate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells. Additionally, pretreatment with the inhibitor of either ERK1/2 or transcriptional factor specificity protein 1 (SP1) can suppress the CYP2E1 expression, and alleviate the oxidative damage caused to these cells. In conclusion, our findings demonstrate that CYP2E1 overexpression plays a crucial role in 2-CE induced oxidative damage of rat astrocytes, and that CYP2E1 expression is upregulated partially through the activation of the ERK1/2 and SP1 signaling pathways by ROS generated during CYP2E1-mediated 2-CE metabolism. This study provides novel information that can be used in elucidating the mechanism by which 1,2-DCE induces brain edema.

A systematic evaluation of microRNAs in regulating human hepatic CYP2E1

Cytochrome P450 2E1 (CYP2E1) is an important drug metabolizing enzyme for processing numerous xenobiotics in the liver, including acetaminophen and ethanol. Previous studies have shown that microRNAs (miRNAs) can suppress CYP2E1 expression by binding to the 3′-untranslated region (3′-UTR) of its transcript. However, a systematic analysis of CYP2E1 regulation by miRNAs has not been described. Here, we applied in silico, in vivo, and in vitro approaches to investigate miRNAs involved in the regulation of CYP2E1. Initially, potential miRNA binding sites in the CYP2E1 mRNA transcript were identified and screened using in silico methods. Next, inverse correlations were found in human liver samples between the expression of CYP2E1 mRNA and the levels of two miRNA species, hsa-miR-214-3p and hsa-miR-942-5p. In a HepG2-derived CYP2E1 over-expression cell model, hsa-miR-214-3p exhibited strong suppression of CYP2E1 expression by targeting the coding region of its mRNA transcript, but hsa-miR-942-5p did not inhibit CYP2E1 levels. Electrophoretic mobility shift assays confirmed that hsa-miR-214-3p recruited other cellular protein factors to form stable complexes with specific sequences present in the CYP2E1 mRNA open reading frame. Transfection of HepaRG cells with hsa-miR-214-3p mimics inhibited expression of the endogenous CYP2E1 gene. Further, hsa-miR-214-3p mimics partially blocked ethanol-dependent increases in CYP2E1 mRNA and protein levels in HepG2 cells and they reduced the release of alanine aminotransferase from CYP2E1-overexpressing HepG2 cells exposed to acetaminophen. These results substantiate the suppressing effect of hsa-miR-214-3p on CYP2E1 expression.

The induction of cytochrome P450 2E1 by ethanol leads to the loss of synaptic proteins via PPARα down-regulation.

Ethanol, one of the most commonly abused substances throughout history, is a substrate and potent inducer of cytochrome P450 2E1 (CYP2E1). Our previous study showed that brain CYP2E1 was induced by chronic ethanol treatment and was associated with ethanol-induced neurotoxicity in rats. We therefore investigated the possible mechanism of brain CYP2E1 involvement in ethanol-induced neurodegeneration. Compared with the controls, chronic ethanol treatment (3.0g/kg, i.g., 160days) significantly increased CYP2E1 mRNA levels in the rat cortex, but the mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and the pre- and post-synaptic proteins (synaptophysin, SYP, and drebrin1, DBN1) were decreased. Ethanol treatment dose-dependently induced CYP2E1 mRNA expression, and CYP2E1 overexpression exacerbated the ethanol-induced neurotoxicity. Pretreatment with p38 inhibitor (SB202190) and ERK1/2 inhibitor (U0126) attenuated the induction of CYP2E1 mRNA and protein levels by ethanol; however, no change was observed with JNK inhibitor pretreatment. Ethanol exposure or CYP2E1 overexpression significantly decreased PPARα, SYP, and DBN1 expression as indicated by the data from real-time RT-PCR, Western blotting and immunocytochemistry. The activation of PPARα by WY14643 increased the activity of the SYP and DBN1 promoters and attenuated the inhibition of these genes by ethanol. The specific siRNA for CYP2E1 significantly attenuated the ethanol-induced inhibition of PPARα, SYP and DBN1 mRNA levels. These results suggest that the induction of CYP2E1 by ethanol may be mediated via the p38 and ERK1/2 signaling pathways in neurons but not via the JNK pathway. The CYP2E1-PPARα axis may play a role in ethanol-induced neurotoxicity via the alteration of the genes related with synaptic function.

Protective effect of Pyropiayezoensis glycoprotein on chronic ethanol consumption-induced hepatotoxicity in rats.

The present study investigated the protective effect of Pyropiayezoensis glycoprotein (PYGP) against chronic ethanol consumption‑mediated hepatotoxicity in rats. Male Sprague-Dawley rats (n=20; 6weeks old) were randomly divided into four groups. The rats in each group were treated for 30days with the following: i)CON group, distilled water only; ii)EtOH group, 20% ethanol 3.7g/kg/BW; iii)EtOH+150group, 20%ethanol 3.7g/kg/BW+PYGP 150mg/kg/BW; iv)EtOH+300 group, 20% ethanol 3.7g/kg/BW+PYGP 300mg/kg/BW. EtOH, PYGP and water were orally administered. The rats were sacrificed after 30days, and blood and liver samples were collected for analysis. Treatment with ethanol caused significant elevation of serum levels of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). Furthermore, inhibition of the antioxidant defense system in the liver, including glutathione (GSH), glutathione peroxidase (GSH‑px) and catalase (CAT) was observed. However, co‑administration with PYGP recovered the antioxidant defense system, and the serum levels of GOT and GPT. PYGP was shown to attenuate ethanol toxicity via the inactivation of mitogen‑activated protein kinases (MAKPs). PYGP suppressed the overexpression of cytochrome P4502E1 (CYP2E1), inducible nitric oxide synthase and cyclooxygenase‑2. These results suggested that the protective effect of PYGP was associated with antioxidant activities, MAPKs and the CYP2E1 signaling pathway.

Hyperinsulinemia shifted energy supply from glucose to ketone bodies in early nonalcoholic steatohepatitis from high-fat high-sucrose diet induced Bama minipigs

The minipig can serve as a good pharmacological model for human subjects. However, the long-term pathogenesis of high-calorie diet-induced metabolic syndromes, including NASH, has not been well described in minipigs. We examined the development of metabolic syndromes in Bama minipigs that were fed a high-fat, high-sucrose diet (HFHSD) for 23 months, by using histology and serum biochemistry and by profiling the gene expression patterns in the livers of HFHSD pigs compared to controls. The pathology findings revealed microvesicular steatosis, iron overload, arachidonic acid synthesis, lipid peroxidation, reduced antioxidant capacity, increased cellular damage, and inflammation in the liver. RNA-seq analysis revealed that 164 genes were differentially expressed between the livers of the HFHSD and control groups. The pathogenesis of early-stage NASH was characterized by hyperinsulinemia and by de novo synthesis of fatty acids and nascent triglycerides, which were deposited as lipid droplets in hepatocytes. Hyperinsulinemia shifted the energy supply from glucose to ketone bodies, and the high ketone body concentration induced the overexpression of cytochrome P450 2E1 (CYP2E1). The iron overload, CYP2E1 and alcohol dehydrogenase 4 overexpression promoted reactive oxygen species (ROS) production, which resulted in arachidonic and linoleic acid peroxidation and, in turn, led to malondialdehyde production and a cellular response to ROS-mediated DNA damage.

Ethanol potentiates hepatitis B virus replication through oxidative stress-dependent and -independent transcriptional activation

Excessive alcohol intake accelerates disease progression in chronic hepatitis B virus (HBV) infection, but the mechanisms by which ethanol worsens the prognosis of chronic hepatitis B (CHB) are not fully understood. The aim of this study was to investigate whether HBV replication is augmented by alcohol or alcohol-induced cytochrome p450 2E1 (CYP2E1), and if so, whether oxidative stress is involved in the process. Ethanol treatment promoted HBV replication in HepAD38 cells that permit the conditional viral replication. Luciferase reporter assays confirmed that HBV core, preS1 and preS2/S promoter activities were augmented by ethanol. Ethanol did not induce oxidative stress in HepAD38 cells with minimal expression of CYP2E1. However, over-expression of CYP2E1 induced oxidative stress and amplified transcriptional activation of HBV by ethanol. Antioxidant glutathione treatment attenuated CYP2E1-mediated augmentation of HBV replication in ethanol-treated cells. In conclusion, ethanol enhances transcriptional activity of HBV promoters in human hepatoma cells in an oxidative stress-independent manner; and CYP2E1-mediated oxidative stress potentiates the ethanol-induced transactivation of HBV.

Knockdown of Cytochrome P450 2E1 Inhibits Oxidative Stress and Apoptosis in the cTnT R141W Dilated Cardiomyopathy Transgenic Mice

Cytochrome P450 2E1 (CYP2E1) is a cytochrome P450 enzyme that catalyzes the metabolism of toxic substrates. CYP2E1 is upregulated in heart disease, including the dilated cardiomyopathy (DCM) mouse model. Here, knockdown of CYP2E1 significantly ameliorated the dilated left ventricle, thin wall, and dysfunctional contraction in the cTnT(R141W) and adriamycin-induced DCM mouse models. Interstitial fibrosis, poorly organized myofibrils, and swollen mitochondria with loss of cristae were improved in the myocardium of α-myosin heavy chain (MHC)-cTnT(R141W)×CYP2E1-silence double-transgenic mice when compared with the cTnT(R141W) transgenic mice. Oxidative stress, the activation of caspase 3 and caspase 9, the release of cytochrome c, and the apoptosis in the myocardium were significantly decreased in double-transgenic mice compared with the cTnT(R141W) transgenic mice. In summary, the expression of CYP2E1 is upregulated in heart disease and might be induced by hypoxemia in cardiomyopathy. The overexpression of CYP2E1 can enhance the metabolism of endogenous ketones to meet the energy demand of the heart in certain disease states, but the overexpression of CYP2E1 can also increase oxidative stress and apoptosis in the DCM heart. Knockdown or downregulation of CYP2E1 might be a therapeutic strategy to control the development of DCM after mutations of cTnT(R141W) or other factors, because DCM is the third most common cause of heart failure and the most frequent cause of heart transplantation.

Increased oxidative stress and toxicity in ADH and CYP2E1 overexpressing human hepatoma VL-17A cells exposed to high glucose

High glucose mediated oxidative stress and cell death is a well documented phenomenon. Using VL-17A cells which are HepG2 cells over-expressing alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1) and control HepG2 cells, the association of ADH and CYP2E1 with high glucose mediated oxidative stress and toxicity in liver cells was investigated. Cell viability was measured and apoptosis or necrosis was determined through caspase-3 activity, Annexin V-propidium iodide staining and detecting decreases in mitochondrial membrane potential. Reactive oxygen species, lipid peroxidation and the formation of advanced glycated-end products were assessed. The levels of several antioxidants which included glutathione, glutathione peroxidase, catalase and superoxide dismutase were altered in high glucose treated VL-17A cells. Greater toxicity was observed in VL-17A cells exposed to high glucose when compared to HepG2 cells. Oxidative stress parameters were greatly increased in high glucose exposed VL-17A cells and apoptotic cell death was observed. Inhibition of CYP2E1 or caspase 3 or addition of the antioxidant trolox led to significant decreases in high glucose mediated oxidative stress and toxicity. Thus, the over-expression of ADH and CYP2E1 in liver cells is associated with increased high glucose mediated oxidative stress and toxicity.

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).

Expression of CYP2E1 increases oxidative stress and induces apoptosis of cardiomyocytes in transgenic mice

Cytochrome P450 2E1 (CYP2E1) is an effective generator of reactive oxygen species. Marked expression of CYP2E1 occurs in the heart and it is known to be regulated in the course of progression of myocardial ischemia and cardiomyopathy. We provide evidence that the expression of CYP2E1 is strongly up-regulated in cTnT(R141W) transgenic mice with dilated cardiomyopathy. Heart tissue-specific CYP2E1 transgenic mice were produced to study the effects of CYP2E1 overexpression on the heart. Increased mortality, chamber dilation and contractile dysfunction, as well as myocyte disarray, interstitial fibrosis, ultrastructural degeneration with myofibrillar disorganization and mitochondria damage, were observed in CYP2E1 transgenic mice and cTnT(R141W) transgenic mice. In addition, levels of H(2) O(2) and malondialdehyde were increased and levels of glutathione and total antioxidant capability were strongly reduced in CYP2E1 transgenic mice and cTnT(R141W) transgenic mice. Myocyte apoptosis was significantly increased by 19-fold in CYP2E1 transgenic mice and by 11-fold in cTnT(R141W) transgenic mice, respectively, compared to wild-type mice. Mitochondrial-dependent apoptotic signal transduction events, such as cytochrome c release from mitochondria into the cytosol and the expression of cleaved (active) caspases 3 and 9, were significantly increased in CYP2E1 transgenic mice and cTnT(R141W) transgenic mice. These results demonstrate that CYP2E1 over-expression produces apoptosis and that the up-regulation of CYP2E1 in cTnT(R141W) transgenic mice also correlates with apoptosis in this model.

Oxidative stress and regulation of anti‐oxidant enzymes in cytochrome P4502E1 transgenic mouse model of non‐alcoholic fatty liver

Reactive oxygen species produced by cytochrome P4502E1 (CYP2E1) are believed to play a role in pathophysiology of non-alcoholic fatty liver disease (NAFLD). However, little is known about the expression, protein content and activity of anti-oxidant enzymes and the role of inducible nitric oxide synthase (iNOS), a source of reactive nitrogen species, in NAFLD. In the present study, we evaluate gene expression, protein content and activity of anti-oxidant enzymes, and iNOS, in a CYP2E1 overexpressing model of non-alcoholic fatty liver (NAFL). Non-transgenic (nTg) and CYP2E1 transgenic (Tg) mice were fed rodent chow for 8 months. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver triglycerides, malondialdehyde and protein carbonyls were measured. Gene expression of NF-E2-related factor (Nrf2), superoxide dismutase-1, -2 (SOD-1,2), catalase (CAT), glutathione peroxidase (GPx), heme oxygenase-1 (HO-1) and iNOS were determined. Protein content, activity and nitrosylation of the enzymes were also measured. Tg mice had greater CYP2E1 activity and histological liver injury. MDA and protein carbonyls were increased, indicating insufficient anti-oxidant response. Gene expression of Nrf2, CAT, GPx, HO-1 and iNOS were significantly increased. Protein content and enzyme activities of most anti-oxidant enzymes were not correspondingly increased. iNOS activity and nitrosylation of CAT and SOD was greater in Tg mice liver. Hepatocyte-specific CYP2E1 overexpression results in increased oxidative stress and nitrosative stress. Several anti-oxidant enzymes are upregulated. Failure of corresponding increase in total protein and activity of anti-oxidant enzymes suggests modification/degradation, possibly by nitrosylation, due to increased iNOS activity in a CYP2E1 overexpressing NAFL mouse model.

Overexpression of liver-specific cytochrome P4502E1 impairs hepatic insulin signaling in a transgenic mouse model of nonalcoholic fatty liver disease

Cytochrome P4502E1 (CYP2E1) expression in the liver is increased in nonalcoholic fatty liver disease. The aim of this study was to determine whether CYP2E1 overexpression in the liver interferes with insulin signaling pathways in a mouse model of nonalcoholic fatty liver disease. Male mice containing the human CYP2E1 transgene under control of the mouse albumin enhancer-promoter (Tg) and control, nontransgenic mice were fed a diet containing 20% calories from fat for 8 months ad libitum. Liver injury was measured by histology and alanine aminotransferase. Malondialdehyde and protein carbonyls were measured as markers of oxidative stress. Total and phosphorylated proteins involved in the insulin signaling cascade were measured by western blotting. Tg mice had higher fasting insulin, and greater hepatic fat accumulation and histological liver injury. Malondialdehyde and protein carbonyls were increased in Tg mice liver indicating increased oxidative stress. Tyrosine phosphorylation of insulin receptor substrates 1 and 2, and serine phosphorylation of PKB/Akt, were significantly decreased in Tg mice. Serine phosphorylation of glycogen synthase kinase 3alpha was decreased in Tg mice and liver glycogen content was decreased correspondingly. Serine phosphorylation of the transcription factor Fox01a was decreased, and expression of the enzyme phosphoenolcarboxykinase was increased in Tg mice. Hepatocyte-specific overexpression of CYP2E1 increased hepatic oxidative stress in the liver, fasting insulin, and histological liver damage. CYP2E1 overexpression reduced hepatic insulin signaling and reduced glycogen storage and increased glucose synthesis. Overall, this study suggests an association of hepatic CYP2E1 with increased oxidative stress, increased systemic insulin resistance, decreased insulin signaling in the liver and increased hepatic fat accumulation.

CYP2E1 overexpression inhibits microsomal Ca 2+-ATPase activity in HepG2 cells

Cytochrome P450 2E1 (CYP2E1) is a microsomal enzyme that generates reactive oxygen species during its catalytic cycle. We previously found an important role for calcium in CYP2E1-potentiated injury in HepG2 cells. The possibility that CYP2E1 may oxidatively damage and inactivate the microsomal Ca 2+-ATPase in intact liver cells was evaluated, in order to explain why calcium is elevated during CYP2E1 toxicity. Microsomes were isolated by differential centrifugation from two liver cell line: E47 cells (HepG2 cells transfected with the pCI neo expression vector containing the human CYP2E1 cDNA, which overexpress active microsomal CYP2E1), and control C34 cells (HepG2 cells transfected with the pCI neo expression vector alone, which do not express significantly any cytochrome P450). The Ca 2+-dependent ATPase activity was determined by measuring the accumulation of inorganic phosphate from ATP hydrolysis. CYP2E1 overexpression produced a 45% decrease in Ca 2+-dependent ATPase activity (8.6 nmol Pi/min/mg protein in C34 microsomes versus 4.7 nmol Pi/min/mg protein in microsomes). Saturation curves with Ca 2+ or ATP showed that CYP2E1 overexpression produced a decrease in V max but did not affect the K m for either Ca 2+ or ATP. The decrease in activity was not associated with a decrease in SERCA protein levels. The ATP-dependent microsomal calcium uptake was evaluated by fluorimetry using fluo-3 as the fluorogenic probe. Calcium uptake rate in E47 microsomes was 28% lower than in C34 microsomes. Treatment of E47 cells with 2 mM N-acetylcysteine prevented the decrease in microsomal Ca 2+-ATPase found in E47 cells. These results suggest that CYP2E1 overexpression produces a decrease in microsomal Ca 2+-ATPase activity in HepG2 cells mediated by reactive oxygen species. This may contribute to elevated cytosolic calcium and to CYP2E1-potentiated injury.

Inhibition of the mitochondrial permeability transition by cyclosporin A prevents pyrazole plus lipopolysaccharide-induced liver injury in mice

Previous results showed that pyrazole potentiates lipopolysaccharide (LPS)-induced liver injury in mice. Mechanisms involved the overexpression of cytochrome P450 2E1 (CYP2E1), oxidative stress, and activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). The current study was carried out to test the hypothesis that the mitochondria permeability transition (MPT) plays a role in this pyrazole plus LPS toxicity. Mice were injected intraperitoneally with pyrazole for 2 days, followed by a challenge with LPS with or without treatment with cyclosporin A (CsA), an inhibitor of the MPT. Serum alanine aminotransferase and aspartate aminotransferase were increased by pyrazole plus LPS treatment, and CsA treatment could attenuate these increases. CsA also prevented pyrazole plus LPS-induced hepatocyte necrosis. Formation of 4-hydroxynonenal protein adducts and 3-nitrotyrosine protein adducts in liver tissue was increased by the pyrazole plus LPS treatment, and CsA treatment blunted these increases. Swelling, cytochrome c release from mitochondria to the cytosol, and lipid peroxidation were increased in mitochondria isolated from the pyrazole plus LPS-treated mice, and CsA treatment prevented these changes. CsA did not prevent the increased levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), pp38 MAPK, and p-JNK2. In conclusion, although CsA does not prevent elevations in upstream mediators of the pyrazole plus LPS toxicity (iNOS, TNF-α, CYP2E1, MAPK), it does protect mice from the pyrazole plus LPS-induced liver toxicity by preventing the MPT and release of cytochrome c and decreasing mitochondrial oxidative stress. These results indicate that mitochondria are the critical targets of pyrazole plus LPS in mediating liver injury.

Enhanced phytoremediation of volatile environmental pollutants with transgenic trees.

Small, volatile hydrocarbons, including trichloroethylene, vinyl chloride, carbon tetrachloride, benzene, and chloroform, are common environmental pollutants that pose serious health effects. We have developed transgenic poplar (Populus tremula x Populus alba) plants with greatly increased rates of metabolism and removal of these pollutants through the overexpression of cytochrome P450 2E1, a key enzyme in the metabolism of a variety of halogenated compounds. The transgenic poplar plants exhibited increased removal rates of these pollutants from hydroponic solution. When the plants were exposed to gaseous trichloroethylene, chloroform, and benzene, they also demonstrated superior removal of the pollutants from the air. In view of their large size and extensive root systems, these transgenic poplars may provide the means to effectively remediate sites contaminated with a variety of pollutants at much faster rates and at lower costs than can be achieved with current conventional techniques.