Stimulation and proliferation of primary rat hepatic stellate cells by cytochrome P450 2E1-derived reactive oxygen species.

The alcohol-inducible cytochrome P450 2E1 (CYP2E1) is expressed mainly in hepatocytes and generates reactive oxygen species (ROS). To better understand how hepatic stellate cells (HSC) become activated in the presence of oxidative stress and evaluate whether CYP2E1-derived ROS activate stellate cells, we coincubated primary stellate cells with HepG2 cells, which do (E47 cells) or do not (C34 cells) express CYP2E1. Morphologic changes and loss of lipid droplets were more apparent in the stellate cells cocultured with E47 cells. There was a more pronounced increase in alpha-smooth muscle actin (alpha-sma), intracellular and secreted collagen type I protein, and intra- and extracellular H(2)O(2) and lipid peroxidation products in stellate cells coincubated with E47 cells. Expression of collagen in stellate cells did not change when cocultured with HepG2 cells expressing a different P450, CYP3A4. Stellate cells cultured on Matrigel expressed increased alpha-sma and collagen when incubated with E47 cells. The increase in collagen production by coculture with E47 cells was prevented by antioxidants, by CYP2E1 inhibitors, and by transfected antisense CYP2E1. The addition of arachidonic acid plus ferric nitrilotriacetate (Fe-NTA), agents that potentiate oxidative stress, further induced collagen protein in the E47 coculture. Stellate cell proliferation was greater in the E47 coculture, and this was partially abrogated by catalase and vitamin E. These results show that hepatocytes containing CYP2E1 release diffusible mediators including ROS, which can activate HSC. Thus, besides perturbing the homeostasis of hepatocytes, CYP2E1-derived diffusible oxidants may also interact with stellate cells and contribute to hepatic fibrosis.     

Steatosis in chronic hepatitis C: association with increased messenger RNA expression of collagen I,tumor necrosis factor-alpha and cytochrome P450 2E1

BACKGROUND: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. METHODS: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. RESULTS: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. CONCLUSION: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV.     

Inhibitory effect of a soluble transforming growth factor beta type II receptor on the activation of rat hepatic stellate cells in primary culture

BACKGROUND/AIMS: Oxidative stress, including the generation of reactive oxygen species (ROS) that acts as a signaling mediator for transforming growth factor (TGF)-beta, plays a key role in hepatic fibrosis. Hepatic stellate cells (HSCs) produce and respond to TGF-beta in an autocrine manner with increased collagen expression. It has previously been reported that the adenovirus-mediated overexpression of a soluble receptor against the extracellular domain of the TGF-beta type II receptor prevents hepatofibrogenesis in vivo, although its inhibitory role and mechanism in HSC activation remains to be elucidated. METHODS: In this study, we report on an examination of the actual role of TGF-beta inhibition on oxidative stress and the activation of cultured rat HSCs, using the adenovirus-mediated soluble TGF-beta type II receptor. RESULTS: This soluble receptor secreted from the adenovirus-infected cells binds to TGF-beta. Infection of HSCs with this adenovirus attenuated intracellular levels of TGF-beta1 mRNA and protein, NADH oxidative activity, ROS generation and lipid peroxidation, and prevented HSC activation. CONCLUSIONS: These findings suggest that this adenovirus-mediated soluble TGF-beta receptor may lead to an interruption of the TGF-beta autocrine loop in activated HSC, in part, by inhibiting oxidative stress.     

Adenovirus-mediated expression of Cu/Zn- or Mn-superoxide dismutase protects against CYP2E1-dependent toxicity

CYP2E1 induction by ethanol is one mechanism by which ethanol creates oxidative stress in the liver. The superoxide dismutases (SODs) are an important antioxidant enzyme defense system against reactive oxygen species (ROS). To investigate the protective role of SOD against CYP2E1-dependent toxicity, a transfected HepG2 cell line overexpressing CYP2E1 (E47 cells) was infected with adenoviral vectors containing Cu/Zn-SOD complementary DNA (cDNA) (Ad.SOD1) and Mn-SOD cDNA (Ad.SOD2). Forty-eight hours after infection, intracellular levels and activity of Cu/Zn-SOD and Mn-SOD were increased about 2- and 3-fold, respectively. Localization of the overexpressed Cu/Zn-SOD in the cytosol and Mn-SOD in the mitochondria was confirmed by assaying the levels and activity of SOD in the corresponding isolated fractions. Arachidonic acid (AA) plus iron-induced cell death was partially prevented in both Ad.SOD1- and Ad.SOD2-infected E47 cells. Overexpression of Cu/Zn-SOD and Mn-SOD also partially protected E47 cells from the increase in reactive oxygen production and lipid peroxidation and the loss of mitochondrial membrane potential induced by AA and iron. Infection with Cu/Zn-SOD and Mn-SOD also protected the E47 cells against AA toxicity or buthionine sulfoximine (BSO)-dependent toxicity. CYP2E1 levels and catalytic activity were not altered by overexpression of Cu/Zn-SOD or Mn-SOD. Cu/Zn-SOD in the cytosol and Mn-SOD in mitochondria each are capable of protecting HepG2 cells expressing CYP2E1 against cytotoxicity induced by pro-oxidants. In conclusion, these enzymes may be useful in the prevention or improvement of liver injury produced by agents known to be metabolized by CYP2E1 to reactive intermediates and to cause oxidative stress.     

Cytochrome P4502E1 is present in rat pancreas and is induced by chronic ethanol administration

Background—The mechanisms responsible for theinitiation of alcoholic pancreatitis remain elusive. However, there isan increasing body of evidence that reactive oxygen species play a rolein both acute and chronic pancreatitis. In the liver, cytochromeP4502E1 (CYP2E1, the inducible ethanol metabolising enzyme) is one of the proposed pathways by which ethanol induces oxidative stress.
Aims—To determine whether CYP2E1 is present inthe pancreas and, if so, whether it is inducible by chronic ethanol feeding.
Methods—Eighteen male Sprague-Dawley rats werepair fed liquid diets with or without ethanol as 36% of energy forfour weeks. CYP2E1 levels were determined by western blotting ofmicrosomal protein from both pancreas and liver. Messenger RNA (mRNA)levels for CYP2E1 were quantified using dot blots of total pancreatic RNA.
Results—CYP2E1 was found in the pancreas.Furthermore, the amount of CYP2E1 was greater in the pancreas of ratsfed ethanol compared with controls (mean increase over controls5.1-fold, 95% confidence intervals 2.4 to 7.7, p<0.02). In the liver,induction by ethanol of CYP2E1 was similar (mean increase over controls 7.9-fold, 95% confidence intervals 5.2 to 10.6, p<0.005). Pancreatic mRNA levels for CYP2E1 were similar in ethanol fed and control rats.
Conclusions—CYP2E1 is present in the rat pancreasand is inducible by chronic ethanol administration. Induction ofpancreatic CYP2E1 is not regulated at the mRNA level. The metabolism ofethanol via CYP2E1 may contribute to oxidative stress in the pancreas during chronic ethanol consumption.

Keywords:cytochrome P4502E1; rat pancreas; chronic ethanoladministration

     

Modulation of Oxidative Stress by Alcohol

It is well established that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in causing acute and chronic alcohol toxicity. Biochemical signs of oxidative damage can be detected in experimental animals exposed to ethanol as well as in alcoholic patients. Ethanol-induced oxidative stress is the consequence of the combined effect of an increased production of ROS by the mitochondria and the alcohol-inducible cytochrome P-4502E1 (CYP2E1) and the impairment of antioxidant defenses. Furthermore, by promoting the activation of Kuppfer cells, ethanol causes the release of cytokines, ROS, and RNS. The mechanisms by which oxidative damage contribute to alcohol toxicity include direct hepatocellular damage, induction of apoptosis, and the stimulation of collagen deposition by hepatic stellate cells. In addition, lipid peroxidation products and, particularly acetaldehyde-malondialdehyde adducts, along with immune reactions triggered by oxidative stress, might also contribute to perpetuate hepatic inflammation. The implication of oxidative stress in alcohol liver damage gives a rationale to the clinical application of therapies aimed to prevent or reduce ethanol-induced oxidative damage by antioxidant compounds.     

Ethanol-induced oxidative stress via the CYP2E1 pathway disrupts adiponectin secretion from adipocytes

Background: Adipose tissue is an important target for ethanol action. One important effect of ethanol is to reduce the secretion of adiponectin from adipocytes; this decrease is associated with lowered circulating adiponectin in rodent models of chronic ethanol feeding. Adiponectin is an insulin‐sensitizing, anti‐inflammatory adipokine; decreased adiponectin activity may contribute to tissue injury in response to chronic ethanol. Here, we investigated the role of cytochrome P450 2E1 (CYP2E1) and oxidative stress in the mechanism for impaired adiponectin secretion from adipocytes in response to ethanol. Methods: Male Wistar rats were fed a liquid diet containing ethanol as 36% of calories or pair‐fed a control diet for 4 weeks. 3T3‐L1 adipocyte cultures, expressing CYP2E1 or not, were exposed to ethanol or 4‐hydroxynonenal (4‐HNE). Results: Chronic ethanol feeding to rats suppressed the secretion of adiponectin from isolated epididymal adipocytes. Ethanol feeding induced the expression of CYP2E1 in adipocytes and increased markers of oxidative stress, including 4‐HNE and protein carbonyls. Because adiponectin is posttranslationally processed in the endoplasmic reticulum and Golgi, we investigated the impact of ethanol on the redox status of high‐density microsomes. Chronic ethanol decreased the ratio of reduced glutathione to oxidized glutathione (4.6:1, pair‐fed; 2.9:1, ethanol‐fed) in high‐density microsomes isolated from rat epididymal adipose tissue. We next utilized the 3T3‐L1 adipocyte‐like cell model to interrogate the mechanisms for impaired adiponectin secretion. Culture of 3T3‐L1 adipocytes overexpressing exogenous CYP2E1, but not those overexpressing antisense CYP2E1, with ethanol increased oxidative stress and impaired adiponectin secretion from intracellular pools. Consistent with a role of oxidative stress in impaired adiponectin secretion, challenge of 3T3‐L1 adipocytes with 4‐HNE also reduced adiponectin mRNA expression and secretion, without affecting intracellular adiponectin concentration. Conclusions: These data demonstrate that CYP2E1‐dependent reactive oxygen species production in response to ethanol disrupts adiponectin secretion from adipocytes.     

Removal of glutathione produces apoptosis and necrosis in HepG2 cells overexpressing CYP2E1

BACKGROUND: Previous studies have shown that addition of ethanol, iron, or arachidonic acid to HepG2 cells expressing CYP2E1 produced a loss in cell viability and caused apoptosis. These effects were enhanced when cellular reduced glutathione (GSH) levels were lowered by treatment with buthionine sulfoximine (BSO). Overexpression of CYP2E1 in HepG2 cells could produce toxicity even in the absence of added toxin after BSO treatment. Studies were carried out to characterize this CYP2E1-and BSO-dependent toxicity. METHODS: HepG2 cells expressing CYP2E1 were treated with BSO for 1 to 4 days, and various parameters associated with apoptosis and cell viability were assayed. RESULTS: Treatment of cells expressing CYP2E1 (E47 cells) with BSO resulted in apoptosis as well as necrosis. The apoptosis and necrosis were independent of each other. No toxicity was found with control HepG2 cells or HepG2 cells expressing CYP3A4 instead of CYP2E1 under these conditions. The antioxidant trolox partially prevented the apoptosis and necrosis, whereas diallylsulfide, a CYP2E1 inhibitor, was fully protective. The activity of caspase 3, but not caspases 1, 8, or 9, was increased in the BSO-treated E47 cells, and an inhibitor of caspase 3 prevented apoptosis. Damage to mitochondria appears to play a role in the CYP2E1- and BSO-dependent toxicity, because mitochondrial membrane potential was decreased and cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition, prevented the apoptosis and the necrosis. The fall in membrane potential was prevented by trolox and diallylsulfide, suggesting damage to the mitochondria by CYP2E1-derived reactive oxygen species. CONCLUSIONS: These results indicate the critical role of GSH in protecting against CYP2E1-mediated oxidative stress and that mitochondria may be a target for CYP2E1-derived reactive oxygen species, and suggest that interactions between CYP2E1, mitochondria, and altered GSH homeostasis may play a role in alcohol-induced liver injury.     

Relationship between oxidative stress levels and activation state on a hepatic stellate cell line.

BACKGROUND/AIMS: Oxidative stress plays an important role in liver fibrosis. Under pathological conditions, hepatic stellate cells (HSC) undergo an activation process, developing a myofibroblast-like phenotype from the lipocyte phenotype. In this study, we determined the levels of oxidative stress and proliferation in different activation states of an experimental model of mouse HSC, the GRX cell line. These cells can be induced in vitro to display a more activated state or a quiescent phenotype. METHODS/RESULTS: We observed increased oxidative damage and higher levels of reactive oxygen species, measured by thiobarbituric acid reactive species and 2',7'-dichlorofluorescein diacetate, respectively, and diminished catalase activity in activated cells. Activation decreased proliferation and increased the number of cells in G2/M. Antioxidants N-acetylcysteine and Trolox varied in their capacity to correct the oxidative stress and proliferation status. CONCLUSIONS: The differences in physiological functions of stellate cell phenotypes suggest a relationship between oxidative stress levels and activation state.     

细胞色素酶P4502E1介导的氧化应激对人肝星状细胞的活化作用

目的 研究细胞色素酶P4502E1(CYP2E1)介导的氧化应激对人肝星状细胞的活化作用. 方法 以CYP2E1为目的 基因,将CYP2E1表达载体PCI-CYP2E1及空载体PCI-neo转染到人肝癌细胞株HepG2细胞,分别为HepG2/CYP2E1,HepG2/PCI.检测HepG2/CYP2E1,HepG2/PCI及正常HepG23组细胞上清液中丙二醛(MDA)含量.将上述3种细胞分别与人肝星状细胞LX2共培养48 h,分别命名为CYP2E1/LX2组,PCI/LX2组,HepG2/LX2组,提取RNA、上清液,用羟脯氨酸(Hyp)试剂盒检测3组LX2上清液中Hyp含量,用逆转录多聚酶链反应检测LX2细胞内Ⅰ型胶原、基质金属蛋白酶2(MMP2)的mRNA水平,用酶联免疫吸附试验检测3组上清液中LX2分泌的Ⅰ型前胶原羧基肽(PICP)蛋白水平,用明胶酶谱法检测3组上清液中LX2分泌的MMP2酶活性.组间比较用单因素方差分析进行统计学分析. 结果 (1)HepG2/CYP2E1细胞、阴性对照组HepG2细胞及HepG2/PCI细胞的MDA值分别为(6.51±0.25)nmol/ml、(3.07±0.29)nmol/ml和(2.57±0.29)nmol/ml,F值为22.66,P值均<0.01.(2)HepG2/CYP2E1、HepG2、HepG2/PCI 3组细胞培养液中Hyp含量分别为(35.24±3.52)nmol/ml、(24.50±1.37)nmol/ml和(17.77±2.58)nmol/ml,F值为58.89,P值均<0.01; 3组LX2细胞Ⅰ型胶原mRNA水平表达,差异无统计学意义.CYP2E1/LX2组、阴性对照组HepG2组及PCI/LX2组LX2分泌的PICP蛋白吸光度值分别为540.01±11.38、262.57±15.61和231.59±12.76,F值为124.97,P值均<0.01; 3组LX2细胞内MMP2 mRNA水平表达、MMP2的酶活性,差异无统计学意义. 结论 CYP2E1可引起氧化应激,CYP2E可增加LX2细胞外Hyp的合成和分泌.CYP2E1活化了肝星状细胞,可促进其细胞外PICP合成与分泌.     

Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: mechanisms and pathophysiological role

Due to the worldwide surge in obesity and type 2 diabetes, the increased incidence of nonalcoholic fatty liver disease (NAFLD) is a major concern for the public health. Indeed, NAFLD encompasses a large spectrum of conditions ranging from fatty liver to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis in some patients. A better understanding of the mechanisms involved in fatty liver and its progression into NASH is important in order to develop efficient drugs able to alleviate these liver diseases. Although numerous investigations pointed to reactive oxygen species (ROS) as key players in the progression of fatty liver to NASH, their exact source is still uncertain. Besides the mitochondrial respiratory chain, cytochrome P450 2E1 (CYP2E1) has recently emerged as another potentially important cause of ROS overproduction. Indeed, higher hepatic CYP2E1 expression and activity have been frequently observed in the context of obesity and NAFLD. It is currently unknown why CYP2E1 is enhanced in these dysmetabolic diseases, although increased hepatic levels of fatty acids and insulin resistance might play a role. Nonetheless, higher hepatic CYP2E1 could play a significant role in the pathophysiology of NASH by inducing lipid peroxidation and oxidative damage of key cellular components. Moreover, CYP2E1-mediated overproduction of ROS could promote hepatic insulin resistance, which can further aggravate fatty liver. Since a significant amount of CYP2E1 can be located within liver mitochondria, higher levels of CYP2E1 in NAFLD could also have detrimental effects on mitochondrial function. Finally, increased CYP2E1 activity during NAFLD could enhance the susceptibility of some patients to the hepatotoxicity of different xenobiotics through the CYP2E1-mediated generation of harmful reactive metabolites.     

Salutary effect of kallistatin in salt-induced renal injury, inflammation, and fibrosis via antioxidative stress

An inverse relationship exists between kallistatin levels and salt-induced oxidative stress in Dahl-salt sensitive rats. We further investigated the role of kallistatin in inhibiting inflammation and fibrosis through antioxidative stress in Dahl-salt sensitive rats and cultured renal cells. High-salt intake in Dahl-salt sensitive rats induced elevation of thiobarbituric acid reactive substances (an indicator of lipid peroxidation), malondialdehyde levels, reduced nicotinamide-adenine dinucleotide phosphate oxidase activity, and superoxide formation, whereas kallistatin gene delivery significantly reduced these oxidative stress parameters. Kallistatin treatment improved renal function and reduced kidney damage as evidenced by diminished proteinuria and serum urea nitrogen levels, glomerular sclerosis, tubular damage, and protein cast formation. Kallistatin significantly decreased interstitial monocyte-macrophage infiltration and the expression of tumor necrosis factor-alpha, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Kallistain also reduced collagen fraction volume and the deposition and expression of collagen types I and III. Renal protection by kallistatin was associated with increased NO levels and endothelial NO synthase expression and decreased p38 mitogen-activated protein kinase, extracellular signal-regulated kinase phosphorylation, and transforming growth factor-beta1 expression. Moreover, kallistatin attenuated tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression via inhibition of reactive oxygen species formation and p38 mitogen-activated protein kinase and nuclear factor-kappaB activation in cultured proximal tubular cells. Kallistatin inhibited fibronectin and collagen expression by suppressing angiotensin II-induced reactive oxygen species generation and transforming growth factor-beta1 expression in cultured mesangial cells. These combined findings reveal that kallistatin is a novel antioxidant, which prevents salt-induced kidney injury, inflammation, and fibrosis by inhibiting reactive oxygen species-induced proinflammatory cytokine and transforming growth factor-beta1 expression.     

Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells

Stem cells of highly regenerative organs including blood are susceptible to endogenous DNA damage caused by both intrinsic and extrinsic stress. Response mechanisms to such stress equipped in hematopoietic stem cells (HSCs) are crucial in sustaining hematopoietic homeostasis but remain largely unknown. In this study, we demonstrate that serial transplantation of human HSCs into immunodeficient mice triggers replication stress that induces incremental elevation of intracellular reactive oxygen species (ROS) levels and the accumulation of persistent DNA damage within the human HSCs. This accumulation of DNA damage is also detected in HSCs of clinical HSC transplant patients and elderly individuals. A forced increase of intracellular levels of ROS by treatment with a glutathione synthetase inhibitor aggravates the extent of DNA damage, resulting in the functional impairment of HSCs in vivo. The oxidative DNA damage activates the expression of cell-cycle inhibitors in a HSC specific manner, leading to the premature senescence among HSCs, and ultimately to the loss of stem cell function. Importantly, treatment with an antioxidant can antagonize the oxidative DNA damage and eventual HSC dysfunction. The study reveals that ROS play a causative role for DNA damage and the regulation of ROS have a major influence on human HSC aging.