CYP2E1 Overexpression Research Articles

Generation and Characterization of CYP2E1-Overexpressing HepG2 Cells to Study the Role of CYP2E1 in Hepatic Hypoxia-Reoxygenation Injury.

The mechanisms of hepatic ischemia/reperfusion (I/R) injury, which occurs during liver transplantation or surgery, are poorly understood. The purpose of the current study was to generate and characterize a HepG2 cell line with a stable overexpression of CYP2E1 to investigate the role of the enzyme in hypoxia/reperfusion (H/R) injury in an ex vivo setting. GFP-tagged CYP2E1 and control clones were developed, and their gene expression and protein levels of GFP and CYP2E1 were determined using RT-PCR and ELISA/Western blot analysis, respectively. Additionally, the CYP2E1 catalytic activity was determined by UPLC-MS/MS analysis of 6-hydroxychlorzoxazone formed from the chlorzoxazone substrate. The CYP2E1 and control clones were subjected to hypoxia (10 h) and reoxygenation (0.5 h), and cell death and reactive oxygen species (ROS) generation were quantitated using LDH and flow cytometry, respectively. Compared with the control clone, the selected CYP2E1 clone showed a 720-fold increase in CYP2E1 expression and a prominent band in the western blot analysis, which was associated with a 150-fold increase in CYP2E1 catalytic activity. The CYP2E1 clone produced 2.3-fold more ROS and 1.9-fold more cell death in the H/R model. It is concluded that the constitutive CYP2E1 in the liver may play a detrimental role in hepatic I/R injury.

CYP2E1 plays a suppressive role in hepatocellular carcinoma by regulating Wnt/Dvl2/β-catenin signaling

ObjectiveKnowledge of the role of CYP2E1 in hepatocarcinogenesis is largely based on epidemiological and animal studies, with a primary focus on the role of CYP2E1 in metabolic activation of procarcinogens. Few studies have directly assessed the effects of CYP2E1 on HCC malignant phenotypes.MethodsThe expression of CYP2E1 in HCC tissues was determined by qRT-PCR, western blotting and immunohistochemistry. Overexpression of CYP2E1 in HCC cell was achieved by lentivirus transfection. The function of CYP2E1 were detected by CCK-8, wound healing, transwell assays, xenograft models and pulmonary metastasis model. TOP/FOPFlash reporter assay, western blotting, functional rescue experiments, Co-immunoprecipitation and reactive oxygen species detection were conducted to reveal the underlying mechanism of the tumor suppressive role of CYP2E1.ResultsCYP2E1 expression is down-regulated in HCC tissues, and this downregulation was associated with large tumor diameter, vascular invasion, poor differentiation, and shortened patient survival time. Ectopic expression of CYP2E1 inhibits the proliferation, invasion and migration and epithelial-to-mesenchymal transition of HCC cells in vitro, and inhibits tumor formation and lung metastasis in nude mice. Mechanistic investigations show that CYP2E1 overexpression significantly inhibited Wnt/β-catenin signaling activity and decreased Dvl2 expression in HCC cells. An increase in Dvl2 expression restored the malignant phenotype of HCC cells. Notably, CYP2E1 promoted the ubiquitin-mediated degradation of Dvl2 by strengthening the interaction between Dvl2 and the E3 ubiquitin ligase KLHL12 in CYP2E1-stable HCC cells. CYP2E1-induced ROS accumulation was a critical upstream event in the Wnt/β-Catenin pathway in CYP2E1-overexpressing HCC cells.ConclusionsThese results provide novel insight into the role of CYP2E1 in HCC and the tumor suppressor role of CYP2E1 can be attributed to its ability to manipulate Wnt/Dvl2/β-catenin pathway via inducing ROS accumulation, which provides a potential target for the prevention and treatment of HCC.Graphical

Unveiling the Role of Cytochrome P450 (2E1) in Human Brain Specifically in Parkinson's Disease - Literature Review.

In recent years, the significance of cytochrome P450 enzymes (CYPs) has expanded beyond their role in the liver. Factors such as genetics, environmental toxins, drug biotransformation and underlying diseases mediate the expression of these enzymes. Among the CYP enzymes, CYP2E1, a well-recognized monooxygenase enzyme involved in the metabolism of various endogenous and exogenous substances, plays a crucial role in the brain concerning the development of Parkinson's disease. The expression of CYP2E1 varies in different brain regions making certain regions more vulnerable than others. CYP2E1 expression is inducible which generates tissuedamaging radicals leading to oxidative stress, mitochondrial dysfunction and ultimately neurodegeneration. Less is understood about the role of CYP2E1 in the central nervous system, therefore the purpose of the study was to investigate the relationship between the expression and activity of CYP2E1 enzyme relevant to Parkinson's disease and to identify whether an increase in the expression of CYP2E1 is associated with neurodegeneration. The objectives of the study were achieved by implicating an unsystematic integrative literature review approach in which the literature was qualitatively analysed, critically evaluated and a new theory with an overall view of the mechanism was presented. The contribution of CYP2E1 in the development of Parkinson's disease was found to be significant as the negative effects of CYP2E1 overshadowed its protective detoxifying role. Overexpression of CYP2E1 seems detrimental to dopaminergic neurons, therefore, to overcome this, a synthetic biochemical is required, which paves the way for further research and development of valuable biomolecules.

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

Vitamin E Attenuates FasL-Induced Apoptotic Death of Dendritic Cells Through PI3K Signalling

Protective Effect of Curcumin on Acrylamide-Induced Hepatic and Renal Impairment in Rats: Involvement of CYP2E1

As a chemical extensively used in industrial areas and formed during heating of carbohydrate-rich foods and tobacco, acrylamide (ACR) has been demonstrated to exert a variety of systemic toxic effects including hepatotoxicity and nephrotoxicity. In the present study, we investigated the effect of curcumin, a natural polyphenolic compound in a popular spice known as turmeric, on the hepatic and renal impairment caused by ACR exposure to 40 mg/kg for 4 weeks in rats. The administration of curcumin at doses of 50 and 100 mg/kg to ACR-intoxicated rats significantly decreased the serum levels of alanine transaminase, aspartate transaminase, creatinine, and urea; improved the histological changes of liver and kidney caused by ACR; reduced the number of apoptotic cells; as well as relieved ACR-induced hepatic and renal oxidative stress. Moreover, curcumin inhibited the CYP2E1 overexpression induced by ACR in the liver and kidney tissues. Therefore, curcumin could be applied as a potential strategy for the intervention of ACR-induced systemic toxicity. The inhibition of CYP2E1 might be involved in the protection of curcumin against ACR-induced hepatotoxicity and nephrotoxicity.

CYP2E1 changes the biological function of gastric cancer cells via the PI3K/Akt/mTOR signaling pathway.

The present study investigated the role of cytochrome P450 family 2 subfamily E polypeptide 1 (CYP2E1) in the development and progression of gastric cancer (GC). The expression levels of CYP2E1 in MGC-803 GC cells and normal GES-1 cells were investigated via western blotting, and it was identified that the expression of CYP2E1 was different between GES-1 and MGC-803 cells. CYP2E1 was overexpressed in MGC-803 cells using a lentiviral vector GV358. Cell Counting Kit-8, flow cytometry, cell migration and Matrigel invasion assays suggested that overexpression of CYP2E1 promoted the proliferation and invasion, and inhibited the apoptosis of GC cells. The relationship between CYP2E1 expression and key signaling molecules in the PI3K/Akt/mTOR signaling pathway was assessed. Reverse transcription-quantitative PCR analysis showed that mTOR mRNA expression was significantly increased after overexpression of CYP2E1 (P<0.05). Western blotting results showed that overexpression of CYP2E1 upregulated the expression of phosphorylated (p)-Akt, p-mTOR and p-p70 ribosomal protein S6 kinase (P70S6K; Ser371) proteins (P<0.05). To further investigate the relationship between CYP2E1 and the PI3K/Akt/mTOR signaling pathway in GC cells, MGC-803 cells were treated with the PI3K inhibitor LY294002, and changes in the expression levels of PI3K, AKT, mTOR, P70S6K and CYP2E1 were observed. The present results showed that LY294002 downregulated the expression of PI3K, CYP2E1, AKT, mTOR and P70S6K (P<0.05). Therefore, changes in the biological function of GC cells induced by CYP2E1 overexpression may be via the PI3K/Akt/mTOR signaling pathway.

Investigating the CYP2E1 Potential Role in the Mechanisms Behind INH/LPS-Induced Hepatotoxicity.

Tuberculosis (TB) is one of the oldest infectious diseases that affected humankind and remains one of the world’s deadliest communicable diseases that could be considered as global emergency, but the discovery and development of isoniazid (INH) in the 1950s paved the way to an effective single and/or combined first-line anti-TB therapy. However, administration of INH induces severe hepatic toxicity in some patients. Previously, we establish a rat model of INH hepatotoxicity utilizing the inflammatory stress theory, in which bacterial lipopolysaccharide (LPS) potentially enhanced INH toxicity. These enhancing activities ranged between augmenting the inflammatory stress, oxidative stress, alteration of bile acid homeostasis, and CYP2E1 over-expression. Although pre-treatment with dexamethasone (DEX) helped overcome both inflammatory and oxidative stress which ended-up in alleviation of LPS augmenting effects, but still minor toxicities were being detected, alongside with CYP2E1 over expression. This finding positively indicated the corner-stone role played by CYP2E1 in the pathogenesis of INH/LPS-induced liver damage. Therefore, we examined whether INH/LPS co-treatment with CYP2E1 inhibitor diallyl sulfide (DAS) and DEX can protect against the INH/LPS-induced hepatotoxicity. Our results showed that pre-administration of both DAS and DEX caused significant reduction in serum TBA, TBil, and gamma-glutamyl transferase levels. Furthermore, the histopathological analysis showed that DAS and DEX could effectively reverse the liver lesions seen following INH/LPS treatment and protect against hepatic steatosis as indicated by absence of lipid accumulation. Pre-treatment with DAS alone could not completely block the CYP2E1 protein expression following INH/LPS treatment, as appeared in the immunoblotting and immunohistochemistry results. This is probably due to the fact that the combined enhancement activities of both INH and LPS on CYP2E1 protein expression levels might resist the blocking probabilities of DAS. In the meantime, addition of DEX to the DAS/INH/LPS combination caused a significant reduction in CYP2E1 protein expression as revealed by the immunoblotting and fading coloration in immunohistochemistry results. Thus, addition of DEX and DAS together caused strong protection against INH/LPS-induced hepatic damage. These findings reveal the potential therapeutic value of combining DAS and DEX with INH in TB management for reducing the potential risk and incidences of hepatotoxicity.

Danshen (Salvia miltiorrhiza) water extract inhibits paracetamol-induced toxicity in primary rat hepatocytes via reducing CYP2E1 activity and oxidative stress.

This study aimed to investigate the protective effects of Danshen (Salvia miltiorrhiza) water extract (DSE) and its major phenolic acid components against CYP2E1-mediated paracetamol (APAP)-induced hepatic toxicity. The protection and underlying mechanisms were detected in CYP2E1 overexpression primary rat hepatocytes by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alamar blue assay, CYP2E1 inhibition assay and glutathione assay. After APAP treatment, DSE (0.06-1 mg/ml) significantly increased cell viability in MTT assay. Two major components danshensu (8.2-130.5 μm) and salvianolic acid B (Sal B; 3.3-53.5 μm) mainly contributed to this protection, but rosmarinic acid, protocatechuic aldehyde and Sal A did not. Alamar blue assay showed that DSE, danshensu and Sal B maintained mitochondrial metabolic activity. DSE inhibited CYP2E1 (Ki = 1.46 mg/ml) in a mixed mode in rat liver microsomes in vitro; DSE decreased APAP-induced total glutathione depletion and preserved redox status (GSH/GSSG ratio) in hepatocytes. Danshensu and Sal B did not inhibit CYP2E1 or decrease total glutathione depletion, but preserved redox status. DSE protected hepatocytes against APAP-induced injury via maintenance of mitochondrial metabolic activity, CYP2E1 inhibition, reduction of total glutathione depletion and preservation of redox status. Danshensu and Sal B were mainly responsible for this protection.

ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes

Tetrandrine, an alkaloid with a remarkable pharmacological profile, induces oxidative stress and mitochondrial dysfunction in hepatocytes; however, mitochondria are not the direct target of tetrandrine, which prompts us to elucidate the role of oxidative stress in tetrandrine-induced mitochondrial dysfunction and the sources of oxidative stress. Rat primary hepatocytes were isolated by two-step collagenase perfusion. Mitochondrial function was evaluated by analyzing ATP content, mitochondrial membrane potential (MMP) and the mitochondrial permeability transition. The oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS) and glutathione (GSH). ROS scavengers largely attenuated the cytotoxicity induced by tetrandrine in rat hepatocytes, indicating the important role of ROS in the hepatotoxicity of tetrandrine. Of the multiple ROS inhibitors that were tested, only inhibitors of CYP450 (SKF-525A and others) reduced the ROS levels and ameliorated the depletion of GSH. Mitochondrial function assays showed that the mitochondrial permeability transition (MPT) induced by tetrandrine was inhibited by SKF-525A and vitamin C (VC), both of which also rescued the depletion of ATP levels and the mitochondrial membrane potential. Upon inhibiting specific CYP450 isoforms, we observed that the inhibitors of CYP2D, CYP2C, and CYP2E1 attenuated the ATP depletion that occurred following tetrandrine exposure, whereas the inhibitors of CYP2D and CYP2E1 reduced the ROS induced by tetrandrine. Overexpression of CYP2E1 enhanced the tetrandrine-induced cytotoxicity. We demonstrated that CYP450 plays an important role in the mitochondrial dysfunction induced by the administration of tetrandrine. ROS generated by CYP450, especially CYP2E1, may contribute to the mitochondrial dysfunction induced by tetrandrine.

Cytochrome P4502E1 inhibitor, chlormethiazole, decreases lipopolysaccharide‐induced inflammation in rat Kupffer cells with ethanol treatment

To investigate the role of Cytochrome P4502E1 in sensitizing Kupffer cells to lipopolysaccharide (LPS)-mediated inflammation after ethanol induction. Sprague-Dawley rats were fed a liquid ethanol diet, control diet or ethanol diet supplemented with CYP2E1 inhibitor, chlormethiazole (CMZ), for 4 weeks. Hepatic CYP2E1 protein, nuclear factor-kappa B (NF-κB) p65 protein and tumor necrosis factor (TNF)-α mRNA were measured. In vitro, isolated Kupffer cells from control rats were exposed to ethanol with different CMZ concentration; CYP2E1 expression and reactive oxygen species (ROS) generation were compared. The identified CMZ concentration was further utilized to evaluate the role of CYP2E1 on the sensitization of ethanol-induced Kupffer cell to LPS. The effect of LPS alone was tested in controlled Kupffer cells without ethanol. TNF-α, nuclear NF-κB p65 and cytoplasm IκB-α were monitored for all groups. Ethanol feeding increased hepatic CYP2E1 level, nuclear accumulation of NF-κB p65 and TNF-α expression in rats. These changes were inhibited by CMZ supplementation. In cultured Kupffer cells, increased CYP2E1 content and ROS production by in vitro ethanol induction were dose-dependently inhibited by CMZ. Compared with LPS alone, the ethanol induction group produced significantly more TNF-α, nuclear NF-κB p65 and less cytoplasm IκB-α under LPS stimuli. CMZ abolished the effects of ethanol on LPS-stimulated NF-κB translocation and TNF-α generation in Kupffer cells. In cultured Kupffer cell, using CMZ as inhibitor, ethanol-induced CYP2E1 overexpression was proved to contribute to the sensitization of Kupffer cells to LPS stimuli, with amplification of ROS production and activation of NF-κB, resulting in increased TNF-α production.

Fermented papaya preparation halts the progression of non-alcoholic steatohepatitis in rats

Non-alcoholic steatohepatitis (NASH) can progress to cirrhosis or hepatocellular carcinoma. Oxidative stress is implicated in NASH progression. Fermented papaya preparation (FPP) has oxygen radical scavenging activity and is effective in oxidative stress-related diseases. We investigated the effects of FPP on NASH progression using a rat NASH model. Plasma biochemical parameters and lipid peroxidation in the liver were elevated in NASH rats. Histologically, the liver of NASH rats showed liver fibrosis, mitochondrial dysfunction and over-expression of microsomal CYP2E1. Myeloperoxidase activity and nuclear factor-kappaB activation were also markedly increased in NASH. Oral administration of FPP ameliorated these changes in NASH rats. These results suggest that FPP halts NASH progression through its anti-oxidative and antiinflammatory properties.

Over-Expression of CYP2E1 mRNA and Protein: Implications of Xenobiotic Induced Damage in Patients with De Novo Acute Myeloid Leukemia with inv(16)(p13.1q22); CBFβ-MYH11

Environmental exposure to benzene occurs through cigarette smoke, unleaded gasoline and certain types of plastic. Benzene is converted to hematotoxic metabolites by the hepatic phase-I enzyme CYP2E1, and these metabolites are detoxified by the phase-II enzyme NQO1. The genes encoding these enzymes are highly polymorphic and studies of these polymorphisms have shown different pathogenic and prognostic features in various hematological malignancies. The potential role of different cytochrome p450 metabolizing enzymes in the pathogenesis of acute myeloid leukemia (AML) in an area of active interest. In this study, we demonstrate aberrant CYP2E1 mRNA over-expression by quantitative real-time polymerase chain reaction in 11 cases of de novo AML with inv(16); CBFβ-MYH11. CYP2E1 mRNA levels correlated with CBFβ-MYH11 transcript levels and with bone marrow blast counts in all cases. CYP2E1 over-expression correlated positively with NQO1 mRNA levels (R2 = 0.934, n = 7). By immunohistochemistry, CYP2E1 protein was more frequently expressed in AML with inv(16) compared with other types of AML (p < 0.001). We obtained serial bone marrow samples from two patients with AML with inv(16) before and after treatment. CYP2E1 mRNA expression levels decreased in parallel with CBFβ-MYH11 transcript levels and blast counts following chemotherapy. In contrast, CYP1A2 transcript levels did not change in either patient. This is the first study to demonstrate concurrent over-expression of CYP2E1 and NQO1 mRNA in AML with inv(16). These findings also suggest that a balance between CYP2E1 and NQO1 may be important in the pathogenesis of AML with inv(16).

Protective effect of fucoidan against acetaminophen-induced liver injury

Fucoidan, a sulfated polysaccharide extracted from various brown seaweeds, possesses a wide range of pharmacological properties. In this study, we investigated the protective effect of fucoidan on acetaminophen-induced acute liver injury in rats. Liver injury was induced by administration of acetaminophen (800 mg/kg, i.p.) and fucoidan was administered (100 mg kg, p.o.) 2 h before and after acetaminophen administration. After 24 h, co-treatment of fucoidan ameliorated liver damage and cell death induced by acetaminophen. Acetaminophen induced the overexpression of CYP2E1, one of the metabolizing enzymes of acetaminophen, but cotreatment with fucoidan suppressed its increased expression of CYP2E1. Fucoidan also reduced the hepatic apoptosis induced by acetaminophen exposure as shown in the protein expression of Bax, Bcl-2, and cleaved caspase-3. The anti-oxidative effect of fucoidan was observed from the increase of the production and expression of glutathione, superoxide dismutase, and glutathione peroxidase, both of which were decreased by acetaminophen. Also, fucoidan decreased the expression of inflammatory mediators, including tumor necrosis factoralpha, interleukin 1 beta, and inducible nitric oxide synthase. Taken together, the data demonstrate the hepato-protective effects of fucoidan against acetaminophen-induced liver injury via anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.

Ethanol-induced oxidative stress is associated with EGF receptor phosphorylation in MCF-10A cells overexpressing CYP2E1

Breast cancer is the most common cancer and the second leading cause of cancer-related mortality worldwide. The etiology of breast cancer is very diverse and 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 unknown. CYP2E1 is known to metabolize ethanol and produce reactive oxygen species (ROS), including superoxide in epithelial cells. Therefore, in the present studies, we investigated whether there is an increase in ROS following overexpression of CYP2E1 in MCF-10A cells. We found that 30 and 100mM EtOH increased ROS levels after 2h treatment in CYP2E1 overexpressing cells. Based on these results and our previous studies with ROS-producing chemicals, we also examined epidermal growth factor receptor (EGFR) activation following exposure to ethanol. We found that there was an increase in phosphorylation of pY1086 EGFR after 18h EtOH treatment in CYP2E1 overexpressing cells. These studies support a hypothesis that EtOH might increase human mammary cell activation, via an EGFR-dependent signaling mechanism associated with oxidative stress.

The impact of CYP2E1 on the development of alcoholic liver disease as studied in a transgenic mouse model

CYP2E1 metabolizes ethanol, generates reactive oxygen species, and is suggested to be important for development of alcoholic liver disease. The present study aims to evaluate the role of CYP2E1 in combination with ethanol for development of alcoholic liver disease using mice transgenic for the human CYP2E1 gene. Changes in hepatic gene expression were monitored in controls and mice transgenic for human CYP2E1, treated with ethanol or isocaloric dextrose intragastrically for 4 weeks, and related to pathology using Affymetrix microarrays and TaqMan RealTime PCR. Presence of the CYP2E1 transgene increased liver injury and increased expression of stress related genes. Microarray analyses revealed the expression of structural genes, particularly cytokeratin 8 and 18, to be highly related to pathology. This in vivo study confirms several findings regarding CYP2E1 and alcohol previously found only in vitro. These results provide in vivo evidence that CYP2E1 overexpression aggravates hepatic injury, and suggest that expression of cytokeratins 8 and 18 can be considered as biomakers for the progression of alcoholic liver disease.

Overexpression of CYP2E1 enhances sensitivity of HepG2 cells to Fas-mediated cytotoxicity

Hepatocellular carcinoma (HCC) has been reported to be resistant to Fas-mediated apoptosis. In present study, experiments were conducted to investigate the potential effects of CYP2E1 overexpression on susceptibility of HCC to Fas-mediated cytotoxicity. HCC cell line HepG2 was infected with Ad-CYP2E1 to enhance the expression of CYP2E1, followed by treatment with low toxic dose of recombinant human Fas ligand (FasL, 0.5ng/ml) in the presence of Actinomycin D (Act D, 125ng/ml). High level of Fas expression was found in HepG2 cells. Its protein level and distribution kept unchanged after different treatments. Compared with control, CYP2E1 expressed HepG2 cells were more sensitive to FasL plus Act D. The sensitivity was elevated in a multiplicity of infection (m.o.i)-dependent manner, which was dramatically suppressed by CYP2E1 inhibitor diallyl disulfide (DAS) (P

Overexpression of CYP2E1 induces HepG2 cells death by the AMP kinase activator 5′-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR)

5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a phylogenetically conserved serine/threonine protein kinase. AMPK may inhibit cell growth and proliferation and also regulates apoptosis. 5'-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) is a cell-permeable AMPK activator. Activation of AMPK with AICAR has been shown to induce apoptosis of the rat hepatoma cell line FTO2B cells and almost completely inhibited HepG2 cells growth. In this study, a HepG2 cell line, which was transfected with a vector containing human CYP2E1 cDNA (E47 cells), was treated with AICAR. Cell proliferation was blocked, and apoptosis and necrosis were elevated as assessed by cellular morphology, DNA content assay, and lactate dehydrogenase leakage. AICAR treatment significantly increases CYP2E1 activity (20-fold) and expression (5.5-fold) in E47 cells. Iodotubericidin, which inhibits the conversion of AICAR to its activated form AICAR monophosphate, the antioxidants trolox and MnTMPyP, and 4-methylpyrazole, an inhibitor of CYP2E1, all can protect the E47 cells from AICAR-induced necrosis. Production of intracellular reactive oxygen species was increased by AICAR treatment in E47 cells. The cytotoxicity mechanism of AICAR in E47 cells is suggested to include AMPK activation, p53 phosphorylation, p21 expression, overexpression of CYP2E1, and intracellular ROS accumulation.

W1865 N-Acetyl Cysteine Ameliorates Liver Injury in Patients with Erythropoietic Protoporhyria

mice. mRNA expression of Nrf2 was increased by 80% (p<0.02 vs. nTg) while mRNA expression of its cytosolic repressor protein, Keap1, was increased by 180% (p<0.006 vs. nTg). Expression of the antioxidant enzymes CAT, HO-1, GPx, GST and TRX were significantly increased in liver of the Tg mice. Expression of SOD 1 and SOD 2 was not increased in Tg mice. Conclusion: Overexpression of CYP2E1 in hepatocytes in the setting of a high fat diet increases ER stress which leads to increased expression of Nrf2 as well as multiple antioxidant systems regulated by Nrf2. Nevertheless, markers of oxidative stress are increased in CYP2E1 Tg mice indicating insufficient overall antioxidant response. Increased expression of Keap1 in Tg mice may limit the ability of Nrf2 to translocate to the nucleus thereby limiting the antioxidant response and contributing to increased oxidative liver injury.

Synergistic toxicity induced by prolonged glutathione depletion and inhibition of nuclear factor-kappaB signaling in liver cells

TNF-α, GSH depletion and CYP2E1 are factors that play an important role in alcoholic liver disease. Activation of NF-κB prevents hepatocyte damage caused by TNF-α. This work describes the effect of NF-κB inhibition on toxicities caused by GSH depletion or arachidonic acid (AA) treatment in liver cells, and evaluates the possible influence of CYP2E1 overexpression. Cells were exposed to the NF-κB inhibitor BAY11-7082, in the absence or presence of l-buthionine sulfoximine (BSO) to block GSH synthesis. BSO toxicity was higher in CYP2E1-expressing E47 HepG2 cells compared to control cells; the incubation with BAY11-7082 potentiated BSO toxicity in both cell lines comparably. Several other agents which suppress activation of NF-κB increased BSO toxicity in E47 cells. NF-κB inhibition, however, did not sensitize E47 cells to AA toxicity. Suppressing activity of NF-κB also potentiated BSO, but not AA toxicity, in isolated rat hepatocytes. BAY11-7082 plus BSO induced a greater p38 MAPK activation as compared to BAY11-7082 or BSO alone, and a p38 MAPK inhibitor protected against the synergistic toxicity. In summary, inhibition of NF-κB sensitizes liver cells to toxicity linked to GSH depletion, probably accelerating the processes of thiol homeostasis deregulation and induction of apoptosis through a mechanism mediated by p38 MAPK.

Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: Role of nonoxidative metabolism

Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100-800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs. VA-13 cells incubated with ethanol appears to be mediated by release of mitochondrial cytochrome c via activation of caspase-9 and caspase-3. These results strongly support our hypothesis that diminished hepatic ADH activity facilitates nonoxidative metabolism of ethanol and the products of ethanol nonoxidative metabolism cause apoptosis in HepG2 cells via intrinsic pathway.