CYP2E1-overexpressing Cells Research Articles

Nicotinamide riboside attenuates alcohol induced liver injuries via activation of SirT1/PGC-1α/mitochondrial biosynthesis pathway

BackgroundNicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD+) precursor which is present in foods such as milk and beer. It was reported that NR can prevent obesity, increase longevity, and promote liver regeneration. However, whether NR can prevent ethanol-induced liver injuries is not known. This study aimed to explore the effect of NR on ethanol induced liver injuries and the underlying mechanisms. MethodsWe fed C57BL/6 J mice with Lieber-DeCarli ethanol liquid diet with or without 400 mg/kg·bw NR for 16 days. Liver injuries and SirT1-PGC-1α-mitochondrial function were analyzed. In in vitro experiments, HepG2 cells (CYP2E1 over-expressing cells) were incubated with ethanol ± 0.5 mmol/L NR. Lipid accumulation and mitochondrial function were compared. SirT1 knockdown in HepG2 cells were further applied to confirm the role of SirT1 in the protection of NR on lipid accumulation. ResultsWe found that ethanol significantly decreased the expression and activity of hepatic SirT1 and induced abnormal expression of enzymes of lipid metabolism in mice. Both in vivo and in vitro experiments showed that NR activated SirT1 through increasing NAD+ levels, decreased oxidative stress, increased deacetylation of PGC-1α and mitochondrial function. In SirT1 knockdown HepG2 cells, NR lost its ability in enhancing mitochondrial function, and its protection against lipid accumulation induced by ethanol. ConclusionsNR can protect against ethanol induced liver injuries via replenishing NAD+, reducing oxidative stress, and activating SirT1-PGC-1α-mitochondrial biosynthesis. Our data indicate that SirT1 plays an important role in the protection of NR against lipid accumulation and mitochondrial dysfunctions induced by ethanol.

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.

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.

Chronic oxidative stress sensitizes hepatocytes to death from 4-hydroxynonenal by JNK/c-Jun overactivation

Sustained activation of the c-Jun NH(2)-terminal kinase (JNK) signaling pathway mediates the development and progression of experimental diet-induced nonalcoholic fatty liver disease (NAFLD). Delineating the mechanism of JNK overactivation in the setting of a fatty liver is therefore essential to understanding the pathophysiology of NAFLD. Both human and experimental NAFLD are associated with oxidative stress and resultant lipid peroxidation, which have been proposed to mediate the progression of this disease from simple steatosis to steatohepatitis. The ability of oxidants and the lipid peroxidation product 4-hydroxynonenal (HNE) to activate JNK signaling suggested that these two factors may act synergistically to trigger JNK overactivation. The effect of HNE on hepatocyte injury and JNK activation was therefore examined in cells under chronic oxidant stress from overexpression of the prooxidant enzyme cytochrome P450 2E1 (CYP2E1), which occurs in NAFLD. CYP2E1-generated oxidant stress sensitized a rat hepatocyte cell line to death from normally nontoxic concentrations of HNE. CYP2E1-overexpressing cells underwent a more profound depletion of glutathione (GSH) in response to HNE secondary to decreased gamma-glutamylcysteine synthetase activity. GSH depletion led to overactivation of JNK/c-Jun signaling at the level of mitogen-activated protein kinase kinase 4 that induced cell death. Oxidant stress and the lipid peroxidation product HNE cause synergistic overactivation of the JNK/c-Jun signaling pathway in hepatocytes, demonstrating that HNE may not be just a passive biomarker of hepatic oxidant stress but rather an active mediator of hepatocellular injury through effects on JNK signaling.

CYP2E1 overexpression alters hepatocyte death from menadione and fatty acids by activation of ERK1/2 signaling.

Chronic oxidative stress induced by overexpression of the cytochrome P450 isoform 2E1 (CYP2E1) has been implicated in hepatocyte injury and death. However, the mechanism by which CYP2E1 overexpression may promote cell death is unknown. Acute oxidative stress activates mitogen-activated protein kinases (MAPK), suggesting that chronic oxidant generation by CYP2E1 may regulate cellular responses through these signaling pathways. The effect of CYP2E1 overexpression on MAPK activation and their function in altering death responses of CYP2E1-overexpressing hepatocytes were investigated. Chronic CYP2E1 overexpression led to increased extracellular signal-regulated kinase 1/2 (ERK1/2) activation constitutively and in response to oxidant stress from the superoxide generator menadione. CYP2E1-overexpressing cells were resistant to menadione toxicity through an ERK1/2-dependent mechanism. Similar to menadione, the polyunsaturated fatty acid (PUFA) arachidonic acid (AA) induced an increased activation of ERK1/2 in hepatocytes that overexpressed CYP2E1. However, CYP2E1-overexpressing cells were sensitized to necrotic death from AA and the PUFA gamma-linolenic acid, but not from saturated or monounsaturated fatty acids. Death from PUFA resulted from oxidative stress and was blocked by inhibition of ERK1/2, but not p38 MAPK or activator protein-1 signaling. CYP2E1 expression induced ERK1/2 activation through increased epidermal growth factor receptor (EGFR)/c-Raf signaling. Inhibition of EGFR signaling reversed CYP2E1-induced resistance to menadione and sensitization to AA toxicity. In conclusion, chronic CYP2E1 overexpression leads to sustained ERK1/2 activation mediated by EGFR/c-Raf signaling. This adaptive response in hepatocytes exposed to chronic oxidative stress confers differential effects on cellular survival, protecting against menadione-induced apoptosis, but sensitizing to necrotic death from PUFA.

Increased cytotoxicity of carbon tetrachloride in a human hepatoma cell line overexpressing cytochrome P450 2E1.

Cytotoxic free radicals generated during the metabolism of carbon tetrachloride by cytochrome P450 2E1 (CYP2E1) are thought to cause hepatotoxicity. Here, the cytotoxic effects of carbon tetrachloride in a liver cell line expressing CYP2E1 (HLE/2E1) are compared with those in the mother cell line (HLE). The effects of carbon tetrachloride on the gene expression of HSP70, a potential marker of oxidative stress, were also examined. The viability of HLE/2E1 cells after exposure to carbon tetrachloride was significantly decreased compared with that of HLE cells. Northern blot analysis revealed that the HSP70 mRNA level was significantly increased after carbon tetrachloride treatment in both cell lines, while the magnitude of its increase was much greater in HLE/2E1 cells than in HLE cells. These results suggest that the oxidative stress induced by CYP2E1 plays an important role in the increase in cytotoxicity of carbon tetrachloride in CYP2E1-overexpressing cells.

CYP2E1-dependent toxicity and up-regulation of antioxidant genes.

Induction of cytochrome P450 2E1 (CYP2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. Glutathione (GSH) is critical in preserving the proper cellular redox balance and for its role as a cellular protectant. The goal of the present study was to characterize the GSH homeostasis in human hepatocarcinoma cells (HepG2-E47 cells) that overexpress CYP2E1. Toxicity in the E47 cells was markedly enhanced after GSH depletion by buthionine sulfoximine (BSO) treatment. The antioxidant trolox partially prevented the apoptosis and necrosis, while diallylsulfide, a CYP2E1 inhibitor, was fully protective. Damage to mitochondria appears to play a role in the CYP2E1- and BSO-dependent toxicity. CYP2E1-overexpressing cells showed increases in total GSH levels, GSH synthetic rate and in gamma-glutamylcysteine synthetase (GCS) mRNA. This GCS increase was due to transcriptional activation of the GCS gene and could be blocked by certain antioxidants. Activity, protein and mRNA levels for other antioxidants such as catalase, alpha- and microsomal glutathione transferases were also increased in the E47 cells. Up-regulation of these antioxidant genes may reflect an adaptive mechanism to remove CYP2E1-derived oxidants. These oxidants are diffusable and were able to elevate collagen type I protein in a co-culture system consisting of the E47 cells + rat hepatic stellate cells. Such interactions between CYP2E1, mitochondria and altered GSH homeostasis, and elevation of collagen levels, may play a role in alcohol-induced liver injury.

CYP2E1 Overexpression in HepG2 Cells Induces Glutathione Synthesis by Transcriptional Activation of γ-Glutamylcysteine Synthetase

Induction of CYP2E1 (cytochrome P450 2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. CYP2E1 is a loosely coupled enzyme; formation of reactive oxygen species occurs even in the absence of added substrate. GSH is critical for preserving the proper cellular redox balance and for its role as a cellular protectant. Since cells must maintain optimal GSH levels to cope with a variety of stresses, the goal of this study was to characterize the GSH homeostasis in human hepatocarcinoma cells (HepG2) that overexpress CYP2E1. This study was prompted by the finding that toxicity in CYP2E1-overexpressing cells was markedly enhanced after GSH depletion by buthionine sulfoximine treatment. CYP2E1-overexpressing cells showed a 40-50% increase in intracellular H(2)O(2); a 30% increase in total GSH levels; a 50% increase in the GSH synthesis rate; and a 2-fold increase in gamma-glutamylcysteine synthetase heavy subunit (GCS-HS) mRNA, the rate-limiting enzyme in GSH synthesis. This GCS-HS mRNA increase was due to increased synthesis since nuclear run-on assays showed increased transcription in CYP2E1-expressing cells, and the GCS-HS mRNA decay after actinomycin D treatment was similar in CYP2E1-expressing cells and empty vector-transfected cells. The facts that treatment with GSH ethyl ester almost completely prevented the increase in GCS-HS mRNA and decreased H(2)O(2) levels and that transient transfection with catalase (but not manganese-superoxide dismutase) produced a decrease in GCS-HS mRNA only in CYP2E1-expressing cells suggest a possible role for H(2)O(2) in the induction of GCS-HS gene transcription. In contrast to results with HepG2 cells expressing CYP2E1, no increase in GCS-HS mRNA was found with a HepG2 cell line engineered to express human cytochrome P450 3A4. In summary, CYP2E1 overexpression in HepG2 cells up-regulates the levels of reduced GSH by transcriptional activation of GCS-HS; this may reflect an adaptive mechanism to remove CYP2E1-derived oxidants such as H(2)O(2).