Kavitha Swaminathan

Apoptosis in HepG2 cells exposed to high glucose

Hyperglycemia which characterizes diabetes, leads to several abnormalities in the cellular pathways. We examined the toxicity of glucose in human hepatoma HepG2 cells. HepG2 cells when incubated with 50mM glucose for 72h showed altered morphology i.e. presence of detached and shrunken rounded cells. Glucose treated HepG2 cells also exhibited a significant decrease in viability. Caspase-3 activity and Annexin V staining were significantly increased in glucose treated HepG2 cells, suggesting an apoptotic mode of cell death. Glucose induced apoptosis in HepG2 cells was a consequence of increased oxidative stress as evidenced by the increased reactive oxygen species (ROS) level, lipid peroxidation, protein carbonyl and 3-nitrotyrosine adduct formation. The intracellular antioxidant glutathione was found to be increased in HepG2 cells treated with glucose, possibly to aid the cells to overcome the persistent oxidative stress elicited by glucose in HepG2 cells. N-Acetyl cysteine, a precursor of glutathione and an antioxidant was effective in reversing the morphological changes, increasing the viability, decreasing the ROS level and 4-hydroxynonenal and 3-nitrotyrosine adduct formation, thus validating the role of oxidative stress as a major mechanism for glucose induced apoptosis in HepG2 cells. These results suggest that glucose induces apoptosis in liver cells through increased oxidative stress.

Hyperglycemia-induced mitochondrial alterations in liver.

The increasing prevalence of diabetes continues to be a major health issue world wide. Liver injury is highly relevant in diabetic subjects and Type 2 diabetes mellitus is an acknowledged risk factor for non-alcoholic fatty liver disease (NAFLD). Chronic hyperglycemia is an important feature of diabetes and hyperglycemia induced mitochondrial dysfunction in liver holds importance in context to NAFLD. Some of the hyperglycemia induced changes in mitochondria include decreased oxidative phosphorylation, increased oxidative stress and ultra structural abnormalities. The following review identifies the mechanisms through which hyperglycemia causes mitochondrial dysfunction in liver.

Inhibition of CYP2E1 leads to decreased malondialdehyde–acetaldehyde adduct formation in VL-17A cells under chronic alcohol exposure

AIM Ethanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde-acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated. MAIN METHODS Chronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor - diallyl sulfide or ADH inhibitor - pyrazole or ADH and CYP2E1 inhibitor - 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis. KEY FINDINGS Inhibition of CYP2E1 with 10 μM diallyl sulfide or ADH with 2mM pyrazole or ADH and CYP2E1 with 5mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100 mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury. SIGNIFICANCE CYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.

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.

In vitro evidence for chronic alcohol and high glucose mediated increased oxidative stress and hepatotoxicity.

BACKGROUND Hyperglycemia or alcoholism can lead to impaired liver functions. Cytochrome P450 2E1 (CYP2E1) is elevated in hyperglycemia or alcoholism and plays a critical role in generating oxidative stress in the cell. METHODS In the present study, we have used VL-17A cells that overexpress the alcohol metabolizing enzymes [alcohol dehydrogenase (ADH) and CYP2E1] to investigate the toxicity due to ethanol (EtOH) plus high glucose. Toxicity was assessed through viability assay and amount of acetaldehyde adduct formation. Oxidative stress parameters included measuring reactive oxygen species (ROS) levels and malondialdehyde adduct formation. Apoptosis was determined through caspase-3 activity, Annexin V- Propidium iodide staining, and changes in mitochondrial membrane potential. The effects of antioxidants and specific inhibitors of ADH and CYP2E1 on cell viability and ROS levels were also studied. RESULTS When present together, EtOH plus high glucose-treated VL-17A cells exhibited greater oxidative stress and toxicity than other groups. Apoptosis was observed in liver cells treated with the toxins, and the EtOH plus high glucose-treated VL-17A cells exhibited apoptosis to the largest extent. A distinct and graded increase in CYP2E1 level occurred in the different groups of VL-17A cells. Further, antioxidants or inhibitors of ADH and CYP2E1 were effective in decreasing the observed oxidative stress and toxicity. CONCLUSIONS The combined oxidative insult due to alcohol plus high glucose leads to greater liver injury, which may prove to be a timely warning for the injurious effects of alcohol consumption in diabetics.

Inhibition of CYP2E1 leads to decreased advanced glycated end product formation in high glucose treated ADH and CYP2E1 over-expressing VL-17A cells

BACKGROUND In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure. METHODS In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied. RESULTS Inhibition of CYP2E1 with 10μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation. CONCLUSIONS Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1. GENERAL SIGNIFICANCE The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.

Elevated glutathione level does not protect against chronic alcohol mediated apoptosis in recombinant human hepatoma cell line VL-17A over-expressing alcohol metabolizing enzymes – Alcohol dehydrogenase and Cytochrome P450 2E1

Chronic consumption of alcohol leads to liver injury. Ethanol-inducible Cytochrome P450 2E1 (CYP2E1) plays a critical role in alcohol mediated oxidative stress due to its ability to metabolize ethanol. In the present study, using the recombinant human hepatoma cell line VL-17A that over-expresses the alcohol metabolizing enzymes-alcohol dehydrogenase (ADH) and CYP2E1; and control HepG2 cells, the mechanism and mode of cell death due to chronic ethanol exposure were studied. Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells. Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells. Chronic ethanol mediated cell death in VL-17A cells was predominantly apoptotic, with increased oxidative stress as the underlying mechanism. Chronic ethanol exposure of VL-17A cells resulted in 1.1- to 2.5-fold increased levels of ADH and CYP2E1. Interestingly, the level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation to the persistent and overwhelming oxidative stress. In conclusion, the increased GSH level may not be sufficient enough to protect VL-17A cells from chronic alcohol mediated oxidative stress and resultant apoptosis.

Bio-informatics based analysis of genes implicated in alcohol mediated liver injury.

Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.

The effects of changes in glutathione levels through exogenous agents on intracellular cysteine content and protein adduct formation in chronic alcohol-treated VL17A cells

Abstract Alcohol-mediated liver injury is associated with changes in the level of the major cellular antioxidant glutathione (GSH). It is interesting to investigate if the changes in intracellular GSH level through exogenous agents affect the intracellular cysteine content and the protein adduct formation indicative of oxidative insult in chronic alcohol treated liver cells. In VL-17A cells treated with 2 mM N-acetyl cysteine (NAC) or 0.1 mM ursodeoxycholic acid (UDCA) plus 100 mM ethanol, an increase in cysteine concentration which was accompanied by decreases in hydroxynonenal (HNE) and glutathionylated protein adducts were observed. Pretreatment of 100 mM ethanol treated VL-17A cells with 0.4 mM buthionine sulfoximine (BSO) or 1 mM diethyl maleate (DEM) had opposite effects. Thus, altered GSH level through exogenous agents may either potentiate or ameliorate chronic alcohol-mediated protein adduct formation and change the cysteine level in chronic alcohol treated VL-17A cells. The gene expression of non-treated and ethanol-treated hepatocytes in 2 microarray datasets was also compared to locate differentially expressed genes involved in cysteine metabolism. The study demonstrates that increased protein adducts formation and changes in cysteine concentration occur under chronic alcohol condition in liver cells which may increase alcohol-mediated oxidative injury.

Chronic ethanol and high glucose inducible CYP2E1 mediated oxidative stress leads to greater cellular injury in VL-17A cells: a potential mechanism for liver injury due to chronic alcohol consumption and hyperglycemia

Diabetes, characterized by the presence of inherent oxidative stress, may be further complicated by the additional oxidative stress generated due to the metabolism of alcohol. This study focuses on the roles of alcohol and high glucose inducible ADH and CYP2E1, both of which function as ethanol metabolizing enzymes, in promoting oxidative stress and cellular damage under chronic alcohol plus hyperglycemic conditions in VL-17A cells over-expressing ADH and CYP2E1. A specific CYP2E1 inhibitor, diallyl sulfide, proved to be more effective in decreasing the toxicity in VL-17A cells exposed to chronic alcohol plus high glucose than the specific ADH inhibitor pyrazole and the dual ADH and CYP2E1 inhibitor 4-methyl pyrazole. Furthermore, the greatest decrease in several parameters for oxidative stress such as ROS level, lipid peroxidation, protein carbonyl and protein aldehyde adduct formation and apoptosis was observed in the diallyl sulfide plus chronic alcohol plus high glucose treated VL-17A cells. In addition, specific inhibition of CYP2E1 with phenethyl isothiocyanate or CYP2E1 siRNA led to significant restoration of viability, and decrease in oxidative stress and apoptosis. Thus, the inducibility of CYP2E1 with both ethanol and high glucose leads to greater oxidative stress and cellular dysfunction in liver.