Publications by authors named "Aparajita Dey"

28 Publications

  • Page 1 of 1

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

Toxicol Mech Methods 2017 Feb 21;27(2):128-135. Epub 2016 Dec 21.

a Life Science Division , AU-KBC Research Centre, MIT Campus of Anna University , Chennai , India.

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.
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http://dx.doi.org/10.1080/15376516.2016.1268229DOI Listing
February 2017

GSH protects against oxidative stress and toxicity in VL-17A cells exposed to high glucose.

Eur J Nutr 2015 Mar 23;54(2):223-34. Epub 2014 Apr 23.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai, 600044, India.

Purpose: The deficiency of glutathione (GSH) has been linked to several diseases. The study investigated the role of GSH as a protective factor against hyperglycemia-mediated injury in VL-17A cells treated with 50 mM glucose.

Methods: The cell viability and different oxidative stress parameters including glyoxalase I activity were measured.

Results: GSH supplementation with 2 mM N-acetyl cysteine (NAC) or 0.1 mM ursodeoxycholic acid (UDCA) increased the viability, GSH level and the GSH-dependent glyoxalase I activity in 50 mM glucose-treated VL-17A cells. Further, pretreatment of 50 mM glucose-treated VL-17A cells with NAC or UDCA decreased oxidative stress (levels of reactive oxygen species and protein carbonylation), apoptosis (caspase 3 activity and annexin V-propidium iodide positive cells) and glutathionylated protein formation, a measure of oxidative stress. GSH depletion with 0.4 mM buthionine sulfoximine (BSO) or 1 mM diethyl maleate (DEM) potentiated the decrease in viability, glyoxalase I activity and increase in oxidative stress and apoptosis, with decreased GSH levels in 50 mM glucose-treated VL-17A cells.

Conclusion: Thus, changes in GSH levels with exogenous agents such as NAC, UDCA, BSO or DEM modulate hyperglycemia-mediated injury in a cell model of VL-17A liver cells.
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http://dx.doi.org/10.1007/s00394-014-0703-2DOI Listing
March 2015

Modulation of GSH with exogenous agents leads to changes in glyoxalase 1 enzyme activity in VL-17A cells exposed to chronic alcohol plus high glucose.

Food Funct 2014 Feb;5(2):345-58

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai-600044, India.

Gluthathione (GSH) is a major cellular antioxidant. The present study utilizing VL-17A cells exposed to chronic alcohol plus high glucose investigated the changes in oxidative stress, toxicity, and glyoxalase 1 activity as a detoxification pathway due to changes in GSH level through GSH supplementation with N-acetyl cysteine (NAC) or ursodeoxycholic acid (UDCA) and its depletion through buthionine sulfoximine (BSO) or diethyl maleate (DEM). Glyoxalase 1 plays an important role in detoxification of methylglyoxal which is formed as a precursor of advanced glycated end products formed due to high glucose mediated oxidative stress. Significant changes in glyoxalase 1 activity utilizing methylglyoxal or glyoxal as substrates occurred with NAC or UDCA or BSO or DEM supplementation in chronic alcohol plus high glucose treated VL-17A cells. NAC or UDCA administration in chronic alcohol plus high glucose treated VL-17A cells increased viability and decreased ROS levels, lipid peroxidation and 3-nitrotyrosine adduct formation. Similarly, GSH depletion with BSO or DEM had an opposite effect on the parameters in chronic alcohol plus high glucose treated VL-17A cells. In conclusion, modulation of GSH with NAC or UDCA or BSO or DEM leads to significant changes in oxidative stress, glyoxalase 1 enzyme activity and toxicity in chronic alcohol plus high glucose treated VL-17A cells.
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http://dx.doi.org/10.1039/c3fo60354gDOI Listing
February 2014

The role of antioxidants and other agents in alleviating hyperglycemia mediated oxidative stress and injury in liver.

Food Funct 2013 Aug;4(8):1148-84

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai 600044, India.

Several antioxidants and agents having similar antioxidant effects are known to exert beneficial effects in ameliorating the injurious effects of hyperglycemia on liver in different diabetic in vitro and in vivo models. The review deals with some of the agents which have been shown to exert protective effects on liver against hyperglycemic insult and the various mechanisms involved. The different classes of agents which protect the diabetic liver or decrease the severity of hyperglycemia mediated injury include flavonoids, catechins, and other polyphenolic compounds, curcumin and its derivatives, certain vitamins, hormones and drugs, trace elements, prototypical antioxidants and amino acids. Some of the pronounced changes mediated by the antioxidants in liver exposed to hyperglycemia include decreased oxidative stress, and alterations in carbohydrate and lipid metabolism. Other mechanisms through which the agents ameliorate hyperglycemia mediated liver injury include decrease in oxidative DNA and protein damage, restoration of mitochondrial structural and functional integrity, decrease in inflammation and improved insulin signaling. Thus, antioxidants may prove to be an important mode of defense in maintaining normal hepatic functions in diabetes.
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http://dx.doi.org/10.1039/c3fo30317aDOI Listing
August 2013

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

Biochim Biophys Acta 2013 Oct 23;1830(10):4407-16. Epub 2013 May 23.

AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, India.

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.
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http://dx.doi.org/10.1016/j.bbagen.2013.05.022DOI Listing
October 2013

Cytochrome P450 2E1: its clinical aspects and a brief perspective on the current research scenario.

Authors:
Aparajita Dey

Subcell Biochem 2013 ;67:1-104

AU-KBC Research Centre, Anna University, MIT Campus, Chromepet, Chennai, Tamil Nadu, 600044, India,

Research on Cytochrome P450 2E1 (CYP2E1), a key enzyme in alcohol metabolism has been very well documented in literature. Besides the involvement of CYP2E1 in alcohol metabolism as illustrated through the studies discussed in the chapter, recent studies have thrown light on several other aspects of CYP2E1 i.e. its extrahepatic expression, its involvement in several diseases and pathophysiological conditions; and CYP2E1 mediated carcinogenesis and modulation of drug efficacy. Studies involving these interesting facets of CYP2E1 have been discussed in the chapter focusing on the recent observations or ongoing studies illustrating the crucial role of CYP2E1 in disease development and drug metabolism.
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http://dx.doi.org/10.1007/978-94-007-5881-0_1DOI Listing
August 2014

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

Life Sci 2013 Mar 24;92(6-7):325-36. Epub 2013 Jan 24.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai-600044, India.

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.
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http://dx.doi.org/10.1016/j.lfs.2012.12.014DOI Listing
March 2013

Foxp3+ regulatory T cells among tuberculosis patients: impact on prognosis and restoration of antigen specific IFN-γ producing T cells.

PLoS One 2012 19;7(9):e44728. Epub 2012 Sep 19.

Cellular Immunology Division, Department of T.I.I., All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) and programmed death-1 (PD-1) molecules have emerged as pivotal players in immune suppression of chronic diseases. However, their impact on the disease severity, therapeutic response and restoration of immune response in human tuberculosis remains unclear. Here, we describe the possible role of Treg cells, their M. tuberculosis driven expansion and contribution of PD-1 pathway to the suppressive function of Treg cells among pulmonary tuberculosis (PTB) patients. Multicolor flow cytometry, cell culture, cells sorting and ELISA were employed to execute the study. Our results showed significant increase in frequency of antigen-reactive Treg cells, which gradually declined during successful therapy and paralleled with decline of M. tuberculosis-specific IL-10 along with elevation of IFN-γ production, and raising the IFN-γ/IL-4 ratio. Interestingly, persistence of Treg cells tightly correlated with MDR tuberculosis. Also, we show that blocking PD-1/PD-L1 pathway abrogates Treg-mediated suppression, suggesting that the PD-1/PD-L1 pathway is required for Treg-mediated suppression of the antigen-specific T cells. Treg cells possibly play a role in dampening the effector immune response and abrogating PD-1 pathway on Treg cells significantly rescued protective T cell response, suggesting its importance in immune restoration among tuberculosis patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044728PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3446959PMC
March 2013

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

Integr Biol (Camb) 2012 May 22;4(5):550-63. Epub 2012 Mar 22.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai-600044, India.

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.
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http://dx.doi.org/10.1039/c2ib00155aDOI Listing
May 2012

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

Alcohol Clin Exp Res 2012 Jun 6;36(6):1004-12. Epub 2012 Feb 6.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai, India.

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.
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http://dx.doi.org/10.1111/j.1530-0277.2011.01697.xDOI Listing
June 2012

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

Gene 2012 Feb 25;494(1):130-9. Epub 2011 Nov 25.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai, India.

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.
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http://dx.doi.org/10.1016/j.gene.2011.11.031DOI Listing
February 2012

Cytochrome P450 2E1 and hyperglycemia-induced liver injury.

Cell Biol Toxicol 2011 Aug 1;27(4):285-310. Epub 2011 Apr 1.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai, India.

Cytochrome P450 2E1 (CYP2E1), a microsomal enzyme involved in xenobiotic metabolism and generation of oxidative stress, has been implicated in promoting liver injury. The review deals with the changes in various cellular pathways in liver linked with the changes in regulation of CYP2E1 under hyperglycemic conditions. Some of the hepatic abnormalities associated with hyperglycemia-mediated induction of CYP2E1 include increased oxidative stress, changes in mitochondrial structure and function, apoptosis, nitrosative stress, and increased ketone body accumulation. Thus, changes in regulation of CYP2E1 are associated with the injurious effects of hyperglycemia in liver.
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http://dx.doi.org/10.1007/s10565-011-9188-4DOI Listing
August 2011

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.

Toxicol In Vitro 2011 Jun 23;25(4):969-78. Epub 2011 Mar 23.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai 600 044, India.

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.
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http://dx.doi.org/10.1016/j.tiv.2011.03.006DOI Listing
June 2011

Hyperglycemia-induced mitochondrial alterations in liver.

Life Sci 2010 Aug 17;87(7-8):197-214. Epub 2010 Jun 17.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai, India.

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.
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http://dx.doi.org/10.1016/j.lfs.2010.06.007DOI Listing
August 2010

Apoptosis in HepG2 cells exposed to high glucose.

Toxicol In Vitro 2010 Mar 3;24(2):387-96. Epub 2009 Nov 3.

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai 600 044, India.

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.
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http://dx.doi.org/10.1016/j.tiv.2009.10.020DOI Listing
March 2010

Hyperglycemia induced changes in liver: in vivo and in vitro studies.

Curr Diabetes Rev 2009 May;5(2):67-78

Life Science Division, AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai-600044, India.

Diabetes, characterized by chronic hyperglycemia, has reached serious epidemic proportions. It is also not infrequent to find increased incidence of liver injury in diabetics and hyperglycemia plays an important role in promoting liver injury through several mechanisms. The following review identifies the pathways through which hyperglycemia causes changes in liver of various animal models and liver cell culture models, and elucidates the mechanisms and consequences of hyperglycemia induced liver injury in humans. Some of the pathways which are hyperglycemia driven include increased oxidative and nitrosative stress, activation of stress signaling pathways and increased cytokine levels, impairment of protective mechanisms such as the expression of molecular chaperones and proteosome activity, and dysregulation of glucose and lipid metabolism. Thus, hyperglycemia induced changes in the liver's cellular environment in in vitro and in vivo models have been documented extensively in the literature.
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http://dx.doi.org/10.2174/157339909788166864DOI Listing
May 2009

Induction of cytochrome P450 2E1 [corrected] promotes liver injury in ob/ob mice.

Hepatology 2007 Jun;45(6):1355-65

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

Unlabelled: Cytochrome P450 2E1 (CYP2E1) activates several hepatotoxins and contributes to alcoholic liver damage. Obesity is a growing health problem in the United States. The aim of the present study was to evaluate whether acetone- or pyrazole-mediated induction of CYP2E1 can potentiate liver injury in obesity. CYP2E1 protein and activity were elevated in acetone- or pyrazole-treated obese and lean mice. Acetone or pyrazole induced distinct histological changes in liver and significantly higher aminotransferase enzymes in obese mice compared to obese controls or acetone- or pyrazole-treated lean mice. Higher caspase-3 activity and numerous apoptotic hepatocytes were observed in the acetone- or pyrazole-treated obese mice. Increased protein carbonyls, malondialdehyde, 4-hydroxynonenal-protein adducts, elevated levels of inducible nitric oxide synthase, and higher 3-nitrotyrosine protein adducts were found in livers of acetone- or pyrazole-treated obese animals, suggesting elevated oxidative and nitrosative stress. Liver tumor necrosis factor alpha levels were higher in pyrazole-treated animals. The CYP2E1 inhibitor chlormethiazole and iNOS inhibitor N-(3-(aminomethyl)-benzyl) acetamidine abrogated the toxicity and the oxidative/nitrosative stress elicited by the induction of CYP2E1.

Conclusion: These results show that obesity contributes to oxidative stress and liver injury and that induction of CYP2E1 enhances these effects.
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http://dx.doi.org/10.1002/hep.21603DOI Listing
June 2007

S-adenosyl methionine protects ob/ob mice from CYP2E1-mediated liver injury.

Am J Physiol Gastrointest Liver Physiol 2007 Jul 19;293(1):G91-103. Epub 2007 Apr 19.

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

Pyrazole treatment to induce cytochrome P-450 2E1 (CYP2E1) was recently shown to cause liver injury in ob/ob mice but not in lean mice. The present study investigated the effects of S-adenosyl-l-methionine (SAM) on the CYP2E1-dependent liver injury in ob/ob mice. Pyrazole treatment of ob/ob mice for 2 days caused necrosis, steatosis, and elevated serum transaminase and triglyceride levels compared with saline ob/ob mice. Administration of SAM (50 mg/kg body wt ip every 12 h for 3 days) prevented the observed pathological changes as well as the increase of apoptotic hepatocytes, caspase 3 activity, and serum TNF-alpha levels. SAM administration inhibited CYP2E1 activity but not CYP2E1 content. The pyrazole treatment increased lipid peroxidation, 4-hydroxynonenal and 3-nitrotyrosine protein adducts, and protein carbonyls. These increases in oxidative and nitrosative stress were prevented by SAM. Treatment of ob/ob mice with pyrazole lowered the endogenous SAM levels, and these were elevated after SAM administration. Mitochondrial GSH levels were very low after pyrazole treatment of the ob/ob mice; this was associated with elevated levels of malondialdehyde and 4-hydroxynonenal and 3-nitrotyrosine protein adducts in the mitochondria. All these changes were prevented with SAM administration. SAM protected against pyrazole-induced increase in serum transaminases, necrosis, triglyceride levels, caspase-3 activity, and lipid peroxidation even when administered 1 day after pyrazole treatment. In the absence of pyrazole, SAM lowered the slightly elevated serum transaminases, triglyceride levels, caspase-3 activity, and lipid peroxidation in obese mice. In conclusion, SAM protects against and can also reverse or correct CYP2E1-induced liver damage in ob/ob mice.
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http://dx.doi.org/10.1152/ajpgi.00004.2007DOI Listing
July 2007

Geldanamycin, an inhibitor of Hsp90 increases cytochrome P450 2E1 mediated toxicity in HepG2 cells through sustained activation of the p38MAPK pathway.

Arch Biochem Biophys 2007 May 8;461(2):275-86. Epub 2007 Mar 8.

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA.

Cytochrome P450 2E1 (CYP2E1) can mediate reactive oxygen species (ROS) induced cell death through its catalytic processes. Heat shock protein 90 (Hsp90) is an important molecular chaperone which is essential for cellular integrity. We previously showed that inhibition of Hsp90 with Geldanamycin (GA), an inhibitor of Hsp90 increased CYP2E1 mediated toxicity in CYP2E1 over-expressing HepG2 cells (E47 cells) but not in C34-HepG2 cells devoid of CYP2E1 expression. The aim of the present study was to test the hypothesis that the potentiation of CYP2E1 toxicity in E47 cells with GA may involve changes in mitogen activated protein kinase signal transduction pathways. GA was toxic to E47 cells and SB203580, an inhibitor of p38 MAPK prevented this decrease in viability. The protective effects of SB203580 were effective only when SB203580 was added before GA treatment. GA activated p38 MAPK in E47 cells and this activation was an early and a sustained event. GA elevated ROS levels and lipid peroxidation and lowered GSH levels in E47 cells and these changes were blunted or prevented by treatment with SB203580. Apoptosis was increased by GA and prevented by pre-treatment with SB203580. The loss in mitochondrial membrane potential in E47 cells after GA treatment was also decreased significantly with SB203580 treatment. The activity and expression of CYP2E1 and Hsp90 levels were not altered by SB203580. In conclusion, the inhibition of Hsp90 with GA increases the toxicity of CYP2E1 in HepG2 cells through an early and sustained activation of the p38 MAPK pathway.
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http://dx.doi.org/10.1016/j.abb.2007.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1942044PMC
May 2007

Evidence for cytochrome P450 3A expression and catalytic activity in rat blood lymphocytes.

Life Sci 2006 Sep 9;79(18):1729-35. Epub 2006 Jun 9.

Developmental Toxicology Division, Industrial Toxicology Research Centre, M.G. Marg, P.O. Box 80, Lucknow-226001, India.

Freshly isolated peripheral blood lymphocytes from control rats were found to catalyze the N-demethylation of erythromycin, known to be mediated by cytochrome P450 3A (CYP3A) isoenzymes in rat liver. Pretreatment of rats with dexamethasone (100 mg/kgx3 days, i.p.), a CYP3A inducer, resulted in 3-4-fold increase in the activity of erythromycin demethylase (EMD) in freshly isolated peripheral blood lymphocytes. This increase in the enzyme activity was found to be associated with an increase in the rate of the reaction and affinity of the substrate towards the enzyme. Significant inhibition of the EMD activity on in vitro addition of ketoconazole, a specific CYP3A inhibitor in liver and polyclonal antibody raised against rat liver CYP3A have suggested that EMD activity in blood lymphocytes is catalyzed primarily by CYP3A isoenzymes. Further, immunoblot analysis with polyclonal antibody raised against rat liver CYP3A revealed significant immunoreactivity, co-migrating with the liver isoenzyme, indicating constitutive expression of CYP3A in blood lymphocytes. Pretreatment with dexamethasone was found to significantly increase the expression of CYP3A protein in freshly isolated rat blood lymphocytes, as observed with liver. Likewise, significant CYP3A mRNA detected in control rat blood lymphocytes has further demonstrated constitutive expression of CYP3A isoenzymes in blood lymphocytes. Furthermore, several fold increase in CYP3A mRNA expression following pretreatment with dexamethasone showed similarities in the regulation of CYP3A isoenzymes in rat blood lymphocytes with the liver enzyme. The data suggest that the blood lymphocytes can be used to monitor tissue expression of CYP3A isoenzymes and validate the suitability of lymphocytes as surrogates of CYP status in less accessible target tissues.
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http://dx.doi.org/10.1016/j.lfs.2006.06.006DOI Listing
September 2006

Geldanamycin, an inhibitor of Hsp90, potentiates cytochrome P4502E1-mediated toxicity in HepG2 cells.

J Pharmacol Exp Ther 2006 Jun 2;317(3):1391-9. Epub 2006 Mar 2.

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, Box 1603, One Gustave L Levy Place, New York, NY 10029, USA.

Cytochrome P450 2E1 (CYP2E1) potentiates oxidative stress-mediated cell death. Heat shock proteins (Hsps) modulate the stability and function of numerous proteins. We examined the effect of geldanamycin (GA), an inhibitor of Hsp90, on CYP2E1-mediated toxicity in transfected HepG2 cells overexpressing CYP2E1 (E47 cells). Basal expression of CYP2E1 and Hsp90 was higher in E47 cells compared with control C34 cells, which do not express CYP2E1. Treatment with GA resulted in significant toxicity to E47 cells compared with C34 cells. An enhanced loss of E47 cell viability was also observed using two different inhibitors of Hsp90, herbimycin A and radicicol. Treatment of E47 cells with GA caused depletion of glutathione coupled to an increase in reactive oxygen species level and lipid peroxidation. These effects of GA were more pronounced in the E47 than the C34 cells. The antioxidants trolox and N-acetylcysteine prevented the increased reactive oxygen species accumulation and resultant loss of viability. GA caused increased caspase 3 activity and Annexin V staining in E47 cells, suggesting an apoptotic mode of cell death. A decrease in mitochondrial membrane potential was observed in GA-treated HepG2 cells, and mitochondrial permeability transition inhibitors prevented the cytotoxicity of GA. These results suggest that Hsp90 is protective against CYP2E1-dependent oxidant stress and loss of cell viability in HepG2 cells.
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http://dx.doi.org/10.1124/jpet.106.101808DOI Listing
June 2006

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1.

Arch Biochem Biophys 2006 Mar 8;447(2):155-66. Epub 2006 Feb 8.

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA.

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.
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http://dx.doi.org/10.1016/j.abb.2006.01.013DOI Listing
March 2006

Alcohol and oxidative liver injury.

Hepatology 2006 Feb;43(2 Suppl 1):S63-74

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY, USA.

Acute and chronic ethanol treatment has been shown to increase the production of reactive oxygen species, lower cellular antioxidant levels, and enhance oxidative stress in many tissues, especially the liver. Ethanol-induced oxidative stress plays a major role in the mechanisms by which ethanol produces liver injury. Many pathways play a key role in how ethanol induces oxidative stress. This review summarizes some of the leading pathways and discusses the evidence for their contribution to alcohol-induced liver injury. Many of the seminal reports in this topic have been published in Hepatology , and it is fitting to review this research area for the 25th Anniversary Issue of the Journal.
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http://dx.doi.org/10.1002/hep.20957DOI Listing
February 2006

Evidence for cytochrome P450 2E1 catalytic activity and expression in rat blood lymphocytes.

Life Sci 2005 Jul;77(10):1082-93

Developmental Toxicology Division, Industrial Toxicology Research Centre, P.O. Box 80, M.G. Marg, Lucknow-226 001, U.P., India.

Studies initiated to characterize cytochrome P450 2E1(CYP2E1) in freshly isolated rat blood lymphocytes revealed significant mRNA of CYP2E1 in control blood lymphocytes. RT-PCR studies have shown that as observed in liver, acute treatment of ethanol (single oral dose of 0.8 ml/kg b.wt, i.p), resulted in increase in the mRNA expression of CYP2E1 in freshly isolated rat blood lymphocytes. Western blotting studies using polyclonal antibody raised against rat liver CYP2E1 demonstrated significant immunoreactivity, comigrating with the liver isoenzyme, in freshly isolated control rat blood lymphocytes. Similar to that seen in liver, pretreatment of ethanol was found to produce an increase in the CYP2E1 isoenzyme in the blood lymphocytes. Blood lymphocytes were also found to catalyze the CYP dependent N-demethylation of N-nitrosodimethylamine (NDMA), which like in liver increased 2-3 fold following pretreatment of rats with known CYP2E1 inducers. Kinetic studies have further shown significant increase in the apparent Vmax and the affinity towards the substrate in rat blood lymphocytes indicating that as observed in liver, the increase in mRNA and protein expression following exposure to CYP2E1 inducers is associated with the increased catalytic activity of CYP2E1 in freshly isolated rat blood lymphocytes. The data indicating similarities of the blood lymphocyte CYP2E1 with the liver enzyme suggest that lymphocyte CYP2E1 levels in freshly isolated rat blood lymphocytes could be used to monitor tissue enzyme levels.
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http://dx.doi.org/10.1016/j.lfs.2005.01.021DOI Listing
July 2005

CYP450, COX-2 and Obesity Related Renal Damage.

Toxicol Mech Methods 2005 ;15(2):125-36

Vascular Biology Center and Department of Physiology, Medical College of Georgia, Augusta, Georgia, USA.

Abstract The number of obese people in the world is growing rapidly worldwide and has reached epidemic status. Obesity is often associated with the clustering of metabolic and cardiovascular risk factors that contribute to metabolic syndrome or syndrome X. Likewise, metabolic syndrome and its associated traits are major contributing factors to the increase in nephropathy and end stage renal disease. The specific factors that link the metabolic syndrome traits to the progression of nephropathy remain largely unexplored. Recent studies have demonstrated that an imbalance between cyclooxygenase-2 (COX-2) and cytochrome P450 (CYP450) arachidonic acid metabolizing enzymes in the kidney may contribute to the renal damage associated with obesity. Along these lines, COX-2 inhibition decreases renal cytokine levels and glomerular injury in obese rats. Peroxisome proliferators-activated receptors (PPARs) are transcription factors that also contribute to chronic kidney disease in obesity and metabolic syndrome. Intriguingly, interactions between PPARs and arachidonic acid metabolites could be key determinants of renal damage in metabolic syndrome patients. Therefore, there is a promising future for pharmacological agents that manipulate COX-2 and CYP450 metabolites and PPARs to treat obesity related nephropathy.
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http://dx.doi.org/10.1080/15376520590918856DOI Listing
October 2012

Decreased epoxygenase and increased epoxide hydrolase expression in the mesenteric artery of obese Zucker rats.

Am J Physiol Regul Integr Comp Physiol 2005 Jan 2;288(1):R188-96. Epub 2004 Sep 2.

Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500, USA.

Previous studies suggest that epoxyeicosatrienoic acids (EETs) are vasodilators of the mesenteric artery; however, the production and regulation of EETs in the mesenteric artery remain unclear. The present study was designed 1) to determine which epoxygenase isoform may contribute to formation of EETs in mesenteric arteries and 2) to determine the regulation of mesenteric artery cytochrome P-450 (CYP) enzymes in obese Zucker rats. Microvessels were incubated with arachidonic acid, and CYP enzyme activity was determined. Mesenteric arteries demonstrate detectable epoxygenase and hydroxylase activities. Next, protein and mRNA expressions were determined in microvessels. Although renal microvessels express CYP2C23 mRNA and protein, mesenteric arteries lacked CYP2C23 expression. CYP2C11 and CYP2J mRNA and protein were expressed in mesenteric arteries and renal microvessels. In addition, mesenteric artery protein expression was evaluated in lean and obese Zucker rats. Compared with lean Zucker rats, mesenteric arterial CYP2C11 and CYP2J proteins were decreased by 38 and 43%, respectively, in obese Zucker rats. In contrast, soluble epoxide hydrolase mRNA and protein expressions were significantly increased in obese Zucker rat mesenteric arteries. In addition, nitric oxide-independent dilation evoked by acetylcholine was significantly attenuated in mesenteric arteries of obese Zucker rats. These data suggest that the main epoxygenase isoforms expressed in mesenteric arteries are different from those expressed in renal microvessels and that decreased epoxygenases and increased soluble epoxide hydrolase are associated with impaired mesenteric artery dilator function in obese Zucker rats.
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http://dx.doi.org/10.1152/ajpregu.00018.2004DOI Listing
January 2005

Altered kidney CYP2C and cyclooxygenase-2 levels are associated with obesity-related albuminuria.

Obes Res 2004 Aug;12(8):1278-89

Vascular Biology Center, Medical College of Georgia, 1120 Fifteenth Street, Augusta, GA 30912, USA.

Objective: To determine cytochrome P450 (CYP450) and cyclooxygenase (COX) expression and metabolite regulation and renal damage in the early stages of obesity-related hypertension and diabetes.

Research Methods And Procedures: Obese and lean Zucker rats at 10 to 12 weeks of age were studied. Blood pressure was measured in the conscious state using radiotelemetry. Blood glucose levels and body weight were measured periodically. Protein expression of CYP450 and COX enzymes in the kidney cortex, renal microvessels, and glomeruli was studied. The levels of CYP450 and COX metabolites in urine were measured, and urinary albumin excretion, an indicator of kidney damage, was measured.

Results: Body weight and blood glucose averaged 432 +/- 20 grams and 105 +/- 5 mg/dl, respectively, in obese Zucker rats as compared with 320 +/- 8 grams and 91 +/- 5 mg/dl, respectively, in age-matched 10- to 12-week-old lean Zucker rats. Renal microvascular CYP4A and COX-2 protein levels were increased 2.3- and 17.0-fold, respectively, in obese Zucker rats. The protein expression of CYP2C11 and CYP2C23 was decreased 2.0-fold in renal microvessels isolated from obese Zucker rats when compared with lean Zucker rats. The urinary excretion rate of thromboxane B(2) was increased significantly in obese Zucker as compared with lean Zucker rats (22.0 +/- 1.8 vs. 13.4 +/- 1.0 ng/d). Urinary albumin excretion, an index of kidney damage, was increased in the obese Zucker rat at this early age.

Discussion: These results suggest that increased CYP4A and COX-2 protein levels and decreased CYP2C11 and CYP2C23 protein levels occur in association with microalbuminuria during the onset of obesity-related hypertension and type 2 diabetes.
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http://dx.doi.org/10.1038/oby.2004.162DOI Listing
August 2004

Rofecoxib decreases renal injury in obese Zucker rats.

Clin Sci (Lond) 2004 Dec;107(6):561-70

Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA.

The present study tested the hypothesis that altered vascular regulation of arachidonic acid enzymes in obese Zucker rats contributes to renal damage. Protein expression of CYP450 (cytochrome P450) and COX (cyclo-oxygenase) enzymes in renal microvessels was studied in obese and lean Zucker rats at 20-21 weeks of age. Body weight and blood glucose averaged 649+/-13 g and 142+/-10 mg/dl in obese Zucker rats compared with 437+/-10 g and 111+/-5 mg/dl in age-matched lean Zucker rats. Renal microvascular CYP4A and COX-2 protein levels were increased and CYP2C protein levels decreased in obese Zucker rats. TX (thromboxane) B2 excretion was 2-fold higher and PG (prostaglandin) E2 excretion significantly lower in obese Zucker rats. Additional studies investigated the ability of the COX-2 inhibitor, rofecoxib, to slow the progression of renal injury in obese Zucker rats. Rofecoxib treatment decreased urinary PGF2alpha and 8-isoprostane levels in obese Zucker rats. Renal microvessel mRNA expression of pro-inflammatory chemokines was decreased in COX-2-inhibitor-treated obese Zucker rats. Urinary albumin excretion, an index of kidney damage, averaged 95+/-11 mg/day in vehicle-treated and 9+/-1 mg/day in rofecoxib-treated obese Zucker rats. Glomerulosclerosis, characterized by mesangial expansion, tubulo-interstitial fibrosis and extracellular matrix accumulation, was prominent in obese Zucker rats compared with a lack of damage in age-matched lean Zucker rats and rofecoxib-treated obese Zucker rats. These results suggest that altered vascular arachidonic acid enzymes contribute to the renal damage, and that COX-2 inhibition decreases glomerular injury in obese Zucker rats.
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http://dx.doi.org/10.1042/CS20040125DOI Listing
December 2004