Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, Box 1603, One Gustave L. Levy Place, New York, NY 10029, USA.
Archives of Biochemistry and Biophysics (Impact Factor: 3.02). 02/2007; 457(2):252-63. DOI: 10.1016/
Source: PubMed


Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (alpha-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by alpha-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or alpha-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.

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Available from: Andres Caro, Jul 09, 2015
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    • "Lipid peroxidation may cause damage of the plasma membrane and an increase in the number of cytosolic-free calcium ions. This can result in the change in the verapamil and nifedipine-sensitive Ca 2+ channels [8] or an increased possibility of arachidonic acidinduced toxicity of CYPE1-expressing cells [9]. This increase in cytosolic Ca 2+ concentration can be prevented by vitamin E; moreover, we have also demonstrated that vitamin E may prevent bleb formation and the loss of protein thiols in tertbutyl hydroperoxide-(TBH-) treated hepatocytes [10] [11]. "
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    ABSTRACT: We investigated the effect of vitamin E on membrane protein thiols under oxidative stress, which we induced by treating hepatocytes with tert-butyl hydroperoxide (TBH) for 60 mins. Those cells which we pretreated with vitamin E formed fewer blebs (22.3% compared to 60.0% in nonvitamin E-treated cells) and maintained cytosolic calcium concentration and the number of membrane protein thiols instead of showing the usual symptoms in cells undergoing oxidative stress. Dithiothreitol (DTT) also commonly reduces bleb formation in hepatocytes affected by TBH. However, our experiments clearly demonstrate that DTT does not prevent the changes in cytosolic calcium and membrane protein thiols in the blebbing cells. Consequently, we decided to pretreat cells with both DTT and vitamin E and found that the influence of TBH was entirely prevented. These findings may provide us with a new aspect for investigating the mechanism of bleb formation under oxidative stress.
    Full-text · Article · May 2010 · BioMed Research International
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    • "Damage and death are due to excess stimulation of Ca 2+ -sensitive targets, which are numerous and concern key cellular functions: many enzymes that control supramolecular assembly, or degrading nucleic acids, lipids or proteins are Ca 2+ -sensitive. Among them are m-calpains, activated by high Ca 2+ levels and implicated in cell death and in many neurological disturbances [20]; lipoxygenases, and a set of Ca 2+ activated enzymes modifying arachidonic acid (AA) that are major actors of the inflammatory response, and also involved in apoptotic pathways [21]; phospholipases A2, which liberate AA from phospholipids, thus favoring, in the presence of high [Ca 2+ ]mt mitochondria stress or collapse [22]; a set of DNAses, one of which historical interest for being the enzyme responsible for apoptotic laddering [23]. A major form of damage is caused by the intervention of mitochondria that, taking up the excess of cytosolic Ca 2+ for scavenging purposes, may be subjected to stress and even collapse if it exceeds a physiological threshold, therefore increasing cell damage (see below) [14]. "
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    ABSTRACT: Ca(2+) is an important second messenger participating in many cellular activities; when physicochemical insults deregulate its delicate homeostasis, it acts as an intrinsic stressor, producing/increasing cell damage. Damage elicits both repair and death responses; intriguingly, in those responses Ca(2+) also participates as second messenger. This delineates a dual role for Ca(2+) in cell stress, making difficult to separate the different and multiple mechanisms required for Ca(2+)-mediated control of cell survival and apoptosis. Here we attempt to disentangle the two scenarios, examining on the one side, the events implicated in deregulated Ca(2+) toxicity and the mechanisms through which this elicits reparative or death pathways; on the other, reviewing the role of Ca(2+) as a messenger in the transduction of these same signaling events.
    Full-text · Article · Mar 2010 · International Journal of Cell Biology
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    • "Increased ALD has been described in conditions where CYP2E1 is over-expressed (Korourian et al., 1999; Butura et al., 2009). CYP2E1 has been associated with increased liver pathology score; lipid peroxidation; expression of mRNAs for the cytokine tumor necrosis factor alpha (TNFα) and the mitogen-activated protein (MAP) kinase ERK; and increased hepatocyte necrosis and apoptosis in response to TNFα and arachidonic acid (Butura et al, 2009; Lu and Cederbaum, 2008; Wu and Cederbaum, 2008; Caro and Cederbaum, 2007; Fang et al., 1998) and with stellate cell activation (Nieto et al., 2002). On the other hand, CYP2E1 inhibition has been reported to inhibit both ethanol-induced alterations in cytokine expression and liver pathology (Morimoto et al., 1995; Albano et al., 1996; Fang et al. 1998; Gouilon et al., 2000). "
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    ABSTRACT: The importance of ethanol metabolism in the development of alcoholic liver disease remains controversial. The present study examined the effects of selective inhibition of the cytochrome P450 enzyme CYP2E1 compared with the inhibition of overall ethanol metabolism on the development of alcoholic steatohepatitis. Adult male Sprague-Dawley rats were fed via total enteral nutrition for 45 days with or without 10-12g/kg/d ethanol. Some groups were given 200mg/kg/d of the CYP2E1 inhibitor diallyl sulfide (DAS). Other groups were treated with 164mg/kg/d of the alcohol dehydrogenase (ADH) inhibitor 4-methylpyrazole (4-MP) and dosed at 2-3g/kg/d ethanol to maintain similar average urine ethanol concentrations. Liver pathology scores and levels of apoptosis were elevated by ethanol (P<.05) but did not differ significantly on cotreatment with DAS or 4-MP. However, liver triglycerides were lower when ethanol-fed rats were treated with DAS or 4-MP (P<.05). Serum alanine aminotransferase values were significantly lower in ethanol-fed 4-MP-treated rats indicating reduced necrosis. Hepatic oxidative stress and the endoplasmic reticulum (ER) stress marker tribbles-related protein 3 were increased after ethanol (P<.05); further increased by DAS but partly attenuated by 4-MP. Both DAS and 4-MP reversed ethanol increases in the cytokine, tumor necrosis factor-alpha (TNF-alpha), and the chemokine CXCL-2 (P<.05). However, neither inhibitors prevented ethanol suppression of interleukins IL-4 or IL-12. Moreover, neither inhibitors prevented ethanol increases in tumor growth factor-beta mRNA. Ethanol and DAS additively induced hepatic hyperplasia (P<.05). These data suggest that a significant proportion of hepatic injury after ethanol exposure is independent of alcohol metabolism. Ethanol metabolism by CYP2E1 may be linked in part to triglyceride accumulation, to induction of TNF-alpha, and to chemokine production. Ethanol metabolism by ADH may be linked in part to oxidative and ER stress and necrotic injury.
    Full-text · Article · Mar 2010 · Alcohol (Fayetteville, N.Y.)
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