The results presented in this work suggest that AA-induced cell death in liver cells overexpressing CYP2E1 is mediated by release of calcium from intracellular stores and subsequent activation of PLA2: i) a partial depletion of ionomycin-sensitive calcium stores together with increased cytosolic calcium was detected prior to toxicity; ii) AA-mediated toxicity was prevented after depletion of intracellular calcium, but not after removal of extracellular calcium alone; iii) activation of PLA2 paralleled toxicity, both events were inhibited by PLA2 inhibitors and by depletion of intracellular calcium, but not after removal of extracellular calcium alone. Oxidative stress and lipid peroxidation in CYP2E1-expressing HepG2 cells is suggested to be the initiator of AA-induced intracellular calcium release, as α -tocopherol restored the amount of calcium in the ER, inhibited lipid peroxidation, PLA2 activation and cytotoxicity without altering CYP2E1 levels. CYP2E1 expression was critical for AA toxicity, as AA-treated C34 cells, which do not express CYP2E1, did not show increased lipid peroxidation, activation of PLA2 or cytotoxicity. These events are summarized in and further discussed below.
Role of calcium in AA-dependent toxicity in CYP2E1-expressing liver cells. See Discussion for details.
We propose that increased calcium derived from intracellular stores activates PLA2, which in turn increases membrane damage through phospholipid hydrolysis: PLA2 inhibitors which decrease 3H-AA-phospholipid hydrolysis prevent the AA toxicity, without affecting CYP2E1 activity or lipid peroxidation. Thus, the increase in PLA2 activity is downstream of CYP2E1, and the CYP2E1 plus AA-induced lipid peroxidation. It is important to note that although the sustained increase in intracellular Ca2+ produced by AA was blunted in SMEM compared to MEM, AA toxicity and activation of PLA2 was similar in MEM and SMEM. Hence, the extracellular Ca2+, in contrast to stored Ca2+, does not play a significant role in the AA toxicity or activation of PLA2 activity.
We hypothesize that AA+CYP2E1-induced lipid peroxidation, increased intracellular calcium and PLA2 activation are events that converge on mitochondria, inducing mitochondrial damage (decreased mitochondrial membrane potential which is blocked by PTP and PLA2 inhibitors) and ultimately, cell necrosis. Each of these, the elevated calcium, the enhanced lipid peroxidation, and the increased PLA2 activity which is likely to promote mitochondrial membrane damage, likely contribute to the mitochondrial dysfunction. The fact that toxicity is not blocked in SMEM, but almost completely blocked in calcium-depleted cells suggests that intracellular calcium stores are critical in AA+CYP2E1-dependent toxicity. Since AA toxicity in SMEM is still blocked by α -tocopherol and PLA2 inhibitors, release of stored calcium through lipid peroxidation and activation of PLA2 is still operative in SMEM similar to results in MEM. This is supported by the lack of inhibition of 3HAA-phospholipid hydrolysis in SMEM, and the practically complete inhibition of 3HAA release in calcium-depleted cells. These results were mainly obtained with HepG2 cells overexpressing CYP2E1 (E47 cells), but the basic aspects of this mechanism were replicated using primary rat hepatocytes overexpressing CYP2E1.
Thapsigargin and calcium ionophores are classical inducers of the ER stress response through ER calcium depletion [46
]. Conditioning with thapsigargin or calcium ionophores prevented oxidative stress-dependent cell death in renal epithelial cells, through induction of ER stress proteins [47
]. There are few studies on the ability of ER stress protein induction to provide cytoprotection in the liver [46
]. After induction of ER stress, no protection against agents associated with oxidative stress (menadione, tert-butyl hydroperoxide) was observed in hepatoma cells (HepG2 and H4IIE), suggesting that protection afforded by the ER stress response may be cell type-specific [48
]. Induction of ER stress does not seem to play a role in cytoprotection by pretreatment with thapsigargin and ionomycin in our model, as two common markers of ER stress (calreticulin and GRP94) were not induced after complete calcium depletion in E47 HepG2 cells. We conclude that inhibition of PLA2 activation is the key factor to consider in the cytoprotection by ER calcium depletion in our model.
Research into the mechanism of oxidative cell injury is shifting away from the central association of cytosolic calcium with lethal injury to focus on adverse effects of oxidants on mitochondria [49
]. Our results suggest that in AA plus CYP2E1-dependent oxidative injury, influx of extracellular calcium does not play a central role in toxicity, but that activation of phospholipase A2, promotion of the mitochondrial permeability transition and loss of mitochondrial function is a general pathway for toxicity.
Nanji et al. [50
] reported CYP2E1 induction, increased lipid peroxidation and elevated phospholipase A2 activity in rats treated with corn oil (unsaturated fat) plus ethanol for 1 month (intragastric infusion model), with respect to controls fed glucose instead of ethanol. Alcoholic liver injury developed only in the corn oil–ethanol-fed groups [50
]. Increased oxidative stress and lipid peroxidation are critical pathogenic events for alcoholic liver disease [23
]. In addition, ethanol administration in vivo
increased the calcium content in the liver of rats and mice [52
]. Our results suggest a possible connection between these reported events in alcoholic liver disease, which might lead to liver injury: alcohol+ unsaturated fat; increased CYP2E1; lipid peroxidation; increased intracellular calcium; phospholipase A2 activation; liver injury.
In summary, we 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 the 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. The basic signaling pathway proposed is described in : AA and CYP2E1 activity leads to increased oxidative stess and lipid peroxidation, which induces calcium release from intracellular stores and activation of phospholipase A2, leading to cell necrosis through mitochondrial impairment.