Oxygen free radicals have been considered to be responsible for the pathogenesis of carcinogenesis, aging, ischemia/reperfusion injury and tissue injuries by certain xenobiotics and anticancer drugs¹⁾. All aerobic cells generate, enzymatically or nonenzymatically, oxygen free radicals such as superoxide, hydrogen peroxide and, probably, hydroxyl radicals during normal and abnormal metabolic processes from the metabolism of exogenous drugs and toxins. At the same time, the abundant antioxidant defenses of most cells prevent oxygen free radical-induced cell injury. Nevertheless, when the rate of oxygen free radical generation is increased and/or the antioxidant defenses of the cells are weakened, oxidative cell injury would result²⁾.

Exposure of isolated hepatocytes to oxidants, such as t-butylhydroperoxide (t-BHP) or H₂O₂, results in peroxidation of membrane lipids and a rapid loss of cell viability^3–6). Lipid peroxidation has been recognized to be an important mediator of oxygen free radical-induced cell injury. Nevertheless, the role of lipid peroxidation in hepatocyte injury is controversial. Masaki et al. reported that t-BHP causes cell death by a mechanism that depends on the peroxidation of cellular lipids in cultured hepatocytes⁶⁾. By contrast, Rush et al. reported that lipid peroxidation does not play a critical role in the acute toxicity of t-BHP in isolated hepatocytes³⁾. t-BHP can be metabolized to free radicals by iron to result in the formation of the t-butyl alkoxyl radical. This radical can initiate the peroxidation of cellular lipids which is responsible for the loss of cell viability. Alternatively, the t-butyl alkoxyl radical may cause cell injury by lipid peroxidation-independent mechanism. In the latter case, lipid peroxidation could be induced as a consequence rather than as a cause of cell death or as epiphenomenon accompanying lethal attack on the cell⁶⁾. Thus, the role of lipid peroxidation in the underlying mechanism of t-BHP-induced cell injury is not clearly defined.

Studies in vitro have shown that oxidants induce an increase in intracellular Ca²⁺ concentration in myocytes⁷⁾ and hepatocytes^4,8). This rise in intracellular Ca²⁺ mediates the cell injury associated with an acute oxidative stress^5,9). Several studies demonstrated that the mobilization of Ca²⁺ from intracellular stores or an inhibition of the Ca²⁺ extrusion pump of the plasma membrane are the major mechanisms responsible for the elevated cytosolic Ca²⁺ concentration^5,10). On the other hand, Ca²⁺ fluxes in hepatocytes seem to be, at least in part, regulated by Ca²⁺ channels^11,12), and the cytoprotective effect of Ca²⁺ channel blockers has been documented by various heptotoxins^13,14). However, it has not been known that Ca²⁺ channel blockers exert a protective effect against oxidant-induced liver cell injury.

Elevated intracellular Ca²⁺ by oxidants may initiate a cascade of signaling leading to activation of phospholipase A₂(PLA₂) resulting in cell injury⁹⁾. In fact, previous in vitro studies have also showed that PLA₂ inhibitors attenuated oxidant-induced cell injury in renal cells¹⁵⁾. However, it is unclear whether similar results could appear in hepatocytes.

This study was undertaken to determine whether Ca²⁺ channel blockers, modulation of external Ca²⁺ and PLA₂ inhibitors affect t-BHP-induced cell injury in rat liver slices. The present study demonstrated that LDH release and lipid peroxidation induced by t-BHP are significantly prevented by Ca²⁺ channel blockers or PLA₂ inhibitors, and oxidant-induced cell injury does not necessarily result from lipid peroxidation.

The exposure of t-BHP to liver slices resulted in an increase of lipid peroxidation in a dose-dependent manner (Fig. 1A). Similar results were observed in LDH release (Fig. 1B). Thus, there is close correlation between t-BHP-induced lipid peroxidation and LDH release (Fig. 2).

In order to determine whether antioxidant could prevent lipid peroxidation as well as t-BHP-induced cell injury as estimated by LDH release, the effect of a phenolic antioxidant, DPPD, was examined. As shown in Fig. 3, 20mM DPPD exerted a siginficant protective effect against lipid peroxidation and LDH release caused by 1mM t-BHP. However, the extent of protective effect on LDH release was less than that on lipid peroxidation. t-BHP-induced lipid peroxidation was completely prevented by DPPD, whereas t-BHP-induced LDH release was partially (although significantly) reduced.

t-BHP reacts with ferrous iron to produce a more potent oxidant, the t-butyl alkoxyl radical^6,8). Thus, the effect of iron chelator was examined to ascertain if iron chelator could prevent both lipid peroxidation and LDH release by t-BHP. Slices were pretreated for 10min with 2mM deferoxamine before treatment of t-BHP. The results depicted in Fig. 4 indicated that the lipid peroxidation and LDH release induced by t-BHP were siginficantly decreased by pretreatment of deferoxamine.

Fig. 5 shows the effect of a sulfhydryl reducing agent, DTT, and GSH on t-BHP-induced lipid peroxidation and LDH release. Addition of 2mM DTT completely protected against the lipid peroxidation and LDH release caused by 1 mM t-BHP. Likewise, both t-BHP-induced lipid peroxidation and LDH release were markedly prevented by 2mM GSH.

Sippel et al. reported in liver cells that Ca²⁺ channel blockers exert a protective effect against cell death by 98/202 which causes cell injury through a disturbance of intracellular calcium homeostasis¹⁴⁾. Therefore, effects of Ca²⁺ channel blockers on t-BHP-induced lipid peroxidation and LDH release were examined. As shown in Fig. 6A, t-BHP-induced lipid peroxidation was partially but significantly reduced by addition of diltiazem, nifedipine or verapamil.

In order to determine whether depletion of external Ca²⁺ affects t-BHP-induced cell injury, effects of Ca²⁺-free medium and the external Ca²⁺ chelator EGTA on t-BHP-induced lipid peroxidation and LDH release was examined. As shown in Fig. 7, when slices were exposed to Ca²⁺-free medium in the absence of t-BHP, there was a siginficant increase in LDH release and a partial but nonsignificant increase in lipid peroxidation. However, both lipid peroxidation and LDH release induced by t-BHP rather decreased in the Ca²⁺-free medium as compared with those in the normal medium, although the difference was nonsignificant. The results depicted in Fig. 8 indicated that t-BHP-induced lipid peroxidation was not significantly altered by the addition of 2mM EGTA, whereas t-BHP-induced LDH release was significantly reduced by EGTA.

Since previous in vitro studies have showed that PLA₂ activation plays a role in the pathogenesis of cell injury by oxidants or ischemia in various cell types^15,18–20), it was examined whether if t-BHP-induced lipid peroxidation and LDH release could be protected by PLA₂ inhibitors. The results are depicted in Fig. 9. Mepacrine and butacaine at 0.25mM concentration exerted a significant protective effect against both t-BHP-induced lipid peroxidation and LDH release. The treatment of liver slices with PLA₂ inhibitors in the absence of t-BHP did not induce liver cell toxicity (data not shown).

Although there is an increasing recognition of the importance of oxygen free radicals in cell injury, the exact mechanisms or sequence of events by which cells sustain such injury are not clearly defined. Although lipid peroxidation has been considered to be an important mediator of certain deleterious effects of oxygen free radicals in cells, it is not clear whether the cell injury with acute oxidative stress resulted totally from lipid peroxidation. In liver cells, Masaki et al. proposed that the lipid peroxidation plays a critical role in t-BHP-induced cell injury⁶⁾. However, Rush et al. observed that the antioxidant completely blocked the formation of MDA in hepatocytes exposed to t-BHP but had no effect on cell injury or the morphological changes, suggesting that lipid peroxidation does not play an important role in the toxicity of t-BHP³⁾. As shown in Fig. 10, lipid peroxidation could appear as a consequence of cell injury rather than a cause of cell injury.

In the present study, DPPD completely prevented t-BHP-induced lipid peroxidation, whereas t-BHP-induced LDH release was partially reduced by the same concentration of DPPD(Fig. 3). t-BHP-induced lipid peroxidation was not altered by the addition of an external Ca²⁺ chelator EGTA, but LDH release was significantly reduced(Fig. 8). These results may indicate that lipid peroxidation is not a primary mediator for t-BHP-induced cell injury in hepatocytes. This supports the reports of Farber et al. that oxidant-induced cell injury can develop in the absence of detectable lipid peroxidation²¹⁾.

Since the cytotoxicity by oxidants is associated with oxidation of the sulfhydryl group, the sulfhydryl reducing agents protect against oxidant-induced cell injury²¹⁾. GSH has been also known to provide a marked protection against oxidant-induced cell injury²²⁾. As expected, in the present study, DTT and GSH significantly decreased t-BHP-induced lipid peroxidation as well as LDH release (Fig. 5).

Although the in vivo and in vitro studies have reported that Ca²⁺ channel blockers attenuate the hepatocellular damage by various hepatotoxins^{13,14,23–25)}, it has not been known that Ca²⁺ chennal blockers are benefical on oxidant-induced liver cell injury. In the present study, verapamil, diltiazem and nifedipine exerted significant protective effect against t-BHP-induced lipid peroxidation and LDH release (Fig. 6). However, it is unclear that such effects are associated with reduction in the influx of extracellular Ca²⁺ and changes in intracellular Ca²⁺ concentration were not determined in the present study. Since nonspecific action of Ca²⁺ channel blockers have been suggested to involve membrane stabilizing effect^26,27), these agents could exert protective effect without inducing alterations in Ca²⁺ influx. Thus, the precise mechanisms of protective effect by Ca²⁺ channel blockers remain to be determined.

Although the oxidative stress has been reported to be associated with the mobilization of Ca²⁺ from intracellular stores^5,10), several studies have proposed that increased Ca²⁺ influx across the plasma membrane is essential for the pathogenesis of cell injury and death induced by various chemical agents (Schanne et al., 1979; Kane et al., 1980). In the present study, it was examined whether modulation of external Ca²⁺ affects t-BHP-induced liver cell injury. When control slices untreated with t-BHP were incubated in the Ca²⁺-free medium for 60min, LDH release significantly increased (Fig. 7). If oxidant-induced cell injury was not affected by Ca²⁺ depletion, cell injury would be increased by both t-BHP and Ca²⁺ depletion as compared with t-BHP alone. However, the present study indicated that t-BHP-induced lipid peroxidation and LDH release did not more increase in the Ca²⁺-free medium than those in the normal Ca²⁺ medium. When slices were treated with t-BHP in the presence of EGTA, LDH release but not lipid peroxidation induced by t-BHP significantly decreased (Fig. 8). These results indicate that the influx of external Ca²⁺ across the plasma membrane may play a role in oxidant-induced liver cell injury. The induction of cell injury by Ca²⁺ depletion was demonstrated in other previous studies²⁸⁾.

Several in vitro studies have also reported that oxidant-induced cell injury is prevented by PLA₂ inhibitors in liver cells^29,30). The present study showed that t-BHP-induced lipid peroxidation and LDH release also decreased by mepacrine and butacaine(Fig. 9). These results suggest that oxidant-induced toxicity of liver cells may be, at least in part, associated with PLA₂ activation.