Effect of Ca2+ channel blockers, external Ca2+ and phospholipase A2 inhibitors on t-butylhydroperoxide-induced lipid peroxidation and toxicity in rat liver slices

Abstract

Objectives: This study was undertaken to examine the effect of oxidant on lipid peroxidation and lethal cell injury in rat liver slices.

Methods: t-Butylhydroperoxide (t-BHP) was employed as a model of an oxidant. The lipid peroxidation and lethal cell injury were estimated by measuring the formation of malondialdehyde (MDA) and lactate dehydrogenase (LDH) release, respectively.

Results: t-BHP increased lipid peroxidation and LDH release in a dose-dependent manner over concentrations of 0.5-10 mM. t-BHP-induced lipid peroxidation was completely prevented by an antioxidant, N,N-diphenyl-p-phenylenediamine (DPPD), but LDH release was partially decreased. Both t-BHP-induced lipid peroxidation and LDH release were significantly protected by iron chelator, deferoxamine, sulfhydryl reducing agent, dithiothreitol and glutathione. Ca2+ channel blockers, verapamil, diltiazem and nifedipine exerted a significant protective effect against t-BHP-induced lipid peroxidation and LDH release. By contrast, addition of external Ca2+ chelator, ethylene glycol bis(b-aminoethyl ether)-N,N-tetraacetic acid (EGTA) did not alter t-BHP-induced lipid peroxidation, whereas t-BHP-induced lethal cell injury was significantly prevented. Phospholipase A2 (PLA2) inhibitors, mepacrine and butacaine produced a partial protective effect.

Conclusions: These results suggest that t-BHP induces cell injury by lipid peroxidation-dependent and -independent mechanisms which can be partially prevented by Ca2+ channel blockers and PLA2 inhibitors.

Fig. 1.

Effect of various concentrations of t-BHP on lipid peroxidation (A) and LDH release (B) in rat liver slices. Data are mean ± SE of four experiments. *p<0.05, **p<0.01 compared with the control in the absence of t-BHP.

Fig. 2.

Relationship between t-BHP-induced lipid peroxidation and LDH release. Data are obtained from Fig. 1.

Fig. 3.

Effect of DPPD on t-BHP-induced lipid peroxidation (A) and LDH release (B). Liver slices were treated with 1 mM t-BHP for 60 min at 37°C in the presence or absence of 20 mM DPPD. Data are mean ± SE of four experiments. *p<0.05, **p<0.01 compared with t-BHP alone.

Fig. 4.

Effect of iron chelator on t-BHP-induced lipid peroxidation (A) and LDH release (B). Liver slices were treated with 1 mM t-BHP for 60 min at 37°C in the presence or absence of 2 mM deferoxamine. Data are mean ± SE of four experiments. *p<0.05, **p<0.01 compared with t-BHP alone.

Fig. 5.

Effect of DTT and GSH on t-BHP-induced lipid peroxidation (A) and LDH release (B). Liver slices were treated with 1 mM t-BHP for 60 min at 37°C in the presence or absence of 2 mM DTT or GSH. Data are mean ± SE of four experiments. **p<0.01 compared with t-HP alone.

Fig. 6.

Effect of Ca²⁺ channel blockers on t-BHP-induced lipid peroxidation (A) and LDH release (B). Liver slices were treated with 1 mM t-BHP for 60 min at 37°C in the presence or absence of Ca²⁺ channel blockers (0.1 mM), verapamil(Ver), diltiazem (Dil) or nifedipine (Nif). Data are mean ± SE of five experiments. *p<0.05 compared with t-BHP alone.

Fig. 7.

Effect of external Ca²⁺ depletion on t-BHP-induced lipid peroxidation (A) and LDH release (B). Liver slices were treated with 1 mM t-BHP for 60 min at 37°C in the normal or Ca²⁺-free medium. Data are mean ± SE of four experiments. *p<0.05 compared with the control of normal Ca²⁺ concentration.

Fig. 8.

Effect of external Ca²⁺ chelator on t-BHP-induced lipid peroxidation (A) and LDH release (B). Liver slices were treated with 1 mM t-BHP for 60 min at 37°C in the presence or absence of 2 mM EGTA. Data are mean ± SE of four experiments. *p<0.05 compared with t-BHP alone.

Fig. 9.

Effect of PLA2 inhibitor on t-BHP-induced lipid peroxidation (A) and LDH release (B). Liver slices were treated with 1 mM t-BHP for 60 min at 37°C in the presence or absence of 0.25 mM mepacrine (Mepa) or butacaine (Buta). Data are mean ± SE of four experiments. *p<0.05 compared to t-BHP alone.