The role of thiols in mitochondrial susceptibility to iron and tert-butyl hydroperoxide-mediated toxicity in cultured mouse hepatocytes.

Cultured hepatocytes derived from the newborn mutant c14CoS/c14CoS mouse (14CoS/14CoS cells) have 3-fold higher levels of reduced glutathione (GSH) and greater resistance to menadione toxicity than hepatocytes derived from the wild-type cch/cch mouse (ch/ch cells). Therefore, we used these cell lines to examine mechanisms of oxidative stress produced by iron and tert-butyl hydroperoxide (TBHP). Both cell types were resistant to 25 microM Fe2+ toxicity in the absence of added TBHP. However, in the presence of Fe2+, striking differences in susceptibility to TBHP toxicity between the cell types were observed. With 25 microM Fe2+, ch/ch cells showed TBHP concentration-dependent toxicity, with total lethality at 500 microM; in contrast, 14CoS/14CoS cells were completely resistant to the lethal effects of this concentration of TBHP. Concentration-dependent TBHP-mediated increases in cytosolic Ca2+, pH, and GSSG/GSH ratios, and decreases in GSH levels, were evident in ch/ch cells. 14CoS/14CoS cells exhibited concentration-dependent TBHP-mediated changes in GSH and GSSG/GSH ratios, but cytosolic Ca2+ and pH remained at control levels. Mitochondrial GSH pools were also diminished by TBHP, although there was no selective depletion; mitochondrial GSH remained at about 14% of total cellular GSH. Both cell types exhibited the same time-dependent decrease in plasma membrane protein thiols and a time-dependent increase in plasma membrane protein carbonyls. However, only ch/ch cells displayed a time-dependent depletion of mitochondrial protein thiols, concomitant with an increase in mitochondrial protein carbonyls, while 14CoS/14CoS cells were resistant to such changes. All of the effects produced by TBHP were prevented by desferoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)