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 C-14CoS/C-14CoS m ouse (14CoS/14CoS cells) have 3-fold higher levels of reduced glutathi one (GSH) and greater resistance to menadione toxicity than hepatocyte s derived from the wild-type C-ch/C-ch mouse (ch/ch cells). Therefore, we used these cell lines to examine mechanisms of oxidative stress pr oduced by iron and tert-butyl hydroperoxide (TBHP). Both cell types we re resistant to 25 mu M Fe2+ toxicity in the absence of added TBHP. Ho wever, in the presence of Fe2+, striking differences in susceptibility to TBHP toxicity between the cell types were observed. With 25 mu M F e2+, ch/ch cells showed TBHP concentration-dependent toxicity, with to tal lethality at 500 mu M; in contrast, 14CoS/ 14CoS cells were comple tely resistant to the lethal effects of this concentration of TBHP. Co ncentration-dependent TBHP-mediated increases in cytosolic Ca2+, pH, a nd GSSG/GSH ratios, and decreases in GSH levels, were evident in ch/ch cells. 14CoS/14CoS cells exhibited concentration-dependent TBHP-media ted changes in GSH and GSSG/GSH ratios, but cytosolic Ca2+ and pH rema ined at control levels. Mitochondrial GSH pools were also diminished b y TBHP, although there was no selective depletion; mitochondrial GSH r emained 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. Howev er, only ch/ch, cells displayed a time-dependent depletion of mitochon drial protein thiols, concomitant with an increase in mitochondrial pr otein carbonyls, while 14CoS/14CoS cells were resistant to such change s. All of the effects produced by TBHP were prevented by desferoxamine . These results suggest that 14CoS/14CoS and ch/ch cells both exhibit an iron-dependent response to TBHP, producing an oxidant stress and pl asma membrane damage. However, ch/ch cells, but not 14CoS/14CoS cells, display elevated levels of cytosolic Ca2+ and pH that precede mitocho ndrial protein oxidation and subsequent cell death.