Metallothionein and zinc as potential antioxidants in radical-induced lipid peroxidation in cultured hepatocytes.

Rat hepatocytes were isolated by a two-step collagenase perfusion technique and introduced to the hydroxyl radical (OH)-generating xanthine-xanthine oxidase-iron (X/XO/Fe) system. The amount of thiobarbituric acid reactive substances (TBA) and thiobarbituric acid bound malondialdehyde (TBA-MDA) were assayed in homogenates after different phases of cultivation. The effects on lipid peroxidation of supplemented metallothionein (MT) ranging from 25 to 75 microM and zinc ranging from 14.5 to 77.8 microM, as well as the effect of a Zn-pretreatment for 18 h were investigated. The addition of X/XO/Fe resulted in a 3 to 4-fold increase in the levels of TBA and TBA-MDA. These results show that X/XO/Fe initiated the lipid peroxidation in the hepatocyte cell system. High doses of supplemented MT inhibited the production of TBA and TBA-MDA. Neither Zn nor the Zn-pretreatment, which resulted in an increase of intracellular MT, had any effect on TBA and TBA-MDA levels. This study suggests that MT can act as an antioxidant in high concentrations via the cysteinyl groups of the protein. The postulated protective effects of Zn via its release from the oxidized MT can be ruled out.