ROLES FOR OXIDATIVE STRESS AND POLY(ADP-RIBOSYL)ATION IN THE KILLING OF CULTURED-HEPATOCYTES BY METHYL METHANESULFONATE

The mechanisms by which the methylating agent methyl methanesulfonate (MMS) kills cultured hepatocytes were studied. In an amino-acid-free K rebs-Ringer buffer (KRB), 1 mM MMS depleted the cells of glutathione ( GSH) within 1-2 hr. Lipid peroxidation, as measured by the accumulatio n of malondialdehyde (MDA), followed, and over 70% of the cells died w ithin 3-4 hr. The iron chelator deferoxamine and the antioxidant N,N'- diphenyl-1,4-phenylenediamine (DPPD) prevented lipid peroxidation and death of the hepatocytes without any effect on the loss of GSH. 3-Amin obenzamide (ABA), a poly(ADP-ribose) polymerase inhibitor, also preven ted the cell killing by attenuating the loss of GSH. Ir. a culture med ium containing amino acids and antioxidants (Williams' E medium, WEM), 1 mM MMS killed the hepatocytes more slowly, with 70% of the cells dy ing 8-12 hr after treatment. Lipid peroxidation accompanied the loss o f viability. Deferoxamine and DPPD inhibited lipid peroxidation, while only partially protecting against the cell killing. ABA offered more protection and reduced the decline of GSH and decreased lipid peroxida tion. ABA also reduced the extent of the depletion of both NAD and ATP that accompanied the cell killing by MMS in WEM. These data indicate that MMS killed the hepatocytes by different mechanisms depending on t he culture conditions. In KRB, the toxicity of MMS was a consequence o f oxidative cell injury that follows the depletion of GSH. In WEM, bot h oxidative injury and the action of poly(ADP-ribose) polymerase in re sponse to DNA single-strand breaks contributed to the loss of viabilit y.