Disassociation of oxidant-induced ATP depletion and DNA damage from early cytotoxicity in LLC-PK1 cells.

To determine the mechanism(s) of oxidant-mediated cell lysis in renal tubular epithelial cells, we determined ATP depletion, DNA damage, lipid peroxidation, and cytotoxicity in LLC-PK1 cells exposed to 500 microM hydrogen peroxide for 1 h with and without inhibitors of lipid peroxidation including a lazaroid compound, 2-methylaminochroman (2-MAC), and Trolox, a vitamin E analog. ATP levels were determined by luciferin-luciferase, DNA damage by the alkaline unwinding technique, lipid peroxidation by the generation of malondialdehyde, and early cytotoxicity (5 h) by the release of 51Cr, whereas late cytotoxicity (24 h) was determined by release of [3H]leucine from prelabeled cells. Cells exposed to 500 microM hydrogen peroxide demonstrated significant (P < 0.01) ATP depletion, DNA damage, and lipid peroxidation, followed by cell death at 5 h. Concentrations of 0.1-25 microM 2-MAC or 25-500 microM Trolox each markedly and significantly (P < 0.01) inhibited lipid peroxidation and early cytotoxicity but had little to no effect on ATP depletion or DNA damage. Thus oxidant-stressed cells remained intact for several hours despite significant ATP depletion and DNA damage when lipid peroxidation was inhibited with the antioxidant compounds. At 24 h, 2-MAG and Trolox had lost their protective effect, suggesting that mechanisms other than lipid peroxidation play a role in later cytotoxicity. We conclude that ATP depletion and DNA damage are not the primary mediators of early cytotoxicity following oxidant stress, whereas lipid peroxidation plays an central role in mediating early cytotoxicity following oxidant injury.