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, lip id peroxidation, and cytotoxicity in LLC-PK1 cells exposed to 500 mu M hydrogen peroxide for 1 h with and without inhibitors of lipid peroxi dation including a lazaroid compound, 2-methylaminochroman (2-MAC), an d Trolox, a vitamin E analog. ATP levels were determined by luciferin- luciferase, DNA damage by the alkaline unwinding technique, Lipid pero xidation by the generation of malondialdehyde, and early cytotoxicity (5 h) by the release of Cr-51, whereas late cytotoxicity (24 h) was de termined by release of [H-3]leucine from prelabeled cells. Cells expos ed to 500 eta M hydrogen peroxide demonstrated significant (P < 0.01) ATP depletion, DNA damage, and lipid peroxidation, followed by cell de ath at 5 h. Concentrations of 0.1-25 mu M 2-MAC or 25-500 mu M: Trolox each markedly and significantly (P < 0.01) inhibited lipid peroxidati on and early cytotoxicity but had little to no effect on ATP depletion or DNA damage. Thus oxidant-stressed cells remained intact for severa l hours despite significant ATP depletion and DNA damage when lipid pe roxidation was inhibited with the antioxidant compounds. At 24 h, 2-MA C and Trolox had lost their protective effect, suggesting that mechani sms other than lipid peroxidation play a role in later cytotoxicity. W e conclude that ATP depletion and DNA damage are not the primary media tors of early cytotoxicity following oxidant stress, whereas lipid per oxidation plays an central role in mediating early cytotoxicity follow ing oxidant injury.