ROLE OF REACTIVE OXYGEN METABOLITES IN DNA-DAMAGE AND CELL-DEATH IN CHEMICAL HYPOXIC INJURY TO LLC-PK1 CELLS

Hypoxia is considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, generall y considered a feature of apoptosis, to be almost entirely responsible for DNA damage in hypoxic injury to renal tubular epithelial cells. T he role of reactive oxygen metabolites in endonuclease-induced DNA dam age and cell death in chemical hypoxic injury has not been previously examined. LLC-PK-(1) cells exposed to chemical hypoxia with antimycin A resulted in enhanced generation of intracellular reactive oxygen spe cies as measured by oxidation of a sensitive fluorescent probe, 2',7'- dichlorofluorescin diacetate. Superoxide dismutase, a scavenger of sup eroxide radical, significantly reduced the fluorescence induced by ant imycin A and provided significant protection against chemical hypoxia- induced DNA strand breaks (as measured by the alkaline unwinding assay ). Pyruvate, a scavenger of hydrogen peroxide, provided significant pr otection against chemical hypoxia-induced DNA strand breaks and DNA fr agmentation (as measured by agarose gel electrophoresis). The interact ion between superoxide anion and hydrogen peroxide in the presence of a metal catalyst leads to generation of other oxidant species such as hydroxyl radical. Hydroxyl radical scavengers, dimethylthiourea, salic ylate, and sodium benzoate, and two metal chelators, deferoxamine and 1,10-phenanthroline, also provided marked protection against DNA stran d breaks and DNA fragmentation. These scavengers of reactive oxygen me tabolites and metal chelators provided significant protection against cell death as measured by trypan blue exclusion and lactate dehydrogen ase release. Taken together, these data indicate that reactive oxygen species play an important role in the endonuclease activation and cons equent DNA damage, as well as cell death in chemical hypoxic injury to renal tubular epithelial cells.