ROLE OF ENHANCED CERAMIDE GENERATION IN DNA-DAMAGE AND CELL-DEATH IN CHEMICAL HYPOXIC INJURY TO LLC-PK1 CELLS

Background. Ceramide has been implicated to be a second messenger in t he cell signaling pathway involved in cell growth, proliferation, and apoptotic cell death. However, there is little information of a role o f ceramide in DNA damage and cell death in hypoxic injury known to ind uce necrotic cell death. Methods. Ceramide generation was measured in LLC-PK1 cells exposed to chemical hypoxia with a mitochondrial electro n transport inhibitor, antimycin A and glucose deprivation. The effect of inhibition of ceramide generation on chemical hypoxia-induced DNA damage and cell death and the effect of exogenous ceramide on cellular injury were also determined. Results. Chemical hypoxia resulted in a rapid increase in ceramide production prior to any evidence of DNA dam age and cell death in LLC-PK1 cells. The inhibitor of ceramide synthas e, fumonisin B1, provided a marked protection against chemical hypoxia -induced DNA strand breaks, DNA fragmentation and cell death. Fumonisi n B1 did not affect adenosine triphosphate (ATP) depletion induced by antimycin A, suggesting that fumonisin B1 does not alter cellular upta ke of antimycin A. We confirmed the ability of ceramide synthase inhib itor, fumonisin B1, to suppress chemical hypoxia-induced ceramide gene ration. Exposure of LLC-PK1 cells to synthetic ceramide, C2- and C6-ce ramide, but not C2-dihydroceramide, the structural analog of C2-cerami de, resulted in DNA strand breaks, DNA fragmentation and cell death in a dose- and time-dependent manner similar to the effect of chemical h ypoxia. Conclusions. Our data indicate that ceramide is a key modulato r for DNA damage and cell death in chemical hypoxia to renal tubular e pithelial cells.