TNF-a and sphingolipid enhanced mitochondrial gene transcript levels and Superoxide production

TNF-a and its potential second messeners, the sphingoüpids coramide and sphingosiiie, can cause cytotoxicity and apoptosis, possibly through their effects on mitochondrial functions. The purpose of this study was to investigate the effects of these agents on the expression of selected mitochondrial respiratory chain protein mKNAs and Superoxide production in HepG2 cells and cul tured rat hepatocytes. In both these cell types, ceramide and sphingosine suppressed cell growth. Similar to TNF-a, they caused a concentration-dependent increase in mRNA levels of nuclear genome-encoded ATPase subunit E and mitorhrondria genome-encoded NADH dehydrogenase subunit 1. cytochrome b. cytochrome c oxidase subunits I, II and III, ATPase subunit 6, mitochromlrial transcript precursor and 16S rRNA. To determine whether these sphingolipid affected mitochondrial function, isolated rat liver mitochondria were treated with ceramide or sphingosine, and Superoxide production was measured us ing lucigenin-derived chemiluminescence. At 30 M, both increased Superoxide generation up to 50% over control. Treatment of both cell types with these sphingoüpids caused a concentration-/time-dependent increase in Superoxide production to levels up to 4-fold greater than controls, a pattern similar to thai seen for increases in levels of the mitochondrial gene transcripts. Additional studies are under way to examine the possible invoivment of these processes in sphingolipid-mediated apoptosis. Supported by grants R01 (,'A 36701 M.D.Y.). and R01 GM 46897 (K.T.M.); J.Q.C. is supported by T32 KS 07141.