The effect of non-genotoxic carcinogens, phenobarbital and clofibrate, on the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis

Phenobarbital and clofibrate, two non-genotoxic carcinogens, have beeninvestigated regarding the relationship between reactive oxygen species,antioxidant enzyme expression and apoptosis in primary cultures of rathepatocytes. Low toxicity concentrations, 200 and 100 microg/ml forphenobarbital and clofibrate respectively, were used to examine theireffect on spontaneous or transforming growth factor beta1(TGFbeta1)-induced apoptosis and on the expression of antioxidant defenceenzymes (superoxide dismutases and catalase). The increased incidence ofapoptotic nuclei was visualized in TGFbeta1-treated cultures with thefluorescent dye Hoechst 33258 and was quantified under all experimentalconditions by measurement of the hypodiploid peak in DNA histogramsobtained by flow cytometry. Both substances, when added separately tohepatocyte cultures and incubated for 24 and 48 h, significantly diminishedspontaneous apoptosis and exhibited a slight suppression ofTGFbeta1-induced apoptosis. Endogenous peroxide production by hepatocytesincreased with TGFbeta1, phenobarbital or clofibrate and the increase wasgreater with phenobarbital and in the presence of TGFbeta1 with both drugs.Gene expression of catalase and Mn- and Cu,Zn superoxide dismutases (SOD)was evaluated by northern blot analysis of hepatocytes incubated in thepresence of phenobarbital or clofibrate with or without TGFbeta1 and thefollowing differences were detected: phenobarbital induced a significantdecrease in both dismutases (to 56%, P < 0.05, and 55%, P < 0.05, forMn- and Cu,Zn-SOD respectively) and a 2-fold increase (P < 0.01) incatalase; clofibrate induced a slight decrease in both SODs and a 4-foldincrease (P < 0.05) in catalase; TGFbeta1 significantly decreased to 37%(P < 0.05) expression of catalase while not significantly affectingexpression of both SODs. We conclude that inhibition of spontaneousapoptosis induced by either phenobarbital or clofibrate is accompanied byincreases in the endogenous levels of peroxides and by significantinduction of catalase gene expression. Furthermore, the lack of effect ofboth compounds on TGFbeta1-induced apoptosis could be a consequence of theinability of these two compounds to counteract the depressing effect ofTGFbeta1 on expression of catalase.