Sex Differences in Rat Hepatic Cytolethality of the Protein Kinase C Inhibitor Safingol: Role of Biotransformation

Abstract

Safingol [(2S,3S)-2-amino-1,3-octadecanediol], a sphingosine analog that inhibits protein kinase C, was developed to treat dermatoses and cancer. Preclinical toxicology studies performed to assess the effects of safingol showed that 6 weeks of dermal application over 10% of body surface area caused dose-dependent increases in serum enzymes and hepatic histopathological changes associated with liver damage in female rats. Liver toxicity was not seen in male rats at the same doses. Plasma safingol concentrations were similar in male and female rats following topical exposure. The underlying mechanism(s) for the sex differences in toxicity in rats were examined using isolated hepatocytes. Anin vitromodel of male versus female differences in safingol·HCl-induced hepatotoxicity was established using a suspension/culture technique. Concentrations of safingol·HCl which produced cytolethality in 50% of the hepatocytes were 125 and 48 μMfor male and female rat hepatocytes, respectively. Cytolethality was time-, concentration-, and cell number-dependent. Inhibition of cytochrome P450in vitrowith 1-phenylimidazole increased safingol·HCl-induced cytolethality in male but not female hepatocytes, suggesting that male rat hepatocytes have a cytochrome P450 isoenzyme which metabolizes safingol·HCl to an inactive metabolite thus reducing hepatotoxicity. Furthermore,in vivopretreatment with the CYP4A-inducing agent, clofibrate, protected both male and female hepatocytes from cytolethality. The results of this study indicate that the sex differences seen in hepatotoxicity could be due to differences in biotransformation such that female rat hepatocytes either lack or have a reduced constitutive level of a cytochrome P450 isoenzyme that metabolizes safingol to a nontoxic metabolite. In addition, safingol [bnproduced hepatocyte cell death without inflammationin vivo,and a “ladder-like” DNA fragmentation patternin vitro,consistent with an apoptotic mechanism of cell death.