EFFECT OF CULTURE CONDITIONS PRIOR TO EXPOSURE ON CADMIUM CYTOTOXICITY IN PRIMARY RAT HEPATOCYTES

Abstract

Primary rat hepatocytes area useful research tool for direct investigation of hepatotoxic effects of chemicals or drugs at the cellular level. USEPA's reevaluation of current human health risk assessment models for thetoxicmetal cadmium has identified a need for the development of new models that include in vitro toxicity and kinetic data. The EC50 for cadmium obtained in rat hepatocyte cultures in 96-well plates (EC50 [96]=3.2 0.8muM) was found to be significantly different from that obtained in 6-well cultures (EC50 [6]=14.8+/-0.7muM). When medium depth in 96-well plates was adjusted to reflect the same depth as that in 6-well plates prior to, during, and following cadmium exposure, an EC50 was observed that was similar to that obtained in 6-well plates (EC50 [96*]=13.2+/-0.9muM). Hepatocytes incubated in 96-well plates with various depths of culture medium, from 1 mm (34muL/well)to 6 mm (200muL/well), exhibited a significant decrease in viability and attached cell protein with greater medium depths. Assessment of various biochemical markers of hepatic function indicated that hepatocytes cultured at 2-mm medium depths were similar to those in vivo. Metal analysis indicated that the amount of cadmium entering hepatocytes was independent of treatment. Thus, differences in toxicity were not a result of differences in cadmium uptake.This study indicated that in vitrotest systems should beoptimized and validated prior to use in mechanistic and kinetic studies, if they are to be considered by regulatory agencies.