Hepatotoxicity studies with primary cultures of rat liver cells

Summary A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and norethindrone. Caffeine was evaluated as an agent thought to be relatively nontoxic to liver. Cytotoxicity was evaluated by phase-contrast microscopy of cellular morphology and by measurement of leakage of intracellular enzymes arginosuccinate lyase (ASAL), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and acid phosphatase (AP)] into the culture medium. Hepatic cultures were treated with each of the agents in concentrations ranging from 5×10⁻⁶ to 1×10⁻³ m and for durations from 1 to 24 hr. ASAL was found to be the most sensitive in predicting early cell injury and AP the least sensitive; the other three enzymes tested were intermittent in value and equally sensitive in evaluating cytotoxicity. Treatment of the cultures with tetracycline (5×10⁻⁴ m) for 6 hr resulted in ASAL leakage that was 400% of control values; and norethindrone (5×10⁻⁴ m) for 6 hr caused a 250% increase relative to controls. The hepatotoxic agents demonstrated a dose- and timedependence of cytotoxicity in the cultures. In contrast, caffeine was relatively nontoxic to the cultures. Part of this investigation was presented orally at the 17th Annual Meeting of the Society of Toxicology, San Francisco, March 13, 1978.