Cytotoxicity of acetaminophen and papaverine in primary cultures of rat hepatocytes

Abstract

Primary cultures of hepatocytes obtained from postnatal Sprague-Dawley rats were grown in arginine-deficient medium to inhibit fibroblastic overgrowth and to selectively isolate relatively pure cultures of parenchymal hepatocytes. The cultures were grown on collagen-coated lens paper which freely floated in the medium and thus facilitated the exchange of nutrients and waste products between the cells and the culture medium. This system of primary hepatocytes was used to study the cytotoxicity of acetaminophen, papaverine, nitrofurantoin, and sodium salicylate. Cytotoxicity was monitored by measurement of leakage of intracellular enzymes into the culture medium: argininosuccinate lyase (ASAL), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and acid phosphatase (AP). Cultures were treated with each of the agents in concentrations ranging from 5 × 10 −6 to 5 × 10 −3 m and for durations from 1 to 24 hr. ASAL was found to be most sensitive in predicting early cell injury and AP the least sensitive. GPT, GOT, and LDH were intermittent in value and equally sensitive in evaluating cytotoxicity. No sign of cytotoxicity was observed within 3 hr of treating cultures with different concentrations of acetaminophen and papaverine. At 6 hr a significant increase in leakage of enzymes into the culture medium was observed relative to controls. After the first 3 hr the hepatotoxic agents demonstrated a time- and dose-dependence of cytotoxicity. Treatment of the cultures with acetaminophen and papaverine (5 × 10 −5 m) for 12 hr resulted in ASAL leakage that was 360 and 260% of control values, respectively. In contrast, nitrofurantoin and sodium salicylate were only minimally toxic to the cultured cells. The time lag observed in the cytotoxicity of acetaminophen and papaverine may be related to the period required for metabolite(s) accumulation and saturation of detoxifying systems of the cultured liver cells.