Characterization of Chemically Induced Hepatotoxicity in Collagen Sandwiches of Rat Hepatocytes

It has been shown that hepatocytes cultured in a collagen sandwich configuration maintain cell viability, morphology, and drug metabolizing activities for several weeks. The purpose of this study was to characterize chemically induced general toxicity in this system by exposing hepatocytes to eight different hepatotoxic compounds. Cell function and viability was measured by analyzing the secretions of urea and albumin and the release of lactate dehydrogenase. Significant decreases in urea and albumin secretions were detected after treatments with 32 nM aflatoxin B(1) and 1 mM doses of cadmium and the alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS). However, no significant toxicity could be measured following exposures to 5 mM carbon tetrachloride, 1 mM N, N-dimethylformamide (DMF), 1 mM vinyl acetate, and 1 mM acetaminophen. Western blots of cell lysates showed that hepatocytes maintained CYP1A, 2B, 3A2 but gradually lost CYP2E1, which is the main metabolic enzyme for acetaminophen, carbon tetrachloride, and DMF. The metabolites of acetaminophen were identified using liquid chromatography and electrospray mass spectrometry. It was determined that the hepatocytes converted most of the acetaminophen to the glucuronide and sulfate metabolites and only formed a small amount of the glutathione adduct. This research shows that the collagen sandwich culture system can only be used selectively for detecting hepatotoxicity and for identifying major metabolites of xenobiotic compounds.