The use of primary cultured rat hepatocytes for the assessment of xenobiotic effects on biotransformation

By Schepers, G. - Aschmann, C. - Morchel, S.

An in vitro test protocol is reported, which, using primary cultured rat hepatocytes, allows for the screening of xenobiotic effects on biotransformation as well as on basal cellular functions. O-Deethylation of 7-ethoxycoumarin (7-EC) and subsequent conjugation of the metabolite 7-hydroxycoumarin (7-HC) with sulphate or glucuronic acid are determined, as representative parameters for the hepatic biotransformation. Cell viability is examined by measuring cellular ATP content and leakage of lactate dehydrogenase. With respect to immediate and delayed effects on biotransformation reactions, the standard test protocol includes exposure to xenobiotics for 1, 24 and 48 hours. Different response patterns could be demonstrated for the solvents dimethylformamide (DMF) and dimethylsulphoxide (DMSO), and the chlorinated phenols, pentachlorophenol (PCP) and hexachlorophene (HCP), which are known to uncouple mitochondrial respiration. Short-term incubation with the solvents resulted in decreased 7-EC-O-deethylation without signs of cytotoxicity. PCP and HCP inhibited 7-EC-O-deethylation and 7-HC-conjugation, affecting sulphate and glucuronide formation differently. 24-hour exposures to PCP and HCP resulted in decreased 7-ethoxycoumarin-O-deethylase activity, which correlated with diminished cell viability, while DMSO and DMF enhanced 7-EC-O-deethylation at sub-cytotoxic concentrations. After exposure for 48 hours to the solvents, enzyme induction was even more pronounced.