A Microtiterplate-Based Screening Assay to Assess Diverse Effects on Cytochrome P450 Enzyme Activities in Primary Rat Hepatocytes by Various Compounds

Abstract

During the development of potential drugs it is useful to identify pharmacological and/or toxicological side effects of a compound as early as possible in order to exclude them from further development for reasons of time and cost. Activation or inactivation of members of the cytochrome P450-dependent monooxygenase system (CYP450) might indicate potential undesired effects of a given compound. However, results using CYP450 assay systems are often inconsistent because of different experimental settings. Therefore, it was the goal of the present study to optimize the CYP450 assay in primary rat hepatocytes with respect to the time point of addition of and duration of exposure to alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF) as well as trans-resveratrol (RES), which have well-described stimulatory and inhibitory effects on CYP450 enzymes of the 1A and 2B family, respectively. Hepatocytes were also treated with putative lipoxygenase (LOX)/cyclooxygenase (COX) inhibitors with unknown impact on CYP450 enzyme activity in order to detect potential side effects. Cells were cultured for up to 7 days on 96-well microtiter plates, and enzyme activity was determined by a conventional fluorescence spectroscopy assay. ANF and BNF, given to the cells after 4 days of culture, stimulated CYP1A and 2B activities significantly in a concentration-dependent fashion after long-term exposure for at least 1 day. However, during short-term exposure for 1-6 h, CYP1A activity was inhibited, while CYP2B was increased weakly by ANF but not BNF. RES inhibited CYP1A activity during short- and long-term exposure without affecting CYP2B activity. From the results it was concluded that primary rat hepatocytes should be cultured for at least 3-4 days but no longer prior to the assay. The assay should be performed at two different time points of exposure, i.e., 6 h for short-term and 24 h for long-term exposure. The compounds under investigation should be applied at two different concentrations, e.g., at one time and 10 times higher concentrations, which should be oriented to the ED50, provided it is known for the respective substance. Under these assay conditions the LOX/COX inhibitors tested activated CYP1A enzyme activity in long-term but instead inhibited it in short-term experiments. CYP2B activity was stimulated during short- and long-term exposure. These results indicated drug side effects recommending exclusion of the compounds from the drug developmental process. Hence, in order to assess the pharmacological potential of novel compounds it is adequate to perform both short- and long-term experiments to concisely describe the effect of a compound on the CYP450 system.