Microassay for rapid measurement of 7-ethoxyresorufin-O-deethylase activity in intact fish hepatocytes
摘要
In vitro assays using isolated fish cells are increasingly used to measure the induction of cytochrome P4501A (CYP1A) and its associated enzyme activity, 7-ethoxyresorufin-O-deethylase (EROD). Compared to permanent cell lines, primary hepatocyte cultures represent an in vitro system that is closer to the in vivo situation, and, therefore, may be a particularly suitable model to study physiological and xenobiotic regulation of CYP1A expression. The application of conventional EROD assays to primary hepatocyte cultures, however, suffers from some disadvantages such as the labour-intensity and the need for high cell numbers per sample. In this report, we present an approach for assessment of EROD activity in teleost hepatocytes cultured in 48-well plates without the need for preparation of microsomes. The assay is based on the method of (Kennedy, S. W. Lorenzen, A., James, C. A. and Collins, B. T. (1993) Anal. Biochem. 211, 1102-112.) buy employs live instead of dead cells. For EROD detection, 7-ethoxyresorufin is added directly to the live cells and its metabolism to resorufin is observed by repeated measurements over 20 min in a fluorescence plate reader. The assay does not require the addition of exogenous NADPH. Resorufin utilization through cytoplasmic DT-diaphorase has to be inhibited by addition of dicoumarol. Disturbance by conjugate formation accounts to not more than 20%of total resorufin formed. After measurement, the cells can be washed and, after continued incubation, can be used for further assays.