Microassay for rapid measurement of 7-ethoxyresorufin-O-deethylase activity in intact fish hepatocytes

被引:39
作者
Behrens, A
Schirmer, K
Bols, NC
Segner, H
机构
[1] UFZ Helmholtz Ctr Environm Res, Dept Chem Ecotoxicol, D-04318 Leipzig, Germany
[2] Univ Waterloo, Dept Biol, Waterloo, ON N2L 3G1, Canada
关键词
D O I
10.1016/S0141-1136(97)00039-1
中图分类号
X [环境科学、安全科学];
学科分类号
08 ; 0830 ;
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. Ir? 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, 102-112.) but 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. (C) 1998 Elsevier Science Ltd. All rights reserved.
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页码:369 / 373
页数:5
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