Biological effects of contaminants: Microplate method for measurement of ethoxyresorufin-O-deethylase (EROD) in fish.

Abstract

Interest in the use of mixed function oxidase (MFa) as a monitoring tool for measuring the effects of pollntants derives from basic research carried out over the past twenty years (see review in Payne et al., 1987). The MFa system catalyses the degradation of both endogenous and exogenous lipophilic substrates to polar water-soluble products which are more easily excreted. It is present at relatively low activity in wild fish and its activity increases dramatically, apparently to enhance the degradation and clearance of offending compounds. This suggests that the activity of the MFa system in naturally contaminated organisms might be a measure of the degree of chemical contamination. There have been a number of field studies in which elevated MFa activity in fish was found to be associated with contamination by hydrocarbons (payne et al., 1987). The MFa system requires molecular O 2 and NADPH and involves a co-binding protein: cytochrome P-450. In marine fish, two of the model reactions of the MFa-system, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-a-deethylase (BRaD) (see Figure I), have been studied most intensively (Spies et al., 1982; Payne et al., 1987; Ellenton et al., 1985; Luxon et al., 1987). Both AHH and ERaD, of which ERaD is the more specific, are catalyzed by the cytochrome P-450IA sub-family (Nebert and Gonzalez, 1987). In mammals, the cytochrome P-450IA sub-family contains two isozymes, namely, cytochrome P-450IA1 and cytochrome P-450IA2, but in fish only the former appears to be inducible by contaminants (Nebert and Gonzalez, 1987). The cytochrome P-450IA1 isozyme in fish can be induced by polycyclic aromatic hydrocarbons (P AHs), polychlorinateddibenzodioxins (PCDDs), and certain polychlorinated biphenyls (PCBs) that have a planar configuration. This has been reported many times in laboratory studies (Jimenez et al., 1988), as well as in mesocosms (Addison and Edwards, 1988; Stegeman et al., 1988) and field studies (Spies et al., 1982; Luxon et al., 1987; Addison and Edwards, 1988). This makes the measurement of ERaD activity a good means of evaluating fish response to PAH contamination. It has recently been shown that specific isoforms of this protein are involved in the metabolism of xenobiotics. Therefore, an increase in this specific isoform is good evidence of the induction of the MFa system by contaminants. Ethoxyresorufin-a-deethylase (ERaD) activity is a specific assay for the xenobiotically inducible form of cytochrome P-450, thus making measurement of this activity in fish liver a good means of evaluating fish response to PAH contamination. The method described here has been adapted from the techniques described by Burke and Mayer (1974) and Klotz and Stegeman (Klotz et al., 1985) for routine measurements of ERaD in flatfish. The method uses microplate technology and is suitable for use in the field on research vessels and in the laboratory.