INDUCTION OF CYTOCHROME-P450 1A IN FISH TREATED WITH 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN OR CHEMICALLY CONTAMINATED SEDIMENT

Mirror carp were exposed to Rotterdam Harbor sediment, highly contaminated with polychlorodibenzo-p-dioxins (PCDDs), polychlorodibenzofurans (PCDFs), and polychlorobiphenyls (PCBs) (0.5 mug 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD] equivalents per kilogram dry weight). In two additional separate experiments rainbow trout (Oncorhynchus mykiss) and mirror carp (Cyprinus carpio) received a single intraperitoneal injection of approximately 0.01, 0.03, 0.06, 0.3, 0.6, or 3.0 mug TCDD per kilogram body weight. In all three experiments induction of hepatic P450 IA was measured with immunochemical and enzymatic methods. The polyclonal antibodies anti-cod (Gadus morhua), anti-perch (Perca fluviatilis), and anti-rainbow trout P450 1A all cross-reacted with the P450 1A orthologue of the carp and rainbow trout. In most cases high correlations were found between 7-ethoxyresorufin O-deethylation (EROD) activity and cytochrome P450 1A protein contents, the latter measured by the enzyme-linked immunosorbent assay (ELISA) and protein blot methods. However, the correlations between EROD activity and P450 1A protein levels were higher within the separate sampling periods (i.e., 3, 6, and 12 weeks after dosage) than with the total data set, especially in the dose-effect study with the rainbow trout. This was probably caused by a difference in time-dependent relationships between P450 1A protein content and EROD enzyme activity: 12 weeks after dosage the P450 1A protein was still increased, although EROD activity had returned to background level. In addition, there were higher correlations of the EROD activity and P450 1A protein content with total P450 content in rainbow trout and carp treated with a single dose of TCDD, than with total P450 content in carp exposed to contaminated sediment. In our study, the ELISA method appeared to be more useful than the protein blot technique, because the ELISA is faster and has higher reproducibility. In addition, in all our experiments EROD activity showed a higher induction than the P450 IA protein, indicating a higher sensitivity of the EROD assay. Our results strongly indicated that determination of the P450 1A protein content and EROD activity provides complementary information. Thus we recommended the use of both the ELISA and the EROD activity assay in order to understand the nature of P450 1A induction,