Effect of copper chloride in vitro and in vivo on the hepatic erod activity in the fishDicentrarchus labrax

Abstract

The effect of copper chloride was studied on the hepatic microsomal 7-ethoxyresorufin-O-deethylase (EROD) activity of the fish Dicentrarchus labrax intraperitoneally injected with benzo[a]pyrene (BaP). In vitro experiments showed that copper significantly decreased EROD activity, and IC50 was estimated at 50 μg Cu/L. The apparent Michaelis constant (Km) of cytochrome P4501A was constant, whereas maximum velocity (Vmax) decreased as a function of copper added to the incubation medium. (Km) of nicotinamide adenine dinucleotide phosphate [NADPH]-cytochrome P450 reductase increased as a function of copper concentration, whereas Vmax remained constant. Absorption spectra showed that the amount of cytochrome P420s increased as a function of copper concentrations added to the medium at the expense of cytochrome P450s. The injection of copper and BaP (in vivo experiments) to fish decreased EROD activity compared with the injection of BaP alone. An increase of immunoquantified CYP1A content measured by Western blotting (intense band of 55–60 kDa molecular weight) was noted in microsomes of fish injected with BaP compared with controls. In the case of fish treated with copper and BaP, the band was less intense and accompanied by another band of lower molecular weight. The destruction of the native P450s spectrophotometrically measured in the presence of copper implied that the catalytic activity would be diminished. This was confirmed by decreased EROD activity after either in vitro additions or in vivo treatment with copper. Moreover, immunodetection experiments suggested that the decrease of the catalytic activity resulted more from cytochrome P450s loss than from direct inhibition of EROD activity by copper.