Effect of storage conditions and subcellular fractionation of fish liver on cytochrome p-450-dependent enzymatic activities used for the monitoring of water pollution

Abstract

Hepatic cytochrome P-450-dependent monooxygenase activities of fish, especially 7-ethylresorufin O-deethylase activity (EROD activity) have been proposed as possible indicators of water pollution. Nevertheless there is a need for comparisons between different methods of storage and of subcellular fractionation of the liver with regard to absolute activity and responsiveness of EROD activity to pollution. In this report the livers of nase ( Chondrostoma nasus) were collected in PCB-polluted and reference areas of the River Rhône. They were stored in liquid nitrogen either after slicing or after homogenization in phosphate buffer containing 20% glycerol; subcellular fractionations were performed at the laboratory. Results demonstrate the deleterious effect of freezing slices of liver on EROD activity and point out the protective effect of homogenizing in buffer containing 20% glycerol (a cryoprotector) prior freezing. Besides, we observed a 3-fold higher EROD activity in the microsomal fraction as compared to postmitochondrial fraction, demonstrating the interest of subcellular fractionation to increase the sensitivity of the assay. Finally, we did not notice any difference between methods of storage and of subcellular preparation with regard to the rate of induction of EROD activity between polluted and reference areas. We conclude that in most cases postmitochondrial fraction originating from liver homogenized in buffer containing 20% glycerol prior freezing in liquid nitrogen is a useful preparation to measure EROD activity as a pollution indicator.