Spectral properties of substrate-cytochrome P-450 interaction and catalytic activity of xenobiotic metabolizing enzymes in isolated rainbow trout liver cells.
A method for the isolation of intact viable rainbow trout liver cells in high numbers is described. The technique involves perfusion of collagenase through the liver. A major part of the cytochrome P-450 in isolated liver cells was present in the oxidized non-substrate bound form. It was observed that 7-ethoxycoumarin was rapidly taken up by the liver cells and bound to cellular cytochrome P-450. The substrate binding spectrum for isolated trout liver cells was slightly modified compared with that obtained with trout liver microsomes. The microsomal affinity of 7-ethoxycoumarin, calculated as the apparent spectral dissociation constant (ks), was elevated 11-fold after fish were treated with beta-naphthoflavone, indicating a qualitative alteration in the nature of the constitutive cytochrome P-450. The metabolism of 7-ethoxycoumarin in isolated liver cells was found to be of a comparable rate to that obtained in liver microsomes. Pretreatment of fish with Clophen A50 or beta-naphthoflavone significantly increased the content of cytochrome P-450 and elevated the rate of 7-ethoxycoumarin deethylation in isolated liver cells. Furthermore, the rate of conjugation of 7-hydroxycoumarin was significantly elevated in liver cells isolated from beta-naphthoflavone treated fish when compared with the control rate. In isolated liver cells, 90% of the 7-hydroxycoumarin formed from deethylation of 7-ethoxycoumarin was further metabolized to conjugated products. However, in beta-naphthoflavone of Clophen A50 treated fish the fraction of conjugated metabolites was markedly decreased, indicating a changed balance between cytochrome P-450 dependent reactions and conjugation reactions in the cell.
D000818:Animals; D002470:Cell Survival; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D005260:Female; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D008297:Male; D013053:Spectrophotometry; D014337:Trout; D014468:Umbelliferones / Q000378:metabolism
D003374:Coumarins; D014468:Umbelliferones; C017299:7-ethoxycoumarin; C031477:7-hydroxycoumarin; D003577:Cytochrome P-450 Enzyme System