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Spectral properties of substrate cytochrome p 450 interaction and catalytic activity of xenobiotic metabolizing enzymes in isolated rainbow trout salmo gairdneri liver cells

, : Spectral properties of substrate cytochrome p 450 interaction and catalytic activity of xenobiotic metabolizing enzymes in isolated rainbow trout salmo gairdneri liver cells. Biochemical Pharmacology 34(9): 1407-1414

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. 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 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. 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. In .beta.-naphthoflavone or clophen A50 treated fish the fraction of conjugated metabolites was markedly decreased, indicating a changed balance between cytochrome P-450 dependent reactions and conjugation reactios in the cell.

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