Ethoxyresorufin O-deethylase activity in intact human cells

Abstract

The cytochrome P450IA ( P-450) family can be induced in both primary and continuous cell culture, as well as in lymphocytes, by polycyclic aromatic and halogenated hydrocarbons. Ethoxyresorufin O-deethylase (EROD) activity has been correlated with both cytochrome P450IA1 and arylhydrocarbon hydroxylase activity in rat hepatocytes. This activity can be assessed in intact cells in culture after induction with benzo[ a]anthracene by measuring the production of resorufin and its subsequent release to the medium. EROD activity was observed in intact cells of LS174T, a human colon tumour cell line, at a level (14 pmol/min/mg protein) comparable with that in microsomal preparations (24 pmol/min/mg) and lysed cells (13 pmol/min/mg). Furthermore, this cell line showed time- and concentration-dependent production of resorufin. α-Naphthoflavone (5 μ m), a P450IA inhibitor, reduced the resorufin production to 5% of control levels, but metyrapone (10 μ m) had no significant effect. Dicumarol (10 μ m), an inhibitor of quinone oxidoreductase, had only a small effect on the production of resorufin by the LS174T cell line (111% of control). Another human colon tumour cell line, DiFi, also showed benzo[ a]anthracene-inducible resorufin production. This intact cell assay has the advantage of allowing the use of cells for preliminary assays without their destruction, and may be of use in quickly screening large numbers of xenobiotics for the ability to induce P450IA1 in a human cell line.