Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture

Abstract

The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout ( Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin- O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in concentrations ranging from 10 −6 to 10 −12 M. After 24 h, TCDD showed a maximal induction at a concentration of 10 −9 M while BNF showed a maximal induction at a concentration of 10 −7 M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 μM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6β and 16β and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.