7-Ethoxyresorufin O-deethylase induction in cultured gill epithelial cells from rainbow trout

Abstract

Induction of the cytochrome P4501A (CYP1A)-mediated enzyme activity 7-ethoxyresorufin O-deethylase (EROD) was measured in cultured respiratory epithelial cells from rainbow trout ( Oncorhynchus mykiss) gills. Monolayers of adherent cells were exposed to the inducers β-naphthoflavone (β-NF), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB#126) and benzo[ k]fluoranthene (B[k]F). EROD activity was either measured directly in the adherent cells after exposure to inducer, or the exposed adherent cells were dislodged by trypsination and EROD activity was measured in suspended cells. A time course study of the induction caused by β-NF showed that the EROD activity remained at a similar level from 24 to 72 h after addition of the compound. A second time course study was performed using all four substances. For β-NF, B[k]F and TCDD the induction was similar throughout the time period and for PCB#126 maximal induction was reached at 24 h, followed by a decrease. Concentration–response relationships for EROD induction were established for the two different methods by exposing the cells to 0.036, 0.36 and 3.6×10 −6 M β-NF. When measured in suspended cells, EROD activity increased at the two lowest concentrations. At the highest concentration no further increase was detected. The adherent cells showed a different concentration–response to the inducer, with an increase at the low concentration, a maximum at 0.36×10 −6 M, and a decrease at the highest concentration. The maximal EROD activity was about two times higher in adherent cells than in suspended cells. All further measurements were therefore performed on adherent intact cells. The potencies and efficacies of the different compounds to induce EROD were compared in a set of experiments using concentrations ranging from 10 −11 to 10 −6 M. In all cases, EROD activity increased with increasing concentrations up to a maximal level, and thereafter the activity decreased. TCDD caused maximal EROD activity at a concentration of 10 −9 M, and all other inducers caused maximal EROD activity at a concentration of 10 −7 M. The maximal EROD activities obtained with B[k]F, β-NF and PCB#126 were about 80, 80 and 40%, respectively, of the maximal activity obtained with TCDD. The relative potencies compared with TCDD were 0.008 for B[k]F, 0.0035 for PCB#126 and 0.002 for β-NF, based on EC 50 values determined from the concentration–response curves.