CYP1A inhibition in fish gill filaments: A novel assay applied on pharmaceuticals and other chemicals

Abstract

The gill filament 7-ethoxyresorufin O-deethylase (EROD) assay was originally developed as a biomarker for cytochrome P4501A (CYP1A) induction by Ah-receptor agonists in water. In this study, the assay was adapted to measure inhibition of CYP1A activity in fish gill filaments ex vivo. The experiments were carried out using gill arch filaments from β-naphthoflavone (βNF)-exposed three-spined stickleback ( Gasterosteus aculeatus). Candidate CYP1A inhibitors were added to the assay buffer. Nine selected pharmaceuticals and five known or suspected CYP1A-modulating chemicals were examined with regard to their ability to reduce EROD activity in gill filaments. Ellipticine, a well characterized CYP1A inhibitor, was the most effective inhibitor of the compounds tested. At a concentration in the assay buffer of 1 μM the antifungal azoles ketoconazole, miconazole and bitertanol, and the plant flavonoid acacetin reduced gill EROD activity by more than 50%, implying IC50 values below 1 μM. These compounds have previously been shown to inhibit EROD activity in liver microsomes from fish and mammals at similar concentrations. The proton pump inhibitor omeprazole reduced the gill EROD activity by 39% at 10 μM. It is concluded that the modified gill filament EROD assay is useful to screen for waterborne pollutants that inhibit catalytic CYP1A activity in fish gills.