The zebrafish gill model: induction of CYP1A, EROD and PAH adduct formation

Zebrafish (Danio rerio) is a common model species in fish toxicology, and the zebrafish gill is potentially useful in screening waterborne pollutants. We have previously developed a gill-based ethoxyresorufin-O-deethylase (EROD) assay, and proposed gill EROD activity as a biomarker for exposure to waterborne aryl hydrocarbon receptor (AHR) agonists. In this study we modified the gill EROD assay for use in zebrafish. We used immunohistochemistry to localize CYP1A induction, and microautoradiography to localize irreversible binding of the prototypic polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA) in zebrafish gills. Gill filament and liver microsomal EROD activities were measured after waterborne exposure of zebrafish and rainbow trout to benzo[a]pyrene (BaP) or beta-naphthoflavone (betaNF). The results showed considerably lower relative EROD induction by betaNF (1microM) in zebrafish than in rainbow trout, both in gills (13-fold versus 230-fold compared to control) and in liver (5-fold versus 320-fold compared to control). The induced hepatic EROD activity was similar in the two species, whereas the basal activity was considerably higher in zebrafish than in rainbow trout. In zebrafish gills, betaNF enhanced DMBA adduct formation and CYP1A immunostaining. Ellipticine blocked DMBA adduct formation and EROD activity following betaNF exposure but had no effect on CYP1A immunostaining. A notable finding was that the localization of DMBA adducts differed from that of CYP1A protein in betaNF-induced fish; CYP1A immunoreactivity was evenly distributed in the gills whereas DMBA adduction was confined to the leading edges of the filaments and the gill rakers, i.e., structures being highly exposed to DMBA-containing inhaled water. The results show that the modified method is suitable for determination of gill EROD activity in zebrafish, although rainbow trout seems more sensitive. They also imply that the sites of DMBA adduct formation in zebrafish gills are markedly influenced by kinetic factors.