Dynamics of BNF-induced in vivo ethoxyresorufin-O-deethylase (EROD) activity during embryonic development of medaka (Oryzias latipes)

This study aimed to characterize quantitatively the temporal basal and induced ethoxyresorufin-O-deethylase (EROD) activity as indicator of cytochrome P4501A (CYP1A) function during embryonic development of medaka (Oryzias latipes). For this purpose, non-invasive methods over fluorescence images of the whole embryo (non-organ-specific [NOS] EROD activity) or specifically of the gallbladder (organ-specific [OS] EROD activity) were used. To induce this EROD activity, embryos were continuously exposed to β-naphthoflavone (BNF; 0.005, 0.05, 0.5, 5 μg/L). Analytical chemistry suggested no signs of BNF dissipation. Mean fluorescence intensity values for EROD induction increased with BNF concentration throughout embryonic development. Significant increments in the NOS activity were seen from exposures to ≥0.5 μg BNF/L as early as 2 days post-fertilization (dpf), and in the OS EROD activity as soon as the gallbladder was conspicuous (i.e. 4 dpf). Morphometric in vivo analysis of the gallbladder during embryonic development did not indicate significant dilation after BNF treatment suggesting normal hepatic bile formation. The conditions optimized in this study using intact embryos should allow the quantitation of EROD activity induced by specific chemicals, mixtures and environmental samples in terms of BNF-equivalents, offering a proper estimation of their potency. These results demonstrate the utility of medaka in a fish embryo test for a non-invasive CYP1A analysis expressed as EROD activity, fitting in the three R principles for the minimization of animal use in ecotoxicology evaluations and that are among the objectives of the European Community regulation for the Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH).

► Quantitation using non-invasive fluorescence imaging in whole embryo or gallbladder.
► Fluorescence values increased over development and with inducer at ≥0.5 μg BNF/L.
► Significant fluorescence induction in whole embryo from day 2 after fertilization.
► Significant induction in gallbladder seen as soon as it is discernible.