A non-destructive BFCOD assay for in vivo measurement of cytochrome P450 3A (CYP3A) enzyme activity in fish embryos and larvae.

There is increasing interest in quantifying the exposure and effects of anthropogenic contaminants in fish. Determination of exposures in wild fish is routinely performed, but methods to investigate potential effects are less established. One of the most relevant approaches would be the use of in vivo assays, but existing assays are often limited to in vitro determination of enzyme activity. Many pharmaceuticals and some persistent pollutants activate, and are metabolized by cytochrome P4503A (CYP3A), which make it a relevant and desirable target for biomarker research. We altered the established 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylation (BFCOD) in vitro protocol for CYP3A activity determination, developing a rapid and inexpensive method to measure in vivo (and in ovo) CYP3A activity in two fish systems: Gulf killifish (Fundulus grandis) and zebrafish (Danio rerio) early life stages. Even with very low concentrations of 7-benzyloxy-4-trifluoromethyl coumarin (BFC, 0.06 µM or 20 µg/L), we were able to detect significant induction in CYP3A activity in embryos of F. grandis, as well as in larvae of D. rerio in response to benzo[a]pyrene (BaP) and fluoranthene (FL) exposures. Because of concerns regarding the possible contribution of CYP1A to BFCOD activity from previous research, we have used a CYP1A post-translational inhibitor (FL) in order to calculate the contribution of CYP1A to the BFCOD assay. We also dosed with benzo[k]fluoranthene (BkF) and showed significant induction of CYP1A activity, with no concurrent increase in CYP3A activity. In this paper, we have taken an established in vitro CYP3A activity assay, and utilized the reaction in a novel way to allow for the non-destructive determination of CYP3A. In summary, we describe a sensitive, cheap, fast and easy modified BFCOD assay for in ovo and in vivo determination of CYP3A activity for use in moderate throughput early-life-stage fish experiments.

Development of Betta splendens embryos and larvae reveals variation in pigmentation patterns.

Vertebrate pigmentation provides an ideal system for studying the intersections between evolution, genetics, and developmental biology. Teleost fish, with their accessible developmental stages and intense and diverse colours produced by chromatophores, are an ideal group for study. We set out to test whether Betta splendens is a good model organism for studying the evolution and development of diverse pigmentation. Our results demonstrate that B. splendens can be bred to produce large numbers of offspring with easily visualized pigment cells. Depending on the colour of the parents, there was variation in larval pigmentation patterns both within and between breeding events. In juveniles the developing adult pigmentation patterns showed even greater variation. These results suggest that B. splendens has great potential as a model organism for pigmentation studies.