In vitro CYP1A activity in the zebrafish: temporal but low metabolite levels during organogenesis and lack of gender differences in the adult stage

Abstract

The zebrafish (Danio rerio) is increasingly used as a screening model for acute, chronic and developmental toxicity. More specifically, the embryo is currently investigated as a replacement of in vivo developmental toxicity studies, although its biotransformation capacity remains a point of debate. As the cytochrome P450 1 (CYP1) family plays an important role in the biotransformation of several pollutants and drugs, a quantitative in vitro protocol was refined to assess gender- and age-related CYP1A activity in the zebrafish using the ethoxyresorufin-o-deethylase (EROD) assay. Microsomal protein fractions were prepared from livers of adult males and females, ovaries and whole embryo homogenates of different developmental stages. A large biological variation but no gender-related difference in CYP1A activity was observed in adult zebrafish. Embryos showed distinct temporal but low CYP1A activity during organogenesis. These in vitro data raise questions on the bioactivation capacity of zebrafish embryos in developmental toxicity studies.

In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development

“…Similar to our findings, these authors thus suggested a difference in function or expression of drug-metabolizing enzymes between larval and adult zebrafish [ 21 ]. Finally, in contrast to the in vitro CYP1A activity that has been reported for the whole zebrafish embryo as early as at five hpf [ 30 ] and at eight hpf [ 29 ], BOMR was not metabolized by these earliest embryonic stages in our study. The presence of CYP activity in the early zebrafish embryo before its basic body plan has been established, is maternally derived [ 17 ] and disappears quickly.…”

“…During measurement, the temperature was kept at 28 °C which is within the zebrafish’s optimal water temperature range of 26–28.5 °C [ 5 ]. The same temperature was utilized for the controls as, in a previous literature report [ 30 ], similar CYP activities could be detected for HLM at 28.5 °C and 37 °C, respectively. The concentration of resorufin (nM)—a metabolite of BOMR—produced at each time point was determined from a standard curve that had been established by using the pure fluorescent metabolite (Vivid ® Red Fluorescent Standard, P2874, Thermo Fisher Scientific).…”

“…Our data are also in accordance with earlier in vitro and in vivo studies on CYP1A activity. Indeed, in vitro assessment of basal CYP1A activity in zebrafish embryos and larvae using 7-ethoxyresorufin- O -deethylase (EROD) also demonstrated low levels of metabolite formation around 72 and 96 hpf, with even lower levels around 120 hpf [ 29 , 30 ]. Even after CYP induction, activity levels remained low and a similar temporal trend was observed for basal versus induced CYP1 activity [ 20 , 29 , 31 ].…”

“…Only one sex was used for liver collection as hepatic CYP1A activity was shown to be independent of gender in zebrafish [ 30 ]. Six batches of adult female zebrafish between six months and one year of age were utilized for the preparation of zebrafish liver microsomes.…”

“…Besides the identification of CYPs in adult zebrafish, Goldstone et al also demonstrated distinct temporal patterns of CYP expression over the course of zebrafish development [ 17 ]. In addition to the research of Goldstone et al, other in vitro and in vivo studies have already been performed on the expression and activity of CYP1 and, to a lesser extent, CYP3 enzymes in adult and developing zebrafish [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. However, results from these studies are inconclusive and in some cases even contradictory.…”