Investigating the application of a nitroreductase-expressing transgenic zebrafish line for high-throughput toxicity testing

Abstract

Nitroreductase enzymes are responsible for the reduction of nitro functional groups to amino functional groups, and are found in a range of animal models, zebrafish (Danio rerio) excluded. Transgenic zebrafish models have been developed for tissue-specific cell ablation, which use nitroreductase to ablate specific tissues or cell types following exposure to the non-toxic pro-drug metronidazole (MTZ). When metabolized by nitroreductase, MTZ produces a potent cytotoxin, which specifically ablates the tissue in which metabolism occurs. Uses, beyond tissue-specific cell ablation, are possible for the hepatocyte-specific Tg(l-fabp:CFP-NTR)^s891 zebrafish line, including investigations of the role of nitroreductase in the toxicity of nitrated compounds. The hepatic ablation characteristics of this transgenic line were explored, in order to expand its potential uses. Embryos were exposed at 48, 72, or 96 hours post fertilization (hpf) to a range of MTZ concentrations, and the ablation profiles were compared. Ablation occurred at a 10-fold lower concentration than previously reported. Embryos were exposed to a selection of other compounds, with and without MTZ, in order to investigate alternative uses for this transgenic line. Test compounds were selected based on: their ability to undergo nitroreduction, known importance of hepatic metabolism to toxicity, and known pharmaceutical hepatotoxins. Selected compounds included nitrated polycyclic aromatic hydrocarbons (nitro-PAHs), the PAHs retene and benzo[a]pyrene, and the pharmaceuticals acetaminophen and flutamide. The results suggest a range of potential roles of the liver in the toxicity of these compounds, and highlight the additional uses of this transgenic model in toxicity testing.

Keywords: nitrated polycyclic aromatic hydrocarbon; nitroreductase; pharmaceuticals; tissue ablation; transgenic; zebrafish.

Fig. 1

Comparison of incrossed (homozygous positive) and outcrossed (heterozygous) Tg(l-fabp:CFP-NTR)^s891 zebrafish. Embryos were exposed to either DMSO or 10 mM metronidazole (MTZ) at 48, 72, or 96 hpf until imaging at 120 hpf. Presence of hepatocytes is indicted by green fluorescence.

Fig. 2

Dose-response of Tg(l-fabp:CFP-NTR)^s891 zebrafish embryos exposed to metronidazole (MTZ) at 96 hpf, and imaged at 120 hpf, at a broad (a) and refined (b) range of MTZ concentrations. Ablation of hepatocytes was assessed based on visible green fluorescent signal, where a decreasing fluorescent signal indicates hepatocyte ablation.

Fig. 3

Time course of tissue ablation (a) and regeneration (b) using 10 mM metronidazole (MTZ) in the hepatocytes of Tg(l-fabp:CFP-NTR)^s891 zebrafish. For the ablation study, embryos were dosed at 96 hpf with 10 mM MTZ, and ablation was evaluated by visible fluorescence, following continuous exposure until the indicated time point. For the regeneration study, embryos were exposed to 10 mM MTZ from 96 to 120 hpf, then rinsed and moved to clean media, with imaging at the indicated time points after MTZ removal. The presence of hepatocytes is indicated by green fluorescence.

Fig. 4

Windows of exposure in wild-type and Tg(l-fabp:CFP-NTR)^s891 embryos exposed to 1-nitropyrene (a), 1-aminopyrene (b), and 9-nitrophenanthrene (c). Embryos were exposed at 6 hpf with evaluations at 120 hpf. Bar height indicates incidence of each individual endpoint, where red dots indicate statistical significance. Toxicity profiles for exposures at other time points are shown in SI 2.

Fig. 5

Co-exposures with 10 mM metronidazole (MTZ) in Tg(l-fabp:CFP-NTR)^s891 and wild-type zebrafish embryos. Structures of all compounds tested are shown in (a). Embryos were exposed to MTZ at 48 hpf, and to 1% DMSO (1.5% DMSO-exposed animals were phenotypically indistinguishable) (b), retene (c), benzo[a]pyrene (B[a]P) (d), acetaminophen (APAP) (e), or flutamide (f) at 72 hpf. Additionally, wild-type embryos were exposed to flutamide (g). Imaging and evaluations were done for all animals at 120 hpf. Concentrations tested but not shown resulted in 100% mortality both in the presence and absence of MTZ.