Teratogenicity of Ochratoxin A and the Degradation Product, Ochratoxin α, in the Zebrafish (Danio rerio) Embryo Model of Vertebrate Development

Abstract

Ochratoxins, and particularly ochratoxin A (OTA), are toxic fungal-derived contaminants of food and other agricultural products. Growing evidence supports the degradation of OTA by chemical, enzymatic and/or microbial means as a potential approach to remove this mycotoxin from food products. In particular, hydrolysis of OTA to ochratoxin α (OTα) and phenylalanine is the presumptive product of degradation in most cases. In the current study, we employed the zebrafish (Danio rerio) embryo, as a model of vertebrate development to evaluate, the teratogenicity of OTA and OTα. These studies show that OTA is potently active in the zebrafish embryo toxicity assay (ZETA), and that toxicity is both concentration- and time-dependent with discernible and quantifiable developmental toxicity observed at nanomolar concentrations. On the other hand, OTα had no significant effect on embryo development at all concentrations tested supporting a decreased toxicity of this degradation product. Taken together, these results suggest that ZETA is a useful, and highly sensitive, tool for evaluating OTA toxicity, as well as its degradation products, toward development of effective detoxification strategies. Specifically, the results obtained with ZETA, in the present study, further demonstrate the toxicity of OTA, and support its degradation via hydrolysis to OTα as an effective means of detoxification.

Keywords: detoxification; mycotoxins; ochratoxin A; ochratoxin α; teratogenicity; zebrafish.

Figure 1

Hydrolysis of the amide bond in OTA (1) to yield OTα (2) and phenylalanine as considered the primary degradation/detoxification pathway for ochratoxins.

Figure 2

Chromatogram (HPLC-FL) of the hydrolysis of OTA to OTα. Shown are OTA standard (A), and subsequent hydrolysis product (B); for comparison, the chromatogram of the reference standard of OTα (C) as the presumptive hydrolysis product is shown. The identity of hydrolysis product was confirmed as OTα by LC-MS (see inset for pane (B)) based on the molecular ion (MH⁺ 255 m/z, Full Scan) and Selected Reaction Monitoring (SRM) mass transition (255 > 211 m/z). Apparent degradation to OTα was also observed in the OTA standard (see (A)).

Figure 3

Dose and time dependence of OTA and OTα teratogenicity in ZETA. Shown are averages and standard deviations (n = 3) of the percent deformed embryos (5 per well/replicate) at a range of concentrations at 1 (A), 2 (B), 4 (C) and 5 (D) days post-fertilization. Activity is statistically compared to solvent (i.e., “MeOH”) only controls (n = 3); statistically significant differences are indicated at: (a) p < 0.05; (b) p < 0.01; and (c) p < 0.005. No significant difference was observed between MeOH-only and untreated (“E3” i.e., test medium alone) controls as shown.

Figure 4

Representative development dysfunction of zebrafish embryos exposed to OTA and OTα at 1 and 3 days post-fertilization. Shown are embryos exposed to 0.5 µM OTA (A) compared to solvent (i.e., MeOH) only controls (B) at 1 dpf; and embryos exposed to 0.5 µM (C) and 0.1 µM (D) OTA, and 2.5 µM OTα (E), compared to solvent, i.e., MeOH, only controls (F), at 3 dpf.

Figure 5

Representative development dysfunction of zebrafish embryos exposed to OTA and OTα at 5 days post-fertilization. Shown are embryos exposed to 0.5 µM (A), 0.25 µM (B), 0.1 µM (C) and 0.05 µM (D) OTA, as well as 2.5 µM OTα (E), compared to solvent-only (i.e., MeOH) controls (F).

Figure 6

Effect of OTA on zebrafish hatching rate. Shown, for a representative exposure, is percent unhatched eggs/embryos at 5 days post-fertilization for 0.25, 0.1 and 0.05 µM OTA compared to solvent only (“MeOH”) and solvent-free medium (“E3”). At OTA > 0.25 µM, all embryos were dead or severely deformed (and not hatched). Error bars represent standard deviations (n = 2).

Figure 7

Dose and time dependence of OTA and OTα lethality in ZETA. Shown are averages and standard deviations (n = 3) of the percent mortality (5 per well/replicate) at a range of concentrations at 1 (A), 2 (B) and 3 (C) days post-fertilization. Activity is statistically compared to solvent (i.e., MeOH) only controls (n = 3); statistically significant difference are indicated at: (a) p < 0.05; (b) p < 0.01; and (c) p < 0.005. No significant difference was observed between MeOH-only and untreated (i.e., E3 test medium alone) controls as shown.