Impact of Mycotoxin Contaminations on Aquatic Organisms: Toxic Effect of Aflatoxin B1 and Fumonisin B1 Mixture

Abstract

(1) Background: Multiple contaminations of several mycotoxins have been detected in human and veterinary food and feed worldwide. To date, a number of studies on the combined effects of mycotoxins have been conducted on cell and animal models, but very limited studies have been done on aquatic organisms. (2) The purpose of the present study was to evaluate the combined toxic effects of Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1) on zebrafish (Danio rerio) embryos. (3) Results: Our results showed that the combination of AFB1 and FB1 at nontoxic concentrations exerted a negative effect on the lethal endpoints analyzed, such as survival, hatching, and heart rate. In addition, the mixture of mycotoxins caused an increase in the levels of enzymes and proteins involved in the antioxidant process, such as superoxide dismutase (SOD) and catalase (CAT), both in terms of protein levels and gene expression, as well as an increase in the levels of the detoxification enzymes glutathione s-transferases (GST) and cytochromes P450 (CYP450). Furthermore, we showed that the mycotoxin mixture induced an increase in pro-apoptotic proteins such as bax and caspase 3, and at the same time reduced the gene expression of the anti-apoptotic bcl-2 protein. Finally, a significant decrease in thyroid function was observed in terms of triiodothyronine (T3), thyroxine (T4), and vitellogenin (VTG) levels. (4) Conclusion: We can say that the mixture of mycotoxins carries a greater risk factor than individual presences. There is a greater need for effective detoxification methods to control and reduce the toxicity of multiple mycotoxins and reduce the toxicity of multiple mycotoxins in feed and throughout the food chain.

Keywords: combined toxicity; mycotoxins; oxidative stress.

Figure 1

Morphology. Effects of AFB1 0.05 mg/kg, FB1 0.1 mg/kg and mixture on morphological changes (A), score and body length at 96 hpf. Larva at 96 hph of CTRL (A), AFB1 (B), FB1 (C), AFB1 + FB1 (D) groups. Morphology Score (E), body length (F) of zebrafish larvae treated. Data are expressed as means ± SEM; * p < 0.05 versus CTRL.

Figure 2

ELISA assay. Effects of exposure to AFB1 0.05 mg/kg, FB1 0.1 mg/kg singly and in mixture on thyroid hormones levels (A) Triiodothyronine (T3); (B) Thyroxine(T4), as well as (C) Vitellogenin (VTG), the egg yolk precursor protein important in early life stage development of embryo/larvae, levels at 96 hpf. Data are expressed as means ± SEM; * p < 0.05 versus CTRL.

Figure 3

ELISA assay. Effects of exposure to AFB1 0.05 mg/kg, FB1 0.1 mg/kg singly and in mixture on antioxidant and detoxification enzymes activity at 96 hpf. CAT (A), SOD (B), GST (C), CYP450 (D). Content of malondialdehyde, a marker of lipid peroxidation, at 96 hpf after single and mixed exposure of AFB1 and FB1 (E). Data are expressed as means ± SEM; * p < 0.05 versus CTRL; ** p < 0.01 versus CTRL.

Figure 4

qPCR analysis. Effects of exposure to AFB1 0.05 mg/kg, FB1 0.1 mg/kg singly and in mixture on the expression of oxidative stress-related sod1, cat, and gstp2 genes (A) and apoptosis-related caspase3, bax, and bcl-2 genes (B) at 96 hpf. The results are expressed as mean of data from three independent experiments. The expression levels of mRNA are represented as the fold change from the CTRL group. * p < 0.05, ** p < 0.01, *** p < 0.001 versus CTRL.