ZnO-NPs alleviate aflatoxin B1-induced hepatoxicity in ducklings by promoting hepatic metallothionein expression

Abstract

Aflatoxin B₁ (AFB₁) is a mycotoxin widely present in animal feed and human food, posing a serious threat to animal and human health. This study was aim to illustrate the mechanism of the protective role of MT against AFB₁-induced hepatotoxicity, as well as to explore the feasibility of enhancing the tolerance of poultry to AFB₁ by upregulating the expression of hepatic MT. After being exposed to AFB₁ (50 ng/kg) primary duckling hepatocytes, the cell viability, the antioxidant index (SOD and GPx) and the mRNA levels of MT downstream genes (PTGR, p53, TrxR, AR and Bcl-2) significantly (p < 0.05) decreased, while the intracellular formation of (AFBO)-DNA adduct content, apoptosis, and MDA content significantly (p < 0.05) increased. Interestingly, overexpression of MT in primary duckling hepatocytes markedly (p < 0.05) reversed the detrimental impact of AFB₁ and increased the expression of MT downstream genes. HepG2 cells were applied to study the mechanism how MT works to relieve the hepatic toxicity of AFB₁. The ZnO-NPs (20 μg/mL) + AFB₁ (20 μg/mL) group significantly (p < 0.05) increased the cell viability, the expression of NRF2, NQO1 and SOD, and expression of MT and MTF-1, as well as significantly (p < 0.05) decreased LDH, ROS and apoptotic rate, comparing with the AFB₁ group. While joint treatment with AFB₁ and ZnO-NPs, the hepatic toxicity exerted by AFB₁ alone was reversed, along with the translocation of MTF-1 from the cytoplasm to the nucleus and upregulated its expression. Duckling trails were further carried out. A total number of 96 1-day-old healthy Cherry Valley commercial ducklings were randomly allocated according to a 2 by 2 factorial arrangement of treatments with the main factors including oral administration of AFB₁ (0 vs. 40 μg/kg) and dietary supplementation of ZnO-NPs (0 vs. 60 mg/kg) for 7 days. It showed that AFB₁ exposure caused body weight loss (p < 0.05), impaired liver structure and failure in hepatic function (activity of ALT, AST and concentration of TP and GLU) (p < 0.05), and decreases in antioxidant capacity(activity of SOD, CAT and concentration of GSH) (p < 0.05), along with the decrease in hepatic concentration of Zn, increase in expression of apoptosis-related genes and protein CAS3 and mRNA Bcl-2 expression (p < 0.05), and suppressed mRNA levels of antioxidant-related genes MT, SOD1, NRF2, and NQO1 (p < 0.05). In accordance with the cell test, dietary supplementation with ZnO-NPs mitigated the toxicity exerted by AFB₁. In conclusion, ZnO-NPs has the protective effects against AFB₁-induced hepatocyte injury by activating the expression of MTF-1 and the ectopic induction of MT expression, providing detailed information on the detoxification ability of MT on AFB₁.

Keywords: Aflatoxin B(1); Ducklings; Liver; Metal-regulatory transcription factor-1; Metallothionein; ZnO-NPs.