Effects of natural antioxidants on aflatoxin B1 hepatotoxicity in cattle: targeted in vitro post-transcriptional studies

Aflatoxin B1 (AFB1) is a natural feed and food contaminant from Aspergillus flavus and A. parasiticus. AFB1 is a group I carcinogen for humans, but most of food-producing species are differently susceptible. In dairy industry, AFB1 and its derivative “AFM1” are concerns for food safety for the related economic losses and their possible presence in milk and dairy food-products [1]. Nevertheless, a full understanding of AFB1 molecular toxicity in cattle is still lacking. AFB1 is mostly hepatotoxic, but it causes also oxidative stress and the modulation of several other biological pathways. In the past decade, the dietary supplementation with natural antioxidants (AOs), or food by-products that contain a fair amount of them, has been considered among the strategies to mitigate the presence and toxicity of mycotoxins [2]. Therefore, studies aiming at clarifying the AFB1 molecular mechanisms of toxicity in cattle, and the potential protective role of natural AOs, should be conducted. Recently, the protective role of four natural AOs, i.e. curcumin (CUR), curcuminoids (CUM), resveratrol (RES), and quercetin (QUE) has been investigated in a foetal bovine hepatocyte cell line (BFH12), exposed to AFB1, by measuring cytotoxicity and transcriptional changes. Overall, these AOs reversed the AFB1-dependent toxicity, albeit to a variable degree. Most relevant transcriptional effects were observed in molecular pathways associated with antioxidant and anti-inflammatory response, cancer, and drug metabolism [3,4]. In the present study, confirmatory-targeted post-transcriptional studies were executed, using BFH12 and the same experimental settings described elsewhere [3,4]. Briefly, cells were pre-incubated for 24 hrs with the cytochrome P450 (CYP) inducer PCB126 and, then, for 16 hrs with CUR and CUM (10 μM), QUE and RES (30 μM). Lastly, cells were incubated for further 48h with 3.6 μM AFB1, either alone or in combination with aforementioned AOs concentrations. We measured the amount of malondialdehyde (MDA, a marker of oxidative stress), and the catalytic activities of the antioxidant enzyme diaphorase (NQO1) and CYP3A. Two commercial colorimetric kits were used for MDA and NQO1 evaluation, while a commercial fluorescence kit that allows the direct measurement of the enzyme activity on cell monolayers was selected to for CYP3A assessment. The obtained results corroborated former transcriptional data, highlighting a significant protective role of these AOs. Additionally, RES was confirmed as the most effective AO, followed by CUM, CUR, and QUE. Present results confirm AOs might represent potentially useful dietary supplements to protect cattle against aflatoxicosis. Funding: research supported by the Ministry of Education, University and Research (2015NL8JWS_003) and University of Padua (DOR1917008) to M.D. and M.P. respectively.