Food contaminants and animal health: a whole transcriptomic in vitro approach to unveil aflatoxin B1 toxic effects in cattle

Aflatoxin B1 (AFB1) is a widespread human carcinogenic food contaminant. Many food-producing species, including ruminants, are differently susceptible to such mycotoxin. The metabolism of AFB1 occurs mostly in the liver where it undergoes bioactivation, with the production of a carcinogenic and genotoxic epoxide derivative, and other metabolites, like the hydroxylated carcinogenic derivative aflatoxin M1 (AFM1) and aflatoxicol [1]. Dairy cows fed with AFB1-contaminated feedstuffs may excrete AFM1 into the milk, thus representing a public health concern. Despite the numerous studies about the presence of AFB1 and AFM1 in dairy milk and food-products, little information is available on the overall toxic effects of AFB1 in bovine liver. A recent in vitro study on a bovine foetal hepatocyte cell line (BFH12), pre-incubated with a known CYP1A inducer (PCB126), described for the first time the hepatic transcriptional perturbations elicited by AFB1 [1]. To better characterise the molecular mechanisms involved in AFB1 hepatotoxicity, we exposed BFH12 cells for 48 hrs to increasing AFB1 concentrations (0.9, 1.8, 3.6 M). Whole-transcriptomic changes were measured by RNA-seq. The enrichment analysis identified pathways linked to stress signals and inflammatory response, oxidative stress, cancer, apoptosis, cellular reorganization, and drug metabolism. In particular, genes coding for antioxidant (i.e., NQO1) and phase II enzymes (i.e., UGT1A1 and GSTs) were downregulated in a dose-dependent manner. A similar behaviour was observed for NRF2, a master regulator of the antioxidant response. However, the BTB Domain and CNC Homolog 1 (BACH1) gene, a repressor of genes involved in the oxidative stress response, was upregulated at the highest AFB1 concentration. A similar trend was noticed for the small MAF BZIP Transcription Factors (sMAFs). Both NRF2 and BACH1 form heterodimers with sMAFs; once bound to DNA response elements, they activate (NRF2) or repress (BACH1) the transcription of cytoprotective genes. Moreover, sMAFs may form homodimers and lead to downregulation of target antioxidant genes [2]. Interestingly, TLR2 (a pattern recognition receptor) was dose-dependently upregulated by AFB1; this might be suggestive of a role in AFB1-related inflammatory response. Indeed, TLR2 might trigger an intracellular signalling cascade involving a kinase (p38 MAPK), which allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, with the consequent release of pro-inflammatory cytokines (e.g., IL-6). IL-6, in its turn, could affect the metabolism inhibiting CYP expression [3]. In the present study IL-6 and CYP2B6 showed a dose dependent opposite behaviour (induced the first, inhibited the second). Further studies (e.g., flow cytometry, immunoblotting, AFB1 metabolite profiling) are envisaged to better decipher the molecular mechanisms involved in cattle liver response to AFB1.