Protective effects of quercetin towards aflatoxin B1-induced hepatotoxicity in cattle: a whole transcriptomic in vitro study

Introduction: Aflatoxin B1 (AFB1) is a natural feed and food contaminant. In the dairy industry, AFB1 and its derivatives are of concern for the related economic losses and their possible presence in milk and dairy food products. Oxidative stress is one of the prominent toxic effects of AFB1, thus, dietary supplementation with natural antioxidants is a valid strategy to mitigate AFB1 toxicity. In this respect, quercetin (QUE) is a promising bioactive compound. Here we assessed the protective role of QUE in bovine foetal hepatocyte-derived cells (BFH12) exposed to AFB1, by measuring the QUE impact on AFB1-induced cytotoxicity and related whole- transcriptional changes. Methods: to increase cells responsiveness to AFB1, monolayers were pre-treated with 1 nM PCB126 for 24h and then exposed to 3.6 μM AFB1, alone or in combination with sub-cytotoxic concentrations of QUE (10, 20, 30 μM). Cytotoxicity was then assessed. For the whole-transcriptome study, we exposed cells to 30 μM QUE, 3.6 μM AFB1, or their combination. Cells pre-treated with PCB126 and exposed to DMSO were used as control (CTRL). Total RNA was isolated, 12 tagged RNA-seq libraries were prepared and sequenced (50 bp single-end). Reads were quality checked, trimmed and mapped to cow reference genome using validated pipelines. EdgeR and clusterProfiler were used to identify differentially expressed genes (DEGs) and perform the functional enrichment analysis, respectively. Results: 30 μM QUE significantly reduced AFB1-induced cytotoxicity by 13.39%. As to RNA-seq, the differential expression analysis comparing QUE vs CTRL and QUE+AFB1 vs AFB1 identified 1028 and 1890 DEGs, respectively. QUE alone affected the expression of genes related to steroids metabolism, inflammation, immunity, and P450-mediated drug metabolism. When comparing QUE+AFB1 vs AFB1, top upregulated genes possess antiapoptotic and antioxidant activity (e.g. SEMA5A, TF), while top downregulated genes play a role in inflammation and cancer progression (e.g. CXCL5, IL6). If comparing these results with the mRNA changes induced in cells exposed to AFB1 alone, all the top-10 genes upregulated by the cotreatment were inhibited by AFB1; on the opposite, all the top-10 downregulated genes were induced by AFB1 alone. Conclusions: we showed the QUE potential to mitigate AFB1-induced toxicity and the underneath molecular mechanisms. If the beneficial effects will be confirmed in vivo, QUE-supplemented diets might be adopted to improve health status of cattle exposed to AFB1-contaminated feed.