FURTHER MOLECULAR INSIGHTS INTO AFLATOXIN B1 MECHANISTIC TOXICOLOGY IN CATTLE HEPATOCYTES: AN INTEGRATED APPROACH USING RNA-SEQUENCING AND CRISPR/CAS9 SYSTEM

Aflatoxin B1 (AFB1) is a food and feed contaminant metabolized mostly in the liver and resulting in acute and chronic toxicity, therefore it is considered as a global concern with negative impacts on health and economy. Cattle are moderately susceptible to AFB1, but the underlying mechanisms of toxicity has not been fully investigated yet. Hence, in the present thesis an integrated approach to explore in vitro AFB1 mechanistic toxicology in cattle liver was applied. In chapter I we exposed the bovine foetal hepatocyte (BFH12) cell line to three sub-cytotoxic AFB1 concentrations (i.e., 0.9, 1.8, 3.6 µM). LC-MS/MS investigations proved that BFH12 cells metabolized AFB1 in a dose-dependent manner, producing its main derivatives AFM1 and AFL. Cell death assessment showed that AFB1 was cytotoxic for BFH12 cells, mostly via necrosis rather than apoptosis events. RNA-seq analyses suggested a core toxicity mechanism in response to AFB1, with the involvement of genes mainly associated with inflammation and oxidative stress response, besides those correlated to carcinogenesis and drug metabolism. Subsequent confirmatory analysis allowed to propose a putative AFB1 response pathway that correlated oxidative stress and inflammatory response through the toll-like receptor 2 activation. Then, the specific role played by CYP1A1 and CYP3A74 in AFB1 metabolism and hepatotoxicity was investigated in chapters II and III. In this respect, we generated CYP1A1 and CYP3A74 CRISPR/Cas9 knockout (KO) BFH12 cell lines. To fully characterized the physiological role of CYP1A1 and CYP3A74 in the cell, we performed a transcriptome analysis. As to CYP1A1 KO cells, the dysregulation of genes involved in cellular defence against toxic and carcinogenic compounds was revealed, suggesting a faulty mechanism of detoxification. Additionally, the impairment of cell cycle regulation and cell metabolic activity was highlighted, as genes related to iron, lipid and retinoid homeostasis were dysregulated upon CYP1A1 deletion. As to CYP3A74 KO, crashed cell cycle regulation, mainly linked to carcinogenesis events, was pointed out, together with an impact on inflammation and immune system signalling pathways. Changes in metabolic functions were also noted, particularly the disruption of lipid, cysteine and creatine metabolism. Overall, such results highlighted the fundamental role of CYP1A1 and CYP3A74 in maintaining BFH12 cell homeostasis. CYP1A1 and CYP3A74 KO BFH12 cells were then used (chapter IV) to dissect CYP1A1- and CYP3A74-mediated molecular mechanism of AFB1 hepatotoxicity in cattle. Firstly, in silico evaluations were conducted by molecular docking; the resulting conformational predictions of AFB1 into CYP1A1 apo-model suggested the formation of both epoxide metabolites and AFM1, while docking of AFB1 onto CYP3A74 model advised the possible production of 8,9-exo-epoxide and AFQ1. To biologically confirm these predictions, engineered cells were treated with AFB1 and the biotransformation pattern was determined by LC-MS/MS. In CYP1A1 KO cells, despite the still active AFB1 metabolism, the production of AFM1 was completely abolished, confirming the role of CYP1A1 in AFB1 9α-hydroxylation. Conversely, in CYP3A74 KO cells AFB1 metabolism strongly decreased. As a consequence, AFB1-mediated cytotoxicity was significantly reduced in CYP3A74 KO cells compared to controls, suggesting a major role of CYP3A74 in AFB1 epoxidation. RNA-seq analysis showed that AFB1 affected only few pathways in the deleted cell lines (i.e., cell proliferation in CYP1A1 KO cells, inflammation and cell cycle regulation in CYP3A74 KO cells) compared to naïve one, letting us hypothesize a higher resistance of engineered cells to AFB1. Moreover, the signalling pathway postulated in chapter I was inhibited in cells deleted for CYP1A1. Overall, this integrated approach allowed to gain new insights into AFB1 metabolism in cattle liver.