Study on Aflatoxin B1 mechanistic toxicology in cattle liver based on molecular docking and transcriptional effects in CYP1A1 and CYP3A74 knockout BFH12 cells

Aflatoxin B1 (AFB1) is a pro-carcinogen food and feed contaminant metabolized by cytochrome P450 (CYP) isoenzymes. In human liver, CYP1A and CYP3A are responsible for its bioactivation into the carcinogenic AFB1-8,9-epoxide as well as the formation of AFM1 and AFQ1 derivatives. Cattle is exposed to AFB1 through the ingestion of contaminated feed and may release AFM1 in milk, thus posing a great concern to animal and human health. Despite this, the knowledge about CYP isoenzymes involved in cattle AFB1 biotransformation is still scanty. In the present study we dissected the role played by bovine CYP1A1 and CYP3A74 in AFB1 hepatotoxicity, combining molecular docking, LC-MS/MS metabolite profiling and transcriptomic investigations in CRISPR/Cas9 edited, i.e. CYP1A1 and CYP3A74 knockout (KO), bovine liver cells. AFB1 molecular docking into CYP1A1 protein model suggested the formation of AFB1 8,9-endo- and 8,9-exo-epoxide and AFM1, while docking into CYP3A74 advised the formation of AFB1 8,9-exo-epoxide and AFQ1. To confirm these predictions, engineered cells were exposed to 0.9 and 1.8 µM AFB1. LC-MS/MS analyses showed the abolished production of AFM1 in CYP1A1KO cells, confirming bovine CYP1A1 involvement in AFM1 synthesis; by contrast, in CYP3A74KO cells we observed an indirect accumulation of AFB1 associated to a decreased cytotoxicity, thus suggesting a major contribution of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved a lower cytotoxic effect induced by AFB1 in the engineered cells compared to naïve ones. Overall, this study broadens the knowledge on CYP1A1- and CYP3A74-mediated AFB1 metabolism and its mechanistic toxicology in bovine liver.