Novel insights on the role of bovine CYP1B1 in aflatoxin B1 mechanistic toxicology: molecular docking and CRISPR/Cas9 approaches

Aflatoxin B1 (AFB1) is a mycotoxin occurring in food and feed, affecting human and animal health. In human and bovine liver, AFB1 undergoes oxidative metabolism through cytochromes P450 (CYPs), mostly resulting in the production of carcinogenic derivatives, i.e. AFB1-8,9-epoxide (AFBO) and AFM1. Dairy cattle are one of the most important food-producing species worldwide; whenever exposed to AFB1-contaminated feed, an increasing of AFM1 release in dairy food products is observed, thus representing a One Health concern. Recently, the involvement of human CYP1B1 in AFB1 metabolism, beyond CYP1A2 and CYP3A4, was reported, too. However, the role played by bovine CYP1B1 (bCYP1B1) is unknown. This study aimed to investigate the contribution of bCYP1B1 in AFB1 biotransformation. Preliminary AFB1 molecular docking into bCYP1B1 model suggested the formation of both AFBO and AFM1 derivatives. To validate this computational prediction, a CYP1B1 CRISPR/Cas9 knockout (KO) bovine liver cell line (BFH12) was generated and fully characterized. Engineered cells exposed to subcytotoxic concentrations of AFB1 showed a significant reduction of AFM1 production, along with a marked decrease of the cytotoxicity, supporting the earlier in silico predictions. Additionally, the transcriptomic effects of AFB1 in bCYP1B1KO vs wild type cells were investigated by RNA-seq. Results obtained highlighted a counteracting effect of bCYP1B1KO on cell proliferation and apoptosis as well as on inflammation and oxidative stress induced by AFB1. Overall, this integrated approach shed a light on bCYP1B1 role in AFB1 hepatic metabolism.