Unveiling the role of bovine CYP1A1 and CYP3A28 in AFB1 metabolism: molecular docking and CRISPR/Cas9-mediated genetic knockout in BFH12 cells

Introduction: Cytochromes P450 (CYPs) represent a multigene family of monooxygenase proteins involved in the biotransformation of endogenous compounds and xenobiotics. Aflatoxin B1 (AFB1) is a mycotoxin occurring in several food and feed commodities. In human liver, AFB1 is metabolized by CYP1A and CYP3A isoforms in different carcinogenic and toxic metabolites, such as AFB1-exo-8,9-epoxide (AFBO) and AFM1, and relatively nontoxic derivatives (AFQ1 and AFP1) (1). In cattle, CYP1A1 and CYP3A28 seem crucial for AFB1 metabolism, but their specific role has not been thoroughly investigated yet (2). In this study, AFB1 molecular docking into the abovementioned bovine CYPs was performed. Moreover, the generation of CYP1A1 and CYP3A28 knockout (KO) BFH12 cell lines was exploited to elucidate the role of these two CYPs in AFB1 metabolism. Materials and Methods: Homology modelling using human CYP1A1 (PDB:4I8V) and CYP3A4 (PDB:5TE8) in complex with alpha-naphthoflavone or midazolam, respectively, was preliminarily conducted to build bovine CYP1A1 and CYP3A28 models; then, docking of AFB1 with Glide was performed into these models (Schrödinger Maestro, v12.8). The CRISPR/Cas9-induced genetic KO of CYP1A1 and CYP3A28 was achieved in BFH12 cells using RNP-complex approach. The gene deletion was confirmed by Sanger sequencing, qPCR and/or immunoblotting. Therefore, the cytotoxic effects of AFB1 on native and CYP1A1/CYP3A28 KO BFH12 cells was evaluated using the WST-1 reagent. Results: Docking of AFB1 onto CYP1A1 resulted in two relevant binding modes. In the first one, a hydrogen bond (i.e., ASN226-ring A) and π- π stacking interactions (i.e., PHE228-ring B and PHE127-ring C) were showed, suggesting the formation of the endo-epoxide metabolite. The second pose (π- π stacking interactions between PHE127-ring B) suggests the production of exo-epoxide derivative. Docking of AFB1 onto CYP3A28 resulted in SER119-ring A hydrogen bond, possibly allowing the formation of AFQ1 derivative. CYP1A1 and CYP3A28 CRISPR/Cas9-mediated deletion was confirmed by Sanger sequencing. In engineered cells, CYP1A1 apoprotein was reduced of ~80% and CYP3A28 mRNA expression was completely ablated. Compared to native cells, AFB1 cytotoxicity was significantly reduced in CYP1A1 KO cells, while it was unmodified in CYP3A28 KO cells, corroborating docking predictions and suggesting the main involvement of CYP1A1 in AFB1 bioactivation. Conclusions: This study, through the integration of molecular docking and genetic KO approaches, laid the groundwork to decipher the role played by CYP1A1 and CYP3A28 in AFB1 metabolism and bioactivation. Further studies (i.e., AFB1 metabolite profiling and RNA-seq analysis) are envisaged to implement the knowledge on AFB1 fate in bovine liver.