New insights into aflatoxin B1 mechanistic toxicology in cattle liver: an integrated approach using molecular docking and biological evaluation in CYP1A1 and CYP3A74 knockout BFH12 cell lines

Abstract

Aflatoxin B1 (AFB1) is a pro-carcinogenic compound bioactivated in the liver by cytochromes P450 (CYPs). In mammals, CYP1A and CYP3A are responsible for AFB1 metabolism, with the formation of the genotoxic carcinogens AFB1-8,9-epoxide and AFM1, and the detoxified metabolite AFQ1. Due to climate change, AFB1 cereals contamination arose in Europe. Thus, cattle, as other farm animals fed with grains (pig, sheep and broiler), are more likely exposed to AFB1 via feed with consequent release of AFM1 in milk, posing a great concern to human health. However, knowledge about bovine CYPs involved in AFB1 metabolism is still scanty. Therefore, CYP1A1- and CYP3A74-mediated molecular mechanisms of AFB1 hepatotoxicity were here dissected. Molecular docking of AFB1 into CYP1A1 model suggested AFB1 8,9-endo- and 8,9-exo-epoxide, and AFM1 formation, while docking of AFB1 into CYP3A74 pointed to AFB1 8,9-exo-epoxide and AFQ1 synthesis. To biologically confirm these predictions, CYP1A1 and CYP3A74 knockout (KO) BFH12 cell lines were exposed to AFB1. LC-MS/MS investigations showed the abolished production of AFM1 in CYP1A1 KO cells and the strong increase of parent AFB1 in CYP3A74 KO cells; the latter result, coupled to a decreased cytotoxicity, suggested the major role of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved lower AFB1-induced cytotoxic effects in engineered cells versus naïve ones. Overall, this study broadens the knowledge on AFB1 metabolism and hepatotoxicity in cattle, and it provides the weight of evidence that CYP1A1 and CYP3A74 inhibition might be exploited to reduce AFM1 and AFBO synthesis, AFB1 toxicity, and AFM1 milk excretion.

Keywords: Aflatoxin B1; Bovine liver; CYP1A1; CYP3A74; Molecular docking; RNA-seq.

Fig. 1

AFB1 structure (a) and docking poses of AFB1 against CYP1A1 (b, c) and CYP3A74 (d, e) models. CYPs backbone is shown in grey ribbon, while hydrophobic residues that surround the ligand are figured in violet. Black dashes indicated hydrogen bonds, while yellow and red dashes indicated π–π stacking and π–cation interactions, respectively

Fig. 2

Amount of AFB1 (a), AFL (b), and AFM1 (c) detected by LC–MS/MS in the medium of CTL, CYP1A1 KO, and CYP3A74 KO cells exposed to 0.9 and 1.8 µM AFB1 and normalized to the total protein content of the respective cell monolayers. Data are expressed as ng of analyte per µg of total protein, as the mean ± mean standard error (SEM) of three biological replicates. Cytotoxicity evaluation (d) of increasing AFB1 concentrations in CTL, CYP1A1 KO, and CYP3A74 KO cells using WST-1 reagent. Data are expressed as the mean percentage of dead cells relative to that of cells exposed to the vehicle only (0.1% DMSO) ± SEM of three biological replicates, each performed in sextuplicate. Statistical analysis: one-way ANOVA followed by Dunnett’s multiple comparisons test; *: p < 0.05, **: p < 0.01, and ***: p < 0.001