The aflatoxin B₁‐fumonisin B₁toxicity in BRL‐3A hepatocytes is associated to induction of cytochrome P450 activity and arachidonic acid metabolism

Abstract

Human oral exposure to aflatoxin B₁ (AFB₁ ) and fumonisin B₁ (FB₁ ) is associated with increased hepatocellular carcinoma. Although evidence suggested interactive AFB₁ -FB₁ hepatotoxicity, the underlying mechanisms remain mostly unidentified. This work was aimed at evaluating the possible AFB₁ -FB₁ interplay to induce genetic and cell cycle toxicities in BRL-3A rat hepatocytes, reactive oxygen species (ROS) involvement, and the AFB₁ metabolizing pathways cytochrome P450 (CYP) and arachidonic acid (ArAc) metabolism as ROS contributors. Flow cytometry of stained BRL-3A hepatocytes was used to study the cell cycle (propidium iodide), ROS intracellular production (DCFH-DA, HE, DAF-2 DA), and phospholipase A activity (staining with bis-BODIPY FL C11-PC). The CYP1A activity was assessed by the 7-ethoxyresorufin-O-deethylase (EROD) assay. Despite a 48-h exposure to FB₁ (30 μM) not being genotoxic, the AFB₁ (20 μM)-induced micronucleus frequency was overcome by the AFB₁ -FB₁ mixture (MIX), presumably showing toxin interaction. The mycotoxins blocked G1/S-phase, but only MIX caused cell death. Overall, the oxidative stress led these alterations as the pretreatment with N-acetyl-l-cysteine reduced such toxic effects. While AFB₁ had a major input to the MIX pro-oxidant activity, with CYP and ArAc metabolism being ROS contributors, these pathways were not involved in the FB₁ -elicited weak oxidative stress. The MIX-induced micronucleus frequency in N-acetyl-l-cysteine pretreated cells was greater than that caused by AFB₁ without antioxidants, suggesting enhanced AFB₁ direct genotoxicity probably owing to the higher CYP activity and ArAc metabolism found in MIX. The metabolic pathways modulation by AFB₁ -FB₁ mixtures could raise its hepatocarcinogenic properties.