Effects of AFB1 and FB1, alone or combined, on the activity of aryl hydrocarbon receptor and CYP1A

MARY VERÓNICA S.; VALDEHITA ANA; NAVAS JOSÉ M; RUBINSTEIN HÉCTOR R.; FERNANDEZ-CRUZ MARÍA LUISA

Rotterdam

Congreso; 7th Conference of The World Mycotoxin Forum and the XIIIth IUPAC International Symposium on Mycotoxins and Phycotoxins; 2012

IUPAC - International Union of Pure and Applied Chemistry

  The activation of aryl hydrocarbon receptor (AhR) induces metabolizing enzymes, such as cytochrome P450 1A (CYP1A). Aflatoxin B1 (AFB1) genotoxicity is associated with its biotransformation by CYP1A to mutagenic AFB1-exo-8,9-epoxide. Co-exposure to AFB1 and fumonisin B1 (FB1) is a frequent situation in nature, and has been associated with a high incidence of human hepatocellular carcinoma.   Aim: To assess the ability of AFB1 and FB1, alone or as a mixture, to induce AhR and CYP1A.   Materials and Methods: -Rat hepatoma cell lines H4IIE and DR-CALUX were exposed to AFB1 (0.6-30 μM), FB1 (3-150 μM), or several AFB1-FB1 mixtures, for 0.5-24 h. Rat spleen mononuclear cells (SMC) were incubated with AFB1 (20 μM), FB1(10 μM) and MIX (20 μM AFB1+10 μM FB1) for 24 h. -CYP1A1 activity in H4IIE. The 7-ethoxyresorufin-O-deethylase (EROD) assay was used. -AhR activity in DR-CALUX (H4IIE cell line transfected with luciferase as reporter gene under control of dioxin responsive elements, which are in promoter of AhR target genes). The luminescence was quantified in a liquid scintillation counter. -Measurement of CYP1A and AhR mRNA levels by real time RT-PCR in H4IIE and SMC.   Results and Conclusions: Exposure of H4IIE and DR-CALUX to different concentrations of AFB1 or several AFB1-FB1 mixtures for 24 h resulted in significant increases of EROD and AhR activity, greater for the mixtures, despite FB1 did not produce changes in activity of CYP1A1 or AhR. AFB1, alone or combined with FB1 significantly stimulated CYP1A mRNA levels, reaching maximal expression at 4 h, while FB1 slightly raised at 8 h treatment. Again, the highest levels were induced by the mixture. In SMC, AFB1, FB1 and MIX enhanced CYP1A mRNA levels, reaching maximal values at 4, 8 and 4 h, respectively. MIX produced significant highest expression of CYP1A.  These changes were preceded by enhancement in AhR expression, since the AFB1, FB1 and MIX increased AhR mRNA levels, with a maximal effect at 2, 4 and 2 h, respectively. These results indicate that a positive interaction occurred between both mycotoxins in the induction of CYP1A and AhR, and might suggest that AFB1-FB1 mixtures could raised AFB1 bioactivation, generation of its mutagenic metabolite, and transformation of hepatocytes in malignant cells.