The AFB1-FB1 mixture enhances the oxidative stress induced by the individual mycotoxins in rat hepatocytes

MARY VERÓNICA S.; THEUMER MARTÍN G.; RUBINSTEIN HÉCTOR R.

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 natural co-contamination with AFB1 and FB1 especially in corn and rice has been reported as a worldwide problem and associated with a high incidence of human hepatocellular carcinoma. However, little is known about the interaction of these mycotoxins regarding their toxic and carcinogenic properties. Aim: To assess the cellular oxidative status changes induced by AFB1 and FB1 individually or as a mixture in rat hepatocytes.   Materials and Methods: -Cell culture: BRL-3A rat liver cell line that was exposed to 20 μM AFB1, 30 μM FB1 and AFB1-FB1 mixture (MIX= 20 μM AFB1 + 30 μM FB1) for 0.5, 4, 24 h, at 37ºC in 5% CO2. To investigate the role of CYP450 and Arachidonic acid (AA) metabolism in ROS generation, cells were pre-incubated with 5 μM ciprofloxacin (CYP450 1A and 3A inhibitor) or 0.01 μM dexamethasone (AA metabolism inhibitor) for 30 min. -Measurement of intracellular reactive oxygen species (ROS), superoxide radical anion (O2−) and reactive nitrogen species (RNS): The 2',7'-dichlorofluorescin diacetate, hydroethidine and 4,5-diaminofluorescein probes were used to determine ROS, O2− and RNS intracellular contents, respectively, by flow cytometry. -Detection of protein and lipid oxidation: Oxidative damage of proteins was determined by measuring the levels of carbonyl groups, using the spectrophotometric DNPH method; lipid peroxidation was determinate by measuring the MDA level, using TBA test, with subsequent separation and quantification of the MDA-TBA adducts by HPLC.   Results and Conclusions: At short times, changes in the oxidative status of BRL-3A exposed to MIX were detected. This treatment produced increased levels of ROS and O2−. At long times, AFB1 and MIX raised RNS levels, and all treatments increased ROS levels, being MIX which produced the greatest changes. The increase of ROS generation induced by mycotoxins, alone or combined, was prevented by ciprofloxacin and to a lesser extent by dexamethasone, suggesting that this mechanism is mainly dependent on CYP450. Biomolecular oxidative damage was mainly caused by MIX, demonstrating a synergistic action between AFB1 and FB1, since individual mycotoxins produced null or little changes in the oxidation of proteins and lipids, respectively. This work demonstrates that MIX potentiates the actions of individual mycotoxins in the oxidative stress induction. This mechanism could mediate the AFB1 and FB1 individual and combined hepatotoxicities.