Biochemical Pathways Involved In The Reactive Oxygen Species Generation Induced By Aflatoxin B1 And Fumonisin B1 In Rat Spleen Mononuclear Cells

VERÓNICA S. MARY, MARTÍN G. THEUMER AND HÉCTOR R. RUBINSTEIN

Mendoza

Congreso; ISM Conference 2011; 2011

Sociedad Latinoamericana de Micología y Sociedad Internacional de Micología

Biochemical Pathways Involved In The Reactive Oxygen Species Generation Induced By AFB1 And FB1 In Rat Spleen Mononuclear Cells   Verónica S. MARY, Martín G. THEUMER* and Héctor R. RUBINSTEIN.   CIBICI-CONICET, Dpto Bioq. Clínica, Fac. de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba Capital, Argentina. hectorru@fcq.unc.edu.ar     Aflatoxin B1 (AFB1) and fumonisin B1 (FB1), the main toxins synthesized by fungi of the genus Fusarium and Aspergillus, respectively, are common contaminants of food and produce carcinogenic and immunotoxic effects. These mycotoxins frequently coexist in nature, therefore, it is important to know the effects induced by both-toxins mixtures. Previously, we have observed increases in the reactive oxygen species (ROS) generation in spleen mononuclear cell (SMC) exposed to AFB1 and/or FB1 for 0.5 or 24 h, and the main changes were produced by the mixture. In cells various enzyme systems produce ROS, including the mitochondrial electron transport chain, the NADPH oxidase, the complex cytochrome CYP450 and arachidonic acid (AA) metabolism. Aim: To study contribution of potential biochemical pathways involved in the production of ROS, induced in SMC, by AFB1 and FB1 individually or as a mixture.   Materials and Methods: -Cell culture: SMC of male inbred Wistar rats (8 weeks old) were cultured in the presence or absence of 20µM AFB1, 10µM FB1 and MIX (20µM AFB1 + 10µM FB1) for 0.5 or 24 h, at 37ºC in 5% CO2. To investigate the role of each of the pathways of ROS generation above mentioned, SMC were pre-incubated with or without 1 μM rotenone (Mitochondrial electron-transport-chain inhibitor of complexes I), 2 μM diphenyleneiodonium (DPI is NADPH oxidase inhibitor), 50 μM ciprofloxacin (CYP450 1A2 isoenzyme inhibitor) or 0.01 μM dexamethasone (AA metabolism inhibitor) for 30 min, under the same conditions. -Measurement of intracellular ROS: The reduced form of 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to determine the ROS intracellular content by flow cytometry. The probe was added to the cultures 0.5 or 24 h after the toxins additions, and then the incubation lasted for another 20 minutes at 37ºC in 5% CO2. We determined the percentage of fluorescent cells (ie cells that incorporated and oxidized the probe, due to increased oxidative cellular state), and the mean fluorescence intensity, as a parameter to estimate the extent of ROS accumulation per cell.   Results and Conclusion: The increase of ROS production induced by AFB1 and FB1 was significantly inhibited by rotenone and ciprofloxacin. Instead DPI was able to significantly decreased the FB1 induced ROS production, but did not modify the oxidative stress generated by AFB1. In addition, dexamethasone only significantly reduced the ROS accumulation stimulated by AFB1. ROS generation induced by the mixture was prevented by all inhibitors tested. Our studies demonstrated that ROS production by AFB1 is dependent on mitochondrial electron transport, AA metabolism and CYP450 1A2, and by FB1 is dependent on NADPH oxidase, mitochondrial electron transport, and CYP450 1A2. Furthermore, all these biochemical pathways contribute in the ROS generation induced by both-toxins mixture, so that the oxidative stress generated results from the addition of the mechanisms of action of individual toxins.