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,
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
-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
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.