87. ROS production mechanisms induced by AFB1 and FB1in rat spleen mononuclear cells

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

Mendoza

Congreso; Strategies to reduce the impact of mycotoxins in Latin America in a global context; 2011

International Society for Mycotoxicology (ISM) and The Latin American Society for Mycotoxicology (SLAM)

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1), the main toxins synthesized by toxicogenic fungi of the genus fusarium spp. and Aspergillus spp., respectively, are common contaminants of food and produce carcinogenic and immunomodulators effects (Theumer et. al., 2010 and 2003). These mycotoxins frequently coexist in nature (González Pereyra ML et. al., 2008) therefore, it is important to know the effects induced by both-toxins mixture. Previously, we have observed increases in the ROS generation in SMC exposed to AFB1 and/or FB1 for 0.5 or 24 h, and the main changes were produced by the mixture (Mary et. al., 2009). In this work, we studied different sources of ROS using the following inhibitors: rotenone (mitochondrial electron transport chain inhibitor), diphenyleneiodonium (DPI) (NADPH oxidase inhibitor), ciprofloxacin (CYP450 1A2 isoenzyme inhibitor) and dexametasone (phospholipase A2 activity inhibitor). Aim: To study potential mechanisms 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 Wistar inbred rats, 8 weeks old, were cultured in the presence or absence of AFB1 (20µM), FB1 (10µM) and MIX (AFB1 20µM + FB1 10µM) for 0.5 or 24 h, at 37°C in 5% CO2. SMC were pre-incubated with or without rotenone (1 μM), DPI (1 μM), dexamethasone (0.01 μM) or ciprofloxacin (35 μM) for 30 min, under the same conditions. -Measurement of intracellular Reactive Oxygen Species (ROS): The reduced form of 2´,7´-dichlorofluorescin (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 addition, and then the incubation lasted for another 20 minutes at 37°C in 5% CO2. We determined the percentage of fluorescent cells (ie cells that incorporated the DCFH and oxidized it), and the mean fluorescence intensity (MFI) as a parameter to estimate the extent of ROS accumulation per cell (Kang J. et. al., 2003). Results and Discussion: The main alterations in the ROS accumulation were found in SMC exposed for 50 min to the mycotoxins, where increases in the percentages of fluorescent cells and MFI were observed in all the treatments, however, FB1 and MIX induced higher rise than AFB1. In a previous work we observed that the toxins individually or as in combination increased the ROS production, being the highest ROS accumulation observed in the later group. Together, these observations suggest that the initial changes in the oxidative status of SMC exposed to the mixture of toxins are mainly driven by the FB1, and that longer time of exposure are needed to evidence the contribution of AFB1 to ROS accumulation. MIX is the only condition that produced O2− increases (time 30min). In a previous study, no changes were observed in this parameter when SMC were incubated for 4 and 24 h with the toxins, probably as a consequence of the short half life of this metabolite. Conclusion: These results show that AFB1 and FB1 induce oxidative stress in rat SMC, however, the mixture of both-toxins produces major changes than the toxins individually. Furthermore, a differential behavior was found for O2− and total ROS accumulation. References: -González Pereyra ML, Pereyra CM, Ramírez ML, Rosa CA, Dalcero AM, Cavaglieri LR. Lett Appl Microbiol. 2008; 46(5):555-61. -Mary VS, Theumer MG, Rubinstein HR. III Congreso Internacional de Ciencia y Tecnología de los Alimentos. Córdoba Capital, Argentina, 2009. -Theumer MG, Lopez AG, Masih DT, Chulze SN, Rubinstein HR. Toxicology. 2003; 186:159-170.