CIBICI   14215
CENTRO DE INVESTIGACION EN BIOQUIMICA CLINICA E INMUNOLOGIA
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Oxidative stress in rat spleen mononuclear cells exposed to AFB1 and FB1: time-course accumulation of ROS.
Autor/es:
MARY, VS; THEUMER, MG; RUBINSTEIN HR
Lugar:
Mérida
Reunión:
Congreso; VI Latinamerican Congress of Mycotoxins, II International Symposium on Fungal and Algal Toxins in Industry.; 2010
Institución organizadora:
Sociedad Latinoamericana de Micotoxicología.
Resumen:
Background: Aflatoxins and fumonisins are synthesized by fungi of the genus Aspergillus and Fusarium, respectively. Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are the most important in their groups for their impact on food and immunotoxic and carcinogenic effects (Guindon KA et. al., 2008; Silva LJ et. al., 2009). Co-exposure to AFB1 and FB1 is a situation that occurs frequently in nature, in relation to individual exposure to these compounds (González Pereyra ML et. al., 2008). Although the alterations induced by these mycotoxins, when administered individually, are partially characterised, little is known about the effects induced by both-toxins mixtures. Previously, we have observed different immunobiological effects (Theumer MG et. al., 2003). and alteration of cellular oxidative state after a long-time (24 h) exposure of male Wistar rats spleen mononuclear cells (SMC) to AFB1 and FB1 individually or as a mixture.( Mary VS et. al., 2009). Aim: To study the early oxidative status changes induced in SMC exposed to 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 20 or 50 min, at 37°C in 5% CO2.  -Apoptosis: Assessed by the propidium iodide staining and the subsequent analysis by flow cytomety.     -Measurement of intracellular Reactive Oxygen Species (ROS) and superoxide radical anion (O2−): The reduced form of 2',7'-dichlorofluorescin (DCFH), and Hydroethidine (HE) were used to determine the ROS and O2− intracellular contents, respectively. The probes were added to the cultures along with (time 0) or 30 min after the toxins addition, and then the incubation lasted for another 20 minutes at 37°C in 5% CO2.  ROS and O2− generation was analyzed by flow cytometry. We determined the percentage of fluorescent cells (ie cells that incorporated the DCFH or HE probes and oxidized them), and the mean fluorescence intensity (MFI) as a parameter to estimate the extent of ROS or O2− accumulation per cell (Kang J. et. al., 2003; Zielonka J et. al., 2008). Results and Discussion: Cytotoxicity was not evidenced up to 24 h of culture in presence of the toxins individually or as a combination. 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:           Guindon KA, Foley JF, Maronpot RR, Massey TE. Toxicol Appl Pharmacol. 2008; 227(2):179-83. 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. Silva LJ, Lino CM, Pena A. Toxicon. 2009; 54(4):390-8. Theumer MG, Lopez  AG, Masih DT, Chulze SN, Rubinstein HR. Toxicology. 2003; 186:159-170.