Mechanisms of induction of apoptosis in liver from rats intoxicated with AFB1, FB1 and with a mixture of both toxins. Correlation with in vitro studies.

THEUMER MG,; LÓPEZ AG,; CÁNEPA MC,; RUBINSTEIN HR.

Córdoba, Argentina

Congreso; Advances in research on toxicogenic fungi and mycotoxins in South America ensuring food and feed safety in a myco-globe context; 2006

sixth framework programme

Objetives: To study the possible induction of apoptosis in hepatocytes exposed in vivo and in vitro to aflatoxin B1 (AFB1), fumonisin B1 (FB1), and to a mixture of both toxins; evaluating the caspases (Cas-X) activities, enzymes involved in this type of cell death. Methods: Animals: Male Wistar rats (n=6 per group). AFB1: Sigma (USA). FB1: was produced by Fusarium verticillioides MRC826 in maize. Separation and quantification of FB1 were done as previously described (1, 2). For in vivo studies, rats were fed 90 days with the following experimental diets: mycotoxins-free (Control), with 40 ppb of AFB1 (EDAFB1), with 100 ppm of FB1 (EDFB1), and with the mixture of AFB1 (40 ppb)+FB1 (100 ppm)(EDM). After the rat necropsies, a portion of the liver was fixed for the Tunel assay, and the remaining tissue was homogenized. For in vitro studies, normal hepatocytes obtained as described (3), were treated in 20h-cultures with AFB1 (TAFB1), FB1 (TFB1), and with the mixture of toxins (TM). The Cas activities (3, 8, and 9) were measured in homogenates from livers of intoxicated rats, and from hepatocytes exposed in vitro to the mycotoxins, by a colorimetric test. Results: Apoptosis and mitosis, as well as increases in Cas-8, Cas-9 and Cas-3 activities, were found in livers from rats fed the EDM; while no changes were observed in the EDAFB1 or EDFB1 groups. In in vitro studies, the TAFB1 and the TM induced hepatocyte apoptosis, and Cas-8, Cas-9 and Cas-3 activation. The TFB1 did not modify the Cas activities. Conclusions: The compensatory regenerative response of the hepatocyte loss by apoptosis could be one of the early events in the hepatocellular carcinoma development by consumption of the EDM. AFB1+FB1 appear to induce apoptosis by more than one mechanism involving Cas activation. References:                                                             1. Voss, K. A., et al. 1990. Mycopathologia 112:81-92.              2. Shephard, G. S., et al. 1990. Journal of Liquid Chromatography 13:2077-2087. 3. Seglen, P. O. 1976. Methods Cell Biol 13:29-83.