REDUCTION OF THE OCHRATOXIN A-INDUCED CYTOTOXICITY IN VERO CELLS BY ASPARTAME

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and hum an food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. Recentl y lipid peroxidation induced by OTA has been reported. OTA, a structur al analogue of phenylalanine, inhibits protein synthesis by competitio n with phenylalanine in the phenylalanine-tRNA aminoacylation reaction , constituting the main mechanism of OTA-induced cytotoxicity. Since i t seems impossible to avoid contamination of foodstuffs by toxigenic f ungi, investigation is required for preventing the toxicity of OTA. An attempt to prevent its toxic effect, mainly the inhibition of protein synthesis, has been made using aspartame (L-aspartyl-L-phenylalanine methyl ester) a structural analogue of both OTA and phenylalanine. Pro tein synthesis was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (10-100 mu M). After 24 h incubat ion, protein synthesis was inhibited by OTA in a concentration depende nt manner (the 50% inhibitory concentration, IC50, was c. 14.5 mu M). Aspartame (A(19)), at tenfold higher concentrations than OTA (100-1000 mu M), was found to partially protect against the OTA-induced inhibit ion of protein synthesis in Vero cells, and more efficiently when adde d 24 h prior to the toxin (IC50 34 mu M) than together (IC50 22 mu M). AS expected A(19)(250 mu M) prevented the OTA-induced leakage of cert ain enzymes, including lactate dehydrogenase, gamma-glutamyl transfera se, alkaline phosphatase, into the culture medium, and the concomitant decrease of their intracellular activity in OTA (25 mu M)-treated cel ls. In order to investigate the effect of aspartame (A(19)) on OTA-pro tein binding as explanation of the above results, the mycotoxin time- and concentration-dependent binding to human samples was studied in st atic diffusion cells with two compartments separated by a dialysis mem brane. When A(19) (34 mu M) was added to the upper compartment contain ing plasma before installing OTA (50, 250, 1240 mu M) in the lower one , OTA binding was largely prevented (95-98%). When A19 (34 mu M) was a dded to the lower compartment simultaneously with the toxin (50, 250, 1240 mu M), for the lowest concentration of OTA, the same efficiency w as shown in preventing OTA binding, but at the two high concentrations A19 seemed less efficient.