The cytotoxic effects of aflatoxin B1 and fumonisin B1 on cultured human cells.
Van der Stok, Mary Elizabeth.
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Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1), potentially cytotoxic and carcinogenic mycotoxins are common contaminants of agricultural commodities in South Africa and thus could be detrimental to the human immune system. Many of the cytotoxic effects of AFB1 require its bioactivation to an epoxide, which will bind covalently to macromolecules to form protein and DNA adducts. Fumonisin B1 is a competitive inhibitor of sphingosine and sphinganine N aceyltransferase, which are key components in the pathways for sphingolipid biosynthesis. Accumulation of free sphingoid bases, which are both cytotoxic and mitogenic, could provide a plausible explanation for the toxicity and carcinogenicity of FB1. The cytotoxic effects of AFB1 and FB1 on normal human lymphocytes, individually and in combination were assessed using the methylthiazol tetrazolium (MTT) bioassay. Two different methods of treatment were used, the treatment of isolated normal human lymphocytes for 12, 24, 48, 72 and 96 hours and whole blood treated for 12 hours. Flow cytometry and fluorescent microscopy were used to determine whether AFB1 and FB1 (5uM and 50uM), individually or in combination, were capable of inducing apoptosis, necrosis or nuclear fragmentation in isolated lymphocytes and whole blood treated for 12 hours. DNA damage was evaluated using the comet assay. The results showed that AFB1routinely induced higher levels of cytotoxicity in isolated lymphocytes than FB1. In the combination treatment, the mitogenic properties of FB1 appeared to partially counteract the cytotoxic effect exerted by AFB1. When whole blood was treated with the same concentration and ratio of toxin, FB1 was shown to be more cytotoxic than AFB1. The combination treatment of whole blood was shown to be cytotoxic in a dose dependent manner. The toxins appeared to exert a greater cytotoxic effect, when treated in combination than individually at higher concentrations. Aflatoxin B1 induced increased levels of apoptosis and necrosis in isolated lymphocytes while treatment with the FB1 resulted in increased levels of apoptosis at both concentrations. Treatment with the combination also resulted in increased levels of apoptosis. The levels of apoptosis were reduced in whole blood lymphocytes when compared to isolated lymphocytes. However, treatment with AFB1 and FB1 resulted in increased levels of apoptosis. Both AFB1 and FB1 are capable of inducing nuclear fragmentation. Treatment with FB1 (5uM and 50uM) resulted in greater degree of fragmentation than AFB1. The most nuclear fragmentation was induced by the 5uM combination treatment. The 50uM combination treatment of isolated lymphocytes induced the most DNA damage. As both toxins are common contaminants and have been known to coexist, this could be a potential area of concern for public health.