Effects of combinations of Fusarium mycotoxins on the inhibition of macromolecular synthesis, malondialdehyde levels, DNA methylation and fragmentation, and viability in Caco-2 cells.

We studied the interactive effects of either binary or tertiary mixtures of Fusarium mycotoxins, deoxynivalenol (DON), zearalenone (ZEA), and fumonisin B1 (FB1) on the human intestinal cell line, Caco-2, using the endpoints including malonedialdehyde (MDA) production, inhibition of protein and DNA syntheses, DNA methylation, DNA fragmentation, and cell viability as measured by the neutral red (NR) test. The mixtures of mycotoxins reduce cellular viability in increasing order: [FB1+ZEA]<[FB1+DON]<[ZEA+DON]<[FB1+DON+ZEA] in NR test. Because FB1 antagonizes the effects of estrogenic Zearalenone, FB1 was assayed against estradiol. In NR assay, mixture of FB1 and estradiol and/or ZEA improves Caco-2 cells viability in contrast to individual effects. Mixtures of ZEA or FB1 and DON, display synergistic effects in lipid peroxidation. The ability of the toxins to inhibit DNA synthesis is 45%, 70%, and 43% for 10 microM of ZEA, DON, and FBI, respectively. Their binary mixtures (at 10 microM each), inhibit DNA synthesis by 35%, 62%, and 65%, far less than additive effects. Surprisingly, the tertiary mixture (10 microM each) only inhibits DNA synthesis by 25%. ZEA, DON, and FB1 induce DNA fragmentation individually. However, mixtures of these mycotoxins always damage DNA to a greater extent. Each individual mycotoxin (10 microM) raises the percentage of 5-methylcytosine (m5dC) in DNA from 4.5% to 9%, while the combination does not increase this rate any further. Altogether, the data indicate that mixtures of Fusarium toxins are able to induce lipid peroxidation, DNA damage, DNA fragmentation, DNA methylation, and cytotoxicity in Caco-2 cells, and suggest a potential promoter effect in human intestinal cells.

Ochratoxin A in human blood in Abidjan, Côte d'Ivoire.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates a diversity of foods including cereals; cereals-derived foods; dry fruits; beans; cocoa; coffee; beer; wine; and foodstuffs of animal origin mainly poultry, eggs, pork and milk, including human breast milk. OTA is nephrotoxic to all animal species studied so far and most likely to humans, who show the longest half-life for elimination of this toxin among all species examined. Among other toxic effects, OTA is teratogenic, immunotoxic, genotoxic, mutagenic and carcinogenic, all of which lead to life-threatening pathologies through several molecular pathways. In Côte d'Ivoire, preliminary surveys conducted by us have proven from 1998 to 2004 the reality of ochratoxin A-contamination of foodstuffs. To assess OTA in human blood, the immunoaffinity columns were used along with HPLC for separation and fluorimetric quantification of blood samples collected in Abidjan from two categories of people: apparently healthy donors (n=63) and nephropathy patients undergoing dialysis (n=39). Among healthy donors, 34.9% show OTA concentrations ranging from 0.01 - 5.81 microg/l with a mean value of 0.83 microg/l, whereas, among nephropathy patients undergoing dialysis 20.5% are OTA positive in a range of 0.167-2.42 microg/l and a mean value of 1.05. Although the sex ratio is 0.82 (46 females for 56 males) ochratoxin A contamination is equally distributed in both sexes. Nephropathy patients undergoing dialysis appear, however, less frequently contaminated than healthy donors (20.5 versus 34.9%) and show higher OTA concentrations (higher mean value, p=0.01). Ochratoxin A concentrations found in human blood reflect concentrations previously detected in cereals and peanuts according to the eating habits and diets of people in Côte d'Ivoire. But, the prevalence of ochratoxin A in blood of nephropathy people undergoing dialysis appears lower than expected from the frequency of OTA contamination in cereals and peanuts. Pearson chi(2)-test indicates that among OTA-positive individuals renal dialysis and age are important modalities for consideration.

Comparative study of cytotoxicity and oxidative stress induced by deoxynivalenol, zearalenone or fumonisin B1 in human intestinal cell line Caco-2.

Fusarium species infestations of cereals crops occur worldwide. Fusarium toxins such as, deoxynivalenol (DON), zearalenone (ZEN) and fumonisin B1 (FB1) have been shown to cause diverse toxic effects in animals and also suspected of disease causation in humans. From the literature and mechanistic point of view, DON binds to the ribosomal peptidyl-transferase and inhibits protein synthesis specifically and DNA synthesis consequently. ZEN known to be genotoxic, binds to 17-beta-estradiol receptors, induces lipid peroxidation, cell death and inhibits protein and DNA synthesis. FB1 disrupts sphingolipid metabolism, induces lipid peroxidation altering the cell membrane and causing cell death. We intended to compare DON, ZEN and FB1 (1-150 microM) cytotoxic effect and the pathways leading to cell death and related to oxidative stress and macromolecules syntheses in a human intestinal cell line in order to tentatively classify them according to their respective potential toxicity. The comparison reveals that all three mycotoxins bear, at variable degree, the capability of inducing lipid peroxidation (MDA production) and could be classified above 10 microM in decreasing potency order FB1>DON>ZEN. This effect seems to be related to their common target that is the mitochondria as revealed by MTT test and seemingly not related to sphingoids accumulation concerning FB1. DON and ZEN also adversely affect lysosomes in contrast to FB1. The three mycotoxins inhibit protein synthesis with respective IC50 of 5, 8.8 and 19 microM for DON, FB1 and ZEN confirming that protein synthesis is a specific target of DON. DNA synthesis is inhibited by DON, ZEN and FB1 with respective IC50 of 1.7, 10 and 20 microM. However at higher concentrations DNA synthesis seems to be restored for FB1 and DON suggesting a promoter activity. Altogether the potency of the three mycotoxins in macromolecules inhibition is DON>ZEN>FB1 in Caco-2 cells. It appears then that FB1 acts rather through lipid peroxidation while DON affects rather DNA and protein synthesis.