E.E. Creppy

Foodstuffs and human blood contamination by the mycotoxin ochratoxin A: correlation with chronic interstitial nephropathy in Tunisia

Ochratoxin A (OTA) has been detected in high amounts in human blood samples collected in nephrology departments in Tunisia from nephropathy patients under dialysis, especially those categorised as having a chronic interstitial nephropathy of unknown aetiology. These represent 12–26.1% of all chronic renal failure patients. To clarify the situation, food and blood samples were collected from nephropathy patients and controls, (with no familial case of nephropathy). The OTA assay showed very different scales of OTA food and blood contamination from 0.1 to 16.6 μg/kg and 0.1–2.3 ng/ml, respectively, in controls and healthy individuals and 0.3–46 830 μg/kg for food and 0.7–1136 ng/ml for blood in nephropathy patients. The disease seems related to OTA blood levels and food contaminations, since the control group was significantly different from the nephropathy group (p<0.005) for both food and blood ochratoxin A contamination. Combined with data published already, the results emphasize the likely endemic aspect of this OTA-related nephropathy occurring in Tunisia and possibly in other countries of northern Africa. This nephropathy is very similar to Balkan endemic nephropathy.

Co-occurrence of aflatoxin B1, fumonisin B1, ochratoxin A and zearalenone in cereals and peanuts from Côte d’Ivoire

Abstract This survey examined 30 samples of rice (nu2009=u200910), maize (nu2009=u200910) and peanuts (nu2009=u200910) from Côte d’Ivoire for aflatoxin B1, fumonisin B1 and zearalenone using immunoassays, and ochratoxin A using a validated HPLC method with fluorescence detection. In Côte d’Ivoire, as in other countries, several mycotoxins are present in the same commodities. These mycotoxins are from different structural families: aflatoxin B1, fumonisin B1, zearalenone and ochratoxin A, normally produced by fungal species from Aspergillus, Penicillium and Fusarium genera. Some samples contained four mycotoxins (86%). Four peanuts samples did not show ochratoxin A (14%), whereas they contained aflatoxin B1 concentrations above the EU regulatory limits. Concentrations of ochratoxin A, zearalenone and fumonisin B1 were low and may not cause problems per se; however, fears remain that the tolerable daily intake may be exceeded due to eating habits and synergistic effects could be important with the combination of several mycotoxins. Investigations in this direction are underway, together with isolation and characterization of the fungal species involved.

Effect of superoxide dismutase and catalase on the nephrotoxicity induced by subchronical administration of ochratoxin A in rats

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other molds. It is a natural contaminant of mouldy food and feed. OTA has a number of toxic effects, the most prominent being nephrotoxicity. Furthermore, OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. OTA inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction. Recently, lipid peroxidation induced by OTA has been reported, indicating that the lesions induced by this mycotoxin could be also related to oxidative pathways. It was then interesting to study effects of the superoxide dismutase (SOD) and catalase on the nephrotoxicity induced by OTA in rats. The two enzymes (20 mg/kg body weight each) were given to rats by subcutaneous injection, every 48 h, 1 h before gavage by OTA (289 micrograms/kg b.w. every 48 h), for 3 weeks. SOD and catalase prevented most of the nephrotoxic effects induced by ochratoxin A, observed as enzymuria, proteinuria, creatinemia and increased urinary excretion of OTA. Altogether these results indicate (i) that superoxide radicals and hydrogen peroxide are likely to be involved in the damaging processes of OTA in vivo, (ii) that SOD and catalase might be used for prevention of renal lesions in cases of ochratoxicosis.

DNA breaks and cell cycle arrest induced by okadaic acid in Caco-2 cells, a human colonic epithelial cell line.

Abstract. Okadaic acid (OA) is a shellfish toxin produced by dinoflagellates, in mussels. It is a potent tumour promoter and represents a potential threat to human health even at low concentrations. OA targets mainly the gastrointestinal tract in acute poisoning, causing diarrhoea. Therefore the present investigations were designed to study the ability of okadaic acid to induce cytotoxicity and DNA lesions in a human colonic cell line (Caco-2). Incubation of Caco-2 cells with OA (3.75–60xa0ng/ml, i.e 4.6×10–3–7.5×10–2xa0µM) causes a significant reduction in cell viability. Moreover, okadaic acid inhibits protein and DNA synthesis with, respectively, IC50 of 16 and 6.5xa0ng/ml after 24xa0h incubation. It also provokes cell cycle arrest, characterised by an increase in the number of S phase cells, correlated with a significant decrease in G0/G1 phase cells at high concentration. One of the main results obtained in these investigations is the apoptosis induced by OA in Caco-2 cells of intestinal origin, shown by DNA laddering in agarose gel electrophoresis (250–1000 base pairs). OA also induces clastogenic effects evaluated by DNA fragmentation analysis using the method of Higuchi and Aggarwal (52% for 60xa0ng/ml) and comet assay (increase of the frequency of comets and their tails length). Therefore, the cell death induced by OA seems clearly to be concentration-dependent after 24xa0h of incubation. The cytotoxic properties of okadaic acid and its ability to damage DNA result in cell death, mainly by apoptosis. Since consumption of shellfish contaminated with acceptable okadaic acid concentrations exposes colonic cells to harmful concentrations of this toxin, the possibility that OA would display its toxic effects on intestinal cells in vivo should be evaluated in human primary intestinal cells and human intestinal slices for cytotoxic effects, DNA fragmentation and apoptosis.

Regional selectivity to ochratoxin A, distribution and cytotoxicity in rat brain.

Abstract Ochratoxin A (OTA) a chlorodihydro-isocoumarin linked through an amide bond to phenylalanine, is a mycotoxin found as a contaminant in foodstuffs and shown to be nephrotoxic, teratogenic, immunosuppressive, genotoxic, mutagenic and carcinogenic in rodents. Ochratoxin A is known to induce teratogenic effects in neonates (rats and mice) exposed in utero, characterised by microcephaly and modification of the brain levels of free amino acids. Since OTA has been found to accumulate in the brain according to the duration of exposure to doses in the range of natural contamination of feedstuffs, experiments were designed to determine more precisely the structural target of OTA in the brain. After intracerebral injection, OTA (403 ng/10 μl) was not found in the following parts of the brain : the frontal cortex (FC), striatum (ST), ventral mesencephalon (VM) and the cerebellum (CB) in contrast to the rest of the brain, probably due to the detection limit of 0.1 ng/g of tissue. However lactate dehydrogenase (LDH) was increased in extracellular space in the VM to a greater extent than in the rest of the brain, indicating that this structure could be one of the targets of OTA in the brain. Contents of free amino acids were morever similarly modified in the VM and in the rest of the brain. Male rats were given OTA (289 μg/kg per 24 h) by gastric intubation for 8 days and the main brain structures analysed for OTA content and cytotoxicity. OTA was found in the following structures in decreasing order: rest of the brain (50.3%), cerebellum (34.4%), VM (5.1%), striatum (3.3%) and hippocampus (2.9%) of the total OTA amount found in the brain, which represents 0.022% to 0.028% of the given dose. Interestingly cytotoxicity as measured by lactate dehydrogenase (LDH) release in the extracellular space was much more pronounced in the VM, hippocampus, and striatum than in the cerebellum, whereas no cytotoxicity was observed in the rest of the brain. Similarly deoxyribonuclease (DNase) activity in relation to possible necrotic cells was increased in the VM and cerebellum. Altogether these results designated the ventral mesencephalon, hippocampus, striatum and cerebellum as the main OTA-targets in the brain of adult rats and excluded the rest of the brain.

Reduction of the ochratoxin A-induced cytotoxicity in Vero cells by aspartame

Abstract Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and human food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. Recently lipid peroxidation induced by OTA has been reported. OTA, a structural analogue of phenylalanine, inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction, constituting the main mechanism of OTA-induced cytotoxicity. Since it seems impossible to avoid contamination of foodstuffs by toxigenic fungi, 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. Protein synthesis was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (10–100u2009μM). After 24u2009h incubation, protein synthesis was inhibited by OTA in a concentration dependent manner (the 50% inhibitory concentration, IC50, was c.␣14.5u2009μM). Aspartame (A19), at tenfold higher concentrations than OTA (100–1000u2009μM), was found to partially protect against the OTA-induced inhibition of protein synthesis in Vero cells, and more efficiently when added 24u2009h prior to the toxin (IC50u200934u2009μM) than together (IC50u200922u2009μM). As expected A19(250u2009μM) prevented the OTA-induced leakage of certain enzymes, including lactate dehydrogenase, γ-glutamyl transferase, alkaline phosphatase, into the culture medium, and the concomitant decrease of their intracellular activity in OTA (25u2009μM)-treated cells. In order to investigate the effect of aspartame (A19) on OTA-protein binding as explanation of the above results, the mycotoxin time- and concentration-dependent binding to human samples was studied in static diffusion cells with two compartments separated by a dialysis membrane. When A19 (34u2009μM) was added to the upper compartment containing plasma before installing OTA (50, 250, 1240u2009μM) in the lower one, OTA binding was largely prevented (95–98%). When A19 (34u2009μM) was added to the lower compartment simultaneously with the toxin (50, 250, 1240u2009μM), for the lowest concentration of OTA, the same efficiency was shown in preventing OTA binding, but at the two high concentrations A19 seemed less efficient.

Characterization of bolesatine, a toxic protein from the mushroom Boletus satanas Lenz and it's effects on kidney cells

Protein, DNA, and RNA syntheses were assayed in Madin Darby canine kidney cells (MDCK) treated by bolestaine, a toxic glycoprotein from the mushroom Boletus satanas (single chain, Mr 63,000 +/- 3000, pI 8.3 +/- 0.1, disulphide intrachain bridge) previously shown to be an inhibitor of in vitro protein synthesis. Cellular protein and DNA syntheses are inhibited in a dose-dependent manner after 24 h of incubation, whereas the uptake of the labelled precursors of proteins, DNA and RNA biosyntheses into the cells is not affected. The IC50 of bolesatine for protein synthesis is 0.14 microM in the cell culture medium. RNA synthesis is not inhibited at this concentration. The IC50 for DNA synthesis is 0.32 microM. When galactose is added to the culture medium, it decreases or even abolishes the toxic effects, indicating that it prevents the toxin from binding on the membrane and penetrating inside the cells. The profiles of polysomes in MDCK cells treated with bolesatine, compared to the untreated ones, show an increasing pool of polysomes indicating that the toxin acts on the peptidyl elongation step in the protein synthesis.

Preliminary survey of ochratoxin A in millet, maize, rice and peanuts in Côte d'Ivoire from 1998 to 2002.

In a preliminary study, samples of millet (n=33) maize (n=41), rice (n=10) and peanuts (n=10) from Côte dIvoire were analysed for ochratoxin A (OTA) by HPLC with fluorimetric detection, followed by confirmation by cleavage of the OTA molecule using carboxypeptidase with HPLC separation and fluorimetric quantification of the released ochratoxin alpha (OTα). With the exception of four samples of peanuts, all samples showed OTA contamination, ranging from 3 to 1738 μg/kg. All cereals were contaminated and the OTA concentrations were in the range of 17-204 μg/kg for millet, 3-1738 μg/kg for maize, 9-92 μg/kg for rice and 0.6-64 μg/kg for peanuts, depending on the year of harvest. Most of the samples would not be accepted according to the EU regulatory limits for this mycotoxin. Following this survey, research for other mycotoxins and the evaluation of the exposure of the population is under-way.

Effect of piroxicam on the nephrotoxicity induced by ochratoxin A in rats

Ochratoxin A (OTA) is a mycotoxin which contaminates animal feed and human food and is nephrotoxic for all animal species studied so far. It binds to plasma proteins and is transported into target organs, especially the kidney. An attempt to prevent its toxic effects has been made using piroxicam, a non-steroidal anti-inflammatory drug (NSAID). Piroxicam also binds strongly to plasma proteins and our hypothesis is that this drug could stop OTA-binding and transport into target organs, thereby preventing its nephrotoxicity. Our experiments on rats given OTA (289 micrograms/kg/48 h for 2 weeks) show that piroxicam prevents the enzymuria induced by OTA and increases renal elimination of OTA. In vivo, piroxicam could prove useful in preventing the chronic effects of ochratoxin A, mainly nephrotoxicity, at doses 5 mg/kg/48 h, which were not found to be nephrotoxic in experimental animals.

Some effects of ochratoxin A, a mycotoxin contaminating feeds and food, on rat testis

In previous toxicokinetic studies, ochratoxin A (OTA) was found in testicles of laboratory animals. Therefore its possible adverse effects on the rat testis have been investigated. Male Wistar rats 300 g +/- 20 g, randomised in groups of 5 animals, were given OTA: 289 micrograms/kg in 0.1 M NaHCO3 every 48 h for 2, 4, 6 and 8 weeks, by gastric intubation. This corresponds to a contamination of 2 ppm/day in the feeds. Controls were given only the vehicle. Some testicular enzymic activities and the stages of association of germinal cells (according to Leblond and Clermont) were determined. Some of these enzymic activities vary with the ochratoxin A poisoning in the homogenate of testicles: alpha-amylase, from 1905 +/- 145 units/g to 3190 +/- 128 units/g, alkaline phosphatase from 259 +/- 20 units/g to 323 +/- 15 units/g, gamma-glutamyltransferase (gamma GT), from 170 +/- 59 units/g to 900 +/- 65 units/g after 8 weeks. In adult rats, the increase of these enzymes, especially gamma GT, may be associated with an impairment of spermatogenesis and an accumulation of premeiotic germinal cells induced by OTA. Concerning the number of association stages of germinal cells, a decrease is observed for stages I and VII whereas stages XII and XIII increase after OTA treatment, indicating also a possible impairment of spermatogenesis. All these changes seem to be due to an earlier modification of the androgen status, the testosterone level in testes being 10.4 +/- 3.6 micrograms/g of protein after 3 weeks OTA treatment instead of 5.3 +/- 1 micrograms/g in control animals.