Anne-Marie Betbeder

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 (n = 10), maize (n = 10) and peanuts (n = 10) 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.

Ochratoxin A in human blood in relation to nephropathy in Tunisia.

The determination of ochratoxin A (OTA) in human blood in Tunisian populations is underway. The range of conta mination is between 0.7 to 7.8 ng ml-1 for the general popu lation and 12 to 55 ng ml-1 for people suffering from chron ic renal failure. It appears that 21 to 64% of people suffer ing from nephropathy are OTA positive with a detection limit of 1ng ml-1. This situation prompted us to search for possible association of OTA contamination and nephropa thy resembling Balkan endemic nephropathy. The classifi cation of the ill population into chronic interstitial nephropathy (CIN), chronic glomerular nephropathy (CGN), chronic vascular nephropathy (CVN) and others, indicated that the largest is the CIN group which is signifi cantly different from the other groups, and from the con trol (P < 0.005). Furthermore, it presented the highest OTA mean values (25 to 59 ng ml-1) compared with the control, CGN, CVN and other groups (6 to 18 ng ml-1) according to the designated region in Tunisia. The rural population seems to be more exposed to ochratoxins in Tunisia, as has been previously reported in the Balkans and Western Europe. Altogether, these results emphasise that in Tunisia an endemic ochratoxin-related nephropathy is probably occurring.

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.

Formation of ochratoxin a metabolites and DNA-adducts in monkey kidney cells

Monkey kidney cells (named Vero cells) were incubated with increasing doses of ochratoxin A (10-100 microM). The inhibiting concentration 50% (IC50) on protein synthesis was about 14 microM in the presence of 5% fetal calf serum and 37 microM in the presence of 10% fetal calf serum. Some metabolites of ochratoxin A, including the chlorinated dihydroisocoumarin moiety of OTA (OT alpha), 4-[S]-hydroxy-OTA and 4-[R]-hydroxy-OTA were detected by HPLC in the mixture of cell homogenate after a 24 h incubation with 10 and 25 microM of OTA. Using the 32P-postlabelling method, several DNA-adducts, similar to those formed in mouse kidney after OTA treatment, were detected in monkey kidney cells. Thus, Vero cells are suitable for genotoxic and cytotoxic studies in relation to the metabolism of nephrotoxic xenobiotics such as OTA.

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 (10–100 μM). After 24 h incubation, protein synthesis was inhibited by OTA in a concentration dependent manner (the 50% inhibitory concentration, IC50, was c.␣14.5 μM). Aspartame (A19), at tenfold higher concentrations than OTA (100–1000 μM), was found to partially protect against the OTA-induced inhibition of protein synthesis in Vero cells, and more efficiently when added 24 h prior to the toxin (IC50 34 μM) than together (IC50 22 μM). As expected A19(250 μM) 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 (25 μM)-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 (34 μM) was added to the upper compartment containing plasma before installing OTA (50, 250, 1240 μM) in the lower one, OTA binding was largely prevented (95–98%). When A19 (34 μM) was added to the lower compartment simultaneously with the toxin (50, 250, 1240 μM), 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.

Ochratoxin A levels in human plasma and foods in Lebanon

Ochratoxin A (OTA) is a widespread mycotoxin which contaminates food such as cereals, beer, coffee, wine and products of animal origin. OTA is known for its nephrotoxic, immunotoxic and carcinogenic properties. The prevalence of OTA in human blood and foodstuffs has been investigated in many countries. In this study, exposure of the Lebanese population to OTA was evaluated and the contamination of the most commonly consumed foods in Lebanon by OTA was assessed. Plasma samples from healthy individuals and also cereals and beer samples obtained from markets were collected from the different regions of Lebanon. OTA was detectable in 33% of tested plasma samples (n=250) with a concentration ranging from 0.1 to 0.87 ng/mL and a mean of 0.17±0.01 ng/mL. No sex and age differences were found. The frequency of OTA-positive plasma samples obtained in the South of Lebanon and in the Bekaa valley (50 and 47%, respectively) was significantly higher compared to plasma samples obtained in the Beirut/Mount Lebanon region (19%). Food analyses showed that wheat, burghul and beer were contaminated with a mean value of 0.15±0.03 μg/kg, 0.21±0.04 μg/kg and 0.19±0.12ng/mL, respectively. These data suggest that the Lebanese population is exposed to OTA through food ingestion at concentrations lower than the tolerable daily intake.

Selective toxicity of ochratoxin A in primary cultures from different brain regions

Abstract Ochratoxin A (OTA) is a mycotoxin produced by moulds from the Aspergillus and Penicillium genera. It is a natural contaminant of a wide variety of both human and animal foodstuffs. Via dietary intake, OTA passes into the blood of both humans and animals and accumulates in several organs, such as the kidney and the brain with selective toxicity in the ventral mesencephalon and in the cerebellum. In order to confirm the regional selectivity to OTA cytotoxicity in rat brain, investigations were designed to study the mechanism of the cytotoxicity of OTA in primary cultures of the above-mentioned structures (ventral mesencephalon and cerebellum), and to compare their sensitivity to the toxin. Protein and DNA synthases, lactate dehydrogenase (LDH) release and production of malondialdehyde (MDA) were assayed in astrocytes and neurones of the selected structures in the presence of OTA. After 48 h incubation, OTA (10–150 μM) induced an inhibition of protein and DNA syntheses in a concentration-dependent manner with a selective higher toxicity in the cells of the ventral mesencephalon [50% inhibitory concentrations (IC50) of protein and DNA syntheses were 14 ± 2 μM for neurones and 40 ± 5 μM for astrocytes] compared to the cerebellum values (24 ± 7 μM for neurones and 69 ± 9 μM for astrocytes). In parallel, a significant increase in levels of MDA and LDH release were noted. Altogether these results indicate that OTA is also a neurotoxic substance in addition to its well-documented nephrotoxicity and that the effects are likely to be restricted within particular structures of the brain.

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.