Edmond E. Creppy

Update of survey, regulation and toxic effects of mycotoxins in Europe.

The most frequent toxigenic fungi in Europe are Aspergillus, Penicillium and Fusarium species. They produce aflatoxin B1 transformed into aflatoxin M1 found in the milk, as well as Ochratoxins and Zearalenone, Fumonisin B1, T-2 toxin, HT-2 toxin and deoxynivalenol (vomitoxin), which are of increasing concern in human health. These mycotoxins are under continuous survey in Europe, but the regulatory aspects still need to be set up and/or harmonised at European level. They are found in foodstuffs and are not destroyed by normal industrial processing or cooking since they are heat-stable. Some of their metabolites are still toxic and may be involved in human diseases. Their toxic effects (liver, kidney and hematopoetic toxicity, immune toxicity, reproduction toxicity, foetal toxicity and teratogenicity, and mainly carcinogenicity) are mostly known in experimental models, the extrapolation to humans being always inaccurate. The inaccuracy of extrapolation to humans may be explained by the lack of adequate food consumption data, lack of knowledge about relative health risks associated with specifically proposed limits and by the possibility of synergism with other mycotoxins present in the same food commodities. Other pathological causes are viral hepatitis, immune or hormonal deficiencies or organ dysfunction. Even when a specific biomarker of a given mycotoxin is identified in humans, it remains difficult to establish the relation with a given illness, because of genetic polymorphism and the possible beneficial influence of diet, and because other environmental toxicants may well interfere. The acceptable daily intake limits are mostly based on animal data and may be too high, due to the differences in the sensitivity of different animal species. The prevention involves first reduction of mycotoxin levels in foodstuffs and further increasing the intake of diet components such as vitamins, antioxidants and substances known to prevent carcinogenesis.

Oxythiamine and dehydroepiandrosterone induce a G1 phase cycle arrest in Ehrlich's tumor cells through inhibition of the pentose cycle

Transketolase (TK) reactions play a crucial role in tumor cell nucleic acid ribose synthesis utilizing glucose carbons, yet, current cancer treatments do not target this central pathway. Experimentally, a dramatic decrease in tumor cell proliferation after the administration of the TK inhibitor oxythiamine (OT) was observed in several in vitro and in vivo tumor models. Here, we demonstrate that pentose cycle (PC) inhibitors, OT and dehydroepiandrosterone (DHEA), efficiently regulate the cell cycle and tumor proliferation processes. Increasing doses of OT or DHEA were administered by daily intraperitoneal injections to Ehrlichs ascites tumor hosting mice for 4 days. The tumor cell number and their cycle phase distribution profile were determined by DNA flow histograms. Tumors showed a dose dependent increase in their G0‐G1 cell populations after both OT and DHEA treatment and a simultaneous decrease in cells advancing to the S and G2‐M cell cycle phases. This effect of PC inhibitors was significant, OT was more effective than DHEA, both drugs acted synergistically in combination and no signs of direct cell or host toxicity were observed. Direct inhibition of PC reactions causes a G1 cell cycle arrest similar to that of 2‐deoxyglucose treatment. However, no interference with cell energy production and cell toxicity is observed. PC inhibitors, specifically ones targeting TK, introduce a new target site for the development of future cancer therapies to inhibit glucose utilizing pathways selectively for nucleic acid production.

DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E.

Zearalenone (ZEN) is a non-steroidal oestrogenic mycotoxin produced by several Fusarium species growing on cereals. ZEN and its metabolites bind to human oestrogen receptors and hence display oestrogenic and anabolic properties. Several lines of investigation suggest that ZEN may be genotoxic in vivo. ZEN damages DNA in Bacillus subtilis recombination tests, and it induces sister chromatid exchange and chromosomal aberration in CHO cells. ZEN also induces DNA-adduct formation in mouse tissues and SOS repair process in lysogenic bacteria. In the present study, ZEN genotoxicity has been confirmed in three cell-lines, Vero, Caco-2 and DOK at concentrations of 10, 20 and 40 microM. Under these conditions, ZEN induces concentration-dependent DNA fragmentation resulting in DNA laddering patterns on agarose gel electrophoresis. This observation is consistent with apoptosis, which was confirmed by observations of formation of apoptotic bodies. Moreover, ZEN induces cell cycle arrest in the three cell-lines characterised by an increase of the number of cells in the G2/M phase of the cell cycle. Vitamin E (25 microM) added simultaneously with ZEN partially reduces DNA fragmentation and apoptotic body formation after 24h incubation. Vitamin E may act by maintaining prolonged cell cycle arrest during which time DNA repair takes place.

Differential DNA adduct formation and disappearance in three mouse tissues after treatment with the mycotoxin ochratoxin A

Ochratoxin A (OTA) is a mycotoxin which has been implicated in Balkan endemic nephropathy, a disease characterized by a high incidence of urinary tract tumors. It induces DNA single-strand breaks and has been shown to be carcinogenic in two rodent species. For a better understanding of the OTA genotoxic effect, OTA-DNA adduct formation and disappearance has been measured using the 32P-post-labelling method after oral administration of 2.5 mg/kg of OTA to mice. In kidney, liver and spleen, several modified nucleotides were clearly detected in DNA, 24 h after administration of OTA, but their level varied significantly in a tissue and time dependent manner over a 16-day period. Total DNA adducts reached a maximum at 48 h when 103, 42 and 2.2 adducts per 10(9) nucleotides were found respectively in kidney, liver and spleen, indicating that kidney is the main target of the genotoxicity and likely carcinogenicity of OTA. The major adduct differed between kidney and liver. All adducts disappeared in liver and spleen 5 days after compound administration, whereas some adducts persisted for at least 16 days in the kidney. Some adducts were organ specific. The finding that the adducts are not quantitatively and qualitatively the same in the three organs examined is likely due to differences of metabolism in these organs, leading to different ultimate carcinogens and may also result from differences in the efficiency of repair processes.

Zearalenone induces modifications of haematological and biochemical parameters in rats

Zearalenone produced by the fungus Fusarium roseum causes important perturbations in the gestation cycle of the rat with hormonal disorders and infertility. In order to find out other eventual toxic effects, female rats were given intraperitoneally (i.p.) (1.5, 3 and 5 mg/kg) zearalenone in sterile olive oil. Forty-eight hours later, some blood parameters changed (hematocrit, MCV, the number of platelets and WBC) as well as some biochemical markers such as aminotransferases (ALT, AST), alkaline phosphatase (ALP), serum creatinine, bilirubin, indicating liver toxicity, and likely impairment of blood coagulation process.

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.

Cytotoxicity of fumonisin B1: implication of lipid peroxidation and inhibition of protein and DNA syntheses

Abstract The effects of fumonisin B1 (FB1) from Fusarium moniliforme on lipid peroxidation and protein and DNA syntheses were studied in monkey kidney cells (Vero cells). FB1 was found to be a potent inducer of malondialdehyde (MDA), one of the secondary products formed during lipid peroxidation. At 0.14 μM (0.1 μg/ml), FB1 induced 0.496 ± 0.1 nmoles of MDA/mg protein, compared to the control level 0.134 ± 0.01 nmoles of MDA/mg protein (P < 0.005). No inhibition of protein or DNA synthesis was observed at this concentration of FB1. Inhibition of protein and DNA syntheses was observed at FB1 concentrations >14 μM (10 μg/ml) with an IC50 of 33 μM for both protein synthesis and DNA synthesis. These results indicate that lipid peroxidation is a very sensitive cellular response to the mycotoxin fumonisin B1 observed at concentrations lower than that required to inhibit cellular synthesis of macromolecules, protein and DNA. This oxidative damage induced by FB1 concentrations encountered in naturally contaminated foodstuffs and feed might lead to mutagenicity and genotoxicity.

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.

Ochratoxin a and human chronic nephropathy in Tunisia: is the situation endemic?

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that is being increasingly considered as the main causal agent of Balkan endemic nephropathy (BEN), a fatal kidney disease associated with the end stage of urothelial tumours. However, despite the considerable amount of data, it is still controversial whether OTA plays a causative or only a subordinate role in the induction of this human nephropathy. Tunisia for years had to confront a very similar human nephropathy, which is tentatively called chronic interstitial nephropathy of unknown cause. This study tends firstly to consolidate the suspected link between this Tunisian chronic interstitial nephropathy (CIN) of unknown cause and the presence of OTA in the blood and food of such patients, and second to enlighten the endemic character of this particular nephropathy. Therefore, in four consecutive inquiries, performed within the period 1991-2000, blood and food OTA contaminations were assayed and compared for 954 nephropathy patients and 205 healthy subjects from the Tunisian general population. This survey was also designed to show that, although the whole population is likely to be exposed to OTA, specific people living in conditions showing similarities with the Balkans do have a kidney disease apparently linked to ochratoxin in food. The results showed that the highest incidences were found in patients with CIN of unknown cause. Indeed, the percentages of OTA-positive samples ranged from 93% to 100%, whereas it was only from 62% to 82% in healthy subjects. Mean OTA concentrations were also higher in patients with CIN of unknown cause than in controls (44.4±-19 mg/L to 55.6±-19 mg/L as opposed to 1.22±-1.2 mg/L to 3.35±-2.32 mg/L, respectively). This study emphasizes further the implication of OTA on this particular human nephropathy and underlines the probable causative role of OTA in the onset of this disease. It is important to note that the highest levels of food OTA contamination were found in the group presenting with CIN of unknown cause, indicating that, similar to the case in the Balkans, people are exposed to OTA essentially by their food.

Sister chromatid exchange frequency in cultured isolated porcine urinary bladder epithelial cells (PUBEC) treated with ochratoxin A and alpha.

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha (OT-alpha) were investigated, to examine their potency to induce sister chromatid exchages (SCE) in cultured poricine urinary baldder epithedial cells (PUBEC) (primary cluture). Serum-free cultured PUBEC were incubated for 5 h with either OTA or OT-alpha, respectively, and subsequently cutured in the presence of 5-bromo-2-deoxyuridine (BrdU). After two cell cycles, mitosis was inhibited by the colchicine derivative Colcemid, cells were fixed and chromosomes were prepared for SCE analysis. For OTA, a dose-dependent increase in SCE frequency was measured in concentrations between 100 pM and 100 nM OTA. At 100nM OTA, SCE frequency increased by about 41%, compared to the base SCE level (7.27 SCEs per chromosome set, solvent control). Higher concentrations of OTA were cytotoxic. The metabolite OT-alpha also increased SCE frequency, but at higher concentrations. At a concentration of 10μM OT-alpha, an increase of about 55% was detected. OT-alpha showed no cytotoxic effect. There results indicate that OTA is genotoxic in this in vitro system, which represents the urinary bladder epithelium, a target organ of OTA in vivo. It could also be shown that OT-alpha, which is said to be non-toxic, is genotoxic in this assay at higher concentrations.