Evaluation of Cytotoxicity and Cell Death Induced In Vitro by Saxitoxin in Mammalian Cells.

Since the cyanotoxin saxitoxin (STX) is a neurotoxin and induces ecological changes in aquatic environments, a potential risk to public and environmental health exists. However, data on STX-mediated cytotoxic and genotoxic effects are still scare. In order to gain a better understanding of the effects of this toxin, the cytotoxic and genotoxic potential of STX was examined in two mammalian cell lines. Neuro 2A (N2A), a neuroblastoma mouse cell line, and Vero cell line, derived from Vero green monkey kidney cells, were exposed to several concentrations of STX ranging from 0.5 to 64 nM to determine cell viability, induction of apoptosis (DNA fragmentation assay), and formation of micronuclei (MN) (cytokinesis-block micronucleus assay; CBMN) following 24 h of incubation. The half maximal effective concentration (EC50) values for STX calculated in cell viability tests were 1.01 nM for N2A and 0.82 nM for Vero cells. With increasing STX concentration there was evidence of DNA fragmentation indicating apoptosis induction in Vero cells with a 50% increase in DNA fragmentation compared to control at the highest STX concentration tested (3 nM). The results demonstrated no significant changes in the frequency of micronucleated binucleated cells in N2A and Vero cells exposed to STX, indicating the absence of genotoxicity under these test conditions. There was no apparent cellular necrosis as evidenced by a lack of formation of multinucleated cells. In conclusion, data reported herein demonstrate that STX produced death of both cell types tested through an apoptotic process.

DNA-adducts in subjects exposed to urban air pollution by benzene and polycyclic aromatic hydrocarbons (PAHs) in Cotonou, Benin.

Air pollution effect on humans represents a major public health problem. Exposure to genotoxic compounds in the ambient air is evaluated using different biomarkers. In the present study we assessed DNA-adducts levels in apparently healthy people living and working in the city of Cotonou (Benin) in which exposure to air pollutants such as benzene and polycyclic aromatic hydrocarbons (PAHs) mainly benzo(a)pyrene has been evidenced. Rural inhabitants were enrolled as control group. Taxi-motorbike drivers, street food vendors, and gasoline salesmen were recruited in Cotonou whereas suburban residents were recruited in Godomey, 12 km from Cotonou. We found that taxi-motorbike drivers, roadside residents, street vendors, taxi-motor-bike drivers and gasoline sellers had significantly higher levels of DNA-adducts than suburban and village inhabitants (P < 0.001; post hoc, LSD). Means values were 24.6 ± 6.4, 23.78 ± 6.9, 34.7 ± 9.8, and 37.2 ± 8.1 in the exposed groups versus 2.1 ± 0.6 and 3.1 ± 0.8 adducts/10(8) nucleotides, in the two control groups, respectively. We did not find any significant difference within the high exposure groups and inside low exposure subgroups (namely suburban residents and villagers) because the mean individual exposure values to both PAHs and benzene were similar among subjects exposed in the city of Cotonou and those in suburban and village areas. However, there is significant interindividual variations in adducts levels that may reflect variation of genetic susceptibility factors. Ranges of adduct level/10(8) nucleotides were: 1-69, 1-76, 3-169, 4-124, 0-9, 0-8 adducts/10(8) for taxi-motorbike drivers, roadside residents, street vendors, gasoline sellers, suburban and village inhabitants, respectively. Our study demonstrated a clear-cut elevated level of DNA adducts in city residents than in none exposed people (or very low exposure levels people) and designate these city residents groups as people at risks for the chronic diseases possibly caused by benzene and PAHs.

The cytotoxicity and genotoxicity of okadaic acid are cell-line dependent.

Okadaic acid (OA) is a polyether fatty acid produced mainly by dinoflagellates causing diarrhoeic shellfish poisoning (DSP) in humans. To resolve the controversies concerning its genotoxicity in vitro, we have investigated eventual specific cellular response in DOK, Caco-2 (Deltap53/p53(-)), HepG-2 and C6 glioma cells using the DNA damage detection test (3d DNA repair test: nucleotide excision repair (NER) and base excision repair (BER)), caspase-3-triggered apoptosis, neutral red (NR) and lactate dehydrogenase (LDH) release tests. At low concentrations of OA (10nM), cytotoxicity measured by LDH release is more marked in DOK cells, indicating necrotic cell death that occurs only slightly in HepG-2 cells. At the same concentration, caspase-3 activation-dependent apoptosis and DNA damage caused by OA were only detected in HepG-2 cells. This apoptosis appears to be p53 gene dependent. Cell death occurs in the other cell types only by necrosis at OA concentrations amended to cultures. Among the tested cell lines, HepG-2 cells are the most sensitive to OA (10-50nM) at 12 and 72h as revealed by the NR test. The 3D test shows that only HepG-2 cells bear damaged DNA at tested concentrations. It is concluded that the genotoxicity of OA is chiefly cell type dependent and concentration dependent, giving sense to controversial genotoxicity data found in the literature.

Evaluation of the cytotoxicity and genotoxicity of extracts of mussels originating from Moroccan Atlantic coast, in human colonic epithelial cells Caco-2.

Industrial processing of phosphates generates chemical wastes which are, without any treatment, discharged directly into the Atlantic Ocean at Jorf Lasfar (JL), located 120 km south of Casablanca (Morocco) were shellfish are also collected by people without any control. Marine bivalves concentrate these pollutants by filtration and serve as vectors in human's exposure. The objective of this study was to test and compare in vitro on human intestinal cells (Caco-2) the cytotoxicity and genotoxicity of mussels (Mytilus galloprovincialis) extracts (either hydrophilic or lipophilic) collected at two coastal sites; JL (neighboring a phosphate processing plat-form) and Oualidia (OL) (a vegetable growing area) located 160 km south of Casablanca (i.e. 40 km south of JL). Using Caco-2 cells, the following end-points have been evaluated, cytotoxicity as measured by MTS test, inhibition of cellular macromolecules syntheses (DNA and protein) and genotoxicity evaluated by DNA fragmentation in agarose gel electrophoresis. The results indicated, that hydrophilic and lipophilic OL mussels extracts are cytotoxic and inhibit cellular macromolecules syntheses. Moreover these extracts damage the DNA in Caco-2 cells. The lipophilic JL mussels extract is cytotoxic, inhibits cellular macromolecules syntheses, and damages the DNA in Caco-2 cells whereas the hydrophilic extract of JL mussels fails to inhibit protein synthesis and does not damage the DNA. This extract rather enhances protein synthesis, suggesting possible metallothioneins induction by metal ions. Altogether these in vitro data indicate that mussels collected from OL could be more harmful than those from JL even though the later is closer to the pollution site than OL. Nevertheless consumption of mussels from all these areas may present a risk for humans. Epidemiological studies will be needed for global risk assessment in humans living in these areas especially those consuming see food regularly.

Effect of polyhexamethylene biguanide on rat liver.

Polyhexamethylene biguanide (PHMB), an amphiphilic polymeric biocide, increased liver tumor incidence in male and female rats at 1000 and 1500 mg/L in drinking water, but not at 500 mg/L in previous studies. In another study, PHMB administered in diet at 4000 mg/kg was negative for hepatocellular tumors. The present studies evaluated bioavailability and distribution of PHMB administered in drinking water and diet and possible modes of action (MOA). PHMB in drinking water was unpalatable during the first 3 days, resulting in markedly decreased food consumption and decreased body weight. Ki-67 labeling index was increased in hepatocytes and endothelial cells dose responsively with PHMB administered in drinking water but not diet. Vitamin E had no effect on this. There was no cytotoxicity by histopathology or serum enzymes, and no increase in cytokines TNFα, IL-1α or NF-κB. Focal iron deposition in sinusoidal lining cells was detected. Microarray analyses were non-contributory. No effect on CAR or PPARα activation was detected. 14C-PHMB administered at 500, 1000, or 1500 mg/L in the drinking water or 4000 mg/kg in the diet was nearly completely absorbed and excreted in urine, with some fecal excretion. The hypothesized MOA for liver tumors induced by PHMB in drinking water is: 1) severe dehydration and starvation because of unpalatability, followed by ingestion with rapid absorption and urinary excretion; 2) increased hepatocyte proliferation; and 3) induction of hepatocellular foci and tumors. The PHMB-induced rat hepatocellular tumors are unlikely to pose a human cancer risk. However, the actual MOA has not been determined.

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.

A quinoxaline 1,4-di-N-oxide derivative induces DNA oxidative damage not attenuated by vitamin C and E treatment.

Some anticancer compounds are pro-drugs which give rise to toxic species through enzymatic reduction. The quinoxaline-di-N-oxide derivative Q-85 HCl (7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride) is a bioreductive compound selectively toxic in hypoxia. Due to the possibility of secondary tumors the study of the genotoxic capability of antitumoral drugs is very important. The aim of this study was to assess the ability of Q-85 HCl to produce reactive oxygen species (ROS) and oxidative DNA damage in Caco-2 cells, both in hypoxia and in well-oxygenated conditions. Secondly, we attempted to evaluate the effect of vitamins C and E under hypoxic and normoxic conditions, in order to determine if these antioxidant substances modify Q-85 HCl effect in hypoxic cells or possibly exert a protective action in normal cells. Caco-2 cells were treated with Q-85 HCl for 2h, at high concentrations in normoxia (0.1-5 microM) and at low concentrations in hypoxia (0.002-0.1 microM). In normoxia, a dose-related significant increase in intracellular ROS level was evident; in hypoxia all the concentrations produced very high level of ROS. Just after the treatment and 24h later, oxidative DNA damage was evaluated by the modified comet assay after post-digestion of the cells with formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (Endo III). Q-85 HCl treatment evoked a significant dose-dependent increase in the total comet score of the cells both in hypoxia and normoxia, indicating that this compound or some metabolite is able to oxidize purine and pyrimidine bases. After 24h DNA damage caused by the compound was completely repaired with only one exception: cells treated with the highest concentration of Q-85 HCl in hypoxia and post-digested with FPG. Vitamin C (5-100 microM) and vitamin E (500-400 microM) did not have a pro-oxidant effect in Caco-2 cells. Treatment of cells with vitamin C (10 microM) or vitamin E (100 microM) did not significantly reduce oxidative DNA damage in hypoxia and normoxia. In conclusion, the use of these vitamins would not hinder toxicity against hypoxic cells, but a protective effect in normoxic cells was not evident.

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.

Survey of air pollution in Cotonou, Benin--air monitoring and biomarkers.

Exposure to genotoxic compounds present in ambient air has been studied in Cotonou, Benin, a city where two-stroke motorbikes are the major form of transportation and gasoline quality is poor. Personal monitoring and biomarkers were used to assess the exposure. Non-smoking taxi-moto drivers (city) and village residents were the study subjects. Benzene exposure was significantly higher in the city, as compared to the village (76.0+/-26.8 microg/m(3) versus 3.4+/-3.0, p=0.0004). Urinary excretion of benzene and S-phenylmercapturic acid (S-PMA) were also highest in subjects living in the city, whereas 1-hydroxypyrene was not different. The level of total polycyclic aromatic hydrocarbons (PAHs), associated with particles, ranged from 76.21 to 103.23 in Cotonou versus 1.55 ng/m(3) for the village. Determination of DNA damage in lymphocytes showed that subjects from the city had elevated number of lesions compared to subjects in the village in terms of bulky DNA adducts, 8-hydroxy-2'-deoxyguanosine and 5-methylcytosine, whereas DNA fragmentations analysed by alkaline gel electrophoresis was not different between the subjects. In conclusion, this study shows that air pollution is pronounced in Cotonou, Bénin and is associated with elevated levels of DNA damage in residents of the city compared to people living in a non-polluted rural village.

Comparative study of Domoic Acid and Okadaic Acid induced-chromosomal abnormalities in the Caco-2 cell line.

Okadaic Acid (OA) the major diarrheic shellfish poisoning (DSP) toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA), the major Amnesic Shellfish Poisoning (ASP) toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN) arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA) and domoic acid (DA) to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH) using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.

Upholding science in health, safety and environmental risk assessments and regulations.

A public appeal has been advanced by a large group of scientists, concerned that science has been misused in attempting to quantify and regulate unmeasurable hazards and risks.1 The appeal recalls that science is unable to evaluate hazards that cannot be measured, and that science in such cases should not be invoked to justify risk assessments in health, safety and environmental regulations. The appeal also notes that most national and international statutes delineating the discretion of regulators are ambiguous about what rules of evidence ought to apply. Those statutes should be revised to ensure that the evidence for regulatory action is grounded on the standards of the scientific method, whenever feasible. When independent scientific evidence is not possible, policies and regulations should be informed by publicly debated trade-offs between socially desirable uses and social perceptions of affordable precaution. This article explores the premises, implications and actions supporting the appeal and its objectives.

[Comparative evaluation of the toxicity induced by mussels (Mytilus galloprovincialis) from two sites of the Moroccan Atlantic coast in mice]. / Evaluation comparée de la toxicité des moules (Mytilus galloprovincialis) de deux sites du littoral atlantique marocain sur des souris.

Atlantic coast in mice. Preliminary studies showed that seawater contains heavy metals from domestic, agricultural and industrial wastes. Marine bivalves concentrate these pollutants by filtration and serve as vectors in human exposure. The objective of this study was to determine the concentration of heavy metals; cadmium (Cd); chromium (Cr), and lead (Pb) in mussels (Mytilus galloprovincialis) collected in two coastal sites; Jorf Lasfar (JL) (neighbouring a phosphate processing platform) and Oualidia (OL) (a vegetable growing area) located at 120 and 190 km south of Casablanca, respectively. Another objective was to test and compare the toxicity of these mussels on mice. The results indicated the presence of heavy metals (Cd, Cr, and Pb) in mussels at different concentrations, depending on the collection period. Higher concentrations were obtained at JL than at OL: for example, Cd concentrations were 80 +/- 15 to 199 +/- 28 versus 23 +/- 5 microg/g mussel dry weight, respectively. Cramming with mussel powder did not increase Cd, Cr, or Pb concentration in either liver or kidneys of treated mice. The relative kidney weights were reduced. Increased glucose urea was observed in animals' urine. Treatment with mussels from OL induced significant reduction (20%) in mice body weight, together with an increase in creatinuria. These results indicate that mussels collected from OL are more harmful than those obtained from JL are. All these mussels should not be recommended for human consumption.

Cytotoxicity of abnormal Savda Munziq aqueous extract in human hepatoma (HepG2) cells.

Abnormal Savda Munziq (ASMq) is a traditional Uighur medicinal herbal preparation commonly used to treat diseases such as diabetes, cardiovascular diseases, chronic asthma and especially digestive cancer. Earlier studies have shown that ASMq is a free radical scavenger and could prevent mitochondrial and DNA oxidative damage. In this study, we tested the effects of aqueous extract of ASMq on human hepatoma cells (HepG2) to explore the possible mechanism of its putative anticancer properties. Aqueous extract of ASMq was tested on HepG2 proliferation (MTT assay) at 72 h, cell viability at 48 h (neutral red assay), lactate dehydrogenase release over 48 or 72 h as a measure of cytoplasmic leakage, lipid peroxidation (malondialdehyde-thiobarbituric acid adducts) at 48 h, and incorporation of [3H]-leucine, [3H]-thymidine and [3H]-uridine into cellular protein, DNA and RNA, respectively, at 24 or 48 h to assess the inhibition effects to cellular macromolecule synthesis. Our results showed a significant (P < 0.05) time- and concentration-dependent inhibition of HepG2 proliferation and viability, with increased cytoplasmic leakage, and time- and concentration-dependent inhibition of protein, DNA and RNA synthesis. No lipid peroxidation was found at these concentrations. The results of the present study suggest that the putative anticancer mechanisms of ASMq may at least involve cytotoxicity.

How much should we involve genetic and environmental factors in the risk assessment of mycotoxins in humans?

Despite consented efforts in prevention, mycotoxins remain a problem of human health concern in several parts of the world including developed countries. Within the same range of toxins concentrations in the blood some people develop a disease while others do not. Could this inequality in front of mycotoxins effects be explained by environment factors and/or genetic predisposition? Among recent advances in environmental health research Correlation between chronic diseases and mycotoxins in humans deserves attention through several questions: Are genetic factors involved in disease causation of mycotoxins? How much are these factors currently taken into account for mycotoxins risk assessment and how much should we involve them? Answers are still to come. Genetic and environment factors deserve therefore more attention when dealing with regulatory limits, since among the general population, those who are at risk and will develop specific diseases are likely those bearing genetic predispositions. We have addressed these questions for the specific case of ochratoxin A in humans by investigating in Tunisia, county of Jelma, in four rural families forming a household of 21 persons all exposed to ochratoxin A in diet. Our results confirm that ochratoxin A induces chronic tubular nephropathy in humans and mainly point at those having the HLA haplotype A3, B27/35, DR7 to be more sensitive to the disease for quantitatively similar or lower exposure. Persons with such haplotype were found to bear chronic interstitial nephropathy with tubular karyomegalic cells while others were apparently healthy. Godin et al. (1996) in France have also found in sibling (a sister and her brother from urban area) that have similar HLA haplotype B35-patern, OTA-related renal tubulopathy with mild proteinuria including beta2-microglobulinuria. Several mechanisms are discussed that could be put ahead to explain how the HLA haplotype could lead to tubular cells lyses and renal failure. In the mean time it is urgent to search for mass screening biomarkers for mycotoxins in humans and related genetic factors to set-up more appropriate regulation.

Synergistic effects of fumonisin B1 and ochratoxin A: are in vitro cytotoxicity data predictive of in vivo acute toxicity?

Contamination of food and feeds by mycotoxins is a major problem of human and animals health concern which is also extremely detrimental to economy. Mycotoxins producing moulds may produce a diversity of toxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, tremorgenic toxins and ergot alkaloids. Although toxicological, environmental and epidemiological studies have addressed the problem of these toxins one by one, more than one mycotoxin are found usually in the same contaminated commodities. That rises the incommensurable problem of multi-toxicosis in which the respective metabolites are also involved. These mycotoxins bear potential toxicity leading to acute and chronic effects in humans and animals, depending on species. The mechanisms that lead to toxic effects, such as immune toxicity, and carcinogenicity are complexe. The risk assessment for humans potentially exposed to multi-mycotoxins suffers very much from the lack of adequate food consumption data. Furthermore, for a given mycotoxin synergism and antagonism with other mycotoxins found in the same food commodities are not taken into account. The case of combination of ochratoxin A (OTA) and fumonisin B1 (FB1) has been addressed in the present paper with the purpose of predicting the in vivo toxicity using a simple in vitro test, i.e. neutral red uptake, in three different cell-lines, C6 glioma cells, Caco-2 cells and Vero cells. Using the equation of [ATLA 27 (1999) 957], in vivo toxicity (LD50) is in adequation with the in vitro data, (IC50 values) for both toxins as well as for the combination of 10 microM OTA and variable concentrations of FB1 (10-50 microM). A synergistic effect is prouved in vitro that is in line with some in vivo data from the literature. Such simple in vitro test may thus help predicting in vivo toxicity of combinations of mycotoxins naturally occurring in foodstuffs.

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.

Comparative study of the toxic effects of fumonisin B1 in rat C6 glioma cells and p53-null mouse embryo fibroblasts.

The present experiments have been carried out in order to study (comparatively) oxidative stress and its consequences (i.e. modifications of DNA bases and/or DNA fragmentation), cell cycle progression (through two generations) and apoptosis in C6 glioma cells (with normal p53 status) and p53-null mouse embryonic fibroblasts (MEF) after incubation with fumonisin B(1) (FB(1)). Further endpoints, including protein and DNA syntheses as well as cytotoxicity, have been also studied. The results show that FB(1) (incubation) produced a significant increase of malondialdehyde (MDA) production (suggestive of lipid peroxidation) which was prevented by antioxidant agents in both cell types. Moreover, FB(1) induced a significant and dose-related increase of 8-OH-dG and DNA fragmentation in both C6 glioma and MEF cells. Unlike MEF cells, apoptotic C6 glioma cells were observed after FB(1) incubation. Moreover, suppression of cell cycle progression was observed in C6 glioma but not in MEF cell incubated with FB(1). The results suggest a possible loss of protective mechanisms (such as p53-dependent apoptosis and cell cycle arrest) in FB(1)-damaged MEF cells and confirm that cells lacking of mechanisms governed by p53 gene would be more susceptible to neoplastic cascade or mutation following DNA lesions induced by this mycotoxin.

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.

Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin mainly produced by Fusarium graminaerum, found as a world-wide contaminant mainly of corn and wheat. Previous studies have demonstrated that among several other effects on animals and humans, ZEN also displays hepatotoxicity, immunotoxicity and nephrotoxicity. ZEN is mainly known as a hormonal disrupter due to its estrogenic activities and consequent toxicity for reproduction. Furthermore, mutagenic and genotoxic proprieties of ZEN were disclosed recently, the molecular mechanisms of which are not yet well understood. In the present study, the genotoxic potential of ZEN was evaluated using genotoxicity tests: the 'cytokinesis block micronucleus assay' in Vero monkey kidney cells and the 'in vivo mouse bone marrow micronucleus assay'. In cultured cells treated with 5, 10 and 20 microM ZEN, the frequency of binucleated micronucleated cells (BNMN) was assessed in 1000 binucleated cells and in mice given oral doses of 10, 20 and 40 mg/kg bw, the frequency of polychromatic erythrocytes micronucleated (PCEMN) in bone marrow cells was assessed in 2000 polychromatic erythrocytes (PCE). The potential prevention of ZEN-induced effects by 25 microM Vitamin E (Vit E) was also evaluated. In vivo, doses of 10, 20 and 40 mg/kg bw ZEN representing, respectively 2, 4 and 8% of the LD50 (LD50 of ZEN in mice is 500 mg/kg bw), were administered to animals either with or without pre-treatment with Vit E (216.6 mg/kg bw) in order to evaluate its preventive potential.ZEN was found to induce micronuclei (MN) in a dose-dependent manner in cultured Vero cells as well as in mouse bone marrow cells. The present data emphasise the likely clastogenic pathway among the molecular mechanisms that underlay the ZEN-induced genotoxicity. Vit E was found to prevent partially-from 30 to 50%-these toxic effects, most likely acting either as a structural analogue of ZEN or as an antioxidant.

Fungal flora and ochratoxin a production in grapes and musts from france.

Eleven samples of grapes and musts used in red table wines were investigated for the occurrence of potential ochratoxin A (OTA)-producing molds. From these samples, 59 filamentous fungi and 2 yeasts were isolated. Among the 30 genera isolated, Deuteromycetes were the most frequent (70%) followed by Ascomycetes (10%). Six of the eleven grapes samples were contaminated by potentially ochratoxinogenic strains (Penicillium chrysogenum and Aspergillus carbonarius). When cultivated in vitro on solid complex media, the 14 strains of A. carbonarius produced OTA. No other species produced OTA under the same conditions. Among must samples, eight of eleven were found to be contaminated by OTA (concentrations from <10 to 461 ng/L). There is a strong correlation between the presence of ochratoxin-producing strains on grapes and OTA in musts. These findings should be connected with the OTA contamination of human blood in these areas and in France.