In vitro and in vivo induction of chromosome aberrations by alpha- and beta-zearalenols: comparison with zearalenone.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by Fusarium fungi. It contaminates different components of the food chain and can cause serious economic and public health problems. The major metabolites of ZEN in various animal species are alpha- and beta-zearalenol (α-, ß-ZOL). Some in vivo studies have shown that these two metabolites are as toxic as the mother molecule (ZEN), but other investigations have demonstrated that α- and ß-ZOL are less toxic than ZEN. Thus, the aim of the present study was to evaluate cytotoxicity and genotoxicity of α- and ß-ZOL in vivo, in mouse bone-marrow cells and in vitro, in cultured HeLa cells, and to compare it with ZEN. ZEN showed the same cytotoxicity as α-ZOL and both are more cytotoxic than ß-ZOL. Genotoxicity of ZEN and its derivatives was assessed by the chromosome aberration assay. Our results show that ZEN as well as α- and ß-ZOL increased the percentage of chromosome aberrations in mouse bone-marrow cells and in HeLa cells. In the two systems, ZEN and α-ZOL exhibited the same range of genotoxicity and both were more genotoxic than ß-ZOL. Furthermore, our results show that either ZEN or its two metabolites inhibited cell viability in a dose-dependent manner. We conclude that biotransformation of ZEN may be considered as only a partial detoxification pathway since the resulting metabolites remain relatively toxic.

Single and combined genotoxic and cytotoxic effects of two xenobiotics widely used in intensive aquaculture.

Several chemicals are used in aquaculture to prevent or to treat disease outbreaks. These substances are mainly administered by two different routes: by prolonged immersion or by mixing into the diet. In the case of intensive aquaculture, the chemicals that are most frequently applied by immersion are formaldehyde (FA) 37% and oxytetracycline (OTC). The first is highly effective against most protozoa, as well as some of the most common parasites such as monogenetic trematodes. OTC presents a large spectrum of antibacterial activities and is used to treat systemic bacterial infections that affect fish. Under therapeutic use, FA (37%) is applied prophylactically at 200ml/m(3), whereas OTC is used curatively at 40g/m(3). The goal of the present study is to assess genotoxic and cytotoxic effects associated with exposure of the European sea bass (Dicentrarchus labrax) to FA37% and OTC under the same conditions as those applied in intensive aquaculture systems. To this end the micronucleus (MN) assay was applied in erythrocytes. Our results show that both tested chemicals present genotoxic and cytotoxic potential following a time-dependent pattern. Remarkably, the combined treatment induces a cumulative effect, which is particularly pronounced after 15 days of exposure. This suggests the critical hazards associated with exposure to FA and OTC when applied or released together.

Isothiocyanate from the Tunisian radish (Raphanus sativus) prevents genotoxicity of Zearalenone in vivo and in vitro.

Zearalenone (ZEN) is a naturally occurring contaminant of animal feed that has been implicated in several mycotoxicoses in farm livestock. Recently some information has become available indicating that ZEN caused cancer or at least increased its prevalence, although the mechanism of action is unknown. Many papers mentioned that exposure to ZEN results in genotoxicity and DNA damage. Therefore, we investigated the chemo-preventive role of 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) extracted from Tunisian Raphanus sativus (radish) on the cytogenetic effect of ZEN in Balb/c mice and in in vitro cultures of mouse lymphocytes isolated from mouse spleen. We determined chromosome aberrations and micronuclei as well as the mitotic index and DNA fragmentation following ZEN treatment alone or in combination with MTBITC. This report is the first to provide evidence of a statistically significant decrease of structural chromosome aberrations and micronuclei associated with an augmentation of the mitotic index and prevention of DNA fragmentation in all mice treated with ZEN-MTBITC and in mouse lymphocyte cultures. The MTBITC alone was safe and succeeded in reducing the toxicity of ZEN by counteracting its deleterious effect, thus protecting against the genotoxicity and clastogenicity from ZEN.

Tunisian radish extract (Raphanus sativus) enhances the antioxidant status and protects against oxidative stress induced by zearalenone in Balb/c mice.

Radish (Raphanus sativus) is a food plant known worldwide. From antiquity it has been used in folk medicine as a natural drug against many toxicants. Zearalenone (zen) is a non-steroidal estrogenic mycotoxin present in corn and food mixture for farm animals and it is hepatotoxic, hematotoxic, immunotoxic, nephrotoxic and genotoxic. The objectives of the present study were to assess the biological activity of radish extract and to evaluate the protective role of radish extract against the toxicity of zen in female Balb/c mice. Animals were divided into seven groups and treated orally for 10 days as follows: a control, an olive oil group, groups treated with radish extract alone (5, 10 and 15 mg kg(-1) b.w.), a group treated with zen (40 mg kg(-1) b.w.) and a group treated with zen plus the lowest dose of radish extract. The results indicate that radish extract improved the antioxidant status and had no significant effects on hematological and biochemical parameters tested or histology of the liver and kidney. Treatment with zen results in a significant increase in ALT, AST, ALP, BILT, BILD, CRE accompanied with significant changes in most of hematological parameters and the antioxidant enzyme activities, co-treatment of zen and the radish extract results in a significant reestablishment of hematological, serum biochemical parameters, and the histology of the liver and kidney. These findings suggest that radish extract is safe and can be overcome or, at least, significantly diminish zen effects.

Toxicities induced in cultured cells exposed to zearalenone: apoptosis or mutagenesis?

Zearalenone (ZEN) is a fusarotoxin produced mainly by Fusarium graminearum in temperate and warm countries. ZEN has several adverse effects on humans and animals. It has a strong estrogenic activity associated with hyperestrogenism and leads to several physiological alterations in the reproductive tract. Even though the mutagenic and genotoxic proprieties of ZEN have been described recently, its molecular mechanisms of action are not completely understood. The aim of this study was to determine the involvement of other possible mechanisms in ZEN-induced toxicities. Each of the following toxicities, cytotoxicity, cell cycle perturbation, genotoxicity, and mutagenicity, was monitored in Vero cells exposed to ZEN. Our results showed that ZEN-reduced cell viability correlated to cell cycle perturbation-induced DNA fragmentation, resulting in DNA-laddering patterns on agar gel electrophoresis. This observation is consistent with apoptosis, which was confirmed by induction of apoptotic bodies. Moreover, ZEN induced in a concentration-dependant manner the formation of micronuclei and chromosome aberrations. This apparent contradiction between the apoptotic and mutagenic effects of ZEN can be explained by the modification of normal cellular regulation inducing apoptotic or antiapoptotic factors resulting from a lack of or an incorrect DNA-reparation in relation to cell exposure to the toxin.

Preventive role of aluminosilicate clay against induction of micronuclei and chromosome aberrations in bone-marrow cells of Balb/c mice treated with Zearalenone.

Zearalenone (ZEN) is a potent estrogenic metabolite produced by some Fusarium species. No treatment has been successfully employed to remove ZEN contamination in foods. This study was conducted to evaluate the ability of hydrated sodium calcium aluminosilicate (HSCAS) to protect Balb/c mice against cytotoxicity and genotoxicity induced by ZEN. HSCAS was given via the oral route, either alone or simultaneously with a toxic intra-gastric dose of ZEN. The experimental approach comprised treatments of seven groups of mice. The first three groups received 400, 600 or 800 mg/kg bw of HSCAS. Two experimental groups received, respectively, ZEN alone (40 mg/kg bw, representing 8% of the LD(50)) and ZEN in combination with HSCAS at 400 mg/kg bw. The two control groups received distilled water and olive oil, respectively. The positive control groups received colchicine (4 mg/kg bw) for the micronucleus assay and mitomycin C (1mg/kg bw) for the chromosome aberration test. Forty-eight hours after treatment, the femur and tibia were dissected out and analyzed. The results show that ZEN was cytotoxic and genotoxic to Balb/c mice, as indicated by the increase in the frequencies of micronucleated polychromatic erythrocytes (PCEMN) and of chromosomal aberrations in bone-marrow cells. The simultaneous intra-gastric administration of HSCAS with ZEN resulted in a reduction in the number of PCEMN and a decrease of the chromosomal aberration frequency, and an increase in the number of polychromatic erythrocytes (PCE) in bone-marrow cells, compared with those in the group treated with ZEN alone. It could be concluded that HSCAS itself was safe and efficient in the prevention of the toxic effects of ZEN in the gastrointestinal tract.

Preventive role of phyllosilicate clay on the Immunological and Biochemical toxicity of zearalenone in Balb/c mice.

Zearalenone (ZEN), a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. ZEN and its metabolites have anabolic activities and induced severe stress on liver, kidney and immune system. The aims of the current study were twofold: (1) to investigate the changes in serum biochemical, immunological parameters and histological picture of spleen in ZEN-treated Balb/c mice and (2) to evaluate the safety and efficacy of HSCAS to ameliorate the deleterious effects of ZEN. The results indicated that a single dose of ZEN (40 mg/kg bw) significantly reduced total cholesterol, HDL, LDL, triglycerides, total protein, albumin, total count of WBCs, immunoglobulin profile (Ig A and Ig G) and T-cells subtypes (CD3+, CD4+, CD8+ and CD56+). Whereas, it significantly increased uric acid and urea and induced degenerative changes in the spleen tissues. Mice treated with HSCAS alone (400 mg/kg bw) were comparable to the control regarding all the tested parameters. While HSCAS at levels 600 and 800 mg/kg bw caused changes in some tested biochemical parameters. The combined treatment of ZEN and the lowest tested dose of HSCAS (400 mg/kg bw) showed a significant improvement of the immunological, biochemical and histological parameters. It could be concluded that HSCAS was effective in the protection against the hazards of ZEN at a dose as low as 400 mg/kg bw. These results supported our hypothesis that HSCAS tightly-bind and immobilized ZEN resulted in reduction of toxin bioavailability in animal's gastrointestinal tract.

The protective effect of hydrated sodium calcium aluminosilicate against haematological, biochemical and pathological changes induced by Zearalenone in mice.

Hydrated sodium calcium aluminosilicate (HSCAS), an anticaking agent for mixed feed, was added alone or simultaneously with a toxic Zearalenone (ZEN) dose to balb/c mice and was evaluated for its ability to restore damages induced by ZEN. The latter is a mycotoxin produced by fusarium genera; it is mainly known to induce several toxic effects such as hepatotoxicity, immunotoxicity and nephrotoxicity on animals and humans. The experimental approach consisted of eight treatments of six mice each by 400 mg/kg bw or 5 g/kg bw of HSCAS. Two experimental groups have received respectively ZEN alone at 40 (8% of LD50) and at 500 mg/kg bw (LD50). Two other groups have received ZEN at 40 or 500 mg/kg bw combined respectively with HSCAS at 400 mg/kg bw and 5 g/kg bw. The control groups received water or olive oil. Forty-eight hours after treatment, blood samples were collected for haematological and serum biochemical parameters measurements. ZEN treatment significantly increased hematocrit, haemoglobin, white blood cells: lymphocytes, eosinophils, neutrophils, monocytes and the most of biochemical serum parameters; it significantly reduced platelets and induced degenerative changes in the hepatic and renal tissues; while, the mixture of HSCAS with ZEN induced a reestablishment of haematological parameters, levels of serum biochemical enzyme activities and histological pictures of both liver and kidney. It also prevented general toxicity of ZEN. This was observed by the shift of LD50 for this toxin. Thus, our data strongly suggested that deleterious effects of ZEN could be overcome or, at least, significantly were diminished by HSCAS. Moreover, this sorbent by itself did not show any toxic effects.

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.

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.