Hassen Bacha

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.

Different apoptotic pathways induced by zearalenone, T-2 toxin and ochratoxin A in human hepatoma cells.

Mycotoxins, secondary metabolites produced by moulds, have been shown to cause diverse toxic effects in animals and are also suspected of disease causation in humans. The present study compares the molecular mechanisms of the toxicity of zearalenone (ZEN), T-2 toxin and ochratoxin A (OTA) in human hepatoma cells HepG2. The three mycotoxins-induced a caspase-dependent mitochondrial apoptotic pathway. The mitochondrial alterations include: bax relocalisation into the mitochondrial outer membrane, loss of the mitochondrial transmembrane potential, PTPC opening, and cytochrome c (but not AIF) release. In the presence of ZEN and T-2 toxin, reactive oxygen species (ROS) level was highly increased at an early stage even before mitochondrial alterations were observed, whereas OTA-induced only O(2)(-) generation among total ROS. This ROS production appears as a consequence of mitochondrial alterations. HepG2 cell treatment with the p53 inhibitor pifithrin-alpha (PFT) and western blot analysis suggested that both ZEN and OTA, but not T-2 toxin, trigger a p53-dependent apoptotic pathway. These results clearly point to a central role of mitochondria in the apoptotic process induced by ZEN, T-2 toxin and OTA and provide new insights into the molecular mechanisms by which these mycotoxins might promote hepatotoxicty.

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.

Cactus (Opuntia ficus-indica) cladodes prevent oxidative damage induced by the mycotoxin zearalenone in Balb/C mice

Zearalenone (ZEN) is one of the most widely distributed fusarial mycotoxins which is encountered at high incidence in many foodstuffs. ZEN was associated with different reproductive disorders in animals. Several in vivo studies have shown that ZEN is hepatotoxic, haematotoxic and causes several alterations of immunological parameters. Furthermore, evidence of its cytotoxicity and genotoxicity has recently emerged from several reports. The aim of the current study was (i) to find out whether oxidative stress could be relevant for ZEN induced toxicity in vivo using Balb/c mice and (ii) to evaluate the safety and efficacy of cactus cladodes Opuntia ficus to prevent the deleterious effects of ZEN. To this end, the effect of a single dose of ZEN (40 mg/kg b.w.) alone and with extract of cactus cladodes (25, 50 and 100 mg/kg b.w.) on the induction of oxidative stress was monitored in kidney and liver by measuring the MDA level, the protein carbonyls generation, the catalase activity and the expression of the heat shock proteins (Hsp). Our results clearly showed that ZEN induced significant alterations in all tested oxidative stress markers. Oxidative damage seems to be a key determinant of ZEN induced toxicity in both liver and kidney of Balb/c mice. The combined treatment of ZEN with the lowest tested dose of cactus extracts (25 mg/kg b.w.) showed a total reduction of ZEN induced oxidative damage for all tested markers. It could be concluded that cactus cladodes extract was effective in the protection against ZEN hazards. This could be relevant, particularly with the emergent demand for natural products which may counteract the detrimental effects of oxidative stress and therefore prevent multiple human diseases.

Individual and combined effects of ochratoxin A and citrinin on viability and DNA fragmentation in cultured Vero cells and on chromosome aberrations in mice bone marrow cells.

Ochratoxin A (OTA) and citrinin (CTN) are two common contaminant mycotoxins which can occur jointly in a wide range of food commodities. Both mycotoxins have several toxic effects but share a significant nephrotoxic and carcinogenic potential since OTA and CTN were reported to be responsible for naturally occurring human and animal kidney diseases and tumors. Considering the concomitant production of OTA and CTN, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Therefore, the aim of the present study was to investigate, in vivo and in vitro, whether DNA damage is enhanced by combination of both mycotoxins as compared to their effect separately. To this end, we have assessed their effects individually or combined on cell proliferation and DNA fragmentation in cultured Vero cells and in vivo by monitoring the induction of chromosome aberrations. Our results clearly showed that cultured renal cells respond to OTA and CTN exposure by a moderate and weak inhibition of cell proliferation, respectively. However, when combined, they exert a significant increase in inhibition of cell viability. Similar results were found for the investigated genotoxicity endpoints (DNA fragmentation and chromosome aberrations). Altogether, our study showed that OTA and CTN combination effects are clearly synergistic. The synergistic induction of DNA damage observed with OTA and CTN taken concomitantly could be relevant to explain the molecular basis of the renal diseases and tumorogenesis induced by naturally occurring mycotoxins.

Cytotoxicity and genotoxicity induced by aflatoxin B1, ochratoxin A, and their combination in cultured Vero cells.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are important food‐borne mycotoxins that have been implicated in human health. In this study, independent and combinative toxicities of AFB1 and OTA were tested in cultured monkey kidney Vero cells. The experiments reported here were conducted to evaluate the effect of these toxins on cell viability followed by the determination of cell death pathways, using the quantification of DNA fragmentation and the expression of p53 and bcl‐2 protein levels. Our results showed that AFB1 and OTA caused a marked decrease of cell viability in a dose‐dependent manner. Under the same conditions, these mycotoxins increased fragmented DNA levels. In addition, p53 was activated in response to DNA damage and the expression of the antiapoptotic factor bcl‐2 decreased significantly. According to these data, AFB1 and OTA seemed to be involved in an apoptotic process. Moreover, combined AFB1 and OTA induced all the toxicities observed with the mycotoxins separately. Therefore, this combination was classified as an additive response of the two mycotoxins.

The mycotoxin Zearalenone induces apoptosis in human hepatocytes (HepG2) via p53-dependent mitochondrial signaling pathway

Zearalenone (Zen) is a fusarial mycotoxin commonly found in several food commodities worldwide. It is frequently implicated in reproductive disorders and exerts several genotoxic effects in vivo and in vitro. In response to DNA damage, cells may undergo an intricate network of different pathways including apoptosis. Meanwhile, data regarding the induction of apoptosis after Zen exposure are limited. Thus, the aim of this study was to demonstrate whether Zen-induced DNA damage can lead to apoptosis as a stress response and which pathways are undertaken. Our results clearly show that Zen reduces cell proliferation in HepG2 cells in a dose-dependent manner as attested by the MTT assay (IC50%, 100microM). The analysis of propidum iodide uptake has shown that the amount of necrotic cells was about 6% among 55% of dead cells (at 120microM of Zen). The involvement of apoptosis as a major cause of Zen-induced cell death was further confirmed but results of caspase-3 activity showed a Zen-dose dependant increase. Furthermore, results of microarrays analysis have shown that Zen induced an upregulation of ATM and p53 genes family. ATM pathway responds primarily to DNA double-strand breaks and has been involved in the activation and stabilization of p53. The activation of p53 was accompanied by an upregulation of GADD45 to arrest the cell cycle and to allow the repair mechanisms to take place. In addition, results of genes profiling as well as western-blotting analysis showed that Zen increased the ratio of pro-apoptotic factors/anti-apoptotic factors which led to the loss of mitochondrial potential, Bax translocation and cytochrome c release. Once released, cytochome c activates caspase 9 which in turn activates caspase-3 and enhances apoptosis. In summary, these data suggested that Zen induced apoptosis in a dose-dependent manner in HepG2 cells via a p53-dependent mitochondrial pathway.

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.

Hsp70 expression as biomarkers of oxidative stress: mycotoxins' exploration.

The environment represents a key contributor to human health and disease. Exposure to many substances such as pollutants, toxins and chemicals, has detrimental effects on health and are considered to contribute substantially to most diseases of major public health significance. Environmental diseases as mycotoxicosis are those in general aroused or exacerbated by exposure to environmental stressors as mycotoxins. These hazardous compounds are secondary metabolites produced by fungi and occurred simultaneously in food, feed and raw materials. The present investigation was conducted to assess if (i) Hsp70 induction, a parameter of protective and adaptive response, is a systematic biomarker to mycotoxin intoxications and (ii) all mycotoxins undergo oxidative stress in there toxic signalling pathways, as the omnipresent process playing a role in the initiation or progression of numerous disorders. Overall, observations to date evoke that Hsp70 can act as biomarkers of oxidative injury instead they are not systematic to mycotoxin exposure.

Pathway of deoxynivalenol-induced apoptosis in human colon carcinoma cells.

The mycotoxin, deoxynivalenol (DON), is generally detected in cereal grains and grain-based food products worldwide. Therefore, DON has numerous toxicological effects on animals and humans. The present investigation was conducted to determine the molecular aspects of DON toxicity on human colon carcinoma cells (HT 29). To this aim, we have monitored the effects of DON on (i) cell viability, (ii) Heat shock protein expressions as a parameter of protective and adaptive response, (iii) oxidative damage and (iv) cell death signalling pathway. Our results clearly showed that DON treatment inhibits cell proliferation, did not induce Hsp 70 protein expression and reactive oxygen species generation. We have also demonstrated that this toxin induced a DNA fragmentation followed by p53 and caspase-3 activations. Finally, our findings suggested that oxidative damage is not the major contributor to DON toxicity. This mycotoxin induces direct DNA lesions and could be considered by this fact as a genotoxic agent inducing cell death via an apoptotic process.