Théophile A. Mobio

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.

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.

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.

Prevention by vitamin E of DNA fragmentation and apoptosis induced by fumonisin B1 in C6 glioma cells

Abstract Fumonisin B1 (FB1), produced by the fungus Fusarium moniliforme, belongs to a class of sphingosine analogue mycotoxins that occur widely in the food chain. Epidemiological studies have associated consumption of Fusarium moniliforme-contaminated food with human oesophageal cancer in China and South Africa. FB1 also causes equine leucoencephalomalacia. Evidence for induction of apoptosis by FB1 was first obtained when C6 glioma cells were incubated with fumonisin B1 (3–27 μM) causing DNA fragmentation profiles showing DNA laddering in gel electrophoresis and apoptotic bodies revealed by chromatin staining with acridine orange and ethidium bromide. Further confirmation experiments and comet assays have been performed under similar conditions. The results of the comet test show that FB1 at 9 and 18 μM induces respectively 50 ± 2% and 40 ± 1% of cells with a comet with an increased tail length of 93 ± 9 μm and 102 ± 17 μm respectively. Under these concentrations, FB1 induced DNA fragmentation and laddering and many apoptotic bodies. Pre-incubation of the cells with vitamin E (25 μM) for 24 h before FB1 (18 μM) significantly reduced DNA fragmentation and apoptotic bodies induced by FB1.

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 humans 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.