Fatma Bensassi

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.

Oxidative Stress, Biochemical and Histopathological Alterations in the Liver and Kidney of Female Rats Exposed to Low Doses of Deltamethrin (DM): A Molecular Assessment

OBJECTIVE To evaluate histopathological alterations of the liver and kidney of female rats exposed to low doses of DM and its potential genotoxic activity. METHODS Female Wistar rats were randomly assigned to control (3 groups, 6 rats in each) and treatment groups (3 groups, 6 rats in each). They were subjected to subcutaneous injections of DM (at doses of 0.003, 0.03, and 0.3 mg/kg bw/d) after 30, 45, and 60 d, respectively. RESULTS Significant alterations were recorded in liver parenchyma induced by hepatic vacuolization, fragmented chromatin in nuclei, dilatation of sinusoids and congestions. Lesions within proximal and distal tubules were observed in the kidneys. Tissue congestions and severe alterations within glomeruli were visible. DM as a pyrethroid insecticide induced significant increase (P≤0.05) of plasma MDA concentrations after 45 d. A significant increase (P≤0.05) in plasma ALT (after 45 and 60 d) and AST (after 60 d) concentrations was recorded as compared to controls. During the whole experimental period the toxic agent provoked significant DNA damages (P≤0.05), especially in the dominance of classes 3 and 4 of obtained comet. CONCLUSION DM even at a very low dose displays harmful effects by disrupting hepatic and renal function and causing DNA damages in puberscent female rats. Low doses of DM are hepatotoxic and nephrotoxic.

Involvement of mitochondria-mediated apoptosis in deoxynivalenol cytotoxicity

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates cereal crops and harmfully affects the gastrointestinal tract. Since it is well known that mitochondria play a central role in apoptosis triggered by many stimuli, an effort was made to examine whether DON-induced cytotoxicity occurs through mitochondria-mediated apoptotic pathway. The intestinal system being one of the primary targets of mycotoxins, the human colon carcinoma cell line HCT116 was used in this study. Using flow cytometric analyses and immunofluorescence, we showed that DON at 100 μM induced a mitochondria-dependent apoptotic pathway associated with opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (ΔΨm), downstream generation of O₂·⁻ and cytochrome c release. The DON-induced apoptosis was accompanied by an activation of caspase 9 and 3, as demonstrated by Western blot and caspase activity assay. In addition, by taking advantage of HCT116 cells invalidated for Bax, we showed that this pro-apoptotic protein favored mitochondrial alterations induced by the mycotoxin. Besides, incubation of purified mitochondria with DON indicated that this mycotoxin does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Altogether, our results indicate that mitochondria-related caspase-dependent apoptotic pathway is involved in this in vitro model of DON induced-cytotoxicity.

Cell death induced by the Alternaria mycotoxin Alternariol.

Mycotoxins are unavoidable contaminants of most foods and feeds, and some are known to be detrimental to human health. It is thus worthwhile to understand how cells of the intestinal system, one of the primary targets of these toxins, respond to their toxic effects. In this study, human colon carcinoma cells were used to elucidate the cell death mode and the pathways triggered by Alternariol (AOH), the most important mycotoxin produced by Alternaria species, which are the most common mycoflora infecting small grain cereals worldwide. Treatment of cells with AOH resulted in a loss of cell viability by inducing apoptosis. AOH-induced apoptosis was mediated through a mitochondria-dependent pathway, characterized by a p53 activation, an opening of the mitochondrial permeability transition pore (PTP), a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O(2)(*-) and caspase 9 and 3 activation. Besides, deficiency of the pro-apoptotic protein Bax partially protected cells against AOH-induced mitochondrial alterations. In addition, experiments performed on purified mitochondria indicated that AOH does not directly target this organelle to induce cell death. Our results demonstrate for the first time that AOH-induced cytotoxicity is mediated by activation of the mitochondrial pathway of apoptosis in human colon carcinoma cells.

In vitro investigation of toxicological interactions between the fusariotoxins deoxynivalenol and zearalenone

It is expected that humans are exposed to combined mycotoxins, which occur simultaneously in the food items, than to individual compounds and that can increase their potential toxicity. Considering this coincident production, deoxynivalenol (DON) and zearalenone (ZEN) as they are produced by several Fusarium species, can interfere at a cellular level. Therefore, these two toxins were chosen to study their interactive effects on human colon carcinoma cells (HCT116), using the endpoints including cell viability, cell cycle analysis, mitochondrial transmembrane potential (ΔΨm) determination and permeability transition pore (PTP) opening. Our results showed that DON and ZEN caused a marked decrease of cell viability in a dose-dependent manner, mediated by an activation of the mitochondrial apoptotic process; characterized by PTP opening and the loss of ΔΨm. Nevertheless, combined DON and ZEN reduced all the toxicities observed with the mycotoxins separately. Therefore, the combination of the two mycotoxins appears as a sub-additive response.

Presence of ochratoxin A in Tunisian blood nephropathy patients. Exposure level to OTA.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates cereals and different food compounds. OTA presents a wide range of toxic effects, especially nephrotoxicity. It is also considered to be the main causal agent of Balkan Endemic Nephropathy (BEN) which is similar to the Chronic Interstitial Nephropathy with unknown aetiology seen in Tunisia. In this study, we attempted to confirm the relationship between OTA blood levels and the development of renal pathology. Hence, serum OTA levels were measured in several groups of patients having different renal diseases: a group presenting Chronic Interstitial Nephropathy (CIN) with unknown aetiology, a group presenting Chronic Interstitial Nephropathy (CIN) with known aetiology, a group presenting Chronic Glomerular Nephropathy (CGN), and a group presenting Chronic Vascular Nephropathy (CVN). Each group was compared to a healthy control group. In the healthy group, 49% of individuals showed OTA concentrations ranging from 1.7 to 8.5 ng/ml, with a mean value of 3.3±1.5 ng/ml. However, among nephropathic patients, the group with CIN of unknown aetiology showed the highest incidence (76%), ranging from 1.8 to 65 ng/ml with a mean value of 18±7 ng/ml. Even in the healthy group, the calculated Daily Intake (DI) ranged from 5.0 to 24.9 ng/kgb.w./day when compared to the recommended DI by the scientific committee on foods of 5 ng/kgb.w./day, indicating a high degree of exposure to OTA in the Tunisian population. Our study confirms the involvement of this nephrotoxic mycotoxin, present at high blood levels in the Tunisian population, in the outcome of this particular human nephropathy (CIN with unknown aetiology) which is similar to BEN.

First report ofAlternaria species associated with black point of wheat in Tunisia

Durum wheat (Triticum turgidum) is one of the most important cereal crops in the world and the staple food for the Tunisian population. The main growing areas of durum wheat are located in the Northern part of the country, which is characterised by a sub humid climate favourable for fungal spoilage of grain such asAlternaria species. These species are able to produce plant pathogenic as well as toxic metabolites. For mycological analysis, the agar test was used. After mycelial growth, theAlternaria species were inoculated on potato carrot agar media. A PCR-based assay followed by a sequencing step allowed the identification ofAlternaria alternata; Alternaria tenuissima and Alternaria japonica from grains.

Mechanism of Alternariol monomethyl ether-induced mitochondrial apoptosis in human colon carcinoma cells.

Alternariol monomethyl ether (AME) is a major mycotoxin produced by fungi of the genus Alternaria and a common contaminant of food products such as fruits and cereals worldwide. AME can cause serious health problems for animals as well as for humans. In this study, human colon carcinoma cells (HCT116) were used to explore the mechanisms of cell death induced by AME. Exposure of HCT116 cells to AME resulted in significant cytotoxicity manifested by a loss in cell viability mainly mediated by activation of apoptotic process. AME activated the mitochondrial apoptotic pathway evidenced by the opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (ΔΨm) downstream generation of O(2)(-), cytochrome c release and caspase 9 and 3 activation. Experiments conducted on isolated organelles indicated that AME does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Moreover, no difference was observed in Bax-KO cells in comparison to parental cells, suggesting that the pro-apoptotic protein Bax is not involved in AME-induced mitochondrial apoptosis. Our findings demonstrate for the first time that AME induces cell death in human colon carcinoma cells by activating the mitochondrial pathway of apoptosis.

Combined effects of alternariols mixture on human colon carcinoma cells.

Abstract Mycotoxins are naturally occurring contaminants encountered at high levels in a wide variety of agricultural products intended for human and animal consumptions. Various Alternaria mycotoxins may occur simultaneously in small grain cereals. Considering the concomitant production of alternariol (AOH) and alternariol monomethyl ether (AME), it is expected that humans and animals are exposed to the mixture rather than to individual compounds. Therefore, we studied the interactive effects of binary mixture of alternariols (AOH and AME) on the human intestinal cell line, HCT116 cells. Exposure of HCT116 cells to low cytotoxic alternariols doses, resulted in a moderate cytotoxicity manifested by a loss in the cell viability mediated by an activation of the mitochondrial apoptotic process, associated with the opening of mitochondrial permeability transition pore (PTP) and the loss of the mitochondrial transmembrane potential (ΔΨm). However, when combined, they exert a significant increase in their toxic potential. Altogether, our study showed that AOH and AME combination is obviously additive.

Comparative cytotoxicity of the possibly occurring mycotoxins in wheat on the basis of a mycological analysis

Abstract A mycological survey was carried out at the Northern part of Tunisia on freshly harvested durum wheat in 2007, as wheat is the basic staple food for the Tunisian population. A total of 65 samples was analysed for their contamination with pathogenic and saprophytic fungal species and isolation frequencies and relative densities of the fungal species were recorded. Our results showed that Alternaria species were observed in all the prospected samples and Fusarium spp. were present in about 70% of fields whereas the incidence of the genus Penicillium was low. In the light of the mycological analysis, we evaluated the cytotoxicity of the possible occurring mycotoxins in such crops using the MTT bioassay. For this purpose, four mycotoxins — Deoxynivalenol (DON), Alternariol (AOH), Ochratoxin A (OTA) and Citrinin (CTN) — were screened for their toxicity in human colon carcinoma cell line HT 29. The viability assay showed significant variation in IC50 concentrations between the tested mycotoxins.