Khira Maaroufi

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.

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.

Comparative Study on in Vitro Effects of Homocysteine Thiolactone and Homocysteine on HUVEC Cells: Evidence for a Stronger Proapoptotic and Proinflammative Homocysteine Thiolactone

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However the underlying mechanisms responsible for endothelial cell injury with increased plasma concentration of homocysteine or homocysteine derivatives remains still incompletely elucidated. In this study, we investigated the ability of homocysteine (Hcy) and homocysteine thiolactone (HcyT) to induce cell death and IL-8 secretion in primary human umbilical vein endothelial cells (HUVEC). Hcy and HcyT were both cytotoxic and capable of promoting cell death, as measured by caspase-3 activation and DNA fragmentation. ELISA assays clearly demonstrated that Hcy and HcyT strongly activated IL-8 release. Furthermore, our results showed that HcyT was much more efficient than Hcy in activating caspase-3 or in inducing IL-8 secretion. The use of antioxidants such as vitamin C and vitamin E strongly but not completely reduced programmed cell death and chemokine release suggesting that other pathways different than reactive oxygen species are also involved. This study suggests that Homocysteine derivatives like HcyT might possess stronger cytotoxicity and pro-inflammatory properties and that Hcy derivatives levels should therefore be more taken into account during diagnostics.

Hyperhomocysteinaemia, methylenetetrahydrofolate reductase polymorphism and risk of coronary artery disease.

Background: Hyperhomocysteinaemia is an independent, graded risk factor for coronary artery disease (CAD). The methylenetetrahydrofolate reductase (MTHFR) polymorphism is associated with hyperhomcysteinaemia and may therefore influence individual susceptibility to CAD. We have investigated this risk factor in a Tunisian Arab population. Methods: Polymerase chain reaction-restriction fragment length polymorphism analysis was used to detect the C677T and A1298C variants of the MTHFR gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed as the number of affected vessels. Plasma total homocysteine (tHcy) concentration was determined using a direct chemiluminescence assay. Results: MTHFR CC, CT and TT genotype frequencies in the CAD group were significantly different from those observed in the control group (49%, 35% and 16% versus 48.3%, 45.8% and 5.8%, respectively; P=0.031). However, MTHFR AA, AC and CC genotypes frequencies in the CAD group were not significantly different from the control group ( P = 0.568). Patients with CAD showed higher plasma tHcy concentrations than patients without CAD (15.86±8.63 μmol/L versus 11.90 ± 3.25 μmol/L, P<0.001). There was no association between the MTHFR polymorphisms and the number of stenosed vessels. Patients with the MTHFR TT genotype had higher plasma tHcy, serum creatinine, cholesterol and triglyceride concentrations than patients with the MTHFR CC genotype. Conclusions: The C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinaemia, lipid dysregulation and the presence of CAD in this Tunisian Arab population.

Karyomegaly of tubular cells as early stage marker of the nephrotoxicity induced by ochratoxin A in rats

Cases of karyomegaly were described by Sclare30 and by Mihatch31 in patients affected with tubular-interstitial nephropathy. The Karyomegalic cells showed enlarged nuclei with accumulation of genetic material. No aetiology was suggested. Our study of rats experimentally intoxicated by ochratoxin A, a well-known nephrotoxic compound, indicates the presence of karyomegaly with alteration of the tubular tissue. In control animals no karyomegalic cells were detected. These observations suggest that karyomegaly with megacytosis may be caused by the nephrotoxic ochratoxin A in the kidney. In addition abnormal mitosis together with karyomegalic cells were observed at an earlier stage of the intoxication (30 days) suggesting possible regeneration if the OTA insults are stopped. After 90 days of treatment, the degeneration increased and only karyomegalic and apoptotic-like cells were observed indicating that the regeneration no longer occurs and that the degeneration becomes irreversible.

Effect of acute cadmium exposure on metal accumulation and oxidative stress biomarkers of Sparus aurata.

Cadmium (Cd) is a non-essential metal which could be toxic in traces for aquatic species. Increasing Cd concentrations in sea water is mainly related to waste waters provided from growing industrial and agricultural activities. The present study investigated the accumulation of Cd (0.5mg/L) in different tissues of Sparus aurata under a short term exposure (2, 4 and 24h) using the atomic absorption. This work investigated also the impact of the metal on oxidative stress biomarkers and acetyl cholinesterase activity (AchE). Our results showed that Cd accumulation in different tissues depends largely on the length of the exposure period to the metal. Indeed, the highest concentrations were obtained after 24h. Cd accumulation in tissues was in the following order: intestines > liver > gills > dorsal muscle. Cadmium administration increased significantly catalase activity (CAT), glutathione level (GSH) and malondialdehyde production (MDA) after 24h of exposure. In contrast, AchE activity was decreased after the same period of exposure to the metal. There were no significant changes in oxidative stress biomarkers after 2 and 4h of exposure, except for superoxide dismutase (SOD) activity which attained the highest level after 4h. These results suggest that short-term exposure of Sparus aurata to Cd (0.5mg/L) induced an important metal accumulation in intestine and a notable oxidative stress response.

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.

Hyperhomocysteinaemia and parameters of antioxidative defence in Tunisian patients with coronary heart disease

Abstract Background An imbalance between oxidative damage and antioxidative protection in association with the pathophysiology of atherosclerosis has been suggested. The aim of this study was to test the parameters of antioxidative defence and to assess their association with hyperhomocysteinaemia and the severity of coronary heart disease (CHD) in Tunisian patients. Methods The study population included 100 patients with CHD and 120 healthy controls. The severity of CHD was expressed as the number of affected vessels. Superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity and total antioxidant status (TAS) concentrations were measured using commercially available methods. Plasma total homocysteine (tHcy) concentration was determined by direct chemiluminescence assay. Serum zinc (Zn) was measured by a colorimetric method. Results Compared with healthy control subjects, patients with CHD had significantly lower activities of SOD (P < 0.01), GPx (P < 0.001), and serum Zn concentrations (P < 0.001) and significantly higher tHcy concentration (P < 0.001). However TAS concentrations were not significantly different between the groups. SOD and GPx activities were negatively correlated with tHcy concentration (P < 0.05, P < 0.001, respectively). Patients with hyperhomocysteinaemia showed a lower GPx and SOD activities than patients with normohomocysteinaemia. Antioxidant enzyme activities tended to be decreased in CHD patients presenting with 0- to 3-vessel stenosis. Conclusions This study indicates that low activity of GPx, SOD and Zn concentration are associated with CHD patients. We hypothesize that hyperhomocysteinaemia and low antioxidant enzyme activities may increase the extent of CHD.

Combined cytotoxicity and genotoxicity of a marine toxin and seafood contaminant metal ions (chromium and cadmium).

Algal bloom with consequent production of marine toxins contaminating bivalves is increasing in costal regions worldwide because of sea water quality worsening. Contamination of seafood by diarrheic shellfish poisoning toxins (DSP) together with metals is frequently reported, a phenomenon not fully explained yet. In this context, metal ions were assayed in clams collected from the banned area of Boughrara, Tunisia, contaminated by Gymnodinium and other algae such as Dinophysis sp, accumulated by these bivalves. The presence of toxic metals ions such as Chromium (Cr) and Cadmium (Cd) in meat, shells, and water released by the clams prompted us to experiment in Caco‐2 intestinal cell line toxic effects of these heavy metals ions in combination with okadaic acid, one DSP present in clams to assess the potential global toxicity. Cr and Cd produce additive effects in (i) reactive oxygen species production, (ii) cytotoxicity as assessed by the mitochondrial activity testing method (MTT test), and (iii) DNA lesions evaluated by agarose gel electrophoresis and acridine orange staining. Exaggerated DNA fragmentation is observed, suggesting an overloading of repair capacity of Caco‐2 cells. The apoptosis suggested by a DNA fragment sizing (180–200 bp) in agarose gel and mechanisms underlying these additive effects in Caco‐2 cells still need to be more comprehensively explained.

Endothelial nitric oxide synthetase, methylenetetrahydrofolate reductase polymorphisms, and cardiovascular complications in Tunisian patients with nondiabetic renal disease.

OBJECTIVES The relationship between endothelial nitric oxide synthase (eNOS), methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and cardiovascular disease (CVD) in Tunisian patients with chronic renal disease (CRD) has not been examined. We investigated (a) the relationship of these gene polymorphisms with the presence and the severity of renal disease, and (b) their relationships with CVD in these patients. DESIGN AND METHODS We used PCR-RFLP analysis to detect the eNOS G894T, MTHFR C677T and A1298C variants in 100 patients with CRD and in 120 healthy controls. RESULTS MTHFR C677T and A1298C polymorphisms were not associated with the presence of renal disease. However, we found that eNOS G894T polymorphism was associated with the presence and severity of renal disease and with CVD in CRD patients (P=0.028, P=0.018, P=0.016 respectively). We showed that 894T allele was an independent risk factor of severity of renal disease and the incidence of CVD (P=0.01 and P<0.01 respectively). CONCLUSION The G894T polymorphism of the eNOS gene is associated with severity of renal disease. Presence of the 894T allele aggravated renal damage and increased the incidence of CVD in Tunisian CRD patients.