Does the phycotoxin Okadaic acid cause oxidative stress damages and histological alterations to seabream (Sparus aurata)?

Okadaic Acid (OA) is a marine toxin responsible for DSP (Diarrheic Shellfish Poisoning) in humans produced by dinoflagellate. The genotoxic and cytotoxic effects of OA have been well reported in mammalian experimental animals and in vitro cultured cells. However, there are no available investigations regarding the involvement of the oxidative stress pathways in OA toxicity, especially on aquatic animals such as fish. In this context, we aimed in the present work to demonstrate whether OA (7.5 µg/ml) induces oxidative stress and histopathological damages in the fish species Sparus aurata under short term exposure (2 h, 4 h and 24 h). To this end, we have assessed lipid peroxidation and anti-oxidative stress response in liver tissue, and finally ultrastructural changes were investigated in hepatic and gills tissues. Our results clearly showed that OA induced significant enhancement in all tested parameters in a time dependent manner and seems to be a strong inducer of oxidative stress in aquatic animals. The data of the present study indicate also that histology is a successful tool to reveal OA impact on liver and gill tissues of Sparus aurata since the animal showed vascular dilation and hepatocellular membrane disintegration in liver and hypertrophy in secondary lamellae and necrotic aspect in the primary lamellae in gill tissue.

Circadian variation in murine hepatotoxicity to the antituberculosis agent «Isoniazide¼.

The circadian time is an important process affecting both pharmacokinetics and pharmacodynamics of drugs. Consequently, the desired and/or undesired effects vary according to the time of drug administration in the 24 h scale. This study investigates whether the toxicity in liver as well as oxidative stress varies according to the circadian dosing-time of isoniazid (INH) in mice. A potentially toxic INH dose (120 mg/kg) was injected by i.p. route to different groups of animals at three different circadian times: 1, 9, and 17 Zeitgeber time (ZT). INH administration at 1 ZT resulted in a maximum hepatotoxicity assessed by the significant increase in both serum transaminase (ALAT: alanine aminotransferase) and (ASAT: aspartate aminotransferase) and antioxidant enzyme activities (catalase: CAT and superoxide dismutase: SOD). The highest malondialdehyde (MDA) level indicating an induction of lipid peroxidation resulting in oxidative damage was also observed at 1 ZT. Liver histopathology from INH groups at 9 ZT and at 1 ZT showed moderate to severe cytoplasma vacuolation, hepatocyte hypertrophy, ballooning, and necrosis. The circadian variation in INH toxicity may help realize a chronotherapy protocol in humans based on the selection of the best time associated to optimal tolerance or least side effects.

Lead accumulation pattern and molecular biomarkers of oxidative stress in seabream (Sparus aurata) under short-term metal treatment.

The present work aimed to look at the distribution and accumulation pattern of Lead (Pb) within the fish body after 2 h, 4 h and 24 h of waterborne exposure to the metal at 0.75 mg/L. Tests of lead acute toxicity and bioaccumulation were performed in the common fish species Sparus aurata. In our study, we assessed the oxidative stress damages extent after 2 h, 4 h and 24 h of exposure to lead using the enzymatic stress biomarkers: Superoxide Dismutase (SOD), Catalase (CAT) and Gluthathione (GSH). The lipid peroxidation (LPO) was also investigated by dosing Malondyhaldéhyde (MDA) quantities in the liver tissue. The acute neurotoxicity of Pb was evaluated in the dorsal white muscle using the Acethylcholenesterase (AchE) activity. The liver tissue accumulates preferentially the metal, followed by the intestines, the gills and finally the dorsal muscle. The antioxidant response failed to prevent the lipid peroxidation and the neurotoxic effect of lead after 24 h of exposure.

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.

Aflatoxin determination in commonly consumed foods in Tunisia.

BACKGROUND: To investigate natural aflatoxin occurrence, a total of 180 samples of different foods widely consumed in Tunisia were analysed by an in-house-validated high-performance liquid chromatography method including affinity column clean-up and post-column bromination techniques. RESULTS: The method used appeared to be rapid, selective and reproducible, and its performances were established. Detection limits were 0.05 ng g(-1) for aflatoxin B1 and 0.025 ng g(-1) for aflatoxins B2, G1 and G2. Aflatoxins were detected in all investigated commodities except rice, with an overall contamination frequency of 34.4% and concentrations ranging from 0.1 to 40.6 ng g(-1). Aflatoxin B1 was found in all contaminated samples. Sorghum, spices and nuts were most contaminated. CONCLUSION: This study has provided an effective analytical method for the reliable determination of aflatoxins in food samples. Over one-third of the samples investigated were contaminated with aflatoxins. Sorghum, spices and nuts were most contaminated, whereas rice showed no contamination.

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.

Hyperhomocysteinemia, paraoxonase concentration and cardiovascular complications in Tunisian patients with nondiabetic renal disease.

OBJECTIVES: Hyperhomocysteinemia is associated with an increased risk of cardiovascular diseases. We determine homocysteine levels (Hcy), paraoxonase (PON1) concentration and their relationship on cardiovascular complications in patients with chronic renal disease (CRD). DESIGN AND METHODS: The study population included 100 CRD patients and 120 healthy controls. Renal function was assessed using the eGFR by the MDRD study equation. Patients were considered to have CRD when the eGFR was <60 mL/min/1.73 m(2). Hcy concentrations were determined by direct chemiluminescence assay. PON1 concentration was measured spectrophotometrically using phenylacetate as a substrate. RESULTS: We found an increased Hcy levels and a decreased eGFR and PON1 concentration in CRD patients compared to the control group (P<0.001, P<0.001, P<0.01 respectively). Patients with cardiovascular complications showed an increased Hcy levels and a lower PON1 concentration than patients without cardiovascular complications (P<0.001, P<0.01 respectively). CONCLUSION: We showed that hyperhomocysteinemia and low PON1 concentration are associated with CRD and markedly associated in patients with cardiovascular complications. Additional effects contribute to the severity of renal disease and increase the incidence of cardiovascular disease.

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.

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.

Hyperhomocysteinemia, paraoxonase activity and risk of coronary artery disease.

OBJECTIVES: Paraoxonase-1 (PON1) detoxifies homocysteine thiolactone (HcyT) in human blood and could thus delay the development of atherosclerosis. We investigated (a) PON1 activity and polymorphisms, and (b) the relationship between PON1 activity, homocysteine (Hcy) and the severity of CAD patients in Tunisian population. DESIGN AND METHODS: We used PCR-RFLP analysis to detect the Q192R and L55M variants of the PON1 gene in 100 patients with CAD and in 120 healthy controls. Paraoxonase activity was measured spectrophotometrically using phenylacetate as a substrate. Total plasma homocysteine concentrations were determined by direct chemiluminescence assay. RESULTS: We found an increased Hcy level in CAD patients compared to the control group (15.86+/-8.63 vs. 11.9+/-3.25 micromol/L respectively, P<0.001), and a decrease in PON1 activity in CAD patients as compared to the control group (117+/-56 vs. 181+/-73 U/mL respectively, P<0.001). PON1 Q192R and L55M polymorphisms were not associated with the presence of CAD (P=0.592, P=0.294, respectively). However, we found that PON1 activity is lower with the PON1 192RR than with PON1 192QQ genotypes in the study population. Furthermore, there were no association between PON1 L55M polymorphism and PON1 activity. We showed a significant decrease in PON1 activity in CAD patients presenting 0- to 3-vessel stenosis (155+/-39; 135+/-36; 103+/-22; 77+/-24 U/mL, respectively; P<0.001). CONCLUSION: In this study, we showed that low PON1 activity is associated with the PON1 192RR genotypes and associated with the severity of CAD in the Tunisian population. We hypothesize that high level of Hcy together with low PON1 activity results in an increased plasma HcyT plasma concentration leading to protein N-homocysteinylation and the development and progression of atherosclerosis.

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 micromol/L versus 11.90 +/- 3.25 micromol/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.

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.

Hyperhomocysteinemia, endothelial nitric oxide synthase polymorphism, and risk of coronary artery disease.

BACKGROUND: Hyperhomocysteinemia is an independent, graded risk factor for coronary artery disease (CAD). The G894T variant of endothelial nitric oxide synthase (eNOS) was postulated to be associated with hyperhomocysteinemia and could influence individual susceptibility to CAD. The aims of this study were to investigate (a) the relationship of the eNOS G894T polymorphism with the presence and the severity of CAD and (b) the possible relationship between hyperhomocysteinemia and the eNOS G894T variant for the risk of CAD severity in a Tunisian population. METHODS: We used PCR with restriction fragment length polymorphism analysis to detect the G894T variant of the eNOS gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed by the number of affected vessels. Total plasma homocysteine concentrations were determined by direct chemiluminescence assay. RESULTS: The frequencies of the eNOS GG, GT, and TT genotypes in the CAD group were significantly different from those in the control group (45%, 44%, and 11% vs 60%, 35.8% and 4.2%, respectively; P = 0.035). There was no association between the eNOS G894T genotype frequencies and the number of stenosed vessels (P = 0.149). In the CAD group, the coexistence of the 894 GT or TT genotypes and hyperhomocysteinemia led to an increased risk of CAD severity. CONCLUSION: The G894T polymorphism of the eNOS gene is associated with the presence of CAD, and in conjunction with hyperhomocysteinemia, increased the risk of CAD severity in a Tunisian population.

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 microg/L to 55.6 +/- 19 microg/L as opposed to 1.22 +/- 1.2 microg/L to 3.35 +/- 2.32 microg/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.