http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, 2014; 24(2): 151–160 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2013.869781

RESEARCH ARTICLE

Khaled Bellassoued1,2,3, Fatma Makni-Ayadi2, Jos Van Pelt3, and Abdelfattah Elfeki1 1

Animal Ecophysiology Laboratory, Department of Life Sciences, Sciences Faculty of Sfax, Sfax, Tunisia, 2Biochemistry Laboratory, CHU Habib-Bourguiba of Sfax, Sfax, Tunisia, and 3Liver Research Facility/Labo Hepatology, University Hospital Gasthuisberg, Leuven, Belgium

Abstract

Keywords

Aims: The present study was aimed to assess the cytotoxic effects of not-yet identified compounds present in organ extracts of Sarpa salpa, collected in autumn, the period with a peak in health problems. Methods: The toxicity was assessed by mouse bioassay of extract of the fish’s organs. Wistar rats received daily extracts of different organs of S. salpa by gastric gavage for 7 d (0.3 ml of extract/ 100 g body weight, BW). The dose of tissue extracts of viscera, liver, brain and flesh of S. salpa administered to rats were as follows: 17.2, 31.3, 205, 266 mg/100g BW, respectively. No deaths occurred during the period of treatment. Results: The lethal dose (LD50%) determined for the crude ciguatoxin (neurotoxins) extracts of viscera, liver, brain and flesh of S. salpa were as follows: 1.2, 2.2, 14.4, 18.6 g/kg mouse, respectively. Changes in locomotor activity during the first 2 h and failure breathing and no evident signs of gastrointestinal problems were recorded. We observed: (1) Induction of oxidative stress, indicated by an increase in lipid peroxidation (TBARS) in groups that received extracts of liver (þ490%) or viscera (þ592%). Accompanied by a significant decrease in antioxidant enzyme activities (SOD, CAT, GPx) in liver tissue by 15%, 17%, 18% (LT: animals receiving liver extracts) and by 19%, 22%, 22% (VT: animals receiving viscera extracts), respectively. In contrast the administration of extracts of flesh and brain induced an increase in antioxidant enzyme activities (SOD, CAT, GPx) in liver tissue by 15%, 19%, 15% (FT: flesh extract) and 18%, 55%, 55% (BT: brain extract), respectively; (2) A significant increase in total metallothionein levels in liver tissue was recorded in (FT), (BT), (LT) and (VT) by 55%, 88%, 255% and 277%, respectively, (3) The histological findings confirmed the biochemical results. Conclusions: Liver and especially visceral part of S. salpa presented toxicity, which clearly indicates the danger of using this fish as food.

Antioxidant status, ciguatoxins, hepatotoxicity, histological studies, rats, S. salpa

Introduction Sarpa salpa, also known as salema porgy, is a species of bream which is an herbivorous sea fish that preferentially feeds on the seagrass Posidonia oceanica through the year (Peirano et al., 2001; Prado et al., 2008) and is used for human consumption in the Mediterranean region (e.g. Tunisia, region Gulf of Gabes). Due to its low cost, this fish is predominantly on the menu of the lower income classes. The consumption of the S. salpa is however inadvisable in certain periods of the year because it causes a hallucinatory syndrome and central nervous system disorders. Most poisonings involving S. salpa consumption have been reported in spring and summer (Chevaldonne, 1990; Raikhlin-Eisenkraft et al., 1989). An important observation in this context is the presence of Address for correspondence: Dr. Abdelfattah Elfeki, Animal Physiology Laboratory, Life Sciences Department, Sfax Faculty of Science, University of Sfax, BP1171, 3000 Sfax, Tunisia. Tel: (+216) 74 268 858. Fax: (+216) 74 274 437. E-mail: [email protected]

History Received 1 October 2013 Revised 4 November 2013 Accepted 8 November 2013 Published online 16 December 2013

ciguateric species that live as epiphytes on P. oceanica leaves (Ben Brahim et al., 2010) and that are co-ingested by the S. salpa as part of their diet (Velimirov, 1984). With respect to this, the Gulf of Gabes is a threatened biotope mainly due to the pressure of anthropogenic expansion and dumping of large quantities of phosphogypsum and other chemical products that severely impacted benthic habitats (Hamza-Chaffai et al., 1999). It has been shown that epiphytes of seagrass are sensitive to environmental changes (Giovannetti et al., 2010). For example, various studies reported increases in epiphyte biomass parallel with nutrient enrichment (Armitage et al., 2006), eutrophication (Frankovich et al., 2009) and water quality (Meric et al., 2005). This has led to a substantial proliferation of microalgae and particularly of toxic dinoflagellates in the Gulf of Gabes (Turki et al., 2006). Proliferation of such undesirable microalgae has been shown to result in increasing problems in both coastal and estuarine environments (Leong & Taguchi, 2005; Smayda, 1997). For instance, ciguatera food poisoning increases due to the presence in fish

20 14

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

Hepatotoxicity in rats induced by the poisonous dreamfish (Sarpa salpa)

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

152

K. Bellassoued et al.

toxins produced by the benthic alga Gambierdiscus toxicus and other coral microalgae, most of them belonging to the genera: Prorocentrum, Ostreopsis and Amphidinum. Another less common form of poisoning is ichthyoallyeinotoxism that is characterized by the development of central nervous system disturbances, especially hallucinations and nightmares (Chateau-Degat, 2003; Halstead, 1988). Several of the toxins increase to dangerous levels for humans during their transmission through herbivorous and carnivorous fish (Vaillant et al., 2001). The liver is the key organ involved in metabolic and detoxifying processes. It is continuously exposed to high levels of endogenous and exogenous oxidants that are the byproducts of many biochemical pathways. In rat, the intracellular oxidant production is higher in liver than in other tissues (Bejma et al., 2000; Ji, 1999). Hepatic damage, such as caused by microcystins (toxic blooms of cyanobacteria), can be measured by some clinical enzyme markers, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AP) (Solter et al., 2000). Since a prolonged administration of toxins provokes the dysfunction of mitochondrial and mono-oxygenase systems, these alterations may be accompanied by an increased generation of reactive oxygen species (ROS) (Ito et al., 2000). When the enhanced ROS production exceeds the antioxidant defense and repair capacity this could lead to oxidative stress and cell damage (Zegura et al., 2003). Lipid peroxidation can be either a cause for or an effect of the reactions that produce toxicity (Halliwell & Gutteridge, 1986). A study by Gehringer et al. (2004) has shown that some marine toxins provoke the production of ROS and lipid peroxides as well as DNA damage and antioxidant enzymes activity in hepatocytes by multi-specific bile acid transporters. Moreover, this results in an increase in oxidative stress, as evidenced by the enhanced rates of thiobarbituric acid reactive substances (TBARS), which is further accompanied by a concomitant decrease in the rates of scavenging enzyme superoxide dismutases (SOD), catalase (CAT) and glutathione peroxidase activity (GPx) in the liver, kidney, and spleen (Ding et al., 1998). The present study was carried out first, the toxicity was investigated by mouse bioassay of compounds present in the extract of fish organs collected in autumn and secondly, in Wistar rats that were daily received by gastric gavage, for 7 d, with extracts from the different organs of S. salpa to mimick human consumption. To evaluate the toxicity of the treatment, clinical symptoms and mortality was recorded. Since some marine toxins had been found to induce an oxidative stress (Davies et al., 1987; Saoudi et al., 2008), we also investigated lipid peroxidation and changes in the activity of antioxidant enzymes in rat liver.

Materials and methods Fish collection The study was conducted off the Island of Kerkennah (Gulf of Gabes, southeast Tunisia). This archipelago is characterized by extensive P. oceanica seagrass meadows (Hamza et al., 2000). Specimens of S. salpa (Linnaeus, 1758) were collected between 2008 and 2010 in autumn season. Immediately after

Toxicol Mech Methods, 2014; 24(2): 151–160

capture, the fish were dissected: the flesh, brain, liver and viscera were removed, rinsed with ice-cold saline, and stored at 80  C until further analysis. Lipid-soluble extracts preparations from fish organs and LD50 determination Samples for toxicity assay were prepared as follows: the flesh (including dark muscle) or organs (50–100 g) was thawed and cooked at 70  C for 15 min in a water bag to denature proteins to enhance extraction efficiency during homogenization. Samples were cooled to room temperature, minced and extracted twice with acetone (3 l/kg flesh) using an explosionproof homogenizer. The acetone filtrate was dried in a rotor evaporator at 55  C water bath and 556 mbar vacuum, re-dissolved in 90% of aqueous methanol (0.5 l/kg flesh) and extracted twice with hexane (1:1, v/v) to remove impurities from the aqueous methanol phase. The aqueous methanol portion was dried in rotor evaporator at 55  C water bath and 337 mbar vacuum, re-dissolved again in 25% of aqueous ethanol (0.5 l/kg flesh) and extracted with diethyl ether (1:1, v/v) 3 times. The extraction of lipophilic toxin from fish tissues was performed by subsequent liquid–liquid partitioning (separator funnel) as described by Lewis (2003). Diethyl ether extracts were concentrated by using rotor evaporator, re-dissolved in a known volume of chloroform–methanol (97:3, v/v) for quantification and were dried under nitrogen gas. The protein extracts were stored at 80  C prior to testing. In order to study the toxicity of marine toxin expected to be present in the samples, lipid-soluble extracts of the samples were analyzed using a mouse bioassay that was previously described by Vernoux (1994) and Lewis (1995, 2003). Mouse bioassay experiments were carried out using seven groups of male mice weighing 18–22 g (eight animals per group) purchased from the Central Pharmacy of Tunisia (SIPHAT, Tunisia). Animals were housed in a controlled environment (22  3  C, 54–56% humidity, a 12-h/12-h light– dark cycle). Mice were fed with a commercial balanced diet (SICO, Sfax, Tunisia) and drinking water was offered ad libitum. The body weight (BW) of the mice at the start of the experiment was measured. The ether-soluble extract was suspended in 1% Tween 60/0.9% saline at different concentrations, sonicated at 37  C for 5–10 min, 0.8 ml (0.04 ml/1 g of mouse) was administered by intraperitoneal (i.p) injection and assayed in duplicate. Control mice were administered the same volume of 1% Tween 60/0.9% saline only. The mice were closely monitored at 1 h interval for 3–5 h after sample injection. Symptoms of intoxication including hypothermia (rectal body temperature below 33  C), diarrhoea, reduced locomotor activity and time of death of the mice (if this occurred within the first 24 h) were recorded. Symptoms or signs of intoxication in mice, other than the above mentioned, were rejected in this experiment to avoid subjective bias (Hoffman et al., 1983; Lewis, 1995). The diethyl ether extract containing marine toxin was quantified using the principle of the dose versus time-to-death relationship equation log (MU) ¼ 2.3 log (1 þ 1/T), where MU is the number of mouse units (one MU ¼ LD50 dose for a 20 g mouse) and T is survival time in hours of each mouse (Lewis, 1995, 2003; Lewis & Sellin, 1993).

DOI: 10.3109/15376516.2013.869781

Evaluation of clinical and functional parameters following exposure to marine toxin(s) in a rat model

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

Animals and treatments Males Wistar rats weighing about 130–135 g were purchased from the Central Pharmacy of Tunisia (SIPHAT, Tunisia). They were housed at 22  3  C with light/dark periods of 12 h and a minimum relative humidity of 40%. The animals had free access to commercial pellet diet (SICO, Sfax, Tunisia) and water ad libitum. The general guidelines for the use and care of living animals in scientific investigations were followed (Council of European communities, 2010). The handling of the animals was approved by the Tunisian Ethical Committee for the Care and Use of laboratory animals. After acclimatizing to the laboratory conditions for 1 week, 45 rats were divided into 5 groups that were daily received by gastric gavage; – for the control group (C) with 0.3 ml of a 1% Tween 60/0.9% saline solution /100 g of BW, – for the flesh treated group (FT) with 0.3 ml of flesh extract/100 g BW, – for the brain treated group (BT) with 0.3 ml of brain extract/100 g BW, – for the liver treated group (LT) 0.3 ml of liver extract/100 g BW and – for the viscera treated group (VT) with 0.3 ml of viscera extract/100 g BW. The dose of ciguatoxin extracts of viscera, liver, brain and flesh of S. salpa administered to rats were as follows: 17.2, 31.3, 205 and 266 mg/100 g BW, respectively. The doses of extract organs 0.3 ml/100 g of BW, chosen in our experiment, represent 1/7 of LD50% for each extract organs. At the end of treatment, five animals from each group were rapidly sacrificed by decapitation to avoid stress. The liver tissue was immediately removed and dissected over ice-cold glass slides. The livers were collected and a part was homogenized (10% w/v) with an Ultra Turrax homogenizer in ice-cold, 1.15% KCl–0.01 M sodium, potassium phosphate buffer. Homogenates were centrifuged at 10 000 g for 20 min at 4  C. The resulting supernatants were used for immediate lipid peroxidation and protein oxidation determination. Homogenate aliquots were stored at 80  C for further biochemical assays. Other parts of these livers were fixed in 10 % buffered formalin and processed for paraffin sectioning and histological studies. Biochemical assays Biochemical markers in plasma The activities of plasma ALT and AP were determined using standard enzymatic kits (ALT/TGP A03020 and AP A03000, Biotrol, France). Protein quantification Protein content in liver was determined according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. Lipid peroxidation The liver malondialdehyde concentrations (marker for lipid peroxidation) were determined spectrophotometrically according to Draper & Hadley (1990). Briefly, an aliquot of liver extract supernatant was mixed with 1 ml of 5% trichloroacetic acid and centrifuged at 2500  g for 10 min.

Hepatotoxicity in rats induced by (Sarpa salpa)

153

An amount of 1 ml of thiobarbituric acid reagent (0.67%) was added to 500 ml of supernatant and heated at 90  C for 15 min. The mixture was then cooled and measured for absorbance at 532 nm using a spectrophotometer (Jenway UV-6305, Essex, England). The malondialdehyde values were calculated using 1,1,3,3-tetraethoxypropane as standard and expressed as nmoles of malondialdehyde/g of tissue. Determination of liver antioxidant enzyme activities Catalase (CAT) activity was measured according to Aebi (1984). A total of 20 ml livers homogenate (about 1.5 mg proteins) were added to 1 ml phosphate buffer (0.1 M, pH 7) containing 100 mM H2O2. Rate of H2O2 decomposition was followed by measuring the decrease in absorbance at 240 nm for 1 min. The enzyme activity was calculated using an extinction coefficient of 0.043 mM1 cm1 and expressed in international units (I.U.), i.e. in mmoles H2O2 destroyed/min/ mg protein, at 25  C.  Superoxide dismutase (SOD) activity was estimated according to Beyer & Fridovich (1987). The reaction mixture contained 50 mM of tissue homogenates in potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine, 2 mM riboflavin and 75 mM nitro blue tetrazolium (NBT). The developed blue color in the reaction was measured at 560 nm. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of NBT by 50% and the activity was expressed as units/mg of protein.  Glutathione peroxidase (GPx) activity was determined using a commercial kit (Ref. No. RS 505; Randox Ltd, Crumlin, United Kingdom). GPx catalyzes the oxidation of GSH by cumene hydroperoxide. In the presence of GSH reductase and NADPH, the oxidized GSH is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADPþ. The decrease in absorbance at 340 nm was measured (Randox Ltd). The enzyme activity was expressed as nmoles of GSH oxidized/min/ mg protein. Liver total metallothionein (MT) MT levels in liver were determined using a spectrophotometric assay for MT using Ellman’s reagent [0.4 mM (DTNB) in 100 mM KH2PO4] at pH 8.5 in a solution containing 2 M NaCl and 1 mM EDTA (Viarengo et al., 1997). In brief, aliquots were homogenized in 3 volumes of 0.5 M sucrose, 20 mM Tris-HCl buffer, pH 8.6, with added 0.006 mM leupeptine, 0.5 mM PMSF (phenylmethylsulphonyl fluoride), and 0.01% 2-mercaptoethanol. The homogenate was then centrifuged at 15 000 g for 30 min at 4  C. The obtained supernatant was treated with ethanol/chloroform as described by Viarengo et al. (1997) in order to obtain the MT-enriched pellet. The MT pellet was resuspended in HCl/EDTA in order to remove metal ions still bound to the MT. Finally, 2 M NaCl was added to the solution to facilitate thiol interactions with DTNB by reducing the interaction of divalent metals with the apothionein. Histological studies Liver samples for histological examination by light microscopy were fixed in 10% of formalin and processed in a series

154

K. Bellassoued et al.

Toxicol Mech Methods, 2014; 24(2): 151–160

of graded ethanol solutions. They were then embedded in paraffin, serially sectioned at 5 mm and stained with hematoxylin–eosin.

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

TUNEL assay After deparaffinization and rehydration, tissue sections were incubated with 0.1% (v/v) Triton X-100 for 2 min on ice, followed by washing of the slides twice in PBS (0.8 mM CaCl2 2H2O, 2.6 mM KCl, 1.4 mM KH2PO4, 0.4 mM MgCl2 6H2O, 136 mM NaCl, 8 mM Na2HPO4, pH 7.2). The specimens were then incubated for 1 h at 37  C in a solution consisting of 1 mM cobalt chloride, 140 mM sodium cacodylate and terminal deoxyribonucleotidyl transferase (TdT) at a final concentration of 0.1 U/ml to insert biotin-16-dUTP at the 30 -ends of DNA fragments. A streptavidin-peroxidase complex and 3-amino-9-ethylcarbazole served as the detection system for biotin. Sections were lightly counterstained with hematoxylin and mounted in glycerin jelly. Negative control included omission of TdT from the labeling mixture. Statistical analysis The data were analyzed using the statistical package program Stat view 5 Soft Ware for Windows (SAS Institute, Berkley, CA). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Fisher’s protected least significant difference (PLSD) test as a post-hoc test for comparison between groups. All values were expressed as mean  SE. Differences were considered significant if p50.05.

Results LD50 determination of marine toxin accumulated in the organs of S. salpa After extraction with diethyl ether, we obtained the crude ciguatoxins (neurotoxins) from 50 g of fish sample: viscera Table 1. Concentration of toxicity expressed in mouse units per 100 g tissue estimated in fish organs of S. salpa collected from the Island of Kerkennah (Gulf of Gabes; South East Tunisia) during autumn (2008–2010). Concentration of toxicity (MU/100 g tissue)

DL 50% g/kg of mouse

48.25  0.85 24  0.40 2.87  0.025 0.75  0.004

1.217 2.195 14.395 18.645

Viscera extract Liver extract Brain extract Flesh extract

Values are average  SE; n ¼ 8.

(viscera except liver) 1.16  0.20 g (mean  SE); liver 1.02  0.15 g (mean  SE); brain 0.5  0.11 g (mean  SE); flesh (including dark muscle) 0.69  0.20 g (mean  SE). For the LD50 determination, we used six experimental groups and one control group, each with eight mice. Affected mice exhibited typical signs of neurotoxicity disorders including hypothermia (rectal body temperature 533  C; trembling), a significantly reduced locomotor activity during the first 2 h and failure breathing and no evident signs of gastrointestinal problems (e.g. diarrhoea). Results are given in Table 1. The difference between toxin concentrations of the liver, viscera and brain extracts was significant as compared to flesh extract, also a significant difference (p50.05) between the liver and viscera organ since concentrations of toxins in organs are defined in ascending order: flesh, brain, liver and viscera. Evaluation of clinical symptoms and body, liver weights following exposure to marine toxins in a rat model No death and a significantly reduced locomotor activity during the first 2 h and failure breathing and no evident signs of gastrointestinal problems (e.g. diarrhoea) were observed in rats treated with extracts from the organs of S. salpa. After 7 d of treatment, the total body and liver weights of rats treated with liver extracts LT and those treated with viscera extract VT were found to be lower than that of controls (Table 2). In the groups of FT and BT rats, no significant change of the weight of the organs was observed. Only the BW was reduced, as compared to controls, but to a lesser extent than in the LT and VT groups. ALT and AP activities in blood plasma Serum ALT and AP activities, frequently used as indicators of hepatocellular damage, were examined. Table 3 illustrates that the activities of ALT and AP in the plasma of LT and VT rats underwent a significant (p50.05) decrease of 35%, 49% and 26%, 40% respectively, as compared to normal rats. Conversely, the activities of ALP and AP in the plasma of FT and BT rats were noted to undergo significant (p50.05) increases of 12%, 11% and 17%, 16% respectively, as compared to control animals. Estimation of lipid peroxidation levels (TBARS) TBARS concentration increased by 490% and 592% in liver tissue of LT and VT rats. The increase of the lipid

Table 2. Effects of tissue extract of flesh, liver, brain and viscera of S. salpa (0.3 ml/100 g, v/w) on body and liver weights of control and treated rats after 7 d of treatment. Parameters and groups Body weights (g) Liver weights (g)

Control

FT group

BT group

LT group

VT group

170.20  3.00 8.68  0.43

162.97  3.06* 7.44  0.37

162.83  1.88* 7.35  0.43

142.40  6.95* 6.74  0.54*

137.26  6.39*,# 5.70  0.36*,#

FT, Flesh treated group; BT, brain treated group; LT, liver treated group; VT, viscera treated group. Values are mean  SE; n ¼ 6. *p  0.05 significant from control. #p  0.05: FT, BT groups versus LT, VT groups.

Hepatotoxicity in rats induced by (Sarpa salpa)

DOI: 10.3109/15376516.2013.869781

peroxidation appeared generally to be higher in VT and LT rats, in the liver. Conversely, no significant changes were observed in liver TBARS levels of FT and BT rats as compared to the controls animals (Figure 1).

155

277%, respectively. The increase in MT levels appeared generally to be higher in VT and LT rats, in the liver, when compared to those of FT and BT groups (Figure 2). Liver histological changes

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

Enzymatic antioxidant status in liver As shown in Table 4, extracts of liver and viscera of S. salpa induced a significant decrease (p50.05) of SOD activity in liver of LT and VT rats by 15% and 17% respectively as compared to the control animals. Moreover, the treatment led also to a decrease of CAT activity by 18%, 19% and GPx activity by 22%, 22% in liver of LT and VT rats, respectively. Conversely, extracts of flesh and brain of S. salpa induced an increase of SOD, CAT and GPx activities, in liver of FT and BT rats by 15%, 19%, 15%, 18% and 55%, 55%, respectively. Liver total metallothionein (MT) Total MT protein content was evaluated in the liver of rats treated with extracts of S. salpa organs. A significant increase in MT levels in comparison with to the control animals was registered in FT, BT, LT and VT rats by 55%, 88%, 255% and Table 3. Effects of tissue extract of flesh, brain, liver and viscera of S. salpa (0.3 ml/100 g, v/w) on alanine aminotransferase (ALT) and alkaline phosphatase (AP) activities in plasma of control and treated rats. Parameter and treatments Exposure duration (7 d)

Controls FT BT LT VT

ALT (U/L)

AP (U/L)

39.8  1.46 44.8  0.86* 44.2  1.36* 25.8  1.20*,# 20.2  0.73*,#

309.6  13.29 362  15.42* 361.4  15.0* 226.4  1.83*,# 185.4  10.54*,#

FT, flesh treated group; BT, brain treated group; LT, liver treated group; VT, viscera treated group. Values are average  SE; n ¼ 6. *p  0.05 significant from control. #p  0.05 FT, BT groups versus VT, LT groups; ALT and AP as U/L.

Figure 1. Effects of tissue extracts of flesh, brain, liver and viscera of S. salpa (0.3 ml/100 g, v/w) on the liver TBARS levels of treated rats versus control rats. FT, flesh treated group; BT, brain treated group; LT, liver treated group; VT, viscera treated group. *p50.05: significant from control; #p50.05: FT, BT groups versus VT, LT groups.

Upon histological examination of control rats, liver tissue presented normal histoarchitecture (Figure 3A). Hepatocytes were polyhedral, with round nuclei and sizes roughly uniform, except for a few binucleated cells. Similar observations were registered in FT and BT rats. Necrosis in liver cells were clearly observed in all animals LT and VT rats (Figure 3D and E) when compared to controls. TUNEL observations Nick end-labeling for detection of apoptotic liver cells revealed a high percentage of TUNEL-positive cells in liver Table 4. Effects of tissue extract of flesh, brain, liver and viscera of S. salpa (0.3 ml/100 g, v/w) on the liver SOD, CAT and GPx activities of treated rats versus control rats after 7 d of treatment. Groups

SODa

CATb

GPxc

Control FT BT LT VT

23.72  0.61 27.47  1.06* 28.24  1.11* 20.11  0.48*,# 19.62  0.42*,#

326.43  9.83 375.87  15.67* 385.92  19.79* 265.88  21.82*,# 261.71  23.28*,#

1.07  0.04 1.66  0.19* 1.66  0.17* 0.83  0.064*,# 0.83  0.075*,#

FT, flesh treated group; BT, brain treated group; LT, liver treated group; VT, viscera treated group. Values are expressed as mean  SD for six animals in each group. *p50.05 significant from control. #p50.05 FT, BT groups versus VT, LT groups. a Superoxide dismutase: units/mg protein. b Catalase: mmoles H2O2 degraded/min/mg protein. c Glutathione peroxidase: nmoles of GSH/min/mg protein.

Figure 2. Effects of tissue extracts of flesh, brain, liver and viscera of S. salpa (0.3 ml/100g, v/w) on the liver MT level of treated rats versus control rats. FT, flesh treated group; BT, brain treated group; LT, liver treated group; VT, viscera treated group. *p50.05: significant from control; #p50.05: FT, BT groups versus VT, LT groups.

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

156

K. Bellassoued et al.

Toxicol Mech Methods, 2014; 24(2): 151–160

Figure 3. Photomicrographs showing histological changes in liver in different groups; Control group (A); Flesh and brain treated groups (B); (C) and liver and viscera treated groups (D); (E) showed several abnormalities (indicated by arrows). (A) Histological picture showed normal liver tissue. Hematoxylin and eosin staining, 400. !: Cell necrosis.

of rats treated following liver and viscera extracts of S. salpa received by gastric gavage (Figure 4D–E). The density of the apoptotic cells was more significant in VT than LT. Conversely, FT and BT rats did not show the apoptotic cells in livers (Figure 4B and C).

Discussion Mouse bioassay (AOAC, 1980; Lewis, 2003) remains the accepted regulatory method for detection and quantification of many marine toxins in suspect samples, so as to protect the health of the public. The assay may also quantify lethal and sublethal doses of marine toxins that are found in coral fish extracts (Louzao et al., 2004). These toxins (especially neurotoxins, e.g. ciguatoxins, brevotoxin) structure cannot be destroyed through cooking, refrigeration and weak acid treatments (Brusle´, 1997; Guzma´n-Pe´rez & Park, 2000). It is

also well known that even the application of temperature up to 120  C would not reduce the toxicity of ciguateric fish (Pottier et al., 2002a,b). In our study, standard laboratory criteria include hypothermia (rectal body temperature below 533  C, trembling), symptoms of intoxication (reduced locomotors activity, respiratory failure). These are objective parameters to determine the presence of toxin in fish organs. Three clinical reports were published about possible ciguatera poisoning in humans after consumption of fish caught in the eastern Mediterranean. One case involved the S. salpa (Spanier et al., 1989), two other reports, one recently published, involved rabbitfishes, Siganus sp. (Herzberg, 1973). Within a few hours, specific signs of poisoning occur including delirium, visual and/or auditory hallucinations (often involving animals), depression and feelings of impending death with reactive tachycardia and hyperventilation and disturbed

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

DOI: 10.3109/15376516.2013.869781

Hepatotoxicity in rats induced by (Sarpa salpa)

157

Figure 4. Photomicrographs of section of liver in different groups; Control group (A); flesh and brain treated groups (B); (C) and liver and viscera treated groups (D); (E) stained by TUNEL technique (magnification 400). Liver was fixed by direct immersion in a 4% paraformaldehyde in 0.1 M phosphate buffer. Serial sections (5 mm) were mounted on gelatin-coated glass slides cut and stained using the TUNEL technique (see Materials and Methods section). !: Apoptotic cells (arrows).

behavior. If they are able to sleep, patients classically report terrifying nightmares (De Haro et al., 2003). Ciguatoxins are produced by Gambierdiscus toxicus epiphytic dinoflagellates living on macroalgae and other substrates in tropical areas. Other dinoflagellates have also been suspected (e.g. Amphidinium carterae, Coolia monotis, Prorocentrum lima, P. concavum, P. rhathymum and Ostreopsis siamensis, Swift & Swift, 1993). Therefore, we noticed that the toxicity in the flesh and brain of S. salpa were lower than the toxicity in the viscera and liver. Shellfish exhibiting any detectable level of toxicity by mouse bioassay are considered potentially unsafe for human consumption. In practice, a value of 20 MU/100 g (USFDA, 2005) is considered the guidance level at or above

which shellfish are prohibited from harvest. Moreover, we noticed that the cytotoxic compounds present in different organs of this fish can pose a threat to human health and is a source of intoxication especially in the visceral part. This study demonstrated the effectiveness of the mouse bioassay to determine the edibility of the studied fish. Humans usually consume the fish S. salpa, but it can pose a threat to their health. For this reason, the collection of S. salpa was done in autumn season when the toxicity of this animal is the highest (Bellassoued et al., 2013). The present study, to determine the safety of S. salpa organs for human consumption, toxicological evaluation is carried out in various experimental animals to predict toxicity and to provide guidelines for selecting a safe organ in humans.

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

158

K. Bellassoued et al.

After 7 d of treatment, the total body and livers weight of rats treated with liver extracts LT and those treated with viscera extract VT were found to be lower than that of control. Changes in BW are a valuable indicator in evaluating the toxicity of extract organs of S. salpa. The slight drop in weight after 7 d of treatment seen in all the treated groups could be a normal physiological and adaptational responses (mediated by the extract organs of S. salpa), thus decreasing the appetite and thereby lowering caloric intake by the animals (Tubaro et al., 2004). The increase in antioxidant enzyme activities (SOD, CAT, GPx) without accompanied changes in TBARS levels in liver of rats treated with flesh and brain extracts, can originate from a low accumulation of ciguatoxins originated from epiphytic toxic phytoplanktons that live on the P. oceanica leaves to the fish organs by grazing (Bellassoued et al., 2013). It is more plausible that the toxic effects on humans are due especially to flesh and sometimes to brain consumption. Cellular antioxidant systems have demonstrated a great adaptation to oxidative stress in order to counteract the excessive ROS production (Alvarez & Boveris, 1993; Sureda et al., 2004). The toxicity in the flesh and brain of S. salpa were lower than the toxicity in the viscera and liver. Shellfish exhibiting any detectable level of toxicity by mouse bioassay are considered potentially unsafe for human consumption (Bellassoued et al., 2013). Consequently, the difference between this result and the decrease in antioxidant enzyme activities (SOD, CAT, GPx) with concomitant increase in TBARS levels after liver and viscera extracts treatment. Our results showed that the increase of the lipid peroxidation appeared generally to be higher in VT and LT rats, in the liver. This modification could be due to several factors, since LPO products were consequences of higher oxygen free radicals. Indeed, treatment induced, in liver of the rats, a decrease in antioxidant enzyme activities (SOD, CAT, GPx), which probably explains the concomitant increase in TBARS levels. These results confirm previous findings that had shown an association between T-2 toxin and increased oxidative stress in experimental animals (Sehata et al., 2005). Extracts of liver and especially visceral part toxicity resulted in an oxidative stress. Indeed, treatment induced, in liver of the rats, a decrease in antioxidant enzyme activities (SOD, CAT, GPx), which probably explains the concomitant increase in TBARS levels. These effects are not specific, since they are reported in numerous toxicity studies (Davies et al., 1987; Saoudi et al., 2008), but they confirm the presence of ciguatoxins in the S. salpa extracts. Total MT can be induced by various factors such as organic chemicals and metals (Dunn et al., 1987). MT synthesis is known for being a response to various pathological and physiological agents; the present investigation also revealed a noticeable increase in MT content in LT, BT, FT and especially VT rats 7 d after treatment. We hypothesized that these effects could be related to ciguatoxin. This phenomenon could equilibrate the deleterious effects of ciguatoxins on hepatic MT synthesis. Ciguatoxins accumulate in the muscle of fish and are carried up throughout the food chain. They also occur in the liver and viscera at concentrations estimated to be 10 times higher than in the muscle (Vernoux, 1994). Generally, most of

Toxicol Mech Methods, 2014; 24(2): 151–160

the consumption of P. oceanica (approximately 75%) has been attributed to S. salpa (Cebria´n et al., 1996), although the relative importance of grazing by fish varies strongly both spatially and temporally (Alcoverro et al., 1997; Peirano et al., 2001; Prado et al., 2008; Tomas et al., 2005). The interaction between herbivores and seagrass can be mediated by epiphytes (Young et al., 2005), at least in part, because seagrasses do not appear to be an attractive food source (Hereu, 2006) as the presence of phenolics in them proves to be a source of chemical deterrents (McMillan, 1984). The proliferation in the Gulf of Gabes of unwanted microalgae has been widely shown to be an increasing problem in both coastal and estuarine environments (Leong & Taguchi, 2005; Smayda, 1997), causing significant overfishing of demersal resources, thus degrading benthic habitats (Turki et al., 2006) and shellfish poisoning (Matsuoka et al., 2003). In Tunisia (1994), toxic blooms cause the death of fish in the Gulf of Gabes (Hansen et al., 2004), inciting the authorities to launch a regular monitoring in shellfish areas. Ciguatoxins are produced by dinoflagellates contaminate more generally shellfish. They are powerful inhibitors of serine; threonine phosphatases. The observed damage may be due to the fact that the liver being the first target of acute toxicity and the first organ exposed to everything that is absorbed in the small intestine, may metabolize foreign substances to highly reactive metabolites which may be hepatotoxic (Stohs, 1995). In addition, because of the short duration of treatment, the alterations might be incipient and reversible. The determination of serum enzyme activity can be a great value for the detection of liver cells death as well as changes in metabolic function. In this regard, we noted a significant reduction of serum transaminase activities ALT and AP in rats treated with a liver and viscera extracts of S. salpa after 7 d of treatment. The reduction of serum transaminase (ALT, AP) activities was more significant in VT than LT. The decrease of ALT serum activity could be explained by the failure of enzyme defense system to overcome the influx of toxin accumulation in LT and VT groups, which promoted the lipid peroxidation, modulation of DNA, alteration in gene expression and cell death. It appears to comprise a cellular exhaustion of enzymatic activities (ALT and AP) after treatment. Similar results were reported in okadaic acid treated mice that revealed a disturbance of AST and ALT levels in plasma (Tubaro et al., 2003, 2004). The toxicity exerted by liver and viscera extracts of S. salpa was confirmed from histological sectioning which indicated various degrees of hepatocellular necrosis in the LT and VT rats. Oncotic necrosis is typically the consequence of acute metabolic perturbation as occurs in ischemia/reperfusion or acute drug-induced hepatotoxicity (Kerr et al., 1972; Searle et al., 1987). Histopathological alterations observed dissociation and necrosis of hepatocytes after acute exposure to cyanotoxins. Ding et al. (2000) indicated that cyanotoxin ROS generation may play an important role in the disruption of microfilaments structure in rat hepatocytes. Several techniques are now available to investigate the proapoptotic effects of test compounds (Bejma et al., 2000). The cell morphology characteristic for apoptotic cells can be observed by microscopy. TUNEL (Terminal deoxynucleotidyl

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

DOI: 10.3109/15376516.2013.869781

transferase-mediated dUTP Nick End Labeling) is a fluorescence-based technique used to detect DNA fragmentation (Lockshin & Zakeri, 2004). Apoptosis and necrosis are usually considered separate entities, but an alternate view is emerging that apoptosis and necrosis are frequently the same initiating factors and signaling pathways. Rather than being separate entities, apoptosis and necrosis in their pure form may represent extremes on a continuum of cell death (Lemasters, 1999, 2005). The presence of cells death in the liver proves the effect of ciguatoxin or neurotoxin accumulated among organs of S. salpa as a pro-apoptotic compound of cell death. Numerous marine natural products are potent modulators of programmed cell death. The majority of the compounds described in the review specifically induce apoptosis. The neurotoxin domoic acid which was described earlier in the present review as a pro-apoptotic compound can simultaneously induce apoptosis (characterized by shrunk cells) and oncosis (characterized by swollen cells) (Mayer, 2009).

Conclusions Overall, the findings of the current study indicate that the toxicity levels observed in fish organs are decreasing in the following order: viscera, liver, brain and flesh. Moreover, the viscera, liver and edible parts of S. salpa collected along the Island of Kerkennah can be contaminated with cytotoxic compounds able to induce an oxidative stress at the level of liver. The histological findings confirmed the biochemical results. The danger of using this fish as food will depend by the amount of fish ingested or ciguatoxins accumulated among organs. Hence, the liver and especially visceral part of this fish can pose a threat to human health and consumption should for this reason be dissuaded. The results of these animal experiments are indicative but additional studies are needed with regard to extrapolating the data to humans.

Declaration of interest The authors have no conflict of interests. The authors alone are responsible for the content and writing of this article. This research was supported by the Tunisian Ministry of Higher Education and Scientific Research and the Tunisian Ministry of Public Health.

References Aebi H. (1984). Catalase in vitro. Method Enzymol 105:121–6. Alcoverro T, Duarte CM, Romero J. (1997). The influence of herbivores on Posidonia oceanica epiphytes. Aquat Bot 130:93–104. Alvarez S, Boveris A. (1993). Induction of antioxidant enzymes and DTdiaphorase in human blood mononuclear cells by light stress. Arch Biochem Biophys 305:247–51. AOAC. (Association of Official Analytical Chemists). (1980). Paralytic shellfish poison biological method. Official methods of analysis. Washington, DC: AOAC, 289–99. Armitage AR, Frankovich TA, Fourqurean JW. (2006). Variable responses within epiphytic and benthic microalgal communities to nutrient enrichment. Hydrobiologia 569:423–35. Bejma J, Ramires P, Ji LL. (2000). Free radical generation and oxidative stress with ageing and exercise: differential effects in the myocardium and liver. Acta Physiol Scand 169:343–51. Bellassoued K, Hamza A, Van Pelt J, Elfeki A. (2013). Seasonal variation of Sarpa salpa fish toxicity, as related to phytoplankton consumption, accumulation of heavy metals, lipids peroxidation level

Hepatotoxicity in rats induced by (Sarpa salpa)

159

in fish tissues and toxicity upon mice. Environ Monit Assess 185: 1137–50. Ben Brahim M, Hannechi I, Hamza A, et al. (2010). Variability in the structure of epiphytic assemblages of Posidonia oceanica in relation to human interferences in the Gulf of Gabes. Tunisia. Mar Environ Res 70:411–21. Beyer Jr WF, Fridovich I. (1987). Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal Biochem 161:559–66. Brusle´ J. (1997). In Ciguatera fish poisoning: a review. Sanitary and economic aspects. Paris: Les Editions INSERM. 147 p. Cebria´n J, Duarte CM, Marba` N, et al. (1996). Herbivory on Posidonia oceanica: magnitude and variability in the Spanish Mediterranean. Mar Ecol Prog Ser 130:147–55. Chateau-Degat ML. (2003). Marine toxins: appearance of health problem. Vertigo 4:1–11. Chevaldonne P. (1990). Ciguatera and the saupe, Sarpa salpa, in the Mediterranean: a possible misinterpretation. J Fish Biol 37: 503–4. Council of European Communities. (2010). Council instructions about the protection of living animals used in scientific investigations. Off J Euro Commun (JO 10/63/CEE) L276:1–33. Davies KJA, Delsignore ME, Lin SW. (1987). Protein damage and degradation by oxygen radicals. Modification of amino acids. J Biol Chem 262:9902–7. De Haro L, Pommier P, Valli M. (2003). Emergence of imported ciguatera in Europe: report of 18 cases at the poison control centre of Marseille. J Toxicol Clin Toxicol 41:927–30. Ding WX, Shen HM, Ong CN. (2000). Microcystic cyanobacteria extract induces cytoskeletal disruption and intracellular glutathione alteration in hepatocytes. Environ Health Perspect 108:605–9. Ding WX, Shen HM, Zhu HG, Ong CN. (1998). Studies on oxidative damage induced by cyanobacteria extract in primary cultured rat hepatocytes. Environ Res 78:12–18. Draper HH, Hadley M. (1990). Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 186:421–31. Dunn MA, Blalock TL, Cousins RJ. (1987). Metallothionein. Proc Soc Exp Biol Med 185:107–19. Frankovich TA, Armitage AR, Wachnicka AH, et al. (2009). Nutrient effects on seagrass epiphyte community structure in Florida Bay. J Phycol 45:1010–20. Gehringer MM, Shephard EG, Downing TG, et al. (2004). An investigation into the detoxification of microcystin-LR by the glutathione pathway in Balb/c mice. Int J Biochem Cell Biol 36: 931–41. Giovannetti E, Montefalcone M, Morri C, et al. (2010). Early warning response of Posidonia oceanica epiphyte community to environmental alterations (Ligurian Sea, NWMediterranean). Mar Pollut Bull 60: 1032–9. Guzma´n-Pe´rez SE, Park DL. (2000). Ciguatera toxins: chemistry and detection. In Botana LM, ed. Seafood and freshwater toxins: pharmacology, physiology and detection. New York (NY): Marcel Dekker, 401–18. Halliwell B, Gutteridge JM. (1986). Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 246:501–4. Halstead BW. (1988). Poisonous and venomous marine animals. Princetown (NJ): Darwin Press Inc., 683–6. Hamza A, Bouain A, El Abed A. (2000). Observations sur la floraison et la fructification de la phane´rogame marine Posidonia oceanica (Linneaus) Delile sur les coˆtes du Golfe de Gabe`s (Tunisie). Me´soge´e 58:93–9. Hamza-Chaffai A, Amiard JC, Cosson RP. (1999). Relationship between metallothionein and metals in a natural population of clam Ruditapes decussates from Sfax coast, a non-linear model using Box-Cox transformation. Comp Biochem Phys 123:153–63. Hansen G, Erard-Le Denn E, Daugbjerg N, Rodriguez F. (2004). Karenia selliformis responsible for the fish-kills in the Gulf of Gabes, Tunisia 1994. In Book of abstract international conference on harmful algae N 11. Cape Town, South Africa, November 15–19. Hereu B. (2006). Depletion of palatable algae by sea urchins and fishes in a Mediterranean subtidal community. Mar Ecol-Prog Ser 313: 95–103. Herzberg A. (1973). Toxicity of Siganus luridus [Rueppell] on the Mediterranean coast of Israel. Aquaculture 2:89–91.

Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by University Of Alabama Birmingham on 12/21/14 For personal use only.

160

K. Bellassoued et al.

Hoffman PA, Granade HR, Mcmillan JP. (1983). The mouse ciguatoxin bioassay: a dose-response curve and symptomatology analysis. Toxicon 21:363–9. Ito E, Satake M, Ofuji K, et al. (2000). Multiple organ damage caused by a new toxin azaspiracid, isolated from mussels produced in Ireland. Toxicon 38:917–30. Ji LL. (1999). Antioxidants and oxidative stress in exercise. Proc Soc Exp Biol Med 222:283–92. Kerr JF, Wyllie AH, Currie AR. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–57. Lemasters JJ. (2005). Dying a thousand deaths: redundant pathways from different organelles to apoptosis and necrosis. Gastroenterology 129: 351–60. Lemasters JJV. (1999). Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis. Am J Physiol 276:G1–6. Leong SCY, Taguchi S. (2005). Optical characteristics of the harmful dinoflagellate Alexandrium tamarense in response to different nitrogen sources. Harmful Algae (4):211–19. Lewis RJ. (1995). Detection of ciguatoxins and related benthic dinoflagellate toxins: in vivo and in vitro methods. In Hallegraeff GM, Anderson DM, Cembella AD, eds. Manual on harmful marine microalgae IOC manuals and guides No. 33. France: UNESCO, 135–61. Lewis RJ. (2003). Detection of toxins associated with ciguatera fish poisoning. In Hallegraeff GM, Anderson DM, Cembella AD, eds. Manual on harmful marine microalgae, IOC manuals and guides No. 33. France: UNESCO, 267–77. Lewis RJ, Sellin M. (1993). Recovery of ciguatera from fish flesh. Toxicon 311:1333–6. Lockshin RA, Zakeri Z. (2004). Apoptosis, autophagy, and more. Int J Cell Biol 36:2405–19. Louzao MC, Vieytes MR, Yasumoto T, Botana LM. (2004). Detection of sodium channel activators by a rapid fluorimetric microplate assay. Chem Res Toxicol 17:572–8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951). Protein measurement with Folin phenol reagent. J Biol Chem 193:265–75. Matsuoka K, Joyce LB, Kotani Y, Matsuyama Y. (2003). Modern dinoflagellate cysts in hypertrophic coastal waters of Tokyo Bay. Japan J Plankton Res 25:1441–70. Mayer A. (2009). Special issue on marine toxins. Mar Drugs 7:19–23. McMillan C. (1984). The condensed tannins (Proanthocyanidins) in seagrasses. Aquat Bot 20:351–7. Meric S, Nicola ED, Iaccarino M, et al. (2005). Toxicity of leather tanning wastewater effluents in sea urchin early development and in marine microalgae. Chemosphere 61:208–17. Peirano A, Niccolai I, Mauro R, Bianchi CN. (2001). Seasonal grazing and food preference of herbivores in a P. oceanica meadow. Sci Mar 65:367–74. Pottier I, Vernoux JP, Jones A, Lewis RJ. (2002a). Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies. Food Addit Contam 19:1034–42. Pottier I, Vernoux JP, Jones A, Lewis RJ. (2002b). Characterisation of multiple Caribbean ciguatoxins and congeners in individual specimens of horse-eye jack (Caranx latus) by high-performance liquid chromatography/mass spectrometry. Toxicon 40:929–39. Prado P, Farina S, Tomas F, et al. (2008). Marine protection and meadow size alter fish herbivory in seagrass ecosystems. Mar Ecol Prog Ser 371:11–21.

Toxicol Mech Methods, 2014; 24(2): 151–160

Raikhlin-Eisenkraft B, Finkelstein Y, Spanier E. (1989). Ciguatera like poisoning in the Mediterranean. Vet Hum Toxicol 30:352–3. Saoudi M, Abdelmouleh A, Kammoun W, et al. (2008). Toxicity assessment of puffer fish Lagocephalus lagocephalus from the Tunisian coast. C R Biol 331:611–16. Searle J, Harmon BV, Bishop CJ, Kerr JFK. (1987). The significance of cell death by apoptosis in hepatobiliary disease. J Gastroenterol Hepatol 2:77–96. Sehata S, Kiyosawa N, Atsumi F, et al. (2005). Microarray analysis of T-2 toxin-induced liver, placenta and fetal liver lesions in pregnant rats. Exp Toxicol Pathol 57:15–28. Solter P, Liu Z, Guzman R. (2000). Decreased hepatic ALT synthesis is an outcome of subchronic microcystin-LR toxicity. Toxicol Appl Pharmacol 164:216–20. Smayda TJ. (1997). Harmful algal blooms: their ecophysiology and general relevance to phytoplankton blooms in the sea. Limnol Oceanogr 42:1137–53. Spanier E, Finkelstein Y, Raikhlin-Eisenkraft B. (1989). Toxicity of the saupe, Sarpa salpa (Linnaeus, 1758), on the Mediterranean coast of Israel. J Fish Biol 34:635–6. Stohs SJ. (1995). The role of free radicals in toxicity and disease. J Basic Clin Physiol Pharmacol 6:205–28. Sureda A, Batle JM, Tauler P, et al. (2004). Neutrophil tolerance to oxidative stress induced by hypoxia/reoxygenation. Free Radic Res 38:1003–9. Swift AE, Swift TR. (1993). Ciguatera. J Toxicol Clin Toxicol 31:1–29. Tomas F, Turon X, Romero J. (2005). Seasonal and small-scale spatial variability of herbivory pressure on the temperate seagrass Posidonia oceanica. Mar Ecol Prog Ser 301:95–107. Tubaro A, Sosa S, Altinier G, et al. (2004). Short-term oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice. Toxicon 43: 439–45. Tubaro A, Sosa S, Carbonatto M, et al. (2003). Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxin in mice. Toxicon 41:783–92. Turki S, Harzallah A, Sammari C. (2006). Occurrence of harmful dinoflagellates in two different Tunisian ecosystems: the lake of Bizerte and the gulf of Gabes. Cah Biol Mar 47:1–7. USFDA. (2005). National Shellfish Sanitation Program Guide for the Control of Molluscan Shellfish. Washington (DC): US Food and Drug Administration, Center for Food Safety and Applied Nutrition. Vaillant V, Caumes E, De Valk H, et al. (2001). Intoxication alimentaire a` la ciguatera: savoir l’e´voquer meˆme en l’absence de voyage. Bulletin Epide´miologique Hebdomadaire, 38 p. Velimirov B. (1984). Grazing of Sarpa salpa L. on Posidonia oceanica and utilization of soluble compounds. In Boudouresque CF, Grissac AJD, Olivier J, eds. First International Workshop on Posidonia oceanica Beds. Marseille: GIS Posidonie, 381–7. Vernoux JP. (1994). The mouse ciguatoxin bioassay: directions for use to control fish for consumption. Memoir Queensland Museum 34:625–9. Viarengo A, Ponzano E, Dondero F, Fabbri R. (1997). A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs. Mar Environ Res 44:69–84. Young EB, Lavery PS, Van Elven B, et al. (2005). Nitrate reductase activity in macroalgae and its vertical distribution in macroalgae epiphytes of seagrass. Mar Ecol Prog Ser 288:103–14. Zegura B, Sedmak B, Filipic M. (2003). Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon 41:41–8.