Effects of malathion and cadmium on ...

PDF-OUTPUT Fish Physiol Biochem (2006) DOI 10.1007/s10695-006-0041-2

Effects of malathion and cadmium on acetylcholinesterase activity and metallothionein levels in the fish Seriola dumerilli J. Jebali M. Banni H. Guerbej E.A. Almeida A. Banaoui H. Boussetta

Received: 13 July 2005 / Accepted: 5 March 2006 Springer-Verlag 2006

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Abstract The potential use of acetylcholinesterase (AChE) and metallothionein (MT) responses as biomarker of organophosphorous (OPs) and trace metal were assessed in fish Seriola dumerilli exposed to 0, 4, 6 mg/kg of malathion for 2, 7 and 13 days, and to 0, 50, 100, 250 µg/Kg of Cd for 2 days. Brain AChE was significantly inhibited after 2 and 7 days of malathion exposure, in a dose–response manner, but no inhibition was observed after 13 days of exposure. When exposed to Cd for 2 days, S. dumerelli presented an increase in AChE activity at a concentration of 50µg/Kg, but a strong and dose-dependent AChE inhibition at 100 and 250 µg/Kg. Cd treatment

J. Jebali Æ M. Banni Æ H. Boussetta (*) Laboratoire de Biochimie et de Toxicologie Environnementale, Ecole Supe´rieure d’Horticulture et d’Elevage, Chott-Marie`m 4042, Tunisia e-mail: [email protected] Tel.: +216-73-348-546/544 Fax: +216-73-348-691 H. Guerbej Centre de Monastir, Institut National des Sciences et Technologie de la Mer, INSTM, Monastir, Tunisia E.A. Almeida Departamento de Bioqu ´mica, Instituto de Qu´mica, Universidade de Sao Paulo, CP 26077, 05513-970 Sao Paulo, Brasil A. Banaoui Laboratoire Eau et Environnement, Faculte´ des sciences, Universite´ Ibn zohr, Agadir, Maroc

also caused a rapid increase in MTs concentration in liver, even at the lower concentration. Our experiments indicate that the measurement of hepatic MT concentration and brain AChE activity in S. dumerilli would be useful biomarkers of OP and Cd exposure and/or effects.

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Keywords Acetylcholinesterase Æ Biomarkers Æ Cadmium Æ Malathion Æ Metallothionein Æ Metals Æ Organophosphorous Æ Seriola dumerilli

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Introduction

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Marine littoral ecosystem and estuaries becomes an area of concern because it constitutes the final receptacle of anthropogenic discharge of pollutants. Many pollutants including pesticides and heavy metals reaches the marine environmental through rivers, the atmosphere, agricultural and industrial runoffs, threatening species inhabiting this ecosystem (Crane et al. 2002; Banni 2004). The organophosphorus pesticide (OP) malathion is a common insecticide used widely in agriculture (Lundebye et al. 1997). In spite of replacement of organochlorine (OC), excessive use of OPs in the treatment of phytopathologies of cereals in the north of Tunisia have threaten Bizerte lagoon ecosystem, especially at the raining season (Dellali et al. 2001). The primary toxic action of OPs on organisms is the irreversible inhibition of the acetylcholinesterase

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(AChE), which hydrolyzes the acetylcholine (ACH) into choline and acetic acid at the cholinergic synapses and neuromuscular junctions. OPs block the hydrolysis of ACH, leading to excessive accumulation of ACH and disruption of nerve function (PenaLlopis et al. 2003). In addition to OPs, other classes of environmental contaminants like polycyclic aromatic hydrocarbons (PAH) (Sturm et al. 1999) and heavy metals (Rodriguez-Fuentes and Gold-Bouchot 2000), have the potential to decrease AChE activity in exposed organisms. Thus, the measurement of brain or muscle AChE in natural or caged fish would be a useful tool in the diagnostic of exposure to such compounds (Xavier et al. 1998). Ventura et al. (2002) suggested that fish hepatic AChE activity is very low when compared to brain, muscle and gills, suggesting that the evaluation of AChE inhibition by OPs would be better observed in these tissues. Cadmium is a common contaminant in the aquatic environment and has no biological role, and its mechanism of action in organisms is well understood (Marchi et al. 2000). Romeó et al. (2000) reported that exposure to Cd causes cellular injury and reduced lysosomal stability in Dicentrarchus labrax. Other authors reported that Cd exposure causes alterations in the activities of various enzymes (Viarengo et al. 1997a, b), induces lipid peroxidation (Vaglio and Landdriscina 1999), and induces the expression of hepatic metallothioneins (MTs) (Hamza-Chaffai et al. 1995; Romeó et al. 2002; Kraemer et al. 2005, in press), in different fish species. Thus, the measurement of MTs could constitute an attractive biomarker of trace metal exposure. The aim of this work was to study the effects of malathion and Cd exposure on brain AChE activity, and of Cd on hepatic MTs content in fish Seriola dumerilli, in order to evaluate the potential use of AChE activity and MTs levels as sensitive biomarkers of toxicity of these compounds in this fish.

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Materials and methods

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Animal treatment

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Specimens of S. dumerilli (600–800 g, and 37– 40 cm) were purchased from the aquaculture farm of the Institut Nationale des Sciences et Technologie de la Mer (Monastir, Tunisia). They were acclimated for

15 days to laboratory conditions in a 1,000 l aquarium filled with artificial seawater maintained constantly at 16 C, and continually water changing. After the acclimation period, fishes were intraperitoneally injected (0.2 µg/Kg) with cadmium and malathion. Cadmium (CdCl2) was diluted in NaCl 9 and then adjusted to the desired concentrations (0, 50, 100, 250 µg/Kg). Malathion was diluted in corn oil and then diluted to obtain the concentrations injected in the fishes (0, 4, 6 mg/kg). After 2, 7 and 13 days of exposure, fishes were sacrificed. The liver and brain were removed, washed briefly in ice-cold homogenizing buffer and frozen at 80 C. In previous experiments we observed that fishes having a small weight body (less than 100 g) presents a good tolerance for the exposure to contaminants diluted at nominal concentrations in water, contrary to those fishes with higher body weight. Fishes having great body weight (more 200 g) placed in aquarium of 280 l for example, could clearly modify the physicochemical properties of marine water in aquarium along time (after 6, 12 h, etc.). In preliminary studies with fishes Dicentrarchus labrax (100–150 g) exposed to CdCl2 (1 mg/l) for 48 h, we observed that dissolved O2 decreased dramatically and water pH increased with time of exposure (unpublished data). Physicochemical parameters are needed to be in optimal conditions in laboratory exposure experiments. Thus, as the fishes used in the present work weighed about 700 g, we preferred the intraperitoneally injection of contaminants instead of its dilution in water. The concentrations used for cadmium and malathion were chosen as suggested in some published works, and were very lower than DL50. Cadmium concentrations (50, 100, 250µg/Kg) are sublethal (DL50=3 mg/kg for D. labrax and Sparus aurata) and usually used in studies of acute toxicity in some fishes in our laboratory (D. labrax and S. aurata). Same concentrations were also used by George et al. (1996). The malathion concentration (4 and 6 mg/kg) are considered sublethal according to Rodr´guezAriza et al. (1999).

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Biologic parameter measurements

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The total body length, body and liver weight were measured. Body condition factor (CF) was calculated as total body weight (g)·100/(total body length, cm3)

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and liver somatic index (LSI), expressed as total liver weight (g) as percent of total body weight (g) (Al-Arabi and Gokøyr 2002).

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Measurement of AChE activity, MT levels and protein content

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Brain tissue of S. dumerilli was homogenized in Tris buffer, 0.1 M, pH7 .5. The homogenate obtained was centrifuged at 9,000·g for 30 min at 4 C. The supernatant was removed and used to determine AChE activity. AChE activity was spectrophotometrically determined according to Ellman method (Ellman et al. 1961), by measuring the increasing in absorbance of the sample at 412 nm in the presence of 1 mM acetylthiocholine as substrate and 0.1 mM 5,5,-dithiobis2-dinitrobenzoic acid (DTNB). The enzymatic reaction rate was conducted against a blank without substrate for each activity measurement. In order to subtract the spontaneous hydrolysis of the substrate a second blank was performed without sample. Each AChE activity measurement was performed in duplicate. AChE activity is expressed as nmol of developed product per minute per mg of proteins. MT content was evaluated in duplicate in liver homogenates according to the spectrophotometric method described in Viarengo et al. (1997b) based on cystein titration and using reduced glutathione as standard. Protein content was estimated by the Bradford method (Bradford 1976), using bovine serum albumin as standard.

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Statistical analysis

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Statistical analyses were performed with SP SS/PC (SP SS, Microsoft, and Redmond, WA). Significant differences between means were determined using one-way ANOVAs and the Duncan’s test for multiple range comparison with a significance level established at P < 0.05.

LSI and body length, between control and treated control fishes (data not shown). However, the condition factor was significantly higher (P < 0.05) in controls compared to fishes treated with 4 and 6 mg/ kg of malathion, after 13 days.

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Determination of AChE activity

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Table 1 shows the dose–response relationship between malathion concentrations and brain AChE activity in S. dumerilli. Malathion treatment resulted in a substantial and concentration-dependent decrease in AChE activity, after 2 and 7 days of the treatment, compared to controls. After 13 days, no differences were observed between treated and control fishes.

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Effect of CdCl2 on AChE activity

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Brain AChE activity of S. dumerilli injected with CdCl2 is shown in Fig. 1. The basal activity level in controls was 164.13 10.85 nmol/min/mg protein. A significant increase in AChE activity was detected in the brain of fishes treated with 50µg/Kg, but fishes injected with 100 and 250µg/Kg of Cd, presented a dramatic decrease in AChE activity (37–85%, respectively), when compared to controls.

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MT concentration

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Treatment of S. dumerilli for 2 days with 50 and 100µg/Kg of CdCl2 increased 4.96 and 5.86 times, respectively, the levels of hepatic MTs in respect to controls (Fig. 2). With 250 µg/Kg of Cd, the MTs

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Table 1 Evolution of brain AChE activity of Seriola dumerilli treated once with 0, 4, 6 mg/kg of malathion. All values are expressed as mean SD, (n=6) n AChE (nmol/min/mg Days Malathion concentration protein) (mg/kg) 02

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Results

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General indices

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No differences were observed in general indices of fish S. dumerilli, such as body weight, liver weight,

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251.64 181.83 160.89 141.22 96.25 69.16 190.19 172.52 211.76

13.59c 4.01b 4.45a 22.02c 23.66 0.74a 24.03 6.48 3.02

Different letters indicates statistical differences between treatments (P < 0.05)


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Cd (µg / Kg) Fig. 1 Effects of different concentrations of CdCl2 on brain AChE activity after 2 days of exposure (mean SD; n=6). Different letters indicates statistical differences between different treatments (P < 0.05)

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Cd (µg / Kg) Fig. 2 Metallothionein (MTs) content in Seriola dumerilli liver after 2 days of treatment with different concentration of CdCl2 (mean SD; n=5). Different letters indicates statistical differences between different treatments (P < 0.05)

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levels maintained similar to the levels observed with 100 µg/kg.

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Discussion

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Pollution biomarkers are useful descriptors of field situations that allow the identification of chemical stressors and their potential ecological risk (Banni et al. 2005). Due to relative lack of information about the use of biochemical systems as pollution biomarkers in S. dumerilli, a time and dose response study between OPs, Cd and AChE activity was performed. Intraperitoneal injection, different from the natural contamination, may not necessarily simulate the same biological responses as a direct exposure to OPs and heavy metals in field, but this route of xenobiotc input could be considered as a preliminary study on the effects of Cd and malathion on brain AChE of S. dumerilli. The great AChE inhibition after 2 and

7 days of treatment with malathion agrees with several studies on the inhibitory effects of this OP (Johnston 1995; Dutta et al. 1995; Pathiratne and George 1998). Varo` et al. (2003) reported that the predominant cholinesterase (ChE) in brain of fish seems to be AChE and observed that the ‘‘in vivo’’ exposure of D. labrax with 1 mg/l of dichlorvus (OP) for 96 h decrease dramatically the ChE activity in brain (76%). In a preliminary study, we observed that S. dumerilli injected by a single intraperitoneally dose of 18 mg/kg of body weight of malathion causes mortality in four of six fishes, after 2 days (data not shown). In general, 20% or greater depression in AChE activity in several species of birds, fishes or invertebrates can be an indicative of exposure to pesticides (Day and Scott 1990; Banni et al. 2003). Lundbaye et al. (1997) reported that crab (Carcinus moenas) exposed to 2 mg/l of the OP dimethoate had a 30% reduction in hemolymph AChE activity. The lack of AChE inhibition after 13 days in S. dumerilli seen in the present work would indicate the total detoxification of malathion after this period and the recuperation of AChE activity, probably due to de novo synthesis. De la Torre et al. (2002) noted that the inhibition of fish brain AChE can be detected soon after the beginning of ‘‘in field’’ exposure to OPs but the time required for recovery of basal values after transfer fishes to unpolluted media can take several weeks. Interestingly, we noted that AChE activity decreased drastically in both control and treated fishes after 7 days of exposure, and increased after 13 days. The reasons for these oscillations in AChE activities along time are unknown, but are probably due to natural fluctuations of the enzyme due to biological rhythms; the effects of malathion on AChE activity were observed as well. It has been known that AChE activity is more sensitive for OPs and carbamate pesticides than other contaminants, but the inhibition of this enzyme have been also used to indicate the exposure and/or effects of other contaminants in fishes. It has been shown that the addition of crude oil to brain homogenate in amounts equivalent to sediment concentration inhibited AChE activity in fishes (Rodriguez-Fuentes and Gold-Bouchot 2000). Minier et al. (2000) reported that muscle AChE of flounder from polluted sites



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with high level of PAH was inhibited by 40%. Also, a reduction of 40% of brain AChE was observed in Mullus barbatus from three pollutes sites of Salento Apulia (Italy), related with presence of great variety of compounds (PAH, heavy metals and pesticides) present in the sediment (Lionetto et al. 2003). The current work demonstrated that Cd treatment in S. dumerilli results in significant induction of AChE activity at low doses (50 µg/kg), and a strong and dose-dependent AChE inhibition with 100 and 250 µg/kg of Cd. Gill et al. (1991) also found that Cd exposure produced an activation of AChE in Barbus conchonius. On the contrary, De la Torre et al. (2000) reported no differences in AChE activity after 14 and 33 days of Cd exposure in Cyprinus carpio. The sublethal concentration of cadmium at 100–250 µg/kg did not causes direct mortality after 2 days exposure but could give rise to several effects at molecular and cellular level if the exposure persist, affecting tissues, organs and organisms and leading to death. Romeó et al. (2000) reported that fishes Dicentrarchus labrax exposed to same sublethal concentrations of cadmium for 2 days presented an increase in lipid peroxidation levels, a high destabilization of lysosomal membrane, and also a significant inhibition of catalase activity. Viarengo et al. (1997a) reported that cadmium toxicity results mainly on binding to SH residues of proteins and indirectly thought the alteration of lysosomal membrane leading to increase of protein catabolism. In the present study, Cd administration to S. dumerilli resulted in a rapid increase of MTs content, which reached a plateau at 100 lg/kg. Similar results were found by De Smet et al. (2001) during Cd exposure with the common carp Cyprinus carpio. MTs are known to be induced immediately after the exposure of animals to metals (Gagne´ et al. 1990), controlling both the kinetics of bioaccumulation and the manifestation of toxic effects via the reduction of the metal’s accessibility by binding these metals for posterior excretion. Then, the induction of MTs by Cd observed in the present work would represent an important defense mechanism against the toxic effects of this metal. Baudrimont et al. (2003) observed that depuration of Cd in the bivalve Corbicula fluminea was correlated to rapid decrease of MTs concentration, indicating the role of MTs on metal detoxification. Thus, it would be expected that MTs induction could protect against the inhibitory

effect of Cd on AChE activity. However, our experiments showed an inverse correlation between MTs content and brain AChE in S. dumerilli, indicating that even the highest level of MTs induced by Cd was not sufficient to protect organisms against Cd effects. Taking into account the inhibition of brain AChE activity by malathion and Cd, and the induction of hepatic MTs by Cd in S. dumerilli, we conclude that these biochemical systems are promising biomarkers of exposure and effects of OPs and metals in field studies.

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Acknowledgements A special thanks to the Tunisian Ministry of Scientific Research and technology ‘‘Unite´ de Recherche en Biochimie et Toxicologie Environnementale’’ who allowed this study to be carried out. This research was supported by ‘‘Cooperation Inter-Universitaire Franco-Tunisienne (CMCU)’’ within the framework of the project (04G0907), Tunisian Ministry of Scientific Research and Technology. E. A. Almeida is a recipient of CNPq—Brasil fellowships (201391/03-1).

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