Chemosphere 77 (2009) 902906

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Chemosphere
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Mixture toxicity assessment of cadmium and benzo[a]pyrene in the sea worm Hediste diversicolor
M. Banni a,*, Z. Bouraoui a, C. Clerandeau b, J.F. Narbonne b, H. Boussetta a,*
a b

Laboratoire de Biochimie et de Toxicologie Environnementale, Institut Suprieur Agronomique, Chott-Mariem, Sousse, Tunisia Laboratoire de Physico-Toxico Chimie des Systmes Naturels, Universit Bordeaux I, France

a r t i c l e

i n f o

a b s t r a c t
In the present study, Hediste diversicolor biotransformation and anti-oxidant responses to acute exposure to cadmium (Cd) and to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated. Worms were submitted to 0.2, 0.4 and 1 lM of each contaminant and to their mixture during a period of test of 48 h. Following biological responses were measured: (1) NADPH cytochrome c reductase (NADPH cyt c) activity, as phase I biotransformation parameter; (2) gluthathione-S-transferase (GST) activity as a phase II conjugation enzyme, (3) catalase activity as anti-oxidant response and (4) malondialdehyde accumulation (MDA) as lipid peroxydation marker. The cholinergic system was evaluated using the acetylcholinesterase activity (AChE). Exposure to the mixture resulted in low dose level additive effects on the investigated biomarkers. However, worms exposed to 1 lM of the single compounds and to their mixture exhibited the highest MDA accumulation and the lowest enzymatic biomarkers activities suggesting severe toxicological effects. These data should be carefully considered in view of the biological effects of mixture pollutants and particularly in marine sediment ecosystems. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 22 June 2009 Received in revised form 16 August 2009 Accepted 23 August 2009 Available online 15 September 2009 Keywords: Cadmium Benzo[a]pyrene Mixture Biomarkers Hediste diversicolor

1. Introduction Concern on the biological sublethal effects of toxic chemicals present in the marine sediment environment has risen in recent years and increasing efforts to nd methods for an early evaluation of the potential toxicity of these pollutants have been made (Griscom et al., 2000; Lee et al., 2000). It is becoming clear that chemical analysis cannot take into account issues such as mixture toxicity and the environmental conditions (such as sediment structure and chemical absorption, temperature, pH, etc.) determining chemical bioavailability. The best integrators of these complex effects are the exposed organisms themselves. Among sediment living organisms, sea-worms such Hediste diversicolor, are considered to be of particular interest, as they are almost ubiquitous and because of their ecological role in sediment biocenosis (Grant et al., 1989; Saiz-Salinas and Francs Zubillaga, 1997; Bouraoui et al., 2009). The importance of worms in testing the adverse effects of chemicals on sediment fauna has been described in many recent works (Sandrini et al., 2008; Bouraoui et al., 2009; Ferreira-Cravo et al., 2009).

* Corresponding author. Address: Laboratory of Biochemistry and Ecotoxicology, Higher Institute of Agronomy 4042, Chott-Mariem, Sousse, Tunisia. Tel.: +216 73 327 544; fax: +216 73 327 591. E-mail address: [email protected] (M. Banni). 0045-6535/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.08.041

Cadmium (Cd) is a heavy metal commonly used in environmental studies because it is highly toxic (Lane and Morel, 2000), wildly distributed in the environment and can adversely affect the organisms at relatively low exposure concentrations (Waisberg et al., 2003). Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds in the marine environment and in general, they appear at the highest concentrations in most urbanized costal areas, as a signature of human activity or as a result of industrial processes (Phillips, 1981; Christensen et al., 2002). Benzo[a]pyrene (B[a]P), a model PAHs compound, is widely distributed in various environmental media (Koyama et al., 2004). The use of biomarkers has been reported to be very informative about the organisms stress response to individual toxicants and mixtures (Svendsen et al., 2004; Gastaldi et al., 2007; Adam et al., 2009). NADPH cytochrome c reductase is a phase I biotransformation enzyme (CYP-dependent monooxygenase) playing a main role in the detoxication of organics xenobiotics (Arun and Subramanian, 2003). Glutathione-S-transferase (GST) is a phase II enzyme involved in the metabolism of lipophilic organic contaminants. This enzyme also plays a role in cellular protection against oxidative stress (Michel et al., 1998). Catalase (CAT) is a well-known anti-oxidant enzyme, its activity increasing in organisms submitted to oxidative stress (Durou et al., 2007). One of the well-known lipid peroxidation products is malondialdehyde (MDA), this markers was usually used to evaluate the state of lipid peroxidation of the membrane (Alexandrova and Bochev, 2005). Acetylcholinesterase

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(AChE) is an enzyme essential to the correct transmission of nerve impulses. Its inhibition is directly linked with the mechanisms of toxic action of some pesticides (Galgani and Bocquen, 1991). Aquatic organisms are invariably exposed to pollutants mixtures. Toxicities associated with exposure to chemical mixtures are often difcult to delineate due to the complexity of both the characteristics of the mixture itself, the chemical-to-chemical differences in toxicokinetic and mechanism(s) of toxic action. In this work a set of biomarkers was applied in order to assess the pollutant-induced stress in the sea worm H. diversicolor. Worms were exposed over a period of 48 h to different concentrations of inorganic Cd and B[a]P, as representative models of heavy metals and organic xenobiotics. 2. Material and methods 2.1. Animal treatment Specimens of the polychaete H. diversicolor, with a mass comprised between 0.4 and 0.6 g were collected from Teboulba (Tunisia) that was previously reported to be a clean site (Banni et al., 2005, 2007; Bouraoui et al., 2009). The animals together with the surrounding sediments were put in open polyethylene bottles and maintained at 14 C to 16 C in isothermal containers, Once in the laboratory the worms were separated from sediment, cleaned from debris then placed in glass petri dishes at 14 C with aerated clean seawater to ambient photoperiod regimes for 3 d, this acclimatation period was used for excluding specimens with exoskeleton or skin infections. After this period, worms were exposed for 48 h to 0.2, 0.4 and 1 lM of Cd (CdCl2) or B[a]P and to their equimolar mixture. Two control groups were considered, the rst one was maintained in clean sea water (absolute control) and the second one was exposed to 100 lL/L dimethyl sulfoxide (DMSO) (B[a]P and mixture control groups). After 48 h of exposure, worms were sacriced, washed briey in ice-cold buffer and conserved in liquid nitrogen until analysis. 2.2. Biochemical analyses A pool of three worms per sample were homogenized (1:5, w/v) in phosphate buffer 100 mM, pH 7.5, NaCl (2.5%). Homogenates were then centrifuged at 9000g for 30 min (4 C). The supernatant (S9 fraction) of each sample was stored at 20 C, for no longer than a week, until enzyme activity determinations. Subsequently, the supernatant was centrifuged at 100.000g for 50 min at 4 C. The pellet was resuspended in 10 mM HEPES, pH 7.4, containing 250 mM sucrose in 20% glycerol, to obtain a microsomal fraction. This microsomal suspension was used for NADPH cytochrome c activity measurements. Total protein content in the homogenate was measured following the Bradford method (Bradford, 1976), at 595 nm, using bovine serum albumin as standard. 2.3. NADPH cytochrome c determination NADPH cytochrome c activity was determined according to Hayes (1982). Reaction mixture contained the stock microsomal enzyme, 20 mM NADPH and 10 mM of cytochrome c. The specic activity was determined by spectrophotometric method at 550 nm. The results were expressed as nmoles cytochrome c reduced/min/mg proteins. 2.4. Glutathion-S-transferase determination GST activity was assayed by the method described by Habig et al. (1974) using the 1-chloro-2,4-dinitrobenzene (CDNB) as

substrate, and GSH (1 and 4 mM nal concentration, respectively), in 100 mM sodium phosphate buffer, pH 7.5. All GST activity assays were realized in conditions of linearity with respect to incubation time. The results were expressed as nmole produced/min/mg proteins. 2.5. Catalase determination Catalase activity was determined by the method of Claiborne (1985) measuring the rate of enzymatic decomposition of H2O2 determined as absorbance decrements at 240 nm. The assay mixture consisted of 750 lL of sodium phosphate buffer (0.1 M, pH 7.5 and 25 C), 200 lL solution of 0.5 mM H2O2 and 50 lL of cytosolic fraction. Results were expressed as lmol H2O2 consumed/ min/mg proteins. 2.6. Malondialdehyde accumulation Lipid peroxidation was estimated in terms of thiobarbituric acid reactive species (TBARS) with use of 1,1,3,3-treaethyloxypropane as a standard. The reaction was determined at 532 nm, using TBA reagent as described by Buege and Aust (1978). MDA content was expressed as nmoles equivalent MDA/mg proteins. 2.7. Acethylcholinesterase determination Acetylcholinesterase activity was determined according to the method described by Ellman et al. (1961). Reaction mixture contained 0.1 M sodium phosphate buffer (pH 7.5), 8 mM DNTB and the stock cytosolic solution containing acetylcholinesterase fractions. After pre-incubation, the reaction was started by the addition of 8.25 mM acetylthiocholine (AtChl) as substrate. Acetylcholinesterase activity was determined by kinetic measurement at 420 nm. Results were expressed as nmole (AtChl) hydrolised/min/mg proteins. 2.8. Statistical analysis The results for biomarker measurements are presented as means SD of 10 pools of three samples each. Statistica Software, version 6.0 computer software package (Statsoft. Inc., 2002) was used for statistical analysis. The normality of the distribution was tested using the ShapiroWilk test. To assess multiple comparisons, a parametric one-way analysis of variance (ANOVA) was performed on data, with a Tukeys test. 3. Results Following the experimental period, no signicant mortality was observed during all the exposure time either to the single compounds or to their equimolar mixture. Fig. 1 reported the effect of the tested single compounds and their mixture on the phase I enzyme NADPH cytochrome c reductase activity. Our data indicated a signicant increase in the phase I enzyme with all the B[a]P tested concentrations. The maximum activity was recorded in worms exposed to 0.4 lM with up to 1.25 0.18 nmole/min/ mg proteins (Fig. 1). Cadmium exposure resulted also in an increase of the NADPH cyt c activity in worms exposed to 0.2 and 0.4 lM, however no effect was registered in animals exposed to 1 lM of the heavy metal (Fig. 1). A pronounced increase of the NADPH cyt c activity was observed in worms exposed to 0.2 lM of the mixture with up to 3.85 0.71 nmole/min/mg proteins (Fig. 1). The response of the phase II enzyme GST activity is shown in Fig. 2. GST activity was signicant in worms exposed to the

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5 4.5 nmole/min/mg proteins 4 3.5 3 2.5 2 1.5 1 0.5 0 Control 0.2 M 0.4 M 1 M

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200 180 mole/min/mg proteins 160 140 120 100 80 60 40 20 0 Control 0.2 M 0.4 M

* a b

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Fig. 1. Acute effect of B[a]P, Cd and their mixture on NADPH cytochrome c reductase activity in worms H. diversicolor after 48 h exposure. Control worms were maintained in clean sea water (for cadmium treated worms) or exposed to DMSO (for B[a]P expose worms). Measurements represent the mean SD (n = 10 pools of three animals each). P < 0.05: signicantly different, Tukeys test ANOVA multiple comparison test versus control; aP < 0.05: signicantly different versus B[a]P treated group; bP < 0.05: signicantly different versus Cd treated animals.

Fig. 3. Acute effect of B[a]P, Cd and their mixture on catalase activity in worms H. diversicolor after 48 h exposure. Control worms were maintained in clean sea water (for cadmium treated worms) or exposed to DMSO (for B[a]P expose worms). Measurements represent the mean SD (n = 10 pools of three animals each). P < 0.05: signicantly different, Tukeys test ANOVA multiple comparison test versus control; aP < 0.05: signicantly different versus B[a]P treated group; b P < 0.05: signicantly different versus Cd treated animals.

80 70 nmole/min/mg proteins 60 50 40 30 20 10 0 Control 0.2 M 0.4 M

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2 1.8 1.6 nmole/mg proteins 1.4 1.2 1 0.8 0.6 0.4 0.2 0

B[a]P Cd Mix

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Fig. 2. Acute effect of B[a]P, Cd and their mixture on glutathione-S-transferase activity in worms H. diversicolor after 48 h exposure. Control worms were maintained in clean sea water (for cadmium treated worms) or exposed to DMSO (for B[a]P expose worms). Measurements represent the mean SD (n = 10 pools of three animals each). P < 0.05: signicantly different, Tukeys test ANOVA multiple comparison test versus control; aP < 0.05: signicantly different versus B[a]P treated group; bP < 0.05: signicantly different versus Cd treated animals.

Fig. 4. Acute effect of B[a]P, Cd and their mixture on MDA accumulation in worms H. diversicolor after 48 h exposure. Control worms were maintained in clean sea water (for cadmium treated worms) or exposed to DMSO (for B[a]P expose worms). Measurements represent the mean SD (n = 10 pools of three animals each). P < 0.05: signicantly different, Tukeys test ANOVA multiple comparison test versus control; aP < 0.05: signicantly different versus B[a]P treated group; b P < 0.05: signicantly different versus Cd treated animals.

different concentrations of B[a]P and Cd. In both cases the maximum activation was recorded in worms exposed to 0.4 lM with respectively 40.01 2.52 nmole/min/mg proteins and 34.72 3.27 nmole/min/mg proteins (Fig. 2). While a clear and pronounced increase of the GST activity was registered in worms exposed to 0.2 lM of the mixture (64.19 5.93 nmole/min/mg proteins) no effect was observed in worms exposed to 1 lM of the two tested compounds when compared to control (Fig. 2). The results relative to the catalase activity are reported in Fig. 3. Our data show that CAT activity signicantly increased in animals exposed to 0.2 lM and 0.4 lM of the two single compounds reaching a maximum of 93.56 10.48 lmole/min/mg proteins and 101.76 7.19 lmole/min/mg proteins respectively in worms exposed to 0.4 lM B[a]P and 0.4 lM Cd. No signicant effect was observed in worms exposed to 1 lM of the two single compounds (Fig. 3). Interestingly, a higher increase of CAT activity was recorded in worms exposed to 0.2 lM of the mixture (162.43 20.01 lmole/min/mg proteins) when compared to control animals (74.76 5.27 lmole/min/mg proteins). However, a signicant decrease of the catalase activity was observed in animals exposed to 0.4 lM of the mixture when compared to control.

Fig. 4 represents the MDA levels in worms exposed to Cd, B[a]P and their mixture. A higher and signicant accumulation of MDA was registered in animals exposed to the single compounds with a maximum of 1.21 0.16 nmole/mg proteins in worms exposed to 1 lM B[a]P and 1.39 0.22 nmole/mg proteins in animals exposed to 1 lM Cd. The highest MDA accumulation was recorded in worms exposed to 1 lM of the mixture with up to 1.48 0.22 nmole/mg proteins. The response of AChE activity is reported in Fig. 5. AChE activity was inhibited in worms exposed to 1 lM of the two single compounds and to their equimolar mixture (Fig. 5). However exposure to 0.2 lM Cd resulted in a signicant increase of AChE activity reaching a value of 29.20 4.20 nmole/mn/mg proteins when compared to control value (21.85 2.82 nmole/mn/mg proteins). The highest AChE activity was recorded in worms exposed to 0.2 lM of the mixture (31.96 3.76 nmole/mn/mg proteins). 4. Discussion In the natural environment, aquatic organisms and particularly sediment living fauna are most often exposed to a large range of

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40 35 nmole/mg proteins 30 25 20 15 10 5 0 Control 0,2 M 0,4 M

B[a]P Cd Mix

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Fig. 5. Acute effect of B[a]P, Cd and their mixture on AChE activity in worms H. diversicolor after 48 h exposure. Control worms were maintained in clean sea water (for cadmium treated worms) or exposed to DMSO (for B[a]P expose worms). Measurements represent the mean SD (n = 10 pools of three animals each). P < 0.05: signicantly different, Tukeys test ANOVA multiple comparison test versus control; aP < 0.05: signicantly different versus B[a]P treated group; b P < 0.05: signicantly different versus Cd treated animals.

potentially toxic chemicals at the same time (Saiz-Salinas and Francs Zubillaga, 1997; Bouraoui et al., 2009). Aquatic animals are thus invariably exposed to contaminant equimolar mixtures, whose individual components are likely to produce different lifehistory responses within the same organisms and/or interact producing additive, synergistic or antagonistic toxic effects (Barata et al., 2006). However, the vast majority of available toxicity data deals with the effects of single pure chemicals. The effects of cadmium and benzo[a]pyrene on marine organisms was largely documented (Gomez-Mendikute and Cajaraville, 2003; Alsop et al., 2007) however, and to our knowledge, no studies investigated the acute response of the sea worm H. diversicolor to mixture contaminant exposure. In the present study worms were exposed to a relatively high Cd and B[a]P concentration since in the aquatic environment, PHAs accumulate mainly in the sediment, and sediment ingesting in faunal organisms such as H. diversicolor are potentially exposed to high PAHs both from interstitial water and food (Neff, 1985). Moreover these concentrations were reported to be sublethal for terrestrial (Gastaldi et al., 2007) and marine worms (Sandrini et al., 2008). The present work reported the acute effects of three concentrations of Cd, B[a]P and their equimolar mixture on H. diversicolor using a multimarker approach comprising a set of enzymatic and lipid peroxydation markers. In this experiment, the B[a]P was dissolved in DMSO and Cd in (NaCl) 9. The results indicated no signicant difference (P < 0.05) of the investigated biomarkers in worms exposed to DMSO or to clean sea water. The cytochrome P-450 mixed function monooxygenases are particularly important in transforming xenobiotics to reactive intermediates which are often more toxic than the parent compounds (Arun and Subramanian, 2003). The level of NADPH cyt c red was identied in worms to increase after exposure mainly to hydrocarbons compounds (Christensen et al., 2002). In the present work, exposure to the lowest level of BaP/Cd equimolar mixture (0.2 lM each chemical) caused a clear synergic increase of NADPH cyt c red activity, suggesting a high increase of the metabolisation processes. However, the phase I enzyme activity in worms exposed to the higher dose levels tested (0.4 and 1 lM) appeared more compliant with the response addition model (Bliss, 1939). A similar pattern was also observed for worm phase II conjugation, measured as GST activity which showed a maximum at the lowest dose level and further weaker effects. The equimolar mixture toxicity effect of cadmium and B[a]P on phase I and phase II enzymes is well described in vertebrates. In deed, Benedetti et al. (2007) showed that the marked

EROD induction caused by B[a]P was completely suppressed by co-exposure to Cd in the Antarctic sh Trematomus bernacchii. Jensen and Krkje (2008) described a possible inhibitory effect of a ternary mixtures (0.7 lM Cd/B[a]P/Tetrachlorobiphenyl) on B[a]P induced CYP1A in rat hepatoma cells. To our knowledge this is the rst report of the equimolar mixture B[a]P and Cd effect on detoxication enzymes in worms. The additive effect of B[a]P and Cd on the phase I and the Phase II enzymes described in this works could be probably due to the relatively low tested concentrations (0.2 lM) and also to the worms metabolizing system specicities. Several environmental pollutants can become toxic through the induction of oxidative stress. Despite the large number of studies which describe the relationship between the exposure to Cd and/ or B[a]P and the anti-oxidant defense system, for several species of animals, only a small number of these studies were performed using polychaete as model. It is well-known that the displacement of iron and copper from various intracellular sites by Cd increases the concentration of ionic iron and copper. This causes oxidative stress through the Fenton reaction, producing hydroxyl radical species that are believed to initiate lipid peroxidation. It is also well established that PAHs compounds like B[a]P are able to create an oxidative stress status in the cell by enhancing the production of oxygen reactive species like hydrogen peroxide (Gastaldi et al., 2007). Our results demonstrate that, in H. diversicolor, exposure to sublethal concentrations of Cd and B[a]P induced signicant changes in CAT activity and MDA accumulation. Interestingly, coexposure to 0.2 lM Cd/B[a]P resulted in an additive effect on the activation of the CAT activity, while exposure to 1 lM of the single compounds and to their mixture maintained the CAT effect at the control level. Indeed, the outcome of the 0.4 lM mixture on CAT activity appeared compliant with an antagonism, as no effects could be determined. The MDA accumulation followed an increasing trend being always signicantly different from the control. This could act in favor of a pronounced toxic effect in worms treated with the two investigated compounds. The maximum MDA accumulation was registered in worms exposed to 1 lM of the mixture. Recent works reported the cytotoxic effects of Cd and B[a]P on worms (Gastaldi et al., 2007; Sandrini et al., 2008). In deed and as suggested by our data Sandrini et al. (2008) described a higher increase of the reactive oxygen and nitrogen species as well as a signicant decrease of the anti-oxidant enzymes in worms exposed to 1 lM Cd. A clear evidence of oxidative perturbations was observed in the Antarctic sh T. bernacchii co-treated with sublethal Cd and B[a]P concentrations resulting in a reduced capability to absorb hydroxyl radicals (Benedetti et al., 2007). AChE activity of different marine species has been reported to be modulated by environmental contaminants other than pesticides, such as metals and PAHs (Gill et al., 1990; Guilhermino et al., 2000). Data discussed in the present work show an inhibition of the acetylcholinesterase enzyme in worms exposed to 1 lM of the two single compounds and to their equimolar mixture (B[a]P + Cd). However, exposure to 0.2 lM increased signicantly (P < 0.05) the AChE activity in Cd-treated worms and also in animals co-exposed to Cd and B[a]P. The latter nding suggest an independent effect of Cd on acetylcholinesetrase enzyme rather than that of the PAH hydrocarbon in worms at a concentration of 0.2 lM. To our knowledge the inhibitory effect on AChE activity was only reported in mussels exposed to higher B[a]P concentrations (Akcha et al., 2000). Several authors demonstrated that in sh exposure to low Cd concentration could increase AChE activity (Gill et al., 1990; Jebali et al., 2006). 5. Conclusion The present study demonstrated that sub-chronic exposure to a BaP/Cd mixture resulted in low dose level additive effects on

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sublethal enzymatic responses of the sea worm H. diversicolor. Conversely, at higher dose, and in particular at the highest level tested (1 lM) the joint exposure of the two chemicals elicited severe toxicological effects, often resulting in the suppression of the detoxifictive responses. Despite these results, additional studies are required with the aim to evaluate further toxicological endpoints and assess deviations from mixture toxicity reference model systems using an appropriate experimental design. Acknowledgements This work was supported by founds from Ministre de lEnseignement Suprieur et de la Recherche Scientique; UR. Biochimie et Toxicologie Environnementale and the project Cooperation Inter- Universitaire Franco-Tunisienne (CMCU) N 04G0907. References
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