Enzymatic inhibition and morphological changes in Hoplias malabaricus from dietary exposure to lead(II) or methylmercury

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Ecotoxicology and Environmental Safety 67 (2007) 82–88 www.elsevier.com/locate/ecoenv

Enzymatic inhibition and morphological changes in Hoplias malabaricus from dietary exposure to lead(II) or methylmercury Joa˜o Ricardo Maleres Alves Costaa, Maritana Melaa, Helena Cristina da Silva de Assisb, E´milien Pelletierc, Marco Antonio Ferreira Randia, Ciro Alberto de Oliveira Ribeiroa, a

Departamento de Biologia Celular, Universidade Federal do Parana´, C.P. 19031, CEP 81.531-990, Curitiba, PR, Brazil b Departamento de Farmacologia, Universidade Federal do Parana´, C.P. 19031, CEP 81.531-990, Curitiba, PR, Brazil c Institut des Sciences de la Mer de Rimouski, 310 alle´e des Ursulines, Rimouski, Que´bec G5L 3A1, Canada Received 24 October 2005; received in revised form 17 March 2006; accepted 25 March 2006 Available online 6 June 2006

Abstract Neotropical fish traı´ ra (Hoplias malabaricus) were used to investigate the effects of dietary doses of metals through individual exposures to either inorganic lead(II) or methylmercury, respectively, 21 mg Pb2+ g1 w.w. and 75 ng H3C-Hg+ g1 w.w., every 5 days, for 70 days (14 doses). The erythrocyte d-aminolevulinic acid dehydratase (ALAd) activity was inhibited after 14 doses of Pb2+and H3C–Hg+. The muscle cholinesterase (ChE) activity was inhibited after 14 doses of H3C-Hg+. Damage in cytoskeleton and nuclei were observed after exposure to inorganic lead. Individuals exposed to H3C-Hg+ showed the presence of atypical granules and vesicles, cytoplasm disorganization, and mitochondria damages in hepatocytes also after 14 doses. The present results demonstrate that erythrocyte ALAd and muscle ChE activities can be used as long-term biomarkers of sublethal, subchronic, and trophic exposures to Pb2+, and H3C-Hg+ in fish. Also the morphological aspects described in the present work confirm the toxicity of both studied metals. r 2006 Elsevier Inc. All rights reserved. Keywords: Methylmercury; Inorganic lead(II); d-Aminolevulinic acid dehydratase activity; Cholinesterase activity; Hepatocytes ultrastructure; Hoplias malabaricus

1. Introduction The use of biomarkers as measured biological responses in organisms is important to simplify and reduce costs of biological monitoring, especially in aquatic ecosystems. dAminolevulinic acid dehydratase (E.C. 4.2.1.24; ALAd) catalyzes the synthesis of one molecule of porphobilinogen from two molecules of d-aminolevulinic acid (d-ALA) and is involved in the metabolic synthesis of the heme group, an important compound in eucaryotic cells (Hodson et al., 1984). The ALAd activity is used as a biomarker for lead exposure in humans and aquatic organisms and may be a valuable biomarker for oxidative stress in hematological systems exposed to lead (Hodson et al., 1977; Goyer and Clarkson, 2001; Gurer-Orhan et al., 2004).

Corresponding author. Fax: +55 41 3266 2042.

E-mail address: [email protected] (C.A. Oliveira Ribeiro). 0147-6513/$ - see front matter r 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2006.03.013

Cholinesterase (ChE) enzymes (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)) hydrolyze the neurotransmitter acetylcholine in cholinergic synapses of both vertebrates and invertebrates, and no attempt was made to distinguish between the two homologous groups in vertebrates (Massoulie´ et al., 1993), but there is evidence that AChE is most important in fish muscle (Lundin, 1962; Abou-Donia and Menzel, 1967; Coppage, 1971; Habig et al., 1988; Leibel, 1988). Heavy metals are among the first environmental pollutants recognized as nonspecific anticholinesterase agents and have been described to inhibit ChE activities in fish and invertebrates (Olson and Christensen, 1980; Gill et al., 1990, 1991; Schmidt and Ibrahim, 1994; Labrot et al., 1996; Payne et al., 1996; Najimi et al., 1997; Silva de Assis, 1998; Guilhermino et al., 1998; Cajaraville et al., 2000; Rabitto et al., 2005). Morphological damages in specific organs express health conditions and represent the time-integrated endogenous and exogenous impacts on the organism (Chavin, 1973;

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Rabitto et al., 2005). According to Pacheco and Santos (2002), liver is considered an important target organ to investigate the effects of dietary pollutants exposure. In addition, many aspects of hepatic morphological alterations related to metal uptake in fish have been previously described as very useful tools in toxicological studies (Holm et al., 1991; Oliveira Ribeiro et al., 1996; Rabitto et al., 2005; Hongxia et al., 1998; Stentiford et al., 2003; Rabitto et al., 2005). Lead is one of the most commonly used metals in industry and its toxicity is important partly due to its persistence in the environment (Ogunseitan et al., 2000; Gurer-Orhan et al., 2004). Over the past 2 decades in Brazil, data assessing lead exposure showed a wide distribution and toxic effects of this metal. Contamination of children living close to a primary lead refinery located in the Ribeira River valley (states of Sa˜o Paulo and Parana´) was verified (Paoliello et al., 2002). Some have wide acquaintance with H3C-Hg+ neurotoxicity, but mercury contamination of aquatic ecosystems, mainly in the Amazon region, is an important concern in Brazil due to ongoing gold mining activities. The microbial transformation of Hg2+ into H3C-Hg+ increases toxicity to aquatic organisms, and the whole mechanism of its toxicity is not completely known (Shanker et al., 2004). Traı´ ra, Hoplias malabaricus (Bloch), is a freshwater carnivorous fish widely distributed in South America. This species is an interesting biological model for experimental study of dietary exposure to contaminants due to its voracious behavior, its ability to adapt to experimental conditions, and its food chain position. In addition, H. malabaricus have one of the greatest tolerances to food deprivation recorded, surviving for periods of up to 180 days without reduction in metabolic rates (oxygen uptake) (Rios et al., 2005). Although fish have been considered very acceptable organisms for pollution monitoring in aquatic ecosystems (Van der Oost et al., 2003), few studies using South American freshwater fish are available (Akaishi et al., 2004; Rabitto et al., 2005). Enzymatic inhibition (erythrocyte ALAd and muscle ChE activities) and morphological damages on liver ultrastructure are discussed here in relation to their feasibility in fish exposure to metals assessment. The use of those parameters as sensitive and long-term biomarkers for sublethal, subchronic, and trophic exposures to Pb2+ and H3C-Hg+ was investigated in H. malabaricus. 2. Material and methods 2.1. Experimental design Thirty-five mature fish (H. malabaricus: 110.8749 g) were obtained from northwestern Parana´, in southern Brazil. Prior to the experiment, fish were acclimated to experimental conditions for 30 days (1 fish for each 30 L aquarium in dechlorinated tap water, T ¼ 2172 1C, 12 h:12 h photoperiod). The food supply was young, live individuals of Astyanax sp. from the Iguac- u River basin (Parana´, southern Brazil). Each study fish

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(H. malabaricus) was fed one individual (approximately 10% of the wet weight) every 5 days to ensure ingestion. The feeding rate was similar to that used by Rios et al. (2005) for H. malabaricus: 2% of the wet weight per day. Eight fish were contaminated with Pb2+ [from Pb(NO3)2, neutral aqueous solution], 9 fish were contaminated with H3C-Hg+ [from H3CHg  Cl, diluted in 1 mN HCl aqueous solution], and 18 fish were kept as controls. Contaminants, administered every 5 days, were injected intraperitonealy in the food fish (Astyanax sp. with 10% of the wet weight of each test fish). The volume of the contaminant was adjusted according to the individual weight of each test fish (H. malabaricus) to obtain a standardized ingestion doses of 21 mg Pb2+ g1 (Rabitto et al., 2005) and 75 ng H3C-Hg+ g1 (Oliveira Ribeiro et al., 1999) wet weight. The prey item was not force-fed to individual fish. For Pb2+, the dose administered was calculated based on a concentration factor of 3.0  103 g L1, proposed for secondary consumers and dietary contamination (Vighi, 1981), while the average total lead concentration in the Ribeira river, Registro, state of Sa˜o Paulo, Brazil, was 27.88 mg L1 between 1978 and 1997, as measured by the local water company (CETESB). The concentration permitted by Brazilian Legislation, CONAMA Resolution No. 20 (BRASIL, 1986) is 30.00 mg L1. For methyl mercury, the dose administered was calculated based on the data from Amazon impacted areas studies (Oliveira Ribeiro et al., 1999). The control groups were fed on prey fish injected with distilled water (controls for Pb2+) or 1 mN HCl aqueous solution (controls for H3CHg+). All the test fish fed on 14 doses (complete ingestion of the prey item). At the end of the experiment all individuals were anesthetized with 0.02% MS222 (ethyl-ester-3-aminobenzoic acid, Sigma), blood samples were collected from the caudal artery with heparinized syringes, and muscle tissue samples were excised from the axial muscle. After 70 days blood samples were collected from Pb2+ and H3C-Hg+ treatments (14 doses) and from controls. Blood was stored in liquid nitrogen until ALAd activity measurements. Axial muscle samples collected from the H3C-Hg+ treatment and control groups were stored at 20 1C prior to ChE activity measurements.

2.2. Erythrocyte ALAD activity measurement The ALAd activity was evaluated by photometry modified for microplates following Hodson et al. (1977). Erythrocytes were hemolyzed by 0.5% Triton X-100 in 0.1 M, pH 6.3, phosphate buffer (NaH2PO4/ Na2HPO4), without (blank) or with (test) the substrate d-aminolevulinic hydrochloride (d-ALA.HCl) at 4 1C. To improve sensitivity (unchanged reaction velocity) and ensure available d-ALA (saturation) in the reaction medium, 4 mM d-ALA as final substrate concentration was used. Enzymatic incubation was carried out at 37 1C for 1 h under the following conditions: 4 mM d-ALA as final substrate concentration, 300 mL of final volume, and blood dilution 1:12 (v/v) in reaction medium. The reaction was stopped with an ice bath, followed by precipitation by adding 700 mL of 99.45 mM HgCl2 in 40 mg mL1 trichloroacetic acid (TCA). Deproteination (HgCl2/TCA) was carried out with blank tubes before the incubation to avoid any reaction with endogenous substrate. After centrifugation at 4 1C, 5000g for 5 min the supernatant (100 mL) was pipeted with 100 mL of the Ehrlich reagent: 18.18 mg mL1 dimethylaminobenzaldehyde, 3.18 mg mL1 HgCl2, 76.36% (v/v) glacial acetic acid, and 18.18% (v/v) perchloric acid at 70%. Before measuring the experimental samples, several assays were conducted with fish blood samples to standardize the methodology (i.e., substrate Km and optimal temperature). After precisely 15 min of color reaction, absorption of the product (Ehrlich salt) was measured at 550 nm on a Tecan Sunshine microplate photometer. Hemoglobin concentration from each blood sample was measured following Beutler et al. (1995). Blood samples were measured at least three times in different reactions for ALAd activity, and mean values were calculated as OD (discounted blanks) corrected by the dilution factor of the Ehrlich reagent on microplates (D ¼ 2) and as function of the hemoglobin concentration (g dL1) to normalize the samples.

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2.3. Muscle ChE activity measurement Muscle samples were thawed and homogenized at 4 1C in 0.1 M potassium phosphate buffer, pH 7.5. Homogenates were centrifuged at 10,000g for 10 min at 4 1C for enzyme activity measurement, following Ellman et al. (1961) and adapted for microplates (Silva de Assis, 1998). The final concentration of the substrate acetylthiocoline iodide was 9 mM, and that for the color reagent (5,5-dithio-bis-2-nitrobenzoic acid) was 0.5 mM. Protein content of each sample was measured following Bradford (1976) using bovine serum albumin as a standard. The enzymatic activity was expressed as (nmol of colored product) (min)1 (mg of total protein)1.

2.4. Electron microscopy procedures For ultrastructure investigations, liver samples were preserved in 2% glutaraldehyde, 2% paraformaldehyde, 5 mM CaCl2, 20 mM NaCl dissolved in 0.1 M cacodylate buffer (pH 7.2–7.4) fixative solution at room temperature for 2 h. The specimens were postfixed in 1% osmium tetroxide in the same buffer for 1 h, dehydrated in a graded series of ethanol, propylene oxide, and embedded in PoliEmbed 812 DER736 resin (Polysciences). The ultra-thin sections were contrasted by uranil acetate (5%) and lead citrate (Reynolds) and observed in a JEOL TEM 1200 EXII.

2.5. Statistical procedures

Fig. 1. Saturation-binding curve (rectangular hyperbola from Michaelis–Menten equation) of erythrocyte ALAd reaction from Hoplias malabaricus: Km ¼ 0.3653 mM d-ALA; r2 ¼ 0:9167, n ¼ 7, 8 reactions (d-ALA concentrations) per fish sample, after incubation at 37 1C for 1 h. Relative activity: every OD value as a function of the maximum OD measurement (reaction in a specific d-ALA concentration) for each fish sample. Error bars: 95% confidence interval. Inset graph is the Lineweaver–Burke plot (linear regression, inverse of the Michaelis–Menten equation).

ALAd data were analyzed using the software GraphPad PRISM (v. 3.0). A saturation-binding curve (rectangular hyperbola from the Michaelis–Menten equation) was calculated to show the enzyme velocity of the reaction as a function of substrate concentration. The Lineweaver–Burke plot and a polynomial third-order curve fitting of optimal temperature also illustrate enzyme activity. Hemoglobin concentration and ALAd activity were analyzed by one-way analysis of variance (ANOVA), using Dunnett’s test to compare experimental treatments with the control, and significance was determined at 5%. To fit parameter assumptions of homoscedasticity (Bartlett’s test) and normality (Shapiro–Wilks’ test), a logarithmic transformation of ChE data stabilized the variance and provided a normal distribution. The logtransformed ChE data were analyzed by ANOVA; the critical significance level was set at 5%.

3. Results Experimental concentrations were not lethal for the tested individuals, as no mortality was observed during exposure to either Pb2+ or H3C-Hg+. The ALAd activity assay is a quite simple method to obtain evidence of subchronic Pb2+ exposure and to compare groups, even if the results are expressed by relative values of OD. Substrate km from blood of H. malabaricus was 0.3653 mM d-ALA (r2 ¼ 0:9167, n ¼ 7, eight reactions (d-ALA concentrations) per fish sample) (Fig. 1). A final d-ALA concentration of 4.0 mM was used and this value is quite similar to that used by Hodson et al. (1977), 4.257 mM, to four teleost species. According to the same authors, the use of pH 6.3 in the incubating medium was close to the optimal value reached previously (pH 6.2) for those teleosts. After acclimation, the erythrocyte ALAd activity of H. malabaricus increased with temperature up to at least 56 1C (n ¼ 6, Fig. 2). The temperature selected for the assays of

Fig. 2. Temperature standard curve obtained for erythrocyte ALAd activity from Hoplias malabaricus (n ¼ 6): polynomial third-order curve fitting. The OD values were transformed to normalize residuals as a function of the OD measurements after 1 h of incubation at 37 1C, the temperature used for all test measurements.

ALAd activity was 37 1C, for 1 h of incubation. Either preincubating with phago DNase (0.25 mg mL1, 1 h at 37 1C) or without (1 h at 37 1C) affected significantly the OD results (n ¼ 5; data not shown). Therefore, both preincubating procedures increase the blood volume after the partial hemolyzed sample is thawed out (196 to 4 1C). Dietary and subchronic exposure of H. malabaricus to both Pb2+ and H3C-Hg+ resulted in erythrocyte ALAd activity inhibition (Dunnett’s test, Po0.01) (Fig. 3). Also an inhibition of muscular ChE occurred (Dunnett’s test,

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the most affected structure in the cytosol due to the rough endoplasmic reticulum dispersion after both Pb2+ (Fig. 5B and C) and H3C-Hg+ (Fig. 5E) exposure compared to control individuals (Fig. 5A). Also the presence of atypical granules and vesicles in the cytoplasm were observed only in individuals exposed to H3C-Hg+, suggesting disturbance in the metabolic storage (Fig. 5D and E). 4. Discussion

Fig. 3. Erythrocyte ALAd relative activity in Hoplias malabaricus exposed to 21 mg Pb2+ g1 and 75 ng H3C-Hg+ g1 wet weight, administered every 5 days under controlled conditions. Raw data present mean7SEM and both treatments differ from control (Dunnett’s test: **Po0.01). OD: absorbance at 550 nm after incubation at 37 1C for 1 h (t); D: dilution factor of the Ehrlich reagent on microplates (D ¼ 2); [HB]: hemoglobin concentration (g dL1).

Fig. 4. Cholinesterase activity in muscle of Hoplias malabaricus after 14 ingested doses of 75 ng H3C-Hg+ g1 wet weight, administered every 5 days under controlled conditions. Raw data present mean7SEM and ilustrate differences from control activity: *Po0.05 after logarithm transformation.

Po0.05) after trophic and subchronic exposure to H3CHg+. The mean values of muscle ChE activity registered were 88.7876.25 nmol min1 mg1 protein for the H3CHg+-treated group and 138.98717.30 nmol min1 mg1 protein for the control group (Fig. 4). Both Pb2+-(Rabitto et al., 2005) and H3C-Hg+-exposed groups presented severe damages in hepathocytes after 14 doses or 70 days of exposure time. Despite the cytoplasm disorganization of hepathocytes observed in individuals exposed to Pb2+ (previous data) the damages identified after H3C-Hg+ exposure suggest a more severe effect than damage in Pb-exposed fish. The cytoskeleton appears to be

The daily dietary exposure of 75 ng H3C-Hg+ g1 w.w. over 70 days consistently inhibited erythrocyte ALAd activity in H. malabaricus. This is the first experimental study to demonstrate an effect of H3C-Hg+ on erythrocyte ALAd activity at low concentrations in dietary exposure. Mercury is a ubiquitous pollutant that disrupts many biochemical pathways, while its ALAd inhibition in fish is not completely known. Recently, experiments with rats in vivo demonstrated reduced kidney ALAd activity after subcutaneous injection with 17 mmol HgCl2 kg1 (Perottoni et al., 2004a, b). Mercury interaction with sulfhydryl groups in combination with mercury-induced oxidative damage (Stohs and Bagchi, 1995) may explain these results and those described in the current work or due to the oxidative damage on hematopoietic tissues, also an effect already reported for H3C-Hg+ (Stohs and Bagchi, 1995; Cestari et al., 2004; Oliveira Ribeiro et al., 2006). The ALAd activity is a very sensitive in vitro indicator for a variety of metals due to its high dependency on sulfhydryl groups present in the active site, but until now only lead had been reported as an effective in vivo inhibitor (Hodson et al., 1984) in fish species. In addition to the effects of H3C-Hg+, our results also confirm the inhibition of ALAd by Pb2+ in H. malabaricus after trophic, subchronic experimental exposure. As a consequence of H3C-Hg+ and lead exposure the DNA damage and altered sulfhydryl homeostasis have been reported due to the depletion of glutathione, protein-bound sulfhydryl groups and enhanced lipid peroxidation, resulting in the production of reactive oxygen species (Stohs and Bagchi, 1995). In a similar experiment, H. malabaricus exposed to Pb2+ dietary doses showed strong evidence of genetic damage in the head kidney such as chromatid gaps and breaks, chromosome fragments, and pericentric inversions (Cestari et al., 2004). According to the same authors, frequency of chromosomal aberrations increased in H. malabaricus exposed to 4 and 8 doses and high DNA damage in red blood cells were evidenced after 13 doses by comet assay. The d-ALA (precursor of heme group) accumulation is involved with oxidative damage due to reactive species production (Demasi et al., 1996). In this way, an explanation for the DNA damages after Pb2+ exposure can be supported also by ALAd inhibition. The present data reinforce the evidence presented by Gurer-Orhan et al. (2004) and Perottoni et al. (2004a, b) that ALAd activity evaluation is a promising indicator of lead-induced oxidative damage in blood and other tissues. In this way,

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Fig. 5. Ultrastructural effects of Pb2+ and H3C-Hg+ in liver of Hoplias malabaricus after trophic and subchronic exposure. (A) Control group; observe the organization of rough endoplasmic reticulum (arrow). Scale bar, 0.5 mm. (B) Complete disorganization of cytoplasm is observed after Pb2+ exposure. Scale bar, 2 mm. In detail (C) is shown the total dispersion of endoplasmic reticulum (arrows). Scale bar, 2 mm. (D) Hepatocyte after H3C-Hg+ exposure showing also a cytoplasm disorganization and the presence of atypical granules such as lysosomes (arrows). Scale bar, 2 mm. (E) Observe the presence of lipid vacuoles (arrows) in the cytosol, not observed in the control group. Scale bar, 2 mm.

the ALAd activity may be a new sensitive index and a valuable biomarker of oxidative stress in lead-exposed hematological systems (Goyer and Clarkson, 2001; Cestari et al., 2004; Gurer-Orhan et al., 2004), a biochemical indicator of lead (Johansson-Sjobeck and Larsson, 1979; Martin and Black, 1996, 1998; Burden et al., 1998; Campana et al., 2003) and H3C-Hg+ exposure to fish. Rabitto et al. (2005) in a preliminary study showed that H. malabaricus exposed to Pb2+ revealed a tendency for muscular ChE inhibition with a time-dependent decrease in activity (4 and 8 dietary doses), but a significant inhibition of ChE activity occurred after Pb2+ exposure to 14 dietary doses (21 mg g1 w.w.). Here we demonstrated that H3CHg+ can also inhibit the activity of muscular ChE in H. malabaricus, corroborating literature data on other fish species (Galgani and Bocquene´, 1990; Schmidt and Ibrahim, 1994; Labrot et al., 1996; Payne et al., 1996; Guilhermino et al., 1998; Cajaraville et al., 2000, Rabitto et al., 2005). The AChE activity is strongly inhibited by even low concentrations of organophosphate and carbamate pesticides and so has been widely used as a specific biomarker for these compounds (Galgani and Bocquene´, 1990, Sturm et al., 1999, Guilhermino et al., 2000). Because AChE is

involved in critical neural and neuromuscular functions, it may be more important than nonspecific esterases (BChE). However, this idea awaits further study (Payne et al., 1996). The mechanism of ChE inhibition by metals is not clear. It is generally accepted that metals may deactivate enzymes by binding to their specific groups (Viarengo, 1989; Simkiss et al., 1993). A hypothesis for the inhibition is that the metal binds at the anionic site of ChEs (Guilhermino et al., 1998). Hence, with the metal at anionic sites, acetylcholine cannot bind properly to the enzyme and cannot be degraded. This hypothesis suggests that differences in the potential for some metals to inhibit AChE will be explained by properties such as ionic size, capacity of complex formation, electronegativity, and reduction potential (Guilhermino et al., 1998; Grippo and Heath, 2003). Lead is more than twice as effective as H3C-Hg+ in muscular ChE inhibition, suggesting that different inhibitory mechanisms are involved (Rabitto et al., 2005). The known metal–ion-induced oxidative damage should be connected with the presumed broken macromolecular structure of cytoskeleton, whereas the exceeding reactive oxygen species results from Pb2+ and H3C-Hg+ exposures (Stohs and Bagchi, 1995). In fact, Rabitto et al. (2005) showed that Pb2+-induced hepathocyte changes in H.

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malabaricus are a time-related effect suggesting structural and functional changes in cytoskeleton dynamics, with consequences for location of organelles and vesicle movements into the cytoplasm. The presence of structures such as lysosomes and lipid vacuoles in the cytoplasm was attributed to H3C-Hg+ exposure. The mitochondria autophagy was described by Rabitto et al. (2005) in hepatocytes of H. malabaricus after dietary exposure to Pb2+, but was not observed here for H3C-Hg+ exposure. 5. Conclusion The erythrocyte ALAd activity is affected not just by Pb2+, as previously reported for fish species, but also by H3C-Hg+. We demonstrated also that H3C-Hg+ can inhibit the activity of muscular ChE in H. malabaricus after low doses and diet exposure, as was previously shown for organophosphate and carbamate pesticides. According to the current results, due to its high-level trophic position and wide geographical distribution in South America, we are suggesting the use of H. malabaricus erythrocyte ALAd and muscle ChE activities as exposure biomarkers in biomonitoring programs of freshwater areas impacted by lead and mercury. Also the morphological aspects described confirm the toxicity of both studied metals. Acknowledgments This work was in part supported by CNPq and CAPES (Brazilian Agencies for Science and Technology), the National Science and Engineering Research Council of Canada and the Secretary of Science and Technology of Parana´ State—Brazil. The authors thank the technical assistance from the Electron Microscopy Center of Federal University of Parana´. References Abou-Donia, M.B., Menzel, D.M., 1967. Fish brain cholinesterase its inhibition by carbamates and automatic assay. Comp. Biochem. Physiol. 21, 99–108. Akaishi, F.M., Silva de Assis, H.C., Jakobi, S.C.G., Eiras-Stofella, D.R., St-Jean, S.D., Courtenay, S.C., Lima, E.F., Wagener, A.L.R., Scofield, A.L., Oliveira Ribeiro, C.A., 2004. Morphological and neurotoxicological findings in tropical freshwater fish (Astyanax sp.) after waterborne and acute exposure to water soluble fraction (WSF) of crude oil. Arch. Environ. Contam. Toxicol. 46, 244–253. Beutler, E., Liechtman, M.A., Coller, B.S., Kipps, T.J., 1995. Measurement of hemoglobin. In: Beutler, E., Liechtman, M.A., Coller, B.S., Kipps, T.J. (Eds.), Willians Hematology, fifth ed. McGraw-Hill, New York, pp. 9–10. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein dyebinding. Anal. Biochem. 72, 248–254. BRASIL, 1986. Resoluc- a˜o do CONAMA No. 20, de 18 de junho de 1986. Dia´rio Oficial da Repu´blica Federativa do Brasil, Brası´ lia, pp. 72–89. Burden, V.M., Sandheinrich, M.B., Caldwell, C.A., 1998. Effects of lead on the growth and d-aminolevulinic acid dehydratase activity of juvenile rainbow trout, Oncorhynchus mykiss. Environ. Pollut. 101, 285–289.

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