Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults

Comparative Biochemistry and Physiology, Part C 152 (2010) 338–345

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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c

Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults☆ Inês Domingues a,⁎, Rhaul Oliveira a, Joana Lourenço a, Cesar Koppe Grisolia b, Sónia Mendo a, A.M.V.M. Soares a a b

CESAM & Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal Departamento de Genética e Morfologia, Universidade de Brasília, Brazil

a r t i c l e

i n f o

Article history: Received 7 January 2010 Received in revised form 28 May 2010 Accepted 31 May 2010 Available online 8 June 2010 Keywords: Biomarkers Embryotoxicity Genotoxicity Potassium dichromate Zebrafish

a b s t r a c t The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC50 = 145.7 mg/L) than adults (96 h-LC50 = 39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Zebrafish (Danio rerio) has been widely used as a model organism in studies of ecotoxicology, not only to assess the effects of different types of chemicals and their risk to the environment but also to understand their modes of action. As environmental concentrations of contaminants are very often in the sub-lethal range, several tools, capable of detecting sub-organismic effects, have been developed to allow a better assessment of the risks posed by chemicals in more realistic scenarios. Zebrafish tests focusing on behavior, histology, molecular markers and endocrine disruption have been showing promising results. However, risk characterization is better achieved if assessed at the different life stages of an organism as physiology, ecology, behavior and response to chemicals varies according with life stages. In the case of zebrafish, early life stage assays have been increasingly used to assess the toxicity of chemicals and waste waters, showing a great potential due to the wide variety of

☆ Ethical considerations: The procedures described in the present paper respect national and international safety regulations and ethical principles for animal welfare. ⁎ Corresponding author. Departamento de Biologia, Universidade de Aveiro, 3810193 Aveiro, Portugal. Tel.: + 351 234 370 350; fax: + 351 234 372 587. E-mail address: [email protected] (I. Domingues). 1532-0456/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2010.05.010

endpoints that can be incorporated in the test (Scholz et al., 2008). Some of the advantages of using zebrafish embryos are: 1) the high egg yield of this species affords large quantities of eggs that can be used in a simple experimental set up (using 24-wells microplates) involving low volumes of test substances; 2) eggs are transparent, allowing the monitoring of the entire organogenesis which is completed within the first 48 h of development; and 3) embryo tests proved to be very informative allowing the study of a wide range of sub-lethal endpoints including developmental (evaluating anomalies and delays of embryo development including hatching) and biochemical parameters (Oliveira et al., 2009). Moreover, as it has been proved that there exists a strong correlation in acute toxicity between early life stages and adults, fish embryo tests have been proposed, mainly for ethical reasons, as a surrogate for tests with adult organisms, at this level (Lammer et al., 2009). However such correlation is still to be verified at sub-lethal levels. For this reason it is important to compare the sensitivity of endpoints such as behavior, biochemical and genotoxicity markers in different life stages. In the present work we aim to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers as endpoints. Biochemical markers have been widely used in aquatic environment to detect the effects of natural and anthropogenic stressors. Their high sensitivity allows the identification of a potential hazard before it is verified at higher levels of biological

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organization, being an important tool in the prevention of risk. In addition, they can also provide information on chemical modes of action. The biomarkers selected for this work include cholinesterase (EC 3.1.1.8, ChE), an important enzyme in the maintenance of normal nerve function (Olsen et al., 2001) which is known to be affected by some metals (Guilhermino et al., 1996); glutathione S-transferase (EC 2.5.1.18, GST), a family of enzymes with a key role in the general biotransformation of xenobiotics and endogenous substances (Hyne and Maher, 2003) and lactate dehydrogenase (EC 1.1.1.27, LDH) which is involved in the carbohydrate metabolism (Diamantino et al., 2001). The test substance used in this work, chromium (VI) or hexavalent chromium (Cr6+), unlike the trivalent chromium, is not common in nature, rather being the result of anthropogenic activity. It is very often used as chemical model in ecotoxicity studies for two main reasons: (i) its importance as a pollutant that threatens especially aquatic ecosystems and (ii) its value as a metallic element in reference toxicants used in standard toxicity tests (e.g. Daphnids (OECD, 2004), rainbow trout, fathead minnow, Daphnia spp, Ceriodaphnia dubia, Selenastrum capricornutum and three spined stickleback (EC, 1990)). Cr (VI) is used in industry for wood preservation, leather tanning, metal finishing, in pigments, refractory processes, among others (Barnhart, 1997), and very often Cr (VI) rich wastes are used as a filling material in dumps where it easily reaches groundwaters by leaching and seepage. Tanning industry, in particular, strongly contributes to chromium aquatic pollution, being estimated that in India alone about 2000 to 3200 tons of elemental chromium escapes into the environment every year (Zayed and Terry, 2003). Cr (VI) acts as a strong oxidizing agent in fish leading to alteration in the levels of oxidative stress enzymes such as catalase and glutathione reductase (Roberts and Oris, 2004; Begum et al., 2006; Farag et al., 2006; Lushchak et al., 2008), it can cause histological and morphological alterations in kidneys, liver and gills (Begum et al., 2006; Farag et al., 2006; Abbas and Ali, 2007; Mishra and Mohanty, 2008) and it is genotoxic, inducing micronucleus, nuclear abnormalities, DNA damage and single strands or double strands breaks (De Lemos et al., 2001; Zhu et al., 2004; Matsumoto et al., 2006; Normann et al., 2008). Alterations on the metabolism and hematological indices as well as behavioral disturbances were also reported in many studies (Begum et al., 2006; Abbas and Ali, 2007; Oner et al., 2008). Table S1 shows results from studies where effects of Cr (VI) in fish were investigated. In this work, a comparison between responses of biomarkers in early life stages and adults, exposed to Cr (VI), will be done. Moreover, endpoints such as embryo development and behavioral effects (early life stages) or genotoxicity markers such as micronuclei and comet assay (adults) will be also evaluated, allowing the selection of the most sensitive and informative test and endpoints. Toxicity parameters obtained (LC50) will be useful as indicative values for other tests with zebrafish where potassium dichromate is used as reference substance.

2. Materials and methods 2.1. Test organisms The zebrafish (Danio rerio) facility established at the Department of Biology, University of Aveiro (Portugal) provided all organisms (zebrafish eggs and adults) used in the present study. In the zebrafish facility, organisms are maintained in carbon-filtered water complemented with salt “Instant Ocean Synthetic Sea Salt”, at 27.0 ± 1 °C and under a 16:8 h light: dark photoperiod cycle (conductivity: 550 ± 50 μS, pH: 7.5± 0.5 and dissolved oxygen N95% saturation). This water was used as dilution water in the preparation of test solutions in all assays performed. Temperature and photoperiod conditions mentioned above were used in all assays. Adult fish are fed twice daily with commercially available artificial diet (ZM 400 Granular) and brine shrimp.

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2.2. Test chemical and preparations of test solutions Potassium dichromate (K2Cr2O7) (Riedel-de Haen analytical grade, CAS number 7778-50-9) was used as the source of chromium (VI). Stock solutions were prepared by dissolving potassium dichromate in water and test solutions were obtained by diluting the stock. Chromium (VI) concentrations in the test solutions were measured by ICP (Inductively Coupled Plasma Spectrometry) at the beginning of each assay. Cr (VI) concentrations mentioned during the text refer to the measured concentrations. 2.3. Early life stages assay The assay was based on the OECD draft guideline on Fish Embryo Toxicity (FET) Test (OECD, 2006) and on the embryo test described by Fraysse et al. (2006). Zebrafish eggs were collected within 30 min after natural mating, rinsed in water and checked under a stereomicroscope (Stereoscopic Zoom Microscope-SMZ 1500, Nikon Corporation). Unfertilized or injured eggs or with irregularities during cleavage were discarded. Seventy-two eggs per treatment (12 replicates) were selected and distributed in 24-wells microplates. Eggs were placed in each well individually with 2 mL of test solution. The following concentrations of Cr (VI) were used: 0, 21, 49, 75, 93, 116 and 120 mg/L. Test run for 6 days. Embryos and larvae were observed daily under a stereomicroscopy (magnification used for observations was 70× for eggs and 40× for larvae). In the embryo phase the following parameters were evaluated: egg coagulation, otolith formation, eye and body pigmentation, somite formation, heart-beat, tail circulation, detachment of the tail-bud from the yolk sac and hatching. After hatching, larvae heart-beat, oedemas, tail malformations, behaviour and mortality were observed and reported. A second test was performed for collection of larvae for biomarkers analyses. Test ended at day 4 and 10 clusters of eight larvae were snap-frozen in microtubes with 0.4 mL of the adequate buffer (described ahead). Concentrations used were the same except for the last concentration that was skipped due to high mortality rates previously observed. Samples were stored at −80 °C until enzymatic analysis. 2.4. Adult fish assay The assay using adult fish followed the OECD Guideline TG 203 (OECD, 1992) in semi static test conditions. Adult zebrafish of similar length and age (2 ± 1 cm, 6 months old) were selected for the test. Three groups of eight Danio rerio were exposed per treatment in aquariums with 4 L of test solution. The following concentrations of Cr (VI) were used:0, 21, 32, 42, 53, 64 mg/L. Test run for 96 h, fish were not fed during the test period and mortality was daily recorded. A second test using a similar design was run to allow the use of organs for biomarker analysis. Three groups of 12 Danio rerio were used per treatment. The following concentrations of Cr (VI) were used: 0, 21, 32 and 42 mg/L. At the end of the test, the number of dead fish was recorded and the living fish were sacrificed in ice by decapitation. Heads, muscles and gills were isolated and snap-frozen in microtubes with 0.5 mL of the adequate buffer and used for ChE, LDH and GST determinations, respectively. Samples were stored at −80 °C until enzymatic analysis. 2.5. Biomarkers determinations Assays were performed to analyse ChE, GST and LDH activities on larvae and adults of Danio rerio. All collected samples were immediately frozen at −80 °C in adequate buffer until analysis as explained in the previous sections. On the day of enzymatic analysis, samples were defrosted on ice and homogenised (Ystral GmbH D-7801). For ChE analyses, heads of adults or clusters of eight larvae per concentration were homogenised on ice on potassium phosphate

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buffer (0.1 M, pH 7.2). The supernatant obtained after the centrifugation of the homogenate (4 °C, 3000 g, and 4 min) was removed and used as enzyme extract for ChE activity determination. Total ChE activity was determined at 414 nm according to the Ellman's method (Ellman et al., 1961), adapted to microplate (Guilhermino et al., 1996) using 0.05 mL of homogenate and 0.25 mL of the reaction solution (1 mL of 10 mM 5.50-dithiobis-2-nitrobenzoic acid solution with sodium hydrogen carbonate, 0.2 mL of 0.075 M acetylcholine solution and 30 mL of 0.1 M phosphate buffer). For the LDH determinations the dorsal muscle of adults or clusters of eight larvae were homogenised in Tris–NaCl buffer (0.1 M, pH 7.2), on ice. Samples were centrifuged (4 °C, 3000 g, 5 min) and the supernatant was removed and used for the enzymatic determination. LDH activity was measured at 340 nm, by continuously monitoring (during 5 min) the decrease in absorbance due to the oxidation of NADH, following the methodology described by Vassault (1983) with the modifications introduced by Diamantino et al. (2001). Activity determinations were made using 40 μL of sample, 250 μL of NADH (0.24 mM) and 40 μL of pyruvate (10 mM). For the GST determinations a pair of gills or clusters of eight larvae was homogenised in potassium phosphate buffer (0.1 M, pH 6.5). After centrifugation (4 °C, 9000 g, 30 min) the supernatant was used to the enzymatic determination. GST activity was determined at 340 nm by monitoring the increase in the absorbance during 5 min, following the general methodology described by Habig and Jakoby (1981), adapted to the microplate reader by Frasco and Guilhermino (2002) and using 0.1 mL of homogenate and 0.2 mL of the reaction mixture (reduced 10 mM glutathione (GSH) and 60 mM 1-chloro-2.4dinitrobenzene). Enzymatic activities were determined in quadruplicate and expressed as nanomoles of substrate hydrolysed per minute per mg of protein. Protein concentration in samples was determined in quadruplicate by the Bradford method (Bradford, 1976), at 595 nm, using γ-globulin as standard. A Labsystem Multiskan EX microplate reader was used for all biochemical determinations.

Visual scoring of cellular DNA on each slide was based on the categorization of 100 randomly-selected cells. The comet-like formations were visually graded into five classes, depending on DNA damage level adapted from Garcia et al. (2004): undamaged—no tail visible (class 0); low damage—tails with low fluorescence and head still round and bright (class 1); medium damage—head and tail equally bright (class 2); high damage—small head, and a long and very bright tail (class 3); extreme damage—very long tail, while head is no longer round (class 4). Each cell was assigned a value (from 0 to 4) according to its comet class, and the overall score for 100 cells ranged from 0 (100% of comets being in class 0) to 400 (100% of comets in class 4) (Garcia et al., 2004). The number of comets in each class was counted, and average DNA damage (DD) was calculated as follows:

2.6. Zebrafish micronucleus (MN) and nuclear abnormalities (NA) assay

3. Results

This assay followed the same procedure of the adult assay but with fewer treatments: 0, 34 and 48 mg/L. The test was carried out as described by Hooftman and Raat (1982) for fish erythrocytes. Peripheral blood was obtained by cardiac puncture with a heparinized syringe and immediately smeared. After fixation in ethanol for 15 min, slides were left to air-dry and then were Giemsa stained at concentration of 5%. Three thousand erythrocyte cells with complete cytoplasm were scored per fish for MN analysis. The criteria for the identification of fish micronucleated erythrocytes were as follows: (a) MN should be smaller than one-third of the main nuclei, (b) MN must not touch the main nuclei, (c) MN must be a nonrefractive, circular or ovoid chromatin body showing the same staining pattern as the main nucleus (Al-Sabti and Metcalfe, 1995). One thousand cells were scored for NA analysis. Several types of NAs are defined according to Carrasco et al. (1990): cells with two nuclei are considered as binuclei, blebbed nuclei present a relatively small evagination of the nuclear membrane, which contains euchromatin; evaginations larger than the blebbed nuclei which can have several lobes are classified as lobed nuclei; nuclei with vacuoles and appreciable depth into a nucleus that does not contain nuclear material are notched nuclei. In this work NA types were not discriminated.

3.1. Embryotoxicity of Cr (VI)

2.7. Comet assay Fish were exposed as described in the adult assay but with fewer treatments Cr (VI): 21, 32 and 42 mg/L of Cr (VI). The assay was conducted in whole blood under yellow light, to prevent UV-induced DNA damage, and performed as described by Nogueira et al. (2006).

DD =

1n1 + 2n2 + 3n3 + 4n4 Σ

=100

where n1–n4 is the number of comets in classes 1–4 and Σ is the sum of all counted comets (n1 + n2 + n3 + n4). DD is expressed in arbitrary units (Collins et al., 1995). Positive controls were always included, and consisted of cells exposed for 1 h to H2O2 before the layering of cells. 2.8. Statistical analysis Sigma Stat 3.1 statistical package was used for statistical analyses (SPSS, 2004). One-way ANOVA was performed except when data did not pass the Kolmogorov Smirnov normality test and therefore a Kruskal–Wallis test was performed. If significant results were found, the Dunett or Dunn's test was used to verify differences between tested concentrations and control. Data ofclass 1 and 2 comet  p ffiffiffi frequencies were transformed log x + 1 , to attain normality. Lethal concentrations (LCx) for early stages and adults were calculated using MiniTab (Minitab, 2000). All statistical analyses were performed with a significance level of 0.05.

In the present study fertilized zebrafish eggs were exposed for 144 h to several concentrations of Cr (VI). The control group presented a normal embryo development as described by Kimmel et al. (1995). Cr (VI) showed low toxicity for embryos while inside the egg. Fig. 1 shows mortality of organisms during the exposure period and it is evident that effect of Cr (VI) was very low on the first days and increased drastically after hatching (see Table 1 for LC50 values). Embryo development parameters (otolith formation, eye and body pigmentation, somite formation, heart-beat, tail circulation, detachment of the tail-bud from the yolk sac) where neither affected. Embryos started to hatch at day 2 (10–29% of the eggs) and on day 3 100% of the embryos had hatched in all treatments. No differences were observed on the hatching rate among concentrations (day 2: one-way ANOVA, F6, 83 = 2.05, p = 0.069; day 3: Kruskal–Wallis, H = 8.49, p = 0.204). Seventy-two hours after the beginning of the exposure, some larvae presented a general malformation characterized by under size and bowing of vertebral column (Fig. 2B). This effect was even more pronounced at concentrations of 93 and 120 mg/L (9.6 and 14.1% respectively). After 72 h, mortality reached 26% at the highest concentration. At 96 h larvae presented a series of behavioural disturbances characterized by a difficulty in keeping a normal upright position in water; larvae were constantly moving the fins in order to avoid lying in one side, but still they were floating upside down and were unable to swim down to the bottom (Figs. 2D and 3). This behavior was observed at concentrations higher than 21 mg/L (Kruskal–Wallis, H = 39.264, p b 0.001). Mortality rate was 54% at the highest

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Fig. 1. Cumulative mortality of Zebrafish embryo and larvae observed for several concentrations of potassium dichromate tested over the exposure period 6 days (144 h) of the test. Values are mean ± standard error. Percentages of hatching at 48 and 72 h (for all concentrations) are indicated in the graph.

concentration and a LC50 of 145.7 mg/L (95% CI = 126.1–179.7) was calculated (Table 1, Fig. 1). The effects of Cr (VI) on ChE, LDH and GST activities in larvae are presented in Fig. 4 (D, E and F): ChE presented a dose-dependent inhibition pattern (one-way ANOVA: F5, 38 = 6.13; p b 0.001, see Fig. 4D); GST activity was drastically inhibited in the three last concentrations (Kruskal–Wallis: H = 35.83; p b 0.001, see Fig. 4E) and LDH activity was increased in larvae exposed to the concentrations of 21 and 75 mg/L and inhibited at the highest concentration tested (116 mg/L) (one-way ANOVA: F5, 40 = 50.35; p b 0.001, see Fig. 4F).

the different “comet” classes was generally affected by Cr (VI) concentrations (Fig 5C). Frequency of class 1 comets was affected by Cr (VI) concentrations (one-way ANOVA: F3, 17 = 7.99; p = 0.002) being more abundant in the control; frequency of class 2 comets was not affected by Cr (VI) concentrations (one-way ANOVA: F3, 17 = 2.49; p = 0.102); frequency of class 3 and 4 comets was affected by Cr (VI) (one-way ANOVA: F3, 17 = 9.78; p b 0.001 and one-way ANOVA: F3, 17 = 8.83; p = 0.002 respectively) being more abundant at the highest concentration of 48 mg/L. 4. Discussion

3.2. Cr (VI) toxicity in adults The 96 h-LC50 value for adult Danio rerio was 39.4 mg/L (95% CI: 32.1–47.2) (Table 1). In vivo effects of Cr (VI) on ChE, LDH and GST activities are presented in Fig. 4 (A, B and C). No alterations on the enzymatic levels were observed for ChE or LDH (ChE: one-way ANOVA: F3, 21 = 0.32; p = 0.81, see Fig. 4A and LDH: ANOVA: F3, 21 = 0.7; p = 0.59, see Fig. 4C). GST activity presented a dose-dependent inhibition pattern (one-way ANOVA: F3, 20 = 4.29; p = 0.02, see Fig. 4B). Effects of Cr (VI) on the number of micronuclei, nuclear abnormalities and DNA integrity are presented in Fig. 5. The number of micronuclei found on fish erythrocytes was affected by the highest Cr (VI) concentration tested (one-way ANOVA: F2, 17 = 7.92; p = 0.04) but not the number of total nuclear abnormalities (one-way ANOVA: F2, 17 = 1.37; p = 0.284, Fig. 4A). DNA integrity was affected (one-way ANOVA: F3, 17 = 29.06; p b 0.001) with all concentrations exhibiting higher DNA damage than the control (see Fig. 5B). The frequency of

Table 1 LC50 and LC10 values for zebrafish early life stages (fertilized eggs) and adults (2 ± 1 cm length) exposed to chromium (VI) using Probit analysis.

Early life 24h 48h 72h 96h 120h 144h Adults 48h 96h

LC50 (mg/L)

95% CI (confidence interval)

LC10 (mg/L)

stages n.d. n.d. 680.33 362.42 245.17 176.15

n.d. n.d. 441.1–2549.6 253.4–14758.0 177.0–430.5 85.1–218.4

98.48 96.31 127.16 88.42 79.56 88.46

26.9–146.8 33.1–139.9 65.0–164.8 0.1–145.4 2.6–130.6 7.9–134.9

141.38 112.76

130.3–149.1 86.1–129.9

126.37 70.94

101.6–135.0 31.1–90.6

95% CI

n.d.—not determined (due to mathematical reasons or inappropriate data).

Zebrafish early life stage assays have been increasingly used in ecotoxicology not only to complement information given by adult assay but also as a substitute to that test. In the present study, 96 hLC50 values of 145.7 mg/L for early life stages and 39.4 mg/L for adults were calculated (see Table 1). Differences in the lethality of a chemical between early life stages and adults may be attributed, among other factors, to the interference of the chorion in the embryos. This acts as a barrier especially to lipophilic compounds (Braunbeck et al., 2005), avoiding the entrance of the chemical and protecting the embryo. Therefore, differences can also be observed between the pre and post hatching period. In our work mortality or effects on embryo development, before hatching, were negligible. In post-hatched larvae mortality levels increased and malformations and equilibrium disturbances could be observed, in agreement with the work of Braunbeck and Lammer (2006). Low embryotoxicity of Cr (VI) to fish eggs comparatively to post-hatched embryos (larvae) has been reported by several authors (see Table S1). Nguyen and Janssen (2001, 2002) worked with Clarias gariepinus and Danio rerio and calculated separately LOEC values for embryos and larvae finding, in both cases, a higher sensitivity of larvae; Krejci and Palikova (2006) worked with the common carp Cyprinus carpio and found no differences between the 120 h-LC50 and the 48 h-post hatching LC50 contributing to the idea that embryos, while inside the egg, are well protected against Cr (VI) effects. They also suggested that the previtelline membrane of fish eggs may constitute a barrier, preventing Cr (VI) to accumulate in the embryos before hatching. No hatching delay was observed in our experiments. Similar results were observed in the above mentioned works and also by Dave et al. (1987). Concerning development abnormalities, work performed with Clarias gariepinus and Cyprinus carp describe anomalies in post-hatched larvae including problems mainly related to the vertebral column (abnormal

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Fig. 2. Developmental and behavioral anomalies observed during the embryo/larvae assay. Plate A shows a larvae from control at 72 h of exposure while plate B shows a larva from the concentration of 75 mg/L with general malformation. Plate C shows a larva from control at 96 h of exposure while plate D shows a larva from the concentration of 49 mg/L with equilibrium disturbances.

body axis and bowing of vertebral column) but also with head, fins, yolk sac and heart (Nguyen and Janssen, 2002; Krejci and Palikova, 2006). In our work, 72 h larvae also presented spine deformities in agreement with the previous data. It was also possible to verify that equilibrium disturbances after 96 h of exposure showed to be a very sensitive parameter (with a 96 h-LOEC of 49 mg/L) in agreement with the study of Braunbeck and Lammer (2006) with potassium chromate, using zebrafish embryos with and without chorion. Cr (VI) concentrations selected to test effects on biomarkers activities are between the LC10 and LC50 values for larvae and adults. To our knowledge, the biomarkers analyzed have not been measured before in zebrafish larvae exposed to Cr (VI). The results obtained showed that, for the tested ranges, larvae seemed to be more sensitive than adults, since a response was observed for the three biomarkers while in adults a response was only observed for GST. In the work of Oliveira et al. (2009) in which zebrafish was exposed to Triclosan, biomarkers measured on larvae also showed a higher responsiveness than adults. ChEs are a family of enzymes important for neurotransmission, being responsible for the degradation of the neurotransmitter

Fig. 3. Percentage of larvae with behavioral anomalies at 96 h. An asterisk (*) represents significant differences from control (Dunn's method).

acetylcholine in the cholinergic synapses. In our work, ChE of larvae was inhibited in a dose-dependent way, suggesting disruption of the nervous system which may cause adverse effects on several functions including respiration, feeding and behavior (Cunha et al., 2007). Although ChEs are known to be inhibited by compounds like organophosphates and carbamate pesticides (Olsen et al., 2001) many authors such as Frasco et al. (2005) suggest that ChEs can be inhibited by metals as well. Examples can be found in the literature such as the work of Costa et al. (2007), where the ChE of the muscle of the fish Hoplias malabaricus was inhibited by dietary exposure to methylmercury and the work of Silva and Pathiratne (2008) where ChE of Nile tilapia was inhibited by cadmium both in vivo and in vitro. However, in our work, at concentrations close to LC50 values, head ChE of adults was not inhibited as expected. Compounds biotransformation mechanisms in organisms are divided into two phases: in phase I, the enzyme system P450-MO is involved, catalyzing a variety of oxidative reactions and converting the initial substance into more water-soluble compounds. In phase II, the enzymes of the GSTs' family play a central role in the detoxification of both xenobiotics and endogenic compounds that conjugate with glutathione. Therefore, induction of GST activity has been used as a biomarker of exposure to xenobiotics with electrophilic centers. However, as Kostaropoulos et al. (2005) described, contradictory results have been obtained after exposure to metals, with increases and decreases in the activity being observed depending on the test organism and tissue analyzed. In literature, gill GST activity was induced in the fish Pomatoschistus microps under exposure to mercury and copper (Vieira et al., 2009) and was inhibited in Channa punctatus under exposure to a mixture of metals (copper, cadmium, iron and nickel) (Pandey et al., 2008). In our work a dose-dependent inhibition of GST activity was observed for adults and larvae which may be the result of the relatively high concentrations tested. LDH is a key enzyme in the anaerobic pathway of energy production, responsible for the catalyses of the interconversion of pyruvate to lactate in glycolysis, and has been used as general biomarker of stress in fish

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Fig. 4. Variation of biomarkers activities (mean value ± standard error) on zebrafish larvae and adults after 96 h exposure to potassium dichromate: A, B and C: ChE, GST and LDH activities on adults; D, E and F: ChE, GST and LDH activities on larvae. Asterisks mean significantly different from control treatment (Dunett test P b 0.05 after 1-way ANOVA, or Dunn's test after one-way ANOVA on Ranks).

(Almeida et al., 2001; Osman et al., 2007; Vieira et al., 2008). At the lowest concentrations tested, a significant increase of LDH activity in larvae was observed in agreement with the work of Gill et al. (1990) with mercury in Puntius conchonius. However, at higher concentrations, an inhibition of larvae LDH activity was observed. Variations of LDH reflect metabolic alterations in the larvae exposed to Cr (VI). Several works indicate that many fish suffer a decrease on LDH levels after exposure to metals. Some examples are the (post hatching) larvae of the African catfish (Clarias gariepinus) exposed to lead nitrate (Osman et al., 2007), the fish Channa punctatus after exposure to mercuric chloride (Sastry and Rao, 1984) and the fish Oreochromis niloticus exposed to cadmium (Almeida et al., 2001). LDH activity measured in adult zebrafish exposed to Cr (VI) was not affected. The enzymes analyzed, which are involved in neurological and detoxification processes and energy production were affected by the exposure to Cr (VI), especially on larvae, providing a first clue about the chemical mode of action. LDH seemed to be the most sensitive with differences being detected at the lowest concentration tested

(LOEC = 21 mg/L), followed by GST (LOEC = 75 mg/L) and ChE (LOEC= 116 mg/L). In early stages, Cr (VI) can also affect the nervous system as proved by the equilibrium disturbances observed (LOEC= 49 mg/L). The responsiveness of the biomarkers analyzed in larvae reinforces the idea of the usefulness of early life stages assay in the assessment of chemicals effects. Although concentrations of Cr (VI) tested are not commonly found the environment, this constitutes a first step in the understanding of how these biomarkers can be used to assess environmental risk. Further experiments should consider more realistic scenarios, focusing on long term exposures to assess chronic effects, influence on biomarkers activities and their relationships. Other biomarkers, such as oxidative stress enzymes should also be used to complement information. In adults, for the concentration range between the 96 h-LC10 and LC50 only GST responded (LOEC =42 mg/L), perhaps because in adults longer exposure times are required for effects to be detectable. However, genotoxicity biomarkers analyzed (micronucleous and comet assay) seemed to be very sensitive (LOEC=48 mg/L for MN and 21 mg/L for comets). Micronuclei are cytoplasmatic chromatin

Fig. 5. Endpoints of genotoxicity. Graph A: Total number of micronuclei and nuclear abnormalities found per treatment group. Graph B: DNA Damage. Graph C: Comets of each class found per treatment group. Comet-like formations were visually graded into five progressive classes (0, undamaged to 4, extreme damage), depending on DNA damage level. Results are expressed as mean ± standard error. An asterisk (*) represents significant differences from control (Dunnet method following one-way ANOVA).

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masses with the appearance of small nuclei that arise from chromosome fragments or intact whole chromosomes lagging behind in the anaphase stage of cell division. Therefore, their presence in cells is indicative of structural and/or numerical chromosomal aberrations. Nuclear abnormalities, other than micronuclei, are also considered to be indicators of genotoxic damage complementing MN scoring (Cavas and ErgeneGozukara, 2005). The comet assay (single cell gel electrophoresis (SCGE) assay) is a method to detect DNA strand breaks at single cell level (Hartmann et al., 2003) indicating a loss of DNA integrity (Matsumoto et al., 2006; Nogueira et al., 2006). The combinations of both techniques can contribute more accurately to the toxicity assessment of chemical compounds. Genotoxic effects of Cr (VI) are well documented and are caused by reactive oxygen species (ROS) that directly attack nucleophilic centers in the DNA macromolecules (Schmitt et al., 2005). In fish exposed to hexavalent chromium, an induction in the number of micronucleus was observed in Hypostomus plecotomus, Cyprinus carpio, Oryzias latipes and Pimephales promelas (De Lemos et al., 2001; Zhu et al., 2004; Goodale et al., 2008; Normann et al., 2008) confirming in this way Cr (VI) clastogenic potential, which was also observed in our work. Two studies where Oreochromis niloticus was exposed to chromium contaminated effluents showed increased frequencies of micronucleous and nuclear abnormalities (Cavas and Ergene-Gozukara, 2005; Matsumoto et al., 2006). DNA damage analyzed through the comet assay was observed in all concentrations tested in the present work. Similar results were obtained by other authors where two fish species (Oreochromis niloticus and Leuciscus cephalus) were exposed to chromium contaminated effluents (Matsumoto et al., 2006; Ergene et al., 2007). Since there are already techniques to develop the comet assay in fish larvae (Kosmehl et al., 2006), this should be included in further studies with early life stages for the test with potential genotoxic compounds.

5. Conclusions Zebrafish eggs proved to have lower sensitivity to Cr (VI) than adults, in terms of lethality, probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave some clear indications on Cr (VI) mode of action. Cr (VI) seems to be neurotoxic (inhibited ChE), to inhibit GST activity and interfere with cellular metabolic activity (changes in LDH activity). The biomarkers incorporated in the early life stages assay seem to be a valuable tool for the analysis of chemicals effects and modes of action; moreover, early life stages assay also contributed with relevant information regarding anomalies in larvae development and behavior, especially at 96 h where Cr (VI) seemed to cause swimming and equilibrium disturbances. In this way, embryo/larvae assay was indicative of possible effects of Cr (VI) on population dynamics of D. rerio as it would affect significantly embryos and larvae, originating non-surviving fish and short-term life fish. Further research should focus on the use of biomarkers to assess long term effects which are more relevant ecologically.

Acknowledgements Authors acknowledge Fundação para Ciência e Tecnologia (FCTPortugal) for the financial support through the grant SFRH/BPD/ 31752/2006 and University of Aveiro. Authors also want to thank the four reviewers for their comments, which substantially improved this manuscript.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.cbpc.2010.05.010.

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