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Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO
Chemosphere 56 (2004) 659–666 www.elsevier.com/locate/chemosphere
Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO A.V. Hallare
a,b,*
€hler a, R. Triebskorn , H.-R. Ko
a,c
a
c
Animal Physiological Ecology Section, Zoological Institute, University of T€ubingen, Konrad-Adenauer-Str 20, D-72072, T€ubingen, Germany b Department of Biology, CAS, University of the Philippines, Padre Faura, Manila 1000, Philippines Steinbeis Transfer-Center for Ecotoxicology and Ecophysiology, Kreuzlingerstraße 1, D-72108, Rottenburg, Germany Received 13 August 2003; received in revised form 18 February 2004; accepted 21 April 2004
Abstract One of the most frequently detected pharmaceuticals in environmental water samples is the anti-rheumatic drug, diclofenac. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species are lacking up to now. To determine the developmental toxicity and proteotoxicity of this drug on the growing fish embryos, eggs of zebrafish were exposed to six concentrations of diclofenac (0, 1, 20, 100, 500, 1000, and 2000 lg l1 ) using DMSO as solvent. Early life stage parameters such as egg and embryo mortality, gastrulation, somite formation, movement and tail detachment, pigmentation, heart beat, and hatching success were noted and described within 48- and 96-h of exposure. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in both the diclofenac-treated and respective DMSO controls. Results showed no significant inhibition in the normal development until the end of 96 h for all exposure groups. However, there was a delay in the hatching time among embryos exposed to 1000 and 2000 lg l1 . Late-hatched embryos (108 h) did not differ morphologically from normally hatched embryos. The mortality and average heart rate data did not show significant differences for all embryos in both diclofenac-treated and DMSO control groups. No significant malformations were likewise noted among all developing embryos throughout the exposure period. The levels of heat shock proteins in diclofenac-treated and control embryos did not differ significantly. DMSO control embryos, on the other hand, showed a concentration-dependent increase in hsp 70 levels. We suggest possible modulating effect of diclofenac in DMSOtriggered expression of stress proteins and this might have a possible repercussion on the use of DMSO as solvent in any toxicity assay. Since the present data indicate no significant embryotoxicity and proteotoxicity induced by diclofenac and due to the fact that the concentrations of diclofenac used in the present study is up to 2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose a hazard to early-life stages of zebrafish. 2004 Elsevier Ltd. All rights reserved. Keywords: Embryotoxicity; Heat shock proteins; Diclofenac; DMSO; Zebrafish
*
Corresponding author. Tel.: +49-7071-7573557; fax: +49-7071-7573560. E-mail address: [email protected] (A.V. Hallare).
0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.04.007
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1. Introduction The presence of pharmaceuticals in the environment is an emerging environmental issue. Unlike other classes of substances (e.g. metals, pesticides, PCB’s, and nutrients), the environmental fate and ecotoxicological effects of many pharmaceuticals are poorly understood (Halling-Soerensen et al., 1998; Boxall et al., 2000; Jones et al., 2001). One of the most frequently detected pharmaceuticals in environmental water samples is the antirheumatic drug, Diclofenac (Jux et al., 2002; Weigel et al., 2002; Koutsouba et al., 2003). Diclofenac belongs to non-steroidal anti-inflammatory drugs (NSAIDs), widely used for treating a variety of conditions including menstrual pain, stiffness caused by arthritis and gout, or pain after surgery or childbirth (Chan et al., 2001). After being used in human therapy, this drug finds its way to municipal sewage treatment plants (STPs), where it is not completely eliminated and is often discharged as contaminants to the receiving waters. Based on the recent European Union Draft Guideline III/5504/94 (EU, 2001), the ‘predicted environmental concentration’ (PEC) for Central European surface waters is set at 0.54 lg l1 . However, Ternes (2001) has reported values of up to 1.2 lg l1 . Thus, there has been a steadily growing concern among ecotoxicologists (e.g. Buchberger, 2002; Heberer, 2002; Andreozzi et al., 2003; Koutsouba et al., 2003) concerning the possible implications of this drug on non-target organisms in freshwater environment. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species as well as its proteotoxic potentials in the growing fish embryos are lacking up to now. The early life stage (ELS) test using zebrafish embryos is currently one of the most widely used tools for investigating the detrimental effects of aquatic pollutants in fish. Several authors consider early life stage to be the most sensitive (McKim, 1977; Eaton et al., 1978; Kristensen, 1995; Luckenbach et al., 2001), though, this may not necessarily be true for all compounds and species. The growing embryos offer many diverse endpoints to determine sublethal effects. The zebrafish, Danio rerio, is a small, freshwater, aquarium species which is easy to grow and maintain in different environments, has a short generation time, and breeds almost all year round. Much has also been written about the development (e.g. Hisaoka and Battle, 1958; Westerfield, 1998) and ecotoxicology of this species (e.g. Laale, 1977; Groth et al., 1993; Herrmann, 1993; Ensenbach and Nagel, 1997). The species could, therefore, serve as an excellent model for studying embryotoxic effects induced by pollutants. Heat shock proteins or, more generally, stress proteins belong to a group of highly conservative proteins which are involved in protein assembly, correct folding and translocation of other cellular proteins. Under
conditions of environmental stress, serious cellular impairments such as degradation of protein or synthesis of aberrant proteins might occur. Organisms respond to proteotoxicity with the expression of stress proteins which are able to repair partly denatured proteins. Because the accumulation of these heat shock proteins has been linked to the intensity of stress, these proteins have been regarded as a suitable biomarker in assessing reactions of biota to environmental and physiological stressors (Hightower, 1991; Sanders, 1993; K€ ohler et al., 1996; Triebskorn et al., 1997). Heat shock proteins are categorized based on molecular weights. Hsp 70 is probably the best characterized and best studied of the stress protein family. The present study attempts to determine if diclofenac affects the development of an indicator species, Danio rerio, and to investigate the possible proteotoxic potential of this drug to the growing embryos.
2. Methodology 2.1. Origin and maintenance of parental fish Danio rerio adults were obtained from a commercial dealer and were kept in 25-l full glass aquaria with the following conditions: 26 ± 1 C, with a 12-h/12-h light/ dark cycle. They were fed with frozen red mosquito larvae from an uncontaminated source, alternatively with commercially available artificial diet (TetraMine flakes), twice daily. On the evening before spawning was required, several rectangular mesh wire boxes were laid on the bottom of the aquaria to collect the eggs the following morning. Spawning was triggered once the light was turned on and was completed within 30 min. 2.2. Toxicant and exposure procedures The eggs were collected and rinsed several times with tap water. To start exposure immediately, the eggs were transferred to various exposure chambers. At around 2– 4 h postfertilization, only the fertilized eggs (blastula stage) were selected and transferred to each of the glass petri dishes (10 per plate) containing different concentrations of diclofenac dissolved in DMSO at 26 C: [1 lg l1 diclofenac + 0.00002% DMSO (v/v)]; [20 lg l1 diclofenac + 0.0004% DMSO(v/v)]; 100 lg l1 diclofenac + 0.002% DMSO (v/v)]; [500 lg l1 ) diclofenac + 0.01% DMSO (v/v)]; [1000 lg l1 diclofenac + 0.02% DMSO (v/v)]; and [2000 lg l1 diclofenac + 0.04% DMSO (v/v)] as well as DMSO controls containing only the respective concentrations of DMSO. Reconstituted water (ISO, 1984) served as the over-all control (0 lg l1 ). Occasional stirring as well as replacement of the medium were done daily to ensure even distribution of
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the chemical. The entire experiment was conducted in triplicate with a total of 120 eggs per treatment group. 2.3. Embryo-larval toxicity test The development of blastula eggs was monitored at specified time points (t ¼ 2–4, 12, 24, 36, 48, 60, 72, 84, and 96 h), and in between defined intervals (12 h < t < 24 h; 36 h < t < 48 h), throughout the 96 h of exposure after fertilization. Then observation was extended until the time of hatching for the different exposure groups. Endpoints used for assessing the effects of diclofenac included egg and embryo mortality, gastrulation, somite formation, movement, tail detachment, pigmentation, heartbeat and circulation, and hatching success. Malformations and inhibitory tendencies were also noted and described among the juveniles from both control and treated groups using a stereomicroscope (·8–50) connected to a camera device. 2.4. Hsp 70 measurement All 96-h embryos were frozen in liquid nitrogen and stored at )20 C for subsequent analysis. The frozen embryos (8–10 embryos pooled for one sample) were ultrasonically homogenized for 5 s in 20 ll of extraction buffer (80 mM potassium acetate, 4 mM magnesium acetate, 20 mM Hepes pH 7.5) the volume of which was adjusted based on the animal’s body weight. The homogenized samples were then centrifuged (12 min in 20 000g at 4 C). The total protein concentration in each supernatant was determined according to the method of Bradford (1976). Constant protein weights (10 lg of total protein per lane) were loaded on a minigel SDSPAGE (12% acrylamide: 0.12% bisacrylamide (w/v), 150 at 80 V, 900 at 120 V). Protein was transferred to nitrocellulose by semi-dry blotting, and the filter was blocked for 2 h in 50% horse serum in tris[hydroxymethyl] amino methane (Tris)-buffered saline (TBS) (50 mM Tris pH 5.7, 150 mM NaCl). After washing in TBS, monoclonal antibody (mouse anti-human hsp70; Dianova, FRG, dilution 1:5000 in 10% horse serum/TBS) was added, and the sample was then incubated at room temperature overnight. After repeated washing in TBS for 5 min, the nitrocellulose filter was incubated with the second antibody goat anti-mouse IgG (H + L) coupled to peroxidase (Dianova, dilution 1:1000 in 10% horse serum/TBS) at room temperature for 2 h. The nitrocellulose filter was then washed again in TBS for 5 min and then antibody complex was detected by 4chloro(1)naphthol and 0.015% H2 O2 in 30 mM Tris pH 8.5 containing 6% methanol. The grey value intensity of the hsp 70 band in the immunoblots was quantified by densitometric image analysis (Herolab E.A.S.Y., Germany).
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2.5. Statistical analysis Treatment effects on the developmental parameters and stress protein levels were determined using one-way analysis of variance (ANOVA). Where parameter assumptions of normality and homogeneity of variance were met, ANOVA was followed by Dunnett’s test to compare the treatment means with respective controls. Where the assumptions were not met, data were analyzed using a suitable non-parametric test (Wilcoxon’s rank test). Significant difference occurs for a given parameter when p < 0:05. The entire statistical analysis was carried out using JMP Version 3.2.6 Statistical Software (SAS).
3. Results 3.1. Early life stage parameters Danio rerio eggs developed like those of all teleost fish. The observed developmental events included blastulation, gastrulation, completion of somites and optic cup formation, spontaneous contraction and tail detachment, retinal pigmentation, heart beating, body pigmentation, and commencement of hatching (Fig. 1). In the present study no detectable developmental differences were observed in zebrafish embryos subjected to the control condition, diclofenac plus DMSO, and DMSO alone. All embryos developed almost synchronously. There was, however, a tendency towards a delay in hatching at relatively higher diclofenac concentrations (1000 and 2000 lg l1 ) within the 96 h postfertilization exposure period. The larvae ultimately hatched beyond 96 h and were not different morphologically from the normally hatched embryos. DMSO alone did not cause any delay in hatching. The over-all hatching success rate did not differ significantly for all exposure groups (Fig. 2). No significant mortality or malformations were observed in zebrafish embryos exposed to the different treatment groups. At 48 h, the time point at which various critical developmental events were expected to have taken place, the growth and survival of embryos were not affected at any of the applied diclofenac and DMSO concentrations as well as in the control. Abnormalities in the form of edema, eye defect, and tail defect were found in both control and treated embryos and found to be rather minimal (<5%) (Fig. 3). The average heart rate (48 h) was also found to be unaffected by the tested nominal concentrations of diclofenac and DMSO controls (Fig. 4). The mortality and abnormality trends were maintained until the end of the 96-h exposure period. 3.2. Stress protein response Diclofenac did not significantly induce expression of the heat shock protein 70 relative to the control.
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Fig. 1. Time-specific stages in the early development of zebrafish: (1) blastulation; (2) completion of gastrulation; (3) completion of somites and optic cup formation; (4) spontaneous movement and tail detachment; (5–7) retinal pigmentation, heart beating, and body pigmentation; and (8) commencement of hatching.
Fig. 2. Hatching success rate (%) in zebrafish embryos exposed to (a) control and diclofenac-treated and (b) DMSO alone. Data were based on the original number of embryos at the beginning of the study.
However, 96-h embryos exposed to DMSO alone showed a concentration-dependent increase in hsp 70 expression (Fig. 5). Since the tested diclofenac concentrations contain DMSO in comparable concentrations, it is apparent that diclofenac has a modulating effect on DMSO-induced expression of the stress proteins.
4. Discussion The purpose of the present study was to investigate the effects of diclofenac on survival, hatching, and stress protein responses in zebrafish embryos. Zebrafish appeared to be very suitable for assessing sublethal effects of pharmaceuticals since embryos develop synchronously even when taken from different batches. Thus, any possible retardation of development due to the drug could be clearly and distinctly observed. Our data clearly indicate that environmentally relevant concentrations of diclofenac apparently do not
cause detrimental effects on the early life stages of zebrafish, if they were exposed via the water only. In the experiment, we actually used concentrations which are up to 2000-fold higher than what is currently measured in Central European rivers. Thus, the present situation does not seem to call for immediate concern. Except for an apparent delay in hatching at the latter part of a 96-h exposure period, the development of zebrafish embryos proceeded rather normally in both the control and exposure groups. Late-hatched embryos did not exhibit significant morphological difference compared to earlierhatched embryos. The over-all hatching success rates did not differ significantly among the different exposure groups. Furthermore, no differences were observed in either mortality or incidence of malformations between the treated and control embryos. These indicate that the concentration thresholds at which diclofenac could potentially cause teratogenic or cytotoxic effects were not reached in the present study (McKim, 1985; von Westernhagen, 1988). Similar findings were reported by
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Fig. 3. (a) Viability and (b) malformations in zebrafish embryos exposed to various concentrations of diclofenac and respective DMSO controls for 48 and 96 h.
Heart rate (beats/min) (48 h)
130.0 125.0 120.0 115.0 110.0 105.0 Diclofenac/DMSO
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Fig. 4. Average heart rate (beats per minute) among embryos exposed to diclofenac and respective DMSO controls for 48 h. Column 0 is the reconstituted water control for both diclofenac and DMSO treatment groups and does not contain diclofenac nor DMSO.
Henschel et al. (1997) who reported high EC50 values for fish embryos for several related pharmaceuticals such as salicylic acid (37 000 lg l1 ) and clofibrinic acid (86 000 lg l1 ). These levels are 30–80-fold greater than the highest concentration used in the present study which was only 2000 lg l1 . Other human drugs such as valpromide, methylhexanoic acid, pentenoic acid, and diethylacetic acid were also found to behave similarly as diclofenac, i.e. weak inhibition or no effect on zebrafish development (Herrmann, 1993). However, the same au-
thor had reported pronounced retardation in zebrafish embryo development when exposed to valproic acid. A very recent experiment conducted on Hydra vulgaris, a freshwater invertebrate (Pascoe et al., 2003), showed no negative effects of related pharmaceuticals (ibuprofen, paracetamol, acetylsalicylic acid, amoxicillin, bendroflumethiazide, furosemide, atenolol, diazepam, digoxin, and amlodipine) on survival, feeding, and bud formation at concentrations up to 1000 lg l1 . Cleuvers (2003) exposed daphnids, chlorophyte, and macrophyte to
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Average Hsp 70 Grey Values
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Respective DMSO controls (DMSO alone)
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Fig. 5. Induction of heat shock protein 70 in 96-h old embryos after exposure to diclofenac and respective DMSO controls. Asterisks indicate significant differences ( , p < 0:05; , p < 0:01). Column 0 is the reconstituted water control for both diclofenac and DMSO treatment groups and does not contain diclofenac nor DMSO.
environmentally relevant concentrations of major pharmaceuticals and concluded that acute effect stemming from single substances in the aquatic environment is very unlikely. In mammalian systems, on the other hand, exposure to high concentrations of diclofenac (7500 lg l1 ) resulted in pronounced embryotoxicity to rat whole embryo culture (Chan et al., 2001). The question of why zebrafish embryos showed no reactions to diclofenac treatment needs to be satisfied. As stated earlier, the concentrations used in the present investigation were not sufficient to cause developmental effects. There is a possibility for metabolites to be formed outside the developing embryo, e.g. in natural sediments, and may cause toxicity in natural systems. Thus, indirect adverse effects of the parent compound, diclofenac, cannot be excluded in the field. Whereas early life stages of fish are usually regarded as the most sensitive stage in fish development (McKim, 1977; Eaton et al., 1978; Kristensen, 1995; Luckenbach et al., 2001), the present study with diclofenac in zebrafish embryos suggests that chorion of the eggs [diameter 1.5–3.0 l (Laale, 1977)] may act as a protectant by reducing the incorporation of the toxicant into the embryo (Fent and Meier, 1992). However, with the inherent liphophilicity of diclofenac (log Pow ¼ 4:4), it may behave similarly with other organic contaminants such as musk xylene (log Pow ¼ 4:9) and musk ketone (log Pow ¼ 4:3). These compounds are taken up by the zebrafish embryos during a 96-h exposure period. However, neither musk xylene nor musk ketone have demonstrated any embryotoxic effect (Chou and Dietrich, 1999). Wiegand et al. (2000) have reported that atrazine (log Pow ¼ 2:3) passes the chorion of zebrafish eggs readily and accumulated in the embryos after 24-h exposure. However, atrazine was eliminated from the embryos between 24 and 48 h, due to a detoxication pathway via the glutathione S-transferases (GST) system transforming atrazine to a more hydrophilic GSH conjugates. Whether
the relative resistance of zebrafish embryos to diclofenac may be due to the faster diffusion rate or faster elimination rate (as in nitromusks) or to a possible involvement of the GST system (as in atrazine) is not yet clear as of the moment. Therefore, further research on the pharmacokinetics of this drug is needed to provide a definitive answer. While previous studies have documented increased susceptibility of newly hatched larvae to toxicants as compared to eggs (Eaton et al., 1978; Shazili and Pascoe, 1986), the present study showed that newly hatched larvae were equally insensitive as the embryos. No larval aberrations were observed after hatching. No apparent proteotoxicity was likewise observed in the embryos as a result of exposure to diclofenac. This might indicate that the levels of diclofenac used in the study are not within a specified range that would elicit stress protein responses. These results corroborate very well with the embryotoxicity data since no developmental difference was observed in all groups and therefore no stress was shown by the developing embryos. The insignificant mortality and pathological abnormalities in the ELS-test may likewise reflect the absence of stress among the embryos during the course of development. This does not mean, however, that diclofenac is not toxic to zebrafish embryos when higher concentrations are used. In fact, exposure of zebrafish embryos to >100 mg l1 diclofenac caused 100% mortality among embryos within 24-h exposure (own data, not shown). One interesting finding in the present study is the ability of the solvent DMSO to induce a concentrationdependent increase in hsp 70 production. However, when used as solvent in combination with diclofenac, the characteristic induction of hsp was not observed. It is apparent that diclofenac displays a modulating effect on DMSO induction of the stress protein. The possible mechanisms of the interaction between diclofenac and DMSO (both considered as pharmaceuticals) as regards
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to hsp 70 induction remains to be shown. This will have potential consequence for the use of DMSO as solvent in toxicity assays. DMSO has already been reported to be inappropriate as a solvent in any in vitro study due to its inherent genotoxicity (Herbold et al., 1998). Thus, the use of DMSO as a solvent needs to be reviewed further. Additional studies are also needed to investigate the combined toxicity of diclofenac with other parameters such as physical and chemical factors and to a wide variety of species before we can fully evaluate the ecological effects of this drug in aquatic ecosystem. To conclude, since the concentrations of diclofenac used in the present study is about 1–2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose any hazard to early life stages of the model fish species, Danio rerio, if maternal exposure is excluded. This conclusion is made despite the fact that zebrafish is a tropical species and thus have a much faster (10-fold) embryonal development (Chou and Dietrich, 1999) than indigenous cold water species. In order to take this factor into account, we are currently conducting similar studies using a representative cold water species, brown trout embryos.
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Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO A.V. Hallare
a,b,*
€hler a, R. Triebskorn , H.-R. Ko
a,c
a
c
Animal Physiological Ecology Section, Zoological Institute, University of T€ubingen, Konrad-Adenauer-Str 20, D-72072, T€ubingen, Germany b Department of Biology, CAS, University of the Philippines, Padre Faura, Manila 1000, Philippines Steinbeis Transfer-Center for Ecotoxicology and Ecophysiology, Kreuzlingerstraße 1, D-72108, Rottenburg, Germany Received 13 August 2003; received in revised form 18 February 2004; accepted 21 April 2004
Abstract One of the most frequently detected pharmaceuticals in environmental water samples is the anti-rheumatic drug, diclofenac. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species are lacking up to now. To determine the developmental toxicity and proteotoxicity of this drug on the growing fish embryos, eggs of zebrafish were exposed to six concentrations of diclofenac (0, 1, 20, 100, 500, 1000, and 2000 lg l1 ) using DMSO as solvent. Early life stage parameters such as egg and embryo mortality, gastrulation, somite formation, movement and tail detachment, pigmentation, heart beat, and hatching success were noted and described within 48- and 96-h of exposure. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in both the diclofenac-treated and respective DMSO controls. Results showed no significant inhibition in the normal development until the end of 96 h for all exposure groups. However, there was a delay in the hatching time among embryos exposed to 1000 and 2000 lg l1 . Late-hatched embryos (108 h) did not differ morphologically from normally hatched embryos. The mortality and average heart rate data did not show significant differences for all embryos in both diclofenac-treated and DMSO control groups. No significant malformations were likewise noted among all developing embryos throughout the exposure period. The levels of heat shock proteins in diclofenac-treated and control embryos did not differ significantly. DMSO control embryos, on the other hand, showed a concentration-dependent increase in hsp 70 levels. We suggest possible modulating effect of diclofenac in DMSOtriggered expression of stress proteins and this might have a possible repercussion on the use of DMSO as solvent in any toxicity assay. Since the present data indicate no significant embryotoxicity and proteotoxicity induced by diclofenac and due to the fact that the concentrations of diclofenac used in the present study is up to 2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose a hazard to early-life stages of zebrafish. 2004 Elsevier Ltd. All rights reserved. Keywords: Embryotoxicity; Heat shock proteins; Diclofenac; DMSO; Zebrafish
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Corresponding author. Tel.: +49-7071-7573557; fax: +49-7071-7573560. E-mail address: [email protected] (A.V. Hallare).
0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.04.007
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1. Introduction The presence of pharmaceuticals in the environment is an emerging environmental issue. Unlike other classes of substances (e.g. metals, pesticides, PCB’s, and nutrients), the environmental fate and ecotoxicological effects of many pharmaceuticals are poorly understood (Halling-Soerensen et al., 1998; Boxall et al., 2000; Jones et al., 2001). One of the most frequently detected pharmaceuticals in environmental water samples is the antirheumatic drug, Diclofenac (Jux et al., 2002; Weigel et al., 2002; Koutsouba et al., 2003). Diclofenac belongs to non-steroidal anti-inflammatory drugs (NSAIDs), widely used for treating a variety of conditions including menstrual pain, stiffness caused by arthritis and gout, or pain after surgery or childbirth (Chan et al., 2001). After being used in human therapy, this drug finds its way to municipal sewage treatment plants (STPs), where it is not completely eliminated and is often discharged as contaminants to the receiving waters. Based on the recent European Union Draft Guideline III/5504/94 (EU, 2001), the ‘predicted environmental concentration’ (PEC) for Central European surface waters is set at 0.54 lg l1 . However, Ternes (2001) has reported values of up to 1.2 lg l1 . Thus, there has been a steadily growing concern among ecotoxicologists (e.g. Buchberger, 2002; Heberer, 2002; Andreozzi et al., 2003; Koutsouba et al., 2003) concerning the possible implications of this drug on non-target organisms in freshwater environment. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species as well as its proteotoxic potentials in the growing fish embryos are lacking up to now. The early life stage (ELS) test using zebrafish embryos is currently one of the most widely used tools for investigating the detrimental effects of aquatic pollutants in fish. Several authors consider early life stage to be the most sensitive (McKim, 1977; Eaton et al., 1978; Kristensen, 1995; Luckenbach et al., 2001), though, this may not necessarily be true for all compounds and species. The growing embryos offer many diverse endpoints to determine sublethal effects. The zebrafish, Danio rerio, is a small, freshwater, aquarium species which is easy to grow and maintain in different environments, has a short generation time, and breeds almost all year round. Much has also been written about the development (e.g. Hisaoka and Battle, 1958; Westerfield, 1998) and ecotoxicology of this species (e.g. Laale, 1977; Groth et al., 1993; Herrmann, 1993; Ensenbach and Nagel, 1997). The species could, therefore, serve as an excellent model for studying embryotoxic effects induced by pollutants. Heat shock proteins or, more generally, stress proteins belong to a group of highly conservative proteins which are involved in protein assembly, correct folding and translocation of other cellular proteins. Under
conditions of environmental stress, serious cellular impairments such as degradation of protein or synthesis of aberrant proteins might occur. Organisms respond to proteotoxicity with the expression of stress proteins which are able to repair partly denatured proteins. Because the accumulation of these heat shock proteins has been linked to the intensity of stress, these proteins have been regarded as a suitable biomarker in assessing reactions of biota to environmental and physiological stressors (Hightower, 1991; Sanders, 1993; K€ ohler et al., 1996; Triebskorn et al., 1997). Heat shock proteins are categorized based on molecular weights. Hsp 70 is probably the best characterized and best studied of the stress protein family. The present study attempts to determine if diclofenac affects the development of an indicator species, Danio rerio, and to investigate the possible proteotoxic potential of this drug to the growing embryos.
2. Methodology 2.1. Origin and maintenance of parental fish Danio rerio adults were obtained from a commercial dealer and were kept in 25-l full glass aquaria with the following conditions: 26 ± 1 C, with a 12-h/12-h light/ dark cycle. They were fed with frozen red mosquito larvae from an uncontaminated source, alternatively with commercially available artificial diet (TetraMine flakes), twice daily. On the evening before spawning was required, several rectangular mesh wire boxes were laid on the bottom of the aquaria to collect the eggs the following morning. Spawning was triggered once the light was turned on and was completed within 30 min. 2.2. Toxicant and exposure procedures The eggs were collected and rinsed several times with tap water. To start exposure immediately, the eggs were transferred to various exposure chambers. At around 2– 4 h postfertilization, only the fertilized eggs (blastula stage) were selected and transferred to each of the glass petri dishes (10 per plate) containing different concentrations of diclofenac dissolved in DMSO at 26 C: [1 lg l1 diclofenac + 0.00002% DMSO (v/v)]; [20 lg l1 diclofenac + 0.0004% DMSO(v/v)]; 100 lg l1 diclofenac + 0.002% DMSO (v/v)]; [500 lg l1 ) diclofenac + 0.01% DMSO (v/v)]; [1000 lg l1 diclofenac + 0.02% DMSO (v/v)]; and [2000 lg l1 diclofenac + 0.04% DMSO (v/v)] as well as DMSO controls containing only the respective concentrations of DMSO. Reconstituted water (ISO, 1984) served as the over-all control (0 lg l1 ). Occasional stirring as well as replacement of the medium were done daily to ensure even distribution of
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the chemical. The entire experiment was conducted in triplicate with a total of 120 eggs per treatment group. 2.3. Embryo-larval toxicity test The development of blastula eggs was monitored at specified time points (t ¼ 2–4, 12, 24, 36, 48, 60, 72, 84, and 96 h), and in between defined intervals (12 h < t < 24 h; 36 h < t < 48 h), throughout the 96 h of exposure after fertilization. Then observation was extended until the time of hatching for the different exposure groups. Endpoints used for assessing the effects of diclofenac included egg and embryo mortality, gastrulation, somite formation, movement, tail detachment, pigmentation, heartbeat and circulation, and hatching success. Malformations and inhibitory tendencies were also noted and described among the juveniles from both control and treated groups using a stereomicroscope (·8–50) connected to a camera device. 2.4. Hsp 70 measurement All 96-h embryos were frozen in liquid nitrogen and stored at )20 C for subsequent analysis. The frozen embryos (8–10 embryos pooled for one sample) were ultrasonically homogenized for 5 s in 20 ll of extraction buffer (80 mM potassium acetate, 4 mM magnesium acetate, 20 mM Hepes pH 7.5) the volume of which was adjusted based on the animal’s body weight. The homogenized samples were then centrifuged (12 min in 20 000g at 4 C). The total protein concentration in each supernatant was determined according to the method of Bradford (1976). Constant protein weights (10 lg of total protein per lane) were loaded on a minigel SDSPAGE (12% acrylamide: 0.12% bisacrylamide (w/v), 150 at 80 V, 900 at 120 V). Protein was transferred to nitrocellulose by semi-dry blotting, and the filter was blocked for 2 h in 50% horse serum in tris[hydroxymethyl] amino methane (Tris)-buffered saline (TBS) (50 mM Tris pH 5.7, 150 mM NaCl). After washing in TBS, monoclonal antibody (mouse anti-human hsp70; Dianova, FRG, dilution 1:5000 in 10% horse serum/TBS) was added, and the sample was then incubated at room temperature overnight. After repeated washing in TBS for 5 min, the nitrocellulose filter was incubated with the second antibody goat anti-mouse IgG (H + L) coupled to peroxidase (Dianova, dilution 1:1000 in 10% horse serum/TBS) at room temperature for 2 h. The nitrocellulose filter was then washed again in TBS for 5 min and then antibody complex was detected by 4chloro(1)naphthol and 0.015% H2 O2 in 30 mM Tris pH 8.5 containing 6% methanol. The grey value intensity of the hsp 70 band in the immunoblots was quantified by densitometric image analysis (Herolab E.A.S.Y., Germany).
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2.5. Statistical analysis Treatment effects on the developmental parameters and stress protein levels were determined using one-way analysis of variance (ANOVA). Where parameter assumptions of normality and homogeneity of variance were met, ANOVA was followed by Dunnett’s test to compare the treatment means with respective controls. Where the assumptions were not met, data were analyzed using a suitable non-parametric test (Wilcoxon’s rank test). Significant difference occurs for a given parameter when p < 0:05. The entire statistical analysis was carried out using JMP Version 3.2.6 Statistical Software (SAS).
3. Results 3.1. Early life stage parameters Danio rerio eggs developed like those of all teleost fish. The observed developmental events included blastulation, gastrulation, completion of somites and optic cup formation, spontaneous contraction and tail detachment, retinal pigmentation, heart beating, body pigmentation, and commencement of hatching (Fig. 1). In the present study no detectable developmental differences were observed in zebrafish embryos subjected to the control condition, diclofenac plus DMSO, and DMSO alone. All embryos developed almost synchronously. There was, however, a tendency towards a delay in hatching at relatively higher diclofenac concentrations (1000 and 2000 lg l1 ) within the 96 h postfertilization exposure period. The larvae ultimately hatched beyond 96 h and were not different morphologically from the normally hatched embryos. DMSO alone did not cause any delay in hatching. The over-all hatching success rate did not differ significantly for all exposure groups (Fig. 2). No significant mortality or malformations were observed in zebrafish embryos exposed to the different treatment groups. At 48 h, the time point at which various critical developmental events were expected to have taken place, the growth and survival of embryos were not affected at any of the applied diclofenac and DMSO concentrations as well as in the control. Abnormalities in the form of edema, eye defect, and tail defect were found in both control and treated embryos and found to be rather minimal (<5%) (Fig. 3). The average heart rate (48 h) was also found to be unaffected by the tested nominal concentrations of diclofenac and DMSO controls (Fig. 4). The mortality and abnormality trends were maintained until the end of the 96-h exposure period. 3.2. Stress protein response Diclofenac did not significantly induce expression of the heat shock protein 70 relative to the control.
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Fig. 1. Time-specific stages in the early development of zebrafish: (1) blastulation; (2) completion of gastrulation; (3) completion of somites and optic cup formation; (4) spontaneous movement and tail detachment; (5–7) retinal pigmentation, heart beating, and body pigmentation; and (8) commencement of hatching.
Fig. 2. Hatching success rate (%) in zebrafish embryos exposed to (a) control and diclofenac-treated and (b) DMSO alone. Data were based on the original number of embryos at the beginning of the study.
However, 96-h embryos exposed to DMSO alone showed a concentration-dependent increase in hsp 70 expression (Fig. 5). Since the tested diclofenac concentrations contain DMSO in comparable concentrations, it is apparent that diclofenac has a modulating effect on DMSO-induced expression of the stress proteins.
4. Discussion The purpose of the present study was to investigate the effects of diclofenac on survival, hatching, and stress protein responses in zebrafish embryos. Zebrafish appeared to be very suitable for assessing sublethal effects of pharmaceuticals since embryos develop synchronously even when taken from different batches. Thus, any possible retardation of development due to the drug could be clearly and distinctly observed. Our data clearly indicate that environmentally relevant concentrations of diclofenac apparently do not
cause detrimental effects on the early life stages of zebrafish, if they were exposed via the water only. In the experiment, we actually used concentrations which are up to 2000-fold higher than what is currently measured in Central European rivers. Thus, the present situation does not seem to call for immediate concern. Except for an apparent delay in hatching at the latter part of a 96-h exposure period, the development of zebrafish embryos proceeded rather normally in both the control and exposure groups. Late-hatched embryos did not exhibit significant morphological difference compared to earlierhatched embryos. The over-all hatching success rates did not differ significantly among the different exposure groups. Furthermore, no differences were observed in either mortality or incidence of malformations between the treated and control embryos. These indicate that the concentration thresholds at which diclofenac could potentially cause teratogenic or cytotoxic effects were not reached in the present study (McKim, 1985; von Westernhagen, 1988). Similar findings were reported by
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Fig. 3. (a) Viability and (b) malformations in zebrafish embryos exposed to various concentrations of diclofenac and respective DMSO controls for 48 and 96 h.
Heart rate (beats/min) (48 h)
130.0 125.0 120.0 115.0 110.0 105.0 Diclofenac/DMSO
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Fig. 4. Average heart rate (beats per minute) among embryos exposed to diclofenac and respective DMSO controls for 48 h. Column 0 is the reconstituted water control for both diclofenac and DMSO treatment groups and does not contain diclofenac nor DMSO.
Henschel et al. (1997) who reported high EC50 values for fish embryos for several related pharmaceuticals such as salicylic acid (37 000 lg l1 ) and clofibrinic acid (86 000 lg l1 ). These levels are 30–80-fold greater than the highest concentration used in the present study which was only 2000 lg l1 . Other human drugs such as valpromide, methylhexanoic acid, pentenoic acid, and diethylacetic acid were also found to behave similarly as diclofenac, i.e. weak inhibition or no effect on zebrafish development (Herrmann, 1993). However, the same au-
thor had reported pronounced retardation in zebrafish embryo development when exposed to valproic acid. A very recent experiment conducted on Hydra vulgaris, a freshwater invertebrate (Pascoe et al., 2003), showed no negative effects of related pharmaceuticals (ibuprofen, paracetamol, acetylsalicylic acid, amoxicillin, bendroflumethiazide, furosemide, atenolol, diazepam, digoxin, and amlodipine) on survival, feeding, and bud formation at concentrations up to 1000 lg l1 . Cleuvers (2003) exposed daphnids, chlorophyte, and macrophyte to
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Average Hsp 70 Grey Values
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Respective DMSO controls (DMSO alone)
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Fig. 5. Induction of heat shock protein 70 in 96-h old embryos after exposure to diclofenac and respective DMSO controls. Asterisks indicate significant differences ( , p < 0:05; , p < 0:01). Column 0 is the reconstituted water control for both diclofenac and DMSO treatment groups and does not contain diclofenac nor DMSO.
environmentally relevant concentrations of major pharmaceuticals and concluded that acute effect stemming from single substances in the aquatic environment is very unlikely. In mammalian systems, on the other hand, exposure to high concentrations of diclofenac (7500 lg l1 ) resulted in pronounced embryotoxicity to rat whole embryo culture (Chan et al., 2001). The question of why zebrafish embryos showed no reactions to diclofenac treatment needs to be satisfied. As stated earlier, the concentrations used in the present investigation were not sufficient to cause developmental effects. There is a possibility for metabolites to be formed outside the developing embryo, e.g. in natural sediments, and may cause toxicity in natural systems. Thus, indirect adverse effects of the parent compound, diclofenac, cannot be excluded in the field. Whereas early life stages of fish are usually regarded as the most sensitive stage in fish development (McKim, 1977; Eaton et al., 1978; Kristensen, 1995; Luckenbach et al., 2001), the present study with diclofenac in zebrafish embryos suggests that chorion of the eggs [diameter 1.5–3.0 l (Laale, 1977)] may act as a protectant by reducing the incorporation of the toxicant into the embryo (Fent and Meier, 1992). However, with the inherent liphophilicity of diclofenac (log Pow ¼ 4:4), it may behave similarly with other organic contaminants such as musk xylene (log Pow ¼ 4:9) and musk ketone (log Pow ¼ 4:3). These compounds are taken up by the zebrafish embryos during a 96-h exposure period. However, neither musk xylene nor musk ketone have demonstrated any embryotoxic effect (Chou and Dietrich, 1999). Wiegand et al. (2000) have reported that atrazine (log Pow ¼ 2:3) passes the chorion of zebrafish eggs readily and accumulated in the embryos after 24-h exposure. However, atrazine was eliminated from the embryos between 24 and 48 h, due to a detoxication pathway via the glutathione S-transferases (GST) system transforming atrazine to a more hydrophilic GSH conjugates. Whether
the relative resistance of zebrafish embryos to diclofenac may be due to the faster diffusion rate or faster elimination rate (as in nitromusks) or to a possible involvement of the GST system (as in atrazine) is not yet clear as of the moment. Therefore, further research on the pharmacokinetics of this drug is needed to provide a definitive answer. While previous studies have documented increased susceptibility of newly hatched larvae to toxicants as compared to eggs (Eaton et al., 1978; Shazili and Pascoe, 1986), the present study showed that newly hatched larvae were equally insensitive as the embryos. No larval aberrations were observed after hatching. No apparent proteotoxicity was likewise observed in the embryos as a result of exposure to diclofenac. This might indicate that the levels of diclofenac used in the study are not within a specified range that would elicit stress protein responses. These results corroborate very well with the embryotoxicity data since no developmental difference was observed in all groups and therefore no stress was shown by the developing embryos. The insignificant mortality and pathological abnormalities in the ELS-test may likewise reflect the absence of stress among the embryos during the course of development. This does not mean, however, that diclofenac is not toxic to zebrafish embryos when higher concentrations are used. In fact, exposure of zebrafish embryos to >100 mg l1 diclofenac caused 100% mortality among embryos within 24-h exposure (own data, not shown). One interesting finding in the present study is the ability of the solvent DMSO to induce a concentrationdependent increase in hsp 70 production. However, when used as solvent in combination with diclofenac, the characteristic induction of hsp was not observed. It is apparent that diclofenac displays a modulating effect on DMSO induction of the stress protein. The possible mechanisms of the interaction between diclofenac and DMSO (both considered as pharmaceuticals) as regards
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to hsp 70 induction remains to be shown. This will have potential consequence for the use of DMSO as solvent in toxicity assays. DMSO has already been reported to be inappropriate as a solvent in any in vitro study due to its inherent genotoxicity (Herbold et al., 1998). Thus, the use of DMSO as a solvent needs to be reviewed further. Additional studies are also needed to investigate the combined toxicity of diclofenac with other parameters such as physical and chemical factors and to a wide variety of species before we can fully evaluate the ecological effects of this drug in aquatic ecosystem. To conclude, since the concentrations of diclofenac used in the present study is about 1–2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose any hazard to early life stages of the model fish species, Danio rerio, if maternal exposure is excluded. This conclusion is made despite the fact that zebrafish is a tropical species and thus have a much faster (10-fold) embryonal development (Chou and Dietrich, 1999) than indigenous cold water species. In order to take this factor into account, we are currently conducting similar studies using a representative cold water species, brown trout embryos.
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