- Email: [email protected]
Accumulation and elimination of cadmium in larval stage zebrafish following acute exposure
ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 66 (2007) 44–48 www.elsevier.com/locate/ecoenv
Short communication
Accumulation and elimination of cadmium in larval stage zebrafish following acute exposure Carlyn J. Matza, Ronald G. Trebleb, Patrick H. Kronea, a
Department of Anatomy and Cell Biology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5 b Department of Chemistry and Biochemistry, University of Regina, 3737 Wascana Parkway, Regina, Saskatchewan, Canada S4S 0A2 Received 29 July 2005; received in revised form 28 October 2005; accepted 5 November 2005 Available online 10 January 2006
Abstract A number of recent studies have examined the impact of acute cadmium exposure on early zebrafish development at the morphological, cellular, and molecular levels. However, no information on the accumulation and elimination of cadmium during early life stages of zebrafish development has been available. Here we have quantified cadmium accumulation in larval zebrafish (Danio rerio) by graphite furnace atomic absorption spectroscopy following short-term acute exposure and recovery periods. Zebrafish (80 h postfertilization) were exposed to various concentrations of cadmium (0.2, 1.0, 5.0, 25, 125 mM) for 3 h. Cadmium accumulation in larvae increased with exposure concentration. After exposure at 5.0, 25, and 125 mM cadmium, the fish were allowed to recover in freshwater for 0, 12, or 24 h. Cadmium content did not show a statistically significant decrease over the recovery period when exposed to 5.0 or 25 mM cadmium, whereas significant losses over the recovery period were observed following 125 mM exposure. These results suggest that the larval zebrafish decrease total cadmium body burden only following relatively high short-term acutely toxic exposures. r 2005 Elsevier Inc. All rights reserved. Keywords: Zebrafish larvae; Cadmium; Bioaccumulation
1. Introduction Cadmium (Cd), a heavy metal with limited biological function (Lane and Morel, 2000), is widely distributed in the environment as a result of natural and anthropogenic activities. Cd is a common pollutant in surface waters and can cause adverse effects on fish and other organisms inhabiting these bodies of water (Perceval et al., 2004; Gravel et al., 2005). Accordingly, the effect of Cd on aquatic ecosystems has been and continues to be an active area of research (e.g., Ciutat et al., 2005; Riddell et al., 2005; Stanley et al., 2005). While numerous studies have examined the effects of cadmium on juvenile and adult fish and fish cells, the mechanisms of action of cadmium in early life stages is only beginning to be addressed. Furthermore, while the long biological half-life of Cd allows it to readily bioaccumulate in exposed organisms (e.g., Ke and Wang, 2001; Savinov et al., 2003), very little Corresponding author. Fax: +1 306 966 4298.
E-mail address: [email protected] (P.H. Krone). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.11.001
about the uptake and elimination of cadmium during embryonic and larval stages of fish development is known. Elucidation of Cd accumulation is important for mechanistic interpretation of its toxic effects. Zebrafish offer many advantages for toxicological assessment of embryonic and larval stages including small size, high reproductive potential, transparent embryos, and well-described development (reviewed in Hill et al., 2005). Larval zebrafish have been utilized to study the effects of many environmental pollutants including cadmium (Blechinger et al., 2002), insecticides (Levin et al., 2004), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (Mattingly et al., 2001). The adverse effects of Cd on developing zebrafish have been investigated at the whole-body, cellular, and molecular levels. These effects include ectopic apoptosis (Chan and Cheng, 2003), morphological deformities due to altered gene expression (Cheng et al., 2000), abnormal somitogenesis (Chow and Cheng, 2003), and induction of heat shock protein 70 gene expression (hsp70; Blechinger et al., 2002). In the latter study, we demonstrated using a live transgenic zebrafish model that a stress-responsive
ARTICLE IN PRESS C.J. Matz et al. / Ecotoxicology and Environmental Safety 66 (2007) 44–48
hsp70/eGFP transgene acts as an accurate indicator of cellspecific induction of hsp70 gene activation following acute cadmium exposure during larval development. Furthermore, this transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early life stage toxicity and is sensitive enough to detect cadmium at doses below the median adverse effect concentration and the median lethal concentration. Here we have examined the accumulation and elimination of cadmium in whole zebrafish larvae under comparable acute exposure and recovery regimes. 2. Materials and methods 2.1. Animal care and embryo collection Adult wild-type zebrafish were obtained from a local pet store and maintained at 28 1C in carbon-filtered tap water, with a photoperiod of 14 h. Embryos were collected and staged using standard procedures as outlined in Westerfield (1995). After collection embryos and larvae were reared in 25-mL petri dishes with system water changes daily.
2.2. Reagent and treatment solutions Cadmium chloride hemipentahydrate (CdCl2 2.5 H2O; CAS No. 779078-5) was purchased from J.T. Baker Inc. (Phillipsburg, NJ). A 1 mM Cd stock solution was prepared in triple-distilled water. Treatment solutions are made from dilutions of the stock in carbon-filtered tap water, and exposures are conducted in sterile 25-mL petri dishes.
2.3. Cadmium treatment and sample preparation For the acute exposures, larval zebrafish were placed in 25-mL plastic petri dishes containing 0.2, 1.0, 5.0, 25, or 125 mM Cd (0.02, 0.11, 0.56, 2.8, or 14.1 mg/mL Cd, respectively) as described previously (Blechinger et al., 2002). Untreated controls were included with all exposure groups. Exposures began when the newly hatched larvae were 80 h postfertilization (hpf) for a duration of 3 h at 28 1C. After the exposure, the larvae were rinsed several times in fresh system water to remove nonaccumulated Cd. At least three swirling rinses at 50 times dilution per rinse were performed. For recovery experiments, larvae from each Cd exposure group and control were then placed in unused petri dishes containing fresh system water to ensure no exposure to residual Cd adsorbed to original treatment dishes. Following the exposure period (i.e., no recovery) or recovery period (12 or 24 h) the larvae were transferred to preweighed microcentrifuge tubes (10 larvae per tube). Excess water was removed and the wet weight of the larvae was determined. Then 1 mL of 5% HNO3 was added to each tube and the tubes were placed in a 65 1C water bath for 12 h to digest the tissue. The digests were allowed to settle within the tubes, and the supernatants were removed to be analyzed for Cd content. Each replicate (n) represents one set of 10 larvae; 7–15 replicates (n ¼ 7215) were analyzed and an average Cd content was calculated for each data set.
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Cd reference standard solution and analysis of the reference material TORT-2 lobster hepatopancrease (NRC-CNRC, Ottawa, Canada). From the Cd concentration of the digested sample, the ng of Cd per g wet weight of fish tissue was determined and used for data analysis.
2.5. Data treatment Data were normalized against average values obtained for untreated controls. One-way ANOVA with Tukey–Kramer multiple comparisons posttest was performed using GraphPad InStat version 3.05 (GraphPad Software, San Diego, CA). The limit of significance was set at Po0:05 throughout.
3. Results 3.1. General observations No mortality or nonlethal effects were observed in any of the treatment or control groups over the course of the exposures and/or recovery periods. This is most likely due to the short length of the exposure periods. We have previously demonstrated that the LC50 and EC50 of Cd for zebrafish larvae determined using a 96-h acute exposure beginning at 72 hpf are 18.8 and 1.7 mM Cd, respectively (Blechinger et al., 2002), whereas the same study found no increase in mortality following a 3-h pulse exposure. 3.2. Three-hour pulse exposures and recovery Accumulation of Cd by larval zebrafish increased with the Cd concentration of treatment solutions (Figs. 1 and 2). The Cd content in control samples averaged below 25 ng/g wet weight for exposure and recovery time points, representing any spectral interference during quantification and potential background Cd content. All samples generated following the pulse exposures had greater Cd content than control samples. Following a 3-h pulse exposure, Cd uptake was significantly different from
2.4. Cd quantification Cd concentrations were measured using a graphite furnace atomic absorption spectrophotometer (Varian SpectraAA 220Z) with Zeeman background correction; 6 mL of digest sample and 6 mL 2% m/v NH4H2PO4 (matrix modifier; Baker Analyzed A.C.S. Reagent; J.T. Baker) were hot injected at 85 1C. Operating conditions were 12 s at 95 1C and 15 s at 120 1C to dry the sample, 15 s at 500 1C, and 3 s at 2000 1C. Each sample was analyzed in duplicate. Data accuracy was monitored using quality control samples prepared from Fischer certified
Fig. 1. Whole-body Cd content of 80-hpf larval zebrafish following 3-h exposure to 0.2, 1.0, or 5.0 mM Cd. Mean7SE (n ¼ 7215 replicates of 10 larvae each). **Po0:001. Data were normalized against values obtained for untreated controls.
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C.J. Matz et al. / Ecotoxicology and Environmental Safety 66 (2007) 44–48
Fig. 2. Whole-body Cd content versus length of recovery period of 80-hpf larval zebrafish following 3-h Cd exposure at 5.0, 25, or 125 mM Cd. Mean7SE (n ¼ 7215 replicates of 10 larvae each). Data were normalized against values obtained for untreated controls.
controls (Po0:001) when the developing zebrafish were exposed to 1.0, 5.0, 25, and 125mM, with average total body burdens of 69.2, 151.3, 512.8, and 11,100 ng Cd/g wet weight relative to control samples, respectively. While exposure to 0.2 mM Cd resulted in an average total body burden of 13.3 ng Cd/g relative to control, this value was not statistically significant. Recovery periods of 12 and 24 h did not result in a change in Cd present in larval zebrafish following a 3-h pulse exposure at 5.0 mM Cd (Fig. 2). A consistent decrease in total Cd content was observed following exposure at 25 mM Cd; however, the loss was not statistically significant (Fig. 2). In contrast, Cd levels dropped significantly during recovery from a pulse exposure to 125 mM Cd (Fig. 2). Whole larval cadmium burden declined 2.5-fold within 12 h of recovery (Po0:001) and was reduced approximately 15-fold (Po0:05) by 24 h of recovery in freshwater. However, total cadmium levels were still approximately 10-fold higher in the 125 mM exposed larva compared to those exposed to 5.0 mM Cd. 4. Discussion For this investigation, 80-h-old hatched zebrafish larvae were exposed to a 3-h acute pulse of cadmium at concentrations comparable to those used in previous studies that examined molecular and morphological endpoints (Blechinger et al., 2002; Chow and Cheng, 2003). As expected, accumulation of Cd by larval zebrafish during the 3-h pulse exposure increased with concentration of the treatment solution (Fig. 1). Cd accumulation by unhatched zebrafish embryos exposed to acute Cd concentrations for 48 h has been reported to be concentration dependent (Hallare et al., 2005). Chow and Cheng (2003) exposed zebrafish embryos (also prior to hatch from chorion) to 100 mM Cd during gastrulation and segmentation
(4–24 hpf) but found substantially less Cd accumulation than that in our present study. This difference is likely due to the presence of the chorion, a membrane envelope surrounding the egg, which is capable of acting as a barrier to Cd transfer to the developing embryo (Rombough and Garside, 1982). The protective effect of the chorion is evident in observations that hatched larvae are more susceptible to Cd than unhatched embryos (Hallare et al., 2005; S.R. Blechinger and P.H. Krone, unpublished data). Once hatched from the chorion, larval stage zebrafish readily accumulate Cd from their environment most likely leading to an increased sensitivity to Cd. The routes of Cd uptake in larval zebrafish have yet to be determined. Previous studies with fully developed zebrafish have demonstrated that, following exposure to waterborne Cd, uptake occurs mainly at the gills via facilitated diffusion through Ca2+-transporting chloride cells (Wicklund Glynn et al., 1994) and this process can become saturated (Wicklund Glynn, 1996). Cd accumulation in the olfactory sensory system has also been reported following aqueous exposure, suggesting that some Cd uptake occurred at the sensory neurons exposed to the surrounding environment (reviewed in Tjalve and Henriksson (1999)). These are both locations at which we have observed hsp70/eGFP transgene activation following exposure to sublethal doses of Cd in our previous work (Blechinger et al., 2002). Thus, it is likely that both of these pathways are involved in Cd accumulation observed in the present study with larval zebrafish. Our results indicate that larval fish exposed to a high acute concentration of Cd are able to significantly reduce their body burden after as little as 12 h in freshwater. Jackson et al. (2003) reported that the rate of elimination of Cd by Menidia beryllina was also highest in fish that accumulated the most Cd and rapid elimination occurred within the first 24 h. Rapid elimination of Cd suggests that not all Cd initially acquired following high acute doses is tightly bound to tissues as it is readily removed from the body. A previous study with adult carp reported that a decrease in Cd content during the depuration period was associated with rapid loss of Cd from muscle tissue, whereas loss from the kidney and liver was much slower. This suggests that during depuration Cd is transferred from the muscle to the liver and kidney for excretion (de Conto Cinier et al., 1999). In the present study, elimination via the liver and kidney is likely the means for decreasing total Cd body burden. By 50 hpf the liver of a developing zebrafish is a separate and distinct tissue, and by 72 hpf the liver is vascularized and presumably functional beyond this stage (Field et al., 2003). Furthermore, the relatively simple pronephros kidney of zebrafish is formed and functional by 48 hpf (reviewed in Drummond, 2003). Thus, during the exposure (80–83 hpf) and recovery periods (83–107 hpf) utilized in this study, the liver and kidney of the developing zebrafish are physiologically functional and likely responsible for metabolism and excretion of Cd, respectively.
ARTICLE IN PRESS C.J. Matz et al. / Ecotoxicology and Environmental Safety 66 (2007) 44–48
We have previously reported that 80-h-old zebrafish larvae activate an hsp70/eGFP transgene in a dose- and tissue-specific manner following similar acute cadmium exposures (Blechinger et al., 2002). While larvae exposed to 0.2 mM Cd activated the transgene only in cells of the skin and gills, we were unable to detect any significant increase in total body cadmium in similarly exposed larvae in the present study. This suggests that these cells may activate the stress response at lower acute concentrations as a result of their direct exposure to waterborne cadmium. Previous studies have similarly shown that adult fish tissues directly exposed to waterborne Cd such as the olfactory epithelium also exhibit more severe lesions than internal tissues such as the gut epithelium (Stromberg et al., 1983). In our previous study with the transgenic indicator strain, we also showed that hsp70/eGFP was strongly activated in the liver and kidney within 24 h following exposure to a 3-h pulse of 125 mM Cd, whereas the transgene was not activated in these organs during a 24-h recovery period following exposure to 5 mM cadmium. Interestingly, larvae exposed to 5 mM Cd in the present study did not reduce their total cadmium burden during a 24-h recovery, and larvae exposed to 25 mM Cd displayed a consistent but statistically insignificant trend toward Cd elimination. In contrast, 93% of the cadmium was eliminated from larvae during recovery from the 125 mM concentration. This suggests that a relationship exists between the concentrations of cadmium capable of eliciting a stress response within the organs of metal metabolism (liver) and excretion (kidney) in zebrafish larvae and those at which the mechanisms of detoxification and elimination begin to function. 5. Conclusion The results of this study show that larval zebrafish readily accumulate waterborne Cd in a dose-dependent manner following 3-h acute exposures and that the body burden can be significantly decreased after a 12-h recovery period in freshwater following high-dose exposure. This rapid elimination suggests that not all Cd acquired during a short-term acute exposure is tightly bound to tissues. In contrast, no Cd is eliminated during a 24-h recovery period following acute exposure to a more moderate Cd dose. Finally, no significant increase in total body cadmium occurs at low acute exposure concentrations that have been shown to be capable of activating a stressresponsive transgene in cells of the skin and gills in earlier studies. Acknowledgments The authors thank Drs. M. Wickstrom and L.E. Doig, Toxicology Centre, University of Saskatchewan for use of the GFAAS and technical support. This work was supported by an NSERC discovery grant to PHK, and a Canada Graduate Scholarship to CJM.
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References Blechinger, S.R., Warren Jr., J.T., Kuwada, J.Y., Krone, P.H., 2002. Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line. Environ. Health Perspect. 110, 1041–1046. Chan, P.K., Cheng, S.H., 2003. Cadmium-induced ectopic apoptosis in zebrafish embryos. Arch. Toxicol. 77, 69–79. Cheng, S.H., Wai, A.W.K., So, C.H., Wu, R.S.S., 2000. Cellular and molecular basis of cadmium-induced deformities in zebrafish embryos. Environ. Toxicol. Chem. 19, 3024–3031. Chow, E.S.H., Cheng, S.H., 2003. Cadmium affects muscle type development and axon growth in zebrafish embryonic somitogenesis. Toxicol. Sci. 73, 149–159. Ciutat, A., Cerino, M., Mesmer-Dudons, N., Anschutz, P., Boudou, A., 2005. Cadmium bioaccumulation in Tubificidae from the overlying water source and effects on bioturbation. Ecotoxicol. Environ. Saf. 60, 237–246. de Conto Cinier, C., Petit-Ramel, M., Faure, R., Garin, D., Bouvet, Y., 1999. Kinetics of cadmium accumulation and elimination in carp Cyprinus carpio tissues. Comp. Biochem. Physiol. C 122, 345–352. Drummond, I., 2003. Making a zebrafish kidney: a tale of two tubes. Trends Cell Biol. 13, 357–365. Field, H.A., Ober, E.A., Roeser, T., Stainier, D.Y.R., 2003. Formation of the digestive system in zebrafish. I. Liver morphogenesis. Dev. Biol. 253, 279–290. Gravel, A., Campbell, P.G.C., Hontela, A., 2005. Disruption of the hypothalamo-pituitary-interrenal axis in 1+ yellow perch (Perca flavescens) chronically exposed to metals in the environment. Can. J. Aquat. Sci. 62, 982–990. Hallare, A.V., Schirling, M., Luckenbach, T., Kohler, H.R., Triebskorn, R., 2005. Combined effects of temperature and cadmium on developmental parameters and biomarker responses in zebrafish (Danio rerio) embryos. J. Therm. Biol. 30, 7–17. Hill, A.J., Teraoka, H., Heideman, W., Peterson, R.E., 2005. Zebrafish as a model vertebrate for investigating chemical toxicity. Toxicol. Sci. 86, 6–19. Jackson, C.S., Sneddon, J., Heagler, M.G., Lindow, A.G., Beck, J.N., 2003. Use of flame atomic absorption spectrometry and the effect of water chemistry for the study of the bioaccumulation of cadmium in Menidia beryllina (cope), the tidewater silverside. Microchem. J. 75, 23–28. Ke, C., Wang, W.X., 2001. Bioaccumulation of Cd, Se, and Zn in an estuarine oyster (Crassostrea rivularis) and a coastal oyster (Saccostrea glomerata). Aquat. Toxicol. 56, 33–51. Lane, T.W., Morel, F.M.M., 2000. A biological function for cadmium in marine diatoms. Proc. Natl. Acad. Sci. USA 97, 4627–4631. Levin, E.D., Swain, H.A., Donerly, S., Linney, E., 2004. Developmental chlorpyrifos effects on hatchling zebrafish swimming behaviour. Neurotoxicol. Teratol. 26, 719–723. Mattingly, C.J., McLAchlan, J.A., Toscano Jr., J.A., 2001. Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AhR) function in developing zebrafish (Danio rerio). Environ. Health Perspect. 109, 845–849. Perceval, O., Couillard, Y., Pinel-Alloul, B., Gigue`re, A., Campbell, P.G.C., 2004. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses. Aquat. Toxicol. 69, 327–345. Riddell, D.J., Culp, J.M., Baird, D.J., 2005. Behavioural responses to sublethal cadmium exposure within an experimental aquatic food web. Environ. Toxicol. Chem. 24, 431–441. Rombough, P.J., Garside, E.T., 1982. Cadmium toxicity and accumulation in eggs and alevins of Atlantic salmon Salmo salar. Can. J. Zool. 60, 2006–2014. Savinov, V.M., Gabrielsen, G.W., Savinov, T.N., 2003. Cadmium, zinc, copper, arsenic, selenium and mercury in seabirds from the Barents Sea: levels, interspecific and geographical differences. Sci. Total Environ. 306, 133–158. Stanley, J.K., Brooks, B.W., La Point, T.W., 2005. A comparison of chronic cadmium effects on Hyalella azteca in effluent-dominated
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stream mesocosms to similar laboratory exposures in effluent and reconstituted hard water. Environ. Toxicol. Chem. 24, 902–908. Stromberg, P.C., Ferrante, J.G., Carter, S., 1983. Pathology of lethal and sublethal exposure of fathead minnows, Pimephales promelas, to cadmium: a model for aquatic toxicity assessment. J. Toxicol. Environ. Health 11, 247–259. Tjalve, H., Henriksson, J., 1999. Uptake of metals in the brain via olfactory pathways. Neurotoxicology 20, 181–196.
Westerfield, M., 1995. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio). University of Oregon Press, Eugene. Wicklund Glynn, A., 1996. The concentration dependency of branchial intracellular cadmium distribution and influx in the zebrafish (Brachydanio rerio). Aquat. Toxicol. 35, 47–58. Wicklund Glynn, A., Norrgren, L., Mussener, A., 1994. Differences in uptake of inorganic mercury and cadmium in the gills of zebrafish, Brachydanio rerio. Aquat. Toxicol. 30, 13–26.
Ecotoxicology and Environmental Safety 66 (2007) 44–48 www.elsevier.com/locate/ecoenv
Short communication
Accumulation and elimination of cadmium in larval stage zebrafish following acute exposure Carlyn J. Matza, Ronald G. Trebleb, Patrick H. Kronea, a
Department of Anatomy and Cell Biology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5 b Department of Chemistry and Biochemistry, University of Regina, 3737 Wascana Parkway, Regina, Saskatchewan, Canada S4S 0A2 Received 29 July 2005; received in revised form 28 October 2005; accepted 5 November 2005 Available online 10 January 2006
Abstract A number of recent studies have examined the impact of acute cadmium exposure on early zebrafish development at the morphological, cellular, and molecular levels. However, no information on the accumulation and elimination of cadmium during early life stages of zebrafish development has been available. Here we have quantified cadmium accumulation in larval zebrafish (Danio rerio) by graphite furnace atomic absorption spectroscopy following short-term acute exposure and recovery periods. Zebrafish (80 h postfertilization) were exposed to various concentrations of cadmium (0.2, 1.0, 5.0, 25, 125 mM) for 3 h. Cadmium accumulation in larvae increased with exposure concentration. After exposure at 5.0, 25, and 125 mM cadmium, the fish were allowed to recover in freshwater for 0, 12, or 24 h. Cadmium content did not show a statistically significant decrease over the recovery period when exposed to 5.0 or 25 mM cadmium, whereas significant losses over the recovery period were observed following 125 mM exposure. These results suggest that the larval zebrafish decrease total cadmium body burden only following relatively high short-term acutely toxic exposures. r 2005 Elsevier Inc. All rights reserved. Keywords: Zebrafish larvae; Cadmium; Bioaccumulation
1. Introduction Cadmium (Cd), a heavy metal with limited biological function (Lane and Morel, 2000), is widely distributed in the environment as a result of natural and anthropogenic activities. Cd is a common pollutant in surface waters and can cause adverse effects on fish and other organisms inhabiting these bodies of water (Perceval et al., 2004; Gravel et al., 2005). Accordingly, the effect of Cd on aquatic ecosystems has been and continues to be an active area of research (e.g., Ciutat et al., 2005; Riddell et al., 2005; Stanley et al., 2005). While numerous studies have examined the effects of cadmium on juvenile and adult fish and fish cells, the mechanisms of action of cadmium in early life stages is only beginning to be addressed. Furthermore, while the long biological half-life of Cd allows it to readily bioaccumulate in exposed organisms (e.g., Ke and Wang, 2001; Savinov et al., 2003), very little Corresponding author. Fax: +1 306 966 4298.
E-mail address: [email protected] (P.H. Krone). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.11.001
about the uptake and elimination of cadmium during embryonic and larval stages of fish development is known. Elucidation of Cd accumulation is important for mechanistic interpretation of its toxic effects. Zebrafish offer many advantages for toxicological assessment of embryonic and larval stages including small size, high reproductive potential, transparent embryos, and well-described development (reviewed in Hill et al., 2005). Larval zebrafish have been utilized to study the effects of many environmental pollutants including cadmium (Blechinger et al., 2002), insecticides (Levin et al., 2004), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (Mattingly et al., 2001). The adverse effects of Cd on developing zebrafish have been investigated at the whole-body, cellular, and molecular levels. These effects include ectopic apoptosis (Chan and Cheng, 2003), morphological deformities due to altered gene expression (Cheng et al., 2000), abnormal somitogenesis (Chow and Cheng, 2003), and induction of heat shock protein 70 gene expression (hsp70; Blechinger et al., 2002). In the latter study, we demonstrated using a live transgenic zebrafish model that a stress-responsive
ARTICLE IN PRESS C.J. Matz et al. / Ecotoxicology and Environmental Safety 66 (2007) 44–48
hsp70/eGFP transgene acts as an accurate indicator of cellspecific induction of hsp70 gene activation following acute cadmium exposure during larval development. Furthermore, this transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early life stage toxicity and is sensitive enough to detect cadmium at doses below the median adverse effect concentration and the median lethal concentration. Here we have examined the accumulation and elimination of cadmium in whole zebrafish larvae under comparable acute exposure and recovery regimes. 2. Materials and methods 2.1. Animal care and embryo collection Adult wild-type zebrafish were obtained from a local pet store and maintained at 28 1C in carbon-filtered tap water, with a photoperiod of 14 h. Embryos were collected and staged using standard procedures as outlined in Westerfield (1995). After collection embryos and larvae were reared in 25-mL petri dishes with system water changes daily.
2.2. Reagent and treatment solutions Cadmium chloride hemipentahydrate (CdCl2 2.5 H2O; CAS No. 779078-5) was purchased from J.T. Baker Inc. (Phillipsburg, NJ). A 1 mM Cd stock solution was prepared in triple-distilled water. Treatment solutions are made from dilutions of the stock in carbon-filtered tap water, and exposures are conducted in sterile 25-mL petri dishes.
2.3. Cadmium treatment and sample preparation For the acute exposures, larval zebrafish were placed in 25-mL plastic petri dishes containing 0.2, 1.0, 5.0, 25, or 125 mM Cd (0.02, 0.11, 0.56, 2.8, or 14.1 mg/mL Cd, respectively) as described previously (Blechinger et al., 2002). Untreated controls were included with all exposure groups. Exposures began when the newly hatched larvae were 80 h postfertilization (hpf) for a duration of 3 h at 28 1C. After the exposure, the larvae were rinsed several times in fresh system water to remove nonaccumulated Cd. At least three swirling rinses at 50 times dilution per rinse were performed. For recovery experiments, larvae from each Cd exposure group and control were then placed in unused petri dishes containing fresh system water to ensure no exposure to residual Cd adsorbed to original treatment dishes. Following the exposure period (i.e., no recovery) or recovery period (12 or 24 h) the larvae were transferred to preweighed microcentrifuge tubes (10 larvae per tube). Excess water was removed and the wet weight of the larvae was determined. Then 1 mL of 5% HNO3 was added to each tube and the tubes were placed in a 65 1C water bath for 12 h to digest the tissue. The digests were allowed to settle within the tubes, and the supernatants were removed to be analyzed for Cd content. Each replicate (n) represents one set of 10 larvae; 7–15 replicates (n ¼ 7215) were analyzed and an average Cd content was calculated for each data set.
45
Cd reference standard solution and analysis of the reference material TORT-2 lobster hepatopancrease (NRC-CNRC, Ottawa, Canada). From the Cd concentration of the digested sample, the ng of Cd per g wet weight of fish tissue was determined and used for data analysis.
2.5. Data treatment Data were normalized against average values obtained for untreated controls. One-way ANOVA with Tukey–Kramer multiple comparisons posttest was performed using GraphPad InStat version 3.05 (GraphPad Software, San Diego, CA). The limit of significance was set at Po0:05 throughout.
3. Results 3.1. General observations No mortality or nonlethal effects were observed in any of the treatment or control groups over the course of the exposures and/or recovery periods. This is most likely due to the short length of the exposure periods. We have previously demonstrated that the LC50 and EC50 of Cd for zebrafish larvae determined using a 96-h acute exposure beginning at 72 hpf are 18.8 and 1.7 mM Cd, respectively (Blechinger et al., 2002), whereas the same study found no increase in mortality following a 3-h pulse exposure. 3.2. Three-hour pulse exposures and recovery Accumulation of Cd by larval zebrafish increased with the Cd concentration of treatment solutions (Figs. 1 and 2). The Cd content in control samples averaged below 25 ng/g wet weight for exposure and recovery time points, representing any spectral interference during quantification and potential background Cd content. All samples generated following the pulse exposures had greater Cd content than control samples. Following a 3-h pulse exposure, Cd uptake was significantly different from
2.4. Cd quantification Cd concentrations were measured using a graphite furnace atomic absorption spectrophotometer (Varian SpectraAA 220Z) with Zeeman background correction; 6 mL of digest sample and 6 mL 2% m/v NH4H2PO4 (matrix modifier; Baker Analyzed A.C.S. Reagent; J.T. Baker) were hot injected at 85 1C. Operating conditions were 12 s at 95 1C and 15 s at 120 1C to dry the sample, 15 s at 500 1C, and 3 s at 2000 1C. Each sample was analyzed in duplicate. Data accuracy was monitored using quality control samples prepared from Fischer certified
Fig. 1. Whole-body Cd content of 80-hpf larval zebrafish following 3-h exposure to 0.2, 1.0, or 5.0 mM Cd. Mean7SE (n ¼ 7215 replicates of 10 larvae each). **Po0:001. Data were normalized against values obtained for untreated controls.
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Fig. 2. Whole-body Cd content versus length of recovery period of 80-hpf larval zebrafish following 3-h Cd exposure at 5.0, 25, or 125 mM Cd. Mean7SE (n ¼ 7215 replicates of 10 larvae each). Data were normalized against values obtained for untreated controls.
controls (Po0:001) when the developing zebrafish were exposed to 1.0, 5.0, 25, and 125mM, with average total body burdens of 69.2, 151.3, 512.8, and 11,100 ng Cd/g wet weight relative to control samples, respectively. While exposure to 0.2 mM Cd resulted in an average total body burden of 13.3 ng Cd/g relative to control, this value was not statistically significant. Recovery periods of 12 and 24 h did not result in a change in Cd present in larval zebrafish following a 3-h pulse exposure at 5.0 mM Cd (Fig. 2). A consistent decrease in total Cd content was observed following exposure at 25 mM Cd; however, the loss was not statistically significant (Fig. 2). In contrast, Cd levels dropped significantly during recovery from a pulse exposure to 125 mM Cd (Fig. 2). Whole larval cadmium burden declined 2.5-fold within 12 h of recovery (Po0:001) and was reduced approximately 15-fold (Po0:05) by 24 h of recovery in freshwater. However, total cadmium levels were still approximately 10-fold higher in the 125 mM exposed larva compared to those exposed to 5.0 mM Cd. 4. Discussion For this investigation, 80-h-old hatched zebrafish larvae were exposed to a 3-h acute pulse of cadmium at concentrations comparable to those used in previous studies that examined molecular and morphological endpoints (Blechinger et al., 2002; Chow and Cheng, 2003). As expected, accumulation of Cd by larval zebrafish during the 3-h pulse exposure increased with concentration of the treatment solution (Fig. 1). Cd accumulation by unhatched zebrafish embryos exposed to acute Cd concentrations for 48 h has been reported to be concentration dependent (Hallare et al., 2005). Chow and Cheng (2003) exposed zebrafish embryos (also prior to hatch from chorion) to 100 mM Cd during gastrulation and segmentation
(4–24 hpf) but found substantially less Cd accumulation than that in our present study. This difference is likely due to the presence of the chorion, a membrane envelope surrounding the egg, which is capable of acting as a barrier to Cd transfer to the developing embryo (Rombough and Garside, 1982). The protective effect of the chorion is evident in observations that hatched larvae are more susceptible to Cd than unhatched embryos (Hallare et al., 2005; S.R. Blechinger and P.H. Krone, unpublished data). Once hatched from the chorion, larval stage zebrafish readily accumulate Cd from their environment most likely leading to an increased sensitivity to Cd. The routes of Cd uptake in larval zebrafish have yet to be determined. Previous studies with fully developed zebrafish have demonstrated that, following exposure to waterborne Cd, uptake occurs mainly at the gills via facilitated diffusion through Ca2+-transporting chloride cells (Wicklund Glynn et al., 1994) and this process can become saturated (Wicklund Glynn, 1996). Cd accumulation in the olfactory sensory system has also been reported following aqueous exposure, suggesting that some Cd uptake occurred at the sensory neurons exposed to the surrounding environment (reviewed in Tjalve and Henriksson (1999)). These are both locations at which we have observed hsp70/eGFP transgene activation following exposure to sublethal doses of Cd in our previous work (Blechinger et al., 2002). Thus, it is likely that both of these pathways are involved in Cd accumulation observed in the present study with larval zebrafish. Our results indicate that larval fish exposed to a high acute concentration of Cd are able to significantly reduce their body burden after as little as 12 h in freshwater. Jackson et al. (2003) reported that the rate of elimination of Cd by Menidia beryllina was also highest in fish that accumulated the most Cd and rapid elimination occurred within the first 24 h. Rapid elimination of Cd suggests that not all Cd initially acquired following high acute doses is tightly bound to tissues as it is readily removed from the body. A previous study with adult carp reported that a decrease in Cd content during the depuration period was associated with rapid loss of Cd from muscle tissue, whereas loss from the kidney and liver was much slower. This suggests that during depuration Cd is transferred from the muscle to the liver and kidney for excretion (de Conto Cinier et al., 1999). In the present study, elimination via the liver and kidney is likely the means for decreasing total Cd body burden. By 50 hpf the liver of a developing zebrafish is a separate and distinct tissue, and by 72 hpf the liver is vascularized and presumably functional beyond this stage (Field et al., 2003). Furthermore, the relatively simple pronephros kidney of zebrafish is formed and functional by 48 hpf (reviewed in Drummond, 2003). Thus, during the exposure (80–83 hpf) and recovery periods (83–107 hpf) utilized in this study, the liver and kidney of the developing zebrafish are physiologically functional and likely responsible for metabolism and excretion of Cd, respectively.
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We have previously reported that 80-h-old zebrafish larvae activate an hsp70/eGFP transgene in a dose- and tissue-specific manner following similar acute cadmium exposures (Blechinger et al., 2002). While larvae exposed to 0.2 mM Cd activated the transgene only in cells of the skin and gills, we were unable to detect any significant increase in total body cadmium in similarly exposed larvae in the present study. This suggests that these cells may activate the stress response at lower acute concentrations as a result of their direct exposure to waterborne cadmium. Previous studies have similarly shown that adult fish tissues directly exposed to waterborne Cd such as the olfactory epithelium also exhibit more severe lesions than internal tissues such as the gut epithelium (Stromberg et al., 1983). In our previous study with the transgenic indicator strain, we also showed that hsp70/eGFP was strongly activated in the liver and kidney within 24 h following exposure to a 3-h pulse of 125 mM Cd, whereas the transgene was not activated in these organs during a 24-h recovery period following exposure to 5 mM cadmium. Interestingly, larvae exposed to 5 mM Cd in the present study did not reduce their total cadmium burden during a 24-h recovery, and larvae exposed to 25 mM Cd displayed a consistent but statistically insignificant trend toward Cd elimination. In contrast, 93% of the cadmium was eliminated from larvae during recovery from the 125 mM concentration. This suggests that a relationship exists between the concentrations of cadmium capable of eliciting a stress response within the organs of metal metabolism (liver) and excretion (kidney) in zebrafish larvae and those at which the mechanisms of detoxification and elimination begin to function. 5. Conclusion The results of this study show that larval zebrafish readily accumulate waterborne Cd in a dose-dependent manner following 3-h acute exposures and that the body burden can be significantly decreased after a 12-h recovery period in freshwater following high-dose exposure. This rapid elimination suggests that not all Cd acquired during a short-term acute exposure is tightly bound to tissues. In contrast, no Cd is eliminated during a 24-h recovery period following acute exposure to a more moderate Cd dose. Finally, no significant increase in total body cadmium occurs at low acute exposure concentrations that have been shown to be capable of activating a stressresponsive transgene in cells of the skin and gills in earlier studies. Acknowledgments The authors thank Drs. M. Wickstrom and L.E. Doig, Toxicology Centre, University of Saskatchewan for use of the GFAAS and technical support. This work was supported by an NSERC discovery grant to PHK, and a Canada Graduate Scholarship to CJM.
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