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Combined effects of temperature and cadmium on developmental parameters and biomarker responses in zebrafish... Article in Journal of Thermal Biology · January 2005 DOI: 10.1016/j.jtherbio.2004.06.002
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Journal of Thermal Biology 30 (2005) 7–17 www.elsevier.com/locate/jtherbio
Combined effects of temperature and cadmium on developmental parameters and biomarker responses in zebrafish (Danio rerio) embryos A.V. Hallarea,b,, M. Schirlinga, T. Luckenbacha, H.-R. Ko¨hlera, R. Triebskorna,c a
Animal Physiological Ecology, Zoological Institute, Faculty of Biology, University of Tu¨bingen, Konrad-Adenauer-Str. 20., D-72072 Tu¨bingen, Germany b Department of Biology, CAS, University of the Philippines, Padre Faura, Manila 1000, Philippines c Steinbeis Transfer-Center for Ecotoxicology and Ecophysiology, Blumenstr. 13, 72108, Rottenburg, Germany Received 5 March 2004; received in revised form 12 May 2004; accepted 16 June 2004
Abstract To determine the interactions between temperature and cadmium on zebrafish (Danio rerio) development, fertilized eggs were exposed to combinations of three temperature levels (21 1C, 26 1C, and 33 1C) and six cadmium concentrations (0, 0.25, 0.5, 2.0, 5.0, and 10.0 mg/L). Endpoints used included LC50 value (48 h), developmental rate, mortality, heart rate, hatching success, liver histopathology, embryo abnormalities, and heat shock protein (hsp) induction. Results showed a significant acceleration in the developmental rate with increasing temperature and irrespective of the presence of cadmium. Data on LC50 and ELS-test revealed that simultaneous exposure to both cadmium ions and cold stress (21 1C) was highly detrimental to growing embryos, causing a pronounced mortality and a significant reduction in average heart rate and embryo hatchability. In contrast, no similar reactions to cadmium were observed in pre-hatched embryos exposed to both control (26 1C) and high temperature (33 1C), and this can be explained by the significantly higher expression of hsp (hsp70) in embryos at these temperatures. Upon hatching, however, the larvae showed increased sensitivity to cadmium. The severity of malformations in the post-hatched larvae was in the order: hot cadmium stress4cold cadmium stress4cadmium stress alone4no stress at all. Liver histopathology as well as depletion in glycogen reserves exhibited greater severity with increasing cadmium concentration, irrespective of temperature. The present study confirms that temperature effectively confounds cadmium toxicity and needs to be considered for the accurate prediction and assessment of cadmium-induced toxicity in fish. r 2004 Elsevier Ltd. All rights reserved. Keywords: Temperature; Cadmium; Biomarker; Liver histopathology; Heat shock proteins; Embryotoxicity; Zebrafish; Danio rerio
1. Introduction Corresponding author. Animal Physiological Ecology,
Zoological Institute, Faculty of Biology, University of Tu¨bingen, Konrad-Adenauer-Str. 20., D-72072 Tu¨bingen, Germany. Tel.: +49-7071-757-3557; fax: +49-7071-757-3560. E-mail address:
[email protected] (A.V. Hallare).
Aquatic organisms such as fish are, in most cases, exposed to multitudes of stressors that are either natural or anthropogenically introduced into the environment. In the present study, temperature, a potent physical stressor, and cadmium, a highly toxic chemical stressor,
0306-4565/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2004.06.002
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A.V. Hallare et al. / Journal of Thermal Biology 30 (2005) 7–17
were combined to determine their concerted effects on the early development and biomarker responses in zebrafish (Danio rerio). Most studies on fish embryotoxicity were focused on the general effects of a single toxicant on developmental parameters and have ignored the possible confounding influence of other environmental stressors such as temperature. The accurate prediction of pollutant toxicity lies heavily on the consideration of other variables that act in concert with the toxicant. Although there has been a few studies which attempted to investigate the possible interaction of temperature and heavy metal toxicity, these were conducted using adult stages of fish or invertebrates (Leung et al., 2000; Del Ramo et al., 1987; Cairns et al., 1975). Studies which elucidate the potential harm of these two stressors, singly or in combination, to the early life stages of a tropical fish species are still lacking. The developing fish embryo is generally considered to be the most sensitive stage in the life cycle of a fish. Previous studies revealed that the sensitivity of fish embryos and larvae to chemicals is far greater than that of adults (Luckenbach et al., 2001; Eaton et al., 1978; McKim, 1977; Rosenthal and Alderdice, 1976; Skidmore, 1965). In early life stage test, toxicant effects on ontogenesis and growth can be examined through many diverse endpoints. The gathered data could then be used for estimating pollutant toxicity not only at individual but also at the level of fish populations (Luckenbach et al., 2001; Triebskorn et al., 2001; Ensenbach and Nagel, 1997). In addition to developmental parameters, other biomarkers can also be investigated in the growing embryos. The induction of stress proteins in whole embryos as well as the histological changes in the developing liver are very suitable biomarkers to reveal embryotoxic potential in fish (Strmac and Braunbeck, 1999; Schwaiger et al., 1997; Triebskorn et al., 1997). The present study, therefore, is an attempt to assess the possible influence of temperature on cadmium-induced toxicity and multi-level biomarker responses in the developing zebrafish embryos.
occasionally supplemented with frozen red mosquito larvae from an uncontaminated source. Spawning was achieved, as described by Westerfield (1998), following sudden illumination within 30 min. 2.2. Exposure conditions
2. Methods
Eggs were collected using rectangular mesh wire boxes (12 24 cm) which had been previously placed at the bottom of each aquarium. The eggs were rinsed several times with tap water. To determine the acute LC50, 20 blastula eggs (2 h post-fertilization) were transferred to 9-cm diameter styrene petri dishes containing 50 mL of each of the cadmium concentrations (0, 5, 10, 25, 50, 75, and 100 mg/L of Cd) at different temperatures (21 1C, 26 1C, and 33 1C). Embryos were exposed for 48 h. To minimize the influence of transfer of embryos on dilution, around 30 eggs were transferred first to a preliminary dish containing the respective exposure medium before final transfer of only 20 fertilized eggs to the test solutions. This was also done to ensure the immediate exposure of the embryos. The LC50 values with 95% confidence intervals were estimated using probit analysis. In another experiment to determine effects of temperature and cadmium on developmental parameters, 20 blastula eggs (2 h post-fertilization) were transferred to 9-cm styrene petri dishes containing 50 ml of each of the cadmium concentrations (0, 0.25, 0.5, 2, 5, and 10 mg/L of Cd) at different temperatures (21 1C, 26 1C, and 33 1C). The embryos were exposed until the time of hatching. Cadmium solutions were prepared from high grade CdCl2 which had been dissolved in reconstituted water (REKO water, ISO, 1984). Evaporation from the dishes was controlled by placing a cover on each of them. The medium was also renewed daily to ensure that the concentration of cadmium, though not a volatile substance, was maintained in the dish. The experimental subset with 26 1C and 0 mg/L of Cd served as the overall control. Three replicates were made for each of the treatment and control groups. The exposure portion of the experiment alone was repeated five times and distributed over a 2-month period. New set of zebrafish embryos and test solutions were used in each round.
2.1. Origin and maintenance of parental fish
2.3. Cadmium accumulation in the embryos
Sexually mature zebrafish (D. rerio) were obtained from a local hatchery which also maintains a temperature of 26 1C. The fish used for the present study were kept in 25-L glass aquaria. The following control conditions were maintained: temperature 2670.8 1C, dissolved oxygen of 6 mg/L, total hardness of 25 mg/L as Ca ion, pH of 8.270.2 and 12 h light/12 h dark photoperiod. The fish were fed with a commercially available artificial diet (TetraMinTM flakes) twice daily,
Embryos exposed to 0, 2, and 10 mg/L of Cd at all temperature levels were selected. Five pools of embryos (each with approximately 250 mg total fresh weight) from each group were dried at 90 1C for 24 h until constant mass was achieved. The samples were then digested in 200 mL of concentrated HNO3 for 12 h at room temperature, followed by another 12 h at 80 1C, until a clear solution was obtained. After adding 1.0 mL of double-distilled water, the cadmium concentrations in
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the samples were determined by atomic absorption spectrophotometry (Perkin-Elmer 5000, HGA-500) at l ¼ 228:8 nm with GEM conversion. 2.4. Embryo and larval toxicity test The development of zebrafish eggs was followed for the first 48 hours and then observation was extended until the time of hatching for each cadmium/temperature treatment group. Endpoints used for assessing temperature effects and cadmium toxicity included egg and embryo mortality, gastrulation, somite formation, optic cup formation, movement, tail detachment, retinal and body pigmentation, heartbeat and circulation, and hatching success. Morphological aberrations in the posthatched embryos were also noted and described for all treatment and control groups. 2.5. Liver histology and carbohydrate content For histological examination, eight post-hatch larvae (8-day old) from 0, 2, and 10 mg/L Cd groups at all temperatures were fixed in Bouin’s solution, dehydrated in an ethanol series and embedded in Techno-Vit. Hematoxylin/eosin and Periodic Acid Schiff (PAS) stains were carried out on 2 mm sections. A total of 64 sections per animal per treatment group were analyzed for liver histopathology. In addition, histological alterations in the liver as well as changes in liver glycogen content were qualitatively described. The nucleus-to-cell ratio were quantified by means of a computer-based morphometrical program (Openlab 2.0). 2.6. Stress protein (hsp70) expression Control and treated 48-h old zebrafish embryos were frozen in liquid nitrogen and stored at 20 1C for subsequent analysis. The frozen embryos were then homogenized by sonification for 5 s in an ice-cold 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 homogenates were then centrifuged for 5 min at 20000g at 4 1C. The total protein concentrations in each supernatant were determined according to the method of Bradford (1976). Constant protein weights (10 mg of total protein per lane) were analyzed by minigel SDS-PAGE (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 filter was then incubated at
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room temperature overnight. After repeated washing in TBS for 2 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. After repeated washing for 5 min in TBS, the antibody crossreaction was detected using 4-chloro(1)naphthol and 0.015% H2O2 in 30 mM Tris pH 8.5 containing 6% methanol. The grey value intensity of the hsp 70 bands in the immunoblots were quantified by densitometric image analysis (Herolab E.A.S.Y., Germany). 2.7. Statistical analysis The data for developmental parameters and other biomarkers were analyzed by analysis of variance. 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 nonparametric test (Wilcoxon’s rank test). The entire statistical analysis was carried out using JMP Version 3.2.6 Statistical Software (SAS). Data were not significant for p40:05 and were significant for pp0:05 (*), pp0:01 (**), pp0:001 (***).
3. Results 3.1. Early life parameters 3.1.1. LC50 values for 48-hour embryos The combination of cold temperature and cadmium exposure exerted the highest toxicity to embryos after 48 h (Fig. 1). Mean LC50 values obtained at low temperature (21 1C; 4.75 mg/L) were significantly lower (po0:001) compared with those obtained at control temperature (26 1C; 30.1 mg/L) and at higher temperature (33 1C; 46.8 mg/L). The means and variances in the LC50 values also increased with temperature.
Fig. 1. Estimated LC50 values (mg/L) with 95% confidence interval for cadmium in zebrafish embryos after 48 h exposure at different temperatures. ***(pp0:001).
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3.1.2. Cadmium content in 48-h-old zebrafish embryos The uptake of cadmium by zebrafish embryos was concentration-dependent. At 10.0 mg/L Cd group, embryos have accumulated more cadmium at higher temperature (Fig. 2). 3.1.3. Developmental rate Compared to the control (26 1C), the rate of development at 33 1C was found to be twice as fast, whilst that at 21 1C it was only half as fast. This trend occurred regardless of the presence of cadmium in the medium. The completion of gastrulation, completion of somites, formation of optic cups, spontaneous contraction and tail detachment, heart beat and circulation, retinal and body pigmentation, and hatching occurred at the fastest rate in surviving embryos exposed at the highest temperature (33 1C).
Egg Cadmium Uptake (microgram/g dry wt)
3.1.4. Egg/embryo mortality Zebrafish embryos kept at 21 1C for 48 h showed more than 50% mortality starting at 5.0 mg/L Cd. This high mortality rate was not observed for similar Cd concentrations at 26 1C and 33 1C (pp0:001). Also, the
10 9 8 7 6 5 4 3 2 1 0
21˚C 26˚C 33˚C
0
10 2 Cadmium Concentration (mg/L)
mortalities observed at 26 1C and 33 1C were not found to be significantly different from each other. At 21 1C, a typical concentration-effect relationship was observed (pp0:05) (Fig. 3). In addition, the frequency of embryo deaths in this group were distributed throughout the 48-h exposure period. Exogastrulation (or swelling of the yolk sac) preceding actual deaths was observed almost exclusively at 21 1C. In contrast, significant mortalities at 26 1C and 33 1C occurred only within the time window of 8–16 hours of exposure and no more deaths were observed until hatching. At the time of hatching, however, embryos showed again increased sensitivity to cadmium regardless of temperature. 3.1.5. Average heart rate and circulation The onset of heart beating occurred earlier among embryos kept at 33 1C (Fig. 4). The average heart rate values were also observed to be greater at this temperature. At different cadmium concentrations, the heart rate values at 26 1C and 33 1C did not vary significantly from one another and from the control (0 mg/L Cd). Embryos kept at low temperature (21 1C) registered lower average heart rate values irrespective of cadmium concentrations. The onset of blood circulation also occurred earlier at the highest temperature (33 1C). Blood circulation of early developmental stages (p48 hours) was not affected by low-level cadmium. 3.1.6. Hatching success Embryos exposed to higher temperature hatched first. Cadmium, on the other hand, did not apparently affect onset of hatching at any given temperature (Fig. 5). In terms of hatching success, cadmium was found to reduce significantly the number of hatched embryos at 21 1C in a concentration-dependent manner. A decrease in hatching rate due to cadmium was also noted at 26 1C,
Average Heart Rate (beats/min)
Fig. 2. Uptake of cadmium by zebrafish eggs after 48 h exposure at different temperatures. Values are the mean of five samples. 180.00 160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00
33˚C
24h
26˚C
48h
21˚C 72h
0
0.25
0.5
2.0
5.0
10.0
Cadmium Concentration (mg/L) Fig. 3. Mortality rates in zebrafish embryos exposed to cadmium at different temperatures. Highest mortality rate among embryos exposed to cold stress (21 1C). Mortality rates at 26 1C and 33 1C were not significantly different. Typical concentration-dependent mortality was observable only at 21 1C. *(pp0:05); **(pp0:01); NS (not significant).
Fig. 4. Onset of heart beat and average heart rate in zebrafish embryos exposed to cadmium at different temperatures. Earlier onset of heart beating occurred among embryos exposed at 33 1C. No significant difference in average heart rate values among embryos exposed at 26 1C and 33 1C. Reduction in average heart rate values in relation to the control was observed only at 21 1C.
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though not concentration-dependent. In these cases, the larvae were found trapped within the chorion and never hatched. Cadmium did not affect the onset nor the rate of hatching at 33 1C. 90
21˚C
26˚C
33˚C
% Cumulative Hatching
80 70 60 50
mg/L Cd 0.00 0.25 0.50 2.0 * 5.0 + 10.0
40 30 20 10 0 48
60
*arrow(onset of hatching)
72
84
96
108
120
144
168
Hours After Fertilization
Fig. 5. Onset of hatching and hatching success rate in zebrafish embryos exposed to cadmium at different temperature. Earlier onset of hatching occurred among embryos exposed at 33 1C. No significant difference in hatching success between 26 1C and 33 1C. Concentration-dependent reduction in hatching success was observed only at 21 1C.
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3.1.7. Morphological abnormalities During the earliest stages of development (o48 h), the observed abnormalities, especially at 21 1C, included blastodermal lesions and exogastrulation (Fig. 6b,c). Upon hatching, clear macroscopic aberrations included acute heart and head edema, curvature of the CNS, weak pigmentation, helical bodies, hooked tail, tail degeneration, blistering of fins, immobilization and abnormal body posture, and dwarf bodies were observed in embryos from all treated groups (Fig. 6d–l). No incidence of malformations was observed in control embryos (26 1C, 0 mg/L Cd). Exogastrulation was found exclusively in pre-hatched embryos exposed at cold temperature (21 1C), and the frequency of occurrence was not significantly increased by cadmium. Heart edema was one of the most dominant abnormalities observed but head edema was found more often at 33 1C. In contrast to pre-hatched embryos, cadmium showed greater toxicity among post-hatched embryos, in a dose-dependent way and aggravated by extreme (low or high) temperatures. Severity of observed malformations was in decreasing order: hot cadmium stress, cold cadmium stress, cadmium stress alone, no stress at all (Table 1). Prior to their death the larvae exhibited vertebral flexures, reduced swimming activity, cessation of heart activity and subsequent immobilization.
Fig. 6. Morphological abnormalities in zebrafish embryos exposed to varying cadmium levels at different temperatures. (a) Normal 8day old post-hatch larvae with straight body and normal pigmentation, (b) exogastrulated embryo, (c) blastodermal lesions, (d) acute cardiac edema, (e) head edema, (f) enlarged view of edema and eye abnormality, (g) helical body, (h) degenerate tail, (i) blistering of fins, (j) spiral nervous system, (k) hooked tail and (l) tail deformity. Some of the affected larvae also showed combinations of morphological defects. Greater severity of malformations was observed at temperature extremes (33 1C and 21 1C) than at control temperature (26 1C).
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Table 1 Semi-quantitative matrix of morphological aberrations and inhibitory tendencies during zebrafish embryo development when exposed to cadmium at different temperature Temperature and cadmium levels
Average severity of morphological aberrations and inhibitory tendencies observed
Spirality of the CNS
Helical bodies Hooked tail and tail degeneration
Weak pigmentation
Fin blistering
Dwarf larvae
Slow movement and abnormal body posture
21 1C 0 mg/L 0.25 mg/L 0.5 mg/L 2.0 mg/L 5.0 mg/L 10.0 mg/L
+ () +++ () ++ () + (,) ++ (,) +++ (,)
— ++ ++ ++ +++ +++
— — — + ++ ++
— + + + ++ ++
— + + + ++ ++
— — + + + +
— — + ++ ++ +++
— — — — + ++
+ + + + ++ ++
— — + ++ +++ +++
26 1C 0 mg/L 0.25 mg/L 0.5 mg/L 2.0 mg/L 5.0 mg/L 10.0 mg/L
— + + () + () ++ () ++ ()
— — — — + ++
— — — — — +
— — — + + +
— — — + ++ ++
— — — — + +
— — — + + ++
— — — — — +
— — — — — +
— — + + + ++
33 1C 0 mg/L 0.25 mg/L 0.5 mg/L 2.0 mg/L 5.0 mg/L 10.0 mg/L
— — — — +() ++()
— +++ +++ +++ +++ +++
— + + ++ ++ ++
— + ++ ++ ++ +++
— + ++ +++ +++ +++
— + + ++ ++ ++
— — — — ++ ++
— — — — — —
— — — + + ++
— + ++ ++ ++ +++
Severity of observed malformations was in decreasing order: hot cadmium stress, cold cadmium stress, cadmium stress alone, and no stress at all. Assessment values: () no alterations, (+) slight alterations, (++) moderate alterations, and (+++) marked alterations.
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Acute edema of the head
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Exogastrulation () Acute edema and blastodermal of the heart lesions ()
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3.2. Histological response in the liver The liver of 8-day-old control larvae showed hexagonal-shaped hepatocytes each with centrally located nuclei (Fig. 7). Each nucleus exhibited only a small amount of heterochromatin and a prominent nucleolus. The most characteristic responses to cadmium stress were the relative increase in nuclear size and expansion of heterochromatin fields (Fig. 7d). The chromatin material of a control hepatocyte was usually localized at the center of the nucleus (Figs 7a–c), whereas in treated liver cells the chromatin was scattered within the nucleus (Fig. 7d). Cases of bleeding (entry of blood cells into the liver stroma) were observed especially in embryos exposed to hot cadmium stress (33 1C) (Fig. 7f). The universal appearance of increased vacuolar degeneration and enlargement of connective tissue compartments (fibrosis) of the liver were also observed in Cd-treated embryos (Fig. 7e). There was a significant increase in the nucleus-to-cell ratio for cadmium-treated
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embryos (2.0 and 10 mg/L Cd) with respect to the control (0 mg/L Cd) at 26 1C and 33 1C, but not at 21 1C (Fig. 8). Whereas huge glycogen fields were detectable in the liver of 8-day-old untreated larvae of zebrafish, exposure to increasing cadmium resulted in a conspicuous depletion of glycogen (Fig. 9).
3.3. The hsp70 response The induction of hsp70 was found to be significantly higher in embryos exposed to heat stress (33 1C) and exposure to an increasing cadmium concentrations did not result in further significant hsp70 expression (Fig. 10). The hsp70 levels in embryos exposed to cold stress (21 1C) remained at low levels and a concentration–effect relationship could not be established. In contrast, cadmium-triggered induction of hsp70 was very evident in embryos exposed at 26 1C and was found to be concentration-dependent.
Fig. 7. Changes in liver histology of 8-day old zebrafish larvae after exposure to cadmium. (a—c) Control liver at 21 1C, 26 1C, and 33 1C, respectively. Chromatin materials were found at the center of the nucleus. (d) Case of increased nuclear size and the scattering of chromatin materials (261 C, 10.0 mg/L Cd) (arrows), (e) Destruction of normal histology, atrophy of cells, vacuolar degeneration, and enlargement of connective tissue septa (fibrosis) (asterisks) (21 1C, 2.0 mg/L Cd). (f) Case of extensive bleeding (entry of erythrocytes) of liver stroma (arrows) (33 1C, 10.0 mg/L Cd). HE. Length of scale bar is 10 mm.
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4. Discussion 4.1. Early life parameters Our data clearly support the general notion that temperature acts as a single, potent physical stressor of fish embryo development. This is consistent with previous data showing positive correlations between temperature and developmental rates for many species (Cairns et al., 1975; Blaxter, 1992; Petersen and MartinRobichaud, 1995). Since the development of surviving embryos exposed at normal (26 1C) and high (33 1C) temperatures proceeded synchronously, irrespective of increasing cadmium concentrations, it is obvious that even the highest level of heavy metal used in the present study (10 mg/L) does not affect the rate of zebrafish development. Similar observation was noted by Rom-
Fig. 8. A significant increase in nucleus-to-cell ratio of hepatocytes of Cd-treated embryos exposed at 26 1C and 33 1C. *(pp0:05); **(pp0:01); NS (not significant).
bough and Garside (1982) who showed that cadmium (p27 mg/L) had no significant effect on the speed of development of Atlantic salmon embryos. However, under low temperature (21 1C) regime, the significantly high mortality rate is apparently due to the combined effects of cold stress and the dissolved chemical. Firstly, only those embryos exposed to cold stress showed pronounced (450%) mortalities in a matter of 48 h and secondly, these observed mortalities showed greater severity with increasing cadmium concentrations. One possible explanation for the observed high mortality could be the gradual change in the chorion structure
Fig. 10. Induction of the stress protein (hsp70) by zebrafish embryos after 48-hour exposure to cadmium at different temperature. The hsp70 expression was found to be highest in embryos exposed to heat stress (33 1C) irrespective of cadmium. Embryos exposed to cold stress (21 1C) showed little hsp70 production and the increase due to exposure to cadmium stress did not differ significantly from being exposed to cold stress alone. Embryos at normal temperature (26 1C) showed elevated hsp70 levels in response to higher cadmium levels. *(pp0:05); ** (pp0:01); NS (not significant).
Fig. 9. Mobilization and depletion in glycogen content in the liver of Cd-treated embryos. (a) Without cadmium, (b) with cadmium. PAS stain. Length of scale bar is 10 mm.
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when exposed to cold stress making it more permeable to metal ions. We actually observed that the chorion became slightly brittle and sticky so that it adhered closely to the bottom of the dish. Atomic absorption spectrophotometry (AAS) data proved that embryos could accumulate cadmium even at low temperature (21 1C), an observation that conflicts with previous findings which showed that the uptake of metal occurred mainly at normal and higher temperature (Rombough and Garside, 1982; Woodworth and Pascoe, 1982). The mortalities, therefore, may be caused by the accumulated cadmium which intensifies the stress that was already sensed by the embryos as a result of exposure to cold. The general observation of greater susceptibility of larvae (post-hatched) as compared to embryos (prehatched) (Shazili and Pascoe, 1986; Eaton et al., 1978; Skidmore, 1965) was noted at 26 1C and 33 1C. However, at 21 1C, both the pre- and post-hatched embryos showed equal susceptibility, implicating the additional difficulty experienced by the embryos due to cold stress. The phenomenon of exogastrulation (swelling of the yolk sac) was shown to be associated with exposure to cold stress rather than cadmium. It is not yet clear what causes the yolk to swell at low temperature and further investigation is necessary to provide a definitive answer. Our data show that the physiological processes such as heart beating and the phenomenon of hatching were clearly affected by temperature. Cadmium, on the other hand, was found to reduce the hatching success only at 21 1C and 26 1C. In this study, we observed that some larvae had managed to hatch but had acquired malformed helical bodies. The hatching failure, as described in previous studies, may be due to various mechanisms that include (1) the diminished activity of the embryo and abnormal distribution of the hatching enzyme (Rosenthal and Alderdice, 1976); (2) the deactivation of proteolytic enzyme by the toxic metal (Hagenmaier, 1974); or (3) the inability of the emerging larvae to break through the non-digestible outer part of the egg shell (Sinha and Kanamadi, 2000). The various morphological aberrations observed in the present study are considered primarily as an effect of cadmium since these abnormalities were not observed in untreated embryos at any given temperature. The observed malformations can be classified as relatively unspecific responses to a number of pollutants (Strmac and Braunbeck, 1999). Edema observed shortly after hatching was the most prominent morphological malformation in response to cadmium, although it has also been found in response to other inorganic or organic pollutants (Fent and Meier, 1992; Guiney et al., 1990). It is characterized by leakiness of endothelial vessels supplying the yolk sac as a result of cardio-vascular dysfunction (Guiney et al., 1990), or it could be interpreted as an indicator of metabolic or osmotic
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disruptions possibly caused by mitochondrial malfunction due to the chemical (Sinha and Kanamadi, 2000). The appearance of both cardiac and head edema in embryos kept at 33 1C indicates that the toxicity is more intense at higher temperature. Mechanisms responsible for spinal deformations and tail malformations due to cadmium have been described in previous studies. Muramoto (1985) observed cadmium-induced damage in the vertebral column of the carp and related this effect to a depletion in calcium and phosphorus level. Recently, Cheng et al. (2000) attributed the spinal deformities to a reduction in both myosin and myotome formation necessary for the normal development of a healthy musculo-skeletal system. Tail malformations such as tail erosion and bent tail can be explained genetically as resulting from inability of Cd-treated embryos to express the evenskipped 1 gene, which is important during tail extension (Cheng et al., 2000) or developmentally by altered cell migration of the precursors of the somitic mesoderm in the trunk (Ho and Kane, 1990). Tail erosion and helical bodies were also reported by Ozoh (1979) in larvae exposed to lead ions. The reduced activity and swimming movements observed in larvae can be attributed to the disruption of neuromuscular coordination (Fent and Meier, 1992). In the present study, blistering or necrosis of fins was observed in cadmium-exposed embryos subjected to low temperature. This malformation could be similar to that observed by Ozoh (1979) in zebrafish embryos exposed to lead; both malformations were characterized by outgrowths or epitheliomas. Billiard et al. (1999) reported that the etiologic agent of fin rot was Cytophaga spp (Myxobacteria), which grows in cold water conditions. Fin rot appears to be induced more by cold stress rather than by cadmium. 4.2. Liver histology and glycogen storage The use of the fish liver as a monitor organ is wellestablished in ecotoxicology, and changes in hepatocytes are useful biomarkers to trace environmental pollution caused by chemicals (Gernho¨fer et al., 2001; Schramm et al., 1998; Triebskorn et al., 1997). In the present study, changes in hepatocyte histology were observed. The relative increase in nuclear size and expansion of heterochromatin fields indicate that the nucleus represents one major target for the toxic action of cadmium (Strmac and Braunbeck, 1999). The invasion of liver stroma by blood cells in cadmium-treated embryos is probably an unspecific stress symptom (Schramm et al., 1998). The observed damage in liver histology became more severe with increasing cadmium, irrespective of temperature. Our data also showed a reduction in glycogen in cadmium-treated liver. Similar findings were reported by Leung et al. (2000), who found that cadmium significantly reduced the glycogen content in
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the digestive gland of dogwhelk, regardless of temperature. Bucher et al. (1992) reported elevated glucose and lactate levels and a reduction in liver glycogen content in male bullheads after exposure to treated paper mill effluents. The decline in glycogen could be considered as an unspecific stress symptom induced by toxicants and represents a major physiological trade-off in combating toxicity of pollutants (Leung et al., 2000; Strmac and Braunbeck, 1999; Schwaiger et al., 1997). Biochemically, this glycogen depletion is also related to a reduction in cellular ATP contents in hepatocytes (Strmac and Braunbeck, 1999). 4.3. The hsp (hsp70) Response Since its discovery, the induction of hsp (hsp70) has been regarded as a suitable biomarker in assessing reactions of biota to environmental and physiological stressors (Ko¨hler et al., 2001; Triebskorn et al., 1997; Sanders, 1993). Our results showed that hsp70 induction is very pronounced among zebrafish embryos exposed to hot stress (33 1C). The significant increase in hsp70 production observed in zebrafish embryos exposed to higher temperature supports the previous suggestions that hsp70 belong to a common group of heat shock or general stress proteins similar to metallothioneins (Van Cleef-Toedt et al., 2001). When combined with cadmium, hsp70 production does not differ significantly from that of the control. This suggests that hsp70 in zebrafish embryos is primarily induced by increased temperature (Fader et al., 1994) and remains unaffected by exposure to another stress, cadmium. The relatively high levels of stress proteins at 33 1C provided the prehatched embryos with greater resistance or recovery from heat stress or protection from cadmium, as evidenced by the high LC50 value. On the other hand, the relatively low levels of stress proteins in pre-hatched embryos kept at 21 1C could provide yet another explanation for the high mortality. It could be possible that the metabolic machinery of embryos exposed to cold stress (21 1C) could not induce production of hsp (Fader et al., 1994) even if subjected to the increasing levels of cadmium. Moreover, since zebrafish are not native to cold habitats they will not be able to synthesize hsps at lower temperatures compared with fish adapted to cold habitats (Koban et al., 1991). On the other hand, the observed concentration-dependent increase in the levels of hsps at 26 1C has provided resistance to embryos against cadmium stress. Overall, the present study validates that temperature is a very potent modifier of cadmium toxicity on early life parameters and biomarker responses in zebrafish embryos. Exposure of embryos to temperature that deviates from the optimum (26 1C) negatively affected their development. For pre-hatched embryos, exposure to warm temperature (33 1C) may have allowed them to
generate more stress proteins and this may have protected them initially from both cadmium and heat stress. After hatching, however, the larvae showed an increased sensitivity to cadmium. Based on the nature of the stress protein response (Ko¨hler et al., 2001), the production of hsp could have reached its maximum just prior to the time of hatching and thus, the pathological action of both stressors would have overriden the capability of cells to generate more hsps. Embryos exposed at 21 1C failed to generate stress proteins and this explains their high susceptibility to the heavy metal challenge. Temperature has been shown to act as a potent regulator of cadmium-induced toxicity, thus, consideration of this factor in assessing cadmium toxicity is necessary to make accurate prediction of its effects on fish and other aquatic life.
Acknowledgments We gratefully acknowledge the Deutsche Akademisches Austauschdienst (DAAD) for the financial support of this project. Special thanks to Heidi Casper, Andreas Heyd, Anna Ko¨hler, and Eva Pfefferle for the technical support and to Becca Eza and Sean O’Brien for their invaluable proof reading assistance and language preview.
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