Environ. Sci. Technol. 2007, 41, 5143-5148
Cell Death, Stress-Responsive Transgene Activation, and Deficits in the Olfactory System of Larval Zebrafish Following Cadmium Exposure CARLYN J. MATZ AND PATRICK H. KRONE* Department of Anatomy and Cell Biology and Toxicology Group, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5
Cadmium (Cd) is a well-described environmental pollutant known to have adverse effects in fish, including behavioral deficits. We have previously reported the development of an in vivo system that utilizes hsp70 gene activation as a measure of acute 3 h cadmium toxicity in whole living transgenic zebrafish larvae carrying a stably integrated hsp70enhanced green fluorescent protein (eGFP) reporter gene. Here, we report that activation of this transgene in olfactory epithelium of zebrafish larvae during 96 h sublethal Cd exposure is predictive of cadmium-induced cell death, altered histological and surface organization of the epithelium, and changes in olfactory dependent behavior. The transgene is first activated in the olfactory epithelium at concentrations below those giving rise to significant defects, but exhibits a more robust response following exposure to Cd at concentrations that begin to cause significant cell death, morphological alterations, and behavioral deficits. Further, the data show that Cdinduced olfactory deficits reported previously in juvenile and adult fish can also occur during larval stages of fish development, and that such behavioral deficits are closely associated with cell death and structural alterations in the olfactory epithelium.
Introduction Cadmium (Cd) is a toxic metal with limited biological function (1) and is a common pollutant in aquatic ecosystems due to natural and anthropogenic activities. The biological effects of Cd exposure are well described, including induction of metallothionein (2) and heat shock protein 70 (hsp70) expression (reviewed in refs 3, 4), apoptosis, and necrosis (5-8). Clinical studies have demonstrated that Cd exposure impairs olfactory function in factory workers chronically exposed to Cd (9-11). While olfactory dysfunction due to waterborne Cd exposure has also been reported in juveniles and adults of several fish species (12, 13), any potential modifications or defects in the olfactory epithelium at the molecular, cellular, or tissue level were not examined in these studies. Fish olfactory systems are particularly sensitive to waterborne pollutants since the olfactory sensory epithelium is directly exposed to the aqueous environment, and ac* Corresponding author phone: 1-306-966-4089; fax: 1-306-9664298; e-mail:
[email protected]. 10.1021/es070452c CCC: $37.00 Published on Web 06/13/2007
2007 American Chemical Society
cumulation of Cd and other metals in olfactory neurons has been reported (14). Any impairment of fish olfactory systems has the potential to impact the survival of fish species, as a variety of fish behaviors such as feeding and foraging, reproduction, and predator avoidance rely heavily on olfactory cues (15, 16). Heat shock proteins (hsp’s) are a family of highly conserved molecular chaperones that aid in the proper folding and repair of proteins as well as the targeting of proteins for degradation. The 70 kDa subfamily of hsps (hsp70) is the most widely studied and is an attractive biomarker of exposure for a number of reasons. Expression of hsp70 is induced in response to a wide variety of environmental pollutants, hsp70 is highly conserved across diverse phyla, and increased expression of hsp70 is directly linked to a stressed state within the cell (17). While traditional methods for detection and measurement of hsp70 expression allow for accurate detection of toxicant impact, they are of more limited value for assessing toxicity or examining mechanisms in intact organisms. The creation of stable transgenic organisms in which hsp gene promoters are linked to a reporter gene construct such as green fluorescent protein (gfp) have allowed for rapid detection of cells responding to a stressor in a living organism. Importantly, continued monitoring of the tissue and cell specific, toxicant-induced stress response in real-time is possible when using such fluorescent reporter genes. Zebrafish offer many advantages to researchers examining early life stage toxicology including small size, high reproductive potential, transparent embryos and larvae, and well described development (18, 19). The toxic effects of Cd on developing zebrafish have been studied at the whole body, cellular, and molecular levels. Reported effects include ectopic apoptosis in embryos (6), altered gene expression causing morphological deformities (20), abnormal somitogenesis (21), and induction of hsp70 (22). In the latter study, we reported the creation of a stable transgenic line of zebrafish that express enhanced green fluorescent protein (eGFP) under the control of the hsp70 promoter. Expression of the hsp70/ eGFP reporter construct was demonstrated to be an accurate and reproducible indicator of cell-specific induction of hsp70, and was expressed in a dose-dependent and tissue-specific fashion following acute Cd exposures in larval zebrafish. In the present study, we have examined hsp70/eGFP expression in zebrafish larvae during sublethal continuous Cd exposures over a 96 h time period in zebrafish larvae, and show it is predictive of cell death and morphological alterations in the olfactory epithelium, as well as deficits in olfactory-dependent behaviors.
Materials and Methods Animals. Adult wild-type zebrafish were purchased from a local pet store and maintained in an Aquatic Habitats Stand Alone System (Apopka, FL) in carbon-filtered tap water, with a photoperiod of 14 h of light and 10 h of darkness. The line of stable transgenic hsp70/eGFP zebrafish was also maintained in this manner. For embryo collection, 6-8 zebrafish were placed into 4.5 L glass aquaria lined with glass marbles and maintained at 28 °C with the same light-cycle as described above. After spawning embryos were collected and reared in 25 mL petri dishes with system water changes daily. Embryos were staged and maintained using standard procedures (23) at 28 °C with a 14 h photoperiod prior to and during all experimental exposures. Reagent and Treatment Solutions. Cadmium chloride hemi-pentahydrate (CdCl2‚2.5 H2O; CAS no. 7790-78-5) was VOL. 41, NO. 14, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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purchased from J.T. Baker Inc. (Phillipsburg, NJ). A 1 mM Cd stock solution was prepared in triple distilled water. Treatment solutions were made from dilutions of the stock in carbon-filtered tap water. Cd Exposures. A range of sublethal Cd concentrations (0.5, 1.0, and 5.0 µM Cd) were chosen that lie well below the previously reported 96 h LC50 (median lethal concentration) of 18.8 µM Cd for larval stage zebrafish, and near the 96 h EC50 (median combined adverse effect concentration) of 1.7 µM based on morphological indicators (22). For Cd exposures, 72 hpf (hours post fertilization) zebrafish larvae were placed in petri dishes and continuously exposed to Cd for 96 h, as previously described in Blechinger et al. (22). For assessment of lethality and nonlethal morphological effects, 9 replicates of 25 larvae each were used. For all other analyses described below, a minimum of 6 replicates of 25-30 larvae were used. Solutions were changed and observations of morphological effects were made daily; dead larvae were recorded and removed. All exposures were done with wild-type larvae unless indicated otherwise (i.e., hsp70/eGFP transgenic larvae). hsp70/eGFP Detection. Transgenic larvae exposed continuously to Cd were observed at 0, 4, 8, 12, 24, 48, 72, and 96 h of the exposure period for expression of the hsp70/eGFP reporter gene. Fluorescence in whole, living larvae was detected using a Nikon Y-FL epifluorescence attachment on a Nikon Eclipse E600 photomicroscope (Nikon, Tokyo, Japan) as previously described in Blechinger et al. (22). Cell Death Detection (TUNEL Assay). Cell death was assessed using the terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assays.After the 96 h exposure period, the larvae were rinsed and fixed in 4% paraformaldehyde. The assay was performed according to manufacturer’s instructions (Roche Diagnostic Canada, Laval, PQ), and samples were viewed with fluorescence microscopy as described above. Histology. After the exposure period, larvae were rinsed and then fixed in paraformaldehyde. Fixed larvae were oriented and embedded in 1% agarose and processed in JB-4 methacrylate (Polysciences Inc., Warrington, PA). Sections of 5.5 µm thickness were stained with methylene blue-azure II stain (24). Scanning Electron Microscopy. After the exposure period, larvae were rinsed and then fixed in paraformaldehyde. Larvae were dehydrated in a graded series of washes in PBST/ acetone and stored in acetone. Dehydrated larvae were critical point dried with CO2, mounted onto stubs, and gold coated. Samples were examined using a Phillips 505 scanning electron microscope. Behavior Assays. Olfactory function was assayed by measuring the aversion response to L-cysteine (L-cys), using a procedure described by Vitebsky et al. (25) with modifications (see Figure S1 of the Supporting Information). Following the 96 h exposure period, larvae were transferred to fresh system water (i.e., no Cd) and assessed for motility via contact stimulus. Nonmotile larvae were removed and not included in behavior testing. At least 6 trials (15-20 larvae/trial) were performed for each exposure concentration and stimulus tested (distilled water and L-cys). For statistical analysis, an arcsine transformation was applied to the proportion of fish responding to the added stimulus for each trial and one-way ANOVA with StudentNewman-Keuls post-hoc tests were performed using GraphPad InStat version 3.05 (GraphPad Software, San Diego CA).
Results hsp70/eGFP Expression. General observations of the gross effects of continuous Cd exposure on developing zebrafish larvae are summarized in Table S1 of the Supporting Information. The low mortality rates and percentage of 5144
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FIGURE 1. Induction of hsp70/eGFP expression (eGFP fluorescence) in zebrafish larvae during continuous Cd exposure. Maximal reporter gene expression in the olfactory epithelium was observed after 48 h of Cd exposure (B-D). After 96 h exposure, hsp70/eGFP expression had decreased in this tissue (F-H), especially in the 5 µM Cd treated group (H). Unexposed transgenic fish of the same age are shown in panels A and E. Note: lens expression of the reporter gene is due to constitutive hsp70 expression. oe - olfactory epithelium, g is gill, s is skin. Scale bar represents 250 µm. morphological effects were as expected based on the LC50 and EC50 reported by Blechinger et al. (22). As shown in Figure 1, Cd-induced expression of the reporter construct in larval zebrafish was dose-dependent with expression occurring in cells of the gills, skin and olfactory epithelium. Constitutive lens expression of hsp70 and hsp70/eGFP occurs as a part of normal eye development (26-28), and is not due to any toxicant-induced stress. Relatively weak fluorescence and a lower number of expressing cells were observed in the olfactory epithelium in the 0.5 and 1.0 µM Cd exposure groups. At these lower Cd concentrations, eGFP fluorescence was first observed after 12 h of continuous Cd exposure, peaked by 48 h, and weakened by 72 and 96 h (Figure 1; a full exposure profile is shown in Figure S2 of the Supporting Information). In contrast, exposure at 5.0 µM Cd resulted in a greater number of cells expressing the reporter gene within the olfactory epithelium. Expression was first observed at 8 h, with maximal expression between 24 and 48 h of exposure and the signal again weakening by the 72 and 96 h timepoints (Figure 1). The circular rosette organization of the olfactory sensory neurons is clearly evident in these larvae (oe in panel D). These expression patterns were observed in 100% of larvae in each treatment group. At all time points and concentrations, fluorescence was not observed in any control larvae other than the expected lens signal. Cell Death Detection and Histological Analysis. Cell death (TUNEL) assays were performed to detect the presence of dying cells in the olfactory epithelium after 96 h of continuous Cd exposure. This exposure period was chosen as it represented the time point of the greatest observed decrease in eGFP fluorescence. Larvae exposed to 5.0 µM Cd contained a large number of TUNEL-positive, dying cells in
FIGURE 2. Panel A. Cd induced cell death in the olfactory epithelium of zebrafish larvae following 96 h exposure as assessed using fluorescent TUNEL assay. TUNEL-positive cells (arrowheads) were observed in the olfactory epithelium in larvae exposed to 5 µM Cd for 96 h (iii). No cell death was observed in untreated fish (i), nor fish exposed to 1 µM Cd (ii). Scale bar represents 250 µm. Panel B. Histopathology of zebrafish larvae olfactory epithelium following 96 h Cd exposure. Abnormal histopathology was observed in 1.0 (ii) and 5.0 (iii) µM Cd exposure groups. Note that the cells are not as densely packed as in the control tissue. In addition, there are irregular areas of cell loss (arrowheads) and small, round, densely stained cell structures in these areas. Scale bar represents 20 µm. the olfactory epithelium following a 96 h exposure period (Figure 2A, iii). The circular organization and anatomical location of the TUNEL labeling clearly corresponds to the olfactory pits expressing hsp70/eGFP in the transgenic larvae. In contrast, no cell death was detectable in larvae exposed to lower Cd concentrations of 0.5 (not shown) and 1.0 µM (Figure 2A, ii). The histopathological effects of 96 h continuous Cd exposure on the olfactory system were again found to be dose-dependent. Histology of unexposed larvae (Figure 2B, i) and 0.5 µM Cd exposed larvae (not shown) were similar. Cells forming the layers of the olfactory epithelium were densely packed, and no overt histological alterations were present in these groups. At the highest Cd exposure group (5.0 µM Cd), abnormal histopathology was clearly evident (Figure 2B, iii), which corresponded with the olfactory epithelial cell death detectable in larvae of the same exposure group. Staining indicated irregular clear areas of cellular loss, and the overall presence of fewer and less densely arranged cells. Small, round, darkly stained structures were apparent in the areas of cellular loss. These structures were not observed in any sections of unexposed control larvae olfactory tissue, and most likely represent cellular debris. Histology of olfactory tissue from larvae exposed to 1.0 µM Cd represented a transition between control and 5.0 µM Cd exposure groups. The abnormal histopathology was less severe in the 1.0 µM Cd exposed larvae with less extensive areas of cellular loss and fewer small, round, darkly stained structures (Figure 2B, ii). Topographical Analysis. The surface of the olfactory epithelium contains two major types of ciliated cells that can be discerned using SEM (29). Around the outer edges of the olfactory pit are the long-ciliated, nonsensory cells, whereas the central area of the olfactory pit contains the shorter sensory ciliated cells critical for olfaction (Figure 3, panel B). Larvae exposed to 5.0 µM Cd exhibited fewer of the olfactory sensory cilia compared to control and the other exposure groups (Figure 3, compare panels F, G, to B-E). In these larvae, large areas of the floor of the olfactory pit containing few, if any, cilia were interspersed with areas
containing cilia. Further, the entire olfactory pit would often appear collapsed and misshapen in these larvae. In contrast, the entire floor of the olfactory pit in control embryos is covered with cilia (panel B, C). Exposure at 0.5 µM (not shown) and 1.0 µM Cd had no significant discernible effect on surface appearance of the olfactory epithelium. Behavior Analysis. Behavior assays were performed to functionally assess the impact of Cd exposures on the olfactory system of zebrafish larvae. The functional impact of Cd exposures on zebrafish larvae was found to be dosedependent (Figure 4). Larvae exposed to 5.0 µM Cd for 96 h had the lowest average response to the L-cys stimulus. The average proportion of 5.0 µM Cd exposed larvae displaying aversion to L-cys was 0.51, which was less than 60% of the average proportion of unexposed control larvae responding to the same stimulus. Larvae that had been exposed to 1.0 µM Cd also had a significantly lower response to L-cys with an average proportion response of 0.74, compared to the control larvae average proportion response of 0.88. Although the average proportion response to L-cys for larvae in the lowest exposure group (0.5 µM Cd) was lower than observed with control larvae, the difference was not found to be significant. Also, no significant difference was observed for the average proportion response to the distilled water control stimulus across all exposure and control groups of larvae.
Discussion Previous behavioral studies have correlated Cd exposure with olfactory dysfunction in juvenile and adult banded kokopu (12) and rainbow trout, (13), but did not address potential mechanisms by which this may occur. The data presented here demonstrate that zebrafish exhibit deficits in olfactorydependent behavior following Cd exposure (Figure 4), and that such deficits can occur during the larval stages of fish development. Importantly, our data also show that in larval zebrafish, these deficits are likely due, at least in part, to cell death within the olfactory epithelium (Figure 2) and changes in the ciliated sensory cells within the olfactory pit (Figure 3). Furthermore, cells of the olfactory epithelium mount a stress response following Cd exposure at concentrations VOL. 41, NO. 14, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Behavioral response of zebrafish larvae to L-cysteine following 96 h Cd exposures. Impact of Cd exposure on the olfactory system was assessed by measuring the aversion response to an L-cys stimulus, relative to a distilled water stimulus. Exposure to Cd resulted in a decreased response to the L-cys stimulus, indicating that olfactory function has been impaired. Data shown represents average of at least six trials (∼15 fish/trial). Error bars represent standard error. *P < 0.05, ***P < 0.001.
FIGURE 3. Surface topography of zebrafish larvae olfactory epithelium following 96 h cadmium exposure. Ciliated, nonsensory cells (nsc), located around the outer edges of the olfactory pit, were unaffected by cadmium exposure. In contrast, exposure to 5.0 µM Cd (F and G) clearly impacted the ciliated sensory cells (sc) located in the central area of the olfactory pit (inside the area outlined by the ciliated, nonsensory cells), with large regions devoid of sensory cilia. Inset boxes indicate area shown in following panel. Scale bars represent A ) 100 µm, B-G ) 10 µm. below those causing a significant response in other cell types (Figure 1). This finding indicates that activation of the hsp70/ eGFP reporter gene can serve as an indicator of cadmium exposures that lead to disruption of the olfactory epithelium and subsequent deficits in olfactory-dependent behaviors. Such a tool could prove valuable given the time-consuming and laborious nature of behavioral assays. Most studies on behavioral deficits induced by exposure to Cd and other metals have focused on juvenile and adult fish. Recently, Carreau and Pyle (30) reported that exposure of fathead minnows to copper during the first 5-7 days of embryonic development prior to hatching, followed by a return to clean water, caused deficits in chemosensory function once these fish reached the juvenile stage at 84-96 days. The authors did not examine the olfactory system at the cellular or tissue level, so it is not known if specific olfactory system defects were present either during exposure or once the fish had reached the juvenile stage. Development 5146
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of the olfactory system has not been well studied in fathead minnow, so it is difficult to determine whether the exposure period they used includes the comparable stages of olfactory system development we have examined here. Regardless, while the impact of olfaction on larval fish survival has not been well studied, the appearance of olfactory deficits at larval stages is likely to have significant consequences on feeding and other behaviors. Embryonic zebrafish develop olfactory sensory neurons well before taste buds, and by hatching at day 3-4 have a reasonably well developed olfactory system (16). This also corresponds with the time at which amino acids (known olfactory and feeding stimulants) are able to elicit physiological changes within the olfactory bulb (31), and larval zebrafish begin to modify swimming behavior in response to amino acids (32). Additionally, larvae of reef fish such as Ostorhinchus doederleini demonstrate a specific olfactory preference for water from the home reef on which they are obtained (33), suggesting that previously reported odor preferences for specific water sources in adults may be established in larval stages. Several metals, including Cd, are known accumulate in olfactory sensory neurons in juvenile and adult fish following waterborne metal exposures (10, 13, 19). Once Cd has entered the olfactory sensory neurons, it is transported along the axons of the olfactory nerve accumulating in the nerve termini in the anterior part of the olfactory bulb (34). Severe necrosis has been reported to occur in the olfactory epithelium of juvenile fathead minnows following 96 h acute exposures to over 100 µM Cd (35), while exposure to 1 µM Cd for longer periods altered the fine structure of the cilia on olfactory sensory neurons in adult Alburnus alburnus (36). Thus, it is possible that the behavioral effects reported in juvenile and adult fish are due to alterations in the olfactory epithelium at the cellular and organizational levels. In our study, we were able to link the activation of the stress response, appearance of Cd-induced cell death and defects in the organization of the olfactory epithelium to subsequent behavioral deficits in larval fish. This is, to our knowledge, the first demonstration linking Cd-dependent behavioral deficits with molecular and cellular changes in the olfactory epithelium. Interestingly, copper also been shown to cause organizational changes in the olfactory epithelium and sensory cell disruption during more chronic exposures of adult and juveniles in a number of fish species (37-39). Thus,
it will be interesting to determine if the molecular and cellular response of the olfactory epithelium to Cd in larval zebrafish that we have reported here also occurs following exposure to other metals causing olfactory defects and behavioral deficits. Our previous studies with the hsp70/eGFP transgenic line of zebrafish indicated that expression of the transgene was also induced by acute 3 h Cd exposure in a dose-dependent fashion with more tissues affected at higher concentrations of 25 and 125 µM Cd (22). Expression of hsp70/eGFP in the gills and olfactory epithelium in the present study is not unexpected, as both tissues are sites of Cd uptake (40, 41), and both activated the transgene following acute exposures in our previous study. However, eGFP fluorescence was also observed in both the liver and pronephros following acute 3 h exposure to 125 µM Cd (22), neither of which expressed the transgene in the present study despite substantially longer 96 h exposure periods. This suggests that cells in these tissues are not as sensitive to lower dose Cd exposure as the olfactory epithelium, or that Cd is more efficiently eliminated and does not build up to toxic levels during lower dose exposures. Since Cd is known to accumulate in olfactory sensory neurons of fish, toxic effects in these cells would be expected at lower doses. Studies have shown Cd accumulation in mammalian olfactory systems and subsequent deficits in function (reviewed in 10). This suggests that the transgenic zebrafish system should find use in assessing mechanistic effects of Cd on olfactory function, and possibly in future risk assessment strategies. Transgenic organisms are gaining popularity for evaluation and assessment of environmental pollutants. Controlling the expression of a reporter protein or enzyme such as GFP (22, 42), luciferase (43, 44), or lacZ (45, 46) with a stress protein promoter allows for rapid detection of target tissues and cells, and assessment of toxicity of specific compounds or complex mixtures. GFP reporter systems are advantageous as they do not require the addition of exogenous substrates for detection, and observations are made in whole, living organisms. Stable transgenic lines, such as the hsp70/eGFP zebrafish used in this study, are preferential as the foreign DNA has stably inserted into the genome and, thus, the transgene is present in every cell of an individual. This ensures that the transgene will be expressed in a consistent and reproducible fashion between individuals, and between experiments. Although transient transgenics are much easier to generate, the foreign DNA construct is present in only a portion of the cells, and in different cells of different embryos, resulting in mosaic patterns of expression. While transient systems have found use in biomonitoring (47), conclusions regarding tissue-specificity of gene expression and mechanisms of toxicant action are more difficult to make. This is particularly true of developing embryos and larvae, in which the characteristics of any one cell population are often changing over relatively short periods of time. As the utility of stable transgenic reporter systems expands, they should find increased use in routine biomonitoring, toxicological assessments, and mechanistic investigations. In particular, they should prove especially valuable in monitoring cell fate and function in individual fish in real time following contaminant exposure.
Acknowledgments We thank X. Wu for assistance with SEM analysis, and K. Yuen and T. Evans for assistance with histology. This work was supported by an NSERC Discovery grant to P.H.K., and a Canada Graduate Scholarship to C.J.M.
Supporting Information Available A schematic diagram and description of the behavior assay; data table of lethality and nonlethal effects per treatment
group; and, a complete profile of hsp70/eGFP expression during the 96 h exposure period. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review February 21, 2007. Revised manuscript received April 24, 2007. Accepted May 3, 2007. ES070452C
