Cadmium exposure reduces the density of a specific ionocyte subtype in developing zebrafish

Chemosphere 244 (2020) 125535

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Cadmium exposure reduces the density of a specific ionocyte subtype in developing zebrafish Preeti H. Dave, Raymond W.M. Kwong* Department of Biology, York University, Toronto, Ontario, Canada

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Early life stage of zebrafish is sensitive to Ca2þ disruption by NOECrange of Cd.
Cd exposure specifically inhibits the development of Ca2þ-transporting ionocytes.
The compensatory regulation of Ca2þ-transporting ionocytes is attenuated by Cd.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 June 2019 Received in revised form 30 November 2019 Accepted 2 December 2019 Available online 6 December 2019

The present study examined the effects of waterborne cadmium (Cd) exposure on ionic balance and ionocyte density in developing zebrafish (Danio rerio) (0e4 days post-fertilization). Fish exposed to 1 or 10 mg Cd/L exhibited an increase in whole body Cd level. Exposure to 10 mg Cd/L also significantly reduced whole body content of Ca2þ, but not other major ions (e.g., Naþ, Kþ and Mg2þ). Such reduction was accompanied by a decrease in the density of Ca2þ-transporting ionocytes, the Naþ/Kþ-ATPase-rich cells (NaRCs). However, the densities of other ionocyte subtypes (e.g., Naþ-transporting ionocytes) remained unchanged after exposure to 10 mg Cd/L. The potential interactive effects between water chemistry and Cd exposure on ionocyte density were examined further in Cd-exposed larvae acclimated to different water NaCl or Ca2þ levels. The results demonstrated that NaRC density increased in fish acclimated to low Ca2þ water, presumably increasing Ca2þ uptake for maintaining Ca2þ homeostasis. However, Cd exposure completely abolished the increased NaRC density in low water Ca2þ environments. The increased NaRCs over development was also reduced in Cd-exposed larvae. In conclusion, our study suggested that Cd exposure reduces the density of NaRCs and suppresses the compensatory regulation of NaRCs during acclimation to low water Ca2þ level. These inhibitory effects by Cd exposure ultimately disrupt Ca2þ balance in the early life stages of zebrafish. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Cadmium Ionic regulation Ionocyte Zebrafish

1. Introduction

* Corresponding author. Department of Biology, York University, 4700 Keele Street, Toronto, ON, M3J 1P3, Canada. E-mail address: [email protected] (R.W.M. Kwong). https://doi.org/10.1016/j.chemosphere.2019.125535 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

Cadmium (Cd) is a global environmental pollutant commonly found in metal-contaminated aquatic ecosystems. In clean waters, Cd level is normally below 0.1 mg/L, but its level can increase over 10 to 100 times in Cd-polluted environments (Sabti et al., 2000; US EPA, 2016). Cd elicits its acute toxic effect in aquatic animals

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primarily by a disruption in ionic balance, particularly Ca2þ (Wood et al., 2012). Specifically, at the site of absorption, the ionic form of Cd (Cd2þ) competes with Ca2þ for uptake via the epithelial Ca2þ channel (ECaC) at the apical membrane (Galvez et al., 2006; Niyogi and Wood, 2004; Verbost et al., 1987) and inhibits the activity of the Ca2þ-ATPase at the basolateral membrane (Verbost et al., 1988), ultimately leading to hypocalcemia. In addition to disruption in Ca2þ homeostasis, Cd may also affect the regulation of other major ions. For example, in European eels (Anguilla) and streaked prochilod (Prochilodus lineatus), Cd exposure has been shown to reduce the branchial activity of carbonic anhydrase and Naþ/Kþ-ATPase (Lionetto et al., 1998; da Silva and Martinez, 2014). These enzymes are known to play important roles in promoting Naþ uptake in the gill of freshwater fish (reviewed by Guh et al., 2015; Hwang and Chou, 2013; Kwong et al., 2014a). In larval tilapia (Oreochromis mossambicus), Cd exposure has been found to decrease both Ca2þ and Kþ contents (Hwang et al., 1995). A reduction in whole body Naþ level has also been reported in larval zebrafish (Danio rerio) exposed to Cd (Alsop and Wood, 2011). In larval fish before the gill is fully developed, the skin of the yolk sac is the major site for the acquisition of ions. In zebrafish larvae, five different subtypes of ion transporting cells (also called ionocytes) have been identified in the skin of the yolk sac (reviewed by Guh et al., 2015). Among these 5 ionocytes, the predominant sites for Naþ uptake are the Hþ-ATPase-rich cells (HRCs) (Esaki et al., 2006; Kumai and Perry, 2011; Shih et al., 2012) and the Naþ-Cl--cotransporter expressing cells (NCCCs) (Chang et al., 2013; Kwong and Perry, 2016; Wang et al., 2009). Ca2þ uptake occurs primarily via the Naþ/Kþ-ATPase-rich cells (NaRCs) (ECaC and Ca2þATPase are expressed in a subset of NaRCs) (Kumai et al., 2015; Lin and Hwang, 2016; Pan et al., 2005). The transcription factors forkhead box I3a (foxi3a) and foxi3b are thought to be the master regulators of ionocyte specification and differentiation in developing zebrafish (Esaki et al., 2006; Hsiao et al., 2007). Considering the importance of the ionocytes in regulating epithelial ion transport, we hypothesized that in larval fish, the influence of Cd exposure on ionic balance is primarily owing to the disruption in ion regulation via the integumentary ionocytes. In the present study, we investigated the effects of exposure to an environmentally-relevant level of Cd (0e10 mg Cd/L) on whole body ionic balance and functional regulation of ionocyte (e.g., changes in water chemistry, mRNA levels of foxi3a and foxi3b), using larval zebrafish as a model organism. Findings from our study suggested that Cd exposure specifically inhibited the development and the density of Ca2þ-transporting ionocytes (NaRCs), leading to dysregulation in whole body Ca2þ balance in larval zebrafish. To our knowledge, this is the first study demonstrating the effects of Cd on suppressing ionocyte development in the early life stages of fish. 2. Materials and methods 2.1. Animals Adult zebrafish of the strain Tüpfel long fin (Danio rerio) were maintained in a recirculating system (Aquaneering, CA, USA) at 28  C, and were subjected to a constant 14 h light: 10 h dark photoperiod. Fertilized embryos were collected and transferred to 50 mL Petri dishes (<30 embryos per dish) until use. Exposure was performed either in system water (water chemistry is summarized in Table 1) or in artificial freshwater (detailed below) in an incubator set at 28  C. For clarity, the condition of each experiment was also specified in the figure legends. The experiments were performed in compliance with guidelines of the Canadian Council of Animal Care and after the approval of the York University Animal

Table 1 Summary of the water chemistry in system water. Ion

Concentrations (mg/L)

Naþ Mg2þ Kþ Ca2þ

107.73 ± 2.24 7.96 ± 0.20 0.48 ± 0.03 5.35 ± 0.97

Metals

Concentrations (mg/L)

Zn2þ Cu2þ Mn2þ Co2þ

130.70 ± 78.16 21.95 ± 6.84 12.37 ± 4.39 0.09 ± 0.11

Water hardness (as CaCO3 in mg/L) pH

150 7.6

Ion and metal levels were measured using ICP-MS. Values are mean ± SEM from four independent measurements.

Care Committee (2017-2 R1). 2.2. Exposure regime Embryos at 0 day post-fertilization (dpf) were transferred to either Petri dishes or multi-well plates (depending on the experiments; detailed below) containing 0, 1, or 10 mg Cd/L. Larvae in the same dish or well were considered as one replicate (N ¼ 1). The exposure waters were prepared by adding Cd from a stock solution [as Cd(NO3)2$4H2O] to the system water or to the artificial freshwater (AFW; to test the effects of varying ionic compositions, described below). The pH of the exposure waters was adjusted to 7.6 with the addition of KOH. During the experiments, the water was changed daily with the addition of Cd, and the larvae were sampled at 2, 3 or 4 dpf, depending on the experiments. The measured total Cd levels in the exposure waters are shown in Table 2. To evaluate the interactive effects of water ionic composition and Cd exposure on ionocyte density, larvae were raised in AFW containing 0 or 10 mg Cd/L at varying NaCl (0.08, 0.8, and 8 mM NaCl) or Ca2þ (0.025, 0.25, and 2.5 mM Ca2þ) levels. The AFW was prepared by adding CaSO4$2H2O, NaCl, MgSO4, K2HPO4, and KH2PO4 in MilliQ water. The levels of Mg2þ and Kþ were maintained at 0.16 and 0.3 mM, respectively, and pH was adjusted to 7.6. Fish were exposed to these waters starting at 0 dpf, and waters were changed daily. Samples were collected at 4 dpf. 2.3. Ion and metal analysis Whole body ion and metal levels were measured in 4 dpf larvae following exposure to 0, 1, and 10 mg Cd/L. In this experiment, twenty fish from the same Petri dish were pooled as one replicate, and a total of 4 replicates per treatment (N ¼ 4) were prepared for the analyses. Following euthanization with an overdose of buffered tricaine methanesulfonate (MS-222), the fish were briefly washed in deionized water and then dried at 65  C overnight. The samples were digested with 6 N HNO3 at 65  C for 48 h, and then diluted and filtered (0.45 mm). Subsequently, the samples were measured using Table 2 Measured total Cd level in exposure water.

Measured [Cd] (mg/L)

Control

1 mg Cd/L

10 mg Cd/L

0.06 ± 0.01

0.87 ± 0.06

7.66 ± 0.28

Cd levels were measured using ICP-MS. Values are mean ± SEM from two independent measurements.

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an inductively coupled plasma mass spectrometry (ICP-MS; Water Quality Centre, Trent University). The exposure waters were also measured using ICP-MS.

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Rad). The expression levels of foxi3a and foxi3b were normalized to that of the ef1a1, and the data were expressed relative to the control (0 mg Cd/L). Primer sequences used are summarized in Supplementary Table 1.

2.4. Whole-mount immunohistochemistry and cell counting Whole-mount immunohistochemistry was performed to labelled different ionocyte subtypes in developing zebrafish. Larvae were exposed to 0 or 10 mg Cd/L in multi-well plates, and at least 10 larvae per treatment were analyzed for this experiment. Methods for whole-mount immunostaining of NCC (to label NCCCs) and NKA (to label NaRCs) were performed as described previously (Kwong and Perry, 2016). HRCs were labelled either by the use of the vital dye concanavalin-A (described below) or by immunostaining of carbonic anhydrase 2 like a (CA2-like a) [this protein is specifically expressed in HRCs (Lin et al., 2008)]. For immunostaining, larvae were first fixed with 4% paraformaldehyde (PFA) and then washed with phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBST). The fish were then gradually dehydrated with methanol and stored at 20  C until use. After rehydration with PBST, the samples were treated with 150 mM Tris$HCl (pH 9.0) at 65  C for 15 min, washed with PBST, and then blocked with 3% bovine serum albumin in PBST plus 0.8% Triton-X at room temperature for 2 h. The samples were then incubated with anti-NCC (Kwong and Perry, 2016) or anti-NKA (a5; Developmental Studies Hybridoma Bank) antibodies at 4  C overnight. Subsequently, the fish were washed several times with PBST and incubated with an Alexa Fluor 488- or 564-coupled secondary antibodies (Invitrogen) for 2 h in the dark at room temperature. In separate experiments, concanavalin-A (Con-A; Invitrogen) was also used to visualize HRCs. Con-A is a lectin protein that selectively binds to a-mannopyranosyl and a-glucopyranosyl residues and several previous studies have shown that Con-A specifically labelled HRCs (Esaki et al., 2006; Kwong and Perry, 2015; Lin et al., 2005). Fish were exposed to 50 mg/ml of Con-A for 30 min in the dark. The fish were then washed, fixed in 4% PFA, and stored at 4  C until imaging. The images were acquired using a fluorescence microscope (Leica). Densities of HRCs, NaRCs, and NCCCs were determined by averaging the number of cells present in five randomly selected 100 mm2 squares on the skin of the yolk sac. For HRCs, only the immunostaining with CA2-like a was shown in the figures. 2.5. ddPCR analysis of foxi3a and foxi3b The expression levels of foxi3a and foxi3b were measured using droplet digital PCR (ddPCR) technologies. Following exposure to 0 or 10 mg Cd/L, larvae at 4 dpf were collected for total RNA extraction (twenty fish from the same Petri dish were pooled as one replicate, and a total of 4 replicates per treatment was analyzed). RNA extraction was conducted using the GeneJET RNA Purification Kit (ThermoScientific), according to the manufacturer’s protocol. RNA yield was quantified using Synergy™ LX Multi-Mode Reader (BioTek). 1 mg of total RNA was then converted to cDNA using the iScript™ cDNA synthesis kit (Bio-Rad). Samples for ddPCR analysis were prepared using the QX200™ ddPCR™ EvaGreen® supermix (Bio-Rad), according to the manufacturer’s guidelines. In brief, the samples were emulsified into nanolitre-sized droplets using the QX200™ ddPCR™ droplet generator (Bio-Rad). The emulsified samples were then amplified using a deep-well thermocycler (Bio-Rad). The PCR conditions were: enzyme activation for 5 min at 95  C, followed by 40 cycles of 30 s at 95  C and 1 min at 60  C. Subsequently, signal stabilization was performed at 4  C for 5 min and then at 90  C for 5 min. Droplets were analyzed using the QX200™ droplet reader (Bio-

2.6. Statistical analysis Statistical analyses were performed using Sigmaplot® (Systat Software, Inc., USA). All data were first checked for the assumption of normality and equal variance. If both assumptions were met, data were then analyzed using parametric tests. If either one or both of the assumptions failed, data were transformed via logarithmic or square root transformation. A non-parametric analysis was performed if these transformations still failed to restore normality or equal variance. Specific details of the statistical analyses used for each dataset were reported in the figure legends/ table captions. Data are reported as means ± SEM, and P  0.05 was taken as the level of significance.

3. Results 3.1. Whole body metal and ion content Whole body Cd level in fish increased with increasing water Cd concentrations (Fig. 1A). Cd exposure did not affect whole body contents of other trace metals tested (e.g., Zn2þ, Cu2þ, Mn2þ and Co2þ) (Supplementary Table 2). Results from one-way ANOVA revealed that whole body Naþ, Mg2þ, and Kþ levels were not significantly affected by Cd exposure (Fig. 1B, D and E). However, Ca2þ level was substantially reduced in fish exposed to 10 mg Cd/L (Fig. 1C). Exposure to 10 mg Cd/L did not significantly affect the survival rate of larvae (e.g., cumulative survival rate at 4 dpf: control, 94.4 ± 0.0%; 10 mg Cd/L, 90.7 ± 3.7%; data not shown). 3.2. Effects of Cd exposure on ionocyte density Fig. 2A to C are representative images showing the immunostaining of HRC, NaRC, and NCCC in the skin of larval zebrafish at 4 dpf. HRC and NCCC density remained unchanged following Cd exposure (Fig. 2D and F). In contrast, a significant decrease in NaRC density was observed in fish exposed to 10 mg Cd/L (Fig. 2E). 3.3. Interactive effects of Cd exposure and changes in water ion levels on ionocyte density Cd exposure and changes in water NaCl levels did not affect the density of HRCs in 4 dpf larvae (Fig. 3A). For NaRC density, a twoway ANOVA analysis revealed that there was a significant interaction between Cd exposure and changes in water Ca2þ levels. Subsequent post hoc analysis indicated that the density of NaRCs was increased with decreasing water Ca2þ levels (Fig. 3B). However, these changes were not observed when the fish were exposed to 10 mg Cd/L. Exposure to Cd in different water NaCl levels did not affect the density of NCCCs (Fig. 3C). Fig. 3D shows representative images of the effects of Cd exposure on the density of NaRCs in 4 dpf larvae acclimated to different water Ca2þ levels. The effects of varying water NaCl levels on NaRC density were also examined in 4 dpf larvae (Fig. 4). Results from two-way ANOVA analysis revealed that NaRC density was significantly affected by Cd exposure, but not by water NaCl levels. Additionally, there was no interactive effect between Cd exposure and water NaCl levels on NaRC density.

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Fig. 1. Whole body Cd and ion contents in larval zebrafish following Cd exposure. Effects of Cd exposure on whole body contents of (A) Cd, (B) Naþ, (C) Ca2þ, (D) Mg2þ, and (E) Kþ in larval zebrafish. Water chemistry is summarized in Table 1. Bars labelled with different letters represent a statistical difference (One-way ANOVA followed by a post hoc Holm-Sidak test). Data are mean ± SEM, N ¼ 4 per treatment (each replicate consisted of a pooled sample of 20 larvae).

3.4. Effects of Cd exposure on NaRC density during development The effects of Cd exposure on the density of NaRCs were examined in 2e4 dpf larvae (Fig. 5A). A two-way ANOVA analysis revealed that there was a significant difference among developmental ages and Cd exposure. However, no interactive effect

between these two variables was observed. Results from unpaired student’s t-tests (i.e., 0 vs 10 mg Cd/L at each development age) suggested that Cd exposure significantly reduced the density of NaRCs at all ages tested (Fig. 5A). Findings from ddPCR analysis indicated that Cd exposure did not affect mRNA expression levels of foxi3a and foxi3b at 4 dpf (Fig. 5B). Fig. 5C are immunofluorescent

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Fig. 2. Densities of Hþ-ATPase-rich cells (HRCs), Naþ/Kþ-ATPase-rich cells (NaRCs), and Naþ-Cl--cotransporter expressing cells (NCCCs) in larval zebrafish following Cd exposure. Representative fluorescent images showing (A) HRCs, (B) NaRCs, and (C) NCCCs on the yolk sac of larval zebrafish at 4 dpf. Scale bar ¼ 100 mm. Quantitative analysis of (D) HRC, (E) NaRC and (F) NCCC densities in fish exposed to Cd. Water chemistry is summarized in Table 1. Bars labelled with different letters represent a statistical difference (One-way ANOVA followed by a post hoc Holm-Sidak test). Data are mean ± SEM, N ¼ 14e21 per treatment.

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Fig. 3. Densities of Hþ-ATPase-rich cells (HRCs), Naþ/Kþ-ATPase-rich cells (NaRCs), and Naþ-Cl--cotransporter expressing cells (NCCCs) in Cd-exposed larval zebrafish at different water NaCl or Ca2þ levels. Densities of (A) HRCs, (B) NaRCs, and (C) NCCCs in 4 dpf zebrafish exposed to Cd at varying water NaCl or Ca2þ levels (See Section 2.2 for ionic compositions). In (B), bars labelled with different letters represent a statistical difference between different Ca2þ levels at 0 mg Cd/L; bars labelled with an asterisk represent a statistical difference between 0 and 10 mg Cd/L within the same Ca2þ treatment (Two-way ANOVA followed by a post hoc Holm-Sidak test). The box indicates the results from the two-way ANOVA analysis. Data are mean ± SEM, N ¼ 10 per treatment (D) Representative fluorescent images showing the density of NaRCs following exposure to Cd in different water Ca2þ levels. Scale bar ¼ 100 mm.

images demonstrating the effects of Cd exposure on the density of NaRCs in larval zebrafish at 2, 3, and 4 dpf.

4. Discussion Waterborne Cd is known to disrupt Ca2þ balance in freshwater fish primarily by competing with Ca2þ for uptake. In the present

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Fig. 4. Density of Naþ/Kþ-ATPase-rich cells (NaRCs) in Cd-exposed larval zebrafish at different water NaCl levels. Densities of NaRCs in 4 dpf zebrafish exposed to 0 or 10 mg Cd/L in different water NaCl levels. The box indicates the results from a two-way ANOVA analysis where there was a significant effect of Cd exposure on NaRC density. Data are mean ± SEM, N ¼ 10e13 per treatment.

study, we demonstrated that larval zebrafish exposed to Cd exhibited a reduction in whole body Ca2þ content. This reduction appeared to be associated with a decreased density of the Ca2þtransporting ionocytes, the NaRCs. We also observed that Cdexposed larvae failed to regulate NaRC density during acclimation to different water Ca2þ levels, suggesting the inhibitory effects of Cd on the compensatory regulation of NaRCs. Findings from the present study revealed a novel toxic action of Cd on ionocyte development in the early life stages of fish. Many studies have demonstrated that in embryonic or larval fish, exposure to a high level of Cd (e.g., 100 mg/L) results in developmental defects and mortality (Cao et al., 2010; Chow et al., 2008; Williams and Holdway, 2000; Witeska et al., 2014). However, little is known about the effects of exposure to low Cd level on the early life stages of freshwater fish (Hansen et al., 2002; LizardoDaudt and Kennedy, 2008). Similar to adult fish, the acute toxicity of waterborne Cd in larval fish appears to be associated with a disruption in ionic balance (Alsop and Wood, 2011; Chang et al., 1997; Hwang et al., 1995). For example, in larval tilapia (Oreochromis mossambicus), exposure to  100 mg Cd/L for 4 days resulted in a decrease in both Ca2þ and Kþ contents (Hwang et al., 1995). It was also suggested that in larval tilapia, the toxic effects of Cd was unlikely due to the accumulation of Cd, but rather owing to the inhibition of Ca uptake and thereby dysregulation in Ca balance (Chang et al., 1997, 1998). Interestingly, a reduction in whole body Naþ level, but not Ca2þ level, was observed in 7 dpf zebrafish following 40 h exposure to 1200 mg Cd/L (i.e., water Ca2þ level ¼ 1.35 mM) (Alsop and Wood, 2011). In the current study, we observed that exposure to 1 or 10 mg Cd/L from 0 dpf increased whole body Cd level in larval zebrafish at 4 dpf. We also found that fish exposed to 10 mg Cd/L exhibited a significant reduction in whole body content of Ca2þ, but not other major ions (e.g., Naþ, Kþ and Mg2þ). These findings suggested that Ca2þ disturbance by Cd exposure was likely the major mechanism of the toxic effect of Cd at the early life stages of zebrafish. To understand the potential effects of Cd on ionocyte development, the density of ionocytes following Cd exposure was examined. In larval zebrafish before the gill becomes functional, ion transport occurs predominantly through ionocytes found in the skin of the yolk sac. Specifically, HRCs (i.e., via Naþ/Hþ exchanger)

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and NCCCs (i.e., via Naþ-Cl- cotransporter) mediate the apical uptake of Naþ, while NaRCs mediate the uptake of Ca2þ (i.e., via ECaC) (Guh et al., 2015; Hwang and Chou, 2013; Lewis and Kwong, 2018). In the present study, we found that Cd exposure did not affect HRC and NCCC densities at 10 mg Cd/L. However, a substantial reduction (about 30% decrease) in NaRC density was observed in fish exposed to 10 mg Cd/L. Previous studies have shown that ionocyte specification and differentiation in developing zebrafish are regulated by the transcription factors foxi3a and foxi3b (Esaki et al., 2006; Hsiao et al., 2007). For example, high foxi3a expression coupled with low foxi3b expression was found to promote HRC differentiation, whereas low foxi3a expression coupled with high foxi3b expression promoted NaRC differentiation (Hsiao et al., 2007). However, the present study showed that neither foxi3a nor foxi3b mRNA level was affected by Cd exposure, suggesting the inhibitory effects of Cd on NaRC density was unlikely via the foxi3-regulatory pathways. In developing zebrafish, several hormones are known to regulate Ca2þ homeostasis, such as the parathyroid hormone (PTH) and stanniocalcin (STC) (Lin and Hwang, 2016). In particular, STC has also been shown to negatively regulate the density of ionocoytes in larval zebrafish (Chou et al., 2015). STC is a hypocalcemic hormone which reduces Ca2þ absorption by reducing ECaC expression (Tseng et al., 2008). A reduction in stc mRNA level has also been reported in zebrafish larvae acclimated to low water Ca2þ environments (Kwong et al., 2014b; Tseng et al., 2008). Interestingly, a recent study has shown that exposure of zebrafish embryos to 0.9 mg Cd/L for 48 h reduced the mRNA level of stc (Sonnack et al., 2017), potentially increasing Ca2þ absorption and ionocyte density. However, our results suggested that Cd exposure had opposite effects on Ca2þ regulation and NaRC density in 4 dpf larvae. Similarly, a decrease in Ca2þ uptake and a reduction in ecac mRNA expression have also been reported in larval zebrafish acutely exposed to Cd (Alsop and Wood, 2011; Liu et al., 2012). Therefore, it seemed that the early inactivation of stc by Cd exposure was unable to promote Ca2þ balance by increasing Ca2þ absorption and ionocyte development. In addition to possible direct competition between Cd and Ca2þ for absorption (Alsop and Wood, 2011; Chang et al., 1997; Galvez et al., 2006; Niyogi and Wood, 2004; Verbost et al., 1987), results from the present study suggested that the reduction in the uptake and whole body content of Ca2þ in developing fish could be due to the reduced NaRC density by Cd exposure. The interactive effects of Cd exposure and water chemistry on ionocyte density were investigated further in Cd-exposed larvae acclimated to different water ionic compositions. The results demonstrated that changes in water NaCl levels did not affect HRC, NCCC, and NaRC densities. Cd exposure also did not affect HRC and NCCC densities in different water NaCl levels. Analyses using a twoway ANOVA indicated that the inhibitory effects of Cd exposure on NaRC density was not dependent on water NaCl level, but rather on water Ca2þ level. A recent study has also shown that densities of HRCs and NCCCs were not influenced by water Naþ and Cl levels in larval zebrafish (Dai et al., 2014). Many studies have reported that larval zebrafish acclimated to low Ca2þ water exhibited an increase in ecac expression, Ca2þ uptake and number of NaRCs, presumably to maintain Ca2þ balance in the body (Kumai et al., 2015; Kwong et al., 2014b; Lin et al., 2011, 2012; Pan et al., 2005; Tseng et al., 2008). The increased density of NaRCs in low Ca2þ water is associated with an increase in the number of pre-existing NaRCs to undergo cell proliferation (Dai et al., 2014). In agreement with these studies, we observed that larval zebrafish exposed to low Ca2þ water exhibited a significant elevation in NaRC density. More importantly, our results also demonstrated that Cd exposure completely attenuated the increased NaRCs density in low Ca2þ water, indicating the suppressive effects of Cd on the compensatory regulation of NaRCs. To further investigate whether Cd exposure

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Fig. 5. Effects of Cd exposure on the density of Naþ/Kþ-ATPase-rich cells (NaRCs) and foxi3a and foxi3b mRNA levels in larval zebrafish. (A) Densities of NaRCs at different developmental ages after Cd exposure. Compared to control larvae (0 mg Cd/L), NaRC densities in Cd-exposed larvae were significantly lower at all the developmental ages examined (denoted by an asterisk) (two-tailed Student’s t-test). Data are mean ± SEM, N ¼ 10 per treatment. (B) ddPCR analysis of foxi3a and foxi3b mRNA expression levels in 4 dpf zebrafish larvae. The mRNA levels of foxi3a and foxi3b were normalized by that of ef1a1, and the data were expressed relative to the control (0 mg Cd/L). Data are mean ± SEM, N ¼ 4 per treatment (each replicate consisted of a pooled sample of 20 larvae). No statistical difference was observed between 0 and 10 mg Cd/L treatments (two-tailed Student’s t-test). (C) Representative fluorescent images showing the effects of Cd exposure on the density of NaRCs in larval zebrafish at different developmental ages. Scale bar ¼ 100 mm. Water chemistry for these experiments is reported in Table 1.

affected NaRCs over development, NaRC density in 2, 3 and 4 dpf larvae were examined (i.e., zebrafish begin to hatch at 2 dpf). The results suggested that NaRC density increased with increasing developmental ages, particularly between 3 and 4 dpf. This increase was likely due to an increased demand for Ca2þ during development. However, the results also indicated that NaRC density was substantially reduced by Cd exposure in all the developmental ages tested. Therefore, the decreased whole body Ca2þ content by Cd exposure was, at least in part, associated with the continued reduction in the number of NaRCs over development. Interestingly, a previous study has shown that Cd exposure stimulates cell proliferation in a zebrafish liver cell line (Chen et al., 2014), suggesting that Cd may elicit different effects in different cell types. Future studies should address the mechanism underlying the effects of Cd on cell proliferation, and the cytotoxicity of Cd in other ionregulatory organs in developing fish.

In conclusion, the present study showed that exposure to a low level of Cd reduced whole body Ca2þ content, but not other major ions, in larval zebrafish. Such reduction was likely due to a specific inhibition in the development of the Ca2þ-transporting ionocytes, the NaRCs. We also demonstrated that Cd exposure reduced the density and the compensatory regulation of NaRCs. To our knowledge, this study provides the first in vivo evidence of the specific inhibitory effects of Cd on NaRC density in developing fish. However, the precise mechanism underpinning such effects is yet to be elucidated. Cd uptake occurs primarily via ECaC expressed in NaRC, therefore it is possible that accumulation of Cd in NaRCs resulted in cellular damages in these cells. Several endocrine factors are known to regulate the development of ionocytes in larval zebrafish, (e.g., cortisol, isotocin, parathyroid hormone-1, insulin-like growth factor-1) (Chou et al., 2011; Cruz et al., 2013; Dai et al., 2014; Hsiao et al., 2007; Kwong and Perry, 2015). Whether Cd inhibits NaRC

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development by interacting with these signaling pathways warrants further investigations. 5. Grants The research was supported by the Canada Research Chairs Program and a Discovery Grant (05984) from the Natural Sciences and Engineering Research Council of Canada (NSERC) to R.W.M. Kwong. Author contribution Preeti Dave: performed the experiment, analyzed the data, and wrote the manuscript. Raymond Kwong: acquired funding, conceptualized the experiment, and revised the manuscript. Declaration of competing interest The authors declare that there are no conflicts of interest. Acknowledgements We thank Janet Fleites for her technical support in zebrafish husbandry. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125529. References Alsop, D., Wood, C.M., 2011. Metal uptake and acute toxicity in zebrafish: common mechanisms across multiple metals. Aquat. Toxicol. 105, 385e393. Cao, L., Huang, W., Liu, J., Yin, X., Dou, S., 2010. Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 151, 386e392. Chang, M.-H., Lin, H.-C., Hwang, P.P., 1997. Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus. Fish Physiol. Biochem. 16, 459e470. Chang, M.H., Lin, H.C., Hwang, P.P., 1998. Ca2þ uptake and Cd2þ accumulation in larval tilapia (Oreochromis mossambicus) acclimated to waterborne Cd2þ. Am. J. Physiol. Regul. Integr. Comp. Physiol. 274. Chang, W.-J., Wang, Y.-F., Hu, H.-J., Wang, J.-H., Lee, T.-H., Hwang, P.-P., 2013. Compensatory regulation of Naþ absorption by Naþ/Hþ exchanger and NaþCl- cotransporter in zebrafish (Danio rerio). Front. Zool. 10, 46. Chen, Y.Y., Zhu, J.Y., Chan, K.M., 2014. Effects of cadmium on cell proliferation, apoptosis, and proto-oncogene expression in zebrafish liver cells. Aquat. Toxicol. 157, 196e206. Chou, M.-Y., Hung, J.-C., Wu, L.-C., Hwang, S.-P.L., Hwang, P.-P., 2011. Isotocin controls ion regulation through regulating ionocyte progenitor differentiation and proliferation. Cell. Mol. Life Sci. 68, 2797e2809. Chou, M.-Y., Lin, C.-H., Chao, P.-L., Hung, J.-C., Cruz, S.A., Hwang, P.-P., 2015. Stanniocalcin-1 controls ion regulation functions of ion-transporting epithelium other than calcium balance. Int. J. Biol. Sci. 11, 122e132. Chow, E.S.H.S.H., Hui, M.N.Y.N.Y., Lin, C.C.C., Cheng, S.H.H., 2008. Cadmium inhibits neurogenesis in zebrafish embryonic brain development. Aquat. Toxicol. 87, 157e169. Cruz, S.A., Lin, C.-H., Chao, P.-L., Hwang, P.-P., 2013. Glucocorticoid receptor, but not mineralocorticoid receptor, mediates cortisol regulation of epidermal ionocyte development and ion transport in zebrafish (Danio rerio). PLoS One 8, e77997. da Silva, A.O.F., Martinez, C.B.R., 2014. Acute effects of cadmium on osmoregulation of the freshwater teleost Prochilodus lineatus: enzymes activity and plasma ions. Aquat. Toxicol. 156, 161e168. Dai, W., Bai, Y., Hebda, L., Zhong, X., Liu, J., Kao, J., Duan, C., 2014. Calcium deficiencyinduced and TRP channel-regulated IGF1R-PI3K-Akt signaling regulates abnormal epithelial cell proliferation. Cell Death Differ. 21, 568e581. Esaki, M., Hoshijima, K., Kobayashi, S., Fukuda, H., Kawakami, K., Hirose, S., 2006. Visualization in zebrafish larvae of Naþ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a. AJP Regul. Integr. Comp. Physiol. 292, R470eR480. Galvez, F., Wong, D., Wood, C.M., 2006. Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout. Am. J. Physiol. Integr. Comp. Physiol. 291, R170eR176. Guh, Y.-J., Lin, C.-H., Hwang, P.-P., 2015. Osmoregulation in zebrafish: ion transport

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