Evidence of Common Cadmium and Copper Uptake Routes in

Subscriber access provided by TRENT UNIV

Article

Evidence of common cadmium and copper uptake routes in zebrafish Danio rerio Irina Komjarova, and Nicolas R. Bury Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5032272 • Publication Date (Web): 07 Oct 2014 Downloaded from http://pubs.acs.org on October 16, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

Environmental Science & Technology

1

Evidence of common cadmium and copper uptake routes in zebrafish Danio

2

rerio

3 4 5

I.Komjarova* and N.R. Bury

6 7 8

King’s College London, Division of Diabetes and Nutritional Science, Franklin Wilkins

9

Building, 150 Stamford Street, London SE1 9NH, UK.

10 11

*Corresponding author: [email protected]

12 13 14

1

ACS Paragon Plus Environment


15

Page 2 of 26

Abstract

16 17

Cadmium and copper accumulations in gills of zebrafish were measured

18

during a 48 h exposure to 0.025 µM 106Cd and 0.05 or 0.5 µM 65Cu as a single metal

19

or their mixtures. The gill transcript levels of genes involved in the transport of Cu

20

(CTR1 and ATP7A), Na (NHE-2), Ca (ECaC), divalent metals (DMT1) and Zn (ZIP8)

21

were also compared between treatments at 24 and 48 h. Cd uptake was significantly

22

suppressed in the presence of Cu, indicating interaction between Cu and Cd at

23

uptake sites, but Cu uptake was unaffected by Cd. The decrease in Cd accumulation

24

rates in the presence of Cu was associated with an increase in transcript abundance

25

of ECaC at 24h and DMT1 at 48h, and a decrease in Zip8 transcript levels, all known

26

as routes for Cd uptake. Fish exposed to 0.5 µM

27

ATP7a transcript abundance, suggesting that Cu is removed from the gill and is

28

transferred to other organs for detoxification. A reduction in gill CTR1 transcript

29

abundance was observed during the Cu-Cd exposure; this may be a regulatory

30

mechanism to reduce Cu loading if Cu is entering the gills by other uptake routes,

31

such as ECaC and DMT1.

65

Cu show an increase in gill

32 33 34 35 36 37 38 39 2


Page 3 of 26


40 41

Introduction

42 43

Understanding the environmental toxicology of metals is essential for setting

44

adequate site specific water quality criteria. In natural environments, aquatic

45

organisms are constantly exposed to a variety of metals via the water and diet.

46

Some of the trace metals, such as Cu, Fe, and Zn are essential for the health of

47

most organisms as they are integral components of enzymes and other molecular

48

complexes.1 Intracellular concentrations of these metals are tightly regulated by

49

complex uptake/excretion mechanisms. Other metals, such as Cd and Pb, are toxic

50

to living organisms even at low exposure concentrations and tend to accumulate in

51

the body.2 Due to anthropogenic activities metals are present in surface waters as

52

mixtures and often at elevated concentrations. Single metal toxicity studies that are

53

currently used to derive water quality standards do not accurately represent the

54

processes occurring in complex natural environments. Consequently, current

55

standards may over- or underestimate toxicity.

56 57

Some essential-nonessential metal interactions, such as Cd-Ca and Cu-Na are well

58

studied in fish.3,4 In particular, Cd is known to enter gill tissue via Ca2+ channels

59

present on the apical cell membrane.5 Even at low concentrations Cd competes with

60

Ca ions for the uptake sites causing acute hypocalcaemia.3 The uptake of Cu is

61

known to interfere with Na uptake resulting in the impairment of branchial Na

62

influxes.4 Based on a current understanding of metal transport across the cell

63

membrane, Cd and Cu do not share common uptake routes. Nevertheless, the

64

presence of Cd-Cu interactions has been demonstrated for several aquatic 3



Page 4 of 26

65

organisms during aqueous and dietary exposures even at low concentrations.6-7

66

Studies show that proteins responsible for the transport of essential metals, such as

67

Fe and Zn, may also be involved in the uptake of non-essential elements.8-10 Cooper

68

et al11 proposed that in addition to a well characterized Ca uptake pathway, Cd may

69

be taken up via a divalent metal transporter (DMT1), a protein which delivers Fe to

70

the cell. Dalton et al10 showed that a mouse zinc importer ZIP8 also transports Cd.

71

Other studies indicate the existence of Cd-Cu, Cd-Pb or Pb-Zn interactions, which

72

cannot be explained by simple competition for binding sites and most likely involve

73

unknown

74

compartmentalization/ efflux processes.13

common

uptake

routes7,12

or

alterations

in

intracellular

75 76

To better understand the relationship between Cd and Cu uptake, this study utilises

77

stable isotopes and molecular approaches to investigate the accumulation of these

78

metals in gills of adult zebrafish Danio rerio. The stable isotope technique enables

79

simultaneous exposure of organisms to several metals and allows separate

80

detection of newly added and background metals in the exposure medium and

81

organisms.14-16 This feature is particularly important for studying uptake processes of

82

essential metals, such as Cu, when small amounts of newly accumulated metals are

83

masked by a naturally relatively high metal content of the tissue. Zebrafish were

84

exposed to

85

OECD water at environmentally relevant metal concentrations and gill metal burdens

86

were assessed over a 48 h period. The transcript abundance of genes involved in

87

transport of Cu (CTR1, ATP7a), Na (NHE-2), Ca (ECaC), divalent metals (DMT1)

88

and Zn (ZIP8) were compared between the treatments at 24 and 48 h of exposure.

89

By comparing the metal uptake profiles with changes in relative transcript abundance

106

Cd and

65

Cu as single metals and their mixtures in medium hard

4


Page 5 of 26


90

levels, a further aim was to identify potential transporters involved in Cu and/or Cd

91

uptake during a metal mixture exposure and suggest a possible uptake pathway.

92 93

Experimental Section

94 95

Test Organisms

96

Adult zebrafish, Danio rerio, (~ 1 g) were obtained from a local supplier and

97

maintained in 150 L glass tanks containing 100−150 fish per aquarium at 26 ± 1 °C

98

under a 12 h light/12 h dark regime. Fish were fed with the commercial tropical fish

99

food twice a day on a 1% body mass daily ration. The aquaria were equipped with

100

trickling filters and constantly aerated. The water was checked routinely for NH4+,

101

NO2−, and NO3−. The medium was renewed if the concentration of any of these ions

102

exceeded 5, 2, or 200 µM, respectively. The rearing and test media of the aquaria

103

(pH=7) were prepared according to the OECD guidelines for testing chemicals and

104

contained 2 mM CaCl22H2O, 0.5 mM NaHCO3, 0.5 mM MgSO47H2O and 0.077 mM

105

KCl dissolved in deionised water. Fish were acclimatized to the test waters for at

106

least 20 days. Feeding was stopped 12 h prior to the start of experiments to allow

107

sufficient time for gut depuration and no food was provided during the experiment to

108

avoid metal leaching into the exposure solution.

109 110

Metal exposure, sample collection and measurement

111

Twenty four hours prior to conducting the experiments 5L polypropylene

112

aquaria were filled with freshly prepared test medium and spiked with individual

113

metals (0.003 and 0.025 µM

114

µM

106

Cd - 0.05 µM

65

106

Cd , 0.05 and 0.5 µM 65Cu) or their mixtures (0.025

Cu and 0.025 µM

106

Cd - 0.5 µM

65

Cu) to achieve metal 5



Page 6 of 26

115

equilibration in the solution. These concentrations are in the range of the UK

116

Environmental Quality Standards (Cd EQS 0.045 µM and Cu EQS 0.08 to 0.625

117

µM)17. Total metal concentrations in the exposure media were measured at the

118

beginning and at the end of the experiment and were within 100-104% (106Cd) and

119

90-112 % (65Cu) of the nominal values (Supporting Information Table S1). No

120

was detected in Cu-only exposure solutions. Organisms were randomly distributed

121

among experimental and control containers with five aquaria per condition and seven

122

fish per aquarium. At 4, 24, 30 and 48 h five fish were randomly sampled for metal

123

analysis. These fish were allowed to swim in fresh test water without metal additions

124

for 10 to 15 min to wash off any metals weakly bound to the surface, thereafter they

125

were blotted dry and sacrificed following UK Home Office Schedule 1 procedure. The

126

gills of each fish were carefully dissected, dried to a constant weight at 60°C,

127

weighed and digested with 200 µL of 69% nitric acid (Ultrapur, Merck) at room

128

temperature for 48 h, to enable complete digestion of the soft gill tissue. Water

129

samples (10mL) were collected from each aquarium at the beginning and at the end

130

of experiments, acidified with 100 µL of 69% nitric acid and kept for total metal

131

measurements. Gill digests, water samples and procedural blanks were spiked with

132

yttrium as an internal standard and analysed by ICP-MS (Perkin Elmer Sciex Elan

133

6100 DRC, 3 replicates per sample). The instrument was recalibrated every 25

134

samples to account for any signal drift. Isotope concentrations in the samples (µg/L)

135

were determined from calibration curves constructed for each isotope of interest. Gill

136

106

137

parallel, at 24 and 48 h an additional five fish were randomly sampled for transcript

138

abundance analysis. Each fish was dissected on a cold surface, the gills were

139

immediately placed in an Eppendorf tube containing 0.5 mL ice-cold TRI-reagent®,

Cd and

106

Cd

65

Cu loadings were calculated on a µmole·kg dry weight (dw)-1 basis. In

6


Page 7 of 26


140

homogenized and stored at -80 °C until the preparation of total RNA. All surfaces

141

and the instruments used at the sample collection and disruption stages were treated

142

with RNaseOut™ to minimise RNA degradation.

143 144

RNA extraction and RT-PCR analysis

145

Homogenised gills samples were defrosted and incubated in TRI-reagent® for

146

5 minutes at room temperature and total RNA was isolated from gills according to

147

manufacturer instructions. Briefly, nucleic acids were extracted with 100 µL of

148

chloroform and precipitated with 250 µL isopropanol. The pellet was washed twice

149

with ice cold 75% ethanol, re-suspended in 20 µL RNAase free water and DNAse

150

treated with Turbo DNA-free kit (Ambion®). An aliquot of each extract was used to

151

assess the purity of the RNA preparation and its concentration using a Nanodrop

152

ND-100 spectrophotometer. All extracts with 260/280 ratio between 1.95 and 2.2

153

were considered acceptable and RNA integrity was additionally evaluated by running

154

samples on 1% agarose TAE gel. Samples with 260/280 ratios below 1.95 were

155

precipitated with EtOH/CH3COONa overnight, re-suspended in RNAase free water

156

and re-analysed to ensure they met the acceptable 260/280 ratio. cDNA was

157

synthesised from 2 µg of total RNA (20 µL final volume) using RNA-to-cDNA

158

Ecodry™ premix kit from Clontech. Subsequently, 2 µL of cDNA template (1:40

159

dilution, 10 µL final reaction volume) was amplified in triplicates with 5 µL SYBR

160

Premix Ex Taq™ II Master Mix (Clontec), 0.4 µmole·L-1 each primer pair (IDT) and

161

ROX (1:50 final dilution) as a reference dye using ABI Prism 7900 HT RT-PCR

162

sequence detection system. The initial denaturation step (95˚C for 30s) was followed

163

by 40 cycles of the amplifying stage (95˚C for 5s, 55˚C for 34s). Non-template (with

164

no cDNA added) and reverse transcriptase negative (with reverse transcriptase 7



Page 8 of 26

165

omitted during cDNA synthesis) control samples were included in each PCR run to

166

ensure there was no amplification due to genomic DNA contamination. The primers

167

for

168

(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

169

determined using calibration curves generated from a serial dilution of a pooled

170

cDNA sample and these varied between 101 and 115 %.

171

dissociation curve was run and showed that each primer pair produced a single

172

amplicon, indicating absence of primer dimer formation or non-specific amplification.

173

Transcript abundance levels were determined from relative standard curves

174

prepared by serial dilutions of pooled cDNA samples and normalised to transcript

175

abundance levels of a reference gene EF1 α in the same sample (β-actin, EF1α,

176

ubiquitin and ribosomal protein L13a were evaluated as potential references genes

177

and EF1α was the most stable transcript in our experimental conditions). The

178

changes in relative transcript abundance levels in treatment groups were then

179

expressed as fold change (FC) relative to those in the control (non-exposed) group.

each

gene

(Table

1)

were

designed

Primer

using

amplification

NCBI

software

efficiencies

were

After 40 cycles a

180 181

Statistics

182

The statistical significance of the changes in transcript abundance levels (based on

183

fold changes, FC±SD, p

Evidence of Common Cadmium and Copper Uptake Routes in

Recommend Documents