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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
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 3 of 26
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 5 of 26
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 7 of 26
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
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
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 3 of 26
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 5 of 26
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
ACS Paragon Plus Environment
Page 7 of 26
Environmental Science & Technology
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
ACS Paragon Plus Environment
Environmental Science & Technology
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
