Subscriber access provided by UNIVERSITY OF CONNECTICUT
Article
Chronic dietary selenomethionine exposure induces oxidative stress, dopaminergic dysfunction, and cognitive impairment in adult zebrafish (Danio rerio) Mohammad Naderi, Arash Salahinejad, Ankur Jamwal, Douglas P. Chivers, and Som Niyogi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03937 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 6, 2017
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 30
Environmental Science & Technology
Chronic dietary selenomethionine exposure induces oxidative stress, dopaminergic dysfunction, and cognitive impairment in adult zebrafish (Danio rerio)
1 2 3 4 5 6 7 8 9 10
*
Mohammad Naderi1 , Arash Salahinejad1, Ankur Jamwal1, Douglas P. Chivers1, Som Niyogi1,2 1
Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada 2 Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK S7N 5B3, Canada
11
* Corresponding Author: Mohammad Naderi
12
Email:
[email protected] 13
14
15
16
17
18
19
20
21
22
23
24
ACS Paragon Plus Environment
Environmental Science & Technology
25
Abstract
26
The present study was designed to investigate the effects of chronic dietary exposure to selenium
27
(Se) on zebrafish cognition and also to elucidate possible mechanism(s) by which Se exerts its
28
neurotoxicity. To this end, adult zebrafish were exposed to different concentrations of dietary L-
29
selenomethionine (control, 2.3, 9.7, 32.5 or 57.7 µg Se/g dry weight) for 30 days. Cognitive
30
performance of fish was tested using a latent learning paradigm in a complex maze. In addition,
31
we also evaluated oxidative stress biomarkers and the expression of genes involved in
32
dopaminergic neurotransmission in the zebrafish brain. Fish treated with higher dietary Se doses
33
(32.5 and 57.5 µg Se/g) exhibited impaired performance in the latent learning task. The impaired
34
learning was associated with the induction of oxidative stress and altered mRNA expression of
35
dopamine receptors, tyrosine hydroxylase, and dopamine transporter genes in the zebrafish brain.
36
Collectively, our results illustrate that cognitive impairment in zebrafish could be associated with
37
Se-induced oxidative stress and altered dopaminergic neurotransmission in the brain.
38
Keywords:
39
Selenium, Zebrafish, Learning and Memory, Dopamine, Oxidative stress
40
41
42
43
44
45 2 ACS Paragon Plus Environment
Page 2 of 30
Page 3 of 30
Environmental Science & Technology
46
Introduction:
47
Learning and memory are fundamental higher brain functions taking place at the synaptic level
48
to encode experiential information, which enables animals to generate adaptive behaviors
49
essential for their survival. A wide range of environmental contaminants have been shown to
50
interfere with different aspects of the central nervous system (CNS), which consequently
51
manifests as learning and memory deficits in animals.1 In recent years there has been an
52
increasing level of concern about essential trace elements since not only their deficiency may
53
cause abnormal neurological functions, but their excessive intake due to environmental
54
contamination may also lead to neurological dysfunctions.
55
Selenium (Se) is a naturally occurring metalloid element found in trace amounts in soil, water,
56
animals, and plants. The anthropogenic redistribution of Se through agricultural runoff, coal
57
combustion, metal mining and oil refining activities results in elevated concentrations of Se in
58
surface waters.2 Although Se can be extremely toxic to fish, much of its toxicity has been
59
characterized in terms of its reproductive and developmental effects,3 and virtually nothing is
60
known about its neuro-behavioral effects in fish. The biological functions of Se are primarily
61
implemented through its incorporation into selenoproteins. Mammalian studies have
62
demonstrated an indispensable role of Se in the maintenance of optimal brain functions via redox
63
regulation 4, as selenoproteins play a crucial role in neurodevelopment, 5 neuroprotection, 6 and
64
signal transduction.
65
supranutritional levels of Se contribute to adverse health effects. 8 For example, imbalance in Se
66
concentrations in the brain has been implicated in the pathophysiology of several neurological
67
diseases
9-10
7
Although beneficial at optimum levels, both insufficient and
and cognitive decline.
11
There is now a general consensus that the primary
3 ACS Paragon Plus Environment
Environmental Science & Technology
Page 4 of 30
68
mechanism underlying Se toxicity is oxidative stress,12-13 which in turn is a major contributor to
69
the neurodegeneration of the brain leading to cognitive decline.14
70
Among divergent outcomes of Se neurotoxicity, interaction with and perturbation of several
71
neurotransmitter systems has received increasing attention in recent years.15-16 Dopamine (DA) is
72
the predominant catecholamine neurotransmitter in vertebrates and has been implicated in a
73
plethora of processes such as learning and memory, locomotion, emotion and neuroendocrine
74
secretion. Degeneration of dopaminergic neurons is believed to mediate neuropathological
75
conditions such as Parkinson’s disease and schizophrenia, and oxidative stress has been
76
implicated as the major pathogenic process in all of them.17-18 Indeed, biosynthesis, transport,
77
and metabolism of DA are all strongly linked to oxidative stress (reviewed in ref
78
dopaminergic neurons intrinsically bear a high oxidative load, even a moderate level of oxidative
79
stress can trigger a cascade of events that may eventually lead to the dysfunction of the
80
dopaminergic system and its related neurological functions. Selenium has been found to affect
81
the dopaminergic system in mammals.20-22 Although early life-stage exposure to SeMet was
82
found to impair spatial learning in adult zebrafish23, whether this effect is mediated by the
83
disruption of the dopaminergic system has never been investigated.
84
In the natural environment, Se exposure to fish occurs predominantly via diet and in the form of
85
selenomethionine (SeMet).24 The primary goal of the present study was therefore to investigate
86
the effects of chronic dietary Se exposure on learning and memory in adult zebrafish (Danio
87
rerio), with a particular focus on the dopaminergic system. Zebrafish possess a conserved
88
dopaminergic projection pathway that is homologous to the mammalian midbrain dopaminergic
89
system.25 Moreover, we previously established the role of DA receptors in different forms of
90
learning and memory in this species.26-27 Adult zebrafish exhibit robust learning ability in a 4 ACS Paragon Plus Environment
19
). Since
Page 5 of 30
Environmental Science & Technology
91
variety of paradigms, making them a powerful model for neurobehavioral studies.28 It has been
92
demonstrated that older adult zebrafish (2 years old) are more prone to neurobehavioral effects of
93
neurotoxicants in comparison to younger adults.29 On the other hand, it has been suggested that
94
juvenile zebrafish (younger than 4 weeks of age) do not possess fully developed brain functions
95
necessary for acquisition of different learning tasks.30 With that in mind, 6 months old adult
96
zebrafish were used in the present study. Subjects were exposed to different environmentally
97
relevant concentrations of SeMet, and then trained in a latent learning of spatial task. To
98
elucidate possible biochemical and molecular mechanisms underlying Se neurotoxicity,
99
oxidative stress and expressions of different genes associated with DA receptor activity (D1 and
100
D2 receptors), as well as DA biosynthesis (tyrosine hydroxylase 1 (TH)), reuptake (dopamine
101
transporter (SCL6A3)), and metabolism (monoamine oxidase (MAO)) were quantified in
102
zebrafish telencephalon. This brain region is the main target of DA projections from the posterior
103
tuberculum (homologous to the midbrain dopaminergic system of mammals), and plays a pivotal
104
role in various forms of learning and memory in fish.31-32 Expression of two immediate early
105
genes (IEGs; brain-derived neurotrophic factor (BDNF) and early growth response1 (EGR-1))
106
was also assessed in the telencephalon. These genes are involved in neural plasticity processes
107
related to learning and memory, and their expressions are commonly used as markers of neuronal
108
activity in the brain of vertebrates, including zebrafish.33-34
109
Materials and Methods:
110
Fish
111
Zebrafish (wild-type, 6 months old) were procured from the in house stock of the R.J.F. Smith
112
Center for Aquatic Ecology of the University of Saskatchewan, and maintained in the aquatic
113
facility of the Dept. of Biology. A total number of 280 adult zebrafish (0.54±0.30g and
5 ACS Paragon Plus Environment
Environmental Science & Technology
114
3.54±0.07cm) were used in this study and were randomly distributed into twenty 30 l glass
115
aquaria (14 fish/aquarium) supplied with filtered and de-chlorinated Saskatoon tap water (total
116
hardness 150 mg/l as CaCO3, alkalinity 120 mg/l as CaCO3, pH 8) at 27±1◦C under a 12:12 h
117
light:dark cycle, and fed with fish flake food (Nutrafin Max flakes, Germany).
118
Diet Preparation and Experimental Design
119
Fish were fed four times per day (5% body weight/day ration) with control food or food spiked
120
with different nominal concentrations of Se (3, 10, 30, and 60 µg/g dw; added as Seleno-L-
121
methionine (purity >98%), Sigma-Aldrich, USA). These Se concentrations are environmentally
122
relevant since a similar range of Se concentrations has been reported in aquatic invertebrates and
123
prey fish species collected from Se-impacted sites.35-38 Different nominal concentrations of
124
SeMet were dissolved in deionized distilled water, mixed with flake food, and freeze-dried using
125
a freeze dryer (Labconco, USA) for 48 h. Control diet was treated the same way without any
126
added SeMet. Triplicate diet samples (500 mg each) were taken from each batch of food for total
127
Se analysis. Fish (56 fish per treatment, equally divided in 4 replicates per treatment) were
128
exposed to either control diet or SeMet spiked food for a period of 30 days. This exposure period
129
was selected on the basis of previous observations demonstrating chronic effects of trace
130
elements in zebrafish brains.39-40 Fish were allowed to feed for 1 hr, after which excess food was
131
siphoned out. A 75% exchange of water was carried out in each tank every day. On day 15,
132
water samples (n=3 per treatment) were collected 3 hr post feeding in order to determine
133
dissolved Se concentrations.
134
Latent Learning Task
135
After the first 14 days of experimental exposure, training trials were started in which fish from
136
each treatment were trained in a latent learning task for subsequent 16 days. Latent learning is a 6 ACS Paragon Plus Environment
Page 6 of 30
Page 7 of 30
Environmental Science & Technology
137
complex cognitive task in which fish incidentally learn about the spatial construction of a
138
complex maze by exploring and wandering, in the absence of a reward.
139
shown to perform well in this paradigm.27 The maze was composed of a start chamber, a reward
140
chamber, and two tunnels which connect these two chambers to each other (Supporting
141
Information (SI), Figure S1). As described elsewhere,27 the training sessions were conducted
142
each day by placing groups of 14 fish in the start chamber and releasing them after 30s. Fish that
143
demonstrated normal swimming behavior (no erratic movement, jumping, and/or freezing) were
144
only used in the training sessions. Fish were then allowed to explore the maze for 30 min in
145
absence of a reward. During the training trials, only one tunnel was kept open: either the right or
146
the left tunnel. Among the four replicate groups of fish assigned to each treatment, two groups
147
were trained in the maze with the right tunnel open, and other two groups were trained with the
148
left tunnel open. In order to evaluate learning performance of fish after 16 days of training, probe
149
trials were conducted. Due to differential mortality among treatments, the number of fish tested
150
in the probe trial was not equal for each treatment. The number of fish used in the probe was 53,
151
56, 52, 44, and 36 in the control, 3, 10, 30 and 60 µg/g dietary Se treatments, respectively.
152
During the probe trial, a single fish was placed in the start chamber while both the right and the
153
left tunnels were open, and the reward chamber contained 6 stimulus fish (fish to which the
154
zebrafish would respond in the test) as a reward. Given the highly social nature of zebrafish, the
155
sight of conspecifics (or shoaling) has potent rewarding properties and thus triggers a response.42
156
Learning performance of subjects was monitored for 10 min using an HD webcam (Logitech
157
c310, USA) mounted above the maze. Video footages were analyzed using image processing and
158
vision techniques utilizing MATLAB (Academic version R2015a) and the parameters of interest
159
were extracted. To evaluate latent learning performance in zebrafish, five behavioral variables
7 ACS Paragon Plus Environment
41
Zebrafish have been
Environmental Science & Technology
160
were quantified: the latency to leave the start chamber, the time spent in the correct versus
161
incorrect tunnel, the latency to enter the reward chamber, the time spent in the reward chamber,
162
and locomotion (total distance traveled by fish). After the completion of probe trials, fish were
163
euthanized by 10 mg/l of Aquacalm within 2 min (Syndel Laboratories, Canada). Whole-brains
164
were dissected out from male and female zebrafish and pooled separately. Briefly, the dorsal
165
surface of the brain was exposed. Then, the optic nerves and the medulla at the beginning of the
166
spinal cord were cut and the brain was removed. Subsequently, the telencephalon was dissected
167
out from a subset of brains, using a fine forceps and iridectomy scissors under a dissecting
168
microscope that was equipped with an Axiocam camera (Zeiss, Germany). Brain and whole-
169
body samples were stored at −80 ℃ until further analysis. Oxidative stress responses were
170
assessed in the whole-brain, since it is generally accepted that reactive oxygen species (ROS)
171
generated in different brain regions can affect dopaminergic neurons. More importantly, ROS
172
generated from the auto-oxidation of DA shows widespread toxicity not only in DA neurons but
173
also in other brain regions.43 In contrast, the expression of genes related to dopaminergic
174
neurotransmission and IEGs was evaluated specifically in the zebrafish telencephalon, as these
175
genes can also express in other parts of the brain that have no cognitive functions.34
176
Measurement of Selenium
177
Total Se concentrations in water, food and whole-body of fish were measured using a GF-AAS
178
(PerkinElmer AAnalyst 800, USA) as described previously.44 Briefly, water samples (n=3) were
179
treated with 0.2% (v/v) concentrated nitric acid and stored in 4 ℃ until Se analysis. Food and
180
tissue samples (n=3) were weighed and transferred into borosilicate glass vials with
181
polypropylene screw tops (Metal free, EPA certified, VWR, Canada). Then, 1N nitric acid was
182
added to vials (1 to 5 mass (g): volume (ml) ratio) and kept at 60 Co for 48h. Digested samples
8 ACS Paragon Plus Environment
Page 8 of 30
Page 9 of 30
Environmental Science & Technology
183
were removed and centrifuged at 15,000 g for 4 min. Supernatants were collected and stored at
184
4°C until Se analysis. The quality control and assurance of the analysis were maintained using
185
appropriate method blanks and sample duplicates, a certified Se standard, and a reference
186
material (DOLT-4; National Research Council of Canada). The recovery percentage of Se in the
187
reference material was 96%.
188
Biochemical Assays
189
Glutathione (GSH) is considered as the most abundant antioxidant in aerobic cells, and it plays a
190
critical role in protection of the brain against oxidative stress. The ratio of reduced GSH to
191
oxidized GSH (GSSG) is employed to gauge the degree of oxidative stress in cells.45 The
192
concentration of GSH and GSSG in zebrafish brain (pools of three, n=4 per treatment) was
193
measured using a fluorometric method as described previously.13 GSH content was measured in
194
a final reaction mixture volume of 200 µl, which contained 180 µl of phosphate–EDTA buffer
195
(0.1 M sodium phosphate–0.005 M EDTA, pH 8.0), 10 µl of o-phthalaldehyde (OPT, 100 µg per
196
100 µl methanol) and 10 µl of sample. The final reaction mixture for GSSG measurement
197
contained 140 µl of 0.1 N NaOH, 20 µl of OPT, and 40 µl of sample. The fluorescence intensity
198
was measured in a multimode microplate reader (Varioskan Flash, Thermo Fisher Scientific,
199
Finland) at excitation and emission wavelengths of 350 nm and 450 nm, respectively. The
200
Bradford method was employed for quantification of the protein content in the samples (n=4).46
201
The GSH and GSSH content was expressed as µg per mg of protein. Lipid peroxidation (LPO),
202
another index of oxidative stress, was measured by the determination of malondialdehyde
203
(MDA) content, using a commercially available assay kit (Abcam, USA). Each replicate was a
204
pool of 2–3 brain samples and 5 replicates per treatment were used for LPO measurement.
205
Gene Expression Measurement 9 ACS Paragon Plus Environment
Environmental Science & Technology
Page 10 of 30
206
The expression of the of following genes was assessed, based on their involvement in cognitive
207
functions and their localization in zebrafish telencephalon as reported previously:34,
208
receptor (DRD1), D2 receptors (DRD2b, DRD2c, DRD3, DRD4a, DRD4b subtypes), TH,
209
SCL6A3, MAO, BDNF and EGR-1. Total RNA was isolated from a pool of 4 telencephalons
210
(n=4 per treatment) using the RNeasy Mini Kit (Qiagen, Germany), which included a DNase
211
treatment according to the manufacturer's protocol. The RNA concentrations and purity were
212
verified on a Nanodrop spectrophotometer (NanoDrop, Thermo Scientific, USA) using the
213
absorbance ratios at wavelengths of 260 and 280 nm, respectively. Subsequently, the cDNA was
214
synthesized from 1µg total RNA using a QuantiTect Reverse Transcription® kit (Qiagen,
215
Germany). Quantitative real-time PCR was performed on an iCycler Thermal Cycler (Bio-Rad,
216
USA). The 20 µl reaction mixture contained 10 µl of SYBR Green PCR Master Mix
217
(SensiFAST, SYBR No-ROX Kit, Bioline, USA), 0.8 µl of each forward and reverse primers,
218
and 2 µl of the cDNA and nuclease-free water. PCRs were performed in triplicate. The mRNA
219
expression of each target gene was normalized to β-actin as the house-keeping gene. The relative
220
quantification of gene expression among treatment groups were analyzed by the 2−∆∆ct method.49
221
The sequences of primers are presented in Table S1.
222
Statistical Analysis
223
Data were analyzed using SPSS software (version 23.0, IBM SPSS Inc., USA) and presented as
224
mean ± the standard error of the mean. The data were checked for normality and homogeneity of
225
variance using the Kolmogorov–Smirnov one-sample test and Levene's test, respectively.
226
Statistical analysis was performed on parametric data using one way analysis of variance
227
(ANOVA) with Tukey's multiple comparisons. The Welch's test followed by the Games–Howell
228
post hoc test was used if the equality of variance assumption was rejected. When nonparametric
10 ACS Paragon Plus Environment
47-48
D1
Page 11 of 30
Environmental Science & Technology
229
tests were required, statistical analysis was performed using the Kruskal–Wallis test with Dunn's
230
post hoc analysis. In addition, a chi-square test (with Bonferroni's correction) was used to
231
determine significant differences in mortality among the treatment groups. The alpha level was
232
set at 0.05 except when a Bonferroni's correction was applied (α = 0.016). No sex-specific
233
differences in learning performance of fish and the expression of dopaminergic genes were
234
found, and therefore the data were pooled for sexes.
235
Results:
236
Selenium Concentrations
237
The measured Se concentrations in water, food and whole-body of fish are outlined in Table S2.
238
Concentrations of dissolved Se in water samples differed significantly among fish aquaria
239
(F4,10=56.54, p
