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