Investigation of developmental neurotoxic effects of exposure to a combination of methylmercury and chlorpyrifos
Annica Forslund
Degree project inbiology, Master ofscience (2years), 2017 Examensarbete ibiologi 30 hp tillmasterexamen, 2017
Biology Education Centre and Institution ofEnvironmental Toxicology, Uppsala University Supervisor: Henrik Viberg
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Table of contents
ACKNOWLEDGMENTS 3
ABSTRACT 5
INTRODUCTION 6
METHYL MERCURY 6
CHLORPYRIFOS 7
COMBINATION STUDIES OF METHYL MERCURY AND CHLORPYRIFOS 8
BRAIN GROWTH SPURT 9
NEUROPROTEINS 9
AIM 13
MATERIAL AND METHODS 14
ANIMALS 14
TREATMENT AND CHEMICALS 14
PROTEIN ANALYSIS 15
STATISTICAL ANALYSIS 17
RESULTS 17
DISCUSSION 21
REFERENCES 25
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Acknowledgments
I would like to thank Henrik Viberg for being a dedicated supervisor and also Iwa Lee for trying to teach me everything she knows. I would also like to thank the whole department Environmental Toxicology at the Evolutionary Biology Centre in Uppsala for making my master thesis such a pleasant experience but especially Jan Örberg and Björn Brunström for being supporting teachers during my master studies.
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Abstract
There are a lot of chemicals in the environment and some of them are
neurotoxic and can cause harm at small doses, especially if exposure occurs during brain development. For humans the brain are developing most rapidly around birth and is therefore considered as the most critical period to be exposed to neurotoxic agents. Both methyl mercury (MeHg) and chlorpyrifos (CPF) is known to be developmental neurotoxic agents, however little is known about potential synergistic effects. If they elicit developmental effects synergistically they can cause harm at concentrations earlier considered safe.
A behavioral study was performed by Viberg et al (unpublished) which
detected hyperactivity in adult male mice after exposure to MeHg and CPF at PND 10 in concentrations that were too low to detect behavioral alterations by the compounds separately. It is important to detect by which mechanisms MeHg and CPF elicit their developmental neurotoxicity since then it is possible to get an understanding if there could be even more chemicals with similar properties which interact synergistically. In this study the levels of six neuroproteins which are considered important for developing a fully
functional nervous system were examined in both hippocampus and cerebral cortex.
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Introduction
One in six children in the US have a developmental disability, often
neurobehavioral, some studies are reporting an increase but it is difficult to tell if that is the case or if it is due to increased awareness and improved
diagnostics (Boyle et al., 2011; Rutter, 2005). However, it is still believed that in some cases the disabilities are due to industrial chemicals in the
environment, it has been suggested that roughly 3% of all major
developmental disabilities are due to toxic chemicals and agents and about 25% are due to a combination of genetic and environmental factors (National Research Council, 2000).
Methyl mercury
Methyl mercury (MeHg) is an organometal, which occurs naturally but is considered to be an environmental contaminant as human emissions have caused it to rise well over background levels (Roos-Barraclough et al., 2002).
MeHg is a known neurotoxicant and accumulates in the central nervous system (CNS) by forming a complex with cysteine, the complex closely resembles a large neutral amino acid called methionine. By mimicking
methionine the complex tricks the large neutral amino acid carrier to transport it through the blood–brain barrier (Clarkson and Magos, 2006). MeHg can also pass the placental barrier and be excreted via breast milk (Grandjean et al., 1994; Vahter et al., 2000). Results from animal studies have found
disturbances in memory, learning, emotional behavior and motor coordination after developmental exposure to MeHg (Franco et al., 2006; Onishchenko et al., 2007; Sakamoto et al., 2002). In the 1950s, an epidemic started in
Minimata, Japan, where persons were showing symptoms of acute MeHg poisoning. The epidemic was due to consumption of fish, from Minimata bay,
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that was MeHg-contaminated via the water from a nearby plastic plant. Later on children to mothers, who prior had showed little or no toxic effect, showed spasticity, blindness and mental retardation as adolescence/adults (Harada, 1995). Several studies have been investigating mechanisms of how MeHg causes developmental neurotoxicity and it appears to be very complex. It has been found that MeHg causes oxidative stress, microtubule disruption, and altered neurotransmission (Cagiano et al., 1990; Johansson et al., 2007, Stringari et al., 2006; Vogel et al., 1985). A better understanding of how MeHg causes developmental neurotoxicity is still needed for reliable
biomarkers and risk assessments (Johansson et al., 2007). Human exposure to methyl mercury today is mostly due to consumption of fish or other marine animals. There are several sources to mercury vapor in the environment, and the major anthropogenic source is stationary combustion, which is responsible for 65 % of the emissions caused by human activity (Pacyna et al., 2006).
There are also natural sources like volcanos, soil and water surfaces (Clarkson and Magos, 2006).
Chlorpyrifos
Chlorpyrifos (CPF) is an organophosphorous (OP) insecticide used world- wide both indoors and outdoors. OP pesticides are generally known to cause acute toxicity by inhibiting acetylcholinesterase (AChE). Today, many studies have been made investigating the effects of CPF on the developing brain.
Developmental neurotoxicity has been detected after exposure to doses below acute systemic toxicity. Behavioral changes in adolescence and adulthood after developmental exposure to CPF have been seen in rats and mice (Aldridge et al., 2005; Icenogle et al., 2003; Levin et al., 2001; Levin et al., 2002; Lee 2015). Mechanisms suspected to cause the delayed toxicity are,
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disruption of neuronal cell replication, myelination, gliogenesis, axonogenesis, synaptogenesis and generation of oxidative stress (Crumpton et al., 2000; Dam et al., 1999; Garcia et al., 2002). Since CPF targets gliogenesis, axonogenesis, and synaptogenesis a higher vulnerability in the neonatal brain than prenatal, is indicated (Qiao et al., 2002). In the US, CPF has been restricted for indoor use after studies indicating that CPF exposure to children could be a risk.
One study performed measured residues in air and on surfaces in a home, 24 hours after a 0.5 percent Dursban@ broadcast application for fleas. Results from the study indicated that the total absorbed dose for an infant could reach 1.2-5.2 times the human No Observable Effect Level (NOEL) (Fenske et al., 1990).
Combination studies of methyl mercury and chlorpyrifos
There are countless of chemicals in the environment, however most studies only investigate the toxicity of one chemical at a time, although chemicals may interact. Many combination studies have focused on compounds with similar structures and effects, such as two organophosphates. This study focuses on MeHg and CPF, which are structurally dissimilar and known to cause neurotoxicity by different mechanisms. Dissimilar chemicals often cause synergistic or antagonistic effects. If synergistic effects occurs, chemicals can cause harm at lower concentrations than recognized before, which is important to consider for future risk assessments.
Few combination studies have been published on MeHg and CPF. The ones found have used the amphipod Hyalella Azteca as a model organism and the relevance for humans can be questioned, however there have been some
interesting findings. It was found that MeHg and CPF interact additively, with
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survival as endpoint, and also that the elimination rate of MeHg becomes significantly reduced after exposure to MeHg combined with CPF, compared to MeHg alone (Steevens and Benson, 2001). Another publication found that MeHg protects Hyalella azteca from the AChE inhibition by CPF. The same study describes a formation of a CPF-MeHg complex detected by thin-layer chromatography, which is very interesting (Steevens and Benson, 2000). A study performed by Viberg et al. (unpublished) detected hyperactivity in adult fmale mice after neonatal exposure to MeHg and CPF, during a critical period of brain development.
Brain growth spurt
There is an important period in the mammalian brain development called the brain growth spurt (BGS), which is characterized by dendritic and axonal growth, synaptogenesis, proliferation of glia cells and myelination. During the BGS the brain is extra sensitive for toxic insults (Davison and Dobbing,
1968). The BGS occurs at different times during development in different species. In humans, the period starts around the third trimester and spans throughout the first 2 years of life with a peak just before birth. In mice, the BGS spans throughout the first 3-4 weeks of life and peaks around postnatal day 10 (PND 10) (Dobbing and Sands, 1978). The events occurring during BGS is dependent on the expression of several neuroproteins, and these proteins have also been recognized to be important for normal brain development and function.
Neuroproteins
Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) is present in neurons throughout the brain, almost 1% of total protein in the brain is CaMKII
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(Erondu and Kennedy, 1985). The levels of CaMKII increases dramatically during brain development. In mice it was found that between PND 1 and PND 28 the levels of CaMKII increased 25-fold, with the most rapid increase
between PND 7 and 14 (Viberg et al., 2008). It has been observed that
exposure to perfluorohexane sulfonate, PFOS, PFOA, Bisphenol A, PBDE 99, PBDE 209, chlorpyrifos, carbaryl and endosulfan affects the levels of CaMKII (Lee and Viberg, 2013; Viberg and Lee, 2012; Viberg and Eriksson, 2011;
Johansson et al., 2009; Viberg et al., 2008; Lee et al., 2015; Lee et al., 2015;
Lee, 2015). It is primarily localized in synapses and in the postsynaptic density (PSD). The PSD is a huge protein complex in the postsynaptic membrane (Elias et al., 2006). CaMKII regulates several neuronal functions probably both enzymatic and structural, however most focus have been on its involvement in basic synaptic processes important for learning and storing memories (Erondu and Kennedy, 1985). CaMKII plays an important part in long-term potentiation (LTP), which is an activity-dependent strengthening of synaptic transmission. The ability to alter synaptic strength in an activity- dependent manner is what makes it possible for synapses to store information during development (Shonesy et al., 2014). CaMKII-deficient mice displayed impairments in ability to store memories and learning (Silva et al., 1992).
Growth associated protein-43 (GAP-43) is a protein that belongs to a small group of axonally transported “growth-associated” proteins (Skene et al., 1986). The presence of GAP-43 peaks during brain development and then declines during maturation. In mice GAP-43 peaks on PND 7 in hippocampus and on PND 10 in cortex (Viberg et al., 2008). It has been observed that
exposure to perfluorohexane sulfonate, PFOS, PFOA, PBDE 99, PBDE 209, chlorpyrifos and carbaryl alters the levels of GAP43 (Lee and Viberg, 2013;
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Viberg and Eriksson, 2011 Johansson et al., 2009, Viberg et al., 2008;
Sachana et al., 2005; Lee et al., 2015). GAP-43 is believed to be required with a small number of other growth associated proteins during axonal elongation during development or axonal regeneration in the peripheral nerve system in adult mammals (Skene et al., 1986). GAP-43 is frequently used as a marker for sprouting, since it is localized in the growth cone membranes and
immature synaptic terminals. Growth cones are essential during development since they elongate growing axons by membrane addition and guide them by recognition of target cells (Skene et al., 1986). In a study with GAP-43-null mice, only 5-10% survived weaning and they showed impairments in strength, coordination, balance, hyperactivity, reduced anxiety and abnormal reflexes were observed, which indicates that normal levels of GAP-43 is essential for development of a functional nervous system (Metz et al., 2004).
Glutamate receptor 1 (GluR1) is a subunit of the α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptor (AMPAR). The AMPAR is an ionotropic glutamate receptor, which consist of four subunits (GluR1-4).
AMPAR trafficking is believed to be essential for synaptic plasticity, and phosphorylation of AMPARs are necessary for two of the basic synaptic
processes LTP and long-term depression (LTD). To investigate the importance of GluR1 phosphorylation in synaptic plasticity mice with knock-in mutations at the GluR1 phosphorylation sites were constructed. The mice showed
difficulties memorizing spatial learning tasks indicating that GluR1 is important for retention of memories (Lee et al., 2003). It has been observed that exposure to endosulfan and cypermethrin alters the levels of GluR1 (Lee et al., 2015).
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Postsynaptic density-95 protein (PSD-95) is member of the membrane
associated guanylate kinase (MAGUK) family. PSD-95 is a scaffold protein in the PSD, the major scaffold proteins influences the structure and function of the PSD. Therefore play PSD-95 an important part in regulating synaptic transmission and the maturation of glutamatergic synapses (Ehrlich et al., 2007). A study with PSD-95 mutant mice shows disruption of AMPAR
mediated synaptic transmission and LTD (Carlisle et al., 2008). A reduction of PSD-95 in dentate gyrus has also been linked to schizophrenia and mood disorder, the reduction probably inhibits the flow of information to other hippocampal regions (Toro and Deakin, 2005).
Synaptophysin is one of the most abundant proteins in the membranes of presynaptic vesicles (Wiedenmann and Franke, 1985). Its exact function is unknown, but since it is a major protein of the synaptic vesicles it has been believed to play an essential role in synaptic transmission. A study with synaptophysin-deficient mice was made to investigate the formation and function of vesicles. The synaptophysin-deficient mice showed no visible abnormalities and the study concluded that synaptophysin are not essential for formation of vesicles and synaptic transmission (Eshkind and Leube, 1995).
However, another study found that synaptophysin-deficient mice expressed increased exploratory behavior and disturbances in learning and memory. This indicates that synaptophysin can be partially compensated for by other
synaptic vesicle related proteins for synaptic transmission, but important for the efficacy of the synaptic vesicle cycle (McMahon et al., 1996; Schmitt et al., 2009). Synaptophysin increases dramatically during brain development. In mice, the levels of synaptophysin increased 14-fold between PND 1 and PND 28, with the most rapid increase between PND 7 and 14 (Viberg, 2008). It has
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been observed that exposure to perfluorohexane sulfonate, PFOS, PFOA, Bisphenol A, PBDE 99, PBDE209 and chlorpyrifos alters the levels of synaptophysin (Lee and Viberg, 2013; Viberg and Lee, 2012; Viberg and Eriksson, 2011; Johansson et al., 2009; Viberg 2009; Lee et al., 2015).
Tau is a neuronal microtubule-associated protein (MAP) present primarily in axons in both the brain and in the peripheral nervous system. Tau is highly expressed during brain development and peaks around PND 7 in mice (Viberg, 2008). It has been observed that exposure to perfluorohexane
sulfonate, PFOS, PFOA, PBDE 209, carbaryl, endosulfan, cypermethrin and ionizing radiation alters the levels of tau (Lee and Viberg, 2013; Johansson et al., 2009; Viberg, 2009; Lee et al., 2015; Lee et al., 2015; Buratovic et al., 2014) Tau´s role in neurons is complex, although its major biological function, during development, is to organize microtubule assembly and
control that formed microtubules stay stable by binding to them. Microtubule formation and stability is crucial for axonal transport in neurons (Wang and Liu, 2008). Tau-deficient mice show hyperactivity, muscle weakness and learning impairment in contextual fear test (Ikegami et al., 2000). The effects of tau depletion were suspected to cause severe neuropathological damage, however it seems like elimination of tau causes only disruption of small- calibre axons. Large-calibre axons appears to compensate with increased expression of other microtubule-associated proteins like MAP1A (Harada et al., 1994).
Aim
The aim of this study was to investigate if a single neonatal exposure to MeHg, CPF or the combination of MeHg and CPF, during a critical period of
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brain development, can cause alterations in the levels of CaMKII, GAP-43, GluR1, PSD-95, synaptophysin and tau.
Material and methods Animals
Pregnant NMRI mice were purchased from Scanbur, Sollentuna, Sweden and were housed individually in plastic cages in a room with an ambient
temperature of 22 °C and a 12/12 h cycle of light and dark. The animals were supplied with standardized pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. The day of birth was assigned as PND 0; the size of the litters was adjusted to 10–14 mice, within the first 48 h after birth, by the killing of excess pups. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986
(86/609/EEC), after approval from the local ethical committees (Uppsala University and Agricultural Research Council) and by the Swedish Committee for Ethical Experiments on Laboratory Animals, approval number C185/9.
Treatment and chemicals
Methyl mercuric chloride (CAS no. 115-09-3, CH3ClHg, Merck, KEBO, Sweden) and chlorpyrifos (purity >99%, CAS no. 2921-88-2, C12H11NO2, Sigma-Aldrich, Stockholm, Sweden) were dissolved in an egg lecithin (Merck, Darmstadt, Germany) and peanut oil (Oleum arachidis) mixture (1:10), which were sonicated with water to yield a 20% (w/w) fat emulsion vehicle containing; 0.04 mg MeHg/ml, 0.5 or 1.0 mg chlorpyrifos/ml, and two mixtures with 0.5 or 1.0 mg chlorpyrifos/ml combined with 0.04 mg MeHg/ml respectively. At PND 10, male mice were randomly picked from different
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litters for each treatment group and exposed to 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW combined with 0.4 mg MeHg/kg BW, as a single oral dose by gavage, via a metal gastric tube. These doses correspond to an administration volume of 10 ml/kg for each treatment group and control mice received 10 ml/kg of the 20% fat emulsion vehicle. On PND 11, 24 hours after exposure, the mice were sacrificed by decapitation. The brains were dissected immediately on an ice- cold glass plate and the cerebral cortex and hippocampus were collected and snapfrozen in liquid N2 and stored at −80 °C until analyzed.
Protein analysis
Cerebral cortex and hippocampus were homogenized in ice cold RIPA cell lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 20 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% sodium deoxycholate) with the addition of 0.5% protease inhibitor cocktail (Protease Inhibitor Cocktail Set III, Calbiochem). The
homogenates were centrifuged at 14,000 x g for 10 min at 4 °C, afterwards the supernatant was collected and stored at -80 °C. To determine the total protein concentration in each homogenate a BCA protein assay was performed
(Pierce).
The total protein amount for each sample was 4 µg for CaMKII and GAP-43, 3 µg for GluR1, 5 µg for PSD-95, 3 µg for synaptophysin and 3.5 µg for tau.
Before each slot-blot analysis the homogenates were diluted with sample buffer (120 mM KCl, 20 mM NaCl, 2 mM NaHCO3, 2 mM MgCl2, 5 mM HEPES, pH 7.4, 0.05% Tween 20, 0.2% NaN3) to a total volume of 200 µl, and then boiled for 5 min to denaturate the proteins. The diluted samples were
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added to a nitrocellulose membrane (0.45 mm, Bio-Rad) soaked in 1% TBS buffer (0.9% NaCl, 42.1 mM Tris–HCl and 7.5 mM Tris–Base) in duplicates by using a Bio-Dot SF microfiltration apparatus (Bio-Rad). Afterwards the membrane was dried at 37 °C for 10 min, fixed in a 25% isopropanol and 10%
acetic acid solution for 15 min, washed in 1% TBS buffer and incubated in 1%
SDS in TBS for 30 min at room temperature to unblock epitopes. Then, the membrane incubated in blocking solution (1% TBS containing 5% non-fat dry milk and 0.03% Tween-20) for 1 h at room temperature. After blocking, the membrane was incubated overnight at 4 °C in blocking solution with a
primary antibody specific for one of the neuroproteins. The primary antibodies used were a mouse monoclonal CaMKII antibody (Millipore, 05-532, 1:5000), a rabbit monoclonal GAP-43 antibody (Millipore, AB5220, 1:10,000), a rabbit monoclonal GluR1 antibody (Millipore, AB1504, 1:1000), a mouse
monoclonal PSD-95 antibody (Millipore, MABN68, 0.1 µl/ml), a mouse
monoclonal synaptophysin antibody (Calbiochem, 573822, VWR, 1:5000) and a mouse monoclonal tau antibody (Santa Cruz, 32274, AH Diagnostics,
1:1000).
The next day the membrane was washed again with 1% TBS and incubated for one hour at room temperature with a horseradish peroxidase-conjugated secondary antibody against mouse (KPL, 074-1806, 1:20,000) or rabbit (KPL, 074-1506, 1:20,000). To detect the immunoreactive bands an enhanced
chemiluminescent substrate (Pierce, Super Signal West Dura) was added and for visualization of the bands LAS-1000 (Fuji Film, Tokyo, Japan) was used.
The intensity of the bands was quantified using IR-LAS 1000 Pro (Fuji Film).
The results are expressed as percentage of protein detected in the treatment groups compared to protein detected in the controls.
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Statistical analysis
The chemiluminescent data from the study were analyzed by performing a one-way ANOVA and pairwise testing with Newman-Keul´s post hoc test (GraphPad Prism 5.01, GraphPad Software, San Diego, CA).
Results
Male mice were exposed on PND 10 to a single oral dose of 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW combined with 0.4 mg MeHg/kg BW respectively. On PND 11, 24 hours later, the mice were euthanized and cortex and hippocampus were collected.
CaMKII, GAP-43, GluR1, PSD-95, synaptophysin and tau were quantified in both brain regions. There were eight animals per treatment group in this study.
No significant decrease or increase of protein-levels were detected compared to the controls for any treatment group or protein (Fig. 1-6).
18 CaMKII
Hippocamp us
Cortex 0
50 100 150
200 Control
MeHg CPF low CPF high MeHg+CPF low MeHg+CPF high
Brain region protein level (% of control)
Figure 1 Protein levels of CaMKII for each treatment group compared to control (mean ± SD, n: 8). Male mice exposed to one single oral dose of 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW both combined with 0.4 mg MeHg/kg BW or a 20% fat emulsion vehicle on PND10. No statistical significance was detected using one-way ANOVA and pairwise testing with Newman-Keuls Multiple Comparison Test; hippocampus (P value: 0.9847, F: 0.1301) and cortex (P value: 0.9274, F:
0.2692).
GAP-43
Hippocamp us
Cortex 0
50 100
150 Control
MeHg CPF low CPF high MeHg+CPF low MeHg+CPF high
Brain region protein level (% of control)
Figure 2. Protein levels of GAP-43 for each treatment group compared to control (mean ± SD, n: 8). Male mice exposed to one single oral dose of 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW both combined with 0.4 mg MeHg/kg BW or a 20% fat emulsion vehicle on PND10. No statistical significance was detected using one-way ANOVA and pairwise testing with Newman-Keuls
Multiple Comparison Test; hippocampus (P value: 0.1839, F: 1.590) and cortex (P value: 0.9535, F: 0.2165).
19 GluR1
Hippocamp us
Cortex 0
50 100 150 200
250 Control
MeHg CPF low CPF high MeHg+CPF low MeHg+CPF high
Brain region protein level (% of control)
Figure 2 Protein levels of GluR1 for each treatment group compared to control (mean ± SD, n: 8). Male mice exposed to one single oral dose of 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW both combined with 0.4 mg MeHg/kg BW or a 20% fat emulsion vehicle on PND10. No statistical significance was detected using one-way ANOVA and pairwise testing with Newman-Keuls Multiple Comparison Test; hippocampus (P value: 0.8147, F: 0.4446) and cortex (P value: 0.6119, F:
0.7203).
PSD-95
Hippocamp us
Cortex 0
50 100
150 Control
MeHg CPF low CPF high MeHg+CPF low MeHg+CPF high
Brain region protein level (% of control)
Figure 3 Protein levels of PSD-95 for each treatment group compared to control (mean ± SD, n: 8). Male mice exposed to one single oral dose of 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW both combined with 0.4 mg MeHg/kg BW or a 20% fat emulsion vehicle on PND10. No statistical significance was detected using one-way ANOVA and pairwise testing with Newman-Keuls Multiple Comparison Test; hippocampus (P value: 0.4455, F: 0.9727) and cortex (P value: 0.9522, F:
0.2194).
20 Synaptophysin
Hippocamp us
Coretx 0
50 100 150 200
250 Control
MeHg CPF low CPF high MeHg+CPF low MeHg+CPF high
Brain region protein level (% of control)
Figure 4 Protein levels of Synaptophysin for each treatment group compared to control (mean ± SD, n: 8).
Male mice exposed to one single oral dose of 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW both combined with 0.4 mg MeHg/kg BW or a 20% fat emulsion vehicle on PND10. No statistical significance was detected using one-way ANOVA and pairwise testing with Newman- Keuls Multiple Comparison Test; hippocampus (P value: 0.5712, F: 0.7780) and cortex (P value: 0.8322, F:
0.4199).
Tau
Hippocamp us
Cortex 0
50 100 150 200 250
Control MeHg CPF low CPF high MeHg+CPF low MeHg+CPF high
Brain region protein level (% of control)
Figure 5 Protein levels of Tau for each treatment group compared to control (mean ± SD, n: 8). Male mice exposed to one single oral dose of 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW both combined with 0.4 mg MeHg/kg BW or a 20% fat emulsion vehicle on PND10. No statistical significance was detected using one-way ANOVA and pairwise testing with Newman-Keuls Multiple Comparison Test; hippocampus (P value: 0.1388, F: 1.776) Cortex (P value: 0.4775, F: 0.9200).
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Discussion
A study performed by Viberg et al. (unpublished) detected hyperactivity in adult male mice after neonatal exposure to MeHg and CPF. In this study the levels of six neuroproteins were measured 24 hours after the same kind of exposure as in the behavioral study. This was done to examine which mechanisms MeHg and CPF act by to cause the behavioral effects.
Male mice were exposed to a single oral dose of 0.4 mg MeHg/kg BW, 5 or 10 mg chlorpyrifos/kg BW and 5 or 10 mg chlorpyrifos/kg BW combined with 0.4 mg MeHg/kg BW respectively on PND 10. The mice were
euthanized on PND 11, 24 hours after exposure. The brains were dissected immediately and cerebral cortex and hippocampus were collected. Cerebral cortex is interesting to examine due to its importance for cognitive functions and hippocampus is interesting due to its role in learning. CaMKII, GAP-43, GluR1, PSD-95, synaptophysin and tau are all believed to be important for developing a fully functional nervous system. The levels of the neuroproteins were investigated by using a slot-blot method.
There were no significant changes found in the levels of the examined
neuroproteins after the exposures to MeHg, CPF or the combination of them.
However, it has earlier been observed that the combination of MeHg and CPF can cause hyperactivity which indicates that the combination does affect the developing brain. Why no significant changes were found in this study can be due to MeHg and CPF may act by other mechanisms, changes could also be masked by the high standard deviations.
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Another study analyzed the levels of the same neuroproteins as this one after exposure to 5 mg CPF/kg BW with the same slot-blot method, however it seems to be discrepancies between the results. The other study found a decrease of CaMKII in the hippocampus and a decrease of synaptophysin in the cerebral cortex (Lee et al., 2015). It is hard to say what caused the
discrepancy however it might be due to the high standard deviations in this study, especially in the cerebral cortex samples. In yet another study where axon outgrowth was observed there was also a decrease in GAP-43 after exposure to CPF (Sachana et al., 2005).
The high standard deviations made it hard to detect any possible difference in protein levels, especially in the cerebral cortex. The reason to the high
variance of those samples are discussible, the hippocampus samples displayed in general a normal variance and therefore it can be assumed that there should not be something wrong with the method and the laboratory work in general.
However, the brain regions were homogenized at different time’s maybe some samples thawed or became contaminated or in some other way got their
proteins degraded. Since the controls displayed the abnormally high variance it should not be due to the treatments.
Many studies have been performed investigating mechanisms of how MeHg causes developmental neurotoxicity and it appears to be very complex.
However, many of them investigates toxicity of prenatal exposure even though MeHg is known to accumulate in astrocytes which are developed during the BGS. MeHg-induced neurotoxicity can cause hyperactivation of the glutamatergic system (Allen et al., 2002, Farina et al., 2002), disruption of proteins (Giordano and Costa, 2012), disruption of cholinergic system (Coccini
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el al.,) and oxidative stress (Stringari et al., 2006). There have been combination studies performed with MeHg and other agents to study the effects on the developing brain. They have detected synergistic interactions of MeHg with PCB 153, PBDE 99 or gamma radiation in the low-dose range(Eriksson et al., 2010; Fischer et al., 2008a; Fischer et al., 2008b). It has also been found that MeHg hyperphosphorylates tau in cortex but not in hippocampus in adult mice (Fujimura et al., 2009). There have also been studies made which detected that OP pesticides too can hyperphosphorylate tau (Torres-Altoro et al., 2011). The antibodies used in this study do not distinguish between the different phosphorylated forms of tau and therefore it is still possible that tau can play a role in causing toxicity since hyperphosphorylated tau aggregates and causes neuronal cell death. Taupathies of this kind is the same as in Alzheimer’s disease and frontotemporal dementia (Fujimura et al., 2009).
A mixture of chlorpyrifos and carbaryl has been investigated (Lee et al., 2015;
Lee, 2015). Mice behavior were analyzed after exposure to chlorpyrifos and carbaryl individually and also a combination of them. Altered behavior were detected at 2 and 4 months after exposure of chlorpyrifos, carbaryl and the combination on PND10. It seemed like the synergistic effects of the
combination was driven by high chlorpyrifos doses. AChE inhibition was also examined, the inhibition was around 10% for the combination but also for chlorpyrifos and carbaryl individually, therefore it is believed that AChE inhibition probably isn´t the cause for the synergistic effect. However, a cholinergic susceptibility test was assessed through a nicotine-induced
behavior test and the mice exposed to the combination did not respond as the controls which indicates that another cholinergic pathway could be involved for this combination (Lee et al., 2015; Lee, 2015). MeHg have also been found
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to disrupt cholinergic pathways. One study observed an 44% increase in mAChR density in hippocampus of rats 14 days after exposure (Coccini et al., 2000)
Even though this study didn´t detect changes in neuroprotein levels there are discrepancies with other studies which indicates that changes in neuroprotein levels can still be a possible mechanism for MeHg and CPF to elicit
synergistic toxicity. Possible other mechanisms could be oxidative stress, tau hyperphosphorylation or altered cholinergic susceptibility. Since our brain is so complex it doesn’t seem unlikely that MeHg and CPF could act through several pathways to cause neurotoxic effects.
It is difficult to try to hypothesize how MeHg and CPF can cause
neurodevelopmental damage together by comparing the toxicity they elicit separately since MeHg and CPF could possibly form a complex that have completely different properties. A complex of MeHg and CPF was detected by Steevens and Benson (1999, 2000) by thin-layer chromatography. They also studied the exposure of MeHg and CPF in the amphipod Hyalella azteca where they detected additive toxicity, partial protection against AChE inhibition, decreased elimination of MeHg and no MeHg-induced oxidative damage. These observations could indicate some changes in properties for the complex compared to MeHg and CPF independently. However, the toxicity detected in these studies are in an amphipod and the relevance for mice and human can be questioned, but for now it is what have been published
regarding the toxicity of the mixture.
25
As mentioned earlier behavioral disturbances have been observed in adult mice after exposure to MeHg combined with CPF, which indicates that the mix should affect the developing brain in some way, although, the
mechanisms behind the disturbances remains unidentified, at least for a while longer. Additional studies investigating this issue are needed for the future, possibly investigating several pathways, it would be useful to unravel the details about MeHg and CPF suggested synergistic interaction. The knowledge could lead to a greater understanding of the interactions of
chemicals in the environment and if other OP pesticides similar to CPF could interact with MeHg in the same way.
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