Neurotoxicity
in
mice
after
exposure
during
early
brain
development
A
study
of
insecticides
chlorpyrifos
and
cypermethrin
Sara
Thedvall
Degree project inbiology, Master ofscience (2years), 2016 Examensarbete ibiologi 45 hp tillmasterexamen, 2016
Table of Contents Preface ... 1 Abstract ... 2 1. Introduction ... 3 1.1 Organophosphorus insecticides ... 3 1.1.1 Chlorpyrifos ... 4 1.2 Pyrethroid insecticides ... 5 1.2.1 Cypermethrin ... 6 1.3. Brain development ... 7
1.3.1 Brain Growth Spurt (BGS) ... 7
1.3.2 Cerebral cortex and hippocampus ... 9
1.4. Neuroproteins ... 9 1.4.1. CaMKII ... 9 1.4.2. GAP43 ... 10 1.4.3. GluR1 ... 11 1.4.4. PSD-95 ... 11 1.4.5. Synaptophysin ... 12 1.4.6. Tau ... 12 2. Objectives ... 13
3. Materials and methods ... 14
3.1. Chemicals ... 14
3.2. Animals and treatment ... 14
3.3. Biochemical assays ... 15
3.4. Statistical analysis ... 16
4. Results ... 17
4.1. Effects on neuroprotein levels in cerebral cortex ... 17
4.1.1. Neonatal ... 17
4.1.2. Adult ... 18
4.2. Effects on neuroprotein levels in hippocampus ... 20
Preface
This master degree project was carried out at the Department of Environmental Toxicology, within the Department of Organismal Biology, Uppsala University, Sweden, and at Statens Veterinärmedicinska Anstalt (SVA) in Uppsala, Sweden.
Abstract
1. Introduction
Pesticides are the collective term for compounds such as herbicides, fungicides, rodenticides and insecticides, and are mixtures used to prevent, repel or abolish pests. These are chemicals ubiquitously used in agriculture, household products, for industrial purposes, as well as public health, and the world’s expenditure was estimated to more than $39.4 billion in 2007, measuring a staggering 2,36 billion kg (EPA 2016a,b,c, EPA 2011). In the environment, humans are continuously exposed to chemicals, and even though certain persistent organic pollutants (POPs) have been removed or banned from use, their metabolites and residues can still be detected in human adipose tissue and milk, and in the environment (water, soil and sediment) (Aulakh et al. 2007, Singh et al. 2008, Stockholm Convention 2012, Bedi et al. 2013, Toan et al. 2013, Vukavić et al. 2013). This means that fetuses encounter pesticides indirectly via the mother, neonates via the mother’s milk, and neonates as well as adults directly through ingestion, inhalation and dermal contact. This can cause adverse effects, since the central nervous system (CNS) is extremely vulnerable at certain critical periods during the development. Exposure during that delicate time to certain chemicals can cause permanent neurotoxic damage and affect the levels of proteins and behavior in the adult animal, and this has for instance been reported for PBDE 99 and PBDE 209, OPs, DDT, bisphenol A, PFHxS, and PCBs (Eriksson et al. 1992, Ahlbom et al. 1995, Eriksson & Fredriksson 1998, Eriksson et al. 2002, Viberg et al. 2003, Viberg et al. 2008, Viberg & Eriksson 2011, Viberg et al. 2011, Viberg & Lee 2012, Lee & Viberg 2013, Viberg et
al. 2013).
Many insecticides are designed to target the nervous system of insects. The insect CNS is in some ways similar to the mammalian, and insecticides can thus adversely affect humans as well (Klaassen & Watkins 2010). Insecticides are often classified based on their chemical structure, mode of action and/or target insect. Some of the most common insecticide families used are the organic pollutants organophosphates (OPs) and pyrethroids (EPA 2016b).
1.1 Organophosphorus insecticides
OP agents (Figure 1) are compounds first developed during the Second World War for chemical warfare, as engine flame-retardants and as pesticides, and half of today’s global insecticide use is represented by OPs (reviewed in Casida & Quistad 2004, Jiang
et al. 2010, reviewed in John & Shaike 2015). They mainly elicit their neurotoxicity via
bioactivated metabolites, and affect the cholinergic system by inhibiting the brain enzyme acetylcholinesterase (AChE) in the CNS and PNS (peripheral nervous system) (Eaton et al. 2008, Klaassen & Watkins 2010, Abreu-Villaça et al. 2011).
OPs block this reaction by phosphorylating the hydroxyl groups of the serine amino acid of AChE. The enzyme is then inhibited and can no longer hydrolyze the ACh released into the synapses (Walker et al. 2012). ACh is thus accumulated in the synaptic cleft, and the cholinergic receptors on the postsynaptic membrane become overstimulated, leading to tremors, motor dysfunction and death (Klaassen & Watkins 2010, Walker et al. 2012).
Figure 1. The general structure of OP insecticides, where the S-moiety is exchanged for an O-moiety
during bioactivation, and X is the leaving group. Adapted from Klaassen & Watkins (2010).
Phosphorylated AChE can be reactivated by slowly being hydrolyzed by water, however this process cannot occur if the AChE-OP complex has aged. During aging, an alkyl group leaves the AChE-OP complex (dealkylation), and the enzyme inhibition of AChE becomes permanent (Rodríguez-Castellanos & Sanchez-Hernandez 2007, Klaassen & Watkins 2010).
Some common OPs are chlorpyrifos, diazinon and parathion (reviewed in Flaskos 2012), where the focus in this study will be on the first one.
1.1.1 Chlorpyrifos
Chlorpyrifos (Figure 2), (chemical name O,O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate), one of the most studied OPs, was introduced in 1965 for agricultural use and to help keep homes and pets free from fleas, ticks, termites and cockroaches (Eaton et al. 2008). It is a chlorinated OP with a broad spectrum, and even though it has been withdrawn from many domestic products and industrial uses, due to toxicity concerns for children, animals and the environment, the world consumption is still increasing (reviewed in John & Shaike 2015).
In humans and rodents chlorpyrifos is readily detoxified and excreted, and is in itself not very dangerous (Sams et al. 2004). However, chlorpyrifos has several metabolites, which are broken down via the biotransformation steps desulfuration, dearylation and dealkylation (Foxenberg et al. 2006, Croom et al. 2010, reviewed in Flaskos 2012). During desulfuration CYP2B6 replaces the oxygen with sulfur (P=O moiety to P=S), converting chlorpyrifos into the much more toxic chlorpyrifos-oxon, which is the main agent inhibiting AChE (Tang et al. 2001, Klaassen & Watkins 2010, reviewed in Flaskos 2012).
Exposure to chlorpyrifos-oxon results in symptoms such as salivation, lacrimation, urinating and defecating (SLUD) in mice, and a distressed gastrointestinal tract (and previous symptoms) in humans, cellular aberrations (for instance abnormal cell differentiation), problems in DNA synthesis and decreased synaptogenesis in rats (exposure at gestational day [gd] 9.5) when the inhibition has exceeded 70%. Some of these symptoms even occur at subtoxic doses, causing atypical social behavior, memory deficiencies, increased locomotor activity, and decreased anxiety in the perinatal rodent (Roy et al. 1998, Duysen et al. 2001, Huff et al. 2001, Ricceri et al. 2006, reviewed in Braquenier et al. 2010).
1.2 Pyrethroid insecticides
Synthetic insecticides are ubiquitously used compounds in agriculture, for animal husbandry and in public health, for instance against tse-tse flies (WHO 1989, Walker et
al. 2012). They have been on the market for more than 40 years and account for more
than a 30% of the worldwide usage (reviewed in Patel et al. 2006). Synthetic insecticides were developed off of the naturally toxic compound pyrethrin, which is found in the flowers of Chrysanthemum cinerariifolium “Dalmatian daisy”, and production started in Dalmatia (Croatia) in the 1840’s (Casida 1980).
These insecticides exert their neurotoxicity by disturbing (binding to) the voltage-gated Na+ channels on the cell membrane, which are responsible for creating action potentials
by producing the inward Na+ current. This makes the Na+ channels stay open longer, and close slower than normal, leaving the membrane depolarized for a longer time (Motomura & Narahashi 2001, reviewed in Ray & Fry 2006, reviewed in Farag et al. 2007). The extended activation time of the Na+ channels leads to a prolonged Na+ current flow over the membrane, which causes uncontrolled, repetitive firing of several action potentials from a single stimulus. This causes twitches, seizures and muscular tremors (reviewed in Farag et al. 2007, Walker et al. 2012).
Type I pyrethroid insecticides yield short Na+ tail currents and thus cause less complex symptoms: an amplified startle response, uncoordinated dorsal twitching, ataxia, hyperexcitation and fine tremor. The tremor increases the metabolic rate and causes metabolic fatigue and the thermoregulation to fail, which leads to hyperthermia. These two things usually lead to death. The effects produced by type I pyrethroid insecticides resembles those by DDT (Verschoyle & Aldridge 1980, Wright et al. 1988, reviewed in Ray & Fry 2006).
The poisoning symptoms of type II pyrethroid insecticides are more complex as they yield longer Na+ tail currents, which causes effects such as salivation, coarse tremor, convulsions, incoordination, seizures, exaggerated opening of the jaw, paralysis, inability to extend hind limbs causing a swaying walk, repetitive and involuntary and jerky movements and finally death (Barnes & Verschoyle 1973, Wright et al. 1988, reviewed in Ray & Fry 2006).
Compared to OPs, pyrethroid insecticides are rapidly biodegraded, have a lower toxicity for mammals (insects are three times more sensitive because of their slower metabolic rate, and lower body temperature), and are much more efficient against their target insect. One of the most common pyrethroids, and the most used type II pyrethroid is cypermethrin (Song & Narahashi 1996, reviewed in Patel et al. 2006).
1.2.1 Cypermethrin
Cypermethrin, (Figure 3), (chemical name [cyano-(3-phenoxyphenyl)methyl]3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane-1-carboxylate), is a broad spectrum and fast-acting toxin, and is used in particular against the insect order Lepidoptera (butterflies and moths), on vegetable crops, cotton and fruit (reviewed in Bhunya & Pati 1988, reviewed in Patel et al. 2006, PubChem 2016). This insecticide is lipophilic, and thus accumulates in tissues. It readily crosses the blood-brain barrier (BBB) into the brain, where it mediates its neurotoxicity by inhibiting Na+ channels and the GABA (γ-aminobutyric acid) receptor, causing hyper-excitation and convulsions (Ramadan et al. 1988a, reviewed in Patel et al. 2006, Singh et al. 2012).
Cypermethrin also prevents neurons from taking up Ca2+ and inhibits monoamine oxidase, the enzyme responsible for breaking down certain neurotransmitters. This causes the typical type II pyrethroid symptoms (Ramadan et al. 1988b, Rao & Rao 1993).
Studies in mice (in vivo and in vitro) have shown that cypermethrin also causes damage by free radicals in liver, brain and red blood cell tissues, as well as chromosomal aberrations, micronuclei formation in human lymphocytes, and sister chromatid exchange in mouse bone marrow. People who are exposed to cypermethrin through their work, are shown to have lymphocyte DNA damage (Chauhan et al. 1997, Kale et
al. 1998, Giray et al. 2000, Ündeğer & Bașaran 2002, reviewed in Wang et al. 2010).
1.3. Brain development
1.3.1 Brain Growth Spurt (BGS)
The development of the brain is a highly complex and delicate procedure, where the various pathways and regions carefully follow a set program to make sure the product is finished in time and is intact and working as it should (Dobbing & Smart 1974). The development can be divided into two critical periods, where the vulnerability to teratogenesis is different, and the outcome is determined by the timing of xenobiotic exposure (Klaassen & Watkins 2010).
Embryogenesis is the first critical period, and it takes place during the first trimester of human gestation, and mid-gestation for mice (gd 9-9.5 out of a gestational period of 20-21 days). Organogenesis occurs during this period and the neural tube and neural crest are formed (from which the brain develop). This is the first main step for all vertebrates (reviewed in Guerri 1998, DeSesso et al. 1999, Rice and Barone 2000). Tight junctions and the BBB also develop during this period, and can keep many unwanted molecules out of the brain by the time of birth (Engelhardt 2003, Kniesel et al. 1996). Exposure to xenobiotics during this stage usually leads to death or grave malformations. Each organ has its own peak of vulnerability, and thus the timing of exposure causes different malformations depending on what structure is forming at that time. Malformations in eyes, face and brain are common (Klaassen & Watkins 2010).
The BGS is a period characterized by a significant increase in brain weight, due to a very high rate of development and proliferation of glial cells (astro- and oligodendro-), which allows the brain to mature to its adult size and structure (Guerri 1998, Purves et
al. 2012). Some processes occurring during BGS are neurite outgrowth (sprouting and
arborization), new neuronal connections, synaptogenesis (at the peak of BGS about 1.8 million synapses are generated every second), myelination, and glial proliferation. Pruning, which is a programmed loss of synapses not working correctly (about 100.000 synapses are lost per second at the peak of BGS), also occurs (Davison & Dobbing 1968, Kolb & Whishaw 1989, Kolb 1995, Eliot 1999). Also developed at this stage are sensory and locomotor abilities (Bolles & Woods 1964). For the brain to develop properly and correctly, several neuroproteins with specific neurological functions are needed, and during BGS, numerous neuroproteins are also developed. Xenobiotic exposure during this stage usually leads to growth retardation and functional deficits (Klaassen & Watkins 2010).
Figure 4. The starting point and duration of the brain growth spurt is different in different species.
Adapted and modified from Davison & Dobbing (1968). Illustration Sara Thedvall.
As stated before, the starting point and duration of brain developmental events are different for different brain regions and species, and this must be considered when extrapolating from rodents to humans. However, it is possible to extrapolate and make interspecies comparisons since the BGS identifies the age in terms of brain growth
be ignored, since the consequences of exposure/injury are linked to the developmental age (Dobbing & Sands 1979, Semple et al. 2013).
1.3.2 Cerebral cortex and hippocampus
Cerebral cortex and hippocampus are two brain regions associated with behavior and cognitive processes, like memory and learning, including long-term potentiation (LTP) (Purves et al. 2012).
The biggest part of the cerebral cortex is known as neocortex, and is the part of the cortex covering the cerebral hemispheres. It is comprised of grey matter, and is in mammals divided into six distinct layers (laminae), made up by different cell types (Purves et al. 2012). Some layers have short intracortical connections, and some contain the largest and longest cells in the brain, connecting to the spinal cord and the other hemisphere (reviewed in Molnár & Cheung 2006, Purves et al. 2012). Gyrification, the folding of the cerebral cortex, is a major difference between humans (prominent) and rodents (lacking). The sulcal and gyral regions created give the human brain the possibility to grow a much larger surface than the skull normally would allow. The cerebral cortex is responsible for handling arriving informational input to the primary sensory cortex and output from motor cortex, social behavior, emotional and cognitive processes, decision-making, memory and LTP, as well as perception, thoughts, language and consciousness (Purves et al. 2012).
Hippocampus has three distinct cellular layers, and is shaped like a seahorse, hence the name. It is situated more dorsal in the mouse brain than in the human brain. Hippocampus is a brain region important for spatial navigation and working memory (McEwen 1995), as well as formation of new memories and LTP (Hevroni et al. 1998, reviewed in Giap et al. 2000, Cryan & Holmes 2005, Purves et al. 2012).
1.4. Neuroproteins
1.4.1. CaMKII
Calcium-calmodulin-dependent protein kinase II (CaMKII) is activated through a conformational change freeing CaMKII from its inactive conformation, when the intracellular Ca2+ concentration increases, causing calcium ions to bind to calmodulin (CaM), and the Ca2+/CaM complex binds to CaMKII. This neuroprotein is a multifunctional protein kinase (Anderson 2010, Anderson et al. 2011, reviewed in Yao
et al. 2011), located on both sides of the synaptic cleft, and plays a role in various
extinction via reduced synapse efficacy), anxiety, and aggression in mice (Mayford et
al. 1995, Giese & Mizuno 2013, Frankland et al. 2001, Lisman et al. 2002, Hasegawa et al. 2009, Pi et al. 2010).
Viberg et al. (2008) studied mice ontogeny (development between fertilization and sexual maturity) of CaMKII, and found that CaMKII levels dramatically increases from PND 1-28 in both cerebral cortex and hippocampus. The neuroprotein has its peak rate between PND 7-14, suggesting that it is an important protein for the processes of BGS (peaks PND 10). In different studies the CaMKII levels have been shown to both decrease and increase from its normal levels after neonatal exposure to xenobiotics, for instance PBDE 99 and 209, chlorpyrifos, carbaryl, endosulfan, bisphenol A and ketamine (Viberg et al. 2008, Viberg & Eriksson 2011, Viberg & Lee 2012, Buratovic
et al. 2014a, Lee et al. 2015a,b).
1.4.2. GAP43
Growth-associated protein 43 (GAP43) is a polypeptide located in axonal growth cones (Meiri et al. 1986), whose functions involve navigating the growth cones (axonal pathfinding), neuronal outgrowth, synaptic plasticity, as well as elongation and stabilizing of axonal branches (Denny 2006). GAP43 is also important for serotonergic (5-HT) innervation of many brain regions and projections from the brainstem to cerebral cortex and hippocampus, and it is very abundant in areas where memories are formed (such as frontal cortex and hippocampus) (Donovan et al. 2002). When the intracellular Ca2+ concentration is low, GAP43 is bound to CaM, but as soon as the concentration rises, GAP43 is phosphorylated by PKC, and CaM is let go (to create CaMKII). GAP43 phosphorylation leads to LTP, spatial memory and learning (Neve et
al. 1998, Hulo et al. 2002, Lovinger et al. 1986). It may also contribute to endocytosis,
neurotransmitter release and recycling of synaptic vesicles (Denny 2006).
The lack of GAP43 causes serotonin to be deficient in some brain regions and overexpressed in others, leading to developmental disorders, such as depression, anxiety, schizophrenia and autism in humans (Hen 1996, Mann 1998, reviewed in Donovan et al. 2002). An increase in cortical GAP43 levels has been found in schizophrenic individuals, and a disrupted cortical map and autistic-like features have been seen in GAP43-lacking mice (Maier et al. 1999, Perrone-Bizzozero et al. 1996, Weickert et al. 2001, Zaccaria et al. 2010).
1.4.3. GluR1
Glutamate receptor 1 (GluR1) is a subunit of the AMPA (alfa-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor located on the postsynaptic side of the synaptic cleft. It is an ionotropic (ligand-gated ion channel) receptor, which mediates the Na+ flow over the membrane (in small measures also the Ca2+ flow). It plays an important role in fast synapse excitatory neurotransmissions, dendrite growth, synaptic plasticity, and in memory and spatial learning (Pellegrini-Giampietro et al. 1997, Sheng & Kim 2002, Song & Huganir 2002, Lee et al. 2003, reviewed in Sanderson et al. 2008, Zhou
et al. 2008). LTP is linked to phosphorylated GluR1 and LTD to the dephosphorylated
neuroprotein (Barria et al. 1997, Lee et al. 2000, Kameyama et al. 1998, Lee et al. 1998). Abnormal decreases of GluR1 has been linked to schizophrenia, Alzheimer’s disease, and mood disorders (Beneyto et al. 2007, Eastwood et al. 1995, Wakabayashi
et al. 1999). In schizophrenic individuals GluR1 protein levels have been found in early
endosomes in dorsolateral prefrontal cortex (Hammond et al. 2010), and elevated GluR1 mRNA expression has been found in the same brain area (Dracheva et al. 2005). The ontogeny of GluR1 in rat hippocampus was studied by Ritter et al. (2002), and they found that the neuroprotein level does not change over time. Chan et al. (2003) studied the GluR1 mRNA in rat striatum at PND 7, PND 21 and adult age and found that the levels stayed the same for all age groups. The GluR1 level has been shown to both decrease and increase in cerebral cortex, as well as in hippocampus after exposure to different xenobiotics. In a study by Lee et al. (2015b) the neonatal GluR1 level increased in hippocampus after neonatal exposure to endosulfan, and after neonatal exposure to cypermethrin the level increased in cerebral cortex, but decreased in hippocampus of adults.
1.4.4. PSD-95
Postsynaptic density protein-95 (PSD-95) is an abundant PSD scaffolding/anchoring protein belonging to the membrane-associated guanylate kinase (MAGUK) family – a family known to take part in synaptic plasticity, and stabilization of cytoskeletal elements. It is concentrated at glutamatergic synapses on the postsynaptic membrane, where it clusters to form a supporting framework (Cho et al. 1992, Kistner et al. 1993, El-Husseini et al. 2000, Kim & Sheng 2004, Funke et al. 2005, Cheng et al. 2006). At synapses it regulates the synaptic strength by binding to AMPA glutamate receptors, and the information storage for memory and learning takes place in the PSD. PSD-95 might also participate in synapse development (Rao et al. 1998, Chen et al. 2000, Malenka & Bear 2004, Elias et al. 2006, Bats et al. 2007). Overexpression of PSD-95 causes the young synaptic contacts to mature and stabilize faster, and the amount and size of dendritic spines increases (El-Husseini et al. 2000).
1.4.5. Synaptophysin
Synaptophysin, an integral protein located in the axonal terminals, is the substrate of CaMKII on the presynaptic side of the synaptic cleft (Jahn et al. 1985, Lynch 2004). It was discovered in 1985, and is involved in informational exchange between neurons through initiation of the neurotransmitter release, recycling, exocytosis (for instance SNARE assembly) and endocytosis of synaptic vesicles, and LTP (Wiedenmann & Franke 1985, Jahn et al. 1985).
Viberg (2009) studied the ontogeny of synaptophysin in mice, and found that synaptophysin increases continuously in both hippocampus and cerebral cortex during the period PND 1-28. In different studies, the synaptophysin levels have been shown to both decrease and increase in cerebral cortex, as well as in hippocampus after neonatal exposure to xenobiotics, for instance PBDE 99 and 209, chlorpyrifos, bisphenol A and ionizing radiation () (Viberg & Eriksson 2011, (Viberg & Lee 2012, Buratovic et al. 2014a,b, Lee et al. 2015a).
1.4.6. Tau
The roles of tubule-associated unit (tau), being a phosphoprotein in a family of microtubule-associated proteins, are to stimulate the assembly of microtubules, thus supporting neuronal outgrowth, stabilize and sustain the already existing microtubules, and support neuronal polarity (Caceres & Kosik 1990, reviewed in Wang & Liu 2008). By controlling microtubule formation, tau also indirectly controls the cell shape, as well as cellular processes, like mitosis and meiosis (reviewed in Weingarten et al. 1975). Tau overexpression can be linked to atypical phosphorylation, which in turn can be linked to microtubule detachment and destabilization, and to protein clusters called paired helical filaments (PHF), which are the main constituent of neurofibrillary tangles (NFT) – a distinctive marker of Alzheimer’s disease (AD) (Goedert et al. 1991, Goedert 1993, Dickson et al. 1995).
Viberg (2009) also studied the ontogeny of tau in untreated mice, and tau first increases and then decreases just before the peak of the BGS, peaking in hippocampus between PND 3-7 and in cerebral cortex between PND 7-10.
2. Objectives
3. Materials and methods
3.1. ChemicalsThe emulsion mixes given on PND 10, was mixed from 1 g egg lecithin (from Merck, Darmstadt, Germany, CAS no. 8002-43-5), and 10 ml peanut oil (Oleum arachidis). Chlorpyrifos and cypermethrin were added, to yield the appropriate concentrations for the different doses (Table 3). The mixtures were then sonicated with water to yield a 20% fat emulsion, to imitate the fat content of mouse milk (~14%).
Chlorpyrifos (C9H11Cl3NO3PS, CAS no. 2921-88-2) and cypermethrin (C22H19Cl2NO3,
CAS no. 52315-07-8) were purchased from Sigma-Aldrich, and mouse monoclonal antibodies α-CaMKII and PSD-95 from EMD Millipore Corporation, Temecula, CA, USA, mouse monoclonal antibody synaptophysin from EMD Millipore Corporation, San Diego, CA, USA, and mouse monoclonal antibody tau from Santa Cruz Biotechnology INC, Santa Cruz, CA, USA. Rabbit polyclonal antibodies GAP43 and GluR1 were purchased from EMD Millipore Corporation, Temecula, CA, USA.
3.2. Animals and treatment
Pregnant NMRI-mice were bought from B&K, Germany. The dams were housed individually in a plastic cage, in a room with a 12 h light/12 h dark cycle, and free access to food pellets and water. The litters contained around 12 pups, but only males were used in this study (n = 6 per exposure group). The day of birth was called PND (postnatal day) 0, and on PND 10 the neonatal males were orally exposed to a single dose via a metal gastric tube (emulsion vehicle, 0.5 mg/kg BW chlorpyrifos, 10.0 mg/kg BW cypermethrin or combination 0.5 mg/kg BW + 10.0 mg/kg BW cypermethrin) (Table 1). 50% of all males were euthanized 24 h after exposure to the insecticides. When the remaining animals reached sexual maturity (at about four weeks of age) the males and females were separated, the males housed in a different room, and the females euthanized. The remaining males were euthanized 4 months after exposure to the insecticides. All males were dissected, cerebral cortex and hippocampus removed, frozen in liquid nitrogen and stored in -80° C until homogenizing and protein analysis.
Table 1. The different doses of chlorpyrifos and cypermethrin used for exposure on PND 10. Total
volume administered for all treatments was 10 ml/kg BW. Treatment Dose
20% fat emulsion vehicle (control)
Cypermethrin 0.5 mg/kg BW Chlorpyrifos 10.0 mg/kg BW
3.3. Biochemical assays
Homogenizing: The brain regions (cerebral cortex and hippocampus) were homogenized in 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), and then the homogenate was centrifuged for 10 min in 4° C at 14.000 g. The supernatant was saved and stored in -80° C.
Bicinchoninic Acid Assay (BCA protein assay): To determine the total protein concentration in the samples, BCA protein assay was used. A 96-well plate was prepared with RIPA buffer (blank), standards (0.5 mg/ml std, 1.0 mg/ml std and 2.0 mg/ml std), as well as the samples in triplicates. To all wells BCA reagent mix was added (PierceTM BCA Protein Assay Kit from Thermo Fisher, Rockford, IL, USA) and
the plate was incubated for 30 minutes at 37° C. The 96-well plate was subsequently measured in a microplate reader (Perkin Elmer precisely VICTOR3 1420 Multilabel counter) at 562 nm. When the protein content had been analyzed (R2 > 0.98) the sample volume to use in the Slot Blots could be calculated (for GluR1 and Synaptophysin 3 µg protein were used, for GAP43 and CaMKII 4 µg, for tau 3.5 µg and for PSD-95 5 µg protein).
Slot Blot: The supernatant was diluted with sample buffer ((120 mM KCl, 20 mM NaCl, 2 mM NaHCO3, 2 mM MgCl2 [6H2O], 5 mM HEPES [pH 7.4], 0.05% Tween-20,
0.2% NaN3)in 2 ml Eppendorf SafeSeal tubes to a total volume of 200 ml. This mixture
was boiled for 5 min and then added to a nitrocellulose membrane using a Bio-Dot SF microfiltration apparatus (Bio-Rad). After being dried for 10 min at 37° C, the proteins were fixed to the membrane by a solution containing 25% isopropanol, 10% acetic acid and for 15 min on a flip board and then washed in TBS (NaCl [0.9%], 42.1 mM Tris-HCl, 7.5 mM Tris-Base). In order to open up the epitopes of the proteins to allow antibodies to bind, 30 min incubation with SDS solution (10% SDS stock, TBS) followed, then a second wash in TBS. The membranes were then incubated for 1 h in blocking-buffer (5% non-fat dry milk and 0.03% Tween-20) to block unoccupied binding sites on the membrane and reduce non-specific binding of the antibodies (preventing high background), and then incubated overnight at 4° C in a mixture of blocking-buffer and the primary antibodies (α-CaMKII, GAP43, GluR1, PSD-95, synaptophysin and tau).
3.4. Statistical analysis
To identify significant outliers, Graphpad Quickcalcs was used
4. Results
4.1. Effects on neuroprotein levels in cerebral cortex
4.1.1. Neonatal
Slot blots analyses of CaMKII, GAP43, GluR1, PSD-95, synaptophysin and tau in the cerebral cortex 24 hours after exposure to one single oral dose of 20% fat emulsion vehicle, 0.5 mg/kg BW cypermethrin, 10.0 mg/kg BW chlorpyrifos, or a combination of 0.5 mg/kg BW cypermethrin and 10.0 mg/kg BW chlorpyrifos, on PND 10, are presented in Figure 6.
Figure 6. Protein levels in cerebral cortex 24 hours after neonatal exposure to one single oral dose of 20%
fat emulsion, 10.0 mg chlorpyrifos/kg BW, 0.5 mg cypermethrin/kg BW or a combination of 10.0 mg chlorpyrifos/kg BW and 0.5 mg cypermethrin/kg BW on PND 10. Statistical differences are shown as: a = significantly different from control (p ≤ 0.05), c = significantly different from cypermethrin (p ≤ 0.05), d = significantly different from the combination of chlorpyrifos and cypermethrin (p ≤ 0.05). A = significantly different from control (p ≤ 0.01), C = significantly different from cypermethrin (p ≤ 0.01). A* = significantly different from control (p ≤ 0.001). The height of the bars represents mean ± SD from 5-6 animals.
Significant changes were shown in five out of six neuroproteins: CaMKII (p ≤ 0.012), GAP43 (p ≤ 0.0448), GluR1 (p ≤ 0.0434), PSD-95 (p ≤ 0.0001) and tau (p ≤ 0.0001) in the neonatal cerebral cortex, 24h after exposure to chlorpyrifos, cypermethrin or the combination.
addition, the levels of CaMKII were significantly higher in chlorpyrifos exposed animals compared to the animals exposed to cypermethrin.
Mice exposed to chlorpyrifos also showed a 33% increase for GAP43 compared to the control, but no change was seen after cypermethrin or combination exposure.
In mice exposed to cypermethrin a 210% increase was seen for GluR1 compared to the control, but no change was seen after chlorpyrifos and combination exposure.
Mice exposed to chlorpyrifos also showed significantly changed levels of PSD-95 compared to the control, and so did the mice exposed to the combination, with increases of 97% and 51% respectively. In addition, the levels of PSD-95 were significantly higher in chlorpyrifos exposed animals compared to the animals exposed to both cypermethrin and the combination.
The neuroprotein levels of tau changed significantly for mice exposed to chlorpyrifos, cypermethrin, as well as the combination compared to the control, with increases of 640%, 508% and 517% respectively.
There were no significant changes of the neuroprotein synaptophysin in mice 24 hours after exposure to chlorpyrifos, cypermethrin or the combination compared to the control (p ≤ 0.1086).
4.1.2. Adult
Figure 7. Protein levels in cerebral cortex 4 months after neonatal exposure to one single oral dose of
20% fat emulsion, 10.0 mg chlorpyrifos/kg BW, 0.5 mg cypermethrin/kg BW or a combination of 10.0 mg chlorpyrifos/kg BW and 0.5 mg cypermethrin/kg BW on PND 10. Statistical differences are shown as: a = significantly different from control (p ≤ 0.05). A = significantly different from control (p ≤ 0.01), C = significantly different from cypermethrin (p ≤ 0.01). D = significantly different from the combination of chlorpyrifos and cypermethrin (p ≤ 0.01). The height of the bars represents mean ± SD from 5-6 animals.
Significant changes were shown in three out of six neuroproteins: GluR1 (p ≤ 0.009), synaptophysin (p ≤ 0.0102) and tau (p ≤ 0.0005) in the adult cerebral cortex, 4 months after exposure to chlorpyrifos, cypermethrin or the combination.
The neuroprotein levels of GluR1 changed significantly for mice exposed to chlorpyrifos, cypermethrin, as well as the combination compared to the control, with increases of 69%, 65% and 74% respectively.
In mice exposed to the combination, a 53% increase was seen for synaptophysin compared to the control, but no change was seen after chlorpyrifos and cypermethrin exposure.
In mice exposed to chlorpyrifos a 303% increase was seen for tau compared to the control, but no change was seen after cypermethrin and combination exposure. In addition, the levels of tau were significantly higher in chlorpyrifos exposed animals compared to the animals exposed to cypermethrin and the combination.
4.2. Effects on neuroprotein levels in hippocampus
4.2.1. Neonatal
Slot blots analyses of CaMKII, GAP43, GluR1, PSD-95, synaptophysin and tau in the cerebral cortex 24 hours after exposure to one single oral dose of 20% fat emulsion vehicle, 0.5 mg/kg BW cypermethrin, 10.0 mg/kg BW chlorpyrifos, or a combination of 0.5 mg/kg BW cypermethrin and 10.0 mg/kg BW chlorpyrifos, on PND 10, are presented in Figure 8.
Figure 8. Protein levels in cerebral cortex 24 hours after neonatal exposure to one single oral dose of 20%
fat emulsion, 10.0 mg chlorpyrifos/kg BW, 0.5 mg cypermethrin/kg BW or a combination of 10.0 mg chlorpyrifos/kg BW and 0.5 mg cypermethrin/kg BW on PND 10. The height of the bars represents mean ± SD from 5-6 animals.
4.2.2. Adult
Slot blots analyses of CaMKII, GAP43, GluR1, PSD-95, synaptophysin and tau in the cerebral cortex 4 months after exposure to one single oral dose of 20% fat emulsion vehicle, 0.5 mg/kg BW cypermethrin, 10.0 mg/kg BW chlorpyrifos, or a combination of 0.5 mg/kg BW cypermethrin and 10.0 mg/kg BW chlorpyrifos, on PND 10, are presented in Figure 9.
Figure 9. Protein levels in cerebral cortex 4 months after neonatal exposure to one single oral dose of
20% fat emulsion, 10.0 mg chlorpyrifos/kg BW, 0.5 mg cypermethrin/kg BW or a combination of 10.0 mg chlorpyrifos/kg BW and 0.5 mg cypermethrin/kg BW on PND 10. The height of the bars represents mean ± SD from 5-6 animals.
5. Discussion
Xenobiotics are very common in today’s world, exposing individuals throughout their lifetime. Pesticides are used in agriculture, in medicine and at home, thus exposing humans indirectly and directly through the environment and food (EPA 2016c). Neonates are more sensitive than adults, due to the vulnerable developmental state of the brain, raising concerns for their future well-being (Davison & Dobbing 1968). During the BGS, the brain goes through many quick changes leading to the final mature, organized structure, such as axonal and dendritic outgrowth, synaptogenesis and myelination. Together these events make it possible for neurological functions, such as learning, memory and behavior, to be established (Purves et al. 2012), and toxic insults during the delicate key brain development events can cause cognitive impairments in the adult (Eriksson 1997).
In the present study, neonatal male NMRI mice were exposed on PND 10 (peak of BGS) to a single oral dose of the insecticides chlorpyrifos (10 mg/kg BW), cypermethrin (0.5 mg/kg BW) or the combination of the two, and six neuroproteins, important for normal brain development and cognition, were studied in the neonatal and adult brain. The results of the study demonstrated disturbed neurodevelopment through altered neuroprotein levels in mouse cerebral cortex, in neonatal (five out of six proteins tested) as well as in adult mice (three out of six), compared to the control mice. The pesticides had no apparent effect in the hippocampus.
The neonatal chlorpyrifos-exposed animals showed a significant increase of cortical CaMKII (99%). CaMKII is necessary for LTP, and overexpression of CaMKII can lead to LTD, anxiety and aggression (Mayford et al. 1995, Giese & Mizuno 2013, Frankland
et al. 2001, Lisman et al. 2002, Hasegawa et al. 2009, Pi et al. 2010). Compared to a
mechanism of action as OPs (inhibit AChE), and Lifshitz et al. (1999) show that exposure to carbamates and OPs gives similar symptoms in children, making it possible for the mice in this study to also would have had behavior impairments. Impaired spontaneous motor behavior, and changed GAP43 levels in adult mice has previously been seen after neonatal exposure to PBDE 99, ketamine, PFOS, and PFOA (Eriksson
et al. 2002, Viberg et al. 2008, Johansson et al. 2009, Viberg & Eriksson 2011). The
scaffolding protein PSD-95 also increased after the neonatal exposure of chlorpyrifos (97%). This neuroprotein takes part in synaptic plasticity, stabilization and synaptic strength, and overexpression increases the amount of dendritic spines and matures the synaptic contacts (El-Husseini et al. 2000). The last neuroprotein to increase due to neonatal chlorpyrifos-exposure was tau (640%), which assembles and stabilizes microtubules, plays a role in neuronal outgrowth, and overexpression is linked to atypical phosphorylation (Caceres & Kosik 1990, Goedert et al. 1991, Goedert 1993, Dickson et al. 1995, reviewed in Wang & Liu 2008). Johansson et al. (2009) exposed mice neonatally to PFOS and PFOA, which also induced increased tau levels in cerebral cortex, and they showed in an earlier study (Johansson et al. 2008) that these compounds could cause altered spontaneous behavior in mice, such as decreased habituation, and hyperactivity. Buratovic et al. (2014b) exposed neonatal mice to ionizing radiation of 500 mGy and saw significantly increased cortical tau levels, that persisted in adulthood, leading to cognitive dysfunction, impaired spontaneous behavior (locomotion, rearing and total activity were measured) and reduced ability to habituate in a novel home environment. In another study by Buratovic (2016b) mice neonatally exposed to ketamine + 200 mGy also demonstrated increased cortical tau levels, and behavioral aberrations as adults. This suggests that the neonatal mice exposed to chlorpyrifos could have had an enhanced neurite outgrowth, more and bigger dendritic spines, and axonal sprouting, combined with overly mature and stable synapses. However microtubule detachment and destabilization might also have occurred due to an abnormal phosphorylation. It is also possible that these animals suffered from memory and learning impairments, anxiety attacks and aggressive behavior. Overexpression of GAP43 and tau can, later in life cause disorders such as schizophrenia and Alzheimer’s disease (Goedert et al. 1991, Goedert 1993, Dickson et
al. 1995, Hen 1996, Mann 1998, reviewed in Donovan et al. 2002).
found a significant increase of cortical tau, and impaired memory and learning, as well as reduced habitation in adult mice. Lee & Viberg (2013) also saw an adult increase in cortical tau after neonatal exposure to PFHxS, and the study by Viberg et al. (2013) also showed behavioral changes in the adult mice after neonatal exposure to PFHxS. In the adult chlorpyrifos exposed animals, CaMKII, GAP43 and PSD-95 alterations turned out to be transient, meaning they all reverted back to normal levels, and were no longer detectable in the adult brain.
Neonatal mice exposed to cypermethrin showed a significant increase in GluR1 (210%) and tau (508%) compared to the control, 24 hours after exposure. This suggests that these neonatal mice may experience outgrowth of longer, but less stable dendrites due to detaching microtubules, and could have early signs of schizophrenia and Alzheimer’s disease. Lee et al. (2015b) reported increased hippocampal GluR1 and tau levels, and increased cortical tau levels after neonatal endosulfan exposure, as well as increased cortical tau levels after neonatal exposure to cypermethrin, leading to impaired spontaneous behavior in adult mice.
The adult mice neonatally exposed to cypermethrin also showed a significant cortical increase in GluR1 (65%) compared to the control, 4 months after exposure. This shows that cypermethrin can cause a permanent increase of GluR1, in the mouse cerebral cortex, suggesting that these mice could have schizophrenic-like symptoms, however the change in tau was transient. Cypermethrin has previous been shown to significantly increase cortical GluR1, and cause altered spontaneous behavior in adult mice after neonatal exposure (Lee et al. 2015b), further strengthening the results from the present study.
The individual compounds have been shown to cause effects in protein levels, and such effects were seen for mice exposed to the combination of chlorpyrifos and cypermethrin as well. The neonates showed a significant increase in PSD-95 (51%) and in tau (517%) compared to the control, 24 hours after exposure. This suggests that these mice may develop long, big, and unstable dendrites due to detaching microtubules, but mature and stable synapses, leading to signaling problems for the information receiving cell, as discussed above.
(Viberg et al. 2011). In studies by Lee et al. (2015a,b), neonatal synaptophysin levels decreased in cerebral cortex after chlorpyrifos exposure, and adult GluR1 increased in cerebral cortex after neonatal cypermethrin exposure, but both exposures led to adult behavioral and cognitive aberrations. The increased levels of PSD-95 in the neonates exposed to the combination of chlorpyrifos and cypermethrin, was no longer visible in the adult mice. This transient effect was also seen in tau.
If comparing the effects of chlorpyrifos and cypermethrin, it seems like chlorpyrifos is much more toxic for these neuroproteins after a single exposure at PND 10, since CaMKII and PSD-95 increased significantly in neonatal mice exposed to chlorpyrifos compared to the mice exposed to cypermethrin, and PSD-95 also significantly increased for chlorpyrifos compared to the combination, 24 hours after the exposure. In addition, in the adult mice tau was the only neuroprotein still significantly increased in cerebral cortex in adult mice, after neonatal exposure to chlorpyrifos compared to the cypermethrin-, and the combination-exposed animals.
Four protein levels were significally different from the combination exposures compared to the control; neonatal PSD-95 and tau, and adult GluR1 and synaptophysin. Neonatal tau and adult GluR1 does not show significant protein level changes between the combination and the separate exposures, but neonatal PSD-95, and adult synaptophysin do suggesting a tendency towards an interaction for the last two proteins mentioned. PSD-95 shows a tendency (p ≤ 0.05) towards an antagonistic interaction, and synaptophysin a tendency (p ≤ 0.01) towards an additive interaction. Additionally, for synaptophysin there was no significant effects between the separate insecticides and the control, but there was for the combination and the control, showing that two doses without a separate effect can cause one in a combined exposure.
Even though two neonatal protein levels and two adult protein levels significantly increased for the combination exposure, none of them showed a clear interaction for the chlorpyrifos + cypermethrin combination. This could be because chlorpyrifos and cypermethrin have very different mechanisms of action, thus diminishing the effect of the other insecticide (Klaassen & Watkins 2010). But since there is a tendency towards an interaction for these two insecticides, and that interactions has previously been seen affecting the spontaneous behavior and ability to habituate in several previous studies (co-exposure of PBDE 99 and MeHg, PCB 153 and MeHg, gamma radiation and paraquat, ionizing radiation and ketamine) more studies are needed in this area (Fischer
et al. 2008a,b, Buratovic 2016a,b, Lee 2015).
PFHxS (Lee et al. 2015ab, Buratovic et al. 2014b, Lee & Viberg 2013).
In contrast to the transient protein effects, in some cases the effects were permanent or heightened over time, in the adult brain compared to the neonatal. It is possible that the permanent changes were too severe to overcome. For the heightened effects and effects only seen in adults, it is possible to argue that our exposure of the animals, as well as the euthanization, are snapshots in time. As the endogenous protein levels are developing during the neonatal period, it is possible that a fluctuation in the protein levels at that particular time was not picked up, even though the protein might be over-producing at that time. In this study this happened for GluR1 and synaptophysin protein levels, and similar new increases in cortical GluR1 and synaptophysin have been seen for cypermethrin, and bisphenol A (Viberg & Lee 2012, Lee et al. 2015b). The tau protein level for chlorpyrifos stayed permanent in this study, just like in a previous study where mice were irradiated with 500mGy (Buratovic et al. 2014b).
6. Conclusion
A single oral exposure to chlorpyrifos, cypermethrin or the combination of the two, during the peak of BGS, can cause developmental neurotoxic effects, manifested as altered levels of certain neuroproteins crucial for normal brain development and cognition, both in neonates and in adulthood.
Altered levels of certain neuroproteins have also been seen for other compounds, such as PBCs, DDT, PCBEs, PFOS, PFOA, ketamine, endosulfan, carbaryl and IR – compounds that might not share the same chemical structure or acute mode of action, as chlorpyrifos and/or cypermethrin, indicating that the BGS is a highly susceptible period in the development of the brain, and thus needs to be studied more.
It cannot be stated for sure that the animals we investigated would have shown aberrant behaviour as adults, since it was not tested. However, since neuroproteins, important for the brain to develop properly, were significantly altered in this study, and since both chlorpyrifos and cypermethrin previously have caused behavioural aberrations (Lee et
al. 2015), I think it is not very far-fetched to believe the mice in this study also would
7. Acknowledgements
This has been a year filled with great opportunities, new experiences, and new skills, which I will carry with me into the future. Therefore, I would like to express my gratitude to my supervisor, Henrik Viberg. Thank you for letting me conduct my master thesis under your supervision, teaching me so much about animal handling and how these projects are conducted, and answering all my questions. To Sonja Buratovic, thank you for answering all sorts of questions and teaching me the methods. Additionally, to everyone at the department, thank you for a year filled with interesting conversations, great company and many laughs. I also want to thank my family and friends for all the coffee pots you have brewed, and for all the love and support you have given me (and a special thanks to you, Tommy Dżus, for being sharp-eyed)! Lastly, I want to thank the three-digit numbers of mice needed for this project, and my multichannel pipette: without you this project would not have been possible, and my workdays much longer.
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