Neonatal Exposure to Paracetamol Affects the Levels of Important Neuroproteins in Developing Mouse Brain

UPTEC K 15003 Examensarbete 30 hp 18 Februari 2015

Neonatal exposure to paracetamol affects the levels of important neuroproteins in developing mouse brain

Carin Johansson Fall 2014 Abstract Neonatal exposure to paracetamol affects the levels of important neuroproteins in developing mouse brain Carin Johansson

Teknisk- naturvetenskaplig fakultet UTH-enheten Paracetamol is one of the most frequently used drugs against pain and fever in both adults and children. It has earlier been shown that neonatal paracetamol Besöksadress: administration can cause altered adult spontaneous behavior and cognitive dysfunction Ångströmlaboratoriet Lägerhyddsvägen 1 in mice. There is also evidence from epidemiological studies showing association Hus 4, Plan 0 between prenatal exposure to paracetamol and adverse outcomes later in life. These adverse effects may have been produced by changes in proteins important for normal Postadress: brain development, during the brain growth spurt. Box 536 751 21 Uppsala Mice were exposed to either paracetamol, cannabinoid receptor type 1 (CB1R) agonist Win 55212-2 or the combination of both substances on postnatal day 10 Telefon: (PND10). Slot blot analysis was used to determine the levels of the neuroprotein 018 – 471 30 03 markers CaMKII, GAP-43, GluR1, PSD95, tau and synaptophysin in both frontal and

Telefax: parietal cortex. Analysis showed a significant decrease in protein levels for GluR1, 018 – 471 30 00 PSD95 and synaptophysin in parietal cortex for mice, neonatally exposed to paracetamol. These results support earlier findings and we conclude, that paracetamol Hemsida: acts as a neurological toxin during brain development. http://www.teknat.uu.se/student

Handledare: Henrik Viberg Ämnesgranskare: Per Eriksson Examinator: Margareta Hammarlund-Udenaes ISSN: 1650-8297, UPTEC K15003

Neonatal exposure to paracetamol affects the levels of important neuroproteins in developing mouse brain

CARIN JOHANSSON MASTER THESIS FALL 2014

UPPSALA UNIVERSITY

Carin Johansson MASTER THESIS Uppsala University

ABSTRACT Paracetamol is one of the most frequently used drugs against pain and fever in both adults and children. It has earlier been shown that neonatal paracetamol administration can cause altered adult spontaneous behavior and cognitive dysfunction in mice. There is also evidence from epidemiological studies showing association between prenatal exposure to paracetamol and adverse outcomes later in life. These adverse effects may have been produced by changes in proteins important for normal brain development, during the brain growth spurt. Mice were exposed to either paracetamol, cannabinoid receptor type 1 (CB1R) agonist Win 55212-2 or the combination of both substances on postnatal day 10 (PND10). Slot blot analysis was used to determine the levels of the neuroprotein markers CaMKII, GAP-43, GluR1, PSD95, tau and synaptophysin in both frontal and parietal cortex. Analysis showed a significant decrease in protein levels for GluR1, PSD95 and synaptophysin in parietal cortex for mice, neonatally exposed to paracetamol. These results support earlier findings and we conclude, that paracetamol acts as a neurological toxin during brain development.

Carin Johansson MASTER THESIS Uppsala University

POPULÄRVETENSKPLIG SAMMANFATTNING Paracetamol är ett av de mest använda receptfria läkemedlen mot smärta och feber, för både vuxna och barn. Det har, i studier gjorda på möss, visat sig att paracetamol-administration kan orsaka förändring av spontanbeteende och ge kognitiva funktionsnedsättningar. Epidemiologiska studier har visat association mellan prenatal paracetamol exponering och negativa effekter senare i livet. Effekterna kan ha uppstått som följd av förändringar av specifika neuroproteiner, vilka är viktiga för normal utveckling av hjärnan.

Möss exponerades för paracetamol, cannabinoid receptor typ 1 (CB1R) agonist Win 55212-2 eller båda substanserna på 10 dagen efter födseln. För att mäta nivåerna av neuroproteinerna av intresse CaMK2, GAP-43, GluR1, PSD-95, tau and synaptophysin användes Slot blot analys. Hjärnregionerna som undersöktes var frontal och parietal cortex.

Analyserna visade på signifikant nedreglerade nivåer av proteinerna GluR1, PSD-95 and synaptophysin i parietal cortex. Dessa resultat stödjer tidigare forskning och slutsatsen är att paracetamol verkar som ett neurologiskt toxin då fostrets hjärna utvecklas.

Carin Johansson MASTER THESIS Uppsala University

TABLE OF CONTENT

Neonatal exposure to paracetamol affects the levels of important neuroproteins in developing mouse brain

ABSTRACT 1

POPULÄRVETENSKAPLIG SAMMANFATTNING PÅ SVENSKA 2

INTRODUCTION Paracetamol 4

Recent studies on developmental neurotoxicity of paracetamol 4

Brain growth spurt 5

Biomarkers 6

Paracetamol and Win 55212-2 7

PROJECT AIM 11

MATERIAL AND METHOD Methods and animals 12

Guidelines for pain management 12

Chemicals and exposure 12

Protein assay 13

Statistical Analysis 13

RESULT 15

DISCUSSION 19

CONCLUSION AND OUTLOOK

Conclusion 21

Outlook 21

ACKNOLEDGEMENT 22

REFERENCES 23

Carin Johansson MASTER THESIS Uppsala University

INTRODUCTION

Paracetamol Paracetamol (N-acetyl-p-aminophenol, CAS number 103-90-2) is a commonly bought over-the- counter drug and is marketed as an analgesic and antipyretic drug (Feldkamp et al. 2010). Furthermore paracetamol is commonly used medication during pregnancy. When recommended doses of the drug are taken it is considered safe to take for pregnant women (Bonati et al. 1990).

Figure 1. Paracetamol (N-acetyl-p-aminophenol, CAS number 103-90-2). Recent studies on developmental neurotoxicity of paracetamol In two recent epidemiological studies maternal paracetamol consumption during pregnancy was associated with neuro-toxicological effects on their outcomes.

In one study prenatal exposure to paracetamol was associated with adverse neurodevelopmental effects that can cause dysfunctional behaviour, such as attention deficit hyperactivity disorder (ADHD) and hyperkinetic disorder (HKD) in children (Liew et al. 2014). In another epidemiological study, long term prenatal exposure ≥ 28 days to paracetamol have shown to be associated with poor gross motor functioning, delayed age starting to walk, poor communication skills, externalizing and internalizing behaviour problems and an active temperament. Short term prenatal exposure <28 days of paracetamol have shown to be associated with poor gross motor functioning and several other negative development outcomes (Brandlistuen et al. 2013).

As previously mentioned, the effects of paracetamol exposure during brain development have also been evaluated in mice. Ten day old mice were treated in two ways; one group with a single dose of 30 mg paracetamol/kg body weight and the other group with, 4 hours in between, 30 mg paracetamol/body weight twice. The study shows that mice neonatally injected with 2 doses of paracetamol (2x30mg paracetamol/kg body weight), displayed altered locomotor activity on exposure to novel home cage area and fail to acquire spatial learning in adulthood. Mice that were neonatally exposed to 2x30 mg paracetamol/kg bw also failed to exhibit paracetamol-induced anxiolytic and antinociceptive behaviour as adults (Viberg et al. 2014).

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Brain growth spurt Many new development features take place during the fetal and neonatal life, including establishment of neural connections, maturation of dendritic and axonal outgrowth and synaptogenesis and proliferation of glia cells with accompanying myelinisation and acquisition of many motor and sensor abilities (Bolles and Woods 1964, Davison and Dobbing 1966, Dobbing and Sands 1979, Kolb and Whishaw 1989).

When brain weight is plotted against age, the brain grows through a sigmoid trajectory. “The transient period of rapid growth illustrated by this type of inflection is known as the brain growth spurt (BGS). And there is reason to believe that this may be a period of enhanced vulnerability nutritional and other growth restriction”. When extrapolation is done it is important to take into account that timing for the BGS is different in relation to birth in various species. A rough categorization can be made of various species of prenatal, perinatal and postnatal BGS developers (Dobbing and Sands 1979).

For rats, mice and other rodents the BGS is neonatal and spanning the first 3-4 weeks in life, to about postnatal day 28. For humans the BGS begins during the third trimester of pregnancy and is ongoing until approximatively 2 years of age. During this period in life the brain is going through great neurological development (Semple et al. 2013).

Figure 2. Brain growth spurt and its difference between spices.

Carin Johansson MASTER THESIS Uppsala University

Biomarkers Neurotypic and gliotypic proteins can be used as biomarkers, due to the fact that they are sensitive indicators of time- and region-specific effects of chemicals on the developing nervous system (O'Callaghan and Miller 1988, Garcia et al. 2003).

Calmodulin-dependent protein kinase II (CaMKII) is a protein kinase which is widely expressed in neuronal tissue (Erondu and Kennedy 1985) and plays an important role in several important processes such as synaptogenesis (Kazama et al. 2003, Kazama et al. 2007), apoptosis (Heist and Schulman 1998), long-term potentiation (Lisman and Goldring 1988) and axonal and dendritic arborisation (Zou and Cline 1999). Studies on rats have shown that CaMKII expression during the first four weeks of life increased and that the increase was roughly the same in cortex, hippocampus and whole brain. The steepest increase of CaMKII concentration in the brain was between PND 7-14. The CaMKII concentration boost coincides with the peak for brain development (Kelly et al. 1987, Polli et al. 1990, Sugiura and Yamauchi 1992, Viberg et al. 2008).

Growth Associated Protein 43 (GAP-43) is a presynaptic protein and plays an important role in guiding the growth of axons and the creation of new axonal connections during the development of central nervous system (CNS) and regeneration in the parasympathetic nervous system (PNS). GAP- 43 is commonly used as a biomarker for axonal sprouting and growth, dependent on its characteristics and pattern (Oestreicher et al. 1997). Studies performed on rats indicate that there is an increase in GAP-43 concentration in hippocampus and cortex the first two weeks of life. The concentration gradually decreases during the years to come (Shughrue and Dorsa 1994). The development profiles between GAP-43 and CaMKII varies over time. GAP-43 increased in the early postnatal period peaked during the second postnatal week and after this GAP-43 decreased (Oestreicher et al. 1997).

Glutamate receptor 1 (GluR1) is the most expressed α-Amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptor subunit in combination with Glutamate receptor 2 (GluR2) in the hippocampus and neocortex region (Wenthold et al. 1996). The AMPA receptors are tetrameric or pentameric complexes consisting of four homologous subunits GluR1–GluR4 (Seeburg 1993, Hollmann and Heinemann 1994). Phosphorylations of GluR1 sites are crucial for the synaptic plasticity and it has been shown to play an important role for spatial memory (Lee et al. 1998).

Postsynaptic density protein 95 (PSD95) is a postsynaptic protein and is associated with cytoskeletal elements at synapses and receptors. It plays a central role for neural functions and is concentrated at glutamatergic synapses (El-Husseini et al. 2000). It has been found that the PSD95 levels increase the most between PND 8 and 18, during which the development of synaptogenesis is occurring most frequently also coinciding with the BGS (Aghajanian and Bloom 1967). This may indicate that PSD95 is involved in the synaptogenesis or other important processes of the BGS (Cho et al. 1992).

Carin Johansson MASTER THESIS Uppsala University

Synaptophysin is an integral membrane glycoprotein (Wiedenmann and Franke 1985, Wiedenmann et al. 1986). It is one of the most abundant of the transmembrane glycoproteins expressed in presynaptic vesicles. Synaptophysin is likely involved synaptic vesicle endocytosis and serves as an adaptor protein for other synaptic vesicles. The detailed function of the protein is not for the time being fully explored (Gordon et al. 2011, Kwon and Chapman 2011). Also interesting is that the synaptophysin levels increase the most between PND 7 and PND 10 and during this time period roughly most of the neonatal brain growth occurs (Viberg 2009).

Tau is primarily localized in axons and is a neuronal microtubule binding protein. It is thought to build short cross-bridges between axonal microtubules (Harada et al. 1994) and is fundamentally important for axonal transport and maintenance of normal morphology (Vila-Ortiz et al. 2001, Wang and Liu 2008). Increases of Tau protein during the BGS have been shown and when the BGS is over the Tau protein concentration decreases. The protein peak occurs between PND 7 and PND 10 (Viberg 2009).

Paracetamol and Win 55212-2 Paracetamol can easily distribute throughout the different body fluids. It penetrates the blood brain barrier and crosses the placenta with ease (Levy et al. 1975). The peak concentration after oral administration is reached within two to three hours in humans (Bannwarth et al. 1992, Anderson et al. 1998). Paracetamol undergoes the first pass metabolism and its bioavailability is 60 to 89 percent for oral administration (Rawlins 1977, Borin and Ayres 1989). The approximate half-life of the substance in plasma is two hours. The lowest toxic dose in adults is 7.5 g and for children 150 mg/kg bw (Bizovi and Smilkstein 2002). Paracetamol has a ceiling effect at the oral dose of 1g; the analgesic activity does not increase when it reaches its ceiling dosage. The analgesic-antipyretic effect, of one dose, last for three to four hours (Skoglund et al. 1991).

Paracetamol is mainly metabolized through conjugation with sulphuric acid, glucuronide acid and through Cytochrome (CYP) enzymes (Miners et al. 1984). There is also a small fraction which is metabolized to a p-aminophenol, which, in turn is metabolized through the enzyme fatty acid amide hydrolase (FAAH) to N-arachidonoyl-phenolamine (AM404) (Zygmunt et al. 2000). AM404 have been shown to raise extracellular endogenous cannabinoid levels in the brain, probably through activation of cannabinoid receptor type 1 (CB1) (Högestätt et al. 2005, Bertolini et al. 2006, Ottani et al. 2006, Mallet et al. 2008). The mechanism is not completely clear but two studies are inclined in the same direction. Paracetamol metabolite AM404 seem to be responsible for the analgesic mechanism and is thought to be similar to Δ9-tetrahydrocannabinol (Δ9-THC), the active ingredient in cannabis and it is probably through this mechanism paracetamol gets its analgesic effect (Umathe et al. 2009). This has been shown in a study were paracetamol-induced analgesia was prevented by blocking the CB1 receptor (Bertolini et al. 2006, Ottani et al. 2006, Dani et al. 2007, Mallet et al. 2008, Ruggieri et al. 2008).

Carin Johansson MASTER THESIS Uppsala University

Paracetamol is almost unanimously considered to have no anti-inflammatory effect and it does not cause gastrointestinal damage or induce cardio renal effects unlike nonsteroidal anti-inflammatory drugs (NSAIDs) (Bertolini et al. 2006). Paracetamol is not considered to be a NSAID substance due to the fact that it possesses very little anti-inflammatory activity. Several studies suggest the possibility that the site of action of paracetamols anti-nociceptive effect may be the central nervous system (CNS) (Ottani et al. 2006).

Inhibition of cyclooxygenase (COX) is found to give paracetamol its antipyretic effect (Flower and Vane 1972). This inhibition is probably what gives paracetamol its NSAID characteristics. However, it has also been suggested that paracetamol does not affect the COX-2 dependent pathways (Graham and Scott 2005). Instead paracetamol blocks COX activity by reducing the conversion of the active oxidized form of the enzymes to an inactive form. Still evidence that paracetamol is dependent on COX, and by this gives analgesic and antipyretic effects, is lacking (Ouellet and Percival 2001, Boutaud et al. 2002, Lucas et al. 2005).

Win 55212-2 ((R)-(+)-[2, 3-Dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo [1, 2, 3-de]-1, 4- benzoxazin-6-yl]-1-napthalenylmethanone; CAS number 131543-23-2) is a CB1-receptor agonist (Felder et al. 1995). Studies on rats have shown that prenatal exposure to the substance give rise to permanent alterations of cortical glutamatergic function and learning difficulties as an adult. BDNF levels are significantly reduced by Win 55212-2 in the hippocampus and frontal cortex which results in decreased activation of pathways linked to glutamate and neurotropin receptor signalling (Fernández-Ruiz et al. 2000, Maj et al. 2007).

Figure 3. Win 55212-2 ((R)-(+)-[2, 3-Dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo [1, 2, 3-de]-1, 4-benzoxazin-6-yl]- 1-napthalenylmethanone; CAS number 131543-23-2) The substance induced cortical and reduced basal extracellular glutamate levels in offspring. This has been seen in in-vitro studies on rats with prenatal exposure to the substance. The results indicate that repeated prenatal exposure to Win 55212-2 enhances N-methyl-D-aspartate (NMDA) receptor activity loss in the offspring. Exposure to Win 55212-2is linked with cognitive dysfunction and gives rise to significant impairment of glutamatergic neurotransmission in hippocampus and cerebral cortex. These brain regions are crucial for memory and learning (Harkany et al. 2007, Maj et al. 2007).

Carin Johansson MASTER THESIS Uppsala University

Evidence that maternal exposure to Win 55212-2 induces short and long term deterioration of cognitive capacities in the offspring was revealed. It was demonstrated that prenatal exposure to Win 55212-2, at a dose not associated with toxicity, caused learning difficulties and changes in emotional reactivity in the offspring.(Ferraro et al. 2009).

For the moment the mechanism (could be several mechanisms) for the neurochemical interaction and/or the signalling pathways between NMDA and cannabinoid receptors are not completely revealed (Ferraro et al. 2009).

Carin Johansson MASTER THESIS Uppsala University

Figure 4. Proposed mechanism of paracetamol. Source of revised figure (Bertolini et al. 2006).

Carin Johansson MASTER THESIS Uppsala University

PROJECT AIM The aim with this project was to investigate if and how neonatal administration of paracetamol, Win 55212-2 or the combination of both substances, on PND 10, affected the protein markers CaMKII, GAP-43, GluR1, PSD95, synaptophysin and tau.

This task was performed by using brain tissue from neonatal mice to analyse the levels of protein markers with Western blot and Slot blots in hippocampi and cortices from mice neonatally exposed to paracetamol, Win 55212-2 or the combination of the both substances.

Carin Johansson MASTER THESIS Uppsala University

METHOD AND MATERIAL

Methods and animals Pregnant Naval Medical Research Institute (NMRI) mice were purchased from B&K (Sollentuna, Sweden) and were housed individually in plastic cages in a room with an ambient temperature of 22C and 12/12-h cycle of light and dark. The animals were supplied with standardized pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. Experiments were carried out in accordance with European Communities Council directive (86/609/EEC), after approval from the local ethical committees (Uppsala University and Agricultural research council) and the Swedish Committee for Ethical Experiments on Laboratory Animals, approval number C 32/14.

Chemicals and exposure Paracetamol (Perfalgan, 10 mg/mL Fresenius Kabi) and Win 55212-2 (Sigma –Aldrich). Stock solutions for subcutaneous injections of both paracetamol and Win 55 212-2 was made: 6 mg/ml and 0.2 mg/ml respectively. On postnatal day 10, male pups were administrated 30+30 mg (four hours apart) paracetamol/kg bw or 1 + 1 mg Win 55212-2/kg bw, (4 hours apart) or vehicle (0.9% NaCl) in volume of 5 mL/kg by subcutaneous injection in the neck. The recommended dose for suppositories containing paracetamol is 12.5 mg/kg bw in new born and toddlers (FASS, 2014). The recommended dose for Paracetamol injection fluid is 7.5 mg paracetamol/kg bw, where maximum daily dose should not exceed 30 mg/kg bw (FASS, 2014). Our doses are higher than the recommended doses, but in the same range, and the exposure occurs on 1 day only, in contrast to what is often common in daily life.

Male mice were sacrificed by decapitation at postnatal day 11. Brains were dissected on an ice-cold glass plate. Hippocampus, frontal and parietal cortex from all male mice (control, paracetamol, WIN

55212-2 and combination) were immediately placed in liquid nitrogen (N2) and stored at −80 °C until assayed.

Guidelines for pain management Astrid Lindgren Hospital for Children proposes the fallowing doses, when administrating via intravenous injection; for children 0 to 2 months of age 10-15 mg/kg bw ×4 and for children older than 2 months of age 15-20 mg/kg bw ×4. The loading dose for intravenous injection is 20 mg/kg bw to newly born and 30 mg/kg bw to children older than 2 months of age.

Carin Johansson MASTER THESIS Uppsala University

Protein assays The method consist of three steps; homogenization of mouse brain region, bicinchoninic acid assay (BCA) to evaluate the content of protein in sample and finally Slot blot to measure the level of protein.

The brain regions were homogenized in a 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) to which 0.5% protease inhibitor cocktail (Protease Inhibitor Cocktail set III, Calbiochem) was added. Homogenates were centrifuged at 14,000 × g at 4 °C for 15 min and the supernatant was analyzed for protein content using the BCA method (Pierce). Homogenates were stored at −80 °C.

To estimate the total protein concentration in a solution BCA was used (Wiechelman et al. 1988). The assay consists of two steps. First the cupric sulphate pentahydrate in the BCA stock solution is reduced by the peptide bonds. Then the bicinchoninic acid chelate reacts with Cu2+ and the reaction product formed gives the change of colour. The reaction product absorbs light (wavelength 562 nm) (Smith et al. 1985, Olson and Markwell 2001). An absorption spectra of known solution is used for comparison, to quantify the total amount of protein in the solution(Noble and Bailey 2009).

To show that the antibodies are suitable for Slot blot, Western blot and SDS-PAGE had previously been performed on the primary antibodies. If the primary antibody detected only one band in homogenates from PND 10 mouse brain at the appropriate molecular weight the primary antibody was considered specific for the protein intended (Viberg et al. 2008, Viberg 2009).

A total of 3 μg protein for synaptophysin and GluR1, 3.5 μg for tau 4 μg of protein for CaMKII and GAP-43 and 5µg for PSD95 were diluted in 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 final volume of 200 μl. Duplicates of each sample were applied to a nitrocellulose membrane (0.45 μm, BioRad) using a Bio-Dot SF microfiltration apparatus (BioRad). Membranes were fixed in 25% isopropanol and 10% acetic acid solution for 15 min, washed and subsequently blocked for 1 h in 5% non-fat dry milk and 0.03% Tween-20 at room temperature.

Incubation of membranes with primary mouse monoclonal synaptophysin antibody (1:10 000) (Calbiochem, 573822), primary rabbit polyclonal GluR1 antibody (1:1000) (Upstate Millipore, AB1504), primary mouse monoclonal Tau antibody (1:1000) (Santa Cruz, 32274), primary mouse monoclonal CaMKII antibody (1:5000) (Upstate Millipore, 05-552), primary rabbit polyclonal GAP- 43 antibody (1:10 000) (Chemicon Millipore, AB5220) or primary mouse monoclonal PSD-95 antibody (0, 1µg/mL) (Upstate Millipore, MABN68) took place overnight at 4 °C. A horseradish peroxidase conjugate secondary antibody against mouse (KPL 074-1806, 1:20,000) or rabbit (KPL 074-1506, 1:20.000) was used to detect immunoreactivity. Immunoreactive bands were traced using

Carin Johansson MASTER THESIS Uppsala University an enhanced chemiluminescent substrate (Pierce, Super Signal West Dura) and imaged with a LAS- 1000 (Fuji Film, Tokyo, Japan). Band intensity was quantified using IR-LAS 1000 Pro (Fuji Film).

Statistical analysis The comparisons of the levels of CaMKII, GAP-43, GluR1, PSD-95, synaptophysin and tau protein between controls, paracetamol, Win 55212-2 and the combination of the both-treated animals were analyzed using a one-way ANOVA with Newman – Keuls multiple comparison test (Prism, version 5.04).

Carin Johansson MASTER THESIS Uppsala University

RESULTS

Effects on CaMKII protein levels in the neonatal frontal cortex and parietal cortex. There were no significant treatment effects of paracetamol, Win 55212-2 or the combination on CaMKII level in the frontal cortex (p = 0. 8493) or the parietal cortex (p = 0. 5418).

See Figure 5 and 6.

Effects on GAP-43 protein levels in the neonatal frontal cortex and parietal cortex. There were no significant treatment effects of paracetamol, Win 55212-2 or the combination on GAP- 43 levels in the frontal cortex (p = 0. 5306) or the parietal cortex (p = 0. 1313).

See Figure 5 and 6.

Effects on GluR1 protein levels in the neonatal frontal cortex and parietal cortex. There were no significant treatment effects of paracetamol, Win 55212-2 or the combination on GluR1 levels in the frontal cortex (p= 0. 4470). See Figure 5.

There was a significant effect of the treatment with paracetamol on the GluR1 level in parietal cortex (p= 0. 0181). The levels of GluR1 were decreased (p < 0. 05) by 33.336% in the parietal cortex of animals exposed to paracetamol compared to the control group. See Figure 6.

There were no significant treatment effects of Win 55212-2 or the combination compared to the control group on GluR1 levels in the parietal cortex. See Figure 6.

Effects on PSD95 protein levels in the neonatal Frontal cortex and Parietal cortex. There were no significant treatment effects of paracetamol, Win 55212-2 or the combination on PSD95 level in the frontal cortex (p= 0. 4501). See Figure 5.

There was a significant effect of the treatment with paracetamol on the PSD95 level in parietal cortex (p= 0. 0182). The level of PSD95 was decreased (p < 0, 05) by 22.504% in the parietal cortex of animals exposed to paracetamol compared to the control group. See Figure 6.

There were no significant treatment effects of Win 55212-2 or the combination compared to the control group on PSD95 level in the parietal cortex. See Figure 6.

Effects on synaptophysin protein levels in the neonatal Frontal cortex and Parietal cortex. There were no significant treatment effects of paracetamol, Win 55212-2 or the combination on synaptophysin level in the frontal cortex (p= 0, 4735). See Figure 5.

There was a significant effect of the treatment with paracetamol on the synaptophysin level in parietal cortex (p= 0. 0067). The level of synaptophysin was decreased (p < 0.05) by 36.76% in the parietal cortex of animals exposed to paracetamol compared to the control group. See Figure 6.

Carin Johansson MASTER THESIS Uppsala University

There were no significant treatment effects of Win 55212-2 or the combination compared to the control group on synaptophysin level in the parietal cortex. See Figure 6.

Effects on tau protein levels in the neonatal Frontal cortex and Parietal cortex. There were no significant treatment effects of paracetamol, Win 55212-2 or the combination of the both on tau level in the frontal cortex (p= 0.6981) or the parietal cortex (p= 0.5219).

See Figure 5 and 6.

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Figure 5: Frontal cortex, the proteins CaMKII, GAP-43, GluR1, PSD95, synaptophysin and tau showed no significant changes in any of the three groups vs. control group. The data were subjected to one-way ANOVA with Newman- Keuls test. The height of the bars represents the mean value ± SD, expressed as percent of control.

Carin Johansson MASTER THESIS Uppsala University

Figure 6: Parietal cortex, the proteins CaMKII, GAP-43 and tau showed no significant changes in any of the three groups vs. control group. The data were subjected to one-way ANOVA with Newman-Keuls test. The height of the bars represents the mean value ± SD, expressed as percent of control. GluR1, PSD95 and synaptophysin showed significant changes in the parietal cortex of animals exposed to paracetamol compared to the control group. There were no significant changes of Win55212-2 or the combination compared to the control group on GluR1, PSD95 and synaptophysin level in the parietal cortex. (** p<0.05) Statistical difference on level of neuroproteins.

Carin Johansson MASTER THESIS Uppsala University

DISCUSSION The current study was conducted to investigate the mechanism of paracetamol developmental neurotoxicity, possibly mediated through activation of the CB1-receptor. We therefore investigated the protein expression of six important neuroproteins in mice, neonatally exposed to paracetamol, Win55212-2 or a combination of both.

We showed significant decreased protein expression levels for GluR1 in parietal cortex in mice neonatally exposed to paracetamol. Several studies have showed that the GluR1 plays a crucial role for neuronal plasticity and regulates, amongst many things, the synaptic connections between neurons (Hebb 1952, Bliss and Lømo 1973, Morris et al. 1986, Martin et al. 2000). The efficacy of synaptic neuro-transmission is crucial for connection of the brain regions and for memory and learning (Mayer and Westbrook 1987, Dingledine et al. 1988, Monaghan et al. 1989). GluR1 knockout mice has displayed alterations in the glutamate systems, which increased their locomotor activity (Viggiano 2008). Mice, exposed neonatally to paracetamol, also showed increased adult locomotor activity (Viberg et al. 2014). These studies together indicates that GluR1 is important for memory and learning and that the mice exposed neonatally to paracetamol show the same impairment of locomotor activity and memory deficits, as the GluR1 knockout mice. This may indicate that paracetamol induced down regulation of GluR1 during brain development leads to alteration of cognitive functions and memory.

We also showed significant decreased protein expression levels for PSD95 in parietal cortex in mice neonatally exposed to paracetamol. PSD95 is important in coupling the NMDA receptor to pathways that are associated with bidirectional synaptic plasticity and learning (Migaud et al. 1998). In the same study, PSD95-knockout mice displayed difficulty in remembering spatial recognition when exposed to the same maze several times, which indicate that the long-term potentiation (LTP) is affected (Migaud et al. 1998). Mice, neonatally exposed to paracetamol, have also shown altered behavior and learning deficits (Viberg et al. 2014). This, together with the result presented in this master thesis, suggest that the neurotoxic effect of paracetamol might be through reduction of PSD95 levels.

Furthermore, decreased protein levels for synaptophysin was shown in parietal cortex in mice neonatally exposed to paracetamol. A reduction in synaptophysin levels has previously been shown to be associated with decreased synaptic connectivity, eventual cell loss in the hippocampal region and subsequent functional deficit in synaptic transmission in mice (Sun et al. 2007). It has also been shown in behavioral tests on synaptophysin knockout mice that these mice have impairments in learning and memory, which can be indicated by reduces novelty recognition and spatial learning (Umathe et al. 2009). Paracetamol as well as other pharmaceuticals and environmental toxins, such as ketamine and highly brominated PBDEs, causing cognitive and memory deficits, have also shown alterations in synaptophysin levels (Viberg et al. 2008, Viberg et al. 2008, Viberg et al. 2014). These studies together show that synaptophysin is crucial for many features during development of the brain. The synaptophysin knockout mice and the mice, neonatally exposed to paracetamol display similar

Carin Johansson MASTER THESIS Uppsala University learning and memory deficits. The reduction in synaptophysin level might therefore explain, at least in part, the memory and learning deficits previously shown after neonatal paracetamol exposure.

Taken together, these studies show that mice having decreased levels of neuroproteins GluR1, PSD95 and synaptophysin have difficulties to perform in maze-test and also have memory deficits. This indicates that the memory and learning deficits shown after neonatal paracetamol administration might be caused by affecting these neuroproteins. Furthermore epidemiological studies have shown, as previously mentioned, that exposure to paracetamol during pregnancy can result in ADHD-like or HKD-like behavior in children (Brandlistuen et al. 2013, Liew et al. 2014). The animal and epidemiological studies show that administration of paracetamol during brain development may give rise to neurotoxic effects that could result in adverse outcomes in the offspring later in life.

CaMKII, GAP-43 and tau are also of importance for normal brain development. However after neonatally paracetamol exposure none of these neuroproteins were effected in this study. These neuroproteins have in other studies shown to be effected after exposure to other substances such as ketamine and PDBs, the mice have in these studies displayed adverse cognitive and behavioral deficits (Viberg et al. 2008, Viberg et al. 2008). These neuroproteins may therefore not be involved in the neurotoxic effect of paracetamol.

There were however no effect on neuroprotein expression in frontal cortex. Frontal cortex and parietal cortex have similar function, but differ in detail (Baddeley 1992, Funahashi and Kubota 1994, Culham 2002)). There are many possible explanations for why we do not see an effect in the frontal cortex but in parietal cortex after neonatal paracetamol exposure. For example, it is possible that development of these brain regions does not occur at the same time (Dobbing and Sands 1973, Giedd et al. 1999, Andersen 2003) and might therefore have different periods of vulnerability. The micro distribution of a substance may also differ between different brain regions, which have been shown for perfluoroktansulfonat (PFOS) (Greaves et al. 2013). It has also been shown that neuroprotein effects can differ between brain areas, for example between hippocampus and cortex (Viberg et al. 2008).

No significant effect was shown on protein levels in mice exposed to Win55212-2. Win55212-2 acts, as previously mentioned, as an endocannabinoid on the CB1-receptor (Felder et al. 1995). In several animal studies, done in pregnant rats (gestation day 5 to 20), Win55212-2 exposure has shown to give adverse cognitive and behavioral outcomes in the offspring (Maj et al. 2007, Ferraro et al. 2009, Saez et al. 2014). Mice neonatally exposed to paracetamol also display cognitive and memory deficits later in life (Viberg et al. 2014). We did however not see an effect on the protein levels in this exposure group, which indicates that Win55212-2 does not have the same neurotoxic mechanism as paracetamol.

We did not see a significant effect on the protein levels in mice exposed to the combination of paracetamol and Win55212-2 and we did not see an effect on the protein levels in mice exposed to

Carin Johansson MASTER THESIS Uppsala University

Win55212-2. However we did see an effect in the paracetamol exposure group. It could be that paracetamol and Win 55212-2 have a competitive effect on the binding site; Win55212-2 activates the CB1-receptor and hence hinders neurotoxicity of paracetamol. An example of competitive binding to a specific receptor is alpha-actins binding to the NMDA-receptor, alpha-actin competes with Ca2+/Calmodulin for binding to the NMDA receptor C-subunit (Wyszynski et al. 1997). It could also be that, when paracetamol is given together with Win55212-2, Win55212-2 acts as a protecting agent on the CB1-receptor. This could be why we do not see a neurotoxic effect in the combination exposure group. Furthermore, other proteins, which are not examined in this study, may have been affected by paracetamol exposure. Brain derived neurotropic factor (BDNF) is an example of a biomarker that could be used, and has previously been shown to be affected after paracetamol exposure (Viberg et al. 2014). Finally, the method used in this study may not be sensitive enough to detect an actual difference in protein levels. Taken together there are several possible reasons for the lack of efficacy of the combination exposure group, in this piece, we speculated on possible reasons for the given result.

CONCLUSION AND FUTURE STUDIES

Conclusion In conclusion, this master thesis shows that neonatal paracetamol administration causes significant down-regulation of three important neuroproteins synaptophysin, GluR1 and PSD-95 after paracetamol administration on PND 10. All of the proteins are crucial for correct brain development during the brain growth spurt and if not the right amount of these proteins are present during this vulnerable time period developmental impairment may occur. This master thesis, together with previous studies showing altered spontaneous behavior after paracetamol administration, indicate that altered protein levels may in part be responsible for the neurotoxicity recently shown as behavioral disturbances and cognitive impairments. Further studies on the possible mechanism are however warranted.

Future studies Due to lack of time the effect on protein levels in the hippocampus was excluded from this master thesis project. Hippocampus is associated with learning and memory and it has been stated that hippocampus is especially important in LTP. LTP is dependent on the NMDA-receptors and last for several hours (Bliss and Collingridge 1993). Therefore it would be of great importance to also perform protein assays on these proteins in this brain region.

Carin Johansson MASTER THESIS Uppsala University

ACKNOWLEDGEMENT I would like to thank, Henrik Viberg, Gaëtan Philippot, Iwa Lee, Stefan Hallgren, Sonja Buratovic and Per Eriksson for excellent help, discussions, support and commitment throughout the project. Also I would like to thank my parents for support throughout my education and my room buddy Andreas Eriksson, and of course all of the staff at the institution for a great last term at Uppsala University.

Carin Johansson MASTER THESIS Uppsala University

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