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. 1 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. 2 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 3 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 anti- nociceptive behaviour as adults (Viberg et al. 2014). 4 Carin Johansson MASTER THESIS Uppsala University 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. 5 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