Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1919 Neurodevelopmental Consequences of Exposure to Paracetamol (Acetaminophen) and Related Drugs Experimental studies in mice GAËTAN PHILIPPOT ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0913-2 UPPSALA urn:nbn:se:uu:diva-407311 2020 Dissertation presented at Uppsala University to be publicly examined in Ekmansalen, Evolutionsbiologisk centrum, Norbyvägen 14-18, 752 36 Uppsala, Friday, 15 May 2020 at 13:26 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Vesna Jevtovic-Todorovic (Department of anesthesiology, University of Colorado). Abstract Philippot, G. 2020. Neurodevelopmental Consequences of Exposure to Paracetamol (Acetaminophen) and Related Drugs. Experimental studies in mice. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1919. 64 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0913-2. Paracetamol (acetaminophen) is the analgesic pharmaceutical most commonly used during pregnancy and early life. While therapeutic doses of paracetamol are considered harmless during these periods, recent findings in both humans and rodents suggest a link between developmental exposure to paracetamol and behavioral consequences later in life. Paracetamol has a known interaction with the cannabinoid receptor type 1 (CB1R) and the cyclooxygenase (COX) system; both interactions have the potential to induce developmental neurotoxicity (DNT). Central to this thesis is the use of the neonatal mouse, in which the potential DNT of paracetamol was examined after a single day’s exposure during a critical period of brain development called the brain growth spurt (BGS). This thesis investigates whether behavioral consequences can be induced by paracetamol exposure at different timepoints during the BGS and if male and female mice are equally affected. Further, it compares these effects with those of two other pharmaceuticals with analgesic properties: ibuprofen and Δ9-tetrahydrocannabinol (THC). These pharmaceuticals were included because both these drugs have pharmacodynamic similarities with paracetamol; THC, like paracetamol, interacts with the CB1R and ibuprofen, like paracetamol, interacts with the COX system. Paracetamol exposure on postnatal day (PND) 3 and 10 affected adult spontaneous behavior and habituation capability in both male and female mice. These periods are comparable, in terms of brain development, to the beginning of the third trimester and the time around birth, respectively, in humans. Exposure on PND 19, comparable to the development stage of a two-year-old human child, did not induce any adult behavioral changes. PND 10 exposure to THC, but not ibuprofen, affected adult spontaneous behavior and habituation. In addition, simultaneous exposure to a CB1R agonist enhanced the DNT of paracetamol. Interestingly, early-life exposure to both paracetamol and THC decreased transcript levels of genes encoding a receptor involved in neurogenesis and increased markers of oxidative stress. This may indicate that the two substances share common features in their respective mechanisms of DNT. This thesis provides new evidence from a human-relevant experimental design indicating that single-day exposure to paracetamol during the peak of the BGS is sufficient to affect adult spontaneous behavior, memory, learning, and cognitive function in mice. Although the high paracetamol use during pregnancy and early life is based on its advantages over other painkillers, the need for a balanced risk assessment based on the best professional judgement must be prioritized. Keywords: paracetamol (acetaminophen), developmental neurotoxicology, delta9- tetrahydrocannabinol (THC), ibuprofen, endocannabinoid system, neonatal mice, brain growth spurt, behavior Gaëtan Philippot, Department of Organismal Biology, Environmental toxicology, Norbyvägen 18A, Uppsala University, SE-752 36 Uppsala, Sweden. © Gaëtan Philippot 2020 ISSN 1651-6214 ISBN 978-91-513-0913-2 urn:nbn:se:uu:diva-407311 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-407311) To mom and sis In loving memory of Annie & Opa List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Philippot, G., Gordh, T., Fredriksson, A., Viberg, H. (2017) Adult neurobehavioral alterations in male and female mice fol- lowing developmental exposure to paracetamol (acetamino- phen): characterization of a critical period. J Appl Toxicol 37:1174–1181. II Philippot, G., Nyberg, F., Gordh, T., Fredriksson, A., Viberg, H. (2016) Short-term exposure and long-term consequences of neonatal exposure to delta(9)-tetrahydrocannabinol (THC) and ibuprofen in mice. Behav Brain Res 307:137–144. III Philippot, G., Hallgren, S., Gordh, T., Fredriksson, A., Fredriks- son, R., Viberg, H. (2018) A Cannabinoid Receptor Type 1 (CB1R) Agonist Enhances the Developmental Neurotoxicity of Acetaminophen (AAP). Toxicol Sci 166: 203–212. IV Philippot, G., Forsberg, E., Tahan, C., Viberg, H., Fredriksson, R. (2019) A Single δ9-Tetrahydrocannabinol (THC) Dose Dur- ing Brain Development Affects Markers of Neurotrophy, Oxida- tive Stress, and Apoptosis. Front Pharmacol 10:1156. V Philippot, G., Mhajar, Y., Viberg, H., Buratovic, S., Fredriksson, R. Paracetamol (acetaminophen) and its effect on the developing mouse brain: short-term oxidative response and long-term effects on memory, learning and cognitive flexibility. In manuscript. Reprints were made with permission from the respective publishers. Additional Publications Philippot, G., Stenerlöw, B., Fredriksson, A., Sundell-Bergman, S., Eriksson, P., Buratovic, S. (2019) Developmental effects of neonatal fractionated co- exposure to low-dose gamma radiation and paraquat on behaviour in adult mice. J Appl Toxicol. 9:582–589. Contents Introduction ................................................................................................... 11 Evaluated substances ................................................................................ 12 Paracetamol (acetaminophen) .............................................................. 12 Ibuprofen ............................................................................................. 13 Δ9-tetrahydrocannabinol (THC)........................................................... 14 Brain development ................................................................................... 15 A vulnerable period during brain development: the brain growth spurt ..................................................................................................... 15 Endocannabinoid system ..................................................................... 17 Brain-derived neurotrophic factor and its receptor tropomyosin receptor kinase B ................................................................................. 18 Aims .............................................................................................................. 19 Specific aims ............................................................................................ 19 Materials and methods .................................................................................. 20 Animals .................................................................................................... 20 Doses ........................................................................................................ 20 Behavioral tests ........................................................................................ 21 Spontaneous motor activity and habituation in a novel home environment ......................................................................................... 21 Morris water maze ............................................................................... 23 Biochemical analyses ............................................................................... 24 Quantitative polymerase chain reaction (qPCR) ................................. 24 Slot blot ................................................................................................ 25 Enzyme-linked immunosorbent assay (ELISA) .................................. 25 Results and discussion .................................................................................. 26 Adult behavior assessment following neonatal exposures to paracetamol, THC, and ibuprofen ............................................................ 27 Paracetamol ......................................................................................... 27 THC and ibuprofen .............................................................................. 32 Effect of co-exposure to the CB1R agonist WIN on paracetamol- induced DNT ....................................................................................... 34 Biochemical assessment following neonatal exposure to paracetamol and THC ................................................................................................... 37 Paracetamol as a potential developmental neurotoxicant ......................... 40 Considerations for the evaluation of developmental neurotoxic data .. 40 Parallels between experimental studies and existing epidemiology .... 42 Concluding remarks and future perspectives ................................................ 44 Populärvetenskaplig sammanfattning ........................................................... 46 Acknowledgment .........................................................................................