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 ...... 49 References ...... 51 Abbreviations
ADHD Attention deficit hyperactivity disorder AKT Protein kinase B AM404 N-arachidonoylaminophenol AOP Adverse outcome pathway ASD Autism spectrum disorder BAX Bcl-2-like protein 4 BBB Blood brain barrier BDNF Brain-derived neurotrophic factor BGS Brain growth spurt BSA Body surface area CB1R Cannabinoid receptor type 1 CNS Central nervous system COX Cyclooxygenase DNT Developmental neurotoxicity ECS Endocannabinoid system ELISA Enzyme-linked immunosorbent assay FAAH Fatty acid amide hydrolase HED Human equivalent dose IR Infrared MAPK/ERK Mitogen-activated protein kinase/extracellular signal–regulated kinase mTOR Mammalian target of rapamycin MWM Morris water maze NAPQI N-acetyl-p-benzoquinone imine NMRI Naval Medical Research Institute NSAID Nonsteroidal anti-inflammatory drugs PI3K Phosphoinositide-3 kinase PND Postnatal day PRAC Pharmacovigilance Risk Assessment Committee qPCR Quantitative polymerase chain reaction THC Δ9-tetrahydrocannabinol TRKB Tropomyosin receptor kinase B TRPV1 Transient receptor potential vanilloid 1 WIN WIN 55,212-2
Introduction
During brain development, a small layer of cells must develop into a complex system consisting of billions of precisely organized cells, within a tightly con- trolled time frame. This can make the developing brain more susceptible to toxic insults than the adult brain. Metaphorically, the developing brain can be regarded as a car – the faster it goes, the greater the damage if you crash. Even though the brain is one of the most well-protected systems of the body, against physical damage by the cranium and against chemical insults by the blood brain barrier (BBB), there are substances that we are exposed to with the abil- ity to reach the brain and affect brain development. Exposure to such sub- stances can be involuntary, for example when they are in the form of environ- mental contaminants. However, there are other substances with the potential to affect brain development that we deliberately expose ourselves or our chil- dren to. This thesis focuses on one such group, namely the analgesics. Expo- sure of pregnant women, neonates (including premature babies), and young children to analgesics is common. Despite this, there is often a lack of knowledge regarding the safety of these drugs, since pregnant women and young children are traditionally left out of clinical trials. This thesis primarily investigated the developmental effects and conse- quences of neonatal exposure to paracetamol (acetaminophen). In addition, two other substances were investigated: ibuprofen and Δ9-tetrahydrocanna- binol (THC). Both ibuprofen and THC are of clinical relevance, as they are used during pregnancy; they also have mechanistic features similar to those of paracetamol and can therefore be used from a comparative perspective. Both paracetamol and ibuprofen are well-established over-the-counter analgesics, while THC is mainly known as the psychoactive ingredient in cannabis and as an illegal drug; however, more and more attention is directed at its medicinal properties. In parallel, various formulations containing THC have been legal- ized around the world. The neurotoxic effects of these substances on brain development were investigated in mice during a defined period of neonatal brain development called the brain growth spurt (BGS).
11 Evaluated substances Paracetamol (acetaminophen) Paracetamol, also known as acetaminophen (Fig. 1), is a widely used non- prescription drug with antipyretic and analgesic properties and forms part of the initial step of the World Health Organization’s analgesia ladder [1]. Mar- keted internationally in the 1950s as an analgesic replacement for the ne- phrotoxic phenacetin [2], paracetamol use in children soon increased. By 1985, it had almost completely replaced aspirin, which was avoided because of concerns about the risk of Reye’s syndrome [3, 4]. Unlike aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), paracetamol is considered to possess very weak anti-inflammatory properties and does not produce gas- trointestinal damage or cardiorenal effects [2]. Furthermore, paracetamol ex- hibits poor anti-platelet actions, in contrast to most NSAIDs [5]. Taken to- gether, this makes paracetamol, at therapeutic doses, superior to NSAIDs re- garding safety in treatment of pain and fever, especially during pregnancy, in toddlers/children and patients with concomitant cardiovascular or gastrointes- tinal diseases.
Figure 1. Molecular structure of paracetamol.
Paracetamol intake during pregnancy is common, reaching prevalence rates up to 49% and 61% in Northern America and Northern/Western Europe, re- spectively [6]. A high paracetamol prevalence is also observed during early life, with the majority of neonates and toddlers having been medicated with paracetamol [7, 8]. Paracetamol reaches the fetal or neonatal brain across the placenta [9] and BBB [10] and might therefore affect brain development. Like the NSAIDs, paracetamol has an effect on prostaglandins through in- teraction with the cyclooxygenase (COX) system [11]. Despite its similarities with the NSAIDs (i.e., its antipyretic/analgesic actions and its effect on the COX system), the complete mode of action of paracetamol is not fully known. Interestingly, paracetamol undergoes a metabolic transformation to form the bioactive N-acylphenolamine (AM404) in the central nervous system (CNS). Paracetamol is first metabolized in the liver to form p-aminophenol and sub- sequently conjugated with arachidonic acid to form AM404. This is catalyzed by fatty acid amide hydrolase (FAAH) in the nervous system (Fig. 2) [12].
12 Figure 2. A simplified illustration of the formation of the paracetamol metabolite AM404 and its activity in the CNS (based on information from a review by Bertolini and colleagues [2]).
It has been shown that AM404 interacts with the cannabinoid receptor type 1 receptor (CB1R) (directly and indirectly) [13, 14] as well as the transient re- ceptor potential vanilloid 1 (TRPV1) (Fig. 2) [15, 16]. In recent years, maternal intake of paracetamol has been linked to adverse neurodevelopmental outcomes later in life such as attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), lower IQ, and language delay [17-26]. The unclear mechanism of action, the fact that it is the first-line treatment against pain and fever during pregnancy and early life, and the accumulating evidence of its developmental effects warrant fur- ther investigations into the neurodevelopmental effects of paracetamol.
Ibuprofen Ibuprofen (Fig. 3), belonging to the NSAIDs, is a non-selective COX inhibitor with analgesic, antipyretic, and anti-inflammatory activity [27]. It is also one of the over-the-counter drugs for treating pain and fever that is most frequently used during pregnancy and early life [28]. Ibuprofen acts on the COX system [27], thereby affecting prostaglandin synthesis. Interaction with COX-2 causes its therapeutic effects. COX-2 is also involved in important brain ac-
13 tivities, such as synaptic activity and plasticity, hippocampal long-term poten- tiation, and maturation of the brain [29, 30]. Like paracetamol, ibuprofen crosses the BBB [31].
Figure 3. Molecular structure of ibuprofen.
In the epidemiology studies of developmental exposure to paracetamol, ibu- profen was ruled out as a confounding cause for the observed neurotoxic ef- fects [18]. It is nevertheless important to evaluate the potential developmental neurotoxicity (DNT) of ibuprofen (1), since it is used during pregnancy and early life, and (2) to compare the potential effects with those of other analge- sics, e.g., paracetamol.
Δ9-tetrahydrocannabinol (THC) Being one of the most used illegal substances, THC (Fig. 4) has over the last years garnered increased attention due to its medical properties, together with a growing opinion that this drug is relatively harmless. Among pregnant women (US population), the prevalence of past-year cannabis use has in- creased over the last decade, reaching 12% in 2014 [32]. While the use of other illicit drugs usually decreases during pregnancy, cannabis use often per- sists throughout pregnancy [33]. Use of medical cannabis also occurs in the pregnant population, usually as an anti-emetic treatment for normal nau- sea/vomiting (prevalence 50–90%) or the more severe and sometimes life- threatening condition hyperemesis gravidarum (prevalence 1–2%) [34]. THC is a highly hydrophobic substance and crosses both the placenta [35] and the BBB [36], and – after maternal consumption – is present in milk of breast- feeding mothers [37]. In the brain, THC acts on the CB1R [38-40].
Figure 4. Molecular structure of THC.
14 There is already a large body of evidence concerning the potential cognitive and affective side effects associated with cannabis exposure during brain de- velopment. In animal studies, pre- and perinatal cannabinoid exposures have been shown to affect locomotor activity, memory, social interaction, and emo- tional functions [41-47]. Following developmental exposure to cannabinoids in humans, similar effects have been observed: altered cognition and executive functions, as well as higher levels of depression and anxiety during adoles- cence [42, 48-51]. It has also been shown that activation of CB1R, by both exogenous and endogenous ligands, inhibited the formation of new synapses in vitro [52]. However, it is obvious that ligands of the endocannabinoid system (ECS) also have therapeutic potential, as indicated by the increasing ambition to le- galize the recreational use of cannabis and, perhaps mainly, the current devel- opment of drugs that alter endocannabinoid signaling. Investigating the po- tency of THC as a neurotoxic drug during development is therefore important, especially when the use of CB1R agonists is likely to increase in the future.
Brain development A vulnerable period during brain development: the brain growth spurt The developing brain is inherently much more susceptible to injury caused by toxic agents than the adult brain. During mammalian brain development, there are many critical periods of increased vulnerability; these periods span from early gestation to adolescence and are often characterized by sensitivity to xe- nobiotic insults [53, 54]. One of these is the BGS [55]. In humans, this period occurs during the last trimester of pregnancy and continues up to approxi- mately 2 years of age [55]. It is characterized by an increase in brain weight and a series of rapid and fundamental changes, including maturation of axonal and dendritic growth, glial multiplication, myelination, and neurochemical processes [55-57]. The BGS also exists in mice; however, the corresponding period is mainly postnatal. It starts at birth and continues for the next 3–4 weeks, peaking around postnatal day (PND) 10 (Fig. 5) [55].
15 Figure 5. Brain growth rate trajectories during the BGS in mice and humans. Adapted from Davison and Dobbing [55].
This is also a period when many new motoric and sensory abilities are ac- quired [58] and when spontaneous motor behavior peaks [59]. Comparing hu- mans and mice, the time scale and occurrence relative to birth differ, but the chronology of key events is similar [60]. In terms of these events, PNDs 3, 10 and 20 in mice are comparable to week 32 of gestation, the time around birth, and 2 years of age in humans, respectively (Fig. 6) [61]. Exposure to xenobi- otics during the BGS can lead to irreversible changes in adult brain function, with a critical window around PND 10 having been identified in mice [62, 63]. The majority of DNT studies are focused on in utero exposure, and do not take into account the very important period of rapid brain development (i.e., the BGS) occurring postnatally in research animals (and perinatally in humans). The mouse neonate is a good model for pinpointing different human developmental stages.
16 Figure 6. Timeline for neurodevelopment in mice and humans during the critical pe- riod of the BGS. Key developmental processes (thickness schematically illustrates relative magnitude and/or intensity) in humans during different periods of the BGS (adapted and inspired by Semple and colleagues [61]).
Endocannabinoid system The endocannabinoid system (ECS) is present from the earliest stages of brain development [64]. This system consists (mainly) of the CB1R and the canna- binoid receptor type 2 receptor [39, 65, 66]. The two major endogenous lig- ands are N-arachidonyl ethanolamine (anandamide) [65] and 2-arachidonoyl glycerol (2-AG) [67]. There are five main enzymes involved in the ECS which are responsible for both biosynthesis and inactivation of the ligands. One of these enzymes is FAAH [68-71]. The ECS is an important signaling system during brain development and is involved in both structural and functional aspects of neural development [72- 75]. The major receptor of the ECS in the brain is the CB1R, which is one of the most highly expressed inhibitory G-protein coupled receptors of the brain [66, 76]. The CB1R is localized in axon terminals of GABAergic, glutama- tergic, serotonergic, noradrenergic, and dopaminergic neurons of the brain [77-82], and suppresses neurotransmitter release when activated [80]. The mo- lecular logic of endocannabinoid signaling is retrograde signaling, from post- synapse to pre-synapse, where CB1R activation occurs at the pre-synapse. During brain development, the ECS is also involved in progenitor cell pro- liferation and differentiation, neural migration, synaptogenesis, and correct neurite and axonal outgrowth [72-74, 83, 84]. Although the Cb1r mRNA lev- els remain stable from early postnatal period through adolescence to adulthood [85], receptor binding seems to reach a peak in the second week of postnatal
17 life in mice [85, 86]. The ECS is primarily known as the target system of ille- gal/recreational use of cannabis, but has over the last decade emerged as an interesting target for pharmacotherapy [87].
Brain-derived neurotrophic factor and its receptor tropomyosin receptor kinase B Owing to their diverse roles in normal brain development and adult plasticity, growth factors have become integral components of many hypotheses on early life xenobiotic exposures and adverse effects later in life. A fundamental group of growth factors, referred to as the neurotrophins, is expressed within the brain: nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 [88]. The neurotrophins are instrumental in many neurodevelopmental processes and have been under scrutiny in re- search concerning psychiatric disorders [88, 89]. Much of the research has focused on BDNF, as this molecule is differentially expressed across the brain, following a developmental trajectory. BDNF is a high affinity ligand to tro- pomyosin receptor kinase B (TRKB) [90, 91]. Trkb mRNA is expressed in many regions of the brain in neonatal mice, with the strongest expression in the thalamus and cerebral cortex. However, the hippocampus is the only re- gion where Trkb mRNA expression increases during postnatal development [92]. It has been shown that BDNF is involved in long-term memory [93, 94]. In the hippocampus, BDNF plays an important role in the regulation of hippo- campal neurogenesis [95, 96] and decreased BDNF activity in this brain re- gion leads to hippocampal-dependent memory deficits [97, 98]. Interestingly, the ECS transactivates TRKB during brain development [83]. BDNF levels are decreased in the brains of mice lacking the CB1R [99], whereas activation of CB1R increases BNDF levels in rodents [100], further indicating a link between BDNF/TRKB and the ECS.
18 Aims
The overall aim of this thesis was two-fold. Primarily, this work investigates the neurotoxicity of developmental exposure to the commonly used analgesic paracetamol. Considering the widespread exposure to this analgesic drug dur- ing brain development, it is important to increase the understanding of possi- ble adverse neurodevelopmental outcomes later in life. Secondarily, this work investigates the neurotoxic effects of developmental exposure to two other mechanistically related drugs: ibuprofen and THC. To address these objec- tives, a neonatal animal model was used to simulate both pre- and perinatal exposure that is relevant to humans.
Specific aims • To investigate whether induction of the DNT effect of paracetamol de- pends on when, during a critical period of neonatal brain development in mice, paracetamol exposure occurs.
• To investigate whether male and female mice are equally affected by ne- onatal paracetamol exposure.
• To investigate whether co-exposure to CB1R agonist WIN 55,212-2 (WIN) increases the susceptibility to paracetamol neurotoxicity in mice.
• To investigate and compare the neurodevelopmental effects of paraceta- mol to those of THC and ibuprofen on adult behavior in mice.
• To compare the biochemical effects following neonatal exposure to para- cetamol and THC, to get insights into the developmental neurotoxicolog- ical mechanisms of these drugs.
19 Materials and methods
In this thesis, a number of different materials and methods were used to inves- tigate the effects of neonatal exposure to different substances in mice. In order to investigate biochemical effects of neonatal exposures, both gene expression and protein expression were measured in the neonatal mouse brain. Adult mice were used to study behavioral effects of neonatal exposure. A more detailed description of the materials and methods used in the respective studies is pre- sented in the individual papers. The following section describes the essentials of the materials and methods used in these papers.
Animals Experiments were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), after approval from the local ethical committee (Uppsala University and Agricultural Research Council). Pregnant Naval Medical Research Institute (NMRI) mice were purchased from Scanbur, Sollentuna, Sweden, and maintained individually in plastic cages. The pregnant NMRI mice were checked for birth once daily, and after giving birth, the litters were adjusted to 10–12 pups (containing both sexes) by euthanizing excess pups. In general, mice were exposed on PND 10 (except in Paper I, where some mice were exposed on PND 3 or PND 19) subcutane- ously in the scruff. In cases where biochemical measurements were done on neonatal mice, animals from different litters were randomly exposed. In cases where the animals were examined in adulthood, the entire litter was exposed.
Doses A previous study in mice identified that PND 10 exposure to paracetamol at 30 + 30 mg/kg (4 h apart) affected adult cognitive function, whereas 30 mg/kg as a single dose did not [101]. Using the body surface area (BSA) normaliza- tion method [102], this dose roughly corresponds to a human equivalent dose (HED) of 4.9 mg/kg. In brief, the calculation is based on each species having a different converting factor (Km), expressed as body weight (kg) divided by BSA (m2), which is used to convert the dose (mg/kg) in one species to dose
20 (mg/kg) in another species. To obtain a HED, the following equation can be used: mg mg Animal HED =Animal dose × kg kg Human
These dose comparisons were made using the guidelines of the US Depart- ment of Health and Human Services, Food and Drug Administration (http://www.fda.gov/downloads/Drugs/Guidance/UCM078932.pdf). Recom- mended doses of paracetamol in neonates and toddlers (and even preterm ne- onates) are 7.5–15 mg/kg up to 4 times a day [103].
Behavioral tests Spontaneous motor activity and habituation in a novel home environment The spontaneous behavior test is used for studies on exploration, habituation, and locomotor activity. This test explores the ability to merge sensory input (e.g., a new home cage) into motor output (e.g., activity). Activity is expected to decrease as the stimulus becomes familiar. This process is called habitua- tion and is one of the most basic forms of learning and memory. Habituation is a non-associative form of learning, in which both the frontal cortex and hippocampus play critical roles [104]. Disruption of normal habit- uation capability is considered a disturbance in cognitive function [105-107]. Aberrations in normal behavior in a new home environment can be manifested as reduced explorative initiatives and/or subsequent failure to reduce activity.
Figure 7. Conceptual sketch of a mouse in a home cage during recording of sponta- neous behavior.
In this thesis, two equipments to measure spontaneous behavior were used: one, the Rat-o-Matic, had been in use for a long time before this work began,
21 and was used in Papers I, II, and III, and the other, the Infrared Actimeter, came into use during the course of this work and was used in Paper V. While they differ in some aspects, the principle is the same in these two methods, which both explore animals’ behavior in a home environment (Fig. 7). Thus, both methods give information on the animals’ spontaneous behavior and ha- bituation capability.
Rat-O-Matic This method was used to evaluate the spontaneous behavior of an animal when introduced to a new home cage. Mice were subjected to 60 min spontaneous behavior testing in an automated device consisting of 12 cages (40×25×15 cm) placed within two series of infra-red beams (at two different heights, low and high level) (Rat-O-Matic, ADEA Electronic AB, Uppsala, Sweden) as de- scribed in more detail by Fredriksson et al. [108]. Recordings were made of three different variables: locomotion, rearing, and total activity.
• Locomotion: Counting took place when the mouse moved horizontally through the low-level grid of infra-red beams.
• Rearing: Registered when the mouse moved vertically, thereby interrupt- ing a single high-level beam, at a rate of four counts per second. The num- ber of counts obtained was proportional to the time spent rearing.
• Total activity: All types of vibrations caused by the mice, i.e., those caused by animal movements (e.g., locomotion and rearing), shaking (tremors), and grooming, were registered by a pickup (a needle mounted on a lever with a counterweight), connected to the edge of the test cage.
22 Figure 8. Schematic histogram of normal activity in a novel home cage, encompass- ing locomotion, rearing, and total activity, by time spent in cage.
In the assessment of the motor activity from these three variables, the activity during the 60 min of recording was sub-divided into three 20 min intervals. A schematic histogram of normal activity in a novel home cage during 60 min sub-divided into three 20 min intervals is shown in Fig. 8, in agreement with results from control animals in numerous previous studies [62, 101].
Infrared (IR) Actimeter The Panlab Infrared (IR) Actimeter (Actitrack, Panlab, Barcelona, Spain) al- lows the study of spontaneous locomotor activity. The system is composed of a two-dimensional (X and Y axes) square frame. Each frame has 16 x 16 in- frared beams for subject detection, with activities in four cages (40×25×15 cm) analyzed simultaneously. In contrast to the Rat-O-Matic equipment, the Actimeter equipment allowed recordings of the distance travelled. This varia- ble was used to assess spontaneous behavior and habituation in the new home cage. To reach baseline activity, mice needed a total of 90 min to habituate, where the activity was sub-divided into three time-intervals consisting of 30 min each.
Morris water maze The Morris water maze (MWM) is a method used in neuroscience to study memory and spatial learning. The test was developed in 1981 by Richard G. Morris [109, 110]. It has become one of the most common tools in neurosci- ence and can be used to investigate cognitive functions (such as memory and
23 learning) [111] and cognitive flexibility (adjustment from older to newer thinking) [112]. An animal is placed in an open water container, with its head at the edge to avoid method errors. A platform is hidden just below the water surface (Fig. 9). Visual cues (e.g., different shapes or colors) are placed near the maze, clearly visible to the test subject. The animal then has to navigate to locate the submerged platform. There are many methodological variations of the MWM task for different applications.
Figure 9. Conceptual sketch of a mouse in the MWM during behavior recordings.
To evaluate the effects of neonatal exposure to paracetamol on both cognitive function and flexibility in adulthood, mice were first tested for spatial learning abilities for four consecutive days with the platform in a fixed position. On the fifth day, the platform was re-located to test the mice’s re-learning abilities. The mice were given five trials/day.
Biochemical analyses Quantitative polymerase chain reaction (qPCR) To quantify the transcription levels of specific genes, real-time quantitative polymerase chain reaction (qPCR) was used. The concept of this method is to (1) extract RNA from the target tissue, (2) transcribe mRNA into cDNA, and then (3) amplify cDNA in a cyclic process of denaturation, annealing (of gene- specific primer pairs), and elongation. Thus, a great number of DNA segments is produced, making it possible to semi-quantitatively compare the amount of
24 transcripts of a specific gene between, e.g., control animals and exposed ani- mals.
Slot blot Representing a simplification of the Western blot method, the Slot blot method is used to semi-quantitatively detect proteins. The technique offers significant savings in time, since gel electrophoresis is not needed; however, the speci- ficity of antibodies for the protein intended has to be evaluated with Western blot procedure.
Enzyme-linked immunosorbent assay (ELISA) Another way of quantifying a specific protein is to use an enzyme-linked im- munosorbent assay (ELISA). In brief, (sandwich) ELISA employs an affinity tag-labeled capture antibody and a reporter-conjugated detector antibody, which “captures” the specific protein in solution, i.e., the protein is analyzed in its natural state. This entire complex (capture antibody/analyte/detector an- tibody) is then immobilized thanks to the immune affinity of anti-tag antibod- ies coating the wells in the experimental set-up.
25 Results and discussion
In this thesis, the DNTs of commonly used drugs were evaluated using a mouse model. At the beginning of the work, it was important to identify whether neonatal exposure to the specific drugs affected the adult phenotype. In other words, it was a priority to first answer the question of whether expo- sure of the selected drugs, at relevant doses, had any effect on the behavior of mice later in life. When, and if, a behavioral effect was identified in adulthood, the focus of the work shifted to trying to identify which biochemical mecha- nisms that were potentially responsible. These questions were evaluated in five different papers (Table 1).
Table 1. Overview of DNT studies. Paper Substance(s) Exposure Tissue Adult behavioral Biochemical day(s) endpoint(s) endpoint(s) I paracetamol PNDs 3, 10, Spontaneous or 19 behavior Habituation II THC PND 10 Spontaneous ibuprofen behavior Habituation III paracetamol PND 10 Frontal cortex Spontaneous Gene WIN 55,212-2 Parietal cortex behavior expression Hippocampus Habituation Protein expression IV THC PND 10 Frontal cortex Gene Parietal cortex expression Hippocampus Protein expression V paracetamol PND 10 Hippocampus Memory, learning Gene Cognitive expression flexibility Spontaneous behavior
26 Adult behavior assessment following neonatal exposures to paracetamol, THC, and ibuprofen Paracetamol In Paper I, an investigation of the critical period for the DNT of paracetamol was performed. A previous study had shown that the peak of the BGS was a time sensitive to paracetamol (30 + 30 mg/kg, 4 h apart) exposure in mice; in that study, PND 10 exposure in male mice induced altered spontaneous be- havior, reduced working memory, and affected paracetamol-induced anxio- lytic and analgesic effects in adulthood. The aim in Paper I was to investigate whether the induction of these effects depended on when the exposure oc- curred during the BGS, and if males and females were affected differently. Therefore, male and female mice were exposed to paracetamol (30 + 30 mg/kg, 4 h apart) on either PND 3, 10, or 19.
Figure 10. Spontaneous behavior (only locomotion shown) in adult male and female mice after neonatal exposure to two doses of either vehicle (control) or 30 mg para- cetamol/kg, 4 h apart, on PND 10. The statistical differences are indicated as (**) if they are significant vs. controls, p ≤ 0.01. The heights of the bars represent the means ± SD values of 10 animals. For statistical methods, see Paper I.
Paracetamol exposure on PND 10 altered adult spontaneous behavior and ha- bituation patterns (Fig. 10) in both males and females. The findings in males were in good agreement with previous findings [101]. In terms of key devel- opmental processes, PND 10 corresponds to the time around birth in humans (Fig. 6) [61]. It was also shown that paracetamol exposure on PND 3 affected adult spon- taneous behavior and altered habituation rates. Neurodevelopmental processes around PND 3 in mice are comparable to those occurring in the beginning of
27 the third trimester in humans (Fig. 11) [61]. These findings are in line with existing epidemiology, where an association between in utero exposure to pa- racetamol and adverse behavioral outcomes later in life has been reported [18- 26, 113]. Interestingly, in one of the epidemiological studies, there was a stronger association between paracetamol exposure during the third trimester and adverse behavioral outcome than for exposures during the first or the sec- ond trimester [21]. However, exposure to paracetamol on PND 19 did not af- fect adult behavior in mice (Fig. 11). In terms of brain development, PND 19 in mice is comparable to age 2 years in humans [61]. Using the same animal model, it has previously been shown that both flame retardants and irradiation induce similar effects when exposure occurs early during the BGS (PND 3) [114, 115].
Figure 11. Neurodevelopmental timelines in mice and humans. The illustration shows when the mice received paracetamol, whether this exposure affected their adult spontaneous behavior and habituation (neurotoxic effect indicated in green; lack of neurotoxic effect indicated by a red cross), and what neurodevelopmental pe- riod this time point may correspond to in humans. Information on corresponding time periods between mice and humans came from Semple and colleagues [61].
The behavioral alterations observed in these studies (i.e., the home cage spon- taneous behavior changes) were manifested as a time-dependent change in ac- tivity. In brief, the animals neonatally exposed to paracetamol displayed lower activity compared with control animals during the first 20 min of recording. Then, during the last 20 min of recording, these animals displayed hyperac- tivity compared with control animals. These behavioral alterations were noted for both horizontal (locomotor activity) and vertical movements (rearing). Ex- ploration in the horizontal plane, defined as a degree of movement from one location to the next, is a commonly used variable assessing habituation to a new environment. The exploration in the vertical plane, also a commonly used marker of the response to environmental novelty, is slightly more reliable in the assessment of hippocampal-dependent learning and memory [116]. There are both similarities and differences between the vertical and horizontal move- ments. For instance, both variables can be used to assess spontaneous behavior and habituation, and thus a significant disruption of both variables gives a more robust indication of habituation disruption than an effect on only one variable. However, rearing has some advantages over locomotion in assess- ments of spatial learning, as the mouse gains information of a third dimension
28 while rearing, and it might therefore be considered a response to more distal cues [116]. The potential difference in vulnerability between males and females was evaluated in Paper I following neonatal paracetamol exposure (on either PND 3, 10, or 19). It is known that the developing mammalian brain is sexually dimorphic. For example, one of the target brain regions studied in this thesis, the hippocampus, is considered sexually dimorphic [117-119]. Furthermore, it has recently been shown that there is a sex-dependent effect on language development in humans following developmental exposure to paracetamol, as in utero exposure to paracetamol was associated with language delay in girls, but not in boys [17]. Both animal and human studies have found that parace- tamol has anti-androgenic properties, hence indicating endocrine disturbances [120-124]. Also, in the pregnant mouse, paracetamol exposure decreases pro- gesterone levels [113]. However, as shown in Paper I, there were no observed differences between sexes in the neurotoxic effects induced on either PND 3 or 10. Potential sex differences following single-day exposure to both envi- ronmental contaminants and irradiation have previously been investigated and sex-related differences in adult cognitive function were absent [114, 125, 126], in line with the findings presented herein. In Paper V, the aim was to evaluate the potential impact of PND 10 para- cetamol exposure on adult memory, learning, and cognitive flexibility. For this reason, the MWM was used. By having the submerged platform fixed at the same location for four consecutive days, the animals’ learning and memory abilities could be assessed. Then, by having the submerged platform relocated on the fifth day, an evaluation on the animals’ cognitive flexibility was made. In short, cognitive flexibility refers to the brain’s ability to transition from thinking about one concept to another, or perhaps more accurately, replacing an old paradigm with a new one. Control mice improved their latency to find the platform during the spatial acquisition period, i.e., during the first four days (trail 1–20). However, mice that were exposed to paracetamol on PND 10 had significantly increased latency to find the submerged platform on day 3 and day 4 compared with controls (Fig. 12). These findings indicate that mice neonatally exposed to paracetamol have decreased ability in spatial learning and memory compared with control animals. A similar impairment in spatial learning and memory during the acquisition period has been ob- served following co-exposure to ketamine and irradiation on PND 10 [127]. As presented in Paper I, neonatal exposure to paracetamol affected habitua- tion patterns. The findings from the MWM in Paper V are in accordance with these findings, since habituation is considered one of the simplest forms of learning, and disruption of normal habituation capability, like disruption of memory and learning, is considered a disturbance in cognitive function [105- 107]. Following re-location of the submerged platform on the fifth day, mice were forced to re-learn a new paradigm (the platform had moved). Here, mice
29 neonatally exposed to paracetamol had an increased latency (over the five tri- als) to find the relocated platform compared with control animals (Fig. 12). In other words, mice exposed to paracetamol during brain development had a decreased ability to re-learn a new task previously learned, thus indicating de- creased cognitive flexibility. According to these findings, it seems that both learning in a constant environment and adapting flexibly to a changing one are affected by developmental exposure to paracetamol.
Figure 12. Swim maze performance in adult male mice exposed to paracetamol (30 + 30 mg/kg, 4 h apart) on PND 10. Latencies (s) to locate the platform were meas- ured during the four-day acquisition period and during the re-learning period on the fifth day. Statistical differences are indicated as * if p ≤ 0.05 and ** if p ≤ 0.01. Bars represent means ± SD of 10–12 mice. For statistical methods, see Paper II.
The effects of developmental exposure to paracetamol on adult behavior in rats have been evaluated by another research group. In short, they showed that in utero and early life exposure to paracetamol has effects on both social be- havior, spatial memory, motor performance, and exploration [128, 129]. How- ever, these findings are somewhat puzzling, as rats that received the lower doses of paracetamol displayed a greater impact compared with rats that re- ceived higher doses. In the same study, there were no observed effects during the acquisition period in the MWM, in contrast to findings presented here. One possible explanation for these differences is the different exposure schemes used. In their study, exposure occurred continuously for several weeks (daily dose: 5–15 mg/kg×day), while exposure occurred on a single day in our study.
30 However, in our study, the exposure occurred during a defined critical period of brain development on PND 10, and with a slightly higher dose (60 mg/kg×day). This could indicate that there may be a certain paracetamol plasma level required to induce major behavioral changes later in life. Another – and perhaps more likely – reason for the differing results is that two different animal models were used. A general rule of thumb is that smaller animals have faster metabolisms. However, there are some exceptions: rats have a faster metabolism than both mice and humans as regards paracetamol [130]. As a result, humans are more similar to mice than rats when it comes to paracetamol pharmacokinetics. This may be another explanation for the observed differ- ences. The developmental effects of paracetamol on social behavior are also in- teresting, as endocannabinoids, including anandamide, mediate social behav- ior in rodents [131]. Developmental effects on social behavior have been ob- served following an acute dose of paracetamol (100 mg/kg), which in turn also increased endocannabinoid levels in the frontal cortex [132]. Acute exposure to a high dose paracetamol (~300 mg/kg) in adult mice affected spatial memory [133]. However, a much lower acute dose (~15 mg/kg) improved spatial learning in the same study. It seems that both acute exposures in adult- hood and developmental exposures to paracetamol have an impact on memory, learning, social behaviors, and cognitive function. However, there are some uncertainties regarding which doses and timings of these that induce these changes. In addition to the MWM study in Paper V, spontaneous behavior was rec- orded again, using a new equipment (see Materials and methods for further details on the differences between the two home cage spontaneous behavior assessments). Using the IR Actimeter, behavioral assessment was done based on the distance travelled instead of the number of beam breaks. During the last 30 min of behavioral recordings, the finding that mice neonatally exposed to paracetamol displayed hyperactivity compared with controls was replicated – in this case manifested as a greater distance travelled compared with control mice. On the other hand, the hypoactivity previously observed during the first time period (Papers I and II) was absent, as paracetamol animals and controls had travelled equal distances. The fact that the mice had a decreased explora- tion activity during the first time period is somewhat puzzling. This decreased activity could hypothetically be due to freezing behaviors, as a response to stress, fear, and anxiety. However, a potential paracetamol-induced increase in sensitivity to fear and anxiety seems less likely, owing to the previous ob- servation that these animals did not spend less time in the open arms of the elevated plus maze compared with control animals [101]. Nonetheless, stress is a delicate factor to consider when assessing behavior. The level of stress has an impact on exploratory behavior, with high levels of stress suppressing motor activity. However, stress up to moderate levels is expected to be re- quired to motivate exploration in the first place [116]; therefore, differences
31 in locomotor behavior are expected when moving up and down along the stress axis. The two home cage settings used in this thesis may induce slightly different stress levels in the mice and, consequently, affect the activity during the first period of spontaneous behavior recordings. Regardless of what method was used, neonatal paracetamol exposure seemed to affect explorative behavior, as measured using both the old (i.e., Rat-O-Matic) and the new (i.e., IR Actimeter) equipment, strengthening the conclusion that PND 10 exposure to paracetamol affects adult spontaneous behavior and habituation capability.
THC and ibuprofen Two other drugs were included in the thesis: THC and ibuprofen. Both these drugs are important to evaluate from a DNT perspective owing to their rela- tively high exposure frequency during pregnancy [28, 32]. However, they were also included because they have pharmacodynamic similarities with pa- racetamol; THC, like paracetamol, interacts with the ECS, and ibuprofen, like paracetamol, interacts with the COX system. Therefore, we exposed male mice to different doses of these drugs on PND 10 to compare their potential DNT with the DNT of PND 10 exposure to paracetamol. When the mice reached early adulthood, those that had received a single dose of THC (either 10 or 50 mg THC/kg) showed altered spontaneous behavior and decreased habituation capability (Fig. 13). These effects increased in a dose-related man- ner, where neonatal exposure to 50 mg THC/kg gave a significantly larger alteration of spontaneous behavior and habituation capability during adult- hood than 10 mg THC/kg. However, mice that had received a THC dose of 2 mg/kg did not display any behavioral alterations compared with controls (Fig. 13). Firstly, this illustrates the potency of THC as a developmental neurotox- icant, as a single dose of THC on PND 10 was enough to alter adult behavior. There are several DNT studies on rodents showing that perinatal exposure to cannabinoids affects memory and learning later in life [41, 42]; however, most of these studies (if not all) used either several days/weeks of prenatal canna- binoid exposure or both pre- and postnatal cannabinoid exposures. While these exposures could well be relevant in a human setting, where consumption of cannabinoids (most often maternal smoking of cannabis) likely lasts for prolonged periods, it should be kept in mind that these rodent exposures prob- ably correspond to several months of cannabinoid exposure in humans in terms of brain developmental processes.
32 Figure 13. Spontaneous behavior (only locomotion shown) in adult male mice after PND 10 exposure to THC and ibuprofen. The statistical differences are indicated as (A) if different vs. control, (B) if different vs. 2 mg THC/kg, and (C or c) if different vs. 10 mg THC/kg. Uppercase letter if p ≤ 0.01 and its lowercase equivalent if p ≤ 0.05. The heights of the bars represent the means ± SD values of 12 animals. For statistical methods, see Paper II.
In contrast to paracetamol and THC, exposure to ibuprofen on PND 10 did not induce any adult behavioral alterations in the mice – not even following a PND 10 dose of 140 mg/kg (Fig. 13). Ibuprofen is one of the most used painkillers during pregnancy [28] and, like paracetamol, affects COX activity. It is there- fore interesting that, in our animal model, neonatal ibuprofen exposure did not affect adult behavior. These results, together with the results from the parace- tamol and THC exposures, give further information on the possible mecha- nism of paracetamol, since paracetamol has effects on both COX activity and on the CB1R [9, 11]. Whether it is interaction of paracetamol with the CB1R that causes the neurotoxicity is still difficult to assess from our studies; how- ever, it seems less likely that the neurotoxicity is due to interaction with COX, since developmental exposure to ibuprofen did not affect adult cognitive per- formance following PND 10 exposure. In one of the aforementioned epidemi- ology studies of developmental exposure to paracetamol, ibuprofen was ruled out as a possible confounding cause of the developmental neurotoxic effects [18], in good agreement with the observation that PND 10 exposure to ibu- profen did not affect the mice behavior later in life.
33 Effect of co-exposure to the CB1R agonist WIN on paracetamol- induced DNT Neonatal exposure to relevant doses of paracetamol (Paper I) and THC (Pa- per II), but not ibuprofen (Paper II), affected adult spontaneous behavior and habituation rates in a new home environment. This was shown for both loco- motor and rearing activity. Single-day neonatal paracetamol exposure also af- fected learning and memory, together with cognitive flexibility, in the MWM (Paper V). Since both paracetamol and THC interact with the ECS, and both paracetamol and ibuprofen interact with the COX system, further interest was directed towards the involvement of the ECS in the neurotoxic mechanism of paracetamol. In Paper III, we investigated whether the vulnerability of the developing brain to paracetamol increased if an increased endocannabinoid tone was induced. To investigate the influence of CB1R activation on parace- tamol-induced neurotoxicity at peak growth rate of the developing mouse brain, we exposed 10-day-old mice to either paracetamol, the CB1R agonist WIN, or different combinations of the two substances. This study shows that neonatal co-exposure to WIN and clinically relevant doses of paracetamol al- tered spontaneous behavior and reduced habituation rates in the adult mouse. This conclusion is based on the observation that mice that received a single dose of WIN (1 mg/kg) or a single dose of paracetamol (30 mg/kg, single dose) did not display any adult behavioral alterations, showing that these par- ticular doses were insufficient to cause adult behavioral changes (Fig. 14). However, when co-exposing the animals to both these substances, a behav- ioral alteration was detected in adulthood, similar to what had been observed following exposure to two doses of paracetamol (30 + 30 mg/kg, 4 h apart) (Fig. 14). In addition, by combining two doses of paracetamol (30 + 30 mg/kg) with a single dose of WIN (1 mg/kg) on PND 10, an even more pronounced behavioral alteration was observed compared with the effects in mice that had received either 30 + 30 mg paracetamol/kg or 30 mg paracetamol/kg + 1 mg WIN/kg (Fig. 14). This indicates that endocannabinoid signaling during the BGS plays an important role in development and that the ECS is an important modulator of paracetamol sensitivity. The essential role of an increased endo- cannabinoid tone has also been shown for ethanol sensitivity [134], as rats co- exposed to a CB1R agonist and ethanol on PND 7 had increased novel object recognition memory compared with rats that only received ethanol, an effect that was abolished with a CB1R antagonist.
34 Figure 14 Spontaneous behavior (only locomotion shown) in adult male mice after PND 10 exposure to paracetamol and the CB1R agonist WIN. The statistical differ- ences are indicated as (A) if different vs. control; (B) if different vs. “paracetamol” group; (C) if different vs. “2 × paracetamol” group; (D) if different vs. “WIN” group; (E or e) if different vs. “paracetamol/WIN” group. Uppercase letter if p ≤ 0.01 and its lowercase equivalent if p ≤ 0.05. The heights of the bars represent the means ± SD values of nine animals. For statistical methods, see Paper III.
Taken together, early-life single-day exposure to relevant doses of both para- cetamol and THC, but not ibuprofen, affected spontaneous behavior and al- tered habituation rates in adult mice (Fig. 15). Additionally, the effects of pa- racetamol were enhanced in animals co-exposed to a CB1R agonist. It was also shown that memory, learning, and cognitive flexibility were affected by neonatal paracetamol exposure (Fig. 15).
35 Figure 15. Summary of principal behavioral findings in adult mice following PND 10 exposure to paracetamol, ibuprofen, or THC. Co-exposure to the CB1R agonist WIN enhanced the adverse behavioral effects of paracetamol later in life.
36 Biochemical assessment following neonatal exposure to paracetamol and THC A detailed understanding of molecular mechanisms responsible for an adverse functional effect is, in general, essential in DNT studies. Indeed, the functional changes are a consequence of some molecular events, and those could become important for risk assessment. This holds true not only in drug development, but also in the process of post-marketing surveillance, as new potential asso- ciations between drugs on the market and adverse effects need confirmatory mechanistic explanations. The concept of the adverse outcome pathway (AOP), which is relevant to risk assessment, has been introduced [135]. In brief, the AOP is a chronological sequence of documented and causally con- nected key events within one or more specific sequences, linking a molecular initiating event with an adverse outcome. Considering the high exposures to paracetamol during brain development and the consequences associated with developmental exposure to this analge- sic, there is little information available on the possible molecular mechanism responsible for the adverse effect discussed previously. Since the molecular mechanism behind the therapeutic effects of paracetamol is not fully known, it becomes difficult to predict the possible mechanism(s) responsible for its DNT. What is known on the mechanism of action (as mentioned in the intro- duction) is that paracetamol interacts with the ECS, the COX system, and TRPV1. For its analgesic effect in mice, the CB1R is essential [136]. Owing to the phenotype following neonatal exposure to paracetamol and THC, in which effects on memory, learning and habituation were identified, this thesis focused on the following brain regions, which are all involved in those processes: the hippocampus, the frontal cortex, and the parietal cortex. The hippocampus is highly involved in learning and memory formation and the frontal cortex in learning and memory storage [137]. Both the frontal cor- tex and the hippocampus play important roles in the integration of sensory input into motor output, i.e., in the process of habituation [104, 138]. The pa- rietal cortex is implicated in spatial navigation and decision-making, espe- cially for visually based decision-making [139]. In Paper III, decreased levels of Trkb transcripts were observed in the hip- pocampus 24 h after PND 10 exposure to paracetamol or to the CB1R agonist WIN, and co-exposure to both these substances; however, there was no differ- ence in transcript levels between mice exposed to each of these drugs alone and mice co-exposed to both drugs. In Paper IV, a single dose of THC (50 mg/kg) also decreased Trkb gene expression in the hippocampus 24 h after exposure. Exposure to THC on PND 10 also decreased transcript levels of Trkb in the frontal cortex and the parietal cortex. TRKB, and its ligand BDNF, are highly important in neuronal function, as they are required for both neu- ronal survival and differentiation during brain development and in synaptic
37 and behavioral plasticity in mature neurons, including hippocampal-depend- ent memory [140-142]. In line with the observed effects on Trkb transcript levels presented in Papers III and IV, there is a known interaction between the ECS and the BDNF-TRKB pathway, most notably during brain develop- ment [83, 143]. BDNF levels are decreased in the brains of mice lacking the CB1R [99], whereas activation of the CB1R increases BNDF levels in rodents [100]. BDNF is a member of the neurotrophin family, which is crucial for neuronal survival, development, function maintenance, and plasticity in the CNS [144, 145]. The influence of cannabinoids on BDNF levels has been in- dicated, as healthy human controls showed higher serum levels of BDNF than light cannabis smokers after THC administration [143]. TRKB conditional knockout animals display impaired spatial learning [146], in line with the re- sults presented in Paper V. The role of the ECS during brain development is complex. The question arises as to the role that the ECS fulfills during CNS development. Endocan- nabinoid signaling has a certain control over migration and axon pathfinding during brain development [74], possibly through its interaction with the TRKB receptor [83]. Furthermore, the CB1R is enriched in axonal growth cones of GABAergic interneurons during development [72], suggesting its in- volvement in synaptogenesis. Endocannabinoid signaling also affects prolif- eration, self-renewal, and neuronal differentiation [74, 147, 148]. In good agreement with these observations, prenatal exposure to the CB1R agonist WIN alters migration of early-born glutamatergic neurons and GABAergic interneurons in the rat cerebral cortex [149]. ECS is important in the modula- tion of synaptic transmission in the developing hippocampus [150]. In Paper III, co-exposure to WIN and paracetamol on PND 10 decreased cerebral cor- tical transcript levels of the endocannabinoid-metabolizing enzyme FAAH; however, paracetamol or WIN alone had no effect on Faah transcript levels compared with the transcript levels in controls. FAAH is an enzyme essential in endocannabinoid signaling [151] and a decrease in FAAH levels is sug- gested to increase anandamide concentrations resulting in increased CB1R ac- tivation. At a cellular level, activation of CB1R inhibits adenylyl cyclase ac- tivity, decreases cellular calcium entry through inhibition of voltage-gated cal- cium channels [76], and activates potassium channels through the modulation of cAMP concentrations [152]. It is expressed at presynaptic sites and, upon stimulation by an agonist (endo- or exogenous), it regulates both inhibitory and excitatory neurotransmission, through its control over both GABA [153] and glutamate [154] release in hippocampus. CB1R stimulation triggers the activation of the mitogen-activated protein kinase/extracellular signal–regu- lated kinase (MAPK/ERK) and phosphoinositide-3 kinase (PI3K)/protein ki- nase B (Akt) signaling pathways. In both hippocampal slices and in living mice, anandamide and THC both activated ERK; in living mice, accumulation of ERK was shown in the nuclei of pyramidal cells in CA1 and CA3 regions
38 [155]. Increased phosphorylation of Akt was also observed in mouse hippo- campus after THC exposure, where the THC-induced phosphorylation of Akt was abolished both by blocking of CB1R with the antagonist rimonabant and by PI3K inhibition with wortmannin [156]. An important downstream target of the PI3K/Akt pathway is the mammalian target of rapamycin (mTOR) [157, 158], which regulates neuronal plasticity, modifies synaptic strength [159, 160], and regulates translational rate [161, 162]. Whether developmental ex- posure to clinically relevant doses of paracetamol affects these aforemen- tioned pathways, like THC, remains to be investigated. Interestingly, it has already been shown that mTOR signaling in mouse hippocampus is modulated by THC exposure [163]. The previously discussed potentiating effects of PND 7 CB1R activation on ethanol-induced memory deficits in mice appear to affect ERK and Akt phosphorylation, as well as to induce neurodegeneration – an effect that was restored following CB1R blockade [134]. Furthermore, this effect might be age-dependent, as exposure to a combination of THC and ethanol had an ag- gravating effect on neurodegeneration on PNDs 4, 7, and 10, but not on PNDs 2 and 14, compared with when THC and ethanol were administered alone [164]. Interestingly, this CB1R-related increase in ethanol vulnerability fol- lows the trajectory of brain development during the BGS. It has also been shown that developmental exposure to other types of chemicals, e.g., the in- secticide chlorpyrifos, has an impact on the ECS [165]. Chlorpyrifos has been shown to alter adult spontaneous behavior following PND 10 exposure [166]. While apoptosis is an essential part of brain development, by naturally re- moving non-functioning neurons, xenobiotic interference during brain devel- opment (e.g., by antagonists of NMDARs, agonists of GABAA receptors, and sodium channel blockers) has the potential to trigger unnatural and widespread apoptosis [167-170]. In Paper IV, increased levels of the pro-apoptotic pro- tein bcl-2-like protein 4 (BAX) were found in the frontal cortex in mice 24 h after PND 10 THC exposure (10 mg/kg). On the other hand, no effects were found on BAX levels in the parietal cortex or hippocampus. Furthermore, the effect of THC on apoptosis has been shown to be CB1R-mediated [171]. Neu- ral death has been indicated following acute exposure to higher doses of para- cetamol (in vitro: 1–2 mM; in vivo: 250–500 mg/kg) in rats [172]; however, it is not yet known whether clinically relevant doses of paracetamol can also induce apoptosis. In Paper IV, an increase in the transcriptional ratio of Nrf2/Keap1 was shown in the parietal cortex and in the hippocampus 24 h after exposure to 50 mg/kg of THC. This suggests that the single dose of THC caused oxidative damage, which could be one of the underlying reasons for the behavioral man- ifestations observed in Paper II following the same exposure. In Paper V, an increase in the transcriptional ratio of Nrf2/Keap1 was also observed in the hippocampus following paracetamol exposure, although this was 6 h after ex-
39 posure. High doses of paracetamol have been shown to increase reactive oxy- gen species formation leading to mitochondrial dysfunction [173]. Lower therapeutic doses also seem to lead to oxidative stress-related gene responses in humans [174]. In summary, neonatal exposure to both paracetamol and THC decreased transcript levels of Trkb and increased the transcriptional ratio of Nrf2/Keap1 (Fig. 16). PND 10 exposure to THC also increased BAX. Co-exposure to WIN and paracetamol effected transcript levels of Faah in the frontal cortex (nei- ther paracetamol nor WIN alone affected Faah transcript levels) (Fig. 16). Effects on neurotrophic signaling and oxidative stress may therefore both play a part in the AOP of both paracetamol and THC, following exposure at a time point corresponding to late pregnancy/early life.
Figure 16. Summary of principal biochemical findings following PND 10 exposure to paracetamol and THC. Both paracetamol and THC affected Trkb gene expression and increased expression of oxidative response genes. Co-exposure to the CB1R ag- onist WIN increased transcript levels of the endocannabinoid enzyme Faah, while PND 10 THC exposure induced the proapoptotic protein BAX.
Paracetamol as a potential developmental neurotoxicant Considerations for the evaluation of developmental neurotoxic data Modelling human DNT Animal experimentation has been one of the building blocks of biological and biomedical research, particularly in the fields of clinical medicine, pharma- cology, and toxicology. Modeling human diseases, especially those associated with the brain, is nonetheless very challenging, owing to the limited under- standing of how well the brain development and pathological processes of model organisms correspond to those in humans. Rats and mice are by far the most used animals in DNT studies, especially when it comes to assessment of behavioral endpoints.
40 When comparing mice to humans, there are important factors to consider in potential interspecies variation, such as differences in vulnerability, com- pensatory mechanisms, pace of developmental processes, and pharmacokinet- ics. To correctly choose doses and accurately estimate human relevance in relation to a drug, it is essential to be able to translate the findings from mice to humans. Firstly, due to the pharmacokinetic differences between rodents and humans, doses are often difficult to translate (or choose). While doses are often reported as mg/kg body weight, hence taking differences in body weight between animals into account, some factors in basic kinetic function differ dramatically between species. Fortunately, there are translational methods available. One of these translational methods, which correlates well across several mammalian species, is the BSA normalization method, previously dis- cussed under Materials and methods. It has been shown that this method cor- relates well with several parameters of biology, including oxygen utilization, caloric expenditure, basal metabolism, blood volume, circulating plasma pro- teins, and renal function (summarized in [175]). A second difficulty when translating data from mice to humans is how to compare the exposure time. Humans have a relatively long lifespan, where developmental processes are relatively slow, whereas these processes are expected to have a faster pace and be more compressed in mice. This is especially true during brain development, where mice are expected to have narrower, and perhaps more critical, win- dows of increased vulnerability during brain development compared with hu- mans. In humans, key neurodevelopmental processes last longer compared with those in mice; thus, weeks of exposure in mice may correspond to months in humans. However, the fundamentals of key developmental processes in hu- mans and mice are considered similar, e.g., the chronology of neurodevelop- mental processes [60, 61], making it possible to define fairly similar develop- mental stages between these species. A third concern when evaluating adverse effects of therapeutic drugs is that most experiments are performed on animals lacking the physiological or psychological condition that motivates the use of the specific drug in the first place. In the case of paracetamol in this study, the mice lacked pain and fever, a situation rarely found when humans use parace- tamol. This is an important matter, as pain itself can induce pathological events that can potentially cause DNT [176].
Identification of DNT compounds Regulatory authorities approve drugs for the market only after the pre-market- ing stage. In their assessment, the benefits should outweigh the risks of con- suming the drug. The regulatory authorities often approve marketing based on rigorous, well-made, randomized, and controlled trials. Even if a drug is con- sidered safe for its intended use, this is not equivalent to a zero risk. In most cases, the clinical trials are not large enough, diverse enough, and do not last long enough to get all the necessary information. Following marketing of a drug, there is also a potential risk that it interacts with other drugs, foods, or
41 environmental factors. Continuous post-marketing surveillance and evalua- tion is therefore of high importance – this holds true regarding data from both human and animal models.
Parallels between experimental studies and existing epidemiology In health risk assessments, epidemiological studies benefit from their direct relevance to human populations (in their natural environment). On the other hand, these types of studies can be biased and confounded by unknown factors influencing associations. By use of animal models, e.g., rodents, the environ- mental influences become more controlled and causal relationships between, e.g., xenobiotic exposures and different outcomes, are often more easily es- tablished. The main drawback of animal studies, as mentioned earlier, is the limited relevance to human health. There is therefore an important interplay between experimental and epidemiological studies, as neither is sufficient on its own, but they can serve as important complements to each other. Which shared features can then be summarized from the existing epidemiology and the findings presented in this thesis? As previously mentioned, there is a rapidly increasing body of epidemio- logical evidence suggesting that prenatal exposure to paracetamol alters neu- rodevelopment, showing associations with ADHD, ASD, lower IQ, and language delay [17-26]. Neurodevelopmental disorders such as autism and ADHD are common and can cause lifelong disability, yet their causes are largely unknown. The potential effect of paracetamol during brain develop- ment is consistent with the idea that ADHD and ASD are multifactorial and at least in part result from the cumulative load of environmental contributors [177]. In Papers I and III, a reduced activity (in a new home environment) during the first 20 min of testing was found in adult mice neonatally exposed to pa- racetamol compared with controls. In line with this observation, it has been shown that ASD patients display hypo-responsiveness to novel stimuli [178, 179]. Furthermore, prolonged paracetamol exposure late in pregnancy in- creased the risk of ADHD and ASD symptoms [180]. The timing of exposure also seems to be of essence in mice. In Paper I, paracetamol exposure during neurodevelopmental time points corresponding to late pregnancy/early life and the beginning of the third trimester in humans altered adult habituation and spontaneous behavior in mice. On the other hand, exposure during a time point corresponding to a two-year-old child did not affect adult behaviors in mice. Additionally, in Paper III (which also replicated findings from Viberg et al. [101]), a prolonged exposure of two doses, 4 h apart, affected adult be- havior in mice, while a single dose did not. The timing and duration of para- cetamol exposure seems to be important in both mice and humans.
42 PND 10 exposure to both paracetamol (Paper III) and THC (Paper IV) affected transcript levels of the neurotrophic receptor TRKB. Effects on its ligand BDNF have also been found after developmental exposure to paraceta- mol in rodents [101, 129]. In humans, dysregulation of BDNF has been found to be involved in ASD and ADHD [181, 182]. Furthermore, single-nucleotide variants of BDNF have been found to be associated with increased risk of ADHD [183, 184]. In Paper V, an oxidative response was noted following PND 10 exposure to paracetamol. In Paper IV, a similar response, indicating oxidative stress, was also noted after exposure to THC. Oxidative stress and inflammatory re- sponses have been implicated in the etiology of ADHD and ASD, as indicated by differential expression of pro-inflammatory cytokines [185, 186]. Interest- ingly, therapeutic doses of paracetamol seemed to activate oxidative stress- related gene responses, similar to those observed following higher doses, in humans [174]. Using rodent models, both developmental and adult exposures to relevant doses of paracetamol have been shown to affect various neurotransmitter sys- tems: serotonergic, noradrenergic, glutamatergic, and dopaminergic systems [128, 129, 187-189]. These systems have been associated with ASD and ADHD etiology [190-193]. Altered endocannabinoid neurotransmission could potentially also be affected by paracetamol exposure. The CB1R is activated by the paracetamol metabolite AM404 [12, 13] and is involved in the devel- opment of ASD [194-196]. In addition, the ECS has certain control over other neurotransmitter systems [77-82]; thus, more attention should be directed at CB1R. It is possible that other transmitter systems are affected when parace- tamol affects the ECS, which may explain the diverse effects paracetamol seems to have on other neurotransmitter systems.
43 Concluding remarks and future perspectives
In this thesis, early-life single-day exposures to clinically relevant doses of paracetamol were shown to affect adult spontaneous behavior, habituation ca- pability, memory, learning, and cognitive flexibility in mice. The effect of PND 10 exposure to paracetamol on adult spontaneous behavior and habitua- tion was enhanced by co-exposure to the endocannabinoid activator WIN. Ex- posure to the commonly used recreational drug THC, which also is an endo- cannabinoid activator, caused alterations in spontaneous behavior and habitu- ation. On the other hand, the COX inhibitor ibuprofen did not cause adult be- havioral changes. With the data presented herein, it is difficult to assess if it is the interaction of paracetamol with the CB1R that causes the observed neurotoxicity. How- ever, it seems less likely that the DNT is due to COX inhibition, since devel- opmental exposure to ibuprofen did not affect adult behavior. Both THC and paracetamol interact with the ECS. Furthermore, PND 10 exposure to both these substances 1) affected adult spontaneous behavior and habituation rates and 2) decreased transcript levels of Trkb and increased markers for oxidative stress. There is therefore a possibility that paracetamol and THC share im- portant events in their respective AOP. The negative effect of PND 10 para- cetamol exposure on adult spontaneous behavior and habituation rates was enhanced by simultaneous exposure to the CB1R agonist WIN, indicating a critical role of the ECS during brain development. There are several hypotheses regarding the AOP of paracetamol DNT: (1) excess toxic N-acetyl-p-benzoquinone imine (more known as NAPQI) for- mation in the CNS, (2) oxidative stress, (3) altered levels of BDNF, (4) effects on endocannabinoid signaling, (5) effects on prostaglandin synthesis, and (6) endocrine disruption; for further information, read the review by Bauer and colleagues [180]. Based on the observations presented herein, the possible pa- racetamol/AM404/CB1R/BDNF/TRKB-mediated mechanism of neurotoxi- city needs to be further investigated. In addition, the potential effects on the TRPV1 receptor should not be overlooked in the future, though these are not discussed in this thesis. As evidence of neurodevelopmental effects of paracetamol is accumulat- ing, the safety of using paracetamol during pregnancy is currently being dis- cussed. However, the European Medicines Agency’s Pharmacovigilance Risk Assessment Committee (PRAC) still agrees with the PRAC Rapporteur’s
44 view from 2015 [197] that a causal relationship between paracetamol expo- sure during pregnancy and neurodevelopmental outcomes could not be estab- lished. However, as of 2019, the PRAC concluded that although the studies are “inconclusive,” the summary of product characteristics should be amended by including information that paracetamol-containing products should, during pregnancy, be used at the lowest effective dose for the shortest possible time and at the lowest possible frequency [198]. The need for mechanistic insight is of highest priority. Accumulating data on the negative effects of developmental exposure to paracetamol call for a continuous risk assessment, which should be prioritized, considering the high exposure to paracetamol during brain development. Many substances that are regarded as safe and are sold over-the-counter may be used in combination with (or co-exposed to) other pharmaceuticals/drugs of abuse and environmental contaminants. While the high paracetamol use during pregnancy is based on the many advantages of paracetamol over other painkillers, with NSAIDs having a less favorable risk-benefit profile in preg- nant women, a balanced assessment based on best professional judgement concerning the toxicity of paracetamol to the developing human brain should be prioritized.
45 Populärvetenskaplig sammanfattning
Paracetamol (den verksamma substansen i till exempel Alvedon) är det mest använda smärtstillande och febernedsättande läkemedlet hos gravida och barn. Läkemedlet marknadsfördes internationellt på 1950-talet och dess användning ökade snabbt. Vid rekommenderade doser har paracetamol en mildare biverk- ningsprofil jämfört med till exempel ibuprofen (den verksamma substansen i till exempel Ipren) eller acetylsalicylsyra (den verksamma substansen i till ex- empel Treo). Detta gör att paracetamol betraktats som det säkraste alternativet när det gäller behandling av smärta och feber, särskilt under graviditet och hos yngre barn. Trots att paracetamol betraktas som ofarligt för dessa grupper har det på senare tid visats ett samband mellan dess intag under hjärnans utveckl- ing och en förhöjd risk att diagnostiserats med bland annat ADHD och aut- ismliknande symptom senare i livet. I motsats till vad man kan förvänta sig av ett väletablerat läkemedel är pa- racetamols verkningsmekanism ännu inte helt kartlagd. Precis som för ibupro- fen och acetylsalicylsyra påverkar paracetamol syntesen av prostaglandiner, vilka är pro-inflammatoriska, hormonlika ämnen vilka ger symtom som feber och värk vid sjukdom. På senare tid har det dock visat sig att paracetamol också interagerar med ett helt annat system, det så kallade endocannabinoida systemet. Som namnet antyder är det samma system som påverkas av till ex- empel cannabis. Det har visat sig att denna interaktion är nödvändig för att förmedla paracetamols smärtstillande egenskaper. Samma system är också känt för att vara känslig för störningar under hjärnans utveckling. Denna avhandling syftade till att undersöka effekter orsakade av intag av kliniskt relevanta doser av paracetamol under en känslig period av hjärnans utveckling hos den nyfödda musen. Vidare undersöktes även effekterna av två andra substanser under samma utvecklingsfas: ibuprofen och THC (den psy- koaktiva substansen i cannabis). Utvärderingen av ibuprofen och THC är av klinisk relevans eftersom de båda används under graviditet; ibuprofen används trots att det inte är rekommenderat och THC är den mest använda olagliga drogen under graviditet. Deras verkningsmekanismer har likheter med para- cetamol och kan därför användas ur ett jämförande perspektiv. På senare tid har mer uppmärksamhet riktats mot THCs medicinska egenskaper. Parallellt har olika formuleringar som innehåller THC legaliserats över världen, vilket innebär att användandet av THC kan komma att öka framöver. För att svara på frågan huruvida paracetamol, THC och ibuprofen kan or- saka kognitiva störningar detekterbara senare i livet doserades nyfödda möss
46 för dessa substanser under en enda dag. Nyföddhetsperioden hos möss karak- täriseras av en snabb tillväxt och utveckling av hjärnan. Det har visat sig att möss är mycket känsliga för exponering för främmande substanser under denna utvecklingsperiod. Hos möss sträcker sig denna period från födseln och 3-4 veckor framåt. Denna utvecklingsfas återfinns även hos människa. Där påbörjas den emellertid redan under slutet av graviditeten och fortsätter under barnets första levnadsår. Denna avhandling visar att exponering för paracetamol, under den korta perioden av snabb hjärnutveckling hos möss, leder till förändrat spontanbete- ende och försämrad anpassningsförmåga till nya miljöer senare i livet. Dessa effekter uppkom efter att både 3 och 10 dagar gamla musungar doserades – en effekt som visade sig i både hanar och honor. Vad gäller de processer som sker i hjärnan är dessa två tidpunkter hos möss jämförbara med början av sista tredjedelen av graviditeten och tiden runt födseln hos oss människor. Dosering av paracetamol hos 19 dagar gamla möss, jämförbart med ett tvåårigt barn med avseende på hjärnans utvecklingsprocesser hos människa, orsakade emel- lertid inte några beteendeförändringar i vuxen ålder. Vidare påverkar dosering av 10 dagar gamla möss för paracetamol också minne och inlärning. Studierna i denna avhandling visar även att möss som exponerades för THC när de var unga hade förändrat spontanbeteende och försämrad anpassnings- förmåga till nya miljöer. Däremot återfanns ingen påverkan efter tidig expo- nering för ibuprofen. Detta skulle kunna ses som en indikation på att det en- docannabinoida systemet är involverat i paracetamols utvecklingsneurotox- iska effekter. För att testa det endocannabinoida systemets påverkan på para- cetamols negativa effekter på hjärnans utveckling doserades 10 dagar gamla möss samtidigt för både paracetamol och en substans som aktiverar det en- docannabinoida systemet. Detta orsakade en förvärring av de negativa effek- terna jämfört med de möss som fick dessa substanser var för sig, vilket kan tolkas som ytterligare en indikation på hur viktigt det endocannabinoida sy- stemet är under hjärnans utveckling – och för paracetamols utvecklingsneuro- toxiska effekter. Vad som orsakar dessa långtidseffekter efter dosering av paracetamol un- der hjärnans utveckling är som tidigare nämnt inte känt. I denna avhandling iakttogs en minskad aktivitet av en gen som är viktig under hjärnans utveckl- ing en kort tid efter dosering av paracetamol. Samma gen visade sig minska i aktivitet även efter dosering av THC. Eftersom både paracetamol och THC påverkar det endocannabinoida systemet, samt att de hämmar denna för hjär- nan utvecklingsfrämjande gen, indikerar detta på att både paracetamol och THC kan ha gemensamma drag i sina respektive negativa verkningsmekan- ismer. Det visade sig också att både paracetamol och THC orsakade oxidativ stress i hjärnan, en process som potentiellt kan skada hjärnans celler. I korthet visar studierna i denna avhandling att den neonatala musen är känslig för intag av rekommenderade doser av paracetamol. Eftersom THC hade en liknande påverkan på spontanbeteende och anpassningsförmåga till
47 nya miljöer, men inte ibuprofen, pekar detta på att det är mer sannolikt att det endocannabinoida systemet kan bidra till paracetamols negativa effekter på hjärnans utveckling. Vidare visar denna avhandling tydligt att dessa bestående förändringar i beteende uppkommer efter endast en dags dosering av parace- tamol när mössen var yngre, vilket understryker att det kan finnas korta käns- liga perioder under hjärnans utveckling. Med tanke på hur många som tar pa- racetamol under graviditet och det första levnadsåret, samt att det är det enda rekommenderade smärtstillande läkemedlet för dessa målgrupper, är den fort- satta forskningen och riskbedömningen kring dess användning av stor bety- delse.
48 Acknowledgment
Först vill jag tacka min nuvarande huvudhandledare Robert Fredriksson. Forskningsmässigt har du lärt mig så mycket, både praktiskt och teoretiskt. Du har alltid haft en stor tillit till mig och mina idéer vilket gjort att jag kunnat växa som forskare. Din ledarstil är en inspirationskälla. Otroligt ödmjuk och otroligt kunnig. Jag vill även tacka min första huvudhandledare Henrik Viberg för att du bjöd in mig i akademiska världen genom att ta in mig som doktorand och tro på mig. Att ha haft en så kunnig och omtänksam handledare har varit ett enormt privilegium. Jag är också sjukt glad att vi fortfarande ses med jämna mellanrum för en vindaloo-lunch. Stort tack till er båda som gjort att jag under de senaste fem åren haft det roligaste jobb man kan tänka sig!
Per Eriksson. Min biträdande handledare och personen bakom hela expone- ringsmodellen. Utan dig hade denna avhandling aldrig existerat. Tack för att du alltid ställt upp. Sonja Buratovic. Min doktorandkollega och senare biträdande handledare. Tack för att du har stöttat och hjälpt mig under arbetets gång. Det var även du som lärde mig köra de flesta av beteendetesten – ovärderligt!
Anders Fredriksson. Du hjälpte mig mycket i arbetets början. Det var du som möjliggjorde alla de fina beteenderesultaten.
Johan Rimbaud Svensson. En av mina närmsta vänner och tidigare medar- betare på miljötoxikologi (där han en gång i tiden kallades för ”the chosen one”). Du har hjälpt mig otroligt mycket både som polare och med forsk- ningen.
Tack till: Anna (för att du hade pli på studenterna när jag uppenbarligen inte hade det), Sofie H (för dina inspirerande labb-skills), Erica (för att du alltid har snus till mig och för att du tog hand om examensjobbare när jag var pap- paledig), Kimia (för att vi kunnat planera så spännande nya forskningsprojekt) och Stefan (för att du hjälpte mig med både qPCR och hiphopkarriären).
49 Björn Brunström. Ämnesansvarig professor under större delen av min dok- torandperiod. Speciellt tack för att du hjälpt mig under senare delen av arbetet. Tack! Joëlle Rüegg. Ämnesansvarig professor under mitt sista halvår, tack för hjäl- pen inför disputationen!
Mathias RA. Stort tack för att du visade hur kul forskning är samt att du hjälpte mig skriva min första artikel.
Ett stort tack till mina tidigare examensjobbare Carin, Caroline, Yasser och Mariam. Ni har alla bidragit till denna avhandling på ett otroligt sätt! Tack också till Rekha och Frida som jag delat kontor med under senaste året.
Stort tack till alla andra nuvarande och tidigare medarbetare på avdelningarna för Miljötoxikologi och för Molekylär Neurofarmakologi.
Tack Sepand för att du alltid ställt upp för mig, oavsett vad. Du är en riktig ”copain”!
Även Matte och Dave ska ha ett stort tack för att ni gett mig feedback på några av mina arbeten och för att vi kunnat ses för lunch (ofta) och träna (mindre ofta) tillsammans. Jag vill tacka det gamla ”biologgänget” (Matte, Nisse, Stina, Johan, Tobbe, Groom) + Tomas och Jeremy som jag startade hela detta Uppsalaäventyr tillsammans med. Till mina barndomspolare Chris, Jonas, Felipe, Anders, Jimmie, Richard, Alfonse, Kalle och resten av ÄC vill jag också säga tack för att ni påminner mig om att livet är så mycket mer än bara jobbet!
Stort tack till min mamma Paula – du har alltid gött mitt forskningsintresse! Dessutom har du alltid ställt upp för mig på ett sätt som jag en dag hoppas kunna göra för mina barn. Du är den starkaste personen jag känner och är min största förebild!
Jag vill passa på att tacka min syster Coralie och hennes familj (Calle och Pascal). Ser så mycket fram emot vår nästa familjesemester!
Till sist vill jag tacka min underbara fru Karin och mina två älskade barn Émile och Julie – jag älskar er mest i hela världen!
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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1919 Editor: The Dean of the Faculty of Science and Technology
A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology”.)
ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-407311 2020