This thesis investigated the developmental neurotoxic effects of neonatal exposure to highly brominated diphenyl ethers (PBDEs) and perfluorinated compounds (PFCs), alone or in combinations, during a critical period of the brains’ rapid growth and development, in mice. The compounds investigated were the decaBDE (PBDE 209), nonaBDE (PBDE 206), octaBDE (PBDE 203), heptaBDE (PBDE 183), and three PFCs, PFOS, PFOA, and PFDA. PBDEs and PFCs have been identified as emerging classes of persistent environmental compounds, present in wildlife as well as humans, and present at higher levels in infants/children, compared to older persons. Individuals can be exposed to these compounds throughout her/his lifetime and newborn/children can be exposed to toxicants both via the mothers’ milk and directly via ingestion and inhalation. The brain growth spurt (BGS) is defined by rapid growth and developmental of the brain. For rodents (mice and rats), the BGS is postnatal spanning the first 3-4 weeks after birth. In humans this period begins during the third trimester of pregnancy and continues throughout the first two years of life. It has been shown that several environmental toxicants can induce permanent disorders in brain function when administered to the neonatal mouse, during the BGS. This thesis shows that highly brominated PBDEs, including PBDE 209, PBDE 206, and PBDE 203 can cause developmental neurotoxic effects, when given directly to the neonatal mouse. Of the investigated PFCs, PFOS and PFOA were shown to cause similar effects as the PBDEs. Furthermore, PBDE 209 and PFOA can at low doses interact and enhance the neurotoxic effects in mice. Effects in the adult animal included; deranged spontaneous behavior, reduced or lack of habituation, decreased learning and memory abilities, and increased susceptibility of the cholinergic system. Both classes of compounds were shown to affect proteins (CaMKII, GAP-43, synaptophysin, and tau) important for neuronal growth and synaptogenesis in the neonatal mouse brain.

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Radiological methods for screening, diagnostics and therapy are often used in healthcare; however, it has recently been reported that developmental exposure to low-dose ionizing radiation (IR) causes neurotoxicity. Environmental chemicals also have the potential to affect the developing brain and the concomitant effects caused by IR and chemicals are of high interest today. We therefore aim to investigate if low-dose IR can interact with the known neurotoxicant paraquat to induce neurotoxicity in the neonatal mouse model. Using the same model, we also aim to investigate if fractionated low-dose IR can be as neurotoxic as higher acute doses. Male mice were exposed to a single dose of paraquat (0.2 or 0.02 mg/kg) on postnatal day 10 and 11. Two hours following paraquat exposure, mice were whole body irradiated with 100 or 300 mGy gamma radiation (137 Cs). Behavioural observations were performed at 2 and 3 months of age. Following behavioural testing, we evaluated striatal dopaminergic gene transcription. Animals co-exposed to IR and paraquat generally displayed altered spontaneous behaviour compared to controls and single agent exposed mice. Stronger effects by combined exposure were also observed on adult memory and learning. However, dopaminergic gene transcript levels remained unchanged by treatment. Co-exposure to low-dose IR and paraquat can interact to exacerbate neurotoxic effects and to impair cognitive function. Furthermore, fractionation of the radiation dose was observed to be as potent as higher acute exposure for induction of developmental neurotoxicity.

Acetaminophen (AAP; also known as paracetamol) is the most used and only recommended analgesic and antipyretic among pregnant women and young children. However, recent findings in both humans and rodents suggest a link between developmental exposure to AAP and adverse neurobehavioral effects later in life. We hypothesized that the cannabinoid receptor type 1 (CB1R) may be involved in the developmental neurotoxicity of AAP, owing to its interaction with the endocannabinoid system. Here we test if CB1R agonist WIN 55 212-2 (WIN) and AAP can interact when exposure occurs during a neurodevelopmental stage known for increased growth rate and for its vulnerability to AAP exposure. We exposed male NMRI mice on postnatal day 10 to different combinations of AAP and WIN. Adult mice, neonatally co-exposed to AAP and WIN, displayed a significant lack of habituation in the spontaneous behavior test, when compared with controls and single agent exposed mice. These adult adverse effects may at least in part be explained by a reduction of transcript levels of hippocampal synaptophysin (Syp) and tropomyosin receptor kinase B (Trkb), and cerebral cortical fatty acid amide hydroxylase (Faah), 24h after exposure. These findings are consistent with our hypothesis that AAP and WIN can interact when exposure occurs during early postnatal brain development in mice. Assuming our results are relevant for humans, they raise concerns on AAP safety because it is the only recommended analgesic and antipyretic during pregnancy and early life.

(Br J Anaesth. 2018;120:546–554) The “brain growth spurt” (BGS) during fetal development is a vulnerable period characterized by synaptogenesis, dendritic arborization, extensive myelination, and chemical changes. Exposure to ionizing radiation (IR) may have detrimental effects on this period of development. In experimental rodent models, a single neonatal IR dose of 0 to 100 mGy during the BGS period induced modification in behavior of the mice, with a proposed developmental neurotoxicity at 350 mGy, and alteration in cerebral cortex tau levels at 500 mGy. In addition, rodent neonatal exposure to ketamine has been shown to cause acute apoptotic neurodegeneration, alterations of essential neuroproteins, induction of hyperactive phenotype, and cognitive impairment. As pediatric patients often undergo radiotherapy or sedation/anesthesia before or during radiologic procedures, the authors of the present study explored whether IR doses in the range of exposure associated with computerized tomography (CT) scans could interact with low doses of ketamine to induce neurotoxicity in the developing mouse brain.

Paracetamol (acetaminophen) is a widely used non-prescription drug with analgesic and antipyretic properties. Among pregnant women and young children, paracetamol is one of the most frequently used drugs and is considered the first-choice treatment for pain and/or fever. Recent findings in both human and animal studies have shown associations between paracetamol intake during brain development and adverse behavioral outcomes later in life. The present study was undertaken to investigate if the induction of these effects depend on when the exposure occurs during a critical period of brain development and if male and female mice are equally affected. Mice of both sexes were exposed to two doses of paracetamol (30 + 30 mg kg-1 , 4 h apart) on postnatal days (PND) 3, 10 or 19. Spontaneous behavior, when introduced to a new home environment, was observed at the age of 2 months. We show that adverse effects on adult behavior and cognitive function occurred in both male and female mice exposed to paracetamol on PND 3 and 10, but not when exposed on PND 19. These neurodevelopmental time points in mice correspond to the beginning of the third trimester of pregnancy and the time around birth in humans, supporting existing human data. Considering that paracetamol is the first choice treatment for pain and/or fever during pregnancy and early life, these results may be of great importance for future research and, ultimately, for clinical practice. Copyright © 2017 John Wiley & Sons, Ltd.

This thesis examines the developmental neurotoxic effects of exposure to low-dose ionizing radiation (IR), alone or together with xenobiotics, during a critical period of neonatal brain development in mice.During mammalian brain development there is a period called the brain growth spurt (BGS), which involves extensive growth and maturation of the brain. It is known that neonatal exposure during the BGS to xenobiotics can have a negative impact on neonatal brain development, resulting in impaired cognitive function in the adult mouse. In humans, the BGS starts during the third trimester of pregnancy and continues for approximately 2 years in the child. The present thesis has identified a defined critical period, during the BGS, when IR can induce developmental neurotoxicity in mice. The observed neurotoxicity was not dependent on sex or strain and manifested as altered neurobehaviour in the adult mouse. Furthermore, fractionated dose exposures appear to be as potent as a higher acute dose. The cholinergic system can be a target system for developmental neurotoxicity of IR, since alterations in adult mouse cholinergic system susceptibility were observed. Co-exposure to IR and nicotine exacerbated the behavioural disturbances and cholinergic system dysfunction. Furthermore, co-exposure with the environmental agent paraquat has indicated that the dopaminergic system can be a potential target. In this thesis, clinically relevant doses of IR and a sedative/anesthetic agent (ketamine) were shown to interact and exacerbate defects in adult mouse neurobehaviour, learning and memory, following neonatal exposure, at doses where the single agents did not have any impact on the measured variables. This indicates a shift in the dose-response curve for IR, towards lower doses, if exposure occurs during the neonatal brain development. In addition, co-exposed mice, showing cognitive defects, expressed elevated levels of tau protein in the cerebral cortex. Furthermore, exacerbation of neurochemical deviations were observed following co-exposure compared to irradiation alone.Further investigations of neurotoxic effects following fractionated or acute low-dose IR, modelling the clinical situation during repeated CT scans or levels of radiation deposited in non-target tissue during radiotherapy, and possible interaction effects with xenobiotics, is of great importance in the field of radioprotection.

Neonatal exposure to acetamiophen (paracetamol) and CB1R agonist shos an additive adverese neurodevelopmental effects

Development of the brain includes periods which can be critical for its normal maturation. The present study investigates specifically vulnerable peri-/postnatal periods in mice which are essential for understanding the etiology behind radiation induced neurotoxicity and functional defects, including evaluation of neurotoxicity between sexes or commonly used laboratory mouse strains following low/moderate doses of ionizing radiation (IR).Male Naval Medical Research Institute (NMRI) mice, whole body irradiated to a single 500 mGy IR dose, on postnatal day (PND) 3 or PND 10 showed an altered adult spontaneous behaviour and impaired habituation capacity, whereas irradiation on PND 19 did not have any impact on the studied variables. Both NMRI and C57bl/6 male and female mice showed an altered adult spontaneous behaviour and impaired habituation following a single whole body irradiation of 500 or 1000 mGy, but not after 20 or 100 mGy, on PND 10.The present study shows that exposure to low/moderate doses of IR during critical life stages might be involved in the induction of neurological/neurodegenerative disorder/disease. A specifically vulnerable period for radiation induced neurotoxicity seems to be around PND 3-10 in mice. Further studies are needed to investigate mechanisms involved in induction of developmental neurotoxicity following low dose irradiation. (C) 2016 Elsevier B.V. All rights reserved.

PURPOSE:To investigate whether neonatal exposure to fractionated external gamma radiation and co-exposure to radiation and nicotine can affect/exacerbate developmental neurotoxic effects, including altered behavior/cognitive function and the susceptibility of the cholinergic system in adult male mice. MATERIALS AND METHODS:Neonatal male Naval Medical Research Institute (NMRI) mice were irradiated with one 200 mGy fraction/day and/or exposed to nicotine (66 μg/kg b.w.) twice daily on postnatal day (PND) 10, 10-11, 10-12 or 10-13 (nicotine only). At 2 months of age the animals were tested for spontaneous behavior in a novel home environment, habituation capacity and nicotine-induced behavior. RESULTS:Fractionated irradiation and co-exposure to radiation and nicotine on three consecutive days disrupted behavior and habituation and altered susceptibility of the cholinergic system. All observed effects were significantly more pronounced in mice co-exposed to both radiation and nicotine. CONCLUSIONS:The fractionated irradiation regime affects behavior/cognitive function in a similar manner as has previously been observed for single-dose exposures. Neonatal co-exposure to radiation and nicotine, during a critical period of brain development in general and cholinergic system development in particular, enhance these behavioral defects suggesting that the cholinergic system can be a target system for this type of developmental neurotoxic effects.

Both Δ(9)-tetrahydrocannabinol (THC) and ibuprofen have analgesic properties by interacting with the cannabinoid receptor type 1 (CB1R) and the cyclooxygenase (COX) systems, respectively. Evaluation of these analgesics is important not only clinically, since they are commonly used during pregnancy and lactation, but also to compare them with acetaminophen, with a known interaction with both CB1R and the COX systems. Short-term exposure of neonatal rodents to acetaminophen during the first weeks of postnatal life, which is comparable with a period from the third trimester of pregnancy to the first years of postnatal life in humans, induces long-term behavioral disturbances. This period, called the brain growth spurt (BGS) and is characterized by series of rapid and fundamental changes and increased vulnerability, peaks around postnatal day (PND) 10 in mice. We therefore exposed male NMRI mice to either THC or ibuprofen on PND 10. At 2 months of age, the mice were subjected to a spontaneous behavior test, consisting of a 60min recording of the variables locomotion, rearing and total activity. Mice exposed to THC, but not ibuprofen, exhibited altered adult spontaneous behavior and habituation capability in a dose-dependent manner. This highlights the potency of THC as a developmental neurotoxicant, since a single neonatal dose of THC was enough to affect adult cognitive function. The lack of effect from ibuprofen also indicates that the previously seen developmental neurotoxicity of acetaminophen is non-COX-mediated. These results might be of importance in future research as well as in the ongoing risk/benefit assessment of THC.

BACKGROUND:Numerous animal studies have shown that all commonly used intravenous anaesthetic drugs and volatile agents may cause neuronal apoptosis following exposure in early life. Most studies have focussed on detecting increased apoptosis but their methods are not always readily transferrable to humans. The lipid formulation of etomidate represents an alternative to the currently established intravenous anaesthetic agents but there is no animal or human data on apoptosis or long-term behavioural changes. The aim of our study was to investigate the effects of etomidate on cerebral neuronal apoptosis and long-term behavioural effects using an established mouse model that represents the clinically relevant period of anaesthesia during early infancy in humans.METHODS:Six groups of 10 day old mice (P10) were injected with either etomidate 0.3, 3 or 10 mg/kg, propofol 60 mg/kg, ketamine 50 mg/kg or placebo only. Apoptosis in the cerebral cortex and hippocampus was assessed 24 h after treatment (activated caspase-3). Late behavioural effects were tested at 2 months of age (spontaneous activity in a new environment).RESULTS:No evidence was found of differences in activated caspase 3-concentrations among the study groups. Significant late behavioural changes were only observed in the ketamine group.CONCLUSION:A single dose of etomidate in early infant mice at P10 did not produce evidence of cerebral apoptosis or impaired adult motor behaviour.

Developmental neurotoxicity of industrial chemicals and pharmaceuticals have been of growing interest in recent years due to the increasing reports of neuropsychiatric disorders, such as attention deficit hyperactivity disorder (ADHD) and autism. Exposure to these substances during early development may lead to adverse behavior effects manifested at a later phase of life. Pesticides are a wide group of chemicals which are still actively used and residues are found in the environment and in food products. The present study investigated the potential developmental neurotoxic effects of two different types of pesticides, endosulfan and cypermethrin, after a single neonatal exposure during a critical period of brain development. Ten-day-old male NMRI mice were administrated an oral dose of endosulfan or cypermethrin (0.1 or 0.5 mg/kg body weight, respectively). Levels of proteins were measured in the neonatal and adult brain, and adult behavioral testing was performed. The results indicate that both pesticides may induce altered levels of neuroproteins, important for normal brain development, and neurobehavioral abnormalities manifested as altered adult spontaneous behavior and ability to habituate to a novel home environment. The neurotoxic behavioral effects were also presentseveral months after the initial testing, indicating long-lasting or even persistent irreversible effects. Also, the present study suggests a possible link between the altered levels of neuroprotein and changes in behavior when exposed during a critical period of brain development.

In recent times, an increased occurrence of neurodevelopmental disorders, such as neurodevelopmental delays and cognitive abnormalities has been recognized. Exposure to pesticides has been suspected to be a possible cause of these disorders, as these compounds target the nervous system of pests. Due to the similarities of brain development and composition, these pesticides may also be neurotoxic to humans. We studied two different pesticides, chlorpyrifos and carbaryl, which specifically inhibit acetylcholinesterase (AChE) in the nervous system. The aim of the study was to investigate if the pesticides can induce neurotoxic effects, when exposure occurs during a period of rapid brain growth and maturation. The results from the present study show that both compounds can affect protein levels in the developing brain and induce persistent adult behavior and cognitive impairments, in mice neonatally exposed to a single oral dose of chlorpyrifos (0.1, 1.0 or 5 mg/kg body weight) or carbaryl (0.5, 5.0 or 20.0 mg/kg body weight) on postnatal day 10. The results also indicate that the developmental neurotoxic effects induced are not related to the classical mechanism of acute cholinergic hyperstimulation, as the AChE inhibition level (8–12%) remained below the threshold for causing systemic toxicity. The neurotoxic effects are more likely caused by a disturbed neurodevelopment, as similar behavioral neurotoxic effects have been reported in studies with pesticides such as organochlorines, organophosphates, pyrethroids and POPs, when exposed during a critical window of neonatal brain development.

Substances with analgesic properties, tetrahydrocannabinol (THC) and Ibuprofen, affect behaviour and cognitive function differently after developmental exposure

Maternally and lactionally transferred persistent organic pollutants may interfere with CNS development. Here, 10-day-old male mice were exposed to single oral doses of PFOS (perflourooctanosulphonate) or PBDE 99 (2,2',4,4',5-penta-bromodiphenyl ether), and examined for changes in cholinergic gene transcription in the CNS 24h and 7 weeks later. 24h after exposure qPCR analyses revealed decreased transcription of nAChR-β2 and AChE in cortex, and increased mAChR-5 in hippocampus of PFOS treated mice. Neonatal PFOS treatment altered spontaneous behaviour at 2 months of age but did not affect gene transcription in adults. At 2 months of age neonatally PBDE 99 treated mice had altered spontaneous behaviour, and cortical transcription of AChE, nAChR-α4, nAChR-β2 and mAChR-5 were elevated. Our results indicate that PFOS and PBDE 99 affects the developing central cholinergic system by altering gene transcription in cortex and hippocampus, which may in part account for mechanisms causing changes in spontaneous behaviour.

Neonatal paracetamol (acetaminophen) exposure during a defined and critical period of brain development causes altered spontaneous behavior in both male and female adult mice

This thesis aims to investigate the developmental neurotoxic effects of low-dose exposure to ionizing radiation, alone or together with nicotine, during a defined critical period of neonatal brain development in mice. Investigation of neurotoxic effects following fractionated or acute low-dose radiation, resembling the clinical situation during repeated CT scans or radiation delivered to non-target tissue during radiotherapy, and possible interaction effects with other agents, is of great importance for risk and safety evaluation.During mammalian brain development there are defined critical periods for induction of developmental neurotoxic effects. One of these critical periods is called the brain growth spurt (BGS) and involves extensive growth and maturation of the brain. It is known that neonatal exposure during the BGS to low doses of radiation, as well as nicotine, can have a negative impact on neonatal brain development, resulting in impaired cognitive function in the adult mouse. The present studies have shown that developmental neurotoxicity following low-dose irradiation can be induced during the same critical period of brain development as previously has been shown for chemicals. The observed neurotoxicity was manifested as altered spontaneous behaviour and habituation capacity, independent of sex, as well as elevated levels of an Alzheimer-related neuroprotein in the adult mouse. Furthermore, fractionated dose regimes seem to be as potent for induction of neurotoxicity and behavioural disturbances as an equivalent single acute dose. The cholinergic system can be a target system for developmental neurotoxicity of ionizing radiation, either alone or in combination with the cholinergic agent nicotine. Co-exposure to ionizing radiation and nicotine exacerbated the behavioural disturbances and cholinergic system dysfunction observed in these studies.Further studies on developmental neurotoxic effects of low-dose neonatal irradiation and interaction with medical drugs and environmental pollutants are important for the field of radioprotection.

Polybrominated diphenyl ethers (PBDEs), used as flame retardants in polymer products, are reported to cause developmental neurotoxic effects in mammals. The present study have investigated neurotoxic effects arising from neonatal exposure to PBDE 209, including alterations in sex differences, spontaneous behaviour, learning and memory, neuroproteins and altered susceptibility of the cholinergic system in adults.Three-day-old NMRI mice, of both sexes, were exposed to PBDE 209 (2,2',3,3',4,4',5,5',6,6'-decaBDE at 0, 1.4, 6.0 and 14.0 mu mol/kg b.w.). At adult age (2-7 months) a similar developmental neurotoxic effects in both male and female mice were seen, including lack of or reduced habituation to a novel home environment, learning and memory defects, modified response to the cholinergic agent's paraoxon (males) and nicotine (females) indicating increased susceptibility of the cholinergic system. The behavioural defects were dose-response related and persistent. In mice of both sexes and showing behavioural defects, neuroprotein tau was increased. (C) 2014 Elsevier B.V. All rights reserved.