Low-Dose Ionizing Radiation Induces Neurotoxicity in the Neonate : Acute or fractionated doses and interaction with xenobiotics in mice

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 Low-Dose Ionizing Radiation in Mice : Developmental Neurotoxic Effects of Single and Fractionated Doses and Interaction with Nicotine

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.

Low-Dose Ionizing Radiation Induces Neurotoxicity in the Neonate : Acute or fractionated doses and interaction with xenobiotics in mice

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.

Low-Dose Ionizing Radiation Induces Neurotoxicity in the Neonate : Acute or fractionated doses and interaction with xenobiotics in mice

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.

DNA integrity under alkaline conditions : An investigation of factors affecting the comet assay

The effect of pH on DNA integrity was assessed using a three-step approach. The comet assay was used on a whole genome level, with three different protocols: neutral (no alkaline unwinding), flash (pH 12.5 with 2.5 min unwinding), and the conventional alkaline protocol (pH>13 with 40 min unwinding). Real-time quantitative PCR (RT-qPCR) was then used to study the isolated DNA, revealing that gene amplification decreased with increasing pH, indicating DNA degradation. Specially designed molecular beacons were used to examine DNA at the molecular level, with or without alkali-labile site (ALS) insertions. At pH 12.5, fluorescence in the hairpins with ALS started to increase after 30 min, while at pH> 13, this increase was already observed after 5 min, indicating a significant increase in DNA strand breaks. Liquid chromatography analysis was also used, demonstrating that the hairpins remained intact up to pH 10, even after 1 h exposure, whereas, at pH 12.5, partial conversion into strand breaks occurred after 30 min. At pH> 13, the hairpins were almost completely degraded after 30 min. The flash protocol effectively detects DNA single- and double-strand breaks and identified these damages after 2.5 min of alkaline treatment at pH 12.5. When the hairpins were exposed to pH 12.5 for 60 min, ALS were converted to strand breaks, demonstrating the sensitivity of this approach to detect changes in DNA structure. These findings indicate that pH poses a substantial risk to DNA integrity, leading to significantly higher background levels of DNA damage compared to conditions closer to neutrality. Our study demonstrates the importance of understanding the influence of pH on DNA stability and provides insights into risks associated with alkaline environments, especially at pH> 13

Perfluorinated chemicals (PFOA) can, by interacting with highly brominated diphenyl ethers (PBDE 209) during a defined period of neonatal brain development, exacerbate neurobehavioural defects

Perfluorinated compounds (PFCs) and polybrominated diphenyl ethers (PBDEs) are ubiquitous persistent environmental compounds, present in humans and at higher levels in infants/children than in adults. This study shows that co-exposure to pentadecafluorooctanoic acid (PFOA) and 2,2 ',3,3 ',4,4 ',5,5 ',6,6'-decaBDE (PBDE 209) can significantly exacerbate developmental neurobehavioural defects. Neonatal male NMRI mice, 3 and 10 days old, were exposed perorally to PBDE 209 (1.4 or 8.0 mu mol/kg bw), PFOA (1.4 or 14 mu mol/kg bw), co-exposed to PBDE 209 and PFOA (at the given doses), or a vehicle (20% fat emulsion) and observed for spontaneous behaviour in a novel home environment when 2 and 4 months old. The behavioural defects observed included hyperactivity and reduced habituation indicating cognitive defects. This interaction appears most likely dependent on the presence of PBDE 209 and/or its metabolites together with PFOA, during a defined critical period of neonatal brain development, corresponding to the perinatal and newborn period in humans

Paracetamol (Acetaminophen) and its Effect on the Developing Mouse Brain

Paracetamol, or acetaminophen (AAP), is the most commonly used analgesic during pregnancy and early life. While therapeutic doses of AAP are considered harmless during these periods, recent findings in both humans and rodents suggest a link between developmental exposure to AAP and behavioral consequences later in life. The aim of this study is to evaluate the impact of neonatal exposure to clinically relevant doses of AAP on adult spontaneous behavior, habituation, memory, learning, and cognitive flexibility later in life using a mouse model. Markers of oxidative stress, axon outgrowth, and glutamatergic transmission were also investigated in the hippocampus during the first 24 h after exposure. In addition, potential long-term effects on synaptic density in the hippocampus have been investigated. In a home cage setting, mice neonatally exposed to AAP (30 + 30 mg/kg, 4 h apart) on postnatal day 10 displayed altered spontaneous behavior and changed habituation patterns later in life compared to controls. These mice also displayed reduced memory, learning and cognitive flexibility compared to control animals in the Morris water maze. An increase of markers for oxidative stress was observed in the hippocampus 6 h after AAP exposure. As AAP is the first choice treatment for pain and/or fever during pregnancy and early life, these results may be of great importance for risk assessment. Here we show that AAP can have persistent negative effects on brain development and suggest that AAP, despite the relatively low doses, is capable to induce acute oxidative stress in the hippocampus.Authors and title in the list of papers of Gaëtan Philippot's thesis: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</p

Ionising Radiation Immediately Impairs Synaptic Plasticity-Associated Cytoskeletal Signalling Pathways in HT22 Cells and in Mouse Brain : An In Vitro/In Vivo Comparison Study

Patients suffering from brain malignancies are treated with high-dose ionising radiation. However, this may lead to severe learning and memory impairment. Preventive treatments to minimise these side effects have not been possible due to the lack of knowledge of the involved signalling pathways and molecular targets. Mouse hippocampal neuronal HT22 cells were irradiated with acute gamma doses of 0.5 Gy, 1.0 Gy and 4.0 Gy. Changes in the cellular proteome were investigated by isotope-coded protein label technology and tandem mass spectrometry after 4 and 24 hours. To compare the findings with the in vivo response, male NMRI mice were irradiated on postnatal day 10 with a gamma dose of 1.0 Gy, followed by evaluation of the cellular proteome of hippocampus and cortex 24 hours post-irradiation. Analysis of the in vitro proteome showed that signalling pathways related to synaptic actin-remodelling were significantly affected at 1.0 Gy and 4.0 Gy but not at 0.5 Gy after 4 and 24 hours. We observed radiation-induced reduction of the miR-132 and Rac1 levels; miR-132 is known to regulate Rac1 activity by blocking the GTPase-activating protein p250GAP. In the irradiated hippocampus and cortex we observed alterations in the signalling pathways similar to those in vitro. The decreased expression of miR-132 and Rac1 was associated with an increase in hippocampal cofilin and phospho-cofilin. The Rac1-Cofilin pathway is involved in the modulation of synaptic actin filament formation that is necessary for correct spine and synapse morphology to enable processes of learning and memory. We suggest that acute radiation exposure leads to rapid dendritic spine and synapse morphology alterations via aberrant cytoskeletal signalling and processing and that this is associated with the immediate neurocognitive side effects observed in patients treated with ionising radiation

Combined Treatment with Low-Dose Ionizing Radiation and Ketamine Induces Adverse Changes in CA1 Neuronal Structure in Male Murine Hippocampi

In children, ketamine sedation is often used during radiological procedures. Combined exposure of ketamine and radiation at doses that alone did not affect learning and memory induced permanent cognitive impairment in mice. The aim of this study was to elucidate the mechanism behind this adverse outcome. Neonatal male NMRI mice were administered ketamine (7.5 mg kg(-1)) and irradiated (whole-body, 100 mGy or 200 mGy, Cs-137) one hour after ketamine exposure on postnatal day 10. The control mice were injected with saline and sham-irradiated. The hippocampi were analyzed using label-free proteomics, immunoblotting, and Golgi staining of CA1 neurons six months after treatment. Mice co-exposed to ketamine and low-dose radiation showed alterations in hippocampal proteins related to neuronal shaping and synaptic plasticity. The expression of brain-derived neurotrophic factor, activity-regulated cytoskeleton-associated protein, and postsynaptic density protein 95 were significantly altered only after the combined treatment (100 mGy or 200 mGy combined with ketamine, respectively). Increased numbers of basal dendrites and branching were observed only after the co-exposure, thereby constituting a possible reason for the displayed alterations in behavior. These data suggest that the risk of radiation-induced neurotoxic effects in the pediatric population may be underestimated if based only on the radiation dose

The cognitive defects of neonatally irradiated miceare accompanied by changed synaptic plasticity,adult neurogenesis and neuroinflammation

Background/purpose of the study: Epidemiological evidence suggests that low doses of ionising radiation(≤1.0 Gy) produce persistent alterations in cognition if the exposure occurs at a young age. The mechanismsunderlying such alterations are unknown. We investigated the long-term effects of low doses of total body gammaradiation on neonatally exposed NMRI mice on the molecular and cellular level to elucidate neurodegeneration.Results: Significant alterations in spontaneous behaviour were observed at 2 and 4 months following a single 0.5or 1.0 Gy exposure. Alterations in the brain proteome, transcriptome, and several miRNAs were analysed 6–7months post-irradiation in the hippocampus, dentate gyrus (DG) and cortex. Signalling pathways related to synapticactin remodelling such as the Rac1-Cofilin pathway were altered in the cortex and hippocampus. Further, synapticproteins MAP-2 and PSD-95 were increased in the DG and hippocampus (1.0 Gy). The expression of synapticplasticity genes Arc, c-Fos and CREB was persistently reduced at 1.0 Gy in the hippocampus and cortex. Thesechanges were coupled to epigenetic modulation via increased levels of microRNAs (miR-132/miR-212, miR-134).Astrogliosis, activation of insulin-growth factor/insulin signalling and increased level of microglial cytokine TNFαindicated radiation-induced neuroinflammation. In addition, adult neurogenesis within the DG was persistentlynegatively affected after irradiation, particularly at 1.0 Gy.Conclusion: These data suggest that neurocognitive disorders may be induced in adults when exposed at a youngage to low and moderate cranial doses of radiation. This raises concerns about radiation safety standards andregulatory practices