Developmental effects of neonatal fractionated co-exposure to low-dose gamma radiation and paraquat on behaviour in adult mice.

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.

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, 137Cs) 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.

Total body exposure to low-dose ionizing radiation induces long-term alterations to the liver proteome of neonatally exposed mice.

Tens of thousands of people are being exposed daily to environmental low-dose gamma radiation. Epidemiological data indicate that such low radiation doses may negatively affect liver function and result in the development of liver disease. However, the biological mechanisms behind these adverse effects are unknown. The aim of this study was to investigate radiation-induced damage in the liver after low radiation doses. Neonatal male NMRI mice were exposed to total body irradiation on postnatal day 10 using acute single doses ranging from 0.02 to 1.0 Gy. Early (1 day) and late (7 months) changes in the liver proteome were tracked using isotope-coded protein label technology and quantitative mass spectrometry. Our data indicate that low and moderate radiation doses induce an immediate inhibition of the glycolysis pathway and pyruvate dehydrogenase availability in the liver. Furthermore, they lead to significant long-term alterations in lipid metabolism and increased liver inflammation accompanying inactivation of the transcription factor peroxisome proliferator-activated receptor alpha. This study contributes to the understanding of the potential risk of liver damage in populations environmentally exposed to ionizing radiation.

Developmental neurotoxic effects of two pesticides: Behavior and biomolecular studies on chlorpyrifos and carbaryl.

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 5mg/kg body weight) or carbaryl (0.5, 5.0 or 20.0mg/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.

Exposure to lidocaine in early life does not cause negative long-term behavioural changes in mice.

BACKGROUND: The local anaesthetic lidocaine is widely used in the neonatal intensive unit to treat seizures in premature babies. However, other antiepileptics administered during early development in various animal models have shown negative long-term behavioural effects. Since no long-term behavioural data so far exist regarding lidocaine exposure at an early age, we decided to perform this extended follow-up study using a sensitive behavioural test. METHODS: Neonatal mice received a subcutaneous administration of saline or one dose of lidocaine (0.5, 4, or 12 mg kg-1) on postnatal day 10 (P10; peak of the Brain Growth Spurt). A well-established test to detect long-term behavioural alterations was conducted at 2 and 6 months of age, corresponding to early and late adulthood in humans. RESULTS: All animal survived to later testing. No signs of acute toxicity were observed. Lidocaine exposure did not result in any negative behavioural effects during habituation to a new home environment at any of the two studied time points, compared to saline placebo. CONCLUSIONS: Lidocaine does not by itself produce any negative long-term behavioural effects in mice exposed in early life (P10) despite long-term follow-up. This is reassuring regarding the current practice of treating seizures in premature babies with intravenous lidocaine.

Long-term effects of acute low-dose ionizing radiation on the neonatal mouse heart: a proteomic study.

Epidemiological studies establish that children and young adults are especially susceptible to radiation-induced cardiovascular disease (CVD). The biological mechanisms behind the elevated CVD risk following exposure at young age remain unknown. The present study aims to elucidate the long-term effects of ionizing radiation by studying the murine cardiac proteome after exposure to low and moderate radiation doses. NMRI mice received single doses of total body (60)Co gamma-irradiation on postnatal day 10 and were sacrificed 7 months later. Changes in cardiac protein expression were quantified using isotope-coded protein label and tandem mass spectrometry. We identified 32, 31, 66, and 34 significantly deregulated proteins after doses of 0.02, 0.1, 0.5, and 1.0 Gy, respectively. The four doses shared 9 deregulated proteins. Bioinformatics analysis showed that most of the deregulated proteins belonged to a limited set of biological categories, including metabolic processes, inflammatory response, and cytoskeletal structure. The transcription factor peroxisome proliferator-activated receptor alpha was predicted as a common upstream regulator of several deregulated proteins. This study indicates that both adaptive and maladaptive responses to the initial radiation damage persist well into adulthood. It will contribute to the understanding of the long-term consequences of radiation-induced injury and developmental alterations in the neonatal heart.

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 µmol/kg bw), PFOA (1.4 or 14 µmol/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.

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.

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.

Developmental effects of fractionated low-dose exposure to gamma radiation on behaviour and susceptibility of the cholinergic system in mice.

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.

Neonatal exposure to whole body ionizing radiation induces adult neurobehavioural defects: Critical period, dose--response effects and strain and sex comparison.

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.

Developmental neurotoxic effects of two pesticides: Behavior and neuroprotein studies on endosulfan and cypermethrin.

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.

Developmental exposure to the polybrominated diphenyl ether PBDE 209: Neurobehavioural and neuroprotein analysis in adult male and female mice.

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 14.0µmol/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.

Neonatal exposure to a moderate dose of ionizing radiation causes behavioural defects and altered levels of tau protein in mice.

Medical use of ionizing radiation (IR) has great benefits for treatment and diagnostic imaging, but procedures as computerized tomography (CT) may deliver a significant radiation dose to the patient. Recently, awareness has been raised about possible non-cancer consequences from low dose exposure to IR during critical phases of perinatal and/or neonatal brain development. In the present study neonatal NMRI mice were whole body irradiated with a single dose of gamma radiation (0; 350 and 500 mGy) on postnatal day 10 (PND 10). At 2 and 4 months of age, mice of both sexes were observed for spontaneous behaviour in a novel home environment. The neuroproteins CaMKII, GAP-43, synaptophysin and total tau in male mouse cerebral cortex and hippocampus were analysed 24h post-irradiation and in adults at 6 months of age exposed to 0 or 500 mGy on PND 10. A significantly dose-response related deranged spontaneous behaviour in 2- and 4-month-old mice was observed, where both males and females displayed a modified habituation, indicating reduced cognitive function. The dose of 350 mGy seems to be a tentative threshold. Six-month-old male mice showed a significantly increased level of total tau in cerebral cortex after irradiation to 500 mGy compared to controls. This demonstrates that a single moderate dose of IR, given during a defined critical period of brain development, is sufficient to cause persistently reduced cognitive function. Moreover, an elevation of tau protein was observed in male mice displaying reduced cognitive function.

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.

The cognitive defects of neonatally irradiated mice are 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 mechanisms underlying such alterations are unknown. We investigated the long-term effects of low doses of total body gamma radiation 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 0.5 or 1.0 Gy exposure. Alterations in the brain proteome, transcriptome, and several miRNAs were analysed 6-7 months post-irradiation in the hippocampus, dentate gyrus (DG) and cortex. Signalling pathways related to synaptic actin remodelling such as the Rac1-Cofilin pathway were altered in the cortex and hippocampus. Further, synaptic proteins MAP-2 and PSD-95 were increased in the DG and hippocampus (1.0 Gy). The expression of synaptic plasticity genes Arc, c-Fos and CREB was persistently reduced at 1.0 Gy in the hippocampus and cortex. These changes 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 TNFα indicated radiation-induced neuroinflammation. In addition, adult neurogenesis within the DG was persistently negatively affected after irradiation, particularly at 1.0 Gy. CONCLUSION: These data suggest that neurocognitive disorders may be induced in adults when exposed at a young age to low and moderate cranial doses of radiation. This raises concerns about radiation safety standards and regulatory practices.

Dose-dependent behavioral disturbances after a single neonatal Bisphenol A dose.

Bisphenol A is widely used in polymer products for food and beverage packaging, baby bottles, dental sealants, and fillings, adhesives, protective coatings, flame retardants, water supply pipes, and compact discs, and is found in the environment and in placental tissue, fetuses and breast milk. We have recently reported that neonatal exposure to other environmental pollutants can induce persistent aberrations in spontaneous behavior and also affect learning and memory functions in the adult animal. Furthermore, recent reports indicate that pre- and perinatal exposure to Bisphenol A can induce neurotoxic effects. The present study indicates that a single exposure to Bisphenol A on postnatal day 10 can alter adult spontaneous behavior and cognitive function in mice, effects that are both dose-response related and long-lasting/irreversible. Earlier studies on neonatal exposure to persistent organic pollutants (POPs) have shown the cholinergic system to be a target of neurotoxicity, but here only minor effects on the nicotine-induced behavior was seen. Furthermore, Morris swim-maze and the elevated plus-maze did not reveal any effects on spatial learning and anxiety-like behaviors. The present findings show similarities with effects earlier reported after pre- and perinatal exposure to Bisphenol A, and also with effects seen after a single postnatal exposure to other POPs, such as PBDEs, PCBs and PFCs.