Exposure to tungsten particles via inhalation triggers early toxicity marker expression in the rat brain.

OBJECTIVE: Our work is focused on tungsten, considered as an emerging contaminant. Its environmental dispersion is partly due to mining and military activities. Exposure scenario can also be occupational, in areas such as the hard metal industry and specific nuclear facilities. Our study investigated the cerebral effects induced by the inhalation of tungsten particles. METHODS: Inhalation exposure campaigns were carried out at two different concentrations (5 and 80 mg/m3) in single and repeated modes (4 consecutive days) in adult rats within a nose-only inhalation chamber. Processes involved in brain toxicity were investigated 24 h after exposure. RESULTS AND DISCUSSION: Site-specific effects in terms of neuroanatomy and concentration-dependent changes in specific cellular actors were observed. Results obtained in the olfactory bulb suggest a potential early effect on the survival of microglial cells. Depending on the mode of exposure, these cells showed a decrease in density accompanied by an increase in an apoptotic marker. An abnormal phenotype of the nuclei of mature neurons, suggesting neuronal suffering, was also observed in the frontal cortex, and can be linked to the involvement of oxidative stress. The differential effects observed according to exposure patterns could involve two components: local (brain-specific) and/or systemic. Indeed, tungsten, in addition to being found in the lungs and kidneys, was present in the brain of animals exposed to the high concentration. CONCLUSION: Our data question the perceived innocuity of tungsten relative to other metals and raise hypotheses regarding possible adaptive or neurotoxic mechanisms that could ultimately alter neuronal integrity.

Renal toxicity and biokinetics models after repeated uranium instillation.

During nuclear fuel processing, workers can potentially be exposed to repeated inhalations of uranium compounds. Uranium nephrotoxicity is well documented after acute uranium intake, but it is controversial after long-term or protracted exposure. This study aims to analyze the nephrotoxicity threshold after repeated uranium exposure through upper airways and to investigate the resulting uranium biokinetics in comparison to reference models. Mice (C57BL/6J) were exposed to uranyl nitrate (0.03-3 mg/kg/day) via intranasal instillation four times a week for two weeks. Concentrations of uranium in urines and tissues were measured at regular time points (from day 1 to 91 post-exposure). At each exposure level, the amount of uranium retained in organs/tissues (kidney, lung, bone, nasal compartment, carcass) and excreta (urine, feces) reflected the two consecutive weeks of instillation except for renal uranium retention for the highest uranium dose. Nephrotoxicity biomarkers, KIM-1, clusterin and osteopontin, are induced from day 4 to day 21 and associated with changes in renal function (arterial fluxes) measured using non-invasive functional imaging (Doppler-ultrasonography) and confirmed by renal histopathological analysis. These results suggest that specific biokinetic models should be developed to consider altered uranium excretion and retention in kidney due to nephrotoxicity. The threshold is between 0.25 and 1 mg/kg/day after repeated exposure to uranium via upper airways.

Development of an adverse outcome pathway for radiation-induced microcephaly via expert consultation and machine learning.

BACKGROUND: Brain development during embryogenesis and in early postnatal life is particularly complex and involves the interplay of many cellular processes and molecular mechanisms, making it extremely vulnerable to exogenous insults, including ionizing radiation (IR). Microcephaly is one of the most frequent neurodevelopmental abnormalities that is characterized by small brain size, and is often associated with intellectual deficiency. Decades of research span from epidemiological data on in utero exposure of the A-bomb survivors, to studies on animal and cellular models that allowed deciphering the most prominent molecular mechanisms leading to microcephaly. The Adverse Outcome Pathway (AOP) framework is used to organize, evaluate and portray the scientific knowledge of toxicological effects spanning different biological levels of organizations, from the initial interaction with molecular targets to the occurrence of a disease or adversity. In the present study, the framework was used in an attempt to organize the current scientific knowledge on microcephaly progression in the context of ionizing radiation (IR) exposure. This work was performed by a group of experts formed during a recent workshop organized jointly by the Multidisciplinary European Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE) associations to present the AOP approach and tools. Here we report on the development of a putative AOP for congenital microcephaly resulting from IR exposure based on discussions of the working group and we emphasize the use of a novel machine-learning approach to assist in the screening of the available literature to develop AOPs. CONCLUSION: The expert consultation led to the identification of crucial biological events for the progression of microcephaly upon exposure to IR, and highlighted current knowledge gaps. The machine learning approach was successfully used to screen the existing knowledge and helped to rapidly screen the body of evidence and in particular the epidemiological data. This systematic review approach also ensured that the analysis was sufficiently comprehensive to identify the most relevant data and facilitate rapid and consistent AOP development. We anticipate that as machine learning approaches become more user-friendly through easy-to-use web interface, this would allow AOP development to become more efficient and less time consuming.

Brain accumulation of inhaled uranium in the rat depends on aerosol concentration, exposure repetitions, particle size and solubility.

A rostro-caudal gradient of uranium (U) in the brain has been suggested after its inhalation. To study the factors influencing this mapping, we first used 30-min acute inhalation at 56 mg/m3 of the relatively soluble form UO4 in the rat. These exposure parameters were then used as a reference in comparison with the other experimental conditions. Other groups received acute inhalation at different concentrations, repeated low dose inhalation of UO4 (10 exposures) or acute low dose inhalation of the insoluble form UO2. At 24 h after the last exposure, all rats showed a brain U accumulation with a rostro-caudal gradient as compared to controls. However, the total concentration to the brain was greater after repeated exposure than acute exposure, demonstrating an accumulative effect. In comparison with the low dose soluble U exposure, a higher accumulation in the front of the brain was observed after exposure to higher dose, to insoluble particles and following repetition of exposures, thus demonstrating a dose effect and influences of solubility and repetition of exposures. In the last part, exposure to ultrafine U particles made it possible to show 24 h after exposure the presence of U in the brain according to a rostro-caudal gradient. Finally, the time-course after exposure to micronic or nanometric U particles has revealed greater residence times for nanoparticles.

Evaluation of the Nose-to-Brain Transport of Different Physicochemical Forms of Uranium after Exposure via Inhalation of a UO4 Aerosol in the Rat.

BACKGROUND: Health-risk issues are raised concerning inhalation of particulate pollutants that are thought to have potential hazardous effects on the central nervous system. The brain is presented as a direct target of particulate matter (PM) exposure because of the nose-to-brain pathway involvement. The main cause of contamination in nuclear occupational activities is related to exposure to aerosols containing radionuclides, particularly uranium dust. It has been previously demonstrated that instilled solubilized uranium in the rat nasal cavity is conveyed to the brain via the olfactory nerve. OBJECTIVE: The aim of this study was to analyze the anatomical localization of uranium compounds in the olfactory system after in vivo exposure to a polydisperse aerosol of uranium tetraoxide (UO4) particles. METHODS: The olfactory neuroepithelium (OE) and selected brain structures-olfactory bulbs (OB), frontal cortex (FC), hippocampus (HIP), cerebellum (Cer), and brainstem (BS)-were microdissected 4 h after aerosol inhalation via a nose-only system in adult rats. Tissues were subjected to complementary analytical techniques. RESULTS: Uranium concentrations measured by inductively coupled plasma mass spectrometry (ICP-MS) were significantly higher in all brain structures from exposed animals compared with their respective controls. We observed that cerebral uranium concentrations followed an anteroposterior gradient with typical accumulation in the OB, characteristic of a direct olfactory transfer of inhaled compounds. Secondary ion mass spectrometry (SIMS) microscopy and transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy (TEM-EDX) were used in order to track elemental uranium in situ in the olfactory epithelium. Elemental uranium was detected in precise anatomical regions: olfactory neuron dendrites, paracellular junctions of neuroepithelial cells, and olfactory nerve tracts (around axons and endoneural spaces). CONCLUSION: These neuroanatomical observations in a rat model are consistent with the transport of elemental uranium in different physicochemical forms (solubilized, nanoparticles) along olfactory nerve bundles after inhalation of UO4 microparticles. This work contributes to knowledge of the mechanistic actions of particulate pollutants on the brain. https://doi.org/10.1289/EHP4927.

In vivo animal studies help achieve international consensus on standards and guidelines for health risk estimates for chronic exposure to low levels of tritium in drinking water.

Existing and future nuclear fusion technologies involve the production and use of large quantities of tritium, a highly volatile, but low toxicity beta-emitting isotope of hydrogen. Tritium has received international attention because of public and scientific concerns over its release to the environment and the potential health impact of its internalization. This article provides a brief summary of the current state of knowledge of both the biological and regulatory aspects of tritium exposure; it also explores the gaps in this knowledge and provides recommendations on the best ways forward for improving our understanding of the health effects of low-level exposure to it. Linking health effects specifically to tritium exposure is challenging in epidemiological studies due to high uncertainty in tritium dosimetry and often suboptimal cohort sizes. We therefore argued that limits for tritium in drinking water should be based on evidence derived from controlled in vivo animal tritium toxicity studies that use realistically low levels of tritium. This article presents one such mouse study, undertaken within an international collaboration, and discusses the implications of its main findings, such as the similarity of the biokinetics of tritiated water (HTO) and organically bound tritium (OBT) and the higher biological effectiveness of OBT. This discussion is consistent with the position expressed in this article that in vivo animal tritium toxicity studies carried out within large, multi-partner collaborations allow evaluation of a great variety of health-related endpoints and essential to the development of international consensus on the regulation of tritium levels in the environment. Environ. Mol. Mutagen. 59:586-594, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Biomarkers for Uranium Risk Assessment for the Development of the CURE (Concerted Uranium Research in Europe) Molecular Epidemiological Protocol.

Despite substantial experimental and epidemiological research, there is limited knowledge of the uranium-induce health effects after chronic low-dose exposures in humans. Biological markers can objectively characterize pathological processes or environmental responses to uranium and confounding agents. The integration of such biological markers into a molecular epidemiological study would be a useful approach to improve and refine estimations of uranium-induced health risks. To initiate such a study, Concerted Uranium Research in Europe (CURE) was established, and involves biologists, epidemiologists and dosimetrists. The aims of the biological work package of CURE were: 1. To identify biomarkers and biological specimens relevant to uranium exposure; 2. To define standard operating procedures (SOPs); and 3. To set up a common protocol (logistic, questionnaire, ethical aspects) to perform a large-scale molecular epidemiologic study in uranium-exposed cohorts. An intensive literature review was performed and led to the identification of biomarkers related to: 1. retention organs (lungs, kidneys and bone); 2. other systems/organs with suspected effects (cardiovascular system, central nervous system and lympho-hematopoietic system); 3. target molecules (DNA damage, genomic instability); and 4. high-throughput methods for the identification of new biomarkers. To obtain high-quality biological materials, SOPs were established for the sampling and storage of different biospecimens. A questionnaire was developed to assess potential confounding factors. The proposed strategy can be adapted to other internal exposures and should improve the characterization of the biological and health effects that are relevant for risk assessment.

Concerted Uranium Research in Europe (CURE): toward a collaborative project integrating dosimetry, epidemiology and radiobiology to study the effects of occupational uranium exposure.

The potential health impacts of chronic exposures to uranium, as they occur in occupational settings, are not well characterized. Most epidemiological studies have been limited by small sample sizes, and a lack of harmonization of methods used to quantify radiation doses resulting from uranium exposure. Experimental studies have shown that uranium has biological effects, but their implications for human health are not clear. New studies that would combine the strengths of large, well-designed epidemiological datasets with those of state-of-the-art biological methods would help improve the characterization of the biological and health effects of occupational uranium exposure. The aim of the European Commission concerted action CURE (Concerted Uranium Research in Europe) was to develop protocols for such a future collaborative research project, in which dosimetry, epidemiology and biology would be integrated to better characterize the effects of occupational uranium exposure. These protocols were developed from existing European cohorts of workers exposed to uranium together with expertise in epidemiology, biology and dosimetry of CURE partner institutions. The preparatory work of CURE should allow a large scale collaborative project to be launched, in order to better characterize the effects of uranium exposure and more generally of alpha particles and low doses of ionizing radiation.

Chronic uranium contamination alters spinal motor neuron integrity via modulation of SMN1 expression and microglia recruitment.

Consequences of uranium contamination have been extensively studied in brain as cognitive function impairments were observed in rodents. Locomotor disturbances have also been described in contaminated animals. Epidemiological studies have revealed increased risk of motor neuron diseases in veterans potentially exposed to uranium during their military duties. To our knowledge, biological response of spinal cord to uranium contamination has not been studied even though it has a crucial role in locomotion. Four groups of rats were contaminated with increasing concentrations of uranium in their drinking water compared to a control group to study cellular mechanisms involved in locomotor disorders. Nissl staining of spinal cord sections revealed the presence of chromatolytic neurons in the ventral horn. This observation was correlated with a decreased number of motor neurons in the highly contaminated group and a decrease of SMN1 protein expression (Survival of Motor Neuron 1). While contamination impairs motor neuron integrity, an increasing number of microglial cells indicates the trigger of a neuroinflammation process. Potential overexpression of a microglial recruitment chemokine, MCP-1 (Monocyte Chimioattractant Protein 1), by motor neurons themselves could mediate this process. Studies on spinal cord appear to be relevant for risk assessment of population exposed via contaminated food and water.

Intranasal exposure to uranium results in direct transfer to the brain along olfactory nerve bundles.

AIMS: Uranium olfactory uptake after intranasal exposure raises some concerns for people potentially exposed to airborne radionuclide contamination as the brain could be a direct target for these contaminants. A model of nasal instillation was used to elucidate the transport mechanisms of uranium to the brain and to map its localization. METHODS: Increasing concentrations of depleted uranium containing solutions were instilled in the nasal cavity of adult male rats. Uranium concentrations were measured using inductively coupled plasma-mass spectrometry (ICP-MS) 4 h after instillation. Olfactory neuroepithelium cytoarchitecture was studied using immunohistochemistry experiments. Secondary ion mass spectrometry (SIMS) microscopy was performed to localize uranium in the olfactory system. RESULTS: ICP-MS analyses showed a frontal accumulation of uranium in the olfactory bulbs associated with a smaller increase in more caudal brain regions (frontal cortex, hippocampus and cerebellum). Uranium concentrations in the olfactory bulbs do not reach a saturation point. Olfactory nerve bundle integrity is not affected by uranium as revealed by immunohistochemistry. SIMS microscopy allowed us to show that uranium localization is mainly restricted to the olfactory neuroepithelium and around olfactory nerve bundles. It is subsequently detected in the olfactory nerve layer of the olfactory bulb. DISCUSSION: These results suggest the existence of a transcellular passage from the mucosa to the perineural space around axon bundles. Uranium bypasses the blood brain barrier and is conveyed to the brain via the cerebrospinal fluid along the olfactory nerve. Future studies might need to integrate this new contamination route to assess uranium neurotoxicity after nasal exposure.

Serum-free medium provides a clinically relevant method to increase olfactory ensheathing cell numbers in olfactory mucosa cell culture.

There is much evidence to suggest that transplanted olfactory ensheathing cells may ameliorate the functional deficits associated with injuries to the nervous system, especially the spinal cord. For clinical implementation of this strategy it will be necessary to derive large numbers of these cells from an accessible and, preferably, autologous source, implying that olfactory mucosa would be ideal. Although olfactory ensheathing cells can be derived from olfactory mucosa, in routine culture conditions the proportion of these cells is unacceptably low for clinical purposes. This study compared the effect of culturing dissociated olfactory bulb and olfactory mucosa in two different media: one containing serum and one serum free. The results indicate that olfactory ensheathing cell proportion, and absolute cell numbers, is greatly increased in serum-free conditions. Further analysis suggests that serum-free medium has a differential effect on contaminating fibronectin-positive and p75-positive cells from olfactory bulb and olfactory mucosa. This study demonstrates that serum-free culture conditions provide a simple and useful means of deriving a sufficient number of olfactory ensheathing cells for transplantation and reveals a difference in biological behavior of the cells contained within olfactory bulb and olfactory mucosa.

Contrasting effects of basic fibroblast growth factor and epidermal growth factor on mouse neonatal olfactory mucosa cells.

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) affect proliferation and survival of many cell types, but their role in the maintenance of olfactory mucosa cells remains unclear. In the neonatal mouse olfactory mucosa, cell proliferation mainly occurs in the neuroepithelium and, to a lesser extent, in the lamina propria. To establish whether bFGF and EGF affect proliferation and/or survival of these cells, we isolated olfactory mucosa cells from the neonatal mouse and cultured them as free-floating spheres under bFGF or EGF stimulation. Our data demonstrate that bFGF is a mitogen for the rapidly dividing cells (olfactory neuronal precursors and olfactory ensheathing cells), and also a survival factor for both slowly and rapidly dividing cells of the olfactory mucosa. In contrast, EGF appears to be primarily a survival factor for both the olfactory stem and precursor cells.

Calponin is expressed by fibroblasts and meningeal cells but not olfactory ensheathing cells in the adult peripheral olfactory system.

Olfactory ensheathing cells (OECs), the principal glial cells of the peripheral olfactory system, have many phenotypic similarities with Schwann cells of the peripheral nervous system. This makes reliably distinguishing these two cells types difficult, especially following transplantation into areas of injury in the central nervous system. In an attempt to identify markers by which these two cells types can be distinguished, a recent proteomic analysis of fetal OECs and adult Schwann cells identified the actin-binding protein calponin as a potential marker expressed by OECs but not Schwann cells. Since many studies designed with the translational goal of autologous transplantation in mind have used adult OECs, this study examined the expression of calponin by adult OECs, both in vivo within the peripheral olfactory system and in vitro. Calponin colocalized with strongly fibronectin positive fibroblasts in the olfactory mucosa (OM) and meningeal cells in the olfactory bulb (OB) but not with S100beta or neuropeptide-Y positive OECs. In tissue culture, calponin was strongly expressed by fibronectin-expressing fibroblasts from OM, sciatic nerve and skin and by meningeal cells from the OB, but not by p75(NTR)- and S100beta-expressing OECs. These data, supported by Western blotting, indicate that calponin can not be used to distinguish adult OECs and Schwann cells.

Serum-Free Medium Provides a Clinically Relevant Method to Increase Olfactory Ensheathing Cell Numbers in Olfactory Mucosa Cell Culture.

There is much evidence to suggest that transplanted olfactory ensheathing cells may ameliorate the functional deficits associated with injuries to the nervous system, especially the spinal cord. For clinical implementation of this strategy it will be necessary to derive large numbers of these cells from an accessible and, preferably, autologous source, implying that olfactory mucosa would be ideal. Although olfactory ensheathing cells can be derived from olfactory mucosa, in routine culture conditions the proportion of these cells is unacceptably low for clinical purposes. This study compared the effect of culturing dissociated olfactory bulb and olfactory mucosa in two different media: one containing serum and one serum free. The results indicate that olfactory ensheathing cell proportion, and absolute cell numbers, is greatly increased in serum-free conditions. Further analysis suggests that serum-free medium has a differential effect on contaminating fibronectin-positive and p75-positive cells from olfactory bulb and olfactory mucosa. This study demonstrates that serum-free culture conditions provide a simple and useful means of deriving a sufficient number of olfactory ensheathing cells for transplantation and reveals a difference in biological behavior of the cells contained within olfactory bulb and olfactory mucosa.

Comparison of cell populations derived from canine olfactory bulb and olfactory mucosal cultures.

OBJECTIVE: To evaluate the numbers and proportions of olfactory ensheathing cells (OECs) in cell cultures derived from the olfactory bulb (OB) and olfactory mucosa of dogs. ANIMALS: 7 dogs. PROCEDURES: OB tissue and olfactory mucosa from the nasal cavity and frontal sinus were obtained from euthanatized dogs and prepared for cell culture. At 7, 14, and 21 days of culture in vitro, numbers and proportions of OECs, astrocytes, and fibroblasts were determined via immunocytochemistry. Antibody against the low-affinity nerve growth factor receptor p75 was used to identify OECs, antibody against glial fibrillary acidic protein was used to identify astrocytes, and antibody against fibronectin was used to identify fibroblasts. RESULTS: Cultured OECs derived from the olfactory mucosa of the nasal cavity and frontal sinus had similar characteristics. However, whereas OECs in the OB cell cultures constituted approximately 50% of the cells at 7 days and approximately 75% at 21 days the proportion of OECs in cultures derived from both mucosal types was much lower, with approximately 40% OECs at 7 days and approximately 25% at 21 days. Analysis of OEC numbers revealed that these changes were accompanied by corresponding decreases and increases in the population of cells with fibronectin receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Although olfactory mucosal cell cultures yielded a sufficient number of OECs for spinal cord transplantation procedures in dogs, modification of culture conditions would be required to ensure that the derived cell population contained a sufficient proportion of OECs.

Local synthesis and dual actions of progesterone in the nervous system: neuroprotection and myelination.

Progesterone (PROG) is synthesized in the brain, spinal cord and peripheral nerves. Its direct precursor pregnenolone is either derived from the circulation or from local de novo synthesis as cytochrome P450scc, which converts cholesterol to pregnenolone, is expressed in the nervous system. Pregnenolone is converted to PROG by 3beta-hydroxysteroid dehydrogenase (3beta-HSD). In situ hybridization studies have shown that this enzyme is expressed throughout the rat brain, spinal cord and dorsal root ganglia (DRG) mainly by neurons. Macroglial cells, including astrocytes, oligodendroglial cells and Schwann cells, also have the capacity to synthesize PROG, but expression and activity of 3beta-HSD in these cells are regulated by cellular interactions. Thus, Schwann cells convert pregnenolone to PROG in response to a neuronal signal. There is now strong evidence that P450scc and 3beta-HSD are expressed in the human nervous system, where PROG synthesis also takes place. Although there are only a few studies addressing the biological significance of PROG synthesis in the brain, the autocrine/paracrine actions of locally synthesized PROG are likely to play an important role in the viability of neurons and in the formation of myelin sheaths. The neuroprotective effects of PROG have recently been documented in a murine model of spinal cord motoneuron degeneration, the Wobbler mouse. The treatment of symptomatic Wobbler mice with PROG for 15 days attenuated the neuropathological changes in spinal motoneurons and had beneficial effects on muscle strength and the survival rate of the animals. PROG may exert its neuroprotective effects by regulating expression of specific genes in neurons and glial cells, which may become hormone-sensitive after injury. The promyelinating effects of PROG were first documented in the mouse sciatic nerve and in co-cultures of sensory neurons and Schwann cells. PROG also promotes myelination in the brain, as shown in vitro in explant cultures of cerebellar slices and in vivo in the cerebellar peduncle of aged rats after toxin-induced demyelination. Local synthesis of PROG in the brain and the neuroprotective and promyelinating effects of this neurosteroid offer interesting therapeutic possibilities for the prevention and treatment of neurodegenerative diseases, for accelerating regenerative processes and for preserving cognitive functions during aging.

Developmental expression of genes involved in neurosteroidogenesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase in the rat brain.

In the central nervous system, neurosteroids, in particular progesterone, have neurotrophic and neuroprotective effects. We thus decided to study the developmental expression of 3beta-hydroxysteroid-dehydrogenase/Delta(5)-Delta(4) isomerase (3betaHSD), an enzyme that converts pregnenolone to progesterone, in the male rat brain at 0, 7, 14, and 70 d after birth. 3betaHSD mRNA was widely distributed throughout the brain, as shown by in situ hybridization. At all ages, the same cerebral structures were labeled, but the intensity of the hybridization signal constantly decreased during postnatal development. As the hippocampus is of particular interest because of its neuronal plasticity, we chose to quantify the changes in 3betaHSD mRNA levels as well as progesterone and pregnenolone concentrations in this structure. Quantitative in situ hybridization confirmed a decrease in the expression of 3betaHSD mRNA with progressing age, as revealed by a significant reduction in the density of silver grains per cell in the CA1 layer. This decrease was confirmed by semiquantitative RT-PCR on hippocampal samples. Concentrations of hippocampal pregnenolone and progesterone measured by gas chromatography/mass spectrometry were highest on the day of birth and lower at the other ages. Plasma concentrations of these steroids were lower than those in the hippocampus, suggesting that they may have been mostly synthesized in situ since the day of birth. These results demonstrate variations in the expression of a gene coding for an enzyme critically involved in progesterone synthesis in the hippocampus throughout postnatal development.