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.

Multigenerational Exposure to Uranium Changes Sperm Metabolome in Rats.

Male infertility is a major public health issue that can be induced by a host of lifestyle risk factors such as environment, nutrition, smoking, stress, and endocrine disruptors. Regarding the human population exposed to uranium, it is necessary to explore these effects on male reproduction in multigenerational studies. The sensitivity of mass spectrometry (MS)-based methods has already proved to be extremely useful in metabolite identification in rats exposed to low doses of uranium, but also in human sperm. We applied this method to rat sperm over three generations (F0, F1 and F2) with multigenerational uranium exposure. Our results show a significant content of uranium in generation F0, and a reduction in the pregnancy rate only in generation F1. Based on principal component analysis (PCA), we observed discriminant profiles between generations. The partial least squares discriminant analysis (PLS-DA) of the 48 annotated variables confirmed that parental exposure of generation F0 (during both the preconceptional and prenatal periods) can have metabolic effects on spermatozoa for the next two generations. Metabolomics applied to epididymal spermatozoa is a novel approach to detecting the multigenerational effects of uranium in an experimental model, but could be also recommended to identify potential biomarkers evaluating the impact of uranium on sperm in exposed infertile men.

Early Metabolomic Markers of Acute Low-Dose Exposure to Uranium in Rats.

Changes in metabolomics over time were studied in rats to identify early biomarkers and highlight the main metabolic pathways that are significantly altered in the period immediately following acute low-dose uranium exposure. A dose response relationship study was established from urine and plasma samples collected periodically over 9 months after the exposure of young adult male rats to uranyl nitrate. LC-MS and biostatistical analysis were used to identify early discriminant metabolites. As expected, low doses of uranium lead to time-based non-toxic biological effects, which can be used to identify early and delayed markers of exposure in both urine and plasma samples. A combination of surrogate markers for uranium exposure was validated from the most discriminant early markers for making effective predictions. N-methyl-nicotinamide, kynurenic acid, serotonin, tryptophan, tryptamine, and indole acetic acid associated with the nicotinate-nicotinamide and tryptophan pathway seem to be one of the main biological targets, as shown previously for chronic contaminations and completed, among others, by betaine metabolism. This study can be considered as a proof of concept for the relevance of metabolomics in the field of low-dose internal contamination by uranium, for the development of predictive diagnostic tests usable for radiotoxicological monitoring.

Renal adaptive response to exposure to low doses of uranyl nitrate and sodium fluoride in mice.

BACKGROUND: Despite their differences in physicochemical properties, both uranium (U) and fluoride (F) are nephrotoxicants at high doses but their adverse effects at low doses are still the subject of debate. METHODS: This study aims to improve the knowledge of the biological mechanisms involved through an adaptive response model of C57BL/6 J mice chronically exposed to low priming doses of U (0, 10, 20 and 40 mg/L) or F (0, 15, 30 and 50 mg/L) and then challenged with acute exposure of 5 mg/kg U or 7.5 mg/kg NaF. RESULTS: We showed that an adaptive response occurred with priming exposures to 20 mg/L U and 50 mg/L F, with decreased levels of the biomarkers KIM-1 and CLU compared to those in animals that received the challenge dose only (positive control). The adaptive mechanisms involved a decrease in caspase 3/7 activities in animals exposed to 20 mg/L U and a decrease in in situ VCAM expression in mice exposed to 50 mg/L F. However, autophagy and the UPR were induced independently of priming exposure to U or F and could not be identified as adaptive mechanisms to U or F. CONCLUSION: Taken together, these results allow us to identify renal adaptive responses to U and F at doses of 20 and 50 mg/L, probably through decrease apoptosis and inflammatory cell recruitment.

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.

Multigenerational exposure to uranium changes morphometric parameters and global DNA methylation in rat sperm.

There is increasing evidence that environmental exposures early in fetal development influence phenotype and give rise to disease risk in the next generations. We previously found that lifelong exposure to uranium, an environmental contaminant, induced subtle testicular and hormonal defects; however, its impact on the reproductive system of multiple subsequent generations was unexplored. Herein, rats were exposed to a supra-environmental and non-nephrotoxic concentration of natural uranium (U, 40 mg·L-1 of drinking water) from postnatal life to adulthood (F0), during fetal life (F1), and only as the germ cells from the F1 generation (F2). General parameters (reproductive indices, epididymal weight) and sperm morphology were assessed in the three generations. In order to identify the epigenetic effects of U, we analyzed also the global DNA methylation profile and described for the first time the mRNA expression levels of markers involved in the (de)methylation system in rat epididymal spermatozoa. Our results showed that the F1 generation had a reduced pregnancy rate. Despite the sperm number being unmodified, sperm morphology was affected in the F0, F1 and F2 generations. Morphometric analysis for ten parameters was detailed for each generation. No common parameter was detected between the three generations, but the head and the middle-piece were always modified in the abnormal sperms. In the F1 U-exposed generation, the total number of abnormal sperm was significantly higher than in the F0 and F2 generations, suggesting that fetal exposure to uranium was more deleterious. This effect could be associated with the pregnancy rate to produce the F2 generation. Interestingly, global DNA methylation analysis showed also hypomethylation in the sperm DNA of the last F2 generation. In conclusion, our study demonstrates that uranium can induce morphological sperm defects and changes in the DNA methylation level after multigenerational exposure. The epigenetic transgenerational inheritance of U-induced reproductive defects should be assessed in further experiments.

In Vivo Comparison of the Phenotypic Aspects and Molecular Mechanisms of Two Nephrotoxic Agents, Sodium Fluoride and Uranyl Nitrate.

Because of their nephrotoxicity and presence in the environment, uranium (U) and fluoride (F) represent risks to the global population. There is a general lack of knowledge regarding the mechanisms of U and F nephrotoxicity and the underlying molecular pathways. The present study aims to compare the threshold of the appearance of renal impairment and to study apoptosis and inflammation as mechanisms of nephrotoxicity. C57BL/6J male mice were intraperitoneally treated with a single dose of U (0, 2, 4 and 5 mg/kg) or F (0, 2, 5, 7.5 and 10 mg/kg) and euthanized 72 h after. Renal phenotypic characteristics and biological mechanisms were evaluated by urine biochemistry, gene/protein expression, enzyme activity, and (immuno)histological analyses. U and F exposures induced nephrotoxicity in a dose-dependent manner, and the highest concentrations induced severe histopathological alterations as well as increased gene expression and urinary excretion of nephrotoxicity biomarkers. KIM-1 gene expression was induced starting at 2 mg/kg U and 7.5 mg/kg F, and this increase in expression was confirmed through in situ detection of this biomarker of nephrotoxicity. Both treatments induced inflammation as evidenced by cell adhesion molecule expression and in situ levels, whereas caspase 3/7-dependent apoptosis was increased only after U treatment. Overall, a single dose of F or U induced histopathologic evidence of nephrotoxicity renal impairment and inflammation in mice with thresholds under 7.5 mg/kg and 4 mg/kg, respectively.

Applying a multiscale systems biology approach to study the effect of chronic low-dose exposure to uranium in rat kidneys.

Purpose: To examine the effects of low-dose exposure to uranium with a systems biology approach, a multiscale high-throughput multi-omics analysis was applied with a protocol for chronic exposure to the rat kidney. Methods: Male and female rats were contaminated for nine months through their drinking water with a nontoxic solution of uranyl nitrate. A multiscale approach enabled clinical monitoring associated with metabolomic and transcriptomic (mRNA and microRNA) analyses. Results: A sex-interaction effect was observed in the kidney, urine, and plasma metabolomes of contaminated rats. Moreover, urine and kidney metabolic profiles correlated and confirmed that the primary dysregulated metabolisms are those of nicotinate-nicotinamide and of unsaturated fatty acid biosynthesis. Upstream of the metabolic pathways, transcriptomic profiles of the kidney reveal gene activity focused on gene regulation mechanisms, cell signaling, cell structure, developmental processes, and cell proliferation. Examination of epigenetic post-transcriptional gene regulation processes showed significant dysregulation of 70 micro-RNAs. The multi-omics approach highlighted the activities of the cells' biological processes on multiple scales through analysis of gene expression, confirmed by changes observed in the metabolome. Conclusion: Our results showed changes in multi-omic profiles of rats exposed to low doses of uranium contamination, compared with controls. These changes involved gene expression as well as modifications in the transcriptome and the metabolome. The metabolomic profile confirmed that the main molecular targets of uranium in kidney cells are the metabolism of nicotinate-nicotinamide and the biosynthesis of unsaturated fatty acids. Additionally, gene expression analysis showed that the metabolism of fatty acids is targeted by processes associated with cell function. These results demonstrate that multiscale systems biology is useful in elucidating the most discriminative pathways from genomic to metabolomic levels for assessing the biological impact of this low-level environmental exposure, i.e. the exposome.

Low dose of uranium induces multigenerational epigenetic effects in rat kidney.

PURPOSE: A protocol of chronic exposure to low dose of uranium was established in order to distinguish the sexual differences and the developmental process that are critical windows for epigenetic effects over generations. METHODS: Both male and female rats were contaminated through their drinking water with a non-toxic solution of uranyl nitrate for 9 months. The exposed generation (F0) and the following two generations (F1 and F2) were examined. Clinical monitoring, global DNA methylation profile and DNA methyltransferases (DNMTs) gene expression were analyzed in kidneys. RESULTS: While the body weight of F1 males increased, a small decrease in kidney and body weight was observed in F2 males. In addition, global DNA hypermethylation profile in kidney cells was observed in F1 and F2 males. qPCR results reveal a significant increase of methyltransferase genes expression (DNMT1 and DNMT3a) for F2 females. CONCLUSIONS: In the field of public health policy and to raise attention to generational effects for the risk assessment of the environmental exposures, low doses of uranium do not imply clinical effects on adult exposed rats. However, our results confirm the importance of the developmental windows' sensitivity in addition to the sexual dimorphisms of the offspring.

Chronic Exposure to Uranium from Gestation: Effects on Behavior and Neurogenesis in Adulthood.

Uranium exposure leads to cerebral dysfunction involving for instance biochemical, neurochemical and neurobehavioral effects. Most studies have focused on mechanisms in uranium-exposed adult animals. However, recent data on developing animals have shown that the developing brain is also sensitive to uranium. Models of uranium exposure during brain development highlight the need to improve our understanding of the effects of uranium. In a model in which uranium exposure began from the first day of gestation, we studied the neurobehavioral consequences as well as the progression of hippocampal neurogenesis in animals from dams exposed to uranium. Our results show that 2-month-old rats exposed to uranium from gestational day 1 displayed deficits in special memory and a prominent depressive-like phenotype. Cell proliferation was not disturbed in these animals, as shown by 5-bromo-2'deoxyuridine (BrdU)/neuronal specific nuclear protein (NeuN) immunostaining in the dentate gyrus. However, in some animals, the pyramidal cell layer was dispersed in the CA3 region. From our previous results with the same model, the hypothesis of alterations of neurogenesis at prior stages of development is worth considering, but is probably not the only one. Therefore, further investigations are needed to correlate cerebral dysfunction and its underlying mechanistic pathways.

Endocrine effects of lifelong exposure to low-dose depleted uranium on testicular functions in adult rat.

Environmental toxicant exposure can induce disorders in sex steroidogenesis during fetal gonad development. Our previous study demonstrated that chronic adult exposure to a supra environmental concentration of depleted uranium (DU) does not impair testicular steroidogenesis in rats. In this study, we investigated the effects of lifelong exposure (embryo - adult) to low-dose DU (40 or 120mgL-1) on adult rat testicular steroidogenesis and spermatogenesis. A significant content of uranium was detected in testis and epididymis in the DU 120mgL-1 group and the assay in epididymal spermatozoa showed a significant content in both groups. No major defect was observed in testicular histology except a decrease in the number of basal vacuoles in the DU groups. Moreover, plasma Follicle-Stimuling Hormone [FSH] and Luteinizing Hormone [LH] levels were increased only in the DU 120mgL-1 group and intratesticular estradiol was decreased in both groups. Testosterone level was reduced in plasma and testis in the DU 40mgL-1 group. These modulations could be explained by an observed decrease in gene expression of luteinizing hormone receptor (LHR), and enzymes involved in steroid production and associated signal transduction (StAR, cyp11a1, cyp17a1, 3ßhsd, 17ßhsd, TGFß1, AR). Several genes specific to germ cells and cell junctions of the blood-testis barrier were also modulated. In conclusion, these data show that fetal life is a critical window for chronic uranium exposure and that the endocrine activities of low-dose uranium could disrupt steroidogenesis through the hypothalamic-pituitary-testicular axis. Further investigation should be so useful in subsequent generations to improve risk assessment of uranium exposure.

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.

Low-concentration uranium enters the HepG2 cell nucleus rapidly and induces cell stress response.

This study aimed to compare the cell stress effects of low and high uranium concentrations and relate them to its localization, precipitate formation, and exposure time. The time-course analysis shows that uranium appears in cell nuclei as a soluble form within 5 min of exposure, and quickly induces expression of antioxidant and DNA repair genes. On the other hand, precipitate formations began at the very beginning of exposure at the 300-µM concentration, but took longer to appear at lower concentrations. Adaptive response might occur at low concentrations but are overwhelmed at high concentrations, especially when uranium precipitates are abundant.