Passage of uranium through human cerebral microvascular endothelial cells: influence of time exposure in mono- and co-culture in vitro models.

PURPOSE: Depleted uranium (DU) has several civilian and military applications. The effects of this emerging environmental pollutant on human health raise some concerns. Previous experimental studies have shown that uranium (U) exposure can disturb the central nervous system. A small quantity of U reaches the brain via the blood, but the effects on the blood-brain barrier (BBB) remain unclear. MATERIALS AND METHODS: In the present work, two cell culture models were exposed to DU for different times to study its cytotoxicity, paracellular permeability and extracellular concentration of U. The well-known immortalized human cerebral microvascular endothelial cells, hCMEC/D3, were cultured on the filter in the first model. In the second model, human primary cells of pericytes were cultured under the filter to understand the influence of cell environment after U exposure. RESULTS: The results show that U is not cytotoxic to hCMEC/D3 cells or pericytes until 500 µM (1.6 Bq.L-1). In addition, acute or chronic low-dose exposure of U did not disturb permeability and was conserved in both cell culture models. However, U is able to reach the brain compartment. During the first hours of exposure, the passage of U to the abluminal compartment was significantly reduced in the presence of pericytes. Electronic microscopy studies evidenced the formation of needlelike structures, like urchin-shaped precipitates, from 1 h of exposure. Analytical microscopy confirmed the U composition of these precipitates. Interestingly, precipitated U was detected only in endothelial cells and not in pericytes. U was localized in multilamellar or multivesicular bodies along the endo-lysosomal pathway, suggesting the involvement of these traffic vesicles in U sequestration and/or elimination. CONCLUSIONS: We show for the first time the in vitro passage of U across a human cerebral microvascular endothelial cells, and the intracellular localization of U precipitates without any cytotoxicity or modification of paracellular permeability. The difference between the results obtained with monolayers and co-culture models with pericytes illustrates the need to use complex in vitro models in order to mimic the neurovascular unit. Further in vivo studies should be performed to better understand the passage of U across the blood-brain barrier potentially involved in behavioral consequences.

Development of whole brain versus targeted dentate gyrus irradiation model to explain low to moderate doses of exposure effects in mice.

Evaluation of the consequences of low to moderate doses of ionizing radiation (IR) remains a societal challenge, especially for children exposed to CT scans. Appropriate experimental models are needed to improve scientific understanding of how exposure of the postnatal brain to IR affects behavioral functions and their related pathophysiological mechanisms, considering brain complex functional organization. In the brain, the dorsal and ventral hippocampal dentate gyrus can be involved in distinct major behavioral functions. To study the long term behavioral effects of brain exposure at low to moderate doses of IR (doses range 0.25-1 Gy), we developed three new experimental models in 10-day-old mice: a model of brain irradiation and two targeted irradiation models of the dorsal and ventral dentate gyrus. We used the technological properties of the SARRP coupled with MR imaging. Our irradiation strategy has been twofold endorsed. The millimetric ballistic specificity of our models was first validated by measuring gamma-H2AX increase after irradiation. We then demonstrated higher anxiety/depressive-like behavior, preferentially mediate by the ventral part of the dentate gyrus, in mice after brain and ventral dentate gyrus IR exposure. This work provides new tools to enhance scientific understanding of how to protect children exposed to IR.

DNA methylation and potential multigenerational epigenetic effects linked to uranium chronic low-dose exposure in gonads of males and females rats.

INTRODUCTION: An increased health problem in industrialised countries is the contemporary concern of public and scientific community as well. This has been attributed in part to accumulated environmental pollutants especially radioactive substances and the use of nuclear power plants worldwide. However, the outcome of chronic exposure to low doses of a radionuclide such as uranium remains unknown. Recently, a paradigm shift in the perception of risk of radiotoxicology has emerged through investigating the possibility of transmission of biological effects over generations, in particular by epigenetic pathways. These processes are known for their crucial roles associated with the development of several diseases. OBJECTIVE: The current work investigates the epigenetic effect of chronic exposure to low doses of uranium and its inheritance across generations. Materials and Methods To test this proposition, a rodent multigenerational model, males and females, were exposed to a non-toxic concentration of uranium (40mgL-1 drinking water) for nine months. The uranium effects on were evaluated over three generations (F0, F1 and F2) by analysing the DNA methylation profile and DNMT genes expression in ovaries and testes tissues. RESULTS: Here we report a significant hypermethylation of testes DNA (p <0.005) whereas ovaries showed hypomethylated DNA (p <0.005). Interestingly, this DNA methylation profile was significantly maintained across generations F0, F1 and F2. Furthermore, qPCR results of both tissues imply a significant change in the expression of DNA methyltransferase genes (DNMT 1 and DNMT3a/b) as well. CONCLUSION: Altogether, our work demonstrates for the first time a sex-dependance and inheritance of epigenetic marks, DNA methylation, as a biological response to the exposure to low doses of uranium. However, it is not clear which type of reproductive cell type is more responsive in this context.

Exposure to depleted uranium during development affects neuronal differentiation in the hippocampal dentate gyrus and induces depressive-like behavior in offspring.

The developing brain is known to be sensitive to uranium (U) and exposure to this element during postnatal brain development results in behavioral disorders in adulthood. Moreover, we have previously shown that U exposure during gestation and lactation affects neurogenesis, in particular neural cell proliferation and cell death. In this study, we investigated whether exposure to depleted U (DU) affects neuronal differentiation during prenatal and postnatal brain development. We assessed in situ expression of specific genes involved in neuronal differentiation and expression of neuronal protein markers. The effects of DU on neurobehavioral function were investigated in parallel. Neuronal differentiation involves many signaling pathways that regulate the balance between cell proliferation and the transition to neuronal differentiation. In the present study pregnant rats were exposed from gestational day (GD) 1 throughout lactation to postnatal day (PND) 21. Using in situ hybridization, our results show decreased expression of Wnt3a in the hippocampal neuroepithelium in GD 13 embryos from DU exposed dams and decreased expression of Notch1 and increased expression of Mash1 in the hippocampal and dentate neuroepithelia of GD 18 fetuses from DU exposed dams. Expression of the NeuroD and NeuroD2 genes was not modified in the hippocampal neuroepithelium of GD18 fetuses from DU exposed dams. There was no change in the expression of any of these genes in the dentate gyrus of PND 5 pups from DU exposed dams. No change in nestin or doublecortin immunestaining was observed in the prenatal or early postnatal stages. However, the number of doublecortin-positive cells increased in the granular cell layer of PND 21 pups from DU exposed dams. Finally, depressive-like behavior was induced in PND21 rats, without modification of locomotor and exploratory activities or of spatial memory. In conclusion, these results showed that exposure of pregnant and lactating rats to DU affects brain development by causing disturbed cell proliferation and neuronal differentiation at the prenatal stage. Moreover, this exposure increased the pool of immature neurons in the dentate gyrus and induced depressive-like behavior in neonatal rats. Therefore, these data strongly suggest that exposure to DU during gestation and lactation affects brain development in embryos, fetuses and neonates with behavioral consequences in the offspring.

Assessment of the Central Effects of Natural Uranium via Behavioural Performances and the Cerebrospinal Fluid Metabolome.

Natural uranium (NU), a component of the earth's crust, is not only a heavy metal but also an alpha particle emitter, with chemical and radiological toxicity. Populations may therefore be chronically exposed to NU through drinking water and food. Since the central nervous system is known to be sensitive to pollutants during its development, we assessed the effects on the behaviour and the cerebrospinal fluid (CSF) metabolome of rats exposed for 9 months from birth to NU via lactation and drinking water (1.5, 10, or 40 mg·L(-1) for male rats and 40 mg·L(-1) for female rats). Medium-term memory decreased in comparison to controls in male rats exposed to 1.5, 10, or 40 mg·L(-1) NU. In male rats, spatial working memory and anxiety- and depressive-like behaviour were only altered by exposure to 40 mg·L(-1) NU and any significant effect was observed on locomotor activity. In female rats exposed to NU, only locomotor activity was significantly increased in comparison with controls. LC-MS metabolomics of CSF discriminated the fingerprints of the male and/or female NU-exposed and control groups. This study suggests that exposure to environmental doses of NU from development to adulthood can have an impact on rat brain function.

Cell proliferation and cell death are disturbed during prenatal and postnatal brain development after uranium exposure.

The developing brain is more susceptible to neurotoxic compounds than adult brain. It is also well known that disturbances during brain development cause neurological disorders in adulthood. The brain is known to be a target organ of uranium (U) exposure and previous studies have noted that internal U contamination of adult rats induces behavioral disorders as well as affects neurochemistry and neurophysiological properties. In this study, we investigated whether depleted uranium (DU) exposure affects neurogenesis during prenatal and postnatal brain development. We examined the structural morphology of the brain, cell death and finally cell proliferation in animals exposed to DU during gestation and lactation compared to control animals. Our results showed that DU decreases cell death in the cortical neuroepithelium of gestational day (GD) 13 embryos exposed at 40mg/L and 120mg/L and of GD18 fetuses exposed at 120mg/L without modification of the number of apoptotic cells. Cell proliferation analysis showed an increase of BrdU labeling in the dentate neuroepithelium of fetuses from GD18 at 120mg/L. Postnatally, cell death is increased in the dentate gyrus of postnatal day (PND) 0 and PND5 exposed pups at 120mg/L and is associated with an increase of apoptotic cell number only at PND5. Finally, a decrease in dividing cells is observed in the dentate gyrus of PND21 rats developmentally exposed to 120mg/L DU, but not at PND0 and PND5. These results show that DU exposure during brain development causes opposite effects on cell proliferation and cell death processes between prenatal and postnatal development mainly at the highest dose. Although these modifications do not have a major impact in brain morphology, they could affect the next steps of neurogenesis and thus might disrupt the fine organization of the neuronal network.

Influence of environmental enrichment and depleted uranium on behaviour, cholesterol and acetylcholine in apolipoprotein E-deficient mice.

Alzheimer's disease is associated with genetic risk factors, of which the apolipoprotein E (ApoE) is the most prevalent, and is affected by environmental factors that include education early in life and exposure to metals. The industrial and military use of depleted uranium (DU) resulted in an increase of its deposition in some areas and led to a possible environmental factor. The present study aims to ascertain the effects on the behaviour and the metabolism of cholesterol and acetylcholine of ApoE-/- mice exposed to enriched environment (EE) and exposed to DU (20 mg/L) for 14 weeks. Here we show that ApoE-/- mice were unaffected by the EE and their learning and memory were similar to those of the non-enriched ApoE-/- mice. ApoE-/- mice showed a significant decrease in total (-16 %) and free (-16 %) cholesterol in the entorhinal cortex in comparison to control wild-type mice. Whatever the housing conditions, the exposure to DU of ApoE-/- mice impaired working memory, but had no effect on anxiety-like behaviour, in comparison to control ApoE-/- mice. The exposure of ApoE-/- mice to DU also induced a trend toward higher total cholesterol content in the cerebral cortex (+15 %) compared to control ApoE-/- mice. In conclusion, these results demonstrate that enriched environment does not ameliorate neurobehaviour in ApoE-/- mice and that ApoE mutation induced specific effects on the brain cholesterol. These findings also suggested that DU exposure could modify the pathology in this ApoE model, with no influence of housing conditions.

Influence of depleted uranium on hepatic cholesterol metabolism in apolipoprotein E-deficient mice.

Depleted uranium (DU) is uranium with a lower content of the fissile isotope U-235 than natural uranium. It is a radioelement and a waste product from the enrichment process of natural uranium. Because of its very high density, it is used in the civil industry and for military purposes. DU exposure can affect many vital systems in the human body, because in addition to being weakly radioactive, uranium is a toxic metal. It should be emphasized that, to be exposed to radiation from DU, you have to eat, drink, or breathe it, or get it on your skin. This particular study is focusing on the health effects of DU for the cholesterol metabolism. Previous studies on the same issue have shown that the cholesterol metabolism was modulated at molecular level in the liver of laboratory rodents contaminated for nine months with DU. However, this modulation was not correlated with some effects at organs or body levels. It was therefore decided to use a "pathological model" such as hypercholesterolemic apolipoprotein E-deficient laboratory mice in order to try to clarify the situation. The purpose of the present study is to assess the effects of a chronic ingestion (during 3 months) of a low level DU-supplemented water (20 mg L(-1)) on the above mentioned mice in order to determine a possible contamination effect. Afterwards the cholesterol metabolism was studied in the liver especially focused on the gene expressions of cholesterol-catabolising enzymes (CYP7A1, CYP27A1 and CYP7B1), as well as those of associated nuclear receptors (LXRα, FXR, PPARα, and SREBP 2). In addition, mRNA levels of other enzymes of interest were measured (ACAT 2, as well as HMGCoA Reductase and HMGCoA Synthase). The gene expression study was completed with SRB1 and LDLr, apolipoproteins A1 and B and membrane transporters ABC A1, ABC G5. The major effect induced by a low level of DU contamination in apo-E deficient mice was a decrease in hepatic gene expression of the enzyme CYP7B1 (-23%) and nuclear receptors LXRα (-24%), RXR (-32%), HNF4α (-21%) when compared to unexposed ones. These modifications on cholesterol metabolism did not lead to increased disturbances that are specific for apolipoprotein E-deficient mice, suggesting that chronic DU exposure did not worsen the pathology in this experimental model. In conclusion, the results of this study indicate that even for a sensitive pathologic model the exposure to a low dose of DU has no relevant impact. The results confirm the results of our first study carried out on healthy laboratory rodents where a sub-chronic contamination with low dose DU did not affect in vivo the metabolism of cholesterol.

[Uranium: properties and biological effects after internal contamination]. / Uranium: propriétés et effets biologiques après contamination interne.

Uranium is a radionuclide present in the environment since the origin of the Earth. In addition to natural uranium, recent deposits from industrial or military activities are acknowledged. Uranium's toxicity is due to a combination of its chemical (heavy metal) and radiological properties (emission of ionizing radiations). Acute toxicity induces an important weight loss and signs of renal and cerebral impairment. Alterations of bone growth, modifications of the reproductive system and carcinogenic effects are also often seen. On the contrary, the biological effects of a chronic exposure to low doses are unwell known. However, results from different recent studies suggest that a chronic contamination with low levels of uranium induces subtle but significant levels. Indeed, an internal contamination of rats for several weeks leads to detection of uranium in many cerebral structures, in association with an alteration of short-term memory and an increase of anxiety level. Biological effects of uranium on the metabolisms of xenobiotics, steroid hormones and vitamin D were described in the liver, testis and kidneys. These recent scientific data suggest that uranium could participate to increase of health risks linked to environmental pollution.

Different pattern of brain pro-/anti-oxidant activity between depleted and enriched uranium in chronically exposed rats.

Uranium is not only a heavy metal but also an alpha particle emitter. The main toxicity of uranium is expected to be due to chemiotoxicity rather than to radiotoxicity. Some studies have demonstrated that uranium induced some neurological disturbances, but without clear explanations. A possible mechanism of this neurotoxicity could be the oxidative stress induced by reactive oxygen species imbalance. The aim of the present study was to determine whether a chronic ingestion of uranium induced anti-oxidative defence mechanisms in the brain of rats. Rats received depleted (DU) or 4% enriched (EU) uranyl nitrate in the drinking water at 2mg(-1)kg(-1)day(-1) for 9 months. Cerebral cortex analyses were made by measuring mRNA and protein levels and enzymatic activities. Lipid peroxidation, an oxidative stress marker, was significantly enhanced after EU exposure, but not after DU. The gene expression or activity of the main antioxidant enzymes, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), increased significantly after chronic exposure to DU. On the contrary, oral EU administration induced a decrease of these antioxidant enzymes. The NO-ergic pathway was almost not perturbed by DU or EU exposure. Finally, DU exposure increased significantly the transporters (Divalent-Metal-Transporter1; DMT1), the storage molecule (ferritin) and the ferroxidase enzyme (ceruloplasmin), but not EU. These results illustrate that oxidative stress plays a key role in the mechanism of uranium neurotoxicity. They showed that chronic exposure to DU, but not EU, seems to induce an increase of several antioxidant agents in order to counteract the oxidative stress. Finally, these results demonstrate the importance of the double toxicity, chemical and radiological, of uranium.

Comparison of the effects of enriched uranium and 137-cesium on the behaviour of rats after chronic exposure.

PURPOSE: A radionuclide that accumulates in the central nervous system is likely to exert both a chemical and a radiological effect. The present study aimed at assessing the behavioral effect of two radionuclides previously shown to accumulate in the central nervous system after chronic exposure--uranium and cesium. MATERIALS AND METHODS: Rats were exposed for 9 months to drinking water contaminated with either enriched uranium at a dosage of 40 mg U x l(-1) or 137-cesium at a dosage of 6500 Bq x l(-1), which correspond to the highest concentrations measured in some wells in the south of Finland (uranium) or in the milk in Belarus in the year following the Chernobyl accident (137-cesium). RESULTS: At this level of exposure, 137-cesium had no effect on the locomotor activity measured in an open-field, on immobility time in a forced swimming test, on spontaneous alternation in a Y-maze and on novel object exploration in an object recognition test. Enriched uranium exposure specifically reduced the spontaneous alternation measured in the Y-maze after 3 and 9 months exposure although it did not affect the other parameters. CONCLUSION: Enriched uranium exposure altered the spatial working memory capacities and this effect was correlated with previously described accumulation of uranium in the hippocampus which is one of the cerebral areas involved in this memory system.

Parental exposure to enriched uranium induced delayed hyperactivity in rat offspring.

Several recent reports suggest that chronic exposure to uranium could induce behavioural effects in adult rats. As the immature brains are known to be more susceptible to toxic effects, rats were observed in an open field, in a Y-maze and in an elevated plus-maze at 2, 5 and 9 months old after exposure to enriched uranium (40 mg l-1) during gestation and lactation. The rats exposed to enriched uranium showed a significant decrease in alternation in the Y-maze at 2 months old which reflects a slight decrease in the spatial working memory capacities as previously described in adult rats. However, the main result was a delayed hyperactivity in the rats exposed to enriched uranium, which appeared to a slight extent at 5 months old and was more evident at 9 months old. Although this effect could not be directly explained by some uranium accumulation in the target organs, this experiment showed that early exposure to enriched uranium can induce a very late effect on the rat behaviour and that such studies should not be restricted to the effects observed on young rats.

Chronic ingestion of uranyl nitrate perturbs acetylcholinesterase activity and monoamine metabolism in male rat brain.

Recent animal studies have shown that uranium can reach the brain after chronic exposure. However, little information is available on the neurological effects of chronic long-term exposure to uranium. In the present study, the effects during 1.5, 6 and 9-month periods of chronic ingestion of uranyl nitrate (UN) in drinking water (40 mg of uranium per litre) on cholinergic acetylcholinesterase (AChE) activity and on dopaminergic and serotoninergic metabolisms were investigated in several areas of male Srague Dawley rat brains. Uranium brain accumulation and distribution was also investigated after 1.5 and 9 months. Both after 1.5, 6 and 9 months of exposure, AChE activity was unaffected in the striatum, hippocampus and frontal cortex. Nevertheless, AChE activity was transitionally perturbed in the cerebellum after 6 months of exposure. After 1.5 months of exposure, DA level increased in hypothalamus. After 6 months of exposure, a tiny but significant modification of the DAergic turnover ratio was detected in the frontal cortex. And after 9 months, UN produced a significant decrease in the 5HIAA level and the 5HTergic turn-over ratio in the frontal cortex and also a decrease in the DOPAC level and DAergic turn-over ratio in the striatum. Uranium brain accumulation was statistically significant in striatum after 1.5 months and in striatum, hippocampus and frontal cortex after 9 months of exposure. Although neurochemical changes did not always correlated with increased accumulation of uranium in specific areas, these results suggest that chronic ingestion of UN can cause chronic and progressive perturbations of physiological level of neurotransmitter systems. Considering previous reports on behavioural uranium-induced effects and the involvement of neurotransmitters in various behavioural processes, it would be crucial to determine whether these neurochemical disorders were accompanied by neurobehavioral deficits even at 40 mg of uranium per litre exposure.

The brain is a target organ after acute exposure to depleted uranium.

The health effects of depleted uranium (DU) are mainly caused by its chemical toxicity. Although the kidneys are the main target organs for uranium toxicity, uranium can also reach the brain. In this paper, the central effects of acute exposure to DU were studied in relation to health parameters and the sleep-wake cycle of adult rats. Animals were injected intraperitoneally with 144+/-10 microg DU kg-1 as nitrate. Three days after injection, the amounts of uranium in the kidneys represented 2.6 microg of DU g-1 of tissue, considered as a sub-nephrotoxic dosage. The central effect of uranium could be seen through a decrease in food intake as early as the first day after exposure and shorter paradoxical sleep 3 days after acute DU exposure (-18% of controls). With a lower dosage of DU (70+/-8 microg DU kg-1), no significant effect was observed on the sleep-wake cycle. The present study intends to illustrate the fact that the brain is a target organ, as are the kidneys, after acute exposure to a moderate dosage of DU. The mechanisms by which uranium causes these early neurophysiological perturbations shall be discussed.

Effect of U and 137Cs chronic contamination on dopamine and serotonin metabolism in the central nervous system of the rat.

Following the Chernobyl accident, the most significant problem for the population of the former Soviet Union for the next 50-70 years will be chronic internal contamination by radionuclides. One of the few experiments carried out in this field reported that neurotransmitter metabolism in the central nervous system of the rat was disturbed after feeding with oats contaminated by 137Cs for 1 month. The present study assessed the effect of chronic contamination by depleted U or 137Cs on the metabolism of two neurotransmitters in cerebral areas of rats. Dopamine and serotonin were chosen because their metabolism has been shown to be disturbed after external irradiation, even at moderate doses. Dopamine, serotonin, and some of their catabolites were measured by high-pressure liquid chromatography coupled with an electrochemical detector in five cerebral structures of rats contaminated over a 1-month period by drinking water (40 mg U.L -1 or 6500 Bq 137Cs.L -1). In the striatum, hippocampus, cerebral cortex, thalamus, and cerebellum, the dopamine, serotonin, and catabolite levels were not significantly different between the control rats and rats contaminated by U or 137Cs. These results are not in accordance with those previously described.

Nitric oxide voltammetric measurements in the rat brain after gamma irradiation.

The effects of a lethal gamma irradiation were investigated on cerebral NO-ergic system by using a voltammetric method in freely moving rats. It is reported that the cortical NO concentration increases right from the end of the radiation exposure (15 Gy) and reaches a maximal magnitude (+120%) 24 h later. A dose-effect relationship from 2 to 15 Gy for gamma-ray exposure has also been observed. The effects, obtained with either an NO synthase inhibitor nonselective for the different NO synthase isoforms or an NO synthase inhibitor selective for the constitutive isoform, suggest that the radiation-induced increase in NO is likely to be dependent on the inducible NO synthase isoform. Moreover, experiments performed under ex vivo conditions showed that the cortical mRNA level for Ca(++)-independent NO synthase, the brain NOS activity, and urinary nitrites/nitrates increased significantly 24 h after gamma-ray exposure. These results demonstrate that a supralethal whole-body irradiation alters the NO-ergic pathways. The increase in NO obtained under such conditions might constitute a good index of central nervous system radiosensitivity during the acute phase of the radiation syndrome.

Voltammetric measurement of blood nitric oxide in irradiated rats.

UNLABELLED: PURPOSE. To investigate the effect of blood nitric oxide (NO) as a mediator of the neurovascular syndrome in rats following gamma-irradiation. MATERIAL AND METHODS: Using a voltametric method together with a carbon fibre based sensor, NO measurements were carried out in sham-irradiated and irradiated animals either in blood from the abdominal aorta or in blood samples from the heart. RESULTS: In in vitro conditions, properties of the probe were not altered by the ionizing radiation. Significant increases of +17% and +25.6% were observed in the voltametric signal height at 90 min and 24 h respectively after a 15 Gy gamma-ray exposure. These effects were followed on days 3 and 4 by a progressive decrease in the signal height of 7% and 18% respectively. Dose-effect relationships were observed at 90 min and 24 h after exposure to gamma-rays in the range of 3-15 Gy. Finally, the NO dependence on the measured voltametric signal was controlled by using inhibitors of the NO synthase (NOS) and by performing nitrate assays. CONCLUSIONS: Specific blood NO voltametric measurements are possible. Functional changes associated with NO after gamma-ray exposure are discussed.