Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations
Introduction
Natural uranium is a ubiquitous element in the environment, resulting in human population exposure to low but unavoidable concentrations from air, water and food (ATSDR, 2013). Natural uranium is a weakly radioactive element, its adverse health effects are mainly due to its chemical toxicity rather than its radiotoxicity (Keith et al., 2008; ATSDR, 2013). Once ingested or inhaled, uranium circulates in the blood stream as soluble hexavalent uranium (uranyl) resulting in its accumulation in particular in bones and kidneys where severe acute toxic effects were documented (Keith et al., 2008). High uranium exposures occur mostly in some occupational settings, typically for workers from uranium processing industries but also from phosphate fertilizer industry. Noteworthy, relatively high chronic exposure levels may also be found in geographical areas with elevated levels of naturally occurring uranium (Frisbie et al., 2009, 2013; Bjørklund et al., 2017, 2020). Regardless the route of exposure, a small amount of uranium is able to reach the brain and may exert neurotoxic effects (Fitsanakis et al., 2006; Houpert et al., 2007; Dinocourt et al., 2015). Rodent models exposed to uranium showed neurobehavioral changes such as increased locomotor activity, perturbation of the sleep-wake cycle, decreased memory, and increased anxiety. At the molecular level, such neurological alterations were caused by the disruption of the acetylcholine, serotonin, and dopamine neurotransmitter systems (Bussy et al., 2006; Barber et al., 2007; Dinocourt et al., 2015). We recently reported the alteration of monoamine oxidase B expression in human dopaminergic cells exposed to natural uranium (Carmona et al., 2018). The dopaminergic system was also impaired after chronic exposure of C. elegans to depleted uranium leading to the specific degeneration of dopaminergic neurons (Lu et al., 2020). Further studies are needed to better understand the mechanisms of uranium-induced neurotoxicity on dopaminergic neurons. Such data are required to evaluate in particular whether long-term exposure to low uranium concentrations could affect neurological functions in humans.
Human SH-SY5Y cell line was selected as a study model. Human SH-SY5Y cells are frequently used as human in vitro neuronal model since these cells may acquire a neuronal dopaminergic phenotype after a differentiation procedure (Presgraves et al., 2004). We selected a 7-day temporal window of continuous soluble uranyl exposure in order to approach in vitro the conditions inducing molecular effects reflecting more continuous rather than acute exposures. In these experimental conditions, we previously evidenced the intracellular isotopic fractionation of natural uranium, suggesting the existence of a high affinity transporter protein for uranyl uptake in SH-SY5Y dopaminergic cells (Paredes et al., 2016). We also found a significant alteration of dopamine catabolic pathway beginning at non-cytotoxic (10 μM) uranyl continuous exposure (Carmona et al., 2018). We addressed the question of uranium intracellular distribution using micro-PIXE (Particle Induced X-ray Emission) imaging, a technique applied with a spatial resolution of 2 μm and a limit of detection of about 10 μg.g−1 for uranium imaging. Micro-PIXE imaging revealed the uranium localization in cytoplasmic regions, particularly observable after continuous exposure to sub-cytotoxic (125 μM) and slightly toxic (250 μM) uranyl concentrations (Carmona et al., 2018). The nature of these uranium-rich cytoplasmic regions and their existence for lower concentration exposures have yet to be determined.
The aim of the present study was to shed light on the mechanisms of uranium distribution in SH-SY5Y neuron-like cells after non cytotoxic continuous uranyl exposure. We assessed uranium subcellular distribution after continuous 7-day exposure to a non-cytotoxic uranyl concentration (10 μM) selected on the basis of our previously reported cytotoxicity results (Carmona et al., 2018). We applied Synchrotron X-ray Fluorescence (SXRF) imaging, a technique with high spatial resolution (300 nm at Nanoscopium beamline, SOLEIL synchrotron) and high analytical element sensitivity (< 1 μg.g−1 per 300 nm pixel). We performed a cellular correlative microscopy approach by coupling SXRF imaging to epifluorescence microscopy for the identification of organelles and/or proteins fluorescently labeled (Roudeau et al., 2014; Carmona et al., 2019; Das et al., 2019). Previous studies using transmission electron microscopy (TEM) indicated that after acute exposure to high uranium concentrations (> 300 μM) of alveolar macrophages, renal, or bone cells, uranium precipitates forming large needle-shape structures within cytoplasmic vesicles such as lysosomes (Hengé-Napoli et al., 1996; Mirto et al., 1999; Carrière et al., 2008; Pierrefite-Carle et al., 2017). We thus designed our study in order to evaluate, at lower non-cytotoxic concentration, the possible uranium co-localization with the cellular vesicular pathway (early endosomes, late endosomes, and lysosomes). In addition, we investigated the potential interaction of uranium with fetuin-A, a high affinity uranium-binding protein (Basset et al., 2013).
Section snippets
Reagents and solutions
Eagle’s minimum essential medium (EMEM, 30–2003; ATCC), F12 medium (21765−029; Life Technologies), fetal bovine serum (FBS) (30–2020; ATCC), and penicillin/streptomycin (15070−063; Gibco-Thermo Fisher Scientific) solutions were used to prepare the culture medium for cell growth and exposure experiments. TrypLE Express 1×/EDTA (12605−010; Gibco-Thermo Fisher Scientific) was used for the trypsinization of cells. Phosphate buffered saline (PBS) (pH 7.4) free of CaCl2 and MgCl2 (10010−015; Gibco-
Uranium distribution
In our previous work (Carmona et al., 2018), we determined the cytotoxicity thresholds of soluble hexavalent uranium (uranyl) on SH-SY5Y differentiated cells for a continuous 7-day exposure. We found that exposure to 250 μM uranyl resulted in 15 % of cell viability inhibition, while exposure to 125 μM was considered the limit below which no cytotoxic effects were observable. We selected a concentration of 10 μM, far from the cytotoxicity threshold, to study neurotoxic effects of uranyl at
Conclusions
In the last years, the uranium toxicology field has focused on the impact of uranium on the nervous system (Dinocourt et al., 2015). However, the complex interaction between uranium and the human nervous system is far from being elucidated. In this context, we investigated the uranium intracellular distribution in neuron-like cells differentiated in a dopaminergic phenotype. In a previous study we revealed the alteration of the monoamine degradation pathway after uranium continuous exposure at
Funding
This project has received financial support from the CNRS through the MITI interdisciplinary programs, the Nuclear Toxicology program of CEA, and from CNRS-IN2P3.
CRediT authorship contribution statement
Asuncion Carmona: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Francesco Porcaro: Methodology, Formal analysis, Investigation, Writing - original draft. Andrea Somogyi: Resources, Investigation, Writing - review & editing. Stéphane Roudeau: Investigation, Writing - review & editing. Florelle Domart: Investigation. Kadda Medjoubi: Resources, Investigation. Michel Aubert: Investigation. Hélène Isnard:
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We acknowledge SOLEIL for provision of synchrotron radiation facilities and we would like to thank NANOSCOPIUM staff for assistance in using the beamline. The authors are grateful to Dr. Marta Garcia Cortes for the characterization of the Alexa Fluor® 488 labeled fetuin-A solution.
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Current address: Departament de Dinàmica de la Terra i de l'Oceà. Facultat de Ciències de la Terra, Universitat de Barcelona, C/ Marti Franques s/n, 08028 Barcelona, Spain.