Analytical performance specifications for trace elements in biological fluids derived from six countries federated external quality assessment schemes over 10 years.

OBJECTIVES: This article defines analytical performance specifications (APS) for evaluating laboratory proficiency through an external quality assessment scheme. METHODS: Standard deviations for proficiency assessment were derived from Thompson's characteristic function applied to robust data calculated from participants' submissions in the Occupational and Environmental Laboratory Medicine (OELM) external quality assurance scheme for trace elements in serum, whole blood and urine. Characteristic function was based on two parameters: (1) ß - the average coefficient of variation (CV) at high sample concentrations; (2) α - the average standard deviation (SD) at low sample concentrations. APSs were defined as 1.65 standard deviations calculated by Thompson's approach. Comparison between OELM robust data and characteristic function were used to validate the model. RESULTS: Application of the characteristic function allowed calculated APS for 18 elements across three matrices. Some limitations were noted, particularly for elements (1) with no sample concentrations near analytical technique limit of detection; (2) exhibiting high robust CV at high concentration; (3) exhibiting high analytical variability such as whole blood Tl and urine Pb; (4) with an unbalanced number of robust SD above and under the characteristic function such as whole blood Mn and serum Al and Zn. CONCLUSIONS: The characteristic function was a useful means of deriving APS for trace elements in biological fluids where biological variation data or outcome studies were not available. However, OELM external quality assurance scheme data suggests that the characteristic functions are not appropriate for all elements.

External quality assessment schemes for inorganic elements in the clinical laboratory: Lessons from the OELM scheme.

Measurements of inorganic elements in clinical laboratories produce results used for the diagnosis, the treatment and the monitoring of deficiencies or overloads. The main objective of External Quality Assessment Schemes is to verify, on a regular frequency, that clinical laboratory results correspond to the quality requirement for patient care. Therefore, External Quality Assessment Schemes represent an essential component of a laboratory's quality management system. However, External Quality Assessment Schemes within the same analytical field remain heterogeneous for different reasons such as samples, determination of assigned value, acceptable limits, content of the reports. The aim of this review was to describe and illustrate some major critical aspects of External Quality Assessment Schemes based on Occupational and Environmental Laboratory Medicine external quality assessment scheme experience.

Investigating the toxic effects induced by iron oxide nanoparticles on neuroblastoma cell line: an integrative study combining cytotoxic, genotoxic and proteomic tools.

Nanomaterials have gained much attention for their use and benefit in several fields. Iron Oxide Nanoparticles (IONPs) have been used in Biomedicine as contrast agents for imaging cancer cells. However, several studies reported the potential toxicity of those nanoparticles in different models, especially in cells. Therefore, in our present study, we investigated the effects of IONPs on the SH-SY5Y neuroblastoma cell line. We carried out cytotoxic and genotoxic studies to evaluate the phenotypic effects, and proteomic investigation to evaluate the molecular effects and the mechanisms by which this kind of NPs could induce toxicity. Our results showed that the use of three different sizes of IONPs (14, 22 and 30 nm) induced cell detachment, cell morphological changes, size, and concentration-dependent IONP internalization and cell mortality. IONPs induced slight genotoxic damage assayed by modified comet assay without affecting cell cycle, mitochondrial function, membrane integrity, intracellular calcium level, and without inducing ROS generation. All the studies were performed to compare also the effects of IONPs to the ferric iron by incubating cells with equivalent concentration of FeCl3. In all tests, the NPs exhibited more toxicity than the ferric iron. The proteomic analysis followed by gene ontology and pathway analysis evidenced the effects of IONPs on cytoskeleton, cell apoptosis, and cancer development. Our findings provided more information about IONP effects on human cells and especially on cancer cell line.

Zinc deficiency promotes endothelin secretion and endothelial cell migration through nuclear hypoxia-inducible factor-1 translocation.

Zinc is involved in the expression and function of various transcription factors, including the hypoxia-inducible factor-1 (HIF-1). HIF-1 and its target gene endothelin-1 (ET-1) are activated by intermittent hypoxia (IH), one of the main consequences of obstructive sleep apnea (OSA), and both play a key role in the cardiovascular consequences of IH. Because OSA and IH are associated with zinc deficiency, we investigated the effect of zinc deficiency caused by chelation on the HIF-1/ET-1 pathway and its functional consequences in endothelial cells. Primary human microvascular endothelial cells (HMVEC) were incubated with submicromolar doses of the zinc-specific membrane-permeable chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylene diamine (TPEN, 0.5 µM) or ET-1 (0.01 µM) with or without bosentan, a dual ET-1-receptor antagonist. HIF-1α expression was silenced by transfection with specific siRNA. Nuclear HIF-1 content was assessed by immunofluorescence microscopy and Western blot. Migratory capacity of HMVEC was evaluated with a wound-healing scratch assay. Zinc chelation by TPEN exposure induced the translocation of the cytosolic HIF-1α subunit of HIF-1 to the nucleus as well as an HIF-1-mediated ET-1 secretion by HMVEC. Incubation with either TPEN or ET-1 increased endothelial wound-healing capacity. Both HIF-1α silencing or bosentan abolished this effect. Altogether, these results suggest that zinc deficiency upregulates ET-1 signaling through HIF-1 activation and stimulates endothelial cell migration, suggesting an important role of zinc in the vascular consequences of IH and OSA mediated by HIF-1-ET- signaling.

Utility of macrophages in an antitumor strategy based on the vectorization of iron oxide nanoparticles.

Many solid tumors and their metastases are still resistant to current cancer treatments such as chemo- and radiotherapy. The presence of a small population of Cancer Stem Cells in tumors is held responsible for relapses. Moreover, the various physical barriers of the organism (e.g. blood-brain barrier) prevent many drugs from reaching the target cells. In order to alleviate this constraint, we suggest a Trojan horse strategy consisting of intravascular injection of macrophages loaded with therapeutic nanoparticles (an iron nanoparticle-based solution marketed under the name of FERINJECT®) to bring a high quantity of the latter to the tumor. The aim of this article is to assess the response of primary macrophages to FERINJECT® via functional assays in order to ensure that the macrophages loaded with these nanoparticles are still relevant for our strategy. Following this first step, we demonstrate that the loaded macrophages injected into the bloodstream are able to migrate to the tumor site using small-animal imaging. Finally, using synchrotron radiation, we validate an improvement of the radiotherapeutic effect when FERINJECT®-laden macrophages are deposited at the vicinity of cancer cells and irradiated.

Nanoparticles in foods? A multiscale physiopathological investigation of iron oxide nanoparticle effects on rats after an acute oral exposure: Trace element biodistribution and cognitive capacities.

Iron Oxide Nanoparticles (IONPs) are used in several fields of application, mainly in the biomedical field for their magnetic properties and in food additive known as "E172" for their colour. In the present investigation, we focused on IONP effects on Wistar rat following acute oral exposure. We performed a multiscale physiopathological investigation in order to elucidate potential toxic effects linked to IONP ingestion, especially on cognitive capacities, trace element distribution, blood constituents, organ functions, organ structure and iron deposit. We demonstrated that oral exposure to IONPs induces disturbances of certain parameters depending on the dose. Interestingly, the histopathological examination evidenced inflammatory effects of IONPs in the liver with iron deposits in hepatocytes and Kuppfer cells. Neurobehavioral examination showed that oral exposure to IONPs did not affect nor rat emotions, exploration and locomotion capacities, nor spatial reference memory status. Furthermore, oral administration of IONPs did not disrupt the trace element homeostasis nor in the liver neither in the stomach. Altogether, our study evidenced low signs of toxicity, but some effects lead us to a careful use of these NPs. Thereby, their use in foods should be further studied to better evaluate the potential toxic risks of the oral exposure to IONPs.

Treatment of multiple brain metastases using gadolinium nanoparticles and radiotherapy: NANO-RAD, a phase I study protocol.

INTRODUCTION: Occurrence of multiple brain metastases is a critical evolution of many cancers with significant neurological and overall survival consequences, despite new targeted therapy and standard whole brain radiotherapy (WBRT). A gadolinium-based nanoparticle, AGuIX, has recently demonstrated its effectiveness as theranostic and radiosensitiser agent in preclinical studies. The favourable toxicity profile in animals and its administration as a simple intravenous injection has motivated its use in patients with this first in human study. METHODS AND ANALYSIS: The NANO-RAD study is a phase I, first in human injection, monocentric, open-label, dose-escalation study to investigate the safety, the tolerability and the spectrum of side effects of AGuIX in combination with WBRT (30 Gy, 10 fractions of 3 Gy) for patients with multiple brain metastases. Five dose escalation cohorts are planned: 15, 30, 50, 75 and 100 mg/kg. A total of 15-18 patients will be recruited into this trial. The primary objective is to determine the maximum-tolerated dose of AGuIX nanoparticles combined with WBRT for the treatment of multiple brain metastases. Toxicity will be assessed using the National Cancer Institute Common Toxicity Criteria V.4.03. Secondary objectives are pharmacokinetic profile, distribution of AGuIX in metastases and surrounding healthy tissue visualised by MRI, intracranial progression-free survival and overall survival. Intracranial response will be determined according to Response Evaluation Criteria in Solid Tumour Criteria V.1.1 comparing MRI performed prior to treatment and at each follow-up visits. ETHICS AND DISSEMINATION: Approval was obtained from the ethics committee Sud Est V, France (Reference number 15-CHUG-48). The study was approved by the French National Agency for the Safety of Medicines and Health Products (ANSM) (Reference number 151519A-12). The results will be published in peer-reviewed journals or disseminated through national and international conferences. TRIAL REGISTRATION NUMBER: NCT02820454; Pre-results.

Impact of maternal low-level cadmium exposure on glucose and lipid metabolism of the litter at different ages after weaning.

Cadmium (Cd) is a metal which may participate in the development of type II diabetes even if Cd exposure levels are mild. However, experimental studies focusing on daily environmentally relevant doses are scarce, particularly for glucose metabolism of the offspring of chronically exposed mothers. The aim is to measure the impact of maternal low level Cd exposure on glucose and lipid metabolism of offspring. Female rats were exposed to 0, 50 or 500 µg.kg-1.d-1 of CdCl2, 21 days before mating and during 21 days of gestation and 21 days of lactation. Pups exposure was organized in 3 groups (control, Cd1, Cd2) according to renal dams' Cd burden. Parameters of glucose and lipid metabolisms were measured for the pups on post-natal day 21, 26 and 60. Maternal Cd exposure led to significant amounts of Cd in the liver and kidney of pups. At weaning, insulin secretion upon glucose stimulation was unchanged, but the removal of circulating glucose was slower for pups born from the lowest impregnated dams (Cd1). Five days after, glucose tolerance of all groups was identical. Thus, this loss of insulin sensitivity was reversed, in part by increased adiponectin secretion for the Cd1 group. Furthermore, pups from dams accumulating the highest levels of Cd (Cd2) exhibited a compensatory increased insulin pancreatic secretion, together with increased circulating non-esterified fatty acids, indicating the establishment of insulin resistance, 2 months after birth. This study has demonstrated the influence of maternal exposure to low levels of Cd on glucose homeostasis in the offspring that might increase the risk of developing type II diabetes later in life.

A New Patient-Derived Metastatic Glioblastoma Cell Line: Characterisation and Response to Sodium Selenite Anticancer Agent.

Glioblastoma multiform (GBM) tumors are very heterogeneous, organized in a hierarchical pattern, including cancer stem cells (CSC), and are responsible for development, maintenance, and cancer relapse. Therefore, it is relevant to establish new GBM cell lines with CSC characteristics to develop new treatments. A new human GBM cell line, named R2J, was established from the cerebro-spinal fluid (CSF) of a patient affected by GBM with leptomeningeal metastasis. R2J cells exhibits an abnormal karyotype and form self-renewable spheres in a serum-free medium. Original tumor, R2J, cultured in monolayer (2D) and in spheres showed a persistence expression of CD44, CD56 (except in monolayer), EGFR, Ki67, Nestin, and vimentin. The R2J cell line is tumorigenic and possesses CSC properties. We tested in vitro the anticancer effects of sodium selenite (SS) compared to temozolomide TMZ. SS was absorbed by R2J cells, was cytotoxic, induced an oxidative stress, and arrested cell growth in G2M before inducing both necrosis and apoptosis via caspase-3. SS also modified dimethyl-histone-3-lysine-9 (H3K9m2) levels and decreased histone deacetylase (HDAC) activity, suggesting anti-invasiveness potential. This study highlights the value of this new GBM cell line for preclinical modeling of clinically relevant, patient specific GBM and opens a therapeutic window to test SS to target resistant and recurrent GBM.

Sub-acute intravenous exposure to Fe2O3 nanoparticles does not alter cognitive performances and catecholamine levels, but slightly disrupts plasma iron level and brain iron content in rats.

Engineered nanomaterials are used in various applications due to their particular properties. Among them, Iron Oxide Nanoparticles (Fe2O3-NPs) are used in Biomedicine as theranostic agents i.e. contrast agents in Magnetic Resonance Imaging and cancer treatment. With the increasing production and use of these Fe2O3-NPs, there is an evident raise of Fe2O3-NPs exposure and subsequently a higher risk of adverse outcomes for the environment and Human. In the present paper, we investigated the effects of an intravenous daily Fe2O3-NPs exposure on Wistar rat for one week. As results, we showed that several hematological parameters and transaminase (ALT and AST) levels as well as organ histology remained unchanged in treated rats. Neither the catecholamine levels nor the emotional behavior and learning / memory capacities of rats were impacted by the sub-acute intravenous exposure to Fe2O3-NPs. However, iron level in plasma and iron content homeostasis in brain were disrupted after this exposure. Thus, our results demonstrated that Fe2O3-NPs could have transient effects on rat but the intravenous route is still safer that others which is encouraging for their use in medical and/or biological applications.

Impact of chronic and low cadmium exposure of rats: sex specific disruption of glucose metabolism.

BACKGROUND: Several epidemiological and animal studies suggest a positive association between cadmium (Cd) exposure and incidence of type 2 diabetes, but the association remains controversial. Besides, the experimental data have mainly been obtained with relatively high levels of Cd, over various periods of time, and with artificial routes of administration. OBJECTIVES: Do environmental exposures to Cd induce significant disruption of glucose metabolism? METHODS: Adults Wistar rats were exposed for three months to 0, 5, 50 or 500 µg.kg-1.d-1 of CdCl2 in drinking water. Relevant parameters of glucose homeostasis were measured. RESULTS: Cd accumulated in plasma, kidney and liver of rats exposed to 50 and 500 µg.kg-1.d-1, without inducing signs of organ failure. In rats drinking 5 µg.kg-1.d-1 for 3 months, Cd exposure did not lead to any significant increase of Cd in these organs. At 50 and 500 µg.kg-1.d-1 of Cd, glucose and insulin tolerance were unchanged in both sexes. However, females exhibited a significant increase of both fasting and glucose-stimulated plasma insulin that was assigned to impaired hepatic insulin extraction as indicated by unaltered fasting C-peptide plasma levels. CONCLUSIONS: Glucose homeostasis is sensitive to chronic Cd exposure in a gender-specific way. Moreover, this study proves that an environmental pollutant such as Cd can have, at low concentrations, an impact on the glucose homeostatic system and it highlights the importance of a closer scrutiny of the underlying environmental causes to understand the increased incidence of type 2 diabetes.

Intranasal instillation of iron oxide nanoparticles induces inflammation and perturbation of trace elements and neurotransmitters, but not behavioral impairment in rats.

Over the last decades, engineered nanomaterials have been widely used in various applications due to their interesting properties. Among them, iron oxide nanoparticles (IONPs) are used as theranostic agents for cancer, and also as contrast agents in magnetic resonance imaging. With the increasing production and use of these IONPs, there is an evident raise of IONP exposure and subsequently a higher risk of adverse outcome for humans and the environment. In this work, we aimed to investigate the effects of sub-acute IONP exposure on Wistar rat, particularly (i) on the emotional and learning/memory behavior, (ii) on the hematological and biochemical parameters, (iii) on the neurotransmitter content, and (vi) on the trace element homeostasis. Rats were treated during seven consecutive days by intranasal instillations at a dose of 10 mg/kg body weight. The mean body weight increased significantly in IONP-exposed rats. Moreover, several hematological parameters were normal in treated rats except the platelet count which was increased. The biochemical study revealed that phosphatase alkaline level decreased in IONP-exposed rats, but no changes were observed for the other hepatic enzymes (ALT and AST) levels. The trace element homeostasis was slightly modulated by IONP exposure. Sub-acute intranasal exposure to IONPs increased dopamine and norepinephrine levels in rat brain; however, it did not affect the emotional behavior, the anxiety index, and the learning/memory capacities of rats.

Mitochondrial Morphology and Function of the Pancreatic ß-Cells INS-1 Model upon Chronic Exposure to Sub-Lethal Cadmium Doses.

The impact of chronic cadmium exposure and slow accumulation on the occurrence and development of diabetes is controversial for human populations. Islets of Langerhans play a prominent role in the etiology of the disease, including by their ability to secrete insulin. Conversion of glucose increase into insulin secretion involves mitochondria. A rat model of pancreatic ß-cells was exposed to largely sub-lethal levels of cadmium cations applied for the longest possible time. Cadmium entered cells at concentrations far below those inducing cell death and accumulated by factors reaching several hundred folds the basal level. The mitochondria reorganized in response to the challenge by favoring fission as measured by increased circularity at cadmium levels already ten-fold below the median lethal dose. However, the energy charge and respiratory flux devoted to adenosine triphosphate synthesis were only affected at the onset of cellular death. The present data indicate that mitochondria participate in the adaptation of ß-cells to even a moderate cadmium burden without losing functionality, but their impairment in the long run may contribute to cellular dysfunction, when viability and ß-cells mass are affected as observed in diabetes.

Reactive oxygen species levels control NF-κB activation by low dose deferasirox in erythroid progenitors of low risk myelodysplastic syndromes.

Anemia is a frequent cytopenia in myelodysplastic syndromes (MDS) and most patients require red blood cell transfusion resulting in iron overload (IO). Deferasirox (DFX) has become the standard treatment of IO in MDS and it displays positive effects on erythropoiesis. In low risk MDS samples, mechanisms improving erythropoiesis after DFX treatment remain unclear. Herein, we addressed this question by using liquid cultures with iron overload of erythroid precursors treated with low dose of DFX (3µM), which corresponds to DFX 5 mg/kg/day, an unusual dose used for iron chelation. We highlight a decreased apoptosis rate and an increased proportion of cycling cells, both leading to higher proliferation rates. The iron chelation properties of low dose DFX failed to activate the Iron Regulatory Proteins and to support iron depletion, but low dose DFX dampers intracellular reactive oxygen species. Furthermore low concentrations of DFX activate the NF-κB pathway in erythroid precursors triggering anti-apoptotic and anti-inflammatory signals. Establishing stable gene silencing of the Thioredoxin (TRX) 1 genes, a NF-κB modulator, showed that fine-tuning of reactive oxygen species (ROS) levels regulates NF-κB. These results justify a clinical trial proposing low dose DFX in MDS patients refractory to erythropoiesis stimulating agents.

Anticancer properties of sodium selenite in human glioblastoma cell cluster spheroids.

Glioblastoma (GBM) is the most common type of primary tumor of the central nervous system with a poor prognosis, needing the development of new therapeutic drugs. Few studies focused on sodium selenite (SS) effects in cancer cells cultured as multicellular tumor spheroids (MCTS or 3D) closer to in vivo tumor. We investigated SS anticancer effects in three human GBM cell lines cultured in 3D: LN229, U87 (O(6)-methyguanine-DNA-methyltransferase (MGMT) negative) and T98G (MGMT positive). SS absorption was evaluated and the cytotoxicity of SS and temozolomide (TMZ), the standard drug used against GBM, were compared. SS impacts on proliferation, cell death, and invasiveness were evaluated as well as epigenetic modifications by focusing on histone deacetylase (HDAC) activity and dimethyl-histone-3-lysine-9 methylation (H3K9m2), after 24h to 72h SS exposition. SS was absorbed by spheroids and was more cytotoxic than TMZ (i.e., for LN229, the IC50 was 38 fold-more elevated for TMZ than SS, at 72h). SS induced a cell cycle arrest in the S phase and apoptosis via caspase-3. SS decreased carbonic anhydrase-9 (CA9) expression, invasion on a Matrigel matrix and modulated E- and N-Cadherin transcript expressions. SS decreased HDAC activity and modulated H3K9m2 levels. 3D model provides a relevant strategy to screen new drugs and SS is a promising drug against GBM that should now be tested in GBM animal models.