Exploring the Depths of Corrosion: A Novel GE-XANES Technique for Investigating Compositionally Complex Alloys.

In this study, we propose the use of nondestructive, depth-resolved, element-specific characterization using grazing exit X-ray absorption near-edge structure spectroscopy (GE-XANES) to investigate the corrosion process in compositionally complex alloys (CCAs). By combining grazing exit X-ray fluorescence spectroscopy (GE-XRF) geometry and a pnCCD detector, we provide a scanning-free, nondestructive, depth-resolved analysis in a sub-micrometer depth range, which is especially relevant for layered materials, such as corroded CCAs. Our setup allows for spatial and energy-resolved measurements and directly extracts the desired fluorescence line, free from scattering events and other overlapping lines. We demonstrate the potential of our approach on a compositionally complex CrCoNi alloy and a layered reference sample with known composition and specific layer thickness. Our findings indicate that this new GE-XANES approach has exciting opportunities for studying surface catalysis and corrosion processes in real-world materials.

Investigation of polycapillary half lenses for quantitative confocal micro-X-ray fluorescence analysis.

The use of polycapillary optics in confocal micro-X-ray fluorescence analysis (CMXRF) enables the destruction-free 3D investigation of the elemental composition of samples. The energy-dependent transmission properties, concerning intensity and spatial beam propagation of three polycapillary half lenses, which are vital for the quantitative interpretation of such CMXRF measurements, are investigated in a monochromatic confocal laboratory setup at the Atominstitut of TU Wien, and a synchrotron setup on the BAMline beamline at the BESSY II Synchrotron, Helmholtz-Zentrum-Berlin. The empirically established results, concerning the intensity of the transmitted beam, are compared with theoretical values calculated with the polycap software package and a newly presented analytical model for the transmission function. The resulting form of the newly modelled energy-dependent transmission function is shown to be in good agreement with Monte Carlo simulated results for the complete energy regime, as well as the empirically established results for the energy regime between 6 keV and 20 keV. An analysis of possible fabrication errors was conducted via pinhole scans showing only minor fabrication errors in two of the investigated polycapillary optics. The energy-dependent focal spot size of the primary polycapillary was investigated in the laboratory via the channel-wise evaluation of knife-edge scans. Experimental results are compared with data given by the manufacturer as well as geometric estimations for the minimal focal spot size. Again, the resulting measurement points show a trend in agreement with geometrically estimated results and manufacturer data.

Catalogue of dose rate constants for more than 400 radionuclides in terms of ambient dose H∗ and comparison of figures to ambient dose equivalent H∗(10).

In 2020, the International Commission on Radiation Units & Measurements ICRU has released Report 95 on "Operational Quantities for External Radiation Exposure". This publication introduced a new measurand, namely ambient dose H∗, as the operational quantity for external exposure to be applied in the future replacing ambient dose equivalent H∗(10). It should be noted that this change will make it necessary to adjust previously used constants and coefficients or at least to review them with regard to the new measurand. Therefore, this recommendation represents a significant cut in implementation of radiation protection systems. The revision of all previously valid factors makes it possible to develop a consistent data set. The combination of experience gained in the last decades with more extensive data sets (e.g. on the spectra of individual radionuclides) with comparatively large computing power allows the definition of optimized data collections serving as input for Monte Carlo methods. Therefore, this publication is intended to lay a suitable foundation consisting of dose rate constants for more than 400 radionuclides concerning the future measurand ambient dose H∗ and to indicate differences to values related to the ambient dose equivalent H∗(10) used nowadays.

A novel approach towards the calculation of dose rate constants for ambient dose equivalent H∗(10) by including low energy x-rays.

The nuclide-specific dose rate constant, formerly called gamma ray constant, is one of the most important quantities in practical radiation protection dosimetry. For radiation sources with known radionuclide composition and activity, the expected dose rates at various distances can easily be calculated with reasonable approximations. In addition, they serve as a planning basis for the design of shielding of radiation application rooms and facilities. In this study, dose rate constants were calculated using the most recent conversion coefficients and the most suitable spectral data for more than 400 radionuclides using different calculation approaches. In addition this paper provides a critical review of currently published dose rate constants for the ambient dose equivalent H∗(10).

X-ray Fluorescence Techniques for Element Abundance Analysis in Wine.

The elemental composition has been extensively used to characterize wine and to find correlations with environmental and winemaking factors. Although X-ray fluorescence (XRF) techniques offer many advantages, they have been rarely used for wine analysis. Here, we show the comparison of wine elemental composition results obtained by total reflection X-ray fluorescence (TXRF) and energy dispersive X-ray fluorescence (EDXRF) for elements K, Ca, Mn, Fe, Cu, Zn, Br, Rb, and Sr. The results obtained by TXRF and EDXRF have been additionally verified by inductively coupled plasma-mass spectrometry. The important analytical features of XRF techniques in wine science have been described, the preservation of volatile elements (e.g., Br) being one of their main advantages. In addition, we have shown that XRF techniques offer an optimal analytical approach for building large data sets containing highly reliable and reproducible results of elemental abundances in wines, corresponding soils, and grape juice. Such data sets are especially important for the geographic authentication of wine. This has been shown for 37 Austrian and Croatian wines collected together with respective soils from selected wine regions. The element abundances in soil reflect in a large portion in grape juice and finished wine suggesting that the contribution of the soil, that is, the plant uptake capacity expressed as c i(wine)/c i(soil) concentration factors, can be a highly discriminating factor for wine fingerprinting. This indeed has been proved in the present study in comparison to discrimination based only on wine element abundances. We have identified Fe, Zn, Br, Rb, and Sr as the best discriminator elements for the geographical authentication of wine. The study opens a new perspective in extending the application of XRF techniques as a cost-effective analytical tool for creating large databases of soil, grape juice, and wine element abundances for the evaluation of soil characteristics and other environmental parameters on wine composition.

Cross-modality imaging of bisphosphonate-treated murine jawbones.

In this proof-of-principle study, we established and implemented a cross-modality imaging (CMI) pipeline to characterize and compare bisphosphonate (BIS)-treated jawbones of Sprague-Dawley rats after tooth extraction after physical therapies (photobiomodulation and extracorporeal shockwave therapy (PBMT and ESWT)). We showcase the feasibility of such a CMI approach and its compatibility across imaging modalities to probe the same region of interest (ROI) of the same jawbone. Jawbones were imaged in toto in 3D using micro-Computed Tomography to identify ROIs for subsequent sequential 2D analysis using well-established technologies such as Atomic Force Microscopy and Scanning Electron Microscopy, and recent imaging approaches in biomedical settings, such as micro-X-Ray Fluorescence Spectroscopy. By combining these four modalities, multiscale information on the morphology, topography, mechanical stiffness (Young's modulus), and calcium, zinc and phosphorus concentrations of the bone was collected. Based on the CMI pipeline, we characterized and compared the jawbones of a previously published clinically relevant rat model of BIS-related osteonecrosis of the jawbone (BRONJ) before and after treatment with BISs, PBMT and ESWT. While we did not find that physical therapies altered the mechanical and elemental jawbone parameters with significance (probably due to the small sample size of only up to 5 samples per group), both ESWT and PBMT reduced pore thicknesses and bone-to-enamel distances significantly compared to the controls. Although focused on BIS-treated jawbones, the established CMI platform can be beneficial in the study of bone-related diseases in general (such as osteoarthritis or -porosis) to acquire complementary hallmarks and better characterize disease status and alleviation potentials.

Multimodality imaging beyond CLEM: Showcases of combined in-vivo preclinical imaging and ex-vivo microscopy to detect murine mural vascular lesions.

In imaging, penetration depth comes at the expense of lateral resolution, which restricts the scope of 3D in-vivo imaging of small animals at micrometer resolution. Bioimaging will need to expand beyond correlative light and electron microscopy (CLEM) approaches to combine insights about in-vivo dynamics in a physiologically relevant 3D environment with ex-vivo information at micrometer resolution (or beyond) within the spatial, structural and biochemical contexts. Our report demonstrates the immense potential for biomedical discovery and diagnosis made available by bridging preclinical in-vivo imaging with ex-vivo biological microscopy to zoom in from the whole organism to individual structures and by adding localized spectroscopic information to structural and functional information. We showcase the use of two novel imaging pipelines to zoom into mural lesions (occlusions/hyperplasia and micro-calcifications) in murine vasculature in a truly correlative manner, that is using exactly the same animal for all integrated imaging modalities. This correlated multimodality imaging (CMI) approach includes well-established technologies such as Positron Emission Tomography (microPET), Autoradiography, Magnetic Resonance Imaging (microMRI) and Computed Tomography (microCT), and imaging approaches that are more novel in the biomedical setting, such as X-Ray Fluorescence Spectroscopy (microXRF) and High Resolution Episcopic Microscopy (HREM). Although the current pipelines are focused on mural lesions, they would also be beneficial in preclinical and clinical investigations of vascular diseases in general.

Relation of Metal-Binding Property and Selective Toxicity of 8-Hydroxyquinoline Derived Mannich Bases Targeting Multidrug Resistant Cancer Cells.

Resistance to chemotherapeutic agents is a major obstacle in cancer treatment. A recently proposed strategy is to target the collateral sensitivity of multidrug resistant (MDR) cancer. Paradoxically, the toxicity of certain metal chelating agents is increased, rather than decreased, by the function of P-glycoprotein (Pgp), which is known to confer resistance by effluxing chemotherapeutic compounds from cancer cells. We have recently characterized and compared the solution's chemical properties including ligand protonation and the metal binding properties of a set of structurally related 8-hydroxyquinoline derived Mannich bases. Here we characterize the impact of the solution stability and redox activity of their iron(III) and copper(II) complexes on MDR-selective toxicity. Our results show that the MDR-selective anticancer activity of the studied 8-hydroxyquinoline derived Mannich bases is associated with the iron deprivation of MDR cells and the preferential formation of redox-active copper(II) complexes, which undergo intracellular redox-cycling to induce oxidative stress.

Development and In Vivo Application of a Water-Soluble Anticancer Copper Ionophore System Using a Temperature-Sensitive Liposome Formulation.

Liposomes containing copper and the copper ionophore neocuproine were prepared and characterized for in vitro and in vivo anticancer activity. Thermosensitive PEGylated liposomes were prepared with different molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) in the presence of copper(II) ions. Optimal, temperature dependent drug release was obtained at 70:30 DPPC to HSPC weight ratio. Neocuproine (applied at 0.2 mol to 1 mol phospholipid) was encapsulated through a pH gradient while using unbuffered solution at pH 4.5 inside the liposomes, and 100 mM HEPES buffer pH 7.8 outside the liposomes. Copper ions were present in excess, yielding 0.5 mM copper-(neocuproine)2 complex and 0.5 mM free copper. Pre-heating to 45 °C increased the toxicity of the heat-sensitive liposomes in short-term in vitro experiments, whereas at 72 h all investigated liposomes exhibited similar in vitro toxicity to the copper(II)-neocuproine complex (1:1 ratio). Thermosensitive liposomes were found to be more effective in reducing tumor growth in BALB/c mice engrafted with C26 cancer cells, regardless of the mild hyperthermic treatment. Copper uptake of the tumor was verified by PET/CT imaging following treatment with [64Cu]Cu-neocuproine liposomes. Taken together, our results demonstrate the feasibility of targeting a copper nanotoxin that was encapsulated in thermosensitive liposomes containing an excess of copper.

Total reflection X-ray fluorescence analysis of elemental composition of herbal infusions and teas.

BACKGROUND: The elemental composition of herbal infusions and teas has not been sufficiently investigated. It could potentially be used for defining fingerprints for individual herbal / tea infusions, differentiation of botanical families, detecting the influence of packaging, and other purposes. The objective of this study was to determine the elemental composition, including the trace element content, of various herbal infusions and teas by means of total reflection X-ray fluorescence analysis (TXRF), with a chemometrics approach using principal component analysis (PCA). RESULTS: This study determined the elemental composition of various herbal infusions and teas, including trace elements, using total reflection X-ray fluorescence (TXRF). The methodology for the sample preparation was established, including the multiple-steepings procedure for the two tea samples (Oolong and Pu-erh). Data from 29 samples were collected. We hypothesized that the elemental content of infusions could reflect certain features, such as the influence of processing and the type of tea. CONCLUSION: A chemometric approach (PCA) was applied, and differences between teas and herbal infusions were found. This was further corroborated by explicit differentiation of one botanical family, Theaceae. The influence of packaging (tea bags) on herbal material was identified. The three types of tea (Camellia sinensis) appeared to be separated with PCA, and elemental concentrations in Pu-erh changed with multiple steepings.

Detection and imaging of gadolinium accumulation in human bone tissue by micro- and submicro-XRF.

Gadolinium-based contrast agents (GBCAs) are frequently used in patients undergoing magnetic resonance imaging. In GBCAs gadolinium (Gd) is present in a bound chelated form. Gadolinium is a rare-earth element, which is normally not present in human body. Though the blood elimination half-life of contrast agents is about 90 minutes, recent studies demonstrated that some tissues retain gadolinium, which might further pose a health threat due to toxic effects of free gadolinium. It is known that the bone tissue can serve as a gadolinium depot, but so far only bulk measurements were performed. Here we present a summary of experiments in which for the first time we mapped gadolinium in bone biopsy from a male patient with idiopathic osteoporosis (without indication of renal impairment), who received MRI 8 months prior to biopsy. In our studies performed by means of synchrotron radiation induced micro- and submicro-X-ray fluorescence spectroscopy (SR-XRF), gadolinium was detected in human cortical bone tissue. The distribution of gadolinium displays a specific accumulation pattern. Correlation of elemental maps obtained at ANKA synchrotron with qBEI images (quantitative backscattered electron imaging) allowed assignment of Gd structures to the histological bone structures. Follow-up beamtimes at ESRF and Diamond Light Source using submicro-SR-XRF allowed resolving thin Gd structures in cortical bone, as well as correlating them with calcium and zinc.

Unshielding Multidrug Resistant Cancer through Selective Iron Depletion of P-Glycoprotein-Expressing Cells.

Clinical evidence shows that following initial response to treatment, drug-resistant cancer cells frequently evolve and, eventually, most tumors become resistant to all available therapies. We compiled a focused library consisting of >500 commercially available or newly synthetized 8-hydroxyquinoline (8OHQ) derivatives whose toxicity is paradoxically increased rather than decreased by the activity of P-glycoprotein (Pgp), a transporter conferring multidrug resistance (MDR). Here, we deciphered the mechanism of action of NSC297366 that shows exceptionally strong Pgp-potentiated toxicity. Treatment of cells with NSC297366 resulted in changes associated with the activity of potent anticancer iron chelators. Strikingly, iron depletion was more pronounced in MDR cells due to the Pgp-mediated efflux of NSC297366-iron complexes. Our results indicate that iron homeostasis can be targeted by MDR-selective compounds for the selective elimination of multidrug resistant cancer cells, setting the stage for a therapeutic approach to fight transporter-mediated drug resistance. SIGNIFICANCE: Modulation of the MDR phenotype has the potential to increase the efficacy of anticancer therapies. These findings show that the MDR transporter is a "double-edged sword" that can be turned against resistant cancer.

Multimodal imaging of undecalcified tissue sections by MALDI MS and µXRF.

Matrix-assisted laser desorption/ionisation mass spectrometric imaging (MALDI MSI) is a technique that provides localized information on intact molecules in a sample. Micro X-ray fluorescence (µXRF) imaging allows the examination of the spatial distribution of elements in a sample without any morphological changes. These methods have already been applied separately to different tissues, organs, plants and bacterial films, but, to the best of our knowledge, they have yet to be coupled in a multimodal analysis. In this proof-of-principle study, we established and tested sample preparation strategies, allowing the multimodal analysis of lipids (sphingomyelin and phosphatidylcholines) and elements relevant to bone structures as calcium, phosphorous and sulphur in the very same sample section of a chicken phalanx without tissue decalcification. The results of the investigation of such parameters as adhesive tapes supporting tissue sections, and the sequence of the imaging experiments are presented. We show specific lipid distributions in skin, cartilage, muscle, nail, and the intact morphology of bone by calcium and phosphorus imaging. A combination of molecular and elemental imaging was achieved, thus, providing now for the first time the possibility of gathering MALDI MSI and µXRF information from the very same sample without any washing steps omitting therefore the analytical artifacts that inevitably occur in approaches using consecutive tissue sections. The proposed combination can benefit in research studies regarding bone diseases, osteoporosis, osteoarthritis, cartilage failure, bone/tendon distinguishing, where elemental and lipid interaction play an essential role.

Metal transport capabilities of anticancer copper chelators.

In the present study, several Cu chelators [2,2'-biquinoline, 8-hydroxiquinoline (oxine), ammonium pyrrolidinedithiocarbamate (APDTC), Dp44mT, dithizone, neocuproine] were used to study Cu uptake, depletion and localization in different cancer cell lines. To better understand the concentration dependent fluctuations in the Cu intracellular metal content and Cu-dependent in vitro antiproliferative data, the conditional stability constants of the Cu complex species of the investigated ligands were calculated. Each investigated chelator increased the intracellular Cu content on HT-29 cells causing Cu accumulation depending on the amount of the free Cu(II). Copper accumulation was 159 times higher for Dp44mT compared to the control. Investigating a number of other transition metals, intracellular accumulation of Cd was observed only for two chelators. Intracellular Zn content slightly decreased (cca. 10%) for MCF-7 cells, while a dramatic decrease was observed on MDA-MB-231 ones (cca. 50%). A similar decrease was observed for HCT-116, while Zn depletion for HT-29 corresponded to cca. 20%. The IC50 values were registered for the investigated four cell lines at increasing external Cu(II) concentration, namely, MDA-MB-231 cells had the lowest IC50 values for Dp44mT ranging between 7 and 35 nM. Thus, Zn depletion could be associated with lower IC50 values. Copper depletion was observed for all ligands being less pronounced for Dp44mT and neocuproine. Copper localization and its colocalization with Zn were determined by µ-XRF imaging. Loose correlation (0.57) was observed for the MCF-7 cells independently of the applied chelator. Similarly, a weak correlation (0.47) was observed for HT-29 cells treated with Cu(II) and oxine. Colocalization of Cu and Zn in the nucleus of HT-29 cells was observed for Dp44mT (correlation coefficient of 0.85).

Increased zinc accumulation in mineralized osteosarcoma tissue measured by confocal synchrotron radiation micro X-ray fluorescence analysis.

Abnormal tissue levels of certain trace elements such as zinc (Zn) were reported in various types of cancer. Little is known about the role of Zn in osteosarcoma. Using confocal synchrotron radiation micro X-ray fluorescence analysis, we characterized the spatial distribution of Zn in high-grade sclerosing osteosarcoma of nine patients (four women/five men; seven knee/one humerus/one femur) following chemotherapy and wide surgical resection. Levels were compared with adjacent normal tissue. Quantitative backscattered electron imaging as well as histological examinations was also performed. On average, the ratio of medians of Zn count rates (normalized to calcium) in mineralized tumor tissue was about six times higher than in normal tissue. There was no difference in Zn levels between tumor fraction areas with a low fraction and a high fraction of mineralized tissue, which were clearly depicted using quantitative backscattered electron imaging. Moreover, we found no correlation between the Zn values and the type of tumor regression according to the Salzer-Kuntschik grading. The underlying mechanism of Zn accumulation remains unclear. Given the emerging data on the role of trace elements in other types of cancer, our novel results warrant further studies on the role of trace elements in bone cancer. Copyright © 2016 The Authors. X-Ray Spectrometry published by John Wiley & Sons Ltd.

Fast and direct screening of copper in micro-volumes of distilled alcoholic beverages by high-resolution continuum source graphite furnace atomic absorption spectrometry.

HR-CS-GFAAS methods were developed for the fast determination of Cu in domestic and commercially available Hungarian distilled alcoholic beverages (called pálinka), in order to decide if their Cu content exceeds the permissible limit, as legislated by the WHO. Some microliters of samples were directly dispensed into the atomizer. Graphite furnace heating programs, effects/amounts of the Pd modifier, alternative wavelengths (e.g., Cu I 249.2146nm), external calibration and internal standardization methods were studied. Applying a fast graphite furnace heating program without any chemical modifier, the Cu content of a sample could be quantitated within 1.5min. The detection limit of the method is 0.03mg/L. Calibration curves are linear up to 10-15mg/L Cu. Spike-recoveries ranged from 89% to 119% with an average of 100.9±8.5%. Internal calibration could be applied with the assistance of Cr, Fe, and/or Rh standards. The accuracy of the GFAAS results was verified by TXRF analyses.

Uptake and toxicity of nano-ZnO in the plant-feeding nematode, Xiphinema vuittenezi: the role of dissolved zinc and nanoparticle-specific effects.

Nanoparticulate ZnO is one of the most commonly applied nanomaterials. As ZnO is more soluble than many other oxide nanoparticles, its toxicity beyond the nanoparticle-specific effects can be attributed to the dissolved ionic zinc. The investigation of uptake and toxicity of nano-ZnO in the plant-feeding nematode, Xiphinema vuittenezi, which was used in previous studies as a biological model organism, was aimed. The establishment of the role of dissolved zinc and nanoparticle-specific effects in the toxicity was also the objective of our study. Zn uptake was found to be significantly higher for bulk and nano-ZnO than for ZnSO4 solution; however, treatments caused loss of potassium in the worms in a dissolved-zinc-dependent manner. The toxicity was the lowest for bulk ZnO, and it was very similar for nano-ZnO and ZnSO4 solution. Accordingly, the toxicity of ZnO nanoparticles is a combination of dissolved-zinc-caused toxicity and nanoparticle-specific effects.

Iron overload of human colon adenocarcinoma cells studied by synchrotron-based X-ray techniques.

Fast- and slow-proliferating human adenocarcinoma colorectal cells, HT-29 and HCA-7, respectively, overloaded with transferrin (Tf), Fe(III) citrate, Fe(III) chloride and Fe(II) sulfate were studied by synchrotron radiation total-reflection X-ray spectrometry (TXRF), TXRF-X-ray absorption near edge structure (TXRF-XANES), and micro-X-ray fluorescence imaging to obtain information on the intracellular storage of overloaded iron (Fe). The determined TfR1 mRNA expression for the investigated cells correlated with their proliferation rate. In all cases, the Fe XANES of cells overloaded with inorganic Fe was found to be similar to that of deliquescent Fe(III) sulfate characterized by a distorted octahedral geometry. A fitting model using a linear combination of the XANES of Tf and deliquescent Fe(III) sulfate allowed to explain the near edge structure recorded for HT-29 cells indicating that cellular overload with inorganic Fe results in a non-ferritin-like fast Fe storage. Hierarchical cluster analysis of XANES spectra recorded for Fe overloaded HT-29 and HCA-7 cells was able to distinguish between Fe treatments performed with different Fe species with a 95% hit rate, indicating clear differences in the Fe storage system. Micro-X-ray fluorescence imaging of Fe overloaded HT-29 cells revealed that Fe is primarily located in the cytosol of the cells. By characterizing the cellular Fe uptake, Fe/S content ratios were calculated based on the X-ray fluorescence signals of the analytes. These Fe/S ratios were dramatically lower for HCA-7 treated with organic Fe(III) treatments suggesting dissimilarities from the Tf-like Fe uptake.

µXRF Elemental Mapping of Bioresorbable Magnesium-Based Implants in Bone.

This study investigated the distribution of the elemental constituents of Mg-based implants at various stages of the degradation process in surrounding bone tissue, with a focus on magnesium (Mg), as the main component of the alloy, and yttrium (Y), due to its potential adverse health effects. The measurements were performed on the implant-bearing thin sections of rat bone in a time series of implant degradation between one and 18 months. Micro X-ray fluorescence analysis (µXRF) with a special spectrometer meeting the requirements for the measurements of low-Z elements was used. It was found that the migration and accumulation behaviour of implant degradation products is element-specific. A sharp decrease in Mg was observed in the immediate vicinity of the interface and no specific accumulation or aggregation of Mg in the adjacent bone tissue was detected. By contrast, Y was found to migrate further into the bone over time and to remain in the tissue even after the complete degradation of the implant. Although the nature of Y accumulations must still be clarified, its potential health impact should be considered.

Complex forming competition and in-vitro toxicity studies on the applicability of di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) as a metal chelator.

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is a potential candidate in chelation therapy as an iron chelator. This study showed that a combined treatment with 2µM easily available Fe(II), Cu(II) and Zn(II) each and 5µM Dp44mT on eight different cancer cell lines resulted in a 10-40-fold increase in the intracellular Cu content compared to control samples. The uptake of Cu and Cu-dependent cytotoxicity strictly depend on the Cu concentration of the culture medium. Even as low concentration of Dp44mT as 0.1µM can transport high amounts of copper inside the cells. The Cu accumulation and toxicity through Dp44mT can hardly be influenced by Fe. Copper uptake and toxicity triggered by 2µM extracellular Cu(II) and 5µM Dp44mT could not be influenced by Fe(II) extracellular concentrations even 50-times higher than that of Cu(II). A 50-times higher Co(II) extracellular concentration hindered the Cu(II) uptake almost completely and a 10-times higher Co(II) concentration already decreased the Dp44mT-mediated Cu toxicity. Conditional complex stability constant determinations for Dp44mT with Cu(II), Co(II), Fe(II), Ni(II) and Zn(II) revealed that the metal-to-ligand ratio is 1:1 in [Cu(II)Dp44mT] complex, while for Co(II), Fe(II) and Ni(II) is 1:2. The highest stability constant was obtained for Cu(II) (lg ß=7.08±0.05) and Co(II) (lg ß2=12.47±0.07). According to our results, Dp44mT in combination with Cu is highly toxic in vitro. Therefore, the use of Dp44mT as an iron chelator is limited if biologically available Cu is also present even at low concentrations.