Effects of subacute cadmium exposure and subsequent deferiprone treatment on cadmium accumulation and on the homeostasis of essential elements in the mouse brain.

INTRODUCTION: Cadmium (Cd) is а hazardous multi-organ toxin. In this study, we provide the first results about the effect of oral administration of deferiprone (DFP) on Cd accumulation and on the homeostasis of essential elements in the brain of Cd-exposed mice. METHODS: Adult Institute of Cancer Research (ICR) male mice were randomized into four experimental groups: untreated controls - administered distilled water for 28 days; Cd-exposed group - exposed to 18 mg/kg body weight (b.w.) Cd(II) acetate for 14 days followed by the administration of distilled water for two weeks; Cd + DFP (low dose) - Cd-intoxicated mice subsequently treated with 19 mg/kg b.w. DFP for two weeks; and Cd + DFP (high dose) - Cd-exposed mice administered high-dose DFP (135 mg/kg b.w.) for 14 days. Brains were subjected to inductively coupled plasma-mass spectrometry (ICP-MS) and histological analysis. RESULTS: The results revealed that exposure of mice to Cd for 14 days significantly increased Cd concentration and significantly decreased magnesium (Mg), phosphorus (P), and zinc (Zn) contents in the brain compared to untreated controls. This effect was accompanied by necrotic-degenerative changes in both the cerebrum and cerebellum. Oral administration of low-dose DFP to Cd-exposed mice decreased the concentration of the toxic metal in the brain by 16.37% and restored the concentration of the essential elements to normal control values. Histological analysis revealed substantially improved cerebral and cerebellar histoarchitectures. In contrast, oral administration of high-dose DFP increased Cd content and significantly decreased selenium (Se) concentration in the brain. Necrotic neurons and Purkinje cells were still observed in the cerebral and cerebellar cortices. CONCLUSION: The results demonstrated that oral administration of DFP at low doses has a better therapeutic potential for the treatment of Cd-induced brain damage compared to high doses.

Current and emerging mass spectrometry methods for the preclinical development of metal-based drugs: a critical appraisal.

This Trends article highlights the multiple ways in which the state-of-the-art molecular mass spectrometry can support the preclinical development of novel metal-based anticancer drugs. Examples from the recent literature-beyond routine characterization applications-are presented to illustrate what analytical and experimental design challenges are to be addressed to facilitate the translation of promising drug candidates to clinical practice.

Introducing N-, P-, and S-donor leaving groups: an investigation of the chemical and biological properties of ruthenium, rhodium and iridium thiopyridone piano stool complexes.

A series of 15 piano-stool complexes featuring either a RuII, RhIII or IrIII metal center, a bidentate thiopyridone ligand, and different leaving groups was synthesized. The leaving groups were selected in order to cover a broad range of different donor atoms. Thus, 1-methylimidazole served as a N-donor, 1,3,5-triaza-7-phosphaadamantane (pta) as a P-donor, and thiourea as a S-donor. Additionally, three complexes featuring different halido leaving groups (Cl, Br, I) were added. Leaving group alterations were carried out with respect to a possible influence on pharmacokinetic and pharmacodynamic parameters, as well as the cytotoxicity of the respective compounds. The complexes were characterized via NMR spectroscopy, X-ray diffraction (where possible), mass spectrometry, and elemental analysis. Cytotoxicity was assessed in 2D cultures of human cancer cell lines by microculture and clonogenic assays as well as in multicellular tumor spheroids. Furthermore, cellular accumulation studies, flow-cytometric apoptosis and ROS assays, DNA plasmid assays, and laser ablation ICP-MS studies for analyzing the distribution in sections of multicellular tumor spheroids were conducted. This work demonstrates the importance of investigating each piano-stool complexes' properties, as the most promising candidates showed advantages over each other in certain tests/assays. Thus, it was not possible to single out one lead compound, but rather a group of complexes with enhanced cytotoxicity and activity.

Mercury, silver, selenium and other trace elements in three cyprinid fish species from the Vaal Dam, South Africa, including implications for fish consumers.

The levels of Cr, Cu, Zn, Se, Ag, Cd, Hg, and Pb were determined in muscle and liver samples from 30 specimens of fish belonging to the species Labeobarbus aeneus, Labeobarbus kimberleyensis, and Labeo umbratus from the Vaal Dam. Health risks for human fish consumers were estimated using the target hazard quotient (THQ), the Se:Hg-ratio, and Se health benefit value (Se HBV). This is the first comprehensive report on Hg levels in fish from this lake. Mean concentrations ranging from 0.247-0.481 mg/kg dw in muscle and from 0.170-0.363 mg/kg dw in liver clearly show a contamination with this element. Although levels in muscle did not exceed maximum allowances for human consumption, a calculated THQ of 0.12 and 0.14 for the two Labeobarbus species, respectively, showed a potential risk due to additive effects. All Se:Hg-ratios as well as Se HBVs clearly suggested positive effects for fish consumers. Levels of Cu were remarkably high in the liver of L. umbratus, calling for further investigation on this species. Cadmium levels were above the maximum allowances for fish consumption in the liver of all three species (means between 0.190 and 0.460 mg/kg dw), but below the LOD in all muscle and intestine samples. This is also the first report of Ag in fish from South Africa. Levels were below the LOD in muscle, but well detectable in liver; they varied significantly between the two Labeobarbus species (0.054 ±â€¯0.030 and 0.037 ±â€¯0.016 mg/kg dw) compared to L. umbratus (1.92 ±â€¯0.83 mg/kg dw) and showed a positive correlation with Cu levels (63.7 ±â€¯17.0; 70.3 ±â€¯9.0 and 1300 ±â€¯823 mg/kg dw), possibly due to similar chemical affinities to metallothioneins. The detected Ag levels can serve as a basis to monitor the development of this new pollutant in aquatic environments in South Africa and worldwide.

Biological activity of PtIV prodrugs triggered by riboflavin-mediated bioorthogonal photocatalysis.

We have recently demonstrated that riboflavin (Rf) functions as unconventional bioorthogonal photocatalyst for the activation of PtIV prodrugs. In this study, we show how the combination of light and Rf with two PtIV prodrugs is a feasible strategy for light-mediated pancreatic cancer cell death induction. In Capan-1 cells, which have high tolerance against photodynamic therapy, Rf-mediated activation of the cisplatin and carboplatin prodrugs cis,cis,trans-[Pt(NH3)2(Cl)2(O2CCH2CH2CO2H)2] (1) and cis,cis,trans-[Pt(NH3)2(CBDCA)(O2CCH2CH2CO2H)2] (2, where CBDCA = cyclobutane dicarboxylate) resulted in pronounced reduction of the cell viability, including under hypoxia conditions. Such photoactivation mode occurs to a considerable extent intracellularly, as demonstrated for 1 by uptake and cell viability experiments. 195Pt NMR, DNA binding studies using circular dichroism, mass spectrometry and immunofluorescence microscopy were performed using the Rf-1 catalyst-substrate pair and indicated that cell death is associated with the efficient light-induced formation of cisplatin. Accordingly, Western blot analysis revealed signs of DNA damage and activation of cell death pathways through Rf-mediated photochemical activation. Phosphorylation of H2AX as indicator for DNA damage, was detected for Rf-1 in a strictly light-dependent fashion while in case of free cisplatin also in the dark. Photochemical induction of nuclear pH2AX foci by Rf-1 was confirmed in fluorescence microscopy again proving efficient light-induced cisplatin release from the prodrug system.

Studies of KP46 and KP1019 and the Hydrolysis Product of KP1019 in Lipiodol Emulsions: Preparation and Initial Characterizations as Potential Theragnostic Agents.

BACKGROUND: Lipiodol (iodized poppy seed oil) accumulates predominately in the tumor rather than in the liver tissue [1, 2]. Therefore, mixing anticancer drugs with Lipiodol may enhance the antitumor effect by increasing the local drug concentration. OBJECTIVE: In this pilot study, we made use of Lipiodol as a potential carrier of three promising antitumor metal complexes (tris(8-quinolato)gallium(III) (KP46), tetrachlorobis(indazole)ruthenate(III) (KP1019) and the hydrolysis product of KP1019, mer,trans-[RuCl3(H2O)(Hind)2]. METHODS: The stability of the drugs in Lipiodol and the release profile into the aqueous phase were examined independently by three different analytical techniques (high pressure liquid chromatography, HPLC; atom absorption spectroscopy, AAS; and electron spray ionization mass spectrometry, ESI-MS). RESULTS: The complexes were stable and remained in the Lipiodol emulsion over 3 days. In contrast to KP1019 and KP46, evaluation of Lipiodol emulsions of mer,trans-[RuCl3 (H2O) (Hind) 2] was not possible due to the insolubility of the compound in Lipiodol. KP1019 released rapidly into the aqueous phase in the first week and after 1 month it was not possible to detect the complex in the emulsion. KP46 showed a gradual release with the time resulting in the release of about 6.4 % of KP46 into the aqueous phase after 1 month of incubation. CONCLUSION: The initial results show that Lipiodol can be successfully employed as a carrier of anticancer Ru- or Ga-complexes. Furthermore, advantages can overcome the poor water solubility of the metal complexes, opening new perspectives for the use of Lipiodol emulsions in molecular imaging and cancer therapy as theragnostic agents.

Fast High-Resolution Laser Ablation-Inductively Coupled Plasma Mass Spectrometry Imaging of the Distribution of Platinum-Based Anticancer Compounds in Multicellular Tumor Spheroids.

Multicellular tumor spheroid models serve as an important three-dimensional in vitro cell model system as they mimic the complex tumor microenvironment and thus have contributed to valuable assays in drug discovery studies. In this study, we present a state-of-the-art laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) setup for high spatial resolution elemental imaging of multicellular tumor spheroids and an approach to account for variations in cell density. A low dispersion LA-ICPMS setup was employed, providing accelerated throughput and high sensitivity and permitting a lateral image resolution down to ∼2.5 µm for phosphorus and platinum in HCT116 colon cancer spheroids upon treatment with the clinically used anticancer drug oxaliplatin. Phosphorus was introduced as scalar to compensate for differences in cell density and tissue thickness and the Pt/P ratios together with the high resolution adopted in our approach allows the differentiation of platinum accumulation within each part of the morphology of the tumor spheroids (layers of proliferating, quiescent, and necrotic cells).

Distinct activity of the bone-targeted gallium compound KP46 against osteosarcoma cells - synergism with autophagy inhibition.

BACKGROUND: Osteosarcoma is the most frequent primary malignant bone tumor. Although survival has distinctly increased due to neoadjuvant chemotherapy in the past, patients with metastatic disease and poor response to chemotherapy still have an adverse prognosis. Hence, development of new therapeutic strategies is still of utmost importance. METHODS: Anticancer activity of KP46 against osteosarcoma cell models was evaluated as single agent and in combination approaches with chemotherapeutics and Bcl-2 inhibitors using MTT assay. Underlying mechanisms were tested by cell cycle, apoptosis and autophagy assays. RESULTS: KP46 exerted exceptional anticancer activity at the nanomolar to low micromolar range, depending on the assay format, against all osteosarcoma cell models with minor but significant differences in IC50 values. KP46 treatment of osteosarcoma cells caused rapid loss of cell adhesion, weak cell cycle accumulation in S-phase and later signs of apoptotic cell death. Furthermore, already at sub-cytotoxic concentrations KP46 reduced the migratory potential of osteosarcoma cells and exerted synergistic effects with cisplatin, a standard osteosarcoma chemotherapeutic. Moreover, the gallium compound induced signs of autophagy in osteosarcoma cells. Accordingly, blockade of autophagy by chloroquine but also by the Bcl-2 inhibitor obatoclax increased the cytotoxic activity of KP46 treatment significantly, suggesting autophagy induction as a protective mechanism against KP46. CONCLUSION: Together, our results identify KP46 as a new promising agent to supplement standard chemotherapy and possible future targeted therapy in osteosarcoma.

LA-ICP-MS imaging in multicellular tumor spheroids - a novel tool in the preclinical development of metal-based anticancer drugs.

A novel application of advanced elemental imaging offers cutting edge in vitro assays with more predictive power on the efficacy of anticancer drugs in preclinical development compared to two dimensional cell culture models. We propose LA-ICP-MS analysis of multicellular spheroids, which are increasingly being used as three dimensional (3D) models of tumors, for improving the in vitro evaluation of anticancer metallodrugs. The presented strategy is very well suited for screening drug-tumor penetration, a key issue for drug efficacy. A major advantage of tumor spheroid models is that they enable us to create a tissue-like structure and function. With respect to 2D culture on the one hand and in vivo models on the other, multicellular spheroids thus show intermediate complexity, still allowing high repeatability and adequate through-put for drug research. This strongly argues for the use of spheroids as bridging models in preclinical anticancer drug development. Probing the lateral platinum distribution within these tumor models allows visualizing the penetration depth and targeting of platinum-based complexes. In the present study, we show for the first time that spatially-resolved metal accumulation in tumor spheroids upon treatment with platinum compounds can be appropriately assessed. The optimized LA-ICP-MS setup allowed discerning the platinum localization in different regions of the tumor spheroids upon compound treatment at biologically relevant (low micromolar) concentrations. Predominant platinum accumulation was observed at the periphery as well as in the center of the spheroids. This corresponds to the proliferating outermost layers of cells and the necrotic core, respectively, indicating enhanced platinum sequestration in these regions.

Antagonistic effects of selenium and lipid peroxides on growth control in early hepatocellular carcinoma.

UNLABELLED: Activation of the activator protein 1 (AP-1) transcription factor as well as increased serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-8 predict poor prognosis of patients with hepatocellular carcinomas (HCCs). Moreover, HCC patients display reduced selenium levels, which may cause lipid peroxidation and oxidative stress because selenium is an essential component of antioxidative glutathione peroxidases (GPx). We hypothesized that selenium-lipid peroxide antagonism controls the above prognostic markers and tumor growth. (1) In human HCC cell lines (HCC-1.2, HCC-3, and SNU398) linoleic acid peroxide (LOOH) and other prooxidants enhanced the expression of VEGF and IL-8. LOOH up-regulated AP-1 activation. Selenium inhibited these effects. This inhibition was mediated by glutathione peroxidase 4 (GPx4), which preferentially degrades lipid peroxides. Selenium enhanced GPx4 expression and total GPx activity, while knock-down of GPx4 by small interfering RNA (siRNA) increased VEGF, and IL-8 expression. (2) These results were confirmed in a rat hepatocarcinogenesis model. Selenium treatment during tumor promotion increased hepatic GPx4 expression and reduced the expression of VEGF and of the AP-1 component c-fos as well as nodule growth. (3) In HCC patients, increased levels of LOOH-related antibodies (LOOH-Ab) were found, suggesting enhanced LOOH formation. LOOH-Ab correlated with serum VEGF and IL-8 and with AP-1 activation in HCC tissue. In contrast, selenium inversely correlated with VEGF, IL-8, and HCC size (the latter only for tumors smaller than 3 cm). CONCLUSION: Reduced selenium levels result in accumulation of lipid peroxides. This leads to enhanced AP-1 activation and consequently to elevated expression of VEGF and IL-8, which accelerate the growth of HCC. Selenium supplementation could be considered for investigation as a strategy for chemoprevention or additional therapy of early HCC in patients with low selenium levels.

Gallium in cancer treatment.

Gallium (Ga) is the second metal ion, after platinum, to be used in cancer treatment. Its activities are numerous and various. It modifies three-dimensional structure of DNA and inhibits its synthesis, modulates protein synthesis, inhibits the activity of a number of enzymes, such as ATPases, DNA polymerases, ribonucleotide reductase and tyrosine-specific protein phosphatase. Ga alters plasma membrane permeability and mitochondrial functions. Ga salts are taken up more efficiently and more specifically by tumour cells when orally administered. New compounds have been prepared: Ga maltolate, doxorubicin-Ga-transferrin conjugate and Tris(8-quinolinolato)Ga(III), which show interesting activities. Ga toxicity is well documented in vitro and in vivo in animals. In humans, the oral administration Ga is less toxic, and allows a chronic treatment, allowing an improvement of its bioavailability in tumours, by comparison with the parenteral use. The anticancer activity of Ga salts has been demonstrated but other effects have also been noted such as many bone effects that could be useful in bone metastatic patients. Its has also been shown that a long period of administration could induce tumour fibrosis. Ga is synergistic with other anticancer drugs. Although not as potent as platinum in vitro, the anticancer activity of Ga should not be ignored, but the schedule of administration still needs to be optimised and new compounds are now under clinical investigations.