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The effects of encapsulating the cytotoxic dinuclear trithiolato-bridged arene ruthenium complex [(η6 -p-MeC6 H4 iPr)2 Ru2 (μ2 -S-p-C6 H4 tBu)3 ]Cl (DiRu-1) within the apoferritin (AFt) nanocage were investigated. The DiRu-1-AFt nanocarrier was characterized by UV/Vis spectroscopy, ICP-MS, CD and X-ray crystallography. In contrast to previously reported Au- and Pt-based drug-loaded AFt carriers, we found no evidence of direct interactions between DiRu-1 and AFt. DiRu-1-AFt is cytotoxic toward immortalized murine BALB/c-3T3 fibroblasts transformed with SV40 virus (SVT2) and human epidermoid carcinoma A431 malignant cells, and exhibits moderate selectivity for these cancer cells over normal BALB/c-3T3 cells. DiRu-1-AFt triggers the production of reactive oxygen species, depolarization of mitochondrial membrane potential, and induces cell death via p53-mediated apoptosis. Comparison between our data and previous results suggests that the presence of specific interactions between a metal-based drug and AFt within the protein cage is not essential for drug encapsulation.

Tumor resistance to chemotherapy represents an important challenge in modern oncology. Although platinum (Pt)-based drugs have demonstrated excellent therapeutic potential, their effectiveness in a wide range of tumors is limited by the development of resistance mechanisms. One of these mechanisms includes increased cisplatin sequestration/efflux by the copper-transporting ATPase, ATP7B. However, targeting ATP7B to reduce Pt tolerance in tumors could represent a serious risk because suppression of ATP7B might compromise copper homeostasis, as happens in Wilson disease. To circumvent ATP7B-mediated Pt tolerance we employed a high-throughput screen (HTS) of an FDA/EMA-approved drug library to detect safe therapeutic molecules that promote cisplatin toxicity in the IGROV-CP20 ovarian carcinoma cells, whose resistance significantly relies on ATP7B. Using a synthetic lethality approach, we identified and validated three hits (Tranilast, Telmisartan, and Amphotericin B) that reduced cisplatin resistance. All three drugs induced Pt-mediated DNA damage and inhibited either expression or trafficking of ATP7B in a tumor-specific manner. Global transcriptome analyses showed that Tranilast and Amphotericin B affect expression of genes operating in several pathways that confer tolerance to cisplatin. In the case of Tranilast, these comprised key Pt-transporting proteins, including ATOX1, whose suppression affected ability of ATP7B to traffic in response to cisplatin. In summary, our findings reveal Tranilast, Telmisartan, and Amphotericin B as effective drugs that selectively promote cisplatin toxicity in Pt-resistant ovarian cancer cells and underscore the efficiency of HTS strategy for identification of biosafe compounds, which might be rapidly repurposed to overcome resistance of tumors to Pt-based chemotherapy.

In the context of the vascular effects of hydrogen sulfide (H

Tumor resistance to chemotherapy represents an important challenge in modern oncology. Although platinum (Pt)-based drugs have demonstrated excellent therapeutic potential, their effectiveness in a wide range of tumors is limited by the development of resistance mechanisms. One of these mechanisms includes increased cisplatin sequestration/efflux by the copper-transporting ATPase, ATP7B. However, targeting ATP7B to reduce Pt tolerance in tumors could represent a serious risk because suppression of ATP7B might compromise copper homeostasis, as happens in Wilson disease.To circumvent ATP7B-mediated Pt tolerance we employed a high-throughput screen (HTS) of an FDA/EMA-approved drug library to detect safe therapeutic molecules that promote cisplatin toxicity in the resistant ovarian carcinoma cell line IGROV-CP20. Using a synthetic lethality approach we identified and validated three hits (Tranilast, Telmisartan and Amphotericin B) that could reduce cisplatin resistance. All three drugs induced Pt-mediated DNA damage and inhibited either expression or trafficking of ATP7B in a tumor-specific manner. Global transcriptome analyses showed that Tranilast and Amphotericin B affect expression of genes operating in several pathways that confer tolerance to cisplatin. In the case of Tranilast, these included key molecular players operating in the distribution of platinum to different intracellular compartments. In particular, Tranilast was found to suppress ATOX1 and, as a consequence, ATOX1-mediated trafficking of ATP7B in response to cisplatin.Considering the well-known safety profiles of Tranilast, Telmisartan and Amphotericin B, these drugs emerge as potential candidates that might be used for the rapid development of new therapeutic strategies to overcome resistance of tumors to Pt-based chemotherapy.

The products of the reaction between cisplatin (CDDP) and the model protein hen egg white lysozyme (HEWL) at 20, 37 and 55 °C in pure water were studied by UV-Vis absorption spectroscopy, intrinsic fluorescence and circular dichroism, dynamic and electrophoretic light scattering and inductively coupled plasma mass spectrometry. X-ray structures were also solved for the adducts formed at 20 and 55 °C. Data demonstrate that high temperature facilitates the formation of CDDP-HEWL adducts, where Pt atoms bind ND1 atom of His15 or NE2 atom of His15 and NH1 atom of Arg14. Our study suggests that high human body temperature (fever) could increase the rate of drug binding to proteins thus enhancing possible toxic side effects related to CDDP administration.

In the context of the vascular effects of hydrogen sulfide (H2S), it is known that this gaseous endogenous biological modulator of inflammation, oxidative stress, etc. is a potent vasodilator. Chronic renal failure, a common disease affecting the aging population, is characterized by low levels of H2S in plasma and tissues, which could mediate their typical hypertensive pattern, along with other abnormalities. Lanthionine and homolanthionine, natural non-proteinogenic amino acids, are formed as side products of H2S production. Also in consideration of the intrinsic difficulties in H2S measuring, these compounds have been proposed as reliable and stable markers of H2S synthesis. However, in the setting of chronic renal failure patients on hemodialysis, they represent typical retention products (without ruling out the possibility of an increased intestinal synthesis) and prospective novel uremic toxins. Here, a method utilizing liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring ion mode has been developed and evaluated for the determination of these key H2S metabolites in plasma, by using a triple quadrupole mass spectrometer.

Aim: A Pt(II)-terpyridine compound, bearing two piperidine substituents at positions 2 and 2′ of the terpyridine ligand (1), is highly cytotoxic and shows a mechanism of action distinct from cisplatin. 1 has been incorporated within the ferritin nanocage (AFt). Materials & methods: Spectroscopic and crystallographic data of the Pt(II)–AFt nanocomposite have been collected and in vitro anticancer activity has been explored using cancer cells. Results: Pt(II)-containing fragments bind His49, His114 and His132. Pt(II)–AFt nanocomposite is less cytotoxic than 1, but it is more toxic than cisplatin at high concentrations. The Pt(II)–AFt nanocomposite triggers necrosis in cancer cells, as free 1 does. Conclusion: Pt(II)–AFt nanocomposites are promising vehicles to deliver Pt-based drugs to cancer cells.

Cisplatin (CDDP) can be encapsulated within the central cavity of reconstituted (apo)ferritin, (A)Ft, to form a drug-loaded protein of potential great interest for targeted cancer treatments. In this study, the interactions occurring between cisplatin and native horse spleen Ft in CDDP-encapsulated AFt are investigated by high-resolution X-ray crystallography. A protein bound Pt center is unambiguously identified in AFt subunits by comparative analysis of difference Fourier electron density maps and of anomalous dispersion data. Indeed, a [Pt(NH3)2H2O](2+) fragment is found coordinated to the His132 residue located on the inner surface of the large AFt spherical cage. Remarkably, Pt binding does not alter the overall physicochemical features (shape, volume, polarity/hydrophobicity and electrostatic potential) of the outer surface of the AFt nanocage. CDDP-encapsulated AFt appears to be an ideal nanocarrier for CDDP delivery to target sites, as it possesses high biocompatibility and can be internalized by receptor mediated endocytosis, thus carrying the drug to tumor tissue with higher selectivity than free CDDP.

Auoxo3, a cytotoxic gold(iii) compound, was encapsulated within a ferritin nanocage. Inductively coupled plasma mass spectrometry, circular dichroism, UV-Vis absorption spectroscopy and X-ray crystallography confirm the potential-drug encapsulation. The structure shows that naked Au(i) ions bind to the side chains of Cys48, His49, His114, His114 and Cys126, Cys126, His132, His147. The gold-encapsulated nanocarrier has a cytotoxic effect on different aggressive human cancer cells, whereas it is significantly less cytotoxic for non-tumorigenic cells.

The encapsulation of Pt and Au-based anticancer agents within a protein cage is a promising way to enhance the selectivity of these potential drugs. Here a cytotoxic organometallic compound containing platinum(II) and gold(I) has been encapsulated within a ferritin nanocage (AFt). Inductively plasma coupled mass spectrometry data, collected to evaluate the amount of Pt and Au within the cage, indicate disruption of the starting heterobimetallic complex upon encapsulation within the nanocage. The drug-loaded protein (Pt(II)/Au(I)-AFt) has been characterized by UV–Vis spectroscopy, circular dichroism and X-ray diffraction analysis. Data indicate that the protein maintains its fold upon encapsulation of the metallodrug and that Au(I) and Pt(II)-containing fragments are encapsulated within the AFt cage, with Au(I) ion that binds the side chain of Cys126 and Pt(II) in the bulk, respectively. The in vitro cytotoxicity of Pt(II)Au(I)-AFt, as well as that of the free heterobimetallic complex, has been comparatively evaluated on human cervix and breast cancer cells and against cardiomyoblasts and keratinocytes non-tumorigenic cells. Our data demonstrate that it is possible to obtain a protein nanocarrier containing both Pt and Au atoms starting from a bimetallic compound, opening the way for the design and development of new potential drugs based on protein nanocarriers. Previous article in issue

In AD 79 the town of Herculaneum was suddenly hit and overwhelmed by volcanic ash-avalanches that killed all its remaining residents, as also occurred in Pompeii and other settlements as far as 20 kilometers from Vesuvius. New investigations on the victims' skeletons unearthed from the ash deposit filling 12 waterfront chambers have now revealed widespread preservation of atypical red and black mineral residues encrusting the bones, which also impregnate the ash filling the intracranial cavity and the ash-bed encasing the skeletons. Here we show the unique detection of large amounts of iron and iron oxides from such residues, as revealed by inductively coupled plasma mass spectrometry and Raman microspectroscopy, thought to be the final products of heme iron upon thermal decomposition. The extraordinarily rare preservation of significant putative evidence of hemoprotein thermal degradation from the eruption victims strongly suggests the rapid vaporization of body fluids and soft tissues of people at death due to exposure to extreme heat.

Two anticancer gold(iii) compounds, Au2phen and Auoxo4, were encapsulated within a ferritin nanocage. The gold-compound loaded proteins were characterized by UV-Vis spectroscopy, inductively coupled plasma mass spectrometry and circular dichroism. X-ray crystallography shows that the compounds degrade upon encapsulation and gold(i) ions bind Ft within the cage, close to the side chains of Cys126. The gold-encapsulated nanocarriers are cytotoxic to human cancer cells. Au(i)-loaded Ft, obtained upon the encapsulation of Au2phen within the cage, induces oxidative stress activation, which finally leads to apoptosis in MCF-7 cells.

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses. Several studies have suggested that free D-Asp has a crucial role in N-methyl D-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free D-Asp, L-Asp and N-methyl D-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing L-Asp, D-Asp and N-methyl D-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for D-Asp, 0.46 pg/μl for L-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for D-Asp, 1.41 pg/μl for L-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of D-Asp, L-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free D-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.

Dialysis patients display a high cardiovascular mortality, the causes of which are still not completely explained, but are related to uremic toxicity. Among uremic toxins, homocysteine and cysteine are both substrates of cystathionine beta-synthase and cystathionine gamma-lyase in hydrogen sulfide biosynthesis, leading to the formation of two sulfur metabolites, lanthionine and homolanthionine, considered stable indirect biomarkers of its production. Hydrogen sulfide is involved in the modulation of multiple pathophysiological responses. In uremia, we have demonstrated low plasma total hydrogen sulfide levels, due to reduced cystathionine gamma-lyase expression.Plasma hydrogen sulfide levels were measured in hemodialysis patients and healthy controls with three different techniques in comparison, allowing to discern the different pools of this gas. The protein bound (the one thought to be the most active) and acid-labile forms are significantly decreased, while homolanthionine, but especially lanthionine, accumulate in the blood of uremic patients. The hemodialysis regimen plays a role in determining sulfur compounds levels, and lanthionine is partially removed by a single dialysis session. Lanthionine inhibits hydrogen sulfide production in cell cultures under conditions comparable to in vivo ones. We therefore propose that lanthionine is a novel uremic toxin. The possible role of high lanthionine as a contributor to the genesis of hyperhomocysteinemia in uremia is discussed. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Background Comparison of toxicogenomic data facilitates the identification of deregulated gene patterns and maximizes health risk prediction in human. Results Here, we performed phenotypic anchoring on the effects of acute exposure to low-grade polluted groundwater using mouse and zebrafish. Also, we evaluated two windows of chronic exposure in mouse, starting in utero and at the end of lactation. Bioinformatic analysis of livers microarray data showed that the number of deregulated biofunctions and pathways is higher after acute exposure, compared to the chronic one. It also revealed specific profiles of altered gene expression in all treatments, pointing to stress response/mitochondrial pathways as major players of environmental toxicity. Of note, dysfunction of steroid hormones was also predicted by bioinformatic analysis and verified in both models by traditional approaches, serum estrogens measurement and vitellogenin mRNA determination in mice and zebrafish, respectively. Conclusions In our report, phenotypic anchoring in two vertebrate model organisms highlights the toxicity of low-grade pollution, with varying susceptibility based on exposure window. The overlay of zebrafish and mice deregulated pathways, more than single genes, is useful in risk identification from chemicals implicated in the observed effects. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1067) contains supplementary material, which is available to authorized users.

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.