M. Milagros Gómez-Gómez

Elemental Bioimaging in Kidney by LA–ICP–MS As a Tool to Study Nephrotoxicity and Renal Protective Strategies in Cisplatin Therapies

A laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 μm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters. Cu and Zn distribution maps revealed a significant displacement in cells by Pt, as compared to control tissues. A dramatic decrease in the Pt accumulation in the cortex was observed when cilastatin was coadministered with cisplatin, which can be related to its nephroprotective effect. Excellent imaging reproducibility, sensitivity (LOD 50 fg), and resolution (down to 8 μm) were achieved, demonstrating that LA-ICP-MS can be applied as a microscopic metal detector at cellular level in certain tissues. A simple and quick approach for the estimation of Pt tissue levels was proposed, based on tissue spiking.

Cilastatin Attenuates Cisplatin-Induced Proximal Tubular Cell Damage

A major area in cancer therapy is the search for protective strategies against cisplatin-induced nephrotoxicity. We investigated the protective effect of cilastatin on cisplatin-induced injury to renal proximal tubular cells. Cilastatin is a specific inhibitor of renal dehydrodipeptidase I (DHP-I), which prevents hydrolysis of imipenem and its accumulation in the proximal tubule. Primary cultures of proximal cells were treated with cisplatin (1–30 μM) in the presence or absence of cilastatin (200 μg/ml). Apoptosis and mitochondrial injury were assessed by different techniques. Cisplatin uptake and DNA binding were measured by inductively coupled plasma spectrometry. HeLa cells were used to control the effect of cilastatin on the tumoricidal activity of cisplatin. Cisplatin increased cell death, apoptotic-like morphology, caspase activation, and mitochondrial injury in proximal tubular cells in a dose- and time-dependent way. Concomitant treatment with cilastatin reduced cisplatin-induced changes. Cilastatin also reduced the DNA-bound platinum but did not modify cisplatin-dependent up-regulation of death receptors (Fas) or ligands (tumor necrosis factor α, Fas ligand). In contrast, cilastatin did not show any effects on cisplatin-treated HeLa cells. Renal DHP-I was virtually absent in HeLa cells. Cilastatin attenuates cisplatin-induced cell death in proximal tubular cells without reducing the cytotoxic activity of cisplatin in tumor cells. Our findings suggest that the affinity of cilastatin for renal dipeptidase makes this effect specific for proximal tubular cells and may be related to a reduction in intracellular drug accumulation. Therefore, cilastatin administration might represent a novel strategy in the prevention of cisplatin-induced acute renal injury.

Top-down mass spectrometric approach for the full characterization of insulin-cisplatin adducts.

The interaction of the antitumor drug cisplatin with insulin was studied using a top-down mass spectrometric approach. In vitro incubations were prepared under acidic and physiological conditions at different insulin/cisplatin molar ratios for different incubation times. Size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICPMS) analysis enabled the specific detection of platinum containing species attributed to the binding of the drug to the protein. Further analysis through matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanoelectrospray ionization mass spectrometry using a linear ion trap (nESI-LIT-MS) allowed the identification of platinated mono-, di-, and even triadducts in the incubations. Platinum binding sites were identified by CID-MS(n) as B chain N-terminus, His5, and probably His10 residues, which turned out to be the same, regardless of the incubation conditions. Evidence on the binding of Pt to B chain Cys7 was also observed. Working with the LIT zoom scan mode provides enough resolution to discern the isotopic pattern for both precursor and fragment ions, allowing the differentiation of platinum-containing ions. The elucidation of platinum binding sites in a native protein through a top-down approach has been performed for the first time with this type of instrument.

2-Deoxy-d-glucose cooperates with arsenic trioxide to induce apoptosis in leukemia cells: Involvement of IGF-1R-regulated Akt/mTOR, MEK/ERK and LKB-1/AMPK signaling pathways

While the anti-tumor efficacy of 2-deoxy-D-glucose (2-DG) is normally low in monotherapy, it may represent a valuable radio- and chemo-sensitizing agent. We here demonstrate that 2-10 mM 2-DG cooperates with arsenic trioxide (ATO) and other antitumor drugs to induce apoptosis in human myeloid leukemia cell lines. Using ATO and HL60 as drug and cell models, respectively, we observed that 2-DG/ATO combination activates the mitochondrial apoptotic pathway, as indicated by Bid-, and Bax-regulated cytochrome c and Omi/HtrA2 release, XIAP down-regulation, and caspase-9/-3 pathway activation. 2-DG neither causes oxidative stress nor increases ATO uptake, but causes inner mitochondria membrane permeabilization as well as moderate ATP depletion, which nevertheless do not satisfactorily explain the pro-apoptotic response. Surprisingly 2-DG causes cell line-specific decrease in LKB-1/AMPK phosphorylation/activation, and also causes Akt/mTOR/p70S6K and MEK/ERK activation, which is prevented by co-treatment with ATO. The use of kinase-specific pharmacologic inhibitors and/or siRNAs reveals that apoptosis is facilitated by AMPK inactivation and restrained by Akt and ERK activation, and that Akt and ERK activation mediates AMPK inhibition. Finally, 2-DG stimulates IGF-1R phosphorylation/activation, and co-treatment with IGF-1R inhibitor prevents 2-DG effects on Akt, ERK and AMPK, and facilitates 2-DG-provoked apoptosis. In summary 2-DG elicits IGF-1R-mediated AMPK inactivation and Akt and ERK activation, which facilitates or restrain apoptosis, respectively. 2-DG-provoked AMPK inactivation increases the apoptotic efficacy of ATO, while in turn ATO-provoked Akt and ERK inactivation may increase the efficacy of 2-DG as anti-tumor drug.

Calibration and use of the Chemcatcher® passive sampler for monitoring organotin compounds in water

An integrative passive sampler (Chemcatcher) consisting of a 47 mm C18 Empore disk as the receiving phase overlaid with a thin cellulose acetate diffusion membrane was developed and calibrated for the measurement of time-weighted average water concentrations of organotin compounds [monobutyltin (MBT), dibutyltin (DBT), tributlytin (TBT) and triphenyltin (TPhT)] in water. The effect of water temperature and turbulence on the uptake rate of these analytes was evaluated in the laboratory using a flow-through tank. Uptake was linear over a 14-day period being in the range: MBT (3-23 mL day(-1)), DBT (40-200 mL day(-1)), TBT (30-200 mL day(-1)) and TPhT (30-190 mL day(-1)) for all the different conditions tested. These sampling rates were high enough to permit the use of the Chemcatcher to monitor levels of organotin compounds typically found in polluted aquatic environments. Using gas chromatography (GC) with either ICP-MS or flame photometric detection, limits of detection for the device (14-day deployment) for the different organotin compounds in water were in the range of 0.2-7.5 ng L(-1), and once accumulated in the receiving phase the compounds were stable over prolonged periods. Due to anisotropic exchange kinetics, performance reference compounds could not be used with this passive sampling system to compensate for changes in sampling rate due to variations in water temperature, turbulence and biofouling of the surface of the diffusion membrane during field deployments. The performance of the Chemcatcher was evaluated alongside spot water sampling in Alicante Habour, Spain which is known to contain elevated levels of organotin compounds. The samplers provided time-weighted average concentrations of the bioavailable fractions of the tin compounds where environmental concentrations fluctuated markedly in time.

Speciation analysis of platinum antitumoral drugs in impacted tissues

Chemical compounds containing platinum have been employed since 1978 as drugs to beat certain type of tumours. Nevertheless, besides of their exceptional antitumoral properties, these drugs also have important deleterious side effects, such as, nephrotoxicity and ototoxicity. A study of Pt accumulation and a speciation analysis has been performed by ICP-MS in samples from kidney and inner ear in a controlled population of Wistar rats treated with, either, cisplatin, carboplatin or oxaliplatin. The results on Pt accumulation point out to drug structure and not only to Pt content as the responsible for the alteration of organ functionality. Speciation studies in the samples from kidney and inner ear were performed coupling two-dimensional liquid chromatography (2D-LC) to ICP-MS. Size exclusion (SEC) and anion exchange fast protein liquid chromatography (FPLC) was employed for 2D orthogonal separation. After these separations, free drug peaks were not observed in any of the samples. The binding of Pt to biomolecules was demonstrated by SEC and, independently of the drug used, Pt eluted as two main bands with molecular weights of 12kDa and 25-65kDa for inner ear samples, and as two different bands with 20kDa and 50-60kDa in the samples from kidney. However, the relative band intensity presented important differences for the three drugs. Using the same chromatographic conditions, it was shown that a metallothionein (MT) standard eluted in the same position as some of the cytosolic Pt-biomolecules. High Pt-containing fractions eluting from the SEC column were analysed by anion exchange FPLC after a preconcentration step. Among the different preconcentration methods tested, sample focusing on the head of the FPLC column shows main advantages. In this way, the separation by 2D chromatography of the high molecular Pt-species has been considerably improved.

SEC-ICP-MS and ESI-MS as tools to study the interaction between cisplatin and cytosolic biomolecules

Most of cisplatin’s citotoxic properties are due to the interaction of the drug with DNA. However, other biological molecules present in the cell cytosol, such as MT (metallothionein) and GSH (glutathione), are potential targets for cisplatin and have been related to its side-effects or with the cellular resistance mechanisms to the drug. Experiments simulating physiological conditions have been performed to study the specific cisplatin metabolites which interact with GSH and MT and to characterize the different drug–biomolecule adducts over time. A combination of size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and electrospray ionization-mass spectrometry (ESI-MS) techniques has been used to provide sensible multi-elemental detection and structural information of the species of interest. Time dependent transformation of 10 μM cisplatin at neutral pH (7.4) produces different concentrations of the mono-aquo and oligomeric derivatives, as could be confirmed by ESI-MS. No di-aquo derivative was seen to be produced under these conditions at any of the incubation times used. Cisplatin and the oligomeric derivative were incubated with GSH and MT at different drug:biomolecule ratios. Adducts from cisplatin–GSH (1:500) and from cisplatin–MT (1:10) incubations were characterized by SEC-ICP-MS. While both GSH and MT reacted with cisplatin producing different compounds, only GSH reacted with the oligomeric derivative of cisplatin. SEC-ICP-MS experiments showed that, under neutral pH conditions, Cd atoms remained bound to the cisplatin:MT adducts, but Zn atoms were lost. Results were compared with those obtained by in vitro and in vivo experiments with rat kidney, liver and inner ear cytosols.

Characterization of Pt-protein complexes by nHPLC–ESI-LTQ MS/MS using a gel-based bottom-up approach

The suitability of in-gel digestion for the characterization of Pt-binding proteins by gel-based bottom-up MS approaches has been evaluated regarding the preservation of Pt-protein bonds during the process. Standard proteins (albumin, transferrin, carbonic anhydrase, myoglobin and cytochome c) incubated with cisplatin were separated by nrSDS-PAGE and in-gel trypsin-digested. The whole in-gel digestion protocol included treatment with reagents such as: ammonium bicarbonate, acetonitrile, formic acid, trypsin as enzyme and alternatively, dithiotreitol and iodoacetamide as reducing and alkylating agents. Digests were analyzed by nHPLC-ESI-LTQ-MS/MS and Pt-peptides were recognized in all the proteins studied on the basis of their isotopic pattern. Only when the reducing and alkylating reagents were used, the amount of detectable Pt-peptides decreased due to the high reactivity of thiol containing reagents towards Pt. Furthermore, the repeated use of acetonitrile could lead to the replacement of ligands originally attached to Pt by CN(-), but does not affect the Pt-protein binding. Platinum-binding sites on the proteins were elucidated from the CID-MS/MS fragmentation spectra and assessed by evaluation of protein structures. Several histidines, cysteines and methionines were identified as platinum binding sites in the different standard proteins. Results were in accordance to those obtained with in-solution digestions.

LA-ICP-MS and nHPLC-ESI-LTQ-FT-MS/MS for the analysis of cisplatin–protein complexes separated by two dimensional gel electrophoresis in biological samples

A method for the analysis of Pt–protein complexes in biological samples, previously subjected to cisplatin treatment, has been developed. Proteins were separated by gel electrophoresis, and those bound to Pt were detected with high sensitivity by LA-ICP-(SF)-MS. Pt-containing spots were in-gel digested with trypsin, and the peptides produced identified using nHPLC-ESI-LTQ-FT-MS/MS. The influence of protein separation conditions, staining and gel processing prior to laser ablation on Pt–protein bonds preservation have been evaluated using standard proteins incubated with cisplatin. 2-DE separation under non-reducing conditions followed by either Coomassie blue brilliant or silver staining is appropriate for Pt–protein complexes, achieving a good separating resolution of the proteins in biological samples. Direct LA-ICP-MS analysis of glycerol-treated dried gels for Pt–protein monitoring resulted in better sensitivity, more reliable relative Pt signals and a simpler and less time-consuming approach compared to the analysis of blotted membranes. Ablation of gels allowed tackling protein identification of Pt-spots in the remaining non-ablated material in the gel, making it unnecessary to run several gels in parallel for separate Pt detection and protein identification. By using this approach, Pt coordinated to proteins, such as α-2-macroglobulin, transferrin, albumin or hemoglobin, was detected in the serum from a rat treated in vivo with cisplatin after nrSDS-PAGE separation. Furthermore, the first complete LA-ICP-MS metalloprotein contour map in a 2-DE gel has been produced, in this case for the detection of Pt–protein complexes in renal proximal tubule epithelial cells (RPTECs) incubated with cisplatin. Several proteins were identified in those spots containing Pt, which may have a connection with the drug-induced nephrotoxicity mainly affecting this cell type in the kidney.

Application of Chemcatcher passive sampler for monitoring levels of mercury in contaminated river water

A passive sampler (Chemcatcher) consisting of a 47 mm Emporetrade mark chelating disk (CHE) with iminodiacetic groups as the receiving phase overlaid with a diffusion membrane was developed and calibrated for the monitoring of Hg in water. Three different diffusion membranes including cellulose acetate (CA), polyethersulphone (PS) and cellulose dialysis membrane (D) were tested. The best performance was obtained with the CHE-PS tandem. The effective sampling rate of the device (R(s), L day(-1)) is defined as the equivalent volume of water extracted per unit time, and is analyte specific and can be determined experimentally in a flow-through tank. Effects of water temperature and turbulence on the uptake rate of Hg were assessed under controlled laboratory conditions. Sampling rates were in the range of 0.029-0.091 L day(-1). An increase in sampling rate with turbulence was demonstrated. The detection limit of the sampler obtained in flowing waters ranged between 2.2 and 2.9 ng L(-1)Hg. The performance of Chemcatcher was tested alongside spot water sampling in a 14-day field deployment at two locations on the Valdeazogues River, Almadén, Spain. In general, the Hg concentration estimated by the Chemcatcher was lower than that found in spot water samples collected over the same period. This may be explained by the behaviour of this sampler that measures only the labile fraction of Hg in water, and this will exclude some species. However, Chemcatcher preconcentrates Hg allowing its determination in some places where its concentration is below the detection limit of spot sampling.