Untargeted metabolomics analysis of serum and urine unveils the protective effect of cilastatin on altered metabolic pathways during cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) is one of the most serious complications of cisplatin anticancer therapies. Cilastatin is a highly promising nephroprotective agent to eventually enter clinical use, but its biochemical mechanism is still not fully understood. We have employed an untargeted metabolomics approach based on capillary electrophoresis mass spectrometry (CE-MS) analysis of serum and urine from an in vivo rat model, to explore the metabolic pathways involved in cisplatin-induced AKI and cilastatin nephroprotection. A total of 155 and 76 identified metabolites were found to be significantly altered during cisplatin treatment in urine and serum, respectively. Most of these altered metabolites were either partially or totally recovered by cilastatin and cisplatin co-treatment. The main metabolic pathways disturbed by cisplatin during AKI involved diverse amino acids metabolism and biosynthesis, tricarboxylic acids (TCA) cycle, nicotinate and nicotinamide metabolism, among others. Cilastatin was proved to protect diverse cisplatin-altered pathways involving metabolites related to immunomodulation, inflammation, oxidative stress and amino acid metabolism in proximal tubules. However, cisplatin-altered mitochondrial metabolism (especially, the energy-producing TCA cycle) remained largely unprotected by cilastatin, suggesting an unresolved mitochondrial direct damage. Multivariate analysis allowed effective discrimination of cisplatin-induced AKI and cilastatin renoprotection based on metabolic features. A number of potential serum and urine biomarkers could also be foreseen for cisplatin-induced AKI detection and cilastatin nephroprotection.

Human intake assessment of triclosan associated with the daily use of polypropylene-made antimicrobial food packaging.

In this work, we aimed to evaluate human intake of triclosan (TCS) associated with real-life use of different brands of Microban™ microwave-safe food packaging. Calculations were based on: TCS migration data (under the worst-case foreseeable conditions), MPs abundance and TCS bioaccessibility from microplastics (MPs), leached from containers under microwave heating. Bioaccessibility studies were performed with in vitro digestion of MPs, followed by liquid-liquid extraction of TCS from digestive fluids and LC-QqQ-MS analysis yielding values of 46 ± 9%. The estimated weekly intake (EWI) of TCS ranged between 11 and 42 µg/kg body weight/week, with migration being the largest contribution (0.6-2.3 mg/week), compared to leaching of MPs (75-300 µg/week). These values represent a significant source of human exposure to TCS, emphasizing the need to harmonize the ban of TCS in food contact materials worldwide and improve compliance testing of food contact articles, particularly those marketed through online sales platforms.

Lipid imaging for visualizing cilastatin amelioration of cisplatin-induced nephrotoxicity.

Nephrotoxicity is a major limitation to cisplatin antitumor therapies. Cilastatin, an inhibitor of renal dehydropeptidase-I, was recently proposed as a promising nephroprotector against cisplatin toxicity, preventing apoptotic cell death. In this work, cilastatin nephroprotection was further investigated in a rat model, with a focus on its effect on 76 renal lipids altered by cisplatin, including 13 new cisplatin-altered mitochondrial cardiolipin species. Lipid imaging was performed with MALDI mass spectrometry imaging (MALDI-MSI) in kidney sections from treated rats. Cilastatin was proved to significantly diminish the lipid distribution alterations caused by cisplatin, lipid levels being almost completely recovered to those of control samples. The extent of recovery of cisplatin-altered lipids by cilastatin turned out to be relevant for discriminating direct or secondary lipid alterations driven by cisplatin. Lipid peroxidation induced by cisplatin was also shown to be reduced when cilastatin was administered. Importantly, significant groups separation was achieved during multivariate analysis of cortex and outer-medullary lipids, indicating that damaged kidney can be discerned from the nephroprotected and healthy groups and classified according to lipid distribution. Therefore, we propose MALDI-MSI as a powerful potential tool offering multimolecule detection possibilities to visualize and evaluate nephrotoxicity and nephroprotection based on lipid analysis.

MALDI-LTQ-Orbitrap mass spectrometry imaging for lipidomic analysis in kidney under cisplatin chemotherapy.

Imaging techniques for mapping molecular distributions in tissue sections can reveal valuable information on biomolecules involved in relevant biochemical processes. A method has been developed for comprehensive, reproducible and sensitive lipid imaging by matrix-assisted laser/desorption ionization-LTQ-Orbitrap mass spectrometry in kidney sections, showing the benefits of exact mass determination. Matrix deposition parameters for positive and negative lipid ion imaging using different matrices such as 2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9-AA) or α-cyano-4-hydroxycinnamic acid (CHCA) have been optimized for the broadest detection and identification of renal lipids. The combination of 9-AA and DHB was found as the most suitable for negative and positive ion mode lipid imaging, respectively. Lipid mapping and related identification strategies and limitations have also been discussed. Production of 100-µm resolution images was proved to be enough for discerning lipid distribution in kidney substructures. Imaging reproducibility was assessed on parallel kidney slices with time. This method has been applied to the lipidomics analysis on kidney sections from rats treated with the antitumor drug cisplatin and compared to healthy control rats. Up to 66 different renal lipids out of 450 extracted ion images (mainly phospholipid species, in addition to sulfatides and cholesterol sulfate) have been found and identified showing a modified distribution pattern due to cisplatin-induced nephrotoxicity. These lipid species reflect either topographic, signaling or structural processes in damaged kidney and could potentially be used for nephrotoxicity assessment or as therapeutic targets. This is, to our knowledge, the first imaging lipidomics study for nephrotoxicity assessment of cisplatin chemotherapy.

A shotgun approach for the identification of platinum-protein complexes.

A shotgun approach including peptide-based OFFGEL-isoelectric focusing (IEF) fractionation has been developed with the aim of improving the identification of platinum-binding proteins in biological samples. The method is based on a filter-aided sample preparation (FASP) tryptic digestion under denaturing and reducing conditions of cisplatin-, oxaliplatin-, and carboplatin-protein complexes, followed by OFFGEL-IEF separation of the peptides. Any risk of platinum loss is minimized throughout the procedure due to the removal of the reagents used after each stage of the FASP method and the absence of thiol-based reagents in the focusing buffer employed in the IEF separation. The platinum-peptide complexes stability after the FASP digestion and the IEF separation was confirmed by size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The suitability of peptide-based OFFGEL-IEF fractionation for reducing the sample complexity for further nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) analysis has been demonstrated, allowing the detection of platinum-containing peptides, with significantly lower abundance and ionization efficiency than unmodified peptides. nLC-MS/MS analysis of selected OFFGEL-IEF fractions from tryptic digests with different complexity degrees: standard human serum albumin (HSA), a mixture of five proteins (albumin, transferrin, carbonic anhydrase, myoglobin, and cytochrome-c) and human blood serum allowed the identification of several platinum-peptides from cisplatin-HSA. Cisplatin-binding sites in HSA were elucidated from the MS/MS spectra and assessed considering the protein three-dimensional structure. Most of the potential superficial binding sites available on HSA were identified for all the samples, including a biologically relevant cisplatin-cross-link of two protein domains, demonstrating the capabilities of the methodology.

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.

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.

Biospeciation of tungsten in the serum of diabetic and healthy rats treated with the antidiabetic agent sodium tungstate.

It is known that oral administration of sodium tungstate preserves the pancreatic beta cell function in diabetic rats. Healthy and streptozotocin-induced diabetic rats were treated with sodium tungstate for one, three or six weeks, after which the species of W in serum, were analysed. An increase in serum W with treatment time was observed. After six weeks, the serum W concentration in diabetic rats (70 mg L(-1)) was about 4.6 times higher than in healthy specimens. This different behaviour was also observed for Cu accumulation, while the Zn pattern follows the contrary. The patterns observed in the retention of Cu and Zn may be attributable to a normalization of glycaemia. The speciation analysis of W was performed using 2D separations, including an immunoaffinity packing and a SEC (Size Exclusion Chromatography) column coupled to an ICP-MS (Inductively Coupled Plasma Mass Spectrometry) for elemental detection. Ultrafiltration data together with SEC-ICP-MS results proved that around 80% of serum W was bound to proteins, the diabetic rats registering a higher W content than their healthy counterparts. Most of the protein-bound W was due to a complex with albumin. An unknown protein with a molecular weight higher than 100 kDa was also found to bind a small amount of W (about 2%). MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) analysis of the desalted and concentrated chromatographic fractions confirmed albumin as the main protein bound to tungstate in rat serum, while no binding to transferrin (Tf) was detected. The interaction between glutathione and W was also evaluated using standard solutions; however, the formation of complexes was not observed. The stability of the complexes between W and proteins when subjected to more stringent procedures, like those used in proteomic methodologies (denaturing with urea or SDS, boiling, sonication, acid media, reduction with ß-mercaptoethanol (BME) or DTT (dithiotreitol) and alkylation with iodoacetamide (IAA), was also evaluated. Our results indicate that the stability of the complexes between W and proteins is not too high enough to remain unaltered during protein separation by SDS-PAGE in denaturing and reducing conditions. However, the procedures for in-solution tryptic digestion and for ESI-MS analysis in MeOH/H(2)O/with 0.1% formic acid could be used for protein identification without large loss of binding between W and proteins.

Novel insights into the bottom-up mass spectrometry proteomics approach for the characterization of Pt-binding proteins: The insulin-cisplatin case study.

The characterization of the interaction of platinum drugs with proteins has been previously performed using bottom-up proteomics approaches (enzymatic digestion followed by MS analysis). Nevertheless, the study of the stability of the Pt-protein bonds along the whole process has been obviated for the moment. Herein the suitability of the treatments implied during enzymatic digestion of Pt-protein adducts has been evaluated, focusing on the stability of the Pt bonds. Insulin-cisplatin adducts were generated in vitro and separated from unreacted cisplatin by HPLC, the separation being checked by HPLC-ICP-MS. The chromatographically isolated Pt-insulin adducts have been proved to resist overnight digestion including treatment with Urea, DTT, IAA and trypsin in a Tris buffer. Direct analysis of the peptides generated by nESI-LIT MS allowed the determination of Pt-binding sites in insulin as: B Chain N-terminus, His5, His10, Cys7, Cys19 and A Chain Cys6, Cys7, Cys20. Results have been compared to a previous top-down approach, indicating that more complete information can be obtained with the bottom-up approach. Reactivity of free cysteines has been proved to prevail to N-donor groups, but when cysteines participate in disulfide bonds, their reactivity is comparable to N-donor sites (N-terminus, His). Preliminary results indicate that the use of High Intensity Focused Ultrasound for accelerating the enzymatic digestions is compatible with preserving Pt-protein bonds, allowing a reduction in the total digestion time to 5 min. Pt-containing peptides were fragmented and sequenced by CID, and results were compared with those obtained by the use of ETD, being CID spectra far more informative.

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.