María Milagros Gómez-Gómez

The use of cisplatin chemotherapy is often limited by the occurrence of various side effects, with renal toxicity being one of the most serious. In the present work, a single cell ICP-MS (scICP-MS) methodology was optimised to evaluate the cellular uptake of cisplatin in the presence of three potential nephroprotectors such as chitosan-stabilised selenium nanoparticles (Ch-SeNPs), selenomethionine (SeMet) and methionine (Met). Human telomerase reverse transcriptase-immortalised renal proximal tubular epithelial cells (RPTEC/TERT1) and human cervical cancer cells (HeLa) were employed with this aim. In both cell lines, a decrease in the intracellular Pt levels when using SeMet and Met as coadjuvants was revealed, involving less toxicity in renal cells but no reduction in the anticancer effect after measurement of cell viability by MTT assays. In contrast, Ch-SeNPs had no effect on the internalisation of the Pt-drug but enhanced its antitumour efficacy with no additional damage to kidney cells. This would allow decreasing cisplatin doses which would in turn reduce nephrotoxicity risk. Se determination by scICP-MS was also done to study the cell uptake of the selenocompounds, in addition to transmission electron microscopy (TEM) analysis of Ch-SeNPs internalisation. The effects of both SeMet and Ch-SeNPs were confirmed despite the Pt-drug was shown to induce a decrease in cell uptake. Results were compared by two different scICP-MS settings (a conventional introduction system and a special configuration for intact cells), as well as with the classical digestion-based bulk analysis. Our results demonstrate the potential of scICP-MS for metallomic cellular studies to improve cisplatin-based therapies. Graphical Abstract

The use of cisplatin chemotherapy is often limited by the occurrence of various side effects, with renal toxicity being one of the most serious. In the present work, a single cell ICP-MS (scICP-MS) methodology was optimised to evaluate the cellular uptake of cisplatin in the presence of three potential nephroprotectors such as chitosan-stabilised selenium nanoparticles (Ch-SeNPs), selenomethionine (SeMet) and methionine (Met). Human telomerase reverse transcriptase-immortalised renal proximal tubular epithelial cells (RPTEC/TERT1) and human cervical cancer cells (HeLa) were employed with this aim. In both cell lines, a decrease in the intracellular Pt levels when using SeMet and Met as coadjuvants was revealed, involving less toxicity in renal cells but no reduction in the anticancer effect after measurement of cell viability by MTT assays. In contrast, Ch-SeNPs had no effect on the internalisation of the Pt-drug but enhanced its antitumour efficacy with no additional damage to kidney cells. This would allow decreasing cisplatin doses which would in turn reduce nephrotoxicity risk. Se determination by scICP-MS was also done to study the cell uptake of the selenocompounds, in addition to transmission electron microscopy (TEM) analysis of Ch-SeNPs internalisation. The effects of both SeMet and Ch-SeNPs were confirmed despite the Pt-drug was shown to induce a decrease in cell uptake. Results were compared by two different scICP-MS settings (a conventional introduction system and a special configuration for intact cells), as well as with the classical digestion-based bulk analysis. Our results demonstrate the potential of scICP-MS for metallomic cellular studies to improve cisplatin-based therapies. Graphical Abstract

Nephrotoxicity is a major complication of cisplatin-based chemotherapy, leading to acute kidney injury in ca. 30% of patients, with no preventive intervention or treatment available for clinical use. Cilastatin has proved to exert a nephroprotective effect for cisplatin therapies in in vitro and in vivo models, having recently entered clinical trials. A deeper understanding at the molecular level of cisplatin-induced renal damage and the effect of potential protective agents could be key to develop successful nephroprotective therapies and to establish new biomarkers of renal damage and nephroprotection. A targeted lipidomics approach, using LC-MS/MS, was employed for the quantification of 108 lipid species (comprising phospholipids, sphingolipids, and free and esterified cholesterol) in kidney cortex and medulla extracts from rats treated with cisplatin and/or cilastatin. Up to 56 and 63 lipid species were found to be altered in the cortex and medulla, respectively, after cisplatin treatment. Co-treatment with cilastatin attenuated many of these lipid changes, either totally or partially with respect to control levels. Multivariate analysis revealed that lipid species can be used to discriminate renal damage and nephroprotection, with cholesterol esters being the most discriminating species, along with sulfatides and phospholipids. Potential diagnostic biomarkers of cisplatin-induced renal damage and cilastatin nephroprotection were also found.

The quest for internal standards useful in MALDI imaging studies goes on to get not only lateral distribution but also reliable relative quantitative information. We developed a method based on application of matrix and dual internal standards to allow intra- and intersample normalization of lipids intensities in kidney sections of control and cisplatin-treated Wistar rats. An inkjet printer was used to deposit a custom-prepared ink with DHB as MALDI matrix, a primary lipids-based internal standard, and a spiked lanthanide as a secondary internal standard. We applied different laser energy and varied the amounts of matrix-internal standards mixture to evaluate the normalization potential of the internal standards. Successful correction of intensity artifacts caused by instrumental drifts was possible, but not those resulting from uneven matrix application. ICP-MS absolute quantification of the lanthanide in the printed layer ensured the reproducibility of the matrix and internal standards application with RSD of 10–15%. Internal standard-normalized data allowed intrasample modification of the MALDI image to make it compatible with the optical image. Normalization to internal standards corrected a 2-fold difference in lipids intensity, which allowed a meaningful comparison of tissue lipids in control and cisplatin-treated kidneys. More importantly, normalization of lipid relative abundances based on the same adduct type (H⁺, Na⁺, and K⁺) for analyte and internal standard corrected for different ionization efficiencies showing a realistic signal level and enabling reliable comparison of different samples on relative quantitative basis.