RESUMEN
Cisplatin (CDDP) stands out as an effective chemotherapeutic agent; however, its application is linked to the development of significant adverse effects, notably nephro- and ototoxicity. The human organic cation transporter 2 (hOCT2), found in abundance in the basolateral membrane domain of renal proximal tubules and the Corti organ, plays a crucial role in the initiation of nephro- and ototoxicity associated with CDDP by facilitating its uptake in kidney and ear cells. Given its limited presence in cancer cells, hOCT2 emerges as a potential druggable target for mitigating unwanted toxicities associated with CDDP. Potential strategies for mitigating CDDP toxicities include competing with the uptake of CDDP by hOCT2 or inhibiting hOCT2 activity through rapid regulation mediated by specific signaling pathways. This study investigated the interaction between the already approved cationic drugs disopyramide, imipramine, and orphenadrine with hOCT2 that is stably expressed in human embryonic kidney cells. Regarding disopyramide, its influence on CDDP cellular transport by hOCT2 was further characterized through inductively coupled plasma isotope dilution mass spectrometry. Additionally, its potential protective effects against cellular toxicity induced by CDDP were assessed using a cytotoxicity test. Given that hOCT2 is typically expressed in the basolateral membrane of polarized cells, with specific regulatory mechanisms, this work studied the regulation of hOCT2 that is stably expressed in Madin-Darby Canine Kidney (MDCK) cells. These cells were cultured in a matrix to induce the formation of cysts, exposing hOCT2 in the basolateral plasma membrane domain, which was freely accessible to experimental solutions. The study specifically tested the regulation of ASP+ uptake by hOCT2 in MDCK cysts through the inhibition of casein kinase II (CKII), calmodulin, or p56lck tyrosine kinase. Furthermore, the impact of this manipulation on the cellular toxicity induced by CDDP was examined using a cytotoxicity test. All three drugs-disopyramide, imipramine, and orphenadrine-demonstrated inhibition of ASP+ uptake, with IC50 values in the micromolar (µM) range. Notably, disopyramide produced a significant reduction in the CDDP cellular toxicity and platinum cellular accumulation when co-incubated with CDDP. The activity of hOCT2 in MDCK cysts experienced a significant down-regulation under inhibition of CKII, calmodulin, or p56lck tyrosine kinase. Interestingly, only the inhibition of p56lck tyrosine kinase demonstrated the capability to protect the cells against CDDP toxicity. In conclusion, certain interventions targeting hOCT2 have demonstrated the ability to reduce CDDP cytotoxicity, at least in vitro. Further investigations in in vivo systems are warranted to ascertain their potential applicability as co-treatments for mitigating undesired toxicities associated with CDDP in patients.
Asunto(s)
Quistes , Ototoxicidad , Humanos , Animales , Perros , Transportador 2 de Cátion Orgánico , Proteínas de Transporte de Catión Orgánico/metabolismo , Cisplatino/metabolismo , Disopiramida , Calmodulina/metabolismo , Imipramina , Orfenadrina , Células de Riñón Canino Madin Darby , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Cancer treatment with platinum compounds is an important achievement of modern chemotherapy. However, despite the beneficial effects, the clinical impact of these agents is hampered by the development of drug resistance as well as dose-limiting side effects. The efficacy but also side effects of platinum complexes can be mediated by uptake through plasma membrane transporters. In the kidneys, plasma membrane transporters are involved in their secretion into the urine. Renal secretion is accomplished by uptake from the blood into the proximal tubules cells, followed by excretion into the urine. The uptake process is mediated mainly by organic cation transporters (OCT), which are expressed in the basolateral domain of the plasma membrane facing the blood. The excretion of platinum into the urine is mediated by exchange with protons via multidrug and toxin extrusion proteins (MATE) expressed in the apical domain of plasma membrane. Recently, the monofunctional, cationic platinum agent phenanthriplatin, which is able to escape common cellular resistance mechanisms, has been synthesized and investigated. In the present study, the interaction of phenanthriplatin with transporters for organic cations has been evaluated. Phenanthriplatin is a high affinity substrate for OCT2, but has a lower apparent affinity for MATEs. The presence of these transporters increased cytotoxicity of phenanthriplatin. Therefore, phenanthriplatin may be especially effective in the treatment of cancers that express OCTs, such as colon cancer cells. However, the interaction of phenanthriplatin with OCTs suggests that its use as chemotherapeutic agent may be complicated by OCT-mediated toxicity. Unlike cisplatin, phenanthriplatin interacts with high specificity with hMATE1 and hMATE2K in addition to hOCT2. This interaction may facilitate its efflux from the cells and thereby decrease overall efficacy and/or toxicity.
RESUMEN
In recent decades, a significant amount of anthropogenic gadolinium has been released into the environment as a result of the broad application of contrast agents for magnetic resonance imaging (MRI). Since this anthropogenic gadolinium anomaly has also been detected in drinking water, it has become necessary to investigate the possible effect of drinking water purification on these highly polar microcontaminats. Therefore, a novel highly sensitive method for speciation analysis of gadolinium is presented. For that purpose, the hyphenation of hydrophilic interaction liquid chromatography (HILIC) and inductively coupled plasma-mass spectrometry (ICP-MS) was employed. In order to enhance the detection power, sample introduction was carried out by ultrasonic nebulization. In combination with a novel HILIC method using a diol-based stationary phase, it was possible to achieve superior limits of detection for frequently applied gadolinium-based contrast agents below 20pmol/L. With this method, the contrast agents Gd-DTPA, Gd-DOTA and Gd-BT-DO3A were determined in concentrations up to 159pmol/L in samples from several waterworks in a densely populated region of Germany alongside the river Ruhr as well as from a waterworks near a catchment lake. Thereby, the direct impact of anthropogenic gadolinium species being present in the surface water on the amount of anthropogenic gadolinium in drinking water was shown. There was no evidence for the degradation of contrast agents, the release of Gd(3+) or the presence of further Gd species.
Asunto(s)
Técnicas de Química Analítica/métodos , Cromatografía Liquida , Medios de Contraste/análisis , Medios de Contraste/química , Agua Potable/química , Ríos/química , Contaminantes Químicos del Agua/análisis , Monitoreo del Ambiente , Gadolinio DTPA/análisis , Gadolinio DTPA/química , Alemania , Compuestos Heterocíclicos/análisis , Compuestos Heterocíclicos/química , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas , Compuestos Organometálicos/análisis , Compuestos Organometálicos/química , Contaminantes Químicos del Agua/químicaRESUMEN
The interaction of mercury species with human erythrocytes is studied to investigate possible high molecular binding partners for mercury species. Human blood hemolysate was spiked with methylmercury and investigated by means of liquid chromatography (LC) coupled to electrospray ionization time of flight mass spectrometry (ESI-ToF-MS) and inductively coupled plasma mass spectrometry (ICP-MS). Beside adduct formation of mercury species with hemoglobin, the main compound of the erythrocytes, mercury binding to the enzyme carbonic anhydrase was revealed. Due to an enzymatic digest of the protein-mercury adduct, the binding site at the free thiol group of the protein was identified. These results indicate that carbonic anhydrase might play a role in mercury toxicity.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Cromatografía Liquida/métodos , Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Hemólisis , Compuestos de Metilmercurio/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Anhidrasas Carbónicas/química , Humanos , Masculino , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismoRESUMEN
Thyroxine (T4) is one of the major thyroid hormones, which regulates cellular metabolism, central nervous system development, body temperature, reproduction and growth. The simulation of oxidation reactions of T4 may provide further information about the fate of T4 in cells without using laborious in vitro and susceptible in vivo tests. In this study, oxidation products of T4, generated inside an electrochemical (EC) cell, were separated and identified by on-line EC/liquid chromatography/electrospray ionization-mass spectrometry (EC/LC/ESI-MS). In another experimental setup, the electrogenerated metabolites were separated by LC, subsequently mixed with a compensating countergradient (cg), and finally introduced into an inductively coupled plasma-mass spectrometer (ICP-MS). The gradient compensation was achieved by an additional pump module which generated a reversed gradient to the analytical gradient used for the separation. This setup enabled a constant composition of the LC eluent flowing into the plasma so that stable plasma conditions and a uniform response over the complete elution time could be achieved. Combined with identification information from online-coupled EC/LC/ESI-MS, robust and reliable quantification of T4 and its oxidation products was accomplished by on-line coupled EC/cgLC/ICP-MS, with LOD of 33nM of iodine.
Asunto(s)
Tiroxina/análisis , Cromatografía Liquida/métodos , Oxidación-Reducción , Espectrometría de Masa por Ionización de Electrospray/métodos , Tiroxina/metabolismoRESUMEN
One of the most common setups for elemental bioimaging, the hyphenation of a laser ablation (LA) system and an inductively coupled plasma mass spectrometer (ICP-MS), was expanded by adding full scan mass spectrometric information as another dimension of information. While most studies deal with the analysis of typically not more than up to 10 isotopes per scan cycle, a fast scanning quadrupole mass analyzer was utilized to record the full mass spectrum of interest in this work. Mass-to-charge ratios from 6 to 250 were observed within one cycle. Besides the x- and y-position on the ablated sample and the intensity, the m/z-ratio served as fourth variable for each pixel of the obtained data, closing thereby the gap between "inorganic" and "organic" mass spectrometric imaging techniques. The benefits of this approach include an improved control of interferences, the discovery of unexpected elemental distributions, the possibility to plot isotopic ratios, and to integrate the intensities of a certain number of mass channels recorded for each isotope, thus virtually increasing sensitivity. The respective data are presented for dried droplets as well as embedded animal and human tissue slices. Limits of detection were calculated and found to be in accordance with counting statistics. A dedicated software macro was developed for data manipulation prior to common evaluation and image creation.
Asunto(s)
Espectrometría de Masas/métodos , Imagen Molecular/métodos , Animales , Análisis Químico de la Sangre , Café/química , Humanos , Riñón/química , Ratones , Modelos BiológicosRESUMEN
The combined use of elemental bioimaging and speciation analysis is presented as a novel means for the diagnosis of nephrogenic systemic fibrosis (NSF), a rare disease occurring after administration of gadolinium-based contrast agents (GBCA) for magnetic resonance imaging (MRI), in skin samples of patients suffering from renal insufficiency. As the pathogenesis of NSF is still largely unknown particularly with regard to the distribution and potential retention of gadolinium in the human organism, a skin biopsy sample from a suspected NSF patient was investigated. The combination of inductively coupled plasma mass spectrometry (ICP-MS), laser ablation (LA) ICP-MS for quantitative elemental bioimaging, and hydrophilic interaction liquid chromatography (HILIC) ICP-MS for speciation analysis allowed one to unambiguously diagnose the patient as a case of NSF. By means of ICP-MS, a total gadolinium concentration from 3.02 to 4.58 mg/kg was determined in the biopsy sample, indicating a considerable deposition of gadolinium in the patient's skin. LA-ICP-MS revealed a distinctly inhomogeneous distribution of gadolinium as well as concentrations of up to 400 mg/kg in individual sections of the skin biopsy. Furthermore, the correlation between the distributions of phosphorus and gadolinium suggests the presence of GdPO4 deposits in the tissue section. Speciation analysis by means of HILIC-ICP-MS showed the presence of the intact GBCA Gd-HP-DO3A eight years after the administration to the patient. The concentration of the contrast agent in the aqueous extract of the skin biopsy was found to be 1.76 nmol/L. Moreover, evidence for the presence of further highly polar gadolinium species in low concentrations was found.
Asunto(s)
Espectrometría de Masas , Imagen Molecular , Dermopatía Fibrosante Nefrogénica/diagnóstico , Adulto , Calcio/metabolismo , Medios de Contraste/efectos adversos , Medios de Contraste/análisis , Medios de Contraste/química , Femenino , Gadolinio DTPA/efectos adversos , Gadolinio DTPA/química , Humanos , Dermopatía Fibrosante Nefrogénica/inducido químicamente , Dermopatía Fibrosante Nefrogénica/metabolismo , Dermopatía Fibrosante Nefrogénica/patología , Fósforo/metabolismo , Piel/patología , Solubilidad , Agua/análisisRESUMEN
RATIONALE: Two different approaches to improve the limits of detection (LODs) in elemental bioimaging have been developed. They both consider the fact that for the widely applied quadrupole-based instruments, metals in the mass range <100 u are analyzed with the best figures of merit in the kinetic energy discrimination (KED) mode; much better LODs are achieved for some metalloids and nonmetals by the introduction of more reactive gases, e.g., oxygen, into the collision/reaction cell (CRC). METHODS: While the first approach simultaneously utilizes two inductively coupled plasma mass spectrometry (ICP-MS) detectors hyphenated to one laser ablation (LA) system, the second is based on a single ICP-MS instrument with fast cell mode switching (CMS) of the CRC between individual line scans. RESULTS: Both methods were evaluated concerning their respective improvements by the analysis of rat brain samples. The utilization of two detectors showed improved LODs compared with conventional KED-only analysis in dependency on the gas flow splitting ratio, e.g., for sulfur by about 3.5 orders of magnitude. CMS provided even better results with a further improvement by a factor of 1.6. CONCLUSIONS: As a CRC with a small inner volume was used, fast cell gas switches at the end of every line prevented issues related to the reproducibility of the laser ablation stage for the CMS approach. Linear interpolation was found to be a valuable tool without affecting the spatial resolution of the images. In addition, a software macro is presented, which facilitates data evaluation.
Asunto(s)
Espectrometría de Masas/métodos , Imagen Molecular/métodos , Animales , Química Encefálica , Femenino , Histocitoquímica , Límite de Detección , Ratas , Ratas WistarRESUMEN
The toxicologically most relevant mercury (Hg) species for human exposure is methylmercury (MeHg). Thiomersal is a common preservative used in some vaccine formulations. The aim of this study is to get further mechanistic insight into the yet not fully understood neurotoxic modes of action of organic Hg species. Mercury species investigated include MeHgCl and thiomersal. Additionally HgCl2 was studied, since in the brain mercuric Hg can be formed by dealkylation of the organic species. As a cellular system astrocytes were used. In vivo astrocytes provide the environment necessary for neuronal function. In the present study, cytotoxic effects of the respective mercuricals increased with rising alkylation level and correlated with their cellular bioavailability. Further experiments revealed for all species at subcytotoxic concentrations no induction of DNA strand breaks, whereas all species massively increased H2O2-induced DNA strand breaks. This co-genotoxic effect is likely due to a disturbance of the cellular DNA damage response. Thus, at nanomolar, sub-cytotoxic concentrations, all three mercury species strongly disturbed poly(ADP-ribosyl)ation, a signalling reaction induced by DNA strand breaks. Interestingly, the molecular mechanism behind this inhibition seems to be different for the species. Since chronic PARP-1 inhibition is also discussed to sacrifice neurogenesis and learning abilities, further experiments on neurons and in vivo studies could be helpful to clarify whether the inhibition of poly(ADP-ribosyl)ation contributes to organic Hg induced neurotoxicity.
Asunto(s)
Astrocitos/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Mutágenos/toxicidad , Timerosal/toxicidad , Astrocitos/metabolismo , Línea Celular , Roturas del ADN/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli Adenosina Difosfato Ribosa/análisis , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
A new method for elemental bioimaging with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was developed and applied to study the uptake of manganese (Mn) in Caenorhabditis elegans (C. elegans). C. elegans is a well-established model organism in neuroscience, genetics and genomics, which has been extensively studied to decipher mechanisms of heavy metal induced toxicity. Knowledge about the distribution of manganese (Mn) and other metals in this organism will be helpful in elucidating pathways and mechanisms of transport, distribution and excretion. The LA-ICP-MS method requires limited sample preparation and can be used rapidly and easily to visualize the Mn distribution in C. elegans. Due to thorough optimization of the analytical parameters, intense Mn signals in C. elegans wild-type (WT) and mutants were obtained at a spatial resolution as small as 4 µm, thus proving the suitability of LA-ICP-MS to study the uptake of metals in C. elegans.
Asunto(s)
Caenorhabditis elegans/metabolismo , Manganeso/metabolismo , Animales , Transporte Biológico , Caenorhabditis elegans/química , Terapia por Láser , Manganeso/análisis , Espectrometría de MasasRESUMEN
Two different analytical approaches, external calibration and isotope dilution analysis both using flow-injection inductively coupled plasma mass spectrometry, have been developed and applied to determine the intracellular platinum concentration after Cisplatin incubation of two different medulloblastoma cell lines (UW228 and DAOY). As the internal or isotopically enriched standard was already used for cell lysis, maximum accuracy of the results was obtained, whereas a new home-built and inert injection system dramatically lowered carry-over effects and analyte loss. With limits of the detection well below 0.4µgL(-1) and typical relative standard deviations of 2%, a strong correlation between the cell viability in MTT assays and the incorporated amount of Pt could be shown, which was subsequently normalized to the protein content of the samples. DAOY cells did significantly ingest more Pt and showed a higher mortality, which supports the fact that transporter expression needs to be taken into account in order to obtain meaningful results.
Asunto(s)
Cisplatino/farmacología , Análisis de Inyección de Flujo/métodos , Espacio Intracelular/metabolismo , Meduloblastoma/metabolismo , Platino (Metal)/metabolismo , Espectrofotometría Atómica/métodos , Calibración , Línea Celular Tumoral , Humanos , Espacio Intracelular/efectos de los fármacos , Meduloblastoma/patologíaRESUMEN
In order to reveal the time-depending mercury species uptake by human astrocytes, a novel approach for total mercury analysis is presented, which uses an accelerated sample introduction system combined on-line with an inductively coupled plasma mass spectrometer equipped with a collision/reaction cell. Human astrocyte samples were incubated with inorganic mercury (HgCl2), methylmercury chloride (MeHgCl), and thimerosal. After 1-h incubation with Hg(2+), cellular concentrations of 3 µM were obtained, whereas for organic species, concentrations of 14-18 µM could be found. After 24 h, a cellular accumulation factor of 0.3 was observed for the cells incubated with Hg(2+), whereas the organic species both showed values of about 5. Due to the obtained steady-state signals, reliable results with relative standard deviations of well below 5 % and limits of detection in the concentration range of 1 ng L(-1) were obtained using external calibration and species-unspecific isotope dilution analysis approaches. The results were further validated using atomic fluorescence spectrometry.
Asunto(s)
Astrocitos/metabolismo , Cloruro de Mercurio/análisis , Compuestos de Metilmercurio/análisis , Espectrofotometría Atómica/métodos , Timerosal/análisis , Calibración , Técnicas de Cultivo de Célula , Células Cultivadas , Diseño de Equipo , Humanos , Límite de Detección , Cloruro de Mercurio/metabolismo , Isótopos de Mercurio/análisis , Compuestos de Metilmercurio/metabolismo , Estándares de Referencia , Reproducibilidad de los Resultados , Soluciones , Espectrometría de Fluorescencia , Espectrofotometría Atómica/instrumentación , Relación Estructura-Actividad , Timerosal/metabolismo , Factores de TiempoRESUMEN
In this study, the cellular uptake of the second generation photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP) was investigated using laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) at a spatial resolution of 10 µm. To achieve high sensitivity, the photosensitizer was tagged with palladium. As a tumor model system, a 3D cell culture of the TKF-1 cell line was used. These tumor spheroids were incubated with the Pd-tagged photosensitizer embedded in poly(lactic-co-glycolic acid) (PLGA) nanoparticles to investigate the efficiency of nanoparticle based drug delivery. An accumulation of the drug in the first cell layers of the tumor spheroid was observed. In the case of nanoparticle based drug delivery, a significantly more homogeneous distribution of the photosensitizer was achieved, compared to tumor spheroids incubated with the dissolved photosensitizer without the nanoparticular drug delivery system. The infiltration depth of the Pd-tagged photosensitizer could not be increased with rising incubation time, which can be attributed to the adsorption of the photosensitizer onto cellular components.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/administración & dosificación , Paladio/química , Fármacos Fotosensibilizantes/administración & dosificación , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Humanos , Ácido Láctico/química , Espectrometría de Masas/métodos , Imagen Molecular/métodos , Estructura Molecular , Nanopartículas/química , Neoplasias/metabolismo , Neoplasias/patología , Compuestos Organometálicos/química , Compuestos Organometálicos/metabolismo , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/metabolismo , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Porfirinas/administración & dosificación , Porfirinas/química , Porfirinas/metabolismo , Reproducibilidad de los Resultados , Esferoides Celulares/patologíaRESUMEN
The distribution of different chemical elements from a nanosilver-coated bone implant was visualized, combining the benefits of two complementary methods for elemental bioimaging, the nondestructive micro X-ray fluorescence (µ-XRF), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Challenges caused by the physically inhomogeneous materials including bone and soft tissues were addressed by polymer embedding. With the use of µ-XRF, fast sample mapping was achieved obtaining titanium and vanadium signals from the metal implant as well as phosphorus and calcium signals representing hard bone tissue and sulfur distribution representing soft tissues. Only by the use of LA-ICP-MS, the required high sensitivity and low detection limits for the determination of silver were obtained. Metal distribution within the part of cancellous bone was revealed for silver as well as for the implant constituents titanium, vanadium, and aluminum. Furthermore, the detection of coinciding high local zirconium and aluminum signals at the implant surface indicates remaining blasting abrasive from preoperative surface treatment of the nanosilver-coated device.
Asunto(s)
Prótesis de Cadera , Terapia por Láser/métodos , Espectrometría de Masas/métodos , Nanopartículas del Metal/análisis , Espectrometría por Rayos X/métodos , Espectrofotometría Atómica/métodos , Animales , Perros , Prótesis de Cadera/normas , Plata/análisisRESUMEN
A novel quantification approach for tissue imaging using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) based on tissue embedding in cold-curing resins (Technovit 7100) is presented. With respect to massive side effects on cisplatin, the platinum distribution at different time intervals after cisplatin treatment of mice was determined quantitatively in different tissues including cochlea, testis and kidney. For this purpose, cold-curing resin blocks spiked with different amounts of platinum acetyl acetonate prior to curing were ablated after sectioning at 5 µm thickness and were analysed using ICP-MS after microwave digestion. High spatial resolution and limits of detection in the low ppb range (8 µg kg(-1)) were achieved using a simple and efficient sample preparation. External calibration using the Technovit 7100 standards proved to yield precise and reproducible quantification results. The distribution and retention behaviour of cisplatin in the organs was investigated using the new calibration method.
Asunto(s)
Diagnóstico por Imagen/métodos , Terapia por Láser , Especificidad de Órganos , Platino (Metal)/metabolismo , Polímeros/química , Espectrofotometría Atómica , Animales , Calibración , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía FluorescenteRESUMEN
The application of gadolinium(Gd)-based contrast agents to support medical examinations by magnetic resonance imaging (MRI) results in a large input of Gd into the environment. The long-term effects of the anthropogenic Gd anomaly, especially on aqueous ecosystems, are mostly unknown. The identification and quantification of Gd-based contrast agents in the aquatic environment requires the use of powerful methods of speciation analysis. Therefore, a method employing the hyphenation of hydrophilic interaction liquid chromatography (HILIC) and inductively coupled plasma sector field mass spectrometry (ICP-SFMS) with sample introduction as dry aerosol generated by desolvation was developed. The desolvation resulted in improved limits of detection for the predominantly used contrast agents well below 0.10 nmol/L. This method was subsequently used for the analysis of Gd species in surface waters. Samples from a nature reserve in the city of Münster (Germany), into which the effluent from the city's main wastewater treatment plant enters the environment, were examined. The contrast agents Gd-DTPA, Gd-DOTA and Gd-BT-DO3A were identified and quantified in constant ratios in those samples. The concentrations were found in a range from 0.59 nmol/L for Gd-DOTA up to 3.55 nmol/L for Gd-BT-DO3A. As a result of mass balancing, the contrast agent concentration was found to account for 74-89% of total Gd concentrations, possibly indicating the presence of further Gd species. Nevertheless, there was no direct indication of species transformation by transmetallation reactions resulting in such Gd species. The determination of REE patterns by means of ICP-MS confirmed the results of speciation analysis showing significant Gd anomalies.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Medios de Contraste/análisis , Gadolinio DTPA/análisis , Compuestos Heterocíclicos/análisis , Compuestos Organometálicos/análisis , Contaminantes Químicos del Agua/análisis , Agua Dulce/química , Alemania , Interacciones Hidrofóbicas e Hidrofílicas , Límite de Detección , Espectrometría de Masas/métodos , Análisis de RegresiónRESUMEN
Whereas inorganic arsenic is classified as a human carcinogen, risks to human health related to the presence of arsenosugars in marine food are still unclear. Since studies indicate that human inorganic arsenic metabolites contribute to inorganic arsenic induced carcinogenicity, a risk assessment for arsenosugars should also include a toxicological characterization of their respective metabolites. Here we assessed intestinal bioavailability of the human arsenosugar metabolites oxo-DMAA(V), thio-DMAA(V), oxo-DMAE(V), thio-DMAE(V) and thio-DMA(V) in relation to arsenite in the Caco-2 intestinal barrier model. Whereas arsenite and thio-DMA(V) caused barrier disruption at concentrations ≥10 µM, all other metabolites did not cause a barrier leakage, even when applied at 50 times higher concentrations than arsenite and thio-DMA(V). The transfer studies point to a strong intestinal bioavailability of thio-DMA(V) and thio-DMAE(V), whereas oxo-DMAA(V), thio-DMAA(V) and oxo-DMAE(V) passed the in vitro intestinal barrier only to a very small extent. Detailed influx and efflux studies indicate that arsenite and thio-DMA(V) cross the intestinal barrier most likely by passive diffusion (paracellular) and facilitated (transcellular) transport. LC-ICP-QMS based arsenic speciation studies during the transfer experiments demonstrate transfer of thio-DMA(V) itself across the intestinal barrier and suggest metabolism of thio-DMA(V) using the in vitro intestinal barrier model to its oxygen-analogue DMA(V). In the case of arsenite no metabolism was observed. In summary the two arsenosugar metabolites thio-DMA(V) and thio-DMAE(V) showed intestinal bioavailability similar to that of arsenite, and about 10-fold higher than that reported for arsenosugars (Leffers et al., Mol. Nutr. Food Res., 2013, DOI: 10.1002/mnfr.201200821) in the same in vitro model. Thus, a presystemic metabolism of arsenosugars might strongly impact arsenic intestinal bioavailability after arsenosugar intake and should therefore be considered when assessing the risks to human health related to the consumption of arsenosugar-containing food.
Asunto(s)
Arseniatos/química , Arseniatos/farmacocinética , Ácido Cacodílico/análogos & derivados , Monosacáridos/química , Monosacáridos/farmacocinética , Arsenitos/química , Disponibilidad Biológica , Células CACO-2 , Ácido Cacodílico/química , Carcinógenos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Difusión , Relación Dosis-Respuesta a Droga , Humanos , Intestinos/efectos de los fármacos , Oxígeno/química , PermeabilidadRESUMEN
RATIONALE: Biological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved analysis with simultaneous elemental and molecular mass spectrometry (MS) and promises therefore localization, identification and quantification of metal or heteroelement-containing species in biological samples such as tissue sections. METHODS: A UV laser ablation (LA) system is hyphenated in parallel both with an elemental and a molecular mass spectrometer via flow splitted transfer lines to simultaneously obtain data from both of the mass spectrometers. Elemental MS was performed using inductively coupled plasma (ICP)-MS, whereas atmospheric pressure chemical ionization (APCI)-MS with an orbitrap mass analyzer was utilized for molecular MS. RESULTS: Simultaneous elemental and molecular MS imaging with high lateral resolution down to 25 µm was presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin in thin tissue sections. For molecular MS, target compounds were identified by their exact masses and by characteristic fragment ions. CONCLUSIONS: The first simultaneous elemental and molecular MS imaging approach based on laser ablation sampling was introduced for spatially resolved speciation analysis. The combination of the advantages of LA-ICP-MS such as low detection limits and high spatial resolution with information on the chemical identity promises not only localization of metals, but also identification of metal species in biological samples. Therefore, this novel technique opens up new possibilities to address complex challenges in life science research.
Asunto(s)
Riñón/química , Ganglios Linfáticos/química , Espectrometría de Masas/métodos , Imagen Molecular/métodos , Animales , Terapia por Láser , Espectrometría de Masas/instrumentación , Ratones , Microtomía , Imagen Molecular/instrumentación , Coloración y EtiquetadoRESUMEN
RATIONALE: The functions and properties of compounds are not only specified by their chemical structures, but also by their location inside a sample. Mass spectrometry is a powerful tool for imaging, whereby the kind of sample and compound depend on the used sampling and ionization methods. The developed laser ablation mass spectrometry method delivers high resolution images of small molecules in native samples. METHODS: A UV laser ablation (LA) system was combined with an atmospheric pressure chemical ionization (APCI) mass spectrometer. The spatially resolved sampling was performed by focusing the 213 nm laser beam onto a sample. The fine aerosol generated by the ns pulsed laser irradiation was then transported to the APCI mass spectrometer by a nitrogen stream. In the APCI source, post-ionization was accomplished by a corona discharge. The resulting ions were detected with an orbitrap mass spectrometer. RESULTS: The properties of the novel LA-APCI-MS setup are demonstrated by spatially resolved analysis of several samples including tablets, TLC plates and dried droplets. The target compounds are detected with high spatial and mass resolution. For higher molecular weight compounds like thyroxine, fragmentation was observed, whereas small molecules like caffeine stayed intact. CONCLUSIONS: LA-APCI-MS is introduced as an ambient molecular mass spectral imaging method for molecules with high resolution in space and mass. The combination of two independent instruments offers flexible ion source and mass analyzer exchange and therefore LA-APCI-MS opens up new possibilities for molecular imaging under ambient conditions.
Asunto(s)
Acetaminofén/química , Cafeína/química , Imagen Molecular/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Tiroxina/química , Cromatografía en Capa Delgada , Terapia por Láser , Imagen Molecular/instrumentación , Peso Molecular , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Comprimidos/químicaRESUMEN
This study focuses on the identification of the products that are formed upon binding of therapeutically relevant platinum complexes to proteins like ß-lactoglobulin A (LGA), human serum albumin (HSA), or human hemoglobin (HB). The respective proteins were incubated with the platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin. LGA was selected as the model protein in addition to the two most abundant blood proteins HSA and HB. In case of the model protein, the effect of free thiol groups on the affinity of cisplatin, carboplatin, and oxaliplatin was investigated by means of liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS). The reduced form of LGA, which contains four free thiol groups more than the native LGA, shows a much higher affinity to the platinum-based drugs. By means of liquid chromatography coupled to inductively coupled plasma mass spectrometry, the reaction behavior of the platinum-based drugs towards HSA and HB was investigated under different conditions considering the chloride concentration (4 or 100 mM) and the incubation time (24 and 48 h). In case of carboplatin, less than 6 % protein-bound platinum was detected. However, both cisplatin and oxaliplatin display a high affinity to the proteins investigated. Further information was obtained by means of LC/ESI-ToF-MS. In case of oxaliplatin, the complex [Pt(DACH)](2+) (DACH=C(6)N(2)H(14)) was identified interacting with HSA and HB. For cisplatin, different results were observed for the two proteins. The complex [Pt(NH(3))(2)Cl](+) interacted predominantly with HSA and [Pt(NH(3))(2)](2+) with HB.