RESUMO
The combined use of fluorescence-activated cell sorting (FACS) and single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) is reported, for the first time, in this work. It is applied to evaluate the differences between the cellular uptake of ultrasmall iron oxide nanoparticles (FeNPs) loaded with cisplatin(IV) prodrug (FeNPs-Pt(IV)) and cisplatin regarding cell viability. For this aim, FACS is applied to separate viable, apoptotic, and necrotic A2780 ovarian cancer cells after exposing them to the nanotransported prodrug and cisplatin, respectively. The different sorted cell populations are individually analyzed using quantitative SC-ICP-MS to address the intracellular amount of Pt. The highest Pt intracellular content occurs in the apoptotic cell population (about 2.1 fg Pt/cell) with a narrow intercellular distribution when using FeNPs-Pt(IV) nanoprodrug and containing the largest number of cells (75% of the total). In the case of the cisplatin-treated cells, the highest Pt content (about 1.6 fg Pt/cell) could be determined in the viable sorted cell population. The combined methodology, never explored before, permits a more accurate picture of the effect of the intracellular drug content together with the cell death mechanisms associated with the free drug and the nanotransported prodrug, respectively, and opens the door to many possible single-cell experiments in sorted cell populations.
Assuntos
Antineoplásicos , Neoplasias Ovarianas , Pró-Fármacos , Humanos , Feminino , Cisplatino/química , Pró-Fármacos/química , Linhagem Celular Tumoral , Neoplasias Ovarianas/tratamento farmacológico , Citometria de Fluxo , Antineoplásicos/químicaRESUMO
A systematic investigation on the cellular uptake, intracellular dissolution, and in vitro biological effects of ultra-small (<10 nm) iron hydroxide adipate/tartrate coated nanoparticles (FeAT-NPs) was carried out in intestinal Caco-2, hepatic HepG2 and ovarian A2780 cells, and the nucleotide excision repair (NER) deficient GM04312 fibroblasts. Quantitative evaluation of the nanoparticles uptake, as well as their transformation within the cell cytosol, was performed by inductively coupled plasma mass spectrometry (ICP-MS), alone or in combination with high performance liquid chromatography (HPLC). The obtained results revealed that FeAT-NPs are effectively taken up in a cell type-dependent manner with a minimum dissolution after 3 h. These results correlated with no effects on cell proliferation and minor effects on cell viability and reactive oxygen species (ROS) production for all the cell lines under study. Moreover, the comet assay results revealed significant DNA damage only in GM04312 cells. In vivo genotoxicity was further studied in larvae from Drosophila melanogaster, using the eye-SMART test. The obtained results showed that FeAT-NPs were genotoxic only with the two highest tested concentrations (2 and 5 mmol·L−1 of Fe) in surface treatments. These data altogether show that these nanoparticles represent a safe alternative for anemia management, with high uptake level and controlled iron release.
Assuntos
Nanopartículas , Neoplasias Ovarianas , Animais , Biotransformação , Células CACO-2 , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Humanos , Ferro/farmacologia , Larva/metabolismo , Nanopartículas Magnéticas de Óxido de Ferro , Nanopartículas/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Drought stress in plants causes differential expression of numerous genes. One of these differentially expressed genes in rice is a specific amidohydrolase. We characterized this amidohydrolase gene on the rice chromosome 12 as the first plant guanine deaminase (OsGDA1). The biochemical activity of GDA is known from tea and coffee plants where its catalytic product, xanthine, is the precursor for theine and caffeine. However, no plant gene that is coding for GDA is known so far. Recombinant OsGDA1 converted guanine to xanthine in vitro. Measurement of guanine and xanthine contents in the OsGDA1 knockout (KO) line and in the wild type Tainung 67 rice plants also suggested GDA activity in vivo. The content of cellular xanthine is important because of its catabolic products allantoin, ureides, and urea which play roles in water and nitrogen stress tolerance among others. The identification of OsGDA1 fills a critical gap in the S-adenosyl-methionine (SAM) to xanthine pathway. SAM is converted to S-adenosyl-homocysteine (SAH) and finally to xanthine. SAH is a potent inhibitor of DNA methyltransferases, the reduction of which leads to increased DNA methylation and gene silencing in Arabidopsis. We report that the OsGDA1 KO line exhibited a decrease in SAM, SAH and adenosine and an increase in rice genome methylation. The OsGDA1 protein phylogeny combined with mutational protein destabilization analysis suggested artificial selection for null mutants, which could affect genome methylation as in the KO line. Limited information on genes that may affect epigenetics indirectly requires deeper insights into such a role and effect of purine catabolism and related genetic networks.
Assuntos
Guanina Desaminase , Oryza , Amidoidrolases/genética , Amidoidrolases/metabolismo , Secas , Epigenoma , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/metabolismoRESUMO
Purpose. Echocardiography assessment from apical five-chamber view (A5CV) is the standard technique for aortic stenosis (AS) grading. Data on non-apical views, such as right parasternal (RPV), subcostal (SCV) and suprasternal notch (SSNV), is scarce and constitutes the aim of our study. Methods. We designed an observational study that included patients with AS recruited prospectively in whom the stenosis was graded by echocardiography from A5CV and non-apical view. The value of non-apical views in up-grading the stenosis severity (primary objective), the prognostic relevance of such reclassification and the feasibility and reproducibility of non-apical views assessment (secondary objectives) was evaluated. Results. Feasibility of AS appraisal from RPV, SCV and SSNV was 78%, 81% and 56%, respectively (SCV vs SSNV, p = .009). AS were up-graded from non-apical views according to peak gradient, mean gradient, area and indexed area by 24%, 17%, 24% and 22%, respectively (p < .0001). Non-apical views reclassified from non-severe to severe AS, from low gradient severe to high gradient severe AS and from non-critical to critical AS 19%, 23% and 3% of cases (p < .0001). The 4-years hard cardiac events rate was 41% in patients with non-severe AS, 67% in patients with severe AS from non-apical views, 68% in patients with severe AS from A5CV and 80% in patients with severe AS from A5CV and non-apical views (p < .001). Reproducibility of AS evaluation from non-apical views was fair to excellent (intraclass correlation coefficients: SSNV = 0.44, RPV = 0.61, SCV = 0.92). Conclusion. Assessment of AS from non-apical views is feasible, reproducible and valuable over A5CV; its use is encouraged.
Assuntos
Estenose da Valva Aórtica , Índice de Gravidade de Doença , Estenose da Valva Aórtica/diagnóstico por imagem , Ecocardiografia , Humanos , Reprodutibilidade dos TestesRESUMO
One of the limitations in the use of cisplatin is its low penetration into cells. In addition, some cells develop the so called resistance, a multifactorial event that decreases significantly the intracellular cisplatin concentration. To circumvent these limitations, recent studies are focused on the use of nanocarriers that permit, among others, to achieve higher drug uptake. In this work, ferritin is evaluated as a nanostructured cisplatin-delivery system in cell models of ovarian cancer. One of the key aspects is the characterization of the encapsulated product, and for this aim, a battery of analytical techniques, including size exclusion chromatography (SEC) coupled to UV detection and to inductively coupled plasma mass spectrometry (ICP-MS) together with transmission electron microscopy (TEM), is conducted. Higher level of incorporation occurs when using initial concentrations of the Fe-containing form of the protein at 10 mg/mL and 1 mg/mL cisplatin solution. The incorporation of the free and encapsulated cisplatin is addressed in A2780 and A2780CIS, sensitive and cisplatin-resistant cell lines, respectively, showing a significantly higher uptake of the encapsulated form. These values ranged from 5- to 9-fold in the sensitive line and 2-4 in the resistant model, being always more pronounced at the lower doses. Functionality of the drug after encapsulation is addressed by monitoring the presence of Pt in DNA and normalizing DNA concentration through simultaneous P and Pt measurements by ICP-MS. Time elapsed between exposure and Pt detection in DNA proved to be critical in the encapsulated model, showing the slower drug release mechanism from the ferritin nanocage that could be advantageously used for a controlled therapy. Graphical abstract.
Assuntos
Antineoplásicos/administração & dosagem , Cisplatino/administração & dosagem , Portadores de Fármacos/química , Ferritinas/química , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
Cells are able to precisely control the amount of iron they acquire in the form of transferrin (TF)-bound iron by modulating the synthesis of transferrin receptor 1 (TfR1). In tumor cells, elevated TfR1 seems to be related to poorer outcome for patients. Thus, the direct measurement of this biomarker in breast cancer tissues and cells might serve as a prognosis biomarker. In this work, we have used Nd-labeled antibodies to tag the TfR1 present on the cell surface of two cell models of breast cancer with different malignancy (MCF7 and MDA-MB 231). For this aim, monoclonal antibody anti-TfR1 is first labeled with a polymeric chelator (MAXPAR) with the subsequent incorporation of several isotopic 143Nd atoms. The characterization of the labeled antibody revealed a stoichiometry of 21 Nd atoms per antibody molecule that can be used for further quantification experiments. This antibody is used for cell tagging followed by single-cell analysis using inductively coupled plasma mass spectrometry (ICP-MS) detection. In this case, cell introduction is conducted using a high-efficiency nebulizer and spray chamber to achieve transport efficiencies of up to 55% for cells. Quantitative results revealed a number of receptors per cell significantly higher in the case of the most malignant phenotype (MDA-MB-231). Absolute and relative TfR1 concentration values are obtained in individual cells for the first time using the proposed system.
Assuntos
Anticorpos Monoclonais , Neoplasias da Mama/metabolismo , Espectrometria de Massas/métodos , Análise de Célula Única/métodos , Antígenos CD , Linhagem Celular Tumoral , Feminino , Humanos , Receptores da TransferrinaRESUMO
Epigenetic alterations are increasingly implicated in the initiation and progression of cancer. Genome-wide (global) hypomethylation seems to occur in early neoplasia and is a feature of genomic DNA derived from solid tumour tissues like ovarian cancer. Thus, analytical methods that provide sensitive and quantitative information about cytosine methylation in DNA are currently required. In this work, we compare two different anion-exchange columns for the separation of methylated cytosine from the other DNA nucleotides: a silica-based (Tracer Extrasil SAX) column and a polystyrene/divinyl benzene-based (Mono-Q™) column. Under the optimised conditions, linearity range, precision and detection limits of the developed high-performance liquid chromatography (HPLC) method were evaluated and compared using conventional ultraviolet (UV) absorbance detection at 270 nm. Good separation of the five target nucleotides, including 5-methyl-2'-deoxycytidine monophosphate (5mdCMP) and 2'-deoxycytidine monophosphate (dCMP) was achieved on the Mono-Q™ column with a gradient elution of ammonium acetate buffer (1 M, pH 6.9) at a flow rate of 1 mL min(-1). The coupling of this column to inductively coupled plasma mass spectrometry (ICP-MS) permitted also phosphorous ((31)P) specific detection of the nucleotides. Both detection systems offered adequate analytical performance characteristics, with detection limits of 30 and 40 µg L(-1) for 5mdCMP by HPLC-UV and HPLC-ICP-MS, respectively. However, the latter method allowed the determination of the global DNA methylation level (%) without the need for external calibration. Different genomic DNA samples were analysed including calf thymus DNA and DNA from two human cancer cell lines (adenocarcinoma epithelial A549 and ovarian carcinoma A2780) using the proposed strategy. In the line A2780, the cisplatin-sensitive and cisplatin-resistant variants were analysed, finding no significant differences in the methylation percentage after treatment with cisplatin.
Assuntos
Antineoplásicos/farmacologia , Cromatografia por Troca Iônica/métodos , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Ovarianas/genética , Resinas de Troca Aniônica/química , Linhagem Celular Tumoral , Cromatografia por Troca Iônica/instrumentação , Metilação de DNA , Desoxicitidina/química , Desoxicitidina/genética , Desoxicitidina/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismoRESUMO
Analytical methods allowing sensitive determination of reduced homocysteine (rHcy), one of the so-called biothiols, in human serum is a topic of growing interest due to its close relation to several human disorders, mainly cardiovascular diseases. Although most widely used analytical strategies to determine total Hcy involve derivatization by means of fluorescent labels, this work proposes the use of ebselen, a Se-containing labelling agent to derivatize the reactive sulfhydryl group of the Hcy molecule in its "free" reduced form, which is more likely to play different roles in disease pathogenesis. Optimization of the derivatization and separation conditions by high-performance liquid chromatography (HPLC) to isolate the excess of derivatizing reagent is carried out here using UV/VIS detection. Further, the study of the Se labelling reaction by electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides a stoichiometry of the derivative of 1:1. The main advantage of using ebselen as a labelling agent is the presence of the Se atom in the molecule that allows the use of inductively coupled plasma mass spectrometry (ICP-MS) as a sensitive and selective Se detector. The coupling of HPLC with ICP-MS provided excellent features for the determination of Se-derivatized rHcy (detection limit of 9.6 nM) in real samples. Quantification was accomplished by using post-column isotope dilution (ID) of Se in serum samples, after precipitation of the main serum proteins. Quantitative results for "free" rHcy turned out to be around 0.18-0.22 µM in serum samples from healthy individuals that could be directly analyzed without sample preconcentration.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Homocisteína/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Homocisteína/análise , Humanos , Limite de Detecção , Oxirredução , Espectrometria de Massas em Tandem/métodosRESUMO
Detection of specific DNA sequences is nowadays an important tool in many scientific areas such as forensic science or clinical diagnosis. Although numerous approaches have been suggested for this challenging analysis, certain limitations still remain. In order to overcome these disadvantages, novel and alternative methodologies are required. In this work, we present a strategy based on elemental (lanthanide) labelling of DNA probes followed by size-exclusion chromatography (SEC) coupled with inductively coupled plasma-mass spectrometry (ICP-MS) for monitoring and determining complementary oligonucleotide sequences (oligonucleotide targets) and for visualising the corresponding hybridisation processes. The synthesis and characterisation of the DNA probes are described in detail. SEC was found to be suitable to discriminate between the DNA probe and the hybridised product. Using labelling of different probes with different lanthanides, multiplexed detection of the sought DNA sequences was possible as demonstrated here on three DNA probes (derivatised with Eu, Tb, and Ho, respectively). The achievable detection limits were in the range between 5 and 11 fmol absolute.
Assuntos
Cromatografia em Gel/métodos , Sondas de DNA/química , DNA/química , Espectrometria de Massas/métodos , Hibridização de Ácido Nucleico/métodos , Sequência de Bases , Elementos da Série dos Lantanídeos/químicaRESUMO
Aortic stenosis (AS) is the most prevalent degenerative valvular disease in western countries. Transthoracic echocardiography (TTE) is considered, nowadays, to be the main imaging technique for the work-up of AS due to high availability, safety, low cost, and excellent capacity to evaluate aortic valve (AV) morphology and function. Despite the diagnosis of AS being considered straightforward for a very long time, based on high gradients and reduced aortic valve area (AVA), many patients with AS represent a real dilemma for cardiologist. On the one hand, the acoustic window may be inadequate and the TTE limited in some cases. On the other hand, a growing body of evidence shows that patients with low gradients (due to systolic dysfunction, concentric hypertrophy or coexistence of another valve disease such as mitral stenosis or regurgitation) may develop severe AS (low-flow low-gradient severe AS) with a similar or even worse prognosis. The use of complementary imaging techniques such as transesophageal echocardiography (TEE), multidetector computed tomography (MDTC), or cardiac magnetic resonance (CMR) plays a key role in such scenarios. The aim of this review is to summarize the diagnostic challenges associated with patients with AS and the advantages of a comprehensive multimodality cardiac imaging (MCI) approach to reach a precise grading of the disease, a crucial factor to warrant an adequate management of patients.
RESUMO
Cisplatin is a chemotherapeutic drug widely used in the treatment of several tumours, but this chemotherapy presents problems in terms of side-effects and patient resistance. The detection and determination of cisplatin-induced adducts and the relationship with the physiological or clinical effects of this drug under different repair conditions could be a good measure to assess patient's response to such chemotherapy. A new methodological approach to detect and quantify cisplatin adducts by use of high-performance liquid chromatography with inductively coupled plasma mass-spectrometric detection (HPLC-ICP-MS) and isotope-dilution analysis (IDA), is evaluated for its application in vivo, under different repair conditions. This analysis is combined with the use of the Comet assay, which detects DNA strand-breaks, and the w/w(+) SMART assay, which monitors induction of somatic mutation and recombination in Drosophila melanogaster in vivo under different conditions of nucleotide-excision repair. Results show that (i) cisplatin induces in Drosophila several adducts not detected in mammals. The two most abundant cisplatin-induced adducts, identified by electrospray-mass spectrometry as G monoadduct and G-G intrastrand cross-links, were quantified individually; (ii) cisplatin induces higher levels of G monoadducts and G-G cross-links in NER-proficient than in NER-deficient cells; (iii) the level of adducts correlates with their biological consequences, both in terms of DNA strand-breaks (tail-moment values), and of somatic mutation and recombination (frequency of mosaic eyes and clones in 10(4) cells), when the repair status is considered. This work demonstrates the validity and potential of the adduct detection and quantification methodology in vivo, and its use to correlate adducts with their genetic consequences.
Assuntos
Cisplatino/toxicidade , Adutos de DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Animais , Cromatografia Líquida de Alta Pressão , Ensaio Cometa , Quebras de DNA , Reparo do DNA , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Espectrometria de Massas , Mutação , Técnica de Diluição de RadioisótoposRESUMO
Cisplatin is one of the most effective chemotherapeutic agents, although its clinical use is limited by severe renal toxicity. This toxicity seems to be related to the accumulation of the drug in kidney tissues, leading to renal failure. For this reason, several compounds have been evaluated to ameliorate the nephrotoxicity induced by cisplatin. In the present investigation, we report the effect of the oral administration of selenomethionine before intraperitoneal cisplatin treatment. The preadministration of this Se species has been shown to have an important effect in reducing renal damage induced by cisplatin by increasing the excreted urea and improving creatinine clearance. Quantification of the level of DNA--cisplatin adducts in kidney and liver tissues was carried out by postcolumn isotope dilution analysis using liquid chromatography-inductively coupled plasma (LC-ICP-MS) as speciation set up. The level of DNA--cisplatin adducts in rats given Se-methionine in the drinking water before cisplatin administration was considerably lower in kidney tissues with respect to the animals drinking only water. Such effects were not observed in liver tissue. Initial speciation studies of Pt and Se conducted in kidney tissues of exposed animals by HPLC-ICP-MS have revealed the presence of cisplatin as part of a complex with Se-methionine, which can be eventually excreted into urine. This Pt--Se complex could explain the observed reduction of the kidney damage in Se-methionine-treated animals.
Assuntos
Antineoplásicos/toxicidade , Antioxidantes/uso terapêutico , Cisplatino/toxicidade , Adutos de DNA/metabolismo , Nefropatias/induzido quimicamente , Selenometionina/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Peso Corporal , Cisplatino/análise , Cisplatino/metabolismo , Cisplatino/farmacologia , Creatinina/sangue , Creatinina/urina , Adutos de DNA/análise , Interações Medicamentosas , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/tratamento farmacológico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Modelos Moleculares , Platina/análise , Platina/metabolismo , Ratos , Ratos Wistar , Selênio/análise , Selênio/metabolismo , Selenometionina/farmacologiaRESUMO
A simple CE method has been developed for the separation and determination of thyroxine (T4) enantiomers in pharmaceutical formulations. The method was based on ligand-exchange mechanism using a Cu(II)/L-proline complex as chiral selector. The effects of different parameters affecting separation such as chiral selector concentration, organic additive, buffer pH and temperature were investigated. A baseline separation of the two enantiomers was obtained at a Cu(II)/L-proline ratio of 1:8 in a borate buffer (15 mmol/L, pH 9.6) containing 10% v/v acetonitrile. Under the optimized conditions, precision linearity range and detection limits of the developed enantioselective CE method were evaluated and compared using two different detection systems: conventional UV detection at 226 nm and iodine (127I)specific detection ("chiral speciation") with ICP-MS. Both methodologies show adequate analytical performance characteristics with detection limits around 0.30 microg/mL for each enantiomer of T4. Finally, a levothroid pharmaceutical formulation sample was successfully analyzed using both developed methods CE-UV and CE-ICP-MS.
Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Espectrofotometria Ultravioleta , Tiroxina/química , Concentração de Íons de Hidrogênio , Ligantes , Estereoisomerismo , TemperaturaRESUMO
The antitumoral effect of cisplatin [cis-diamminodichloroplatinum(II)] in mammals is related to its binding to DNA components. However, there is a lack of specific chemical methods to selectively detect those adducts formed in vivo at low concentrations. In this work, a new sensitive and selective method of determining cisplatin-DNA adducts based on the use of element-selective mass spectrometry is proposed, and the method is then applied to detect cisplatin adducts induced in vivo in somatic cells of Drosophila melanogaster. The bioanalytical strategy proposed here allows the determination of the most important DNA adduct formed between adjacent guanine units of the same DNA strand with cisplatin, and it is based on the coupling of capillary liquid chromatography (cap-LC) to inductively coupled plasma mass spectrometry (ICP-MS). This set-up allows the simultaneous monitoring of the Pt (from the drug) and P (from the DNA components) present in these adducts, once they have been cleaved by enzymatic hydrolysis of the DNA samples. Using this instrumental set-up, the adducts of cisplatin formed in vivo when D. melanogaster flies are exposed to different cisplatin concentrations can be detected and their concentration determined. The results obtained show a direct correlation between the concentration of cisplatin adducts, the induced genotoxic damage (measured as DNA strand breaks using the Comet assay) and the cisplatin concentration. [figure: see text] The work illustrates the complementary use of bioanalytical and biological information to study cisplatin interactions with DNA is vivo at biologically relevant concentrations of the drug.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cisplatino/análise , Cisplatino/química , Adutos de DNA/análise , Adutos de DNA/química , Drosophila melanogaster/genética , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Mutagênicos/toxicidade , Animais , Ensaio Cometa , Drosophila melanogaster/efeitos dos fármacos , Feminino , Masculino , Estrutura MolecularRESUMO
Cisplatin, one of the most extensively used metallodrugs in cancer treatment, presents the important drawback of patient resistance. This resistance is the consequence of different processes including those preventing the formation of DNA adducts and/or their quick removal. Thus, a tool for the accurate detection and quantitation of cisplatin-induced adducts might be valuable for predicting patient resistance. To prove the validity of such an assumption, highly sensitive plasma mass spectrometry (ICP-MS) strategies were applied to determine DNA adduct levels and intracellular Pt concentrations. These two metal-relative parameters were combined with an evaluation of biological responses in terms of genomic stability (with the Comet assay) and cell cycle progression (by flow cytometry) in four human cell lines of different origins and cisplatin sensitivities (A549, GM04312, A2780 and A2780cis), treated with low cisplatin doses (5, 10 and 20 µM for 3 hours). Cell viability and apoptosis were determined as resistance indicators. Univariate linear regression analyses indicated that quantitation of cisplatin-induced G-G intra-strand adducts, measured 1 h after treatment, was the best predictor for viability and apoptosis in all of the cell lines. Multivariate linear regression analyses revealed that the prediction improved when the intracellular Pt content or the Comet data were included in the analysis, for all sensitive cell lines and for the A2780 and A2780cis cell lines, respectively. Thus, a reliable cisplatin resistance predictive model, which combines the quantitation of adducts by HPLC-ICP-MS, and their repair, with the intracellular Pt content and induced genomic instability, might be essential to identify early therapy failure.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacocinética , Adutos de DNA/análise , Adutos de DNA/genética , Instabilidade Genômica/efeitos dos fármacos , Humanos , Espectrometria de Massas , Neoplasias/genética , Neoplasias/patologiaRESUMO
The development of a rapid, precise and accurate analytical method for the determination of methylmercury in tuna fish samples is described. The method is based on the use of isotope dilution GC/MS with electron impact ionization, a widespread technique in routine testing laboratories. A certified spike containing (202)Hg-enriched methylmercury was used for the isotope dilution of the samples. After extraction of the methylmercury from the sample, methylmercury was propylated using sodium tetrapropyl borate in SPME vials and the analytes were sampled from the headspace for 15 min. For isotope measurements, the molecular ion (MePrHg(+)) was used in the SIM mode. Five molecular ions were monitored, corresponding to the (198)Hg, (199)Hg, (200)Hg, (201)Hg and (202)Hg isotopes. The detection at masses corresponding to (198)Hg was used to correct for m + 1 contributions of (13)C from the organic groups attached to the mercury atom on the (199)Hg, (200)Hg, (201)Hg and (202)Hg masses with simple mathematical equations, and the concentration of methylmercury was calculated on the basis of the corrected (200)Hg/(202)Hg isotope ratio. The (202)Hg-enriched methylmercury spike was applied, with satisfactory results, to the determination of methylmercury in the certified reference material BCR 464. The method was successfully applied to the determination of methylmercury in tuna fish samples, and the obtained results were included in the CCQM-P39 interlaboratory exercise, organized by the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) with excellent agreement between our results and the average obtained by the other participants.
Assuntos
Carne/análise , Compostos de Metilmercúrio/análise , Atum/metabolismo , Algoritmos , Animais , Cromatografia Gasosa-Espectrometria de Massas , Indicadores e Reagentes , Radioisótopos de Mercúrio , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: We determined, for packed red blood cells (PRBC) and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethylarginine (LNMMA). BACKGROUND: ADMA and LNMMA are near equipotent NOS inhibitors forming blood's total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined. METHODS: We measured total (free and protein incorporated) ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis. RESULTS: In vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM) and LNMMA (58.9 ± 28.9 µM) that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma. CONCLUSION: The compelling physiological ramifications are that regardless of storage age, 1) PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2) PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate risk for iatrogenic NOS inhibition upon transfusion.
Assuntos
Inibidores Enzimáticos/metabolismo , Eritrócitos/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Catalase/metabolismo , Humanos , Óxido Nítrico/metabolismo , Proteólise , Ratos , Temperatura , Fatores de TempoRESUMO
The development of a rapid, precise and accurate speciation method for the simultaneous determination of mono-, di- and tributyltin in environmental samples is described. The method is based on using isotope dilution gas chromatography/mass spectrometry (GC/MS) with electron ionization, a widely used technique in routine testing laboratories. A mixed spike containing (119)Sn-enriched monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) was used for the isotope dilution of the samples. Five molecular ions were monitored for each analyte, corresponding to the (116)Sn, (117)Sn, (118)Sn, (119)Sn and (120)Sn isotopes. The detection at masses corresponding to (116)Sn and (117)Sn were used to correct for m + 1 and m + 2 contributions of (13)C from the organic groups attached to the tin atom on the (118)Sn, (119)Sn and (120)Sn masses with simple mathematical equations and the concentrations of the butyltin compounds were calculated based on the corrected (118)Sn/(119)Sn and (120)Sn/(119)Sn isotope ratios. The (119)Sn-enriched multispecies spike was applied with satisfactory results to the simultaneous determination of MBT, DBT and TBT in three certified reference materials: two sediments, PACS-2 and BCR 646, and the mussel tissue CRM 477. The method was compared with a previously published GC/inductively coupled plasma MS isotope dilution procedure, developed in our laboratory, by injecting the same samples into both instruments. Comparable analytical results in terms of precision and accuracy are demonstrated for both atomic and molecular mass spectrometric detectors. Thus, reliable quantitative organotin speciation analysis can be achieved using the more widespread and inexpensive GC/MS instrument.
Assuntos
Poluição Ambiental/análise , Sedimentos Geológicos/química , Compostos Orgânicos de Estanho/análise , Compostos de Trialquitina/análise , Animais , Bivalves/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Compostos Orgânicos de Estanho/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Compostos de Trialquitina/químicaRESUMO
The feasibility of developing a single-step clean-up, enrichment procedure for organochlorine pesticides (OCPs) in fish tissue samples based on solid-phase microextraction (SPME) was investigated. The general analytical methodology developed combines conventional solid-liquid extraction of the OCPs from the sample using an organic solvent with SPME over the organic extract followed by gas chromatography-electron-capture detection (GC-ECD) analysis. Experimental SPME conditions such as extraction time, temperature and matrix effects were optimised. Under optimised conditions, precision, linearity range, detection limits and accuracy were evaluated. Detection limits obtained for fish tissue samples were in the range of 0.1-0.7 ng g(-1). Good recoveries (over 70% in all cases) were achieved from samples spiked at a concentration level of 10 ng g(-1). The accuracy of the developed SPME-GC-ECD procedure in real samples has been verified by analysing, using the standard addition method, a certified reference material (CRM 430, OCPs in pork fat) with satisfactory results.
Assuntos
Cromatografia Gasosa/métodos , Peixes , Hidrocarbonetos Clorados , Inseticidas/análise , Animais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A method for enantioselective determination of bromocyclen enantiomers in fish tissue has been developed. The enantiomers were resolved by capillary gas chromatography (GC) using a commercial chiral column (CP-Chirasil-Dex CB) and a temperature program from 50 degrees C (held for 1 min), raised to 140 degrees C at 40 degrees C min(-1) and then raised at 0.2 degrees C min(-1) to 155 degrees C. This enantioselective gas chromatographic separation was combined with a clean-up/enrichment procedure based on solid-phase microextraction (SPME). Under SPME optimized conditions, precision, linearity range and detection limits of the developed SPME-enantioselective GC procedure were evaluated and compared using two different detection systems: a classical electron-capture detection (ECD) and an element specific detection using inductively coupled plasma mass spectrometry (ICP-MS). The SPME-GC-ECD method exhibited an excellent sensitivity, with detection limits of 0.2 ng L(-1) for each enantiomer of bromocyclen. Although ICP-MS offered poorer detection limits (7 ng L(-1) as Br, equivalent to 36 ng L(-1) of each enantiomer) than conventional ECD detector, it proved to be clearly superior in terms of selectivity. The relative potential and performance of the two compared methods for real-life analysis has been illustrated by the determination of enantiomers of bromocyclen in spiked tissue extracts of trout.