Cassiana Seimi Nomura

Diethyldithiocarbamate induces apoptosis in neuroblastoma cells by raising the intracellular copper level, triggering cytochrome c release and caspase activation.

Dithiocarbamates are nitrogen- and sulfur-containing compounds commonly used in pharmacology, medicine and agriculture. The molecular effects of dithiocarbamates on neuronal cell systems are not fully understood, especially in terms of their ability to accumulate copper ions inside the cell. In this work, the molecular effects of N,N-diethyldithiocarbamate (DEDTC) were studied in human SH-SY5Y neuroblastoma cells to determine the role of copper in the DEDTC toxicity and the pathway trigged in cell by the complex Cu-DEDTC. From concentration-dependent studies, we found that 5 μM of this compound induced a drastic decrease in viable cells with a concomitant accumulation in intracellular copper resulted from complexation with DEDTC, measured by atomic absorption spectroscopy. The mechanism of DEDTC-induced apoptosis in neuronal model cells is thought to occur through the death receptor signaling triggered by DEDTC-copper complex in low concentration that is associated with the activation of caspase 8. Our results indicated that the mechanism of cell death involves cytochrome c release forming the apoptosome together with Apaf-1 and caspase 9, converting the caspase 9 into its active form, allowing it to activate caspase 3 as observed by immunofluorescence. This pathway is induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions present in the culture medium and transports them into the cell, suggesting that the DEDTC by itself was not able to cause cell death and the major effect is from its copper-complex in neuroblastoma cells. The present study suggests a role for the influence of copper by low concentrations of DEDTC in the extracellular media, the absorption and accumulation of copper in the cell and apoptotic events, induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions.

An environmentally friendly analytical procedure for nickel determination by atomic and molecular spectrometry after cloud point extraction in different samples

Cloud point extraction (CPE) was employed for separation and preconcentration prior to the determination of nickel by graphite furnace atomic absorption spectrometry (GFAAS), flame atomic absorption spectrometry (FAAS) or UV-Vis spectrophotometry. Di-2-pyridyl ketone salicyloylhydrazone (DPKSH) was used for the first time as a complexing agent in CPE. The nickel complex was extracted from the aqueous phase using the Triton X-114 surfactant. Under optimized conditions, limits of detection obtained with GFAAS, FAAS and UV-Vis spectrophotometry were 0.14, 0.76 and 1.5 μg L−1, respectively. The extraction was quantitative and the enrichment factor was estimated to be 27. The method was applied to natural waters, hemodialysis concentrates, urine and honey samples. Accuracy was evaluated by analysis of the NIST 1643e Water standard reference material.

Cell Penetrating Peptide (CPP)-Conjugated Desferrioxamine for Enhanced Neuroprotection: Synthesis and in Vitro Evaluation

Iron overload causes progressive and sometimes irreversible damage due to accelerated production of reactive oxygen species. Desferrioxamine (DFO), a siderophore, has been used clinically to remove excess iron. However, the applications of DFO are limited because of its inability to access intracellular labile iron. Cell penetrating peptides (CPPs) have become an efficient delivery vector for the enhanced internalization of drugs into the cytosol. We describe, herein, an efficient method for covalently conjugating DFO to the CPPs TAT(47-57) and Penetratin. Both conjugates suppressed the redox activity of labile plasma iron in buffered solutions and in iron-overloaded sera. Enhanced access to intracellular labile iron compared to the parent siderophore was achieved in HeLa and RBE4 (a model of blood-brain-barrier) cell lines. Iron complexes of both conjugates also had better permeability in both cell models. DFO antioxidant and iron binding properties were preserved and its bioavailability was increased upon CPP conjugation, which opens new therapeutic possibilities for neurodegenerative processes associated with brain iron overload.

Total determination and direct chemical speciation of Hg in fish by solid sampling GF AAS

Simple and rapid analytical procedures for the direct determination of total Hg and for direct chemical speciation of Hg2+ and methylmercury (MHg) in fish were proposed after careful optimization of the heating program and the addition of appropriate chemical modifiers by solid sampling graphite furnace atomic absorption spectrometry (SS-GF AAS). The total determination was done using Ir film and 5 μg Pd + 3 μg Mg solution as permanent and co-injected chemical modifiers, respectively. For direct chemical speciation, 5 μg Pd + 3 μg Mg or 5 μg Pd + 3 μg Mg + 0.5% w/v Triton X-100 solutions were used as co-injected chemical modifiers. The accuracy of both procedures was verified using the certified reference material (CRM) of tuna fish (CE-464), lobster hepatopancreas (TORT-2) and dogfish (DOLT-2). The certified and the found concentrations were statistically concordant (Students t-test). The total Hg concentration in the tuna fish candidate reference material produced in our laboratory was 2.66 ± 0.15 mg kg−1. In the sample ground in a cryogenic mill, the Hg2+ concentrations were 0.553 and 0.559 mg kg−1 and by the difference between the total Hg and Hg2+ concentration was obtained the MHg concentration, 5.11 and 4.73 g kg−1, using Pd + Mg and Pd + Mg + Triton respectively. These results reveal a high concentration of the methylated species that it is most toxic Hg species.

Direct Determination of Potentially Toxic Elements in Rice by SS-GF AAS: Development of Methods and Applications

The development of methods for direct determinations of Al, Cd, and Pb in rice by SS-GF AAS is presented. Heating program optimization associated with the use of an adequate chemical modifier containing Pd + Mg allowed direct analysis against aqueous calibrations. The obtained LOD values were 114.0, 3.0, and 16.0 μg kg⁻¹ for Al, Cd, and Pb, respectively. Important parameters associated with a solid sampling analysis were investigated, such as minimum and maximum sample mass size and analyte segregation. Seventeen rice samples available in São Paulo City were analyzed, and all of them presented analyte mass fractions less than the maximum allowed by legislation. The influences of rice washing and the cooking procedure were also investigated. The washing procedure diminished the Al and Pb total mass fractions, indicating an exogenous grain contamination. The cooking procedure diminished the Cd total mass fraction. Rice cooking using an aluminum container did not cause a significant increase in the Al mass fraction in the rice, indicating no translocation of this element from container to food. In general, coarse rice presented higher levels of Al when compared to polished or parabolized rice.

Cu(GlyGlyHis) effects on MCF7 cells: copper uptake, reactive oxygen species generation and membrane topography changes.

Elevated levels of copper have been detected in various types of human cancer cells, such as breast cancer cells, and a number of mechanisms have been proposed to explain the action and influence of copper on tumor progress. In this work, we found that stimulating the proliferation of mammary epithelial MCF7 cells with the high-redox-potential copper complex Cu (GlyGlyHis) is associated with the copper-induced intracellular generation of reactive oxygen species (ROS) that induces lipid peroxidation and causes increased roughness of external cell membranes, which leads to the formation of larger cell domes. The results presented herein provide new insights into the molecular link between copper and the proliferation of breast cancer cells and, consequently, into the mechanism by which changes in redox balance and ROS accumulation regulates cell membrane roughness.

Desferrioxamine-caffeine (DFCAF) as a cell permeant moderator of the oxidative stress caused by iron overload

Desferrioxamine (DFO) is a potent iron chelator used in the treatment of iron overload (IO) disorders. However, due to its low cell permeability and fast clearance, DFO administration is usually prolonged and of limited use for the treatment of IO in tissues such as the brain. Caffeine is a safe, rapidly absorbable molecule that can be linked to other compounds to improve their cell permeability. In this work, we successfully prepared and described DFO-caffeine, a conjugate with iron scavenging ability, antioxidant properties and enhanced permeation in the HeLa cell model.

Deferasirox-TAT(47–57) peptide conjugate as a water soluble, bifunctional iron chelator with potential use in neuromedicine

Deferasirox (DFX), an orally active and clinically approved iron chelator, is being used extensively for the treatment of iron overload. However, its water insolubility makes it cumbersome for practical use. In addition to this, the low efficacy of DFX to remove brain iron prompted us to synthesize and evaluate a DFX-TAT(47–57) peptide conjugate for its iron chelation properties and permeability across RBE4 cell line, an in vitro model of the blood–brain barrier. The water-soluble conjugate was able to remove labile iron from buffered solution as well as from iron overloaded sera, and the permeability of DFX-TAT(47–57) conjugate into RBE4 cells was not affected compared to parent deferasirox. The iron bound conjugate was also able to translocate through the cell membrane.

Feasibility of Using AAS for the Characterization of a Tuna Fish Candidate Reference Material for Total Hg and Methyl-Hg Measurement

This work describes the evaluation of several parameters for preparing a tuna fish candidate reference material (RM) in order to measure its total Hg and methyl-Hg mass fractions by cold vapor atomic absorption spectrometry (CV-AAS) and solid sampling graphite furnace atomic absorption spectrometry (SS-GF AAS). Best results were acquired after extraction with toluene and L-cysteine for 15 min at 80 °C. The parameters investigated were stability of chemical composition during production, homogeneity and analyte segregation. No analyte segregation was observed and the material was shown to be stable even when stored at 50 °C for 10 months. Total Hg mass fraction was 3.03 ± 0.22 µg g-1 by CV AAS, 2.99 ± 0.43 µg g-1 by SS-GF AAS and 2.95 ± 0.15 µg g-1 by direct mercury analyser (DMA). Methyl-Hg mass fraction was 2.49 ± 0.13 µg g-1 by CV AAS and 2.44 ± 0.46 µg g-1 by SS-GF AAS.

Internal standard fused glass beads for high silicon content sample analysis by laser-induced breakdown spectrometry

The direct analysis of high silicon content samples by Laser-Induced Breakdown Spectrometry (LIBS) is a good alternative, since the decomposition of samples with refractory characteristics is not an easy task. However, poor precision and difficulty in performing quantitative analysis due to particle size effects, heterogeneity and matrix effects are challenges which need to be overcome. An interesting approach to overcome these issues enabling quantitative measurements of Al, Ca, Fe, Mg, Si and Ti in a variety of samples by LIBS is the use of the borate fusion process for sample preparation. Results for fused glass beads were compared to those obtained for samples pressed into pellets, which is the most common sample preparation procedure for analysis by LIBS. The linearity of calibration curves (R2) using different certified reference materials (CRM) pressed into pellets was worse (R2 = 0.2105–0.9276) than that observed for those fused into glass beads (R2 = 0.5640–0.9778), but this still shows a lack of linearity. To minimize strong absorption effects caused by the matrix composition and improve the linearity, accuracy and precision of results for fused glass beads, B and Li, present in the flux, were used as internal standards. Better results were achieved for Fe, Mg and Si when the B emission line was used as an internal standard, while the Li emission line worked better for the measurement of Al, Ca and Ti. The accuracy of the method was evaluated by analyzing a CRM of soil (NIST SRM 2711) and relative errors of around 8% were found, indicating that internal standardization enables quantitative measurement by LIBS in those matrices.