Interests in the determination of different arsenic species in natural waters is caused by the fact that toxic effects of arsenic are connected with its chemical forms and oxidation states. In determinations of water samples inorganic arsenate (As(III), As(V)), methylated metabolities (MMAA, DMAA) and other organic forms such as AsB, AsC, arsenosugars or arsenic containing lipids have the most importance. This article provides information about occurrence of the dominant arsenic forms in various water environments. The main factors controlling arsenic speciation in water are described. The quantification of species is difficult because the concentrations of different forms in water samples are relatively low compared to the detection limits of the available analytical techniques. Several hyphenated methods used in arsenic speciation analysis are described. Specific advantages and disadvantages of methods can define their application for a particular sample analysis. Insufficient selectivity and sensitivity of arsenic speciation methods cause searching for a new or modifications already existing techniques. Some aspects of improvement and modifications of the methods are highlighted.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent toxic isomer in the dioxin-like family. Due to its resistance to metabolic degradation, this ubiquitous environmental pollutant readily accumulates in multiple organs. Cathepsin B is a lysosomal cysteine protease playing an essential role in the intracellular protein turnover. Alterations in its expression, activity, and localization may facilitate the development of many pathologies, including cancer. TCDD, due to its extremely lipophilic nature, may diffuse through biological membranes and affect lysosomal enzymes, including cathepsins. Therefore, in this study we performed two enzymatic assays, spectrofluorimetry and gelatin zymography, in order to evaluate the effect of TCDD on purified bovine cathepsin B. We showed that the dioxin decreases the enzyme's activity in a dose-dependent manner. The reversibility of TCDD-induced inhibition of the protease was also examined, suggesting that TCDD does not bind covalently to the enzyme's active site, acting rather as a reversible inhibitor.
Aim: Matrix metalloproteinases, particularly MMP-2 and MMP-9, are the active factors influencing the structure and properties of the extracellular matrix. This effect is enhanced in metastasis. Hence, it is necessary to enrich the knowledge of the relation between the accumulation and distribution of the enzymes in cells and the intensity of their secretion. Material/Methods: The study was conducted on three cell lines of dermal origin: normal line of human dermal fibroblasts (NHDF) and two melanoma lines (BM and B16F10). The results were obtained with substrate zymography, immunofluorescence microscopy and detergent-complemented fluorimetric assay. Results: All the studied cell lines revealed relatively rich content of MMP-2 (latent and active) with random intracellular localization. Nevertheless, the enzyme secretion was differentiated in various types of cells. The most intensive proMMP-2 secretion was obtained for NHDF, relatively lower for BM, and the lowest for B16F10 line. Zymographic detection of the active form of MMP-2 was restricted to NHDF and BM cell lines. On the other hand, differentiating usage of detergents Brij 35P and Triton X-100 evidenced an active fraction of MMP-2 present in cells of all studied lines. Triton X-100-generated lysis of cells of high MMP-2 secretion (NHDF, BM) revealed the presence of intracellular inhibitors. Conclusions: From the obtained results it can be concluded that the active form of MMP-2 is localized pericellularly in studied cells, being a minor fraction of a total amount of cellular MMP-2. The rest of the enzyme content is located deeper in cell cytoplasm in a latent form. The activity of the pericellular fraction of the enzyme can be measured with detergent-complemented fluorimetric assay, constituting a potentially useful tool for evaluating the enzyme distribution.
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