Investigation of occurrence of aromatic amines in municipal wastewaters using passive sampling.

Aromatic amines (AAs) are human-made compounds known for their mutagenic properties, entering surface waters from various sources, often originating as transformation products of dyes or pesticides. Despite their low concentrations in surface waters, AAs can exhibit mutagenicity. Our study focused on evaluating three passive samplers (PSs) for enriching these compounds from influent and effluent of a wastewater treatment plant (WWTP) in Brno, Czech Republic. The PSs tested included variants containing AttractSPE™ SDB-RPS sorbent disk, one with and one without a diffusive agarose hydrogel layer, and a modified Speedisk (Bakerbond Speedisk® H2O-Philic). PSs were deployed in wastewater (WW) for one to four weeks in various overlapping combinations, and the uptake of AAs to PSs was compared to their concentrations in 24-hour composite water samples. A targeted LC/MS analysis covered 42 amines, detecting 11 and 13 AAs in daily composite influent and effluent samples, respectively. In the influent, AAs ranged from 1.5 ng L-1 for 1-anilinonaphthalene to 1.0 µg L-1 for aniline, and the highest concentration among all measured amines was observed for cyclohexylamine at 2.9 µg L-1. In the effluent, concentrations ranged from 0.5 ng L-1 for 1-anilinonaphthalene to 88 ng L-1 for o-anisidine. PSs demonstrated comparable accumulation of amines, with integrative uptake up to 28 days in both influent and effluent and detection of up to 23 and 27 amines in influent and effluent, respectively; altogether 34 compounds were detected in the study. Sampling rates (Rs) were estimated for compounds present in at least 50 % of the samples and showing <40 % aqueous concentration variability, with robustness evaluated by comparing values for compounds in WWTP influent and effluent. Although all devices performed similarly, hydrogel-based PS exhibited superior performance in several criteria, including time integration and robustness of sampling rates, making it a suitable monitoring tool for AAs in WW.

Factors affecting diffusion of polar organic compounds in agarose hydrogel applied to control mass transfer in passive samplers.

Diffusive hydrogel-based passive sampler (HPS) based on diffusive gradients in thin films (DGT) is designed for monitoring polar organic contaminants in the aquatic environment. DGT technique controls the compound's overall uptake rate by adding a hydrogel layer of known thickness, which minimizes the importance of the resistive water boundary layer in the compound uptake process. In this work, we investigated several factors which may influence the diffusion of a range of aquatic contaminants in 1.5% agarose hydrogel. Diffusion in hydrogel was tested using the sheet stacking method. We demonstrated that a thin nylon netting incorporated into the diffusive hydrogel for mechanical strengthening does not significantly affect the diffusion of 11 perfluoroalkyl compounds. Further, we investigated the effect of pH in the range from 3 to 11 on the diffusion of a range of 39 aromatic amines (AAs) -36 aromatic, 2 aliphatic, and azobenzene in hydrogel. AAs were chosen as representatives of compounds with pH-dependent dissociation in water. Analysis of variance showed no significant difference in mean diffusion coefficient log D value at five pH values. The demonstration that the diffusion coefficient D and thus the sampling rate Rs are independent on pH simplifies the interpretation of data from field studies because we can neglect the influence of pH on the Rs. log D values (m2 s-1) of tested AAs ranged from to - 9.77 for 3,3'-dimethylbenzidine to - 9.19 for azobenzene. A negative correlation of log D with molar mass (log M) and molecular volume (log Vm) was observed (R = - 0.57 and - 0.56, respectively). The diffusion coefficient presents a critical parameter for the sampling rate estimation of HPS. Theoretical sampling rates Rs of AAs were calculated for a HPS using the average D values. Theoretical Rs values calculated for AAs at 22°C ranged from 29 mL day-1 for 3,3'-dimethylbenzidine to 106 mL day-1 for 2-aminopyridine. Our calculated values of Rs are in the same range as those already published for a range of low-molecular polar organic contaminants, which supports the possibility of deriving sampler performance parameters in the field from laboratory-derived diffusivity of analytes in hydrogel.

Infrared Laser Desorption of Intact Nanoparticles for Digital Tissue Imaging.

We report a new technique for the digital mapping of biomarkers in tissues based on desorption and counting intact gold nanoparticle (Au NP) tags using infrared laser ablation single-particle inductively coupled plasma mass spectrometry (IR LA SP ICP MS). In contrast to conventional UV laser ablation, Au NPs are not disintegrated during the desorption process due to their low absorption at 2940 nm. A mass spectrometer detects up to 83% of Au NPs. The technique is demonstrated on mapping a proliferation marker, nuclear protein Ki-67, in three-dimensional (3D) aggregates of colorectal carcinoma cells, and the results are compared with confocal fluorescence microscopy and UV LA ICP MS. Precise counting of 20 nm Au NPs with a single-particle detection limit in each pixel by the new approach generates sharp distribution maps of a specific biomarker in the tissue. Advantageously, the desorption of Au NPs from regions outside the tissue is strongly suppressed. The developed methodology promises multiplex mapping of low-abundant biomarkers in numerous biological and medical applications using multielemental mass spectrometers.

Disulfiram increases the efficacy of 5-fluorouracil in organotypic cultures of colorectal carcinoma.

Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.

Anti-cancer effects of wedelolactone: interactions with copper and subcellular localization.

Wedelactone (WL), a plant polyphenolic derivative of coumestan, represents a promising anti-cancer agent. The underlying mechanisms of its action are not fully understood and appear to involve interplay with copper ions. Herein, we examined coordination and redox interactions of WL with Cu2+ in phosphate buffer (pH 7), and in two breast cancer cell lines. EPR, UV-Vis and fluorescence spectroscopy showed that WL and Cu2+ build a coordination complex with 2 : 1 stoichiometry and distorted tetrahedral geometry. WL showed strong fluorescence that was quenched by Cu2+. The sequestration of the intracellular copper pool with neocuproine led to a significant drop in the cytotoxic effects of WL, whereas the co-application of Cu2+ and WL and the formation of an extracellular complex suppressed both the cytotoxic effects of WL and copper loading. Fluorescence microscopy showed that WL is mainly localized in the cytosol and significantly less in the nuclei. WL fluorescence was stronger in cells pretreated with neocuproine, implying that the complex of WL and Cu2+ is formed inside the cells. WL caused a two-fold increase in the lysosomal level of copper as well as copper-dependent lysosome membrane permeabilization. On the other hand, the protective effects of overexpression of thioredoxin 1 imply that WL exerts the main oxidative impact inside the nucleus. The interactions of WL with copper may be essential for therapeutic performance and selectivity against cancer cells, taking into account that a number of cancer types, including breast cancer, exhibit increased intratumoral copper levels or altered copper distribution.

Wedelolactone Acts as Proteasome Inhibitor in Breast Cancer Cells.

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells.