Determination of benzotriazoles in water samples by concurrent derivatization-dispersive liquid-liquid microextraction followed by gas chromatography-mass spectrometry.

Concurrent acetylation-dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC-MS) has been proposed for the sensitive determination of five polar benzotriazolic compounds (1H-benzotriazole, BTri; 4 and 5-methyl-1H-benzotriazole, 4-TTri and 5-TTri; 5,6-dimethyl-1H-benzotriazole, XTri; and 5-chloro-1H-benzotriazole, 5-ClBTri) in water samples. Under optimized conditions, samples (10 mL volume) were combined with 1 mL of Na2HPO4 (8%, w/v) and mixed with the ternary acetylation-microextraction mixture, consisting of 100 µL of acetic anhydride, 1.5 mL of acetonitrile and 60 µL of toluene. Thus, analytes were simultaneously acetylated and transferred to the dispersed droplets of toluene. The proposed methodology achieved limits of quantification (LOQs) between 0.007 ng mL(-1) and 0.080 ng mL(-1), enrichment factors between 93 and 172 times, good reproducibility, with relative standard deviations lower than 10%, and linearity with determination coefficients above 0.9991 for all compounds in the range between LOQs and 20 ng mL(-1). Pseudo-external calibration, with fortified ultrapure water samples submitted to the acetylation-DLLME procedure, proved to be adequate for the accurate quantification of complex aqueous matrices such as surface or wastewater, providing recoveries comprised between 86% and 112%. BTri, 4-TTri and 5-TTri were measured in environmental samples up to a concentration of 1.9 ng mL(-1) for BTri in raw wastewater.

The thiazole derivative CPTH6 impairs autophagy.

We have previously demonstrated that the thiazole derivative 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) induces apoptosis and cell cycle arrest in human leukemia cells. The aim of this study was to evaluate whether CPTH6 is able to affect autophagy. By using several human tumor cell lines with different origins we demonstrated that CPTH6 treatment induced, in a dose-dependent manner, a significant increase in autophagic features, as imaged by electron microscopy, immunoblotting analysis of membrane-bound form of microtubule-associated protein 1 light chain 3 (LC3B-II) levels and by appearance of typical LC3B-II-associated autophagosomal puncta. To gain insights into the molecular mechanisms of elevated markers of autophagy induced by CPTH6 treatment, we silenced the expression of several proteins acting at different steps of autophagy. We found that the effect of CPTH6 on autophagy developed through a noncanonical mechanism that did not require beclin-1-dependent nucleation, but involved Atg-7-mediated elongation of autophagosomal membranes. Strikingly, a combined treatment of CPTH6 with late-stage autophagy inhibitors, such as chloroquine and bafilomycin A1, demonstrates that under basal condition CPTH6 reduces autophagosome turnover through an impairment of their degradation pathway, rather than enhancing autophagosome formation, as confirmed by immunofluorescence experiments. According to these results, CPTH6-induced enhancement of autophagy substrate p62 and NBR1 protein levels confirms a blockage of autophagic cargo degradation. In addition, CPTH6 inhibited autophagosome maturation and compounds having high structural similarities with CPTH6 produced similar effects on the autophagic pathway. Finally, the evidence that CPTH6 treatment decreased α-tubulin acetylation and failed to increase autophagic markers in cells in which acetyltransferase ATAT1 expression was silenced indicates a possible role of α-tubulin acetylation in CPTH6-induced alteration in autophagy. Overall, CPTH6 could be a valuable agent for the treatment of cancer and should be further studied as a possible antineoplastic agent.