Mass Spectrometry Imaging Reveals Lipid Changes Associated with the Cell Proliferation of Liver Tumor Spheroids Treated with Triclosan

Triclosan (TCS), an antimicrobial additive, has been widely used in many daily consumer products. Although it is banned from using in soap products in 2016 by the USA Food and Drug Administration, it still can be used in many other products such as cosmetics, toys, mouthwash and toothpaste. Because of its ubiquitous characteristics, there is a growing concern over the impact of TCS on human health. Previous studies have reported that exposure to TCS could promote the development of hepatocellular carcinoma. However, little is known about the related mechanisms at the metabolic levels. In cancer research, multicellular tumor spheroids (MCTSs) are sphere-shaped cellular colonies, and which are considered to be the most appropriate in vitro cell model to test toxicities of chemicals because of their ability to mimic the main characteristics of solid tumors. To the best of our knowledge, no studies have tested the effect of TCS on the development of liver MCTS. Hence, in this work, liver MCTSs were exposed to TCS, and mass spectrometry imaging (MSI) was used to explore the variations of abundance and distribution of lipids in MCTS between exposure and control groups. The results revealed that treatment of 6 μmol/L TCS could significantly promote the growth of liver MCTS and induce the lipid disorders in MCTS. Segmentation analysis of MCTS showed that there were three microregions including the outer proliferative area, the middle quiescent area and the inner necrotic area in MCTS. Further MSI data revealed that the abundance of 27 lipids including 19 glycerophospholipids (GPs), 3 glycerolipids (GLs) and 5 sphingolipids (SPs) significantly changed in MCTS between treated and untreated groups. In GPs, a total of 16 up-regulated lipids located in the outer proliferative area of MCTS. In GLs, two up-regulated lipids located in the outer proliferative area, while one down-regulated lipid distributed in the inner area. In SPs, all five lipids with decreased levels located in the inner area. All these data suggested that the enhanced growth of liver MCTS induced by treatment of TCS may through promoting the growth of cells in the outer region of MCTS and inhibiting the death of cells in the inner region of MCTS. This result may offer a better understanding for the mechanisms of the effects of environmental pollutants on cancer progression

Determination of p-Phenylenediamine Antioxidants in Zebrafish Embryos by Gas Chromatography-Tandem Mass Spectrometry with Ultrasonic Extraction

p-Phenylenediamine antioxidants (PPDs) are rubber antioxidants that can suppress oxidation, heat, or light radiation to delay the aging of polymer components and extend the service life of rubber products. In recent years, PPDs have been found in the environment, and their ecological risks remain largely unknown, which cause widespread concern. In this work, a method of gas chromatography-tandem mass spectrometry (GC-MS/MS) with ultrasonic extraction was developed for the determination of PPDs in zebrafish embryos. In order to have good performance, extraction solvents and sorbents were optimized. Under optimal conditions, embryonic samples were combined with 5 mL of dichloromethane and ultrasonically extracted for 15 min. The extraction procedure was repeated twice, after that the resulting extracts were purified by 0.1 g primary secondary amine (PSA). Based on internal standard method, the analytes were detected using GC-MS/MS in selective reaction monitoring (SRM) mode with optimal parameters. The results showed that the chromatographic peaks of analyte were completely separated, and the baseline was smooth. The correlation coefficient (R2) of each analyte exceeded 0.99 in the concentration range of 0.1-200 μg/L. Method detection limits (MDLs) and method quantification limits (MQLs) were in the range of 0.38-0.68 ng/g and 1.28-2.28 ng/g, respectively. Except for the average recovery of N,N′-bis(methylphenyl)-1,4-benzenediamine (DTPD), which was higher than 120%, the average recoveries of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), N-phenyl-N′-cyclohexyl-p-phenylenediamine (CPPD) and N-isopropyl-N′-phenyl-1,4-phenylenediamine (IPPD) were 73.1%-109%, and the intra-day and inter-day relative standard deviations (RSDs) were 2.97%-15.0% and 4.76%-18.8%, respectively. In order to demonstrate the feasibility of this method, it was applied to detect PPDs in zebrafish embryos exposed to PPDs. The results indicated that PPD concentrations were 12.5-16.4 ng/g. After calculation, the measured bioconcentration factors of each analyte within 48 h were 1.70-33.6 L/kg. These values were much lower than the corresponding predicted bioconcentration factors, because PPD concentrations in vivo had not reached equilibrium during the exposure time of 48 h. This method is simple, sensitive, and suitable for the detection of PPDs in zebrafish embryos, which facilitates future bioaccumulation and toxicity mechanism studies for a comprehensive assessment of the ecological risks of PPDs