ABSTRACT
@#In this paper, chemical derivatization-high performance liquid chromatography was used to determine the potential genotoxic impurities chloroacetyl chloride and chloroacetic acid, respectively, in the raw material of azintamide.Derivatization was carried out using 2-nitrophenylhydrazine followed by the determination.Separation was performed on a Thermo Syncronis C18 column (250 mm × 4.6 mm, 5 μm), with mobile phase consisting of 0.1% phosphoric acid in water (A) and acetonitrile(B) by gradient elution, at a flow rate of 1 mL/min.The column temperature was 40 °C and the detection wavelength was 226 nm.The blank solvent, derivatization reagent, and azintamide did not interfere with the peak of the test substance, and the target component was well separated from the others.For impurities chloroacetyl chloride and chloroacetic acid, the limits of detection (LOD) were 7.5 ng/mL and 15 ng/mL respectively. There was a good linear relationship between the integral area and the concentration in the range of 30-300 ng/mL.The sample recovery rate was in the range of 87.37% ~ 109.75%.The two methods established in this study have good specificity, good precision, high sensitivity and simple operation, which can be used for the trace determination of potential genotoxic impurities chloroacetyl chloride and chloroacetic acid in the raw material of azintamide.
ABSTRACT
Resumen Pocos son los trabajos enfocados en la producción biotecnológica y el desarrollo de herramientas analíticas en torno a la Lentinuia edodes, seta comestible con potencial para el desarrollo de nutracéuticos. Por esto, en esta investigación, se estudió la producción de biomasa del hongo en el tiempo, mediante fermentación en estado líquido, y se seleccionaron las condiciones que permitieron la obtención de extractos para la aplicación de herramientas para análisis proteómico. Los métodos de extracción de proteínas, ácido tricloroacético (TCA) - acetona y TCA - acetona - fenol, fueron comparados en términos del rendimiento de extracción y los perfiles de separación usando electroforesis en 1D (SDS-PAGE) y 2D (IEF-SDS PAGE). Se determinó que a los 10 días de crecimiento se obtiene la mayor producción de biomasa y proteína total. La extracción con TCA - acetona - fenol presentó un mayor rendimiento, resolución y número de bandas en la electroforesis 1D. En 2DE, los dos métodos permitieron la extracción de proteínas con puntos isoeléctricos en el rango de pH 3-10, pero el método TCA - acetona - fenol conllevó a una extracción diferencial, favoreciendo el rango de masa de 33 a 113 kDa. Estos resultados se constituyen en una primera aplicación de técnicas de separación electroforética para futuros estudios proteómicos.
Abstract Few are the investigations focused on the biotechnological production and the development of analytical tools about Lentinuia edodes, an edible mushroom which has a potential for being used in the development of nutraceutical products. For this reason, in this research, the production of biomass of the mushroom over time by liquid state fermentation (LSF) was studied. Then, the conditions that allow obtaining protein extracts for the application of tools for proteomic analysis were selected. Trichloroacetic acid (TCA) - acetone and TCA - acetone - phenol were the two protein extraction methods which were compared in terms of extraction yield and separation profiles in 1D (SDS-PAGE) and 2D (IEF-SDS PAGE) electrophoresis. The highest production of biomass and total protein content was obtained after 10 days of LSF. Protein extraction with TCA - acetone -phenol presented the highest yield, resolution and number of bands in 1D electrophoresis. In 2DE the two methods allowed the extraction of proteins with isoelectric points in pH 3-10 range but, the TCA - acetone - phenol method favored a differential extraction of proteins in the range of 33 to 113 kDa. These results constitute a first application of electrophoretic separation techniques for future proteomic studies.
Resumo Lentinuia edodes é um cogumelo comestível com potencial para o desenvolvimento de nutracêuticos. Entretanto, os trabalhos voltados para a produção biotecnológica e o desenvolvimento de ferramentas analíticas que permitem aprofundar sua composição são incipientes. Nesta pesquisa, a produção de biomassa do fungo ao longo do tempo por meio de fermentação no estado líquido foi estudada e as condições que permitem obter extratos para a aplicação de ferramentas para análise proteômica foram selecionadas. Os métodos de extração de proteínas usados foram ácido tricloroacético (TCA) - acetona e TCA - acetona - fenol e comparada em termos de rendimento de extracção e perfis de separação utilizando electroforese 1D (SDS-PAGE) e 2D (IEF-SDS PAGE). Foi determinado que após 10 dias de crescimento, a maior produção de biomassa e proteína total foi obtida. A extração com TCA - acetona - fenol apresentou maior rendimento, maior resolução e número de bandas em eletroforese 1D. No 2DE os dois métodos permitiram a extração de proteínas com pontos isoelétricos na faixa de pH 3-10, mas o método TCA - acetona - fenol levou a uma extração diferencial, favorecendo a faixa de 33 a 113 kDa. Estes resultados constituem uma primeira aplicação de técnicas de separação eletroforética para futuros estudos proteômicos.
ABSTRACT
Objective@#The aim of this study was to investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells.@*Methods@#16HBE cells were exposed to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mmol/L chloroacetic acid for 24 h in vitro. The cytotoxicity induced by chloroacetic acid was assessed by CCK-8 and LDH assays. Cell apoptosis was detected by Annexin V-FITC and PI staining. The protein expression levels of phosphorylation of p38, ERK1/2 and JNK were determined by western blotting. 16HBE cells were pretreated with MAPK signaling pathway specific inhibitors including SB203580, U0126 and SP600125 for 1 h, and these cells were subsequently treated with 2.5 mmol/L chloroacetic acid for 24 h. The expressions of p-p38, p-ERK1/2 and p-JNK as well as the changes of cell viability and apoptosis were measured after pretreated with inhibitors for 1 h.@*Results@#The cell viability by CCK-8 and LDH methods gradually reduced in a dose-dependent manner when chloroacetic acid concentrations elevated (P<0.05) , and their correlation coefficients were -0.902 and -0.825, respectively. The detection efficiency of CCK-8 assay significantly increased compared with LDH assay (P<0.05) . The cell apoptosis rates, which were (17.2±4.0) %, (24.6± 4.2) %, (39.3 ± 5.7) % in 1.5, 2.0, 2.5 mmol/L chloroacetic acid-treated groups, were higher than that of the control group[ (5.6 ± 3.0) %] (P<0.05) . There was a time-or dose-dependent change in the protein expressions of p-p38, p-ERK1/2 and p-JNK. Compared with the control, the levels of p-p38 had 2.1 and 2.6-fold increases in 16 and 24 h treated groups (P<0.01) , while the levels of p-ERK1/2 distinctly decreased by 37% and 52% (P<0.01) . In comparison with the control group, the expressions of p-p38 had 1.9 and 2.6-fold increases in 1.5 and 2.5 mmol/L treatment groups (P<0.01) , whereas the expressions of p-ERK1/2 significantly decreased by 40% and 50% (P<0.01) . No significant change was observed in p-JNK protein expression between the chloroacetic acid-treated and control groups. In comparison with the vehicle control and the exposed group, p-p38, p-ERK1/2, p-JNK protein expressions significantly declined in the inhibitor controls and inhibitor groups. Compared with the controls, the cell survival rates had significant reductions of 28%, 18%, 36% and 26% respectively in chloroacetic acid treated group, SB203580 group, U0126 group and SP600125 group, and the apoptosis rates in the abovementioned groups were 7, 4, 8 and 7 times. Compared with chloroacetic acid-treated group, the cell viability increased by 14% in SB203580 group and decreased by 11% in U0126 group, and the cell apoptosis rates decreased by 36% in SB203580 group and increased by 18% in U0126 group (P<0.05) . But no significant changes were observed in cell viability and apoptosis between SP600125 and chloroacetic acid-treated group.@*Conclusion@#Chloroacetic acid might activate p38 MAPK signaling pathway and inhibit ERK1/2 MAPK signaling pathway. The signaling pathways of p38 and ERK1/2 MAPK are involved in 16HBE cell apoptosis induced by chloroacetic acid, but JNK is not involved in chloroacetic acid-induced 16HBE cell apoptosis.
ABSTRACT
OBJECTIVE: To explore the toxicity of 1,2-dichloroethane( 1,2-DCE) and its metabolites on human astrocytes( HAs). METHODS: Different doses of 1,2-DCE( 5. 00,10. 00,25. 00,50. 00 and 100. 00 mmol/L),2-chlorohydrins( 5. 00,25. 00,50. 00,100. 00 and 200. 00 mmol/L),2-chloroacetaldehyde( 1. 00,5. 00,10. 00,20. 00 and 50. 00 mmol / L) and chloroacetic acid( 0. 01,0. 05,0. 10,0. 50 and 1. 00 mmol / L) were used for treating HAs in vitro during their logarithmic phase. After 24 hours of culture,the morphology of HAs was observed by fluorescent inverted phase contrast microscope. The survival rate and the inhibition ratio of HAs were detected by CCK-8 colorimetry to estimate the50% inhibiting concentration in 24 hours( 24 h-IC50). The apoptosis of HAs was tested by double-labeling and flow cytometry using Annexin Ⅴ-fluorescein isothiocyanate and propidium iodide. RESULTS: The morphology of HAs changed in varying degrees after 24 hours exposure to 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid. The changes included smaller size of cells,pseudopodia tapering,increased intracellular particles and suspension of circular cells and decreased transparency of cells. With the increasing does of 1,2-DCE,2-chlorohydrins,2-chloroacetaldehyde and chloroacetic acid exposure,the survival rates of HAs decreased( P < 0. 01),while its inhibition ratios increased( P <0. 01). They all showed dose-effect relationship. 24 h-IC50 of the above 4 chemicals were 56. 25,235. 00,26. 43 and1. 38 mmol / L,respectively. The 1,2-DCE,2-chlorohydrins and chloroacetic acid could induce the apoptosis of HAs and the apoptosis rate of HAs was positively correlated with the 3 kinds of chemicals( P < 0. 01). CONCLUSION: 1,2-DCE and its metabolites 2-chloroacetaldehyde,2-chlorohydrins and chloroacetic acid can lead to toxic damage and induce the apoptosis of HAs. Chloroacetic acid has the strongest toxicity among the metabolites.