Effects of CWAF chemically enhanced water accommodated sub-fractions on peroxisome proliferator-activated receptor PPAR gamma binding.

CWAF (COREXIT water-accommodated fraction) was fractionated using solid phase extraction using Silica cartridges. Four fractions were collected: 50:50 water:ethanol, methanol, dichloromethane and hexane. Fractions were then dried down and resuspended in DMSO (dimethyl sulfoxide) to be used for ligand binding assays. HEK293T cells were triple transfected with PPARγ (peroxisome proliferator-activated receptor gamma) ligand binding domain expression vectors, firefly luciferase reporter vectors and renilla luciferase control vectors. Cells were then exposed to positive and negative control ligands (Rosiglitazone and DMSO respectively) and serial dilutions of DOSS (dioctyl sodium sulfosuccinate), ICP (inductively coupled plasma):PG (propylene glycol):DOSS, ICP:PG and Span80 treatments. Activation of the PPARγ binding domain was observed in DOSS containing treatments only, exhibiting a dose response curve indicating obesogenic potential DOSS.采用硅胶柱（Silica cartridges）固相萃取对CWAF（COREXIT水相分级物，COREXIT water-accommodated fraction）进行分级分离，共收集4组洗脱组分：体积比50:50的水-乙醇混合液、甲醇、二氯甲烷及正己烷。所得组分经蒸干处理后，以二甲基亚砜（DMSO, dimethyl sulfoxide）重悬，用于配体结合实验。将HEK293T细胞采用三重转染策略，转染PPARγ（过氧化物酶体增殖物激活受体γ，peroxisome proliferator-activated receptor gamma）配体结合结构域表达载体、萤火虫荧光素酶报告载体及海肾荧光素酶对照载体。随后向细胞施加阳性与阴性对照配体（分别为罗格列酮Rosiglitazone与DMSO），并设置系列稀释的DOSS（二辛基琥珀酸磺酸钠，dioctyl sodium sulfosuccinate）、ICP（电感耦合等离子体，inductively coupled plasma）-PG（丙二醇，propylene glycol）-DOSS、ICP-PG及司盘80（Span80）处理组。实验结果显示，仅在含DOSS的处理组中可观测到PPARγ配体结合结构域的激活，且该激活呈现剂量反应曲线，表明DOSS具有致肥胖潜能。