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.

CWAF（COREXIT水相级分，COREXIT water-accommodated fraction）采用硅胶固相萃取柱（Silica cartridges）进行分级分离，共收集得到四个级分：体积比50:50的水-乙醇混合液、甲醇、二氯甲烷及正己烷。随后各级分经氮吹浓缩至干后，重悬于二甲基亚砜（dimethyl sulfoxide，DMSO）中，用于配体结合实验。将HEK293T细胞以三重转染方式，转染过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma，PPARγ）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。随后将细胞分别暴露于阳性对照配体罗格列酮（Rosiglitazone）、阴性对照配体二甲基亚砜，以及系列稀释的琥珀酸二辛酯磺酸钠（dioctyl sodium sulfosuccinate，DOSS）、ICP:PG:DOSS混合液、ICP:PG混合液与司盘80（Span80）处理体系中。实验结果显示，仅在含DOSS的处理组中观测到PPARγ结合结构域的激活，且呈现典型剂量反应曲线，表明DOSS具备致肥胖潜能。