Renaud Helen J, Rutter Allison, Winn Louise M
Department of Pharmacology and Toxicology, Queen's University, Kingston, ON, Canada.
Methods Mol Biol. 2012;889:253-63. doi: 10.1007/978-1-61779-867-2_15.
Quantification of embryonic metabolic capacity is an important tool in developmental toxicology research. Bioactivation of xenobiotics into reactive intermediates often contributes to embryo toxicity; thus, identification and quantification of these toxic metabolites is essential to gain further understanding of developmental toxicity. This chapter uses the environmental chemical benzene as a model xenobiotic to describe the detection of both metabolites and reactive oxygen species (ROS) in fetal liver. Briefly, mice are bred and the presence of a vaginal plug in a female mouse indicates gestational day 1. On the desired gestational day, pregnant dams are exposed to benzene followed by sacrifice at the desired time-point after exposure. Using gas chromatography coupled to mass spectrometry, the detection of benzene metabolites can be achieved. Additionally, we describe the measurement of ROS by flow cytometry using the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein diacetate, which readily diffuses into cells and, upon oxidation by any ROS, is converted to the highly fluorescent, negatively charged carboxydichlorofluorescein, which remains trapped within the cells.
胚胎代谢能力的量化是发育毒理学研究中的一项重要工具。外源性物质生物活化形成反应性中间体往往会导致胚胎毒性；因此，识别和量化这些有毒代谢物对于进一步了解发育毒性至关重要。本章以环境化学物质苯作为典型外源性物质，描述胎儿肝脏中代谢物和活性氧（ROS）的检测。简而言之，饲养小鼠，雌性小鼠出现阴道栓表明妊娠第1天。在期望的妊娠日，将怀孕的母鼠暴露于苯，然后在暴露后的期望时间点处牺牲。使用气相色谱-质谱联用技术，可以实现苯代谢物的检测。此外，我们描述了使用荧光探针5-（及-6）-氯甲基-2'，7'-二氯荧光素二乙酸酯通过流式细胞术测量ROS，该探针易于扩散到细胞中，并且在被任何ROS氧化后，会转化为高荧光、带负电荷的羧基二氯荧光素，其会被困在细胞内。
