Assessment of Xenobiotic Biotransformation Including Reactive Oxygen Species Generation in the Embryo Using Benzene as an Example

Abstract

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.