Development of an Assay to Detect Toxicity Using a Fluorescent Probe as an Indicator of Metabolic Activation by the Enzyme Cytochrome P450

Abstract

Due to its protective role in the body, the liver has been credited with the responsibility of detoxification of ingested foreign compounds. Although, the demonstration of the liver as a protective agent has been tempered by the possibility that some detoxifying processes may yield harmful products. For this reason, it would be beneficial to experimentally expose drug candidates with the potential to cause hepatic injury as the result of metabolic idiosyncrasy. Therefore, the ability to test whether or not a drug will form a dangerous intermediate during biotransformation in the liver is necessary information early on in the drug development process. The pivotal component of phase I, or the toxifying phase of the biotransformation process is the enzyme cytochrome P450. During phase I reactions, multiple forms of cytochrome P450 may increase or decrease the systemic toxicity of the compound while at the same time causing hepatic injury due to the oxidative reactions of phase I yielding reactive, hepatotoxic metabolic products. The danger of such reactive metabolic products lies in the formation of an electrophilic intermediate via sulfation during phase II metabolism. Any compound containing an electrophilic center post phase II reactions is capable of reacting with electron-rich sites (i.e. nucleophilic centers) of DNA, RNA, or other proteins of the cell. Numerous in vitro strategies have been developed in an attempt to further explore the role cytochromes P450 play in the activation of xenobiotics. This study focuses on the role conjugation with glutathione plays in detoxifying reactive intermediates produced by the P450 enzyme. Because several toxic chemicals undergo glutathione conjugation during metabolism, in many cases it would be advantageous to monitor glutathione levels during that process. Therefore an assay to test the likelihood of a drug forming a reactive intermediate was developed based on glutathione conjugation with the products of phase I reactions. In the case of this study, we wanted to develop an assay to detect the formation of a reactive intermediate by utilizing the ability of compound X to selectively bind with GSH. Toward this end, we used induced rat microsomes containing an increased concentration of cytochrome P450 enzymes. Therefore if a new drug characteristically forms a reactive intermediate upon reaction with cytochrome P450 present in the induced microsomes, the yielded product will then react with any GSH present. GSH conjugation with any reactive intermediate produced renders it incapable of reacting with compound X. Compound X left unbound exhibits no fluorescence due to intramolecular quenching by its chlorine group. Hence, in the absence of a reactive intermediate, GSH supplied to the system readily reacts with compound X to produce a conjugate that fluoresces at 475 nm when excited around 406 nm. A reduction in the fluorescent signal is expected for those compounds producing a harmful intermediate whereas a retained signal is expected for “safer” compounds