Cytochrome P450 Gene Regulation: Reporter Assays to Assess Aryl Hydrocarbon Receptor (HLHE76, AhR) Activation and Antagonism

Abstract

Many xenobiotics, including environmental contaminants such as polycyclic aromatic hydrocarbons, dietary substances such as flavonoids, and therapeutic agents such as omeprazole, have the ability to activate the aryl hydrocarbon receptor (AhR, HLHE76). Identifying agents with this capability has not only been a focus for toxicologists/environmentalists but also for pharmaceutical companies. In the pharmaceutical arena, screening for new molecular entities that activate AhR is applicable for two reasons. First, it can predict drug–drug interactions (DDIs) associated with increased expression of specific drug metabolizing enzymes and transporters. Second, AhR can be an effective target for the development of therapeutic agents, particularly those that antagonize the receptor. Several in vitro techniques can be employed to screen for AhR activation, but some of the most effective are the cell-based transactivation assays. These in vitro assays have several advantages: activation of a species-specific nuclear receptor (NR) can be detected, there is a high degree of reproducibility between experiments, they are cost- and time-efficient, and are amenable to either medium or high throughput screening. The technique described here involves the assessment of human, monkey, rat, murine, and canine AhR activation in species-specific cell lines that express the endogenous receptor. Moreover, methods for identifying antagonists of the AhR are described. The cell lines containing AhR from various species are stably transformed with the reporter luciferase linked to dioxin response elements (DREs) and a promoter from the CYP1A2 gene.