Designing a whole cell bioreporter to show antioxidant activities of agents that work by promotion of the KEAP1–NRF2 signaling pathway

Abstract

The major signaling pathway in human cells is related to the antioxidant defense system. The main component of this system is a transcription factor, Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). It regulates this system in different cellular situations under stimulation by oxidative stress or antioxidants. Thus, detecting the stimulation of NRF2 via a screening strategy may enable us to discover stimulating agents of NRF2-related signaling pathway. With this in mind, we designed a whole cell bioreporter containing the NRF2 response elements that are inserted in a luciferase vector, immediately upstream of a luciferase gene whose promoter has been removed. This bioreporter is activated by stimulators such as 3H-1,2-dithiole-3-thione (D3T), butyl hydroxyanisole (BHA) and ascorbic acid reacting as antioxidant agents. It was observed that the regulatory region of the NRF2 gene, which is identified by NRF2 protein, is located inside its coding region. This designed bioreporter can detect the presence of antioxidant agents. It also exhibits a significant linear correlation over different doses of these agents ranging from 0.8 to 80\u2009μM for ascorbic acid, 0.1 to 100\u2009μM for D3T, and 0.1 to 100\u2009μM for BHA. This detection system is proven to be more sensitive than Real-time PCR, suggesting it to be a highly sensitive system among the available methods.