Cyp2a5 Promoter‐based Gene Reporter Assay: A Novel Design of Cell‐based Bioassay for Toxicity Prediction

Abstract

The murine cytochrome P450 2a5 (Cyp2a5) gene is regulated by complex interactions of various stress-activated transcription factors (TFs). Elevated Cyp2a5 transcription under chemical-induced stress conditions is achieved by interplay between the various TFs - including as aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 wild-type (Nrf2) - at the 'stress-responding' cluster of response elements on the Cyp2a5 promoter, as well as through mRNA stabilization mediated by interaction of the stress-activated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with the 3'-UTR of the CYP2A5 mRNA. We designed a unique toxicity pathway-based reporter assay to include regulatory regions from both the 5' and the 3' untranslated regions of Cyp2a5 in a luciferase reporter plasmid to reflect in vivo responses to chemical insult. Human breast cancer MCF-7 cells were stably transfected with pGL4.38-Cyp2a5_Wt3k (wild-type) or mutant - pGL4.38-Cyp2a5_StREMut and pGL4.38-Cyp2a5_XREMut - reporter gene to monitor chemical-induced cellular response mediated by AhR and Nrf2 signalling. The recombinant cells were treated with representative of AhR agonist, polycyclic aromatic hydrocarbons, brominated flame retardant, fluorosurfactant, aromatic organic compound and metal, to determine the sensitivity of the Cyp2a5 promoter-based gene reporter assays to chemical insults by measuring the LC and EC of the respective chemicals. The three assays are sensitive to sublethal cellular responses of chemicals, which is an ideal feature for toxicity pathway-based bioassay for toxicity prediction. The wild-type reporter responded well to chemicals that activate crosstalk between the AhR and Nrf2, whilst the mutant reporters effectively gauge cellular response driven by either Nrf2/StRE or AhR/XRE signalling. Thus, the three gene reporter assays could be used tandemly to determine the predominant toxicity pathway of a given compound.

Toxicology Meets Pharmacodynamics and Pharmacokinetics – New Concepts, Models and In Vitro Approaches and Tools

“…EU‐ToxRisk financed by Horizon2020 is a large project aiming at regulatory relevant models for some end‐points to assess toxicity in liver, kidneys, lungs and intestine . Gene reporter‐based in vitro cellular tests are sensitive to detect toxicity pathways and especially useful to detect predominant toxicity mechanisms of mixtures . The three gene reporter assays developed monitor chemical‐induced cellular response mediated by AhR and Nef2 signalling .…”

“…Gene reporter‐based in vitro cellular tests are sensitive to detect toxicity pathways and especially useful to detect predominant toxicity mechanisms of mixtures . The three gene reporter assays developed monitor chemical‐induced cellular response mediated by AhR and Nef2 signalling .…”