Initial results in the development of a reporter cell line for toxicology studies at gene expression level: Activation of the electrophile-responsive element by copper and methyl parathion

Abstract

Induction of many genes encoding detoxifying enzymes and antioxidant proteins is mediated through a common mechanism, which is controlled by electrophile-responsive elements (EpRE)within the regulatory region of those genes. Copper and methyl parathion are environmental pollutants known to induce the expression of EpRE-mediated genes. In order to evaluate the molecular response triggered by these pollutants, a stable cell line was produced, which carries a transgene comprised of the green fluorescent protein (GFP) reporter gene under transcriptional control of the mouse glutathione-S-transferase (gst1)electrophile-responsive element fused to the mouse metallothionein (mt1) minimal promoter. The rat HTC hepatoma cells were transfected with the EpREmt–GFP construct and successfully selected with G418 antibiotic. EpREmt–GFP HTC cells were treated with 0.002 mg L 1, 0.02 mg L 1, 0.2 mg L 1 and 2mg L 1 copper sulfate and 0.001 mg L 1,0.01 mg L 1, 0.1 mg L 1 and 1 mg L 1 methyl parathion for 48 h. GFP expression was directly quantified in living cells using a microplate fluorimeter. GFP expression was significantly increased in higher concentrations of both pollutants, showing a 1.80- and 2.58-fold induction of GFP at 2 mg copper L 1 and 1 mg methyl parathion L 1, respectively (p < 0.01). The results obtained in the present study demonstrate that the EpREmt–GFP HTC cell line can be an interesting model for further development for the study of the cellular response to aquatic pollutants as well as a new tool for environmental monitoring at the molecular level.