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.
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 2 mg 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 2mg copper L(-1) and 1mg 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.
Programa de Pós-graduação em Ciências Fisiológicas, Fisiologia Animal Comparada, Fundação Universidade Federal do Rio Grande, CP 474, Rio Grande, RS, Brazil.
Daniela Volcan
DV
Almeida
Gilma Santos
GS
Trindade
Laura A
LA
Geracitano
Daniela Martí
DM
Barros
José Maria
JM
Monserrat
Luis Fernando
LF
Marins
D000818:Animals; D045744:Cell Line, Tumor; D003300:Copper / Q000633:toxicity*; D054750:Ecotoxicology / Q000379:methods*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008743:Methyl Parathion / Q000633:toxicity*; D051379:Mice; D051381:Rats; D015203:Reproducibility of Results; D018675:Toxicity Tests / Q000379:methods*; D019076:Transgenes / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000633:toxicity*
D014874:Water Pollutants, Chemical; D049452:Green Fluorescent Proteins; D008743:Methyl Parathion; D003300:Copper