Stimulation of troutCYP1A gene expression in mouse HEPA-1 cells by 3-methylcholanthrene

Abstract

TroutCYP1A-CAT expression construct was generated by cloning approximately 3.5 Kb 5' flanking DNA of trout liverCYP1A gene in front of CAT gene at pCAT-basic vector. Hepa 1 cells, which are known to contain a functional arylhydrocarbon receptor' were transfected with troutCYP1A-CAT using lipofectin. 3-Methylcholanthrene (1 nM) was added into hepa 1 cells in culture in order to examine if 5' flanking DNA of troutCYP1A gene could interact with mouse transactivating factors to bring about transcription of the chloramphenicol acetyltransferase(CAT) reporter gene. The level of CAT protein was measured by CAT ELISA and the level of CAT mRNA was determined by RTPCR. The treatment of 1 nM 3-methylcholanthrene resulted in two fold increases in CAT protein as well as CAT mRNA compared to untreated control hepa 1 cells. These data indicate that arylhydrocarbon receptors of mouse hepa 1 cells are functional to activate exogenously transfected troutCYP1A-CAT construct in terms of both transcription and translation of CAT. We also examined the effect of 3-methylcholanthrene on endogeneouscyp1a1 activity in hepa 1 cell. 3-Methylcholanthrene (1 nM) treatment to hepa 1 cells transfected with troutCYP1A-CAT construct stimulated the level of cyp1a1 mRNA by two folds and the activity of ethoxyresorufin-O-deethylase by two fold compared to that of control cells. In this study we reported that troutCYP1A-CAT reporter gene expression construct could be expressed by 3-methylcholanthrene treatment in mouse hepa 1 cells. Thus troutCYP1A-CAT could serve as a good model to study the mechanism of regulation ofCYP1A1 gene expression.