ALPHA,BETA-UNSATURATED ALDEHYDES INCREASE GLUTATHIONE-S-TRANSFERASE MESSENGER-RNA AND PROTEIN - CORRELATION WITH ACTIVATION OF THE ANTIOXIDANT RESPONSE ELEMENT

A series of alpha,beta-unsaturated aldehydes was evaluated to determin e if these compounds could mediate inducible expression of glutathione S-transferase (GST) through the 5'-flanking antioxidant response elem ent (ARE). The ARE from rGST A1 was subcloned into a luciferase report er construct and used to transiently transfect rat Clone 9 hepatoma ce lls. Transfected cells were treated with 4-hydroxy-trans-2-nonenal (4- HNE), trans-2-hexenal (t-2-HE), 2-propenal (acrolein, 2-PE), and ethac rynic acid (EA), a control compound also containing an alpha,beta-unsa turated carbonyl moiety. Each compound was evaluated for cytotoxicity to construct dosing regimens in transfection studies. IC507 values for growth inhibition were measured using [4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide. IC50 values in Clone 9 cells were: 4-HNE, 6.3 +/- 0.7 mu M; t-2-HE, 16.0 +/- 0.7 mu M; 2-PE, 2.2 +/- 0.4 mu M; and EA, 38.0 +/- 1.6 mu M. A dose-dependent increase in luciferase act ivity was observed in transfected cells with all four compounds tested , indicating that alpha,beta-unsaturated aldehydes function as direct activators of the ARE, To determine whether or not the observed promot er activation led to increased transcriptional and translational induc tion of GST, cells were treated with the various compounds and assayed for increases in GST mRNA, protein, and enzyme activity. Studies in C lone 9 cells revealed increased steady-state message for GST A1 and A4 , increased GST A4-4 protein by Western blotting, and increased GST ac tivity toward 1-chloro-2,4-dinitrobenzene in response to treatment wit h all four compounds evaluated, Collectively, these studies demonstrat e that EA and certain alpha,beta-unsaturated aldehydes produced as a r esult of cellular membrane lipid peroxidation are activators of the AR E and efficient inducers of GST A1-1 and A4-4, (C) 1998 Academic Press .