Violet Daniel

Transcription factors AP-1 and NF-κB have been implicated in the inducible expression of a variety of genes in response to oxidative stress. Recently, based on the observation that butylated hydroxyanisole (BHA) and pyrrolidine dithiocarbamate (PDTC) induce AP-1 binding activity and AP-1-dependent gene expression and assuming that these compounds exert an antioxidant effect, it was claimed that AP-1 is an antioxidant-responsive factor. To determine whether AP-1 can be responsive to both oxidant and antioxidant, we examined the nature of BHA and PDTC inducing activity. Using EPR spectroscopy to detect semiquinone radicals, we demonstrate the autoxidation of BHA metabolite tert-butylhydroquinone (TBHQ) to tert-butylquinone. The kinetics of TBHQ-mediated generation of ·OH radicals were monitored in intact hepatoma HepG2 cells by EPR spin trapping technique. Exogenous catalase inhibited the rate and amount of ·OH radical formation and the induction of AP-1-mediated glutathione S-transferase (GST) Ya gene expression by BHA and TBHQ, thus indicating the intermediate formation of H2O2 in the metabolism of these chemicals. Furthermore, we show that the induction of AP-1 and NF-κB activities and GST Ya gene expression by BHA and TBHQ is due to a pro-oxidant activity, since this induction was inhibited by thiol compounds N-acetyl cysteine and GSH. Similarly, induction of AP-1 and GST Ya gene expression by PDTC was inhibited by N-acetyl cysteine and GSH. The present findings do not support the notion that the induction of AP-1 by BHA, TBHQ, or PDTC is an antioxidant response and demonstrate that both AP-1 and NF-κB activities are induced by oxygen radicals. Transcription factors AP-1 and NF-κB have been implicated in the inducible expression of a variety of genes in response to oxidative stress. Recently, based on the observation that butylated hydroxyanisole (BHA) and pyrrolidine dithiocarbamate (PDTC) induce AP-1 binding activity and AP-1-dependent gene expression and assuming that these compounds exert an antioxidant effect, it was claimed that AP-1 is an antioxidant-responsive factor. To determine whether AP-1 can be responsive to both oxidant and antioxidant, we examined the nature of BHA and PDTC inducing activity. Using EPR spectroscopy to detect semiquinone radicals, we demonstrate the autoxidation of BHA metabolite tert-butylhydroquinone (TBHQ) to tert-butylquinone. The kinetics of TBHQ-mediated generation of ·OH radicals were monitored in intact hepatoma HepG2 cells by EPR spin trapping technique. Exogenous catalase inhibited the rate and amount of ·OH radical formation and the induction of AP-1-mediated glutathione S-transferase (GST) Ya gene expression by BHA and TBHQ, thus indicating the intermediate formation of H2O2 in the metabolism of these chemicals. Furthermore, we show that the induction of AP-1 and NF-κB activities and GST Ya gene expression by BHA and TBHQ is due to a pro-oxidant activity, since this induction was inhibited by thiol compounds N-acetyl cysteine and GSH. Similarly, induction of AP-1 and GST Ya gene expression by PDTC was inhibited by N-acetyl cysteine and GSH. The present findings do not support the notion that the induction of AP-1 by BHA, TBHQ, or PDTC is an antioxidant response and demonstrate that both AP-1 and NF-κB activities are induced by oxygen radicals.

The current knowledge about the structure of GST genes and the molecular mechanisms involved in regulation of their expression are reviewed. Information derived from the study of rat and mouse GST Alpha-class, Ya genes, and a rat GST Pi-class gene seems to indicate that a single cis-regulatory element, composed of two adjacent AP-1-like binding sites in the 5'-flanking region of these GST genes, is responsible for their basal and xenobiotic-inducible activity. The identification of Fos/Jun (AP-1) complex as the trans-acting factor that binds to this element and mediates the basal and inducible expression of GST genes offers a basis for an understanding of the molecular processes involved in GST regulation. The induction of expression of Fos and Jun transcriptional regulatory proteins by a variety of extracellular stimuli is known to mediate the activation of target genes via the AP-1 binding sites. The modulation of the AP-1 activity may account for the changes induced by growth factors, hormones, chemical carcinogens, transforming oncogenes, and cellular stress-inducing agents in the pattern of GST expression. Recent observations implying reactive oxygen as the transduction signal that mediates activation of c-fos and c-jun genes are presently considered to provide an explanation for the induction of GST gene expression by chemical agents of diverse structure. The possibility that these agents may all induce conditions of oxidative stress by various pathways to activate expression of GST genes that are regulated by the AP-1 complex is discussed.

Cultured mouse lymphosarcoma cells are killed on exposure to 0.1 mM N(6),O(2')-dibutyryl-adenosine 3':5'-cyclic monophosphate. A population of cells resistant to the killing effect of dibutyryl cyclic AMP at concentrations as high as 1 mM was selected. The growth characteristics of the resistant cells were similar to those of the sensitive parental line. However, the resistant cells contain less cytoplasmic cyclic AMP-binding proteins and decreased cyclic AMP-stimulated protein kinase activity. It is proposed that transition from sensitivity to resistance to dibutyryl cyclic AMP in lymphoma cells is connected with a modification of the cyclic AMP-binding protein, which appears to be the regulatory subunit of the cyclic AMP-activated protein kinase.

Induction of glutathione S-transferase (GST) Ya gene expression by a variety of chemical agents is mediated by a regulatory element composed of two adjacent AP-1-like binding sites and activated by the Fos/Jun heterodimeric complex (AP-1). We have previously shown that the induction of GST Ya gene expression and of AP-1 binding activity is regulated by intracellular glutathione (GSH) levels. To study the role of reactive oxygen species in the induction of AP-1 activity and GST Ya gene expression and their effect on intracellular GSH levels, we have exposed hepatoma cells to adriamycin and two synthetic quinones, Qcb and Qn, with different capacities to generate oxygen radicals. The kinetics of quinone-mediated generation of hydroxyl radicals were monitored in intact cells by a spin trapping technique and EPR spectral measurements. We find that quinones which can chelate Fe(III) ions, adriamycin and Qcb, are more effective in hydroxyl radical production than the nonchelating quinone Qn. Furthermore, we show that the induction of AP-1 binding activity and GST Ya gene expression by these quinones correlates with their oxygen radical production, adriamycin and Qcb being stronger inducers that Qn. The present study indicates that the AP-1-mediated induction of GST Ya gene expression is part of the response to oxidative stress. A transient increase by 2.5-fold in the intracellular GSH level was observed 30 min after exposure of cells to quinone and was followed by a rapid depletion of GSH. This increase in the GSH level represents an induction of GSH synthesis since it was blocked by buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase.(ABSTRACT TRUNCATED AT 250 WORDS)