Glutathione S-transferase (GST) Ya subunit gene expression is induced in mammalian tissues by two types of chemical agents: (i) planar aromatic compounds (e.g., 3-methylcholanthrene, g3-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) and (i) electrophiles (e.g., trans-4-phenyl-3-buten-2-one and dimethyl fumarate) or compounds easily oxidized to electrophiles (e.g., tert-butylhydroquinone).To study the mechanism of this induction, we have introduced deletions in the 5' flanking region of a mouse GST Ya subunit gene, fused it to the coding sequence for chloramphenicol acetyltransferase (CAT) To elucidate the mechanisms of regulation of GST Ya subunit gene expression by the two types of inducers, we have previously isolated a mouse GST Ya gene (7, 8) and have shown the presence of xenobiotic responsive elements in the 5' flanking region (9). In the present study, we demonstrate that this region contains between nucleotides -754 and -713 an inducible element that activates Ya gene transcription in cis in response to a variety of inducers such as 3-methylcholanthrene, p-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), tert-butylhydroquinone, and trans4phenyl-3-buten-2-one. These results raise the question of the mechanisms proposed for the regulation of GST gene expression by planar aromatic and electrophilic inducers (4,5). Our results show that, in order to function as Ya gene inducers, the planar aromatics have to be metabolized by the cytochrome P1-450 system into electrophilic compounds. In this paper we bring evidence that the inducible expression of GST Ya subunit gene is controlled by a single electrophile-responsive element (EpRE), which is activated exclusively by an electrophilic signal. Protein (Weizmann Institute). The cell lines were propagated as described (9). Nuclear and cytosolic extracts were prepared from the cell lines as described by Dignam et al. (10), and DNase I protection patterns (footprint assays) have been described (9).Transfections. The plasmid constructions containing fragments of the 5' flanking region of the GST Ya subunit gene Abbreviations: GST, glutathione S-transferase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; CAT, chloramphenicol acetyltransferase; EpRE, electrophile-responsive element. *Phase I xenobiotic metabolizing enzymes (e.g., cytochrome P1-450) introduce by oxidation or reduction functional groups into chemical compounds. 6258The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Transcription factors AP-1 and NF-kappaB 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-kappaB 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-kappaB activities are induced by oxygen radicals.

An electrophile-responsive element (EpRE) in the 5' flanking region of the mouse glutathione S-transferase Ya subunit gene was recently found to be responsible for the induction of gene expression by xenobiotics that contain or acquire by metabolism an electrophilic center. We now find that this EpRE is composed of two adjacent 9-base-pair motifs related in sequence to the AP-1 binding site, a transcriptional enhancer originally identified as the phorbol 12-myristate 13-acetate (PMA) response element and known to be regulated by the binding of protein products of c-jun and c-fos genes. Synthetic oligonucleotides representing each of the AP-1-like binding sites of the EpRE and the AP-1 site consensus sequence were prepared and assayed for their enhancer activity and inducibility by tert-butylhydroquinone, ,B-naphthoflavone, and PMA. Single AP-1-like sequences showed a lower enhancer activity than an AP-1 consensus sequence and no inducibility.Two adjacent AP-1-like sites were found to act synergistically and to confer inducibility beyond that observed for a single AP-1 consensus sequence. Examination of the PMA-responsive region of a number of genes shows the presence of adjacent AP-1-like sites and indicates that the structure of the EpRE found in the Ya gene may occur more generally and may be important in regulating the magnitude of the electrophilic response. The present study demonstrates the binding and transactivation of the EpRE by Jun and Fos and indicates that the AP-1 site is part of the EpRE. The induction by PMA or tert-butylhydroquinone appears to be independent of protein kinase C activity since it is not affected by inhibitors of this enzyme.Induction of the electrophile-metabolizing enzymes glutathione transferases, glucuronosyltransferases, and NAD-(P)H:quinone reductase is considered a major mechanism of protection against mutagens, carcinogens, and other toxic compounds (1-3). Compounds that induce these enzymes include the diphenols, isothiocyanates, thiocarbamates, unsaturated dicarboxylic acids, etc., which contain or acquire by metabolism electrophilic centers (2). Many of these inducers are Michael reaction acceptors characterized by olefinic or acetylenic bonds rendered electrophilic by conjugation with electron-withdrawing substituents and share common structural features with glutathione transferase substrates (4). As a consequence the potency of inducers was found to parallel their efficiency as substrates for glutathione transferases (5).In a recent study of a mouse glutathione S-transferase (GST) Ya subunit gene, we have demonstrated (6) that a single cis-regulatory element in the 5' flanking region, between nucleotides (nt) -754 and -714 from the start of transcription, is responsible for Ya gene induction by compounds that are electrophilic (e.g., trans-4-phenyl-3-buten-2-one, dimethyl fumarate) or by compounds that are easily oxidized into electrophilic (tert-butylhydroquinone) and planar aromatic (e.g., 3-methylcholanthrene, P-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p...

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