Profiles of antioxidant/electrophile response element (ARE/EpRE) nuclear protein binding and c-Ha-ras transactivation in vascular smooth muscle cells treated with oxidative metabolites of benzo[a]pyrene

doi:10.1016/S0006-2952(00)00439-1

Biochemical Pharmacology

Volume 60, Issue 9, 1 November 2000, Pages 1285-1296

https://doi.org/10.1016/S0006-2952(00)00439-1 Get rights and content

Abstract

Activation of nuclear protein binding to the antioxidant/electrophile response element (ARE/EpRE) by benzo[a]pyrene (BaP) in vascular smooth muscle cells (vSMCs) is associated with transcriptional deregulation of c-Ha-ras. This response may be mediated by oxidative intermediates of BaP generated during the course of cellular metabolism. To test this hypothesis, the profile of ARE/EpRE protein binding and transactivation elicited by BaP was compared with that of 3-hydroxy BaP (3-OH BaP) (0.03 to 3.0 μM), BaP 7,8-dihydrodiol (BaP 7,8-diol) (0.03 to 3.0 μM), BaP 3,6-quinone (BaP 3,6-Q) (0.0003 to 3.0 μM), and H₂O₂ (25 to 100 μM). Specific protein binding to the consensus c-Ha-ras ARE/EpRE was observed in vSMCs treated with all BaP metabolites at concentrations considerably lower than those required for the parent compound. H₂O₂, a by-product of BaP 3,6-Q redox cycling, also increased binding to the ARE/EpRE. Treatment of vSMCs with oxidative BaP metabolites or H₂O₂ transactivated the c-Ha-ras promoter in all instances, but the response was consistently half of the maximal induction elicited by BaP. Similar proteins cross-linked specifically to the consensus c-Ha-ras ARE/EpRE sequence in cells treated with BaP or its oxidative intermediates. The protein binding profile in the c-Ha-ras promoter was similar to that in the NADPH:quinone reductase gene (NQO₁) and the glutathione S-transferase Ya gene (GSTYa) promoters, but the relative abundance of individual complexes was promoter-specific. We conclude that oxidative intermediates of BaP mediate activation of nuclear protein binding to ARE/EpRE and contribute to transcriptional de-regulation of c-Ha-ras in vSMCs.

Section snippets

Reagents

BaP (98% purity) was obtained from the Aldrich Chemical Co., and BaP 3,6-Q (≥98% purity by HPLC), 3-hydroxy BaP (≥99% purity by HPLC), and BaP 7,8-diol (≥98% purity by HPLC) were obtained from the National Cancer Institute Chemical Carcinogen Reference Standard Repositories (c/o Midwest Research Institute). All other chemicals were purchased from the Sigma Chemical Co. unless otherwise noted. BaP, 3-OH BaP, BaP 7,8-diol, and BaP 3,6-Q stock solutions were prepared in DMSO, and H₂O₂ stock

Activation of protein binding to c-Ha-ras ARE/EpRE by BaP metabolites

Previous work in this laboratory has shown that BaP induces specific protein binding to hHa-ras ARE/EpRE [8]. To assess the possible role of BaP metabolites in the activation of protein binding, the effects of 3-OH BaP, BaP 7,8-diol, and BaP 3,6-Q in vSMCs were examined. Cells were challenged for 3 hr with individual metabolites at concentrations comparable to or lower than those required to activate ARE/EpRE protein binding by the parent compound. The range of concentrations examined was based

Discussion

In this report we showed that oxidative intermediates of BaP activate protein binding to the ARE/EpRE and up-regulate transcription of reporter plasmids containing the ARE/EpRE sequence directly upstream of c-Ha-ras regulatory sequences. The threshold and concentration-dependent profiles of protein binding and transactivation in response to 3-OH BaP, BaP 7,8-diol, and BaP 3,6-Q suggest that oxidative intermediates participate in the deregulation of c-Ha-ras gene expression by BaP in vSMCs.

Acknowledgements

This work was supported, in part, by NIEHS Grants 04849 to K. S. R. and Center Grant ES 09106. K. P. M. is a predoctoral fellow on NIEHS Training Grant T32 ES 07273.

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^∗: Present address: Schering Plough Research Institute, Lafayette, NJ 07848.

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Profiles of antioxidant/electrophile response element (ARE/EpRE) nuclear protein binding and c-Ha-ras transactivation in vascular smooth muscle cells treated with oxidative metabolites of benzo[a]pyrene

Abstract

Section snippets

Reagents

Activation of protein binding to c-Ha-ras ARE/EpRE by BaP metabolites

Discussion

Acknowledgements

Growth-related signaling as a target of toxic insult in vascular tissue

Life Sci

Further investigations of the capacity of polynuclear aromatic hydrocarbons to elicit atherosclerotic lesions

J Toxicol Environ Health

An ultrastructural comparison of carcinogen-associated and spontaneous aortic lesions in the cockerel

Am J Pathol

Proliferative responses of quail aortic smooth muscle cells to benzo[a]pyreneImplications in PAH-induced atherogenesis

Toxicology

Benzo[a]pyrene enhances atherosclerosis in White Carneau and Show Racer pigeons

Arterioscler Thromb

Interference with PKC-related mitogenic signal transduction in vascular smooth muscle cells

Arch Biochem Biophys

Modulation of protooncogene expression in rat aortic smooth muscle cells by benzo[a]pyrene

Arch Biochem Biophys

Identification of benzo[a]pyrene-inducible cis-acting elements within c-Ha-ras transcriptional regulatory sequences

Mol Pharmacol

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in chicken aortasMonooxygenation, bioactivation to mutagens, and covalent binding to DNA in vitro

Toxicol Appl Pharmacol

Induction of cytochrome CYPIA1 and formation of toxic metabolites of benzo[a]pyrene by rat aortaA possible role in atherogenesis

Proc Natl Acad Sci USA

Kinetics, activation, and induction of aortic mono-oxygenases—Biotransformation of benzo[a]pyrene

Biochem Pharmacol

Benzo[a]pyrene inhibits protein kinase C activity in subcultured rat aortic smooth muscle cells

Chem Biol Interact

Activation of c-Ha-ras by benzo[a]pyrene in vascular smooth muscle cells involves redox stress and aryl hydrocarbon receptor

Mol Pharmacol

Benzo[a]pyrene metabolism, activation and carcinogenesisRole and regulation of mixed-function oxidases and related enzymes

Physiol Rev

Benzo[a]pyrene dione–benzo[a]pyrene diol oxidation—reduction couples; Involvement in DNA damage, cellular toxicity, and carcinogenesis

J Toxicol Environ Health

Benzo[a]pyrenedione/benzo[a]pyrenediol oxidation-reduction couples and the generation of reactive reduced molecular oxygen

Biochemistry

Metabolic conversion of benzo[a]pyrene by Syrian hamster liver microsomes and binding of metabolites to deoxyribonucleic acid

J Med Chem

High-pressure liquid chromatographic analysis of benzo(a)pyrene metabolism and covalent binding and the mechanism of action of 7,8-benzoflavone and 1,2-epoxy-3,3,3-trichloropropane

Cancer Res

The rat liver glutathione S-transferase Ya subunit geneCharacterization of the binding properties of a nuclear protein from HepG2 cells that has high affinity for the antioxidant response element

Biochemistry

Oxy radicals and antioxidative responses in cancer12th Sapporo Cancer Seminar

Cancer Res

The antioxidant responsive element

J Biol Chem

Transcriptional regulation of a rat liver glutathione S-transferase Ya subunit gene

J Biol Chem