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

doi:10.1016/0300-483X(92)90143-3

Toxicology

Volume 74, Issues 2–3, September 1992, Pages 243-258

https://doi.org/10.1016/0300-483X(92)90143-3 Get rights and content

Abstract

Repeated exposure of avian and rodent species to benzo(a)pyrene (BaP) has been associated with the development of aortic lesions of atherosclerotic etiology. Because the occurence of these lesions may involve alterations in the regulation of smooth muscle cell (SMC) growth, the present studies were conducted to evaluate the proliferative responses of quail aortic SMCs to BaP treatment in vivo and in vitro. Measurements of [³H]thymidine incorporation and cell growth were conducted in cultured aortic SMCs isolated from male Japanese quail treated with 10 mg/kg BaP or vehicle weekly for 10 weeks or in naive aortic SMCs exposed in vitro to BaP (0.003–30 μM). Inhibition of DNA synthesis was observed in primary and early passage cultures of aortic SMCs isolated from BaP-treated quail relative to controls. Continued propagation of these cultures yielded a population of BaP cells which proliferated at faster rates than controls. The proliferative phenotype induced by BaP was first observed after the tenth passage and preserved in all subsequent passages tested. In vitro growth of SMCs from BaP-treated animals was serum- and anchorage-dependent. A 24-h exposure of cycling SMC cultures to BaP (0.003–30 μM) was associated with a dose-dependent decrease in DNA synthesis and significant delay in the progression of SMCs through the cell cycle. A time-course study revealed that maximal inhibition of DNA synthesis occurred 10 h after addition of 3 μM BaP to cycling cultures of SMCs. As seen in SMCs isolated from BaP-treated quail, serial subculture of SMCs exposed to 0.3 μM BaP in vitro for 24 h yielded a fast-growing population of cells. In these cultures, expression of the proliferative phenotype was observed after the fifth passage. These data suggest that BaP induces the expression of a proliferative phenotype in aortic SMCs characterized by enhanced serum responsiveness. This phenotypic modulation may contribute to the initiation and/or progression of vascular lesions of atherosclerotic etiology induced by BaP.

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Benzo(a)pyrene (BaP), a ubiquitous environmental pollutant known to cause many diseases including atherosclerosis, induces a dose-dependent reduction in the levels of the inducible Hsp70. To explore the mechanism underlying the reduction of Hsp70, we measured the levels of Hsp70, cytoplasmic and nuclear heat shock factor 1 (HSF1) in porcine aortic endothelial cells using Western blot, and then further characterized the binding ability of HSF1 and heat shock element (HSE) by electrophoretic mobility shift assay. We found that when porcine aortic endothelial cells were treated by 0.1–10 μM of BaP for 24 h, there was a significant reduction of Hsp70, cytoplasmic and nuclear HSF1 and the binding rate of HSF1 and HSE at 5, 10 μM of BaP but less effective at lower concentrations. The effect of BaP on the Hsp70 expression level was markedly attenuated by co-treatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). Staurosporine (STP), an inhibitor of PKC, blocked the effect of PMA treatment in combination with BaP. These results suggest that BaP might inhibit Hsp70 levels by reducing the expression of HSF1 and decreasing binding of HSF1 and HSE via PKC-dependent signaling pathways that might be involved in the regulation of Hsp70 gene expression under BaP.
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Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the Western world. Its incidence has been increasing lately in developing countries. Several lines of evidence support a role for oxidative stress in atherogenesis. Growing evidence indicates that chronic and acute overproduction of reactive oxygen species (ROS) under pathophysiologic conditions is integral in the development of cardiovascular diseases (CVD). ROS mediate various signaling pathways that underlie vascular inflammation in atherogenesis from the initiation of fatty streak development through lesion progression to ultimate plaque rupture. Various animal models of oxidative stress support the notion that ROS have a causal role in atherosclerosis and other cardiovascular diseases. Human investigations also support the oxidative stress hypothesis of atherosclerosis. Oxidative stress is the unifying mechanism for many CVD risk factors, which additionally supports its central role in CVD. A main source of ROS in vascular cells is the reduced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase system. This is a membrane-associated enzyme, composed of five subunits, catalyzing the one-electron reduction of oxygen, using NADH or NADPH as the electron donor. This system is an important target for genetic investigations. Identification of groups of patients with genetically prone or resistant of oxidative stress is therefore an obvious target of investigation. A better understanding of the complexity of cellular redox reactions, development of a new class of antioxidants targeted to specific subcellular sites, and the phenotype–genotype linkage analysis for oxidative stress will likely be avenues for future research with regards to the broader use of pharmacological therapies in the treatment and prevention of CVD.
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T-cadherin, a glycosylphosphatidylinositol-modified cadherin subtype, is highly expressed in cardiac and vascular tissues. Neither the functions nor regulation of T-cadherin in these tissues is understood. We have cloned rat T-cadherin cDNA encoding the full length amino acid sequence. The 5^′ untranslated nucleotide sequences of rat, mouse, and human T-cadherin contain a conserved GCGTG motif which constitutes the invariant core sequence of dioxin- or xenobiotic-regulatory elements. These elements function as target sites for aryl hydrocarbon receptor/aryl hydrocarbon nuclear translocator (AhR/ARNT) in genes regulated by this transcription factor. Using cultures of rat aortic smooth muscle cells this study presents data revealing T-cadherin as a putative target gene for negative regulation of expression through AHR signalling. Prototypic AHR agonists benzo[a]pyrene (BaP) or 7,12-dimethylbenzanthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) repressed T-cadherin mRNA levels. Repression was antagonized by the cognate AHR antagonist α-naphthoflavone (α-NF). Repression was insensitive to inhibitors of gene transcription (actinomycin D) or de novo protein synthesis (cycloheximide), suggesting AHR/ARNT functions directly in transcriptional repression of T-cad. Regulation of adhesion proteins through the AHR pathway may represent a novel mechanism of action by atherogenic polycyclic aromatic hydrocarbons.
Differential expression of ribosomal L31, Zis, gas-5 and mitochondrial mRNAs following oxidant induction of proliferative vascular smooth muscle cell phenotypes
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Treatment of cultured vascular smooth muscle cells (vSMCs) with benzo(a)pyrene (BaP), a prooxidant present in the particulate phase of tobacco smoke, induces highly proliferative (i.e. atherogenic) phenotypes. Critical early target genes in vSMCs have been identified, but patterns of gene expression following repeated cycles of carcinogen treatment in vivo have yet to be evaluated. In the present study, male Sprague–Dawley rats (175–200 g) were given weekly injections of BaP (10 mg/kg) for 8 weeks to induce atherogenic phenotypes. At the end of this atherogenic regimen, vSMCs were established in serial culture and monitored for patterns of proliferative activity and gene expression. vSMCs isolated from BaP-treated animals (hence forth referred to as BaP cells) exhibited constitutively increased growth rates, and marked enhancement of proliferation in response to serum mitogens. Differential display polymerase chain reaction (DD-PCR) and Northern blot analyses revealed that mRNAs for ribosomal protein L31 and Zis genes were suppressed, while gas-5 and mitochondrial mRNAs were overexpressed in BaP cells relative to control mRNA populations. In situ hybridization experiments in vascular tissue confirmed these alterations in vivo. This is the first report linking expression of these genes to proliferative dysregulation during the course of experimentally-induced atherogenesis.
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Citation Excerpt :
The enzyme is of carcinogenic importance because it catalyzes the bioactivation of many procarcinogenic polyaromatic hydrocarbons (such as B[a]P [8–10]) and certain heterocyclic aryl amines (such as PhIP) [11] to their ultimate reactive intermediates. These activities of the enzyme have also been implicated in other disorders, such as cardiovascular diseases [12,13]. CYP1A1 induction by TCDD is mediated by the binding of TCDD to the AhR.
The levels and activities of pulmonary microsomal CYP1A1 and CYP1A2 in 40-day-old male and female, and 120-day-old male offspring of pregnant rats treated with five weekly 0.1 μg/kg doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during gestation and lactation were compared with those in age-matched offspring of untreated dams. The CYP1A1-preferential activity, ethoxyresorufin O-deethylase (EROD), was comparably induced 5.3- and 6.4-fold in 40-day-old male and female offspring, respectively, but was not induced in 120-day-old male offspring, of TCDD-treated dams. Similarly, CYP1A1 protein was induced in 40-day-old female or male offspring of untreated dams but was undetectable in 120-day-old offspring of untreated or treated dams. CYP1A2 activity, as measured by the bioactivation of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to mutagens in the Ames assay, was elevated 11.1- and 5.5-fold in 40-day-old female and male offspring, respectively, of TCDD-treated dams, but was unaffected by TCDD exposure in 120-day-old offspring. CYP1A2 protein was undetectable in 40-day-old male or female offspring of untreated dams or in 120-day-old male offspring of treated or untreated dams; it was detected in 40-day-old offspring of treated dams, at a level that was higher in females than in males. The results show that gestational and lactational exposure to TCDD causes long-lasting and gender-preferential induction of CYP1A1 as well as CYP1A2 in the lungs of rat offspring.

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

Abstract

Toxicology

Life Sci.

Toxicol. Appl. Pharmacol.

Atherosclerosis

Mutat. Res.

Air Quality Guidelines of Europe

Towards a role for promutagenic lesions in carcinogenesis

Bladder cancer and occupational exposure to polycyclic aromatic hydrocarbons

Int. J. Cancer

Effect of carcinogens on chicken atherosclerosis

Cancer Res.

Effect of 3-methylcholanthrene on the development of aortic lesions in mice

Cancer Res.

Cytochrome P-450 monooxygenase system: Localization in smooth muscle of rabbit aorta

Mol. Pharmacol.

Benzo(a)pyrene metabolism in bovine aortic endothelial and bovine lung fibroblast-like cell cultures

Cancer Res.

The ultrastructure of spontaneous and experimentally-induced lesions. II. The spontaneous plaque in the chicken

Lab. Invest.

Arterial smooth muscle: A multifunctional mesenchymal cell

Arch. Pathol. Lab. Med.

Localization of PDGF-protein in macrophages in all phases of atherogenesis

Science