Residual Oil Fly Ash Induces Cytotoxicity and Mucin Secretion by Guinea Pig Tracheal Epithelial Cells via an Oxidant-Mediated Mechanism

doi:10.1006/taap.1999.8886

Toxicology and Applied Pharmacology

Volume 163, Issue 3, 15 March 2000, Pages 221-230

https://doi.org/10.1006/taap.1999.8886 Get rights and content

Abstract

Inhalation of ambient air particulate matter (PM) is associated with pulmonary injury and inflammation. Using primary cultures of guinea pig tracheal epithelial (GPTE) cells as an in vitro model of airway epithelium, we examined effects of exposure to suspensions of six different emission and ambient air PM samples: residual oil fly ash (ROFA) from an electrical power plant; fly ash from a domestic oil burning furnace (DOFA); ambient air dust from St. Louis (STL), Ottawa (OT), and Washington, DC (WDC); and volcanic ash from the eruption of Mount Saint Helens (MSH) in 1980. Effects of these particulates on cell viability (assessed via LDH assay), secretion of mucin (measured by a monoclonal antibody-based ELISA), and steady-state mRNA levels of the mucin gene MUC2 were determined. ROFA was the most toxic of the dusts tested, as it significantly increased LDH release following a 24-h incubation with 50 μg/cm² ROFA. ROFA also enhanced MUC2 mRNA after 4-h exposure, and mucin secretion after 8 h. ROFA-induced mucin secretion and cytotoxicity were attenuated by the oxidant scavenger, dimethylthiourea (DMTU). ROFA exposure also depleted cells of glutathione (GSH). Relatedly, depletion of intracellular GSH by treatment of the cells with buthionine sulfoxamine (BSO) also provoked mucin secretion, as well as enhancing the secretory effect of ROFA when the two agents were added together. L-NMA, the nitric oxide synthase (NOS) inhibitor, did not affect ROFA-induced mucin secretion. Of the soluble transition metals in ROFA (nickel, iron, vanadium), only vanadium individually, or combinations of the metals containing vanadium, provoked secretion. The results suggest ROFA enhances mucin secretion and generates toxicity in vitro to airway epithelium via a mechanism(s) involving generation of oxidant stress, perhaps related to depletion of cellular antioxidant capacity. Deleterious effects of inhalation of ROFA in the respiratory tract in vivo may relate to these cellular responses. Vanadium, a component of ROFA, may be important in generating these reactions.

References (50)

M.E. Anderson
Determination of glutathione and glutathione disulfide in biological samples
Methods Enzymol.
(1985)
A.R. Byrne et al.
Vanadium in foods and human body fluids and tissues
Sci. Total Environ.
(1978)
K. Dreher et al.
Soluble transition metals mediate the acute pulmonary injury and airway hyperreactivity induced by residual oil fly ash particles
Chest
(1996)
S.H. Gavett et al.
Metal and sulfate composition of residual oil fly ash determines airway hyperreactivity and lung injury in rats
Environ. Res.
(1997)
G.E. Hatch et al.
Inhalable particles and pulmonary host defense: In vivo and in vitro effects of ambient air and combustion particles
Environ. Res.
(1985)
R.J. Keller et al.
Importance of hydroxyl radical in the vanadium-stimulated oxidation of NADH
Free Radic. Biol. Med.
(1989)
R.J. Keller et al.
Vanadium and lipid peroxidation: Evidence for involvement of vanadyl and hydroxyl radical
Arch. Biochem. Biophys.
(1988)
D. Li et al.
Cloning of the amino-terminal and 5′-flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts
J. Biol. Chem.
(1998)
M.H. Oster et al.
Vanadium treatment of diabetic Sprague-Dawley rats results in tissue vanadium accumulation and pro-oxidant effects
Toxicology
(1993)
N.B. Parker et al.
Hydrogen peroxide causes dimethylthiourea consumption while hydroxyl radical causes dimethylsulfoxide consumption in vitro
Free Radic. Biol. Med.
(1985)

L.G. Rochelle et al.

Concurrent measure of reactive oxygen and nitrogen species in airway epithelial cells in vitro

Free Radic. Biol. Med.

(1998)

P. Southorn et al.

Free radicals in medicine. II. Involvement in human disease

Mayo Clin. Proc.

(1988)

M. Whiteman et al.

Thiourea and dimethylthiourea inhibit peroxynitrite-dependent damage: Nonspecificity as hydroxyl radical scavengers

Free Radic. Biol. Med.

(1997)

K.B. Adler et al.

Platelet-activating factor provokes release of mucin-like glycoproteins from guinea pig respiratory epithelial cells via a lipoxygenase-dependent mechanism

Am. J. Respir. Cell Mol. Biol.

(1992)

K.B. Adler et al.

Characterization of guinea pig tracheal epithelial cells maintained in biphasic organotypic culture: Cellular composition and biochemical analysis of released glycoconjugates

Am. J. Respir. Cell Mol. Biol.

(1990)

K.B. Adler et al.

Hypersecretion of mucin in response to inflammatory mediators by guinea pig tracheal epithelial cells in vitro is blocked by inhibition of nitric oxide synthase

Am. J. Respir. Cell Mol. Biol.

(1995)

K.B. Adler et al.

Oxygen metabolites stimulate release of high-molecular-weight glycoconjugates by cell and organ cultures of rodent respiratory epithelium via an arachidonic acid-dependent mechanism

J. Clin. Invest.

(1990)

L.A. Cohn et al.

Antioxidant properties of guinea pig tracheal epithelial cells in vitro

Am. J. Physiol. (London)

(1994)

D.L. Costa et al.

Bioavailable metals in particulate matter mediate cardiopulmonary injury in healthy and compromised animal models

Environ. Health Perspect.

(1997)

M. Ding et al.

Vanadate-induced activation of activator protein-1: Role of reactive oxygen species

Carcinogenesis

(1999)

D.W. Dockery et al.

Acute respiratory effects of particulate air pollution

Annu. Rev. Public Health.

(1994)

K.L. Dreher et al.

Soluble transition metals mediate residual oil fly ash induced acute lung injury

J. Toxicol. Environ. Health.

(1997)

J.A. Dye et al.

Injury of rat tracheal epithelia cultures by exposure to residual oil fly ash (ROFA) involves generation of hydroxyl radical-like reactive oxygen species

Am. J. Respir. Crit. Care Med.

(1996)

J.A. Dye et al.

Epithelial injury induced by exposure to residual oil fly-ash particles: Role of reactive oxygen species

Am. J. Respir. Cell Mol. Bio.

(1997)

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However, in patients with inflammatory airway diseases, mucus hypersecretion from metaplastic and hyperplastic goblet cells likely contributes to mucus obstruction of airways.43 Mucus secretion is stimulated by several inflammatory stimuli such as the following: (1) oxidants including tobacco smoke,44 residual oil fly ash,45 nitric oxide,46 and superoxide via PGF2α47; (2) proteases48 including neutrophil elastase,49,50 and P aeruginosa proteases51; and (3) cytokines including TNF-α52 and platelet activating factor.53 Common signaling intermediates for many stimuli include G protein-coupled receptor activation, protein kinase C activation, tyrosine phosphorylation, and phospholipase activation.54,55
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¹: To whom correspondence should be addressed at North Carolina State University, College of Veterinary Medicine, Department of Anatomy, Physiological Sciences and Radiology, Raleigh, NC 27606. Fax: (919) 513-6465. E-mail: [email protected].

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Regular ArticleResidual Oil Fly Ash Induces Cytotoxicity and Mucin Secretion by Guinea Pig Tracheal Epithelial Cells via an Oxidant-Mediated Mechanism

Abstract

Methods Enzymol.

Sci. Total Environ.

Chest

Environ. Res.

Environ. Res.

Free Radic. Biol. Med.

Arch. Biochem. Biophys.

J. Biol. Chem.

Toxicology

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Mayo Clin. Proc.

Free Radic. Biol. Med.

Platelet-activating factor provokes release of mucin-like glycoproteins from guinea pig respiratory epithelial cells via a lipoxygenase-dependent mechanism

Am. J. Respir. Cell Mol. Biol.

Characterization of guinea pig tracheal epithelial cells maintained in biphasic organotypic culture: Cellular composition and biochemical analysis of released glycoconjugates

Am. J. Respir. Cell Mol. Biol.

Hypersecretion of mucin in response to inflammatory mediators by guinea pig tracheal epithelial cells in vitro is blocked by inhibition of nitric oxide synthase

Am. J. Respir. Cell Mol. Biol.

Oxygen metabolites stimulate release of high-molecular-weight glycoconjugates by cell and organ cultures of rodent respiratory epithelium via an arachidonic acid-dependent mechanism

J. Clin. Invest.

Antioxidant properties of guinea pig tracheal epithelial cells in vitro

Am. J. Physiol. (London)

Bioavailable metals in particulate matter mediate cardiopulmonary injury in healthy and compromised animal models

Environ. Health Perspect.

Vanadate-induced activation of activator protein-1: Role of reactive oxygen species

Carcinogenesis

Acute respiratory effects of particulate air pollution

Annu. Rev. Public Health.

Soluble transition metals mediate residual oil fly ash induced acute lung injury

J. Toxicol. Environ. Health.

Injury of rat tracheal epithelia cultures by exposure to residual oil fly ash (ROFA) involves generation of hydroxyl radical-like reactive oxygen species

Am. J. Respir. Crit. Care Med.

Epithelial injury induced by exposure to residual oil fly-ash particles: Role of reactive oxygen species

Am. J. Respir. Cell Mol. Bio.

Regular Article
Residual Oil Fly Ash Induces Cytotoxicity and Mucin Secretion by Guinea Pig Tracheal Epithelial Cells via an Oxidant-Mediated Mechanism