Biochimica et Biophysica Acta (BBA) - General Subjects
ReviewMechanisms of the acute effects of inhaled ozone in humans☆
Introduction
Unlike its congener, “triplet” dioxygen, 3O2, whose oxidizing power is held in check by the fact that its two unpaired electrons have the same spin state and which therefore requires input of activation energy to convert the spin state one of these electrons to allow them to become paired, inhaled O3 (O3) is a powerful, highly reactive non-physiologic oxidant whose immediate effects are limited to the respiratory tract. Its mechanisms of action are therefore of particular interest to inhalation toxicologists and pulmonary medicine specialists but also of interest to the redox biology community.
Other than the periodic Integrated Science Assessment documents prepared by EPA/NCEA which are designed to provide an exhaustive review and assessment of published peer-reviewed scientific literature ultimately relevant to the determination and setting of human health-based National Ambient Air Quality Standard(s) (NAAQS) for tropospheric O3, there seem not to have been reviews that focus on the mechanisms underlying the multiple acute pulmonary responses of healthy humans to O3 inhalation since the critical, synthetic review in 2000 by Mudway and Kelly [1]. The present paper attempts to summarize and speculate about newer work on inhaled O3 dosimetry and on mechanisms responsible for the acute effects of controlled O3 exposure in healthy human volunteers. These mechanisms involve many complex areas of active investigation and this writer has neglected many important contributions and has undoubtedly also been unaware of many others.
Effects of chronic O3 exposure and observational (epidemiologic) studies will not be addressed. Nor will extra-pulmonary effects or O3 interactions with other inhaled air pollutants or biologically active materials (e.g., allergens, endotoxin). The remarkable features of even relatively intense experimental exposures to O3 concentrations as high as 600 ppb(v) combined with exercise-induced increased minute ventilation for one or more hours, which have involved thousands of volunteers over the past half century include:
- -
full reversibility of acute responses
- -
the absence of clinically apparent pulmonary edema
- -
the absence of long-term development of chronic airways or parenchymal lung disease.
The absence of durable clinically significant features has permitted investigators to ethically continue to study in human volunteers the effects of controlled O3 exposures that exceed NAAQS, currently set at 70 ppb averaged over an 8-hour period.
The major acute responses of the human respiratory tract to inhalation of 100–600 ppb O3 for 1–4 h, typically conducted in a protocol that calls for the intermittent use of exercise-induced ventilation (25–60 L(BTPS)/min), are:
- 1.
Variably decreased vital capacity, sometimes with a reduction as high as 50% of the pre-exposure value, accompanied by substernal discomfort and cough, maximal at the end of exposure and regressing progressively over a period of hours post-exposure.
- 2.
A variable degree of neutrophilic inflammation of the lower airways, detectable at 1 h post-exposure but maximal at about 6 h, which clears more slowly than does the change in vital capacity. The subject remains afebrile. Interestingly the intensity of the neutrophilia does not correlate with the degree of acute decrease in vital capacity. The development of acute inflammation implies that apart from the immediate oxidative stress of O3 itself there is a secondary inflammation-related oxidant stress.
- 3.
Increased bronchial reactivity to inhaled bronchoconstrictors (e.g., methacholine, histamine), maximal immediately post-exposure.
- 4.
A surprisingly modest degree of bronchoconstriction, even in asthmatic volunteers.
Clinical evidence of pulmonary edema does not seem to develop following exposures up to 600 ppb despite evidence of airways inflammation and increased airways permeability (increased protein in bronchoalveolar lavage); more rapid uptake of inhaled aerosolized 99mTc-labeled DTPA [2] and appearance of bronchiolar club cell-specific protein CC16 in blood plasma [3]. However, other than a recent study of healthy young volunteers exposed to 100 and 200 ppb O3 in which DLCO and DLNO were not affected [4], modern imaging techniques or diffusing capacity measurements to look for subtle parenchymal effects appear not to have been used.
Section snippets
How does inhaled O3 provoke functional and inflammatory changes in the respiratory tract?
Despite its relative insolubility in aqueous media about 80% of inhaled O3 is deposited in the respiratory tract. Expired O3 during an exposure is limited to the air derived from the anatomic dead space [5]. Since O3 is taken up in the nose the dose to the lower airways is increased by preventing nasal respiration (using nose clips) or by exercise sufficient to automatically convert inspiration from the nasal to the oral route. The longitudinal distribution of O3 uptake along the lower airways
Products of phospholipid ozonation and their biological activity
Thus, purified products from ozonized 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC) were used to examine the responses of cultured BEAS-2B human immortalized bronchial epithelial cells. All seven products were predicted by the Criegee mechanism. Various products caused dose-related activation of cPLA2 (with arachidonate release), of PLC and of PLD [55]. These data extended the findings of Samet et al. [52] and of Wright et al. [53] who had exposed BEAS-2B cells directly to O3.
Products of cholesterol ozonation
Ozonation in lung lining fluid or in cell membranes of the single C5C6 double bond in cholesterol initially forms an unstable primary ozonide. This compound then gives rise to a variety of products such as cholesterol epoxide and 5,6 secosterol [61], [62]. These O3-derived oxysterols increase NFkB activity and are pro-inflammatory (increased IL-6 and IL-8 expression). The liver X receptor (LXR) in human bronchial epithelial cells is adducted by secosterol A through its lysine groups and this
O3 and lipid peroxidation
Although the primary site of ozonation of lipid substrates in ASL appears to be CC bonds, their ozonation-derived products are capable of causing lipid peroxidation in cells by activation of enzymatic pathways (PLA2, cyclooxygenase, lipoxygenase) and by production of radicals that abstract H atoms from allylic or, especially, bisallylic methylene groups in polyunsaturated fatty acids. Lipid peroxidation in the presence of oxygen is initiated by H atom abstraction (“initiation”) to generate a
Non-enzymatic lipid peroxidation and iso-prostanes
Unlike cyclooxygenase or lipoxygenase-catalyzed peroxidation, the products of non-enzymatic peroxidation are not stereospecific and can involve unsaturated sn-1 or sn-2 fatty acids that are still part of a phospholipid. In the case of polyunsaturated fatty acids such as arachidonate further reaction with oxygen to generate an endoperoxide and formation of a cyclopentane ring gives rise to isomers of prostaglandins called isoprostanes (reviewed by G. Milne, Q. Dai and L. Roberts [65]).
How does inhaled O3 affect lung function?
Perhaps the best-known acute respiratory effect of O3 inhalation in humans is the variable decrease in vital capacity due almost entirely to a decrease in inspiratory capacity. This change is not attributable to a change in lung mechanics or to weakness of the respiratory muscles [75]. It is caused by involuntary, neurally mediated inhibition of inspiration, often accompanied by substernal (tracheal) pain and cough on inspiration. Passannante et al. [76] showed that the loss of inspiratory
How does inhaled O3 cause acute airways inflammation
Although the neuropeptides secreted by the arborization of activated C fibers have pro-inflammatory effects, and activation of non-neural TRPA1 cation channels in epithelial cells may also have inflammatory effects, the principal mechanism responsible for acute O3-provoked neutrophilic lung inflammation has generally been thought to involve activation of the important transcriptional regulator, NF-kB. Recent studies, however, have pointed to other mechanisms involving macrophage toll-like
Glutathione-S-transferases (GST), NADPH-quinone-oxidoreductase-1 (NQO1), and O3
Cytosolic glutathione transferases are dimeric. Each monomer has an N-terminal GSH-binding/thiolate-activating domain and a hydrophobic C-terminal domain at which substrate electrophiles are conjugated with GSH and thus detoxified [135], [136]. Among the multiple antioxidant enzymes that use GSH as a reductant the families of cytoplasmic GST enzymes are considered to be important for detoxification of electrophiles such as 4-hydroxy-nonenal (HNE) which are significant PUFA ozonation products.
Repair of oxidized membrane phospholipids in lung
Given the reactive affinity of O3 for CC double bonds as well as the ability of its products (e.g., POPC) to activate cPLA2 and also to be associated with non-enzymatic lipid peroxidation and isoprostane generation, the lack of irreversible lung injury following relevant O3 exposures in human volunteers suggests that in addition to mechanisms involving lipoxins, resolvins, protectins and maresins for non-phlogistic clearing of established inflammation (see review by C. Serhan [153]) the lung
Final thoughts
This review has attempted to summarize how inhaled ozone, a potent environmental oxidant with particular affinity for CC bonds, causes acute decrements in lung function (with little bronchoconstriction in humans) and, by other pathways, acute neutrophilic airways inflammation. Borrowing concepts from Fessler and Summer [43], unsaturated airway phospholipids and cholesterol serve as sensors and ultimately as second messengers (relays) of ozone-provoked oxidant stress – a concept originally
Transparency Document
References (156)
- et al.
Ozone and the lung: a sensitive issue
Mol. Asp. Med.
(2000) How far does ozone penetrate into the pulmonary air/tissue boundary before it reacts?
Free Radic. Biol. Med.
(1992)- et al.
A model of the regional uptake of gaseous pollutants in the lung. I. The sensitivity of the uptake of ozone in the human lung to lower respiratory tract secretions and exercise
Toxicol. Appl. Pharmacol.
(1985) - et al.
Incorporation and disappearance of oxygen-18 in lung from mice exposed to 1 ppm 18O3
Toxicol. Appl. Pharmacol.
(1989) - et al.
Kinetics of ozone reaction with uric acid, ascorbic acid, and glutathione at physiologically relevant conditions
Arch. Biochem. Biophys.
(2006) - et al.
Modeling the interactions of ozone with pulmonary epithelial lining fluid antioxidants
Toxicol. Appl. Pharmacol.
(1998) - et al.
Lung epithelial cells release ATP during ozone exposure: signaling for cell survival
Free Radic. Biol. Med.
(2005) - et al.
Purinergic signaling and kinase activation for survival in pulmonary oxidative stress and disease
Free Radic. Biol. Med.
(2006) - et al.
Vitamin supplementation does not protect against symptoms in ozone-responsive subjects
Free Radic. Biol. Med.
(2006) - et al.
Antioxidant-mediated augmentation of ozone-induced membrane oxidation
Free Radic. Biol. Med.
(2005)
Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence
Free Radic. Biol. Med.
How do nutritional antioxidants really work: nucleophilic tone and para-hormesis versus free radical scavenging in vivo
Free Radic. Biol. Med.
NAD(P)H:quinone oxidoreductase (NQO1) polymorphism, exposure to benzene, and predisposition to disease: a HuGE review
Genet. Med.
In vivo determination of surface tension in the horse trachea and in vitro model studies
Respir. Physiol.
Lipid hydroperoxide generation, turnover, and effector action in biological systems
J. Lipid Res.
The cascade mechanism to explain ozone toxicity: the role of lipid ozonation products
Free Radic. Biol. Med.
What does ozone react with at the air/lung interface? Model studies using human red blood cell membranes
Arch. Biochem. Biophys.
Ozone does not react with human erythrocyte membrane lipids
Arch. Biochem. Biophys.
Reaction of ozone with sulfhydryls of human erythrocytes
Arch. Biochem. Biophys.
Reaction of ozone with phospholipid vesicles and human erythrocyte ghosts
Arch. Biochem. Biophys.
Ozone stimulates release of platelet activating factor and activates phospholipases in guinea pig tracheal epithelial cells in primary culture
Toxicol. Appl. Pharmacol.
Identification of oxidized phospholipids in bronchoalveolar lavage exposed to low ozone levels using multivariate analysis
Anal. Biochem.
Lipid ozonation products activate phospholipases A2, C, and D
Toxicol. Appl. Pharmacol.
Regulation of C/EBPbeta and resulting functions in cells of the monocytic lineage
Cell. Signal.
Pathways of cholesterol oxidation via non-enzymatic mechanisms
Chem. Phys. Lipids
The isoprostanes—25 years later
Biochim. Biophys. Acta
Comparison of formation of D2/E2-isoprostanes and F2-isoprostanes in vitro and in vivo—effects of oxygen tension and glutathione
Arch. Biochem. Biophys.
Ozone-induced increase in exhaled 8-isoprostane in healthy subjects is resistant to inhaled budesonide
Free Radic. Biol. Med.
Nonenzymatic free radical-catalyzed generation of 15-deoxy-Delta(12,14)-prostaglandin J(2)-like compounds (deoxy-J(2)-isoprostanes) in vivo
J. Lipid Res.
Tmem100 is a regulator of TRPA1-TRPV1 complex and contributes to persistent pain
Neuron
Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants, in pulmonary epithelial cells
Chem. Biol. Interact.
The contribution of transient receptor potential ankyrin 1 (TRPA1) to the in vivo nociceptive effects of prostaglandin E(2)
Life Sci.
Ozone exposure increases respiratory epithelial permeability in humans
Am. Rev. Respir. Dis.
Clara cell protein as a biomarker for ozone-induced lung injury in humans
Eur. Respir. J.
Cardiovascular effects of ozone in healthy subjects with and without deletion of glutathione-S-transferase M1
Inhal. Toxicol.
Bronchoscopic determination of ozone uptake in humans
J. Appl. Physiol.
Longitudinal distribution of ozone absorption in the lung: effects of respiratory flow
J. Appl. Physiol.
Progress in assessing air pollutant risks from in vitro exposures: matching ozone dose and effect in human airway cells
Toxicol. Sci.
Proteomic analysis of the airway surface liquid: modulation by proinflammatory cytokines
Am. J. Phys. Lung Cell. Mol. Phys.
Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?
Am. J. Phys. Lung Cell. Mol. Phys.
Analysis of the proteome of human airway epithelial secretions
Proteome Sci.
Structure and function of the polymeric mucins in airways mucus
Annu. Rev. Physiol.
Oxidation increases mucin polymer cross-links to stiffen airway mucus gels
Sci. Transl. Med.
Kinetics of ozonation. 5. Reactions of ozone with carbon-hydrogen bonds
J. Am. Chem. Soc.
Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids
Am. J. Phys.
The uric acid transporter SLC2A9 is a direct target gene of the tumor suppressor p53 contributing to antioxidant defense
Oncogene
Mechanisms regulating airway nucleotides
Subcell. Biochem.
Airway purinergic responses in healthy, atopic nonasthmatic, and atopic asthmatic subjects exposed to ozone
Inhal. Toxicol.
Identification of vitamin C transporters in the human airways: a cross-sectional in vivo study
BMJ Open
Antioxidant responses to acute ozone challenge in the healthy human airway
Inhal. Toxicol.
Cited by (0)
- ☆
This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.