Review
Mechanisms of the acute effects of inhaled ozone in humans

https://doi.org/10.1016/j.bbagen.2016.07.015Get rights and content

Highlights

  • O3 causes acute respiratory morbidity, functional decrements and inflammation

  • O3 effects are mediated largely by products of its reaction with unsaturated lipids

  • O3-induced lung function decrements involve activation of unmyelinated sensory nerves

  • O3 induces inflammation through activation of macrophage and epithelial receptors

  • Acute lung effects of O3 are normally reversible but many health concerns remain

Abstract

Ambient air ozone (O3) is generated photochemically from oxides of nitrogen and volatile hydrocarbons. Inhaled O3 causes remarkably reversible acute lung function changes and inflammation. Approximately 80% of inhaled O3 is deposited on the airways. O3 reacts rapidly with Cdouble bondC double bonds in hydrophobic airway and alveolar surfactant-associated phospholipids and cholesterol. Resultant primary ozonides further react to generate bioactive hydrophilic products that also initiate lipid peroxidation leading to eicosanoids and isoprostanes of varying electrophilicity. Airway surface liquid ascorbate and urate also scavenge O3. Thus, inhaled O3 may not interact directly with epithelial cells.

Acute O3–induced lung function changes are dominated by involuntary inhibition of inspiration (rather than bronchoconstriction), mediated by stimulation of intraepithelial nociceptive vagal C-fibers via activation of transient receptor potential (TRP) A1 cation channels by electrophile (e.g., 4-oxo-nonenal) adduction of TRPA1 thiolates enhanced by PGE2-stimulated sensitization.

Acute O3-induced neutrophilic airways inflammation develops more slowly than the lung function changes. Surface macrophages and epithelial cells are involved in the activation of epithelial NFkB and generation of proinflammatory mediators such as IL-6, IL-8, TNFa, IL-1b, ICAM-1, E-selectin and PGE2. O3-induced partial depolymerization of hyaluronic acid and the release of peroxiredoxin-1 activate macrophage TLR4 while oxidative epithelial cell release of EGFR ligands such as TGFa or EGFR transactivation by activated Src may also be involved. The ability of lipid ozonation to generate potent electrophiles also provides pathways for Nrf2 activation and inhibition of canonical NFkB activation. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

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

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    the absence of clinically apparent pulmonary edema

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    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 C5double bondC6 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 Cdouble bondC 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 Cdouble bondC 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 Cdouble bondC 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

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    This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

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