AOP ID and Title:

Short Title: Oxidative stress [MIE] Leading to Decreased Lung Function [AO]

Authors

Karsta Luettich, Philip Morris Products S.A., Philip Morris International R&D, Neuchatel, Switzerland

Hasmik Yepiskoposyan, Philip Morris Products S.A., Philip Morris International R&D, Neuchatel, Switzerland

Monita Sharma, PETA Science Consortium International e.V., Stuttgart, Germany

Frazer Lowe, Broughton Nicotine Services, Earby, Lancashire, United Kingdom

Damien Breheny, British American Tobacco (Investments) Ltd., Group Research and Development, Southampton, United Kingdom
 

Status

Abstract

This AOP evaluates one of the major processes known to be involved in regulating efficient mucociliary clearance (MCC)—ciliary function. MCC is a key aspect of the innate immune defense against airborne pathogens and inhaled chemicals and is governed by the concerted action of its functional components, the cilia and the airway surface liquid (ASL), which is composed of mucus and periciliary layers (Bustamante-Marin and Ostrowski, 2017). Disturbances in any of the processes regulating ciliary function can cause MCC dysfunction. Impaired MCC is linked to airway diseases such as chronic obstructive pulmonary disease (COPD) or asthma, both of which are characterized by decreased lung function and bear a significant risk of increased morbidity and mortality. 

Background

With a surface area of ~100 m² and ventilated by 10,000 to 20,000 liters of air per day (National Research Council, 1988; Frohlich et al., 2016), the lungs are a major barrier that protect the body from a host of external factors that enter the respiratory system and may cause lung pathologies. Mucociliary clearance (MCC) is a key aspect of the innate immune defense against airborne pathogens and inhaled particles and is governed by the concerted action of its functional components, the cilia and the airway surface liquid (ASL), which comprises mucus and the periciliary layer (Bustamante-Marin and Ostrowski, 2017). In healthy subjects, ≥10 mL airway secretions are continuously produced and transported daily by the mucociliary escalator. Disturbances in any of the processes regulating ASL volume, mucus production, mucus viscoelastic properties, or ciliary function can cause MCC dysfunction and are linked to airway diseases such as chronic obstructive pulmonary disease (COPD) or asthma, both of which bear a significant risk of increased morbidity and mortality. The mechanism by which exposure to inhaled toxicants might lead to mucus hypersecretion and thereby impact pulmonary function has already been mapped in AOP148 on decreased lung function. However, whether an exposure-related decline in lung function is solely related to excessive production of mucus is debatable, particularly in light of the close relationship between mucus, ciliary function, and efficient MCC. To date, no single event has been attributed to MCC impairment, and it is likely that events described in this AOP as well as in AOP148 have to culminate to lead to decreased lung function.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Stressors

Acrolein

Acrolein, a ubiquitous environmental pollutant, is a highly reactive unsaturated aldehyde that exerts toxicity through several mechanism, including oxidative stress (Moghe et al., 2015). Acrolein exposure decreased CFTR-mediated Cl− transport in primary murine nasal septal epithelia, in human bronchial epithelial cells grown in monolayers and in human Calu-3 lung cancer cells (Alexander et al., 2012; Raju et al., 2013), transiently reduced CBF at low concentrations (0.5‒1 mM) and induced ciliostasis at high concentrations (> 1 mM) in rabbit tracheal epithelial cells (Romet et al., 1990), and significantly increased mucin production in rats (Chen et al., 2013; Liu et al., 2009; Borchers et al., 1998; Wang et al., 2009). In addition, exposure of Fischer rats to acrolein caused a left shift in the quasi-static compliance curves and increased lung volumes, indicative of airway obstruction (Costa et al., 1986).

Ozone

Tracheas of Wistar rats exposed to 1.5 ppm ozone for 1 h/day for 3 days exhibited reduced CFTR protein expression. Similarly, at 4 hours following a 30-min exposure to ozone, CFTR mRNA and protein were down-regulated in 16HBE14o- cells. At 24 hours post-exposure, a reduction in forskolin-stimulated CFTR Cl− conductance was observed (Qu et al., 2009).

Continuous, exposure of human nasal epithelial cells to different concentrations of ozone at 37°C for up to 4 weeks slightly (but not significantly) reduced CBF in healthy mucosa (7.1% at 500 µg/m³ and 10.3% at 1000 µg/m³), and significantly in chronically inflamed mucosa (20.5/16.4%) at 2 weeks. During the third and fourth week of exposure at these higher concentrations CBF was significantly reduced in both healthy (after 3 weeks: 18.7/37.5%; after 4 weeks: 11.1/33.3%) and chronically inflamed mucosa (after 3 weeks: 33.8/26.8%; after 4 weeks: 21.4/38.6%). Low ozone concentrations (100 µg/m³) appeared to not have an effect on CBF (Gosepath et al., 2000).

Acute exposure (2 h) of adult ewes to 1.0 ppm ozone significantly reduced tracheal mucus transport velocity (TMV) at 40 min and 2 h post-exposure. Repeated exposure to 1.0 ppm ozone for 5 hper day, for 4 consecutive days showed a progressively significant decrease in TMV on the first and second days, and stabilized over the third and fourth days, around values ranging from -42% to -55% of the initial baseline. TMV remained depressed even after the end of exposure, persisting up to 5 days post-exposure (Allegra et al., 1991).

Acute exposure of healthy young adult subjects (aged 19 to 35 years, non-smokers) to 0.06 ppm ozone for 6.6 h resulted in a 1.71 + 0.50% (mean + SEM) decrease in FEV1 and a 2.32 + 0.41% decrease in FVC compared with air exposure (Kim et al., 2011).

A US-based study found inverse associations between increasing lifetime exposure to ozone (estimated median: 36 ppm; interquartile range 29–45 ppm; range 19–64 ppm) and FEF75 and FEF25–75 in adolescents (aged 18–20 years) (Tager et al., 2005).
 

Cigarette smoke

CFTR transcript and protein levels were reduced in human Calu-3 lung cancer cells exposed to the gas phase of cigarette smoke (Cantin et al., 2006b), human immortalized bronchial epithelial 16HBE14o- cells treated with 10% cigarette smoke extract (Hassan et al., 2014; Rasmussen et al., 2014; Xu et al., 2015), differentiated primary human bronchial epithelial cells exposed to whole cigarette smoke (Sloane et al., 2012; Hassan et al., 2014), and in airways of smokers compared to non-smokers (Dransfield et al., 2013). Following exposure to cigarette smoke, Cl⁻ conductance (i.e., CFTR-mediated Cl⁻ transport) decreased in primary human bronchial epithelial cells grown in monolayers (Lambert et al., 2014), differentiated primary human bronchial epithelial cells (Schmid et al., 2015; Chinnapaiyan et al., 2018), and nasal respiratory and intestinal epithelia of A/J mice (Raju et al., 2013; Raju et al., 2017).
In the lower airways, healthy smokers and smokers with chronic obstructive pulmonary disease (COPD) showed reduced CFTR-dependent Cl⁻ transport, whereas COPD former smokers showed an intermediate response to chloride-free isoproterenol solution compared to non-smokers. Similarly, amiloride-sensitive lower airway potential difference was also lower in healthy smokers and COPD smokers than in healthy non-smokers. This was linked to reduced CFTR protein levels in the airways of smokers compared to non-smokers, although there were no significant differences between healthy and COPD subjects (Dransfield et al., 2013). CFTR-dependent Cl⁻ conductance as measured by nasal potential difference was also significantly reduced in healthy and COPD smokers compared to healthy non-smokers or to former smokers with COPD (Sloane et al., 2012). In addition, healthy never-smokers had higher mean sweat chloride concentrations than COPD smokers and COPD former smokers (Raju et al., 2013; Courville et al., 2014).

Multiple studies showed that exposure of primary human bronchial epithelial cells, either undifferentiated or differentiated at the air-liquid interface, to cigarette smoke decreased ASL height (Hassan et al., 2014; Lambert et al., 2014; Raju et al., 2016; Rasmussen et al., 2014; Schmid et al., 2015). Treatment of immortalized bronchial epithelial 16HBE14o- cells with 10% cigarette smoke extract for 48 hours also resulted in a significant reduction in ASL height (Xu et al., 2015).

Treatment of human sinonasal epithelial cells with cigarette smoke condensate significantly reduced forskolin-stimulated CBF (Cohen et al., 2009). CBF was also decreased in differentiated normal human bronchial epithelial cells exposed to whole cigarette smoke (Schmid et al., 2015), in cilia-bearing explant adenoid tissues treated with 5 and 10% cigarette smoke extract (Wang et al., 2012), in hamster oviducts treated with various mainstream cigarette smoke fractions (Knoll et al., 1995), and in nasal epithelial cells fom smokers with moderate and severe chronic obstructive pulmonary disease (COPD) (Yaghi et al., 2012).

Whole cigarette smoke exposure or treatment with cigarette smoke extract of normal human bronchial epithelial cells significantly lowered FoxJ1 mRNA and protein levels (Milara et al., 2012; Brekman et al., 2014; Valencia-Gattas et al., 2016; Ishikawa and Ito, 2017). Cigarette smoke extract treatment of normal human bronchial epithelial cells also reduced the expression of cilia-related transcription factor genes, including FOXJ1, RFX2, and RFX3, as well as that of cilia motility and structural integrity genes regulated by FOXJ1, including DNAI1, DNAH5, DNAH9, DNAH10, DNAH11, and SPAG6 (Brekman et al., 2014).

Exposure of human bronchial epithelial cells cultured at the air-liquid interface to cigarette smoke extract during differentiation significantly shortened the average cilia length compared to untreated cultures, and treatment of differentiated cultures prevented elongation of cilia seen in untreated cultures
(Brekman et al., 2014). Whole smoke exposure of mouse tracheal epithelial cells differentiated at the air-liquid interface resulted in cilia shortening and also complete loss of cilia at 24 h post-exposure (Lam et al., 2013). Cilia length was also reduced in mouse nasal septal epithelial cells treated with cigarette smoke condensate (Tamashiro et al., 2009). Cilia length was reduced in endobronchial biopsies and airway brushings of smokers compared to nonsmokers (Leopold et al., 2009) and in COPD smokers compared to healthy smokers and nonsmokers (Hessel et al., 2014). In adults with adults with chronic sputum production, current and former smokers had a higher frequency of axonemal ultrastructural abnormalities than non-smokers and controls (Verra et al., 1994).

Nasomuciliary clearance time (determined by saccharin transit test) was significantly higher in smokers than in non-smokers and correlated positively with cigarettes per day and packs/year index (Proença et al., 2011; Baby et al., 2014; Yadav et al., 2014; Habesoglu et al., 2012; Pagliuca et al., 2015; Xavier et al., 2013; Dülger et al., 2018; Solak et al., 2018; Polosa et al., 2021).

Smoking decreased pulmonary function including forced vital capacity (FVC), forced expiratory volume in one second (FEV1) and FEF25–75 (Kuperman and Riker, 1973;  Ashley et al., 1975, Tantisuwat and Thaveeratitham, 2014, Gold et al., 1996; Broekema et al., 2009).

Nitrogen dioxide

Exposure of human bronchial epithelial cells to 100, 400, and 800 ppb NO₂ decreased CBF by 2.3±1.7% , 3.0±2.5%, and 6.8±1.7% respectively, which was not significant relative to incubator controls. Exposure of the cells to 2000 ppb NO₂ significantly decreased CBF by 14.2±2.5% in comparison with controls (Devalia et al., 1993).

Diesel engine exhaust

Incubation of human primary bronchial epithelial cells differentiated at the air-liquid interface with Diesel exhaust particles (DEP; 100 µg/mL = 16.26 ng/mL phenanthrene, 3.65 ng/mL fluoranthene, 2.53 ng/mL pyrene) attenuated CBF in a time- and dose-dependent manner. Exposure to 10 µg/mL DEP decreased CBF by 40% (Q1 = 19, Q3 = 46) from baseline after 24-h incubation. Similarly, exposure to 50 µg/mL DEP, filtered DEP solution, or 100 µg/mL DEP decreased CBF by 51% (Q1 = 49, Q3 = 56), 33% (Q1 = 26, Q3 = 36), and 73% (Q1 = 65, Q3 = 83), respectively, from baseline after 24-h incubation. Changes in CBF started to become significant at 4 h with 50 µg/mL DEP and at 2 h with 100 µg/mL DEP compared to untreated cultures (Bayram et al., 1998).

Overall Assessment of the AOP

The experimental evidence to support the biological plausibility of the KERs from MIE to AO is moderate to strong overall for the AOP presented here, while there is a moderate concordance of dose-response relationships. The weakest evidence is for the KER of decreased CFTR function leading to decreased ASL height (KERC), due to both insufficient experimental evidence of causality and a scarcity of quantitative data on dose-related responses and temporal concordances. In terms of essentiality, we have rated all of the KEs as either moderate or high.

AOPs such as this one can play a central role in risk assessment strategies for a wide variety of regulatory purposes by providing mechanistic support to an integrated approach to testing and assessment (IATA; (Clippinger et al., 2018)). IATAs are flexible frameworks that can be adapted to best address the regulatory question or purpose at hand. More specifically, this AOP can be applied to the risk assessment of inhaled toxicants, by enabling the development of testing strategies through the assembly of existing information and the generation of new data where they are currently lacking. Targeted approaches to fill data gaps can be developed using new approach methodologies (NAMs) informed by this AOP.

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

All KE proposed in this AOP network occur and are measurable in several species, including frogs, mice, rats, guinea pigs, ferrets, cats, dogs, cows, monkeys, and humans. The majority of the supporting empirical evidence derives from studies in rodent and human systems, and experimental findings in animals appear to be highly translatable to humans.

Data regarding the applicability of KE to all life-stages from birth to adulthood are available for the MIE (Oxidative Stress), KE2 (Cilia Beat Frequency, Decreased), KE3 (Mucociliary Clearance, Decreased), and AO (Decreased Lung Function), and indicate that they apply to all life stages. It is also worth noting here that age-dependent decreases in CBF, MCC, and lung function have been demonstrated in several species (e.g., guinea pigs, mice, and humans) and reflect normal physiological aging processes (Bailey et al., 2014; Grubb et al., 2016; Ho et al., 2001; Joki and Saano, 1997; Paul et al., 2013; Sharma and Goodwin, 2006).

Gender-specific data relevant to the AOP network are not as widely available as species-specific data, and to our knowledge, the role of gender has not been systematically evaluated for all KE described here. Informative evidence on gender differences stems from patients with chronic pulmonary diseases, such as cystic fibrosis, asthma, COPD, and bronchiectasis, that are characterized by decreased lung function. Considering the importance of efficient MCC—brought about by the interactions of ciliary function, ASL homeostasis and mucus properties—for normal physiological function, we consider this AOP applicable to both genders.

Essentiality of the Key Events

The definition of essentiality implies that the modulation of upstream KEs impacts the downstream KEs in an expected fashion. If blocked or failing to occur, the KEs in the current AOP will not necessarily stop the progression to subsequent KEs. Due to the complex biology of motile cilia formation and function, ASL homeostasis, mucus properties and MCC, the KEs and AO may be triggered because of alternative pathways or biological redundancies. However, when exacerbated, the KEs promote the occurrence of downstream events eventually leading to the AO. The causal pathway starting from the exposure to oxidants and leading to decreased lung function involves parallel routes with KEs, each of which is sufficient to cause the downstream KE to occur. Based on the evidence we judge the key events MIE (Oxidative Stress), KE2 (Cilia Beat Frequency, Decreased), and KE3 (Mucociliary Clearance, Decreased) as highly essential. 

Weight of Evidence Summary

We judge the overall biological plausibility of this AOP as strong. The KER Oxidative stress leading to decreased CBF is supported by multiple studies across different species with ample empirical evidence reflecting both dose-response and time concordance. The KER Decreased CBF leading to decreased MCC lacks this expanse of empirical evidence, or the evidence does not fully support the causality between the KE even though the relationship is logical and plausible. 

Quantitative Consideration

Overall, our quantitative understanding of the AOP network is moderate.

There is robust evidence that provides an insight into several KER presented here, and the dose response and temporal relationship between the two KE in question are well described and quantified for different stressors across different test systems (Oxidative stress leading to decreased CFTR function, Decreased CFTR function leading to decreased ASL height; Decreased FOXJ1 protein leading to decreased motile cilia length/number; Decreased motile cilia length/number leading to decreased cilia beating frequency; Decreased cilia beat frequency leading to decreased MCC; Increased mucus viscosity leading to decreased MCC; Oxidative stress leading to decreased cilia beat frequency). 

In some instances, we are less confident in our quantitative understanding. For example, for the KER Decreased ASL height leading to decreased cilia beat frequency, empirical evidence supporting causality between the two KE is lacking as is quantitative evidence. Dose response data as well as data supportive of the KE causality are limited for the KER Decreased MCC leading to decreased lung function. Similarly, the KER Decreased ASL height leading to increased mucus viscosity is supported by only a small number of studies, most of which evaluated the KEs in parallel, without interrogating dose responses and/or time responses. 

Considerations for Potential Applications of the AOP (optional)

Given the individual and public health burden of the consequences of lung function impairment, gaining a greater understanding of the underlying mechanisms is extremely important in the risk assessment of respiratory toxicants. An integrated assessment of substances with the potential to be inhaled, either intentionally or unintentionally, could incorporate inhalation exposure and dosimetry modelling to inform an in vitro approach with appropriate exposure techniques and cell systems to assess KEs in this AOP (EPA’s Office of Chemical Safety and Pollution Prevention, 2019). Standardization and robustness testing of assays against explicit performance criteria using suitable reference materials can greatly increase the level of confidence in their use for KE assessment (Petersen et al., In Press). Much of the empirical evidence that supports the KERs in the qualitative AOP described here was obtained from in vitro studies using well-established methodologies for biological endpoint assessment. Being chemical agnostic, this AOP can be applied to a variety of substances that share the AO. For example, impaired MCC and decreased lung function have a long-known relationship with smoking, but little is known about the consequences of long-term use of alternative inhaled nicotine delivery products such as electronic cigarettes and heated tobacco products. This AOP can form the basis of an assessment strategy to evaluate the effects of exposure to aerosol from these products based on the KEs identified here. 

References

Bailey, K.L., Bonasera, S.J., Wilderdyke, M., Hanisch, B.W., Pavlik, J.A., DeVasure, J., et al. (2014). Aging causes a slowing in ciliary beat frequency, mediated by PKCε. Am. J. Physiol. Lung Cell. Mol. Physiol. 306, L584-L589.

Bustamante-Marin, X.M., and Ostrowski, L.E. (2017a). Cilia and Mucociliary Clearance. Cold Spring Harb. Persp. Biol. 9, a028241. 

Clippinger, A.J., Allen, D., Behrsing, H., BéruBé, K.A., Bolger, M.B., Casey, W., et al. (2018). Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity. Toxicol. In Vitro 52, 131-145.

EPA’s Office of Chemical Safety and Pollution Prevention (2019). "FIFRA Scientific Advisory Panel Meeting Minutes and Final Report No. 2019-01 Peer Review on Evaluation of a Proposed Approach to Refine the Inhalation Risk Assessment for Point of Contact Toxicity: A Case Study Using a New Approach Methodology (NAM) December 4 and 6, 2018 FIFRA Scientific Advisory Panel Meeting". U.S. Environmental Protection Agency).

Frohlich, E., Mercuri, A., Wu, S., and Salar-Behzadi, S. (2016). Measurements of Deposition, Lung Surface Area and Lung Fluid for Simulation of Inhaled Compounds. Front. Pharmacol. 7, 181. 

Grubb, B.R., Livraghi-Butrico, A., Rogers, T.D., Yin, W., Button, B., and Ostrowski, L.E. (2016). Reduced mucociliary clearance in old mice is associated with a decrease in Muc5b mucin. Am. J. Physiol. Lung Cell. Mol. Physiol. 310, L860-L867.

Ho, J.C., Chan, K.N., Hu, W.H., Lam, W.K., Zheng, L., Tipoe, G.L., et al. (2001). The effect of aging on nasal mucociliary clearance, beat frequency, and ultrastructure of respiratory cilia. Am. J. Respir. Crit. Care Med. 163, 983-988.

Joki, S., and Saano, V. (1997). Influence of ageing on ciliary beat frequency and on ciliary response to leukotriene D4 in guinea-pig tracheal epithelium. Clin. Exp. Pharmacol. Physiol. 24, 166-169.

National Research Council (1988). Air Pollution, the Automobile, and Public Health. Washington, DC: The National Academies Press.

Paul, P., Johnson, P., Ramaswamy, P., Ramadoss, S., Geetha, B., and Subhashini, A. (2013). The effect of ageing on nasal mucociliary clearance in women: a pilot study. ISRN 2013, 598589.

Petersen, E.J., Sharma, M., Clippinger, A.J., Gordon, J., Katz, A., Laux, P., et al. (2021). Use of Cause-and-Effect Analysis to Optimize the Reliability of In Vitro Inhalation Toxicity Measurements Using an Air–Liquid Interface. Chem. Res. Toxicol. 34, 1370–1385.

Sharma, G., and Goodwin, J. (2006). Effect of aging on respiratory system physiology and immunology. Clin. Interv. Aging 1, 253-260. 

Appendix 1

List of MIEs in this AOP

Event: 1392: Oxidative Stress

Short Name: Oxidative Stress

AOPs Including This Key Event

Stressors

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Oxidative stress is produced in, and can occur in, any species from bacteria through to humans.

Key Event Description

Oxidative stress is defined as an imbalance in the production of reactive oxygen species (ROS) and antioxidant defenses. High levels of oxidizing free radicals can be very damaging to cells and molecules within the cell.  As a result, the cell has important defense mechanisms to protect itself from ROS. For example, Nrf2 is a transcription factor and master regulator of the oxidative stress response. During periods of oxidative stress, Nrf2-dependent changes in gene expression are important in regaining cellular homeostasis (Nguyen, et al. 2009) and can be used as indicators of the presence of oxidative stress in the cell.

In addition to the directly damaging actions of ROS, cellular oxidative stress also changes cellular activities on a molecular level. Redox sensitive proteins have altered physiology in the presence and absence of ROS, which is caused by the oxidation of sulfhydryls to disulfides (2SH àSS) on neighboring amino acids (Antelmann and Helmann 2011). Importantly Keap1, the negative regulator of Nrf2, is regulated in this manner (Itoh, et al. 2010).

Protection against oxidative stress is relevant for all tissues and organs, although some tissues may be more susceptible. For example, the brain possesses several key physiological features, such as high O2 utilization, high polyunsaturated fatty acids content, presence of autooxidable neurotransmitters, and low antioxidant defenses as compared to other organs, that make it highly susceptible to oxidative stress (Halliwell, 2006; Emerit and al., 2004; Frauenberger et al., 2016).

How it is Measured or Detected

Oxidative Stress. Direct measurement of ROS is difficult because ROS are unstable. The presence of ROS can be assayed indirectly by measurement of cellular antioxidants, or by ROS-dependent cellular damage:

-    Detection of ROS by chemiluminescence (https://www.sciencedirect.com/science/article/abs/pii/S0165993606001683)

-    Detection of ROS by chemiluminescence is also described in OECD TG 495 to assess phototoxic potential.

-    Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html). 

-    TBARS. Oxidative damage to lipids can be measured by assaying for lipid peroxidation using TBARS (thiobarbituric acid reactive substances) using a commercially available kit. 

-    8-oxo-dG. Oxidative damage to nucleic acids can be assayed by measuring 8-oxo-dG adducts (for which there are a number of ELISA based commercially available kits),or  HPLC, described in Chepelev et al. (Chepelev, et al. 2015).

Molecular Biology: Nrf2. Nrf2’s transcriptional activity is controlled post-translationally by oxidation of Keap1. Assay for Nrf2 activity include:
-    Immunohistochemistry for increases in Nrf2 protein levels and translocation into the nucleus; 

-    Western blot for increased Nrf2 protein levels; 

-    Western blot of cytoplasmic and nuclear fractions to observe translocation of Nrf2 protein from the cytoplasm to the nucleus; 

-    qPCR of Nrf2 target genes (e.g., Nqo1, Hmox-1, Gcl, Gst, Prx, TrxR, Srxn), or by commercially available pathway-based qPCR array (e.g., oxidative stress array from SABiosciences);

-    Whole transcriptome profiling by microarray or RNA-seq followed by pathway analysis (in IPA, DAVID, metacore, etc.) for enrichment of the Nrf2 oxidative stress response pathway (e.g., Jackson et al. 2014);

-    OECD TG422D describes an ARE-Nrf2 Luciferase test method;

-    In general, there are a variety of commercially available colorimetric or fluorescent kits for detecting Nrf2 activation.

References

Antelmann, H., Helmann, J.D., 2011. Thiol-based redox switches and gene regulation. Antioxid. Redox Signal. 14, 1049-1063.

Chepelev, N.L., Kennedy, D.A., Gagne, R., White, T., Long, A.S., Yauk, C.L., White, P.A., 2015. HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in Cultured Cells and Animal Tissues. J. Vis. Exp. (102):e52697. doi, e52697.

Emerit, J., Edeas, M., Bricaire, F., 2004. Neurodegenerative diseases and oxidative stress. Biomed. Pharmacotherapy. 58(1): 39-46.

Frauenberger, E.A., Scola, G., Laliberté, V.L.M., Duong, A., Andreazza, A.C., 2015. Redox modulations, Antioxidants, and Neuropsychitrica Disorders. Ox. Med. Cell. Longevity. Vol. 2016, Article ID 4729192.

Halliwell, B., 2006. Oxidative stress and neurodegeneration: where are we now? J. Neurochem. 97(6):1634-1658.

Itoh, K., Mimura, J., Yamamoto, M., 2010. Discovery of the negative regulator of Nrf2, Keap1: a historical overview. Antioxid. Redox Signal. 13, 1665-1678.

Jackson, A.F., Williams, A., Recio, L., Waters, M.D., Lambert, I.B., Yauk, C.L., 2014. Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan. Toxicol. Applied Pharmacol.274, 63-77.

Nguyen, T., Nioi, P., Pickett, C.B., 2009. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J. Biol. Chem. 284, 13291-13295.

OECD (2018), Test No. 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264229822-en.

OECD (2019), Test No. 495: Ros (Reactive Oxygen Species) Assay for Photoreactivity, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/915e00ac-en.

List of Key Events in the AOP

Event: 1908: Cilia Beat Frequency, Decreased

Short Name: CBF, Decreased

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Evidence for Perturbation by Stressor

Cigarette smoke

Treatment of human sinonasal epithelial cells with cigarette smoke condensate for 3 minutes significantly reduced forskolin-stimulated CBF (Cohen et al., 2009). CBF was also decreased in differentiated normal human bronchial epithelial cells exposed to whole cigarette smoke (Schmid et al., 2015), in cilia-bearing explant adenoid tissues treated with 5 and 10% cigarette smoke extract (Wang et al., 2012), in hamster oviducts treated various mainstream cigarette smoke fractions (Knoll et al., 1995), and in nasal epithelial cells fom smokers with moderate and severe chronic obstructive pulmonary disease (COPD) (Yaghi et al., 2012).

Acetaldehyde

A concentration-dependent decrease in CBF has been observed after treatment with aldehydes. For example inhibition of cilia ATPase activity was observed after treatment with acetaldehyde, in ciliated bovine bronchial epithelial cells (Sisson et al., 1991). 

Acrolein

Acrolein, an aldehyde in the gas phase of cigarette smoke, induced ciliostasis at high concentrations (> 1 mM), after 5 min of treatment, and cellular necrosis after 3 hr. However, at lower concentrations (from 0.5‒1 mM), acrolein transiently reduced the CBF to 4 Hz (Romet et al., 1990).

Nicotine

Normal human bronchial epithelial cells exposed to aerosolized nicotine showed decreased CFTR and BK conductance, CBF, ASL volume, and decreased expression of FOXJ1 and KCNMA1 (Garcia-Arcos et al., 2016). 

Ozone

Continuous, exposure of human nasal epithelial cells to different concentrations of ozone at 37°C for up to 4 weeks slightly (but not significantly) reduced CBF in healthy mucosa (7.1% at 500 µg/m3 and 10.3% at 1000 µg/m3), and significantly in chronically inflamed mucosa (20.5/16.4%) at 2 weeks. During the third and fourth week of exposure at these higher concentrations CBF was significantly reduced in both healthy (after 3 weeks: 18.7/37.5%; after 4 weeks: 11.1/33.3%) and chronically inflamed mucosa (after 3 weeks: 33.8/26.8%; after 4 weeks: 21.4/38.6%). Low ozone concentrations (100 µg/m3) appeared to not have an effect on CBF (Gosepath et al., 2000).

 

Sex hormone

Female hormones, i.e. progesterone and estrogen, have been shown to have direct effect on CBF, i.e., progesterone reduces CBF, 17β-estradiol and progesterone receptor antagonists counteract progesterone effects, but estradiol alone has also been shown to have no effect on CBF. However, the mechanism by which these hormones modulate CBF is yet to be elucidated (Jain et al., 2012; Jia et al., 2011).

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Age-dependent decreases in CBF have been demonstrated in several species (e.g. guinea pigs, mice, and human) (Bailey et al., 2014; Grubb et al., 2016; Ho et al., 2001; Joki and Saano, 1997; Paul et al., 2013). In a study with 46 healthy subjects with a wide age distribution (mean 42, range 19–81 years), age was found to be negatively associated with airway clearance of inhaled 6-μm Teflon particles (Svartengren et al., 2005).

Female hormones, i.e. progesterone and estrogen, have been shown to have direct effect on CBF, i.e., progesterone reduces CBF, 17β-estradiol and progesterone receptor antagonists counteract progesterone effects, but estradiol alone has also been shown to have no effect on CBF. However, the mechanism by which these hormones modulate CBF is yet to be elucidated (Jain et al., 2012; Jia et al., 2011).

Key Event Description

Cohesive beating of cilia lining the upper and lower respiratory tract is critical for efficient MCC. CBF is influenced by several factors including changes in the physical and chemical properties of the ASL (especially the periciliary fluid), structural modulation in the cilia, concentration of cyclic nucleotides cAMP and cGMP, and intracellular calcium (Ca²⁺). Formation of cyclic nucleotides such as cGMP is mediated by nitric oxide (NO), which is released by an enzyme family of nitric oxide synthases (NOSs) when the substrate L-arginine (L-Arg) is transformed to L-citrulline. NO activates its receptor protein, soluble guanylate cyclase (sGC), which catalyzes formation of cGMP from guanosine triphosphate (GTP). cGMP then activates protein kinase G (PKG) which has been implicated in the regulation of CBF (Jiao et al., 2011; Li et al., 2000). NO-dependent stimulation of CBF has also been associated with an increase in cAMP-dependent protein kinase A (PKA) (Di Benedetto et al., 1991; Lansley et al., 1992; Salathe et al., 1993; Sanderson and Dirksen, 1989; Schmid et al., 2007; Sisson et al., 1999; Uzlaner and Priel, 1999). An increase in intracellular endogenous cAMP was observed after treatment with isobutyl-1-methylxanthine that also increased CBF (Tamaoki et al., 1989). cAMP accumulation in the airway cilia has been shown to be dependent on Ca2+–calmodulin-dependent PDE1A and indirectly regulates CBF (Kogiso et al., 2018). Increase in CBF after treatment with NO substrate, L-arginine and inhibition of CBF by a NOS inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME) further provides evidence for the role of NO in increasing CBF (Jiao J. et al., 2011; Sisson J. H., 1995; Uzlaner and Priel, 1999; Yang et al., 1997). 
Modulation of CBF is not always accompanied by changes in cAMP levels. PKC activators, phorbol 12-myristate 13-acetate and L-o~-dioctanoylglycerol have been shown to decrease CBF in a concentration- and time-dependent manner in rabbit tracheal epithelial cells (Kobayashi et al., 1989). CBF has been shown to decrease after exposure to inhaled oxidants such as cigarette smoke across different species. A study with 120 subjects showed a significant decrease in nasal CBF following exposure to tobacco smoke (Agius et al., 1998). Exposure to cigarette smoke extract lead to reduction in forskolin-induced CBF in human sinonasal epithelium (Cohen et al., 2009) and  isoproterenol- and methacholine-induced CBF in human adenoid tissues (Wang et al., 2012). This decrease in CBF and unresponsiveness to beta-agonist stimulation occurs in parallel to PKC activation and has been shown to be dependent on the duration of exposure to cigarette smoke in mice (Simet et al., 2010). Normal human bronchial epithelial cells exposed to aerosolized nicotine showed decreased CFTR and BK conductance, impaired CBF, ASL volume, and decreased expression of FOXJ1 and KCNMA1 (Garcia-Arcos et al., 2016). 
A concentration-dependent decrease in CBF has been observed after treatment with aldehydes. For example inhibition of cilia ATPase activity was observed after treatment with acetaldehyde, in ciliated bovine bronchial epithelial cells (Sisson et al., 1991). Acrolein, an aldehyde in the gas phase of cigarette smoke, induced ciliostasis at high concentrations (> 1 mM), after 5 min of treatment, and cellular necrosis after 3 hr. However, at lower concentrations (from 0.5‒1 mM), acrolein transiently reduced the CBF to 4 Hz (Romet et al., 1990).
 

How it is Measured or Detected

There is no standardized method for measuring CBF. Digital high-speed video imaging with a manual count of CBF in slow motion video play is the most commonly used method for CBF measurement (Kim et al., 2011; Peabody et al., 2018). Photometry and video-microscopy have been used to measure CBF in vitro and ex vivo, including in ciliated bovine bronchial epithelial cells (Allen-Gipson et al., 2011; Sisson et al., 2003; Uzlaner and Priel, 1999), normal human bronchial epithelial cells (Feriani et al., 2017), human nasal epithelial cells (Dimova et al., 2005; Min et al., 1999b), human nasal ciliated epithelium (nasal brushings) (Agius et al., 1998), and mouse tracheal rings (Simet et al., 2010).
CBF measurement in vitro generally involves mounting the tissue at the air-liquid interface on a stage followed by microscopic analysis and acquisition of images and/or video recordings of beating cilia. For in vivo and ex vivo measurements, Doppler optical coherence tomography (D-OCT) can also be applied, a mesoscopic non-contact imaging modality that provides high-resolution tomographic images and detects micromotion simultaneously (Jing et al., 2017). D-OCT has been used to quantitatively measure CBF in ex vivo rabbit tracheal cultures (Lemieux et al., 2015).

References

Agius, A. M., L. A. Smallman, and A. L. Pahor (1998). Age, smoking and nasal ciliary beat frequency. Clin. Otolaryngol. Allied Sci. 23, 227-230.

Allen-Gipson, D.S., Blackburn, M.R., Schneider, D.J., Zhang, H., Bluitt, D.L., Jarrell, J.C., et al. (2011). Adenosine activation of A(2B) receptor(s) is essential for stimulated epithelial ciliary motility and clearance. Am. J. Physiol. Lung Cell. Mol. Physiol. 301, L171-L180.

Bailey, K.L., Bonasera, S.J., Wilderdyke, M., Hanisch, B.W., Pavlik, J.A., Devasure, J., et al. (2014). Aging causes a slowing in ciliary beat frequency, mediated by PKCε. Am. J. Physiol. Lung Cell. Mol. Physiol. 306, L584-L589.

Cohen, N.A., Zhang, S., Sharp, D.B., Tamashiro, E., Chen, B., Sorscher, E.J., et al. (2009). Cigarette smoke condensate inhibits transepithelial chloride transport and ciliary beat frequency. Laryngoscope 119, 2269-2274.

Di Benedetto, G., Manara-Shediac, F.S. and Mehta, A. (1991). Effect of cyclic AMP on ciliary activity of human respiratory epithelium. Eur. Respir. J. 4, 789-795.

Dimova, S., Maes, F., Brewster, M.E., Jorissen, M., Noppe, M. and Augustijns, P. (2005). High-speed digital imaging method for ciliary beat frequency measurement. J. Pharmacy Pharmacol 57, 521-526.

Feriani, L., Juenet, M., Fowler, C.J., Bruot, N., Chioccioli, M., Holland, S.M., et al. (2017). Assessing the Collective Dynamics of Motile Cilia in Cultures of Human Airway Cells by Multiscale DDM. Biophys. J. 113, 109-119.

Garcia-Arcos, I., Geraghty, P., Baumlin, N., Campos, M., Dabo, A.J., Jundi, B., et al. (2016). Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner. Thorax 71, 1119-1129.

Gosepath, J., Schaefer, D., Brommer, C., Klimek, L., Amedee, R.G., and Mann, W.J. (2000). Subacute Effects of Ozone Exposure on Cultivated Human Respiratory Mucosa. Am. J. Rhinol. 14, 411-418. 

Grubb, B.R., Livraghi-Butrico, A., Rogers, T.D., Yin, W., Button, B. and Ostrowski, L.E. (2016). Reduced mucociliary clearance in old mice is associated with a decrease in Muc5b mucin. Am. J. Physiol. Lung Cell. Mol. Physiol. 310, L860-L867.

Ho, J.C., Chan, K.N., Hu, W.H., Lam, W.K., Zheng, L., Tipoe, G.L., et al. (2001). The Effect of Aging on Nasal Mucociliary Clearance, Beat Frequency, and Ultrastructure of Respiratory Cilia. Am. J. Respir. Crit. Care Med. 163, 983-988.

Jain, R., Ray, J.M., Pan, J.-H. and Brody, S.L. (2012). Sex hormone-dependent regulation of cilia beat frequency in airway epithelium. Am. J. Respir. Crit. Care Med. 46, 446-453.

Jia, S., Zhang, X., He, D.Z., Segal, M., Berro, A., Gerson, T., et al., 2011. Expression and Function of a Novel Variant of Estrogen Receptor–α36 in Murine Airways. Am. J. Respir. Cell Mol. Biol. 45, 1084-1089.

Jiao, J., Wang, H., Lou, W., Jin, S., Fan, E., Li, Y., et al. (2011). Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells. Exp. Cell Res. 317, 2548-2553.

Jing, J.C., Chen, J.J., Chou, L., Wong, B.J.F. and Chen, Z. (2017). Visualization and Detection of Ciliary Beating Pattern and Frequency in the Upper Airway using Phase Resolved Doppler Optical Coherence Tomography. Sci. Rep. 7, 8522-8522.

Joki, S. and Saano, V. (1997). Influence of ageing on ciliary beat frequency and on ciliary response to leukotriene D4 in guinea‐pig tracheal epithelium. Clin. Exp. Pharmacol. Physiol. 24, 166-169.

Kim, W., Han, T.H., Kim, H.J., Park, M.Y., Kim, K.S. and Park, R.W. (2011). An Automated Measurement of Ciliary Beating Frequency using a Combined Optical Flow and Peak Detection. J. Healthc. Inform. Res. 17, 111-119.

Knoll, M., Shaoulian, R., Magers, T. and Talbot, P. (1995). Ciliary beat frequency of hamster oviducts is decreased in vitro by exposure to solutions of mainstream and sidestream cigarette smoke. Biol. Reprod. 53, 29-37.

Kobayashi, K., Tamaoki, J., Sakai, N., Chiyotani, A. and Takizawa, T. (1989). Inhibition of ciliary activity by phorbol esters in rabbit tracheal epithelial cells. Lung 167, 277-284.

Kogiso, H., Hosogi, S., Ikeuchi, Y., Tanaka, S., Inui, T., Marunaka, Y., et al. (2018). [Ca(2+) ]i modulation of cAMP-stimulated ciliary beat frequency via PDE1 in airway ciliary cells of mice. Exp. Physiol. 103, 381-390.

Lansley, A.B., Sanderson, M.J. and Dirksen, E.R. (1992). Control of the beat cycle of respiratory tract cilia by Ca2+ and cAMP. Am. J. Physiol. 263, L232-242.

Lemieux, B.T., Chen, J.J., Jing, J., Chen, Z. and Wong, B.J.F. (2015). Measurement of ciliary beat frequency using Doppler optical coherence tomography. Int. Forum Allergy Rhinol. 5, 1048-1054.

Li, D., Shirakami, G., Zhan, X. and Johns, R.A. (2000). Regulation of ciliary beat frequency by the nitric oxide-cyclic guanosine monophosphate signaling pathway in rat airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 23, 175-181.

Min, Y.-G., Ohyama, M., Lee, K.S., Rhee, C.-S., Oh, S.H., Sung, M.-W., et al. (1999). Effects of free radicals on ciliary movement in the human nasal epithelial cells. Auris Nasus Larynx 26, 159-163.

Paul, P., Johnson, P., Ramaswamy, P., Ramadoss, S., Geetha, B. and Subhashini, A.S. (2013). The Effect of Ageing on Nasal Mucociliary Clearance in Women: A Pilot Study. ISRN Pulmonology 2013, 5.

Peabody, J.E., Shei, R.-J., Bermingham, B.M., Phillips, S.E., Turner, B., Rowe, S.M., et al. (2018). Seeing cilia: imaging modalities for ciliary motion and clinical connections. Am. J. Physiol. Lung Cell. Mol. Physiol. 314, L909-L921.

Romet, S., Dubreuil, A., Baeza, A., Moreau, A., Schoevaert, D. and Marano, F. (1990). Respiratory tract epithelium in primary culture: Effects of. Toxicol. In Vitro 4, 399-402.

Salathe, M., Pratt, M.M. and Wanner, A. (1993). Cyclic AMP-dependent phosphorylation of a 26 kD axonemal protein in ovine cilia isolated from small tissue pieces. Am. J. Respir. Cell Mol. Biol. 9, 306-314.

Sanderson, M.J. and Dirksen, E.R. (1989). Mechanosensitive and beta-adrenergic control of the ciliary beat frequency of mammalian respiratory tract cells in culture. Am. Rev. Respir. Dis. 139, 432-440.

Schmid, A., Sutto, Z., Nlend, M.-C., Horvath, G., Schmid, N., Buck, J., et al. (2007). Soluble Adenylyl Cyclase Is Localized to Cilia and Contributes to Ciliary Beat Frequency Regulation via Production of cAMP. J. Gen. Physiol. 130, 99-109.

Schmid, A., Baumlin, N., Ivonnet, P., Dennis, J.S., Campos, M., Krick, S., et al. (2015). Roflumilast partially reverses smoke-induced mucociliary dysfunction. Respir. Res. 16, 135.

Simet, S.M., Sisson, J.H., Pavlik, J.A., Devasure, J.M., Boyer, C., Liu, X., et al. (2010). Long-term cigarette smoke exposure in a mouse model of ciliated epithelial cell function. Am. J. Respir. Cell Mol. Biol. 43, 635-640.

Sisson, J.H. (1995). Ethanol stimulates apparent nitric oxide-dependent ciliary beat frequency in bovine airway epithelial cells. Am. J. Physiol. 268, L596-600.

Sisson, J.H., May, K. and Wyatt, T.A. (1999). Nitric oxide-dependent ethanol stimulation of ciliary motility is linked to cAMP-dependent protein kinase (PKA) activation in bovine bronchial epithelium. Alcohol Clin. Exp. Res. 23, 1528-1533.

Sisson, J.H., Stoner, J., Ammons, B. and Wyatt, T. (2003). All‐digital image capture and whole‐field analysis of ciliary beat frequency. J. Microsc. 211, 103-111.

Sisson, J.H., Tuma, D.J. and Rennard, S.I. (1991). Acetaldehyde-mediated cilia dysfunction in bovine bronchial epithelial cells. Am. J. Physiol. 260, L29-36.

Svartengren, M., Falk, R. and Philipson, K. (2005). Long-term clearance from small airways decreases with age. Eur. Respir. J. 26, 609-615.

Tamaoki, J., Kondo, M. and Takizawa, T. (1989). Effect of cAMP on ciliary function in rabbit tracheal epithelial cells. J. Appl. Physiol. 66, 1035-1039.

Uzlaner, N. and Priel, Z. (1999). Interplay between the NO pathway and elevated [Ca(2+)](i) enhances ciliary activity in rabbit trachea. J. Physiol. 516, 179-190.

Wang, L.F., White, D.R., Andreoli, S.M., Mulligan, R.M., Discolo, C.M. and Schlosser, R.J. (2012). Cigarette smoke inhibits dynamic ciliary beat frequency in pediatric adenoid explants. Otolaryngol. Head Neck Surg. 146, 659-663.

Yaghi, A., Zaman, A., Cox, G. and Dolovich, M.B. (2012). Ciliary beating is depressed in nasal cilia from chronic obstructive pulmonary disease subjects. Respir. Med. 106, 1139-1147.

Yang, B., Schlosser, R.J. and Mccaffrey, T.V. (1997). Signal transduction pathways in modulation of ciliary beat frequency by methacholine. Ann. Otol. Rhinol. Laryngol. 106, 230-236.

Event: 1909: Mucociliary Clearance, Decreased

Short Name: MCC, Decreased

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Evidence for Perturbation by Stressor

Sulfur dioxide

SO₂ exposure of dogs dose-dependently decreased CBF and also caused a marked decrease in mean bronchial mucociliary clearance (from 53.7 ± 5.7% to 32.8 ± 7.7%) after 90 min (Yeates et al., 1997). In guinea pig tracheas, SO₂ exposure affected CBF, albeit non-significantly, and mucociliary activity (Knorst et al., 1994).

Formaldehyde

Treatment of frog palate epithelium with different concentrations of formaldehyde induced significant decreases in CBF and MCC (Fló-Neyret et al., 2001; Morgan et al., 1984). Exposure of F344 rats to formaldehyde caused epithelial adaptation of the nasal epithelium, effectively reducing the number of ciliated cells (and hence cilia beating activity) through squamous metaplasia. At the same time, formaldehyde exposure resulted in “ciliastasis” or loss of ciliary activity in a concentration- and exposure duration-dependent manner as well as in a slowing of mucus flow rates (Morgan et al., 1986). 

PM10

Incubation of frog palates with PM10 from Sao Paolo, Brazil, for up to 120 min decreased mucociliary transport at concentrations ≥1000 pg/m³ (Macchione et al., 1999).

Nitric oxide

In New Zealand white rabbits exposed to 3 ppm NO₂ for 24 h, the average CBF decreased from 764 beats/min to 692 beats/min and the transport velocity decreased from 5.23 mm/min to 3.03 mm/min (Kakinoki, 1998).

Ozone

Acute exposure (2 h) of adult ewes to 1.0 ppm ozone significantly reduced tracheal mucus transport velocity (TMV) at 40 min and 2 h post-exposure. Repeated exposure to 1.0 ppm ozone for 5 hper day, for 4 consecutive days showed a progressively significant decrease in TMV on the first and second days, and stabilized over the third and fourth days, around values ranging from -42% to -55% of the initial baseline. TMV remained depressed even after the end of exposure, persisting up to 5 days post-exposure (Allegra et al., 1991).
 

Cigarette smoke

Nasomuciliary clearance time (determined by saccharin transit test) was significantly higher in smokers than in non-smokers 8 h after smoking (16 ± 6 min vs 10 ± 4 min) and insignicantly higher immediately after smoking (11 ± 6 min vs 10 ± 4 min). Nasomuciliary clearance time correlated positively with cigarettes per day and packs/year index (Proença et al., 2011).

In a small Indian cross-sectional study, the mean nasomuciliary clearance (determined by saccharin transit test) in smokers was significantly higher than that of nonsmokers (481.2 ± 29.83 s vs 300.32 ± 17.4 s). In addition, mean nasomuciliary clearance increased as the duration of smoking increased (NMC in smoking <1 year = 492.25 ± 79.93 s, NMC in smoking for 1-5 years = 516.7 ± 34.01 s, and NMC in smoking >5 years = 637.5 ± 28.49 s) (Baby et al., 2014).

Nasomuciliary clearance (determined by saccharin transit test) in active and passive smokers was significantly higher than in non-smokers (23.08 ± 4.60 min; 20.31 ± 2.51 min vs 8.57 ± 2.12 min) (Yadav et al., 2014).

Nasomuciliary clearance (determined by saccharin transit test) was significantly higher in active smokers than in passive smokers and non-smokers (23.59 ± 12.41 min vs 12.6 ± 4.67 min; 6.4 ± 1.55 min) (Habesoglu et al., 2012).

Nasomuciliary clearance time (determined by saccharin transit test) in smokers was significantly higher than in former smokers and non-smokers (15.6 min vs 11.77 min and 11.71 min, respectively) (Pagliuca et al., 2015).

Moderate and heavy smokers had higher saccharin transit test times than light smokers and non-smokers, and there was a positive correlation between STT and cigarettes/day (Xavier et al., 2013).

The median nasal mucociliary clearance time (determined by saccharin transit test) was significantly higher in smokers (who smoked a mean of 20.6 cigarettes (median: 20) per day) than in  nonsmokers (12 (interquartile range: 5–33) min vs 9 (interquartile range: 4–12) min) (Dülger et al., 2018). 

Nasal mucociliary clearance time (determined by saccharin transit test) in smokers was significantly higher than in non-smokers (536.19 ± 254.81 s vs 320.43 ± 184.98 s) and correlated with the numbers of cigarettes per day, pack-years and smoking duration (Solak et al., 2018).

Current smokers had a median (IQR) mucociliary clearance transit time (determined by saccharin transit test) of 13.15 (9.89–16.08) min, which was significantly longer compared with that of never smokers at 7.24 (5.73–8.73) min, former smokers at 7.26 (6.18–9.17) min, exclusive e-cigarette users at 7.00 (6.38–9.00) min, and exclusive heated tobacco product users at 8.00 (6.00–8.00) min (Polosa et al., 2021).

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Key Event Description

In healthy adults, tracheal mucus movement varies from 4 to >20 mm/min (Stannard and O'Callaghan, 2006), whereas mucociliary clearance (MCC) in the small airways is slower due to the lower number of ciliated cells (fewer cilia) and their shorter length (Foster et al., 1980; Iravani, 1969; Wanner et al., 1996).
Since optimal MCC is dependent in multiple factors, including cilia number and structure as well as ASL and mucus properties, any disturbances of these can lead to impaired MCC. While high humidity or infection can enhance MCC, long-term exposure to noxious substances (e.g. cigarette smoke) lead to decreased mucus clearance from the airways. In most instances this is reflected by decreased mucus transport rates or velocities.
 

How it is Measured or Detected

In humans, MCC has been assessed traditionally following inhalation of radio-labeled particles such as 99Tcm-labeled polystyrene particles, resin particles or serum albumin and following their clearance at regular intervals by radioimaging using gamma cameras (Agnew et al., 1986; Kärjä et al., 1982). Taking into account inhalation volumes and flow rates, lung airflow, particle deposition and retention, clearance rates can be calculated and effects of e.g. drugs on MCC can be examined. Alternatively, since MCC occurs at a similar rate in the nose to that in trachea and bronchi (Andersen and Proctor, 1983; Rutland and Cole, 1981) and for ease of use, measurements of MCC can be restricted to that of nasal MCC only. Probably one of the simplest methods is the saccharin transit test (STT). For this test, a small particle of saccharin is placed behind the anterior end of the inferior turbinate. The saccharin will be transported by mucociliary action toward the nasopharynx, where its sweet taste is perceived. When MCC is impaired, saccharin transit times will increase, with a 10- to 20-minute delay being considered a clinical sign of decreased MCC. Using the same principle, the test can also be performed or complemented with dyes such as indigo carmine or methylene blue (Deborah and Prathibha, 2014).

In experimental animals, MCC has been evaluated by gamma-scintigraphy (Greiff et al., 1990; Hua et al., 2010; Read et al., 1992), fluorescence videography/fluoroscopy (in explanted tracheas etc.) (Grubb et al., 2016; Rogers  et al., 2018), or by 3D-SPECT (Ortiz Belda et al., 2016). Direct observation of particle movement across airway epithelia to determine mucus velocity or transport rates by using a fiberoptic bronchoscope may be helpful when working in larger animals such as dogs (King, 1998).
In vitro, freshly excised frog palate preparations have been used to assess cilia function and mucociliary transport by videomicroscopy (Macchione et al., 1995; Macchione et al., 1999; Trindade et al., 2007). Murine and human nasal, bronchial and small airway epithelial models grown at the air-liquid interface are also suitable in vitro test systems for determining mucus transport by tracing inert particle movement with a set-up similar to that used for assessing CBF (Benam et al., 2018; Fliegauf et al., 2013; Knowles and Boucher, 2002; Sears et al., 2015).
 

References

Agnew, J., Sutton, P., Pavia, D. and Clarke, S. (1986). Radioaerosol assessment of mucociliary clearance: towards definition of a normal range. Brit. J. Radiol. 59, 147-151.

Allegra, L., Moavero, N., and Rampoldi, C. (1991). Ozone-induced impairment of mucociliary transport and its prevention with N-acetylcysteine. Am. J. Med. 91, S67-S71.

Andersen, I. and Proctor, D. (1983). Measurement of nasal mucociliary clearance. Eur. J. Respir. Dis. Suppl. 127, 37-40.

Baby, M.K., Muthu, P.K., Johnson, P., and Kannan, S. (2014). Effect of cigarette smoking on nasal mucociliary clearance: A comparative analysis using saccharin test. Lung India 31, 39-42. 

Benam, K.H., Vladar, E.K., Janssen, W.J. and Evans, C.M. (2018). Mucociliary defense: emerging cellular, molecular, and animal models. Ann. Am. Thorac. Soc. 15, S210-S215.

Deborah, S. and Prathibha, K., 2014. Measurement of nasal mucociliary clearance. Clin. Res. Pulmonol. 2, 1019.

Dülger, S., Akdeniz, Ö., Solmaz, F., Şengören Dikiş, Ö., and Yildiz, T. (2018). Evaluation of nasal mucociliary clearance using saccharin test in smokers: A prospective study. Clin. Respir. J. 12, 1706-1710. 

Fliegauf, M., Sonnen, A.F.P., Kremer, B. and Henneke, P. (2013). Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection. PloS ONE 8, e59925.

Fló-Neyret, C., Lorenzi-Filho, G., Macchione, M., Garcia, M.L.B. and Saldiva, P.H.N. (2001). Effects of formaldehyde on the frog's mucociliary epithelium as a surrogate to evaluate air pollution effects on the respiratory epithelium. Braz. J. Med. Biol. Res. 34, 639-643.

Foster, W., Langenback, E. and Bergofsky, E. (1980). Measurement of tracheal and bronchial mucus velocities in man: relation to lung clearance. J. Appl. Physiol. 48, 965-971.

Greiff, L., Wollmer, P., Erjefält, I., Pipkorn, U. and Persson, C. (1990). Clearance of 99mTc DTPA from guinea pig nasal, tracheobronchial, and bronchoalveolar airways. Thorax 45, 841-845.

Grubb, B.R., Livraghi-Butrico, A., Rogers, T.D., Yin, W., Button, B. and Ostrowski, L.E. (2016). Reduced mucociliary clearance in old mice is associated with a decrease in Muc5b mucin. Am. J. Physiol. Lung Cell. Mol. Physiol. 310, L860-L867.

Habesoglu, M., Demir, K., Yumusakhuylu, A.C., Sahin Yilmaz, A., and Oysu, C. (2012). Does passive smoking have an effect on nasal mucociliary clearance? Otolaryngol Head Neck Surg. 147, 152-156.

Hua, X., Zeman, K.L., Zhou, B., Hua, Q., Senior, B.A., Tilley, S.L., et al. (2010). Noninvasive real-time measurement of nasal mucociliary clearance in mice by pinhole gamma scintigraphy. J. Appl. Physiol. 108, 189-196.

Iravani, J. (1969). Zum Mechanismus der Ortsabhängigkeit der Flimmeraktivität im Bronchialbaum/Location-Dependent Activity of the Ciliary Movement in the Bronchial Tree and its Possible Mechanism. In: Habermann E. et al. (eds) Naunyn Schmiedebergs Archiv für Pharmakologie. Springer, Berlin, Heidelberg.

Kakinoki Y, Ohashi Y, Tanaka A, Washio Y, Yamada K, Nakai Y, Morimoto K. (1998). Nitrogen dioxide compromises defence functions of the airway epithelium. Acta Oto-Laryngol. 118, 221-226.

Kärjä, J., Nuutinen, J. and Karjalainen, P. (1982). Radioisotopic Method for Measurement of Nasal Mucociliary Activity. Arch. Otolaryngol. 108, 99-101.

King, M. (1998). Experimental models for studying mucociliary clearance. Eur. Respir. J. 11, 222-228.

Knorst, M.M., Kienast, K., Riechelmann, H., Müller-Quernheim, J. and Ferlinz, R. (1994). Effect of sulfur dioxide on mucociliary activity and ciliary beat frequency in guinea pig trachea. Int. Arch. Occup. Environm. Health 65, 325-328.

Knowles, M.R. and Boucher, R.C. (2002). Mucus clearance as a primary innate defense mechanism for mammalian airways. J. Clin. Invest. 109, 571-577.

Macchione, M., Guimarães, E., Saldiva, P. and Lorenzi-Filho, G. (1995). Methods for studying respiratory mucus and mucus clearance. Braz. J. Med. Biol Res. 28, 1347.

Macchione, M., Oliveira, A.P., Gallafrio, C.T., Muchão, F.P., Obara, M.T., Guimarães, E.T., et al. (1999). Acute effects of inhalable particles on the frog palate mucociliary epithelium. Environm. Health Persp. 107, 829-833.

Morgan, K., Patterson, D. and Gross, E. (1986). Responses of the nasal mucociliary apparatus of F-344 rats to formaldehyde gas. Toxicol. Appl. Pharmacol. 82, 1-13.

Morgan, K.T., Patterson, D.L. and Gross, E.A. (1984). Frog palate mucociliary apparatus: structure, function, and response to formaldehyde gas. Fund. Appl. Toxicol. 4, 58-68.

Ortiz Belda, J.L., Ortiz, A., Milara Payá, J., Armengot Carceller, M., Sanz García, C., Compañ Quilis, D., et al. (2016). Evaluation of Mucociliary Clearance by Three Dimension Micro-CT-SPECT in Guinea Pig: Role of Bitter Taste Agonists. Plos ONE 11, e0164399.

Pagliuca, G., Rosato, C., Martellucci, S., De Vincentiis, M., Greco, A., Fusconi, M., et al. (2015). Cytologic and functional alterations of nasal mucosa in smokers: temporary or permanent damage? Otolaryngol Head Neck Surg 152, 740-745.

Proença, M., Xavier, R.F., Ramos, D., Cavalheri, V., Pitta, F., and Ramos, E.C. (2011). Immediate and short term effects of smoking on nasal mucociliary clearance in smokers. Revista Portuguesa de Pneumologia (English Edition) 17), 172-176.

Read, R.C., Roberts, P., Munro, N., Rutman, A., Hastie, A., Shryock, T., et al. (1992). Effect of Pseudomonas aeruginosa rhamnolipids on mucociliary transport and ciliary beating. J. Appl. Physiol. 72, 2271-2277.

Rogers, T.D., Ostrowski, L.E., Livraghi-Butrico, A., Button, B. and Grubb, B.R., 2018. Mucociliary clearance in mice measured by tracking trans-tracheal fluorescence of nasally aerosolized beads. Sci. Rep. 8, 1-12.

Rutland, J. and Cole, P.J. (1981). Nasal mucociliary clearance and ciliary beat frequency in cystic fibrosis compared with sinusitis and bronchiectasis. Thorax 36, 654-658.

Sears, P.R., Yin, W.-N. and Ostrowski, L.E. (2015). Continuous mucociliary transport by primary human airway epithelial cells in vitro. Am. J. Physiol. Lung Cell. Mol. Physiol. 309, L99-L108.

Solak, I., Marakoglu, K., Pekgor, S., Kargin, N.C., Alataş, N., and Eryilmaz, M.A. (2018). Nasal mucociliary activity changes in smokers. Konuralp Med. J. 10, 269-275.

Stannard, W. and O'callaghan, C. (2006). Ciliary function and the role of cilia in clearance. J. Aerosol Med. 19, 110-115.

Trindade, S.H.K., De Mello Júnior, J.F., De Godoy Mion, O., Lorenzi-Filho, G., Macchione, M., Guimarães, E.T., et al. (2007). Methods for Studying Mucociliary Transport. Braz. J. Otorhinolaryngol. 73, 704-712.

Wanner, A., Salathe, M. and O'riordan, T.G. (1996). Mucociliary clearance in the airways. Am. J. Respir. Crit. Care Med. 154, 1868-1902.

Xavier, R.F., Ramos, D., Ito, J.T., Rodrigues, F.M., Bertolini, G.N., Macchione, M., et al. (2013). Effects of cigarette smoking intensity on the mucociliary clearance of active smokers. Respiration 86, 479-485. 

Yadav, J., and Kaushik, G. (2014). K Ranga R. Passive smoking affects nasal mucociliary clearance. J. Indian Acad. Clin. Med. 15, 96-99.

Yeates, D.B., Katwala, S.P., Daugird, J., Daza, A.V. and Wong, L.B. (1997). Excitatory and inhibitory neural regulation of tracheal ciliary beat frequency (CBF) activated by ammonia vapour and SO2. Ann. Occup. Hyg. 41, 736-744.

List of Adverse Outcomes in this AOP

Event: 1250: Decrease, Lung function

Short Name: Decreased lung function

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Evidence for Perturbation by Stressor

Ozone

Acute exposure of healthy young adult subjects (aged 19 to 35 years, non-smokers) to 0.06 ppm ozone for 6.6 h resulted in a 1.71 + 0.50% (mean + SEM) decrease in FEV1 and a 2.32 + 0.41% decrease in FVC compared with air exposure (Kim et al., 2011).

A US-based study found inverse associations between increasing lifetime exposure to ozone (estimated median: 36; interquartile range 29–45; range 19–64) and FEF75 and FEF25–75 in adolescents (aged 18–20 years) (Tager et al., 2005).

Nitric oxide

In a Dutch cross-sectional study in school children (aged 7–13 years), NOx exposure from industrial emissions per interquartile range of 7.43 μg/m³  had a significantly lower percent predicted peak expiratory flow (PEF) (-3.67%, 95%CI -6.93% to -0.42%). Children exposed to NOx (per interquartile range of 7.43 μg/m³) also had a significantly lower percent forced vital capacity (FVC) and percent predicted 1-s forced expiratory volume (FEV1) (− 2.73 95%CI -5.21 to -0.25) (Bergstra et al., 2018). 

The  European Study of Cohorts for Air Pollution Effects (ESCAPE), a meta-analysis of 5 cohort studies on the association of air pollution with lung function, found that a 10 μg/m³ increase in NO₂ exposure was associated with lower levels of FEV1 (−14.0 mL, 95% CI −25.8 to −2.1) and FVC (−14.9 mL, 95% CI −28.7 to −1.1), and an increase of 20 μg/m³ in NOx exposure was associated with a lower level of FEV1, by −12.9 mL (95% CI −23.87 to −2.0) and of FVC, by −13.3 mL (95% CI −25.9 to −0.7) (Adam et al., 2015).

Cigarette smoke

A smoking history of > 20 pack-years decreased pulmonary function including forced vital capacity (FVC), forced expiratory volume in one second (FEV1), FEV1/FVC, and forced expiratory flow at 25–75% (FEF25–75%) (Kuperman and Riker, 1973).

In the Framingham Heart Study, cigarette smoking showed an inverse association with FVC and FEV1% (Ashley et al., 1975).

In the international Seven Countries Study, there was a dose-effect relationship between pack-years and forced expiratory volume in 0.75 s (FEV0.75) in continuous smokers without chronic bronchitis (Pelkonen et al., 2006).

In 34 male subjects aged between 15–18 years who smoked FVC was lower than in an age-matched male group that did not smoke. The most common duration of cigarette smoking was 1–3 years (47%) and the maximal number of cigarettes smoked per day was less than or equal to 10 cigarette(s) per day (88%) (Tantisuwat and Thaveeratitham, 2014). 

A dose–response relation was found between smoking and lower levels of FEV1/FVC and FEF25–75 in children between 10-18 years of age (Gold et al., 1996).

In a study of 147 asthmatics, FEV1%predicted was significantly lower in ex-smokers and current smokers compared with never-smokers (Broekema et al., 2009).

In a 6-year longitudinal study in Japanese-American men, FEV1 was lowest in current smokers (2702 mL) and in former smokers (2817 mL) at baseline. These 2 groups experienced a steeper annual decline in FEV1 (-34.4 and -22.8 mL/year, respectively, adjusted by height and age at baseline) compared with never-smokers (-20.3 mL/year) (Burchfiel et al., 1995).

Diesel engine exhaust

In a study of 733 adult females who had lived in the Tokyo metropolitan area for more than 3 years, the higher the level of air pollution, the more significantly the FEV1 was reduced (Sekine et al., 2004).

In a study in 29 healthy subjects, exposure to DE inside diesel-powered trains for 3 days was associated with reduced lung function (Andersen et al., 2019).

In workers who tested diesel engines in an assembly unit of a manufacturing plant, FEV1, FEV1/FVC, FEV25-75 and MEF were significantly reduced compared to non-exposed workers (Zhang et al., 2017).

PM10

A Taiwanese study in 1016 children between 6 and 15 years of age reported that lifetime exposure to to 25–85 μg/m³ PM10 were associated with lower FEV1, FVC, and FEF25-75 (Tsui et al., 2018).

The Swiss Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) found that an increase of 10 μg/m³ in annual mean concentration of PM10 was associated with 3.4% lower FVC and 1.6% lower FEV1 (Ackermann-Liebrich et al., 1997).

In the Health Survey for England, a 10 mg/m³ difference in PM10 across postcode sectors was associated with a lower FEV1 by 111 mL,  independent of active and passive smoking, social class, region and month of testing (Forbes et al., 2009). 

A 7 μg/m³ increase in five year means of PM10 (interquartile range) was associated with a 5.1% (95% CI: 2.5%–7.7%) decrease in FEV1, a 3.7% (95% CI: 1.8%–5.5%) decrease in FVC in the German SALIA study (Schikowski et al., 2005).

The ESCAPE project, a meta-analysis of 5 European cohorts/studies from 8 countries, reported that an increase of 10 μg/m³ in PM10 was associated with a lower level of FEV1 (−44.6 mL, 95% CI:−85.4– −3.8) and FVC (−59.0 mL, 95% CI: −112.3– −5.7) (Adam et al., 2015).

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Pulmonary function tests reflect the physiological working of the lungs. Therefore, the AO is applicable to a variety of species, including (but not limited to) rodents, rabbits, pigs, cats, dogs, horses and humans, independent of life stage and gender.

Key Event Description

Lung function is a clinical term referring to the physiological functioning of the lungs, most often in association with the tests used to assess it. Lung function loss can be caused by acute or chronic exposure to airborne toxicants or by an intrinsic disease of the respiratory system. 

Although signs of cellular injury are typically exhibited first in the nose and larynx, alveolar-capillary barrier breakdown may ultimately arise and result in local edema (Miller and Chang, 2003). Clinically, bronchoconstriction and hypoxia are seen in the acute phase, with affected subjects exhibiting shortness of breath (dyspnea) and low blood oxygen saturation, and with reduced lung function indices of airflow, lung volume and gas exchange (Hert and Albert, 1994; and How it is Measured or Detected;). When alveolar damage is extensive, the reduced lung function can develop into acute respiratory distress syndrome (ARDS). This severe compromise of lung function is reflected by decreased gas exchange indices (PaO₂/FIO₂ ≤200 mmHg, due to hypoxemia and impaired excretion of carbon dioxide), increased pulmonary dead space and decreased respiratory compliance (Matthay et al., 2019). Acute inhalation exposures to chemical irritants such as ammonia, hydrogen chloride, nitrogen oxides and ozone typically cause local edema that manifests as dyspnea and hypoxia. In cases where a breakdown of the alveolar capillary function ensues, ARDS develops. ARDS has a particularly high risk of mortality, estimated to be 30-40% (Gorguner and Akgun, 2010; Matthay et al., 2018; Reilly et al., 2019).

Lung function decrease due to reduction in lung volume is seen in pulmonary fibrosis, which can be linked to chronic exposures to e.g. silica, asbestos, metals, agricultural and animal dusts (Meltzer and Noble, 2008; Cheresh et al., 2013; Cosgrove, 2015; Trethewey and Walters, 2018). Additionally. decreased lung function occurs in pleural disease, chest wall and neuromuscular disorders, because of obesity and following pneumectomy (Moore, 2012). Decreased lung function can also be a result of narrowing of the airways by inflammation and mucus plugging resulting in airflow limitation. Decreased lung function is a feature of obstructive pulmonary diseases (e.g. asthma, COPD) and linked to a multitude of causes, including chronic exposure to cigarette smoke, dust, metals, organic solvents, asbestos, pathogens or genetic factors.

 

How it is Measured or Detected

Pulmonary function tests are a group of tests that evaluate several parameters indicative of lung size, air flow and gas exchange. Decreased lung function can manifest in different ways, and individual circumstances, including potential exposure scenarios, determine which test is used. The section outlines the tests used to evaluate lung function in humans (https://www.nhlbi.nih.gov/health-topics/pulmonary-function-tests, accessed 22 March 2021) and in experimental animals.

Lung function tests used to evaluate human lung function

The most common (“gold standard”) lung function test in human subjects is spirometry. Spirometry results are primarily used for diagnostic purposes, e.g. to discriminate between obstructive and restrictive lung diseases, and for determining the degree of lung function impairment. Specific criteria for spirometry tests have been outlined in the American Thoracic Society (ATS) and the European Respiratory Society (ERS) Task Force guidelines (Graham et al., 2019). These guidelines consist of detailed recommendations for the preparation and conduct of the test, instruction of the person tested, as well as indications and contraindications, and are complemented by additional guidance documents on how to interpret and report the test results (Pellegrino et al., 2005; Culver et al., 2017).

Spirometry measures several different parameters during forceful exhalation, including:

• Forced expiratory volume in 1 s (FEV1), the maximum volume of air that can forcibly be exhaled during the first second following maximal inhalation
• Forced vital capacity (FVC), the maximum volume of air that can forcibly be exhaled following maximal inhalation
• Vital capacity (VC), the maximum volume of air that can be exhaled when exhaling as fast as possible
• FEV1/FVC ratio
• Peak expiratory flow (PEF), the maximal flow that can be exhaled when exhaling at a steady rate
• Forced expiratory flow, also known as mid-expiratory flow; the rates at 25%, 50% and 75% FVC are given
• Inspiratory vital capacity (IVC), the maximum volume of air that can be inhaled after a full expiration

A reduced FEV1, with normal or reduced VC, normal or reduced FVC, and a reduced FEV1/FVC ratio are indices of airflow limitation, i.e., airway obstruction as seen in COPD (Moore, 2012). In contrast, airway restriction is demonstrated by a reduction in FVC, normal or increased FEV1/FVC ratio, a normal spirometry trace and potentially a high PEF (Moore, 2012).

Lung capacity or lung volumes can be measured using one of three basic techniques: 1) plethysmography, 2) nitrogen washout, or 3) helium dilution. Plethysmography consists of a series of sequential measurements in a body plethysmograph, starting with the measurement of functional residual capacity (FRC), the volume of gas present in the lung at end-expiration during tidal breathing. Once the FRC is known, expiratory reserve volume (ERV; the volume of gas that can be maximally exhaled from the end-expiratory level during tidal breathing, i.e., the FRC), vital capacity (VC; the volume change at the mouth between the positions of full inspiration and complete expiration), and inspiratory capacity (IC; the maximum volume of air that can be inhaled from FRC) are determined, and total lung capacity (TLC; the volume of gas in the lungs after maximal inspiration, or the sum of all volume compartments) and residual volume (RV; the volume of gas remaining in the lung after maximal exhalation) are calculated (Weinstock and McCannon, 2017).

The other two techniques used to measure lung volumes—helium dilution and nitrogen washout—are based on the principle of conservation of mass: [initial gas concentration] x [initial volume of the system] = [final gas concentration] x [final volume of the system]. The nitrogen washout method is based on the fact that nitrogen is present in the air, at a relatively constant amount. The subject is given 100% oxygen to breathe, and the expired gas, which contains nitrogen in the lung at the beginning of the test, is collected. When no more nitrogen is noted in the expirate, the volume of air expired and the entire amount of nitrogen in that volume are measured, and the initial volume of the system (FRC) can be calculated. In the helium dilution method, a known volume and concentration of helium is inhaled by the subject. Helium, an inert gas that is not absorbed significantly from the lungs, is diluted in proportion to the lung volume to which it is added. The final concentration of helium is then measured and FRC calculated (Weinstock and McCannon, 2017).

Measurements of lung volumes in humans are technically more challenging than spirometry. However, they complement spirometry (which cannot determine lung volumes) and may be a preferred means of lung function assessment when subject compliance cannot be reasonably expected (e.g. in pediatric subjects) or where forced expiratory maneuvers are not possible (e.g. in patients with advanced pulmonary fibrosis). There are recommended standards for lung volume measurements and their interpretation in clinical practice, issued by the ATS/ERS Task Force (Wanger et al., 2005; Criée et al., 2011).

Finally, indices of gas exchange across the alveolar-capillary barrier are tested by diffusion capacity of carbon monoxide (DLCO) studies (also referred to as transfer capacity of carbon monoxide, TLCO). The principle of the test is the increased affinity of hemoglobin to preferentially bind carbon monoxide over oxygen (Weinstock and McCannon, 2017). Complementary to spirometry and lung volume measurements, DLCO provides information about the lung surface area available for gas diffusion. Therefore, it is sensitive to any structural changes affecting the alveoli, such as those accompanying emphysema, pulmonary fibrosis, pulmonary edema, and ARDS. Recommendations for the standardization of the test and its evaluation have been outlined by the ATS/ERS Task Force (Graham et al., 2017). An isolated reduction in DLCO with normal spirometry and in absence of anemia suggests an injury to the alveolar-capillary barrier, as for example seen in the presence of pulmonary emboli or in patients with pulmonary hypertension (Weinstock and McCannon, 2017; Lettieri et al., 2006; Seeger et al., 2013). Reduced DLCO together with airflow obstruction (i.e., reduced FEV1) indicates lung parenchymal damage and is commonly observed in smokers and in COPD patients (Matheson et al., 2007; Harvey et al., 2016), whereas reduced DLCO with airflow restriction is seen in patients with interstitial lung diseases (Dias et al., 2014; Kandhare et al., 2016).

Lung function tests used to evaluate experimental animal lung function

Because spirometry requires active participation and compliance of the subject, it is not commonly used in animal studies. However, specialized equipment such as the flexiVent system (SCIREQ^®) are available for measuring FEV, FVC and PEF in anesthetized and tracheotomized small laboratory animals. Other techniques such as plethysmography or forced oscillation are increasingly preferred for lung function assessment in small laboratory animals (McGovern et al., 2013; Bates, 2017).

In small laboratory animals, plethysmography can be used to determine respiratory physiology parameters (minute volume, respiratory rate, time of pause and time of break), lung volume and airway resistance of conscious animals. Both whole body and head-out plethysmography can be applied, although there is a preference for the latter in the context of inhalation toxicity studies, because of its higher accuracy and reliability (OECD, 2018a; Hoymann, 2012).

Gas diffusion tests are not frequently performed in animals, because reproducible samplings of alveolar gas are difficult and technically challenging (Reinhard et al., 2002; Fallica et al., 2011). Modifications to the procedure employed in humans have, however, open possibilities to obtain a human-equivalent DLCO measure or the diffusion factor for carbon monoxide (DFCO)—a variable closely related to DLCO, which can inform on potential structural changes in the lungs that have an effect on gas exchange indices (Takezawa et al., 1980; Dalbey et al., 1987; Fallica et al., 2011; Limjunyawong et al., 2015).

 

Regulatory Significance of the AO

Established regulatory guideline studies for inhalation toxicity focus on evident clinical signs of systemic toxicity, including death, or organ-specific toxicity following acute and (sub)chronic exposure respectively. In toxicological and safety pharmacological studies with airborne test items targeting the airways or the lungs as a whole, lung function is a relevant endpoint for the characterization of potential adverse events (OECD, 2018a; Hoymann, 2012). Hence, the AO “decreased lung function” is relevant for regulatory decision-making in the context of (sub)chronic exposure (OECD, 2018b; OECD, 2018c).

Regulatory relevance of the AO “decreased lung function” is evident when looking at the increased risk of diseases in humans following inhalation exposure, and because of its links to other comorbidities and mortality.

To aid diagnosis and monitoring of fibrosis, current recommendations include both the recording of potential environmental and occupational exposures as well as an assessment of lung function (Baumgartner et al., 2000). The latter typically confirms decreased lung function as demonstrated by a loss of lung volume. As the disease progresses, dyspnea and lung function worsen, and the prognosis is directly linked to the decline in FVC (Meltzer and Noble, 2008).

Chronic exposure to cigarette smoke and other combustion-derived particles results in the development of COPD. COPD is diagnosed on the basis of spirometry results as laid out in the ATS/ERS Task Force documents on the standardization of lung function tests and their interpretation (Pellegrino et al., 2005; Culver et al., 2017, Graham et al., 2019). Rapid rates of decline in the lung function parameter FEV1 are linked to higher risk of exacerbations, increased hospitalization and early death (Wise et al., 2006; Celli, 2010). Reduced FEV1 also poses a risk for serious cardiovascular events and mortality associated with cardiovascular disease (Sin et al., 2005; Lee et al., 2015).

 

References

Appendix 2

List of Key Event Relationships in the AOP