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Oxidative Stress leads to CBF, Decreased
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Oxidative stress Leading to Decreased Lung Function||adjacent||High||High||Karsta Luettich (send email)||Open for comment. Do not cite|
Life Stage Applicability
|All life stages|
Key Event Relationship Description
Because the lung interfaces with the external environment, it is frequently exposed to airborne oxidant gases and particulates, and thus prone to oxidant-mediated cellular damage (Ciencewicki et al., 2008). Oxidant stress—through the action of exogenous and endogenous free radicals, such as super oxides, hydroxyl radicals, and hydrogen peroxides—is a common factor in lung inflammation and various respiratory diseases. The presence of redox-sensitive proteins in motile cilia suggests that oxidant stresses may impact ciliary function negatively (Price and Sisson, 2019). Indeed, exposure of human or rodent ciliated airway epithelial cells to hydrogen peroxide, acetaldehyde, ozone or cigarette smoke—all of which are known to cause oxidative stress—decreases CBF in a dose- and time-dependent manner (Bayram et al., 1998; Burman and Martin, 1986; Gosepath et al., 2000; Hastie et al., 1990; Helleday et al., 1995; Kienast et al., 1994; Knorst et al., 1994a; Min et al., 1999; Simet et al., 2010).
Evidence Collection Strategy
Evidence Supporting this KER
Experimental studies in vitro have shown that exposure of ciliated respiratory cells directly or indirectly to sources of oxidative stress leads to decreased CBF (Burman and Martin, 1986; Wilson et al., 1987; Feldman et al., 1994; Yoshitsugu et al., 1995; Min et al., 1999), which can be reversed by treatment with antioxidants (Schmid et al., 2015). Cigarette smoke condensate, a known inducer of oxidative stress, also causes a decrease in CBF in vitro (Cohen et al., 2009), while, in human subjects exposed to different oxygen levels, oxygen stress causes a decrease in nasal CBF (Stanek et al., 1998).
One mode of antimicrobial defense in the airway epithelium is generation of free radicals by neutrophils and monocytes/macrophages. Some microbes have also been shown to produce oxidants in significant amounts, e.g. H2O2 production by pneumococcus. Several studies have shown that oxidants, irrespective of the source (microbial or host-derived) inhibit ciliary function. Additionally, there is a large body of experimental evidence demonstrating that exposures to environmental oxidants, including volatile aldehydes, peroxides, sulfur dioxide, nitric dioxide and Diesel exhaust particles have a detrimental impact on ciliary function. Therefore, this KER is highly plausible.
Treatment with H2O2 causes a dose-dependent decline in ciliary beat frequency (CBF) in tracheal rings from male Sprague-Dawley rats (Burman and Martin, 1986).
Exposure of nasal ciliated epithelial cells to enzymatically generated oxidants (xanthine/xanthine oxidase) was accompanied by ciliary slowing, which was maximal at the end of the experiment (4 h). After 4 h, the reduction in CBF was 37.4%. Catalase alone, or in combination with superoxide dismutase (SOD), completely protected the epithelial strips from oxidant-mediated ciliary dyskinesia. Exposure of respiratory epithelium to glucose/glucose oxidase resulted in similar effects to those obtained with the xanthine/xanthine oxidase system, with 38% and 57% CBF reduction after 4 h in systems containing 25 and 100 mU of glucose oxidase, respectively. Treatments with H2O2 and HOCl at concentrations of ≥100 µM caused dose-dependent ciliary dyskinesia after 4 h (Feldman et al., 1994).
Marked ciliary slowing was observed after exposure of human nasal epithelial cells to free radicals within the first 5 min. There was a significant difference in CBF between experimental and control groups. Pretreatment with 300 U/mL SOD or with 5 mM 3-ABA prevented CBF changes. (Min et al., 1999).
Pyocyanin dissolved in PBS produced gradual slowing of CBF in strips of normal human nasal ciliated epithelium without recovery. Ciliary dyskinesia was observed only late in the experiments when ciliostasis and epithelial disruption were also noted. In contrast, 1-hydroxyphenazine produced rapid onset of ciliary slowing, dyskinesia, and immediate ciliostasis but also some recovery of ciliary beating during the experiment (Wilson et al., 1987).
Baseline CBF was analyzed in human sinonasal epithelial cells for 4 min (time 0) followed by administration of either cigarette smoke condensate (CSC; not further specified) or DMSO for 3 min. Forskolin was then administered to the apical surface and stimulated CBF measured. Following addition of forskolin, stimulated CBF was significantly decreased in the CSC-exposed group compared to DMSO controls (Cohen et al., 2009).
Treatment of fully differentiated normal human bronchial epithelial cells with 100 nM roflumilast raised the CBF at 1 h after exposure. Subsequent air or cigarette smoke exposure increased CBF significantly in roflumilast-treated cultures (Schmid et al., 2015).
In superficial mucosae of the inferior nasal turbinates from non-smoking healthy volunteers exposed to three different oxygen concentrations—21%, 60% and 95%—for 2 h, CBF dose-dependently increased. Extending exposure to extreme oxygen concentrations to 4 h, however, decreased CBF, which is indicative of “oxygen stress” (Stanek et al., 1998).
Within 2 min of exposure of human respiratory epithelial cell monolayers to 10 µM H2O2, a decrease in CBF was observed. All cilia stopped moving within 10 min without obvious surface structural change in the ciliated cells. Catalase significantly reduced the ciliotoxic effect of H2O2 (Yoshitsugu et al., 1995).
Uncertainties and Inconsistencies
Several studies show that oxidants decrease CBF which can be reversed by addition of antioxidants, suggesting a direct effect. However, there is evidence suggesting that oxidant-mediated decreases in CBF cannot be prevented by addition of antioxidants. For example, a polycyanin-induced decrease in CBF in human nasal epithelium could be reversed by treatment with isobutylmethylxanthine and forskolin, both of which increase intracellular cAMP, and also by the cAMP analog dibutyryl cAMP, while antioxidants did not seem to have any effect on CBF (Kanthakumar et al., 1993). Like polycyanin, two other P. aeruginosa toxins, 1-hydroxyphenazine (1-HP) and rhamnolipid reduced CBF which was associated with a decrease in intracellular adenosine nucleotides (Kanthakumar et al., 1996).
Inconsistent with several studies, there are studies that suggest that exposure to cigarette smoke does not inhibit CBF. A study involving 56 human subjects (27 non-smokers and 29 smokers) showed no differences in CBF between the 2 groups. However, a decrease in nasal mucociliary clearance was observed in smokers who exhaled smoke through their noses (Stanley et al., 1986).
While several studies have shown age dependence of CBF, there is evidence that suggests otherwise (Agius et al., 1998).
Known modulating factors
|Modulating Factor (MF)||MF Specification||Effect(s) on the KER||Reference(s)|
|CFTR genetic dysfunction / cystic fibrosis||Mutations in CFTR gene and decreased CFTR levels lead in reduced Cl–channel activity and to compromised fluid and electrolyte transport, consequently to reduced airway surface liquid levels, thus hindering coordinated ciliary beating.||Decrease in CFTR protein activity decreases CBF (see also KERs 2440 and 2441).||Van Goor et al., 2009|
Quantitative Understanding of the Linkage
Several studies in various species, including humans and rodents, provide evidence in support of this KER. The empirical evidence confirms both a dose- and time-dependence between the upstream KE/MIE and the downstream KE. Our quantitative understanding of this KER is therefore strong.
Treatment of human nasal ciliated epithelial cells with 0.4 mM xanthine + 100 mU/mL xanthine oxidase—producing 159 ± 4.0 µM/h H2O2—decreased CBF by ca. 1 Hz at 1 h and ca. 2.5 Hz (37.4%) at 4 h. Catalase alone (500 U/mL), or in combination with SOD (300 U/mL ) completely protected the cells from oxidant-mediated ciliary dyskinesia (Feldman et al., 1994).
Treatment of human nasal ciliated epithelial cells with 5 mM glucose + 25 mU/mL glucose oxidase— producing 114 ± 7.7 µM/h H2O2—decreased CBF by ca. 2 Hz at 1 h and ca. 4 Hz (38%) at 4 h. The decline in CBF was even larger with 57% (approx. 6 Hz) at 4 h when 100 mU/mL glucose oxidase was used (producing 322 ± 11.5 µM/h H2O2). Catalase alone (500 U/mL) completely protected the cells from oxidant-mediated ciliary dyskinesia (Feldman et al., 1994).
Treatment of human nasal ciliated epithelial cells with H2O2 at concentrations ≥100 µM dose-dependently decreased CBF in human nasal ciliated epithelial cells, with 100 µM causing a 22.4% reduction and the maximal decrease (51.6%) seen with 500 µM H2O2 at 4 h. Adding 100 mU/mL MPO to 150 µM H2O2 enhanced the H2O2-mediated decrease in CBF (control:11.7 ±0.6 Hz; H2O2: 8.2 ± 1.1 Hz, 30% decrease; H2O2 + MPO: 5.4±0.2 Hz, 53.8% decrease). (Feldman et al., 1994).
Treatment of human nasal ciliated epithelial cells with HOCl at concentrations ≥100 µM dose-dependently decreased CBF in human nasal ciliated epithelial cells, with 100 µM causing a 26.1% reduction and 500 µM causing the maximal decrease (100%) at 4 h (Feldman et al., 1994).
Treatment of human nasal epithelial cells with 0.4 mM xanthine + 100 mU/mL xanthine oxidase decreased CBF by ca. 50% within 2 min. Addition of 300 U/mL SOD abolished this effect (Min et al., 1999).
Treatment of human nasal epithelial cells with 10 mM H2O2 decreased CBF to 36.5 ±4.4% of baseline within 5 min, with a maximal decrease in CBF of 100% seen after 10 min, whereas 1 mM H2O2 had no effect on CBF. Treatment of human nasal ciliated epithelial cells with 0.8 mM xanthine + 100 mU/mL xanthine oxidase transiently increased CBF by 12.1±1.0% from baseline. When xanthine concentration was increased to 4 and 8 mM, CBF decreased by 26.8±1.7 and 25.6±1.5%, respectively (Yoshitsugu et al., 1995).
Treatment of bovine ciliated bronchial epithelial cells with acetaldehyde, an oxidative stressor, decreased CBF in a dose-dependent manner. Significant slowing of ciliary beating by ca. 50% was observed with concentrations as low as 15-30 µM, and ciliary beating was completely abrogated at concentrations > 250 µM. Ciliary beating also decreased following treatment with 15-30 µM propionaldehyde (40-50% of control), butyraldehyde (35-65% of control), isobutyraldehyde (20-40% of control), and benzaldehyde (80-90% of control) (Sisson et al., 1991).
Exposure of rabbit tracheal explants to formaldehyde dose-dependently decreased CBF. At 66 µg formaldehyde/cm3, CBF decreased from 12.6 to 11.8 Hz; at 33 µg formaldehyde/cm3, CBF decreased from 13.0 to 10.9 Hz (Hastie et al., 1990).
Exposure of guinea pig trachea to SO2 at concentrations of 2.5-12.5 ppm for 30 min dose-dependently decreased CBF. Exposure to 2.5 ppm SO2 caused a small, non-significant decrease in mean CBF, and exposure to 5 ppm SO2 caused a 45% decrease. The greatest decrease (72 %) in mean CBF was recorded after exposure to 12.5 ppm SO2 (Knorst et al., 1994a).
Exposure of human nasal epithelial cells (cultured in Ringer’s solution) to SO2 at concentrations of 2.5-12.5 ppm for 30 min dose-dependently decreased CBF. Exposure to 2.5 ppm yielded a 42.8% decrease, whereas exposure to 12.5 ppm yielded a 96.5% decrease in CBF (Kienast et al., 1994).
A 20-min exposure to NO2, a known air pollutant, at concentrations of 1.5 or 3.5 ppm did not affect CBF in healthy human subjects at 45 min post-exposure (Helleday et al., 1995).
Exposure of human bronchial epithelial cells from healthy volunteers to 10, 50, and 100 µg/mL Diesel exhaust particles (DEP) significantly decreased CBF by 15.9%, 31.0%, and 55.5%, respectively, from baseline after 24 h (Bayram et al., 1998).
A 4-week exposure of human nasal epithelial cells to 100 µg/m3 ozone had no effect on CBF, whereas 5- and 10-times that concentration significantly decreased CBF (-11.1% at 500 µg/m3; -33.3% at 1000 µg/m3) (Gosepath et al., 2000).
Baseline CBF in tracheal rings from C57Bl/6 mice exposed to cigarette smoke (whole body exposure to mainstream and sidestream cigarette smoke via inhalation from 1R1 reference cigarettes, at 150 mg/m3 total particulate matter for 2 h/day, 5 days/week, for up to 1 year) for 1.5 to 3 months was slightly, but not significantly, increased (∼1 Hz). After 6 months of smoke exposure, however, baseline CBF significantly decreased (∼2–3 Hz) (Simet et al., 2010).
Treatment of human nasal ciliated epithelial cells with 0.4 mM xanthine + 100 mU/mL xanthine oxidase decreased CBF over time, with a noticeable decrease by ca. 1 Hz at 1 h and a maximal decrease of 37.4% reached at 4 h (Feldman et al., 1994).
Treatment of human nasal ciliated epithelial cells with 5 mM glucose + 25 mU/mL glucose oxidase decreased CBF by ca. 2 Hz at 1 h and a maximal decrease of ca. 4 Hz (38%) at 2 h, that did not change until the end of the experiment at 4 h. When 100 mU/mL glucose oxidase was used, CBF decreased by ca. 2 Hz at 1 h, 4 Hz at 2 h, 5.5 Hz at 3 h, reaching a maximum of 57% (approx. 6 Hz) at 4 h (Feldman et al., 1994).
Treatment of human nasal epithelial cells with 0.4 mM xanthine + 100 mU/mL xanthine oxidase decreased CBF maximally by ca. 50% within 2 min, after which it began to increase again, reaching approx. 80% of the baseline value after 30 min (Min et al., 1999).
Treatment of human nasal ciliated epithelial cells with 0.8 mM xanthine + 100 mU/mL xanthine oxidase transiently increased CBF by 12.1±1.0% from baseline within 15 s, after which it rapidly returned to baseline levels (within 30 min). When xanthine concentrations were increased to 4 and 8 mM, CBF decreased by 26.8±1.7 and 25.6±1.5%, respectively (Yoshitsugu et al., 1995).
Treatment of bovine ciliated bronchial epithelial cells with acetaldehyde reduced CBF rapidly, with a significant drop in CBF occurring within 30 s and a maximal decrease by 3 min (Sisson et al., 1991).
Exposure of rabbit tracheal explants to formaldehyde time-dependently decreased CBF: At 66 µg/cm3, CBF decreased from 12.6 to 11.8 Hz immediately upon addition of HCHO to complete cessation of beating by 10 min. At 33 µg/cm3, CBF decreased from 13.0 to 10.9 Hz by 30 min (Hastie et al., 1990).
At 24 h following a 4-h exposure of healthy human subjects to 3.5 ppm NO2, there was a significant elevation in CBF from 12.4±0.9 Hz (at baseline, pre-exposure) to 13.8±0.8 Hz (Helleday et al., 1995).
Exposure of human bronchial epithelial cells to DEP significantly decreased CBF from 2 h onward after incubation with 50 to 100 µg/mL DEP and from 6 hours onward after incubation with 10 µg/mL DEP (Bayram et al., 1998).
A 4-week exposure of human nasal epithelial cells to ozone significantly reduced CBF, with effects becoming noticeable at the higher concentrations (-7.1% at 500 µg/m3; -10.3% at 1000 µg/m3) after 2 weeks of exposure and a maximal decrease after 4 weeks (-11.1% at 500 µg/m3; -33.3% at 1000 µg/m3) (Gosepath et al., 2000).
Known Feedforward/Feedback loops influencing this KER
Domain of 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).
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).
Agius, A.M., Smallman, L.A., and Pahor, A.L. (1998). Age, smoking and nasal ciliary beat frequency. Clin. Otolaryngol. Allied Sci. 23, 227-230.
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.
Bayram, H., Devalia, J.L., Khair, O.A., Abdelaziz, M.M., Sapsford, R.J., Sagai, M., et al. (1998). Comparison of ciliary activity and inflammatory mediator release from bronchial epithelial cells of nonatopic nonasthmatic subjects and atopic asthmatic patients and the effect of diesel exhaust particles in vitro. J. Allergy Clin. Immunol. 102, 771-782.
Burman, W.J., and Martin, W.J. (1986). Oxidant-Mediated Ciliary Dysfunction: Possible Role in Airway Disease. Chest 89, 410-413.
Ciencewicki, J., Trivedi, S., and Kleeberger, S.R. (2008). Oxidants and the pathogenesis of lung diseases. J. Allergy Clin. Immunol. 122, 456-470.
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.
Feldman, C., Anderson, R., Kanthakumar, K., Vargas, A., Cole, P.J., and Wilson, R. (1994). Oxidant-mediated ciliary dysfunction in human respiratory epithelium. Free Radic. Biol. Med. 17, 1-10.
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.
Hastie, A.T., Patrick, H., and Fish, J.E. (1990). Inhibition and recovery of mammalian respiratory ciliary function after formaldehyde exposure. Toxicol. Appl. Pharmacol. 102, 282-291.
Helleday, R., Huberman, D., Blomberg, A., Stjernberg, N., and Sandstrom, T. (1995). Nitrogen dioxide exposure impairs the frequency of the mucociliary activity in healthy subjects. Eur. Respir. J. 8, 1664-1668.
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.
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.
Kanthakumar, K., Taylor, G., Cundell, D., Dowling, R., Johnson, M., Cole, P., et al. (1996). The effect of bacterial toxins on levels of intracellular adenosine nucleotides and human ciliary beat frequency. Pulm. Pharmacol. 9, 223-230.
Kanthakumar, K., Taylor, G., Tsang, K., Cundell, D., Rutman, A., Smith, S., et al. (1993). Mechanisms of action of Pseudomonas aeruginosa pyocyanin on human ciliary beat in vitro. Infect. Immun. 61, 2848-2853.
Kienast, K., Riechelmann, H., Knorst, M., Schlegel, J., Müller-Quernheim, J., Schellenbergt, J., et al. (1994). An experimental model for the exposure of human ciliated cells to sulfur dioxide at different concentrations. The clinical investigator 72, 215-219.
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. Environ. Health 65, 325-328.
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.
Price, M.E., and Sisson, J.H. (2019). Redox regulation of motile cilia in airway disease. Redox. Biol. 27, 101146-101146.
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., Tuma, D.J., and Rennard, S.I. (1991). Acetaldehyde-mediated cilia dysfunction in bovine bronchial epithelial cells. Am. J. Physiol. 260, L29-36.
Stanek, A., Brambrink, A., Latorre, F., Bender, B., and Kleemann, P. (1998). Effects of normobaric oxygen on ciliary beat frequency of human respiratory epithelium. Br. J. Anaesth. 80, 660-664.
Stanley, P., Wilson, R., Greenstone, M., MacWilliam, L., and Cole, P. (1986). Effect of cigarette smoking on nasal mucociliary clearance and ciliary beat frequency. Thorax 41, 519-523.
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Wilson, R., Pitt, T., Taylor, G., Watson, D., MacDermot, J., Sykes, D., et al. (1987). Pyocyanin and 1-hydroxyphenazine produced by Pseudomonas aeruginosa inhibit the beating of human respiratory cilia in vitro. J. Clin. Investig. 79, 221-229.
Yoshitsugu, M., Matsunaga, S., Hanamure, Y., Rautiainen, M., Ueno, K., Miyanohara, T., et al. (1995). Effects of oxygen radicals on ciliary motility in cultured human respiratory epithelial cells. Auris Nasus Larynx 22, 178-185.