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Oxidative Stress leads to CFTR Function, 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 [MIE] Leading to Decreased Lung Function [AO]||adjacent||High||High||Karsta Luettich (send email)||Open for comment. Do not cite|
Life Stage Applicability
|All life stages||High|
Key Event Relationship Description
Exposure to inhaled oxidants (such as cigarette smoke) leads to decreased CFTR gene and protein expression as well as CFTR internalization, thereby reducing or abolishing open probabilities, short-circuit currents and subsequently ASL height/volume (Cantin et al., 2006a; Cantin et al., 2006b; Clunes et al., 2012; Rasmussen et al., 2014; Sloane et al., 2012). Decreased CFTR mRNA expression was previously linked to reduced mRNA stability following treatment with tert-butylhydroquinone (BHQ) (Cantin et al., 2006a) as well as increased intracellular Ca2+, which is thought to activate protein kinases, thereby decreasing transcription rates (Bargon et al., 1992a; Bargon et al., 1992b; Rasmussen et al., 2014). Other evidence suggests that the STAT1 pathway is involved in CFTR down-regulation following ozone exposure or in the presence of interferon-γ (Kulka et al., 2005; Qu et al., 2009). In addition, transcriptional activation of an antioxidant response element in the CFTR promoter by Nrf2 was shown to regulate CFTR gene expression in airway epithelial cells under oxidative stress conditions, leading to an upregulation of transcript levels in the short-term, but a decline in the long-term (Zhang et al., 2015). On the post-transcriptional level, CFTR function was shown to be affected in multiple ways due to oxidative stress: For example, cell surface CFTR expression was drastically diminished in airway epithelial cells following cigarette smoke exposure, involving a change in protein solubility and trafficking to a perinuclear aggresome-like structure rather than to lysosomes or the proteasome (Bodas et al., 2017; Clunes et al., 2012). ATP depletion as a surrogate for lung ischemia also resulted in decreased CFTR expression at the plasma membrane. This was shown to be a consequence of irreversible actin filament depolymerization, which resulted in loss of cell polarity and relocalization of CFTR to the cytoplasm (Brézillon et al., 1997) .
Evidence Supporting this KER
Inducers of oxidative stress such as cigarette smoke reduced CFTR expression at both the RNA (Cantin et al., 2006a; Cantin et al., 2006b; Qu et al., 2009; Rennolds et al., 2010) and protein (Cantin et al., 2006b; Qu et al., 2009; Rennolds et al., 2010; Sloane et al., 2012; Hassan et al., 2014; Rasmussen et al., 2014; Xu et al., 2015) level in vitro. CFTR protein expression was lower in the airways of smokers compared to non-smokers (Dransfield et al., 2013). In some of these studies, an accompanying decrease in Cl– conductance was also observed (Qu et al., 2009; Rennolds et al., 2010; Sloane et al., 2012). There are many studies that support a direct link between oxidative stress and decreased CFTR function in vitro, ex vivo, in vivo and in human subjects. Human primary epithelial cells and cell lines of respiratory epithelial origin have consistently decreased conductance of Cl– and other ions following exposure to cigarette smoke and other oxidants (Cantin et al., 2006b; Schwarzer et al., 2008; Raju et al., 2013; Lambert et al., 2014; Schmid et al., 2015; Raju et al., 2016; Chinnapaiyan et al., 2018), which could be reversed upon antioxidant treatment (Raju et al., 2013; Lambert et al., 2014; Schmid et al., 2015). Similar observations were made under hypoxic conditions (Brézillon et al., 1997; Zhang et al., 2013; Woodworth, 2015). Antioxidants could also increase Cl– conductance and anion transport in the absence of oxidant treatment or hypoxia induction in human and murine respiratory cells in vitro and in ex vivo tissues (Azbell et al., 2010; Alexander et al., 2011; Conger et al., 2013). Healthy smokers and smokers with COPD have reduced Cl– conductance (Sloane et al., 2012; Dransfield et al., 2013) and increased sweat chloride concentrations (Raju et al., 2013; Courville et al., 2014).
A link between CFTR and chronic bronchitis, which shares some of the features of cystic fibrosis lung disease, was proposed more than 20 years ago. Gene association studies, however, found no clear link between CFTR genotype and COPD except for hereditary disseminated bronchiectasis (Artlich et al., 1995; Joos et al., 2002), leading to the ion channel being of more interest in cystic fibrosis than chronic bronchitis research for some time. More recent evidence points toward a mechanism of acquired CFTR dysfunction in the context of cigarette smoking, which remains the leading risk factor for COPD. Multiple studies clearly demonstrated that CFTR Cl− conductance is significantly inhibited following cigarette smoke exposure in vitro and in vivo, resulting in reductions of ASL height/volume which ultimately impair MCC (Cantin. et al., 2006b; Clunes et al., 2012; Courville et al., 2014; Dransfield et al., 2013; Hassan et al., 2014; Raju et al., 2013; Rasmussen et al., 2014; Sailland et al., 2017; Schmid et al., 2015). Although there appear to be different schools of thought as to how cigarette smoke modulates CFTR (directly or indirectly), the available evidence on the inhibitory effects of cigarette smoke on CFTR anion transport is conclusive. Considering the similarities between cigarette smoke exposure-related oxidative stress in the airways and oxidative stress arising from e.g. the exposure to other inhalation toxicants and pathogens, the described mechanisms are likely to apply to other stressors eliciting an imbalance in the lung’s redox state. Moreover, studies with antioxidants such as NAC, resveratrol, genistein and hesperidin confirm the role of oxidative stress in modulating CFTR function (Alexander et al., 2011; Conger et al., 2013; Raju et al., 2013; Woodworth, 2015; Zhang et al., 2013). Taken together, the described cause-effect relationship is biologically plausible, and our confidence in biological plausibility is strong.
Uncertainties and Inconsistencies
There is currently only limited knowledge about the mechanism by which oxidative stress may affect CFTR expression and function. At least one study indicates that CFTR channel gating is decreased following exposure to acrolein, possibly by protein carbonylation (Raju et al., 2013). Other studies reports that cell surface CFTR expression was drastically diminished in airway epithelial cells following cigarette smoke exposure, involving a change in protein solubility and trafficking to a perinuclear aggresome-like structure rather than to lysosomes or the proteasome (Bodas et al., 2017; Clunes et al., 2012b).
CFTR Gene & Protein Expression
In Calu-3 cells exposed to cigarette smoke (drawn into a syringe and injected into the 5-L exposure chamber at a rate of 35 mL/min) for 10 min every 2 h for a total of four exposures CFTR mRNA expression decreased by approx. half, from 0.87 ± 0.03 to 0.47 ± 0.03 units (CFTR/GAPDH ratio; northern blot). CFTR protein expression decreased from 2.07 ± 0.24 to 1.24 ± 0.14 units (CFTR/actin ratio; western blot) (Cantin et al., 2006b).
Exposure of fully differentiated primary human bronchial epithelial cells (HEBCs) to 30 puffs of whole smoke from 2 cigarettes (generated according to ISO standard) every day for 5 days (total exposure 120 hr) resulted in a ca. 40% reduction in CFTR expression. Exposure of 16HBE14o- airway epithelial cells to cigarette smoke extract (CSE; smoke from one non-filtered Camel cigarette was bubbled using a peristaltic pump apparatus into 10 mL of complete culture media, defined as 100%) led to a dose-dependent decrease in CFTR protein expression which, with approx. –70% from baseline, was significant for concentrations ≥ 10% (Hassan et al., 2014).
Apical treatment of fully differentiated primary HBECs with 2% CSE (not further described) for 24 h resulted in approx. 30% reduction in CFTR mRNA expression, 50% reduction in total CFTR protein and 30% reduction in CFTR protein cell surface expression (Sloane et al., 2012).
Treatment of 16HBE14o- immortalized HBECs with 10% CSE (smoke from one non-filtered Camel cigarette was bubbled using a peristaltic pump apparatus into 10 mL of complete culture media, defined as 100%) resulted in more than 50% reduction of CFTR total and cell surface protein expression. Co-treatment with 10 mM NAC prevented this decrease in CFTR expression (0.5 mM NAC was not effective) (Xu et al., 2015).
Treatment of fully differentiated primary HBECs with 2% CSE (bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 min; defined as 100%) for 24 h decreased total CFTR expression and cell surface CFTR expression by approx. 20 and 25%, respectively (Raju et al., 2016a).
CFTR protein expression was significantly lower in endobronchial biopsies of healthy smokers (33 [12-54] pack-years; CFTR/tubulin ratio: 0.70), smokers with COPD (52.5 [35-147] pack-years; CFTR/tubulin ratio: 0.50-0.88), and former smokers (45 [39-80] pack-years; CFTR/tubulin ratio: 1.75) with COPD than in healthy non-smokers (0 pack-years; CFTR/tubulin ratio: 4.09 – 4.49) (Dransfield et al., 2013).
Exposure of Calu-3 lung cancer cells to 50 µM cadmium sulfate for 1 or 3 days significantly reduced CFTR protein expression by approx. 20 and 50%, respectively, whereas exposure to lower doses of cadmium (2 µM) for 3 days did not affect CFTR protein levels (Rennolds et al., 2010).
At 4 hours post-exposure to 1.5 ppm ozone for 30 min, CFTR mRNA and protein expression were more than 2-fold decreased (CFTR/GAPDH ratio: 1.35±0.3 (control) vs 0.46±0.07; CFTR/actin ratio: 0.81±0.02 vs 0.33±0.02) in 16HBE14o- airway epithelial cells (Qu et al., 2009).
CFTR Channel Function
Treatment of T84 cells with 100 µM tert-butylhydroquinone (BHQ) for 6 h caused oxidative stress, evidenced by a significant increase in intracellular glutathione concentrations (44.8 ± 0.6 vs. 32.3 ± 0.3 nmol/106 cells at 0 hr) and reduced peak cAMP-dependent 125I effluxes in a dose-dependent manner; however, only at BHQ concentrations ≥ 300 µM was the reduction significant (Cantin et al., 2006a).
In murine nasal septal epithelia grown at the air-liquid interface, resting Cl− secretion was maximally stimulated by 100 µM acrolein (15.9±2.2 vs. 2.4±0.8 μA/cm2 (control)), whereas forskolin-sensitive ion current was inhibited by 300 μM (13.3±1.2 vs. 19.9±1.0 μA/cm2 (control); max. and significant) and completely eliminated by 500 μM acrolein (Alexander et al., 2012).
Following exposure of primary HBECs to 3.2 μg/mL acrolein for 24 h, forskolin-stimulated ion currents were inhibited by approx. 50%. Noticeable inhibition occurred at concentrations above 2 μg/ml, and maximum inhibition was seen with ca. 5 μg/mL acrolein; higher concentrations did not further decrease ion currents. Repeated exposure to 10 ng/mL acrolein for 7 days also resulted in an approx. 50% decrease in forskolin-stimulated ion currents (Raju et al., 2013).
Exposure of fully differentiated primary HBECs to whole smoke from four 3R4F reference cigarettes (generated according to ISO standard 3308; Vitrocell VC10 exposure system) resulted in a significant decrease in Cl− currents (2.1±0.26 vs 4.8±0.71 μA/cm2 vs 4.8 ± 0.71 μA/cm2 (control)) (Schmid et al., 2015).
Resveratrol dose-dependently increased CFTR-mediated anion transport in murine sinonasal epithelial cells, with no effect seen for 50 μM, maximal increase (while not affecting total stimulation) seen for 100 μM, and slight inhibitory effects seen for concentrations ≥200 μM (Alexander et al., 2011; Woodworth, 2015). Forskolin-stimulated Cl− currents increased in murine (14.2±1.5 vs. 0.8±0.2 mA/cm2 (control)), human (17.4±.7 vs. 1.0±0.2 mA/cm2 (control)(Woodworth, 2015), 15.69±2.66 vs. 2.49±0.98 mA/cm2 (control)(Alexander et al., 2011)) and porcine (6.8±0.3 vs. 1.1±0.3 mA/cm2 (control)) sinonasal epithelial cells following treatment with 100 μM resveratrol. Resveratrol treatment (100 μM) also restored CFTR Cl− transport in hypoxic murine sinonasal epithelial cells (11.51±0.23 vs. 0.2±0.05 mA/cm2 (control)) and human sinonasal epithelial cells (10.8±0.7 vs. 0.3±0.05 mA/cm2 (control)) (Woodworth, 2015; Zhang et al., 2013). In C57B/L6 mice perfused with 100 μM resveratrol, mean nasal potential difference polarization increased to −4.0±1.87 mV (vs. −0.93±1.69 mV, control), which was slightly higher than results with forskolin (−1.65±1.78 mV), but not significant (Alexander et al., 2011).
Genistein (50 μM) enhanced basal CFTR-mediated anion transport in human sinonasal epithelial cells (23.1±1.8 vs. 0.7±0.2 μA/cm2 (control)), but had no effect on forskolin-sensitive current (Conger et al., 2013).
Hesperidine dose-dependently increased CFTR-mediated anion transport in murine sinonasal epithelial cells, with the maximum responses seen for 2 mM (16.67±0.43 µA/cm2) which was not further investigated due to the precipitation of hesperidine. Instead, 1 mM hesperidine was used as it also significantly increased CFTR Cl− currents (mouse cells: 13.51±0.77 vs. 4.4±0.66 µA/cm2 (control); human cells: 12.28±1.08 vs. 0.69±0.32 µA/cm2 (control)). In C57B/L6 mice perfused with 1 mM hesperidine, mean nasal potential difference polarization was increased (−2.3±1.0 mV vs. −0.8±0.8 mV (control)), similar to results with forskolin (−1.9±1.4 mV) (Azbell et al., 2010).
Basal lower airway potential difference (LAPD) was lower in healthy smokers (-7.71±0.88 mV; 33 [12-54] pack-years) and COPD smokers (-7.33±1.30 mV; 52.5 [32-147] pack-years) than in former smokers with COPD (no data; 45 [39-80] pack-years) or healthy non-smokers (-12.61±1.94 mV)(Dransfield et al., 2013).
Healthy smokers, current and former smokers with COPD had lower mean sweat chloride than healthy non-smokers (51.3±4.4 [31.08±14 pack-years], 41.9±3.4 [38±19 pack-years], and 39.0±5.4 [44±19 pack-years], respectively, vs. 53.6±3.3 mmol/L). The association between sweat chloride and pack-years was significant (Courville et al., 2014).
Healthy smokers and smokers with COPD exhibited significantly lower nasal potential difference than healthy non-smokers in response to isoproterenol (-6.3±1.4 [33.2; 10-78 pack-years] and -8.0±2.0 [55.1; 35-78 pack-years], respectively, vs. -15.2±2.7 mV (control)) (Sloane et al., 2012).
Treatment of wild-type CFTR expressing HEK293 cells with 1% CSE (bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 min; defined as 100%) decreased CFTR channel-open probability by 59% (Raju et al., 2016a).
Single channel recordings in apical membrane patches of murine sinonasal epithelial cells demonstrated that 100 µM resveratrol significantly enhanced channel-open probability (0.329± 0.116 vs. 0.119±0.059 NPo/N (control)) (Woodworth, 2015).
In T84 cells exposed to 100 µM BHQ, intracellular GSH levels were significantly increased from 6 hours onwards (from 32.3±0.3 to 44.8±0.6 nmol/106 cells), up to 24 hours (from 45.5±0.9 to 94.9±2.5 nmol/106 cells), whereas CFTR mRNA expression was significantly decreased over time (from 8.2±0.8 to 1.8±0.3 CFTR/GAPDH mRNA ratio at 6 hours). As a consequence, CFTR protein expression was significantly decreased following treatment of T84 cells with 100 µM BHQ for 24 hours (from 101.3±4.6 to 84.7±4.1 CFTR/actin protein, density as % control) (Cantin et al., 2006a).
Exposure of T84 cells to cigarette smoke condensate (CSC; prepared by drawing 35 mL/min cigarette smoke for 5 min into a syringe and injecting this smoke into a tonometer containing 10 mL culture media; defined as 100%) for 10 min resulted in a significant increase in GCLC mRNA expression (from 0.69±0.08 to 2.44± 0.25 GCLC/GAPDH mRNA ratio) and decrease in CFTR mRNA expression (from 0.57±0.04 to 0.21±0.02 CFTR/GAPDH mRNA ratio) at 6 hours post-exposure and a significant increase in GSH at 24 hours post-exposure (from 41.7±1.0 to 89.8±1.5 nmol/mg protein) (Cantin et al., 2006b).
Exposure of immortalized 16HBE14o- airway epithelial cells to 10% cigarette smoke extract (CSE; cigarette smoke from one non-filtered cigarette was bubbled using a peristaltic pump apparatus into 10 mL of complete culture media; defined as 100%) led to an approx. 50% decrease in CFTR mRNA expression and approx. 70% decrease in CFTR protein expression after 24 hours, with little change at later time points (Hassan et al., 2014).
Treatment of Calu-3 lung cancer cells with 50 µM cadmium sulfate significantly reduced CFTR protein expression after 1 day, with maximum decrease observed after 3 days (54 ± 5% of control). Exposure to lower doses of cadmium (2 µM) required 5-day treatment before CFTR protein levels were affected (Rennolds et al., 2010).
Treatment of fully differentiated primary HBECs with 2% CSE (bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 minutes; defined as 100%) for 24 hours decreased total CFTR expression and cell surface CFTR expression by approx. 20 and 25%, respectively, but treatment for 20 minutes did not. A 50% reduction in CFTR channel activity occurred immediately after addition of CSE and lasted for at least 20 minutes (Raju et al., 2016a).
Calu-3 lung cancer cells exposed to cigarette smoke (drawn into a syringe and injected into the 5-L exposure chamber at a rate of 35 mL/min) for 10 min every 2 h for a total of four exposures were loaded with 125I for 1 hour, prior to stimulation with isobutyl methylxanthine, forskolin, and dibutyryl cAMP. cAMP-dependent anion efflux was significantly decreased compared to controls within 3 min of stimulation and remained significantly decreased for a further 3 min (Cantin et al., 2006b).
In untreated monolayers of CFTR-corrected CFBE41o- airway epithelial cells, cAMP-stimulated Cl– currents remained at stimulated levels for 2 to 3 h, whereas currents were inhibited by 86.0±5.8 and 40.0±2.7% in cells treated with 100 μM pyocyanin or 100 μM H2O2, respectively, in the same time period. The effect of pyocyanin occurred at a faster rate than that of H2O2; washout of the compounds partly restored cAMP-stimulated Cl− currents (Schwarzer et al., 2008).
Exposure of fully differentiated primary HBECs to whole smoke (3R4F reference cigarette; inExpose exposure system) resulted in a time-dependent decrease in CFTR short-circuit currents, with significant differences (approx. 15% reduction) from control after 10 min of exposure. Maximal reduction (ca. 50%) was seen after 30 min of exposure (Lambert et al., 2014).
Culture of fully differentiated primary human and murine sinonasal epithelial cells (HSNECs; MSNECs) in 1% O2 for 12 or 24 h significantly reduced CFTR-mediated (forskolin-sensitive) Cl− current: HSNECs, 19.55±0.56 mA/cm2 (12 h); 17.67±1.13 mA/cm2 (24 h) vs. 25.49±1.48 mA/cm2 (control); MSNECs, 13.55±0.46 mA/cm2 (12 h); 12.75±0.07 mA/cm2 (24 h) vs. 19.23±0.18 mA/cm2 (control). Transfer of cultures to physiologic O2 conditions (21%) restored CFTR ion currents (HSNECs, 25.12±1.24 mA/cm2) after 24 h (Woodworth, 2015).
Known modulating factors
CFTR has also been implicated in transmembrane glutathione transport (Linsdell and Hanrahan, 1998; Roum et al., 1993). Multiple studies suggest that oxidative injury of the lungs, e.g. following inhalation exposures or infections, can be effectively counteracted, if not prevented, by CFTR-mediated elevations of ASL glutathione levels (Day et al., 2004; Gould et al., 2010; Jungas et al., 2002; Kariya et al., 2007; Velsor et al., 2001). The antioxidant properties of glutathione may temporarily delay the acquisition of CFTR dysfunction by neutralizing reactive oxygen species that would otherwise contribute to downregulation of gene and protein expression.
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Available evidence indicates that this KER is applicab le to human, mouse and pig, independent of life stage and gender.
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