Extracellular superoxide quantification can be performed exploiting the ability of the superoxide to reduce ferricytochrome c. Ferricytochrome c reduction can be monitored measuring absorbance at 550 nm, that corresponds to the absorbance maximum of the reduced form of this protein (Fridovich, 1970; Quick et al., 2000). So, the higher the amount of superoxide, the higher the signal recorded. To determine the nmoles of superoxide produced, it should be run an identical sample that contains superoxide dismutase (SOD) for every experimental condition. Then, the difference in absorbances in the absence and presence of SOD corresponds to superoxide contribution. It is recommended to measure the two samples in parallel because also other molecules can reduce ferricytochrome c (i.e., glutathione (GSH) and ascorbate). Moreover, catalase (CAT) can be included to eliminate unwanted hydrogen peroxide (H₂O₂)-mediated reactions from the assay (Quick et al., 2000; Nauseef, 2013).

Dihydroethidium (DHE), also known as hydroethidine (HE), is a fluorogenic probe. Its superoxide-mediated oxidation generates the fluorescent product 2-hydroxyethidium (2-E⁺OH) (λ_excitation = ∼ 500 nm, λ_emission = ∼ 600 nm), while other ROS or reactive nitrogen species (RNS) do not form the same fluorescent product when reacting with DHE (Zhao et al., 2003; Zielonka and Kalyanaraman, 2010). The increase in fluorescence intensity is directly proportional to superoxide amount. The specificity of the detection can be confirmed by exploiting the competition between DHE and SOD (Michalski et al., 2013).

MitoSOX Red is a derivative of HE, synthesized to detect superoxide in mitochondria. The mitochondrial localization is driven by a hexyl triphenylphosphonium cation, covalently bonded to DHE (Robinson et al., 2006).

Another method applied to investigate superoxide levels is based on the ability of the anion generated during the xanthine oxidase-mediated reaction to oxidize epinephrine. This event initiates a sequence of reactions that terminates with the formation of the coloured product adrenochrome. The change of absorbance at 485 nm as a consequence of adrenochrome generation is indicative of superoxide formation (Chen and Thakker, 2002; Alhasan and Njus, 2008).

Frequently MitoSOX measurements are associated with staining with 5-(and -6)-carboxy-2′,7′-dichlorodihydrofluoresceine diacetate (DCFH-DA) for the evaluation of H₂O₂ basal content. Specifically, DCFH-DA is a membrane permeable non-fluorescent probe that diffuses into the cells, and it is activated into 2′,7′-dichlorodihydrofluoresceine (DCFH) by esterase-mediated deacetylation. The removal of the acetate groups generates a polar molecule that remains trapped into the cell. Then, DCFH is oxidized to DCF by intracellular H₂O₂, generating the fluorophore DCF (λ_excitation = 488 nm, λ_emission = 530 nm). Therefore, following H₂O₂-mediated oxidation of DCF, measurements of the fluorescence intensity can be predictive of the H₂O₂ produced by the cells (Ubezio and Civoli, 1994; Reiniers et al., 2017).

Extracellular H₂O₂ can be quantified by exploiting its ability to oxidize susceptible probes in the presence of a peroxidase (Nauseef, 2013).

Scopoletin is a fluorescent agent that is bleached when oxidized, whereas HVA, phenol red, and ADHP exhibit increased fluorescence upon oxidation. HVA is a substituted phenol that, when oxidized, produces a fluorescent dimer (λ_ex = 321 nm, λ_em = 421 nm) (Thannickal and Fanburg, 1995). HRP-mediated oxidation of phenol red by H₂O₂ results in the formation of a compound measurable at 610 nm absorbance, with a linear relationship between H₂O₂ concentration and absorbance (Pick and Keisari, 1980).

ADHP is a colorless, highly stable, and nonfluorescent substrate that, upon oxidation, becomes a highly fluorescent molecule named resorufin (λ_ex = 561 nm, λ_em = 585 nm). Although ADHP oxidation by other types of ROS has been described, this occurs at a lower yield than H₂O₂-mediated oxidation. Moreover, the ADHP-induced increase in fluorescence is inhibitable by CAT, confirming its specificity for H₂O₂ detection. Compared to scopoletin, ADHP offers advantages including (i) a low background that barely changes over time and (ii) higher sensitivity due to its high fluorescent yield (Mohanty et al., 1997).

Another fluorescent probe to measure H₂O₂ is the 2,2′-dihydroxy-biphenyl-5,5′ diacetate, the stable dimer generated from the oxidation of the p-hydroxyphenylacetic acid (pHPA) by the Complex I. This, in turn, had been oxidised by the H₂O₂-consuming HRP (Schick et al., 1997).

Early investigations into the oxidative burst mechanisms in CF revealed notable differences in immune cell activity, even among carriers of a single CFTR mutation (heterozygotes). Utilizing the ferricytochrome c reduction assay for SOD-inhibitable superoxide quantification, Regelmann et al. observed that monocytes isolated from CF heterozygotes, when stimulated with concanavalin A, exhibited a significantly higher release of superoxide compared to monocytes from healthy controls after a 3-min measurement period. This heightened oxidative burden extends beyond immune cells to the airway epithelial cells, which are primary sites of CFTR dysfunction (Regelmann et al., 1991). Studies employing CFBE41o-cells have corroborated these findings, demonstrating elevated superoxide levels.

For instance, ferricytochrome c reduction and adrenochrome assay measured significantly higher levels of superoxide anion in CFBE41o-cells compared to WT controls in two independent studies (Atlante et al., 2016; de Bari et al., 2018). Moreover, MitoSOX Red probe, in combination with the mitochondrial Complex I inhibitor rotenone, pinpointed this mitochondrial complex as a principal source of the augmented superoxide production in these CF cells (Atlante et al., 2016). Furthermore, treatment with Lumacaftor, a CFTR corrector facilitating the trafficking of misfolded F508del-CFTR protein, led to a reduction of superoxide levels in CFBE41o-cells, suggesting that while Lumacaftor primarily addresses CFTR protein processing, its therapeutic efficacy might also stem from mitigating oxidative damage and subsequent inflammatory responses (de Bari et al., 2018). Analogously, it was discovered that CFTR corrector therapy can modulate a metabolic shift detected with MitoSOX as an elevation of mitochondrial ROS production associated with increased mitochondrial respiration (Jarosz-Griffiths et al., 2024).

Exposure of primary HBE cells to Pseudomonas aeruginosa quinolone resulted in a significant increase in superoxide production (Abdalla et al., 2017). Another research using MitoSOX Red has specifically identified augmented mitochondrial superoxide production in CF macrophages, where altered mitochondrial ROS levels are implicated in diminished bacterial clearance and heightened inflammation (Hamilton et al., 2021). Studies utilizing MitoSOX Red have shown increased mitochondrial superoxide levels in IB3-1 cells and primary cells expressing F508del-CFTR, particularly following infection with P. aeruginosa, thereby suggesting a connection between bacterial challenge and mitochondrial oxidative stress in CF airways (Rimessi et al., 2015; 2020). Notably, earlier research using MitoSOX also demonstrated significantly elevated mitochondrial ROS in CFTR-deficient IB3-1 cells compared to control cells (Velsor et al., 2006). Beyond airway cells and bacterial interactions, DHE-based investigations have indicated increased general intracellular superoxide in the pancreas of CF pigs, a finding linked to endothelial dysfunction within the CF context (O’Malley et al., 2022).

Investigations into H₂O₂ and related ROS have employed various methodologies, including the use of DCFH-DA and its derivatives. The assessment of general intracellular ROS using DCFH-DA has produced varied outcomes contingent on cell type and experimental context. For example, DCFH-DA assay measured an increased oxidative burst in peripheral blood neutrophils from CF patients (Frühwirth et al., 1998), while no significant differences in total intracellular ROS between CF and WT mouse macrophages were detected at baseline or following bacterial challenge (Hamilton et al., 2021). Conversely, DCFH-DA oxidation indicated increased intracellular H₂O₂ levels in CFTR-deficient IB3-1 cells (Velsor et al., 2006), although no differences in bulk oxidative capabilities, were noted in homozygous F508del-CFTR nasal epithelial cells (Schwarzer et al., 2007). Methodological nuances concerning probe application and detection further influence the interpretation of these findings (Clauzure et al., 2021).

Peroxidase-mediated assays offer alternative approaches to quantify H₂O₂. Clinical research has utilized techniques like the pHPA coupled with HRP to detect elevated H₂O₂ levels in the EBC of children with CF during acute pulmonary infections, with these levels decreasing following antibiotic treatment (Jöbsis et al., 2000). In cellular models, the Amplex Red^® assay can directly quantify accumulated H₂O₂, as shown in studies measuring its release rate into the apical solution of CF HNE cells grown on permeable supports after viral infection (Schwarzer et al., 2007). While the primary focus remains on CF-relevant systems, techniques for measuring intracellular ROS have also been applied in studies investigating the effects of compounds on oxidative status in other cell types, such as platelets, in inflammatory contexts, highlighting the broader applicability of these methodologies in understanding redox biology (Gziut et al., 2013). These diverse approaches underscore the varied strategies employed to quantify and comprehend the role of H₂O₂ and related oxidants in the complex pathology of CF and associated inflammatory processes.

The Thiobarbituric Acid Reactive Substances (TBARS) assay is an old but commonly used test to study lipid peroxidation by exploiting the product malondialdehyde (MDA) as a marker of the reaction. This assay is based on the ability of the MDA to react with the thiobarbituric acid (TBA) under acidic condition, leading to the formation of MDA-TBA2 adducts called TBARS. TBARS are characterized by a red color pigment, that can be measured both using visible wavelength spectrophotometry at 532 nm and fluorescence (λ_excitation = 370 nm, λ_emission = 420 nm). Actually, this method presents some limitations: (i) not all lipid peroxidation reactions lead to the formation of MDA; (ii) some molecules other than MDA can react with TBA, producing adducts with similar absorption to TBARS; (iii) MDA is not generated exclusively through lipid peroxidation; (iv) the experimental conditions required for MDA reaction with TBA (high temperature and low pH) may cause artefactual formation of lipid peroxidation products, eventually producing misleading data (Janero et al., 1990; Jentzsch et al., 1996). It is reasonable to suggest that TBARS test is associated with data from other methods to study lipid peroxidation, or that the implementation of TBARS test proposed by Jentzsch in 1996 is adopted. Specifically, to avoid lipid peroxidation reactions dependent on experimental settings, authors proposed to exclude oxygen or add high amounts of butylhydroxytoluene (BHT) (Jentzsch et al., 1996).

Hydroperoxides can be estimated with the ferrous ion oxidation xylenol orange (FOX) methods. Two version of this method are described: FOX1 is used for the determination of low levels of lipid hydroperoxides in aqueous phase, while FOX2 is applied in case of lipid phase. FOX1 is described as more sensitive than FOX2 (Wolff, 1994). Briefly, in dilute acid, lipid hydroperoxides oxidises ferrous to ferric ion. Xylenol orange is a fluorochrome acid dye with high selective chelating properties for ferric ions. Their interactions produce a coloured complex that can be detected with a spectrophotometer at 560 nm. Thus, it is possible to indirectly measure hydroperoxide concentration by monitoring ferric ions generation (Banerjee et al., 2003).

Cis-parinaric acid (PnAc) is a naturally fluorescent fatty acid, applied as a structural analogue of membrane lipids. Its chemical and physical properties make it appropriate as a probe for lipid peroxidation. In fact, when excited at ∼ 320 nm, this chromophore provides for a natural fluorescence at ∼ 420 nm, which is decreased by oxidative stimuli. Thus, the greater lipid peroxidation, the less fluorescence is detected (Laranjinha et al., 1992; Steenbergen et al., 1997).

BODIPY™ 581/591 C11 undecanoic acid is a lipid peroxidation sensor that localizes to membranes. Upon its oxidation, a shift of the fluorescence emission happens. Specifically, to the reduced form corresponds an emission peak at ∼590 nm, while to the oxidised form the peak is shifted to ∼510 nm. This assay allows a ratiometric analysis of membrane lipid oxidation (Drummen et al., 2002; Wiernicki et al., 2020; Stockert, 2021).

A derivative from BODIPY™ 581/591 C11 called MitoPerOx has been developed in 2012 to detect mitochondrial lipid hydroperoxides. In this probe the BODIPY fluorophore was conjugated via a dienyl group to a phenyl group, and, similarly to MitoSOX, the specificity for the inner membrane of the mitochondria (facing the matrix) is given by a triphenylphosphonium lipophilic cation, whose uptake is driven by the mitochondrial membrane potential. Analogously to C11-BODIPY, probe oxidation induces a shift of the fluorescence emission peak from ∼590 to ∼520 nm (Prime et al., 2012; Oh et al., 2022).

Oxidative stress is characterized by the accumulation of ROS, which can induce damages to the mitochondrial respiratory chain, influence Ca²⁺ homeostasis, and impair mitochondrial functionality. The Seahorse XF (Extracellular Flux) technology is an advanced analytical platform designed for real-time, label-free, and non-destructive evaluation of cellular bioenergetics. This system enables the simultaneous measurement of oxygen consumption rate (OCR), reflecting mitochondrial oxidative phosphorylation, and extracellular acidification rate (ECAR), indicative of glycolytic activity. Together, these metrics delineate the two principal adenosine triphosphate (ATP)-generating pathways in viable cells, which may be affected by oxidative stress.

The methodology employs specialized multi-well plates seeded with adherent or suspended cell cultures. A sensor cartridge equipped with biosensors for dissolved oxygen and hydrogen ion concentration is positioned above the wells, establishing transient, isolated microenvironments. Within these microenvironments, fluctuations in oxygen tension and pH levels - driven by cellular metabolic processes - are continuously monitored. Through timed injections of pharmacological modulators (e.g., metabolic inhibitors, activators or substrates), the assay facilitates a comprehensive investigation of mitochondrial and glycolytic functionality. Key parameters assessed include basal and maximal respiratory capacity, ATP-linked respiration, spare respiratory capacity, basal glycolysis, and glycolytic reserve. This dynamic, multi-parametric approach yields a detailed bioenergetic profile, enabling rigorous characterization of cellular metabolic adaptations under controlled experimental conditions (Ferrick et al., 2008).

Markers of lipid peroxidation, notably TBARS and MDA, are crucial indicators of oxidative damage to cellular membranes and are frequently evaluated in CF to assess the impact of oxidative stress. A substantial body of research reports elevated TBARS and MDA levels in diverse biological samples from CF patients compared to healthy controls. These include plasma (Brown and Kelly, 1994; Portal B. et al., 1995; Back et al., 2004; Bennemann et al., 2022), and white blood cells, even in patients considered clinically stable, indicating a chronic inflammatory and oxidative state (Olveira et al., 2013). Conversely, one study noted initially lower MDA levels in CF patients that increased with age and clinical deterioration and observed an increase in lipid peroxidation (TBARS and FOX1) following antibiotic administration, highlighting potential variability influenced by disease stage and treatment (Lagrange-Puget et al., 2004). In CF animal models, increased TBARS have been demonstrated in the lung tissue of Cftr-KO mice, reflecting localized oxidative damage within affected organs (Velsor et al., 2001), although TBARS in BAL fluid (BALF) were below detection limits in both Cftr-KO and WT mice.

The FOX1 method was notably employed by Hull and colleagues and revealed a significant elevation in lipid hydroperoxide concentrations in the ELF of CF patients exhibiting inflammation, compared to both the control group and the CF group without inflammation. Crucially, lipid hydroperoxide levels in CF patients without inflammation were similar to those in healthy controls (Hull et al., 1997). This provided direct evidence of increased lipid peroxidation in the CF airway and strongly suggested that this elevation is a consequence of the pulmonary inflammation characteristic of CF, rather than a direct, inflammation-independent outcome of the primary CF defect.

PnAc enabled the demonstration of an exacerbated lipid peroxidation in the mitochondrial membranes of CFBE41o-cells compared to 16HBE14o-cells, thereby adding to the cumulative evidence of increased oxidative damage at the mitochondrial level in CF (Atlante et al., 2016).

Furthermore, a pivotal study by Maniam et al. demonstrated the increased susceptibility to ferroptosis of IB3-1cells in comparison to control epithelial cells. In fact, BODIPY™ 581/591 C11 revealed heightened levels of lipid peroxidation in the CF cell line and provided compelling evidence that CF airway epithelial cells are predisposed to accumulate lipid peroxides, a key driver of ferroptosis (Maniam et al., 2021).

Overall, the consistent finding of elevated lipid peroxidation markers across various studies and sample types provides robust evidence of heightened oxidative stress in CF, contributing to cellular dysfunction and disease progression.

Immune cells, such as macrophages and neutrophils, exhibit significant metabolic reprogramming in the context of CF, profoundly influencing their inflammatory responses and ability to effectively clear pathogens. Seahorse XF analysis has proven instrumental in comprehensively characterizing these altered metabolic profiles in CF immune cells. For instance, investigations into defective immunometabolism in CF macrophages have utilized the Seahorse Extracellular Flux analyzer, revealing impaired oxygen consumption, both at baseline and following infection with challenging pathogens like Burkholderia cenocepacia (Hamilton et al., 2021). Further applying Seahorse extracellular flux assays, studies have demonstrated that the heightened mitochondrial respiration observed in CF cell lines, including HBE cells expressing CFTR^{F508del/F508del}, can be remarkably normalized by triple CFTR modulator therapy through mechanisms involving calcium (Jarosz-Griffiths et al., 2024). Additionally, utilizing Seahorse XF assays to delve into the immunometabolism of monocyte-derived macrophages (MDMs), research has shown that exposure to secondhand e-cigarette vapor significantly impacts macrophage metabolic profiles in both CF and non-CF cells; notably, vape exposure blunted macrophage basal respiration, an effect partially rescued by CFTR modulator therapy (ETI), while concurrently increasing glycolytic metabolism, a change unaffected by ETI treatment (Wisniewski et al., 2025).

In CF, increased amount of 8-OHdG was measured both in nuclear DNA (Velsor et al., 2001; Velsor et al., 2001) and mitochondrial DNA (Velsor et al., 2006) extracted from lung tissues of Cftr-KO mice and compared to WT. Analogously, urinary level of 8-OHdG was significantly higher in CF children than in age-matched controls and this event was independent of clinical status (Brown et al., 1995).

Another application consists in the measurement of 8-oxoGua in the DNA of P. aeruginosa strains. From these studies it emerged that hypermutable P. aeruginosa strains have more 8-oxoGua upon exposure to polymorphonucleates (PMN) and this mechanism is proposed to be linked to an increased antibiotic resistance. For this reason, authors suggest an antioxidative therapy for patients with CF to diminish oxidative stress, thus reducing the risk of developing antibiotic resistance (Ciofu et al., 2005; Mandsberg et al., 2009).

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separates complex protein mixtures by isoelectric point and molecular weight, enabling analysis of thousands of protein spots in a single gel (Kavallaris and Marshall, 2005). This technique has contributed to CF research through BALF proteome studies, revealing low molecular weight protein differences between CF patients and controls (von Bredow et al., 2001). Its enhanced version, two-dimensional difference gel electrophoresis (2D-DIGE), allows simultaneous quantitative comparison of multiple samples using distinct fluorescent labels, as demonstrated in CF serum proteomics investigations (Charro et al., 2011). Despite these historical contributions, gel-based approaches exhibit notable limitations that have prompted their progressive replacement by direct mass spectrometry (MS)-based methodologies for oxidative stress research. These traditional techniques are labor-intensive with restricted dynamic range for detecting low-abundance proteins and provide suboptimal resolution for proteins with extreme molecular weights or high hydrophobicity, including CFTR (Charro et al., 2011). Contemporary CF research has consequently shifted toward MS-centric methodologies offering superior sensitivity, throughput, and capabilities for identifying specific post-translational modifications essential for comprehensive oxidative stress analysis.

MS has established itself as the preeminent analytical platform in modern proteomics research, offering exceptional sensitivity, specificity, and throughput for protein identification and quantification. Shotgun proteomics, primarily utilizing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), provides a robust methodology for comprehensive protein identification in complex biological matrices. This “bottom-up” approach begins with enzymatic protein digestion (typically using trypsin) to generate peptides, which undergo liquid chromatographic separation before mass spectrometric analysis. The analytical workflow involves initial measurement of peptide mass-to-charge ratios (m/z) in MS1 scans, followed by fragmentation of selected peptides to produce tandem mass spectra (MS/MS) containing sequence information, which are subsequently matched against protein databases for identification (Nimer and Abdel Rahman, 2023). Multidimensional Protein Identification Technology (MudPIT) represents an advanced shotgun proteomics implementation, incorporating multiple orthogonal chromatographic separation stages prior to MS analysis, significantly enhancing proteome coverage through improved peptide mixture resolution (Rauniyar et al., 2014).

Quantitative proteomics enables determination of relative or absolute protein abundance variations across distinct biological conditions, utilizing either label-free or label-based approaches.

Label-free quantification (LFQ) compares protein abundance based on MS signal intensity or spectral counts without isotopic labeling (Li et al., 2012). This methodology offers simplified sample preparation and versatility across various sample types, providing extensive proteome coverage. LFQ has been widely implemented in CF research, including investigations of cellular models (Rauniyar et al., 2014), BALF metaproteomics (Kruk et al., 2024), and plasma proteomics in analogous fibrotic pulmonary conditions characterized by oxidative stress (Saraswat et al., 2020). However, LFQ may exhibit lower quantitative accuracy and reproducibility compared to label-based techniques, particularly when measuring subtle alterations in oxidative stress markers or antioxidant enzymes (Li et al., 2012).

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) involves metabolic labeling where cells incorporate “light” or “heavy” (stable isotope-enriched) essential amino acids during protein synthesis. Samples from different conditions are combined early in processing, with relative protein abundance determined by heavy-to-light peptide signal ratios (Peters-Hall et al., 2015). SILAC delivers high accuracy in cell culture experiments by minimizing downstream processing variability (Harsha et al., 2008), but remains primarily limited to metabolically active, culturable cells, constraining its applicability for direct patient sample analysis.

Isobaric labeling techniques including iTRAQ (isobaric Tags for Relative and Absolute Quantitation) and TMT (Tandem Mass Tags) involve chemical tagging of peptides post-digestion. These tags possess identical masses, enabling co-elution during chromatography and identical m/z in MS1 scans. Upon fragmentation, each tag releases a unique “reporter ion” whose intensity reflects the peptide’s original abundance (Li et al., 2012). These methods allow multiplexing of 8–16+ samples simultaneously, enhancing throughput and statistical power through concurrent analysis of multiple conditions. However, they may experience “ratio compression” from interfering peptides, potentially underestimating true protein abundance fold changes.

Selection among these quantitative approaches depends on experimental design, sample characteristics, required analytical depth, and quantitative precision needs. SILAC proves optimal for investigating oxidative stress mechanisms in controlled cell culture models, while TMT or LFQ may be preferred for larger patient cohorts or clinical samples, considering their respective strengths in quantifying redox-related proteins in CF.

Targeted proteomics, predominantly utilizing Multiple Reaction Monitoring (MRM) or Selected Reaction Monitoring (SRM) mass spectrometry, represents a hypothesis-driven methodology for precise and sensitive quantification of pre-selected proteins through their representative peptides. Unlike discovery-based approaches, MRM/SRM selectively monitors specific precursor ion-to-fragment ion transitions unique to target peptides (McShane et al., 2014). This high selectivity enables quantification within complex biological matrices with minimal sample fractionation. Quantitative accuracy is enhanced through incorporation of stable isotope-labeled synthetic peptides as internal standards (Liquid Chromatography-Stable Isotope Dilution-MRM MS or LC-SID-MRM MS), a methodology successfully applied for absolute quantification of CFTR protein using signature peptides as full-length protein surrogates (McShane et al., 2014). Targeted proteomics serves a critical function in translational research, bridging discovery-phase findings with clinical applications. Following identification of candidate oxidative stress biomarkers through discovery proteomics, MRM/SRM provides robust, reproducible, and high-throughput quantification essential for validation in larger clinical cohorts. This crucial validation determines the utility of potential oxidative stress biomarkers for CF diagnosis, patient stratification, disease progression monitoring, and therapeutic response assessment. The capability for absolute quantification, demonstrated for CFTR (McShane et al., 2014), provides particular value for establishing precise physiological or pathological concentrations of key proteins involved in oxidative stress responses.

Redox proteomics provides powerful tools for investigating oxidative stress molecular impact in CF by identifying and quantifying proteins with specific oxidative or nitrosative post-translational modifications (PTMs). Protein carbonylation, an irreversible PTM introducing carbonyl groups, serves as a critical biomarker of severe oxidative protein damage in CF. These modifications result from direct ROS attack on amino acids (proline, arginine, lysine, threonine) or indirectly via reactive aldehydes from lipid peroxidation (MDA, 4-hydroxynonenal), or through reactions with reducing sugars and advanced glycation end-products (Pinzaru et al., 2023). With elevated protein carbonyls documented in CF patients (Causer et al., 2020), identifying specifically affected proteins is essential for understanding functional consequences. Traditional detection employs 2,4-dinitrophenylhydrazine (DNPH) derivatization, forming stable adducts quantifiable spectrophotometrically or via anti-DNPH antibody Western blotting (Madian and Regnier, 2010). For proteomic identification, DNPH derivatization can be coupled with 2D-PAGE and MS. Advanced approaches utilize biotin hydrazide or other tagged hydrazides for carbonyl derivatization, enabling avidin-based affinity chromatography enrichment before LC-MS/MS identification of modified proteins and specific carbonylation sites (Madian and Regnier, 2010). Profiling the “carbonylome” in CF provides a molecular fingerprint of irreversible oxidative damage, revealing vulnerable cellular pathways and potential therapeutic targets (Starosta et al., 2006). Cysteine thiols are highly susceptible to redox-based PTMs like S-nitrosylation and S-glutathionylation, which are critical for cellular signaling and stress responses. S-nitrosylation involves covalent attachment of nitric oxide (NO) to cysteine thiols, forming reversible S-nitrosothiols (SNOs) (Kimura, 2020). While essential for physiological signaling, excessive NO production in CF airway inflammation can dysregulate S-nitrosylation, contributing to pathology (Kimura, 2020). Proteomic identification typically employs “biotin-switch” assays or differential labeling techniques, followed by enrichment and MS identification (Wolhuter and Eaton, 2017). S-glutathionylation forms mixed disulfide bonds between protein cysteine thiols and GSH (Townsend, 2007), serving protective, regulatory, and signaling functions. In CF, CFTR dysfunction compromises GSH transport and homeostasis, altering S-glutathionylation patterns (Pinzaru et al., 2023). Proteomic approaches for S-glutathionylated protein identification involve trapping glutathionylated peptides or using isotopically labeled GSH derivatives for enrichment before MS-based identification and quantification (Harsha et al., 2008).

Redox proteomics continues to advance, offering increasingly detailed insights into protein structure, dynamics, and modifications under oxidative stress. Fast Photochemical Oxidation of Proteins (FPOP) stands out as an innovative MS-based foot printing technique utilizing highly reactive hydroxyl radicals to map solvent-accessible protein surfaces (McKenzie-Coe et al., 2021). In FPOP, hydroxyl radicals are generated on a microsecond timescale via laser-induced photolysis of H₂O₂. These radicals rapidly and irreversibly modify solvent-exposed amino acid side chains. Subsequent MS analysis identifies the sites and extent of these modifications, providing residue-level resolution of protein conformation, ligand binding sites, and interaction interfaces. FPOP is exceptionally suited for studying dynamic processes like protein folding and conformational changes, as its rapid labeling kinetics can “capture” transient structural states (Johnson et al., 2019). A significant recent advancement, In-Cell FPOP of Membrane Proteins (IC-FPOMP), enables foot printing of integral membrane proteins, such as CFTR, within their native live-cell environment. This addresses the challenge of structurally studying membrane proteins. Although specific applications of FPOP to CF-related oxidative damage are not yet extensively documented, its capabilities are highly promising. Since hydroxyl radicals are key damaging species in biological oxidative stress, FPOP can mimic and map proteins’ most susceptible sites to this oxidative attack. This could be instrumental in investigating how the chronic oxidative environment in CF alters the conformation of CFTR or other critical airway proteins, or how CFTR mutations affect protein structure and solvent accessibility, potentially predisposing them to oxidative damage (Johnson et al., 2019; Sun et al., 2025). While current redox proteomics in CF often focuses on identifying which proteins are modified (e.g., carbonylated, glutathionylated), techniques like FPOP offer the potential to delve deeper into how these modifications occur - identifying specific residues targeted by ROS - and how these modifications, or the oxidative environment itself, impact protein structure, dynamics, and interactions (Johnson et al., 2019; Sun et al., 2025).

Supplementary Table S3 summarizes the proteomic methodologies discussed, highlighting their principles and applications in CF oxidative stress research.

Comprehensive lipidomic investigations in CF have revealed multifaceted lipid dysregulation intimately connected with oxidative stress mechanisms. Significant phospholipid profile alterations characterize CF pathology, with diminished phosphatidylcholine and lysophosphatidylcholine species in patient’s plasma correlating with respiratory disease severity (Guerrera et al., 2009; Ollero et al., 2011). Recent advanced methodologies have identified specific oxidized phospholipid species (PC 40:5;O and PC 36:5;O), providing direct evidence of membrane oxidative modification (Zardini Buzatto et al., 2021). Concurrently, ceramide metabolism exhibits complex dysregulation, with apparent contradictions reflecting tissue-specific pathophysiology; some investigations report reduced ceramide concentrations in plasma (Guilbault et al., 2008; Wojewodka et al., 2011), while others demonstrate accumulation of long-chain ceramide species in pulmonary tissues (Veltman et al., 2021). Notably, elevated ratios of long-chain to very long-chain ceramide species in BALF correlate with inflammatory markers and lung disease severity, mechanistically linked to enhanced bacterial susceptibility and epithelial apoptosis (Zhang et al., 2008; Becker et al., 2010). Essential fatty acid imbalance constitutes another consistent aberration in CF, characterized by decreased linoleic acid and docosahexaenoic acid levels alongside relative arachidonic acid elevation, promoting pro-inflammatory eicosanoid production (Kleme and Levy, 2015; Drzymala-Czyz et al., 2024). Supporting this paradigm, elevated urinary 8-iso-prostaglandin F2α serves as a reliable biomarker of increased lipid peroxidation, correlating with pulmonary dysfunction (Ciabattoni et al., 2000; Veltman et al., 2021). Additional alterations manifest in ether-linked phospholipids, cholesterol esters, and glycosylated sphingolipids, with particular disruption of membrane cholesterol distribution crucial for CFTR protein trafficking (Dei Cas et al., 2020; Liessi et al., 2020; Cui et al., 2021). Therapeutic CFTR modulator therapies demonstrate promising effects on these lipid abnormalities. ETI enhances mutated CFTR protein expression while simultaneously remodeling sphingolipid composition (Dobi et al., 2025), increasing dihydrosphingolipids via modulation of Δ4-desaturase enzymes (Liessi et al., 2023; Ciobanu et al., 2024), and beneficially altering fatty acid metabolism (Veltman et al., 2021). Clinically, these interventions improve lipoprotein profiles, increasing anti-atherogenic HDL cholesterol while decreasing atherogenic LDL particles (Yuzyuk et al., 2023; Lonabaugh et al., 2024). Complementary antioxidant supplementation (vitamin E, GSH, β-carotene) effectively mitigates oxidative stress-induced lipid peroxidation, reducing isoprostane excretion and enhancing LDL oxidation resistance (Ciabattoni et al., 2000; Cantin et al., 2007; Veltman et al., 2021). These findings collectively establish the lipidomic profile as both a mechanistic framework and therapeutic target in CF, highlighting the critical intersection between lipid metabolism, oxidative stress and CFTR function in the disease pathophysiology.

Two main approaches for transcriptomics are microarray and RNA-sequencing (RNA-Seq).

Transcriptome profiling is a valuable tool in CF when studying the pathophysiology of the disease or when looking for new therapeutical targets (McKiernan et al., 2014; Kormann et al., 2017). Intriguingly, transcriptomic analysis has not been applied only to identify differences in gene expression between cells with or without a functional CFTR (Kamei et al., 2019; Declercq et al., 2021), but also to characterize the transcriptional activity of each cell type component of the lung epithelia (Zoso et al., 2019; Sun and Zhou, 2025) and to study pathogen virulence in CF patients (Worgall et al., 2005; Drevinek et al., 2008; Rossi et al., 2018; Irvine et al., 2019). Regulation of genes involved in the antioxidant response in CF have been identified in several studies, both with microarrays and RNA-Seq.

For example, Declercq and colleagues have performed transcriptomic analysis to study the effect on gene expression of CFTR depletion. In particular, they have used complementary models of CFTR silencing and blockade (by the CFTR inhibitor, CFTRinh-172) in freshly isolated human umbilical vein endothelial cells (HUVECs). Then, they have validated key findings in vitro and in vivo in Cftr-KO mice and ex vivo in CF patient-derived endothelial cells. Transcriptomics revealed that CFTR knock down cells are characterized by an upregulation of oxidative stress response-related genes like those expressing for GSH consuming enzymes (i.e., GPX1, GPX4), SOD1 and 2, peroxiredoxin and thioredoxins. These results were confirmed by flow cytometry. Moreover, LC-MS analysis followed to prove the depletion of GSH and NADPH, that confirmed the sustained oxidative stress. Further validation analyses were conducted with DCF and MitoSOX (Declercq et al., 2021). Similar results were obtained with a transcriptomic analysis following the pollution-caused CFTR decrease in 16HBE14o-cells. What has emerged is a pattern similar to that observed in CF: a diminished amount of CFTR leads to an upregulation of SOD1 and a downregulation of CAT. These events together induce the accumulation of H₂O₂ within the cells. Moreover, an upregulation of HMOX1 favours the accumulation of Fe²⁺ that can react with H₂O₂ in a Fenton-like reaction thus producing the highly reactive ROS hydroxyl radical. This worsens the oxidative stress and finally leads to DNA damages (Stermann et al., 2022).

A different application of transcriptomics in CF consists in the study of gene regulation in pathogens typically infecting CF patients, for the identification of mechanisms involved in their adaptation and virulence. For example, transcriptomic analysis of a strain of P. aeruginosa, isolated from a mouse model of chronic infection, but originally from a CF patient, was performed to identify transcriptional modifications responsible for the phenotypic change from mucoid to small colonies vescicles (SCV). The latter phenotype is typical of lung adaptation and loss of functionality in CF patients. Authors have performed RNA-seq and discovered that, among all the genes regulated in the phenotypic change, half of those upregulated are involved in the response to oxidative stress (Irvine et al., 2019). Analogously, another important pathogen in CF, the B. cenocepacia, isolated from CF sputum have activated genes that confer protection against ROS, as demonstrated with microarray-based studies (Drevinek et al., 2008). As demonstrated by the previous examples, transcriptional analysis can help in the identification of virulence genes of pathogens that represent the principal cause of mortality in CF patients. Resistance to oxidative stress in these pathogens help them survive the attack of neutrophils. The identification of these genes is a step forward to a possible therapy in case of antibiotic resistance. When performing transcriptomics, it is important to validate the results with studies of protein amount or enzyme activity, because of regulatory mechanism that could intervene after transcription.

The ABTS protocol evaluates antioxidant efficacy through the neutralization of pre-generated ABTS·⁺ radical cations (Deadman, 1973). These chromogenic radical species, produced via ABTS oxidation using potassium persulfate or comparable oxidizing agents, demonstrate pronounced light absorption across the visible range, particularly at 734 nm wavelength. Sample antioxidants interact with ABTS·⁺ through either single electron transfer (SET) or hydrogen atom transfer (HAT) pathways, leading to radical quenching and a proportional reduction in measured absorbance (Deadman, 1973). Quantification typically employs Trolox (water-soluble vitamin E derivative) as reference standard, with results expressed as Trolox Equivalent Antioxidant Capacity (TEAC) units. This analytical approach demonstrates broad applicability across both water-soluble and fat-soluble antioxidant compounds (Deadman, 1973).

FRAP analysis determines sample reducing capacity by measuring electron donation potential (Benzie and Strain, 1996). Within acidic environments (pH 3.6), antioxidant molecules facilitate the reduction of ferric-2,4,6-Tris(2-pyridyl)-s-triazine (Fe³⁺-TPTZ) complexes to their ferrous counterparts (Fe²⁺-TPTZ). This chemical transformation produces a distinctive blue chromophore (Benzie and Strain, 1996). Spectrophotometric monitoring occurs at 593 nm, where absorbance increases correlate directly with reducing antioxidant concentrations. Quantification employs either ferrous ion equivalents or Trolox standardization. Notably, FRAP methodology predominantly captures electron-transfer mechanisms while potentially underestimating hydrogen atom transfer-based radical scavenging processes (Benzie and Strain, 1996).

DPPH analysis represents an established approach for evaluating antioxidant radical-neutralizing capabilities (Brand-Williams et al., 1995). The method employs stable DPPH· synthetic radicals, characterized by purple coloration and maximum absorption at 517 nm. Antioxidant exposure leads to DPPH· reduction to its non-radical state (DPPH-H) through hydrogen atom acceptance (HAT mechanism) or electron acquisition (SET mechanism) (Brand-Williams et al., 1995). This transformation results in decreased 517 nm absorbance, monitored either kinetically or at predetermined endpoints. Data presentation includes percentage radical scavenging calculations or standardization against reference antioxidants such as ascorbic acid or Trolox (Brand-Williams et al., 1995). This methodology proves particularly useful for rapid in vitro screening of free radical interception capacity.

BAP testing utilizes commercially available assay systems frequently implemented in clinical diagnostic environments for evaluating collective reducing potential in biological matrices including serum and plasma (Celi et al., 2010). The procedure involves sample-mediated reduction of proprietary ferric-chromogen complexes, generating colored ferrous products (Celi et al., 2010). Spectrophotometric quantification (wavelengths typically 505–520 nm depending on kit specifications) measures color intensity proportional to sample reducing capacity. Standardization against predetermined references (commonly ferrous ion-based) provides rapid systemic antioxidant potential assessment (Celi et al., 2010). While offering high-throughput convenience for clinical laboratories, BAP analysis provides composite measurements without identifying individual antioxidant contributors.

TRAP analysis specifically quantifies sample effectiveness in neutralizing peroxyl radicals (R-OO·), offering critical information regarding cellular protection against lipid peroxidation processes (Uotila et al., 1994). Systematic peroxyl radical production occurs via controlled thermal breakdown of radical initiators, particularly 2,2′-Azobis(2-amidinopropane) dihydrochloride (ABAP) (Uotila et al., 1994). The methodology tracks oxidative damage inflicted upon target molecules or detection probes. When employing fluorescent probes such as R-phycoerythrin, the analysis measures fluorescence signal deterioration resulting from peroxyl radical-induced oxidation. This fluorescence monitoring typically utilizes excitation wavelengths spanning 488–565 nm with emission detection ranging 575–675 nm (commonly centered around 580 nm). Alternatively, when chemiluminescent probes are utilized, direct measurement of light emission decay provides the analytical signal (Uotila et al., 1994).

Dysregulation of the key antioxidant GSH is a widely investigated hallmark of CF. In peripheral blood, CF patients consistently exhibit altered GSH status: reduced intracellular levels in neutrophils and lymphocytes (Lands et al., 1999; Tirouvanziam et al., 2006), the latter correlating with lung function, and diminished plasma GSH and GSH:GSSG ratios (Roum et al., 1993; Lagrange-Puget et al., 2004; Innis et al., 2007). Despite persistent oxidative stress markers (Olveira et al., 2013), interventions like N-acetylcysteine or specific supplements show some restorative promise (Tirouvanziam et al., 2006; Innis et al., 2007). The airway presents a more complex GSH profile; while BALF from CF patients and CFTR-deficient models typically shows depleted GSH, exacerbated by infection (Gould et al., 2010; Kettle et al., 2014; Dickerhof et al., 2017), sputum studies have surprisingly reported higher total GSH (Dauletbaev et al., 2004). Furthermore, one murine study found no GSH:GSSG alterations in lung tissue or BALF (Velsor et al., 2001), highlighting compartmental intricacies. Nevertheless, aerosolized GSH (Roum et al., 1999) and hypertonic saline (Gould et al., 2010) can augment airway GSH. Cellular investigations reveal further nuances. CFTR deficiency is linked to markedly lower mitochondrial GSH (Velsor et al., 2006) and often reduced intracellular GSH in epithelial cells, which can be partially restored by CFTR modulators like lumacaftor (de Bari et al., 2018). However, contrasting findings of normal or even slightly higher intracellular GSH in certain CF cell lines exist (Bergamini et al., 2009). Promisingly, compounds such as γ-glutamylcysteine and whey protein hydrolysates can effectively increase intracellular GSH in CF bronchial epithelial cells (Grey et al, 2003; Vilela et al., 2006; Hewson et al., 2020).

Altered vitamin status, linked to malabsorption and pulmonary disease severity, is a well-documented challenge in CF (Wysocka-Wojakiewicz et al., 2022). Deficiencies in fat-soluble vitamins (A, D, E, K) are particularly prevalent. Consistently low plasma vitamin A and E necessitate supplementation (Brown and Kelly, 1994; Portal B. C. et al., 1995; Benabdeslam et al., 1999; Lai et al., 2022), while highly prevalent vitamin D deficiency, impacting bone and potentially lung health, faces repletion challenges despite supplementation efforts (Lai et al., 2022; Francalanci et al., 2023; Farahbakhsh et al., 2024). Impaired vitamin K status, especially with pancreatic insufficiency, also risks coagulopathy and compromised bone mineralization (Krzyzanowska et al., 2015). Notably, CFTR modulator therapies variably affect these vitamins: lumacaftor/ivacaftor increased plasma vitamin A but moderately decreased the vitamin E/cholesterol ratio (Sommerburg et al., 2021), whereas ETI boosted vitamins A and D, leaving levels of E and K reportedly stable (Hergenroeder et al., 2023).

Though less universally deficient, water-soluble vitamins can be suboptimal and play distinct roles in CF. Vitamin C, for instance, may activate defective CFTR in vitro (Fischer et al., 2004), and its supplementation can positively influence vitamin E levels, suggesting oxidative stress mitigation (Traber et al., 2022). Among B vitamins, while 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation showed potential cellular benefits (Scambi et al., 2009), paradoxically high, often supra-physiological, serum B12 levels are frequently observed in highly supplemented children with pancreatic insufficiency (Maqbool et al., 2014). Early studies indicated generally adequate riboflavin (B2) and pyridoxine (B6) levels in treated patients (Congden et al., 1981). However, low plasma concentrations of B6’s active form, pyridoxal 5′-phosphate, are a common abnormality (Faraj et al., 1986). The metabolic importance of folate (Scambi et al., 2009) is also recognized, and rare isolated vitamin B2 deficiencies have been documented in specific clinical contexts (McCabe, 2001).

Few studies have employed TAC methodologies to highlight alterations in antioxidant capacity in CF. Notably, Langley and colleagues reported that despite elevated plasma concentrations of individual antioxidants like ascorbic acid, uric acid, and sulfhydryl groups compared to healthy controls, the CF patients exhibited a significantly reduced TRAP (Langley et al., 1993). Furthermore, they observed a strong negative correlation between TRAP values and high plasma ascorbic acid levels in CF patients, suggesting that high concentrations of ascorbic acid might exert a pro-oxidant effect in the context of CF, thereby diminishing the overall extracellular antioxidant defense (Langley et al., 1993).

Expanding on these observations, serum total antioxidant capacity using ABTS and FRAP assays in a cohort of CF patients was studied (Galiniak et al., 2022). Results indicated a significantly lower overall serum TAC by the FRAP assay in CF patients compared to controls, although the ABTS assay did not show this overall difference (Galiniak et al., 2022). This study further demonstrated that reduced TAC was associated with increased CF disease severity. However, TAC levels did not differ significantly among CF patients based on the presence or type of bacterial infection (Galiniak et al., 2022). The Galiniak et al. study also found that TAC measured by ABTS correlated negatively with age and positively with FEV1.

Erythrocyte lysates commonly assess intracellular SOD activity (predominantly Cu/Zn-SOD/SOD1), normalized to hemoglobin content for comparability (Matkovics et al., 1982). Plasma or serum measures extracellular SOD (EC-SOD/SOD3), though plasma may contain SOD1 released from lysed cells, affecting interpretation (Laskowska-Klita and Chelchowska, 2001; Olveira et al., 2013). Sputum and BALF provide direct insight into airway antioxidant defenses, with sputum typically processed into a soluble phase (“sol”) for enzyme analysis. However, sputum’s inherent complexity (mucus, cells, bacteria, mediators) challenges assay standardization, necessitating meticulous sample processing to preserve enzyme activity and minimize interference (Dosanjh, 2008). SOD activity in CF exhibits varied, compartment-specific alterations reflecting complex redox imbalance. Initial research demonstrated increased SOD activity in erythrocytes of CF children and heterozygous parents compared to controls, potentially representing an adaptive response to systemic oxidative stress (Laskowska-Klita and Chelchowska, 2001). Conversely, more recent studies in bronchiectasis patients, including CF cohorts, reported decreased plasma SOD activity, potentially from increased consumption due to heightened extracellular superoxide, reduced synthesis, or impaired EC-SOD release (Olveira et al., 2013). Animal models support tissue-specific changes, with decreased pancreatic Cu/Zn-SOD activity observed in CF porcine models (O’Malley et al., 2022). These varying observations highlight the critical distinction between enzyme concentration and actual catalytic activity, as evidenced in other inflammatory conditions where plasma SOD1 concentration increased while total activity remained unchanged (Sciskalska et al., 2020). Within airway secretions, CF sputum sol attenuates superoxide radical production by phorbol-stimulated control neutrophils in a time- and concentration-dependent, heat-labile manner, suggesting inhibitory enzymatic factors (Dosanjh, 2008). This effect is not mere scavenging; its inhibition by exogenous SOD indicates CF sol interferes with superoxide generation or availability. Paradoxically, while reducing superoxide, CF sputum can impair neutrophil bactericidal capacity (Fantone et al., 2021; 2023), partly linked to reduced phagosomal ROS production, essential for bacterial clearance. Investigations using CF cell lines reveal intrinsic SOD alterations linked to CFTR dysfunction. Pancreatic (CFPAC-1) and tracheal (CFT-2) CF cell lines exhibited reduced Mn-SOD and Cu/Zn-SOD expression compared to controls (Rottner et al., 2011). Despite similar EC-SOD expression, its enzymatic activity was significantly reduced in CF cells. Treatment with SOD mimetic (MnTMPyP) reduced the increased apoptosis sensitivity of CF cells, implicating superoxide-mediated oxidative stress (Rottner et al., 2011). These findings collectively highlight that impaired SOD function in CF can result from reduced expression or diminished catalytic activity, underscoring the necessity of functional assays beyond mere protein detection.

In the context of CF, the presence and activity of CAT in airway secretions like sputum and BALF are significant for local H₂O₂ detoxification. Studies have quantified both CAT concentration and activity in CF sputum supernatants (Worlitzsch et al., 1998). Furthermore, tissue and cell lysates, particularly from bronchiolar epithelium which is a major site of CAT expression in healthy lungs, are highly relevant for investigating CAT’s contribution to lung pathology, including that observed in CF (Odajima et al., 2010). The status of CAT activity in CF patient blood has been investigated, yielding conflicting reports. Older studies indicated increased erythrocyte CAT activity in children with CF and their heterozygous parents compared to controls (Matkovics et al., 1982). Similarly, a study in adults with bronchiectasis, including a CF subgroup, reported increased plasma CAT (Olveira et al., 2013), potentially reflecting compensatory upregulation against chronic systemic oxidative stress (Galli et al., 2012). Conversely, a more recent study in CF children found significantly lower CAT activity after confounder adjustment (Laskowska-Klita and Chelchowska, 2001). These discrepancies likely stem from differences in patient populations, methodologies, or the specific blood compartment analyzed (erythrocytes vs. plasma), noting that plasma CAT is usually low, with increases potentially indicating cell leakage during inflammation (de Camargo et al., 2021). CF airways feature intense inflammation and high H₂O₂ production from phagocytes (Galli et al., 2012). CAT protein and activity are detectable in CF sputum, with high levels reported in some studies (Worlitzsch et al., 1998; Dauletbaev et al., 2005), contributing significantly to local H₂O₂ detoxification within the lumen (Worlitzsch et al., 1998). Despite high oxidant production, exhaled breath condensate H₂O₂ levels in CF patients are often similar to controls (Worlitzsch et al., 1998; Ho et al., 1999). This paradox is likely due to efficient H₂O₂ scavenging by CAT and other antioxidants in airway lining fluid and sputum (Worlitzsch et al., 1998; Mishra and Imlay, 2012). CF sputum extracts can neutralize H₂O₂ and protect cells in vitro, a protective effect sustained even after CAT inactivation by inhibitors (Dauletbaev et al., 2005). This highlights the crucial role of other antioxidant systems, particularly GPxs and reduced thiols, in CF sputum H₂O₂ neutralization (Dauletbaev et al., 2005). Conversely, viral infections like RSV decrease lung Cat in mice (Hosakote et al., 2011; Ansar et al., 2020), while chronic bacterial infection, specifically P. aeruginosa colonization, is associated with lower systemic CAT in CF children (Bennemann et al., 2022), suggesting infections can compromise CAT activity systemically or locally. Tissue-specific CAT status in CF also varies. The CF porcine pancreas showed no significant difference in CAT activity compared to non-CF animals (O’Malley et al., 2022). This contrasts with lung fibrosis models, which share some features with chronic CF lung damage, where decreased CAT activity, mRNA, and protein are observed, notably in bronchiolar epithelium (Odajima et al., 2010; Betsuyaku et al., 2013). Studies in acatalasemic mice demonstrated increased susceptibility to bleomycin-induced lung injury and fibrosis, highlighting CAT’s protective role in mitigating fibrotic lung disorders (Odajima et al., 2010).

Studies highlight GPx’s substantial role, with abundant reduced thiols, in detoxifying H₂O₂ in CF sputum, particularly when CAT is inhibited or overwhelmed (Dauletbaev et al., 2004). Assessing GPx activity in tissue homogenates (e.g., lung, pancreas) or cell lysates (e.g., airway epithelial cells) is vital for understanding its contribution to CF pathology in affected organs. Glutathione peroxidase (GPx) activity in the blood of CF patients has shown some variability across studies. Older findings reported normal whole blood GPx in infants and children with CF despite lower whole blood selenium levels, suggesting potential compensatory mechanisms or non-critically limiting selenium (Ward et al., 1984). In contrast, a more recent study in CF children found significantly lower systemic GPx activity after adjusting for confounders (Bennemann et al., 2022). Given the common pancreatic insufficiency in CF leading to selenium malabsorption, reduced GPx activity is plausible (Winklhofer-Roob et al., 1998; Baker, 2008; Zheng and Mostamand, 2023), particularly for plasma GPx (GPx3), which is closely linked to selenium availability (Ottaviano et al., 2009). The glutathione system (GSH and GPx) is a major pulmonary antioxidant defense. Studies on CF sputum report surprisingly high levels of total GSH (predominantly reduced GSH) and GPx activity (Dauletbaev et al., 2004). Median total GSH in CF sputum was significantly higher than in non-CF controls, with over 90% as GSH in CF compared to <50% in controls (Dauletbaev et al., 2004). This abundance contributes to sputum’s H₂O₂-detoxifying capacity, sustained even after CAT inactivation, highlighting the GPx system’s importance in airway secretions (Dauletbaev et al., 2005). These high sputum GSH levels starkly contrast with reports of low GSH in BALF from the lower airways of CF children (Kettle et al., 2014; Dickerhof et al., 2017). This suggests compartmentalization: while expectorated sputum may be GSH-rich, the epithelial lining fluid of smaller, distal airways appears deficient (Gao et al., 1999; Kettle et al., 2014). This deficiency is likely linked to CFTR dysfunction affecting apical GSH transport (Kariya et al., 2007; Kettle et al., 2014). Viral infections, like RSV, also decrease lung GPx activity in mice (BALF) (Hosakote et al., 2011; Ansar et al., 2020). Efforts to augment airway GSH via inhalation have been explored. However, a large randomized controlled trial of inhaled GSH in CF patients increased sputum GSH but yielded no clinically relevant improvements in lung function, exacerbations, or oxidative stress markers (Griese et al., 2013). This outcome suggests that delivering GSH to bulk sputum may not correct the underlying oxidative imbalance or restore GPx-mediated protection effectively, possibly because GSH does not reach the critical distal airway site, local GPx activity is limited (e.g., by selenium), or the oxidant burden is overwhelming. Direct data on GPx activity in CF cellular or specific tissue models (beyond blood/airway secretions) are limited. A study on the CF porcine model found no significant difference in pancreatic GPx activity between CF and non-CF animals (O’Malley et al., 2022). However, research using CF airway epithelial cell lines (e.g., IB3-1) has shown decreased GPx4 levels under specific externally induced pro-oxidative stress (Maniam et al., 2021).

Beyond the well-characterized antioxidant enzymes such as SOD, GPx, and CAT, variations in other enzymatic activities also contribute to the complex redox imbalance and metabolic dysfunction observed in CF. GR, crucial for maintaining the pool of reduced GSH, has shown varied alterations depending on the cell type or tissue examined; studies have reported GR activity to be unvaried in erythrocytes of CF children (Carmagnol et al., 1983), while intracellular GR activity was significantly higher in lung tissue from Cftr-KO mice (Velsor et al., 2001), and conversely, significantly decreased in CF bronchial epithelial cells compared to WT controls (de Bari et al., 2018). Another enzyme involved in GSH conjugation and detoxification, Glutathione S-Transferase (GST), was found to be significantly increased in erythrocytes from children with CF (Carmagnol et al., 1983). In the context of mitochondrial function and its susceptibility to oxidative stress, aconitase, a key enzyme in the Krebs cycle, has demonstrated altered activity in CF models, including a notable loss of mitochondrial aconitase activity in FABP Cftr-KO mice and a decrease in CFTR-deficient lung cells, highlighting its vulnerability to oxidative damage in CF (Velsor et al., 2006). Concurrently, fumarase, another Krebs cycle enzyme, showed comparable or unvaried activity in these models, suggesting selective impact on aconitase (Velsor et al., 2006). Furthermore, γ-glutamyltransferase (γ-GT), involved in GSH metabolism, was not significantly altered in the lung tissue of Cftr-KO mice (Velsor et al., 2001). In immune cells, specifically activated neutrophils from CF patients, increased peroxidase activity, likely reflecting elevated MPO activity, is observed in response to stimuli such as P. aeruginosa (Yoo et al., 2014), contributing to the heightened oxidative burst.