Differences in Treatment Response in Bronchial Epithelial Cells from Idiopathic Pulmonary Fibrosis (IPF) Patients: A First Step towards Personalized Medicine?

Idiopathic pulmonary fibrosis (IPF) has a detrimental prognosis despite antifibrotic therapies to which individual responses vary. IPF pathology is associated with oxidative stress, inflammation and increased activation of SRC family kinases (SFK). This pilot study evaluates individual responses to pirfenidone, nintedanib and SFK inhibitor saracatinib, markers of redox homeostasis, fibrosis and inflammation, in IPF-derived human bronchial epithelial (HBE) cells. Differentiated HBE cells from patients with and without IPF were analyzed for potential alterations in redox and profibrotic genes and pro-inflammatory cytokine secretion. Additionally, the effects of pirfenidone, nintedanib and saracatinib on these markers were determined. HBE cells were differentiated into a bronchial epithelium containing ciliated epithelial, basal, goblet and club cells. NOX4 expression was increased in IPF-derived HBE cells but differed on an individual level. In patients with higher NOX4 expression, pirfenidone induced antioxidant gene expression. All drugs significantly decreased NOX4 expression. IL-6 (p = 0.09) and IL-8 secretion (p = 0.014) were increased in IPF-derived HBE cells and significantly reduced by saracatinib. Finally, saracatinib significantly decreased TGF-ß gene expression. Our results indicate that treatment responsiveness varies between IPF patients in relation to their oxidative and inflammatory status. Interestingly, saracatinib tends to be more effective in IPF than standard antifibrotic drugs.

Diffusion-weighted MR imaging within 24 h post-injury after traumatic spinal cord injury: a qualitative meta-analysis between T2-weighted imaging and diffusion-weighted MR imaging in 18 patients.

STUDY DESIGN: Only few studies have been published about diffusion-weighted imaging (DWI) within 24 h of traumatic spinal cord injury (tSCI). OBJECTIVES: The purpose of this study was to compare the imaging findings from conventional magnetic resonance imaging (MRI) and DWI in seven tSCI patients with findings in the existing literature. METHODS: Seven patients with tSCI at neurologic levels C2-T10 were examined with conventional MRI and DWI within 24 h post-injury. DWI was obtained with a b-factor of 1000 s mm(-2). American Spinal Injury Association (ASIA) scores and Spinal Cord Independence Measurement (SCIM) II item 12 after 12 months were collected. In addition, MEDLINE was searched from 1995 to 2010 to identify clinical tSCI studies reporting on MRI, DWI and apparent diffusion coefficient maps within 24 h post-injury to perform a meta-analysis. Images obtained with a b-factor of 1000 s mm(-2) were compared with lower b-factors. Differences were calculated using χ (2) tests. RESULTS: No associations were identified between the images of the seven tSCI patients and ASIA or SCIM II scores. Eighteen SCI patients (11 from the retrieved publications) were included in the meta-analysis. The detection rates of hyperintense signals on T2-weighted and DW imaging did not show significant differences at 94 and 72%, respectively. In addition, there were no significant differences in detection rates or diffusion abnormalities between subjects in whom DW images were obtained with a maximum b-factor of 1000 or <1000 s mm(-2). CONCLUSION: Our analysis suggests that T2-weighted and DW imaging have comparable detection rates for spinal cord damage in tSCI patients within 24 h post-injury.

[Death of an infant following 'craniosacral' manipulation of the neck and spine]. / Overleden zuigeling na 'craniosacrale' manipulatie van hals en wervelkolom.

A healthy 3-month-old girl died after manipulation of the cervical and thoracolumbar spine by a so-called craniosacral therapist. During persistent forced deep flexion of the neck and spine, the infant developed faecal incontinence, atonia and apnoea followed by an asystole. A physical examination, additional MRI studies and an autopsy indicated that the infant probably died as a consequence of local neurovascular lesions of the cervical spine or a mechanically-induced respiratory problem. This is the second reported case of an infant dying after forced manipulations of the neck. Until there is scientific evidence for the effectiveness and safety of forced manipulations of the vertebral column, we advise against this treatment in neonates and infants.

DUOX1 silencing in lung cancer promotes EMT, cancer stem cell characteristics and invasive properties.

Dual oxidase 1 (DUOX1) is an oxidant-generating enzyme within the airway epithelium that participates in innate airway host defense and epithelial homeostasis. Recent studies indicate that DUOX1 is suppressed in lung cancers by epigenetic silencing, although the importance of DUOX1 silencing in lung cancer development or progression is unknown. Here we show that loss of DUOX1 expression in a panel of lung cancer cell lines is strongly associated with loss of the epithelial marker E-cadherin. Moreover, RNAi-mediated DUOX1 silencing in lung epithelial cells and the cancer cell line NCI-H292 was found to result in loss of epithelial characteristics/molecular features (altered morphology, reduced barrier function and loss of E-cadherin) and increased mesenchymal features (increased migration, anchorage-independent growth and gain of vimentin/collagen), suggesting a direct contribution of DUOX1 silencing to epithelial-to-mesenchymal transition (EMT), an important feature of metastatic cancer. Conversely, overexpression of DUOX1 in A549 cells was capable of reversing EMT features. DUOX1 silencing in H292 cells also led to enhanced resistance to epidermal growth factor receptor tyrosine kinase inhibitors such as erlotinib, and enhanced levels of cancer stem cell (CSC) markers CD133 and ALDH1. Furthermore, acquired resistance of H292 cells to erlotinib resulted in enhanced EMT and CSC features, as well as loss of DUOX1. Finally, compared with control H292 cells, H292-shDUOX1 cells displayed enhanced invasive features in vitro and in vivo. Collectively, our findings indicate that DUOX1 silencing in lung epithelial cancer cells promotes features of EMT, and may be strongly associated with invasive and metastatic lung cancer.

Substance P primes the formation of hydrogen peroxide and nitric oxide in human neutrophils.

Substance P (SP), a neurotransmitter of the central and peripheral nervous system, has been implicated as a mediator of the pulmonary inflammatory response through its stimulatory effects on neutrophils. We investigated the role of SP in priming the production of reactive oxygen species by human neutrophils with the cytochrome c reduction assay and by flow cytometry using the intracellular oxidizable probe dichlorofluorescein. We also investigated SP-induced formation of nitrite and nitrate as an index of nitric oxide (NO) production. Our results indicate that SP primes two distinct pathways with respect to the induction of reactive oxygen species in the human neutrophil: the production of superoxide anion and hydrogen peroxide by the calmodulin-dependent NADPH oxidase, and the generation of NO by a constitutive NO synthase. Preincubation of neutrophils with inhibitors of calmodulin and NO synthase diminished the oxidative response in an additive fashion. These results give insight into distinct signal transduction pathways in the SP-primed neutrophil with respect to the formation of superoxide anion, hydrogen peroxide, and NO.

Role of reactive oxygen species in intestinal diseases.

It is well known that reactive oxygen metabolites are generated during several pathologies, and that they are able to disturb many cellular processes and eventually lead to cellular injury. After intestinal ischemia, reactive oxygen species are produced when the ischemic tissue is reperfused. The enzyme xanthine oxidase is thought to play a key role in this process. As a result of this oxygen radical production, the permeability of the endothelium and the mucosa increases, allowing infiltration of inflammatory leukocytes into the ischemic area. Moreover, reactive oxygen species are also indirectly involved in leukocyte activation. In turn, these inflammatory cells respond with the production of oxygen radicals, which play an important role in the development of tissue injury. Thus, intestinal ischemia and reperfusion evokes an inflammatory response. Also during chronic intestinal inflammatory diseases, reactive oxygen metabolites are proposed to play an important role in the pathology. Scavenging of reactive oxygen species will thus be beneficial in these disorders.

Aromatic hydroxylation and nitration of phenylalanine and tyrosine by peroxynitrite. Evidence for hydroxyl radical production from peroxynitrite.

Peroxynitrite is a highly reactive species, generated from superoxide and nitric oxide. Some effects of peroxynitrite are ascribed to the molecule itself, but decomposition products of the protonated form, peroxynitrous acid, may account for much of its reactivity in biological systems. Suggested products include highly-reactive hydroxyl radicals, but thermodynamic calculations have been used to claim that free hydroxyl radicals cannot be formed from peroxynitrite. We utilized aromatic hydroxylation of phenylalanine as a specific detector of hydroxyl radicals, and found that incubation of phenylalanine with peroxynitrite leads to a small amount of p-, m- and o-tyrosine, specific products of attack by this radical. Products of nitration of phenylalanine and tyrosine were also detected, as was dityrosine. Peroxynitrite decomposition generates several reactive species, including some that can nitrate aromatic rings. Formation of nitro-aromatic compounds may be a useful marker of peroxynitrite generation in biological systems.

Molecular mechanisms of damage by excess nitrogen oxides: nitration of tyrosine by gas-phase cigarette smoke.

Nitric oxide (nitrogen monoxide, .NO) plays important physiological roles, but an excess can be toxic. .NO is present in cigarette smoke (CS) at up to 500 ppm, and probably represents one of the greatest exogenous sources of .NO to which humans are exposed. We show here that gas-phase CS is capable of converting tyrosine to 3-nitrotyrosine (3-NO2-Tyr) and dityrosine, to an extent dependent on time of exposure and pH. Glutathione, ascorbic acid and uric acid decreased the CS-induced formation of 3-NO2-Tyr and dityrosine. We suggest that nitrogen oxides in CS can modify proteins in the respiratory tract and may contribute to CS toxicity.

Oxidative damage by ozone and nitrogen dioxide: synergistic toxicity in vivo but no evidence of synergistic oxidative damage in an extracellular fluid.

Inhalation of ozone (O3) and/or nitrogen dioxide (.NO2) is associated with the development of inflammation in the respiratory tract and various alterations in pulmonary functions. Respiratory tract lining fluids (RTLFs) represent the first biological fluids coming into contact with these inhaled toxicants. Using plasma as a surrogate for RTLFs, we have previously shown that O3 [Cross, Motchnik, Bruener, Jones, Kaur, Ames and Halliwell (1992) FEBS Lett. 298, 269-272] and .NO2 [Halliwell, Hu, Louie, Duvall, Tarkington, Motchnik and Cross (1992) FEBS Lett. 313, 62-66] are both capable of depleting antioxidants and damaging proteins and lipids. O3 particularly damages proteins, whereas .NO2 induces the peroxidation of lipids and nitrates aromatic amino acids. It has been reported that O3 and .NO2 cause synergistic toxicity in rodents [Gielzleichter, Witschi and Last (1992) Tox. Appl. Pharmacol. 116, 1-9]. In the present chapter, we review evidence showing that combined exposure of these two oxidant gases to human plasma fails to exert synergistic oxidative damage to plasma constituents, and in fact, O3 and .NO2 antagonize each other's actions. We conclude that the potentiating effect of these two gases on morbidity and mortality in rodents represents a complex interactive biological effect rather than a simple synergistic oxidative effect in extracellular fluids.

Dietary antioxidants and cigarette smoke-induced biomolecular damage: a complex interaction.

Epidemiologic evidence suggests that cigarette smoking is a major risk factor for chronic obstructive pulmonary diseases such as chronic bronchitis and emphysema, for carcinogenesis, and for cardiovascular disease. However, the precise mechanisms of these effects are incompletely understood. The gas phase of cigarette smoke contains abundant free radicals including nitric oxide. Hence, cigarette smoke may induce some of its damaging effects by free radical mechanisms. We report that exposure of plasma, a model for respiratory tract lining fluids, to gas-phase cigarette smoke causes depletion of antioxidants, including ascorbate, urate, ubiquinol-10, and alpha-tocopherol, and a variety of carotenoids, including beta-carotene. Gas-phase cigarette smoke induced some lipid peroxidation, as measured by cholesteryl linoleate hydroperoxide (18:2OOH) formation. Ascorbate was effective in preventing 18:2OOH formation. In contrast to the low concentrations of lipid hydroperoxides measured (< 1 mumol/L), protein carbonyl formation, a measure of protein modification, increased by approximately 400 mumol/L after nine puffs of cigarette smoke. Reduced glutathione inhibited protein carbonyl formation, whereas other plasma antioxidants, including ascorbate, were ineffective. alpha, beta-Unsaturated aldehydes (acrolein and crotonaldehyde) in cigarette smoke may react with protein -SH and -NH2 groups by a Michael addition reaction that results in a protein-bound aldehyde functional group. Gas-phase cigarette smoke is capable of converting tyrosine to 3-nitrotyrosine and dityrosine, indicating free radical mechanisms of protein damage by nitrogen oxides. Aldehydes and nitrogen oxides in cigarette smoke may be significant contributors to biomolecular damage, and endogenous antioxidants can attenuate some of these adverse effects.

Oxidants, nitrosants, and the lung.

The respiratory tract is subjected to a variety of environmental stresses, including oxidizing gases, particulates, and airborne microorganisms, that together, may injure structural and functional lung components and thereby jeopardize the primary lung function of gas exchange. To cope with such various environmental threats, the lung has developed elaborate defense mechanisms that include inflammatory-immune pathways as well as several antioxidant systems. These defense systems operate largely in extracellular spaces, thus protecting underlying bronchial and alveolar epithelial cells from injury, although these cells themselves are also active participants in such (inflammatory) defense mechanisms. Although potentially harmful, oxidants are increasingly recognized as pathophysiologic mediators produced primarily by inflammatory-immune cells as a host defense mechanism, but also by various other cell types as an intracellular mediator in various cell responses, thus affecting inflammatory-immune processes or inducing resistance. The molecular mechanisms and signaling pathways involved in such processes are the focus of much current investigation. Nitric oxide, a messenger molecule produced by many lung cell types, also modulates oxidant-mediated processes, thereby giving rise to a new family of reactive nitrogen species ("nitrosants") with potentially unique signaling properties. The complex role of oxidants and nitrosants in various pathophysiologic processes in the lung have confounded the design of therapeutic approaches with antioxidant substrates. This review discusses current knowledge regarding extracellular antioxidant defenses in the lung, and oxidant/nitrosant mechanisms operating under inflammatory-immune conditions and their potential contribution to common lung diseases. Finally, some recent developments in antioxidant therapeutic strategies are discussed.

Modulation of oxidative stress in the gastrointestinal tract and effect on rat intestinal motility.

The amounts of different factors, which are involved in oxygen free radical production or in protection against oxygen radicals, were determined in different parts of the gastrointestinal tract (GI-tract). Glutathione and superoxide dismutase were present in lower amounts within the small intestine compared with the stomach and the large intestine. In the small intestine glutathione peroxidase and catalase both prevailed in the intestinal muscle compared to the mucosa, whereas in the large intestine both enzymes are equally distributed among the mucosa and muscle. Xanthine oxidase was mainly present in the small intestinal mucosa. Taken together, these results suggest that the large intestine is better provided with protective enzymic and non-enzymic factors against oxidative stress than the small intestine. The protective capacity of different intestinal preparations against lipid peroxidation in liver microsomes was assessed, and particularly the mucosal fractions from the small intestine showed a marked protection against lipid peroxidation, which is not easily explained with the presence of the enzymes measured in this study. Pretreatment of intestinal segments with hydrogen peroxide or cumene hydroperoxide resulted in a damaged contractile response of the longitudinal smooth muscle to methacholine in all parts of the GI-tract, expressed in a lower pD2 value and a decreased maximal response. Pretreatment with these peroxides also decreased contractions after depolarization with K+. The large intestine is more sensitive to hydrogen peroxide and cumene hydroperoxide than the small intestine, which parallels with the sensitivity to lipid peroxidation. The results obtained with hydrogen peroxide also correlate well with the catalase activity in the various segments of the intestine. In conclusion, oxidative stress in the GI-tract alters intestinal motility, especially in the large intestine. Probably this does not occur at the level of muscarinic receptors.

Oxidants, antioxidants, and respiratory tract lining fluids.

Respiratory tract lining fluids (RTLFs) are a heterogeneous group of substances covering the respiratory tract epithelial cells (RTECs) from nasal mucosa to alveoli. Antioxidant contained in the RTLFs can be expected to provide an initial defense against inhaled environmental toxins. The major antioxidants in RTLF include mucin, uric acid, protein (largely albumin), ascorbic acid, and reduced glutathione (GSH). RTLF antioxidants can be augmented by such processes as transudation/exudation of plasma constituents; RTEC secretory processes, including glandular mucus secretion; and cellular antioxidants derived from lysis of RTECs and of inflammatory cells. The antioxidant composition of RTLFs and their role in modulating normal and pathophysiologic RTEC functions under conditions of oxidative stress are yet to be fully characterized.

Oxidative stress and antioxidants at biosurfaces: plants, skin, and respiratory tract surfaces.

Atmospheric pollutants represent an important source of oxidative and nitrosative stress to both terrestrial plants and to animals. The exposed biosurfaces of plants and animals are directly exposed to these pollutant stresses. Not surprisingly, living organisms have developed complex integrated extracellular and intracellular defense systems against stresses related to reactive oxygen and nitrogen species (ROS, RNS), including O3 and NO2. Plant and animal epithelial surfaces and respiratory tract surfaces contain antioxidants that would be expected to provide defense against environmental stress caused by ambient ROS and RNS, thus ameliorating their injurious effects on more delicate underlying cellular constituents. Parallelisms among these surfaces with regard to their antioxidant constituents and environmental oxidants are presented. The reactive substances at these biosurfaces not only represent an important protective system against oxidizing environments, but products of their reactions with ROS/RNS may also serve as biomarkers of environmental oxidative stress. Moreover, the reaction products may also induce injury to underlying cells or cause cell activation, resulting in production of proinflammatory substances including cytokines. In this review we discuss antioxidant defense systems against environmental toxins in plant cell wall/apoplastic fluids, dead keratinized cells/interstitial fluids of stratum corneum (the outermost skin layer), and mucus/respiratory tract lining fluids.

Downregulation of COX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line.

Flavonoids are natural polyphenolic compounds ubiquitously present in the plant kingdom. They are reported to exhibit numerous beneficial health effects. In the present study, we demonstrate the potential effects of different flavonoids on cytokines mediated cyclooxygenase-2 and inducible nitric oxide synthase expression and activities in A549 cell line using quercetin, amentoflavone and flavanone. Our data revealed that quercetin, at 50 micro M concentration inhibited PGE(2) biosynthesis by A549 very strongly with little effect on COX-2 mRNA and protein expression. Unlike quercetin, amentoflavone inhibited both PGE(2) biosynthesis and COX-2 mRNA and protein expression strongly. In another set of experiment, quercetin inhibited iNOS protein expression completely without affecting iNOS mRNA expression. In contrast, amentoflavone although exerted no inhibitory effect on iNOS mRNA expression, did inhibit weakly iNOS protein expression. Flavanone had no inhibitory effect on either enzyme at the same concentration. Taken together, our data indicated that amentoflavone and quercetin differentially exerted supression of PGE(2) biosynthesis via downregulation of COX-2/iNOS expression.

Hydrogen peroxide reduces beta-adrenoceptor function in the rat small intestine.

Incubation of isolated rat intestinal segments with hydrogen peroxide (H2O2) led to a decreased beta-adrenoceptor response. The maximal relaxation induced by isoprenaline was lowered while the EC50 remained unaffected. The effect of H2O2 in the small intestine increased slightly from duodenum to ileum. In the ileum, 10(-4) M H2O2 led to a 10% decrease of the maximal relaxation due to isoprenaline and 1 mM decreased the maximal response to about 50%. We further investigated the level at which the isoprenaline response was impaired. The relaxation caused by the stable cAMP analog, dibutyryl-cAMP, or by the adenylate cyclase activator, forskolin, was not affected or affected less than by isoprenaline. When the response to isoprenaline was expressed relative to the maximal response to dibutyryl-cAMP or forskolin, pretreatment with H2O2 led to a decreased isoprenaline response relative to the response to dibutyryl-cAMP or forskolin. This might indicate that exposure to H2O2 leads to a disturbance in receptor-mediated cAMP production. The adenylate cyclase unit is probably not affected since the response to forskolin is relatively resistant to H2O2. Our conclusion is that pretreatment of isolated intestinal segments with H2O2 leads to disturbed beta-adrenoceptor coupling, probably due to altered membrane integrity.

HA autoreceptor assay with superfused slices of rat brain cortex and electrical stimulation.

Slices of rat brain cortex previously loaded with [3H]histamine ([3H]HA) via de novo synthesis from [3H]histidine released tritiated histamine ([3H]HA) Ca2+ dependently in a superfused system. Both electrical field stimulation and high levels of K+ ions elicited this release. The extent of release depended on stimulation intensity. Rather strong stimuli, either by high frequency or longer stimulation, were required to elicit sufficient HA release for proper assessment of the concentration-dependence of release inhibition by drugs. The system showed marked depletion (less response per pulse) upon long-continued or successive stimulations. HA added to the superfusion medium inhibited the release evoked by stimulation at frequencies up to 10 Hz or with 30 mM K+ but not the release at higher frequencies or with 45 mM K+. The inhibition was mediated by H3 receptors, was concentration-dependent (pD2 = 7.4) and was complete at 10(-6) M. The H2 agonist impromidine antagonized the inhibition competitively (pA2 = 7.1). It is concluded that this assay in a superfusion system with electrical stimulation is suitable for the assessment of H3 receptor activity of drugs.

Lipoic and dihydrolipoic acids as antioxidants. A critical evaluation.

A detailed evaluation of the antioxidant and pro-oxidant properties of lipoic acid (LA) and dihydrolipoic acid (DHLA) was performed. Both compounds are powerful scavengers of hypochlorous acid, able to protect alpha 1-antiproteinase against inactivation by HOCl. LA was a powerful scavenger of hydroxyl radicals (OH.) and could inhibit both iron-dependent OH. generation and peroxidation of ox-brain phospholipid liposomes in the presence of FeCl3-ascorbate, presumably by binding iron ions and rendering them redox-inactive. By contrast, DHLA accelerated iron-dependent OH. generation and lipid peroxidation, probably by reducing Fe3+ to Fe2+. LA inhibited this pro-oxidant action of DHLA. However, DHLA did not accelerate DNA degradation by a ferric bleomycin complex and slightly inhibited peroxidation of arachidonic acid by the myoglobin-H2O2 system. Under certain circumstances, DHLA accelerated the loss of activity of alpha-antiproteinase exposed to ionizing radiation under a N2O/O2 atmosphere and also the loss of creatine kinase activity in human plasma exposed to gas-phase cigarette smoke. Neither LA nor DHLA reacted with superoxide radical (O.2-) or H2O2 at significant rates, but both were good scavengers of trichloromethylperoxyl radical (CCl3O2.). We conclude that LA and DHLA have powerful antioxidant properties. However, DHLA can also exert pro-oxidant properties, both by its iron ion-reducing ability and probably by its ability to generate reactive sulphur-containing radicals that can damage certain proteins, such as alpha 1-antiproteinase and creatine kinase.

Tobacco-related diseases. Is there a role for antioxidant micronutrient supplementation?

It is clear that smoking causes an increase in free radicals, reactive nitrogen and oxygen species (RNS and ROS, respectively), and that cigarette smoking is associated with increases in the incidence and severity of several diseases including atherosclerosis, cancer, and chronic obstructive lung disease. Although there is still no unequivocal evidence that oxidative stress is a contributor to these diseases or that an increased intake of antioxidant nutrients is beneficial, the observation that smokers have lower circulating levels of some of these nutrients, raises concern. This article discusses the possible links between the observed oxidant-induced damage related to tobacco smoking, effects on cellular mechanisms, and their potential involvement in the causation and enhancement of disease processes.

Cigarette smoke impairs neutrophil respiratory burst activation by aldehyde-induced thiol modifications.

Exposure to airborne pollutants such as tobacco smoke is associated with increased activation of inflammatory-immune processes and is thought to contribute to the incidence of respiratory tract disease. We hypothezised that cigarette smoke (CS) could synergize with activated inflammatory/immune cells to cause oxidative injury or result in the formation of unique reactive oxidants. Isolated human neutrophils were exposed to gas-phase CS, and the production of nitrating and chlorinating oxidants following neutrophil stimulation was monitored using the substrate 4-hydroxyphenylacetate (HPA). Stimulation of neutrophils in the presence of CS resulted in a reduced oxidation and chlorination of HPA, suggesting inhibition of NADPH oxidase or myeloperoxidase (MPO), the two major enzymes involved in inflammatory oxidant formation. Peroxidase assays demonstrated that neutrophil MPO activity was not significantly affected after CS-exposure, leaving the NADPH oxidase as a likely target. The inhibition of neutrophil oxidant formation was found to coincide with depletion of cellular GSH, and a similar modification of critical cysteine residues, such as those in NADPH oxidase components, might be involved in reduced respiratory burst activity. As alpha,beta-unsaturated aldehydes such as acrolein have been implicated in thiol modifications by CS, we exposed neutrophils to acrolein prior to stimulation, and observed inhibition of NADPH oxidase activation in relation to GSH depletion. Additionally, translocation of the cytosolic components of NADPH oxidase to the membrane, a necessary requirement for enzyme activation, was inhibited. Protein adducts of acrolein (or related aldehydes) could be detected in several neutrophil proteins, including NADPH oxidase components, following neutrophil exposure to either CS or acrolein. Alterations in neutrophil function by exposure to (environmental) tobacco smoke may affect inflammatory/infectious conditions and thereby contribute to tobacco-related disease.