Anti oxidants in resp medicine

1. OXIDATIVE STRESS AND
ANTIOXIDANTS IN RESPIRATORY
DISEASES
DR ANUSHA CM

2. INTRODUCTION
• Molecular oxygen is a prerequisite to life of all aerobic organisms.
• High concentrations of oxygen or its metabolites, referred to as reactive
oxygen species (ROS), have the potential to cause cellular injury and
contribute to disease pathogenesis.
• The most damaging forms of ROS are free radicals.
• A free radical, by definition, refers to any chemical species containing one
or more unpaired electrons in their atomic or molecular orbitals.
• Unpaired electron(s) give considerable reactivity to free radical species,
which can trigger chemical reactions that damage cellular constituents of
living organisms.

3. • O2 can form the superoxide anion radical (O2
- ) upon addition of an electron;
thus, overcoming this restraint and making it a highly reactive species.
• When two free radicals share their unpaired electrons, nonradical species of
lower reactivity are generated.
• Thus, ROS constitute both free radicals and nonradicals

4. NITRIC OXIDE
• Nitric oxide (NO•) is another small gaseous molecule that serves as an
important signaling molecule in diverse physiological processes, including
vasorelaxation and immune regulation during chronic inflammation in the
lung.
• The regulated production of NO• by lung cells is critical for homeostasis of
the lung
• The reaction of NO• with (O2
- ) to form reactive nitrogen species (RNS),
such as peroxynitrite (ONOO–), may contribute to the pathophysiology of
chronic lung diseases.

5. • ROS and RNS together play important roles in regulation of cell
proliferation, differentiation, and survival
• ROS/RNS can inactivate enzymes including antiproteases, induce
apoptosis, regulate cell proliferation, and modulate the immune-
inflammatory system in the lungs and other tissues
• ROS/RNS have been implicated in initiating inflammatory responses in the
lungs through the activation of transcription factors, protein kinase
pathways, chromatin remodeling, and gene expression of
proinflammatory mediators.
• Under normal physiological conditions, the balance between generation
and elimination of ROS/RNS maintains the functional integrity of redox-
sensitive signaling cascades regulating cellular phenotypes.

6. • Redox homeostasis of cells/tissues is maintained by the regulated balance
of oxidant production and antioxidant systems, both enzymatic and
nonenzymatic
• An increase in oxidant generation in excess of the capacity of
cells/tissues to detoxify or scavenge reactive species leads to OXIDATIVE
STRESS
• Oxidative stress typically causes damage to cellular components in an
indiscriminant manner.
• Such states are accelerated in the presence of transition metals, such as
iron and copper, and/or specific monooxygenases or oxidases.

7. An imbalance between reactive oxygen species and antioxidants can lead to elevated
stress. A, Normally, there are sufficient antioxidants in the respiratory tract such that the
production of a small amount of reactive oxygen species is inconsequential.
B, If either antioxidants are diminished or production of reactive oxygen species is
increased (eg, during an asthma exacerbation), the balance of antioxidants and reactive
oxygen species is tipped toward oxidative stress.

9. SOURCES OF ROS/RNS
• The primary ROS include superoxide anion (O2
- ), hydrogen peroxide
(H2O2), and hydroxyl radical (•OH), which can be generated from both
enzymatic and nonenzymatic sources.
• Normal metabolic processes in all cells are the major source of reactive
oxygen species.
• The human lung is constantly exposed to ambient air, which may contain
environmental toxins capable of inducing ROS generation.
• ROS may also be generated by electron transfer reactions in the
mitochondria and endoplasmic reticulum (ER), from xenobiotics, and a
range of metabolic enzymes that catalyze oxidation reactions

10. • Additional major sources of superoxide include the membrane oxidases,
such as the cytochrome P450 and b5 families of enzymes in the
endoplasmic reticulum2 and the reduced nicotin- amide adenine
dinucleotide phosphate oxidases of phagocytic cells.
• Direct exposure to environmental air is an additional source of reactive
oxygen species exposure that is unique to the airways
• For instance, cigarette smoke inhalation results in increased exposure to
both superoxide and hydrogen peroxide.
• Cigarette smoke–mediated lung damage might also be a result of
increased exposure to nitric oxide and nitrites.

11. • Other sources of environmental oxidants include air
pollution, which contains ozone.
• Ionizing radiation is an efficient method of producing
reactive oxygen species in the laboratory but is a minor
contributor to reactive oxygen species in vivo

12. ENZYMATIC SOURCES OF ROS/RNS

13. CONTD…

14. ENZYMATIC SOURCES OF RNS
• An important RNS in the lung is NO•, which is endogenously generated by
specific nitric oxide synthases (NOSs).
• NOS enzymes metabolize L-arginine to NO• and L-citrulline via a five
electron oxidation reaction which requires a dimeric enzyme, oxygen,
NADPH and the cofactors, flavin adenine dinucleotide (FAD), flavin
mononucleotide (FMN), tetrahydrobiopterin (BH4), calmodulin, and iron
protoporphyrin.
• There are three different NOS forms, all of which are expressed in the
lung, the inducible form iNOS or NOS2, neuronal NOS or NOS1, and the
endothelial NOS enzyme NOS3

15. • The NOS1 and NOS3 enzymes are calcium dependent and produce
picomolar levels of NO, while nanomolar levels are generated by the
calcium-independent iNOS.
• NO• synthesis by iNOS is regulated by the availability of the substrate L-
arginine and cofactor BH4.
• Uncoupling of NOS enzymes can contribute to the formation of the O2
- .
• It has also been reported that iNOS can specifically bind to cyclooxgenase-
2 (COX-2) and S-nitrosylate COX-2, upregulate its catalytic activity and
enhance prostaglandin E2 production.
• RNS may also mediate lipid peroxidation, protein oxidation and nitration,
enzyme inactivation, or even cell necrosis.

16. SUPEROXIDE DISMUTASES
• It participates in the generation of other reactive metabolites, H2O2, •OH,
and ONOO–.
• SODs represent a key defense against reactive species generated during
normal metabolism or inflammatory states.
• SODs are ubiquitous enzymes that catalyze the dismutation of O2
- radical
to the weaker oxidant, H2O2.
• EC-SOD normally protects the lungs against fibrosis by preventing
oxidative degradation of the matrix and by binding type I and type IV
collagen via its heparin/matrix binding domain.

17. • Three mammalian SOD isozymes:
1. The intracellular copper-zinc SOD (CuZn-SOD)
2. The mitochondrial manganese SOD (Mn-SOD)
3. Extracellular SOD (EC-SOD)

19. Reactive oxygen species and antioxidant enzymes in airways. A, Environmental sources include
ozone (O3) and hydrogen peroxide (H2O2) from air pollution and cigarette smoke. Intracellular
sources include mitochondrial respiration, xanthine oxidase (XO), and P450 and cytochrome b5
enzymes; hydrogen peroxide is a product of superoxide dismutases (SOD), such as Cu,Zn SOD
and mitochondrial DOS (Mn- SOD), as well as other oxidases, such as those found in
peroxisomes. Extracellular sources of reactive oxygen species include membrane oxidases
(Mox) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases of
eosinophils (EOS) and neutrophils (PMN).

20. Antioxidants found in airways include the SODs, glutathione peroxidase (Gpx)
and catalase (Cat), thrioredoxin (Trx), and the low-molecular-weight
antioxidants glutathione (GSH), vitamin C (ascorbate), and vitamin E (α-
tocopherol

21. The major endogenous ROS/RNS and the primary mechanisms for their
generation are summarized

22. ENZYMATIC MECHANISM TO DETOXIFY
ROS /RNS

23. IMMUNE SOURCES AND ROS/RNS
REGULATION
1. IMMUNE CELLS
• NO• participates in pathogen killing by macrophages.
• It also delays fusion of phagosomes with lysosomes to form a functional
phagolysosome, which enhances antigen processing/presentation of
macrophages.
• Macrophages scavenge endogenous dying cells.
• Phagocytosis of dying cells requires the secretion of alarmins by dying cells
to attract and preactivate phagocytes; these signals by dying cells ensures
specific recognition and phagocytosis/efferocytosis, all of which involve
redox regulation.

24. 2. ENDOTHILIAL CELLS
• The importance of ECs in inflammation-induced vascular dysfunction is
dependent on their ability to produce and respond to ROS and RNS.
• Targeting ROS-quenching enzymes catalase and SOD , it alleviates toxic
effects of excessive ROS and suppresses proinflammatory mechanisms,
including endothelial cytokine activation and barrier disruption.
• Pulmonary EC-derived ROS play a pivotal role in EC activation and
function.
• Alterations in EC phenotype contribute to vascular tone, permeability, and
inflammatory responses and, thus, have been implicated in lung diseases,
including pulmonary hypertension, ischemia-reperfusion (IR) injury, and
adult respiratory distress syndrome.

25. 3. FIBROBLASTS
• Fibroblasts and fibroblast-like mesenchymal cells participate in innate
immunity and in tissue repair.
• Epithelial-to-mesenchymal transition (EMT) is a process regulating cell
plasticity, which allows epithelial cells to lose their polarity and specialized
junctional structures, to undergo cytoskeletal reorganization, and to
acquire morphological and functional features of mesenchymal like cells.
• Myofibroblasts are the primary “effector” cells in tissue remodeling and
pulmonary fibrosis.
• Activation of the NADPH oxidase isoform, NOX4, mediates generation of
H2O2, myofibroblast differentiation, contractility, and ECM production in
response to TGF-β1.

26. OXIDATIVE STRESS IN LUNG
DISORDERS

27. 1. ASTHMA
• ROS and RNS have been implicated in the pathogenesis of asthma.
• Dysregulation in pathways that lead to oxidative stress or its defense may
contribute to the initiation and severity of asthma.
• Leukocyte activation with induction of NADPH oxidase and production of
O2
- and H2O2 correlate negatively with FEV1 in asthmatic subjects.
• An increase in ROS production is inversely correlated with FEV1.
• Airway inflammation-associated oxidative stress in asthma may induce
oxidative modifications of proteins or lipids.
• Increased numbers of eosinophils and neutrophils in association with
higher expression of peroxidases and other markers of eosinophil
activation are found in bronchoalveolar lavage fluid (BAL) and bronchial
tissues of asthmatics

28. • ROS can decrease β-adrenergic function in lungs and sensitize airway
smooth muscle to acetylcholine-induced contraction.
• H2O2 activates mitogen-activated kinases in tracheal myocytes and
stimulates contraction of tracheal smooth muscle cells.
• ROS also stimulates mucin secretion, contributes to Th2 cell
differentiation, and promotes T cell proliferation via arginase and NADPH
oxidase pathways.
• Proinflammatory cytokines are elevated during airway inflammation,
which activate oxidases leading to increases in ROS, the targets of which
include receptor kinases, phosphatases, phospholipids, or nonreceptor
tyrosine kinases

29. • Exhaled NO• is increased in asthmatics and is associated with airway
inflammation.
• It is primarily iNOS that contributes to exhaled NO•.
• Induction of iNOS is observed at both transcriptional and translational
levels principally in steroid-naïve patients.
• Following antigen challenge, levels of NO• decrease, while nitrate
increases without perturbing levels of nitrite and SNO(S-nitrosothiols).
• Persistent increases in ROS and NO• lead to RNS formation and
subsequent oxidation and nitration of proteins, which contributes to the
dysregulation of airway inflammation in asthma
• High levels of ROS may overwhelm antioxidant defenses, causing
significant loss of antioxidant activity in asthma

30. • Global loss of SOD activity, due to SOD deficiency, loss of circulating SOD
activity, or inactivation of SOD via oxidative modification reflects the increased
oxidative stress in asthmatic patients.
• Oxidative modification-mediated reduction of catalase activity is also observed
in asthmatics.
• Although airway glutathione is increased in asthmatic patients, the ratio of
oxidized to reduced glutathione is elevated reflecting an oxidizing
microenvironment.
• Inhalation of exogenous ROS and RNS from exposures to environmental
pollutants including ozone, diesel exhaust particles, and oxidant components
of tobacco smoke, all contribute to additive oxidative stress, airway
hyperreactivity, and inflammation in asthma.
• Numerous cytokines, such as TNF-α, heparin-bound epidermal growth factor,
fibroblast growth factor 2, angiotensin II, serotonin, and thrombin, are found
in the lung during inflammation and activate oxidases that lead to increases in
reactive oxygen species in cell culture.

31. THERAPEUTIC IMPLICATIONS
• There are 2 strategies for treating oxidative stress asthma:
1. reducing exposure to reactive oxygen species
2. augmenting antioxidant defenses
• Reducing exposure to environmental oxidants, such as nitrites and ozone,
might decrease asthmatic exacerbations through the attenuation of the
activity of pulmonary inflammatory cells
• For instance, ozone decreases FEV1 by 12.5% compared with filtered air,
and children playing sports (hence more outdoor exposure) have a higher
prevalence of asthma in areas with high concentrations of environmental
ozone.
• Augmentation of existing antioxidant defenses with catalytic antioxidants
might also be useful in attenuating asthma and other respiratory disorders

32. 2.EMPHYSEMA
• Important contributing factors to the pathobiology of emphysema include
inflammation, alveolar epithelial cell injury/apoptosis, protease–
antiprotease and oxidant–antioxidant imbalances.
• Oxidative stress caused by cigarette smoke inhalation contributes to the
pathogenesis of emphysema.
• Cigarette smoke contains , •OH, and H2O2.
• ROS are also generated by the chronic inflammation, characteristic of
emphysema and that persists even after smoking cessation.
• Oxidative stress originating from constituents of cigarette smoke or
products of inflammatory cells can overcome the antioxidative capacity of
lung tissues and diminish antiprotease defenses

33. • A major consequence of oxidative stress is the activation of the
transcription factor nuclear factor-κB (NF-κB), which activates
transcription of proinflammatory cytokines
• Cigarette smoke also inhibits histone deacetylase, further promoting the
release of proinflammatory cytokines.
• Therefore, oxidant injury and lung inflammation act in concert to increase
alveolar destruction or compromise maintenance and repair of alveolar
structure.
• Antioxidant defenses are determinants of susceptibility to emphysema.
• A protective role for Nrf2, a transcription factor that regulates multiple
critical antioxidant enzymes, has been identified in pulmonary
emphysema.

34. • SOD mimetics abrogate alveolar cell apoptosis and emphysema in mouse
models.
• This blockade of apoptosis prevents oxidative stress and emphysema
further supporting the link between oxidative stress and apoptosis

35. 3. PULMONARY FIBROSIS
• IPF is characterized by exuberant ECM deposition, tissue contraction, and
apoptosis resistance of myofibroblasts, alongside apoptosisprone and
aberrantly differentiated alveolar type 2 cells.
• This loss of epithelial–mesenchymal homeostasis and communication is
central to the pathogenesis of IPF.
• Myofibroblasts are key effector cells in tissue remodeling and fibrosis,
typically contained in fibroblastic foci, which are a pathological hallmark of
IPF.
• Chronic inflammation, aberrant wound healing, and degenerative aging
processes have all been proposed as contributing to the pathogenesis of
IPF

36. • Oxidative stress is common to these processes and is implicated in IPF
pathogenesis.
• ROS/RNS released by phagocytes are involved in lung tissue damage in
interstitial lung diseases.
• IPF and pneumoconiosis may explain the high levels of hydroxyl radical
and superoxide anion concentrations in these diseases.
• Elevated oxidative and nitrosative stress in idiopathic pulmonary fibrosis
has also been demonstrated by increased levels of H2O2 in expired breath
condensate, increased eosinophilic mediators and myeloperoxidase
concentrations in BAL, enhanced levels of lipid peroxidation products
(such as 8-isoprostane) in BAL and condensate and elevated nitric oxide
concentrations in exhaled breath.

37. 4.PULMONARY ARTERIAL
HYPERTENSION
• Increased expression of ROS-generating enzymes, uncoupling of NOS
enzymes, and mitochondrial dysfunction all contribute to the oxidative
stress in PAH.
• Upstream dysregulation of ROS/NO• redox homeostasis impairs vascular
tone, which then triggers the activation of antiapoptotic and mitogenic
pathways, leading to cell proliferation and obliteration of the vasculature.
• Decreases in ROS inhibit a O2
- sensitive K+ channel leading to pulmonary
vascular constriction.
• ROS derived from the NOX2 and NOX4 isoforms are associated with
medial thickening, disordered proliferation and migration, impaired
angiogenesis, and disturbed fibrinolysis

38. • Xanthine oxidoreductases (XORs), including xanthine dehydrogenase (XD)
and xanthine oxidase (XO) are increased in idiopathic PAH patients.
• In hypoxia dependent PAH, hypoxia increases the expression of TGF-β1
and NOX4 expression.
• TGF-β–induced NOX4 expression and NOX4-mediated ROS production
have been implicated in PASMC (pulmonary artery smooth muscle cells)
proliferation.
• TGF-β1 also induces proangiogenic effects by upregulating VEGF.
• Nitrosative stress with increased nitrated eNOS is an early contributor to
the development of PAH. The vasodilatory effects of cGMP are mediated
through protein kinase G (PKG).
• This nitration-dependent reduction in PKG activity is observed in lungs of
patients with PAH. Nitration of carnitine acetyltransferase, an enzyme
that maintains normal mitochondrial function is another indicator of early
nitrosative stress in PAH

39. 5. ACUTE RESPIRATORY DISTRESS
SYNDROME
• ARDS is characterized by sudden onset, impaired gas exchange, and an
increase in pulmonary capillary permeability.
• Oxidative damage by ROS and RNS has been implicated in the pulmonary
vascular endothelial damage that characterizes ARD.
• The high inspiratory concentrations of oxygen required to achieve
adequate arterial oxygenation, infection, or extrapulmonary inflammation
lead to increased ROS production. This, combined with decreased
antioxidant capacity of tissues resulting from consumption of the natural
antioxidants leads to cellular damage and loss of vasomotor control.
• Measurements of antioxidant concentrations have revealed an oxidant–
antioxidant imbalance in ARDS patients

40. • The production of toxic levels of ROS and RNS not only leads to damage of
key molecules in cells but can signal changes in cellular responses such as
proliferation, apoptosis, and necrosis.
• H2O2 has been detected in the exhaled breath, while MPO and oxidized
α1-antitrypsin have been detected in BAL of ARDS patients.
• Nitration and oxidation of alveolar space proteins including the surfactants
have been identified ex vivo in patient samples with ARDS.
• Overabundance of ROS also induces adhesion molecules and cytokines
that contribute to endothelial injury.

41. CONCLUSION
• The lungs are exposed to exogenous oxidants from the environment, in
addition to endogenous generation of ROS/RNS from resident and
recruited inflammatory cells.
• Several measures of oxidative stress have been used to estimate oxidative
stress within the lungs; however, current approaches do not adequately
differentiate between different oxidative mechanisms and are used as
biomarkers of oxidative damage.
• Further investigations are needed to discover biomarkers that correlate
and differentiate between various types of oxidative injury
• A better understanding of factors that influence individual
susceptibilitywill also be useful in risk stratification of patients.
Investigations on how early life exposures to oxidants impact airway
morphology, immune function, and the airway epigenome may also aid in
determining susceptibility, disease expression, and progression

42. THANK YOU