Free Radicals in Various Human Diseases

This article throws light upon the top eight roles of free radicals in various human diseases. Some of the roles are: 1. Atherosclerosis 2. Ischemia and Reperfusion Injury 3. Lung Diseases 4. Theory of Aging 5. Hypertension 6. Diabetes 7. Malignant Diseases 8. Inflammation and Arthritis

Free Radicals: Role # 1.

Atherosclerosis:

Free radicals have a profound link with athero­sclerosis. The development of atherosclerosis is a multifactorial process in which both elevated plas­ma cholesterol levels and proliferation of smooth cells play a central role.

Atherogenesis is an Alter­ation of the Artery Wall that Includes Two Major Phases:

1. Adhesion of monocytes to the endothelium, their migration to the sub endothelial space and differentiation into macrophages. These cells oxidized low density lipoproteins and through this process they are transformed into foam cells.

2. Vascular smooth muscle cell migration from the media into the intima and their prolifera­tion with the formation of atherosclerotic plaques.

Reactive oxygen species have been implicat­ed as key processes of atherosclerosis, including oxidative modification of low density lipoprotein and endothelial dysfunction, thereby promoting a vascular inflammatory response. Angiotensin II is a major stimulator of vascular reactive oxygen species production.

Angiotensin II stimulates the production of the ROS from both arterial smooth muscle cells and endothelial cells. Thus, there is substantial evidence implicating that vascular NADPH oxidase stimulated, at least in part, by angiotensin II is a major vascular source of ROS in atherosclerosis.

More recent data also show that ROS is criti­cally involved in angiotensin II inducing pro-in­flammatory effects in vascular cells. In endotheli­al cells, the expression of the leucocyte adhesion molecules VCAM-1 is potently induced by angi­otensin II. These data support the hypothesis that ROS formation is critical for vascular inflammation – basic patho­genetic mechanism of atherosclerosis.

Recently studies suggest that oxidative modi­fication of low density lipoprotein is a critical fac­tor for atherogenesis and designated as the “bad actor in the free radical hypothesis of atheroscle­rosis”.

LDL may be oxidatively modified by all major cell types of the arterial wall, including endothelial cells, smooth muscle cells, and macrophages via their extracellular release of reactive oxygen species (ROS).

Hydroxyl radical (thus formed) may initiate the peroxidation of long-chai n polyunsaturated fatty acids within LDL molecule, giving rise to conjugated dienes and lipid hydroperoxy radicals (LOO^•).

This process is self-propagating, such that LOO^• can attack adjacent fatty acids until complete fatty acid chai n fragmentation occurs. A number of highly reactive products then accumulate in the LDL particle, including malondialdehyde and lysophosphatides. These products interact with the amino side chain of the apoprotein B 100 and modify it to form new epitopes that are not recognized by the LDL receptor.

OxLDL is avidly taken up by sub-endothelial macrophages via the “scavenger” receptor pathway which does not recognize native, unmodified LDL. Through the scavenger receptor, unlimited amounts of modified LDL are ingested by the monocyte/macrophage, which is now a “foam cell” in the arterial intima. The “foam cell” recruits more monocyte/ macrophages to convert in foam cells (Figure 31.2).

Accumulation of LDL-laden foam cells beneath arterial endothelium lays the foundation for the “fatty streak“, the earliest histopathological evidence of the development of atherosclerotic plaque. Oxidized LDL also stimulates the release of monocyte-derived TNFa and IL-iβ, leading to smooth muscle cell proliferation.

Elaboration of collagen and elastin by smooth muscle cells lays the foundation for plaque formation and ultimately fibrosis.

Lipid peroxides also inhibit synthesis of prostacyclin, an antiplatelet-aggregation substance, which can result in platelet adherence and aggregation. Platelets release growth factors, subsequently leading to smooth muscle cell proliferation and migration to intima. Besides, aggregation of platelets lays the foundation for formation of thrombus.

Free Radicals: Role # 2.

Ischemia and Reperfusion Injury:

Myocardial ischemia occurs when myocardial oxygen demand exceeds oxygen supply. Unless reversed, this situation results in cell injury, and clinical event is myocardial infarction.

Reperfusion of the ischemic myocardium can restore oxygen and substrates to the ischemic myocardial cells, but this process may create another form of myo­cardial damage, termed “reperfusion injury“.

Evidence suggests that damage to the myo­cardial cell induced by the cycle of ischemia and reperfusion may be due, in part, to the generation of toxic, reactive oxygen species such as superoxide radical, hydrogen peroxide, and the hydroxyl radical.

The active involvement of free radicals in the ischemia-reperfusion damage is demonstrat­ed by direct and indirect experimental evidences.

Direct evidences arise from the possibility of meas­uring radicals in myocardial tissue by electron spin resonance (ESR) and spin trapping methodology; indirect evidences by the measurement of the products of free radical attack on biological substrates (usual­ly malondialdehyde as a measure of lipid peroxi­dation extent), and intracellular and extracellular antioxidant capacity.

Reactive oxygen radicals in reperfusion injury can originate from intracellular sources, such as mitochondria and xanthine oxidase, or from extra­cellular sources, such as neutrophils and macro­phages.

The rapid decrease of oxygen in ischemic tis­sue causes a switch from oxidative to anaerobic metabolism. Within minutes of the onset of ischemia, energy demands exceed the heart’s ca­pacity to synthesize ATP anaerobically. Energy depletion has fundamental importance in the gen­esis of subsequent injurious events.

Lactate and un-buffered hydrogen ions accumulate in tissue leading to the rapid change in tissue acid-base sta­tus. The failure of all energy dependent mecha­nisms leads to the deterioration of membrane ion gradients, opening of selective and unselective ion channels and equilibration of most intracellular and extracellular ions.

As a consequence of this “anoxic depolarization“, potassium ions leave the cell, sodium chloride and calcium ions enter. Cel­lular accumulation of ions causes formation of cytotoxic edema. Intracellular Ca²⁺ overload can also set off a cascade of events which may lead to the formation of ROS.

The elevated Ca concen­tration activates proteases that can convert xan­thine dehydrogenase to xanthine oxidase. During re-oxygenation, xanthine oxidase can use O₂ as an electron acceptor, leading to formation of su­peroxide anion (O.^−•) and hydrogen peroxide (H₂O₂), which can react to produce hydroxyl rad­icals (OH^•).

These reactive species are responsi­ble for the tissue damage. Xanthine oxidase pro­duction of oxygen free radicals plays a major part in generating tissue damage seen in ischemia/ reperfusion injury (Figure 31.3).

Free Radicals: Role # 3.

Lung Diseases:

There is increasing evidence that ROS can be of importance of pathophysiology of several lung diseases like adult respiratory distress syndrome (ARDS), activated polymorphonuclear leucocytes, hyperactivity in asthmasis, oedema, lung fibrosis, emphysema etc.

Lung injury induced by air pol­lutant, mineral dust, cigarette smoke, herbicides and anticancer agents since, at least partly, to be mediated by free radicals, these agents can affect epithecial, nasothecial and fibroblastic cells.

These cells may rupture, releasing proteolytic enzymes and chemolytic mediators causing infiltration of other cells such as neutrophils, thus initiating an inflammatory process leading to an increase gen­eration of ROS.

One may divide ROS-induced oxidative dam­age into two categories: those which occur at rath­er high oxidative challenge and those occurring at relatively low oxidative challenge.

Those events occurring at high oxidative challenge and which generally lead to acute cell and tissue injury are probably mediated through the hydroxyl radical. Such events include lipid peroxidation as well as protein sulfhydryl oxidation, which may lead to membrane destruction and altered enzyme activ­ity respectively.

An example related to the lung is the inactivation of a₁-antitrypsin through an oxi­dative modification of a methionine moiety in this enzyme.

This inactivation may contribute to the development of emphysema, since a antitrypsin inhibits the effects of several proteolytic enzymes, such as elastase and collagenase. ROS can also cause oxidative DNA damage of various types, cytoskeletal damage and disturbed calcium home­ostasis.

With the limited oxidative stress, events such as modification of receptor activity and signalling as well as release of endogenous mediators (in­cluding AA metabolites), may occur. These events can lead to over expression of normal physiolog­ical processes and are probably more important than direct oxidative damage.

In the lung, AA re­lease and metabolism to eicosanoid agents can lead to vaso- and bronchoconstriction as well as to the development of oedema.

Thus, the exposure of the lung to hydrogen peroxide and other hydro peroxide or systems gen­erating O₂^• or H₂O₂ (xanthine/xanthine oxidase; glucose/glucose oxidase) has been shown to re­sult in vaso- and bronchoconstriction as well as development of oedema.

Free Radicals: Role # 4.

Theory of Aging:

A free radical theory of aging was proposed by Harman in 1956. Multicel­lular organisms generally undergo qualitative changes with time (aging) that are associated with progressive degeneration of biological functions, increased susceptibility to diseases and increased probability of death within a given time period.

The widely popular free radical theory of aging states that the age-related degen­erative process is to a large extent the consequence of free radical damage.

Genetic evidence linking oxidative stress to life span has been obtained for different animal species. Harman et al also proposed a slow progressive damage caused by un-scavenged free radicals leading to failure of normal self-recognition mechanisms.

Autoantibodies may be produced leading to ac­cumulation of phagocytes ultimately resulting in autoimmunity. Cutler, 1984 supported the theory by his findings of a direct correlation between SOD activity and lifespan.

SOD extracted from liver of older rats was found to be less effective and thermo-labile as compared to SOD extracted from younger rats. This theory may also explain the in­creased survivality of cold blooded animals at low temperatures as fewer radicals are produced at low temperature due to lower metabolic activity.

In 1978, Nohl and Hegner described an age- related increase in the release of O₂^−• and H₂O₂ from respiring mitochondria. In the same paper, it was also reported that the age-related stimulation of mitochondrial oxy­gen activation was responsible for an imbalance between pro-oxidants and antioxidants.

The exist­ence of this so-called ‘oxidative stress’ situation was surmised from the observed accumulation of oxidative damage to the structure and function of mitochondria isolated from old animals. Many papers have been published since that time which all supports the idea that in ageing the organism has to face an increasing imbalance between pro-oxidant formation and antioxidant defense sys­tems.

Free Radicals: Role # 5.

Hypertension:

Up to date experimental evidence indicates that reactive oxygen species (ROS) play an important pathophysiological role in the development of hypertension. This is due, in large part, to ^•O₂ excess (oxidative stress) and decreased NO bioa­vailability in the vasculature and kidneys and to ROS-mediated cardiovascular remodeling.

In human hypertension, biomarkers of systemic oxidative stress are elevated.

Treatment with superoxide dismutase (SOD) mimetics or antioxidants improves vascular and re­nal function, regresses vascular remodeling, and reduces blood pressure (BP).

Vascular ROS are produced in endothelial, adventitial, and vascu­lar smooth muscle cells (VSMCs) and derived pri­marily from NAD (P) H oxidase: 2O₂ + NAD (P) H → 2O₂⁻ + NAD(P) + H⁺.

Vascular NAD (P) H oxidase comprises at least 4 components: cell membrane-associated p22phox and gp91 phox (or gp91 phox [nox2] homologues, nox1 and nox4), and cytosolic subunits, p47phox and p67phox. Vascular NAD(P)H oxidase is regulated by humoral (cytokines, growth factors, and vasoac­tive agents) and physical factors (stretch, pulsatile strain, and shear stress).

Physiologically, ROS are produced in a control­led manner at low concentrations and function as signalling molecules to main­tain vascular integrity by regulating endothelial function and vascular contraction-relaxation.

Un­der pathological conditions, increased ROS bio- activity leads to endothelial dysfunction, increased contractility, VSMC growth, monocyte invasion, lipid peroxidation, inflammation, and increased deposition of extracellular matrix proteins, impor­tant factors in hypertensive vascular damage.

Impaired endothelium-mediated vasodilation in hypertension has been linked to decreased NO bioavailability. This may be secondary to de­creased NO synthesis or to increase NO degra­dation because of its interaction with ^•O₂ to form ONOO⁻.

Vasomotor tone is also modulated through direct ROS effects on calcium ion. Whereas VSMC ^•O₂ is associated pri­marily with vasoconstriction, endothelial H₂O₂ has been described as an endothelium-derived re­laxing factor.

Molecular processes underlying ROS-induced vascular changes involve activation of redox-sensitive signalling pathways. Superoxide anion and H₂O₂ stimulate mitogen-activated protein kinas­es, tyrosine kinases, and transcription factors (NFkB, AP-1, and HIF-1) and inactivate protein tyrosine phosphatases.

ROS also increase calcium ion and up-regulate proto-oncogene and proinflam­matory gene expression. These processes occur through oxidative modification of proteins by altering important amino acid resi­dues, by inducing protein dimerization, and by interacting with metal complexes such as Fe-S moieties.

Free Radicals: Role # 6.

Diabetes:

Elevated ROS levels have also been implicated in diabetes meliitus. In this case oxidative stress is associated with a pro-oxidative shift of the glutathione redox state in the blood. Hyperglycemia is a hallmark of both noninsulin-dependent (type 2) and insulin-dependent diabetes meliitus (type 1).

Elevated glucose levels are associated with in­creased production of ROS by several different mechanisms. In cultured bovine aortic endothelial cells, hyperglycemia was shown to cause increased ROS production at the mitochon­drial complex II.

Several independent strategies that ameliorate mitochon­drial ROS production were shown to prevent some of the typical secondary complications of the dis­ease, including the activation of protein kinase C or NF-kB and the formation of advanced glycation end products.

In addition, superoxide is generated by the process of glucose auto-oxidation that is associat­ed with the formation of glycated proteins in the plasma of diabetic patients.

The interaction of advanced glycation end prod­ucts with corresponding cell surface receptors stimulates ROS production and decreases intrac­ellular glutathione levels.

The increase in ROS production contributes to the de­velopment of diabetic complications such as atherosclerosis and other vascular complications. In addition, hyperglycemia enhances cell-mediated low-density lipoprotein (LDL) peroxidation in endothelial cells.

Treatment with anti­oxidants ameliorates diabetic complications in­cluding the dysfunction of endothelial cells or in­creased platelet aggregation.

In addition to O₂⁻, hyperglycemia also stimu­lates the synthesis of NO via increased enzymatic activity of endothelial and inducible isoforms of NOS. However, the NO generated in diabetic vasculature is rapidly scavenged by omnipresent O₂⁻ to form peroxynitrite [OONO⁻] at a rate of 6.7 x 109 ms⁻¹.

This rate is three times faster than the reaction between O₂⁻ and SOD. Hence, the formation of OONO” is a double- edged sword; on one hand potentially deleterious O₂ ⁻ is neutralised, on the other hand the most po­tent vasodilator NO is consumed and OONO⁻ is produced as a result.

It is therefore easy to comprehend why OONO⁻ itself has been suggested as both a toxic compound eliciting tis­sue damage as well as a protective molecule im­proving cellular and organ vitality.

OONO⁻ has been shown to increase insulin secretion, DNA damage and cell death in human and rat islets of Langerhans. It has also been linked to attenuation of vascular responses in diabetic and preeclamptic human placentas.

A recent report has also demonstrated that OONO⁻ contributes to the destruc­tion of pancreatic islet beta-cells of NOD mice developing autoimmune diabetes, suggesting that OONO⁻ may play a pivotal role in the initiation of insulin-dependent diabetes meliitus (IDDM).

Proposed general theory of hyperglycemia resulting in the pathophysiology of diabetes via generation of ROS is presented diagrammatically in the Figure 31.4.

Free Radicals: Role # 7.

Malignant Diseases:

ROS are potential carcinogens because they fa­cilitate mutagenesis, tumor promotion and pro­gression. The growth-promoting effects of ROS are related to redox-responsive signalling cas­cades.

A placebo-controlled clinical study of pa­tients with previous adenomatous colonic polyps, i.e., a group with an increased risk for colon can­cer and increased proliferative index of colonic crypts, revealed a significant decrease in the pro­liferative index after treatment with N-acetyl- cysteine.

A pro-oxidative shift in the plasma thiol/disulfide redox state has been observed in patients with various types of advanced malignancies. This shift is reminiscent of similar changes in diabetes mellitus, old age and intensive physical exercise.

Because cancer patients commonly have de­creased glucose clearance capacity and, in addition, abnormally high glycolytic activity and lactate production, it is reasonable to assume that the observed pro- oxidative shift is mediated by an increased and basically uncontrolled availability of mitochon­drial energy substrate. Oxidative stress has many diverse cellular ef­fects.

As depicted in Figure 31.5, this ubiquitous stress can cause mutagenicity, cytotoxicity, and stimulate changes in gene expression. Furthermore, these effects are likely to in­terplay in the development of carcinogenesis by oxidants.

Mutations induced by oxidants may in­itiate carcinogenesis; oxidative modification of the genetic material may also participate in the pro­gression of benign to malignant neoplasms. Alter­ation of the pattern of gene expression by oxidants may function in the stimulation of the initiated cell during tumour promotion. Further, oxidant-induced toxicity in the normal.

Free Radicals: Role # 8.

Inflammation and Arthritis:

In recent years it has become increasingly appar­ent that, in man, free radicals play a role in varie­ty of normal regulatory systems, the deregulation of which may play an important role in inflamma­tion.

Inflammation represents the normal response to mechanical, chemical or infectious tissue inju­ry. Whether acute or chronic, inflammation is dependent upon regulated humoral and cellular responses and if unchecked, leads to the genera­tion of inflammatory diseases.

The physiological function which may be perturbed in inflamma­tion, include: the oxidative modification of low density lipoprotein, the oxidative inactivation of alpha-1-protease inhibitor, DNA damage/repair and heat shock protein synthesis. During phago­cytosis neutrophils, eosinophil’s and mononuclear phagocytes consume increased amount of oxy­gen, i.e., the process termed as respiratory burst.

Activation results in increased NADPH produc­tion and the generation of superoxide anion radi­cal, hydrogen peroxide, hydroxyl radical and hypochlorous acid (HOCI); these ROS are capable of damaging cell membranes and wide variety of biomolecules. At the sites of inflammation, in­creased ROS activates neutrophil NADPH oxidase and/or the uncoupling of a variety of redox sys­tem.

It has been proved that free radicals which were generated through the induction of DNA strand breaks activates transcription of genes c- fos, c-myc, c-jun and β-actin which are responsi­ble for cell growth, differentiation and develop­ment.

At low doses, ROS stimulate growth of fibroblast and epithelial cells in culture and thus play a role early in inflamma­tion by promoting fibrosis and wound healing. The inflammation also takes place via an indirect route, through the lipid peroxidation products which promotes collagen synthesis.

NFkB is an important transcription factor in inflammatory system because it controls the tran­scription of a number of cytokinin genes like IL-2, TNF α, etc. Recently, it has been shown that free radical generating systems also damage a variety of cellular and plasma proteins, e.g., immunoglob­ulin C (Figure 31.6) and a-1-proteinase inhibitor which are well documented examples, relevant to inflammation.