-The role of Reactive Oxygen Species and Al)tioxidants inPeriodontal Tissue destruction

Dr Rahul NadkarniPG student

Dr Narayan WalavalkarProfessor

Department of Periodontics

Periodontitis is a term used to describe an inflammatoryprocess, initiated by the plaque biofi1m,that leads to loss ofperiodontal attachment to the root surface and adjacentalveolar bone and which ultimately results in tooth loss.The inflammatory and immune responses to the bacteriathat colonize the periodontal and associated tissues involvethe systemic circulation and ultimately the peripheralsystems of the body. This creates a complex bi-directionalseries of host- microbial interactions involving cellular andhumoral factors and networks of cytokines, chemokines,and growth factors. It is believed that while the primaryetiological agent is specific, predominantly gram negativeanaerobic or facultative bacteria within the sub gingivalbiofilm, the majority of periodontal tissue destruction arecaused by an inappropriate host response to thosemicroorganisms and their products. More specifically, aloss of homeostatic balance between proteolytic enzymes(e.g. neutrophil elastase) and their inhibitors (e.g. al-antitrypsin) and reactive oxygen species (ROS) and theantioxidant defence systems that protect and repair vitaltissue, cell, and molecular components is believed to beresponsible. The larger upward shifts in the pro-oxidant/antioxidant ratio intracellularly bring about directdamage to vital biomolecules and structures, cellmembrane damage and dysfunction, and cell death (bynecrosis or accelerated apoptosis), and extracellularlycause direct connective tissue damage (both mineralizedand unmineralized) and damage to extracellular matricesand their components.Definition -Free radicals have been defined as any species capable ofindependent existence that contain one or more unpairedelectrons. They are, by nature, highly reactive and diversespecies, capable of extracting electrons and therebyoxidizing a variety ofbiomo1ecules vital to cell and tissuefunction. (Halliwell 1991)ROS is a term that has become more popular because itencompasses other reactive species which are not trueradicals but are nevertheless capable of radical formation inthe intra- and extracellular environments. ( Battino etal.1999)Antioxidants are defined as those substances which whenpresent at low concentrations, compared to those of anoxidizable substrate, will significantly delay or inhibit

oxidation of that substrate. Innormal physiology there is adynamic equilibrium between ROS activity andantioxidant defense capacity and when that equilibriumshifts in favour ofROS, either by a reduction in antioxidantdefenses or an increase in ROS production or activity,oxidative stress results. (Halliwell 1989).Oxidative stress was defined as a disturbance in the pro-oxidant-antioxidant balance in favor of the former, leadingto potential damage. (Sies 1991).The redox potential is a measure (in volts) of the affinity ofa substance for electrons, relative to hydrogen.Within the gingival crevice/pocket a low redox potential isregarded as essential for the growth and survival ofsubgingival anaerobes, whereas within cells and tissues areducing environment (low redox potential) is protectiveagainst oxidative stress. There is therefore an apparentconflict in developing future therapeutic strategies forperiodontitis which are based on redox biology, because :maintaining a low redox status to protect host cells andtissues from oxidative stress is conducive to encouraginggrowth and survival of anaerobes. However, it is vital tounderstand that the body is compartmentalized. Bacteriaare not intracellular pathogens (unlike viruses) andtherefore maintaining a low redox state within a cell maynot have relevance to a high redox state within theperiodontal pocket/gingival crevice. A key example ofcompartmentalized differences in antioxidant compositionwas highlighted by (Brock et al.) who demonstrated thatthe antioxidant composition of gingival crevicular fluiddiffered substantially from the plasma and salivacompartments, with reduced glutathione (GSH) being amajor antioxidant within gingival crevicular fluid, whereasuric acid predominates �� saliva and plasmaExogenous sources of oxygen radicals include heat,trauma, ultrasound, ultraviolet light, ozone, smoking,exhaust fumes, radiation, infection, excessive exercise, andtherapeutic drugs.Endogenous sources are primarily:Bi-products of metabolic pathways - electron leakage frommitochondrial electron transport systems formingsuperoxide;Functional generation by host defense cells (phagocytes)and cells of the connective tissues (osteoclasts andfibroblasts).

Free Radicals And Reactive Oxygen Species

Free Radicals Reactive Oxygen SpeciesSuperoxide Hydrogen PeroxideHydroxyl Hypochlorous Acid

Perhydroxyl Singlet OxygenHydroperoxyl Ozone

AlkoxylAryloxylPeroxyl

ArylperoxylAcyloxyl

Acylperoxyl

Types Of Tissue Damage ByReactive Oxygen Species.

1. Protein Damage2. Lipid Peroxidation3. Dna Damage

Superoxide Hydroxyl Radical

Functional production ofsuperoxide involves

activation of the HM:P (orNADPH-oxidase) shunt. ).This process comprises theso-called respiratory burstwithin polymorphonuclear

leukocytes. Superoxide forms.initially and then

spontaneously dismutates tohydrogen peroxide.

Hydrogen peroxide may alsoundergo Fenton reactions in

the presence of Fe2+ or Cu2+ions, forming the most potent

of all oxygen radicals, thehydroxyl radical (oOH).

Hydrogen Peroxide

Hydrogen peroxide is a weakROS, the potential of which

to cause tissue damage islimited to its interaction with

transition metal ions viaFenton chemistry or

ultraviolet light, when itforms the more potenthydroxyl radical. The

principal enzymes chargedwith removal of hydrogen

peroxide are the antioxidantenzymes catalase, which

predominantly actsintracellularly and

glutathione peroxidase,which operates within

mitochondria andextracellularly.

The hydroxyl (·OH)radical and the relatedperhydroxyl radical

(H02) are the most potentspecies known to cause

damage and destruction toan array of cellular and. tissue components.

Specifically, damage mayaffect cellular and

extracellular targets.Targets include:

• Lipids• Proteins

• Carbohydrates• DNA

Antioxidant Defence SystemsThe preventative antioxidants function by enzymatic removal of superoxide and hydrogen peroxide or by sequestrationof divalent metal ions, preventing Fenton reactions and subsequent hydroxyl radical formation. The chain-breakingantioxidants are the most important within extracellular fluids. The lipid soluble antioxidants (a-tocopherol and thecarotenoids) act at the cell membrane level and protect against lipid peroxidation, whereas the water-soluble scavengersare more important within the extracellular tissue fluids. It is important to recognize however, that several antioxidantshave dual and sometimes triple actions. For example, ascorbate acts as a chainbreaking or scavenging antioxidant aswell as a preventative antioxidant by virtue of its ability to recycle a-tocopherol (vitamin E) from its oxidized form andby its ability to bind metal ions, respectively. Similarly, the intracellular enzymes (superoxide dismutase, catalase,glutathione peroxidase) are regarded by some as preventative antioxidants. The efficacy of an antioxidant depends�����

Its location (intra- vs. extracellular or cell membrane bound);The nature ofthe ROS-challenge;Other antioxidant species important in co-operative interactionsOther environmental conditions (e.g. pH, oxygen tension).

The role of Reactive OXY~Jc:mSpecies and Antloxldants in Periodontal Tissue destruction 24- - - -

Carotenoids

SuperoxideDismutase

Different types of superoxide exist,• Superoxide .. dismutase 1 - aCu2+/Zn2+-dependent enzymefound within the cytosol;• Superoxide dismutase 2 - theMn2+-dependent enzyme locatedwithin the mitochondria;• Superoxide dismutase 3 -extracellular enzyme, found at lowlevels extracellularly.

o 2 + 0 2 + 2H+ SOD 02 +- -H202

Catalase

Catalase contains heme- bound iron andis mainly located in perioxisomes. Itremoves H202 with great efficacy. 1tdismutates H202 to form water and O2.

Ascorbic Acid(Vitamin C)

A-Tocopherol(Vitamin E)

The role of vitamin C as an antioxidantin inflammatory diseases is summarizedas:• scavenging water-soluble peroxylradicals;• scavenging superoxide andhypochlorous acid;• prevention of damage mediated byhydroxyl radicals• decreases heme breakdown and Fe2+release thereby preventing Fentonreactions;• re-forms a-tocopherol from its radical;• protects against ROS-release fromcigarette smoke.

Vitamin E is generally regarded as themost important and effective lipid-soluble antioxidant in vivo, vital tomaintaining cell membrane integrityagainst lipid peroxidation by peroxylradical scavenging. It requires otherantioxidant species to re-constitutevitamin E, the most effective being thereduced form of co enzyme QlO(ubiquinol) in the lipid environment andascorbic acid in the aqueous phase.Carotenoids include lycopene, a-

carotene, b-carotene, lutein,cryptoxanthine, retinol (vitamin AI),dehydroretinol (vitamin A2).Carotenoids are lipophilic and higherplasma concentrations have been shownto protect against various inflammatoryand malignant diseases.

CoenzymeQI0

Co-enzyme Q 10 exists in an oxidizedform (ubiquinone or CoQ) and a reducedCo-enzyme Q 10 exists in an oxidized

form (ubiquinone or CoQ) and a reducedform (ubiquinol or CoQH2), both ofwhich possess antioxidant activity. Co-enzyme Q 10 deficiency has beendemonstrated in the gingival tissues ofperiodontitis subjects.Uric acid is one of the major radicalscavengers within plasma, urine, andsaliva. Its antioxidant activities include:• scavenger of singlet oxygen, hydroxylradicals• binding of divalent metal ionspreventing Fenton chemistry.

Uric Acid

The most researched polyphenols are thewater-soluble catechin, epigallocatechingallate, and the po lyphenol, Quercetin.

Polyphenols Polyphenols function by:• Radical scavenging, terminating lipidperoxidation, iron chelation, sparingvitamin E, restoration of vitamin C.

Glutathione is a non-essential tri-peptidein that it can be synthesized within thecell. Glutathione exists in oxidized

Glutathione (GSSG) and reduced (GSH) forms andGSH is a Ubiquitous thiol that plays amajor role in human physiology andpathology.

Role Of Reactive Oxygen Species In PeriodontalTissue DamageChronic inflammatory conditions are generally thought tobe associated with increased oxidative stress, withphagocytes (particularly the neutrophil) being implicatedin disease pathogenesis because of the generation of theoxidative burst during phagocytosis and killing.Originally, ROS were thought to be directly microbicidalbut recent evidence indicates that their role is to establishan environment in the phagocytic vacuole suitable forkilling and digestion by enzymes released into the vacuolefrom cytoplasmic granules. Significant ROS generationby neutrophils requires a minimum oxygen tension ofabout 1% and a pH of7.0-7.5. Both these conditions arefound within periodontal pockets, indicating that chronicor excess ROS production is possible at this important siteof periodontal tissue damage.

Plaque bacteria and their products are an obvious sourceof factors that could stimulate neutrophils infiltrating theperiodontal tissues. Enhanced ROS generation byperipheral neutrophils from patients with both chronicand aggressive disease can be stimulated with opsonizedbacteria associated with periodontal disease

(Fusobacterium nucleatum, Actinobacillusactinomycetemcomitans). Further studies have shownthat several isolates of two strains of fusobacteria (F.nucleatum and F. necrophorum) can stimulate significantROS generation, cytokine [interleukin-l b (IL-l b), TNF-a, IL-8] and elastase production by neutrophils isolatedfrom healthy individuals. This group has also shown thatF. nucleatum, in the absence of plasma, could alsostimulate large amounts of ROS production and inducelipid peroxidation in vitro. That F. nucleatum might bepivotal in neutrophil-dependent, ROS induced tissuedamage within the periodontium is also supported by thefinding that phagocytosis of F. nucleatum inducessignificantly greater ROS generation than phagocytosis ofPorphyromonas gingivalis orA. actinomycetemcomitans.

A variety of pro-inflammatory cytokines (TNF-a,granulocyte-macrophage colony stimulating factor,granulocyte colony-stimulating factor, IL-8, IL-l, IL-6),growth factors (e.g. platelet activating factor), andlipopolysaccharide have been shown to have a primingeffect on the human neutrophil oxidative burst both invitro and in vivo. TNF-a can prime for ROS generation byneutrophils from patients with chronic and aggressiveperiodontitis as well as periodontally healthy individuals.Cytokines can modulate the respiratory burst activity ofneutrophils and have a role in determining oxidative stresslocally within the tissues.

ConclusionOxidative stress lies at the heart of the periodontal tissuedamage that results from host-microbial interactions,

either as a direct result of excess ROS activity/antioxidardeficiency or indirectly as a result of the activation credox-sensitive transcription factors and the creation ofpro-inflammatory state. A body of literature suppor1peripheral blood neutrophil hyper reactivity in chroniand aggressive forms of periodontitis, with respect to totsFcc-receptor-mediated ROS generation. This, togethewith the evidence for compromised plasma antioxidarcapacity, independent of smoking, suggests an underlyinenvironment of oxidative stress within periodontitipatients. Considerable evidence has emerged over the las2 years that oxidative stress and depressed antioxidarfunction are features of periodontal tissues and fluids iperiodontitis subjects. This concept has led to search fcappropriate antioxidant therapy in inflammatory diseaseIt may become necessary to deliver antioxidantselectively to cell types in inflammatory diseases likperiodontitis to achieve normal cell function.

References:1. The role of reactive oxygen and antioxidant species

periodontal tissue destruction. Periodontology 2000; 200'Vol. 43; 160-232.

2. Assessment of Total Antioxidant Capacity and the Use cVitamin C in the Treatment of Non-Smokers with ChroniPeriodontitis. J Periodontol; November 2010; Vol 81; 154~1554.

3. Oxidative injury and inflammatory periodontal diseases: thchallenge of antioxidants to free radicals and reactive oxygespecies. CritRev OraIBiol Med; 1999; Vol 10; 458-476.

4. Role of antioxidants as an adjunct in periodontal therapy.Academy Adv Dental Research. 2011; Vo12; 9-16

The role of Reactive Oxygen Specie" and Antioxidants ill Periodontal Tissue destruction 25- - - -- - - -~-- - - - -