Dr Harshavardhan Patwal

INTRODUCTIONPERIODONTAL DISEASE IS AN

INFLAMMATORY CONDITIONIT HAS A MICROBIAL ETIOLOGYTHE HOST RESPONSE ALSO HAS AN

IMPORTANT ROLE TO PLAYTHE ROLE OF OXIDATIVE STRESS IN

INFLAMMATORY CONDITIONS HAS BEEN WELL UNDERSTOOD AND DOCUMENTED

ROS AND FREE RADICALSFREE RADICALS HAVE BEEN IMPLICATED

IN THE PATHOGENESIS OFRHEUMATOID ARHRITIS( Mc cord, 1974)ARDS (Tate and Repine , 1983)AIDS ( Droge, 1988)

WHAT ARE FREE RADICALS? A FREE RADICAL IS ANY SPECIES

CAPABLE OF INDEPENDENT EXISTANCE THAT CONTAINS ONE OR MORE UNPAIRED ELECTRONS

( HALLIWELL ,1991)

IS THERE A DIFFERENCE BETWEEN FREE RADICALS AND ROS?

ROS ARE MOLECULES SUCH AS H2O2 AND HOCl AND SINGLET OXYGEN WHICH WHILE NOT FREE RADICALS BY THEMSELVES ARE ABLE TO INITIATE RADICAL FORMATION IN INTRACELLULAR AND EXTRACELLULAR ENVIRONMENTS

( HALLIWELL AND GUTERIDGE, 1990)

ARE ROS AND FREE RADICALS INITIATORS OF TISSUE DAMAGE?THESE ROS CAN CAUSE TISSUE DAMAGE

DIRECTLY AND BY INDIRECT MECHANISMS.THEY CAN CAUSE

LIPID PEROXIDATION AND THEREBY ACTIVATE CYCLOOXYGENASE AND LIPOXYGENASE

PROTEIN DAMAGE TO HYALURONIC ACID AND PROTEOGLYCANS ( Bartold et al, 1984)

OXIDATION OF IMPORTANT ENZYMES SUCH AS ALPHA 1 ANTITRYPSIN

DNA DAMAGE

DO ROS HAVE ANY ROLE ON TRANSCRIPTION FACTORSTHIS IS AN IMPORTANT MECHANISM. THE ROS

MOLECULES DEPLETE INTRACELLULAR THIOL COMPOUNDS . BECAUSE OF THIS, THERE IS AN INDUCTION AND STIMULATION OF REDOX SENSITIVE TRANSCRIPTION FACTORS AP1 AND NF KB WHICH CAUSES PRODUCTION OF PRO INFLAMMATORY CYTOKINES IL 1 AND TNF BETA.

BALANCE BETWEEN ROS AND ANTIOXIDANTS

THE MAJOR ROS MOLECULESSUPEROXIDE ANION

HYDROXYL RADICAL

NITROUS OXIDE

HYDROGEN PEROXIDE

HYPOCHLOROUS ACID

SINGLET OXYGEN

SUPEROXIDE ANIONFORMED BY AN ADDITION OF AN

ELECTRON TO THE OXYGEN MOLECULE

THESE ELECTRONS CAN LEAK FROM THE MITOCHONDRIA FROM THE RESPIRATORY CHAIN

O2 + e- O2-. ( FRIDOVICH , 1989 )

BUT THE MOST IMPORTANT SOURCE OF SUPEROXIDE ANIONS IN THE PERIODONTIUM IS THROUGH THE NADPH OXIDASE SHUNT OR HEXOSE MONO PHOSPHATE PATHWAY

2NADPH + 2O2 2NADP+2H+ + 2O2.-

2O2.- +2H+ SOD 1O2 + H2O2

O2.- + H2O2 .OH +OH-+ O2

METAL CATALYSED HABER WEISS REACTION

2H2O2 2H2O + O2 CATALASE

CATALASE MEDIATED UTILIZATION OF H2O2 IS LARGELY INTRACELLULAR.

Fe or Cu ions

WHEN NADPH OXIDASE SHUNT FAILS TO WORK THERE IS A FAILURE OF PRODUCTION OF O2-. WHICH CAUSES A CONDITION CALLED CHRONIC GRANULOMATOUS DISEASE

IN THIS CONDITION THE NEUTROPHILS ENGULF THE BACTERIA BUT CANNOT OPSONISE THEM

SIMILARILY AN ABSENCE OF THE ENZYME CATALASE RESULTS IN A CONDITION CALLED ACATALASIA WHICH ALSO CAUSES PERIODONTAL DESTRUCTION DUE TO THE LACK OF MITIGATION OF INTRACELLULAR H2O2

THE ROLE OF CATALASE IN THE EXTRA CELLULAR ENVIRONMENT IS PERFORMED BY GLUTATHONE PEROXIDASE WHICH IS A SELENIUM DEPENDANT ENZYME WHICH OXIDISES REDUCED GLUTATHIONE INTO ITS OXIDISED FORM

2GSH + H2O2 GSSG + 2H2O

SUPEROXIDE ANION CAN CAUSE BONE

DESTRUCTION

HAS BEEN LOCALISED IN THE RUFFLED BORDER OF RESORBING BONE

MOROVER IT CAN ALSO GIVE RISE TO H2O2, 1O2 AND HOCL AND OH-.

THE HYDROXYL RADICALMOST LETHAL ROS MOLECULECAN BE RELEASED BY THE HABER WEISS

REACTIONO2.- +H2O2 .OH+ OH-

+O2ALTERNATELY IT CAN BE RELEASED BY A

TRANSITION METAL DEPENDANT FENTON REACTION

H2O2 + Fe2+ Fe3+ + .OH+ OH-

Fe or Cu ions

CONSEQUENCE OF HYDROXYL RADICAL RELEASEDNA STRAND BREAKS – BREIMER et al 1991BASE HYDROXYLATIONS – JACKSON et al 1988MALIGNANT TRANSFORMATION AND CELL DEATHLIPID PEROXIDATION OF PHOSPHOLIPIDS WITH

ARACHIDONIC ACID BEING THE TARGETLIPID HYDROPEROXIDES CAN DECOMPOSE TO

FORM CYTOTOXIC ALDEHYDES – ESTERBAEUR et al 1988

RELEASE OF PROSTANOIDS LIKE PG F2 ALPHA LIKE ISOPROSTANES INVOLVED IN LYMPHOCYTE ACTIVATION, BONE RESORPTION AND VASODILATION

LIPID PEROXIDATION END PRODUCTS RESULT IN DYSFUNCTION OF Ca 2+ ATPase WHICH CAUSES OPENING OF Ca CHANNELS CAUSING THE EXCESSIVE ACCUMULATION OF INTRACELLULAR CALCIUM

THIS CAUSES CELL DAMAGE

DAMAGE OF ENZYMES , GLYCOPROTEINS AND MEMBRANE BOUND RECEPTORS CAN ALSO OCCUR

FREE MOVEMENT OF H2O2 ACROSS MEMBRANES ALSO CAUSES FENTON REACTIONS TO OCCUR EVERYWERE IN THE CELL

NITRIC OXIDENITRIC OXIDE CAN BE SYNTHESISED FROM L-

ARGININE BY A FAMILY OF ENZYMES CALLED NITRIC OXIDE SYNTHASES.

TYPE I IS THE BRAIN ENZYME b NOS

TYPE 2 IS INDUCIBLE FORM SYNTHESISED BY MACROPHAGES

TYPE 3 IS ENDOTHELIAL NITRIC OXIDE SYNTHASE

PEROXYNITRITE ANIONMACROPHAGE DERIVED I NOS IS

IMPORTANT AS THE NO SYNTHESISED REACTS WITH SUPEROXIDE TO FORM PEROXYNITRITE ANION

NO. + O2.- ONOO-

IT IS NOT A TRUE FREE RADICAL BUT HAS PROFOUND EFFECTS

ACTIVITIES OF PEROXYNITRITE ANIONLIPID PEROXIDATIONGLUTATHIONE DEPLETION BY OXIDATIONNITROTYROSINE FORMATION WHICH WILL

INHIBIT SODDNA DAMAGE BY NITROSILATION,

DEAMINATION, OXIDATIONCELLULAR NECROSISAPOPTOSIS

HYDROGEN PEROXIDERELEASED BY BACTERIA AND BY NADPH

OXIDASE SHUNTHYDROGEN PEROXIDE CAN OXIDISE NF

KB AND CAN RESULT IN PROINFLAMMATORY CYTOKINE RELEASE

INCREASE ADHESION MOLECULE EXPRESSION

INDUCE APOPTOSISMODULAE PLATELET AGGREGATION

HYPOCHLOROUS ACIDIS FORMED BY THE ACTION OF

MYELOPEROXIDASE ON H2O2CAN CAUSE DISRUPTION OF PROTEIN

FUNCTIONSACTIVATES NEUTROPHIL COLLAGENASEOXIDISES ALPHA 1 ANTITRYPSINCELL LYSIS

SINGLET OXYGENNOT A TRUE FREE RADICAL BUT UNSTABLE

CAUSED BY INPUT OF ENERGY INTO OXYGEN MOLECULE THAT REVERSES THE SPIN OF ONE OF THE OUTERMOST UNPAIRED ELECTRONS FROM A PARALLEL SPIN INDUCING INSTABILITY

CAN CAUSE LIPID PEROXIDATION

HOW DO ROS CAUSE TISSUE DAMAGETHEY AFFECT IMPORTANT BIOLOGICAL

MOLECULES

LIPIDS

PROTEINS

DNA

EFFECTS OF ROS ON LIPIDSTHIS HAS BEEN DESCRIBED BY HALLIWEL IN

3 STAGES

INITIATION

PROPAGATION

TERMINATION

THE PROCESS IS CALLED LIPID PEROXIDATION

DETAILS ABOUT LIPID PEROXIDATIONTHE HYDROXYL OR PEROXYNITRITE RADICAL

ATTACKS A PUFA IN THE LIPID MEMBRANE ( INITIATION) AND ABSTRACTS A HYROGEN ATOM FORMING A CARBON CENTERED RADICAL L.

THE LATTER MAY REARRANGE TO FORM A CONJUGATED DIENE OR MAY COMBINE WITH ANOTHER PUFA SIDE CHAIN RADICAL TO FORM A COVALENT BOND THUS DISRUPTING MEMBRANE STRUCTURE AND FUNCTION

HOWEVER IT MAY ALSO COMBINE WITH OXYGEN TO FORM A LIPID PEROXYL RADICAL WHICH ( LOO.) WHICH MAY ATTACK A PUFA ( PROPAGATION)

THIS MAY GENERATE ANOTHER CARBON CENTERED RADICAL AND LOOH

THIS MAY KEEP ON CONTINUING WITH HUNDRED OF MOLECULES GETTING INVOLVED WITH ACCUMULATION OF TOXIC PRODUCTS OF LIPID PEROXIDATION

TERMINATION IS BROUGHT ABOUT BY LIPID SOLUBLE SCAVENGER VITAMIN E

PROTEIN DAMAGE BY ROSRADICAL ATTACK MAY ATTACK C=C BONDS

CREATING CARBON CENTERED RADICAL INTERMEDIATES.

2 SPECIES FORMED BY HOMOLYTIC FISSION OF THE C=C BOND WILL TAKE AN ELECTRON EACH AND CONTINUE THE PROCESS

THIOL GROUPS ON PROTEINS ARE TARGETS FOR ROS ACTIVITY

EFFECTS ON PROTEINS

PROTEIN FOLDING AND UNFOLDINGPROTEIN FRAGMENTATION AND

POLYMERISATIONPROTEASE DEGRADATION OF THE

MODIFIED PROTEINFORMATION OF PROTEIN RADICALSFORMATION OF PROTEIN BOUND ROSFORMATION OF STABLE END PRODUCTS

DNA DAMAGE BY ROSMECHANISMS OF DAMAGE DY ROS ON DNA

INCLUDESTRAND BREAKS BASE PAIR MUTATIONSFORMATION OF 8 HYDROXYGUANINE WHICH

WILL FORM THE NUCLEOSIDE 8 OHDGDELETIONINSERTIONNICKINGSEQUENCE AMPLIFICATIONHYDROXYL RADICALS ATTACK ALL 4 BASES TO

FORM A CHARACTERISTIC DNA FINGER PRINT

EFFECT OF ROS ON THE CELL

ROS INDUCTION OF TRANSCRIPTION FACTORSNORMAL REGULATION OF MEMBRANE

RECEPTOR BOUND SIGNALLING CASCADES DEPEND ON LOCALLY GENERATED ROS

THESE OXIDISE OR REDUCE GLUTATHIONE TO CAUSE POST TRANSLATIONAL MODIFICATION OF PROTEINS

NF KB AND AP 1THEY ARE TRANSCRIPTION FACTORS

IN THE NON ACTIVATED STATE NF KB IS BOUND TO AN INHIBITORY PROTEIN IKB

H2O2 BINDS TO ACTIVATE IKB KINASE WHICH PHOSPHORYLATES 2 CRITICAL SEREINE RESIDUES IN IKB

THIS FREES THE NF KB THAT TRANSLOCATES TO THE NUCLEUS AND SWITCHES ON IL 1 , TNF A GENES

AP 1AP1 IS A HETEROGENEOUS GROUP OF

DIMERIC TRANSCRIPTION FACTORS JUN, FOS AND ATF

API IS ALSO AFEECTED BY ROS

GENES UNDER THE CONTROL OF NF KB AND AP1

CENTRAL ROLE OF OXIDATIVE STRESS IN PERIODONTAL PATHOGENESIS

METHODS TO MEASURE THE BIOMARKERS OF OXIDATIVE DAMAGEFREE RADICALS HAVE EXTREMELY SHORT

HALF LIVES IN VIVOIN VITRO SYSTEMS CALLED SPIN TRAPS

ARE USED TO MEASURE THESE RADICALS EX VIVO SPIN TRAPS AREASCORBIC ACID WHICH BECOMES SEMI

DEHYDROASCORBATE AROMATIC TRAPS SUCH AS SALICYLATES

AND PHENYLALANINEURATE WHICH IS OXIDISED TO ALLANTOIN

BIOMARKERS OF LIPID PEROXIDATIONCONJUGATED DIENES

THIOBARBITURIC ACID REACTIVE SUBSTANCES( MALONDIALDEHYDE)

ISOPROSTANES

ETHANE / PENTANE

BIOMARKERS OF PROTEIN DAMAGEPROTEIN CARBONYL RADICAL ASSAY

AOPP

BIOMARKERS TO MEASURE DNA DAMAGE8 OH DG MEASUREMENT

COMET ASSAY TO MEASURE STRAND BREAKS

METHODS EMPLOYEDMASS SPECTROSCOPYELISAHPLC

EVIDENCES TO SHOW ELEVATED ROS IN PERIODONTAL DISEASEPANJAMURTHY ET AL 2005: ELEVATED

LEVELS OF THIOBARBITURIC ACID REACTIVE SUBSTANCES IN PLASMA AND RBC OF PATIENTS WITH CPD

TSAI CC ET AL 2005: MALONDIALDEHYDE LEVELS RAISED IN GCF AND SALIVA OF PATIENTS WITH CPD

NOUROOZ ZADEH ET AL 1994: ELEVATED HYDROPEROXIDE LEVELS IN PATIENTS WITH PAPILLON LE FEVRES SYNDROME MEASURED BY FOX 2 ASSAY

VOLOZHIN AL ET L 2001 : EXHALED AIR CONTAINS VOLATILE HYDROCARBONS , SHORT CHAIN FATTY ACIDS AND ALDEHYDE ELEVATED IN CPD PATIENTS

DI PAOLA ET AL 2005: EXPRESSION AND IMMUNOHISTOCHEMICAL DETECTION OF NITROTYROSINE IN LIGATURE INDUCED PERIODONTITIS IN RODENTS

SAWAMOTO ET AL 2005: ELEVATED LEVELS OF 8 OH DG IN SALIVA OF PERIODONTITIS SUBJECTS

THANK YOU.