Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Oxidative stress from oxidative metabolism causes base damage, as well as strand breaks in DNA. Base damage is mostly indirect and caused by reactive oxygen species (ROS) generated, e.g. O2− (superoxide radical), OH (hydroxyl radical) and H2O2 (hydrogen peroxide).Further, some reactive oxidative species act as cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signaling. Dr Harshavardhan Patwal , Chemically, oxidative stress is associated with increased production of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses, such as glutathione.The effects of oxidative stress depend upon the size of these changes, with a cell being able to overcome small perturbations and regain its original state. However, more severe oxidative stress can cause cell death and even moderate oxidation can trigger apoptosis, while more intense stresses may cause necrosis. Production of reactive oxygen species is a particularly destructive aspect of oxidative* stress. Such species include free radicals and peroxides. Some of the less reactive of these species (such as superoxide) can be converted by oxidoreduction reactions with transition metals or other redox cycling compounds (including quinones) into more aggressive radical species that can cause extensive cellular damage.Most long-term effects are caused by damage to DNA

1. Dr Harshavardhan Patwal

2. INTRODUCTION
PERIODONTAL DISEASE IS AN INFLAMMATORY
CONDITION
IT HAS A MICROBIAL ETIOLOGY
THE HOST RESPONSE ALSO HAS AN IMPORTANT
ROLE TO PLAY
THE ROLE OF OXIDATIVE STRESS IN
INFLAMMATORY CONDITIONS HAS BEEN WELL
UNDERSTOOD AND DOCUMENTED

3. ROS AND FREE RADICALS
FREE RADICALS HAVE BEEN IMPLICATED IN THE
PATHOGENESIS OF
RHEUMATOID ARHRITIS( Mc cord, 1974)
ARDS (Tate and Repine , 1983)
AIDS ( Droge, 1988)

4. WHAT ARE FREE RADICALS?
A FREE RADICAL IS ANY SPECIES CAPABLE OF
INDEPENDENT EXISTANCE THAT CONTAINS ONE
OR MORE UNPAIRED ELECTRONS
( HALLIWELL ,1991)

5. 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)

6. 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

7. DO ROS HAVE ANY ROLE ON
TRANSCRIPTION FACTORS
THIS 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.

9. BALANCE BETWEEN ROS AND
ANTIOXIDANTS

10. THE MAJOR ROS MOLECULES
SUPEROXIDE ANION
HYDROXYL RADICAL
NITROUS OXIDE
HYDROGEN PEROXIDE
HYPOCHLOROUS ACID
SINGLET OXYGEN

11. SUPEROXIDE ANION
FORMED 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 )

12. 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

13. 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

14. 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

15. 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

16. 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-.

17. THE HYDROXYL RADICAL
MOST LETHAL ROS MOLECULE
CAN BE RELEASED BY THE HABER WEISS
REACTION
O2.- +H2O2 .OH+ OH-+O2
ALTERNATELY IT CAN BE RELEASED BY A
TRANSITION METAL DEPENDANT FENTON
REACTION
H2O2 + Fe2+ Fe3+ + .OH+ OH-
Fe or Cu ions

18. CONSEQUENCE OF HYDROXYL
RADICAL RELEASE
DNA STRAND BREAKS – BREIMER et al 1991
BASE HYDROXYLATIONS – JACKSON et al 1988
MALIGNANT TRANSFORMATION AND CELL DEATH
LIPID PEROXIDATION OF PHOSPHOLIPIDS WITH
ARACHIDONIC ACID BEING THE TARGET
LIPID 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

19. 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

20. NITRIC OXIDE
NITRIC 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

21. PEROXYNITRITE ANION
MACROPHAGE 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

22. ACTIVITIES OF PEROXYNITRITE
ANION
LIPID PEROXIDATION
GLUTATHIONE DEPLETION BY OXIDATION
NITROTYROSINE FORMATION WHICH WILL INHIBIT
SOD
DNA DAMAGE BY NITROSILATION, DEAMINATION,
OXIDATION
CELLULAR NECROSIS
APOPTOSIS

23. HYDROGEN PEROXIDE
RELEASED BY BACTERIA AND BY NADPH
OXIDASE SHUNT
HYDROGEN PEROXIDE CAN OXIDISE NF KB AND
CAN RESULT IN PROINFLAMMATORY CYTOKINE
RELEASE
INCREASE ADHESION MOLECULE EXPRESSION
INDUCE APOPTOSIS
MODULAE PLATELET AGGREGATION

24. HYPOCHLOROUS ACID
IS FORMED BY THE ACTION OF
MYELOPEROXIDASE ON H2O2
CAN CAUSE DISRUPTION OF PROTEIN
FUNCTIONS
ACTIVATES NEUTROPHIL COLLAGENASE
OXIDISES ALPHA 1 ANTITRYPSIN
CELL LYSIS

25. SINGLET OXYGEN
NOT 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

26. HOW DO ROS CAUSE TISSUE
DAMAGE
THEY AFFECT IMPORTANT BIOLOGICAL
MOLECULES
LIPIDS
PROTEINS
DNA

27. EFFECTS OF ROS ON LIPIDS
THIS HAS BEEN DESCRIBED BY HALLIWEL IN 3
STAGES
INITIATION
PROPAGATION
TERMINATION
THE PROCESS IS CALLED LIPID PEROXIDATION

28. DETAILS ABOUT LIPID
PEROXIDATION
THE 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)

29. 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

31. PROTEIN DAMAGE BY ROS
RADICAL 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

32. EFFECTS ON PROTEINS
PROTEIN FOLDING AND UNFOLDING
PROTEIN FRAGMENTATION AND
POLYMERISATION
PROTEASE DEGRADATION OF THE MODIFIED
PROTEIN
FORMATION OF PROTEIN RADICALS
FORMATION OF PROTEIN BOUND ROS
FORMATION OF STABLE END PRODUCTS

34. DNA DAMAGE BY ROS
MECHANISMS OF DAMAGE DY ROS ON DNA INCLUDE
STRAND BREAKS
BASE PAIR MUTATIONS
FORMATION OF 8 HYDROXYGUANINE WHICH WILL
FORM THE NUCLEOSIDE 8 OHDG
DELETION
INSERTION
NICKING
SEQUENCE AMPLIFICATION
HYDROXYL RADICALS ATTACK ALL 4 BASES TO FORM
A CHARACTERISTIC DNA FINGER PRINT

35. EFFECT OF ROS ON THE CELL

36. ROS INDUCTION OF
TRANSCRIPTION FACTORS
NORMAL REGULATION OF MEMBRANE
RECEPTOR BOUND SIGNALLING CASCADES
DEPEND ON LOCALLY GENERATED ROS
THESE OXIDISE OR REDUCE GLUTATHIONE TO
CAUSE POST TRANSLATIONAL MODIFICATION
OF PROTEINS

37. NF KB AND AP 1
THEY 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

38. AP 1
AP1 IS A HETEROGENEOUS GROUP OF DIMERIC
TRANSCRIPTION FACTORS JUN, FOS AND ATF
API IS ALSO AFEECTED BY ROS

39. GENES UNDER THE CONTROL OF
NF KB AND AP1

40. CENTRAL ROLE OF OXIDATIVE STRESS IN
PERIODONTAL PATHOGENESIS

41. METHODS TO MEASURE THE
BIOMARKERS OF OXIDATIVE DAMAGE
FREE RADICALS HAVE EXTREMELY SHORT HALF
LIVES IN VIVO
IN VITRO SYSTEMS CALLED SPIN TRAPS ARE
USED TO MEASURE THESE RADICALS
EX VIVO SPIN TRAPS ARE
ASCORBIC ACID WHICH BECOMES SEMI
DEHYDROASCORBATE
AROMATIC TRAPS SUCH AS SALICYLATES AND
PHENYLALANINE
URATE WHICH IS OXIDISED TO ALLANTOIN

42. BIOMARKERS OF LIPID
PEROXIDATION
CONJUGATED DIENES
THIOBARBITURIC ACID REACTIVE
SUBSTANCES( MALONDIALDEHYDE)
ISOPROSTANES
ETHANE / PENTANE

43. BIOMARKERS OF PROTEIN
DAMAGE
PROTEIN CARBONYL RADICAL ASSAY
AOPP

44. BIOMARKERS TO MEASURE DNA
DAMAGE
8 OH DG MEASUREMENT
COMET ASSAY TO MEASURE STRAND BREAKS

45. METHODS EMPLOYED
MASS SPECTROSCOPY
ELISA
HPLC

46. EVIDENCES TO SHOW ELEVATED
ROS IN PERIODONTAL DISEASE
PANJAMURTHY 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

47. 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

48. THANK YOU.