BRIEF ABOUT REACTIVE OXYGEN SPECIES

1. REACTIVE OXYGEN SPECIES
DR AYANA DINACHANDRAN
1 ST YEAR PG
DEPT PERIODONTICS

2. INTRODUCTION
 Antioxidants are defined as those substances which when present at
low concentrations, compared to those of an oxidizable substrate, will
significantly delay or inhibit oxidation of that substrate (Halliwell B,
Gutteridge JM 1992)
 Free radicals have been defined as any species capable of independent
existence that contain one or more unpaired electrons. (Halliwell B. 1991)
 Oxidative stress was defined by Sies (1991) as a disturbance in the pro-
oxidant–antioxidant balance in favor of the former, leading to potential
damage.
 REACTIVE OXYGEN SPECIES(ROS)-Chemically reactive molecules
containing oxygen Eg: peroxide , superoxide , hydroxyl radical etc.

3. FREE RADICALS

4. – The redox potential is a measure (in volts) of the affinity of a substance for electrons, relative to
hydrogen. (Ower PC, Ciantar M et al 1997)
– Substances more strongly electronegative than hydrogen (i.e. capable of oxidizing hydrogen) have
positive redox potentials and are oxidants.
– Substances less electronegative than (i.e. capable of reducing) hydrogen have negative redox
potentials and are reducing agents.
– Oxidation and reduction reactions always go together and are termed redox reactions.
– Within the gingival crevice/pocket a low redox potential is regarded as essential for the growth and
survival of subgingival anaerobes.

7. ChappleandMatthew’smodificationofBattinoetal’s
review

8. SOURCES OF FREE RADICALS AND ROS
Internal Sources
• Mitochondria
• Xanthine oxidase
• Phagocytes
• Reactions involving
iron and transitional
metals
• Peroxisomes
• Arachidonate
pathways
• Ischaemia/reperfusion
• inflammation
External Sources
• Cigarette smoke
• Environmental
pollutants
• Radiations
• Ultraviolet light
• Ozone
• Industrial solvents
Physiological Factors
• Mental status
• Disease conditions

9. Origins and formation of ROS and oxygen radicals
Exogenous:-
– Heat, trauma, ultrasound, ultraviolet light,
ozone, smoking, exhaust fumes, radiation,
infection, excessive exercise, and
therapeutic drugs
Endogenous:-
– Bi-products of metabolic pathways Oxidative
metabolic transformation
 Mitochondrial respiratory chain
 Oxygen burst (respiratory burst) during
phagocytosis
 Enzymatic reactions (oxygenases, oxidases)
– Functional generation by host defense cells

10. SUPEROXIDE ANION

11. THE HYDROXYL RADICAL
• Most lethal ROS molecule.
• CAN BE RELEASED BY THE HABER WEISS
REACTION
O2.- +H2O2
Feor Cu ions
.OH+ OH-+ O2
• ALTERNATELY IT CAN BE RELEASED BYA
TRANSITION METAL DEPENDANT FENTON
REACTION
H2O2 + Fe2+ Fe3+ + .OH+ OH-

12. NITRIC OXIDE
• Nitric oxide can be synthesized from L- arginine by a family of enzymes called nitric
oxide synthases.
There are three forms:
• Type 1 nitric oxide synthase – brain enzyme (bNOS);
• Type 2 nitric oxide synthase – inducible enzyme
(iNOS), found in macrophages;
• Type 3 nitric oxide synthase – endothelial cell enzyme
(eNOS).

13. PEROXYNITRITE ANION
• Macrophage-derived inducible nitric oxide synthase
when released simultaneously with superoxide it
forms the reactive nitrogen species peroxynitrite
anion (Beckman JS et al 1990).
NO. + O2.- ONOO-
• While peroxynitrite is not a true radical .
• DNA damage:- By Nitrosilation, Deamination And
Oxidation

14. HYDROGEN PEROXIDE
– Released by bacteria and by NADPH oxidase shunt.
– Hydrogen peroxide can oxidise NF κB and can result in pro-
inflammatory cytokine release
– Increase adhesion molecule expression
– Induce apoptosis
– Modulate platelet aggregation

15. HYPOCHLOROUS ACID
Is formed by the actionof myeloperoxidase on
H2O2
Can cause disruption of protein functions
Activates neutrophil collagenase
Oxidises alpha-1 antitrypsin
Cell lysis
SINGLET OXYGEN
– Not a true free radical.
– Can cause lipid peroxidation

16. ROS CAUSE TISSUE
DAMAGE
THEY AFFECT IMPORTANT BIOLOGICAL
MOLECULES
– LIPIDS
– PROTEINS
– DNA

17. EFFECTS OF ROS ON LIPIDS

18. PROTEIN DAMAGE BY ROS

19. DNA DAMAGE BY ROS
Mechanisms of DNA damage by peroxynitrite and
hydroxyl radicals include:
• strand breaks;
• base pair mutations (purine and pyrimidine
bases);
• conversion of guanine to 8-hydroxyguanine Which
is measured as a marker of DNA damage as the
nucleioside 8-hydroxydeoxyguanosine .
• deletion
• iinsertion;
• nicking
• Sequenceamplification.
– Hydroxyl radicals cause damage to all four bases
and create a characteristic DNA fingerprint.

20. M o d i f i c a t i o n o f a a ,
f r a g m e n t a t i o n a n d
a g g r e g a t i o n o f p r o t e i n s
L i p i d p e r o x i d a t i o n D N A d a m a g e
M e m b r a n e d a m a g e
L o s s o f m e m b r a n e
i n t e g r i t y
D a m a g e t o C a 2 +
a n d o t h e r
i o n t r a n s p o r t s y s t e m s
I n a b i l i t y t o m a i n t a i n
n o r m a l i o n g r a d i e n t s
A c t i v a t i o n / d e a c t i v a t i o n o f
v a r i o u s e n z y m e s
A l t e r e d g e n e
e x p r e s s i o n
D e p l e t i o n o f A T P
23
L i p i d s P r o t e i n s D N A
C e l l i n j u r y /
C e l l d e a t h

21. 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 CHRONIC PERIODONTITIS.
– TSAI CC ET AL 2005: MALONDIALDEHYDE LEVELS RAISED
IN GCF AND SALIVA OF PATIENTS WITH CHRONIC
PERIODONTITIS.
– NOUROOZ ZADEH ET AL 1994: ELEVATED HYDROPEROXIDE
LEVELS IN PATIENTS WITH
PAPILLON LE FEVRES SYNDROME MEASURED BY FOX 2 ASSAY

22. – VOLOZHIN AL ET L 2001: EXHALED AIR CONTAINS VOLATILE HYDROCARBONS ,
SHORT CHAIN FATTYACIDS AND ALDEHYDE ELEVATED IN CHRONIC
PERIODONTITIS. PATIENTS.
– DI PAOLAET AL 2005: EXPRESSION AND IMMUNOHISTOCHEMICAL
DETECTION OF NITROTYROSINE IN LIGATURE INDUCED PERIODONTITIS IN
RODENTS.
– SAWAMOTO ET AL 2005: ELEVATED LEVELS OF 8 OH DEOXYGUANOSINE IN
SALIVA OF PERIODONTITIS SUBJECTS

23. 27
ROLE OF ROS IN
PERIODONTAL
TISSUE DAMAGE
HALLIWELL’S POSTULATES
1. ROS or the oxidative damage caused must be
present at site of injury
2. Time course of ROS formation or the oxidative
damage caused should occur before or at the
same time as tissue injury
3. direct application of ROS over a relevant time
course to tissues at concentrations found in
vivo should reproduce damage similar to that
observed in the diseased tissue
4. removing or inhibiting ROS formation should
decrease tissue damage to an extent related to
their antioxidant action in vivo

24. EFFECT OF ROS ON PERIODONTAL
TISSUE AND COMPONENT :
1. Gingival cells
2. Bone
3. Ground substance
4. Collagen
5. DNA

25. GINGIVAL CELLS :
 ROS generated using a neutrophil myeloperoxidase, chloride, glucose, and glucose oxidase
system caused lysis of epithelial targets that could be inhibited by azide and catalase. (Altman
et al. 1992).
 This observation has important implications for disease pathogenesis but evidence that in vivo
levels of ROS production by neutrophils in periodontitis cause such an effect are lacking.

26. Bone resorption:
 Certain ROS (superoxide and hydrogen peroxide) activate osteoclasts (Bax et
al.1992, Hall et al. 1995)
 and promote osteoclast formation (Garrett et al. 1990).
 Osteoclasts produce ROS at the ruffle border/bone interface, suggesting a
more direct role in resorption.(Key et al. 1994).

27.  Such a direct role in bone resorption in periodontitis is supported by the finding that hydroxyl
radicals and hydrogen peroxide can degrade alveolar bone proteoglycans in vitro. (Moseley et al.
1998)
GROUND SUBSTANCE DEGRADATION:
• Moseley and co-workers in 1998 have investigated the effects of ROS on
glycosaminoglycans and proteoglycans present in the soft and calcified
tissues of the periodontium
• All glycosaminoglycans undergo a variable degree of chain depolymerization
and residue modification (especially in the presence of hydroxyl radicals).
• Sulfatedglycosaminoglycans were more resistant to ROS degradation than the
non-sulfated glycosaminoglycan hyaluronan

28.  Chondroitin sulfate proteoglycans from alveolar bone  susceptible to
damage by hydroxyl radicals, caused degradation of both the core
proteins and glycosaminoglycan chains.
 Hydrogen peroxide caused more selective damage with core proteins
being more susceptible than glycosaminoglycan chains.

29.  Evidence suggests that ROS at physiological levels can selectively damage proteoglycans
associated with both the soft periodontal tissues and alveolar bone.
Collagen:
 ROS effects on type I collagen in vitro including direct fragmentation and polymerization
as well as producing oxidative modifications, rendering the molecule more prone to proteolysis.
 The structure of collagen, with its high proline/ hydroxyproline content, is particularly
susceptible to damage by ROS.

30.  Superoxide anions and hydroxyl radicals  cleave collagen into small
peptides at proline and hydroxyproline residues, liberating
hydroxyproline-containing peptides. (Monboisse et al. 1998)
 Periodontal disease is associated with levels of superoxide dismutase-
1 (found in the cytoplasm and nuclei of cells) in gingival extracts, there
are no studies of the extracellular isoenzyme (superoxide dismutase-3)
in periodontitis.

31. DNA DAMAGE
 Only one published report investigating DNA damage in gingival tissues in
periodontal health and disease.(Sugano et al. 2000).
 PCR found deletions within mitochondrial DNA only in samples from
periodontitis patients. Once damaged, oxidative stress within the cell can be
amplified because of decreased expression of proteins critical for electron
transport, leading to cell death.


32. ANTIOXIDANT DEFENSE SYSTEMS
Antioxidants can be categorized by several
methods

33. – BASED ON THEIR SOLUBILITY

34. – BASED ON THEIR STRUCTURALDEPENDENTS
– BASED ON THEIR ORIGIN/SOURCE, E.G. DIETARY OR NON-DIETARY SOURCES

35. ASCORBIC ACID (VITAMIN C)
▪ Vitamin C is an essential nutrient, with a recommended daily intake of 40-
60mg.
▪ It has ability to regenerate α-tocopherol from its radical
▪ GCF levels of ascorbate have been reported to be 3 times higher than those
of plasma (Meyle & Kapitza, 1990) & it has been shown to prevent
activation of neutrophil derived GCF collagenase (Suomalainen et al, 1991).

36. α-TOCOPHEROL (VITAMIN E)
▪ Located within the Cell Membrane Phospholipids, & is a major chain
breaking Antioxidant, preventing Lipid Peroxidation.

37. ▪ According to Brock (2005), Vitamin E:
o Inhibits protein kinase C, & subsequent platelet aggregation ;
o Inhibits nitric oxide production by vascular endothelium ;
o Inhibits superoxide production by Macrophages & PMNs.
 LIMITATIONS:
o Limited mobility within cell membranes;
o Lack of water solubility (many ROS are generated in the aqueous phase)

38. CAROTENOIDS (VITAMIN A)
▪ Carotenoids like β-carotene have long double bonds to attract & quench radical
attack (Krinsky,1989).
▪ Vitamin A is controversial as antioxidant because its behaviour depends upon the
oxygen tension of the immediate environment (Omenn et al, 1996).
▪ Till present, no clear evidence has emerged of antioxidant role of Vitamin A.
▪ Waerhaug (1967), found no such relationship in an epidemiological study in
Srilanka.

39. CO-ENZYME Q10
▪ Exists in oxidized form (ubiquinone or CoQ) and a reduced form (ubiquinol or
CoQH2), both of which possess antioxidant activity.
▪ CoQ is a vital component of mammalian cell mitochondria & performs an imp
function in electron transport system.
▪ It’s a strong antioxidant, and its deficiency has been demonstrated in pts with
periodontitis (Hasen et al, 1976; Littaru et al, 1971), but currently there is lack of
sufficient intervention studies, to substantiate its benefit.

40. GLUTATHIONE & CYSTEINE
▪ Glutathione is a non essential tripeptide, however its constituent amino acids
are essential, and obtained through diet.
▪ Exists in Oxidized (GSSG) & Reduced (GSH) forms.
▪ GSH is an imp antioxidant (radical scavenger), which removes H2O2.
▪ The antioxidant property of glutathione is because of its central Thiol (-SH)
containing cysteine amino acid.

41. REFERENCE :
• Chapple IL, Matthews JB. The role of reactive oxygen and antioxidant species in
periodontal tissue destruction. Periodontol 2000. 2007 Feb 1;43(1):160-232.
• Dr B M Bhasurai , Dr Ridhima Mahajan , Dr Shubhangi Rajbhoj , Pooja shah .
Reactive oxygen species and its role in periodontal disease . IOSR 2014
• Bax BE, Alam AS, Banerji B,Bax CM, Bevis PJ, Stevens CR, Moonga BS,
Blake DR, Zaidi M.Stimulation of osteoclastic bone resorption by
hydrogen peroxide.BiochemBiophys Res Commun. 1992 Mar
31;183(3):1153-8.