Reactive Oxygen Species in periodontal disease destruction by DR SUHANI GOEL

1. SEMINAR
ROLE OF REACTIVE OXYGEN AND ANTI-OXIDANT
SPECIES IN PERIODONTAL TISSUE DESTRUCTION
PRESENTED BY-DR SUHANI
GOEL

2. CONTENTS
– INTRODUCTION
– TERMINOLOGIES
– IS THERE A DIFFERENCE BETWEEN FREE RADICALS AND ROS?
– THE MAJOR ROS MOLECULES
– Origins and formation of ROS and oxygen radicals
– HOW DO ROS CAUSE TISSUE DAMAGE
– ROS INDUCTION OF TRANSCRIPTION FACTORS
– CENTRAL ROLE OF OXIDATIVE STRESS IN PERIODONTAL PATHOGENESIS
– METHODS TO MEASURE THE BIOMARKERS OF OXIDATIVE DAMAGE
– EVIDENCES TO SHOW ELEVATED ROS IN PERIODONTAL DISEASE
– ANTIOXIDANT DEFENSE SYSTEMS
– CONCLUSION
– REFERENCES

3. INTRODUCTION
– Primary etiologic agent of periodontal disease is subgingival microbial plaque biofilm . (Haffajee AD,
Socransky SS. 1994) 1
– The majority of periodontal destruction is caused by an inappropriate host response to micro-organisms
and their products also has an important role to play. (Lamster IB, Novak MJ. 1992). 2
The current status suggests that loss of homeostatic balance:- 3
– Between proteolytic enzymes (e.g. neutrophil elastase) and their inhibitors (e.g. α1-antitrypsin),and
– Reactive oxygen species (ROS) and the antioxidant defense systems
– Pro-inflammatory and anti inflammatory cytokines

4. – .
Antioxidant defense systems help in restoring the
intracellular redox balance and their effects upon
gene transcription and cell signaling.
The larger upward shifts in the pro-oxidant/
antioxidant ratio intracellularly and
extracellularly bring about direct damage to
vital biomolecules and structures of the cell
(Fig. 1).
The basis for such dysregulation is in part genetic (38–82%) (Michalowicz BS et al. 1991) and in part
the result of environmental factors (e.g. smoking) (Palmer RM et al 2005).
Source :-Chapple IL, Matthews JB. The role of reactive oxygen and antioxidant species
in periodontal tissue destruction. Periodontology 2000. 2007 Feb 1;43(1):160-232.

5. TERMINOLOGY
– Free radicals have been defined as any species capable of independent existence that contain one or more
unpaired electrons. (Halliwell B. 1991) 4
– Oxidative stress was defined by Sies (1991) 6 as a disturbance in the pro-oxidant–antioxidant balance in
favor of the former, leading to potential damage.
NOTE:-They are capable of extracting electrons and thereby oxidizing a variety of biomolecules
vital to cell and tissue function
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) 5

6. – 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) 7
– 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

7. Most ROS have extremely short half-lives 10-9- 10-6. (Pryor 1986)
Source :-Chapple IL, Matthews JB. The role of reactive oxygen and antioxidant species in periodontal tissue
destruction. Periodontology 2000. 2007 Feb 1;43(1):160-232.

8. 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) 8

9. 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
– Protein damage to hyaluronic acid and proteoglycans ( Bartold et al, 1984) 9
– Oxidation of important enzymes such as α-1 antitrypsin (proteolytic enzyme inhibitor)
– DNA damage (Varani et al 1990) 10
– Stimulation of pro-inflammatory cytokine release by monocytes and macrophages, by depleting
intracellular thiol compounds and activating nuclear factor κB (Staal et al. 1990) 11

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

11. Origins and formation of ROS and oxygen radicals 3
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

12. SUPEROXIDE ANION 12
– Mitochondria are an important source of metabolism-
derived ROS in vivo.
– Functional production of superoxide involves activation of
the hexose-monophosphate (or NADPH-oxidase) shunt,
– 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
Source :-Chapple IL, Matthews JB. The role of reactive oxygen and
antioxidant species in periodontal tissue destruction. Periodontology
2000. 2007 Feb 1;43(1):160-232.

13. 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
– Superoxide dismutase enzyme (SOD) (McCord & Fridovich 1969)13 , they remove superoxide from tissues.
– When NADPH oxidase shunt fails to work there is a failure of production of O2-. which causes a condition
called chronic granulomatous disease. 14 Curnette JT & Babior BM (1987)
 In this condition the neutrophils engulf the bacteria but cannot opsonise them.

14. Hydrogen peroxide undergoes Fenton reaction.
O2.- + H2O2 .OH + OH- + O2
METAL CATALYSED HABER WEISS REACTION
2H2O2 2H2O + O2
CATALASE
CATALASE ENZYME REMOVES HYDROGEN PEROXIDE.
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. 15 Delgado W & Calderon R. (1979)
Fe or Cu ions

15. THE HYDROXYL RADICAL 3
– 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

16. CONSEQUENCE OF HYDROXYL RADICAL
RELEASE
Cellular targets include:
• Lipids – via lipid peroxidation
• Carbohydrates – depolymerizes
mucopolysaccharides;
• Protein damage – forms (protein
hydroperoxides) (16,17)
• DNA –damage
• Oxidation Of Antiproteases – eg. capable of
inactivating α1-antitrypsin by oxidation of
methionine 18
– Extracellular targets include:
• Extracellular matrix components – in
particular proteoglycan and constituent
glycosaminoglycan chain-degradation ;
• Collagens and structural proteins – proline
sites are prone to hydroxyl radical- mediated
degradation.

17. NITRIC OXIDE 3
– 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).

18. PEROXYNITRITE ANION 3
– Macrophage-derived inducible nitric oxide synthase when
released simultaneously with superoxide it forms the
reactive nitrogen species peroxynitrite anion (Beckman
JS et al 1990). 19
NO. + O2.- ONOO-
– While peroxynitrite is not a true radical .
– DNA damage:- By Nitrosilation, Deamination And
Oxidation
Source :-Chapple IL, Matthews JB. The role of reactive oxygen and antioxidant species
in periodontal tissue destruction. Periodontology 2000. 2007 Feb 1;43(1):160-232.

19. HYDROGEN PEROXIDE
– Released by bacteria and by NADPH oxidase shunt.
Unless concentrations exceed 50μM the cytotoxicity of hydrogen peroxide is limited and its biological
significance is more as a cell signaling molecule.
– Hydrogen peroxide can oxidise NF κB and can result in pro-inflammatory cytokine release
– Increase adhesion molecule expression
– Induce apoptosis
– Modulate platelet aggregation

20. HYPOCHLOROUS ACID 3
 Is formed by the action of myeloperoxidase on H2O2
 Can cause disruption of protein functions
 Activates neutrophil collagenase
 Oxidises alpha-1 antitrypsin
 Cell lysis
SINGLET OXYGEN 3
– Not a true free radical.
– Can cause lipid peroxidation

21. HOW DO ROS CAUSE TISSUE DAMAGE
THEY AFFECT IMPORTANT BIOLOGICAL MOLECULES
– LIPIDS
– PROTEINS
– DNA

22. EFFECTS OF ROS ON LIPIDS
This has been described by Halliwel in 3 stages. 20
INITIATION:- hydroxyl or peroxynitrite radical attacks a
PUFA in the lipid membrane.
PROPAGATION:- combine with oxygen to form a lipid peroxyl
radical which ( LOO.) which may attack a PUFA.
TERMINATION:- is brought about by lipid soluble scavenger
vitamin E.
THE PROCESS IS CALLED LIPID PEROXIDATION Source :-Chapple IL, Matthews JB. The role of reactive oxygen and antioxidant species
in periodontal tissue destruction. Periodontology 2000. 2007 Feb 1;43(1):160-232.

23. PROTEIN DAMAGE BY ROS
The effects of ROS on proteins are summarized simply in Fig.
7 (adapted from Dean et al.) 21 and include:
• protein folding or unfolding (which may or may not be
reversible);
• protein fragmentation and polymerization reactions;
• protease degradation of the modified protein;
• formation of protein radicals;
• formation of protein-bound ROS;
• formation of stable end products e.g. carbonyl-compounds
such as oxo-acids or aldehydes (e.g. alanine to acetaldehyde).
22
Source :-Chapple IL, Matthews JB. The role of reactive oxygen and antioxidant species
in periodontal tissue destruction. Periodontology 2000. 2007 Feb 1;43(1):160-232.

24. DNA DAMAGE BY ROS 3
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 , 23
Which is measured as a marker of DNA damage as :-
• deletions;
• insertions;
• nicking;
• sequence amplification.
– Hydroxyl radicals cause damage to all four bases and create a characteristic DNA
fingerprint.

25. EFFECT OF ROS ON THE CELL

26. 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 .

27. NF κB AND AP 1 3
– 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 serine residues in IKB. 24
– This frees the NF KB that translocates to the nucleus and switches on IL 1 , TNF-α genes

28. Activating protein-1 (AP 1) 3
– AP1 IS A HETEROGENEOUS GROUP OF DIMERIC TRANSCRIPTION FACTORS JUN, FOS AND
ATF
AP-1 IS ALSO AFEECTED BY ROS
– Activating protein-1 activity is regulated via an increase in transcription of Jun and Fos genes as well
as by post-translational phosphorylation of Jun and Fos proteins. 25
– By mitogen activated protein kinases, protein kinase A, and protein kinase C.
– Phosphorylation alters the DNA binding capacity of the proteins and modulates their transcriptional
activity .

29. GENES UNDER THE CONTROL OF NF KB
AND AP1 3

30. CENTRAL ROLE OF OXIDATIVE STRESS IN
PERIODONTAL PATHOGENESIS

31. EVIDENCES TO SHOW ELEVATED ROS IN PERIODONTAL
DISEASE
– PANJAMURTHY ET AL 2005: 27 ELEVATED LEVELS OF THIOBARBITURIC ACID REACTIVE
SUBSTANCES IN PLASMA AND RBC OF PATIENTS WITH CHRONIC PERIODONTITIS.
– TSAI CC ET AL 2005: 28 MALONDIALDEHYDE LEVELS RAISED IN GCF AND SALIVA OF
PATIENTS WITH CHRONIC PERIODONTITIS.
– NOUROOZ ZADEH ET AL 1994: 29 ELEVATED HYDROPEROXIDE LEVELS IN PATIENTS WITH
PAPILLON LE FEVRES SYNDROME MEASURED BY FOX 2 ASSAY

32. – VOLOZHIN AL ET L 2001: 30 EXHALED AIR CONTAINS VOLATILE HYDROCARBONS , SHORT
CHAIN FATTY ACIDS AND ALDEHYDE ELEVATED IN CHRONIC PERIODONTITIS. PATIENTS.
– DI PAOLA ET AL 2005: 31 EXPRESSION AND IMMUNOHISTOCHEMICAL DETECTION OF
NITROTYROSINE IN LIGATURE INDUCED PERIODONTITIS IN RODENTS.
– SAWAMOTO ET AL 2005: 32 ELEVATED LEVELS OF 8 OH DEOXYGUANOSINE IN SALIVA OF
PERIODONTITIS SUBJECTS

33. ANTIOXIDANT DEFENSE SYSTEMS 3
Various ANTI-OXIDANT
classification systems exit.
Antioxidants can be categorized
by several methods:
MODE OF FUNCTION
LOCATION OF ACTION

34. – BASED ON THEIR SOLUBILITY

35. – BASED ON THEIR STRUCTURAL DEPENDENTS
– BASED ON THEIR ORIGIN/SOURCE, E.G. DIETARY OR NON-DIETARY SOURCES

36. BALANCE BETWEEN ROS AND ANTIOXIDANTS

37. Ascorbic acid (vitamin C) 3
•Its activities may be summarized as:
• Scavenging water-soluble peroxyl radicals;
• Scavenging superoxide and perhydroxyl radicals;
• Prevention of damage mediated by hydroxyl radicals on uric acid;
• Decreases heme breakdown and subsequent Fe2+ Release thereby preventing Fenton reactions;
• Scavenger of singlet oxygen and hydroxyl radicals;
• Re-forms α-tocopherol from its radical;
• Protects against ROS-release from cigarette smoke.
Vitamin C is an essential nutrient and plasma levels are approximately 30– 60μM but reduced in smokers.

38. α- Tocopherol (vitamin E) 3
Vitamin E possesses anti-inflammatory as well as antioxidant properties and these were reviewed by Brock
(2005) :
• Inhibition of protein kinase C and subsequent platelet aggregation;
• Inhibition of nitric oxide production by vascular endothelium;
• Inhibition of superoxide production by macrophages and neutrophils33, by inhibition of p47phox
phosphorylation during NADPH oxidase activation .
– Reduced form of co-enzyme Q10 (ubiquinol) in the lipid environment and ascorbic acid in the aqueous
phase will reconstruct vitamin E.

39. CAROTENOIDS 3
– Carotenoids are tetraterpines with over 600 variants., some are.
• lycopene;
• a-carotene;
• b-carotene;
• lutein;
• cryptoxanthine;
• retinol (vitamin A1);
• dehydroretinol
(Vitamin A2) Derived only from the diet (green vegetables, tomatoes, fruits), lycopene predominates in
plasma.
ß-carotene levels and intake are reduced in smokers, whereas others such as lycopene appear unaffected by
smoking.

40. Co-enzyme Q10 3
– Exists in an oxidized form (ubiquinone or CoQ) and a reduced form (ubiquinol or CoQH2), both of which
possess antioxidant activity.
– Hansen IL, Iwamoto Y, Kishi T, Folkers K, Thompson LE. 1976 34
Co-enzyme Q10 co-enzyme Q10 deficiency has been demonstrated in the gingival tissues of periodontitis
subjects.
– Exact mechanism is not known

41. URIC ACID
Normally uric acid is oxidized to allantoin enzymatically or by hydroxyl radicals but the enzymatic route
does not occur in humans,
Therefore allantoin formation is used as a marker of urate oxidation by ROS (measured as
allantoin:urate ratio).
POLYPHENOLS
Battino et al. 35 proposed that the polyphenolic flavenoids are absorbed following dietary intake of, in
particular, vegetables, red wine, and tea are helpful in inflammatory diseases.
No current data regarding exact mechanism.

42. Glutathione (GSH) 3
– It can be synthesized within the cell; however, its constituent
amino acids are “essential” and obtained through the diet.
– It is essential to the glutathione peroxidase antioxidant enzyme
system, which removes hydrogen peroxide by converting two GSH
molecules to one GSSG molecule and water .
– Is one of the most vital intracellular antioxidant scavengers .
– It is important to the preservation and restoration of other
antioxidant species, e.g. vitamin C and vitamin E.

43. CONCLUSION
– Factors to account for when contemplating antioxidant approaches to therapy
– • There should be evidence of excess ROS production associated with the presence of disease
(ideally locally to the diseased tissues)
• There should be evidence of ROS-mediated tissue damage either by:
(a) Direct effects of ROS activity (biomarkers) measured locally;
(b) Indirect effects of ROS activity via hyperinflammation as a result of local redoxsensitive
transcription factor activity and subsequent imbalances of pro- and antiinflammatory cytokine
behavior
(c) There should be clear mechanistic links between oxidative stress, the observed tissue damage
and the mode of activity of the candidate antioxidant;
• Supplementation with the antioxidant should reduce the incidence of disease at the affected site or
tissues;

44. REFERENCES
1.) Haffajee AD, Socransky SS. Microbial aetiological agents of destructive periodontal diseases. Periodontol 2000 1994: 5: 78–111.
2.) Lamster IB, Mmandella RD, Zove SM, Harper DS. The polyamines putrescine, spermidine, and spermine in human gingival crevicular fluid. Arch Oral Biol 1987:
32: 329– 333.
3.) 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.
4.) Halliwell B. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med 1991: 91 (Suppl. 3C): 14S–22S.
5.) Halliwell B, Gutteridge JMC. Oxygen free radicals and iron in relation to biology and medicine. Arch Biochem Biophys 1986: 246: 501–514.
6.) Sies H, Murphy ME. Role of tocopherols in the protection of biological systems against oxidative damage. J Photochem Photobiol B Biol 1991: 8: 211–224.
7.) Ower PC, Ciantar M, Newman HN, Wilson M, Bulman JS. The effects on chronic periodontitis of a subgingivally placed redox agent in a slow release device. J
Clin Periodontol 1995: 22: 494–500.
8.) Halliwell B, Gutteridge JMC. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 1990: 186: 1–85.
9.) Bartold PM, Thonard JC. The effect of oxygen‐derived free radicals on gingival proteoglycans and hyaluronic acid. J Periodontol Res 1984 Jul 1;19(4):390-400.
10.) Varani J, Fligiel SE, Till GO, Kunkel RG, Ryan US, Ward PA. Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical.
Laboratory investigation; a journal of technical methods and pathology. 1985 Dec;53(6):656-63.
11.) Staal FJ, Roederer M, Herzenberg LA. Intracellular thiols regulate activation of nuclear factor kappa B and transcription of human immunodeficiency virus.
Proceedings of the National Academy of Sciences 1990 1;87(24):9943-7.
12.) Fridovich I. Superoxide dismutases. An adaptation to a paramagnetic gas. J Biol Chem. 1989 May 15;264(14):7761-4.
13.) McCord JM, Fridovich I. Superoxide dismutase an enzymic function for erythrocuprein (hemocuprein). Journal of Biological chemistry 1969 25;244(22):6049-
55.
14.) Curnette, J. T & Babior, B. M. (1987) Chronic granulomatous disease. Advances in Human Genetics 16, 229-245.

45. 15.) Delgado, W & Calderon, R. Acatalasia in 2 Peruvian siblings. J Oral Pathol 1979; 358-368.
16.) Fu S, Hick LA, Sheil MM, Dean RT. Structural identification of valine hydroperoxides and hydroxides on radical damaged amino acid, peptide,
and protein molecules. Free Radic Biol Med 1995: 19: 281–292.
17.) Kopoldova J, Liebster J, Babicky A. The mechanism of the radiation chemical degradation of amino acids. V. Radiolysis of norleucine, leucine
and isoleucine in aqueous solution. Int J Appl Radiat Isot 1963: 14: 455–460.
18.) Mohsenin V, Gee JL. Oxidation of alpha 1-protease inhibitor: role of lipid peroxidation products. J Appl Physiol 1989: 66: 2211–2215.
19.) Beckman JS, Beckman JW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for
endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A 1990: 87: 1620–1624.
20.) Halliwell B. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med 1991: 91 (Suppl. 3C): 14S–
22S.
21.) Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 1997: 324: 1–18.
22.) Dakin HD. Comparative studies of the mode of oxidation of phenyl derivatives of fatty acids by the animal organism and by hydrogen
peroxide. J Biol Chem 1908: 4: 419–453.
23.) Box HC, Freund HG, Budzinski E, Wallace JC, Maccubbin AE. Free radical-induced double base lesions. Radiat Res 1995: 141: 91–94.
24.) Fratelli M, Goodwin LO, Orom UA, Lombardi S, Tonelli R, Mengozzi M, Ghezzi P. Gene expression profiling reveals a signalling role of
glutathione in redox regulation. Proc Natl Acad Sci U S A 2005: 102: 13998–14003.
25.) Yates S, Rayner TE. Transcription factor activation in response to cutaneous injury: role of AP-1 in re-epithelialisation. Wound Repair Regen
2002: 10: 5–15.
26.) Duthie SJ, Ma A, Ross MA, Collins AR. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res
1996: 56: 1291–1295.
27.) Panjamurthy K, Manoharan S, Ramachandran CR. Lipid peroxidation and antioxidant status in patients with periodontitis. Cell Mol Biol Lett
2005: 10: 255–264.

46. 28.) Tsai CC, Chen HS, Chen SL, Ho YP, Ho KY, Wu YM, Hung CC. Lipid peroxidation: a possible role in the induction
and progression of chronic periodontitis. J Periodontal Res 2005: 40: 378–384.
29.) Nourooz-Zadeh J, Tajaddini-Samadi J, Wolff SP. Measurement of plasma hydroperoxide concentrations by the
ferrous oxidation-xylenol orange assay in conjunction with triphenylphosphine. Anal Biochem 1994: 220: 403–409.
30.) Volozhin AI, Petrovich IuA, Filatova ES, Barer GM, Fomina OL, Volozhina SA, Dieva SV. Volatile compounds in air
and saliva in healthy people and in periodontitis and gingivitis patients. Stomatologiia (Mosk) 2001: 80: 9–12 (in
Russian).
31.) Di Paola R, Mazzon E, Zito D, Maiere D, Britti D, Genovese T, Cuzzocrea S. Effects of Tempol, a membrane-
permeable radical scavenger, in a rodent model periodontitis. J Clin Periodontol 2005: 32: 1062–1068.
32.) Sawamoto Y, Sugano N, Tanaka H, Ito K. Detection of periodontopathic bacteria and an oxidative stress marker
in saliva from periodontitis patients. Oral Microbiol Immunol 2005: 20: 216–220.
33.) Azzi A, Ricciarelli R, Zingg JM. Non-antioxidant molecular functions of a-tocopherol (vitamin E). FEBS Lett 2002:
519:8–10.
34.) Hansen IL, Iwamoto Y, Kishi T, Folkers K, Thompson LE. Bioenergetics in clinical medicine. IX. Gingival and
leucocytic deficiencies of coenzyme Q10 in patients with periodontal disease. Res Commun Chem Pathol
Pharmacol 1976: 14: 729–738.
35.) Battino M, Bullon P, Wilson M, Newman H. Oxidative injury and inflammatory periodontal diseases: the
challenge of anti-oxidants to free radicals and reactive oxygen species. Crit Rev Oral Biol Med 1999: 10: 458–476.