reacive oxygen species, anti oxidants

2. Introduction
Definitions
Reactive oxygen and antioxidant species in biology and medicine
Atomic and molecular oxygen – basic principles
Origin and formation of ROS and oxygen radicals
Superoxide and nitric oxide
Hydrogen peroxide
The hydroxyl radical
Mechanisms of tissue damage
Protein damage
Lipid peroxidation
DNA damage
Antioxidant defense systems
Classification of antioxidants
Ascorbic acid (vitamin C)
α-Tocopherol (vitamin E)
Carotenoids
Co-enzyme Q10
Uric acid
Polyphenols
Glutathione

3. • Pathophysiology of periodontal destruction caused by ROS
• Measuring ROS and oxidative stress/ damage in biological samples
Biomarkers of lipid peroxidation
Biomarkers of DNA damage
Biomarkers of protein damage
• Evidence for the presence and role of ROS in periodontal tissue damage
• Conclusion

4. Introduction
The majority of periodontal tissue destruction is caused by an
inappropriate host response to those microorganisms and their
products.
More specifically, a loss of homeostatic balance between
• Proteolytic enzymes & their inhibitors
• Reactive oxygen species (ROS) and the antioxidant
defense systems
• pro inflammatory and anti inflammatory cytokines

5. ROS are produced in animals and humans under various physiological and
pathological conditions
They act in phagosomes as agents toxic to micro-organisms, fungi, parasites,
and neoplasmic cells
While most ROS have extremely short life they can cause substantial
damage to tissue and cellular components, e.g., cellular phospholipid,
nucleic acid, protein, carbohydrate, and enzymes

6. Definitions
Free radicals Any species capable of independent
existence that contain one or more
unpaired electrons.
Antioxidants Those substances which when present at
low concentrations, compared to those of
an oxidizable substrate, will significantly
delay or inhibit oxidation of that substrate
by radical. Halliwell B, Gutteridge JM 1992
Halliwell B.,1991

7. Oxidative stress A disturbance in the pro-oxidant–
antioxidant balance in favor of the former,
leading to potential damage., Sies H 1991
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

9. Reactive oxygen and antioxidant species in biology and medicine

11. Different radicals

12. Different reactive oxygen species

13. 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
Functional generation by host defense cells

15. O2 + e- = O2-
Atomic and molecular oxygen – basic principles:
O2- + e- = H2O2
H2O2 + e- = •OH + OH-
•OH + e- = H2O

16. Superoxide radicle formation

17. Superoxide establishes a pro-inflammatory state in a variety of ways, such as
triggering nuclear factor-kB transcription of pro-inflammatory cytokines

18. Nitric oxide formation
Nitric oxide is 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).
Endothelial cell nitric oxide synthase causes smooth muscle relaxation within
blood vessels

19. Macrophage-derived inducible nitric oxide synthase is of interest when
released simultaneously with superoxide it forms the reactive nitrogen
species peroxynitrite anion
NO. + O2- = ONOO-
While peroxynitrite is not a true radical it is now believed to be responsible for
many of the cytotoxic effects

20. Activities include
• Lipid peroxidation;
• Glutathione depletion by oxidation;
• Nitrotyrosine formation which may inhibit superoxide dismutase
activity
• DNA damage by nitrosilation, deamination and oxidation;
• High concentrations cause rapid cellular necrosis
• Low concentrations cause apoptosis

21. Hydrogen peroxide
• Hydrogen peroxide is a weak ROS, the potential of which to cause
tissue damage is limited to its interaction with transition metal ions
undergo “Fenton reaction” in the presence of Fe2+or Cu2+ ions, forming the
most potent of all oxygen radicals, the hydroxyl radical (•OH).
Fe+2 + H2O2 ----------> Fe+3 + °OH + -OH
The dismutation of hydrogen peroxide also gives rise to the ROS called
singlet oxygen ( 'O2 )
Removal of singlet oxygen is achieved by carotenoid pigments, which
will absorb the energy of singlet oxygen and release heat

22. • Increase adhesion molecule expression
• Cause cell proliferation
• Induce apoptosis
• Modulate platelet aggregation.
• Unless concentrations exceed 50μM the cytotoxicity of hydrogen
peroxide is limited and its biological significance is more as a cell
signaling molecule.

23. The principal enzymes charged with removal of hydrogen peroxide are the
antioxidant enzymes catalase, which predominantly acts intracellularly
Glutathione peroxidase, which operates within mitochondria and
extracellularly, thioredoxin-linked peroxidases.
Hydrogen peroxide is also ingested at high concentrations in tea and coffee
and is thought to diffuse into oral mucosal cells
It is also produced by oral bacteria and salivary hydrogen peroxide is used
by the salivary peroxidase system to oxidize thiocyanate into antimicrobial
products.

24. Hydroxyl radical
The hydroxyl (•OH) radical and the related perhydroxyl radical (HO2-) are
the most potent species known to cause damage and destruction to an
array of cellular and tissue components. Specifically, damage may affect
cellular and extracellular targets.
Cellular targets
• lipids
• carbohydrates
• protein damage
• DNA– damage
• oxidation of anti-proteases
• low molecular weight species
Extracellular targets
Extracellular matrix component like collagens and structural proteins

25. Mechanisms of tissue damage
Protein damage
Lipid peroxidation
DNA damage

26. Antioxidant defense systems
Antioxidants can be categorized by several methods:
Based on their mode of function

27. Based on their location of action

28. Based on their solubility

29. Based on their structural dependents

30. Based on their origin/source

31. The preventative antioxidants function by enzymatic removal of superoxide
and hydrogen peroxide or by sequestration of divalent metal ions, preventing
Fenton reactions and subsequent hydroxyl radical formation
Lactoferrin is probably more important than transferrin within the periodontal
tissues, given the dominance of the neutrophil infiltrate and the recognition of
high levels of lactoferrin within gingival crevicular fluid.
The chain-breaking antioxidants are the most important within extracellular
fluids
Low molecular weight species donate electrons before becoming oxidized,
requiring subsequent reduction or replacement to maintain the body’s
antioxidant capacity

32. The lipid soluble antioxidants (a-tocopherol and the carotenoids) act at the
cell membrane level and protect against lipid peroxidation, whereas the
water-soluble scavengers are more important within the extracellular tissue
fluids.
However, several antioxidants have dual and sometimes triple actions.
ascorbateEg:
The efficacy of an antioxidant depends upon:
• its location (intra- vs. extracellular or cell membrane bound);
• the nature of the ROS-challenge;
• other antioxidant species important in co-operative
interactions.
• other environmental conditions (e.g. pH, oxygen tension).

33. Anti-oxidants
• Ascorbic acid (vitamin C)
• α-Tocopherol (vitamin E)
• Carotenoids
• Co-enzyme Q10
• Uric acid
• Polyphenols
• Glutathione

34. General functions of anti oxidants
• Scavenging water-soluble peroxyl radicals;
• Scavenging superoxide and perhydroxyl radicals;
• Prevention of damage mediated by hydroxyl radicals on uric acid;
• Scavenger of hypochlorous acid;
• Decreases heme breakdown and subsequent Fe2+ Release thereby
preventing Fenton reactions;
• Scavenger of singlet oxygen and hydroxyl radicals;
• Re-forms a-tocopherol from its radical;
• Protects against ROS-release from cigarette smoke.

35. Ascorbic acid (vitamin C)
Vitamin C is an essential nutrient and plasma levels are approximately 30–
60μM but reduced in smokers
Gingival crevicular fluid levels are reported to be three-fold higher than
plasma levels
Vitamin C is an essential nutrient with a recommended daily intake of 40–60
mg

36. Ascorbate is converted by radical attack to the ascorbyl radical, which then
breaks down to dehydroascorbate
Dehydroascorbate can be converted back to ascorbate directly by reduced
GSH or by the NAD-semi-dehydroascorbate reductase enzyme system, which
also utilizes GSH.
These systems are intracellular and thus ascorbate within the extracellular
fluids is rapidly depleted (oxidized) in conditions of oxidative stress unless
adequate GSH levels are present

37. α-Tocopherol (vitamin E)
Vitamin E is generally regarded as the most important and effective lipid-
soluble antioxidant in vivo, vital to maintaining cell membrane integrity
against lipid peroxidation
Its antioxidant behavior is the result of a single phenolic OH group, which
when oxidized gives rise to the vitamin E (tocopheryl) radical
Reduced form of co-enzyme Q10 (ubiquinol) in the lipid environment and
ascorbic acid in the aqueous phase will reconstruct vitamin E.

38. Carotenoids
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
Like many other extracellular antioxidants, b carotene levels and intake
are reduced in smokers, whereas others such as lycopene appear
unaffected by smoking
β-carotene is efficient at scavenging singlet oxygen (1O2) and other
carotenoid antioxidant activities include the scavenging of peroxyl radicals

39. Co-enzyme Q10 exists in an oxidized form (ubiquinone or CoQ) and a reduced
form (ubiquinol or CoQH2), both of which possess antioxidant activity
Co-enzyme Q10
co-enzyme Q10 deficiency has been demonstrated in the gingival tissues of
periodontitis subjects
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
Pharmacol1976:14: 729–738
Exact mechanism is not known

40. 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. 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.

41. Glutathione (GSH)
Glutathione is a non-essential tri-peptide. 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

42. Biomarkers
The tissue destruction can be assessed by measuring the levels of markers
for lipid peroxidation, protein damage, DNA damage, and
anti-oxidants
Biomarkers of lipid peroxidation
• Conjugated dienes,
• Thiobarbituric acid reactive substances (notably MDA),
• Isoprostanes,
• Ethane/pentane,
• Other volatile hydrocarbons

43. Thiobarbituric-acid-reactive
substances
Non specific
MDA (melondialdehide) Can be measured directly
or by high-pressure liquid
chromatography
Acrolein more cytotoxic than MDA and may
be a better biomarker
Isoprostanes Best biomarkers of lipid peroxidation

44. Biomarker for protein damage
• The carbonyl assay measures protein carbonyl (PC) groups
• PC has a major advantage over lipid peroxidation product as the marker
of oxidation stress as Oxidized proteins are more stable
• PC forms early and circulates in blood for longer period
• But carbonyls are not specific biomarkers of ROS damage
• Acrolein • Protein-bound aldehyde that has been widely
used to measure oxidative damage

45. Biomarkers of DNA damage
• Products of hydroxyl radical attack on DNA bases (purines) and pyrimidines)
and carbohydrate moieties (deoxyribose)
• can be measured by various methods (high-
pressure liquid chromatography: Gas or liquid),
liquid chromatography or antibody methods
• No individual reaction product should be used as
the sole index of DNA damage
• But despite this, 8-hydroxydeoxyguanosine is
frequently used as a biomarker for DNA damage.

46. Patho-physiology of periodontal tissue destruction

47. Respiratory burst

48. Evidence for the presence and role of ROS in periodontal tissue damage
The majority of tissue destruction in periodontitis is considered to be the result of an
aberrant inflammatory/immune response to microbial plaque adjacent to the
gingival margin and to involve prolonged release of neutrophil enzymes and ROS.
Most published work in the periodontal literature has focused on markers of ROS
reactions with lipids.
Till date, only thiobarbituric acid reactive substances and MDA have been
investigated in chronic periodontitis
All the published studies have suggested that patients with chronic periodontitis
have higher levels of lipid peroxidation than periodontally healthy controls.

49. The GCF concentrations of MDA/4-hydroxyalkanal were 200-to-400-fold
higher than the respective saliva concentrations in patients with chronic
periodontitis, which reflected a substantially higher amount of ROS activity
(thus lipid peroxidation) in GCF than saliva, but total anti-oxidant capacity
was comparable in GCF than saliva
Akalin FA, Baltacioğlu E, Alver A, Karabulut E. Lipid peroxidation levels and total oxidant
status in serum, saliva and gingival crevicular fluid in patients with chronic periodontitis. J
Clin Periodontol 2007;34:558-65

50. Wei et al. measured the lipid peroxidation levels, total oxidant status, and
superoxide dismutase in serum, saliva, and GCF in chronic periodontitis
patients before and after periodontal therapy.
Found that the levels of total oxidative status (TOS) and super oxide
dismutase (SOD) values were significantly higher in the chronic
periodontitis group than in the healthy control group (P < 0.05)
Post-periodontal therapy, serum, saliva, and GCF ,TOS and SOD levels
significantly decreased compared to basal levels (P < 0.05),
Wei D, Zhang XL, Wang YZ, Yang CX, Chen G. Lipid peroxidation levels, total oxidant
status and superoxide dismutase in serum, saliva and gingival crevicular fluid in chronic
periodontitis patients before and after periodontal therapy. Aust Dent J
2010;55:70-8.

51. In a recent study, Diacron-reactive oxygen metabolites (D-ROM),
anti-oxidant potential, C-reactive protein (CRP), interleukin-6,
and lipid profiles were determined with high-sensitivity
assays in serum.
Patients with severe periodontitis exhibited higher D-ROM levels and lower
total anti-oxidant capacity compared with healthy control individual and D-
ROM levels were positively correlated with CRP.
D’Aiuto F, Nibali L, Parkar M, Patel K, Suvan J, Donos N. Oxidative stress, systemic
inflammation, and severe periodontitis.
J Dent Res 2010;89:1241-6

52. The majority of published data on oxidative damage to DNA has been
reported by a Japanese group who investigated 8 hydroxydeoxyguanosine
levels in saliva by enzyme-linked immunosorbent-assay.
These studies demonstrated that levels of 8-hydroxydeoxyguanosine in
samples from subjects with chronic periodontitis were significantly higher
than those from periodontally healthy controls
Takane M, Sugano N, Iwasaki H, Iwano Y, Shimizu N, Ito K. New biomarker evidence of
oxidative DNA damage in whole saliva from clinically healthy and periodontally diseased
individuals.
J Periodontol 2002;73:551-4

53. 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
• 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;

54. • Supplementation with the antioxidant should reduce disease recurrence;
• Subjects with the disease should have a demonstrable local deficiency of
the antioxidant, or of total antioxidant capacity
• Subjects without disease should have no antioxidant deficiency
• Restoration of the antioxidant level locally should improve clinical
measures of disease
• Adjunctive use of the antioxidant with traditional therapies should provide
improved treatment outcomes over non-surgical therapy alone;
• Markers of local ROS activity should decrease with antioxidant therapy.

55. CONCLUSION
Oxidative stress lays at the heart of the periodontal tissue damage that results
from host–microbial interactions, either as a direct result of excess ROS
activity/antioxidant deficiency or indirectly as a result of the activation of
redox-sensitive transcription factors and the creation of a pro-inflammatory
state
It is now evident that significant relations are present between
oxidant status and periodontal status
But it is still not clear whether this event is the cause or a result of
periodontitis

56.  As we know Smoking, infection, UV light, high temperature, etc., play an
important role in generation of free radicals, so one should avoid exposure
to these agents.
 Consumption of nutrients with anti-oxidant ability like Vitamin-C and E, β-
carotene, selenium, and manganese should be encouraged, as they help
in fighting oxidative insults to the periodontal tissue.
 Adjunctive use of anti-oxidants with traditional therapies should be
considered to improve treatment outcome of various surgical and non-
surgical periodontal therapies
So…..

57. References
• The role of reactive oxygen and antioxidant species in periodontal tissue
destruction., Periodontology 2000, Vol. 43, 2007, 160–232
• Reactiveoxygenspeciesand antioxidantsininflammatory diseases.,
JClinfenodontcil1997;24:2S7-296
• Oxidative injury and inflammatory periodontal diseases: the challenge of anti-
oxidants to free radicals and reactive oxygen species, t Rev Oral Biol Med,
10(4):458-476 (1999)
• Butyrate induces reactive oxygen species production and affects cell cycle
progression in human gingival fibroblasts, J Periodont Res 2013; 48: 66–73
• Reactive oxygen species in periodontitis., Journal of Indian Society of
Periodontology- Vol 17, Issue 4, Jul-Aug 2013
• Relationship Between Periodontal Condition and Plasma Reactive Oxygen
Metabolites in Patients in the Maintenance Phase of Periodontal Treatment, J
Periodontol 2008;79:2136-2142.
• Short-Term Effects of Non-Surgical Periodontal Treatment on Plasma Level of
Reactive Oxygen Metabolites in Patients With Chronic Periodontitis., J
Periodontol 2009;80:901-906.