ROS, periodontal diseases

1. REACTIVE OXYGEN SPECIESAND ITS
ROLE IN PERIODONTALDISEASES
Dr.T.Hudson Jonathan

2. Contents
• Intoduction
• Origin of reactive oxygen species and free radicals
• Superoxide and nitric oxide
• Hydrogen peroxide
• Mechanism of tissue damage
• Measuring reactive oxygen species
• Effect of ROS on periodontal tissues and components
• Biomarkers for tissue destruction
• Biomarkers of lipid peroxidation

3. • Biomarkers of DNA damage
• Biomarkers for protein damage
• Antioxidant defense system
• Measuring antioxidant status
• Evidence of tissue destruction by ROS in periodontitis
• Antioxidant status in periodontitis
• Conclusion
• References

4. Introduction

5. • PMNs encounter the bacterial challenge by producing proteolytic
enzymes and O2 by oxidative burst.

6. Origin of reactive oxygen species and free radicals
• It is well known that free radicals and ROS are essential for
many biologic processes. They are produced as superoxide ions
by neutrophils at the site of infection.
• Free radicals have been defined as any species capable of
independent existence that contain one or more unpaired
electrons. [Halliwell 1991] .

7. • They are by nature, highly reactive and diverse species, capable
of extracting electrons and thereby oxidizing a variety of
biomolecules vital to cell and tissue functions, which not only
include oxygen-free radicals, but also nitrogen and chlorine
species.
• ROS is a term that has become more popular because it
encompasses other reactive species which are not true radicals
but are nevertheless capable of radical formation in the
intra-and extra-cellular environments.

8. (Praveen Dahiya et al 2014)
Free radicals and reactive oxygen species (ROS)
Tissue injury
Stimulate the growth of
fibroblasts and epithelial cells
Low concentrations High concentrations

9. Periodontology 2000,vol 43, Chapple & Matthews, 2007

10. Exogenous sources Endogenous sources
1. Heat
2. Trauma
3. ultrasound
4. ultraviolet light
5. ozone
6. smoking
7. exhaust fumes
8. radiation
9. infection
10. excessive exercise and
therapeutic drugs
1. By-products of metabolic
pathways – electron leakage
from mitochondrial electron
transport systems forming
superoxide;
2. Functional generation by host
defence cells (phagocytes) and
cells of the connective tissues
(osteoclasts and fibroblasts).
Sources of free radicals and ROS

11. Superoxide and nitric oxide
• 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).

12. • Endothelial cell nitric oxide synthase causes smooth muscle
relaxation
• Type II nitric oxide + superoxide peroxynitrite anion
(Beckman et al,
1990).

13. • While peroxynitrite is not a true radical it is now believed to be
responsible for many of the cytotoxic effects. (Castro et al,
1994).
• These activities include:
• lipid peroxidation;
• glutathione depletion by oxidation;
• high concentrations cause rapid cellular necrosis
• low concentrations cause apoptosis

14. • Superoxide establishes a pro-inflammatory state in a variety of
ways, such as triggering nuclear factor-κB.
• Other examples include:
• endothelial cell damage
• increased vascular permeability
• neutrophil chemotaxis
• lipid peroxidation
Chapple et al. The role of reactive oxygen and antioxidant species in
periodontal tissue destruction. Perio2000, Volume 43, 2007

15. 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
via ultraviolet light, when it forms the more potent hydroxyl radical.
• Unless concentrations exceed 50 μM the cytotoxicity of hydrogen
peroxide is limited and its biological significance is more as a cell
signaling molecule.
• It also play a role as a second messenger in nuclear factor- κB
activation

16. • Where inflammation is present it may:
• increase adhesion molecule expression
• cause cell proliferation;
• induce apoptosis
• modulate platelet aggregation
• 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, and the
thioredoxin linked peroxidases (Halliwell et al 2000)

17. • Superoxide forms initially and then either spontaneously
dismutates to hydrogen peroxide (a rapid reaction at pH 7.4) or
is actively converted to hydrogen peroxide by one of three
superoxide dismutase enzyme systems.
• Superoxide dismutase (SOD) has been localized within human
periodontal ligament and may represent an important defense
mechanism within gingival fibroblasts against excess
superoxide release.

18. Effect of ROS on periodontal tissues and components
• Gingival cells
• Bone
• Ground substance
• Collagen
• DNA

19. Gingival cells
• Direct damaging effects of ROS on gingival cells have received little
attention.
• 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.

20. Bone resorption
• Although the effects of ROS on bone resorption have not been
studied in periodontal disease it has been shown that 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).

21. • 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)

22. Ground substance degradation
• A study by 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).
• Sulfated glycosaminoglycans were more resistant to ROS degradation
than the non-sulfated glycosaminoglycan hyaluronan.

23. • Chondroitin sulfate proteoglycans from alveolar bone were
particularly susceptible to damage by hydroxyl radicals, which
caused degradation of both the core proteins and
glycosaminoglycan chains.
• By contrast, hydrogen peroxide caused more selective damage
with core proteins being more susceptible than
glycosaminoglycan chains.
• A similar pattern of ROS damage is said to occur with
proteoglycans isolated from gingival soft tissue. (Waddington et
al. 2000)

24. Collagen
• ROS have a variety of 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.

25. • Superoxide anions and hydroxyl radicals are able to cleave
collagen into small peptides at proline and hydroxyproline
residues, liberating hydroxyproline-containing peptides.
(Monboisse et al. 1998)
• Although periodontal disease is associated with increased 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

26. DNAdamage
• There appears to be 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.

27. Measuring reactive oxygen species
• There are currently no gold standard methods for measuring
anti-oxidant capacity or ROS-mediated tissue damage in human
biology.
• Free radicals and other reactive species have extremely short half-lives
in vivo 10-6 -10-9 s and simply cannot be measured directly.
• All systems utilize different indices of measurement and the specificity
of the biomarker employed will dictate the measurement obtained,
which differs between assays and between different biological samples
and their components.

28. • In vitro systems, called spin traps, are used to measure radical species
but there are currently no suitable spin traps/probes available for in vivo
measurement of ROS production in the human.
• So, measurement of ROS is done by measuring the concentration of
biomarkers of tissue destruction.

29. Biomarkers for tissue destruction
• 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.

30. • The tissue destruction can be assessed by measuring the levels of
markers for lipid peroxidation, protein damage, DNA damage,
and anti-oxidants.

31. BIOMARKERS
• Conjugated dienes,
• Thiobarbituric acid
reactive substances
(notably MDA),
• Isoprostanes,
• ethane/pentane, and
• other volatile
hydrocarbons.
• 8-hydroxydeoxyguan
osine
• Protein carbonyl
(PC)
PROTEIN DAMAGE
DNA DAMAGE
LIPID PEROXIDATION

32. Biomarkers of lipid peroxidation
• Thiobarbituric-acid-reactive substances have become obsolete
as a measure of ROS damage[Halliwell et al 2004] because
they lack specificity for ROS activity, being formed by
mechanisms other than lipid peroxidation.
• MDA can be measured directly or by high-pressure liquid
chromatography and is more specific to ROS activity than
thiobarbituric-acid-reactive substances.

33. • Another aldehyde formed by lipid peroxidation is acrolein,
which is more cytotoxic than MDA and may be a better
biomarker.[Uchida 2003]
• Isoprostanes form the following peroxidation of the
polyunsaturated fatty acid side chains of lipids and are currently
regarded as the best biomarkers of lipid peroxidation (e.g.,
F2-isoprostanes).[Roberts et al 2002]
• Ethane and pentane may be useful biomarkers of oxidative
stress, particularly ethane,[Dale et al 2003] but their collection
and measurement are cumbersome and technically challenging.

34. Biomarkers of DNAdamage
• 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.[Dizdaroglu et al 2002]
• No individual reaction product should be used as the sole index
of DNA damage, [Halliwell et al 2004] but despite this,
8-hydroxydeoxyguanosine is frequently used as a biomarker for
DNA damage.

35. Biomarker for protein damage
• The carbonyl assay measures protein carbonyl (PC) groups are
formed as relatively stable end-products of protein oxidation by
ROS.
• PC has a major advantage over lipid peroxidation product as
the marker of oxidation stress. Oxidized proteins are more
stable. PC forms early and circulates in blood for longer period.

36. • But carbonyls are not specific biomarkers of ROS damage
because protein-bound aldehydes and glycated proteins are also
measured.[Halliwell et al 2004]
• Indeed acrolein is a protein-bound aldehyde that has been
widely used to measure oxidative damage. At best, carbonyl
levels provide average measures of protein damage by ROS in
human tissues and fluids.

37. Anti-oxidant defense system
• Anti-oxidants are “those substances which when present in
lower concentration are compared to those of oxidizable
substrate, will significantly delay or inhibit oxidation of that
substrate.”

38. Human body
Endogenous antioxidant Enzymatic oxidant
Vitamin A, C, E,
and B, carotene,
superoxide dismutase, catalase,
and myeloperoxidase
• This antioxidant system functions to detoxify ROS and modify them to form less
reactive species.

40. • The efficacy of an anti-oxidant depends upon:
• Its location (intra-.vs. extracellular or cell membrane bound),
• The nature of the ROS-challenge,
• Other anti-oxidant species important in co-operative interactions and
• Environmental conditions (e.g., pH, oxygen tension).

41. ANTI-OXIDANTS PROTECTIVE MECHANISM AGAINST FREE
RADICALS
I. Prevention of formation of free radicals
II. Interception of free radicals by scavenging the reactive metabolites
and converting them to less reactive molecules
III. Facilitates the repair of damage caused by free radicals
IV. Providing favorable environment for the effective functioning of
other anti-oxidants.

42. Antioxidant status in periodontitis
• There are only few studies that have investigated total anti-oxidant
capacity in serum/plasma from periodontitis patients and controls.
The results of these studies demonstrated significantly lower total
anti-oxidant capacity in serum and plasma samples from periodontitis
subjects.[Chapple et al 2002]
• Panjamurthy et al.2005 found lower plasma vitamin C, vitamin E,
and glutathione (GSH) in periodontitis patients even after adjusting
for protein levels, whereas anti-oxidant enzyme levels were raised,
which might be considered as a protective response to oxidative
stress.

43. Measuring antioxidant status
• Large number of studies from literature have suggested that an
imbalance in the level of free radicals, ROS, and anti-oxidant
play an important role in initiation and progression of
periodontal disease.
• The body’s anti-oxidant systems are highly integrated and
complex and while the study of individual systems and species
greatly improves our understanding of their role in human
diseases, it ignores their co-operative activities and may present
a picture that does not accurately represent the in vivo situation.

44. • They also work in concert through redox cycling reactions,
regenerating each other from their respective radical
species.Free radicals, ROS, and anti-oxidants status can be
measured in serum, saliva, and gingival crevicular fluid (GCF).
• But their levels in saliva and GCF provide a more accurate
status of periodontal disease activity. Total antioxidant capacity
(TAOC) has therefore been developed to reduce the costly and
time-consuming task of measuring individual anti-oxidant
species.

45. Evidence of tissue destruction by ROS in periodontitis
• 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.

46. • All the published studies have suggested that patients with
chronic periodontitis have higher levels of lipid peroxidation
than periodontally healthy controls.
• Thiobarbituric acid reactive substances were raised in patients
both systemically in plasma and erythrocytes and locally in
tissue homogenates[Panjamurthy et al 2005]. MDA was also
found to be raised in GCF and saliva of patients compared to
controls.Similar results were reported by Akalin in 2007

47. • The GCF concentrations of MDA/4-hydroxyalkanal reported by
Tsai et al.[2005] were 200-to-400-fold higher than the
respective saliva concentrations, 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.[Brock et al 2004]
• Wei et al.2010 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.

48. • It is found that the levels of Total oxidative status (TOS) and
Superoxide dismutase (SOD) values were significantly higher in
the chronic periodontitis group than in the healthy control group
(P < 0.05), and levels significantly decreased in
Post-periodontal therapy, but only MDA in GCF.
• In a recent study, Diacron-reactive oxygen metabolites
(D-ROM), anti-oxidant potential,CRP, interleukin-6, and lipid
profiles were determined with high-sensitivity assays in serum.

49. • Severe Periodontitis patients 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 et al 2010]
• 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 that resulted in significantly higher than
those from periodontally healthy controls.[Takane et al 2002]

50. • Moore et al.1994 determined that the predominant anti-oxidant
component of saliva was uric acid (>70% of anti-oxidant
activity).
• Tsai et al.2005 found that salivary GSH concentrations were
significantly reduced in periodontitis subjects relative to controls
and after treatment they found an increase in GSH
concentrations.

51. • But Kim et al 2010 in their research found that the total
anti-oxidant level in the saliva was higher in the patients with
severe chronic periodontitis than the healthy or gingivitis
control before scaling and root planning.
• Whereas SOD activity of the periodontitis patients was lower
than the control at each time-point.

52. • Guarnieri et al.1991 demonstrated spontaneous generation of
superoxide in the GCF of periodontitis subjects, but found no
difference in antioxidant scavenging capacity between cases and
controls.
• Brock et al.2004 demonstrated a significantly lower total
anti-oxidant capacity in periodontitis subjects relative to age and
sex-matched controls.

53. • Pavlica et al 2004. confirmed the above data in their study of
miniature poodle dogs and found significant negative
correlations between serum and GCF total anti-oxidant capacity
and gingival inflammation.
• Linden et al.2009 concluded that low serum levels of a number
of carotenoids, in particular, β-cryptoxanthin and β-carotene,
were associated with an increased prevalence of periodontitis in
this homogenous group of 60-70-year-old Western European
men.

54. • A recent study reported that chronic periodontitis is significantly
associated with lower levels of plasma TAOC and the
non-surgical periodontal therapy seems to reduce the oxidative
stress. However, the adjunctive use of vitamin C still needs
further investigation.[Abou sulaiman et al 2010]

55. Conclusion
• It is now evident that significant relations are present between
oxidant status and periodontal status, and that oxidative stress
may play an important role in the pathology of periodontitis and
the associated tissue damage.
• So the key message includes:
(1) Smoking, infection, UV light, high temperature, etc., play an
important role in generation of free radicals, so one should avoid
exposure to these agents.

56. (2) 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.
(3) Adjunctive use of anti-oxidants with traditional therapies
should be considered to improve treatment outcome of various
surgical and non-surgical periodontal therapies.

57. References
• Carranza’s clinical periodontologyn – 12th edition
• Chapple et al. The role of reactive oxygen and antioxidant
species in periodontal tissue destruction. Perio2000, Volume
43, 2007
• Bhusari et al. Reactive Oxygen Species& Its Role in
Periodontal Disease. J Dent Med Sci, Volume 13, 2014
• Dahiya et al, Reactive oxygen species in periodontitis. J Ind Soc
Periodontol, Volume 17, 2013

58. • Waddington et al. Reactive oxygen species: A potential role in
the pathogenesis of periodontalm diseases. Oral diseases
(2000);6:138-51.
• Liu et al.Systemic Oxidative Stress Biomarkers in Chronic
Periodontitis: A Meta-Analysis. Disease Markers, 2014
• Greabu et al,Hydrogen Sulfide, Oxidative Stress and
Periodontal Diseases: A Concise Review. Antioxidants 2016
• Bullon et al,Metabolic Syndrome and Periodontitis: Is
Oxidative Stress a Common Link?. J Dent Res, 2009

59. Thank you