2. Contents
1.Introduction
2.Reactive Oxygen Species
3.Free Radicals
4.Sources of free radicals and ROS
5.Oxidative stress
6.Mechanisms of tissue injury
7.Measurements of ROS and oxidative stress or damage in biological samples
8.Role of ROS in periodontal tissue damage
9.Local presence of ROS in periodontal disease
10.Antioxidant defense system
11.Conclusion
2
3. INTRODUCTION
3
• Periodontal Diseases :
Primary etiological agent is specific, predominantly gram negative anaerobic or
facultative bacteria within the subgingival biofilm
Majority of periodontal tissue destruction ------ An inappropriate host response to
those microorganisms and their products
4. Reactive Oxygen Species? – Ke Cui et al 2004
4
ROS : is a collective term that includes all reactive forms of oxygen, including
both radical and non radical species that participate in the initiation and/or
propagation of chain reaction.
Free radicals represent a class of highly reactive intermediate chemical entities
whose reactivity is derived from the presence of unpaired electron in their
structure, which are capable of independent existence for very brief interval of
time
7. Some characteristics of ROS
7
ROS Symbol Properties
Superoxide radical O2
•- poor oxidant
Hydroperoxyl
radical
HO2
• stronger oxidant than O2
•-
Hydrogen
peroxide
H2O2 oxidant, diffuses across membranes
Hydroxyl radical OH• extremely reactive, diffuses only to very low distance
Alkoxyl radical LO• less reactive than OH•, but more than peroxyl
Peroxyl radical LOO• weak oxidant, high diffusability
Singlet oxygen 1O2 powerful oxidizing agent
Chapple and Matthew’s modification of Battino et al’s review
10. Oxidative Stress? – Sies H, 1991
▪ As a disturbance in the pro-oxidant and antioxidant balance in favour of the
former, leading to potential damage.
10
Antioxidants
• Those substances when present at low concentrations, compared to those of an
oxidizable substrate, will significantly delay or inhibit oxidation of that
substrate.
12. SUPEROXIDE ANION (O2
.-)
▪ Formed chemically by addition of an extra electron to O2 molecule
O2 + e- O2
.-
▪ This reaction can occur due to two reasons:
1) Electron leak from their carriers within the respiratory chain of
mitochondria.
2) Functional Source, when activated phagocytes (PMNLs,
macrophages) produce superoxide as an antibacterial agent.
13.
14. ▪ This Superoxide can spontaneously dismutase in aqueous solution to
form Hydrogen Peroxide & Singlet Oxygen.
O2
.- + O2
.- + 2H+ 1O2 + H2O2
▪ Superoxide can also be converted to the more potent Hydroxyl Radical
(OH.), by reaction with Hydrogen Peroxide, catalyzed by metal ions
(Haber-Weiss reaction)
O2
.- + H2O2 OH. + OH- + O2
Fe or Cu ions
15. ▪ So how does body deals with this Superoxide Free Radical….?
1) This Superoxide is removed from the tissues by spontaneous
dismutation reaction to form Hydrogen Peroxide
2) By enzyme Superoxide Dismutase (SOD) which catalyses conversion
of Superoxide to Hydrogen Peroxide
2O2 + 2H+ O2 + H2O2
SOD
16. ▪ All this Hydrogen Peroxide formed is removed by another enzyme called
Catalase.
2H2O2 2H2O + O2
▪ Both SOD & Catalase are largely found intracellularly, but are present in very
small amounts in the extracellular fluid also.
▪ The role of Catalase in the extracellular fluid is performed by Glutathione
Peroxidase (GSH-Px), which reduces Hydrogen Peroxide.
2GSH + H2O2 GSSG + 2H2O
Catalase
GSH-Px
17. HYDROXYL RADICAL (OH.)
▪ Most reactive radical
▪ Formation occurs by
1) Haber-Weiss reaction
O2
.- + H2O2 OH. + OH- + O2
2) Fenton reaction
H2O2 + Fe2+ (or Cu2+) OH- + OH. + Fe3+ (or Cu3+)
▪ Thus proteins that sequester iron or copper (albumin, cerruloplasmin, haptoglobin,
lactoferrin, transferrin) are extremely imp Antioxidants.
Fe or Cu ions
18. HYDROGEN PEROXIDE (H2O2)
▪ Can be produced directly by the Bacteria
▪ And by Phagocytes from the NADPH Oxidase Shunt by dismutation of
Superoxide spontaneously or catalysed by SOD.
▪ It is weak oxidant, however can lead to the development of highly toxic
Hydroxyl Radical.
▪ Removed from cells by the action of antioxidants like Catalase &
selenium dependant Glutathione Peroxidase.
19. SINGLET OXYGEN (1O2)
▪ Not a true radical as it does not contain an unpaired electron
▪ Formed by an input of energy to molecular oxygen, which reverses the spin
direction of one of the outermost unpaired electron away from a parallel spin,
causing instability.
▪ Capable of initiating lipid peroxidation from side chains of PUFA.
20. NITROUS OXIDE (NO)
▪ Can be produced by Macrophages as well as Vascular Endothelium
▪ It is synthesized from L-arginine by a family of enzymes called Nitric
Oxide Synthetases (NOS), which are in three forms.
1) Type I NOS – brain enzyme (bNOS).
2) Type II NOS – inducible enzyme found in macrophages (iNOS).
3) Type III NOS – endothelial cell enzyme (eNOS)
21. ▪ Endothelial cell nitric oxide causes Vasodilatation.
▪ Macrophage derived nitric oxide when released simultaneously with
Superoxide, forms new reactive nitrogen species, Peroxynitrite Anion
(ONOO-).
NO. + O2
- ONOO-
▪ While not a true radical Peroxynitrite is now believed to be
responsible for many cytotoxic effects, previously attributed to nitric
oxide & superoxide.
26. DNA
Damage ▪ Strand breaks
▪ Base pair mutations
▪ Conversion of guanine to 8-hydroxyguanine
which is measured as a marker of DNA
damage as the nucleoside 8-hydroxyl deoxy
guanosine
▪ deletions
▪ insertions
▪ Nicking
▪ Sequence amplification
26
27. 27
Modification of aa,
fragmentation and
aggregation of proteins
Lipid peroxidation DNA damage
Membrane damage
Loss of membrane
integrity
Damage to Ca2+
and other
ion transport systems
Inability to maintain
normal ion gradients
Activation/deactivation of
various enzymes
Altered gene
expression
Depletion of ATP
Lipids Proteins DNA
Cell injury/
Cell death
29. ROLE OF ROS IN
PERIODONTAL
TISSUE DAMAGE
29
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
30. 30
Title Author’s
Name
Journal
and year
Method
ology
Author’s conclusion
How
neutrophils
kill
microbes?
Anthony
W. Segal
Annual
Review
of
Immuno
logy,
Vol 23,
2005
Review The role of ROS is to
establish an environment in
the phagocytic vacuole
suitable for killing and
digestion by enzymes
released into the vacuole
from cytoplasmic granules
31. Local presence of ROS in periodontal disease
31
Title Author’s Name Jornal and
year
Methodology Author’s conclusion
Lipid
peroxidation: a
possible role in
the
induction and
progression
of chronic
periodontitis
Tsai et al Journal of
periodontal
research
2005
Cross sectional
study
Gingival crevicular
fluid and saliva
were collected from
13 patients and
9 healthy control
subjects during the
preliminary study,
and from 21 patients
during the
subsequent study.
Lipid peroxidation
level, GSH level and
GPx activity
were determined by
spectrophotometric
assay.
The increased levels of lipid
peroxidation may play a role in
the inflammation and
destruction of the periodontium
in periodontitis.
32. 32
Title Author’s Name Jornal and year Methodology Author’s conclusion
Locally delivered
antioxidant gel as an
adjunct to
nonsurgical therapy
improves measures
of oxidative stress
and periodontal
disease
Chandra et al Journal of
periodontal
and implant
science,
2013
Thirty-one subjects
participated in this study.
In the pretreatment phase,
the ROS levels in pockets
≥ 5 mm were measured
by flow cytometry. Three
sites in each subject were
randomly assigned into
each of the following
experimental groups:
sham group, only scaling
and root planing (SRP)
was done; placebo group,
local delivery of placebo
gel after SRP; and
lycopene group, local
delivery of lycopene gel
after SRP. Clinical
parameters included
recording site-specific
measures of GCF 8-
OHdG, plaque, gingivitis,
probing depth, and
clinical attachment level
From this trial conducted over a period
of 6 months, it was found that locally
delivered lycopene seems to be effective
in reducing the measures of oxidative
stress and periodontal disease.
33. 33
Title Author’s Name Jornal and
year
Methodology Author’s conclusion
Parameters of
oxidative stress in
saliva from
patients with
aggressive and
chronic
periodontitis
Acquier et al Redox Rep,
2016
Eighty subjects were
divided into two
groups: 20 patients
with AgP and 20
patients with CP with
their 20 corresponding
matched controls,
based on clinical
attachment loss
(CAL), probing pocket
depth
(PPD), and bleeding
on probing (BOP).
Saliva reactive oxygen
species (ROS), lipid
peroxidation were
measured by luminol-
dependent
chemiluminescence
assay respectively.
ROS was high in AgP in
comparison to CGP. In AgP, a
strong and positive correlation
was observed between ROS and
CAL and PPD
34. 34
Title Author’s Name Jornal and
year
Methodology Author’s conclusion
Chronic
Periodontitis in
Type 2 Diabetes
Mellitus: Oxidative
Stress as a
Common Factor in
Periodontal Tissue
Injury
Patil et al Journal of
clinical and
diagnostic
research, 2016
The study comprised of
total 100 subjects among
which 25 were normal
healthy controls, 25
were gingivitis patients,
25 were chronic
periodontitis patients
(CP) and 25 were having
chronic periodontitis
with type 2 diabetes (CP
with DM). ROS levels
were determined as
MDA
(Malondialdehyde) and
antioxidant status as
plasma total antioxidant
capacity (TAC), vitamin
C and erythrocyte
Superoxide dismutase
(SOD) and catalase
activity.
There is increased oxidative stress in
chronic periodontitis with and
without type 2 diabetes indicating a
common factor involvement in tissue
damage. More severe tissue
destruction in periodontitis is
associated with excessive ROS
generation which is positively
correlated in type 2 diabetic subjects.
35. ANTIOXIDANT DEFENSE SYSTEM
▪ Complex and various classification systems exists.
▪ Can be categorized by several methods
1) Their mode of function
36. 2) According to location of action (intracellular, extracellular, or cell
membrane)
40. 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).
41. ▪ It is a powerful scavenger of Hypochlorous Acid, Superoxide, Singlet
Oxygen & Hydroxyl Radical & protects against oxidants in cigarette smoke
(Haliwell, 1990).
▪ Other relevant effects recently reported include the ability to reduce C-
reactive-protein-mediated expression of monocyte adhesion molecules
(Rayment et al, 2003) and the ability to decrease pro-inflammatory gene
expression via effects on the nuclear factor-kB transcription factor (Grifiths
et al, 2001).
42. α-TOCOPHEROL (VITAMIN E)
▪ Located within the Cell Membrane Phospholipids, & is a major chain
breaking Antioxidant, preventing Lipid Peroxidation.
43. ▪ 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)
44. ▪ Accelerated gingival wound healing was demonstrated in rat model with vitamin E
supplementation (Kim & Shklar, 1983).
▪ Goodson & Bowles (1973) demonstrated that patients with periodontitis rinsing
mouths with vitamin E daily for 21 days experienced a significant decrease in GCF
flow compared with control group.
▪ Asman et al. (1994) demonstrated in a rat model that the combination of vitamin E
and selenium was protective against ROS-induced collagen degradation.
45. 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.
46. ▪ It has been suggested that vitamin A in toothpaste would be useful in
treating periodontitis, since fewer deep pockets and reduced gingival
bleeding were found following its use (Trykowsky et al., 1994).
47. 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.
48. 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.
49. ▪ Also imp to the immune system, regulates IL-2 dependant T-lymphocyte
proliferation (Suthanthiran et al, 1990).
▪ It is also important in the preservation & restoration of other antioxidant
species ,e.g. Vitamin C & Vitamin E.
▪ Increasing cystolic cysteine (& thus GSH), concentrations of monocyte &
macrophages blocks ROS mediated activation of NF-kB, & thus pro-
inflammatory cytokine production (Schreck et al. 1991).
50. ▪ T.denticola, B.intermedius, & P.gingivalis are capable of metabolizing
cysteine to form Hydrogen Sulphide (H2S) (Persson et al, 1990).
▪ Also P. micros, & F. nucleatum can readily degrade GSH to form H2S
(Carlsson et al, 1993, 1994).
51. ▪ Intracellular levels are high (1-10mM), & extracellular are low (1-4uM).
▪ It is imp to note, however that GCF levels of glutathione are high, in
millimolar (Chappel et al, 2002), which has lead to the hypothesis that it acts
as an innate & fundamental defense strategy at exposed epithelial surfaces.
▪ Also its levels are increased in Smokers as compared to Non-smokers
(Rahman et al, 1999). This may as a protective mechanism, as cigarette
smoke contains many oxidants.
53. References
▪ Chapple I, Matthews J. The role of reactive oxygen and antioxidant species in
periodontal tissue destruction. Periodontology 2000, Vol 43, 2007, 160-232
▪ Chapple ILC. Role of free radicals and antioxidants in the pathogenesis of the
inflammatory periodontal diseases. J Clin Pathol Mol Pathol 1996: 49: M247-M255.
▪ Chapple ILC. Reactive oxygen species and antioxidants in inflammatory diseases. J
Clin Periodontol 1997: 24: 287–296.
▪ Segal A, How neutrophils kill microbes? Annu Rev Immunol 2005: 23
▪ Tsai et al. Lipid peroxidation: a possible role in the induction and progression of
chronic periontitis. J Periodont Res. 2005
▪ Chandra et al. Locally delivered antioxidant gel as an adjunct to nonsurgical therapy
improves measures of oxidative stress and periodontal disease. J Periodontol
Implant Sci, 2013
53
Editor's Notes
More specifically, a loss of homeostatic balance between proteolytic enzymes and their inhibitors and reactive oxygen species (ROS) and the antioxidant defense systems that protect and repair vital tissue, cell, and molecular components is believed to be responsible.
The basis for such dysregulation is in part genetic (38–82%) and in part the result of environmental factors (e.g. smoking)
It is ironic that oxygen, which is an indispensable element for life can, under certain situations, have severe deleterious effects on the human body. Most of the potentially harmful effects of the oxygen are due to the formation and activity of number of chemical compounds, known as reactive oxygen species (ROS), which have tendency to donate oxygen to other substances.
so ROS encompasses other reactive species which are not true radicals but are nevertheless capable of radical formation in intra and extra cellular environment
Any species capable of independent existence that contain one or more unpaired electrons
homolytic cleavage of the covalent bond of a normal molecule, with each fragment retaining one of the paired electrons (i.e., homolytic fission), requiring a high energy input
loss of a single electron from a normal molecule
"electron transfer “ --- gain of electron
The addition of one electron to oxygen results in the formation of the superoxide anion
The addition of a second electron results in the formation of the ROS hydrogen peroxide (H2O2)
The addition of a third electron results in the formation of the hydroxyl radical (•OH)
The addition of a fourth electron results in the formation of water (H2O)
The loss of homeostatic balance between ROS and their antioxidant defense systems that protect and repair vital tissue, cell, and molecular components is believed to be responsible for Periodontal tissue destruction.
Functional production of superoxide involves activation of the hexose-monophosphate (or NADPH-oxidase) shunt, which shunts glucose-6- phosphate from the glycolysis pathway and utilizes molecular oxygen and NADPH to form the superoxide radical anion (O2 ). This process comprises the so-called respiratory burst within neutrophilic polymorphonuclear leukocytes (neutrophils) and is stimulated by a variety of mitogens/ antigens/cytokines and other mediators such as granulocyte–macrophage colony-stimulating factor
Superoxide dismutase (SOD) has been localized within human periodontal ligament and may represent an important defense mechanism within gingival fibroblasts against excess superoxide release.
superoxide dismutase 1 – a Cu2+/Zn2+-dependent enzyme found within the cytosol
Superoxide dismutase 2 , which is manganese-dependent enz in mitochondria, functions to remove the superoxide radicals that form.
superoxide dismutase 3 – extracellular enzyme, found at low levels extracellularly.
Once formed, hydrogen peroxide acts as a substrate for neutrophil myeloperoxidase, which converts it to hypochlorous acid (HOCl) another ROS.
This reaction is one of the most important in free radical biology .It gives rise to site-specific hydroxyl radical formation because the uncharged hydrogen peroxide can diffuse across lipid membranes, and Fenton reactions may therefore convert a low activity and largely weak ROS to the potent hydroxyl radical in close proximity to vital cell structures and macromolecules (e.g. DNA).
Normally soluble ferrous iron (Fe2+) is not present in vivo, being bound tightly to proteins, but it can be produced within cells (e.g. mitochondria or cytosol) by the action of superoxide on ferric iron (Fe3+) within iron storage proteins
The dismutation of hydrogen peroxide also gives rise to the ROS called singlet oxygen.
These 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
Fig: The broad range of biological activities that may arise from the interaction of nitric oxide, superoxide, and peroxynitrite were summarized by Cuzzocrea et al.
Superoxide establishes a pro-inflammatory state in a variety of ways, such as triggering nuclear factor-kappa Beta transcription of pro-inflammatory cytokines
FR & ROS lead to modification of enzyme structure leading to impaired function, or non-function. protein folding or unfolding (which may or may not
be reversible); protein fragmentation and polymerization reactions; Also seen is formation of protein radicals, formation of protein-bound ROS, & formation of stable end products like carbonyl compounds or aldehydes
Oxidative attack on proteins results in site-specific amino acid modification, fragmentation of the peptide chain, aggregation of cross linked reaction products, altered electrical charges and increased susceptibility to proteolysis [Farr and Kogoma, 1991].
Polyunsaturated fatty acids (PUFAs), because of their multiple double bonds are excellent targets for ROS. Figure illustrates one sequence of events, initiated
by a hydroxyl radical, which gives rise to the lipid peroxidation chain reaction, consisting of three steps:
Initiation, Propagation, Termination.
Initiation – FR (OH.) abstracts a hydrogen atom from PUFA (LH) to form Carbon Centered Radical on lipid itself (L.)
Propagation - L. reacts rapidly with oxygen to form a Lipid Peroxy Radical (LO2 .). Such a radical can in turn abstract a H-atom from another PUFA, to form Lipid Hydropeoxide (LOOH).
Termination: Propagation continues until two FR combine with each other to terminate the chain.
Hence a single initiation can result in the conversion of several PUFA into Lipid Hydroperoxides
Termination is most effectively brought about by the lipid-soluble radical scavenger vitamin E (a-tocopherol), which is vital to membrane integrity
Products of lipid peroxidation include a variety of bioactive molecules:
• conjugated dienes; lipid peroxides; aldehydes, e.g. malondialdehyde, which is an example of a thiobarbituric acid reactive substance; acrolein; isoprostanes, e.g. F2-isoprostanes from arachidonic acid (8-iso-PGF2) ; neuroprostanes (F4-isoprostanes); volatile hydrocarbons, e.g. pentane, ethane
Both DNA & RNA are highly susceptible.Both nuclear & mitochondrial DNA are targets.The FR involved are hydroxyl, nitrite & peroxynitrite.
All of this mechanisms this can lead to Malignant Transformation or Cell Death
Free radicals and other reactive species have extremely short half-lives in vivo (10)6–10)9 s) and simply cannot be measured directly. 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, because of their unknown toxicity. Ex vivo spin traps can be used and these would include:
ascorbic acid – which forms semi-dehydroascorbate when it traps a radical;
• aromatic traps – such as salicylates and phenylalanine. However, such traps lack specificity for the OH radical and peroxynitrite and quantification is not deemed possible;
• urate – which is oxidized to allantoin, which can be measured in various fluids (e.g. plasma, urine,cerebrospinal fluid) in diseases whose onset and course are associated with oxidative stress.
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. 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.
As with vitamin C, a-tocopherol levels in plasma are significantly compromised in smokers.
Oxidative stress lies 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 (NF-kB) and the creation of a pro-inflammatory state.
On the basis of above study following conclusions can be drawn: 1.Free radicals are very harmful to human health and can cause several degenerative diseases like diabetes, cancer, atherosclerosis, hypertension etc. 2. Various kinds of antioxidants particularly from natural sources such as enzymes, tocopherol, carotenoids, ascorbic acid, polyphenols etc. inhibit the cellular damage mainly through free radical scavenging property.
