free radical cell injury for Pathology students

1. Dr.
Tareni
Das
1

2. WHAT IS FREE RADICAL?
 Free radicals are chemical species that have a
single unpaired electron in outer orbit.
 Free radicals initiate autocatalytic reaction
 FR are very unstable and very reactive bcoz they
tend to catch an electron to molecules(oxidation).
 Their lifetime is very short (from milliseconds to
nanoseconds
 FR are produced by an electron transfer that
requires high energy input
 When reacting with other endogenous origin or
molecules, a FR can form new radicals.
Dr.
Tareni
Das
2

3. REACTIVE SPECIES
 ROS (reactive oxygen species)
Free radicals
superoxide, O2
· -
hydroxyl radical, OH ·
peroxyl, ROO ·
alkoxyl, RO ·
hydroperoxyl, HO2
Dr.
Tareni
Das
3

4. CONTD..
Particals, which are not free radicals
hydrogen peroxide, H2O2 (Fenton´s reaction)
hypochlorous acid, HClO
ozone, O3
singlet oxygen, 1O2
Dr.
Tareni
Das
4

5. CONTD…
 RNS (reactive nitrogen species)
Free radicals
nitrogen(II) oxide, NO .
nitrogen(IV) oxide, NO2
.
Particals, which are not free radicals
nitrosyl, NO+
nitrous acid, HONO
nitogen(III) oxide, N2O3
peroxynitrite, ONOO -
alkylperoxinitrite, ROONO
Dr.
Tareni
Das
5

6. DIFFERENT CAUSES OF FORMATION OF
FREE RADICALS
 Absorption of radiant energy.
 Enzymatic metabolism of chemicals or drugs. For
ex, carbon tetrachloride can generate [CCl3]*
which cause autooxidation of the polyenic fatty
acid present within membrane phospholipids.
 Some metals which accept or donate e-. For ex,
Cu & Fe (Fenton reaction).
Dr.
Tareni
Das
6

7. CONTD…
 The redox reactions occur during normal
metabolism. For ex, in respiration, molecular
oxygen is reduced to water by accepting
4 electrones. During this process small amount of
toxic intermediates are formed.
 Nitric Oxide (NO) can act as a free radical and
converted into highly reactive peroxynitrate
anion (ONOO-) as well as NO2* and NO3 -.
Normally, NO can be produced by endothelial,
neurons, macrophages etc.
Dr.
Tareni
Das
7

8. CONTD..
 These can be produced by activity of variety of
oxidative enzymes like b5 oxidase, NADPH
oxidase, xanthine oxidase, mpo in different sites
of cell like Endoplasmic reticulum, plasma
membrane, cytosol, peroxisome, lysosome
 special cells (leukocytes)
 superoxide creation by NADP-oxidase
 2 O2 + NADPH → 2 O2•- + NADP+ + H+
 After that,H2O2 can be transformed into HOCL,
which is very active for antigen degradation.
 During oxidation of hemoglobin and myoglobulin
Dr.
Tareni
Das
8

9. CONTD…
 ROS Formation During Ischaemia Reperfusion
Reperfusion Injury
 a transient period of ischemia causes the death of
some cells and injury to others.
 these injured cells are "at risk" due to metabolic
changes, but are not yet dead and may be able to
survive.
 reperfusion can induce new damaging processes
that cause death in cells that might otherwise
recover.
Dr.
Tareni
Das
9

10. CONTD…
mechanisms of reperfusion injury
 -new O2 free radicals from reoxygenation of
injured parenchymal / endothelial cells (from
damaged mitochondria, altered oxides, damaged
antioxidants) and from leukocytes (“spill over”).
 marked influx of new Ca2+ due to membrane /
ion pump damage.
 -initial ischemic injury leads to 2o inflammation
following reperfusion ! additional injury.
Dr.
Tareni
Das
10

11. CONTD..
 Other Ways of ROS Production
 Other processes involved in ROS production
during exercise are increased central
temperature, catecholamine and lactic acid,
which has the ability to convert O2•– into OH•
Dr.
Tareni
Das
11

12. Dr.
Tareni
Das
12

13. IMPORTANT FREE RADICALS
 Superoxide Ion
 O2•–is created with the addition of one electron
on dioxygen and becomes highly reactive.
 O2 + e O2•–
 Fenton’s reaction is an iron-salt-dependent
decomposition of dihydrogen peroxide, generating
the highly reactive hydroxyl radical. It occurs in
the presence of ferrous ions (Fe2+) and O2•–.
Iron is mainly present in tissues in a ferric ion
state (Fe3+). The reaction is called the Haber-
Weiss reaction.
Dr.
Tareni
Das
13

14. CONTD…
 A) O2•-+ H+ → O2• H
 b )O2 H + O2•- + H+ → H2O2 + O2
 c )Fe3+ + O2•- → Fe2+ + O2
 d )Fe2+ + H2O2 → Fe3+ + OH + OH
 Hydrogen Peroxide
 The above Equation summarises the first and
the secondstages of Fenton’s reaction (equations
2a and 2b). This reaction forms hydrogen
peroxide(H is associated 2O2) in an acid
environment and is catalysed by the superoxide
dismutase(SOD) enzyme
Dr.
Tareni
Das
14

15. CONTD
 2 O2•- + 2 H+ → H2O2 + O2
 H2O2 is not a FR because it has no unpaired
electron, but it is considered a ROS because of its
toxicity and its capacity to cause ROS formation.
 In leukocytes, myeloperoxydase (MPO)
transformH2O2 in hypochlorous acid (HOCL),
one of the strongest physiological oxidants and a
powerful antimicrobial agent.
Dr.
Tareni
Das
15

16. CONTD…
 Hydroxyl Radical
 Hydroxyl radical (OH•) is the end product of
Fenton’s reaction
 It is also produced by hydrolysis of water by
ionising radiation.
 OH• is a very reac-tive and very toxic ROS and
there is no specific antioxidant against this FR.
 This FR causes lipid peroxidation and protein
oxidation.
Dr.
Tareni
Das
16

17. CONTD…
 Singlet oxygen
 It is oxygen in which one electron is shifted to
higher orbit
Dr.
Tareni
Das
17

18. BIOLOGICAL EFFECT OF REACTIVE SPECIES
Positive effects
 immunity phenomenon
 as cell messengers
 enzyme activation
 in drug detoxification
 or in facilitating glycogen repletion
 increased strength of muscle contraction
,
Dr.
Tareni
Das
18

19. NEGATIVE EFFECTS
 Lipid Peroxidation: Polyunsaturated fatty
acid of membrane is attacked repeatedly by free
radicals to form highly
destructive polyunsaturated fatty acid (PUFA)
radicals like lipid hydroperoxy radicals
and lipid hypoperoxides. This is termed
as lipidperoxidation. These lipids are widely
spreaded to other part of membrane that
is lipidperoxidation takes place at adjoining part
of membrane causing damage to entire cell
membrane.

Dr.
Tareni
Das
19

20. CONTD…
 2. Oxidation of protein: Free radical causes CI by
oxidation of protein macromolecules of cell causing
cross linkage in the amino acid sequences of protein
and fragmentation of polypeptides.
3. Effect on DNA damage: Free radical breaks
DNA fragments to single strand, so there will be
formation of DNA which is defective. Replication of
this DNA is not possible and there by cell death may
occur.
 4. Cytoskeleton Damage: Free radicals interfere
with mitochondrial aerobic phosphorylation and
decreases synthesis of ATP leading to cytoskeleton
damage.
Dr.
Tareni
Das
20

21. IMPLICATION OF FR IN MUSCULAR FATIGUE
 alteration of the mitochondrial functions
 Contractile protein, calcium pump altered
 Enzyme inactivation
 Iron release
 Inhibition of Ca21-ATPase activity in SE
Dr.
Tareni
Das
21

22. Dr.
Tareni
Das
22

23. FREE RADICAL –BIOCHEMICAL
CHANGES
 -peroxidation of membrane of membrane lipid
 -DNA –sugar component affected by abstraction
and base by addition
 -depletion of NADH pool
 -PROTEIN- disulphide linkage of cystine
 -biological activity of enzyme lost
 -malondialdehyde-advanced lipoxidation end
product
 -depolymerization of hyaluronic acid
 -enzymes-ALT, AST-Liver
 CK-skeletal muscle
 Amylase-pancreas
Dr.
Tareni
Das
23

24. ASSESSMENT OF FREE RADICAL
ACTIVITY
Determination of endogenous
antioxidant level
 Concentration of antioxidant vitamin E, A, C, zn,
folate,
 Cellular activity of antioxidant enzymes like
glutathione reductase, superoxide dismutase,
catalase, glutathione peroxidise
 GSH is rapidly oxidised to GSSH and exported
from cell, so ratio of GSSH to square of GSH is a
good measure of free radical injury.
Dr.
Tareni
Das
24

25. CONTD…
Measurement of products of oxidised
macromolecules
 Assessment of lipid peroxidation by analysis of
lipid peroxides, isoprostane, diene conjugate,
breakdown products of lipid like malonaldehyde,
ethane, pentane
 Assessing ROS induced protein oxidation like
protein carbonyl products, loss of free thiol group,
nitration of protein bound tyrosine group
Dr.
Tareni
Das
25

26. CONTD..
 DNA base oxidation product-5-OH-cytosine, 8-
OH-Guanine and Adenine, thymine glycol,
 Urinary excreation of 8-OH guanosine is
important marker
Dr.
Tareni
Das
26

27. CONTD…
DIRECT DETECTION FREE RADICAL
 Electron spin resonance
 Spin trapping technique
 Absorption spectroscopy
Dr.
Tareni
Das
27

28. VARIOUS TESTS
The FORT Test
 FORT (Free Oxygen Radicals Testing) is a
colorimetric test based on the properties of an
amine derivative employed as chromogen,ChNH2
(4-Amino-N-ethyl-isopropylanilinehydrochloride)
to produce a fairly long-lived radical cation.
 When sample is added to a ChNH2 solution, the
coloured radical cation of the chromogen is
formed and the absorbance at 505 nm, which is
proportional to the concentration of hydroperoxyl
molecules, is associated to the oxidative status of
the sample.
Dr.
Tareni
Das
28

29. THE FORD TEST
FORD (Free Oxygen Radicals Defence)
 It is a colorimetric test based on the ability of
antioxidants present in plasma to reduce a
preformed radical cation.
 The principle of the assay is that at an acidic pH
(5.2) and in the presence of a suitable oxidant
solution (FeCl3), 4-Amino-N,Ndiethylaniline,the
FORD chromogen, can form a stable and colored
radical cation.
Dr.
Tareni
Das
29

30. CONTD..
 Antioxidant molecules (AOH) present in the
sample which are able to transfer a hydrogen
atom to the FORD chromogen radical cation,
reduce it quenching the colour and producing a
decolouration of the solution which is
proportional to their concentration in the sample
 Chromogen(uncolored) + oxidant (Fe3+) H+ →
Chromogen.+(purple)
 Chromogen.+(purple) + AOH → Chromogen+
(uncolored) + AO
Dr.
Tareni
Das
30

31. D-ROMS TEST
 In the d-ROMs test, ROMs (Reactive Oxygen
Metabolites, mainly hydroperoxides, ROOH) of a
blood sample, in presence of iron (that is released
from plasma proteins by an acidic buffer) are able to
produce alkoxyl (RO.) and peroxyl (ROO.) radicals,
according to the Fenton’s reaction.
 Such radicals, in turn, are able to oxidize an alkyl-
substituted aromatic ammine (A-NH2, that is
dissolved in chromogenic mixture) thus transforming
them in a pink-colored derivative ([A-NH2.]+),
accordingly to the reactions (the first two for
alkoxylradicals and the others two for peroxyl
radicals) which is photometrically quantified
 ROOH + Fe2+ →RO· + Fe3++ OHRO· + A-NH2 →
RO- + [A-NH2·]+ROOH + Fe3+ → ROO· + Fe2+ +
H+ROO· + A-NH2 → ROO· + [A-NH2·]+
Dr.
Tareni
Das
31

32. CONTD
 The intensity of developed color is directly
proportional to the level of ROMs, according to the
Lambert-Beer’s law.
 The d-ROMs test is based on spectrophotometer
studies on increases in red colour intensity after the
addition of a small quantity of human blood to a
solution of N,N-diethylparaphenylendiamine
(chromogen), buffered to pH 4.8. Such colouring is
attributed to the formation, via oxidation, of the
cation radical of the amine which formationis due to
alkoxyl and peroxyl radicals.
 These latter derive from the reaction of the Fe2+and
Fe3+ ions released by proteins in acidic conditions as
created in vitro.
Dr.
Tareni
Das
32

33. BAP TEST
 BAP (Biological Antioxidant Power) test is based
on the capacity of a colored solution, containing a
source of ferric (Fe3+) ions adequately bound to a
special chromogenic substrate, to a decolour
when Fe3+ ions are reduced to ferrous ions
(Fe2+), as it occurs by adding
reducing/antioxidant system, i.e. a blood plasma
sample.
 Therefore, in the BAP test, a small quantity of
blood plasma (10 μl) to be tested is dissolved in a
coloured solution, which has been previously
obtained by mixing a source of ferric ions (i.e.
ferric chloride, FeCl3) with a special chromogenic
substrate (i.e. a thiocyanate derivative).
Dr.
Tareni
Das
33

34. CONTD
 After a short incubation (5 min), at 37°C, such
solution will decolor and the intensity of this
chromatic change will be directly proportional to
the capacity of plasma to reduce, during the
incubation, ferric ions (initially responsible for
the color of solution) to ferrous ions, according to
these reactions:
 FeCl3 + AT(uncolored)→FeCl3 – AT(colored)
FeCl3 – AT(colored) + BP(e-)→FeCl2 +
AT(uncolored) + BP
 AT(uncolored) is a thiocyanate derivative (uncolored)
 FeCl3-AT(colored) is the colored complex of ferric chloride with
the thiocyanatederivative;
Dr.
Tareni
Das
34

35. CONTD
 BP(e-) is a molecule of blood plasma barrier with
reducing/electron giving/antioxidant activity
against ferric ions;
 BP is the oxidized form of BP(e-);
 FeCl2 is the ferrous chloride obtained by
thereducing activityof BP(e-).
 By photometrically assessing the intensity of
decoloration, the concentrations of reduced ferric
ions can be adequately determined thus allowing
a measurement of reducing capacity or
antioxidant potential of tested blood plasma.
Dr.
Tareni
Das
35

36. ANTIOXIDANT SYSTEM
 Any molecule capable of deactivating free radical
 IDEAL ANTIOXIDANTS
 Readily absorbed
 Quench free radicals
 Chelate redox metal
 Work at aqueous and membrane domain
 Effect gene expression in positive way
Dr.
Tareni
Das
36

37. ANTIOXIDANT SYSTEM
3 levels-inhibition of production the abundance of
RONS, capture of radicals, correction
mechanism of destroyed biomoleculs
Dr.
Tareni
Das
37

38. 1. ENDOGENNOUS ANTIOXIDANTS
 nonenzymatic
- fixed in membranes ( -tocopherol, -
caroten, coenzym Q 10)
- out of membranes (ascorbate, transferrin,
bilirubin)
 enzymes (cytochrome c,SOD, GSHPx, catalase)
Dr.
Tareni
Das
38

39. 2. EXOGENNOUS ANTIOXIDANTS
 FR scavengers
 Trace elements
 Drugs and compounds influence to FR
metabolism
Dr.
Tareni
Das
39

40. ENZYMES DEFENCE MECHANISM
Dr.
Tareni
Das
40

41. SUPEROXID DISMUTASE
2O2
. - + 2H+  H2O2 + O2
SOD - is present in all oxygen-metabolizing cells,
different cofactors (metals)
an inducible in case of superoxide overproduction
Mn 2+ SOD (SOD1)
tetramer
matrix mitochondria
lower stability then Cu, Zn - SOD
Dr.
Tareni
Das
41

42. CU 2+/ZN 2+ SOD (SOD 2)
dimer, Cu = redox centr
cytosol, intermitochondrial space
hepatocyt, brain, erytrocyte
high stability, catalysation at pH 4,5-9,5
Dr.
Tareni
Das
42

43. GLUTATHION PEROXIDASES
elimination of intracellular hydroperoxides and
H2O2
2 GSH + ROOH  GSSH + H2O + ROH
 Cytosolic GSH - glutathionperoxidasa (
 Extracelullar GSH – glutathionperoxidasa
 Phospholipidhydroperoxide GSH - peroxidase
Dr.
Tareni
Das
43

44. CATALASE
2 H2O2  2 H2O + O2
High affinity to H2O2 : peroxisomes hepatocytes
mitochondria, cytoplasm of erytrocytes
Tetramer with Fe, needs NADPH
Dr.
Tareni
Das
44

45. HIGH-MOLECULA ENDOGENNOUS
ANTIOXIDANTS
• Transferrin
• Ferritin
• Haptoglobin
• Hemopexin
• Albumin
Dr.
Tareni
Das
45

46. LOW-MOLECULE ENDOGENNOUS ANTIOXIDATS
I
collagen synthesis
dopamine to
epinephrine
conversion
reduction agent
Fe absorption
antioxidant = reduction
O2
· - OH ·, ROO·, HO2
·
tocopheryl radical
regeneration
localise in membranes
produces
hydroperoxides, which
are changes by
GSHPx
Alfa-tocopherol
Dr.
Tareni
Das
46

47. LOW-MOLECULE ENDOGENNOUS ANTIOXIDATS
II
 Ubiquinone (coenzyme Q)
Electron carrier in respisratory chain
Co-operates with tocopheryl
 Carotenoides, -caroten, vitamin A
removing the radicals from lipids
Dr.
Tareni
Das
47

48. LOW-MOLECULE ENDOGENNOUS ANTIOXIDATS
III
 Glutathione (GSH, GSSG)
in all mammalian cells (1-10 mmol/l)
important redox buffer
2 GSH  GSSG + 2e- + 2H+
ROS elimination, stabilisation in reduction form (
SH- groups, tocopheryl and ascorbate
regeneration)
substrate of glutathione peroxidases
Dr.
Tareni
Das
48

49. LOW-MOLECULE ENDOGENNOUS ANTIOXIDATS
IV
 Lipoic acid (lipoate)
Tocopheryl and ascorbate regeneration
 Melatonin
Lipophilic ; hydroxyl radicals scavenger
 Low-molecule endogennous antioxidats V
 Uric acid (urates)
 Bilirubin
 Flavonoids
Dr.
Tareni
Das
49

50. TRACE ELEMENTS INFLUENCE TO FR
METABOLISM
 Selenium
 Influence to vitamin E resorption, part of
selenoproteins
 of Se = insufficient immun. Respons,
erytrocytes hemolysis, methemoglobin synthesis
 Zinc
 Cell membrane stabilisation
Fe antagonist
Dr.
Tareni
Das
50

51. OXIDATIVE STRESS
 The contemporary definition of oxidative stress
has been refined to account for two different
mechanistic outcomes, macromolecular damage,
and disruption of thiol redox circuits, which leads
to aberrant cell signaling and dysfunctional redox
control .
 Macromolecular damage is usually considered in
terms of oxidative mechanisms linked to free
radicals.
Dr.
Tareni
Das
51

52.  Oxidative stress reflects an imbalance between
the systemic manifestation of reactive oxygen
species and a biological system's ability to readily
detoxify the reactive intermediates or to repair
the resulting damage
 In biological system, mostly non radical oxidants
are produced
Dr.
Tareni
Das
52

53. HYPOTHESIS RELATED TO OXIDATIVE
STRESS
 MITOHORMESIS HYPOTHESIS”
 In mitohormesis, sublethal mitochondrial stress
is proposed to produce beneficial outcomes
through mitochondrial generation of reactive
oxygen species, which serve as signaling
elements for cytoprotection.
 However, the mitohormesis hypothesis does not
discriminate between free radical and nonradical
mechanisms
Dr.
Tareni
Das
53

54. THE REDOX HYPOTHESIS
 In an attempt to clearly delineate the non radical
complement to free radical theories that have
dominated oxidative stress research, scientist
formulated a “redox hypothesis” with four postulates:
 All biological systems contain redox elements [e.g.,
redox-sensitive cysteine, Cys, residues] that function
in cell signaling, macromolecular trafficking, and
physiological regulation.
 Organization and coordination of the redox activity of
these elements occurs through redox circuits
dependent on common control nodes (e.g., thioredoxin,
GSH).

Dr.
Tareni
Das
54

55. CONTD..
 The redox-sensitive elements are spatially and
kinetically insulated so that “gated” redox
circuits can be activated by translocation/
aggregation and/or catalytic mechanisms
 Oxidative stress is a disruption of the function of
these redox circuits caused by specific reaction
with the redox-sensitive thiol elements, altered
pathways of electron transfer, or interruption of
the gating mechanisms controlling the flux
through these pathways.
Dr.
Tareni
Das
55

56. OXIDATIVE STRESS AS A BIOLOGICAL
MODULATOR AND AS A SIGNAL
OXIDATIVE STRESS
 Reactive oxygen
species
 Ischemia
 Inflammation
 Radiation
 Ultraviolet light
 Anticancer drugs
 Heavy metals
 Cytokines
Glutathione system
Thio redoxin system
Dr.
Tareni
Das
56

57. MKK3/6-p38 pathway
Tyrosine kinase
Src family
Syk/ZAP-70 family
EGF receptors
Protein kinase C
MAP kinase cascade
MEK-ERK pathway
SEK1-JNK pathway
MKK3/6-p38 pathway
Activation of
transcription factors
AP-1
NF-_B
Nrf2
CELLULAR RESPONSES
Activation
Proliferation
Inflammatory
reaction
Stress
protection
Death
Dr.
Tareni
Das
57

58. Neurological
Alzheimers Disease
Parkinson‘s Disease
Endocrine
Diabetes
Gastrointestinal
Acute Pancreatitis
Dr.
Tareni
Das
58

59. FREE RADICAL CELL INJURY VS HYPOXIC
CELL INJURY
 Due to decrease O2 supply
 Cell membrane
damage mainly by
calcium dependant
phospholipase, ATP
loss
 Protease degrade
protein
 Endonuclease break
down DNA
 Toxic activated oxygen
species
 lipid peroxidation
 Cross linking of
protein
 Direct interaction
with thymine
produces single strand
break
Hypoxic cell injury Free radical cell injury
Dr.
Tareni
Das
59

60. CONTD…
 1st point of attack of
hypoxia is oxidative
phosphorylation
 protection-oxygen
restoration
 Initiates autocatalytic
reaction with lipid,
protein, DNA,
carbohydrate
 Very damaging in
presence of oxygen
Dr.
Tareni
Das
60

61. Dr.
Tareni
Das
61