Free Radicals: A cellular way to understand

Free Radicals

1. FREE RADICALS
Dr. ARUN KUMAR
SCIENTIST- I
MAHAVIR CANCER SANSTHAN

2. Free radicals and antioxidants
•What is „free radical“?
•Reactive oxygen and nitrogen species (RONS)
•Are the RONS always dangerous?
•Well known term „oxidative stress“ - what is
it?
•Antioxidants - types and appearance
•Markers of oxidative stress
•Disorders Associated with Oxidative stress

3. Free radical - what is it?
Atom: proton, neutron, electronic shell (orbital)
Free radical
• particles with an unpaired electron spinning
around the nucleus. (can be atom, ions,
molecule).
•tend to reach equilibrium, plucks an electron
from the nearest intact molecule.
•most of biomoleculs are not radicals

4. Simply say , our welt is composed of atom . And
there is a special principle that is if only there are two
or more electron fit together , their periphery electron
must be in pairs , if not , they need to find another
electron to make themselves steady. Free radicals is
any atom or molecule which has an
"unpaired electron " in the outer ring .
Free radicals
Definition

5. Free radical and “science”
•chemist
during the thirties - there is superoxide
•biochemist
during the sixties - make a discovery of
superoxid dismutase (SOD)
•doctor
free radicals are associated with many
disorders

6. Mechanism of radical reactions
Radicals are highly reactive species
Three distinc steps
•initiation (homolytic covalent bonds cleavage)
•propagation (chain propagation)
•termination

7. ROS (reactive oxygen species)
Free radicals
superoxide, O
2
· -
hydroxyl radical, OH
·
peroxyl, ROO ·
alkoxyl, RO
·
hydroperoxyl, HO
2
·
Particals, which are not
free radicals
hydrogen peroxide, H
2
O
2
(Fenton´s reaction)
hypochlorous acid, HClO
ozone, O
3
singlet oxygen,
1
O
2

9. RNS (reactive nitrogen species)
Free radicals
nitrogen(II) oxide, NO
.
nitrogen(IV) oxide, NO
2

.
Particals, which are not free
radicals
nitrosyl, NO
+
nitrous acid, HONO
nitogen(III) oxide, N
2O
3
peroxynitrite, ONOO
-
alkylperoxinitrite, ROONO

10. The main sources of free radicals
membranes enzymes and/or coenzymes with
flavine structures, hem coenzymes, enzymes
containing Cu atom in an active site
1. respiratory chain mitochondria : mainly
superoxide and then H
2O
2
•approx 1- 4% O
2 entres into resp. chain
(mainly complexes I a III)

14. The main sources of free radicals II
2. Endoplasmic reticulum
superoxide creation (by cytochrome P- 450)
3. special cells (leukocytes)
superoxide creation by NADP-oxidas
4. hemoglobin to methemoglobin oxidation
(erytrocyte is „full“ of antioxidants)

15. Free radicals physiological function
Used by oxides a oxygenes
•cytochromoxidase (toxic intermediates, H
2O
2
and superoxide, are bound to an enzymu)
•monoxygenases - activate O
2 in liver ER or in
adrenal gland mitochondria ; hydroxylation

16. Free radicals physiological function II
ROS a RNS against bacteria
•enzyme complex NADPH-oxidase of leukocytes
•myeloperoxidase - catalysis of the following
reaction
H
2O
2 + Cl
-
+ H
+
= HClO + H
2O

17. Free radicals physiological function
III
•Signal molecules
first messenger  second messenger  information
net
This info net function is affected by the redox state of
cells
•redox state : antioxidant capacity, reduction
equivalent availlability, RONS rate
 ROS: second messenger

19. Immunity vs. regulation
a massive production of ROS as immunity
instrument
x
an induction of the changes low concentration
ROS, which are probably regulation
mechanism

22. Where are the free radicals in existence ?
1 from chimneys and cars
2 heat ,burning and illumination
3 chemistry reaction
4 cooking , smoking and facing- painting

23. The function of free radicals
 There are various types of free radicals and they
exist everywhere . They have different structure
characters ,when they integrate with different
element , the function are also different.
helping transfer the energy.
Kill the bacteria and parasites .
 Participate in debar toxin.

24. Some diseases caused by free radicals
inflammation
Tumour
blood diseases

25. The attack approach by free radicals
destroy the cell membrane
make blood serum antiparallel loose
active
Trauma gene lead to the cell aberrance

26. The attack is beginning with the cell membranes .Cell
membranes is elasticity and flexible ,so it’s easy to
loose electron , and easy to attacked by free radicals .
Once it seize electron by free radicals ,it will loose all
it’s function , leading to heart and blood diseases .
Ever more serious the gene’s molecule structure will
destroy ,which lead to gene mutation ,sequentially
arose the whole life chaos.

27. Every cell contains an enormous set of molecules called DNA
which provide chemical instructions for a cell to function. This
DNA is found in the nucleus of the cell, which serves as the
"command center" of the cell, as well as in the mitochondria. The cell
automatically fixes much of the damage done to nuclear DNA.
However, the DNA in the mitochondria cannot be readily fixed.
Therefore, extensive DNA damage
accumulates over time and shuts
down mitochondria, causing the
cells to die and the organism to
age.
Hence, this free radical generation
process can disrupt all levels of
cell function. This is why free
radical damage is thought to be
such a basic mechanism of tissue
injury. It damages us at the cellular
level.

28. Antioxidant defence system
3 levels
inhibition of production the abundance of RONS
capture of radicals (scavengers, trappers, quenchers)
correction mechanism of destroyed biomoleculs

30. As vitamin E is fat soluble it can protect the cell
membrane from radical damage.
Laboratory experiments suggest that this
process involves the water soluble vitamin C.
After donating a hydrogen atom to a reactive
oxygen-centred radical, the vitamin E radical
can be converted back to vitamin E by reaction with vitamin C.
  
                                                                                                                                                   

31. Antioxidants and scavengers review
1. Endogennous antioxidants
•enzymes (cytochrome c,SOD, GSHPx, catalase)
•nonenzymatic
- fixed in membranes ( -tocopherol, -
caroten, coenzym Q
10)
- out of membranes (ascorbate, transferrin,
bilirubin)

32. Antioxidants and scavengers review II
2. Exogennous antioxidants
•FR scavengers
• trace elements
•drugs and compounds influence to FR
metabolism

33. Enzymes defence mechanism

35. Superoxid dismutase
(EC 1.15.1.1, SOD)
2O
2
. -
+ 2H
+
 H
2O
2 + O
2
SOD - is present in all oxygen-metabolizing
cells, different cofactors (metals)
an inducible in case of superoxide overproduction

36. Superoxid dismutase
Mn
2+
SOD (SOD1)
tetramer
matrix mitochondria
lower stability then Cu, Zn - SOD

37. Superoxid dismutase
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

38. Glutathion peroxidases
elimination of intracellular hydroperoxides
and H
2O
2
2 GSH + ROOH  GSSH + H
2
O + ROH
•cytosolic GSH - glutathionperoxidasa (EC
1.11.1.9, cGPx)
•extracelullar GSH - glutathionperoxidasa
(eGSHPx)
•phospholipidhydroperoxide GSH -
peroxidase (EC 1.11.1.12, PHGPx)

39. Catalase
(EC 1.11.1.6, KAT)

2 H
2O
2  2 H
2O + O
2
high affinity to H
2O
2 : peroxisomes
hepatocytes mitochondria, cytoplasm of
erytrocytes
tetramer with Fe, needs NADPH

40. High-molecula endogennous
antioxidants
• transferrin
• ferritin
• haptoglobin
• hemopexin
• albumin

41. Low-molecule endogennous antioxidats I
Ascorbate (vitamin C)
collagen synthesis
dopamine to epinephrine
conversion
reduction agent
Fe absorption
antioxidant = reduction O
2
· -
OH
·
, ROO·, HO
2
·
tocopheryl radical
regeneration
prooxidant
Alfa-tocopherol a vitamin E
localise in membranes
produces hydroperoxides,
which are changes by
GSHPx

42. Ascorbic acid and its metabolites

43. Low-molecule endogennous antioxidants II
•ubiquinone (coenzyme Q)
electron carrier in respisratory chain
co-operates with tocopheryl
•carotenoides, -caroten, vitamin A
removing the radicals from lipids

44. 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

45. Low-molecule endogennous antioxidats IV
•Lipoic acid (lipoate)
PDH cofactor
tocopheryl and ascorbate regeneration
•melatonin
lipophilic ; hydroxyl radicals scavenger

46. Low-molecule endogennous antioxidats V
•uric acid (urates)
•bilirubin
•flavonoids

47. 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

48. Oxidative stress
Equilibrium failure between creation and
a elimination of RONS leads to
oxidative stress

Be carefull - this equilibrium can be
disbalance in both sides

49. Oxidative damage to lipid
Damage
•unsaturated bonds loss
•arising of reactive
metabolites (aldehydes)
Sequel
•changes in fluidity and
permeability of
membranes
•membranes integral
enzymes are
influenced

51. The peroxidation of linoleic acid

52. Oxidative damage to proteins
Damage
•agregation, fragmentation
and cleveage
•reaction with hem iron
ion
•functional group
modification
Sequel
•changes in: enzymes
activity, ions transport
•proteolysis

53. Oxidative damage to DNA
Damage
•saccharide ring
cleveage
•bases modification
•chain breakeage
Sequal
•mutation
•translation mistakes
•protoesynthesis
inhibition

54. Oxidative stress markers
Free radicals detection
•very difficult, because of chem-phys.
properties
Oxidative stress products detection
•more simple, a wide range of techniques

55. Oxidative stress markers II
Lipoperoxidation markers:
malondialdehyde (MDA), conjugated diens,
isoprostanes
Oxidative damage to protein markers :
protein hydroperoxides
Oxidative damage to DNA :
modified nucleosides

56. Antioxidants determination
ascorbate
tocopheryl
SOD
GSHPx
glutathion

57. Disorders Associated with
Oxidative stress
Neurological
Alzheimers Disease
Parkinson‘s Disease
Endocrine
Diabetes
Gastrointestinal
Acute Pancreatitis

58. Disorders Associated with
Oxidative stress
Others conditions
Obesity
Air Pollution
Toxicity
Inflammation

59. Malondialdehyde (MDA) Assay :
Draper & Hadley (1990)
Reactions Blank (ml) Test (ml)
TCA (10%) 2.5
2.5
Serum - 0.5
Incubated for 15 minutes at 90
0
C
Cooled at R.T and centrifuged at 3000 rpm for 10 minutes
and taken the 2ml supernatant
Supernetant - 2.0
TBA (0.675%) 1.0 1.0
Incubated for 15 minutes at 90
0
C
Taken O.D at 532 nm

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