Jude: The Acts of the Apostate: High Handed Sins (vv.5-7).pptx
A Student's Prayer
1. A Student’s Prayer
Father, I have knowledge, so I pray
you'll show me now
How to use it wisely
and find a way somehow
To make the world I live in
a little better place,
And make life, with its problems,
a bit easier to face...
Grant me faith and courage
and put purpose in my days,
And show me how to serve Thee
in the most effective ways
So all my education,
my knowledge and my skill,
May find their true fulfillment
as I learn to do Thy will...
And may I ever be aware
in everything I do
That knowledge comes from learning -
And wisdom comes from You.
1
2. Oxygen Metabolism
and Oxygen Toxicity
NOEL MARTIN S. BAUTISTA, MD, DPPS, MBAH
Department of Biochemistry, Molecular Biology and Nutrition
3. Oxygen Metabolism
and Toxicity
Properties of Oxygen / O2
Properties of ROS
Major Sources of ROS in the Cell
Oxygen Radical Reactions with Cellular
Components
Cellular Defenses Against Oxygen Toxicity
3
4. Oxygen: The Element of Life
one of the most abundant
elements on this planet
earth's crust (46.6% by
weight), oceans (86% by
weight), atmosphere (21% by
volume)
comes from the Greek stems
oxys, "acid," and gennan, "to form
or generate, " literally means “acid
former”
term introduced by Lavoisier, who
noticed that compounds rich in
oxygen (eg, SO2) when dissolved
in water generate acids
4
5. Oxygen: Chemistry
colorless, odorless diatomic
molecule with the formula O2
two oxygen atoms are
bonded
bond has a bond order of
two, and is thus often
simplified in description as a
double bond
5
6. Oxygen: Chemistry
chemical element with the
chemical symbol O
atomic number 8
valency of 2
8 neutrons, 8 protons, 8 electrons
6
7. Oxygen: Chemistry
electron configuration of the
molecule has two unpaired electrons
occupying two molecular orbitals
orbitals are classified as anti-
bonding, so the diatomic oxygen
bond is weaker than the diatomic
nitrogen bond, where all bonding
molecular orbitals are filled
unpaired electrons are commonly
associated with high reactivity in
chemical compounds
7
8. Properties of Oxygen
biradical molecule
2 single electrons in
different orbitals with
parallel spins
high tendency to form
toxic reactive oxygen
species (ROS)
8
9. Oxygen: Toxicity
Most of the damaging effects
of oxygen can be explained
by oxygen free radicals
- Gershman and Gilbert, 1954
9
10. O2: Radical Nature
radicals - molecules that possess a single
unpaired electron in an orbital
highly reactive and can initiate chain
reactions
Paired Electrons
Unpaired Electron
10 Stable Molecule Free Radical
12. O2: Reduction Products
O2 is capable of
accepting 4
electrons, reducing it to
water
4-electron reduction
steps for O2
progressively generate
superoxide, hydrogen
peroxide, and the
hydroxyl radical plus
12 water
14. ROS: Properties
major oxygen metabolites produced by
one-electron reduction of oxygen
react indiscriminately by extracting
electrons from other molecules
include oxygen ions, free radicals and
peroxides
levels can increase dramatically with
environmental stress resulting to
significant damage to cell structures
(oxidative stress)
14
16. ROS: Superoxide Anion
O2-
can be formed from
free O2 by donation of
an electron to another
free radical
highly reactive but has
limited lipid solubility
and cannot diffuse far
from site of origin
contains one additional
unpaired electron
16
17. ROS: Superoxide Anion
reacts non-
enzymatically with
hydrogen peroxide
in the Haber Weiss
reaction to generate
other ROS (hydroxyl
and hydroperoxy
radicals)
17
18. ROS:
Superoxide Anion
Sources:
produced by the
ETC
other sites
18
19. ROS: Hydrogen Peroxide
H 2O 2
contains two
additional paired
electrons
formed by two-
electron reduction of
oxygen
not a free
radical, but a weak
oxidizing agent
19
20. ROS: Hydrogen Peroxide
classified as ROS
because it can
generate the
hydroxyl free
radical by reaction
with a transition
metal (Fe2+) in the
non-enzymatic
Fenton Reaction
20
21. ROS: Hydrogen Peroxide
lipid soluble and thus can
diffuse into and through cell
membranes
dismutation Reaction
2O2- + 2H+ H2O2 + O2
precursor of the powerful
oxidizing
agent, hypochlorous acid
(HOCl)
21
22. ROS:
Hydroxyl Radical
OH•
most reactive species in
attacking biological
molecules
produced by H2O2 in the
presence of Fe++ or Cu+
(Fenton Reaction) or via
the Haber-Weiss reaction
one of its damaging
immediate effects is the
initiation of lipid
peroxidation
22
23. ROS: Organic Radicals
R•, RO•, R-S•
organic free radical produced from RH by
superoxide or •OH attack by extracting
electrons
RH can be the carbon or a double bond in
fatty acid (resulting in –C• =C-) or RSH
(resulting in R-S•)
23
24. ROS:
Organic Peroxide Radical
RCOO•
organic peroxyl radicals, such as occurs
during lipid degradation (also denoted as
LOO•)
important reaction because the primary
molecules that undergo this chemistry are
the PUFAs
Allylic carbonyl radicals are generated;
organic peroxyl radical participates in a
chain reaction of lipid oxidations cell
membrane damage and death
24
25. ROS: Hypochlorous Acid
(Hypochlorite)
HOCl
produced in neutrophils (respiratory burst) to
destroy invading organisms; toxicity via
halogenation and oxidation reactions
generated by myeloperoxidase on Cl- ions
in the presence of H2O2
H2O2 + Cl- HOCl + OH-
can lead to formation of more toxic ROS (OH•)
HOCl + O2- •OH + Cl- + O2
HOCl + Fe2+ •OH + Cl- + Fe3+
25
26. ROS: Singlet Oxygen
high energy species of oxygen
molecule with anti-parallel spins
no unpaired electrons, but one
orbital is completely empty
highly reactive
can react with organic
conjugated double bonds to form
endoperoxides, dioxetanes and
hydroperoxides
produced at high-oxygen
tensions from absorption of UV
light
Decays rapidly, not significant
26
27. ROS: Nitric Oxide
NO
free radical produced endogenously by nitric
oxide synthase
endothelium derived relaxing factor
synthesized from arginine via action of nitric
oxide synthetase
binds to metal ions
combines with O2 or other oxygen-containing
radicals to produce additional RNOS
example: peroxynitrite (strong oxidizing agent)
O2-• + NO• ONOO-
27
29. A. Coenzyme Q
major source of superoxide
ETC “leaks” free radicals at
CoQ
The one-electron reduced
form of CoQ (CoQH•) is
free within the membrane
and can accidentally
transfer an electron to
dissolved O2, thereby
forming the superoxide
29
30. B. Respiratory Burst
process by which
phagocytic cells
consume large
amounts of oxygen
during phagocytosis
and release ROS
major source of
superoxide
anion, hydrogen
peroxide, hydroxyl
radical, and
hypochlorite
(HOCl), nitric oxide
30 (NO) and other free
32. B. Respiratory Burst
1. NADPH Oxidase
catalyzes the transfer of
an electron from NADPH
to O2 to form superoxide
activation of NADPH
oxidase initiates the
respiratory burst at the
cell membrane
superoxide
32
34. B. Respiratory Burst
3. Myeloperoxidase
formation of
hypochlorous acid
from H2O2 is
catalyzed by
myeloperoxidase
hypochlorous acid is a
powerful toxin that
destroys bacteria
within seconds
through halogenation
and oxidation
reactions
34
35. B. Respiratory Burst
5. Nitric Oxide
Synthase
generates NO
NO reacts
rapidly with
superoxide to
generate
peroxynitrite, whi
ch forms
additional RNOS
35
36. C. Oxidases, Oxygenases
and Peroxidases
oxidases, peroxidases and oxygenases in
the cell bind O2 and transfer single
electrons to it via a metal
free radical intermediates of these
reactions may be accidentally released
hydrogen peroxide and lipid peroxides are
generated enzymatically as major reaction
products by a number of oxidases present
in peroxisomes, mitochondria and the
endoplasmic reticulum
36
37. C. Oxidases, Oxygenases
and Peroxidases
Examples:
Cytochrome P450 enzymes – major source of free
radicals “leaked” from reactions
Monoamine oxidase oxidatively degrades the
neurotransmitter dopamine and generates H2O2
Peroxisomal fatty acid oxidase generates H2O2
rather than FAD (2H) during the oxidation of very long
chain fatty acids
Xanthine oxidase, an enzyme of purine degradation
that can reduce O2 to O2- or H2O2 in the cytosol;
major contributor to ischemia-reperfusion injury
Lipid peroxides are formed enzymatically as
intermediates in the synthesis of many eicosanoids
37
39. Exogenous Sources:
Ionizing Radiation
electromagnetic radiation
generate primary radicals by
transferring their energy to
cellular components such as
water
its high energy level can split
water into hydroxyl and hydrogen
radicals radiation damage to
skin, mutations, cancer and cell
death
may generate organic radicals
through direct collision with
organic cellular components
39
40. Exogenous Sources: Drugs
appear to increase free radical production
in the presence of increased oxygen
tensions (hyperoxia)
antibiotics (quinoid groups or bound
metals)
antineoplastics
(bleomycin, anthracyclines, methotrexate)
40
41. Exogenous Sources:
Tobacco Smoking
tobacco smoke contains enormous amount of
oxidant material (aldehydes, epoxides, peroxides,
NO and semiquinones) that may cause damage to
the alveoli
micro-hemorrhages causes iron deposition in the
smokers’ lung tissue formation of the lethal
hydroxyl radical from H2O2 (Fenton reaction)
elevated amounts of neutrophils found in the lower
respiratory tract of smokers increased formation
of free radicals
smoke oxidants deplete intracellular antioxidants
41
43. Exogenous Sources:
Alcohol Consumption
excessive alcohol ingestion
induce oxidative reactions in the liver
43
44. Exogenous Sources:
Inorganic Particles
asbestos, quartz, silica (mineral dust)
Leads to cell rupture lung injury
↑ production of ROS
44
45. Exogenous Sources:
Inorganic Particles
Phagocytosis by
pulmonary macrophages
Release proteolytic enzymes
and chemotactic factors inflammation
Increased free radical and
ROS production
45
46. Exogenous Sources:
Gases (Ozone)
not a free radical; highly potent
oxidizing agent
contains two unpaired electrons and
degrades under physiological conditions
to hydroxyl radicals
photo-dissociation of chlorofluorocarbons
(aerosol sprays) can lead to chlorine
radicals Cl•
46
50. A. Membrane Attack
formation of lipid and lipid
peroxy radicals
free radical auto-oxidation
requires an initiator
(e.g., hydroxyl radical from
Fenton reaction) to begin
the chain reaction
50
51. Lipid Peroxidation: 1. Initiation
initiated by a hydroxyl or other radical that
extracts a hydrogen atom from
polyunsaturated lipid (LH), thereby forming
a lipid radical (L•)
51
52. Lipid Peroxidation:
2. Propagation
free radical chain
reaction is
propagated by
reaction with O2,
forming the lipid
peroxy radical (LOO•)
and lipid peroxide
(LOOH)
52
53. Lipid Peroxidation:
3. Degradation
rearrangements of the single electron result in
degradation of the lipid
malondialdehyde, one of the compounds formed,
is soluble and appears in blood and urine
(marker of free radical damage)
53
54. Lipid Peroxidation:
4. Termination
chain reaction can be terminated by Vitamin E
and other lipid-soluble antioxidants that donate
single electrons.
two subsequent reduction steps form a
stable, oxidized antioxidant
54
55. Lipid Peroxidation: Effects
invariably changes or damages lipid
molecular structure
the aldehydes that are formed can cross-
link with proteins
disrupts the cohesive lipid bilayer
arrangement and stable structural
organization
disruption of mitochondrial membrane
integrity may result in further free radical
production
55
56. B. Proteins and Peptides
proline, histidine, arginine, cysteine and
methionine: most susceptible to OH• attack and
oxidative damage
protein may fragment or residues may cross-link
damaged cysteine residues cross-link and form
aggregates that prevent their degradation
oxidative damage increases the susceptibility of
other proteins to proteolytic digestion
free radical attack and oxidation of the cysteine
sulfhydryl residues of glutathione increases
oxidative damage throughout the cell
56
57. C. DNA Damage
oxygen-derived free
radicals are a major
source of DNA damage
non-specific binding of
Fe2+ to DNA facilitates
localized production of
the hydroxyl radical
(Fenton reaction)
causes strand breaks
and base alterations in
the DNA
57
63. A. Anti-Oxidant Scavenging
Enzymes
Superoxide Dismutase
primary defense against oxidative stress
speeds up dismutation of O2- H2O2
and O2
Exists as three isoenzyme forms
Cu+-Zn2+ – cytosol
Mn2+ – mitochondria
Cu+-Zn2+ – extracellular
activity of Cu+-Zn2+ SOD is by
chemicals or conditions (such as
hyperbaric oxygen) that increase the
production of superoxide
63
64. A. Anti-Oxidant Scavenging
Enzymes
Catalase
heme-containing enzyme catalyzing
dismutation of hydrogen peroxide into
water and oxygen
found principally in the peroxisomes and
cytosol and microsomal fraction of the
cell
highest activities are found in tissues
with a high peroxisomal content (kidney
and liver)
in the immune system, catalase serves
to protect the cell against its own
respiratory burst
64
66. Glutathione
-glutamylcysteinyl-
glycine
one of body’s
principal substances
against oxidative
damage
sulfhydryl group
oxidized to a
disulfide, transferring
electrons to H2O2 to
produce water
66
67. A. Anti-Oxidant Scavenging
Enzymes
Glutathione Peroxidase
one of principal means of
protection against oxidative
damage
catalyzes the reduction of H2O2
and lipid peroxides (LOOH) by
glutathione
reactive sulfhydryl groups
reduce H2O2 to water and lipid
peroxides to non-toxic alcohols
two glutathione molecules are
oxidized to form a single
molecule, glutathione disulfide
(GSSG)
67
68. A. Anti-Oxidant Scavenging
Enzymes
Glutathione Peroxidase
sulfhydryl reactions are also
oxidized in non-enzymatic chain
terminating reactions with
organic radicals
within cells, found principally in
the cytosol and
mitochondria, and are a major
means for removing H2O2
produced outside of
peroxisomes
contribute to our dietary
requirement for selenium and
account for the protective effect
of selenium in the prevention of
free radical injury
68
69. A. Anti-Oxidant Scavenging
Enzymes
Glutathione Reductase
reduces oxidized glutathione (GSSG) back to the
reduced form
contains an FAD and catalyzes transfer of electrons
from NADPH to the disulfide bond of GSSG
NADPH is essential for protection against free
radical injury; major source is the pentose
phosphate pathway (HMP)
69
70. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
convert free radicals to a nonradical
nontoxic form in nonenzymatic reactions
mostly are antioxidants
compounds that neutralize free radicals by
donating a hydrogen atom (with its one
electron) to the radical
reduce free radicals but themselves are
oxidized
70
71. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
common structural feature: conjugated double
bond system
a system of atoms covalently bonded with alternating
single and multiple bonds (e.g., C=C-C=C-C)
enables the electrons to be delocalized over the
whole system and so be shared by many atoms
delocalized electrons may move around the whole
system
71
72. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
common structural feature: conjugated
double bond system
may also be an aromatic ring
example: phenol
(C6H5OH, benzene
with hydroxyl group)
72
73. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
Vitamin E
-tocopherol
lipid soluble
efficient antioxidant and
terminator of free
radical chain reactions;
radical trap
little pro-oxidant activity;
most widely distributed;
most potent anitoxidant
73
74. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
Vitamin E
primarily to
protect against
lipid peroxidation
donates single
electrons to lipid
peroxyl radicals
(LOO•) to form
stable lipid
peroxide (LOOH)
74
75. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
Vitamin C
oxidation-reduction
coenzyme (collagen
synthesis, etc)
water soluble
circulates unbound in
blood, extracellular
fluid
important role in free
radical defense –
regenerate reduced
Vitamin E
75
76. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
L-ascorbate donates single electrons to
free radicals / disulfides in 2 steps
reacts also with superoxide, H2O2,
hypochlorite, hydroxyl and peroxyl radicals
and NO2
76
77. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
Carotenoids
-carotene
compounds with
functional oxygen-
containing substituents
on the rings
“chain-breaking”
antioxidants
“quench” singlet O2
“health benefits” of diets
high in fruits /
vegetables
77
81. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
Flavonoids
found in red
wine, green
tea, chocolates, other
plant-derived foods
group of structurally
similar compounds with
2 spatially separate
aromatic rings
81
82. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
Flavonoids
exhibit the same
ring structure
differ in ring
substituents (=O, -
OH and OCH3)
eg. Quercetin
fruit skins
antioxidant activity
82
83. B. Nonenzymatic Antioxidants
(Free Radical Scavengers)
Flavonoids
contribute to free radical defenses in various
ways:
free radical scavengers by donating electrons to
superoxide or lipid peroxy radicals
stabilize free radicals by complex-formation
inhibit enzymes responsible for superoxide anion
production
regeneration of reduced Vitamin E
efficient chelators of Fe and Cu (Fenton reaction)
e.g. Quercetin – effective in Fe chelation
83
85. Time to Take Five
NATIONAL INSTITUTES OF HEALTH
National Cancer Institute
Department of Health and Human
Services
Public Health Service
85
86. Count 'Em Up!
What's a serving of fruits and vegetables?
A serving is:
1 medium fruit or 1/2 cup of small or cut-up fruit
3/4 cup of 100% fruit juice
1/4 cup dried fruit
1/4 cup raw or cooked vegetables
1 cup raw leafy vegetables (such as lettuce, spinach)
1/2 cup cooked beans or peas (such as lentils, pinto
beans, kidney beans)
86
87. C. Endogenous Antioxidants
Uric Acid
formed from degradation of purines
and is released into the extracellular
fluids, including blood, saliva and
lung-lining fluid
with protein thiols, accounts for the
major free radical trapping capacity
of plasma
directly scavenge hydroxyl
radicals, oxyheme oxidants formed
between the reaction of hemoglobin
and peroxy radicals and peroxy
radicals themselves
87
88. C. Endogenous Antioxidants
Melatonin
nonenzymatic free radical scavenger that donates an
electron to “neutralize” free radicals
reacts with ROS and RNOS to form addition products
(“suicidal transformations”)
hydrophilic/hydrophobic; can pass through
membranes and the blood brain barrier
88
89. D. Metal Chelators
bind Fe and Cu
disable them from participating in Fenton
reaction ↓ OH• production
ferritin – multi-subunit protein shell
surrounding a Fe+3 core
transferrin – binds Fe+3
ceruloplasmin – converts Fe+2 to Fe+3
albumin – binds Cu+2 tightly and Fe+2 weakly
89
90. E. Compartmentation
various defenses against ROS are found in
different subcellular compartments
location of free radical defense enzymes
matches the type and amount of ROS generated
in each subcellular compartment
Separation of species and sites involved in ROS
generation from the rest of the cell
Fe being tightly bound to the storage
protein, ferritin, cannot react with ROS.
enzymes that produce H2O2 are sequestered in
peroxisomes
90
92. F. Repair Mechanisms
Repair mechanisms for:
DNA
removal of oxidized fatty
acids from membrane
lipids
oxidized amino acids
through protein
degradation and re-
synthesis of new proteins
92
93. Thank you very much!
NOEL MARTIN S. BAUTISTA, MD, DPPS, MBAH
Department of Biochemistry, Molecular Biology and Nutrition
