Detail metabolism of xenobiotics including generation of ROS, it's removal and anti oxidant mechanism including reperfusion injury

1. Metabolism of Xenobiotics
Dr Abhra Ghosh

2. FREE
RADICALS
• Molecule with an unpaired electron
• Highly reactive
• Short span of life
• Either oxidizing or reducing
• Activity depends on electron potential

3. ONE
ELECTRON
POTENTIAL
LIST
REDOX COUPLE E0/mV
HO- +2310
Aliphatic alkoxy radical +1600
Alkyl peroxyl radical +1000
GS-/GSH +920
PUFA- +600
Urate +590
Tocopherol +480
H2O2 +320
Ascorbate +282
Fe+3/Fe+2 +120
O2/O2
-2 -330

4. OXYGEN –
A DOUBLE
EDGED
SOWRD
• Oxygen is important to sustain life
• Required by all living organisms during respiration
• Produces ROS
• Most common one is superoxide radicals

5. WHY
OXYGEN IS
PRONE TO
PRODUCE
SUPEROXIDE
RADICAL
• Molecular oxygen is a paramagnetic molecule
• Contains two unpaired electrons with parallel spins
• Reduction of O2 requires direct insertion of electrons
• It requires inversion of spin of electron
• It is added in successive steps

6. REACTIVE
OXYGEN
SPECIES
• Univalent reduction of molecular oxygen produces O2
-
• O2 + e-
• O2
- + e- + 2H+
• O2
- + H2O2
• O2
- + H+
• NO + O2
-
O2
- (Superoxide)
H2O2
OH* + ‘O2
Fe+2
(Free hydroxy radical+
singlet oxygen)
HOO* (Hydroperoxy radical)
ONOO- (Peroxynitrite)
OH*

7. SOURCES
OF ROS IN
CELLS
1. Leakage from electron transport in mitochondria
2. Reactions in peroxisomes
3. Cytochrome P450 in endoplasmic reticulum
4. Respiratory burst
5. Exogenous sources

8. LEAKAGE FROM
ELECTRON
TRANSPORT
CHAIN IN
MITOCHONDRIA
Complex I
FMN
Fe-S
Complex II
FAD
Fe-S
Ubiquinone
cycle
Complex III
Cyt b
Fe-S
O2
-
Cyt c
ComplexIV
H2O2

9. FENTON
REACTION
HABER WEISS
REACTION
H2O2 + Fe+2 Fe+3 + OH* + OH-
Fe+3 + O2
-
Cu+
Fe+2 + O2

10. REACTIONS IN
PEROXISOMES
Fatty acyl CoA
Delta
enoyl-CoA
FAD
FADH2
Oxidase
O2
H2O2

11. CYTOCHROME
P450 IN
ENDOPLASMIC
RETICULUM
• Cytochromes are enzyme system required
in hydroxylation
Substrate
Substrate
- OH
NADPH + H+ + O2
Cyto P450
NADP+ + H2O + O2
-
H2O2OH*

12. RESPIRATORY
BRUST
NADPH + H+
NADP+
O2
H2O2 OH*Fe+2
HOCl*
Bactericidal
Phagocyte
Oxidase

13. EXOGENOUS
SOURCES
• Smoking, air pollution
• Ultraviolet and X ray radiation
• Drugs, pesticides etc
• Industrial toxins

14. EFFECTS OF
ROS
• PROTEINS
• 3 types of damage –
1. Protein fragmentation
2. Protein aggregation
3. Protein protein cross linking
• Most susceptible amino acids are –
Proline, histidine, arginine, cysteine, methionine

15. EFFECTS OF
ROS
• LIPIDS
• Initiation:
LH + OH*
L* (Lipid radical) + H2O
• Propagation:
L* + O2
LOO* + LH
LOO* (Lipid peroxyl)
LOOH (Lipid peroxide) + L*
• Termination:
LOO* + L*
L* + Vitamin E
Vitamin E* + L*
LOOH + LH
LH + Vit E*
Vit E + LH

16. EFFECTS OF
ROS
• NUCLEIC
ACID
• Involves nuclear DNA and mitochondria DNA
• Causes base alterations and strand breaks
• Binding of iron initiates production of OH*
• Mitochondrial DNA is more susceptible

17. DEFENCE
AGAINST
ROS
1. Antioxidants
a. Endogenous
- Enzymes
- Non enzymes
b. Exogenous
- Nutritional elements
2. Compartmentalization in cells
3. Damage repair mechanisms

18. ANTIOXIDANT
ENZYMES
1. SOD
• Helps in dismutation of superoxide radical
2O2
- + 2H+
• Two isoenzymes
• Mitochondrial - Mn+2 dependent
• Cytosolic – Cu-Zn dependent
• Exercise acts as a positive effector
H2O2 + O2

19. ANTIOXIDANT
ENZYMES
2. CATALASE
• Heme containing enzyme
• High Km value
• Destroy H2O2
H2O2 H2O + O2

20. ANTIOXIDANT
ENZYMES
3. GLUTATHIONE
PEROXIDASE
• Acts where H2O2 conc. is less
• Low Km value
• Require presence of Se
• Require presence of Reduced glutathione
H2O2 2H2O
GSH GS-SG
Glu Perox

21. NON
ENZYMATIC
ANTIOXIDANTS
• Transferrin, Ferritin
• Ceruloplasmin
• Albumin
• Glutathione
• Uric acid
• Bilirubin
• Ubiquinone

22. NUTRITIONAL
ANTIOXIDANTS
1. VITAMIN E
• Lipophilic free radical scavenger
• Terminator of propagation stage
• Active at high pO2
LOO* + TocOH
LOO* + ToCO*
ToCO* + Vit C
LOOH + ToCO*
LOOH + ToC
TocOH + Vit C (oxidized)
Vit C (reduced)
GSH

23. NUTRITIONAL
ANTIOXIDANTS
2. VITAMIN C
• Works in aqueous environment
• Hydrophilic, chain terminating
• Regenerates other antioxidant molecules

24. NUTRITIONAL
ANTIOXIDANTS
3. VITAMIN A
4. SELENIUM
• Chain breaking antioxidant
• Acts at low pO2
• Acts as cofactor for Glu Perox, Deiodinase
• Synergistic with Vit E

25. OTHER
DEFENCE
MECHANISMS
1. Compartmentation in cell
2. Repair mechanism

26. ANTIOXIDANTS
AS
PRO-OXIDANTS
• Vitamin at high cellular concentration
• Vit C reacts with oxygen to form superoxide
• Also reacts with Cu to form hydroxyl radical
• Vit A at high pO2 act as autocatalytic prooxidant
• Vit E form lipid soluble free radical

27. REPERFUSION
TISSUE INJURY
Ischemia Cellular death Release of Ca+2
Activate phospholipasesPerfusion ↑ O2
Cell membrane
fragmentation
Generation of ROS Cell death & release of
more calcium
↑ Xanthine oxidase