Nico Wanandy UNSW mechanism of antioxidant for the skin - ICNACS 2013, Jakarta

1. School of Biotechnology and Biomolecular Science
Mechanism of Antioxidant for the Skin
Dr Nico Wanandy and Dr Helder Marçal
24/10/2013

2. Skin: Epidermis
The source of oxidative stress
I. Exogenous – γ-irradiation, UV irradiation, drugs, xenobiotics, and toxin
metabolism
Sunlight (UV irradiation):
1. UVC (180-280 nm):
• Absorbed primarily by the atmospheric ozone layer
• Penetrate the skin to depth of approximately 60-80 μm
• Enormous energy and are mutagenic in nature
• Can damage DNA molecules directly
2. 5% UVB (280–314 nm):
• Penetrates epidermis and dermis (depth 160-180 μm)
• Oxidative stress, DNA damage, premature aging of the skin
3. 90-95% UVA (315-400 nm) – “Aging Ray”
• Penetrates deeper into the epidermis and dermis of the skin (depth 1000
μm)
• Barely excite DNA molecule directly
• Generate singlet oxygen (O•) and hydroxyl radical (OH•)
• Damaging to cellular macromolecules: proteins, lipids and DNA
II. Endogenous – metabolic pathways, mitochondrial respiration, oxidative
burst, phagocytosis, enzyme activities, aging and diseases

3. Reactive Oxygen Species

4. Proposed Mechanisms of the Oxidative Damage in the
Skin Following Exposure to UV Irradiation
Kang, S., et al. (2003). J Invest Dermatol 120(5): 835-841.

5. Essential antioxidants (Required vitamins):
1.
2.
3.
Vitamin C: kiwi fruit
Vitamin A precursor (β-carotene): carrot
Vitamin E (α-tocopherol): vegetable oils, nuts,
green leaves
Ratnam, D. V., et al. (2006). J Control Release 113(3): 189-207.
Other antioxidants:
1. Carotenoids: Lutein (yellow things): corn, squash, egg yolk
2. Flavonoids  Flavanols: Catechin, Epicathechin,
Epigallocathechin, Epigallocatechin gallate: chocolate, tea, apples
3. Flavonoids  Anthocyanidins (coloured fruit and vegetables):
a. Pelargonidin (red)
b. Cyanidin (purple)
c. Delphinidin (blue)
d. Peonidin (red)

6. Reactions Leading to the Formation of ROS
• Primary ROS :
• Superoxide radical (O2•−) created by a premature
electron ‘leak’ to oxygen in the electron transport
phase of aerobic metabolism.
• Secondary ROS: The unpaired electron in the valence
shell of the superoxide radical makes it reactive and it
subsequently reacts with other molecules to form
secondary radicals such as:
• Hydroxyl radical (OH•)
• Hydrogen peroxide (H2O2)
• Peroxyl radical (LOO•)
• Alternatively, It can also be split to form singlet
oxygen (O•).
• Under normal conditions the removal of ROS is regulated
by antioxidant enzymes such as:
• Superoxide dismutase (SOD)
• Glutathione peroxidase (GPx)
• Catalase (CAT)
Ferreira, I. C., et al. (2009). Curr Med Chem 16(12): 1543-1560.
Flora, S. J. (2009). Oxid Med Cell Longev 2(4): 191-206.
Lipid peroxidation
Haber-Weiss reactions
Fenton reactions

7. Targets of Free Radicals
Benov, L. and A. F. Beema (2003). Free Radic Biol Med 34(4): 429-433.
Dizdaroglu, M., et al. (2002). Free Radic Biol Med 32(11): 1102-1115.
Halliwell, B. and S. Chirico (1993). Am J Clin Nutr 57(5 Suppl): 715S-724S.
Lobo, V., et al. (2010). Pharmacogn Rev 4(8): 118-126.
Valko, M., et al. (2004). Mol Cell Biochem 266(1-2): 37-56.

8. Mechanisms of Oxidative Cellular Damage
and Cellular Defense against ROS
Superoxide
dismutase
(SOD)
Catalase
Glutathione
peroxidase
(GPx)
Garcia-Fernandez, M., et al. (2008). Endocrinology 149(5): 2433-2442.

9. Non-Enzymatic Antioxidant
“Some antioxidants perform this function by being oxidised themselves, thus performing a rate limiting role in initiation, propagation
and termination of radical chain reactions where the resulting ‘antioxidant radical’ is less reactive. Antioxidants differ in their efficacy
against differing substrates; some are potent free radical scavengers whilst others have stronger metal chelation effects, for example,
carotenoids are particularly effective at inhibiting the oxidation caused by singlet oxygen” - Niki & Noguchi (2000).
“Dietary antioxidants: substances which can (sacrificially) scavenge reactive oxygen/nitrogen (ROS/RNS) to stop radical chain
reactions, or can inhibit the reactive oxidants from being formed in the first place” - Huang, Ou, and Prior (2005):

10. Example of Non-Enzymatic Antioxidant Mechanism
Glutathione: γ-Glutamyl-Cysteinyl-Glycine:
a) The reduced form (GSH) is a strong antioxidant that
protects cells against damage caused by free radicals
and it recycles Vitamin C and E, so that they again
become active as antioxidants after been used in
antioxidant processes.
b) Serves as substrate/cofactor in GSH-linked enzymes
I. Glutathione Transferase (GST)
II. Glutathione Peroxidase (GPx)
c) The oxidised form (GSSG) is catalysed by Glutathione
Reductase (GSR) back to GSH using NADPH as
reductant
d) Glutathione is employed by the white blood cells as a
source of energy used for lymphoproliferation
(glutathione may help increase the resistance to
bacterial and viral infections)
e) Glutathione is a natural purifier (high concentrations
are found in the liver for detox purposes)

11. ARE/EpRE-mediated Regulation of γ-GCS (γ-glutamyl cysteine synthetase)
Cytoplasm
Under quiescent conditions, Keap1
sequestered and repressed Nrf2
Nucleus
ARE: Antioxidant Response Element
EpREs: Electrophil Response Element
Nrf2: NF-E2-related factor 2 (transcription factor) – master regulator of antioxidant response
Keap1: Kelch-like ECH-associated protein 1
Moskaug, J. O., et al. (2005). Am J Clin Nutr 81(1 Suppl): 277S-283S.

12. Defense Network in vivo Against Oxidative Stress
e.g. phenolic
compound
Niki, E. (2010). Free Radic Biol Med 49(4): 503-515.

13. Eukaryotic Cell Anatomy
MnSOD (SOD2) - tetramer
GPx – tetramer or monomer
GSR – homodimer
GST – homodimer or heterodimer
CAT – homotetramer
Cu-ZnSOD (SOD1) – homodimer
GPx – tetramer or monomer
GSR – homodimer
GST – homodimer or heterodimer
Extracellular
Cu-ZnSOD (SOD3) – tetramer

14. Damaged Mitochondria: Intramitochondrial / Intracellular source of ROS
Mitochondria is very susceptible to oxidative damage
because:
1. Close proximity to electron transport chain
2. Continuously exposed to ROS generated during
oxidative phosphorylation (it is estimated that up to 4%
of the oxygen consumed by cells is converted to ROS
under physiological condition)
3. Mitochondria has limited capacity of DNA repair
strategies and the lack of protection by histones
The downstream effect of mitochondrial damage:
1. Problem for ATP-dependent DNA synthesis and repair
mechanism
2. Mitochondrial disruption also resulted in the increase
rate of program cell death (apoptosis)
“Oxidative stress exerts deleterious effects on mitochondria function by directly impairing oxidative phosphorylation
through direct attack of proteins or membrane lipids. Mitochondrial damage produces mitochondrial dysfunction
decreasing MMP and ATP synthesis and increasing ROS production” – Morón and Castilla-Cortázar (2012)
Morón, Ú. M. and I. Castilla-Cortázar (2012).

15. Interaction of ROS and Mitogenic Cascade
NADPH Oxidase
O2
Peroxiredoxin
H2O2
O2-
Oxidative Stress
Sirtuins
FOXO
SMADs
ROS
Stress
MnSOD (reduced ROS)

16. Apoptosis Pathway
ROS

17. Three Modes of Action of Botanical Antioxidants
UV Irradiation
Afaq, F. and H. Mukhtar (2006). Exp Dermatol 15(9): 678-684.

18. Alleviating Effects of Natural Extracts on
Exogenous H2O2
Preliminary in vitro study using:
•
•
•
•
•
Human Adult Fibroblast (HAF)
Routinely passaged in DMEM + 10% FBS
24 hr incubation with natural extract (0.5 mM)
2 hr incubation with H2O2 (25 mM)
Interrogating cell viability via:
• LDH assay
• Cellular respiration

19. LDH Assay
A measure of membrane integrity of the cells subjected to an agent
Aim
• Identify any levels that may induce detrimental and
undesirable levels/result

20. LDH Activity
60
IU/L/mg protein
50
40
30
20
10
0

21. Aerobic Cellular Respiration
Aerobic Cellular Respiration happens in
Mitochondria. Three main reactions are involved:
1. Glycolysis occurs in cytoplasm of mitochondria
(requires 2 ATP to start/ makes 2 ATP)
2. Krebs Cycle occurs in matrix of mitochondria
(makes 2 ATP)
3. Electron Transport Chain occurs in mitochondria;
makes majority of ATP (32 ATP)
Out of 38 ATP Produced - energy of 2 ATP required
to start the process.

22. Oxygen Consumption Rate [OCR]
140
120
100
80
60
40
20
0

23. Cell Metabolic Function [NAD+]
6000
ng/mg protein
5000
4000
3000
2000
1000
0

24. Energy Availability (ATP)
20000
18000
nM/mg protein
16000
14000
12000
10000
8000
6000
4000
2000
0

25. Conclusion
Results:
The addition of H2O2:
• ↑ LDH activity
• ↓ Cellular respiration
• The natural extracts did not negatively affecting LDH activity and cellular
respiration
• The natural extract in the H2O2-challenged cells demonstrated that
cellular respiration and ATP were maintained whilst reducing the LDH
activity.
The damaging effect of exogenous H2O2 can be alleviated when cells were
subjected to natural extracts suggesting that the extracts contribute to
cellular endogenous protection from exogenous H2O2.

26. Acknowledgement
• SOHO Global Health
• Laboratory members:
• Dr. Helder Marçal
• Dr. Nady Braidy
• Mr. Alfonsus Alvin
• Ms. Sonia Ho
• Mr. Rodman Chan
• Ms. Hayley Cullen
–
temulawak extract

30. Thank You