Mechanism of Antioxidant for the Skin

Dr Nico Wanandy and Dr Helder Marçal24/10/2013

School of Biotechnology and Biomolecular Science

Skin: EpidermisThe 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

Reactive Oxygen Species

Kang, S., et al. (2003). J Invest Dermatol 120(5): 835-841.

Proposed Mechanisms of the Oxidative Damage in the Skin Following Exposure to UV Irradiation

Ratnam, D. V., et al. (2006). J Control Release 113(3): 189-207.

Essential antioxidants (Required vitamins):

1. Vitamin C: kiwi fruit2. Vitamin A precursor (β-carotene): carrot3. Vitamin E (α-tocopherol): vegetable oils, nuts,

green leaves

Other antioxidants:1. Carotenoids: Lutein (yellow things): corn, squash, egg yolk2. Flavonoids Flavanols: Catechin, Epicathechin,

Epigallocathechin, Epigallocatechin gallate: chocolate, tea, apples3. Flavonoids Anthocyanidins (coloured fruit and vegetables):

a. Pelargonidin (red)b. Cyanidin (purple)c. Delphinidin (blue)d. Peonidin (red)

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.

Reactions Leading to the Formation of ROS

Lipid peroxidation

Fenton reactionsHaber-Weiss reactions

• 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)

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.

Targets of Free Radicals

Mechanisms of Oxidative Cellular Damageand Cellular Defense against ROS

Garcia-Fernandez, M., et al. (2008). Endocrinology 149(5): 2433-2442.

Glutathioneperoxidase(GPx)

Catalase

Superoxide dismutase

(SOD)

“Some antioxidants perform this function by being oxidised themselves, thus performing a rate limiting role in initiation, propagationand termination of radical chain reactions where the resulting ‘antioxidant radical’ is less reactive. Antioxidants differ in their efficacyagainst 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 chainreactions, or can inhibit the reactive oxidants from being formed in the first place” - Huang, Ou, and Prior (2005):

Non-Enzymatic Antioxidant

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 enzymesI. 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)

ARE/EpRE-mediated Regulation of γ-GCS (γ-glutamyl cysteine synthetase)

Moskaug, J. O., et al. (2005). Am J Clin Nutr 81(1 Suppl): 277S-283S.

ARE: Antioxidant Response ElementEpREs: Electrophil Response ElementNrf2: NF-E2-related factor 2 (transcription factor) – master regulator of antioxidant responseKeap1: Kelch-like ECH-associated protein 1

Under quiescent conditions, Keap1 sequestered and repressed Nrf2

Nucleus

Cytoplasm

Niki, E. (2010). Free Radic Biol Med 49(4): 503-515.

Defense Network in vivo Against Oxidative Stress

e.g. phenolic compound

Eukaryotic Cell Anatomy

MnSOD (SOD2) - tetramer GPx – tetramer or monomerGSR – homodimerGST – homodimer or heterodimer

Cu-ZnSOD (SOD1) – homodimerGPx – tetramer or monomerGSR – homodimerGST – homodimer or heterodimer

ExtracellularCu-ZnSOD (SOD3) – tetramer

CAT – homotetramer

Damaged Mitochondria: Intramitochondrial / Intracellular source of ROS

“Oxidative stress exerts deleterious effects on mitochondria function by directly impairing oxidative phosphorylationthrough direct attack of proteins or membrane lipids. Mitochondrial damage produces mitochondrial dysfunctiondecreasing MMP and ATP synthesis and increasing ROS production” – Morón and Castilla-Cortázar (2012)

Morón, Ú. M. and I. Castilla-Cortázar (2012).

Mitochondria is very susceptible to oxidative damage because:1. Close proximity to electron transport chain2. 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

mechanism2. Mitochondrial disruption also resulted in the increase

rate of program cell death (apoptosis)

Interaction of ROS and Mitogenic Cascade

NADPH Oxidase

Peroxiredoxin

H2O2

O2

O2-

MnSOD (reduced ROS)

Stress

SMADs

FOXO

Sirtuins

Oxidative Stress

ROS

ROS

Apoptosis Pathway

UV Irradiation

Afaq, F. and H. Mukhtar (2006). Exp Dermatol 15(9): 678-684.

Three Modes of Action of Botanical Antioxidants

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

LDH AssayA measure of membrane integrity of the cells subjected to an agent

• Identify any levels that may induce detrimental and undesirable levels/result

Aim

0

10

20

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50

60

IU/L

/mg

prot

ein

LDH Activity

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.

0

20

40

60

80

100

120

140

Oxygen Consumption Rate [OCR]

0

1000

2000

3000

4000

5000

6000

ng/m

g pr

otei

nCell Metabolic Function [NAD+]

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

nM/m

g pr

otei

nEnergy Availability (ATP)

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.

Acknowledgement

• SOHO Global Health – temulawak extract

• Laboratory members:• Dr. Helder Marçal• Dr. Nady Braidy• Mr. Alfonsus Alvin• Ms. Sonia Ho• Mr. Rodman Chan• Ms. Hayley Cullen

Thank You