Nico wanandy unsw mechanism of antioxidant for the skin


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

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Nico wanandy unsw mechanism of antioxidant for the skin

  1. 1. School of Biotechnology and Biomolecular Science Mechanism of Antioxidant for the Skin Dr Nico Wanandy and Dr Helder Marçal 24/10/2013
  2. 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. 3. Reactive Oxygen Species
  4. 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. 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. 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. 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. 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. 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. 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. 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. 12. Defense Network in vivo Against Oxidative Stress e.g. phenolic compound Niki, E. (2010). Free Radic Biol Med 49(4): 503-515.
  13. 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. 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. 15. Interaction of ROS and Mitogenic Cascade NADPH Oxidase O2 Peroxiredoxin H2O2 O2- Oxidative Stress Sirtuins FOXO SMADs ROS Stress MnSOD (reduced ROS)
  16. 16. Apoptosis Pathway ROS
  17. 17. Three Modes of Action of Botanical Antioxidants UV Irradiation Afaq, F. and H. Mukhtar (2006). Exp Dermatol 15(9): 678-684.
  18. 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. 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. 20. LDH Activity 60 IU/L/mg protein 50 40 30 20 10 0
  21. 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. 22. Oxygen Consumption Rate [OCR] 140 120 100 80 60 40 20 0
  23. 23. Cell Metabolic Function [NAD+] 6000 ng/mg protein 5000 4000 3000 2000 1000 0
  24. 24. Energy Availability (ATP) 20000 18000 nM/mg protein 16000 14000 12000 10000 8000 6000 4000 2000 0
  25. 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. 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
  27. 27. Thank You