老化與抗氧化能力 及其相關分子檢測

1. 老化與抗氧化能力及其相關分子檢測 Dr.曾婉芳

2. Oxidative stress

3. Oxidative Stress • Reactive oxygen species (ROS) • ROS and oxidative stress • Antioxidant system • Oxidative damage • Oxidative stress and apoptosis • Oxidative stress and aging • Oxidative stress and cancer • ROS as signaling molecules

4. Reactive oxygen species (ROS) • ROS • OH. (hyroxyl radical) • O2-. (superoxide radical) • H2O2 (hydrogen peroxide) • NO. (nitric oxide) • Oxidative stress • Oxidative damage

6. Toxic effects of ROS • Protein oxidation • Lipid peroxidation • Nucleic acids damage • Double-strand DNA breaks • Single-strand DNA breaks • Change DNA bases • 8-oxoguanine • Thymine glycol

9. Lipid peroxidation • Measure the malondialdehyde formed • Lipid peroxidation is a chain reaction. • Each fatty acyl moiety that undergoes peroxidaion generate a radical that can initiate another peroxidation reaction.

11. Intracellular sources of free radicals • Mitochondrial electron transport system • Superoxide radical and semiquinone radical • Microsomal (ER) electron transport system • Superoxide radical and H2O2 • Arachidonic acid metabolism • Reactions within peroxisome • Superoxide radical and H2O2

12. Intracellular sources of free radicals • In cytosol • Xanthine oxidase oxidizes xanthine and generates H2O2 • Amino acid oxidases generates H2O2 as their ordinary products

14. H2O2 and O2-. may diffuse from their subcellular sites of production and affect the whole cell • H2O2 can cross biological membranes

15. NO.synthesis

16. Reactive nitrogen species (RSN) • Inactivation of respiratory chain complexes; inhibition of protein and DNA synthesis • RNS are reduced or inactivated through the generation of a disulfur bond between two glutathione molecules to form oxidized glutathione

17. Dietary oxidants • Generation of ROS • ROS are reduced or inactivated through the generation of a disulfur bond between two glutathione molecules to form oxidized glutathione

18. Xenobiotics • Man-made compounds with chemical structures foreign to a given organism • Induce cancer • Glutathione is involved in the conjugation of epoxides to less toxic compounds that will be eventually excreted

19. Antioxidative system • Antioxidant • Glutathione, GSH • Vitamin C, E • Cysteine • Protein-thiol • Cerutoplasmin: important in reducing Fe3+ release from ferritin • Antioxidative enzyme

20. Glutathione (GSH)

22. Antioxidative enzyme • Catalase • Superoxide dismutase • Glutathione peroxidase • Glutathione reductase • Gluththione S-transferase • Glucose-6-phosphate dehydrogenase • DT-diaphorase

23. Catalase (EC 1.11.1.6) • 2H2O2 2H2O＋O2 catalase • A homotetrameric haeminenzyme, 240 kD • Subunit 60 kD • Four ferriprotoporphyrin groups • One of the most efficient enzymes known • It is so efficient that it cannot be saturate by H2O2 at any concentration

24. Superoxide dismutase (SOD. EC 1.15.1.1) • Human SOD • Cytosolic CuZn-SOD • Mitochondrial SOD: MnSOD • Extracellular SOD • 2O2-.＋ 2H＋H2O2 ＋ O2 superoxide dismutase

25. Manganese SOD (MnSOD) • A homotetramer (96 kDa) containing one manganese atom per subunit • Cycles from Mn(III)–Mn(II) and back to Mn(III) during the dismutation of superoxide

26. Cytosolic CuZn-SOD • Two identical subunits of about 32 kDa • Each containing a metal cluster, the active site, constituted by a copper and a zinc atom bridged by a common ligand: His 61 • Inactivation of copper- and zinc-containing SOD by H2O2 is the consequence of several sequential reactions

27. Inactivation of cytosolic CuZn-SOD by H2O2 • Reduction of the active site Cu(II) to Cu(I) by H2O2 • Oxidation of the Cu(I) by a second H2O2, thus generating a powerful oxidant, which may be Cu(I)O, Cu(II)OH or Cu(III) • Oxidation of the histidine, causing loss of SOD activity

28. Extracellular superoxide dismutase (EC-SOD) • A secretory, tetrameric, copper and zinc containig glycoprotein • High affinity for certain glycosaminogycans such as heparin and heparan sulfate • In the intersticial spaces of tissues • In extracellular fluids, accounting for the majority of the SOD activity of plasma, lymph, and synovial fluid

29. EC-SOD • Not induced by its substrate or other oxidants (xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, a-naphthoflavone, hydroquinone, catechol, Fe2+, Cu2+, buthionine sulphoximine, diethylmaleate, t-butyl hydroperoxide, cumene hydroperoxide, selenite, citiolone and high oxygen partial pressure) • Its regulation in mammalian tissues primarily occurs in a manner coordinated by cytokines, rather than as a response of individual cells to oxidants

30. Nickel superoxide dismutase(Ni-SOD) • Purified from the cytosolic fraction of Streptomyces sp. and Streptomyces coelicolor • Four identical subunits of 13.4 kDa, stable at pH 4.0–8.0, and up to 70°C

31. Glutathione peroxidase(GP, EC 1.11.1.19) glutathione peroxidase ROOH  ROH＋H2O 2GSH GSSG

32. Glutathione peroxidase (GP) • GP contains covalently bound Se (selenium) in the form of selenocysteine

33. GPX isoenzymes • Cytosolic GPX (cGPX) • Mitochondrial GPX (GPX1) • found in most tissues • Predominantly present in erythrocytes, kidney, and liver • Phospholipid hydroperoxide glutathione peroxidase GPX4 (PHGPX) • Cytosolic GPX2 (GPX-G1) • Extracellular GPX3 (or GPX-P) • GPX5 • Expressed specifically in mouse epididymis, Selenium-independent

34. GPX • cGPX and GPX1 reduce fatty acid hydroperoxides and H2O2 at the expense of GSH • Cytosolic GPX2 (GPX-G1) and extracellular GPX3 (GPX-P) are poorly detected in most tissues except for the gastrointestinal tract and kidney, respectively.

35. GPX1 • 80 kD, contains one selenocysteine (Sec) residue in each of the four identical subunits, which is essential for enzyme activity • The principal antioxidant enzyme for the detoxification of H2O2 has for a long time been considered to be GPX, as catalase has much lower affinity for H2O2 than GPX

36. PHGPX • Found in most tissues • Highly expressed in renal epithelial cells and testes • Located in both the cytosol and the membrane fraction • Directly reduce the phospholipid hydroperoxides, fatty acid hydroperoxides, and cholesterol hydroperoxides that are produced in peroxidized membranes and oxidized lipoproteins

37. Tissue-specific functions of individual glutathione peroxidases • All glutathione peroxidases reduce hydrogen peroxide and alkyl hydroperoxides at expense of GSH • Four glutathione peroxidases isozymes 1.Classical glutathione peroxidase (cGPx) 2. Gastrointestinal glutathione peroxidases (GI-GPx) 3.Plasma GPx (pGPx) 4. Phospholipid hydroperoxide glutathione peroxidases (PHGPx)

38. Classical glutathione peroxidase (cGPx) • Ubiquitously distributed • Reduces only soluble hydroperoxides, such as H2O2, and some organic hydroperoxides, such as hydroperoxyl fatty acids, cumene hydroperoxide, or t-butyl hydroperoxide

39. Gastrointestinal glutathione peroxidases(GI-GPx) • Expressed in gastrointestinal tract • Provides a barrier against hydroperoxides derived from the diet or from metabolism of ingested xenobiotics • Substrate specificity is similar to that of cGPx

40. Plasma GPx (pGPx) • Expressed in tissues in contact with body fluids, e.g., kidney, ciliary body, and maternal/fetal interfaces

41. Phospholipid hydroperoxide glutathione peroxidases (PHGPx) • Protects membrane lipids • Reduces hydroperoxides of more complex lipids like phosphatidylcholine hydroperoxide • Reduces hydroperoxo groups of thymine, lipoproteins, and cholesterol esters • Unique in acting on hydroperoxides integrated in membranes • Silence lipoxygenases • Becomes an inactive structural component of the mitochondrial capsule during sperm maturation

42. Glutathione reductase (GR) glutathione reductase GSSG＋H＋ 2GSH NADPH NADP＋

43. Glucose-6-phosphate dehydrogenase (G6PD) glucose-6-phosphate dehydrogenase, Mg2+ Glucose-6-phosphate  6-phosphoglucono-δ-lactone NADP＋NADPH

44. DT-diaphorase • NAD(P)H：(quinone acceptor) oxidoreductase (EC 1.6. 99.2) • In cytosol • Two electron transfer of quinone compounds Quinone  Hydroquinone

45. Glutathione S-transferase (GST) • Detoxification of toxic compounds (RX) to increase the solubility of the compound • The less toxic derivative of the original compound can then be excreted in the urine

46. Detoxification by glutathione S-transferase (GST)

47. Heme oxygenase • Heme  biliverdin bilirubin • A major stress protein induced in cells response to oxidant stress • Bilirubin is an efficient plasma or serum scavenger of singlet 1O2, O2-., and peroxy radicals

48. Oxidants as stimulators of signal transduction • Oxidants • Superoxide • Hydrogen peroxide • Hydroxyl radicals • Lipid hydroperoxides

49. ROS act as second messengers • Ligand-receptor interactions produce ROS and that antioxidants block receptor-mediated signal transduction led to a proposal that ROS may be second messengers

50. Reactive oxygen species (ROS) as second messengers • Generation of ROS by cytokines Ligand ROS Tumor necrosis factor- H2O2/HO Interleukin 1 H2O2/O2- Transforming growth Factor-1 H2O2 Platelet derived growth factor H2O2 Insulin H2O2 Angiotension II H2O2/O2- Vitamin D3 O2- Parathyroid hormone O2-