Oxidative Stress: an Introduction

Oxidative Stress: an Introduction Helmut Sies Institute for Biochemistry and Molecular Biology I, Heinrich-Heine-University Düsseldorf, Germany sies@uni-duesseldorf.de

Oxidative Stress: Reactive Oxygen Species, Antioxidants Nutritional (dietary) Oxidative Stress: Postprandial Oxidative Stress Total Antioxidant Capacity (TAC) Plasma Redox Potentials, Glutathione Photooxidative Stress: Carotenoids, Flavonoids Optimum Dose of Antioxidants?

Michaelis, L.(1939) Cold Spring Harbor Symp., 33-49

Das Sauerstoff übertragende Ferment: Cytochrome-Oxidase Otto Warburg (1883-1970) Kaiser-Wilhelm-Institut für Zellphysiologie, Berlin-Dahlem

Pathways of Hydrogen in the Living Organisation Angewandte Chemie 70 (1958) 552-570

In: Metabolic Compartmentation, Sies, H., ed. (1982) pp. 205-231, Academic Press, London

Single cell image of rogfp1 targeted to HeLa cell mitochondria obtained by fluorescence microscopy The rogfp1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near - 360 mv (assuming mitochondrial ph 8.0 at 37 C). Hanson GT et al (2004) J. Biol. Chem. 279, 13044-13053

Biological Half-Lives of Reactive Oxygen Species (ROS) (= Prooxidants) Sies, H. (1993) Eur. J. Biochem. 215: 213-219

Antioxidants in Biological Systems Enzymatic Nonenzymatic Superoxide Dismutases Ascorbate (Vitamin C) Glutathione Peroxidases Tocopherols (Vitamin E) Catalase Quinone Reductases Flavonoids Carotenoids Urate Glutathione (GSH)

Oxidative Stress Oxidative Stress Prooxidant - Antioxidant Balance Disturbance in favor of the prooxidants Pro- Oxidants Anti- Oxidants H. Sies (1985) in: Oxidative Stress, Academic Press, London, pp.1-8; (1986) Angewandte Chemie Int. Ed. 25, 1058-1071

Janus Oxidative Stress denotes a disturbance in the prooxidant/antioxidant balance in favor of the prooxidants, leading to potential damage In: Oxidative Stress (1985) Sies, H., Academic Press

Hydroperoxide Metabolism in Mammalian Systems Chance, Sies and Boveris (1979) Physiol. Revs. 58: 527-605

Oxidative Stress An imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage. Sies, H, Jones, D.P. in: Encyclopedia of Stress (2007), Vol. 3, 45-48 (Fink, G., ed.), Elsevier

Metabolic Fine-Tuning through Modulation by Dietary Micronutrients Postprandial Oxidative Stress Hyperglycemia Hypertriglyceridemia oxidizable Lipids oxidized Lipids Reactive Oxygen Species (ROS) ox- LDL Protection by Antioxidant Micronutrients e.g. Dietary Flavanols Endothelial Dysfunction Diabetic complications Atherosclerosis Sies, H., Stahl, W., Sevanian, A. (2005) J. Nutr. 135, 969-972

In industrialized societies, a significant part of the day is spent in the postprandial state Unsaturated fatty acids incorporated into LDL are suitable targets for oxidation, and oxidized LDL are an atherogenic factor. Lipid hydroperoxides already present in the diet are absorbed and contribute to the prooxidant load.

Effect of a Single High-Fat meal on Endothelial Function in Healthy Subjects Vogel RA, Corretti MS, Plotnick GD (1997) Am J Cardiol 79, 350-354

Vasoprotective endothelial effects of a standardized grape product in humans High-Fat Meal + Grape Product (standardized) High-Fat meal Chaves AA et al (2009) Vasc Pharmacol 50, 20-26

Red wine mitigates the postprandial increase of LDL susceptibility to oxidation. Natella et al (2001) Free Radic Biol Med. 30:1036-1044

Postprandial oxidative stress is attenuated when dietary antioxidants are supplied together with a meal rich in oxidized or oxidizable lipids. Ingestion of dietary polyphenols, e.g. from wine, cocoa, or tea, improves endothelial dysfunction and lowers the susceptibility of LDL lipids to oxidation.

High-flavanol cocoa drink ameliorates endothelial function in vivo through improved bioactivity and bioavailability of NO Flow-mediated dilation (%) 7 6 5 4 3 2 1 * RNO in plasma (nmol/l) 45 40 35 30 25 20 15 10 5 * 0 before 2 h after before 2 h after low-flavanol cocoa drink high-flavanol cocoa (17 mg/100ml) drink (176mg/10 0ml) 0 before 2 h after before 2 h after low-flavanol high-flavanol cocoa drink cocoa drink C. Heiss et al (2003) JAMA 290, 1030-1031

The issue of TAC Is there a use in non-compositional assays of antioxidant capacity?

A pioneering paper... Quantitative measurement of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation. The important contribution made by plasma proteins. Keith Ingold Wayner, Burton, Ingold, Locke (1985) FEBS Lett 187:33-7

followed by a flurry of assay methods... TRAP, TEAC, ORAC, FRAP...: Total Radical-trapping Antioxidant Parameter (TRAP) Trolox Equivalent Antioxidant Capacity (TEAC) Oxygen Radical Absorbance Capacity (ORAC) Ferric Reducing Antioxidant Power (FRAP)... Generic: TAC, Total Antioxidant Capacity

Transfer to the Public...

Note: TAC applied to in vivo: neither Total nor the real Antioxidant Capacity is measured what is measured by TAC is mainly ascorbate, urate and other minor components; in vivo antioxidant enzyme support is not assessed in isolated fluids out of steady-state

% Erhola et al. 1997 Wayner et al. Tubaro et al. 1987 Benzie & Strain 1998 1999 Courtesyof G. Bartosz, Lodz

Upshot: TAC in vitro (e.g. in foods or extracts): OK TAC in vivo (e.g. in plasma): not OK Publication of such data is encouraged Publication of such data is discouraged See: Total antioxidant capacity: appraisal of a concept. Sies, H. (2007) J Nutr. 137:1493-5

Sies, H. (2007) J Nutr 137, 1493-1495

Glutathione (L-γ-glutamyl-L-cysteinyl-glycine) GSH Transferases GSH Peroxidases GSSG Reductase γ Glutamyl Transpeptidase Sies, H. (1999) FRBM 27, 916-921

GLUTATHIONE What does it do? Roles in: signal transduction gene expression; protein folding cell proliferation apoptosis

Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple Oxidation-Reduction Potential (E(h)) of GSSG/2GSH correlates with the biological status of the cell: Proliferation E(h) approx. -240 mv Differentiation E(h) approx. -200 mv Apoptosis E(h) approx. -170 mv Schafer F, Buettner G (2001) Free Rad Biol Med 30, 1191-1212

Meier et al (1989) Biochem. J. 263, 539-545

Cytokines also regulate production of NO via intracellular signal pathways after Kroencke (2002)

Light Electronic Excitation Sensitizer Photooxidative Stress Sensitizer* ROS Singlet Oxygen LIPIDS DNA PROTEIN

Sens hν 1 Sens* ISC Car 3 Sens* Energytransfer 3 3 O2 Car* Reaction rate constant: Quenching of 1 O 2 10 9 (M -1 s -1 ) ß-Carotene 8 Lutein 8 Zeaxanthin 10 Lycopene 15 4-OH-ß-Carotene 9 Cryptoxanthin 9 Capsorubin 8 Sens + 1 O 2 Car

Carotenoids and Oxocarotenoids Polyene Structure The essential function of carotenoids is to prevent harmful photooxidative reactions There would be no photosynthesis were it not for the presence of carotenoids : Plants would get sunburnt!

Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Second-order rate constant near diffusion limit Di Mascio et al (1989) Arch. Biochem. Biophys. 274, 532-538

Irradiated Skin: Reddening 24 h after UV-Exposition 1 2 3 4 5 a value

Lycopene protects against UV-induced erythema Serum Lycopene Skin Carotenoids a Value Serum Lycopene (µm) 1,00 0,75 0,50 0,25 * * Skin Carotenoids inside arm (nmol/g) 0,6 0,4 0,2 Erythema - -a value 8 6 4 2 ns * day 0 wk 4 wk 10 day 0 wk 4 wk 10 day 0 wk 4 wk 10 n = 10; p = 0.002 Stahl et al (2001) J. Nutr. 131:1449-1451

Dietary tomato paste protects against ultraviolet light-induced erythema in humans Stahl et al (2001) J Nutr 131: 1449-1451

Cocoa Polyphenols: Flavanols Cocoa with high flavanol content protects against UV-induced erythema 6 Low flavanol High flavanol -a-value 5 4 3 2 * * 1 0 0 6 12 week 0 6 12 *significantly different from wk 0; p < 0.05 Heinrich et al (2006) J. Nutr. 136, 1565-1569

High-frequency ultrasound scan skin density at week 0 after 12 weeks of consuming high flavanol cocoa beverage Heinrich et al (2006) J. Nutr. 136, 1565-1569

Skin surface profiles at week 0 after 12 weeks of consuming high flavanol cocoa beverage Corresponding top view of skin at week 0 after 12 weeks Heinrich et al (2006) J. Nutr. 136, 1565-1569

Is there an optimum dose?

UV-Induced Lipid Peroxidation in Skin Fibroblasts 2,00 1,80 Lycopene prooxidant TBARS/Protein [pmol/µg] 1,60 1,40 1,20 1,00 0,80 0,60 0,40 antioxidant Control: lycopene -free cells exposed to UV-light; TBARS 0.98 ± 0.11 pmol MDA/µg protein 0,20 0,00 0,00 0,10 0,20 0,30 0,40 0,50 Lycopene/Protein [pmol/µg] Eichler et al (2002) Photochem. Photobiol. 75, 503-506

Odds ratios for peripheral arterial disease by serum selenium levels Curves represent adjusted odds ratios (solid line) Bleys J et al (2009) Amer J Epidemiol 169: 996-1003

Acknowledgements Wilhelm Stahl Tankred Schewe Cristina Polidori Lars-Oliver Klotz Institut für Umweltmedizinische Forschung, Heinrich-Heine-Universität Düsseldorf Deutsche Forschungsgemeinschaft Ernst-Jung-Stiftung für Medizin National Foundation for Cancer Research (NFCR), Bethesda