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A Hypothesis for the Physiological Antioxidant Action of the Salicylates.

A Hypothesis for the Physiological Antioxidant Action of the Salicylates. I. Francis Cheng Department of Chemistry University of Arizona Tucson, Arizona 85721 Tel. (520) 621-6340 ifcheng@u.arizona.edu. Seminar Outline. A brief history of the salicylates

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A Hypothesis for the Physiological Antioxidant Action of the Salicylates.

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  1. A Hypothesis for the Physiological AntioxidantAction of the Salicylates. I. Francis Cheng Department of Chemistry University of Arizona Tucson, Arizona 85721 Tel. (520) 621-6340 ifcheng@u.arizona.edu

  2. Seminar Outline • A brief history of the salicylates • Accepted model for acetylsalicylic (aspirin) action. • Weakness of accepted model. • Hypothesis for salicylate action. • Experiments. • Discussion. • Proposed Studies.

  3. History of Aspirin • Plant Based Product • Folk remedy for centuries, known to relieve pains and fevers. • 1828 - active ingredient isolated by Johann Buchner. • Found effective for fevers, inflammation, and pains but found to cause stomach irritation. • 1898 - Felix Hofmann (Bayer) synthesizes and tests Acetylsalicylic Acid (Aspirin) • Just as effective but less irritating than salicylic acid.

  4. Accepted model for acetylsalicylic action. • Proposed in the 1970's - John Vane (1982 Nobel Prize) • Irreversible inactivation of Prostaglandin Synthase Action. -Key enzyme in the arachidonic acid cascade -Prostaglandins are local hormones that regulate inflammation blood clotting • PG consists of two components, Aspirin works on cyclooxygenase. -by acetylation of serine residue. • Inhibition of Cyclooxygenase results in reduction of inflammation. Nature-New Biology 264 (1971) pp84-90.

  5. Weakness of the acetylation explanation. • Vane's Theory Describes The Action of Aspirin • But, How Does Salicylic Acid Exert Its Medicinal Action? • Lacks an Acetyl Group! • Pharmacological Literature Indicates That Salicylic Acid Exerts Anti-inflammatory Action Almost as Potent As Acetylsalicylic Acid. • Yet Salicylic Acid Lacks an Acetyl Group That Forms the Center Piece of Vane's Theory for Acetylsalicylic Acid

  6. Other Weaknesses of the Acetylation Mechanism. • Does not explain other documented medicinal effects of aspirin. • Aspirin acts as a chemopreventative for...... • Heart and circulatory diseases • Parkinson’s and Alzheimer’s diseases • Cancers • Cataracts • All of the above may be due to oxidative damage by oxygen containing free radicals.

  7. Formation of Activated Oxygen • O2.- and H2O2 released asRespiration by-products, [H2O2] = 10-7 [O2.-] = 10-11 • Also, Inflammation response(pathogen defense) by white blood cells • Physiological oxidative damage linked to chronic inflammation Physiological Reviews, 59 (1979) pp527-605.

  8. Goal of Respiration. (CH2O)n + O2 = nCO2 + nH2O I n c r e a s i n g R e d u c i n g P o w e r ( - ) ( C H O ) ( s u g a r s ) o ' 2 n E ( p H 7 ) . - + 1 e - 0 . 4 5 v o l t s O O 2 2 Redox Potential + 2 e G = n F E D + O + 2 H H O 0 . 3 0 V 2 2 2 + 4 e + O + 4 H 2 H O 0 . 8 2 V 2 2 ( + ) I n c r e a s i n g O x i d i z i n g P o w e r • H2O2 & O2.- are known as “activated oxygen species”

  9. Dangers of Activated Oxygen Species • Hydrogen peroxideFenton Reaction H2O2 + FeII(L)n = FeIII(L)n + HO- + HO. HO. + e- = HO- Eo = 1.8 volts • Superoxide ionDisproportionation to H2O2 O2.- + O2.- + 2H+ = H2O2 + O2 Reducing agent for Fenton rxn. O2.- + FeIII(L)n = FeII(L)n + O2 Reduces Fe3+(insoluble) to Fe2+ (soluble) physiological evidence indicates that O2.- is may be more toxic than H2O2.

  10. Hydroxyl Radical Damage to Biological Molecules Results in ..... • Denaturation of lens proteins cataracts • DNA strand breakage damage to genes aging cancers mitochondrial dysfunction • Fatty acid cross linking circulatory diseases • Damage to nervous system Parkinson’s Alzheimer’s diseases • Summary Hydroxyl radicals are the likely source of physiological oxidative damage -Scientific American, December 1992, pp131-141.

  11. Iron complexes and activated oxygen are conspirators in the oxidative damage to physiological components • FeII[complex] + H2O2 = FeIII[complex] + HO- + HO. • Fe and disease origins Recently Discovered Statistical Implications in - Heart Diseases - Strokes - Cancers - Cataracts - Alzheimer’s - Parkinson’s • Key Point Ailments due to active oxygen forms and iron are closely linked Bioelectrochemistry and Bioenergetics, 18 (1987) pp105-116.Ibid, 18 (1987) pp3-11. Biochemistry, 31 (1992) pp11255-11264. Circulation, 86 (1992) pp803-811. New England Journal of Medicine,320 (1989) 1012. Iron and Human Disease, CRC Press, Boca Raton, FL, 1992.

  12. Migration of Fe Under Conditions of Oxidative Stress Fe Containing Enzymes H2O2 + O2.- Oxidized Ligands Fe2+ + ATP, citrate + H2O2 + O2.- Fe(L) + HO. Fe(L)

  13. Hypothesized Antioxidant Properties of Salicylates. • Aspirin may play a role in the moderation of physiological oxidative damage. • Hypothesized because of aspirin’s ability to act as a chemopreventative of many diseases associated with oxidative damage. Free Radicals in Biology and Medicine9, (1990) 299.

  14. Proposed Route of Antioxidant Action for Aspirin. (literature) • Salicylates act as Hydroxyl Radicals Scavengers.

  15. Problems with Radical Scavenging Hypothesis. • Physiological concentration of aspirin (10-4 M) cannot compete with the oxidative damage to cellular components. • Most organics (physiological components) will react with HO. at the same rate as salicylates k = 1010M-1 s-1 (diffusion limited kinetics). • Acetaminophen is a more effective hydroxyl radical scavenger. k = 1.5 x 1010 M-1 s-1 lacks - chemopreventative effects - anti-inflammation Summaryradical scavenging alone cannot explain the antioxidant characteristics of salicylates.

  16. Alternative Hypothesis forSalicylate Antioxidant Behavior. • Key Point Salicylates moderate iron activity rather than HO radical scavenging. Salicylate may aid in one or more of the following antioxidant actions I) Redox deactivation of Fe2+/3+ (observed in vitro) II) Superoxide Dismutase Action. III) Catalase Action.

  17. Proposed Hypothesis (Continued) I)Storage and Transport of Fe. Redox Deactivation Requires Fenton Inactive Forms (shift Fe2+/3+ threshold to thermodynamically unfavorable potentials) • Animals (Humans) - Ferritin, Transferrin • Plants & Bacteria - Siderophores II)Superoxide Dismutase (SOD) Action. O2.- + 2H+ + e- = H2O2 III)Catalase Action. 2H2O2 = 2H2O + O2

  18. Salicylate as an inhibitor of Fenton processes.Redox Deactivation of Fe2+/3+ • Salicylates as chelation agent of iron ions. -may be plantsiderophores - iron transport agents • Exact structure may vary with pH Hand book of Chemical Equilibria in Analytical Chemistry, Chichester, U.K., Ellis Horwood Limited, 1985, p163. log B3 = 35.5

  19. Outline of Experimental Section. • Electrochemistry - cyclic voltammetry experiments Tells us something about thermodynamic ability to drive Fenton reaction. • DNA oxidations via Fenton reaction. Examine the ability of salicylates to prevent the degradation of calf thymus DNA via Fenton reaction.

  20. FeII[sal] FeIII[sal] + e- Redox Potential of Fe-Sal Indicates that it is a Fenton Inactive Complex. Cyclic voltammogram of iron-salicylate (0.5 mM Ferric Nitrate with 2.0 mM Salicylate) at pH 7.2, 0.05 M phosphate buffer with a potential sweep rate of 5 mV/sec. The electrodes consisted of a 0.071 cm2 wax impregnated graphite disk with a Ag/AgCl, saturated KCl reference (0.197 volts vs. SHE). Potential versus SHE -0.4 0.4 Eredox = 0.370 volts vs. SHE at pH 7.2 FeII[sal] e- + FeIII[sal]

  21. Salicylate chelates iron into a Fenton inactive form • Thermodynamics of the Fenton Reaction Stronger Reducing Agents (-) } EFe[EDTA] EOxidases EOxygenases Fenton Active E0Fenton = 0.307 volts x EFe-sal = 0.370 volts Fenton Inactive

  22. Evidence for Fenton Reaction Inertness of Fe-salicylate from Cyclic Voltammetry experiments. • Electrochemical electrocatalytic wave for FeIII(EDTA) reduction in the presence of H2O2 Electrode:FeIII(EDTA) + e = FeII(EDTA) 0.090 volts SHE Solution:FeII(EDTA) + H2O2 = FeIII(EDTA) + HO- + HO. • Results in enhanced electroreduction current for FeIII(EDTA) wave, no electro-oxidation wave for FeII(EDTA)

  23. Cyclic Voltammetry of FeII/III [EDTA] in the Absence and Presence of H2O2 -0.7 Potential vs. Ag/AgCl m A 0.4 A) 0.1 mM FeIII(EDTA) B) +10 mM H2O2. Potential sweep rate = 5 mV/sec pH 7.2 0.05 M phosphate buffer with a potential sweep rate of 5 mV/sec 0.071 cm2 wax impregnated graphite disk Ag/AgCl, saturated KCl reference (0.197 volts vs. SHE). A Current B A 1.0 m

  24. Results of H2O2 electrocatalytic voltammetry. Potential H2O2 Reduction CuI(EDTA) 0.450 volts No FeII(sal)30.370 No H2O2 = HO- + HO.0.307 ---- FeII(EDTA) 0.090 Yes CuI(sal)20.050 Yes • Important Predictions.If Redox Deactivation Hypothesis Works Then…. • Salicylate acts as an Antioxidant for Fe but not Cu. • EDTA acts as an Antioxidant for Cu but not Fe.

  25. Important Predictions (continued). • If radical scavenging is the predominate mechanism for salicylate antioxidant action then….. • Salicylate (k =1010 M-1s-1) • will act as a antioxidant for both Fe and Cu • EDTA (k = 109 M-1s-1) • will act as a antioxidant for both Fe and Cu.

  26. DNA as a Probe for Hydroxyl Radical Production. • DNA Strand is an efficient chelator of iron and copper ions. Binding Constant 1012 Primarily through phosphate residues • DNA-FeII ,- CuI complexes participates in Fenton type chemistries. • DNA degradation by .OH (or other oxidizing products) leads to attack on deoxyribose residues which releases bases from strands. Adenine, Thymine, Guanine, Cytosine • Products are easily quantifiable by HPLC. UV detection at 254 nm Key Point - DNA strand is a convenient probe for detection of hydroxyl radical. JACS 1992, 114, pp2303-2312.

  27. DNA Incubation Studies. • Fe-DNA complexEredox{FeII/III(DNA)} = -0.10 volts SHE FeIII(DNA) + Ascorbate = FeII(DNA) + Deoxyascorbate FeII(DNA) + H2O2 = FeIII(DNA) + HO- + HO. Conditions0.1 mM Fe(NO3)3, 1.0 mM ascorbate, and 7.8 mM H2O2 DNA (0.2 mM in base pairs), 120 minutes • Incubation of DNA with Fe-EDTA FeIII(EDTA) + Ascorbate = FeII(EDTA) + Deoxyascorbate FeII(EDTA) + H2O2 = FeIII(EDTA) + HO- + HO. Conditions0.1 mM Fe(NO3)3, 0.4 mM EDTA, 1.0 mM ascorbate, and 7.8 mM H2O2,DNA(0.2 mM in base pairs), 120 minutes

  28. HPLC chromatogram following incubation of calf thymus (CT) DNA A)salicylate absent. B)0.4 mM salicylate present. Salicylate retards oxidative DNA damage due to Fenton type processes Retention times; Guanine, 1.09 mins.; Thymine, 1.44 mins.; Adenine 2.35 mins Separation conditions:50/1 water to methanol mobile phase, C18 reversed phase Zorbex cartridge column, absorbance detection at 254 nm.

  29. (Thousands) HPLC Detector Response HPLC incubation results 100 80 Thymine 60 Adenine • DNA Incubation with… A) 0.1 mM Fe(NO3)3B) 0.1 mM Fe[EDTA] C) 0.1 mM Fe(NO3)3 and D) 0.1 mM Fe[EDTA] and 0.4 mM salicylate 0.4 mM salicylate • Salicylate decreases oxidative DNA damage due to Both Fe-DNA and Fe(EDTA) complexes 40 20 0 A B C D

  30. Salicylates may compete for Fe chelation with oxidized EDTA EDTA hydroxyl radical scavenging rate, k = 109 M-1 s-1 Under inflamed conditions Fe undergoes migration due to oxidative attack of low molecular weight ligands

  31. Summary of DNA Incubation Experiments. Incubation-10 Minutes Damage to CT-DNA Control 0.5 mM Ascorbate NO 5.0 mM H2O2 + 0.1 mM Fe(EDTA) YES + 0.1 mM Cu(EDTA) NO + 0.1 mM Fe(salicylate) NO + 0.1 mM Cu(salicylate) YES Confirms Redox deactivation hypothesis

  32. Summary of DNA Incubation Experiments Excess Ligand (salicylate or EDTA) Incubation 10 minutes Damage to CT-DNA Control 0.5 mM Ascorbate NO 5.0 mM H2O2 + 0.1 mM Cu(salicylate) YES + 10.0 mM salicylate + 0.1 mM Fe(EDTA) YES + 50.0 mM EDTA Indicates that radical scavenging is not an important mechanism.

  33. Incubation Results with Aspirin • Acetylsalicylic acid cannot chelate iron • slowly hydrolyzes to salicylic acid (t1/2 = 20 min.) • Radical scavenging rates; aspirin = salicylate Incubation 10 minutes CT-Damage Control 0.5 mM Ascorbate NO 5.0 mM H2O2 + 0.1 mM Fe(NO3)3 YES + 0.4 mM aspirin

  34. Release of adenine with incubation time for controls, and presence of salicylate, and aspirin. • Adenine Release • Less than 10 minutes aspirin = control • Greater than 60 minutes aspirin = salicylic acid • Results consistent with acetylsalicylic acid to salicylic acid Control HPLC Detector Response (254 nm) Salicylic Acid Acetylsalicylic Acid 0 20 40 100 Incubation Time (min)

  35. Outline of Discussion • Role of pH in the Fenton Reaction • Implications in inflammation and cancer • pH and the FeII/III[salicylate] redox potential • This is a key feature in salicylate’s antioxidant ability

  36. The role of H+ activity and physiological oxidative damage. • Fenton Reaction is pH sensitive H2O2 + e- = HO- + HO. EFenton = 0.732 -(0.059 pH) where [H2O2] = [HO.] = 1 at pH 7.2 EFenton = 0.307 volts SHE at pH 5.5 EFenton = 0.408 volts SHE • Fenton threshold becomes more facile with decreasing pH. • Important consideration Inflamed, damaged, or tumorous tissues may reach pH’s as low as 3.5

  37. FeII/III[salicylate] potential is pH dependent. 2 EFe(sal) = 0.793 - (0.059 pH) • Measured by Cyclic Voltammetry Potential (volts vs. SHE) 1 0 2 4 6 8 10 pH

  38. pH dependence may be due to HO- complexation • FeIII(sal)n + HO- = FeIIIOH(sal)n • FeIIIOH(sal)n + e- = FeII(sal)n + HO- III RT [ Fe ( - OH )( sal ) ] n 0 E = E + ln - II nF [ Fe ( sal ) ][ HO ] n • E = const - 0.059 pH • E = 0.793 - 0.059 pH

  39. Fenton threshold and the FeII/III(sal) redox potential 2 1.5 • FeII/III(sal) redox potential closely parallels EoFenton • Remains just slightly thermodynamically uphill • Why does salicylic acid not seek to maximize Fe deactivation? • By increasing FeII/III potential E0Fe-Sal = 0.793 - 0.059pH Potential (volts SHE) 1 0.5 E0Fenton = 0.732 - 0.059pH 0 0 2 4 6 8 10 pH

  40. HypothesisPossible Significance of the close parallel of Fe II/III(sal)n and Standard State Fenton threshold. 2 • Superoxide Dismutation. O2.- + 2H+ + e- = H2O2 Eo = 1.77 volts E = 1.77 + 2(0.059)pH • Salicylic acid may seek to maximize SOD activity with a minimum of Fenton type reactivity. S u p e r o x i d e D i s m u t a t i o n Zone I 1.5 1 EFe-Sal 0.5 Zone II Fenton Threshold Zone III 0 0 2 4 6 8 10 pH

  41. ThermodynamicSuppression of HO. Production by Salicylate. • Reduction: H2O2 + e = HO- + HO. • Oxidation: FeII(sal)n = FeIII(sal)n + e • Ecell = Ered - Eox Eox = 0.793 - 0.0591pH Calculate equilibrium value for product/reactant ratio @ pH 7 (Ecell= 0) • Healthy Tissue Maintains [H2O2] = 10-9 - 10-7 (Physiological Reviews, 59 (1979) p564.) Salicylic acid is a modest suppression agent of HO.

  42. Thermodynamic Analysis of Superoxide Dismutase Activity of Iron-Salicylate Reduction: O2.- + 2H+ + e- = H2O2 Oxidation: FeII[sal] = FeIII[sal] + e- . - O ] [ 2 Ecell = Ered - Eox Ered = 1 . 77 - 0 . 118 pH + 0 . 0591 log [H2O2] Eox = 0.793 - 0.0591 pH @ pH 7 E Spontaneous until cell . - [ O ] 2 - 10 = 2 . 94 x 10 [ H O ] 2 2 Salicylic acid may be an excellent suppression agent of O2.-

  43. Equilibrium SOD and Fenton Ratios vs. Iron Chelate Redox Potential Equilibrium values (from Nernst equation) for SOD action and Fenton reaction moderation as a function of the redox potential of FeII/III transition of a chelate. pH 7 10 10 FeII/III[salicylate] Fenton Rxn Moderation 5 5 SOD Action 0 0 -5 -5 -10 -10 -15 -15 -20 -20 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 Redox Potential of Chelated Iron (SHE)

  44. Conclusions • Antioxidant Action via Suppression of Fenton Reaction. • Redox inactivation, E = 0.793 - 0.059pH, rather than HO. radical scavenging • DNA Oxidation Studies with Fe2+/3+and Cu1+/2+ with salicylate and EDTA.

  45. Future Research • Binding constant data, function of pH, potentiometric titrations • Crystal structure of iron-salicylate complex • Superoxide dismutase (SOD) action. • Catalase action H2O2 + 2H+ + 2e- = 2H2O H2O2 = O2 + 2H+ + 2e- 2H2O2 = 2H2O + O2 -qualitatively observed during DNA oxidation studies. • Prediction of Structure-Activity Relationships -antioxidant characteristics of other NSAID, (ibuprofen) -increase activity of salicylates -quick screen for antioxidant characteristics of newly isolated natural products • Collaborative Research -physiological Studies

  46. Quantitative Structure-Activity Relationships (QSAR) for Salicylates and Derivatives (Anti-inflammatory action) Rule 1.Substitution on either the carboxyl or the phenolic hydroxyl groups affect activity. Rule 2.Placing the phenolic hydroxyl group meta or para to the carboxyl group abolishes activity. Rule 3.Substitution of halogen atoms on the aromatic ring enhances potency. Rule 4.Substitution of aromatic rings meta to the to the carboxyl and para to the phenolic hydroxyl groups increases anti-inflammatory activity.

  47. Rule 1. Substitution on either the carboxyl or the phenolic hydroxyl groups affect activity. • May Affect Chelation of Fe ions. • Binding Constant to Fe • Rate of hydrolysis to salicylate

  48. Rule 2. Placing the phenolic hydroxyl group meta or para to the carboxyl group abolishes activity. • Meta and Para derivatives are not Fe chelators Bidentate Chelation Site C O O H C O O H C O O H H O O H O H Salicylic Acid 3-hydroxyl benzoic acid 5-hydroxyl benzoic acid

  49. Rule 3. Substitution of halogen atoms on the aromatic ring enhances potency.Rule 4. Substitution of aromatic rings meta to the to the carboxyl and para to the phenolic hydroxyl groups increases anti-inflammatory activity. • Increases electron withdrawing ability of salicylate raises FeII/III potential C O O - e I I F e O • May improve Fenton deactivation

  50. If Fe chelation correlates to QSAR anti-inflammatory rules ???Anti-inflammatory action = Antioxidant action???

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