A Hypothesis for the Physiological Antioxidant Action of the Salicylates'

1
A Hypothesis for the Physiological
AntioxidantAction of the Salicylates.
I. Francis Cheng Department of Chemistry Universit
y of Arizona Tucson, Arizona 85721 Tel. (520)
621-6340 ifcheng_at_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
• Parkinsons and Alzheimers 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 as Respiration
  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
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• H2O2 O2.- are known as activated oxygen
  species

9
Dangers of Activated Oxygen Species

• Hydrogen peroxide Fenton Reaction
• H2O2 FeII(L)n FeIII(L)n HO- HO.
• HO. e- HO- Eo 1.8 volts
• Superoxide ion Disproportionation 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 Parkinsons
• Alzheimers 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

• FeIIcomplex H2O2 FeIIIcomplex HO-
  HO.
• Fe and disease origins
• Recently Discovered Statistical Implications in
  
• - Heart Diseases - Strokes - Cancers -
  Cataracts
• - Alzheimers - Parkinsons
• 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. Circulati
  on, 86 (1992) pp803-811.
• New England Journal of Medicine, 320 (1989)
  1012. Iron and Human Disease, CRC Press, Boca
  Raton, FL, 1992.

12
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 aspirins ability to act
  as a chemopreventative of many diseases
  associated with oxidative damage.
• Free Radicals in Biology and Medicine 9,
  (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 plant siderophores - 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
Redox Potential of Fe-Sal Indicates that it is a
Fenton Inactive Complex.
Potential versus SHE
0.4
-0.4
Eredox 0.370 volts vs. SHE at pH 7.2
FeIIsal e- FeIIIsal

• 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).

21
Salicylate chelates iron into a Fenton inactive
form

• Thermodynamics of the Fenton Reaction

Stronger Reducing Agents (-)

EFeEDTA 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

• 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).

Current
B
1.0
m
A
24
Results of H2O2 electrocatalytic voltammetry.
Potential H2O2 Reduction CuI(EDTA) 0.450
volts No FeII(sal)3 0.370 No H2O2 HO-
HO. 0.307 ---- FeII(EDTA) 0.090 Yes CuI(sal)
2 0.050 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 complex EredoxFeII/III(DNA) -0.10
  volts SHE
• FeIII(DNA) Ascorbate FeII(DNA)
  Deoxyascorbate
• FeII(DNA) H2O2 FeIII(DNA) HO- HO.
• Conditions 0.1 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.
• Conditions 0.1 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
HPLC incubation results
100
80
Thymine
60
Adenine
40
20
0
A B C D

• DNA Incubation with
• A) 0.1 mM Fe(NO3)3 B) 0.1 mM FeEDTA
• C) 0.1 mM Fe(NO3)3 and D) 0.1 mM FeEDTA and
  0.4 mM salicylate 0.4 mM salicylate
• Salicylate decreases oxidative DNA damage due to
• Both Fe-DNA and Fe(EDTA) complexes

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/IIIsalicylate redox potential
• This is a key feature in salicylates 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
  pHs as low as
• 3.5

37
FeII/IIIsalicylate potential is pH dependent.
2
EFe(sal) 0.793 - (0.059 pH)
Potential (volts vs. SHE)
1
0
2
4
6
8
10
pH

• Measured by Cyclic Voltammetry

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
-

(
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n
0
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• E const - 0.059 pH
• E 0.793 - 0.059 pH

39
Fenton threshold and the FeII/III(sal) redox
potential
2
1.5
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

• 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

40
Hypothesis Possible Significance of the close
parallel of Fe II/III(sal)n and Standard State
Fenton threshold.
2
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Zone I

• 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.

1.5
1
EFe-Sal
0.5
Zone II
Fenton Threshold
Zone III
0
0
2
4
6
8
10
pH
41
Thermodynamic Suppression 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 _at_ 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 FeIIsal FeIIIsal e-
-
.
O


2
Ecell Ered - Eox
Ered
pH

-

1
77
0
118
0
0591
.
.
.
log
H2O2
Eox 0.793 - 0.0591 pH
_at_ pH 7
E
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cell
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x
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94
10



H
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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/IIIsalicylate
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 Fe2/3and 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
???Anti-inflammatory action Antioxidant
action???
If Fe chelation correlates to QSAR
anti-inflammatory rules
51
Other anti-inflammatory agents

• All of the following NSAIDs are iron chelation
  agents.
• Iron chelation may play a role in their medicinal
  action.

C
l
N
O
H
O
C
O
O
H
C
O
N
H
2
O
N
N
H
O
H
N
H
N
C
H
R
R
3
S
C
H
1
3
3
O
O
Salicylamide
H
C
O
C
H
C
O
O
H
R
3
2
2
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N-ayrlanthranilic Acids
52
Acknowledgments
Seton Hall University Graduate Students
(M.S.) Andris Amolins Chris
Zhao Undergraduates Malgorzata
Galazka Leon Doneski University of Arizona
Dr. Quintus Fernando Dr. Paul
Oram Equipment Ciba-Giegy Union-Camp FM
C