Download Spring 1997 - University of Idaho

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
[email protected]
1
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.
2
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.
O
OH
O
O
OH
OH
pKa = 3.0
O
CH3
pKa =3.5
 1898 - Felix Hofmann (Bayer)
synthesizes and tests Acetylsalicylic
Acid (Aspirin)
 Just as effective but less irritating than
salicylic acid.
3
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.
4
Weakness of the acetylation explanation.
O
OH
O
O
O
OH
CH3
OH
Aspirin
Salicylic Acid
 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 Antiinflammatory 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
5
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.
6
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.
7
Goal of Respiration. (CH2O)n + O2 = nCO2 + nH2O
Increasing
Reducing
Pow er
(-)
(CH 2 O ) n
(sugars)
E
+ 1e
Redox
Potential
DG = nFE
O2
+ 2e
.O2
o'
(pH7)
- 0.45 volts
O 2 + 2H
+
H2O 2
0.30 V
+ 4e
O 2 + 4H
(+)
+
2H 2O
0.82 V
Increasing
O xidizing
Po wer
 H2O2 & O2.- are known as “activated oxygen species”
8
Dangers of Activated Oxygen Species
 Hydrogen peroxide
Fenton Reaction
H2O2 + FeII(L)n = FeIII(L)n + HO- + HO.
HO. + e- = HO-
 Superoxide ion
Eo = 1.8 volts
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.
9
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.
10
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
- Alzheimer’s
- Strokes
- Parkinson’s
- Cancers
- Cataracts
 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.
11
Migration of Fe Under Conditions of Oxidative Stress
H2O2 + O2.Fe Containing Enzymes
Oxidized
Ligands
Fe2+
+ ATP, citrate
Fe(L) + HO.
+ H2O2 + O2.Fe(L)
12
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 Medicine 9, (1990) 299.
13
Proposed Route of Antioxidant Action for Aspirin.
(literature)
 Salicylates act as Hydroxyl Radicals Scavengers.
COOH
+ HO.
COOH
A)
HO
+
OH
OH
COOH
B)
OH
OH
k  1010 M-1 s-1
Xenobiotica, 18 (1988) pp459-470.
14
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
Summary
radical scavenging alone cannot explain the antioxidant characteristics of
salicylates.
15
Alternative Hypothesis for
Salicylate 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.
16
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
17
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
18
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.
19
Redox Potential of Fe-Sal Indicates that it is a
Fenton Inactive Complex.
FeII[sal]
FeIII[sal] + e-
Potential versus SHE
-0.4
0.4
Eredox = 0.370 volts vs. SHE at pH 7.2
FeII[sal]
e- + FeIII[sal]
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).
20
Salicylate chelates iron into a Fenton
inactive form
 Thermodynamics of the Fenton Reaction
Stronger
Reducing
Agents (-)
Fenton Active
x
Fenton Inactive
}
EFe[EDTA]
EOxidases
EOxygenases
E0Fenton = 0.307 volts
EFe-sal = 0.370 volts
21
Evidence for Fenton Reaction Inertness of Fesalicylate from Cyclic Voltammetry experiments.
 Electrochemical electrocatalytic wave for FeIII(EDTA)
reduction in the presence of H2O2
Electrode:
FeIII(EDTA) + e = FeII(EDTA)
Solution:
FeII(EDTA) + H2O2 = FeIII(EDTA) + HO- + HO.
0.090 volts SHE
 Results in enhanced electroreduction current for FeIII(EDTA)
wave, no electro-oxidation wave for FeII(EDTA)
22
Cyclic Voltammetry of FeII/III [EDTA] in the
Absence and Presence of H2O2
-0.7
Potential vs. Ag/AgCl
0.4 mA
A
A) 0.1 mM FeIII(EDTA)
B) +10 mM H2O2.
Current
B
1.0 mA
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).
23
Results of H2O2 electrocatalytic voltammetry.
CuI(EDTA)
FeII(sal)3
Potential
0.450 volts
0.370
H2O2 Reduction
No
No
H2O2 = HO- + HO.
0.307
----
FeII(EDTA)
CuI(sal)2
0.090
0.050
Yes
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.
24
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.
25
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.
O3PO
O
Base
+ .OH
H
OPO3
O3PO
O
Base
.
OPO3
JACS 1992, 114, pp2303-2312.
H2O
O3PO
O
Base
-PO4
-H
HO
OPO3
O3PO
O
Base
O
+ Base
H
O
H
O
26
DNA Incubation Studies.
 Fe-DNA complex
Eredox{FeII/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
27
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.
28
HPLC Detector Response
(Thousands)
HPLC incubation results
100
80
Thymine
60
Adenine
40
20
0
A
B
 DNA Incubation with…
C
D
A) 0.1 mM Fe(NO3)3
B) 0.1 mM Fe[EDTA]
C) 0.1 mM Fe(NO3)3 and
0.4 mM salicylate
D) 0.1 mM Fe[EDTA] and
0.4 mM salicylate
 Salicylate decreases oxidative DNA damage due to
Both Fe-DNA and Fe(EDTA) complexes
29
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
30
Summary of DNA Incubation Experiments.
Control
Incubation-10 Minutes
Damage to CT-DNA
0.5 mM Ascorbate
5.0 mM H2O2
NO
+ 0.1 mM Fe(EDTA)
+ 0.1 mM Cu(EDTA)
YES
NO
+ 0.1 mM Fe(salicylate)
+ 0.1 mM Cu(salicylate)
NO
YES
Confirms Redox deactivation hypothesis
31
Summary of DNA Incubation Experiments
Excess Ligand (salicylate or EDTA)
Control
Incubation 10 minutes
Damage to CT-DNA
0.5 mM Ascorbate
5.0 mM H2O2
NO
+ 0.1 mM Cu(salicylate)
+ 10.0 mM salicylate
YES
+ 0.1 mM Fe(EDTA)
+ 50.0 mM EDTA
YES
Indicates that radical scavenging is not an important mechanism.
32
Incubation Results with Aspirin
 Acetylsalicylic acid cannot chelate iron
– slowly hydrolyzes to salicylic acid (t1/2 = 20 min.)
– Radical scavenging rates; aspirin = salicylate
Control
Incubation 10 minutes
CT-Damage
0.5 mM Ascorbate
5.0 mM H2O2
NO
+ 0.1 mM Fe(NO3)3
+ 0.4 mM aspirin
YES
33
Release of adenine with incubation time for
controls, and presence of salicylate, and aspirin.
 Adenine Release
– Less than 10 minutes
– Greater than 60 minutes
aspirin = control
aspirin = salicylic acid
HPLC Detector Response (254 nm)
 Results consistent with acetylsalicylic acid to salicylic acid
Control
Salicylic Acid
Acetylsalicylic Acid
0
20
40
Incubation Time (min) 100
34
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
35
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
36
FeII/III[salicylate] potential is pH dependent.
Potential (volts vs. SHE)
2
EFe(sal) = 0.793 - (0.059 pH)
1
0
2
4
6
8
10
pH
 Measured by Cyclic Voltammetry
37
pH dependence may be due to HO- complexation
 FeIII(sal)n + HO- = FeIIIOH(sal)n
 FeIIIOH(sal)n + e- = FeII(sal)n + HOIII
RT
[
Fe
( - OH )( sal ) n ]
0
E=E +
ln
II
nF [ Fe ( sal ) n ][ HO ]
E = const - 0.059 pH
E = 0.793 - 0.059 pH
38
Fenton threshold and the FeII/III(sal) redox potential
Potential (volts SHE)
2
1.5
E0Fe-Sal = 0.793 - 0.059pH
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
39
Hypothesis
Possible Significance of the close parallel of Fe II/III(sal)n and
Standard State Fenton threshold.
2
Superoxide Dismutation
 Superoxide Dismutation.
Zone I
1.5
O2.- + 2H+ + e- = H2O2
Eo = 1.77 volts
1
EFe-Sal
E = 1.77 + 2(0.059)pH
 Salicylic acid may seek to maximize
SOD activity with a minimum of
Fenton type reactivity.
0.5
Zone II
Fenton Threshold
Zone III
0
0
2
4
6
8
10
pH
40
Thermodynamic Suppression of HO.
Production by Salicylate.
 Reduction: H2O2 + e = HO- + HO.
 Oxidation: FeII(sal)n = FeIII(sal)n + e
 Ecell = Ered - Eox
Ered
[ H 2 O2 ]
= 0.732 - 0.0591 pH + 0.0591log
[ HO. ]
Eox = 0.793 - 0.0591pH
Calculate equilibrium value for product/reactant ratio @ pH 7 (Ecell= 0)
[ HO . ]
= 0.0928
[ H 2O 2]
 Healthy Tissue Maintains [H2O2] = 10-9 - 10-7
(Physiological Reviews, 59 (1979) p564.)
Salicylic acid is a modest suppression agent of HO.
41
Thermodynamic Analysis of Superoxide Dismutase
Activity of Iron-Salicylate
Reduction: O2.- + 2H+ + e- = H2O2
Oxidation: FeII[sal] = FeIII[sal] + eEcell = Ered - Eox
.[O 2 ]
Ered = 1.77 - 0.118 pH + 0.0591 log
[H2O2]
Eox = 0.793 - 0.0591 pH
@ pH 7 Ecell Spontaneous until
[ O2.- ]
= 2.94 x10- 10
[ H 2 O2 ]
Salicylic acid may be an excellent suppression agent of O2.42
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
.
[ HO ]
log
[ H 2 O2 ]
5
5
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)
SOD Action
[O2.- ]
log
[ H 2 O2 ]
43
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.
44
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+ + 2e2H2O2 = 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
45
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
46
activity.
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
O
OH
O
O
OH
OH
salicylic acid
O
CH3
acetylsalicylic acid
47
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
COOH
COOH
COOH
HO
OH
OH
Salicylic Acid
3-hydroxyl benzoic acid
5-hydroxyl benzoic acid
48
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
COO
e-
Fe II
O
 May improve Fenton deactivation
49
If Fe chelation correlates to QSAR
anti-inflammatory rules
???
Anti-inflammatory action = Antioxidant action
???
50
Other anti-inflammatory agents
 All of the following NSAID’s are iron chelation agents.
 Iron chelation may play a role in their medicinal action.
OH
COOH
CONH2
R3
Cl
O
N
NH
OH
N
O
R1
S
O
Salicylamide
R2
Mefenamic Acid, R1 = R2 = CH3, R3 = H
Meclofenamic Acid, R1 = R3 = Cl, R3 = CH3
Flufenamic Acid, R1 = R3 = H, R3 = CF3
N
H
CH3
N
CH3
O
H3CO
Piroxicam
CH2COOH
Indomethacin
N-ayrlanthranilic Acids
51
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
FMC
52