455
European Journal of Pharmacology, 36 (1976) 455--458
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands Short c o m m u n i c a t i o n COMPETITIVE OXIDATION OF 6-HYDROXYDOPAMINE BY OXYGEN AND H Y D R O G E N P ER OX I D E Y.-O. LIANG, R.M. WIGHTMAN and R.N. ADAMS Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, U.S.A.
Received 3 February 1976, accepted 24 February 1976
Y.-O. LIANG, R.M. WIGHTMAN and R.N. ADAMS, Competitive oxidation of 6-hydroxydopamine by oxygen and hydrogen peroxide, European J. Pharmacol. 36 (1976) 455--458. Simultaneous oxygen electrode and conventional polarographic measurements show the net concentration of hydrogen peroxide produced by air oxidation of 6-hydroxydopamine is considerably less than that predicted from the known stoichiometry of the reaction. This is due to competitive oxidation of 6-hydroxydopamine by the generated hydrogen peroxide. The presence of ascorbic acid in this reaction also results in significant decreases of hydrogen peroxide under most conditions. The implications of these results to the molecular mechanism of 6-hydroxydopamine neurotoxicity are discussed. Hydrogen peroxide
6-Hydroxydopamine
Polarographic measurements
1. Introduction The molecular mechanism responsible for the remarkable n e u r o t o x i c i t y of 6-hydroxyd o p a m in e (6-OHDA) is still unclear. In a series of thorough studies (Heikkila and Cohen, 1972; Cohen and Heikkila, 1974; Cohen et al., 1975), it has been proposed t hat the in vivo oxidation of 6-OHDA generates hydrogen peroxide and th at the latter (or superoxide or h y d r o x y l radicals derived from the peroxide) is the effective n eu r o to xi n. This is based on the in vitro oxidation by air which oxidizes 6-OHDA to the p-quinone (6-Q) and yields peroxide as in reaction (1):
Ho~NH2
+ 02
D, O
Ho ~'.~...,-'oH
6-OHDA
~
NN2+ H202
Ho ~'-~,..,~ o +
02 -~ 6-Q
+
H202
(1)
We examined this reaction by simultaneous oxygen electrode and polarographic techniques and our interest was piqued by the fact t ha t we consistently f o u n d far less H2 02 generated than called for by reaction (1). This loss was far
Neurotoxicity
greater than could be account ed for by the natural instability of aqueous H2 02 solutions. A t horough study ensued and the results are summarized herein. T hey are important, we believe, for t hey place the peroxide hypothesis in a new quantitative framework.
2. Materials and m et hods The 6-OHDA (Regis) and ascorbic acid (Sigma) were used as received and all ot her chemicals were reagent grade. All studies were made in citrate--phosphate buffer (pH 7.4) at 25°C. The oxygen c o n t e n t of the solutions was m o n i t o r e d with a Beckman Oxygen Analyser. The polarographic measurements were made with a PAR 174 polarograph (Princeton Applied Research) using a conventional dropping mercury electrode (dme). The dme polarography m o n i t o r e d 0 2 , H2 0 2 , 6 - O H D A , 6-Q and ascorbic acid (AA). The reaction vessel was arranged to allow simultaneous measurements with the Beckman analyser and polarograph.
456
All potentials are referred to the saturated calomel electrode. Polarography provides a convenient method for determining dissolved 0 2 . At pH 7.4 an air-saturated solution gives two reduction waves with half wave potentials (Ev,) at --0.02 and --0.88 V, corresponding to the reduction of O2 to H2 02 (first wave) and further reduction of H2 02 to H2 O (second wave), respectively (Brezina and Zuman, 1958). Removal of 02 by thorough nitrogen bubbling removes both of these waves. The Beckman Oxygen Analyser and the polarographic method gave identical results for percent 02 saturation. Hydrogen peroxide in a nitrogen-bubbled pH 7.4 buffer shows only one reduction wave at the expected - 0 . 8 8 V and the height of this wave is directly proportional to H2 02 concentration. If dissolved 02 is also present, a correction for the H2 02 contributed by the second oxygen wave can be made. The relative concentrations of 6-OHDA and 6-Q can be determined from their respective anodic and cathodic waves at the dme (E~ = - 0 . 2 0 V). Where the 6-OHDA and excess 02 waves overlap, the 02 can be ascertained via the Beckman Analyser (which can only respond to molecular 02 ). For these experiments where ascorbic acid is present, the oxidation wave of AA overlaps that of 6-OHDA and 0 2 . However, the 6-Q and H2 02 concentrations can still be determined since their reduction waves are not obscured.
Y.-O. L I A N G E T AL. TABLE 1 H202 p r o d u c t i o n in air-saturated s o l u t i o n s as a funct i o n o f 6 - O H D A c o n c e n t r a t i o n *. Initial conc. ** (raM)
F i n a l conc. (raM)
6-OHDA
6-Q
H202
Ratio H202/6-Q
0.10 0.20 0.30 0.40 0.60 0.60 ** 1.20 1.20 **
0.10 0.20 0.29 0.26 0.23 0.29 0.31 0.47
0.10 0.18 0.26 0.23 0.20 0.22 0.17 0.10
1.00 0.90 0.89 0.88 0.86 0.75 0.54 0.21
* C i t r a t e - - p h o s p h a t e buffer, p H 7.4, air-saturated. ** Dissolved O2 c o n c e n t r a t i o n initially ca. 0.25 m M (prevailing air s a t u r a t i o n values) in all cases. *** R e m e a s u r e d 5 rnin a f t e r p r e c e d i n g e n t r y in table.
As shown in table 1, only if the concentration of starting 6-OHDA is considerably less than that of dissolved O2 is the stoichiometry of reaction (1) realized with respect to H202 production. When the initial concentration of 6-OHDA approaches or exceeds that of 0 2 , the yield of H2 O2 decreases markedly (columns 3 and 4 of table 1). As reported earlier, this is clearly due to the generated H2 0 2 , itself an effective oxidant for 6-OHDA, reacting as (Liang et al., 1975): 6-OHDA + H202 -' 6-Q + 2 H20
3. Results
The air-saturated pH 7.4 buffer solutions were ca. 0.25 mM in 02 as determined polarographically and by the Beckman Analyser. To such solutions were added varying amounts of 6-OHDA and the reaction was followed by monitoring the 02 via the Beckman Analyser. When the reaction was essentially complete, as evidenced by a very low r~te of 02 consumption, the concentrations of 6-Q and H2 02 were determined polarographically. (The H2 02 concentrations were appropriately corrected as mentioned above.)
(2)
Thus, as available 02 is consumed, the H2 02 generated begins to oxidize the remaining 6-OHDA and the result is far less net H2 02 than expected from reaction (1). In substantiation of this important point, it should be noted (column 2, table 1) that the maximum amount of 6-Q formed at high starting concentrations of 6-OHDA is not ca. 0.25 mM as predicted from the available 02 in reaction (1). Instead, its value is almost twice that (ca. 0.47 mM), which corresponds to the 6-Q produced by the sum of reactions (1) and (2). (Exact quantitative relationships are difficult since some air always leaked in during the time
C O M P E T I T I V E O X I D A T I O N O F 6 - O H D A BY 0 2 A N D H 2 0 2
course of the measurements and there are small uncertainties in the polarographic measurements.) Similar results were obtained even if air was continuously bubbled through the solution during the experiment, b u t were more variable due to inconsistencies in bubbling rates. In a second style of experiment, 6-OHDA was again added to air-saturated buffer, b u t in these cases the 6-OHDA concentration was fixed at 0.2 mM. When the rate of oxygen consumption was essentially zero, varying amounts of AA were added. The AA enhanced the 02 consumption as indicated by Heikkila and Cohen (1972), since it rapidly re-reduces 6-Q and thus 'recycles' the 6-OHDA oxidation by 0 2 . However, as seen in table 2, the steady state concentration of experimentally measured H2 02 remained essentially the same. With large excesses of AA the actual H2 O2 concentration is decreased from that obtained in the absence of AA. With 10 mM AA, if one waits ca. 10 min, the AA will re-reduce all 6-Q and eventually consume all H2 O2 (last entry of table 2) by the recycling process. Finally, it can be readily shown that H2 O2 alone is an effective oxidant for 6-OHDA by mixing the two in thoroughly deoxygenated
TABLE 2 H 2 0 2 p r o d u c t i o n b y air o x i d a t i o n o f 6 - O H D A as a function o f a s c o r b a t e acid c o n c e n t r a t i o n . Initial c o n c . * (raM)
F i n a l conc. ( m M )
Ascorbate
6-Q
H2 0 2
0 2 4 6 6 ** 8 8 ** 10 10 **
0.20 0.18 0.17 0.14 0.10 0.11 0.08 0.08 0.00
0.18 0.23 0.27 0.23 0.10 0.26 0.20 0.19 0.00
* All s o l u t i o n s c i t r a t e - - p h o s p h a t e b u f f e r , p H 7.4 air-saturated, c o n t a i n i n g fixed 0.2 m M 6-OHDA. ** R e m e a s u r e d 10 rain a f t e r p r e c e d i n g e n t r y in table.
457
buffer. The appearance of 6-Q and disappearance of H2 02 can be followed polarographically. Even with equimolar ratios of 6-OHDA to H2 0 2 , virtually all the H2 02 is consumed. The rate of the H2 02 oxidation is, however, approximately 10--40 times slower than 02 (air) oxidation.
4. Discussion The present results show that the net concentration of H2 02 produced in the air oxidation of 6-OHDA is considerably less than that predicted by the stoichiometry of reaction (1). As O: is consumed and thus H2 02 increases, oxidation by the latter becomes competitive and H2 02 is consumed. Furthermore, the addition of ascorbic acid, while increasing the rate of 02 consumption, also results in significant decreases of H2 02 under most conditions. It is, of course, always questionable to extrapolate such results to the microscopic CNS environment. However, Sachs and Jonsson (1975) have estimated the intraneuronal concentration of 6-OHDA required for neurotoxicity to be as high as 20--50 mM (calculations on 6-OHDA concentration per gram). The intracellular 02 concentration is variable b u t values of a b o u t 10--20% of that of air-saturated solutions (hence, a b o u t 0.03 mM) have been suggested (Cater, 1966). Finally, the gross ascorbate concentration of mammalian brain is a b o u t 2--3 mM (Allison and Stewart, 1971). Under such conditions in vitro, tables 1 and 2 show the steady state concentration of H202 far less than that predicted by equation (1). It is reasonable to conclude a similar low concentration of H2 02 would exist in the neurotoxic action, unless one assumes some catalytic in vivo effect which prevents H2 02 from oxidizing 6-OHDA.
Acknowledgement The support o f this w o r k b y T h e N a t i o n a l Science F o u n d a t i o n via G r a n t GP 3 2 8 4 6 is gratefully a c k n o w l edged.
458
References Allison, J.H. and M.A. Stewart, 1971, Quantitative analysis of ascorbic acid in tissues by gas--liquid chromatography, Anal. Biochem. 43,401. Brezina, M. and P. Zuman, 1958, Polarography in Medicine, Biochemistry and Pharmacology (Interscience, N e w York). Cater, D.B., 1966, in: Symposium on Oxygen Measurements in Blood and Tissues, eds. Payne and Hill (Churchill, London) p. 167. Cohen, G. and R.E. Heikkila, 1974, The generation of hydrogen peroxide, superoxide radical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related cytotoxic agents, J. Biol. Chem. 249, 2447.
Y.-O. LIANG ET AL.
Cohen, G., B. Allis, B. Winston, C. Mytilineou and R. Heikkila, 1975, Prevention of 6-hydroxydopamine neurotoxicity, European J. Pharmacol. 33, 217. Heikkila, R. and G. Cohen, 1972, Further studies on the generation of hydrogen peroxide by 6-hydroxydopamine: Potentiation by ascorbic acid, Mol. Pharmacol. 8,241. Liang, Y.-O., R.M. Wightman, P. Plotsky and R.N. Adams, 1975, Oxidative interactions of 6-hydroxydopamine with C N S constituents, in: Proceedings of the Symposium, 'Chemical Tools in Catecholamine Research', Vol. I, (Elsevier, Amsterdam) p. 15. Sachs, C. and G. Jonnson, 1975, Mechanisms of action of 6-hydroxydopamine, Biochem. Pharmacol. 24, 1.
European Journal of Pharmacology, 36 (1976) 455--458
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands Short c o m m u n i c a t i o n COMPETITIVE OXIDATION OF 6-HYDROXYDOPAMINE BY OXYGEN AND H Y D R O G E N P ER OX I D E Y.-O. LIANG, R.M. WIGHTMAN and R.N. ADAMS Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, U.S.A.
Received 3 February 1976, accepted 24 February 1976
Y.-O. LIANG, R.M. WIGHTMAN and R.N. ADAMS, Competitive oxidation of 6-hydroxydopamine by oxygen and hydrogen peroxide, European J. Pharmacol. 36 (1976) 455--458. Simultaneous oxygen electrode and conventional polarographic measurements show the net concentration of hydrogen peroxide produced by air oxidation of 6-hydroxydopamine is considerably less than that predicted from the known stoichiometry of the reaction. This is due to competitive oxidation of 6-hydroxydopamine by the generated hydrogen peroxide. The presence of ascorbic acid in this reaction also results in significant decreases of hydrogen peroxide under most conditions. The implications of these results to the molecular mechanism of 6-hydroxydopamine neurotoxicity are discussed. Hydrogen peroxide
6-Hydroxydopamine
Polarographic measurements
1. Introduction The molecular mechanism responsible for the remarkable n e u r o t o x i c i t y of 6-hydroxyd o p a m in e (6-OHDA) is still unclear. In a series of thorough studies (Heikkila and Cohen, 1972; Cohen and Heikkila, 1974; Cohen et al., 1975), it has been proposed t hat the in vivo oxidation of 6-OHDA generates hydrogen peroxide and th at the latter (or superoxide or h y d r o x y l radicals derived from the peroxide) is the effective n eu r o to xi n. This is based on the in vitro oxidation by air which oxidizes 6-OHDA to the p-quinone (6-Q) and yields peroxide as in reaction (1):
Ho~NH2
+ 02
D, O
Ho ~'.~...,-'oH
6-OHDA
~
NN2+ H202
Ho ~'-~,..,~ o +
02 -~ 6-Q
+
H202
(1)
We examined this reaction by simultaneous oxygen electrode and polarographic techniques and our interest was piqued by the fact t ha t we consistently f o u n d far less H2 02 generated than called for by reaction (1). This loss was far
Neurotoxicity
greater than could be account ed for by the natural instability of aqueous H2 02 solutions. A t horough study ensued and the results are summarized herein. T hey are important, we believe, for t hey place the peroxide hypothesis in a new quantitative framework.
2. Materials and m et hods The 6-OHDA (Regis) and ascorbic acid (Sigma) were used as received and all ot her chemicals were reagent grade. All studies were made in citrate--phosphate buffer (pH 7.4) at 25°C. The oxygen c o n t e n t of the solutions was m o n i t o r e d with a Beckman Oxygen Analyser. The polarographic measurements were made with a PAR 174 polarograph (Princeton Applied Research) using a conventional dropping mercury electrode (dme). The dme polarography m o n i t o r e d 0 2 , H2 0 2 , 6 - O H D A , 6-Q and ascorbic acid (AA). The reaction vessel was arranged to allow simultaneous measurements with the Beckman analyser and polarograph.
456
All potentials are referred to the saturated calomel electrode. Polarography provides a convenient method for determining dissolved 0 2 . At pH 7.4 an air-saturated solution gives two reduction waves with half wave potentials (Ev,) at --0.02 and --0.88 V, corresponding to the reduction of O2 to H2 02 (first wave) and further reduction of H2 02 to H2 O (second wave), respectively (Brezina and Zuman, 1958). Removal of 02 by thorough nitrogen bubbling removes both of these waves. The Beckman Oxygen Analyser and the polarographic method gave identical results for percent 02 saturation. Hydrogen peroxide in a nitrogen-bubbled pH 7.4 buffer shows only one reduction wave at the expected - 0 . 8 8 V and the height of this wave is directly proportional to H2 02 concentration. If dissolved 02 is also present, a correction for the H2 02 contributed by the second oxygen wave can be made. The relative concentrations of 6-OHDA and 6-Q can be determined from their respective anodic and cathodic waves at the dme (E~ = - 0 . 2 0 V). Where the 6-OHDA and excess 02 waves overlap, the 02 can be ascertained via the Beckman Analyser (which can only respond to molecular 02 ). For these experiments where ascorbic acid is present, the oxidation wave of AA overlaps that of 6-OHDA and 0 2 . However, the 6-Q and H2 02 concentrations can still be determined since their reduction waves are not obscured.
Y.-O. L I A N G E T AL. TABLE 1 H202 p r o d u c t i o n in air-saturated s o l u t i o n s as a funct i o n o f 6 - O H D A c o n c e n t r a t i o n *. Initial conc. ** (raM)
F i n a l conc. (raM)
6-OHDA
6-Q
H202
Ratio H202/6-Q
0.10 0.20 0.30 0.40 0.60 0.60 ** 1.20 1.20 **
0.10 0.20 0.29 0.26 0.23 0.29 0.31 0.47
0.10 0.18 0.26 0.23 0.20 0.22 0.17 0.10
1.00 0.90 0.89 0.88 0.86 0.75 0.54 0.21
* C i t r a t e - - p h o s p h a t e buffer, p H 7.4, air-saturated. ** Dissolved O2 c o n c e n t r a t i o n initially ca. 0.25 m M (prevailing air s a t u r a t i o n values) in all cases. *** R e m e a s u r e d 5 rnin a f t e r p r e c e d i n g e n t r y in table.
As shown in table 1, only if the concentration of starting 6-OHDA is considerably less than that of dissolved O2 is the stoichiometry of reaction (1) realized with respect to H202 production. When the initial concentration of 6-OHDA approaches or exceeds that of 0 2 , the yield of H2 O2 decreases markedly (columns 3 and 4 of table 1). As reported earlier, this is clearly due to the generated H2 0 2 , itself an effective oxidant for 6-OHDA, reacting as (Liang et al., 1975): 6-OHDA + H202 -' 6-Q + 2 H20
3. Results
The air-saturated pH 7.4 buffer solutions were ca. 0.25 mM in 02 as determined polarographically and by the Beckman Analyser. To such solutions were added varying amounts of 6-OHDA and the reaction was followed by monitoring the 02 via the Beckman Analyser. When the reaction was essentially complete, as evidenced by a very low r~te of 02 consumption, the concentrations of 6-Q and H2 02 were determined polarographically. (The H2 02 concentrations were appropriately corrected as mentioned above.)
(2)
Thus, as available 02 is consumed, the H2 02 generated begins to oxidize the remaining 6-OHDA and the result is far less net H2 02 than expected from reaction (1). In substantiation of this important point, it should be noted (column 2, table 1) that the maximum amount of 6-Q formed at high starting concentrations of 6-OHDA is not ca. 0.25 mM as predicted from the available 02 in reaction (1). Instead, its value is almost twice that (ca. 0.47 mM), which corresponds to the 6-Q produced by the sum of reactions (1) and (2). (Exact quantitative relationships are difficult since some air always leaked in during the time
C O M P E T I T I V E O X I D A T I O N O F 6 - O H D A BY 0 2 A N D H 2 0 2
course of the measurements and there are small uncertainties in the polarographic measurements.) Similar results were obtained even if air was continuously bubbled through the solution during the experiment, b u t were more variable due to inconsistencies in bubbling rates. In a second style of experiment, 6-OHDA was again added to air-saturated buffer, b u t in these cases the 6-OHDA concentration was fixed at 0.2 mM. When the rate of oxygen consumption was essentially zero, varying amounts of AA were added. The AA enhanced the 02 consumption as indicated by Heikkila and Cohen (1972), since it rapidly re-reduces 6-Q and thus 'recycles' the 6-OHDA oxidation by 0 2 . However, as seen in table 2, the steady state concentration of experimentally measured H2 02 remained essentially the same. With large excesses of AA the actual H2 O2 concentration is decreased from that obtained in the absence of AA. With 10 mM AA, if one waits ca. 10 min, the AA will re-reduce all 6-Q and eventually consume all H2 O2 (last entry of table 2) by the recycling process. Finally, it can be readily shown that H2 O2 alone is an effective oxidant for 6-OHDA by mixing the two in thoroughly deoxygenated
TABLE 2 H 2 0 2 p r o d u c t i o n b y air o x i d a t i o n o f 6 - O H D A as a function o f a s c o r b a t e acid c o n c e n t r a t i o n . Initial c o n c . * (raM)
F i n a l conc. ( m M )
Ascorbate
6-Q
H2 0 2
0 2 4 6 6 ** 8 8 ** 10 10 **
0.20 0.18 0.17 0.14 0.10 0.11 0.08 0.08 0.00
0.18 0.23 0.27 0.23 0.10 0.26 0.20 0.19 0.00
* All s o l u t i o n s c i t r a t e - - p h o s p h a t e b u f f e r , p H 7.4 air-saturated, c o n t a i n i n g fixed 0.2 m M 6-OHDA. ** R e m e a s u r e d 10 rain a f t e r p r e c e d i n g e n t r y in table.
457
buffer. The appearance of 6-Q and disappearance of H2 02 can be followed polarographically. Even with equimolar ratios of 6-OHDA to H2 0 2 , virtually all the H2 02 is consumed. The rate of the H2 02 oxidation is, however, approximately 10--40 times slower than 02 (air) oxidation.
4. Discussion The present results show that the net concentration of H2 02 produced in the air oxidation of 6-OHDA is considerably less than that predicted by the stoichiometry of reaction (1). As O: is consumed and thus H2 02 increases, oxidation by the latter becomes competitive and H2 02 is consumed. Furthermore, the addition of ascorbic acid, while increasing the rate of 02 consumption, also results in significant decreases of H2 02 under most conditions. It is, of course, always questionable to extrapolate such results to the microscopic CNS environment. However, Sachs and Jonsson (1975) have estimated the intraneuronal concentration of 6-OHDA required for neurotoxicity to be as high as 20--50 mM (calculations on 6-OHDA concentration per gram). The intracellular 02 concentration is variable b u t values of a b o u t 10--20% of that of air-saturated solutions (hence, a b o u t 0.03 mM) have been suggested (Cater, 1966). Finally, the gross ascorbate concentration of mammalian brain is a b o u t 2--3 mM (Allison and Stewart, 1971). Under such conditions in vitro, tables 1 and 2 show the steady state concentration of H202 far less than that predicted by equation (1). It is reasonable to conclude a similar low concentration of H2 02 would exist in the neurotoxic action, unless one assumes some catalytic in vivo effect which prevents H2 02 from oxidizing 6-OHDA.
Acknowledgement The support o f this w o r k b y T h e N a t i o n a l Science F o u n d a t i o n via G r a n t GP 3 2 8 4 6 is gratefully a c k n o w l edged.
458
References Allison, J.H. and M.A. Stewart, 1971, Quantitative analysis of ascorbic acid in tissues by gas--liquid chromatography, Anal. Biochem. 43,401. Brezina, M. and P. Zuman, 1958, Polarography in Medicine, Biochemistry and Pharmacology (Interscience, N e w York). Cater, D.B., 1966, in: Symposium on Oxygen Measurements in Blood and Tissues, eds. Payne and Hill (Churchill, London) p. 167. Cohen, G. and R.E. Heikkila, 1974, The generation of hydrogen peroxide, superoxide radical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related cytotoxic agents, J. Biol. Chem. 249, 2447.
Y.-O. LIANG ET AL.
Cohen, G., B. Allis, B. Winston, C. Mytilineou and R. Heikkila, 1975, Prevention of 6-hydroxydopamine neurotoxicity, European J. Pharmacol. 33, 217. Heikkila, R. and G. Cohen, 1972, Further studies on the generation of hydrogen peroxide by 6-hydroxydopamine: Potentiation by ascorbic acid, Mol. Pharmacol. 8,241. Liang, Y.-O., R.M. Wightman, P. Plotsky and R.N. Adams, 1975, Oxidative interactions of 6-hydroxydopamine with C N S constituents, in: Proceedings of the Symposium, 'Chemical Tools in Catecholamine Research', Vol. I, (Elsevier, Amsterdam) p. 15. Sachs, C. and G. Jonnson, 1975, Mechanisms of action of 6-hydroxydopamine, Biochem. Pharmacol. 24, 1.
