Autoxidation Of 6-hydroxydopamine Research Articles

Antioxidant properties of dimethyl sulfoxide and its viability as a solvent in the evaluation of neuroprotective antioxidants

Introduction Dimethyl sulfoxide is an amphiphilic compound whose miscibility with water and its ability to dissolve lipophilic compounds make it an appreciated solvent in biomedical research. However, its reported antioxidant properties raise doubts about its use as a solvent in evaluating new antioxidants. The goal of this investigation was to evaluate its antioxidant properties and carry out a comparative study on the antioxidant properties of some known neuroprotective antioxidants in the presence and absence of dimethyl sulfoxide. Methods The antioxidant properties of dimethyl sulfoxide were studied in rat brain homogenates by determining its ability to reduce both lipid peroxidation (TBARS formation) and protein oxidation (increase in protein carbonyl content and decrease in free thiol content) induced by ferrous chloride/hydrogen peroxide. Its ability to reduce the production of hydroxyl radicals by 6-hydroxydopamine autoxidation was also estimated. The same study was also performed with three known antioxidants (α-phenyl- N- tert-butylnitrone; 2-methyl-2-nitrosopropane; 5,5-dimethyl-1-pyrroline N-oxide) in the presence and absence of dimethyl sulfoxide. Results Our results showed that dimethyl sulfoxide is able to reduce both lipid peroxidation and protein carbonyl formation induced by ferrous chloride/hydrogen peroxide in rat brain homogenates. It can also reduce the production of hydroxyl radicals during 6-hydroxydopamine autoxidation. However, it increases the oxidation of protein thiol groups caused by ferrous chloride/hydrogen peroxide in rat brain homogenate. Discussion Despite the here reported antioxidant and pro-oxidant properties of dimethyl sufoxide, the results obtained with α-phenyl- N- tert-butylnitrone, 2-methyl-2-nitrosopropane, and 5,5-dimethyl-1-pyrroline N-oxide corroborate the antioxidant properties attributed to these compounds and support the potential use of dimethyl sulfoxide as a solvent in the study of the antioxidant properties of lipophilic compounds. Conclusion Dimethyl sulfoxide is a very useful solvent that may be used at relatively low concentrations in the development of new antioxidants with neuroprotective properties.

Effects of aluminum and zinc on the oxidative stress caused by 6-hydroxydopamine autoxidation: relevance for the pathogenesis of Parkinson’s disease

Aluminum and zinc have been related to the pathogenesis of Parkinson’s disease (PD), the former for its neurotoxicity and the latter for its apparent antioxidant properties. 6-Hydroxydopamine (6-OHDA) is an important neurotoxin putatively involved in the pathogenesis of PD, its neurotoxicity often being related to oxidative stress. The potential effect of these metals on the oxidative stress induced by 6-OHDA autoxidation and the potential of ascorbic acid (AA), cysteine, and glutathione to modify this effect were investigated. Both metals, particularly Al 3+, induced a significant reduction in ⋅OH production by 6-OHDA autoxidation. The combined action of AA and a metal caused a significant and sustained increase in ⋅OH generation, particularly with Al 3+, while the effect of sulfhydryl reductants was limited to only the first few minutes of the reaction. However, both Al 3+ and Zn 2+ provoked a decrease in the lipid peroxidation induced by 6-OHDA autoxidation using mitochondrial preparations from rat brain, assessed by TBARS formation. In the presence of AA, only Al 3+ induced a significant reduction in lipid peroxidation. After intrastriatal injections of 6-OHDA in rats, tyrosine hydroxylase immunohistochemistry revealed that Al 3+ reduces 6-OHDA-induced dopaminergic lesion in the striatum, which corroborates the involvement of lipid peroxidation in 6-OHDA neurotoxicity and appears to discard the participation of this mechanism on PD by Al 3+ accumulation. The previously reported antioxidant properties of Zn 2+ appear to be related to the induction of Zn 2+-containing proteins and not to the metal per se.

Reaction of Oxygen with 6-Hydroxydopamine Catalyzed by Cu, Fe, Mn, and V Complexes: Identification of a Thermodynamic Window for Effective Metal Catalysis

In the autoxidation of 6-hydroxydopamine, we investigated the reactivity of metals and metal complexes with a range of abilities to catalyse the reaction with oxygen. Comparing the catalytic effectiveness of aquo metals at pH 7.4, copper accelerated autoxidation 61-fold, iron 24-fold, manganese 7.3-fold, and vanadium 5.7-fold. Copper was thus the most effective catalyst despite being the weakest oxidant, indicating reduction of oxygen as rate limiting. EDTA, which decreases the reduction potential of Fe(III)/Fe(II), increased catalysis by iron 74% to almost that of aquo copper. Conversely, EDTA inhibited catalysis by copper, manganese, and vanadium. Desferrioxamine strongly inhibited catalysis by all of the metals. Histidine prevented catalysis by copper, accelerated catalysis by iron (43%), and had little effect on catalysis by manganese or vanadium. ADP and phytate inhibited catalysis by iron and manganese (50% or more), accelerated catalysis by vanadium (10–27%), and had no effect on catalysis by copper. The effects of the ligands largely reflected their influence on the reduction potential of the metal. Accordingly, addition of NaBr, which increases the reduction potential of Cu(II)/Cu(I), inhibited by 50%. In contrast, Na2SO4 augmented catalysis by copper 3-fold. Consistent with effects of OH− on reduction potentials and on metal coordination to 6-hydroxydopamine, an increase in pH to 8.0 decreased catalysis by copper and iron, but increased that of manganese 10-fold. In conclusion, the catalytic effectiveness of the metal–ligand complexes are largely attributable to their reduction potential, with steric accessibility playing secondary roles. The results delineate a window of catalytically effective potentials suitable for facile reduction and reoxidation by oxygen. By extension the results identify factors determining the pro- and antioxidant roles of ligands in metal mediated reduction of oxygen.

Impairment of human neutrophil oxidative burst by polychlorinated biphenyls: inhibition of superoxide dismutase activity.

We report evidence of a novel mechanism by which polychlorinated biphenyls might act as potent inducers of inflammation. Aroclor 1242 (A1242), a polychlorinated biphenyl mixture, and 2,2',4,4'-tetrachlorobiphenyl (PCB47), a constituent of A1242 that produces the same patterns of effects, impaired the oxidative burst of human neutrophils by inhibiting the antioxidant enzyme superoxide dismutase, which converts O2- to H2O2. Pre-incubation of neutrophils with A1242 or PCB47 before stimulation with phorbol 12-myristate 13-acetate heightened the respiratory burst, producing a significant increase in intracellular O2- production along with a significant decrease in H2O2 production compared with unexposed agonist-stimulated neutrophils. This was also evident in a physiologically relevant situation in which neutrophils pre-incubated with A1242 were subsequently stimulated with a combination of N-formyl-L-methionyl-L-leucyl-L-phenylalanine and tumor necrosis factor-alpha. Incubation of bovine copper-zinc superoxide dismutase (EC 1.15.1.1) with A1242 or PCB47 in a cell-free system reversed the enzyme-mediated inhibition of 6-hydroxydopamine autoxidation, indicating that polychlorinated biphenyls inhibited superoxide dismutase activity. Low superoxide dismutase activity in neutrophils leads to imbalances between production of free radicals and antioxidant defense mechanisms, which can in turn induce tissue damage and hasten the onset of neutrophil apoptosis.

Modulation of 6-hydroxydopamine oxidation by various proteins

The spontaneous autoxidation of the neurotoxin 6-hydroxydopamine proceeds by a free radical chain reaction involving the superoxide anion radical and produces the corresponding chromogen 6-hydroxydopamine quinone and hydrogen peroxide. The rate of this reaction is increased in the presence of ceruloplasmin and peroxidase, and reduced by superoxide dismutase, catalase, and DT-diaphorase. We report some explanations of why these proteins may increase or reduce the rate of autoxidation of 6-hydroxydopamine.

On the Use of 2,4,5-Trihydroxyphenethylamine as Peroxidase Substrate

Abstract The rate of autoxidation of 2,4,5-trihydroxyphenethylamine (6-hydroxydopamine) and the sensitivity of this autoxidation to increase by peroxidases was studied. The autoxidation of 6-hydroxydopamine proceeds by a free radical chain reaction involving O2 and produces the corresponding chromogen 6-hydroxydo-paminequinone and hydrogen peroxide. The yield of quinone produced increased with increasing pH in the range 5 → 9. The ability of peroxidases to increase the autoxidation of 6-hydroxydopamine at pH 5.5 has been used as sensitive assay for these enzymes. The results are tabulated and discussed.

Effect of nickel on oxygen free radical metabolism

The effect of nickel on superoxide dismutase activity (SOD), as well as on rate of hydroxydopamine oxidation, was studied in vitro since lipid peroxidation has been implicated in cell damage by nickel, whose toxicity and carcinogenicity are well established. Nickel strongly inhibits SOD activity. The degree of inhibition is directly proportion to the nickel concentration (tested range 0.066 to 0.33 microgram/mL in the reaction mixture); to the substrate concentration (tested range 0.4 x 10 4M to 1.1 x 10 4M 6-hydroxydopamine); and to reaction mixture. Autoxidation of 6-hydroxydopamine was increased by nickel concentrations higher than 15 micrograms/mL. The combination of excessive oxygen free radical production and inhibition of their elimination by inhibition of SOD activity may contribute to the nickel toxicity that has been reported in industrial accidents, as well as to the high incidence of cancer occurring in nickel workers. It may also contribute to many complications in uremic patients, in whom increased serum nickel levels were reported to be in a similar range to those inhibiting SOD.

6-Hydroxydopamine releases iron from ferritin and promotes ferritin-dependent lipid peroxidation

Iron was released from ferritin by the catecholamine analog, 6-hydroxydopamine. Iron release was more efficient under nitrogen than in air, suggesting that the hydroquinone has the major role in the process. Superoxide dismutase, alone or in combination with catalase, strongly inhibited 6-hydroxydopamine oxidation and greatly enhanced the amount of ferritin iron release. Catalase alone had a similar, but lesser effect. Iron released from ferritin accelerated the autoxidation of 6-hydroxydopamine. This occurred by a mechanism that was inhibited by a combination of catalase and a chelator, and to a lesser extent by superoxide dismutase. 6-Hydroxydopamine was a good promoter of metalcatalysed lipid peroxidation, and ferritin-iron participated in the process. Superoxide dismutase, and to a lesser extent catalase, stimulated peroxidation catalysed by adventitious levels of iron, but in the presence of ferritin, each enzyme was inhibitory. It appears that the greatly enhanced iron release seen under these conditions accelerated the autoxidation of 6-hydroxydopamine so that less was available to participate in peroxidative reactions. However, when 6-hydroxydopamine autoxidation was prevented by a combination of superoxide dismutase and catalase, lipid peroxidation was also inhibited, suggesting that some intermediate of autoxidation is a further requirement for the process.

Intermediates in the aerobic autoxidation of 6-hydroxydopamine: Relative importance under different reaction conditions

Autoxidation of 6-hydroxydopamine (6-OHDA) proceeds through a balanced network of: transition metal ions, superoxide, hydrogen peroxide, hydroxyl radicals, and other species. The contribution of each to the reactions mechanism varies dramatically depending upon which scavengers are present. The contribution of each propagating intermediate increases when the involvement of others is diminished. Thus, superoxide (which is relatively unimportant when metal ions can participate) dominates the reaction when transition metal ions are bound (especially at higher pH), and it becomes essential in the simultaneous presence of catalase plus chelators. Transition metal ions participate more if superoxide is excluded; hydrogen peroxide becomes more important if both ·O 2 − and metal ions are excluded; and hydroxyl radicals contribute more to the reaction mechanism if both H 2O 2 and ·O 2 − are excluded. Superoxide dismutase inhibited strongly, by two distinct mechanisms: a high affinity mechanism (< 13% inhibition) at catalytically effective concentrations, and a low affinity mechanism (almost complete inhibition at the highest concentrations) which depends upon both metal binding and catalytic actions. In the presence of DETAPAC catalytic concentrations of superoxide dismutase inhibited by over 98%. Conversely, metal cheating agents inhibited strongly in the presence of superoxide dismutase. When present alone they stimulated (like EDTA), inhibited (like desferrioxamine), or had little effect (like DETAPAC). Catalase which stimulated slightly but consistently (<5%) when added alone, inhibited 100% in the presence of superoxide dismutase + DETAPAC. However, in the absence of DETAPAC, catalase decreased inhibition by superoxide dismutase, yielding a 100% increase in reaction rate. Hydroxyl scavengers (formate, mannitol or glucose) alone produced little or no (<10%) inhibition, but inhibited by 30% in the presence of catalse + superoxide dismutase. Paradoxically, they stimulated the reaction in the presence of catalse + superoxide dismutase + DETAPAC.

Interactions between metals, ligands, and oxygen in the autoxidation of 6-hydroxydopamine: Mechanisms by which metal chelation enhances inhibition by superoxide dismutase

Transition metal ions and Superoxide participate in different autoxidations to a variable extent. In the reaction of 6-hydroxydopamine (6-OHDA) with oxygen at pH 7.0 or 8.0, addition of 5 to 300 U/ml Superoxide dismutase inhibited autoxidation by up to 96% at the highest concentrations. Superoxide dismutase at concentrations of 5–20 U/ml inhibited by less than 40% when present alone, but inhibited by over 99% in the presence of desferrioxamine or histidine. EDTA also enhanced the inhibition by 20 U/ml Superoxide dismutase to 86%, even though EDTA accelerated the autoxidation of 6-OHDA when present alone or with desferrioxamine. In contrast, other ligands, such as ADP or phytic acid, had little or no effect on inhibition by Superoxide dismutase. Proteins such as albumin, cytochrome oxidase, or denatured Superoxide dismutase also enhanced inhibition by active Superoxide dismutase from less than 40% to over 90%. Evidently, in the presence of redox active metals, autoxidation occurs by inner sphere electron transfer, presumably within a ternary 6-OHDA · metal · oxygen complex. This mechanism does not involve free O ⨪ 2 and is not inhibited by Superoxide dismutase. On the other hand, the presence of certain ligands (including proteins) diminishes the ability of trace metals to exchange electrons with 6-OHDA or oxygen by an inner sphere mechanism. These ligands render autoxidation dependent on propagation by O ⨪ 2 and therefore inhibitable by Superoxide dismutase. Previously conflicting reports that superoxide dismutase alone inhibits 6-OHDA autoxidation are thus explicable on the basis that at sufficient concentration the apoprotein coordinates trace metals in such a way to preclude inner sphere metal catalysis.

Effects of scavengers of oxygen free radicals on the anaerobic oxidation of 6-hydroxydopamine by H2O2

Abstract H 2 O 2 , a product of the aerobic autoxidation of 6-hydroxydopamine, is also consumed as a reactant, contributing progressively more to the oxidation as the concentration of O 2 becomes limiting H 2 O 2 is a less effective oxidant than O 2 , since the anaerobic peroxidatic oxidation of 6-hydrodopamine is slower than the aerobic oxidation by three orders of magnitude. The anaerobic peroxidation was inhibited by the hydroxyl scavengers mannitol (13–40%), glucose (41–62%) and benzoate (15–100%), implying a catalytic role for . OH. -9e strongly inhibitory action of desferrioxamine (76–91%), regardless of which other scavengers were present, suggests a specific role for iron in the reaction, despite the use of Chelex 100-treated buffers. Further addition of diethylenetriaminepentaacetate (DTPA), benzoate or formate to the desferrioxamine-treated reactions resulted in complete inhibition. In contrast, the presence of DTPA alone, accelerated the reaction by 160%. This acceleration is in part due to stimulation by DTPA of production of . OH (by Fenton-type reactions), since it was partially prevented by the hydroxyl scavengers benzoate (32% inhibition) and glucose (41%). Thus, DTPA inhibits the participation of metals other than iron, but potentiates the catalytic role of iron, in the reduction of hydrogen peroxide. The semidehydromannitol radical can reduce the DTPA-Fe 3+ chelate directly, since mannitol further accelerated the DTPA-stimulated peroxidation (by 55%). Superoxide dismutase also accelerated the reaction (by 57–84%). This activation was seen regardless of which other scavengers were present. These effects are explained in terms of potentiating or moderating interactions among the reactive intermediates which propagate the overall reaction.

Redox state of cytochrome C in the presence of the 6-hydroxydopamine/oxygen couple: Oscillations dependent on the presence of hydrogen peroxide or superoxide

The reduction of ferricytochrome c in the presence of 6-hydroxydopamine/O 2 mixtures was examined under various reaction conditions. As the autoxidation of 6-hydroxy-dopamine progressed to completion, there were fluctuations in the net redox reactivity between reducing and oxidizing steady states. This was reflected in a sequence of damped oscillations in the redox state of cytochrome c. Corresponding to the time when 6-hydroxydopamine was 75–100% exhausted, reoxidation of the ferrocytochrome c occurred (prevented by catalase or catalase plus Superoxide dismutase). After the H 2O 2, in turn, was mostly consumed, the next phase commenced in which the cytochrome c became reduced for a second time. This reductive phase was 52% inhibited by superoxide dismutase. In the subsequent and final phase of the process, a progressive oxidation of cytochrome c lasting at least 24 h was observed. Of the initial reduction of ferricytochrome c, at most 37% can be attributed to direct reduction by 6-hydroxydopamine or its semiquinone. This initial net reduction of cytochrome c was inhibited 51% by superoxide dismutase and 41% by catalase. However, since either catalase or superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine by at least as much as it slowed the reduction of cytochrome c, their effects in slowing the reduction of cytochrome c resulted largely from the decreased production of those free radicals which reduce ferricytochrome c, and only in part from accelerated removal. Elimination of the actions of transition metal ions (whether by passage of the buffer solutions through Chelex 100 resins or by addition of desferrioxamine to the reaction medium) slowed both the reoxidation and rereduction by up to 96%. Addition of mannitol decreased the rate of the first reoxidation by 25% and increased the rate of the rereduction by 7%. In general, the oscillations are explicable in terms of changes in the steady state levels of O 2 and H 2O 2, with metal ions playing a major role and hydroxyl radicals a minor role in both the reoxidation and rereduction.

6-Hydroxydopamine does not reduce molecular oxygen directly, but requires a coreductant

The autoxidation of 6-hydroxydopamine (6HODA) was virtually blocked ( k 2 < 10 −15M −1 S −1 at pH 8.0, ionic strength 0.04) by the simultaneous presence of diethylenetriaminepentaacetic acid (DTPA), catalase, and superoxide dismutase (SOD). No quinone product or oxygen consumption was detectable after 12 min under these conditions. Thus, if 6HODA is to react with molecular oxygen at a measurable rate, some other redox species is required as a coreductant. The subsequent addition of formate or mannitol proved capable of overcoming the total inhibition induced by the mixture of catalase, SOD, and DTPA. The simplest interpretation of the data is that most of the autoxidation of 6HODA, as commonly observed, involves successive reduction of a series of metal-bound species of oxygen; the actual transfer of electrons occurring within a ternary reductant-metal-oxygen transition state.

Inhibition of iron-stimulated catecholamine degradation by the iron-chelators DETAFAC and Desferal: Potentially useful laboratory agents

The addition of ferrous sulfate to phosphate buffer at pH 7.4 brought about a large increase in the rate of autoxidation of dopamine or norepinephrine. The iron-chelating agents diethylenetri-aminepentaacetic acid (DETAPAC) and desferroxamine (Desferal) inhibited both the baseline as well as the iron-stimulated autoxidation of these catecholamines. Lesser inhibitory effects were observed with another iron-chelating agent, namely EDTA. In other experiments, DETAPAC or Desferal had little effect on the autoxidation of 6-hydroxydopamine in the absence of added ferrous sulfate, but both inhibited the ferrous sulfate-stimulated autoxidation. In contrast, both the baseline and ferrous sulfate-catalyzed rate of oxidation of 6-hydroxydopamine were greatly stimulated by EDTA addition. DETAPAC and Desferal appear to be useful experimental tools that may be added to solutions containing catecholamines to prevent their degradation.

Effects of superoxide dismutase and catalase on catalysis of 6-hydroxydopamine and 6-aminodopamine autoxidation by iron and ascorbate

The effects of superoxide dismutase and catalase on autoxidation of 6-hydroxydopamine and 6-aminodopamine in several chemical environments were studied. Inhibition by superoxide dismutase of autoxidation of 6-hydroxydopamine to its p-quinone required the presence of metal chelators, EDTA or diethylenetriamine pentaacetic acid (DETAPAC). A “lag” period in 6-hydroxydopamine autoxidation in the presence of superoxide dismutase couid be prevented by carrying out autoxidation in a mixture of 6-hydroxydopamine and its p-quinone, conditions in which adequate levels of the semiquinone are available for reaction with O 2. Catalase potentiated the inhibitory effect of superoxide dismutase in the presence of EDTA but had no effect in the presence of DETAPAC. Superoxide dismutase and catalase had no effect on the initial rate of 6-aminodopamine autoxidation to the p-quinone imine or on later intracyclization and polymer formation. Iron chelated by EDTA functioned as a catalyst in 6-hydroxydopamine autoxidation, making the reaction independent of superoxide as shown by the lack of effect of superoxide dismutase. the presence of iron-EDTA resulted in bleaching of the p-quinone product and the consumption of the H 2O 2 formed during autoxidation. Catalase had no effect on the rate of 6-hydroxydopamine autoxidation but completely prevented bleaching of the p-quinone, probably by preventing formation of hydroxyl radical by a Fenton reaction between iron-EDTA and H 20 2. Ethanol, which scavenges hydroxyl radical but not superoxide of H 2O 2, similarly prevented bleaching of the p-quinone with no other effects on autoxidation. Iron-EDTA also catalyzed 6-aminodopamine autoxidation with associated consumption of H 2O 2. Superoxide dismutase, catalase and ethanol had no effect on 6-aminodopamine autoxidation in the presence or absence of iron-EDTA, showing the independence of the kinetics of 6-aminodopamine autoxidation and polymerization from its products, superoxide, H 20 2 and hydroxyl radical. Chelation by DETAPAC prevented the effects of iron on 6-hydroxydopamine and 6-aminodopamine autoxidation. Autoxidation of 6-hydroxydopamine in the presence of ascorbate exhibited a lag phase followed by a linear phase of autoxidation. Superoxide dismutase, catalase and ethanol had no effect on 6-hydroxydopamine or 6-aminodopamine autoxidation in the presence of ascorbate. Autoxidation of 6-hydroxydopamine or 6-aminodopamine in the combined presence of iron-EDTA and ascorbate showed the full effects of both additions, except that 6-hydroxydopamine autoxidation exhibited no lag phase and the iron-catalyzed autoxidation of ascorbate occurred simultaneously with the 6-hydroxydopamine or 6-aminodopamine reactions.

On the Nature of Biochemically Generated Hydroxyl Radicals Studies using the Bleaching of p‐Nitrosodimethylaniline as a Direct Assay Method

An efficient scavenger for radiolytically generated hydroxyl (OH) radicals, p-nitrosodimethylaniline, was used to try to substantiate the presence of this oxygen radical species in several biochemical systems. Most of these systems which were investigated had previously been assumed to generate OH radicals, e.g. the autoxidation of 6-hydroxydopamine, the hydroxylating system NADH/phenazine methosulfate, and the oxidation of xanthine or acetaldehyde by xanthine oxidase. We did not observe inhibition of the bleaching of p-nitrosodimethylaniline in oxygenated solutions by other scavengers of OH radicals nor, in the case of xanthine/xanthine oxidase, by catalase and superoxide dismutase. We therefore conclude that, under biochemical conditions as opposed to radiolysis or photolysis, no freely diffusable OH radicals are formed. Rather, a strongly oxidizing OH-analogous complex is considered to represent the p-nitrosodimethylaniline-detectable species formed under these conditions.

The stimulation of 6-hydroxydopamine autoxidation by bivalent copper: Potential importance in the neurotoxic process

Copper sulfate (Cu 2+, 10 −6M to 10 −5M) stimulated the rate of autoxidation of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) as judged both by absorbancy measurements at 490 nm and oxygen consumption. This stimulation of 6-OHDA autoxidation by Cu 2+ was prevented by the copper chelating agent EDTA. In other experiments, Cu 2+ stimulated the rates of autoxidation of dopamine and norepinephrine as well as the neurotoxic agents 6-aminodopamine, 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine. It is suggested that intraneuronal levels of copper, by virtue of controlling the rates of autoxidation reactions, might be important in vivo.

A sensitive assay for superoxide dismutase based on the autoxidation of 6-hydroxydopamine

A simple, sensitive spectrophotometric assay system for superoxide dismutase (SOD) has been developed. This assay is based on the inhibitory effects of SOD on the initial rate of 6-hydroxydopamine autoxidation. The inhibition of 6-hydroxydopamine autoxidation was virtually linear to an SOD concentration of approximately 100 ng of SOD/ml (about a 50% inhibition at 100 ng/ml; there was a greater inhibition at higher SOD concentrations). With this assay system it was determined that SOD levels in rat brain, liver, and spinal cord were 84, 660, and 56 μg of SOD/g of tissue, respectively. These results agree very well with results obtained by other assays.

The Generation of Hydrogen Peroxide, Superoxide Radical, and Hydroxyl Radical by 6-Hydroxydopamine, Dialuric Acid, and Related Cytotoxic Agents

Hydrogen peroxide, superoxide radical, and hydroxyl radical were all formed during the autoxidation of four cytotoxic agents, namely, 6-hydroxydopamine, 6-aminodopamine, 6,7-dihydroxytryptamine, and dialuric acid. Ascorbic acid and 5-hydroxydopamine, two autoxidizable agents of biological interest but without similar cytotoxic actions, were used as negative controls. The results tend to implicate O2- and ·OH, as well as H2O2, in the molecular mechanisms for cytotoxicity. Production of H2O2 was measured with an oxygen electrode. Accumulation of H2O2 was apparent from the return to solution of approximately one-half of the consumed oxygen after the addition of catalase. Superoxide radicals were detected by reduction of cytochrome c, with inhibition by superoxide dismutase. Although O2- radicals were observed previously by other means during the autoxidation of 6-hydroxydopamine, there was no clear effect in the cytochrome c system. This may have been due, in part, to a previously described scavenging of O2- by 6-hydroxydopamine itself. Hydroxyl radicals were detected by reaction with β-methylthiopropionaldehyde to form ethylene. Catalase or superoxide dismutase inhibited ethylene production. These results point to a reaction between H2O2 and O2- (Haber-Weiss reaction) as a major source of the ·OH radicals. Ethylene production was inhibited by known ·OH trappers, such as benzoate and ethanol, and by a variety of other agents. Catechol and the catecholamines, dopamine and norepinephrine, were very potent inhibitors of ethylene production. The effectiveness of these agents may have been due to a combined action of blocking ·OH formation as well as accelerating its removal. Superoxide dismutase had a biphasic effect in the 6-hydroxydopamine system, inhibitory at early times but causing increased accumulation of ethylene at later times. An explanation for this latter phenomenon may be based on a reaction between O2- and ·OH, as described by other investigators.

6-Hydroxydopamine: evidence for superoxide radical as an oxidative intermediate.

Superoxide dismutase inhibited the autoxidation of 6-hydroxydopamine as measured by the rate of formation of a quinone and the rate of oxygen consumption. These observations demonstrate the formation of the superoxide radical during the autoxidation process. This finding may be relevant to the mechanism of adrenergic nerve terminal degeneration caused by 6-hydroxydopamine.