Monika Z. Wrona

Electrochemical oxidation of 5-hydroxytryptamine in aqueous solution at physiological pH

The electrochemical oxidation of the indolic neurotransmitter 5-hydroxytryptamine (5-HT) has been studied in aqueous solution at physiological pH using a pyrolytic graphite electrode. The initial step in the oxidation involves a 2e,1H+ reaction leading to a reactive carbocation intermediate (28b). This intermediate is attacked by 5-HT in an ion-substrate reaction to give 5,5′-dihydroxy-4,4′-bitryptamine (1) as the major product along with the C(4)OC(5′) bridged dimer 9. Alternatively cation 28b is attacked by water to yield, ultimately, tryptamine-4,5-dione (5). Many additional reactions occur between primary dimers 1 and 9 and carbocation 28b such that a very complex mixture of ultimate products are formed. A total of 15 products have been isolated and partially or completely characterized. It is possible that the oxidation reaction pathways and product profile elucidated might have a bearing on, as yet hypothetical, aberrant oxidation reactions of 5-HT in the central nervous system which could play a role in the etiology of mental illnesses such as diseases of the Alzheimers type.

Interactions of 5-hydroxytryptamine with oxidative enzymes

Peroxidase (EC 1.11.1.7)/H2O2, ceruloplasmin (human type X)/O2, and tyrosinase (EC 1.14.18.1)/O2 all oxidized the indolic neurotransmitter 5-hydroxytryptamine (5-HT) in the physiological pH domain. Peroxidase/H2O2 oxidized 5-HT at pH values down to about 2.5. All oxidation reactions generated complex mixtures of products which included at least one known neurotoxin, tryptamine-4,5-dione. In general, the enzymatic oxidation pathways paralleled the in vitro electrochemical oxidation of 5-HT which has permitted suggestions to be made concerning the probable mechanisms of the enzyme-mediated reactions.

Hydroxyl radical-mediated oxidation of serotonin: potential insights into the neurotoxicity of methamphetamine.

Abstract: When incubated with a hydroxyl radical (HO•)‐generating system (ascorbic acid/Fe2+‐EDTA/O2/H2O2), 5‐hydroxytryptamine (5‐HT; serotonin) is rapidly oxidized initially to a mixture of 2,5‐, 4,5‐, and 5,6‐dihydroxytryptamine (DHT). The major reaction product is 2,5‐DHT, which at physiological pH exists as its keto tautomer, 5‐hydroxy‐3‐ethylamino‐2‐oxindole (5‐HEO). Rapid autoxidation of 4,5‐DHT gives tryptamine‐4,5‐dione (T‐4,5‐D), which reacts with the C(3)‐centered carbanion of 5‐HEO to give 3,3′‐bis(2‐aminoethyl)‐5‐hydroxy‐[3,7′‐bi‐1H‐indole]‐2,4′,5′‐3H‐trione (7). The latter slowly cyclizes to 3′‐(2‐aminoethyl)‐1′,6′,7′,8′‐tetrahydro‐5‐hydroxyspiro[3H‐indole‐3,9′‐[9H]pyrrolo[2,3‐f]quinoline]‐2,4′,5′(1H)‐ trione (9). A minor amount of T‐4,5‐D dimerizes to give 7,7′‐bi‐(5‐hydroxytryptamine‐4‐one) (7,7′‐D). In the presence of GSH, the reaction of T‐4,5‐D with 5‐HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T‐4,5‐D to give 7‐S‐glutathionyltryptamine‐4,5‐dione (11). The results of this investigation suggest that 5,6‐DHT, 5‐HEO, 7, and 9 are products unique to the HO•‐mediated oxidation of 5‐HT. Thus, the observation of other investigators that 5,6‐DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO• formation. However, the present in vitro study indicates that 5,6‐DHT is a rather minor, unstable product of the HO•‐mediated oxidation of 5‐HT and suggests that detection of 5‐HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO• formation. Furthermore, one or more of the intermediates and major products of oxidation of 5‐HT by HO• might, in addition to 5,6‐DHT, contribute to the MA‐induced degeneration of serotonergic neurons.

Electrochemical oxidation of tryptophan

Abstract The electrochemical oxidation of tryptophan at graphite electrodes has been studied in aqueous solutions over a wide pH range. The single voltammetric oxidation peak of tryptophan is due to an irreversible 2 e− reaction giving an extremely reactive methylene-imine intermediate. This intermediate, being highly electrophilic, is attacked by nucleophiles present in the reaction solution. Nucleophilic attack by water leads to two more intermediates which can be detected by thin-layer spectroelectrochemistry and cyclic voltammetry. Further chemical and/or electrochemical reactions of these intermediates lead to formation of two isomers of 2-carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-(2,3b)-indole, oxindolylalanine, dioxindolylalanine and kynurenine. Mechanisms rationalizing the formation of these products are proposed. Two other products appear to be oligomeric or polymeric compounds.

Electrochemical oxidation of N-methylated derivatives of uric acid

Abstract The electrochemical oxidation of a number of N-methylated uric acids has been studied by linear and cyclic sweep voltammetry, coulometry and thin-layer spectroelectrochemistry. The observed results support the view that the electrooxidation is a 2e reaction to give a very unstable diimine primary product. This is rapidly hydrated to give an imine-alcohol which is further hydrated to give a uric acid-4,5-diol derivative which subsequently fragments to the various products.

5,5′‐Dihydroxy‐4,4′‐Bitryptamine: A Potentially Aberrant, Neurotoxic Metabolite of Serotonin

Abstract: Previous investigators have detected unknown oxidized forms of 5‐hydroxytryptamine (5‐HT) in the CSF of Alzheimers disease (AD) patients. Furthermore, an unidentified autoxidation product of this neurotransmitter is an inhibitor of acetylcholinesterase (AChE), an enzyme compromised in the Alzheimer brain. In this study it is demonstrated that the major product of autoxidation of 5‐HT is 5,5′‐dihydroxy‐4,4′‐bitryptamine (DHBT). Central administration of DHBT to mice at a dose of 40 μg (free base) evokes profound behavioral responses, which persist until the animals die (∼24 h). One hour after central administration of DHBT, the levels of norepinephrine, dopamine, 5‐HT, and acetylcholine and their metabolites in whole brain are greatly elevated. Disturbances to the catecholaminergic and serotonergic systems were still evident shortly before the death of animals. DHBT is also shown to be a noncompetitive inhibitor of AChE in vitro. These observations suggest that if DHBT is formed as an aberrant metabolite of 5‐HT in the human brain, it could potentially be neurotoxic and contribute to the neuronal degeneration and other neurochemical and neurobiochemical changes associated with AD or perhaps other neurodegenerative diseases.

7-S-Glutathionyl-tryptamine-4,5-dione: A possible aberrant metabolite of serotonin

Tryptamine-4,5-dione (Compound 1) is an in vitro oxidation product of 5-hydroxytryptamine (5-HT). Recent evidence has suggested that aberrant oxidations of 5-HT occur in the central nervous system of individuals with Alzheimers disease (AD). In the event that Compound 1 is formed as a result of oxidation of 5-HT within serotonergic nerve terminals or axons, it would be expected to be rapidly conjugated by intraneuronal glutathione (GSH) to give 7-S-glutathionyl-tryptamine-4,5-dione (Compound 2). When injected into the brains of laboratory mice, Compound 2 was lethal (LD50 = 21 micrograms) and evoked hyperactivity for the first 30 min following drug administration. Particularly during this hyperactive phase Compound 2 caused a statistically significant decrease in whole brain levels of norepinephrine and 5-HT. Levels of dopamine were also decreased while whole brain concentrations of its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were increased significantly. In the presence of GSH, NADPH and ascorbic acid, Compound 2 redox cycled in reactions that catalyzed the oxidation of these cellular reductants by molecular oxygen and formed H2O2 as a byproduct. Compound 2 also reacted with molar excesses of GSH to form more structurally complex glutathionyl conjugates. Several of these conjugates have been isolated and their structures determined using spectroscopic methods. It is conceivable that one or more of these conjugates might serve as analytical markers in a search for evidence in support of the hypothesis that aberrant oxidations of 5-HT occur in the Alzheimer brain. The redox cycling properties of Compound 2 and its facile reactions with cellular nucleophiles such as GSH may represent mechanisms that contribute to the toxicity of this drug.

Investigation of the enzymic and electrochemical oxidation of uric acid derivatives.

The electrochemical oxidation of a number of N-methylated uric acids at the pyrolytic graphite and gold electrodes has been compared to their enzymic oxidation with type VIII peroxidase and H2O2. Spectral, electroanalytical and kinetic evidence supports the conclusion that for all compounds the electrochemical and enzymic reactions proceed by identical mechanisms.

Putative oxidative metabolites of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline of potential relevance to the addictive and neurodegenerative consequences of ethanol abuse

Ethanol is metabolized in the brain by catalase/H2O2 to yield acetaldehyde and by an ethanol-inducible form of cytochrome P450 (P450 IIE1) in a reaction that yields oxygen radicals. Within the cytoplasm of serotonergic axon terminals these metabolic pathways together provide conditions for the endogenous synthesis of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline (1), by reaction of acetaldehyde with unbound 5-hydroxytryptamine (5-HT), and for the oxygen radical-mediated oxidation of this alkaloid. The major initial product of the hydroxyl radical (HO.)-mediated oxidation of 1 in the presence of free glutathione (GSH), a constituent of nerve terminals and axons, is 8-S-glutathionyl-1-methyl-1,2,3,4-tetrahydro-beta-carboline-5,6-dione (6). When administered into the brains of mice, 6 is a potent toxin (LD50 = 2.9 microg) and evokes episodes of hyperactivity and tremor. Compound 6 binds at the GABA(B) receptor and evokes elevated release and turnover of several neurotransmitters. Furthermore, the GABA(B) receptor antagonist phaclofen attenuates the behavioral response caused by intracerebral administration of 6. These observations suggest that 6 might be an inverse agonist at the GABA(B) receptor site. Accordingly, it is speculated that ethanol drinking might potentiate formation of 6 that contributes to elevated release of several neurotransmitters including dopamine (DA) and endogenous opioids in regions of the brain innervated by serotonergic axon terminals. Subsequent interactions of DA and opioids with their receptors might be related to the initial development of dependence on ethanol. Redox cycling of 6 (and of several putative secondary metabolites) in the presence of intraneuronal antioxidants and molecular oxygen to produce elevated fluxes of cytotoxic reduced oxygen species might contribute to the degeneration of serotonergic pathways. Low levels of 5-HT in certain brain regions of the rat predisposes these animals to drink or augments drinking. Accordingly, 6, formed as a result of ethanol metabolism in the cytoplasm of certain serotonergic axon terminals, might contribute to the initial development of dependence on ethanol, by mediating DA and opioid release, and long-term preference and addiction to the fluid as a result of the progressive degeneration of these neurons.

5‐Hydroxy‐3‐Ethylamino‐2‐Oxindole Is Not Formed in Rat Brain Following a Neurotoxic Dose of Methamphetamine: Evidence that Methamphetamine Does Not Induce the Hydroxyl Radical‐Mediated Oxidation of Serotonin

Abstract: Oxygen radicals have been implicated in the neurodegenerative and other neurobiological effects evoked by methamphetamine (MA) in the brain. It has been reported that shortly after a single large subcutaneous dose of MA to the rat, the serotonergic neurotoxin 5,6‐dihydroxytryptamine (5,6‐DHT) is formed in the cortex and hippocampus. This somewhat controversial finding suggests that MA potentiates formation of the hydroxyl radical (HO•) that oxidizes 5‐hydroxytryptamine (5‐HT) to 5,6‐DHT, which, in turn, mediates the degeneration of serotonergic terminals. A major and more stable product of the in vitro HO•‐mediated oxidation of 5‐HT is 5‐hydroxy‐3‐ethylamino‐2‐oxindole (5‐HEO). In this investigation, a method based on HPLC with electrochemical detection (HPLC‐EC) has been developed that permits measurement of very low levels of 5‐HEO in rat brain tissue in the presence of biogenic amine neurotransmitters/metabolites. After intracerebroventricular administration into rat brain, 5‐HEO is transformed into a single major, but unknown, metabolite that can be detected by HPLC‐EC. One hour after administration of MA (100 mg/kg s.c.) to the rat, massive decrements of 5‐HT were observed in all regions of the brain examined (cortex, hippocampus, medulla and pons, midbrain, and striatum). However, 5‐HEO, its unidentified metabolite, or 5,6‐DHT were not detected as in vivo metabolites of 5‐HT. MA administration, in particular to rats pretreated with pargyline, resulted in the formation of low levels of N‐acetyl‐5‐hydroxytryptamine (NAc‐5‐HT) in all brain regions examined. These results suggest that MA does not potentiate the HO•‐mediated oxidation of 5‐HT. Furthermore, the rapid MA‐induced decrease of 5‐HT might not only be related to oxidative deactivation of tryptophan hydroxylase, as demonstrated by other investigators, but also to the inhibition of tetrahydrobiopterin biosynthesis by NAc‐5‐HT. The massive decrements of 5‐HT evoked by MA are accompanied by small or no corresponding increases in 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) levels. This is due, in part, to the relatively rapid clearance of 5‐HIAA from the brain and monoamine oxidase (MAO) inhibition by MA. However, the loss of 5‐HT without corresponding increases in its metabolites point to other mechanisms that might deplete the neurotransmitter, such as oxidation by superoxide radical anion (O2•−), a reaction that in vitro does not generate 5‐HEO or 5,6‐DHT but rather another putative neurotoxin, tryptamine‐4,5‐dione. One hour after administration, MA evokes large depletions of norepinephrine (NE) throughout the brain but somewhat smaller decrements of dopamine (DA) that are restricted to the nigrostriatal pathway. Furthermore, MA evokes a major shift in the metabolism of both NE and DA from the pathway mediated by MAO to that mediated by catechol‐O‐methyltransferase. The profound and widespread effects of MA on the noradrenergic system, but more anatomically localized influence on the dopaminergic system, suggests that NE in addition to DA, or unusual metabolites of these neurotransmitters, might play roles in the neurodegenerative effects evoked by this drug.