Virginia R. Florang

Protein Reactivity of 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopamine Metabolite, Is Dependent on Both the Aldehyde and the Catechol

Dopamine (DA) has been implicated as an endogenous neurotoxin to explain selective neurodegeneration, as observed for Parkinsons disease (PD). However, previous work demonstrated that 3,4-dihydroxyphenylacetaldehyde (DOPAL) was more toxic than DA. DOPAL is generated as a part of DA catabolism via the activity of monoamine oxidase, and the mechanism of DOPAL toxicity is proposed to involve protein modification. Previous studies have demonstrated protein reactivity via the aldehyde moiety; however, DOPAL contains two reactive functional groups (catechol and aldehyde), both with the potential for protein adduction. The goal of this work was to determine whether protein modification by DOPAL occurs via a thiol-reactive quinone generated from oxidation of the catechol, which is known to occur for DA, or if the aldehyde forms adducts with amine nucleophiles. To accomplish this objective, the reactivity of DOPAL toward N-acetyl-lysine (NAL), N-acetyl-cysteine (NAC), and two model proteins was determined. In addition, several DOPAL analogues were obtained and used for comparison of reactivity. Results demonstrate that at pH 7.4 and 37 degrees C, the order of DOPAL reactivity is NAL >> NAC and the product of NAL and DOPAL is stable in the absence of reducing agent. Moreover, DOPAL will react with model proteins, but in the presence of amine-selective modifiers citraconic anhydride and 2-iminothiolane hydrochloride, the reactivity of DOPAL toward the proteins is diminished. In addition, DOPAL-mediated protein cross-linking is observed when a model protein or a protein mixture (i.e., mitochondria lysate) is treated with DOPAL at concentrations of 5-100 microM. Protein cross-linking was diminished in the presence of ascorbate, suggesting the involvement of a quinone in DOPAL-mediated protein modification. These data indicate that DOPAL is highly reactive toward protein nucleophiles with the potential for protein cross-linking.

Products of Oxidative Stress Inhibit Aldehyde Oxidation and Reduction Pathways in Dopamine Catabolism Yielding Elevated Levels of a Reactive Intermediate

Dopamine (DA) has been implicated as an endogenous neurotoxin to explain the selective neurodegeneration as observed for Parkinsons disease (PD). In addition, oxidative stress and lipid peroxidation are hypothesized culprits in PD pathogenesis. DA undergoes catabolism by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) via aldehyde dehydrogenase (ALDH). As a minor and compensatory metabolic pathway, DOPAL can be reduced to 3,4-dihydroxyphenylethanol (DOPET) via cytosolic aldehyde or aldose reductase (AR). Previous studies have found DOPAL to be significantly more toxic to DA cells than DA and that the major lipid peroxidation products, that is, 4-hydroxynonenal (4HNE) and malondialdehyde (MDA), potently inhibit DOPAL oxidation via ALDH. The hypothesis of this work is that lipid peroxidation products inhibit DOPAL oxidation, yielding aberrant levels of the toxic aldehyde intermediate. To test this hypothesis, nerve growth factor-differentiated PC6-3 cells were used as a model for DA neurons. Cell viability in the presence of 4HNE and MDA (2-100 microM) was measured by MTT assay, and it was found that only 100 microM 4HNE exhibited significant cytotoxicity. Treatment of cells with varying concentrations of 4HNE and MDA resulted in reduced DOPAC production and significant elevation of DOPAL levels, suggesting inhibition of ALDH. In cells treated with 4HNE that exhibited elevated DOPAL, there was a significant increase in DOPET. However, elevated DOPET was not observed for the cells treated with MDA, suggesting MDA to be an inhibitor of AR. Using isolated cytosolic AR, it was found that MDA but not 4HNE inhibited reductase activity toward DOPAL, surprisingly. These data demonstrate that the oxidative stress products 4HNE and MDA inhibit the aldehyde biotransformation step of DA catabolism yielding elevated levels of the endogenous neurotoxin DOPAL, which may link oxidative stress to selective neurodegeneration as seen in PD.

Inhibition and Covalent Modification of Tyrosine Hydroxylase by 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopamine Metabolite

Parkinsons disease (PD) is a neurodegenerative disorder marked by the selective loss of dopaminergic neurons, leading to a decrease of the neurotransmitter dopamine (DA). DA is metabolized by monoamine oxidase to 3,4-dihydroxyphenyacetaldehyde (DOPAL). While the mechanism of pathogenesis of PD is unknown, DOPAL has demonstrated the ability to covalently modify proteins and cause cell death at concentrations elevated from physiologic levels. Currently, the identities of protein targets of the aldehyde are unknown, but previous studies have demonstrated the ability of catechols and other DA-catabolism products to interact with and inhibit tyrosine hydroxylase (TH). Given that DOPAL is structurally related to DA and is a highly reactive electrophile, it was hypothesized to modify and inhibit TH. The data presented in this study positively identified TH as a protein target of DOPAL modification and inhibition. Furthermore, western blot analysis demonstrated a concentration-dependent decrease in antibody recognition of TH. DOPAL in cell lysate significantly inhibited TH activity as measured by decreased l-DOPA production. Inhibition of TH was semi-reversible, with the recovery of activity being time and concentration-dependent upon removal of DOPAL. These data indicate DOPAL to be a reactive DA-metabolite with the capability of modifying and inhibiting an enzyme important to DA synthesis.

Relative inhibitory potency of molinate and metabolites with aldehyde dehydrogenase 2: Implications for the mechanism of enzyme inhibition

Molinate is a thiocarbamate herbicide used as a pre-emergent in rice patty fields. It has two predominant sulfoxidation metabolites, molinate sulfoxide and molinate sulfone. Previous work demonstrated an in vivo decrease in liver aldehyde dehydrogenase (ALDH) activity in rats treated with molinate and motor function deficits in dogs dosed chronically with this compound. ALDH is an enzyme important in the catabolism of many neurotransmitters, such as dopamine. Inhibition of this enzyme may lead to the accumulation of endogenous neurotoxic metabolites such as 3,4-dihydroxyphenylacetaldehyde, a dopamine metabolite, which may account for the observed neurotoxicity. In this study, the relative reactivity of molinate and both of its sulfoxidation metabolites toward ALDH was investigated, as well as the mechanism of inhibition. The ALDH activity was monitored in two different model systems, human recombinant ALDH (hALDH2) and mouse striatal synaptosomes. Molinate sulfone was found to be the most potent ALDH inhibitor, as compared to molinate and molinate sulfoxide. The reactivity of these three compounds was also assessed, using N-acetyl Cys, model peptides, and hALDH2. It was determined that molinate sulfone is capable of covalently modifying Cys residues, including catalytic Cys302 of ALDH, accounting for the observed enzyme inhibition.

Aldehyde dehydrogenase inhibition generates a reactive dopamine metabolite autotoxic to dopamine neurons

The neurotransmitter dopamine (DA) is important for numerous biological functions, including control of movement. Oxidation of DA to highly toxic and reactive species has been hypothesized to contribute to the selective neurodegeneration observed in Parkinsons disease (PD). DA catabolism is initiated by oxidative deamination via monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL). Such metabolism can be problematic as it greatly increases the toxicity of DA by production of DOPAL, known to be a toxic and reactive intermediate. DOPAL undergoes carbonyl metabolism primarily via aldehyde dehydrogenase (ALDH) enzymes to a less toxic acid product. Previous studies from our laboratory have shown that cellular ALDH enzymes are sensitive towards products of oxidative stress and lipid peroxidation, which are thought to be elevated during PD pathogenesis. Inhibition of ALDH and the resulting accumulation of DOPAL are concerning as DOPAL is toxic to dopaminergic cells, readily modifies proteins and causes protein aggregation. In addition, pesticides with association between exposure and PD incidence can interfere with DA metabolism and trafficking and/or ALDH activity, directly or indirectly, yielding elevation of DOPAL. Therefore, impairment of carbonyl metabolism is a potential mechanistic link between cellular insult and generation of a toxic and reactive intermediate endogenous to dopamine neurons.

Cellular Localization of Dieldrin and Structure–Activity Relationship of Dieldrin Analogues in Dopaminergic Cells

The incidence of Parkinsons disease (PD) correlates with environmental exposure to pesticides, such as the organochlorine insecticide, dieldrin. Previous studies found an increased concentration of the pesticide in the striatal region of the brains of PD patients and also that dieldrin adversely affects cellular processes associated with PD. These processes include mitochondrial function and reactive oxygen species production. However, the mechanism and specific cellular targets responsible for dieldrin-mediated cellular dysfunction and the structural components of dieldrin contributing to its toxicity (toxicophore) have not been fully defined. In order to identify the toxicophore of dieldrin, a structure-activity approach was used, with the toxicity profiles of numerous analogues of dieldrin (including aldrin, endrin, and cis-aldrin diol) assessed in PC6-3 cells. The MTT and lactate dehydrogenase (LDH) assays were used to monitor cell viability and membrane permeability after treatment with each compound. Cellular assays monitoring ROS production and extracellular dopamine metabolite levels were also used. Structure and stereochemistry for dieldrin were found to be very important for toxicity and other end points measured. Small changes in structure for dieldrin (e.g., comparison to the stereoisomer endrin) yielded significant differences in toxicity. Interestingly, the cis-diol metabolite of dieldrin was found to be significantly more toxic than the parent compound. Disruption of dopamine catabolism yielded elevated levels of the neurotoxin, 3,4-dihydroxyphenylacetaldehyde, for many organochlorines. Comparisons of the toxicity profiles for each dieldrin analogue indicated a structure-specific effect important for elucidating the mechanisms of dieldrin neurotoxicity.

Catechol and aldehyde moieties of 3,4-dihydroxyphenylacetaldehyde contribute to tyrosine hydroxylase inhibition and neurotoxicity

Parkinsons disease (PD) is a progressive neurodegenerative disorder which leads to the selective loss of dopaminergic neurons. This causes a decrease in the important neurotransmitter dopamine (DA), which is essential for coordinated movement. Previous studies have implicated the monoamine oxidase metabolite of DA, 3,4-dihydroxphenylacetaldehyde (DOPAL), in the pathogenesis of PD and have shown it to be a reactive intermediate capable of protein modification. DOPAL also has demonstrated the ability to cause mitochondrial dysfunction and lead to significant inhibition of the rate-limiting enzyme in DA synthesis, tyrosine hydroxylase (TH). The current study was undertaken to investigate four analogs of DOPAL, including a novel nitrile analog, to determine how the structure of DOPAL is related to its toxicity and inhibition of TH. Both mitochondrial function and inhibition of TH in cell lysate were investigated. Furthermore, a novel whole cell assay was designed to determine the consequence to enzyme action when DOPAL levels were elevated. The results presented here demonstrate that changes to DOPAL structure lead to a decrease in toxicity and inhibition of enzyme activity as compared to the parent compound. Furthermore, the production of superoxide anion but not hydrogen peroxide increased in the presence of elevated DOPAL. These results reveal the toxicity of DOPAL and demonstrate that both the catechol and aldehyde are required to potently inhibit TH activity.

Antioxidant-Mediated Modulation of Protein Reactivity for 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopamine Metabolite.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is an endogenously produced toxic aldehyde. It is a bifunctional electrophile implicated in the loss of dopaminergic cells concomitant with Parkinsons disease and neurodegeneration. DOPAL is known to react with proteins and amino acids such as N-acetyl lysine (NAL); oxidation of the catechol moiety to the quinone of DOPAL increases this reactivity. Here, we demonstrate the ability of the antioxidants N-acetylcysteine, glutathione, and ascorbic acid to mitigate the reactivity of DOPAL with proteins and amino acids in a dose-dependent fashion. Conversely, Trolox did not lessen the observed reactivity with proteins. Interestingly, use of tricine, a buffer and reducing agent, in these systems also decreased the reactivity of DOPAL with amines, yielding tricine-derived free radical species. Modification of amines with aldehydes typically involves Schiff base chemistry; however, the observance of free radicals suggests that an oxidative step is involved in the reaction of DOPAL with lysine. Furthermore, while Schiff base formation is usually optimal at pH 5, the reaction rate of DOPAL with NAL is negligible at pH 5 and is enhanced under basic conditions (e.g., pH 9). Conditions of high pH are also favorable for catechol auto-oxidation, known to occur for DOPAL. The antioxidant-mediated protection demonstrated here suggests that oxidative stress may impart cellular vulnerability to protein modification by DOPAL. Therefore, depleted antioxidants and increased levels of lipid peroxidation products, known to prevent the detoxifying metabolism of DOPAL, may present a survival challenge to dopaminergic cells targeted in Parkinsons disease.