Jonathan A. Doorn

Nitrative and Oxidative Stress in Toxicology and Disease

Persistent inflammation and the generation of reactive oxygen and nitrogen species play pivotal roles in tissue injury during disease pathogenesis and as a reaction to toxicant exposures. The associated oxidative and nitrative stress promote diverse pathologic reactions including neurodegenerative disorders, atherosclerosis, chronic inflammation, cancer, and premature labor and stillbirth. These effects occur via sustained inflammation, cellular proliferation and cytotoxicity and via induction of a proangiogenic environment. For example, exposure to the ubiquitous air pollutant ozone leads to generation of reactive oxygen and nitrogen species in lung macrophages that play a key role in subsequent tissue damage. Similarly, studies indicate that genes involved in regulating oxidative stress are altered by anesthetic treatment resulting in brain injury, most notable during development. In addition to a role in tissue injury in the brain, inflammation, and oxidative stress are implicated in Parkinsons disease, a neurodegenerative disease characterized by the loss of dopamine neurons. Recent data suggest a mechanistic link between oxidative stress and elevated levels of 3,4-dihydroxyphenylacetaldehyde, a neurotoxin endogenous to dopamine neurons. These findings have significant implications for development of therapeutics and identification of novel biomarkers for Parkinsons disease pathogenesis. Oxidative and nitrative stress is also thought to play a role in creating the proinflammatory microenvironment associated with the aggressive phenotype of inflammatory breast cancer. An understanding of fundamental concepts of oxidative and nitrative stress can underpin a rational plan of treatment for diseases and toxicities associated with excessive production of reactive oxygen and nitrogen species.

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.

4-Hydroxy-2-nonenal adduction of extracellular signal-regulated kinase (Erk) and the inhibition of hepatocyte Erk-Est-like protein-1-activating protein-1 signal transduction.

4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation (LPO) product formed during oxidative stress. 4-HNE is highly reactive toward cellular nucleophiles and is implicated in the evolution of numerous pathologies associated with oxidative stress and LPO. Recent evidence suggests that chronic prooxidant exposure results in the loss of extracellular signal-regulated kinase (Erk)-1/2 phosphorylation in vivo, a signaling pathway associated with cellular proliferation, survival, and homeostasis. Immunodetection and molecular analysis were used in this study to evaluate the hypothesis that 4-HNE modification of Erk-1/2 inhibits constitutive Erk-Est-like protein (Elk)-1-activating protein (AP)-1 signaling. Primary rat hepatocytes treated with subcytotoxic, pathologically relevant concentrations of 4-HNE demonstrated a concentration-dependent loss of constitutive Erk-1/2 phosphorylation, activity, and nuclear localization. These findings were consistent with iron-induced intracellular LPO, which also resulted in a concentration-dependent decrease in hepatocyte Erk-1/2 phosphorylation and activity. 4-HNE and iron-induced inhibition of Erk-1/2 was inversely correlated with the accumulation of 4-HNE-Erk-1/2 monomer adducts. 4-HNE treatment of hepatocytes decreased nuclear total and phosphorylated Erk-1/2, Elk-1, and AP-1 phosphorylation as well as cFos and cJun activities. The cytosolic modification of unphosphorylated Erk-1/2 was evaluated in vitro using molar ratios of inactive Erk-2 to 4-HNE consistent with increasing oxidative stress in vivo. Liquid chromatography combined with tandem mass spectrometry confirmed monomer adduct formation and identified the major adduct species at the histidine 178 residue within the kinase phosphorylation lip. These novel results show that the formation of 4-HNE-Erk-1/2 monomer-adducts results in the inhibition of Erk-Elk-AP-1 signaling in hepatocytes and implicates the His 178 residue with the mechanism of inhibition.

Oxidation of 3,4-dihydroxyphenylacetaldehyde, a toxic dopaminergic metabolite, to a semiquinone radical and an ortho-quinone

The oxidation and toxicity of dopamine is believed to contribute to the selective neurodegeneration associated with Parkinson disease. The formation of reactive radicals and quinones greatly contributes to dopaminergic toxicity through a variety of mechanisms. The physiological metabolism of dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL) via monoamine oxidase significantly increases its toxicity. To more adequately explain this enhanced toxicity, we hypothesized that DOPAL is capable of forming radical and quinone species upon oxidation. Here, two unique oxidation products of DOPAL are identified. Several different oxidation methods gave rise to a transient DOPAL semiquinone radical, which was characterized by electron paramagnetic resonance spectroscopy. NMR identified the second oxidation product of DOPAL as the ortho-quinone. Also, carbonyl hydration of DOPAL in aqueous media was evident via NMR. Interestingly, the DOPAL quinone exists exclusively in the hydrated form. Furthermore, the enzymatic and chemical oxidation of DOPAL greatly enhance protein cross-linking, whereas auto-oxidation results in the production of superoxide. Also, DOPAL was shown to be susceptible to oxidation by cyclooxygenase-2 (COX-2). The involvement of this physiologically relevant enzyme in both oxidative stress and Parkinson disease underscores the potential importance of DOPAL in the pathogenesis of this condition.

In Vitro and in Silico Characterization of Peroxiredoxin 6 Modified by 4-Hydroxynonenal and 4-Oxononenal

Peroxiredoxin 6 (PRX6) belongs to the 1-Cys class of peroxiredoxins and is recognized as an important antioxidant protein in tissues such as cardiac muscle, skin, and lung. Preliminary in vivo proteomic data have revealed that PRX6 is adducted by 4-hydroxynonenal (4HNE) in the livers of rats chronically fed an ethanol-containing diet. The goals of this study were to evaluate the in vitro effect of aldehyde adduction on PRX6 peroxidase activity, identify specific sites of aldehyde modification using mass spectrometry, and predict conformational changes due to adduction using molecular modeling. PRX6 was found to be resistant to inactivation via aldehyde modification; however, Western blots of adducted protein revealed that both 4HNE and 4-oxononenal (4ONE) caused extensive cross-linking, resulting in high molecular mass species. Tandem mass spectrometry (ESI-LC-MS/MS) analysis demonstrated multiple sites of modification, but adduction of the active site Cys47 was not observed. Molecular modeling simulations indicated that adduction at Cys91 results in a change in protein active site conformation, which potentially restricts access of 4-HNE to Cys47. The Cys91-Lys209 cross-linked adducts could provide the conformational changes required to inactivate the protein by either restricting access to electrophiles or preventing important amino acid interactions within the catalytic triad.

Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells.

Hepatic oxidative stress and lipid peroxidation are common features of several prevalent disease states, including alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), a common component of the metabolic syndrome. These conditions are characterized in part by excessive accumulation of lipids within hepatocytes, which can lead to autocatalytic degradation of cellular lipids giving rise to electrophilic end products of lipid peroxidation. The pathobiology of reactive lipid aldehydes remains poorly understood. We therefore sought to investigate the effects of 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) on the transport and secretion of very low-density lipoprotein using HepG2 cells as a model hepatocyte system. Physiologically relevant concentrations of 4-HNE and 4-ONE rapidly disrupted cellular microtubules in a concentration-dependent manner. Interestingly, 4-ONE reduced apolipoprotein B-100 (ApoB) secretion while 4-HNE did not significantly impair secretion. Both 4-HNE and 4-ONE formed adducts with ApoB protein, but 4-HNE adducts were detectable as mono-adducts, while 4-ONE adducts were present as protein-protein cross-links. These results demonstrate that reactive aldehydes generated by lipid peroxidation can differ in their biological effects, and that these differences can be mechanistically explained by the structures of the protein adducts formed.

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.