Terrence Gavin

Molecular Mechanisms of 4-Hydroxy-2-nonenal and Acrolein Toxicity: Nucleophilic Targets and Adduct Formation

Acrolein and 4-hydroxy-2-nonenal (HNE) are byproducts of lipid peroxidation and are thought to play central roles in various traumatic injuries and disease states that involve cellular oxidative stress, for example, spinal cord trauma, diabetes, and Alzheimers disease. In this review, we will discuss the chemical attributes of acrolein and HNE that determine their toxicities. Specifically, these aldehydes are classified as type 2 alkenes and are characterized by an alpha,beta-unsaturated carbonyl structure. This structure is a conjugated system that contains mobile pi-electrons. The carbonyl oxygen atom is electronegative and can promote the withdrawal of mobile electron density from the beta-carbon atom causing regional electron deficiency. On the basis of this type of electron polarizability, both acrolein and HNE are considered to be soft electrophiles that preferentially form 1,4-Michael type adducts with soft nucleophiles. Proteomic, quantum mechanical, and kinetic data will be presented, indicating that cysteine sulfhydryl groups are the primary soft nucleophilic targets of acrolein and HNE. This is in contrast to nitrogen groups on harder biological nucleophiles such as lysine or histidine residues. The toxicological outcome of adduct formation is not only dependent upon residue selectivity but also the importance of the targeted amino acid in protein function or structure. In attempting to discern the toxicological significance of a given adduct, we will consider the normal roles of cysteine, lysine, and histidine residues in proteins and the relative merits of corresponding adducts in the manifestations of diseases or toxic states. Understanding the molecular actions of acrolein and HNE could provide insight into many pathogenic conditions that involve initial cellular oxidative stress and could, thereby, offer new efficacious avenues of pharmacological defense.

Application of the Hard and Soft, Acids and Bases (HSAB) Theory to Toxicant–Target Interactions

Many chemical toxicants and/or their active metabolites are electrophiles that cause cell injury by forming covalent bonds with nucleophilic targets on biological macromolecules. Covalent reactions between nucleophilic and electrophilic reagents are, however, discriminatory since there is a significant degree of selectivity associated with these interactions. Over the course of the past few decades, the theory of Hard and Soft, Acids and Bases (HSAB) has proven to be a useful tool in predicting the outcome of such reactions. This concept utilizes the inherent electronic characteristic of polarizability to define, for example, reacting electrophiles and nucleophiles as either hard or soft. These HSAB definitions have been successfully applied to chemical-induced toxicity in biological systems. Thus, according to this principle, a toxic electrophile reacts preferentially with biological targets of similar hardness or softness. The soft/hard classification of a xenobiotic electrophile has obvious utility in discerning plausible biological targets and molecular mechanisms of toxicity. The purpose of this perspective is to discuss the HSAB theory of electrophiles and nucleophiles within a toxicological framework. In principle, covalent bond formation can be described by using the properties of their outermost or frontier orbitals. Because these orbital energies for most chemicals can be calculated using quantum mechanical models, it is possible to quantify the relative softness (σ) or hardness (η) of electrophiles or nucleophiles and to subsequently convert this information into useful indices of reactivity. This atomic level information can provide insight into the design of corroborative laboratory research and thereby help investigators discern corresponding molecular sites and mechanisms of toxicant action. The use of HSAB parameters has also been instrumental in the development and identification of potential nucleophilic cytoprotectants that can scavenge toxic electrophiles. Clearly, the difficult task of delineating molecular sites and mechanisms of toxicant action can be facilitated by the application of this quantitative approach.

Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective

Aldehydes are electrophilic compounds to which humans are pervasively exposed. Despite a significant health risk due to exposure, the mechanisms of aldehyde toxicity are poorly understood. This ambiguity is likely due to the structural diversity of aldehyde derivatives and corresponding differences in chemical reactions and biological targets. To gain mechanistic insight, we have used parameters based on the hard and soft, acids and bases (HSAB) theory to profile the different aldehyde subclasses with respect to electronic character (softness, hardness), electrophilic reactivity (electrophilic index), and biological nucleophilic targets. Our analyses indicate that short chain aldehydes and longer chain saturated alkanals are hard electrophiles that cause toxicity by forming adducts with hard biological nucleophiles, e.g., primary nitrogen groups on lysine residues. In contrast, α,β-unsaturated carbonyl derivatives, alkenals, and the α-oxoaldehydes are soft electrophiles that preferentially react with soft nucleophilic thiolate groups on cysteine residues. The aldehydes can therefore be grouped into subclasses according to common electronic characteristics (softness/hardness) and molecular mechanisms of toxicity. As we will discuss, the toxic potencies of these subgroups are generally related to corresponding electrophilicities. For some aldehydes, however, predictions of toxicity based on electrophilicity are less accurate due to inherent physicochemical variables that limit target accessibility, e.g., steric hindrance and solubility. The unsaturated aldehydes are also members of the conjugated type-2 alkene chemical class that includes α,β-unsaturated amide, ketone, and ester derivatives. Type-2 alkenes are electrophiles of varying softness and electrophilicity that share a common mechanism of toxicity. Therefore, exposure to an environmental mixture of unsaturated carbonyl derivatives could cause “type-2 alkene toxicity” through additive interactions. Finally, we propose that environmentally derived aldehydes can accelerate diseases by interacting with endogenous aldehydes generated during oxidative stress. This review provides a basis for understanding aldehyde mechanisms and environmental toxicity through the context of electronic structure, electrophilicity, and nucleophile target selectivity.

Molecular Mechanism of Acrylamide Neurotoxicity: Lessons Learned from Organic Chemistry

Background: Acrylamide (ACR) produces cumulative neurotoxicity in exposed humans and laboratory animals through a direct inhibitory effect on presynaptic function. Objectives: In this review, we delineate how knowledge of chemistry provided an unprecedented understanding of the ACR neurotoxic mechanism. We also show how application of the hard and soft, acids and bases (HSAB) theory led to the recognition that the α,β-unsaturated carbonyl structure of ACR is a soft electrophile that preferentially forms covalent bonds with soft nucleophiles. Methods: In vivo proteomic and in chemico studies demonstrated that ACR formed covalent adducts with highly nucleophilic cysteine thiolate groups located within active sites of presynaptic proteins. Additional research showed that resulting protein inactivation disrupted nerve terminal processes and impaired neurotransmission. Discussion: ACR is a type-2 alkene, a chemical class that includes structurally related electrophilic environmental pollutants (e.g., acrolein) and endogenous mediators of cellular oxidative stress (e.g., 4-hydroxy-2-nonenal). Members of this chemical family produce toxicity via a common molecular mechanism. Although individual environmental concentrations might not be toxicologically relevant, exposure to an ambient mixture of type-2 alkene pollutants could pose a significant risk to human health. Furthermore, environmentally derived type-2 alkenes might act synergistically with endogenously generated unsaturated aldehydes to amplify cellular damage and thereby accelerate human disease/injury processes that involve oxidative stress. Conclusions: These possibilities have substantial implications for environmental risk assessment and were realized through an understanding of ACR adduct chemistry. The approach delineated here can be broadly applied because many toxicants of different chemical classes are electrophiles that produce toxicity by interacting with cellular proteins.

Acrylamide-Induced Nerve Terminal Damage : Relevance to Neurotoxic and Neurodegenerative Mechanisms

Acrylamide (ACR) has demonstrable neurotoxic effects in animals and humans that stem from its chemical behavior as a soft electrophilic alpha,beta-unsaturated carbonyl compound. Evidence is presented that the nerve terminal is a primary site of ACR action and that inhibition of neurotransmission mediates the development of neurological deficits. At the mechanistic level, recent proteomic, neurochemical, and kinetic data are considered, which suggest that ACR inhibits neurotransmission by disrupting presynaptic nitric oxide (NO) signaling. Nerve-terminal damage likely mediates the neurological complications that accompany the occupational exposure of humans to ACR. In addition, the proposed molecular mechanism of synaptotoxicity has substantial implications for the pathogenesis of Alzheimers disease and other neurodegenerative conditions that involve neuronal oxidative stress and the secondary endogenous generation of acrolein and other conjugated carbonyl chemicals.

Molecular mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by α,β-unsaturated carbonyl derivatives.

α,β-Unsaturated carbonyls make up an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these data gaps, we incubated purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with acrylamide (ACR), acrolein, or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration- and time-dependent manner. The rank order of enzyme inhibition (K(I)) (i.e., ACR ≪ MVK < acrolein) was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pK(a)-lowering microenvironment (pK(a) = 6.03), and at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate state. These data suggest a general cytotoxic mechanism in which electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, because chemical derivatization causes loss of protein function.

Type-2 alkenes mediate synaptotoxicity in neurodegenerative diseases

Synaptic dysfunction appears to be an early pathogenic event in Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease. Although the molecular mechanism of this synaptotoxicity is not known, evidence suggests that these diseases are characterized by a common pathophysiological cascade involving oxidative stress, lipid peroxidation and the subsequent liberation of alpha,beta-unsaturated carbonyl derivatives such as acrolein and 4-hydroxy-2-nonenal (HNE). A diverse body of in vivo and in vitro data have shown that these soft electrophilic chemicals can cause nerve terminal damage by forming Michael-type adducts with nucleophilic sulfhydryl groups on presynaptic proteins. Therefore, the endogenous generation of acrolein and HNE in oxidatively stressed neurons of certain brain regions might be mechanistically related to the synaptotoxicity associated with neurodegenerative conditions. In addition, acrolein and HNE are members of a large class of structurally related chemicals known as the type-2 alkenes. Chemicals in this class (e.g., acrylamide, methylvinyl ketone, and methyl acrylate) are pervasive pollutants in human environments and new research has shown that these alpha,beta-unsaturated carbonyl derivatives are also toxic to nerve terminals. In this review, we provide evidence that the regional synaptotoxicity, which develops during the early stages of many neurodegenerative diseases, is mediated by endogenous generation of acrolein and HNE. Based on a presumed common nerve terminal site of action, we propose that the onset and progression of this neuropathogenic process is accelerated by environmental exposure to other type-2 alkenes.

Synaptosomal toxicity and nucleophilic targets of 4-hydroxy-2-nonenal.

4-Hydroxy-2-nonenal (HNE) is an aldehyde by-product of lipid peroxidation that is presumed to play a primary role in certain neuropathogenic states (e.g., Alzheimer disease, spinal cord trauma). Although the molecular mechanism of neurotoxicity is unknown, proteomic analyses (e.g., tandem mass spectrometry) have demonstrated that this soft electrophile preferentially forms Michael-type adducts with cysteine sulfhydryl groups. In this study, we characterized HNE synaptosomal toxicity and evaluated the role of putative nucleophilic amino acid targets. Results show that HNE exposure of striatal synaptosomes inhibited 3H-dopamine membrane transport and vesicular storage. These concentration-dependent effects corresponded to parallel decreases in synaptosomal sulfhydryl content. Calculations of quantum mechanical parameters (softness, electrophilicity) that describe the interactions of an electrophile with its nucleophilic target indicated that the relative softness of HNE was directly related to both the second-order rate constant (k2) for sulfhydryl adduct formation and corresponding neurotoxic potency (IC50). Computation of additional quantum mechanical parameters that reflect the relative propensity of a nucleophile to interact with a given electrophile (chemical potential, nucleophilicity) indicated that the sulfhydryl thiolate state was the HNE target. In support of this, we showed that the rate of adduct formation was related to pH and that N-acetyl-L-cysteine, but not N-acetyl-L-lysine or β-alanyl-L-histidine, reduced in vitro HNE neurotoxicity. These data suggest that, like other type 2 alkenes, HNE produces nerve terminal toxicity by forming adducts with sulfhydryl thiolates on proteins involved in neurotransmission.

β-DICARBONYL ENOLATES: A NEW CLASS OF NEUROPROTECTANTS

J. Neurochem. (2011) 116, 132–143.

ROLE OF THE Nrf2-ARE PATHWAY IN ACRYLAMIDE NEUROTOXICITY

Acrylamide (ACR) intoxication is associated with selective nerve terminal damage in the central and peripheral nervous systems. As a soft electrophile, ACR could form adducts with nucleophilic sulfhydryl groups on cysteine residues of kelch-like erythroid cell-derived protein with CNS homology-associated protein 1 (Keap1) leading to dissociation of the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 activation of the antioxidant-responsive element (ARE) and subsequent upregulated gene expression of phase II detoxification enzymes and anitoxidant proteins might provide protection in neuronal regions with transcriptional capabilities (e.g., cell body). In contrast, non-transcriptional cell regions (axons, nerve terminals) might be vulnerable to electrophile-induced damage. To test this possibility, immunoblot analysis was used to measure protein products of Nrf2-activated ARE genes in nerve terminals and in cytosolic/nuclear factions of neuronal cell bodies isolated from rats intoxicated at two different ACR dose-rates; i.e., 50mg/kg/d×10 days, 21mg/kg/d×38 days. To detect possible differences in cell-specific induction, the cytoprotective response to ACR intoxication was determined in hepatic cells. Results show that control brain and hepatic cell fractions exhibited distinct subcellular distributions of Nrf2, Keap1 and several ARE protein products. ACR intoxication, however, did not alter the levels of these proteins in synaptosomal, brain cytoplasm or liver cell fractions. These data indicate that ACR was an insufficient electrophilic signal for ARE induction in all subcellular fractions tested. Because a cytoprotective response was not induced in any fraction, nerve terminal vulnerability to ACR cannot be ascribed to the absence of transcription-based defense mechanisms in this neuronal region.