In vivo neurophysiological assessment of in silico predictions of neurotoxicity: Citronellal, 3,4-dichloro-1-butene, and benzyl bromoacetate.

Neurotoxicants may be widespread in the environment and can produce serious health impacts in the human population. Screening programs that use in vitro methods have generated data for thousands of chemicals. However, these methods often do not evaluate repeated or prolonged exposures, which are required for many neurotoxic outcomes. Additionally, the data produced by such screening methods may not include mechanisms which play critical biological roles necessary for in vivo neurotoxicity. The Hard and Soft Acids and Bases (HSAB) in silico model focuses on chemical structure and electrophilic properties which are important to the formation of protein adducts. A group of structurally diverse chemicals have been evaluated with an in silico screening approach incorporating HSAB parameters. However, the predictions from the expanded chemical space have not been evaluated using in vivo methods. Three chemicals predicted to be cumulative toxicants were selected for in vivo neurotoxicological testing. Adult male Long-Evans rats were treated orally with citronellal (CIT), 3,4-dichloro-1-butene (DCB), or benzyl bromoacetate (BBA) for 8 weeks. Behavioral observations were recorded weekly to assess motor function. Peripheral neurophysiological measurements were derived from nerve excitability (NE) tests which involved compound muscle action potentials (CMAPs) in the tail and foot, and mixed nerve action potentials (MNAPs) in the tail. Compound nerve action potentials (CNAPs) and nerve conduction velocity (NCV) in the tail were also quantified. Peripheral inputs into the central nervous system were examined using somatosensory evoked potentials recorded from the cortex (SEPCTX) and cerebellum (SEPCEREB). CIT or BBA did not result in significant alterations to peripheral nerve or somatosensory function. DCB reduced grip-strength and altered peripheral nerve function. The MNAPs required less current to reach 50% amplitude and had a lower calculated rheobase, suggesting increased excitability. Increased CNAP amplitudes and greater NCV were also observed. Novel changes were found in the SEPCTX with an abnormal peak forming in the early portion of the waveforms of treated rats, and decreased latencies and increased amplitudes were observed in SEPCEREB recordings. These data contribute to testing an expanded chemical space from an in silico HSAB model for predicting cumulative neurotoxicity and may assist with prioritizing chemicals to protect human health.

Acute in vitro effects on embryonic rat dorsal root ganglion (DRG) cultures by in silico predicted neurotoxic chemicals: Evaluations on cytotoxicity, neurite length, and neurophysiology.

The Hard-Soft Acid and Base hypothesis can be used to predict the potential bio-reactivity (electrophilicity) of a chemical with intracellular proteins, resulting in neurotoxicity. Twelve chemicals predicted to be neurotoxic were evaluated in vitro in rat dorsal root ganglia (DRG) for effects on cytotoxicity (%LDH), neuronal structure (total neurite length/neuron, NLPN), and neurophysiology (mean firing rate, MFR). DRGs were treated acutely on days in vitro (DIV) 7 (1-100 µM) with test chemical; %LDH and NLPN were measured after 48 h. 4-cyclohexylhexanone (4-C) increased %LDH release at 50 (29%) and 100 µM (56%), citronellal (Cit) and 1-bromopropane increased %LDH at 100 µM (22% and 26%). 4-C, Cit, 2,5 Hexanedione (2,5Hex), phenylacetylaldehyde (PAA) and 2-ethylhexanal decreased mean NLPN at 48 h; 50 and 100 µM for 4-C (28% and 60%), 100 µM Cit (52%), 100 µM 2,5- Hex (37%) 100 µM PAA (41%) and 100 µM for 2-ethylhexanal (23%). Separate DRG cultures were treated on DIV 14 and changes in MFR measured. Four compounds decreased MFR at 50 or 100 µM: Acrylamide (-83%), 3,4-dichloro-1-butene (-93%), 4-C (-89%) and hexane (-79%, 50 µM). Changes in MFR and NLPN occurred in absence of cytotoxicity. While the current study showed little cytotoxicity, it gave insight to initial changes in MFR. Results provide insight for future chronic exposure experiments to evaluate neurotoxicity.

Application of the hard and soft, acids and bases (HSAB) theory as a method to predict cumulative neurotoxicity.

Xenobiotic electrophiles can form covalent adducts that may impair protein function, damage DNA, and may lead a range of adverse effects. Cumulative neurotoxicity is one adverse effect that has been linked to covalent protein binding as a Molecular Initiating Event (MIE). This paper describes a mechanistic in silico chemical screening approach for neurotoxicity based on Hard and Soft Acids and Bases (HSAB) theory. We evaluated the applicability of HSAB-based electrophilicity screening protocol for neurotoxicity on 19 positive and 19 negative reference chemicals. These reference chemicals were identified from the literature, using available information on mechanisms of neurotoxicity whenever possible. In silico screening was based on structural alerts for protein binding motifs and electrophilicity index in the range of known neurotoxicants. The approach demonstrated both a high positive prediction rate (82-90 %) and specificity (90 %). The overall sensitivity was relatively lower (47 %). However, when predicting the toxicity of chemicals known or suspected of acting via non-specific adduct formation mechanism, the HSAB approach identified 7/8 (sensitivity 88 %) of positive control chemicals correctly. Consequently, the HSAB-based screening is a promising approach of identifying possible neurotoxins with adduct formation molecular initiating events. While the approach must be expanded over time to capture a wider range of MIEs involved in neurotoxicity, the mechanistic nature of the screen allows users to flag chemicals for possible adduct formation MIEs. Thus, the HSAB based toxicity screening is a promising strategy for toxicity assessment and chemical prioritization in neurotoxicology and other health endpoints that involve adduct formation.

Enolate-forming compounds provide protection from platinum neurotoxicity.

Cisplatin (CisPt) and other platinum (Pt)-based antineoplastic drugs (e.g., carboplatin, oxaliplatin) are highly effective and widely used in the treatment of solid tumors in both pediatric and adult patients. Although considered to be life-saving as a cancer treatment, Pt-based drugs frequently result in dose-limiting toxicities such as chemotherapy-induced peripheral neuropathies (CIPN). Specifically, irreversible damage to outer hair cells and injury of sensory neurons are linked to profound sensorineural hearing loss (ototoxicity), which complicates tumor management and can lead to a poor clinical prognosis. Given the severity of CIPN, substantial effort has been devoted to the development of neuroprotective compounds, regardless clinical results have been underwhelming. It is noteworthy that Pt is a highly reactive electrophile (electron deficient) that causes toxicity by forming adducts with nucleophilic (electron rich) targets on macromolecules. In this regard, we have discovered a series of carbon-based enol nucleophiles; e.g., N-(4-acetyl-3,5-dihydroxyphenyl)-2-oxocytclopentane-1-carboxamide (Gavinol), that can prevent neurotoxicity by scavenging the platinum ion. The chemistry of enol compounds is well understood and mechanistic research has demonstrated the role of this chemistry in cytoprotection. Our cell-derived data were corroborated by calculations of hard and soft, acids and bases (HSAB) parameters that describe the electronic character of interacting electrophiles and nucleophiles. Together, these observations indicate that the respective mechanisms of Pt neurotoxicity and antitumor activity are separable and can therefore be affected independently.

Mechanisms of soft and hard electrophile toxicities.

Electron-deficient chemicals (electrophiles) react with compounds that have one or more unshared valence electron pairs (nucleophiles). The resulting covalent reactions between electrophiles and nucleophiles (e.g., Michael addition, SN2 reactions) are important, not only to Organic Chemistry, but also to the fields of Molecular Biology and Toxicology. Specifically, covalent bond formation is the operational basis of many critically important cellular processes; e.g., enzyme function, neurotransmitter release, and membrane-vesicle fusion. Given this context it is understandable that these reactions are also relevant to Toxicology, since a significant number of xenobiotic chemicals are toxic electrophiles that can react with endogenous nucleophilic residues. Therefore, the purpose of this Review is to discuss electrophile-nucleophile chemistry as it pertains to cell injury and resulting organ toxicity. Our discussion will involve an introduction to the Hard and Soft, Acids and Bases (HSAB) theory of Pearson. The HSAB concept provides a framework for calculation of quantum chemical parameters that classify the electrophile and nucleophile covalent components according to their respective electronic nature (softness/hardness) and reactivity (electrophilicity/nucleophilicity). The calculated quantum indices in conjunction with corroborative in vivo, in chemico (cell free) and in vitro research can offer an illuminating approach to mechanistic discovery. Accordingly, we will provide examples that demonstrate how this approach has been used to discern mechanisms and sites of electrophile action.

Phloretin cytoprotection and toxicity.

Phloretin (Phl) is a dihydrochalcone flavonoid with significant cytoprotective properties; e.g., free radical trapping, electrophile scavenging. Based on this, it has been suggested that Phl might be useful in the treatment of pathogenic processes and prevention of drug toxicities. Therefore, we determined the ability of Phl to provide route- and dose-dependent hepatoprotection in a mouse model of acetaminophen (APAP) overdose. Intraperitoneal (i.p.) administration of Phl produced a bimodal effect; i.e., the highest dose (2.40 mmol/kg) did not prevent APAP-induced lethality, whereas lower doses (0.2-0.4 mmol/kg) afforded modest hepatoprotection. When given alone, the highest i.p. Phl dose was lethal within 24 h, whereas the lower doses were not toxic. Oral Phl (0.40-2.40 mmol/kg) did not prevent APAP-induced hepatotoxicity. The highest oral dose given alone (2.4 mmol/kg) produced 64% lethality, whereas lower doses were not lethal. This toxicity profile was reflected in a study using APAP-exposed isolated mouse hepatocytes, which showed that the Phl pharmacophores, 1,3,5-trihydroxyacetophenone (PG) and 2',4',6'-trihydroxyacetophenone (THA) where protective. Corroborative cell free studies showed that polyphenol protectants prevented glutathione loss mediated by the APAP metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Thus, in spite of possessing cytoprotective attributes, Phl was generally toxic in our APAP models. These and earlier findings suggest that Phl is not a candidate for drug design. In contrast, we have found that the enol-forming pharmacophores, THA and PG, are potential platforms for pharmacotherapeutic development.

Enolate-Forming Compounds as a Novel Approach to Cytoprotection.

Evidence from laboratory studies and clinical trials suggests that plant-derived polyphenolic compounds such as curcumin, resveratrol, or phloretin might be useful in the treatment of certain diseases (e.g., Alzheimer's disease) and acute tissue injury states (e.g., spinal cord trauma). However, despite this potential, the corresponding chemical instability, toxic potential, and low bioavailability of these compounds could limit their ultimate clinical relevance. We have shown that pharmacophores of curcumin (e.g., 2-acetylcyclopentanone) and phloretin (e.g., 2',4',6'-trihydroxyacetophenone; THA) can provide cytoprotection in cell culture and animal models of oxidative stress injury. These pharmacophores are 1,3-dicarbonyl and polyphenol derivatives, the enol groups of which can ionize in biological solutions to form an enolate. This carbanionic moiety can chelate metal ions and, as a nucleophile, can scavenge toxic electrophiles (e.g., acrolein, 4-hydroxy-2-nonenal, and N-acetyl-p-benzoquinone imine) involved in many pathogenic conditions. Aromatic derivatives such as THA can also trap free oxygen and nitrogen radicals and thereby provide another layer of cytoprotection. The multifunctional character of these enolate-forming compounds suggests an ability to block pathogenic processes (e.g., oxidative stress) at several steps. The purpose of this review is to discuss research supporting our theory that enolate formation is a significant cytoprotective property that represents a platform for development of pharmacotherapeutic approaches to a variety of toxic and pathogenic conditions. Our discussion will focus on mechanism and structure-activity studies that define enolate chemistry and their corresponding relationships to cytoprotection.

Joint toxic effects of the type-2 alkene electrophiles.

Human populations are exposed to complex environmental mixtures of acrolein, methylvinyl ketone (MVK) and other type-2 alkenes. Many members of this chemical class are electrophiles that possess a common molecular mechanism of toxicity; i.e., protein inactivation via formation of stable cysteine adducts. Therefore, acute or chronic exposure to type-2 alkene mixtures could represent a health risk due to additive or synergistic interactions among component chemicals. Despite this risk, there is little experimental information regarding the joint effects of type-2 alkenes. In the present study we used sum of toxic units (TUsum = ∑TUi) to assess the relative toxicity of different type-2 alkene mixtures. These studies involved well characterized environmental type-2 alkene toxicants and included amide (acrylamide; ACR), ketone (methyl vinyl ketone; MVK), aldehyde (2-ethylacrolein; EA) and ester (methyl acrylate; MA) derivatives. In chemico analyses revealed that both binary and ternary mixtures could deplete thiol groups according to an additive joint effect at equitoxic and non-equitoxic ratios; i.e., TUsum = 1.0 ± 0.20. In contrast, analyses of joint effects in SNB19 cell cultures indicated that different permutations of type-2 alkene mixtures produced mostly synergistic joint effects with respect to cell lethality; i.e., TUsum < 0.80. A mixture of ACR and MA was shown to produce joint toxicity in a rat model. This mixture accelerated the onset and development of neurotoxicity relative to the effects of the individual toxicants. Synergistic effects in biological models might occur when different cellular proteomes are targeted, whereas additive effects develop when the mixtures encompasses a similar proteome.

Enolate-Forming Phloretin Pharmacophores: Hepatoprotection in an Experimental Model of Drug-Induced Toxicity.

Drug-induced toxicity is often mediated by electrophilic metabolites, such as bioactivation of acetaminophen (APAP) to N-acetyl-p-benzoquinone imine (NAPQI). We have shown that APAP hepatotoxicity can be prevented by 2-acetylcyclopentanone (2-ACP). This 1,3-dicarbonyl compound ionizes to form an enolate nucleophile that scavenges NAPQI and other electrophilic intermediates. In this study, we expanded our investigation of enolate-forming compounds to include analyses of the phloretin pharmacophores, 2',4',6'-trihydroxyacetophenone (THA) and phloroglucinol (PG). Studies in a mouse model of APAP overdose showed that THA provided hepatoprotection when given either by intraperitoneal injection or oral administration, whereas PG was hepatoprotective only when given intraperitoneally. Corroborative research characterized the molecular pharmacology (efficacy, potency) of 2-ACP, THA, and PG in APAP-exposed isolated mouse hepatocytes. For comparative purposes, N-acetylcysteine (NAC) cytoprotection was also evaluated. Measurements of multiple cell parameters (e.g., cell viability, mitochondrial membrane depolarization) indicated that THA and, to a lesser extent, PG provided concentration-dependent protection against APAP toxicity, which exceeded that of 2-ACP or NAC. The enolate-forming compounds and NAC truncated ongoing APAP exposure and thereby returned intoxicated hepatocytes toward normal viability. The superior ability of THA to protect is related to multifaceted modes of action that include metal ion chelation, free radical trapping, and scavenging of NAPQI and other soft electrophiles involved in oxidative stress. The rank order of potency for the tested cytoprotectants was consistent with that determined in a parallel mouse model. These data suggest that THA or a derivative might be useful in treating drug-induced toxicities and other conditions that involve electrophile-mediated pathogenesis.

Reactions of electrophiles with nucleophilic thiolate sites: relevance to pathophysiological mechanisms and remediation.

Electrophiles are electron-deficient species that form covalent bonds with electron-rich nucleophiles. In biological systems, reversible electrophile-nucleophile interactions mediate basal cytophysiological functions (e.g. enzyme regulation through S-nitrosylation), whereas irreversible electrophilic adduction of cellular macromolecules is involved in pathogenic processes that underlie many disease and injury states. The nucleophiles most often targeted by electrophiles are side chains on protein amino acids (e.g. Cys, His, and Lys) and aromatic nitrogen sites on DNA bases (e.g. guanine N7). The sulfhydryl thiol (RSH) side chain of cysteine residues is a weak nucleophile that can be ionized in specific conditions to a more reactive nucleophilic thiolate (RS(-)). This review will focus on electrophile interactions with cysteine thiolates and the pathophysiological consequences that result from irreversible electrophile modification of this anionic sulfur. According to the Hard and Soft, Acids and Bases (HSAB) theory of Pearson, electrophiles and nucleophiles can be classified as either soft or hard depending on their relative polarizability. HSAB theory suggests that electrophiles will preferentially and more rapidly form covalent adducts with nucleophiles of comparable softness or hardness. Application of HSAB principles, in conjunction with in vitro and proteomic studies, have indicated that soft electrophiles of broad chemical classes selectively form covalent Michael-type adducts with soft, highly reactive cysteine thiolate nucleophiles. Therefore, these electrophiles exhibit a common mechanism of cytotoxicity. As we will discuss, this level of detailed mechanistic understanding is a necessary prerequisite for the rational development of effective prevention and treatment strategies for electrophile-based pathogenic states.

2-Acetylcyclopentanone, an enolate-forming 1,3-dicarbonyl compound, is cytoprotective in warm ischemia-reperfusion injury of rat liver.

We have previously shown that 2-acetylcyclopentanone (2-ACP), an enolate-forming 1,3-dicarbonyl compound, provides protection in cell culture and animal models of oxidative stress. The pathophysiology of ischemia-reperfusion injury (IRI) involves oxidative stress, and, therefore, we determined the ability of 2-ACP to prevent this injury in a rat liver model. IRI was induced by clamping the portal vasculature for 45 minutes (ischemia phase), followed by recirculation for 180 minutes (reperfusion phase). This sequence was associated with substantial derangement of plasma liver enzyme activities, histopathological indices, and markers of oxidative stress. The 2-ACP (0.80-2.40 mmol/kg), administered by intraperitoneal injection 10 minutes prior to reperfusion, provided dose-dependent cytoprotection, as indicated by normalization of the IRI-altered liver histologic and biochemical parameters. The 2-ACP (2.40 mmol/kg) was also hepatoprotective when injected before clamping the circulation (ischemia phase). In contrast, an equimolar dose of N-acetylcysteine (2.40 mmol/kg) was not hepatoprotective when administered prior to reperfusion. Our studies to date suggest that during reperfusion the enolate nucleophile of 2-ACP limits the consequences of mitochondrial-based oxidative stress through scavenging unsaturated aldehyde electrophiles (e.g., acrolein) and chelation of metal ions that catalyze the free radical-generating Fenton reaction. The ability of 2-ACP to reduce IRI when injected prior to ischemia most likely reflects the short duration of this experimental phase (45 minutes) and favorable pharmacokinetics that maintain effective 2-ACP liver concentrations during subsequent reperfusion. These results provide evidence that 2-ACP or an analog might be useful in treating IRI and other conditions that have oxidative stress as a common molecular etiology.

Toxic neuropathies: Mechanistic insights based on a chemical perspective.

2,5-Hexanedione (HD) and acrylamide (ACR) are considered to be prototypical among chemical toxicants that cause central-peripheral axonopathies characterized by distal axon swelling and degeneration. Because the demise of distal regions was assumed to be causally related to the onset of neurotoxicity, substantial effort was devoted to deciphering the respective mechanisms. Continued research, however, revealed that expression of the presumed hallmark morphological features was dependent upon the daily rate of toxicant exposure. Indeed, many studies reported that the corresponding axonopathic changes were late developing effects that occurred independent of behavioral and/or functional neurotoxicity. This suggested that the toxic axonopathy classification might be based on epiphenomena related to dose-rate. Therefore, the goal of this mini-review is to discuss how quantitative morphometric analyses and the establishment of dose-dependent relationships helped distinguish primary, mechanistically relevant toxicant effects from non-specific consequences. Perhaps more importantly, we will discuss how knowledge of neurotoxicant chemical nature can guide molecular-level research toward a better, more rational understanding of mechanism. Our discussion will focus on HD, the neurotoxic γ-diketone metabolite of the industrial solvents n-hexane and methyl-n-butyl ketone. Early investigations suggested that HD caused giant neurofilamentous axonal swellings and eventual degeneration in CNS and PNS. However, as our review will point out, this interpretation underwent several iterations as the understanding of γ-diketone chemistry improved and more quantitative experimental approaches were implemented. The chemical concepts and design strategies discussed in this mini-review are broadly applicable to the mechanistic studies of other chemicals (e.g., n-propyl bromine, methyl methacrylate) that cause toxic neuropathies.

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,ß-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,ß-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.

Protective properties of 2-acetylcyclopentanone in a mouse model of acetaminophen hepatotoxicity.

Our previous research showed that enolates formed from 1,3-dicarbonyl compounds, such as 2-acetylcyclopentanone (2-ACP), could provide protection in cell culture models from electrophile- or oxidative stress-induced toxicity. In the present study, we evaluated the protective abilities of 2-ACP in a mouse model of acetaminophen (APAP) hepatotoxicity. Results show that oral APAP overdose (500 mg/kg) was nearly 90% lethal within 72 hours and that the resulting hepatotoxicity was associated with substantial changes in plasma liver enzyme activities, histopathological indices, and markers of hepatocyte oxidative stress. 2-ACP administered intraperitoneally 20 minutes before APAP completely prevented lethality over a 7-day observation period. This effect was dose-dependent (0.80-2.40 mmol/kg) and was correlated with normalization of measured parameters. Nearly complete protection was afforded when 2-ACP was administered 20 minutes post-APAP, but not 60 minutes after intoxication. Although intraperitoneal administration of N-acetylcysteine (NAC) was not effective over a broad dose range (2.40-7.20 mmol/kg), temporal studies indicated that intraperitoneal NAC was hepatoprotective when injected 60 minutes after APAP intoxication. Because of a loss of function in stomach acid, oral administration of 2-ACP was associated with modest APAP protection. In contrast, NAC administered orally provided dose-dependent (0.80-2.40 mmol/kg) protection against APAP hepatotoxicity. In chemico studies and quantum mechanical calculations indicated that 2-ACP acted as a surrogate nucleophilic target for the reactive electrophilic APAP metabolite N-acetyl-p-benzoquinone imine. Our findings suggest that 2-ACP or a derivative might be useful in treating acquired toxicities associated with electrophilic drugs and metabolites or environmental toxicants.

Protein targets of acrylamide adduct formation in cultured rat dopaminergic cells.

Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate groups on cysteine residues of neuronal proteins. Identifying specific proteins targeted by ACR can lead to a better mechanistic understanding of the corresponding neurotoxicity. Therefore, in the present study, the ACR-adducted proteome in exposed primary immortalized mesencephalic dopaminergic cells (N27) was determined using tandem mass spectrometry (LTQ-Orbitrap). N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics identified a total of 15,243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys groups. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity, diabetes, inflammation, nerve degeneration and atherosclerosis. These results provide detailed information regarding the ACR-adducted proteome in a dopaminergic cell line. The catalog of affected proteins indicates the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the corresponding neurotoxic mechanism.

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,ß-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.

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

α,ß-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,ß-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,ß-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.

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.