Molecular Mechanism of Acrylamide Neurotoxicity: Lessons Learned from Organic Chemistry

1Acrylamide (ACR) is a water-soluble alkene used in the production of polymers and gels that have various commercial applications. For example, polyacrylamide preparations are used in the cosmetic, paper, and textile industries; in ore processing; and as soil conditioners and flocculants for wastewater treatment. Coincidental with the burgeoning industrial use of ACR monomer in the 1950s, it was quickly realized that cumulative neurotoxicity characterized by ataxia, skeletal muscle weakness, cognitive impairment, and numbness of the extremities was a potential outcome of occupational exposure (;;; reviewed by;;; ). Early research involving laboratory animals showed that exposure to ACR monomer produced a neurotoxicity syndrome that resembled the neurological symptoms of human intoxication (reviewed by;; ). Morphological studies conducted during the late 1960s and early 1970s suggested that both human and experimental ACR neurotoxicities were associated with cerebellar Purkinje cell death and degeneration of distal axons and nerve terminals in the peripheral and central nervous systems (PNS and CNS, respectively) (reviewed by;; ). In addition to characteristic neurotoxicity in adult humans and animals, there is more recent experimental evidence, albeit controversial, that prenatal and perinatal exposure of rodent pups to ACR causes neurodevelopmental toxicity (e.g.,;; ).Whereas the majority of research indicates selective targeting of nervous tissue, rodent studies have also suggested that ACR causes reproductive toxicity [e.g., decreased litter size, DNA strand breaks] and an increased incidence of certain tumors [e.g., mammary gland fibroadenomas in female rats, tunica vaginalis mesotheliomas in male rats]. However, to date, there is little evidence that these experimental non-neurotoxic consequences have human relevance.

2Thus, the majority of evidence suggests that ACR exposure across broad daily dose-rates causes selective neurotoxicity in humans and laboratory animals. The early morphological descriptions of ACR neuropathy provided a framework for subsequent research that attempted to decipher the molecular mechanisms of neurotoxicity (reviewed by;;;; ). Although many putative mechanisms and sites of ACR action were tested, for example, inhibition of Na+/K+-ATPase and the resulting reverse operation of the axolemmal Na+/Ca2+-exchanger, reduced fast axonal transport and inactivation of enzymes involved in neuronal energy production, the identification of a necessary and sufficient neurotoxic process remained elusive. However, these early hypotheses were not developed within the framework that xenobiotics can produce toxicity by interacting directly with specific sites on cellular macromolecules (e.g., enzymes) and that this interaction is dictated by the chemical nature of the toxicant (; Coles 1984–1985;; see also; ). Therefore, by understanding toxicant chemistry, plausible molecular-level sites and mechanisms of action can be predicted. In this review, we discuss the chemical nature of ACR (a soft electrophile) and how this determines the corresponding sites of protein adduction (soft nucleophilic sulfhydryl thiolates on cysteine residues). Basic recognition of the chemistry of toxicant–target reactions has led the development and testing of a rational mechanistic hypothesis of ACR neurotoxicity (see below). Although this review focuses on ACR, the proposed algorithm is broadly applicable to many different classes of chemical neurotoxicants, for example, heavy metals, quinones, and unsaturated aldehyde derivatives. In the following section, we provide a brief historical overview of ACR neurotoxicity in humans and laboratory animals.

3Daily exposure of laboratory animals (rodents, rabbits, primates, dogs, cats, and guinea pigs) to a broad range of ACR dose-rates (0.5–50 mg/kg/day) is associated with neurological deficits that resemble human neurotoxicity. Our early research was based on the contemporary concept that ACR produced central–peripheral distal axon degeneration and, accordingly, we focused on possible axonal sites of action [e.g., axolemmal Na+/K+-ATPase (;,,; reviewed by )]. However, results from quantitative morphometric studies of the peripheral nerve suggested that axon degeneration was an epiphenomenon specifically related to lower ACR dose-rates. Silver stain analyses of CNS tissue from ACR-intoxicated rats subsequently confirmed this dose-rate phenonmenon (,,; however, see ) and showed that regardless of exposure level, ACR intoxication was associated with selective nerve terminal degeneration in broad CNS regions. Therefore, these findings, in conjunction with data from earlier morphological, electrophysiological, and neurochemical studies (reviewed by ) provided observational evidence that ACR disrupted neurotransmission. Accordingly, we proposed that nerve terminals were a primary site of ACR action and that neurotoxicity was a consequence of impaired synaptic transmission in the PNS and CNS.

4In formulating possible molecular mechanisms of presynaptic toxicity, we considered the fact that ACR was an electrophile that might produce neurotoxicity by binding to nucleophilic cysteine sites on proteins. In support of this possibility, it was recognized that the activities of many nerve terminal proteins were regulated by the ionization of specific cysteine sulfhydryl groups to highly reactive thiolates. We therefore hypothesized that ACR adduction of these regulatory residues might cause presynaptic toxicity, although some contemporary research did not support this idea (e.g., ). Nonetheless, ensuing studies showed that ACR disrupted presynaptic neurotransmitter release, membrane re-uptake and vesicular storage by selectively forming adducts with cysteine residues on specific proteins involved in these processes, for example, N-ethylmaleimide (NEM)–sensitive factor (release), the dopamine membrane transporter (re-uptake) and the vesicular monoamine transporter (vesicular storage). Experimental evidence that ACR did not alter protein synthesis, energy production, or axonal transport indicated that presynaptic toxicity was a direct toxicant effect (reviewed by ). Whereas these data implied a central role for cysteine adduction in ACR neurotoxicity, it was not clear how such adduct formation might cause protein dysfunction and why nerve terminals were selectively vulnerable to the effects of protein adduction. This latter concern was particularly germane because most proteins contain at least one cysteine residue and ACR has been reported to form adducts with a variety of neuronal and non-neuronal proteins (e.g.,; ). As a consequence, it could not be assumed that adduct formation at a given cysteine residue had toxicological relevance. In the next section we discuss the adduct chemistry of ACR and show how this chemistry is related to the production of nerve terminal toxicity.

5ACR is a three-carbon α,β−unsaturated carbonyl derivative and is a member of a large chemical class known as type-2 alkenes. Members of this class are characterized by a conjugated system formed when an electron-withdrawing group (e.g., the carbonyl group) is linked to an alkene carbon (Figure 1). The pi electrons in these conjugated systems are highly polarizable (mobile), and the carbonyl group of ACR withdraws electron density from the alkene to form an electron deficient (electrophilic) site at the β-carbon. As an electrophile, ACR, like many xenobiotic chemicals and/or their metabolites, causes cytotoxicity by forming covalent bonds with electron-rich (nucleophilic) residues on biological macromolecules (e.g., enzymes, DNA). Because ACR is an amide derivative, it does not undergo Schiff base formation with nucleophiles, but can form Michael-type adducts with nucleophiles via second-order addition reactions to the β-carbon. Electrophiles do not react arbitrarily with nucleophiles. Instead, these interactions exhibit a significant degree of selectivity as predicted by the hard and soft, acids and bases (HSAB) theory of. Accordingly, electrophilic and nucleophilic molecules are classified as being either soft (relatively polarizable) or hard (relatively non-polarizable) and, based on this principle, toxic electrophiles will react selectively with biological targets of comparable softness or hardness. The unsaturated carbonyl structure of ACR is a soft electrophile that will preferentially form Michael-type adducts with soft nucleophiles, which in biological systems are sulfhydryl side-chains on cysteine residues. In contrast, although nitrogen groups on lysine (ε-amino groups) and histidine (imidazole ring) residues are also nucleophilic, these are harder sites and are therefore less favored targets for ACR adduction (see below).

6The relative softness or hardness of an electrophile can be determined from the respective energies of the outermost or frontier molecular orbitals (FMOs). Because small molecule FMO energies can be calculated using various quantum mechanical models, HSAB parameters such as softness (σ) and hardness (η) of an electrophile are readily computed. With respect to covalent reactions, relative softness (σ) reflects the ease with which electron redistribution occurs during adduct (covalent bond) formation. Thus, the softer the electrophile (larger σ value), the faster it will accept electron density from a donating nucleophile. The values of σ and η also can be used in an algorithm to calculate the electrophilic index (ω) of a toxicant, the magnitude of which reflects the relative propensity of the electrophile to form an adduct with a given nucleophile. Indeed, substantial evidence suggests that σ and ω are determinants of the chemical reactions that mediate electrophile toxicity. Whereas this is true for the majority of type-2 alkene electrophiles, physiochemical characteristics such as steric hindrance imposed by tertiary structure, solubility, and acid–base equilibrium can influence predictions of toxic potency based on HSAB parameters (reviewed by,, ). For example, Table 1 shows that when σ and ω values were calculated for a series of type-2 alkenes, the corresponding values were only qualitatively related to the second-order rate constants (k2) for type-2 alkene adduction of cysteine sulfhydryl groups and to the respective magnitudes of in vitro synaptosomal dysfunction [toxic potency or half maximal inhibitory concentration (IC50)]. This lack of correspondence is due to the slower than predicted reaction rate for 4-hydroxy-2-nonenal (HNE), that is, in the absence of HNE, the type-2 alkene σ values in Table 1 are closely correlated to the corresponding k2 values (r2 = 0.92; see ). The slower adduct reaction is attributable to steric hindrance imposed by the bulky (–C5H11) alkyl tail of HNE. Such discrepancies are expected because the HSAB algorithms incorporate electronic components but not three-dimensional features of chemical structure that can influence the toxicological outcome. Nonetheless, it is evident that ACR is a relatively weak electrophile (low ω value) that slowly forms adducts with cysteine residues (slow second-order reaction rate; Table 1).

7The weak electrophilic character of ACR seems inconsistent with the well-documented ability of this chemical to cause significant neurotoxicity. However, the second-order reaction rate for the formation of ACR-cysteine adducts is governed not only by the relative concentrations of each reactant but also by the electrophilicity of the electron acceptor (ACR, see above) and the relative nucleophilicity of the electron donor (cysteine sulfhydryl group). Thus, the nucleophilic strength of the sulfhydryl target can affect the energy of the transition state for adduct formation and hence the magnitude of the corresponding rate constant (k2). As indicated above, soft electrophiles such as ACR preferentially react with soft nucleophiles. The softness of a nucleophile reflects its relative ability to rapidly transfer electron density to the electrophile. In aqueous environments, sulfhydryl groups on cysteine residues exist in a pH-dependent equilibrium that determines the respective concentrations of the protonated thiol (RSH) and non-protonated thiolate (RS–) forms. Corresponding calculations of nucleophilic softness (σ; Table 2) indicate that the thiolate is substantially softer than the thiol. The side chain nitrogen nucleophiles of histidine and lysine residues, as well as the protonated ε-amino group nitrogen of lysine, are also harder moieties than the sulfhydryl thiolate (Table 2). Based on the HSAB premise of soft–soft interactions, these data identify the sulfhydryl thiolate state of cysteine residues as the preferred target of ACR. The extent to which a given nucleophile will react with ACR can be predicted by calculating the nucleophilicity index (ω–). This HSAB-derived parameter utilizes the hardness (η) and chemical potential (μ) of both ACR (electrophile) and possible nucleophilic amino acid targets. The significantly lower ω– values for the harder nucleophiles (Table 2) indicate that ACR targets soft cysteine thiolate sites. This type of calculation also demonstrates that, relative to ACR, acrolein is a softer and more electrophilic type-2 alkene that reacts much faster with sulfhydryl thiolates (Table 2). The thiolate predilection of ACR and other type-2 alkenes based on HSAB calculations has been experimentally confirmed using proteomic and in chemico approaches.

8Both in vivo and in vitro proteomic studies (e.g.,;;; reviewed by ) have indicated that ACR and other type-2 alkenes impair protein function by reacting with specific cysteine residues on cellular proteins. For example, ACR inhibits presynaptic Na+-dependent dopamine transporter function by reacting with Cys342; NEM forms adducts with Cys254 and thereby inhibits presynaptic vesicle (H+)-ATPase activity; and HNE adduct formation at Cys280 inhibits mitochondrial SIRT3 (SIRT3) activity. However, it is unclear why these specific residues were targeted and, because the functional importance of these cysteines is not known, the toxicological relevance of this adduct formation is uncertain. The preceding discussion suggests that such targeting might reflect the interaction of these type-2 alkenes with the highly nucleophilic sulfhydryl thiolate state of cysteine residues. However, the pKa of the sulfhydryl side chain is approximately 8.4 and therefore, at intracellular pH ranges (7.0–7.4), these groups exist mostly in the non-nucleophilic thiol state (Table 2). Nonetheless, sulfhydryl thiolate groups can be found in cysteine-centered catalytic triads and other microenvironments that significantly reduce side chain pKa values. The ionization of these sulfhydryl groups, and therefore the corresponding nucleophilicity, is determined by proton shuttling that occurs among basic (histidine, arginine, lysine) and acidic (aspartate, glutamate) amino acid residues that are brought into proximity via the tertiary structure of the protein, for example, the arginine357-cysteine121-aspartate355 motif of methionine adenosyl-transferase. Thus, although the majority of sulfhydryl groups in proteins exist primarily (> 90%) in the nonreactive thiol state; those present in catalytic triads are ionized to a much greater extent and, consequently, will react significantly faster with electrophiles. This concept is exemplified by the ryanodine-responsive calcium-release channel of skeletal muscle, where 1 of 50 cysteine residues is reactive because of its presence in a catalytic triad. Cysteine catalytic triads are often located within the active sites of many critical nerve terminal enzymes (e.g., NEM-sensitive factor, vesicular monoamine transporter). The highly nucleophilic sulfhydryl thiolate sites regulate enzyme activity by acting as acceptors for redox modulators such as nitric oxide (NO) or by playing a direct role in corresponding catalytic activity (reviewed by;; ). Thus, it should be evident that adduction of the triad sulfhydryl thiolate will have substantial implications for protein function and subsequent presynaptic toxicity (see below).

9To investigate the possibility that ACR targeted cysteine residues in catalytic triads, we determined the effects of selected type-2 alkenes on the activity of recombinant human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a regulatory cysteine-centered (Cys152) catalytic triad. Consistent with HSAB concepts, the softness (σ) and electrophilicity (ω) values for ACR and the other type-2 alkenes tested [acrolein, methylvinyl ketone (MVK)] were related to the corresponding second-order rate constants (log k2) and potencies (log KI) for GAPDH inhibition (Table 3). Tandem mass spectrometry was used to quantify the adduct formation associated with graded concentrations of ACR. Results indicated that lower in vitro concentrations of ACR inhibited GAPDH activity by selectively forming adducts with Cys152 in the active site of this enzyme, whereas at higher concentrations ACR also reacted with Cys156 and Cys247. Calculations using the PROPKA program (Jensen Research Group, Copenhagen, Denmark) revealed a pKa of 6.03 for Cys152, whereas the pKa values for Cys156 and Cys247 were higher. Furthermore, we found that GAPDH inhibition by the selected type-2 alkenes was pH-dependent, which also indicated thiolate mediation. These data suggest that Cys152 of GAPDH exists in a pKa-lowering microenvironment and that ACR inhibited enzyme function by preferentially forming irreversible Michael-type adducts with this highly nucleophilic sulfhydryl thiolate site. In general, cysteine thiolates contained within catalytic triads function as acceptors for electrophilic mediators of redox signaling [e.g., NO, hydrogen peroxide (H2O2)] and, therefore, ACR adduction of these sites might impair protein function by disrupting this neuromodulatory signaling.

10The preceding discussion suggests that ACR and other type-2 alkenes preferentially form irreversible adducts with sulfhydryl thiolate groups that also function as acceptors for NO and other redox neuromodulators, for example, the thiolate of Cys152 on GAPDH is an NO acceptor. NO is a biological electrophile that forms reversible adducts with sulfhydryl thiolate groups (S-nitrosylation) on proteins. NO signaling transiently decreases synaptic strength by reversibly inhibiting the function of several proteins involved in the synaptic vesicle cycle, for example, NEM-sensitive factor (release), the dopamine membrane transporter (re-uptake) and the vesicular monoamine transporter (vesicular storage). It is highly significant that NO and ACR have similar inhibitory effects on protein function and that the NO-sensitive proteome exhibits substantial overlap with the ACR-adducted proteome. This correspondence suggests that ACR mimics the protein effects of the redox neuromodulators (inactivation) by reacting with thiolate acceptors in catalytic triads. In contrast, the resulting irreversible blockade of redox signaling causes loss of NO-directed neuromodulation and ensuing synaptic toxicity.

11NO signaling, however, is characteristic of most cell types and ACR will form adducts with the thiolate acceptor sites of these non-neuronal cells. The proposed NO-based mechanism of ACR-induced synaptotoxicity therefore lacks nerve terminal specificity. Nonetheless, several unique anatomical and functional characteristics predispose this neuronal region to cumulative electrophilic attack. Specifically, neurotransmission is a complex multistep process that is highly regulated by NO signaling (reviewed by ) and, therefore, ACR disruption of this pathway is likely to have significant consequences for presynaptic function. Furthermore, because the nerve terminal is anatomically separated from the cell body, it is devoid of transcriptional or translational capacity. Thus, unlike the cell body, the nerve terminal lacks the ability to initiate transcription-based reparative or cytoprotective responses, for example, the Nrf2-Keap1 antioxidant response. In the absence of machinery for protein synthesis, maintenance of the presynaptic proteome is dependent on cell body protein manufacturing and subsequent anterograde axonal transport. Correspondingly, as a mechanism to limit material expenditure and increase efficiency, the turnover rates of many nerve terminal proteins are exceptionally slow relative to those of proteins in the nerve cell body or other cell types. Thus, presynaptic proteins inactivated by cysteine adduct formation will be replaced slowly and will consequently accumulate as the rate of adduct formation exceeds the rate of removal by protein turnover. In contrast, dysfunctional adduct-inactivated proteins with short half-lives will not accumulate because they are rapidly replaced by the turnover process. Indeed, our proteomic studies have demonstrated a presynaptic buildup of cysteine adducts that is progressive and closely correlated to the development of ACR neurological symptoms. Furthermore, we have provided evidence that CNS nerve terminal dysfunction occurs at a cumulative adduct level of 350–500 pg cysteine adduct/μg protein. This reflects the minimal exposure threshold below which neurotoxicity does not occur because this level of adduct formation (i.e., < 350 pg cysteine adduct/μg protein) does not affect synaptic processes. As intoxication continues and adduct formation exceeds this threshold, the pool of dysfunctional proteins increases proportionately and the related presynaptic processes are progressively disabled, leading to the characteristic cumulative neurotoxicity of ACR.

12The preceding discussion indicates that several anatomical and neurophysiological attributes render nerve terminals selectively vulnerable to dysfunction via cumulative electrophilic attack. However, as mentioned above, ACR is a type-2 alkene and therefore shares a common mechanism of toxicity with other structurally related members of this chemical class (e.g., acrolein, MVK, NEM). Although selective neurotoxicity is a clearly defined outcome in ACR-exposed human cohorts, similar exposure to other members of this class is associated with systemic toxicity, that is, cardiovascular, respiratory, hepatic or renal toxicity. This diversity of toxic responses is not related to mechanistic differences among members of this chemical family, rather it is due to variations in electrophilic reactivity that correspondingly influence toxicokinetics and tissue distribution. Thus, highly electrophilic type-2 alkenes such as acrolein (Table 1) rapidly form adducts with protein sulfhydryl thiolate groups at systemic sites of absorption. Adduct formation is not only the mechanism of acrolein toxicity, but also restricts the corresponding tissue distribution. As a consequence of this restriction, the toxic manifestations of acrolein and other reactive electrophiles are characteristic of the absorption site; for example, acrolein inhalation produces pulmonary toxicity, whereas systemic administration is associated with hepatic or vascular toxicity. In contrast, as a weak water-soluble electrophile, ACR slowly forms thiolate adducts and is therefore less susceptible to the limiting influence of systemic “adduct buffering.” Accordingly, ACR has a large volume of distribution and readily crosses the blood-brain barrier. Based on theoretically similar CNS accessibilities, systemic exposure to methyl acrylate, ethyl methacrylate, or other weak type-2 alkene electrophiles (Table 1) should also cause selective neurotoxicity. Indeed, the results of both human and animal studies suggest that exposure to these chemicals can produce ACR-like neurotoxicity. Clearly, relative softness (σ) and electrophilicity (ω) determine not only the toxicodynamic character of ACR and other type-2 alkenes (i.e., amino acid targets and mechanisms of toxicity), but also tissue distribution and corresponding toxic manifestations.

13The preceding discussion indicates that the most toxicologically relevant targets of ACR are those nerve terminal proteins that turnover slowly and are importantly involved in neurotransmitter release, storage, and re-uptake. Whereas the adduct chemistry of ACR has been considered through the perspective of nerve terminal damage, future research might confirm an alternative site of neuronal (or glial) action. Regardless of the identified site, a confluence of evidence stemming from early in chemico studies (e.g., ) to recent proteomic research (e.g., ) suggests that the mechanism of toxicity will involve the soft–soft covalent interactions of ACR with cysteine thiolate groups.

14As stated above, ACR is used to manufacture polymers that have broad commercial, industrial, and agricultural applications and, therefore, occupational or accidental intoxication was considered historically to be the primary cause of acquired neurotoxicity. However, other sources of significant daily ACR exposure are now recognized, that is, air/water pollution, cigarette smoke, and diet. Although it has been estimated that the human body burden from these sources can be up to 30 μg ACR/kg/day (Food and Agriculture Organization of the United Nations and the World Health Organization 2005), the neurotoxicological significance of this exposure level is questionable. However, as indicated above, ACR is a member of the type-2 alkene chemical class, which is a large group of structurally related compounds used extensively in the manufacturing, agricultural, polymer, and pharmaceutical industries. As a result, human exposure to the type-2 alkenes is ubiquitous and potentially harmful because many of these compounds are well-documented toxicants. Specifically, unsaturated aldehydes and carbonyls (acrolein, acrylonitrile, MVK) are significant components of air pollution, automobile exhaust, and smoke from cigarette, wood, and coal combustion. At least 36 different unsaturated aldehydes (mostly type-2 alkenes) have been found in the U.S. water supply, often at levels exceeding maximal recommended concentrations. In fact, with the exception of heavy metals, aldehdyes are considered to be the major contaminants in drinking water (reviewed by;;;;; ). There is experimental evidence that the toxic consequences of environmental exposure are mediated by type-2 alkenes (Andre et al. 2008;;; ). Finally, over 300 type-2 alkenes are natural constituents (e.g., acrolein, crotonaldehyde) of various foods and additional carbonyl, aldehyde, and ketone derivatives are produced during cooking fats, oils, and sugars. Based on dietary consumption alone, it is estimated that the α,β-unsaturated aldehyde burden in humans is nearly 200 μg/kg-body wt/day.

15Human populations are therefore exposed to complex type-2 alkene mixtures, the chemical composition and corresponding concentrations of which depend on several variables including geographical location, personal habits (diet, tobacco usage), and occupation. Of particular concern, research has shown that these environmental toxicants produce cell damage via a common molecular mechanism, that is, protein dysfunction through formation of Michael-type adducts with sulfhydryl groups on specific cysteine residues (e.g.,;;,,,;; ). Thus, although the environmental concentrations of any particular unsaturated compound might not be sufficient to cause toxicity, continuous low-level exposure to a mixture of type-2 alkenes might be toxicologically significant.

16In addition to the environmental prevalence of the type-2 alkenes, acrolein, HNE, 4-oxy-2-nonenal (ONE), and other members of this chemical class are produced endogenously during membrane lipid peroxidation associated with cellular oxidative stress. There is growing evidence that these endogenous type-2 alkenes play a pathogenic role in disease processes and traumatic tissue injuries that have oxidative stress as a molecular etiology, for example, stroke, atherosclerosis, Alzheimer’s disease, spinal cord trauma, and diabetes. Therefore, based on their common toxic mechanism, environmentally derived type-2 alkenes might act either synergistically or additively with endogenously generated unsaturated aldehydes. This interaction could amplify the extent of cellular damage and thereby accelerate development of the disease/injury process. That this idea has toxicological plausibility is suggested by epidemiological and experimental research indicating an association between environmental toxicant exposure (e.g., pesticides, heavy metals, industrial chemicals) and an increase in the incidence and severity of many human diseases. With specific reference to environmental type-2 alkene exposure, research has shown that dietary consumption of acrolein exacerbates myocardial ischemic injury and atherosclerosis in mice by interacting with endogenous unsaturated aldehydes generated during ongoing oxidative stress. On the basis of these studies it has been proposed that chronic environmental exposure to unsaturated aldehydes is a significant risk factor for cardiovascular diseases. Similarly, we have suggested that environmental exposure to a mixture of weak type-2 alkene electrophiles (e.g., ACR, methyl acrylate, ethyl methacrylate) could accelerate the progressive nerve terminal demise associated with Alzheimer’s disease (reviewed by, ). In support of this, there is now considerable evidence that the Alzheimer’s disease pathogenic mechanism involves neuronal oxidative stress with subsequent generation of highly reactive type-2 alkene derivatives including acrolein, HNE, and ONE. Furthermore, evidence suggests that nerve terminal dysfunction in relevant brain regions precedes neurodegeneration and is a primary pathophysiological event in Alzheimer’s disease (reviewed by;;;; ). Thus, presynaptic dysfunction in Alzheimer’s disease could be mediated by both environmental and endogenous type-2 alkenes (e.g.,;,,;; ). Along these lines, subchronic human exposures to environmental matrices that contain significant type-2 alkene concentrations such as air pollution or cigarette smoke are associated with an increased incidence of neurodegenerative conditions (e.g.,;;;;;; ). Whereas other toxicant classes in these complex matrices could contribute to the corresponding neuropathogenic processes, the type-2 alkenes are distinguished by their exogenous prevalence, their common toxic mechanism, and their endogenous role in oxidative stress (see above). Despite this growing evidence, the potential for toxic synergy among members of the type-2 alkene class has largely gone unrecognized. As a result, risk assessment has been based on analyses of individual unsaturated carbonyls and their respective toxicities. However, from both a research and risk management perspective, future toxicological considerations should include the interactive potential of these chemicals.

17Early studies of ACR neurotoxicity involved observational research designed to define cell-level sites of action, for example, axon versus nerve terminal. Subsequent research was directed toward determining corresponding molecular mechanisms and, accordingly, numerous mechanistic scenarios were proposed and subsequently tested. Nonetheless, whether the selected neurophysiological parameter tested was a rational and therefore toxicologically plausible target could not be determined because significant mechanistic ambiguity existed at the chemical and molecular levels. However, mechanistic investigations were significantly advanced by recognizing the specific electrophilic nature of ACR and understanding the implications of this electronic character on the selective nucleophile targeting that determines the corresponding covalent adduct chemistry. Thus, we realized that ACR was a soft electrophile that preferentially formed adducts with soft nucleophilic sites on macromolecules. This pointed to the soft, highly nucleophilic thiolate states of cysteine residues in protein catalytic triads as toxicologically relevant molecular targets. Because thiolate sulfhydryl groups on proteins acted as regulatory acceptors for electrophilic mediators of redox signaling (e.g., NO), we ultimately provided evidence that ACR reduced neurotransmission at central and peripheral synapses by disrupting these signaling pathways. Also critical was the recognition that the relevant electronic characteristics defining the chemical basis for ACR toxicity were shared by other α,β-unsaturated carbonyl derivatives and possibly the entire type-2 alkene chemical class. This is a potentially significant realization because the type-2 alkenes are a unique group of structurally related unsaturated carbonyl, aldehyde, and ketone derivatives that are well-documented environmental toxicants and/or endogenous mediators of disease/injury processes associated with cellular oxidative stress. Based on their demonstrated common mechanism of toxicity, we propose that environmental exposure to a mixture of type-2 alkenes could represent a significant human health risk. Furthermore, these exogenously derived toxicants could interact synergistically with endogenous unsaturated aldehydes and thereby accelerate the onset and development of atherosclerosis, diabetes, Alzheimer’s disease, and other pathogenic conditions that have cellular oxidative stress as a molecular etiology. Thus, in this review we have described a relatively detailed mechanistic scenario for ACR neurotoxicity. This level of comprehension was achieved through understanding the principles of organic chemistry that govern the covalent interactions of electrophilic toxicants with their nucleophilic targets. Because many toxicants are electrophiles of varying softness and reactivity (e.g., methylmercury; N-acetyl-p-benzoquinone imine, 2,5-hexanedione) a similar approach could be used to identify rational nucleophilic targets on biological macromolecules.