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PerspectiVe Potentialities and Pitfalls Accompanying Chemico-Pharmacological Strategies against Endogenous Electrophiles and Carbonyl Stress Philip C. Burcham Pharmacology and Anaesthesiology Unit, School of Medicine and Pharmacology, the UniVersity of Western Australia, Nedlands, WA 6009, Australia ReceiVed NoVember 8, 2007
The use of powerful analytical technologies to detect endogenous carbonyls formed as byproducts of oxidative cell injury has revealed that these species contribute to many human diseases. As electrophiles, they are attacked by reactive centers in cell macromolecules to form adducts, the levels of which serve as useful biomarkers of oxidative cell injury. Because the pathobiological significance of such damage is often unclear, the possibility of using low molecular weight drugs as exploratory sacrificial nucleophiles to intercept reactive carbonyls within cells and tissues is appealing. This perspective highlights the potential benefits of using carbonyl scavengers to evaluate the significance of endogenous carbonyls in particular diseases but also canvasses a number of challenges confronting this therapeutic strategy. Chief among the latter is the task of confirming that carbonyl sequestration underlies any suppression of disease symptoms elicited by these multipotent reagents, an issue needing clarification if these compounds are to command consideration as drug interventions in humans. Other problems include adverse consequences of reactions between carbonyl scavengers and important endogenous carbonyls (e.g., neurotoxicity due to pyridoxal depletion), as well as the potential for drugs to form ternary complexes with carbonylated cell proteins, raising the prospect of immunotoxicological outcomes. Strategies for moving carbonyl sequestering reagents from the laboratory bench to a clinical testing environment are discussed within the context of the search for new treatments for spinal cord injury, one of the most debilitating medical conditions sustainable by humans. This condition seems an appropriate test case for assessing carbonyl sequestering drugs given growing evidence for noxious carbonyls in the wave of neuronal cell death that follows traumatic injury to the spinal cord. Contents 1. Introduction 2. Electrophilic Carbonyls and Human Disease 3. Potential of Carbonyl Scavengers as Biological Probes 4. Pitfalls Accompanying Use of Carbonyl Scavengers 4.1. Biological Relevance of Data from Cell-Free Systems 4.2. Need for Druglike Physicochemical Properties 4.3. Robust Criteria for In Vivo Carbonyl Scavenging Activity 4.4. Side Effects Due to Nonspecific Carbonyl Depletion 4.5. Drug Reactions with Carbonyl-Adducted Proteins 4.6. Metabolic Fate of Scavengers 4.7. Cytoprotective Actions Unrelated to Carbonyl Sequestration 5. Moving Carbonyl Scavengers from the Test Tube to the Clinical Testing Laboratory 6. Disease Focus: Spinal Cord Injury and Carbonyl Stress 7. Conclusion
1. Introduction 779 780 780 781 781 781 782 782 782 782 783 783 783
The opening essay in the recent Future of Toxicology series published by CRT articulated a key challenge facing modern toxicology in terms of its standing within the broader biomedical research community (1). This concern centers on a perceived feeling among nontoxicologists that knowledge gained from longstanding efforts to characterize risks accompanying exposure to xenobiotics is of questionable etiological relevance to the diseases that take the greatest toll on 21st century public health. On this view, toxicologists are guilty of preoccupying themselves with chemicals that are of dubious significance to obesity, diabetes, neurodegeneration, and other conditions that afflict aging populations. Countering this perception, Liebler argued for expanding the horizons of the toxicological enterprise to include noxious chemicals of endogenous origin, contending that the conceptual tools and methodologies of molecular toxicology are well-suited to investigating the role of endogenous electrophiles in common diseases (1). While this approach deservedly finds growing support among toxicologists, it may nonetheless be subject to its own conceptual challenges and epistemic uncertainties.
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Table 1. Selected Common Diseases in Which Endogenous Carbonyls Have Been Implicateda disease Alzheimer’s disease
atherosclerosis diabetes (type II)
obesity
tissue human human human human human human human human human human human human human human human
brain brain CSF CSF brain brain plaques plaques plaques urine kidney serum plasma plasma plasma
carbonyl compound
form detected and analytical methodology
literature ref
ACR CRO 3-DG glyoxal 4-HNE MDA ACR 4-HNE MDA ACR ACR, MDA, 4-HNE, AGE methyl glyoxal DHA MDA 4-HNE
protein-adducted (immunochemical detection) protein-adducted (immunochemical detection) protein-adducted (LC-MS/MS) protein-adducted (LC-MS/MS) DNA adducts (LC-MS) free carbonyl (TBARS) protein-adducted (immunochemical detection) protein-adducted (immunochemical detection) protein-adducted (immunochemical detection) amino acid-adducted (immunochemical detection) protein-adducted (immunochemical detection) protein-adducted (immunochemical detection) free carbonyl free carbonyl protein-adducted (immunochemical detection)
52 53 54 55 55 56 57 58 59 60 61 62 63 64 65
a Abbreviations: AGE, advanced glycation end products; ACR, acrolein; CRO, crotonaldehyde; DHA, dehydroascorbate; 3-DG, 3-deoxyglucosone; 4-HNE, 4-hydroxynonenal; MDA, malondialdehyde.
2. Electrophilic Carbonyls and Human Disease A large body of data associates production of reactive carbonyls with many human diseases (2). Carbonyls form during the oxidative fragmentation of unsaturated membrane lipids, sugars, amino acids, and nucleic acids. As numerous structurally diverse carbonyl compounds form under prooxidative conditions, use of the umbrella term “carbonyl stress” denotes a cellular environment displaying either heightened formation and/ or diminished detoxication of a range of endogenous carbonyls. Because they possess one or more electron-deficient centers, carbonyl compounds alkylate nucleophilic residues in proteins, DNA, RNA, and other cellular targets. Such chemistry underlies the broad spectrum of effects seen upon exposing cells to carbonyl compounds, including disruption of signaling pathways, perturbation of the levels of hundreds of mRNA transcripts, mutagenesis of gene targets, induction of adaptive response pathways, and activation of apoptotic cell death (3). Given the importance of macromolecular adduction to these outcomes, much effort has been devoted to characterizing reactions between endogenous electrophiles and their cellular targets, in the expectation that chemically modified amino acids or DNA bases will serve as useful biomarkers of exposure to these species. Table 1 lists several diseases in which either free endogenous carbonyls or carbonyl-derived macromolecular adducts have been detected. The table is highly selective, intended only to convey the range of carbonyl compounds that have been associated with several diseases that are of emerging importance in industrialized countries. For accounts of the role of carbonyl stress in specific disorders, one should consult other sources (2). Despite the body of data either documenting noxious effects of carbonyl compounds in vitro or confirming their participation in particular clinical conditions, key questions remain concerning their etiological role in many disorders (4). For example, is the production of carbonyl compounds and the macromolecular damage that they cause directly involved in the pathogenesis of a given disease, or is it peripheral to the main processes mediating cell and tissue dysfunction? Moreover, could it be that the technological innovations that enable detection of low levels of endogenous electrophiles within biological samples have mesmerized us to exaggerate the medical significance of these substances? Answering such questions is obviously relevant to the quest for new therapeutic strategies for these disorders but also seems germane to the uncertainty surrounding the future of toxicology and its position within the medical research endeavor. Undoubtedly, the currency of knowledge
Figure 1. Potential for carbonyl-reactive drug probes to clarify roles of endogenous carbonyls in disease pathogenesis. Note that the two scenarios are opposite poles of a continuum of possible contributions of carbonyl stress to disease mechanisms.
concerning the levels of carbonyl-adducted macromolecules in diseased tissues increases greatly if the pathobiological significance of such damage is demonstrable. In the remainder of this perspective, I address the use of carbonyl-reactive drugs to explore the significance of reactive carbonyls in human disease.
3. Potential of Carbonyl Scavengers as Biological Probes The use of chemical probes of the biological relevance of carbonyl stress involves selecting compounds based on their physicochemical properties and most importantly their reactivity with carbonyl compounds (5, 6). This is quite different from conventional concepts in pharmacology, according to which most drugs elicit their effects via transient interactions with membrane-spanning receptors that modulate cellular signaling and effector pathways. In contrast, carbonyl scavengers should ideally show minimal activity at drug receptors, ensuring that unwanted pharmacological effects do not complicate their in vivo use. Rather, the administration of carbonyl scavengers proceeds in the expectation that they rapidly sequester carbonyl compounds within cells, blocking adduction of macromolecules and any downstream consequences of such damage. As depicted in Figure 1, assuming the doses used suppress carbonyl stress in both instances, carbonyl scavengers may help clarify the mechanistic role of carbonyl stress in specific diseases. In the first scenario, because carbonyl production plays a causal role within the pathobiological continuum, carbonyl scavenger
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Chem. Res. Toxicol., Vol. 21, No. 4, 2008 781 Table 2. Representative Carbonyl-Scavenging Compoundsa scavenger
nucleophilic center(s)
target carbonyls
aminoguanidine bisulfite tri-butyl hydroxylamine carnosine glucosamine glycyl-histidyl-lysine (GHK) hydralazine OPB-9195 metformin N-acetyl cysteine D-penicillamine phenelzine pyridoxamine
hydrazino amine sulfur atom secondary nitrogen imidazole nitrogen (His), terminal amine primary amine primary amine, lysine -amine hydrazino amine hydrazino amine (after acetanilide hydrolysis?) primary amine thiol thiol hydrazino amine primary amine
FA, 4-HNE, MDA, 3-DG, etc. ACR, CRO 3-AP ACR, HEX, 4-HNE, MDA MDA 4-HNE ACR GA, MG, 3-DG, MDA, 4-HNE GA, MG ACR ACR, ACET, GA, MG 3-AP 9- and 13-KODE, MDA, MG
ref 12, 16, 68 69, 71 72 11 73 74 75 10, 8 78,
18, 66 67 70
76, 77 79
a
Abbreviations: AGE, advanced glycation end products; ACET, acetaldehyde; ACR, acrolein; 3-AP, 3-aminopropanal; CRO, crotonaldehyde; DHA, dehydroascorbate; 3-DG, 3-deoxyglucosone; FA, formaldehyde; GA, glyoxal; HEX, trans-hexenal; 4-HNE, 4-hydroxynonenal; KODE, keto-octadecadienoic acid; MDA, malondialdehyde; MG, methyl glyoxal.
administration is expected to suppress the respective disease end points. Under the second scenario, because carbonyl production is an epiphenomenon of the fundamental disease process, carbonyl scavengers will have little effect on disease symptoms. In the former case, the clear mechanistic relevance of carbonyl formation may justify further exploitation of carbonyl scavengers as therapeutic interventions in the disease of interest. Such an expectation is driving a growing interest in the identification of efficient scavengers for a range of endogenous carbonyl compounds (5–8). To date, the scope of carbonyl-sequestering chemistries tested is quite conventional, including compounds comprising nucleophiles directed against the carbonyl group (e.g., hydrazine, amine, and bisulfite) as well as those targeting the double bond of R,β-unsaturated carbonyl compounds (e.g., thiol-containing nucleophiles) (Table 2).
4. Pitfalls Accompanying Use of Carbonyl Scavengers Using biologically inert, chemically reactive drugs to intercept toxic endogenous carbonyls within the body is attractive from the standpoint of experimental biology and a clinical intervention perspective. Nonetheless, a number of significant obstacles confront this approach. In the following section, I highlight a number of these problems, drawing from the work of others as well as studies from my own laboratory relating to scavengers of acrolein, among the most reactive and toxic carbonyls of endogenous origin. 4.1. Biological Relevance of Data from Cell-Free Systems. The development of carbonyl scavengers has often focused on either structural characterization of products formed during reactions between scavengers and carbonyls in cell-free systems or upon evaluating their ability to block carbonylinduced adduction of model macromolecules in test tube reactions. In addition, it is common to evaluate compounds for their ability to suppress the toxicity of carbonyl compounds in cultured cells (9–11). However, because of the chemical complexity of the intracellular environment, which presents opportunities for multiple competing reactions, the fact that a drug traps a given carbonyl compound in a cell-free system is no guarantee that it will exhibit comparable properties within biological systems. An important consideration is that the reactivity of a scavenger with a given carbonyl must compete favorably with the rate of adduction of the preferred intracellular target for that carbonyl (12, 13). For example, the nucleophilic antihypertensive hydralazine was an efficient scavenger of acrolein in a cell-free system and appeared to inhibit adduction of a model protein (14). However, while hydralazine strongly suppressed acrolein-mediated toxicity in cultured cells, cyto-
protection was unrelated to levels of acrolein-hydralazine conjugates in culture media (15). Cytoprotective drug concentrations also failed to suppress carbonylation of cellular proteins during acrolein-mediated toxicity, suggesting that hydralazine scavenges acrolein poorly within cells (15). Moreover, despite blocking cross-linking of a model protein in test tube systems (15), in acrolein-treated A549 lung epithelial cells, hydralazine did not inhibit cross-linking of Hsp90, a known target for reactive carbonyls (16). Comparison of rates of acrolein scavenging by different scavengers to that of the reaction of acrolein with various nucleophilic amino acids suggested that an ability to compete with the adduction of cysteinesthe preferred cellular target for acroleinswas most predictive of the ability to suppress protein carbonylation and protein crosslinking in A549 cells (16). While its pronounced electrophilicity may exaggerate these considerations for acrolein relative to less reactive carbonyls, these findings nonetheless imply that mechanistic studies in biologically relevant test systems should be an early priority in research programs directed toward identifying new carbonyl scavengers. 4.2. Need for Druglike Physicochemical Properties. Compounds likely to succeed as drug candidates are typically comparatively small molecules that are sufficiently lipid soluble to partition into the lipid phase of biphasic oil–water systems while at the same time possessing enough water solubility to allow dissolution and drug transport (17). Lipophilic character is particularly important for drugs administered via the oral route, as they must cross a succession of lipid membranes to reach their target receptors in anatomically remote tissues. Having appropriate numbers of H-bond acceptors and H-bond donors is particularly important for orally administered agents since these factors govern drug polarity and lipophilicity and thus strongly influence oral bioavailability (17). Such considerations are particularly important for oral carbonyl scavengers intended for use in neurodegenerative diseases such as Alzheimer’s (Table 1), since in these patients drugs must also cross the blood-brain barrier to target carbonyl overproduction within diseased CNS sites. Because building carbonyl sequestering reactivity into carbonyl scavengers requires the introduction of nucleophilic centers such as primary amines, these groups change the balance of H-bond acceptors and donors within the molecule, typically increasing hydrophilicity at the expense of lipophilicity and thereby compromising the central distribution of the drug. While problems of this type are potentially surmountable by using biolabile substituents to mask ionizable groups (i.e., prodrugs), such issues seem likely to increase the complexity of drug discovery efforts in this area.
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4.3. Robust Criteria for In Vivo Carbonyl Scavenging Activity. Adherence to rigorous criteria when testing compounds in animal models would greatly improve the search for efficacious carbonyl scavengers. One leading criterion would be confirmation of diminished levels of free carbonyls in body fluids or target tissues of drug-treated subjects. Obtaining estimates of free concentrations may be difficult to achieve in the case of strongly electrophilic carbonyls, which react rapidly with tissue constituents. Nevertheless, in recent work, modest doses of the carbonyl scavenger aminoguanidine diminished urinary levels of formaldehyde, a reactive carbonyl formed during the oxidation of methylamine, an endogenous semicarbazide-sensitive amine oxidase substrate (18). Aminoguanidine is of particular interest as a carbonyl scavenger due to its ability to attenuate a number of diabetic complications in rodents including stiffening of vascular beds, atherosclerosis, and renal injury (19). A second criterion is that any beneficial effects of carbonyl scavengers should be accompanied by detection of drug-carbonyl conjugates in body fluids of treated subjects and that the concentrations of such conjugates should be dose-related to reductions in disease severity. Getting such data could be challenging if drug-carbonyl conjugates undergo rapid metabolic processing in the liver, a problem that has confounded our efforts to detect acrolein-hydralazine conjugates in blood from allyl alcohol-intoxicated mice that received hepatoprotective doses of hydralazine (20). Nevertheless, such complications may not apply to all possible carbonyl/scavenger combinations (18). For example, conjugates formed during the reaction of the carbonyl scavenger pyridoxamine with peroxidized lipids were detected in the urine of streptozocin-induced diabetic and hyperlipidemic rats that received pyridoxamine in their drinking water (21). A third criterion is the demonstration that dosing with carbonyl scavengers diminishes macromolecular adduction by carbonyls in target tissues. Early studies that evaluated aminoguanidine as an inhibitor of protein glycation in diabetic rats clearly recognized the value of this type of data (22). In recent work in cultured smooth muscle cells, antibody- and MS-based strategies allowed confirmation that carbonyl scavengers attenuated adduction of individual protein targets during exposure to reactive dicarbonyl compounds (23). Likewise, recent testing of hydralazine as a carbonyl scavenger in cholesterol-fed atherosclerotic rabbits revealed a lowering of levels of 4-hydroxynonenal adducts in a ≈180 kDa protein within the intima of rabbit aorta (24). This promising outcome was accompanied by a suppression of atherosclerotic plaque formation (24). To date, few studies have evaluated all three of the above criteria within a single experimental protocol. The adoption of such concerted approaches to confirming mechanism of drug action may improve chances of obtaining robust insights during the testing of carbonyl scavengers in vivo. 4.4. Side Effects Due to Nonspecific Carbonyl Depletion. Neurological complications in the form of peripheral neuritis and neuropathy can plague chronic treatment with a diverse range of carbonyl-reactive pharmaceutical agents, including hydrazino drugs (e.g., isoniazid, phenelzine, hydralazine, and carbidopa), substituted hydroxylamines (e.g., Dcycloserine), and sulfhydryl compounds (e.g., D-penicillamine) (25). These toxicities reflect the reactivity of these drugs with pyridoxal, a carbonyl-containing cofactor needed for the expression of various enzymes involved in the biosynthesis of neurotransmitters such as dopamine, GABA, and serotonin (25). Neurological deficits also occur in individuals with inborn
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defects that promote the accumulation of nucleophilic, pyridoxaldepleting metabolites (26). To avoid such problems, future work might explore the challenging possibility of developing carbonyl-scavenging compounds that react preferentially with toxic carbonyls while showing lesser reactivity with pyridoxal. Alternatively, pyridoxine coadministration can help minimize neuropathic complications in patients receiving nucleophilic drugs, but this compound can present its own spectrum of problematic neurological side effects. 4.5. Drug Reactions with Carbonyl-Adducted Proteins. The carbonyl compounds that are most important as disease mediators are typically bifunctional electrophiles, possessing either two electrophilic carbonyl groups (e.g., glyoxal) or a carbonyl group in addition to an R,β-unsaturated bond (e.g., 4-hydroxynonenal). Macromolecular modification by these species can thus proceed via two steps. In the initial step, a nucleophilic center in protein or DNA attacks one electrophilic group possessed by the carbonyl to form Michael addition or Schiff base adducts. Subsequently, the remaining electrophile can react with neighboring nucleophiles to form either cyclic adducts (e.g., propanoadducts formed by R,β-unsaturated aldehydes in DNA) or cross-linked species [e.g., propanal adducts formed by Michael addition of acrolein to histidine residues react with terminal lysine groups to form imino cross-links (27)]. In the presence of carbonyl scavengers, however, primary adducts may react with nucleophilic drugs to form ternary drug-carbonyl-protein complexes. This was first shown for the endogenous carbonyl scavenger carnosine, which reacts with carbonyl groups introduced during protein glycation (28). In the case of protein modification by acrolein, we found that Michael adducts are readily attacked by carbonyl scavengers in a reaction we term “protein adduct trapping” (29). Using an antibody directed against these species, extensive adduct trapping was detected in acrolein-pretreated cells that were treated with cytoprotective concentrations of hydralazine (29). Surprisingly, this ability to trap Michael adducted proteins did not inhibit the formation of cross-linked Hsp90 proteins in acroleinexposed cells (16). Using mass spectrometry to monitor reaction products formed in a model peptide following reaction with acrolein in the presence and absence of bisulfite, we similarly detected adduct trapping by this highly efficient carbonyl scavenger (16). While the cellular consequences of these adducttrapping reactions are unknown, it is possible they may elicit unwanted toxicological outcomes including the induction of autoimmune responses due to presentation of drug-labeled adducts as neoantigens to antibody-producing cells. As lupus erythematosus is a classic complication of hydralazine therapy, it seems likely that adduct-trapping reactions may contribute to this immunotoxicological syndrome. It may be prudent to evaluate carbonyl scavengers for their abilities to generate ternary drug-carbonyl-protein complexes as well as the immunogenicity of these species. 4.6. Metabolic Fate of Scavengers. Unfortunately, the same nucleophilic center that confers reactivity with carbonyl compounds may ensure that carbonyl scavengers are rapidly metabolized in vivo. Xenobiotics containing nucleophilic heteroatoms are often excellent substrates for conjugative metabolism in the liver (e.g., glucuronidation, N-acetylation, sulfonation, amino acid conjugation, etc.). For example, hydralazine exhibits a plasma half-life of less than 1 h, reflecting rapid clearance via N-acetylation, while nonenzymatic hydrazone formation with pyruvate is another significant systemic fate of the drug (30). D-Penicillamine, a thiol-containing scavenger that traps a range of carbonyl compounds, also exhibits an elimina-
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tion half-life of approximately 1 h due to conjugative metabolism and disulfide formation (31). While such considerations may not necessarily preclude successful use of these drugs as carbonyl scavengers, the design of in vivo dosing regimens should take into account the pharmacokinetic properties of scavengers. Alternatively, the use of slow release formulations or organ-directed drug delivery methods may help resolve these concerns. 4.7. Cytoprotective Actions Unrelated to Carbonyl Sequestration. In addition to facilitating reactions with carbonyl compounds, the electron density of the nucleophilic center possessed by carbonyl scavengers may confer other bioactivities, including activity as antioxidants or metal-chelating agents (32). Likewise, the inhibitory action of aminoguanidine against nitric oxide synthase (NOS) needs to be taken into account during the in vivo use of this compound (e.g., NOS inhibition may attenuate production of damaging peroxynitrite species) (33). Of course such ancillary properties may well be beneficial in a carbonyl scavenger since they may confer additional cytoprotection during oxidative cell injury. However, a multiplicity of actions can obscure clarification of the role of carbonyl sequestration in any disease suppression observed in animals. To address such issues, study designs could include evaluation of multiple end points of drug action (e.g., biomarkers of carbonyl sequestration along with markers of antioxidant actions such as superoxide production, protein oxidation, isoprostane levels, etc.).
5. Moving Carbonyl Scavengers from the Test Tube to the Clinical Testing Laboratory The preceding discussion highlighted a number of obstacles confronting efforts to develop drugs for use as chemical scavengers of noxious electrophiles. Rather than discouraging exploration of this experimental approach, frank identification of potential pitfalls will hopefully clarify future work in this field. Indeed, the likelihood of useful returns from ongoing effort in this therapeutic area is reinforced by findings from recent clinical trials, which suggested that two classic carbonylsequestering agents, aminoguanidine and pyridoxamine (Table 2), exhibit at least partial efficacy in patients with diabetesrelated kidney disease, a spectrum of conditions in which carbonyl stress features strongly (34, 35). In the latest report, two multicenter phase 2 studies provided promising results during the evaluation of two closely related pyridoxamine dosing regimens (either 50 mg twice daily for 24 weeks or escalating 250 mg twice daily for 24 weeks) for their ability to preserve renal function in diabetic nephropathy patients. Comparison of changes in serum creatinine levels in placebo-treated controls to pyridoxamine-treated subjects suggested pyridoxamine partially preserved renal function, but most importantly, from the perspective of the third criterion specified in section 4.2 above, these functional outcomes were accompanied by diminished plasma levels of two markers of glycoxidative protein damage, CML (carboxymethyllysine) and CEL (carboxyethyllysine) (36). The pyridoxamine treatments seemed well-tolerated by patients, but because these trials occurred over a comparatively short time frame, it is premature to assess the impact of toxicity problems such as those highlighted in sections 4.3 and 4.4 above that might occur under chronic drug-dosing conditions. Perhaps carbonyl-trapping drugs can be moved forward as a therapeutic class by focusing more on diseases in which key confounders highlighted in section 4 are of diminished significance. These might include disorders for which the evidence of a pathobiological role for carbonyls is strong, where long-
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term drug therapy should not be necessary (to avoid chronic pyridoxal depletion in the CNS), and where local drug administration to diseased tissues is feasible (to avoid problems relating to first-pass clearance of drugs after oral administration). To close this perspective on a positive note, I will now explore the rationale for testing carbonyl scavengers against a devastating health condition in which emerging research suggests that toxic carbonyls may play a significant role, namely, spinal cord injury.
6. Disease Focus: Spinal Cord Injury and Carbonyl Stress Because the spinal cord is the conduit through which sensory and motor signals flow between the brain and the body, physical trauma to this network leads to some of the most debilitating injuries sustained by humans (e.g., paraplegia, tetraplegia). Because of aging, forward projections suggest that spinal cord injury (SCI) will be increasingly prevalent in coming decades (37). Because the efficacy of existing treatments for SCI is quite limited, there is a clear need for new therapeutic approaches. Several distinct phases of disease progression are identifiable in SCI. The first involves direct trauma to the spinal cord and includes membrane shearing, blood vessel damage, and edema (38, 39). The full clinical picture of SCI is not explained by such immediate damage but instead involves spread of injury centrifugally from the lesion site. This secondary phase features waves of necrotic and apoptotic cell death and involves deleterious biochemical changes that persist for days or even weeks. Such processes greatly expand the zone of pathology, culminating during the final chronic stage in the formation of cysts comprising complex mixtures of astrocytes, Schwann cells, inflammatory cells, and axons in various stages of myelination (38–40). The chronic phase of SCI can last for months or years and includes apoptosis, demyelination, glial scarring, and Wallerian degeneration. The recognition that secondary processes strongly influence the severity of SCI has inspired a large body of experimental work that explores a range of drug- and biotechnology-based therapies. A number of therapeutic approaches are feasible, but strategies that limit cell death within the damaged spinal cord are particularly attractive because these may preserve the functional capacity of the tissue. Multiple mechanisms contribute to neuronal death following SCI. Axonal Ca2+ dyshomeostasis is one factor due to opening of voltage-dependent calcium channels, impairment of ATPdependent Ca2+ efflux, and disrupted Ca2+ sequestration by cell organelles. Ca2+ overload accelerates neuronal death by inducing overproduction of reactive nitrogen species, mitochondrial damage, and activating proteases and endonucleases. A related neuropathic process involves the excitatory amino acid glutamate, the extracellular levels of which increase strongly after SCI. Glutamate excitotoxicity kills cells by activating ionotropic receptors and eliciting depolarization and osmotic stress but also by exacerbating Ca2+ influx via voltage-dependent calcium channels. Glutamate antagonists have shown promise as therapeutic interventions against SCI in animals, but outcomes in humans are less positive, partly reflecting specificity issues due to the broad role of glutamate in the CNS (41). Several pathological processes also trigger an overproduction of reactive oxygen species in the early stages of SCI. These include reperfusion of ischemic tissue and xanthine oxidase activation, redox cycling of neurotransmitters, invasion of neutrophils, mitochondrial uncoupling, and release of redoxactive metals (39–41). Reactive oxygen species are especially damaging to the white matter of the spinal cord, which is rich in oxidation-sensitive lipids. One early change in SCI tissue is
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a strong release of polyunsaturated lipids (e.g., arachidonic acid) following phospholipase A2 activation (42). Metabolism of liberated arachidonic acid generates a number of bioactive eicosanoids within the damaged spinal cord (39). Lipids within white matter are also prone to oxidative fragmentation, forming a range of toxic carbonyls that amplify tissue damage in SCI (e.g., malondialdehyde and 4-hydroxynonenal) (43–45). A growing body of work suggests that acrolein, one of the most toxic lipid-derived carbonyls, deserves close attention as a mediator of tissue damage in SCI. For example, Western blotting using antibodies against acrolein-modified proteins revealed a significant increase in acrolein adducts in spinal cord proteins from rats 4 h after compression injury, with damage peaking at 24 h and remaining elevated relative to controls 1 week postinjury (46). Consistent with a role for this diffusible and highly reactive substance in the secondary wave of cell death that follows initial trauma to the spinal cord, acrolein adduction occurred not only on proteins from the primary injury site (T10 to T11) but also in adjacent spinal cord segments (T8-T9 and T12-T13) (46). Comparable trends occurred in levels of 4-hydroxynonenal-modified proteins in the same spinal cord segments (46). A role for acrolein in SCI is strengthened by new findings made using MS to identify post-translational modifications on the signaling protein GDP-dissociation inhibitor-2 (GDI-2) in rat spinal cord proteins following contusion injury (47). GDI-2 was selected for analysis because the authors found several-fold induction of this protein within a few hours after SCI (47). Intriguingly, acrolein adduction on lysine-174 was the only post-translational modification detected on GDI-2 proteins from rats 72 h after SCI (47). In other work, acrolein exposure produced changes in axonal conduction in isolated guinea pig spinal cord strips that resemble those seen in SCI (48). Acrolein exposure also elicited marked changes to the morphology and cytoskeleton of chick dorsal root ganglion cells and sympathetic ganglion cells (49). Given these findings concerning the role of carbonyl stress in SCI pathogenesis, nucleophilic drugs that block the toxicity of acrolein and related carbonyls deserve careful investigation as potential neuroprotective treatments in SCI (50). In addition to such mechanistic justifications, there are several other compelling reasons for why SCI is a promising candidate for testing carbonyl-scavenging drugs. These include animal models that reproduce many features of human SCI, clear behavioral and functional end points that confirm preservation of neural tissue by drugs, and a large database of experimental therapies for comparative purposes; local drug delivery to the lesion is feasible, and finally, chronic drug therapy is unlikely to be required, reducing the likelihood of neurological or immunological side effects. At the very least, testing carbonyl scavengers that exhibit good reactivity toward acrolein, 4-hydroxynonenal, malondialdehyde, and other lipid-derived carbonyls in a wellvalidated model of SCI would allow clarification of the role of carbonyl stress in this important human disease, discriminating between a causal role in disease pathogenesis and an indirect association of limited relevance to disease outcomes (Figure 1). Intriguingly, a new study reports that ex vivo treatment with the acrolein scavenger hydralazine attenuated spinal cord neurodegeneration in guinea pigs subjected to compression trauma to induce SCI (51). Exposure to 500 µM hydralazine suppressed the onset of irreversible cell injury, as assessed by measuring the leakage of lactate dehydrogenase from injured spinal cords, while also diminishing acrolein-mediated protein adduction in damaged tissue (51). Although the drug concentration used was high, the authors suggest that such concentrations
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may be achievable in vivo by using localized administration to deliver drugs to damaged spinal cords. Clearly, these findings highlight the need for thorough evaluation of carbonyl scavengers in rodent SCI models that allow evaluation of complex behavioral and neurological end points.
7. Conclusion In recent decades, the application of powerful analytical technologies has confirmed that reactive carbonyl compounds form during many health disorders and that macromolecular damage induced by these species often accumulates in the respective diseased tissues. Given that carbonyl compounds are toxic and can disrupt a wide range of cell processes, it seems reasonable to assume that these substances contribute to the pathogenesis of these conditions. Nevertheless, in many instances, definitive proof for such a direct involvement is presently lacking. A drug-based strategy for ablating exposure to these substances such as is outlined in this perspective may provide one means of assessing the significance of endogenous carbonyls in human disease. Before such a goal can be achieved, greater attention to fundamental mechanism of action considerations concerning the actions of carbonyl scavengers in biological systems will be required if this therapeutic strategy is to progress beyond its present largely experimental state.
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