Rate of Neurotoxicant Exposure Determines Morphologic Manifestations of Distal Axonopathy

Toxicology and Applied Pharmacology 167, 75– 86 (2000) doi:10.1006/taap.2000.8984, available online at http://www.idealibrary.com on

CONTEMPORARY ISSUES IN TOXICOLOGY Rate of Neurotoxicant Exposure Determines Morphologic Manifestations of Distal Axonopathy Richard M. LoPachin,* ,1 Ellen J. Lehning,* Lisa A. Opanashuk,* and Bernard S. Jortner† *Department of Anesthesiology, Albert Einstein College of Medicine/Montefiore Medical Center, 111 E. 210th Street, Bronx, New York 10467-2490; and †Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0442 Received January 19, 2000; accepted June 7, 2000

and Schaumburg, 1974b, 1980; Spencer et al., 1980). Depending upon the chemical toxicant, these swellings contain accumulations of cytoskeletal neurofilaments, fragments of the smooth endoplasmic reticulum, and degenerating mitochondria (Fullerton and Barnes, 1966; Prineas, 1969; Suzuki and Pfaff, 1973; Spencer and Schaumburg, 1977; Sills et al., 1998; Weiner and Jortner, 1999). Axonal atrophy and segmental demyelination have been observed frequently and are considered secondary phenomena (Berger and Schaumburg, 1995; Brown et al., 1978). Because of the retrograde progression of degeneration, this type of toxic nerve injury was termed a “dying back” neuropathy by Cavanagh (1964). However, to emphasize the coincident involvement of central and peripheral nervous system regions and the distal expression of morphologic changes at the axon level, Spencer and Schaumburg (1977) suggested the term “central–peripheral distal axonopathy.” Clinically, toxic axonopathies are associated with skeletal muscle weakness of the extremities (hands, lower legs, and feet) and uncoordinated gait (ataxia). Although there is little direct evidence for a causal relationship, it has been largely assumed that axon damage mediates much of the behavioral toxicity and neurodysfunctional manifestations associated with toxic exposure (e.g., see LeQuesne, 1980; Spencer et al., 1980). Among the compounds that produce distal axonopathy, acrylamide (ACR) and 2,5-hexanedione (HD) are recognized as prototypical agents. ACR is a water soluble ␣,␤-unsaturated carbonyl compound that forms polyacrylamides during radicalinitiated polymerization reactions. Polymerized ACR is used in the preparation of laboratory electrophoresis gels and has broad application in various chemical industries; e.g., water purification, ore processing, paper, fabric, and textile industries (Spencer and Schaumburg, 1974a; EPA, 1988). HD is the common active ␥-diketone metabolite of n-hexane and methyl-n-butyl ketone (Krasavage et al., 1980; Couri and Milks, 1982). These parent hexacarbons are used extensively in the fabric industry and have been involved in numerous outbreaks of human

Rate of Neurotoxicant Exposure Determines Morphologic Manifestations of Distal Axonopathy. LoPachin, R. M., Lehning, E. J., Opanashuk, L. A., and Jornter, B. S. (2000). Toxicol. Appl. Pharmacol. 167, 75– 86. Exposure to a variety of agricultural, industrial, and pharmaceutical chemicals produces nerve damage classified as a central– peripheral distal axonopathy. Morphologically, this axonopathy is characterized by distal axon swellings and secondary degeneration. Over the past 25 years substantial research efforts have been devoted toward deciphering the molecular mechanisms of these presumed hallmark neuropathic features. However, recent studies suggest that axon swelling and degeneration are related to subchronic low-dose neurotoxicant exposure rates (i.e., mg toxicant/ kg/day) and not to the development of neurophysiological deficits or behavioral toxicity. This suggests these phenomena are nonspecific and of uncertain pathophysiologic relevance. This possibility has significant implications for research investigating mechanisms of neurotoxicity, development of exposure biomarkers, design of risk assessment models, neurotoxicant classification schemes, and clinical diagnosis and treatment of toxic neuropathies. In this commentary we will review the evidence for the dose-related dependency of distal axonopathies and discuss how this concept might influence our current understanding of chemical-induced neurotoxicities. © 2000 Academic Press

Exposure of humans and laboratory animals to numerous compounds used in the pharmaceutical, chemical, and agricultural industries (Table 1) produces nerve damage traditionally classified as a distal axonopathy (Griffin, 1992). The primary morphologic characteristic of this neuropathy is distal retrograde degeneration of central and peripheral nerve axons (Berger and Schaumburg, 1995). Degeneration is often preceded by multifocal paranodal giant axonal swellings (Spencer 1 To whom correspondence should be addressed at Montefiore Medical Center, Anesthesia Research–Moses 7, 111 E. 210th St., Bronx, NY 104672490. Fax: (718) 515-4903; E-mail: [email protected].

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TABLE 1 Pharmaceutical, Industrial, and Agricultural Chemicals that Induce Distal Axonopathies Acrylamide n-Hexane p-Bromophenylacetylurea Zinc pyridinethione Triorthocresylphosphate

Isoniazid Carbon disulfide Disulfiram Leptophos 2,5-Hexanedione

neuropathy following subchronic occupational exposure (Spencer and Schaumburg, 1980; Spencer et al., 1980). ACR and HD have been the focus for a majority of previous studies investigating biochemical mechanisms of toxic distal axonopathies. Design of this research was based on the assumption that, irrespective of route or dosing pattern, a given neurotoxicant acted via a single mechanism and that intoxication produced classic morphologic indices of axonopathy (e.g., see hypothesis proposed by LoPachin and Lehning, 1994 and associated supporting research). It was also assumed that the distal axon was the site of neurotoxicant action and that axonopathy was the pathognomonic manifestation of a corresponding primary mechanism (Spencer and Schaumburg, 1976; Lowndes and Baker, 1980). Nerve injury in the form of axon degeneration and/or swelling was presumably responsible for ensuing neurological deficits. Accordingly, over the past two decades, substantial research effort has been devoted to deciphering biochemical mechanisms of axonal swelling and degeneration. For example, we proposed that the primary mechanism of ACR was inhibition of axolemmal Na ⫹/K ⫹– ATPase activity and secondary toxic Ca 2⫹ entry via reverse Na ⫹–Ca 2⫹ exchange. Resulting intraaxonal Ca 2⫹ accumulation mediated secondary axon swelling and degeneration (see LoPachin and Lehning, 1994, 1997b for details). With respect to ␥-diketone-induced giant axonal swellings, substantial research suggested direct chemical modification of neurofilament (NF) cytoskeletal proteins with resulting paranodal accumulation (DeCaprio, 1985; Graham et al., 1995). However, a growing body of evidence now indicates that the traditional morphologic features of distal axonopathy are expressed differentially depending upon rate of neurotoxicant exposure (e.g., g toxicant/kg body wt/day). Thus, we (Lehning et al., 1998) and others (e.g., Crofton et al., 1996) have found that ACR-induced peripheral axon degeneration occurs primarily during subchronic induction of neurotoxicity with lower daily doses, whereas higher dosing rates produce subacute onset of deficits in the absence of degeneration. The presumed hallmark of ACR axonopathy (i.e., axon degeneration) is, therefore, a conditional response associated with subchronic, lower dose intoxication. Moreover, the role of axon degeneration in ACR neurotoxicity becomes less clear when one considers the fact that classic neurobehavioral deficits (e.g., hindlimb weakness, foot splay, gait abnormalities) can develop in its absence. The

possibility that rate of intoxication determines axonopathic characteristics and the emerging lack of pathophysiological clarity contradict accepted principles and definitions of toxic axonopathies. Therefore, the purpose of this commentary is to describe the influence of dosing rate on neuropathic manifestations of axonopathies and to discuss how resulting new information might impact our understanding of biochemical mechanisms and sites of neurotoxicant action. We will also discuss the implications of exposure-dependent axonopathic expression on design of mechanism-based research and on clinical evaluation and detection of chemical intoxication. In the following sections we will focus on research related to the prototypical chemicals ACR and HD. ACRYLAMIDE-INDUCED DISTAL AXONOPATHY

Over the past 25 years, substantial hypothesis development and corresponding research efforts have been directed toward elucidating the mechanism of ACR-induced axon degeneration. Notable hypotheses have included inhibition of axonal energy production; changes in the rate, quantity, and deposition of anterograde transported materials; and disruption of cytoskeletal structure and function (see review by LoPachin and Lehning, 1994). Based on the role of transmembrane ion shifts in cell injury (Trump et al., 1979), we surmised that ACR might cause distal axonopathy by disrupting subaxonal distribution of Ca 2⫹, K ⫹, and Na ⫹ (Lehning et al., 1998; LoPachin et al., 1992, 1993). In supporting studies, electron probe x-ray microanalysis (EPMA) was used to measure percent water and elemental concentrations of Na, K, P, Cl, Mg, and Ca in axon ultrastructural compartments (e.g., axoplasm, mitochondria) of rat peripheral nerve cryosections (for methodological details see LoPachin and Gaughan, 1999). Previous investigations of ACR and other chemicals (e.g., HD, carbon disulfide) employed different routes of administration and dosing schedules based on the assumption that a neurotoxicant acted by a single mechanism irrespective of dosing paradigm (see reviews by Spencer and Schaumburg, 1974a; LeQuesne, 1980; Lowndes and Baker, 1980; LoPachin and Lehning, 1994). As a corollary, we (LoPachin et al., 1992) suggested that a mechanistically relevant effect would be expressed regardless of neurotoxicant route or dosing rate, whereas nonspecific events would be manifest on a conditional basis and would develop independent of neurotoxicity. Accordingly, rats were exposed to ACR in drinking water (2.8 mM ⫻ 30 days) or were injected ip (50 mg/kg/day ⫻ 10 days) and subaxonal elemental concentrations and distributions were measured by EPMA (LoPachin et al., 1992, 1993). Findings from this initial study provided the first indication that the axonopathic effects of ACR might be routedependent; i.e., swollen and degenerating axons were relatively rare consequences of subacute ip ACR intoxication. Nonetheless, swollen tibial axons from animals exposed to ACR by either route exhibited a complete derangement of elemental

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distribution and water content. This pattern of axoplasmic elemental derangement was similar to that associated with in situ peripheral nerve transection, ischemia, and in vitro exposure of nerves to anoxia and ouabain (LoPachin and Lehning, 1997a). A common mechanistic theme for these neuropathic conditions has been reduction of axolemmal Na ⫹/K ⫹–ATPase activity and we therefore proposed that ACR-induced loss of ion homeostasis and osmoregulation involved reduced Na ⫹ pump activity in distal axons (LoPachin and Lehning, 1994; 1997a). Subsequently, we examined the effects of ACR intoxication on enzymatic Na ⫹ pump activity in rat peripheral nerve. Animals were exposed to ACR by either the oral (2.8 mM ⫻ 30 days) or ip (50 mg/kg/day ⫻ 10 days) route and enzyme activity was measured in homogenates of whole tibial nerve (Lehning et al., 1994). These routes and corresponding doses have been used in earlier research concerning ACR axonopathy (e.g., Burek et al., 1980; O’Shaughnessy and Losos, 1986; Tanii and Hashimoto, 1983). Although both dosing protocols produced moderate levels of behavioral neurotoxicity (e.g., hindlimb muscle weakness, foot splay, gait abnormalities), subchronic oral administration of ACR caused a significant reduction in Na ⫹/K ⫹–ATPase activity, whereas no change in enzyme function was observed in the subacute ip treatment group (Fig. 1). Despite indications of a route-related enzymatic effect, we rationalized that the use of whole-nerve homogenates containing mostly unaffected Schwann cell Na ⫹ pump isotype might have precluded detection of smaller axonal changes in the subacute ip treatment group. To detect possible differential effects of ACR on Na ⫹/K ⫹– ATPase isotypes, we used EPMA to measure Rb ⫹ transport in individual myelinated axons and Schwann cells (Lehning et al., 1997a,1998). Rb ⫹ is a K ⫹ tracer that has been used to measure Na ⫹/K ⫹–ATPase activity in nervous tissue and can be quantitated by EPMA (see discussion in Lehning et al., 1997a). In tibial nerve segments isolated from moderately affected rats exposed to oral ACR (2.8 mM ⫻ 34 days), we found reduced axolemmal Na ⫹ pump activity and early signs of axon degeneration (Lehning et al., 1998). Kinetic analysis revealed that, although apparent K m was not affected (i.e., K m control ⫽ 4.5 mM vs K m ACR ⫽ 4.3 mM), apparent V max was decreased significantly (i.e., V max control ⫽ 14.3 mmol Rb/kg dry wt/min vs V max ACR ⫽ 4.6 mmol Rb/kg dry wt/min). As oral ACR intoxication continued (49 days, severe toxicity), complete inhibition of Rb ⫹ uptake and axonal accumulation was evident in conjunction with widespread frank axon degeneration (Lehning et al., 1998). In contrast, nerves from moderate-toseverely affected ip-treated animals (50 mg ACR/kg/day ⫻ 11 days) exhibited neither enzymatic nor structural alterations; i.e., Rb ⫹ transport was not affected and swollen or degenerating fibers were not evident. Thus, these results confirmed our earlier enzyme studies (Lehning et al., 1994) and demonstrated that, although both neurotoxicant treatment paradigms produced comparable neurobehavioral deficits, subchronic oral

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FIG. 1. Effects of ip (50 mg/kg/day ⫻ 10 days) or oral (2.8 mM in drinking water ⫻ 34 days) ACR exposure on Na ⫹/K ⫹–ATPase (A) and Mg 2⫹–ATPase (B) activities in rat whole tibial nerve homogenates. These exposure paradigms produced subacute or subchronic onset of moderate behavioral neurotoxicity, respectively (see Lehning et al., 1994 for details). Data are expressed as mean enzyme activity ⫾ SEM. Number within bar represents statistically significant (p ⬍ 0.05) percent difference in oral vs control enzyme activities.

intoxication was associated with defective Na ⫹/K ⫹–ATPase function and classic morphologic indices of distal axonopathy whereas peripheral nerves from the subacute exposure group were devoid of axonopathic and enzymatic changes. The finding that classic distal axonopathy developed only when ACR was administered on a subchronic basis suggested that corresponding morphologic characteristics and underlying mechanisms were neither necessary nor sufficient pathophysiological events. This is clearly contrary to historical perceptions of ACR as a prototypical chemical that produces neurotoxicity mediated by distal axonopathy (Spencer and Schaumburg, 1974a,b; LeQuesne, 1980; LoPachin and Lehning, 1994). Because morphologic observations were made during microprobe analysis of unfixed, unstained tibial nerve cryosections with relatively low spatial resolution, we conducted a comprehensive longitudinal study using quantitative morphometric analyses of conventionally fixed tibial nerve from oral and ip ACR-treated rats (Fig. 2; Lehning et al., 1998). Oral ACR exposure (2.8 mM in drinking water ⫻ 15, 26, 34, and 49 days) produced subchronic, progressive neu-

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FIG. 2. Posterior tibial nerve cross sections are presented from a control rat (A) and from rats intoxicated with ACR by oral ingestion (B; 2.8 mM in drinking water ⫻ 34 days) or by ip injection (C; 50 mg/kg/ day ⫻ 11 days). The oral and ip treatment schedules produced moderate and moderate-to-severe neurotoxicity, respectively (see Lehning et al., 1998 for details). Note that in nerves from orally intoxicated rats (B) abundant atrophied fibers are clearly evident in addition to numerous degenerating profiles, whereas the nerve section from ip-treated rats exhibits few morphologic changes (C). Both treatment schedules produced only rare swollen axons (*, C). Toluidine blue-safranin stain. Original magnification 750⫻.

robehavioral toxicity and typical axonopathic changes; i.e., axon atrophy, swelling, and degeneration (Figs. 2B and 3A). These findings confirmed earlier qualitative impressions (LoPachin et al., 1992, 1993; Lehning et al., 1998) and further implicated a causal relationship between axon degeneration and K ⫹ (Rb ⫹) transport deficits (see below). In contrast, during ip ACR intoxication (50 mg/kg/day ⫻ 5, 8, and 11 days), behavioral neurotoxicity developed but was not associated with alterations in tibial axon morphology (Figs. 2C and 3B). This finding is supported by several previously published studies that also reported an absence of axonopathy following subacute ACR intoxication (Abou-Donia et al., 1993; O’Shaughnessy and Losos, 1986; Yoshimura et al., 1992). It is possible that during subacute intoxication axon degeneration is manifest in other nervous tissue regions. However, a survey of PNS (proximal and distal sciatic nerve, distal sural nerve) and CNS (dorsal spinal column) areas from ip-intoxicated rats did not reveal axonopathic changes (Crofton et al., 1996; Lehning et al., 1998, unpublished data). Alternatively, it is possible that degeneration is a late event that did not develop within our ip treatment paradigm (i.e., 5–11 days) but would have occurred if exposure had continued (i.e., ⬎11 days). It is important to note, however, that the longest exposure group (i.e., 11 days) included severely affected animals exhibiting hindlimb paralysis. Morphologic assessment of these rats indicated no neuropathic changes and, therefore, late or delayed onset degeneration during subacute intoxication is doubtful. Considered together, the above findings suggested that subchronic oral ACR dosing schedules produced behavioral neurotoxicity associated with classically defined distal axonopathy. In contrast, high-dose ACR exposure via ip administration caused subacute neurotoxicity in the absence of axon morphologic changes. Crofton et al. (1996) showed that axon swelling and degeneration in PNS and CNS were not related to expression of neurotoxicity but were instead a function of daily ip dosing rate: i.e., low-dose subchronic ACR exposure produced axonopathy, whereas axonal changes were not associated with the subacute neurotoxicity produced by high-dose ip administration. Our documentation of the route-dependent nature of ACR axonopathy is in agreement with the dose-rate effects reported by Crofton et al. (1996). In fact, pharmacokinetic analyses of plasma ACR/metabolite levels (Barber et al., 2000) show that ip and oral dosing represent high and low daily exposure rates, respectively. Thus, the neuropathic outcome of ACR intoxication is determined by daily dosing rate, which has significant implications for corresponding neurotoxicologic relevance. Because ACR behavioral neurotoxicity (e.g., gait abnormalities, hindlimb weakness) can develop fully in the absence of axon swelling and degeneration, these morphologic features and their underlying mechanisms are considered epiphenomena exclusively related to subchronic dosing rates. Our studies suggest that axon swelling and degeneration are mediated by a reduction in axolemmal Na ⫹/K ⫹–ATPase activity

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FIG. 3. Frequency distribution of myelinated axon area (␮m 2) in posterior tibial nerve from rats exposed to ACR by oral (A; 2.8 mM in drinking water ⫻ 34 days) or ip (B; 50 mg/kg/day ⫻ 11 days) administration. These treatment schedules produced moderate and moderate-to-severe neurotoxicity, respectively (see Lehning et al., 1998 for details). Numbers in parenthesis are mean axon area ⫾ SEM. *Statistically different (p ⬍ 0.05) from corresponding control. Sample size is approximately 3000 axons for pooled age-matched controls and 1200 –1600 axons for the treated groups. Axon atrophy associated with oral ACR intoxication is indicated by leftward shift in axon area distribution and corresponding statistically significant decrease in mean area.

that leads to axoplasmic Na ⫹ elevation and subsequent Ca 2⫹ influx via reverse Na ⫹–Ca 2⫹ exchange. Ca 2⫹ influx eventually overwhelms axonal buffering mechanisms and initiates an autodestructive sequence that culminates in distal axon degeneration (LoPachin et al., 1989, 1990; Lehning et al., 1995a; 1996; see also LoPachin and Lehning, 1994, 1997a; Stys, 1998). However, we suggest this degenerative scenario is secondary and in effect only during protracted, long-term treat-

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ment paradigms involving low daily doses of ACR. In the ip model, our studies found no evidence of axonopathy in any region of the peripheral axis. Although normal in appearance, axons from subacutely intoxicated rats might be irreversibly injured and in a predegenerative, dysfunctional state. However, when animals were at a moderate-to-severely affected level, axolemmal Na ⫹/K ⫹–ATPase activity and subaxonal K ⫹ (Rb ⫹) regulation were both similar to control, as were transmembrane mitochondrial ion gradients and aerobic energy metabolism (Lehning et al., 1998; see also Medrano and LoPachin, 1989; Sickles et al., 1990). These axonal processes are highly vulnerable to metabolic, biochemical, and membrane disruptions and are, therefore, considered sensitive indices of injury (Lees, 1991; LoPachin and Lehning, 1997a). Our combined data (see also Lehning et al., 1997b) suggest that distal axons from tibial nerve of ip-intoxicated rats are structurally and functionally intact, which indicates an absence of injury. What does our observation of differential axonopathic expression mean with respect to primary mechanisms and sites of ACR action? The fact that ACR-induced neurobehavioral toxicity can be manifest in the absence of apparent axon injury suggests that axons are not a primary, neurotoxicologically relevant site of action. If this is the case, what anatomical or functional substrate mediates neurotoxicity? A major area of investigation has been the effects of ACR on fast anterograde axonal transport. Supporting studies have shown reductions in rate and quantity of material conveyed by fast transport during ACR intoxication (e.g., Bradley and Williams, 1973; Harris and Gulati, 1994; Sickles, 1989; Sumner et al., 1976). However, other studies have failed to demonstrate a direct effect of ACR on either transport or corresponding molecular components (e.g., Brat and Brimijoin, 1993; Harry, 1992; Martenson et al., 1995; Sidenius and Jakobsen, 1983). Moreover, our research suggests distal peripheral axons in ip-intoxicated animals are structurally and functionally normal, which implies adequate transport and delivery of material. Alternatively, direct nerve cell body compromise might be responsible for distal axon degeneration (Cavanagh, 1964). Indeed, early morphologic studies suggested sensory and motor cell body remodeling (e.g., eccentric nuclei, changes in Nissl and mitochondrial volume) might be a direct neuropathogenic effect of ACR (Jones and Cavanagh, 1986; Sterman, 1982). However, other morphometric and functional evidence indicated that observed changes represent a repair response (e.g., Bisby and Redshaw, 1987; Gold et al., 1991, 1992; Sterman, 1982; 1984; Sterman and Sposito, 1985). Furthermore, if perikaryal function was significantly compromised, secondary distal axon damage should have occurred during subacute exposure (Crofton et al., 1996; Lehning et al., 1998). Since neither axons nor cell bodies appear to be injured in rats expressing obvious clinical neurotoxicity, the principal neuroanatomical site of ACR action might be elsewhere. We are currently investigating the possi-

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bility that the primary mechanism of ACR neurotoxicity involves nerve terminal dysfunction or axonal– glial uncoupling. ␥-DIKETONE PERIPHERAL NEUROPATHY

␥-Diketone neuropathy is classified as a distal axonopathy and is characterized by giant axonal swellings in CNS and PNS (Spencer and Schaumburg, 1980; Spencer et al., 1980). These swellings contain NF masses, which has prompted the hypothesis that this axonopathy is caused by direct ␥-diketone chemical modification of neurofilaments. ␥-Diketone pyrrolation of ⑀-amino groups on NF lysyl residues and secondary autooxidation of pyrrole rings lead to formation of covalent NF–NF crosslinks (see reviews by Graham et al., 1995; LoPachin and Lehning, 1997b). Neurofilaments are thought to undergo chemical modification as they progress along the axonal axis and, eventually, crosslinked NFs accumulate at narrow distal nodes of Ranvier where their proximodistal movement is impeded. Development of swelling presumably initiates axonal degeneration and subsequent behavioral deficits (e.g., gait abnormalities, hindlimb weakness). During x-ray microanalysis of frozen peripheral nerve sections (proximal sciatic, distal tibial) from 2,5-HD-intoxicated rats, qualitative morphologic observations suggested that giant axonal swellings were primarily a product of subchronic oral exposure. In contrast, axon atrophy was a prominent morphologic response regardless of the dosing route; i.e., oral vs ip (LoPachin et al., 1994a). These findings disagreed with the historical view of HD axonopathy; i.e., distal axonal swellings are a hallmark characteristic and are a direct consequence of the primary biochemical mechanism of action (Graham et al., 1995; LoPachin and Lehning, 1997b; Spencer and Schaumburg, 1980; Spencer et al., 1980). To confirm these qualitative observations, an initial quantitative morphometric analysis was conducted to characterize spatiotemporal expression of axonal swelling, atrophy, and degeneration in conventionally fixed peripheral nerves of HDintoxicated rats (Lehning et al., 1995b). For these studies, HD was delivered by either oral (0.4% in drinking water) or ip (400 mg/kg/day) administration and morphologic changes were quantitated in proximal sciatic and distal posterior tibial nerves (see Lehning et al., 1995b for details). These doses and routes of intoxication were selected based on their use in previous investigations of diketone neuropathy (e.g., Anthony et al., 1983; Spencer and Schaumburg, 1977; Monaco et al., 1989a,b). Results showed that oral intoxication produced subchronic development of neurotoxicity associated with axonal swelling and abundant atrophy. In contrast, the predominant neuropathic change produced by subacute ip exposure was axonal atrophy with rare swelling. However, neither route produced notable axon degeneration as had been reported previously (e.g., Spencer and Schaumburg, 1980; Spencer et al., 1980). These early morphometric studies suggested that, irrespective of route or length of exposure, axon atrophy was the

predominant neuropathic effect of ␥-diketone intoxication, whereas swelling was a conditional response and consequently of unclear pathophysiological importance (Lehning et al., 1995b; LoPachin and Lehning, 1997b). To investigate our anomolous findings in greater detail, we conducted a comprehensive quantitative morphometric analysis of HD-induced peripheral axonopathy (Figs. 4 and 5; Lehning et al., 2000). Because daily dosing rate appears to be a primary determinant of axonopathic expression (Crofton et al., 1996; Lehning et al., 1998) and because the impact of dose rate could not be assessed using different routes of administration (i.e., oral vs ip; Lehning et al., 1995b), a single route (gavage) was used to intoxicate rats with HD at four different exposure rates (100, 175, 250, and 400 mg/kg/day). Morphometric parameters were quantitated at several sites along the peripheral nerve axis (5th lumbar spinal nerve to tibial nerve) and at multiple behavioral endpoints (unaffected to severely affected). Our findings (Lehning et al., 2000) indicated that dosing rates of 100 –250 mg/kg/day produced relatively slow onset neurotoxicity and giant axonal swellings in sciatic and tibial regions (Fig. 5A). However, swollen axons were infrequent (⬍3% of the fiber population examined) and neither the number nor corresponding size changed as a function of developing neurotoxicity. The 400 mg/kg/day dose produced rapid onset behavioral toxicity (12 days) but did not cause axon swelling at any peripheral nerve level examined (Fig. 5B). These data are consistent with our earlier findings (Lehning et al., 1995b) where subchronic oral treatment exclusively produced a small population of swollen axons that appeared early during intoxication and then decreased in both size and number as HD exposure continued. In contrast to the conditional expression of swellings, axon atrophy was the prevalent neuropathologic feature irrespective of dosing rate (100 – 400 mg/kg/day). Atrophy was widespread among axon size classes (i.e., small, medium, and large diameter fibers) and appeared initially in distal nerve regions and then moved in a retrograde direction as intoxication continued. Moreover, the development of atrophy in distal nerve segments preceded the onset of behavioral deficits and temporally correlated with changes in peripheral nerve conduction velocity and latency (Lehning et al., 2000). Axon degeneration was noted only in tibial nerve as a late-onset effect (i.e., 307 days) of the lowest dosing rate (100 mg/kg/day). Considered together, our data suggest that, like ACR, dose rate significantly influences the morphologic characteristics of ␥-diketone axonopathy. Thus, long-term exposure to low daily dosing; i.e., 0.4% HD in drinking water or 100 –250 mg/kg/day via gavage, is a critical factor for expression of HD-induced axon swellings (Lehning et al., 1995b, 2000). A similar observation was made by Krasavage et al. (1980). These investigators reported that the frequency of axonal swellings induced by a series of hexacarbon congeners (e.g., n-hexane, HD, 5-hydroxy-2-hexanone, 2,5-hexanediol) was correlated with length of oral exposure and inversely related to respective serum HD

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concentrations or to neurotoxic potencies. For example, a 6.6 mmol/kg/day daily gavage dose of HD achieved a mean peak serum concentration of 569 ⫾ 98 ␮g/ml and required 17 ⫾ 1 days to reach hindlimb weakness. This protocol produced few tibial nerve swellings. In contrast, an equimolar gavage dose of the less potent parent hexacarbon methyl n-butyl ketone was associated with a serum HD concentration of 111 ⫾ 12 ␮g/ml and required 55 ⫾ 4 days to reach the same endpoint. However, this subchronic protocol produced abundant axonal swellings (see Table 1 from Krasavage et al., 1980). Length of intoxication and not hexacarbon potency therefore appears to be the primary determinant of axon swelling in ␥-diketonetreated animals. Based on their findings, the authors concluded that “axonal swelling may not be a prerequisite for axonal dysfunction and is possibly a secondary phenomenon.” Our results showed that neurotoxicity can develop in the absence of swelling (i.e., during 400 mg/kg/day ip or gavage treatment) and, when expressed during subchronic HD exposure, the frequency and magnitude of swellings either do not change or decrease despite advancing neurotoxicity. These attributes are not consistent with a pathophysiologically relevant parameter. Accordingly, we have suggested that axonal swellings associated with ␥-diketone neuropathy are a nonspecific consequence of subchronic exposure and are not a morphologic representation of the principal biochemical mechanism (LoPachin and Lehning, 1997b). The mechanism of axon swelling is not understood, although recent research in our laboratory suggests that direct chemical modification of neurofilaments is not involved as has been suggested (Chiu et al., 2000). Since neurofilamentous axonal swellings can occur in association with a number of neuropathic conditions (see Glass and Griffin, 1991; Maxwell et al., 1997; Ochs et al., 1997; Smith et al., 1999), HD-induced swellings might be a generalized neuronal stress response. Regardless of the HD dosing schedule (subchronic, subacute) or route (gavage, ip, drinking water), atrophy was the predominant morphologic response of peripheral axons to intoxication. The atrophy associated with subacute HD was restricted to distal sciatic and tibial nerve regions, which suggests atrophy might develop initially in distal axon regions and then move in a proximal direction as exposure continues. Our studies were not the first to show that axonal atrophy accompanied HD intoxication. Early research examining hexacarbon neuropathy recognized atrophy as a significant morphologic component (Brown et al., 1978; Spencer and Schaumburg,

FIG. 4. Posterior tibial nerve cross sections are presented from a control rat (A) and from rats intoxicated with HD by gavage administration at either a lower (B; 175 mg/kg/day ⫻ 91 days) or higher (C; 400 mg/kg/day ⫻ 22

days) daily dosing rate. Both treatment schedules produced moderate levels of neurotoxicity (see Lehning et al., 2000 for details). Note that in nerves from subchronically exposed low-dose-rate rats (B; 175 mg/kg/day) occasional swollen axons are evident (arrows) among abundant atrophied fibers (arrowheads), whereas the nerve section from subacutely exposed higher dose-rate rats exhibits primarily axon atrophy (C; 400 mg/kg/day). Toluidine bluesafranin stain. Original magnification 475⫻.

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been suggested that atrophy is a secondary consequence of swelling due to diminished downflow of NFs (Brown et al., 1978; Spencer et al., 1980; Monaco et al., 1989a,b). However, our studies show atrophy can evolve independent of axonal swelling; i.e., ip intoxication produces atrophy in the absence of swelling (Fig. 5). This indicates that these morphologic phenomena are independent and mechanistically distinct. In addition, loss of axon caliber occurs commensurate with the development of neurotoxicity, appears to mediate peripheral nerve conduction deficits, and is expressed regardless of rate or route of intoxication. These characteristics suggest that axonal atrophy is a mechanistically essential and specific component of ␥-diketone axonopathy. Our combined data show that HD neuropathy is characterized predominantly by axon atrophy whereas swelling and degeneration appear to be conditionally expressed. We are currently investigating the possibility that atrophy produced by ␥-diketones does not involve direct chemical interactions at axonal sites but rather occurs via a secondary mechanism possibly involving disrupted target-derived trophic signaling (Gold and Spencer, 1993). Thus, an awareness of how dose rate influences neuropathic expression has helped redefine ␥-diketone neuropathy and has suggested new areas of mechanistic exploration. CONCLUSIONS

FIG. 5. Frequency distribution of myelinated axon area (␮m 2) in posterior tibial nerve from rats intoxicated with HD by gavage administration at either a lower (A; 175 mg/kg/day ⫻ 91 days) or higher (B; 400 mg/kg/day ⫻ 22 days) daily dosing rate. Both treatment schedules produced moderate levels of neurotoxicity (see Lehning et al., 2000 for details). Numbers in parenthesis are mean axon area ⫾ SEM. *Statistically different (p ⬍ 0.05) than corresponding control. Sample size is approximately 5000 axons for pooled age-matched controls and approximately1600 axons for the treated groups. Axon atrophy associated with both the lower (A; 175 mg/kg/day) and higher (B; 400 mg/kg/day) daily dosing rates is indicated by leftward shifts in axon area distributions relative to control dispersion and corresponding statistically significant decreases in mean areas. Swollen axons expressed only during lowdose-rate exposure (A; 175 mg/kg/day) are indicated by the rightward dispersion of axon areas that extend beyond corresponding control distributions.

1980; Spencer et al., 1980). More recent papers have reported loss of axon caliber in CNS white matter tracts (Monaco et al., 1989a; Pyle et al., 1992; Yagi, 1994) and peripheral nerves (Lehning et al., 1995b; LoPachin et al., 1994a; Monaco et al., 1988; Rosenberg et al., 1987) of HD-intoxicated rats. It has

ACR and HD are considered to be prototypical among chemicals that produce a distal axonopathy characterized by axon swelling and/or degeneration. However, research has shown that classically defined axonopathy is expressed only during subchronic, low-dose exposure paradigms (Table 2). Higher dosing rates produce either no structural changes (ACR) or retrograde atrophy (HD; Table 2). Thus, ataxia, muscle weakness, and other indices of neurotoxicity manifest irrespective of dosing rate, whereas axonal swelling and/or degeneration are dependent upon rate of intoxication. We therefore suggest these neuropathic attributes are epiphenomena related to protracted, low-dose exposure schedules. This proposal has significant conceptual, experimental, and clinical implications. Clearly, axonal swelling, degeneration, or any morphologic change induced by a given neurotoxicant cannot a priori be considered pathognomonic or neurotoxicologically relevant. Research is needed to define morphologic expression of the axonopathy as it relates to dosing rate and to identify possible functional consequences; e.g., nerve conduction abnormalities, disrupted neurotransmission. The definition of distal axonopathy should be modified to incorporate the concept that axonopathic characteristics manifest as a function of dosing rate. Accordingly, the classification scheme for toxic chemicals (see Berger and Schaumburg, 1995) also will need revision. The axonopathic characteristics suggested an axonal site of action, which has been the driving presumption of the past three decades of research. However, the finding that toxicants

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TABLE 2 Influence of Neurotoxicant Dose Rate on Morphologic, Molecular, Behavioral, and Electrophysiologic Parameters Parameter Acrylamide Behavioral a Morphometric a

Molecular b

2,5-Hexanedione Behavioral c Morphometric d

Molecular e Electrophysiologic f

Lower daily dose rates

Higher daily dose rates

Subchronic neurotoxicity Widespread axon degeneration Axon atrophy Axon swelling Reduced Na ⫹/K ⫹–ATPase activity Reduced axonal Rb ⫹ transport Deranged subaxonal ion content

Subacute neurotoxicity No axon degeneration No axon atrophy Rare axon swelling Normal enzyme function Normal Rb ⫹ transport Normal ion distribution

Subchronic neurotoxicity Rare axon degeneration Retrograde axon atrophy Giant axon swellings Decreased axonal K ⫹ and water Schwann cell ion derangement Decreased motor amplitude Decreased nerve conduction Increased motor latency

Subacute neurotoxicity No axon degeneration Retrograde axon atrophy Rare axon swelling Decreased axonal K ⫹ and water Normal Schwann cell ion content Normal amplitude Decreased nerve conduction Increased motor latency

a

Behavioral and morphometric data (rat peripheral nerve axis) from Lehning et al. (1998). Molecular results from LoPachin et al. (1992) (EPMA of rat tibial nerve), Lehning et al. (1994) (enzyme analysis of tibial nerve Na ⫹/K ⫹–ATPase activity), and Lehning et al. (1998) (EPMA of Rb ⫹ transport in rat tibial nerve). c Behavioral data from Lehning et al. (1995, 2000). d Morphometric data from Lehning et al. (1994, 2000) (analyses of rat peripheral nerve axis). e Molecular results from LoPachin et al. (1994a,b) (EPMA of rat tibial nerve). f Electrophysiologic data from Lehning et al. (2000) (analyses of rat tibial, caudal, and sural nerves). b

such as ACR can produce neurotoxicity without axonopathy suggests that nonaxonal sites of action such as nerve terminals, cell bodies, or glial cells might be relevant and should be considered in future studies of this and other chemicals. Identifying primary sites and mechanisms of neurotoxicant action is critically important for developing efficacious pharmacotherapies for toxic axonopathies. Thus, pharmacological intervention directed toward axon degeneration would be of unclear benefit to ACR intoxication, since research has shown that neurotoxicity can proceed without fiber loss. Finally, clinical diagnosis of toxic exposure is dependent upon classification of the neuropathy according to analysis of electrophysiologic deficits and nerve biopsies with assessment of morphologic features. An awareness of how exposure rate can influence morphologic axonopathic characteristics is necessary for accurate diagnosis; i.e., axon degeneration or swelling cannot be viewed as absolute diagnostic criterion for neurotoxicant exposure. The finding that certain morphologic and functional defects can be variably expressed could indicate that a given toxicant produces neurotoxicity through multiple dose-dependent pathways. However, our observation that these defects are selectively linked to low-dose subchronic exposure schedules and not to neurotoxicity (Table 2) suggests that length of exposure is the critical determinant of corresponding expression. This

temporal dependency and lack of correlation with neurotoxicity suggests these changes are nonspecific in nature. Therefore, we favor the scenario that neurotoxicants have a single, primary mechanism that mediates neurotoxicity regardless of dosing rate; e.g., ACR inhibition of neurotransmission, ␥-diketone induction of axon atrophy, and dysfunction via interruption of neurotrophic signaling (see above). During subchronic, low-dose exposure schedules, secondary mechanisms and their effects develop; e.g., ACR inhibition of Na ⫹ pump activity followed by axon degeneration; axon swelling via ␥-diketone neurofilament crosslinking. It is perhaps counterintuitive that low neurotoxicant doses should be associated with such dramatic axonopathic changes as degeneration and swelling. There are several potential explanations for these apparent time-dependent phenomena. It is possible that subchronic effects are produced by differential conversion of protoxicant to an active metabolite that subsequently mediates damage. Consistent with this possibility, research has shown that a greater percentage of administered ACR is converted to its epoxide metabolite glycidamide when rats are intoxicated with lower (5 mg/kg/day ip), as opposed to higher (50 mg/kg/day ip), dosing rates (see Barber et al., 2000; Bergmark et al., 1991). Glycidamide has been associated with induction of axon degeneration (Abou-Donia et al., 1993) and, therefore, might be responsible for fiber loss at lower ACR dosing rates. Alternatively, relative

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vulnerability of certain nerve cells or neurophysiologic processes could determine rate of pathophysiologic development. Thus, nerve cells highly vulnerable to ACR (e.g., Purkinje fibers; Cavanagh and Gysbers, 1983) would succumb rapidly to exposure at any dose rate, whereas more resistant cells, possibly peripheral nerves, would require more prolonged neurotoxicant exposure. Vulnerability or the ability to resist toxic injury is likely determined by multiple cellular attributes, including target density, energy production, xenobiotic metabolism, and inherent repair/regeneration capacity (Reuhl and Lowndes, 1992). Regardless, axon degeneration, swelling, and other time-dependent events cannot be considered of primary pathophysiologic importance since neurotoxicity can be manifest in their absence; i.e., during subacute high-dose intoxication. If these time-dependent events are truly epiphenomenon, then primary mechanisms of neurotoxicity cannot be discerned if the experimental design is based on traditional concepts of toxic axonopathies. Rather, investigators should consider dose rate a guiding principle for deciphering primary vs secondary pathophysiological events. ACKNOWLEDGMENT

Brown, M. J., DeJesus, P. V., Pleasure, D. E., and Asbury, A. K. (1978). Nerve conduction slowing precedes demyelination in experimental n-butyl ketone (MBK) neuropathy. In Proceedings of the IVth International Congress on Neuromuscular Disease (A. Aguayo and G. Kanpati, Eds.), pp. 276 –301. Excerpta Medica, Amsterdam. Burek, J. D., Albee, R. R., Beyer, J. E., Bell, T. J., Carreon, R. M., Morden, D. C., Wade, C. E., Hermann, E. A., and Gorzinski, S. J. (1980). Subchronic toxicity of acrylamide administered to rats in drinking water followed by up to 144 days of recovery. J. Environ. Pathol. Toxicol. 4, 157–182. Cavanagh, J. B. (1964). The significance of the “dying-back” process in experimental and human neurological disease. Int. Rev. Exp. Pathol. 3, 219 –267. Cavanagh, J. B., and Gysbers, M. F. (1983). Ultrastructural features of the Purkinje cell damage caused by acrylamide in the rat: A new phenomenon in cellular neurophysiology. J. Neurocytol. 12, 413– 437. Chiu, F. C., Opanashuk, L. A., He, D. K., Lehning, E. J., and LoPachin, R. M. (2000). ␥-Diketone peripheral neuropathy. II. Neurofilament subunit content. Toxicol. Appl. Pharmacol. 165, 127–140. Couri, D., and Milks, M. (1982). Toxicity and the metabolism of the neurotoxic hexacarbons n-hexane, 2-hexanone and 2,5-hexanedione. Annu. Rev. Pharmacol. Toxicol. 22, 145–166. Crofton, K. M., Padilla, S., Tilson, H. A., Anthony, D. C., Raymer, J. H., and MacPhail, R. C. (1996). The impact of dose rate on the neurotoxicity of acrylamide: The interaction of administered dose, target tissue concentrations, tissue damage, and functional effects. Toxicol. Appl. Pharmacol. 139, 163–176.

The authors’ research described in this review was supported by NIH Grants RO1 ES07912 and ES03830 from NIEHS to R.M.L.

DeCaprio, A. P. (1985). Molecular mechanism of diketone neurotoxicity. Chem.–Biol. Interact. 54, 257–270.

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