Structural basis of γ-diketone neurotoxicity: Non-neurotoxicity of 3,3-dimethyl-2,5-hexanedione, a γ-diketone incapable of pyrrole formation

TOXICOLOGYANDAPPLIEDPHARMACOLOGY

84,36-44(1986)

Structural Basis of y-Diketone Neurotoxicity: Non-neurotoxicity of 3,3DimethyL2,5hexanedione, a y-Diketone Incapable of Pyrrole Formation’ L. M. SAYRE,*T' C. M. SHEARSON,* T.~ONGMONGKOLRIT,~ R. MEDORI,? ANDP. GAMBETTI~ *Department

of Chemistry, Case Western Received

and tDivision of Neuropathology, Reserve University, Cleveland, July 8, 1985; accepted

December

Institute of Pathology, Ohio 44106 17, 1985

Structural Basis of y-Diketone Neurotoxicity: Non-neurotoxicity of 3,3-Dimethyl-2,5-hexanedione, a y-Diketone Incapable of Pyrrole Formation. SAYRE, L. M., SHEARSON, C. M.. WONGMONGKOLRIT, T.,MEDORI, R., ANDGAMBETTI,P. (1986). Toxicol. Appl. Pharmacol. 84, 36-44. The chronic exposure to ydiketones results in the formation of giant neurofilament (NF)-containing axonal enlargements, followed by axonal degeneration in peripheral axons. Based on the specific ability of ydiketones to react with primary amino groups to form pyrroles, and the observation of such reaction with NF protein in vitro and with other proteins in vivo, it has been proposed that pyrrole formation at primary amino groups of NF protein is responsible for the neurotoxicity of ydiketones. We have tested this hypothesis through an investigation of the neurotoxicity in rats of 3,3dimethyl-2,5-hexanedione (3,3-DMHD), a ydiketone which is incapable of forming pyrroles. 3,3-DMHD was found to produce only a slight alteration of axonal caliber and no clinical neurotoxicity after up to I2 weeks of administration, at a dose over 20 times that for which its isomer 3,4-dimethyl-2,5-hexanedione (3,4-DMHD) produced massive focal NFcontaining axonal enlargements and complete paralysis in 4 weeks. These results support the view that the pyrrole-forming capability of ydiketones is the initial molecular event in the pathogenesis of r-diketone neurotoxicity. Q 1986 Academic PRS, hc.

Chronic exposure to the industrial solvents n-hexane and methyl n-butyl ketone (MnBK) results in motor and sensory deficits, which are associated with axonal degeneration in the peripheral nervous system and to a lesser extent in spinal cord and brain (Spencer et al., 1980a). 2,5-Hexanedione (2,5-HD, I), which itself serves a variety of minor commercial roles as a synthetic intermediate, solvent, gasoline additive, and tanning agent, has been shown to be the ultimate metabolite responsible for neurotoxicity of both n-hexane and MnBK, and of other related metabolites of nhexane (2-hexanol, 2,5-hexanediol, and 5-hydroxy-2-hexanone) (Abou-Donia et al., 1982;

Couri and Milks, 1982; Di Vicenzo et al., 1980; Krasavage et al., 1980; Spencer et al., 1980b; Spencer and Griffin, 1982). The neuropathy associated with 2,5-HD as well as with n-hexane and other precursors is characterized morphologically by the massive focal accumulations of lo-nm neurofilaments (NF) in distal regions of the axon at sites just proximal to nodes of Ranvier. A virtually identical distal axonopathy is produced by CS? (Seppalainen and Haltia, 1980) and a similar type is observed in acrylamide intoxication (Le Quesne, 1980; Miller and Spencer, 1985). A different type of neuropathy is produced by exposure to P&3’-iminodipropionitrile (IDPN) (Chou and Hartmann, 1965; Clark et al., 1980; Griffin and Price, 1980; Griffin et al., 1982; Yokoyama et al., 1980) and by an analog of 2,5-HD, 3,4-dimethyl-2,5-hexane-

’ Supported by NIH Grants NS 18714, NS 22688 (L.M.S.), and NS 14509 (P.G.). ’ To whom correspondence should be addressed. 004 I -008X/86

$3.00

Copyright 0 1986 by Academic Press, Inc. All rigbu of reproduction in any form reserved.

36

-y-DIKETONE

+4vt+TY 1

2,5-HD

37

NEUROTOXICITY

2

3,4-DMHD

dione (3,4-DMHD, 2) (Anthony et al., 1983a,c; Griffin et al., 1984), as well as by administration of aluminum in the cistemae magna or intraperitoneally at the level of the medulla (Bizzi et al., 1984; Troncoso et al., 1982; Wisniewski et al., 1984), wherein the NF-containing axonal enlargements form proximally near the cell body, often at the first node of Ranvier. It is of great interest that the chemically induced axonopathies resemble those observed in inherited or acquired human neurological diseases, such as “giant axonal neuropathies” in the case of the distal enlargements and amyotrophic lateral sclerosis (ALS) in the case of proximal enlargements (Griffin et al., 1983a). For this reason, numerous studies have been directed at establishing the pathogenetic mechanisms associated with these toxic chemicals. Early attempts to define the structural basis of 2,5-HD neurotoxicity demonstrated that neuropathies develop upon administration of 2,5-heptanedione and 3,6-octanedione, but not 2,3-hexanedione, 2,4-hexanedione, 3,5heptanedione, or 2,6-heptanedione (Spencer et al., 1978; O’Donoghue and Krasavage, 1980) thus establishing the absolute requirement for a y-spacing of the two keto groups. The current consensus regarding this structural dependence, originally proposed by DeCaprio and Weber (1980), is that y-diketones transform the primary amino groups (lysine sidechains) of NF proteins into pyrrole groups. Several studies have now confirmed the formation of pyrroles in reactions of y-diketones with proteins both in vitro and in vivo (Anthony et al., 1983b; DeCaprio et al., 1982, 1983; Graham et al., 1982, 1984). However, it has yet to be established whether these pyrroles are in fact responsible for the clinical neurotoxicity and formation of NF-containing axonopathies associated with y-diketone in-

3 3.3~DMHD

4 3-MHD

toxication. To address this question, we prepared the geminal dimethyl derivative of 2,5-HD, 3,3-dimethyl-2,5-hexanedione (3,3DMHD, 3), which retains the y-spacing between keto groups, but, on account of the geminal dimethyl substitution, is incapable of forming pyrroles. We chronically exposed animals to this compound to investigate its potential to produce clinical symptoms and axonal alterations associated with neurotoxicity. The effects of 3,3-DMHD were compared to those of the isomeric vicinal dimethyl compound (3,4-DMHD, 2), as well as to 2,5-HD and the monomethyl analog, 3-methyl-2,5hexanedione (3-MHD, 4) which we have recently found to produce NF-containing axonal enlargements at more proximal locations to the cell body than those produced by 2,5-HD (Monaco et al., 1984). METHODS Compounds. 3,3-Dimethyl-2,5-hexanedione was prepared from 4,4-dimethyl-S-nitro-2-hexanone by the Nef reaction (Black, 1972). Our procedure involved refluxing in 95% ethanol with 1.5 equivalents of NaOH followed by workup with 3~ HCl. The starting nitroketone was prepared via a Michael reaction by the method of Smith and Kohlhase (1956), modified according to the instructions of Clark and Cork ( 1982). We employed a mixture of cesium fluoride and tetramethylammonium hydroxide as catalyst for this latter reaction. 2,5-HD and 3,4-DMHD were obtained commercially (Aldrich) and the latter was purified by fractional vacuum distillation. After purification, GLC and 200 MHz NMR showed that all diketones used were >95% the desired compound. Compound administration. Twenty-three male SpragueDawley rats (6-7 weeks old, 190-220 g) were used for the experiments. Seven rats received 3,3-DMHD (2.8 or 3.5 mmol/kg/day); 6 received 2,5-HD (3.5 mmol/kg/day); 3 received 3-MHD (1 .O mmol/kg/day); and 7 received 3,4DMHD (0. I5 mmol/kg/day), with isotonic saline as a carrier. Five animals used as controls received saline only. Equal volumes of saline solutions of the toxins, or saline alone, were injected intraperitoneally daily, 5 days/week

SAYRE ET AL.

38

for a period extending up to 12 weeks or until the rats were severely incapacitated. Clinical studies. The mean body weight was measured at weekly intervals and the animals were watched for the development of neurotoxic signs. The first appearance of obvious weakness and the onset of hindlimb paralysis, as demonstrated by abnormal gait, were noted. The Student I test was used for determining the level of statistical significance. Morphological studies. Experimental and control animals were perfused through the ascending aorta with 0.2 M sodium cacodylate buffer, pH 7.4, followed by 5% (w/ v) glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, with 0.03% CaClz at 37°C. The sciatic systemincluding the roots was dissected out. Segments, 3-mm in length, were obtained and after additional overnight fixation were postfixed in 2% osmium tetroxide, dehydrated, and embedded in “Spur? plastic medium. Thick plastic sections (1 pm) cut from segments O-3 mm, 30-33 mm, 6063 mm, 90-93 mm, and 120-123 mm from the spinal cord were used to determine the cross-sectional area of each axon according to the method described previously (Medori et al., 1985). Statistical analysis on the data was performed by the unpaired Student t test. In some of the experimental animals, liver, kidneys, and testes were removed, weighed, embedded in paraffin, and examined histologically.

RESULTS For the three y-diketones that can form pyrroles, but not for 3,3-DMHD, postmortem examination revealed varying levels of(i) focal necrosis of the liver; (ii) infarcts, focal necrosis, and atrophy of the kidneys; and (iii) liquefication or atrophy of the testes. These changes increased with increasing dosage regimen, apparently independent of structure of the r-diketone. Testicular atrophy has previously been noted by several investigators to accompany 2,5-HD neurotoxicity in rats (Anthony et al., 1983b; Cavanagh and Bennetts, 198 1; Gillies et al., 1981; Krasavage et al., 1980), and ketones in general are known to cause kidney and liver damage when administered in high dosages to experimental animals (Browning, 1965). As shown in Fig. 1, all of the y-diketones caused initial weight loss compared to control. This effect was probably due to a general systemic toxicity associated with the fairly high doses of compounds employed. In the case of

3,3-DIMETHYL-2,5-HD (2.8-3.5 mmol/kg) NO NELJROTOXICITY

DKY 27.6+21: 3-METHYL-2.5~HD(1.0

mmol/kg)

(3.5

mmal/kg)

( weeka

)

0123450780Oll

Length

ot lnkdxtlon

FIG. 1. Effects of various y-diketones on body weight, and the onset of hindlimb paralysis. 3,3-Dimethyl-2,5-HD (2.8-3.5 mmol/kg/day) had no effect on body weight after an initial decrease during the first 2 weeks of administration, whereas body weight continued to decrease in animals receiving 2,5-HD (3.5 mmol/kg/day), 3-methyl-2,5-HD (1 .Ommol/k&lay), and 3,4dimethyl-2,5-HD (0.15 mmol/ kg/day). While following administration of 3,4dimethyl2.5-HD, 3-methyl-2,5-HD, and 2,5-HD, hindlimb paralysis appeared between Day 28 and 38 of administration (arrows), no evidence of neurotoxicity was observed in 3,3-dimethyl-2,5-HD-treated rats up to 11 weeks of administration.

2,5-HD, 3,4-DMHD, and 3-MHD, the weight loss was constant and continual, and eventually was accompanied by the onset of clinical neurotoxicity, as first indicated by the appearance of hindlimb weakness, followed by definite paralysis. These signs appeared at different times according to the drug administered. Following 3,4-DMHD (0.15 mmol/kg) or 3-MHD (1 .O mmol/kg), the hindlimb paralysis consistently appeared at approximately the 28th day of intoxication; whereas following 2,5-HD (3.5 mmol/kg), it appeared at the 38th day. Hindlimb weakness preceded the paralysis by 2-4 days in the case of 3,4-DMHD and 3-MHD, or 7-10 days in the case of 2,5-HD. From Fig. 1 it is possible to estimate that the dimethyl compound 3,4-DMHD and the

y-DIKETONE

NEUROTOXICITY

39

monomethyl compound 3-MHD are, respec- 3 1% smaller than the control at I23 mm. This tively, 23 and 3.5 times as neurotoxic as 2,5- proximal enlargement and distal shrinking of HD on a molar basis in terms of clinical axons observed for the non-neurotoxic 3,3symptoms. This is consistent with the report DMHD are reminiscent of what has been reby Anthony et al. (1983a,c) that 3,4-DMHD ported for its neurotoxic isomer 3,4-DMHD is 20-30 times more potent than 2,5-HD. In (Anthony et al., 1983a,c). To permit a quancontrast, no hindlimb weakness or paralysis titative comparison between the two comnor other neurotoxic signs were ever detected pounds, a morphometric study was performed in 3,3-DMHD-treated rats, even though this on one animal treated with a total of 6.3 compound was administered at a dosage (2.8mmol/kg 3,4-DMHD (22-24 times less than 3.5 mmol/kg) 21 times higher than that em- the total amount of 3,3-DMHD). Our results ployed for its isomer (3,4-DMHD (Fig. 1). For reveal that in contrast to the small increase in 3,3-DMHD, the initial weight loss stabilized axonal size seen at 3 mm from the spinal cord after 2 weeks, such that the mean weight of with 3,3-DMHD, axons from the 3,4-DMHDthese animals became statistically different treated animal had a mean cross-sectional area from that of the animals treated with the other twice as large as in the control. In addition, y-diketones (Fig. 1). for 3,4-DMHD, the progressive reduction in The morphometric study (Table 1) revealed axonal size with increasing distance from the that after 54-59 days of administration of a cord was more pronounced than in the case total of 137- 15 1 mmol/kg of 3,3-DMHD, the of 3,3-DMHD. All along the sciatic system of cross-sectional area of axons from roots of the the 3,CDMHD-treated animal, occasional sciatic system, 3 mm from the spinal cord, degenerating and clusters of regenerating axwas increased approximately 19% over that of ons were observed, along with markedly enthe control. The relative size of axons from larged axons. Such changes were not present the 3,3-DMHD-treated animals diminished at in animals exposed to 3,3-DMHD. increasing distances from the cord, becoming smaller than the control at 63 mm, and was DISCUSSION TABLE

1

MEAN CROSS-SECTIONALSIZE(pm2) OF AXONS OF THE SCIATIC SYSTEM IN 3,3-DMHD AND 3,4-DMHD INTOXICATION

Distance from the spinal cord (mm) 3 33 63 93 123

3,3DMHD” 25.2 14.6 11.7 9.1 6.0

f 2.1 c + 1.7 + I.0 rt_ 0.9 k lSd

Control” 21.1 f 2.5 12.6 10.6 9.4 8.7

+ f + +

0.6 0.7 0.8 1.2

3,4DMHDb 43.4 12.9 8.4 7.6 5.7

n Data based on measurement of 3400 axons from six animals, three of which receiving a total dose of 137- I5 1 mmol/kg, and three controls. b Data based on measurement of 850 axons from one animal receiving a total dose of 6.3 mmol/kg. c Statistically different from control (p < 0.05). d Statistically different from control (p < 0.0 I).

Two distinct biochemical hypotheses have been advanced to explain the axonal changes produced by 2,5-HD and other y-diketones. On the basis of the observation that 2,5-HD inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other glycolytic enzymes in vitro (Sabri et al., 1979a,b), it was suggested (Spencer et al., 1979; Sabri and Spencer, 1980) that the resulting disruption of intermediary metabolism would cause an impairment of axonal transport of NFs, which would in turn accumulate focally in the distal regions of the axons. On the other hand, Graham et al. (1982, 1984) and DeCaprio et al. (1982, 1983) have suggested that 2,5-HD acts directly on NF, modifying the chemical structure of these organelles. The administration of 2,5-HD has recently been found to accelerate the transport of NF

40

SAYRE

ET

subunits and of two other polypeptides, but not other polypeptide constituents of the slow transport (Monaco et al., 1985). This selective effect is inconsistent with a defect of energy metabolism, which should indiscriminately affect entire components of axonal transport. In addition, the relatively high concentrations of 2,5-HD required to inhibit glycolytic enzymes in vitro are unlikely to be achieved in vivo during 2,5-HD intoxication (Graham and AbouDonia, 1980). Moreover, the rapid reorganization of NFs and microtubules (MTs) following intraneural injection of 2,5-HD (Griffin et al., 1983b; Zagoren et al., 1983) is more consistent with a direct effect of y-diketones on cytoskeletal components rather than with an indirect effect on energy metabolism. Thus, although inhibition of glycolysis is unlikely to be the primary mechanism responsible for the formation of neurofilamentous axonopathies, recent work showing that relatively low concentrations of 2,5-HD are capable of retarding glycolysis after prolonged exposure (Sabri, 1984a,b) suggests that this effect may be in part responsible for the axonal degeneration that occurs secondary to NF accumulation in y-diketone intoxication. The first attempt to rationalize the required y-spacing in diketone neurotoxicity in terms of a chemical mechanism was the proposal by Graham ( 1980) that the y-diketone could undergo reversible Schiff base condensations with two lysine c-amino groups on different NF (or related) proteins to give intermolecular diimine crosslinks, which, because of the yspacing, could subsequently undergo oxidation to a non-hydrolyzable conjugated enediimine (Scheme 1). The latter functionality

b NH2+

AL.

was thought to be responsible for the orange chromophore observed when 2,5-HD was incubated with proteins. In contrast, DeCaprio and Weber ( 1980) suggested that the condensation of y-diketones with primary amino groups of proteins would result in the formation of pyrroles. Subsequent studies by Graham et al. (1982) identified the orange chromophore as arising from pyrrole autoxidation rather than from a conjugated enediimine. In addition, the evidence cited by Graham and Abou-Donia ( 1980) in support of the notion that the irreversible inhibition of GAPDH by 2,5-HD was due to a reaction with enzyme amino rather than sullhydryl groups, suggests that pyrrole formation is probably responsible for this effect as well. Although it is currently thought that pyrrole formation is responsible for y-diketone neurotoxicity, the fact that extensive pyrrole formation is observed throughout the intoxicated animal (DeCaprio et al., 1982,1983) leads one to question why pyrrole formation would be selectively neurotoxic. In the present study, we tested the role of pyrrole formation in ydiketone neurotoxicity by determining the effect of 3,3-DMHD. This compound would theoretically be capable of covalently modifying proteins through the formation of a diimine crosslink (Scheme 2, Path A) or a 5hydroxy-2-pyrroline (5, Scheme 2, Path B). However, these reactions would be reversible, and the quaternary carbon center created by the gem-dimethyl substitution precludes the otherwise irreversible last step of pyrrole formation (dehydration of 5, Scheme 2, Path B). We found that 3,3-DMHD exhibited no clinical neurotoxicity (at up to at least 12 weeks)

4 0

SCHEME

1.

y-DIKETONE NEUROTOXICITY

SCHEME

at a dose over 20 times greater than that for which the isomeric compound 3,4-DMHD produced hindlimb paralysis in 4 weeks. Our findings, therefore, strongly support pyrrole formation as an obligatory step in the expression of y-diketone neurotoxicity. Assuming that y-diketone intoxication results in pyrrole formation on NF (or related) proteins, the selective neurotoxicity of these compounds is probably a consequence of low turnover rate of NF compared to most other proteins (Lasek et al., 1984; Graham et al., 1982). The cause of the small, yet statistically significant, alteration of axonal caliber seen for 3,3-DMHD is presently unknown. One possibility is that at the high dosage of 3,3-DMHD employed, one or more reversible reactions with NF and/or other cytoskeletal proteins (Scheme 2) could slow their rate of transport. These organelles would then be retained proximally while fewer are transported distally. Because of the close relationship between axonal size and number of NF, this uneven longitudinal distribution of NF and other cytoskeletal components may result in an increase in axonal caliber proximally and a decrease distally. A similar chain of events may occur in streptozotocin-induced diabetes, a condition also characterized by proximal enlargement and distal attenuation of axons (Medori et al., 1985). Another possibility is that the high dosage of 3,3-DMHD employed may be sufficient to produce some inhibition of glycolytic enzymes, observed in vitro for high concentrations of both 2-hexanone and 2,5-

41

2.

HD (Sabri et al., 1979a), and that the corresponding reduction of energy metabolism retards axonal transport and results in the observed changes in axonal caliber. Alternatively, these changes might result from a pyrroleforming impurity present at a low level (<5%) in the 3,3-DMHD used (at high dosages) in our study. How pyrrole formation at lysine c-amino groups can lead to the formation of axonal enlargements with accumulation of NF remains an open question. Graham et al. (1982) have proposed that pyrrole autoxidation results in intermolecular crosslinking of NFs. Although this theory is supported by the finding that y-diketones induce crosslinking of NF protein in vivo (DeCaprio and O’Neill, 1985) as well as in vitro (Graham et al., 1984; DeCaprio and O’Neill, 1985), it is not clear if such crosslinking is a causative factor in the pathogenetic process (DeCaprio, 1985). First, if covalent crosslinking of NF (and related) proteins is the common denominator by which CSp and IDPN produce identical neuropathological morphologies to those of the y-diketones, it is not clear how the former agents can participate in crosslinking reactions. Second, the notorious crosslinking agent glutaraldehyde is apparently non-neurotoxic (Spencer et al., 1978). Third, crosslinking of NF should cause a progressive slow-down of NF transport in the axonal segment proximal to the NFcontaining enlargements, whereas it was found that transport of NF and of two NF-associated polypeptides in this region is accelerated

42

SAYRE

(Gambetti et al., 1986) following administration of 2,5-HD (Monaco et al., 1985), 3-MHD (Monaco et al., 19X4), and carbon disulfide (Papolla et al., 1984). We recently presented a hypothesis (Sayre ef al., 1985), based on the published structure of NF subunits (Geisler et al., 1983, 1984, 1985a,b), that rationalizes the effect of all chemicals known to produce NF accumulations as a result of the alteration of charged amino acid side-chains in special regions of NF subunits (or possibly other cytoskeletal proteins) characterized by high proportions of basic and acidic amino acids. These highly charged regions are believed to be involved in intermolecular multivalent coulombic interactions with other cytoskeletal entities such as microtubules,microtubule-associated proteins, and actin microfilaments. Charge alteration could disrupt the intricate cytoskeletal meshwork, leading to an accelerated transport and ultimately focal axonal accumulation of NF. This notion is consistent with the observed rapid dissociation between NF and MT upon intraneural injection of 2,5-HD (Griffin et al., 1983b; Zagoren et al., 1983). According to our hypothesis, the effect of pyrrole formation in y-diketone neurotoxicity is to alter the physicochemical properties of, rather than to crosslink, NF and/or other cytoskeletal proteins, as originally suggested by DeCaprio et al. ( 1982). Carbon disulfide, IDPN, acrylamide, and aluminum ion are all capable (either directly or after metabolism) of entering into reactions which induce chargealtering physicochemical changes of NF protein in a manner distinct from that of the pyrrole-forming y-diketones, but producing the same ultimate effect (Sayre et al., 1985). Although recent studies suggest that the average extent of covalent modification of NF protein in 2,5-HD intoxication is quite low (DeCaprio and O’Neill, 1985), those amino acid residues which are modified may play a critical role in stabilizing the supramolecular cytoskeletal framework. The finding that successive methyl substitution on 2,5-HD, generating first 3-MHD and

ET

AL.

then 3,4-DMHD, is accompanied by a continuous shift of the location of NF-containing axonal enlargements from distal to intermediate and then to proximal positions (Anthony et al., 1985~; Sayre et al., 1985), provides strong evidence that distal and proximal axonopathies are related by a common pathogenetic mechanism. According to our “chargealteration” hypothesis, the shift in location of the enlargements can be explained by the monotonic increase in hydrophobicity of the resulting pyrrole upon successive methyl substitution on 2,5-HD. In conclusion, the results presented here demonstrate that it is possible to gain insight into mechanistic aspects associated with chemically induced neuropathies through a structure-activity approach. Information gained in such studies is also likely to lead to a better understanding of the molecular basis of the naturally occurring neuropathies. REFERENCES ABUO-D~NIA,

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ANTHONY, D. C., BOEKELHEIDE.K., ANDGRAHAM, D. G. (1983a). The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,Shexanedione. 1. Accelerated clinical neuropathy is accompanied by more proximal axonal swellings. To.xicol. Appl. Pharmacol. 71, 362-311. ANTHONY, D. C., BOEKELHEIDE, K., ANDERSON, C. W.. ANDGRAHAM, D. G. (1983b). The effect of 3,4dimethyl substitution on the neurotoxicity of 2,Shexanedione. II. Dimethyl substitution accelerates pyrrole formation and protein crosslinking. Toxicol. Appl. Pharmacol. 71, 372-382.

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DECAPRIO, A. P., AND O’NEILL, E. A. (1985). Alterations in rat axonal cytoskeletal proteins induced by in vifro and in vivu 2,5-hexanedione exposure. Toxicol. Appl. Pharmacol.

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