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Inhibition of sterologenesis but not glycolysis in 2,5-hexanedione-induced distal axonopathy in the rat
I OYICOI
oCY
%ND
APPI.IED
PHARMACOI
OGY
59, 287-292
(1981)
Inhibition of Sterologenesis but Not Glycolysis 2,5-Hexanedione-Induced Distal Axonopathy in the Rat PETERJ.GILLIES,'
RONALD M.NORTON,
in
AND JAMES S. Bus
inhibition Axonopathy
of Sterologenesis but Not Glycolysis in 2.5Hexanedione-Induced Distal in the Rat. GILLIES. P. J.. NORTON, R. M., AND BLIS, J. S. (1981). Tu;c,,/. Appl. Phrmacol. 59, 287-292. If glycolysis is inhibited in distal axonopathy. there should be a concomitant inhibition of lipogenesis from glucose. To investigate this possibility. lipogenesis from [‘“Clglucose and [“HIacetate was studied in sciatic nerves incubated with iodoacetate. a known inhibitor of glycolysis, in sciatic nerves incubated with 2.5hexanedione, a putative inhibitor of glycolysis, and in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy induced by 2.5hexanedione. Nerves incubated with 1.0 mM iodoacetate, in comparison with untreated nerves, exhibited decreased incorporation of ]“C]glucose into sterols + diacylglycerols (33.fold), free fatty acids (14-fold), triacylglycerols (27-fold). and phospholipids (21-fold). In addition, these nerves exhibited decreased incorporation of [YH]acetate into sterols + diacylglycerols (30-fold), free fatty acids (2-fold), triacylglycerols (23-fold), and phospholipids (12.fold). In contrast, the incorporation of [“Cjglucose into sterols + diacylglycerols. free fatty acids, and triacylglycerols was not affected by 1.0 mM 2.5.hexanedione. Compared to untreated nerves. nerves incubated with I .O mM 2,5-hexanedione exhibited a small decrease (15%) in the incorporation of [IV]glucose into phospholipids. Nerves from rats given 1% 2,5-hexanedione in the drinking water for 6 weeks, in comparison with those from pair-fed control rats, exhibited decreased (45%) incorporation of [“Clglucose and [3H]acetate into digitonin-precipitable sterols. Nerves from 2,5-hexanedione-treated and pair-fed control rats exhibited similar incorporation of [“C]glucose and [JH]acetate into free fatty acids, triacylglycerols, and phospholipids. The data indicate that while sterologenesis is inhibited in distal axonopathy. glycolysis is not.
Hexacarbon-induced distal axonopathy has frequently been studied in rats given 2.5 hexanedione in the drinking water (Spencer and Schaumburg, 1975; Powell et al., 1978; Krasavage et cl/., 1980). The morphological changes induced by 2,5hexanedione, e.g., giant axonal swelling, focal accumulation of neurofilaments, and paranodal myelin retraction. are similar to those observed in ’ Present address, to which reprint be sent: E. I. DuPont de Nemours Haskell Laboratory of Toxicology Medicine, Newark. Del. 19711.
animals and humans exposed to n-hexane and methyl n-butyl ketone (Spencer et trl., 1980). This mode1 has also been used to investigate biochemical changes in distal axonopathy. Cholesterol biosynthesis has been reported to be inhibited in sciatic nerves from rats fed 2,5-hexanedione (Gillies et al., 1980a). The site of inhibition in sterologenesis was found to be between acetyl-CoA and mevalonate and likely involved the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (Gillies et ul., 1980b). The inhibition of sterologenesis may
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GILLIES
be related to the decreased microviscosity in sciatic nerves that is reported to precede the development of clinical signs of peripheral neuropathy in 2,5-hexanedione-treated rats (Couri and Nachtman, 1979). In addition to sterologenesis, there is indirect evidence that glycolysis may also be inhibited in 2,5-hexanedione-induced distal axonopathy. Purified preparations of phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and glycerol-3-phosphate dehydrogenase have been reported to be inhibited by high concentrations of 2,5hexanedione (Spencer and Schaumburg, 1978; Sabri et al., 1979a, b). Since dithiothreitol prevented this inhibition, it was suggested that 2,5-hexanedione reacts with sulfhydryl groups of key enzymes in glycolysis. These studies formed the experimental basis of the hypothesis that the biochemical basis of hexacarbon-induced distal axonopathy is an inhibition of glycolysis (Spencer et al., 1979). If glycolysis is inhibited in distal axonopathy, then lipogenesis from glucose should also be inhibited. To investigate this possibility, lipogenesis from [14C]glucose and [“HIacetate was studied in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy induced by 2,5-hexanedione. METHODS Animals
and Diets
Male Fischer-344 rats (Charles River Company, Wilmington, Mass.: weight, 187 2 5 g) were randomly divided into two groups, six animals per group. The experimental group of rats (HD rats) received 1% 2.5 hexanedione (Eastman Kodak, Rochester, N.Y.; redistilled before use) in the drinking water for 6 weeks; food and water were available ad libitum. Control (PFC) rats were pair-fed to the HD rats; water was available ad libitum.
Incubation
of Sciatic
Nerves
Rats were sacrificed by cervical dislocation. ments (3-4 cm; weight, 35 it 2 mg) of sciatic
Segnerve
ET AL. were carefully excised, stripped of adhering tissue, and incubated in 2.0 ml of Krebs-Ringer-bicarbonate buffer, pH 7.4, that contained 100 units and 100 pg/ml of penicillin G (sodium salt) and streptomycin sulfate (Sigma Chemical Company, St. Louis. MO.), respectively, and 10 &i each of D-[IC(U)]glucose (New England Nuclear, Boston: sp act. 13.9 mCi/mmol; radiochemical purity, 99%) and [3H]acetate (sodium salt) (New England Nuclear: sp act. 140.0 mCi/mmol; radiochemical purity, 99%). In experiments in which 1 .O mM iodoacetate (Sigma) or 1.0 mM 2.5hexanedione was added to the incubation medium. penicillin G and streptomycin sulfate were omitted. Incubations were carried out in 25-ml Erlenmeyer flasks, gassed with O,/CO, (95/5%), stoppered. and slowly agitated in a water bath at 37.5”C. After 3 hrs, the nerves were removed from the incubate, rinsed in 1% glucose plus 1% sodium acetate, and then homogenized in 20 vol of chIoroform:methanol ( 1: I).
Extraction
oj’ Lipids
The nerve homogenate was centrifuged at 3008 for 5 min, and the supemate decanted and adjusted to 2: 1 chloroform:methanol. The homogenate residue was extracted repeatedly with chloroform:methanol (2:l) until radioactivity was no longer detected in the supemate. A phase separation in the combined supemates was induced by the addition of 0.2 vol of 0.73% sodium chloride and the lower phase Folchwashed (Folch et al., 1957). The lower phase was washed with theoretical upper phase that contained 0.29% sodium chloride, 1% glucose, and 1% sodium acetate, followed by upper phase that contained only 0.29% sodium chloride, until radioactivity was no longer detected in the washes. The lower phase was evaporated to near-dryness under nitrogen and the lipid extract dissolved in chloroform:methanol (2: I). Digitonin (0.5%, w/v, in 50% ethanol) was added to an aliquot of total lipid extract reconstituted in acetone:ethanol (1: 1). Sterol digitonides were washed with acetone:ether (1:2), then dissolved in pyridine and water, and the sterols were extracted into ether (Sperry and Webb, 1950). The ether extract was evaporated to dryness under nitrogen and the radioactivity in the samples was determined. Saponijcation
of Lipids
Phospholipids and triacylglycerols. isolated, identified, and eluted from thin-layer chromatographic plates, were saponified in 82% ethanolic-11% potassium hydroxide at 65°C for 2 hr. Recovery of lipids from tic plates was greater than 90%. The saponification mixture was cooled and diluted 1:l with water. Nonsaponifiable lipids were extracted into n-hexane
EFFECT
OF 2,5-HEXANEDIONE TABLE
EFFECT
OF IODOACETATE AND [3H]A~~~~~~
ON
289
LIPOGENESIS
1
ON THE INCORPORATION OF [YJGLUCOSE INTO LIPIDS BY SCIATIC NERVE”
Incorporation
(dpmimg
wet weight)”
[“ClGlucose Lipid
fractions
Sterols + diacylglycerols Free fatty acids Triacylglycerols Phospholipids n Control nerves were 2.5 &i/ml [Wlglucose and h Values are the means * Significantly different
Control 1024 42 752 1960
+ t + 2
Treated 29 2 128 37
Chromutogruphy
The lipid extract was fractionated on a precoated silica gel 60 thin-layer chromatography plate (EM Laboratories Inc., Elmsford, N.Y.; layer thickness, 0.25 mm) in a solvent system consisting of nhexane-diethyl ether-glacial acetic acid (14650~8). Lipid bands were visualized by iodine vapor and identified by comparison of R, values with those of lipid standards (Sigma). Neutral lipids were eluted from the thin-layer chromatography plate with chloroform; phospholipids were eluted according to the method of Skipski (Skipski et al., 1964). The incorporation of radioactivity into lipid was determined by standard dual-label liquid scintillation counting techniques. Quench corrections were made by the external standardization method.
Strrtisticul
31 2 10* ND* 28 + 7* 94 2 7’
Control 1667 97 164 893
t + + t
Treated 135 3 55 59
56 2 6* 55 rt 6* ND* 76 2 9*
incubated for 3 hr at 37.5”C in Krebs-Ringer-bicarbonate buffer that contained [3H]acetate; in addition, treated nerves were incubated with 1.0 mM iodoacetate. f SE of six rats: ND, not detectable. from control values by Students’ independent I test, p < 0.05.
(3 x 10 ml): the n-hexane extracts were backwashed with 5 ml of water. The saponification mixture was then adjusted to pH 1.5 by the addition of 6 N HCl, and the free fatty acids were extracted into diethyl ether (3 x 10 ml); the diethyl ether extracts were backwashed with 5 ml of water at pH 1.5. [‘4C]Glycerol incorporated into phospholipids and triacylglycerols was determined by counting the radioactivity remaining in the saponification mixture after the n-hexane and diethyl ether extractions.
Thin-Layer
[3H]Acetate
Andyses
Students’ t test for comparing unpaired samples was used; p values were derived from a two-tailed table of Students’ values for t. The level of significance was chosen as p < 0.05.
RESULTS Ejyect of Iodoacetate and 2,5-Hexanedione on the Incorporation of [14C]Glucose and [3H]Acetate into Lipids by Sciatic Nerve
Nerves were incubated with 1.0 mM iodoacetate to determine what effects an inhibition of glycolysis would have on lipogenesis from [14C]glucose. Lipogenesis from [3H]acetate was examined in the same nerves to determine whether or not iodoacetate could inhibit lipogenesis directly as well as indirectly subsequent to an inhibition of glycolysis. The incorporation of [“Clglucose and [3H]acetate into lipid was significantly decreased by iodoacetate (Table 1). Incorporation of [‘“Clglucose was decreased into sterols + diacylglycerols (33fold), free fatty acids (lCfold), triacylglycerols (27-fold), and phospholipids (21fold). Similarly, incorporation of [3H]acetate was decreased into sterols + diacylglycerols (30-fold), free fatty acids (Zfold), triacylglycerols (23-fold), and phospholipids (1Zfold). Lipogenesis from [ 14C]glucose was also examined in nerves incubated with 1.0 mM 25hexanedione. In contrast to that of iodoacetate, the incorporation of [‘“Clglucase into sterols + diacylglycerols, free
290
GILLIES TABLE
EFFECT OF 25HEXANEDIONE OF [%]GLUCOSE INTO
2
LIPIDS
ON THE INCORPORATION BY SCIATIC NERVE”
Incorporation (dpm/mg wet weightY Lipid fractions Sterols + diacylglycerols Free fatty acids Triacylglycerols Phospholipids
Control 1691 85 685 3958
t k k +
137 4 148 86
Treated 1414 74 802 3326
+ 5 2 r
135 4 194 115*
u Control nerves were incubated for 3 hr at 37.5”C in Krebs-Ringer-bicarbonate buffer that contained 5 &i/ml of [“Clglucose; in addition, treated nerves were incubated with 1.0 mrvt 2,5-hexanedione. b Values are the means k SEM of six rats. * Significantly different from control values by Students’ independent f test, p < 0.05.
ET AL.
into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats. Incorporation of [3H]acetate was significantly decreased into both sterols + diacylglycerols (42%) and digitonin-precipitable sterols (46%) in HD compared to PFC rats. [3H]Radioactivity in digitonin-precipitable sterols accounted for all of the radioactivity in the combined sterol + diacylglycerol fraction. Incorporation of [“HIacetate into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats. The distribution of 3H and ‘C radioactivity in the acyl chain and glycerol moieties of triacylglycerols and phospholipids was not significantly different in HD and PFC rats (Table 3).
DISCUSSION fatty acids, and triacylglycerols was not affected by 2,5-hexanedione; the incorporation of [14C]glucose into phospholipids was slightly decreased (15%) (Table 2). Effect of Chronic Exposure to 2,5-Hexanedione on the Incorporation of [‘4C]Glucose and r3H]Acetate into Lipids by Sciatic Nerve
Rats given 1% 2,5-hexanedione for 6 weeks exhibited everted and flat foot placement, hindlimb weakness, and ataxia. These clinical signs of neuropathy were first discernible after 4 weeks and were clearly evident after 6 weeks. Similar changes were not observed in PFC rats. Lipogenesis from [14C]glucose and [3H]acetate was examined in sciatic nerves from HD and PFC rats; the data are presented in Table 3. Incorporation of [14C]glucose was significantly decreased into digitoninprecipitable sterols (45%) in HD compared to PFC rats. 14C Radioactivity in digitoninprecipitable sterols accounted for 30-40% of the total 14C radioactivity incorporated into the combined sterol + diacylglycerol fraction. The incorporation of [l*C]glucose
If glycolysis is inhibited in distal axonopathy, there should be a concomitant inhibition of lipogenesis from glucose. This possibility was investigated in sciatic nerves incubated with iodoacetate, a known inhibitor of glycolysis, in sciatic nerves incubated with 2,5-hexanedione, a putative inhibitor of glycolysis, and in sciatic nerves from rats exhibiting clinical signs of distal axonopathy. In these studies, lipogenesis from both [‘Clglucose and [“HIacetate was examined; these dual-label studies permitted the differentiation of effects on glycolysis vs lipogenesis. Iodoacetate inhibits glyceraldehyde-3-phosphate dehydrogenase in glycolysis; inhibition of this enzyme would be expected to decrease the formation of glycerol-3-phosphate and acetyl-CoA available for lipid biosynthesis. This prediction was substantiated by the observation that the incorporation of [ ‘Clglucose into sterols, free fatty acids, triacylglycerols, and phospholipids was markedly decreased by iodoacetate (Table 1). The incorporation of 13H]acetate into these same lipids was also significantly decreased by iodoacetate.
EFFECT
OF 2JHEXANEDIONE
ON
TABLE INCORPORATION
OF [‘“C]GLUCOSE FROM RATS
291
LIPOGENESIS
3
AND [$H]ACETATE INTO LIPIDS WITH DISTAL AXONOPATHY”
Incorporation
(dpm/mg
BY SCIATIC
wet weight)”
[“CjGlucose
Lipid Sterols
+ diacylglycerols
Digitonin-precipitable Free
Pair-fed controls
fractions
fatty
acids
sterols
NERVES
[“HIAcetate
2JHexanedione treated
Pair-fed controls
2.5Hexanedione treated
1153 5 145
878 2 128
1067 rt 84
SO4 k 46
278 t 15*
935 k 71 186 i
615 2 38* 506 i
31*
98 2 7
85 2 6
15
232 2 22
Triacylglycerols Fatty acids Glycerol
2748 k 41.5 278 t 83 2824 + 326
3441 2 499 190 k 50 3428 2 414
415 c 79 269 2 54 62 2 15
419 lr 32 234 2 25 60t 13
Phospholipids Fatty acids Glycerol
2152 k 171 883 k 101 1799 2 195
2142 ? 96 640 t 25 2109 r 172
1041 L 90 826 ” 71 311 * 49
1115 t 63 901 2 54 290 k 42
” Rats received 1% 2,5-hexanedione in the drinking water for 6 weeks. Control rats did not receive 2,5-hexanedione and were pair-fed to the treated rats. Sciatic nerves were incubated for 3 hr at 37.5”C in Krebs-Ringer-bicarbonate buffer that contained 5 &i/ml [“HIacetate and [‘YJglucose. Incorporation of ],‘H]acetate and [“C]glucose into the acylchain and glycerol moieties of triacylglycerols and phospholipids was determined on separate aliquots of the lipid fractions from which incorporation into total triacylglycerols and phospholipids was determined. ” Values are the means ? SE of six rats. * Significantly different from control values by Students’ independent t test, p < 0.05.
Whether or not the inhibition of lipogenesis from [3H]acetate is a direct effect of iodoacetate or a consequence of the inhibition of glycolysis remains to be elucidated. These studies illustrate the changes in lipid metabolism that would be expected in sciatic nerves if glycolysis were inhibited by compounds that react with sulfhydryl groups. It has been proposed that the biochemical basis of hexacarbon-induced distal axonopathy is the reaction of neurotoxic hexacarbons with sulfhydryl groups of key enzymes in glycolysis (Spencer& al., 1979). The data presented in Table 2 clearly indicate, however, that 2,5-hexanedione does not react with sulfhydryl groups to the extent that it inhibits lipogenesis from glucose in intact sciatic nerves. Little, if any, physiological significance can be attached to the observation that the incorpora-
tion of [“Clglucose into phospholipids was decreased by 1.O mM 2,5-hexanedione since (i) the concentration of 2,5-hexanedione used in this study was lOO-fold greater than that found in sciatic nerves from rats exposed to 1000 ppm of n-hexane for 6 hours or given 1% 2,5-hexanedione in drinking water for 6 weeks (Gillies et al., 1980bl and (ii) phospholipid biosynthesis was not inhibited in 2,5-hexanedione-induced distal axonopathy (Table 31. In addition, in a previous study it was reported that 1.0 mM 2,5-hexanedione did not inhibit lipogenesis from acetate in sciatic nerve (Gillies et rtl., 1980b). These observations are consistent with the report that 2,5-hexanedione is 10,000 times less potent than such classical sulfhydryl inhibiting reagents aspchloromercuribenzoate and N-ethylmaleimide (Graham and Abou-Donia, 1980). Lipogenesis from [ 14C]glucose and [“HI-
292
GILLIES
acetate was examined in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy. Since rats receiving 2,5hexanedione consume less food (Gillies er al., 1980a), and lipid and carbohydrate metabolism are influenced by caloric intake (Romosos and Leveille, 1974), it was essential to use control nerves from animals pair-fed to 2,5-hexanedione-treated animals in these studies. Failure to use nerves from pair-fed control animals may account for the reported decrease in phosphofructokinase activity in brain homogenates, and decreased oxygen uptake in sciatic nerves, from 2,5-hexanedione-treated rats (Sabri et al., 1979b; Couri and Nachtman, 1979). Compared to nerves from PFC rats, nerves from HD rats exhibited a selective inhibition of sterologenesis from [14C]glucase and [3H]acetate (Table 3). Since the incorporation of [14C]glucose into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats (Table 3), the inhibition of sterologenesis from [‘Qglucose cannot be attributed to an inhibition of glycolysis, but must be attributed to a biochemical lesion that occurs after the formation of acetyl-CoA. This is consistent with the reported inhibition of sterologenesis between acetate and mevalonate in sciatic nerves from HD rats. (Gillies et al., 1980b). In summary, the data presented in this paper indicate that while sterologenesis is inhibited in 2,5-hexanedione-induced distal axonopathy, glycolysis is not. REFERENCES COURI, D., AND NACHTMAN, M. P. (1979). Biochemical and biophysical studies of 2,5hexanedione neuropathy. Neurotoxicology 1, 269-283. FOLCH, J., LEES, M.. AND SLOANE-STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol.
Chem.
226, 497-509.
GILLIES, P. J., NORTON. R. M.. AND Bus, J. S. (1980a). Effect of 2,5hexanedione on lipid biosynthesis in sciatic nerve and brain of the rat. Toxicol.
Appl.
Pharmacol.
54, 210-216.
GILLIES, P. J., NORTON, R. M., WHITE, E. L., AND BUS, J. S. (1980b). Inhibition of sciatic nerve
ET AL.
sterologenesis in hexacarbon-induced distal axonopathy in the rat. Toxicol. Appl. Pharmacol. 54, 217-222. GRAHAM, D. G., AND ABOU-DONIA, M. B. (1980). Studies of the molecular pathogenesis of hexane neuropathy. I. Evaluation of the inhibition of glyceraldehyde-3-phosphate dehydrogenase by 2,5hexanedione. J. Toxicol. Environ. Health 6, 621631. KRASAVAGE, W. J., O’DONAGHUE, J. L., DIVINCENZO. G. D., AND TERHAAR, C. J. (1980). The relative neurotoxicity of methyl n-butyl ketone, n-hexane and their metabolites. Toxicol. Appl. Pharmacol. 52,433-441. POWELL, H. C.. KOCH, T., GARRETT, R., AND LAMPERT. P. W. (1978). Schwann cell abnormaiities in 2,5-hexanedione neuropathy. J. Neurocytol. 7, 517-528. ROMSOS, D. R., AND LEVEILLE, G. A. (1974). Effect of diet on activity of enzymes involved in fatty acid and cholesterol synthesis. Advan. Lipid Res. 12, 97- 146. SABRI, M. I.. MOORE, C. L., AND SPENCER, P. S. (1979a). Studies on the biochemical basis of distal axonopathies. I. Inhibition of glycolysis by neurotoxic hexacarbon compounds. J. Neurochem. 32, 683-689. SABRI, M. I.. EDERLE, K., HOLDSWORTH. C. E.. AND SPENCER, P. S. (1979b). Studies on the biochemical basis of distal axonopathies. II. Specific inhibition of fructose-6-phosphate kinase by 2,5hexanedione and methyl butyl ketone. Neurotoxicology. 1, 285-297. SKIPSKI, V. P., PETERSON, R. F.. AND BARCLAY, M. (1964). Quantitative analysis of phospholipids by thin layerchromatography. Biochem. J. 90,374-378. SPENCER, P. S., COURI, D., AND SCHAUMBURG. H. H. (1980). n-Hexane and methyl n-butyl ketone. In Experimentul Clinical Neurotoxicology (P. S. Spencer and H. H. Schaumburg, eds.), pp. 456475. Williams and Wilkins, Baltimore. SPENCER, P. S., SABRI, M. I., SCHAUMBURG, H. H.. AND MOORE, C. L. (1979). Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies? Ann. Neural. 5, 501-507. SPENCER, P. S., AND SCHAUMBURG, H. H. (1975). Experimental neuropathy produced by 2,5-hexanedione, a major metabolite of the neurotoxic industrial solvent methyl n-butyl ketone. J. Neural. Neuurosurg.
Psych&.
38, 771-775.
SPENCER, P. S., AND SCHAUMBURG, H. H. (1978). Pathobiology of neurotoxic axonal degeneration. In Physiology and Pathobiology of Axons (S. G. Waxman, ed.), pp. 265-282. Raven Press, New York. SPERRY, W. M.. AND WEBB, M. (1950). A revision of the Schoenheimer-Sperry method for cholesteroi determination. J. Biol. Chem. 187, 97-106.
oCY
%ND
APPI.IED
PHARMACOI
OGY
59, 287-292
(1981)
Inhibition of Sterologenesis but Not Glycolysis 2,5-Hexanedione-Induced Distal Axonopathy in the Rat PETERJ.GILLIES,'
RONALD M.NORTON,
in
AND JAMES S. Bus
inhibition Axonopathy
of Sterologenesis but Not Glycolysis in 2.5Hexanedione-Induced Distal in the Rat. GILLIES. P. J.. NORTON, R. M., AND BLIS, J. S. (1981). Tu;c,,/. Appl. Phrmacol. 59, 287-292. If glycolysis is inhibited in distal axonopathy. there should be a concomitant inhibition of lipogenesis from glucose. To investigate this possibility. lipogenesis from [‘“Clglucose and [“HIacetate was studied in sciatic nerves incubated with iodoacetate. a known inhibitor of glycolysis, in sciatic nerves incubated with 2.5hexanedione, a putative inhibitor of glycolysis, and in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy induced by 2.5hexanedione. Nerves incubated with 1.0 mM iodoacetate, in comparison with untreated nerves, exhibited decreased incorporation of ]“C]glucose into sterols + diacylglycerols (33.fold), free fatty acids (14-fold), triacylglycerols (27-fold). and phospholipids (21-fold). In addition, these nerves exhibited decreased incorporation of [YH]acetate into sterols + diacylglycerols (30-fold), free fatty acids (2-fold), triacylglycerols (23-fold), and phospholipids (12.fold). In contrast, the incorporation of [“Cjglucose into sterols + diacylglycerols. free fatty acids, and triacylglycerols was not affected by 1.0 mM 2.5.hexanedione. Compared to untreated nerves. nerves incubated with I .O mM 2,5-hexanedione exhibited a small decrease (15%) in the incorporation of [IV]glucose into phospholipids. Nerves from rats given 1% 2,5-hexanedione in the drinking water for 6 weeks, in comparison with those from pair-fed control rats, exhibited decreased (45%) incorporation of [“Clglucose and [3H]acetate into digitonin-precipitable sterols. Nerves from 2,5-hexanedione-treated and pair-fed control rats exhibited similar incorporation of [“C]glucose and [JH]acetate into free fatty acids, triacylglycerols, and phospholipids. The data indicate that while sterologenesis is inhibited in distal axonopathy. glycolysis is not.
Hexacarbon-induced distal axonopathy has frequently been studied in rats given 2.5 hexanedione in the drinking water (Spencer and Schaumburg, 1975; Powell et al., 1978; Krasavage et cl/., 1980). The morphological changes induced by 2,5hexanedione, e.g., giant axonal swelling, focal accumulation of neurofilaments, and paranodal myelin retraction. are similar to those observed in ’ Present address, to which reprint be sent: E. I. DuPont de Nemours Haskell Laboratory of Toxicology Medicine, Newark. Del. 19711.
animals and humans exposed to n-hexane and methyl n-butyl ketone (Spencer et trl., 1980). This mode1 has also been used to investigate biochemical changes in distal axonopathy. Cholesterol biosynthesis has been reported to be inhibited in sciatic nerves from rats fed 2,5-hexanedione (Gillies et al., 1980a). The site of inhibition in sterologenesis was found to be between acetyl-CoA and mevalonate and likely involved the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (Gillies et ul., 1980b). The inhibition of sterologenesis may
requests should & Co., Inc.. and Industrial
287
0041.008x/81/080287-06$02.00/O Copyrlghi All right,
$, 1981 by Academic Press. Inc. of reproductmn in any form reserved.
288
GILLIES
be related to the decreased microviscosity in sciatic nerves that is reported to precede the development of clinical signs of peripheral neuropathy in 2,5-hexanedione-treated rats (Couri and Nachtman, 1979). In addition to sterologenesis, there is indirect evidence that glycolysis may also be inhibited in 2,5-hexanedione-induced distal axonopathy. Purified preparations of phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and glycerol-3-phosphate dehydrogenase have been reported to be inhibited by high concentrations of 2,5hexanedione (Spencer and Schaumburg, 1978; Sabri et al., 1979a, b). Since dithiothreitol prevented this inhibition, it was suggested that 2,5-hexanedione reacts with sulfhydryl groups of key enzymes in glycolysis. These studies formed the experimental basis of the hypothesis that the biochemical basis of hexacarbon-induced distal axonopathy is an inhibition of glycolysis (Spencer et al., 1979). If glycolysis is inhibited in distal axonopathy, then lipogenesis from glucose should also be inhibited. To investigate this possibility, lipogenesis from [14C]glucose and [“HIacetate was studied in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy induced by 2,5-hexanedione. METHODS Animals
and Diets
Male Fischer-344 rats (Charles River Company, Wilmington, Mass.: weight, 187 2 5 g) were randomly divided into two groups, six animals per group. The experimental group of rats (HD rats) received 1% 2.5 hexanedione (Eastman Kodak, Rochester, N.Y.; redistilled before use) in the drinking water for 6 weeks; food and water were available ad libitum. Control (PFC) rats were pair-fed to the HD rats; water was available ad libitum.
Incubation
of Sciatic
Nerves
Rats were sacrificed by cervical dislocation. ments (3-4 cm; weight, 35 it 2 mg) of sciatic
Segnerve
ET AL. were carefully excised, stripped of adhering tissue, and incubated in 2.0 ml of Krebs-Ringer-bicarbonate buffer, pH 7.4, that contained 100 units and 100 pg/ml of penicillin G (sodium salt) and streptomycin sulfate (Sigma Chemical Company, St. Louis. MO.), respectively, and 10 &i each of D-[IC(U)]glucose (New England Nuclear, Boston: sp act. 13.9 mCi/mmol; radiochemical purity, 99%) and [3H]acetate (sodium salt) (New England Nuclear: sp act. 140.0 mCi/mmol; radiochemical purity, 99%). In experiments in which 1 .O mM iodoacetate (Sigma) or 1.0 mM 2.5hexanedione was added to the incubation medium. penicillin G and streptomycin sulfate were omitted. Incubations were carried out in 25-ml Erlenmeyer flasks, gassed with O,/CO, (95/5%), stoppered. and slowly agitated in a water bath at 37.5”C. After 3 hrs, the nerves were removed from the incubate, rinsed in 1% glucose plus 1% sodium acetate, and then homogenized in 20 vol of chIoroform:methanol ( 1: I).
Extraction
oj’ Lipids
The nerve homogenate was centrifuged at 3008 for 5 min, and the supemate decanted and adjusted to 2: 1 chloroform:methanol. The homogenate residue was extracted repeatedly with chloroform:methanol (2:l) until radioactivity was no longer detected in the supemate. A phase separation in the combined supemates was induced by the addition of 0.2 vol of 0.73% sodium chloride and the lower phase Folchwashed (Folch et al., 1957). The lower phase was washed with theoretical upper phase that contained 0.29% sodium chloride, 1% glucose, and 1% sodium acetate, followed by upper phase that contained only 0.29% sodium chloride, until radioactivity was no longer detected in the washes. The lower phase was evaporated to near-dryness under nitrogen and the lipid extract dissolved in chloroform:methanol (2: I). Digitonin (0.5%, w/v, in 50% ethanol) was added to an aliquot of total lipid extract reconstituted in acetone:ethanol (1: 1). Sterol digitonides were washed with acetone:ether (1:2), then dissolved in pyridine and water, and the sterols were extracted into ether (Sperry and Webb, 1950). The ether extract was evaporated to dryness under nitrogen and the radioactivity in the samples was determined. Saponijcation
of Lipids
Phospholipids and triacylglycerols. isolated, identified, and eluted from thin-layer chromatographic plates, were saponified in 82% ethanolic-11% potassium hydroxide at 65°C for 2 hr. Recovery of lipids from tic plates was greater than 90%. The saponification mixture was cooled and diluted 1:l with water. Nonsaponifiable lipids were extracted into n-hexane
EFFECT
OF 2,5-HEXANEDIONE TABLE
EFFECT
OF IODOACETATE AND [3H]A~~~~~~
ON
289
LIPOGENESIS
1
ON THE INCORPORATION OF [YJGLUCOSE INTO LIPIDS BY SCIATIC NERVE”
Incorporation
(dpmimg
wet weight)”
[“ClGlucose Lipid
fractions
Sterols + diacylglycerols Free fatty acids Triacylglycerols Phospholipids n Control nerves were 2.5 &i/ml [Wlglucose and h Values are the means * Significantly different
Control 1024 42 752 1960
+ t + 2
Treated 29 2 128 37
Chromutogruphy
The lipid extract was fractionated on a precoated silica gel 60 thin-layer chromatography plate (EM Laboratories Inc., Elmsford, N.Y.; layer thickness, 0.25 mm) in a solvent system consisting of nhexane-diethyl ether-glacial acetic acid (14650~8). Lipid bands were visualized by iodine vapor and identified by comparison of R, values with those of lipid standards (Sigma). Neutral lipids were eluted from the thin-layer chromatography plate with chloroform; phospholipids were eluted according to the method of Skipski (Skipski et al., 1964). The incorporation of radioactivity into lipid was determined by standard dual-label liquid scintillation counting techniques. Quench corrections were made by the external standardization method.
Strrtisticul
31 2 10* ND* 28 + 7* 94 2 7’
Control 1667 97 164 893
t + + t
Treated 135 3 55 59
56 2 6* 55 rt 6* ND* 76 2 9*
incubated for 3 hr at 37.5”C in Krebs-Ringer-bicarbonate buffer that contained [3H]acetate; in addition, treated nerves were incubated with 1.0 mM iodoacetate. f SE of six rats: ND, not detectable. from control values by Students’ independent I test, p < 0.05.
(3 x 10 ml): the n-hexane extracts were backwashed with 5 ml of water. The saponification mixture was then adjusted to pH 1.5 by the addition of 6 N HCl, and the free fatty acids were extracted into diethyl ether (3 x 10 ml); the diethyl ether extracts were backwashed with 5 ml of water at pH 1.5. [‘4C]Glycerol incorporated into phospholipids and triacylglycerols was determined by counting the radioactivity remaining in the saponification mixture after the n-hexane and diethyl ether extractions.
Thin-Layer
[3H]Acetate
Andyses
Students’ t test for comparing unpaired samples was used; p values were derived from a two-tailed table of Students’ values for t. The level of significance was chosen as p < 0.05.
RESULTS Ejyect of Iodoacetate and 2,5-Hexanedione on the Incorporation of [14C]Glucose and [3H]Acetate into Lipids by Sciatic Nerve
Nerves were incubated with 1.0 mM iodoacetate to determine what effects an inhibition of glycolysis would have on lipogenesis from [14C]glucose. Lipogenesis from [3H]acetate was examined in the same nerves to determine whether or not iodoacetate could inhibit lipogenesis directly as well as indirectly subsequent to an inhibition of glycolysis. The incorporation of [“Clglucose and [3H]acetate into lipid was significantly decreased by iodoacetate (Table 1). Incorporation of [‘“Clglucose was decreased into sterols + diacylglycerols (33fold), free fatty acids (lCfold), triacylglycerols (27-fold), and phospholipids (21fold). Similarly, incorporation of [3H]acetate was decreased into sterols + diacylglycerols (30-fold), free fatty acids (Zfold), triacylglycerols (23-fold), and phospholipids (1Zfold). Lipogenesis from [ 14C]glucose was also examined in nerves incubated with 1.0 mM 25hexanedione. In contrast to that of iodoacetate, the incorporation of [‘“Clglucase into sterols + diacylglycerols, free
290
GILLIES TABLE
EFFECT OF 25HEXANEDIONE OF [%]GLUCOSE INTO
2
LIPIDS
ON THE INCORPORATION BY SCIATIC NERVE”
Incorporation (dpm/mg wet weightY Lipid fractions Sterols + diacylglycerols Free fatty acids Triacylglycerols Phospholipids
Control 1691 85 685 3958
t k k +
137 4 148 86
Treated 1414 74 802 3326
+ 5 2 r
135 4 194 115*
u Control nerves were incubated for 3 hr at 37.5”C in Krebs-Ringer-bicarbonate buffer that contained 5 &i/ml of [“Clglucose; in addition, treated nerves were incubated with 1.0 mrvt 2,5-hexanedione. b Values are the means k SEM of six rats. * Significantly different from control values by Students’ independent f test, p < 0.05.
ET AL.
into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats. Incorporation of [3H]acetate was significantly decreased into both sterols + diacylglycerols (42%) and digitonin-precipitable sterols (46%) in HD compared to PFC rats. [3H]Radioactivity in digitonin-precipitable sterols accounted for all of the radioactivity in the combined sterol + diacylglycerol fraction. Incorporation of [“HIacetate into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats. The distribution of 3H and ‘C radioactivity in the acyl chain and glycerol moieties of triacylglycerols and phospholipids was not significantly different in HD and PFC rats (Table 3).
DISCUSSION fatty acids, and triacylglycerols was not affected by 2,5-hexanedione; the incorporation of [14C]glucose into phospholipids was slightly decreased (15%) (Table 2). Effect of Chronic Exposure to 2,5-Hexanedione on the Incorporation of [‘4C]Glucose and r3H]Acetate into Lipids by Sciatic Nerve
Rats given 1% 2,5-hexanedione for 6 weeks exhibited everted and flat foot placement, hindlimb weakness, and ataxia. These clinical signs of neuropathy were first discernible after 4 weeks and were clearly evident after 6 weeks. Similar changes were not observed in PFC rats. Lipogenesis from [14C]glucose and [3H]acetate was examined in sciatic nerves from HD and PFC rats; the data are presented in Table 3. Incorporation of [14C]glucose was significantly decreased into digitoninprecipitable sterols (45%) in HD compared to PFC rats. 14C Radioactivity in digitoninprecipitable sterols accounted for 30-40% of the total 14C radioactivity incorporated into the combined sterol + diacylglycerol fraction. The incorporation of [l*C]glucose
If glycolysis is inhibited in distal axonopathy, there should be a concomitant inhibition of lipogenesis from glucose. This possibility was investigated in sciatic nerves incubated with iodoacetate, a known inhibitor of glycolysis, in sciatic nerves incubated with 2,5-hexanedione, a putative inhibitor of glycolysis, and in sciatic nerves from rats exhibiting clinical signs of distal axonopathy. In these studies, lipogenesis from both [‘Clglucose and [“HIacetate was examined; these dual-label studies permitted the differentiation of effects on glycolysis vs lipogenesis. Iodoacetate inhibits glyceraldehyde-3-phosphate dehydrogenase in glycolysis; inhibition of this enzyme would be expected to decrease the formation of glycerol-3-phosphate and acetyl-CoA available for lipid biosynthesis. This prediction was substantiated by the observation that the incorporation of [ ‘Clglucose into sterols, free fatty acids, triacylglycerols, and phospholipids was markedly decreased by iodoacetate (Table 1). The incorporation of 13H]acetate into these same lipids was also significantly decreased by iodoacetate.
EFFECT
OF 2JHEXANEDIONE
ON
TABLE INCORPORATION
OF [‘“C]GLUCOSE FROM RATS
291
LIPOGENESIS
3
AND [$H]ACETATE INTO LIPIDS WITH DISTAL AXONOPATHY”
Incorporation
(dpm/mg
BY SCIATIC
wet weight)”
[“CjGlucose
Lipid Sterols
+ diacylglycerols
Digitonin-precipitable Free
Pair-fed controls
fractions
fatty
acids
sterols
NERVES
[“HIAcetate
2JHexanedione treated
Pair-fed controls
2.5Hexanedione treated
1153 5 145
878 2 128
1067 rt 84
SO4 k 46
278 t 15*
935 k 71 186 i
615 2 38* 506 i
31*
98 2 7
85 2 6
15
232 2 22
Triacylglycerols Fatty acids Glycerol
2748 k 41.5 278 t 83 2824 + 326
3441 2 499 190 k 50 3428 2 414
415 c 79 269 2 54 62 2 15
419 lr 32 234 2 25 60t 13
Phospholipids Fatty acids Glycerol
2152 k 171 883 k 101 1799 2 195
2142 ? 96 640 t 25 2109 r 172
1041 L 90 826 ” 71 311 * 49
1115 t 63 901 2 54 290 k 42
” Rats received 1% 2,5-hexanedione in the drinking water for 6 weeks. Control rats did not receive 2,5-hexanedione and were pair-fed to the treated rats. Sciatic nerves were incubated for 3 hr at 37.5”C in Krebs-Ringer-bicarbonate buffer that contained 5 &i/ml [“HIacetate and [‘YJglucose. Incorporation of ],‘H]acetate and [“C]glucose into the acylchain and glycerol moieties of triacylglycerols and phospholipids was determined on separate aliquots of the lipid fractions from which incorporation into total triacylglycerols and phospholipids was determined. ” Values are the means ? SE of six rats. * Significantly different from control values by Students’ independent t test, p < 0.05.
Whether or not the inhibition of lipogenesis from [3H]acetate is a direct effect of iodoacetate or a consequence of the inhibition of glycolysis remains to be elucidated. These studies illustrate the changes in lipid metabolism that would be expected in sciatic nerves if glycolysis were inhibited by compounds that react with sulfhydryl groups. It has been proposed that the biochemical basis of hexacarbon-induced distal axonopathy is the reaction of neurotoxic hexacarbons with sulfhydryl groups of key enzymes in glycolysis (Spencer& al., 1979). The data presented in Table 2 clearly indicate, however, that 2,5-hexanedione does not react with sulfhydryl groups to the extent that it inhibits lipogenesis from glucose in intact sciatic nerves. Little, if any, physiological significance can be attached to the observation that the incorpora-
tion of [“Clglucose into phospholipids was decreased by 1.O mM 2,5-hexanedione since (i) the concentration of 2,5-hexanedione used in this study was lOO-fold greater than that found in sciatic nerves from rats exposed to 1000 ppm of n-hexane for 6 hours or given 1% 2,5-hexanedione in drinking water for 6 weeks (Gillies et al., 1980bl and (ii) phospholipid biosynthesis was not inhibited in 2,5-hexanedione-induced distal axonopathy (Table 31. In addition, in a previous study it was reported that 1.0 mM 2,5-hexanedione did not inhibit lipogenesis from acetate in sciatic nerve (Gillies et rtl., 1980b). These observations are consistent with the report that 2,5-hexanedione is 10,000 times less potent than such classical sulfhydryl inhibiting reagents aspchloromercuribenzoate and N-ethylmaleimide (Graham and Abou-Donia, 1980). Lipogenesis from [ 14C]glucose and [“HI-
292
GILLIES
acetate was examined in sciatic nerves from rats exhibiting clinical signs of peripheral neuropathy. Since rats receiving 2,5hexanedione consume less food (Gillies er al., 1980a), and lipid and carbohydrate metabolism are influenced by caloric intake (Romosos and Leveille, 1974), it was essential to use control nerves from animals pair-fed to 2,5-hexanedione-treated animals in these studies. Failure to use nerves from pair-fed control animals may account for the reported decrease in phosphofructokinase activity in brain homogenates, and decreased oxygen uptake in sciatic nerves, from 2,5-hexanedione-treated rats (Sabri et al., 1979b; Couri and Nachtman, 1979). Compared to nerves from PFC rats, nerves from HD rats exhibited a selective inhibition of sterologenesis from [14C]glucase and [3H]acetate (Table 3). Since the incorporation of [14C]glucose into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats (Table 3), the inhibition of sterologenesis from [‘Qglucose cannot be attributed to an inhibition of glycolysis, but must be attributed to a biochemical lesion that occurs after the formation of acetyl-CoA. This is consistent with the reported inhibition of sterologenesis between acetate and mevalonate in sciatic nerves from HD rats. (Gillies et al., 1980b). In summary, the data presented in this paper indicate that while sterologenesis is inhibited in 2,5-hexanedione-induced distal axonopathy, glycolysis is not. REFERENCES COURI, D., AND NACHTMAN, M. P. (1979). Biochemical and biophysical studies of 2,5hexanedione neuropathy. Neurotoxicology 1, 269-283. FOLCH, J., LEES, M.. AND SLOANE-STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol.
Chem.
226, 497-509.
GILLIES, P. J., NORTON. R. M.. AND Bus, J. S. (1980a). Effect of 2,5hexanedione on lipid biosynthesis in sciatic nerve and brain of the rat. Toxicol.
Appl.
Pharmacol.
54, 210-216.
GILLIES, P. J., NORTON, R. M., WHITE, E. L., AND BUS, J. S. (1980b). Inhibition of sciatic nerve
ET AL.
sterologenesis in hexacarbon-induced distal axonopathy in the rat. Toxicol. Appl. Pharmacol. 54, 217-222. GRAHAM, D. G., AND ABOU-DONIA, M. B. (1980). Studies of the molecular pathogenesis of hexane neuropathy. I. Evaluation of the inhibition of glyceraldehyde-3-phosphate dehydrogenase by 2,5hexanedione. J. Toxicol. Environ. Health 6, 621631. KRASAVAGE, W. J., O’DONAGHUE, J. L., DIVINCENZO. G. D., AND TERHAAR, C. J. (1980). The relative neurotoxicity of methyl n-butyl ketone, n-hexane and their metabolites. Toxicol. Appl. Pharmacol. 52,433-441. POWELL, H. C.. KOCH, T., GARRETT, R., AND LAMPERT. P. W. (1978). Schwann cell abnormaiities in 2,5-hexanedione neuropathy. J. Neurocytol. 7, 517-528. ROMSOS, D. R., AND LEVEILLE, G. A. (1974). Effect of diet on activity of enzymes involved in fatty acid and cholesterol synthesis. Advan. Lipid Res. 12, 97- 146. SABRI, M. I.. MOORE, C. L., AND SPENCER, P. S. (1979a). Studies on the biochemical basis of distal axonopathies. I. Inhibition of glycolysis by neurotoxic hexacarbon compounds. J. Neurochem. 32, 683-689. SABRI, M. I.. EDERLE, K., HOLDSWORTH. C. E.. AND SPENCER, P. S. (1979b). Studies on the biochemical basis of distal axonopathies. II. Specific inhibition of fructose-6-phosphate kinase by 2,5hexanedione and methyl butyl ketone. Neurotoxicology. 1, 285-297. SKIPSKI, V. P., PETERSON, R. F.. AND BARCLAY, M. (1964). Quantitative analysis of phospholipids by thin layerchromatography. Biochem. J. 90,374-378. SPENCER, P. S., COURI, D., AND SCHAUMBURG. H. H. (1980). n-Hexane and methyl n-butyl ketone. In Experimentul Clinical Neurotoxicology (P. S. Spencer and H. H. Schaumburg, eds.), pp. 456475. Williams and Wilkins, Baltimore. SPENCER, P. S., SABRI, M. I., SCHAUMBURG, H. H.. AND MOORE, C. L. (1979). Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies? Ann. Neural. 5, 501-507. SPENCER, P. S., AND SCHAUMBURG, H. H. (1975). Experimental neuropathy produced by 2,5-hexanedione, a major metabolite of the neurotoxic industrial solvent methyl n-butyl ketone. J. Neural. Neuurosurg.
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SPENCER, P. S., AND SCHAUMBURG, H. H. (1978). Pathobiology of neurotoxic axonal degeneration. In Physiology and Pathobiology of Axons (S. G. Waxman, ed.), pp. 265-282. Raven Press, New York. SPERRY, W. M.. AND WEBB, M. (1950). A revision of the Schoenheimer-Sperry method for cholesteroi determination. J. Biol. Chem. 187, 97-106.