STUDIES OF THE MOLECULAR PATHOGENESIS

OF HEXANE NEUROPATHY. I. EVALUATION OF THE

INHIBITION OF GLYCERALDEHYDE-3-PHOSPHATE
DEHYDROGENASE BY 2,5-HEXANEDIONE

Doyle G. Graham, Mohamed B. Abou-Donia

Departments of Pathology and Pharmacology, Duke

University Medical Center, Durham, North Carolina

Inhibition of the sulfhydryi enzyme glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) by 2,5-hexanedione (2,5-HD) was found to be irreversible, proceeding via a
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reversible enzyme-inhibitor intermediate, while acetone was a weak reversible inhibi-

tor. Comparison of 2,5-HD and acetone with p-chloromercuribenzoate (PCMB) and
N-ethylmaleimide (NEM) demonstrated that the former are not significant sulfhydryi
reagents, since they must be present at more than 104 times higher concentrations
than PCMB or NEM to effect measurable inhibition of this enzyme. Thus it is unlikely
that inhibition of GAPDH by 2,5-HD has any significance in the molecular
pathogenesis of hexane neuropathy. The irreversibility of 2,5-HD inhibition, on the
other hand, suggests that 2,5-HD reacts with amino groups rather than sulfhydryi
groups on proteins. This reaction is proposed as the molecular lesion in hexane
neuropathy.

INTRODUCTION
Degeneration of peripheral and central nervous system axons after
exposure to hexane is a problem of industrial exposure and inhalant abuse
(Herskowitz et al., 1971; Towfighi et al., 1976). Like humans, rodents
exposed to hexane develop progressive distal hind limb paralysis (Schaum-
burg and Spencer, 1976). The structural correlate of this paralysis is axonal
degeneration, which begins with multifocal swelling of axons due to the
accumulation of large numbers of 10-nm neurofilaments (Spencer and
Schaumburg, 1977a, 1977b). This lesion is also seen in inherited giant
axon neuropathy in humans and in acrylamide and carbon disulfide
intoxication (Spencer and Schaumburg, 1978; Schaumburg et al., 1974;
Prineas, 1969; Szendzikowski et al., 1974). Chronic exposure to the
hexane metabolites methyl /7-butyl ketone (MnBK), 2,5-hexanediol, and
2,5-hexanedione (2,5-HD) results in the same clinical and pathological state
(Mendell et al., 1974; Allen et al., 1975; Spencer and Schaumburg, 1975,
1976; Spencer et al., 1975,1978).
In recent publications Spencer and co-workers (Spencer et al., 1979;

We wish to thank Drs. D. B. Menzel, I. Fridovich, and K. Boekelheide for helpful suggestions.
Requests for reprints should be sent to Doyle G. Graham, Department of Pathology, Duke
University Medical Center, Durham, North Carolina 27710.
621

Journal of Toxicology and Environmental Health, 6:621-631, 1980

Copyright © 1980 by Hemisphere Publishing Corporation
0098-4108/80/030621-11$2.25
 622 D. G. GRAHAM AND M. B. ABOU-DONIA

Sabri et al., 1979a, 1979b) posulated that MnBK, 2,5-HD, acrylamide, and
carbon disulfide impair energy synthesis in the axon through inhibition
of the sulfhydryl enzymes glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and phosphofructokinase (PFK). According to their hypothesis
this defect in energy metabolism reduces axonal transport, which then leads to
axonal degeneration. They observed that the concentrations of MnBK and
2,5-HD required for inhibition of GAPDH were in the range 10-50 mM
and that preincubation of these agents with GAPDH was required before
any inhibition occurred (Sabri et al., 1979a).
The present study was designed to test this hypothesis and was
stimulated by our previous experience with sulfhydryl enzymes and with
reversible versus irreversible enzyme inhibition (Graham et al., 1977, 1978;
Abou-Donia and Rosen, 1977). The concentrations of sulfhydryl reagents
such as p-chloromercuribenzoate (PCMB), /V-ethylmaleimide (NEM), and
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various quinone species required for significant inhibition of sulfhydryl

enzymes are in the low micromolar range—1000 to 10,000 times less than
those of 2,5-HD and MnBK (Graham et al., 1977, 1978). In addition, the
observation by Sabri et al. (1979a) that preincubation was required for
inhibition raised the possibility that MnBK and 2,5-HD are not reversible
inhibitors of these enzymes, but that the inhibition is irreversible. If this is
the case, Michaelis-Menten kinetics cannot appropriately be applied and
the conclusion from the Lineweaver-Burke plot of their data that MnBK
and 2,5-HD yield a "mixed type of inhibition" (mixed competitive and
noncompetitive) is not valid.
In this study we compared the nonneurotoxic ketone acetone with its
dimer, 2,5-HD, as inhibitors of crystalline GAPDH. The inhibition was
contrasted with that of two different sulfhydryl reagents, the known
reversible inhibitor PCMB and the irreversible inhibitor NEM. The activity
of GAPDH was assayed as described by Park et al. (1961); GAPDH is
stripped of NAD by treatment with charcoal and functions as an esterase,
hydrolyzing p-nitropheny! acetate. This hydrolysis proceeds via a thioester
of the active site cysteine, Cys-149 (Harris et al., 1963), and is thus very
sensitive to inhibition by sulfhydryl reagents.

METHODS

Acetone (99.5%) was obtained from Matheson, Coleman, and Bell,

Norwood, Ohio; 2,5-HD (acetonylacetone, 97%) from Aldrich Chemical
Co., Milwaukee, Wise; and PCMB (C 4378), NEM (E 3876), and crystal-
line rabbit muscle GAPDH (G 5126) from Sigma Chemical Co., Saint
Louis, Mo.
GAPDH was treated twice with charcoal to remove bound NAD, then
dialyzed twice against 1000-fold volumes of 20 m/W Tris, 1 m/W EDTA, pH
8.0, to remove (NH 4 ) 2 SO 4 . GAPDH activity was recorded at 31°C by
following the hydrolysis of p-nitrophenyl acetate at 400 nm with a Gilford
 2,5-HEXANEDlONE IN HEXANE NEUROPATHY 623

recording spectrophotometer (Park et al., 1961). All components of the

reaction mixture (2.9 ml)..were preincubated for 5 min at 31 °C to record
baseline hydrolysis of p-nitrophenyl acetate and the reaction was started
with the addition of GAPDH in 0.1 ml. The final concentration of
GAPDH was 0.37 jiM [e\°js = 8 . 1 5 (Murdoch and Koeppe, 1964); absor-
bance ratio A2&0/A260 ~ 2.13]. The rate of p-nitrophenyl acetate hydro-
lysis in the presence of inhibitors was compared with that of uninhibited
GAPDH.

RESULTS
When GAPDH was assayed in the presence of acetone, slight inhibition
was observed at 25 and 50 m/W. The degree of inhibition was the same at
the beginning of incubation as after 15 min at 31 °C, suggesting that the
inhibition was reversible (Fig. 1). By contrast, there was enhanced
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inhibition of GAPDH by 2,5-HD with increasing incubation time at 31°C,

characteristic of irreversible enzyme inhibition. In Fig. 1 it can be seen
that the inhibition lines for 2,5-HD intercept the y axis at points below
100% of control activity (log 100% = 2), revealing that the enzyme is
inhibited at zero time. Inhibition at zero time, as defined by Aldridge
(1950), results from the intermediate production of a reversible enzyme-
inhibitor complex. The progression of inhibition with incubation time,
with the lines becoming roughly parallel, indicates saturation of the
enzyme by irreversibly bound inhibitor. As shown by Aldridge (1950),
Main (1964, 1969), and Abou-Donia and Rosen (1977), this phenomenon
can be explained by the kinetic scheme:

E + I ^ = t = i El — ^ — E l ' (1)
I *" t
*/
where E is the free enzyme; I the inhibitor; El the intermediate reversible
complex, whose formation is controlled by the equilibrium constant Ka
(= k-1/k1); and El' the enzyme bound irreversibly by inhibitor, whose rate
of formation is governed by k2. Finally, k; (—k2/Ka) is the rate constant
governing the overall rate of inhibition, the bimolecular rate constant.
As derived by Main (1964, 1969), k2 and Ka can be calculated from
the relation between progressive inhibition and time and inhibitor concen-
tration that is valid when

[I] > [ E ] k-i >k2

Thus

1 1 dt k2 1
(2)
[II 2. 303 d log V Ka Ka
 624 D. G. GRAHAM AND M. B. ABOU-DONIA

25 mM n
2 0-
• 1 » ± ' 50mM g

* 2.0'
__^__5mM .~

I ,8- —_a^lOmM 2
— • - . 0
* 0.

5'
-1 1.6

—1—1—1—1— 1 1
0 3 6 9 12 15 18
Incubation Time ( minutes)

FIGURE 1. Inhibition of GAPDH by acetone and 2,5-HD. GAPDH was assayed in the presence of
acetone or 2,5-HD at the concentrations given. The least mean squares fit is drawn for each.
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Preincubation of GAPDH with 2,5-HD for 5, 10, and 15 min in the absence of p-nitrophenyl acetate
gave the same rates of inhibition as seen in the presence of substrate. The correlation coefficient (r)
for the plot of log % remaining activity versus time of incubation for no preincubation and 5, 10,
and 15 min preincubation was +0.97.

where [I] is the concentration of inhibitor, t the incubation time, and d

log V the change in the velocity of the reaction caused by inhibition.
When 1/[l] is plotted against dt/2.303d log V, a straight line is obtained
with slope kh y-axis intercept —1//C0) and x-axis intercept 1//?2-
As shown in Fig. 2, the kinetics of inhibition of GAPDH by 2,5-HD
were compatible with the Main equation, Eq. (2). The value of the
dissociation constant, Ka, was 62.8 mM; that of the rate constant for

200 -

- 100 -

100

2.303 fl log V
-50L

FIGURE 2. Main plot of inhibition of GAPDH by 2,5-HD. The change in velocity with incubation
time was calculated from the data in Fig. 1 for each concentration of 2,5-HD. Correlation
coefficient r = +0.99.
 2,5-HEXANEDIONE IN HEXANE NEUROPATHY 625

TABLE 1. Inhibition of GAPDH

Inhibitor Inhibition Time for 50%

concentration at zero time inhibition
Inhibitor (m/W) (%) (min)

2,5-HD 5 9.9 > 100.0

10 17.5 21.6
20 30.0 8.5
30 43.2 3.0
Acetone 25 3.4 -
50 11.6 -
PCMB 0.00102 38.3 —
0.00170 70.2 —
0.00340 87.0 —
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irreversible binding, k2, was 0.15 min ' ; and the bimolecular rate
constant, kit was 2.32 M~x min" 1 .
In Fig. 1 the intercept on t h e y axis gives the amount of inhibition at
zero time, a measure of the reversible phase of inhibition. Table 1 shows
that the inhibition at zero time by 2,5-HD is more than twice as great as
that by acetone. Table 1 also presents the inhibition of GAPDH by the
sulfhydryl reagent PCMB, which, like acetone, inhibited GAPDH to the
same degree at zero time as after 15 min incubation at 31 °C. On a molar
basis, however, the inhibition of GAPDH by PCMB was greater than that
by 2,5-HD or acetone by a factor of more than 10 4 .
The effectiveness of NEM as an inhibitor of GAPDH was likewise
much greater than that of 2,5-HD or acetone. As shown in Fig. 3, the
time course of NEM inhibition was different from that of 2,5-HD. There
was an initial rapid loss of enzyme activity, which extrapolated to 100%
activity at zero time, followed by a lower rate of inhibition. At 31 °C, the
rate inhibition by high concentrations of NEM was too fast to measure by
these techniques, so that micromolar concentrations of NEM were
employed. Since the concentration of active-site sulfhydryl groups
(4 X 0.37 ixM = 1.48 juA7) was in the range of NEM concentrations tested,
the departure from pseudo-first-order kinetics was the result of mutual
depletion of enzyme and inhibitor from the reaction (Webb, 1963).
Assuming that the initial reaction between NEM and GAPDH occurred
with the active-site Cys-149 (Harris et al., 1963), resulting in a stoichio-
metric depletion of the NEM concentration with loss of enzyme activity,
the reduction in NEM concentration was 20.3 ±1.4% (mean ± SE) in the
first minute of reaction with the five levels of NEM employed. In a
bimolecular reaction with constant [I] the time for 50% inhibition, f o .s, is
related to the concentration of inhibitor by

1
(3)
 626 D. G. GRAHAM AND M. B. ABOU-DONIA

[b) (c)

1.0 / 00 -

80 -
' /
60
0.5
40

20
n 1 1 1 1 I i
0
I i i
3 6 9 12 D 1 2 3 4
Incubation Time (mm) T
0.5 (min) tNEM] ((iMI

FIGURE 3. Inhibition of GAPDH by NEM. (a) The time course of NEM inhibition, shown here at
an NEM concentration of 1.7 v-M, discloses initial rapid inhibition followed by a slower progressive
loss of GAPDH activity, (£>) Extrapolation of the rate of inhibition in the first minute of the
reaction at 31°C yields the time required for .50% inhibition. Here the reciprocal of NEM
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concentration is plotted against fn-s (r=+0.99). (c) If the lower rate of GAPDH inhibition
presented in (a) was extrapolated to zero time, a measure of the amount of inhibition in the rapid
phase of reaction between GAPDH and NEM was obtained. Significant inhibition was achieved at
low micromolar concentrations of NEM during this period.

In a plot of 1/[l] against 10 5 , the slope is kn the bimolecular rate

constant (Aldridge, 1950). In Fig. 3b, a straight line was obtained for the
first minute of reaction of NEM with GAPDH, consistent with the
conclusion that the reaction is bimolecular. The value of k,- from these
data was 1.7 X 10 s AT1 min" 1 .
If the low rate of GAPDH inhibition by NEM in Fig. 3o is extra-
polated to zero time, the net inhibition achieved by the fast reaction
between NEM and GAPDH can be calculated. Figure 3c shows that
significant inhibition .of GAPDH was achieved in the early minutes of
reaction at low micromolar concentrations of NEM.

DISCUSSION
This study shows that while 2,5-HD is an irreversible inhibitor of
GAPDH, its inhibition is distinguished from that of the sulfhydryl reagents
PCMB and NEM by two features. First, the concentrations of 2,5-HD
required to effect comparable levels of inhibition are more than 10 4 times
greater than those of PCMB or NEM. Second, the bimolecular rate
constant /?,- for the reaction between GAPDH and 2,5-HD is 10 s times less
than that for the reaction of GAPDH with NEM. Thus, if 2,5-HD inhibits
GAPDH through its reactivity as a sulfhydryl reagent, it is an exceedingly
weak one, and it is unlikely that the initial biochemical event in hexane
neuropathy is inhibition of GAPDH and PFK by 2,5-HD, as proposed
earlier (Spencer et al., 1979; Sabri et al., 1979a, 1979b). Another feature
one would expect to see if 2,5-HD significantly impaired glycolysis is
hemolysis, such as occurs when patients have reduced levels of a glycolytic
 2,5-HEXANEDIONE IN HEXANE NEUROPATHY 627

enzyme such as pyruvate kinase (Oski and Diamond, 1963). That

hemolysis has not been documented in patients or experimental animals
must be taken as additional evidence against the energy deficiency
hypothesis in hexane neurotoxicity.
On the other hand, the present study does point to a significant
difference between nonneurotoxic acetone and its neurotoxic dimer
2,5-HD, which may be a clue to the molecular pathogenesis of hexane
neuropathy. While both are only weak inhibitors of GAPDH, the inhibi-
tion by 2,5-HD is irreversible and that by acetone is reversible. In
considering the possible reactive groups on proteins that could result in
reversible binding to acetone but irreversible binding to 2,5-HD, it is clear
that sulfhydryl groups are not candidates. The reaction of a ketone with a
sulfhydryl group is reversible, as shown in Fig. 4. A hemithioacetal can be
formed, but reactions leading to irreversibility are not available. This
reaction between sulfhydryl groups and ketones may, in fact, explain how
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dithiothreitol protected GAPDH from inhibition by 2,5-HD and MnBK in

the experiments reported by Sabri et al. (1979a).
As proposed in Fig. 5, reaction of 2,5-HD with amino groups on
proteins could account for the difference between 2,5-HD and acetone as
GAPDH inhibitors. A monoketone such as acetone can react reversibly
with an amino group to form a Schiff base or imine. A diketone such as
2,5-HD could react with one or two amino groups. The 2,5 spacing of the
ketones in 2,5-HD could in theory result in the equilibria shown, both of
which would yield a conjugated Schiff base. Such a compound would be
expected to be stable (Chio and Tappel, 1969) and thus yield the kinetics
of irreversible binding.
These reaction sequences would be consistent with the kinetics of
2,5-HD inhibition of GAPDH. The initial binding of 2,5-HD with one or
two amino groups could represent the reversible phase of binding in Eq.
(1), while the equilibria leading to stable conjugated dienes and Schiff
bases would represent the irreversible phase. It is noteworthy that
inactivation of GAPDH occurs when pyridoxal 5'-phosphate reacts with
Lys-191 or Lys-212 (Forcina et al., 1971).

0 OH
R-C-R'+R'"-SH^R-C-R'
s
R-
FIGURE 4. Reaction of ketones with sulfhydryl groups. Reaction of a ketone with a single
sulfhydryl group reversibly yields a hemithioacetal. If sulfhydryl groups are in free solution rather
than fixed to proteins, the reversible reaction of a ketone with two sulfhydryl groups can result in
a thioacetal.
 628 D. G. GRAHAM AND M. B. ABOU-DONIA

This hypothesis might also explain why 2,5-HD is neurotoxic but

2,4-hexanedione and 3,5-heptanedione are not (Spencer et al., 1978).
While the latter diketones could react with one or two amino groups and
imine-enamine isomerization yield conjugated Schiff bases, these Schiff
bases would be in equilibrium only with the nonconjugated Schiff base,
which could react with water t o generate the diketone. Support for the
neurotoxic specificity of 2,5-diketones is gained from the recent observa-
tion that 2,5-heptanedione also results in neurotoxicity (Katz et al.,
1980).
An observation that may bear on the toxicity of 2,5-diketones and the
lack of neurotoxicity of 2,4- or 3,5-diketones is the greater water
solubility of the former. We found that 2,4-hexanedione and 2,4-
pentanedione, like MnBK, were not soluble in water at the concentrations
at which 2,5-HD inhibited GAPDH, whereas 2,5-HD and acetone mixed
with water in all proportions. As illustrated in Fig. 6, ketone-enol
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isomerization with 2,4- or 3,5-diketones could result in a six-membered

ring with internal hydrogen bonding, a reaction that would limit the
hydration of these c o m p o u n d s . A much less likely seven-membered ring
would be formed with 2,5-diketones. The oxygens of 2,5-diketones would
thus be much more likely to hydrate, whether in the ketone or the enol

Acetone 2,5-Hexanedione 2,5-Hexanedione

0 0 (} 0 0
CH3-C-CH3 CH 3 -C-CH 2 -CH 2 -C-CH 3 CH 3 -C-CH 2 -CH 2 -C-CH 3
»R-NH2 t*2R-NH2
0 |^-»2H 2 0
R R R R
N N 0 N N
CH3-C-CH3 CH 3 -C-CH 2 -CH 2 -C-CH 3 CH 3 -C-CH 2 -CH 2 -C-CH 3

R R R
NH OH NH NH
CH3 -C=CH - CH = C - C H 3 CH3 -C=CH - CH =C-CH 3

R R R
N OH NH N
C H 3 - C - C H = CH-CH-CH 3 CH 3 -CH-CH=CH-C-CH 3

FIGURES. Proposed reaction of acetone and 2,5-HD with amino groups of proteins. A mono-
ketone such as acetone or MnBK could form an imine (Schiff base) in the reversible reaction at the
left. A diketone could react with one or two amino groups. The equilibria shown for 2,5-
hexanedione require imine-enamine and/or ketone-enol isomerization and migration of double
bonds through reversible dissociation of protons to yield in each case a conjugated Schiff base.
 2,5-HEXANEDIONE IN HEXANE NEUROPATHY 629

0 0
II II
R-C-CH2-C-R"

o^ 11 --o
I II
R ^c •s' R
H
FIGURE 6. Internal hydrogen bonding in 2,4- or 3,5-diketones. Ketone-enol isomerization yields a
stable six-membered ring through hydrogen bonding.
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configuration. Indeed, hydration of ketones would be expected to retard

Schiff base formation and may partially explain the low rate of reaction
between GAPDH and 2,5-HD.
We propose, therefore, that 2,5-HD is toxic to axons by virtue of both
its high water solubility and its capacity to form stable conjugated Schiff
bases with amino groups of proteins. In a separate communication we will
show that conjugated Schiff bases are formed when 2,5-HD reacts with
GAPDH and other proteins and that through reaction with two amino
groups intramolecular and intermolecular cross-linking occurs (D. G.
Graham, B. R. Shaw, R. G. Richards, M. B. Abou-Donia, in preparation).
The latter events would be in theory result in neurofilament aggregation,
which we postulate to be the molecular event leading to axonal degeneration
in hexane neuropathy.

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Received November 10, 1979

Accepted December 29, 1979
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