Effect of disulfiram administration on rat brain glutathione metabolism

Alcohol, Vol. 11, No. 1, pp. 7-10, 1994 Copyright © 1994ElsevierScienceLtd Printed in the USA. All rights reserved 0741-8329/94 $6.00 + .00

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Effect of Disulfiram Administration on Rat Brain Glutathione Metabolism S. N . N A G E N D R A ,

.1 K. T A R A N A T H S H E T T Y / f K. M A M A T H A B. S. S R I D H A R A R A M A R A O *

RAO*

AND

*Department o f Neurochemistry, National Institute o f Mental Health and Neuro Sciences, Bangalore 560 029, India ~Laboratory o f Neurochemistry, National Institute o f Neurological Disorders and Stroke, Bethesda, M D 20892 R e c e i v e d 25 M a r c h 1993; A c c e p t e d 16 J u l y 1993 NAGENDRA, S. N., K. TARANATH SHETTY, K. MAMATHA RAO AND B. S. SRIDHARA RAMA RAO.

Effect of disulfiram administration on rat brain glutathione metabolism. ALCOHOL ll(1) 7-10, 1994.- Chronic administration of disulfiram (DS) to rats was found to affect glutathione (GSH) metabolism. Glutathione was measured in the rat brain following DS administration. Reduced glutathione was decreased significantly (1.52 + 0.3 #tool/g; p < 0.001), with a concomitant increase in oxidised glutathione (GSSG) content (0.12 + 0.013 #mol/g;p < 0.001) in the brain as a consequence of DS treatment. However, total glutathione (GSH + GSSG) content of the experimental group did not show any appreciable change. Similar changes were observed in the liver following chronic DS treatment. Brain glutathione reductase (GR) activity was found to be significantly depleted (100 ± 0.16 #mol/min/mg protein), hut glutathione peroxidase (GP) activity was not affected in rats chronically treated with DS. It is reported that the treatment with DS decreases the GSH content, with a concomitant increase in GSSG level, and perturbs the GSH/GSSG redox status, inducing an oxidative stress on the brain. Glutathione reductase implicated in maintaining GSH/GSSG homeostasis by replenishing GSH is also affected by DS potentiating the oxidative damage of the tissue. This effect of DS on glutathione metabolism in the brain would explain some of its known neurotoxic effects. Disulfiram

Glutathione

Glutathione reductase

D I S U L F I R A M (tetraethylthiuram disulfide) (DS), an extensively used pharmacological deterrent against alcohol abuse, is reported to have neurotoxic effects with respect to the nervous system (17). The literature review (16) reveals that DS and its metabolite carbon disulfide have neurotoxic effects. In spite of several reports regarding the adverse effects of DS on the nervous system, the precise biochemical basis o f neurotoxic effects associated with DS treatment has yet to be evaluated. While the metabolic pathways o f DS are reasonably well worked out, it is not very certain as to whether the parent c o m p o u n d per se or its metabolite(s) are responsible for neurotoxicity. DS undergoes a rapid metabolic reduction that gives rise to diethyldithiocarbamate (DDC), a sulfhydryl compound known for its ability to chelate divalent metal ions in general, and copper in particular (2). Because o f its structural relationship with oxidised glutathione (GSSG), the metabolic reduction of DS to D D C is invariably linked to glutathione

Oxidative stress

Neurotoxicity

reductase (GR) per se, or with " G R system" (7,13). In this regard, a recent report from our laboratory has clearly demonstrated that GR as such does not act upon DS (13). Further, it was also shown that G S H per se could reduce DS to D D C through a sulphydryl group exchange reaction. Glutathione (GSH), an endogenous sulfhydryl compound, plays an important role in protecting cells from oxidative stress (15). Glutathione could exist both in reduced (GSH) and oxidised (GSSG) form, with the concentration of G S H usually severalfold higher than that of GSSG. Under normal circumstances, GSSG is rapidly reduced to G S H by G R using reduced nicotinamide adenine dinucleotide phosphate ( N A D P H ) as cofactor. Hence the change in the G S H / G S S G ratio could serve as an index o f redox state of the tissue (18,19). The highly lipophilic character of DS implies the possibility o f this exogenous disulfide having an easy accessibility to tissues that are rich in lipids, including nervous tissues. Further, the obser-

J Requests for reprints should be addressed to S. N. Nagendra, Department of Pharmacology & Toxicology, School of Pharmacy, The University of Kansas, 5064, Mallot, Lawrence, KS 66045-2205.

8

NAGENDRA ET AL.

vation that DS could be nonenzymatically reduced by GSH with a stoichiometry of 1 : 2 (13) implies that the DS treatment could deplete tissue GSH content. DS was found to be reduced by GSH in blood and that reaction did not deplete intracellular GSH levels (9). However, GSH content was found to be decreased with a constant increase in GSSG content in isolated hepatocytes following DS treatment (14). Thus ambiguity with respect to G S H / G S S G homeostasis as a result of DS treatment still exists. Further, glutathione reductase is implicated in the reduction of GSSG to GSH in an NADPH-dependent process and plays an important role in maintaining G S H / G S S G cellular homeostasis at a steady level. The present report deals with findings on the effect of chronic administration of DS on rat brain and liver glutathione content and glutathione reductase activity with special reference to its relevance to the neurotoxic effects of the drug. MATERIALS AND METHODS Male Sprague-Dawley rats weighing 150-180 g were orally administrated with DS (12 m g / k g body weight) dissolved in olive oil for 60 days. Control animals received only olive oil. After the experimental period the animals were ether-anaesthetised and perfused with ice cold saline through the heart, and both liver and brain were removed. The blood-free tissues were immediately homogenised (1 : 10 w/v) in ice cold 0.4 M perchloric acid (PEA) containing 5 mM ethylenediaminetetraacetic acid (EDTA) and centrifuged at 1500 x g for 15 min. Glutathione reductase, disulfiram, N A D P H , glutathione disulfide, CM-cellulose, 2' ,5 '-ADP-sepharose, dithioerythreitol, 5,5 '-dithiobis(2-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM) were purchased from Sigma Chemical Company (St. Louis). All other reagents used were of analytical reagent grade.

Total GSH was estimated spectrophotometrically by enzymatic reduction of GSSG to GSH using GR in the presence of N A D P H and DTNB. Aliquots of PCA extract of liver and brain tissues were adjusted to pH 7.4 +__ 0.1 by adding an appropriate volume of 0.3 M Tris base and were mixed with 5 mM DTNB in the assay buffer. The reaction was initiated by adding 0.34 mM N A D P H in 1 ml assay volume containing 2.0 U of GSSG reductase. GSH concentrations were determined by the method of Ellman (~4~2 ~= 1.36 x 103) (6L

Assay of GSSG GSSG was measured by the method of Tietze (21) as modified by Adams et al. (1). An aliquot of the tissue extract supernatant was mixed with 10 mM N-ethylmaleimide (NEM) in 100 mM Tris/HCl, pH 7.4, containing 5 mM EDTA, to remove endogenous GSH. The sample was mixed and centrifuged at 2000 x g for 10 min. The solution was passed over a Sep-Pak, C-18 cartridge (Water Associates, Framingham, MA) to remove unreacted NEM. The cartridge was rinsed once with an equal volume of buffer, the final pH being 7.4. In separate experiments, we confirmed the complete removal of NEM, as reported by Adams et al. (t), by measuring the absorbance of NEM at 315 nm. The assays were performed with 1 ml of this elute, to which DTNB, NADPH, and GSSG reductase were added in a total assay volume of 2 ml. The rate of colour development was monitored at 412 nm at ambient temperature. Difference in total GSH and GSSG value was taken as GSH content of tissue. The recovery of added GSSG and GSH was in the range of 95-98%, and the presence of tissue did not alter the recovery of GSSG. Absorbance and velocity measurements were made on a KONTRON (UVIKON) double-beam spectrophotometer with a thermostarted cell compartment maintained at 25 *C.

Statistical Evaluation of Results Assay o f GR Rat brain/liver glutathione reductase was purified with some modifications of the method described by Carlberg and Mannervik (3). The gel filtration steps were totally replaced by dialysis against the appropriate buffers, and chromatography on CM-cellulose (3). A single chromatography was performed on 2',5'-ADP-sepharose. After eluting the affinity column with 0.5 M Tris/HCl, pH 7.4, the glutathione reductase was eluted with a linear gradient (0-0.5 mM) of N A D P H in 0.05 mM Tris/HCl, p H 7.4. GR activity was measured using the method described by Goldbcrg and Spooner (8). The specific activity of glutathione reductase in the brain and liver was 140 and 210 /zmol/min/mg protein, respectively, when assayed at 2 mM GSSG, 0.5 mM N A D P H in Tris/HCl buffer, pH 7.4, containing 1 mM EDTA at 25°C.

Assay of GP A portion of rat brain/liver was homogenised (1 : 5 w/v) in 0.1 M Tris/HCl, p H 7.4 and centrifuged at 2000 x g for 10 min. The supernatant was used for the G P enzyme assay as described by Wendel (22). The protein content was determined by the method of Lowry et al. (11).

The significances of the results were determined using Student's t test. In all the experiments the findings were considered significant only when p < 0.05. The minimum number of animals in control and experimental groups were six each. RESULTS

Glutathione Reductase Activity in the Rat Brain~Liver The enzyme activity in rat brain was found to be decreased following chronic DS administration. Thus, a significant decrease in brain GR (100 + 16 tLmol/min/mg protein; p < 0.05) activity was observed in experimental rats as compared to the control group (140 +_ 32/~mol/min/mg protein). However, in liver the decrease in GR activity o f DS-administered animals was not statistically significant(Fig. 1).

Glutathione Peroxidase Activity The enzyme activity in the b r a i n and liver did not change significantly following chronic DS administration as compared to control animals.

GSH and GSSG Content in the Brain~Liver Assay of Total Glutathione (GSH + GSSG) Total glutathione was measured in the rat brain and liver based essentially on the method of Adams et al. (l).

Rats administered with DS for 60 days were found to have a significant (p < 0.001) decrease in brain GSH content (1.52 +_ 0.03 #mol/g) as compared to the control group (1.95

EFFECT OF DS ADMINISTRATION ON BRAIN GLUTATHIONE CONTENT

300

9

served in GSSG content in the experimental group (0.97 _+ 0.05 ~mol/g; p < 0.001) as compared to control animals (0.026 _+ 0.008/~mol/g).

300

(n:6)

DISCUSSION

c E Z

200

200 © or" [3_

E

(n:6)

klJ

100

~ 100 0 -r n £3 ,< Z

CON ~-

TEST P<0'05

TEST CON ~ N5.

BRAIN

LIVER

FIG. 1. Glutathione reductase activity in the rat liver and brain after disulfiram administration (12 mg/kg body weight) for 60 days. Values are means ± SD. Glutathione reductase was assayed in the tissue as described. Numbers in parentheses indicate the number of experiments.

+_ 0.02/~mol/g). However, no significant difference between total glutathione (GSH + GSSG) content of the brains of the experimental group (1.95 ,+ 0.04 ~moles/g as GSH) was seen as compared to controls (1.97 ,+ 0.04/~mol/g) (Table 1). On the other hand, a concomitant increase in brain GSSG content of the experimental group (0.12 _+ 0.013/~mol/g) was noted as compared to the control group (0.016 _+ 0.004 /~mol/g; p < 0.001). Consequently, a decrease in the GSH/GSSG ratio (12.8) (Table 1) in the brain was noted after DS administration when compared to the control group (122). Similar changes with respect to GSH and GSSG content of the liver were also noted following DS administration (Table 1). Liver GSH content was decreased in experimental animals (4.51 _+ 0.12 /~mol/g; p < 0.001) as compared to the control group (5.47 + 0.08 /zmol/g). Further, a concomitant increase was ob-

Disulfiram is an extensively used pharmacological agent in the treatment of alcoholism. In the present study GSH/GSSG homeostasis, which is so vital for cellular viability, was found to be affected by chronic DS administration to rats. Glutathione metabolism directed by two enzymes (viz., glutathione reductase and peroxidase system) serves to detoxify a major portion of cellular hydroperoxides generated by the oxidative metabolism. The enzyme GR replenishes the GSH level, which in turn has an important role in protecting ceils from oxidative stress (15). Glutathione could exist both in oxidised and reduced forms, with the concentration of GSH usually severalfold higher than that of GSSG in normal circumstances (5). Hence the change in the GSH/GSSG ratio could serve as an index of the oxidative stress of the tissue (19). The highly lipophilic character of DS gives it easy accessibility to the cellular compartments in general, and particularly the lipidrich tissues such as the nervous system and adipose tissue. Further, being an oxidising compound, DS could easily oxidisc the endogenous -SH compounds such as GSH, cysteine, and so on to their corresponding disulfhydryl compounds. The in vitro studies on this aspect, as shown earlier, have clearly established the stoichiometry of 1 : 2 between DS and GSH in the -SH group exchange-mediated reduction of DS. This implies that DS administration could lead to depletion of cellular GSH content resulting in a state of oxidative stress and is in accordance with the reported depletion of GSH by DS which oxidised critical protein sulfhydryl groups causing the rupture of cell membrane and eventual hemolysis (10). Further, as shown in Table 1, chronic administration of DS caused a significant depletion of GSH content in both the liver and the brain, with a concomitant increase of GSSG content. Thus it is evident that DS administration could result in a state of oxidative stress wherein the GSH/GSSG ratio is significantly decreased without any change in the total glutathione content. In this regard the present observation of decreased GSH/GSSG ratio is in agreement with the significant depletion of GSH content following DS administration as reported by Ohno et al. (14) and could possibly explain the rapid disappearance of DS in the circulation as reported by earlier workers (4,7). The observation that the GSH/GSSG ratio was decreased even in the brain following DS administration may be associ-

TABLE 1 GLUTATHIONECONTENTOF RAT LIVER AND BRAIN FOLLOWING DISULFIRAM ADMINISTRATION(12 rng/kg body weight) FOR 60 DAYS Liver

Total glutathione (GSH + GSSG) GSH GSSG Ratio

Brain

Control(n = 10)

Exp. (n = 10)

Control(n= 10)

Exp. (n = 10)

5.500 ± 0.080 5.470 ± 0.080 0.026 ± 0.008 210.4

5.48 ± 0.07 4.51 ± 0.12" 0.97 ± 0.05* 4.65*

1.970 + 0.04 1.950 + 0.02 0.016 ± 0.004 122.0

1.95 ± 0.04 1.52 + 0.03* 0.12 ± 0.013• 12.8"

Values (#mol/g) are expressed as mean ± SD. *p < 0.001 (Student's t test).

10

NAGENDRA ET AL.

ated with some of the neurotoxic effects associated with DS treatment as secondary to the neuronal oxidative stress caused by the drug. As a result of GSH depletion following DS therapy, the detoxification of some of the cytotoxins such as aminoquinones and aminochromes (12) that are formed during the metabolism of neurotransmitters may be severely affected. Glutathione reductase, an NADPH-dependent enzyme, is important in replenishing the cellular content of GSH from GSSG. Therefore, the circumstances where GR activity is affected/altered would adversely affect the GSH content, thereby exposing the tissues to oxidative stress. As discussed earlier, DS treatment was found to have caused a significant decrease in GSH content with a concomitant increase in GSSG content, indicating that the decreased GSH/GSSG ratio was a sign of cellular oxidative stress. The observed decrease in GSH may be due to the effect of DS on GR by itself. Thus the studies on GR in animals treated with DS have also indicated a significant decrease in GR in the brain (Fig. 1). However, in the liver the observed changes in GR activity were not significant. Since GR has 10 cysteine residues per monomer (3), it is likely that some of the -SH groups may form a mixed disulfide as a result of DS reduction rendering the enzyme less active. This may be particularly true in view of the reported disulfide exchange reaction of this enzyme with a number of thiols (20). Thus, the observed decrease in brain GSH content may be

due to the synergistic effect of nonenzymatic reduction of DS through -SH group exchange reaction, wherein for every molecule of DS reduced two molecules of GSH are oxidised, and the effect of DS on GR per se, resulting in the decrease of enzyme activity responsible for the replenishment of GSH. The observed differential effect with respect to GR activity in the liver and brain may be because of the differential redox potential status of the liver and brain to sustain the oxidative stress. During the present study, involving chronic administration of DS to rats, a significant decrease in reduced glutathione content of the brain has been noted in the experimental group as compared to control rats. However, the total glutathione (GSH + GSSG) content of tissue was not altered significantly. The liver/brain GSH levels decreased, while an increase of GSSG content was found, indicating a decrease in ratio of GSH/GSSG in the brain following DS administration (Table 1). Similar changes with respect to GSH and GSSG were also observed in the livers of animals in the experimental group. Glutathione reductase, implicated in maintaining GSH/GSSG homeostasis, was found to be affected by chronic DS treatment which would eventually expose the tissues to possible oxidative stress. These findings indicate that some of the neurotoxic effects of DS administration are due to the tissue oxidative stress mediated through altered GSH/GSSG ratio brought about by DS metabolism.

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