Month: October 2019

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Experiments were carried out to establish the role of glutathione reductase (GR), if any, in the metabolic conversion of disulfiram (DS) to diethyldithiocarbamate (DDC). It was observed that, under standard assay conditions, whereas DS was incorporated as a substrate instead of oxidised glutathione (GSSG), the enzymes from both human liver extract and yeast sources failed to reduce the parent compound, implying that glutathione reductase perse do not reduce disulfiram. However, the incorporation of disulfiram into an assay system comprising of GSSG, NADPH and reductase resulted in DS reduction to DDC. Further, the observation, that the GR assay system devoid of either GSSG or NADPH was found to lack DS reducing ability, implies that GSH as a reaction product of GR system is responsible for the reduction of DS to DDC. The results of in-vitro experiments indicated that GSH perse could reduce DS to DDC nonenzymatically, with a stoichiometric relationship of 2:1. Thus it is inferred that GR perse do not reduce DS, whereas GSH, as an intermediary metabolite of GR system, brings about non-enzymatic reduction of DS via a sulfhydral group exchange reaction.

The prophylactic deterrent effect of disulfiram (DS) has been attributed to its ability to exacerbate sympathetic function. Though there are reports to indicate that DS administration could as well affect the neurotransmitter metabolism, few reports implicate the possibility of central nervous system (CNS) mediated anticraving effect of the drug. The present study involving the oral administration of DS to rats for 45 days has clearly shown a significant increase in 5-HT (815.4 ± 74.7 ng/g, P < 0.01) and 5-HIAA (506.1 ± 86.3 ng/g, P < 0.02) contents in brain when compared to control rats. The observed increase in 5-HT and 5-HIAA content was found to correlate (ζ = 0.89) with the concomitant increase in brain tryptophan content (4.15 ± 1.05 nmol/g, P < 0.001) following DS administration. Further, the study on peripheral tryptophan content has shown an increase in both total and free fraction (ultrafiltrate) of plasma, which in turn was found to have an inverse relationship (ζ = −0.94, P < 0.05) with the decrease in liver tryptophan content following DS administration. Thus the observed increase in brain 5-HT level is attributed to the ability of DS to mobilise peripheral tryptophan for 5-HT synthesis in CNS. As there are reports to imply the hyposerotonergic function as responsible for craving, the present findings, that DS could enhance the 5-HT metabolism in brain, may partially explain the CNS mediated anticraving effect of DS.

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 μmol/g; p < 0.001), with a concomitant increase in oxidised glutathione (GSSG) content (0.12 ± 0.013 μmol/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 μmol/min/mg protein), but 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.

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 mumol/g; p < 0.001), with a concomitant increase in oxidised glutathione (GSSG) content (0.12 +/- 0.013 mumol/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 mumol/min/mg protein), but 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.

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca2+, Mg(2+)-ATPase activity. In vitro studies indicated that DS (ID50 = 20 microM) produced a dose-dependent inhibition of Ca2+, Mg(2+)-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca2+, Mg(2+)-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID50 = 20 microM) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca2+, Mg(2+)-ATPase specific activity was observed when treated with DS (30 microM) and EGTA (100 microM) respectively. The remaining 25-30% of total activity is suggested to be of Mg(2+)-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70-75% of total Ca2+, Mg(2+)-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca2+, Mg(2+)-ATPase activity could affect its function of maintaining intracellular Ca2+ concentration. This could contribute to the deleterious effects on CNS.

Differential development of monoamine oxidase (MAO) isoenzymes in rat whole brain is described in postnatally developing Sprague-Dawley rats. Total MAO and isoenzyme activity was measured using nonspecific and specific substrates. Total MAO activity measured using tyramine, increased postnatally up to 24 weeks of age and attained a plateau afterward. The increase in total MAO activity was significant at all age groups (18 days to 36 months) investigated as compared to new born rats. MAO-A and MAO-B activities were measured using octopamine and benzylamine respectively. We also observed a marginal increase of MAO-A activity and a significant increase of MAO-B activity upon development. Furthermore, at 12 weeks of age, MAO-B activity increased by 10-fold as compared to new born and was consistent up to 36 months of age. The qualitative localization of the enzyme activity on non SDS-PAGE by nitroblue tetrazolium staining confirmed the increase of MAO-B during the development. It is suggested that the maturational increase of total MAO activity in brain is predominantly due to the increase of MAO-B isoenzyme.

Abstract

Although disulfiram used as a pharmacological agent in the treatment of alcoholism is reported to act on both peripheral and
central nervous systems with several adverse effects, the ncurotoxic property of the drug has not been properly elucidated. We
observed that the chronic administration of the drug to rats significantly inhibited synaptosomal (Na+,K+)-ATPase and basal
Mg2+-ATPase activities. Further, the uptake of y-aminobutyric acid and e-glutamate which rely on the energy provided by this
system was depleted following chronic drug administration. Similar findings were observed when the isolated synaptosomes were
treated with the drug in an in vitro system. Further, treatment of synaptosomes with ouabain, a known inhibitor of
(Na+,K +)-ATPase resulted in significant depletion of 3H-GABA and e-[3H]glutamate uptake into synaptosomes indicating the
importance of the enzyme in the uptake mechanism. However, diethyldithiocarbamate, a major metabolite of disuifiram did not
elicit any change in either the enzyme activity or the uptake of these neurotransmitters. On the basis of these evidences, we
suggest that the chronic disulfiram administration attenuated the neurotransmitter uptake mechanism and resulted in higher
cxtracellular concentration of glutamate that could lead to glutamate-induced neurotoxicity.

Introduction

The uptake of excitatory neurotransmitter L-gluta-
mate (L-GIu) and inhibitory neurotransmitter y-amino

butyric acid (GABA) into synaptosomes exclusively rely
on the energy provided by (Na +,K ÷)-ATPase system of
the brain. Indeed the neurotransmission by amino acids
is terminated by high affinity uptake of the transmitters
in neurons and glial cells. In fact it was shown that an
impairment of the Na+-dependent glutamate uptake
capacity, which occurs during energy failure of the
brain such as ischemia and anoxia (Sanchez-Prieto and
Gonzalez, 1988; Silverstein et al., 1986), results in
excess amounts of extracellular glutamate. Further
C1–dependent glutamate uptake was observed in
synaptic membranes and glial cells (Cho and
Bannai,1984; Zaczek et al., 1987), and the treatment of
(Na+,K+)-ATPase with ouabain affected Cl–dependent uptake in synaptic membranes (Koyama et al.,
1993). More recently a carrier-mediated release and

uptake of GABA and glutamate has also been re-
ported (see review by Levi and Raiteri, 1993). Over the

last few years much interest has been focussed upon
neurotoxic properties of neuroexcitatory amino acid
glutamate. Although the concept of an excitatory

mechanism as the basis of toxic effects is still contro-
versial, there can be no doubt to date as to potential of glutamate as a neurotoxin.

Among the compounds that augment neurotoxicity,
we discovered tetraethylthiuram disulfide (disulfiram)
an extensively used pharmacological agent for the
treatment of alcoholism. Although, the disulfiram has
been known to cause neurotoxicity (Laplane et al.,
1992; Simonian et al., 1992), the precise mechanism of

its neuropathological role is yet to be elucidated. How-
ever, the electrophysiological findings suggest that

disulfiram induces degeneration of the axon and basal
ganglia. These neuropathies occur even in the absence
of disulfiram ethanol reaction suggesting that factors other than the inhibition of aldehyde dehydrogenase and dopamine fl-hydroxylase are involved in the neuro- toxic effects of disulfiram. Experimental studies have shown that serotonergic (Fukumori et al., 1980; Nagen-
dra et al., 1993), dopaminergic (Molinengo et al., 1991), noradrenergic and cholinergic (Nilsson et al., 1987) functions are affected by disulfiram. It has been re-
ported that disulfiram modulates Mg2+-ATPase-de – pendent uptake of monoamines in chromaffin granular
membranes (Schlichter et al., 1975). Disulfiram also
interacts with plasma membranes affecting its integrity, and may alter the native structure of proteins by form-
ing internal S-S bonds (Brien and Loomis, 1985; Lauri-
alt and O’Brien, 1991). Thus, we expect disulfiram to

follow the same cellular mechanism that alters the
membrane integrity and affects membrane transport
eventually leading to the neurotoxic effect. We assume

that the development of cell injury/death is a conse-
quence of membrane perturbation and transport mech-
anism caused by disulfiram.

In the present study we have examined the effects of
brief and chronic treatments with disulfiram on
(Na+,K+)-ATPase activity and the uptake of GABA
and L-GIu into the brain synaptosomes.

Materials and methods


L-[3,4-aH]glutamate (specific activity 5.19 Ci/mmol)
and y-[2,3-3H]aminobutyric acid (specific activity 94.2
Ci/mmol) were purchased from DuPont-New England

Nuclear (USA). All other chemicals used were of ana-
lytical grade including disulfiram, diethyldithio-
carbamate and ouabain obtained from Sigma Chemical

Co. (St.Louis, MO, USA).

Abstract

We examined the brain oxidative stress which accompanies 30 min of bilateral carotid artery ligation (BCAL) in terms of changes in brain levels of glutathione; reduced (GSH) and oxidized (GSSG) forms and the exacerbation of oxidative stress by disulfiram (DSF). These results indicate that BCAL alone decreases GSH content and limits glutathione reductase (GR) activity, and these changes were enhanced by DSF pretreatment. Similar observations were recorded with DSF alone. GR activity (74.3 ± 4.0 µmol min–1 mg–1 tissue; p < 0.001) and GSH content (1.23 ± 0.06 µmol min–1 g–1 tissue; p < 0.001) was attenuated in rats subjected to synergistic effect of BCAL and DSF with a concomitant increase of GSSG (0.006 ± 0.006 µmol min–1 g–1 tissue; p < 0.001). Recovery of GSH/GSSG level and GR activity during reperfusion following 30 min BCAL was considerably delayed (96 h) in the BCAL and DSF group as compared to the recovery time of 24 h in the group subjected to BCAL-reperfusion alone. Perturbation of GSH/GSSG homeostasis as a result of BCAL was augmented by DSF. These findings clearly demonstrate central nervous system oxidative stress due to a BCAL-DSF synergistic effect. Based on the results obtained with this model, we conclude that DSF increases brain oxidative stress and this may be detrimental to alcoholics who might drink and develop an acetaldehyde-induced hypotension while taking DSF

Abstract

S-Methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO) and sulfone (DETC-MeSO2) both inhibit rat liver low Km aldehyde dehydrogenase (ALDH2) in vitro and in vivo (Nagendra et al., Biochem Pharmacol 47: 1465-1467, 1994). DETC-MeSO has been shown to be a metabolite of disulfiram, but DETC-MeSO2 has not. Studies were carried out to further investigate the inhibition of ALDH2 by DETC-MeSO and DETC-MeSO2. In an in vitro system containing hydrogen peroxide and horseradish peroxidase, the rate of DETC-MeSO oxidation corresponded to the rate of DETC-MeSO2 formation. Carbamoylation of GSH by both DETC-MeSO and DETC-MeSO2 was observed in a rat liver S9 fraction. Carbamoylation of GSH was not observed in the presence of N-methylmaleimide. In in vitro studies, DETC-MeSO and DETC-MeSO2 were equipotent ALDH2 inhibitors when solubilized mitochondria were used, but DETC-MeSO was approximately four times more potent than DETC-MeSO2 in intact mitochondria. In studies with rats, the dose (i.p. or oral) required to inhibit 50% ALDH2 (ED50) was 3.5 mg/kg for DETC-MeSO and approximately 35 mg/kg for DETC-MeSO2, approximately a 10-fold difference. Furthermore, maximum ALDH2 inhibition occurred 1 hr after DET(-MeSO administration, whereas maximal ALDH2 inhibition occurred 8 hr after DETC-MeSO2 dosing. DETC-MeSO is, therefore, not only a more potent ALDH2 inhibitor than DETC-MeSO2 in vivo, but also in vitro when intact mitochondria are utilized. The in vitro results thus support the in vivo findings. Since oxidation of DETC-MeSO can occur both enzymatically and non-enzymatically, it is possible that DETC-MeSO2 is formed in vivo. DETC-MeSO2, however, is not as effective as DETC-MeSO in inhibiting ALDH2, probably because it has difficulty penetrating the mitochondrial membrane. Thus, even if DETC-MeSO2 is formed in vivo from DETC-MeSO, it is the metabolite DETC-MeSO that is most likely responsible for the inhibition of ALDH2 after disulfiram

administration.PMID: 9586946  DOI: 10.1016/s0006-2952(97)00513-3

Abstract

Although disulfiram used as a pharmacological agent in the treatment of alcoholism is reported to act on both peripheral and central nervous systems with several adverse effects, the neurotoxic property of the drug has not been properly elucidated. We observed that the chronic administration of the drug to rats significantly inhibited synaptosomal (Na+,K+)-ATPase and basal Mg2+-ATPase activities. Further, the uptake of γ-aminobutyric acid and l-glutamate which rely on the energy provided by this system was depleted following chronic drug administration. Similar findings were observed when the isolated synaptosomes were treated with the drug in an in vitro system. Further, treatment of synaptosomes with ouabain, a known inhibitor of (Na+,K+)-ATPase resulted in significant depletion of 3H-GABA and l-[3H]glutamate uptake into synaptosomes indicating the importance of the enzyme in the uptake mechanism. However, diethyldithiocarbamate, a major metabolite of disulfiram did not elicity any change in either the enzyme activity or the uptake of these neurotransmitters. On the basis of these evidences, we suggest that the chronic disulfiram administration attenuated the neurotransmitter uptake mechanism and resulted in higher extracellular concentration of glutamate that could lead to glutamate-induced neurotoxicity.