Disulfiram facilitates ataxin-3 nuclear translocation and potentiates the cytotoxicity in a cell model of SCA3.

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine-encoding CAG repeat in the ATXN3 gene encoding the protein ataxin-3. The nuclear presence of polyglutamine-expanded ataxin-3 is of critical importance for the pathogenesis of SCA3. Disulfiram, an FDA-approved drug for alcoholism, has also garnered attention in cancer treatment. However, it has shown toxicity in the nervous system. Bearing this in mind, we treated cells expressing ataxin-3 with disulfiram to measure several pathogenic cascades of SCA3, including aggregate formation, soluble ataxin-3 expression and nuclear localization of ataxin-3 and the cytotoxicity, which assess the direct effect of disulfiram on SCA3 cell models. To our knowledge, this is direct evidence that disulfiram elevated the nuclear localization of polyglutamine-expanded ataxin-3 and enhanced the cytotoxicity in a cell model of SCA3. Furthermore, disulfiram did not affect the aggregate formation of polyglutamine-expanded ataxin-3 at least at a single dose. Our findings repurpose disulfiram as a modulator of ataxin-3 nuclear transport that aggravates the pathology of SCA3, which is a new target for disulfiram. This study also represents an important example of determining novel side effects in pre-existing drugs. This study suggests that caution may be warranted when this compound is used to treat alcohol abuse or cancer in patients carrying a SCA3-causing mutation.

Sensory-motor axonal polyneuropathy involving cranial nerves: An uncommon manifestation of disulfiram toxicity.

Disulfiram (tetraethylthiuram disulfide) has been used for the treatment of alcohol dependence. An axonal sensory-motor polyneuropathy with involvement of cranial pairs due to disulfiram is exceedingly rare. The authors report a unique case of an extremely severe axonal polyneuropathy involving cranial nerves that developed within weeks after a regular dosage of 500mg/day disulfiram. To the authors best knowledge, such a severe and rapidly-progressive course has never been described with disulfiram dosages of only 500mg/day.

Effects of disulfiram and dopamine beta-hydroxylase knockout on cocaine-induced seizures.

The antialcoholism drug disulfiram has shown recent promise as a pharmacotherapy for treating cocaine dependence, probably via inhibition of dopamine beta-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE). We previously showed that DBH knockout (Dbh -/-) mice, which lack NE, are susceptible to seizures and are hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, suggesting that disulfiram might exacerbate cocaine-induced seizures (CIS) by inhibiting DBH. To test this, we examined CIS in wild-type and Dbh -/- mice following administration of disulfiram or the selective DBH inhibitor nepicastat. We found that Dbh genotype had no effect on CIS probability or frequency, whereas disulfiram, but not nepicastat, increased the probability of having CIS in both wild-type and Dbh -/- mice. Both disulfiram and nepicastat increased CIS frequency in wild-type but not Dbh -/- mice. There were no genotype or treatment effects on serum cocaine levels, except for an increase in disulfiram-treated Dbh -/- mice at the highest dose of cocaine. These results suggest that disulfiram enhances CIS via two distinct mechanisms: it both increases CIS frequency by inhibiting DBH and increases CIS frequency in a DBH-independent manner.

Can experimental paradigms and animal models be used to discover clinically effective medications for alcoholism?

Evaluating medications in animal laboratory paradigms can reveal whether the compound is effective in an established alcoholism model, at clinically relevant doses and exposure conditions, when administered orally (or transdermally) and without serious limiting side effects. Positive outcomes constitute a possible discovery for relevance to alcoholism and, under favorable marketing conditions, encourage further development. Medication testing using animal models of alcoholism might also guide clinical testing by discriminating clinically effective from clinically ineffective compounds. This ability rests on whether there are tests or, more reasonably, batteries of tests having this discriminative ability. The present paper examines this possibility. Effects of naltrexone and acamprosate in animal paradigms which model behavioral aspects of alcoholism are reviewed and compared with the effects of compounds which have limited effects in alcoholics. It is not clear at present whether any single paradigm or combination of paradigms differentiates clinically effective from clinically limited compounds. Steps are suggested to improve the use of preclinical laboratory tests to predict which compounds are likely to be effective medications for reducing drinking and sustaining abstinence in human alcoholics.

N,N-diethyldithiocarbamate produces copper accumulation, lipid peroxidation, and myelin injury in rat peripheral nerve.

Previous studies have demonstrated the ability of the dithiocarbamate, disulfiram, to produce a peripheral neuropathy in humans and experimental animals and have also provided evidence that N,N-diethyldithiocarbamate (DEDC) is a proximate toxic species of disulfiram. The ability of DEDC to elevate copper levels in the brain suggests that it may also elevate levels of copper in peripheral nerve, possibly leading to oxidative stress and lipid peroxidation from redox cycling of copper. The study presented here investigates the potential of DEDC to promote copper accumulation and lipid peroxidation in peripheral nerve. Rats were administered either DEDC or deionized water by ip osmotic pumps and fed a normal diet or diet containing elevated copper, and the levels of metals, isoprostanes, and the severity of lesions in peripheral nerve and brain were assessed by ICP-AES/AAS, GC/MS, and light microscopy, respectively. Copper was the only metal that demonstrated any significant compound-related elevations relative to controls, and total copper was increased in both brain and peripheral nerve in animals administered DEDC on both diets. In contrast, lesions and elevated F2-isoprostanes were significantly increased only in peripheral nerve for the rats administered DEDC on both diets. Autometallography staining of peripheral nerve was consistent with increased metal content along the myelin sheath, but in brain, focal densities were observed, and a periportal distribution occurred in liver. These data are consistent with the peripheral nervous system being more sensitive to DEDC-mediated demyelination and demonstrate the ability of DEDC to elevate copper levels in peripheral nerve. Additionally lipid peroxidation appears to either be a contributing event in the development of demyelination, possibly through an increase of redox active copper, or a consequence of the myelin injury.

Toxicity study of an antidipsotropic Chinese herbal mixture in rats: NPI-028.

OBJECTIVES: To determine the potential toxicity and safety of the Chinese herbal medicine NPI-028 in rats following subchronic (3-month) exposure via daily oral consumption. DESIGN: Subchronic toxicity was evaluated in four groups of rats (n = 10 per group) receiving NPI-028 orally at a dose of either 0.0 (normal diet control), 0.5, 1.0, or 2.0 g/kg, ingested as part of their daily diet for 3 months. NPI-028 was incorporated into powdered rat chow diet as a specific percent of the total diet provided each day. The primary active isoflavone content of NPI-028 (puerarin) used in the rat diet was also determined. OUTCOME MEASURES: Subchronic toxicity was assessed over a 3-month period by biweekly measurement of water and food intake, weight gain, and visual inspection for maintenance of grooming and normal behavior. At the end of the study period rats were euthanized and blood was obtained for hematologic and chemical analysis. Organs were removed for histopathologic examination. RESULTS: Rats in all three NPI-028 dose groups were similar to the control group in weight gain, food intake, and water intake over the study period. Hematology, blood chemistries, and organ histology in rats at all three NPI-028 doses did not significantly differ from control rats. Minor exceptions were elevated urea nitrogen values at all NPI-028 doses, and increased triglyceride and thyroid-stimulating hormone values in the lowest NPI-028 dose-treated group. Puerarin (used as a dietary isoflavone marker) content of NPI-028 was 26 mg/g dry weight. CONCLUSIONS: NPI-028 ingested orally at doses up to 2.0 g/kg per day in the rat diet for up to 3 months resulted in normal growth with no changes in hematologic or hepatic parameters, and only minor alterations in renal and blood chemistry parameters. There was no evidence of abnormal histology. These data suggest the long-term daily oral consumption of NPI-028 as a part of the daily diet for 3 months, at the doses studied, is safe in rats. Thus, NPI-028 may potentially be safe for clinical use as an antidipsotropic agent.

Disulfiram produces a non-carbon disulfide-dependent schwannopathy in the rat.

Disulfiram is a dithiocarbamate drug used for alcohol aversion therapy that produces a distal sensorimotor peripheral neuropathy in certain individuals. Because carbon disulfide, a disulfiram metabolite, produces a peripheral neuropathy clinically similar to disulfiram, it has been postulated that disulfiram neuropathy results from CS2 release in vivo. The current study evaluated the morphological changes produced by disulfiram and the contribution of CS2-mediated protein cross-linking to disulfiram-induced neuropathy. Male Sprague-Dawley rats were administered 1% w/w disulfiram in their feed for 2, 4, 5, or 7 wk, and erythrocyte spectrin, hemoglobin, and neurofilament preparations were isolated and the extent of cross-linking assessed by SDS-PAGE, RP-HPLC, and Western blotting, respectively. Spinal cord and peripheral nerve sections were obtained from separate treated animals and assessed by light and electron microscopy. Significant protein cross-linking was only detected in neurofilament preparations obtained after 7 wk of exposure. Morphological changes were observed after 4 wk exposure and consisted of vacuoles within the Schwann cell cytoplasm and segmental demyelination. No neurofilamentous axonal swellings were detected and no significant changes were observed in the CNS. Because disulfiram neuropathy lacks both the morphological changes and intermolecular cross-linking characteristic of CS2, we conclude that disulfiram neuropathy is not mediated by the axonal toxicant CS2; instead, disulfiram appears to be a primary Schwann cell toxicant. Recognition of a diethylcarbamoyl adduct on globin and axonal proteins presents a novel putative neurotoxic mechanism for disulfiram.

Sister-chromatid exchanges induced by disulfiram in bone marrow and spermatogonial cells of mice treated in vivo.

Disulfiram is a widely used drug to treat alcoholism due to its capacity to inhibit the metabolism of acetaldehyde; however, its genotoxic potential is not well known. Thus, the aim of this investigation was to determine whether the chemical may induce sister-chromatid exchanges (SCEs) in an in vivo study using mouse bone marrow and spermatogonial cells. We used doses of 200, 400 and 800 mg/kg body weight and compared the obtained data with the values determined in a negative control group as well as with a positive control group (cyclophosphamide, 50 mg/kg). The results in both systems indicated a weak genotoxic response by the chemical. In the case of bone marrow, a significant SCE level was achieved only with the high tested dose, but in spermatogonial cells the three doses tested showed a significant difference with respect to the negative control. No significant alterations in the mitotic index or in the cell proliferation kinetics were observed in somatic cells. Concerning the effect of cyclophosphamide, an increase in the level of SCEs was observed in both types of cells, reaching more than three times the values obtained in their respective control groups.

Disulfiram and diethyldithiocarbamate intoxication affects the storage and release of striatal dopamine.

Acute intoxication and chronic therapy with the alcohol consumption deterrent dithiocarbamate disulfiram have been associated with several neurological complications perhaps involving the impairment of neurotransmitter pathways. In this study we have tested the hypothesis that dopaminergic malfunction is a critical component in disulfiram-evoked neurotoxicity. Disulfiram antagonized the in vitro striatal binding of [3H]tyramine, a putative marker of the vesicular transporter for dopamine, and the uptake of [3H]dopamine into striatal synaptic vesicles, with inhibitory constants (Ki) in the range of reported blood dithiocarbamate levels in treated alcoholics. Furthermore, disulfiram provoked a loss of radioactivity from [3H]dopamine-preloaded striatal vesicles, when added directly to the incubation mixture. Several metal-containing fungicide analogs were also potent displacers of specifically bound [3H]tyramine. Diethyldithiocarbamate (DDC), the major metabolite of disulfiram, had none of these effects. The intraperitoneal injection of a high dose of disulfiram and DDC into rats, mimicking acute intoxication, induced in vivo overflow of striatal dopamine from both a reserpine-sensitive (vesicular) and an alpha-methyl-p-tyrosine-sensitive (cytoplasmic) pool. The vesicular component of in vivo dopamine release resulted mainly from a direct activity of disulfiram, on the organelles (interaction with the carrier for dopamine plus membrane permeabilization) and indirectly through the mediation of serotonergic 5-HT3 receptors. DDC acted poorly at the vesicle membrane, and the in vivo releasing effect of dopamine was only partially prevented by the inhibition of 5-HT3 receptors, thus suggesting the role of additional mechanisms. It is concluded that disulfiram intoxication may acutely disrupt dopamine balance, an effect probably underlying some of the central neurotoxic, extrapyramidal symptoms associated with dithiocarbamate overdose.

Comparative effects of two dithiocarbamates disulfiram and thiram, on adrenal catecholamine content and on plasma dopamine-beta-hydroxylase activity.

Both disulfiram (tetraethylthiuram disulfide), an alcohol aversive drug, and thiram (tetramethyl-thiuram disulfide), a widely used pesticide, significantly increased the dopamine pool in the adrenal glands of dosed rats. The dopamine increase was detectable within 4 h of oral dosing with 100 mg/kg of either dithiocarbamate and peaked 24 h later at 10 times control values. In control rats the dopamine turnover was 0.51 h-1 as calculated by the assumed first order decline of dopamine after a single injection of alpha-methyl-p-tyrosine (alpha-MT, 400 mg/kg i.p.) resulting in a dopamine-beta-hydroxylase (DBH) activity of 0.73 nmol/h per pair of adrenals. In the adrenals of rats pretreated with thiram and then injected with alpha-MT, the adrenal dopamine content did not significantly decline, indicating that thiram reduced the conversion of dopamine to noradrenaline, eventually leading to the observed dopamine increase. Plasma DBH activity was significantly reduced 4 h and 24 h after dosing with thiram, but was unchanged after treatment with disulfiram. The determination of plasma DBH activity could be a marker to monitor the effect of thiram on catecholamine metabolism in occupationally exposed workers but not that of disulfiram in abstinent alcoholics.

[A preclinical study on the safety of a new antialcoholic drug inmecarb]. / Doklinicheskoe issledovanie bezopasnosti novogo protivoalkogol'nogo preparata inmekarba.

A preclinical study on safety of a new antialcoholic drug inmecarb was carried out on three species of experimental animals (rats, guinea pigs, dogs) receiving the drug at different doses including a subtoxic one (1/5 of LD50) for 6 months. The authors concluded that on the basis of a relatively low toxicity and the absence of specific toxicity and long-term side effects the drug may be recommended for clinical use as an antialcoholic agent.

[Effect of teturam on the development of the offspring of alcoholized animals]. / Vliianie teturama na razvitie potomstva alkogolizirovannykh zhivotnykh.

During experiments on rats it was found that in alcoholized animals teturam on the whole did not potentiate and in some cases even attenuated the toxic effect of alcohol on the offspring development. The data confirm the idea about necessity of studying toxicity of drugs under the conditions corresponding to their clinical use.

[Chronic alcoholic intoxication in animals as a model for the study of the safety of new anti-alcohol agents]. / Khronicheskaia alkogol'naia intoksikatsiia u zhivotnykh kak model' dlia izucheniia bezopasnosti novykh protivoalkogol'nykh sredstv.

Experiments on 200 noninbred male rats have demonstrated that daily administration of ethanol per os in a dose of 8 g/kg (2/3 LD50) for a month produced pathological changes in organs and systems of the body, which were similar to the manifestations of chronic alcoholic intoxication in man. It is concluded that the model under consideration may be used during preclinical study of the safety of new antialcoholic agents.