thiourea and 2-4-dithiobiuret

2,4-Dithiobiuret (DTB) causes ascending motor weakness when given chronically to rodents. In muscles of animals with DTB-induced weakness, quantal release of acetylcholine (ACh) is impaired. We examined in detail the structural changes that occurred at neuromuscular junctions and their associated Schwann cells of extensor digitorum longus (EDL) muscles of male rats treated with DTB to the onset of muscle weakness, 5-8 days. Our objective was to assess the involvement of the Schwann cells and to determine the most likely primary targets of DTB. At the onset of muscle weakness, nerve terminals exhibited some enlarged regions, but did not sprout. Terminal Schwann cells became flatter and expanded to cover most of the endplate. The extent of invasion of the synaptic cleft by Schwann cell processes was not significantly different from controls; extension of Schwann cell sprouts away from the junction was not seen. Thus, the morphology of the Schwann cells, although clearly affected by DTB, does not suggest that they contribute directly to the physiological defects of DTB-treated terminals. Abnormal tubulovesicular structures or tangles of neurofilaments were clustered in the centers of about 25% of treated terminals. Fewer synaptic vesicles occupied the region opposite the postsynaptic folds. Vesicle volumes were variable and included some very large vesicles, corresponding with the variable MEPP amplitudes reported previously for terminals of DTB-treated rodents. The postsynaptic area stained by rhodamine-labeled alpha-bungarotoxin expanded with terminal swelling, apparently by unpleating of the postsynaptic folds. No loss of ACh receptors or spread of ACh receptors beyond terminal boundaries was detected. Morphometric data are consistent with the conclusion that DTB affects, either directly or indirectly, vesicular release of ACh and the subsequent vesicular recycling process.

Topics: Animals; Bungarotoxins; Coloring Agents; Evoked Potentials; Male; Mice; Mice, Inbred ICR; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Muscle Weakness; Muscle, Skeletal; Nerve Endings; Neuromuscular Junction; Nitroblue Tetrazolium; Pyridinium Compounds; Quaternary Ammonium Compounds; Rats; Rats, Sprague-Dawley; Schwann Cells; Thiourea

Chronic treatment of rodents with 2,4-dithiobiuret (DTB) induces a neuromuscular syndrome of flaccid muscle weakness that mimics signs seen in several human neuromuscular disorders such as congenital myasthenic syndromes, botulism, and neuroaxonal dystrophy. DTB-induced muscle weakness results from a reduction of acetylcholine (ACh) release by mechanisms that are not yet clear. The objective of this study was to determine if altered release of ACh during DTB-induced muscle weakness was due to impairments of synaptic vesicle exocytosis, endocytosis, or internal vesicular processing. We examined motor nerve terminals in the triangularis sterni muscles of DTB-treated mice at the onset of muscle weakness. Uptake of FM1-43, a fluorescent marker for endocytosis, was reduced to approximately 60% of normal after either high-frequency nerve stimulation or K(+) depolarization. Terminals ranged from those with nearly normal fluorescence ("bright terminals") to terminals that were poorly labeled ("dim terminals"). Ultrastructurally, the number of synaptic vesicles that were labeled with horseradish peroxidase (HRP) was also reduced by DTB to approximately 60%; labeling among terminals was similarly variable. A subset of DTB-treated terminals having abnormal tubulovesicular profiles in their centers did not respond to stimulation with increased uptake of HRP and may correspond to dim terminals. Two findings suggest that posttetanic "slow endocytosis" remained qualitatively normal: the rate of this type of endocytosis as measured with FM1-43 did not differ from normal, and HRP was observed in organelles associated with this pathway- coated vesicles, cisternae, as well as synaptic vesicles but not in the tubulovesicular profiles. In DTB-treated bright terminals, end-plate potential (EPP) amplitudes were decreased, and synaptic depression in response to 15-Hz stimulation was increased compared with those of untreated mice; in dim terminals, EPPs were not observed during block with D-tubocurarine. Nerve-stimulation-induced unloading of FM1-43 was slower and less complete than normal in bright terminals, did not occur in dim terminals, and was not enhanced by alpha-latrotoxin. Collectively, these results indicate that the size of the recycling vesicle pool is reduced in nerve terminals during DTB-induced muscle weakness. The mechanisms by which this reduction occurs are not certain, but accumulated evidence suggests that they may include defects in either or both exocytosi

Topics: Animals; Exocytosis; Fluorescent Dyes; Horseradish Peroxidase; Male; Mice; Mice, Inbred ICR; Nerve Endings; Neuromuscular Junction; Oxidation-Reduction; Pyridinium Compounds; Quaternary Ammonium Compounds; Spider Venoms; Staining and Labeling; Synaptic Transmission; Synaptic Vesicles; Thiourea; Time Factors

In previous studies, 2,4-dithiobiuret (DTB) caused a delayed onset neuromuscular weakness in rats which was associated with decreased quantal content, alterations in postsynaptic ion channel properties, and abnormalities in nerve terminal ultrastructure. The latter include features typical of degenerating or diseased motor endplates as well as a marked proliferation of smooth endoplasmic reticulum (SER), swelling of mitochondria and evidence for a decreased in intraterminal calcium concentrations at early stages of intoxication (Jones, 1989, Acta Neuropathol. 78:72). These in vivo studies do not allow us to distinguish between the initial effects of DTB on the nerve terminal and those evolving as a result of disuse or secondary to its action on the muscle fiber or Schwann cells. To begin to distinguish between primary and secondary effects of DTB, we examined DTB-treated rat PC12 cells for comparable changes. The direct effects of DTB on PC12 cells included signs of general toxicity. Cell death in sparsely- plated cultures increased from 8-9% in controls to 13.7% at 10 microM for 24 hr exposure, and continued to increase in a concentration-dependent fashion to 25% mortality at 25 microM. However, between 25 and 100 microM there was little additional increase in mortality. 10 to 40 microM DTB slightly decreased the ability of both differentiated and undifferentiated cells to adhere to a substrate. This effect was independent of cell mortality. In moderately-differ-entiated cells having processes up to 10 cell diameters and several varicosities, concentrations of DTB as high as those invoking increased cell mortality and comparable to those affecting the rat neuromuscular junction did not cause abnormalities in the structure of the SER. No masses of tubulovesicular profiles were seen with transmission electron microscopy, and large changes in the quantity or distribution were not detected at the light microscope with the fluorescent stains DiOC6 or rhodamine B. Other signs of neuronal degeneration (blebbing of the plasmalemma, large intracellular droplets, mitochondrial abnormalities) preceded or accompanied any evidence for abnormalities in the SER. Thus the effect of DTB on the SER at the rat motor nerve terminal may occur secondary to a more general toxic action on other cell types, or may be dependent on a level of neuronal activity not achieved in sparsely- plated cultures, or may require a greater degree of differentiation of the neuronal cells than p

Topics: Animals; Cell Adhesion; Cell Differentiation; Cell Survival; Endoplasmic Reticulum, Smooth; Glass; Models, Neurological; Nerve Growth Factors; Neurons; Neurotoxins; Paralysis; PC12 Cells; Polylysine; Rats; Thiourea

2,4-Dithiobiuret (DTB) is a sulfonated derivative of urea that is used as a reducing agent in chemical manufacture. Its low acute toxicity to rodents belies a peripherally mediated, delayed-onset muscle weakness which develops during repeated daily exposure. In experiment 1, a standard dose regimen of DTB (0.5 mg/kg per day IP for 5 days) was used to induce motor dysfunction as a way to dissociate peripheral and central influences on a test of cognitive and motor function in rats. Sixteen male rats were trained to perform a Delayed Matching-to-Position/Visual Discrimination (DMTP/VD) task which permits quantification of working memory (matching accuracy), reference memory (discrimination accuracy), and motor function (choice response latency and nosepoke inter-response time, IRT). The first dose of DTB significantly increased matching accuracy; during the following week, DTB reduced matching accuracy, increased choice response latency and nosepoke IRT, and reduced trial completion. Discrimination accuracy remained unaffected. Experiment 2 explored the effects of single administrations of DTB on DMTP/VD. Sixteen other trained rats were divided into two groups with equal matching accuracy. One group received DTB (0.5,1.0, and 2.0 mg/kg, IP) in separate injections at least 1 week apart; the other group received vehicle at the same times. Matching accuracy increased significantly in the treated rats and not in the controls following each dose of DTB. The magnitude of this increase was dose dependent, and lasted from 1 to 8 weeks after each injection. Discrimination accuracy, response latency, nosepoke IRT and trial completion remained unaffected throughout the study. After DTB, matching accuracy was less easily disrupted by scopolamine (0.1-0.3 mg/kg, IP). However, DTB did not alter the rats' response to reducing the distance between the response levers, to reversal of the matching rule to a nonmatching rule, or to challenge with MK-801 (0.05-0.10 mg/kg, IP). These data indicate that acute DTB causes a long-lasting facilitation of working memory in rats in the absence of any of the indications of motor impairment which follow repeated, daily injections of the chemical.

Topics: Animals; Cognition; Injections, Intraperitoneal; Male; Motor Activity; Rats; Thiourea; Time Factors

2,4-Dithiobiuret (DTB) causes a delayed-onset neuromuscular weakness when given chronically to rats. Mechanisms underlying this effect involve disruptions of acetylcholine (ACh) release and possibly effects on the ACh receptor channel complex. Previous experiments demonstrated a decrease in decay time constants for end-plate currents and miniature end-plate currents of muscles from rats exhibiting DTB-induced muscle weakness compared with those of controls. The purpose of the present study was to determine whether the alteration in rise and decay times for synaptic currents was due to direct effects of DTB on ACh receptor channels. Currents carried through single ACh-activated channels were recorded using patch voltage-clamp techniques in G8 mouse myotubes exposed to DTB in their growth medium and from intact hemidiaphragm preparations of rats treated with DTB by examining fluctuations in membrane noise during iontophoresis of agonist. Exposure of myotubes to DTB (1 or 10 microM) decreased the mean channel open time induced by suberyldicholine for short durations of exposure, whereas longer exposures (24-48 h) to DTB were required in order for decreased open times for ACh as an agonist to be observed. In the absence of DTB, closed times for single channels of G8 cells were described by a two-exponential fit reflecting intraburst and interburst closures. At 1 microM DTB, the duration of gaps within bursts and of gaps between bursts increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cells, Cultured; Ion Channels; Iontophoresis; Male; Mice; Muscle, Skeletal; Paralysis; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Thiourea; Time Factors

Chronic administration of 2,4-dithiobiuret (DTB), causes delayed-onset neuromuscular weakness in rats. This effect results from inhibition of quantal release of acetylcholine (ACh) from motor nerve terminals. The effects of noncholinergic neurotransmission are unknown. The purpose of the present study was to examine the presynaptic mechanisms involved in DTB-induced inhibition of ACh release, particularly, the specificity of action of DTB for cholinergic secretion. Differentiated pheochromocytoma (PC12) cells were used to compare the effects of DTB on the content and release of ACh and dopamine (DA) using neurochemical techniques. At concentrations of 50 to 1000 microM, DTB had little or no effect on [3H]choline uptake or on the spontaneous release of endogenous or [3H]ACh, but caused a significant decrease in release of endogenous or [3H]ACh elicited by depolarization with elevated extracellular [K+]. DTB reduced evoked release of ACh without altering cellular levels of ACh or choline, suggesting that DTB acts directly on mechanisms involved in ACh release. These alterations occurred without prominent alterations in [Ca2+]i as measured by fluorescence microscopy of individual PC12 cells loaded with fura-2. Moreover, DTB did not affect the increase of [Ca2+]i of PC12 cells in response to KCl-induced depolarization. alpha-Latrotoxin-stimulated release of ACh was not inhibited by DTB. DTB-induced suppression of depolarization-evoked release of [3H]ACh was associated with an increased level of [3H]ACh in the vesicular pool although the cytosolic pool was unaffected. High concentrations of DTB also reduced depolarization-evoked release of DA and inhibited DA synthesis resulting in a decrease in the readily releasable pool of DA. These effects occurred at higher concentrations and after longer exposures to DTB than were necessary to alter ACh release. Inasmuch as DA synthesis in the PC12 cell has been shown to be modulated by ACh release, this effect on DA release may reflect a consequence of the diminished release of ACh. These results suggest that DTB alters the release of ACh by interrupting either the mobilization and/or release of the vesicular pool of ACh.

Topics: Acetylcholine; Animals; Dopamine; Dopamine Uptake Inhibitors; Fura-2; PC12 Cells; Rats; Spectrometry, Fluorescence; Thiourea

Increasing the daily dose of dithiobiuret (DTB) given to rats from 0.5 to 1 to 5 mg/kg shortened the latency to onset of treadmill failure and associated flaccid muscle tone from 7 to 5 to 3 days, respectively. Death generally did not follow treadmill failure for at least 2-3 days. After 3 consecutive days of treatment with 5 mg/kg, gastrocnemius muscle contractions elicited by high frequency trains of nerve stimulation were lower in peak tension and more susceptible to tetanic fade (rapid tension decline in tetanus) than were contractions from control rats. Conversely, rats given 12 mg/kg of DTB for 3 days did not exhibit treadmill failure, flaccid skeletal muscle tone, or tetanic contractile abnormalities. Additional treatment with this dose for 2-3 days was required to produce treadmill failure which was not accompanied by flaccid muscle tone. Refractoriness to development of toxicity with DTB was limited in its spectrum; decreased feed and water intake, weight loss, diuresis, dehydration, chromodacryorrhea, and production of mucoid feces occurred even in the absence of flaccid muscle tone. Tissue distribution of DTB-derived [14C] determined 3 hr after injection of [14C]-DTB (12 mg/kg) was largely unaffected by prior treatment with unlabeled DTB (12 mg/kg/day x 2 days). Conventional microelectrode recording studies using end-plates of extensor digitorum longus muscles indicated that abnormalities occurred in quantal release of acetylcholine (ACh) after 5mg/kg/day but not 12 mg/kg/day of DTB. Specifically, reduced quantal content, increased amplitude and prolonged decay of miniature end-plate potentials were observed. The mechanism by which large daily doses overcome or prevent the expected development of flaccid muscle tone and depressed release of ACh typically associated with treatment with DTB does not involve compensatory increases in quantal release of ACh, or altered distribution of the compound.

Topics: Animals; Dose-Response Relationship, Drug; Electric Stimulation; Evoked Potentials; Male; Muscle Contraction; Neuromuscular Junction; Rats; Rats, Sprague-Dawley; Reaction Time; Thiourea; Tissue Distribution

Rats treated with small daily doses of 2,4-dithiobiuret (DTB) develop a delayed onset neuromuscular weakness after 4-6 days of treatment. Analysis of quantal release using nerve-muscle preparations taken from rats exhibiting neuromuscular weakness demonstrated a decrease in quantal content of evoked end-plate potentials (EPP), a decrease in the frequency of spontaneously occurring miniature end-plate potentials (MEPP) and a prolongation of rise and decay times for MEPPs. This latter effect is also observed in muscles taken from rats after one large dose of DTB; in which no weakness is observed. Moreover, an increase in the incidence of abnormally large amplitude MEPPs has also been observed after both acute and chronic treatment of rats with DTB. The purpose of the present study was to determine whether the observed prolongation of rise and decay times for the spontaneous synaptic events observed after exposure to DTB can be attributed to a generalized slowing of all synaptic events or whether DTB causes an increase in a distinct population of abnormal synaptic events. End-plate currents and miniature end-plate currents (MEPC) were recorded, using two microelectrode voltage clamp, from hemidiaphragm preparations of rats after acute administration of 25 mg/kg and chronic treatment with 1 mg/kg/day (7-8 days) DTB. Mean MEPC amplitude calculated for all MEPCs was unaffected by DTB treatment; however there was an increase in the incidence of giant MEPCs after both acute and chronic treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Kinetics; Male; Membrane Potentials; Microelectrodes; Motor Endplate; Muscle, Smooth; Neuromuscular Junction; Rats; Synapses; Thiourea; Time Factors

2,4-Dithiobiuret (DTB) exposure causes a delayed onset muscle weakness in rats that has been attributed to depressed neuromuscular transmission. The present study compares the effects of DTB on both sensory and motor function in rats. Adult male Long-Evans hooded rats were exposed to saline, 0.25, 0.5, or 1.0 mg/kg/day DTB, ip, for 5 consecutive days (Days 1-5). Body weights were monitored throughout the experiment. Motor activity was measured for 1 hr in figure-eight mazes on Days 0, 6, 13, and 27. Forelimb and hindlimb grip strength were assessed on Days 6, 13, and 27. Auditory thresholds were determined for 5- and 40-kHz tones using reflex modification of the startle response on Days 0, 7, 14, and 28. Visual function was examined on Day 6 in animals exposed at 0.5 mg/kg/day using flash- and pattern-elicited visual evoked potentials (FEPs and PEPs, respectively). Thermal sensitivity was measured using the hot plate procedure. All motor endpoints were decreased in a dosage- and time-dependent manner; the higher the dosage the longer the effects lasted. There were no effects on any measure of sensory function with the exception of peak N2 of the FEP. Both the amplitude and latency of FEP N2 were altered by DTB exposure. Decreases in body weight were maximal on Day 9 at 1.0 mg/kg/day (20% from control), but recovered by Day 22. Motor activity was suppressed on Day 6 only, whereas grip strength measures were decreased on both Days 6 and 13. Auditory thresholds were not significantly altered; however, baseline startle amplitude was decreased at the highest dosage on Days 7 and 14, but recovered by Day 28. Hot plate latencies were not altered by DTB treatment. These data demonstrate that DTB produces a reversible impairment of motor function, without altering auditory, thermal, or pattern visual function. FEP N2, which is thought to arise from activity generated in the superficial layers of visual cortex, was diminished by DTB treatment, indicating that DTB can alter the function of the CNS, although effects on the motor system are more pronounced.

Topics: Animals; Body Weight; Dose-Response Relationship, Drug; Electroencephalography; Evoked Potentials; Evoked Potentials, Visual; Male; Motor Activity; Motor Neurons; Neurons, Afferent; Psychomotor Performance; Rats; Reflex; Reflex, Startle; Sensory Thresholds; Thiourea

It has been well established that 2,4-dithiobiuret (DTB) intoxication in rats produces a rapidly progressive hindlimb paralysis within days. The cause of this has, until recently, been explained on the basis of a physiological abnormality that involves a prejunctional impairment in the neuromuscular transmission alone. The morphological correlate of the electrophysiological abnormalities has now been provided. This study describes the sequential morphological alterations resulting from a chronic long-term DTB intoxication (1 mg/kg per day, IP) in the rat nervous system up to 48 days. The findings indicate that DTB neurotoxicity evolves as a central peripheral distal axonopathy initially affecting the motor nerve terminals which show accumulation of interconnecting branched tubulovesicular profiles. With continued exposure, nerve terminal swelling and degeneration took place. Similar pathological changes in distal axons were observed progressively involving the small intramuscular nerve bundles close to the nerve terminals. Central nervous system axons in the long descending tracts of spinal cord and the cerebellar vermis showed similar changes but to a lesser extent in later stage of intoxication.

Topics: Animals; Axons; Male; Motor Neurons; Muscles; Nerve Degeneration; Nerve Endings; Nervous System Diseases; Peroneal Nerve; Rats; Rats, Inbred Strains; Sciatic Nerve; Thiourea

Treatment of rats for 5 to 6 days with dithiobiuret (DTB, 1 mg/kg/day, ip) causes a flaccid, ascending neuromuscular weakness which is associated with a decreased end-plate potential (EPP) amplitude, quantal content, miniature end-plate potential (MEPP) frequency, and prolongation of MEPP and EPP rise and decay times. Whereas small daily doses of DTB reliably cause this paresis, a single large dose, approximating the LD50, and far in excess of the cumulative dose given chronically to induce paralysis, causes no apparent muscle weakness. It was of interest to determine whether subtle changes in neuromuscular transmission are produced by DTB under dosing conditions in which gross muscle weakness is not apparent. As such the present study had two goals: first, to determine whether a single large dose of DTB (25 mg/kg, ip) altered neuromuscular transmission at times when the animal did not exhibit paresis; and second, to determine whether bath application of DTB, at concentrations approximating those in the animal following a single large dose, altered junctional transmission at early exposure times. EPPs and MEPPs were recorded, from hemidiaphragms taken 1, 4, 8, or 24 hr following treatment of rats with a single dose of DTB or vehicle or from untreated rats which were exposed to 200 microM or 1.85 mM DTB by bath application. One hour after a single large dose of DTB, EPP amplitude and MEPP frequency and amplitude were all decreased. Rise and decay times for MEPPs were prolonged in muscles taken 4 hr after treatment. By 4, 8, and 24 hr after treatment, EPP amplitude, MEPP amplitude, and MEPP frequency returned toward control levels. Bath application of DTB initially increased EPP amplitude, MEPP amplitude, and MEPP frequency; however, with continued exposure EPP amplitude decreased to below control levels. Block of EPPs occurred after approximately 10 or 37 min of exposure to 1.85 mM or 200 microM DTB, respectively. MEPP frequency also decreased with continued exposure to DTB, yet remained above control levels for the duration of DTB exposure. Bath application of DTB caused a slowing of decay times of MEPPs similar to that observed following in vivo exposure. These results demonstrate that a single large dose of DTB initially induces neuromuscular effects similar to those observed in rats paralyzed following chronic treatment with DTB but these effects, with the exception of effects on rise and decay times of synaptic potentials, tend to reverse by 24 hr fol

Topics: Animals; Dose-Response Relationship, Drug; Evoked Potentials; In Vitro Techniques; Male; Membrane Potentials; Microelectrodes; Neuromuscular Junction; Rats; Rats, Inbred Strains; Synapses; Synaptic Transmission; Thiourea; Time Factors

Daily treatment of rats with 2,4-dithiobiuret (DTB, 1 mg/kg/day, ip) causes a flaccid neuromuscular weakness first observable in the hindlimbs after 5-6 days of treatment. With continued exposure, neuromuscular weakness appears to encompass the other muscles of the body; death is presumed to result from paralysis of respiratory muscles. The purpose of the present study was to investigate the early effects on neuromuscular transmission which precede neuromuscular weakness caused by DTB, particularly as they relate to the apparent differential muscle sensitivity, using conventional intracellular microelectrode recording techniques. Experiments were conducted using the hemidiaphragm muscle isolated from male rats treated for 7-8 days, with 1 mg/kg/day DTB ip, a regimen which resulted in hindlimb, but not diaphragmatic paralysis, or with 0.9% NaCl (1 ml/kg/day) as control. Analysis of quantal content of end plate potentials (EPPs) from hemidiaphragms of DTB-treated rats indicated no difference from control. Exposure of hemidiaphragm preparations from DTB-paralyzed rats to solutions containing elevated Mg2+ and lowered Ca2+ concentrations (6 and 1 mM, respectively) resulted in a decreased quantal content of the EPP compared to that of similarly treated control preparations. When miniature EPPs (MEPPs) were evoked from nerve terminals by elevating [K+]e, in the presence of 6 mM Mg2+ and 1 mM Ca2+, the mean peak frequency evoked by K(+)-induced depolarization was reduced in the DTB-treated group; however, the time at which peak frequency was attained was the same for the control and DTB-treated group. Mean MEPP amplitude but not resting MEPP frequency was altered in the presence of high [Mg2+] for diaphragms of the DTB-treated group. Neither MEPP amplitude nor frequency was altered in diaphragms exposed to normal concentrations of Ca and Mg. Prolongation of rise and decay times of MEPPs occurred from end plates of DTB-treated rats irrespective of whether low [Ca2+]/high [Mg2+] solutions were used. However, these effects were more pronounced when low [Ca2+]/high [Mg2+] solutions were used. Diaphragm-derived end plates of the DTB-treated group were also characterized frequently by the presence of very large amplitude MEPPs with prolonged decay times. The overall percentage of the total population of MEPPs which these abnormal MEPPs made up in the DTB-treated rats was increased dramatically by exposure to low [Ca2+]/high [Mg2+] solutions. Thus, differential muscle

Topics: Animals; Calcium; Diaphragm; Magnesium; Male; Neuromuscular Junction; Rats; Synaptic Transmission; Thiourea

Daily treatment of rats with 2,4-dithiobiuret (DTB, 1 mg/kg/day i.p.) produces a flaccid neuromuscular weakness first observed in the hindlimbs after 5 to 6 days of treatment. This condition is characterized by diminished contractile strength following single shock and tetanic stimulation of the motor nerve, but no effect on contractions evoked by direct muscle stimulation, indicating an apparent impairment of motor axon conduction, junctional transmission or both. The purpose of the present study was to investigate further the neuromuscular depression caused by DTB using conventional intracellular microelectrode recording techniques. All experiments were conducted using the extensor digitorum longus muscle isolated from male rats treated for 6 to 7 days with 1 mg/kg/day i.p. of DTB or with 0.9% NaCl (1 ml/kg/day) as control. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from single junctions of DTB-poisoned or NaCl-treated paired controls. Muscles were transected ("cut muscle") to prevent contraction after peroneal nerve stimulation. EPP amplitude was decreased at the time of observable muscle weakness in DTB-treated rats. Endplate resting membrane potential was not affected. Decreased EPP amplitude was associated with a decrease in mean quantal content. Quantal content was depressed to an equivalent extent in DTB-treated rats when stimulus frequency was increased from 0.5 to 2, 5, 25 and 50 Hz. However, as the stimulus frequency was increased, preparations from DTB-treated rats were characterized by failures of nerve impulses to elicit an EPP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Evoked Potentials; Male; Motor Endplate; Neuromuscular Junction; Paralysis; Rats; Rats, Inbred Strains; Thiourea

2,4-Dithiobiuret was given i.p. to rats for 4 days at a daily dosage of 1 mg/kg and the development of the lesion associated with neuromuscular dysfunction studied in hindlimb lumbrical muscles. The first morphological indication of neurointoxication was the appearance in some motor endplates of masses of branching tubular smooth endoplasmic reticulum (SER) on day 2 which correlated with the initial functional disturbances. By the 3rd day, most motor endplates were distended by accumulations of dense-cored, lucent and synaptic vesicles, abnormally swollen mitochondria, intermediate filaments and branching, tubular SER. Evidence of collateral axonal sprouting was seen first at this time. On days 4 and 5, many motor endplates were markedly enlarged and showed axoplasmic organelle congestion. A significant increase in synaptic vesicle size was noted at these times in some terminals. Interposition of Schwann cell processes between the pre- and postsynaptic membranes and terminal retraction was now evident. Some intramuscular nerves showed hydropic Schwann cell cytoplasm with separation of the outermost myelin lamellae, mitochondrial swelling and adaxonal vacuoles as early as the 1st day. Proliferation and segregation of SER around central cores of neurofilaments was seen in myelinated nerve fibres and preterminals on the 3rd day. At this and later times accumulations of SER and swollen mitochondria were found at sites of axonal varicosities and at the paranodal constrictions at nodes of Ranvier. These ultrastructural data are discussed with regard to reduced terminal Ca2+ content (demonstrated by oxalate-pyroantimonate cytochemistry) and compared with the sequelae of botulinum intoxication.

Topics: Animals; Axons; Calcium; Female; Motor Endplate; Motor Neurons; Muscles; Rats; Rats, Inbred Strains; Thiourea; Time Factors

Inhibition of in vitro translation activity by the chemotherapeutic agent cisplatin has been studied. Peptide synthesis was measured in translation assays prepared from guinea pig and rabbit reticulocyte lysates. There is a concentration- and time-dependent inactivation of translation by cisplatin. Inhibition is nearly maximal after a 30-min exposure. Translation is inhibited by cisplatin at concentrations that have been found after the administration of therapeutic doses. Evidence suggests that the suppression of peptide synthesis is due to an interaction between cisplatin and mRNA. An apparent qualitative difference in the peptide products translated from mRNA exposed to cisplatin is demonstrated. Large peptide products are inhibited to a greater extent than products with faster electrophoretic mobility. Decreased translation activity cannot be explained by digestion of the message by either cisplatin itself or by some contaminant of the drug solution.

Topics: Animals; Cisplatin; Dose-Response Relationship, Drug; Guinea Pigs; Male; Peptide Biosynthesis; Protein Biosynthesis; RNA, Messenger; Sparsomycin; Thiourea

To evaluate the hypothesis that depressed neuromuscular transmission causes dithiobiuret (DTB)-induced muscle weakness in rats, the temporal development of impaired treadmill performance and deficits in the nerve-elicited muscle contractions were compared during daily treatment with the toxicant (DTB, 1 mg/kg/day X 6 days). Diminished treadmill test performance after 4 days of treatment marked the initial detection of impaired motor function. At this time fading (loss of tension during tetanus) of gastrocnemius contractions elicited in response to 100-Hz sciatic nerve stimulation occurred in DTB-treated rats but not in controls. After 5 and 6 days of treatment, treadmill failure became complete, tetanic fade worsened dramatically, and peak contractile tension measured during trains of nerve stimulation (10-250 Hz) decreased progressively. Appearing by Day 6 were marked body weight loss, dehydration, hypothermia, and a depression in serum concentrations of thyroid hormones. Total oxygen content of the blood was not reduced at any time during treatment, and serum concentrations of glucose, sodium, potassium, calcium, chloride, and phosphorus in DTB-treated rats on Day 6 were similar to those of control animals. Therefore, hypoxia, hypoglycemia, or a serum electrolyte imbalance do not initiate or modulate the neuromuscular toxicity. Light microscopic evaluation of liver, kidney, lung, thyroid, and other organs in intoxicated rats was unremarkable and in skeletal muscles and selected sites of brain, spinal cord, and sciatic nerve no morphologically significant lesions were observed. Even when DTB-intoxicated rats were maintained in a state of flaccid muscle weakness for 5 continuous days, peripheral nerve lesions proximal to the intramuscular nerves were not detected. Thus, depressed neuromuscular transmission appears to be the primary cause of the flaccid muscle weakness and no evidence was obtained that nonneural effects of DTB initiate or modulate this effect.

Topics: Animals; Kidney; Liver; Male; Neuromuscular Junction; Neurotoxins; Physical Exertion; Rats; Rats, Inbred Strains; Sciatic Nerve; Thiourea; Time Factors

Daily administration of dithiobiuret (DTB, 1 mg/kg X 6 days, ip) produced delayed onset muscle weakness in rats as indicated by failure in a treadmill test. In nerve-muscle preparations from DTB-intoxicated rats neuromuscular toxicity was manifested as contractile fatigue during tetanic nerve stimulation. As muscle weakness developed, feed intake decreased and the animals lost body weight. Water intake was not altered during this time, but urine output was increased concomitant with the development of muscle weakness and resulted in a state of negative water balance. Daily administration of d-penicillamine (d-PEN) antagonized DTB-induced treadmill failure in a dose-dependent fashion. A daily dose of d-PEN (1 mMol/kg, ip) that completely antagonized treadmill failure also antagonized the contractile fatigue, reduced feed intake, weight loss and negative water balance caused by DTB administration. In rats already intoxicated with DTB, initiating daily d-PEN treatment or discontinuing further DTB administration, caused the animals to recover normal treadmill performance after a latent period of five days. A single dose of d-PEN (1 mMol/kg, iv) was not effective in reversing treadmill failure or contractile fatigue in rats already intoxicated with DTB. Thus, continuous daily administration of d-PEN was necessary for it to be effective. A single dose of d-PEN (1 m Mol/kg, ip) administered one hr after [14C]-DTB (1 mg/kg, ip) did not affect the plasma and tissue concentrations of DTB-derived radioactivity or their corresponding elimination kinetics. Cumulative urinary and fecal excretion of DTB-derived radioactivity were also unaffected by d-PEN administration as were the relative proportions of DTB and two of its metabolites, monothiobiuret and thiuret, in urine. Other agents that produced dose-dependent antagonism of DTB toxicity were diethyldithiocarbamate, disulfiram, cysteamine and 2,2'-dipyridyl. Considering the chemical and biological properties of DTB and its antagonists, a mechanism of antagonism involving an alteration of the thiol-disulfide and/or divalent metal cation status of motor axon terminals is postulated.

Topics: 2,2'-Dipyridyl; Animals; Cysteamine; Disulfiram; Ditiocarb; Edetic Acid; Male; Neuromuscular Diseases; Penicillamine; Pyridines; Rats; Thiourea; Tissue Distribution

Effects of 2,4-dithiobiuret (DTB) treatment in rats on neuromuscular transmission and the disposition of cholinergic substances, acetylcholine (ACh) and choline (Ch), were examined in a combined electrophysiological/biochemical study using an in vitro extensor digitorum longus (EDL) muscle-peroneal nerve preparation. EDL muscle preparations isolated from rats treated with DTB (1 mg/kg/day X 5 days, ip) displayed a 49% depression in the frequency of miniature end-plate potentials (MEPPs) and a 21% depression in mean MEPP amplitude. Statistical analysis of evoked end-plate potentials (EPPs) measured in curarized preparations indicated that the mean quantal content (m) was significantly depressed in EDL muscles from DTB-treated rats. At stimulation rates of 1, 10, 20, and 50 Hz the estimated values of m in EDL preparations from DTB-treated rats were, respectively, 21, 25, 45, and 51% of that in control preparations. Biochemical determinations of ACh and Ch revealed a significant DTB-induced increase in endogenous ACh and Ch content in EDL preparations fixed for extraction of ACh and Ch immediately after dissection from the treated rats. In vitro, however, there were negligible changes in overall ACh synthesis since the total (tissue and medium) tracer ACh (2H4-ACh) synthesized from tracer Ch (2H4-Ch; 10 microM) supplied in the perfusion medium was similar in EDL preparations from DTB-treated and control rats. Also, in EDL muscles from DTB-treated rats the resting release of ACh was not affected, but when exogenous Ch (2H4-Ch) was not supplemented in the medium the evoked release (via peroneal nerve stimulation) of ACh was depressed. Thus, decreases in spontaneous quantal ACh release, as detected in the electrophysiological experiments, were not reflected by changes in the biochemically determined ACh resting release. The biochemical determination of evoked ACh release, however, correlated with the decrease in quantal content detected in the electrophysiological analysis of evoked EPPs when exogenous Ch was not supplemented in the perfusion medium. Significant and consistent increases (two to three times) in both Ch content and efflux occurred in the EDL muscles from DTB-intoxicated rats. These results indicate that DTB induces a prejunctional impairment of neuromuscular transmission that is not specifically directed at ACh synthesis. Rather those processes by which ACh is incorporated into or released from vesicles appear to be altered.

Topics: Acetylcholine; Animals; Choline; Electric Stimulation; Electrophysiology; Evoked Potentials; Injections, Intraperitoneal; Male; Membrane Potentials; Muscles; Neuromuscular Junction; Peroneal Nerve; Rats; Rats, Inbred Strains; Thiourea

Urinary excretion of porphyrin precursors delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) and total porphyrins was measured during intoxication of rats with 2,4-dithiobiuret (DTB), a chemical which produces delayed-onset neuromuscular weakness, in an attempt to ascertain whether or not DTB poisoning in the rat would serve as an animal model of the neurologic symptoms of acute intermittent porphyria. Daily administration of DTB (1 mg/kg/day, i.p.) produced flaccid skeletal muscle weakness first detected after 4 to 5 days of treatment. Onset of skeletal muscle weakness was associated with a significant increase in urinary excretion of ALA. The excretion of PBG and total porphyrin was also increased; however, the increase was not significant. The increase in porphyrins and porphyrin precursors was due to increased urine output which coincided with the onset of neuromuscular weakness; urinary concentrations of ALA, PBG, and porphyrins were not increased by DTB. Measurements of free-erythrocyte protoporphyrin, taken after 7 days of DTB treatment, indicated a significant elevation of free erythrocyte protoporphyrin concentration. The pattern of alterations in the heme precursors associated with DTB-induced paralysis in rats is quite different from that observed in humans afflicted with acute intermittent porphyria. Therefore, we conclude that DTB-induced paralysis in the rat does not represent an accurate animal model of acute intermittent porphyria.

Topics: Aminolevulinic Acid; Animals; Disease Models, Animal; Levulinic Acids; Male; Muscle Hypotonia; Neuromuscular Diseases; Paralysis; Porphobilinogen; Porphyrias; Porphyrins; Rats; Thiourea; Time Factors

Chronic treatment of rats with dithiobiuret (DTB) produces a delayed onset muscle weakness and, as suggested by a preliminary study, a distal axonopathy. An inhibition of glycolysis resulting in an energy deficit has been suggested as a possible mechanism of neurotoxin-induced distal axonopathies. To determine whether chronic DTB administration (1 mg/kg/day X 7 days; sacrificed on Day 7) might be associated with a perturbation of glucose metabolism in neuronal tissues, lactate production was measured in brain and spinal cord homogenates prepared from rats which exhibited hindlimb skeletal muscle weakness. Kinetic and statistical analysis showed that glucose-dependent lactate production in these homogenates was not different from that of controls. Lactate production was also determined in brain homogenates prepared from rats intoxicated with acrylamide (50 mg/kg/day X 9 days; sacrificed on Day 9), p-bromophenylacetylurea (200 mg/kg/day X 2 days; sacrificed on Day 14) or 2,5-hexanedione (400 mg/kg/day X 21 days; sacrificed on Day 21). The results indicated that whereas 2,5-hexanedione produced a slight decrease in Vmax, acrylamide and p-bromophenylacetylurea did not cause changes in glucose-dependent lactate production. These findings do not support the suggestion that an inhibition of glycolysis represents a common biochemical lesion associated with neurotoxins which cause delayed onset muscle weakness and distal axonopathy.

Topics: Acrylamide; Acrylamides; Animals; Central Nervous System; Glucose; Glycolysis; Hexanones; In Vitro Techniques; Kinetics; Lactates; Lactic Acid; Male; Neuromuscular Diseases; Rats; Rats, Inbred Strains; Thiourea; Urea

Daily dosing with 1-3 mg/kg dithiobiuret for 4-5 days causes progressive, generalised muscle weakness which is fatal in about 50% of cases on day 4 or 5. Survivors recover mobility by day 7 and appear normal, although still weak. Striking changes in the motor nerves and motor end plates can be observed before and during the development of this weakness, using the zinc iodide-osmium staining technique. The terminal internodes of intramuscular axons become densely stained: later this may extend back into the main intramuscular (i.m.) nerves, and is often followed by axonal degeneration. Many motor end plates lose their branching form and become globular, and profuse terminal sprouting develops before any nerve degeneration appears. Following axonal degeneration, collateral sprouting becomes prominent, and, within four weeks of beginning the dose regime, restores the normal appearance of the innervation. This pattern of response was observed clearly in the whole mounts of the sternocostalis muscle: similar but less marked changes occurred in the lumbrical muscles, while in the soleus and tibialis anterior muscles, loss of end plates seemed to be a more common response. Despite marked differences in the severity of the functional disability, the i.m. changes were similar in juvenile and adult rats. These changes are related to previous electrophysiological findings on the possible mechanism of action of dithiobiuret.

Topics: Animals; Female; Hindlimb; Neuromuscular Diseases; Neuromuscular Junction; Rats; Rats, Inbred Strains; Thiourea

[14C] Dithiobiuret (DTB)-derived radioactivity is eliminated by adult male rats with an approximate plasma half-life of 8-10 hr. About 65-75% of an i.p. dose appears in the urine within 24 hr after treatment and about 2-4% appears in the feces during the same time period. Less than 1% is recovered as [14C] carbon dioxide in expired air. The elimination kinetics for plasma and the % dose of DTB eliminated as the parent compound and metabolites in urine and feces, respectively, are the same for 1 mg/kg and 25 mg/kg acute treatments and for 1 mg/kg/day chronic treatment. Dose and dosing regimen dependent changes in tissue distribution are evident for most tissues with the thyroid gland, lung, stomach and fat being the most affected. Accumulation of DTB or its metabolites in the thyroid gland is greater than in any other tissue and is saturable at 25 mg/kg. The lung and stomach have a lower concentration of [14C] DTB equivalents than the thyroid, but they show the greatest relative increase in concentrations, about 5 fold, when [14C] DTB is administered chronically. Conversely, fat does not accumulate DTB-derived radioactivity during chronic dosing. These findings suggest that DTB metabolism is altered by changes in dose and dosing regimen.

Topics: Animals; Dose-Response Relationship, Drug; Kinetics; Male; Rats; Thiourea; Tissue Distribution

Topics: 4-Aminopyridine; Acetylcholine; Aminopyridines; Animals; Bungarotoxins; Choline; Hemicholinium 3; Male; Muscular Diseases; Neuromuscular Junction; Rats; Rats, Inbred Strains; Thiourea

Our main objective was to describe the metabolism of dithiobiuret (DTB) in the adult, male rat. Based on the thin-layer chromatographic analysis of urine from animals treated ip with 1 mg/kg of [14C] or [35S] labeled DTB, two pathways for metabolism are proposed. One pathway is reversible and involves the oxidation of DTB to thiuret and the reduction of thiuret back to DTB. The other pathway consists of the desulfuration of DTB to monothiobiuret. The liver appears to desulfurate DTB because DTB-derived [35S] was eliminated from the liver more rapidly (T1/2 = 10 hr) than [14C] (T1/2 = 15 hr). The liver was the only tissue where the elimination kinetics of [35S] and [14C] DTB were different. For all extrahepatic tissues examined and plasma, the elimination of DTB-derived [35S] paralleled that of [14C]. The T1/2 for plasma disappearance of both radiolabeled forms of DTB was approximately 10 hr and the cumulative urinary excretion of DTB-derived [35S] and [14C] was parallel and amounted to about 60% of the dose in 24 hr. DTB-derived radioactivity in urine that co-chromatographed with DTB, monothiobiuret, thiuret and sulfate was quantitated along with that of three uncharacterized metabolites. The presence of these unknown metabolites suggests that DTB metabolism is complex. The present study is the first description of the metabolic fate of DTB in the rat and serves as a starting point for determining whether DTB neurotoxicity is caused by the parent compound or a metabolite.

Topics: Animals; Biotransformation; Carbon Radioisotopes; Liver; Male; Rats; Sulfur Radioisotopes; Thiourea; Thyroid Gland; Time Factors; Tissue Distribution

Weanling, juvenile and adult rats both sexes were treated with 1 mg/kg/day [14C] dithiobiuret (DTB) until they developed skeletal muscle weakness as detected by failure in a rotarod test. The latent period before the onset of weakness was longest in weanling animals of both sexes. The greater resistance of the weanlings, to DTB intoxication, could not be explained by differences in the cumulative body burden or tissue distribution of DTB and its metabolites. The sex of the animal was not a determinant of DTB toxicity. Urine and feces were the major and minor routes, respectively, for elimination of DTB and its metabolites in all animals. However, weanlings excreted relatively less 14C by the urinary route and more by the fecal route when compared to adults. These findings demonstrated the age dependence of DTB neurotoxicity in rats and show that it is not caused by differences in either the cumulative body burden or tissue distribution of DTB-derived [14C].

Topics: Age Factors; Animals; Female; Male; Nervous System; Neuromuscular Junction; Rats; Rodenticides; Sex Factors; Thiourea

Topics: Age Factors; Animals; Cholinesterases; Female; Lethal Dose 50; Male; Neuromuscular Diseases; Neuromuscular Junction; Rats; Sex Factors; Thiourea

Topics: Animals; Electric Stimulation; Electroshock; Histocytochemistry; Male; Muscle Contraction; Muscles; Nervous System; Neuromuscular Junction; Postural Balance; Rats; Sciatic Nerve; Tetany; Thiourea

Topics: Animals; Body Burden; Dose-Response Relationship, Drug; Feces; Male; Rats; Thiourea; Tissue Distribution