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.

Crosslinking of apolipoprotein E by products of lipid peroxidation.

Apolipoprotein E (APOE) genotype and advancing aging are interacting ri sk factors in the expression of late onset and sporadic Alzheimer's Disease (AD). We tested the hypothesis that 2 products of lipid peroxidation, malondialdehyde (MDA) and 4 hydroxy-2-nonenal (HNE), covalently modify APOE and alter its metabolism. In vitro, both HNE and MDA crosslinked purified APOE3 and APOE4. HNE was a more potent crosslinker than MDA, and purified APO3 was more susceptible to crosslinking by HNE than was purified APOE4. In P19 neuroglial cultures, oxidative stress with lipid peroxidation led to increased intracellular accumulation of anti-HNE and anti-APOE immunoreactive proteins of approximately 50 kDa. Intercellular accumulation of the 50 kDa APOE-immunoreactive protein (APOE-50) was not prevented by cyclohexamide, suggesting formation by post-translational mechanisms. In CSF, a 50 kDa APOE-immunoreactive protein co-migrated with proteins most immunoreactive for HNE and MDA adducts, containing NaB3H4-reducible bonds. These proteins were in CSF from adult subjects (with or without dementia), and in AD patients homozygous for APOE3 or APOE4 alleles. These data suggest that HNE covalently crosslinks APOE in P19 neuroglial cultures to form a 50 kDa protein, and that similar modifications of APOE appear to occur in vivo.

Toluene-3,4-dithiol analysis of blood for assessing carbon disulfide exposure.

Carbon disulfide is a neurotoxic compound used in the production of viscose rayon, and is a major decomposition product of dithiocarbamates used in industry, agriculture, and medicine. Methods used currently for assessing exposure to CS2 are limited in their ability to evaluate cumulative exposures and provide useful information for relatively short periods of time after exposure has ended. The present investigation evaluates a method for monitoring CS2 exposure that consists of cleaving the thiocarbonyl function of free CS2 or certain CS2-generated modifications on proteins using toluene-3,4-dithiol. The resulting toluene trithiocarbonate product is then quantified using reverse-phase high-performance liquid chromatography. The sensitivity, dose response, kinetics and specificity of this biomarker in blood were examined in rats administered CS2 by inhalation, intraperitoneal injection, or gavage for acute through subchronic periods. Dithiol reactive functions in plasma and hemolysate demonstrated a linear dose response over a wide range of exposure levels, were dependent upon the duration of exposure, and appeared to have an appropriate sensitivity for evaluating occupational levels of exposure. Elimination rates of dithiol reactive functions may also be dependent upon exposure duration and exhibit different kinetics for plasma and hemolysate suggesting that elimination rates may be useful for estimating cumulative exposure and intervals between exposure and sample procurement. Dithiol analysis, used in conjunction with previously established erythrocyte protein cross-linking biomarkers, may provide a means to characterize the internal dose of CS2 resulting from acute through chronic periods, and may provide insight into the level of CS2-mediated covalent protein modifications occurring within the nervous system.

Characterizing the influence of structure and route of exposure on the disposition of dithiocarbamates using toluene-3,4-dithiol analysis of blood and urinary carbon disulfide metabolites.

Differences in the toxicities observed for dithiocarbamates have been proposed to result from the influence of nitrogen substitution, oxidation state, and route of exposure. To better characterize the fate of dithiocarbmates in vivoas a function of structure and route of exposure, rats were administered equimolar doses of carbon disulfide (CS2), N-methyldithiocarbamate, pyrrolidine dithiocarbamate, N,N-diethyldithiocarbamate, or disulfiram daily for five days, either po or ip, and sequential blood samples obtained. Protein dithiocarbamates formed by the in vivo release of CS2, parent dithiocarbamate, and protein-bound mixed disulfides were assessed in plasma and hemolysate by measuring toluene trithiocarbonate generated upon treatment with toluene-3, 4-dithiol (TdT). To aid in determining the bioavailability of CS2 from the administered dithiocarbamates, the urinary CS2 metabolites, 2-thiothiazolidine-4-carboxylic acid (TTCA) and 2-thiothiazolidin-4-ylcarbonylglycine (TTCG), were also determined. The levels of TdT-reactive moieties detected depended upon both the compound administered and the route of exposure. Parent dithiocarbamates, with the exception of disulfiram, were eliminated from blood within 24 h; but protein associated TdT-reactive moieties persisted and accumulated with repeated exposure, regardless of the route of exposure. N-Methyldithiocarbamate demonstrated the greatest potential to produce intracellular globin modifications, presumably through its unique ability to generate a methylisothiocyanate metabolite. Urinary excretion of TTCA and TTCG was more sensitive than TdT analysis for detecting dithiocarbamate exposure, but TdT analysis appeared to be a better indicator of in vivo release of CS2 by dithiocarbamates than were urinary CS2 metabolites. These data suggest that CS2 is a more important metabolite, following oral exposure, than are other routes of exposure, e.g., inhalation or dermal. In addition, data also suggest that acid stability, nitrogen substitution, and route of exposure are important factors governing the toxicity observed for a particular dithiocarbamate.

Exposure of C57BL/6 mice to carbon disulfide induces early lesions of atherosclerosis and enhances arterial fatty deposits induced by a high fat diet.

Even though atherosclerotic cardiovascular disease (ACVD) is the number one cause of death in the United States, the effects of environmental toxicants on this process are less well studied than the effects of chemicals on the second leading cause of death, cancer. There is considerable epidemiological evidence that workers exposed to carbon disulfide (CS2) have increased rates of ACVD, and there is conflicting evidence of the atherogenic potential of CS2 from animal studies. Chemical modification, such as oxidation of low-density lipoproteins (LDL), is tightly associated with increased LDL uptake by macrophages and the development of arterial fatty streaks. CS2 has been previously demonstrated to modify several proteins in vitro including LDL, and others in vivo through derivatization and covalent cross-linking. To investigate both the capacity of CS2 to induce arterial fatty deposits by itself, and its ability to enhance the rate of fatty deposit formation induced by a western style, high fat diet, groups of 20 female C57BL/6 mice were exposed to 0, 50, 500, or 800 ppm CS2 by inhalation. Half the animals in each group were placed on an atherogenic high fat diet and half on a control diet (NIH-07). Animals were sacrificed after 1, 4, 8, 12, 16, or 20 weeks of exposure, and the rates of fatty deposit formation under the aortic valve leaflets were evaluated. Exposure of mice on the control diet to 500 and 800 ppm CS2 induced a small but significant increase in the rate of fatty deposit formation over non-exposed controls. A more striking result was observed in the animals on the high fat diet. There was marked enhancement of the rate of fatty deposit formation in mice exposed to 500 and 800 ppm over the animals on the high fat diet alone. In addition, there was a small but significant enhancement in mice exposed to 50 ppm over the rate of fatty deposit formation induced by the high fat diet alone. Analysis of erythrocyte spectrin for protein cross-linking revealed a dose-dependent formation of alpha- and beta-heterodimers in animals on both diets. These data demonstrate that CS2 is atherogenic at high concentrations, but more importantly, suggest that, in conjunction with other risk factors, CS2 at relatively low concentrations can enhance atherogenesis.

Electromyographic assessment of the neuromuscular blockade produced in vivo by anatoxin-a in the rat.

The indirectly evoked compound action potentials (ECAP) of the plantar muscles of the rat were used to investigate the pharmacodynamics in vivo of the neuromuscular blockade produced by anatoxin-a. Onset time to maximum depression and the magnitude of maximum depression in amplitude of the ECAP were dose-dependent. The mean maximum percent depression (+/- S.D.) of the ECAP induced by single, supramaximal stimulations of the posterior tibial nerve after i.v. doses of (+)anatoxin-a hydrochloride at 0, 50, 100, 200 and 800 micrograms/kg were 3 (4), 53 (15), 82 (7), 95 (2), and 100 (1), respectively. The ED50 (95% confidence limits) for depression of the ECAP was 47 mg/kg (39-57 micrograms/kg). Rats administered 200 micrograms/kg or less of (+)anatoxin-a hydrochloride had 75% return of the pretoxin amplitude of the ECAP within 93 min. Animals dosed at 800 micrograms/kg did not have return of neuromuscular function and died despite mechanical ventilation, suggesting a lethal mechanism(s) of action in addition to respiratory paralysis. Percent decrements (+/- S.D.) in the amplitude of the fourth ECAP following repetitive stimulation at 10 Hz were 6 (5), 13 (22), 46 (18) and 59 (8) from (+)anatoxin-a hydrochloride given i.v. at 0, 50, 100 and 200 micrograms/kg, respectively. The decrement observed following repetitive stimulation was attributed to a presynaptic site of action. No change in maximal motor nerve conduction velocity or latency of the ECAP was observed after i.v. administration of (+)anatoxin-a hydrochloride at 100 micrograms/kg. LD50 values (95% confidence limits) for anatoxin-a administered i.v. to mice were 386 micrograms/kg (365-408 micrograms/kg, for (+)anatoxin-a hydrochloride and 913 micrograms/kg (846-985 micrograms/kg) for racemic anatoxin-a hydrochloride. No deaths were observed in mice after i.p. administration of (-)anatoxin-a hydrochloride at doses up to 73 mg/kg.

Carbon disulfide neurotoxicity in rats: V. Morphology of axonal swelling in the muscular branch of the posterior tibial nerve and spinal cord.

The study objectives were to examine the morphological progression and dose response of carbon disulfide (CS2) distal axonopathy in the muscular branch of the posterior tibial nerve (MBPTN) and spinal cord. Male and female F344 rats were exposed to 0, 50, 500 or 800 ppm CS2 by inhalation, 6 hours/day, 5 days per week, for 2, 4, 8 or 13 weeks. At 8 weeks, in the MBPTN, single fascicles contained individual swollen axons. By 13 weeks, multiple fascicles had giant swollen axons with thin myelin sheaths and occasional degenerated and regenerated axons. At 8 weeks, in the spinal cord, white matter changes in cervical segments 1 and 2 consisted of prominent multifocal axonal swelling in the fasciculus gracilis nerve tracts. In lumbar segments 1 and 2, multifocal axonal swelling was first present at 8 weeks in the lateral and ventro-medial funiculus. By 13 weeks, axonal swelling was diffuse in the fasciculus gracilis nerve tracts of the cervical spinal cord and the lateral and ventral funiculus nerve tracts in the lumbar spinal cord. Compared to the spinal cord, where axonal swelling was present in rats exposed to 800 and 500 ppm, in the muscular branch of the posterior tibial nerve, axonal swelling was only present at 800 ppm at both 8 and 13 weeks. Electron microscopic examination demonstrated marked accumulations of neurofilaments in swollen axons in the spinal cord and MBPTN. Axonal swelling was not present in the spinal cord at 50 ppm, or in the MBPT at 50 and 500 ppm. Axonal swelling was not present at earlier time points of 2 and 4 weeks in either the spinal cord or MBPTN.

Covalent modification of hemoglobin by carbon disulfide: III. A potential biomarker of effect.

Although the neurotoxicity of CS2 has been recognized for over a century, presently there is no accepted biomarker of effect for CS2 exposure. Previous investigations have supported covalent cross-linking of erythrocyte spectrin as a potential preneurotoxic marker reflective of the biochemical changes occurring within the axon. In the present investigation, the potential of using CS2 promoted modification of hemoglobin as a dosimeter for quantifying exposure to CS2 was evaluated. Liquid chromatography was used to isolate and measure alpha and beta chains of globin in blood obtained from rats exposed to CS2 by inhalation as a function of exposure level and duration. The degree of globin modification was compared to light microscopic and ultrastructural changes in the central and peripheral nervous systems to determine the temporal relationship of globin modification to the structural changes in the axon. Samples obtained from rats exposed to CS2 contained a globin chain not present in control samples. Analysis of the peak corresponding to the new chain using electrospray mass spectrometry was consistent with the generation of a single dithiocarbamate ester or thiourea intramolecular cross-link in the alpha 1 major chain. This altered globin chain was detectable both at the subneurotoxic level of exposure and prior to axonal structural changes at the neurotoxic levels of exposure used. The extent of modification was positively correlated to the exposure level and duration for all conditions examined. These findings support hemoglobin as a potential preneurotoxic biomarker of effect for CS2 possessing several practical advantages relative to the use of CS2-mediated spectrin cross-linking.

Toxicology of selected pesticides, drugs, and chemicals. Pyrethrin and pyrethroid insecticides.

Pyrethroids have a wide spectrum of insecticidal potency, vertebrate toxicity, and environmental stability. The exceptionally high selectivity ratios of pyrethrins and pyrethroids have resulted in their use for insect control in numerous formulations. A primary effect of pyrethroids is to slow the closing of the sodium activation gate in nerve cells. All pyrethroids have essentially the same basic mechanism of action on voltage-dependent sodium channels but differ in the magnitude of effect. Based on clinical signs, electrophysiologic responses, and chemical structure, pyrethroids can be classified as Type I or Type II. Inhibition of the GABAA receptor appears to be an additional mechanism of Type II pyrethroids. Clinical signs in small animals during a pyrethroid toxicosis vary but are generally attributable to neural dysfunction. Treatment consists of decontamination procedures and application of appropriate symptomatic care, including control of seizures if necessary.

Toxicology of selected pesticides, drugs, and chemicals. Short-chain alcohols.

Widespread utilization of short-chain alcohols in solvents and alcoholic beverages provides small animals with numerous opportunities for exposure. Toxicosis most commonly occurs following ingestion but may also arise from inhalation and/or dermal absorption. The actions of short-chain alcohols are believed to result from nonspecific interactions with biomembranes altering the function of membrane-bound proteins, including the GABAA receptor. Mortality in alcohol toxicosis typically occurs because of respiratory and cardiac arrest as a result of profound CNS depression; therefore, general measures for resuscitation prevail in the initial treatment of severe alcohol toxicosis. Metabolism of alcohols alters the redox state in the liver, leading to hypoglycemia and lactic acidosis in some cases. In primates, treatment for methanol toxicosis is aimed at reducing accumulation of formate, thereby diminishing the metabolic acidosis and ocular damage characteristic in these species.

Algae intoxication in livestock and waterfowl.

Blue-green algae toxins include (1) hepatotoxic peptides that are known to be toxic to cattle, dogs, swine, waterfowl, and sometimes other species; (2) a nicotinic agonist neurotoxin that appears to be toxic to a wide range of animal species; (3) a peripheral-acting cholinesterase inhibitor that is very toxic to swine, birds, and dogs; (4) toxins that impair nervous transmission by blocking sodium channels in nerve cells; and (5) lipopolysaccharide endotoxins. This article provides current information on the mechanisms of action of the primary toxins recognized to date as well as on procedures important in the diagnosis and management of some of the more common cyanobacterial toxicoses in livestock and waterfowl.

Covalent cross-linking of erythrocyte spectrin by carbon disulfide in vivo.

Covalent cross-linking of proteins by CS2 has been demonstrated in vitro and represents a potential mechanism for the toxicity of this compound. In the present investigation the ability of CS2 to cross-link proteins covalently in vivo is demonstrated using denaturing polyacrylamide gel electrophoresis. Intraperitoneal injection of CS2 in rats at 2 or 5 mmol/kg for 21 or 42 days produced several high-molecular-weight (approximately 410 kDa) proteins eluted from erythrocyte membranes which were not present in control animals. Limited proteolysis of the high-molecular-weight protein bands, monomeric alpha spectrin, and monomeric beta spectrin using endoproteinase glu-C, followed by peptide mapping on denaturing polyacrylamide gels, showed the high-molecular-weight proteins to be alpha,beta heterodimers. The production of multiple heterodimers exhibiting different distances of migration was consistent with the existence of several preferred sites for cross-linking. Evidence for the presence of dithiocarbamate ester and thiourea cross-linking structures in spectrin dimers was obtained using selective base hydrolysis. No spectrin dimer was detected in control animals, and dimer formation demonstrated a cumulative dose response in CS2-treated rats. The longevity of red blood cells, the cumulative dose response, and the stability of the cross-linking structures endows spectrin cross-linking with the potential to serve as a biomarker of chronic low-level exposures to CS2 and may provide a means to correlate pathological changes with existing methods of CS2 exposure monitoring. The ability of CS2 to covalently cross-link erythrocyte spectrin suggests that CS2 may also cross-link other proteins in vivo and supports covalent cross-linking of proteins as a possible molecular mechanism through which CS2 manifests toxicity. If so, then spectrin cross-linking may parallel cross-linking reactions in the axon and provide a sensitive, preneurotoxic biomarker of this molecular event.

The mechanism of nucleophilic substitution of alkylpyrroles in the presence of oxygen.

The mechanism of oxidation of alkylpyrroles (1a-d) by molecular oxygen in the presence of nucleophiles is explored. Contrary to previous reports, oxidation of these pyrroles resulted in dimers with both the aromatic rings intact. In the presence of additional nucleophiles these pyrroles entered into substitution reactions. With 2-mercaptoethanol the site of substitution on 1a was the 3-position rather than the side chain. The first-order rate constant for this reaction in acetonitrile with excess oxygen was found to be (7.8 +/- 1.2) x 10(-7) s-1. The rate was unaffected by the presence of either BHT or catechol. Replacing hydrogens at all the potential sites of reaction by deuterium (as in 1aD) did not reduce the rate of substitution. However, the product suffered loss of deuterium from all sites. These observations support a mechanism involving the formation of a complex 20 between the pyrrole and triplet oxygen. Electron transfer from the pyrrole to oxygen in the rate-limiting step is followed by the generation of pyrrolylmethyl intermediate 23 that can react with available nucleophiles including unoxidized pyrroles.

Covalent cross-linking of proteins by carbon disulfide.

Carbon disulfide is known to react with amino groups of proteins to generate dithiocarbamates (2). We observed covalent cross-linking of dithiocarbamate-derivatized proteins under physiological conditions which may occur through several mechanisms. Evidence for the structure of these covalent bridges and the reactive intermediate was obtained using 13C NMR spectroscopy in conjunction with specific isotopic labeling. On incubation at 37 degrees C oxidative coupling of dithiocarbamates generated bis(thiocarbamoyl) disulfides (3) which were reduced by cysteine. In addition, an electrophilic isothiocyanate (4) was generated from decomposition of the dithiocarbamate. Nucleophilic addition of sulfhydryl and amine moieties to the isothiocyanate produced dithiocarbamate ester (5) and thiourea linkages (6), respectively. Evidence for the presence of inter- and intramolecular cross-links was obtained using denaturing polyacrylamide gel electrophoresis under reducing conditions. The formation of isothiocyanate in neutral solution, through elimination of sulfhydryl ion, was correlated with increased pKa values of the parent amine of amino acids. Dithiocarbamates derived from terminal amino groups of proteins did not appear to generate isothiocyanate or form thiourea or dithiocarbamate ester. Both the thiourea and the dithiocarbamate ester were stable at reduced pH, whereas in alkaline media the thiourea was stable but dithiocarbamate ester was hydrolyzed. Although the disulfide and ester linkages were formed more rapidly than the thiourea, generation of the latter appeared to be irreversible, leading to its gradual accumulation over a longer period of time. Generation of isothiocyanate by CS2-derived dithiocarbamates and subsequent covalent cross-linking of proteins may provide a molecular mechanism for CS2-induced axonopathy.

The measurement of 2-thiothiazolidine-4-carboxylic acid as an index of the in vivo release of CS2 by dithiocarbamates.

Dithiocarbamates and their disulfides are used extensively as agricultural fungicides, as accelerators of the vulcanization process of rubber in industry, and as therapeutic agents in medicine. The widespread uses of these compounds in agriculture, industry, and medicine provide many avenues of exposure to the human population. Subchronic to chronic exposures to some dithiocarbamates have resulted in the development of neuropathy in humans and experimental animals. Decomposition to CS2 presents a potential mechanism through which the toxicity of dithiocarbamates may be mediated. The purpose of this study was to determine the potential of dithiocarbamates to release CS2 in vivo. The ability to release CS2 was assessed by measuring urinary 2-thiothiazolidine-4-carboxylic acid (TTCA), which is used in industry to measure the exposure of workers to CS2. In this study, rats were housed individually in metabolic cages and given daily equimolar ip or po doses (1.5 mmol/kg) of N,N-diethyldithio-carbamate (DEDC), disulfiram (DS), N-methyldithiocarbamate (NMDC), or CS2 for 5 days, and TTCA was measured in urine collected at 24 h intervals. For each compound administered, TTCA was produced in all of the treated animals and the amount of TTCA eliminated in urine from po administration was significantly greater than that from ip administration. The relative rates of TTCA elimination in urine were DS > DEDC approximately equal to CS2 > NMDC for both routes of administration. Following administration of N,N-diethyl[13C = S] dithiocarbamate, carbon-13 enrichment at the thiocarbonyl carbon of TTCA was demonstrated using 13C NMR. Analysis of urinary TTCA proved to be useful both for establishing the in vivo release of CS2 by dithiocarbamate containing compounds and for evaluating the bioavailability of CS2. The results appear especially relevant to disulfiram, which is given orally for sustained periods in the treatment of alcoholism and has resulted in the development of neuropathy in susceptible individuals.

CS2-mediated cross-linking of erythrocyte spectrin and neurofilament protein: dose response and temporal relationship to the formation of axonal swellings.

Using model proteins, a mechanism for CS2-mediated covalent cross-linking of proteins has been demonstrated previously. The biologic importance of CS2-promoted protein cross-linking is apparent as a possible dosimeter of CS2 exposure and as a potential mechanism to account for the identical neuropathies produced by 2,5-hexanedione and CS2. The present investigation examines the utility of erythrocyte spectrin cross-linking as a biomarker of effect for inhalation exposure to CS2 and examines the ability of CS2 to cross-link neurofilament proteins, a potential neurotoxic target. Rats were exposed to CS2 via inhalation at control, 50-, 500-, and 800-ppm levels for 2, 4, 8, and 13 weeks and spectrin dimer formation was quantified using denaturing gel electrophoresis and densitometry. Neurofilament preparations were also obtained from spinal cords and examined for cross-linking using Western blotting methods. The results obtained for protein cross-linking were compared to morphologic changes in the cervical and lumbar spinal cord using light and electron microscopy. The spectrin dimer exhibited a cumulative dose response and was detectable at both the 50-ppm level employed that did not produce axonal swellings and prior to the development of axonal swellings for the 500- and 800-ppm levels used. Neurofilament protein cross-linking involved all three subunits and the temporal relationship of cross-linking was consistent with a contributing role in the development of axonal swellings. These results establish the sensitivity of spectrin cross-linking for evaluating inhalation exposures and extend the similarities observed for 2,5-hexanedione and CS2 in both clinical settings and in vitro models to their effects exerted on neurofilaments in the axon.

Disulfiram generates a stable N,N-diethylcarbamoyl adduct on Cys-125 of rat hemoglobin beta-chains in vivo.

Disulfiram (DSF) is a drug used in aversion therapy to treat alcoholics and acts by inhibiting mitochondrial low-K(m) aldehyde dehydrogenase. Investigations into the mechanisms for in vivo inactivation suggest that the DSF metabolite S-methyl-N, N-diethylthiocarbamate sulfoxide reacts irreversibly with an active site Cys. This work aimed to determine if DSF generates monothiocarbamate adducts on cysteine residues in vivo by examining hemoglobin. Sprague-Dawley rats were treated with DSF po for 2, 4, and 6 weeks. Rats have four different globin beta-chains, of which three (beta-1-3) contain two cysteine residues each. MALDI-TOF MS analysis of two new globin species from DSF-treated rats collected by HPLC revealed increments of 99 Da above the mass of the unmodified chains (beta-2 and beta-3). In a separate experiment, the globin mixture was digested for 2 h with Glu-C and reanalyzed by MALDI-TOF MS. Results showed a peptide at m/z 2716.3 having a mass 99 Da higher than a known Cys-containing peptide. Subsequently, the Glu-C digest was analyzed using Q-TOF tandem MS, enabling observation of the +4 charge state of the peptide with m/z 2716.3. This peptide was fragmented to produce y-sequence ions that located the modification to Cys-125 (present on both beta-2 and beta-3). Cys-125 is the most reactive of two cysteine residues on these beta-chains. To confirm the structure of the modification, globin was hydrolyzed with 6 N HCl at 110 degrees C for 18 h. The adduct survived these conditions so that S-(N,N-diethylcarbamoyl)cysteine was detected in the hydrolysates of treated rats on the basis of comparison with the tandem MS spectrum of a standard. These results extend the findings of others obtained using glutathione conjugates and demonstrate the ability of DSF to covalently modify Cys residues of proteins in a manner consistent with the production of S-methyl-N, N-diethylthiocarbamate sulfoxide, or sulfone, intermediates.

Characterization of carbon disulfide neurotoxicity in C57BL6 mice: behavioral, morphologic, and molecular effects.

Female C57BL6 mice were exposed to 0 or 800 ppm carbon disulfide (CS2), 6 h/d, 5 d/wk for 20 weeks. The neurologic function of all mice was assessed once at the end of exposures using a functional observational battery. General health effects included a decrease in body weight gain, piloerection, hunched body posture, and ptosis. Treatment-related effects included altered gait (uncoordinated placement of hind limbs and ataxia) and impaired function on an inverted screen test. In addition, rearing and locomotor movement were decreased in treated mice. Focal to multifocal axonal swelling was seen predominantly in the muscular branch of the posterior tibial nerve, and occasionally giant axonal swelling was detected in the lumbar segment of the spinal cord. Electron microscopic examination revealed swollen axons with massive accumulation of neurofilament proteins within the axoplasm. Covalent cross-linking of erythrocyte spectrin (surrogate protein to neurofilament protein) was demonstrated in mice exposed to CS2 but not in mice receiving filtered air. These data provide supportive evidence that covalent cross-linking of neurofilament proteins is a significant feature of the axonal swellings in mice produced by inhalation exposure to CS2.