Prodrugs of Persulfides, Sulfur Dioxide, and Carbon Disulfide: Important Tools for Studying Sulfur Signaling at Various Oxidation States.

Significance: Bioactive sulfur species such as hydrogen sulfide (H2S), persulfide species (R-SnSH, n ≥ 1), hydrogen polysulfide (H2Sn, n ≥ 2), sulfur dioxide (SO2), and carbon disulfide (CS2) participate in various physiological and/or pathological pathways such as vasodilation, apoptosis, inflammation, and energy metabolism regulation. The oxidation state of the individual sulfur species endows them unique biological activities. Recent Advances: There have been great strides made in achieving molecular understanding of the sulfur-signaling processes. Critical Issues: The development of various chemical tools that deliver reactive sulfur species in a controllable manner has played an important role in understanding the different roles of various sulfur species. In this review, we focus on three types of sulfur species, including persulfide, SO2, and CS2. Starting with a brief introduction of their physiological functions, we will then assess the various drug delivery strategies to generate persulfide species, SO2, and CS2 as research tools and potentially as therapeutic agents. Future Directions: Development of donors of various sulfur species that respond to distinct stimulus is critical for this field. Another key to the long-term success of this field is the identification of an area of unmet medical need that can be addressed with these sulfur species.

Carbon Disulfide Induces Embryo Implantation Disorder by Disturbing the Polarization of Macrophages in Mice Uteri.

Carbon disulfide (CS2) induces embryo implantation disorders. Macrophages participate in the process of pregnancy. Therefore, we want to explore the effects of CS2 exposure on polarization and immune function of macrophages in pregnant mice uteri. The exposure times were gestation days 3 (GD3), 4 (GD4), and 5 (GD5), and the observation end points were arranged in a time series after CS2 exposure. The uterine tissues were collected to detect the expression levels of macrophages cytokines (IL-6, IL-12, TGF-ß1, and Vegf-a) and downstream regulatory cytokines of Th1-type (IL-2 and IFN-γ) and Th2-type (IL-10 and IL-4) by flow cytometry, ELISA, and q-PCR. The results showed that, compared with the controls, the ratios of M1/M2 macrophages in the endometrium significantly increased by 96%, 110%, and 177% at the GD4, GD6, and GD7 observation end points after GD3 exposure and increased about 3.88-fold and 2.37-fold at the GD6 and GD7 observation end points after GD4 exposure, respectively. In contrast, the ratio of M1 and M2 macrophages significantly reduced by 53% at the GD5 observation end point after GD3 exposure. Meanwhile, the expression levels of IL-6 were significantly increased about 2.00-fold for mRNA and 1.60-fold for protein at GD4 observation end points after GD3 exposure, and the mRNA levels of IL-12 increased about 3.61-fold at the GD6 observation end points after GD4 exposure. The mRNA levels of TGF-ß1 were significantly decreased by 41%, 25%, and 20% at the GD7 observation end points after exposure at GD3, GD4, and GD5, and the expression levels of Vegf-a mRNA and protein were decreased. Furthermore, the ratio of IL-2/IL4, IL-2/IL-10, IFN-γ/IL-4, and IFN-γ/IL-10 in the uterine tissue was significantly increased at the exposure groups. These findings suggest that the imbalanced polarization of macrophages is the key regulator in the progress of CS2-induced embryo loss.

Combined exposure to carbon disulfide and low-frequency noise reversibly affects vestibular function.

Chronic occupational exposure to carbon disulfide (CS2) has debilitating motor and sensory effects in humans, which can increase the risk of falls. Although no mention of vestibulotoxic effects is contained in the literature, epidemiological and experimental data suggest that CS2 could cause low-frequency hearing loss when associated with noise exposure. Low-frequency noise might also perturb the peripheral balance receptor through an as-yet unclear mechanism. Here, we studied how exposure to a low-frequency noise combined with 250-ppm CS2 affected balance in rats. Vestibular function was tested based on post-rotary nystagmus recorded by a video-oculography system. These measurements were completed by behavioral tests and analysis of the cerebellum to measure expression levels for gene expression associated with neurotoxicity. Assays were performed prior to and following a 4-week exposure, and again after a 4-week recovery period. Functional measurements were completed by histological analyses of the peripheral organs.Nystagmus was unaltered by exposure to noise alone, while CS2 alone caused a moderate 19% decrease of the saccade number. In contrast, coexposure to 250-ppm CS2 and low-frequency noise decreased both saccade number and duration by 33% and 34%, respectively. After four weeks, recovery was only partial but measures were not significantly different from pre-exposure values. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of cerebellar tissue revealed a slight but significant modification in expression levels for two genes linked to neurotoxicity in CS2-exposed animals. However, neither histopathological changes to the peripheral receptor nor behavioral differences were observed. Based on all these results, we propose that the effects of CS2 were due to reversible neurochemical disturbance of the efferent pathways managing post-rotatory nystagmus. Because the nervous structures involving the vestibular function appear particularly sensitive to CS2, post-rotary nystagmus could be used as an early, non-invasive measurement to diagnose CS2 intoxication as part of an occupational conservation program.

Carbon disulfide induces rat testicular injury via mitochondrial apoptotic pathway.

Carbon disulfide (CS2), one of the most important volatile organic chemicals, was shown to have serious impairment to male reproductive system. But the underline mechanism is still unclear. In the present study, we aim to investigate the male germ cell apoptosis induced by CS2 exposure alone and by co-administration with cyclosporin A (CsA), which is the inhibitor of membrane permeability transition pore (MPTP). It was shown that CS2 exposure impaired ultrastructure of germ cells, increased the numbers of apoptotic germ cells, accumulated intracellular level of calcium, elevated ROS level, and increased activities of complexes of respiratory chain. Meanwhile, exposure to CS2 dramatically decreased the mitochondrial transmembrane potential (ΔΨm) and levels of ATP and MPTP opening. Exposure to CS2 can also cause a significantly dose-dependent increase in the expression levels of Bax, Cytc, Caspase-9, and Caspase-3, but decreased the expression level of Bcl-2. Moreover, co-administration of CsA with CS2 can reverse or alleviate the above apoptotic damage effects of CS2 on testicular germ cells. Taken together, our findings suggested that CS2 can cause damage to testicular germ cells via mitochondrial apoptotic pathway, and MPTP play a crucial role in this process.

Modeling cardio-respiratory system response to inhaled CO2 in patients with congestive heart failure.

In this paper we examine a cardiovascular-respiratory model of mid-level complexity designed to predict the dynamics of end-tidal carbon dioxide (CO(2)) and cerebral blood flow velocity in response to a CO(2) challenge. Respiratory problems often emerge as heart function diminishes in congestive heart failure patients. To assess system function, various tests can be performed including inhalation of a higher than normal CO(2) level. CO(2) is a key quantity firstly because any perturbation in system CO(2) quickly influences ventilation (oxygen perturbations need to be more severe). Secondly, the CO(2) response gain has been associated with respiratory system control instability. Thirdly, CO(2) in a short time impacts the degree of cerebral vascular constriction, allowing for the assessment of cerebral vasculature function. The presented model can be used to study key system characteristics including cerebral vessel CO(2) reactivity and ventilatory feedback factors influencing ventilatory stability in patients with congestive heart failure. Accurate modeling of the dynamics of system response to CO(2) challenge, in conjunction with robust parameter identification of key system parameters, can help in assessing patient system status.

Alterations in neurofilaments content and calpains activity of sciatic nerve of carbon disulfide-treated rats.

Chronic exposure to carbon disulfide (CS2) can induce polyneuropathy in occupational worker and experimental animals, but underlying mechanism for CS2 neurotoxicity is currently unknown. In the present study, male Wistar rats were randomly divided into two experimental groups and one control group. The rats in two experimental groups were treated with CS2 by gavage at dosages of 300 and 500 mg/kg per day, respectively, five times per week for 12 weeks. The contents of neurofilament triplet proteins (NF-H, NF-M, NF-L) and two calpain isoforms (m-calpain and u-calpain) in sciatic nerves were determined by immunoblotting. In the meantime, the mRNA levels of NF-H, NF-M and NF-L in spinal cords were quantified by reverse transcriptase-polymerase chain reaction, and the total activity of calpains in sciatic nerves was measured by fluorescence assay. Results showed that the contents of NF-M and NF-L in CS2-treated rats sciatic nerves increased significantly except NF-M in low dose group. The contents and activity of m-calpain and u-calpain in sciatic nerve also demonstrated a significant elevation. Furthermore, the levels of mRNA expression of NFH, NFM and NFL genes were up-regulated consistently in spinal cords of treated rats. These findings suggested that CS2 intoxication was associated with the disruption of neurofilaments homeostasis and activiation of calpains in rat sciatic nerves, which might be involved in the development of CS2-induced peripheral neuropathy.

[Effects of carbon disulfide on blood pressure and electrocardiogram for workers exposed to levels below the national maximum allowable concentration].

OBJECTIVE: To study the effects of carbon disulfide exposure within the national maximum allowable concentration(MAC) on blood pressure and electrocardiogram, and associations with selected factors. METHODS: Workers in a chemical fiber factory were divided into two groups based on the type of work: a high exposure group (HEG) of 821 individuals and a low exposure group (LEG) of 259. The CS2 concentration at workplace was controlled under the national MAC. A set of 250 randomly selected people taking routine physical check-ups in the same period and hospital constituted the control group. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured on the arm, and the pulse pressure (PP) and mean arterial blood pressure (MABP) were calculated based on SBP and DBP. The blood pressure data, along with the results of the routine 12-lead electrocardiography taken at rest and records on gender, age, years of work, type of work, and concentrations of triglycerol, cholesterol, and glucose in blood, were compiled for analyses. Risk factors upon CS2 exposure for the increase of blood pressure and occurrence of electrocardiogram abnormalities were identified and rationalized. RESULTS: Significant difference (P < 0.01) in the average values of SBP, DBP, MABP, and the corresponding abnormality incident rates was found between HEG and LEG, and between HEG and the control group. For both HEG and LEG, the incident rate of DBP abnormality (high DBP) is nearly two times as high as that of SBP. Type of work is the largest risk factor in both the high SBP and high DBP subgroups, with odds ratios (OR) of 2.086 and 2.331 respectively, and high CS2 exposure presents more than double the risk than low exposure. On the incident rate of ECG abnormalities, both exposure groups are significantly different (P < 0.01) to the control group. High SBP in LEG and high DBP in HEG were found to be significant risk factors (OR = 3.531 and 1.638 respectively), while blood glucose appears to be a protective factor (OR = 0.747), appealing to further investigation. Meanwhile, factors like years of work and cholesterol were found to be risk factors in the high SBP subgroup with low exposure, and in the high DBP subgroup with high exposure. Within HEG, high DBP is the only blood pressure-related risk factor found for the incident of left ventricular high voltage (OR = 4.140), as is high SBP for LEG (OR = 4.776). High PP is the only risk factor found for repolarization disturbances within LEG (OR = 20.417). While blood sugar is a risk factor for origin disturbances, it is a protection factor for left ventricular high voltage (OR = 0.633). CONCLUSION: The damage of CS2 done to the cardiovascular system is a gradual process. Both early and very low level exposures are detrimental to the human circulatory system. Below the National MAC limit, the toxic effect of CS2 to the cardiovascular system increases with time and level of exposure. The effect of CS2 on DBP is more significant than on SBP, which indicates that CS2 may affect peripheral resistant blood vessels more than the artery. The abnormalities of ECG of workers exposed to CS2 are not only the result of high blood pressure on the heart, but also of the direct toxicity of CS2 on heart and blood vessels.

Carbon disulfide-induced changes in cytoskeleton protein content of rat cerebral cortex.

To investigate the mechanism of carbon disulfide-induced neuropathy, male wistar rats were administrated by gavage at dosage of 300 or 500 mg/kg carbon disulfide, five times per week for 12 weeks. By the end of the exposure, the animals produced a slight or moderate level of neurological deficits, respectively. Cerebrums of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and centrifuged at a high speed (100,000 x g) to yield a pellet fraction of NF polymer and a corresponding supernatant fraction, which presumably contained mobile monomer. Then, the contents of six cytoskeletal protein (NF-L, NF-M, NF-H, alpha-tubulin, beta-tubulin, and beta-actin) in both fractions were determined by immunoblotting. Results showed that the contents of the three neurofilament subunits in the pellet and the supernatant fraction decreased significantly regardless of dose levels (P<0.01). As for microtubule proteins, in the pellet fraction of cerebrum, the levels of alpha-tubulin and beta-tubulin demonstrated some inconsistent changes. However, in the supernatant fractions, the content of alpha-tubulin and beta-tubulin increased significantly in both two dose groups (P<0.01). In comparison to neurofilament and tubulin proteins, the content of beta-actin changed less markedly, only the supernatant fraction of the high dose group displayed significant increase (P<0.01), but the others remained unaffected. These findings suggested that the changes of cytoskeleton protein contents in rat cerebrum were associated with the intoxication of carbon disulfide, which might be involved in the development of carbon disulfide neurotoxicity.

Interdisciplinary neurotoxicity inhalation studies: carbon disulfide and carbonyl sulfide research in F344 rats.

Inhalation studies were conducted on the hazardous air pollutants, carbon disulfide, which targets the central nervous system (spinal cord) and peripheral nervous system (distal portions of long myelinated axons), and carbonyl sulfide, which targets the central nervous system (brain). The objectives were to investigate the neurotoxicity of these compounds by a comprehensive evaluation of function, structure, and mechanisms of disease. Through interdisciplinary research, the major finding in the carbon disulfide inhalation studies was that carbon disulfide produced intra- and intermolecular protein cross-linking in vivo. The observation of dose-dependent covalent cross-linking in neurofilament proteins prior to the onset of lesions is consistent with this process contributing to the development of the neurofilamentous axonal swellings characteristic of carbon disulfide neurotoxicity. Of significance is that valine-lysine thiourea cross-linking on rat globin and lysine-lysine thiourea cross-linking on erythrocyte spectrin reflect cross-linking events occurring within the axon and could potentially serve as biomarkers of carbon disulfide exposure and effect. In the carbonyl sulfide studies, using magnetic resonance microscopy (MRM), we determined that carbonyl sulfide targets the auditory pathway in the brain. MRM allowed the examination of 200 brain slices and made it possible to identify the most vulnerable sites of neurotoxicity, which would have been missed in our traditional neuropathology evaluations. Electrophysiological studies were focused on the auditory system and demonstrated decreases in auditory brain stem evoked responses. Similarly, mechanistic studies focused on evaluating cytochrome oxidase activity in the posterior colliculus and parietal cortex. A decrease in cytochrome oxidase activity was considered to be a contributing factor to the pathogenesis of carbonyl sulfide neurotoxicity.

A six year follow up study of the subclinical effects of carbon disulphide exposure on the cardiovascular system.

AIMS: A six year prospective cohort study was conducted to clarify whether the current carbon disulphide (CS2) exposure level is low enough to prevent subclinical health impairment and/or to ameliorate health effects due to previous high exposure. This paper describes the effects on the cardiovascular systems. METHODS: The study subjects were 432 male workers exposed to CS2 and 402 non-exposed workers in Japan, all of whom were examined in 1992-93. A total of 251 CS2 exposed, 140 formerly exposed, and 359 non-exposed workers participated in the follow up survey (follow up rate 89.9%) in 1998-99. Mean duration of exposure was 19.3 years at the end the study. Mean CS2 and 2-thiothiazolidine-4-carboxylic acid (TTCA) concentrations were 5.0 ppm and 1.6 mg/g creatinine. Health items examined were serum biochemical indices including lipids and coagulation-fibrinolysis factors, blood pressure, aortic stiffness, ophthalmography, and electrocardiography at rest and after Master's double 2 step test. Potential confounding factors were adjusted for. RESULTS: Incidence of ischaemic findings, defined as Minnesota codes I, IV(1-3), V(1-3) (at rest and after the load), or receiving treatment for ischaemia, was significantly higher in the exposed workers, especially for the spinning/refining workers (adjusted OR 2.1; 95% CI 1.1 to 4.0) or the highest quartile of six year mean TTCA (adjusted OR 3.9; 95% CI 1.8 to 8.7), although the observed increase in risk was diminished when rigorous ECG criteria were applied. Incidence of retinal microaneurysm was increased with marginal significance. Among cardiovascular risk factors we examined, only blood pressure values were significantly increased in the exposed workers. CONCLUSIONS: Increased risk of ischaemic electrocardiogram findings among Japanese viscose rayon workers was observed. Although its clinical significance is to be discussed, the current Japanese occupational exposure limit for CS2, 10 ppm, would be high to prevent subclinical cardiovascular effects in this study population.

Disassociation of carbon disulfide-induced depression of flash-evoked potential peak N166 amplitude and norepinephrine levels.

Exposure to organic solvents frequently causes functional impairment of the central nervous system (CNS). One method to examine the effects of solvent exposure on visual function is flash-evoked potentials (FEPs). Greater knowledge of the role of various neurotransmitters in generating FEP peaks would be beneficial for understanding the basis of neurotoxicant-induced changes. FEP peak N166 is influenced by the psychological construct of arousal, which in turn is believed to be influenced by the function of neurons containing norepinephrine (NE). Because of its known effects on both NE and FEPs, we utilized carbon disulfide (CS2) as a means to examine the possible role of NE in modulating the amplitude of FEP peaks N36 and N166. Our hypothesis was that CS2-induced alterations in cortical NE levels would be correlated with changes in FEP peak N36 and N166 amplitudes. Adult male Long-Evans rats were implanted with electrodes over their visual cortex and allowed to recover. To develop peak N166, FEPs were recorded for two days prior to dosing. On the third day, FEPs were recorded prior to dosing, and one group of animals was sacrificed to serve as pretreatment controls. The remaining animals were dosed ip with 0 (corn oil vehicle; 2 ml/kg), 100, 200, or 400 mg/kg CS2. The treated animals were retested at 1, 4, 8, or 24 h after dosing, immediately sacrificed, and samples of the cortex, cerebellum, striatum, and brain stem were frozen for high performance liquid chromatography (HPLC) analysis of monoamine levels. Treatment with CS2 decreased peak N166 amplitude at 1 h, and peak N36 amplitude was depressed at 4 h, relative to the subject's pretreatment values. Peak latencies were increased, and colonic temperature was decreased by treatment with CS2. Exposure to CS2 depressed NE levels in the cortex, brain stem, and cerebellum 4 h after treatment. Conversely, at 4 h, levels of dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid were increased in the brain stem and cerebellum, and levels of the DA metabolite homovanillic acid were increased in the brain stem. Levels of serotonin were unaffected by CS2 treatment. There was a slight increase in striatal levels of the serotonin metabolite 5-hydroxyindole acetic acid at all times after treatment with CS2. There was no apparent association between the decreases in NE levels and the reductions in amplitudes for peaks N36 and N166. The neurochemical mechanism for CS2-induced reductions in FEP peak amplitudes remains to be determined.

Long-term neuropsychological effects and MRI findings in patients with CS2 poisoning.

OBJECTIVES: To evaluate the long-term neuropsychological effects and magnetic resonance imaging (MRI) findings among retired patients with a history of exposure to carbon disulfide (CS2). MATERIAL AND METHODS: Seventy-four patients with a history of exposure to CS2 were divided into two equal groups according to their level of exposure, and they completed a questionnaire and were evaluated for neuropsychological symptoms using the Korean version of the revised Wechsler Adult Intelligence Scale. Thirty-one MRI images were reviewed retrospectively. RESULTS: There were no statistically significant differences in total, verbal and performance IQs between high- and low-exposure groups. MRI findings revealed a significantly larger number of cerebral lacunae (five of 12 subjects) in the high-exposure group. Periventricular hyperintensities were mostly located in frontal and occipital areas, and white-matter hyperintensities were mostly in frontal and parietal areas. CONCLUSION: The higher prevalence of lacunae in the high-exposure group as revealed by MRI suggests that further MRI studies are needed into long-term neuropsychological effects induced by CS2.

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.

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.

Carbon disulfide neurotoxicity in rats: VI. Electrophysiological examination of caudal tail nerve compound action potentials and nerve conduction velocity.

The effects of subchronic exposure to carbon disulfide (CS2) on ventral caudal tail nerve compound nerve action potential (CNAP) amplitudes and latencies, and nerve conduction velocity (NCV) in rats were examined. Male and female Fischer 344 rats were exposed to 0, 50, 500, or 800 ppm CS2 for 6 hrs/day, 5 days/week. Using separate groups, exposure duration was 2, 4, 8, or 13 weeks. Exposure to 500 or 800 ppm CS2 for 13 weeks decreased NCV compared to the 50 ppm CS2 group. CNAP amplitudes were increased, and peak P1P2 interpeak latency decreased, after exposure to 500 or 800 ppm CS2 for 13 weeks. Most of the changes in NCV and CNAPs were not attributable to differences in tail or colonic temperature. However, the increases in peak P1 amplitude may relate to the proximity of the electrodes to the tail nerves. Assessment of tail nerve morphology after 13 weeks exposure to 800 ppm CS2 revealed only minor changes compared to the extent of axonal swelling and degeneration observed in the muscular branch of the tibial nerve and axonal swelling in the spinal cord. As anticipated, in older animals the NCV increased, the CNAP amplitudes increased, and the CNAP latencies decreased. The biological basis for the changes in CNAPs produced by CS2 is under investigation. Future studies will focus on electrophysiological evaluation of spinal nerve function, to allow better correlation with pathological and behavioral endpoints.

Carbon disulfide neurotoxicity in rats: IV. Increased mRNA expression of low-affinity nerve growth factor receptor--a sensitive and early indicator of PNS damage.

Expression of the low-affinity nerve growth factor receptor (NGF-R) in the peripheral nervous system is regulated by Schwann cell-axonal contact. Steady-state mRNA levels for NGF-R are very low in the mature peripheral nervous system, but are markedly upregulated in sciatic nerve during both primary demyelination (tellurium exposure) and secondary demyelination (Wallerian degeneration). Upregulation also occurs in various subdegenerative axonopathy models where there is axonal atrophy, suggesting its usefulness as a marker for subtle perturbations in normal axon-Schwann cell interactions (Roberson et al., Mol Brain Res 1995; 28:231-238). To further test this hypothesis, we examined NGF-R mRNA expression in sciatic nerves of rats exposed to carbon disulfide (CS2), a toxicant known to cause a distal axonopathy. Adult rats were exposed to CS2 gas (50, 500, or 800 ppm, 6 hr/day, 5 days/wk) for 2-13 weeks. RNA was isolated from sciatic nerves and levels of mRNA for NGF-R determined by Northern blot analysis. NGF-R mRNA expression increased in a dose- and time-dependent manner. Message levels were already increased after 2 wks of exposure to 800 ppm CS2, and increased further with continued exposure. Morphological alterations were not apparent in the sciatic nerve, even at the highest dosage levels with the longest exposure times. Upregulation of NGF-R mRNA is thus an indicator of subtle alterations in the normal axon-Schwann cell relationship and provides a sensitive measure of CS2 neurotoxicity. Assay of this marker may also be useful as a rapid and very sensitive general screen for other compounds which are potentially toxic to the peripheral nervous system.

Carbon disulfide neurotoxicity in rats: VII. Behavioral evaluations using a functional observational battery.

The neurobehavioral consequences of inhalational exposure to carbon disulfide (CS2) were evaluated as part of a joint project between the National Institute of Environmental Health Sciences and the U.S. Environmental Protection Agency. Behavioral changes in rats were measured using a functional observational battery (FOB), which is a series of observations and manipulations designed to assess the neuronal integrity of autonomic, motor, sensory, and integrative functions. Young adult male and female Fischer-344 rats were exposed to one of four CS2 concentrations (0, 50, 500, or 800 ppm, six hours/day, five days/week) and tested at the end of one of several exposure durations (two, four, eight, or 13 weeks). All rats were also tested before exposure began to obtain baseline values. Neuromuscular deficits which were more pronounced in the hindlimbs, e.g., decreased strength and gait alterations, were detected in rats of both sexes. These changes were closely related to CS2 concentration and exposure duration, with mild gait changes evident after only two weeks of exposure. Other effects, mostly observed at 13 weeks, included decreased responsiveness to a visual stimulus and mild tremors. Reactivity in response to handling was generally increased, and excitability in the open field was decreased, in rats tested after the shorter exposures (two and four weeks). Thus, the exposure-concentration and -duration characteristics of the neuromotor syndrome produced by CS2 were detected and defined using the FOB. These studies provide a more complete evaluation of rats under these CS2 exposure conditions, which can then be used to compare with other mechanistic-related endpoints from this collaborative study.

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.