Cardiac sensitization induced by phenobarbitone and prolonged by CS2

The arrhythmogenic effect of 8 g/kg noradrenaline given i.v. was increased in male rats pretreated 1–2 day s earlier with phenobarbitone and starved from the time of the first phenobarbitone injection (80 mg/kg followed by 50 mg/kg 6 h later). Daily exposure to 4.0 mg/l CS₂ (first exposure 24 h after the first phenobarbitone injection) for 4 h prevented the decline in susceptibility on the 3rd and 4th days after phenobarbitone, when the reaction of unexposed rats to noradrenaline returned to normal.     

The turnover of blood glucose and plasma free fatty acids in the rat after acute carbon disulphide intoxication

The turnover of plasma free fatty acids and blood glucose has been measured in starved rats exposed to inhalation of carbon disulphide (2 mg/l) for 15 hr overnight. Measurements were made following single intravenous injection of [U-¹⁴C]palmitic acid or of [5-³H]glucose and [U-¹⁴C]glucose in groups of exposed and control rats. CS₂ intoxication leads to a small but significant decrease in the rate of utilization of plasma free fatty acids and it is suggested that decreased availability of plasma free fatty acids leads to the increased catabolism of amino acids and the relatively large increase in urea production which is seen in starved CS₂-treated rats. Increased urea production is not associated with an increase in the absolute rate of production and utilization of blood glucose as measured using [5-³H]glucose. There is an increase in the fractional turnover of blood glucose, but this is accompanied by a small hypoglycaemia in starved CS₂-treated rats. The fraction of glucose carbon which is recycled is unchanged in treated rats. A comparison has been made between fed and 24 hr-starved rats in their response to exposure to CS₂ (2 mg/l) for 4 hr. The results indicate that in contrast to starved rats, plasma free fatty acid utilization increases when fed rats are exposed to CS₂ and there is no increase in urea production. Inter-relationships between these effects are discussed.     

The in Vivo Toxicity of CS2 to Liver Microsomes: Binding of Labelled CS2 and Changes of the Microsomal Enzyme Activities

Abstract The binding of ³⁵S and ¹⁴C labelled CS₂ to liver microsomes was studied in control and phenobarbitone pretreated rats 3 and 6 hrs after an intraperitoneal injection. The level of hepatic cytochrome P-450, the activities of epoxide hydratase and UDP-glucuronosyltransferase were analyzed in the same animals. The binding of the sulphur label was considerably higher than that of carbon 3 hrs after the injection, the difference being less evident at 6 hrs. The measurable P-450 declined after the CS₂ injection. It was approximately 40 % in the phenobarbitone pretreated rats and 60 % in control rats of the values of animals which were not treated with CS₂. CS₂ did not affect microsomal epoxide hydratase activity, while it increased the measurable activity of UDP-glucuronosyltransferase. The increase was evident 3 hrs after the injection of CS, in the phenobarbitone pretreated rats. It could also be detected in the control animals 6 hrs after the injection. The present data suggest that the change in the measurable P-450 results from the binding of the metabolite(s) of CS₂ to the cytochrome, and its subsequent degradation. The increase in measurable UDP-glucuronosyltransferase activity results probably from the activated perturbation of the structure of microsomal membrane by the metabolites of CS₂ in vivo.     

The binding of CS2 in central nervous system of control and phenobarbitone-pretreated rats

The binding of ³⁵S- and ¹⁴C-labelled Cs₂ in rat central nervous system (CNS) was studied in control and phenobarbitone-pretreated rats in vivo and in vitro. Animals received CS₂ through intraperitoneal injection in olive oil. Samples were taken for analysis 3 and 6 h after the injection.Sulphur atoms were bound to rat brain more highly than carbon atoms in control and phenobarbitone-pretreated rats in vivo. The phenobarbitone pretreatment increased the cerebral binding of sulphur and decreased that of carbon. Main part of the bound sulphur and carbon was detected in the trichloroacetic acid (TCA) precipitable fraction in both test groups. Pretreatment with phenobarbitone or with polychlorinated hydrocarbon (PCB) mixture did not increase significantly the binding of CS₂ sulphur in brain microsomes in vitro.The present findings suggest that a considerable amount of injected CS₂ is retained in the nervous system and that phenobarbitone pretreatment of test subjects may also alter brain metabolism of CS₂.     

Carbon Disulphide Induced Activation of Liver UDP glucuronosyltransferase in Rats Pretreated with Phenobarbitone

Abstract Carbon disulphide (CS₂) exposure has been shown to activate the UDPglucuronosyltransferase of liver microsomes in rats pretreated with phenobarbitone. Now the nature of CS₂ induced activation of the enzymes has been studied further. Phenobarbitone pretreated rats were exposed to 0.15% CS₂ for 2 hrs on two successive days. The activity of UDPglucuronosyltransferase was measured from the liver microsomes after the enzymes was activated by incubation of the microsomes with various concentrations of the detergents Triton X-100, digitonin and cetylpyridinium chloride. The exposed animals showed an increased enzyme activity at all applied concentrations of the detergents; therefore in addition to membrane destruction by CS₂ exposure, some other mechanism must also be involved in the CS₂ induced activation of liver microsomal UDPglucuronosyltransferase. The changes in membrane lipid-protein interactions with 1-anilino-8-naphthalene sulphonate (ANS) were also probed. The CS₂ exposed animals had more high-affinity binding sites for ANS in their liver microsomal membranes, and in addition the quantum yield of ANS fluorescence was enhanced by CS₂. The changes differed from those found after carbon tetrachloride exposure and suggest that, even if the two drugs have some common effects on microsomes, e.g. UDPglucuronosyltransferase activation, P-450 destruction and lipid peroxidation induction, the changes they cause in the microsomal micro-environment differ.     

Behavior and characterization of blood carbon disulfide in rats after inhalation

Studies were undertaken to characterize free and bound CS₂ in the blood of exposed rats. Rats were exposed for 4 hr to CS₂ at concentrations of 0.5, 1.0, 2.0, 3.0, or 4.0 mg/liter. Both free and bound or acid-labile CS₂ (AL CS₂) in the blood increased linearly with inhalation concentration. AL CS₂ in the blood also increased linearly with time when rats were exposed to 2 mg/liter of CS₂ for up to 4 hr. Free CS₂ was eliminated rapidly by a two-exponential, first-order process, with half-times of 8.7 and 55.2 min; AL CS₂ was eliminated similarly but more slowly, with half-times of 2.2 and 42.7 hr. The direct proportionality of blood AL CS₂ concentrations to the inhalation concentrations and exposure time, coupled with the slow AL CS₂ elimination, suggests that blood AL CS₂ may be useful as an indicator of total CS₂ exposure. When rats were exposed to 4 mg/liter of CS₂ for 4 hr, the majority of free and AL CS₂ was in the red blood cells. Dialysis studies with whole blood from CS₂-exposed rats carried out at 4, 26, and 37°C demonstrated the temperature dependence for the release of free and AL CS₂. Plasma and hemolysates of red blood cells from CS₂-exposed rats, dialyzed at 4°C, showed no detectable loss of AL CS₂ from the plasma and only a slight loss from the hemolysate (10%) after 24 hr. These studies indicate that CS₂ binds mainly to macromolecules in blood.     

The effects of carbon disulfide on the reproductive system of the male rat

Two experimental protocols were employed to determine the effects of carbon disulfide (CS₂) on the reproductive system of the male rat. In the first experiment, adult Long Evans hooded rats were exposed to 0,350 or 600 ppm CS₂ vapor for 10 weeks (5 h/day, 5 days/week). CS₂ exposure caused no change in reproductive organ weights nor in plasma gonadotropin levels. However, animals exposed to 600 ppm CS₂ had slightly lower epididymal sperm counts and significantly reduced plasma testosterone levels. In order to determine if monitoring hormone levels and sperm status in the same male over time might increase the sensitivity of detecting a toxic reaction, the second protocol was employed. Male rats were exposed to 0 or 600 ppm CS₂. After 0, 1, 4, 7 and 10 weeks of exposure, males were observed for mating behavior, and ejaculated sperm count and plasma hormone levels were determined. Animals exposed to 600 ppm CS₂ had significantly shorter times to mount and to ejaculate and decreased ejaculated sperm counts. Plasma gonadotropin levels were similar in both groups while plasma testosterone levels were marginally depressed in CS₂-exposed animals in the early weeks. These data indicate that CS₂ is a toxin of the male reproductive system resulting in abnormal coital behavior and decreased sperm counts. The second experimental protocol proves to be a sensitive method for assessing adverse effects in the male reproductive system.     

Effect of cytochrome P450 isozyme induction and glutathione depletion on the metabolism of CS2 to TTCA in rats

 Analysis of 2-thiothiazolidine-4-carboxylic acid (TTCA), a metabolite of carbon disulfide (CS₂), is used in the biological monitoring exposure to CS₂ at work. In order to clarify the metabolic reasons for individual variation in the urinary excretion of TTCA, the latter was studied in rats pretreated with model cytochrome P450 (CYP) enzyme inducers or glutathione (GSH) depletors. Ethanol, phenobarbital (PB) or 3-methylcholanthrene (MC) did not increase 24-h TTCA output following CS₂ inhalation (50 or 500 ppm, 6 h). After oral dosing (10 mg/rat), PB had an inhibiting effect on the excretion rate of TTCA. Tissue GSH depletors phorone, L-buthionine-(RS)-sulfoximine (BSO) and diethylmaleate (DEM) decreased TTCA excretion in rats given an oral dose (10 mg/rat) of CS₂. The initial inhibition by phorone and DEM was reversed after 6 h and from 12 h onward the TTCA in urine exceeded the control level, an effect not seen with BSO. The proportion of CS₂ excreted in urine as TTCA within 24 h was 1.7% in control rats and 1% after BSO treatment, 1.3% after PB, 1.7% after acetone, 1.8% after MC, 2.0% after phorone and 2.5% after DEM treatment. The amount of TTCA in urine increased with the CS₂ dose in a non-linear fashion: 1.6 μmol (50 ppm/6 h) vs. 4.9 μmol (500 ppm/6 h), and 0.2 μmol (1 mg/kg) versus 3.6 μmol (100 mg/kg). It is concluded that induction of different cytochrome P450 isoforms and transient glutathione depletion have only minor effects on the disposition of TTCA in rats following low-level CS₂ exposure persistently low glutathione level as achieved by E.G. BSO, markedly decreased the metabolism of CS₂ to TTCA; these metabolic effectors are unlikely to have a major role in the individual variation of CS₂ metabolism in exposed workers. Received: 14 June 1994/Accepted: 25 August 1994     

Changes in auditory brainstem response in rats chronically exposed to carbon disulfide

The chronic effect of carbon disulfide (CS₂) on the central nervous system (CNS) was studied by examining auditory brainstem responses (ABRs) in female rats (Jcl Wistar) exposed to 200 ppm or 800 ppm CS₂ by inhalation, 6 h a day, 5 days a week, for 15 weeks. Two modes of ABRs evoked by clicks at 61 and 96 dB sound pressure levels (61 dB-ABR and 96 dB-ABR) were recorded during the exposure and for 6 weeks afterwards. Three main components (I, III and V) of ABRs were analyzed from the latencies and differences between latencies of them (interpeak latencies, IPL I–III, IPL III–V and IPL I–V). The latencies of the three components and IPLs of 96 dB-ABR in rats group exposed to 800 ppm of CS₂ were significantly delayed during the exposure period. The delay of latency of component V and IPL III–V and I–V tended to increase with exposure time. At 61 dB-ABR, the changes in the latency of component V, IPL III–V and I–V resembled those at 96 dB-ABR. For the rats group exposed to 200 ppm CS₂, the latency of component I, IPL III–V and I–V at 96 dB-ABR were delayed significantly but transiently during the exposure period. For both groups, recovery from the latencies of the three components and IPLs of ABR was observed by the end of the recovery period. The delayed latencies of ABR observed in rats exposed to 800 ppm CS₂ suggested a conduction dysfunction in the brainstem due to CS₂ exposure. The transient delay of the parameters in the rats group exposed to 200 ppm CS₂ was considered to represent a slight conduction dysfunction.     

Effect of acute exposure to carbon bisulfide vapour upon some components of the hepatic-microsomal enzyme system in rats

Adult female rats were exposed for 8 h to graded carbon-disulfide (CS₂) concentrations between 20 and 400 ppm. It was found that the lipid content of the hepatic microsomal fraction rose significantly due to an increase in phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyeline, lysophosphatidylcholine, cholesterol, triglycerides, diglycerides, and free fatty acids. The alterations occurred quickly and were completely reversible after exposure. Since the microsomal cytochrome P-450 content, as well as the activity of the microsomal NADPH cytochrome c-reductase, remained within the normal range following identical CS₂ exposures, it is assumed that the alteration in the lipid pattern of the endoplasmic reticulum is a causal factor in inhibition of the microsomal-oxidative drug metabolism, induced by identical inhalatory CS₂ doses. It is suggested that the altered lipid pattern impairs the microsomal membranes, thus affecting electron transport and resulting in a dysfunction of the oxidation chain. A slight elevation of the microsomal total protein was observed in relation both to a rise of the microsomal RNA content and an enhanced incorporation of [2,4 -³H]-L-phenylalanine into the liver microsomes after identical CS₂ exposures. It is considered that this finding might represent an unspecific stimulatory reaction.Presented in part at the Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Mainz, March 21, 1971.     

The effect of an equimolar mixture of carbon tetrachloride and carbon disulphide on the liver of the rat

The inclusion of an equimolar mixture of CS₂ in an oral dose of about 4 ld50's of CCl₄ (5 $\text{mmoles}\text{kg}$) to phenobarbitone pretreated, fasted 100 g male rats reduced the amount of liver injury due to the CCl₄ and prevented deaths occurring. At 24 hr after dosing with the CS₂ + CCl₄ mixture, the morphological changes and chemical composition of the liver more closely resembled those due to CS₂ alone rather than those due to CCl₄. Subsequently the liver lesions due to the mixture became those of a typical but non fatal CCl₄ toxicity. Elevation of conjugated dienes in the hepatic microsomal lipids occurred in the CS₂ + CCl₄ group but not in the CS₂ group, and were less than in the rats given CCl₄ alone.The inclusion of CS₂ with ¹⁴CCl₄ dosed orally to rats caused an initial increase followed by a reduction in the uptake of ¹⁴CCl₄ into the liver within 15 min of dosing, reduction in the amount of bound radioactivity in the liver from 50 per cent in the ¹⁴CCl₄ controls to 30 per cent and a decrease in the respiratory excretion over 24 hr of ¹⁴CO₂ derived from the ¹⁴CCl₄ of about 65 per cent, findings which indicate reduced metabolism in the animal of the CCl₄. In addition, the CS₂ + CCl₄ mixture caused a 50 per cent loss of cytochrome P450 from liver microsomes within 5–10 min of dosing, while CS₂ and CCl₄ alone, respectively, caused no change during this period.Since the toxic effect of CCl₄ on the liver is directly related to its metabolism in the organ, the present findings suggest that the protection caused by the inclusion of CS₂ in the mixture is due to reduction of hepatic microsomal oxidative metabolism of the CCl₄ through an enhanced loss of cytochrome P450.A theoretical basis is thus proposed for the beneficial effects on the liver of sheep of using a CS₂ + CCl₄ mixture rather than CCl₄ alone as a drug treatment in the field.     

Activation of brain tyrosine hydroxylase in rats exposed to carbon disulfide and sodium diethyldithiocarbamate.

A time-dependent activation of brain tyrosine hydroxylase (TH) was observed in rats exposed by inhalation daily for 4 hr to carbon disulfide (5 mg/liter). TH activation rose above control after the second day of exposure, reached 140% of control by the fourth day, and declined thereafter. Activation of TH was also observed in rats given sodium diethyldithiocarbamate (dtc), 400 mg/kg ip daily, followed by 4 hr of air gassing. Brain TH activity fell 17% in control rats gassed with air on the same schedule as CS₂-exposed rats. The decrease in brain TH activity was observed in both CS₂-exposed and dtc-treated rats after one treatment, but rose above control on the second day of exposure and remained elevated for 6 days.     

Long-term effects of postnatal amphetamine treatment on striatal protein kinase A activity, dopamine D1-like and D2-like binding sites, and dopamine content

The purpose of the present study was to determine whether exposure to amphetamine during the preweanling period would alter dopaminergic functioning in the dorsal striatum of adult rats. In three experiments, we assessed the effects of repeated amphetamine treatment on striatal protein kinase A (PKA) activity, dopamine (DA) D₁-like and D₂-like binding sites, and DA content. Rats were pretreated with saline or amphetamine (2.5 mg/kg, ip) for 7 consecutive days starting on postnatal day (PD) 11. At PD 90, rats were killed and their dorsal striata (i.e., caudate–putamen) were removed and frozen until time of assay. Amphetamine pretreatment produced long-term reductions in both striatal PKA activity and DA content. Early amphetamine exposure also resulted in an upregulation of D₂-like binding sites, while leaving D₁-like binding sites unaffected. It is likely that the upregulation of D₂-like binding sites was stimulated by the persistent decline in striatal DA levels. Although speculative, it is possible that excess striatal D₂-like receptors were responsible for inhibiting PKA activity through actions on the cAMP signal transduction pathway. The behavioral relevance of these amphetamine-induced neurochemical changes has not yet be determined.     

Sex and age-related differences in aminopyrine N-demethylase activity in DA- and Wistar-strain rat liver microsomes. Effect of ovariectomy

1. Liver microsomal mixed-function oxidase components were studied in Wistar and Dark Agouti (DA) rats (4-45 weeks) with regard to sex- and age-related differences. Total cytochrome P-450 ranged from 0.29 to 1 nmol/mg in Wistar rats and from 0.21 to 1.27 nmol/mg in DA rats, males had higher levels than females (P<0.0025). Cytochrome b₅ ranged between 0.42-1.37 nmol/mg and 0.42-1.56 nmol/mg in Wistar and DA strains, respectively, and NADPH-reductase activity ranged between 14-43 and 11-46 nmol/min per mg (Wistar and DA respectively).

2. Significant age-related differences were found in DA rats with four- to six-fold increase in N-demethylase activity from young to adult rats. Sex-related differences were found in both Wistar- and DA-strain rats, with males having higher (about twice) metabolic activity than females. In contrast, no significant sex- or age-related differences in cytochrome ₅ content, or NADPH-reductase activity, were found.

3. Ovariectomy of 10-13-week-old females did not affect N-demethylase activity, cytochrome P-450, cytochrome b₅ or NADPH-reductase activity in Wistar or DA rats.

4. Cytochrome P-450 content did not correlate (r = 0.35) with aminopyrine N-demethylase activity.

5. Results indicate that sex- and age-related differences are due to changes in the isozymic composition of cytochrome P-450, and that these changes are not subject to oestrogen regulation.     

Variation in the hepatotoxic effects of carbon disulphide between different strains of rat

The hepatotoxic effects of carbon disulphide have been investigated in an outbred (Porton) strain of rat and in 8 inbred strains. Carbon disulphide (1.38 mmoles/kg body weight) was administered intraperitoneally to rats which had been pretreated with phenobarbitone and starved. Livers were taken for analysis 24 h later. A considerable genetic variation in the response of rats to carbon disulphide was observed. The extent of centrilobular hydropic degeneration varied greatly between strains and was accompanied by a high incidence of focal coagulative necrosis in the most susceptible rats. Analysis of variance of the accompanying changes in liver weight and water content and in total liver cytochrome P450 content gave statistical confirmation of a strain effect in the response to carbon disulphide. Highly significant strain x treatment interactions for these parameters were attributable to changes after carbon disulphide treatment rather than variation between phenobarbitone pretreated starved controls. The experiments were repeated in two blocks, 3 months apart. Block effects and interactions were significant in some cases but were quantitatively small and did not obscure a ranking based on histological assessment. AGUS rats were least affected by carbon disulphide whereas PVG and LEW rats showed extensive liver damage. Other strains (WA, Porton, F344, BDIX, WAG) showed a gradation of response between these extremes.     

The effect of carbon disulphide on the stereotypic effect of dopamine agonists

Exposure to CS₂ increased the intensityof apomorphine-induced stereotypy in male rats without increasing the reaction time. With amphetamine, an indirect agonist of dopamine, exposure to CS₂ had a more intensive effect and significantly prolonged the length of reaction.     

Halothane anesthesia enhances the effect of dopamine uptake inhibition on interstitial levels of striatal dopamine

The anesthetic, isoflurane, has been shown to potentiate the ability of the dopamine (DA)-uptake inhibitor, nomifensine, to increase the brain interstitial dopamine level ([DA]_e). Since the effect of the more commenly used anesthetic, halothane, on this system is unknown, we determined [DA]_e by microdialysis in the striatum of rats, conscious or anesthetized with halothane, in the presence of the more selective DA uptake inhibitor, vanoxerine (GBR 12909), or the DA releaser, d-amphetamine. Basal [DA]_e was not changed by halothane. However, in halothane-anesthetized rats, the vanoxerine (3 mg/kg i.v.)-induced DA response increased severalfold compared to the response in conscious rats. The initial peak response to d-amphetamine (1 mg/kg i.v.) did not change, but the late response (1–3 h after injection) was augmented in anesthetized rats. Halothane is believed to increase firing of DA neurons in the substantia nigra and, hence, to release striatal DA. We hypothesize that [DA]_e, is maintained at a normal level during the increased firing by equally increased activity of the DA transporter. However, when the DA transporter is blocked by vanoxerine, the increased DA release is unimpaired and [DA]_e rises. Correspondence to: A. Fink-Jensen at the above address     

A Benchmark Concentration for Carbon Disulfide: Analysis of the NIOSH Carbon Disulfide Exposure Database

A statistical analysis of the NIOSH (National Institute for Occupational Safety and Health) carbon disulfide (CS₂) exposure database was conducted for purposes of establishing a benchmark concentration (BMC) for CS₂. The analysis addressed the effects of CS₂exposure on the peripheral nervous system and on ischemic heart disease risk factors. The BMC is based on models relating response to exposure determined from statistical analysis of the continuous exposure data for individuals recorded in the NIOSH database. The results demonstrate that changes in the responses associated with increases in CS₂exposure at levels represented in the NIOSH database are relatively small after adjustment for confounders. The only response variables that had statistically significant relationships with CS₂were the peroneal nerve MCV (motor conduction velocity) and the peroneal nerve amplitude ratio. Based on these results, BMCs of 16.2 and 18.5 ppm were derived for MCV and amplitude ratio, respectively.     

Involvement of 3′,5′-cyclic adenosine monophosphate in the increase of tyrosine hydroxylase activity elicited by cold exposure

Summary We studied the relationship between changes of 3,5-cyclic adenosine monophosphate (cAMP) and tyrosine hydroxylase (TH) activity in adrenal medulla of rats exposed to cold stress. Exposure of rats to 4° C produced a ten-fold increase of the cAMP content of adrenal medulla in about 30 min. This increase persisted for about one hour; the levels of cAMP returned to control value within 120 min in spite of the continued exposure to 4° C. In rats with monolaterally denervated (splanchnicotomized) adrenal, the exposure to 4° C produced only insignificant changes of cAMP concentration. During the exposure to 4° C we also observed an increase (about two times) of catecholamine turnover rate as measured by ³H-dopamine efflux from adrenal glands. This increased efflux persisted for 6 h of exposure to cold suggesting that the efflux of ³H-dopamine can increase without a simultaneous increase of cAMP concentrations. Exposure of rats to 4° C for two hour also increases (about two times) the TH activity as measured 24 h later. Exposure of the animals to 4° C for a time period longer than two hour (4 or 24 h) failed to produce further increases of TH activity. These results support the concept that the increase of cAMP concentrations in adrenal medulla may play a central role in initiating the chain of biochemical events modulating the synthesis of TH.