Oral administration of dextromethorphan does not produce neuronal vacuolation in the rat brain.

Dextromethorphan is a widely used antitussive agent, also showing increased recreational abuse. Dextromethorphan and its metabolite dextrorphan are non-competitive antagonists at the N-methyl-d-aspartate (NMDA) receptor ion channel. Single doses of some NMDA receptor antagonists produce neuropathologic changes in neurons of the retrosplenial/posterior cingulate cortices (RS/PC), characterized by vacuolation or neurodegeneration. To determine whether dextromethorphan produces these characteristic lesions, dextromethorphan was administered orally either as a single dose of 120mg/kg to female rats, or daily for 30 days at doses of 5-400 mg/(kg day) to male rats and 5-120mg/(kg day) to female rats. Brains were examined microscopically for evidence of neuronal vacuolation (4-6h postdose) and neurodegeneration ( approximately 24 or 48h postdose). Administration of dextromethorphan at 120mg/(kg day) in females, and at > or =150mg/(kg day) in males produced marked behavioral changes, indicative of neurologic effects. Mortality occurred at the highest doses administered. There were no detectable neuropathologic changes following single or repeated oral administration of dextromethorphan at any dose. Administration of MK-801 (9mg/kg) produced both cytoplasmic vacuolation and neuronal degeneration in neurons of the RS/PC cortex. Thus characteristic neuropathologic changes found with more potent NMDA receptor antagonists do not occur following single or repeated oral administration of dextromethorphan.

Promotional activities of the non-genotoxic carcinogen bemitradine (SC-33643).

Bemitradine (SC-33643), a diuretic antihypertensive agent, was studied for its carcinogenicity in a 2-year bioassay in Charles River CD rats via dietary admix at dosages of 50, 150 and 450 mg kg-1 for up to 97 weeks (after which they were followed for eight additional weeks without treatment). Body weights were decreased compared to controls: 5-15% in the female and 10-12% in the male dosage groups by week 105 of the study. Prolactin values were significantly increased in 150 and 450 mg kg-1 females. The compound caused significant increased incidences of liver, thyroid (both sexes) and mammary (females only) neoplasms. The metabolism of bemitradine was studied in both rats and man. Bemitradine and its primary metabolite (SC-36741; desethylbemitradine) were tested and found to be non-genotoxic in Ames, rat primary hepatocyte UDS, CHO/HGPRT, CHO cytogenetics, in vivo mouse micronucleus and mouse lymphoma TK+/- (bemitradine only) assays. Finally, in an altered hepatic foci (Y-glutamyl transpeptidase positive) promotion assay in female Charles River CD rats, bemitradine was found to be a promotor, though not as potent as phenobarbital. We concluded that bemitradine (which has been dropped from development) is a non-genotoxic carcinogen which appears to act by a hormonally modulated promotional activity in inducing tumors in the liver and mammary glands. Tumors seen in the thyroid were probably secondary to the effects of bemitradine on metabolism.

The effects of chronic ingestion of spironolactone on serum thyrotropin and thyroid hormones in the male rat.

Male rats were fed spironolactone admixed with feed at doses of 6, 50, and 200 mg/kg/day for up to 13 weeks. After 13 weeks of treatment, there were dose-related increases in thyroid weight and follicular hypertrophy. Serum thyrotropin (TSH) concentrations were significantly increased throughout the treatment period. Serum thyroxine (T4) and triiodothyronine (T3) were significantly decreased at Weeks 2 and 4, but returned to control concentrations by Week 13. The TSH increase and transient T4 decrease suggested that the thyroid hypertrophy was a compensatory reaction to lowered thyroid hormone levels. To determine the effect of spironolactone ingestion on T4 synthesis and metabolism, male rats were fed spironolactone admixed with feed at 200 mg/kg for 2 weeks. The decrease in T4 was not due to decreased synthesis, since iodide uptake and organification were significantly increased by spironolactone treatment. Since uridine diphosphate glucuronosyl transferase activity was significantly increased by spironolactone treatment, it appears that, by increasing hepatic clearance of T4, spironolactone causes a decrease in the serum concentration of this hormone. The lower T4 level causes a release of feedback inhibition and an increase in TSH resulting in the increase in thyroid gland size and activity.

Age- and sex-related changes in the components of the hepatic microsomal mixed function oxidase system in Sprague-Dawley rats.

1. A study of the sex- and age- (4 to 103 weeks) related changes in liver microsomal mixed-function oxidase components of Sprague-Dawley rats, showed that total cytochrome P-450 ranged from 12.0 to 34.0 nmol/g, peaking between weeks 26 and 39; males had higher levels than females at weeks 4 to 51. Cytochrome b5 ranged from 5.0 to 15.0 nmol/g with inconsistent age- and sex-related differences. 2. NADPH:cytochrome C reductase ranged from 2.6 to 4.8 mumol/min per g, with maximal activity at week 39; males had greater activity at weeks 12 to 78. NADH:cytochrome C reductase ranged from 5.2 to 26.9 mumol/min per g, peaking between weeks 51 and 78; females had greater activity at weeks 12 to 51. 3. This age- and sex-related pattern of changes in these components is specific to the Sprague-Dawley strain. There are slight but significant differences between sexes only in rats less than 1 year old. There was no significant correlation between rates of p-nitroanisole or aniline metabolism and cytochrome P-450 concentrations.

Age- and sex-related changes in epoxide hydrolase, UDP-glucuronosyl transferase, glutathione S-transferase, and PAPS sulphotransferase in Sprague-Dawley rats.

1. Age- and sex-related patterns in the activity of hepatic phase 2 conjugating enzymes have been studied in Sprague-Dawley (CD) rats. 2. Epoxide hydrolase activity with styrene oxide ranged from 75 to 346 nmol/min per g and increased with age in males, peaking at week 78. In females, activity remained constant, about 80 nmol/min per g. 3. UDP-glucuronosyl transferase activity with p-nitrophenol ranged from 480 to 1050 nmol/min per g; maximal activity was at week 39. Males had greater activity at weeks 12 to 39. Activity with chloramphenicol ranged from 3.8 to 10.3 nmol/min per g with no consistent age or sex relationship. 4. Glutathione S-transferase ranged from 9.5 to 23.6 mumol/min per g with p-nitrobenzyl chloride and from 29.1 to 66.8 mumol/min per g with 1-chloro-2,4-dinitrobenzene, peaking at week 51 with both substrates. Males had greater activity from weeks 12 to 78. 5. Reduced glutathione varied between 3.2 and 6.5 mumol/g, with males tending to have higher concentrations than females and older rats (greater than 39 weeks) having slightly lower concentrations. 6. PAPS sulphotransferase ranged from 190 to 338 nmol/min per g, with maximal activity at week 78 with no consistent sex-related differences.

The chronic toxicity of bromovinyldeoxyuridine in beagle dogs.

Bromovinyldeoxyuridine (BVDU), a substituted pyrimidine analog with antiviral activity, was given orally to beagle dogs (6/sex/dosage) at dosages of 0, 5, 12, and 30 mg/kg/day for 52 weeks. Complete physical examinations, including ECG recordings and rectal temperature measurements, and clinical laboratory determinations were performed every 13 weeks. At the end of the dosing period, 4 dogs/sex/dosage were sacrificed and complete gross and microscopic examinations performed. The remaining 2 dogs/sex/dosage were sacrificed following a 13-week recovery period. BVDU had no effect on feed consumption, respiration, body temperature, or heart rate. At 30 mg/kg, males gained less weight than controls. At 12 mg/kg (males) and 30 mg/kg (both sexes) there were slight, but statistically significant decreases in mean corpuscular volume, but no changes in red blood cell (RBC) count, hematocrit, or hemoglobin, and no evidence of reticulocytosis. In males dosed at 30 mg/kg, during the last 6 months of dosing, partial thromboplastin times, serum alanine aminotransferase, and alkaline phosphatase increased, and cholesterol decreased. Histologically, bile ductule hyperplasia and gall bladder epithelial hyperplasia were present at 12 and 30 mg/kg in both sexes at the end of both the dosing and recovery periods. At 30 mg/kg, bone marrow hypocellularity and testicular germ cell atrophy were also present in males. Thus, the liver and gall bladder are the major target organs of chronically administered BVDU in dogs. BVDU causes degenerative and proliferative hepatobiliary damage, and secondarily causes changes in clinical chemical parameters. At higher dosages, there are hypoplastic and degenerative changes in the bone marrow and testes.

Paradoxical effect of cobaltous chloride on carbon disulfide induced hepatotoxicity in rats.

A single acute dose of carbon disulfide (CS2, 5 mmol/kg ip) caused hepatic damage in rats pretreated with phenobarbital. Rats pretreated with phenobarbital and cobaltous chloride (CoCl2, 250 mumol/kg sc) were protected against CS2 induced hepatotoxicity. When single acute doses of CS2 and CoCl2 were given at the same time, however, rats developed a much more severe hepatic lesion than that seen following CS2 alone. Similar cotreatment of CoCl2 with bromobenzene, carbon tetrachloride or thioacetamide did not enhance the hepatotoxicity of these well-studied hepatotoxins. Additionally, other divalent metal salts (CuSO4 and ZnCl2) did not enhance CS2 hepatotoxicity. Hence, the interaction between CS2 and CoCl2 (that results in enhanced CS2 induced hepatic damage) appears to be relatively specific for these two agents. CS2 caused an approximate 50% decrease in hepatic cytochrome P-450 when given alone, but an approximate 85% decrease when given with CoCl2. This observation supports the hypothesis that the breakdown products of cytochrome P-450 heme are responsible for CS2 induced hepatotoxicity. In addition, single doses of CS2 or CoCl2 caused increases of 30 to 60% in hepatic glutathione (GSH), but additive responses were not obtained when the two agents were given at the same time. GSH synthetase and gamma-glutamyl transpeptidase activity were inconsistently changed by these treatments, and did not provide a consistent explanation for the increases in GSH. The enhanced hepatotoxicity of CS2 + CoCl2 is not due to changes in hepatic glutathione metabolism.

Changes in hepatic glutathione concentrations during carbon disulfide induced hepatotoxicity in the rat.

When administered acutely to male Sprague-Dawley rats, carbon disulfide (CS2, 5 mmole/kg ip) caused centrilobular hepatic hydropic degeneration or necrosis. Pretreatment with phenobarbital was a requirement for hepatotoxicity and treatment with SKF 525-A, an inhibitor of microsomal CS2 metabolism, reduced the extent of hepatotoxicity. These results support the hypothesis that the hepatotoxicity of CS2 requires metabolism by the cytochrome P-450 containing monooxygenase system. CS2-induced hepatotoxicity was not accompanied by a decrease in hepatic glutathione, but rather, an increase of approximately 50% which occurred 16 hours after CS2 administration. This increase occurred only when CS2 was given to phenobarbital pretreated rats and was prevented by prior treatment with SKF 525-A. Hence, the delayed increase in glutathione could be related to CS2-induced hepatotoxicity. Additionally, CS2-induced hepatotoxicity was not enhanced by depletion of hepatic glutathione with diethyl maleate (DEM). Many hepatotoxins that require metabolic activation cause decreases in glutathione, and the extent of hepatic damage is increased by agents that deplete hepatic glutathione. Therefore, while CS2-induced hepatotoxicity requires metabolic activation, the effects on hepatic glutathione suggests that the mechanism of CS2-induced hepatotoxicity may be distinct from other "metabolically activated" hepatotoxins.

Oxidative metabolism of carbon disulfide by isolated rat hepatocytes and microsomes.

The oxidative metabolism of carbon disulfide (CS2) was investigated in isolated rat hepatocytes and liver microsomes. In microsomes, CS2 metabolism was increased by phenobarbital pretreatment of the rats and decreased with pretreatment of the rats with cobaltous chloride. In both microsomes and hepatocytes, CS2 metabolism was inhibited by SKF-525A. Carbon dioxide (CO2) was the major volatile metabolite of CS2 in hepatocytes, and carbonyl sulfide (COS) was the major volatile metabolite in microsomal incubations. Addition of cytosol to microsomal incubations shifted the predominant volatile metabolite from COS to CO2 but did not change total volatile metabolite formation. Acetazolamide, a carbonic anhydrase inhibitor, significantly decreased COS metabolism but not CS2 metabolism in isolated hepatocytes or microsomes fortified with dialyzed cytosol. When [18O]H2O was included in incubations of microsomes and CS2, a substantial portion of the resulting COS was [18O] enriched, indicating that the oxygen atom was derived from water. These data are consistent with the hypothesis that CS2 is oxidized predominantly by the cytochrome P-450 containing monooxygenase system, and the product of this reaction is an unstable intermediate which reacts with water to form monothiocarbonate and reactive sulfur species. Monothiocarbonate is the hydrated form of COS. In intact hepatocytes, it is metabolized predominantly to CO2 and hydrogen sulfide. Unmetabolized monothiocarbonate can be dehydrated to COS. The majority of the reactive sulfur species and hydrogen sulfide are oxidized to nonvolatile sulfur compounds, including sulfate, but by different mechanisms.

Protection by zinc against acetaminophen induced hepatotoxicity in mice.

The purpose of this investigation was to assess protection by zinc against acetaminophen induced hepatotoxicity and to evaluate possible mechanisms of protection. Mice were treated with zinc (3 mg/kg, ip) or saline (ip) 48 and 24 hr before and sacrificed 12 hr after acetaminophen administration (375, 500, or 750 mg/kg, po). Liver toxicity was then assessed by histological examination. The incidence of hepatotoxicity was significantly less at 375 and 500 mg/kg of acetaminophen in zinc treated animals. The same dosage of zinc was not hepatoprotective when given 1 hr after acetaminophen. Mice were also treated with 1 to 10 mg/kg of zinc (ip) 48 and 24 hr prior to sacrifice, and metallothionein, cytochrome P-450, glutathione, and UDP-glucuronosyl transferase (GT) were determined in the liver. Metallothionein and UDP-GT were increased and P-450 and glutathione decreased at the higher dosages of zinc; however, only metallothionein was significantly changed at the dosage of zinc (3 mg/kg) used in the hepatoprotection experiments. Further, mice were similarly treated with 3 mg/kg of zinc before administration of 375 mg/kg of [3H]acetaminophen (po) and the amount of acetaminophen and acetaminophen bound to metallothionein were determined in the liver for 0.5 to 24 hr. In addition, after 6 hr the subcellular distribution and covalent binding to protein of acetaminophen were also determined. Zinc treatment had no significant effect in any of the above determinations. These results indicate that zinc protects against acetaminophen induced hepatotoxicity and that the observed protection is probably due to an induced biochemical change, but it is apparently not the result of any of the commonly invoked mechanisms.

Testicular toxicity of a novel 1,4-benzodiazepine (SC-32855) in rats and dogs.

SC-32855, a novel 1,4-benzodiazepine, was administered orally to adult male Beagle dogs (75 mg/kg/day for 10 days) and rats (550 mg/kg/day for 28 days). In both species, SC-32855 caused arrest of spermatogenesis and degeneration of the germinal epithelium, however, it had no effect on Sertoli or Leydig cells. There were no meaningful effects on the weights of the testis, epididymis or prostate in either species. Mean serum luteinizing hormone concentrations in dogs, which were 1.6 +/- 0.2 and 1.5 +/- 0.5 ng/ml prior to dosing, and 3.9 +/- 1.1 and 2.3 +/- 0.5 ng/ml after dosing for the control and treatment groups, respectively, were not significantly changed. Mean serum testosterone concentrations in dogs, which were 1.11 +/- 0.13 and 0.95 +/- 0.10 ng/ml prior to dosing, and 1.05 +/- 0.15 and 1.17 +/- 0.16 ng/ml after dosing for the control and treatment groups, respectively, were also not significantly changed. Further, SC-32855 in vitro did not inhibit the synthesis of testosterone in testicular microsomes isolated from normal rats or dogs. These data indicate that SC-32855 causes testicular damage, which is not secondary to decreased testosterone.

Changes in thyroidal function and liver UDPglucuronosyltransferase activity in rats following administration of a novel imidazole (SC-37211).

SC-37211, an imidazole with antianaerobic activity, was administered po to male rats for 2 weeks at dosages of 20, 60, and 200 mg/kg/day. Histological changes in the thyroid included irregularly shaped follicles and slightly enlarged follicular cells. Serum triiodothyronine (T3) and/or thyroxine (T4) were significantly decreased in treated animals at all dosages; these decreases were not observed following a 2-week recovery period. The half-life of serum [125I]thyroxine was also significantly decreased in rats treated with SC-37211. Morphological changes in the thyroid are likely the result of thyroid-stimulating hormone (TSH) stimulation, a response to decreased serum T3 and T4 concentrations. The decreases in T3 and T4 were not due to decreases in iodide uptake or organification. There were dose-dependent increases in liver weights, liver-to-body weight ratios, and UDPglucuronosyltransferase activity toward p-nitrophenol and T4. Therefore, the decreases in serum T3 and T4 were probably due to an increase in hepatic metabolism rather than to a direct effect of SC-37211 on the thyroid.

Testicular toxicity induced by oral administration of SC-32855, A 1,4-benzodiazepine, in the dog.

A 1,4-benzodiazepine, SC-32855, was administered orally once daily at doses of 30, 100, and 300 mg/kg for two weeks to adult Beagle dogs (1/sex/dose). The 300 mg/kg animals were sacrificed in extremis on day three after showing extreme signs of CNS depression. At necropsy, the testes of the 100 mg/kg male weighed considerably less than those of the control. The testicular weights of the 30 and 300 mg/kg animals were comparable to the weight of the control testes. Histopathologic evaluation of the testes of the 100 mg/kg male revealed degenerative and necrotic changes in the seminiferous tubules and cytoplasmic vacuolation in Leydig cells. In the 30 and 300 mg/kg animals, degenerative and necrotic changes were restricted to the seminiferous tubules.