Low-dose Animals Research Articles

Central nervous system toxicity of cyclosporine in a rat model.

The central nervous system toxicity of cyclosporine, which is known to be neurotoxic clinically, was investigated in a rat model. Munich-Wistar rats were divided into 3 groups for a 2-week protocol. After baseline EEG and behavioral testing, group 1 (control) received a weight-adjusted volume of parenteral cyclosporine vehicle i.p., group 2 (low-dose) received 5 or 10 mg/kg/day i.p., and group 3 (high-dose) received 20 mg/kg/day i.p. Spontaneous behavior was observed, simple sensorimotor testing performed daily, and awake EEG's recorded 3 times per week. Four of 12 high-dose animals died during study, one after a witnessed tonic-clonic seizure, and two after recording of frankly epileptiform EEG's; there were no deaths in control or low-dose animals. Significant EEG abnormalities developed only at high-dose, with frankly epileptiform EEG's and/or seizures seen in 58 +/- 15% of these rats (P = 0.005, different from controls by life-table analysis). Although some high-dose animals demonstrated hyperirritability and dystonic posturing, behavioral changes were subtle, and animals were often still or rocking slightly during recording of frankly epileptiform EEG's. Walking latency and alley escape behaviors were delayed in high-dose rats, the latter correlating with abnormal EEG's. Serum urea nitrogens were mildly elevated in high-dose animals, but serum creatinine, electrolytes, bilirubin, body magnesium stores, and blood pressure remained normal in all groups. Kidneys showed only mild vacuolation histologically. The brain showed only very focal cortical injury sites related to electrode placement, which did not correlate with EEG changes or mortality. These results suggest that there may be a direct effect of cyclosporine on the central nervous system. This model system should prove useful in defining mechanisms of cyclosporine-related neurotoxicity.

Alterations in pulmonary protective enzymes following systemic bleomycin treatment in mice

Repeated bleomycin administration in animals and humans produces significant lung fibrosis. The pathogenesis of this toxicity may be multifactorial, but it appears to be initiated through the production of radical oxygen species by an activated bleomycin-iron-oxygen ternary complex. Protection of lung tissue from bleomycin-induced toxicity may occur through both specific metabolic inactivation of bleomycin by the enzyme bleomycin hydrolase, as well as by such non-specific antioxidants as catalase and the glutathione system. The effect of chronic, systemic administration of bleomycin on the activities and levels of these enzymes and proteins in pulmonary tissue is unknown. C57BL/6 mice were injected subcutaneously with saline, non-fibrogenic (2mg/kg) and fibrogenic (10mg/kg) doses of bleomycin twice-weekly for 6 weeks. Animals were killed at 0, 1.5, 3, and 6 weeks after initiation of bleomycin treatment. Catalase activity was increased more than 50% at 3 weeks in the low-dose animals, and was decreased over 40% at 6 weeks in the high-dose animals. Total lung glutathione levels were unaffected in both groups, although glutathione reductase activity was increased significantly (over 2-fold) at 1.5 and 3 weeks in the high-dose animals. At 6 weeks glutathione reductase was increased 7- and 12-fold in low and high-dose animals respectively. Glutathione peroxidase activity also was elevated more than 2-fold above control values at 6 weeks in both sets of animals. There was no evidence of induction of bleomycin hydrolase activity at any time point. Rather, bleomycin hydrolase activity was decreased significantly to 30 and 40% of control values at 3 and 6 weeks, respectively, in mice receiving the fibrogenic doses of bleomycin. These results demonstrate that chronic, systemic administration of non-fibrogenic and fibrogenic doses of bleomycin produces changes in activity of lung antioxidant defense mechanisms. The early loss of lung bleomycin hydrolase activity may contribute to the pathogenesis of bleomycin-induced pulmonary toxicity following repeated drug exposure.

Hormesis, Gompertz functions, and risk assessment.

A historical survey of the literature indicates that benefits derived from low doses of toxic substances have been reported over many centuries. Hippocrates, Paracelsus, Arndt, Schulz, and Hahnemann (founder of homeopathy) have all reported that low doses of toxic substances may be "stimulatory" or otherwise beneficial. Assessment of mortality data from modern-day bioassay studies indicates that low-dose animal exposure to a variety of toxic agents can, through an unknown mechanism, also induce beneficial changes which promote health and prolong life (longevity hormesis). This nonspecific and apparently reversible phenomenon has been modeled kinetically through use of age-specific mortality rate and a generalized Gompertz function; the basic assumption is that mortality in an interval is a function of the weighted sum of intensities of physiologic injury during that interval. It was assumed that longevity-enhancing hormetic reduction in population injury may be decremented from life-shortening injury produced through the aging process and concomitant toxicity. At low exposure levels, a net reduction in age-specific mortality rate can sometimes be observed. The implications for risk assessment are significant. It is tacitly assumed in generating virtually all estimates of risk that toxic manifestations observed at higher doses are the sole effects elicited at lower doses. This appears to be qualitatively incorrect.

Oral toxicity of trichlorobenzyl chloride and its acid derivative.

The acute rat oral LD50 of 2,4,6-Trichlorobenzyl chloride (TCBC) was determined to be 3075 mg/kg. When male and female rats were administered 1500 and 3000 ppm TCBC in the diet for 3 weeks, marked retardation in weight gain was observed. Reductions in food consumption were noted in both groups of treated male, but not female, rats. All blood chemistry and urinalysis parameters including those indicative of liver injury, were within the range of normal limits. Gross necropsy revealed pale livers in all high-dose rats and the majority of low-dose animals. Gross observations correlated with observation of degenerative hepatic changes (irregularity of cell and nuclear size, cord pattern, edema and cytoplasmic alterations) following microscopic evaluation. Rats and dogs administered up to 100 ppm trichlorobenzoic acid (TCBA) in the diet for 90 days exhibited no effects considered related to treatment.

Systemic distribution of blood flow during T-2 toxin induced shock in swine

Three groups of swine (6 per group) were used to determine hemodynamic and blood flow alterations induced by T-2 toxin. Two groups were dosed at 0.6 or 2.4 mg/kg T-2 toxin, and one group served as a vehicle control (70% ethanol). Organ blood flow was determined at 0 hr (predosing) and at 90-min intervals for 6 hr postdosing using 15-μm diameter radionuclide labeled microspheres injected into the left atrium. Hemodynamic parameters were obtained at the same time points. The infusion of T-2 toxin resulted in reductions in cardiac output. This trend appeared to reverse itself in the low dose animals after 3 hr, whereas in the high dose group, cardiac output continued to decline. Mean aortic pressure (MAP) declined in a dose dependent fashion which tended to parallel the reduction observed in cardiac output. Heart rate was increased in both groups treated with T-2 toxin. Blood flow to the brain, heart, and kidneys decreased following exposure to the toxin. The relative percentage of cardiac output received by these organs, however, was maintained despite the drop in blood flow. Pancreatic and splenic blood flows were the most severely compromised as a result of T-2 toxicosis. Consequently, the percentage of cardiac output going to the pancreas and spleen was dramatically reduced. Adrenal, hepatic, and total gastrointestinal blood flows increased or did not change from control values. As a result, the percentage of cardiac output supplying these organs increased.

Toxicology and carcinogenesis studies of isophorone in F344 rats and B6C3F1 mice

Toxicology and carcinogenesis studies of isophorone were conducted by administering 0, 250, or 500 mg/kg body weight per day by gavage in corn oil to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex, 5 days/week, for 103 weeks. Dosed male rats developed proliferative lesions of the kidney including hyperplasia, adenoma, and adenocarcinoma of the renal tubule, and epithelial hyperplasia of the renal pelvis. Non-proliferative kidney lesions observed in dosed male rats included mineralization, and a more severe nephropathy in low dose animals than in controls or high dose animals. Carcinomas of the preputial gland occurred in high dose male rats. No isophorone-related lesions were observed in female rats. In male mice, isophorone exposure may have been associated with an increase in hepatocellular neoplasms and mesenchymal neoplasms of the integumentum in high dose animals, and with a marginally increased incidence of lymphoma in low dose male mice. In mice, no non-neoplastic lesions in males or females, or neoplastic lesions in females were considered associated with isophorone administration.

Metabolism and disposition of propylene glycol monomethyl ether (PGME) beta isomer in male rats

Male Fischer 344 rats were given a single po dose of approximately 1 or 8.7 mmol/kg of radiolabelled propylene glycol monomethyl ether (PGME) beta isomer (2-methoxy-1-propanol). After dosing, expired air, excreta, and tissues were analyzed for 14 C activity and metabolites in urine were isolated and identified. Approximately 70 to 80% of the 14 C was excreted in urine while about 10 to 20% was eliminated as 14 CO 2 within 48 hr after dosing. The major urinary metabolite was 2-methoxypropionic acid, which accounted for approximately 93 and 79% of the radioactivity in urine from high- and low-dose animals, respectively. A glucuronide conjugate of the PGME beta isomer was also identified in urine; this metabolite accounted for approximately 3 to 4% of the radioactivity in the urine at both dosages. These results indicate that the PGME beta isomer is metabolized via different routes to different types of metabolites in comparison to the PGME alpha isomer. While the two isomers are biotransformed differently, there is a substantial toxicological data base which clearly shows that the commercial grade PGME mixture (2 to 5% beta isomer) has a low degree of biological activity.

Induction of urinary bladder hyperplasia in Sprague-Dawley rats orally administered tri-n-butyl phosphate.

The effects of tri-n-butyl phosphate (TBP) were investigated in the Sprague-Dawley rat over an 18-wk period. Groups of randomized female (average weight [AW] = 206 +/- 10 g) and male (AW = 294 +/- 13 g) rats were divided into low-dose, high-dose, and control groups (12 rats/sex X group). Tri-n-butyl phosphate was administered by gavage once a day for 5 days/wk over an 18-wk period. Low-dose animals received 0.20 g/kg X day throughout the experiment and high-dose animals received 0.30 g/kg X day for the first 6 wk. For the remaining 12 wk, the high-dose level was increased to 0.35 g/kg X day. Histopathological examination of tissues revealed that all test rats examined developed diffuse hyperplasia of the urinary bladder epithelium. Similar changes were not found in the control animals.

Acute trimethyltin intoxication in the monkey ( Macaca fascicularis)

Adult cynomolgus monkeys were administered trimethyltin (TMT) iv in dosages ranging from 0.75 to 4.0 mg TMT/kg and observed for behavioral changes. Animals were subsequently killed for light and electron microscopic examination. TMT showed a dose-related toxicity, with high dose animals (4.0 and 3.0 mg/kg) dying within 24 hr, and low dose animals (0.75 mg/kg) surviving without morphological effects. Animals given 1.10 mg TMT/kg displayed a reproducible clinical course, characterized by tremor, hyperactivity, and ataxia which progressed to stupor and finally unconsciousness. By light microscopy, neuropathology was most pronounced in the CA-3 and CA-4 regions of Ammon's horn. Degenerating pyramidal neurons, micro- and astrogliosis, and neuronophagia were commonly observed. Mild degenerative changes were identified in amygdala, medulla, spinal cord, and Purkinje cells. The fascia dentata remained intact. Ultrastructurally, injured neurons contained accumulations of lysosomes and lysosome-like structures within perikarya and neurites. Demyelination or vascular damage was not observed. Data indicate the monkey to be highly sensitive to TMT, with morphological injury most severe in limbic structures.

Dose-response studies on male reproductive parameters in dogs with nafarelin acetate, a potent LHRH agonist.

Adult male beagle dogs were administered daily subcutaneous injections of either 0.5 or 2.0 micrograms/kg of a potent LHRH agonist, nafarelin acetate, for 44 days. Although there was a rise in the circulating levels of the gonadotropins and of testosterone following the early injections of agonist, continued treatment caused a marked decline in acute response and basal levels of both LH and testosterone and smaller decreases in the acute FSH response. The decline in LH and testosterone was accompanied by decreases in testicular volume, ejaculated sperm count, sperm motility, ejaculate volume, and duration of ejaculation. The decline in these parameters was more rapid at 2.0 micrograms/kg than at 0.5 micrograms/kg. The profile of responses to 2.0 micrograms/kg could be superimposed on that previously shown for the injection of 10.0 micrograms/kg. At the end of treatment, prostate weights were 36% and 68% of vehicle-treated controls for high- and low-dose animals, respectively. Spermatogenesis was absent in the testes of all agonist-treated animals. Over the dose range tested, the dose-response on all parameters was characterized by a slower evolution to the same maximal effect, rather than by a partial effect. If these data can be extrapolated to man, they would suggest that administration of higher dose levels of LHRH agonists than presently reported should be explored.

Neurotoxicity and protein binding of 2,5-hexanedione in the hen

Previous studies in this laboratory have demonstrated 2,5-dimethylpyrrole adduct formation during in vitro exposure of protein amino groups to the neurotoxic n-hexane metabolite 2,5-hexanedione (2,5-HD). The present investigation reports in vivo pyrrole adduct formation in neural and nonneural protein from 2,5-HD-treated animals. Adult, White Leghorn hens were given daily doses of either 200 or 70 mg 2,5-HD/kg, po, for up to 55 or 135 days, respectively. Additional animals were given 70 mg/kg for 63 days and then allowed to recover for 72 more days. Protein separation by gel electrophoresis followed by staining with a pyrrole-specific reagent yielded evidence of widespread adduct formation in protein from serum, liver, kidney, brain, and purified myelin. Binding was particularly strong in serum albumin and myelin basic protein. Quantitation of the adduct in these tissues revealed that its formation reached peak levels at 20 days in high dose and 30 days in low-dose animals. Levels subsequently declined, suggesting the presence of a clearance mechanism capable of removing altered protein during continuing 2,5-HD exposure. Protein from animals on the recovery regimen contained no detectable pyrrole adduct. Pyrrole adduct formation was also detected in neurofilament protein preparations, although protein yields were too low to allow assessment of clearance. Hens at both dosages displayed clinical signs indicative of CNS and PNS neuropathy. Histologic findings included axonal swelling and degeneration in peripheral nerve and some spinal cord nerve tracts. A hypothesis is proposed involving differential clearance of pyrrole adduct from neural vs nonneural tissue to explain the mechanism of action and target organ specificity of 2,5-hexanedione.

Ten-year oral toxicity study with Norlestrin in rhesus monkeys.

The long term effects of the oral contraceptive, Norlestrin, were evaluated in sexually mature female rhesus (Macaca mulatta) monkeys over a 10 year period. Norlestrin, a combination of norethindrone acetate and ethinylestradiol (50:1) was given orally on a continuous cyclic regimen of 21 d of dosing followed by 7 d without treatment. Groups of 16 monkeys each received the drug at dose levels of 0.05, 0.51, and 2.55 mg/kg representing multiples of 1, 10, and 50 times the human dose, respectively. A comparable group of 16 animals remained untreated and served as controls. Selected clinical and laboratory parameters were monitored throughout the study and all animals were necropsied and evaluated for gross and histopathologic changes. All dose levels were well tolerated and survival was not affected. There were no consistent treatment-related alterations in coagulation or other clinical laboratory parameters. Ophthalmologically, macular pigmentary anomalies were observed in all groups. Treatment-associated pathologic findings, representing exaggerated pharmacological responses with superimposed senile changes, including ovarian and uterine atrophy and dilatation of acini and ducts in the mammary gland. Periodic vaginal cytologic examination and mammary gland palpation did not demonstrate drug related changes. A small number of neoplasms was seen in all groups and a granulosa cell carcinoma of the ovary occurred in a control animal. The benign tumors consisted of three cutaneous papillomas: one in a low dose and one in a high dose animal, a uterine leiomyoma in one high dose animal, and a pancreatic duct adenoma in one low dose animal. The results of this study indicate that Norlestrin had no significant toxic manifestations or tumorigenic potential when administered on a cyclic regimen to female rhesus monkeys at levels up to 50 times the human dose for ten yr.

Morphologic and behavioral effects of perinatal glucocorticoid administration

Male rats were infused with one of two doses of dexamethasone or saline on day 4 of life. Body, whole brain, hippocampal, and cerebellar weights were obtained at 10, 20 and 150 days of age, and the ability of the rats to acquire and reverse a spatial discrimination task was assessed at 90 days of age. Body weights and brain weights were severely and permanently suppressed in the high dose group (100 mg/kg), resulting in a 20% decrement from saline-injected controls in adulthood. Low dose (1 mg/kg) animals were transiently slowed in body and brain growth, but differences from controls were no longer detectable at 60 days of age. Both high dose and low dose animals were significantly impaired in acquisition and reversal of a spatial learning task in an open field water maze, and exhibited deficits characteristically seen in animals with hippocampal damage. The animals' swimming performance per se was unimpaired. It appears that even transient interruption of normal interactive sequences of brain development by the alteration of post-natal neurogenesis or gliogenesis by glucocorticoids results in a permanent behavioral deficit.

Possible role of macrophage-like suppressor cells in the anti-tumour activity of BCG.

The i.v. injection of high doses (3 mg) of BCG into C3H mice bearing a transplantable 3-methylcholanthrene-induced fibrosarcoma caused the regression of a significant proportion. This effect was most evident when the BCG was injected on the day of the graft, or 7 days later. The injection of this agent either 14 days before the graft, or in low doses (0.1 or 0.5 mg), or directly into the tumour (i.t.) only prolonged the survival of the animals. Spleen cells from systemic high-dose BCG-treated mice were found to exert a strong nonspecific cytostatic effect in vitro that was not an artefact of the test conditions, and was not expressed by cells from low-dose animals. The cytostatic effect was shown to be caused by cells with the characteristics of macrophages, i.e. they were strongly adherent, unaffected by treatment with anti-Thy 1.2 + C', radioresistant but heat-sensitive, and were detected in BCG-treated "B" mice. The spleens of high-dose BCG-treated mice also contained suppressor cells that were capable of inhibiting the in vitro reactivity of normal T cells to PHA. Like the cytostatic effect, this suppressor activity was not detected in low-dose mice, and the cells responsible had the properties of macrophages; the effect was lost after the removal of adherent cells by sequential exposure to plastic and colloidal iron, but was conserved after treatment with anti-Thy 1.2 + C'. T-cell-deprived animals, such as "B" or nude mice, also developed suppressor-cell activity when treated with systemic high-dose BCG. Close parallels became evident between the in vivo anti-tumour activity of BCG, the in vitro cytostatic effect, and the suppressor-cell activity. We here discuss the possible role of suppressor cells in the mechanism of action of this agent.

Chronic treatments with zotepine, thioridazine, and haloperidol affect apomorphine-elicited stereotypic behavior and striatal 3H-spiroperidol binding sites in the rat.

Apomorphine (AP)-elicited sterotypic behavior and striatal 3H-spiroperidol binding sites were studied in rats given 3 weeks of chronic treatment with one of the following neuroleptic drugs: zotepine (10 or 20 mg/kg/day IP); thioridazine (10 or 20 mg/kg/day IP); haloperidol (2 or 5 mg/kg/day IP). On days 10-12 after the chronic neuroleptic treatment, enhancement of AP-elicited stereotypy was seen in the high- and low-dose haloperidol-treated, as well as in the high-dose thioridazine and zotepine-treated rats when compared to that of saline-injected controls. No significant change in the response to AP was found in the low-dose thioridazine and zotepine-treated animals. Significant increases in the concentration of striatal 3H-spiroperidol binding sites were seen after treatment with all three neuroleptics, both high and low doses. A positive correlation was found between AP-elicited stereotypy and the concentration of striatal 3H-spiroperidol binding sites in the haloperidol-treated and control rats. However, no such correlation was seen after chronic thioridazine and zotepine treatments.

Granuloma pouch assay. III. Enhancement of the carcinogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine.

Proliferation of granulation tissue on the inside of a subcutaneous (s.c.) air pocket was induced in rats by administration of 0.5 ml of 0.25% croton oil (granuloma pouch). Two days after induction of the granuloma, i.e., during the period of maximal cell growth, a single dose of 0.6 mg or 0.1 mg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was administered into the pouch. Fibrosarcomas of various histopathological types developed in 87% of the rats receiving the high dose and in 64% of the low dose animals. The mean latency period was 47.5 and 51.7 weeks respectively. Only one local sarcoma developed in rats treated with 0.6 mg MNNG by the s.c. route, and no tumors were observed in the groups treated with 0.1 mg s.c. The appearance of local sarcomas in the granuloma pouch tissue is correlated with previously reported frequency of point mutations (OUAR and HGPRT-) induced in granuloma fibroblasts with the same doses of MNNG. Possible mechanisms explaining the marked enhancement of the carcinogenic effect of MNNG in the granuloma pouch assay are discussed.

The sensitivity of adrenal responses to synthetic adrenocorticotrophin in the conscious unrestrained calf.

1. Changes in cortisol and corticosterone output and blood flow from the adrenal gland have been determined in the conscious unrestrained calf during I.V. infusions of synthetic adrenocorticotrophin (Synacthen) at 0-5 ng-kg(-1) min- minus 1 (low dose), 5 ng-kg- minus 1 (medium dose), 50 ng-kg- minus 1 min- minus 1 (high dose) and 500 ng-kg- minus 1 min- minus 1 (medium dose), 50 ng-kg- minus 1 min- minus 1 (high dose) and 500 ng-kg- minus 1 min- minus 1(very high dose). 2. Infusions at the low dose produced a rise in adrenal output of both cortisol and corticosterone to maximum values of approximately 100 and 30 ng-kg- minus 1 min- minus 1 respectively. Mean output of both steroids was significantly increased within 5 min, reached a maximum within 10 min and had fallen to resting levels 10 min after the infusion was discontinued. 3. The effects of infusions at both the high and very high doses were closely similar; maximal cortisol outputs were within the range 600-800 ng-kg- minus 1 min- minus 1 and corticosterone 350-500 ng-kg- minus 1 min- minus 1 in both groups. 4. When the infusions were terminated, pronounced differences were observed in the rates at which steroid outputs declined. Basal levels were restored within 10 min following the low dose and within 60 min in medium dose animals, but both cortisol and corticosterone output were still elevated 2 hr after infusion in high dose animals. In calves infused at the very high dose, cortisol output did not fall significantly during 2 hr period. The ratio of cortisol: corticosterone released from the adrenal gland immediately before infusion (3-2 +/- 0-3) approximated to the proportions in which the two steroids were found in the arterial plasma, but fell progressively to a minimum (1-3 +/- 0-1) with increasing doses of Synacthen. Conversely, the ratio of the steroids in the arterial plasma was increased during infusions at the low dose, but not at the higher doses. 6. No significant change in adrenal blood flow occurred during Synacthen infusion in low dose animals despite the increase in steroid output. In medium dose animals blood flow through the gland rose during infusion by approximately 75 per cent while in both high and very high dose animals the flow increased by up to 300 percent. 7. In the three groups in which adrenal hyperaemia occurred, blood flow had fallen to within the resting range 45 min after infusion: in each case this fall was much more rapid than the fall in steroid output. No significant increase in aortic blood pressure or heart rate accompanied infusion of Synacthen, indicating that adrenal hyperaemia was dependent upon vasodilatation with the gland. 8. Administration of cycloheximide (10 mg/kg) by I.V. injection either before or during an infusion of Synacthen, inhibited steroidogenesis without affecting the vasodilator response.

Morphologic effects of hypervitaminosis a on rat submandibular gland

Young male Sprague-Dawley rats were fed oral supplements of vitamin A; either a subtoxic dose of 5000 i.u./day for 16 weeks (10 animals), or a toxic dose of 15,000 i.u./day for 16 days (5 animals). High-dose animals exhibited signs of acute toxicity; low-dose animals demonstrated only less weight gain than controls. Vitamin A sera and liver levels evidenced intoxication. Light and electron microscopy were conducted upon acini, intercalated ducts, and striated-granular ducts of the submandibular gland. Acinar cells evidenced rough endoplasmic reticulum clefting and hyperplasia of both rough endoplasmic reticulum and Golgi apparatus similar to changes produced by pilocarpine or isoproterenol and interpreted as hyperactivity. Also increased were the number of degenerating acinar cells. Striated-granular duct cells were similar to controls except for increased cellular degeneration. Intercalated ducts appeared unaffected. Abnormalities were more pronounced in short-term, toxic hypervitaminosis A than long-term, subtoxic hypervitaminosis A.