Parenteral application of the active metabolite of vitamin D3, 1, 25-dihydroxyvitamin D3, has been anticipated to have remarkable efficacy in secondary hyperparathyroidism. However, in a reproduction seg. I study in rats, poor reproductive performance was reflected in a decrease in the number of matings, implantations and live births. These changes were though reversible after treatment with the compound was discontinued. In order to clarify the mechanism of these reversible toxicities, the following were examined in female rats treated with the D3 metabolite: 1. effect on the estrous cycle (no treatment for 2 weeks, treatment for 3 weeks and recovery for 2 weeks), and 2. effect on the maintenance of pregnancy (treatment for 2 weeks before mating and during the gestation period). In both groups, the levels of calcium, calcitonin, PTH and progesterone in serum were measured, and histopathological examination of the thyroid, parathyroid, ovary and uterus was carried out. The following results were observed: 1) disturbance of the estrous cycle, 2) hypofunctional changes in the corpus luteum in the ovary, and the epithelium, endometrium and uterine gland in the uterus with a decrease in the serum progesterone level and 3) hypercalcemia with a decrease in calcitonin or PTH levels in serum with morphological changes including atrophy and cyst-formation in the parathyroid. However, the above changes were reversible, and recovery was observed after administration of the compound was discontinued. These results indicate that the hypercalcemia caused by 1, 25-dihydroxyvitamin D3 disrupts endocrinological homeostasis which in turn temporarily disrupts the female reproductive system. Furthermore, it was suggested that 1, 25-dihydroxyvitamin D3 itself directly influences on endocrinological organs (hypothalamus, pituitary, parathyroid and thyroid) and reproductive organs (ovary and uterus).
To determine urinary albumin in a minute amount, a gel-filtration high-performance liquid chromatographic procedure was newly established. When urine of a normal rat was eluted isocratically at 0.6 ml/min by 100 mM Na sulfate in 20 mM Na, K-phosphate (pH 7.4), approximately 6 hrs for complete elution of urinary peak-forming substances was needed. Retention time of albumin was found to be 22.9 min. To shorten the analytic time, 100 mM Na sulfate in 20 mM Na, K-phosphate (pH 7.4) was first used during a 30 min period for separation of albumin. A mixture of acetonitrile/the above solvent=3/7 (v/v) was then flushed to wash away the peak-forming substances. By this elution mode, the analytic time could be reduced to 3 hrs. When the validity of this procedure was tested, the detection limit of albumin was 0.04 μg/injection, and a linearity was observed between 0.2 and 50 μg/injection. Rats then received single subcutaneous injections of puromycin aminonucleoside, which is a nephrotoxic agent. The plasma albumin concentrations fell at 5, 10 and 15 days after the administration, and the urinary excretions of albumin rose from the 1st day up to the 15th day. The results denoted that our procedure could be a good evaluative tool for nephrotoxicity studies where albuminuria was manifested.
The gelatin-starch syrup microencapsulation method was applied to subacute toxicity studies of tribromomethane (TBM), dibromochloromethane (DBCM) and bromodichloromethane (BDCM). Groups of Wistar rats (7 males and 7 females) both sexes were given diet containing microcapsules of each of these trihalomethanes (THMs) at the following concentrations: TBM, 0.068, 0.204 and 0.612% in males, and 0.072, 0.217 and 0.651% in females; DBCM, 0.020, 0.062 and 0.185% in males, and 0.038, 0.113 and 0.338% in females; BDCM, 0.024, 0.072 and 0.215% in males, and 0.024, 0.076 and 0.227% in females. Suppression of body weight gain was seen in each high-dose males fed TBM or BDCM and females fed DBCM or BDCM. Histopathologically, hepatic lesions such as vacuolization and swelling of liver cells were significantly noted in both sexes of all groups fed TBM, in both sexes of the middle- and high-dose groups fed DBCM, and in males of the high-dose group and in females of the middle-and high-dose groups fed BDCM. In addition, single cell necroses were observed in males and females fed DBCM and in males fed BDCM. Hepatic cord abnormalities were also noted in males of the high-dose group fed BDCM. Although no increases in serum transaminase activities (ASAT, ALAT) were evident in either sex fed any of the THMs, decreases in triglyceride content, cholinesterase and lactate dehydrogenase activity were observed. Renal lesions reported to occur in gavage studies were not found in the present feeding study. Lowest-observed-adverse-effect-level (LOAEL) of TBM and no-observed-adverse-effect-level (NOAEL) of DBCM and BDCM were determined to be 56.4 mg/kg, 18.3 mg/kg and 20.6 mg/kg, respectively, under the present experimental conditions.
The acute and subchronic toxicity studies on 2, 2'-methyl-enebis (4-ethyl-6-tert-butylphenol) (MBEBP) were conducted using male and female Wistar rats. In acute toxicity test, the LD<50> values were estimated to be greater than 10 g/kg BW by oral and intraperitoneal administration in each sex. In subchronic toxicity test, groups of 10 rats of each sex were fed a diet containing 0.2, 1.0 or 5.0% of MBEBP and examined at 4 and 12 weeks. Body weight gain was significantly depressed at doses of 1.0 and 5.0% in both sexes, but the depression in the 1.0% group was severer than that in the 5.0% group in males. Hematological analysis showed slight but significant decrease of hemoglobin in the 1.0 and 5.0% groups of both sexes. Urine analysis showed no remarkable changes in all treated rats of both sexes. In biochemical analysis of serum, decrease of triglyceride level and cholinesterase activity, and increase of amylase activity were observed in treated rats. Histopathologically, testicular atrophy and decrease of spermatogenesis were observed in male rats fed 1.0 or 5.0% MBEBP for 4 and 12 weeks and vacuolization of parathyroid gland cells was observed in female rats fed 1.0 and 5.0% MBEBP for 12 weeks. In subchronic test, the lowest observable adverse effect levels for MBEBP toxicity were estimated to be 171 mg/kg BW/day in male rats and 180 mg/kg BW/day in female rats.
To examine the mechanism and toxicological significance of thyroidal tumor observed slightly in a long-term rat study with diethofencarb (isopropyl 3, 4-diethoxycarbanilate), male Sprague-Dawley rats were fed diethofencarb in diets at concentrations of 0, 5, 000 or 20, 000 ppm for 3 months. Examinations mainly for thyroid functions including thyroid uptake of <125>I, serum thyroid hormone and thyroid stimulating hormone (TSH) level, hepatic UDP-glucuronyltransferase (UDP-GT) activity and histopathological examination in thyroid were performed at week 13. Decreases of body weights and food consumptions were observed at and above 5, 000 ppm. Under these conditions, decrease of serum free T4 and increase of serum TSH level were observed only at 20, 000 ppm, concurrently with liver weight increase at and above 5, 000 ppm and increase of hepatic UDP-GT activity at 20, 000 ppm. However, no compound related effects were noted in thyroid weight, thyroid uptake of <125>I and gross or histopathological examination in thyroid. These results indicate that the administration of diethofencarb leads to an increase in UDP-GT activity and acceleration of thyroid hormone excretion from the liver. The acceleration causes a decrease in serum free T4 level, triggering the feedback mechanism of the pituitary gland, promotion of TSH release and consequently an increase in serum TSH level. Thus, the slightly higher incidence of thyroid follicular cell tumors observed in the chronic and oncogenicity study with non-genotoxic diethofencarb is considered to be caused by these weak pituitary-thyroid hormonal imbalances. The toxicological significance in humans is extremely low according to the well established facts that the chronic TSH stimulatin would not induce thyroid tumors in humans and humans may be less sensitive than rats in regard to the response to goitrogenic stimuli.