Stress-reactive rats (high-avoidance female rats) have a shorter lifespan than stress-nonreactive rats (low-avoidance female rats).

Although Hatano high-avoidance and low-avoidance rats (HAA and LAA, respectively) have been selectively bred for good versus poor avoidance learning, HAA rats are known to be more reactive to stress than LAA rats. In this study, HAA and LAA female rats were compared during reproductive aging by observing estrous cycles from 8 to 11 months of age. Furthermore, these rats were allowed to live out their natural lifespans, that is, until 24 months of age, in order to compare their survival and to clarify the relationship between reproductive aging and tumor development. At eight months of age, 2 of 35 HAA rats and 20 of 35 LAA rats had abnormal estrous cycles. The median lifespan of the HAA rats (673 days) was shorter than that of the LAA rats (733 days). The incidence of pituitary neoplasia was higher in the HAA rats than in the LAA rats. These results suggest that HAA female rats (i.e., stress-reactive rats) have a shorter lifespan than LAA female rats (i.e., stress-nonreactive rats) and develop pituitary neoplasia, which was one of the causal factors in their accelerated mortality. However, the onset of an age-matched abnormal cycle did not correspond with their lifespan.

Ovarian dysfunction, obesity and pituitary tumors in female mice following neonatal exposure to low-dose diethylstilbestrol.

In a previous study, we found that early life exposure to low-dose diethylstilbestrol (DES) induced early onset of spontaneous abnormalities in estrus cycle and shortened survival in female Sprague-Dawley rats. In order to confirm the repeatability of the previous study, neonates of C57BL/6J mice were orally administered DES at doses of 0.005, 0.05, 0.5 and 5 µg/kg/day, and the aging of their reproductive function was observed. As a result, delayed toxicity on ovarian function was found in females treated with 0.5 µg/kg/day of DES. Concomitantly, the females in the 0.05 µg/kg/day of DES, or greater, groups, had increased body weights and, in the 0.5 µg/kg/day of DES, or greater, groups, had developed pituitary tumors, which were causal factors in their accelerated mortality. Thus, we found that early life exposure to low-dose DES induced early onset of spontaneous abnormalities in estrus cycle not only in female rats but also in female mice.

Ovariectomized mouse uterotrophic assay of 36 chemicals.

The concern over endocrine disruptors prompted international establishment of a strategic framework for the identification of the estrogenic compounds. OECD has launched the Conceptual Framework tool box containing various screening and testing methods including the uterotrophic assay. The (anti)estrogenicity of 36 chemicals suspected to be estrogen-receptor interactive by in silico and/or in vitro screening in the Extended Scheme for Endocrine Disruptor Screening and Testing of the Ministry of Health, Labour and Welfare, Japan, were monitored by the uterotrophic assay using C57BL/6J ovariectomized adult female mice after a 7-day exposure by oral gavage (po) and subcutaneous injection (sc). Ethynyl estradiol was used as reference for agonist and antagonist detection. In addition, Bisphenol A (sc) and Genistein (po) were tested for the comparison to rat assays. Among the 36, 2-[Bis(4-hydroxy-phenyl)methyl]benzylalcohol, 2,2',4,4'-Tetrahydroxybenzophenone, 2,4-Dihydroxybenzophenone, 3,3',5-Triiodothyroacetic acid, New fuchsin and alpha-Naphtholbenzein, showed both estrogenic agonistic and antagonistic activities; first two showed U-shaped dose-response in antagonistic studies. N,N-Diphenyl-p-phenylenediamine, 2,2'-Dihydroxy-4,4'-dimethoxybenzophenone, n-Butyl 4-hydroxybenzoate, and Reserpine were agonistic by sc. Benzo [a] pyrene, Benz [a] anthracene, Dibenz [a,h] anthracene, 2-(2H-Benzotriazol-2-yl)-4,6-di(t-pentyl)phenol, Rosemarinic acid, meta-Thymol, 6-Gingerol, Colchicine, Malachite green base, Fenbuconazole, and Lead acetate were antagonistic. The rest, i.e. n-Heptyl 4-hydroxybenzoate, Tetrazolium violet, Pravastatin sodium salt, Physostigmine, salicylate (1:1), Nordihydroguaiaretic acid, o-Cresolphthalein, 1,3-Dinitrobenzene, C.I. Pigment orange, Tetrabromobis-phenol-A, 2-Hydroxy-4-methoxybenzophenone, Ethylparaben, Propyl p-hydroxybenzoate, Kaempferol, 2-(2-Benzotriazolyl)-p-cresol and Phenolphthalein were negative for both effects. Taking together with in silico/ in vitro screening, the result suggested that the ovariectomized mouse uterotrophic bioassay has sufficient performance comparable to rat for the screening of (anti)estrogenicity of various chemicals.

Delayed reproductive dysfunction in female rats induced by early life exposure to low-dose diethylstilbestrol.

A one-lifespan test was carried out to establish a test protocol for endocrine-disrupting chemicals (EDCs). Diethylstilbestrol was administered by oral gavage to neonatal rats at doses of 0.05, 0.5 and 5 µg/kg/day for 5 days after birth. Abnormal estrous cycles were observed throughout the study in all females from the 5 µg/kg group, and in 40% from the 0.5 µg/kg group from 24 weeks of age. The conception rate of 12-week-old females in the 5 µg/kg group was 0%, and that of the 23-week-old females in the 0.5 µg/kg group was 33.3%. No effect of DES was observed at the first parturition in any group, except for the 5 µg/kg group. However, litter size was significantly reduced in the 0.5 µg/kg group at the second parturition. These results indicated that a prolonged period of observation of reproductive function is necessary to determine EDCs reliably.

Screening of toxicological properties of 4-methylbenzoic acid by oral administration to rats.

Oral toxicity of 4-methylbenzoic acid in male and female Sprague-Dawley rats was profiled through a twenty-eight-day repeated dose toxicity study (the 28-day study) and a screening test for reproductive/developmental toxicities (the reproduction/developmental study) conducted under Organisation for Economic Co-operation and Development (OECD) test guidelines. Daily administration of 4-methylbenzoic acid, at a dose level of 0, 100, 300 or 1,000 mg/kg, did not show any adverse effect on reproductive organs of animals in the 28-day study. In the reproductive/developmental study, however, 1,000 mg/kg/day of the compound reduced epididymal weights and increased incidence of cauda epididymal oligo/azoospermia. While the compound did not affect estrous cycle or mating performances, 1,000 mg/kg of the compound reduced fertility. Furthermore, 300 mg/kg or more of the compound increased pre-implantation loss, which resulted in a decrease in the number of offspring, and reduced body weight gain of the dams during the latter period of gestation. From these results, the no-observed-effect-level (NOEL) for reproductive/developmental toxicities is considered to be 100 mg/kg, whereas 1,000 mg/kg did not show any effect on neonates. In the 28-day study, NOEL is considered to be 300 mg/kg for male and female rats, since 1,000 mg/kg of the compound caused, in both sexes, a few minor changes, such as temporal salivation, a slight increase in food consumption and a moderate increase in blood aspartate aminotransferase (AST) activity. Thus, 4-methylbenzoic acid has the potential for reproductive toxicity, with diverse adverse effects on the epididymis, after repeated administration, observed in the two studies.

Delayed exacerbation of acute myocardial ischemia/reperfusion-induced arrhythmia by tracheal instillation of diesel exhaust particles.

For understanding the relationship between the increased incidence of sudden cardiac death and air pollution, we examined the effects of intratracheal instillation of diesel exhaust particles (DEP) on acute myocardial ischemia/reperfusion-induced arrhythmia in rats. The animals received 1 mg DEP 24-48 h before the ischemia/reperfusion (DEP-pretreated group, DEP-PRE), and were subjected to 3 successive brief ischemia/reperfusion (3 min ischemia followed by 5 min reperfusion) procedures. These were to make the animals tolerant to ischemia/reperfusion-related myocardial deterioration. Thereafter the animals were subjected to a 10-min ischemia followed by a 30-min reperfusion. In the experiments, an increased mortality was observed in the DEP-PRE group compared to the vehicle (0.05% Tween 80-PBS)-treated group. Forty-six percent of the animals in DEP-PRE died during the first 3-min reperfusion period. The animals of other groups were intratracheally instilled with DEP at the beginning of ischemia/reperfusion experiment, or were pretreated with polyethylene glycol-conjugated superoxide dismutase (1000 IU kg(-1), iv). In these animals, incidences of both arrhythmia and mortality were similar to those in the animals treated with the vehicle. In experiments to investigate the effects of DEP on the biochemical and hematological parameters, neutrophil count was elevated by a higher dose (5 mg) of DEP at 24 h after the intratracheal instillation, and oxygen radical production, which was induced by 12-O-tetradecanoylphorbol 13-acetate, was enhanced at 72 h. These results indicate that intratracheal DEP instillation exacerbates short-period ischemia/reperfusion-induced arrhythmia. Delivery and activation of peripheral neutrophils and oxygen radicals produced in neutrophils might participate in this exacerbation. This is the first article that demonstrates the arrhythmogenicity of DEP using intratracheal instillation in rats.