Two-generation reproduction toxicity study in rats with methoxychlor.

A two-generation reproduction toxicity study was conducted in rats with a reference estrogenic pesticide, methoxychlor, to validate the sensitivity and competency of current guidelines recommended by the United States Environmental Protection Agency; Japanese Ministry of Agriculture, Forestry and Fisheries; and Organisation for Economic Co-operation and Development for predicting reproductive toxicity of the test compound based on estrogenic endocrine disrupting effects. Both sexes of SD rats were exposed to methoxychlor in the diet at concentrations of 0, 10, 500 and 1500 ppm for two successive generations. The present study has successfully detected estrogenic activities and reproductive toxicities of methoxychlor, as well as its systemic toxicity. Body weights, body weight gains and food consumption of both sexes of animals were suppressed significantly in the 500 and 1500 ppm groups. Typical reproductive toxicities observed in females of these groups included, but were not limited to, prolonged estrous cycle, reduced fertility, decreased numbers of implantation sites and newborns, decreased ovary weights and/or increased incidences of cystic ovary. Uterine weights of weanlings increased significantly in these groups, suggesting that the sensitivity of this parameter for predicting estrogenic ability of the test compound is comparable to that of the uterotrophic assay. Reproductive toxicities of methoxychlor seemed less potent in males than in females. Methoxychlor delayed preputial separation and significantly reduced sperm counts and reproductive organ weights of males of the 500 and/or 1500 ppm groups; however, most males that failed to impregnate females in the same group showed normal fertility when they were re-mated with untreated females. Neither systemic nor reproductive toxicities appeared in the 10 ppm group.

Fused pulmonary lobes is a rat model of human Fraser syndrome.

Fused pulmonary lobes (fpl) is a mutant gene that is inherited in an autosomal recessive manner and causes various developmental defects, including fusion of pulmonary lobes, and eyelid and digit anomalies in rats. Since these developmental defects closely resemble those observed in patients with Fraser syndrome, a recessive multiorgan disorder, and its model animals, we investigated whether the abnormal phenotypes observed in fpl/fpl mutant rats are attributable to a genetic disorder similar to Fraser syndrome. At the epidermal basement membrane in fpl/fpl mutant neonates, the expression of QBRICK, a basement membrane protein whose expression is attenuated in Fraser syndrome model mice, was greatly diminished compared with control littermates. Quantitative RT-PCR analyses of Fraser syndrome-related genes revealed that Frem2 transcripts were markedly diminished in QBRICK-negative embryos. Genomic DNA sequencing of the fpl/fpl mutant identified a nonsense mutation that introduced a stop codon at serine 2005 in Frem2. These findings indicate that the fpl mutant is a rat model of human Fraser syndrome.

Pathogenetic transition in the morphology of abnormal sperm in the testes and the caput, corpus, and cauda epididymides of male rats after treatment with 4,6-dinitro-o-cresol.

In order to elucidate the pathogenesis of tailless sperm, 4,6-dinitro-o-cresol (DNOC) was administered to Jcl:SD male rats at daily oral doses of 0, 10 or 15mg/kg for 5 days. Sperm were collected from the caput, corpus, and cauda epididymides on days 1, 7 and 14 after the last dosing (D1, D7 and D14, respectively), counted and examined morphologically by phase-contrast and scanning electron microscopy. The incidence of abnormal sperm was significantly increased in the DNOC 15mg/kg group. On D1, peeled sperm (loss of mitochondrial sheath at the proximal end of the middle piece) was frequently observed in the caput epididymides, whereas sperm in the corpus and cauda epididymides had normal morphology. Distribution of the peeled sperm changed as time passed and the corpus epididymides showed a peak incidence on D7. On D14, the highest incidence of abnormal sperm was observed in the cauda epididymides, where the major abnormality was tailless. Similar effects were also found in the 10mg/kg group but were less potent. Transmission electron microscopy of testicular sperm on D1 revealed the presence of elongated spermatids that lacked the mitochondrial sheath at the proximal end of the middle piece, although the round and elongating spermatids looked normal. These results suggest that DNOC exposure of male rats primarily causes partial loss of the mitochondrial sheath in the testicular elongated spermatids, and that the affected sperm become tailless by D14 after reaching the cauda epididymides.

Two-generation reproduction toxicity study in rats with 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT).

DDT, an organochlorine pesticide, has been cited as a representative chemical suspected of having endocrine disrupting effects. In this study, the potential endocrine disrupting activities of p,p'-DDT, a major component of DDT, were investigated in rats in a 2-generation reproduction toxicity study in accordance with the most current test guidelines of the Ministry of Agriculture, Forestry and Fisheries in Japan, Organization for Economic Cooperation and Development (OECD) and United States Environmental Protection Agency (USEPA) with some modifications and additions. p,p'-DDT was given to parental rats at dietary levels of 0, 5, 50 or 350 ppm. Systemic toxicities in the parental animals consisted of tremors and subsequent deaths (females only) and/or pathological alterations of the liver (both sexes of animals) of the 2 higher dose groups. Reproductive and postnatal developmental toxicities were not evident up to the highest dose level except for the decreased pup viability index on postnatal day 21 in the 350 ppm group. Changes in serum estradiol and progesterone levels and/or a delay in male sexual maturation were noted in the 2 higher dose groups in a dose-dependent fashion, suggesting alterations of endogenous endocrine functions. However, these changes never resulted in substantial reproductive disorders.

A two-generation reproductive toxicity study of 2,4-dichlorophenol in rats.

A two-generation reproductive toxicity study was conducted with 2,4-dichlorophenol (2,4-DCP), an agent suspected of exerting endocrine disrupting effects. Wistar-Hannover rats, 24/sex/group, were given diet containing 2,4-DCP at dose levels of 0, 500, 2000 or 8000 ppm to examine the potential effects of the test substance on parental animals and their offspring over 2 successive generations. Neither clear systemic nor reproductive toxicity of 2,4-DCP was apparent in the 500 ppm group. In the 2000 ppm group, mean body weight gain and food consumption of females were lowered significantly during the treatment period. Effects on body weights and food consumption were more serious in the 8000 ppm group, both males and females being significantly affected. Reproductive effects of the test substance were also observed in the 2000 and 8000 ppm groups dose-dependently. Observations included significantly increased uterine weights of F1 and/or F2 female weanlings and reduced numbers of implantation sites and live births of F1 parental females. These results suggest that 2,4-DCP has weak reproductive toxicity, possibly based on endocrine activity. However, the basic mechanisms for apparent estrogenic effects of 2,4-DCP remain to be elucidated.

Comparative studies on the spermatotoxic effects of dinoseb and its structurally related chemicals.

Three dinitrophenolic compounds, dinoseb (DNBP; 7.5 mg/kg b.w.), 4,6-dinitro-o-cresol (DNOC; 4, 7.5, 15 mg/kg b.w.), and 2,4-dinitrophenol (DNP; 7.5, 15, 30 mg/kg b.w.) were administered orally to sexually matured Jcl:SD male rats for 5 consecutive days. Half of the males in each group were necropsied at 3 (D3) and 14 (D14) days after the last dosing, respectively, and examined for the effects of dinitrophenols on spermato-/spermiogenesis. DNBP (7.5 mg/kg), DNOC (15 mg/kg), and DNP (30 mg/kg) caused 1, 5, and 0 deaths, respectively, as well as a decreased body weights during the treatment. Although examinations on D3 revealed no treatment-related alterations, DNBP and DNOC resulted in reduced sperm motility and increased incidence of tailless sperm in the cauda epididymis on D14. DNP also caused slightly increased incidence of tailless sperm on D14. These results demonstrate that DNBP, DNOC, and DNP manifest similar spermatotoxic effects at or around a lethal dose in rats.

Curly vibrissae, a new mutation in the Wistar-derived rat.

A new mutation was identified in the PD (Preaxial Duplication) strain of rats, the main manifestations of which were curly and sparse vibrissae with retarded outer hair growth. As the main characteristic of this mutant rat is abnormally curled appearance of the vibrissae, "curly vibrissae" is proposed as the name of this mutant gene, and "cv" as its symbol. Genetic analyses reveal that the mutant characteristics are inherited as autosomal recessive traits and the cv gene is independent from the pd gene that carried by the original PD colony. The cv/cv homozygous rats have a small number of short and/or curly vibrissae around the nose. The vibrissae on the cheek and/or above the eyes are also short and curled; however, no vibrissa appears on the lower mandible. Although hair growth seems to be retarded, the outer hairs showed nearly normal length by 10 weeks of age. The outer hairs of matured cv/cv rats appear silky and translucent. The adult mutant rats often exhibit loss of hair on the head and/or back. Lactating females usually lose their abdominal hair. Both sexes of cv/cv homozygotes have normal reproductive ability. No internal malformations accompany vibrissa and hair abnormalities.

Effects of dinoseb, 4,6-dinitro-o-cresol, and 2,4-dinitrophenol on rat Sertoli-germ cell co-cultures.

The effects of dinoseb (DNBP), a known testicular toxicant in the rat, on germ cells were investigated in Sertoli-germ cell co-cultures. Two DNBP-related dinitrophenolic compounds, 4,6-dinitro-o-cresol (DNOC) and 2,4-dinitrophenol (DNP), were also examined, as testicular toxicity of these compounds had not been elucidated. Cultures were exposed to each compound (10(-7)-10(-4)M) for 24h and examined for the number and viability of detached cells and morphologic alterations under a light microscope. DNBP significantly increased the number of detached cells (10(-5) and 10(-4)M) and suppressed their viability (10(-6)-10(-4)M). Morphologic observations revealed degenerative alterations in the germ cells and Sertoli cells. Similar effects as observed after DNBP exposure were evident at 10(-4)M DNOC and 10(-4)M DNP. These results demonstrate that DNBP, DNOC, and DNP have in vitro toxicity to these cell populations at high concentration, and suggest the possibility that DNOC and DNP also cause testicular damage in experimental animals and humans.

Historical control data on reproductive abilities and incidences of spontaneous fetal malformations in Wistar Hannover GALAS rats.

Wistar Hannover rats, which have recently been introduced into Japan, are expected to be used in reproductive and developmental toxicity studies, yet the accumulation of background data is insufficient. This paper describes our historical data on the reproductive ability of this strain of rat. Three lots of sexually matured females (40 each) were received from CLEA JAPAN, Inc. with males of the same strain (30 or 36 each) and mated. A total of 47 dams were killed on gestation day 20 to examine their fetuses. The remaining 71 pregnant females were allowed to deliver spontaneously and observed for common reproductive parameters. The mating and fertility indices of females were both 99.2%. Overall mean numbers of implants and live fetuses at cesarean sectioning were 12.5 and 11.5, respectively. Fetal resorptions and deaths occurred at an incidence of 8.6%. Morphological examinations of fetuses revealed low incidences of spontaneous malformations (each one case of double aortic arch and absent cervical vertebral arch) and a variety of common variations. The followings are overall means of major reproductive parameters obtained from females with live birth: no. of implants, 12.5; no. of pups delivered, 11.8; viability index of pups at birth, 99.8%; and days of age at sexual maturation (vaginal opening and preputial separation), 30.3 and 42.8, respectively. Our present observations confirmed a minimal deviation among 3 lots of animals in terms of reproductive abilities. These results suggest that this strain of rat can be used in reproductive and developmental toxicity studies, although the sensitivity to toxicants remains to be elucidated.