Toxicologic/carcinogenic effects of some representative endocrine disrupting chemicals (EDCs) having estrogenic activity, such as alkylphenols, on the female genital organs of rodents, especially rats, are reviewed and discussed, focusing on our recent research. Neonatal treatment of high-dose p-tert octylphenol (t-OP, 100 mg/kg s.c. injection every other day from postnatal day 1 (PND 1) to PND 15) induced various long-term persistent irreversible effects on the female reproductive system of Donryu rats, such as lower gonadotropin levels at prepuberty, inhibition of uterine gland genesis, persistent estrus and polycystic ovaries. The result indicates that neonatal high-dose treatment of estrogenic EDCs can affect gonadotropin secretion during the developmental period of sexual maturation with direct masculinization of the hypothalamic function. Exposure limited to the first 5 days after birth (PNDs 1-5) to 100 mg/kg t-OP, however, caused delayed influence which was characterized by accerelated appearance of atrophic ovary, manifested by early-occurring and long-term continuing persistent estrus after puberty, whereas no abnormalities could be found with regard to growth and differentiation of the reproductive organs and the hypothalamo-pituitary-gonadal control system up to maturation, the influence being caused by delayed modulation of the hypothalamo-pituitary-gonadal control system. The most notable effect on the female reproductive system when normal cycling rats were exposed to high-doses t-OP for 28 days, was disappearance of the estrous cycle, but no clear changes were detected in other parameters such as uterine weight and morphology. These results indicate that the most serious issue with EDCs is the potential effects of prenatal and/or neonatal exposure on rodents. Well or moderately differentiated adenocarcinomas increased in Donryu rats initiated with N-ethyl-N’-nitro-N-nitrosoguanidine, when high-dose t-OP was given subcutaneously during adulthood. Neonatal exposure for PNDs 1-5 to high-dose t-OP also showed promoting effects on uterine adenocarcinoma development. However, in rats given t-OP for PNDs 1-15, uterine tumor malignancy was clearly increased, although there was no significant alteration in the total incidence of adenocarcinomas. The results are very interesting in consideration of the histogenesis of uterine adenocarcinomas. However, maternal exposure to low doses of EDCs such as nonylphenol and bisphenol A at actual human exposure levels by the oral route showed no effects on growth and development of the female reproductive system or uterine carcinogenesis. These results indicate that dietary exposure to low doses of EDCs might not induce any adverse effects on the female genital system in mammals including humans.

View full abstract

Neurotoxic signs suggested by abnormalities of the posterior extremities are sometimes observed in rats and mice given high doses of certain test substances in acute toxicity studies, but abnormal morphological changes are not always detected in the nervous system and skeletal muscles of these animals during routine histopathological examinations. In such cases, toxicities are attributable to the onset of motor endplate neuropathy. In this article, we focus on the ultrastructural changes in neuromuscular junctions induced by several neurotoxic substances, based on the results of a search in the literature and data obtained in our laboratory. The literature survey indicated that nerve endings of the peripheral motor nerves are initially damaged in the neurotoxicity of venoms of snakes such as β-bungarotoxin, notexin and taipoxin, as well as chemically synthesized substances such as DTB, sarin, Vacor and DTBHQ, and various kinds of morphological changes as a result of the destruction of motor endplates could be observed in neuromuscular junctions of the animals exposed to these toxic substances. When paralysis of posterior extremities occurs in rats treated with certain test substances, a part of the lumbrical muscles in the foot pad should be fixed and subjected to electron microscopic examinations to eliminate misjudgments concerning neurotoxicity.

View full abstract

The present study was conducted as part of an international collaborative project organized by the International Life Sciences Institute Health and Environmental Sciences Institute (ILSI-HESI project). Transgenic CB6F1 mice carrying copies of human prototype c-Ha-ras gene, CB6F1-Tg- rasH2 (rasH2) mice, were utilized as an alternative test system to a standard 2-year bioassay for carcinogenicity assessment. The antihypertensive drug reserpine, a non-genotoxic rodent carcinogen (putative non-human carcinogen), was used as a test compound for validation of these transgenic mice. Reserpine was administered to rasH2 mice at dietary concentrations of 0, 2.5, 5 and 10 ppm and to non-transgenic littermates (non-Tg) at dietary concentrations of 0 and 10 ppm for 26 consecutive weeks. The rasH2 and non-Tg mice receiving basal diet alone in the same way acted as a corresponding control. A single intraperitoneal injection of N-methyl- N-nitrosourea (MNU) to rasH2 mice served as a positive control. All surviving animals were sacrificed 26 weeks later and subjected to histopathological examination. Alveolar/bronchiolar adenoma and carcinoma of the lung, hemangiomas and hemangiosarcomas of the spleen, and papilloma of the forestomach were sporadically detected in rasH2 mice fed either the reserpine-containing diet or basal diet. No specific hyperplastic or neoplastic lesions due to reserpine were detected. In MNU-treated mice, however, thymic and/or systemic malignant lymphomas and squamous cell papilloma and/or carcinoma in the forestomach were commonly detected in both males and females with low survival rates suggesting the sensitivity of assay conditions. In conclusion, no carcinogenicity of reserpine was identified in rasH2 mice under the present experimental conditions.

View full abstract

Allelic loss of chromosome 2 is associated with radiation-induced murine acute myeloid leukemia. However, the gene, which contributes mainly to the leukemogenesis in a tumor suppression manner, has not been identified, yet. Expecting predisposition to acute myeloid leukemia, a radiation leukemogenensis experiment was performed with Pax6^Sey3H, one of the small eye mutants. Deletion mapping of Pax6^Sey3H indicated that the deleted segment extended from 106.00 to 111.47 Mb from the centromere with a length of 5.47 Mb on chromosome 2. Six known and seventeen novel genes were located in the segment. Pax6^Sey3H mutants crossed back into C3H/He did not develop hematopoietic tumors spontaneously, but they did after exposure to γ-rays. The final incidence of hematopoietic tumor in mutants (45.2%) was higher than that in normal sibs (26.2%), and the survival curve of mutants shifted toward the left (p<0.05 by the Cox-Mantel test). Mutants developed intestinal tumors spontaneously with long latency as well as showing abnormality in the Wirsung’s duct from young ages. Congenital deletion of the 5.47 Mb segment at the middle region on chromosome 2 alone did not trigger hematopoietic tumors, however, the deletion promoted the development of hematopoietic tumors initiated by radiation. The deletion developed intestinal tumors spontaneously. Radiation exposure at 10 weeks of age did not contribute to the intestinal tumorigenesis.

View full abstract

Hypospadias was induced in male Sprague-Dawley rats by prenatal exposure to 30 mg/kg/day of flutamide from gestational days 14 to 17, or from 18 to 21. Their external genitalia were examined histopathologically and compared to untreated controls. On postnatal day 6, the glans penis of untreated controls was bordered with epithelium. On postnatal day 22, papillae were recognized on the glans penis side, and cornification started close to the tip of the papilla on postnatal day 35. On postnatal day 42 cornification spread to the surface of the glans penis. The cornification progressed from tip to base of the glans penis, and from dorsal to ventral. When cornification reached the base of the glans penis, separation of the double layered epithelia was complete and the animal was considered sexually mature. Flutamide treatment on gestational days 14-17 induced a defect in the ventral half of the glans penis (cleft phallus) and cleft in the ventral prepuce (cleft prepuce) in the male pups, while treatment on gestational days 18-21 induced cleft phallus without apparent abnormalities in the prepuce. The external urethral orifice opened at the ventral end of the glans penis (hypospadias) in both treatment groups. In male pups with cleft phallus, cornification of the dorsal epithelium followed by separation of the prepuce occurred, while separation of the ventral part of glans penis did not occur because epithelium was not formed at the ventral part of the glans penis. Consequently, the onset of puberty was not decided in these animals. These findings indicate that the defect of the ventral half of the phallus is the reason why the time of sexual maturation was not decided, and that there is a difference between the phallus and prepuce in the sensitive period concerning the development of flutamide-induced malformations.

View full abstract

The modifying effects of dietary feeding of powdered broccoli sprout (PBS), which contained a high concentration of sulforaphane, on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also determined the influence of PBS on proliferating cell nuclear antigen (PCNA) index in “normal-appearing” crypts from rats treated with AOM and/or PBS, and activities of detoxifying enzymes of cytochrome P450 (CYP) and glutathione S-transferase (GST) in the liver. Rats were given two weekly subcutaneous injections of AOM (20 mg/kg body wt) to produce ACF. They also received experimental diets containing PBS at a dose of 20 or 100 ppm for 4 weeks, starting 1 week before the first dose of AOM. AOM exposure induced a substantial number of ACF (106 ± 10) at the end of the study (week 4). Dietary administration of PBS caused significant reduction in the frequency of ACF: 20 ppm PBS, 56 ± 11 (47% reduction, P<0.001) and 100 ppm PBS, 64 ± 23 (40% reduction, P<0.001). Immunohistochemically, PBS administration at both doses significantly lowered the PCNA index in “normal-appearing” crypts. In addition, feeding with PBS significantly reduced the activities of CYP 2B and CYP 3A, and slightly increased GST activity in the liver. These findings suggest a possible chemopreventive ability of PBS through alterations to the activities of cell proliferation in cryptal cells in the colon and activities of drug metabolizing enzymes in the liver.

View full abstract

Adenomatous hyperplasia of the rete ovarii was found in a 9-month-old beagle. At necropsy, no abnormalities were observed in the ovaries. Microscopically, there was a proliferation of gland-like tubules in the hilus of the left ovary. The tubules were lined by a single layer of columnar and cuboidal epithelial cells. In places the tubules appeared to radiate from small ductal structures. The lesion was poorly demarcated and blended with the existing tubular architecture of the rete ovarii, and extended diffusely into the adjacent stroma. Immunohistochemistry showed positive reactions for cytokeratin 18 and vimentin, suggesting that the lesion was derived from the rete epithelia with proliferative activity.

View full abstract

Renal Mesenchymal Tumor (RMT) and Nephroblastoma (NB) represent two diagnostically challenging neoplasms of the rat kidney. Both are uncommon tumors reported less frequently than tubule epithelial neoplasms. Excellent guideline criteria exist for both neoplasms. However, in some cases, the diagnosis between RMT and NB may be difficult and occasionally controversial. The most important feature of RMT is the presence of a fibroblastic-like cell which proliferates between tubules and glomeruli. RMT may contain heterogenous tissue such as fibrous, vascular, smooth or striated muscle, cartilage and even bone. On the other hand, NB are usually characterized by the presence of varying patterns of deeply basophilic blastemal cells which are pathognomic for NB. Organoid epithelial differentiation into glomeruloid or epithelial-lined tubules further characterizes NB. The observation of “altered” renal tubule structures in RMT are often a point of confusion with respect to NB diagnosis. In addition, attempts to compare human NB to the rat RMT and NB may be problematic.

View full abstract

Toxicological pathology of lymphoid organs plays an important role in the risk assessment process of immunotoxicants. Identification of chemicals that have the potential to cause injury to the immune system is of considerable public health significance because of the role of the immune system in infectious diseases, hypersensitivity reactions and autoimmune diseases. In assessing immunotoxicity, a two-tier testing system is usually employed in rodents in which the first tier is a general screen for (immuno)toxicity including enhanced histopathology of lymphoid organs and the second tier consists of more specific immune function studies including host resistance tests or mechanistic studies. Studies with the potent immunotoxicants TCDD, TBTO, HCB, azathioprine and cyclosporin A are discussed, which provide data correlating histopathology with immune function changes. This is followed by a discussion of the outcomes of enhanced histopathology investigation in the interlaboratory validation studies with azathioprine, cyclosporin A and HCB in the rat, as well as the results of a recent evaluation of enhanced histopathology in the mouse as an indicator of immunotoxicity. From these studies, that have been the basis for a number of regulatory activities, the following conclusions can be drawn: i) the consistency between histopathology and functional tests, ii) the complimentary information of pathology and immunology observations, and iii) the dependence on standardised protocols and trained toxicologic pathologists to accurately identify and grade microscopical changes in lymphoid organs and tissues.

View full abstract

Brown adipose tissue (BAT), an endocrine tissue, is an important regulator of nonshivering thermogenesis, energy metabolism, mitochondrial biogenesis, extracellular matrix (EMA) homeostasis and signaling, and hibernation. There is a paucity of data about BAT response in laboratory animals because BAT has not been routinely included in regulated safety assessment study protocols. We will present information on the structure, function and pathology of BAT, focusing mainly on younger and older rats from an exploratory study set to study BAT and all other tissues over time, from 6 weeks to 96 weeks of age. The energy supply process is stimulated in mammals mainly by hypothermia and by hypoxia and, in primates, by hypoglycemia. Of these, hypothermia is the main stimulus of brown adipocyte (BA) proliferation. Thermogenesis is achieved in BA in the mitochondria (mt), which are 50 to 100 times more abundant in BA than in any other cell type. The mt response involves a membranous protein in the inner membrane, uncoupling protein (UCP1) or thermogenin. Triiodothyronine (T₃) is implicated in the regulation of the UCP1 gene. UCP allows mt to oxidize substrates rapidly without ADP phosphorylation, thus uncoupling oxidation from phosphorylation and promoting instead the dissipation of oxidation energy as heat. Norepinephrine (NEPI) binds to the β₃-adrenoreceptor in BA leading to activation of lipoprotein lipase (LPL), which stimulates lipolysis and liberates FFA. Both NEPI and insulin (I) stimulate glucose uptake by BA, and NEPI increases UCP in BA. Nitric oxide (NO) stimulates the biogenesis of mt, redistribution of heat generated by the mt and inhibition of BA proliferation. NO also regulates the binding and releasing of oxygen from hemoglobin (Hgb) and the mt pathway of apoptosis. These effects of NO are mediated by peroxisome proliferator-activated gamma receptor coactivator 1alpha (PPARCA). NO is produced by endothelial NO synthase (eNOS) mainly in BA, but also in the hepatocytes and cells of the zona glomerulosa. PPARCA in turn increases the expression of nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (mtTFA). Thus, UCP, T₃, I, NEPI, NO, PPARCA1, cGMP, eNOS, NRF-1 and mtTFA are all parts of an interactive cascade connecting the BA functions through the EMA of all tissues to controlling the processes of EMA homeostasis and signaling, tissue normoxia and normothermia, apoptosis, proliferation, and differentiation of all cells.

View full abstract