Two initiation protocols for multi-organ carcinogenesis bioassays for detection of modifying effects of chemicals were investigated and compared in F344 male rats. Animals in group 1 were treated sequentially with N-diethylnitrosamine (DEN, i.p.), N-methyl-N-nitrosourea (MNU, i.p.), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN, drinking water), MNU (i.g.), and dihydroxy-di-N-propyl-nitrosamine (DHPN, drinking water) during the first 4 weeks (DMBMD treatment). Rats in group 2 were treated in the same manner as group 1 except that N, N'-dimethylhydrazine (DMH, s.c.) was administered instead of MNU (i.g.) (DMBDD treatment). Surviving animals were killed at 20 and 28 weeks after the commencement, seven rats in group 1 and one rat in group 2 being found dead during the experiment. Immunohistochemical examination revealed quantitative values for glutathione S-transfer-ase placental form positive liver foci to be significantly higher in group 2 than in group 1, especially at the week 28 time point. In the case of pepsinogen 1 altered pyloric gland (PAPG) induction in the glandular stomach, quantitative values for group 1 were higher than for group 2. Complete histopathological assessment of hyperplastic and neoplastic lesion yields revealed relatively high incidences in the lung, gastrointestinal tract, kidney, urinary bladder, thyroid, and other organs in both groups at week 28. We conclude that the present DMBDD protocol is superior for introduction in multi-organ carcinogenesis bioassays because of its wide spectrum initiation. The present results suggest the probability of detecting not only carcinogens and tumor promoters but also tumor inhibitors within a relative short period.

View full abstract

Male and female Fischer 344/DuCrj rats were treated with Food Red No. 106 (acid red) mixed in the basal diet at doses of 5.0% and 2.5% by weight in a two year toxicity/carcinogenicity study. The mean cumulative intake of Red 106 over the 106 experimental week duration was computed to be 738g/rat and 356g/rat for males at the 5.0 and 2.5% levels, respectively, while females consumed an average of 523 and 252g/animal in the high and low dose group, respectively. Body and organ weights, hematology, urinalysis, and histopathological evaluations revealed no evidence of adverse effects associated with the compound relative to the untreated controls. The spectrum, incidence, and histology of tumors developing in both treated and control animals were consistent with spontaneous incidences reported in this strain of rat. This study thus indicates that Red 106 is not carcinogenic to F344 rats after 2 years of dietary administration at a maximal level of 5.0% in the basal diet by weight.

View full abstract

To investigate thiophene-induced vascular damage in the rat cerebellum, we employed aged spontaneously hypertensive rats (SHR), which have vascular vulnerability. The animals were given a daily subcutaneous injection of 0.15ml of thiophene for 3 to 6 days. The cerebellum was then studied by light and electron microscopy, horseradish peroxidase (HRP) methods, and the quantitative determination of thiophene at sequential stages after administration. Well-demarcated regions of degeneration appeared, and these spread extensively in the cerebellar rostral folia and/or focally in the caudal folia. In these lesions, tangled villi or oil droplets with an osmiophilic homogeneous substance were found both within and/or outside the lumina of blood vessels, especially capillaries and venules. They were membrane-trophic and located in the degenerated endothelial cells and then disappeared from the lumina at a late stage. Platelet thrombi occupied the lumina of blood vessels in the pia mater and parenchyma from the early to late stage. A lack of reaction product and its extravascular leakage were clearly revealed using HRP. Thiophene and/or its metabolites were detected quantitatively at an early stage in the damaged cerebellum. These lesions were replaced by gliosis and/or cysts in surviving animals. It is concluded that thiophene or its metabolites cause vascular damage and granule cell degeneration, producing circulatory disturbance in aged SHR.

View full abstract

Many cases of hearing loss have been reported in victims of Minamata disease, caused by methylmercury poisoning. Thiophene, like methylmercury, induces cerebellar granule cell degeneration in rats. To investigate the pathophysiology of the central auditory system in thiophene-poisoned rats, spontaneously hypertensive rats stroke resistance (SHR·SR) were given 0.15ml of thiophene daily for 5 or 6 days. After the rats had undergone brainstem recording and analysis of the auditory brainstem response (ABR), they were necropsied for histopathological examination. In the thiophene-poisoned animals, an abnormal ABR pattern like that in methylmercury poisoning appeared. In the acute stage, by 5 days after administration (DAA), the IV wave amplitude was reduced and the interpeak latency (IPL) was prolonged to various degrees. At 5 DAA, the IPL returned to the pretreatment level, earlier than the amplitude. In rats with severe cerebellar injury, a PAS-positive substance was localized in the inferior colliculus, comprising the central nucleus, and especially its ventrolateral portion. The lesions included neuronal necrosis, edema of the neuropil, and axonal degeneration. Proliferated GFAP-positive cells were located close to areas of neuronal loss. Our results suggest that demonstration of ABR is useful for evaluating disturbance of the central auditory system induced by chemicals.

View full abstract

The effects on pancreatic carcinogenesis of cholecystokinin (CCK) and/or proglumide (PGL), antagonist of CCK receptor, were studied in male hamsters initiated with N-nitrosobis (2-hydroxypropyl) amine (BHP). The animals were initially given weekly 500mg BHP/kg body weight subcutaneous injections for 5 times. This was followed by 40IDU/kg body weight by subcutaneous injections of CCK 3 times a week and/or by 1.25% or 2.5% PGL in the diet for 30 weeks. Pancreatic lesions including duct epithelial cell hyperplasia and atypical hyperplasia, intraductal carcinomas, and invasive carcinomas were induced in all groups of hamsters given BHP. However, no significance influence of CCK and/or PGL on the induction of these lesions was observed. Furthermore, CCK and/or PGL did not affect DNA synthesis in their component cells as judged by BrdU immunohistochemistry.

View full abstract

Hyaline droplets in kidney tubules of rats with chronic progressive nephropathy were histopathologically compared with those found in 2, 2, 4-trimethylpentane (TMP) induced α2u-globulin nephropathy. Repeated oral administration of TMP at the dosage of 50mg/kg/day for four weeks caused development of hyaline droplets which were all immunohistochemically positive for α2u-globulin. In control rats (13 to 52 weeks-old), two types of hyaline droplets were observed: one small round and multiple type being deeply eosinophilic and refractile (hyaline droplet type); and the other being slightly eosinophilic, large, and generally single (eosinophilic body type). Both were positive for anti-α2u-globulin immunohistochemical staining. Fifty-two and 109 weeks-old male rats with chronic progressive nephropathy showed “hyaline droplet degeneration” of tubules with hyaline cast formation in the lumina. Immunohistochemical examination revealed such “hyaline droplet degeneration” to be positive for albumin, but negative for α2u-globulin. Under the electron microscope, round, amorphous, and moderately electron-dense phagolysosomes were observed in the tubules of “hyaline droplet degeneration” cases, clearly different from the polyangular and crystalline-like electron-dense phagolysosomes of normal α2u-globulin reabsorption droplets in young rats or TMP induced α2u-globulin accumulation droplets. The present results suggest that the etiology of chronic progressive nephropathy may be somewhat different from that of α2u-globulin induced nephropathy in terms of the causative protein.

View full abstract

Inguinal mammary tissues from each of 3 male and 2 diestrous female rats of both Sprague-Dawley (SD) and Fischer 344 (F344) strains were harvested 30 min after an i.p. injection of bromodeoxyuridine (BrdU) at the ages of 3, 5, 7, 11, 15, and 19 weeks, respectively, examined using whole mount preparations, histologic methods, and histochemical techniques for BrdU, actin and peanut agglutinin (PNA) binding capacity, and compared with the same age and strain specimens of the opposite sex or with each of 13 castrated males of both strains. In SD males aged 7 weeks and F344 males aged 11 weeks, the mammary glands became more florid than in females. A striking predominance was noted in the duct-lobular system of adult male rats, in which many clusters of large, contiguous cuboidal cells exhibiting neither obvious lobuloalveolar orientation nor distinct lumen were observed. The clusters contained many myoepithelial cells in peripheral portions, and their epithelial cells manifested less BrdU labeling, more PNA binding capacity, and moderately fatty change. These findings were confirmed ultrastructurally in the clusters of 22-week-old male rats of both strains. Such a “male type” was more prominent in adult SD than in adult F344 strains. It could not be detected in either strains 4-8 weeks after orchiectomy performed before or after puberty. Therefore, these clusters would not have resulted from proliferating gland cells, rather from severe hypertrophy in ductulus luminal cells, and this sexual dimorphism may depend on sex hormones and strain difference may be related partly to maturation.

View full abstract

Granulosa cell tumors/luteomas of the ovary were induced at very high incidence in the offspring of F344 rats receiving N-nitrosobis (2-oxopropyl) amine (BOP) transplacentally (Jpn. J. Cancer Res., 81: 1077-1080, 1990). In addition to ovarian tumors, atrophic and hyperplastic lesions were also frequently observed in the ovary and uterus of BOP-treated animals, respectively. In order to investigate the relation between granulosa cell tumors and uterine endometrial lesions, the lesions in BOP-treated rats with ovarian tumors were compared to those in the rats without tumors. While no endometrial adenocarcinoma was found in both rat groups, the incidence and degree of atypical, and adenomatous endometrial hyperplasias were slightly high in rats with ovarian tumors, as compared to rats without tumors, while being not significant. In the assay of plasma gonad steroids, hormonal imbalance was clearly detected in BOP-treated animals and the 17β-estradiol: progesterone (E2: P) ratio was higher in BOP-treated rats with ovarian tumors than control rats or treated rats without tumors. These results suggest that a slight association between ovarian tumors and endometrial proliferative lesions exists in rats and hormonal imbalance, particularly an increased E2: P ratio, might play an important role in the association.

View full abstract

From the control groups of five 2-year carcinogenicity studies, we isolated and observed six pituitary adenomas of the pars intermedia in two mice and four rats by histopathological and immunocytochemical techniques for evidence of production of the following proopiomelanocortin (POMC)-derived peptides; adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), β-melanocyte-stimulating hormone (β-MSH), γ-melanocyte-stimulating hormone (γ-MSH), and β-endorphin (β-END). Macroscopically, most adenomas were from slightly to moderately enlarged or colored dark red, and were well separated from the brain and surrounding tissues. Histologically, their cells were scarcely distinguishable from the normal pars intermediate cells, but they often contained abundant eosinophilic cytoplasms, were enlarged and occasionally elongated and arranged in parallel groups. Atypia was not seen regularly, but some mitotic figures were seen. Two large adenomas from rats revealed vacuolated cell nests and follicular structures with colloidal materials. Immunostainings of all adenomas in both mice and rats were positive for all antisera tested and scarcely differed from staining of normal pars intermediate cells except that the intensities among the antisera were not constant and often presented pale and speckled appearances. Since the cytoplasms of the vacuolated cell nests of two rat adenomas also reacted positively for POMC-derived peptides, it was clear that these nests also originated from the pars intermedia. No endocrinological abnormalities in other organs or tissues, including the adrenal glands, were detected in any adenoma-bearing animals. From this evidence, we concluded that the adenomas derived from pars intermedia were endocrinologically active and were producing POMC-related peptides in both mice and rats.

View full abstract

Five-week-old JCR: CD rats were treated with 10% or 1% sodium chloride (NaCl) diet, 10% miso diet, or 10% ethanol in drinking water after a total dose of 20 Gy of X-rays administered to the gastric region in two equal fractions separated by 3 days. One year after the X-irradiation, the animals were sacrificed and quantitatively analysed for pathological changes in the medium- and small-sized arteries of the stomach. Arteritis induced by X-irradiation in rats was enhanced by 10% NaCl, but miso and 1% NaCl did not enhance the development of arteritis. On the other hand no arteritis was observed in the 10% ethanol group by either histological or quantitative analysis. Furthermore, a good correlation was also observed between enlargement of small-sized artery wall and NaCl concentration, especially among the X-irradiated groups.
In another experiment, after a single dose of 20 Gy of X-rays, rats were treated with either 10% NaCl in diet or 10% ethanol in drinking water for 1 year. A higher incidence of arteritis was observed in the X-ray groups but the modified effect observed in the fractionated irradiation groups treated with 10% NaCl or 10% ethanol was not observed.

View full abstract

A spontaneous granular cell tumor of tongue was found in the routine pathological examination of a carcinogenicity study in rats. Histologically, this tumor was characterized by fascicular pattern of spindle cells containing variously sized eosinophilic granules in their cytoplasm and abundant intercellular collagen. Immunohistochemically, the tumor cells were stained positively for S-100 protein, but negatively for GFAP and desmin. Ultrastructurally, the tumor cell was invested by pericytoplasmic basal lamina. Judging from these findings, this granular cell tumor was considered to be derived from Schwann cells or their precursors.

View full abstract