Safety assessment of endocrine-disrupting chemicals (EDCs) is now an important issue because of fear of human exposure. Bisphenol A (BPA), a compound initially synthesized as a chemical oestrogen, is now used as a monomer for the production of polycarbonate plastic products such as baby bottles. In the present experiment, toxicity and effects of BPA administration to maternal rats and response of their offspring to N-nitrosobis(2-hydroxypropyl)amine (BHP) treatment were studied. Growth retardation was observed in maternal rats throughout the period of BPA exposure (total dose of 21 ± 3 grams per rat). Serum TSH was elevated in maternal rats receiving a soybean-devoid diet and offspring of maternal rats with or without BPA but this was not thought to be caused by BPA. Histopathologically, no significant organ toxicity was observed in BPA-treated maternal rats. Examination of cleavage of the balanopreputial gland in males and opening of the vagina in females performed on 5 to 7-week-aged offspring, demonstrated no abnormal differentiation or growth retardation. BPA exposure did not affect BHP-induced carcinogenesis in the thyroid, lung, thymus, esophagus, and liver, location, incidences, and numbers of tumors not differing in offspring born from maternal rats with or without BPA administration. These results indicate that BPA does not induce any tissue injury for maternal or offspring rats, with no effects on target organ carcinogenesis of BHP in offspring.

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In order to assess the effects of genetic factors on spontaneous tumor development in various organs/tissues, especially the reproductive organs of Donryu and F344 rats, F₁ hybrids between these two strains were investigated. Litters were obtained by mating virgin Donryu or F344 females with F344 or Donryu males, respectively. They were weaned at 4 weeks of age and divided into two hybrid groups: Group A, (Donryu female × F344 male) F₁ (male n=49, female n=48) and group B, (F344 female × Donryu male) F₁ (male n=53, female n=56). These two groups of F₁ hybrids were observed with their parents (Donryu male n=19, female n=30; F344 male n=21, female n=29) until 120 weeks of age. The incidences of persistent estrus in both F₁ groups from 8 to 12 months of age were in between those for their Donryu and F344 mothers, being slightly higher in group A than in group B. They increased thereafter and reached about 90% and 80% at 18 months of age in groups A and B, respectively. In females of group A, the incidences of endometrial hyperplasias and adenocarcinomas were comparable with those in the Donryu strain, and higher than those in group B, whose values were in turn elevated as compared to their F344 mothers. In contrast, endometrial stromal polyps and Leydig cell tumors of the testes were observed with similar incidences in both hybrids as well as F344 rats, but at markedly lower levels in the Donryu strain. The frequency of mononuclear cell leukemias was very low in both F₁ groups of each sex. Various tumors were also observed in many other organs/tissues such as the pituitary, thyroid, pancreas, adrenals, mammary and prostate glands and their incidences were relatively high in both groups A and B, but their variation between the F₁ groups was small. These results indicate that development of uterine endometrial hyperplasias/carcinomas in F₁ hybrids might be related to hormonal conditions depending on the genetic background of the mothers, but other tumors are considered to be influenced by other factors. The effects of genetic background on the occurrence of spontaneous tumors in hybrids are very complex and differ not only with the site but also the tumor types.

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The purpose of this study is to determine which combination of choline-deficient L-amino acid-defined (CDAA) diet as an endogenous factor, phenobarbital (PB) as an exogenous factor, and partial hepatectomy (PH), has the most promotive potential on hepatocellular foci formation initiated by diethylnitrosamine (DEN) in Fischer 344 rats. Experimental groups were treated with various combinations of DEN administration, PB administration, PH and CDAA diet. Formation of hepatocellular foci after eight weeks was examined by glutathione S-transferase placental form (GST-P) immunological staining. The focal area was markedly elevated in the CDAA+DEN group in comparison with the CDAA-only group or the CRF-1(basal diet)+DEN group. CDAA diet was shown to have a strong promotion effect on initiated cells by DEN. In the CRF-1+DEN-treated animals, the additional PB and PH treatment elevated the number of small foci, but slightly did the focal area. The effects of the additional PB and PH treatment in the CDAA+DEN-treated animals were not clear as they were masked by the strong promotion effect of CDAA diet. These results showed that CDAA diet was more promotive than the PB and PH treatment in inducing hepatocellular foci formation initiated by DEN in rats, and the additional PB and PH treatment could not be promotive furthermore in the CDAA+DEN-treated animals.

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We studied the time courses of the number of osteoclasts, the expression of osteoblast differentiation markers and TGF-β1 levels in the platelet-poor plasma (PPP) in order to clarify the mechanism of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)-induced bone formation in the mouse. Mice received PEG-rHuMGDF subcutaneously at a suprapharmacological dose (1.0 mg/kg) daily for 5 days (Day of the first injection: Day 1). PEG-rHuMGDF caused a gradual increase of platelet count, with a maximum increase on day 9. Histological analysis showed an increase of reticulin fibers on the inner side of endosteum on day 9, partial osteoid formation on day 11, new and excessive bone formation on day 13. On day 9, at the early stage of reticulin fiber increase, the number of osteoclasts was decreased. On day 11, osteopontin (OPN) mRNA-expression, a marker of osteoblast production, was observed in the reticulin fiber and osteoid near the substantia of bone marrow, whereas OPN mRNA-expressing cells did not express bone Gla protein (BGP) mRNA, a marker of osteoblasts, indicating that the expression of OPN mRNA was induced earlier than that of BGP mRNA during the course of osteogenesis. Additionally, the level of TGF-β1 in the PPP was increased, with about 2.5-fold elevation compared with the vehicle-treated mice at day 9. These findings suggest that PEG-rHuMGDF causes increase of reticulin fibers and osteogenesis by stimulating osteoblast differentiation and inhibiting osteoclast differentiation via an increase of the TGF-β1 level in the bone marrow.

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To clarify whether 17β-estradiol 3-benzoate (EB), methoxychlor (MXC), atrazine (ATR), or bisphenol A (BPA) have any modifying effects on relatively late stages of thyroid carcinogenesis, six-week old female castrated F344 rats in Experiments 1 and 2 first received a single subcutaneous injection of 2000 mg/kg body wt of N-bis (2-hydroxypropyl) nitrosamine (DHPN) and, starting one week later, were given drinking water containing 1000 ppm sulfadimethoxine (SDM) for 8 weeks. Then, in Experiment 1, cholesterol pellets containing 0.5 mg EB were subcutaneously embedded and renewed every 4 weeks for 25 weeks. Controls received basal diet alone. In Experiment 2, rats of the control, MXC, ATR, or BPA group received diet containing no supplement, 1000 ppm MXC, 400 ppm ATR, and 10000 ppm BPA, respectively, for 27 weeks. Thyroid follicular cell hyperplasias, adenomas, and/or carcinomas were induced in all of the groups. In Experiment 1, the incidence of adenomas in the EB group was significantly increased, as compared to the corresponding control group value. In Experiment 2, the incidences of carcinomas in the MXC and BPA groups were significantly lower than in the corresponding control group. Serum estrogen levels and the PCNA labeling index for carcinomas in the EB group were significantly elevated but there was no clear alteration in serum thyroid related hormone levels. Serum T3 levels in the MXC and ATR groups were significantly reduced, whereas serum T4 was increased in these as well as the BPA group. No significant fluctuation in serum TSH levels was observed in these treated groups. The results of the present studies suggest that EB with strong estrogenic activity, but not MXC and BPA with only weak estrogenic activity or ATR, exerts promoting effects on thyroid carcinogenesis in rats.

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In a previous study, we established a 2-stage uterine carcinogenesis model that is useful for detecting modifying effects of endocrine disrupting chemicals (EDCs) featuring administration of N-ethyl-N-nitrosourea (ENU) as an initiator to female heterozygous p53 deficient CBA mice [p53 (+/-) mice]. In addition, we demonstrated that ethinylestradiol with strong estrogenic activity, but not methoxychlor with weak estrogenic activity, showed promoting effects on uterine carcinogenesis. In the present study, to clarify the effects of other EDCs with weak estrogenic activity on development of uterine tumors, female p53 (+/-) CBA mice received an intraperitoneal injection of 120 mg/kg body weight of ENU followed by the diet containing 1% bisphenol A (BpA), 20% roasted-ground soybean (Kinako) (SB) or no further treatment for 26 weeks. Animals of the ENU+BpA and ENU+SB groups showed no significant differences in body weight gain compared to the ENU alone group. Lower values in absolute and relative uterine weights were observed in the ENU+BpA and ENU+SB groups compared to the ENU alone group, but no significant differences were observed in the incidence of uterine endometrial stromal sarcomas and their PCNA labeling indices among the groups. The results in the present study indicate that 1% BpA and 20% SB in diet have no modifying effects on uterine carcinogenesis in p53 (+/-) CBA mice initiated with ENU.

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Flumequine (FL), a quinolone-antibiotic used for veterinary treatment of infections, was found to elicit hepatocellular tumors in a conventional 18-month carcinogenicity study in mice, and hepatocellular necrosis-regeneration cycle was considered to be a possible underlying mechanism. In order to clarify whether FL has any modifying effects on development of hepatocellular proliferation, groups of heterozygous p53 deficient CBA mice [p53(+/-) mice], sensitive to genotoxic carcinogen, of both sexes and their wild-type littermates [p53(+/+) mice] were fed diet containing 4,000 or 0 ppm FL for 26 weeks after an intraperitoneal injection of 5 or 0 mg/kg body weight of dimethylnitrosamine (DMN). Higher incidences of hepatocellular foci were observed in animals receiving FL, with or without DMN-initiation, than in the corresponding control groups in both p53(+/-) and p53(+/+) mice. Incidences and multiplicities of foci were generally similar in p53(+/-) and p53(+/+) mice, but, in the DMN+FL group, multiplicity of foci and their PCNA labeling indices were greater in p53(+/-) mice. There were also small numbers of hepatocellular adenomas and carcinomas in the DMN+FL group of p53(+/-) mice, hepatocellular adenoma in the FL alone group of p53(+/-) mice, and hepatocellular adenomas in the DMN+FL group of p53(+/+) mice. Induction of hepatocellular tumors in these mice within a relatively short period strongly suggests that mechanisms such as direct or indirect damage to DNA might be responsible for the hepatocarcinogenesis of FL.

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In order to examine the effect of 3,5-di-tert-butyl-4-hydroxytoluene (BHT) on hepatocellular population, BHT was given orally to male rats for 7 consecutive days at dose levels of 75 or 450 mg/kg/day. BHT induced hepatocellular proliferation, increase in apoptosis, and elevated immunoreactivity for transforming growth factor (TGF)-β1 during the treatment, and hepatocellular mitoinhibition following the withdrawal. The induction of mitoinhibition, apoptosis, and TGF-β1 might be adaptive changes in response to cell proliferation; thus the homeostasis of hepatocellular population was preserved. Although both the mechanism and the biological significance of mitoinhibition observed following the withdrawal of BHT, after a 7-day treatment period, were not elucidated, its mechanism seemed to be related to the elevation of TGF-β1 immunoreactivity and its biological significance might be similar to those with non-genotoxic hepatocarcinogens in chronic treatments.

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We have shown that the Mini rat, a transgenic rat strain carrying the antisense transgene for rat growth hormone, is a useful model to investigate the role of growth hormone in the expression of hepatic microsomal cytochrome P450s (CYPs). However, the induction of CYP isoforms by hepatic enzyme inducers in Mini rats has not been investigated. We selected two enzyme inducers, 3-methylcholantherene (2 mg/kg/day, i.p.) and phenobarbital (5 mg/kg/day, p.o.), and administered them to male Mini rats and their parental strain, Wistar rats, for 3 days. Enzyme activity assays and immunohistochemistry showed no differences in the expressions of CYP1A and CYP2B between untreated Wistar and Mini rats. 3-Methylcholanthrene administration induced CYP1A in both strains, but the induction was significantly higher in the Mini rat liver than in the Wistar rat liver. Phenobarbital administration increased CYP2B activity in both strains by more than 20-fold over their respective controls. Although there was no difference in the CYP2B immunohistochemistry between the two strains given phenobarbital, the CYP2B activity of phenobarbital-treated Mini rats was significantly higher than that of phenobarbital-treated Wistar rats. These results suggest that growth hormone may play a role in the hepatic enzyme induction.

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A cyanobacterial toxin, microcystin-LR (MCLR), is a potent inhibitor of protein phosphatase that disrupts cytoskeleton network in hepatocytes. Conventional transmission electron and immunoelectron microscopic studies were conducted in the liver from mice that received a single dose of MCLR and were sacrificed at 24 hours after dosing. The two types of death of hepatocytes, necrosis and apoptosis, were observed concomitantly, and the hepatic injuries, therefore, could be classified as a class of mixed apoptotic and oncotic necrosis according to the recommended criterion by the Society of Toxicologic Pathologists. Apoptotic hepatocytes were characterized by ballooning with numerous intracytoplasmic vacuoles in addition to the common features of apoptotic cells referred in the literatures. In non-parenchymal cells, significant changes indicating microcirculatory alterations and inflammatory reactions included activation of endothelial cells and Kupffer cells, widening of sinusoidal endothelial fenestrae, apoptosis of endothelial cells, accumulation of platelets and polymorphonuclear neutrophils, and fibrin deposition. Pre-embedding immunoelectron microscopy with biotinylated anti-microcystin antibody demonstrated that MCLR was heterogeneously apparent in the cytoplasm of hepatocytes and strong immunoreactivity was evident in apoptotic cell/bodies. In non-parenchymal cells, there were no positive reactions except for phagocytic apoptotic bodies in Kupffer cells. These results suggest that liver damages induced by MCLR are ascribed to primary cytotoxic effects of the toxin localized in hepatocytes and secondary microcirculatory alterations and inflammatory reactions involved non-parenchymal cells.

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Zonal lesions in the hepatic lobules observed in drug-treated rats, dogs, and monkeys or spontaneously diseased monkeys were considered in reference to the different concepts of the hepatic lobule in order to understand the significance of zonal lesions of the liver correctly. Lesions due to congestion appeared at the periphery of the portal lobule of Mall's concept. Incipient changes due to severe anemia occurred in a triangular shape at the center of the classical lobule where the apices of primary lobules become confluent as proposed by Matsumoto and Kawakami. Drug-induced centrilobular necrosis appeared evenly around the central venules showing clear contrast with the change observed in anemia. Periportal necrosis was located along the portal tract at the periphery of the classical lobule. None of these lesions match the zones of the acinar concept proposed by Rappaport. The concept of the hepatic lobule is essential to understand zonal lesions correctly.

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6-Sulfanilamidoindazole (6SAI) is a sulfonamide that induces acute, self-limiting arthritis in rats, and it is reported that endotoxin may modify the sensitivity to 6SAI in rats. In this study, detailed pathological changes were examined in lipopolysaccharide (LPS)- hyporesponsive C3H/HeJ (Lps^d) and LPS-normoresponsive C3H/HeN (Lpsⁿ) mice orally administered with 6SAI (500, 1000 mg/kg) daily for 2 wk. There was no joint swelling observed in any animals treated with 6SAI. Histopathologically, 6SAI-treated mice from both strains showed the same changes such as hypertrophy of hepatocytes, mural thickening of hepatic artery, thickening of adrenal cortex, hypertrophy of thyroid epithelium and hypertrophy of intestinal mucosa. In one animal of the high-dose group of C3H/HeJ strain showed focal epicarditis and capsulitis of thymus and lungs. In the synovium of tarsal joints, there were no changes observed in any 6SAI-treated animals. From this study, it is confirmed that hepatic artery, hepatocytes, thyroid epithelium and intestinal mucosa are target sites of 6SAI in mice. 6SAI-induced toxicity in mice is considered not to be correlated with LPS sensitivity.

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Early ultrastructural changes in the dorsal skin epidermis of Wistar-derived hypotrichotic WBN/ILA-Ht rats were examined for up to 12 hours after UVB-irradiation (10 kJ/m²) (12 HAI). The first change appeared at 3 HAI as swelling and/or vacuolation of mitochondria in the spinous and basal cells. The number of keratinocytes with such mitochondrial changes decreased thereafter. At 6 HAI, apoptosis of basal cells developed. These cells showed shrinkage of cell bodies with condensed or fragmented nuclei and dark cytoplasm due to densely packed bundles of tonofilaments. At the same time, keratinocytes with prominent nucleoli were also observed in the basal and suprabasal layers. At 12 HAI, the number of keratinocytes with prominent nucleoli increased, and mild and focal intercellular edema developed mainly in the basal layer. In addition, a small number of apoptotic basal cells were still observed. The present ultrastructural findings supported the histopathological findings previously reported by our research group.

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