Effects of two weeks of food restriction on toxicological parameters in cynomolgus monkeys.

Animals frequently eat less after a test-article treatment in nonclinical toxicological studies, and it can be difficult to distinguish test article-derived toxicities from secondary changes related to this reduced food intake. Therefore, in this study, we restricted the food intake of cynomolgus monkeys (Cambodian, male, n=2 or 3, 48 ± 3 months old) to 25% of the control for two weeks and evaluated the effects on toxicological parameters (general conditions, body weight, electrocardiography, urinalysis, hematology, blood chemistry, bone marrow analysis, pathological examination). After 2 weeks, the monkeys exhibited decreases in bone marrow erythropoiesis (e.g., decreases in reticulocytes and bone marrow erythrocytes), as well as glycogenesis induction (e.g., increase in aspartate aminotransferase (AST)) and malnutrition (e.g., decrease in triglyceride and systemic adipocytes atrophy). Additionally, histopathological analysis revealed granuloma and inflammatory cell infiltration in coronary fat, which had never been found in previous food restriction studies using other animal species. These findings will enable researchers to more accurately evaluate the toxicological risks of test articles that simultaneously induce food intake reduction.

Lipomatosis of axillary lymph nodes in a cynomolgus monkey (Macaca fascicularis).

Lipomatosis of lymph nodes is defined as the replacement of the lymphatic parenchyma by adipose tissue which grows in the node from the hilus toward the cortical zone. In humans, it is considered as part of the normal aging process and is common in obese patients, but there are no reports in non-human primates. In this report, we describe the first case of lymph node lipomatosis in the bilateral axillary lymph nodes of a young adult cynomolgus monkey. Macroscopically, there were no apparent abnormalities in the axillary lymph nodes on either side, and their volumes were unchanged. At the cut surface, pale yellow fat-like tissue was observed in the medullary area. Histopathologically, well differentiated adipocytes replaced a large part of the lymphatic parenchyma in the area from the hilus to the medulla without any malignant findings. Based on these findings, the patient was diagnosed with lipomatosis of the lymph nodes.

Neutrophil Elastase Inhibition Ameliorates Endotoxin-induced Myocardial Injury Accompanying Degradation of Cardiac Capillary Glycocalyx.

Myocardial injury in sepsis may be caused by a burst of several inflammatory mediators, leading to vascular endothelial injuries. However, the contribution of neutrophil elastase (NE) to myocardial injury in sepsis is still unknown. We aimed to evaluate whether endotoxemia-induced myocardial injury is associated with NE. Lipopolysaccharide (LPS) was injected intraperitoneally at a dose of 20 mg/kg into granulocyte-colony-stimulating-factor knockout mice (G-CSF-KO), which have few neutrophils, and littermate control mice. The survival rate of G-CSF-KO mice 48 hours after LPS injection was significantly greater than that of control mice. The serum level of troponin I in G-CSF-KO mice was significantly lower than that in control mice. In addition, the concentration of inflammatory cytokine interleukin-6 (IL-6) was significantly decreased 6 and 12 hours after LPS administration compared with that in control mice. Ultrastructural analysis revealed that vascular endothelial structures and the endothelial glycocalyx in G-CSF-KO mice were clearly preserved. Next, mice were injected with 0.2 mg/kg sivelestat (an NE inhibitor) after LPS administration. The survival rate was significantly higher and the serum level of troponin I was lower in sivelestat-injected mice than in control mice, respectively. Furthermore, IL-6 levels were significantly decreased 6 and 12 hours after LPS administration compared with those in control mice. Vascular endothelial structures and the endothelial glycocalyx in sivelestat-treated mice were clearly preserved at the ultrastructural level. In conclusion, NE is significantly associated with myocardial injury in endotoxemia. Inhibition of NE may be a useful tool for the management of endotoxemia.

Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet.

Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, details of the liver-specific molecular mechanisms responsible for the NAFLD-NASH-HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis-associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.

A crucial role of constitutive androstane receptor (CAR) in liver tumor development by imazalil in mice.

To clarify the major pathway of liver tumor development induced by imazalil (IMA), an imidazole fungicide, male constitutive androstane receptor (CAR)-knockout (CARKO) and wild-type (WT) mice were treated with IMA at 500 ppm in the diet up to 27 weeks after initiation by diethylnitrosamine. After 27 weeks of treatment, neither altered foci nor adenomas were significantly increased in CARKO mice, whereas both eosinophilic altered foci and adenomas were increased in WT mice. After 4 or 13 weeks of IMA treatment, liver hypertrophy was observed at the tumor-inducible dose without differences among genotypes or durations. Analysis of hepatic drug metabolite enzymes, performed after administration of multiple doses during a 1-week period, indicated that pregnane X receptor might be involved in liver hypertrophy because IMA markedly elevated Cyp3a11 and Cyp2b10 expression levels in a dose-dependent manner in both genotypes. Our results demonstrated that the CAR pathway was the main mechanism of liver tumor development induced by IMA. The carcinogenic pathway was different from that of liver hypertrophy.

Involvement of constitutive androstane receptor in liver hypertrophy and liver tumor development induced by triazole fungicides.

We clarified the involvement of constitutive androstane receptor (CAR) in triazole-induced liver hypertrophy and tumorigenesis using CAR-knockout (CARKO) mice. Seven-week-old male CARKO and wild-type (WT) mice were treated with 200 ppm cyproconazole (Cypro), 1500 ppm tebuconazole (Teb), or 200 ppm fluconazole (Flu) in the diet for 27 weeks after initiation by diethylnitrosamine (DEN). At weeks 4 (without DEN) and 13 (with DEN), WT mice in all treatment groups and CARKO mice in Teb group revealed liver hypertrophy with mainly Cyp2b10 and following Cyp3a11 inductions in the liver. Teb also induced Cyp4a10 in both genotypes. Cypro induced slight and duration-dependent liver hypertrophy in CARKO mice. At week 27, Cypro and Teb significantly increased eosinophilic altered foci and/or adenomas in WT mice. These proliferating lesions were clearly reduced in CARKO mice administered both compounds. The eosinophilic adenomas caused by Flu decreased in CARKO mice. The present study indicates that CAR is the main mediator of liver hypertrophy induced by Cypro and Flu, but not Teb. In contrast, CAR played a crucial role in liver tumor development induced by all three triazoles.

Early indicators of delayed adverse effects in female reproductive organs in rats receiving neonatal exposure to 17alpha-ethynylestradiol.

We previously reported that neonatal exposure to 17α-ethynylestradiol (EE) led to delayed adverse effects in which age-related anovulation after sexual maturation was accelerated. To identify early indicators of these adverse effects, female Wistar Hannover GALAS rats received a single EE injection (0, 0.02, 0.2, 2, 20, or 200 µg/kg) within 24 hr of birth. Histopathological changes in ovarian and uterine development were investigated from postnatal day (PND) 14 to 10 weeks of age. Immunohistochemical expression of estrogen receptor alpha (ERα) in the uterus, serum levels of sex-related hormones and gene expression in the hypothalamus were examined. Although neonatal exposure to EE did not affect body growth or ovarian development, serum FSH tended to decrease at doses ≥ 2 µg/kg, and Kiss1 mRNA level in the whole hypothalamus was significantly decreased in all EE-treated groups at PND14.The number of uterine glands at PND21 was suppressed at doses ≥ 20 µg/kg, and ERα expression in the uterine epithelium at estrus stage decreased in a dose-dependent manner at 10 weeks of age. These results demonstrated that the various identified changes that occurred before the appearance of delayed adverse effects could be candidate early indicators.

Inhibitory potential of postnatal treatment with cyclopamine, a hedgehog signaling inhibitor, on medulloblastoma development in Ptch1 heterozygous mice.

Medulloblastomas (MBs) are thought to be derived from granular cell precursors in the external granular layer (EGL) of the developing cerebellum. Heterozygous patched1 (Ptch1) knockout mice develop MBs that resemble those in humans when the sonic hedgehog (Shh) signaling pathway is activated. The present study was conducted to evaluate postnatal effects of a Shh signaling inhibitor, cyclopamine, on the development of MBs in Ptch1 mice. Ptch1 and wild-type mice were treated daily with subcutaneous cyclopamine at 40 mg/kg or vehicle from postnatal day (PND) 1 to PND14, and the subsequent development of MBs and preneoplastic lesions was examined up to week 12 (W12). Proliferative lesions in the cerebellum, MBs, and preneoplastic lesions were only detected in Ptch1 mice. Cyclopamine treatment resulted in a statistically significant reduction in the incidence and/or area of proliferative lesions at PND14 and 21. The trend of decreasing preneoplastic lesions persisted up to W12. At PND7, cyclopamine treatment reduced the width and proliferation of the EGL regardless of genotype. These results indicate that inhibition of Shh signaling during cerebellar development has prolonged inhibitory potential on MB development in Ptch1 mice. This inhibitory potential might be related to inhibition of EGL proliferation, including preneoplastic MB cells.

Effects of pyperonyl butoxide on the female reproductive tract in rats.

This study was investigated the effects of piperonyl butoxide (PBO) on the female reproductive tract. Female Crj:Donryu rats were fed a basal diet containing 5,000, 10,000 or 20,000 ppm PBO for 28 days, and compared with food-restricted rats of comparable body weights to those in the PBO 10,000 or 20,000 ppm groups. Although treatment with 20,000 ppm PBO for 28 days depressed body weight gain, the abnormal estrous cyclicity, mainly prolonged diestrus, was also induced by the PBO treatment which was not correlated with body weight change. 20,000 ppm PBO treatment markedly decreased uterine weights and slightly decreased ovarian weights. 10,000 and 20,000 ppm PBO treatment increased liver weights. These cycle and organ weight changes were linked to atrophic uterus and increased atretic follicles in the ovary. In hormone assays, PBO at both doses reduced serum E2 levels, but did not affect corticosterone levels. An anti-uterotrophic assay showed a slight but significant decrease in absolute uterine weight and a reduction of endometrial epithelium height in the 20,000 ppm group. PBO was positive in an ER α antagonist reporter gene assay, although the activity was much weaker than that of 4-hydroxytamoxifen. These results indicate that high-dose PBO treatment directly induces atrophic changes in the female reproductive tract in rats, and these effects are likely the result of a hypoestrogenic state and the anti-estrogenic activity of PBO.

Delayed effects of neonatal exposure to 17alpha-ethynylestradiol on the estrous cycle and uterine carcinogenesis in Wistar Hannover GALAS rats.

We investigated the delayed effects of neonatal exposure to 17α-ethynylestradiol (EE) on the female reproductive tract using Wistar Hannover GALAS rats. Female pups received single injections of EE (0, 0.02, 0.2, 2, 20, or 200 µg/kg) within 24h after birth and estrous cyclicity was observed until 10 months of age. All animals were treated at 9 weeks of age with the uterine carcinogen, N-ethyl-N'-nitro-N-nitrosoguanidine. Although the vaginal opening was not affected, abnormal cycles were significantly increased from 0.2 µg/kg. Persistent estrus was prominent and the incidence increased age- and dose-dependently. Severity of atypical hyperplasia of the uterus tended to increase from 2 µg/kg. In these groups, serum progesterone level was lowered relative to estradiol level. In conclusion, estrous cyclicity was a sensitive indicator reflecting delayed effects on the female reproductive tract. Early onset of anovulation leading to prolonged estrogen exposure might be a risk factor for uterine carcinogenesis.

Thickened area of external granular layer and Ki-67 positive focus are early events of medulloblastoma in Ptch1⁺/⁻ mice.

Patched1 (Ptch1) encodes a receptor for Sonic hedgehog (Shh) and is major gene related to human medulloblastoma (MB) in the Shh subgroup. MB is thought to arise from residual granule cell precursors (GCPs) located in the external granular layer (EGL) of the developing cerebellum. As the detailed preneoplastic changes of MB remain obscure, we immunohistochemically clarified the derived cell, early events of MBs, and the cerebellar developmental processes of Ptch1(+/-) (Ptch1) mice, an animal model of human MB of the Shh subgroup. In Ptch1 mice, the earliest proliferative lesions were detected at PND10 as focal thickened areas of outer layer of the EGL. This area was composed of GCP-like cells with atypia and nuclei disarrangement. In the latter cerebellar developmental period, GCP-like cell foci were detected at high incidence in the outermost area of the cerebellum. Their localization and morphological similarities indicated that the foci were derived from GCPs in the EGL. There were two types of the foci. A Ki-67-positive focus was found in Ptch1 mice only. This type resembled the GCPs in the outer layer of EGL characterized by having proliferating activity and a lack of neuronal differentiation. Another type of focus, Ki-67-negative, was observed in both genotypes and exhibited many of the same features of mature internal granule cells, suggesting that the focus had no preneoplastic potential. Due to morphological, immunohistochemical characteristics, our results indicate that the focal thickened area of EGL and Ki-67-positive foci are preneoplastic lesions of MB.

Development of an early induction model of medulloblastoma in Ptch1 heterozygous mice initiated with N-ethyl-N-nitrosourea.

Mice heterozygous for the ptch1 gene (ptch1 mice) are known as a valuable model of medulloblastoma, a common brain tumor in children. To increase the incidence and reduce the time required for tumor development, allowing for evaluation of modifier effects on medulloblastoma in a short time, we attempted to develop an early induction model of medulloblastoma in ptch1 mice initiated with N-ethyl-N-nitrosourea (ENU). Ptch1 mice and their wild-type littermates received a single intraperitoneal injection of ENU (10, 50 or 100 mg/kg) on postnatal day 1 (d1) or 4 (d4), and histopathological assessment of brains was conducted at 12 weeks of age. The width of the external granular layer (EGL), a possible origin of medulloblastoma, after injection of 100 mg ENU on d1 or d4 was measured in up to 21-day-old mice. Cerebellar size was apparently reduced at the 50 mg dose and higher regardless of genotype. Microscopically, early lesions of medulloblastomas occurred with a high incidence only in ptch1 mice receiving 10 mg on d1 or d4, but a significant increase was not observed in other groups. Persistent EGL cells and misalignment of Purkinje cells were increased dose-dependently. Although EGL was strikingly decreased after ENU injection, strong recovery was observed in mice of the d1-treated group. In summary, neonatal treatment with ENU is available for the induction of medulloblastoma in ptch1 mice, and 10 mg of ENU administered on d1 appeared to be an appropriate dose to induce medulloblastoma.

Undifferentiated Sarcoma of the Salivary Gland in a Mongolian Gerbil (Meriones unguiculatus).

A subcutaneous mass was found in the lower ventral neck region of a 55-week-old male Mongolian gerbil (Meriones unguiculatus). Histopathologically, the mass involved salivary glands and featured diffuse proliferation of pleomorphic neoplastic cells with large necrotic foci. The lesion was well demarcated from the surrounding tissue, although invasive growth to fibrous septa was occasionally observed. The neoplastic cells were mainly arranged in irregular sheets with severe cellular atypia, round to oval nuclei and varying amounts of eosinophilic cytoplasm. Mitotic figures and multinucleated giant cells were frequent. Immunohistochemical analysis revealed that the neoplastic cells were strongly positive for vimentin and S-100 and negative for NSE, cytokeratin, α-SMA, c-kit, factor VIII, CD34, α-1-antitrypsin, lysozyme and MSR-A. Based on the results, the mass was diagnosed as an undifferentiated sarcoma of the salivary gland. To the best of our knowledge, this is the first report of such a tumor in Mongolian gerbils.

Emphysematous cystitis in a chemically-induced diabetic dog.

Emphysematous cystitis is a rare disorder caused by bacterial infection and characterized by gas accumulation within the bladder wall with cyst formation. This report describes the histopathological characteristics of emphysematous cystitis found in a diabetic female beagle induced by streptozotocin and alloxan. Macroscopically, multiple cyst-like structures were observed on the cut surface of the urinary mucosa. During fixation, small specimens cut from the mucosa floated on the surface of the fixative solution. Histopathologically, multiple cysts were lined with a single layer of flattened cells found to be immunohistochemically positive for vimentin, partially positive for α-smooth muscle actin or macrophage scavenger receptor, class A, and thought to be myofibroblasts, fibroblasts or macrophages. Multinucleated giant cells were observed around the cysts, and gram-negative short bacilli were observed in the lumen of the urinary bladder. From these findings, this case was diagnosed as emphysematous cystitis.

Early effects of tocilizumab on bone and bone marrow lesions in a collagen-induced arthritis monkey model.

To understand the contribution of IL-6/IL-6R to subchondral bone and bone marrow abnormality in RA patients and the effects of tocilizumab on those abnormalities, we evaluated early change in a collagen-induced arthritis (CIA) monkey model with or without a single administration of tocilizumab. Six CIA cynomolgus monkeys received tocilizumab and 3 CIA monkeys received vehicle only. Their interphalangeal joints were analyzed using HE, silver impregnation (SI), or immunohistochemistry (RANKL) staining. The number of osteoclasts increased in the arthritis control but was suppressed in the tocilizumab-treated animals. Osteoblast/stromal cells of the arthritis control monkeys were of monolayer, while in the tocilizumab-treated monkeys, the cells were multi-layer or differentiated osteoblasts, and the meshwork of the reticulum fibers showed recovery in the SI. Hematopoietic marrow was replaced by interstitial fluid and reticulum fibers were eliminated in the arthritic model but showed recovery in the tocilizumab-treated animals. RANKL showed overproduction with arthritis and suppressed with tocilizumab treatment. The evidence indicates that IL-6/IL-6R is involved in subchondral bone and bone marrow change in RA patients. Tocilizumab treatment recovered changes in the CIA monkeys as a result of the co-differentiation between the osteoclasts and the osteoblast/stramal cells, at least partially through the suppression of RANKL overproduction.