Immunohistochemical analysis of extracellular signal-regulated kinase expression in mature and immature bulls' testes and epididymides.

Extracellular signal-regulated kinase (ERK) has been implicated in mammalian testicular and epididymal development. This study aimed to investigate ERK expression in the immature and mature testes and epididymides of bulls. We evaluated ERK expression using immunoblot analysis and immunohistochemistry. Immunoblot analysis revealed that immature bull testes and epididymides had higher phosphorylated ERK (pERK) expression than mature bull testes and epididymides. pERK immunoreactivity was higher in immature epididymides than in immature testes. pERK was localised mostly in spermatogonia, undifferentiated sustentacular (Sertoli) cells, and interstitial (Leydig) cells in immature testes, as well as in some spermatocytes and spermatids in mature testes. In immature epididymides, the body and tail had higher pERK expression than the head, whereas pERK was broadly distributed throughout the stereocilia, basal cells, and connective tissues. pERK distribution in the head of mature epididymides was similar to that in immature epididymides, whereas few connective tissue cells were expressed in the body and tail of mature epididymides. Collectively, these results suggest that ERK is expressed in the testis and epididymis of immature and mature bulls with varying intensities, and the role of ERK in male reproductive organs may include the specific function of its development.

Significance of EZH2 expression in canine mammary tumors.

BACKGROUND: Current studies report that aberrations in epigenetic regulators or chromatin modifications are related to tumor development and maintenance. EZH2 (Enhancer of zeste homolog 2) is one of the catalytic subunits of Polycomb repressive complex 2, a crucial epigenetic regulator. EZH2 has a master regulatory function in such processes as cell proliferation, stem cell differentiation, and early embryogenesis. In humans, EZH2 is linked to oncogenic function in several carcinomas, including breast cancer, and dysregulation of EZH2 has been particularly associated with loss of differentiation and the development of poorly differentiated breast cancer. In our present study, we were interested in determining whether EZH2 is increased in canine mammary tumors, which show similarities to human breast cancer. RESULTS: Investigation of the expression of EZH2 in canine mammary tumors revealed that EZH2 protein was overexpressed in canine mammary carcinomas, as in human breast cancer. In addition, the immunohistochemical expression level of EZH2 was associated with the degree of malignancy in canine mammary carcinoma. This is the first report to describe EZH2 expression in canine mammary tumors. CONCLUSIONS: Because the expression of EZH2 was similar in canine mammary carcinoma and human breast cancer, spontaneous canine mammary tumors may be a suitable model for studying EZH2 and treatment development.

Carcinogenicity of metamifop, a novel herbicide, in Wistar rats following oral administration for 104 weeks.

Metamifop is a novel herbicide with as yet undetermined properties. To assess its carcinogenicity, metamifop was mixed into standard rodent chow and fed to male and female Wistar rats at doses of 10, 100 and 750ppm for 104weeks. The viability/mortality of these rats was not affected by treatment with metamifop. Treatment had no significant effects on clinical parameters, and food consumption. Males and females fed 750ppm of metamifop for 104weeks showed decreases in body weight and body weight gain. Histopathological examination revealed that treatment with metamifop reduced non-neoplastic findings (chronic progressive nephropathy, tubular basophilia, tubular casts, glomerulosclerosis, basophilic and clear cell foci, senile atrophy, and mesothelial hyperplasia) and reduced neoplastic findings (thymoma, pituitary adenoma, and mammary fibroadenoma and adenocarcinoma in females, and mesenteric lymph node hemangioma in males) compared with control groups. Benign granulosa cell tumors were increased in a dose-dependent manner. As metamifop did not show any genotoxic potential, and there was no correlation between ovarian cancer and increased gonadal hormone levels in humans, the granulosa cell tumors observed in female rats fed a high dose of metamifop were considered not relevant to humans.

Lectin histochemistry of the olfactory mucosa of Korean native cattle, Bos taurus coreanae.

BACKGROUND: The olfactory mucosa (OM) is crucial for odorant perception in the main olfactory system. The terminal carbohydrates of glycoconjugates influence chemoreception in the olfactory epithelium (OE). OBJECTIVES: The histological characteristics and glycoconjugate composition of the OM of Korean native cattle (Hanwoo, Bos taurus coreae) were examined to characterize their morphology and possible functions during postnatal development. METHODS: The OM of neonate and adult Korean native cattle was evaluated using histological, immunohistochemical, and lectin histochemical methods. RESULTS: Histologically, the OM in both neonates and adults consists of the olfactory epithelium and the lamina propria. Additionally, using periodic acid Schiff and Alcian blue (pH 2.5), the mucus specificity of the Bowman's gland duct and acini in the lamina propria was determined. Immunohistochemistry demonstrated that mature and immature olfactory sensory neurons of OEs express the olfactory marker protein and growth associated protein-43, respectively. Lectin histochemistry indicated that numerous glycoconjugates, including as N-acetylglucosamine, mannose, galactose, N-acetylgalactosamine, complex type N-glycan, and fucose groups, were expressed at varied levels in the different cell types in the OMs of neonates and adults at varying levels. According to our observations, the cattle possessed a well-developed olfactory system, and the expression patterns of glycoconjugates in neonatal and adult OMs varied considerably. CONCLUSIONS: This is the first study to describe the morphological assessment of the OM of Korean native cattle with a focus on lectin histochemistry. The findings suggest that glycoconjugates may play a role in olfactory chemoreception, and that their labeling properties may be closely related to OM development and maturity.

Time-course changes in the expression levels of miR-122, -155, and -21 as markers of liver cell damage, inflammation, and regeneration in acetaminophen-induced liver injury in rats.

Drug-induced liver injury (DILI) is a significant threat to patient health and a major concern during drug development. Recently, multiple circulating microRNAs (miRNAs) have been reported to be potential biomarkers for DILI. To adapt and validate miRNAs for clinical use, we investigated the time-course changes in miR-122 expression levels in an acetaminophen-induced liver injury model in rats. In addition, miR-155 and miR-21 were evaluated as makers of inflammation and regeneration, respectively, to characterize liver status. Our results revealed that miR-122 is an early and sensitive biomarker of hepatocellular injury at a stage when alanine transaminase, aspartate transaminase, and total bilirubin were not detectable. However, no significant differences in the expression levels of other miRNAs (miR-155 and -21) were observed between treatment and vehicle groups. Collectively, these time-course changes in the expression levels of miRNAs may be useful as markers for clinical decision-making, in the diagnosis and treatment of DILI.

Metformin inhibits early stage diethylnitrosamine­induced hepatocarcinogenesis in rats.

Antitumor effects of metformin have recently emerged despite its original use for type II diabetes. In the present study, the effects of metformin on the development and recurrence of hepatocellular carcinoma (HCC) were investigated using the diethylnitrosamine (DEN)­induced rat model of HCC. Tumor foci were characterized by gross examination and by histopathological characteristics, including proliferation, hepatic progenitor cell content and the expression of hepatocarcinoma­specific molecular markers. Potential target molecules of metformin were investigated to determine the molecular mechanism underlying the inhibitory effects of metformin on chemically induced liver tumorigenesis. The antitumor effects of metformin were increased by the reduction of surface nodules and decreased the incidence of altered hepatocellular foci, hepatocellular adenoma and carcinoma. Also, decreased expression levels of glutathione S­transferase placental form, proliferating cell nuclear antigen and cytokeratin 8 described the inhibitory effects of metformin on HCC. In the present study, Wistar rats receiving treatment with DEN were administered metformin for 16 weeks. In addition, metformin suppressed liver tumorigenesis via an AMPK­dependent pathway. These results suggested that metformin has promising effects on the early stage of HCC in rats. Therefore, metformin may be used for the prevention of HCC recurrence following primary chemotherapy for HCC and/or for high­risk patients, including chronic hepatitis and cirrhosis.

Evaluation of Nonalcoholic Fatty Liver Disease in C57BL/6J Mice by Using MRI and Histopathologic Analyses.

Nonalcoholic fatty liver disease (NAFLD) can lead to cirrhosis, hepatocellular carcinoma, and ultimately death. Magnetic resonance techniques are accurate, noninvasive methods for evaluating hepatic steatosis but, in animals, have not been fully validated against histologic findings. We sought to validate the MRI fat-signal fraction (MRI-FSF) used for diagnosing NAFLD in human nonclinical trials by comparing MRI data with histopathologic findings in C57BL/6J mice (n = 24) fed normal chow (controls) or a methionine- and choline-deficient (MCD) diet to induce NAFLD. Axial T2-weighted fast spin-echo images were used to examine the entire liver. For histopathologic analyses, liver slides were evaluated for hepatic steatosis according to the NAFLD activity score. Pearson correlation coefficient and receiver operating characteristics analyses were performed. According to the fat-fraction signal, the mean percentage of liver fat in mice with induced NAFLD was 57%, which correlated with the histologically determined steatosis grade. The proton-density fat fraction effectively distinguished severe from mild hepatic steatosis, with an AUC of 0.92. Evaluation accuracy decreased when lobular inflammation and hepatocellular ballooning were considered. This study showed strong concurrence between MRI-FSF and histopathologic steatosis in a murine model of NAFLD. MRI-FSF had moderate sensitivity and specificity in this context. These results confirm that the MRI is a useful biomarker of hepatic steatosis in NAFLD in murine model.

Acute treatment with methotrexate induces hippocampal dysfunction in a mouse model of breast cancer.

Methotrexate (MTX) is a well-known cytostatic agent used in adjuvant chemotherapy for breast cancer, that has neurological side effects, including depression and cognitive impairment. We investigated the neurotoxic effects of MTX on the hippocampus and hippocampus-dependent behaviors in breast cancer cell line (FM3A)-inoculated tumor-bearing mice. In addition, we evaluated the changes in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the hippocampus of tumor-bearing mice after treatment with MTX. Depressive-like behavior test (tail-suspension test, TST) and learning and memory tasks (passive avoidance) were administered 24h after MTX (40 mg/kg, i.p.) injection. MTX-treated tumor-bearing mice showed significant depressive-like behaviors and cognitive impairment. Treatment with MTX significantly decreased the number of doublecortin (a marker for immature progenitor neurons)-positive cells in the hippocampal dentate gyrus of tumor-free and tumor-bearing mice. Moreover, treatment with MTX significantly upregulated proinflammatory enzymes, including iNOS and COX-2, in tumor-bearing mice. These findings indicate that the acute neurotoxic effect of MTX leads to hippocampal dysfunction including depressive-like behaviors and memory deficits, which may be related to an inhibition of neurogenesis and an increase of the inflammatory response in the hippocampus of a mouse model of breast cancer.