Metronidazole enhances steatosis-related early-stage hepatocarcinogenesis in high fat diet-fed rats through DNA double-strand breaks and modulation of autophagy.

Nonalcoholic fatty liver disease is a hepatic disorder with deposition of fat droplets and has a high risk of progression to steatosis-related hepatitis and irreversible hepatic cancer. Metronidazole (MNZ) is an antiprotozoal and antimicrobial agent widely used to treat patients infected with anaerobic bacteria and intestinal parasites; however, MNZ has also been shown to induce liver tumors in rodents. To investigate the effects of MNZ on steatosis-related early-stage hepatocarcinogenesis, male rats treated with N-nitrosodiethylamine following 2/3 hepatectomy at week 3 were received a control basal diet, high fat diet (HFD), or HFD containing 0.5% MNZ. The HFD induced obesity and steatosis in the liver, accompanied by altered expression of Pparg and Fasn, genes related to lipid metabolism. MNZ increased nuclear translocation of lipid metabolism-related transcription factor peroxisome proliferator-activated receptor gamma in hepatocytes, together with altered liver expression of lipid metabolism genes (Srebf1, Srebf2, Pnpla2). Furthermore, MNZ significantly increased the number of preneoplastic liver foci, accompanied by DNA double-strand breaks and late-stage autophagy inhibition, as reflected by increased levels of γ-H2AX, LC3, and p62. Therefore, MNZ could induce steatosis-related hepatocarcinogenesis by inducing DNA double-strand breaks and modulating autophagy in HFD-fed rats.

Differential impacts of mineralocorticoid receptor antagonist potassium canrenoate on liver and renal changes in high fat diet-mediated early hepatocarcinogenesis model rats.

Mineralocorticoid receptor (MR)/NADPH oxidase (NOX) signaling is involved in the development of obesity, insulin resistance, and renal diseases; however, the role of this signaling on steatotic preneoplastic liver lesions is not fully elucidated. We determined the effects of the MR antagonist potassium canrenoate (PC) on MR/NOX signaling in hepatic steatosis and preneoplastic glutathione S-transferase placental form (GST-P)-positive liver foci. Rats were subjected to a two-stage hepatocarcinogenesis model and fed with basal diet or high fat diet (HFD) that was co-administered with PC alone or in combination with the antioxidant alpha-glycosyl isoquercitrin (AGIQ). PC reduced obesity and renal changes (basophilic tubules that expressed MR and p22phox) but did not affect blood glucose tolerance and non-alcoholic fatty liver disease activity score (NAS) in HFD-fed rats. However, the drug increased the area of GST-P-positive liver foci that expressed MR and p22phox as well as increased expression of NOX genes (p22phox, Poldip2, and NOX4). PC in combination with AGIQ had the potential of inhibiting the effects of PC on the area of GST-P-positive liver foci and the effects were associated with increasing expression of an anti-oxidative enzyme (Catalase). The results suggested that MR/NOX signaling might be involved in development of preneoplastic liver foci and renal basophilic changes in HFD-fed rats; however, the impacts of PC were different in each organ.

A case of rapid recurrence of apocrine ductal carcinoma originating from the oral scent gland of a Richardson's ground squirrel (Urocitellus richardsonii).

A 3-year-old female Richardson's ground squirrel developed a subcutaneous mass at the left oral angle. Seven days after removal of the mass, the mass recurred and metastasized to the cervical lymph node. Histologically, the primary mass was subdivided by fibrous trabeculae into various-sized neoplastic cell lobules showing a solid growth pattern with frequent mitoses and sometimes forming intracytoplasmic lumina. Large to medium-sized lobules formed a central cyst plugged by comedo necrosis. Neoplastic cells showed infiltrative subcutaneous growth. In the recurrent tumor, tubular structures lacking apparent apocrine secretion appeared within the solid growth portion. Neutrophil infiltration was evident within the tubules and intracytoplasmic lumina. Neoplastic cells were diffusely immunopositive for AE1/AE3 pan-cytokeratin (CK) in all lobules and focally positive for CAM5.2 CK in the lobules forming a central cyst and/or tubular structures, but they entirely lacked positivity for the periodic acid Schiff reaction. Ki-67-positive proliferating neoplastic cells were higher in numbers with the recurrent tumor than with the primary tumor. In addition, phosphorylated c-MYC immunoreactivity was observed in neoplastic cell nuclei, distinctly at the portion of invasive growth. Thus, the present case was diagnosed as apocrine ductal carcinoma originating from the oral scent gland, which typically shows highly aggressive biological behavior.

Spironolactone in Combination with α-glycosyl Isoquercitrin Prevents Steatosis-related Early Hepatocarcinogenesis in Rats through the Observed NADPH Oxidase Modulation.

Administration of the diuretic, spironolactone (SR), can inhibit chronic liver diseases. We determined the effects of SR alone or in combination with the antioxidant α-glycosyl isoquercitrin (AGIQ) on hyperlipidemia- and steatosis-related precancerous lesions in high-fat diet (HFD)-fed rats subjected to a two-stage hepatocarcinogenesis model. Rats were fed with control basal diet or HFD, which was administered with SR alone or in combination with an antioxidant AGIQ in drinking water. An HFD increased body weight, intra-abdominal fat (adipose) tissue weight, and plasma lipids, which were reduced by coadministration of SR and AGIQ. SR and AGIQ coadministration also reduced hepatic steatosis and preneoplastic glutathione S-transferase placental form-positive foci, in association with decrease in NADPH oxidase (NOX) subunit p22phox-positive cells and an increase in active-caspase-3-positive cells in the foci. Hepatic gene expression analysis revealed that the coadministration of SR and AGIQ altered mRNA levels of lipogenic enzymes ( Scd1 and Fasn), antioxidant-related enzymes ( Catalase), NOX component ( P67phox), and anti-inflammatory transcriptional factor ( Pparg). Our results indicated that SR in combination with AGIQ had the potential of suppressing hyperlipidemia- and steatosis-related early hepatocarcinogenesis through the reduced expression of NOX subunits.

Developmental Exposure to Aluminum Chloride Irreversibly Affects Postnatal Hippocampal Neurogenesis Involving Multiple Functions in Mice.

Aluminum (Al) is neurotoxic to adults and also to infants. In this study, we investigated the developmental exposure effect of AlCl3 on postnatal hippocampal neurogenesis. Pregnant mice were administered 0-, 900-, or 1800-ppm AlCl3 via drinking water from gestational day 6 to postnatal day (PND) 21, with their offspring examined on PND 21 and PND 77. On PND 21, GFAP-immunoreactive (+) neural stem cells (NSCs) and p21Cip1/Waf1+ cells were decreased in number in the subgranular zone at 900 and ≥900 ppm, respectively. Pcna transcript level examined at 1800 ppm was decreased in the dentate gyrus. These results suggest induction of compromised cell quiescence that caused impaired self-renewal capacity of NSCs accompanying slowing down of cell cycling, which ultimately resulted in exhaustion of the NSC pool. At 1800 ppm, Reelin+ hilar GABAergic interneurons were also decreased, suggesting a contribution to the NSC reduction. At this dose, TBR2+ or DCX+ progenitor and immature granule cells and PVALB+ interneurons were increased. Moreover, COX-2+ granule cells were increased at ≥900 ppm. These results suggest facilitation of transient progenitor cell proliferation and differentiation during exposure. Moreover, TUNEL+ or Morin-stained granule cells were increased, together with Casp12 transcript upregulation, suggesting induction of Al accumulation-related endoplasmic reticulum stress-mediated granule cell apoptosis. Transcript expression changes on cholinergic and glutamatergic signals and synaptic plasticity suggested contribution to disruptive neurogenesis. The NSC-targeting effects sustained through the adult stage despite no sustained Al-accumulation. These results suggest that developmental AlCl3-exposure irreversibly affects postnatal hippocampal neurogenesis involving multiple functions in mice.

Fluorescence tumor imaging by i.v. administered indocyanine green in a mouse model of colitis-associated colon cancer.

Fluorescence tumor imaging using exogenous fluorescent tumor-targeting agents has potential to improve early tumor detection. The fluorescent contrast agent indocyanine green (ICG) is used in medical diagnostics. The aim of the present study is to investigate the tumor imaging capability and the imaging mechanism of i.v. administered ICG in a mouse model of colitis-associated colon cancer. To do this, an azoxymethane/dextran sodium sulfate-induced colon cancer mouse model was used. Ex vivo imaging experiments were carried out 1 hour after i.v. injection of ICG. The ICG fluorescence was observed in the colon tumor tissues, with sufficient tumor to normal tissue ratio, correlating with tumor malignancy. In the tumor tissues, ICG fluorescence was localized in the vascular interstitial tissue. Immunofluorescence microscopy revealed that tumor cells formed tight junctions normally, suggesting an inability of tumor cellular uptake of ICG. In contrast, tumor tissues increased the CD31-immunoreactive endothelial cell area, and accumulated stromal cells immunoreactive for COX-2 and tumor cell population immunoreactive for inducible nitric oxide synthase. In vivo vascular permeability assay revealed that prostaglandin E2 promoted the endothelial cell permeability of ICG. In conclusion, our data indicated that fluorescence contrast-enhanced imaging following i.v. administered ICG can be applied to the detection of colon tumors in a mouse colitis-associated colon cancer model. The tumor tissue preference of ICG in the present model can be attributed to the enhanced vascular leakage of ICG involving inflammatory mediators, such as COX-2 and inducible nitric oxide synthase, in conjunction with increased tumor vascularity.

Choroid plexus carcinoma with neuronal and glial differentiation in a 7-week-old male Sprague-Dawley rat.

We describe a case of choroid plexus carcinoma arising in the cerebrum of a 7-week-old male Sprague-Dawley rat. The tumor mass occupied the right lateral ventricle of the cerebrum. Histological analyses revealed that the epithelial tumor cells had proliferated in tubular, cribriform, papillary and solid growth patterns in the vicinity of the choroid plexus, with slight invasion into the cerebrum parenchyma. We divided the tumor cells into cuboidal, elongated and intermediate cells. Immunohistochemical studies showed that these tumor cells expressed relatively high levels of cytokeratin AE1/AE3, vimentin and glial fibrillary acidic proteins, and low levels of nestin, oligodendrocyte transcription factor and doublecortin proteins. The present case was diagnosed as a choroid plexus carcinoma with neuronal and glial differentiation.

Intermediate-grade mammary gland adenocarcinoma in an 18-year-old female black leopard (Panthera pardus) with acute pancreatic necrosis and chronic interstitial nephropathy.

An 18-year-old female black leopard (Panthera pardus) showed renal failure, leukocytosis and presence of subcutaneous masses in the lower abdominal region and right shoulder; she eventually died. Histopathological observations included a mammary gland carcinoma with comedo, solid and tubulopapillary patterns in subcutaneous tissue, and highly proliferated tumor cells in systemic organs. The tumor cells were positive for cytokeratin AE1/AE3. The mammary gland tumor was diagnosed as intermediate-grade adenocarcinoma, based on a previously reported histological grading system of feline mammary carcinomas. Chronic interstitial nephritis was estimated to have been ongoing for 5 years, whilst acute necrotic pancreatitis in relation to tumor metastasis could have been the cause of death.

Fluorescence contrast-enhanced proliferative lesion imaging by enema administration of indocyanine green in a rat model of colon carcinogenesis.

The fluorescent contrast agent indocyanine green (ICG) is approved by the Food and Drug Administration for clinical applications. We previously reported that cultured human colon tumor cells preferentially take up ICG by endocytic activity in association with disruption of their tight junctions. The present study explored ICG availability in fluorescence imaging of the colon to identify proliferative lesions during colonoscopy. The cellular uptake of ICG in cultured rat colon tumor cells was examined using live-cell imaging. Colon lesions in rats administered an ICG-containing enema were further assessed in rats with azoxymethane-induced colon carcinogenesis, using in vivo endoscopy, ex vivo microscopy, and immunofluorescence microscopy. The uptake of ICG by the cultured cells was temperature-dependent. The intracellular retention of the dye in the membrane trafficking system suggested endocytosis as the uptake mechanism. ICG administered via enema accumulated in colon proliferative lesions ranging from tiny aberrant crypt foci to adenomas and localized in proliferating cells. Fluorescence endoscopy detected these ICG-positive colonic proliferative lesions in vivo. The immunoreactivity of the tight-junction molecule occludin was altered in the proliferative lesions, suggesting the disruption of the integrity of tight junctions. These results suggest that fluorescence contrast-enhanced imaging following the administration of an ICG-containing enema can enhance the detection of mucosal proliferative lesions of the colon during colonoscopy. The tissue preference of ICG in the rat model evaluated in this study can be attributed to the disruption of tight junctions, which in turn promotes endocytosis by proliferative cells and the cellular uptake of ICG.

Endometrial adenocarcinoma with choriocarcinomatous differentiation in the uterus of a goat.

An 11-year-old female goat had invasive and metastatic endometrial adenocarcinoma in the uterus. There was a notable proliferation of endometrial epithelial cells in a tubular growth pattern, with a desmoplastic response. The endometrial epithelial tumor cells metastasized to the kidney, liver and lung. In contrast to the primary and metastatic tumor cells, pleomorphic tumor cells with a choriocarcinoma-like growth pattern infiltrated the mesometrium. Cell proliferation activity was high in both types of tumor cells. Both types of tumor cells expressed cytokeratins AE1/AE3, 7 and CAM5.2; choriocarcinomatous cells also had positive immunoreactions to human chorionic gonadotropin, human placental alkaline phosphatase and α-inhibin. The present case was diagnosed as endometrial adenocarcinoma with choriocarcinomatous differentiation.

Late effect of developmental exposure to glycidol on hippocampal neurogenesis in mice: Loss of parvalbumin-expressing interneurons.

Developmental exposure to glycidol of rats causes axonal injury targeting axon terminals in dams and transient disruption of late-stage differentiation of hippocampal neurogenesis, accompanying sustained increase in the number of reelin-producing or calretinin-expressing interneurons in offspring. The molecular mechanism of disruptive neurogenesis probably targets the newly generating nerve terminals. We previously found differences between mice and rats in the effects on hippocampal neurogenesis after developmental exposure to the same neurotoxic substances. In the present study, we examined the effects and underlying mechanisms of developmental exposure to glycidol on hippocampal neurogenesis in mice. Glycidol (800 or 1600ppm) was administered in drinking water to mated female mice from gestational day 6 to postnatal day 21. Compared to mice drinking water without glycidol (control), the exposed dams showed axon terminal injury at both concentrations of glycidol. The offspring of the dams that had received 1600ppm glycidol had fewer parvalbumin (PVALB)+ γ-aminobutyric acid (GABA)-ergic interneurons and neuron-specific nuclear protein+ postmitotic neurons in the hilus of the hippocampal dentate gyrus. Thus, exposure of glycidol to adult mice induced axonal degeneration equivalent to that seen in the rat; however, the target mechanism for the disruption of hippocampal neurogenesis by developmental exposure was different from that in rats, with the hilar neuronal population not affected until adulthood. Considering the role of PVALB+ GABAergic interneurons in the brain, developmental glycidol exposure in mice may cause a decline in cognitive function in later life, and involve a different mechanism from that targeting axon terminals in rats.

Pulmonary neuroendocrine tumor in a female wolf (Canis lupus lupus).

A 17-year-old female wolf (Canis lupus lupus) had a right lung mass that was adhered to the thoracic cavity. Histopathological examination revealed that the mass consisted of sheets, cord or ribbon-like structures of monotonous, small, cuboidal cells with round, oval or short-spindle nuclei and scant clear cytoplasm, demarcated by a fine fibrovascular stroma. Focal necrosis, congestion and thrombi were observed. Immunohistochemically, the tumor cells diffusely expressed cytokeratin AE1/AE3, and some expressed chromogranin A, neural cell adhesion molecule (CD56) and thyroid transcription factor-1. The number of proliferating cell nuclear antigen-positive tumor cells was low. A diagnosis of pulmonary neuroendocrine tumor was based on the resemblance to carcinoids.

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats.

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.

Canine mammary minute oncocytomas with neuroendocrine differentiation associated with multifocal acinar cell oncocytic metaplasia.

Two solitary and minute tumors of 1 and 1.5 mm diameter were identified by microscopy in the left fourth mammary gland of a 13-year-old female Labrador Retriever dog, in addition to multiple mammary gland tumors. The former tumors were well circumscribed and were composed of small-to-large polyhedral neoplastic oncocytes with finely granular eosinophilic cytoplasm, and were arranged in solid nests separated by fine fibrovascular septa. Scattered lumina of variable sizes containing eosinophilic secretory material were evident. Cellular atypia was minimal, and no mitotic figures were visible. One tumor had several oncocytic cellular foci revealing cellular transition, with perivascular pseudorosettes consisting of columnar epithelial cells surrounding the fine vasculature. Scattered foci of mammary acinar cell hyperplasia showing oncocytic metaplasia were also observed. Immunohistochemically, the cytoplasm of neoplastic cells of the 2 microtumors showed diffuse immunoreactivity to anti-cytokeratin antibody AE1/AE3, and finely granular immunoreactivity for 60-kDa heat shock protein, mitochondrial membrane ATP synthase complex V beta subunit, and chromogranin A. One tumor also had oncocytic cellular foci forming perivascular pseudorosettes showing cellular membrane immunoreactivity for neural cell adhesion molecule. The tumors were negative for smooth muscle actin, neuron-specific enolase, vimentin, desmin, S100, and synaptophysin. Ultrastructural observation confirmed the abundant mitochondria in the cytoplasm of both neoplastic and hyperplastic cells, the former cells also having neuroendocrine granule-like electron-dense bodies. From these results, our case was diagnosed with mammary oncocytomas accompanied by neuroendocrine differentiation. Scattered foci of mammary oncocytosis might be related to the multicentric occurrence of these oncocytomas.