Mechanisms of Mycotoxin-induced Dermal Toxicity and Tumorigenesis Through Oxidative Stress-related Pathways.

Among the many mycotoxins, T-2 toxin, citrinin (CTN), patulin (PAT), aflatoxin B1 (AFB1) and ochratoxin A (OTA) are known to have the potential to induce dermal toxicity and/or tumorigenesis in rodent models. T-2 toxin, CTN, PAT and OTA induce apoptosis in mouse or rat skin. PAT, AFB1 and OTA have tumor initiating properties, and OTA is also a tumor promoter in mouse skin. This paper reviews the molecular mechanisms of dermal toxicity and tumorigenesis induced in rodent models by these mycotoxins especially from the viewpoint of oxidative stress-mediated pathways.

Malignant sertoli cell tumor in a goose (Anser cygnoides domesticus).

This paper describes the pathologic features of a malignant Sertoli cell tumor found in an adult goose (Anser cygnoides domesticus). At necropsy, in addition to one large tumor mass (15 cm in diameter), multiple small tumor masses were observed over the peritoneum and mesenterium in the coelomic cavity. The large tumor mass was composed of sheets, lobules, and small islands of tumor cells, and elongated tumor cells lying perpendicular to fibrous connective tissue were characteristic. Such histopathologic characteristics were common to all the tumors. The tumor cells were immunohistochemically positive for neuron-specific enolase and S-100, and some tumor cells contained fine intracytoplasmic pigments that stained red by oil red O staining. These findings, taken together with the fact that one testis was markedly atrophied and bore no tumor cells and the other testis was not discernible, the present case was diagnosed as unilateral malignant Sertoli cell tumor arising from the unilateral testis. To our knowledge, this is the first report of Sertoli cell tumor in the goose.

Mechanisms of mycotoxin-induced neurotoxicity through oxidative stress-associated pathways.

Among many mycotoxins, T-2 toxin, macrocyclic trichothecenes, fumonisin B(1) (FB(1)) and ochratochin A (OTA) are known to have the potential to induce neurotoxicity in rodent models. T-2 toxin induces neuronal cell apoptosis in the fetal and adult brain. Macrocyclic trichothecenes bring about neuronal cell apoptosis and inflammation in the olfactory epithelium and olfactory bulb. FB(1) induces neuronal degeneration in the cerebral cortex, concurrent with disruption of de novo ceramide synthesis. OTA causes acute depletion of striatal dopamine and its metabolites, accompanying evidence of neuronal cell apoptosis in the substantia nigra, striatum and hippocampus. This paper reviews the mechanisms of neurotoxicity induced by these mycotoxins especially from the viewpoint of oxidative stress-associated pathways.

Spontaneous cutaneous mast cell tumor with lymph node metastasis in a Richardson's ground squirrel (Spermophilus richardsonii).

A 4-year-old female Richardson's ground squirrel (Spermophilus richardsonii) presented with multicentric nodules arising from the skin of the middle of the tail and lumbosacral regions. Histologically, the nodules were composed of a proliferation of spindloid to pleomorphic cells that sometimes formed sheets and fascicular to storiform patterns. Diffuse infiltration of eosinophils was also noted. The results of immunohistochemistry indicated positive labeling for vimentin, mast cell tryptase, c-kit, and Ki-67. Toluidine blue stain revealed fine, metachromatic, cytoplasmic granules. The histologic diagnosis was mast cell tumor. The neoplasm recurred and metastasized to the right lumbar lymph node 1 month later.

Disseminated histiocytic sarcoma with excessive hemophagocytosis in a cat.

A 10-year-old Japanese domestic cat was presented with anorexia and weight loss. Severe anemia and thrombocytopenia were detected. Abdominal radiography and ultrasonography revealed the presence of multiple masses in the spleen. Cytological analyses of the masses revealed several atypical histiocytic cells and considerable hemophagocytosis. A splenectomy was performed, and the mass was diagnosed as histiocytic sarcoma on the basis of histopathological, cytochemical and immunohistochemical analyses. Further, abnormal hemophagocytosis was observed in the bone marrow. The cat was administered prednisolone and lomustine, and it survived for 107 days after admission. An autopsy revealed the presence of neoplastic histiocytic cells in the bone marrow, liver, pancreatic lymph node and glomeruli. This is the first case of histiocytic sarcoma in a cat to be reported in Japan.

Relation between lipogranuloma formation and fibrosis, and the origin of brown pigments in lipogranuloma of the canine liver.

BACKGROUND: In a previous study we confirmed that canine hepatic lipogranuloma, defined as lesions consisting of small round cells which contain lipid vacuoles and brown pigments in their cytoplasm, was an assembly of Kupffer cells and/or macrophages, and that the cytoplasmic brown pigments in the lesions were hemosiderin and ceroid. However, the pathogenesis of the lesion remains unclear. Kupffer cells (resident macrophages) play a key role in hepatic fibrogenesis due to the production of cytokines including TGF-beta. In the present study, we have examined 52 canine liver samples (age: newborn - 14 years; 25 males and 27 females) and investigated the correlation between lipogranuloma formation and fibrosis as well as the origin of brown pigments of lipogranulomas. RESULTS: Lipogranulomas were detected histopathologically in 23 (44.2%) of the 52 liver samples. No significant correlation was found between the density of lipogranulomas and distribution of collagen type I/III in the liver. Pigmentation of lipogranulomas showed significant correlations with that on both hepatocytes and sinusoidal cells, indicating that pigments of lipogranuloma (hemosiderin and ceroid) might be derived from hepatocytes and Kupffer cells. CONCLUSION: Lipogranulomas are not a contributing factor in hepatic fibrosis, but might be a potential indicator of the accumulation of iron and lipid inside the liver.

Neural progenitor cells are protected against MPTP by MAO-B inhibitors.

Neurotoxic effects of MPTP on the nigrostriatal dopaminergic system are thought to be initiated by 1-methyl-4-phenylpyridinium (MPP+), a metabolite formed by the monoamine oxidase (MAO)-B-mediated oxidation of MPTP. We previously reported that the administration of MPTP induced apoptosis in migrating neuroblasts (neural progenitor cells, NPCs) in adult mice. To determine whether MAO-B is also involved in the neurotoxicity of MPTP to NPCs, this study looked at the effects of MAO B inhibitors, R(-)-deprenyl (deprenyl) and N-(2-aminoethyl)-4-chlorobenzamide (Ro 16-6491), both of which protect the dopaminergic system against MPTP. Few apoptotic cells were found in saline- or MAO-B inhibitor-treated animals but MPTP markedly induced apoptosis in the subventricular zone (SVZ) and rostral migratory stream (RMS) after 1 day. When mice were pretreated with deprenyl or Ro 16-6491, not only nigrostriatal dopamine levels but also NPCs were significantly protected against MPTP. In addition, MPTP-induced apoptosis was found in both juvenile (postnatal 21 days) and older (12 months old) mice, suggesting NPCs to be different from the dopamine system, which has been thought to exhibit age-dependent susceptibility to MPTP.

Neurotoxicity of MPTP to migrating neuroblasts: studies in acute and subacute mouse models of Parkinson's disease.

The acute or subacute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been widely used in C57BL/6 mice to develop models of Parkinson's disease (PD). The loss of dopaminergic neurons is suggested to be mediated by a mechanism of nonapoptotic cell death or by apoptosis. In recent years, the notion that the neurotoxicity of MPTP is restricted to dopaminergic neurons in the substantia nigra (SN) has been challenged. Here, we provide evidence of rapid cell death in the subventricular zone (SVZ) and rostral migratory stream (RMS) in the adult C57BL/6 mouse brain in response to acute or subacute treatment with MPTP. Significant terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) of fragmented DNA was observed at 24 h (or 1 day) after the last injection in the acute model or after the first injection in the subacute model. Ultrastructural analysis confirmed that dying cells displayed an apoptotic morphology. Using a double labeling method, we demonstrated that the phenotype of the cells undergoing apoptosis is that of migrating neuroblasts. This is further supported by evidence of a subsequent loss of migrating neuroblasts. The results raise the possibility that migrating neuroblasts in the SVZ and RMS may be more vulnerable to MPTP than nigrostriatal dopaminergic neurons in the SN, and the death of migrating neuroblasts may be a primary event in the mouse model of PD. Furthermore, our data suggests that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent.

The effect of fasting on hepatic lipid accumulation and transcriptional regulation of lipid metabolism differs between C57BL/6J and BALB/cA mice fed a high-fat diet.

The effects of fasting on hepatic lipid metabolism in mice fed a high-fat diet (HFD) are still unclear. After fasting, the degree of hepatic lipid accumulation differs between HFD-fed C57BL/6J (B6) and BALB/cA (BALB/c) mice. It is not clear whether this difference is due to sensitivity to fasting or HFD. The aim of this study is to elucidate this difference among strains. After nine weeks of HFD feeding, both B6 and BALB/c mice showed moderate hepatic steatosis. However, after a subsequent twenty-hour fast, the hepatic lipid accumulation was markedly decreased in B6 but not in BALB/c mice. Moreover, the mRNA expression of a transcription factor, Srebp1(regulates hepatic lipid metabolism), and its target genes-malic enzyme, acetyl-CoA carboxylase, fatty acid synthase(regulate fatty acid synthesis), and glycerol-3-phosphate acyltransferase(regulates triacylglycerol synthesis)-were more markedly reduced in B6 than BALB/c mice. In conclusion, fasting may modify hepatic lipid accumulation in HFD-fed B6 and BALB/c mice differently. The difference may be partly owing to a marked downregulation of the expression of some lipid-metabolism-related genes in B6 mice. These results suggest that fasting per se has a significant effect on hepatic lipid accumulation in mouse strains. SREBP1 might play a role in this fasting effect.

Hepatic lesions of a standard poodle dog with intrahepatic portosystemic shunt.

A 1-year-and-3-month-old, male standard poodle dog with intrahepatic portosystemic shunt (PSS) was autopsied. Nineteen regions of the liver were prepared for detailed examination, and the distribution of hepatic lesions caused by PSS was studied in the liver of this dog. Histopathologically, the liver revealed a variety of hepatic lesions including lipogranulomas in the hepatic parenchyma, and a ductular reaction and microvascular proliferation in portal areas. The distribution of the lesions was not significantly different among liver regions. It is concluded that, in the present case, hepatic lesions caused by PSS are independent of shunt location, and are distributed equally in the liver.

Histopathological characteristics of hepatic lipogranulomas with portosystemic shunt in dogs.

In the canine liver with portosystemic shunts (PSS), focal lesions consisting of cells with cytoplasmic brown pigments and lipid vacuoles are often observed in the hepatic parenchyma. Termed lipogranulomas, their histopathological characteristics have been little studied. In the present study, we examined liver biopsy samples from 144 dogs (age: 3 months-16 years; 65 PSS and 79 non-PSS cases), and investigated the histopathological characteristics, incidence, and density of lipogranulomas. Lipogranulomas were detected histopathologically in 55.4% of PSS dogs. The lesions were then grouped into 3 types according to the amount of cytoplasmic lipid vacuoles and brown pigments. The pigments were positive for Berlin blue, PAS, and Sudan black B, but negative with the Hall method. The majority of the cells were immunohistochemically positive for macrophage scavenger receptor, class A (MSR-A), while no cells were positive for hepatocyte-antigen and albumin. The cytoplasmic pigments were recognized as electron-dense microgranular materials by electron microscopy. The incidence of lipogranulomas was significantly higher in the PSS group than non-PSS group when dogs less than 1 year old were excluded. The lipogranuloma density in the liver was significantly higher in the PSS group. It is concluded that lipogranulomas are frequently observed in liver biopsies of canine PSS especially in dogs more than 1 year old. The lesions consisted of Kupffer cells and/or macrophages, and the cytoplasmic brown pigments are ceroid and hemosiderin. The pathogenesis of lipogranuloma in PSS needs to be clarified.

Intracranial meningioma with polygonal granular cell appearance in a Chihuahua.

A menigioma with polygonal granular cell proliferation in an 11-year and 8-month-old male Chihuahua is described. The tumor was observed under the dura matter of the right cerebrum. Microscopically, the tumor consisted of solid growth foci of small- or large- sized polygonal cells, with pale-stained nuclei, prominent nucleoli, and fine granular to foamy eosinophilic cytoplasm. Some of the proliferating cells contained variable amounts of cytoplasmic PAS-positive granules. Immunohistochemical analysis revealed that neoplastic cells were positive for vimentin and S-100 protein. Ultrastructurally, the neoplastic cells contained vesicular structures with a few small round-shaped bodies in the cytoplasm. We diagnosed the case as canine meningioma with granular cell appearance.

Hypereosinophilic syndrome in two cats.

Two cats showing chronic vomiting, diarrhea and weight loss were found to have leukocytosis with marked eosinophilia. Both cats were diagnosed with hypereosinophilic syndrome by the findings of increased eosinophils and their precursors in the bone marrow, eosinophilic infiltration into multiple organs, and exclusion of other causes for eosinophilia. Although cytoreductive chemotherapy with hydroxycarbamide and prednisolone was performed, these two cats died 48 days and 91 days after the initial presentation.

Proliferative changes in the adrenal medulla of aged Chinese native pigs.

Four aged retired Chinese native pigs, three females and one male, estimated as over 10-year-old, were subjected to autopsy because of infertility due to aging. Grossly, nodular lesions were found bilaterally in the adrenal medulla of all four pigs. Based on the gross and the histopathological findings, they were diagnosed as either medullary nodular hyperplasia or pheochromocytoma. Immunohistochemically, proliferating cells of all these lesions were immuno-positive for chromogranin-A, indicating adrenal medulla-derived. Ultrastructurally, cytoplasmic neurosecretory granules suggestive of secretion were observed in these proliferating cells. There have been only limited numbers of reports on adrenal medullar proliferative changes including pheochromocytoma in pigs. The present cases will provide a valuable information for the characterization of similar changes in animals and human.

Involvement of sex, strain and age factors in high fat diet-induced obesity in C57BL/6J and BALB/cA mice.

Although a number of obesity animal models have been reported, each model possesses different characteristics of obesity, suggesting care should be taken in choosing an animal model suitable for the experimental purpose. In this report, we fed 4-(young) and 52-week-old (middle-aged) C57BL/6J (B6) and young BALB/cA (BALB/c) mice with a high fat diet (HFD) for 9 weeks, and investigated the clinical and histological characteristics of obesity. In BALB/c mice, males gained more body weight and body fat weight and had higher energy intake than females by HFD feeding. Comparing the effect of HFD feeding between the strains of mice, BALB/c male mice accumulated more hepatic lipid than B6 male mice. In addition, middle-aged B6 mice increased the ratio of fat to body weight and hepatic lipid accumulation more than young mice. In conclusion, the characteristics of obesity induced by HFD feeding were influenced by the sex, strain and age of mice. Sex steroid hormones, hepatic lipid metabolism and systemic metabolism might be involved in these factors. The basic data in this study will be useful for the development of animal models of high fat diet-induced obesity.

Vitamin K deficiency of germfree mice caused by feeding standard purified diet sterilized by gamma-irradiation.

Germfree mice died when they were fed a purified diet of AIN-76 formula sterilized by gamma-irradiation. Vitamin K deficiency was suspected and this study was performed to confirm the cause of the death. Germfree mice were fed purified diets of AIN-76 or AIN-93M formula, which were pelleted and sterilized by gamma-irradiation at a dose of 50 kGy. One half of the mice fed the AIN-76 diet died within two weeks and the surviving animals were also in poor health, while 91% of mice fed the AIN-93M diet survived. No hemorrhage was observed grossly in any organs of the surviving animals. Histologically, degeneration with inflammatory cell infiltration was observed as well as hemorrhage and fibrosis in the heart muscles of mice fed the AIN-76 diet. No microscopic lesions were observed in the other organs. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were extremely prolonged when mice were fed the AIN-76 diet. The animals totally recovered when they were intragastrically administered 1 microg/day of vitamin K(3) from the third day of feeding of the AIN-76 diet, except for PT and APTT which were still slightly longer than in mice fed the AIN-93M diet. The concentration of vitamin K(3) supplied in the AIN-76 diet decreased to an undetectable level after gamma-irradiation, while the AIN-93M diet contained 240 microg/kg of vitamin K(1). These results indicate that the deaths of the germfree mice fed the gamma-irradiated AIN-76 diet were caused by vitamin K deficiency. Vitamin K deficiency may cause fatal degeneration of cardiac muscle cells.

Expression of epidermal growth factor receptor protein in the liver of db/db mice after partial hepatectomy.

Liver regeneration was impaired after partial hepatectomy (PH) in leptin receptor-deficient db/db mice with severe liver steatosis. In the present study, we analyzed the mode of epidermal growth factor receptor (EGFR) protein expression in the liver of 5- and 10-week-old db/db and age-matched control mice. In 5-week-old db/db mice, neither the expression of EGFR protein in the intact liver nor the rate of liver regeneration after PH was significantly different from that in age-matched control mice. However, in 10-week-old db/db mice, the level of EGFR protein expression was very low and liver regeneration was prominently suppressed. Histopathologically, much severer fatty change was observed in the liver of 10-week-old db/db mice than 5-week-old db/db mice. These results suggest that the down-regulation of EGFR protein expression is associated with an impairment of liver regeneration in db/db mice and that the severity of hepatic steatosis plays an indirect role in the impairment of liver regeneration by modifying EGFR expression.

Histopathological characteristics of Kupffer cells and ito cells in the porcine and bovine liver.

We previously reported that no Kupffer cells reacted with the antibody against lysozyme, and Ito cells contained a large cytoplasmic vacuole in the feline liver. In this report, we further examined the characteristics of porcine and bovine hepatic non-parenchymal cells. In the liver of both animals, Kupffer cells were positive for lysozyme, and cytoplasmic vacuoles in Ito cells were small. The histopathological characteristics of porcine and bovine hepatic non-parenchymal cells were different from those of the feline liver.

Measurement of intestinal mucosal permeability in dogs with lymphocytic-plasmacytic enteritis.

Lymphocytic-plasmacytic enteritis (LPE) is a type of canine inflammatory bowel disease (IBD). One of its most probable causes is a defect in the mucosal permeability barrier. In the present study, intestinal permeability in LPE dogs was examinated to evaluate its clinical value. Twenty-nine dogs with LPE diagnosed by clinical and histological examinations were included in this study. Intestinal permeability was evaluated by measuring the ratio of the concentrations of two sugars (lactulose (L) and rhamnose (R)) with different molecular weights in urine samples after oral administration of a solution containing them. Biopsy specimens of duodenum were evaluated according to histological criteria. The urinary L:R ratio in the 29 LPE dogs (1.68 +/- 1.17, mean +/- SD) was significantly higher than that in the 10 healthy control dogs (0.75 +/- 0.38, P<0.01). In the LPE dogs, a weak correlation was observed between the histopathological grading score of the duodenum and the urinary L:R ratio (r=0.408, P<0.05). The urinary L:R ratio in the 20 dogs showing hypoalbuminemia (< 2.5 g/dl) was significantly higher than that in the 9 dogs with normal serum albumin levels > 2.5 g/dl (P<0.01). In conclusion, permeability of the intestinal mucosa as determined by the urinary L:R ratio could be a useful laboratory parameter for evaluating intestinal damage in LPE dogs.

Experimental induction of necrotic enteritis in chickens by a netB-positive Japanese isolate of Clostridium perfringens.

Necrotic enteritis (NE) is one of the most important bacterial diseases in terms of economic losses. Clostridium perfringens necrotic enteritis toxin B, NetB, was recently proposed as a new key virulent factor for the development of NE. The goal of this work was to develop a necrotic enteritis model in chickens by using a Japanese isolate of C. perfringens. The Japanese isolate has been found to contain netB gene, which had the same nucleotide and deduced amino acid sequences as those of prototype gene characterized in Australian strain EHE-NE18, and also expressed in vitro a 33-kDa protein identified as NetB toxin by nano-scale liquid chromatographic tandem mass spectrometry. In the challenge experiment, broiler chickens fed a commercial chicken starter diet for 14 days post-hatch were changed to a high protein feed mixed 50:50 with fishmeal for 6 days. At day 21 of age, feed was withheld for 24 hr, and each chicken was orally challenged twice daily with 2 ml each of C. perfringens culture (109 to 1010 CFU) on 5 consecutive days. The gross necrotic lesions were observed in 90 and 12.5% of challenged and control chickens, respectively. To our knowledge, this is the first study that demonstrated that a netB-positive Japanese isolate of C. perfringens is able to induce the clinical signs and lesions characteristic of NE in the experimental model, which may be useful for evaluating the pathogenicity of field isolates, the efficacy of a vaccine or a specific drug against NE.