Chronic cadmium treatment induces tubular nephropathy and osteomalacic osteopenia in ovariectomized cynomolgus monkeys.

In an attempt to establish a primate model of chronic cadmium toxicosis, we ovariectomized cynomolgus monkeys and treated them with CdCl2 by repeated intravenous injections for 13 to 15 months. The animals showed normocytic-normochromic anemia. The cadmium treatment resulted in increases of urinary enzyme activity indicative of renal tubular degeneration. Histopathology of the kidney revealed renal proximal tubular atrophy accompanied by interstitial fibrosis. Decreased bone mineral density was evident in the trabecular and cortical zones of the lumbar vertebra and femur, with osteoid accumulation around the trabeculae and Haversian canals. Iron deposition at the mineralization front and osteoclasts hyperplasia were indicative of impairment of bone mineralization and an increase of resorption. Blood inorganic phosphorus and 1α,25(OH)2 vitamin D3 levels decreased and urinary deoxypyridinoline level increased in cadmium-treated animals. The renal and bone lesions closely resemble those of itai-itai disease patients, the most severe case of cadmium toxicosis in terms of clinical chemistry and histopathology. Thus, ovariectomized monkeys chronically exposed to cadmium can serve as a primate itai-itai disease model, which is beneficial for developing novel therapeutic methods, investigating the mechanisms of the renal and bone lesions, and establishing more clearly defined criteria for diagnosing the disease.

Thyroid follicular adenoma with accumulation of collagen type IV in a common marmoset (Callithrix jacchus).

A thyroid tumour was identified in a 10-year-old male common marmoset (Callithrix jacchus). The tumour was encapsulated by fibrous connective tissue and compressed the adjacent normal thyroid. The tumour was composed of variably sized and irregularly shaped thyroid follicles lined by a single layer of columnar epithelial cells. Eosinophilic material at the base of the neoplastic cells stained black with periodic acid-methenamine silver and red with periodic acid-Schiff. Immunohistochemistry confirmed that this eosinophilic material was collagen type IV. Ultrastructurally, highly dense and amorphous material was observed at the base of the neoplastic cells. Small vesicles in the basolateral cytoplasm of the neoplastic cells contained similar material to that at the base of the cells. The tumour was diagnosed as a thyroid follicular adenoma with accumulation of collagen type IV. This is the first description of an endocrine tumour with accumulation of collagen type IV in animals.

Hereditary cerebellar degenerative disease (cerebellar cortical abiotrophy) in rabbits.

A pair of rabbits gave birth to a set of littermates (F1) with symptoms of early-onset ataxia. Microscopic examination revealed cerebellar degenerative disease in 5 of 6 littermates. Light microscopy was used to compare the thickness of each cerebellar layer in affected animals in contrast to a normal control. Affected animals showed narrowing of the molecular layer of the vermis, reduced density of Purkinje cell dendrites and irregular thickness in their branchlets, and reduced density of granular cells and scattered pyknotic cells in the granular layer. Pyknotic cells were apoptotic granular cells, confirmed by positive staining using the TUNEL method. Electron microscopy confirmed the thinning of the molecular layer seen by light microscopy and also showed a reduced number of parallel fibers, which indicate granular cells axons, and a reduced number of synaptic junctions between Purkinje and granular cells. Purkinje cells had electron-dense, irregularly shaped cytoplasm with irregularly shaped nuclei, and some of these cells had a central chromatolysis-like region. These findings support a diagnosis of cerebellar cortical abiotrophy, a hereditary condition that causes nerve function impairment leading to early-onset progressive degeneration of the cerebellar cortex.

Malignant amelanotic melanoma behind the left eye in a female Crj:CD(SD)IGS rat: a case report.

A tumor behind the left eye in a female Crj:CD(SD)IGS rat was investigated histopathologically, immunohistopathologically, and electron microscopically. The tumor invaded and destroyed orbital tissues and bones. It consisted of various tumor cells; namely, spindle-shaped, epithelioid, anaplastic melanoma cells, and had prominent eosinophilic cytoplasm and nuclei with a greater variation in size. Immunohistochemically, almost all of the tumor cells were positive for antimelanoma, PNL2 antibody. Ultrastructurally, the tumor cells were rich in small vesicles containing fine granules and filamentous structures. This is the first report describing an amelanotic melanoma in the head of an albino rat.

Ovarian mucinous cystadenoma with smooth muscle proliferation in a cynomolgus monkey (Macaca fascicularis).

An ovarian mucinous cystadenoma was found in a 5-year-old female cynomolgus monkey (Macaca fascicularis). The tumor was composed of various sizes of multilocular cystic glands lined by a single layer of mucin-filled epithelium. Each of these cystic glands was surrounded by a large amount of solid fibrous stroma resembling smooth muscle. The ovarian surface epithelium showed partial invagination into the ovarian cortex, and a transition was observed between the surface epithelium and the mucinous cyst-forming epithelium. Immunohistochemically, the stromal cells were positive for alpha-smooth muscle actin and proliferating cell nuclear antigen. Ultrastructurally, the glandular epithelium had numerous mucinous secretory granules and microvilli. The stromal cells had numerous parallel microfibrils with focal density. It is rare to encounter evidence of a transition from the surface epithelium to the mucinous tumor epithelium and to show stromal smooth muscle proliferation in a mucinous cystadenoma.

A novel immunohistochemical marker of normal and neoplastic melanocytes in formalin-fixed, paraffin-embedded tissues of albino rats.

In albino rats, spontaneous occurrence of melanocytic tumors is rare, with diagnosis difficult. This study evaluated immunoreactivity for PNL2 in normal and neoplastic melanocytes in formalin-fixed and paraffin-embedded tissues of albino rats. The samples consisted of 11 (1.57%) amelanotic melanomas in 700 rats (2 studies), 23 non-melanocytic tumors, and a wide variety of normal tissues. In normal albino rats, PNL2 stained the melanocytes in the iris and choroid of the eyeball and the hair bulb and basal cell layers of the epidermis of the whole body. In amelanotic melanoma, the tumor cells consisted of spindle cells with eosinophilic cytoplasm without melanin granules. PNL2 consistently stained cytoplasm in all amelanotic melanoma cells. In contrast, the nonmelanocytic tumor cells were not labeled. Electron microscopically, neoplastic, and normal melanocytes showed numerous cytoplasmic premelanosomes (stage II melanosome). In conclusion, PNL2 is direct against a fixative- and decalcific-resistant melanocyte-associated antigen, and has high specificity against normal and neoplastic melanocytes of albino rats.

Eosinophilic substance is "not amyloid" in the mouse nasal septum.

An eosinophilic substance (ES) is usually observed in the mouse nasal septum and increases in volume with aging. It has been described as amyloid in textbooks and one report. However, it has been described as "not amyloid" in other reports because there was a negative reaction to Congo red. In this study, the ES was investigated histopathologically and electron microscopically to determine whether it was amyloid or not. The ES was only observed at the interstitium of clear HE-stained nasal glands in the septum, in which 2 kinds of glands were present (dark and clear stained by HE). The volume of the ES was small in young mice and large in older ones. Neither nasal gland degeneration nor inflammation resulted, even if a large amount of the ES was observed. The ES reacted negatively to Congo red but was strongly positive to periodic acid-Schiff reaction with prior diastase treatment. In the electron microscope observation, the ES consisted of amorphous material and collagen, but no nonbranching fibrils. Similar amorphous material was also observed in the nasal gland epithelial cells and was connected to the material in the rough endoplasmic reticulum. The above-mentioned findings indicated that the ES was not amyloid and suggested the ES might consist of not only collagen but also complex carbohydrate, which was produced by the nasal gland epithelial cells.

Neurotoxicity induced by a single oral dose of aniline in rats.

The neurotoxicity of aniline and its age-dependent responses were investigated in male rats. Groups of 6 rats, 4-week-old, were treated once with aniline (500, 750 or 1,000 mg/kg) or olive oil by gavage. Additional groups of 6 rats, 7- or 10-week-old, were treated once with 800 mg/kg of aniline or olive oil. Paralytic gait or hindlimb paralysis was observed between post-treatment days 8 and 15 in two out of six rats receiving 1,000 mg/kg of aniline at 4 weeks of age. On post-treatment day 15, spongy change in the white matter of the spinal cord was observed in all rats receiving 750 or 1,000 mg/kg of aniline at 4 weeks of age. The lateral and ventral columns of the thoracic spinal cord were the most severely affected. Spongy change in the facial nerve and spinal trigeminal tracts of pons and medulla oblongata, and mild degeneration of the peripheral nerves was found in 3 out of 6 rats receiving 1,000 mg/kg of aniline. At the ultrastructural level, the spongy change was due to distention of the myelin sheath and splitting of the intraperiod line. Axons were well preserved in the affected nerve fibers. No abnormalities were seen in the neuronal cell bodies. Although transient cyanosis was observed in all rats receiving 800 mg/kg of aniline at 7- or 10-week-old, as well as in rats receiving 750 or 1,000 mg/kg of aniline at 4-week-old, no treatment-related neurobehavioral or morphologic abnormalities were found in the former. These findings demonstrate the neurotoxicity of orally administered aniline for rats, depending upon the age of the animal at the time of administration.

Intravenous 1alpha, 25[OH]2 vitamin D3 (calcitriol) pulse therapy for bone lesions in a murine model of chronic cadmium toxicosis.

The aim of the present study was to clarify the therapeutic effects of 1alpha, 25[OH]2 vitamin D3 (calcitriol) pulse injection on bone lesions induced in a rat model of chronic cadmium toxicosis. Ovariectomized (OVX) and control-operated (sham-OVX) rats were given repeated intravenous injections of 0.5 mg/kg/day CdCl2 for 70 weeks. The rats were then treated intravenously with 0.02 microg/kg/day calcitriol 3 days per week for 8 weeks. CdCl2 treatment induced increases in osteoid volumes of the femur cortex and trabecula. This change was accompanied by an increase in the volume of iron deposition at the mineralization front of the trabeculae and a reduction in mineral density. Abnormalities of bone metabolic parameters, which were increases in the blood calcium, inorganic phosphorous, bone-specific alkaline phosphatase, parathyroid hormone (PTH) and osteocalcin levels, and in the urine deoxypyridinoline (D-PYR) level, were also induced. Calcitriol treatment increased the blood calcium and inorganic phosphorous levels, and reduced the blood PTH level. Decreases in blood tartrate-resistant acid phosphatase and urine d-PYR levels were also induced indicating that bone resorption was suppressed. The findings indicated that the increased osteoid volume of the cortex and Fe-deposition volume of the trabecula were improved. These effects or improvements were observed in the sham-OVX rats but not in the OVX rats.

Interaction of L-tryptophan with alpha-cyclodextrin: studies with calorimetry and proton nuclear magnetic resonance spectroscopy.

The interaction of L-tryptophan with alpha-cyclodextrin was investigated in a 0.1 M phosphate buffer at pH 7.4 with a LKB 2277 microcalorimeter, using flow mixed mode at 25 degrees C. The thermodynamic parameters for inclusion complex formation obtained are as follows; DeltaG(0) = - 7.03 kJ/mol (K = 17.0), DeltaH(0) = - 9.50 kJ/mol, DeltaS(0) = - 8.3 J/mol K. The driving force for inclusion complex formation was considered to be mainly van der Waals-London dispersion force, and the contribution of hydrogen bonding was secondary in importance. Also, from the measurements of the proton nuclear magnetic resonance spectra and the model building with Corey-Pauling-Koltum atomic models, the probable structures of the complex, together with conformational change of L-tryptophan by complexation, were determined.

Gross and microscopic anatomy of the extraorbital lacrimal gland of the common marmoset (Callithrix jacchus).

PURPOSE: The lacrimal gland is often selected for microscopic examination in toxicologic studies. However, this gland is difficult to find within the orbit in marmosets at necropsy. Therefore, we examined the extraorbital lacrimal glands in marmosets. METHODS: The formalin-fixed craniums of four marmosets were used in a topographic study to confirm location of the lacrimal gland, and the results were applied to a routine toxicologic study in marmosets. RESULTS: The extraorbital lacrimal gland was located on the temporal surface of the zygomaticofrontal process and was covered with the temporalis muscle. The gland was easily detached from the surrounding tissue, and its histologic features were the same as those of the intraorbital lacrimal gland. CONCLUSIONS: The extraorbital lacrimal glands have been reported in some New World monkeys, but to the authors' knowledge, this is the first report in marmosets. Identification and characterization of this gland will be useful for toxicologic studies in marmosets.

Case report: spontaneous hemangiosarcoma in the pancreas of a Fischer rat.

Spontaneous hemangioma or hemangiosarcoma is sometimes found in the viscera and soft tissue of rats and mice. However, there is no report of the tumor occurring in the pancreas of rats. We report a pancreatic hemangiosarcoma in a 109-wk-old, male Fischer 344 rat, which was used in the control group of a carcinogenicity study. The tumor destroyed and compressed the normal pancreatic tissue and displayed a high density in terms of the numerous capillaries and strands of endothelial tumor cells. A reticulin stain revealed a dense network formation. The frequency of proliferating cell nuclear antigen-positive staining showed active proliferation of the tumor cells. Immunohistochemically, some of the tumor cells stained positive with factor VIII-related antigen, and ultrastructurally, Weibel-Palade bodies were rarely observed in the cytoplasm of the tumor cell. From these findings, the tumor was diagnosed as a hemangiosarcoma that occurred naturally in the pancreas.

Anatomical structure and surface epithelial distribution in the nasal cavity of the common cotton-eared marmoset (Callithrix jacchus).

To validate use of the common cotton-eared marmoset (Callithrix jacchus) in inhalation toxicity studies, its nasal morphology was examined. The nasal turbinates each consisted of one maxilloturbinate and one ethmoturbinate: these were more planar in structure than the comparable structures of rodents or dogs. The nasal cavity epithelia comprised squamous epithelium (SE), nasal transitional epithelium (NTE), respiratory epithelium (RE) and olfactory epithelium (OE), listed in order of occurrence from anterior to posterior positions. NTE was distributed as a narrow band lying between SE and RE. OE was limited to the dorsal part of the cavity, which was structurally similar to that of the macaque or man. Overall, this study revealed structural the similarity of the whole nasal cavity in the marmoset to that of macaques or humans. Prediction of nasal cavity changes in man based on extrapolation from experimentally induced changes in the common marmoset therefore seems likely to be feasible, making it a useful animal model for inhalation studies.

Repeat-assessment of 1,4-dioxane in a rat-hepatocyte replicative DNA synthesis (RDS) test: evidence for stimulus of hepatocyte proliferation.

1,4-Dioxane is a nongenotoxic hepatocarcinogen but in our previous replicative DNA synthesis (RDS) studies with the [3H]thymidine (TdR)-technique, it failed to increase hepatocyte RDS values when given by gavage to male F344 rats as a single 2000 mg/kg body weight dose. However, in a current series of trials with TdR, it showed equivocal responses 24 or 48 hr following treatment with 2000 mg/kg in time-course experiments, and positive responses 24 hr following 1000, 1500 and 2000 mg/kg in dose-response experiments. An increased RDS incidence was also observed at the dose of 2000 mg/kg with data for 5-bromo-2'-deoxyuridine (BrdU)-incorporation. These present findings thus support the hypothesis that a capacity to induce cell proliferation may play a key role in 1,4-dioxane hepatocarcinogenesis.

Occurrence of toxicity and cell proliferation after a single gavage administration of chloroform to male F344 rats.

Chloroform, an industrial solvent and one of the most common environmental contaminants which produces carcinogenic effects in the liver and kidney of rodents, is not genotoxic in most traditional bacterial and mammalian test systems. Its carcinogenic potential appears attributable to the sustained cell turnover (regenerative hyperplasia) which results from chronic chloroform toxicity. In this present study, cell proliferation (replicative DNA synthesis, RDS) and histopathological changes in hepatocytes and renal tubular epithelial cells were assessed in male F344 rats following a single gavage chloroform exposure (50, 150 or 500 mg/kg). In addition, biochemical parameters (BUN, GOT, LDH and NAG) were examined using plasma and urine samples. Cell proliferation and histopathological changes (e.g. hypertrophy, necrosis, vacuolation) were only seen at the dose of 500 mg/kg in the liver and kidney. At the same dose, all biochemical markers were increased at the 24 to 48 hr time points. These results obtained are thus in line with earlier findings pointing to epigenetic carcinogenicity.

Testicular yolk sac carcinoma in a calf.

A testicular yolk sac carcinoma (YSCA) was diagnosed in a 28-day-old male Japanese black calf. Macroscopically, the abdominal cavity was filled with reddish gelatinous masses of various sizes. There were no testes in the scrotum or pelvic cavity. Histologically, the masses consisted of the tissues showing a variety of patterns; loose reticular network, pseudopapillary arrangement, festoon, solid nest, and labyrinthine pattern. Tumor cells were round to oval, with single central or polar nuclei with sharply defined nuclear borders and deeply basophilic chromatin. Tumor cells sometimes had glycogen granules, periodic acid-Schiff (PAS)-positive inclusions, or sudanophilic droplets in their cytoplasm. Hyaline matrices were found in extracellular areas and were intensely PAS positive. Immunohistochemically, most of the tumor cells and hyaline matrices had positive reactivity to anti-alpha-fetoprotein and anti-placental-alkaline-phosphatase antibodies. Positive reaction to anti-laminin antibody appeared only in hyaline matrices. This is the first case of a tumor in a domestic animal with histologic and immunohistochemical features analogous to those of human YSCA, endodermal sinus type.

Vascular leiomyoma of the mesentery in a dog.

A 4-year-old male, miniature Schnauzer dog showed two large masses in the mesentery at necropsy. Histological examination of both masses revealed plain smooth muscle tumour cells intermingled with thick-walled blood vessels. The bundles of tumour cells often extended from the periphery of the vessels. Mitotic figures were rare. From these findings, the tumour was diagnosed as a vascular leiomyoma (angiomyoma), previously unreported in animals. The term, vascular leiomyoma, was proposed to describe this tumour in order to avoid confusion with hamartomatous angiomyoma.

Subchronic toxicity of Di(2-ethylhexyl)phthalate in common marmosets: lack of hepatic peroxisome proliferation, testicular atrophy, or pancreatic acinar cell hyperplasia.

To evaluate the toxicological effect, di(2-ethylhexyl)phthalate (DEHP) was administered orally at 100, 500, and 2500 mg/kg to four male and four female marmosets in each group for 13 weeks. Its potentials of hepatic peroxisome proliferation, testicular atrophy, and pancreatic acinar cell hyperplasia were evaluated more closely. Clofibrate, which potently causes peroxisome proliferation in rodents, was administered in like manner at 250 mg/kg as a reference drug. DEHP induced significant suppression of weight gain in males at 2500 mg/kg. However, the increase in liver mass and hypertrophy of hepatocytes were not detected in organ weight measurements or histopathological examination. The number of peroxisomes, volume density, peroxisome morphology, and peroxisomal enzyme activities were not different from those in the control group, though the males treated with 500 and 2500 mg/kg DEHP showed 1.3- and 1.4-fold increases in mean peroxisome volume, respectively. In contrast, clofibrate induced 2.2 (in male)- and 1.9-fold (in female) increases in hepatic cyanide-insensitive acyl CoA oxidation system activity, 1. 2 (in male)- and 1.7-fold (in female) increases in hepatic carnitine-dependent acetyltransferase activity, and 1.8 (in male)- and 3.0-fold (in female) increases of carnitine-dependent palmitoyltransferase activity. Cytochrome P-450 contents tended to increase in all males and females administered 500 and 2500 mg/kg of DEHP and clofibrate associated with the increase in hepatic microsomal protein content, suggesting a relationship with the treatment. The atrophic change in the testis or proliferative change in the pancreatic acinar cells seen in rodents were not seen histopathologically; also, no changes were observed in testes weight, testicular zinc level, blood levels of testosterone and estradiol, pancreas weight, and blood levels of cholecystokinin. Finally, no changes considered to be due to the administration of DEHP were noted in blood chemical examination or pathological examination of other organs.

Gangliocytoma with immature neuronal cell elements in the pituitary of a rat.

A spontaneous pituitary gangliocytoma with abundant, immature neuronal cell elements was found incidentally in a 109-week-old female Fischer 344 rat. The pituitary parenchyma was largely occupied by a tumor nodule with necrotic and hemorrhagic foci and cyst. The tumor was composed of mature ganglion-like (M) cells, small immature ganglion (I) cells and transitional (T) cells, with a fibrillar matrix. The I and T cells were intermingled with the M cells or were arranged in compact clusters, in which the I cells formed perivascular rosette-like structures, sometimes with mitotic figures. Immunohistochemically, all types of tumor cells were positive for neuron-specific enolase, and only the M cells was positive for chromogranin A. This result may be correlated with the degree of cytodifferentiation.

Four-week repeated inhalation study of HCFC 225ca and HCFC 225cb in the common marmoset.

Four male and three female marmosets in each group were exposed to air only, 1000 ppm of HCFC 225ca or 5000 ppm of HCFC 225cb, for 6 h per day for 28 consecutive days. HCFC 225ca caused a slight reduction in body weight. HCFC 225cb occasionally caused somnolence during exposure and vomiting on the first day of exposure. Clinical chemistry findings included a mild reduction of triglyceride, cholesterol and phospholipid levels and increased GOT level in the HCFC 225ca exposure group. HCFC 225cb also caused a reduction of triglyceride levels in some animals. HCFC 225ca caused a slight increase of hepatic carnitine palmitoyltransferase (CPT) activity while HCFC 225cb slightly increased cyanide-insensitive palmitoyl CoA beta-oxidation (FAOS) activity. In the HCFC 225cb exposure group, an increase in cytochrome P-450 content was also observed. HCFC 225ca caused a fatty change in the hepatic cells. Increased incidence of lipid droplets in the hepatic cells and myelin-like bodies in hepatic cells, Kupffer's cells and hepatic blood vessels were observed electron microscopically in the HCFC 225ca exposure group. A proliferation of smooth endoplasmic reticulum was observed in the HCFC 225cb exposure group. Decreased peroxisome volume density in the HCFC 225ca group, and increased volume density in the HCFC 225cb exposed females were seen. However, organ weight measurement and histopathological examination did not reveal hepatomegaly or hypertrophy with either substance. Although slight changes were noticed in peroxisome volume density and in some of the peroxisomal enzyme activities, the changes related to peroxisome proliferation with HCFC 225ca and 225cb were minimal in marmosets compared to those seen in rats. Histopathological examination and hormonal analysis did not reveal any abnormalities in the pancreas or testes.