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.

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.

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.

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.

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.

Iron deposition at mineralization fronts and osteoid formation following chronic cadmium exposure in ovariectomized rats.

To investigate whether chronic exposure of cadmium (Cd) chloride induces osteomalacic lesions similar to Itai-itai disease (IID), ovariectomized rats were injected intravenously with the cadmium at doses of 0.05 and 0.5 mg/kg/day, 5 days per week, for 50 weeks. In six rats in the 0.5 mg/kg group, the administration was continued for up to 70 weeks. In the 0.5 mg/kg group, the plasma concentration of calcium was similar in the treatment and control groups throughout the treatment period. The urinary excretion of calcium increased from 20 weeks and the increase became marked from 40 weeks. Histopathologically, osteoid seams in the femur, tibia, and humerus were increased from 50 weeks, and these changes became prominent at 70 weeks. Hypertrophy and hyperplasia of chief cells in the parathyroid were also observed from 50 weeks. The osteoid morphometry of the trabecular bone of the femur and sternum revealed a dose-dependent increase in osteoid/bone volumes. Roentgenographs of the antebrachial and metacarpal bones taken at 70 weeks showed so-called paper bone. The bone Cd content markedly increased until 25 weeks, but thereafter decreased linearly for up to 70 weeks. In contrast to the Cd content, the iron content decreased until 25 weeks, but thereafter increased until 70 weeks. Undecalcified section of the humerus showed the deposition of iron and formation of osteoid at mineralization fronts. Our data suggest that osteomalacic lesions were caused by chronic Cd intoxication, and that iron, as well as Cd, was involved in osteoid formation.

Measurement of replicative DNA synthesis (RDS) by a 5-bromo-2'-deoxyuridine (BrdU) labeling technique for detection of hepatocyte proliferation.

The hypothesis has been proposed that cell proliferation, or replicative DNA synthesis (RDS) in S-phase cells, is a nongenotoxic (Ames-negative) mechanism involved in tumorigenesis, providing a very useful conceptual basis for carcinogen testing. In this present study, hepatocyte RDS experiments were conducted using 5-bromo-2'-deoxyuridine (BrdU) labeling in combination with histopathological observation, comparing our results with earlier findings for in situ [3H]thymidine (TdR) labeling. The present BrdU data proved to be consistent with the previous TdR data in all but one case. Hepatocyte RDS induction was observed for some chemicals without hepatotoxicity. BrdU labeling in combination with histopathological observation is therefore a reliable approach to assessment of test compound effects in vivo.

Cadmium-induced dental lesions in ovariectomized rats.

The effects of cadmium chloride on both incisor and molar teeth of ovariectomized female rats were studied histopathologically. The rats were injected intravenously with the compound at doses of 1.0 and 2.0 mg/kg, 5 days/wk. Six rats per group were sacrificed at 4, 8, and 13 wk. Discoloration of the incisors was observed in the rats of the 2.0-mg/kg group from 8 wk. Histopathologic examination of the incisor demonstrated decreased iron-containing pigment in ameloblasts and destruction of the enamel organ. These changes were accompanied with accumulation of cadmium and loss of iron in the teeth. Necrosis of the dental pulp occurred from the coronal end of both the incisor and molar teeth extended to the apical, deep portion of the teeth. The dental pulp of the molar teeth, which is shorter than that of the incisor, was mildly affected by cadmium intoxication. These findings suggested that intradental ischemia due to cadmium toxicity may have contributed to the development of the pulpal necrosis.

Chronic cadmium exposure-induced renal anemia in ovariectomized rats.

Cadmium (Cd) chloride was intravenously injected at doses of 0.05 and 0.5 mg/kg/day in ovariectomized rats for 50 weeks, and the chronic Cd exposure-induced nephrotoxicity and anemia were investigated. The rats treated with 0.05 mg/kg Cd showed no apparent hematological, urinary, and histopathological abnormalities. In the 0.5-mg/kg group, renal tubular disorders became marked at 16 weeks, and cortical fibrosis with glomerular dysfunction appeared at 50 weeks. Anemia occurred at 12 weeks in the 0.5-mg/kg group and became increasingly marked with time. The mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were decreased at 12 and 25 weeks; however, the decreases of MCV and MCH disappeared at 50 weeks. A slight decrease in mean corpuscular hemoglobin concentration was noted at 50 weeks. The blood chemistry from the same group revealed a decrease in plasma iron levels and an increase in total iron binding capacity throughout the administration period. The erythropoietin (EPO) level was increased as the hemoglobin level decreased at 12 weeks, whereas the EPO level was not elevated even when the hemoglobin level was decreased at 50 weeks. These findings showed that renal anemia also occurred in addition to the iron deficiency anemia at 50 weeks.

Spontaneous lesions detected in the common cotton-eared marmosets (Callithrix jacchus).

Spontaneous lesions in the common cotton-eared marmosets (Callithrix jacchus), which were maintained in our laboratory or were purchased from a commercial breeder, were examined histopathologically. There were 39 males and 22 females, between the ages of 7 and 100 months. The most interesting finding noted was extramedullary hematopoiesis, which was observed in the mesenteric lymph nodes, spleen, liver, kidney, adrenal gland, and cerebral choroid plexus. Megakaryocytes were frequently observed in the blood vessels of the alveolar wall of the lung. It is important to distinguish extramedullary hematopoiesis which occurs accidentally from that induced by repeated blood sampling or that resulting from administration of chemicals. Extramedullary hematopoiesis was easily distinguishable from inflammation or hematopoietic neoplasms, because of the various cellular elements present, including megakaryocytes. In the weak and dead animals, enterocolitis was found at a high frequency. Other common findings included thymic involution, prominent vacuolation of liver sinusoidal cells, which were probably Ito-cells, and basophilic changes in the renal tubular epithelium. The liver and the kidney are frequently affected by toxic effects of chemicals. Therefore, it is important to distinguish the toxic changes from the spontaneous ones. Proliferation of apocrine glands in the cervical skin was an interesting finding; however, neither inflammatory change nor cellular atypia was noted in this structure. Although its function is unclear, this structure may be a marmoset-specific tissue.

Cadmium-induced osteomalacic and osteopetrotic lesions in ovariectomized rats.

The effects of long-term administration of cadmium (Cd) chloride on the bone were studied using ovariectomized rats. The rats were injected iv with the compound at doses of 1.0 and 2.0 mg/kg, 5 days a week, for 13 weeks. The serum concentrations of calcium and inorganic phosphorus were significantly increased from 8 weeks in the 2.0 mg/kg group. The bone Cd content was gradually increased for 13 weeks in a dose-dependent manner. Calcium and phosphorus contents in the bone, and serum levels of parathyroid hormone and osteocalcin, were not significantly different between Cd-treated and control rats. Histopathologically, chronic Cd nephropathy such as tubular atrophy and interstitial fibrosis was observed with clinical polyuria and increased enzymuria. The skeletal changes were detected mainly in the femur and tibia. In the metaphysis of Cd-treated rats, cancellous bone mass increased with time. This change was detected as an increased opacity by a roentgenogram. In the cortical bone of the midshaft haversian canals were dilated with clearly bordered osteoid seams and showed a motheaten pattern in rats in the 2.0 mg/kg group at 13 weeks. In the present study, we report Cd nephropathy and osteomalacic changes in ovariectomized rats with iv injection of CdCl2 for 13 weeks. Although an involvement of the indirect action of Cd through renal failure could not be ruled out in this experiment, our biochemical and pathological data suggested that osteomalacia was induced by a direct action of Cd on the bone through abnormal calcium homeostasis.

Acute toxicity of cadmium in rats with or without cadmium pretreatment.

The acute toxicity of cadmium (Cd) in male rats was examined with or without Cd pretreatment. Firstly, the metallothionein (MT) contents in the liver and kidney after Cd exposure (2.0 mg/kg, i.v.) were determined. The MT contents in the liver increased immediately to a peak (36.0 +/- 5.5 n mol/g wet tissue) 2 days after Cd exposure and were 55-fold higher than that at 0 day (0.64 +/- 0.25 n mol/g wet tissue). On the other hand, the MT contents in the kidney increased slightly but steadily for 14 days after Cd exposure. In the study for comparison of Cd-induced toxicity, the LD50 value of the Cd-pretreatment group (Group II) was approximately 2-fold higher than that of the non-pretreatment group (Group I). In microscopic findings, differences between rats in Group I and Group II were recognized in the kidney. Cytoplasmic vacuolation of the proximal tubular epithelium in the kidney was observed in Group I, while degeneration or coagulative necrosis in the proximal tubular epithelium was observed in some rats in Group II in addition to the cytoplasmic vacuolation. Because the toxic changes other than in the kidney in Group II were almost equal to or less than that in Group I, in spite of the doubled dosage of Cd, the toxic effects of Cd, except on the kidneys, were considered to be reduced by the pretreatment with Cd.

Granule cell type cerebellar hypoplasia in a beagle dog.

Cerebellar hypoplasia characterized by severe depletion of granule cells and almost intact Purkinje cells was found in a male 19-month-old beagle dog used in a toxicity study. Microscopically, there was a narrow space lacking granule cells between the row of Purkinje cells and the medulla. Gliosis was not seen in any portion of the cerebellum including this space. No significant changes were seen in the Purkinje cells except for occasional cytoplasmic vacuolation. In the molecular layer and medulla, no histopathological abnormalities were observed.

Ovariectomy enhances cadmium-induced nephrotoxicity and hepatotoxicity in rats.

The toxicity of cadmium (Cd) chloride was studied in ovariectomized (OX) female rats and non-OX female rats after intravenous administration of the compound at doses of 2.0 and 3.0 mg/kg for 14 days. Mild hypochromic microcytic anemia developed in all rats treated with Cd, but growth retardation in the OX rats was more prominent than that in the non-OX rats. There was an increase of AST and ALT and a decrease of total cholesterol and the A/G ratio in both OX and non-OX rats treated with Cd. The hepatic and renal Cd concentrations increased in a dose-dependent manner, and the concentrations in both organs on Day 14 were comparable in the 3.0 mg/kg OX group (liver, 270.0 +/- 39.6 micrograms/g; kidney, 121.3 +/- 10.1 micrograms/g) and non-OX group (liver, 277.0 +/- 29.9 micrograms/g; kidney, 100.8 +/- 1.3 micrograms/g). Hepatocyte necrosis developed only in OX rats treated with Cd, and the nephrotoxicity of Cd was also notably enhanced by ovariectomy, since Cd nephropathy affected the proximal convoluted epithelium more severely and more frequently in OX rats than in non-OX rats. BrdU-labeled cells in the renal cortex were increased by approximately 2.7-fold in OX rat (7.4 cells/mm2) over those in the renal cortex in non-OX rat (2.7 cells/mm2). In conclusion, the present study demonstrated that ovariectomy enhanced Cd-induced nephrotoxicity and hepatotoxicity in rats.