Detection of the toxicity of a candidate compound at an early stage of drug development is an emerging area of interest. It is difficult to determine all of the effects of metabolism of a compound using traditional approaches such as histopathology and serum biochemistry. The goal of a metabolomics approach is to determine all metabolites in a living system, with the potential to detect and identify biomarkers involved in toxicity onset. Here, we summarize the metabolic fingerprints for detection and identification of metabolic changes and biomarkers related to drug-induced toxicity using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS).
To elucidate the effect of a large dose of di (2-ethylhexyl) phthalate (DEHP), a plasticizer and peroxisome proliferator-activated receptor-α (PPARα) agonist, on hepatic peroxisomes, we orally administered 1,000 mg/kg/day, once daily, to 3 male and 4 female cynomolgus monkeys for 28 days consecutively. Light-microscopic and electron microscopic examinations of the liver were carried out in conjunction with measurement of the hepatic fatty acid β-oxidation system (FAOS), carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (CPT) activities, which are peroxisomal and/or mitochondrial enzyme activities. Electron microscopically, enlargement of the mitochondria was observed with lamellar orientation of the cristae along the major axis. Although the number of peroxisomes showed a tendency to increase when compared with those in a biopsied specimen before treatment, no abnormality in morphology was observed. A slight increase in CPT activity was noted at termination. No changes were noted in hepatic FAOS or CAT activity. In conclusion, although repeated oral treatment of cynomolgus monkeys with a large dose of DEHP induced a subtle increase in the numbers of peroxisomes with slight enlargements of the mitochondria, this low-sensitivity response to peroxisome proliferators in cynomolgus monkeys was considered to be closer to the response in humans than that in rodents.
We report here the interesting case of a 5-year-old male cynomolgus monkey with goblet cell hyperplasia and thickening of the muscular layer throughout the small intestine without exhibiting any clinical symptoms. Necropsy examination showed diffuse thickening of the intestinal wall from the jejunum to the ileum, with an appearance likened to a rubber tube. Histopathologically, marked thickening was observed in both the mucosal and muscular layers in the jejunum and ileum, and slight thickening was observed in the duodenum. Goblet cell hyperplasia with extension of the circular folds and villi was prominently observed. The mucosal surface was covered with a thick mucus layer containing desquamated mucosal epithelial cells, and both the inner and outer muscular layers were markedly thickened due to smooth muscle hypertrophy. Neither macroscopic nor histopathological examination identified any causative factors, such as infection, enteritis and intestinal stenosis, or obstruction that may have caused development of this lesion. Given these observations, this case may simply be considered of spontaneous goblet cell hyperplasia and muscular layer thickening in the small intestine of a cynomolgus monkey.
A young male Crl:CD (SD) rat with erythroid leukemia that presented with emaciation, abdominal distension and a pale visible mucosal membrane was euthanized at 7 weeks of age. At necropsy, enlargement of liver, spleen and pancreatic lymph node was noted. Analysis of blood smear samples revealed many mono- or binucleated erythroblasts that had PAS-positive vacuoles in the cytoplasm. Histopathologically, neoplastic proliferation of atypical cells was observed in the hepatic sinusoids, splenic red pulp, bone marrow, pancreatic lymph node, kidney and lung. Neoplastic cells showed a round to spindle shape, and some neoplastic cells had deeply stained small nuclei and small cytoplasms and resembled erythroblasts. Immunohistochemically, many neoplastic cells were positive for hemoglobin. To our knowledge, this is the first report of erythroid leukemia in a rat of this age. The observed features were similar to those of pure erythroid leukemia in humans.
A 10-year-old female miniature poodle had a mass in its carpal joint of the left forelimb. The tumor was divided into small multiple lobules by delicate connective tissues, and necroses were found in some of the central lobules. In some connective stromal areas, chondroid and osteoid tissues were formed. The tumor cells were similar to the structure of apocrine gland epithelial cells with apical blebs resembling apocrine secretion and eosinophilic secretary materials within the luminal space, and spindle cells were sometimes found in the basal area of the glandular structure. In some areas, tumor cells invaded in the blood vessels, bone and bone marrow. Immunohistochemically, the tumor cells forming tubulo-acinar to solid structures were intensely positive for cytokeratin and keratin K8/K18, and the spindle cells were positive for vimentin and alpha-smooth muscle actin. This case was diagnosed as a malignant mixed apocrine gland tumor with metastases to the bone and bone marrow.
A male ferret, which was purchased from abroad at 9 months of age, had shown significant weight loss starting at 13 months of age. The ferret subsequently showed decreasing motor activity and recumbency and was euthanized at 14 months of age. At necropsy, a white, quail egg-sized mass was found in the mesentery. Histopathologically, multifocal granulomas consisting of necrotic foci, macrophages, fibroblasts and plentiful fibrous connective tissues were observed in the mesenteric mass. Surrounding the granulomas, inflammatory cell infiltration consisting of neutrophils, lymphocytes and plasmacytes was observed diffusely and significantly. Immunohistochemistry revealed small numbers of macrophages around necrotic foci that were positively stained for anti-mouse feline coronavirus. Electron microscopically, the cytoplasm of the macrophages contained viral particles, which were identified as coronavirus. The histopathological features in this ferret were similar to those in cats with feline infectious peritonitis (FIP). This was the first case in ferrets in Japan.
Spontaneous malignant mesothelioma was found in a 104-week-old male Crj:CD(SD) rat. The tumor was scattered on the surface of the lung, heart, mediastinal pleura and thoracic wall and metastasized to the alveolar septa. Histopathologically, small flattened or cuboidal tumor cells proliferated with stroma, formed almost normal papillary structures and reacted positively to colloidal iron stain and immunohistochemical staining for mesothelin. Round hyalinous stromata were pronounced, which is a characteristic feature, and the possible reason for this is as follows; at first, a small amount of collagen fibers was formed in the center of the clusters of several tumor cells, and then the cell clusters expanded like balloons with an increase in the collagen fibers.
A guinea pig (9-week-old) that had been placed in a control group for a pharmacological test was found to have a single nodule on the surface of the right ventricular wall. In a transverse section of the heart after fixation, a whitish mass was found that extended from the subendocardium to the subepicardium of the right ventricular wall. Histopathological examination revealed a spongy network consisting of vacuolated spaces in the myocardium of the right ventricle extending to the myocardium and subepicardium of the right atrium. The vacuolated space was PAS-positive. Immunohistochemical examinations revealed that the lesions contained striated fibers that were positive for anti-desmin and anti-myoglobin. Electron micrographs revealed the lesions resulting in affected striated muscle fibers and accumulations of many glycogen granules. Based on the findings, the lesions were diagnosed as a cardiac rhabdomyoma. This is the first report of application of immunohistochemical examinations to diagnosis of cardiac rhabdomyoma in the guinea pig.
N-nitrosobis(2-hydroxypropyl)amine (BHP) is a well-known carcinogen and induces tumors in various tissues. In the present paper, a case of olfactory neuroblastoma (ONB) induced in a rat by BHP is described. The tumor was observed in one out of 25 male rats that received 2000 ppm of BHP in drinking water from 6 to 18 weeks of age and were sacrificed at 26 weeks of age. Histologically, the tumor arose in the posterior nasal cavity and consisted of small round cells and elongate cells with scant basophilic cytoplasm. The neoplastic cells proliferated with compartmentalization into the lobules by fibrovascular septa. True rosettes, pseudorosettes and an intercellular fibrillar matrix were occasionally observed. Immunohistochemically, the tumor cells were positive for NF120/200 and β III-tubulin. These results indicate that the present tumor is the first case of ONB induced in a rat by BHP treatment.