Takaki Seki

GC‐MS‐based metabolomics reveals mechanism of action for hydrazine induced hepatotoxicity in rats

Gas chromatography–mass spectrometry (GC‐MS) has great advantages for analyzing organic/amino acids, which are often targets in efficacy and/or toxicity studies. Although GC‐MS has been used for the detection of many metabolic disorders, applications of GC‐MS‐based metabolomics in pharmacology/toxicology are relatively underdeveloped. We intended to investigate applicability of a GC‐MS‐based metabolomics approach for toxicological evaluation, and tried to elucidate the mechanism of hydrazine‐induced hepatotoxicity. Rats were administered hydrazine chloride orally (120 and 240 mg kg−1), and urine, plasma and liver samples were collected at 24 or 48 h post‐dosing. Conventional clinical chemistry and liver histopathology were performed, urine and plasma were analyzed by GC‐MS, and metabolic profiles were assessed using chemometric techniques. Principal component analysis score plots showed clear separation of the groups, indicating dose‐dependent toxicity and recovery. The mechanism of toxicity was investigated based on semi‐quantification data of identified metabolites. Amino acid precursors of glutathione (cystein, glutamate and glycine) and a product of glutathione metabolism (5‐oxoproline) were elevated dose‐dependently, accompanied with elevation of ascorbate levels. In addition, intermediates of the TCA cycle were decreased, whereas participants of the urea cycle and other amino acids were increased. These alterations were associated with histopathological changes such as fatty degeneration and glycogen accumulation. Application of GC‐MS‐based metabolomics revealed that oxidative stress and GSH consumption play important roles in the etiology of hydrazine‐induced hepatotoxicity, demonstrating that this approach is a useful tool in pharmacology and toxicology for screening, elucidating mode of action and biomarker discovery. Copyright

Influences of biofluid sample collection and handling procedures on GC-MS based metabolomic studies

Sample collection procedures of pharmacology and toxicology studies might have a great impact on interpretation of metabolomic study results. Characterization of range variation among sample collection methods is necessary to prevent misinterpretation, as is use of optimal methods in animal experiments to minimize biological/technical variation. Here, we investigated the influence of urine and plasma sample collection and handling procedures on GC-MS based metabolomic studies as follows: for urine, pooling period and tube conditions during collection; for plasma, sampling sites, anesthesia and anticoagulants. Metabolic profiles of urine varied dramatically depending on urine pooling period and tube conditions, underscoring the importance of determining appropriate sampling periods in consideration of diurnal effects and targets of effect/toxicity, and suggesting it would be preferable to keep tubes in metabolic cages under iced conditions for urine sampling. Metabolic profiles of plasma differed depending on blood sampling sites. Anesthesia was not effective in reducing individual variation, although the anesthesia was beneficial in reducing discomfort in rats. In GC-MS based metabolomic studies, we recommend that EDTA be used as anticoagulant in plasma sample preparation, because peaks derived from heparin might overlap with endogenous metabolites, which may induce inter-sample variation. The present study demonstrated that biofluid sample collection and handling procedures provide great impact on metabolic profiles, at the very least for minimizing biological/technical variation, sampling period for urine collection should not be set as a short period, and the use of EDTA is recommended as anticoagulant in preparing plasma for analysis by GC-MS.

Comparison of potential risks of lactic acidosis induction by biguanides in rats

Lactic acidosis has been considered to be a side effect of some biguanides, after phenformin was withdrawn from the market because of its association with lactic acidosis. The potential of lactic acidosis induced by biguanides at human therapeutic exposure levels, however, has not been examined. Then, we compared the risk of lactic acid at doses providing exposure levels comparable to human therapeutic doses. Metformin and phenformin were orally administered to rats for up to 28 days, and plasma drug concentrations and blood lactic acid levels were examined. Metformin did not elevate lactic acid levels at the dose corresponding to higher systemic drug exposure than human therapeutic level, even for repeated doses. In contrast, phenformin elevated lactic acid levels at the dose corresponding to lower exposure than human therapeutic level, and sustained high levels were observed up to 24h post-dose; furthermore, these changes were enhanced by repeated doses. Direct comparison at each rat equivalent dose clearly indicated that lactic acid levels of phenformin were higher than those of metformin. These non-clinical findings suggest that metformin dose not increase lactic acid levels like phenformin does, and therefore may not increase the risk for lactic acidosis at human therapeutic exposure level.

The First Case of Feline Infectious Peritonitis-like Pyogranuloma in a Ferret Infected by Coronavirus in Japan.

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.

Toxicological approach for elucidation of clobazam-induced hepatomegaly in male rats.

Antiepileptic agents are known to cause adverse effects in human liver, including steatosis. Clobazam (CLB), a 1,5-benzodiazepine, is clinically used as an antiepileptic agent. In the previous study, 4-week treatment with CLB induced hepatomegaly in male rats. In the present study, the human risk of hepatomegaly was assessed and the causative mechanism in terms of cell proliferation and apoptosis, oxidative stress, and drug-metabolizing enzyme induction was elucidated by toxicological approach. Male SD rats were treated orally with 400 mg/kg CLB for 1, 3, 7, 14, or 28 days. The 28-day treatment was followed by 7 or 14 days of withdrawal. At the end of each treatment, the liver and plasma of each rat were examined. Liver weight increased from Day 3 of CLB treatment. This increase was mostly accompanied by hepatic centrilobular hypertrophy and proliferation of smooth endoplasmic reticulum (SER), and by an increase in microsomal proteins. Cyp2b1, Cyp3a1, Cyp3a2, and Ugt2b2 mRNA levels in the liver were upregulated as compared to the control group throughout the dosing period. On the other hand, the thiobarbituric acid reactive substance (TBARS) formulation, hepatocyte proliferation, and apoptosis, assumed to play roles in laying groundwork for effective induction of metabolizing enzymes, were increased only at the acute phase of treatment. These results suggested that CLB-induced hepatomegaly in male rats is mainly attributable to microsomal enzyme induction associated with Cyp2b1, Cyp3a1, Cyp3a2, and Ugt2b2 gene upregulation, but does not cause any toxicological concerns.

Establishment and Characterization of Renal Carcinoma Cell Lines from a Bhd Gene Mutant (Nihon) Rat

A germline insertion of a single nucleotide in the rat homologue of the human Birt-Hogg-Dubé (BHD) gene gives rise to dominantly inherited renal cell carcinoma (RCC) in the Nihon rat model. In this study, we established 7 lines (NR cell lines NR22, 24, 32, 45, 49, 54 and 64) from an RCC found in a Nihon rat. All cell lines consisted mainly of round or polygonal cells arranged in a cobblestone-like growth pattern. Cells of NR cell lines had abundant cytoplasm and tight junctions as well as microvilli on electron microscopy and were positive for cytokeratin on immunocytochemistry. Cell lines NR22, 24 and 32 showed rapid growth, whereas the growth of the remaining lines was very slow. While the modal chromosome number of lines NR24, 45 and 54 was 42, the remaining lines exhibited aberrant modal numbers ranging from 70 to 96. All NR cell lines formed tumors at subcutaneous inoculation sites in nude mice, and tumors from lines NR54 and 64 developed pulmonary metastases. All NR cell lines had a germline mutation in the rat Bhd gene in the gene analysis. NR cell lines would prove valuable experimental tools for studies on unique functions of the BHD gene and renal carcinogenesis.

Cystic Cholangioma in the Thoracic Cavity of a Rat

A female congenic rat produced by repeated backcrossing of Nihon rats, a model for hereditary renal cell carcinoma, to Brown Norway rats was necropsied at 24 months of age. At necropsy, a white mass about 1 centimeter in size was observed in the thoracic cavity, and the mass partly adhered to the esophagus and the diaphragm. Histologically, the mass was clearly circumscribed by connective tissue, and consisted of neoplastic cuboidal epithelial cells that showed cystic tubular proliferation. Some islands of well-differentiated hepatocytes and some vessels were observed in the mass. Immunohistochemically, the tumor cells were strongly positive for cytokeratin and partly positive for vimentin but were negative for mesothelin and Von Willebrand Factor. The positive rate for Ki-67 was 2.4%. Based on these histological and immunohistochemical evidences, we diagnosed this tumor as a cystic cholangioma that might have arisen from the ectopic hepatic tissue in the thoracic cavity.

Immunohistochemical and Ultrastructural Analyses of Cytoplasmic Blood Plasma Inclusions of Rat Hepatocytes

In the present study, we investigated the histological, immunohistochemical and ultrastructural characteristics of cytoplasmic blood plasma inclusions that spontaneously occurred in a rat liver. Histologically, a number of cytoplasmic inclusions were observed in the liver of an 8-week-old female SD rat. These inclusions were strongly positive for PAS staining and resistant to diastase digestion. Immunohistochemical analyses revealed that these inclusions were positive for albumin and IgG; however, most of them were negative for LAMP-1 and LAMP-2. Ultrastructurally, the inclusions were surrounded by limiting membranes and composed of moderately electron dense, homogenous materials. These characteristics described here represent valuable information for pathological examination in toxicity studies.

Spontaneous Fibrosarcoma with Pleomorphic Appearance in an Aged Brown Norway Rat

An 18-month-old male Brown Norway (BN) rat showed a grayish-white subcutaneous mass in the right cheek. Histologically, the mass was composed of highly pleomorphic cells producing collagen. Immunohistochemical analysis showed that the tumor cells were strongly positive for vimentin and partially positive for Ki-67; however, they were negative for ED-1, ED-2, S-100, cytokeratin, desmin and myoglobin. Ultrastructurally, the cytoplasms of the tumor cells contained well-developed rough endoplasmic reticulum. Thus, the tumor had no characteristic feature other than collagen production and was diagnosed as a fibrosarcoma.

Contents Vol. 30, 2009

G. Abelev, Moscow V. Barak, Jerusalem R. Begent, London H. Biran, Tel Aviv E. Bombardieri, Milan O.P. Børmer, Oslo R.R. Brentani, São Paulo K. Chester, London E.P. Diamandis, Toronto A. Epenetos, London H.A. Fritsche, Houston, Tex. A. Fuks, Montreal P. Gold, Montreal S. Hammarström, Umeå F. Itoh, Kanagawa J.M. Jessup, Washington, D.C. H. Kato, Ube Y.S. Kim, San Francisco, Calif. M. Kuroki, Fukuoka J.P. Mach, Epalinges s./Lausanne R. Molina, Barcelona B. Pedley, London M.F. Rajewsky, Essen J. Schlom, Bethesda, Md. J.E. Shively, Duarte, Calif. U.-H. Stenman, Helsinki M. Toyota, Sapporo J. Uriel, Paris C. Wittekind, Leipzig H. zur Hausen, Heidelberg Tumor Markers, Tumor Targeting and Translational Cancer Research