Juki Kimura

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.

The antipsychotics haloperidol and chlorpromazine increase bone metabolism and induce osteopenia in female rats

The main objective of this study was to evaluate the effects of the antipsychotic drugs haloperidol (HAL) and chlorpromazine (CPZ) on bone mineral density (BMD) in female rats and to examine the relationship between the effects on bone and reproductive organs or hormone concentrations. Female rats were orally administered HAL (2 or 10 mg/kg) or CPZ (25 or 50 mg/kg) once daily (7 days/week) for 6 months resulting in a significant increase in prolactin. Hyperprolactinemia resulted in enlarged corpora lutea in the ovary, because prolactin has a luteotropic activity. Thus, atrophy in the uterus, epithelial mucification in the vagina and continuous diestrus stages were observed. These events in the reproductive organs induced a decrease in estradiol, elevation of biochemical markers of bone metabolism, significant reductions of BMD in trabecular bone of the femur and decreased trabecular bone in the femur. The bone loss is associated with an increase in bone resorption due to decreased estradiol derived from the luteotropic activity of prolactin. The mechanism of dopamine blockers to induce bone loss in female rats is considered to be rodent specific because the luteotropic effects of prolactin are confined primarily to rodents. Also, it appears that chronic hyperprolactinemia and maintained corpora lutea leading to bone loss are commonly inducible in female rats receiving long-term treatment with antipsychotic drugs possessing dopamine D2 receptor antagonist activity.

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.

Genotoxicity evaluation of benzene, di(2-ethylhexyl) phthalate, and trisodium ethylenediamine tetraacetic acid monohydrate using a combined rat comet/micronucleus assays

As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo alkaline comet assay (comet assay), we examined DNA damage in the liver, stomach, and bone marrow of rats dosed orally three times with up to 2000 mg/kg of benzene, di(2-ethylhexyl) phthalate, and trisodium ethylenediamine tetraacetic acid monohydrate. All three compounds gave negative results in the liver and stomach. In addition, a bone marrow comet and micronucleus analysis revealed that benzene, but not di(2-ethylhexyl) phthalate or trisodium ethylenediamine tetraacetic acid monohydrate induced a significant increase in the median % tail DNA and micronucleated polychromatic erythrocytes, compared with the respective concurrent vehicle control. These results were in good agreement with the previously reported genotoxicity findings for each compound. The present study has shown that combining the micronucleus test with the comet assay and carrying out these analyses simultaneously is effective in clarifying the mechanism of action of genotoxic compounds such as benzene.

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.

Spontaneous Rhabdomyosarcoma in a Common Marmoset (Callithrix jacchus)

The common marmoset (Callithrix jacchus) is now widely used in various research fields, including toxicology. However, information about the background pathology of this species is scarce. Here, we report a case of rhabdomyosarcoma that spontaneously occurred in a common marmoset. A 44-month-old male common marmoset was euthanized due to bilateral hind limb paralysis. At necropsy, a 2×2×5-cm intramuscular mass was observed in the lower right back. Histologically, the mass was mainly composed of interlacing bundles of spindle-shaped tumor cells. Immunohistochemically, the tumor cells were positive for myogenin, desmin, vimentin and alpha-smooth muscle actin. Ultrastructurally, the tumor cells contained bundles of myofilaments with Z-band-like structures. Thus, the tumor was diagnosed as a rhabdomyosarcoma. To our knowledge, this is the first report of spontaneous rhabdomyosarcoma that was definitely diagnosed in the common marmoset.

The effects of clobazam treatment in rats on the expression of genes and proteins encoding glucronosyltransferase 1A/2B (UGT1A/2B) and multidrug resistance‐associated protein-2 (MRP2), and development of thyroid follicular cell hypertrophy

Clobazam (CLB) is known to increase hepatobiliary thyroxine (T4) clearance in Sprague-Dawley (SD) rats, which results in hypothyroidism followed by thyroid follicular cell hypertrophy. However, the mechanism of the acceleration of T4-clearance has not been fully investigated. In the present study, we tried to clarify the roles of hepatic UDP-glucronosyltransferase (UGT) isoenzymes (UGT1A and UGT2B) and efflux transporter (multidrug resistance-associated protein-2; MRP2) in the CLB-induced acceleration of T4-clearance using two mutant rat strains, UGT1A-deficient mutant (Gunn) and MRP2-deficient mutant (EHBR) rats, especially focusing on thyroid morphology, levels of circulating hormones (T4 and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH), and mRNA or protein expressions of UGTs (Ugt1a1, Ugt1a6, and Ugt2b1/2) and MRP2 (Mrp). CLB induced thyroid morphological changes with increases in TSH in SD and Gunn rats, but not in EHBR rats. T4 was slightly decreased in SD and Gunn rats, and T3 was decreased in Gunn rats, whereas these hormones were maintained in EHBR rats. Hepatic Ugt1a1, Ugt1a6, Ugt2b1/2, and Mrp2 mRNAs were upregulated in SD rats. In Gunn rats, UGT1A mRNAs (Ugt1a1/6) and protein levels were quite low, but UGT2B mRNAs (Ugt2b1/2) and protein were prominently upregulated. In SD and Gunn rats, MRP2 mRNA and protein were upregulated to the same degree. These results suggest that MRP2 is an important contributor in development of the thyroid cellular hypertrophy in CLB-treated rats, and that UGT1A and UGT2B work in concert with MRP2 in the presence of MRP2 function to enable the effective elimination of thyroid hormones.

Oral administration of drugs with hypersensitivity potential induces germinal center hyperplasia in secondary lymphoid organ/tissue in Brown Norway rats, and this histological lesion is a promising candidate as a predictive biomarker for drug hypersensitivity occurrence in humans

It is important to evaluate the potential of drug hypersensitivity as well as other adverse effects during the preclinical stage of the drug development process, but validated methods are not available yet. In the present study we examined whether it would be possible to develop a new predictive model of drug hypersensitivity using Brown Norway (BN) rats. As representative drugs with hypersensitivity potential in humans, phenytoin (PHT), carbamazepine (CBZ), amoxicillin (AMX), and sulfamethoxazole (SMX) were orally administered to BN rats for 28days to investigate their effects on these animals by examinations including observation of clinical signs, hematology, determination of serum IgE levels, histology, and flow cytometric analysis. Skin rashes were not observed in any animals treated with these drugs. Increases in the number of circulating inflammatory cells and serum IgE level did not necessarily occur in the animals treated with these drugs. However, histological examination revealed that germinal center hyperplasia was commonly induced in secondary lymphoid organs/tissues in the animals treated with these drugs. In cytometric analysis, changes in proportions of lymphocyte subsets were noted in the spleen of the animals treated with PHT or CBZ during the early period of administration. The results indicated that the potential of drug hypersensitivity was identified in BN rat by performing histological examination of secondary lymphoid organs/tissues. Data obtained herein suggested that drugs with hypersensitivity potential in humans gained immune reactivity in BN rat, and the germinal center hyperplasia induced by administration of these drugs may serve as a predictive biomarker for drug hypersensitivity occurrence.