Kiyoko Bando

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.

Influences of methamphetamine-induced acute intoxication on urinary and plasma metabolic profiles in the rat.

Methamphetamine (MA) is an illicit psychostimulant, and its abuse has become an international public health problem. MA intoxication can cause life-threatening hyperthermia, renal and liver failure, cardiac arrhythmias, and neurological damage. To investigate the relationship between the underlying mechanism of such intoxication and metabolic networks, mass spectrometry-based metabolomics experiments were performed on Sprague-Dawley rats treated with MA at 10mgkg(-1)h(-1) for 4h. Using a combination of gas chromatography-time-of-flight mass spectrometry and capillary electrophoresis-tandem mass spectrometry, global and targeted analyses were performed on biological samples collected during 0-24 and 72-96h (for urine), and at 24 and 96h (for plasma) after the last drug administration. Body temperature and plasma biochemical parameters were also measured to detect abnormal reactions in neuronal and other several tissues. 5-Oxoproline, saccharic acid, uracil, 3-hydroxybutyrate (3-HB), adipic acid, glucose, glucose 6-phosphate, fructose 1,6-bisphosphate, and tricarboxylic acid (TCA) cycle intermediates, such as fumarate, were proposed as potential biomarkers related to MA-induced intoxications. In particular, the observation of decreased TCA cycle intermediates and 3-HB and increased glucose suggested that high doses of MA inhibit biogenic energy production by glycolysis, oxidative phosphorylation via the TCA cycle, and the beta-oxidation of fatty acids. These results may provide not only a clue to clarify the underlying mechanism of diverse intoxication effects, but also biological fluid-based diagnostic and forensic methods with which to objectively demonstrate intoxication without directly determining the drug.

Metabolic profiling of urine and blood plasma in rat models of drug addiction on the basis of morphine, methamphetamine, and cocaine-induced conditioned place preference

The metabolic profiles of urine and blood plasma in drug-addicted rat models based on morphine (MOR), methamphetamine (MA), and cocaine (COC)-induced conditioned place preference (CPP) were investigated. Rewarding effects induced by each drug were assessed by use of the CPP model. A mass spectrometry (MS)-based metabolomics approach was applied to urine and plasma of MOR, MA, and COC-addicted rats. In total, 57 metabolites in plasma and 70 metabolites in urine were identified by gas chromatography–MS. The metabolomics approach revealed that amounts of some metabolites, including tricarboxylic acid cycle intermediates, significantly changed in the urine of MOR-addicted rats. This result indicated that disruption of energy metabolism is deeply relevant to MOR addiction. In addition, 3-hydroxybutyric acid, l-tryptophan, cystine, and n-propylamine levels were significantly changed in the plasma of MOR-addicted rats. Lactose, spermidine, and stearic acid levels were significantly changed in the urine of MA-addicted rats. Threonine, cystine, and spermidine levels were significantly increased in the plasma of COC-addicted rats. In conclusion, differences in the metabolic profiles were suggestive of different biological states of MOR, MA, and COC addiction; these may be attributed to the different actions of the drugs on the brain reward circuitry and the resulting adaptation. In addition, the results showed possibility of predict the extent of MOR addiction by metabolic profiling. This is the first study to apply metabolomics to CPP models of drug addiction, and we demonstrated that metabolomics can be a multilateral approach to investigating the mechanism of drug addiction.

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.

Troglitazone Inhibits Bile Acid Amidation: A Possible Risk Factor for Liver Injury

Troglitazone and pioglitazone were developed as thiazolidinedione-type antidiabetes drugs, but only troglitazone was withdrawn from the markets due to severe liver injury. As both troglitazone and its sulfate metabolite are strong inhibitors of the bile salt export pump (BSEP), troglitazone-induced bile acid (BA) retention is thought to be one of the underlying mechanisms of liver injury. However, pioglitazone is also a strong BSEP inhibitor, indicating other mechanisms may also be involved in troglitazone-induced BA retention. Although retention of hydrophobic BAs (eg, chenodeoxycholic acid [CDCA]: a nonamidated BA) is known to cause hepatocyte injury, little is known about the hepatic conversion of nonamidated, hydrophobic BA species into less toxic hydrophilic BAs (eg, glycochenodeoxycholic acid: amidated BA) as a mechanism of drug-induced liver injury. In this study, we, therefore, investigated the effects of troglitazone and pioglitazone on BA amidation and the role of amidated BAs in troglitazone-associated BA-mediated hepatotoxicity. We also evaluated the intracellular BA composition of human hepatocytes treated with nonamidated BA species (CDCA or deoxycholic acid [DCA]) in the presence of troglitazone or pioglitazone. Amidation of CDCA and DCA was significantly inhibited by troglitazone (IC50: 5 and 3 µmol/l, respectively), but not pioglitazone. Moreover, treatment with troglitazone led to the retention of CDCA and DCA and decrease of glycine-amidation in hepatocytes. From these results, we suggest that troglitazone-induced liver injury might be caused by the accumulation of nonamidated BAs in hepatocytes due to inhibition of BA amidation.

The in vitro pharmacology and non-clinical cardiovascular safety studies of a novel 5-HT 4 receptor agonist, DSP-6952

&NA; The pharmacological activity of DSP‐6952, a novel compound was investigated, compared to that of clinically efficacious gastrointestinal (GI) prokinetic 5‐hydroxytryptamine4 (5‐HT4) receptor agonists. DSP‐6952 had a strong affinity of Ki = 51.9 nM for 5‐HT4(b) receptor, and produced contraction in the isolated guinea pig colon with EC50 of 271.6 nM and low intrinsic activity of 57%, similar to tegaserod and mosapride. In the development of the 5‐HT4 receptor agonists, cardiovascular risk was deliberately evaluated, because some related prokinetics were reported to cause with cardiovascular adverse events, such as ventricular arrhythmias or ischemia. DSP‐6952 showed minimal effects up to 100 &mgr;M in human ether‐a‐go‐go‐related gene (hERG) channels or guinea pig cardiomyocytes. In telemetered conscious monkeys, DSP‐6952 did not affect blood pressure or any electrocardiogram (ECG) up to 180 mg/kg, p.o.; however, DSP‐6952 transiently increased heart rate, as well as in anesthetized dogs. The positive chronotropic effects of DSP‐6952 were completely antagonized by a 5‐HT4 receptor antagonist, and another 5‐HT4 receptor agonist, TD‐5108 also increased heart rate. These effects are considered a class effect seen in clinically developing and marketed 5‐HT4 receptor agonists, and have not been regarded as a critical issue in clinical use. DSP‐6952 did not induce contraction in the rabbit coronary artery up to 100 &mgr;M, which differed from tegaserod or sumatriptan. These results show that DSP‐6952 does not have cardiac ischemic risk via coronary vasoconstriction. In conclusion, DSP‐6952 is a promising GI prokinetic compound with partial 5‐HT4 receptor agonistic activity as well as a favorable cardiovascular safety profile.

Usefulness of Simultaneous Measurement of Plasma Steroids, Including Precursors, for the Evaluation of Drug Effects on Adrenal Steroidogenesis in Rats

The aim of this study was to evaluate the usefulness of simultaneous measurement of plasma steroids, including precursors, for the evaluation of drug effects on adrenal steroidogenesis in vivo. Plasma concentrations of corticosterone and its precursors were examined in rats dosed with compounds that affect adrenal steroidogenesis via different modes of action as well as the relationships of the changes with blood chemistry and adrenal histopathology. Male rats were dosed with tricresyl phosphate, aminoglutethimide, trilostane (TRL), metyrapone (MET), ketoconazole (KET), or mifepristone for 7 days. In the TRL, MET, and KET groups, precursor levels were markedly increased, while there were no significant changes in the corticosterone level, suggesting that the precursors are more sensitive biomarkers to detect the effect on adrenal steroidogenesis. Also, the precursors with increased levels were those that are normally metabolized by the inhibited enzymes, reflecting the modes of action of the compounds. In addition, different patterns of changes were observed in blood chemistry and histopathology, supporting the mechanism suggested by the steroid changes. These results show that simultaneous measurement of plasma steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.

Metabolic Activation of Cholestatic Drug-Induced Bile Acid-Dependent Toxicity in Human Sandwich-Cultured Hepatocytes

We previously reported a cell-based toxicity assay using sandwich-cultured hepatocytes in combination with a titrated amount of human bile acid (BA) species. In this assay, test compound-induced inhibition of BA efflux from sandwich-cultured hepatocytes leads to BA-dependent cell toxicity (BAtox, i.e., cell death due to the accumulation of BAs). Using this assay, we investigated whether 1-aminobenzotriazole (1-ABT; a nonselective cytochrome P450 inhibitor) enhanced or suppressed test compound-induced BAtox. There was a tendency that BAtox of many compounds was enhanced by 1-ABT in human hepatocytes; in contrast, such a tendency was not observed in rat hepatocytes. In particular, 1-ABT tended to enhance BAtox of several compounds (clopidogrel, ticlopidine, everolimus, etc.) in human, whereas 1-ABT tended to enhance BAtox of only ticlopidine in rat. These results indicate that this system can be used to evaluate BAtox while taking into account drug metabolism and the existence of an interspecies difference in the effect of 1-ABT treatment on BAtox.