The prompt and appropriate safety assessment of drug metabolite(s) was mentioned in regulatory guidances such as an International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance, entitled “Guidance on Non-clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals” (ICH M3(R2)) implemented in January 1 of 2011 in Japan, and has become a significant issue in the drug development. Upon release of ICH M3(R2) Step 4, a survey was conducted between March and April 2010 on the safety assessment of drug metabolites in 63 member companies of the Japan Pharmaceutical Manufacturers Association (JPMA). The Pharmacokinetics Team in the Non-Clinical Evaluation Expert Committee in JPMA conducted a questionnaire survey and compiled the results to comprehend how safety of drug metabolites are currently assessed at research-based pharmaceutical companies in Japan. The assessment of “Metabolites in Safety Testing” (MIST) can be divided into three stages based on the research purpose as follows: MIST 1 is a stage of estimating human drug metabolites and predicting their potential risks, MIST 2 is a stage of deciding the necessity for non-clinical safety studies, and MIST 3 is a stage of conducting non-clinical safety studies. In this paper, we propose typical approaches on safety assessment of metabolites that meet the purpose of each stage, considering the current level of scientific technology. Our proposals are based on the results from our survey and a symposium about the safety assessment of drug metabolites at the 37th annual meeting of the Japanese Society of Toxicology held in June 2010.
Oxidative stress induced by tamoxifen (TAM) in male testis and its effects on fertility and early embryo development were investigated. TAM was orally administered for 4 weeks repeatedly to two isogenic male mice strains, inbred strain of C57BL/6J (B6) mice and hybrid strain of C57BL/6J x CBA F1 (B6CBAF1) mice. Oxidative stress in mice testis was measured based on the level of lipid peroxidation (LPO). The LPO level was significantly increased in inbred strain of B6 mice (p < 0.05), but not hybrid strain of B6CBAF1 mice. Paternal exposure to TAM led to a significant decrease in the fertilization rate in B6 mice (p < 0.05), but not their B6CBAF1 counterparts. Interestingly, TAM had no impact on the cell number and apoptosis status in blastocysts. These results indicate that susceptibility to TAM-induced oxidative stress in the testis differs between isogenic mice strains, and genetic variations play an important role in promoting differential degrees of toxic response.
Delayed effects of exposure to small amounts of estrogenic compounds during the critical period of brain sexual differentiation were investigated by subcutaneous treatment of female Sprague-Dawley rats with 0 (vehicle control), 0.08, 0.4, or 2 µg/kg of 17α-ethynylestradiol (EE) on postnatal day (PND) 1. The treatment did not affect growth and development of the treated animals, and the timings of vaginal opening were similar between the EE-treated and control groups. The animals were periodically examined for the estrous cycle from postnatal week (PNW) 8-9 to PNW 32-33. Patterns of the estrous cycle were similar among the groups until PNW 17. None of the control animals showed persistent estrus until PNW 33. The animals treated with 0.4 µg/kg or more EE showed persistent estrus from PNW 20. The alteration was reflected in the number of days judged as proestrus or estrus, and was found to gradually increase in the EE-treated groups. At necropsy on PNW 32-33, ovulation was not confirmed in most EE-treated animals, even on the day of estrus. In addition, sporadic milk accumulations were observed in the mammary gland of the EE-treated animals. Histological evaluation revealed cystic follicle formation in the EE-treated ovaries and also revealed hyperplasia of mammary glands. Furthermore, ovaries from the animals showing persistent estrus lacked corpus luteum, indicating long-term anovulation. These results clearly show that single exposure to EE during the critical period of brain sexual differentiation can exert effects on reproductive functions at a later period in rats.
Glycidol fatty acid esters (GEs) have been identified as contaminants in refined edible oils. Although the possible release of glycidol (G) from GEs is a concern, little is known about the conversion of GEs to G in the human body. This study addressed the toxicokinetics of glycidol linoleate (GL) and G in male Crl:CD(SD) rats and cynomolgus monkeys. Equimolar amounts of GL (341 mg/kg) or G (75 mg/kg) were administered by gavage to each animal. G was found in both species after the G and GL administration, while plasma GL concentrations were below the lower limit of quantification (5 ng/ml) in both species. In rats, the administration of GL or G produced similar concentration-time profiles for G. In monkeys, the Cmax and AUC values after GL administration were significantly lower than those after G administration. The oral bioavailability of G in monkeys (34.3%) was remarkably lower than that in rats (68.8%) at 75 mg/kg G administration. In addition, plasma G concentrations after oral administration at three lower doses of GL or G were measured in both species. In monkeys, G was detected only at the highest dose of G. In contrast, the rats exhibited similar plasma G concentration-time profiles after GL or G administration with significantly higher G levels than those in monkeys. In conclusion, these results indicate that there are remarkable species differences in the toxicokinetics of GEs and G between rodents and primates, findings that should be considered when assessing the human risk of GEs.
In the present study, we isolated and determined the pharmacological characteristics of a novel gene encoding the zinc finger-like protein (ZfLp). The isolated cDNA consisted of 1,581 base pairs that encoded a 526-amino acid protein. The amino acid sequence of ZfLp is 96% identical to that of zinc finger protein 415 isoform 5 (ZNF415-5). Reverse-transcription (RT)-polymerase chain reaction (PCR) analysis revealed that the ZfLp mRNA is expressed in the breast, lung, stomach, small intestine colon and ovary, but not in the liver. When expressed in Xenopus laevis oocytes, ZfLp mediated the high affinity transport of [3H]paclitaxel (taxol) in a sodium-independent manner (Km = 336.7 ± 190.0 nM). The uptake of [3H]paclitaxel (taxol) by ZfLp was trans-stimulated by glutarate and glutathione (GSH). A cis-inhibition experiment revealed that ZfLp-mediated transport of [3H]paclitaxel (taxol) is inhibited by several organic solutes specifically clotrimazole. Using several clotrimazole derivatives, we found that N-benzylimidazole would be a minimum unit for producing the inhibition of ZfLp-mediated drug uptake. Our results may provide insights into the novel role of soluble protein, such as ZNF, in the human body. Our results, therefore, would be expected to facilitate research on the novel role of ZNFs and on the discovery of novel drugs for targeting ZNF-related proteins such as ZfLp.
1-Methylnaphthalene (1-MN), a constituent of the polycyclic aromatic hydrocarbons (PAHs), is a lung carcinogen in mice. However, conventional genotoxicity tests such as the Ames test and sister chromatid exchange (SCE) test have yielded equivocal results. In the present study, the in vivo genotoxicity of 1-methylnaphthalene (1-MN) together with its toxicological profile was investigated in a 13-week repeated dose toxicity study of 1-MN using B6C3F1 gpt delta mice. In the serum biochemistry, significant increases in AST and ALP were observed in males of the 0.15% 1-MN group. From histopathological examination, the incidence of single cell necrosis in the liver was significantly increased in males of the 0.15% 1-MN group; however, no changes were observed in the lungs, the target organ of 1-MN. In an in vivo mutation assay, no changes in mutant frequencies of gpt and red/gam (Spi-) in lung DNA of 1-MN treated mice were observed at 13 weeks. In addition, there were no significant differences in the proliferating cell nuclear antigen (PCNA)-positive ratios in bronchiolar epithelial cells among the groups for either sex. These results suggest that 1-MN at a carcinogenic dose not induce overt toxicity for any organs and has no in vivo genotoxicity in the lungs.
The present study aimed to establish candidate biomarker genes for the early detection of nephrotoxicity in mice, with a particular focus on nephrotoxicity caused by polyene macrolides. Comprehensive gene expression changes were evaluated using microarrays in a mouse model in which acute nephrotoxicity was induced by amphotericin B deoxycholate, trade name Fungizone. The upregulated genes identified through microarray analysis of kidney tissue of Fungizone-treated mice included several genes that have been reported as nephrotoxicity biomarkers in rats, and 14 genes were selected as candidate nephrotoxicity biomarkers. The usefulness of these genes as nephrotoxicity biomarkers in mice was evaluated further through expression profiling under several experimental conditions using real time RT-PCR. Expression of genes encoding kidney injury molecule 1, lipocalin 2, tissue inhibitor of metalloproteinase 1, and secreted phosphoprotein 1 was highly upregulated by Fungizone, nystatin, natamycin, amphotericin B methyl ester, and liposomal amphotericin B, and their area under the ROC curve values were more than 0.95. These genes were more sensitive at detecting nephrotoxicity than traditional clinical chemistry and histopathology parameters. This study provides novel evidence that these nephrotoxicity biomarker genes identified are translatable to mice, and that they are useful for early and sensitive detection of nephrotoxicity.
Volatile organic compounds (VOCs) are the main substances causing multiple chemical sensitivity reactions in human. The effects of single VOCs exposure on airway inflammatory responses in mice lung have been reported. Previous studies have demonstrated the role of reactive oxygen species (ROS) in lung inflammation induced by single VOCs inhalation. However, effects of VOCs exposure on NO signaling and neurological signaling pathways in airway remain less clear. We exposed male Kunming mice to filtered air (0) and four types of VOCs mixture (formaldehyde, benzene, toluene, and xylene) treated air. Group 1 is 1.0, 1.1, 2.0 and 2.0 mg/m3, group 2 is 3.0, 3.3, 6.0 and 6.0 mg/m3, group 3 is 5.0, 5.5, 10.0 and 10.0 mg/m3, group 4 is 10.0, 11.0, 20.0 and 20.0 mg/m3, which respectively corresponded to 10, 30, 50 and 100 times of indoor air quality standard in China 2 hr per day, 5 days per week, for 2 weeks in the whole body exposure chamber. One day following VOCs exposure, we collected lung, bronchoalveolar lavage fluid (BALF) from each mouse and examined oxidative stress markers, cellular infiltration and production of cytokines, neurotrophin and substance P. We found that VOCs exposure influenced significantly NOS activity, GSH, or IL-6 concentration. The number of total cells, macrophages and eosinophils increased significantly in group 4. In addition, the production of interferon-gamma (IFN-γ) and substance P were significantly decreased. In contrast, neurotrophin-3 production in BALF was significantly increased in group 3 and 4. Our findings suggest that NO signaling pathways may induce airway inflammatory in short term VOCs exposure mice and the airway inflammatory response may be modulated by neurological signaling.
The relationship between methylmercury (MeHg) exposure and aquaporin (AQP) expression in the brain is currently unknown. To investigate this, we used a common marmoset model of acute MeHg exposure to examine AQP1, AQP4 and AQP11 gene expression. MeHg (1.5 mg Hg/kg/day p.o.) was given to three marmosets for 14 days, followed by 14 days without. All treated marmosets showed slight akinesia before sacrifice. In the frontal lobe, occipital lobe and cerebellum, total mercury concentrations following MeHg administration were 26.7, 31.4, and 22.6 μg/g, respectively. Slight apoptosis was observed in the occipital lobe. Immunohistochemistry showed increased expression of glial fibrillary acidic protein, its mRNA and Iba1 with MeHg, indicating that neuronal injury activated astrocytes and microglia. There was no significant difference between control and MeHg-administered groups in AQP1 protein or AQP11 mRNA in the frontal lobe, occipital lobe or cerebellum. The ratio of AQP4 mRNA expression in MeHg-administered marmosets to the mean AQR4 expression in the controls (n = 3) were 1.3, 1.5 and 1.2, 1.7, 1.9 and 1.5, and 1.5, 1.6 and 1.2 for the frontal lobe, occipital lobe and cerebellum, respectively. Western blotting showed significantly increased AQP4 protein in the occipital lobe and cerebellum with MeHg administration, but no obvious up-regulation in the frontal lobe. Immunofluorescence analysis with double staining revealed low AQP4 expression in the cell body of reactive astrocytes in the MeHg-administered group. These results indicate that AQP4 expression might be stimulated by MeHg exposure in astrocytes in the occipital lobe and cerebellum, suggesting a role for AQP4 in MeHg neurotoxicity via astrocyte dysfunction.
To examine the contribution of metallothionein (MT) to mercury accumulation in mouse tissues, 129 strain female mice and MT null mice were exposed to metallic mercury vapor at a sub-toxic level, and Hg levels in the brain, kidney and liver were determined on 1, 3 and 7 days after the exposure. After exposure to mercury vapor, significant Hg accumulation was observed in the brains of wild-type and MT-I/II null and MT-III null mice, as well as in the liver and kidneys. No strain difference was observed in the tissue Hg accumulations 24 hr after the exposure except for the kidneys, where the highest accumulation was found in MT-III null mice. Although the brains of MT-III null mice showed slightly higher Hg accumulation than the other two strains, no significant difference was observed except in the cerebrum on Day 7. Gel chromatograms of cerebrum soluble fractions revealed that a significant amount of Hg existed as an MT-bound form in all the mouse strains. On the other hand, MT-bound Hg was found as a minor fraction in soluble fractions of the kidneys and livers in wild-type and MT-III null mice. Despite a significant strain difference in total MT levels in the cerebrum, there was no difference among the three strains in the amount of Hg accumulated in the cerebrum and its distribution rates in MT fractions. The present study demonstrated that brain uptake of Hg0 and its accumulation as Hg2+ did not depend on the amount of MT isoform in the tissue, at least in the early phase.
Methamphetamine (MA) appears to produce neurotoxic effects, in part, through disruptions of energy metabolism. A recent study of the whole-body proteome of Drosophila melanogaster showed many changes in energy metabolism-related proteins, leading us to hypothesize that MA toxicity may cause whole-body disruptions of energy metabolism. To test this, we monitored the response of energy reserves and other metabolites to MA-exposure with and without the addition of dietary glucose. We also monitored changes in feeding behavior, locomotor activity and respiration rates associated with MA-exposure to investigate how MA affects energy balance. We observed that glycogen and triglyceride levels decreased dramatically within 48 hr of MA-exposure, indicating a strongly negative caloric balance. Behavioral assays revealed that MA-treated flies decreased food consumption by 60-80% and exhibited a 2-fold increase in locomotion. Caloric expenditure decreased with MA-exposure, apparently due to a compensatory decrease in resting metabolism, showing that anorexia was the primary driver of the negative caloric balance. Additionally, we observed that glucose supplementation of MA-containing diet increased glycogen reserves by 44% at 48 hr, leading to a commensurate increase in survivorship. We conclude that dietary sugar supplementation enhances survivorship by partially compensating for decreased caloric intake resulting from MA-induced anorexia. The observation that MA produces similar behavioral changes in Drosophila and humans, i.e. increased locomotor activity and anorexia, further supports the use of Drosophila as a model organism for the study of the effects of MA.
An important technology used in toxicogenomic drug discovery research is the microarray, which enables researchers to simultaneously analyze the expression of a large number of genes. To build a database and data analysis system for use in assessing the safety of drugs and drug candidates, in 2002 we conducted a 5-year collaborative study in the Toxicogenomics Project (TGP1) in Japan. Experimental data generated by such studies must be validated by different laboratories for robust and accurate analysis. For this purpose, we conducted intra- and inter-laboratory validation studies with participating companies in the second collaborative study in the Toxicogenomics Project (TGP2). Gene expression in the liver of rats treated with acetaminophen (APAP) was independently examined by the participating companies using Affymetrix GeneChip microarrays. The intra- and inter-laboratory reproducibility of the data was evaluated using hierarchical clustering analysis. The toxicogenomics results were highly reproducible, indicating that the gene expression data generated in our TGP1 project is reliable and compatible with the data generated by the participating laboratories.
Satratoxin H is an important air- and food-borne mycotoxin, which has been implicated in human health damage. Satratoxin H is known to induce apoptosis as well as genotoxicity in PC12 cells. In the present study, we further investigated the mechanism of apoptotic effects of satratoxin H with focus on caspase-3 and poly-ADP-ribose polymerase (PARP) pathway. We also examined whether it induces DNA damage in PC12 cells. In the cells treated with satratoxin H, caspase-3 was cleaved in a time-dependent manner. Furthermore, satratoxin H induced cleavage of PARP, one of the downstream molecules of caspase-3. The cleavage was inhibited by SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor. Satratoxin H, however, had no effect on expression levels of Bax and Bcl-2. Furthermore, the micronucleus assay revealed that satratoxin H induced chromosome break. Also, satratoxin H increased the level of phosphorylation of histone H2A, indicating that it caused DNA double-stranded breaks in PC12 cells. Meanwhile, no genotoxicity was detected with any of treatments carried out in the alkaline comet assay. These results imply that satratoxin H induces genotoxicity by DNA double-stranded break. Our results suggest a considerable potential for the genotoxic risk associated with the presence of satratoxin H.
Orotic acid (OA) is a tumor promoter of experimental liver carcinogenesis initiated by DNA reactive carcinogens, the molecular mechanisms of which have not been fully elucidated. OA increases cell proliferation and decreases apoptosis in serum-starved SK-Hep1 hepatocellular carcinoma cells, which may ascribe to the inhibition of AMP-activated protein kinase (AMPK) phosphorylation and thus activation of mammalian target of rapamycin complex 1 (mTORC1). The effects of OA on mTORC1 activation, cell proliferation, and cell-cycle progression to S and G2/M phases were completely reversed by rapamycin. Activation of AMPK by a constitutively active mutant or aminoimidazole carboxamide ribonucleotide (AICAR) rescued the effects of OA. In conclusion, OA increases the proliferation and decreases the starvation-induced apoptosis of SK-Hep1 cells via mTORC1 activation mediated by negative regulation of AMPK.
Animal models of ototoxicity represent an elementary tool in otolaryngologic research. Such models are usually created via the consecutive injection of ototoxic drugs or the co-administration of ethacrynic acid and low-dose ototoxic drugs. Injection via the round window membrane (RWM) is one approach that allows for local drug delivery into the inner ear. In this study, 47 guinea pigs received an injection of varying doses of cisplatin via the RWM, and data concerning the animals’ auditory brainstem responses, hair cells, and spiral ganglion neurons were analyzed. Our results indicate the high efficiency and generally small reaction of the subjects, suggesting that the application of cisplatin via the RWM is an effective animal model for ototoxicity research.
Methylglyoxal (MG) is an endogenous carbonyl compound that is produced in large quantity under hyperglycemic conditions, which are believed to contribute to the development of diabetic neuropathy. However, the mechanism by which this occurs and the molecular targets of MG are unclear. In the present study, we investigated the effect of MG on transient receptor potential ankyrin 1 (TRPA1) activation in human TRPA1-expressing HEK293 cells. MG activated TRPA1-expressing HEK293 cells, but failed to activate human capsaicin-sensitive transient receptor potential vanilloid 1 (TRPV1)-expressing HEK293 cells or mock-transfected HEK293 cells. MG also induced calcium (Ca2+) influx in a concentration-dependent manner, and the concentration-response curve indicates that the effect of MG has an EC50 of 343.1 ± 17.3 μM. Interestingly, the time course in the intracellular Ca2+ concentration ([Ca2+]i) in human TRPA1-expressing HEK293 showed considerable differences in response to MG and cinnamaldehyde. Furthermore, we examined four endogenous carbonyl compounds, including MG, glyceraldehyde, glycolaldehyde, and glyoxal; only MG notably activated TRPA1-expressing HEK293 cells. These results may provide insight into the TRPA1-mediated effects of MG on diabetic neuropathy.
Chronic ingestion of arsenic is associated with an increased risk of vascular disease such as atherosclerosis. Previously, we showed that arsenite inhibits the synthesis of general proteoglycans (PGs), which are key molecules in the progression of atherosclerosis, in vascular endothelial cells. In the present study, we investigated the effect of several metals on arsenite-induced inhibitory effect in endothelial cells. The results indicate that, after 24-hr incubation, the inhibition of PG synthesis caused by sodium arsenite was protected by bismuth nitrate but not zinc sulfate, manganese chloride, nickel chloride or cobalt chloride. The accumulation of arsenic in the cell layer was significantly decreased by bismuth after 12-hr incubation and that of bismuth was also decreased by arsenite. It was therefore suggested that the protective effect of bismuth against the inhibitory effect of arsenite on PG synthesis in vascular endothelial cells may be due, at least in part, to the decrease in cellular arsenic accumulation.
The Hand1- and Cmya1-ESTs are novel short-term tests for embryotoxic chemicals using genetically engineering mouse ES cells for luciferase reporter gene assays. These ESTs allow convenient determination of differentiation toxicity and cell viability in a short duration with high throughput 96-well microplates for prediction of embryotoxicity of chemicals. To assess the Hand1-EST technical protocol, we firstly compared reporter gene assay and cytotoxicity test data for a representative compound (hydroxyurea) from four different laboratories with tests carried out under the same experimental conditions. Extensive investigations of the Hand1- and Cmya1-ESTs were then performed to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, across the laboratories. The results gave good correspondence in all four laboratories, indicating that transferability, intra-laboratory variability and inter-laboratory variability of the present technical protocols of the ESTs were sufficient to conduct further validation studies.
We analyzed total Hg concentrations in various tissue samples obtained from 7 commercially available fish species. MeHg contents were also estimated for muscle and liver samples by a selective analysis of inorganic Hg. Among the tissues, high Hg accumulations were shown in liver, muscle, heart and spleen throughout all fish species. Carnivorous fish, such as scorpion fish, sea bream and Japanese whiting, tended to show higher Hg accumulations in the muscle, with the highest Hg levels being shown by scorpion fish. Although the liver was expected to show the highest Hg accumulations among tissues throughout all fish species, the highest accumulation in the liver was observed only in scorpion fish. In contrast, the muscle level was significantly higher than the liver in Pacific saury and Japanese whiting. MeHg accumulated in fish is considered to show a sustained increase throughout the life of the fish, due to its long biological half-life. In fact, in the present study, muscle Hg levels in Japanese whiting, Japanese flying fish, and halfbeak showed good correlations with body weights. However, such correlations were not clear in scorpion fish, sea bream, Jack mackerel and Pacific saury. Selective analyses of inorganic Hg levels revealed that most of the Hg (> 95%) in fish muscle existed as MeHg, while the rates of MeHg contents in the liver varied from 56% in scorpion fish to 84% in Jack mackerel. As a result, fish muscle showed the highest MeHg accumulations in all fish species examined. These results suggest that reliable information on total Hg contents in fish muscle might be sufficient to avoid the risk of MeHg exposure caused by eating fish, even when one consumes other tissues such as fish liver.
Cytochrome P450(CYP)s are known to show a sexual dimorphic expression in rat livers. However, the comprehensive analysis for the sex-dependent gene expressions of drug metabolizing enzymes except for CYPs, transporters and nuclear receptors in rat livers and kidneys has not been investigated yet. The purpose of the present study was to identify the novel drug metabolizing and pharmacokinetics (DMPK)-related gene(s) which show the sex difference in the mRNA expressions in rat livers and kidneys. Total RNAs were prepared from livers and kidneys in both male and female rats (Crl:CD(SD) and Crlj:WI). A DNA microarray analysis using a “GeneSQUARE Multiple Assay DNA Microarray Drug Metabolism Gene Expression for Rat” was performed. DMPK-related genes which showed sex differences in the mRNA expression were identified in rat livers or kidneys. Especially, the female dominant expressions of UDP glucuronosyltransferase (UGT) s were seen in rat livers and kidneys. The sex difference of UGT expressions in rats might be one of the causal factors of the sex difference of the biological response to UGT substrates.
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