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Wada K.,The Institute of Environmental Toxicology | Ohnuma A.,The Institute of Environmental Toxicology | Kojima S.,The Institute of Environmental Toxicology | Yoshida T.,The Institute of Environmental Toxicology | Matsumoto K.,The Institute of Environmental Toxicology
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2012

Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method. © 2011 Elsevier B.V.

Wada K.,The Institute of Environmental Toxicology | Yoshida T.,The Institute of Environmental Toxicology | Takahashi N.,The Institute of Environmental Toxicology | Matsumoto K.,The Institute of Environmental Toxicology
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2014

The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2 bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3. h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained. © 2014.

Harada T.,The Institute of Environmental Toxicology | Takeda M.,The Institute of Environmental Toxicology | Kojima S.,The Institute of Environmental Toxicology | Tomiyama N.,The Institute of Environmental Toxicology
Toxicological Research | Year: 2016

Dichlorodiphenyltrichloroethane (DDT) is still used in certain areas of tropics and subtropics to control malaria and other insect-transmitted diseases. DDT and its metabolites have been extensively studied for their toxicity and carcinogenicity in animals and humans and shown to have an endocrine disrupting potential affecting reproductive system although the effects may vary among animal species in correlation with exposure levels. Epidemiologic studies revealed either positive or negative associations between exposure to DDT and tumor development, but there has been no clear evidence that DDT causes cancer in humans. In experimental animals, tumor induction by DDT has been shown in the liver, lung, and adrenals. The mechanisms of hepatic tumor development by DDT have been studied in rats and mice. DDT is known as a non-genotoxic hepatocarcinogen and has been shown to induce microsomal enzymes through activation of constitutive androstane receptor (CAR) and to inhibit gap junctional intercellular communication (GJIC) in the rodent liver. The results from our previously conducted 4-week and 2-year feeding studies of p, p'-DDT in F344 rats indicate that DDT may induce hepatocellular eosinophilic foci as a result of oxidative DNA damage and leads them to hepatic neoplasia in combination with its mitogenic activity and inhibitory effect on GJIC. Oxidative stress could be a key factor in hepatocarcinogenesis by DDT.

Satsuma K.,The Institute of Environmental Toxicology | Masuda M.,The Institute of Environmental Toxicology | Sato K.,The Institute of Environmental Toxicology
Applied and Environmental Microbiology | Year: 2012

O-Demethylation of insecticide methoxychlor is well known as a phase I metabolic reaction in various eukaryotic organisms.Regarding prokaryotic organisms, however, no individual species involved in such reaction have been specified and characterized so far. Here we successfully isolated abacterium that mediates oxidative transformation of methoxychlor, including O-demethylation and dechlorination, from river sediment. The isolate was found to be closely related to Bradyrhizobium elkanii at the 16S rRNA gene sequence level (100% identical). However, based on some differences in the physiological properties of this bacterium, we determined that it was actually a different species, Bradyrhizobium sp. strain 17-4. The isolate mediated O-demethylation of methoxychlor to yield a monophenolic derivative [Mono-OH; 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl) ethane] as the primary degradation product. The chiral high-performance liquid chromatography (HPLC)analysis revealed that the isolate possesses high enantioselectivity favoring the formation of (S)-Mono-OH (nearly 100%). Accompanied by the sequential O-demethylation to form the bis-phenolic derivative Bis-OH [1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane],oxidative dechlorination of the side chain proceeded, and monophenolic carboxylic acid accumulated, followed by the formation of multiple unidentified polar degradation products. The breakdown proceeded more rapidly when reductively dechlorinated (dichloro-form) methoxychlor was applied as the initial substrate. The resultant carboxylic acids and polar degradation products are likely further biodegraded by ubiquitous bacteria. The isolate possibly plays an important role for complete degradation (mineralization) of methoxychlor by providing the readily biodegradable substrates. © 2012, American Society for Microbiology.

Sugitate K.,Agilent Technologies | Saka M.,The Institute of Environmental Toxicology
Journal of Pesticide Science | Year: 2015

Matrix removal ability of several new types of Solid Phase Extraction (SPEs) column was tested. E-HyCu is made of new type of material, chemically-modified carbon fibers, and can remove food components, monoacylglycerols, tocopherols, and sterols, etc. Z-Sep+ and Z-Sep/C18 are also new types of materials that contain zirconium dioxide. Z-Sep+ and Z-Sep/C18 are used for high lipid samples as the dispersive SPE in the QuEChERS method. We evaluated these sorbents as filled up columns. In our previous study, we reported that when the analytical method based on the multiresidue method is applied, monoacylglycerols are the most significant components that cause a matrix enhancement effect on pesticides in food using GC-MS. These new types of SPEs could remove not only monoacylglycerols, but also fatty acids, tocopherols, flavonoids, and sterols. The matrix enhancement effects of approximately 260 pesticides spiked in brown rice extracts pretreated with these SPEs were dramatically reduced. © Pesticide Science Society of Japan.

Boulange J.,Tokyo University of Agriculture and Technology | Kondo K.,The Institute of Environmental Toxicology | Phong T.K.,University of Queensland | Watanabe H.,Tokyo University of Agriculture and Technology
Journal of Pesticide Science | Year: 2012

This paper demonstrates the procedures for probabilistic assessment of a pesticide fate and transport model, PCPF-1, to elucidate the modeling uncertainty using the Monte Carlo technique. Sensitivity analyses are performed to investigate the influence of herbicide characteristics and related soil properties on model outputs using four popular rice herbicides: mefenacet, pretilachlor, bensulfuron-methyl and imazosulfuron. Uncertainty quantification showed that the simulated concentrations in paddy water varied more than those of paddy soil. This tendency decreased as the simulation proceeded to a later period but remained important for herbicides having either high solubility or a high 1st-order dissolution rate. The sensitivity analysis indicated that PCPF-1 parameters requiring careful determination are primarily those involve with herbicide adsorption (the organic carbon content, the bulk density and the volumetric saturated water content), secondary parameters related with herbicide mass distribution between paddy water and soil (1st-order desorption and dissolution rates) and lastly, those involving herbicide degradations. © Pesticide Science Society of Japan.

Wada K.,The Institute of Environmental Toxicology | Fukuyama T.,The Institute of Environmental Toxicology | Nakashima N.,The Institute of Environmental Toxicology | Matsumoto K.,The Institute of Environmental Toxicology
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2015

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) international validation study of in vivo rat alkaline comet assays, we examined cadmium chloride, chloroform, and d,. l-menthol under blind conditions as coded chemicals in the liver and stomach of Sprague-Dawley rats after 3 days of administration. Cadmium chloride showed equivocal responses in the liver and stomach, supporting previous reports of its poor mutagenic potential and non-carcinogenic effects in these organs. Treatment with chloroform, which is a non-genotoxic carcinogen, did not induce DNA damage in the liver or stomach. Some histopathological changes, such as necrosis and degeneration, were observed in the liver; however, they did not affect the comet assay results. d,. l-Menthol, a non-genotoxic non-carcinogen, did not induce liver or stomach DNA damage. These results indicate that the comet assay can reflect genotoxic properties under blind conditions. © 2015 Elsevier B.V.

Yoshida T.,The Institute of Environmental Toxicology | Ohnuma A.,The Institute of Environmental Toxicology | Horiuchi H.,The Institute of Environmental Toxicology | Harada T.,The Institute of Environmental Toxicology
Journal of Toxicologic Pathology | Year: 2011

Chronic lung injury resulting from a variety of different causes is frequently associated with the development of pulmonary fibrosis in humans. Although the etiology of pulmonary fibrosis is generally unknown, several sources of evidence support the hypothesis that a number of environmental and occupational agents play an etiologic role in the pathogenesis of this disease. The agents discussed in this review include beryllium, nylon flock, textile printing aerosols, polyvinyl chloride and didecyldimethylammonium chloride. The authors also describe a variety of animal models, including genetically modified mice, in order to investigate the molecular mechanism of pulmonary fibrosis, focusing on chemokine receptors, regulatory T cells and transforming growth factor-β and bone morphogenetic protein signaling. Overall, we propose the concept of toxicological pulmonary fibrosis as a lung disease induced in response to environmental cues. © 2011 The Japanese Society of Toxicologic Pathology.

BACKGROUND: Linuron is a globally used phenylurea herbicide, and a large number of studies have been made on themicrobial degradation of the herbicide. However, to date, the few bacteria able individually to mineralise linuron have been isolated only from European agricultural soils. An attempt was made to isolate linuron-mineralising bacteria from Japanese river sediment using a uniquely designed river ecosystem model (microcosm) treated with 14C-ring-labelled linuron (approximately 1 mgL-1). RESULTS: A linuron-mineralising bacterium that inhabits river sediment was successfully isolated. The isolate belongs to the genera Variovorax and was designated as strain RA8. Strain RA8 gradually used linuron in basal salt medium (5.2mg L-1) with slight growth. In 15 days, approximately 25% of 14C-linuron was mineralised to 14CO2, with 3,4-dichloroaniline as an intermediate. Conversely, in 100-fold diluted R2A broth, strain RA8 rapidly mineralised 14C-linuron (5.5 mg L-1) and more than 70% of the applied radioactivity was released as 14CO2 within 3 days, and a trace amount of 3,4-dichloroaniline was detected. Additionally, the isolate also degradedmonolinuron,metobromuron and chlorobromuron, but not diuron, monuron or isoproturon. CONCLUSION: Although strain RA8 can grow on linuron, some elements in the R2A broth seemed significantly to stimulate its growth and ability to degrade. The isolate strictly recognised the structural difference between N-methoxy-N-methyl and N,N-dimethyl substitution of various phenylurea herbicides. © 2010 Society of Chemical Industry.

Ohnuma A.,Toho University | Ohnuma A.,The Institute of Environmental Toxicology | Conlon J.M.,United Arab Emirates University | Iwamuro S.,Toho University
Comparative Biochemistry and Physiology - C Toxicology and Pharmacology | Year: 2010

Previous studies led to the isolation of antimicrobial peptides (AMPs) of the brevinin-2, palustrin-2, and ranatuerin-2 families from skin extracts and/or skin secretions of the Japanese mountain brown frog, Rana ornativentris. In the present study, we cloned cDNAs encoding the precursors of brevinin-2Oc, palustrin-2Oa, and ranatuerin-2Ob and -2Oe from skin total RNA preparations from adult R. ornativentris and established a semi-quantitative RT-PCR system to measure the concentrations of these mRNAs. The levels of preprobrevinin-2 and preproranatuerin-2 mRNAs in the skin specimens of developing R. ornativentris larva were detectable only at stages later than the onset of metamorphosis and reached peaks at the stage of metamorphic climax. In contrast, prepropalustrin-2 mRNA was detected prior to the onset of metamorphosis and levels peaked at stages earlier than those of the other two mRNAs. In adult animals, preprobrevinin-2 and preproranatuerin-2 gene transcripts were detected at low levels in the small intestine and skeletal muscle but not in the stomach, liver, or kidney, whereas prepropalustrin-2 gene transcripts were detected at relatively high concentrations in all tissues examined. These results indicate that the expression of amphibian AMP genes is correlated with metamorphosis but is subjected to differential regulation. © 2009 Elsevier Inc. All rights reserved.

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