• Title/Summary/Keyword: Chemical Assay

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Implications for the Predictivity of Cell-Based Developmental Toxicity Assays Developed Two Decades Apart

  • Kawamura, Satoshi;Horie, Nobuyuki;Okahashi, Noriko;Higuchi, Hashihiro
    • Toxicological Research
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    • v.35 no.4
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    • pp.343-351
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    • 2019
  • Many in vitro developmental toxicity assays have been proposed over several decades. Since the late 1980s, we have made intermittent attempts to introduce in vitro assays as screening tests for developmental toxicity of inhouse candidate products. Two cell-based assays which were developed two decades apart were intensively studied. One was an assay of inhibitory effects on mouse ascites tumor cell attachment to a concanavalin A-coated plastic sheet surface (MOT assay), which we studied in the early days of assay development. The other was an assay of inhibitory effects on the differentiation of mouse embryonic stem cell to beating heart cells (EST assay), which we assessed more recently. We evaluated the suitability of the assays for screening in-house candidates. The concordance rates with in vivo developmental toxicity were at the 60% level. The EST assay classified chemicals that inhibited cell proliferation as embryo-toxic. Both assays had a significant false positive rate. The assays were generally considered unsuitable for screening the developmental toxicity of our candidate compounds. Recent test systems adopt advanced technologies. Despite such evolution of materials and methods, the concordance rates of the EST and MOT systems were similar. This may suggest that the fundamental predictivity of in vitro developmental toxicity assays has remained basically unchanged for decades. To improve their predictivity, in vitro developmental toxicity assays should be strictly based on elucidated pathogenetic mechanisms of developmental toxicity.

Antimutagenic Effects of Ginsenoside Rb$_1$, Rg$_1$ in the CHO-K1 Cells by Benzo[a]pyrene with Chromosomal Aberration Test and Comet Assay

  • Kim, Jong-Kyu;Kim, Soo-Jin;Rim, Kyung-Taek;Cho, Hae-Won;Kim, Hyeon-Yeong;Yang, Jeong-Sun
    • Molecular & Cellular Toxicology
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    • v.5 no.2
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    • pp.126-132
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    • 2009
  • The usage and types of chemicals are advancing, specializing, large-scaled increasing, and new chemical exposed workers are concerning to occupational disease. The generation of reactive oxygen in the body from carcinogen, mutation and DNA damage in cancer is protected by natural antioxidants (phytochemicals) with antimutagenic effect. There were many reports of ginsenoside Rb$_1$, Rg$_1$ grievances of the genetic mutation to suppress the effect confirm the genetic toxicity test with chromosomal aberration test and the Comet (SCGE) assay confirmed the suppression effect occurring chromosomal DNA damage. We had wanted to evaluate the compatibility and sensitivity between the chromosomal aberration (CA) test and the Comet assay. We used the CA test and Comet assay to evaluate the anti-genotoxicity of ginsenoside Rb$_1$ and Rg$_1$, in CHO-K1 (Chinese hamster ovary fibroblast) cell in vitro, composed negative control (solvent), positive control (benzo[a]pyrene), test group (carcinogen+variety concentration of ginsenoside) group. The positive control was benzo[a]pyrene (50 $\mu$M), well-known carcinogen, and the negative control was the 1 % DMSO solvent. The test group was a variety concentration of ginsenoside Rb$_1$, Rg$_1$ with 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10%. In chromo-somal aberration test, we measured the number of cells with abnormally structured chromosome. In Comet assay, the Olive tail moment (OTM) and Tail length (TL) values were measured. The ratio of cell proliferation was increased 8.3% in 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10% Rb$_1$ treated groups, and increased 10.4% in 10$^{-10}$%, 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1% Rg$_1$ treated groups. In the CA test, the number of chromosomal aberration was decreased all the Rb$_1$ and Rg$_1$ treated groups. In the Comet assay, the OTM values were decreased in all the Rb$_1$ and Rg$_1$ treated groups. To evaluate the compatibility between CA and Comet assay, we compared the reducing ratio of chromosomal abnormalities with its OTM values, it was identified the antimutagenicity of ginsenoside, but it was more sensitive the CA test than the Comet assay. Ginsenoside Rb$_1$ and Rg$_1$ significantly decrease the number of cells with chromosomal aberration, and decrease the extent of DNA migration. Therefore, ginsenoside Rb$_1$, Rg$_1$ are thought as an antioxidant phytochemicals to protect mutagenicity. The in vitro Comet assay seems to be less sensitive than the in vitro chromosomal aberration test.

Searching of Possible Target Enzymes for Herbicide Development using Commercial Plant-Specific Inhibitors (식물 특정효소저해제의 생물활성 조사에 의한 신규제초제 작용점 탐색)

  • Hwan, In-Taek;Choi, Jung-Sup;Park, Sang-Hee;Lee, Kwan-Hwi;Lee, Byung-Hoi;Hong, Kyung-Sik;Cho, Kwang-Yun
    • The Korean Journal of Pesticide Science
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    • v.5 no.1
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    • pp.36-45
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    • 2001
  • This study was conducted to search new target enzymes of novel herbicide candidate. Total of 107 biochemical inhibitors reported to inhibit over than 100 different plant enzymes were purchased from commercial chemical companies. 15 inhibitors and 34 enzymes were selected by germination assay, seedling assay, wheat leaf disc assay, and whole plant assay. Among them, seven compounds of purine, phehyl-hydrazine, o-phenanthroline, oleylamine, dicyclohexylcarbodiimide, 7,8-benzoquinoline, and aminooxyacetic acid showed high herbicidal activity in the whole plant assay under greenhouse while 7,8-benzoquinone, 8-hydroxyquinoline, 2,2'-dipyridyl, and o-phenanthroline inhibited seed germination of barnyardgrass, rice, and tomato at concentrations of 1.25 to $5{\mu}M$. The compounds of 7,8-benzoquinoline, chlorpromazine, cyanuric fluoride, 4-methylpyrazole, oleylamine, tranylcypromine, and trifluoperazine inhibited the growth of cyanobacteria at 30 to $100{\mu}M$. The compounds of dicyclohexylcarbodiimide and chlorpromazine exhibited whitening effect on tile wheat leaf disc at $100{\mu}M$. These results suggest that the plant-specific enzyme inhibitors which have biological activities may supply the target enzyme for developing new herbicide candidate.

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Evaluation of the Genetic Toxicity of Synthetic Chemical (XVII) -In vitro Mouse Lymphoma Assay and In vitro Supravital Micronucleus Assay with 1, 2-Dichlorobenzene

  • Kim, Youn-Jung;Ryu, Jae-Chun
    • Molecular & Cellular Toxicology
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    • v.3 no.2
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    • pp.113-118
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    • 2007
  • Chlorobenzenes due to their acute toxicity and the capability of bioaccumulating are of great health and environmental concern. Especially, 1, 2-dichlorobenzene (CAS No. 95-50-1) is used for organic synthesis, dye manufacture, as a solvent and for other applications in chemical industry. Adverse effects of 1, 2-dichlorobenzene includes increases in liver and kidney weights and hepatotoxicity. In this study, we evaluated the genetic toxicity of 1, 2-dichlorobenzene with more advanced methods, in vitro mouse lymphoma assay $tk^{+/-}$ gene assay (MLA) and in vitro mouse supravital micronucleus (MN) assay. 1, 2-Dichlorobenzene appeared the significantly positive results and the induction of large mutant colonies only in the presence of metabolic activation system with MLA. But in vitro testing of 1, 2-dichlorobenzene yielded negative results with supravital MN assay. These results suggest that 1, 2-dichlorobenzene may play a mutagen rather than clastogen in vitro mammalian system.

The Effect of Metallothionein on the Activity of Enzymes Invelved in Remival of Reactive Oxygen Species

  • Go, Mun Ju;Kim, Hui Jeong
    • Bulletin of the Korean Chemical Society
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    • v.22 no.4
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    • pp.362-366
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    • 2001
  • To show the effects of metallothionein (MT) on the activity of enzymes involved in the removal of reactive oxygen species, MT has been added to the assay systems of superoxide dismutase (SOD), catalase and peroxidase. We have used assay systems of SOD based on NADPH oxidation and nitrite formation from hydroxylammonium chloride as an assay of superoxide breakdown rate. The two assay systems showed different results at the high concentration of MT. MT showed the scavenging of superoxide in the SOD assay system in the presence and absence of SOD. MT added to the SOD assay system behaved as an activator of SOD, but apo-MT behaved as an inhibitor. When MT was added to the assay system in the presence of a fixed amount of SOD, the breakdown rate of superoxide increased. The effects of MT on the decomposition of hydrogen peroxide and the activity of catalase and peroxidase decomposing hydrogen peroxide were evaluated. MT decreased the activities of catalase and peroxidase. We have concluded that the function of MT as an antioxidant might effect the level of superoxide scavenging and not the level of hydrogen peroxide.

A MALDI-MS-based Glucan Hydrolase Assay Method for Whole-cell Biocatalysis

  • Ahn, Da-Hee;Park, Han-Gyu;Song, Won-Suk;Kim, Seong-Min;Jo, Sung-Hyun;Yang, Yung-Hun;Kim, Yun-Gon
    • Microbiology and Biotechnology Letters
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    • v.47 no.1
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    • pp.69-77
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    • 2019
  • Screening microorganisms that can produce glucan hydrolases for industrial, environmental, and biomedical applications is important. Herein, we describe a novel approach to perform glucan hydrolase screening-based on analysis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) spectra-which involves degradation of the oligo- and polysaccharides. As a proof-of-concept study, glucan hydrolases that could break down glucans made of several glucose units were used to demonstrate the MALDI-MS-based enzyme assay. First, the enzyme activities of ${\alpha}$-amylase and cellulase on a mixture of glucan oligosaccharides were successfully discriminated, where changes of the MALDI-MS profiles directly reflected the glucan hydrolase activities. Next, we validated that this MALDI-MS-based enzyme assay could be applied to glucan polysaccharides (i.e., pullulan, lichenan, and schizophyllan). Finally, the bacterial glucan hydrolase activities were screened on 96-well plate-based platforms, using cell lysates or samples of secreted enzyme. Our results demonstrated that the MALDI-MS-based enzyme assay system would be useful for investigating bacterial glucoside hydrolases in a high-throughput manner.

DNA Damage Effect of Botanical Insecticides Using Chinese Hamster Lung Cells

  • Kim, Areumnuri;Jeong, Mihye;Park, Kyung-Hun;Chon, Kyongmi;Cho, Namjun;Paik, Min Kyoung
    • Korean Journal of Environmental Agriculture
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    • v.34 no.4
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    • pp.350-354
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    • 2015
  • BACKGROUND: Botanical insecticides, especially Azadirachta Indica extract (AIE) and Sophorae radix extract (SRE) are widely used in Agriculture field. In our previous studies on genotoxicity test of AIE and SRE samples, a suspicious clastogenic properties was shown. Herein, we investigated the DNA damage effect of these botanical insecticide samples through the in vitro comet assay. METHODS AND RESULTS: Chinese hamster lung (CHL) fibroblast cell line was used, and methyl methanesulphonate was as positive control. Respective two samples of AIE and SRE were evaluated using Single Cell Gel Electrophoresis (Comet) assay and measured as the Olive tail moment (OTM). Results from this study indicated that all tested AIE and SRE samples did not show DNA damage in comet assay using CHL cells, compared with control. CONCLUSION: AIE and SRE samples used in this study were not cause genetic toxicity and are suitable for use as organic materials.