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Genetic Toxicity Test of o-Nitrotoluene by Ames, Micronucleus, Comet Assays and Microarray Analysis
Lee, Eun-Mi,Lee, So-Youn,Lee, Woo-Sun,Kang, Jin-Seok,Han, Eui-Sik,Go, Seo-Youn,Sheen, Yhun-Yong,Kim, Seung-Hee,Park, Sue-Nie The Korean Society of Toxicogenomics and Toxicopro 2007 Molecular & cellular toxicology Vol.3 No.2
o-Nitrotoluene is used to synthesize artificial dyes and raw materials of urethane resin. In this study, we have carried out in vitro genetic toxicity tests and microarray analysis to understand the underlying mechanisms and the mode of action of toxicity of onitrotoluene. TA1535 and TA98 cells were treated with o-nitrotoluene to test its toxicity by basic genetic toxicity test. Ames and two new in vitro micronucleus and COMET assays were applied using CHO cells and L5178Y cells, respectively. In addition, microarray analysis of differentially expressed genes in L5178Y cells in response to o-nitrotoluene was analyzed using Affymatrix genechip. The result of Ames test was that o-nitrotoluene treatment did not increase the mutations both in base substitution strain TA1535 and in frame shift TA98. o-Nitrotoluene has not increased micronuclei in CHO cells. But onitrotoluene increased DNA damage in L5178Y cell. Two-hundred two genes were initially selected as differentially expressed genes in response to o-nitrotoluene by microarray analysis and forty four genes among them were over 2 times of log fold changed. These forty four genes could be candidate biomarkers of genetic toxic action of o-nitrotoluene related to induction of mutation and/or induction of micronuclei and DNA damage. Further confirmation of these candidate markers related to the DNA damage will be useful to understand the detailed mechanism of action of o-nitrotoluene.
Lee, So-Yeon,Choi, Jung-Yun,Kim, Young-Lim,Jeong, Hye-Sung,Park, Young-Nam,Bae, Jei-Jun,Baek, Sun-Young,Shin, Jin-Ho,Lee, Seok-Ho,Park, Sue-Nie 한림대학교 환경·생명과학연구소 2001 일송 의학ㆍ생명과학 심포지엄 Vol.- No.3
Background: Human papillomavirus-like particles (HPV VLPs) obtained by self-assembly of L1 or L1 and L2 proteins produced by recombinant DNA technology in insect cells are one of promising prophylactic vaccine candidates to prevent genital HPV infection and the subsequent development of cervical cancer. Insect cells such as Sf9 cells are susceptible to arboviruses of which adventitious contamination during the production process may pose safety concern. The objective of the study was to evaluate viral clearance in the currently available purification procedures for HPV16 L1 VLPs produced from recombinant baculovirus-infected Sf9 cells using Japanese encephalitis virus (JEV) as a model. Methods: HPV16 L1 VLPs were purified by the following procedure: detergent lysis using PBS-0.5% NP40, sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. Each purification procedure was evaluated to determine a reduction factor (Rf) for JEV clearance by infectivity titration and a real-time quantitative RT- PCR assay following deliberate addition (spiking) of JEV. The results of the infectivity titration and real-time quantitative RT-PCR assay were expressed as median tissue culture infectious dose (TCID50). Results: Detergent treatment and CsCl density centrifugation were found to be effective in the JEV clearance, which resulted in the reduction of 6.76 and 4.37 log viral infectivity, respectively. The overall Rf for JEV clearance in the purification process of HPV VLPs was 12.76 log viral infectivity. Conclusions: The results of the study suggest that the above purification procedure for HPV VLPs produced from recombinant baculovirus-infected Sf9 cells will be effective in the clearance (inactivation and/or removal) of enveloped arboviruses with a buoyant density of 1.23 g/ml or below.
Park, Sue Nie,Lee, Kyung Ae,Cho, Young Sik,Choe, Yong Kyung,Yoon, Do Young,Kwon, Dur Han,Kang, Jeong Woo,Cho, Cheong Weon,Cho, Min Chul,Shim, Jung Hyun 생화학분자생물학회 2002 BMB Reports Vol.34 No.1
The human papillomavirus E7 pmtein can form a specific complex with a retinoblastoma tumor suppressor gene product (p105-Rb) that results in the release of the E2F transcription factor, which is critical for the growth-deregulation and transforming properties of the viral E7 oncoprotein. In an attempt to apply interaction between the E7 oncoprotein and a target cellular protein Rb for an in vitro screening system for drugs against human papillomavirus infection, we primarily investigated the E7Rb binding through a pull down assay and enzyme-linked immunosorbent assay. The pull down assay showed that both glutathione S-transferase-tagged E7 and His-tagged E7 immobilized on resins specifically produced complexes with bacterially expressed Rb in a dose-dependent manner, as determined by immunoblot analyses. This result coincided with that of an enzyme-linked immunosorbent assay, which is a useful system for the mass screening of potential drugs. Taken together, this screening system (based on the interaction between E7 and Rb) can be a promising system in the development of drugs against cervical cancers caused by human papillomavirus infection.
Yoen Jung Lee,권호정,In-Kwon Choi,신윤용,Sue Nie Park 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.3
To identify specific biomarkers generated upon exposure of L5178Y mouse lymphoma cells to carcinogens, 2-DE and MALDI-TOF MS analysis were conducted using the cellular proteome of L5178Y cells that had been treated with the known carcinogens, 1,2-dibromoethane and O-nitrotoluene and the noncarcinogens, emodin and D-mannitol. Eight protein spots that showed a greater than 1.5-fold increase or decrease in intensity following carci-nogen treatment compared with treatment with noncarci-nogens were selected. Of the identified proteins, we fo-cused on the candidate biomarker ERM-binding phosphoprotein 50 (EBP50), the expression of which was specifically increased in response to treatment with the carcinogens. The expression level of EBP50 was determined by western analysis using polyclonal rabbit anti-EBP50 antibody. Further, the expression level of EBP50 was increased in cells treated with seven additional carcinogens, verifying that EBP50 could serve as a specific biomarker for carcinogens.
Moesin Is a Biomarker for the Assessment of Genotoxic Carcinogens in Mouse Lymphoma
Yoen Jung Lee,권호정,In-Kwon Choi,신윤용,Sue Nie Park 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.2
1,2-Dibromoethane and glycidol are well known genotoxic carcinogens, which have been widely used in industry. To identify a specific biomarker for these car-cinogens in cells, the cellular proteome of L5178Y mouse lymphoma cells treated with these compounds was ana-lyzed by 2-dimen-sional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry (MS). Of 50 protein spots showing a greater than 1.5-fold increase or decrease in intensity compared to control cells on a 2-D gel, we fo-cused on the candidate biomarker moesin. Western analysis using monoclonal rabbit anti-moesin confirmed the identity of the protein and its increased level of expression upon exposure to the carcinogenic compounds. Moesin expression also increased in cells treated with six additional genotoxic carcinogens, verifying that moesin could serve as a biomarker to monitor phenotypic change upon exposure to genotoxic carcinogens in L5178Y mouse lymphoma cells.