High-concentration Epigallocatechin Gallate Treatment Causes Endoplasmic Reticulum Stress-mediated Cell Death in HepG2 Cells

안준익,정경지,고문정,신희정,정혜주,정호상 한국유전체학회 2009 Genomics & informatics Vol.7 No.2

Epigallocatechin gallate (EGCG), a well-known antioxidant molecule, has been reported to cause hepatotoxicity when used in excess. However, the mechanism underlying EGCG-induced hepatotoxicity is still unclear. To better understand the mode of action of EGCG-induced hepatotoxicity, we examined the effect of EGCG on human hepatic gene expression in HepG2 cells using microarrays. Analyses of microarray data revealed more than 1300 differentially expressed genes with a variety of biological processes. Upregulated genes showed a primary involvement with protein-related biological processes, such as protein synthesis, protein modification, and protein trafficking, while downregulated genes demonstrated a strong association with lipid transport. Genes involved in cellular stress responses were highly upregulated by EGCG treatment, in particular genes involved in endoplasmic reticulum (ER) stress, such as GADD153, GADD34, and ATF3. In addition, changes in genes responsible for cholesterol synthesis and lipid transport were also observed, which explains the high accumulation of EGCG-induced lipids. We also identified other regulatory genes that might aid in clarifying the molecular mechanism underlying EGCG-induced hepatotoxicity.

Analysis of Gene Expression in Mouse Spinal Cord-derived Neural Precursor Cells During Neuronal Differentiation

안준익,김수영,고문정,정혜주,정호상 한국유전체학회 2009 Genomics & informatics Vol.7 No.2

The differentiation of neural precursor cells (NPCs) into neurons and astrocytes is a process that is tightly controlled by complicated and ill-defined gene networks. To extend our knowledge to gene networks, we performed a temporal analysis of gene expression during the differentiation (2, 4, and 8 days) of spinal cord-derived NPCs using oligonucleotide microarray technology. Out of 32,996 genes analyzed, 1878 exhibited significant changes in expression level (fold change>2, p<0.05) at least once throughout the differentiation process. These 1878 genes were classified into 12 groups by k-means clustering, based on their expression patterns. K-means clustering analysis revealed that the genes involved in astrogenesis were categorized into the clusters containing constantly upregulated genes, whereas the genes involved in neurogenesis were grouped to the cluster showing a sudden decrease in gene expression on Day 8. Functional analysis of the differentially expressed genes indicated the enrichment of genes for Pax6-NeuroD signaling−TGFb-SMAD and BMP-SMAD−which suggest the implication of these genes in the differentiation of NPCs and, in particular, key roles for Nova1 and TGFBR1 in the neurogenesis/astrogenesis of mouse spinal cord.

Beta-amyloid의 분자생물학

안준익,이용성 한양대학교 의과대학 2001 한양의대 학술지 Vol.21 No.1

One of the characteristic neuropathological hallmarks of Alzheimer's disease(AD)is the extracellular accumulation of B-amyloid peptides(AB)in neuritic plaques. Experimental data indicate that different molecular forms of AB affect a wide array of neuronal and glial functions and thereby may lead to neuronal death in the nervous system. Whereas the fatal outcome of AB overproduction in transgenic cell lines, and of exogenous AB administration in numerous neurotoxicity models is well established, particular facets of a complex molecular cascade by which AB attack neural cells are still elusive. In the present review we summarize recent knowledge on mechanisms of AB aggregation, its role in AB neurotoxicity, and binding of AB peptides to putative neuronal and glial receptors.

국제적 조화를 이룬 안전성약리시험 지침 연구

안준익,차혜진,정호상,김은정,조혜영,김혜수,정은용 한국에프디시규제과학회(구 한국에프디시법제학회) 2009 FDC법제연구 Vol.4 No.2

Moraxella sp. CK-1의 세포외 Autolysins 특성 연구

안준익,김철호,최영길 한국미생물학회 1997 미생물학회지 Vol.33 No.1

남조류 분해세균으로 알려진 Moraxella sp. CK-1의 세포외 자가용균효소의 특성을 규명하였다. Moraxella sp. CK-1은 대수증기식 초기부터 세포외부로 자가용균효소를 분비하며, 이들 효소는 60-$70^{\circ}C$, pH 9.0의 최적 활성 조건을 나타내었다. 이들 효소는 $Na^{+}$, $K^{+}$, $Li^{+}$등의 이온에 의해 활성이 촉진되고, $Ba^{2+}$, $Mg^{2+}$, $Ca^{2+}$ 및 $Mn^{2+}$ 등의 이온에 의해 활성이 억제되며, 특히, $Fe^{2+}$와 $Cu^{2+}$ 이온 존재하에서는 효소의 활성이 거의 나타나지 않았다. Micrococcusluteus 세포를 기질로 이용하여 renaturing, SDS-PAGE를 수행한 결과 Moraxella sp. CK-1은 분자량이 각각 30,32,38 그리고 41 kDa인 4가지 효소를 분비하며, 이중 32와 41 kDa의 효소는 생장 초기부터 사멸기까지 지속적으로 배지에 분포하는 반면, 38 kDa의 효소는 대수증식기 중기 이전, 30 kDa의 효소는 대수증식기 중기 이후에 배지에 분포하였다. M. luteus의 SDS-insoluble peptidoglycan에 효소추출물을 처리하였을 때 반응액의 유리 아미노기 농도가 증가하는 것은 이들 효소가 N-acetylmuramyl-L-ananine amidase 또는 endopeptidase라는 사실을 시사한다. We studied the characteristics of extracellular autolysins from Moraxella sp. CK-1 which has been known to lyse the cyanobacterial cell walls. This bacterium excreted autolysins from the early exponential growth phase. These enzymes showed optimal action condition of 60-$70^{\circ}C$ and pH 9.0. Whereas $Na^{+}$, $K^{+}$ and $Li^{+}$ ions exhibited positive effect on the enzyme activity, $Ba^{2+}$, $Mg^{2+}$, $Ca^{2+}$ and $Mn^{2+}$ ions exhibited negative effect. Especially, $Fe^{2+}$ and $Cu^{2+}$ ions almost completely suppressed the activity. Four extracellular autolysins of 30, 32, 38 and 41 kDa were detected in renaturing SOS-PAGE gel containing 0.2% heat-killed Micrococcus luteus cells as substrate. Among these 4 autolysins, 2 enzymes of 32 and 41 kDa distributed in the culture medium throughout the experimental time, but the 38 kOa enzyme diminished and 30 kOa began to appear at mid-exponential growth phase. When SOS-insoluble peptidoglycan of M. luteus was treated with the autolysins of Moraxella sp. CK-l, the concentration of free amino groups in reaction mixture increased. This indicates that the autolysins are N-acetylmuramyl-L-alanine amidase or endopeptidase.

Effects of Mercuric Chloride on Gene Expression in NRK-52E Cells

안준익,백시연,신희정,고문정,정혜주,정호상 한국유전체학회 2010 Genomics & informatics Vol.8 No.1

Mercuric chloride, a model nephrotoxicant was used to elucidate time- and dose- dependent global gene expression changes associated with proximal tubular toxicity. Rat kidney cell lines NRK-52E cells were exposed for 2, 6 and 12 hours and with 3 different doses of mercuric chloride. Cell viability assay showed that mercuric chloride had toxic effects on NRK-52E cells causing 20% cell death (IC20) at 40μM concentration. We set this IC20 as high dose concentration and 1/5 and 1/25 concentration of LC20 were used as mid and low concentration, respectively. Analyses of microarray data revealed that 738 genes were differentially expressed (more than two-fold change and p<0.05) by low concentration of mercuric chloride at least one time point in NRK-52E cells. 317 and 2,499 genes were differentially expressed at mid and high concentration of mercuric chloride, respectively. These deregulated genes showed a primary involvement with protein trafficking (CAV2, CANX, ORO1B), detoxification (GSTs) and immunity and defense (HMOX1, NQO1). Several of these genes were previously reported to be up-regulated in proximal tubule cells treated with nephrotoxicants and might be aid in promoting the predictive biomarkers for nephrotoxicity.

Gene Expression Analysis for Statin-induced Cytotoxicity from Rat Primary Hepatocytes

고문정,안준익,신희정,김혜수,정혜주,정호성 한국유전체학회 2010 Genomics & informatics Vol.8 No.1

Statins are competitive inhibitors of hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase and used most frequently to reduce plasma cholesterol levels and to decrease cardiovascular events. However, statins also have been reported to have undesirable side effects such as myotoxicity and hepatotoxicity associated with their intrinsic efficacy mechanisms. Clinical studies recurrently reported that statin therapy elevated the level of liver enzymes such as ALT and AST in patients suggesting possible liver toxicity due to statins. This observation has been drawn great attention since statins are the most prescribed drugs and statin-therapy was extended to a larger number of high-risk patients. Here we employed rat primary hepatocytes and microarray technique to understand underlying mechanism responsible for statin-induced liver toxicity on cell level. We isolated genes whose expressions were commonly modulated by statin treatments and examined their biological functions. It is of interest that those genes have function related to response to stress in particular immunity and defense in cells. Our study provided the basic information on cellular mechanism of statin-induced cytotoxicity and may serve for finding indicator genes of statin -induced toxicity in rat primary hepatocytes.

Gene Expression Profiling in C57BL/6 Mice Treated with the Anorectic Drugs Sibutramine and Phendimetrazine and Their Mechanistic Implications

고문정,최효성,안준익,김소영,정호상,정혜주 한국유전체학회 2008 Genomics & informatics Vol.6 No.3

Recently, obesity has become a worldwide public health concern and the use of anorectic drugs has drastically increased. In this study, sibutramine and phendimetrazine, representative marketed anorectics, were repeatedly administered per os on a daily basis into C57BL/6 mice and the effects of these drugs on food intakes, body weight changes and gene expression profiles were monitored for up to following 7 days. Methamphetamine, which has a potent anorectic effect, was used as a positive control. Anorectic effects were sustained only for two days by phendimetrazine or methamphetamine, but for six days by sibutramine. The modulations of gene expressions in the hypothalamus and the striatum were investigated using microarrays on day 2 and day 7 post-administration, which corresponded to the anorectic period and a return of appetite respectively, for all three drugs tested. Differences in overall gene expression profiles in the stratum on day 2 for sibutramine and phendimetrazine seems to reflect difference between the two in terms of the onsets of drug tolerance. According to microarray findings, the Ankrd26 gene appears to have an important anorectic role, whereas the up-regulation of the olfaction system appeared to be involved in the drug tolerance of anorectics. The microarray data presented in this study demonstrates the usefulness of gene expression analysis for gathering information on the efficacy and safety of anorectic drugs.

Comparison of gene expression profiles in drug-withdrawn rats

차혜진,최문지,안준익,전설희,강호일,김은정,성원근,김형수,정호상 대한독성 유전단백체 학회 2016 Molecular & cellular toxicology Vol.12 No.2

The aim of the present study was to compare gene expression profiles in animals treated with abusable drugs. Wistar rats were treated with saline, morphine, chloral hydrate, or pentobarbital twice daily for 14 days. To induce neurotoxicity, a group of rats was treated with kainic acid. 24 hr after withdrawal, and a genome-wide DNA microarray analysis was conducted with the striata. A total of 57 gene expression patterns were altered in the three test groups; 35 of the 157 genes were selected when compared with the kainic acid treated group. Twenty-two of the 24 genes were functionally described in the gene library, and many were involved in long-term potentiation (LTP) and cell adhesion. The results were confirmed by quantitative reverse transcription-polymerase chain reaction analysis. Taken together, the selected genes may subserve identification of physical dependence biomarkers and contribute to determining the molecular mechanisms underlying physical dependence.

Developmental Neurotoxicity in vitro Screening Test Method that Measures Cell Viability and Proliferation using Human Neuroprogenitor Cells

윤남희,김은지,김학,김태성,강남희,윤혜성,안준익 한국동물실험대체법학회 2022 동물실험대체법학회지 Vol.16 No.1

Current developmental neurotoxicity tests are based on in vivo tests, and many kinds of chemicals cannot be evaluated due to restrictions such as the number, cost, and time of animals used in the test. Recently, in vitro test methods for screening test chemicals that may damage the nervous system are being studied. As an alternative test for developmental neurotoxicity, an in vitro test method that measures the endpoint of neurogenesis process is being studied; however, a single in vitro assay cannot completely cover all neurogenesis processes. Therefore, studies are being carried out to combine test methods as endpoints for key events in neurogenesis process and test battery study. In this study, we used human neuroprogenitor cells, ReNcell™ CX, to evaluate developmental neurotoxicity of test substances on cell viability (MTT assay) and proliferation (BrdU assay) by ELISA method. This method has the advantage of being able to quickly and easily evaluates many substances compared to previous methods using fluorescence microscope. First, we tested to optimize the test method using known antiproliferative/ non-antiproliferative chemicals, we were able to obtain 100% accuracy compared to the previous study. This was verified by confirming that it was appropriate to evaluate cell viability and proliferation using a human neural progenitor cells (ReNcell™ CX). In the utilization study, 18 test substances were evaluated. As a result, 11 test substances (Acrylamide, Bisphenol A, Carbamazepine, Chlorpyrifos, Diethylstilbestrol, Perfluorooctanoic Acid, Sodium arsenite, 3,3’,5,5 ‘-Tetrabromobisphenol A, Ibuprofen, Omeprazole, and Verapamil) were judged as substances with developmental neurotoxicity potential, and 7 test substances (Di (2-ethylhexyl) phthalate, Fluconazole, Permethrin, Atropine, Captopril, D-Mannitol, and Dinotefuran) were judged as non-developmental neurotoxicity substances. The sensitivity was 70% (7/10), specificity 50% (4/8), accuracy 61% (11/18), false positive rate 50% (4/8), and false negative rate 30% (3/10). These results show that it can be used as an in vitro screening test for developmental neurotoxicity that can measure and evaluate many substances within a short time compared to the existing in vivo tests. In addition, this test is a single in vitro screening test, and further studies are needed to evaluate it in combination with other in vitro test method.