Ho-Sang Jeong

Dependence Potential of the Synthetic Cannabinoids JWH-073, JWH-081, and JWH-210: In Vivo and In Vitro Approaches.

Synthetic cannabinoids (CBs) such as the JWH series have caused social problems concerning their abuse liability. Because the JWH series produces euphoric and hallucinogenic effects, they have been distributed illegally under street names such as “Spice” and “Smoke”. Many countries including Korea have started to schedule some of the JWH series compounds as controlled substances, but there are a number of JWH series chemicals that remain uncontrolled by law. In this study, three synthetic CBs with different binding affinities to the CB1 receptor (JWH-073, 081, and 210) and Δ9-tetrahydrocannabinol (Δ9-THC) were evaluated for their potential for psychological dependence. The conditioned place preference test (unbiased method) and self-administration test (fixed ratio of 1) using rodents were conducted. Ki values of the three synthetic cannabinoids were calculated as supplementary data using a receptor binding assay and overexpressed CB1 protein membranes to compare dependence potential with CB1 receptor binding affinity. All mice administered JWH-073, 081, or 210 showed significantly increased time spent at unpreferred space in a dose-dependence manner in the conditioned place preference test. In contrast, all tested substances except Δ9-THC showed aversion phenomenon at high doses in the conditioned place preference test. The order of affinity to the CB1 receptor in the receptor binding assay was JWH-210 > JWH-081 >> JWH-073, which was in agreement with the results from the conditioned place preference test. However, no change in self-administration was observed. These findings suggest the possibility to predict dependence potential of synthetic CBs through a receptor binding assay at the screening level.

Diclofenac, a Non-steroidal Anti-inflammatory Drug, Inhibits L-type Ca2+ Channels in Neonatal Rat Ventricular Cardiomyocytes

A non-steroidal anti-inflammatory drug (NSAID) has many adverse effects including cardiovascular (CV) risk. Diclofenac among the nonselective NSAIDs has the highest CV risk such as congestive heart failure, which resulted commonly from the impaired cardiac pumping due to a disrupted excitation-contraction (E-C) coupling. We investigated the effects of diclofenac on the L-type calcium channels which are essential to the E-C coupling at the level of single ventricular myocytes isolated from neonatal rat heart, using the whole-cell voltage-clamp technique. Only diclofenac of three NSAIDs, including naproxen and ibuprofen, significantly reduced inward whole cell currents. At concentrations higher than 3 microM, diclofenac inhibited reversibly the Na(+) current and did irreversibly the L-type Ca(2+) channels-mediated inward current (IC(50)=12.89+/-0.43 microM) in a dose-dependent manner. However, nifedipine, a well-known L-type channel blocker, effectively inhibited the L-type Ca(2+) currents but not the Na(+) current. Our finding may explain that diclofenac causes the CV risk by the inhibition of L-type Ca(2+) channel, leading to the impairment of E-C coupling in cardiac myocytes.

Effects of nortriptyline on QT prolongation: a safety pharmacology study.

Nortriptyline, a second-generation tricyclic antidepressant, is an active metabolite of amitriptyline. Amitriptyline induces QT prolongation and torsades de pointes (TdP), which causes sudden death. We studied the cardiovascular safety of nortriptyline, including QT prolongation risk. We examined the effects of nortriptyline on the cardiovascular system in vivo and in vitro in accordance with the ICH-S7B guideline. We tested its effect on QT interval in conscious telemetered dogs. We also performed in vitro electrophysiological studies on hERG tail currents using stably transfected human embryonic kidney 293 (HEK293) cells. Action potential parameters were studied in isolated rabbit purkinje fibers. Nortriptyline dose-dependently blocked hERG current, with a tail IC50 value of 2.20 ± 0.09 μM (n = 4). In the APD assay, total amplitude, Vmax, and resting membrane potential were not significantly changed by 1 μM nortriptyline, but nortriptyline at 0.3 and 1 μM shortened APD50 and APD90. Nortriptyline did not affect QTcV at 2 or 6 mg/kg, but slightly increased QTcV at 20 mg/kg. In conclusion, it is unlikely that nortriptyline affects the ventricular repolarization process at therapeutic dosages.

Changes of miRNA and mRNA expression in HepG2 cells treated by epigallocatechin gallate

Epigallocatechin gallate (EGCG), a major type of green tea polyphenol, has been reported to cause hepatotoxicity when used in excess. Micro-RNAs (miRNAs) are small non-coding RNA molecules that functions as negative regulators of gene expression. They play an important role in the regulation of genes involved in the cellular and molecular responses to toxicants. To examine miRNA regulatory effect on global gene expression in EGCG-treated HepG2 cells, we performed pair-wise correlation coefficient analysis on expression levels of 22 miRNA and 27,419 mRNA, and observed negative correlations (r< −0.7) between miRNA and mRNA. We identified a total of 234 negative correlated miRNA-mRNA pairs. Gene Ontology analysis on the miRNA-correlated genes revealed significant enrichment in the several biological processes related to organic acid metabolic process, carboxylic acid metabolic process and cellular protein catabolic process. Connectivity map analysis also revealed that the expression signatures of EGCG were very similar to those of polyphenol gossypol which is hepatotoxic to animal and human.

Identification of classifier genes for hepatotoxicity prediction in non steroidal anti inflammatory drugs

Toxicogenomics has the potential to be used for the regulatory decision making to predict toxicity in developing new drugs. We have identified the classifiers for hepatotoxicity prediction in nonsteroidal anti inflammatory drugs (NSAIDs) through analyzing differential gene expression profiles of hepatotoxic and nonhepatotoxic compounds using HepG2 cell. 100 μM of 8 hepatotoxic and 8 nonhepatotoxic NSAIDs were treated to HepG2 cell and the analysis of gene expression changes after 24 h allowed a set of genes to be identified differentiating hepatotoxicants from nonhepatotoxicants by statistical method. The hepatotoxicity prediction model was built using the selected 77 genes. These genes and pathways, commonly regulated by hepatotoxicants, may be indicative of the early characterization of hepatotoxicity and possibly predictive of later hepatotoxicity onset. 4 test compounds including hepatotoxic and nonhepatotoxic NSAIDs were used for validating the prediction model and the accuracy was 100%. Given that the specificity and sensitivity showed 100%, these are the most precise classifiers identified until now.

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

Abstract 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 al-so have been reported to have undesirable side effects such as myotoxicity and hepatotoxicity associated with their intrinsic efficacy mechanisms. Clinical studies re-currently reported that statin therapy elevated the level of liver enzymes such as ALT and AST in patients sug-gesting possible liver toxicity due to statins. This ob-servation 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 respon-sible for statin-induced liver toxicity on cell level. We isolated genes whose expressions were commonly modulated by statin treatments and examined their bio-logical functions. It is of interest that those genes have function related to response to stress in particular im-munity and defense in cells. Our study provided the ba-sic information on cellular mechanism of statin-induced cytotoxicity and may serve for finding indicator genes of statin -induced toxicity in rat primary hepatocytes. Keywords: cytotoxicity, gene expression, hepatocyte, microarray, statin

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

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.

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

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 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

ArrayQue: The comprehensive transcriptome data analysis tool

We have developed microarray analysis pipeline software for covering the entire process of transcriptome data analysis. This software, part of the Korea Toxicogenomics Integrated System (KOTIS), is freely distributed to users who upload their microarray data to the KOTIS database, which is operated under the server system of the National Institute of Toxicological Research (NITR). The uploaded microarray data can be downloaded by users through a web search interface within KOTIS and are used as input data of the analysis software. The software, which consists of four major analysis modules and one meta-analysis module, is connected to a gene-related annotation database through the web. Major analysis modules consist of (1) data import and preprocessing, (2) differentially expressed gene finding, (3) clustering analysis, and (4) classification analysis. A gene-related annotation database provides the biological meanings of the analysis results. A highly standardized analysis flow, from data import to differentially expressed gene finding, can be easily implemented using the interface series of a run wizard. The KOTIS system and analysis software are accessible at http://kotis.nitr.go.kr.

KOTIS: Korea Toxicogenomics Integrated System-The intelligent data warehouse for systems toxicology

KOTIS (Korea Toxicogenomics Integrated System) is the data warehouse for toxicogenomics research. It contains over 1,700 microarray experiments involved in 40 research projects. The main characteristic of KOTIS is that it has quality inspection modules for evaluating the quality of each microarray data. Furthermore, KOTIS automatically selects significant genes from the given experimental sets and provides well-annotated information with users. These powerful functions are very useful because users can easily identify the quality and the characteristics of each experiment without further data analysis. KOTIS is available at http://kotis.nifds.go.kr/.