Han-Seam Choi

Polycyclic aromatic hydrocarbons induce migration in human hepatocellular carcinoma cells (HepG2) through reactive oxygen species‐mediated p38 MAPK signal transduction

Although polycyclic aromatic hydrocarbons (PAHs) are carcinogenic and have been extensively studied with regard to tumor formation, few studies have investigated the involvement of these environmental chemicals in tumor migration and invasion. Polycyclic aromatic hydrocarbons induce reactive oxygen species (ROS) and activate MAPK signal transduction. The p38 signaling transduction pathway, one of the most typical MAPK pathways, plays an essential role in regulating cell migration. Therefore, we investigated whether three PAHs, benzo[a]anthracene (B[a]A), benzo[k]fluoranthene (B[k]F), and indeno[1,2,3‐c,d]pyrene (IND), induce migration in human hepatocellular carcinoma cell line HepG2 through ROS‐mediated p38 MAPK signal transduction. Reactive oxygen species generation and p38 MAPK activity both increased in a dose‐dependent manner and were prevented by SB203580, an inhibitor of p38 MAPK, and N‐acetylcysteine (NAC), a ROS scavenger. Expression of migration‐related genes was also increased by B[a]A, B[k]F, and IND in a dose‐dependent manner and was inhibited by SB203580 and NAC. The migration of HepG2 cells, observed using the Transwell migration assay, also increased in a dose‐dependent manner and was prevented by SB203580 and NAC. Our results indicate that the ROS‐mediated p38 MAPK signaling pathway plays an essential role in the PAH‐induced migration of HepG2 cells. (Cancer Sci 2011; 102: 1636–1644)

Identification of molecular signatures predicting the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs).

Assessing the potential carcinogenicity of human toxins represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk with limited reliability, and are expensive and time-consuming. To identify alternative prediction methods, we evaluated a transcriptomics-based human in vitro model to classify carcinogens by their modes of action. The aim of this study was to determine the transcriptomic response and identify specific molecular signatures of polycyclic aromatic hydrocarbons (PAHs), which can be used as predictors of carcinogenicity of environmental toxins in human in vitro systems. We found that characteristic molecular signatures facilitate identification and prediction of carcinogens. To evaluate the change in gene expression levels, human hepatocellular carcinoma (HepG2) cells were exposed to nine different PAHs (benzo[a]pyrene, dibenzo[a,h]anthracene, 3-methylcholanthrene, naphthalene, chrysene, phenanthrene, benzo[a]anthracene, benzo[k]fluoranthene, and indeno[1,2,3-c,d]pyrene) for 48 h. Unsupervised gene expression analysis resulted in a characteristic molecular signature for each toxin, and a supervised analysis identified 31 outlier genes as distinct molecular signatures distinguishing carcinogens from noncarcinogens. Further analysis and multi-classification revealed 430 genes as surrogate markers for predicting carcinogenic potencies of each PAH with 100% accuracy. Our results suggest that these expression signatures can be used as predictable and discernible surrogate markers for detecting and predicting PAH exposure, and their carcinogenic potential. Furthermore, the use of these markers can be more widely applied in combination with traditional techniques for assessing and predicting toxic exposure to PAHs.

Gene Expression Analysis Identifies DNA Damage-related Markers of Benzo(a)pyrene Exposure in HepG2 Human Hepatocytes

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is carcinogenic to humans. Although the environmental distribution and metabolism of BaP have been reported and many researchers are performing risk-assessment and toxicological studies of BaP by means of physical and chemical measurements, only a few studies have examined the expression of mRNAs and their functions in BaP-induced toxicity. Toxicogenomic technology, a new paradigm in toxicity screening, is a useful approach for evaluating the toxic properties of environmental pollutants. We analyzed gene expression profiles using human oligonucleotide chips and identified genes in human hepatocellular carcinoma (HepG2) cells whose expression changed > 1.5-fold after exposure to BaP. The expression of 4,048 and 3,926 genes was up-and down-regulated > 1.5-fold (P < 0.01), respectively, after exposure. Gene ontology (GO) analysis of these genes revealed significant enrichment in several key biological processes related to DNA damage, including DNA repair, cell cycle arrest, and apoptosis. We also performed a contrastive study of cellular effects in HepG2 cells exposed to BaP, and identified increased expression of related genes, cell cycle arrest, and apoptotic cells. These results suggest that genetic markers of BaP-induced toxicity may be molecular blueprints that can be more widely implemented in combination with more traditional techniques for assessment and prediction.

Analysis of dose-response to hexanal-induced gene expression in A549 human alveolar cells

The problems of analyzing dose effects on gene expression are gaining attention in toxicological research. Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and elucidating their modes of toxic action. In the present study, we focused on determining the dose-dependent alterations of gene expression profiles with hexanal exposure and we identified the possible biomarkers of hexanal in A549 human alveolar cells. A549 cells were exposed to a 5% inhibitory concentration (IC5) and a 20% inhibitory concentration (IC20) of hexanal for 48 h. Through microarray analysis using an oligonucleotide chip, we identified that the gene expression patterns were differentially shown in the control group and the hexanalexposed groups. The hexanal-exposed groups are more sensitive to gene alteration than the control group, and gene expressions are more significantly altered in the IC20 exposure group than in the IC5 exposure group. With clustering analysis of gene expression profiles, we identified 2,929 IC5− and 3,678 IC20-specific genes, and 302 dose-dependently expressed genes. Gene ontology (GO) analysis with 246 annotated genes of the 302 dosedependent expressed genes showed correlation with the key biological processes involved in neurological system processes, immune system development, cell activation, and cell-cell signaling. In conclusion, current study describes alterations in gene expression profiles in response to exposure to different doses of hexanal and related toxic pathways induced by significantly expressed genes. Moreover, novel genes and pathways that could potentially play a role in the prevention of respiratory disease due to aldehydes are identified.

Formation of a 3,4-diol-1,2-epoxide metabolite of benz[a]anthracene with cytotoxicity and genotoxicity in a human in vitro hepatocyte culture system

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that require metabolic activation to exert their carcinogenic effects. This study investigated the 3,4-diol-1,2-epoxide formation of benz[a]anthracene (BA) and its toxic effects in a human in vitro hepatocyte culture system. Both mRNA and protein expression of metabolic enzymes which can activate PAHs to carcinogenic forms increased after BA exposure in HepG2 cells and our quantitative analysis showed that the formation of BA-3,4-diol-1,2-epoxide in medium extracts increased in a time-dependent manner. We also performed several comparative studies which show that much lower concentrations of BA-3,4-diol-1,2-epoxide had stronger cytotoxicity and genotoxicity than higher doses of BA. These results suggest that BA is activated as the major carcinogenic metabolite 3,4-diol-1,2-epoxide, in human in vitro culture systems by metabolic enzymes and that this metabolite has stronger cytotoxic and genotoxic effects than its parent compound.

Transcriptome profile analysis of saturated aliphatic aldehydes reveals carbon number-specific molecules involved in pulmonary toxicity.

In the current study, we aimed to investigate the transcriptomic responses and identify specific molecular signatures of low-molecular-weight saturated aliphatic aldehydes (LSAAs). To evaluate the change in gene expression levels, A549 human alveolar epithelial cells were exposed to six LSAAs (propanal, butanal, pentanal, hexanal, heptanal, and octanal) for 48 h. Clustering analysis of gene expression data show that the low carbon number group (LCG; propanal, butanal, and pentanal) was distinguished from the high carbon number group (HCG; hexanal, heptanal, and octanal). Also, transcriptomic profiling indicates that the LCG exposure group was more sensitive in gene alterations than the HCG group. Supervised analysis revealed 703 LCG specific genes and 55 HCG specific genes. After gene ontology (GO) analysis on LCG specific genes, we determined several key pathways which are known as being related to increase pulmonary toxicity such as cytokine-cytokine receptor interaction and chemokine signaling pathway. However, we did not find pulmonary toxicity-related pathways through GO analysis on HCG specific genes. Genes that are expressed in only the low carbon LSAA exposure group were regarded as biomarkers of aldehyde-induced pulmonary toxicity. In conclusion, this study describes changes in gene expression profiles in the in vitro respiratory system in response to exposure to 6 LSAAs with different carbon numbers and relates these gene alterations to pulmonary toxicity-related pathways. Moreover, novel carbon number-specific genes and pathways can be more widely implemented in combination with the traditional technique for assessment and prediction of exposure to environmental toxicants.

Monitoring of deiodinase deficiency based on transcriptomic responses in SH-SY5Y cells.

Iodothyronine deiodinase types I, II, and III (D1, D2, and D3, respectively), which constitute a family of selenoenzymes, activate and inactivate thyroid hormones through the removal of specific iodine moieties from thyroxine and its derivatives. These enzymes are important in the biological effects mediated by thyroid hormones. The expression of activating and inactivating deiodinases plays a critical role in a number of cell systems, including the neuronal system, during development as well as in adult vertebrates. To investigate deiodinase-disrupting chemicals based on transcriptomic responses, we examined differences in gene expression profiles between T3-treated and deiodinase-knockdown SH-SY5Y cells using microarray analysis and quantitative real-time RT-PCR. A total of 1,558 genes, consisting of 755 upregulated and 803 downregulated genes, were differentially expressed between the T3-treated and deiodinase-knockdown cells. The expression levels of 10 of these genes (ID2, ID3, CCL2, TBX3, TGOLN2, C1orf71, ZNF676, GULP1, KLF9, and ITGB5) were altered by deiodinase-disrupting chemicals (2,3,7,8-tetrachlorodibenzo-p-dioxin, polychlorinated biphenyls, propylthiouracil, iodoacetic acid, methylmercury, β-estradiol, methimazole, 3-methylcholanthrene, aminotriazole, amiodarone, cadmium chloride, dimethoate, fenvalerate, octylmethoxycinnamate, iopanoic acid, methoxychlor, and 4-methylbenzylidene-camphor). These genes are potential biomarkers for detecting deiodinase deficiency and predicting their effects on thyroid hormone production.

Dose-response functional gene analysis by exposure to 3 different polycyclic aromatic hydrocarbons in human hepatocytes

Polycyclic aromatic hydrocarbons (PAHs) are known as carcinogen and have been studied to show modulation of gene expression by exposure to various PAHs. However, few studies have been reported on microarray analysis of dose-response relationships of gene expression patterns. For comprehensive examination of dose-response effects of PAHs on gene expression, we elicited the genes which were changed more than 1.5-fold by analysis of gene expression profiles in human hepatocellular carcinoma (HepG2) cells, exposed for 48 h to nontoxic (NT) and IC20 doses of 3 different PAHs (benzo[a]anthracene (B[a]A), benzo[k]fluoranthene (B[k]F) and indeno[1,2,3-c,d]pyrene (IND)) by using human oligonucleotide chip. Transcriptomic profiling shows different gene expression patterns in NT and IC20 exposure groups and shows higher sensitivity to gene alteration in IC20 exposure group than NT group. Through the clustering analysis of gene expression profiles, we identified 7 up- and 3 down-regulated NT dose specific genes and 401 up- and 562 downregulated IC20 dose specific genes. After Gene Ontology (GO) analysis on IC20 dose specific genes, we determined several key pathways which are known as related to increase hepatotoxicity such as metabolism of xenobiotics by cytochrome P450, Jak-STAT signaling pathway, cytokine-cytokine receptor interaction and complement and coagulation cascade. But we did not find hepatotoxicity-related pathways through GO analysis on NT dose-specific genes. Genes that are expressed in only IC20 exposure group were regarded as biomarker of PAHs-induced hepatotoxicity. In conclusion, this study describes changes in gene expression profiles in hepatocytes in response to exposure to 3 PAHs with different doses and relates these gene expression changes to hepatotoxicity related pathways. Moreover, potential new leads to genes and pathways that could play a role in liver disease prevention by PAHs were identified.

Identification of classifiers for increase or decrease of thyroid peroxidase activity in the FTC-238/hTPO recombinant cell line.

Thyroid peroxidase (TPO) plays an important role in thyroid hormone biosynthesis, as it catalyzes all of the essential steps in iodide organification. TPO activity can be detected using the guaiacol assay; however, this assay is complex and very time-consuming. Therefore, we focused on devising a simplified method using microarrays to detect changes in TPO activity, which is a target for disruption of the thyroid hormone axis. These experiments have systematically assessed the potential utility of transcriptomic end points as enhancements to the guaiacol assay. Previously, we demonstrated that benzophenone-2, benzophenone, perfluorooctane sulfonate, bisphenol A bis ether, and vinclozolin decreased TPO activity, and that dibutyl phthalate, carbaryl, dibenzo(a,h)anthracene, benzo(a)pyrene, and methylmercury increased TPO activity. In this work, we used human oligonucleotide chips to examine changes in the gene expression profile of FTC-238 human follicular thyroid carcinoma cells expressing human recombinant TPO, after exposure of the cells to TPO activity-disrupting agents. We identified 362 classifiers that could predict the effect of the toxicants on TPO activity with about 70% accuracy. These classifiers are potential markers for predicting the effects of chemicals on thyroid hormone production.

Benzo[k]fluoranthene-induced changes in miRNA-mRNA interactions in human hepatocytes

Toxicology studies assessing the risk of environmental toxicants in humans frequently use in vitro systems in combination with transcriptomics to characterize toxic responses. Thus far, changes have mostly been investigated at the mRNA level. Recently, microRNAs (miRNAs) have attracted attention because they are powerful negative regulators of mRNA levels and thus may be responsible for the modulation of important mRNA networks implicated in toxicity. This study aimed to identify possible miRNA-mRNA networks as novel interactions at the gene expression level after exposure to environmental toxicants. Benzo[k]fluoranthene (BF), a polycyclic aromatic hydrocarbon that is ubiquitously distributed throughout the environment, was used. We analyzed mRNA and miRNA profiles in HepG2 cells, a human liver cell line, using a human oligonucleotide chip. Changes in miRNA expression in response to BF, in combination with multiple alterations of mRNA levels, were observed. Many of the altered mRNAs were targets of altered miRNAs. Using gene ontology (GO) and KEGG pathway analysis, we determined the relevance of such miRNA deregulation to carcinogenicity. This revealed five miRNAs that appear to participate in specific BFresponsive pathways relevant to genotoxicity and carcinogenicity, such as DNA damage repair, apoptosis, cancer, VEGF signaling, and Jak-STAT signaling. Our results indicate that miR-146a, miR-365, let-7f, miR-199b-5p, and miR-30c-1* are novel players in the BF response. Therefore, this study demonstrates the added value of an integrated miRNA-mRNA approach for identification of the molecular mechanisms induced by BF in an in vitro human model.