Gi Won Kim

Environmental risk assessment of toxicity exposure: High-throughput expression profiling

Recently, volatile organic compounds (VOCs) in outdoor air pollution have led to major public health problems and the identification of specific minimally invasive biomarkers for assessing environmental toxicant exposure has become increasingly important. However, research into the human health effects of inhalation exposure to VOCs remains insufficient. Using a microarray based approach, we identified and validated characteristic mRNA expression profiles in the human whole blood of workers exposed to VOCs (toluene, ethylbenzene, and xylene), which were then compared with genomic level expressions in workers not exposed to the toxicants. We surveyed 141 workers working in a chemical production factory, of which 66 were not exposed to VOCs. We identified 4384 characteristic discernible exposure indicator mRNAs for toluene, 1296 for ethylbenzene, and 5821 for xylene. Using these, we were able to discern those subjects from the control group to a higher accuracy, sensitivity, and specificity than when using urinary biomarkers. The results showed that altered levels of mRNA can be a reliable, novel, and minimally invasive biological indicator of occupational exposure to VOCs. Future research directions should consider the adverse effects of exposure to VOCs on epigenetic regulation.

Integrative analyses of differential gene expression and DNA methylation of ethylbenzene-exposed workers

Humans can absorb volatile organic compounds (VOCs), such as ethylbenzene (EB), via inhalation resulting in various effects such as hepatotoxicity and even carcinogenicity. Because occupational EB exposure occurs frequently in the synthetic rubber and plastic industry, its toxicity has been previously reported by various in vitro as well as in vivo animal studies. EB has also been classified as a group 2B possible carcinogen by the IARC. However, these studies investigated the toxicity of EB used a much higher dose than expected occupational exposure. Thus the results are likely to be far away from to be the basis for the validation of the toxicity of the EB exposure. Because the previous studies examine only phenotypes, it is still unclear what and how EB affects physiological pathways in the human body. Using microarray platform, we investigated the gene expression profiles and genomic methylation patterns of 66 human blood samples from employees of local plants that use EB. The affected genes were functionally analyzed using the DAVID Tool and IPA. The methylation array demonstrated that, compared to the control group, a total of 1446 genes were hypermethylated and 60 genes were hypomethylated. Among the 378 differentially expressed genes, 12 had proper epigenetic correlation with the altered methylation patterns. Our study demonstrated how EB exposure affects the human genome by investigating the gene expression profiles and DNA methylation profiles of blood cells. We expect that our results will help establish the genomic basis of the effect of EB exposure on humans.

Application of fluorescence melting curve analysis for dual DNA detection using single peptide nucleic acid probe.

Peptide nucleic acid (PNA) is an artificially synthesized polymer. PNA oligomers show greater specificity in binding to complementary DNAs. Using this PNA, fluorescence melting curve analysis (FMCA) for dual detection was established. Genomic DNA of Mycoplasma fermentans and Mycoplasma hyorhinis was used as a template DNA model. By using one PNA probe, M. fermentans and M. hyorhinis could be detected and distinguished simultaneously in a single tube. The developed PNA probe is a dual‐labeled probe with fluorescence and quencher dye. The PNA probe perfectly matches the M. fermentans 16s rRNA gene, with a melting temperature of 72°C. On the other hand, the developed PNA probe resulted in a mismatch with the 16s rRNA gene of M. hyorhinis, with a melting temperature of 44–45°C. The melting temperature of M. hyorhinis was 27–28°C lower than that of M. fermentans. Due to PNAs high specificity, this larger melting temperature gap is easy to create. FMCA using PNA offers an alternative method for specific DNA detection.

Identification of time-dependent biomarkers and effects of exposure to volatile organic compounds using high-throughput analysis.

Volatile organic compounds (VOCs) can be easily taken up by humans, leading to various diseases, such as respiratory system and central nervous system disorders. Environmental risk assessment is generally conducted using traditional tests, which may be time‐consuming and technically challenging. Therefore, analysis of the effects of VOCs, such as toluene, ethylbenzene, and xylene, may be improved by use of novel, high‐throughput methods, such as microarray analysis. In this study, we examined the effects of VOCs exposure in humans on gene expression and methylation using microarray analysis. We recruited participants who had short‐term exposure, long‐term exposure, or no exposure. We then analyzed changes in gene expression in blood samples from these participants. A total of 866 genes were upregulated, while 366 genes were downregulated in the short‐term exposure group. Similarly, in the long‐term exposure group, a total of 852 and 480 genes were up‐ or downregulated, respectively. Hierarchical clustering analysis was used to divide the clustered genes into nine clusters to investigate the expression of variations in accordance with the exposure period. And the methylation microarray was performed at the same time to see whether this expression variation is related to the epigenetic study. Finally, we have 5 genes that were upregulated and 12 genes that were downregulated, gradually and respectively, so these genes are expected to function as biomarkers of the duration of exposure to VOCs. Further research is required to determine the time‐dependent effects of VOCs on epigenetic regulation of gene expression.

Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences.

Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (Tm) value than DNA. Therefore, short PNA probes can have adequate Tm values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity.

Identification of potential biomarkers for xylene exposure by microarray analyses of gene expression and methylation

Xylene is volatile organic compound that has been reported to increase the incidence of cancer and various diseases related to the immune system, cardiovascular systems, respiratory and reproductive organs. However, there are currently few biomarkers in human cases. Using microarray, we analysed 10 participants for the xylene-exposure group and 10 controls that were not exposed to xylene. The two groups were compared in terms of expression levels and methylation patterns. We identified 6 genes that were down-regulated and hyper-methylated, and 132 that were up-regulated and hypo-methylated in the xylene- exposure group compared to control. We sorted out and 28 biomarker candidates were chosen using DAVID. And then, we used IPA to select the significant potential biomarkers in them. We used network analysis and selected 6 significant genes, and these 6 genes showed altered expression and methylation in xylene-exposure group, suggesting that they are suitable potential biomarkers for xylene exposure.

Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes

Abstract Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences.

Association analysis of toluene exposure time with high-throughput mRNA expressions and methylation patterns using in vivo samples.

The emission of volatile organic compounds (VOCs) resulting from outdoor air pollution can contribute to major public health problems. However, there has been limited research on the health effects in humans from the inhalation of VOCs. Therefore, this study conducted an in vivo analysis of the effects of toluene, one of the most commonly used chemicals in many industries, on gene expression and methylation over time using the high-throughput technique of microarray analysis. We separated participants into three groups (control, short-term exposure, and long-term exposure) to investigate the influence of toluene exposure time on gene expression. We then comprehensively analyzed and investigated the correlation between variations in gene expression and the occurrence of methylation. Twenty-six genes were upregulated and hypomethylated, while 32 genes were downregulated and hypermethylated. The pathways of these genes were confirmed to be associated with cell survival and the immune system. Based on our findings, these genes can help predict the effects of time-dependent exposure to toluene on human health. Thus, observations from our data may have implications for the identification of biomarkers of toluene exposure.

Differentiation of Scomber japonicus from Scomber scombrus by using a single locked nucleic acid probe.

Abstract Mackerel is marketed at prices according to the species type, Scomber japonicus and Scomber scombrus. Distinguishing these two species with the naked eye is difficult, and their differentiation becomes more difficult after they are processed by cooking, thereby leading to counterfeiting issues. Thus, in this study, we developed a method to differentiate S. japonicus from S. scombrus by detecting polymorphisms in mitochondrial 16 s rRNA gene by using fluorescence melting curve analysis and locked nucleic acid probes. Our method could distinguish S. japonicus from S. scombrus in a single experiment by using a single probe. The probes developed matched exactly with S. japonicus and had a melting temperature of 64 °C. However, the probes were mismatched with S. scombrus, resulting in a lower melting temperature of 46 °C. The high specificity of the locked nucleic acid probes resulted in this large difference in the melting temperatures.

Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).