Volatile Organic Compounds (VOCs) have been shown to cause nervous system disorders through skin contact or respiration, and also cause foul odors even at low densities in most cases. Also, as a compound itself, VOCs are directly harmful to the environment and to the human body, and may participate ...
Volatile Organic Compounds (VOCs) have been shown to cause nervous system disorders through skin contact or respiration, and also cause foul odors even at low densities in most cases. Also, as a compound itself, VOCs are directly harmful to the environment and to the human body, and may participate in photochemical reactions in air to create secondary pollutants. In this study, HL-60 cells were treated with volatile organic compounds, including ethylbenzene and trichloroethylene, at a value of $IC_50$. Then, the in house-prepared Human HazChem arrayer was utilized in order to compare the gene expression between the two VOCs. After hybridization, 8 upregulated genes and 8 downregulated genes were discovered in the HazChem array. The upregulated genes were identified as SG15, TNFSF10, PRNP, ME1, NCOA4, SRXN1, TXNRD1, and XBP1. The downregulated genes were identified as MME, NRF1, PRARBP, CALCA, CRP, BAX, C7 or f40, and FGFR1. Such results were highly correlated with the quantitative RT-PCR results. The majority of the 16 genes were related with the characteristics of VOCs, including respiratory mechanism, apoptosis, and carcinogenesis-associated genes. Our data showed that our human HazChem array can be used to monitor hazardous materials via gene expression profiling.
Volatile Organic Compounds (VOCs) have been shown to cause nervous system disorders through skin contact or respiration, and also cause foul odors even at low densities in most cases. Also, as a compound itself, VOCs are directly harmful to the environment and to the human body, and may participate in photochemical reactions in air to create secondary pollutants. In this study, HL-60 cells were treated with volatile organic compounds, including ethylbenzene and trichloroethylene, at a value of $IC_50$. Then, the in house-prepared Human HazChem arrayer was utilized in order to compare the gene expression between the two VOCs. After hybridization, 8 upregulated genes and 8 downregulated genes were discovered in the HazChem array. The upregulated genes were identified as SG15, TNFSF10, PRNP, ME1, NCOA4, SRXN1, TXNRD1, and XBP1. The downregulated genes were identified as MME, NRF1, PRARBP, CALCA, CRP, BAX, C7 or f40, and FGFR1. Such results were highly correlated with the quantitative RT-PCR results. The majority of the 16 genes were related with the characteristics of VOCs, including respiratory mechanism, apoptosis, and carcinogenesis-associated genes. Our data showed that our human HazChem array can be used to monitor hazardous materials via gene expression profiling.
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제안 방법
For the selection of the HazChem array gene, the first selection was made on genes through the query search for each of the harmful materials, by means of the NCBI database and the previous literature and data, after which additional selection was conducted using the database ID, accession number, and unigene ID of the initially selected genes (Figure 2). By means of analysis on the selected genes’ functions and pathways, the genes were finally selected at the proportionate rate, which were associated with stimulus response, signal transduction, biosynthesis, morphogenesis, cell death, development, immune response, cell cycle, transport, transcription protein metabolism, and biosynthesis (Table 1).
In this study, we utilized a human HazChem array in order to identify the differentially expressed significant genes induced by ethylenebenzene and trichloroethylene in HL-60 cells. As a result, 8 upregulated genes and 8 downregulated genes were discovered on the basis of 1.
The hybridized slides were scanned with an Axon Instruments GenePix 4000B scanner and the scanned images were analyzed using the software program GenePix Pro 5.1 (Axon, CA) and GeneSpring GX 7.3.1 (Sillicongenetics, CA). Spots that were adjudged as substandard via the visual examination of each slide were flagged and excluded from further analysis.
, UK), using a Genemachine pin-type arrayer. The reproducibility, reliability and accuracy of the HazChem array were assessed using the control materials in accordance with the internal guidelines, after which the microarray experiments were conducted using RNA extracted from the HL-60 cell line.
The two labeled cDNAs were then mixed, placed on a HazChem array Human 300 (GenoCheck, Korea) and covered Each extracted total RNA sample (30 µg) was labeled with Cyanine (CY3) or Cyanine (Cy5) conjugated dCTP(Amersharm, Piscataway, NJ) via a reverse transcription reaction using reverse transcriptase, SuperScrip ll (Invitrogen, Carlsbad, California).
Moreover, it has been reported that the degree of accuracy is higher when using an analysis method that utilizes a few genes selected from analysis on whole gene sets, rather than in the entire gene method itself. This study applies to an in-house generated human HazChem oligonucleotide array, which could be used to discover significant genes that evidence toxicity expression by processing VOCs including ethylbenzene and trichloroethylene in the HL-60 cell line. The results of this study are expected to contribute to the development of method by which VOCs, including ethylbenzene and trichloroethylene might be searched using the genes commonly expressed in both compounds.
대상 데이터
Fold change filters included the requirement that the genes be present in at least 200% of the controls for upregulated genes and lower than 50% of controls for the downregulated genes. The data were clustered groups of genes that behaved similarly across the drug treatment experiments using GeneSpring GX 7.3.1 (Silicongenetics, CA). We utilized an algorithm, based on the Pearson’s correlation, to separate genes evidencing similar patterns24.
The reaction mixtures contained 10 pmol/µL of each primer and 2X SYBR Green PCR Master Mix (PE Applied Biosystems, www.appliedbiosciences. com), which includes the HotStarTaqt DNA-Polymerase in an optimized buffer, the dNTP mix (with dUTP additive), the SYBRs Green I fluorescent dye, and ROX dye as a passive reference.
A 384-well high-throughput analysis was performed using the ABI Prism 7900 Sequence Detection System (PE Applied Biosystems), and white-colored 384-well plates (ABgene, Hamburg, Germany) for the intensification of the fluorescent signals by a factor of three. This system operates using a thermal cycler and a laser which is directed via fiber optics to each of 384 sample wells. The fluorescence emission from each sample was collected by a charge-coupled device-camera and the quantitative data were analyzed with Sequence Detection System software (SDS version 2.
성능/효과
The results obtained using the human HazChem array and the comparison of the gene expression clusterings for ethylbenzene and trichloroethylene compounds showed a high correlation between the gene variances of the two compounds. Also, 8 upregulated genes and 8 downregulated genes were discovered based on the 1.5 fold threshold, and the upregulated genes were identified as ISG15, TNFSF10, PRNP, ME1, NCOA4, SRXN1, TXNRD1, and XBP1, whereas the downregulated genes were identified as MME, NRF1, PRARBP, CALCA, CRP, BAX, C7 or f40, and FGFR1 (Table 2, 3). These significant results were also highly correlated with the results of quantitative RT-PCR.
The cell viability of HL-60 cells after exposure to a range of concentrations of VOCs compounds was determined via an MTT assay. Based on the results of the MTT assay, the 50% cell viability inhibitory concentration (IC50) of each compound was calculated. Dose-dependent cell viability curves were obtained after 3 hrs of exposure to ethylbenzene and trichloroethylene in HL-60 cells, as shown in Figure 1.
In conclusion, although this data is not sufficient to demonstrate the mechanistic identification of gene expression, our results did suggest that the human HazChem array will facilitate the development of an efficient screening system for environmentally hazardous materials at the level of toxicogenomics in the future.
Hierarchical clustering was applied across the two compounds, using a combined list of genes (Figure 3). The results obtained using the human HazChem array and the comparison of the gene expression clusterings for ethylbenzene and trichloroethylene compounds showed a high correlation between the gene variances of the two compounds. Also, 8 upregulated genes and 8 downregulated genes were discovered based on the 1.
5-fold microarray analysis. The upregulated genes were identified as ISG15, TNFSF10, PRNP, ME1, NCOA4, SRXN1, TXNRD1, and XBP1, whereas the downregulated genes were identified as MME, NRF1, PRARBP, CALCA, CRP, BAX, C7 or f40, and FGFR1. However, according to the results of previously published works, it was determined that the majority of responding genes were associated with predominant changes in the expression of several proteins that regulate apoptosis & cellular growth, differentiation & stress response, all of which have been associated with VOCs toxicity15-22.
후속연구
This study applies to an in-house generated human HazChem oligonucleotide array, which could be used to discover significant genes that evidence toxicity expression by processing VOCs including ethylbenzene and trichloroethylene in the HL-60 cell line. The results of this study are expected to contribute to the development of method by which VOCs, including ethylbenzene and trichloroethylene might be searched using the genes commonly expressed in both compounds.
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