Available online at www.tox.or.kr Differential Gene Expression Profiling in Human Promyelocytic Leukemia Cells Treated with Benzene and Ethylbenzene Sailendra Nath Sarma1,2, Youn-Jung Kim1 & further analysis to explore the mechanism of BE Jae-Chun Ryu1,2 induced hematotoxicity. Keywords: Benzene, Ethylbenzene, Oligomicroarray, Gene 1Cellular and Molecular Toxicology Laboratory, Korea Institute of ontology, KEGG pathway, Hematotoxicity Science & Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Korea 2Department of Biopotency/Toxicology Evaluation, University of Benzene and ethylbenzene (BE) are volatile mono- Science & Technology, Daejeon, Korea aromatic hydrocarbons which are commonly found Correspondence and requests for materials should be addressed together in crude petroleum and petroleum products to J. C. Ryu ([email protected]) such as gasoline1. These compounds produced on the scale of megatons per year as bulk chemicals for Accepted 3 November 2008 industrial use as solvents and starting materials for the manufacture of pesticides, plastics, and synthetic fibers2. They are ubiquitous pollutants mainly due to Abstract engine emissions, tobacco smoke and industrial pol- lution. Benzene and ethylbenzene are in the top 50 Benzene and ethylbenzene (BE), the volatile organic chemicals produced and used worldwide. As a conse- compounds (VOCs) are common constituents of quence of their usage, they are most common waste cleaning and degreasing agents, paints, pesticides, material from the industry3. Indoor exposure is thou- personal care products, gasoline and solvents. VOCs ght to be of greater concern because indoor concen- are evaporated at room temperature and most of trations of many pollutants are often higher than them exhibit acute and chronic toxicity to human. those typically encountered outside4. The observed Chronic exposure of benzene is responsible for mye- health effects in association with volatile organic loid leukemia and also ethylbenzene is also recog- compounds (VOC) exposure it seems to be true that nized as a possible carcinogen. To evaluate the BE 5,6 effect on human, whole human genome 35 K oligo- high VOC concentrations mainly cause toxic effects whereas moderate VOC exposure levels trigger infla- nucleotide microarray were screened for the iden- 7 tification of the differential expression profiling. We mmatory reactions in particular in the airways and identified 280 up-regulated and 201 down-regulat- low exposure levels induce alterations in immune re- ed genes changed by more than 1.5 fold by BE ex- activity resulting in a subsequent higher risk for the 8,9 posure. Functional analysis was carried out by using development of allergic reactivity and asthma . DAVID bioinformatics software. Clustering of these Children are a potentially at-risk population be- differentially expressed genes were associated with cause they may be both more exposed to VOCs and immune response, cytokine-cytokine receptor inter- more susceptible to diverse effects than adults. Child- action, toll-like signaling pathway, small cell lung ren can be affected by different sources, pathways, cancer, immune response, apoptosis, p53 signaling and routes of exposure than adults; that children often pathway and MAPKKK cascade possibly constitu- have greater intake of air, food, beverages, soil, and ting alternative or subordinate pathways of hemato- dust per unit body weight and surface area; and that toxicity and immune toxicity. Gene ontology analysis children differ from adults in terms of important pha- methods including biological process, cellular com- rmacokinetic and pharmacodynamic parameters10,11. ponents, molecular function and KEGG pathway thus Blood concentrations of 11 VOCs were measured up provide a fundamental basis of the molecular path- to four times over 2 years in a probability sample of ways through BEs exposure in human lymphoma more than 150 children from two poor, minority neigh- cells. This may provides a valuable information to do borhoods in Minneapolis, Minnesota. Blood levels of 268 Mol. Cell. Toxicol. Vol. 4(4), 267-277, 2008 benzene, carbon tetrachloride, trichloroethene, and 120 m-/p-xylene were comparable with those measured in Benzene Ethylbenzene selected adults from the Third National Health and 100 Nutrition Examination Survey (NHANES III), where- as concentrations of ethylbenzene, tetrachloroethy- 80 lene, toluene, 1,1,1-trichloroethane, and o-xylene were 12 (% of control) two or more times lower in the children . 60 To predict the risk of environmental toxic subst- IC ances or compounds, investigation of toxicological 40 50 mechanisms or toxic biomarkers is noticed more and Cell viability more. Toxicogenomic technologies are more helpful 20 than traditional tools for investigation of these toxic substances. Recent advancements in high throughput 0 0 2 4 6 8 10 12 14 gene profiling techniques such as DNA microarray Concentration (mM) allows simultaneous measurement of mRNA expre- ssion levels of tens of thousands of genes at once Figure 1. Cell viability after the exposure with BE were whereas previously only a handful of genes could be analyzed by the MTT assay. Three mL of HL-60 cells were investigated13. Recently researchers are interested in seeded in 15 mL tube at 1×106 cells/mL in a shaking in- analyzing the expression patterns of functionally re- cubator. After 3 h treatment with BE, MTT solution (5 mg/mL) was added to each tube for 3 h. The reaction was lated genes usually hope to improve the accuracy of stopped by adding DMSO and the absorbance (optical den- their results beyond the boundaries of currently avail- sity) was determined at 540 nm. The change in absorbance able experimental data. One of the most common which is the change in cell death is shown as % of control. problems encountered while deciphering results from Fifty percent inhibition concentration (IC50) values of BE expression profiling experiments is in relating dif- were calculated using SigmaPlot 8.0 statistical analysis soft- ferential expression of genes to molecular functions ware. and cellular processes. A second important problem is that of comparing experiments performed by dif- ferent labs using different microarray platforms, or for analysis of function of genes. Combination of mi- even unrelated techniques. Gene Ontology (GO) data croarray and pathway data may highlight the process provides a novel way to measure the functional re- taking place in the cell providing information on the lationship between gene products14. Gene Ontology process-specific function of the genome. Biological (GO) is now used to describe biological features, since process can be prospected according to combination GO terms are associated with genes, to overcome the of microarray results and biological information18. apparent distance between expression profiles and The objective of this study is the identification of biological comprehension15. function of the genes through the analysis of GO and Benzene and ethylbenzene were used as models of to find potential gene-based markers induced by ben- VOCs, this toxicogenomic study was carried out to zene and ethylbenzene. We have examined the global compare BE-induced gene expression alterations in gene expression profiles in a small number of well human lymphoma cells. We examined differentially matched exposed-control subject pairs. Differentially expressed genes at multiple levels (biological pro- expressed genes were then ranked and selected for cess, pathway, gene) using Gene Ontology (GO) an- further examination using several forms of statistical alysis, pathway mapping and cross-scatter plots. The analysis. Commonly expressed genes with both che- Gene Ontology (GO) analysis of microarray data pro- micals may assist in the identification of potential vides structured and controlled vocabularies and cla- biomarker of BE and may understand molecular toxi- ssifications for several domains of molecular and cological mechanism of benzene and ethylbenzene in cellular biology16. GO is generally used to annotate human lymphoma cell line. the functions of genes and is composed of three do- mains, biological process, cellular components and Cytotoxicity of Benzene and Ethylbenzene in molecular functions. However, it is not deal with the HL-60 Cells relations between pathways and sub-pathways or bet- To determine the 50% inhibition concentration of ween pathways and their related biological pheno- cell proliferation on benzene, and ethylbenzene, mena17. So, the association of Kyoto Encyclopaedia various concentrations of BE were treated to the HL- of Genes and Genomes (KEGG) pathway containing 60 cells. Benzene and ethylbenzene reduced cell information on biological pathways and GO is useful viability gradually at increasing concentrations were Gene Expressional Profiling of BE 269 Table 1. Biological process of up-regulated genes in benzene and ethylbenzene treated HL-60 cell. Common expressed genes in two chemicals marked by bold. GO Benzene Ethylbenzene BCL2, BCL6, CCL2, CCL20, CCL3L1, CCL4, AIM2, BCL3, BCL6, CCL20, CCL23, CCL3L1, CCL4L1, CCL5, CD83, CFHL5, CLECSF12, CCL4, CCL4L1, CCL5, CD83, CLEC4A, CLU, CLECSF5, CRTAM, CXCL10, CXCR4, DAF, CXCL10, CXCL11, CXCR4, DDX58, EBI2, DDIT3, DDX58, DEFB4, EBI2, ELF1, FASLG, ETS1, F3, FASLG, FCGR1A, FTH1, FYN, FCAR, FCAR, FCGR1A, FLJ14466, FLJ45422, GBP2, HLA-A, HMOX1, IFIT1, IFIT2, IFIT3, FTH1, FYN, G1P2, GBP2, HFE, HLA-DMA, IFIT5, IFNA7, IFNB1, IL10, IL18R1, IL18RAP, Immune response HLA-E, HLA-G, HMOX1, IFI16, IFIT1, IFIT2, IL1A, IL27RA,