Available online at www.tox.or.kr

Differential Expression Profiling in 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 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 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. 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, INPPL1, IRF1, IRF7, IRF8, IFIT3, IGLC2, IL18R1, IL18RAP, IL1R1, ISG15, ISGF3G, LAX1, LTA, LTB, LY96, IL1RAP, IL1RL1, IL23A, IL4R, INPPL1, NCR3, NFKB2, OAS3, OASL, PRG3, PTGER4, KIR2DS2, LAX, LGP2, NFIL3, NFKB2, OASL, PTX3, RBM24, RGS1, SFTPD, SLC11A1, PGLYRP2, PTGER4, PTK9L, PTPN22, RELB, SMAD3, SQSTM1, TAP1, TNF, TNFRSF9, RNF125, SLC11A1, SQSTM1, TNF, TNFSF10, TNFSF10, TNFSF13B, TNFSF14, TNFSF7, TNFSF14, XBP1 WAS, XBP1 APOL2, BCL6, C3AR1, CCL20, CCL23, AOC3, BCL6, BMP6, C3AR1, CCL2, CCL20, CCL3L1, CCL4, CCL5, CLU, CXCL10, CCL3L1, CCL4, CCL4L1, CCL5, CFHL5, Inflammatory CXCL11, CXCR4, CYBB, F11R, FPR1, HRH1, CLECSF12, CXCL10, CXCR4, DAF, F11R, response IL10, IL18RAP, IL1A, IRF7, LY96, MIF, NCR3, FPR1, IL17F, IL18RAP, IL1R1, IL1RAP, IL23A, NFKB1, PTX3, RBM24, SERPINA1, TLR8, NOS2A, NR3C1, PLA2G2E, RAC1, ZFP36 TPST1, USP18, ZFP36 IL17F, NFAT5, IGSF4, ELF1, CRTAM, BCL6, IRF1, NFAT5, SFTPD, ISGF3G, BCL3, BCL6, Cytokine production HMOX1, IL1RAP, ASB1 HMOX1, NFKB1, IL27RA, PRG3, TLR8 Cellular defense C3AR1, CCL5, CLECSF5, HLA-G, KIR3DL2, C3AR1, CCL5, FOSL1, LY6H, LY96, NCF2, response NCF2, TCIRG1 TCIRG1, TRA2, TYROBP AHR, AXUD1, BBC3, BCL2, BCL2A1, BCL6, ACVR1, AIFM2, ANGPTL4, ATF5, BAD, BBC3, BID, BIRC2, BIRC3, BIRC7, BTG1, CASP3, BCL2A1, BCL3, BCL6, BIRC3, BTG1, BTG2, CBX4, CCL2, CDKN1A, CIDEC, CRTAM, CBX4, CD38, CDKN1A, CLU, CXCR4, DAPK3, CXCR4, DAPK2, DDIT3, FASLG, FLJ11259, DEDD2, ERN1, FASLG, GADD45A, GADD45B, GADD45A, GADD45B, GCLM, HMOX1, GCH1, GCLM, HIPK2, HMOX1, HSPA1A, Apoptosis HSPA1A, IER3, IFI16, IFIH1, IGSF4, IKIP, IFIH1, IFNB1, IHPK2, IL10, IL1A, LGALS12, JMY, NFKBIA, PHLDA1, PLEKHF1, PMAIP1, LTA, MIF, NFKB1, NFKBIA, NGFR, PHLDA1, PPP1R15A, PRNP, PTK2B, RASSF5, RB1CC1, PHLDA2, PMAIP1, PPP1R15A, PRNP, PTK2B, RTKN, RYBP, SART1, SGK, SHB, SIAH2, SART1, SGK, SMAD3, SQSTM1, STAT1, TNF, SQSTM1, STAT1, STK17B, TNF, TNFAIP3, TNFAIP3, TNFRSF10B, TNFRSF9, TNFSF10, TNFSF10, TNFSF14, TRAF1, TRAF3, TRIB3 TNFSF13B, TNFSF14, TRAF1, TRAF3, TRIB3 BCL2, BCL2A1, BIRC2, BIRC3, BIRC7, CBX4, ATF5, BCL2A1, BIRC3, CBX4, CLU, HMOX1, Anti-apoptosis CCL2, HMOX1, HSPA1A, IER3, PRNP, RTKN, HSPA1A, IL10, IL1A, NFKB1, PRNP, TNF, TNF, TNFAIP3 TNFAIP3, TNFSF13B Regulation of BIRC2, FASLG, HMOX1, MAP3K7IP2, FASLG, HMOX1, LTA, SQSTM1, TNF, I-kappaB kinase/ SQSTM1, TNF, TNFAIP3, TNFSF10 TNFAIP3, TNFRSF10B, TNFSF10, TRAF3IP2 NF-kappaB cascade FTH1, FTHL17, FTHL3, HFE, SFXN3, FTH1 FTHL17 FTHL3 SLC11A1 Iron ion transport SLC11A1 , , , FTL, DUSP16, CXCR4, ITPKB, GADD45B, BIRC7, FGFR1, CXCR4, ITPKB, GADD45B, TNF, MAPKKK cascade TNF, DUSP2, FPR1, RB1CC1, LAX, MAP3K9, CARD9, MAPBPIP, FPR1, MAP3K11, LAX, DUSP4, RAPGEF2 HIPK2, DUSP4 ARTN, BCL6, BIN1, BTG1, BTG2, CALCRL, ACHE, BCL2, BCL6, BTG1, CASP3, CCL3L1, CCL23, CCL3L1, CDKN1A, CDKN2B, CLU, CDKN1A, CDYL2, CLK1, CNTFR, COL8A1, CXCL10, CXCR4, ELF5, ETS1, FGFR1, FOSL1, CRTAM, CXCL10, CXCR4, ELF5, EMP1, FSCN1, FTH1, HMOX1, IFIT1, IFNB1, IGFBP6, EPHB4, FTH1, GLI1, HBEGF, HHIP, HMOX1, IL10, IL1A, IRS2, ISG20, JAG1, KLF4, LTA, Cell proliferation IFI16, IFIT1, IGSF4, IRS2, JAG1, KLF10, MAFG, MAP3K11, MIF, MXD1, NFKBIA, LAMP3, MAD, NFKBIA, PBEF1, PGF, PPAP2A, PLAUR, POLD4, PTK2B, RARRES3, RECQL4, PTK2B, RARRES3, SERTAD1, SH2D2A, SHB, SERTAD1, SFTPD, SH2D2A, SMAD3, TCF19, TCIRG1, TNF, TNFSF14, TRIB1, TSPAN1, TCIRG1, TNF, TNFRSF9, TNFSF13B, TNFSF14, TXNRD1 TP53I11, TXNRD1, WARS 270 Mol. Cell. Toxicol. Vol. 4(4), 267-277, 2008

Table 2. Biological process of down-regulated genes in benzene and ethylbenzene treated HL-60 cell. Common expressed genes in two chemicals marked by bold. GO Benzene Ethylbenzene TRRAP, CCNJ, CCNYL1, SLC12A6, NF1, PLCB1, BUB1, BUB1B, CDKN2C, TADA2L, Regulation of CUL4A, CDC25A, E2F5, , SHC1, TGFB2, DLG1, IL1B, CCNE1, NF1, CDC25A, MKI67, progression VEGFA, LATS1, SLC12A6, EGFR, PRMT5, CDKL1, RUNX3, SMC1A, HEXIM1, MTCP1, through SMC1A, BRCA1, ABL1, PPP2R1A, ANKRD15, GTSE1, ANLN, CDC25C, CCNB1, PML, cell cycle SUFU, CENPF, ANLN, MPKK6, UHMK1, CCNE2, PKMYT1, TRIM13 SHC1, TRIM13 G1/S transition of GFI1, ACVR1B, CUL4A, BCAT1, SKP2, POLE, GPR132, GFI1, CDKN2C, CDCA5, CCNE1 mitotic cell cycle GPR132 DNAH2, UNC5C, TAOK2, LRP5, ROBO3, TOP2B, SLIT1, ABI3, NCAN, AKAP4, TUBB, ATP1A3, CDK5R1, NF1, ITGA4, MSN, MUC2, IL1B, DOCK2, GAB1, CCL25, NF1, TPM1, Cell motility TGFB2, VEGFA, NTN1, CRKL, EGFR, NTN2L, GDNF, NPY, ATP1A1, PALM, TLX3, CENTD3, TPBG, LTB4R2, MAPK8, ARX, F11R, CABP4, LPAL2, GDNF, ITGA6, MAP3K1 APBB2, NF2, LAMA3, MIA3 BUB1, BUB1B, SPAG5, DLG1, CIT, ASPM, DCTN1, TRRAP, SPAG5, PES1, PBRM1, ASPM, CDCA5, NCAPH, CDC25A, NDC80, SMC1A, STAG1, STAG2, ATM, CDC25A, SHC1, LATS1, Mitosis KIF2C, TARDBP, TPX2, GTSE1, ANLN, SMC4, PRMT5, SMC1A, TARDBP, PES1, CDC25C, CNB1, PML, CDCA2, PKMYT1 CENPF, KIF15, CDCA2, SHC1, CDC27 WDR67, PLEKHG6, TBC1D14, PLEKHG5, WDR67 PLEKHG5 HRBL, ARHGEF2, NF1, G3BP2, CDC42EP4, Ras signal , TBC1D13, HRBL, , ABRA, NF1 KIAA1415 ABR TBC1D3G, KIAA1415, SPATA13, RREB1, transduction RASGRP4, , ARHGEF17, , , CENTD3, MRAS PSD, ABR, ARHGEF6, CRKL, TRIO, DDEFL1, ARHGEF7 ARHGEF2, RGS4, RGS11, RAPGEF4, TAOK2, SPRY4, MIER1, NOD1, WDR67, PLEKHG6, SNF1LK2, ACVR2B, FGF18, GEFT, MGC21654, ABR, RASA3, RIC8B, RAMP3, PLEKHG5, ABR CD8A, , KIAA0720, FLJ40244, STARS, HRBL, ARHGEF6, ACVR1B, DDEFL1, SPRY1, NF1 DTX1 Regulation of signal RASGRP4, IL1B, DKFZP434I092, , , IL6ST, TBC1D3G, RGS22, SPATA13, KIAA1219, HRBL TMOD2 transduction , RASA4, , ARHGEF17, TBC1D13, NF1, IL2, ABR, PSD, SHC1, VEGFA, DTX1, CTNNBIP1, PREX1, CENTD3, APOL3, RFP2, TRIO, PPP2R1A, TMOD2, PDCD11, OTUD7B, KIAA1272, FLJ35390 BC1D14, APOL3, ARHGEF7, SHC1, SOCS4, TRIM13 UNC5C, TAOK2, LRP5, ROBO3, CDK5R1, SLIT1, ABI3, FOXC1, IL1B, DOCK2, GAB1, NF1, ITGA4, MUC2, TGFB2, VEGFA, NTN1, Cell migration CCL25, NF1, TOP2B, NTN2L, TLX3, CENTD3, EGFR, LTB4R2, ARX, APBB2, LAMA3, LPAL2, GDNF, ITGA6 MAP3K1, MIA3 Positive regulation THRB, GFI1, ACVR1B, VHL, RUNX1, CD276, THRB, ACIN1, ZNF488, GFI1, WNT7B of cell differentiation CALCA, TGFB2, NKX6-1, ACIN1, WNT7B ABL1, ACVR1B, ANKRD15, ANLN, APBB2, PLCB1, BUB1, BUB1B, SPAG5, WWOX, ASPM, ATM, BANP, BCAT1, BRCA1, CCNJ, FOXC1, TAF1L, TADA2L, MPHOSPH9, GFI1, CCNYL1, CDC25A, CDC27, CDCA2, CDK5R1, DLG1, CIT, ASPM, IL1B, CDCA5, TCF19, CENPF, CUL4A, DCTN1, DMTF1, DUSP13, CCNE1, BRRN1, CDC25A, MKI67, RAD54L, E2F5, E2F7, EGFR, ERBB2IP, FLCN, FSD1L, Cell cycle PRC1, CDKL1, RUNX3, TOP3A, KNTC2, GFI1, GPR132, KCNK17, KIF15, LATS1, MYC, SMC1L1, HIS1, KIF2C, TARDBP, TPX2, PBRM1, PES1, PES1, POLE, PPP2R1A, PRMT5, RACGAP1, GPR132, MTCP1, GTSE1, ANLN, RIF1, SHC1, SHC1, SKP2, SLC12A6, SLC12A6, CDC25C, CCNB1, PML, CCNE2, EXO1, CDCA2, SMC1A, SMC4, SPAG5, STAG1, STAG2, PKMYT1, MCM7, E2F7, RFP2 SUFU, SYCP2, TARDBP, TGFB2, TRIM13, TRRAP, UHMK1, VEGFA ,VHL APBB2, ARX, ATP1A3, CABP4, CDK5R1, ABI3, AKAP4, ATP1A3, CCL25, CENTD3, CRKL, DNAH2, EGFR, F11R, ITGA4, LAMA3, CSPG3, DOCK2, FOXC1, GAB1, GDNF, GDNF, Localization of cell LRP5, LTB4R2, MAP3K1, MAPK8, MIA3, IL1B, ITGA6, LPAL2, NPY, NTN2L, PALM, MSN, MUC2, NTN1, ROBO3, TAOK2, TGFB2, SLIT1, TLX3, TOP2B, TPM1, TUBB TPBG, UNC5C, VEGFA Gene Expressional Profiling of BE 271

tal RNA were subjected to microarray analysis. Gene expression changes were analyzed by comparing with treated and control group using a statistical criteria of 5.0 ›1.5 fold changes with P⁄0.01. In this analysis, 1,007 genes were up-regulated and 919 genes were 4.0 down-regulated by benzene exposure and also 929 genes were up and 1,455 genes were down regulated 3.0 by ethylbenzene individually exposure. Two hundred 2.5 eight genes were up and 201 genes were commonly 2.0 down regulated through benzene and ethylbenzene exposure. 1.5 These genes were classified according to KEGG pathway to analyze molecular mechanism exposed to 1.2 BE. The up and down regulated genes were listed in Table 1 and 2. Differentially changed genes involved in 1) MAPK signaling pathway, 2) Cytokine-cytokine 1.0 receptor interaction, 3) Natural killer cell mediated 0.9 cytotoxicity, 4) Toll-like receptor signaling pathway, 0.8 5) Apoptosis, 6) p53 signaling pathway, 7) Adipo- cytokine signaling pathway, 8) T cell receptor signal- 0.7 ing pathway, 9) Type I diabetes mellitus, 10) Small 0.6 cell lung cancer, 11) Chronic myeloid leukemia, 12) Cell cycle, 13) Hematopoietic cell lineage, 14) Anti- 0.5 gen processing and presentation (Figure 4). In KEGG 0.4 pathway analysis, Cytokine-cytokine receptor inter- 0.3 action, natural killer cell mediated cytotoxicity, toll- like receptor signaling pathway, apoptosis, small cell 0.2 lung cancer, T cell receptor signaling pathway, p53 0.1 signaling pathway, Adipocytokine signaling pathway, 0.0 Type I diabetes mellitus were commonly up regulat- Benzene EthyBenzene ed. We have investigated an enrichment of GO anno- Figure 2. A cluster analysis was carried out using mean of tations in the up-regulated and down-regulated genes differentially expressed genes of each chemical. HL-60 cells related to the toxic effect of BE. The categories of the were individually exposure to IC50 concentration of BE, total RNA was extracted using Trizol reagent and purified with function in differentially expressed genes are pre- RNeasy mini kit according to manufacturer’s instructions. sented in Figure 5. The biological process profiles of Three independent experiments were performed for each up-regulated genes were subdivided in to 208 groups chemical. Rows represent genes and columns represent and also 30 subdivisions for down regulated genes. samples. Red and green blocks respectively represent high The major up-regulated genes subdivisions were and low expression relative to reference RNA, while black apoptosis, cellular defense response, anti-apoptosis, blocks indicate equal expression. regulation of I-kappaB kinase/NF-kappaB cascade, inflammatory response, iron ion transport, cell pro- determined MTT assay. The survival percentage re- liferation, cell development, system development and lative to solvent control (DMSO) was determined as a immune response. As well the major subdivision of percentage of optical density value measured after down-regulated genes were regulation of progression treatment. Dose dependent cell viability curves were through cell cycle, G1/S transition of mitotic cell obtained after 3 hrs exposure to BTX and there inhi- cycle, cell motility, mitosis, Ras protein signal trans- bition concentration of were shown in Figure 1. The duction, Regulation of signal transduction, cell mi- IC50 value for benzene was 8.75 mM and for ethyl- gration, positive regulation of cell differentiation, benzene was 0.99 mM. localization of cell, and cell cycle. Differentially ex- pressed common genes were listed as bold in the Gene Expression Analysis Table 2. Analysis of cellular localization reveals the HL-60 cells were treated with 8.75 mM benzene genes associated with nucleus, cytoplasm, cytosol, and also 0.99 mM ethylbenzene for 3 hrs, and the to- splicesome, , intracellular organelle part 272 Mol. Cell. Toxicol. Vol. 4(4), 267-277, 2008

Figure 3. Venn diagram (A) (B) showed the differentially ex- pressed genes in HL-60 cells exposed with BE for 3 h with IC50 doses. The diagrams were generated from the list of up (A) and down (B) regu- 1007 280 929 919 201 1455 lated genes obtained from the microarray analysis. At least 1.5 fold increase or de- crease genes were considered to generate these diagrams. The numbers in the diagram show the number of gene re- Benzene IC50 Ethylbenzene IC50 Benzene IC50 Ethylbenzene IC50 gulated by BE exposure.

Prion disease Type I diabetes mellitus Inositol phosphate metabolism B cell receptor signaling pathway Adipocytokine signaling pathway Antigen processing and presentation p53 signaling pathway Hematopoietic cell lineage T cell receptor signaling pathway Epithelial cell signaling in Helicobacter pylori infection Small cell lung cancer Apoptosis Toll-like receptor signaling pathway Natural killer cell mediated cytotoxicity Cytokine-cytokine receptor interaction MAPK signaling pathway 0 5 10 15 20 25 30 Number of up-regulated genes

Pathogenic Escherichia coli infection-EHEC Pathogenic Escherichia coli infection-EPEC p53 signaling pathway Cell cycle Neuroactive ligand-receptor interaction 0246810121416 Number of down-regulated genes

Type I diabetes mellitus Glycine, serine and threonine metabolism Adipocytokine signaling pathway Chronic myeloid leukemia Small cell lung cancer p53 signaling pathway T cell receptor signaling pathway Apoptosis Natural killer cell mediated cytotoxicity Toll-like receptor signaling pathway Cytokine-cytokine receptor interaction 0 5 10 15 20 25 30 35 Number of up-regulated genes

Valine, leucine and isoleucine biosynthesis Pantothenate and CoA biosynthesis Dentatorubropallidoluysian atrophy mTOR signaling pathway Pancreatic cancer Small cell lung cancer Phosphatidylinositol signaling system Colorectal cancer GnRH signaling pathway Figure 4. The pathway an- Chronic myeloid leukemia ErbB signaling pathway alysis of differentially expre- Ubiquitin mediated proteolysis ssed genes by benzene (A) Tight junction Insulin signaling pathway and ethylbenzene (B) in HL- 0 5 10 15 20 25 60 cells using KEGG path- Number of down-regulated genes way database. Gene Expressional Profiling of BE 273

A. Biological process Ras protein signal transduction Cytokine production Cell migration I-kappaB kinase/NF-kappaB cascade Anti-apoptosis Mitosis MAPKKK cascade Cell motility Hemopoiesis Regulation of progression through cell cycle Inflammatory response Cell cycle Defense response Cell differentiation Apoptosis Immune response Signal transduction Response to stimulus Biological regulation

0 5 10 15 20 25 0 5 10 15 20 25 30 Percentage of up-regulated genes (%) Percentage of down-regulated genes (%)

B. Cellular component

Lipid raft Vesicle membrane MHC class I protein complex Microtubule Pigment granule Cytoplasmic membrane-bound vesicle Melanosome Membrane-bound vesicle Cell surface Chromosome Soluble fraction Cell junction Integral to plasma membrane Cytoskeleton Non-membrane-bound organelle Plasma membrane Nucleus Nucleus

0 5 10 15 20 25 0 5 10 15 20 25 Percentage of up-regulated genes (%) Percentage of down-regulated genes (%)

C. Molecular function

Interleukine binding Ras guanyl-nucleotide exchange factor Cytokine activity Enzyme activator activity Enzyme binding Ion channel activity Transcription factor binding Protein serine/threonine kinase activity Kinase activity Lipid binding ATP binding Enzyme regulator activity Zinc ion binding Kinase activity Nucleotide binding Signal transducer activity Cation binding Cation binding Ion binding Ion binding 0 5 10 15 20 25 0 5 10 15 20 25 Percentage of up-regulated genes (%) Percentage of down-regulated genes (%)

Figure 5. The distribution of up-and down-regulated genes according to gene ontology categories in HL-60 cells through ben- zene exposure.

and extracellular matrix. And the majority of profiles in molecular function are signal transduction activity, Discussion molecular transduction activity, transcription regula- tor activity, ATP binding activity, enzyme activator Benzene is a well known carcinogen can cause activity, cytokine binding activity and mostly trans- acute myeloid leukemia (AML) and other hematoto- cription activities. xicity and leukemogenicity in human21 and also ethylbenzene is known as possible carcinogen to 274 Mol. Cell. Toxicol. Vol. 4(4), 267-277, 2008

A. Biological process

Cytokine production Cell differentiation MAPKKK cascade Cell migration Anti-apoptosis Ras protein signal transduction I-kappaB kinase/NF-kappaB cascade Mitosis Hemopoiesis Cell motility Inflammatory response Regulation of progression through cell cycle Defense response Regulation of signal transduction Apoptosis Immune response Cell cycle Response to stimulus Regulation of biological process 0 5 10 15 20 25 0 5 10 15 20 25 Percentage of up-regulated genes (%) Percentage of down-regulated genes (%)

B. Cellular component

Lipid raft Cell cortex MHC class I protein complex Microtubule Pigment granule Golgi apparatus part Melanosome Nuclear lumen Cell surface Cytoskeletal part Soluble fraction Organelle membrane Integral to plasma membrane Non-membrane-bound organelle Plasma membrane Organelle part Nucleus Nucleus 0 5 10 15 20 25 0 5 10 15 20 25 Percentage of up-regulated genes (%) Percentage of down-regulated genes (%)

C. Molecular function Kinase activator activity Chemokine activity Ras guanyl-nucleotide exchange factor G-protein-coupled receptor binding ATP binding Growth factor binding Nucleotide binding Protein kinase binding DNA binding Transcription corepressor activity Zinc ion binding Kinase binding Transition metal ion binding Enzyme binding Cation binding Cytokine activity Metal ion binding Transcription factor activity DNA binding Ion binding

0 5 10 15 20 25 0 5 10 15 20 25 Percentage of up-regulated genes (%) Percentage of down-regulated genes (%)

Figure 6. The distribution of up-and down-regulated genes according to gene ontology categories in HL-60 cells through ethyl- benzene exposure.

human. Here in this study, we have demonstrated the Chronic exposure to benzene and ethylbenzene gene expression profiles together with benzene and characteristically causes hematotoxicity and myelo- ethylbenzene using microarray in conjunction with genous leukemia in . The relationships bet- statistical analysis. Earlier, very few studies were ca- ween levels of VOCs in blood and air have not been rried out only with benzene, but this study evaluated well characterized in the general population. Figure 4 the gene expression profiles with BE in human cells showed the KEGG pathway of the genes, Figure 5 lines. and Figure 6 showed the GO analysis of the genes Gene Expressional Profiling of BE 275 which were differentially expressed genes through diction of the mechanism associated with biological, BE exposure in HL-60 cells. Through the KEGG cellular and molecular functions of genes using whole pathway genes were related to functions such as cyto- genome through chemical exposure. kine-cytokine receptor interaction, toll-like signaling pathway, small cell lung cancer, immune response, apoptosis, p53 signaling pathway and MAPKKK Materials & Methods cascade. Cytokines is a chemical messenger that me- diates intercellular communication. The regulation of Chemicals and Culture Media cellular and nuclear functions by cytokines, growth All reagents used were of analytical grade; ben- factors, and peptide or protein hormones is initiated zene, toluene, o-xylene, dimethylsulfoxide (DMSO) through the activation of cell surface receptors (Rc). and 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazo- Cytokines, chemokines, and cellular adhesion mole- lium bromide (MTT) were purchased from Sigma- cules are soluble that play an important re- Aldrich Co. (USA). Culture media and buffer solu- gulatory role in benzene induced hematopoiesis22. tions were purchased from GibcoTM: RPMI-1640, The activated receptor may then interact with other fetal bovine serum and antibiotics (penicillin and strep- cellular components to complete the signal transduc- tomycin). tion process23. The first steps in the generation of the immune response is the recognition by T lymphocytes Cell Culture of peptide fragments (antigens) derived from foreign The human promyelocytic leukemia HL-60 cell pathogens that are presented on the surface of antigen line was purchased from ATCC (CCL-240, USA). presenting cells (APC). T cell receptor (TCR) mediat- The cell lines were maintained in RPMI-1640 supple- ed this event by transduces these extracellular signals mented with 10% heat-inactivated fetal bovine serum by initiating a wide array of intracellular signaling plus 100 U/mL penicillin and 100 μg/mL streptomy- pathways24. Toll-like receptors lead to the synthesis cin, 1 mM sodium pyruvate and 10 mM HEPES at and secretion of pro-inflammatory cytokines and 37�C in 5% CO2 atmosphere. For cell growth the me- lipid mediators, thereby initiating the inflammatory dium was renewed every two or three days at a den- response that recruits both soluble immune compo- sity of 5×105 cells/mL in T75 tissue culture flask. nents and immune cells from the blood25. GO analy- sis showed that genes belongs to the biological pro- Determination of Cell Viability cess of cytokine production, immune responsive, in- MTT assay was performed for the detection of cell flammatory responsive, hemopoiesis, cellular defense viability19. 3 mL of HL-60 cells were seeded at a seed- response, apoptosis and also MAPKKK cascade were ing density of 1×106 cells/mL. Cells were exposed to significantly up-regulated through BE exposure. The various concentrations of benzene, toluene and o- p53-dependent pathways help to maintain genomic xylene in culture medium at 37�C for 3 h exposure stability by eliminating damaged cells either by arres- time at 200 rpm in a shaking incubator. 200 μL of ting them permanently or through apoptosis26. Apo- MTT (5 mg/mL in PBS) solution was added to each ptosis can be triggered by a wide range of stimuli, tube and incubated for 3 h. DMSO was added to each including cell surface receptors like Fas and FasL. It tube and 100 μL/well transferred in 96 well plate. The constitutes a system for the removal of unnecessary, optimal density (OD) of the purple formazan product aged, or damaged cells that is regulated by the inter- was measured at a wavelength of 540 nm. The 50% play of proapoptotic and antiapoptotic proteins of the inhibitory concentration (IC20 &IC50) of cell prolifer- Bcl-2 family27. MAPK signaling is activated by Ras- ation in a particular chemical was defined as the con- family Guanosine triphosphatases (GTPases) and oth- centration that causes 50% reduction in the cell viabil- er protein kinases, and connects extracellular signals ity versus the solvent treated control. The IC20 &IC50 to transcription factors and other effectors that regu- values were directly determined from the linear dose late many cellular programs such as cell proliferation, response curves. cell death and cytoskeleton remodeling28. In conclusion, KEGG and GO analysis showed that RNA Extraction the changes of gene expression pattern were associ- Total RNA was extracted from the HL-60 cells ated with hematotoxicity induced by benzene and treated to IC20 &IC50 doses of benzene, toluene and ethylbenzene exposure. Toxicogenomic approaches o-xylene, respectively, for 3 h using the Trizol reagent improved the prediction of chemical carcinogenicity (Invitrogen, USA) and purified using RNeasy mini kit by considering multiple techniques like microarray. (Qiagen, USA) according to the manufacturer’s inst- GO analysis gives a new way of analysis for the pre- ructions. Genomic DNA was removed using RNase- 276 Mol. Cell. Toxicol. Vol. 4(4), 267-277, 2008 free DNase set (Qiagen, USA) during RNA purifi- Functional Grouping and Clustering cation. RNA quality were assessed with the NanoDrop Analysis ND-1000 spectrophotometer (NanoDrop Technolo- In order to classify the selected genes into groups gies, USA) (260/280 nm ratios) and only samples with a similar pattern of expression, each gene was with an A260/A280 ratio between 1.9 and 2.2 were assigned to an appropriate category according to its considered for suitable use. The RNA quality was main cellular function. The necessary information to checked with the Experion automated electrophoresis categorize each gene was obtained from several data- station (Bio-Rad Laboratories, USA) (28S/18S ratio). bases particularly the database located at http://david. K-562 and U-937 cells were also treated with only abcc.ncifcrf.gov/home.jsp IC50 doses of BTX and extracted the RNA for real time RT-PCR. Acknowledgements Oligonucleotide Microarray Hybridization Gene expression analysis was conducted on the This subject is supported by the Korea Research RNA samples using 35 K whole human genome mic- Foundation grants from Korea Ministry of Environ- roarray (Operon Biotechnologies, Inc. Germany). ment as “The Eco-technopia 21 project”, KIST Core- Competence Program and Korea Research Council of Inhibition concentration 50% (IC50) dose treated BTX samples were performed for oligonucleotide microar- Fundamental Science and Technology to Ryu, J. C. of ray hybridization. Triplicate analysis was performed the Republic of Korea. for each chemical, simultaneously. Labeling and hyb- ridization were performed by instruction of Platinum References Biochip Reagent Kit (GenoCheck Co. Ltd., Korea). This was followed by the coupling of the Cy3 dye for 1. MacDonald, J. A. Evaluating natural attenuation for the controls (DMSO) or Cy5 dye for the treated sam- groundwater cleanup, Environ Sci Technol 34:346-353 ples. Hybridization was performed in a hybridization (2000). oven at 62�C for 12 h. After washing (2×SSC/0.1% 2. Hutchins, S. R., Sewell, G. W., Kovacs, D. A. & Smith, SDS for 2 min at 58�C, 1×SSC for 2 min at RT and G. A. Biodegradation of aromatic hydrocarbons by 0.2×SSC for 3 min at RT), the slide was dried by aquifer microorganisms under denitrifying conditions, centrifugation at 800 rpm for 3 min at RT. Hybridiza- Environ Sci Technol 25:68-76 (1991). tion images on the slides were scanned by ScanArray 3. Nikodinovic, J. et al. The conversion of BTEX com- Lite (PerkinElmer Life Sciences, USA). Scanned pounds by single and defined mixed cultures to medi- images were analyzed with GenePix 3.0 software um-chain-length polyhydroxyalkanoate. Appl Micro- (Axon Instruments, USA) to obtain gene expression bial Biotechnol 80:665-673 (2008). 4. US EPA. Reducing risk: Setting priorities and strate- ratios. gies for environmental protection. The report of the Science Advisory Board Relative Risk Reduction Stra- Microarray Data Analysis tegies Committee. SAB-EC-90-021. US Environmental The fluorescent intensity of each spot was calculat- Protection Agency, Washington, DC, pp. 26 (1990). ed by local median background subtraction. The 5. Koren, H. S. & Devlin, R. B. Human upper respirato- robust scatter-plot smoother LOWESS function was ry tract responses to inhaled pollutants with emphasis used to perform intensity dependent normalization on nasal lavage. Ann NY Acad Sci 641:215-224 (1992). for the gene expression. Scatter plot analysis was 6. Sarma, S. N., Kim, Y. J., Jeon, H. K. & Ryu, J. C. Iden- made by Microsoft Excel 2000. Significance Analy- tification of immune responsive genes on benzene, sis of Microarray (SAM) was performed for the selec- toluene and o-xylene in jurkat cells using 35 k human tion of the genes with significant gene expression oligomicroarray. Mol Cell Toxicol 2:229-235 (2006). changes20. Computing a q-value for each gene assess- 7. Wieslander, G., Norback, D. & Edling, C. Airway ed the statistical significance of the differential expre- symptoms among house painters in relation to expo- sure to volatile organic compounds (VOCS)-a longi- ssion of genes. To determine the q-value, a permuta- tudinal study. Ann Occup Hyg 41:155-166 (1997). tion procedure was used and for each permutation, 8. Diez, U. et al. Redecoration of apartments promotes two-sample t statistics were computed for each gene. obstructive bronchitis in atopy risk infants-results of Genes were considered differentially expressed when the LARS-study. Int J Hyg Environ Health 206:173- logarithmic gene expression ratios in three indepen- 179 (2003). dent hybridizations were more than 1.5 or less than 9. Herbarth, O. et al. Effect of indoor chemical exposure 0.66, i.e., 1.5-fold difference in expression level, and on the development of allergies in newborns-LARS when the q-values were ⁄5. study. Proc Healthy Build 1:281-286 (2000). Gene Expressional Profiling of BE 277

10. Aprea, C., Strambi, M., Novelli, M. T., Lunghini, L. 20. Tusher, V. G., Tibshirani, R. & Chu, G. Significance & Bozzi, N. Biologic monitoring of exposure to orga- analysis of microarrays applied to the ionizing radia- nophosphate pesticides in 195 Italian children. Envi- tion response. Proc Natl Acad Sci USA 98:5116-5121 ron Health Perspect 108:521-525 (2000). (2001). 11. Needham, L. L. & Sexton, K. Assessing children’s 21. Krewski, D. et al. Assessing the health risks of ben- exposure to hazardous environmental chemicals: an zene: a report on the benzene state-of-the-science overview of selected research challenges and com- workshop. J Toxicol Environ Health A 61:307-338 plexities. J Expo Anal Environ Epidemiol 10:611-629 (2000). (2000). 22. Lan, Q. et al. Polymorphisms in cytokine and cellular 12. Sexton, K. et al. Children’s exposure to volatile orga- adhesion molecule genes and susceptibility to hema- nic compounds as determined by longitudinal mea- totoxicity among workers exposed to benzene. Can- surements in blood. Environ Health Perspect 113: cer Res 65:9574-9581 (2005). 342-349 (2005). 23. Luttrell, L. M. et al. G-protein-coupled receptors and 13. Kim, Y. J., Song, M. & Ryu, J. C. Gene expression their regulation: activation of the MAP kinase sig- profiling of human bronchial epithelial (BEAS-2B) naling pathway by G-protein-coupled receptors. Adv cells treated with nitrofunrantoin, a pulmonary toxi- Second Messenger Phosphoprotein Res 31:263-277 cant. Mol Cell Toxicol 3:222-230 (2007). (1997). 14. Xu, T., Du, L. F. & Zhou, Y. Evaluation of GO-based 24. Germain, R. N. & Stefanova, I. The dynamics of T functional similarity measures using S. cerevisiae cell receptor signaling: complex orchestration and the protein interaction and expression profile data. BMC key roles of tempo and cooperation. Annu Rev Immu- Bioinformatics 9:472 (2008). nol 17:467-522 (1999). 15. Volinia, S. et al. GOAL: automated gene ontology 25. Kim, B. H. et al. Anti-inflammatory benzene diamine analysis of expression profiles. Nucleic Acids Res 32: compound inhibited toll-like receptor 4-mediated in- 92-99 (2004). ducible nitric oxide synthase expression and nuclear 16. Ashburne, M. et al. Gene ontology: tool for the unifi- factor-kappa B activation. Biol Pharm Bull 5:908-911 cation of biology. Nat Genet 25:25-29 (2000). (2005). 17. Kushida, T., Takagi, T. & Fukuda K. I. Event ontolo- 26. King, K. L. & Cidlowski, J. A. Cell cycle regulation gy: A pathway-centric ontology for biological process- and apoptosis. Annu Rev Physiol 60:601-617 (1998). es. Pac Symp Biocomput 11:152-163 (2006). 27. Kelekar, A. & Thompson, C. B. Bcl-2-family pro- 18. Te Pas, M. F. et al. Biochemical pathways analysis of teins: the role of the BH3 domain in apoptosis. Trends microarray results: regulation of myogenesis in pig. Cell Biol 8:324-330 (1998). BMC Dev Biol 7:66-80 (2007). 28. Pruitt, K. & Der, C. J. Ras and Rho regulation of the 19. Mosmann, T. Rapid colorimetric assay for cellular cell cycle and oncogenesis. Cancer Lett 171:1-10 growth and survival: Application to proliferation and (2001). cytotoxicity assays. J Immunol Meth 65:55-63 (1983).