1. Evaluating the Effects of Environmental Toxin 4-Nonylphenol and Estrogen on U937 Human Immune Cells Via Microarray Analysis Esop Baek and Celline Kim Manhasset HS Science Research in Cooperation with Dr. Patrick Cadet and Kirk Mantione Neuroscience Research Institute SUNY/Old Westbury

2. Rationale for Study graph below shows how breast cancer compares to other common causes of death in women of all ages. ‡ Source: Surveillance, Epidemiology, and End Results (SEER) Program (www.seer.cancer.gov) SEER*Stat Database: Mortality – All COD, Public-Use With State, Total U.S. (1969–2004), National Cancer Institute, DCCPS, Surveillance Research Program, Cancer Statistics Branch, released April 2007. Underlying mortality data provided by NCHS (www.cdc.gov/nchs).www.seer.cancer.govwww.cdc.gov/nchs -40,000 women affected each year by breast cancer -5 th leading cause of cancer death worldwide (both sexes counted) -Estradiol-the major estrogen in the body Estradiol

3. 4-Nonylphenol C 15 H 24 O -Used as a starting material for surfactants http://ocw.mit.edu/NR/rdonlyres/Health-Sciences-and-Technology/g -Ruthann (03) demonstrated 4-NP to be ubiquitous in US homes

4. Breast Cancer Rates http://ocw.mit.edu/NR/rdonlyres/Health-Sciences-and-Technology/HST-512Spring2004/8E59C8BC-D738-4FE9-9B2D-35AE060522E2/0/chp_microarray.jpg http://ocw.mit.edu/NR/rdonlyres/Health-Sciences-and-Technology/g -Jacquez (03) suggested that LI’s environment may be responsible

5. DNA Microarray Procedure http://ocw.mit.edu/NR/rdonlyres/Health-Sciences-and-Technology/HST- 512Spring2004/8E59C8BC-D738-4FE9-9B2D- 35AE060522E2/0/chp_microarray.jpg http://www.carleton.ca/catalyst/2006s/images/dk-PersMed3.jpg A microarray chip, or DNA chip, is used to analyze DNA sequences

6. Methodology Control (no treatment) Estrogen (5 uM) 4-nonylphenol (5 uM) Cultured Cells, Isolated RNA, RTed to cDNA Data analyzed by Spotfire software RT-PCR performed with ESR2 gene and beta-actin reference gene Applied cDNA to microarray chips. Which were then scanned for detection of gene expression by chemiluminescence U937 Human Immune Cells (ATTC, USA)

7. RNA Isolation and Semi-quantitave RT-PCR GenesPrimer Sequence ERβ-15’-TGAAAAGGAAGGTTAGTGGGAACC-3’ ERβ-25’-TGGTCAGGGACATCATCATGG-3’ β-actin 1 5’ TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3’ β-actin 2 5’ CTA GAA GCA TTG CGG TGG ACG ATG GAG GG-3’ RNA Isolation: 48 hours after the application of the treatments (E2 and 4-NP), the U937 cells were detached from a six-well plate and then pelleted via centrifugation. RNA was isolated using the RNeasy Protect Mini Kit (Qiagen, Stanford, CA).

8. Sample of the 90 Customized Portfolio of Genes (GeneEntrez, GeneOntology) Gene Ontology Database Gene_ID Gene_NameGene_Symbol hCG21449.3;hCG21449.3estrogen receptor 2 (ER beta)ESR2 hCG2016877;hCG2016877peroxiredoxin 5;estrogen-related receptor alphaESRRA;PRDX5 hCG31941.3;hCG31941.3estrogen receptor bining proteinERBP hCG1796030.1;hCG1796030.1 peroxisome proliferative activated receptor, gamma, coactivator 1, beta PPARGC1B hCG28493.2;hCG28493.2retinol dehydrogenase 8 (all-trans)RDH8 hCG1811630.1;hCG1811630.1estrogen receptor 1ESR1 hCG20789.4;hCG20789.4egl nine homolog 2 (C. elegans)EGLN2 hCG21644.4;hCG21644.4;hCG28380.2estrogen-related receptor betaESRRB hCG2015551;hCG2015551DEAD (Asp-Glu-Ala-Asp) box polypeptide 54DDX54 hCG21644.4;hCG28380.2;hCG21644.4estrogen-related receptor betaESRRB hCG24417.4;hCG24417.4RAS-like, estrogen-regulated, growth inhibitorRERG hCG1781443.2;hCG1818318.1;hCG1781443.2UDP glycosyltransferase 2 family, polypeptide B4UGT2B4 hCG1776094.1;hCG1776094.1tripartite motif-containing 47TRIM47 hCG25939.2;hCG25939.2repressor of estrogen receptor activityREA hCG2028796.1;hCG2028796.1breast cancer anti-estrogen resistance 3BCAR3 hCG2010626;hCG2010626RNA binding motif protein 9RBM9 hCG20144.4;hCG20144.4RNA binding motif protein 14RBM14 hCG24804.3;hCG24804.3estrogen-related receptor gammaESRRG hCG1781443.2;hCG1818318.1;hCG1781443.2UDP glycosyltransferase 2 family, polypeptide B4UGT2B4 hCG1796030.1;hCG1796030.1 peroxisome proliferative activated receptor, gamma, coactivator 1, beta PPARGC1B hCG38316.2;hCG38316.2sulfotransferase family 1E, estrogen-preferring, member 1SULT1E1

9. Results and Discussion

10. Figure 2: (A) Scatterplots of gene expression analyzed by DNA microarray. The probe ID’s for each gene is represented by the x-axis and the fold changes in gene expression for the experimental samples based on the fluorescence-based detection signals of the mRNA levels in the control are represented by the y-axis. (B) Remaining genes after filtering by modulation and normalization with the b-actin reference gene. Genes that exhibited 2- fold regulation AB Genes altered with E2 treatment after 48 hours Genes altered with 4-NP treatment after 48 hours Graphical Representation of Significantly Altered Genes Scanned chip containing DNA oligo information. A display of the gene profiling capabilities offered by microarray technology -19,000 genes were detected by chemiluminescence, offering a wide range of data to be observed Signal Responses from Scanned Estrogen Chip

11. # Of Genes Induced By E2 and 4-NP by a > or = 2-Fold Change from the Control E2 4-NP 14,913 (45.0%) 414 (1.2%) 633 (1.9%) All genes from the whole- genome nanochips (33,155) 54 (60.0%) 7 (7.8%) 8 (8.9%) E2 4-NP Genes from customized Portfolio (90) Figure 3: (A) Venn Diagram which shows the number of genes that exhibited significant changes in gene expression after the application of E2 and concentrations of NP. (B) A normalized hierarchical clustering heat map performed in order to similarities of genes and to what extent they were affected. Genes that were downregulated significantly are illustrated by the green while red denotes genes that were upregulated by at least 2-fold. Comparison of Induced Genes overall and within Portfolio Genes -about 45.0% (14,913) exhibited significant changes in gene expression (> 2-fold) by E2 while approximately 1.91% (633)

12. Putative Biomarkers for 4-NP exposure in U937 Cells Table 3 Possible gene expression biomarkers for 4-NP exposure in U937 Immune Cells Gene_ID Gene NameGene Symbol Fold change compared to control hCG28493.2retinol dehydrogenase 8 (all-trans) RDH82.09 hCG1781181.1ankyrin repeat domain 12ANKRD12-2.40 hCG1781443.2UDP glycosyltransferase 2 family, polypeptide B4 UGT2B4-2.98 hCG21449.3estrogen receptor 2 (ER beta) ESR2-2.47 hCG2042581FLJ46358 proteinFLJ463582.78

13. Figure 5: This figure indicates that E2 and NP have the potential to down regulate the estrogen receptor beta (ESR1) and the estrogen receptor related beta (ESRR-β) and up regulate repressor estrogen receptor activity (REA) (at their respective concentrations). ESR1 does not exhibit a significant pattern. These data appear to demonstrate that NP as well as enhanced estrogen levels diminishes estrogen beta activity negatively. Signal to Noise Values For Four genes From the Portfolio/List Signal/Noise Ratio

14. Figure 7: RT-PCR Analysis of Intensity of ESR2 gene expression normalized with the internal control gene beta-actin. This data serves as a validation for the microarray analysis. RT-PCR Gene Expression Profile ESR2 β-actin 1 2 3 4 Figure 6: These gels exhibit expression of the ESR2 gene the β-actin reference gene. Lane 1=control, Lane 2 =E2 5uM, Lane 3=control and Lane 4 = 4-NP 5uM. n=4

15. Figure 4: Gene profiles of BRCA1 and BRCA2. The E2 treament induced a fold repression of more than two fold while the NP treatment had no significant effect. This phenomenon was observed for 23/45 genes in the portfolio involved in the onset of breast cancer. Gene Expressions Profiles of BRCA1 and BRCA2 Relative Gene Expression (Signal of treatment chip/signal of chip treatment) Relative Gene Expression (Signal of treatment chip/signal of chip treatment)

16. Conclusion -4-Nonylphenol elicited significant changes in expression of the portfolio genes

17. Future Studies Evaluating the 5 potential biomarkers with other substances Testing the long-term effects of Estrogen and 4- Nonylphenol Gene_ID Gene NameGene Symbol Fold change compared to control hCG28493.2retinol dehydrogenase 8 (all-trans) RDH82.09 hCG1781181.1ankyrin repeat domain 12ANKRD12-2.40 hCG1781443.2UDP glycosyltransferase 2 family, polypeptide B4 UGT2B4-2.98 hCG21449.3estrogen receptor 2 (ER beta) ESR2-2.47 hCG2042581FLJ46358 proteinFLJ463582.78

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20. Acknowledgements : -Mr. Guastella, Manhasset Science Research -Dr. George B. Stefano, Lab Director -Dr. Patrick Cadet, Mentor -Mr. Kirk Mantione, Microarray Class Instructor -SCA, Summer Studies Scholarship