Esop Baek and Celline Kim Manhasset HS Science Research in Cooperation with

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). Estradiol -40,000 women affected each year by breast cancer -5th leading cause of cancer death worldwide (both sexes counted) -Estradiol-the major estrogen in the body

3. 4-Nonylphenol C15H24O -Ruthann (03) demonstrated 4-NP to be ubiquitous in US homes http://ocw.mit.edu/NR/rdonlyres/Health-Sciences-and-Technology/g -Used as a starting material for surfactants

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

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

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

7. RNA Isolation and Semi-quantitave RT-PCR 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)

9. Results and Discussion

10. Signal Responses fromScanned Estrogen Chip Graphical Representation of Significantly Altered Genes A B Genes altered with 4-NP treatment after 48 hours Genes that exhibited 2-fold regulation Scanned chip containing DNA oligo information. A display of the gene profiling capabilities offered by microarray technology Genes altered with E2 treatment after 48 hours Genes that exhibited 2-fold regulation -19,000 genes were detected by chemiluminescence, offering a wide range of data to be observed 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.

11. Comparison of Induced Genes overall and within Portfolio Genes # Of Genes Induced By E2 and 4-NP by a > or = 2-Fold Change from the Control 4-NP E2 All genes from the whole- genome nanochips (33,155) 14,913 (45.0%) 633 (1.9%) 414 (1.2%) E2 4-NP Genes from customized Portfolio (90) 54 (60.0%) 8 (8.9%) 7 (7.8%) 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. -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

13. Signal to Noise Values For Four genes From the Portfolio/List Signal/Noise Ratio 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.

14. RT-PCR Gene Expression Profile ESR2 β-actin n=4 n=4 n=4 1 2 3 4 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. 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.

15. 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) 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.

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

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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