Evaluating the Effects of Environmental Toxin 4-Nonylphenol and Estrogen on

U937 Human Immune Cells Via Microarray Analysis

Esop Baek and Celline KimManhasset HS Science Research

in Cooperation with Dr. Patrick Cadet and Kirk Mantione

Neuroscience Research Institute SUNY/Old Westbury

Rationale for Studygraph 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).

-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

Estradiol

4-Nonylphenol

C15H24O-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

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

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

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)

RNA Isolation and Semi-quantitave RT-PCR

Genes Primer Sequence

ERβ-1 5’-TGAAAAGGAAGGTTAGTGGGAACC-3’

ERβ-2 5’-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).

Sample of the 90 Customized Portfolio of Genes(GeneEntrez, GeneOntology)

Gene Ontology Database Gene_IDGene_Name Gene_Symbol

hCG21449.3;hCG21449.3 estrogen receptor 2 (ER beta) ESR2

hCG2016877;hCG2016877 peroxiredoxin 5;estrogen-related receptor alpha ESRRA;PRDX5

hCG31941.3;hCG31941.3 estrogen receptor bining protein ERBP

hCG1796030.1;hCG1796030.1peroxisome proliferative activated receptor, gamma, coactivator 1, beta

PPARGC1B

hCG28493.2;hCG28493.2 retinol dehydrogenase 8 (all-trans) RDH8

hCG1811630.1;hCG1811630.1 estrogen receptor 1 ESR1

hCG20789.4;hCG20789.4 egl nine homolog 2 (C. elegans) EGLN2

hCG21644.4;hCG21644.4;hCG28380.2 estrogen-related receptor beta ESRRB

hCG2015551;hCG2015551 DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 DDX54

hCG21644.4;hCG28380.2;hCG21644.4 estrogen-related receptor beta ESRRB

hCG24417.4;hCG24417.4 RAS-like, estrogen-regulated, growth inhibitor RERG

hCG1781443.2;hCG1818318.1;hCG1781443.2 UDP glycosyltransferase 2 family, polypeptide B4 UGT2B4

hCG1776094.1;hCG1776094.1 tripartite motif-containing 47 TRIM47

hCG25939.2;hCG25939.2 repressor of estrogen receptor activity REA

hCG2028796.1;hCG2028796.1 breast cancer anti-estrogen resistance 3 BCAR3

hCG2010626;hCG2010626 RNA binding motif protein 9 RBM9

hCG20144.4;hCG20144.4 RNA binding motif protein 14 RBM14

hCG24804.3;hCG24804.3 estrogen-related receptor gamma ESRRG

hCG1781443.2;hCG1818318.1;hCG1781443.2 UDP glycosyltransferase 2 family, polypeptide B4 UGT2B4

hCG1796030.1;hCG1796030.1peroxisome proliferative activated receptor, gamma, coactivator 1, beta

PPARGC1B

hCG38316.2;hCG38316.2 sulfotransferase family 1E, estrogen-preferring, member 1 SULT1E1

Results and Discussion

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

Genes that exhibited 2-fold regulation

A B

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

# 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 customizedPortfolio(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)

Putative Biomarkers for 4-NP exposure in U937 Cells

Table 3 Possible gene expression biomarkers for 4-NP exposure in U937 Immune Cells

Gene_IDGene Name Gene Symbol Fold change compared to

control

hCG28493.2 retinol dehydrogenase 8 (all-trans)

RDH8 2.09

hCG1781181.1 ankyrin repeat domain 12 ANKRD12 -2.40

hCG1781443.2 UDP glycosyltransferase 2 family, polypeptide B4

UGT2B4 -2.98

hCG21449.3 estrogen receptor 2 (ER beta)

ESR2 -2.47

hCG2042581 FLJ46358 protein FLJ46358 2.78

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

Sig

nal/N

oise

Rat

io

RT-PCR Gene Profile of ESR2

020406080

100120140

Control (NoTreatment)

E2 (5 uM) 4-NP (5 uM)

Treatments

mRN

A/B

-act

in m

RNA

(%)

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 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

n=4 n=4

Gene Expression Profile of BRCA1

00.10.20.30.40.50.60.70.80.91

1.11.21.3

No Treatment(Control

E2 (5µM) 4-NP (5µM)

Treatments

Relativ

e G

en

e E

xp

ressio

n

(sig

nal

of t

reatm

en

t ch

ip/s

ign

al

of

co

ntr

ol

ch

ip)

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 Expression Profile of BRCA2

00.10.20.30.40.50.60.70.80.91

1.11.21.3

No Treatment(Control

E2 (5µM) 4-NP (5µM)

Treatments

Relativ

e G

en

e E

xp

ressio

n

(sig

nal o

f treatm

en

t ch

ip

/sig

nal o

f

co

ntro

l ch

ip

)

Gene Expressions Profiles of BRCA1 and BRCA2

Rel

ativ

e G

ene

Exp

ress

ion

(Sig

nal o

f tr

eatm

ent c

hip/

sign

al o

f ch

ip tr

eatm

ent)

Rel

ativ

e G

ene

Exp

ress

ion

(Sig

nal o

f tr

eatm

ent

chip

/sig

nal o

f ch

ip

trea

tmen

t)

Conclusion

-4-Nonylphenol elicited significant changes

in expression of the portfolio genes

Future Studies

• Evaluating the 5 potential biomarkers with other substances

• Testing the long-term effects of Estrogen and 4-Nonylphenol

Gene_IDGene Name Gene Symbol Fold change compared to

control

hCG28493.2 retinol dehydrogenase 8 (all-trans)

RDH8 2.09

hCG1781181.1 ankyrin repeat domain 12 ANKRD12 -2.40

hCG1781443.2 UDP glycosyltransferase 2 family, polypeptide B4

UGT2B4 -2.98

hCG21449.3 estrogen receptor 2 (ER beta)

ESR2 -2.47

hCG2042581 FLJ46358 protein FLJ46358 2.78

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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