Set, a Putative Oncogene, as a Biomarker for Prenatal Exposure to Bisphenol A

Background: Bisphenol A (BPA), an endocrine disrupting chemical, has been suspected to pose carcinogenic risks. However, likely mechanisms are obscure and there are difficulties to estimating its real significance for cancer development. Methods: We therefore studied BPA-induced proteomic alterations in immune organs of ICR mice offspring that were prenatally exposed to BPA (15 and 300 mg/L of drinking water). We performed 2D-gel analyses of samples, considering differences in spleen, exposure levels, sex, and ages. Results: From proteomic analyses, we found various proteins were up-or down-regulated by BPA. Among them, SET, a putative oncogene and inhibitor of phosphatase 2A, was significantly down-regulated in a BPA dose-dependent manner. We also confirmed down-regulation of SET in western blot and real time PCR analyses. From gene network analysis, SET is predicted to communicate with other genes including CYP17, which is involved in biosynthesis and metabolism of sex-hormones. Conclusions: This study provided evidence that SET can be applied as a new biomarker for prenatal BPA exposure and suggests a potential new mechanism of action in that BPA may disrupt CYP17 via SET.


Introduction
Most of endocrine disrupting chemicals (EDCs) have been suspected as carcinogens, particularly among second generations.In a case of diethylstylbestrol (DES) of EDCs, DES daughters, who were prenatally exposed to DES by mothers, have an increased risk of developing abnormal cells in the cervix and the vagina that are precursors of cancer, i.e. dysplasia, cervical intraepithelial neoplasia, and squamous intraepithelial lesions (Rubin, 2007).
Bisphenol A [2, 2-bis(4-hydroxyphenyl)propane, BPA] has been considered as a potential EDC.Due to its various usages and increased demands, e.g.substitutes to glass, it has been broadly detected in human urine and blood samples, even the placenta of pregnant women and breast milk (Kuroda et al., 2003;Engel et al., 2006;Ye et al., 2006;Lakind and Naiman, 2008;Yi et al., 2010;Yi et al., 2011).Therefore, exposure to BPA is thought to be quite popular in all populations.Particularly, prenatal exposure to BPA has been emphasized to prevent BPA risks for children's health due to their high susceptibility.BPA has been suspected its carcinogenic risks.However, its carcinogenic end points or mechanisms are obscure and there are even some difficulties to estimate or protect its related cancer risks.Therefore, researchers became to study BPA risks with different ways from those before.Among these studies, omics-approaches and second-hit theories are quite persuasive: For example of omicsapproaches, Dolinoy et al. (2007) reported that early developmental exposure to BPA can change offspring phenotype by stably altering the epigenome (2007).In addition, exposure to BPA increased sensitivity of mutagen-induced sister chromatid exchanges in human, although BPA itself is not a mutagen (Yang et al. 2006), and of prostate gland to carcinogens (Prins et al. 2007).Using proteomic approaches, we also reported some BPA-responsive biomarkers, i.e. apolipoprotein A-I precursor (apo-AI), dipeptidylpeptidase III (DPPIII), and vesicle amine transporter 1 (VAT1), particularly in mouse-thymus (Yang et al., 2008).
In the present study, we performed proteomic and systems biological approaches to find out BPAresponsive biomarkers and -carcinogenic mechanisms among prenatally BPA-exposed mice offsprings.

Materials
All of the reagents for two dimensional polyacrylamide

Animal Care and Treatment
Pregnant ICR mice were arrived at Sookmyung Women's University during the first week of pregnancy from Orient Co. (Seoul, Korea) and maintained under a 12-hr light cycle (lights on 07:00-19:00 h) at 21-24 °C and 40-60 % humidity with free access to pellet food (Samtako, Seoul, Korea) and water continuous access to water.
Pregnant mice group were administered different concentrations of BPA, which was dissolved in 0.1 % ethanol in water into 15 and 300 mg/L through glass bottle for 34-36 days, i.e. from day 7 or 7 days of pregnancy to lactation (days 21 or 21 days from delivery).On the other hand, control pregnant mice drunk 0.1% ethanol in water ad libitum.The 3 and 7 weeks-old offsprings were sacrificed to obtain their immune organ, spleens, which were immediately put into dry ice and kept -80 °C until experiments.

Mass Spectrometry
Selected proteins from 2D gel were identified with peptide mass finger printing methods (Yang et al. 2008): In brief, the protein spots were cut from gel by spot cutter (Bio-Rad), destained and dried in a speed vacuum concentrator for 5 min and rehydrated with 20 μl of 50 mM NH 4 HCO 3 containing 0.2 μg modified trypsin for 30 min on ice.After removal of the solution, 30 μl of 50 mM NH4HCO3 was additionally added and digestion was performed with trypsin overnight at 37 °C.After the removal of residual trypsin, the peptides were desalted using C18 nanoscale column.Custommade columns were used to desalt and concentrate the peptide mixture prior to mass spectrometric analysis: A column containing 100-300 μL of Poros reverse phase R2 material (20-30 μm bead size, PerSeptive Biosystems, Framingham, MA) was packed into a constricted GELoader tip (Eppendorf, Hamburg, Germany).A 10 mL syringe was used to force the liquid through the column via the application of gentle air pressure.Thirty μL of the peptide mixture from the digestion supernatant was loaded onto the column, and then washed with 30 μL of 5% formic acid.For the MS/MS analysis, the peptides were eluted by the direct application of 2.5 μL of a solution consisting of 60% methanol, 35% H 2 O, and 5% formic acid, into a precoated borosilicate nanoelectrospray needle (EconoTipTM, New Objective, Woburn, MA).MS/MS of peptides generated by in-gel digestion was performed by nano-ESI on a Q-TOF mass spectrometer (Micromass, Manchester, UK).The source temperature was 80 ℃.A potential of 1 kV was applied to the precoated borosilicate nanoelectrospray needles in the ion source combined with a nitrogen back-pressure of 0-5 psi to produce a stable flow rate (10-30 nL/min).The cone voltage was 40 V.The quadrupole analyzer was used to select precursor ions for fragmentation in the hexapole collision cell.The collision gas was Ar at a pressure of 6-7 x 10-5 mbar and the collision energy was 20-30 V. Product ions were analyzed using an orthogonal TOF analyzer, fitted with a reflector, a micro-channel plate detector and a time-to-digital converter.All MS/MS spectra recorded on tryptic peptides derived from spot were searched against protein sequences from NCBInr and EST databases using the MASCOT search program.

Western Blot Analysis
Eighty μg of each sample protein was loaded on 15% (w/v) SDS polyacrylamide gel and separated by electrophoresis.The gels were transferred to

BPA Exposure Levels
We calculated the real exposure levels of BPA from different concentrations of BPA in drinking water (Table 1).There were no significant differences in body weight or consumption of drinking water due to BPA exposure (ps> 0.05).

Proteomic analysis
We carried out 2D-PAGE to find responsive proteins for BPA exposure in offspring-spleens.Protein spots were statistically evaluated in order to identify significantly regulated spots by BPA-exposure.Comparison of abundant proteins on 2D gels using PDQUEST program revealed that only the Albumin A1 and SET was significantly down-regulated by BPA; Apo A-I was up-regulated by BPA, compared to controls (Table 2; Figure 1-2).These proteins were identified by peptide mass fingerprinting method using ESI-Q-Tof mass spectrometry and database search.

Validation of the Proteins by Western Blots and Realtime PCR
For the validation of the 2D-gel results, we assessed the protein and gene expression levels by western blots polyvinylidene difluoride membranes (Hybond™-P, Amersham Biosiences, Burkinghamshire, UK) and blocked with phosphate-buffered saline/5% skim milk/0.05%Tween 20 for 1.5 hours.Primary antibodies, SET and Apo A-I, were diluted to 1:1000 in blocking buffer and incubated for 2 hrs at room temperature.After washing, membranes were incubated for 1.5 hrs with a horse radish peroxidase-conjugated secondary antibody, and developed with ECL Plus western blotting detection kit (GE Healthcare, Bukinghamshire, UK).

Gene Expression Analysis by Real-time PCR
Total RNAs were isolated from homogenized spleen samples by SV Total RNA Isolation System (Promega).One ug of the RNA sample was used for reverse transcription polymerase chain reaction (RT-PCR) and real time PCR.The RNA molecules were subjected to cDNA synthesis using Applied Biosystems High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA) according to manufacturer's suggested protocol.To quantify SET expression, we used TaqMan fluorogenic real time PCR method with an ABI Prism 7500 Sequence Detection System (Applied Biosystems).The primers and probe were 5'-CCGAAGCTGGACAAGTC-3' (sense) and 5'-AGCTACGCGGGAAAACTACAC-3'(antisense), and FAM5'-ACGACTCGA-AGCTCC-TAMRA, respectively.PCR conditions were as followed: 2 min at 50 °C for AmpErase UNG activation and 10 min at 95 °C for UNG inactivation, followed by 40 cycles of 15 sec at °C for denaturing, and 1 min at 60 °C for annealing and extension.After constructing standard curves, mRNA expression levels of SET was quantified and normalized with respect to expression of 18S rRNA (Applied Biosystems), a reference gene.We performed triplicate realtime PCR analyses for each sample.

Global gene network analysis
Using STRING scores (STRING 9.0), we studied global function of SET and its related proteins.

Statistical Analyses
ANOVA was used to analyze differences in body weights, consumption of water and expression of SET due to BPA exposure.Association between BPA exposure levels and mRNA expression of SET was studied with regression analysis.P-values for all tests were computed by JMP version 4 (SAS Institute, Cary, NC), and p< 0.05 was used to identify significant associations.

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and real-time PCR.We confirmed BPA down-regulates SET protein-expression and this result is consistent with the above 2D gel results.However, we could not confirm BPA-modulation in protein-expression of apo A-I through western blots (Figure 3).Therefore, we further studied whether BPA exposure altered mRNA expression of not apo A-I but SET.Performing the real time-PCR, we found that expression of SET was downregulated in BPA-dose dependent manner, particularly in the spleens of 3 weeks-old female mice (Figure 4).

Discussion
The present study was designed as our serial attempts to find BPA-sensitive proteomic biomarkers and to clarify carcinogenic mechanisms of BPA in spleen.In the present study, BPA showed down regulation of SET and it was consistently confirmed by 2D-gel, western blot and realtime PCR analyses.SET was first described as part of the SET-CAN fusion gene, a putative oncogene associated with acute undifferentiated leukaemia (von Lindern et al., 1992): SE in SET refers to the patient with leukemia containing SET translocation and the T in SET refers to translocation (Simon et al., 2012).
In a case of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the most notorious EDC, it has a two-edged blade: It is carcinogenic and triggering lots of endocrine disorders, however, inhibits estrogen-induced responses in the rodent uterus and mammary tumors and in breast and endometrial cancer cell lines through complex inhibitory aryl hydrocarbon receptor (AhR) -estrogen receptor (ER) crosstalk (Safe and McDougal, 2002).Therefore, some of selective AhR modulators, which have structure-similarities with TCDD and are relatively non-toxic, inhibit ER-positive and ER-negative mammary tumor growth, synergize with tamoxifen to inhibit breast cancer growth and block tamoxifen-induced estrogenic activity in the uterus.BPA is also suspected to have twoedged blade, because it is suspected as a carcinogen and EDC, however, down-regulated oncogenic SET in the present study.SET is suggested as a new treatment target in B-cell malignancies and SET antagonists can represent novel agents for treatment of chronic lymphocytic leukemia and non-Hodgkin lymphoma (Christensen et al., 2011).Therefore, some of new chemicals, which have structure-similarity to BPA but are not harmful, can be potential anticancer medicines for SET-mediated cancers.
On the other hand, SET is expected to interact with CYP17A1 (score, 0.924) from the gene network analysis (Zhang et al., 2001;Belcher et al., 2005;Mellon et al., 2007).CYP17 is known to convert pregnenolone and  progesterone to their 17-alpha-hydroxylated products and subsequently to dehydroepiandrosterone and androstenedione.Therefore, CYP17 may be involved in sexual development during fetal life and at puberty.In addition, a recent study showed that BPA inhibits activity of human and rat testicular steroidogenic enzyme including CYP17A1 (Ye et al., 2011).Therefore, the present study provides a potential new mechanism that BPA disrupts CYP17 via SET.
In the present study, the putative BPA-biomarker, SET, was down-regulated in spleens of not 7 weeks-but 3 weeks-old female mice by BPA prenatal exposure.These results suggest that BPA exposure during pregnancy and lactation may induce the proteomic alteration in immune organ, particularly spleen, in young generation.In addition, the spleen undergoes significant molecular remodeling during puberty, resulting in both age and gender-dependent differences in immune system (Lamason et al., 2006).Therefore, SET can be an early and female selective biomarker in immune system from consideration of age and sex.
In conclusion, our present study provides that SET can be a responsive biomarker for BPA-prenatal exposure with proteomic and gene net work approaches.In addition, interaction between BPA and SET should be further studied to clarify BPA-end points.

Figure 1 .
Figure 1.Narrow pH Range of Isoelectric Focusing on 2D Gel.Homogenized spleen tissue (350 ug) was loaded onto the gel.Dose-dependent spots (#1010, 2013, and 5107) as well as other features were visible by silver staining

Figure 4 .
Figure 4. Effects of BPA Exposure on SET Expression in Spleen of 3-week-old Female Mice (N=9).Expression of SET was decreased in BPA dosedependent manner (R2, 0.23; p=0.04 by regression analysis)

Table 2 . Identification of Responsive Proteins in Mice Spleen
a Age of mice (week); M, male; F, female; h, up-regulated; i, down-regulated Figure 3.