Mol. Hum. Reprod. Advance Access originally published online on January 19, 2008
Molecular Human Reproduction 2008 14(2):97-106; doi:10.1093/molehr/gam091
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Gene expression analysis of human endometrial endothelial cells exposed to op'-DDT
1Department of Women’s and Children’s Health, Uppsala University, SE-751 85 Uppsala, Sweden 2Department of Woman and Child Health, Karolinska Institutet, SE-171 76 Stockholm, Sweden
3 Correspondence address. E-mail: matts.olovsson{at}kbh.uu.se
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The endocrine disrupting chemical o, p'-dichlorodiphenyltrichloroethane (DDT) can affect reproductive organs, tissues and cells in several species. Treatment of human endometrial endothelial cells (HEECs) with 50 µM o,p'-DDT decreased their proliferation compared with the control. Microarray analyses revealed that o,p'-DDT affected biological processes such as the cell cycle, cell division, defence response and lipid and steroid metabolism, in cellular components such as the plasma membrane and chromosomes, with molecular functions involved in signalling, receptor and cytokine activity, confirming the results of the proliferation assay. Expression of five of the most differentially expressed genes identified in the microarray analysis was verified by real-time quantitative reverse transcription polymerase chain reaction in five HEEC cultures obtained from women in the proliferative phase and in five cultures obtained from women in the secretory phase of the menstrual cycle after treatment with o,p'-DDT. The present study supports our previous findings of decreased proliferation and increased cell death in response to o,p'-DDT and may offer important clues to the mechanisms of action of o,p'-DDT.
Key words: endometrial cells/in vitro cell cultures/microarray/o,p'-DDT/reproductive tissue
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The use of dichlorodiphenyltrichloroethane (DDT) has been banned since the 1970s. However, it persists in the environment and can be detected in environmental samples such as sediment, water and pore water from, for example, the Beijing Guan Ting Reservoir (Xue and Xu, 2006). DDT can also be found in many biological and human samples, such as Mediterranean swordfish liver (Storelli and Marcotrigiano, 2006), human breast milk (Kanja et al., 1992; Torres-Arreola et al., 1999), serum (Kanja et al., 1992; Lino and da Silveira, 2006), uterine tissue (Saxena et al., 1987), endometrium (Schaefer et al., 2000), follicular fluid (Trapp et al., 1984), semen (Stachel et al., 1989) and body fat (Kanja et al., 1992; Schaefer et al., 2000).
Commercially available DDT contains 
80% p,p'-DDT and 15–20% o,p'-DDT. p,p'-DDT and the DDT metabolite o,p'-dichlorodiphenyldichloroethylene (DDE) bind weakly to the transmembrane estrogen receptor GPR30 (Thomas and Dong, 2006). DDT and DDT metabolites can also act through estrogen receptor-independent mechanisms, for example by activation of the transcription factor activator protein-1 in human uterine cell lines (Frigo et al., 2002). It is possible that the maternal level of p,p'-DDT and p,p'-DDE during pregnancy can influence the probability of pregnancy in their daughters (Cohn et al., 2003). Data also suggests that DDE may be involved in the occurrence of precocious puberty in girls (Krstevska-Konstantinova et al., 2001; Parent et al., 2005), and that high maternal levels of DDE may be associated with a decreased foetal birthweight and head circumference (Wolff et al., 2007) and possibly also with foetal loss (Longnecker et al., 2005). Sperm motility has been found to be inversely related to the p,p'-DDT concentration in serum (Toft et al., 2006), and occupational paternal DDT exposure may be associated with an increased risk of birth defects (Salazar-Garcia et al., 2004). A recent study has indicated that a small number of CAG repeats in the androgen receptor gene in combination with high serum levels of p, p'-DDE might be related to an increased DNA fragmentation index in sperm (Giwercman et al., 2007). DDE may cause reproductive failure due to eggshell thinning and broken eggs in avian wildlife (Peakall et al., 1973; Lundholm, 1997). DDE increases aromatase activity in cultured human endometrial stromal cells (Holloway et al., 2005). In rats, p,p'-DDE has antiandrogenic effects both in vitro and in vivo, such as a reduced anogenital distance and delayed onset of puberty in male offspring (Kelce et al., 1995). Alligator eggs from the contaminated Lake Apopka in Florida contain high levels of p,p'-DDE (Heinz et al., 1991), and developmental abnormalities of the gonads, steroidogenesis and sex steroid concentrations in hatching alligators have been reported (Guillette et al., 1994, 1995). In humans, however, there is no conclusive evidence of antiandrogenic effects of DDE on the genitalia of male newborns (Longnecker et al., 2002, 2007).
o, p'-DDT binds to both estrogen receptor 
(Nelson, 1974; Kuiper et al., 1998) and β (Kuiper et al., 1998). Further, o, p'-DDT stimulates transcriptional activity from both estrogen receptors (Kuiper et al., 1998), has estrogenic effects in the female reproductive tract (Bitman et al., 1968; Welch et al., 1969; Cecil et al., 1971), inhibits uterine uptake of estradiol (Welch et al., 1969) and is inhibited by substances that block estrogenic effects (Cecil et al., 1971). In ovo exposure to o, p'-DDT and other xeno-estrogens may disturb gonadal development (Blomqvist et al., 2006;Holm et al., 2006) and male sexual behaviour in adult birds (Bryan et al., 1989; Berg et al., 1998). Recent results from our laboratory also indicate that o,p'-DDT influences proliferation and viability of human endometrial endothelial cells (HEECs) in vitro (Bredhult et al., 2007). Taken together, it seems from the above findings as if DDT is an endocrine disrupting chemical that can influence reproductive organs, tissues and cells. In this investigation, we used microarrays to study the effects of o,p'-DDT on the mRNA expression of HEECs cultured in vitro. Since we had previously found that HEECs showed reduced proliferation and increased cell death after exposure to o,p'-DDT (Bredhult et al., 2007), we expected that gene products involved in these processes would be affected, but we were also interested in the expression of estrogen responsive genes and gene products involved in angiogenesis and cellular stress.
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The study was approved by the Ethics Committee at Uppsala University, Sweden (
583, Ups 03-575). Informed consent was obtained before tissue sampling.
Establishment of cell cultures
Endometrial biopsy samples were obtained from 10 hysterectomy specimens and immediately put in sterile phosphate-buffered saline (PBS) (Dulbecco’s w/o calcium, magnesium and sodium bicarbonate; Invitrogen AB, Lidingö, Sweden) or sterile Endothelial cell Basal Medium-2 (EBM®-2; In vitro Sweden AB, Stockholm, Sweden). All women participating in the study were of fertile age and had regular menstrual cycles. The menstrual cycle phase was determined by combining menstrual history data and histological criteria (Noyes et al., 1950). Five women were in the proliferative phase of the menstrual cycle and five were in the secretory phase. The reasons for surgery were benign leiomyomas in all cases and there were no endometrial pathological conditions. None of the study subjects had received any hormone therapy during a period of at least 3 months prior to surgery. The biopsy samples were washed in sterile PBS, and this was followed by a series of brief washings in an iodine solution (Apoteket AB, Stockholm, Sweden), PBS, iodine solution and finally three times in PBS. The biopsy samples were cut into millimetre-sized pieces and thereafter exposed to a sterile-filtered solution of digesting enzymes [2.5 mg/ml collagenase type II (Invitrogen AB); 50 µg/ml deoxyribonuclease II (Sigma-Aldrich Sweden AB, Stockholm, Sweden) and 200 µg/ml hyaluronidase (Sigma-Aldrich Sweden AB)], and the antibiotic gentamicin, 4 mg/ml (Invitrogen AB) in PBS for 2 x 1 h at 37°C in an atmosphere of 5% CO2 in humidified air in a Forma Scientific CO2 incubator (AB Nino Lab, Upplands Väsby, Sweden). After 1 h, the cell suspension was poured through a 100 µm nylon cell strainer (Bergman-Labora, Upplands Väsby, Sweden) into a sterile centrifugation tube containing PBS, and the undigested tissue was re-incubated in fresh enzyme solution for an additional hour. The cell suspension was poured through the cell strainer into the centrifugation tube and centrifuged at 400g for 10 min. The supernatant was discarded and the cell pellet was dissolved in 1–2 ml of sterile 0.1% bovine serum albumin (Sigma-Aldrich Sweden AB) in PBS. The cell suspension was transferred to a smaller sterile tube and 25 µl Dynabeads® CD31 Endothelial Cell (Dynal® Biotech ASA, Oslo, Norway) was added per millilitre cell suspension. The cell suspension was then placed on a shaking rocker for 30 min at 2–8°C. Thereafter, the cell suspension was split into two tubes and two to three times the cell suspension volume of sterile 0.1% BSA–PBS was added. The tubes were then placed in a magnetic holder for 2 min. The first supernatants were transferred to new tubes and put in the magnetic holder once more. All supernatants were subsequently discarded. The cells were suspended in fresh 0.1% BSA–PBS and the procedure was repeated until the supernatant was clear. The endothelial cells were suspended in Microvascular Endothelial Cell Medium-2 (EGMTM-2MV Bulletkit®; In vitro Sweden AB), seeded into culture flasks (25 cm2; Fischer Scientific GTF, Västra Frölunda, Sweden) and incubated at 37°C in an atmosphere of 5% CO2 in humidified air.
The culture medium was changed two to three times a week and the cells were checked repeatedly for growth, using an inverted phase contrast microscope (Nikon Diaphot 300; TeknoOptik AB, Skärholmen, Sweden). At subconfluency in passages 0 and 1, subcultivation of cells was performed by trypsination with Trypsin–EDTA (Gibco, Invitrogen) according to standard procedures.
Test substance
2,2-Bis(o,p-chlorophenyl)-1,1,1-trichloroethane (o,p'-DDT; 99.5% purity; LGC Promochem AB, Borås, Sweden) was purchased and a stock solution with dimethyl sulphoxide (DMSO: Sigma-Aldrich Sweden AB) was prepared. Prior to the experiment, the stock solution was diluted further in the cell culture medium to a concentration of 50 µM. The DMSO content (v/v) of the final solution was 0.09%, and the DMSO content of the control was 0.21%.
In view of the toxicity and health risks, o,p'-DDT and DMSO were handled with great care in a fume hood and all waste was collected and disposed of according to applicable directions.
Cell culture experiments
5-Bromo-2'-deoxyuridine assay
At subconfluency in passage 1, the HEECs were transferred to sterile 96-well plates (Fischer Scientific GTF, Västra Frölunda, Sweden) for the 5-bromo-2'-deoxyuridine (BrdU) assay (Roche Diagnostics Scandinavia AB, Bromma, Sweden). HEECs from all 10 women were used. At subconfluency, cells in 6 wells per cell culture were used for each of the controls and for exposure to 50 µM o,p'-DDT. As additional controls, a background control with addition of culture medium but no BrdU labelling solution was performed, as well as a blank control with cell culture medium and BrdU labelling solution but without cells in the microtitre plate wells. Each well received 100 µl test solution and 10 µl BrdU labelling solution, except for the background control, from which the labelling solution was omitted, and the plates were incubated for 24 h at 37°C in an atmosphere of 5% CO2 in humidified air.
After the 24 h incubation, the culture medium was removed and 200 µl FixDenat solution was added to each well. The plates were incubated at room temperature for 30 min. After the FixDenat had been removed, 100 µl anti-BrdU-POD (monoclonal antibody from mouse-mouse hybrid cells, conjugated with peroxidase) was added to each well and the plates were incubated for 1 h. After removal of the antibody solution, the wells were washed three times with 200 µl washing solution. Finally, 100 µl substrate solution was added to each well and the plates were incubated for 30 min. The colour intensity was measured spectrophotometrically at 405 nm. As stated in the BrdU kit manual, the colour intensity, and hence the absorbance values, directly correlate to the amount of DNA synthesis and number of proliferating cells. The mean value of the blank control was subtracted from all other values to correct for unspecific binding of anti-BrdU-POD to the plastic in the 96-well plates and the mean absorbance values for each treatment and culture were calculated.
Cell culture for RNA isolation
At subconfluency in passage 2, the human HEECs were exposed to 50 µM o,p'-DDT, or fresh culture medium containing 0.21% DMSO, for 24 h. Cells in four culture flasks (25 cm2) per HEEC culture were exposed to o,p'-DDT, and cells in four culture flasks served as control. Eight culture flasks per HEEC culture were used for an additional control set-up, in which the cells in four culture flasks were designated for inclusion in a pool of the five individual controls, so that each individual control could be compared with the control pool. After exposure, the cells were harvested with a 0.25% trypsin–0.1% EDTA solution (PAA, Fisher Scientific GTF, Gothenburg, Sweden) diluted in Dulbecco’s PBS w/o calcium, magnesium and sodium bicarbonate. The cells were centrifuged at 300g for 5 min, the supernatant was removed and the cell pellets were resuspended in refrigerated PBS; the samples were then put on ice in a refrigerator pending isolation of RNA, which was done as soon as possible the same day. Isolated RNA was frozen in aliquots at –70°C until used.
Isolation of RNA
Total RNA was isolated with use of the RNeasy Mini Kit spin protocol (Qiagen AB) and QiaShredder homogenizers (Qiagen AB). The concentration of the isolated RNA was estimated by using the Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies Inc., Rockland, DE, USA) and the quality was determined with the Agilent RNA 6000 Nano Kit (Agilent Technologies Sweden AB, Kista, Sweden) and the Agilent 2100 Bioanalyzer (Agilent Technologies Sweden AB).
Amplification, labelling and hybridization of RNA
Microarrays were produced at the Swegene DNA Microarray Resource Centre, Department of Oncology, Lund University, Sweden (http://swegene.onk.lu.se). Human array-ready oligonucleotide libraries versions 2.1 and 2.1.1, comprising 27 000 unique probes, were obtained from Operon (Operon Biotechnologies, Germany). Probes were dissolved in Corning Universal Spotting solution (Corning, Acton, MA, USA) at a concentration of 24 µM and printed on aminosilane-coated glass slides (UltraGAPS, Corning), using a MicroGrid2 robot (BioRobotics, Cambridgeshire, UK) equipped with MicroSpot 10K pins (BioRobotics). After printing, arrays were left in a desiccator to dry for 48 h, and UV cross-linked (400 mJ/cm2). The arrays were stored under dark and dry conditions, protected from air, until used.
For each individual human endometrial endothelial cell culture originating from women in the proliferative phase of the menstrual cycle, o,p'-DDT-treated cells were compared with untreated cells, and each individual culture control was compared with the common control pool. All in all, 10 microarrays were utilized. For each treatment, 500 ng RNA was amplified and labelled using the Low RNA Input Linear Amplification Kit PLUS, Two-Colour (Agilent Technologies Sweden AB) and then purified using the RNeasy MinElute Cleanup Kit (Qiagen AB). For the amplification and labelling reactions, the polymerase chain reaction (PCR) instrument model PTC-100 (MJ Research Inc., Waltham, MA, USA) was used. Dye swaps were performed between the individuals to avoid dye bias between RNA from o,p'-DDT-treated and untreated HEECs, and from the individual controls and the control pool. After the labelling reaction, the Nanodrop ND-1000 Spectrophotometer was used to measure the incorporation of Cy dye. Pre-hybridization buffer [5x sodium chloride sodium citrate (SSC, VWR International AB, Stockholm, Sweden), 5x Denhardt’s solution (Sigma-Aldrich Sweden AB), 0.02 µg/µl tRNA (Invitrogen AB), 0.5% sodium dodecyl sulphate (SDS, VWR International AB, Stockholm, Sweden) and 50% formamide (Sigma-Aldrich Sweden AB)] was applied to each array under a hybrislip (22 x 60 mm, Sigma-Aldrich Sweden AB). Incubation was performed in a Hybridization Cassette ArrayItTM (TeleChem International Inc., ArrayIt, Sunnyvale, CA, USA) at 42°C for 45 min at 100% humidity in a Heating Circulator model 8201 (PolyScience, Niles, IL, USA). Each array was then transferred to a 50 ml centrifugation tube containing double distilled water. The arrays were washed five times by repeatedly turning the tubes upside down for 30 s each time. Hybridization and post-hybridization washes of the slides were performed using the Pronto! Hybridization Kit (Corning Life Sciences, Noricon AB, Helsingborg, Sweden). The Hybridization Cassette ArrayItTM and Erie Scientific Lifterslip (25 x 60 mm, Histolab, Västra Frölunda, Sweden) were used for hybridization of the slides at 42°C for 18–20 h. After hybridization, the slides were washed in the three wash buffers described in the Pronto! Hybridization Kit. The slides were scanned in a ScanArray Express HT microarray scanner [(Packard BioScience) Perkin Elmer Life Sciences Inc., Boston, MA, USA]. The scanning was performed at two different voltages for the photo multiplier tube to get a wide dynamic range of signals (high or low). The laser value default was 99%. The tiff files from the scanned arrays were gridded using the software Spotreader v 1.3.1 (Niles Scientific, Portola Valley, CA, USA).
Real-time quantitative reverse transcription PCR (real-time qRT-PCR)
RNA isolated from human endometrial endothelial cells originating from all 10 women, both the five in the proliferative phase and the five in the secretory phase, was used. For each sample, 1.1 µg RNA was reverse transcribed for 10 min at 25°C, followed by 15 min at 42°C and 5 min at 99°C with a Taqman Universal master mix (Applied Biosystems, Foster City, CA, USA) containing 1x reverse transcriptase buffer, 5 mM MgCl2, dNTPs (1 mM of each), 1 U/µl RNase inhibitor, 2.5 µM random hexamers and 2.5 U/µl multiscribe reverse transcriptase. RNase free water (Promega) was used for dilutions. Taqman gene expression assays (Applied Biosystems) were performed on 50 ng cDNA/sample for five genes: sestrin 2 (SESN2, assay ID Hs00230241_m1), E2F transcription factor 7 (E2F7, assay ID Hs00403170_m1), Chemokine (C-C motif) ligand 2 (CCL2, assay ID Hs00234140_m1), Copine VII (CPNE7, assay ID Hs00276274_m1) and endothelial lipase (LIPG, assay ID Hs00195812_m1), and verified and normalized against two house-keeping genes, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH, assay ID Hs99999905_m1) and actin beta (ACTB, assay ID Hs99999903_m1), which were considered to be unchanged in the microarray data set. The primer sequence for each primer spanned an exon junction to avoid detection of genomic DNA. Duplicate assays with a reaction volume of 25 µl were performed for each gene and sample in MicroAmpTM optical 96-well reaction plates with barcodes (Applied Biosystems). The reactions were performed with the ABI 7000 PCR machine (Applied Biosystems), with initial incubations for 2 min at 50°C and 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 1 min at 60°C. For each sample and gene, a negative control containing RNA sample without reverse transcriptase was performed, and one negative control without DNA was carried out per master mix.
Statistical analyses
For all statistical analyses, P-values of <0.05 were considered significant.
BrdU assay
The mean values were analysed with the statistical package for the social sciences (SPSS). As the mean values were not normally distributed, the non-parametric Mann–Whitney U-test was applied to compare cultures obtained from women in the proliferative phase of the menstrual cycle with those obtained from women in the secretory phase. The Wilcoxon signed ranks test was used to compare the o,p'-DDT treatment with the control pairwise on an individual basis, both within cultures originating from the proliferative or secretory phase of the menstrual cycle and for cultures originating from both phases of the menstrual cycle.
Microarray data
The BioArray Software Environment (BASE) (Saal et al., 2002) and the Linnaeus Centre of Bioinformatics Data Warehouse (LCB-DWH) (Ameur et al., 2006) were used for microarray data storage and analysis. Data were normalized using local background subtraction and within arrays using the print-tip lowess method (Yang et al., 2002), and spots that were flagged as either bad, empty or not found in the image analysis were filtered out. Duplicate spots were merged and it was required that gene products should be reported in at least four of five arrays. This procedure resulted in a data set with 8731 reporters that was used for further analysis of o,p'-DDT-treated compared with untreated HEECs and in a data set with 9533 reporters that was used for further analysis of each individual control compared with a common control pool.
With the aim of determining whether any of the individual controls differed from a common control pool, data were visualized using principal component analysis, hierarchical clustering and pairwise correlation plots, and the overall standard deviation of the mean M (log2 ratio, fold change) value for all gene products was calculated and compared with that of the corresponding o,p'-DDT data set.
For comparisons between o,p'-DDT-treated and untreated HEECs, hypothesis testing was performed using an empirical Bayes moderated t-test (Smyth, 2004) and the Limma package (Smyth, 2005). Fifty-three gene products with a B statistic value (posterior log odds ratio) 
2.5 and an adjusted P-value 
0.00625 were accepted for further investigation. The method of Benjamini and Hochberg (1995) was used to adjust P-values for multiple testing. The results were visualized by hierarchical clustering using the publicly available software Genesis (Sturn et al., 2002), and the distribution of gene ontology (GO) terms was investigated. By GO, gene products are grouped on the basis of the biological processes, cellular components and molecular functions with which they are associated (Ashburner et al., 2000), and this grouping was accomplished by the use of a hypergeometric test in a GO tool implemented in the LCB Data Warehouse (Ameur et al., 2006).
Gene expression omnibus
The normalized data set for o,p'-DDT-treated HEECs versus control and that for individual controls versus the control pool were deposited in gene expression omnibus, using accession nos. GPL4861
[NCBI GEO]
(Swegene Human 27K microarray platform) and GSE9252
[NCBI GEO]
(gene expression data set).
Real-time quantitative reverse transcription PCR data
The 2
threshold cycle (Ct) method (Livak and Schmittgen, 2001) was used to analyse the mRNA expression in o,p'-DDT-treated HEECs relative to that in untreated HEECs. Ct values were calculated for each gene product and reference gene according to the formula (Ct o,p'-DDT = Ct target – Ct reference) – (Ct untreated = Ct target – Ct reference), where the Ct values were expressed as the mean value of the duplicate assays. In order to obtain the relative mRNA expression, 2–Ct was calculated for each gene product and reference gene. As the 2–Ct values were not normally distributed, the non-parametric Mann–Whitney U-test was used to analyse for possible differences between HEEC cultures obtained from women in the proliferative and secretory phases of the menstrual cycle. The Wilcoxon signed ranks test was used to compare the mRNA expression of the different gene products, related to either ACTB or GAPDH, after o,p'-DDT- and control treatment pairwise on an individual basis.
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BrdU assay
In cultures originating from both the proliferative and secretory phases of the menstrual cycle, treatment with o,p'-DDT decreased the proliferation compared with the control (Fig. 1, pproliferative=0.043 and psecretory=0.043). That is, the menstrual cycle phase in which the human endometrial endothelial cells were collected did not affect the response of the cells to the o,p'-DDT treatment in terms of proliferation. Hence, the mean values of all 10 HEEC cultures were compared without regard to the menstrual cycle phase in which the cells were collected. Treatment with 50 µM o,p'-DDT significantly decreased the HEEC proliferation compared with the control (P = 0.005). The mean absorbance value for o,p-DDT-treated cells was 0.27 [standard deviation (SD) = 0.09], and the mean absorbance value for the control was 0.53 (SD = 0.16).
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Microarray data
Treatment of the human endometrial endothelial cells with o,p'-DDT did not result in major changes in mRNA expression compared with untreated cells, as evident by the low M values. When the data set obtained from o,p'-DDT-treated HEECs had been normalized and filtered, duplicate spots had been merged and hypothesis testing had been performed for reporters found in
4 of 5 arrays, 53 reporters were found to have a posterior log odds ratio value 2.5 and an adjusted P-value 0.00625 and were submitted for the gene ontology analysis. The 53 most differentially expressed gene products in o,p'-DDT-treated cells compared with untreated cells were also submitted for hierarchical clustering (Fig. 2).
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When the individual control samples were compared with a common control pool and the array data were visualized by a principal component analysis, inter-individual variation was found, which is not very surprising, since the HEEC cultures originated from five different individuals.
Gene ontology analysis of gene products influenced by treatment of human endometrial endothelial cells with o,p'-DDT using a hypergeometric significance test
Only the biological processes with GO groups containing at least three gene products with significantly changed expression were considered relevant. These GO groups can be viewed in Table I and include gene products related to the cell cycle, cell division, defence response and lipid and steroid metabolism. Five of the most differentially expressed gene products were chosen for real-time quantitative reverse transcription PCR (real-time qRT-PCR) verification, namely SESN2 and E2F7, which are involved in cell cycle- and cell division-related processes, CCL2, which is involved in defence response, and CPNE7 and LIPG, which are involved in lipid metabolism.
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The cellular components with GO groups containing at least three gene products with significantly changed expression are displayed in Supplementary Table I and include gene products related to the plasma membrane, endosome and chromosome. The molecular functions with GO groups containing at least three gene products with significantly changed expression are presented in Supplementary Table II and include gene products related to signalling, receptor and cytokine activity.
Real-time qRT-PCR data
There were no differences in mRNA expression of SESN2, E2F7, CPNE7, CCL2 or LIPG in o,p'-DDT-treated HEECs compared with untreated HEECs, depending on the phase of the menstrual cycle in which the HEECs were collected. Therefore, all 10 cultures were compared regardless of whether the cells were obtained in the proliferative or the secretory phase of the menstrual cycle. The real-time qRT-PCR data verified the microarray results. Both when ACTB and GAPDH were used as reference genes, the mRNA expression of SESN2 (pACTB=0.007, pGAPDH=0.013), E2F7 (pACTB=0.005, pGAPDH=0.005) and CPNE7 (pACTB=0.005, pGAPDH=0.005) was increased and the mRNA expression of CCL2 (pACTB=0.005, pGAPDH=0.005) and LIPG (pACTB=0.005, pGAPDH=0.005) was decreased in o,p'-DDT-treated compared with untreated HEECs (Fig. 3).
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In this study, we further evaluated the effects of o,p'-DDT on human endometrial endothelial cells using the microarray technique. Since we had previously found that the proliferation and viability of HEECs exposed to o,p'-DDT were reduced (Bredhult et al., 2007), we expected that gene products involved in these processes would be affected, but we were also interested in the expression of estrogen responsive genes and gene products involved in angiogenesis and cellular stress. In the present investigation, o,p'-DDT reduced the cell proliferation in the BrdU assay. With the GO analysis, we found that mRNA levels of genes involved in biological processes such as the cell cycle and cell division were affected, confirming the results from the proliferation assay and our previous study.
Only minor differences in the mRNA expression of specific genes were detected between o,p'-DDT-treated and untreated HEEC, and since samples from only five individuals were analysed with the microarray technique, we found it most significant to address the question as to which biological processes and molecular functions were affected by the o,p'-DDT treatment rather than to focus solely on the individual gene products. The real-time qRT-PCR analysis revealed that o,p'-DDT treatment altered the mRNA expression of the investigated genes in a similar way in HEECs obtained in the proliferative and secretory phases of the menstrual cycle. Since the mRNA expression profiles seemed to be robust and consistent between all 10 HEEC cultures, and since the results of the GO analysis also seem to fit with the biological effects that we previously observed on exposure of HEECs to o,p'-DDT, we consider the gene ontology analysis results to be relevant at least regarding the question of which biological processes and molecular functions are affected.
The most up-regulated mRNA involved in cell cycle processes was SESN2. Expression of SESN2 induced by hypoxia and oxidative stress is probably independent of p53 expression, whereas its induction by DNA damage appears to be p53 dependent (Budanov et al., 2002). Furthermore, over-expression of SESN2 is reported to have a pro-apoptotic effect in many cell types, whereas inducible expression sensitizes MCF-7 cells to DNA-damaging treatments and serum deprivation, but also protects against apoptosis induced by ischaemia or hydrogen peroxide. These findings imply that SESN2 is involved in an intricate regulation of cell viability in response to cellular stress. In the present study, we found that this gene product was up-regulated in HEEC cultures displaying decreased proliferation in response to o,p'-DDT treatment. In a previous study (Bredhult et al., 2007), we also found decreased viability after treatment with o,p'-DDT, mainly due to necrosis, but at higher concentrations this was also due to apoptosis. These findings combined imply that sestrin 2 has a role in the cell response to o, p'-DDT, similar to that reported by Budanov et al. (2002) for sestrin 2-expressing cells.
The second most up-regulated gene product involved in cell cycle processes was E2F7. Over-expression of E2F7 in mouse embryo fibroblasts has been found to result in a decreased proliferation rate and accumulation of cells in G2/M, accompanied by a decreased number of cells in the G1 phase (de Bruin et al., 2003). Other authors (Di Stefano et al., 2003) report a decrease in the proportion of Rat1 cells in the S phase after over-expression of E2F7, an increase in the proportion of U2OS cells in G1 accompanied by a decreased proportion of these cells in the S phase and a decreased colony-forming ability of HeLa cells. It has also been reported that over-expression of E2F7 increases the proportion of U2OS cells in G1, whereas the proportion of cells in G2/M was decreased (Logan et al., 2004). Even though there seem to be some discrepancies in the reports regarding which cell cycle phases are affected by E2F7, there appears to be agreement on the fact that E2F7 is associated with a negative impact on proliferation. This was also observed in the present study, where E2F7 mRNA was up-regulated in o,p'-DDT-treated cells that displayed decreased proliferation. Furthermore, it is possible that high expression of E2F7 in ovarian carcinomas can predict disease-free survival, whereas low E2F7 expression is associated with platinum resistance (Reimer et al., 2007). A high level of mRNA expression of E2F7, compared with that in normal human peritoneal mesothelial cells, has also been found both in the ovarian cancer cell line A2780 and in the breast cancer cell line T47D (Reimer et al., 2006).
The most differentially regulated mRNA involved in defence response was CCL2. CCL2 itself can up-regulate the apoptosis mediator Fas ligand on the protein level in cultured endometrial stromal cells, but does not affect the rate of apoptosis in these cells (Selam et al., 2006). However, the proportion of apoptotic Jurkat T lymphocyte cells increased when these cells were co-cultured with endometrial stromal cells that previously had been treated with CCL2. Cultured endometrial stromal cells can also respond to treatment with CCL2 with increased secretion of vascular endothelial growth factor (Lin and Gu, 2005), which is a proliferative stimulus to vascular endothelial cells.
The second most differentially regulated gene product associated with lipid metabolic processes was LIPG. Endothelial lipase may be involved in angiogenesis, as evident by increased expression in endothelial cells undergoing tube formation, and is expressed in adult human tissues such as placenta, liver, thyroid and testis, suggesting a role in steroidogenesis (Hirata et al., 1999).
Although there were only minor changes in mRNA expression after treatment of HEECs with o,p'-DDT, this does not necessarily mean that the changes are not important for the function and phenotype of a cell. Doubling or halving of the original mRNA expression could lead to significant changes in the function and phenotype of a cell, provided that the gene product has an essential function. And conversely, large changes of less important gene products might not affect the function and phenotype to any major extent. Furthermore, changes in mRNA expression are not necessarily related to changes in protein expression.
Estrogen-responsive gene expression did not appear to be affected by o,p'-DDT treatment of HEECs in vitro, but this treatment had possible effects on lipid and steroid metabolism, which might influence the endocrine response to estradiol and other hormones. Further, o,p'-DDT elicited a decrease in mRNA expression of CCL2, similar to that reported for estrogen (Arici et al., 1999). HEECs express ERβ (Critchley et al., 2001;Krikun et al., 2005) so it is possible that effects mediated through ER cannot be detected in this test system. Some of the gene products that were included in the GO analysis results may also be involved in cellular stress, such as sestrin 2 (Budanov et al., 2002), and in angiogenesis, such as CCL2 (Lin and Gu, 2005) and LIPG (Hirata et al., 1999).
Inter-individual variation among the control samples was found in the microarray analysis, which is not very surprising, since the HEEC cultures originated from five different individuals. However, the HEEC cultures responded to treatment with o,p'-DDT in a similar fashion, as is reflected by the results of the BrdU assay and verified in HEECs originating from women in both the proliferative and secretory phases of the menstrual cycle by the real-time qRT-PCR analysis.
In conclusion, the present study indicates that o,p'-DDT can affect the mRNA expression of genes involved in proliferation, lipid and steroid metabolism and the defence response in HEECs in vitro. These results support our previous findings of decreased proliferation and increased cell death in response to o,p'-DDT (Bredhult et al., 2007) and may be important clues in the search for the mechanisms of action of o,p'-DDT.
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The Swedish Environmental Protection Agency (I-59-02); the Family Planning Fund in Uppsala; The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (216-2005-894); the Swedish Animal Welfare Agency (2005–2285); the Swedish Research Council (73X-20137).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingSupplementary dataAcknowledgementsReferences |
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Supplementary data are available at http://molehr.oxfordjournals.org.
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The authors would like to thank Jacob Berglund, M Sc, and Malin Lundmark, M Sc, Department of Women’s and Children’s Health, Uppsala University, for technical assistance with the cell cultures, Research engineer Elsebrit Ljungström Köhl, Department of Cell and Molecular Biology, Karolinska Institutet, for help with planning of the experiment and manuscript preparation, and for excellent technical assistance with the RNA amplification and microarray wet lab, Bioinformatician Hanna Göransson, Department of Medical Sciences, Uppsala University, for help with planning of the experiment, assistance with the array data processing, array data analysis and manuscript preparation and Annika Eriksson, PhD, Department of Cell and Molecular Biology, Karolinska Institutet, for excellent assistance with planning of and running the real-time qRT-PCR experiment, and manuscript preparation. Microarrays and protocols were obtained from the Swegene DNA Microarray Resource Centre in Lund, supported by the Knut and Alice Wallenberg Foundation through the Swegene consortium. We also want to express our gratitude for being able to use BASE and the LCB-DWH platform at the Linnaeus Center of Bioinformatics, Uppsala University.
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Submitted on October 23, 2007; resubmitted on December 10, 2007; accepted on December 19, 2007.
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