tcdd induces biomarkers for endometriosis in rat ...

TCDD INDUCES BIOMARKERS FOR ENDOMETRIOSIS IN RAT ENDOMETRIUM AND HUMAN ECC-1 CELLS Van Ede KI, Stelloo S, van den Berg M, van Duursen MBM Institute for Risk Assessment Sciences (IRAS), Division Toxicology, Utrecht University, P.O.Box 80177, NL-3508 TD Utrecht, The Netherlands

Introduction Endometriosis is a gynaecologic disorder that affects up to 10% of the women in reproductive age1,2. The etiology of the disease is still poorly understood. The most widely accepted theory is that retrograde menstruation causes implantation of endometrial cells in the abdominal cavity3. However, retrograde menstruation does not lead in all women to the development of endometriosis. Hormone alteration and discrepancy in gene expression between ectopic endometrium in patients with endometriosis and normal endometrium in healthy women may play an important role to increased susceptibility for development of endometriosis. Bulun et al4,5 describes that elevated levels of cyclooxygenase-2 (COX-2) and cytochrome P450 19 (CYP19, aromatase) in ectopic endometrium may lead to upregulation of estrogen receptor (ER) ER-beta, downregulation of ER-alpha and progesterone resistance. Furthermore, the proto-oncogene c-fos was found to be highly expressed in ectopic endometrium from patients with endometriosis6. Environmental endocrine disruptors like dioxins and dioxin-like compounds have often been linked to endometriosis, because of their ability to alter the steroid synthesis or action. A recently published study found a higher risk to develop endometriosis with high plasma concentrations of dioxin-like compounds7. Another study found a significant correlation with PCB blood levels and increased risk for endometriosis8. On the other hand, a large population-based historical cohort study found no significant association between endometriosis and TCDD exposure in Seveso women9. Although human studies are unequivocal, an association seems plausible10. In this study, the role of the aryl hydrocarbon receptor (AhR), the main target for dioxins, has been investigated in relation with the onset and/or prevention of endometriosis. Gene expression of potential biomarkers for endometriosis, COX-2, CYP19, ER-alpha, ER-beta, progesterone receptor (PR) A/B and c-fos, as well as a commonly used biomarker for dioxin exposure, cytochrome P450 1A1 (CYP1A1) were investigated in rat uterus and in a human endometrium carcinoma cell line (ECC-1).

Materials and Methods Chemicals 2,3,7,8-tetrachlorodibenzodioxin (TCDD) was purchased form Wellington Laboratories Inc. (Guelph, Ontario, Canada). Animals Female Sprague-Dawley rats were purchased at 9 weeks of age from Harlan laboratories (Venray, The Netherlands) and allowed to acclimate for 1 week. The rats were housed in standard cages (46x35x19cm) and conditions (temperature 23 ± 2°C, 50% to 60% relative humidity, 12-h dark and light cycle) with free access to food and water. The rats were randomly assigned to 8 groups, 6 groups (3 day study) received a single dose of 0, 0.5, 2.5, 10, or 25µg TCDD/kg body weight and 2 groups (14 day study) received a single dose of 0 or 25µg TCDD/kg body weight by oral gavage in 2ml corn oil (n=6/group). Animals were sacrificed at day 3 or day 14 with CO2/O2. Blood was obtained from the abdominal aorta directly after decease and the uterus was removed, weighed, snap frozen and stored until use at -80°C. All animal

Organohalogen Compounds

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treatments were performed with permission of the Animal Ethical Committee and according to Dutch law on Animal Experiments. Radioimmunoassay A specific radioimmunoassay (DSL-3400, Diagnostic Systems Laboratories, Inc) was preformed to measure the progesterone (P4) levels in rat plasma. Plasma samples were stored at -80°C until P4 was measured according to the manufacturer’s instructions. Cells The ECC-1 cell line was derived from a well-differentiated human endometrial adenocarcinoma11 and was purchased from ATCC-LGC standards. The cells were cultured and maintained according to the manufacturer’s instructions. For experiments, the cells (45000 cells/well) were exposed for 7 days to the vehicle control (0,1% v/v DMSO), 1nM or 10nM TCDD in 12 well-plates. RNA isolation and cDNA synthesis Total RNA from rat uterine tissue or ECC-1 cells was isolated using phenol-chloroform extraction. Purity and concentration of the isolated RNA was determined by measuring the absorbance ratio at 260/280nm and 230/260nm using a Nanodrop 2000 spectrophotometer (Thermo Scientific). RNA was reverse transcribed to complementary DNA (cDNA) using iScriptTM cDNA sythesis kit (Bio-Rad Laboratories). Real time PCR Quantitative real-time PCR analyses were performed on an iQTM Real-Time PCR Detection System (Bio-rad Laboratories). The PCR primers CYP1A1, COX-2, CYP19, ER-alpha, ER-beta, PR A/B, c-fos and β-actine (reference gene) were designed with the Primer designing tool (NCBI) for rat and human. The PCR master mixture contained SYBR Green supermix, 0,4µM forward and reverse primer, and 66,7µg/ml cDNA in a total volume of 25µl. The following program was used for denaturation and amplification of the cDNA: 3 min at 95°C, followed by 40 cycles of 15s at 95°C and 1min at 60°C. Data were analyzed using iCycler iQ Optical System Software (Bio-Rad Laboratories).

Results and discussion Dioxins are often associated with changes in the hormone homeostasis, which may cause clinical diseases like endometriosis. Genes that have been described to play a major role in the development or onset of endometriosis and thus could potentially be used as biomarker, are CYP1A1, COX-2, CYP19, ER-alpha, ER-beta, PR A/B, and proto oncogene c-fos5,6. In the present study, a potential link between activation of the Ah-receptor and gene expression changes of these potential biomarkers in the endometrium have been investigated in vivo and in vitro. As an in vivo model, 10-week old female Sprague Dawley rats were used. Although the rat is generally considered not to be a suitable model for histopathological changes associated with endometriosis, biochemical endpoints that may play an important role in the etiology of endometriosis in humans can still be detected. Rats received a single oral dose in a range from 0 to 25µg TCDD/kg body weight and were sacrificed at day 3. TCDD exposure did not change the relative uterine weight and circulating progesterone levels. However, TCDD exposure resulted in a dose-dependent induction of CYP1A1 mRNA levels in the uterus (see Table 1), indicating the activation of the Ah-receptor. From the endometriosis biomarkers tested, COX-2 and ER-beta mRNA levels were dose dependent increased with a significant 2,5 fold induction of COX-2 at 10µg TCDD/kg body weight. ER-alpha and PR A/B mRNA expression were not affected by TCDD at the doses tested. Gene expression of CYP19 was very low or undetectable in both untreated and treated rats. mRNA levels of c-fos, were significantly increased at the lowest dose of 0.5µg TCDD/kg body weight, after which it dose-dependently decreased again to levels below the control group at a dose of 25µg TCDD/kg body weight. The decrease of c-fos expression in the rat uterus after TCDD exposure has been earlier described by Astroff et al.12


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Fourteen days after TCDD exposure (25µg/kg body weight), the relative uterus weight was significantly decreased to 68,34% ± 15% compared to the vehicle control group. Blood plasma level of progesterone was significantly increased from 8,04 ± 2,32 ng/ml (control group) to 36,17 ± 30,28 ng/ml (treated group). With respect to gene expression, similar changes were found for CYP1A1, ER-alpha, ER-beta and c-fos as seen after 3 days (table 1). In contrast, COX-2 mRNA levels were unaffected and PR A/B gene expression was significantly increased after 14 days. Parallel to the in vivo studies the effect of TCDD on the expression of CYP1A1, COX-2, CYP19, ER-alpha, ER-beta, PR A/B, and proto oncogene c-fos were investigated in the human endometrium carcinoma cell line (ECC1). The ECC-1 cell line contains the AhR, ER and PR, which makes it a potential model to study mechanistic effects of dioxin exposure on steroid function13,14. ECC-1 cells were exposed to DMSO (0,1% v/v), 1 or 10nM TCDD for 7 days. Gene expression was determined by quantitative real-time RT PCR with β-actine as reference gene. After exposure to TCDD, mRNA levels of CYP1A1 concentration-dependently increased compared to vehicle control up to 12 fold at the highest concentration tested (10 nM). An increasing trend was seen for COX-2 and CYP19 expression, although this increase was not statistically significant. Catalytic activity of CYP19 could not be detected (data not shown). Gene expression of PR A/B was significantly decreased (see Table 1). Comparing the in vitro with the in vivo data, different effects were seen for CYP19, ER-beta, PR A/B and c-fos. Differences in gene expression between the rat uterus and human ECC-1 cells could be due to species-differences as SD rats are known to be very responsive to AhR activation as opposed to humans. Also, the rat uterus is healthy endometrium while the ECC1 cells originate from an adenocarcinoma. Furthermore in vivo, TCDD can also interact with the hypothalamuspituitary-gonadal (HPG) axis, which can influence the uterus hormonal environment and might result in differential AhR responses. Although the rats in this study were not synchronized and their phases in estrous cycle were not determined, the gene expression changes upon TCDD exposure were dose-dependent and time-dependent and significantly different from vehicle control-treated rats. Taken together, our data show the potential of TCDD to cause changes in gene expression in the uterus/endometrial cells that have been associated with the onset of endometriosis. Further research should be performed to elucidate the interaction between the Ah-receptor, estrogen receptor, progesterone receptor and the role of CYP19.

Table 1: Gene expression of potential biomarkers for endometriosis in vivo (rat uterus) and in vitro (ECC-1 cells) after exposure to TCDD. Gene

CYP1A1 COX-2 CYP19 ER-alpha ER-beta PR A/B c-fos

Rat uterus (fold induction §) 3 day 14 day 25µg/kg bw 25µg/kg bw * 420,03 * 6,20 1,66 0,96 ND ND 0,76 1,44 * 14,08 * 5,07 0,85 * 2,32 * 0,33 * 0,14

ECC-1 cells (fold induction §§) 7 days 1nM 10nM * 9,02 * 11,63 2,76 5,34 2,09 3,88 1,08 1,24 0,94 1,19 * 0,51 0,55 0,93 1,09

§ Expression is calculated relative to the expression of vehicle control treated rats from the same time group. §§ Expression is calculated relative to the ECC-1 control cells containing 0,1% DMSO. * Significantly different from control group (p < 0,05).


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Acknowledgements This work was supported financially by the European Union Seventh Framework Project SYSTEQ under grant agreement number FP7-ENV-226694. Purchase of compounds was financially made possible by The Dow Chemical Company.

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