DDT and its metabolite DDE alter steroid hormone secretion in human term placental explants by regulation of aromatase activity

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1 Toxicology Letters 173 (2007) DDT and its metabolite DDE alter steroid hormone secretion in human term placental explants by regulation of aromatase activity Anna K. Wójtowicz a,, Tomasz Milewicz b, Ewa Ł. Gregoraszczuk a a Department of Physiology and Toxicology of Reproduction, Chair of Animal Physiology, Institute of Zoology, Jagiellonian University, Ingardena 6, Krakow, Poland b Department of Gynecological Endocrinology, Jagiellonian University, Krakow, Poland Received 19 March 2007; received in revised form 6 June 2007; accepted 12 June 2007 Available online 16 June 2007 Abstract Placental explants were used to compare the effects of two isomers of DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane), p,p -DDT and o,p -DDT and their metabolites p,p -DDE and o,p -DDE (1,1,-dichloro-2,2-bis(p-chlorophenyl)ethylene) on steroid hormone secretion (estradiol (E2) and progesterone (P4)). Explants were treated with 1, 10, 100 ng/ml or 1 g/ml of each compound for 24 h. We found that all investigated compounds at all doses caused reductions of estradiol secretion. Moreover, it was shown that the inhibition of estradiol secretion was due to direct action on aromatase activity. Twenty-four-hour exposure to p,p -DDE, o,p -DDT or o,p -DDE at doses of 100 ng/ml or 1 g/ml increased P4 secretion, suggesting that these compounds act on P450scc. The fluorometric assay confirmed that all investigated compounds inhibited aromatase activity at a concentration of 100 ng/ml. Our findings suggest that by decreasing estradiol secretion with concomitant stimulation of progesterone secretion, DDT could be a factor that influences the outcome of pregnancy Elsevier Ireland Ltd. All rights reserved. Keywords: Human term placenta; DDT; DDE; Progesterone; Estradiol; Aromatase 1. Introduction DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)- ethane), a well-known organochlorine pesticide, is still present in the environment. Moreover, in living cells, DDT is metabolized to DDE (1,1,-dichloro-2,2- bis(p-chlorophenyl)ethylene) and DDD (1,1-dichloro- 2,2-bis(p-chlorophenyl)ethane) (Fox et al., 1998). Because 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane Corresponding author. Tel.: ; fax: address: (A.K. Wójtowicz). (DDD) are degradation and metabolic products of DDT, humans are usually exposed to a mixture of these three compounds. In addition, DDT, DDE, and DDD can each exist in different isomeric forms determined by the chlorine position on the two chlorophenyl rings of the molecule. Technical-grade DDT typically consists of 77% p,p -DDT, 15% o,p -DDT, 4% p,p -DDE, and less than 1% o,p -DDE, p,p -DDD and o,p -DDD (ATSDR, 2002; World Health Organization, 1979). Analyses of maternal adipose tissue, maternal blood serum, umbilical cord serum, mature milk and amniotic fluid indicate circulation of these compounds through all compartments of the maternal body (Longnecker et al., 1997; Covaci et al., 2002; James et al., 2002). It is well known that DDT and its metabolites are able /$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi: /j.toxlet

2 A.K. Wójtowicz et al. / Toxicology Letters 173 (2007) to cross the placental barrier, and that they accumulate in a placental tissue (Sala et al., 2001; Bjerregaard and Hansen, 2000). There have also been epidemiological studies reporting an association between levels of these compounds in maternal blood and miscarriage rate, premature rupture of fetal membranes, preterm birth and fetal development (Dewailly et al., 1993; Gladen et al., 2003; Karmaus and Zhu, 2004). It is well known that human placenta is responsible for the production of a number of hormones necessary for normal fetal development and pregnancy maintenance, like human chorionic gonadotropin and progesterone (Spencer and Bazer, 2004; Pepe and Albrecht, 1995). During late pregnancy, the placenta is the major source of estrogens (Myers and Nathanielsz, 1993; Mitchell and Wong, 1993; Chibbar et al., 1995). There are several possible mechanisms by which DDT and DDE could act as endocrine disruptors: through a steroidogenic pathway as was suggested by Crellin et al. (2001) and Younglai et al. (2004), through receptor-mediated changes in protein synthesis (Kelce et al., 1995) or through their anti-androgenic and estrogenic actions (Andersen et al., 1999). The main enzyme responsible for converting androgens to estradiol is aromatase and it has been detected in placenta. Aromatase has also been reported to be a target for DDE (You et al., 2001; Younglai et al., 2002). Decreased aromatase activity has been described under the influence of DDT in H295R human adrenocortical carcinoma cells (Sanderson et al., 2002), and by lindane and bisphenol- A, pesticides and antifungal drugs (azoles) in human placental JEG-3 cells (Nativelle-Serpentini et al., 2003; Vinggaard et al., 2000; Laville et al., 2006; Trosken et al., 2004, 2006). On the other hand the increase of aromatase activity under the influence of p,p -DDE was reported in cultures of endometrial stromal cells (ESC) and human granulosa cells (Holloway et al., 2005; Younglai et al., 2004). The aim of the present study was to investigate the action of two isomers, p,p -DDT and o,p -DDT, and their metabolites, DDEs, on specific endocrine endpoints (progesterone and estradiol secretion) and to determine the actions of these compounds on aromatase activity and protein expression. 2. Materials and methods 2.1. Reagents Dulbecco s Modified Eagle s Medium (DMEM), heatinactivated fetal bovine serum (FBS), dehydroepiandrosterone (DHEA), TRIS, HEPES, CHAPS, DTT, EDTA, Tween 20, bromophenol blue and DMSO were purchased from Sigma (Chemical Co. St. Louis, MO, USA). DDT compounds (p,p - DDT, o,p -DDT, p,p -DDE and o,p -DDE) were purchased from Reference Standards, EPA, Research Triangle Park, NC, USA. Stock solutions of these test compounds were prepared in DMSO and added to DMEM supplemented with 5% charcoal-stripped FBS. The final concentration of DMSO in the medium was always 0.1%. The nonsteroidal aromatase enzyme system inhibitor, CGS 16949A (fadrozole) [4-(5,6,7,8-tetrahydroimidazol [1,5- ] pyridin/5- yl)benzonitrile monochloride], was generously provided by Ciba-Geigy Ltd., Switzerland Placental explant cultures Placentas were collected at a gynecological hospital in Krakow, Poland where the clinical information on pregnancy outcome was gathered. Collection of placenta and recording of clinical histories followed previously established protocols that were given ethical approval by the local institutional review board. Patients gave their informed consent for the study. Clinical information recorded on each pregnancy included: smoking history, neonatal mortality and pregnancy outcome. Normal term placentas were obtained from non-smoking women undergoing elective Caesarian section with normal pregnancies at term (37 41 weeks of gestation). Placental cotyledons were harvested and immediately placed in ice-cold PBS and transported to the laboratory within 30 min of delivery. Placental tissues were rinsed three times with PBS containing 100 IU/ml penicillin and 100 g/ml streptomycin. Decidual tissue and blood vessels were removed from villous placenta by blunt dissection and the tissue was finally minced into 2 3 mm pieces. Explants with a total wet weight of approximately mg were dispersed to wells in 12-well plates (NUNC) containing 1.5 ml of DMEM supplemented with 5% charcoalstripped FBS, 5 ng/ml dehydroepiandrosterone (DHEA) and penicillin/streptomycin. The explants were incubated in triplicate for 24 h at 37 C in a humidified atmosphere of 95% air and 5% CO Experimental procedure Experiment 1 was designed to study the dose response effect of the test compounds on progesterone secretion and on conversion of dehydroepiandrosterone (DHEA) to estradiol. Placental explants were cultured in DMEM supplemented with 5% FBS and 5 ng/ml of DHEA in the presence of 1, 10, 100 ng/ml or 1 g/ml of p,p -DDT, p,p -DDE, o,p -DDT or o,p - DDE. These concentrations cover the range of concentrations of DDT and DDE reported to be present in serum of a pregnant woman (Farhang et al., 2005; Hamel et al., 2003; Law et al., 2005). After 24 h of culture, at the end of the experiment, media was collected for hormone analyses and tissue was weighed so that the hormone values could be expressed per mg wet weight. Experiment 2 was performed to demonstrate the effect of DDT isomers and their metabolites on (1) aromatase activ-

3 26 A.K. Wójtowicz et al. / Toxicology Letters 173 (2007) ity by measuring direct action on aromatase activity and (2) expression of aromatase protein by immunoblotting. Placental explants were cultured in DMEM containing 5% FBS supplemented with 5 ng/ml DHEA. The experimental cultures were maintained in the presence of 100 ng/ml p,p -DDT or o,p -DDT or their metabolites. After 24 h of exposure, media was discarded and tissue was frozen in liquid nitrogen and stored at 70 C. For aromatase activity measurement and immunoblotting, the tissue was homogenized twice in 50 l of ice-cold lysis buffer containing 50 mm HEPES, 100 mm NaCl, 0.1% CHAPS, 1 mm EDTA, 10% glycerol and 10 mm DTT. The lysates were clarified by centrifugation at rpm at 4 C for 20 min and supernatants were used for the aromatase assay and immunoblotting. The protein concentration of lysates was determined with Bradford reagent (Bio Rad Protein Assay; Bio Rad Laboratories, Munchen, Germany) using bovine serum albumin (BSA) as the standard Hormone analysis The concentrations of progesterone (P4), and estradiol (E2) were determined in the media by EIA using commercially available kits (DiaMetra, Italy) according to the manufacturer s instructions. All samples were run in duplicate in the same assay. Inter- and intrassay coefficients of variation for progesterone kits were 2.9% and 4.8%, respectively. For estradiol kits, inter- and intra-run precision had coefficients of variation of 3.2% and 5.4%, respectively Aromatase (CYP19) activity measurement Aromatase (CYP19) converts C19 androgens to aromatic C18 estrogenic steroids. We estimated the activity of CYP19 enzyme using the fluorometric substrate dibenzylfluorescein (DBF). The fluorescence assay of aromatase activity using dibenzylfluorescein was performed in 96-well plates according to the method of Stresser et al. (2000). The DBF metabolite, fluorescein, was measured using an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Apart from CYP19 (aromatase), DBF is also a substrate for other cytochromes, such as CYP2C8, CYP2C9, CYP2C19, and CYP3A. In order to determine whether aromatase was involved in production of the DBF metabolite, fluorescein, we used the selective aromatase inhibitor, CGS 16949A. Bio Rad Mini Trans-Blott apparatus. Following the transfer, membranes were washed and non-specific binding sites were blocked with 5% dried milk and 0.2% Tween 20 in 0.02 M TBS for 2 h. Then, the membranes were incubated overnight with anti-aromatase antibody (mouse anti-human cytochrome P450 aromatase, MCA2077S, AbD Serotec Ltd., UK) diluted at 1:200 in TBS/Tween at 4 C. After incubation with the primary antibody, the membranes were washed with TBS and 0.02% Tween 20 and incubated for 2 h with horseradish peroxidase-conjugated antibody (anti-mouse IgG- HRP, P-0447, DakoCytomation, Denmark) diluted at 1:500 in TBS/Tween. To control for the amounts of protein that were loaded onto the gel, the membranes were stripped and reprobed with anti- -actin antibody. Signals were detected by chemiluminescence (ECL) using a Western Blotting Luminol Reagent (sc-2048, Santa Cruz Biotechnology) and visualized with the use of a PhosphoImager FujiLas Statistical analysis Data are presented as the mean ± S.E.M. of four independent experiments. Each treatment was repeated four times (n = 4) in triplicate, and, thus, the total number of replicates was 12. The average of the triplets was used for statistical calculation. Statistical analysis was performed using Statistica 6.0. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey honestly significant difference (HSD) multiple range test. 3. Results 3.1. Progesterone secretion In control cultures, progesterone secretion into the medium during 24 h of culture was 0.49 ± 0.03 ng/ml (Fig. 1). A significant increase in progesterone secretion 2.6. Immunoblotting Twenty micrograms of protein was reconstituted in the appropriate amount of sample buffer consisting of 125 nm Tris ph 6.8, 4% SDS, 25% glycerol, 4 mm EDTA, 20 mm DTT and 0.01% bromophenol blue. Samples were separated by 7.5% SDS-polyacrylamide gel electrophoresis in a BIO-RAD Mini-Protean II Electrophoresis Cell and then proteins were transferred to nitrocellulose membranes using a Fig. 1. Effect of increasing concentrations of p,p-ddt, p,p-dde, o,p -DDT and o,p -DDE on progesterone secretion in placental explant cultures after 24 h of exposure. Each point represents the mean ± S.E.M. of four independent experiments, each of which consisted of three replicates per treatment group. (*) indicates statistically (p < 0.05) significant differences between control and experimental groups.

4 A.K. Wójtowicz et al. / Toxicology Letters 173 (2007) Fig. 2. Effect of increasing concentrations of p,p-ddt, p,p-dde, o,p - DDT and o,p -DDE on conversion of DHEA to estradiol in placental explant cultures after 24 h of exposure. Each point represents the mean ± S.E.M. of three independent experiments, each of which consisted of four replicates per treatment group. (*) indicates statistically (p < 0.05) significant differences between control and experimental groups. was noted after the addition of 100 ng/ml p,p -DDE, o,p - DDT or o,p -DDE (1.9-, 2.4-, and 2.2-fold, respectively) and 1 g/ml p,p -DDE, o,p -DDT or o,p -DDE (2.5-, 1.4-, and 1.8-fold, respectively); (p < 0.01). The second isomer of DDT, p,p-ddt, had no effect on progesterone secretion Conversion of DHEA to estradiol All investigated compounds significantly decreased the conversion of DHEA into estradiol (Fig. 2). In control cultures, estradiol secretion into the medium was pg/ml. The decreased level of estradiol in experimental cultures were similar after the addition of 1, 10, 100 ng/ml and 1 g/ml of both DDT isomers: p,p -DDT and o,p - DDT as well as both DDE Effects of DDT isomers and their metabolites, DDEs, on aromatase activity All investigated compounds, p,p -DDT, p,p -DDE o,p -DDT and o,p -DDE, in a concentration of 100 ng/ml, inhibited aromatase activity by 1.8-, 1.5-, 1.6- and 2.3-fold, respectively (p < 0.05) (Fig. 3). We observed that CGS 16949A did not affect control fluorescein production or p,p -DDT-, p,p -DDE-, o,p -DDT -, or o,p -DDE-induced inhibition of fluorescein production Immunoblotting There are differences in the degree of aromatase protein degradation caused by 100 ng/ml of p,p -DDT and Fig. 3. Effect of 100 ng/ml of p,p-ddt, p,p-dde, o,p -DDT and o,p - DDE on aromatase activity in placental explants cultured for 24 h. Each point represents the mean ± S.E.M. of three independent experiments, each of which consisted of four replicates per group. Statistically significant (p < 0.05) differences between groups (control and treated) are indicated with different letters. The same letter indicates no significant difference, with a < b. There were no statistically significant differences between explants treated with the reagents and CGS 16949A. Fig. 4. Effect of 100 ng/ml of p,p-ddt, p,p-dde, o,p -DDT and o,p - DDE on the expression of the aromatase protein in placental explants cultured for 24 h. Representative Western blot of aromatase protein levels in placental tissue homogenates without treatment (control), and following p,p-ddt, p,p-dde, o,p -DDT or o,p -DDE treatment. Blots were stripped and re-probed with anti- -actin antibody to control for the amounts of protein loaded onto the gel. o,p -DDT treatment. In the cases of p,p -DDT and p,p - DDE, the band had a higher intensity than the band from protein lysates of o,p -DDT and o,p -DDE treated explants (Fig. 4). 4. Discussion Progesterone, a major steroid hormone produced by the ovarian corpus luteum and by the placental syncytiotrophoblast during the second trimester, is considered to be essential for the successful maintenance of pregnancy. A disturbance in progesterone synthesis and secretion is a cause of abortion and preterm birth (Spencer and Bazer, 2004; Pepe and Albrecht, 1995). The production of estradiol rises during human pregnancy. Estrogens influence various aspects of placental function and play an important role in parturition (Myers and Nathanielsz, 1993; Mitchell and Wong, 1993; Chibbar et al., 1995). Results of the presented study clearly show that short term exposure to high concentrations of p,p -DDE, o,p - DDT and o,p -DDE stimulated progesterone secretion,

5 28 A.K. Wójtowicz et al. / Toxicology Letters 173 (2007) while p,p -DDT treatment did not have an effect on progesterone secretion. Surprisingly, this data is the first to show action of both isomers of DDT and their metabolites on progesterone secretion by human placental explants. Similar data which shows an increase in basal progesterone production has been obtained with rat granulosa-luteal cells (Nejaty et al., 2001), pig granulosa cells and the stable pig granulosa cell line, JC-410 (Crellin et al., 1999). Crellin et al. (1999) found that changes in progesterone synthesis corresponded with changes in the level of mrna for cytochrome P450scc, an enzyme that is involved in the first step of progesterone synthesis. This mechanism of action was also suggested for PCB 126 and PCB 153 in porcine granulosa cells (Wójtowicz et al., 2005) and for TCDD in testicular steroidogenesis (Kleeman et al., 1990; Moore et al., 1991). Thus, stimulation of P450scc gene expression is a possible mechanism of action for DDT and its metabolites in placental cells. Increased progesterone secretion under the influence of p,p -DDE, o,p -DDT and o,p -DDE was concomitant with decreased conversion of DHEA to estradiol suggesting additional action on aromatase activity. Different situations have been noted in the case of p,p -DDT. Decreased estradiol secretion did not parallel increased progesterone secretion and no effect on progesterone secretion was observed. The most likely mechanism of action is an influence on aromatase activity and a lack of influence on enzymes involved in progesterone synthesis. Results from the presented data clearly show a decrease in aromatase activity in placental cells exposed to all investigated reagents. Decreased aromatase activity under the influence of DDT has been described in H295R human adrenocortical carcinoma cells (Sanderson et al., 2002), and by lindane and bisphenol-a, pesticides and antifungal drugs (azoles) in human placental JEG-3 cells (Nativelle-Serpentini et al., 2003; Vinggaard et al., 2000; Laville et al., 2006; Trosken et al., 2004, 2006). The inhibitory effects of phytoestrogens on aromatase activity in human placental microsomes was first reported by Kellis and Vickery (1984) and then by Lacey et al. (2005). There are some differences in the degree of inhibition of aromatase protein expression by the two DDT isomers despite the same degree of inhibition of aromatase enzyme activity. Our data is in agreement with data of Lacey et al. (2005) who observed decreased aromatase and 3 -HSD activity under the influence of phytoestrogens, without any significant effect on protein expression as determined by Western blots. Another interesting finding is the difference in the degree of protein degradation following treatment with wither p,p - DDT or o,p -DDT. It is possible that p,p -DDT forms an enzyme-inhibitor complex that slows the degradation of the enzyme. This has been suggested as a mechanism for aromatase inhibitor activity (Harada and Hatano, 1998). Moreover, it should be considered that different isomers could modulate ER activity/expression in different ways (Di Lorenzo et al., 2002; Wójtowicz et al., 2007). Further investigations should concentrate on particular stages of placental steroidogenesis at the levels of mrna, protein and enzymatic activity. In conclusion, this data demonstrates the direct effect of DDT and its metabolites on steroid secretion by human placental tissue for the first time. Out data indicates that antiestrogenic action occurs by inhibition of aromatase activity and stimulation of progesterone secretion, which is a well known function of an aromatase inhibitor in ovarian cells (Fortune and Vincent, 1983). Taking into consideration that aromatase activity is important for the maintenance of pregnancy and for regulation of parturition in late pregnancy, inhibition of the activity of this enzyme could impair the outcome of pregnancy. Acknowledgment This work was supported by grant number 2P05D from the State Committee for Scientific Research (KBN; Warszawa, Poland). References Andersen, H.R., Andersson, A.M., Arnold, S.F., Soto, A.M., et al., Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals. Environ. Health Perspect. 107 (Suppl. 1), ATSDR, Agency for Toxic Substances and Disease Registry. Toxicological profile for DDT, DDE, and DDD: update. 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7 30 A.K. Wójtowicz et al. / Toxicology Letters 173 (2007) Wójtowicz, A.K., Kajta, M., Gregoraszczuk, E.L., DDT- and DDE-induced disruption of ovarian steroidogenesis in prepubertal porcine ovarian follicles: a possible interaction with the main steroidogenic enzymes and estrogen receptor b. J. Physiol. Pharmacol., in press. You, L., Sar, M., Bartolucci, E., Ploch, S., Whitt, M., Induction of hepatic aromatase by p,p -DDE in adult male rats. Mol. Cell. Endocrinol. 178, Younglai, E.V., Holloway, A.C., Lim, A.L., Foster, W.G., Synergistic effects between FSH and 1,1-dichloro-2,2-bis(pchlorophenyl)ethylene (p p -DDE) on human granulosa cell aromatase activity. Hum. Reprod. 19, Younglai, E.V., Foster, W.G., Hughes, E.G., Trim, K., Jarrell, J.F., Levels of environmental contaminants in human follicular fluid, serum, and seminal plasma of couples undergoing in vitro fertilization. Arch. Environ. Contam. Toxicol. 43,

Protocol for Western Blotting

Protocol for Western Blotting Protocol for Western Blotting Materials Materials used on Day 3 Protease inhibitor stock: 1 μg/μl pepstatin A in DMSO 200 μm leupeptin in OG Buffer 200 mm PMSF: Freshly made. Ex) 34.8 mg PMSF in 1 ml isopropanol

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