AJP - Endo Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 290: E135-E142, 2006. First published September 6, 2005; doi:10.1152/ajpendo.00356.2005
0193-1849/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/1/E135    most recent
00356.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, K.
Right arrow Articles by Guan, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, K.
Right arrow Articles by Guan, H.

Cadmium reduces 11{beta}-hydroxysteroid dehydrogenase type 2 activity and expression in human placental trophoblast cells

Kaiping Yang, Laura Julan, Fran Rubio, Anju Sharma, and Haiyan Guan

Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, Children's Health Research Institute and Lawson Health Research Institute, Departments of Obstetrics and Gynaecology and Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada

Submitted 2 August 2005 ; accepted in final form 31 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cadmium, a common environmental pollutant and a major constituent of tobacco smoke, has been identified as a new class of endocrine disruptors with a wide range of detrimental effects on mammalian reproduction. During human pregnancy, maternal cadmium exposure, via the environment and/or cigarette smoking, leads to fetal growth restriction (FGR), but the underlying mechanisms are unknown. Although a substantial amount of evidence suggests that cadmium may affect fetal growth indirectly via the placenta, the molecular targets remain to be identified. Given that reduced placental 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD2, encoded by HSD11B2 gene) is causally linked to FGR, the present study was undertaken to examine the hypothesis that cadmium induces FGR in part by targeting placental HSD11B2. Using cultured human trophoblast cells as a model system, we showed that cadmium exposure resulted in a time- and concentration-dependent decrease in 11{beta}-HSD2 activity, such that an 80% reduction was observed after 24-h treatment at 1 µM. It also led to a similar decrease in levels of 11{beta}-HSD2 protein and mRNA, suggesting that cadmium reduced 11{beta}-HSD2 expression. Furthermore, cadmium diminished HSD11B2 promoter activity, indicative of repression of HSD11B2 gene transcription. In addition, the effect of cadmium was highly specific, in that other divalent metals (Zn2+, Mg2+, and Mn2+) as well as nicotine and cotinine (a major metabolite of nicotine) did not alter 11{beta}-HSD2 activity. Taken together, these findings demonstrate that cadmium reduces human placental 11{beta}-HSD2 expression and activity by suppressing HSD11B2 gene transcription. Thus the present study identifies placental 11{beta}-HSD2 as a novel molecular target of cadmium. It also reveals a molecular mechanism by which this endocrine disruptor may affect human placental function and, consequently, fetal growth and development.

glucocorticoid; placenta; fetal growth restriction; environmental toxin

CADMIUM IS WIDELY DISPERSED in the environment mainly as result of pollution from a variety of sources, including mining, smelting, fossil fuel combustion, batteries, paints, and plastics (30, 80). Additionally, one important source of this heavy metal in the environment is tobacco smoke (82). Cadmium has been classified as a human carcinogen by the International Agency for Research on Cancer (76). It has also been identified recently as a new class of endocrine disruptors, and exposure to this metal is linked to a wide range of detrimental effects on mammalian reproduction (29). Cadmium exposure during human pregnancy is associated with low birth weight, spontaneous abortion, and premature delivery (29, 53).

Previous studies in both animals and humans demonstrate that cadmium accumulates in high concentrations in the placenta, and very little crosses the placenta into the fetus (29). This suggests that the placenta is a primary target for this metal during mammalian pregnancy. Indeed, placental structural abnormalities were observed in women who smoked (9, 46, 74) and in various animals exposed to cadmium during pregnancy (16). In perfused human placentas in vitro, cadmium exposure caused decreased secretion of human chorionic gonadotropin (78), a peptide hormone vital to early pregnancy. Furthermore, progesterone levels were inversely correlated with levels of cadmium in placentas of women who smoked during pregnancy (61). There is also direct evidence that cadmium reduces the biosynthesis of progesterone in cultured human trophoblast cells (32, 33, 38). Given that progesterone plays a key role in maintaining myometrial quiescence (11, 13), the cadmium-induced downregulation of placental progesterone synthesis may provide a mechanistic basis for the association between smoking and premature delivery. Although fetal hypoxia, disruption of normal fetoplacental zinc homeostasis, and reduced uteroplacental blood flow have been proposed as potential mechanisms contributing to cadmium-induced fetal growth restriction (FGR) (29, 53, 81), the precise molecular targets of cadmium in the placenta are poorly defined.

Recently, the placental 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD2; encoded by the HSD11B2 gene) enzyme has emerged as a key player in controlling fetal development for the following reasons (8, 79). First, owing to its localization to the syncytiotrophoblast layer of the human placenta (43, 60), the site of maternal-fetal exchange, placental 11{beta}-HSD2 serves as a functional barrier to protect the fetus from exposure to high levels of maternal glucocorticoid by converting maternal cortisol to its inactive metabolite cortisone. Second, placental 11{beta}-HSD2 activity is positively correlated with birth weight (56, 71). Third, FGR is a characteristic feature of 11{beta}-HSD2 deficiency resulting from mutations in the HSD11B2 gene in humans (39). Importantly, reduced placental 11{beta}-HSD2 (both expression and activity) is associated with pregnancies complicated with FGR (17, 35, 54, 56, 68, 69). Therefore, we hypothesized that cadmium induces FGR in part by targeting the placental HSD11B2 gene. In the present study, we tested this hypothesis utilizing cultured human placental trophoblast cells as a model system. We present the first evidence that cadmium, but not nicotine or cotinine (a major metabolite of nicotine), suppresses HSD11B2 gene transcription, leading to reduced 11{beta}-HSD2 expression and activity in cultured human trophoblast cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Overview. In this study, we utilized our established primary human trophoblast cells as a model system in which to sequentially study the effects of cadmium on placental 11{beta}-HSD2 activity, 11{beta}-HSD2 protein, 11{beta}-HSD2 mRNA, and HSD11B2 promoter activity. The primary focus of our study was on placental 11{beta}-HSD2 activity, because it represents the most important biological readout. First, we determined whether the effect of cadmium on placental 11{beta}-HSD2 activity was time and concentration dependent by treating trophoblast cells with CdCl2 for different times and at various concentrations. Second, we treated trophoblast cells with three other divalent metals, ZnCl2, MgCl2, and MnCl2, to determine whether the inhibitory effect of CdCl2 on placental 11{beta}-HSD2 activity was specific to cadmium. Third, we incubated trophoblast cells with nicotine and cotinine (a major metabolite of nicotine), two well-known constituents of tobacco smoke besides cadmium, to determine whether these two compounds had similar effects on placental 11{beta}-HSD2 activity. Fourth, we determined sequentially whether cadmium reduced expression of 11{beta}-HSD2 protein and mRNA. Furthermore, we examined the effect of cadmium on the mRNA expression of heme oxygenase-1 (HO-1; a well-known inducible target of cadmium) to provide evidence that cadmium did not cause global downregulation of gene expression in trophoblast cells. In the last experiment, we performed transient transfection experiments using a luciferase reporter assay to determine whether the effect of cadmium on placental HSD11B2 occured at the transcriptional level.

Placental trophoblast cell cultures. Placental trophoblast cells were isolated using a modification of the method of Kliman et al. (40), as described (28). Ethics approval for procurement of human placentas was obtained from the University of Western Ontario Ethics Board for Health Sciences Research Involving Human Subjects. Briefly, human placentas were obtained from uncomplicated pregnancies (i.e., without maternal or fetal diseases or maternal substance abuse, including smoking) at term after elective cesarean section. Villous tissues were dissected free from fetal membranes and blood vessels, rinsed in 0.9% NaCl2, and digested with 0.125% trypsin and 0.02% deoxyribonuclease-I (Sigma) in DMEM containing 0.05% streptomycin and gentamicin (Invitrogen) three times for 30 min each. The placental cells were loaded onto a 5–70% Percoll gradient at step increments of 5% Percoll, and centrifuged at 2,500 g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml were collected and plated in either 24-well plates (for enzyme activity assay) or 35-mm dishes (for Western blot analysis) at a density of 1.35 x 106 cells/ml in M199 containing 10% FCS (Invitrogen). The cells were maintained at 37°C in humidified 5% CO2-95% air (20% O2) for 48 h. We have shown previously (28) that the isolated cytotrophoblasts will differentiate into syncytiotrophoblasts over 48 h of culture under conditions of the present study. After the end of 48 h, the trophoblast cells (in triplicate) were treated for 24 h (or as indicated otherwise) with various compounds in the medium containing 2% FCS. Controls, also in triplicate, received equivalent volume of vehicle.

Assay of 11{beta}-HSD2 activity: radiometric conversion assay. The level of 11{beta}-HSD2 activity in intact cells at various time points and following different treatment regimens was determined by a radiometric conversion assay, as described previously (28). Briefly, the cells were incubated for 1 h at 37°C in serum-free medium containing ~50,000 cpm [3H]cortisol and 100 nM unlabeled cortisol. At the end of incubation, the medium was collected and steroids were extracted. The extracts were dried and the residues resuspended. A fraction of the resuspension was spotted on a TLC plate that was developed in chloroform-methanol (9:1, vol/vol). The bands containing the labeled cortisol and cortisone were identified by UV light of the cold carriers, cut out into scintillation vials, and counted in Scintisafe Econo 1 (Fisher Scientific). The rate of cortisol-to-cortisone conversion was calculated, and the blank values (defined as the amount of conversion in the absence of cells) were subtracted and expressed as percentage of control. Results are shown as means ± SE.

Protein extraction and Western blot analysis. Western blot analysis was used to determine changes in the level of 11{beta}-HSD2 protein. After treatment with cadmium, both control and cadmium-treated cells were lysed with cold lysis buffer (100 mM NaCl, 50 mM NaF, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 1 mM orthovanadate, and 50 mM Tris·HCl, pH 7.5) for 5 min at room temperature. The total cell lysates were collected with a cell scraper, vortexed vigorously, and centrifuged at 10,000 g for 20 min at 4°C. The supernatant was collected, and the protein content was determined by the Bradford method using a protein assay kit (Bio-Rad Laboratories, Missasauga, ON, Canada) with BSA as a standard.

Western blot analysis was conducted as described previously (28). Briefly, 30 µg of the protein extracts were subjected to a standard 12% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose using a Bio-Rad Mini Transfer Apparatus. The 11{beta}-HSD2 protein was detected on the nitrocellulose filter by use of an ECL Western Blotting Analysis System (Pharmacia) following the manufacturer's instructions. Briefly, the nitrocellulose filter was blocked overnight at 4°C with 10% Blotto in TTBS (0.1% Tween-20 in TBS), and incubated with primary antibody (HUH23; 0.25 µg/ml in TTBS) for 1 h at room temperature. The primary antibody was a polyclonal rabbit anti-human 11{beta}-HSD2 antibody (a generous gift from Dr. Z. Krozowski) (43). After three 5-min washes with TTBS, the filter was incubated with horseradish peroxidase-labeled second antibody (1:5,000 dilution) and developed in ECL detection reagents. The filter was then exposed to X-ray film (Eastman Kodak, Rochester, NY) for 1–5 min. Densitometry was performed on the radiographs, and the level of 11{beta}-HSD2 protein was expressed as percentage of controls.

Assessment of 11{beta}-HSD2 mRNA and HO-1 mRNA: real-time quantitative RT-PCR. To determine whether cadmium-induced concentration-dependent decreases in 11{beta}-HSD2 activity were associated with corresponding reductions in 11{beta}-HSD2 mRNA levels, trophoblast cells were treated for 24 h with various concentrations of CdCl2, and the relative abundance of 11{beta}-HSD2 mRNA was assessed by two-step real-time quantitative RT-PCR (qRT-PCR), as described previously (34). Given that HO-1 is a well-known inducible target of cadmium (51), changes in the level of HO-1 mRNA in cultured trophoblast cells following CdCl2 treatment at various concentrations were also determined as described below.

Briefly, total RNA was extracted from cultured cells using an RNeasy Mini Kit (QIAGEN, Mississauga, ON, Canada) coupled with on-column DNase digestion with the RNase-Free DNase Set (QIAGEN) according to the manufacturer's instructions. One-half microgram of total RNA was reverse transcribed in a total volume of 20 µl using the High Capacity Complimentary Deoxyribonucleic acid (cDNA) Archive Kit(Applied Biosystems, Forest City, CA) following the manufacturer's instructions. For every RT reaction set, one RNA sample was set up without RT enzyme to provide a negative control. Gene transcript levels of 18S rRNA (housekeeping gene) and 11{beta}-HSD2 were quantified separately by TaqMan assays using the TaqMan Universal PCR Master Mix (Applied Biosystems) and the universal thermal cycling parameters (2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C) on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Levels of 18S rRNA were assessed using TaqMan Ribosomal RNA Control Reagents (Applied Biosystems), and those of 11{beta}-HSD2 mRNA using custom-designed TaqMan assays. Primers (60 nM each) and TaqMan MGB probe (200 nM) for human 11{beta}-HSD2 were designed using the Primer Express Software (Applied Biosystems; Table 1), and their optimal concentrations were determined following guidelines developed for Sequence Detection Systems by Applied Biosystems.


View this table:
[in this window]
[in a new window]
  		Table 1. Human 11{beta}-HSD2 TaqMan assay primers and probe and human HO-1 SYBR Green I assay primers

 
The level of HO-1 mRNA was determined by a custom-designed SYBR Green I chemistry-based assay (27). Briefly, primers (300 nM each) for human HO-1 were designed with the same Primer Express Software (Applied Biosystems; Table 1), and the optimal concentrations were determined empirically. The SYBR Green I assay was performed with the SYBR Green PCR Master Mix (Applied Biosystems) and a modified universal thermal cycling condition (2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 10 s each at 95, 60, and 72°C) with the standard disassociation/melting parameters (15 s each at 95, 60, and 95°C) on the ABI Prism 7900HT Sequence Detection System. The specificity of the SYBR Green I assay was verified by performing a melting-curve analysis (single peak with a Tm of 83.7°C) and by subsequent sequencing of the PCR products.

Levels of 18S rRNA, 11{beta}-HSD2 mRNA, and HO-1 mRNA in each RNA sample were quantified by the relative standard curve method (Applied Biosystems). Briefly, standard curves for 18S rRNA, 11{beta}-HSD2 mRNA and HO-1 mRNA were generated by performing a dilution series of the untreated control cDNA. For each RNA sample, the relative amount of 18S rRNA, 11{beta}-HSD2 mRNA and HO-1 mRNA was obtained, and the ratio of 11{beta}-HSD2 mRNA to 18S rRNA as well as the ratio of HO-1 mRNA to 18S rRNA were calculated. For each experiment, the amount of 11{beta}-HSD2 mRNA or HO-1 mRNA at any given concentration of CdCl2 is expressed relative to the amount of transcript present in the untreated control.

Transient transfection and reporter gene assay. To determine whether cadmium altered the rate of HSD11B2 gene transcription, a standard reporter gene assay was performed as described (34). Briefly, the isolated trophoblast cells were plated on 24-well plates and cultured under standard conditions for 48 h. The cells were then cotransfected with 0.8 µg/well of the pGL3-HSD11B2P+330bp and 0.2 µg/well of a cytomegalovirus promoter (pCMV) {beta}-galactosidase plasmid (Promega, Madison, WI), or with 0.8 µg/well pGL3-Basic and 0.2 µg/well pCMV-{beta}-Gal (negative control). All transfections were carried out in serum-free M199 for 1 h using Transfast Transfection Reagent (Promega) at a ratio of 2:1 (transfection reagent to DNA) according to the manufacturer's instructions. At the end of transfection, cells were gently overlaid with fresh medium containing 10% serum and incubated for 4 h. The medium was then replaced with fresh medium containing 2% serum, and the cells were treated with 1.0 µM CdCl2 for 24 h. At the end of treatment, luciferase and galactosidase activities were analyzed using the Luciferase Assay System (Promega) and the {beta}-Galactosidase Enzyme Assay System (Promega), respectively. Luciferase activity was measured using a Lumat LB 9507 luminometer (EG&G Berthold, Bad Wildbad, Germany) and normalized against {beta}-galactosidase activity. Each transfection was performed in triplicate, and a total of three to five independent experiments were carried out.

Reagents and supplies. [1,2,6,7-3H(N)]cortisol (80 Ci/mmol) was purchased from DuPont Canada (Markam, ON). Nonradioactive cortisol and cortisone were obtained from Steraloids (Wilton, NH). CdCl2, ZnCl2, MgCl2, MnCl2, nicotine, and cotinine were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Polyester-backed thin-layer chromatography (TLC) plates were obtained from Fisher Scientific (Nepean, ON). All solvents used were from VWR Canlab (Mississauga, ON, Canada). Cell culture supplies were obtained from either Invitrogen Life Technologies (Burlington, ON, Canada) or Fisher Scientific. General molecular biology reagents were from Invitrogen or Pharmacia Canada (Baie D'Urte, QC, Canada). Oligonucleotides were synthesized (11{beta}-HSD2 primers) by a Pharmacia Gene Assembler and purified with NAP-50 columns (Pharmacia) according to the manufacturer's instructions or purchased (HO-1 primers) from Sigma Genosys (Oakville, ON, Canada).

Statistical analyses. Results are presented as means ± SE of three to five independent experiments (i.e., tissues from different patients), as indicated. Statistical analyses of 11{beta}-HSD2 activity and mRNA as well as HO-1 mRNA data were performed using one-way ANOVA followed by Tukey's post hoc test. 11{beta}-HSD2 protein and promoter activity data were analyzed by a standard Student's t-test. Significance was set at P < 0.05. Calculations were performed using SPSS software version 9.0 (Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of cadmium on 11{beta}-HSD2 activity. To study the effects of cadmium on placental 11{beta}-HSD2 activity, isolated human trophoblast cells were treated with 1.0 µM CdCl2 for different times (3, 6, 12 and 24 h). This treatment resulted in a time-dependent decrease in 11{beta}-HSD2 activity such that a significant reduction (P < 0.05) occurred after 3 h of treatment (Fig. 1A). Furthermore, the cells were also treated for 24 h with various concentrations of CdCl2 (0.25 to 1.0 µM), and this led to a concentration-dependent decrease in 11{beta}-HSD2 activity with a maximal effect at 0.75 µM CdCl2 (20% of control; Fig. 1B).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1. Time- and concentration-dependent effects of CdCl2 on 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}-HSD2) activity. A: time-dependent effects of CdCl2 on 11{beta}-HSD2 activity. Trophoblast cells were incubated with or without 1.0 µM CdCl2 for various periods of time (3, 6, 12, and 24 h). B: concentration-dependent effects of CdCl2 on 11{beta}-HSD2 activity. Trophoblast cells were treated for 24 h with increasing concentrations of CdCl2 (0.25, 0.50, 0.75, and 1.0 µM). At end of treatment, the level of 11{beta}-HSD2 activity in intact cells was determined by standard radiometric conversion assay, as described in MATERIALS AND METHODS. Each data point is expressed as percentage of control (separate controls without CdCl2 were used for each time point in A), and each bar represents mean ± SE of 4 independent experiments each performed in triplicate (*P < 0.05 vs. control).

 
Effects of other divalent metals on 11{beta}-HSD2 activity. To determine whether decreases in placental 11{beta}-HSD2 activity following treatment with CdCl2 were specific to this metal, we treated trophoblast cells for 24 h with three other divalent metals, ZnCl2, MgCl2, and MnCl2, each at 1.0 µM (corresponding to the maximal effective concentration of CdCl2) and 10 µM concentrations. In contrast to cadmium, none of these divalent metals reduced 11{beta}-HSD2 activity (data not shown), indicating that the effect of cadmium on placental 11{beta}-HSD2 is not a general property of divalent metals.

Effects of nicotine and cotinine on 11{beta}-HSD2 activity. Besides cadmium, nicotine and cotinine (a major metabolite of nicotine) are well-known constituents of tobacco smoke (4). To determine whether these two compounds have similar effects on placental 11{beta}-HSD2 activity, trophoblast cells were treated for 24 h with various concentrations of nicotine and cotinine (0.1, 0.5, and 1.0 mM). In contrast to cadmium, nicotine and cotinine, at concentrations up to 1 mM, did not decrease 11{beta}-HSD2 activity in cultured trophoblast cells (data not shown).

Effects of CdCl2 on 11{beta}-HSD2 protein and mRNA. To determine whether the CdCl2-induced decrease in 11{beta}-HSD2 activity was a result of reduced 11{beta}-HSD2 expression, levels of 11{beta}-HSD2 protein and mRNA were assessed by Western blot analysis and qRT-PCR, respectively. As shown in Fig. 2, treatment of trophoblast cells for 24 h with 1.0 µM CdCl2 led to a profound reduction in levels of 11{beta}-HSD2 protein (25% of control). Similar to its effects on 11{beta}-HSD2 activity, cadmium resulted in a concentration-dependent decrease in the steady-state level of 11{beta}-HSD2 mRNA with a maximal effect at 0.75 µM (10% of control; Fig. 3A).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 2. Effects of CdCl2 on 11{beta}-HSD2 protein. Trophoblast cells were treated with 1.0 µM CdCl2 for 24 h. At end of treatment, levels of 11{beta}-HSD2 protein were determined by western blot analysis as described in MATERIALS AND METHODS. A: one representative autoradiograph is shown. B: each bar represents mean ± SE of 4 independent experiments (**P < 0.01 vs. control).

 


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 3. Effects of CdCl2 on 11{beta}-HSD2 mRNA and heme oxygenase-1 (HO-1) mRNA. Trophoblast cells were treated with increasing concentrations of CdCl2 (0.25, 0.50, 0.75, and 1.0 µM) for 24 h. At end of treatment, total cellular RNA was isolated, and the steady-state level of 11{beta}-HSD2 mRNA (A) and HO-1 mRNA (B) was assessed by quantitative RT-PCR, as described in MATERIALS AND METHODS. Each data point is expressed as percentage of control, and each bar represents mean ± SE of 4 independent experiments (*P < 0.05 vs. control).

 
Effects of CdCl2 on HO-1 mRNA. To determine whether cadmium causes global downregulation of gene expression in trophoblast cells, the level of HO-1 mRNA (a well-known inducible target of cadmium) was measured following treatment for 24 h with 1 µM CdCl2, by qRT-PCR. As shown in Fig. 3B, cadmium resulted in a concentration-dependent increase in HO-1 mRNA levels. Furthermore, there was no evidence of CdCl2-induced cytotoxicity, as determined by trypan blue exclusion (data not shown). Collectively, these observations suggest that the inhibitory effects of cadmium on placental 11{beta}-HSD2 are specific and cannot be attributed to global downregulation of gene expression resulting from cytotoxicity.

Effects of CdCl2 on HSD11B2 gene transcription. To determine whether the effect of cadmium on placental HSD11B2 occurs at the transcriptional level, transient transfection experiments were performed. Trophoblast cells were cotransfected with a luciferase construct containing a 330-bp 5'-flanking region of the human HSD11B2 gene (Fig. 4A) and pCMV-{beta}-Gal plasmid DNA. The level of luciferase activity was normalized to that of {beta}-galactosidase and used as an indicator of HSD11B2 promoter activity. Treatment with CdCl2 (1.0 µM) resulted in a 75% reduction in HSD11B2 promoter activity (Fig. 4B).



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 4. Effects of CdCl2 on HSD11B2 gene promoter activity. A: schematic representation of the reporter gene construct. Trophoblast cells were transfected with a luciferase (Luc) reporter gene construct driven by the HSD11B2 promoter and pCMV {beta}-galactosidase (B), as described in MATERIALS AND METHODS. Four hours after transfection, cells were treated with 1.0 µM CdCl2 for 24 h. At end of treatment, luciferase and {beta}-galactosidase ({beta}-Gal) activities were determined, and the ratio of luciferase activity to that of {beta}-galactosidase was calculated and expressed as percentage of control. Each bar represents mean ± SE of 4 independent experiments (**P < 0.01 vs. control).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
FGR affects 5–10% of all pregnancies and is a leading cause of perinatal morbidity and mortality (6, 48). Moreover, accumulating evidence suggests that FGR is also a significant risk factor for obesity, type 2 diabetes, and cardiovascular diseases later in life (58). Of all the identified etiological determinants of FGR in developed countries, the most significant is prenatal exposure to cigarette smoking (42, 63). Furthermore, a dose-dependent relationship exists between the reduction in birth weight and the number of cigarettes smoked per day (12). In the United States, maternal smoking is responsible for 20–30% of low-birth-weight infants and 10% of infant deaths (64, 65). In addition, maternal smoking is a leading risk factor for sudden infant death syndrome in FGR infants (14, 67). The growth-inhibiting effect of tobacco smoke is observed not only in cases of heavy maternal smoking (≥10 cigarettes/day) but also in instances of light (<10 cigarettes/day) and even passive smoking (50). Importantly, recent evidence also suggests that there is a transgenerational effect of maternal smoking such that a mother's prenatal exposure to smoke may affect the birth weight of her offspring (55).

Because tobacco smoke contains several thousand chemicals (21, 70), identification of the causative agents of FGR by cigarette smoking has been a formidable task. One component of tobacco smoke found preferentially to accumulate in the placenta, and investigated extensively in both humans and animal models, is cadmium (29, 53). During human pregnancy, maternal exposure to cadmium is associated with low birth weight and an increased incidence of spontaneous abortion (23, 29, 44, 66). In rodents, maternal cadmium administration during pregnancy leads to FGR (3, 36). Although pharmacokinetic studies have demonstrated that the metal does not readily reach the fetus, it accumulates in high concentrations in the placenta (29), suggesting that the placenta is a primary target by which cadmium influences fetal growth and development. Indeed, both structural and functional abnormalities were observed in placentas of humans and rodents exposed to cadmium during pregnancy (9, 16, 46, 74). Proposed mechanisms by which cadmium leads to FGR include reduced uteroplacental blood flow (7, 10, 47) and impaired placental transport function (15, 26, 49, 59). In particular, there is robust evidence that disruption of normal fetoplacental zinc homeostasis is an important mechanism contributing to cadmium-induced FGR (53). However, the molecular targets within the placenta that are responsible for mediating the growth-inhibiting effect of cadmium remain largely unknown.

Given that reduced placental 11{beta}-HSD2 is causally linked to FGR (54), we hypothesized that cadmium induces FGR in part by downregulating placental 11{beta}-HSD2. As a first step in examining this hypothesis, we studied the effects of cadmium on placental 11{beta}-HSD2 by utilizing cultured human trophoblast cells as a model system. We demonstrated that treatment of trophoblast cells with cadmium led to a time- and concentration-dependent decrease in the level of 11{beta}-HSD2 activity. Furthermore, the cadmium-induced decrease in 11{beta}-HSD2 activity was a consequence of reduced 11{beta}-HSD2 expression, as levels of both 11{beta}-HSD2 protein and mRNA were decreased following treatment with the metal.

In theory, a decrease in the steady-state mRNA level of a given gene can be achieved by reducing the rate of gene transcription and/or by decreasing the mRNA stability. To determine whether the cadmium-induced decrease in 11{beta}-HSD2 mRNA was mediated at the level of HSD11B2 gene transcription, we transiently transfected trophoblast cells with a reporter gene construct driven by the proximal HSD11B2 promoter and examined the effect of cadmium on the reporter gene activity. Our results demonstrated that cadmium reduced HSD11B2 promoter activity by 75%, which was similar in magnitude to the reduction in levels of 11{beta}-HSD2 mRNA, suggesting that the inhibitory effect of cadmium on placental 11{beta}-HSD2 activity and expression was mediated primarily at a transcriptional level. Given that the proximal HSD11B2 promoter contains multiple Sp1 binding sites (1, 57) and that cadmium has been shown to reduce Sp1 binding activity in adult rat alveolar epithelial cells (77), it remains possible that cadmium may suppress HSD11B2 gene transcription by interfering with Sp1 activity in human trophoblast cells. Obviously, this contention warrants future study.

Although the signal transduction pathway involved in mediating the effects of cadmium on placental 11{beta}-HSD2 remains to be defined, two pathways may be proposed, and they are currently under investigation in our laboratory. Given that cadmium activates the extracellular signal-regulated kinase (ERK) in a variety of mammalian cells (76) and that the activation of ERK has been shown to decrease 11{beta}-HSD2 activity in the JEG-3 trophoblast cell line (45), the inhibitory effects of cadmium on placental 11{beta}-HSD2 may be mediated by the ERK pathway. Alternately, cadmium may suppress placental HSD11B2 gene expression through activation of the estrogen receptor (ER). This contention is based on our previous findings that estrogen reduced 11{beta}-HSD2 expression and activity in cultured primary human trophoblast cells (73). Furthermore, cadmium activated ER in breast cancer cells (72) and mimicked the in vivo effects of estrogen in the rat uterus and mammary gland (31).

To determine whether other divalent metals had similar effects on placental 11{beta}-HSD2, we treated trophoblast cells with ZnCl2, MgCl2, and MnCl2. In contrast to cadmium, none of the three metals affected 11{beta}-HSD2 activity, suggesting that the decreased 11{beta}-HSD2 activity and expression were not general effects of heavy metals but were specific effects of cadmium. We also examined the effects of cadmium on the expression of HO-1, a known inducible target of this metal (51), to demonstrate that the decreased placental HSD11B2 gene expression was not due to cadmium-induced global downregulation of gene expression resulting from cytoxicity in trophoblast cells. As expected, cadmium evoked a concentration-dependent increase in the level of HO-1 mRNA. Furthermore, previous studies showed that cadmium, at 10 times higher concentration than that used in the present study, did not cause cytotoxic effects on cultured human trophoblast cells (38). Collectively, these findings suggest that cadmium exerts specific effects on placental 11{beta}-HSD2.

Our present study demonstrates that cadmium reduces placental 11{beta}-HSD2. If this occurs in the human placenta in vivo, it will lead to increased fetal exposure to maternal glucocorticoid and consequent FGR. It is noteworthy that previous studies have provided evidence for cadmium-induced potentiation of glucocorticoid receptor activation (20, 62). If cadmium exerts similar effects on the developing fetus, it would suggest that cadmium might not only result in increased glucocorticoid exposure due to reduced placental 11{beta}-HSD2 but also enhance the response to glucocorticoids, further exacerbating glucocorticoid-induced FGR. To determine whether other key constituents of tobacco smoke have similar effects on placental 11{beta}-HSD2, we treated trophoblast cells with nicotine and cotinine (a major metabolite of nicotine). Neither of the two compounds, at concentrations up to 1 mM, altered placental 11{beta}-HSD2 activity. These findings suggest that cadmium may be a key constituent of tobacco smoke that is involved in mediating the adverse effects of maternal smoking on fetal growth and development.

Human exposure to cadmium occurs primarily through dietary sources, cigarette smoking, and occupational exposure (24, 25). The average daily intake is estimated to be 10–60 µg/day (25, 52). The metal has a biological half-life of 10–30 years (52) and is known to accumulate in the placenta (29). In human placental tissues, the amount of cadmium was 12–535 and 6–270 ng/g in women from high [occupationally exposed or living in areas highly polluted by battery, smelters, or refineries (2, 5, 18)] and low (18, 19, 22, 23, 41) environmental exposures, respectively. Furthermore, mothers who smoked during pregnancy had significantly increased cadmium levels, approximately doubled that for nonsmoking mothers (9, 19, 37, 75). Thus the concentrations of cadmium found in human placenta in the general population (including both smokers and nonsmokers) are within those (0.25–1 µM = 45–180 ng/ml) that reduce placental 11{beta}-HSD2 activity and expression in the present study, supporting a role for the decreased placental 11{beta}-HSD2 in cadmium-induced FGR in the human.

Placental 11{beta}-HSD2 plays a key role in fetal development by protecting the fetus from exposure to high levels of maternal glucocorticoid. Consequently, molecules that downregulate placental 11{beta}-HSD2 can adversely affect fetal growth and contribute to FGR. The results of the present study suggest that cadmium is a candidate for such a role. At concentrations relevant to those in the human placenta in vivo, cadmium reduces placental 11{beta}-HSD2 activity and expression by suppressing HSD11B2 gene transcription. Thus our present study identifies placental 11{beta}-HSD2 as a novel molecular target of cadmium. It also reveals a molecular mechanism by which cadmium and smoking may exert their growth-restricting effects on the developing fetus.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
We thank Dr. Daniel Hardy and Andrea Homan for their technical assistance and helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Yang, Children's Health Research Institute, Rm. A5-132, Victoria Research Laboratories-Westminster Campus, 800 Commissioners Rd. East, London, ON, Canada N6A 4G5 (e-mail: kyang{at}uwo.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Agarwal AK and White PC. Analysis of the promoter of the NAD+ dependent 11 beta-hydroxysteroid dehydrogenase (HSD11K) gene in JEG-3 human choriocarcinoma cells. Mol Cell Endocrinol 121: 93–99, 1996.[CrossRef][Web of Science][Medline]
  2. Baranowska I. Lead and cadmium in human placentas and maternal and neonatal blood (in a heavily polluted area) measured by graphite furnace atomic absorption spectrometry. Occup Environ Med 52: 229–232, 1995.[Abstract/Free Full Text]
  3. Baranski B. Effect of exposure of pregnant rats to cadmium on prenatal and postnatal development of the young. J Hyg Epidemiol Microbiol Immunol 29: 253–262, 1984.[Medline]
  4. Barbieri RL, Gochberg J, and Ryan KJ. Nicotine, cotinine, and anabasine inhibit aromatase in human trophoblast in vitro. J Clin Invest 77: 1727–1733, 1986.[Web of Science][Medline]
  5. Berlin M, Blanks R, Catton M, Kazantzis G, Mottet NK, and Samiullah Y. Birth weight of children and cadmium accumulation in placentas of female nickel-cadmium (long-life) battery workers. 257–262.
  6. Brodsky D and Christou H. Current concepts in intrauterine growth restriction. J Intensive Care Med 19: 307–319, 2004.[Abstract/Free Full Text]
  7. Burton GJ, Palmer ME, and Dalton KJ. Morphometric differences between the placental vasculature of non-smokers, smokers and ex-smokers. Br J Obstet Gynaecol 96: 907–915, 1989.[Web of Science][Medline]
  8. Burton PJ and Waddell BJ. Dual function of 11beta-hydroxysteroid dehydrogenase in placenta: modulating placental glucocorticoid passage and local steroid action. Biol Reprod 60: 234–240, 1999.[Abstract/Free Full Text]
  9. Bush PG, Mayhew TM, Abramovich DR, Aggett PJ, Burke MD, and Page KR. A quantitative study on the effects of maternal smoking on placental morphology and cadmium concentration. Placenta 21: 247–256, 2000.[CrossRef][Web of Science][Medline]
  10. Castro LC, Allen R, Ogunyemi D, Roll K, and Platt LD. Cigarette smoking during pregnancy: acute effects on uterine flow velocity waveforms. Obstet Gynecol 81: 551–555, 1993.[Web of Science][Medline]
  11. Challis JRG, Matthews SG, Gibb W, and Lye SJ. Endocrine and paracrine regulation of birth at term and preterm. Endocr Rev 21: 514–550, 2000.[Abstract/Free Full Text]
  12. Cnattingius S. The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes. Nicotine Tob Res 6: S125–S140, 2004.
  13. Condon JC, Jeyasuria P, Faust JM, Wilson JW, and Mendelson CR. A decline in the levels of progesterone receptor coactivators in the pregnant uterus at term may antagonize progesterone receptor function and contribute to the initiation of parturition. Proc Natl Acad Sci USA 100: 9518–9523, 2003.[Abstract/Free Full Text]
  14. Cooke RW. Smoking, intra-uterine growth retardation and sudden infant death syndrome. Int J Epidemiol 27: 238–241, 1998.[Abstract/Free Full Text]
  15. Derfoul A, Lin FJ, Awumey EM, Kolodzeski T, Hall DJ, and Tuan RS. Estrogenic endocrine disruptive components interfere with calcium handling and differentiation of human trophoblast cells. J Cell Biochem 89: 755–770, 2003.[CrossRef][Web of Science][Medline]
  16. Di Sant'Agnese PA, Jensen KD, Levin A, and Miller RK. Placental toxicity of cadmium in the rat: an ultrastructural study. Placenta 4: 149–163, 1983.[CrossRef][Web of Science][Medline]
  17. Dy J, Sampath-Kumar R, Richardson B, and Yang K. Placental 11beta-hydroxysteroid dehydrogenase type 2 activity is attenuated in pregnancies complicated with intrauterine growth restriction. Xth International Congress on Hormonal Steroids, Quebec City, Canada, 1998.
  18. Fagher U, Laudanski T, Schutz A, Sipowicz M, and Akerlund M. The relationship between cadmium and lead burdens and preterm labor. Int J Gynaecol Obstet 40: 109–114, 1993.[CrossRef][Medline]
  19. Falcon M, Vinas P, Osuna E, and Luna A. Environmental exposures to lead and cadmium measured in human placenta. Arch Environ Health 57: 598–602, 2002.[Web of Science][Medline]
  20. Favatier F and Polla BS. Tobacco-smoke-inducible human haem oxygenase-1 gene expression: role of distinct transcription factors and reactive oxygen intermediates. Biochem J 353: 475–482, 2001.[CrossRef][Web of Science][Medline]
  21. Feng T. Substance abuse in pregnancy. Curr Opin Obstet Gynecol 5: 16–23, 1993.[Web of Science][Medline]
  22. Fiala J, Hruba D, and Rezl P. Cadmium and zinc concentrations in human placentas. Cent Eur J Public Health 6: 241–248, 1998.[Medline]
  23. Frery N, Nessmann C, Girard F, Lafond J, Moreau T, Blot P, Lellouch J, and Huel G. Environmental exposure to cadmium and human birthweight. Toxicology 79: 109–118, 1993.[CrossRef][Web of Science][Medline]
  24. Gartrell MJ, Craun JC, Podrebarac DS, and Gunderson EL. Pesticides, selected elements, and other chemicals in adult total diet samples, October 1980–March 1982. J Assoc Off Anal Chem 69: 146–159, 1986.[Medline]
  25. Gartrell MJ, Craun JC, Podrebarac DS, and Gunderson EL. Pesticides, selected elements, and other chemicals in infant and toddler total diet samples, October 1980–March 1982. J Assoc Off Anal Chem 69: 123–145, 1986.[Medline]
  26. Goodman DR, Fant ME, and Harbison RD. Perturbation of alpha-aminoisobutyric acid transport in human placental membranes: direct effects by HgCl2, CH3HgCl, and CdCl2. Teratog Carcinog Mutagen 3: 89–100, 1983.[CrossRef][Web of Science][Medline]
  27. Guan H, Arany E, van Beek JP, Chamson-Reig A, Thyssen S, Hill DJ, and Yang K. Adipose tissue gene expression profiling reveals distinct molecular pathways that define visceral adiposity in offspring of maternal protein-restricted rats. Am J Physiol Endocrinol Metab 288: E663–E673, 2005.[Abstract/Free Full Text]
  28. Hardy DB and Yang K. The expression of 11 beta-hydroxysteroid dehydrogenase type 2 is induced during trophoblast differentiation: effects of hypoxia. J Clin Endocrinol Metab 87: 3696–3701, 2002.[Abstract/Free Full Text]
  29. Henson MC and Chedrese PJ. Endocrine disruption by cadmium, a common environmental toxicant with paradoxical effects on reproduction. Exp Biol Med (Maywood) 229: 383–392, 2004.[Abstract/Free Full Text]
  30. Jarup L, Berglund M, Elinder CG, Nordberg G, and Vahter M. Health effects of cadmium exposure–a review of the literature and a risk estimate. Scand J Work Environ Health 24, Suppl 1: 1–51, 1998.[Web of Science][Medline]
  31. Johnson MD, Kenney N, Stoica A, Hilakivi-Clarke L, Singh B, Chepko G, Clarke R, Sholler PF, Lirio AA, Foss C, Reiter R, Trock B, Paik S, and Martin MB. Cadmium mimics the in vivo effects of estrogen in the uterus and mammary gland. Nat Med 9: 1081–1084.
  32. Jolibois LS Jr, Burow ME, Swan KF, George WJ, Anderson MB, and Henson MC. Effects of cadmium cell viability, trophoblastic development, and expression of low density lipoprotein receptor transcripts in cultured human placental cells. Reprod Toxicol 13: 473–480, 1999.[CrossRef][Web of Science][Medline]
  33. Jolibois LS Jr, Shi W, George WJ, Henson MC, and Anderson MB. Cadmium accumulation and effects on progesterone release by cultured human trophoblast cells. Reprod Toxicol 13: 215–221, 1999.[CrossRef][Web of Science][Medline]
  34. Julan L, Guan H, van Beek JP, and Yang K. Peroxisome proliferator-activated receptor (delta) suppresses 11(beta)-hydroxysteroid dehydrogenase type 2 gene expression in human placental trophoblast cells. Endocrinology 146: 1482–1490, 2005.[CrossRef][Web of Science][Medline]
  35. Kajantie E, Dunkel L, Turpeinen U, Stenman UH, Wood PJ, Nuutila M, and Andersson S. Placental 11 beta-hydroxysteroid dehydrogenase-2 and fetal cortisol/cortisone shuttle in small preterm infants. J Clin Endocrinol Metab 88: 493–500, 2003.[Abstract/Free Full Text]
  36. Kanter M, Yoruk M, Koc A, Meral I, and Karaca T. Effects of cadmium exposure on morphological aspects of pancreas, weights of fetus and placenta in streptozotocin-induced diabetic pregnant rats. Biol Trace Elem Res 93: 189–200, 2003.[CrossRef][Web of Science][Medline]
  37. Kantola M, Purkunen R, Kroger P, Tooming A, Juravskaja J, Pasanen M, Saarikoski S, and Vartiainen T. Accumulation of cadmium, zinc, and copper in maternal blood and developmental placental tissue: differences between Finland, Estonia, and St. Petersburg. Environ Res 83: 54–66, 2000.[Medline]
  38. Kawai M, Swan KF, Green AE, Edwards DE, Anderson MB, and Henson MC. Placental endocrine disruption induced by cadmium: effects on P450 cholesterol side-chain cleavage and 3beta-hydroxysteroid dehydrogenase enzymes in cultured human trophoblasts. Biol Reprod 67: 178–183, 2002.[Abstract/Free Full Text]
  39. Kitanaka S, Tanae A, and Hibi I. Apparent mineralocorticoid excess due to 11 beta-hydroxysteroid dehydrogenase deficiency: a possible cause of intrauterine growth retardation. Clin Endocrinol (Oxf) 44: 353–359, 1996.[CrossRef][Medline]
  40. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, and Strauss JF, 3rd. Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118: 1567–1582, 1986.[Abstract/Free Full Text]
  41. Korpela H, Loueniva R, Yrjanheikki E, and Kauppila A. Lead and cadmium concentrations in maternal and umbilical cord blood, amniotic fluid, placenta, and amniotic membranes. Am J Obstet Gynecol 155: 1086–1089, 1986.[Web of Science][Medline]
  42. Kramer MS. Socioeconomic determinants of intrauterine growth retardation. Eur J Clin Nutr 52: S29–S33, 1998.
  43. Krozowski Z, MaGuire JA, Stein-Oakley AN, Dowling J, Smith RE, and Andrews RK. Immunohistochemical localization of the 11 beta-hydroxysteroid dehydrogenase type II enzyme in human kidney and placenta. J Clin Endocrinol Metab 80: 2203–2209, 1995.[Abstract]
  44. Kuhnert BR, Kuhnert PM, and Zarlingo TJ. Associations between placental cadmium and zinc and age and parity in pregnant women who smoke. Obstet Gynecol 71: 67–70, 1988.[Web of Science][Medline]
  45. Lanz CB, Causevic M, Heiniger C, Frey FJ, Frey BM, and Mohaupt MG. Fluid Shear stress reduces 11ss-hydroxysteroid dehydrogenase type 2. Hypertension 37: 160–169, 2001.[Abstract/Free Full Text]
  46. Larsen LG, Clausen HV, and Jonsson L. Stereologic examination of placentas from mothers who smoke during pregnancy. Am J Obstet Gynecol 186: 531–537, 2002.[CrossRef][Web of Science][Medline]
  47. Levin AA and Miller RK. Fetal toxicity of cadmium in the rat: decreased utero-placental blood flow. Toxicol Appl Pharmacol 58: 297–306, 1981.[CrossRef][Web of Science][Medline]
  48. Lin CC and Santolaya-Forgas J. Current concepts of fetal growth restriction: part I Causes, classification, and pathophysiology. Obstet Gynecol 92: 1044–1055, 1998.[CrossRef][Web of Science][Medline]
  49. Lin FJ, Fitzpatrick JW, Iannotti CA, Martin DS, Mariani BD, and Tuan RS. Effects of cadmium on trophoblast calcium transport. Placenta 18: 341–356, 1997.[CrossRef][Web of Science][Medline]
  50. Luciano A, Bolognani M, Biondani P, Ghizzi C, Zoppi G, and Signori E. The influence of maternal passive and light active smoking on intrauterine growth and body composition of the newborn. Eur J Clin Nutr 52: 760–763, 1998.[CrossRef][Web of Science][Medline]
  51. Malstrom SE, Jekic-McMullen D, Sambucetti L, Ang A, Reeves R, Purchio AF, Contag PR, and West DB. In vivo bioluminescent monitoring of chemical toxicity using heme oxygenase-luciferase transgenic mice. Toxicol Appl Pharmacol 200: 219–228, 2004.[CrossRef][Web of Science][Medline]
  52. Martin MB, Voeller HJ, Gelmann EP, Lu J, Stoica EG, Hebert EJ, Reiter R, Singh B, Danielsen M, Pentecost E, and Stoica A. Role of cadmium in the regulation of AR gene expression and activity. Endocrinology 143: 263–275, 2002.[Abstract/Free Full Text]
  53. McAleer MF and Tuan RS. Cytotoxicant-induced trophoblast dysfunction and abnormal pregnancy outcomes: role of zinc and metallothionein. Birth Defects Res C Embryo Today 72: 361–370, 2004.[CrossRef][Medline]
  54. McTernan CL, Draper N, Nicholson H, Chalder SM, Driver P, Hewison M, Kilby MD, and Stewart PM. Reduced placental 11beta-hydroxysteroid dehydrogenase type 2 mRNA levels in human pregnancies complicated by intrauterine growth restriction: an analysis of possible mechanisms. J Clin Endocrinol Metab 86: 4979–4983, 2001.[Abstract/Free Full Text]
  55. Misra DP, Astone N, and Lynch CD. Maternal smoking and birth weight: interaction with parity and mother's own in utero exposure to smoking. Epidemiology 16: 288–293, 2005.[CrossRef][Web of Science][Medline]
  56. Murphy VE, Zakar T, Smith R, Giles WB, Gibson PG, and Clifton VL. Reduced 11beta-hydroxysteroid dehydrogenase type 2 activity is associated with decreased birth weight centile in pregnancies complicated by asthma. J Clin Endocrinol Metab 87: 1660–1668, 2002.[Abstract/Free Full Text]
  57. Nawrocki AR, Goldring CE, Kostadinova RM, Frey FJ, and Frey BM. In vivo footprinting of the human 11beta-hydroxysteroid dehydrogenase type 2 promoter: evidence for cell-specific regulation by Sp1 and Sp3. J Biol Chem 277: 14647–14656, 2002.[Abstract/Free Full Text]
  58. Osmond C and Barker DJ. Fetal, infant, and childhood growth are predictors of coronary heart disease, diabetes, and hypertension in adult men and women. Environ Health Perspect 108: 545–553, 2000.[Web of Science][Medline]
  59. Pastrakuljic A, Derewlany LO, and Koren G. Maternal cocaine use and cigarette smoking in pregnancy in relation to amino acid transport and fetal growth. Placenta 20: 499–512, 1999.[CrossRef][Web of Science][Medline]
  60. Pepe GJ, Burch MG, and Albrecht ED. Expression of the 11beta-hydroxysteroid dehydrogenase types 1 and 2 proteins in human and baboon placental syncytiotrophoblast. Placenta 20: 575–582, 1999.[CrossRef][Web of Science][Medline]
  61. Piasek M, Blanusa M, Kostial K, and Laskey JW. Placental cadmium and progesterone concentrations in cigarette smokers. Reprod Toxicol 15: 673–681, 2001.[CrossRef][Web of Science][Medline]
  62. Pinot F, Kreps SE, Bachelet M, Hainaut P, Bakonyi M, and Polla BS. Cadmium in the environment: sources, mechanisms of biotoxicity, and biomarkers. Rev Environ Health 15: 299–323, 2000.[Medline]
  63. Prada JA and Tsang RC. Biological mechanisms of environmentally induced causes of IUGR. Eur J Clin Nutr 52: S21–S28, 1998.
  64. Rasmussen KM and Adams B. Annotation: cigarette smoking, nutrition, and birthweight. Am J Public Health 87: 543–544, 1997.[Free Full Text]
  65. Salihu HM, Shumpert MN, Aliyu MH, Alexander MR, Kirby RS, and Alexander GR. Stillbirths and infant deaths associated with maternal smoking among mothers aged > or =40 years: a population study. Am J Perinatol 21: 121–129, 2004.[CrossRef][Web of Science][Medline]
  66. Salpietro CD, Gangemi S, Minciullo PL, Briuglia S, Merlino MV, Stelitano A, Cristani M, Trombetta D, and Saija A. Cadmium concentration in maternal and cord blood and infant birth weight: a study on healthy non-smoking women. J Perinat Med 30: 395–399, 2002.[CrossRef][Web of Science][Medline]
  67. Schellscheidt J, Jorch G, and Menke J. Effects of heavy maternal smoking on intrauterine growth patterns in sudden infant death victims and surviving infants. Eur J Pediatr 157: 246–251, 1998.[CrossRef][Web of Science][Medline]
  68. Schoof E, Girstl M, Frobenius W, Kirschbaum M, Dorr HG, Rascher W, and Dotsch J. Decreased gene expression of 11beta-hydroxysteroid dehydrogenase type 2 and 15-hydroxyprostaglandin dehydrogenase in human placenta of patients with preeclampsia. J Clin Endocrinol Metab 86: 1313–1317, 2001.[Abstract/Free Full Text]
  69. Shams M, Kilby MD, Somerset DA, Howie AJ, Gupta A, Wood PJ, Afnan M, and Stewart PM. 11Beta-hydroxysteroid dehydrogenase type 2 in human pregnancy and reduced expression in intrauterine growth restriction. Hum Reprod 13: 799–804, 1998.[Abstract/Free Full Text]
  70. Sharara FI, Seifer DB, and Flaws JA. Environmental toxicants and female reproduction. Fertil Steril 70: 613–622, 1998.[CrossRef][Web of Science][Medline]
  71. Stewart PM, Rogerson FM, and Mason JI. Type 2 11 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid and activity in human placenta and fetal membranes: its relationship to birth weight and putative role in fetal adrenal steroidogenesis. J Clin Endocrinol Metab 80: 885–890, 1995.[Abstract]
  72. Stoica A, Katzenellenbogen BS, and Martin MB. Activation of estrogen receptor-alpha by the heavy metal cadmium. Mol Endocrinol 14: 545–553, 2000.[Abstract/Free Full Text]
  73. Sun K, Yang K, and Challis JR. Regulation of 11beta-hydroxysteroid dehydrogenase type 2 by progesterone, estrogen, and the cyclic adenosine 5' -monophosphate pathway in cultured human placental and chorionic trophoblasts. Biol Reprod 58: 1379–1384, 1998.[Abstract/Free Full Text]
  74. Van der Veen F and Fox H. The effects of cigarette smoking on the human placenta: a light and electron microscopic study. Placenta 3: 243–256, 1982.[CrossRef][Web of Science][Medline]
  75. Van Hattum B, de Voogt P, and Copius Peereboom JW. An analytical procedure for the determination of cadmium in human placentae. Int J Environ Anal Chem 10: 121–133, 1981.[Web of Science][Medline]
  76. Waisberg M, Joseph P, Hale B, and Beyersmann D. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192: 95–117, 2003.[CrossRef][Web of Science][Medline]
  77. Watkin RD, Nawrot T, Potts RJ, and Hart BA. Mechanisms regulating the cadmium-mediated suppression of Sp1 transcription factor activity in alveolar epithelial cells. Toxicology 184: 157–178, 2003.[CrossRef][Web of Science][Medline]
  78. Wier PJ, Miller RK, Maulik D, and DiSant'Agnese PA. Toxicity of cadmium in the perfused human placenta. Toxicol Appl Pharmacol 105: 156–171, 1990.[CrossRef][Web of Science][Medline]
  79. Yang K. Placental 11 beta-hydroxysteroid dehydrogenase: barrier to maternal glucocorticoids. Rev Reprod 2: 129–132, 1997.[Abstract]
  80. Zadorozhnaja TD, Little RE, Miller RK, Mendel NA, Taylor RJ, Presley BJ, and Gladen BC. Concentrations of arsenic, cadmium, copper, lead, mercury, and zinc in human placentas from two cities in Ukraine. J Toxicol Environ Health A 61: 255–263, 2000.[CrossRef][Web of Science][Medline]
  81. Zdravkovic T, Genbacev O, McMaster MT, and Fisher SJ. The adverse effects of maternal smoking on the human placenta: a review. Placenta 26: S81–S86, 2005.
  82. Zenzes MT, Krishnan S, Krishnan B, Zhang H, and Casper RF. Cadmium accumulation in follicular fluid of women in in vitro fertilization-embryo transfer is higher in smokers. Fertil Steril 64: 599–603, 1995.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 Hum Reprod UpdateHome page
A. E. Michael and A. T. Papageorghiou
Potential significance of physiological and pharmacological glucocorticoids in early pregnancy
Hum. Reprod. Update, September 1, 2008; 14(5): 497 - 517.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/1/E135    most recent
00356.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, K.
Right arrow Articles by Guan, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, K.
Right arrow Articles by Guan, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.