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Biology of Reproduction 67, 178-183 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Placental Endocrine Disruption Induced by Cadmium: Effects on P450 Cholesterol Side-Chain Cleavage and 3ß-Hydroxysteroid Dehydrogenase Enzymes in Cultured Human Trophoblasts

Motoyuki Kawaia,e, Kenneth F. Swana, Amy E. Greena,d, Deborah E. Edwardsa,d, Mary B. Andersonb,d, and Michael C. Henson1,,a,b,c,d

a Departments of Obstetrics and Gynecology, b Structural and Cellular Biology, c Physiology, and d Interdisciplinary Program in Molecular and Cellular Biology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112-2699 e Developmental Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously suggested that cadmium (Cd), an environmental toxicant and constituent of tobacco smoke, inhibits progesterone secretion in cultured human placental trophoblasts by inhibiting low-density lipoprotein receptor mRNA expression. In the current study, we investigated whether Cd also disrupts progesterone synthesis via P450 cholesterol side-chain cleavage (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), enzymes that play important roles in placental steroidogenesis. Human cytotrophoblasts were purified by density gradient centrifugation and incubated in Dulbecco modified Eagle medium + 10% fetal bovine serum with 0, 5, 10, or 20 µM CdCl2 for 96 h. Cells progressed to syncytiotrophoblastic maturity regardless of treatment. No differences (P > 0.05) in cell protein and lactate dehydrogenase activity were observed between untreated trophoblasts and those treated with CdCl2. However, P450scc and 3ß-HSD mRNA transcript levels declined in a dose-dependent manner (P <0.05) in trophoblasts cocultured with 5, 10, or 20 µM CdCl2. P450scc activity was similarly inhibited (P < 0.05) by CdCl2 treatment, although 3ß-HSD activity was not significantly affected. Coculture with 8-bromo-cAMP enhanced progesterone secretion in untreated cultures but did not reverse the decline in progesterone secretion induced by CdCl2 treatment. CdCl2 failed to influence cAMP content in cultured cells. Collectively, results suggest that P450scc enzyme is another site at which Cd interferes with placental progesterone production. However, it is unlikely that an inhibition of cAMP is involved with the inhibition of progesterone biosynthesis by Cd in human trophoblasts.

environment, placenta, progesterone, toxicology, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because of its presence in tobacco smoke and its widespread commercial use, cadmium (Cd) has become a common environmental pollutant [1]. In human pregnancy, maternal exposure to this trace metal is associated with low birth weight [2, 3] and possibly with an increased incidence of spontaneous abortion [4]. Although pharmacokinetic studies have demonstrated that Cd does not readily reach the fetus, it accumulates in high concentrations in the placenta [5, 6]. In both rodent and human placentae, observable symptoms of Cd toxicity in trophoblast cells [7, 8] included lysosomal vesiculation, nuclear chromatin clumping, and mitochondrial calcification. In perfused human placenta, exposure to Cd precipitated a decline in hCG secretion [7].

The human placental syncytiotrophoblast is responsible for the production of a number of hormones necessary for normal conceptus development and pregnancy maintenance. These hormones include progesterone [9], a steroid that plays a vital role by promoting uterine myometrial quiescence [10]. Cd has been reported to affect steroidogenesis directly both in vivo and in vitro, although differences in experimental methods and the concentrations tested may explain slight variations in results [11–14]. Thus, we have demonstrated that Cd inhibits progesterone secretion in cultured human placental cells [15, 16], an effect that was not simply the result of Cd-induced cell death or the inhibition of syncytial maturation in culture [16, 17]. We propose that the suppression of secretion in cultured trophoblast cells represents a direct effect of Cd on the progesterone biosynthetic pathway. We have previously attributed this effect, at least in part, to a deleterious effect on low-density lipoprotein receptor (LDL-R) mRNA [17]. However, because a number of steroidogenic enzymes are necessary for optimal progesterone production, it is quite possible that this inhibition may be multifaceted, affecting multiple sites.

Among the steroidogenic enzymes expressed by the primate placenta and involved in progesterone formation, cytochrome P450 cholesterol side-chain cleavage (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) are important components. P450scc catalyzes the conversion of cholesterol into pregnenolone, which is then converted into progesterone via 3ß-HSD [18]. Syncytiotrophoblasts, formed in culture (as in vivo) from cytotrophoblast progenitors, adapt to facilitate this transition [19]. The purpose of the present study was to determine the influence of Cd on the expression of mRNA transcripts and enzyme activities of P450scc and 3ß-HSD in cultured human placental cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Placentae and Culture of Trophoblast Cells

Studies were performed with the approval of the Institutional Review Board of the Tulane University Health Sciences Center. Human placentae (n = 18) were obtained at term from uncomplicated pregnancies. Placental villous tissue was enzymatically dispersed, and cytotrophoblasts were purified via density gradient centrifugation according to the method of Kliman et al. [20], as modified in our laboratory [15, 21]. Purified cytotrophoblasts were suspended in Dulbecco modified Eagle medium (DMEM) containing 44 mM sodium bicarbonate, 25 mM Hepes, 25 mM glucose, and 4 mM glutamine (Life Technologies, Grand Island, NY), plus 10% fetal bovine serum (FBS; Life Technologies). The initial cell suspension was adjusted to a final concentration of 106 cells/ml, containing 0, 5, 10, or 20 µM CdCl2 (Sigma, St. Louis, MO). Cells were cultured in 24-well culture plates at 37°C in a humidified atmosphere of 10% CO2 and 90% air for 96 h. In culture, cytotrophoblasts differentiate into morphologically and endocrinologically active syncytiotrophoblasts prior to 96 h as previously described [15].

To assess P450scc and 3ß-HSD activities, trophoblast cells were cultured under conditions (with or without pregnenolone and with or without 25-hydroxycholesterol or aminoglutethimide) designed to assess their respective steps in the progesterone biosynthetic pathway. Because 25-hydroxycholesterol (25-OHC) readily transverses the plasma and mitochondrial membranes, its conversion to progesterone provides an index of P450scc activity [22, 23]. Similarly, because aminoglutethimide is an inhibitor of P450scc-mediated hydroxylation, addition of this compound along with pregnenolone enables progesterone production to serve as an index of 3ß-HSD activity. Therefore, trophoblast cells were cultured in DMEM + 10% FBS for 72 h and then incubated for 24 h with DMEM + 10% steroid-stripped FBS (HyClone, Logan, UT) in the absence or presence of 5, 10, or 20 µM CdCl2. After incubation, trophoblast cells were incubated for 4 h (96–100 h in culture) with DMEM + 10% steroid-stripped FBS, in the absence or presence of 20 µM CdCl2, 90 µM aminoglutethimide (Sigma) and 5 µg/ml pregnenolone (Sigma), or 20 µg/ml 25-OHC (Sigma).

In the same cultures, trophoblast cells were incubated in DMEM + 10% steroid-stripped FBS in the absence or presence of 20 µM CdCl2 for 72–96 h. 8-Bromo-cAMP (1 mM) was then added, in the absence or presence of 20 µM CdCl2, for 48 h (96–144 h in culture). Culture medium from all experiments was stored at -20°C until assayed for progesterone or lactate dehydrogenase (LDH) activity. Cellular protein, solubilized for 24 h in 0.1 N NaOH, was measured according to the method of Bradford [24] using BSA as a standard.

LDH Activity Assessment

LDH activity, as an index of cell injury and death, was assessed in medium following 96 h in culture using an In Vitro Toxicology Assay kit, which is LDH based (Sigma). LDH activity was measured spectrophotometrically (absorbance at 490 nm) as previously described [25].

Progesterone Quantitation

Progesterone concentrations in medium were determined using a commercially available solid-phase RIA (Coat-a-Count; Diagnostic Products Corp., Los Angeles, CA), as described by Kato and Braunstein [26] and used in our laboratory [21]. Progesterone concentrations were normalized with respect to total cellular protein.

Isolation of Total RNA and cDNA Synthesis

Total cellular RNA was isolated according to the methods of Chomczynski and Sacchi [27] and Chirgwin et al. [28], as utilized in our laboratory [29, 30]. Total RNA was extracted from cells pooled from 2 or 4 culture wells using TRIzol reagent (Life Technologies). Concentrations of RNA were quantitated by absorbance at 260 nm (DU640 Spectrophotometer; Beckman Instruments, Fullerton, CA) and stored at -70°C until further use. Complementary DNA was synthesized from 1 µg of total cellular RNA using the SuperScript First-Strand Synthesis System for RT-PCR (Life Technologies).

Quantitation of mRNA Transcripts by Competitive Reverse Transcription Polymerase Chain Reaction

Competitive reverse transcription polymerase chain reaction (RT-PCR) was used to assess the abundance of P450scc, 3ß-HSD, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA transcripts in placental cells cocultured with increasing concentrations of CdCl2, as described previously [29, 30]. Oligonucleotide primers specific for human P450scc [31], 3ß-HSD [32], and GAPDH [33] were synthesized (Midland Certified Reagent Co., Midland, TX). Nucleotide sequences for the P450scc primers were as follows: 5' primer, 5'-GAAAGGAAGTGTTCACCACG-3' and 3' primer, 5'-TAGTGTCTCCTTGATGCTGG-3'. Sequences for the 3ß-HSD primers were as follows: 5' primer, 5'-GGAGACATTCTGGATGAG-3'; and 3' primer, 5'-GTCCTGCAGGGCCCTCAAGGCCAGAATGTG-3'. Sequences for the GAPDH primers were as follows: 5' primer, 5'-TGATGACATCAAGAAGGTGGTGAAG; and 3' primer, 5'-TCCTTGGAGGCCATGTAGGCCAT. The expected sizes of RT-PCR products for P450scc, 3ß-HSD, and GAPDH were 286 base pairs (bp), 545 bp, and 240 bp, respectively. The quantitation of RT-PCR products utilized the Competitive DNA Construction kit (Takara Shuzo Co., Ltd., Otsu, Japan) as previously reported [30]. With this method, one set of primers is used to amplify both the target gene cDNA and competitor DNA, a nonhomologous DNA fragment engineered to contain specific target gene sequences necessary for recognition by the gene-specific primers for the gene of interest [34]. Therefore, the competitor DNA effectively competes with the target cDNA for the same primers. RT-PCR was carried out with 2 µl of both cDNA and competitor DNA according to the GeneAmp PCR Reagent kit protocol (Perkin-Elmer/Cetus, Norwalk, CT). PCR conditions used for P450scc were 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 90 sec. Conditions for 3ß-HSD were 30 cycles of denaturation at 94°C for 60 sec, annealing at 54°C for 60 sec, and extension at 72°C for 90 sec. Conditions for GAPDH were 25 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 60 sec. PCR products were electrophoresed on 2% agarose gels, and band intensities were quantitated using the Alpha Imager 2000 digital analysis system (Alpha Innotech, San Leandro, CA). PCR reactions were accompanied by controls, including GAPDH as a positive cDNA synthesis control and RNA that had not been transcribed into cDNA as a genomic DNA contamination control.

Cyclic AMP Quantitation

Trophoblast cells were cultured in DMEM + 10% FBS for 72 h at 37°C and then incubated in DMEM + 10% FBS with 0, 5, 10, or 20 µM CdCl2 for 30 min. The content of total cellular cAMP in cultured trophoblast cells was measured using the Biotrak Cellular Communication Assay kit (125I-cAMP; Amersham Pharmacia Biotech, Buckinghamshire, U.K.), according to the manufacturer's instructions.

Statistical Analysis

The results of reported laboratory analyses (hormone concentrations, mRNA abundances, etc.) reflect determinations from multiple cultures. Therefore, results represent several cultured human placentae (n = 4 or 5), not simply replicated wells from a single placenta. Data were expressed as means ± SEM and were analyzed by ANOVA or Friedman repeated measures ANOVA on ranks, followed by a Dunnett test or a Student-Newman-Keuls multiple comparison test. SigmaStat statistical software was employed (SPSS, Richmond, CA). Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Viability

As reported in Table 1, coculture with CdCl2 for 96 h did not affect (P > 0.05) the protein content of cultured human trophoblasts, although a slight decline with increasing dosage might be the result of enhanced cell detachment from culture well surfaces. Cell viability was further assessed by quantitation of LDH activity in medium from trophoblasts cocultured with increasing concentrations of CdCl2. No differences in LDH activity were observed between untreated trophoblasts and those treated with 5, 10, or 20 µM CdCl2. No apparent differences were observed in mRNA transcript abundance for the housekeeping gene GAPDH in trophoblasts treated with 0 and 20 µM CdCl2.


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  		TABLE 1. Cellular protein, LDH activity and GAPDH mRNA transcript abundance in cultured human trophoblasts cocultured with 0, 5, 10, 20 µM CdCl2 for 96 h

P450scc and 3ß-HSD mRNAs

Quantitative competitive RT-PCR was performed to determine the effect of CdCl2 on the transcript abundance of P450scc and 3ß-HSD mRNAs in human trophoblasts cultured for 96 h. The expression of P450scc (Fig. 1A) and 3ß-HSD (Fig. 1B) mRNA transcripts declined (P < 0.05) in a dose-dependent manner in trophoblasts cocultured with 5, 10, or 20 µM CdCl2. The abundance of P450scc and 3ß-HSD mRNAs decreased (P < 0.05) approximately 93% and 65%, respectively, in trophoblasts cocultured with 20 µM CdCl2.



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  		FIG. 1. Effects of CdCl2 on P450scc (A) and 3ß-HSD (B) mRNA transcript abundance, as determined by competitive RT-PCR, in human trophoblasts cultured for 96 h. Values represent means ± SEM of 4 separate placental cultures. Different lowercase letters (a, b) indicate significant (P < 0.05) differences

P450scc and 3ß-HSD Enzyme Activities

To determine whether the decline in the abundance of mRNA transcripts for both P450scc and 3ß-HSD in cells cocultured with CdCl2 reflects an inhibition of the activities of both enzymes, 25-OHC and pregnenolone were provided as substrates. Progesterone synthesis (i.e., P450scc activity), as determined following the addition of 25-OHC over a 4-h incubation period, increased (P < 0.05) approximately 220% in cultured human trophoblasts (Fig. 2). Treatment with 20 µM CdCl2 inhibited 25-OHC-stimulated progesterone synthesis by approximately 81% (P < 0.05).



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  		FIG. 2. Effect of CdCl2 on P450scc activity, as determined by the formation of progesterone from 25-OHC, in cultured human trophoblasts. Trophoblasts were incubated for 4 h (96–100 h in culture) with DMEM + 10% steroid-stripped FBS in the absence or presence of 20 µM CdCl2 and/or 20 µg/ml 25-OHC. Values represent the means ± SEM of 4 separate placental cultures. Different lowercase letters (a, b, c) indicate significant (P < 0.05) differences

Basal progesterone production (18.8 ± 2.2 ng h-1 mg-1 cell protein) was inhibited (P < 0.05) by the addition of 90 µM aminoglutethimide (Fig. 3). Following the addition of 5 µg/ml pregnenolone over a 4-h incubation period, a dramatic (approximately 500-fold) increase was observed in progesterone synthesis (i.e., 3ß-HSD activity) in cultured human trophoblasts coincubated with 90 µM aminoglutethimide. However, coculture with CdCl2 did not significantly affect pregnenolone-stimulated progesterone synthesis.



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  		FIG. 3. Effect of CdCl2 on 3ß-HSD activity, as determined by the formation of progesterone from pregnenolone, in cultured human trophoblasts. Trophoblasts were incubated for 4 h (96–100 h in culture) with DMEM + 10% steroid-stripped FBS and 90 µM aminoglutethimide in the absence or presence of CdCl2 and/or 5 µg/ml pregnenolone. Values represent the means ± SEM of 5 separate placental cultures. Different lowercase letters (a, b, c) indicate significant (P < 0.05) differences

8-Bromo-cAMP-Stimulated Progesterone Secretion

Progesterone secretion from cultured trophoblasts increased (P < 0.05) almost 250% over that from untreated controls after coculture with 1 mM 8-bromo-cAMP for 48 h (Fig. 4). However, treatment with 8-bromo-cAMP was not efficacious in counteracting the decline (P < 0.05) in progesterone secretion elicited by coculture with 20 µM CdCl2.



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  		FIG. 4. Effect of 8-bromo-cAMP on CdCl2-inhibited progesterone secretion by cultured human trophoblasts. Trophoblasts were incubated for 48 h (96–144 h in culture) with DMEM + 10% FBS in the absence or presence of 20 µM CdCl2 and/or 1 mM 8-bromo-cAMP. Medium was changed at 24-h intervals. Values represent the means ± SEM of 4 separate placental cultures. Different lowercase letters (a, b, c) indicate significant (P < 0.05) differences

Cyclic AMP Content in Cultured Trophoblasts

The effect of coculture with CdCl2 on total cellular cAMP content in cultured human trophoblasts is shown in Figure 5. Treatment with 5, 10, or 20 µM CdCl2 over a 30-min incubation period did not significantly affect cAMP content. In preliminary experiments, coculture with 5, 10, or 20 µM CdCl2 for 24 h also did not affect cAMP content (data not shown).



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  		FIG. 5. Effect of CdCl2 on cAMP content in cultured human trophoblasts. Trophoblasts were incubated for 72 h in DMEM + 10% FBS and for 30 min with DMEM + 10% FBS in the presence of 5, 10, or 20 µM CdCl2. Values represent the means ± SEM of 3 separate placental cultures


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, cytotrophoblasts differentiated into endocrinologically active syncytiotrophoblasts, as previously demonstrated [15–17, 20, 21, 35]. We previously reported that progesterone production in cultured human trophoblasts was reduced after coculturing with CdCl2, either prior to or during differentiation of cytotrophoblasts into syncytiotrophoblasts or following completion of the differentiation process [15, 16]. In the current study, cytotrophoblasts progressed to syncytiotrophoblastic maturity regardless of treatment, but treatment with CdCl2 following differentiation inhibited 25-OHC-stimulated progesterone secretion. These results suggest that Cd does not interfere with progesterone synthesis/secretion by simply inhibiting morphological change but rather by exerting a direct inhibitory effect on the progesterone biosynthetic pathway. Moreover, no significant differences were observed in cell protein or LDH activity between untreated trophoblasts and those treated with CdCl2, a finding similar to that in a previous report of no significant decline in cell viability with Cd treatment, as assessed by both DNA fragmentation assay and the conversion of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide to formazan [17]. These results suggest that the suppression of progesterone production in treated cultures cannot be attributed to cell death by either apoptosis or necrosis.

P450scc activity, as determined by the formation of progesterone from 25-OHC, was inhibited in cells cocultured with CdCl2, indicating that P450scc enzyme is one site at which Cd may interfere with placental progesterone production. The formation of progesterone from 25-OHC did not reflect P450scc activity directly; its biosynthesis also included the conversion of pregnenolone to progesterone by 3ß-HSD. However, Cd also did not affect pregnenolone-stimulated progesterone secretion in cells treated with 20 µM CdCl2, and treatment with 5 µg pregnenolone precipitated a 500-fold increase in cellular progesterone secretion regardless of CdCl2 treatment. These results suggest that 20 µM CdCl2 did not significantly inhibit 3ß-HSD activity in human trophoblasts.

We recently reported that Cd inhibited the abundance of LDL-R mRNA transcripts in cultured human placental cells in a dose-dependent manner [17]. Because LDL-R is responsible for the uptake of cholesterol by trophoblast cells and is the initial step in progesterone biosynthesis, it may be one of the principal sites at which Cd interferes with placental progesterone production. Another important step in progesterone production is the conversion of cholesterol to pregnenolone via P450scc. In the present study, 25-OHC-stimulated progesterone production, although independent of cholesterol uptake by LDL-R, was inhibited in cells cocultured with CdCl2. These findings strongly suggest that Cd also interferes with progesterone synthesis via a direct inhibition of P450scc activity in cultured human trophoblasts.

Although posttranscriptional regulation might also be at work, the apparent dose-dependent reduction in the abundance of P450scc mRNA transcripts in response to Cd treatment suggested an effect on transcriptional regulation. In this capacity, the hormonal regulation and developmental pattern of P450scc expression are specific to individual steroidogenic tissues. In each case, hormone receptor binding activates a G protein that increases intracellular cAMP, which in turn increases P450scc gene transcription [36, 37]. Ringler et al. [38] reported that cAMP regulates progesterone production in normal human trophoblasts at least in part by regulating the abundance of mRNA transcripts encoding P450scc. Therefore, the regulation of human placental P450scc is considered to occur mainly at the transcriptional level, and Cd may inhibit progesterone production by human trophoblasts via direct interference with P450scc transcription.

Although a decline in 3ß-HSD mRNA transcripts was also observed following coculture with Cd, 3ß-HSD activity, which is abundant in human trophoblasts [39], was not significantly affected. Therefore, perhaps because of the high concentration of 3ß-HSD within human trophoblasts, coculture with the CdCl2 concentrations tested in the present study had no significant effects on activity. The 3ß-HSD protein may be regulated at both transcriptional and posttranscriptional levels. Similarly, treatment of human trophoblasts with progesterone and estradiol increased 3ß-HSD mRNA levels but had no significant effect on protein levels [40], suggesting that 3ß-HSD steady-state mRNA levels in human placenta could be under posttranscriptional regulation. A similar mechanism has been suggested in respect to FSH-induced gene expression in rat granulosa cells [41]. Therefore, Cd may exert a direct effect on 3ß-HSD transcript abundance but not on a posttranscriptional regulatory mechanism.

Others have proposed that the accumulation of P450scc mRNA is mainly controlled by the cAMP-dependent pathway in human trophoblasts [38, 42]. Thus, the human P450scc gene promoter contains consensus sequences that match known positive cAMP-responsive elements [43, 44]. However, in the present study, the cAMP analogue 8-bromo-cAMP was not effective in blunting the decline in progesterone secretion elicited by Cd treatment. In addition, coculture with CdCl2 did not influence the cAMP content in cultured cells, suggesting that cAMP in human trophoblasts may not be involved with the Cd-induced inhibition of progesterone secretion. The possibility still exists, however, that Cd interferes with the downstream cascade of cAMP-protein kinase A-dependent pathway. Kostrzewska and Sobieszek [45] reported that higher Cd concentrations inhibited the phosphorylation of myosin light-chain kinase in the smooth muscle myosin, suggesting that Cd interferes with the phosphorylation of protein kinases. An additional possibility is that the metal may directly affect transcription of P450scc by interfering with the DNA binding zinc finger motif through the substitution of Cd2+ for Zn2+ [46]. Moreover, cations such as Cd2+ have been reported to alter the structure of nucleic acids (DNA, tRNA) and certain enzymes by reacting with their sulfydryl groups [47, 48]. Therefore, one or more mechanisms may allow Cd to interfere with progesterone biosynthesis via a direct effect on the transcriptional machinery for P450scc.

Results of the present study suggest that P450scc, the enzyme responsible for the conversion of cholesterol into pregnenolone, is one site at which Cd interferes with progesterone production in cultured human trophoblasts. The potential for such a specific effect on placental progesterone production by Cd is further supported by recent results from Piasek et al. [49], who determined an almost two-fold increase in Cd, which was commensurate with an almost 50% decline in progesterone, in placentae of women who smoked during pregnancy. In light of the accumulated evidence, therefore, further study is needed to definitively determine the collective mechanisms by which Cd interferes with placental progesterone biosynthesis and to better understand the ramifications of this interference with respect to smoking, environmental exposure, normal placental function, and the maintenance of human pregnancy.


   ACKNOWLEDGMENTS
 
The authors express their sincere gratitude to Dr. April G. O'Quinn and the faculty, attending physicians, and staff of the Department of Obstetrics and Gynecology for their help in obtaining placental tissue and are extremely grateful for the general support of the department for this study. Appreciation is also extended to Shionogi & Co., Ltd. for their support and Mrs. Nathlynn Dellande for her assistance in manuscript preparation.


   FOOTNOTES
 
First decision: 11 November 2001.

1 Correspondence: Michael C. Henson, Department of Obstetrics and Gynecology, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112-2699. FAX: 504 584 1846;michael.henson{at}tulane.edu Back

Accepted: January 31, 2002.

Received: October 25, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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