Bromodichloromethane Inhibits Human Placental Trophoblast Differentiation

Jiangang Chen*, Twanda L. Thirkill*,{dagger}, Peter N. Lohstroh*, Susan R. Bielmeier{ddagger}, Michael G. Narotsky§, Deborah S. Best§, Randy A. Harrison, Kala Natarajan*, Rex A. Pegram, James W. Overstreet*, Bill L. Lasley* and Gordon C. Douglas*,{dagger},1

* Center for Health and the Environment, and {dagger} Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616; {ddagger} Curriculum of Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599; and § Reproductive Toxicology Division and Environmental Toxicology Division, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

*

Received September 16, 2003; accepted November 19, 2003

ABSTRACT

Epidemiological data suggest an association between exposures to bromodichloromethane (BDCM), a trihalomethane found in drinking water as a result of drinking water disinfection, and an increased risk of spontaneous abortion. We previously hypothesized that BDCM targets the placenta and showed that the secretion of chorionic gonadotrophin (CG) was reduced in primary cultures of human term syncytiotrophoblasts exposed to BDCM. In the present study we extend this observation by evaluating the effects of BDCM on the morphological differentiation of mononucleated cytotrophoblast cells to multinucleated syncytiotrophoblast-like colonies. Addition of BDCM to cytotrophoblast cultures inhibited the subsequent formation of multinucleated colonies in a dose-dependent manner, as determined by immunocytochemical staining for desmosomes and nuclei. The effect was seen at BDCM concentrations between 0.02 and 2 mM and was confirmed by quantitative image analysis. Secretion of bioactive and immunoreactive chorionic gonadotropin was also significantly inhibited in a dose-dependent manner under these culture conditions, and cellular levels of CG were also reduced. Trophoblast viability was not compromised by exposure to BDCM. We conclude that BDCM disrupts syncytiotrophoblast formation and inhibits CG secretion in vitro. Although other tissue targets are not ruled out, these data substantiate the idea that BDCM targets the placenta and could have implications for understanding the adverse pregnancy outcomes associated with BDCM exposure in humans.

Key Words: pregnancy; bioactivity; desmosomes; toxicity.

Bromodichloromethane (BDCM) is one of several trihalomethanes found in drinking water as a by-product of disinfection processes (Boorman, 1999Go; Deinzer, 1978Go; Dunnick and Melnick, 1993Go; Swan et al., 1998Go; Toussaint et al., 2001Go). Several adverse effects have been reported for trihalomethanes (including BDCM) based on animal and human studies. There is an association between human exposure to total trihalomethanes in drinking water and increased risk of cancer (Cantor et al., 1999Go; Hildesheim et al., 1998Go; King and Marrett, 1996Go; Villanueva et al., 2001Go). BDCM is also hepatotoxic and nephrotoxic in laboratory animal models (Aida et al., 1992Go; Chu et al., 1982Go; Keegan et al., 1998Go; Thornton-Manning et al., 1994Go; Torti et al., 2001Go).

Women exposed to trihalomethanes via drinking water show an increased risk of small-for-gestational-age (SGA) births, stillbirths, spontaneous abortions, and low birth weight (Bove et al., 1995Go; Dodds et al., 1999Go; Gallagher et al., 1998Go; Kramer et al., 1992Go; Savitz et al., 1995Go; Swan et al., 1998Go; Waller et al., 1998Go). In studies examining the concentration of the individual trihalomethanes, only BDCM exposure was associated with spontaneous abortion (Waller et al., 1998Go) and an increased risk of stillbirth (Dodds et al., 1999Go).

Halocarbon-associated pregnancy loss has been reported in animal studies (Bielmeier et al. 2004Go; Narotsky and Kavlock, 1995Go; Narotsky et al., 1997Go). Full litter resorption (FLR) was observed in F344 rats following BDCM administration by aqueous gavage (Bielmeier et al., 2001Go; Narotsky et al., 1997Go). BDCM-induced reductions in serum luteinizing hormone (LH), indicating an effect on the hypothalamic-pituitary axis resulting in reduced LH secretion, were observed (Bielmeier et al., 2004). Corpora lutea exposed to BDCM in vitro showed reduced responsiveness to chorionic gonadotropin (CG), suggesting a contributory role of the corpora lutea (Bielmeier et al., 2003Go). It is likely that these effects are strain specific, since FLR is not observed in BDCM-exposed Sprague-Dawley (SD) rats (Bielmeier et al., 2001Go; Christian et al., 2001Go; Ruddick et al., 1983Go).

To begin to understand the mechanism of BDCM-induced adverse effects on human pregnancy, we previously postulated that the placenta was a target of BDCM toxicity (Chen et al., 2003aGo). Results reported in that study showed that, when BDCM was added to primary human placental syncytiotrophoblast cultures, secretion of CG was reduced, consistent with the idea that the placenta could be a target in vivo.

The present study extends these in vitro observations and provides more information about the mechanism(s) of action of BDCM on trophoblasts. In the above study, BDCM was added to trophoblasts that had already differentiated into syncytiotrophoblast-like colonies. While this allowed us to investigate the effect of BDCM on CG secretion, the experimental protocol did not provide any information about direct effects of BDCM on the differentiation process itself. Here, we test the idea that BDCM disrupts trophoblast differentiation by adding BDCM to trophoblast cultures during the differentiation process and monitoring the formation of multinucleated syncytiotrophoblast and CG secretion. We have previously shown that the immunocytochemical localization of desmosomes and nuclei provides a convenient indicator of the status of trophoblast morphological differentiation (Douglas and King, 1990Go, 1993Go). Desmosomes are specialized structures that assemble at epithelial cell-cell junctions. The formation of multinucleated trophoblast colonies is believed to occur by cell-cell fusion and is accompanied by a rearrangement of desmosomal proteins and loss of cell-cell borders. The results show that BDCM inhibits trophoblast morphological differentiation and CG secretion in a dose-dependent manner.

MATERIALS AND METHODS

Trophoblast isolation and primary culture.
A detailed description of the procedure used to isolate cytotrophoblast cells from term human placentas has been given previously (Douglas and King, 1989Go). Briefly, the method described by Kliman et al. (1987)Go was modified by the substitution of a continuous Percoll gradient (yielding greater than 95% cytotrophoblast as assessed by intermediate filament immunocytochemistry) and the addition of a final step using immunomagnetic microspheres (Douglas and King, 1989Go) to remove the few remaining vimentin-positive and HLA-positive cells (yielding 100% cytotrophoblast). Absence of vimentin staining was confirmed for each batch of trophoblast cells.

Trophoblast cells do not replicate in culture and were plated in either 24-well cluster dishes or LabTek chamber slides at a density of 400,000 cells/cm2. In order to induce morphological differentiation and produce syncytiotrophoblast-like colonies, the cytotrophoblast cells were cultured in Keratinocyte Growth Medium (KGM; Clonetics Corporation, San Diego, CA) containing 10% fetal calf serum (FCS; Gemini Bio-Products, Woodland, CA). We have previously shown that this medium supports both morphological and biochemical differentiation of cytotrophoblast cells (Douglas and King, 1990Go, 1993Go; Ho et al., 1997aGo). Typically, after 48 h in KGM more than 90% of the culture consists of multinucleated syncytiotrophoblast-like colonies as determined by desmosomal and nuclear staining (Douglas and King, 1993Go). The cells also secrete bioactive CG (Ho et al., 1997aGo).

Exposure of trophoblast cultures to BDCM.
BDCM (98+% purity, stabilized with potassium carbonate, Aldrich Chemical Co. Milwaukee, WI) was prepared in KGM containing 10% FCS and stored at 4°C in amber vials with Teflon-silicon lined caps. (Fisher Scientific, Pittsburgh, PA).

Since our objective was to examine the effects of BDCM on trophoblast differentiation, BDCM was added to cytotrophoblasts that had not yet undergone any visible morphological differentiation. Undifferentiated, mononucleated cytotrophoblast cells were cultured in KGM (with FCS) for 16 h, after which BDCM was added (final target concentrations were 0.5, 20, 200, 0.02, 0.2, and 2 mM). At the time of BDCM addition, the cells had formed adherent colonies, but no multinucleated structures were present based on desmosomal protein immunocytochemistry (see below). The plates were sealed with plate seals and incubated at 37°C under differentiation-inducing conditions (i.e., in KGM) for an additional 48 h. After this time, 290 µl of the medium was collected and sealed in a nonsilanized headspace vial with a Teflon-silicon lined cap and kept at -20°C for determination of BDCM levels (see below). The remaining culture medium was stored at -20°C until used for determination of immunoreactive and bioactive CG levels. The adherent cells were processed for protein determination using a modified Lowry method (Bennett, 1982Go). Other identically treated trophoblast cultures were fixed and processed for morphological evaluation (see below). Additional cultures were exposed to BDCM as above and used for determination of lactate dehydrogenase (LDH) levels (see below).

CG immunoassay.
Levels of immunoreactive CG in culture media were determined by ELISA as previously described (O'Connor et al., 1988Go; Taylor et al., 1992Go). The primary capture antibody was clone B 109, which reacts with intact CG, and the second antibody was clone B 108, which reacts with ß-CG (Canfield et al., 1987Go; O'Connor et al., 1988Go). Immunoreactive CG is expressed as ng/mg cellular protein.

CG bioassay.
Bioactive CG was measured by in vitro bioassay as described previously (Chen et al., 2003bGo; Jia et al., 1991Go). Briefly, clonal human fetal kidney cells (cell line 293) were cotransfected with the LH/CG receptor and a luciferase reporter gene. Fifty microliters of hCG CR 127 standards (provided by R. Canfield, Columbia University, NY), internal controls, and experimental samples, were then added. At the end of the incubation (16 h), the cells were lysed, and the lysates were used for estimation of luciferase activity. LH/CG bioactivity was calculated by reference to a CR 127 standard curve and was expressed as ng/mg cellular protein.

LDH assay.
LDH activity in culture supernatants was determined using the fluorescence-based CytoTox-ONE kit obtained from Promega Corporation, Madison, WI 53711. LDH activity is expressed in arbitrary fluorescence units.

Assessment of trophoblast differentiation.
Differentiation was assessed by measuring the extent of multinucleated trophoblast formation at the end of the culture period (Douglas and King, 1990Go, 1993Go; Ho et al., 1997aGo). Trophoblast cultures (in eight-chamber LabTek slides) were incubated with or without BDCM and cultured under differentiation-inducing conditions as described above. The cells were fixed and permeabilized using ice-cold methanol, after which they were incubated in PBS/gelatin. Cell-cell borders and nuclei were revealed by staining cultures simultaneously with an anti-desmosomal protein antibody (Sigma Chemical Co, St. Louis, MO) (Douglas and King, 1990Go) and 7-aminoactinomycin D (7-AAD) (10 µg/ml) (Scholz et al., 1996Go). The primary anti-desmosomal protein antibody was detected using an Oregon Green-labeled secondary antibody. The slides were mounted using GVA aqueous mounting medium (Zymed, San Francisco, CA) and examined using a Nikon Eclipse E800 epifluorescence microscope. Images were captured using an Optronics DEI750 CCD camera and Adobe Photoshop software. Total numbers of nuclei from three random fields per well were counted using Image Pro software (Media Cybernetics, Silver Spring, MD). At least 150 nuclei were counted per field. Nuclei present as three or more nuclei in a single cell (cells were identified by desmosomal staining at cell-cell borders) were also counted and expressed as a percentage of total nuclei. Cells containing three or more nuclei were defined as multinucleated, and so the latter percentage provided a measure of the extent of multinucleated cell formation.

Immunocytochemical staining for CG.
Cultured adherent cells were fixed in methanol and then stained with a polyclonal antibody against the ß subunit of human CG (N1534; DAKO Corporation, Carpenteria, CA). The monoclonal antibody used for the CG ELISA (see above) was not used for immunocytochemistry because it was not reactive with fixed trophoblast cells. The primary antibody was detected using Oregon Green-labeled goat anti-rabbit Ig secondary antibodies. As a control, other cultures were incubated with a matched rabbit immunoglobulin instead of the primary antibody. Images were captured directly into Photoshop using identical exposure and brightness/contrast settings. Digitized images were analyzed using Image Pro software (Media Cybernetics, Silver Spring, MD). Relative fluorescence intensity was measured by reference to a fluorescence standard curve obtained using fluorescence calibration beads (Molecular Probes, Eugene, OR). Images from at least three random fields per well viewed using a 20x objective were analyzed from each experiment.

Statistical analysis.
All experiments were performed four times, and each experiment used cells obtained from different placentas. Cells were not pooled. Within an experiment, means were obtained from duplicate determinations. The immunoreactivity and bioactivity of hCG under different treatment conditions were compared using ANOVA with repeated measures followed by Student-Newman-Keuls comparison and linear trend post-tests with a significance level of 0.05. ANOVA with repeated measures on rank was performed if the normality assumption was not met. The data are presented as mean ± SEM. The analysis was performed using SPSS software and Prism Software.

RESULTS

Effect of BDCM on the Formation of Syncytiotrophoblast-Like Colonies in Vitro
As we have described in detail previously, cytotrophoblast cells show little morphological differentiation (i.e., desmosomal proteins are retained at cell-cell borders, and the cells remain mononucleated) when maintained in Ham's/Waymouth medium (HWM). However, when cultured in keratinocyte growth medium (KGM) cytotrophoblast cells give rise to multinucleated syncytiotrophoblast-like colonies (i.e., loss of intercellular desmosomal staining and appearance of multiple nuclei in a common cytoplasm). These differences can be seen in the nondifferentiated and differentiated control cultures shown in Figures 1A and 1B, respectively. Note the cobblestone appearance of the non-differentiated culture (maintained in HWM) and the loss of this pattern in the differentiated culture.



View larger version (63K):
[in this window]
[in a new window]
 
FIG. 1. Effect of BDCM on the organization of desmosomal proteins in trophoblast cultures. Cytotrophoblast cells were incubated with or without BDCM under differentiation-inducing conditions (i.e., in KGM) for 48 h and then stained to reveal nuclei (red fluorescence) and desmosomal proteins (green fluorescence) as described in Methods. (A) Control nondifferentiated cytotrophoblast colonies. (B) Control differentiated syncytiotrophoblast-like colonies. The images in the other panels show trophoblasts cultured under differentiation-inducing conditions in the presence of BDCM. The concentrations were 2.0 mM (C); 0.2 mM (D); 0.02 mM (E) and 0.5 nM (F). The images are representative of samples from three separate experiments (cells from three different placentas). The horizontal bar represents 50 µm.

 
Figure 1 also shows the effect of different concentrations of BDCM on the desmosomal/nuclear staining pattern. In contrast to the differentiated control cells (Fig. 1B), when cytotrophoblast cells were incubated in differentiation-inducing medium (KGM) for 48 h in the presence of 2 mM BDCM, there was no reorganization of desmosomal proteins, and the cobblestone desmosomal staining pattern was retained (Fig. 1C). These BDCM-treated cultures resembled control non-differentiated trophoblasts (see Fig. 1A). BDCM also prevented desmosomal protein reorganization when used at concentrations of 0.2 mM and 0.02 mM (Fig. 1D and E), although the effect was not as dramatic and some colonies did show loss of desmosomal protein staining. At 200 nM (not shown) and 0.5 nM BDCM (Fig. 1F), trophoblast cultures appeared similar to untreated differentiated control cultures (i.e., there was extensive re-organization of desmosomal proteins and loss of the cobblestone staining pattern).

To more objectively evaluate these visual impressions, the distribution of nuclei was determined in the stained cultures to provide information about the extent of multinucleated colony formation. Nuclei were counted, and those present as three or more in a common cytoplasm were scored. This was taken as an indicator of multinucleated cell formation. Figure 2 shows the extent of multinucleated cell formation in the presence or absence of BDCM. As we have reported before (Douglas and King, 1993Go; Ho et al., 1997aGo), more than 90% of the cells were multinucleated under differentiation-inducing conditions (KGM). For trophoblasts incubated under non-differentiation-inducing conditions (HWM), less than 20% of cells were multinucleated. For trophoblasts incubated under differentiation-inducing conditions in the presence of 2 mM BDCM, 30% of the cells were multinucleated. It can also be seen from Figure 2 that the number of nuclei sharing a common cytoplasm under differentiation-inducing conditions increased as the BDCM concentration was decreased. The lowest BDCM concentration showing a significant (p < 0.05) effect was 0.2 mM. Trend analysis showed a significant linear trend.



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 2. Effect of BDCM on the formation of multinucleated trophoblast colonies. Cytotrophoblast cells were incubated with or without BDCM under differentiation-inducing conditions for 48 h. The data indicated by 0(K) and 0(H) represent control cultures incubated in KGM or HWM, respectively, in the absence of BDCM. The cultures were then stained to reveal nuclei and desmosomal proteins as described in Methods and Figure 1. Fluorescence images were captured, and the total numbers of nuclei were counted in random fields. At least 150 nuclei were counted in each case. The extent of multinucleated cell (i.e., syncytiotrophoblast) formation was calculated as described in Methods. Results are means ± SEM. from three separate experiments. Asterisks indicate values significantly different (p < 0.05) from untreated controls. Post-ANOVA trend analysis indicates a significant linear trend.

 
Effect of BDCM on Secretion of Immunoreative and Bioactive CG
Since syncytiotrophoblast is the major source of CG, we examined the effect of BDCM on CG secretion during syncytiotrophoblast formation. BDCM was added to trophoblasts before differentiation had occurred, as described above. After 48 h, the culture supernatants were removed for assay of immunoreactive and bioactive CG. The results in Figure 3A show that exposure to BDCM caused a significant dose-dependent decrease in the secretion of immunoreactive CG at all concentrations tested. The highest concentration (2 mM) reduced the secretion of immunoreactive CG by 94%, whereas the lowest concentration tested (0.5 nM) reduced immunoreactive CG secretion by 30%. BDCM also reduced the levels of bioactive CG in culture supernatants from differentiated trophoblast in a dose-dependent manner (Fig. 3B). At 2 mM BDCM, secretion of bioactive CG was reduced by 95%. The lowest concentration showing a significant effect was 20 nM.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 3. Effect of BDCM on the secretion of immunoreactive and bioactive CG. Cytotrophoblasts were cultured for 48 h under differentiation-inducing conditions (i.e., in KGM) in the presence or absence of BDCM. The culture media were collected and analyzed for immunoreactive (A) and bioactive (B) CG as described in the Materials and Methods section. Results are means ± SEM from four separate experiments (four different placentas). The asterisks indicate values that are significantly different (p < 0.05) from the control. Post-ANOVA trend analysis indicates a significant linear trend for both datasets.

 
Immunocytochemical Staining for CG in Trophoblasts Treated with BDCM
Reduced CG secretion could result from disruption of the final stages of the secretory process, disruption of posttranslational processing, or disruption of protein synthesis. The former could be manifest as an accumulation of intracellular CG. We therefore used immunocytochemical staining to examine cellular expression of CG in trophoblasts exposed to BDCM. Trophoblasts were incubated in the presence or absence of BDCM in differentiation-inducing medium as described above. The cells were fixed and stained with a polyclonal antibody against the ß subunit of human CG. This antibody reacts with intact CG and free ß subunit. Control cultures incubated in non-differentiation-inducing medium showed a diffuse fluorescence pattern (Fig. 4A). As expected, control cultures incubated in differentiation-inducing medium showed a much brighter diffuse fluorescence (Fig. 4B). Trophoblast cultures incubated under differentiation-inducing conditions in the presence of 2 mM BDCM also showed a diffuse staining pattern (Fig. 4C), but the fluorescence appeared reduced and the intensity was similar to or less than that of the nondifferentiated control cells. Reduced CG-associated fluorescence was also seen in cultures exposed to BDCM at 0.2 mM and 0.02 mM (Figs. 4D and 4E), although at the latter concentration the fluorescence appeared greater than that at 0.2 mM. At 200 nM BDCM (Fig. 4F), fluorescence intensity appeared greater than at 0.02 mM, but was still less than untreated, differentiated control cultures. At 0.5 nM BDCM (Fig. 4G) fluorescence intensity appeared similar to untreated, differentiated control cultures (see Fig. 4B).



View larger version (115K):
[in this window]
[in a new window]
 
FIG. 4. Immunofluorescence localization of CG in trophoblasts exposed to BDCM. Cytotrophoblasts were cultured for 48 h under differentiation-inducing (KGM) conditions in the presence or absence of BDCM. The cultures were then fixed and stained using an anti-CG antibody as described in the Methods section. (A) Control cells incubated under non-differentiation-inducing conditions (B) Control cells incubated under differentiation-inducing conditions. The other images show cells incubated under differentiation-inducing conditions in the presence of different concentrations of BDCM. The concentrations used were 2.0 mM (C), 0.2 mM (D), 0.02 mM (E), 200 nM (F), and 0.5 nM (G). The last image (H) shows the immunoglobulin control. The horizontal bar represents 50 µm.

 
Visual inspection of these images suggested a dose-related decrease in CG immunofluorescence with increasing concentrations of BDCM. To substantiate this qualitative finding, photographic images from three separate experiments were subjected to computerized image analysis. The results of this analysis are shown in Figure 5. Fluorescence intensity values of colonies exposed to 0.02-2.0 mM BDCM were significantly lower than the control KGM cultures and post hoc analysis indicates a dose response with a significant linear trend.



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 5. Image analysis of CG immunofluorescence. Fluorescence micrographs from the experiment described in Figure 4 were imported into Image Pro software as described in Materials and Methods. Mean green fluorescence intensity values were obtained. Results are means ± SEMs from three separate experiments. At least 125 cell colonies were analyzed in each case. The data indicated by 0(K) and 0(H) represent control cultures incubated in KGM or HWM, respectively, in the absence of BDCM. The asterisks indicate values that are significantly different (p < 0.05) from the differentiated (K) control culture. ANOVA post-tests also showed a significant linear trend.

 
Effect of BDCM on Trophoblast Viability
Cellular protein levels were determined to assess potential BDCM-mediated cell loss/detachment (trophoblasts do not replicate in culture). Levels of cellular protein were not significantly different in BDCM-treated and untreated cultures (Fig. 6A). Potential cytotoxic effects of BDCM were also tested by measuring levels of LDH in culture supernatants following exposure to different concentrations of BDCM. The data in Figure 6B show that LDH activity in culture supernatants was unchanged following exposure of cells to BDCM for 48 h.



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 6. Effect of BDCM on trophoblast viability. Trophoblasts were cultured for 48 h under differentiation-inducing conditions in the presence or absence of BDCM. After removal of culture supernatants, the protein content of the adherent cells was measured (A). Culture supernatants were assayed for LDH activity (B) as described in Materials and Methods. Results are means ± SEM from three separate experiments. ANOVA showed no significant difference between means.

 
DISCUSSION

Difficulties in assessing exposures to trihalomethanes (THMs) in epidemiological studies can result in exposure misclassification biases that could produce significant underestimates of risk as well as attenuated exposure-response trends (Swan et al., 1998Go). Additional means of evaluating and characterizing the reproductive toxicity of these compounds are therefore desirable. The results reported here show that BDCM, a trihalomethane found in drinking water as the result of disinfection processes, directly inhibits the morphological differentiation of mononucleated placental cytotrophoblast cells to multinucleated syncytiotrophoblast-like colonies in vitro. Syncytiotrophoblast formation was inhibited in a dose-dependent manner and was accompanied by no loss of cell viability. Based on image analysis, the lowest concentration to significantly disrupt morphological differentiation was 200 mM. Peak levels of BDCM in human blood after showering range from 0.0013 nM to 0.57 nM (Miles et al., 2002Go). In a previous study (Chen et al., 2003aGo), we showed that BDCM reduced CG secretion by primary cultures of already-differentiated human syncytiotrophoblasts. In the present study, where BDCM was added to cytotrophoblast cultures prior to the onset of differentiation, we also found that BDCM significantly inhibited CG secretion by trophoblast cultures, but the degree of inhibition was much greater. In this study, bioactive CG secretion was reduced at 200 nM, and immunoreactive CG secretion was reduced at 0.5 nM, the lowest concentration tested; this concentration is within the range of BDCM levels found in human blood (Miles et al., 2002Go). Undifferentiated cytotrophoblast cultures produce low levels of CG, and we have previously shown that the formation of syncytiotrophoblast-like cultures from cytotrophoblast cells is accompanied by an increase in the secretion of bioactive CG (Ho et al., 1997aGo). The greater inhibition of CG secretion seen in the present study compared with the previous study is therefore consistent with the inhibition of syncytiotrophoblast formation. These findings substantiate and extend our previous conclusion that BDCM may target the human placenta, and trophoblasts in particular. Since these cells are the sole source of CG during normal human pregnancy and play a major role in the maintenance of the conceptus, a decrease in the amount of bioactive hormone could have adverse effects on pregnancy outcome. However, care must be applied in extrapolation of these in vitro data to the in vivo situation. While every precaution was taken to minimize loss of BDCM by volatilization, it is likely that actual concentrations were lower than the target dose. Similarly, the lack of detailed toxicokinetic data regarding BDCM levels in human blood, particularly with regards peak durations, makes direct comparison difficult. Nonetheless, the in vitro findings reported here are consistent with human epidemiological studies, which suggest increased incidence of spontaneous abortion (Waller et al., 1998Go) and stillbirth (Dodds et al., 1999Go) in women exposed to BDCM.

The previous observation that BDCM inhibited CG secretion when incubated with trophoblasts that had already differentiated suggested that BDCM was affecting CG synthesis and/or secretion. The present data indicate that BDCM also affects CG production by preventing the formation of the major CG-producing cell type. The greater effect of BDCM on CG secretion in the present study may be related to the compound having an effect on both syncytiotrophoblast formation and CG production. A dual effect of BDCM is also suggested by the different dose-response curves that were found for the effects of BDCM on morphological differentiation and CG secretion. The findings are consistent with the observation that CG itself may play an autocrine role in the induction of trophoblast differentiation (Cronier et al., 1994Go, 1995Go; Yang et al., 2003Go). Other compounds known to inhibit trophoblast morphological and biochemical differentiation include colchicine (Douglas and King, 1993Go) and dimethyl sulfoxide (Thirkill and Douglas, 1997Go).

Based on immunocytochemical staining, cellular levels of CG were also significantly reduced by BDCM. The fact that cellular levels were reduced and that there was no intracellular accumulation of CG suggest that production is blocked at earlier stages (transcription or translation) rather than at later stages (exocytosis). However, effects of BDCM on posttranslational CG processing (glycosylation or subunit dimerization) cannot be ruled out. Another possibility is that BDCM affects CG secretion by blocking gonadotropin releasing hormone (GnRH) activity. It is known that human trophoblasts express a GnRH receptor and secrete GnRH (Cheng et al., 2000Go; Lin et al., 1995Go; Wolfahrt et al., 1998Go). If an effect of BDCM on GnRH is demonstrated, this would parallel a mechanism that has been proposed for BDCM-induced pregnancy loss in rodents (Bielmeier et al., 2004Go) (see discussion below).

Given the importance of syncytiotrophoblast in hormone production and nutrient transport in the human, disruption of differentiation could have important implications on maintaining a healthy pregnancy. Thus, the increased incidence of spontaneous abortion (Waller et al., 1998Go) and stillbirth (Dodds et al., 1999Go) in women exposed to BDCM could be related to inhibitory effects of BDCM on trophoblast differentiation. Trophoblast differentiation is a complex process that is poorly understood. Mononucleated cytotrophoblast cells, which produce CG alpha subunits and little or no beta subunit, are believed to fuse, giving rise to multinucleated syncytiotrophoblasts (Enders, 1965Go; Hoshina et al., 1985Go; Kliman et al., 1986Go; Midgley et al., 1963Go). Differentiation likely involves the interplay of several factors that include EGF, cAMP, TGFb, and microtubules (Dakour et al., 1999Go; Douglas and King, 1993Go; Morrish et al., 1987Go, 1991Go, 1998Go, 1996Go). The use of subtraction cDNA libraries and microarray technology has identified many genes whose expression changes during trophoblast differentiation (Aronow et al., 2001Go; Morrish et al., 1996Go). In most cases it is unclear whether the changes in expression reflect cause or effect. BDCM could disrupt trophoblast differentiation at one or more steps, and further studies will be required to elucidate the mechanism. Regardless of the mechanism, the in vitro observations reported here are consistent with earlier in vivo studies which suggest that a decrease in the secretion of bioactive CG is associated with early pregnancy loss (Guo et al., 1999Go; Ho et al., 1997bGo).

Although the current findings indicate that BDCM may target the placenta in humans, they do not rule out other target tissues. Indeed, BDCM-induced pregnancy loss in F344 rats involves both the hypothalamic-pituitary axis (Bielmeier et al., 2004Go) and the corpus luteum (Bielmeier et al., 2003Go). Given the many similarities between human and nonhuman primates with regards reproductive physiology, in vivo studies using a nonhuman primate model would provide more information about the mode of action of BDCM during human pregnancy.

ACKNOWLEDGMENTS

This work was supported by NIH Grants ES06198, ES05707, and P42ES04699. Placental tissue was made available to us through the help and cooperation of the medical and nursing staff at Sutter Memorial Hospital, Sacramento, CA.

NOTES

Disclaimer: The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

REFERENCES

Aida, Y., Takada, K., Uchida, O., Yasuhara, K., Kurokawa, Y., and Tobe, M. (1992). Toxicities of microencapsulated tribromomethane, dibromochloromethane and bromodichloromethane administered in the diet to Wistar rats for one month. J. Toxicol. Sci. 17, 119–133.[Medline]

Aronow, B. J., Richardson, B. D., and Handwerger, S. (2001). Microarray analysis of trophoblast differentiation: Gene expression reprogramming in key gene function categories. Physiol. Genomics 6, 105–116.[Abstract/Free Full Text]

Bennett, J. P. (1982). Solubilization of membrane-bound enzymes and analysis of membrane protein concentration. In Techniques in Lipid and Membrane Biochemistry (T. R. Hesketh, H. L. Kornberg, J. C. Metcalfe, D. H. Northcote, C. I. Pogson, and K. F. Tipton, Eds.), pp. 1–22. Elsevier, Amsterdam.

Bielmeier, S. R., Best, D. S., Guidici, D. L., and Narotsky, M. G. (2001). Pregnancy loss in the rat caused by bromodichloromethane. Toxicol. Sci. 59, 309–315.[Abstract/Free Full Text]

Bielmeier, S. R., Best, D. S., and Narotsky, M. G. (2004). Serum hormone characterization and exogenous hormone rescue of bromodichloromethane-induced pregnancy loss in the F344 rat. Toxicol Sci. (in press).

Bielmeier, S. R., Murr, A. S., Best, D. S., Goldman, J. M., and Narotsky, M. G. (2003). Effects of bromodichloromethane (BDCM) on ex vivo luteal function in the pregnant F344 rat. Toxicologist 72(S-1), 26–27.

Boorman, G. A. (1999). Drinking water disinfection byproducts: review and approach to toxicity evaluation. Environ. Health Perspect. 107(Suppl. 1), 207–217.[ISI][Medline]

Bove, F. J., Fulcomer, M. C., Klotz, J. B., Esmart, J., Dufficy, E. M., and Savrin, J. E. (1995). Public drinking water contamination and birth outcomes. Am. J. Epidemiol. 141, 850–862.[Abstract]

Canfield, R. E., O'Connor, J. F., Birken, S., Krichevsky, A., and Wilcox, A. J. (1987). Development of an assay for a biomarker of pregnancy and early fetal loss. Environ. Health Perspect. 74, 57–66.[ISI][Medline]

Cantor, K. P., Lynch, C. F., Hildesheim, M. E., Dosemeci, M., Lubin, J., Alavanja, M., and Craun, G. (1999). Drinking water source and chlorination byproducts in Iowa. III. Risk of brain cancer. Am. J. Epidemiol. 150, 552–560.[Abstract]

Chen, J., Douglas, G. C., Thirkill, T. L., Lohstroh, P. N., Bielmeier, S. R., Narotsky, M. G., Best, D. S., Harrison, R. A., Natarajan, K., Pegram, R. A., et al. (2003a). Effect of bromodichloromethane on chorionic gonadotrophin secretion by human placental trophoblast cultures. Toxicol. Sci. 76, 75–82.[Abstract/Free Full Text]

Chen, J., Thirkill, T. L., Overstreet, J. W., Lasley, B. L., and Douglas, G. C. (2003b). Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on chorionic gonadotropin secretion by human trophoblasts. Reprod. Toxicol. 17, 87–93.[CrossRef][ISI][Medline]

Cheng, K. W., Nathwani, P. S., and Leung, P. C. (2000). Regulation of human gonadotropin-releasing hormone receptor gene expression in placental cells. Endocrinology 141, 2340–2349.[Abstract/Free Full Text]

Christian, M. S., York, R. G., Hoberman, A. M., Diener, R. M., and Fisher, L. C. (2001). Oral (drinking water) developmental toxicity studies of bromodichloromethane (BDCM) in rats and rabbits. Int. J. Toxicol. 20, 225–237.[CrossRef][ISI][Medline]

Chu, I., Villeneuve, D. C., Secours, V. E., Becking, G. C., and Valli, V. E. (1982). Trihalomethanes: II. Reversibility of toxicological changes produced by chloroform, bromodichloromethane, chlorodibromomethane and bromoform in rats. J. Environ. Sci. Health B 17, 225–240.[ISI][Medline]

Cronier, L., Bastide, B., Herve, J. C., Deleze, J., and Malassine, A. (1994). Gap junctional communication during human trophoblast differentiation: influence of human chorionic gonadotropin. Endocrinology 135, 402–408.[Abstract]

Cronier, L., Bois, P., Herve, J. C., and Malassine, A. (1995). Effect of human chorionic gonadotrophin on chloride current in human syncytiotrophoblasts in culture. Placenta 16, 599–609.[ISI][Medline]

Dakour, J., Li, H., Chen, H., and Morrish, D. W. (1999). EGF promotes development of a differentiated trophoblast phenotype having c-myc and junB proto-oncognene activation. Placenta 20, 119–126.[CrossRef][ISI][Medline]

Deinzer, M. S., Schaumburg, F., Klein, E. (1978). Environmental Health Sciences Center Task Force review on halogenated organics in drinking water. Environ. Health Perspect. 24, 209–239. [ISI]

Dodds, L., King, W., Woolcott, C., and Pole, J. (1999). Trihalomethanes in public water supplies and adverse birth outcomes. Epidemiology 10, 233–237.[ISI][Medline]

Douglas, G. C., and King, B. F. (1989). Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres. J. Immunol. Methods 119, 259–268.[CrossRef][ISI][Medline]

Douglas, G. C., and King, B. F. (1990). Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei. J. Cell Sci. 96, 131–141.[Abstract]

Douglas, G. C., and King, B. F. (1993). Colchicine inhibits human trophoblast differentiation in vitro. Placenta 14, 187–201.[ISI][Medline]

Dunnick, J. K., and Melnick, R. L. (1993). Assessment of the carcinogenic potential of chlorinated water: experimental studies of chlorine, chloramine, and trihalomethanes. J. Natl. Cancer Inst. 85, 817–822.[Abstract]

Enders, A. C. (1965). Formation of syncytium from cytotrophoblast in the human placenta. Obstet. Gynecol. 25, 378–386.[ISI][Medline]

Gallagher, M. D., Nuckols, J. R., Stallones, L., and Savitz, D. A. (1998). Exposure to trihalomethanes and adverse pregnancy outcomes. Epidemiology 9, 484–489. [ISI][Medline]

Guo, Y., Hendrickx, A. G., Overstreet, J. W., Dieter, J., Stewart, D., Tarantal, A. F., Laughlin, L., and Lasley, B. L. (1999). Endocrine biomarkers of early fetal loss in cynomolgus macaques (Macaca fascicularis) following exposure to dioxin. Biol. Reprod. 60, 707–713. [Abstract/Free Full Text]

Hildesheim, M. E., Cantor, K. P., Lynch, C. F., Dosemeci, M., Lubin, J., Alavanja, M., and Craun, G. (1998). Drinking water source and chlorination byproducts. II. Risk of colon and rectal cancers. Epidemiology 9, 29–35.[ISI][Medline]

Ho, H. H., Douglas, G. C., Qiu, Q. F., Thirkill, T. L., Overstreet, J. W., and Lasley, B. L. (1997a). The relationship between trophoblast differentiation and the production of bioactive hCG. Early Pregnancy 3, 291–300. [Medline]

Ho, H. H., O'Connor, J. F., Nakajima, S. T., Tieu, J., Overstreet, J. W., and Lasley, B. L. (1997b). Characterization of human chorionic gonadotropin in normal and abnormal pregnancies. Early Pregnancy 3, 213–224.[Medline]

Hoshina, M., Boothby, M., Hussa, R., Pattillo, R., Camel, H. M., and Boime, I. (1985). Linkage of human chorionic gonadotrophin and placental lactogen biosynthesis to trophoblast differentiation and tumorigenesis. Placenta 6, 163–172.[ISI][Medline]

Jia, X. C., Oikawa, M., Bo, M., Tanaka, T., Ny, T., Boime, I., and Hsueh, A. J. (1991). Expression of human luteinizing hormone (LH) receptor: Interaction with LH and chorionic gonadotropin from human but not equine, rat, and ovine species. Mol. Endocrinol. 5, 759–768.[Abstract]

Keegan, T. E., Simmons, J. E., and Pegram, R. A. (1998). NOAEL and LOAEL determinations of acute hepatotoxicity for chloroform and bromodichloromethane delivered in an aqueous vehicle to F344 rats. J. Toxicol. Environ. Health A 55, 65–75.[CrossRef][ISI][Medline]

King, W. D., and Marrett, L. D. (1996). Case-control study of bladder cancer and chlorination by-products in treated water (Ontario, Canada). Cancer Causes Control 7, 596–604.[ISI][Medline]

Kliman, H. J., Feinman, M. A., and Strauss, J. F., III (1987). Differentiation of human cytotrophoblast to syncytiotrophoblast in culture. Trophobl. Res. 2, 407–421.

Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., and Strauss, J. F., III (1986). Purification, characterization and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118, 1567–1582.[Abstract]

Kramer, M. D., Lynch, C. F., Isacson, P., and Hanson, J. W. (1992). The association of waterborne chloroform with intrauterine growth retardation. Epidemiology 3, 407–413.[ISI][Medline]

Lin, L. S., Roberts, V. J., and Yen, S. S. (1995). Expression of human gonadotropin-releasing hormone receptor gene in the placenta and its functional relationship to human chorionic gonadotropin secretion. J. Clin. Endocrinol. Metab. 80, 580–585.[Abstract]

Midgley, A. R., Pierce, G. B., Deneau, G. A., and Gosling, J. R. G. (1963). Morphogenesis of syncytiotrophoblast in vivo: An autoradiographic demonstration. Science 141, 349–350. [ISI][Medline]

Miles, A. M., Singer, P. C., Ashley, D. L., Lynberg, M. C., Mendola, P., Langlois, P. H., and Nuckols, J. R. (2002). Comparison of trihalomethanes in tap water and blood. Environ. Sci. Technol. 36, 1692–1698.[CrossRef][ISI][Medline]

Morrish, D. W., Bhardwaj, D., Dabbagh, L. K., Marusyk, H., and Siy, O. (1987). Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta. J. Clin. Endocrinol. Metab. 65, 1282–1290.[Abstract]

Morrish, D. W., Bhardwaj, D., and Paras, M. T. (1991). Transforming growth factor {alpha}1 inhibits placental differentiation and human chorionic gonadotropin and human placental lactogen secretion. Endocrinology 129, 22–26.[Abstract]

Morrish, D. W., Dakour, J., and Li, H. (1998). Functional regulation of human trophoblast differentiation. J. Reprod. Immunol. 39, 179–195.[CrossRef][ISI][Medline]

Morrish, D. W., Linetsky, E., Bhardwaj, D., Li, H., Dakour, J., Marsh, R. G., Paterson, M. C., and Godbout, R. (1996). Identification by subtractive hybridization of a spectrum of novel and unexpected genes associated with in vitro differentiation of human cytotrophoblast cells. Placenta 17, 431–441.[ISI][Medline]

Narotsky, M. G., and Kavlock, R. J. (1995). A multidisciplinary approach to toxicological screening: II. Developmental toxicity. J. Toxicol. Environ. Health 45, 145–171.[ISI][Medline]

Narotsky, M. G., Pegram, R. A., and Kavlock, R. J. (1997). Effect of dosing vehicle on the developmental toxicity of bromodichloromethane and carbon tetrachloride in rats. Fundam. Appl. Toxicol. 40, 30–36.[CrossRef][ISI][Medline]

O'Connor, J. F., Schlatterer, J. P., Birken, S., Krichevsky, A., Armstrong, E. G., McMahon, D., and Canfield, R. E. (1988). Development of highly sensitive immunoassays to measure human chorionic gonadotropin, its beta-subunit, and beta core fragment in the urine: application to malignancies. Cancer Res. 48, 1361–1366.[Abstract]

Ruddick, J. A., Villeneuve, D. C., Chu, I., and Valli, V. E. (1983). A teratological assessment of four trihalomethanes in the rat. J. Environ. Sci. Health B 18, 333–349. [ISI][Medline]

Savitz, D. A., Andrews, K. W., and Pastore, L. M. (1995). Drinking water and pregnancy outcome in central North Carolina: source, amount, and trihalomethane levels. Environ. Health Perspect. 103, 592–596.[ISI][Medline]

Scholz, D., Devaux, B., Hirche, A., Potzsch, B., Kropp, B., Schaper, W., and Schaper, J. (1996). Expression of adhesion molecules is specific and time-dependent in cytokine-stimulated endothelial cells in culture. Cell Tissue Res. 284, 415–423. [CrossRef][ISI][Medline]

Swan, S. H., Waller, K., Hopkins, B., Windham, G., Fenster, L., Schaefer, C., and Neutra, R. R. (1998). A prospective study of spontaneous abortion: Relation to amount and source of drinking water consumed in early pregnancy. Epidemiology 9, 126–133.[ISI][Medline]

Taylor, C. A., Jr., Overstreet, J. W., Samuels, S. J., Boyers, S. P., Canfield, R. E., O'Connor, J. F., Hanson, F. W., and Lasley, B. L. (1992). Prospective assessment of early fetal loss using an immunoenzymometric screening assay for detection of urinary human chorionic gonadotropin. Fertil. Steril. 57, 1220–1224.[ISI][Medline]

Thirkill, T. L., and Douglas, G. C. (1997). Differentiation of human trophoblast cells in vitro is inhibited by dimethyl sulfoxide. J. Cell. Biochem. 65, 460–468.[CrossRef][ISI][Medline]

Thornton-Manning, J. R., Seely, J. C., and Pegram, R. A. (1994). Toxicity of bromodichloromethane in female rats and mice after repeated oral dosing. Toxicology 94, 3–18.[CrossRef][ISI][Medline]

Torti, V. R., Cobb, A. J., Everitt, J. I., Marshall, M. W., Boorman, G. A., and Butterworth, B. E. (2001). Nephrotoxicity and hepatotoxicity induced by inhaled bromodichloromethane in wild-type and p53-heterozygous mice. Toxicol. Sci. 64, 269–280.[Abstract/Free Full Text]

Toussaint, M. W., Rosencrance, A. B., Brennan, L. M., Dennis, W. E., Beaman, J. R., Wolfe, M. J., Hoffmann, F. J., and Gardner, H. S., Jr. (2001). Chronic toxicity of bromodichloromethane to the Japanese medaka (Oryzias latipes). Toxicol. Pathol. 29, 662–669.[CrossRef][ISI][Medline]

Villanueva, C., Kogevinas, M., and Grimalt, J. (2001). Chlorination of drinking water in Spain and bladder cancer. Gac. Sanit. 15, 48–53.[Medline]

Waller, K., Swan, S. H., DeLorenze, G., and Hopkins, B. (1998). Trihalomethanes in drinking water and spontaneous abortion. Epidemiology 9, 134–140.[ISI][Medline]

Wolfahrt, S., Kleine, B., and Rossmanith, W. G. (1998). Detection of gonadotrophin releasing hormone and its receptor mRNA in human placental trophoblasts using in-situ reverse transcription-polymerase chain reaction. Mol. Hum. Reprod. 4, 999–1006.[Abstract]

Yang, M., Lei, Z. M., and Rao, Ch. V. (2003). The central role of human chorionic gonadotropin in the formation of human placental syncytium. Endocrinology 144, 1108–1120.[Abstract/Free Full Text]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
78/1/166    most recent
kfh046v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Chen, J.
Articles by Douglas, G. C.
PubMed
PubMed Citation
Articles by Chen, J.
Articles by Douglas, G. C.