* Center for Health and the Environment and
Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616;
Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599;
Reproductive Toxicology Division and
¶ Environmental Toxicology Division, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received June 5, 2003; accepted August 14, 2003
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ABSTRACT |
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Key Words: bioactivity; chlorination; trihalomethanes; pregnancy.
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INTRODUCTION |
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An increased risk of small-for-gestational-age (SGA) births, stillbirths, spontaneous abortions, and low birth weight has been reported for women exposed to TTHM (Bove et al., 1995; Dodds et al., 1999
; Gallagher et al., 1998
; Kramer et al., 1992
; Savitz et al., 1995
; Swan et al., 1998
; Waller et al., 1998
). Waller and colleagues reported an increased risk of spontaneous abortion among women who drank at least five glasses per day of cold tap water with
75 µg TTHM per liter. Stratified analysis further showed that only BDCM exposure was associated with spontaneous abortion (Waller et al., 1998
). In a Canadian study, increased risk of stillbirth was associated with THM consumption, and the strongest association was observed for BDCM exposure (King et al., 2000
); relative risk estimates associated with BDCM exposures were larger for asphyxia-related deaths than for unexplained deaths.
Several animal studies have investigated halocarbon-associated spontaneous abortion. Oral administration of BDCM was shown to cause full-litter resorption (FLR) in F344 rats (Bielmeier et al., 2001; Narotsky and Kavlock, 1995
; Narotsky et al., 1997
). Bielmeier and colleagues (Bielmeier et al., 2001
) showed a 62% incidence of FLR in rats following aqueous gavage treatment with BDCM between gestational days 6 and 10. This encompasses a critical, luteinizing hormone (LH)-dependent period of pregnancy in rats. More recent work indicates that reduced LH secretion (Bielmeier et al., 2002
) and reduced luteal responsiveness to LH (Bielmeier et al., 2003
) may both contribute to BDCM-induced FLR in F344 rats. However, these effects are likely to be strain specific, as several investigators have failed to observe FLR in BDCM-exposed Sprague-Dawley (SD) rats (Bielmeier et al., 2001
; Christian et al., 2001
; Ruddick et al., 1983
). When similar doses (75 mg and 100 mg BDCM/kg day) were administered to SD rats under the same conditions as F344 rats, all of the SD rats maintained their pregnancies (Bielmeier et al., 2001
). Christian and colleagues conducted developmental toxicity studies with BDCM in Crl SD rats and rabbits. Instead of being administered by oral gavage, pregnant rats and rabbits were allowed to drink BDCM-containing water ad libitum. FLR was not observed, and no adverse effects on embryo-fetal viability were found at levels up to 900 ppm (Christian et al., 2001
). The authors postulated that the sensitivity difference might reflect the different reproductive performance and endocrine physiology of the species and strains or the difference in toxicokinetics resulting from the route of exposure.
While further investigation of differences in strain susceptibility could lead to a greater understanding of the toxic mechanism of BDCM, and while rodents provide a valuable model to study the potential adverse effects of environmental hazards, it is important to note both the differences and similarities in reproductive physiology between the rat and the human. In higher primates, including humans, it is the products of conception (placental trophoblast) that support pregnancy. Early pregnancy in higher primates is maintained by luteal secretion of progesterone, which is supported by chorionic gonadotropin (CG), an LH-like glycoprotein, produced by placental syncytiotrophoblast. In contrast, in the rat, luteal maintenance during midpregnancy depends on pituitary LH, which is under the control of the hypothalamus and higher brain centers. Therefore, the target tissues of BDCM-induced pregnancy failure in rats may differ from those in human pregnancy; however, it is possible that parallel mechanisms may exist for LH-mediated toxicity in rats and CG-mediated toxicity in humans.
The above observations suggest that a possible target of BDCM toxicity in higher primates is the placental syncytiotrophoblast. Specifically, it is possible that BDCM disrupts the synthesis and/or secretion of syncytiotrophoblast-derived CG. In the present study, we have tested the idea that BDCM targets trophoblasts by examining the effect of BDCM on CG secretion by primary cultures of human trophoblasts. The results show that BDCM reduces the secretion of immunoreactive and bioactive CG.
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MATERIALS AND METHODS |
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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). We have previously shown that this medium supports both morphological and biochemical differentiation of cytotrophoblast cells (Douglas and King, 1990, 1993
; Ho et al., 1997a
). 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, 1993
). The cells also secrete bioactive CG (Ho et al., 1997a
).
Incubation of syncytiotrophoblast cultures with 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).
Cytotrophoblast cells were cultured in KGM for 48 h to induce morphologic differentiation and produce syncytiotrophoblast-like colonies (see above). The culture medium was removed and fresh KGM with or without BDCM (20 nM, 20 µM, or 2 mM) was added. The plates were sealed with plate seals and incubated at 37°C for an additional 24 h. At the end of the experiment, the culture medium was removed and 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, 1982). 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 (OConnor et al., 1988; Taylor et al., 1992
). The primary capture antibody was clone B 109, which reacts with intact hCG, and the second antibody was clone B 108, which reacts with ß-hCG (Canfield et al., 1987
; OConnor et al., 1988
). Immunoreactive CG was expressed as ng/mg cellular protein.
CG bioassay.
Bioactive CG was measured by in vitro bioassay as described previously (Chen et al., 2003; Jia et al., 1991
). Briefly, clonal human fetal kidney cells (cell line 293) were cotransfected with the LH/CG receptor and a luciferase reporter gene. Fifty-µl 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 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 morphology.
Syncytiotrophoblast cultures (in 8-chamber LabTek slides) were established as described above and then incubated with or without BDCM for an additional 24 h. 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 (Douglas and King, 1990) and 7-aminoactinomycin D (7-AAD) (10 µg/ml) (Scholz et al., 1996
). The primary anti-desmosomal protein antibody was detected using an Oregon Green-labeled secondary antibody. The slides were mounted using glycerol vinyl alcohol (GVA) aqueous mounting medium and examined using a Nikon Eclipse E800 epifluorescence microscope. Images were captured using an Optronics DEI750 CCD camera and Adobe Photoshop software. Identical exposure and brightness level settings were used for test and control samples.
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). Fluorescence intensity was measured by reference to a fluorescence standard curve obtained using fluorescence calibration beads (Molecular Probes, Eugene, OR). Images from at least six random fields viewed using a 20x objective were analyzed from each of four experiments.
Statistical analysis.
All experiments were performed six times, and each experiment used cells obtained from different placentas. Cells from different placentas were not pooled. Within an experiment, means were obtained from duplicate determinations. The immunoreactivity and bioactivity of hCG under different treatment conditions was 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 analyses were performed using SPSS software and Prism Software.
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RESULTS |
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DISCUSSION |
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The most important finding from this study is that BDCM, a major by-product resulting from disinfection of drinking water, appears to target human placental trophoblasts. This conclusion is based on the BDCM dose-dependent reduction in the secretion of immunoreactive and bioactive CG from viable, primary cultures of human placental trophoblasts. These cells are the sole source of CG during normal human pregnancy and play a major role in the maintenance of the conceptus. Given the vital role of CG in maintaining pregnancy, a decrease in the amount of bioactive hormone that is produced by placental trophoblasts could have adverse effects on pregnancy outcome. The lowest effective target concentration of BDCM found in the present study (24 h exposure) was 20 nM, which is 100-fold higher than maximum baseline blood levels and approximately 35-fold higher than the maximum peak level reported in blood after showering (Miles et al., 2002). Compared to median values, the lowest effective concentration in this study was approximately 500- and 80-fold higher than baseline and after-shower blood concentrations, respectively. Blood BDCM concentrations decrease significantly from 10 to 30 min after showering, but concentrations at the latter time point were elevated compared to baseline values (Backer et al., 2000
). While every precaution was taken to minimize loss of BDCM in vitro by volatilization, it is likely that actual concentrations in culture media were lower than the target dose. Clearly, caution must be exercised in extrapolating these in vitro results to the in vivo situation, but nonetheless, they indicate that the placenta could be a target for BDCM. A placental target for BDCM is consistent with epidemiological studies showing increased incidence of spontaneous abortion in women exposed to BDCM. In vivo studies using a nonhuman primate model are needed in order to better understand the physiological consequences of these in vitro observations.
Although the current findings indicate that BDCM may target the placenta in humans, the F344 rat model of BDCM-induced pregnancy loss appears to involve other target tissues. Initially, reduced serum progesterone levels, together with an inability to detect a reduction in serum LH, suggested that BDCM may affect the luteal response to LH during the LH-dependent period of gestation in F344 rats (Bielmeier et al., 2001). However, a more sensitive assay revealed BDCM-induced reductions in serum LH, indicating an effect on the hypothalamic-pituitary axis resulting in reduced LH secretion (Bielmeier et al., 2001
, 2002
). More recently, a contributory role of the corpora lutea has also been implicated; corpora lutea exposed to BDCM in vitro showed reduced responsiveness to CG (Bielmeier et al., 2003
).
Our previous studies have shown that early pregnancy loss in macaques and humans is associated with changes in the serum levels of immunoreactive and bioactive CG (Guo et al., 1999; Ho et al., 1997b
). In these latter studies, early pregnancy loss was associated with a decrease in the secretion of bioactive CG and an increase in the secretion of immunoreactive CG (i.e., a net decrease in the ratio of bioactive [B] to immunoreactive [I] CG). These observations led to the suggestion that a decrease in the secretion of bioactive CG (and a decrease in the B/I ratio) may provide a marker for impending pregnancy failure. In a previous in vitro study we showed that the environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused an increase in the secretion of immunoreactive CG and no change in the secretion of bioactive CG in human trophoblast cultures (Chen et al., 2003
). The net effect was still a decrease in the B/I ratio. In the present in vitro study, both bioactivity and immunoreactivity were decreased to a similar extent such that the B/I ratio was unchanged in cultures exposed to BDCM. If similar findings are obtained from in vivo studies and such studies are associated with early pregnancy loss, then this would suggest that a decrease in the B/I ratio is not the only marker for impending pregnancy failure. Until in vivo studies are carried out with nonhuman primates, further speculation on the significance of the B/I ratio is premature.
Although we were able to detect an effect of BDCM on the secretion of immunoreactive CG, we were not able to show an effect on intracellular CG levels in syncytiotrophoblast cultures based on immunocytochemical staining. This could be related to the use of different antibodies, the lower resolution of the image analysis procedure, and to the fact that the effects on immunoreactive CG secretion, while significant, were modest.
The mechanism by which BDCM perturbs CG secretion is unknown at present. Reduced CG secretion could result from disruption of CG synthesis at the translational or posttranslational level. Since it has been demonstrated that the biological activity of hCG is largely determined by the degree of glycosylation, it is possible that BDCM affects the glycosylation of CG subunits. Another possibility is that BDCM affects subunit dimerization. This could be the result of perturbations in protein folding during processing (Bedows et al., 1993; Furuhashi and Suganuma, 2000
; Ruddon et al., 1996
; Tominga et al., 1991; Wang et al., 1988
, 1989
). It is also possible that BDCM could affect CG secretion indirectly through disruption of gonadotropin releasing hormone (GnRH) activity. GnRH is thought to be a major regulator of placental CG synthesis and secretion (Barnea et al., 1991
; Currie et al., 1992
; Kikkawa et al., 2002
; Siler-Khodr et al., 1987
), and human trophoblasts express a GnRH receptor and secrete GnRH (Cheng et al., 2000
; Lin et al., 1995
; Wolfahrt et al., 1998
). If an effect of BDCM on GnRH is substantiated, this would parallel the proposed mechanism of BDCM-induced pregnancy loss in rodents. Further experiments will be required to determine if the effects of BDCM are due to alterations in the degree of CG glycosylation and/or subunit dimerization, or whether other mechanisms (perhaps involving GnRH) are involved.
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ACKNOWLEDGMENTS |
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NOTES |
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