Bromodichloromethane inhibits human placental trophoblast differentiation.

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.

Effect of bromodichloromethane on chorionic gonadotrophin secretion by human placental trophoblast cultures.

Bromodichloromethane (BDCM) is a trihalomethane found in drinking water as a by-product of disinfection processes. BDCM is hepatotoxic and nephrotoxic in rodents and has been reported to cause strain-specific full-litter resorption in F344 rats during the luteinizing hormone-dependent phase of pregnancy. In humans, epidemiological studies suggest an association between exposure to BDCM in drinking water and increased risk of spontaneous abortion. To begin to address the mechanism(s) of BDCM-induced spontaneous abortion, we hypothesized that BDCM targets the placenta. Primary cultures of human term trophoblast cells were used as an in vitro model to test this hypothesis. Trophoblasts were allowed to differentiate into multinucleated syncytiotrophoblast-like colonies, after which they were incubated for 24 h with different concentrations of BDCM (20 nM to 2 mM). Culture media were collected and assayed for immunoreactive and bioactive chorionic gonadotropin (CG). Cultures exposed to BDCM showed a dose-dependent decrease in the secretion of immunoreactive CG as well as bioactive CG. The lowest effective BDCM concentration was 20 nM, approximately 35-times higher than the maximum concentration reported in human blood (0.57 nM). Trophoblast morphology and viability were similar in controls and cultures exposed to BDCM. We conclude that BDCM perturbs CG secretion by differentiated trophoblasts in vitro. This suggests that the placenta is a likely target of BDCM toxicity in the human and that this could be related to the adverse pregnancy outcomes associated with BDCM.

Effects of exogenous estrogenic agents on pubertal growth and reproductive system maturation in female rhesus monkeys.

Concern has been raised that environmental contaminants with estrogenic properties can alter normal sexual maturation. Monkeys, like humans, undergo a long and complex period of development during adolescence, which makes them important models for understanding exogenous estrogen effects during this period. This study examined the consequences of treatment with estrogenic agents (methoxychlor, MXC, 25 and 50 mg/kg/day; diethylstilbestrol, DES, 0.5 mg/kg/day) given in the peripubertal period (6 months before and after the expected age at menarche) to female rhesus monkeys. These treatments increased estrogen activity of serum as determined with an in vitro estrogen receptor alpha (ERa) transcription assay. DES completely suppressed adolescent growth (weight and height) and menses in a reversible manner; smaller effects of MXC on the timing of growth and menarche were also detected. Both DES and MXC led to premature emergence of a secondary sex characteristic, reddening and swelling of skin, but retarded growth of the nipple. As evaluated by ultrasound after an 8-month recovery period, uterine size was not affected by exogenous estrogen, but there was some indication of increased incidence of ovarian cysts/masses in MXC- and DES-treated groups. Ovarian cyclicity, as reflected in urinary hormone metabolites, demonstrated shorter follicular stages in the MXC-treated monkeys. In conclusion, the data indicate that DES had a striking effect on adolescent maturation and that the estrogenic pesticide MXC also altered development during this period. The pattern of effects across agents and doses may be based on specifics of estrogenic action, such as relative ERalpha and ERbeta binding and activation. Long-term consequences of this disruption of pubertal development are being studied in this cohort of monkeys as adults.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on chorionic gonadotrophin activity in pregnant macaques.

As many as 62% of all human conceptions are lost prior to 12 weeks of pregnancy and it is unknown how many of these losses result from environmental hazards. Previous studies have shown that single doses of 1, 2, and 4 microg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administrated orally to cynomolgus macaques during the peri-implantation period leads to early fetal loss (EFL) within 10-20 days. TCDD induced EFL is associated with a reduction in the biological activity of monkey chorionic gonadotrophin (mCG) but no change in the immunoreactive mCG profile. These studies are consistent with either a direct effect of TCDD on differentiation of the trophoblast and an indirect effect on mCG synthesis, or a direct effect on mCG synthesis and secretion independent of trophoblast development. The present study was designed to test the hypothesis that the action of TCDD is directly on mCG synthesis rather than on the differentiation of the trophoblast. Female macaques (Macaca fascicularis) were treated with a single dose of TCDD (4 microg/kg b.wt.) on Gestational Day 20, a stage of pregnancy following initial trophoblast differentiation and invasion. Circulating mCG concentrations were monitored for the next 6 days. Compared to the controls, the peak level of serum bioactive mCG was lower in the treated group (P<0.05), with a decrease observed on the day following exposure. The bioactive/immunoreactive mCG ratio was also lower in the treated group compared to the controls (P<0.05). There was no difference in serum immunoreactive mCG levels between the groups. Histological evaluation of the embryo-placental unit showed increased apoptosis and vascular congestion after treatment but was otherwise grossly normal. Because exposure of the conceptus to TCDD following differentiation of the trophoblast decreased the bioactivity of circulating mCG, we conclude that the action of TCDD in the placenta is directly on mCG synthesis.

Detection of xenoestrogens in serum after immunoprecipitation of endogenous steroidal estrogens.

In this article we report a simple and efficient method for detecting nonsteroidal estrogens in a biologic sample. This method uses polyclonal antibodies to estradiol (E2) to immunoprecipitate these major biologically active steroidal estrogens, leaving behind the nonsteroidal estrogens, which are then detected in a cell-based transcriptional activation bioassay for estrogen receptor agonist. The immunoprecipitation method efficiently removed 99% of radiolabeled E2 and estrone (E1) from human serum. In experiments in which supraphysiologic concentrations of E2 and E1 to human serum, all of the immunoreactive estrogens were still removed by the immunoprecipitation protocol. We carried out an in vivo validation study of this method in which we treated female macaques with the xenoestrogen nonylphenol (NP), during the late follicular phase of the menstrual cycle. We used blood samples collected before and after treatment to evaluate and characterize endogenous and exogenous serum estrogens. An immunoassay for E2 did not detect the NP in treated monkeys. The cell-based bioassay also did not detect the estrogenic activity of NP because of its saturation by the endogenous serum steroidal estrogens. However, when steroidal estrogens were removed by immunoprecipitation, we detected the estrogenic activity of NP in the bioassay. Thus, this approach is appropriate for detecting exogenous, nonsteroidal estrogens in serum samples.