Pregnancy Loss in the Rat Caused by Bromodichloromethane

Susan R. Bielmeier*,1, Deborah S. Best{dagger}, Dorothy L. Guidici{dagger} and Michael G. Narotsky{dagger}

* Curriculum of Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and {dagger} Reproductive Toxicology Division, National Health and Environmental Effects ResearchLaboratory, Mail Drop 72, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received June 21, 2000; accepted October 30, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bromodichloromethane (BDCM), a trihalomethane, is a by-product of the chlorination of drinking water. In a recent epidemiological study, consumption of BDCM was associated with an increased risk of spontaneous abortion in pregnant women. We have previously shown that BDCM causes pregnancy loss, i.e., full-litter resorption (FLR), in the F344 rat. The mode of action was investigated, with three main findings. First, there was a dramatic difference in sensitivity between F344 and Sprague-Dawley (SD) rat strains. Following aqueous gavage treatment on gestational days (GD) 6–10, F344 rats had a 62% incidence of FLR at 75 mg/kg/day, whereas all SD rats maintained their litters. Second, the critical period encompassed the luteinizing hormone (LH)-dependent period of pregnancy. Rats treated on GD 6–10 at 75 mg/kg/day had a 75% incidence of FLR, but rats treated on GD 11–15 at 75 or 100 mg/kg/day were unaffected. Third, 24 h after a single dose, all dams with FLR had markedly reduced serum progesterone levels; however, LH levels were unaffected. The high FLR rate during the LH-dependent period, the lack of response thereafter, and the reduced progesterone levels without an associated reduction in LH levels suggests that BDCM disrupts luteal responsiveness to LH.

Key Words: bromodichloromethane; disinfection by-product; pregnancy loss; strain differences; progesterone; luteinizing hormone; corpus luteum.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bromodichloromethane (BDCM) is a disinfection by-product, formed as a result of the chlorination of drinking water. This highly volatile compound is one of 4 trihalomethanes (THMs) that are now regulated in drinking water as a group, total trihalomethanes (TTHMs), by the U.S. EPA, with a maximum contaminant level of 80 µg/l (c Protection Agency, 1998). BDCM is classified as a probable human carcinogen, based on an increased incidence of tumors in the kidneys and livers of rats and mice and the large intestine of rats (U.S. Environmental Protection Agency, 1997).

In a recent epidemiological study (Waller et al., 1998Go), consumption of THMs, especially BDCM, in drinking water was associated with an increased risk of spontaneous abortion. The prospective cohort study of over 5000 pregnant women monitored water consumption and use, and estimated THM-exposure levels from utility records. When the results were stratified for individual THMs, BDCM had the highest odds ratio at 3.0 (95%CI = 1.4–6.6) for women who drank at least 5 glasses per day of cold tap water with a TTHM concentration greater than 75 µg/l.

Exposure to high amounts of THMs in drinking water has been associated with deleterious birth outcomes in numerous epidemiological studies. First, a study in central North Carolina reported a modest, but not dose-related, association with TTHMs and spontaneous abortion. However, the association was not apparent when water intake was taken into account (Savitz et al., 1995Go). Second, an increased risk of uterine growth retardation was found, but there was no significant relationship with low birth weight (Kramer et al., 1992Go). In a cross-sectional study, the authors noted the likelihood of neural tube defects was tripled by exposure to TTHMs at levels exceeding 80 µg/l (Bove et al., 1995Go). Lastly, a retrospective cohort study reported an association with high THM exposure in the third trimester and low term-birth weights. (Gallagher et al., 1998Go).

BDCM has been shown to be a reproductive toxicant in rodent bioassays. Klinefelter et al. (1995) reported that BDCM had an effect on sperm motility when given to F344 rats in drinking water (0.62 g/l) for 52 weeks. When averaged for body weights and water consumption, this concentration corresponds to a dose of 39 mg/kg/day. BDCM was also found to cause pregnancy loss, i.e., full-litter resorption (FLR), in F344 rats when dams were given an oral dose of 75 mg/kg/day of BDCM throughout the period of embryonic organogenesis (Narotsky et al., 1997bGo). This effect was an all-or-nothing phenomenon in that the litter was either completely resorbed or appeared normal at term, suggesting a maternally mediated mechanism.

In view of the epidemiological findings of Waller et al. (1998), we pursued the mode of action of BDCM-induced pregnancy loss in rats. Rats were dosed via aqueous gavage during gestation and monitored for FLR. Although the F344 rat has been shown to be susceptible to FLR caused by BDCM (Narotsky et al., 1997bGo) and other low-molecular-weight halocarbons (Narotsky et al., 1993Go), SD rats are being used in many other reproductive studies involving disinfection by-products. Therefore, we compared F344 and SD rats for their sensitivity to BDCM-induced FLR. In addition, the effects of BDCM on serum progesterone and luteinizing hormone (LH) levels during pregnancy were examined.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Bromodichloromethane (98+%, stabilized with potassium carbonate) was obtained from Aldrich Chemical Co. (Milwaukee, WI). BDCM formulations were stored in amber vials with Teflon-lined caps at 4°C. The vehicle was 10% aqueous emulphor EL-620 (ethoxylated castor oil; Rhone Poulenc, Cranbury, NJ) in distilled deionized water. All dosing formulations were prepared at appropriate concentrations to provide the desired dose when administered at 1 ml/kg body weight. Dose volumes were adjusted to daily body weights throughout the dosing period.

Animals and husbandry.
Timed-pregnant F344 and SD rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and Charles River Laboratories (Raleigh, NC), respectively. Animals were between 60 and 120 days old. The day that evidence of mating (copulatory plug or vaginal sperm) was detected was designated gestational day (GD) 0. The animals were individually housed in polycarbonate cages with heat-treated wood shavings supplied as bedding. The animals were provided feed (Purina Lab Chow 5001) and tap water ad libitum and a 12-h light:dark cycle. Room temperature and relative humidity were maintained at 22.2 ± 1.1°C and 50 ± 10%, respectively. Dams were killed by cervical dislocation, pups by decapitation.

Experimental Designs
Strain comparison study.
In order to compare their susceptibilities to BDCM-induced FLR, SD and F344 rats were dosed concurrently on GD 6–10. F344 rats were given doses of 0 and 75 mg/kg/day, based on previous studies, while SD rats were given doses of 0, 75, and 100 mg/kg/day.

Critical period study.
To evaluate the critical period for BDCM to cause FLR, 3 treatment groups, all receiving 75 mg/kg/day, were compared. One group was dosed from GD 6 to 15 as the positive control (Narotsky et al., 1997bGo), while the other 2 groups split the dosing regimen, GD 6–10 and GD 11–15. A negative control group was dosed with 10% emulphor on GD 6–15.

Hormone Profile Studies.
To simplify the hormone profile analysis, we conducted pilot studies with shorter dosing regimens. We found the GD 8–9 period to be sensitive to BDCM-induced FLR (data not shown). Then, to evaluate the progesterone and LH profiles after a single BDCM dose, blood was sampled in 2 experiments. In the first experiment, BDCM (100 mg/kg) was administered on GD 8 or 9, and animals were tail bled once daily for 4 days (GD 9–12). In the second study, rats were given a single dose of 75 or 100 mg/kg on GD 9, and blood was collected at 0, 6, 12, and 24 h after dosing. Control groups for both studies were dosed with vehicle, and blood was collected concurrently with the treated groups.

General.
In each of the experiments, animals were distributed among treatment groups using a non-biased randomization procedure that assured a homogeneous distribution of body weights (Narotsky et al., 1997aGo). During the morning of assigned treatment days, animals received a single BDCM dose of 0-, 75-, or 100-mg/kg body weight in aqueous emulphor via oral gavage.

Maternal body weights were determined on GD 5–16 and 20 for animals initially dosed on GD 6, and GD 7–16, and 20 for those initially dosed on GD 8 or later. All rats were examined throughout the experimental period for clinical signs of toxicity. Beginning on GD 20, the dams were observed periodically to determine the approximate time of parturition. The stage of parturition (complete, in progress, first pup delivered, or blood only) was recorded. Pups were individually examined and weighed on postnatal day (PD) 1 and PD 6. PD 1 was defined as GD 22, independent of the actual time of parturition; hence, pups were examined at the same time post-coitus. After PD 6, the dams and pups were sacrificed. The number of uterine implantation sites was recorded. The uteri of females that did not deliver were stained with 10% (v/v) ammonium sulfide to enhance detection of resorption sites (Narotsky et al., 1997aGo).

Serum collection.
In the hormone profile studies, tail-blood samples were collected after a single dose of BDCM. The blood was collected by sectioning the very tip (last few millimeters) of the tail and stroking the tail to the tip to quicken blood flow. The rats were restrained inside a cloth wrap throughout the entire process. Blood samples were collected in Becton Dickinson Microtainer serum separator tubes (lot #80510), chilled, and then spun at 4°C for 30 min at 1185 x g) with a Beckman GS-6R centrifuge. Finally, the serum was transferred to siliconized microcentrifuge tubes (A. Daigger and Company, Inc.) and stored frozen at –80°C.

Hormone assays.
Serum progesterone levels were quantified utilizing a direct solid phase enzyme-linked immunosorbent assay (ELISA). Progesterone ELISA kits (DRG Diagnostics, Germany) were used as per provided instructions. The serum samples were thawed, agitated, and diluted 1:8 with Dulbecco's phosphate-buffered saline (Gibco BRL, Life Technologies, lot #1013794) before introduction to the ELISA kit. Samples were assayed in duplicate. For quality control, 3 standards (Diagnostics Products Corp., CON6, lot #015) also were analyzed. Absorbance was measured at 450 nm using a Molecular Devices ThermoMax (Columbia, MD) microplate reader spectrophotometer. The intra-assay coefficients of variation involving multiple assays ranged from 0.27% to 7.6%; the interassay coefficients of variation ranged from 0.14% to 3.0%.

LH levels were quantified by radioimmunoassay. Materials were provided by the National Hormone and Pituitary Program: iodination preparation I-8, reference preparation RP-3, and anitsera S-11. The assay was performed according to provided recommendations, with the sensitivity for LH optimized by a 24-h co-incubation of sample and primary antibody prior to the addition of 125I-labeled hormone. The second antibody was goat anti-rabbit gamma globulin (Calbiochem). Iodination materials were radiolabeled with 125I (NEN, DuPont, Boston, MA) using a modification of the chloramine-T method (Greenwood et al., 1963Go). Labeled LH was separated from unreacted iodide by gel filtration chromatography as described by Goldman et al. (1986). The intra-assay coefficient of variation for the LH assay was 3.04%.

Statistical Analyses.
Dams with only one implantation site were considered outliers and were excluded from statistical analyses. Fisher's Exact Test was used to compare the incidences of FLR between appropriate treatment groups. In order to evaluate possible relationships between maternal endpoints (e.g. body weight changes, hormone levels) and subsequent litter survival, treatment groups were divided into subgroups according to litter survival status; i.e., live vs. fully resorbed litters. Maternal body weights, percent body weight changes, and hormone levels were evaluated by analysis of variance (ANOVA) using the General Linear Models (GLM) procedure on SAS (Cary, NC). When a significant treatment effect was detected by ANOVA, Student's t-test on least squares means (LSM) was used to identify individual subgroups that were significantly different from controls, as well as to compare dams with live litters to dams with FLR within the same treatment group. Serum progesterone and LH values also were analyzed with a repeated measures analysis of variance.

For developmental data, the litter was considered the experimental unit of analysis. Prenatal loss was defined as the number of implantation sites minus the number of live pups, divided by the total number of implantations. Postnatal loss was defined as the number of pups on PD 1 minus the number of pups on PD 6, divided by the number of pups on PD 1. The number of implantation sites, litter size, prenatal and postnatal loss, and pup weights were evaluated by GLM; LSM was used to contrast individual treatment and/or litter survival subgroups against controls. The number of live PD-1 pups was used as a covariate in the analyses of pup weights. Similarly, the number of implants was used as a covariate in the analyses of the numbers of live pups. A p value less than 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Strain Comparison Study
In order to compare their sensitivities to BDCM-induced FLR, SD and F344 rats were concurrently dosed on GD 6–10. A 62% FLR rate was observed in the F344 rats given 75 mg/kg/day, whereas all of the SD rats successfully maintained their pregnancies after receiving either 75 or 100 mg BDCM/kg/day (Table 1Go).


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TABLE 1 Incidences of Full-litter Resorption
 
Although the SD rats maintained their pregnancies, they did show similar signs of maternal toxicity compared to the F344 rats that resorbed their litters. The SD rats displayed maternal weight loss in a dose-dependent fashion during the treatment period, but their weights recovered by the end of the pregnancy to match controls (Fig. 1Go). The percent body weight loss after the first day of dosing was comparable between the SD rats and the F344 rats that resorbed their litters (Fig. 2Go). Interestingly, the F344 rats that maintained their pregnancies generally did not lose weight after the first dose; although, they did gain significantly less weight than the controls. The SD and F344 rats also had similar incidences of piloerection: 46% and 43%, respectively. Piloerection usually occurred during the treatment period and, in some rats, continued sporadically throughout gestation. There was a difference in ocular clinical findings for the two strains. One-half (7/14) of the treated F344 rats displayed lacrimation and/or excessive blinking shortly after dosing during the first 2 days of dosing, compared to only 1 of 28 treated SD rats. Lacrimation was not predictive of FLR among F344 rats.



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FIG. 1. Mean maternal body weights for strain comparison study. Rats were dosed with BDCM on GD 6–10 via oral gavage at 0 (circle), 75 (triangle), or 100 (square) mg/kg/day. The Fischer 344 (F344) rats that resorbed their litters are represented by a dotted line.

 


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FIG. 2. Strain comparison of percent change in maternal body weight after first dose, i.e. from GD 6 to 7 (mean ± standard error). Rats were dosed with BDCM on GD 6–10 at 0, 75, or 100 mg/kg/day. Hatched bars represent dams with fully resorbed litters. *p < 0.05, ***p < 0.001, statistically different from respective control group.

 
Critical Period Study
Two different 5-day periods during organogenesis were compared for their susceptibility to BDCM-induced FLR. F344 rats were dosed at 75 mg/kg/day on GD 6–10, GD 11–15, or GD 6–15. FLR was observed only when BDCM administration was on GD 6–10 or GD 6–15. The incidences of FLR were 75% (9 of 12) and 50% (5 of 10) in these respective groups. In contrast, all 13 rats treated with BDCM on GD 11–15 successfully maintained their litters (Table 1Go).

Hormone Profile Studies
Experiment I.
To characterize the progesterone and LH levels following BDCM exposure, rats were given 100 mg/kg on GD 8 or 9 and tail blood samples were collected once daily on GD 9–12. Thus, the first blood collection (GD 9) established baseline levels for the GD-9-treated group and 24-h post-dosing levels for the GD-8-treated group. FLR was observed in 0, 60, and 100% of the control, GD-8, and GD-9 dose groups, respectively (Table 1Go). In all rats that resorbed their litters, progesterone levels were markedly reduced at 24 h after dosing compared to controls and BDCM-treated dams with live litters (Fig. 3AGo). In animals dosed on GD 9, the mean (± standard error) progesterone level decreased from 137.94 ± 11.44 ng/ml to 48.45 ± 23.57 ng/ml in 24 h (n = 9). For the group dosed on GD 8, the mean progesterone level at 24 h after BDCM treatment was 67.01 ± 16.22 ng/ml for the animals that displayed FLR (n = 6); the corresponding control value was 127.19 ± 14.89 ng/ml. At 3 days after dosing, both resorbed groups had markedly reduced progesterone levels that were similar to the 2 nonpregnant animals in the study (data not shown).



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FIG. 3. Serum hormone levels on GD 9–12 following a single BDCM dose. Rats were dosed on GD 8 (triangle) or GD 9 (square) at 100 mg/kg. Solid lines represent dams with live litters, dotted lines represent dams with fully resorbed litters. (A) Serum progesterone concentrations. (B) Serum LH concentrations. *p < 0.05, **p < 0.01, ***p < 0.001 significant difference from controls. Error bars represent standard error from the mean.

 
Unlike progesterone, LH levels were unaffected 24 h after dosing. However, serum LH concentrations were elevated on GD 11–12 in the resorbed groups (Fig. 3BGo). Among the groups that resorbed, the mean serum LH levels rose from about 0.20 ng/ml on GD 10 to near 0.80 ng/ml on GD 11, and remained elevated on GD 12; whereas, the control levels dropped from 0.31 ng/ml to 0.14 ng/ml. These elevated LH levels of the dams with FLR were comparable to the LH levels of the 2 non-pregnant animals in the study (data not shown).

Experiment II.
To further characterize the progesterone and LH profiles, rats were dosed on GD 9 and blood samples were collected at 0, 6, 12, and 24 h after dosing. There was a high incidence of FLR at both dose levels tested: 64% at 75 mg/kg and 90% at 100 mg/kg (Table 1Go).

At 6 h after dosing, progesterone levels peaked for all groups, including the controls (Fig. 4AGo). For dams that had FLR, the animals given 75 mg/kg demonstrated a peak that was significantly reduced compared to controls. For the 100-mg/kg group, the peak was marginally reduced (p = 0.072). At 12 h, progesterone levels of dams that resorbed their litters were significantly reduced compared to controls, in both the 75 and 100 mg/kg groups. At 24 h, progesterone levels were further reduced in dams that displayed FLR. In contrast, progesterone levels remained comparable to controls in treated animals that maintained their litters.



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FIG. 4. Serum hormone levels at 0, 6, 12, and 24 h after a single BDCM dose. Rats were dosed on GD 9 at 0 (circle), 75 (triangle), or 100 (square) mg/kg. Solid lines represent dams with live litters, dotted lines represent dams with fully resorbed litters. (A) Serum progesterone concentrations. (B) Serum LH concentrations. *p < 0.05, **p < 0.01, ***p < 0.001 significantly different from controls. Error bars represent standard error from the mean.

 
LH levels were comparable at all time points for all groups (Fig. 4BGo). A repeated-measures analysis indicated a significant decline in LH levels among all groups over the 24-h period; however, no significant differences were noted among groups.

General Findings
Throughout these studies, the ocular effects in the F344 dams were very consistent: lacrimation or excessive blinking was often noted in animals after the first dose. As stated earlier, this was not observed among the SD rats. The timing of vaginal bleeding was predictive of FLR. With few exceptions, when vaginal bleeding was first observed on GD 11 or 12, the litters resorbed. However, when vaginal bleeding was first documented on GD 13–15, the dams generally maintained their pregnancies.

Also, throughout these studies, surviving litters appeared normal with no effects on pre- or postnatal survival. Litter size and pup weights were comparable between groups, except for two studies. In the first hormone profile study, the BDCM-exposed litters had significantly fewer, heavier pups than control litters. In contrast, in the second hormone profile study, the litters of treated dams had significantly more pups, but with comparable weights compared to controls.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The F344 and SD rat strains showed a dramatic difference in sensitivity to BDCM-induced FLR. Ruddick et al. (1983) also reported a lack of FLR in SD rats given oral BDCM, at doses up to 200 mg/kg/day in corn oil on GD 5–14. However, since their high-dose group had only 10 of 15 females pregnant and the non-gravid uteri were not stained to detect early resorption sites, it is possible that FLR occurred, but went undetected. In the current study, we sought to compare the sensitivities of the F344 and the SD rat strains, with the possibility that future investigation would continue with the SD strain. Since we had observed a high response at 75 mg/kg/day in the F344 rats, we were mainly interested in the sensitivity of SD rats at similar levels. Although BDCM did not cause FLR in SD rats at <=100 mg/kg/day, it remains possible that higher doses would cause FLR in this strain. Nonetheless, the F344 rats are clearly the more sensitive strain and our studies will continue in that strain. This dramatic difference in response between the SD and F344 rats exemplifies the importance of animal strain as a consideration when assessing toxicity.

It is unclear whether the difference in response was due to possible strain differences in reproductive physiology or toxicokinetics. Although the SD rats did not exhibit the ocular effects (lacrimation and/or blinking) after dosing, the maternal body weight data indicate comparable systemic toxicity in the SD and F344 rats, arguing against, although not excluding, a toxicokinetic difference. Also, a separate study with non-pregnant ovariectomized rats showed striking differences in pulsatile peak serum LH levels among F344 and SD rat strains. The F344 rats had peak serum LH levels that were much higher and more erratic than SD rats (J. M. Goldman, personal communication). This underscores the potential impact of strain differences in endocrine physiology, and may be pertinent to the contrasting results of this study. Elucidation of an explanation for varying strain susceptibility could lead to a greater understanding of the mechanism underlying BDCM-induced FLR in F344 rats.

To gain insight into the mode of action of BDCM, 5-day exposures during 2 physiologically different stages of pregnancy were compared. The earlier exposure period, GD 6–10, encompasses the LH-dependent period, whereas the later period, GD 11–15, is LH independent. During GD 7–10, the rat corpora lutea require LH stimulation from the pituitary gland to signal progesterone release (Rothchild, 1981Go; Terkel, 1988Go). After GD 10, placental lactogens are able to maintain the corpora lutea without LH stimulation (Gibori and Richards, 1978Go). The F344 rats demonstrated FLR only when dosed during the LH-dependent period; if dosing began after GD 10, the pregnancies were unaffected by BDCM. The high response during the LH-dependent period and the lack of response thereafter suggest that BDCM may disrupt the pregnancy via an LH-mediated mechanism.

To characterize BDCM's effects on hormones during pregnancy, tail blood samples were collected after a single BDCM dose during the LH-dependent period. In all rats that displayed FLR, serum progesterone levels were significantly reduced at 12 h and further reduced at 24 h after dosing. This decrease in progesterone appears to be a cause, rather than a consequence, of FLR, since the conceptus is not required to maintain progesterone secretion during this stage of pregnancy or pseudopregnancy (Pepe and Rothchild, 1974Go). Because LH from the pituitary is required to signal the corpus luteum to release progesterone during this time of pregnancy, the data support the hypothesis that BDCM disrupts the pituitary-gonadal axis by altering the secretion and/or signal transduction of LH. However, the apparent lack of effect on LH levels during the first 24 h after dosing favors the latter possibility, i.e., that BDCM disrupts luteal responsiveness to LH.

LH levels were unaffected during the first 24 h after dosing; however, LH increased on GD 11 in treated dams that resorbed their litters. Although, this rise in LH is likely a consequence, rather than a cause of FLR, the mechanism is unclear. It may have been due to the decreased serum progesterone levels and/or subsequent lack of negative feedback on LH release. However, if this were the case, the LH rise in the GD-8 group would be expected to occur earlier than in the GD-9-treated group. It is noteworthy that the rise in LH in both groups coincides with the expected dependence on placental lactogens. Thus, the rise in LH may be related to the presumed lack of placental lactogens in these dams.

Evaluation of the LH data is challenging because of the pulsatile release of LH from the pituitary during this stage of pregnancy (Gallo et al., 1985Go). Toxicant-induced changes in serum LH may be difficult to detect because of the fluctuating nature of serum LH levels. Also, the LH levels may be naturally diminishing because the LH-dependent period of pregnancy is nearing its end.

In view of the epidemiological association of BDCM consumption and an increased risk of spontaneous abortion (Waller et al., 1998Go), BDCM-induced pregnancy loss in rats is of particular interest in the risk assessment of drinking water disinfection by-products. It is important to note, however, the endocrinological differences between rats and humans in pregnancy maintenance. In humans, the corpus luteum is essential in establishing and maintaining early pregnancy. Prior to implantation, LH is needed to maintain the post-ovulatory corpus luteum. Upon implantation, human chorionic gonadotropin (hCG) from the syncytiotrophoblast "rescues" the corpus luteum from natural regression, which would occur in the absence of successful implantation. With hCG stimulation, the corpus luteum continues to secrete progesterone. Luteal secretion of progesterone peaks at 5 weeks of gestation then declines. After 8 weeks, the placenta produces and secretes enough progesterone to maintain the conceptus. The placenta continues to sustain the pregnancy until parturition (Martin et al., 1991Go). In the rat, corpora lutea are required for a much longer proportion of the pregnancy, up to GD 17 of a 22-day gestational period (Niswender and Nett, 1988Go). The hormonal requirements of the corpus luteum change as the rat progresses through different gestational stages (Rothchild, 1981Go). Initially, the corpora lutea secrete progesterone autonomously, soon after prolactin from the anterior pituitary is required (Terkel, 1988Go). Prolactin is essential until GD 6 (Niswender and Nett, 1988Go), after which, the corpora lutea also require LH. The corpora lutea are LH-dependent (Rothchild, 1981Go) until GD 11, after that, placental lactogens maintain the corpora lutea (Gibori and Richards, 1978Go). Although there are species differences, rats and humans are similar in that either LH or hCG, acting via the same receptor (Griffin and Ojeda, 1996Go), are required during specific periods to sustain pregnancy.

Although verification is needed and other mechanisms are possible, these studies suggest that BDCM diminishes luteal responsiveness to LH in the F344 rat. If this is due to an effect at the level of the LH/hCG receptor, e.g., inhibition or down-regulation, this finding in the F344 rat could be relevant to the epidemiological findings. That is, in humans, BDCM may be able to affect the signal transduction of hCG. Further research is needed to verify a lack of effect on LH secretion in the F344 rat, to elucidate the mechanism of the strain difference, and to investigate the potential of BDCM to disrupt the signal transduction of LH.


    ACKNOWLEDGMENTS
 
We thank Joan Hedge and Janet Ferrell for assisting with blood collection, Selena Mistich for performing the LH radioimmunoassay, and Judy Schmid for statistical consultation. Funding was provided by the EPA/UNC Toxicology Research Program, Training Agreement CT902908, with the Curriculum in Toxicology, University of North Carolina at Chapel Hill.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 541-4017. E-mail: bielmeier.susan{at}epa.gov. Back

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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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]

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]

Gallo, R. V., Devorshak-Harvey, E., and Bona-Gallo, A. (1985). Pulsatile luteinizing hormone release during pregnancy in the rat. Endocrinology 116, 2637–2642.[Abstract]

Gibori, G., and Richards, J. S. (1978). Dissociation of two distinct luteotropic effects of prolactin: regulation of luteinizing hormone-receptor content and progesterone secretion during pregnancy. Endocrinology 102, 767–774.[ISI][Medline]

Goldman, J. M., Cooper, R. L., Rehnberg, G. L., Hein, J. F., McElroy, W. K., and Gray, L. E., Jr. (1986). Effects of low subchronic doses of methoxychlor on the rat hypothalamic- pituitary reproductive axis. Toxicol. Appl. Pharmacol. 86, 474–483.[ISI][Medline]

Greenwood, F., Hunter, W. M., and Glover, J. S. (1963). The preparation of I-131-labeled human growth hormone of high specific activity. Biochem. J. 89, 114–123.[ISI]

Griffin, J. E., and Ojeda, S. R. (1996). Textbook of Endocrine Physiology, 3rd ed. Oxford University Press, New York.

Klinefelter, G. R., Suarez, J. D., Roberts, N. L., and DeAngelo, A. B. (1995). Preliminary screening for the potential of drinking water disinfection byproducts to alter male reproduction. Reprod. Toxicol. 9, 571–578.[ISI][Medline]

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]

Martin, G. C., Taylor, R. N., and Hoffman, P.G., Jr. (1991). The endocrinology of pregnancy. In Basic and Clinical Endocrinology (F. S. Greenspan, Ed.), Appleton & Lange, Norwalk, CT.

Narotsky, M. G., Hamby, B. T., Mitchell, D. S., and Kavlock, R. J. (1993). Full-litter resorptions caused by low-molecular-weight halocarbons in F-344 rats. Teratology 45, 472–473.

Narotsky, M. G., Brownie, C. F., and Kavlock, R. J. (1997a). Critical period of carbon tetrachloride-induced pregnancy loss in Fischer-344 rats, with insights into the detection of resorption sites by ammonium sulfide staining. Teratology 56, 252–261.[ISI][Medline]

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

Niswender, G. D., and Nett, T. M. (1988). The corpus luteum and its control. In The Physiology of Reproduction (E. Knobil and J. Neill, Eds.), pp. 489–525. Raven Press, New York.

Pepe, G. J., and Rothchild, I. (1974). A comparative study of serum progesterone levels in pregnancy and in various types of pseudopregnancy in the rat. Endocrinology 95, 275–279.[ISI][Medline]

Rothchild, I. (1981). The regulation of the mammalian corpus luteum. Recent Progress in Hormone Research 37, 183–298.[Medline]

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. 18, 333–349.[ISI]

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]

Terkel, J. (1988). Neuroendocrine processes in the establishment of pregnancy and psuedopregnancy in rats. Psychoneuroendocrinology 13, 5–28.[ISI][Medline]

U. S. Environmental Protection Agency (1997). Bromodichloromethane. Intergrated Risk Information System (IRIS) 0213.

U. S. Environmental Protection Agency (1998). EPA Stage 1 Disinfection and Disinfection By-Products Rule. U.S. Office of Water 815-F-98–010, 2.

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