Effect of Atrazine on Implantation and Early Pregnancy in 4 Strains of Rats

A. M. Cummings1, B. E. Rhodes2 and R. L. Cooper

Reproductive Toxicology Division, MD-72, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received May 14, 2000; accepted July 24, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atrazine (ATR) is an herbicide that has been shown to have adverse reproductive effects including alterations in levels of pituitary hormones such as prolactin (prl) and luteinizing hormone (LH) in female LE rats when administered at doses of 200 mg/kg/day for 1 and 3 days. Because the action of prl in promotion of progesterone secretion is essential for the initiation of pregnancy in rats, this study was designed to examine the effect of exposure to ATR during early pregnancy on implantation and short-term pregnancy maintenance. Rats were divided into two groups representing periods of dosing with ATR prior to the diurnal or nocturnal surges of prl. Within each group, four groups consisting of four strains of rats [Holtzman (HLZ); Sprague Dawley (SD); Long Evans (LE); Fischer 344 (F344)] were each further subdivided into four ATR dosages. Rats were dosed by gavage with 0, 50, 100, or 200 mg/kg/day ATR on days 1–8 of pregnancy (day 0 = sperm +). All animals were necropsied on day 8 or 9 of pregnancy. The 200 mg/kg dose of ATR reduced body weight gain in all but one group. Two groups of animals dosed at 100 and 200 mg/kg/day in the nocturnal dosing period showed an increase in percent preimplantation loss, and both of these were F344 rats. HLZ rats were the only strain to show a significant level of postimplantation loss and a decrease in serum progesterone at 200 mg/kg/day both following diurnal and nocturnal dosing. Doses of 100 mg/kg/day also produced postimplantation loss following diurnal and nocturnal dosing, but progesterone levels were decreased only after nocturnal dosing. Alterations in serum LH were seen in several groups. Serum estradiol was significantly increased only in SD rats dosed at the diurnal interval with 200 mg/kg ATR. We conclude that F344 rats are most susceptible to preimplantation effects of ATR and that HLZ rats appear most sensitive to the postimplantation effects of the chemical. LE and SD rats were least sensitive to effects of ATR during very early pregnancy.

Key Words: atrazine; rat; early pregnancy; embryo implantation; strain difference; progesterone; prolactin; hormones.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The herbicide atrazine (ATR) is a member of a family of chloro-s-triazines that are used in contemporary agricultural applications and are frequently found in groundwater at low ppb levels (Gressel et al., 1984Go; Sinclair and Lee, 1992Go; Stevens and Sumner, 1991Go). Recent research has focused on the potential reproductive effects of ATR in rats, including mammary tumor formation in Sprague Dawley (SD) rats (Stevens et al., 1994Go; Wetzel et al., 1994Go), alterations in hormonal profiles (Cooper et al., 2000Go; Simpkins et al., 1998Go), and disruption of ovarian cycling (Cooper et al., 1996Go). As reported by Cooper et al. (2000), the administration of ATR to ovariectomized rats suppressed the estrogen-induced surges of both luteinizing hormone (LH) and prolactin (prl) in a dose-dependent manner in both Long Evans (LE) and SD females. A decrease in LH and prl secretion after ATR exposure was also reported by Simpkins et al. (1998).

Progesterone is essential for the initiation of pregnancy in the rat and human (De Feo, 1967Go; Psychoyos, 1973Go). During the first 10 days of pregnancy in the rat, there are two daily surges of prl, a nocturnal surge with a peak between 0300 and 0500 h, and a diurnal surge peaking between 1700 and 1900 h (Terkel, 1988Go). It is these surges of prl that are luteotropic, leading to the rescue and maturation of the corpora lutea (CL) and to the subsequent dramatic increase in the secretion of the progesterone that, along with ovarian estrogen, directly permits implantation of the embryo in the uterus (Morishige and Rothchild, 1974Go; Smith et al., 1976Go). In fact, previous work has shown that the administration of bromocryptine, a dopamine agonist that inhibits the synthesis and release of pituitary prl (Barrow and Tindall, 1983Go), to rats during gestation day (GD) 1–8 can block implantation, presumably due to its ability to prevent prl secretion and thereby block the necessary rise in serum progesterone (Cummings et al., 1991Go). In the human it is LH that supports CL function prior to implantation and human chorionic gonadotropin (hCG) that exerts postimplantation support of the CL and progesterone secretion (Zeleznik and Fairchild-Benyo, 1994Go).

Effects of ATR on pregnancy in rats have been investigated to a limited extent. According to Infurna et al. (1988), the administration of technical ATR to rats during GD 6–15 had no effect at the 70 mg/kg dose, whereas 700 mg/kg produced maternal mortality. A report by Peters and Cook (1973) states that 1000 or 2000 mg/kg ATR, administered on GD 3, 6, and 9 resulted in embryonic resorption and embryotoxicity. In work reported by Narotsky et al. (1999), exposure of rats to ATR during GD 6–10 produced full litter resorptions at 200 mg/kg. None of these studies investigated the effects of relatively low doses of ATR or effects on implantation.

Our study is based on the potential for ATR to disrupt early pregnancy and implantation in the rat via a suppression of prl surges. The hypothesis was that if ATR suppresses prl during early rat pregnancy, then such a suppression of either the diurnal or nocturnal surge of prl by ATR would impair implantation in rats. The testing of this hypothesis has been done indirectly: ATR was administered to rats during early pregnancy and the impact of the chemical on implantation and pregnancy maintenance until day 9 was assessed. The two different times of ATR exposure used correspond to intervals preceding the diurnal and nocturnal prl surges (Terkel, 1988Go). A previous investigation of strain sensitivity to ATR during the postimplantation interval (Narotsky et al., 1999Go) led us to incorporate four different rat strains. The strains employed were 1) Holtzman (HLZ) rats, which are historically used in physiologically based pregnancy studies (Yochim and De Feo, 1963Go); 2&3) LE and SD, strains that were previously shown to have a sensitivity to ATR with respect to altered estrous cyclicity (Cooper et al., 1995Go); and 4) Fischer 344 (F344), animals that have been used in previous work on effects of ATR on pregnancy (Narotsky et al., 1999Go). Thus, these studies examine the possibility that different strains of rats are differentially sensitive to the effects of potential prl suppression by ATR, leading to adverse effects on pregnancy.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Four strains of rats were employed: HLZ (Harlan, Indianapolis); LE (Charles River, Raleigh, NC; SD (Charles River, Raleigh, NC); and F344 (Charles River, Raleigh, NC). Animals were purchased at 70 days of age and caged in pairs in a room maintained at 20–24°C. Food and water were provided ad libitum. The light:dark cycle was kept at 14:10 with the midpoint of the cycle at 12 N. For experiments in which dosing was during the dark phase, the light cycle was exactly reversed so that the dark portion of the cycle was during the day, and a red light was used while dosing the animals. Rats were permitted to acclimate for 1 week, after which daily vaginal smears were initiated. When rats demonstrated two consecutive regular estrous cycles, they were bred by caging with untreated males on the night of proestrus. Successful mating was confirmed by the presence of sperm in the vaginal smear and the finding of vaginal plugs in the pan under the mating cages. The day of the sperm-positive vaginal smear, corresponding with the day of estrus, was considered day 0. Dosing with ATR commenced on day 1. Because only rats with regular cycles are used and because both vaginal plugs and sperm in smears are used to identify breeding, the historical rate of percent pregnant in our laboratory is 90–95%. Animals were randomized by assigning each to a treatment group at the time that they were identified as pregnant (day 0). Rats were bred in a staggered fashion over time, and the estrous cycles were not synchronized. Because the decision of when to breed a particular animal was based only on her vaginal smear record, this protocol provided an effective randomization process.

Experiment 1.
Rats were dosed by gavage with ATR, following daily weighing, on days 1–8 of pregnancy at 1400 h. This corresponds to a period prior to the diurnal prl surge. Technical ATR (6-chloro-N-ethyl-N`-(1-methylethyl)-1,3,5-triazine-2,4-diamine) was supplied by Novartis (Greensboro, NC) and was of 97.1% purity. ATR was suspended in 1% methyl cellulose and diluted so that animals received 2.5 ml/kg at each dose level. Groups of 8–10 rats received 0 (1% methyl cellulose vehicle), 50, 100, or 200 mg ATR/kg/day. On day 9, rats were decapitated at 1400 h and trunk blood collected. Following separation from red cells, serum was frozen for later assay of LH, progesterone, and estradiol. Serum prl was not measured, as the experimental design (including times and method of euthanasia) precluded the determination of prl surge levels or patterns. At necropsy the following parameters were assessed: body weight, number of implantation sites, number of corpora lutea (CL), ovarian weight, uterine weight, and number of resorptions apparent by day 9.

Experiment 2.
Groups of rats were treated and evaluated exactly as in Experiment 1 except that ATR was administered at 0200 h (prior to the nocturnal surge of prl) and rats were killed at 2100 h on day 8 of pregnancy (19 h after the last dose of ATR).

Radioimmunoassays (RIAs).
Serum estradiol was measured by a double antibody RIA kit obtained from Diagnostic Products (Los Angeles, CA), and serum progesterone was measured using a coat-a-count RIA kit supplied by the same company. The determination of LH was performed using materials supplied by the National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases. The iodination preparation was I-6, the reference preparation was RP-2, and the antisera was S-8. Radiolabeling of the iodination preparations was performed with 125I (New England Nuclear, Boston) using the Chloramine-T method of Greenwood et al. (1963), after which labeled hormone was purified on a BioGel P-60 (Bio-Rad Laboratories, Melville, NY) column. The assay for LH had a sensitivity of 15 pg/tube, and inter-and intra-assay coefficients of variation of 7.7% and 5.9%, respectively.

Statistics.
Parametric data were analyzed by a 3-way ANOVA (dose x strain x time) using the General Linear Models (GLM) procedure of SAS statistical software (SAS Institute, 1985). When the overall ANOVA was significant at p < 0.05, comparisons between groups were made using t-tests (Least Squares Means; SAS). In most cases, only comparisons across dose within strain and time were made. For hormone levels in control groups, comparisons across strain and time were made. Percent data (pre- and postimplantation loss) were analyzed by 3-way ANOVA as above after arcsin square-root transformation. Significant differences between data points were indicated when the p value was less than 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of rats to 200 mg/kg ATR produced a significant decrease in body weight change in all groups except F344 (diurnal dosing) (Table 1Go). Dosing with ATR during the nocturnal interval resulted in significant effects on body weight change at lower doses than were effective when given during the diurnal interval. Despite the use of four strains of rats, two times of dosing, and four dosage levels of ATR, a significant increase in preimplantation loss was seen in only two groups: F344 rats following nocturnal dosing with 100 or 200 mg/kg ATR (Fig. 1Go). In one data set (LE rats, diurnal dosing), ATR treatment appeared to reduce preimplantation loss. HLZ rats alone showed a significant increase in postimplantation loss, at both dosing intervals at 100 and 200 mg/kg ATR (Fig. 2Go). Table 2Go shows the percent pregnant which represents the level of success of breeding in these strains. The table also shows the rarity of Full Litter Resorption (FLR). When the number of implantations per dam was evaluated, treatment groups showing a significant difference from control corresponded to groups that also showed significant or a trend toward significant increases in the percent of preimplantation loss (Table 2Go; Fig. 1Go). HLZ and F344 rats were also the only groups in which a significant change in organ weights was seen. Ovarian weight declined in all ATR-treated HLZ rats after the nocturnal dosing interval and in those treated with 200 mg/kg ATR at the diurnal interval (Table 1Go). Uterine weight was decreased in F344 rats treated in the nocturnal interval with 100 or 200 mg/kg ATR in parallel with significant changes in preimplantation loss (Table 1Go). In no case was there a significant change in the number of CL (data not shown).


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TABLE 1 Effect of Atrazine on Relevant Parameters of Early Pregnancy
 


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FIG. 1. Effect of atrazine on preimplantation Loss. The effect on preimplantation loss of four dose levels of ATR in four strains of rats following either diurnal or nocturnal dosing was evaluated on day 8 or 9 of pregnancy after dosing during days 1–8. Diurnal dosing = 1400 h. Nocturnal dosing = 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE, Long Evans; F344, Fischer 344 rats. Data are expressed as percentages (± SE) calculated as follows: {[(total # CL) – (total # implantation sites)]/# CL} x 100. Data marked with an asterisk (*) are statistically different from vehicle-treated controls within each strain and dosing interval (p < 0.05).

 


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FIG. 2. Effect of atrazine on postimplantation loss. Diurnal dosing = 1400 h. Nocturnal dosing = 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE, Long Evans; F344, Fischer 344 rats. Data are expressed as percent preimplantation loss (± SE) = [(# resorptions)/(total # implantations)] x 100. Data marked with an asterisk (*) are statistically different from vehicle controls within each strain and dosing interval (p < 0.05).

 

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TABLE 2 Effect of Atrazine on Parameters of Early Pregnancy
 
When progesterone was measured on day 9 of pregnancy, HLZ rats alone showed a decrease in serum progesterone at 100 and/or 200 mg/kg ATR in both intervals of dosing (Fig. 3Go). When SD rats were dosed at the 1400 h interval (diurnal) with 200 mg/kg ATR, a significant increase in serum estradiol was seen; in no other treatment groups did this phenomenon appear (Fig. 4Go). Estradiol was, in fact, undetectable in all F344 rats dosed and killed in the afternoon (Fig. 4Go). A significant reduction in baseline serum LH was seen as a consequence of ATR exposure in several cases: 1) HLZ/diurnal dosing/100 and 200 mg/kg ATR; 2) LE/ diurnal dosing/100 mg/kg; 3) LE/ nocturnal and diurnal dosing/ 200 mg/kg; and 4) F344/ nocturnal dosing/200 mg/kg (Fig. 5Go). SD rats exhibited no effect of ATR on LH levels (Fig. 5Go).



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FIG. 3. Effect of atrazine on serum progesterone. Diurnal dosing = 1400 h. Nocturnal dosing = 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE, Long Evans; F344, Fischer 344 rats. Data are expressed as means ± SE. Data marked with an asterisk (*) are statistically different from vehicle-treated controls within each strain and dosing interval with a cutoff of p < 0.05.

 


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FIG. 4. Effect of atrazine on serum estradiol. Diurnal dosing = 1400 h. Nocturnal dosing = 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE, Long Evans; F344, Fischer 344 rats. Data are expressed as means ± SE. Data marked with an asterisk (*) are statistically different from vehicle-treated controls within each strain and dosing interval with a cutoff of p < 0.05.

 


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FIG. 5. Effect of atrazine on serum luteinizing hormone. Diurnal dosing = 1400 h. Nocturnal dosing = 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE, Long Evans; F344, Fischer 344 rats. Data are expressed as means ± SE. Data marked with an asterisk (*) are statistically different from vehicle-treated controls within each strain and dosing interval with a cutoff of p < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The action of ATR to produce a reduction in the amount of weight gained during the first 9 days of pregnancy is an indication of the general toxicity of the chemical and is not a reflection of any change in uterine weight. Such a general toxicity may have contributed to maternal toxicity and the pregnancy loss observed, but the effects of ATR on body weight change are not consistently paralleled by effects on pregnancy. F344 rats appear most sensitive to the preimplantation effects of ATR, and those effects occur only following nocturnal dosing. The increase in preimplantation loss in F344 rats treated in the nocturnal interval with 100 or 200 mg/kg ATR is paralleled by a decrease in uterine weight in those animals alone. HLZ rats showed a trend toward increased preimplantation loss following both dosing intervals. SD and LE rats appear insensitive to effects of ATR on implantation. The apparent high preimplantation loss in control LE rats following diurnal dosing is the result of finding two animals in that group that were not pregnant (zero implantation sites), a situation which is most likely due to failure of the breeding process. The finding of statistically significant preimplantation loss in the F344 group suggests that ATR was successful in reducing prl levels sufficiently to interfere with implantation in that strain following nocturnal dosing. Further work to pursue that possibility is needed. Percent postimplantation loss is an indicator of the degree to which pregnancy maintenance is compromised between implantation (starting on day 4) and day 9 of pregnancy. Such an effect, observed in HLZ rats, suggests that the hormonal mechanisms regulating pregnancy maintenance in rats, including prl, LH, and decidual luteotropin (Morishige and Rothchild, 1974Go) may be most sensitive to disruption in this strain. In addition, in HLZ rats dosed with ATR, there are parallel effects on preimplantation loss, postimplantation loss, and the level of serum progesterone, suggesting that the decrease in serum progesterone may mediate the decrease in implantations and increase in resorptions. An additional observation in HLZ rats is the finding of decreased ovarian weight, a phenomenon that may be related to CL failure and decreased progesterone secretion.

In a related set of studies, Narotsky et al. (1999) examined strain differences in the sensitivity of rats to ATR and bromodichloromethane (BDCM), a by-product of drinking water disinfection. In those experiments, rats were dosed on GD 6–10, a postimplantation, LH-dependent interval, and evaluated following parturition. When the pregnancies of ATR- or BDCM-treated dams were allowed to proceed to term, an all-or-nothing phenomenon of full litter resorptions (FLR) was observed. Whereas BDCM treatment caused FLR in F344 but not SD rats, ATR treatment (200 mg/kg/day) led to comparable incidences of FLR in F344, SD, and LE rats (Narotsky et al., 1999Go). Based on the current data, it is not known whether further evidence of postimplantation loss and/or FLR might have been observed if the pregnancies in which the dams were treated on days 1–8 were allowed to proceed to term. However, such a finding would corroborate data reported by Narotsky et al. (1999). The increase in preimplantation loss observed here is likely the result of preimplantation exposure to ATR producing a suppression of prl levels, which in turn would impair CL maturation, elevation of progesterone secretion, and the promotion of implantation. Such a prl suppression, induced by bromocryptine, has been shown to block implantation in a similar model of early pregnancy exposure in the rat (Cummings et al., 1991Go).

The finding of no effect of ATR on serum progesterone in three out of four strains of rats is surprising in light of the essential role of prl in CL development and progesterone secretion and also in light of the putative action of ATR to alter serum prl (Cooper et al., 2000Go). The measurement of baseline prl at necropsy on day 9 (with euthanasia performed rapidly enough to prevent stress-induced prl elevation) was not considered useful, as it is the twice-daily surges of the hormone that are important for the stimulation of progesterone secretion (Smith et al., 1976Go). In a study such as that reported here, the introduction of an additional variable, that of using a series of sequential necropsies to determine the rise and fall of each prl surge, for each time of dosing, dose level, and strain data point was considered to be unjustifiable with respect to the number of animals required relative to the amount of information obtained. Although it is impossible to state with certainty the effect of ATR on prl during early pregnancy in this study, the data on progesterone suggest either that ATR, at these dosages, had little or no effect on the surges of prl during early pregnancy, or that ATR-induced changes in prl did not affect CL function sufficiently to interfere with pregnancy.

With the exception of one case (F344/diurnal dosing/all dosages), treatment of every strain at both time intervals yielded at least a trend toward reduced baseline serum LH, and this effect was shown to be significant in six cases. This is not a measure of LH surge suppression, but merely an assessment of changes in baseline levels. In two of the six cases of baseline LH suppression, there was also a significant increase in percent postimplantation loss (HLZ/diurnal dosing/100 and 200 mg/kg). In the other four cases, there is usually at least a trend toward increased postimplantation loss that parallels the decline in LH in each group. The number of resorptions detectable on day 9 does not always reflect the full complement of resorptions that might be seen later in pregnancy or near term. In work reported by Narotsky et al. (1999), for example, FLR was detected in rats at term following treatment with ATR during early pregnancy.

Nocturnal but not diurnal dosing with ATR was effective in increasing preimplantation loss in F344 rats. No time-of-dosing effects were observed in other strains for this end point. Both nocturnal and diurnal dosing (independently) produced postimplantation loss in HLZ rats, but no effects on this end point were evident in other strains. Serum LH showed a mixed pattern with respect to effects of dosing interval. In LE rats, dosing at each interval reduced serum LH. However, in HLZ and F344 rats only diurnal or nocturnal (respectively) dosing was effective. The finding of increased serum estradiol was seen in only one case, following diurnal dosing. The apparent elevation of estradiol levels in this one group of SD rats may be simply the result of variability in the data for that hormone. Overall, the effect of different intervals of dosing depended on the end point measured and the strain of rat employed.

A comparison of serum levels of progesterone, estradiol, and LH on day 9 in control animals across strains yields some interesting observations. In animals killed at 1400 h on day 9 (following diurnal dosing), serum progesterone levels were significantly higher in F344 and HLZ animals than in SD or LE rats. At the same necropsy time, estradiol levels in F344 rats were lower (undetectable) than those of the other three strains, and serum LH showed a pattern where F344 and HLZ rats were lowest, SD rats were intermediate, and LE rats were significantly higher than all others. In animals killed at 2100 h on day 8 (following nocturnal dosing), serum progesterone levels were again high in the F344 strain and low in the SD strain, but in LE rats progesterone was high whereas HLZ rats showed low levels of the hormone. At 2100 hr, estradiol levels were much higher in LE rats than in any other strain, and estradiol levels were above detectable levels in F344 rats at this time. Serum LH patterns were similar to those seen at the earlier time: HLZ and F344 rats showed low levels, whereas SD and LE rats showed significantly higher levels of LH. These differences in hormone levels in control animals are a function not only of strain but also of the time of euthanasia, suggesting another caution for the design of toxicological studies that incorporate the measurement of ovarian or pituitary hormones. It is possible that the differences in control hormone levels across strains may play a role in the differential sensitivity of the various strains to the effects of chemicals. For example, LE rats have much higher levels of estradiol than other strains at 2100 h. A connection may exist between this and the apparent resistance of LE rats to effects of ATR on implantation.

Although previous work has suggested that ATR can suppress prl levels under specific conditions unrelated to pregnancy (Cooper et al., 2000Go; Simpkins et al., 1998Go), it is possible that the chemical may not be as effective in altering prl when administered during early pregnancy. The only strain in which ATR produced significant preimplantation loss was F344, and in those animals no significant effect on progesterone levels was observed. On the other hand, it is possible that ATR indeed reduces prl during early pregnancy, but that the minimal amount of prl necessary to sustain CL function and implantation is so low that no outward adverse effect was observed in conjunction with the hormonal effect.

In summary, exposure of rats to ATR during early pregnancy produced no adverse effects in LE or SD rats. HLZ and F344 rats appear most sensitive to the effects of ATR on early pregnancy. Our hypothesis that ATR should produce a preimplantation loss that might be based on the chemical's effect on prl has been shown to be true only for F344 rats. Postimplantation effects, likely to be mediated by LH, were produced by ATR only in HLZ rats. We conclude that the effects of ATR on early pregnancy may be selective with respect to strain and are significant only in the sensitive strains. Further work to delineate effects of ATR on prl in susceptible strains during early pregnancy is necessary.


    ACKNOWLEDGMENTS
 
The authors acknowledge the excellent technical assistance of Keith McElroy, Dorothy Guidici, and Joan Hedge for assistance with the hormone assays. We also thank the National Hormone and Pituitary Agency for the gift of the radioimmunoassay materials for LH.


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

2 Present address: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295. Back

The research presented in this article was funded wholly 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.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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