* Endocrinology Branch,
Gamete and Early Embryo Biology Branch, and
Biostatistics and Research Support Staff, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received May 15, 2000; accepted September 13, 2000
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ABSTRACT |
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Key Words: female reproductive toxicology; puberty; rat; atrazine; vaginal opening; estrous cyclicity.
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INTRODUCTION |
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An integral part of the Tier I Screening Program is focused upon detecting chemicals that alter male and female pubertal development and thyroid function through steroid-mediated mechanisms of action. The Protocol for the Assessment of Pubertal Development and Thyroid Function in Juvenile Female Rats (Goldman et al., 2000; U.S. EPA, 1998b) is currently undergoing testing to evaluate its reliability and reproducibility as such a screen. Marty et al. (1999) evaluated the Female Pubertal Protocol using estrogen and selected pharmaceuticals that are known to alter thyroid function or steroid biosynthesis. Goldman et al. (2000) recently reviewed the physiological and biochemical changes that occur during sexual maturation in the female rat, and also the influence of peripubertal exposures to endocrine disrupting chemicals. While the studies cited in that review used endpoints and dosing paradigms that were chronologically similar to the Female Pubertal Protocol, few investigators have used the protocol to evaluate environmental chemicals. In this study, we report the effects of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on female pubertal development and thyroid function, using the dosing regimen and required endpoints described in the protocol (Goldman et al., 2000
). Some of the optional endpoints in the protocol were also included in the study, to address specific issues related to the proposed mode of action for atrazine.
Atrazine, a chorotriazine herbicide, is used extensively in the United States. Degradation by-products, as well as the parent compound, have been detected in surface and ground water in areas of major usage (Baker, 1998; Rawn et al., 1998). Previous studies have shown that atrazine has adverse effects on the reproductive system in mammals (Stevens et al., 1994
; Wetzel et al., 1994
). Eldridge et al. (1998, 1999a, 1999b) reported an earlier onset of mammary tumors in Sprague-Dawley rats following long-term oral exposure. The premature appearance of persistent estrus in these animals suggested that atrazine may have induced early reproductive senescence. Cooper et al. (1996) reported that atrazine (75300 mg/kg, orally) disrupts estrous cyclicity in adult Long Evans and Sprague-Dawley rats during a 21-day exposure. These authors suggested that the effects on estrous cyclicity were most likely mediated via alterations in the neurotransmitter and hormonal control of gonadal function. Specifically, atrazine has been reported to increase dopamine and reduce norepinephrine concentrations in the hypothalamus (Cooper et al., 1998
), and to diminish the estrogen-induced surge of luteinizing hormone and prolactin in ovariectomized rats following single or multiple (3 and 21 days) doses of atrazine (Cooper et al., 2000
). In addition, Stoker et al. (1999) showed that suckling-induced prolactin release in lactating Wistar females is inhibited by atrazine and that male pups nursed by dams treated with 12.5 or 25 mg/kg atrazine twice per day during PND 19 exhibited an increased incidence of inflammation of the prostate at PND 120.
In the study reported here, we continue to explore the effects of atrazine on pituitary and ovarian function by evaluating the effect of the chemical on female pubertal development. Since atrazine has been reported to alter the release of prolactin and luteinizing hormone from the pituitary, we hypothesized that this compound would alter the onset of puberty. In addition, these studies evaluate the usefulness of the required and selected optional endpoints included in the female pubertal protocol as a screen for chemicals that alter hypothalamic-pituitary function.
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MATERIALS AND METHODS |
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Dosing solutions and procedures.
Atrazine (97.1% purity; a gift from Novartis Crop Protection, Inc., Greensboro, NC) was prepared as a suspension in 1.0% methyl cellulose (M-7140, Lot No. 64H0619, Sigma Chemical Co., St. Louis, MO) in distilled water. Dose groups included 0 (vehicle), 12.5, 25, 50, 100, and 200 mg atrazine/kg bw, which were delivered in a volume of 5.0 ml dosing solution/kg bw. All doses were administered by oral gavage.
Experimental design.
The study was conducted in 2 blocks. Eight treatment groups were included in the first block. The females in 4 of the treatment groups received either 0 (vehicle), 50, 100, or 200 mg/kg (n = 7/treatment group) and were killed on PND 41. Females in the other 4 treatment groups received 0, 50, 100, or 200 mg/kg (n = 7/treatment group) and were evaluated for changes in estrous cyclicity from PND 42149. These same treatment groups were used in a second block (n = 8/treatment group), along with 2 lower dose groups (e.g., 12.5 and 25 mg/kg; n = 15/treatment group; all animals killed on PND 41) to determine the no observed adverse effect level (NOAEL). An additional control group (e.g., pair-fed control; n = 8, killed on PND 41; n = 7, were evaluated for changes in estrous cyclicity from PND 42149), and this group was also included in the second block to evaluate the effects of restricted food intake and lower body weight on the reproductive endpoints. In this group, the amount of food made available for each pair-fed control was dependent upon the amount of food consumed by its respective counterpart in the 200 mg/kg atrazine group on the previous day. Pair-fed controls also received vehicle.
All animals were dosed daily by oral gavage, beginning on PND 22 and continuing through PND 41 (Fig. 1). Body weights were recorded daily and the dose administered each day was adjusted for body weight. For those females killed on PND 41, liver, kidney, adrenal, ovary, uterus, and pituitary weights were recorded. In addition, serum was frozen for triiodothyronine (T3), thyroxin (T4), thyroid stimulating hormone (TSH), and prolactin (PRL). Pituitaries were frozen for PRL assays. Estrous cyclicity was evaluated for the remaining females from PND 42149.
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Histology.
Immediately upon necropsy on PND 41, the uterus, ovaries, and thyroid were placed in formalin for 24 h. The tissues were rinsed and stored in 70% alcohol until embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissues from the pair-fed control, 0 (vehicle) and atrazine 200 mg/kg groups were evaluated by Experimental Pathology Laboratory, Inc. (Research Triangle Park, NC) for pathologic abnormalities and potential treatment-related effects.
Radioimmunoassays.
Serum and pituitary prolactin concentrations and serum thyroid stimulating hormone (TSH) were measured by radioimmunoassay using material supplied by the National Hormone and Pituitary Agency. The iodination preparation, reference preparation, and antisera for PRL and TSH were as follows, respectively: I-6, I-9, RP-3, RP-3, S-9, and S-6. The iodination preparation was radiolabeled with 125I (Dupont/New England Nuclear) by a modification of the chloramine-T methods (Greenwood et al., 1963). Labeled PRL and TSH were separated from free iodide by gel filtration chromatography, and the radioimmunassays were conducted as described by Goldman et al. (1986).
Totals of T3 and T4 were measured using coat-a-count radioimmunoassay kits obtained from Diagnostic Products Corp. (Los Angeles, CA). The detection limits for T3 and T4 were 0.2 and 10 ng/ml, respectively.
Statistical analyses.
Data were evaluated for differences between the replicate studies (e.g., Blocks 1 and 2) and treatment effects by analysis of variance (ANOVA) using the General Linear Model (GLM) procedure (Statistical Analysis System (SAS), SAS Institute, Inc. Cary, NC). Since no significant differences between Block 1 and 2 were observed, data from the replicates were pooled prior to testing for treatment effects. Body weight and vaginal opening data from those females killed on PND 41 were analyzed separately from data collected for females used for the estrous cyclicity observations, in order to maintain a sample size of 15 as recommended in the Female Pubertal Protocol (Goldman et al., 2000). When a significant treatment effect (
= 0.05) was present, Dunnett's test (control vs. each treatment group) or Bonferroni t-tests (multiple comparisons of treatment groups) was used to compare treatment groups. Bartlett's test (GraphPad InStat, GraphPad Software, San Diego, CA) was used to test for homogeneity of variance, and where heterogeneity of variance was evident, the Welch t-test or Kruskal-Wallis Nonparametric Test with Dunn's Multiple Comparison Test were used. Organ weights were analyzed by analysis of covariance (ANCOVA) using the body weight at necropsy as a covariate. Means and adjusted means relative to necropsy body weight were calculated for liver, kidney, adrenal, and pituitary weights. Adjusted means were compared with the control, using a pairwise t-test with the Bonferroni correction. All data are reported as mean ± SE (n).
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RESULTS |
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Age and Body Weight at Vaginal Opening
The age of vaginal opening was significantly delayed following exposure to 50, 100, or 200 mg/kg atrazine, but was unaltered following exposure to 12.5 or 25 mg/kg (Fig. 3A). While vaginal opening occurred in the controls by 32.5 ± 0.51 days (n = 15), the age at vaginal opening was delayed by 3.4, 4.5, or greater than 6.8 days for females in the 50, 100, or 200 mg/kg treatment groups, respectively. In addition, vaginal opening did not occur within the treatment period in 4 of 15 females in the 200 mg/kg group. Body weights at the age of vaginal opening are shown in Figure 3B
and were significantly increased in the 50, 100, and 200 mg/kg treatment groups as compared with the control (103 ± 2.9 g [n = 15]).
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Body Weight Gain and Pubertal Development in the Pair-Fed Group
Figure 4 shows the mean daily food consumption by females in the atrazine 200 mg/kg group (n = 15) as compared with controls (n = 10) that were allowed to eat ad libitum from PND 22 to 41. Food intake was significantly reduced in the atrazine group during the treatment period. To evaluate the effects of decreased food consumption and lower body weight gain during the treatment period on pubertal development, a pair-fed control group was included in Block 2 of the study. Food intake for the group`s females was dependent upon the amount of food consumed each day by their respective mates in the atrazine 200 mg/kg group. Body weights on PND 33 (e.g., the age at vaginal opening for the ad libitum control) and PND 41, as well as, the body weight gain during the treatment period were similar in the pair-fed control and atrazine 200 mg/kg groups (Table 1
). However, these parameters were significantly reduced in the pair-fed control and atrazine 200 mg/kg groups when compared with the vehicle controls fed ad libitum. Lower body weights on PND 33 and body weight gain during the treatment period did not significantly delay the age or body weight at vaginal opening in the pair-fed control group (34.4 ± 0.5 days [n = 15]) as compared with the ad libitum controls (32.8 ± 0.4 days [n = 15]). In contrast, both age and body weight at vaginal opening were significantly increased in the atrazine 200 mg/kg group as compared with the pair-fed control group.
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Tissue weights in the 0 and 200 mg/kg atrazine groups and the pair-fed control groups are shown in Table 4. To keep sample size equal with the pair-fed control (n = 8), only data for the 0 (control) and 200 mg/kg atrazine groups from Block 2 were used for this comparison. While kidney and pituitary weights were significantly reduced in the pair-fed control females as compared with the 0 controls, these weights remained significantly higher than those of the 200 mg/kg atrazine animals. Ovarian and uterine weights were not significantly reduced in the pair-fed females as compared with the controls. However, ovarian and uterine weights (with and without fluid) in the 200 mg/kg atrazine group were significantly lower than those in the pair-fed control group.
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Hormones associated with thyroid function were not significantly altered with atrazine treatment. Serum levels of T3, T4, and TSH were not significantly different from the control (Tables 3 and 4).
Histology
The thyroids from the 0 and 200 mg/kg atrazine groups and the pair-fed controls were evaluated for morphologic changes such as altered follicular epithelial height, the relative abundance and tinctorial characteristics of colloid, the extent of the thyroid vascular supply, and the density, size, and shape of the thyroid follicles. No changes were observed in any of the treatment groups, nor were there any indications of a treatment effect on thyroid hormone secretion.
No pathologic changes of any kind were observed in uterine or ovarian tissues from the control or pair-fed control groups. A decrease in corpus luteum development was observed in the 200 mg/kg atrazine group. This decrease was generally associated with underdevelopment of the reproductive tract on PND 41 in comparison with the controls. In addition, although the average number of atretic follicles (e.g., contained more than 12 cells' worth of karyorrhectic nuclear debris within the follicular antrum or lining cells) per ovary was 1.6-fold higher in the 200 mg/kg group, this increase was not statistically significant (p = 0.07). Uterine hypoplasia (e.g., characterized by one or more of the following, decreased uterine horn diameter, decreased myometrial development, decreased or absent endometrial glands, and/or immature endometrial stroma), was observed in 60% of the 200 mg/kg females and was restricted to those females that demonstrated a marked decrease in corpus luteum development.
Estrous Cyclicity
Estrous cyclicity was monitored in the 0, 50, 100, and 200 mg/kg and pair-fed control groups from the day of vaginal opening through PND 149. The age of the first complete 45 day estrous cycle after vaginal opening was significantly delayed in the 100 and 200 mg/kg atrazine groups as compared with the control (Table 5). A slight delay was also noted during this period for the pair-fed group, but the delay was not significantly different from the control.
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DISCUSSION |
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Data from this study provide a means to evaluate the usefulness of both the required and some of the optional endpoints included in the Female Pubertal Protocol. Of the required endpoints, the age and weight at vaginal opening were the first and perhaps the best indicators of atrazine's effect on pubertal development. Changes in body weight during treatment were also informative since the reduction in body weight in the highest-atrazine treatment group needed to be taken into account when evaluating the data. In this study, the issue of treatment-associated changes in body weight was addressed by using necropsy body weight as a covariate when analyzing tissue weights and by including a pair-fed control group in the second block of the study. The ability to evaluate hypothalamic-ovarian function by monitoring vaginal cytology during the treatment period (PND 2241) was somewhat limited in this study. This is because there is an increased tendency for irregular cycles in all females at the onset of puberty and also an inability of many of the females in the highest-atrazine-treatment group to reach vaginal opening. By extending the observation period for monitoring estrous cyclicity in the study reported here, a more thorough assessment of cyclicity was conducted. The protocol also allowed for the detection of treatment-related changes in kidney, pituitary, adrenal, ovarian, and uterine weights. However, due to the changes associated with different stages of the estrous cycle, the usefulness of uterine weight and serum prolactin was minimal. Although serum T3, T4, and TSH levels and thyroid histology were unaffected by atrazine, these endpoints are valid and add strength to the screening aspect of the protocol.
The effect of endocrine status at necropsy is a factor that should be considered while evaluating tissue weights and hormone concentrations when using this protocol. Changes in hormone concentrations vary with circadian rhythm as well as during the estrous cycle (Esber et al., 1976; Goldman et al., 2000
). Uterine and ovarian weights can vary depending upon the endocrine milieu and endocrine state (e.g., cycling, pseudopregnant, or anestrous) in postpubertal females. Goldman et al. (2000) have discussed these technical issues at length and suggest that these data be blocked according to the particular day of the estrous cycle that each animal was killed. Here, we included the number of animals within each treatment group that displayed vaginal smears indicative of diestrus, proestrus, or estrus. However, the sample size of 1516 animals used in the study was not sufficient for analyzing the data by endocrine status. The observation of lower uterine and ovarian weights in the 200 mg/kg atrazine group at PND 41 in this study was most likely due to the immaturity of the endocrine system. In addition, since the variation in the serum and pituitary prolactin concentrations was greatly inflated by the fact that the females were killed on varying days of the estrous cycle, no significant treatment effect could be observed for this measure.
A primary concern with the Female Pubertal Protocol, as well as for any in vivo test of toxicity, is that systemic toxicity may play a key role in any observed effect. For the most part, this may be associated with a decrease in body weight during treatment. For this reason, we included a group of control females that were pair-fed to match the food consumption and reduction in body weight observed in the females dosed with the highest concentration of atrazine. This was especially important when evaluating the results from the Female Pubertal Protocol, since it is known that lower body weight during the peripubertal period, in both humans and laboratory animals, will delay the onset of puberty and reduce fertility in the adult (Goldman et al., 2000). While it is generally recognized that there is a threshold for body weight and percent body fat that must be achieved prior to the onset of puberty (Bronson, 1987
; Holeham and Merry, 1985
; Wilen and Naftolin, 1978
), the age of vaginal opening in the females observed in this study was not totally dependent upon body weight. For example, some of the animals with lower body weights reached vaginal opening before others with greater body weights. However, it is interesting to note that the average body weight of the pair-fed control females at vaginal opening was only 5% lower than the control, and the age of vaginal opening was not significantly different from the control. Conversely, the average body weight at vaginal opening for the 200 mg/kg atrazine females was approximately 10 and 14% higher than the control or pair-fed groups, respectively.
Another important observation was that estrous cyclicity was altered in the pair-fed control during the first 15-day interval following vaginal opening. Again, it is well documented that reduced body weight/food consumption and stress can disrupt estrous cyclicity (Bates et al., 1982; Warren et al., 1999
). Although the irregularities of the estrous cycles during this observation period are consistent with data reported by Cooper et al. (1996) and Eldridge et al. (1994a), where single or multiple doses of atrazine disrupt estrous cyclicity, the abnormal cycles observed in the 200 mg/kg group in this study cannot be totally dissociated from a possible correlation with reduced body weight and/or stress. However, the fact that cyclicity was also irregular in the100 mg/kg atrazine females with no reduction in body weight, lends support to the hypothesis that atrazine alone can disrupt estrous cyclicity.
The observation period for estrous cyclicity was extended in this study to evaluate posttreatment effects of atrazine on pituitary and ovarian function. No treatment differences were detected in the number of regularly cycling animals from PND 57149. However, since Eldridge et al (1999) have observed early reproductive senescence and increased incidences of mammary tumors in rats, following long-term exposure to atrazine, it would have been interesting to have monitored the pubertal females through reproductive aging. Such a precedence for effects becoming apparent later in life has been set by the increase in prostate inflammation in adult male rats following lactational exposure to atrazine (Stoker et al., 1999).
The identification of the particular mode of action is not a stated purpose of either the male or female pubertal protocols currently under consideration for the Tier-1 Screening Program (U.S. EPA, 1998b). Nevertheless, by evaluating the changes observed in the present study in light of previously published data, some insight into the primary mode of action for atrazine's ability to delay puberty may be gained. Current evidence suggests that atrazine may work through one or several of the endocrine-disrupting mechanisms targeted by the protocol. For example, although this herbicide does not bind to the estrogen receptor directly nor possess any estrogenic activity in vivo (Conner et al., 1996; Eldridge et al., 1994b; Tennant et al., 1994a
), it has been reported to inhibit estrogen-induced 3H-thymidine incorporation and uterine growth in vivo (Tennant et al., 1994b
). This observation is consistent with the pubertal delay in the present study. However, atrazine can suppress the pulsatile release of LH in long-term, ovariectomized females, an effect that occurs in the absence of endogenous or exogenous estrogen (Cooper et al., 1996
). The results of in vitro studies evaluating the effect of atrazine on the estrogen receptor are also mixed. Conner et al. (1996) reported no estrogenic or anti-estrogenic activity of atrazine in several cell lines. In contrast, Tran et al. (1996) reported that atrazine, simazine, and cyanazine all inhibited an estrogen-induced response in yeast cells. However, these data could not be replicated by Graumann et al. (1999) who reported no inhibitive effect of atrazines on 17ß-estradiol-mediated transactivation in yeast. Thus, these observations lead us to conclude that it is unlikely that atrazine alters reproductive development via an estrogen receptor-dependent change.
Importantly, there is a growing line of evidence that atrazine may increase the activity of aromatase, a key enzyme for the conversion of androgens to estrogens. Crain et al. (1997) have observed elevated aromatase activity in the gonadal-adrenal mesonephros in male alligator hatchlings exposed in ovo to atrazine. Sanderson et al. (2000) have recently reported that in vitro exposure to atrazine, simazine, and propazine induces aromatase activity (CYP19) and increases levels of CYP19 mRNA in H295R human adrenocortical carcinoma cells. Indeed, Stoker et al. (2000) report a reduction in intratesticular testosterone and increases in serum estrone and estradiol, following pubertal exposure to 200 mg/kg of atrazine. It is not known whether the changes in steroids in the pubertal males were due to a delay in the onset of pubertal development or an atrazine-induced change in aromatase activity. In the study reported here, estrogen and estrone were not measured because of the variability in hormone concentrations associated with the females being killed on different days of the estrous cycle. However, there was no evidence that atrazine elevated serum estrogen concentrations in the females in this study (e.g., no incidences of advanced vaginal opening, persistent cornified vaginal epithelial cells, or uterotrophic activity were observed). Thus, while an effect on steroidogenesis was not immediately apparent in this study, the issue of a possible change in aromatase activity in those tissues key to neuroendocrine development during puberty should be explored.
The results of this study also support the hypothesis that atrazine delays the onset of puberty by altering hypothalamic-pituitary activity. Atrazine reduces serum LH and prolactin secretion (Cooper et al., 1996, 2000
; Simpkins et al., 1998
). Both of these hormones are important for normal pubertal development. Although serum and pituitary prolactin were measured at necropsy for the females in this study, no significant differences were observed due to the variability associated with prolactin concentrations on different days of the estrous cycle. Reports that atrazine can reduce hypothalamic norepinephrine concentrations (Cooper et al., 1998
) and that intravenous injections of GnRH restore the estrogen-induced secretion of LH in ovariectomized, atrazine-treated female rats (Cooper et al., 2000
) provide additional evidence for a CNSpituitary mode of action. It is well known that neurotransmitters and their regulation of pituitary hormone synthesis and secretion are critical for the onset of puberty and the maintenance of reproductive capability in the adult female (Goldman et al., 2000
). Thus, because the onset of puberty is a transitional period during which there are changes in the signaling within the hypothalamic-pituitary-ovarian axis, the hypothesis that atrazine mediates its effects through the CNS and hormonal control of gonadal function warrants further investigation.
In summary, we report that oral exposure to atrazine from PND 22 through 41 delays vaginal opening in Wistar rats in a dose-dependent manner. The LOAEL for a delay in vaginal opening was 50 mg/kg. As compared with pair-fed control group, the reduced body weight in the 200 mg/kg atrazine group was not solely responsible for the delay in vaginal opening. Fewer regular 4- or 5-day estrous cycles were observed during the first 15-day period following vaginal opening in the 100- and 200 mg/kg atrazine groups as compared with the control, but no differences were detected in any treatment group between PND 57 and 149. While this study was not intended to identify a specific mechanism of action, these data are consistent with a possible effect on the CNS and hormonal control of gonadal function that warrant further investigation. In addition, this study documents the effectiveness of the Female Pubertal Protocol as a method for screening for endocrine-disrupting chemicals, which may not mediate their effect through the estrogen, androgen, or thyroid systems.
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ACKNOWLEDGMENTS |
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NOTES |
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Portions of these data were presented at the 2000 Annual Meeting of the Society of Toxicology, Philadelphia, PA.
1 To whom correspondence should be addressed at MD-72, NHEERL, U.S. EPA, Research Triangle Park, NC 27711. Fax: 919 541-5138. E-mail: laws.susan{at}epa.gov.
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