Endocrinology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received May 30, 2003; accepted August 14, 2003
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ABSTRACT |
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Key Words: atrazine; propazine; metabolites; female; pubertal development; reproductive toxicology.
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INTRODUCTION |
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The environmental fate of ATR can affect the potential for exposure of humans and wildlife to ATR and its by-products. It has been estimated that the surface runoff of ATR usually ranges from 0.5 to 3% of the applied ATR (Snedeker and Clark, 1999). While the half-lives of ATR and its by-products vary with geographic conditions, the predominate forms found in the soil, surface, and ground water are hydroxyatrazine (OH-ATR), (2-chloro-4-amino-6-(ethylamino)-s-triazine (DIA), 2-chloro-4-amino-6-(isopropylamino)-s-triazine (DEA), and, to a lesser extent, 2-chloro-4,6-diamino-s-triazine (DACT) (Buchanan and Hiltbold, 1973
; Eldridge et al., 1994
; Kolpin et al., 1997
; Koskinen and Clay, 1997
; Muir and Baker, 1978
; Sorenson et al., 1993
; Thurman et al., 1998
; Winkelmann and Klaine, 1991
). These biotransformation by-products are a result of microbial degradation, photodegradation, chemical hydrolysis, and plant metabolism.
The metabolism of ATR in animals and humans has been thoroughly studied, and metabolites identical to those found in the environment are also produced in vivo. Major urinary metabolites identified in the adult rat include OH-ATR, DIA, DEA, DACT, and ammeline (Bakke et al., 1972; Bradway and Moseman, 1982
). McMullin et al. (2003)
recently reported that DACT is the major, persistent plasma metabolite in the rat following oral exposure. Finally, the metabolites identified in the urine of occupationally exposed males are DIA, DEA, and DACT (Catenacci et al., 1990
; Ikonen et al., 1988
). Thus, the potential exists for humans and wildlife to be exposed to a number of these chemicals resulting from either environmental sources and/or metabolism following exposure to ATR.
In 1994, ATR and the related s-triazine herbicides, simazine and cyanazine, were placed under a special review by the U.S. EPA based on concerns that ATR was a possible cancer risk to humans (//www.epa.gov/oppsrrd1/reregistration/atrazine/hed_redchap_16apr02.pdf). Subsequently, the Agencys Scientific Advisory Panel (SAP) concluded that the cancer mode of action responsible for the earlier onset of mammary tumors in the Sprague-Dawley rat following chronic exposure to ATR was not relevant to humans (www.epa.gov/scipoly/sap/2000/june27/finalatrazine.pdf). Although ATR might cause adverse effects on hypothalamic-pituitary functions in humans, the hormonal environment conducive to tumor development (i.e., elevated or prolonged exposure to estrogen and prolactin) that is found in Sprague-Dawley rats is not expected to occur in humans. However, the SAP did conclude that the findings on the neuroendocrine mode of action for ATR in the rat did raise a developmental concern for children. These later conclusions were based on recent studies that have shown that ATR can adversely affect reproductive function in the laboratory rat through a neuroendocrine mode of action (Cooper et al., 2000; Das et al., 2001
). Cooper et al. (1996)
reported the disruption of estrous cyclicity in adult Long Evans and Sprague-Dawley rats during a 21-day exposure to ATR (75300 mg/kg, oral gavage). These authors concluded that the effects on estrous cyclicity were most likely mediated via alterations in the neurotransmitter and hormonal control of the gonadal function. Specifically, ATR 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 (LH) and prolactin in ovariectomized rats following single or multiple (3 and 21 days) doses of ATR (Cooper et al., 2000
). The observation that intravenous injections of gonadotropin-releasing hormone (GnRH) restored the estrogen-induced secretion of LH in these animals provided additional evidence for a central nervous system (CNS)-pituitary mode of action. Similarly, significant delays in pubertal development observed in both female (50200 mg/kg [Laws et al., 2000
] and 30100 mg/kg [Ashby et al., 2002
]) and male rats (12.5200 mg/kg [Stoker et al., 2000
]) following exposure to ATR are possibly due to alterations in the hormonal signaling within the hypothalamicpituitaryovarian axis. Recent studies by Stoker et al. (2002)
have shown that at least three chlorinated by-products also delay pubertal development in male rats at doses similar to that of ATR. Together, these data suggest that there is a need to understand the potential for cumulative toxic effects of the chlorotriazines and their biotransformation by-products.
ATR is currently undergoing a re-registration process by the U.S. EPA (www.epa.gov/oppsrrd1/reregistration/atrazine/) and is one of a group of s-triazine pesticides for which the agency is conducting a reassessment of tolerances. In carrying out the tolerance reassessment provisions of the 1996 Food Quality Protection Act (FQPA), the U.S. EPA recently determined that the triazine pesticidesATR, PRO, and simazineand the by-productsDACT, DIA, and DEAshare a common mechanism of toxicity due to their ability to suppress the LH ovulatory surge and produce consequent effects on reproductive function and reproductive development. Thus, for purposes of a cumulative risk assessment (and as part of the tolerance reassessment process for triazine pesticides), these compounds will be considered as a "Common Mechanism Group" (http://www.epa.gov/oppsrrd1/cumulative/triazines/triazinescommonmech.pdf).
To date, there have been few studies that have compared the toxicological effects following equimolar doses of these chemicals. Such studies are needed to provide the necessary data for the cumulative risk assessment of this group of s-triazine compounds. In addition, comparative studies to determine structureactivity relationships between the chlorinated and nonchlorinated by-products are also needed. Thus, the studies reported here were conducted to compare the effects of two by-products of ATR, DACT and OH-ATR (Fig. 1), on female pubertal development and thyroid function in young Wistar rats. A structurally similar s-triazine, propazine (2-chloro-4,6-bis (isopropylamino)-s-triazine; PRO) was also tested to compare its potency with ATR. The Protocol for the Assessment of Pubertal Development and Thyroid Function in the Female Rat (Goldman et al., 2000
) was used for these studies, which was identical to that used in our previous studies with ATR (Laws et al., 2000
). Since this protocol is currently undergoing an extensive validation process by the U.S. EPA for possible use in a Tier I screening battery of the agencys Endocrine Disruptor Screening Program, all endpoints included in the protocol were evaluated to provide additional performance criteria data for the protocol (www.epa.gov/scipoly/oscpendo/). Thus, thyroid hormones were evaluated in these studies even though no previous data suggested that the chlorotriazines would affect thyroid function. To facilitate the comparison of the potency of all of the test chemicals, the dose range for each was equimolar to the doses used for ATR in our previous studies. Thus, it should be noted that the doses used are higher than that which would be expected from environmental exposures. Since DACT is a common chlorinated environmental by-product as well as a human and animal metabolite of ATR and PRO, this study tested the hypothesis that DACT is the active compound for both herbicides. The use of a nonchlorinated by-product, OH-ATR, provides information as to whether the chlorine moiety is a necessary structural component for reproductive toxicity. Finally, this study provides data to evaluate whether environmental exposure to multiple chlorotriazines and their biotransformation by-products could possibly have an additive effect on pubertal development.
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MATERIALS AND METHODS |
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Dosing solutions and procedures.
DACT (purity 96.8%) and OH-ATR (purity 97.1%) were gifts from Syngenta Crop Protection, Inc. (Greensboro, NC). PRO (purity 99.8%) was a gift from Griffin LLC (Valdosta, GA). All of the chemicals were administered by oral gavage in a suspension of 1% methyl cellulose/distilled water (M-7140, Lot No. 64H0619, Sigma Chemical Co., St. Louis, MO) in a volume of 5.0-ml dosing solution/kg body weight. To facilitate the comparison of the potency of each test chemical with that of ATR, the dose ranges for each test chemical were the molar equivalent of ATR (atrazine equimolar dose; AED; see Table 1) and were selected based on results reported in a previous study of ATR on female pubertal development (Laws et al., 2000
). The doses used for the study were as follows: DACT (16.7, 33.8, 67.5, 135 mg/kg); OH-ATR (22.8, 45.7, 91.5, 183 mg/kg); and PRO (13, 26.7, 53, 106.7, 213 mg/kg). The control animals received the 1% methylcellulose.
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Radioimmunoassays.
Serum TSH was measured by radioimmunoassay using material supplied by the National Hormone and Pituitary Agency (e.g., iodination preparation I-9, reference preparation RP-3, and antisera S-6). The iodination material was radiolabeled with 125I (Dupont/New England Nuclear) by a modification of the chloramine-T method (Greenwood et al., 1963). The labeled TSH was separated from the unreacted iodide by gel filtration chromatography as described in Laws et al. (2000). Total triiodothyronine (T3) and thyrotropin (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.
The data from each of the doseresponse experiments for PRO and OH-ATR (n = 15) were analyzed separately by analysis of variance (ANOVA) using the General Linear Model (GLM) procedure (Statistical Analysis System (SAS), SAS Institute, Inc., Cary, NC). The data from the two doseresponse block studies for DACT (n = 78) were initially analyzed separately by ANOVA. The data were then combined to yield a sample nmber of 15/treatment group and analyzed by ANOVA for block, treatment, and blocktreatment interaction effects. In cases where a significant treatment effect (p < 0.05) was observed, the doseresponse data were further evaluated by the Dunnett multiple comparison (control compared with each treatment group). The treatment means for each endpoint were tested for homogeneity of variance using the Bartlett test (GraphPad InStat, GraphPad Software, San Diego, CA), and, where heterogeneity was evident, the Welch t-test or Kruskal-Wallis Nonparametric test with Dunns 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 organ weights for which a significant effect of body weight was observed. 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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DISCUSSION |
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The protocol used in this study is currently undergoing validation by the U.S. EPA for possible inclusion in a Tier I screening battery for the detection of environmental chemicals that can disrupt the homeostasis of estrogen, androgen, and thyroid hormones (Goldman et al., 2000; www.epa.gov/scipoly/oscpendo/). The data from this study demonstrate the ability of the protocol to also detect environmental chemicals that disrupt the endocrine system via another mechanism of action. Earlier reports have shown that ATR does not bind to the estrogen receptor directly, nor does it possess any estrogenic activity in vivo (Conner et al., 1996; Eldridge et al., 1994
; Tennant et al., 1994
). However, it has been clearly demonstrated that ATR can reduce serum LH and prolactin secretion (Cooper et al., 1996
; Simpkins et al., 1998) and that these actions are most likely mediated via an effect on the regulation of pituitary hormone synthesis at the level of the CNS (Cooper et al., 2000
). Thus, the use of this protocol as a screen to detect endocrine disruptors provides a broader scope for identifying chemicals with modes of action that are not necessarily associated with steroidogenesis or steroid receptors.
The age at vaginal opening was the most sensitive endpoint in this study, and it was perhaps the best indicator of the effects of DACT and PRO on pubertal development. The onset of puberty in the female encompasses a period of transition during which there are changes in the signaling within the hypothalamicpituitaryovarian axis (Goldman et al., 2000). Vaginal opening (or vaginal patency) and the occurrence of the first ovulation are dependent on estrogen synthesis and the development of the ability of the female brain to respond to the positive feedback of estrogen (Ojeda and Urbanski, 1994
). The fact that the age of vaginal opening is an apical endpoint is an advantage when using this protocol because it allows for the evaluation of multiple sites of action within the hypothalamicpituitaryovarian axis. Additionally, since the observation is noninvasive, the option to slightly modify the study design prior to necropsy remains available, and the study could be extended to evaluate longer periods of cyclicity or fertility. Within the confines of the pubertal protocol used in the study reported here, doses were selected that were slightly below the maximal tolerated dose (MTD, a dose that causes more than a 10% reduction in body weight), which minimized an effect of lower body weight on the age at vaginal opening. While in this study a 10.4% reduction in body weight was observed for the highest dose of DACT, delayed vaginal opening occurred in the animals treated with the next two lower dose groups for which no reductions in body weights were observed. This observation is in agreement with our earlier report (Laws et al., 2000
) for pair-fed controls for which vaginal opening was unaffected by an 11.6% reduction in body weight after being fed the same daily food intake as consumed by their ATR-treated counterparts.
In summary, this study shows that oral exposure to DACT and PRO from PNDs 2241 delays the age of vaginal opening in Wistar rats in a dose-dependent manner. The LOEL for the delay in vaginal opening for DACT was equimolar to that reported for ATR. The LOEL for PRO was 2-fold higher than that for ATR (e.g., 100 vs. 50 mg/kg, AED). In contrast, the dechlorinated by-product, OH-ATR, appeared to be much less potent and did not alter the onset of puberty at doses equimolar to 200 mg/kg ATR in two doseresponse studies. Importantly, none of the test chemicals had any dose responsive effect on serum thyroid hormone concentrations or histology. Together, these data demonstrate that PRO and DACT can delay reproductive development in the laboratory rat and that the persistent nature of the chlorotriazines in the environment raises the potential for additive reproductive effects in humans and wildlife.
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ACKNOWLEDGMENTS |
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NOTES |
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* To whom correspondence should be addressed at MD-72, NHEERL, U.S. EPA, Alexander Drive, Research Triangle Park, NC 27711. Fax: 919 541-5138. E-mail: laws.susan{at}epa.gov.
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