* Gamete and Early Embryo Biology Branch and
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 September 17, 2001; accepted January 7, 2002
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ABSTRACT |
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Key Words: atrazine; diaminochlorotriazine; deethylatrazine; deisopropylatrazine; preputial separation; hormones; puberty; reproductive tract.
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INTRODUCTION |
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Atrazine has been shown to adversely affect reproductive tissues in the rat, although a specific mechanism or mechanisms of action remain to be identified. In chronic feeding studies using Sprague-Dawley female rats, 400 ppm atrazine (approximately 22.5 mg/kg) causes premature reproductive aging (Eldridge et al., 1999), a condition in which regular ovarian cycles are replaced by the appearance of persistent vaginal cornification and polyfollicular ovaries that continue to secrete estradiol. Atrazine also causes a premature appearance of mammary gland tumors in the Sprague-Dawley female. This result would be expected, as the underlying endocrine milieu of the persistent estrous female (i.e., uninterrupted estrogenic stimulation, low progesterone, and elevated prolactin concentrations) is conducive to the development of such tumors. In both Long-Evans hooded and Sprague-Dawley rats, atrazine (50300 mg/kg by gavage) suppressed the estrogen-induced surge of luteinizing hormone and prolactin (Cooper et al., 1996
, 2000
) after 3 days of treatment.
The suppression of pituitary hormone secretion by atrazine appears to be a direct effect on the CNS and not the pituitary gland. The pulsatile release of GnRH was suppressed by atrazine following oral dosing of 100 or 200 mg/kg (Cooper et al., 1996). We have also shown that the pituitary is still responsive to GnRH following atrazine treatment and that the suppression of prolactin secretion following atrazine treatment is not observed when ectopic pituitary secretion was evaluated (Cooper et al., 2000
). Recently, we found that following oral atrazine exposure in Long-Evans adult females, hypothalamic norepinephrine concentrations were decreased and dopamine concentrations and turnover were increased (Cooper et al., 1998
). Importantly, these changes in hypothalamic GnRH activity and pituitary hormone secretion induced by atrazine are observed both in the presence and absence of endogenous or exogenous estradiol, ruling out any role of the estrogen receptor in this process.
The effects of atrazine on luteinizing hormone and prolactin and the importance of these hormones in the development of the male reproductive system, led us to hypothesize that atrazine would alter reproductive development (i.e., puberty). Indeed, we recently reported that atrazine at doses ranging from 12.5 to 200 mg/kg delayed puberty (from 1.6 to 3 days as compared to controls) in the male rat when administered daily by gavage from PND 23 to 53 according to the Endocrine Disruptors Screening Program (EDSP) Tier 1 pubertal male rat assay (Fig. 1). We also observed decreased prostate and seminal vesicle development, decreased testosterone (PND 45), a trend for a decrease in serum luteinizing hormone and prolactin, and an increase in serum estrone and estradiol in the 200 mg/kg dose group.
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In the current study, we examined the effects of deisopropylatrazine (DIA), deethylatrazine (DEA), and diaminochlorotriazine (DACT) on the progression of puberty in the male rat by exposure to either of the 3 metabolites from PND 23 to 53. This data could then be used to compare the relative potency of the chlorinated metabolites with that of previously published dose-response effects of the parent compound, atrazine.
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MATERIALS AND METHODS |
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Necropsy.
Males were killed on PND 53 or 54. The day before necropsy (PND 52 or 53), males were placed in a holding room adjacent to the room used for necropsy. This room was maintained under the same lighting conditions as the animal room but the location allowed for each animal to be decapitated immediately (routinely less than 15 s after removal from their home cage) to minimize stress-induced changes in hormones. Following decapitation, blood was collected, and the pituitary, adrenal, testes, ventral and lateral prostates, epididymides, seminal vesicles with coagulating gland (with fluid) were removed and weighed. The clotted blood was centrifuged at 1260 x g for 30 min, the serum collected and stored frozen at 80°C for subsequent hormone assays. The anterior pituitary was removed, frozen on dry ice, and stored at 80°C for subsequent hormonal analyses. The epididymides, left testis, and thyroid (with the bracketing trachea) were removed, fixed in 10% neutral buffered formalin for 24 h before transferring to 70% ethanol until later processing for histology (H&E stain).
Radioimmunoassays.
Serum and anterior pituitaries were analyzed for luteinizing hormone (LH), prolactin (PRL), and thyroid-stimulating hormone (TSH) by radioimmunoassay. The assays were performed using the following materials supplied by the National Hormone and Pituitary Agency for LH, PRL, and TSH, respectively: iodination preparation I-9, I-6, I-9; reference preparation RP-3, RP-3, RP-3; and antisera S-11, S-9, S-6. Iodination material was radiolabeled with 125I (Dupont/New England Nuclear) by a modification of the chloramine-T method of Greenwood et al. (1963). Labeled PRL was separated from unreacted iodide by gel filtration chromatography as described previously (Goldman et al., 1986). Sample serum and pituitary homogenate were pipetted with appropriate dilutions to a final assay volume of 500 µl with 100mM phosphate buffer containing 1% bovine serum albumin (BSA). Standard reference preparations were serially diluted for the standard curves. 200 µl of primary antisera in 100mM potassium phosphate, 76.8 mM EDTA, 1% BSA, and 3% normal rabbit serum were pipetted into each assay tube, vortexed, and incubated at 5°C for 24 h. 100 µl of the iodinated hormone were then added to each tube, and the tube was vortexed and incubated for 24 h. Second antibody (Goat Anti-Rabbit Gamma Globulin, Calbiochem, at a dilution of 1 unit/100 µl) was then added, vortexed, and incubated 24 h. The samples were centrifuged at 1260 x g for 30 min and the supernate aspirated and the sample tube, with pellet, was counted on a gamma counter. Historical intra-assay coefficients of variation for the assays were 5.9, 5.3, and 6.2%, and interassay coefficients of variation were 7.7, 7.0, and 7.2% for LH, PRL, and TSH, respectively.
Serum testosterone, total triiodothyronine (T3), and thyrotropin (T4) were measured using Coat-a-Count radioimmunoassay kits obtained from Diagnostic Products Corporation (Los Angeles, CA). The serum estradiol and estrone were measured using kits from Diagnostic Systems Laboratories, Inc. (Webster, TX).
Histology.
To initially screen the tissue samples, only tissues from the highest dose groups and controls were submitted to Experimental Pathology Laboratory, Inc. for processing and histopathological evaluation. Following paraffin embedding, each 46 µm section was stained with hematoxylin and eosin for evaluation. For the thyroid tissues, each slide to be evaluated contained a transverse section of the thyroid gland (bracketing the trachea).
Statistics.
Data were analyzed for age, treatment, and block effects by ANOVA using the General Linear Model (GLM) procedure (Statistical Analysis System [SAS], version 8.1, SAS Institute, Inc., Cary, NC), and for homogeneity of variance using Bartlett's test (GraphPad InStat, GraphPad Software, San Diego, CA). Since no significant age effect was observed for necropsy data obtained on PND 53 and 54, these data were pooled for further analyses. There was a block effect in the serum estradiol concentrations, however, no treatment effects were observed within each block. When significant treatment effects (p < 0.05) were indicated by GLM, the Dunnett's t-test was used to compare each treatment group with the control. Necropsy pituitary, ventral and lateral prostate, testes, adrenal, and epididymal weights were analyzed by ANCOVA and Least Squares mean comparison using the body weight at PND 53 as the covariable. For quantification of linear association between treatments and LH or PRL concentrations, a Pearson correlation test was used (GraphPad, InStat).
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RESULTS |
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Reproductive Tract and Organ Weights
Testicular weights were not different from the controls in any treatment group. In the DIA treatment groups, the ventral prostate weights were significantly reduced by the 50, 100, and 200 AED (Fig. 5). However, the lateral prostate weights were reduced only by 100 and 200 AED (Table 3
). Likewise, the mean seminal vesicle weight was lower than controls at 100 and 200 (Fig. 6
). However, when the PND 53 body weight was entered as a covariable, the 50 AED group of DIA became significant for a decreased seminal vesicle weight as well. Similarly, the epididymis weight was lower at 200 mg/kg but when entered as a covariable with body weight on PND 53, the 100 AED group also became significant (Table 3
).
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Following treatment with DACT, the ventral prostate was significantly reduced by the highest dose group, however, the lateral prostate was unaffected by the DACT treatments (Fig. 5; Table 3
). The seminal vesicles and epididymis weights were reduced in the 100 and the 200 AED groups as compared to controls (Fig. 6
). The epididymis of the 100 and 200 AED groups were significantly decreased above controls, however, when analyzed as a covariable with PND 53 BW, the high dose was no longer significant (Table 3
).
Hormone Analyses
Steroid hormones.
In the DIA group, serum testosterone was significantly decreased at the 100 and the 200 AED doses as compared to the controls (Fig. 7). None of the other treatments significantly affected the mean serum testosterone, although the highest doses of DEA appeared to decrease in a dose-response manner (Control, 2.21 ± 0.23; 25, 1.54 ± 0.32; 50, 1.58 ± 0.28; 100, 1.28 ± 0.58; 200, 1.11 ± 0.58). There was increased variability between individual testosterone levels on PND 53. Thus, it was difficult to assess changes in testosterone, such as in the DEA group.
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Pituitary hormones.
The mean anterior pituitary weight was significantly decreased on PND 53 by the 2 highest doses of DEA and DACT and the highest dose of DIA (data not shown). On PND 53, serum luteinizing hormone showed a significant trend for a dose-dependent decrease from control to the high dose groups in the DIA group (See Table 4; r = 0.947 for LH) and appeared to be lower in the highest dose of DEA. While serum PRL appeared decreased in the 2 highest doses of DEA and DIA, the variability of this hormone on PND 53 made comparisons to the control difficult (Table 4
). Pituitary LH was significantly decreased in the 100 AED of DIA and the 200 AED of DEA as compared to the control means. Pituitary PRL content was significantly decreased in the 100 and 200 AED of DEA and in the 200 AED of DACT treatment group.
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Histology
High dose metabolite and control thyroids, epididymides, and testes were examined histologically by Experimental Pathology Laboratories (Research Triangle Park, NC).
As mentioned, no significant differences between control and high dose treatment groups of the metabolites were observed in the thyroid gland sections. No significant effects were observed in the high dose groups of the testes as compared to controls. However, approximately 25% of the high dose group of DIA, 40% of the high dose group of DACT, and 12.5% of the high dose group of DEA showed minimal hypospermia (defined as rats in which the overall density of spermatazoa within the epididymal ducts appeared to be decreased compared to control samples). This minimal hypospermia rated a 1 on a scale of 1 to 5, with 5 being severe. This observation is probably a result of the length of the delay in puberty in the high dose groups of the atrazine metabolites and the age at sacrifice.
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DISCUSSION |
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All 3 metabolites similarly affected pubertal development in the male rat suggesting that all 3 share a common mode of action as atrazine. For example, all 3 delayed preputial separation. In this regard, DACT was like atrazine, delaying PPS at doses ranging from 12.5 through 200 AED. DIA and DEA were somewhat less potent, with delays in PPS at 25 AED and in the 100 and 200 AED. Similarly, all 3 metabolites affected the reproductive tissue weights, however, there were some differences when compared to atrazine. Atrazine itself did not decrease the lateral prostate weight, as did DIA and DEA at the 2 highest doses (100 and 200 AED). Also, atrazine decreased ventral prostate weight at doses of 25 to 200 mg/kg (Stoker et al., 2000a), while these decreases occurred at higher doses of metabolite exposure (50 to 100 AED for DIA and DEA, respectively; and 200 AED in DACT). In contrast, the effect on seminal vesicle weight was more dramatic following metabolites exposure (e.g., with a decrease in seminal vesicle weight from 25 AED in the DEA group and at 100 and 200 AED for DIA and DACT) than following atrazine (with a significant decrease in the 200 mg/kg dose group). In addition, epididymal weights were decreased in the 200 mg/kg atrazine in the previous study, but when adjusted for body weight the decrease was no longer significant. However, following exposure to the highest dose of the metabolites, epididymal weights were significantly different whether absolute weight was compared or when the data was adjusted for changes in body weight. In summary, these observations strongly suggest that each of the 3 metabolites share a mode of action that is similar to atrazine itself.
The data presented herein, and our previous work with the parent compound also emphasize an important point concerning the effect of atrazine and its metabolites on body weight and delayed puberty. Nutritional status and body weight are known to have effects on reproduction and puberty, which are suggested to reflect the metabolic signals in the brain that serve as indices of the metabolic state. It has also long been suggested that a low body weight may contribute to a delay in puberty (Kennedy and Mitra, 1963), with food restriction resulting in a delay in pubertal onset and refeeding reversing the delay (Kennedy and Mitra, 1963
). The idea that metabolic alterations associated with weight loss or decrease in growth rate are inhibitory to the reproductive system may be related to substances in the body that can alter the release of GnRH such as insulin, amino acids necessary for precursors of neurotransmitter synthesis, and essential fatty acids (Ojeda and Urbanski, 1994
). Therefore, at doses that significantly affect body weight, these nutritional factors may contribute to the delay in pubertal progression.
For each chlorotriazine, we identified doses that resulted in more than a 10% decrease in body weight (similar to a maximum tolerated dose level for this study), doses that significantly reduced body weight but did not exceed 10% and doses that did not modify body weight. Importantly, for each compound we observed significant changes in reproductive development (PPS, organ weight decreases) at doses that were without effects on body weight. Thus, a reduction in body weight is not a prerequisite for identifying the effect of atrazine or its metabolites on reproductive development. A comparison of the relative potencies using the NOAELs for atrazine and the 3 metabolites for selected reproductive tissues and PPS is shown in Table 1.
Although some discrepancies in the dose required to produce a change in tissue weight were noted (i.e., 150 mg/kg atrazine vs. 12.5 AED of DEA to reduce seminal vesicle weight), the dose required to delay PPS was quite similar between the metabolites and the parent compound. Further studies are needed to determine the reason for these differences.
The mode of action for a decrease in testosterone and increase in estrogens, as observed in the 200 mg/kg group of atrazine, was also apparent in this study of the metabolites. DIA significantly suppressed serum testosterone at the 100 and 200 AED and DACT increased serum estrone levels at the same doses. Therefore, it would appear that the metabolites are slightly more effective in this mode of action on steroid hormone release, because 200 mg/kg atrazine was the only dose that significantly decreased intratesticular testosterone (at PND 45, but not PND 53) and increased serum estradiol and estrone concentrations. As was also apparent in the atrazine study, measurements of testosterone on PND 53 are not ideal for statistical comparisons, as mentioned in a recent review of the male pubertal protocol (Stoker et al., 2000b). Normally, testosterone levels rise gradually from PND 20 to 40, and abruptly double by PND 50 (Matsumoto et al., 1986
; Monosson et al., 1999
). For this reason, a more complete evaluation would include a time-point analysis, with testosterone measurements at five-day intervals following the first doses of atrazine (i.e., PND 28, 33, 38, 43, 48). However, it should be pointed out that testosterone-dependent alterations were observed in this study, as in the atrazine study, since it is well known that the cornification of the epithelium of the prepuse and preputial separation in immature rats is regulated by androgens (Marshall, 1966) and that a decrease in testosterone during the juvenile period can delay preputial separation (Lyons et al., 1942
) and reduce the size of the androgen-dependent tissues.
This type of time-point analysis would also be more strategic for evaluating atrazine and metabolites on serum prolactin and luteinizing hormone. From the previous work with atrazine, one would hypothesize that prolactin and luteinizing hormone would be suppressed and that would lead to decreased luteinizing hormone receptors on the Leydig cells and decreased testosterone. However, in this study and in the atrazine study on male pubertal development, there were only trends for a dose-based suppression with no significant changes in mean serum levels of PRL and LH on PND 53. Again, as mentioned, a time-point analysis may have revealed these changes.
It is also interesting to point out that both in the previous atrazine study and in the current study with the metabolites, we observed a nonuniform dose response. This occurred with PPS, ventral prostate and estradiol in the atrazine study, and with PPS and seminal vesicle with some of the metabolites. For example, in the DIA and DEA groups, the 25 AED was significant, but the 50 AED was not. This type of dose response makes the determination of the NOAEL difficult. We did not see this dose response with DACT to the same extent as atrazine, DIA, or DEA. Further investigations are necessary to better characterize this effect, but one interpretation of these results may be that pubertal development is altered through more than one mechanism of action following exposure to the chlorotriazine herbicides.
In the atrazine study, the only effects observed on the thyroid hormones or thyroid histology was an increase in total T3 at the 200 mg/kg dose level. Similarly, there were no effects on these measures following exposure to the 3 chlorinated metabolites.
In summary, the results of the present study clearly demonstrate that the primary chlorinated metabolites of atrazine appear to have a similar mode of action as atrazine when administered in the male pubertal protocol, from PND 23 to 53. All 3 of the metabolites were able to delay preputial separation significantly and decrease the weights of the reproductive tissues associated with androgenic responses. The decrease in testosterone and increase in estrone observed at necropsy in DIA and DACT, respectively, were similar to the changes seen previously following atrazine exposure. In addition, these effects appeared to occur at similar dose ranges as the previous atrazine study, with DACT closely following atrazine's effects on PPS and DIA and DEA following atrazine's effects on the reproductive tissue development. The metabolites and atrazine affected body weight at higher doses. However, this body weight difference was not observed at lower doses that delayed preputial separation in the metabolite data. Thus, body weight difference was not a prerequisite for delaying preputial separation. Together, this data strongly suggests that any evaluations of the potential toxic effects of the chlorotriazines should also include possible exposure to the metabolites produced in the soil.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (919) 5415138. E-mail: stoker.tammy{at}epa.gov.
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