The Effects of Atrazine Metabolites on Puberty and Thyroid Function in the Male Wistar Rat

T. E. Stoker*,1, D. L. Guidici{dagger}, S. C. Laws{dagger} and R. L. Cooper{dagger}

* Gamete and Early Embryo Biology Branch and {dagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently we reported that atrazine (ATR), a chlorotriazine herbicide, alters the onset of puberty in male Wistar rats. In this study, we examined the same reproductive parameters in the developing male rat following a similar exposure to the primary, chlorinated metabolites of atrazine. Intact male Wistar rats were gavaged from postnatal day (PND) 23 through PND 53 and several reproductive endpoints were examined. The doses selected were the molar equivalents to atrazine in our previous work. Deethylatrazine (DEA), deisopropyl-atrazine (DIA), and diaminochlorotriazine (DACT) were administered by gavage at doses equivalent to the atrazine equimolar doses (AED) of 6.25, 12.5, 25, 50, 100, or 200 mg/kg. Preputial separation (PPS) was significantly delayed by DEA at 25, 100, and 200 AED, by DIA at 25, 100, and 200 AED, and by DACT at 12.5 through 200 AED. When the males were killed on PND 53, DEA (100 and 200 AED), DIA (50 through 200 AED), and DACT (200 AED) treatments caused a significant reduction in ventral prostate weight, while only the highest doses of DIA and DEA resulted in a significant decrease in lateral prostate weight. Seminal vesicle weight was reduced by DEA (25, 100, and 200 AED), DIA (100 and 200 AED), and 100 and 200 AED of DACT. Epididymal weights were reduced in the DEA (200 AED), DIA (200 AED), and DACT (100 and 200 AED) treatment groups. Serum testosterone was reduced only in the males receiving the 2 highest doses of DIA. Serum estrone was increased in the 2 highest doses of the DACT group, while serum estradiol was not different in any group. No differences were observed in any of the thyroid measures. In summary, the metabolites of ATR delay puberty in a manner similar to that observed in the previous study testing atrazine. These data also suggest that the 3 chlorinated metabolites are similar to ATR, by affecting the CNS control of the pituitary/gonadal axis and subsequent development of the reproductive tract.

Key Words: atrazine; diaminochlorotriazine; deethylatrazine; deisopropylatrazine; preputial separation; hormones; puberty; reproductive tract.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine) is a chloro-s-triazine herbicide that has been employed extensively in agriculture in the U.S. and worldwide for over 40 years (U.S. EPA, 1994Go). It was first introduced in 1958 and provided farmers with an alternative to 2, 4-D for the control of grasses and other weeds (Gianessi, 1998Go). In the United States, approximately 75 million pounds of atrazine are used each year, which is more than any other agricultural herbicide. Atrazine is used primarily in the growing of corn and is also used extensively for sorghum and sugar cane (U.S. Department of Agriculture, 1990–1994Go). The primary mode of action of atrazine in plants is to inhibit photosynthesis (Gysin and Knuesli, 1960Go).

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., 1999Go), 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 (50–300 mg/kg by gavage) suppressed the estrogen-induced surge of luteinizing hormone and prolactin (Cooper et al., 1996Go, 2000Go) 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., 1996Go). 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., 2000Go). 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., 1998Go). 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. 1Go). 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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FIG. 1. Overview of the EDSTAC 31-day Tier 1 Pubertal Male Rat Assay designed to screen for endocrine-disrupting chemicals for effects on pubertal progression.

 
The metabolic composition of chlorotriazines in the urine of different species has been well studied (see Fig. 2Go). In the adult rat, 5 major types of urinary metabolites have been identified, including (1) 2-hydroxyatrazine, (2) 2-chloro-4-amino-6-(ethylamino)-s-triazine or DIA, (3) 2-chloro-4-amino-6-(isopropylamino)-s-triazine or DEA, (4) 2-chloro-4,6-diamino-s-triazine or DACT, and (5) ammeline (Bakke et al., 1972Go; Bradway and Moseman, 1982Go). In the urine of occupationally exposed males, DIA, DEA, and DACT have been identified (Catenacci et al., 1990Go; Ikonen et al., 1988Go).



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FIG. 2. Atrazine metabolism and major metabolites.

 
Atrazine is relatively persistent in soils (Seiler et al., 1992Go) and has been detected in the surface and groundwater in several areas of the United States where its usage is greatest (Baker, 1988Go) at levels exceeding the Environmental Protection Agency's maximum contaminant level of 3 ppb (Kello, 1989Go). Microbial degradation of atrazine in the soil yields deethylatrazine or DEA (6-chloro-N-[1-methylethyl]-1,3,5-triazine-2,4-diamine) and deisopropylatrazine or DIA (6-chloro-N-ethyl-1,3,5-triazine-2,4-diamine). These microbial degradation products have relatively high mobility and potential to contaminant groundwater (Sorenson et al., 1993Go). In addition, photodegradation of atrazine on the soil surface produces DEA and diaminochlorotriazine (DACT; Koskinen and Clay, 1997Go). Chemical hydrolysis of atrazine to 6-hydroxyatrazine also occurs in surface soil (Harris, 1967Go). Thus, the degradation of atrazine in vivo and in the environment produces many of the same metabolites.

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Timed-pregnant female Wistar rats were purchased from Charles River (Raleigh, NC) and shipped to arrive on gestation day 13. Upon arrival the rats were housed 1 per cage in an AAALAC accredited facility maintained at 22°C and on a 12 h:12 h light:dark cycle (on 0500 h, off 1700 h). Food (Purina laboratory rat chow 5001) and water were provided ad libitum unless otherwise noted. The day of delivery was designated postnatal day (PND) 0. On PND 3, the pups were culled to 8 to 10 per litter to maximize uniformity in growth rates. On PND 22, all male pups were weaned and weighed to the nearest 0.1 g and weight-ranked. Pups were then assigned so that treatment groups exhibited similar body weight means and variances. Littermates were also equally distributed among the treatment groups. After assignment, similarly treated males were housed 2 per cage and weighed daily throughout treatment (Fig. 3Go). All males were sacrificed on PND 53 or 54 for the protocol endpoint measures.



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FIG. 3. Effect of atrazine metabolites on growth from PND 23 to 53. Metabolite treatment indicated as AED. (A) DIA effect on growth; a, p < 0.05 for body weight differences at PND 23, 33, 43, or 53; b, body weight mean exceeded a 10% decrease as compared to the control mean. (B) DEA effect on growth; a, p < 0.05 for body weight differences at PND 23, 33, 43, or 53; b, body weight mean exceeded a 10% decrease as compared to the control mean. (C) DACT effect on growth; a, p < 0.05 for body weight differences at PND 23, 33, 43, or 53; b, body weight mean exceeded a 10% decrease as compared to the control mean.

 
Treatment.
All treatments were administered daily by po gavage from PND 23 to 53 or 54 (for males killed on PND 54) at a volume of 0.5 cc per 100 g body weight between 0800 and 0900 h. All metabolites (Novartis, Greensboro, NC, 97–98% purity) were administered as a suspension in a 1% solution of methyl cellulose (Sigma Chemical, St. Louis, MO). Dose groups were selected based on the previous study with atrazine using the pubertal protocol (Stoker et al., 2000aGo, Table 1Go). Although the 200 mg/kg dose of atrazine decreased body weight more than 10% below controls, the metabolite molar equivalent to this dose was selected as the highest dose level for a comparison to the atrazine dose response study (Tables 1 and 2GoGo). Therefore, groups of rats were treated with DIA (10.4, 20.8, 40.1, 80.3, or 160.9 mg/kg body weight/day), DEA (21.7, 43.4, 86.8, or 173.91 mg/kg body weight/day), or DACT (4.4, 8.4, 16.9, 33.8, 84.3, or 135.2 mg/kg body weight/day) in a suspension of methyl cellulose. For the purposes of this article and to facilitate the comparison to atrazine, all metabolite doses are presented as atrazine equimolar doses (AED; See Table 2Go for dose chart). Control males received the 1% solution of methyl cellulose only. The number of animals per treatment group is noted on the x-axis of Figure 4Go.


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TABLE 1 NOAELs for Decreased Reproductive Tract Weights and for Delayed Preputial Separation following Exposure to Atrazine and Metabolites in the Male Rat Pubertal Protocol
 

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TABLE 2 Atrazine Equimolar Doses
 


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FIG. 4. Effect of atrazine metabolites on postnatal day of preputial separation. Metabolite treatments indicated as atrazine equivalent dose or AED. Mean ± SEM day of PPS; *p < 0.05 as compared to control mean. Numbers under bars indicate the number of males per treatment group.

 
Preputial separation.
The separation of the foreskin of the penis from the glans penis, preputial separation (PPS), is an early reliable marker of the progression of puberty that normally occurs between 40 and 50 days of age, with an average of 43 days, depending on the rat strain (Korenbrot et al., 1977Go). In the present study, PPS was monitored beginning on PND 33 and continued until all males showed separation. All males were monitored daily at approximately the same time each day. A partial separation with a thread of cartilage remaining was recorded as "partial," but only the day of complete separation was used in the data analyses.

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., 1986Go). 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 4–6 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body Weight
The growth of the 2 highest dose groups of each metabolite was significantly reduced as compared to the controls (Fig. 3Go; Table 3Go). We expected the highest doses, which were equimolar to the 200 mg/kg dose of atrazine, to significantly decrease body weight by PND 53. This was confirmed by a greater than 10% decrease in body weight in the highest dose group of each metabolite. However, the mean body weight of the animals receiving the next highest dose of DEA also exceeded this level on PND 53. Although there was a statistically significant decrease in body weight in the 2 highest doses of the metabolites beginning on PND 33 or 43, this decrease did not always exceed 10% (Fig. 3Go; Table 3Go).


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TABLE 3 Mean Weights
 
Preputial Separation
The day of preputial separation was delayed by all 3 metabolites, and a NOAEL level was established for each (Fig. 4Go). In the DIA group, PPS was significantly delayed by the 25, 100, and 200 AED (by 1.2, 1.2, and 3.4 days respectively) with a NOAEL of 12.5. In the DEA groups, PPS was significantly delayed in the 25, 100, and 200 AED (by 1, 2, and 4 days, respectively) with a NOAEL of 12.5. PPS was also significantly delayed by 12.5, 25, 50, 100, and 200 AED of DACT (by 1, 2.3, 1, 4, and 4.2 days, respectively) with a NOAEL of 6.25.

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. 5Go). However, the lateral prostate weights were reduced only by 100 and 200 AED (Table 3Go). Likewise, the mean seminal vesicle weight was lower than controls at 100 and 200 (Fig. 6Go). 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 3Go).



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FIG. 5. Effect of atrazine metabolites on ventral prostate weight at PND 53. Metabolite treatments indicated as AED. Mean ± SEM; *p < 0.05 as compared to control mean.

 


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FIG. 6. Effect of atrazine metabolites on seminal vesicle with coagulating gland weight (with fluid) at PND 53. Metabolite dose indicated as AED. Mean ± SEM; *p < 0.05 as compared to control mean; a, no longer significant when weights were adjusted for body weight on PND 53; b, became significant when weights were adjusted for body weight on PND 53.

 
In the DEA treatment group, the ventral and lateral prostate weights were decreased by the 2 high treatment groups (Fig. 5Go; Table 3Go). The seminal vesicle weights were decreased at 25, 100, and 200 AED (Fig. 6Go). However, when analyzed as a covariate to body weight at PND 53, the 25 AED group was no longer significant. In addition, the epididymis was decreased at 200 AED of DEA (Table 3Go).

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. 5Go; Table 3Go). The seminal vesicles and epididymis weights were reduced in the 100 and the 200 AED groups as compared to controls (Fig. 6Go). 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 3Go).

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. 7Go). 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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FIG. 7. Effect of atrazine metabolites on serum estrone (top) and serum testosterone (bottom) concentrations on PND 53. Metabolite treatments indicated as AED. Mean ± SEM; *p < 0.05 as compared to control mean.

 
Serum estradiol levels were unaffected by any of the metabolite treatment groups (data not shown), as compared to controls. However, serum estrone was significantly increased in the 100 and 200 mg/kg dose group of DACT as compared to controls (Fig. 7Go).

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 4Go; 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 4Go). 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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TABLE 4 Hormone Measures at Necropsy on PND 53
 
Thyroid hormones.
Serum thyroid stimulating hormone, total thyrotropin (T4), and total triiodothyronine (T3) measured on PND 53 were unaffected by any of the metabolite treatment groups (Table 4Go) when compared to the controls. Also, no effects were observed on thyroid histology of the high dose or controls.

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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of this study confirm that the 3 primary chlorinated metabolites of atrazine, deisopropylatrazine (DIA), deethylatrazine (DEA), and diaminochlorotriazine (DACT) are able to delay pubertal progression and reproductive tract development in the Wistar rat. Because atrazine is persistent for extended periods in the environment (Rodriguez and Harkin, 1997Go), there is potential for this herbicide and its microbial degradation metabolites to affect human and wildlife populations. Since current maximum contaminant levels are only set for atrazine, not its metabolites, it is important to determine the in vivo effects of the metabolites. For this reason, we addressed 4 primary issues that are relevant to the risk assessment of atrazine and its metabolites: (1) whether the chlorinated metabolites affected pubertal development in the male rat in a way that was similar to that of atrazine (i.e., same mode of action); (2) whether the effects observed were apparent at doses below the dose that resulted in a 10% decrease in necropsy body weight; (3) whether 1 metabolite was more active in its effects on reproductive development in the male than the other two; and (4) a comparison of effects and NOAELs between atrazine and the metabolites.

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., 2000aGo), 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, 1963Go), with food restriction resulting in a delay in pubertal onset and refeeding reversing the delay (Kennedy and Mitra, 1963Go). 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, 1994Go). 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 1Go.

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., 2000bGo). Normally, testosterone levels rise gradually from PND 20 to 40, and abruptly double by PND 50 (Matsumoto et al., 1986Go; Monosson et al., 1999Go). 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., 1942Go) 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.


    ACKNOWLEDGMENTS
 
The authors express their gratitude to the National Hormone and Pituitary Agency for the gift of the radioimmunoassay materials. We would also like to thank Al Moore, Janet Ferrell, and Keith McElroy for their excellent technical contributions.


    NOTES
 
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed. Fax: (919) 541–5138. E-mail: stoker.tammy{at}epa.gov. Back


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