Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 (e-mail: email@example.com).
ABSTRACT: Atrazine is an herbicide used worldwide to control grasses and weeds. Previous studies have shown that, depending on atrazine's administered dose, exposure of male rats during the early postnatal or peripubertal periods can result in alterations in endocrine function. The gestational period is particularly vulnerable to environmental agents; however, the possible effects of atrazine exposure during this period have received only limited attention. Herein we examine the dose effects of atrazine exposure during Sprague-Dawley rat gestation on the postnatal development of male offspring. Pregnant dams were treated by oral gavage with atrazine at 0 to 100 mg/kg/d from gestational day 14 to parturition. Thereafter, neither the pups nor the dams received atrazine. Atrazine had no effect on the number of live births per dam. Neonatal pup survival was affected, however, with increased pup death seen at doses of 10 mg/kg/d and higher. There was no effect of atrazine on the testosterone concentration within the testes of newborn pups. Anogenital distance, an androgen-dependent process, decreased from the control level at the 75 and 100 mg/kg/d doses, with the decrease reaching significance at 100 mg/kg/d. Preputial separation, also an androgen-dependent process, was delayed significantly compared with that in controls in response to the 50 and 100 mg/kg/d doses. At postnatal day 60, serum testosterone concentrations were reduced significantly from controls in the 50 to 100 mg/kg/d groups. However, these decreases had little effect on seminal vesicle or ventral prostate weights. These results, taken together, are suggestive of antiandrogenic effects of gestational atrazine exposure on male offspring, although for most parameters, the doses used in this study are unlikely to be experienced under any but experimental conditions.
Reports of global decline in the number of spermatozoa produced by men and higher incidence of cryptorchidism, hypospadias, testicular cancer, and reproductive abnormalities in wildlife have generated concern that environmental toxicants may adversely affect human reproductive health (Colborn et al, 1993; Sharpe and Skakkebaek, 1993; Toppari et al, 1996; Gray et al, 2001; Foster, 2006). A recent study of pregnant female rats exposed to the antiandrogen vinclozolin was particularly alarming in this regard; vinclozolin not only caused decreased spermatogenesis and decreased male fertility in the F1 generation, but these effects were reported to be transferred to males of subsequent generations (Anway et al, 2005).
Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is an herbicide used worldwide to control grasses and weeds. Its extensive use and its detection in surface and ground water (Baker, 1998) have made atrazine the subject of a number of studies designed to determine atrazine's possible adverse effects on endocrine parameters and reproductive function in both females and males. Exposure of female rodents to atrazine via food or gavage has been reported to cause lengthening of the estrous cycle, reduced estradiol-induced uterine weight gain, reduced uterine cytosolic progesterone receptor binding, suppression of luteinizing hormone (LH) and prolactin secretion, and early onset of mammary and pituitary tumors (Eldridge et al, 1994a,b; Simic et al, 1994; Tennant et al, 1994; Wetzel et al, 1994; Connor et al, 1996; Cooper et al, 1996, 2000, 2007; Stevens et al, 1999; Stoker et al, 1999). These effects were seen following postnatal exposures and depended upon atrazine dose and stage of the life cycle during which atrazine was administered. There also have been studies of the effects of prenatal exposure to atrazine on female offspring. For example, Rayner et al (2004) reported that administration of atrazine to pregnant rats on gestation days (GD) 15 to 19 resulted in delay of mammary gland development and that its additional administration to lactating dams resulted in delayed vaginal opening in nursing litters. This study suggested that milk-derived factors in addition to transplacental exposure may have effects on the female offspring. Additionally, pregnancy loss following atrazine administration has been reported (Narotsky et al, 2001).
Atrazine also has been shown to have effects on the male reproductive tract. As in the female, the effects have been shown to depend upon atrazine dose and period of the life cycle during which it was administered. When administered during the neonatal or peripubertal period, atrazine at relatively high (≥75 mg/kg/d) doses has been shown to result in decreases in serum levels of LH (Stoker et al, 2000; Trentacoste et al, 2001) and in serum and intratesticular levels of testosterone (Stoker et al, 2000; Trentacoste et al, 2001; Friedmann, 2002). Stoker et al (1999) reported that suckling-induced prolactin release at postnatal days 1 to 4 was reduced in response to postnatal atrazine administered to the dams and that early postnatal exposure of male pups through the dam resulted in increased prostate inflammation in the adult males. Testosterone and its metabolite dihydrotestosterone are known to affect androgen-dependent organ growth (Wilson, 1983). Not surprisingly, therefore, atrazine administration has been reported to suppress the growth of the ventral prostate and seminal vesicles (Stoker et al, 2000; Trentacoste et al, 2001). Another androgen-dependent process, preputial separation, was delayed when atrazine was administered to peripubertal rats (Stoker et al, 2000; Trentacoste et al, 2001). The mechanism by which atrazine exposure elicits these changes is unknown.
It is well established that androgens exert organizational effects on the morphogenesis of specific organs and programming effects on functions and enzyme activities that are expressed later in life (Forest, 1983). Thus, not surprisingly, the latter part of gestation, a time period in which there is significant growth and development of male reproductive organs, is considered to be particularly susceptible to environmental agents. A recent study reported the effects of the exposure of Long-Evans male fetuses to atrazine administered during gestation (Rayner et al, 2007). The authors reported that exposure of male pups to atrazine during gestation and early during postnatal life resulted in delayed preputial separation and that gestational atrazine alone resulted in increased lateral prostate weights in the adults. These results suggested that in Long-Evans rats, gestational atrazine exposure resulted in delayed preputial separation when the male offspring suckled an atrazine-exposed dam. Other than the Rayner et al (2007) study, the possible effects of atrazine exposure during gestation on the male offspring have received little attention.
Herein we examine the effects of exposing Sprague-Dawley male fetuses to increasing doses of atrazine on live births, birth weights, intratesticular and serum testosterone levels, anogenital distance (AGD), preputial separation, and the growth of androgen-dependent organs (prostate, seminal vesicles).
Materials and Methods
Animals and Treatment
Pregnant Sprague-Dawley rats were purchased from Harlan (Indianapolis, Indiana). Timed-pregnant rats typically arrived at the Johns Hopkins animal facility on GD 7 or 10. Rats were housed individually under a 14:10-hour light:dark cycle at room temperature (22°C) with food and water provided ad libitum. Atrazine (98.5% purity; kindly provided by Syngenta Crop Protection, Inc, Greensboro, North Carolina) was suspended in 0.5% carboxy methylcellulose (Sigma-Aldrich, St Louis, Missouri). Control solutions contained vehicle only. Pregnant dams received atrazine or vehicle in a volume of 2.5 mL/kg body weight by daily oral gavage starting on GD 14 and continuing until parturition. The order in which rats were gavaged was rotated each day to minimize stress-related effects on any particular group. Gavage consistently was performed in the morning hours. The atrazine dosages used were 0, 1, 10, 50, 75, and 100 mg of atrazine/kg maternal body weight/d. There were a total of 14 to 16 pregnant dams per dosage group. The day on which pups were born was referred to as postnatal day (PND) 0. All animal protocols were approved by the Johns Hopkins University Institutional Animal Care and Use Committee.
The body weights of pregnant dams were recorded daily from GD 14 to 21. The number of live births per dam was recorded. The male pups from 7 to 8 dams per dosage group (half of the dams) were euthanized by decapitation on PND 0. The testes from all male pups in each given litter (40–50 pups) were snap-frozen in liquid nitrogen and stored at −80°C. For steroid extraction, the testes were thawed, and all testes from a given litter were pooled. The testes were mechanically homogenized in 1.5 mL of phosphate-buffered saline with gelatin, and 4 mL of anhydrous ether was immediately added. Tubes were shaken 3 times for approximately 1 minute each and then stored at −80°C. The ether was decanted into glass tubes, and the tubes were placed in a 50°C water bath until the remaining ether evaporated. The dry tubes were stored at 4°C. Intratesticular testosterone was assayed by radioimmunoassay (RIA) using a testosterone antibody from ICN Pharmaceuticals (Costa Mesa, California) and 3H-testosterone from NEN Life Science Products (Boston, Massachusetts). The sensitivity of the assay was 10 pg/tube. The RIA procedure was as described previously (Turner et al, 1984).
With the remaining 7 to 8 dams per dosage group, the number of pups per dam was culled to 8 (4 males and 4 females) on PND 2. The body weights and AGD were measured for each pup on PND 21. AGD was measured with a dial vernier caliper (Electron Microscopy Sciences, Fort Washington, Pennsylvania), as described previously (Gallavan et al, 1999). An AGD index was calculated as AGD/cube root of body weight. The rationale for this AGD index is that it accounts for the effect of pup size on AGD (Gallavan et al, 1999). An average AGD index was determined per litter; means per treatment group were determined by averaging litter averages. Thus, for statistical analyses of this and other measurements, the average value per litter represented n = 1.
Following AGD measurements on PND 21, the male pups were weaned, and the 4 male pups from a given litter were housed together. Preputial separation, the separation of the foreskin of the penis from the glans penis, is considered to be an early marker of the progression of puberty (Korenbrot et al, 1977). Preputial separation was assessed in each of the 4 pups per litter by manually retracting the prepuce with gentle pressure. Preputial separation was monitored daily beginning on PND 37, at approximately the same time each day, and an average day of preputial separation was determined for each litter. In rats, preputial separation typically occurs between 40 and 50 days, depending on the strain (Korenbrot et al, 1977).
The same 4 male pups per litter from which the AGD and preputial separation data were obtained were euthanized by decapitation on PND 60. Trunk blood was collected and kept on ice. Serum was collected and stored at −20°C. Testes were removed and weighed individually. Each testis was decapsulated, and the tubules were pressed through a 3-mL syringe into a centrifuge tube. The tubes were spun at 6000 × g for 15 minutes at 4°C. The intratesticular fluid, which included fluid from both the seminiferous tubules and interstitial space, was then placed into a 0.5-mL microcentrifuge tube and frozen in liquid nitrogen. For measuring testosterone, steroid was extracted from 10 μL of intratesticular fluid or 100 μL of serum from each animal, as described above. The ventral prostate and seminal vesicles were dissected out and weighed. As above, values obtained from rats of a given litter were averaged.
Values are presented as mean ± SEM. Means were analyzed initially by 1-way analysis of variance. Significant effects were indicated when P < .05. If effects were significant, the means were compared by Dunnett's post hoc test and considered to be significantly different at P < .05. The χ2 test was used to evaluate differences among proportions of live births.
Effects of Atrazine Administration on Pregnant Dam Weight Gain
We first investigated the effect of atrazine administered from GD 14 to parturition on weight gain by the pregnant dams (Figure 1). (For all figures, the control group [atrazine at 0 mg/kg] is designated C, and the 1, 10, 50, 75, and 100 mg/kg groups are designated A1, A10, A50, A75, and A100, respectively.) Maternal body weights were comparable in all groups on GD 14, the start of gavage. On the day before delivery (GD 21), there were no differences in body weights among the C, A1, A10, and A50 groups. By GD 18, however, there were significant reductions in body weights in the A75 and A100 groups compared with the C group. Based on a previous study which indicated that high-dose atrazine during the peripubertal period resulted in reduced food intake and thus reduced weight gain (Trentacoste et al, 2001), we hypothesized that one possible explanation for the decrease in maternal body weight in response to atrazine might be reduced food consumption. Consequently, daily food consumption by the pregnant dams was recorded. No differences in average daily food consumption were noted for the A1 and A10 groups compared with the C group; in the A50, A75, and A100 groups, the dams consumed less food than the C group by approximately 33%, 38%, and 47%, respectively (data not shown).
Live Births, Pup Survival, and Pup Weights Following Atrazine In Utero
The average number of pups born to individual dams in the C and atrazine groups ranged from 11 to 14, with no differences regardless of atrazine dose. However, there were dose-dependent differences in pup survival. Figure 2 shows the percentage of pups that died between PND 0 and PND 2. With increasing atrazine dose, the percentage of dead pups increased from less than 1% for the C and A1 groups to approximately 10% in the A10 and A50 groups and 25% in the A75 and A100 groups. χ2 analysis indicated that the increases in pup death compared with control values reached significance in the A75 and A100 groups. Pup weights in the C, A1, A10, A50, A75, and A100 groups on PND 2 were 8.0 ± 0.4, 7.9 ± 0.2, 8.2 ± 0.3, 7.2 ± 0.2, 6.8 ± 0.3, and 6.6 ± 0.4 g, respectively. Although there was a downward trend, there were no significant differences among these means.
Intratesticular Testosterone Concentrations on PND 0
No significant differences were seen in mean testosterone concentrations within the testicular fluid of PND 0 pups among any of the dosage groups (Figure 3). Interestingly, a 60% increase in intratesticular testosterone concentration was seen in the A10 group, but this increase did not reach statistical significance.
AGD Index on PND 21
No significant differences were seen in the AGD index in female offspring at any atrazine dose (data not shown). Figure 4 shows the AGD index of male offspring on PND 21. An increase was noted in the AGD index in the A10 group compared with the C group that did not reach statistical significance. (A nonsignificant increase in intratesticular testosterone concentration was also noted in the A10 group compared with the C group.) Decreases from the level in the C group occurred in the A75 and A100 groups, reaching significance in the A100 group.
Figure 5 shows the mean age of preputial separation in the atrazine dosage groups. Preputial separation occurred around PND 41 in the C, A1, and A10 groups. Preputial separation was delayed significantly, by about 1 day, in the A50 and A75 groups (P < .05 and P < .01, respectively) and by 2.5 to 3 days in the A100 group (P < .004).
Serum Testosterone Levels and Androgen-Dependent Organs in Adults (PND 60)
The same male pups from which the AGD and preputial separation data were obtained were sacrificed on PND 60. As for all other measures, values from the 4 male pups per litter were averaged, and grand means per treatment group were compared. No significant differences were seen in body (Figure 6A) or testis (Figure 6B) weights, although there was a downward trend for both measures in the A100 group. No significant differences were seen among any dosage groups with respect to seminal vesicle (Figure 6C) or ventral prostate (Figure 6D) weights. Serum testosterone concentrations were significantly reduced from control values in the A75 and A100 groups (Figure 6E). Intratesticular testosterone concentration was reduced significantly in the A50, A75, and A100 groups compared with the C group (Figure 6F).
When administered during the neonatal or peripubertal periods, atrazine, depending upon its dose, has been shown in previous studies to result in decreases in serum levels of LH (Stoker et al, 2000; Trentacoste et al, 2001), serum and intratesticular levels of testosterone (Stoker et al, 2000; Trentacoste et al, 2001; Friedmann, 2002), and ventral prostate and seminal vesicle weights (Stoker et al, 2000; Trentacoste et al, 2001). Additionally, postnatal exposure to atrazine, at doses of 100 to 200 mg/kg/d, has been shown to delay preputial separation, a marker of puberty onset (Stoker et al, 2000; Trentacoste et al, 2001). A recent study (Rayner et al, 2007) reported that exposure of pregnant and early postpartum dams to atrazine at 100 mg/kg/d resulted in preputial separation delays in the male offspring and had effects on the prostates of the adult males. Indeed, there are number of agents in the environment that have been shown to have similar effects on androgen-dependent processes. For example, prenatal exposure to vinclozolin, p,p′-dichlorodiphenyldichloroethylene, or diethylhexyl phthalate (DEHP) has been shown to decrease AGD and androgen-dependent organ weights, increase the percentage of pups with hypospadias and areolas, and increase the number of permanent nipples (Gray et al, 1999). In some cases, agents considered to be antiandrogenic have been shown to act directly on the steroidogenic machinery. For example, dimethoate has been reported to inhibit steroidogenic acute regulatory gene transcription (Walsh et al, 2000), octylphenol to decrease 17α-hydroxylase/lyase activity and interfere with LH binding to its receptor on Leydig cells (Murono et al, 2000), and DEHP to cause declines in the activities of androgen biosynthetic enzymes (Akingbemi et al, 2001).
These are among the observations that led to our current study of the effects of atrazine administered during the gestational period on neonatal and later endocrine and reproductive tract parameters. It should be noted that for our studies, the pups were not treated directly with atrazine but rather were exposed through maternal gavage from GD 14 to parturition and that neither the pups nor the dams were treated with atrazine after parturition.
The administration of atrazine at 1 or 50 mg/kg/d to pregnant dams from GD 14 to parturition did not affect weight gain by the dams; however, administration at 75 or 100 mg/kg/d resulted in significantly reduced weight gain. Rayner and colleagues (2007) also showed reduced weight gain by pregnant Long-Evans dams in response to the administration of 100 mg/kg/d atrazine from GD 15 to 19. Previous studies reported that food restriction during the late stages of gestation and during lactation can lead to reductions in testosterone concentration and thus delays in preputial separation in male offspring (Korenbrot et al, 1977). In our study, however, the 10 and 50 mg/kg/d doses did not affect dam weight or food intake, but there was a 10-fold increase in pup death rate compared with the controls. Although these increases were not significant by χ2 analysis, they nonetheless were substantial. Thus, at least at the 10 and 50 mg/kg/d doses, the apparent increases in pup death were unrelated to decreased dam food intake or weight. The cause of increased pup death is uncertain for any atrazine dose. One possibility is that atrazine, through its effects on prolactin (Stoker et al, 1999; Cooper et al, 2000), might result in altered milk production or quality and in this way have detrimental effects on fetal survival. In this regard, a previous study of prenatal exposure to atrazine on Long-Evans female rats suggested that delays in vaginal opening and mammary gland development in atrazine-exposed females might result from effects of atrazine on milk-derived factors (Rayner et al, 2004).
Preputial separation has been shown to be androgen dependent, normally occurring when androgen levels are rapidly increasing. In our study, there was a significant delay in preputial separation in response to atrazine at 50, 75, or 100 mg/kg/d. A recent study of the effects of atrazine administration during gestation also showed that preputial separation in Long-Evans rats was delayed but only when pups that had been exposed during gestation were also exposed to atrazine during the early postnatal period (Rayner et al, 2007). It is possible that strain differences explain the difference in results. Previous studies reported that atrazine, when administered during the peripubertal period, also caused delays in preputial separation (Stoker et al, 2000; Trentacoste et al, 2001). Whether the delays in preputial separation seen in our study resulted in some way from reduced food intake by the dams receiving the highest (75 and 100 mg/kg) doses, through a direct effect on steroidogenesis, or by some other mechanism was not determined. Interestingly, there was no effect of gestational atrazine of any dose on PND 0 intratesticular testosterone concentrations, suggesting that neonatal testosterone production may not have been affected by atrazine even at the highest doses used in this study. Thus, the mechanism for the delay in preputial separation delay is not known.
The AGD index (AGD/cube root of body weight) has been shown to be a sensitive biomarker of the effects of known antiandrogens such as dibutylphthalate (Mylchreest et al, 2000), flutamide (McIntyre et al, 2001), and finasteride (Bowman et al, 2003). Atrazine administration during gestation had no effect on the AGD index of female offspring. In males, however, the 75 and 100 mg/kg/d doses resulted in AGD index reductions from the control, reaching statistical significance at 100 mg/kg. It should be noted that the reductions in AGD index occurred in the dosage groups (75 and 100 mg/kg/d) in which there was reduced weight gain by the dams.
The same male pups (4 per litter) from which the AGD and preputial separation data were obtained were used to obtain values for serum testosterone concentrations and androgen-dependent organ weights when the rats reached 60 days. Serum testosterone concentrations were reduced significantly from the control at the 75 and 100 mg/kg doses. The mechanism by which gestational administration could have caused these results in the adult is not known. Despite the reduced serum testosterone levels, the weights of the seminal vesicles and ventral prostates in these rats did not differ from their respective controls, indicating that the reductions in serum testosterone were not sufficient to affect these androgen-dependent organs. The observation that serum testosterone levels may be reduced without effects on androgen-dependent organs is not unprecedented; prenatal exposure of rats to mestranol was shown to result in a 50% decrease in serum testosterone levels in adults (PND 120) without reductions in the weights of the seminal vesicles or ventral prostates (Varma and Bloch, 1987).
In conclusion, our data suggest that atrazine, when administered during gestation and depending upon its dose, can have effects in utero that manifest in the neonatal and prepubertal periods, as well as effects that persist into adulthood. In our study, the 10 and 50 mg/kg/d atrazine doses affected neonatal pup death for unknown reasons, but the doses had minimal, if any, effects on intratesticular testosterone concentrations in the neonates or on AGD. Atrazine administration at 50 to 100 mg/kg/d did result in delayed preputial separation and also in reduced intratesticular and serum testosterone concentrations in the adult but did not have effects on seminal vesicle or ventral prostate weights. At this point, it remains unclear whether atrazine, even at high doses, has significant acute or long-term pathophysiologic effects that have relevance to humans or to wildlife.
This work was supported by grants from the National Institutes of Health (ES 013495; V.P. and B.R.Z.) and Novartis Crop Protection, Inc (B.R.Z.).