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From the * Division of Reproductive Biology,
Department of Biochemistry and Molecular Biology, Johns Hopkins University
Bloomberg School of Public Health, Baltimore, Maryland; and the
Department of Biochemistry and Molecular &
Cellular Biology, Georgetown University Medical Center, Washington, DC.
| Correspondence to: Dr Barry R. Zirkin, Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 (e-mail: brzirkin{at}jhsph.edu). |
| Received for publication May 6, 2007; accepted for publication October 31, 2007. |
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Abstract |
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Key words: Gestation, Leydig cells, steroidogenesis, antiandrogen
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).
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Materials and Methods |
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Experimental Design
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 x 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.
Statistical Analysis
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 
2 test was used to evaluate differences among
proportions of live births.
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Results |
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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. 2 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.
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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.
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Preputial Separation
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).
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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).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-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 2 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.
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Footnotes |
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