CIIT Centers for Health Research, P. O. Box 12137, Research Triangle Park, North Carolina 27709-2137
Received June 1, 2001; accepted September 25, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: AGD; nipple retention; linuron; male reproductive development; testicular atrophy; in utero exposure; gestational exposure.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Male rats exposed in utero to antiandrogens often display alterations in androgen-mediated development, as evidenced by decreased anogenital distances (AGD) and retention of areolae and/or nipples, together with clearly adverse responses such as genital malformations and reproductive tract lesions. However, the correlation between end points that signify antiandrogen-mediated perturbations (e.g., decreased AGD and nipple retention) and adverse and irreversible malformations in androgen-dependent development is unclear. Confusion exists about the biological significance of antiandrogen-mediated changes in these end points and how they may be used in risk assessment. Male rats exposed in utero to finasteride, a 5-reductase inhibitor that blocks the conversion of testosterone (T) to dihydrotestosterone (DHT), display decreased AGD at birth (Clark et al., 1993
, 1990
). However, these offspring displayed catch-up growth and adult animals exposed to low doses of finasteride in utero displayed AGDs similar to control animals at adulthood, suggesting that decreases in AGD seen in early postnatal life are transient. These investigators also indicated that areolae/nipples seen in early postnatal rats were temporary (Clark et al., 1990
). In contrast to these previous studies with finasteride, rats exposed to the antiandrogens diethylhexylphthalate, di(n)butyl phthalate, and linuron displayed retained nipples at both PND 13 and at the adult (PND 180270) necropsy (Gray et al., 1999
). Whether antiandrogen-induced changes in these early postnatal end points are predictors and/or indicators of subsequent adverse responses in rat reproductive development is unknown.
Linuron is a pre-and postemergence herbicide applied to suppress broadleaf and grassy weeds. In a 3-generation reproductive study in rats, animals exposed to 625 ppm (31.5 mg/kg/day) in the diet exhibited decreased fertility in the F2a and F3a generations but not the F1 generation (U.S. EPA, 1995
). In this study, linuron also decreased reproductive performance, as evidenced by decreased pup survival at the 625-ppm dose level and decreased pup weights (male and female) at both the 125-ppm (6.25 mg/kg/day) and 625 ppm dose levels (U.S. EPA, 1995
). In a later, 2-generation reproductive toxicity study in rats conforming to relevant guidelines at that time, dietary exposure to 625 ppm (4454 mg/kg/day) of linuron had no effect on fertility (U.S. EPA, 1995
). However, testicular and epididymal pathology (testicular atrophy, intratubular fibrosis, epididymal inflammation, and oligospermia) were observed in the F1 adults but not in the parental generation, no epididymal malformations were reported (U.S. EPA, 1995
). In a recently published, multigenerational study in rats, treatment with 40 mg/kg/day of linuron by gavage delayed the onset of puberty and decreased seminal vesicle and cauda epididymal weights in the F0 generation (Gray et al., 1999
). F1 rats exposed to linuron in utero and via milk during lactation and gavaged after weaning produced fewer pups when mated continuously over 12 breeding cycles (Gray et al., 1999
). Male offspring displayed reduced testicular and epididymal weights and decreased spermatid numbers. Linuron has been reported to be neither teratogenic nor embryo-toxic when administered by gavage to pregnant rats from GD 6 to 15 at dose levels as high as 100 mg/kg/day (Khera et al., 1978
). In a developmental toxicity study in the rat, dams administered linuron in the diet at 625 ppm exhibited decreased body weight and food consumption, increased postimplantation loss, and increased litter and fetal incidences of resorptions (developmental lowest-observable-effect level (LOEL) in rat) (U.S. EPA, 1995
). Rabbits exposed in utero to 100 mg/kg/day from GD 7 to 19 by gavage exhibited skeletal variations of the skull (developmental LOEL in rabbit) (U.S. EPA, 1995
). Late gestational exposure to linuron during androgen-dependent reproductive development has been shown to cause epididymal malformations, hypospadias, and decreases in AGD, and to induce retention of areolae and nipples in male rats (Gray et al., 1999
; McIntyre et al., 2000
). Linuron is a weak competitive androgen receptor antagonist in vitro and induces a positive response in the immature and adult Hershberger assay (Cook et al., 1993
; Lambright et al., 2000
; McIntyre et al., 2000
). Although the exact mechanism is unknown, these data suggest that the antiandrogenic effects observed in linuron-exposed rats are the result of altered androgen-receptor-dependent rat reproductive development.
Male rats exposed to linuron in utero exhibit dose-dependent epididymal abnormalities (epididymal hypoplasia and agenesis). These alterations are associated with ipsilateral testicular atrophy in the adult rat (Gray et al., 1999; Lambright et al., 2000
; McIntyre et al., 2000
).
Whether the observed linuron-mediated testicular lesions in adult rats are a direct effect of linuron on the testes or are secondary to malformations of the epididymides is unclear. Therefore, the objectives of this study were to determine whether (1) linuron-induced changes in AGD and areola/nipple retention in rats are permanent; (2) linuron-induced epididymal malformations are associated with increased testicular size (the result of restricted or obstructed testicular fluid outflow from the testis, causing subsequent seminiferous epithelial degeneration and testicular atrophy); and (3) there is an association between rats that display reproductive tract lesions and either areolae on PND 13 or permanent nipple retention. To test these hypotheses, pregnant rat dams were treated with either corn oil (vehicle control) or 50 mg/kg/day of linuron from GD 12 to 21. This dose level was selected from previous dose-response studies (McIntyre et al., 2000) to maximize effects on the fetus while minimizing any toxicity to the dam. Male offspring were uniquely identified at birth. Male rats were euthanized on PND 35 (low testicular fluid outflow) and 56 (high fluid outflow) (Setchell et al., 1994
), and male offspring were inspected for permanent decreases in AGD and retention of areolae on PND 13 and nipples at necropsy.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment.
Sperm-positive animals, 8 control dams and 20 dams receiving 50 mg linuron/kg/day, were gavaged daily (08001030) from GD 12 to 21 with either 2 ml/kg/day corn oil (Sigma) or 2 ml/kg/day linuron (50 mg/kg/day). This dose level was chosen based on a previous study in which pregnant rat dams receiving 50 mg/kg/day of linuron from GD 12 to 21 displayed limited maternal toxicity (decreased weight gain during treatment). However, adult male offspring exhibited approximately a 12% incidence in epididymal abnormalities and testicular atrophy (McIntyre et al., 2000). Dams were examined daily for clinical signs of toxicity.
Androgen-dependent reproductive end points.
On the day of delivery, which was considered to be postnatal day (PND) 1, pups were counted and examined for signs of clinical toxicity. Pups were uniquely identified by foot tattoo, and the AGD was measured using a dissecting microscope with an eyepiece reticle (accuracy of 0.05 mm). An individual investigator who was unaware of animal exposure measured the AGD for all pups. Pup litter weights (by sex and litter) and individual pup body weights after weaning were collected weekly. On PND 13, a single investigator, who was unaware of animal exposure, inspected male pups for the presence and number of either areolae or nipples. No distinction was made between the retention of an areola or nipple on PND 13.
Necropsy of F1 animals.
Pups were weaned on PND 21, and dams were euthanized by CO2 asphyxiation. The male offspring from one-half of the control and linuron-treated dams were necropsied on PND 35 and the other half on PND 56. Rats were euthanized by decapitation, and trunk blood was collected. Following blood collection, the ventral surface of the animal was shaved for counting the number of nipples, and the AGD was measured with a dial caliper (on PND 56 only). The external genitalia, scrotum, prepuce, and penis were visually inspected. Gross internal examination of the reproductive tract included inspection of the testes, epididymides, vasa deferentia, prostate, seminal vesicles, and coagulating glands. Additionally, the liver, kidneys, and adrenal glands were grossly examined. Body weight and weight of the epididymides and testes were collected. Tissues (right testis and epididymis) were fixed in 10% neutral buffered formalin (for ancillary immunohistochemical studies), processed, paraffin-embedded, sectioned (5 µm), and stained with hematoxylin and eosin. The left testes and epididymides were retained for ancillary studies.
Statistical analysis.
The litter was the experimental unit, unless otherwise noted. Statistical analyses were conducted using JMP (version 4.0.0, SAS Institute, Cary, NC). Retained nipples on PND 35 and 56 were combined and compared to control. Normality (Sharpiro-Wilk) and homogeneity (Bartlett) assumptions were tested prior to analysis. Pups were nested by dam to yield litter means. Either ANOVA or ANCOVA was used to test for significance of treatment effects, and the covariates are defined in the figure legends. If the p value for treatment effects was less than 0.05, post hoc comparisons of either Dunnett's (for ANOVA) or contrasts of least-square means were used to assess the significance of treatment differences. The Bonferroni correction was applied for post hoc ANCOVA analyses, as appropriate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Linuron-Induced Perturbations in AGD, and Retention of Areolae/Nipples
On PND 1, male offspring from linuron-treated dams displayed a significant decrease in the nested litter mean AGD of male offspring by approximately 8% when compared to control animals (Fig. 1). On PND 56, the litter-mean AGD of linuron-exposed animals displayed a statistically significant decrease of 5% relative to control animals (Fig. 1
). On PND 13, the nested litter mean for the number of retained areolae per pup from vehicle-treated control dams had a value of less than one. Male offspring exposed prenatally to linuron exhibited a litter mean of 3.3 areolae per pup (Fig. 2
). At necropsy, male offspring exposed to the vehicle control displayed a low incidence of retained nipples (one control male rat displayed 2 nipples at necropsy with 1 areola present on PND 13), and the nested litter mean was 0.04 nipples per rat. Linuron-exposed animals displayed a nested litter mean of 1.7 nipples per rat, and this increase was significant as compared to vehicle control-exposed animals (Fig. 2
).
|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In our current study, male rats exposed in utero to linuron exhibited a significant decrease in AGD on PND 1 and PND 56. It has been previously demonstrated that rats exposed prenatally to linuron exhibit a decrease in AGD measured shortly after birth, and this magnitude of decrease on PND 1 (8%) is similar to what we previously observed under the same dosing regimen (McIntyre et al., 2000). These data demonstrate that small decreases in AGD as a result of antiandrogen exposure are reproducible from experiment to experiment within a given laboratory. Studies in our laboratory as well as other laboratories have demonstrated that prenatal exposure to vinclozolin and flutamide can also induce permanent decreases in AGD (Gray et al., 1994
, McIntyre et al., 2001
). In contrast, previous investigators utilizing finasteride have suggested that these decreases are, in part, transient (Clark et al., 1990
). In the current study, rats exposed to linuron displayed an 8% decrease (relative to control) in AGD on PND 1 and 5% on PND 56. In addition, we have previously shown that rats exposed to 6.25 mg/kg/day of flutamide exhibit a 43% decrease in AGD on PND 1 but only 29% at adult necropsy (McIntyre et al., 2001
). The discrepancy in magnitude of response of these findings at different ages probably reflects postnatal growth of the rodent perineum and genital area. Exposure to linuron during gestation resulted in an increased number of areolae on PND 13, and this increase correlated with an increased number of nipples at necropsy. This finding confirms and extends the finding of previous investigators that demonstrated in utero exposure to linuron (in addition to other antiandrogens such as dibutyl phthalate, diethylhexyl phthalate, and flutamide), results in permanent retention of nipples (Gray et al., 1999
; McIntyre et al., 2001
). Since the definition of a malformation is traditionally considered to be a permanent structural change that is either rare or life threatening, it could be argued that the presence of nipples and decreased size of the perineum in male rats constitute true malformations and are therefore adverse effects that could be used in risk assessment.
Rats exposed to linuron exhibited abnormal T-mediated development of the epididymis and vas deferens, and the spectrum of target tissues was similar to that reported previously (Gray et al., 1999; Lambright et al., 2000
; McIntyre et al., 2000
). In contrast, male offspring from linuron-treated dams displayed minimal alterations in some DHT-mediated end points such as testicular descent (2 animals from 1 litter) and development of the external genitalia (hypospadias). Gestational exposure to 100 mg/kg/day of linuron to pregnant rats has been shown to induce a low incidence of epispadias, a less severe form of hypospadias, in the male offspring (Lambright et al., 2000
). In our study, prenatal linuron exposure resulted in a 40 and 20% incidence of malformations of the epididymis and vas deferens, respectively. This incidence is higher than that previously reported and likely reflects the steepness of the dose-response curve for linuron-induced teratogenesis (McIntyre et al., 2000
). Nevertheless, this finding confirms our previous report that male rats exposed to linuron in utero display malformations (McIntyre et al., 2000
). This dose level is similar to the developmental LOEL in the rat of 625 ppm, a dose level that increased postimplantation loss and increased fetal incidences of resorptions (U.S. EPA, 1995
).
The relative absence of concomitant testicular and epididymal lesions in linuron-exposed rats on PND 35 correlates with low testicular fluid outflow (Setchell et al., 1994). In contrast, on PND 56 the appearance of both testicular and epididymal lesions is associated with high fluid outflow from the rat testis (Setchell et al., 1994
). These data indicate that linuron-induced testicular atrophy observed previously on PND 100, and in 4 animals in the current study at PND 56, is the progressive outcome of increased intratubular pressure resulting from obstruction of testicular fluid outflow and secondary to malformed epididymides (Lambright et al., 2000
; McIntyre et al., 2000
). Although slight decreases in grossly normal testicular and epididymal weight on PND 56 were observed, suggesting that in utero linuron exposure may affect spermatogenesis directly, these data demonstrate that T-dependent development of the epididymis and vas deferens is the primary target of in utero linuron exposure. Studies are currently under way in our laboratory to determine whether in utero linuron exposure decreases subsequent sperm levels in the adult rat.
A prognostic association between linuron-induced malformations and retained areolae on PND 13 or retained nipples at necropsy was not evident. This finding suggests that linuron-mediated blockade of DHT-dependent nipple regression has a different threshold from that of linuron-mediated alterations in T-mediated reproductive development. This observation brings into question the predictive usefulness and validity of an end point of androgen perturbation in which 20 and 40% of the animals that exhibited malformations did not display either retained areolae or nipples, respectively. Moreover, nipple retention has been shown to be an insensitive indicator of altered T-mediated development, as evidenced by a study examining the effects of gestational exposure to flutamide and subsequent androgen-dependent reproductive development in the male offspring. This study demonstrated that a female-like nipple response would be required before epididymal malformations would be observed in male offspring (McIntyre et al., 2001).
In summary, the current study demonstrates that changes in AGD and nipple retention resulting from prenatal linuron exposure to 50 mg/kg/day are permanent. Testicular atrophy that has been previously reported in linuron-exposed adult animals exhibiting ipsilateral epididymal agenesis is the result of restricted fluid outflow from the testis. Late gestational exposure, coupled with retaining the full male litter complement, may be more sensitive than traditional protocols in detecting teratogenic responses of environmental antiandrogens. Areola retention on PND 13 and nipple retention at necropsy are not predictive of linuron-induced malformations in T-dependent tissues.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed. Fax: (919) 558-1300. E-mail: foster{at}ciit.org.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clark, R. L., Antonello, J. M., Grossman, S. J., Wise, L. D., Anderson, C., Bagdon, W. J., Prahalada, S., MacDonald, J. S., and Robertson, R. T. (1990). External genitalia abnormalities in male rats exposed in utero to finasteride, a 5 -reductase inhibitor. Teratology 42, 91100.[ISI][Medline]
Cook, J. C., Mullin, L. S., Frame, S. R., and Biegel, L. B. (1993). Investigation of a mechanism for Leydig cell tumorigenesis by linuron in rats. Toxicol. Appl. Pharmcol. 119, 195204.[ISI][Medline]
Gray, L. E., Jr., and Kelce, W. R. (1996). Latent effects of pesticides and toxic substances on sexual differentiation of rodents. Toxicol. Ind. Health 12, 515531.[ISI][Medline]
Gray, L. E., Jr., Ostby, J. S., and Kelce, W. R. (1994). Developmental effects of an environmental antiandrogen: The fungicide vinclozolin alters sex differentiation of the male rat. Toxicol. Appl. Pharmacol. 129, 4652.[ISI][Medline]
Gray, L. E., Jr., Ostby, J., Wolf, C., Lambright, C., and Kelce, W. (1998). The value of mechanistic studies in laboratory animals for the prediction of reproductive effects in wildlife: Endocrine effects on mammalian sexual differentiation. Environ. Toxicol. Chem. 17, 109118.[ISI]
Gray, L. E., Jr., Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R. L., and Ostby, J. (1999). Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p`-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol. Ind. Health 15, 94118.[ISI][Medline]
Kelce, W. R., Gray, L. E., Jr., and Wilson, E. M. (1998). Antiandrogens as environmental endocrine disruptors. Reprod. Fertil. Dev. 10, 105111.[ISI][Medline]
Kelce, W. R., Lambright, C. R., Gray, L. E., Jr., and Roberts, K. P. (1997). Vinclozolin and p,p`-DDE alter androgen-dependent gene expression: In vivo confirmation of an androgen receptor-mediated mechanism. Toxicol. Appl. Pharmacol. 142, 192200.[ISI][Medline]
Khera, K. S., Whalen, C., and Trivett, G. (1978). Teratogenicity studies on linuron, malathion, and methoxychlor in rats. Toxicol. Appl. Pharmacol. 45, 435444.[ISI][Medline]
Lambright, C., Ostby, J., Bobseine, K., Wilson, V., Hotchkiss, A. K., Mann, P. C., and Gray, L. E., Jr. (2000). Cellular and molecular mechanisms of action of linuron: An antiandrogenic herbicide that produces reproductive malformations in male rats. Toxicol. Sci. 56, 389399.
LeBlanc, G. A., Bain, L. J., and Wilson, V. S. (1997). Pesticides: Multiple mechanisms of demasculinization. Mol. Cell. Endocrinol. 126, 15.[ISI][Medline]
McIntyre, B. S., Barlow, N. J., and Foster, P. M. D. (2001). Androgen-mediated development in male rat offspring exposed to flutamide in utero: Permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues. Toxicol. Sci. 62, 236249.
McIntyre, B. S., Barlow, N. J., Wallace, D. G., Maness, S. C., Gaido, K. W., and Foster, P. M. D. (2000). Effects of in utero exposure to linuron on androgen-dependent reproductive development in the male Crl:CD(SD)BR rat. Toxicol. Appl. Pharmacol. 167, 8799.[ISI][Medline]
Mylchreest, E., Cattley, R. C., and Foster, P. M. D. (1998). Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl) phthalate: An antiandrogenic mechanism? Toxicol. Sci. 43, 4760.[Abstract]
Mylchreest, E., Wallace, D. G., Cattley, R. C., and Foster, P. M. D. (2000). Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to di(n-butyl) phthalate during late gestation. Toxicol. Sci. 55, 143151.
National Research Council (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC.
Schardein, J. (1993). Hormones and Hormone Antagonists. Dekker, New York.
Setchell, B. P., Maddocks, S., and Brooks, D. E. (1994). The Physiology of Reproduction. Raven Press, Ltd.
Sharpe, R. M., and Skakkebaek, N. E. (1993). Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? [see comments]. Lancet 341, 13921395.[ISI][Medline]
Toppari, J., Larsen, J. C., Christiansen, P., Giwercman, A., Grandjean, P., Guillette, L. J., Jr., Jégou, B., Jensen, T. K., Jouannet, P., Keiding, N., Leffers, H., McLachlan, J. A., Meyer, O., Müller, J., Rajpert-De Meyts, E., Scheike, T., Sharpe, R., Sumpter, J., and Skakkebaek, N. E. (1996). Male reproductive health and environmental xenoestrogens [see comments]. Environ. Health Perspect. 104(Suppl.), 741803.[ISI][Medline]
U.S. EPA (1995). Reregistration Eligibility Decision Document (RED), Linuron. United States Environmental Protection Agency. EPA/NCEPI.
You, L., Casanova, M., Archibeque-Engle, S., Sar, M., Fan, L. Q., and Heck, H. d'A. (1998). Impaired male sexual development in perinatal Sprague-Dawley and Long-Evans hooded rats exposed in utero and lactationally to p,p`-DDE. Toxicol. Sci. 45, 162173.[Abstract]