- Email: [email protected]
Exposure with the environmental estrogen bisphenol a disrupts the male reproductive tract in young mice
Life S&aces, Vol. 65, No. 22, pp. 23514357.1999 copytightQ1999ElswierSciarclnc. F’r&dinthcUSA. Allrightrnxmed 00243205/99/$-see timt malta
PI1 SOO24-3205(99)00502-0
EXPOSURE DISRUPTS
WITH THE ENVIRONMENTAL THE MALE REPRODUCTIVE
ESTROGEN BISPHENOL TRACT IN YOUNG MICE
A
Toshihiro Takao, Wakako Nanamiya, Isao Nagano, Koichi Asaba, Kenji Kawabata* and Kozo Hashimoto Second Department of Internal Medicine, Kochi Medical School, Nankoku 783-8505; *Department of Pathology, Akashi Municipal Hospital, Akashi, Hyogo 673-8501, Japan (Received in final form July 22, 1999)
Summary Environmental estrogens (endocrine disruptive chemicals) have been shown to affect reproduction in wild life and it has been reported that maternal exposure with those chemicals have adverse effects on the male reproductive tract. However, little is known about the potential effects of prepubertal or pubertal exposure with environmental estrogens on the male reproductive tract. Hem we examine plasma hormone levels and histology in the testis of mice following either 4- or g-week oral administration of bisphenol A. Plasma free testosterone levels were dramatically decreased following 8 weeks of bisphenol A treatment compared with control group and morphologically multinucleated giant cells having greater than three nuclei were found in seminiferous tubules in the testis following the g-week No differences in plasma corticosterone and luteintig bisphenol A treatment. Thus, hormone levels were seen between bisphenol A and control groups. exposure with bisphenol A around pubertal period may directly disrupt the male reproductive tract. These facts suggest that more detailed studies will warrant the assessment of the risk to the developing human testis from exposure to bisphenol A and other environmental estrogens in prepubertal and pubertal period. Key Words: bisphenol A, free testosterone, luteinizing hormone, testis
Environmental estrogens are nonsteroidal, man-made chemicals that can enter the body by ingestion or adsorption and mimic the actions of estrogens (l-4). Bisphenol A and alkylphenols have been reported to have estrogenic activity (5-7). Bisphenol A is a monomer of polycarbonate plastics and a constituent of epoxy and polystyrene resins that are extensively used in the food-packaging industry and dentistry (8-11). Bisphenol A increased prolactin gene expression and release following in vitro incubation with pimitary cells (12, 13). In addition, acute administration of bisphenol A induces cfos gene expression and stimulates cell division in the rat uterus and vagina (14). In addition, prolonged exposure to bisphenol A results in hyperplasia, hypertrophy and differentiation in the female reproductive tract epitelium of F344 rats (14). Although recent reports demonstrated that maternal exposure to environmental estrogens significantly affected male reproductive function (1517) and their adult prostate weight relative to control males (18), little is known about the potential and direct effects of prepubertal or pubertal exposure with bisphenol A to the male reproductive Address for Correspondence: Toshihiro Takao, M.D., Ph.D., Second Department of Internal Medicine, Kochi Medical School, Kohasu Okoh-cho, Nankoku 783-8505, JAPAN, Phone: +81888-80-2343, Fax NO. 81-888-80-2344, E-mail: [email protected]
2352
Vol. 65, No. 22, 1999
Bisphenol A on Male RepreduetiveTract
In tbe present study, we evaluated the effects of bisphenol A on the male reproductive tract of young mice following 4- or g-week oral administration with low and high doses by measuring plasma levels of free testosterone (FT), corticosterone (B), luteinizing hormone (LH) and by assessing testicular histology.
Methods Animals.
AU studies were carried out in accordance with the Guide for the Care and Use of MaIe C57BU6 Laboratory AnimaIs as adopted and promulgated by National Institutes of Health. AU animals were mice (5 weeks old) were purchased from JAPAN SLC Inc.(Shizuoka, Japan). housed individually in a light (12 h on/off) and temperature-controIIed room, with food and water available srd l&&l.
BisphenolA trea&nent. Bisphenol A was purchased from Sigma Chemical Co. (St. Louis, MO). Water, 0.005% ethanol (control), bisphenol Bisphenol A was tinally dissolved in 0.005% ethanol. A (0.5 &ml) or bisphenol A (5Opg/ml) were administered p.o.(as drinking water) for 4 or 8 weeks. In order to keep the concentration of bisphenol A stable, we changed the drinking water twice per The beds of mice were kept clean to week although the exact stability of bisphenol A is not known. The mice were sacrificed by decapitation and avoid the exposure of bisphenol A through the skin. One testis per mouse was taken for the histological studies both testes and spleen were weighed. Trunk blood was collected in siliconized 1.5 ml eppendorf tubes with hematoxylin-eosin staining. containing disodium EDTA. Whole blood was centrifuged in a TGMY microfuge for 10 min at 3000 rpm to separate red blood cehs from plasma.
Plasma free testosterone, cortkosterone and luteinizing hormone levels.
Plasma free testosterone, corticosterone and luteinizing hormone levels were measured using commercialIy available RIA and EIA assay kits (DPC Corp., Los Angeles, CA; ICN BiomedicaIs Inc., Costa Mesa, CA and Amersham International plc, Buckinghamshire, England, respectively). The inter- and intra-assay coefficients of variation were 3.7% and 4.0% at 19 pg/mI for free testosterone, 6.5 % at 150 n&nI and 7.1% at 166 ng/ml for corticosterone and were 10.9% at 17.2 ng/ml and 10.7% at 16 rig/ml for LH assay, respectively.
L&a analysi.r. difference test.
Data were analyzed by ANOVA followed by Fisher’s
protected least squares
TABLE 1 Effect of 4- or 8- weeks oral administration of bisphenol A on average water intake, body weight, testis weight and spleen weight in male C57BL6 mouse
~~~1
Bisphenol A Bisphenol A
Control
*p
4 WEEKS
“1
0 Control
2353
Bisphenol A on Male ReproductiveTract
Vol. 65, No. 22, 1999
I
1
I
0.5pglml
8 WEEKS
“1
JOIg/ml
0
1
Control
Fig. 1 Effects of bisphenol A treatment for 4 weeks (left panel) plasma free testosterone levels in male C57BU6 mice. performed as described in the Methods. Data represent * represents significant alterations at p< 0.02 vs control
I
05lglml
50Irg/ml
1
or 8 weeks (right panel) on Bisphenol A treatment was the mean * SEM (n=7). group.
Results The average water intake, body weight, testis and spleen weight in control and bisphenol A treated groups are shown in Table 1. Although average water intake in 4-week treatment of bisphenol A (50 @ml) group was decreased compared with control group, other parameter such as body weight, testis weight, testis weight /body weight, spleen weight and spleen weight / body weight were not different among control, bisphenol A 0.5 pg and 50 ,@ml group both in 4- or 8-week treatment. No differences in all parameters were seen between water and control groups following 4 or 8 weeks of the treatment (data not shown). Bisphenol A (0.5 pg and 50 pg/rnl) tended to decrease plasma free testosterone levels dosedependently following 4 weeks of treatment, however, it did not reach statistical significance. In contrast, plasma free testosterone levels were dramatically decreased following 8 weeks of bisphenol A (50 pg/ml) treatment compared with the control group (Figure 1). No differences in plasma free testosterone were seen between water and control groups following 4 or 8 weeks of treatment (data not shown). Next, we examined the effect of bisphenol A on plasma corticosterone levels. Bisphenol A (0.5 pg and 50 &ml) tended to decrease plasma corticosterone levels following 4 weeks of treatment, however, it did not reach statistical significance (Figure 2). Plasma corticosterone levels were unchanged among bisphenol A (0.5 pg and 5Opglml) and control groups following 8 weeks of treatment. No differences in plasma corticosterone were seen between water and control groups following 4 or 8 weeks of treatment (data not shown). Figure 3 shows morphological changes in testis induced by the S-week treatment with bisphenol A (50 pg/ml). Multinucleated giant cells containing more than three nuclei were seen in the seminiferous tubules of the testis. There were 1.83-cl.22 cells/ 263 213 semineferous tubes/ one testis section in the g-week treatment with bisphenol A (50 pg/rnl) (n=6) and were 0.2 cells/ 283 218 semineferous tubes/ one testis section in the g-week treatment with bisphenol A (0.5 pg/ml) (n=5). There were no multinucleated giant cells in the control group in the g-week treatment with bisphenol A In addition, there were no abnormal morphological changes in any groups receiving the 4-week treatment with bisphenol A.
Bisphenol A on Male Reproductive Tract
2354
Vol. 65, No. 22, 1999
8 WEEKS
4 WEEKS
6 1
Control
’
5Opglml
’
Control
’
0.6
pglrnl
’
Fig. 2 Effects of bisphenol A treatment for 4 weeks (left panel) or 8 weeks (right panel) on plasma corticosterone levels in male C57BU6 mice. Bisphenol A treatment was performed as described in the Methods. Data represent the mean + SEM (n=7).
Fig. 3 Photomicrograph showing morphological changes in the testis of a mate C57BU6 mouse treated for 8 weeks with bisphenol A (50 c/g/ml). A multinucleated giant cell containing more than three nuclei is seen in the seminiferous tubule (arrowhead). To determine whether bisphenol A directly affects the male reproductive tract or modulates the pituitary function, plasma luteinizing hormone (LH) was measured following the 4 or 8 week of treatment regimen. Plasma LH levels were not significantly different among bisphenol A (0.5 pg and 5O~g/rnl) and control groups following either 4 or 8 weeks of treatment (Figure 4). NO differences in plasma LH levels were Seen between water and control groups following 4 or 8 weeks of treatment (data not shown)
Vol. 65, No. 22, 1999
Bisphenol A on Male ReproductiveTract
8 WEEKS
4 WEEKS
Control
’
05 jrg/ml
2355
5Opg/ml
Control
’
0.6
w/ml
5Opg/ml
’
Fig. 4 Effects of bisphenol A treatment for 4 weeks (left panel) or 8 weeks (right panel) on plasma luteinizing hormone levels in male C57BU6 mice. Bisphenol A treatment was Data represent the mean r SEM (n=7). performed as described in the Methods. Discussion A previous report demonstrated that maternal exposure to bisphenol A permanently increased the size of the prepubertal testis and reduced the size of the epididymus (19). In addition, a high dose of bisphenol A significantly decreased efficacy of sperm production by 20% relative to control males. In another study, maternal treatment with a putative environmental estrogen (4-octylphenol) resulted in reduced expression of the messenger RNA and protein for P450c17 in fetal Leydig cells (20) suggesting that the fetal male reproductive tract is very sensitive to maternal exposure to In addition, it has been reported that exposure to 4-octylphenol at a environmental estrogens. concentration of 1000 ,@ from days l-22 after birth resulted in a small but significant reduction in mean testicular size showing that neonatal exposure is also important to the male reproductive tract (21). Although testis weights in the bisphenol A treated group were not different from controls in the present study, plasma free testosterone levels were dramatically decreased following g-week treatment of bisphenol A As the puberty is reached around 6 or 7-week old in the mouse, it is suggested that not only maternal and neonatal exposure but also prepubertal and/or pubertal exposure to a environmental estrogen specifically disrupt male reproductive functions in mice. A recent study demonstrated that 3-day treatment with bisphenol A induced the molecular and morphological alternation in the rat uterus and vagina (14). Morphological data in the present study revealed multinucleated giant cells containing more than three nuclei in the seminiferous tubules following an g-week treatment with bisphenol A. Although the number of muhinucleated giant cells is small in whole seminiferous tubules, it is possible that the prolonged exposure to bisphenol A may induced morphological changes in the spermatogonia and/or primary spermatocyte during spermatogenesis in the testis. This is in agreement with disruption of spermatogenesis in the seminiferous tubules and no developing germ cells when diethylstilbestrol was injected at the day of birth and animals were sacrificed at 42 (pubertal) days of age (22). The mechanism underlying the decreases of free testosterone and the morphological changes in response to bisphenol A is unknown. It is reported that maternal treatment with either a potent synthetic estrogen diethylstilbestrol or a putative environmental estrogen (boctylphenol) resulted in reduced expression of the messenger
2356
Bisphenol A on Male ReproductiveTract
Vol. 65, No. 22, 1999
RNA and protein for P45Oe17 (20). Taken together the fact that plasma corticosterone levels in bisphenol A treated group were not different from the control group in the present study, bisphenol A More detailed might affect the enzymes other than P450 see and 3P-hydroxysteroid dehydrogenase. molecular studies would be needed to clarify this mechanism. In a recent report it was demonstrated that bisphenol A increased prolactin gene expression, release and cell proliferation in anterior pituitary cells albeit at a lOOO- to 5000-fold lower potency than estradiol in vitro and vivo (13), suggesting this environmental estrogen may affect reproductive tract function by affecting pituitary function. The possibility also exists that bisphenol A may affect gonadotropin releasing hormone (GnRH) in the hypothalamus and thus play a role to modulate LH secretion. A study in male sheep demonstrated that the arcuate-ventromedial region and possibly the preoptic area are brain sites at which estrogen acts to reduce GnRH secretion (23). The present data showed that plasma LH levels were unchanged following 4- or &week bisphenol A treatment. Although plasma LH levels were not elevated via feedback mechanism, it is tempting to speculate that bisphenol A affects the male reproductive system at least partly via a direct mechanism in the testis However, during these periods as pituitary function is not fully developed in prepubertal period. more long term effects of bisphenol A on pituitary and/or hypothalamic modulation should not be excluded. In Japan, the Minister of Education reported that 40.1% of public elementary and junior high school use polycarbonate dishes possibly containing bisphenol A for lunch service in 1998 (News of the Minister of Education, http://www.monbu.go.jp/news/00000275/. Although recent studies demonstrated that no detectable bisphenol A was found in the extracts from polycarbonate resins (24) and babv nolvcarbonate bottle (25). more emergent and detailed studies are warranted to assess the possible &k’to the development’of the hum&r testis from exposure to bisphenol A and other environmental estrogens. Acknowledgments We are extremely manuscript.
grateful to Dr. Nathan
M. Appel for comments
and suggestions
on the
References K. S . KORACH, Endocrinology 13 2 2277-2278 (1993). :: R. STONE, Science 265 308-310 (1994). 3. D.M. FRY, Environ Health Perspect 103 165-171(1995). 4. L.J. GUILLE’ITE JR, D.A. CRAIN, A.A. ROONEY and D.B. PICKFORD, Environ Health Perspect 103 157-164 (1995). 5. A.V. KRISHNAN, P. STATHIS, S.F. PERMUCTH, L. TOKES and D. FELDMAN, Endocrinology 132 2279-2286 (1993). 6. A.M. SOTO, H. JUSTICIA, J.W. WRAY and C. SONNENSCHEIN, Environ Health Perspect 92 167-173 (1991). 7. R. WHITE, S. JOBLING, S.A. HOARE, J.P. SUMPTER and M.G. PARKER, Endocrinology 135 175-182 (1994). 8. J.E. BILES, T.P. MCNEAL and T.H. BEGLEY, J Agricul Food Chem 45 4697-4700 (1997). 9. J.E. BILES, T.P. MCNEAL, T.H. BEGLEY and H.C. HOLLIFIELD, J Agricul Food Chem 45 3541-3544 (1997). 10. J.A. BROTONS, M.F. OLEA-SERRANO, M. VILLALOBOS, V. PEDRAZA and N. OLEA, 1995 Environ Health Perspect 103, 608-612 (1995). 11. N. OLEA, R. PULGAR, P. PEREZ, F. OLEA-SERRANO, A. RIVAS, A. NOVILLOFERTRELL, V. PEDRAZA, A.M. SOT0 and C. SONNENSCHEIN, Environ Health Perspect 104 298-305 (1996). 12. N. BENJONATHAN and R. STEINMETZ, Trends Endocrinol Metab 9 124-128 (1998).
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13. R. STEINMETZ, N.G. BROWN, D.L. ALLEN, R.M. BIGSBY and N. BENJONATHAN, Endocrinology 138 1780-1786 (1997). 14. R. STEINMETZ, N.A. MITCHNER, A. GRANT, D.L. ALLEN and R.M. BIGSBY, N. BENJONATHAN, Endocrinology 139 2741-2747 (1998). 15. W. GILL, G. SCHMACHER, M. BIBBO, F. STRAUS and H. SCHOENBERG, J Uroll22 36-39 (1979) 16. J. MCLACHLAN, R. NEWBOLD and B. BULLOCK, Science 190 991-992 (1975). 17. A. WILCOX, D. BAIRD, C. WEINBERG, P. HORNSBY and A. HERBST, N Engl J Med 332 1411-1415 (1995). 18. S.C. NAGEL, F.S. VOM SAAL, K.k THAYER, M.G. DHAR, M. BOECHLER and W.V. WELSHONS, Environ Health Perspect 105 70-76 (1997). 19. F.S. VOM SAAL, P.S. COOKE, D.L. BUCHANAN, P. PAL4NZA, K.A. THAYER, S.C. NAGEL, S. PARMIGIANI and W.V. WELSHONS, Toxic01 Indust Health 14 239-260 (1998). 20. G. MAJDIC, R.M. SHARPE, P.J. O’SHAUGHNESSY and P.T.K. SAUNDERS, Endocrinology 137 1063-1070 (1996). 21. R.M. SHARPE, J.S. FISHER, M.M MILLAR, S. JOBLING and J.P. SUMPTER, Environ Health Perspect 103 1136-1143 (1995). 22. S.A. KHAN, R.B. BALL and W.J.3. HENDRY, Biol Reproduct 58 137-142 (1998). 23. C.J. SCOTT, D.E. KUEHL, S.A. FERREIRA and G.L. JACKSON, Endocrinology 138 3686-3694 (1997). 24. S.R. HOWE and L. BORODINSKY, Food Additiv Contam 15 370-375 (1998). 25. K.A. MOUNTFORT, J. KELLY, S.M. JICKELLS and L. CASTLE, Food Additiv Contam 14 737-740 (1997).
PI1 SOO24-3205(99)00502-0
EXPOSURE DISRUPTS
WITH THE ENVIRONMENTAL THE MALE REPRODUCTIVE
ESTROGEN BISPHENOL TRACT IN YOUNG MICE
A
Toshihiro Takao, Wakako Nanamiya, Isao Nagano, Koichi Asaba, Kenji Kawabata* and Kozo Hashimoto Second Department of Internal Medicine, Kochi Medical School, Nankoku 783-8505; *Department of Pathology, Akashi Municipal Hospital, Akashi, Hyogo 673-8501, Japan (Received in final form July 22, 1999)
Summary Environmental estrogens (endocrine disruptive chemicals) have been shown to affect reproduction in wild life and it has been reported that maternal exposure with those chemicals have adverse effects on the male reproductive tract. However, little is known about the potential effects of prepubertal or pubertal exposure with environmental estrogens on the male reproductive tract. Hem we examine plasma hormone levels and histology in the testis of mice following either 4- or g-week oral administration of bisphenol A. Plasma free testosterone levels were dramatically decreased following 8 weeks of bisphenol A treatment compared with control group and morphologically multinucleated giant cells having greater than three nuclei were found in seminiferous tubules in the testis following the g-week No differences in plasma corticosterone and luteintig bisphenol A treatment. Thus, hormone levels were seen between bisphenol A and control groups. exposure with bisphenol A around pubertal period may directly disrupt the male reproductive tract. These facts suggest that more detailed studies will warrant the assessment of the risk to the developing human testis from exposure to bisphenol A and other environmental estrogens in prepubertal and pubertal period. Key Words: bisphenol A, free testosterone, luteinizing hormone, testis
Environmental estrogens are nonsteroidal, man-made chemicals that can enter the body by ingestion or adsorption and mimic the actions of estrogens (l-4). Bisphenol A and alkylphenols have been reported to have estrogenic activity (5-7). Bisphenol A is a monomer of polycarbonate plastics and a constituent of epoxy and polystyrene resins that are extensively used in the food-packaging industry and dentistry (8-11). Bisphenol A increased prolactin gene expression and release following in vitro incubation with pimitary cells (12, 13). In addition, acute administration of bisphenol A induces cfos gene expression and stimulates cell division in the rat uterus and vagina (14). In addition, prolonged exposure to bisphenol A results in hyperplasia, hypertrophy and differentiation in the female reproductive tract epitelium of F344 rats (14). Although recent reports demonstrated that maternal exposure to environmental estrogens significantly affected male reproductive function (1517) and their adult prostate weight relative to control males (18), little is known about the potential and direct effects of prepubertal or pubertal exposure with bisphenol A to the male reproductive Address for Correspondence: Toshihiro Takao, M.D., Ph.D., Second Department of Internal Medicine, Kochi Medical School, Kohasu Okoh-cho, Nankoku 783-8505, JAPAN, Phone: +81888-80-2343, Fax NO. 81-888-80-2344, E-mail: [email protected]
2352
Vol. 65, No. 22, 1999
Bisphenol A on Male RepreduetiveTract
In tbe present study, we evaluated the effects of bisphenol A on the male reproductive tract of young mice following 4- or g-week oral administration with low and high doses by measuring plasma levels of free testosterone (FT), corticosterone (B), luteinizing hormone (LH) and by assessing testicular histology.
Methods Animals.
AU studies were carried out in accordance with the Guide for the Care and Use of MaIe C57BU6 Laboratory AnimaIs as adopted and promulgated by National Institutes of Health. AU animals were mice (5 weeks old) were purchased from JAPAN SLC Inc.(Shizuoka, Japan). housed individually in a light (12 h on/off) and temperature-controIIed room, with food and water available srd l&&l.
BisphenolA trea&nent. Bisphenol A was purchased from Sigma Chemical Co. (St. Louis, MO). Water, 0.005% ethanol (control), bisphenol Bisphenol A was tinally dissolved in 0.005% ethanol. A (0.5 &ml) or bisphenol A (5Opg/ml) were administered p.o.(as drinking water) for 4 or 8 weeks. In order to keep the concentration of bisphenol A stable, we changed the drinking water twice per The beds of mice were kept clean to week although the exact stability of bisphenol A is not known. The mice were sacrificed by decapitation and avoid the exposure of bisphenol A through the skin. One testis per mouse was taken for the histological studies both testes and spleen were weighed. Trunk blood was collected in siliconized 1.5 ml eppendorf tubes with hematoxylin-eosin staining. containing disodium EDTA. Whole blood was centrifuged in a TGMY microfuge for 10 min at 3000 rpm to separate red blood cehs from plasma.
Plasma free testosterone, cortkosterone and luteinizing hormone levels.
Plasma free testosterone, corticosterone and luteinizing hormone levels were measured using commercialIy available RIA and EIA assay kits (DPC Corp., Los Angeles, CA; ICN BiomedicaIs Inc., Costa Mesa, CA and Amersham International plc, Buckinghamshire, England, respectively). The inter- and intra-assay coefficients of variation were 3.7% and 4.0% at 19 pg/mI for free testosterone, 6.5 % at 150 n&nI and 7.1% at 166 ng/ml for corticosterone and were 10.9% at 17.2 ng/ml and 10.7% at 16 rig/ml for LH assay, respectively.
L&a analysi.r. difference test.
Data were analyzed by ANOVA followed by Fisher’s
protected least squares
TABLE 1 Effect of 4- or 8- weeks oral administration of bisphenol A on average water intake, body weight, testis weight and spleen weight in male C57BL6 mouse
~~~1
Bisphenol A Bisphenol A
Control
*p
4 WEEKS
“1
0 Control
2353
Bisphenol A on Male ReproductiveTract
Vol. 65, No. 22, 1999
I
1
I
0.5pglml
8 WEEKS
“1
JOIg/ml
0
1
Control
Fig. 1 Effects of bisphenol A treatment for 4 weeks (left panel) plasma free testosterone levels in male C57BU6 mice. performed as described in the Methods. Data represent * represents significant alterations at p< 0.02 vs control
I
05lglml
50Irg/ml
1
or 8 weeks (right panel) on Bisphenol A treatment was the mean * SEM (n=7). group.
Results The average water intake, body weight, testis and spleen weight in control and bisphenol A treated groups are shown in Table 1. Although average water intake in 4-week treatment of bisphenol A (50 @ml) group was decreased compared with control group, other parameter such as body weight, testis weight, testis weight /body weight, spleen weight and spleen weight / body weight were not different among control, bisphenol A 0.5 pg and 50 ,@ml group both in 4- or 8-week treatment. No differences in all parameters were seen between water and control groups following 4 or 8 weeks of the treatment (data not shown). Bisphenol A (0.5 pg and 50 pg/rnl) tended to decrease plasma free testosterone levels dosedependently following 4 weeks of treatment, however, it did not reach statistical significance. In contrast, plasma free testosterone levels were dramatically decreased following 8 weeks of bisphenol A (50 pg/ml) treatment compared with the control group (Figure 1). No differences in plasma free testosterone were seen between water and control groups following 4 or 8 weeks of treatment (data not shown). Next, we examined the effect of bisphenol A on plasma corticosterone levels. Bisphenol A (0.5 pg and 50 &ml) tended to decrease plasma corticosterone levels following 4 weeks of treatment, however, it did not reach statistical significance (Figure 2). Plasma corticosterone levels were unchanged among bisphenol A (0.5 pg and 5Opglml) and control groups following 8 weeks of treatment. No differences in plasma corticosterone were seen between water and control groups following 4 or 8 weeks of treatment (data not shown). Figure 3 shows morphological changes in testis induced by the S-week treatment with bisphenol A (50 pg/ml). Multinucleated giant cells containing more than three nuclei were seen in the seminiferous tubules of the testis. There were 1.83-cl.22 cells/ 263 213 semineferous tubes/ one testis section in the g-week treatment with bisphenol A (50 pg/rnl) (n=6) and were 0.2 cells/ 283 218 semineferous tubes/ one testis section in the g-week treatment with bisphenol A (0.5 pg/ml) (n=5). There were no multinucleated giant cells in the control group in the g-week treatment with bisphenol A In addition, there were no abnormal morphological changes in any groups receiving the 4-week treatment with bisphenol A.
Bisphenol A on Male Reproductive Tract
2354
Vol. 65, No. 22, 1999
8 WEEKS
4 WEEKS
6 1
Control
’
5Opglml
’
Control
’
0.6
pglrnl
’
Fig. 2 Effects of bisphenol A treatment for 4 weeks (left panel) or 8 weeks (right panel) on plasma corticosterone levels in male C57BU6 mice. Bisphenol A treatment was performed as described in the Methods. Data represent the mean + SEM (n=7).
Fig. 3 Photomicrograph showing morphological changes in the testis of a mate C57BU6 mouse treated for 8 weeks with bisphenol A (50 c/g/ml). A multinucleated giant cell containing more than three nuclei is seen in the seminiferous tubule (arrowhead). To determine whether bisphenol A directly affects the male reproductive tract or modulates the pituitary function, plasma luteinizing hormone (LH) was measured following the 4 or 8 week of treatment regimen. Plasma LH levels were not significantly different among bisphenol A (0.5 pg and 5O~g/rnl) and control groups following either 4 or 8 weeks of treatment (Figure 4). NO differences in plasma LH levels were Seen between water and control groups following 4 or 8 weeks of treatment (data not shown)
Vol. 65, No. 22, 1999
Bisphenol A on Male ReproductiveTract
8 WEEKS
4 WEEKS
Control
’
05 jrg/ml
2355
5Opg/ml
Control
’
0.6
w/ml
5Opg/ml
’
Fig. 4 Effects of bisphenol A treatment for 4 weeks (left panel) or 8 weeks (right panel) on plasma luteinizing hormone levels in male C57BU6 mice. Bisphenol A treatment was Data represent the mean r SEM (n=7). performed as described in the Methods. Discussion A previous report demonstrated that maternal exposure to bisphenol A permanently increased the size of the prepubertal testis and reduced the size of the epididymus (19). In addition, a high dose of bisphenol A significantly decreased efficacy of sperm production by 20% relative to control males. In another study, maternal treatment with a putative environmental estrogen (4-octylphenol) resulted in reduced expression of the messenger RNA and protein for P450c17 in fetal Leydig cells (20) suggesting that the fetal male reproductive tract is very sensitive to maternal exposure to In addition, it has been reported that exposure to 4-octylphenol at a environmental estrogens. concentration of 1000 ,@ from days l-22 after birth resulted in a small but significant reduction in mean testicular size showing that neonatal exposure is also important to the male reproductive tract (21). Although testis weights in the bisphenol A treated group were not different from controls in the present study, plasma free testosterone levels were dramatically decreased following g-week treatment of bisphenol A As the puberty is reached around 6 or 7-week old in the mouse, it is suggested that not only maternal and neonatal exposure but also prepubertal and/or pubertal exposure to a environmental estrogen specifically disrupt male reproductive functions in mice. A recent study demonstrated that 3-day treatment with bisphenol A induced the molecular and morphological alternation in the rat uterus and vagina (14). Morphological data in the present study revealed multinucleated giant cells containing more than three nuclei in the seminiferous tubules following an g-week treatment with bisphenol A. Although the number of muhinucleated giant cells is small in whole seminiferous tubules, it is possible that the prolonged exposure to bisphenol A may induced morphological changes in the spermatogonia and/or primary spermatocyte during spermatogenesis in the testis. This is in agreement with disruption of spermatogenesis in the seminiferous tubules and no developing germ cells when diethylstilbestrol was injected at the day of birth and animals were sacrificed at 42 (pubertal) days of age (22). The mechanism underlying the decreases of free testosterone and the morphological changes in response to bisphenol A is unknown. It is reported that maternal treatment with either a potent synthetic estrogen diethylstilbestrol or a putative environmental estrogen (boctylphenol) resulted in reduced expression of the messenger
2356
Bisphenol A on Male ReproductiveTract
Vol. 65, No. 22, 1999
RNA and protein for P45Oe17 (20). Taken together the fact that plasma corticosterone levels in bisphenol A treated group were not different from the control group in the present study, bisphenol A More detailed might affect the enzymes other than P450 see and 3P-hydroxysteroid dehydrogenase. molecular studies would be needed to clarify this mechanism. In a recent report it was demonstrated that bisphenol A increased prolactin gene expression, release and cell proliferation in anterior pituitary cells albeit at a lOOO- to 5000-fold lower potency than estradiol in vitro and vivo (13), suggesting this environmental estrogen may affect reproductive tract function by affecting pituitary function. The possibility also exists that bisphenol A may affect gonadotropin releasing hormone (GnRH) in the hypothalamus and thus play a role to modulate LH secretion. A study in male sheep demonstrated that the arcuate-ventromedial region and possibly the preoptic area are brain sites at which estrogen acts to reduce GnRH secretion (23). The present data showed that plasma LH levels were unchanged following 4- or &week bisphenol A treatment. Although plasma LH levels were not elevated via feedback mechanism, it is tempting to speculate that bisphenol A affects the male reproductive system at least partly via a direct mechanism in the testis However, during these periods as pituitary function is not fully developed in prepubertal period. more long term effects of bisphenol A on pituitary and/or hypothalamic modulation should not be excluded. In Japan, the Minister of Education reported that 40.1% of public elementary and junior high school use polycarbonate dishes possibly containing bisphenol A for lunch service in 1998 (News of the Minister of Education, http://www.monbu.go.jp/news/00000275/. Although recent studies demonstrated that no detectable bisphenol A was found in the extracts from polycarbonate resins (24) and babv nolvcarbonate bottle (25). more emergent and detailed studies are warranted to assess the possible &k’to the development’of the hum&r testis from exposure to bisphenol A and other environmental estrogens. Acknowledgments We are extremely manuscript.
grateful to Dr. Nathan
M. Appel for comments
and suggestions
on the
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