Doi:10.1016/j.jsbmb.2005.08.019

Journal of Steroid Biochemistry & Molecular Biology xxx (2005) xxx–xxx The xenoestrogen bisphenol A in the Hershberger assay: androgen receptor regulation and morphometrical reactions indicate no major effects Tsuyuki Nishino , Thilo Wedel , Oliver Schmitt , Katja B¨uhlmeyer , Martin Sch¨onfelder , Christian Hirtreiter , Thorsten Schulz , W. K¨uhnel , H. Michna Institute of Public Health Research, Technical University of Munich, Connollystraße 32, 80809 Munich, Germany Institute of Anatomy, University of Luebeck, Germany Institute of Anatomy, University of Rostock, Germany Institute of Organic Chemistry, University of Regensburg, Germany Received 24 January 2005; accepted 31 August 2005 We evaluated androgen-like effects of bisphenol A (BPA) using orchiectomized Wistar rats. Animals were treated p.o. either with vehicle or with 3, 50, 200, 500 mg/kg bw/day BPA (n = 13) for 7 days. One group was treated s.c. with 1 mg/kg bw/day testosterone propionate (TP).
Flutamide (FL) (3 mg/kg bw/day, p.o.) was used to antagonize androgen effects of the suprapharmacological dose (500 mg/kg bw/day) of BPA.
Androgen-like effects of BPA on prostates and seminal vesicles were assessed by the Hershberger assay, densitometric analysis of androgen receptor (AR) immunoreactivity, cell proliferation-index and a morphometric analysis. Absolute weights of prostates and seminal vesicles were not increased by BPA, whereas the relative weights were increased at higher doses of BPA, most likely due to a decrease in body weight.
Staining intensity for AR immunoreactivity was increased at low but not at higher doses of BPA in comparison to the orchiectomized rats.
BPA at all doses tested did not cause an increase of the cell proliferation-index. Epithelial height and glandular luminal area were increased by low doses of BPA, whereas higher doses caused a decrease of these parameters. The data provide evidence that BPA does not exert major androgenic effects.
2005 Elsevier Ltd. All rights reserved.
Keywords: Bisphenol A; Prostate; Seminal vesicle; Immunohistochemistry; Morphometry; Densitometry; Androgen receptor regulation; PCNA; MIB-5; Proliferation markers Recent in vitro studies demonstrate, in fact, that xeno- biotics can bind with estrogen receptors and activate them, BPA is a chemical monomer used primarily to make resulting in gene expression BPA slightly induced epoxy-resins, polycarbonate (PC) plastic products and the MCF-7 cell proliferation at a level of 0.1 ␮M and maxi- flame retardant tetrabromobisphenol A A number of mum proliferation at 10 ␮M Also in in vivo studies, so-called xenobiotics, including pesticides (p,p-DDT), plas- BPA showed estrogenic activity. Plasma free testosterone lev- ticizers (BPA) and a variety of other industrial chemicals els were dramatically decreased following 8 weeks of BPA (polychlorinated biphenyls) contain a phenolic ring that mim- treatment It was claimed by vom Saal that, the expo- ics the A-ring of estradiol and have been reported to have sure of pregnant mice to extremely low concentrations of hormonal or antihormonal activity Although the level certain xenobiotics, for instance, results in offspring with of exposure to these xenobiotics may be, if any, very low, they lower sperm production, increased prostate size or alters may exert their potential toxicity or endocrine disturbance in maternal behaviour, postnatal growth rate and reproduc- human beings and wildlife.
tive function in female mice work groups, in contrast, found an uterotrophic response (increase in uter- ∗ Corresponding author. Tel.: +49 89 289 24571; fax: +49 89 289 24572.
ine wet weight) at doses up to 100 mg/kg BPA for 3 days E-mail address: [email protected] (T. Nishino).
0960-0760/$ – see front matter 2005 Elsevier Ltd. All rights reserved.

T. Nishino et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2005) xxx–xxx Also Gupta an enhancement of the anogen- stitution (OX) and to a vehicle treated intact control group ital distance and the prostate size of fetuses, when pregnant (Intact). Propylene glycol was purchased from Merck, Darm- CD-1 mice were treated with BPA in the microgram range stadt, Germany. TP and FL were kindly provided by Schering per kg bw/day. BPA induces inappropriate androgen receptor AG, Berlin, Germany. After the treatment animals were sacri- activation and mitogenesis in prostatic adenocarcinoma cells ficed by decapitation and seminal vesicles and prostates were (LNCaP). Takao reported a significant decrease in plasma harvested surgically, weighed and immediately fixed in 4% free testosterone levels at 50 ␮g BPA/ml in drinking water neutral buffered paraformaldehyde for 24 h.
(14 mg/kg/day). No significant effect (although a trend in the same direction) was observed neither after 4 weeks of expo- sure nor after 4 and 8 weeks exposure to 5 ␮g of BPA/ml drinking water (0.14 mg/kg/day) After fixation the specimens (prostate, seminal vesicle) In addition, Kim et al. did not detect any androgenic or were dehydrated in ascending series of alcohol, embed- anti-androgenic activities of BPA in Hershberger assay ded in paraffin and cut in sections of 5 ␮m thickness (10 the BPA doses used were 10–1000 mg/kg/day.
sections per specimen). For the immunohistochemical visu- The present study was carried out to clarify the andro- alization of androgen receptors and proliferation markers genic potential of BPA in a broad dose range from "ultralow", the following primary antibodies were applied using the "pharmacological" to "suprapharmacological" in rats using standard procedure protocols provided by the manufac- the standard Hershberger assay with additionally androgen- turer: anti-androgen-receptor (1:100, sc-815, rabbit poly- clonal, Santa Cruz Biotechnology, CA, USA), anti-androgen- Additional parameters such as morphometric and qualita- receptor (1:100, 554224, mouse monoclonal, BD PharMin- tive data are therefore required to determine the androgenicity gen, Germany), anti-PCNA (1:200, PC-10, mouse mono- of a given substance, in particular if the expected effects are clonal, Novocastra, New Castle, United Kingdom) and anti- of lower degree.
MIB-5 (1:100, M 7248 mouse monoclonal, DakoCytoma- tion, Denmark).
2. Materials and methods
2.4. Densitometry and morphometry 2.1. Animals and housing Intensity of immunohistochemical staining was deter- mined densitometrically, while epithelial height and luminal Wistar rats (male HdrBrHan from Harlan Winkelmann, area of the glandular ducts were measured morphometri- Borchen, Germany), weighing about 150 g (age of 2 weeks) cally (KS 100, KS RUN, Zeiss-Vision, Jena, Germany).
were separated into different groups by randomized proce- Microscopy was performed with an Axiophot light micro- dure. They received tap water and ssniff R 10, laboratory stan- scope (Zeiss, Jena, Germany) equipped with a high resolution dard rat diet (in pellet form) ad libitum (ssniff Spezialdi¨aten scanner camera (Axiocam, Zeiss, Germany).
GmbH, Soest, Germany). Groups of 2–4 animals were kept All images had a uniform size of 1300 × 1030 pixel. Since in Makrolon cages type IV with ssniff bedding (3/4 Faser) at the images were generated by using a 20× objective and 1.0 22 ± 3 ◦C, a relative humidity of 30–70% and artificial 24 h optovar the final resolution of the edge lengths of one pixel light. After acclimatization animals were orchiectomized in the resulting image is 0.32 ␮m. This resolution was large under Ketanest/Rompun—anesthesia (Ketanest 10 mg/kg bw enough for deciding which profile of glandular ductus in the from Parke-Davis, Berlin, Germany and Rompun 2 mg/kg bw field of vision was suitable for densitometric measurements.
from Bayer AG, Leverkusen, Germany).
Gray values were transformed pixel by pixel into optical den- 2.2. Treatment of animals Five measurements were performed within each section.
Five sections were examined per animal resulting in 25 mea- Seven days after orchiectomy animals were partitioned surements for each animal. The mean values were calculated into eight groups (n = 13 in each group). They were treated and compared between the different groups.
p.o. with 3, 50, 200 and 500 mg BPA/kg/day dissolved For morphometric measurements the software package in propylene glycol for 7 days. BPA was purchased from KS 100 3.0 (Zeiss-Vision, Jena, Germany) was used. The Fa. Bayer (PtNr. 97.001/Prod.Nr. 04111095, CasNr. 80-05, epithelial height was determined by using a 40× objective Leverkusen, Germany). Another group of orchiectomized and 1.0 optovar. For the determination of the luminal area animals was treated s.c. with testosterone propionate (TP) a 10× objective and 1.0 optovar was used. The quantitative 1 mg/kg bw in arachis oil.
assessment of proliferating cells was performed using a 40× combination with 500 mg objective and 1.0 optovar. One thousand cells per section sible androgen effects of UNCORRECTED PROOF Flutamide (FL) 3 mg/kg bw p.o. in BPA was used to antagonize pos- the "suprapharmacological" dose were counted excluding those which due to the section did of BPA. These groups were compared to vehicle (propylene not show a nucleus to avoid overestimation of the total cell glycol) treated, orchiectomized rats without any other sub- number. A two-sided t-test at a significance level of p < 0.05

T. Nishino et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2005) xxx–xxx Fig. 1. Comparison of absolute wet weights of the whole prostate in (mg) Fig. 2. Comparison of body weights in (g) between the Intact group, orchiec- between the Intact group, OX group and TP group in relation to the BPA- tomized group (OX) and TP group in relation to the BPA-treated groups (3, treated groups (3, 50, 200, 500 and 500 + FL). Asterisks indicate statistically 50, 200, 500 and 500 + FL). Asterisks indicate statistically significant dif- significant differences (p < 0.05), which refer to the castrated control group.
ferences (p < 0.05), which refer to the castrated control group.
was applied for statistical comparison. Data were depicted as 500 + FL) for seminal vesicles, respectively. In the prostate mean ± standard deviation (S.D.).
tissue the intensity of staining was significantly higher in both the Intact and TP group (t-test, p < 0.05, n = 13) compared to the OX group. BPA at lower doses (3 and 50 mg/kg bw) 3. Results
increased AR immunoreactivity and staining intensity of prostate tissue, but reduced them at higher doses (200 and 3.1. Hershberger assay 500 mg/kg bw). In seminal vesicles, the intensity of AR stain- ing was reduced by orchiectomy in comparison with the Intact In contrast to TP, BPA induced no effect on absolute control. The treatment of orchiectomized animals with BPA weights of prostate (and seminal vesicle (not shown).
showed no dose-dependent effects (not shown).
BPA at high doses of 200 and 500 mg/kg bw caused a decrease Using the anti-androgen-receptor monoclonal antibody in body weights and a significant increase in relative (1:100, 554224, mouse monoclonal, Novocastra, New cas- weights of prostate and seminal vesicle (not shown). A simul- tle, United Kingdom) the staining intensity of AR in the taneous administration of FL had no further effect in BPA prostates revealed the following optical density values: treated animals. Animals treated with 200, 500 and 500 + FL 104 ± 20 (Intact), 56 ± 17 (OX), 77 ± 22 (TP), 99 ± 7 (BPA BPA showed severe signs of gastro-intestinal toxicity.
3), 103 ± 23 (BPA 50), 78 ± 16 (BPA 200), 49 ± 23 (BPA 500) and 36 ± 6 (BPA 500 + FL). Statistical analysis con- 3.2. Densitometric analysis firmed that the intensity of staining was significantly higher in both the intact and TP group (t-test, p < 0.05, n = 13) The staining intensity after incubation with the poly- compared to the OX group (Thus, the clonal antibody (1:100, sc-815, rabbit polyclonal, Santa Cruz data obtained for both antibodies used provide evidence that Biotechnology, CA, USA) directed against AR was 75 ± 21 orchiectomy results in a reduced staining intensity of AR, (Intact), 43 ± 12 (OX), 61 whereas substitution with TP enhances the immunoreactive 57 ± 19 (BPA 50), 48 ± 18 signal of AR. The intensity of staining was significantly 33 ± 10 (BPA 500*FL) for UNCORRECTED PROOF ± 24 (TP), 65 ± 27 (BPA 3), (BPA 200), 39 ± 9 (BPA 500) andthe prostates and 42 ± 17 (Intact), increased in prostate after treatment with lower doses of 11 ± 10 (OX), 19 ± 11 (TP), 8 ± 5 (BPA 3), 11 ± 15 (BPA BPA (3 and 50 mg/kg bw). At 500 mg/kg bw staining inten- 50), 7 ± 3 (BPA 200), 3 ± 1 (BPA 500) and 15 ± 17 (BPA sity was similar to the castrated control, but the combina-

T. Nishino et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2005) xxx–xxx to the Intact and TP group (Whereas orchiectomy caused a considerable decrease of cell prolif- eration, administration of TP could reverse this effect and induced a cell proliferation index similar to the Intact group.
The assessment of both proliferation markers revealed that BPA showed at all doses tested no stimulation of proliferat- ing activity in prostate.
3.4. Morphometry The mean epithelial height of prostate glands measured 17 ± 2 ␮m in both the Intact and TP group, whereas the mean epithelial height was significantly (t-test, p < 0.05, n = 13) decreased to 11 ± 1 ␮m in the OX group. BPA treated groups displayed following data: 14 ± 2 ␮m (BPA 3), 14 ± 2 ␮m (BPA 50), 10 ± 1 ␮m (BPA 200), 9 ± 1 ␮m (BPA 500) and 8 ± 1 ␮m (BPA 500 + FL). Similar significant (t-test, p < 0.05, n = 13) data were obtained for the epithelial height of sem- inal vesicle glands: 18 ± 3 ␮m (Intact), 9 ± 2 ␮m (OX) and 15 ± 2 ␮m (TP), 11 ± 1 ␮m (BPA 3), 11 ± 1 ␮m (BPA 50), 8 ± 1 ␮m (BPA 200), 8 ± 1 ␮m (BPA 500) and 8 ± 1 ␮m (BPA 500 + FL), respectively Fig. 3. Densitometric values of the Intact group, orchiectomized group The mean luminal area of prostate glands was (OX), TP group and the BPA-treated groups (3, 50, 200, 500 and 500 + FL) 131 000 ± 40 000 ␮m2 in the Intact group, 7000 ± 5000 ␮m2 after immunohistochemical staining (monoclonal antibody) of the androgen in the OX group and 87 000 ± 30 000 ␮m2 in the TP receptor in the prostate. Asterisks indicate statistically significant differences group. BPA treated groups showed following data: (p < 0.05), which refer to the castrated control group.
23 000 ± 16 000 ␮m2 (BPA 3), 25 000 ± 12 000 ␮m2 (BPA 50), 15 000 ± 10 000 ␮m2 (BPA 200), 4000 ± 500 ␮m2 (BPA tion of BPA (500 mg/kg bw) with FL significantly reduced 500) and 4000 ± 3000 ␮m2 (BPA 500 + FL). The luminal the staining intensity Absolute organ area in the OX group was significantly (t-test, p < 0.05, n = 13) weights (prostate, seminal vesicle) at 200 and 500 mg/kg/day reduced compared to the Intact and TP group. In seminal vesi- (40/100 fold the NOAEL (no-observed-adverse-effect-level)) cles the mean luminal area measured 113 000 ± 80 000 ␮m2 were not significantly altered (The NOAEL is (Intact), 7000 ± 5000 ␮m2 (OX) and 151 000 ± 86 000 ␮m2 the greatest concentration or amount of a substance e.g.
(TP), 35 000 ± 43 000 ␮m2 (BPA 3), 27 000 ± 20 000 ␮m2 BPA, found by experiment or observation, which causes (BPA 50), 8000 ± 3000 ␮m2 (BPA 200), 4000 ± 1000 ␮m2 no detectable adverse alteration of morphology, functional (BPA 500) and 3000 ± 2000 ␮m2 (BPA 500 + FL), respec- capacity, growth, development, or life span of the target tively. Similarly as observed for the prostates the luminal organism under defined conditions of exposure.
area in the OX group was significantly (t-test, p < 0.05, n = 13) reduced compared to the Intact and TP group ( 3.3. Cell proliferation These morphologic observations clearly reveal that orchiectomy causes a substantial reduction of both epithe- The assessment of cell proliferation markers yielded the lial height and luminal area of the prostate gland and seminal following data in rat prostates: The percentage of immunore- vesicles. If TP is substituted both parameters return to values active epithelial cells for MIB-5 was 85 ± 9% in the Intact similar to those found in the Intact group. Lower doses of group, 9 ± 1% in the OX group and 90 ± 2% in the TP BPA caused an increase in epithelial height and luminal area group. BPA treated groups displayed: 10 ± 1% (BPA 3), of prostate and seminal vesicle, while high doses reduced the 8 ± 1% (BPA 50), 2% (BPA 200), 1% (BPA 500) and 1% epithelial height of prostate significantly in comparison to the (BPA 500 + FL). The percentage of immunoreactive epithe- lial cells for MIB-5 in the OX group was significantly (t-test, p < 0.05, n = 13) reduced compared to the intact and TP group Similar results were obtained for the rela- tive amount of cells immunoreactive for PCNA: 90 ± 9% in the Intact group, 10 ± 2% 4.1. Methodologic approaches TP group, 2% (BPA 3), 4% (BPA 500) and 6 ± 1% UNCORRECTED PROOF in the OX group and 88 ± 9% in the (BPA 50), 2% (BPA 200), 5 ± 1% (BPA 500 + FL). The percentage of 4.1.1. Hershberger assay epithelial cells immunoreactive for PCNA in the OX group Although the Hershberger assay is a valid quantitative was significantly (t-test, p < 0.05, n = 13) reduced compared method for evaluating androgenic or anti-androgenic proper-

T. Nishino et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2005) xxx–xxx Fig. 4. All panels show UNCORRECTED PROOF photographs of the prostate. Zones are described according to McNeal Immunohistochemical staining of androgen receptor (monoclonal antibody) showing the transition zone of the Intact group (A), OX group (B) and TP group (C) in relation to the BPA-treated groups (3, 50, 200,500 and 500 + FL). Original magnification 20×. I–P: Immunohistochemical staining of MIB-5 showing the peripheral zone of the Intact group (I), OX group(J) and TP group (K) in relation to the BPA-treated groups (3, 50, 200, 500 and 500 + FL). Original magnification 10×. Q–X: Immunohistochemical staining ofPCNA showing the transition zone of the intact group (Q), OX group (R) and TP group (S) in relation to the BPA-treated groups (3, 50, 200, 500 and 500 + FL).
Original magnification 10×.

T. Nishino et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2005) xxx–xxx Fig. 4. (Continued ).
ties of substances by measuring the organ weight of seminal advantages of a computer-assisted densitometry are a faster vesicles and prostates, the findings obtained by this assay scoring procedure of sections from large series and a higher provide only limited information on the specificity of the reliability. However, the disadvantage of a semiquantitative observed effects when only the reactions of the organ weights approach is the possibility that relevant signals can easily be are judged: For example, the growth of seminal vesicles can missed, so that comparative studies should be based on rather be stimulated not only by androgens but also by estrogenic substances, well known as a paradoxical effect of estrogens 4.3. Influence of BPA Morphologic and functional analysis of cellular param- eters in male accessory organs may allow a more subtle In this study lower doses of BPA (3 and 50 mg/kg bw/day) and reliable assessment of the (anti-) androgenicity of sub- were found to cause an enhancement in staining intensity of stances; in previous studies we analyzed the regulation of AR in rat prostate. Morphometric data showed that lower tenascin expression Since the amount of nuclear AR doses of BPA cause an increase in epithelial height and lumi- present in the rat prostate has been demonstrated to be influ- nal area of prostate and seminal vesicle, while high doses sig- enced by androgens densitometric analysis of AR- nificantly reduce the epithelial height of prostate. These find- immunoreactive cells in prostates and seminal vesicles was ings are similar to those observed in testosterone propionate performed by using immunohistochemical methods substituted castrated rats. Gupta androgen-like effects of BPA in pregnant CD-1 mice at 0.05 mg/kg/day by 4.2. Densitometric analysis observing an enhancement of the anogenital distance and the size of the prostate in fetuses. The androgen receptor (AR) It has been previously described that the concentrations binding affinity in prostate of fetuses was also increased sig- of biochemically active substances can be estimated from nificantly. In contrast, Kim et al. did not found any androgenic the optical density of the or anti-androgenic activities of BPA in Hershberger assay at a result, we found a mark 10–1000 mg/kg/day AR-positive cells after UNCORRECTED PROOF immunoreactive signal sed decrease in staining intensity of orchiectomy in comparison to the con- BPA at all doses tested exerted no significant effects on trol group. This effect of orchiectomy was mostly reversed absolute weights of prostate and seminal vesicle. High doses by an administration of a pharmacological dose of TP. The of BPA (200 and 500 mg/kg bw/day) caused a significant

T. Nishino et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2005) xxx–xxx Fig. 5. Quantitative comparison of (a) MIB-5-immunoreactive and (b)PCNA-immunoreactive epithelial prostatic cells between the Intact group, Fig. 6. Comparison of epithelial height of (a) prostate and (b) seminal vesicle OX group and TP group in relation to the BPA-treated groups (3, 50, 200, between the Intact group, OX group and TP group in relation to the BPA- 500 and 500 + FL). Asterisks indicate statistically significant differences treated groups (3, 50, 200, 500 and 500 + FL). Asterisks indicate statistically (p < 0.05), which refer to the significant differences (p < 0.05), which refer to the castrated control group.
than to an androgenic effect of this substance. An oral pre- These effects of BPA may UNCORRECTED PROOF castrated control group.
increase in relative weights of prostates and seminal vesicles.
be due rather to a toxicity-related dictable no effect concentration (PNECoral) of 33 mg/kg food significant decrease in body weights and well known gen- has been derived for the secondary poisoning assessment eral side effects, e.g. loss of appetite and diarrhea from a NOAEL of 50 mg/kg bw (based on a reduction in litter T. Nishino et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2005) xxx–xxx cell proliferation in epithelial prostatic cells, but effects on androgen receptor immunoreactivity and epithelial height of prostate and seminal vesicles, glandular area of prostates and seminal vesicles was observed. Overall, in standard devel- opmental studies in rodents, there is no convincing evidence that BPA is a developmental toxicant The estrogenic activity of BPA has been mostly observed in higher doses up to 100 mg/kg bw/day mech- anism concerning the androgen-like effect of the low-doses of BPA on the epithelial cells of the prostate is not clear at the present time. It may be difficult to correlate this BPA- effect with the known estrogenic property of this compound BPA had almost no effect on the seminal vesi- cle. Estrogens have been known to stimulate the development of the fibrous tissue and muscular walls of both prostate and seminal vesicle stimulating the epithelium and secretory activity Moreover, it seems very improbable, that the increase of the immunoreactive AR in the prostatic epithelial cells (not in the seminal vesicle) is up-regulated by estrogens. It is also unlikely that BPA, via an activation of adrenal androgen synthesis (the production of corticosteroid- binding globulin CBG and an activation of adrenal functions, leads to an increased steroidogenesis including androgen pro- duction), stimulates the prostate selectively, although estro- gens have been considered to be one of the controllers of adrenal androgen secretion Further studies on the low- dose effects of BPA using an antiandrogen may be necessary to elucidate the selective effects on the prostate in the rat.
Based on the present data, the densitometric analysis of AR-immunoreactivity and the assessment of both cell mor- phology and cell proliferation proved to be independent and sensitive parameters for the evaluation of androgen effects on prostates and seminal vesicles. The combined application of these parameters may provide an additional tool to test the broad spectrum of endocrine active substances such as endocrine disruptors, which are actually discussed on their potential risk to the environment and humans.
The authors wish to thank the director of the Institute of Anatomy (Univ. Prof. Dr. med. J. Westermann) for his gen- erous support. We gratefully acknowledge Mrs. H. Strauch- mann, Mrs. K. Budler, Mrs. L. Gutjahr, and Mrs. U. M¨uller- Horn for their excellent technical assistance.
Fig. 7. Comparison of luminal glandular area of (a) prostate (␮m2) and (b)seminal vesicle (␮m2) between the Intact group, OX group and TP groupin relation to the BPA-treated groups (3, 50, 200, 500 and 500 + FL). Aster- isks indicate statistically significant differences (p < 0.05), which refer to thecastrated control group.
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