Bisphenol a Inhibits Penile Erection Via Alteration of Histology in the Rabbit

Bisphenol A inhibits penile erection via alteration of histology in the rabbit

DG Moon1, DJ Sung1, YS Kim2, J Cheon1 and JJ Kim1*

1Department of Urology, Korea University College of Medicine, Seoul, Korea; and 2Department of Pathology, Korea University College of Medicine, Seoul, Korea

Despite extensive research into the toxicity of bisphenol A (BPA), no report of its effect on erectile function exists. We performed this study to investigate the effect of BPA on erectile function. New Zealand white rabbits were treated intraperitoneally with 150 mg=kg of BPA every other day for 12 days (cumulative dose of 900 mg=kg). Four and 8 weeks after administration of BPA, the contractions and relaxation of cavernosal tissue strips were signiﬁcantly suppressed in the BPA- treated animals compared to the control animals. Histologically, thickening of tunica albuginea, subtunical deposition of fat and decreased sinusoidal space with consequent increase of trabecular smooth muscle content were observed in the BPA-treated animals. These results suggest that xenoestrogen BPA may affect the erectile function through evident histological changes of the penis. International Journal of Impotence Research (2001) 13, 309–316.

Keywords: xenoestrogen; bisphenol A; erection; penis

Introduction Materials and methods

Bisphenol A (BPA) is an environmental estrogen- Animals and treatments like chemical,1,2 a monomer of polycarbonate plas- tics and a constituent of epoxy and polystyrene resins that are extensively used as food packaging All procedures involving animals were approved by materials for industrial processing and consumer the institutional animal care and use committee and use.3 Xenoestrogens have been shown to affect followed the guidelines set by the Korea University reproduction in wildlife4,5 and may have adverse College of Medicine Animal Research Policies effects on humans6,7 because of their ubiquitous Committee. Male rabbits (New Zealand white, 8 – presence in the environment, resistance to degrada- 12 weeks old, 2.0 – 2.5 kg, Samyuk Hi-Quality tion and potential for accumulation in fat tissues. Laboratory Animal Inc., Osan City, Kyunggi Pro- Human exposure to BPA is not insignificant, as vince, Korea) were treated intraperitoneally with microgram amounts of BPA were detected in liquid 150 mg=kg of BPA dissolved in corn oil every other from canned vegetables8 and in the saliva of patients day for 12 days (cumulative dose of 900 mg=kg). treated with dental sealants.9 Four weeks (group I, N ¼ 15) and 8 weeks (group II, Penile erection depends on the balance and N ¼ 15) after the administration of BPA, the rabbits integration of neurotransmitters, vasoactive sub- were sacrificed by a bolus injection of air into an ear stances, endocrine factors and tissue fibroelastic vein. In five rabbits of both groups, the penis was properties. It can be assumed that antiandrogen, removed and histologically examined. In 10 rabbits estrogen or xenoestrogen may affect erectile func- of both groups, the penis was removed and im- tion. Despite extensive research into the toxicity of mediately placed in chilled Krebs solution. A Krebs BPA, no report of its effect on erectile function solution of the following composition was used exists. We performed this study to investigate the (mM): NaCl 119; KCl 4.6; CaCl2 1.5; MgCl2 1.2; effect of BPA on erectile function. NaHCO3 15; NaH2PO4 1.2; glucose 11. After removal of surrounding muscles and connective tissues, the two corpora cavernosa (CC) were carefully dissected *Correspondence: JJ Kim, Department of Urology, Korea under microscope. Each CC was cut into two University Ansan Hospital, 516, Gojan-dong, Ansan-city, strip preparations measuring approximately Kyunggi-do 425-020, Korea. 16267 mm at the level of proximal part of the CC E-mail: [email protected] Received 15 November 2000; revised 15 March 2001; and pharmacologic strip study was performed. In accepted 20 July 2001 group III (n ¼ 10, young control) and group IV Bisphenol A inhibits penile erection DG Moon et al 310 (n ¼ 10, older control), the same experiment was bubbled with a mixture of 95% O2 and 5% CO2, performed as controls. In the strip study, contrac- resulting in a pH of 7.4. The bath fluid was changed tions were studied with norepinephrine, whereas every 20 min and replaced with fresh Kreb’s solu- relaxations were studied with acetylcholine, L- tion. Silk ligatures were applied at both ends of the arginine and sodium nitroprusside (SNP). All CC strips, which were suspended between two L- experimental drugs were purchased from Sigma formed metal prongs. One of the prongs was Chemical Co. (Sigma Chemical Co., St Louis, connected to a Grass FTO3C force-displacement Missouri, USA). transducer (Grass Instruments Co., Quincy, MA, USA), and the other was attached to a movable unit, allowing precise adjustment of preload tension. Isometric tension was recorded using a Grass 7D Pathologic examination polygraph (Grass Instruments Co., Quincy, MA, USA). Tissue strips were incrementally stretched (1 g tension=stretch), and periodically contracted Grossly, each penis was cut according to a dorsal to 76 ventral axis. All specimens were fixed in 10% with 10 M phenylnephrine. This procedure was neutral buffered formalin, dehydrated in graded repeated until the tissues achieved optimal iso- ethanol and embedded in paraffin. Sections were metric tension. When the amplitude of the contrac- cut at 4mm thickness, placed upon poly-L-lysine tion was within 10% of the previous contraction, coated glass slides, and stained with hematoxylin – that tension was considered optimal for isometric eosin (H&E) and Masson’s trichrome. Tunical contraction. thickness was measured at three levels of the 1. Noradrenaline (NA) was added cumulatively in distal, mid-shaft and proximal part of the penis. order to determine the concentration – response Computerized analysis of the smooth muscle fibers relationship. The contractile responses were was done on a CAS-200 image analyzer (Becton expressed as maximal g tension (mN) after the Dickinson Co., USA) in combination with a light cumulative addition of NA at optimal state for Olympus microscope equipped with a video cam- isometric contraction (n ¼ 22, N ¼ 7). era. The percentage of area of smooth muscle fibers 2. To investigate the effects of relaxants, acetylcho- per standard square area was measured at five line, L-arginine and SNP were added cumula- different fields (magnification 4006) of vision for tively in half-log increments (1078 –1074 M) to two sections in each case. The mean value was used preparations precontracted with 1074 M phe- for each animal. nylnephrine, the concentration producing 50% of maximum contraction.

Measurement of isometric tension Calculations The preparations were transferred to 10 ml organ baths containing Kreb’s solution maintained at 37C Student’s t-test was used for statistical analysis of by a thermoregulated water circuit and continuously paired or unpaired observations. A probability of

Figure 1 Contraction of isometric strips to cumulative dose (1078 –1074 M=ml) of norepinephrine (NE). NE induced contraction of strips in a dose-dependent manner in each group but contraction in BPA-treated animals was signiﬁcantly suppressed compared to that of control animals. Group I: 4 weeks after treatment of BPA: group II, 8 weeks after treatment of BPA; group III, young control; IV; group IV, older control. *P < 0.05; **P < 0.01.

International Journal of Impotence Research Bisphenol A inhibits penile erection DG Moon et al 311 P < 0.05 was accepted as signiﬁcant. When appro- and IV (n ¼ 16, N ¼ 5), respectively. In groups I priate, results are given as mean value and standard (n ¼ 32, N ¼ 10) and II (n ¼ 28, N ¼ 10), a maximum error of the mean. ‘n’ denotes the number of strip effect of 19.87 Æ 3.17 and 6.37 Æ 1.65% was obtained preparations, and ‘N’ denotes the number of in- at a concentration of 1074 M, respectively (Table 2, dividuals. All statistical calculations were made Figure 2). Preparations contracted by 1074 M phe- using N. nylephrine were concentration (1078 –1074 M)-de- pendently relaxed by SNP (Table 2, Figure 3). At a concentration of 1074 M, a maximum effect of Results 69.34 Æ 11.68 and 64.56 Æ 9.54% was obtained in groups III (n ¼ 18, N ¼ 5) and IV (n ¼ 16, N ¼ 5), respectively. In groups I (n ¼ 32, N ¼ 10) and II Pharmacologic strip study (n ¼ 28, N ¼ 10), a maximum effect of 49.34 Æ 9.87

The tension of the preparation was repeatedly adjusted during an equilibration period of approximately 1 h, tension was adjusted, and for the CC (n ¼ 85, N ¼ 18), the final isometric tensions were 4.35 Æ 0.52 mN in controls. NA contracted the preparations in a concentration-dependent manner (Table 1, Figure 1). Threshold concentration was 1077 M. Cumulative concentrations of NA (1078 – 1074 M) produced stable contraction with a maximal amplitude of 16.6 Æ 3.54 mN (n ¼ 18, N ¼ 5) and 14.15 Æ 3.12 mN (n ¼ 16, N ¼ 5) in groups III and IV of control animals, respectively. The contractions to cumulative concentrations of NA were significantly decreased to 4.56 Æ 1.96 mN (n ¼ 32, N ¼ 10) and 2.61 Æ 1.54 mN (n ¼ 28, N ¼ 10) in groups I and II, respectively. Threshold concentration was 361075 74 and 10 M in groups I and II, respectively. Figure 3 Relaxation of precontracted strip to cumulative dose Acetylcholine relaxed the precontracted strips by (1078 –1074 M=ml) of sodium nitroprusside (SNP). SNP induced 1074 M phenylephrine in a dose-dependent fashion relaxation of strips in a dose-dependent manner in each group but (1078 –1074 M). At a concentration of 1074 M, a relaxation in BPA-treated animals was significantly suppressed compared to that of control animals. Group I, 4 weeks after maximum effect of 49.34 Æ 7.57% and 44.56 Æ treatment of BPA; group II, 8 weeks after treatment of BPA; group 6.76% was obtained in Group III (n ¼ 18, N ¼ 5) III, young control; group IV, older control. *P < 0.05; **P < 0.01.

Figure 2 Relaxation of precontracted strip to cumulative dose (1078 –1074 M=ml) of acetylcholine. Acetylcholine induced Figure 4 Relaxation of precontracted strip to cumulative dose relaxation of strips in a dose-dependent manner in each group (1078 –1074 M=ml) of L-arginine. L-Arginine induced relaxation but relaxation in BPA-treated animals was signiﬁcantly sup- of strip in a dose-dependent manner in control group but pressed compared to that of control animals. Group I, 4 weeks relaxation in BPA-treated animals was signiﬁcantly suppressed after treatment of BPA; group II, 8 weeks after treatment of BPA; in BPA-treated animals. Group I, 4 weeks after treatment of BPA; group III, young control; group IV, older control. *P < 0.05; group II, 8 weeks after treatment of BPA; group III, young control; **P < 0.01. group IV, older control. *P < 0.05; **P < 0.01.

International Journal of Impotence Research Bisphenol A inhibits penile erection DG Moon et al 312 and 44.56 Æ 10.34% was obtained at a concentration animals, intracavernosal fibrosis and decreased of 1074 M, respectively. L-Arginine relaxed the sinusoidal space with consequent increase of trabe- precontracted strips by 1074 M phenylephrine in a cular smooth muscle content were also noted in dose-dependent manner (1078 –1074 M). At a con- groups I (Figure 6) and II (Figure 7). The amounts of centration of 1074 M, a maximum effect of smooth muscle in BPA-treated animals were sig- 17.96 Æ 3.45 and 16.87 Æ 2.94% was obtained in nificantly increased to 73.3 – 83.2% compared to groups III (n ¼ 18, N ¼ 5) and IV (n ¼ 16, N ¼ 5), those of control animals (Table 3, P < 0.01). Com- respectively (Table 1, Figure 4). In group I (n ¼ 32, pared to the young rabbit, the amount of smooth N ¼ 10) and II (n ¼ 28, N ¼ 10), a maximum effect of muscle fibers significantly decreased to 15.0% in 5.17 Æ 0.75 and 0% was obtained at a concentration group IV. In group IV, tunical thickness and of 1074 M, respectively. intracavernosal fibrosis were slightly increased but there was no statistical significance compared to group III (Figure 7). Compared to these control Histologic examination animals of groups III and IV, the thickness of tunica albuginea was significantly increased to 0.93 – 1.12 mm and abnormal subtunical depositions of In normal penis of young-aged rabbit, the amount of fat were observed in groups I and II (Table 3, Figures smooth muscle fibers on histomorphometry was 7 and 8). The thickening of tunica appeared to be 33.2% of specimen and tunica albuginea was 0.32 – derived from markedly increased collagen fibers, 0.43 mm in thickness (Table 3). There was no which were evident on Masson’s trichrome stain evidence of fibrosis (Figure 5). In BPA-treated (Figure 8).

Figure 6 Corpus cavernosum of BPA-treated rabbit. (A) Sub- tunical deposition of fat and decreased sinusoidal space with Figure 5 Corpus cavernosum of young-aged rabbit. (A) Rich consequent increase of trabecular smooth muscle content. (A) 8 vascular spaces are well maintained by ﬁbromuscular partitions. weeks after BPA treatment, H&E, reduced from 640. (B) Four H&E, reduced from 620. (B) Section of corpus cavernosum tissue weeks after BPA treatment. Masson’s trichrome stain, reduced stained by trichrome Masson’s technique. Reduced from 640. from 6100.

International Journal of Impotence Research Bisphenol A inhibits penile erection DG Moon et al 313 Discussion arteries and veins, which are surrounded by a collagenous tunica albuginea. Recently, trabecular smooth muscle tone has been accepted as a major The erectile tissue of the human penis is composed contributing factor to trabecular smooth muscle of elastic ﬁbers, collagen ﬁbers, smooth muscles, contractility; relaxation of the smooth muscle of

Figure 7 Corpus cavernosum of BPA-treated rabbit and older rabbit. (A) Rich vascularity and slight ﬁbrosis are seen in older rabbit. (B) Eight weeks after treatment of BPA. Compared to older control, subtunical deposition of fat and decreased sinusoidal space with consequent increase of trabecular smooth muscle content are seen. Masson’s trichrome stain, reduced from 6200.

Figure 8 Changes of tunical thickness after treatment of BPA. Compared to normal control animals, tunical thickness was signiﬁcantly increased in BPA-treated animals. Masson’s trichrome stain, reduced from 640. (A) Young control; (B) older rabbit; (C) 4 weeks after BPA treatment. (D) 8 weeks after BPA treatment.

International Journal of Impotence Research Bisphenol A inhibits penile erection DG Moon et al 314 Table 1 Changes of maximal contraction to vasoactive Holmquist et al reported that castration increase substances after treatment of BPA the relaxation of corpus cavernosum strips to Maximal contraction induced by norepinephrine electrical stimulation through decrease in the neu- ronal release of NA.13 Meanwhile, Baba reported Group I Group II Group III Group IV that tissue strips from intact rabbits showed a greater degree of contraction than those of castrated ani- mN 4.56 Æ 1.96** 2.61 Æ 1.54** 16.6 Æ 3.54 14.15 Æ 3.12 mals, suggesting changes in the adrenergic pathway Data are expressed as mean Æ standard deviation. Asterisks in response to androgen deprivation.14 Castration indicate that scores are significantly different from those of reduces NANC fibers in rat model;15 however, these control animals. *P < 0.05; **P < 0.01. fibers are thought to be decreased in the castrated Group I, 4 weeks after BPA treatment; group II, 8 weeks after BPA 13 treatment; group III, young-aged control; group IV, old-aged model, suggesting differences in the response control. to androgens among species. Recently, Traish et al reported that castration reduces intracavernosal pressure and expression of a1-adrenergic receptor while increasing phosphodiesterase type 5 Table 2 Changes of maximal relaxation to vasoactive substances activity.16 after treatment of BPA Estrogen exhibits potent anti-atherogenic effects Maximal relaxation (%) of precontracted strips through a mechanism which involves endothelium- dependent vasodilation of arteries and veins. This Group Acetylcholine Sodium nitroprusside L-Arginine may be mediated by a direct effect of estrogen on I 19.87 Æ 3.17** 49.34 Æ 9.87* 5.17 Æ 0.75** vascular function or may be induced through 17 II 6.37 Æ 1.65** 44.56 Æ 10.34* 0** modification of lipoprotein metabolism. Case III 49.34 Æ 7.57 69.34 Æ 11.68 17.96 Æ 3.45 et al suggested that estrogen increases the nitric IV 44.56 Æ 6.76 64.56 Æ 9.54 16.87 Æ 2.94 oxide component of endothelium-dependent dila- Data are expressed as mean Æ standard deviation. Asterisks tion and decreases the cyclooxygenase compo- 18 indicate that scores are significantly different from those of nent. In this study, the contraction by NA and control animals. *P < 0.05; **P < 0.01. relaxation by acetylcholine, L-arginine and SNAP Group I, 4 weeks after BPA treatment; group II, 8 weeks after BPA were significantly decreased in the corpus caverno- treatment; group III, young-aged control; group IV, old-aged sum strips of BPA-treated animals compared to control. control animals. Xenoestrogen is not identical to antiandrogen and estrogen but is similar to estrogen in action. Besides the adrenergic and nitrergic the corpus cavernosum is crucial for development of pathways fibrosis may inhibit the contraction and penile erection.10,11 For erection to occur, the penile relaxation of strips. However the xenoestrogenic arteries and sinusoids must be dilated, thereby effects of BPA on the adrenergic and nitric oxide increasing the blood flow into the penis. The pathway should be elucidated. distended sinusoids compress the venules against The effect of estrogen on smooth muscle in the tunica albuginea.12 Contraction of the penile various organs is unpredictable. It has been well smooth muscle necessary for keeping the penis in its demonstrated that 17b-estradiol exhibits hypertro- flaccid state is maintained chiefly by the release of phy of uterine smooth muscle19 but inhibits smooth NA. The cholinergic and nonadrenergic noncholi- muscle cell proliferation in porcine coronary.20 nergic (NANC) neurotransmitter, nitric oxide, plays Fotsis et al reported that the endogenous estrogen a critical role in the control of smooth muscle metabolite 2-methoxyestradiol inhibits angiogenesis relaxation.11,12 in vitro.21 In castrated or estrogen-treated animals,

Table 3 Thickness of tunica and percentage of cavernosal smooth muscle ﬁbers in each group

Group

I II III IV

Thickness of tunica albuginea (mm) Distal 0.93 Æ 0.14* 1.03 Æ 0.11* 0.32 Æ 0.03 0.36 Æ 0.06 Mid 0.99 Æ 0.18* 1.09 Æ 0.13* 0.39 Æ 0.06 0.41 Æ 0.08 Proximal 1.05 Æ 0.29* 1.12 Æ 0.10* 0.43 Æ 0.07 0.46 Æ 0.08 Percentage of intracavernous smooth muscle ﬁbers Percentile (%) 73.25 Æ 11.34* 83.15 Æ 9.67* 33.19 Æ 8.45 15.04 Æ 6.78

Data are expressed as mean Æ standard deviation. Asterisks indicate that scores are signiﬁcantly different from those of control animals. *P < 0.05; **P < 0.01. Group I: 4 weeks after BPA treatment; group II, 8 weeks after BPA treatment; group III, young-aged control; group IV, old-aged control.

International Journal of Impotence Research Bisphenol A inhibits penile erection DG Moon et al 315 the reduction of the glandular lumen is the most was shown that bisphenol A was estrogenic.29 The obvious morphological alteration, accompanied by amount of BPA used in this study was in a toxic an increase in connective tissues.22 In BPA-treated dose range and it is impossible to receive such animals, subtunical deposition of fat was noticed. amounts of BPA in the daily activities of human Shabsigh23 suggested the castration-induced pro- beings. Thus, the effects of small amounts of BPA on grammed smooth muscle cell death in the rat penis human erectile tissue should be determined. The and Traish et al reported the castration-induced aging process exerts a direct effect on all penile decrease of trabecular smooth muscle content in the tissues. Cellular senescence and death lead to rabbit penis.16 increased deposition of connective tissue, which The major difference of this study compared to leads to a decrease in penile distensibility. Chronic previous castration studies is the increase of exposure of low-dose BPA may result in erectile trabecular smooth muscle content. In this study, dysfunction in aging males or may exacerbate the cavernosal smooth muscle was markedly increased aging process with consequent erectile dysfunction. compared to those of control animals and whole It suggests the possibility that certain human cavernosum was replaced with the muscle mass. It subpopulations, eg developing fetuses, prepubertal is also opposed to the concept of venoocclusive children and postmenopausal women, all of whom erectile dysfunction in which reduction or dysfunc- have low estrogen levels, may be vulnerable to the tion of the intracavernous smooth muscle fibers has undesirable effects of BPA and other xenoestrogens. been suggested to evoke erectile dysfunction.24,25 In this study, trabecular smooth muscle content was measured by hitomorphometry. Therefore, BPA may not increase the definite content of smooth muscle Conclusion but reduces individual sinusoidal lumen with consequent increased ratio of smooth muscle con- To our knowledge, this study is the first demonstra- tent on histomorphometry. Although changes of tion concerning the effect of BPA on erectile intracavernosal pressure to vasoactive substances function. The results indicate that erectile tissue were not evaluated in this study, it can be assumed may not be free from the adverse effects of this that the decreased potential cavity of sinusoid with endocrine disruptor. The changes in corpus caver- consequent increased ratio of smooth muscle con- nosum by BPA in this study were difficult to explain tent may inhibit arterial inflow. Additionally, low because of the complexity of hormonal changes expandability of thickened tunica albuginea re- induced by xenoestrogen, but it is evident that BPA stricts the engorgement of the cavernosum, thus induces morphological and functional alterations of prevents penile enlargement and erection. Penile the corpus cavernosum, which appears to be tunica albuginea represents an important part of the sensitive to the estrogenic action of BPA. Further fibrous corpora cavernosa skeleton. Its collagenous studies concerning the effects of BPA on the nitric fibers accompanied by elastic networks are arranged oxide pathway, types of collagen and receptors in in an outer, mainly longitudinal and an inner human erectile tissue should be carried out to circular layer.26 These fibers allow penile elongation identify the more precise effect of BPA on human during tumescence and an increase in girth during erectile function. erection while providing adequate resilience for a rapid return to the relaxed nonerect state at detumescence.27,28 Loss of corporeal elasticity by decreased vascularity with consequent increased Acknowledgment ratio of cavernosal smooth muscle content and thickening of tunica albuginea may be the result of xenoestrogenic effect of BPA, which adversely This study was supported by Korea University affects erectile homodynamic. The corpus caverno- Medical Science Research Center Grant 1999 and sum seems very sensitive to the stimulation by BPA Ministry of Education in Korea. in this study. The mechanisms should be identified in further studies. A directive of the European Union (EU) has established a specific migration limit in food of 0.02 mg=kg for diglycidyl ether References bisphenol A (BADGE; CAS no. 1675-54-3). The presence of the monomer bisphenol A (4,4-isopro- 1 Colborn T, vom Sall FS, Sato AM. Developmental effects of pylidenediphenol, CAS no. 80-05-07) in these coat- endocrine disrupting chemicals in wildlife and humans. ings was considered of lesser importance, and a Environ Health Perspect 1993; 101: 378 – 385. higher tolerance limit (3 mg=kg) for its specific 2 Krishnan AV et al. Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. En- migration was therefore established by the EU docrinology 1993; 132: 2279 – 2286. Commission; however concerns about the toxicity 3 Knaak JB, Sullivan LJ. Metabolism of bisphenol A in the rat. of this compound were heightened recently when it Toxicol Appl Pharmac 1966; 8: 175 – 184.

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International Journal of Impotence Research