International Journal of Impotence Research (2001) 13, 309–316 ß 2001 Nature Publishing Group All rights reserved 0955-9930/01 $15.00 www.nature.com/ijir 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 approxi- mately 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 pre- parations 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 max- imal 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).