A Comparative Study on the Protective Effects of 17Β-Estradiol, Biochanin a and Bisphenol a on Mammary Gland Differentiation and Tumorigenesis in Rats

Indian Journal of Experimental Biology Vol. 44, July 2006, pp. 540-546

A comparative study on the protective effects of 17β-estradiol, biochanin A and bisphenol A on mammary gland differentiation and tumorigenesis in rats

Hong Yin1#, Akihiro Ito1, Dilip Bhattacharjee2* & Masaharu Hoshi2 Departments of Cancer Research1 and Radiation Biophysics2, Research Institute for Radiation Biology and Medicine, Hiroshima University. Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan

Received 18 August 2005; revised 22 March 2006

Mammary tissue differentiation and tumorigenesis were studied in female rats following subcutaneous injection at 2, 4 and 6 days after birth with low or high doses of 17β-estradiol (0.1 or 10 µg; E2), biochanin A (0.1 or 10 mg; BCA) or bisphenol A (0.1 or 10.0 mg; BPA). Half of the rats were killed on day 35 to analyze the terminal end bud (TEB), terminal duct (TD) and alveolar bud (AB) of the mammary tissue. The remaining rats were injected, ip, with a dose of 50 mg/kg of N-nitroso-N-methylurea (MNU) at 7 weeks of age and sacrificed 26 weeks later. The incidence and multiplicity of mammary tumors (MT) decreased among all three different treated groups, dose-dependently. However, the pattern of mammary gland differentiation varied. No significant difference was observed after E2 administration. TEB decreased dose- dependently in BCA treated groups and the number of TD and AB were suppressed significantly in BPA high dose group.

Keywords: Biochanin A, Bisphenol A, 17-β-estradiol, Mammary gland differentiation, Mammary tumorigenesis

Incidence of mammary cancer has been increasing all claving process leached BPA out of the polycarbonate over the world including Japan. Considerable atten- plastics7,8. BPA has been detected binding to estrogen tion has been focused to prevent this by using receptors and inducing estrogenic activity9. Since environmental estrogenic chemicals. Biochanin A BPA is known to have estrogenic activity, its chemo- (BCA), an isoflavone derivative and a precursor of preventive property in tumorigenesis of breast is genistein has chemopreventive effect of breast proposed to be explored. However, the fetal exposure tumourigenesis1. It is capable of effectively to 10-9 M of BPA, currently being consumed by the competing for cytosolic estrogen receptor binding human being, altered the adult reproductive system in sites of rat liver and uterus in vitro2. Genistein, a mice10,11. metabolite of BCA, is structurally similar to estradiol, The physiological development of mammary gland binding weakly to estrogen receptor (ER)3. Neonatal in association with mammary tumorigenesis was administration of genistein inhibited the occurrence of extensively studied12-15. The undifferentiated struc- mammary tumors in animal models4,5. tures like terminal end buds (TEB) are the major Bisphenol A (BPA), a monomer of plastics and target for the mammary carcinogenesis. TEBs are epoxy resins, is widely used in dentistry and food located in the growing fringe of the mammary gland, packaging industry6. Autoclaved culture medium in and with maturation, TEBs regress to terminal ducts polycarbonate plastic flasks was found to cause an (TD) giving rise to alveolar buds (AB), which estrogenic function. It was determined that the auto- comprise type 1 and type 2 lobules. These lobules respond to the hormones of pregnancy by differentia- ______# Present address ting further to type 3 lobules and form functional units National Institute of Biological Sciences, Beijing of the lactating gland. The maturation of terminal end 7, Science Park Road, Zhongguancun Life Science Park, buds to lobules appears to provide a basic protective Beijing 102206 mechanism against chemical carcinogenesis. Phone: 86-10-80726688; Fax: 86-10-80726689 Since N-nitroso-N-methylurea (MNU) is a direct-

*Correspondent author acting carcinogen and has a short half-life in vivo, the Phone: 082 257 5856; E-mail: [email protected] mammary tumorigenesis with MNU in rats has been YIN et al.: ENVIRONMENTAL ESTROGENS ON MAMMARY TUMORIGENESIS 541

extensively used for human breast cancer model. The weeks after birth. Body weights and palpation results induced tumors are primarily estrogen-sensitive and of breast tumors in both sides of cervical, abdominal also histologically quite similar to human breast and inguinal glands were recorded once every 2 cancers16. weeks until 26 weeks after MNU injection. After In the present study, utilizing MNU-induced sacrifice at 26 weeks, all mammary tumors, ovary and mammary tumors in animal model, comparison has uterus were weighed and histological observations been made between the relationship of mammary- made. gland development and mammary tumorigenesis after neonatal administration of BCA and BPA with that of Pathology of mammary tissue⎯In mammary 17β-estradiol, a well established hormone that induces glands, total numbers of terminal end bud (TEB), protection following a short-term exposure against terminal duct (TD) and alveolar bud (AB) were mammary carcinogenesis. counted in 1 mm2 areas under microscope. TEB were composed of 3-6 layers of medium-sized epithelial Materials and Methods cells having scanty cytoplasm, and large and oval Sprague-Dawley CD/Crj pregnant rats purchased nuclei with one or two prominent nucleoli. TD from Charles River Japan Co., Kanagawa, were showed a thin wall and a wide lumen, which was maintained in a climate-controlled room at 24°±2°C lined by a single layer of small cuboidal epithelial on a 12; 12 hr light/dark cycle. Diet and tap water cells and an external layer of myoepithelial cells. AB were available ad libitum. At birth, the offspring were appeared as clusters of 3-5 tubules, each with a sexed and all females were randomly distributed to centrally located lumen surrounded by a layer of 12,13 the dams with enough males to have 10 offspring/ cuboidal epithelial cells . litter (4~6 females/litter). Animals were weaned at 24 days postpartum. All experiments were conducted Results under the guidelines of the “Guide for the care and The body weights of E2 (0.1 μg), BCA (0.1 mg) use of laboratory animals” of Hiroshima University. and BPA (0.1 mg) treated groups decreased signifi- A total of 170 newborn female rats were used for cantly (P<0.05) comparing to that of respective all the experiments and injected (sc) with 17β-estra- control group at the age of 35 day (Table 1). The diol (Sigma Chemicals, Co., St. Louis, USA), or relative pituitary weight of E2 (10 μg) group was Biochanin A (Sigma Chemicals) or Bisphenol A decreased remarkably (P<0.05). The relative weights (Wako Junyaku Co., Osaka, Japan) on day 2, 4 and 6 of thyroid in rats injected with BCA (10 mg), BPA after birth. The doses of E2 were 0.1 μg (for 15 rats) (0.1 and 10 mg) increased markedly comparing with or 10 μg (for 15 rats)/rat/day, while doses of BCA and control. No significant difference was found in the BPA were 0.1 mg (for 20 rats each) or 10 mg (for 20 weights of thymus, ovary and uterus. and 30 rats respectively)/rat/day. For E2, BCA and As compared to control density of TEB in BCA BPA groups 10, 20 and 20 infant rats respectively (10 mg) treated dose group decreased remarkably were kept as controls. At least 10% of the rats died (P<0.01; Table 2). The number of TD and AB in BPA during various phases of the experiments, maximum (10 mg) group showed a significant inhibiting effect being in BPA high dose group. comparing with control typical TEB, TD and AB are On day 35 after births, about 50% of female rats shown in Fig. 1. from each dose groups were sacrificed. Autopsied rats After MNU injection at the age of 7 weeks, all the were carefully observed and the pituitary, thyroid, rats were observed for next 26 weeks. After 26 weeks, thymus, uterus and ovary weighed. Both sides of body weight of both E2 (10 μg) and BPA (10 mg) inguinal mammary glands attached to the skin pelt groups were significantly increased (P<0.05; were removed and fixed in 10% physiological Table 3). The BCA (10 mg) group also showed an formalin/PBS. All tissues were fixed, embedded in obvious increasing tendency, though not significantly. paraffin, cut into 5 μm thick sections, stained by H&E No remarkable differences were found in the relative and observed under the light microscope. organ weights and the relative mammary tumor The rest of the female rats were injected (iv) with weights. MNU (N-nitroso-N-methylurea, Sigma Chemicals, Incidence of mammary tumor was inhibited dose- Co., USA) at a dose of 50 mg/g body weight at 7 dependently in rats given E2, BCA and BPA during 542 INDIAN J EXP BIOL, JULY 2006

almost all the observation period (Fig. 2). Latency Table 2—Density of TEB, TD and AB in the mammary gland period of the mammary tumor development was on 35 day old rats enhanced in E2 (10 μg), BCA (0.1 and 10 mg), and [Values are mean ±SE from 3-7 rats in each group] BPA (10 mg) groups by 2-4 weeks (Table 4). The Group and dose Number per mm2 under microscope incidence of mammary tumors was recorded at 3 TEB TD AB intervals in 10, 20 and 26 weeks after MNU treat- ment. Comparing with related control groups, all the Control 7±4.95 10±4.24 13.9±4.95 treated groups with E2, BCA and BPA treatments E 2 0.1 4±5.66 13.7±9.19 5.7±0.01 showed a similar inhibitory effects on the occurrence (μg) 10 3.3±0.71 13.7±9.19 6.3±4.24 of mammary tumor by palpation method or otherwise BCA 0.1 1.2±2.12 11.5±0.71 8.5±1.41 at week 26 by direct observation. The multiplicity of (mg) 10 0.3±0.71** 10.7±5.66 10.7±5.66 tumor expressed a decreasing tendency in all three BPA 0.1 2.3±1.41 11.7±5.66 6.7±3.54 treated groups (Fig. 3). It could be concluded that (mg) 10 5±6.36 3.7±0.71** 3±4.24* MNU-induced mammary tumors were inhibited by both BCA and BPA administered in infant rats similar Density = number of TEB, TD or AB/mm2 under microscope to E2. P values: *<0.05; **<0.01

Table 1—Body and relative organ weights at 35 day old rats following E2, BCA and BPA treatment in infancy [Values are mean ±SE] Group and dose N Body weight Relative organ weight (mg organ wt/100 g body wt) Control (g) Pituitary Thyroid Thymus Ovary 3 149.8±8.7 5.4±0.1 32.4±3.5 417.8±52.3 62.8±9.2 E 2 0.1 4 135±3.4* 5.8±0.8 30.2±3.9 395.3±36.4 58.0±8.4 (μg) 10 3 148.6±6.6 5±0.1* 30.8±1.6 348.9±10.8 51.9±8.8 BCA 0.1 4 128.2±7.4* 5.5±0.9 40.6±4.7 399.3±61.3 66.4±5.9 (mg) 10 3 134.9±4.6 5.6±0.8 50.7±8.1* 345.9±45.5 67.6±2.3 BPA 0.1 3 132.4±5.5* 6.4±0.9 51.8±4.2** 376.6±28.3 71.4±14.5 (mg) 10 3 141.9±4.3 5.9±1 41.6±2.5* 369.3±10.7 55.8±5.6

N = number of rats P values: *<0.05; **<0.01

Table 3—Body, relative organ weights and incidence of mammary tumor in rats at 26 weeks after MNU injection [Values are mean ±SE] Group and dose N Body weight Relative organ weight Incidence of (g) Ovary Uterus mammary tumor E 2 Control 8 290.1±40.2 56.4±11.8 249±60.5 0.51±1.1 (μg) 0.1 12 327.8±54.9 43.7±7.8 232.8±129.5 0.6±1.1 10 13 338.4±38.8* 60.3±55.2 208.8±61.8 0.7±1.2 BCA Control 17 322.1±48.1 59.9±19.9 212.4±90.7 0.98±1.5 (mg) 0.1 20 311.7±49.8 53.9±25.7 246.7±79.3 0.51±0.7 10 17 351.6±59.2 72.7±26.2 255.2±71.2 1.37±4.8 BPA Control 17 319.5±19.5 71.9±43.4 198±82.6 0.66±1.4 (mg) 0.1 20 313.2±35.3 54.1±21.6 192.5±86.3 0.63±1 10 26 344.8±43.3* 50.8±12.7 242.2±62.8 0.49±0.9

N = number of rats Relative organ weight = mg organ wt/100 g body wt P values: *<0.05, significantly different from respective control value

YIN et al.: ENVIRONMENTAL ESTROGENS ON MAMMARY TUMORIGENESIS 543

Discussion similar pattern on the mammary tumorigenesis except In the present study, effects of estrogen mimic certain minor differences on mammary-gland compounds, biochanin A and bisphenol A were development. compared with 17 β-estradiol on developments of The incidence of mammary tumors expressed a mammary gland and tumorigenecity in rats. The similar pattern among the different chemicals of results revealed that all these chemicals behaved in BCA, BPA and E2 dose-dependently. Recipients of

Fig. 1⎯Photomicrograph (H&E) of mammary gland development in control rats [A: cross section of TEB (×200); B: cross section of TD (×200); C: transverse section of AB (×400]

544 INDIAN J EXP BIOL, JULY 2006

Table 4—Incidence and multiplicity of mammary tumors in rats treated with E2, BCA and BPA Chemicals and doses Latency 10 weeks 20 weeks 26 weeks (w) In Mul In Mul In Mul E 2 Control 6 23.8 0.24 90.5 1.43 95.2 3.62 (μg) 0.1 6 29.63 0.3 57.7 1.31 84.6 2.62 10 8 16 0.32 45.8 0.92 66.7 1.75 BCA Control 6 11.8 0.18 85.3 0.47 82.4 1.88 (mg) 0.1 8 16.7 0.21 29.2 0.46 75 2.17 10 8 9.5 0.1 21 0.26 57.1 1.24 BPA Control 6 36.8 0.42 76.5 1.59 94.1 2.24 (mg) 0.1 6 16.7 0.21 58.3 0.71 79.2 1.42 10 10 7.7 0.08 34.6 0.46 76.9 1.65

Latency is when mammary tumors were observed for the first time after MNU injection In = incidence of mammary tumor (%) Mul = multiplicity, number of mammary tumor per rat

Fig. 3⎯Multiplicity of palpable mammary tumors after MNU Fig. 2⎯Incidence of palpable mammary tumors at various bi- administration at various bi-weekly intervals. [(A) E2 groups; weekly intervals after MNU administration. [(A) E2 groups; Control □ E2 0.1 μg ▲ E2 10 μg (B) BCA groups; Control Control □ E2 0.1 μg ▲ E2 10 μg (B) BCA groups; Control □ □ BCA 0.1 mg ▲ BCA 10 mg (C) BPA groups; Control □ BCA 0.1 mg ▲ BCA 10 mg (C) BPA groups; Control □ BPA BPA 0.1 mg ▲ BPA 10 mg]. Multiplicity is expressed as mean 0.1 mg ▲ BPA 10 mg] tumour number per rat YIN et al.: ENVIRONMENTAL ESTROGENS ON MAMMARY TUMORIGENESIS 545

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