摘要
双酚A(Bisphenol A,BPA)广泛存在于塑料制品中,随着社会生产的迅猛发展,塑料制品的广泛应用,成为人们在生产生活中不可避免接触到的一类环境雌激素。BPA对机体的不良影响,已受到人们的广泛关注。BPA对机体的影响是多方面的,主要包括生殖系统、胚胎发育、神经系统、免疫系统、肿瘤发生等。BPA对雄性生殖系统的影响研究较多,大量动物实验证明,BPA几乎能够引起各种雄性生殖系统异常,对雄性生殖系统的发育和功能有较为广泛的影响。BPA对雌性生殖系统也有影响,BPA与卵巢囊肿、子宫内膜异位症、子宫肌瘤、子宫内膜癌等疾病的发生有密切的关系,并能诱发流产,降低生育率和妊娠率。但目前对BPA对雌性生殖系统影响的研究仍不全面,尤其对其复杂的作用机制的了解仍不够完善。因此,对BPA对小鼠子宫的影响及其分子机制的研究,能够为BPA诱导生殖系统疾病致病机制的研究提供一定的理论和实验依据,以期进一步加深人们对BPA的认识。同时也为BPA诱导肿瘤的预防、治疗及诊断提供依据。
本实验主要采用子宫生长实验、RT-PCR,Western-blot及免疫组化等实验方法,研究了BPA处理后去势小鼠子宫湿重、脏器系数变化,ERa、phospho-ERα、ERβ、phospho-ERβ及凋亡基因Bcl-2/Bax在小鼠子宫中的分布及表达。实验结果表明:
1)BPA具有雌激素样效应,可使去势小鼠子宫总体呈现增殖趋势,子宫肌层增厚,腺体增多,子宫腔增大等一系列形态学变化。
2)BPA可使子宫内雌激素受体表达量增加,可激活ER,磷酸化受体表达量增加,其中子宫内膜、腺上皮ER表达变化明显,表明ER的合成及表达受BPA的调控, BPA的雌激素样效应可能通过ER途径起作用。
3)BPA可明显上调Bcl-2基因的表达,并抑制Bax基因的表达,抑制细胞凋亡,促进细胞增殖,但在子宫内膜,腺上皮又上调Bax基因的表达,BPA诱导子宫增殖的同时可能对子宫内膜、腺上皮等功能性细胞有一定的损害。
Bisphenol A(BPA) is a kind of environmental estrogens which broadly existed in plastic products and it can not be avoided touching in our lives as the development of social production and the wider useing of plastic products. More and more People already pay attention to the adverse effects of BPA. The effects of BPA impacting on organism have many aspects include reproductive system, embryonic development, nervous system, immune system, tumor genesis and so on. There are a lot of researches of BPA on the male reproductive system. Large number of animal experiments proved that BPA may almost cause all male reproductive system abnormalities including the functions and the developments. BPA also could affect the female reproductive system. BPA may related to ovarian cysts, endometriosis, uterine fibroids, endometrial cancer and so on. BPA may induce abortion, reducing fertility and pregnancy. But the study of BPA on the female reproductive system is not comprehensive, especially its complex understanding mechanism. Studying the effects of BPA impacting on mouse uterus and its molecular mechanism will provide the theoretical and experimental basis for the pathogenic mechanism of reproductive system diseases inducing by BPA, deeping people's understanding of BPA. It also will provide the basis for BPA-induced tumor's prevention, treatment and diagnosis.
Using uterine growth experiment, we detected uterine wet weight and organ coefficient of ovariectomized mice treated by BPA. Using RT-PCR, Western-blot and immunohistochemistry methods, we detected gene changes of distribution and expression in the uterus of ovariectomized mice treated by BPA, including ERα, phospho-ERα, ERβ, phospho-ERβ, Bcl-2 and Bax. The results as showed:
1) BPA has estrogen effects which can show the whole uterus in ovariectomized mice proliferation trends: myometrium thickening, glands increasing, uterine cavity increasing.
2) BPA can cause changes of the expression of ER, so its estrogen-like effect may play a role through the ER channels.
3) BPA caused apoptosis gene Bcl-2, Bax expression changes, induced endometrial and glandular epithelial cell apoptosis. BPA can induce proliferation in the uterus ,at the same time it may have some functional cell damage.
引文
黄朝晖,王金福. 2000. ERβ-一种新型的雌激素受体. 2000.生命科学, 12(3): 1296 1
林勇,曾祥贵,吴德生等. 2006.双酚A对原代培养胎鼠脑多巴胺神经元的氧化损伤作用研究.卫生研究, 35(4):19- 23
龙鼎新,李小玲,李勇等. 2002.应用全胚胎培养模型研究双酚A的胚胎毒性.美国中华临床医学杂志, 4(4): 264-266
吕毅,赵淑华,杨湘山等. 2006.双酚A对妊娠小鼠仔鼠免疫功能影响.中国公共卫生, 22(12):1534
裴新荣,李勇,龙鼎新等. 2003.双酚A对小鼠早期胚胎发育毒性的体外实验研究.中国生育健康杂志, 14(1): 34- 37
孙桂英,金志军,杨云纺. 1999.雌激素受体的变异及其临床意义.国外医学妇产科分册, 26(3): 159- 161
王薛君,张玉敏,李海山等. 2005.双酚A对小鼠生殖和发育毒性的研究.中国职业医学, 32(3): 37- 39
徐宏勇,李开宗,付由池等. 2002.雌激素受体和凋亡相关基因bc1-2及bax在人原发性胆囊癌中表达的临床意义.中国普外基础与临床杂志, 9(2): 145- 149
张波,陈道达,壬固斌等. 2003.乳腺癌雌激素受体mRNA的变异剪切与Tamoxifen耐受. 华中科技大学学报(医学版), 32(1): 46
张仁汉,陈茂森,何冰. 1998.雌二醇和三苯氧胺对小鼠肺癌基因表达的影响.中华内科杂志, 37(3): 192
张玉敏,崔金山,段志文等. 2004.环境雌激素双酚A对小鼠生精功能的影响.工业卫生与职业病, 30(5): 296- 299
Adriani W, Seta DD, Dessì-Fulgheri F, et a1. 2003. Altered profiles of spontaneous novelty seeking ,impulsive behavior , and response to D-Amphetamine in rats perinatallyexposed to bisphenol A. Environ Health Perspect ,111 (4) : 395- 401
Aikawa H, Koyama S, Matsuda M, et al. 2004. Relief effect of vitamin A on the decreased motility of sperm and the increased incidence of malformed sperm in mice exposed neonatally to bisphenol A. Cell Tissue Res, 315: 119- 124
Aikawa H, Koyama S, Matsuda M, et al. 2004. Relief effect of vitamin A on the decreased motility of sperm and the increased incidence of malformed sperm in mice exposed neonatally to bisphenol A. Cell Tissue Res, 315(1): 119- 124
Akita M, Yokoyama A, Shimizu S, et al. 2000. Effects of Bisphenol A on cultured rat embryos. Teratology, 62(3): 15.
Al-Hiyasat AS, Darmani H, Elbetieha AM. 2002. Effects of bisphenol A on adult male mouse fertility. Eur J Oral Sci, 110: 163- 167
Al-Hiyasat AS, Darmani H, Elbetieha AM. 2002. Effects of bisphenol A on adult male mouse fertility. Eur J Oral Sci, 110(2): 163- 167
Ankley, Yanase T, Nishi Y, et al. 2001. Saturated FFAs. Palmitic acid and stearic acid, induce apoptosis in human granulosa cells. Endocrinology, 142(8): 3590- 7
Ashby J, Tinwell H, Lefevre PA, et al. 2003. The effect on spermproduction in adult Sprague-Dawley rats exposed by gavage to bisphenol A between postnatal days 91- 97. Toxicol Sci, 74: 129- 138
Ashby J, Tinwell H, Lefevre PA, et al. 2003. The effect on spermproduction in adult Sprague-Dawley rats exposed by gavage to bisphenol A between postnatal days 91- 97. Toxicol Sci, 74(1): 129- 138
Bergeron RM, Thompson TB, Leonard LS, et a1. 1999. Estrogenicity of bisphenol A in a human endometrial carcinoma cell line. Mol cell Endocrinol, 150(1-2):179- 187
Beyer C, Raab H. 1998. Nongenomic effects of oestrogen: embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores. Eur J Neurosci, 10(1): 255- 262
Can A, Semiz O, Cinar O. 2005. Bisphenol-A induces cell cycle delay and alters centrosome and spindle microtubular organization in oocytes during meiosis. Mol Hum Reprod, (6): 389-96.
Caroline M, Markey, Perinaaz R, et al. 2005. Long-term effects of fetal exposure to low doses of the Xenoestrogen bisphenol-A in the Female Mouse Genital Tract. Biol Reprod, 72: 1344- 1351
Caroline M, Markey, Perinaaz R, et al. 2005. Long-term effects of fetal exposure to low doses of the Xenoestrogen bisphenol-A in the Female Mouse Genital Tract. Biol Reprod, 72(6): 1344- 1351
Clarker R, Hilakivi Clarke L, Trock B. 2001. Breast cancer:dietary and environmental oestrogens. Biologist (London), 48(1): 21- 26
Clements J. 2000. The Mouse Lymphoma Assay. Mutat Res, 455(1- 2): 97- 110
Couse JF, Korach KS. 1999. Estrogen receptor null mice :what have we learned and where will they lead us[J ]. Endocrine Rev, 1999, 20 (3): 358- 417.
Deve B J. 2000, CREM: a master-switch regulating the balance between differentiation and apoptosis in male germ cells. Mol Reprod Dev, 56(2 ): 228- 229
Diel P, Schulz T, Smolnikar k, et a1. 2000. Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: estrogenicity profiles and uterotropic activity. J Steroid Biochem Mol Biol, 73(1-2):1- 10
Fujimoto J, Sakaguchi H, Aoki I, et al. 2002. Clinical implication of the expression of estrogen receptor-alpha and beta in primary and metastatic lesions of uterine endometrial cancers[J]. Oncology, 62: 269
Geefer, et al. 1999. Mechanism of apoptosis by EDs. Mol Biol, 11(2): 479- 500
Goumenou A, Panayiotides I, Matalliotakis I, et al. 2001. Bcl-2 andBax expression in human endometriotic and adenomyotic tissues. Eur J Obstet Gynecol Reprod Biol, 99(2): 256
Green P, Lonning PE. 2000. High-does estrogen treatment in male reproductive system heavily exposed to endocrine therapy. Cancer Research, 31(3): 111- 116
Hanstain B, Beckmann MW, Bender HG. 2002. Role of estrogen receptor isoforms in the pathogensis and treatment of endometrial cancer[J]. Zentralbl Gynakol, 124(1): 17
Herath CB, Jin W, Watanabe G, et al. 2004. Adverse effects of environmental toxicants, octylphenol and bisphenol A, on male reproductive functions in pubertal rats. Endocrine, 25(2): 163- 172
Howdeshell KL, Peterman PH, Judy BM, et a1. 2003. Bisphenol A is released from used polycarbonate animal cages into water at room temperature. Environ Health Perspect, 111(9): 1180- 1187
Howdeshell KL, Peterman PH, Judy BM, et a1. 2003. Bisphenol A is released from used polycarbonate animal cages into water at room temperature. Environ Health Perspect, 111(9): 1180- 1187
Inadera H, Hashimoto H, Dong HY, et a1. 2000. WISP-2 as a novel estrogen-responsive gene in human breast cancer cells.Biochem Biophys Res Commun, 275(1):108- 114
Jones R K, Searle R F, Bumer J N, et a1. 1998. Apoptosis and bcl-2 expression in normal human endometrium, endometriosis and adenomyosis. Human Reprod Oxford, 13(12): 3496- 3502
Katzenellenbogen BS, Katzenellbogen JM. 2000. Estrogen receptor transcription and transactivation: Estrogen receptor alpha and estrogen receptor beta: regulation by selective estrogen receptor modulators an d importance in breast cancer. Breast Cancer Res, 2(5): 335- 342
Kavlock R J, Daston G P, Derosa C, et al. 1996. Research needs for the risk assessment of health and environmental effects of endocrine disrupters: a report of the USEPA-sponsored workshop. Environmental Health Perspectives, 104: 715- 740
Kester MH, Bulduk S, Van Toor H, et a1. 2002. Potent inhibition of estrogen sulfotransferase by hydroxylated metabolites of polyhalogenated aromatic hydrocarbons reveals alternative mechanism for estrogenic activity of endocrine disrupters. J Clin Endocrinol Metab, 87(3):1142- 1150
Kester MH, Bulduk S, Van Toor H, et a1. 2002. Potent inhibition of estrogen sulfotransferase by hydroxylated metabolites of polyhalogenated aromatic hydrocarbons reveals alternative mechanism for estrogenic activity of endocrine disrupters. J Clin Endocrinol Metab, 87(3): 1142- 1150
Kester MH, Bulduk S, Van Toor H, et a1. 2002. Potent inhibition of estrogen sulfotransferase by hydroxylated metabolites of polyhalogenated aromatic hydrocarbons reveals alternative mechanism for estrogenic activity of endocrine disrupters. J Clin Endocrinol Metab, 87(3): 1142- 1150
Klotz DM, Hewitt SC, Korach KS, et a1. 2000. Activation of a uterine insulin- like growth factor I signaling pathway by clinical and environmental estrogens: requirement of estrogen receptor- alpha. Endocrinology, 141(9): 3430- 3439
Klotz DM, Hewitt SC, Korach KS, et a1. 2000. Activation of a uterine insulin like growth factor I signaling pathway by clinical and environmental estrogens: requirement of estrogen receptor alpha. Endocrinology, 141(9): 3430- 3439
Kokawa K, Shikone T, Otani T, et a1. 2001. Apoptosis and the expression of bax and bcl-2 in hyperplasia and adenocarcinoma of the Uterine endometrium[J]. Hum Repro, 16(10): 2211- 2218
Kuiper GGJM, Enmark E, Pelto Huikko M, et al. 1996. Cloning of a novel estrogen receptor ecpressed in rat prostate and ovary. Proc Natl Acad Sci USA, 93(12): 5925- 5930
Masaharu Akita, Atsushi Yokoyama, Yukiaki Kuroda. 2002. Study of Alternatives to Testing for Endocrine Disrupters in Rat Whole Embryo Culturetes. Forth World Congress, 8: 11- 15
Matsuo H, Maruo T, Samoto T. 1997. Increased expression of Bcl- 2 protein in human uterine leiomyoma and its up- regulation by progesterone[J]. J Clin Endo Metab, 82(1): 293- 299
Matsuzaki S, Fukaya T, Suzuki T, et al. 1999. Oestrogen receptor alpha and beta mRNA expression in human endometrium throughout the menstrual cycle. Mol Hum Reprod, 5(6): 559- 64
Matsuzaki S, Fukaya T, Uehara S, et al. 2000. Characterization of messenger RNA expression of estrogen receptor-alpha and -beta in patients with ovarian endometriosis. Fertil Steril, 73(6): 1219- 25
Matthews JB, Twomey K, Zacharewski TR. 2001. In vitro and in vivo interaction of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors alpha and beta. Chem Res Toxicol, 14(2): 149- 157
Matthews JB, Twomey K, Zacharewski TR. 2001. In vitro and in vivo interaction of bisphenol A and its metabolite,bisphenol A glucuronide,with estrogen receptors alpha and beta. Chem Res Toxico, 14(2): 149- 157
Matthews JB, Twomey K, Zacharewski TR. 2001. In vitro and in vivo interaction of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors alpha and beta. ChemResToxicol, 14(2): 149- 157
Mizuo K, Narita M, Miyagawa K, et a1. 2004. Prenatal and neonatal exposure to bisphenol A affects the morphinerinduced rewarding effect and hyperlocomotion in mice. Neurosci Lett, 356(2): 95- 98
Moore M M, Honma M, Clements J, et a1. 2003. Mouse Lymphoma Thymidine Kinase Gene Mutation Assay[J].International Workshop on Genotoxicity Tests Workgroup ReportPlymouth, UK 2002. Mutat Res, 540(2): 127- 140
Muramatsu M, Inoue S. 2000. Breakthroughs and views estrogen receptors :How do they control reproductive and nonreproductive functions[J ] . Biochem Biophys Res Commun, 270 (1): 1210
Negishi T, Kawasaki K, Suzaki S, et a1. 2004. Behavioral alterations in response to fear-provoking stimuli and tranylcypromine induced by perinatal exposure to bisphenol A and nonylphenol in male rats. Environ Health Perspect , 112(11) : 1159- 1164
Orimo A, Inouc S, Minowa O, et a1. 1999. Underdeve1opment uterus and reduced estrogen responsiveness in mice with disruption of the es trogen responsive finger protein gene which is a direct target of estrogen receptor. Proc Natl Acad Sci USA, 96(21): 12027- 032
Orimo A, Osorio I, Craig R, et a1. 2002. Tumor-specific regulation of angiogentic growth factors and their receptors during recovery from cytotoxic therapy. Clin Canc Res, 8(4): 1213- 1222
Petter G, Nadi S, Ford S. et al. 2002. Regulation of Fas ligand expression in breast cancer cells by estrogen: Functional differences between estradiol and tamoxifen. Steroid Biochem Mol Biol, 73: 185- 194
Pocar F, Petersion G. 1999, A role for estrogens in the female reproductive system. Nature, 93(2): 509-512
Quesada I, Fuentes E, Viso Leon MC, et a1. 2002. Low doses of the endocrine disruptor bisphenol-A and the native hormone 17beta-estradiol rapidly activate transcription factor CREB. FASEB J, 16(12): 1671- 1673
Quesada I, Fuentes E, Viso—Leon MC, et a1. 2002. Low doses of the endocrine disruptor bisphenol-A and the native hormone 17beta-estradiol rapidly activate transcription factor CREB. FASEB J, 16(12): 1671- 1673
Rubin BS, Murray MK, Damassa DA, et al. 2001. Perinatal exposure to low doses of bisphenol A affects body weight, patterns of estrous cyclicity, and plasma LH levels. Environ Health Perspect, 109(7):675- 680
Saegusa M, Okaysau I. 2002. Changes in estrogen receptor alpha and beta in relation to progesterone receptor and status in normal and malignant endonetriun. Jap J Cancer Res, 9(5): 5l0- 5l8
Sakazaki H, Ueno H, Nakamuro K, et a1. 2002. Estrogen receptor alpha in mouse splenic lymphocytes: possible involvement in immunity. Toxicol Let, 133(2- 3): 221- 229
Sander CC, Green DR, 2001. Estrogen-induced apoptosis in a tamoxifen sutimulated MCF-7 model. Breast Cancer, 34(1): 1246- 1251
Singer CF, Kronsteiner N, Marton E, et al. 2002. Interleukin-1 system and sex steroid receptor gene expression in human endometrial cancer [J]. Gynecol Oncol, 85(3): 423
Tinwell H, Joiner R, Pate I, et a1.2000. Uterotrophic activity of bisphenol A in the immature mouse. Regul roxicol Pharmacol, 32(1): 118- 126
Tohei A, Suda S, Taya K, et al. 2001. Bisphenol A inhibits testicular functions and increases luteinizing hormone secretion in adult male rats. Exp Biol Med (Maywood), 226(3): 216- 221
Toyama Y, Suzuki-Toyota F, Maekawa M, et al. 2004. Adverse effects of bisphenol A to spermiogenesis in mice and rats. Arch Histol Cytol, 67:373- 381
Toyama Y, Suzuki-Toyota F, Maekawa M, et al. 2004. Adverse effects of bisphenol A to spermiogenesis in mice and rats. Arch Histol Cytol, 67(4):373- 381.
Tsutsumi O. 2000. Effects of endocrine disruptors on preimplantation embryo development Nippon Rinsho, 58(12): 2464- 8.
Vaskivuo TE, Stenb?ck F, Tapanainen JS. 2002. Apoptosis and apoptosis-related factors Bcl-2, Bax, tumor necrosis factor-alpha, and NF-kappaB in human endometrial hyperplasia and carcinoma. Cancer, 95(7): 1463- 1471
Xu J, Osuga Y, Yano T, et a1. 2002. Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells. Biochem Biophys Res Commun, 292(2): 456- 462