邻苯二甲酸二丁酯诱导雄性大鼠发生尿道下裂的分子作用机制
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摘要
目的：研究邻苯二甲酸二丁酯(DBP)孕晚期染毒诱导雄性大鼠发生尿道下裂的分子作用机制。
方法：SD雌性大鼠怀孕(GD)第14-18天，称重后每天分别灌胃予大豆油，DBP 500或800mg／kg／day，雄性仔鼠出生后(PND)第1天，鉴别出DBP染毒组中发生尿道下裂的雄性仔鼠，分组为雄性仔鼠对照组(A组)，500mg／kg／day(H)(B组)和800mg／kg／day(H)(C组)。用实时定量逆转录聚合酶链反应(Real-time quantitative RT-PCR)方法检测阴茎组织中sonic hedgehog(Shh)，骨形态发生蛋白4(Bmp4)和雄激素受体(Ar)mRNA表达水平。用Western blotting测量阴茎组织中Ar蛋白表达水平。用放射免疫分析(RIA)方法测定血清睾酮(T)水平。
结果：B组在mRNA(shh，Ar)表达水平和血清T水平较对照组有明显的降低(P＜0.05)，C组在mRNA(Shh，Bmp4，Ar)和蛋白(Ar)表达水平以及血清T水平都较对照组有明显的降低(P＜0.05)。
结论：DBP通过对生殖结节(GT)生长发育早期的Shh信号系统，以及晚期的雄激素信号系统发生作用而影响GT的生长发育，诱导尿道下裂的发生。
Objective: The objective of this study was to investigate molecularmechanisms of hypospadias in male rats exposed to di-n-butyl phthalate(DBP) during late gestation.
Methods: Timed pregnant Sprague-Dawley (SD) rats were givenDBP by gastric intubation at a dose of 0, 500 or 800 mg/kg/day fromgestation day (GD) 14 to 18 after maternal body weight was recordeddaily. On postnatal day (PND) 1, male offspring were allocated to threegroups: control male offspring (group A), 500 mg/kg/day (H) (group B)and 800 mg/kg/day (H) (group C) after identifying control male offspringnot exposed to DBP and hypospadiac male offspring exposed to DBP.Real-time quantitative RT-PCR and Western blotting were used toquantify mRNA of sonic hedgehog (Shh), bone morphogenetic protein 4(Bmp4) and androgen receptor (Ar) and protein of Ar expression in ratpenises of above three groups on PND 1. Serum testosterone (T)concentration of male offspring in above three groups on PND 1 wasdetermined by using T radioimmunoassay (RIA) kit.
Results: mRNA (Shh, Ar) expression in rat penises and serum Tlevel in group B were significantly decreased compared with those incontrol group (P＜0.05). mRNA (Shh, Bmp4, Ar), protein (Ar) expressionin rat penises and serum T level in group C were significantly decreasedcompared with those in control group (P＜0.05).
Conclusions: DBP affects the development of the genital tubercle(GT) by regulating Shh signaling in early development of GT andandrogen signaling in late development of GT, inducing the occurrence ofhypospadias in male rats.
方法：SD雌性大鼠怀孕(GD)第14-18天，称重后每天分别灌胃予大豆油，DBP 500或800mg／kg／day，雄性仔鼠出生后(PND)第1天，鉴别出DBP染毒组中发生尿道下裂的雄性仔鼠，分组为雄性仔鼠对照组(A组)，500mg／kg／day(H)(B组)和800mg／kg／day(H)(C组)。用实时定量逆转录聚合酶链反应(Real-time quantitative RT-PCR)方法检测阴茎组织中sonic hedgehog(Shh)，骨形态发生蛋白4(Bmp4)和雄激素受体(Ar)mRNA表达水平。用Western blotting测量阴茎组织中Ar蛋白表达水平。用放射免疫分析(RIA)方法测定血清睾酮(T)水平。
结果：B组在mRNA(shh，Ar)表达水平和血清T水平较对照组有明显的降低(P＜0.05)，C组在mRNA(Shh，Bmp4，Ar)和蛋白(Ar)表达水平以及血清T水平都较对照组有明显的降低(P＜0.05)。
结论：DBP通过对生殖结节(GT)生长发育早期的Shh信号系统，以及晚期的雄激素信号系统发生作用而影响GT的生长发育，诱导尿道下裂的发生。
Objective: The objective of this study was to investigate molecularmechanisms of hypospadias in male rats exposed to di-n-butyl phthalate(DBP) during late gestation.
Methods: Timed pregnant Sprague-Dawley (SD) rats were givenDBP by gastric intubation at a dose of 0, 500 or 800 mg/kg/day fromgestation day (GD) 14 to 18 after maternal body weight was recordeddaily. On postnatal day (PND) 1, male offspring were allocated to threegroups: control male offspring (group A), 500 mg/kg/day (H) (group B)and 800 mg/kg/day (H) (group C) after identifying control male offspringnot exposed to DBP and hypospadiac male offspring exposed to DBP.Real-time quantitative RT-PCR and Western blotting were used toquantify mRNA of sonic hedgehog (Shh), bone morphogenetic protein 4(Bmp4) and androgen receptor (Ar) and protein of Ar expression in ratpenises of above three groups on PND 1. Serum testosterone (T)concentration of male offspring in above three groups on PND 1 wasdetermined by using T radioimmunoassay (RIA) kit.
Results: mRNA (Shh, Ar) expression in rat penises and serum Tlevel in group B were significantly decreased compared with those incontrol group (P＜0.05). mRNA (Shh, Bmp4, Ar), protein (Ar) expressionin rat penises and serum T level in group C were significantly decreasedcompared with those in control group (P＜0.05).
Conclusions: DBP affects the development of the genital tubercle(GT) by regulating Shh signaling in early development of GT andandrogen signaling in late development of GT, inducing the occurrence ofhypospadias in male rats.
引文
1 Baskin LS. Hypospadias and urethral development. J Urol, 2000, 163:951-956.
2 Manson JM, Carr MC. Molecular epidemiology of hypospadias: review of genetic and environmental risk factors. Birth Defects Res A Clin Mol Teratol, 2003, 67:825-836.
3 Baskin LS, Himes K, Colborn T. Hypospadias and endocrine disruption: is there a connection? Environ Health Perspect, 2001, 109:1175-1183.
4 Canning DA. Hypospadias trends in two US surveillance systems. Rise in prevalence of hypospadias. J Urol, 1999, 161:366.
5 Paulozzi LJ. International trends in rates of hypospadias and cryptochidism. Environ Health Perspect, 1999, 107:297-302.
6 Autian J. Toxicity and health threats of phthalate esters: review of the literature. Environ Health Perspect, 1973, 4:3-26.
7 Mayer FL, Stalling DL, Johnson JL. Phthalate esters as environmental contaminants. Nature, 1972, 238(5364):411-413.
8 Foster PM, Cattley RC, Mylchreest E. Effects of di-n-butyl phthalate (DBP) on male reproductive development in the rat: implications for human risk assessment. Food Chem Toxicol, 2000, 38(1 Suppl):S97-99.
9 Kavlock R, Boekelheide K, Chapin R, et al. NTP Center for the Evaluation of Risks to Human Reproduction: phthalates expert panel report on the reproductive and developmental toxicity of di-n-butyl phthalate. Reprod Toxicol, 2002, 16:489-527.
10 Barlow NJ, Foster PM. Pathogenesis of male reproductive tract lesions from gestation through adulthood following in utero exposure to Di(n-butyl) phthalate. Toxicol Pathol, 2003, 31:397-410.
11 Barlow NJ, Phillips SL, Wallace DG, et al. Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate. Toxicol Sci, 2003, 73:431-451.
12 Yamada G, Satoh Y, Baskin LS, et al. Cellular and molecular mechanisms of development of the external genitalia. Differentiation, 2003, 71:445-460.
13 Klonisch T, Fowler PA, Hombach-Klonischa S. Molecular and genetic regulation of testis descent and external genitalia development. Dev Biol, 2004, 270:1-18.
14 蒋君涛，马隆，昊婷，等．邻苯二甲酸二丁酯致尿道下裂大鼠发育异常和阴茎病理学改变研究．中华实验外科杂志，2006，23：576-578．
15 张炜，袁琳，吴婷，等．邻苯二甲酸二丁酯诱导尿道下裂大鼠模 型的建立及其作用机制．中华实验外科杂志，2005，22：246-248．
16 Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2~(-△△CT) Method. Methods, 2001, 25:402-408.
17 Haraguchi R, Mo R, Hui C, et al. Unique functions of Sonic hedgehog signaling during external genitalia development. Development, 2001, 128:4241-4250.
18 Perriton CL, Powles N, Chiang C, et al. Sonic hedgehog signaling from the urethral epithelium controls external genital development. Dev Biol, 2002, 247:26-46.
19 Duprez D, Fournier-Thibault C, Le Douarin N. Sonic hedgehog induces proliferation of committed skeletal muscle cells in the chick limb. Development, 1998, 125:495-505.
20 Drossopoulou G, Lewis KE, Sanz-Ezquerro JJ, et al. A model for anteroposterior patterning of the vertebrate limb based on sequential long-and short-range Shh signalling and Bmp signalling. Development, 2000, 127:1337-1348.
21 Zhao X, Zhang Z, Song Y, et al. Transgenically ectopic expression of Bmp4 to the Msxl mutant dental mesenchyme restores downstream gene expression but represses Shh and Bmp2 in the enamel knot of wild type tooth germ. Mech Dev, 2000, 99:29-38.
22 Monsoro-Burq A, Le Douarin NM. BMP4 plays a key role in leftright patterning in chick embryos by maintaining Sonic hedgehog asymmetry. Mol Cell, 2001, 7:789-799.
23 Renfree MB, Wilson JD, Shaw G. The hormonal control of sexual development. Novartis Found Symp, 2002, 244:136-152.
24 Mylchreest E, Sar M, Wallace DG, et al. Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate. Reprod Toxicol, 2002,16:19-28.
1 Paulozzi LJ. International trends in rates of hypospadias and cryptochidism. Environ Health Perspect, 1999, 107:297-302.
2 Domenice S, Yumie NM, Correio BA, et al. A novel missense mutation (S 18 N) in the 5'non-HMG box region of the SRY gene in a patient with partial gonadal dysgenesis and his normal male relatives. Hum Genet, 1998, 102:213-215.
3 De Santa Barbara P, Bonneaud N, Boizet B, et al. Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-mullerian hormone gene. MCB, 1998,6653-6665.
4 Olney PN, Kean LS, Graham D, et al. Campomelic syndrome and deleltion of SOX9. Am J Med Genet, 1999, 84:20-24.
5 Bardoni B, Zanaria E, Guioli S, et al. A dosage sensitive locus at chromosome xp21 involved male to female SEX reversal. Nat Genet, 1994, 7:497-501.
6 Wachtel. X-linked sex-reversing genes. Cytogene Cell Gene, 1998, 80:222-225.
7 Klamt B, Koziell A, Poulat F, et al. Frasier syndrome is caused by defective alternative splicing of WTl leading to an altered ratio of WT1 + / - KTS space isoforms. Hum Mol Genet, 1998, 7:709-714.
8 Diller L, Ghahremani M, Morgan J, et al. Constitutional WT1 mutations in Wilms' tumor patients. J Clin Oncol, 1998, 16:3634-3640.
9 Kohler B, Schumacher V, Schulte-Over-berg U, et al. Bilateral Wilms'tumor in a boy with sever hypospadias and cryptochidism due to a heterozygous mutation in the WT1 gene. Pediatr Res, 1999, 45:187-190.
10 Nordenskjold A, Friedman F, TapperPers-son M, et al. Screening for mutation in candidate genes for hypospadias. Urol Res, 1999, 27:249-255.
11 Hiort O, Willenbring H, Allers, et al. Molecular genetic analysis and human chorionic gonadotropin stimulation test in the diagnosis of prepubertal patients with partial 5a-reductase deficiency. Eur J Pediatr, 1996, 155(6):445-451.
12 Kojima Y, Hayashi Y, Mizuno K, et al. Spermatogenesis, fertility and sexual behavior in a hypospadias mouse model. J Urol, 2002, 167(3):1532-1537.
13 Wiener JS, Teague JL, Roth DR, et al. Molecular biology and function of the androgen receptor in genital development. J Urol, 1997, 157:1377-1386.
14 Albers N, Ulrichs C, Gluer S, et al. Etiologic classification of severe hypospadias implications for prognosis and management. J Pediatr, 1997, 131:386-392.
15 Kelce WR, Wilson EM. Environmental antiandrogen: developmental effects, molecular mechanisms, and clinical implications. J Mol Med, 1997, 75:198-207.
16 Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study. Lancet, 2002, 359(9312):1102-1107.
17 Miyagawa S, Buchanan DL, Sato T, et al. Characterization of diethylstilbestrol-induced hypospadias in female mice. Anat Rec, 2002, 266(1):43-50.
18 Silver RI, Rodriguez R, Chang TS, et al. Invitro fertilization is associated with an increased risk of hypospadias. J Urol, 1999, 161(6):1954-1957.
19 Toppari J, Larsen JC, Christiansen P, et al. Male reproductive health and environmental xenoestrogens. Environ Healtlh Perspect, 1996, 104(Supp4):741-803.
20 North K, Golding J. A maternal vegetarian diet in pregnancy is associated with hypospadias. BJU Int, 2000, 85(1): 107-113.
21 Mylchreest E, Cattley RC, Foster PM. Male reproductive tract malformations in rats following gestational and lactational exposure to Di (n2butyl) phthalate: an antiandrogenic mechanism? Toxicol Sci, 1998, 43(1):47-60.
22 Foster PM, Mylchreest E, Gaido KW, et al. Effects of phthalate esters on the developing reproductive tract of male rats. Hum Reprod Update, 2001, 7(3):231-235.
23 Higuchi TT, Palmer JS, Gray LE, et al. Effects of dibutyl phthalate in male rabbits following in utero, adolescent, or postpubertal exposure. Toxicol Sci, 2003, 72(2):301-313.
24 Nakahara H, Shono T, Suita S, et al. Effects of prenatal exposure to phthalate ester on both testicular descent and urogenital development in rats. Fukuoka Igaku Zasshi, 2003, 94(12):331-337.
25 Gray LE, Wolf C, Lambright C, et al. Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health, 1999, 15(1-2):94-118.
26 Gray LE, Ostby J, Furr J, et al. Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol Sci, 2000, 58(2):350-365.
27 Shono T, Suita S, Kai H, et al. The effect of a prenatal androgen disruptor, vinclozolin, on gubernacular migration and testicular descent in rats, J Pediatr Surg, 2004, 39(2):213-216.
28 Baskin LS. Hypospadias and urethral development. Pediatric urology, 2000, 163(3):951.
2 Manson JM, Carr MC. Molecular epidemiology of hypospadias: review of genetic and environmental risk factors. Birth Defects Res A Clin Mol Teratol, 2003, 67:825-836.
3 Baskin LS, Himes K, Colborn T. Hypospadias and endocrine disruption: is there a connection? Environ Health Perspect, 2001, 109:1175-1183.
4 Canning DA. Hypospadias trends in two US surveillance systems. Rise in prevalence of hypospadias. J Urol, 1999, 161:366.
5 Paulozzi LJ. International trends in rates of hypospadias and cryptochidism. Environ Health Perspect, 1999, 107:297-302.
6 Autian J. Toxicity and health threats of phthalate esters: review of the literature. Environ Health Perspect, 1973, 4:3-26.
7 Mayer FL, Stalling DL, Johnson JL. Phthalate esters as environmental contaminants. Nature, 1972, 238(5364):411-413.
8 Foster PM, Cattley RC, Mylchreest E. Effects of di-n-butyl phthalate (DBP) on male reproductive development in the rat: implications for human risk assessment. Food Chem Toxicol, 2000, 38(1 Suppl):S97-99.
9 Kavlock R, Boekelheide K, Chapin R, et al. NTP Center for the Evaluation of Risks to Human Reproduction: phthalates expert panel report on the reproductive and developmental toxicity of di-n-butyl phthalate. Reprod Toxicol, 2002, 16:489-527.
10 Barlow NJ, Foster PM. Pathogenesis of male reproductive tract lesions from gestation through adulthood following in utero exposure to Di(n-butyl) phthalate. Toxicol Pathol, 2003, 31:397-410.
11 Barlow NJ, Phillips SL, Wallace DG, et al. Quantitative changes in gene expression in fetal rat testes following exposure to di(n-butyl) phthalate. Toxicol Sci, 2003, 73:431-451.
12 Yamada G, Satoh Y, Baskin LS, et al. Cellular and molecular mechanisms of development of the external genitalia. Differentiation, 2003, 71:445-460.
13 Klonisch T, Fowler PA, Hombach-Klonischa S. Molecular and genetic regulation of testis descent and external genitalia development. Dev Biol, 2004, 270:1-18.
14 蒋君涛，马隆，昊婷，等．邻苯二甲酸二丁酯致尿道下裂大鼠发育异常和阴茎病理学改变研究．中华实验外科杂志，2006，23：576-578．
15 张炜，袁琳，吴婷，等．邻苯二甲酸二丁酯诱导尿道下裂大鼠模 型的建立及其作用机制．中华实验外科杂志，2005，22：246-248．
16 Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2~(-△△CT) Method. Methods, 2001, 25:402-408.
17 Haraguchi R, Mo R, Hui C, et al. Unique functions of Sonic hedgehog signaling during external genitalia development. Development, 2001, 128:4241-4250.
18 Perriton CL, Powles N, Chiang C, et al. Sonic hedgehog signaling from the urethral epithelium controls external genital development. Dev Biol, 2002, 247:26-46.
19 Duprez D, Fournier-Thibault C, Le Douarin N. Sonic hedgehog induces proliferation of committed skeletal muscle cells in the chick limb. Development, 1998, 125:495-505.
20 Drossopoulou G, Lewis KE, Sanz-Ezquerro JJ, et al. A model for anteroposterior patterning of the vertebrate limb based on sequential long-and short-range Shh signalling and Bmp signalling. Development, 2000, 127:1337-1348.
21 Zhao X, Zhang Z, Song Y, et al. Transgenically ectopic expression of Bmp4 to the Msxl mutant dental mesenchyme restores downstream gene expression but represses Shh and Bmp2 in the enamel knot of wild type tooth germ. Mech Dev, 2000, 99:29-38.
22 Monsoro-Burq A, Le Douarin NM. BMP4 plays a key role in leftright patterning in chick embryos by maintaining Sonic hedgehog asymmetry. Mol Cell, 2001, 7:789-799.
23 Renfree MB, Wilson JD, Shaw G. The hormonal control of sexual development. Novartis Found Symp, 2002, 244:136-152.
24 Mylchreest E, Sar M, Wallace DG, et al. Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate. Reprod Toxicol, 2002,16:19-28.
1 Paulozzi LJ. International trends in rates of hypospadias and cryptochidism. Environ Health Perspect, 1999, 107:297-302.
2 Domenice S, Yumie NM, Correio BA, et al. A novel missense mutation (S 18 N) in the 5'non-HMG box region of the SRY gene in a patient with partial gonadal dysgenesis and his normal male relatives. Hum Genet, 1998, 102:213-215.
3 De Santa Barbara P, Bonneaud N, Boizet B, et al. Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-mullerian hormone gene. MCB, 1998,6653-6665.
4 Olney PN, Kean LS, Graham D, et al. Campomelic syndrome and deleltion of SOX9. Am J Med Genet, 1999, 84:20-24.
5 Bardoni B, Zanaria E, Guioli S, et al. A dosage sensitive locus at chromosome xp21 involved male to female SEX reversal. Nat Genet, 1994, 7:497-501.
6 Wachtel. X-linked sex-reversing genes. Cytogene Cell Gene, 1998, 80:222-225.
7 Klamt B, Koziell A, Poulat F, et al. Frasier syndrome is caused by defective alternative splicing of WTl leading to an altered ratio of WT1 + / - KTS space isoforms. Hum Mol Genet, 1998, 7:709-714.
8 Diller L, Ghahremani M, Morgan J, et al. Constitutional WT1 mutations in Wilms' tumor patients. J Clin Oncol, 1998, 16:3634-3640.
9 Kohler B, Schumacher V, Schulte-Over-berg U, et al. Bilateral Wilms'tumor in a boy with sever hypospadias and cryptochidism due to a heterozygous mutation in the WT1 gene. Pediatr Res, 1999, 45:187-190.
10 Nordenskjold A, Friedman F, TapperPers-son M, et al. Screening for mutation in candidate genes for hypospadias. Urol Res, 1999, 27:249-255.
11 Hiort O, Willenbring H, Allers, et al. Molecular genetic analysis and human chorionic gonadotropin stimulation test in the diagnosis of prepubertal patients with partial 5a-reductase deficiency. Eur J Pediatr, 1996, 155(6):445-451.
12 Kojima Y, Hayashi Y, Mizuno K, et al. Spermatogenesis, fertility and sexual behavior in a hypospadias mouse model. J Urol, 2002, 167(3):1532-1537.
13 Wiener JS, Teague JL, Roth DR, et al. Molecular biology and function of the androgen receptor in genital development. J Urol, 1997, 157:1377-1386.
14 Albers N, Ulrichs C, Gluer S, et al. Etiologic classification of severe hypospadias implications for prognosis and management. J Pediatr, 1997, 131:386-392.
15 Kelce WR, Wilson EM. Environmental antiandrogen: developmental effects, molecular mechanisms, and clinical implications. J Mol Med, 1997, 75:198-207.
16 Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study. Lancet, 2002, 359(9312):1102-1107.
17 Miyagawa S, Buchanan DL, Sato T, et al. Characterization of diethylstilbestrol-induced hypospadias in female mice. Anat Rec, 2002, 266(1):43-50.
18 Silver RI, Rodriguez R, Chang TS, et al. Invitro fertilization is associated with an increased risk of hypospadias. J Urol, 1999, 161(6):1954-1957.
19 Toppari J, Larsen JC, Christiansen P, et al. Male reproductive health and environmental xenoestrogens. Environ Healtlh Perspect, 1996, 104(Supp4):741-803.
20 North K, Golding J. A maternal vegetarian diet in pregnancy is associated with hypospadias. BJU Int, 2000, 85(1): 107-113.
21 Mylchreest E, Cattley RC, Foster PM. Male reproductive tract malformations in rats following gestational and lactational exposure to Di (n2butyl) phthalate: an antiandrogenic mechanism? Toxicol Sci, 1998, 43(1):47-60.
22 Foster PM, Mylchreest E, Gaido KW, et al. Effects of phthalate esters on the developing reproductive tract of male rats. Hum Reprod Update, 2001, 7(3):231-235.
23 Higuchi TT, Palmer JS, Gray LE, et al. Effects of dibutyl phthalate in male rabbits following in utero, adolescent, or postpubertal exposure. Toxicol Sci, 2003, 72(2):301-313.
24 Nakahara H, Shono T, Suita S, et al. Effects of prenatal exposure to phthalate ester on both testicular descent and urogenital development in rats. Fukuoka Igaku Zasshi, 2003, 94(12):331-337.
25 Gray LE, Wolf C, Lambright C, et al. Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health, 1999, 15(1-2):94-118.
26 Gray LE, Ostby J, Furr J, et al. Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol Sci, 2000, 58(2):350-365.
27 Shono T, Suita S, Kai H, et al. The effect of a prenatal androgen disruptor, vinclozolin, on gubernacular migration and testicular descent in rats, J Pediatr Surg, 2004, 39(2):213-216.
28 Baskin LS. Hypospadias and urethral development. Pediatric urology, 2000, 163(3):951.