利谷隆致雄性仔鼠的生殖毒性及其机制研究
摘要
目的:探讨利谷隆致雄性仔鼠的生殖毒性及其机制。
方法:孕期妊娠第13-18天时用花生油溶解利谷隆(0、50、100、150、200mg/kg)灌胃染毒,于孕期妊娠(GD)20天各组解剖数只孕鼠,提取雄性胎鼠尿生殖结节和睾丸做病理切片;于雄性子代出生后28d测量肛殖距(anogenital distance,AGD)和称重;于雄性子代出生后第2天用酶联免疫吸附试验(enzyme-linked immunosorbentassay,ELISA)检测睾酮激素水平;在胚胎20d解剖孕鼠,提取雄性胎鼠睾丸做电镜观察睾丸间质细胞的超微结构的变化、免疫组织化学检测睾丸间质细胞中P450scc、3β-HSD、PCNA蛋白,AR的表达情况,用实时荧光定量的RT-PCR实验检测睾丸间质细胞中P450c17、17β-HSD、PCNA、AR基因表达情况。
结果:1.各组之间雄性仔鼠体重无明显差异。在一定的剂量范围内,随着染毒剂量的增加,用药组AGD随之有缩短的趋势。2.LIN染毒组与空白LIN 0mg/kg对比,病理见睾丸索变细,睾丸内各细胞空泡变性,核固缩增多。3.LIN染毒组雄性子代出生后第2d睾酮激素水平较LIN 0mg/kg降低。4.电镜观察结果:睾丸间质细胞粗面内质网扩张,线粒体肿胀。5.免疫组织化学结果:在睾丸间质细胞中P450scc酶、3β-HSD、PCNA蛋白表达降低(P均＜0.05),AR蛋白表达与LIN 0mg/kg相近(P＞0.05)。6.实时荧光定量的RT-PCR实验显示:在睾丸间质细胞中P450c17基因,17β-HSD、PCNA基因表达减弱(P均＜0.05),AR基因表达与LIN 0mg/kg相近(P＞0.05)。
结论:孕期暴露LIN可透过胎盘屏障,对雄性仔鼠造成如下影响:1.各染毒组与LIN0mg/kg之间体重无差异。在一定的剂量范围内,随着染毒剂量的增加,染毒组AGD随之有缩短的趋势。2.染毒组与空白LIN 0mg/kg对比病理见睾丸索变细,睾丸内各细胞空泡变性,核固缩增多。3.ELISA显示:除染毒组50mg/kg外,其以上剂量可使睾酮激素水平降低,可见睾酮激素分泌水平与染毒剂量之间具有剂量效应关系。4.电镜观察结果证实了LIN能够引起大鼠睾丸间质细胞粗面内质网扩张,线粒体肿胀,从而导致睾酮激素的合成障碍。5.免疫组化显示LIN能够降低睾丸间质细胞中合成睾酮代谢酶P450scc和3β-HSD,PCNA酶蛋白的活性表达,从而影响睾酮的产生。6.实时荧光定量的RT-PCR实验显示:LIN降低了P450c17,17β-HSD、PCNA基因在睾丸间质细胞中在表达,利谷隆对雄性生殖系统的影响可能是通过影响睾酮生成过程中的关键酶造成睾酮生成减少的。
Objective:To explore reproductive toxicity and mechanism of linuron on F1 male rats.
Method:Pregnant female rats were exposed to LIN(LIN was dissolved to peanut oil) at doses of 0,50,100,150 and 200mg/kg by body weight,respectively,during the period of gestation day(GD) 13 to 18.The fetuses of two rats from each group were anatomized on GD20 to extract genital tubercle(GT) and testis to make pathological section.The datas of AGD and weight were observed on PND28.We detected the level of testosterone with ELISA in PND2 on male offspring.Pregnant SD rats were sacrificed on GD 20.we extracted testis to observe the inner constitution change of cell of Leydig by electron microscope.Moreover,we have done experiment in Immunohistochemistry and real time RT-PCR.
Result:1.The body weights were no significant difference between each group. At a certain dose range,the treatment group have the trend to shorten AGD following the dose increases.2.Exposure groups were observed testicular cord thin,cells vacuolar degeneration and karyopyknosis increase compared with control group on pathological section.3.The results shows the level of testosterone step down in PND2 on male offspring.4.The results displaied the distension of rough endoplasmic reticulum and the swell of chondrosome in Leydig by electron microscope.5.The immunohistochemistrical results show the protein expression of P450scc、3β-HSD,and PCNA are shortage(P＜0.05) in Leydig.the protein expression of AR is alike with control(P＞0.05).6.The results display the gene expression of P450c17 degrade,the gene expression of 17β-HSD and PCNA weaken in Leydig in real time RT-PCR(P＜0.05),but the gene expression of AR identical with control.(P＞0.05).
Conclusion:LIN can enter embryo and have adverse affects on F1 male rats:1.The body weights were no significant difference between each group.At a certain dose range, the treatment group have the trend to shorten AGD following the dose increases.2. Exposure groups were observed testicular cord thin、cells vacuolar degeneration and karyopyknosis increase compared with control group on pathological section.3.exposures to 50mg/kg LIN gestation might degrade the level of testosterone in PND2 on male offspring with ELISA.4.LIN displays the distension of rough endoplasmic reticulum and the swell of chondrosome in Leydig by electron microscope.5.LIN can degrade the protein expression of P450scc、3β-HSD and PCNA in Leydig.6.LIN cut down the gene expression of P450c17 degrade,the gene expression of 17β-HSD and PCNA weaken in Leydig in real time RT-PCR.
方法:孕期妊娠第13-18天时用花生油溶解利谷隆(0、50、100、150、200mg/kg)灌胃染毒,于孕期妊娠(GD)20天各组解剖数只孕鼠,提取雄性胎鼠尿生殖结节和睾丸做病理切片;于雄性子代出生后28d测量肛殖距(anogenital distance,AGD)和称重;于雄性子代出生后第2天用酶联免疫吸附试验(enzyme-linked immunosorbentassay,ELISA)检测睾酮激素水平;在胚胎20d解剖孕鼠,提取雄性胎鼠睾丸做电镜观察睾丸间质细胞的超微结构的变化、免疫组织化学检测睾丸间质细胞中P450scc、3β-HSD、PCNA蛋白,AR的表达情况,用实时荧光定量的RT-PCR实验检测睾丸间质细胞中P450c17、17β-HSD、PCNA、AR基因表达情况。
结果:1.各组之间雄性仔鼠体重无明显差异。在一定的剂量范围内,随着染毒剂量的增加,用药组AGD随之有缩短的趋势。2.LIN染毒组与空白LIN 0mg/kg对比,病理见睾丸索变细,睾丸内各细胞空泡变性,核固缩增多。3.LIN染毒组雄性子代出生后第2d睾酮激素水平较LIN 0mg/kg降低。4.电镜观察结果:睾丸间质细胞粗面内质网扩张,线粒体肿胀。5.免疫组织化学结果:在睾丸间质细胞中P450scc酶、3β-HSD、PCNA蛋白表达降低(P均＜0.05),AR蛋白表达与LIN 0mg/kg相近(P＞0.05)。6.实时荧光定量的RT-PCR实验显示:在睾丸间质细胞中P450c17基因,17β-HSD、PCNA基因表达减弱(P均＜0.05),AR基因表达与LIN 0mg/kg相近(P＞0.05)。
结论:孕期暴露LIN可透过胎盘屏障,对雄性仔鼠造成如下影响:1.各染毒组与LIN0mg/kg之间体重无差异。在一定的剂量范围内,随着染毒剂量的增加,染毒组AGD随之有缩短的趋势。2.染毒组与空白LIN 0mg/kg对比病理见睾丸索变细,睾丸内各细胞空泡变性,核固缩增多。3.ELISA显示:除染毒组50mg/kg外,其以上剂量可使睾酮激素水平降低,可见睾酮激素分泌水平与染毒剂量之间具有剂量效应关系。4.电镜观察结果证实了LIN能够引起大鼠睾丸间质细胞粗面内质网扩张,线粒体肿胀,从而导致睾酮激素的合成障碍。5.免疫组化显示LIN能够降低睾丸间质细胞中合成睾酮代谢酶P450scc和3β-HSD,PCNA酶蛋白的活性表达,从而影响睾酮的产生。6.实时荧光定量的RT-PCR实验显示:LIN降低了P450c17,17β-HSD、PCNA基因在睾丸间质细胞中在表达,利谷隆对雄性生殖系统的影响可能是通过影响睾酮生成过程中的关键酶造成睾酮生成减少的。
Objective:To explore reproductive toxicity and mechanism of linuron on F1 male rats.
Method:Pregnant female rats were exposed to LIN(LIN was dissolved to peanut oil) at doses of 0,50,100,150 and 200mg/kg by body weight,respectively,during the period of gestation day(GD) 13 to 18.The fetuses of two rats from each group were anatomized on GD20 to extract genital tubercle(GT) and testis to make pathological section.The datas of AGD and weight were observed on PND28.We detected the level of testosterone with ELISA in PND2 on male offspring.Pregnant SD rats were sacrificed on GD 20.we extracted testis to observe the inner constitution change of cell of Leydig by electron microscope.Moreover,we have done experiment in Immunohistochemistry and real time RT-PCR.
Result:1.The body weights were no significant difference between each group. At a certain dose range,the treatment group have the trend to shorten AGD following the dose increases.2.Exposure groups were observed testicular cord thin,cells vacuolar degeneration and karyopyknosis increase compared with control group on pathological section.3.The results shows the level of testosterone step down in PND2 on male offspring.4.The results displaied the distension of rough endoplasmic reticulum and the swell of chondrosome in Leydig by electron microscope.5.The immunohistochemistrical results show the protein expression of P450scc、3β-HSD,and PCNA are shortage(P＜0.05) in Leydig.the protein expression of AR is alike with control(P＞0.05).6.The results display the gene expression of P450c17 degrade,the gene expression of 17β-HSD and PCNA weaken in Leydig in real time RT-PCR(P＜0.05),but the gene expression of AR identical with control.(P＞0.05).
Conclusion:LIN can enter embryo and have adverse affects on F1 male rats:1.The body weights were no significant difference between each group.At a certain dose range, the treatment group have the trend to shorten AGD following the dose increases.2. Exposure groups were observed testicular cord thin、cells vacuolar degeneration and karyopyknosis increase compared with control group on pathological section.3.exposures to 50mg/kg LIN gestation might degrade the level of testosterone in PND2 on male offspring with ELISA.4.LIN displays the distension of rough endoplasmic reticulum and the swell of chondrosome in Leydig by electron microscope.5.LIN can degrade the protein expression of P450scc、3β-HSD and PCNA in Leydig.6.LIN cut down the gene expression of P450c17 degrade,the gene expression of 17β-HSD and PCNA weaken in Leydig in real time RT-PCR.
引文
[1]Hyun K,Hyung SK,Jae HS et al.Comparison of anti-androgenic activity of flutamide,vinclozoLIN,procymidone,LINuron,and p,p'-DDE in rodent 10-day Hershberger assay.Toxicology 2004;199(2-3):145-159
[2]Yamada G,Satoh Y,Baskin LS,et al.Cellular and molecular mechanisms of development of the external genitalia.International Society of differentiation,2003;71(8):445-460
[3]McIntyre BS,Barlow Nj,Foster PM.Androgen-mediated development in male rat offspring exposed to flutamide in utero:permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues[J].Toxicol Sci,2001;62(2):236-249
[4]Charles S,Patrick B,Beatrice T et al.Environmental xenoestrogens,antiandrogens and disorders of male sexual differentiation.Molecular and Cellular Endocnnology,2001;178(1-2):99-105
[5]McIntyre BS,Barlow NJ,Wallace DG,et al.Effects of in utero exposure to Linuron on androgen-dependent reproductive development in the male Cr1:CD(SD)BR rat.Toxicol Appl Pharmacol,2000;167(2):87-99
[6]Lambright C,Ostby J,Bobseine K,et al.Cellular and Molecular Mechanisms of Action of Linuron:An Antiandrogenic Herbicide that Produces Reproductive Malformations in Male Rats.Toxicological Sciences,2000;56:389-399
[7]Gray LEJ,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;15:94-118.
[8]Mclntyre BS,Barlow Nj,Foster PM.Androgen-mediated development in male rat offspring exposed to flutamide in utero:permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues[J].Toxicol Sci,2001;62(2):236-249
[9]Gray LE,Ostby J,Wolf C,et al.The value of mechanistic studies in laboratory animals for the prediction of reproductive effects in wildlife:Endocrine effects on mammalian sexual differentiation.Environ Toxicol Chem,1998;17(1):109-118
[10]周宏,周新华,吴巍,等.胚胎及生后不同发育时期大鼠睾丸生殖细胞的凋亡.中国组织化学与细胞化学杂志,2002,11(6):146
[11]卢洹,周新华等。ICR小鼠胚胎及生后不同阶段睾丸内生殖细胞的发育,武汉大学学报 2004,25(4):359
[12]Barlow,Norman J,Phillips,et al.Quantitative changes in gene expression in fetal rat testes following exposure to Di(n-butyl) phthalate.2003;73(2):431-441
[13]祝辉,沙家豪,周作民.睾丸中细胞色素P450酶的生物学特性及调控.国外医学计划生育分册,1999;18(3):129-133
[14]Higuchi R,Fockler C,etal.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions.Biotechnology,1993;11(9):1026-1030
[15]Fitch RH,Berrebi AS,Cowell PE.Effects of neonatal hormones on sexual dimorphism in the rat.Brain Research,1990;515:111-116
[16]Rhees RW,Shryne JE,Gorski RA.Onset of the hormone-sensitive perinatal period for sexual differentiation of the sexually dimorphic nucleus of the preoptic area.Journal of Neurobiology,1990;21:781-786
[17]Rhees RW,Shryne JE,Gorski RA.Termination of the hormone-sensitive period for differentiation of the sexually dimorphic nucleus of the preoptic areain male and female rats.Developmental Brain Research,1990;52:17-23
[18]Bloch GJ,Mills R.Prepuberal testosterone treatment of neonatally gonadectomized male rats:defeminization of behavioral and endocrine functionin adulthood.Neuroscience and Biobehavioral Reviews,1995;19:187-200
[19]Tan JA,et al.[J].J BioI Chem,1992;267(7):4456-4466
[20]Anderson KM,Cheung PH,Kell MD.Rapid generation of homologous internal standards and evaluation of data for quantitaion of messenger RNA by competitive polymerase chain reaction.J Pharmacol Toxicol Methods,1997;38:133-140
[21]Luhu A,Waiztu M,Koch A.A rapid and sensitive protocol for competitive reverse transcriprase(CRT) PCR analysis of cellular genes.Brain Pathol,1998;8:13-18
[22]文斌,高巍,费然等.核酸扩增产物的量化酶免疫通用型检测方法.中华医学检验杂志,1999;22:83-86
[23]Actor JK,Limor JR,Hunter RL.A flexible bioluminescent-quantitive polymerase reaction assay for analysis of competitive PCR amplicons.J CLIN Lab Anal,1999;13(1):40-47
[24]Schnell S,Mendoza C.Enzymological considerations for a theoretical description of the quantitative competitive polymerase chain reaction(QC-PCR).J Theor Biol,1997;184(4):433-440
[25]Ke LD,Chen Z,Yung WK.A reliability test of standard-based quantitative PCR:exogenous vs endogenous standards.Mol.Cell Probes,2000;14(2):127-135
[26]Becker K,Pan D,Whitely C.B.Real-time quantitative polymerase chain reaction to assess gene transfer.Hum.Gene Ther,1999;10:2559-2566
[27]Bieche I,etal.Real-time reverse transcription-PCR assay for future nagement of ERBB2-based cLINical apolications.CLIN.Chem,1999;45:1148-1156
[28]Wang X,Li X,etal.Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1 beta mRNA in ischemic brain tolerance.Neurosci.Res,2000;59(2):238-46
[29]Schafers BA,Schlutius BG,Haider SG.Ontogenesis of oxidative reaction of 17beta-hydroxysteroid dehydrogenase and 11 beta-hydroxysteroid dehydrogenase in rat Leydig cells,a histochemical study.Histochem J,2001;33(9-10):585-595
[30]Fitch RH,Berrebi AS,Cowell PE.Effects of neonatal hormones on sexual dimorphism in the rat.Brain Research,1990;515:111-116
[31]祝辉,沙家豪,周作民.睾丸中细胞色素P450酶的生物学特性及调控.国外医学计划生育分册,1999;18(3):129-133
[32]Yanase T,Simpson ER,Waterman MR.17a hydroxylase/17,20-lyase deficiency:from cinical investigation t0 molecular definition.Endoct Rev,.1991;12:91-108
[33]Kater CE,Biglher EG.Disorders of steroid 17a-hydroxylase deficiency Endkcrinol Metab Clin North Am,1994;23:341-357
[34]李岩,张浩,王文冬,等.小剂量氟他胺对大鼠性分化影响的实验研究.四川大学学报(医学版),2006;37(2):258-261
[1]Hrana S,Ranmal S,Ben-Jonathan N,et al.Exposure of newbom male and female rats to environmental estrogens:delayed and sustained hyperoprolactinemia and alterations in estrogen receptor expression[J].Endocrinology,2000,14 1(12):4512-4517.
[2]Tokao T,Nannamiya W,Nazarloo HP,et al.Exposure to the environmental estrogen bisphenol A differentially modulated estrogen receptor-alpha and-beta immunoreactivity and mRNA in male mouse testis[J].Life Sci,2003,72(10):1159-1169.
[3]Hsu PC,Lai TJ,Guo NW.et al.Serum hormones in boys prenatally exposed to p01ychl0rinated biphenyls and dibenzo-furans[J].J Toxicol Environ Health,2005,68(17-18):1447-1456.
[4]Murray TJ,Lea RG,Abramovich DR,et al.Endocrine disrupting chemicals:effects on human male reproductive health[J].Early Pregnancy,2001,5(2):80-92.
[5]IlletteLJ,etal.[J].EnvironHealthPer2spect,1994,102(8):6802688.
[6]ToppariJ,etal.[J].EnvironHealthPerspect,1996,104(Suppl,4):7412803
[7]Kimura F,Funabashi T.Two subgroups of gonadotropin-releasing neurons control gonadotropin secretion in rats.News Physiol Sci,1998;13;225-231
[8]Terasawa E.Cellular mechanism of pulsatile LHRH release.Gen.Comp.Endocrinol,1998;112:283-295
[9]Evans JJ.Modulation of gonadotropin levels by peptides acting at the anterior pituitary gland.Endocrinol Revs,1990;20:46-67
[10]Crowley WF Jr,Whitcomb RW,Jameson JL,et al.Neuroendocrine control of human reproduction in the male.Rec Progr Hormone Res,1991;47:27-67
[11]Bousfield GR,Perry WM,Ward DN.(1994) Gonadotropins:chemistry andbiosynthesis.In;Knobil E & Neill JD ed.The Physiology of Reproduction.2~(nd)edition,New York:Raven Press
[12]Reed L & Pangaro LN(1995) Physiology of the thyroid gland I;synthesis and release,iodine metabolism and binding and transport.In:Becker KL ed.Principles and practice of Endocrinology and Metabolism,2~(nd) edition,Philadelphia,PA:Lippincott,pp.285-291
[13]Philips DIW,Barker DJP,Fall CHD,et al.Elevated plasma cortisol concentrations:an expression for the relationship between low birth weight and adult cardiovascular risk factors.J CLIN Endocrinol Metab,1998;83:757-760
[14]Wilson JD,Lasnitzki I.Dihydrotestosterone formation in fetal tissues of the rabbit and rat[M].Endocrinology,1971;89:659
[15]Griffin J,McPhau IM,Russel D,et al.The androgen resistance syndromes:steroid 5alpha-reductase deficiency,testicular feminization and related disorders.In:The metabolic and molecular bases of inherited disease[M].Edited by.Scriver CR,Beaudet AL,Sly WS,et al.New York;McGraw-Hil,1995,chapt,3,2967
[16]Nielsen M,Bjomsdottir S,Hoyer PE,Byskov AG.Ontogeny of oestrogen receptor alpha in gonads and sex ducts of fetal and newborn mice.J Reprod Fertil,2000;118(1):195-204
[17]Barthold JS,Kryger JV,Derusha AM,Duel BP,Jednak R,Skafar DF.Effects of an environmental endocrine disruptor on fetal development,estrogen receptor(alpha)and epidermal growth factor receptor expression in the porcine male genital tract.JUrol.1999;162(3 Pt 1):864-71
[18]Drea CM,Weldele NG,Forger E M,et al.Androgens and maseuLINization of genitalia in the spotted hyaena(Croeuta crocuta).2.Effects of prenatal anti-androgens.J Reprod Fertil,1998;113:117-127.
[19]Miyagawa S,Buchanan DL,Sato T,et al.Characterization ofdiethylstilbestrol induccd hypospadias in female mice.The Anatomical record,2002;266(1):43-50.
[20]Hellwig J,Vanravenzwaay B,Mayer M.Pre- and postnatal oral toxicity of vinclozoin in wistar and Long-Evans rats.Regul Toxicol Pharmacol,2000;32(1):42-50.
[21]WongCI,etal.[J].JBiolChem,1995,270(34):19998220003.
[22]ManessSC,etal.[J].ToxicolApplPharma2col,1998,151(1):1352142.
[23]TanJA,etal.[J].JBiolChem,1992,267(7):445624466.
[24]KelceWR,etal.[J].Nature,1995,375(6532):5812585.
[25]WongCI,etal.[J].JBiolChem,1995,270(34):19998220003
[26].KuilCW,etal.[J].JBiolChem,1995,270(46):27569227576.
[27]Imperato2McGinley,etal.[J].Endocrinology,1992,131(3):114921156.
[28]KassimNM,etal.[J].JAnat,1997,190(Pt4):5772588.
[29].Imperato2McGinley,etal.[J].Endocrinology,1992,131(3):114921156.
[30]李岩,张浩一,吴德生.氟他胺对仔鼠雄性生殖系统的影响[J].贵州医药,2007,31(8):675-677.
[31]罗艳,栾荣生,朱军,等.尿道下裂与环境污染的关系-Poisson回归模型与GM(1,N)模型结合分析[J].现代预防医学,2005,32(1):1-3.
[32]KelceWR,etal.[J].ToxicolApplPharmacol,1994,126(2):2762285.
[33]KelceWR,etal.[J].ToxicolApplPharmacol,1997,142(1):1922200.
[34]JrGrayLE,etal.[J].ToxicoLINdHealth,1999,15(122):942118.
[35]WongCI,etal.[J].JBiolChem,1995,270(34):19998220003.
[36]KelceWR,etal.[J].ToxicolApplPharmacol,1994,126(2):2762285.
[37]KelceWR,etal.[J].ToxicolApplPharmacol,1997,142(1):1922200.
[38]KelceWR,etal.[J].Nature,1995,375(6532):5812585.
[39]丁瑜,田英.邻苯二甲酸酯雄性生殖毒性的动物实验研究进展[J].国外医学卫生学分册,2008,35(4):202-207.
[40]C.Lambright~*,J.Ostby~*,K.Bobseine~*,V.Wilson,A.K.Hotchkiss,P.C.Mannand L.E.Gray,Jr.~*,1Received January 11,2000;accepted April 24,2000Cellular and Molecular Mechanisms of Action of LINuron:An Antiandrogenic Herbicide that Produces Reproductive Malformations in Male Rats
[41]Rider,-C-V;Furr,-J;Wilson,-V-S;Gray,-L-E Jr.A mixture of seven antiandrogens induces reproductive malformations in rats.Int-J-Androl.2008 Apr;31(2):249-62
[42]袁琳,贺厚光,龚永光等.邻苯二甲酸二丁酯诱发尿道下裂大鼠模型的建立.京医科大学学报(自然科学版),2005:25(5):341-342
[43]侯中林,王红,范凌松,等1邻苯二甲酸二-2-乙基己酯对雄性大鼠的生殖毒性1卫生毒理学杂志,1999,13(2):72-751.
[44]LambrightC,etal.[J].ToxicolSci,2000,56(2):3892399
[45]GrayLE,etal.[J].ToxicolLett,1998,1022103:6772680.
[46]GrayLE,etal.[J].ToxicolLett,1998,1022103:6772680.
[47]JrGrayLE,etal.[J].ToxicoLINdHealth,1999,15(122):942118.
[48]Tokao T,Nannamiya W,Nazarloo HP,et al.Exposure to the environmental estrogen bisphenol A differentially modulated estrogen receptor-alpha and-beta immunoreactivity and mRNA in male mouse testis[J].Life Sci,2003,72(10):1159-1169.
[49]HiortO,etal.[J].EurJPediatr,1994,153(5):3172321.
[50]WilsonJD,etal.[J].EndocrRev,1993,14(5):5772593.
[51]NewboldR.[J].EnvironHealthPerspect,1995,103(Suppl.7):83287.
[52]ManessSC,etal.[J].ToxicolApplPharma2col,1998,151(1):1352142.
[53]KallooNB,etal.[J].JCLINEndocrinolMetab,1993,77(3):6922
[2]Yamada G,Satoh Y,Baskin LS,et al.Cellular and molecular mechanisms of development of the external genitalia.International Society of differentiation,2003;71(8):445-460
[3]McIntyre BS,Barlow Nj,Foster PM.Androgen-mediated development in male rat offspring exposed to flutamide in utero:permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues[J].Toxicol Sci,2001;62(2):236-249
[4]Charles S,Patrick B,Beatrice T et al.Environmental xenoestrogens,antiandrogens and disorders of male sexual differentiation.Molecular and Cellular Endocnnology,2001;178(1-2):99-105
[5]McIntyre BS,Barlow NJ,Wallace DG,et al.Effects of in utero exposure to Linuron on androgen-dependent reproductive development in the male Cr1:CD(SD)BR rat.Toxicol Appl Pharmacol,2000;167(2):87-99
[6]Lambright C,Ostby J,Bobseine K,et al.Cellular and Molecular Mechanisms of Action of Linuron:An Antiandrogenic Herbicide that Produces Reproductive Malformations in Male Rats.Toxicological Sciences,2000;56:389-399
[7]Gray LEJ,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;15:94-118.
[8]Mclntyre BS,Barlow Nj,Foster PM.Androgen-mediated development in male rat offspring exposed to flutamide in utero:permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues[J].Toxicol Sci,2001;62(2):236-249
[9]Gray LE,Ostby J,Wolf C,et al.The value of mechanistic studies in laboratory animals for the prediction of reproductive effects in wildlife:Endocrine effects on mammalian sexual differentiation.Environ Toxicol Chem,1998;17(1):109-118
[10]周宏,周新华,吴巍,等.胚胎及生后不同发育时期大鼠睾丸生殖细胞的凋亡.中国组织化学与细胞化学杂志,2002,11(6):146
[11]卢洹,周新华等。ICR小鼠胚胎及生后不同阶段睾丸内生殖细胞的发育,武汉大学学报 2004,25(4):359
[12]Barlow,Norman J,Phillips,et al.Quantitative changes in gene expression in fetal rat testes following exposure to Di(n-butyl) phthalate.2003;73(2):431-441
[13]祝辉,沙家豪,周作民.睾丸中细胞色素P450酶的生物学特性及调控.国外医学计划生育分册,1999;18(3):129-133
[14]Higuchi R,Fockler C,etal.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions.Biotechnology,1993;11(9):1026-1030
[15]Fitch RH,Berrebi AS,Cowell PE.Effects of neonatal hormones on sexual dimorphism in the rat.Brain Research,1990;515:111-116
[16]Rhees RW,Shryne JE,Gorski RA.Onset of the hormone-sensitive perinatal period for sexual differentiation of the sexually dimorphic nucleus of the preoptic area.Journal of Neurobiology,1990;21:781-786
[17]Rhees RW,Shryne JE,Gorski RA.Termination of the hormone-sensitive period for differentiation of the sexually dimorphic nucleus of the preoptic areain male and female rats.Developmental Brain Research,1990;52:17-23
[18]Bloch GJ,Mills R.Prepuberal testosterone treatment of neonatally gonadectomized male rats:defeminization of behavioral and endocrine functionin adulthood.Neuroscience and Biobehavioral Reviews,1995;19:187-200
[19]Tan JA,et al.[J].J BioI Chem,1992;267(7):4456-4466
[20]Anderson KM,Cheung PH,Kell MD.Rapid generation of homologous internal standards and evaluation of data for quantitaion of messenger RNA by competitive polymerase chain reaction.J Pharmacol Toxicol Methods,1997;38:133-140
[21]Luhu A,Waiztu M,Koch A.A rapid and sensitive protocol for competitive reverse transcriprase(CRT) PCR analysis of cellular genes.Brain Pathol,1998;8:13-18
[22]文斌,高巍,费然等.核酸扩增产物的量化酶免疫通用型检测方法.中华医学检验杂志,1999;22:83-86
[23]Actor JK,Limor JR,Hunter RL.A flexible bioluminescent-quantitive polymerase reaction assay for analysis of competitive PCR amplicons.J CLIN Lab Anal,1999;13(1):40-47
[24]Schnell S,Mendoza C.Enzymological considerations for a theoretical description of the quantitative competitive polymerase chain reaction(QC-PCR).J Theor Biol,1997;184(4):433-440
[25]Ke LD,Chen Z,Yung WK.A reliability test of standard-based quantitative PCR:exogenous vs endogenous standards.Mol.Cell Probes,2000;14(2):127-135
[26]Becker K,Pan D,Whitely C.B.Real-time quantitative polymerase chain reaction to assess gene transfer.Hum.Gene Ther,1999;10:2559-2566
[27]Bieche I,etal.Real-time reverse transcription-PCR assay for future nagement of ERBB2-based cLINical apolications.CLIN.Chem,1999;45:1148-1156
[28]Wang X,Li X,etal.Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1 beta mRNA in ischemic brain tolerance.Neurosci.Res,2000;59(2):238-46
[29]Schafers BA,Schlutius BG,Haider SG.Ontogenesis of oxidative reaction of 17beta-hydroxysteroid dehydrogenase and 11 beta-hydroxysteroid dehydrogenase in rat Leydig cells,a histochemical study.Histochem J,2001;33(9-10):585-595
[30]Fitch RH,Berrebi AS,Cowell PE.Effects of neonatal hormones on sexual dimorphism in the rat.Brain Research,1990;515:111-116
[31]祝辉,沙家豪,周作民.睾丸中细胞色素P450酶的生物学特性及调控.国外医学计划生育分册,1999;18(3):129-133
[32]Yanase T,Simpson ER,Waterman MR.17a hydroxylase/17,20-lyase deficiency:from cinical investigation t0 molecular definition.Endoct Rev,.1991;12:91-108
[33]Kater CE,Biglher EG.Disorders of steroid 17a-hydroxylase deficiency Endkcrinol Metab Clin North Am,1994;23:341-357
[34]李岩,张浩,王文冬,等.小剂量氟他胺对大鼠性分化影响的实验研究.四川大学学报(医学版),2006;37(2):258-261
[1]Hrana S,Ranmal S,Ben-Jonathan N,et al.Exposure of newbom male and female rats to environmental estrogens:delayed and sustained hyperoprolactinemia and alterations in estrogen receptor expression[J].Endocrinology,2000,14 1(12):4512-4517.
[2]Tokao T,Nannamiya W,Nazarloo HP,et al.Exposure to the environmental estrogen bisphenol A differentially modulated estrogen receptor-alpha and-beta immunoreactivity and mRNA in male mouse testis[J].Life Sci,2003,72(10):1159-1169.
[3]Hsu PC,Lai TJ,Guo NW.et al.Serum hormones in boys prenatally exposed to p01ychl0rinated biphenyls and dibenzo-furans[J].J Toxicol Environ Health,2005,68(17-18):1447-1456.
[4]Murray TJ,Lea RG,Abramovich DR,et al.Endocrine disrupting chemicals:effects on human male reproductive health[J].Early Pregnancy,2001,5(2):80-92.
[5]IlletteLJ,etal.[J].EnvironHealthPer2spect,1994,102(8):6802688.
[6]ToppariJ,etal.[J].EnvironHealthPerspect,1996,104(Suppl,4):7412803
[7]Kimura F,Funabashi T.Two subgroups of gonadotropin-releasing neurons control gonadotropin secretion in rats.News Physiol Sci,1998;13;225-231
[8]Terasawa E.Cellular mechanism of pulsatile LHRH release.Gen.Comp.Endocrinol,1998;112:283-295
[9]Evans JJ.Modulation of gonadotropin levels by peptides acting at the anterior pituitary gland.Endocrinol Revs,1990;20:46-67
[10]Crowley WF Jr,Whitcomb RW,Jameson JL,et al.Neuroendocrine control of human reproduction in the male.Rec Progr Hormone Res,1991;47:27-67
[11]Bousfield GR,Perry WM,Ward DN.(1994) Gonadotropins:chemistry andbiosynthesis.In;Knobil E & Neill JD ed.The Physiology of Reproduction.2~(nd)edition,New York:Raven Press
[12]Reed L & Pangaro LN(1995) Physiology of the thyroid gland I;synthesis and release,iodine metabolism and binding and transport.In:Becker KL ed.Principles and practice of Endocrinology and Metabolism,2~(nd) edition,Philadelphia,PA:Lippincott,pp.285-291
[13]Philips DIW,Barker DJP,Fall CHD,et al.Elevated plasma cortisol concentrations:an expression for the relationship between low birth weight and adult cardiovascular risk factors.J CLIN Endocrinol Metab,1998;83:757-760
[14]Wilson JD,Lasnitzki I.Dihydrotestosterone formation in fetal tissues of the rabbit and rat[M].Endocrinology,1971;89:659
[15]Griffin J,McPhau IM,Russel D,et al.The androgen resistance syndromes:steroid 5alpha-reductase deficiency,testicular feminization and related disorders.In:The metabolic and molecular bases of inherited disease[M].Edited by.Scriver CR,Beaudet AL,Sly WS,et al.New York;McGraw-Hil,1995,chapt,3,2967
[16]Nielsen M,Bjomsdottir S,Hoyer PE,Byskov AG.Ontogeny of oestrogen receptor alpha in gonads and sex ducts of fetal and newborn mice.J Reprod Fertil,2000;118(1):195-204
[17]Barthold JS,Kryger JV,Derusha AM,Duel BP,Jednak R,Skafar DF.Effects of an environmental endocrine disruptor on fetal development,estrogen receptor(alpha)and epidermal growth factor receptor expression in the porcine male genital tract.JUrol.1999;162(3 Pt 1):864-71
[18]Drea CM,Weldele NG,Forger E M,et al.Androgens and maseuLINization of genitalia in the spotted hyaena(Croeuta crocuta).2.Effects of prenatal anti-androgens.J Reprod Fertil,1998;113:117-127.
[19]Miyagawa S,Buchanan DL,Sato T,et al.Characterization ofdiethylstilbestrol induccd hypospadias in female mice.The Anatomical record,2002;266(1):43-50.
[20]Hellwig J,Vanravenzwaay B,Mayer M.Pre- and postnatal oral toxicity of vinclozoin in wistar and Long-Evans rats.Regul Toxicol Pharmacol,2000;32(1):42-50.
[21]WongCI,etal.[J].JBiolChem,1995,270(34):19998220003.
[22]ManessSC,etal.[J].ToxicolApplPharma2col,1998,151(1):1352142.
[23]TanJA,etal.[J].JBiolChem,1992,267(7):445624466.
[24]KelceWR,etal.[J].Nature,1995,375(6532):5812585.
[25]WongCI,etal.[J].JBiolChem,1995,270(34):19998220003
[26].KuilCW,etal.[J].JBiolChem,1995,270(46):27569227576.
[27]Imperato2McGinley,etal.[J].Endocrinology,1992,131(3):114921156.
[28]KassimNM,etal.[J].JAnat,1997,190(Pt4):5772588.
[29].Imperato2McGinley,etal.[J].Endocrinology,1992,131(3):114921156.
[30]李岩,张浩一,吴德生.氟他胺对仔鼠雄性生殖系统的影响[J].贵州医药,2007,31(8):675-677.
[31]罗艳,栾荣生,朱军,等.尿道下裂与环境污染的关系-Poisson回归模型与GM(1,N)模型结合分析[J].现代预防医学,2005,32(1):1-3.
[32]KelceWR,etal.[J].ToxicolApplPharmacol,1994,126(2):2762285.
[33]KelceWR,etal.[J].ToxicolApplPharmacol,1997,142(1):1922200.
[34]JrGrayLE,etal.[J].ToxicoLINdHealth,1999,15(122):942118.
[35]WongCI,etal.[J].JBiolChem,1995,270(34):19998220003.
[36]KelceWR,etal.[J].ToxicolApplPharmacol,1994,126(2):2762285.
[37]KelceWR,etal.[J].ToxicolApplPharmacol,1997,142(1):1922200.
[38]KelceWR,etal.[J].Nature,1995,375(6532):5812585.
[39]丁瑜,田英.邻苯二甲酸酯雄性生殖毒性的动物实验研究进展[J].国外医学卫生学分册,2008,35(4):202-207.
[40]C.Lambright~*,J.Ostby~*,K.Bobseine~*,V.Wilson,A.K.Hotchkiss,P.C.Mannand L.E.Gray,Jr.~*,1Received January 11,2000;accepted April 24,2000Cellular and Molecular Mechanisms of Action of LINuron:An Antiandrogenic Herbicide that Produces Reproductive Malformations in Male Rats
[41]Rider,-C-V;Furr,-J;Wilson,-V-S;Gray,-L-E Jr.A mixture of seven antiandrogens induces reproductive malformations in rats.Int-J-Androl.2008 Apr;31(2):249-62
[42]袁琳,贺厚光,龚永光等.邻苯二甲酸二丁酯诱发尿道下裂大鼠模型的建立.京医科大学学报(自然科学版),2005:25(5):341-342
[43]侯中林,王红,范凌松,等1邻苯二甲酸二-2-乙基己酯对雄性大鼠的生殖毒性1卫生毒理学杂志,1999,13(2):72-751.
[44]LambrightC,etal.[J].ToxicolSci,2000,56(2):3892399
[45]GrayLE,etal.[J].ToxicolLett,1998,1022103:6772680.
[46]GrayLE,etal.[J].ToxicolLett,1998,1022103:6772680.
[47]JrGrayLE,etal.[J].ToxicoLINdHealth,1999,15(122):942118.
[48]Tokao T,Nannamiya W,Nazarloo HP,et al.Exposure to the environmental estrogen bisphenol A differentially modulated estrogen receptor-alpha and-beta immunoreactivity and mRNA in male mouse testis[J].Life Sci,2003,72(10):1159-1169.
[49]HiortO,etal.[J].EurJPediatr,1994,153(5):3172321.
[50]WilsonJD,etal.[J].EndocrRev,1993,14(5):5772593.
[51]NewboldR.[J].EnvironHealthPerspect,1995,103(Suppl.7):83287.
[52]ManessSC,etal.[J].ToxicolApplPharma2col,1998,151(1):1352142.
[53]KallooNB,etal.[J].JCLINEndocrinolMetab,1993,77(3):6922