邻苯二甲酸二异壬酯胚胎期和哺乳期染毒对子代雄性大鼠生殖系统的影响
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摘要
[目的]作为环境内分泌干扰物邻苯二甲酸二乙基己脂(DEHP)的替代物,邻苯二甲酸二异壬酯(DINP)已逐渐成为用量最大的增塑剂之一。本研究旨在探索DINP胚胎期及哺乳期染毒对雄性子代生殖系统的影响及其可能机制。[方法]采用两代繁殖实验设计,在Wistar雌性受孕大鼠的GD7(受孕第8天)至PND21(产后第21天),每天经口灌胃DINP,染毒剂量为5、50、500、1 000 mg/kg(体重),玉米油为溶剂对照组,每组由5～6只孕鼠组成。PND2时,测量子代仔鼠的肛殖距(AGD),并计算其AGD指数。在PND4时通过窝标准化操作保持每窝8只仔鼠(4只雌鼠,4只雄鼠)。分别于PND21和PND49,从各剂量组随机抽取10只雄性仔鼠,用ELISA法测定其血清睾酮水平,并解剖观察其睾丸病理变化;计算PND21时睾丸和附睾系数,并用实时定量PCR方法测定睾酮合成途径关键基因的mRNA表达情况。[结果]与溶剂对照组相比较,DINP染毒组新生仔鼠的围生期损失数和雌雄性别比差异无统计学意义(P>0.05),但雄性仔鼠的AGD缩短,分别为(5.15±0.37)、(5.17±0.33)、(4.57±0.38)、(5.16±0.32)mm,500、1 000 mg/kg染毒组雄性仔鼠的AGD指数(2.48±0.19、2.51±0.13)降低。与对照组相比:PND21时, 50 mg/kg及以上的DINP处理组中Star基因mRNA表达量增高,500及1 000 mg/kg处理组中Scarb1的mRNA表达量降低,Lhcgr基因mRNA表达量也在1 000 mg/kg剂量组降低(均P<0.05)。PND49 DINP染毒组大鼠生精细胞和精子减少,间质细胞出现聚集现象,但PND21和PND49雄性仔鼠血清睾酮浓度未发现降低(P>0.05)。[结论]胚胎期及哺乳期DINP暴露对雄性F在其作用机1代大鼠的生殖系统存在影响,睾酮合成过程中关键基因Scarb1、Star和Lhcgr可能制中发挥一定作用。
[Objective] As an alternative to environmental endocrine disruptor diethylhexyl phthalate(DEHP),diisodecyl phthalate(DINP) has gradually become one of the most widely used plasticizers. This study aims to explore the effects of embryonic and lactational DINP exposure on the reproductive system of male offspring and its possible mechanisms.[Methods] In a two-generation reproduction toxicity study, pregnant Wistar rats were treated with 5, 50, 500, and 1 000 mg/kg(body weight) DINP or corn oil(solvent control) by gavage once a day from gestational day 7(GD7) to postnatal day 21(PND21), with 5-6 pregnant rats in each group. On PND2, the anogenital distance(AGD) of the pups was measured and the AGD Index was calculated. On PND4, the litters were culled to 8 pups per litter(4 females and 4 males). On PND21 and PND49, 10 male pups randomly selected from each dose group were measured for serum testosterone levels by ELISA and observed for testicular pathological changes. The testis and epididymis coefficients were calculated on PND21, and the mRNA expressions of key genes in testosterone synthesis pathway were determined by real-time quantitative PCR.[Results] Compared with the solvent control group, the perinatal loss and male-female sex ratio of the newborn rats in the designed DINP groups were not statistically different(P >0.05), but the AGDs of male pups were significantly shortened to(5.15±0.37),(5.17±0.33),(4.57±0.38), and(5.16±0.32) mm in the order of low to high DINP exposure level, respectively, and the AGD indices of the 500 and 1 000 mg/kg dose groups were significantly reduced to(2.48±0.19) and(2.51±0.13), respectively. Compared with the control group, on PND21, the mRNA expression levels of Star were significantly increased in the DINP-treated groups at 50 mg/kg and above, the levels of Scarb1 were significantly decreased in the DINP-treated groups at 500 and 1 000 mg/kg, and the levels of Lhcgr was significantly reduced in the 1 000 mg/kg DINP-treated group(P< 0.05). The spermatogenic cells and spermatozoa decreased and aggregation appeared in the interstitial cells after the DINP treatment on PND49; but there was no significant decrease in serum testosterone concentrations in the male rats on PND21 and PND49(P >0.05).[Conclusion] Embryonic and lactational DINP exposure affects the reproductive system of male F1 rats. Scarb1, Star and Lhcgr, key genes of testosterone synthesis, may play a role in the male reproductive toxicity of DINP.
[Objective] As an alternative to environmental endocrine disruptor diethylhexyl phthalate(DEHP),diisodecyl phthalate(DINP) has gradually become one of the most widely used plasticizers. This study aims to explore the effects of embryonic and lactational DINP exposure on the reproductive system of male offspring and its possible mechanisms.[Methods] In a two-generation reproduction toxicity study, pregnant Wistar rats were treated with 5, 50, 500, and 1 000 mg/kg(body weight) DINP or corn oil(solvent control) by gavage once a day from gestational day 7(GD7) to postnatal day 21(PND21), with 5-6 pregnant rats in each group. On PND2, the anogenital distance(AGD) of the pups was measured and the AGD Index was calculated. On PND4, the litters were culled to 8 pups per litter(4 females and 4 males). On PND21 and PND49, 10 male pups randomly selected from each dose group were measured for serum testosterone levels by ELISA and observed for testicular pathological changes. The testis and epididymis coefficients were calculated on PND21, and the mRNA expressions of key genes in testosterone synthesis pathway were determined by real-time quantitative PCR.[Results] Compared with the solvent control group, the perinatal loss and male-female sex ratio of the newborn rats in the designed DINP groups were not statistically different(P >0.05), but the AGDs of male pups were significantly shortened to(5.15±0.37),(5.17±0.33),(4.57±0.38), and(5.16±0.32) mm in the order of low to high DINP exposure level, respectively, and the AGD indices of the 500 and 1 000 mg/kg dose groups were significantly reduced to(2.48±0.19) and(2.51±0.13), respectively. Compared with the control group, on PND21, the mRNA expression levels of Star were significantly increased in the DINP-treated groups at 50 mg/kg and above, the levels of Scarb1 were significantly decreased in the DINP-treated groups at 500 and 1 000 mg/kg, and the levels of Lhcgr was significantly reduced in the 1 000 mg/kg DINP-treated group(P< 0.05). The spermatogenic cells and spermatozoa decreased and aggregation appeared in the interstitial cells after the DINP treatment on PND49; but there was no significant decrease in serum testosterone concentrations in the male rats on PND21 and PND49(P >0.05).[Conclusion] Embryonic and lactational DINP exposure affects the reproductive system of male F1 rats. Scarb1, Star and Lhcgr, key genes of testosterone synthesis, may play a role in the male reproductive toxicity of DINP.
引文
[1]Directive 2005/84/EC of the European Parliament and of the Council of 14 December 2005[A/OL].[2018-11-01].https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32005L0084&rid=1.
[2]LI L,BU T,SU H,et al.In utero exposure to diisononyl phthalate caused testicular dysgenesis of rat fetal testis[J].Toxicol Lett,2015,232(2):466-474.
[3]GRAY L J J R,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[J].Toxicol Sci,2000,58(2):350-365.
[4]BOBERG J,CHRISTIANSEN S,AXELSTAD M,et al.Reproductive and behavioral effects of diisononyl phthalate(DINP)in perinatally exposed rats[J].Reprod Toxicol,2011,31(2):200-209.
[5]LEE H C,YAMANOUCHI K,NISHIHARA M.Effects of perinatal exposure to phthalate/adipate esters on hypothalamic gene expression and sexual behavior in rats[J].J Reprod Dev,2006,52(3):343-352.
[6]CLEWELL R A,THOMAS A,WILLSON G,et al.A dose response study to assess effects after dietary administration of diisononyl phthalate(DINP)in gestation and lactation on male rat sexual development[J].Reprod Toxicol,2013,35:70-80.
[7]CARBONE S,SAMANIEGO Y A,CUTRERA R,et al.Different effects by sex on hypothalamic-pituitary axis of prepubertal offspring rats produced by in utero and lactational exposure to di-(2-ethylhexyl)phthalate(DEHP)[J].NeuroToxicology,2012,33(1):78-84.
[8]BORCH J,METZDORFF S B,VINGGAARD A M,et al.Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis[J].Toxicology,2006,223(1/2):144-155.
[9]VALERI C,SCHTEINGART H,REY R.The prepubertal testis:biomarkers and functions[J].Curr Opin Endocrinol Diabetes Obes,2013,20(3):224-233.
[10]SHONO T,SHIMA Y,KONDO T,et al.In utero exposure to mono-N-butyl phthalate impairs insulin-like factor 3 gene expression and the transabdominal phase of testicular descent in fetal rats[J].J Pediatr Surg,2005,40(12):1861-1864.
[11]IVELL R,HENG K,NICHOLSON H,et al.Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring[J].Asian J Androl,2013,15(2):261-268.
[12]HANNAS B R,LAMBRIGHT C S,FURR J,et al.Genomic biomarkers of phthalate-induced male reproductive developmental toxicity:a targeted RT-PCR array approach for defining relative potency[J].Toxicol Sci,2012,125(2):544-557.
[13]OECD.OECD Guidelines for the Testing of Chemicals,Section 4.Environment,Health and Safety Publications,Test No.414:Prenatal Developmental Toxicity Study[EB/OL].[2018-11-01].https://www.oecd-ilibrary.org/environment/test-no-414-prenatal-development-toxicitystudy_9789264070820-en.
[14]OECD.OECD Guidelines for the Testing of Chemicals,Section4.Environment,Health and Safety Publications,Test No.421:Reproduction/Developmental Toxicity Screening Test[EB/OL].[2018-11-01].https://www.oecd-ilibrary.org/environment/test-no-414-prenatal-developmenttoxicity-study_9789264070820-en.
[15]BORCH J,LADEFOGED O,HASS U,et al.Steroidogenesis in fetal male rats is reduced by DEHP and DINP,but endocrine effects of DEHP are not modulated by DEHA in fetal,prepubertal and adult male rats[J].Reprod Toxicol,2004,18(1):53-61.
[16]MASUTOMI N,SHIBUTANI M,TAKAGI H,et al.Impact of dietary exposure to methoxychlor,genistein,or diisononyl phthalate during the perinatal period on the development of the rat endocrine/reproductive systems in later life[J].Toxicology,2003,192(2/3):149-170.
[17]SUN Y,SHEN J,ZENG L,et al.Role of autophagy in di-2-ethylhexyl phthalate(DEHP)-induced apoptosis in mouse Leydig cells[J].Environ Pollut,2018,243:563-572.
[18]逯晓波,于涛,马明月,等.邻苯二甲酸二(2-乙基己基)酯对大鼠睾丸发育及StAR、CYP19a1、CYP11a1基因表达的影响[J].卫生研究,2010,39(3):263-267.
[19]ADAMSSON A,SALONEN V,PARANKO J,et al.Effects of maternal exposure to di-isononylphthalate(DINP)and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene(p,p’-DDE)on steroidogenesis in the fetal rat testis and adrenal gland[J].Reprod Toxicol,2009,28(1):66-74.
[20]HANNAS B R,LAMBRIGHT C S,FURR J,et al.Dose-response assessment of fetal testosterone production and gene expression levels in rat testes following in utero exposure to diethylhexyl phthalate,diisobutyl phthalate,diisoheptyl phthalate,and diisononyl phthalate[J].Toxicol Sci,2011,123(1):206-216.
[21]BERGER K,ESKENAZI B,KATHERINE K,et al.Association of prenatal urinary concentrations of phthalates and bisphenol a and pubertal timing in boys and girls[J].Environ Health Perspect,2018,126(9):97004.
[2]LI L,BU T,SU H,et al.In utero exposure to diisononyl phthalate caused testicular dysgenesis of rat fetal testis[J].Toxicol Lett,2015,232(2):466-474.
[3]GRAY L J J R,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[J].Toxicol Sci,2000,58(2):350-365.
[4]BOBERG J,CHRISTIANSEN S,AXELSTAD M,et al.Reproductive and behavioral effects of diisononyl phthalate(DINP)in perinatally exposed rats[J].Reprod Toxicol,2011,31(2):200-209.
[5]LEE H C,YAMANOUCHI K,NISHIHARA M.Effects of perinatal exposure to phthalate/adipate esters on hypothalamic gene expression and sexual behavior in rats[J].J Reprod Dev,2006,52(3):343-352.
[6]CLEWELL R A,THOMAS A,WILLSON G,et al.A dose response study to assess effects after dietary administration of diisononyl phthalate(DINP)in gestation and lactation on male rat sexual development[J].Reprod Toxicol,2013,35:70-80.
[7]CARBONE S,SAMANIEGO Y A,CUTRERA R,et al.Different effects by sex on hypothalamic-pituitary axis of prepubertal offspring rats produced by in utero and lactational exposure to di-(2-ethylhexyl)phthalate(DEHP)[J].NeuroToxicology,2012,33(1):78-84.
[8]BORCH J,METZDORFF S B,VINGGAARD A M,et al.Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis[J].Toxicology,2006,223(1/2):144-155.
[9]VALERI C,SCHTEINGART H,REY R.The prepubertal testis:biomarkers and functions[J].Curr Opin Endocrinol Diabetes Obes,2013,20(3):224-233.
[10]SHONO T,SHIMA Y,KONDO T,et al.In utero exposure to mono-N-butyl phthalate impairs insulin-like factor 3 gene expression and the transabdominal phase of testicular descent in fetal rats[J].J Pediatr Surg,2005,40(12):1861-1864.
[11]IVELL R,HENG K,NICHOLSON H,et al.Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring[J].Asian J Androl,2013,15(2):261-268.
[12]HANNAS B R,LAMBRIGHT C S,FURR J,et al.Genomic biomarkers of phthalate-induced male reproductive developmental toxicity:a targeted RT-PCR array approach for defining relative potency[J].Toxicol Sci,2012,125(2):544-557.
[13]OECD.OECD Guidelines for the Testing of Chemicals,Section 4.Environment,Health and Safety Publications,Test No.414:Prenatal Developmental Toxicity Study[EB/OL].[2018-11-01].https://www.oecd-ilibrary.org/environment/test-no-414-prenatal-development-toxicitystudy_9789264070820-en.
[14]OECD.OECD Guidelines for the Testing of Chemicals,Section4.Environment,Health and Safety Publications,Test No.421:Reproduction/Developmental Toxicity Screening Test[EB/OL].[2018-11-01].https://www.oecd-ilibrary.org/environment/test-no-414-prenatal-developmenttoxicity-study_9789264070820-en.
[15]BORCH J,LADEFOGED O,HASS U,et al.Steroidogenesis in fetal male rats is reduced by DEHP and DINP,but endocrine effects of DEHP are not modulated by DEHA in fetal,prepubertal and adult male rats[J].Reprod Toxicol,2004,18(1):53-61.
[16]MASUTOMI N,SHIBUTANI M,TAKAGI H,et al.Impact of dietary exposure to methoxychlor,genistein,or diisononyl phthalate during the perinatal period on the development of the rat endocrine/reproductive systems in later life[J].Toxicology,2003,192(2/3):149-170.
[17]SUN Y,SHEN J,ZENG L,et al.Role of autophagy in di-2-ethylhexyl phthalate(DEHP)-induced apoptosis in mouse Leydig cells[J].Environ Pollut,2018,243:563-572.
[18]逯晓波,于涛,马明月,等.邻苯二甲酸二(2-乙基己基)酯对大鼠睾丸发育及StAR、CYP19a1、CYP11a1基因表达的影响[J].卫生研究,2010,39(3):263-267.
[19]ADAMSSON A,SALONEN V,PARANKO J,et al.Effects of maternal exposure to di-isononylphthalate(DINP)and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene(p,p’-DDE)on steroidogenesis in the fetal rat testis and adrenal gland[J].Reprod Toxicol,2009,28(1):66-74.
[20]HANNAS B R,LAMBRIGHT C S,FURR J,et al.Dose-response assessment of fetal testosterone production and gene expression levels in rat testes following in utero exposure to diethylhexyl phthalate,diisobutyl phthalate,diisoheptyl phthalate,and diisononyl phthalate[J].Toxicol Sci,2011,123(1):206-216.
[21]BERGER K,ESKENAZI B,KATHERINE K,et al.Association of prenatal urinary concentrations of phthalates and bisphenol a and pubertal timing in boys and girls[J].Environ Health Perspect,2018,126(9):97004.
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