围产期低剂量氯化三丁基锡暴露对子代雄性小鼠的内分泌干扰作用及其机制初探
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摘要
研究背景
有机锡化合物(Organotin compounds, OTs)广泛用于船舶表面防生物附着涂料、杀虫剂、防腐剂(木材、纤维及皮革)、工业杀菌剂、塑料制品稳定剂等。由于应用广泛、环境中残存时间长,OTs已对全球环境(尤其是水体)造成了严重的污染。三丁基锡(Tributyltin, TBT)是OTs中最重要的一种,被认为是迄今为止由人为因素大量引入海水环境中最毒的化学品之一。它是一种确定的内分泌干扰物(ED),已有研究显示TBT对哺乳动物可产生免疫毒性、神经毒性、生殖毒性等。但这些研究所采用的染毒剂量都较大,而许多研究表明ED的危害并不呈线性剂量-反应关系,低剂量时的危害有可能要高于其高剂量时的危害。我们课题组的前期研究发现围产期低剂量氯化三丁基锡(TBTCl)暴露可导致子鼠睾丸中雌激素水平紊乱,附睾中精子数量减少及精子活度降低,睾丸组织出现病理性变化,并伴随着芳香酶含量的下降。目前又(?)TBTCl导致子代雄性小鼠发生以上变化的机制并不清楚,本研究在前期研究的基础上以子代雄性小鼠为主要研究对象,并添加己烯雌酚(DES)组作为阳性对照,对TBTCl影响妊娠结果、子鼠性发育及身体发育和血清中激素水平及其机制在分子和基因水平上做初步的探索。为研究TBTCl对人类的内分泌干扰作用及作用机制提供参考和借鉴。
研究目的：
探讨围产期低剂量TBTCl暴露对妊娠结果、子鼠发育和血清中激素水平的毒性作用,并且对其作用机制做初步的探索。
研究方法：
将受孕的KM小鼠随机分为阴性对照组(玉米油)、10、100μg/kgTBTCl组和阳性对照组(5μg/kg DES),每组6只。母鼠妊娠第6天(GD6)起,经口灌胃染毒,每天在同一时间染毒1次,染毒剂量为1ml/kg,直到哺乳期结束,即子鼠出生第21天(Postnatal day, PND21)。GD0、GD6、PND1和PND21时称量母鼠体重,染毒后开始观察并记录母鼠的流产及死亡情况；母鼠生产后,观察并记录出生子鼠的数量及雌雄比例,观察死亡情况并记录每组子鼠在哺乳期死亡的数量。PNDO、1、2、3、4、6、7、14和21时称量雄性子鼠体重,测量其肛门与生殖器之间的距离(AGD)。PND6时开始观察长毛情况,PND7时开始观察门牙萌生情况,PND12时开始观察睁眼情况,PND13时开始观察睾丸下降情况及PND28时开始观察包皮分离情况并分别记录其日龄。
PND22和100时每组随机抽取10只雄性子鼠(阳性对照取8只),称重后摘眼球取血,离心取血清并保存；分离睾丸、附睾、脑、肝脏、脾脏、肾脏和胸腺(PND100时还取腹侧前列腺、精囊),并称重；将睾丸立即放入液氮中速冻并置于-80℃保存。应用酶联免疫吸附试验方法测定PND22和PND100雄性子鼠血清中雌激素(E2)、睾酮(T)、游离睾酮(F-T)、促卵泡激素(FSH)和黄体生成素(LH)的浓度。采用Real-time PCR法检测PND22子代小鼠睾丸组织中Cypllal、Cyp17a、 Hsd3b2、Hsdl7b3和CyP19五个基因mRNA相对表达量,并用亚硫酸盐直接测序法(BSP)检测调控芳香酶表达的cAMP反应元件结合蛋白、泛素/蛋白酶体亚基α2和雌激素受体β基因启动子区域CpG岛的甲基化情况。
研究结果
剂量组孕鼠的流产(死亡)率与阳性对照组相比显著降低,与阴性对照组相比有增加的趋势；100μg/kg TBTC1组母鼠在PND21时体重较对照组均显著增加,PND1-21(?)PGD0-PND21母鼠体重增加量与对照组相比均显著增加,GD6-PND21母鼠体重增加量较阴性对照组显著升高；剂量组子鼠的数量较对照组有下降的趋势；100μg/kg TBTCl组和阳性对照组幼鼠死亡数与阴性对照组相比均显著增加,而阳性对照组和100ug/kg TBTCl组幼鼠死亡数相比没有统计学意义。
剂量组睾丸下降时间与阳性对照组相比显著提前,而与阴性对照组相比没有差异,阳性对照组与阴性对照组相比显著延迟。PNDO、1时100μg/kg TBTCl组的NAGD较阴性对照组显著降低,体重显著升高；PND3、6、14和21时TBTCl组NAGD与对照组相比有降低的趋势。PND0、1、2、6、7和21时100μg/kg组体重与阴性对照组相比显著增加,PND4-6、PNDO-7、PND14-21、PNDO-21的体重增加量与阴性对照组相比均显著增加。PND22时100μg/kg组睾丸脏器系数较对照组均显著降低；剂量组肝脏脏器系数与对照组相比均显著降低,脾脏重量及脏器系数与对照组相比显著增加,肾脏重量及脏器系数与阴性对照组相比显著增加。PND100时TBTCl组睾丸重量与阴性对照组相比显著升高,而与阳性对照组相比则显著降低；100μg/kg组精囊脏器系数与阴性对照组相比显著降低,肝脏脏器系数与阴性对照组相比显著升高；胸腺脏器系数与阴性对照组相比显著降低,与阳性对照组相比则显著升高。
子代雄性小鼠血清中激素测量结果显示,PND22时100μg/kg组的E2和FSH浓度与对照组相比均显著降低；PND100时100μg/kg组血清中E2的水平与阴性对照组相比仍显著降低,而与阳性对照组相比则没有差异,阳性对照组血清中E2的水平与阴性对照组相比也显著降低。
PND22子代雄性小鼠睾丸组织中类固醇激素合成途径关键酶mRNA(?)目对定量结果显示,10μg/kg组Cyp17a基因mRNA表达量与对照组相比均显著升高,Hsd3b2基因mRNA表达量与阴性对照组相比显著升高,而与阳性对照组相比则没有统计学差异；100μg/kg组Hsd17b(?)基因和(?)Cyp19(?)基因mRNA表达量与阴性对照组相比显著降低,剂量组Cyp19基因mRNA表达量有降低的趋势,而与阳性对照组相比则没有统计学差异；阳性对照组Hsd17b(?)基因和Cyp19(?)基因的mRNA表达量与阴性对照组相比有降低的趋势,但是没有统计学差异；Cypllal基因在各染毒组中mRNA表达量与对照组相比均无统计学差异。
PND22子代雄性小鼠睾丸组织中甲基化克隆测序结果发现只有cAMP反应元件结合蛋白(CREB)启动子区域基因序列发生甲基化改变,而泛素/蛋白酶体亚基α2和雌激素受体β基因启动子区域序列未发现甲基化情况。
结论
围产期低剂量TBTCl暴露导致母鼠流产增多、导致肥胖及食仔,导致雄性子鼠出现雌性化的趋势,并影响子鼠生长和器官发育。TBTCl可通过影响CREB启动子区域基因序列发生甲基化,类固醇激素合成途径中关键酶mRNA的表达水平改变,而导致血清中激素水平紊乱。
Backgroud:
Organotin compounds (OTs) has been widely used as painting in ship surface coating for preventing biofouling, pesticides, preservatives (wood, fiber and leather), industrial microbicides, plastic stabilizer, etc. Due to the wide application and long time existence in the environment, OTs has caused serious pollution on the global environment (especially water). Tributyltin (TBT) is one of the most important one of OTs, and it is considered as one of the most toxic chemicals in the environment intro-duced by man-made factors by far. TBT is a kind of certain Endocrine disruptor (ED), the existed studies have shown that TBT to the mammals can cause immunotoxicity, neurotoxicity and reproductive toxicity, etc. However, the dose of these study used is high, and many studies have shown that the damage of ED is not linear dose response relationship, some time the harm of low doses may be higher than the high doses. Our research group have studied and found that perinatal low doses of TBTCl can disturb the level of estrogen in mice testis, result in the decrease of sperm counts, the sperm motility reduced, the pathological changes of testicular tissue occurred in epididymis and the aromatase levels decreased in testis. While we have not known the mechanism of above changes up to now, so we take the progeny male mice as the main research object on the basis of previous research in this study, add the Diethylstilbestrol (DES) group as the positive control, study the influence of TBTCl on the pregnancy out-comes of the pregnant mice, the sex and body development of the progeny male mice and the sex hormone levels in the serum, and do the preliminary exploration on the mechanisms of which at the molecular and gene level. We hope our study can provide the reference for the study on TBTCl disrupting the human endocrine balance and its mechanism.
Objective:
To discuss the toxic effects of perinatal exposure to low doses of TBTCl to the pregnancy outcomes, progeny male mice growth and sex hormone levels in serum, and do a preliminary exploration on its mechanism.
Methods:
The pregnant KM mice are randomly divided into4groups,6in each group, they are given corn oil as the negative control group,10,100μg/kg TBTC1and5μg/kg DES as the positive control group by gavage from days6of gestation to the end of lacation(Postnatal21day, PND21). We infect one time every day at the same time and the dose is1ml/kg. At the time of GDO, GD6, PND1and PND21the weight of the mothers are weighed, and then we begin to observe and record the condition of the abortion and death of the infected mother. After the mother cub, we begin to observe and record the number of birth and the sex ratio. We also need to record the number of deaths of each group of the offspring in lactation. We weigh the body weight of the offspring on PNDO,1,2,3,4,6,7,14and21, and measure the distance between the anus and genitals (AGD) of the male mice. Beginning to examine the hair growing on PND6, the incisors drop-in on PND7, the eye opening on PND12, the testicular des-cent on PND13and the preputial separation on PND28, and we must record their age respectively.
On PND22and100, respectively,10male offspring mice (The positive control group is8.) were selected in each group randomly and killed after weighed and trunk blood was collected. The blood was let stand at room temperature for2hours and then centrifugaing for the serum and save it. The testicles, epididymis, brain, liver, spleen, kidney and thymus (the ventral prostate and seminal vesicle also were taken on PND100) were separated and weighed for account of viscera coefficient. The testis were immediately put into liquid nitrogen for preservation at-80℃. Using the method of enzyme-linked immunosorbent assay measure the concentration of estro-gen (E2), testosterone (T), free testosterone (F-T), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in serum on PND22and PND100. Adopting the Real-time PCR method to detect the relative transcript level of Cyp11al, Cyp17a, Hsd3b2, Hsd17b3and Cyp19gene mRNA in offspring mice testicular tissue on PND22. And the detection of the methylation pattern of CpG islands in promter region of cAMP response element binding protein, proteasome subunit alpha2and estrogen receptor beta gene who can control aromatase expression by bisulfite sequencing polymerase chain reaction (BSP).
Results:
Comparing with the positive control group, the dosage group's abortion (death) rate of the pregnant mice is significantly decreased, while with the negative control group has an increasing trend. On PND21, the weight of the pregnant mice is signify-cantly higher than the control group, the body weight gain of the pregnant mice is significantly higher than the control group during PND1-21and GD0-PND21, and during GD6-PND21the body weight gain of the pregnant mice is significantly incr-eased compared with the negative control group in100μg/kg TBTCl group. The number of TBTCl group's offspring has a downtrend compared with the control group. In100μg/kg TBTCl and the positive control group the mortality of mouse pup are significantly increased compared with the negative control group, but there is no statistical significance between the positive control group and the100μg/kgTBTCl group.
The time of testicular descent of TBTCl group is significantly ahead of time compared with positive control group, but no statistical differences compared with negative control group, and the testicular descent time of the positive control group is significantly delayed compared with the negative control group. On PND0and1, comparing with the negative control group the NAGD is significantly reduced, at the same time the weight is also significantly increased in100μg/kgTBTCl, while there is only a tendency to reduce compared with control group, but there is no statistical difference on PND3,6,14and21. In100μg/kg TBTCl group the body weight of the offspring is significantly increased compared with negative control group on PNDO,1,2,6,7and21, the body weight gain is significantly increased compared with the negative control group during PND4-6, PND0-7, PND14-21, PND0-21. On PND22the viscera coefficient of testicular is significantly lower than the control group in100μg/kg TBTCl group; In TBTCl group the Viscera coefficient of Liver is signifi-cantly reduced compared with control group, while the weight and viscera coefficient of Kidney are significantly higher than negative control group and the weight and viscera coefficient of Spleen are also higher than the control group. On PND100, the testicular weight of TBTC1group is significantly increased compared with negative control group, but is significantly reduced compared with positive control group; in100μg/kgTBTCl group the viscera coefficient of Seminal vesicle is significantly lower than negative control group, but the liver is significantly higher; The viscera coefficient of Thymus was significantly decreased compared with negative control group, while was significantly increased compared with positive control group; The viscera coefficient of thymus of positive control group is significantly lower than negative control group.
The measurement results of hormone in male offspring mice serum show that in100μg/kg TBTC1group the concentration of E2and FSH are significantly lower than the control group on PND22, and on PND100the serum E2levels is also significantly decreased compared with negative control group, while compared with positive control group their is no statistically difference, and the serum E2level of positive control group is also significantly reduced compared with negative control group.
On PND22, the key enzymes in offspring testis tissue of steroid hormones mRNA relative quantitative results show that the mRNA expression amount of Cyp17a gene is significantly higher than the control group, Hsd3b2gene is signifycantly in-creased compared with negative control group, but their is no significant differences compared with positive control group in10μg/kg TBTC1group; the mRNA express-ion amount of Hsdl7b3and Cypl9genes are significantly lower than negative control group in100ug/kg TBTC1group, and the infected group's mRNA expression amount of Cyp19gene has a tendency to reduce, while their is no significant different-ce compared with positive control group; the mRNA expression amount of Hsdl7b3gene and Cyp19gene in positive control group have a decreased trend compared with negative control group, but no statistical difference; Cypllal gene's mRNA express-ion in the infected group is no statistical difference compared with the control group.
On PND22, through the cloning sequencing comparison we find methylation only exists in the promoter region of cAMP response element binding protein (CREB) gene. However, the promoter region of the Ubiquitin/proteasome subunit alpha2and estrogen receptor beta gene do not find.
Conclusion
Perinatal exposure to low doses of TBTC1can result in the abortion of pregnant mice increased, lead to obesity and eat their own children. What's more, it also makes the male offspring feminized and affects the growth and organ development. Through the study of the mechanism we find in perinatal low-dose TBTC1treatment can influe-nee the methylation happened in the promoter region of CREB, lead to the mRNA ex-pression levels of key enzymes of steroid hormone synthesis change, eventually lead to the serum hormone level disordered.
有机锡化合物(Organotin compounds, OTs)广泛用于船舶表面防生物附着涂料、杀虫剂、防腐剂(木材、纤维及皮革)、工业杀菌剂、塑料制品稳定剂等。由于应用广泛、环境中残存时间长,OTs已对全球环境(尤其是水体)造成了严重的污染。三丁基锡(Tributyltin, TBT)是OTs中最重要的一种,被认为是迄今为止由人为因素大量引入海水环境中最毒的化学品之一。它是一种确定的内分泌干扰物(ED),已有研究显示TBT对哺乳动物可产生免疫毒性、神经毒性、生殖毒性等。但这些研究所采用的染毒剂量都较大,而许多研究表明ED的危害并不呈线性剂量-反应关系,低剂量时的危害有可能要高于其高剂量时的危害。我们课题组的前期研究发现围产期低剂量氯化三丁基锡(TBTCl)暴露可导致子鼠睾丸中雌激素水平紊乱,附睾中精子数量减少及精子活度降低,睾丸组织出现病理性变化,并伴随着芳香酶含量的下降。目前又(?)TBTCl导致子代雄性小鼠发生以上变化的机制并不清楚,本研究在前期研究的基础上以子代雄性小鼠为主要研究对象,并添加己烯雌酚(DES)组作为阳性对照,对TBTCl影响妊娠结果、子鼠性发育及身体发育和血清中激素水平及其机制在分子和基因水平上做初步的探索。为研究TBTCl对人类的内分泌干扰作用及作用机制提供参考和借鉴。
研究目的：
探讨围产期低剂量TBTCl暴露对妊娠结果、子鼠发育和血清中激素水平的毒性作用,并且对其作用机制做初步的探索。
研究方法：
将受孕的KM小鼠随机分为阴性对照组(玉米油)、10、100μg/kgTBTCl组和阳性对照组(5μg/kg DES),每组6只。母鼠妊娠第6天(GD6)起,经口灌胃染毒,每天在同一时间染毒1次,染毒剂量为1ml/kg,直到哺乳期结束,即子鼠出生第21天(Postnatal day, PND21)。GD0、GD6、PND1和PND21时称量母鼠体重,染毒后开始观察并记录母鼠的流产及死亡情况；母鼠生产后,观察并记录出生子鼠的数量及雌雄比例,观察死亡情况并记录每组子鼠在哺乳期死亡的数量。PNDO、1、2、3、4、6、7、14和21时称量雄性子鼠体重,测量其肛门与生殖器之间的距离(AGD)。PND6时开始观察长毛情况,PND7时开始观察门牙萌生情况,PND12时开始观察睁眼情况,PND13时开始观察睾丸下降情况及PND28时开始观察包皮分离情况并分别记录其日龄。
PND22和100时每组随机抽取10只雄性子鼠(阳性对照取8只),称重后摘眼球取血,离心取血清并保存；分离睾丸、附睾、脑、肝脏、脾脏、肾脏和胸腺(PND100时还取腹侧前列腺、精囊),并称重；将睾丸立即放入液氮中速冻并置于-80℃保存。应用酶联免疫吸附试验方法测定PND22和PND100雄性子鼠血清中雌激素(E2)、睾酮(T)、游离睾酮(F-T)、促卵泡激素(FSH)和黄体生成素(LH)的浓度。采用Real-time PCR法检测PND22子代小鼠睾丸组织中Cypllal、Cyp17a、 Hsd3b2、Hsdl7b3和CyP19五个基因mRNA相对表达量,并用亚硫酸盐直接测序法(BSP)检测调控芳香酶表达的cAMP反应元件结合蛋白、泛素/蛋白酶体亚基α2和雌激素受体β基因启动子区域CpG岛的甲基化情况。
研究结果
剂量组孕鼠的流产(死亡)率与阳性对照组相比显著降低,与阴性对照组相比有增加的趋势；100μg/kg TBTC1组母鼠在PND21时体重较对照组均显著增加,PND1-21(?)PGD0-PND21母鼠体重增加量与对照组相比均显著增加,GD6-PND21母鼠体重增加量较阴性对照组显著升高；剂量组子鼠的数量较对照组有下降的趋势；100μg/kg TBTCl组和阳性对照组幼鼠死亡数与阴性对照组相比均显著增加,而阳性对照组和100ug/kg TBTCl组幼鼠死亡数相比没有统计学意义。
剂量组睾丸下降时间与阳性对照组相比显著提前,而与阴性对照组相比没有差异,阳性对照组与阴性对照组相比显著延迟。PNDO、1时100μg/kg TBTCl组的NAGD较阴性对照组显著降低,体重显著升高；PND3、6、14和21时TBTCl组NAGD与对照组相比有降低的趋势。PND0、1、2、6、7和21时100μg/kg组体重与阴性对照组相比显著增加,PND4-6、PNDO-7、PND14-21、PNDO-21的体重增加量与阴性对照组相比均显著增加。PND22时100μg/kg组睾丸脏器系数较对照组均显著降低；剂量组肝脏脏器系数与对照组相比均显著降低,脾脏重量及脏器系数与对照组相比显著增加,肾脏重量及脏器系数与阴性对照组相比显著增加。PND100时TBTCl组睾丸重量与阴性对照组相比显著升高,而与阳性对照组相比则显著降低；100μg/kg组精囊脏器系数与阴性对照组相比显著降低,肝脏脏器系数与阴性对照组相比显著升高；胸腺脏器系数与阴性对照组相比显著降低,与阳性对照组相比则显著升高。
子代雄性小鼠血清中激素测量结果显示,PND22时100μg/kg组的E2和FSH浓度与对照组相比均显著降低；PND100时100μg/kg组血清中E2的水平与阴性对照组相比仍显著降低,而与阳性对照组相比则没有差异,阳性对照组血清中E2的水平与阴性对照组相比也显著降低。
PND22子代雄性小鼠睾丸组织中类固醇激素合成途径关键酶mRNA(?)目对定量结果显示,10μg/kg组Cyp17a基因mRNA表达量与对照组相比均显著升高,Hsd3b2基因mRNA表达量与阴性对照组相比显著升高,而与阳性对照组相比则没有统计学差异；100μg/kg组Hsd17b(?)基因和(?)Cyp19(?)基因mRNA表达量与阴性对照组相比显著降低,剂量组Cyp19基因mRNA表达量有降低的趋势,而与阳性对照组相比则没有统计学差异；阳性对照组Hsd17b(?)基因和Cyp19(?)基因的mRNA表达量与阴性对照组相比有降低的趋势,但是没有统计学差异；Cypllal基因在各染毒组中mRNA表达量与对照组相比均无统计学差异。
PND22子代雄性小鼠睾丸组织中甲基化克隆测序结果发现只有cAMP反应元件结合蛋白(CREB)启动子区域基因序列发生甲基化改变,而泛素/蛋白酶体亚基α2和雌激素受体β基因启动子区域序列未发现甲基化情况。
结论
围产期低剂量TBTCl暴露导致母鼠流产增多、导致肥胖及食仔,导致雄性子鼠出现雌性化的趋势,并影响子鼠生长和器官发育。TBTCl可通过影响CREB启动子区域基因序列发生甲基化,类固醇激素合成途径中关键酶mRNA的表达水平改变,而导致血清中激素水平紊乱。
Backgroud:
Organotin compounds (OTs) has been widely used as painting in ship surface coating for preventing biofouling, pesticides, preservatives (wood, fiber and leather), industrial microbicides, plastic stabilizer, etc. Due to the wide application and long time existence in the environment, OTs has caused serious pollution on the global environment (especially water). Tributyltin (TBT) is one of the most important one of OTs, and it is considered as one of the most toxic chemicals in the environment intro-duced by man-made factors by far. TBT is a kind of certain Endocrine disruptor (ED), the existed studies have shown that TBT to the mammals can cause immunotoxicity, neurotoxicity and reproductive toxicity, etc. However, the dose of these study used is high, and many studies have shown that the damage of ED is not linear dose response relationship, some time the harm of low doses may be higher than the high doses. Our research group have studied and found that perinatal low doses of TBTCl can disturb the level of estrogen in mice testis, result in the decrease of sperm counts, the sperm motility reduced, the pathological changes of testicular tissue occurred in epididymis and the aromatase levels decreased in testis. While we have not known the mechanism of above changes up to now, so we take the progeny male mice as the main research object on the basis of previous research in this study, add the Diethylstilbestrol (DES) group as the positive control, study the influence of TBTCl on the pregnancy out-comes of the pregnant mice, the sex and body development of the progeny male mice and the sex hormone levels in the serum, and do the preliminary exploration on the mechanisms of which at the molecular and gene level. We hope our study can provide the reference for the study on TBTCl disrupting the human endocrine balance and its mechanism.
Objective:
To discuss the toxic effects of perinatal exposure to low doses of TBTCl to the pregnancy outcomes, progeny male mice growth and sex hormone levels in serum, and do a preliminary exploration on its mechanism.
Methods:
The pregnant KM mice are randomly divided into4groups,6in each group, they are given corn oil as the negative control group,10,100μg/kg TBTC1and5μg/kg DES as the positive control group by gavage from days6of gestation to the end of lacation(Postnatal21day, PND21). We infect one time every day at the same time and the dose is1ml/kg. At the time of GDO, GD6, PND1and PND21the weight of the mothers are weighed, and then we begin to observe and record the condition of the abortion and death of the infected mother. After the mother cub, we begin to observe and record the number of birth and the sex ratio. We also need to record the number of deaths of each group of the offspring in lactation. We weigh the body weight of the offspring on PNDO,1,2,3,4,6,7,14and21, and measure the distance between the anus and genitals (AGD) of the male mice. Beginning to examine the hair growing on PND6, the incisors drop-in on PND7, the eye opening on PND12, the testicular des-cent on PND13and the preputial separation on PND28, and we must record their age respectively.
On PND22and100, respectively,10male offspring mice (The positive control group is8.) were selected in each group randomly and killed after weighed and trunk blood was collected. The blood was let stand at room temperature for2hours and then centrifugaing for the serum and save it. The testicles, epididymis, brain, liver, spleen, kidney and thymus (the ventral prostate and seminal vesicle also were taken on PND100) were separated and weighed for account of viscera coefficient. The testis were immediately put into liquid nitrogen for preservation at-80℃. Using the method of enzyme-linked immunosorbent assay measure the concentration of estro-gen (E2), testosterone (T), free testosterone (F-T), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in serum on PND22and PND100. Adopting the Real-time PCR method to detect the relative transcript level of Cyp11al, Cyp17a, Hsd3b2, Hsd17b3and Cyp19gene mRNA in offspring mice testicular tissue on PND22. And the detection of the methylation pattern of CpG islands in promter region of cAMP response element binding protein, proteasome subunit alpha2and estrogen receptor beta gene who can control aromatase expression by bisulfite sequencing polymerase chain reaction (BSP).
Results:
Comparing with the positive control group, the dosage group's abortion (death) rate of the pregnant mice is significantly decreased, while with the negative control group has an increasing trend. On PND21, the weight of the pregnant mice is signify-cantly higher than the control group, the body weight gain of the pregnant mice is significantly higher than the control group during PND1-21and GD0-PND21, and during GD6-PND21the body weight gain of the pregnant mice is significantly incr-eased compared with the negative control group in100μg/kg TBTCl group. The number of TBTCl group's offspring has a downtrend compared with the control group. In100μg/kg TBTCl and the positive control group the mortality of mouse pup are significantly increased compared with the negative control group, but there is no statistical significance between the positive control group and the100μg/kgTBTCl group.
The time of testicular descent of TBTCl group is significantly ahead of time compared with positive control group, but no statistical differences compared with negative control group, and the testicular descent time of the positive control group is significantly delayed compared with the negative control group. On PND0and1, comparing with the negative control group the NAGD is significantly reduced, at the same time the weight is also significantly increased in100μg/kgTBTCl, while there is only a tendency to reduce compared with control group, but there is no statistical difference on PND3,6,14and21. In100μg/kg TBTCl group the body weight of the offspring is significantly increased compared with negative control group on PNDO,1,2,6,7and21, the body weight gain is significantly increased compared with the negative control group during PND4-6, PND0-7, PND14-21, PND0-21. On PND22the viscera coefficient of testicular is significantly lower than the control group in100μg/kg TBTCl group; In TBTCl group the Viscera coefficient of Liver is signifi-cantly reduced compared with control group, while the weight and viscera coefficient of Kidney are significantly higher than negative control group and the weight and viscera coefficient of Spleen are also higher than the control group. On PND100, the testicular weight of TBTC1group is significantly increased compared with negative control group, but is significantly reduced compared with positive control group; in100μg/kgTBTCl group the viscera coefficient of Seminal vesicle is significantly lower than negative control group, but the liver is significantly higher; The viscera coefficient of Thymus was significantly decreased compared with negative control group, while was significantly increased compared with positive control group; The viscera coefficient of thymus of positive control group is significantly lower than negative control group.
The measurement results of hormone in male offspring mice serum show that in100μg/kg TBTC1group the concentration of E2and FSH are significantly lower than the control group on PND22, and on PND100the serum E2levels is also significantly decreased compared with negative control group, while compared with positive control group their is no statistically difference, and the serum E2level of positive control group is also significantly reduced compared with negative control group.
On PND22, the key enzymes in offspring testis tissue of steroid hormones mRNA relative quantitative results show that the mRNA expression amount of Cyp17a gene is significantly higher than the control group, Hsd3b2gene is signifycantly in-creased compared with negative control group, but their is no significant differences compared with positive control group in10μg/kg TBTC1group; the mRNA express-ion amount of Hsdl7b3and Cypl9genes are significantly lower than negative control group in100ug/kg TBTC1group, and the infected group's mRNA expression amount of Cyp19gene has a tendency to reduce, while their is no significant different-ce compared with positive control group; the mRNA expression amount of Hsdl7b3gene and Cyp19gene in positive control group have a decreased trend compared with negative control group, but no statistical difference; Cypllal gene's mRNA express-ion in the infected group is no statistical difference compared with the control group.
On PND22, through the cloning sequencing comparison we find methylation only exists in the promoter region of cAMP response element binding protein (CREB) gene. However, the promoter region of the Ubiquitin/proteasome subunit alpha2and estrogen receptor beta gene do not find.
Conclusion
Perinatal exposure to low doses of TBTC1can result in the abortion of pregnant mice increased, lead to obesity and eat their own children. What's more, it also makes the male offspring feminized and affects the growth and organ development. Through the study of the mechanism we find in perinatal low-dose TBTC1treatment can influe-nee the methylation happened in the promoter region of CREB, lead to the mRNA ex-pression levels of key enzymes of steroid hormone synthesis change, eventually lead to the serum hormone level disordered.
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