多溴联苯醚的生殖发育毒性研究
发布时间：2018-09-04 10:51
【摘要】：多溴联苯醚(Polybrominated Diphenyl Ethers, PBDEs)是新型持久性有机污染物。自Main KM等2007年首次报道隐睾症患儿的患病率与母乳中PBDEs浓度存在关联后,针对PBDEs的生殖毒性成为众多学者关注的研究热点。 目前,关于PBDEs暴露与人类生殖发育之间关系的人群研究资料非常有限。有关PBDEs生殖毒性的动物试验虽然不断增多,但针对青春期暴露于PBDEs对雄性生殖系统影响的系统报道较少,且所用染毒剂量远高于实际生活中人群的暴露水平,缺乏对应于人群暴露水平的低剂量毒性研究资料。 针对上述研究不足,本研究首先通过山东某滨海开发区建立的出生队列,就新生儿PBDEs暴露水平及其影响开展前瞻性研究；然后以人群暴露水平作为剂量参考,建立低剂量染毒动物模型,系统观察青春期前后低剂量BDE-209暴露对雄性生殖系统发育,特别是精子发育的影响；最后通过建立睾丸生殖细胞体外培养体系,探索PBDEs影响生殖健康的可能机制。 第一部分某地新生儿多溴联苯醚暴露水平调查及其生殖发育毒性的研究 目的：探讨新生儿体内PBDEs暴露水平,分析其暴露与新生儿宫内妊娠结局的相关性。 方法：选择山东省某滨海开发区孕妇作为研究对象,追踪随访孕妇妊娠过程及妊娠结局,同时对新生儿脐带血血清PBDEs水平进行测定,并对两者关系进行相关分析。 结果： 新生儿所有血清样本(n=176)均能检测到一种或多种PBDEs同系物。新生儿脐带血清BDE-100(r=0.607, P0.01)、BDE-47(r=0.352, P0.05)、 BDE-99(r=0.412,P0.05)水平与其母亲既往怀孕期间因胎儿不正常选择人工流产呈现明显的正相关。新生儿血清BDE-153水平与新生儿身长呈显著正相关(r=0.452,P0.01)。 结论：山东某滨海开发区新生儿体内普遍存在PBDEs暴露,新生儿体内PBDEs水平与其宫内不良妊娠发育结局存在一定的关联。 第二部分雄性大鼠青春期前后十溴联苯醚暴露的生殖发育毒性研究 目的：探讨青春期前后BDE-209暴露对雄性大鼠性发育、生殖器腺发育、精子发育及生殖内分泌功能的影响。 方法：24只SD雄性大鼠(4周龄)随机分为4组,以不同浓度BDE-209(0、2、10、50mg/kg.d)灌胃染毒,1次/d,连续染毒一个月。染毒结束后,采集样本并分析。 结果： 1、与对照组相比,50mg/kg BDE-209染毒组睾丸抗氧化酶SOD、GSH-Px以及标记酶AKP活性均较对照组明显下降(P0.05),睾丸病理切片可见空泡样变。但各染毒组在体重、肛殖距、阴茎包皮分离时间以及各脏器系数等方面未见明显改变。 2、BDE-209染毒后,精子活力呈现出随BDE-209浓度增高而明显下降的趋势(P0.05),其中50mg/kg组精子活力下降最为明显(P0.05)。BDE-209染毒组精子运动速率、前进运动精子率较对照组也呈现出下降趋势,其中50mg/kg组精子运动速率下降具有统计学意义(P0.05)。与此同时,BDE-209染毒组的精子畸形率较对照组明显增高(P0.05)。但未见BDE-209暴露对精子密度产生显著性改变。 3、BDE-209染毒后,血清睾酮、血清雌二醇、睾丸组织睾酮含量与对照组相比,均未见显著性改变(P0.05)。 结论：在本实验设计剂量下,青春期前后BDE-209暴露对大鼠睾丸的形态功能已产生一定的改变,尤其是对精子分化成熟过程产生了明显影响,引起精子畸形率增加,精子活力下降,精子运动速率降低。 第三部分体外培养睾丸支持细胞暴露于十溴联苯醚的毒性研究 目的：探讨BDE-209对睾丸支持细胞的毒性作用。 方法：选用18天龄SD大鼠,取睾丸支持细胞进行原代培养,给予BDE-209染毒24h,采用MTT法测定细胞浓度,同时利用Annexin V-FITC试剂盒检测支持细胞的凋亡情况。 结果： 1、大鼠睾丸支持细胞在培养36h后进入对数增长期,48-72h是适宜的染毒窗口期。不同浓度BDE-209染毒24h后,随染毒浓度增加,细胞存活率下降,其中当BDE-209浓度≥30μg/ml时,睾丸支持细胞的相对存活率较对照组明显降低(P0.05)。 2、与对照组相比,BDE-209染毒组支持细胞T-AOC、SOD呈现下降趋势,其中10、20μg/ml染毒组T-AOC、SOD活性较对照组明显下降(P0.05)。 3、BDE-209染毒24h后,睾丸支持细胞的凋亡率出现增高趋势,当BDE-209浓度≥20μg/ml时,睾丸支持细胞凋亡率较对照组显著增高(P0.05)。 结论：BDE-209对体外培养的睾丸支持细胞具有细胞毒性作用,BDE-209暴露可引起睾丸支持细胞出现凋亡。
[Abstract]:Polybrominated Diphenyl Ethers (PBDEs) is a new type of persistent organic pollutant. Since the incidence of cryptorchidism in children was first reported by Main KM in 2007, the reproductive toxicity of PBDEs in breast milk has become a research hotspot.
At present, there is very limited data on the relationship between PBDEs exposure and human reproductive development. Animal trials on the reproductive toxicity of PBDEs are increasing, but systematic reports on the effects of pubertal exposure to PBDEs on the male reproductive system are scarce, and the dose used is much higher than the exposure level in real life. Lack of data for low dose toxicity studies corresponding to population exposure levels.
In view of the insufficiency of the above studies, this study firstly carried out a prospective study on the level of PBDEs exposure and its effects in newborns through a birth cohort in a coastal development area of Shandong Province, and then established an animal model of low-dose exposure to BDE-209 before and after puberty, taking the level of population exposure as a dose reference, to systematically observe the effects of low-dose BDE-209 exposure on male puberty. The effects of PBDEs on reproductive system development, especially sperm development, and the possible mechanism of PBDEs on reproductive health were explored by establishing an in vitro culture system of testicular germ cells.
The first part is a survey of the exposure level of polybrominated diphenyl ethers in a newborn and its reproductive toxicity.
Objective: To investigate the level of PBDEs exposure in neonates and analyze the correlation between PBDEs exposure and intrauterine pregnancy outcome.
Methods: The pregnant women in a coastal development area of Shandong Province were selected as the research object. The pregnancy process and pregnancy outcome were followed up. The serum PBDEs levels of umbilical cord blood were measured and the relationship between them was analyzed.
Result:
One or more PBDEs homologues were detected in all neonatal serum samples (n = 176). The levels of BDE-100 (r = 0.607, P 0.01), BDE-47 (r = 0.352, P 0.05), BDE-99 (r = 0.412, P 0.05) in neonatal umbilical cord serum were positively correlated with maternal abnormal selection of induced abortion during previous pregnancy. Neonatal length was positively correlated (r=0.452, P0.01).
Conclusion: PBDEs are ubiquitous in newborns of a coastal development zone in Shandong Province. The level of PBDEs in newborns is related to the adverse pregnancy outcomes.
The second part is about the reproductive and developmental toxicity of ten brominated diphenyl ethers before and after puberty in male rats.
Objective: To investigate the effects of BDE-209 exposure on sexual development, genital gland development, sperm development and reproductive endocrine function in male rats before and after puberty.
METHODS: Twenty-four SD male rats (4 weeks old) were randomly divided into four groups. The rats were given different concentrations of BDE-209 (0,2,10,50 mg/kg.d) by gastric Administration for one month.
Result:
1. Compared with the control group, the activities of SOD, GSH-Px and AKP in testis of 50 mg/kg BDE-209 poisoning group were significantly lower than those of the control group (P 0.05). The pathological section of testis showed vacuole-like changes.
2. After BDE-209 exposure, the sperm motility decreased significantly with the increase of BDE-209 concentration (P 0.05), especially in the 50mg/kg group (P 0.05). Meanwhile, the sperm deformity rate of BDE-209 exposed group was significantly higher than that of the control group (P 0.05). However, there was no significant change in sperm density after exposure to BDE-209.
3. There were no significant changes in serum testosterone, serum estradiol and testosterone content in testicular tissue between BDE-209 and control group (P 0.05).
CONCLUSION: BDE-209 exposure before and after puberty has a certain effect on the morphology and function of rat testis, especially on the process of sperm differentiation and maturation, resulting in an increase in the rate of sperm deformity, a decrease in sperm motility and a decrease in sperm motility.
The third part is the study of the toxicity of Sertoli cells exposed to ten brominated diphenyl ethers in vitro.
Objective: To investigate the toxic effects of BDE-209 on Sertoli cells.
METHODS: Sertoli cells from 18-day-old SD rats were cultured for 24 hours after exposure to BDE-209. MTT assay was used to determine the concentration of Sertoli cells and Annexin V-FITC kit was used to detect the apoptosis of Sertoli cells.
Result:
1. Rat Sertoli cells entered the logarithmic growth phase after 36 hours of culture, and 48-72 hours was the appropriate exposure window period. After 24 hours of exposure to different concentrations of BDE-209, the cell survival rate decreased with the increase of the concentration of BDE-209. The relative survival rate of Sertoli cells decreased significantly when the concentration of BDE-209 was greater than 30 ug/ml (P 0.05).
2. Compared with the control group, the activity of T-AOC and SOD in Sertoli cells of BDE-209 exposed group showed a downward trend. The activity of T-AOC and SOD in 10,20 ug/ml exposed group was significantly lower than that in the control group (P 0.05).
3. After 24 hours of BDE-209 exposure, the apoptotic rate of Sertoli cells increased. When the concentration of BDE-209 was more than 20 ug/ml, the apoptotic rate of Sertoli cells increased significantly (P 0.05).
Conclusion: BDE-209 has cytotoxic effect on Sertoli cells cultured in vitro, and BDE-209 exposure can induce apoptosis of Sertoli cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114
本文编号：2221862
[Abstract]:Polybrominated Diphenyl Ethers (PBDEs) is a new type of persistent organic pollutant. Since the incidence of cryptorchidism in children was first reported by Main KM in 2007, the reproductive toxicity of PBDEs in breast milk has become a research hotspot.
At present, there is very limited data on the relationship between PBDEs exposure and human reproductive development. Animal trials on the reproductive toxicity of PBDEs are increasing, but systematic reports on the effects of pubertal exposure to PBDEs on the male reproductive system are scarce, and the dose used is much higher than the exposure level in real life. Lack of data for low dose toxicity studies corresponding to population exposure levels.
In view of the insufficiency of the above studies, this study firstly carried out a prospective study on the level of PBDEs exposure and its effects in newborns through a birth cohort in a coastal development area of Shandong Province, and then established an animal model of low-dose exposure to BDE-209 before and after puberty, taking the level of population exposure as a dose reference, to systematically observe the effects of low-dose BDE-209 exposure on male puberty. The effects of PBDEs on reproductive system development, especially sperm development, and the possible mechanism of PBDEs on reproductive health were explored by establishing an in vitro culture system of testicular germ cells.
The first part is a survey of the exposure level of polybrominated diphenyl ethers in a newborn and its reproductive toxicity.
Objective: To investigate the level of PBDEs exposure in neonates and analyze the correlation between PBDEs exposure and intrauterine pregnancy outcome.
Methods: The pregnant women in a coastal development area of Shandong Province were selected as the research object. The pregnancy process and pregnancy outcome were followed up. The serum PBDEs levels of umbilical cord blood were measured and the relationship between them was analyzed.
Result:
One or more PBDEs homologues were detected in all neonatal serum samples (n = 176). The levels of BDE-100 (r = 0.607, P 0.01), BDE-47 (r = 0.352, P 0.05), BDE-99 (r = 0.412, P 0.05) in neonatal umbilical cord serum were positively correlated with maternal abnormal selection of induced abortion during previous pregnancy. Neonatal length was positively correlated (r=0.452, P0.01).
Conclusion: PBDEs are ubiquitous in newborns of a coastal development zone in Shandong Province. The level of PBDEs in newborns is related to the adverse pregnancy outcomes.
The second part is about the reproductive and developmental toxicity of ten brominated diphenyl ethers before and after puberty in male rats.
Objective: To investigate the effects of BDE-209 exposure on sexual development, genital gland development, sperm development and reproductive endocrine function in male rats before and after puberty.
METHODS: Twenty-four SD male rats (4 weeks old) were randomly divided into four groups. The rats were given different concentrations of BDE-209 (0,2,10,50 mg/kg.d) by gastric Administration for one month.
Result:
1. Compared with the control group, the activities of SOD, GSH-Px and AKP in testis of 50 mg/kg BDE-209 poisoning group were significantly lower than those of the control group (P 0.05). The pathological section of testis showed vacuole-like changes.
2. After BDE-209 exposure, the sperm motility decreased significantly with the increase of BDE-209 concentration (P 0.05), especially in the 50mg/kg group (P 0.05). Meanwhile, the sperm deformity rate of BDE-209 exposed group was significantly higher than that of the control group (P 0.05). However, there was no significant change in sperm density after exposure to BDE-209.
3. There were no significant changes in serum testosterone, serum estradiol and testosterone content in testicular tissue between BDE-209 and control group (P 0.05).
CONCLUSION: BDE-209 exposure before and after puberty has a certain effect on the morphology and function of rat testis, especially on the process of sperm differentiation and maturation, resulting in an increase in the rate of sperm deformity, a decrease in sperm motility and a decrease in sperm motility.
The third part is the study of the toxicity of Sertoli cells exposed to ten brominated diphenyl ethers in vitro.
Objective: To investigate the toxic effects of BDE-209 on Sertoli cells.
METHODS: Sertoli cells from 18-day-old SD rats were cultured for 24 hours after exposure to BDE-209. MTT assay was used to determine the concentration of Sertoli cells and Annexin V-FITC kit was used to detect the apoptosis of Sertoli cells.
Result:
1. Rat Sertoli cells entered the logarithmic growth phase after 36 hours of culture, and 48-72 hours was the appropriate exposure window period. After 24 hours of exposure to different concentrations of BDE-209, the cell survival rate decreased with the increase of the concentration of BDE-209. The relative survival rate of Sertoli cells decreased significantly when the concentration of BDE-209 was greater than 30 ug/ml (P 0.05).
2. Compared with the control group, the activity of T-AOC and SOD in Sertoli cells of BDE-209 exposed group showed a downward trend. The activity of T-AOC and SOD in 10,20 ug/ml exposed group was significantly lower than that in the control group (P 0.05).
3. After 24 hours of BDE-209 exposure, the apoptotic rate of Sertoli cells increased. When the concentration of BDE-209 was more than 20 ug/ml, the apoptotic rate of Sertoli cells increased significantly (P 0.05).
Conclusion: BDE-209 has cytotoxic effect on Sertoli cells cultured in vitro, and BDE-209 exposure can induce apoptosis of Sertoli cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114
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