对羟基苯甲酸丁酯妊娠哺乳期暴露对雄性子代的生殖内分泌毒性及机制研究
发布时间：2018-08-24 12:01
【摘要】：对羟基苯甲酸酯类物质(parabens, PB)是一类对羟基苯甲酸的烷基化合物,作为防腐抗菌剂广泛应用于食品、化妆品、药品行业。以往对羟基苯甲酸酯类物质被认为是安全有效的防腐抗菌剂,然而近年来研究发现多种对羟基苯甲酸酯类物质具有弱雌激素活性作用。并且研究发现人的生物样品血尿中都检测到对羟基苯甲酸酯类物质的存在,在乳腺癌患者的肿瘤细胞中同样存在该类物质。随着对羟基苯甲酸酯类物质的使用日益广泛,人类接触机会和水平也会逐渐增加,摄入途径日趋复杂,当前对该类物质进行安全性评价,确定其安全限值就显得尤为重要和迫切。由于对羟基苯甲酸丁酯的毒性特征研究并不多,相关的标准也是根据以往少有的研究的毒性数据而确定,对于对羟基苯甲酸酯丁酯(n-butylparaben,n-BP)的毒性和未观察到有害作用水平(no observed adverse effect level, NOAEL)也存在争论,这使得政府机构很难采取适当的监管对策。因而有必要对n-BP的毒性作用特征进行研究,确定毒性参数和安全限值,为制定卫生标准以及对该物质的管理提供科学依据,从而更好地保护人群的健康。 本研究选择选择妊娠哺乳期暴露为敏感模型,探讨n-BP对仔鼠的生殖内分泌毒性作用特征及其潜在的毒性作用机制。胎儿期或是新生儿期过多的生成或暴露于内源性激素,如睾酮(testosterone, T)、雌二醇(17β-estradiol, E2)等,能够产生不良的结局,影响生殖系统的发育和成年期的生殖功能。因此本研究对生命早期雄性n-BP暴露对内源性激素的合成代谢途径的影响。激素需要和相应的激素受体结合才能发挥生理效应,内分泌干扰物可以通过调节靶细胞的激素受体的表达进而介导生殖毒性,本研究同时也探讨激素受体在n-BP介导毒性过程中可能发挥的作用。表观遗传学修饰可能在胎源性疾病发挥重要的机制作用。本研究基于n-BP妊娠哺乳期暴露为研究模型初步探讨其对甲基化水平的影响。 1.对羟基苯甲酸丁酯妊娠哺乳期暴露对雄性大鼠生殖内分泌影响 本研究选择Wistar大鼠为研究对象,妊娠第7天(Gestational day, GD7)到出生后第21天(postnatalday, PND21)染毒n-BP,孕鼠随机分为5组,分别为玉米油对照组,64mg/kg剂量组,160mg/kg剂量组,400mg/kg剂量组,1000mg/kg剂量组。结果显示,n-BP染毒组肛门生殖器距离(anogenital distance, AGD)下降,包皮分离(preputial separation, PPS)时间延迟；在5个不同的时间点血清T显著下降,E2水平显著增加,生殖器官重量不同程度降低；仔鼠成年后附睾尾精子计数和睾丸每日精子生成量显著减少。根据剂量反应关系和统计学差异推测妊娠哺乳期暴露n-BP生殖毒性的最大无作用剂量为160mg/kg/day。本研究结果表明妊娠哺乳期暴露n-BP影响雄性子代生殖内分泌系统。 2.对羟基苯甲酸丁酯干扰子代激素合成及其受体机制探讨 本研究主要通过免疫印迹和荧光定量PCR技术,对睾丸内非受体途径,即T的合成途径分别进行研究。结果显示,n-BP染毒组类固醇激素急性调节蛋白(steroidogenic acute regulatory protein, StAR)的转录和蛋白水平显著减少,细胞色素P450侧链裂解酶(cytochrome cholesterol side chain cleavage, P450scc)转录水平减少而蛋白水平无显著变化。对E2的合成代谢进行研究,发现芳香化酶(Aromatase, Arom)表达显著增加,雌激素磺基转移酶(estrogen sulfotransferase, SultlEl)显著降低。本研究同时还研究了睾丸的受体途径。结果发现雌激素受体(estrogen receptor, ERa)表达相对增加,雄激素受体(androgen receptor, AR)表达减少。 3.对羟基苯甲酸丁酯对子代甲基化表征影响 本研究主要通过荧光定量PCR和重亚硫酸盐修饰后测序技术研究n-BP暴露对睾丸内甲基化转移酶(DNA methltransferases, Dnmts)的转录水平和ERa启动子区域特异甲基化的影响。结果显示,Dnmt3b显著增加,而Dnmtl呈现下降趋势。同时研究发现大鼠睾丸内ERa启动子区域甲基化率呈剂量相关的下降,在1000mg/kg/day具有显著差异。 结论(1)妊娠哺乳暴露n-BP对仔鼠具有生殖发育毒性,确定其NOAEL为160mg/kg/day。(2)n-BP暴露可引起循环雌激素的增加和睾酮的减少,睾丸甾类合成代谢途径功能受到干扰。其机制可能与StAR/Arom/SULT1E1相关。(3) n-BP通过增加睾丸内ERa发挥体内雌激素活性效应,此外还可能拮抗雄激素受体AR的作用。(4)妊娠哺乳期n-BP暴露影响幼成熟期睾丸内甲基化转移酶的水平,从而调控整体基因组的转录水平,抑制或增强下游基因和蛋白的表达。本研究表明ERα的启动子区域甲基化率减少趋势。可能是PND90ERa增加的间接证据。
[Abstract]:Parabens (PB) are alkyl compounds of p-hydroxybenzoic acid, which are widely used as antiseptics in food, cosmetics and pharmaceutical industries. In the past, parabens were considered as safe and effective antiseptics. However, in recent years, many kinds of parabens have been found. P-hydroxybenzoic acid esters are also found in cancer cells of breast cancer patients. With the widespread use of p-hydroxybenzoic acid esters, human exposure opportunities and levels will gradually increase, intake. It is very important and urgent to evaluate the safety of these substances and determine their safety limits. As there are not many studies on the toxicity of butyl p-hydroxybenzoate, the relevant standards are based on the toxicity data of rare studies in the past. For butyl p-hydroxybenzoate (n-butylparaben, n-butylparaben, n-butylben) The toxicity of BP and the level of no observed adverse effect (NOAEL) are also controversial, which makes it difficult for government agencies to take appropriate regulatory measures. It provides scientific basis for better protection of people's health.
In this study, the reproductive endocrine toxicity of n-BP on offspring rats was investigated by using the sensitive model of pregnant and lactating period exposure.Excessive production of or exposure to endogenous hormones, such as testosterone (T), estradiol (E2) during fetal or neonatal period can produce adverse effects. Consequently, this study investigated the effects of early male n-BP exposure on endogenous hormone biosynthesis and metabolism pathways. Hormone needs to bind to the corresponding hormone receptors in order to exert physiological effects. Endocrine disruptors can regulate the expression of hormone receptors in target cells. Epigenetic modifications may play an important role in the pathogenesis of fetal diseases. This study was based on the n-BP exposure during pregnancy and lactation to explore the effects of hormone receptors on methylation levels.
Effects of 1. paraben on reproductive endocrine in male rats during pregnancy and lactation
Wistar rats were randomly divided into five groups: corn oil control group, 64 mg / kg dose group, 160 mg / kg dose group, 400 mg / kg dose group, 1000 mg / kg dose group. The results showed that the distance between anus and genital organs in n-BP poisoning group was higher than that in control group. Angenital distance (AGD) decreased, preputial separation (PPS) delayed, serum T decreased, E2 level increased and reproductive organ weight decreased at 5 different time points, and sperm counts and daily testicular sperm production decreased significantly in adult offspring. The results showed that exposure to n-BP during pregnancy and lactation affected the reproductive endocrine system of male offspring.
Hormone synthesis and receptor mechanism of 2. pairs of n-butyl benzoate interference
The results showed that the transcription and protein levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (c) were significantly decreased in n-BP group. Ytochrome cholesterol side chain cleavage (P450scc) transcription levels decreased but protein levels did not change significantly. Studies on E2 metabolism showed that aromatase (Arom) expression increased significantly and estrogen sulfotransferase (SultlEl) decreased significantly. The results showed that estrogen receptor (ERa) expression was relatively increased, androgen receptor (AR) expression was decreased.
Effect of 3. butyl p hydroxybenzoate on the methylation of progeny
In this study, the effects of n-BP exposure on the transcriptional level of DNA methylation transferases (Dnmts) and the specific methylation of ERa promoter region in testis were studied by fluorescence quantitative PCR and resulfate-modified sequencing. The results showed that Dnmt3b increased significantly while Dnmtl decreased. The methylation rate of ERa promoter in pellets decreased in dose related manner, and there was a significant difference in 1000mg/kg/day.
CONCLUSIONS (1) Exposure to n-BP during pregnancy and lactation has reproductive and developmental toxicity in offspring mice, and its NOAEL is 160mg/kg/day. (2) Exposure to n-BP can increase circulating estrogen and decrease testosterone, and disturb the function of testosterone biosynthesis pathway. The mechanism may be related to StAR/Arom/SULT1E1. (3) n-BP exerts estrogen in vivo by increasing ERa in testis. 4. Exposure to n-BP during pregnancy and lactation affects the level of methyltransferase in the testis of young and mature rats, thereby regulating the transcriptional level of the whole genome and inhibiting or enhancing the expression of downstream genes and proteins. Potential. It may be indirect evidence for the increase of PND90ERa.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
本文编号：2200764
[Abstract]:Parabens (PB) are alkyl compounds of p-hydroxybenzoic acid, which are widely used as antiseptics in food, cosmetics and pharmaceutical industries. In the past, parabens were considered as safe and effective antiseptics. However, in recent years, many kinds of parabens have been found. P-hydroxybenzoic acid esters are also found in cancer cells of breast cancer patients. With the widespread use of p-hydroxybenzoic acid esters, human exposure opportunities and levels will gradually increase, intake. It is very important and urgent to evaluate the safety of these substances and determine their safety limits. As there are not many studies on the toxicity of butyl p-hydroxybenzoate, the relevant standards are based on the toxicity data of rare studies in the past. For butyl p-hydroxybenzoate (n-butylparaben, n-butylparaben, n-butylben) The toxicity of BP and the level of no observed adverse effect (NOAEL) are also controversial, which makes it difficult for government agencies to take appropriate regulatory measures. It provides scientific basis for better protection of people's health.
In this study, the reproductive endocrine toxicity of n-BP on offspring rats was investigated by using the sensitive model of pregnant and lactating period exposure.Excessive production of or exposure to endogenous hormones, such as testosterone (T), estradiol (E2) during fetal or neonatal period can produce adverse effects. Consequently, this study investigated the effects of early male n-BP exposure on endogenous hormone biosynthesis and metabolism pathways. Hormone needs to bind to the corresponding hormone receptors in order to exert physiological effects. Endocrine disruptors can regulate the expression of hormone receptors in target cells. Epigenetic modifications may play an important role in the pathogenesis of fetal diseases. This study was based on the n-BP exposure during pregnancy and lactation to explore the effects of hormone receptors on methylation levels.
Effects of 1. paraben on reproductive endocrine in male rats during pregnancy and lactation
Wistar rats were randomly divided into five groups: corn oil control group, 64 mg / kg dose group, 160 mg / kg dose group, 400 mg / kg dose group, 1000 mg / kg dose group. The results showed that the distance between anus and genital organs in n-BP poisoning group was higher than that in control group. Angenital distance (AGD) decreased, preputial separation (PPS) delayed, serum T decreased, E2 level increased and reproductive organ weight decreased at 5 different time points, and sperm counts and daily testicular sperm production decreased significantly in adult offspring. The results showed that exposure to n-BP during pregnancy and lactation affected the reproductive endocrine system of male offspring.
Hormone synthesis and receptor mechanism of 2. pairs of n-butyl benzoate interference
The results showed that the transcription and protein levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (c) were significantly decreased in n-BP group. Ytochrome cholesterol side chain cleavage (P450scc) transcription levels decreased but protein levels did not change significantly. Studies on E2 metabolism showed that aromatase (Arom) expression increased significantly and estrogen sulfotransferase (SultlEl) decreased significantly. The results showed that estrogen receptor (ERa) expression was relatively increased, androgen receptor (AR) expression was decreased.
Effect of 3. butyl p hydroxybenzoate on the methylation of progeny
In this study, the effects of n-BP exposure on the transcriptional level of DNA methylation transferases (Dnmts) and the specific methylation of ERa promoter region in testis were studied by fluorescence quantitative PCR and resulfate-modified sequencing. The results showed that Dnmt3b increased significantly while Dnmtl decreased. The methylation rate of ERa promoter in pellets decreased in dose related manner, and there was a significant difference in 1000mg/kg/day.
CONCLUSIONS (1) Exposure to n-BP during pregnancy and lactation has reproductive and developmental toxicity in offspring mice, and its NOAEL is 160mg/kg/day. (2) Exposure to n-BP can increase circulating estrogen and decrease testosterone, and disturb the function of testosterone biosynthesis pathway. The mechanism may be related to StAR/Arom/SULT1E1. (3) n-BP exerts estrogen in vivo by increasing ERa in testis. 4. Exposure to n-BP during pregnancy and lactation affects the level of methyltransferase in the testis of young and mature rats, thereby regulating the transcriptional level of the whole genome and inhibiting or enhancing the expression of downstream genes and proteins. Potential. It may be indirect evidence for the increase of PND90ERa.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114
【参考文献】
相关期刊论文 前2条
1 周超;张英姿;;雌激素代谢酶CYP1A1、COMT和SULT基因多态性的研究进展[J];滨州医学院学报;2011年01期
2 郑小梅;伍宁丰;;DNA甲基化作用的生物学功能[J];中国农业科技导报;2009年01期
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