【摘要】：對(duì)羥基苯甲酸酯類物質(zhì)(parabens, PB)是一類對(duì)羥基苯甲酸的烷基化合物,作為防腐抗菌劑廣泛應(yīng)用于食品、化妝品、藥品行業(yè)。以往對(duì)羥基苯甲酸酯類物質(zhì)被認(rèn)為是安全有效的防腐抗菌劑,然而近年來(lái)研究發(fā)現(xiàn)多種對(duì)羥基苯甲酸酯類物質(zhì)具有弱雌激素活性作用。并且研究發(fā)現(xiàn)人的生物樣品血尿中都檢測(cè)到對(duì)羥基苯甲酸酯類物質(zhì)的存在,在乳腺癌患者的腫瘤細(xì)胞中同樣存在該類物質(zhì)。隨著對(duì)羥基苯甲酸酯類物質(zhì)的使用日益廣泛,人類接觸機(jī)會(huì)和水平也會(huì)逐漸增加,攝入途徑日趨復(fù)雜,當(dāng)前對(duì)該類物質(zhì)進(jìn)行安全性評(píng)價(jià),確定其安全限值就顯得尤為重要和迫切。由于對(duì)羥基苯甲酸丁酯的毒性特征研究并不多,相關(guān)的標(biāo)準(zhǔn)也是根據(jù)以往少有的研究的毒性數(shù)據(jù)而確定,對(duì)于對(duì)羥基苯甲酸酯丁酯(n-butylparaben,n-BP)的毒性和未觀察到有害作用水平(no observed adverse effect level, NOAEL)也存在爭(zhēng)論,這使得政府機(jī)構(gòu)很難采取適當(dāng)?shù)谋O(jiān)管對(duì)策。因而有必要對(duì)n-BP的毒性作用特征進(jìn)行研究,確定毒性參數(shù)和安全限值,為制定衛(wèi)生標(biāo)準(zhǔn)以及對(duì)該物質(zhì)的管理提供科學(xué)依據(jù),從而更好地保護(hù)人群的健康。 本研究選擇選擇妊娠哺乳期暴露為敏感模型,探討n-BP對(duì)仔鼠的生殖內(nèi)分泌毒性作用特征及其潛在的毒性作用機(jī)制。胎兒期或是新生兒期過(guò)多的生成或暴露于內(nèi)源性激素,如睪酮(testosterone, T)、雌二醇(17β-estradiol, E2)等,能夠產(chǎn)生不良的結(jié)局,影響生殖系統(tǒng)的發(fā)育和成年期的生殖功能。因此本研究對(duì)生命早期雄性n-BP暴露對(duì)內(nèi)源性激素的合成代謝途徑的影響。激素需要和相應(yīng)的激素受體結(jié)合才能發(fā)揮生理效應(yīng),內(nèi)分泌干擾物可以通過(guò)調(diào)節(jié)靶細(xì)胞的激素受體的表達(dá)進(jìn)而介導(dǎo)生殖毒性,本研究同時(shí)也探討激素受體在n-BP介導(dǎo)毒性過(guò)程中可能發(fā)揮的作用。表觀遺傳學(xué)修飾可能在胎源性疾病發(fā)揮重要的機(jī)制作用。本研究基于n-BP妊娠哺乳期暴露為研究模型初步探討其對(duì)甲基化水平的影響。 1.對(duì)羥基苯甲酸丁酯妊娠哺乳期暴露對(duì)雄性大鼠生殖內(nèi)分泌影響 本研究選擇Wistar大鼠為研究對(duì)象,妊娠第7天(Gestational day, GD7)到出生后第21天(postnatalday, PND21)染毒n-BP,孕鼠隨機(jī)分為5組,分別為玉米油對(duì)照組,64mg/kg劑量組,160mg/kg劑量組,400mg/kg劑量組,1000mg/kg劑量組。結(jié)果顯示,n-BP染毒組肛門生殖器距離(anogenital distance, AGD)下降,包皮分離(preputial separation, PPS)時(shí)間延遲；在5個(gè)不同的時(shí)間點(diǎn)血清T顯著下降,E2水平顯著增加,生殖器官重量不同程度降低；仔鼠成年后附睪尾精子計(jì)數(shù)和睪丸每日精子生成量顯著減少。根據(jù)劑量反應(yīng)關(guān)系和統(tǒng)計(jì)學(xué)差異推測(cè)妊娠哺乳期暴露n-BP生殖毒性的最大無(wú)作用劑量為160mg/kg/day。本研究結(jié)果表明妊娠哺乳期暴露n-BP影響雄性子代生殖內(nèi)分泌系統(tǒng)。 2.對(duì)羥基苯甲酸丁酯干擾子代激素合成及其受體機(jī)制探討 本研究主要通過(guò)免疫印跡和熒光定量PCR技術(shù),對(duì)睪丸內(nèi)非受體途徑,即T的合成途徑分別進(jìn)行研究。結(jié)果顯示,n-BP染毒組類固醇激素急性調(diào)節(jié)蛋白(steroidogenic acute regulatory protein, StAR)的轉(zhuǎn)錄和蛋白水平顯著減少,細(xì)胞色素P450側(cè)鏈裂解酶(cytochrome cholesterol side chain cleavage, P450scc)轉(zhuǎn)錄水平減少而蛋白水平無(wú)顯著變化。對(duì)E2的合成代謝進(jìn)行研究,發(fā)現(xiàn)芳香化酶(Aromatase, Arom)表達(dá)顯著增加,雌激素磺基轉(zhuǎn)移酶(estrogen sulfotransferase, SultlEl)顯著降低。本研究同時(shí)還研究了睪丸的受體途徑。結(jié)果發(fā)現(xiàn)雌激素受體(estrogen receptor, ERa)表達(dá)相對(duì)增加,雄激素受體(androgen receptor, AR)表達(dá)減少。 3.對(duì)羥基苯甲酸丁酯對(duì)子代甲基化表征影響 本研究主要通過(guò)熒光定量PCR和重亞硫酸鹽修飾后測(cè)序技術(shù)研究n-BP暴露對(duì)睪丸內(nèi)甲基化轉(zhuǎn)移酶(DNA methltransferases, Dnmts)的轉(zhuǎn)錄水平和ERa啟動(dòng)子區(qū)域特異甲基化的影響。結(jié)果顯示,Dnmt3b顯著增加,而Dnmtl呈現(xiàn)下降趨勢(shì)。同時(shí)研究發(fā)現(xiàn)大鼠睪丸內(nèi)ERa啟動(dòng)子區(qū)域甲基化率呈劑量相關(guān)的下降,在1000mg/kg/day具有顯著差異。 結(jié)論(1)妊娠哺乳暴露n-BP對(duì)仔鼠具有生殖發(fā)育毒性,確定其NOAEL為160mg/kg/day。(2)n-BP暴露可引起循環(huán)雌激素的增加和睪酮的減少,睪丸甾類合成代謝途徑功能受到干擾。其機(jī)制可能與StAR/Arom/SULT1E1相關(guān)。(3) n-BP通過(guò)增加睪丸內(nèi)ERa發(fā)揮體內(nèi)雌激素活性效應(yīng),此外還可能拮抗雄激素受體AR的作用。(4)妊娠哺乳期n-BP暴露影響幼成熟期睪丸內(nèi)甲基化轉(zhuǎn)移酶的水平,從而調(diào)控整體基因組的轉(zhuǎn)錄水平,抑制或增強(qiáng)下游基因和蛋白的表達(dá)。本研究表明ERα的啟動(dòng)子區(qū)域甲基化率減少趨勢(shì)。可能是PND90ERa增加的間接證據(jù)。
[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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114

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本文編號(hào)：2200764