本文選題：苯并芘 + 多氯聯(lián)苯��； 參考：《復(fù)旦大學(xué)》2012年碩士論文

【摘要】：苯并芘(Benzo(a)pyrene;3,4-Benzypyrene; BaP)是一種多環(huán)芳香族碳?xì)浠衔?它主要存在于煤焦油,汽車尾氣,各類有機(jī)物產(chǎn)生的煙霧,燒烤的食物以及工業(yè)污水中。BaP具有雄性生殖毒性,它可以引起睪丸萎縮、精子數(shù)量及活動(dòng)能力下降,降低曲精小管的長度和重量以及減少血漿和睪丸內(nèi)的睪酮水平。多氯聯(lián)苯(Polychlorinated biphenyls, PCBs)是一類人工合成的工業(yè)有機(jī)化合物,被列為12種最為危險(xiǎn)的持續(xù)性有機(jī)污染物(Persistent organic pollutants, POPs)之一。PCBs的雄性生殖毒性和內(nèi)分泌干擾效應(yīng)已被大量研究證實(shí)。BaP和PCBs經(jīng)常共存于環(huán)境中,且在體外實(shí)驗(yàn)中PCB126可通過增強(qiáng)BaP的氧化損傷毒性而表現(xiàn)出與BaP作用的協(xié)同效應(yīng)。與PCB126同為二惡英類多氯聯(lián)苯的PCB169毒性略低于PCB126,我們的研究假設(shè)PCB169與BaP聯(lián)合暴露具有協(xié)同效應(yīng)。 在BaP的毒作用機(jī)制中,BaP進(jìn)入細(xì)胞微粒體中代謝形成環(huán)氧化苯并芘可與DNA共價(jià)結(jié)合形成DNA加合物可造成DNA損傷,而形成DNA加合物的靶點(diǎn)往往位于DNA的甲基化區(qū)域；另外,也有不少研究證實(shí),BaP暴露可引起DNA甲基化和組蛋白乙�；母淖儭Ｋ�,表觀遺傳修飾的改變很可能是BaP生殖毒性機(jī)制的重要組成部分。 新生期是甲基化形成和維持的關(guān)鍵時(shí)期,也是機(jī)體受化學(xué)物干擾的敏感期。因此,本研究以新生SD大鼠聯(lián)合暴露BaP和PCB169為模型,評價(jià)兩種化學(xué)物的近期和遠(yuǎn)期聯(lián)合生殖毒效應(yīng),同時(shí)探索表觀遺傳機(jī)制在BaP致生殖功能損傷中的作用。 出生當(dāng)天(Postnatal Day0, PNDO),將所有雄性SD大鼠混合后,重新分成10只/窩(組內(nèi)、組間體重變異小于10%)。在PND1,按窩別隨機(jī)分成對照和處理組,每組30只。從PND1開始連續(xù)7天,對照和處理組分別經(jīng)口給予等量的溶劑對照；或BaP5、10和25mg/kg BW;或PCB1690.25mg/kg BW,或PCB1690.25mg/kg聯(lián)合BaP5、10和25mg/kg BW.各組大鼠分別于染毒結(jié)束后24h(PND8)、青春期(PND35)和成年期(PND90),經(jīng)麻醉后處死,收集并保存血液、肝臟、睪丸及附睪。 結(jié)果一新生期大鼠BaP和PCB169聯(lián)合暴露影響成年精子數(shù)量及血清睪酮水平 BaP暴露后,到PND90,每日精子生成量在BaP10mg/kg,25mg/kg下降；PCB1690.25mg/kg暴露亦導(dǎo)致PND90的每日精子生成量降低；在BaP和PCB169聯(lián)合暴露的各個(gè)劑量組,每日精子生成量顯著降低。 BaP暴露后,在PND8和PND35, BaP10,25mg/kg劑量組引起血清中T水平降低,在PND90,僅BaP25mg/kg劑量抑制血清T水平;PCB1690.25mg/kg暴露在PND8, PND35和PND90均未引起血清T水平的變化；BaP和PCB169聯(lián)合暴露后,在PND8和PND35,各個(gè)劑量組的血清T水平均顯著降低,而在PND90, PCB169聯(lián)合BaP10mg/kg和PCB169聯(lián)合BaP25mg/kg劑量組血清T水平受到抑制。 BaP和PCB169對成年期精子數(shù)量和發(fā)育各階段血清睪酮水平的影響無協(xié)同作用。 結(jié)果二新生期大鼠BaP和PCB169暴露干擾Leydig cells中類固醇合成酶的表達(dá) 新生期BaP暴露后,在PND8和PND35, StARmRNA表達(dá)在BaP10mg/kg、25mg/kg劑量組下降,在PND90僅在BaP25mg/kg表達(dá)降低；17β-HSD mRNA的表達(dá)水平僅在PND8的BaP25mg/kg劑量組降低,在PND35和PND90未發(fā)生變化；BaP10mg/kg、25mg/kg劑量在PND8顯著降低P450c17mRNA水平,各劑量在PND35和PND90未引起P450cl7mRNA變化；P450scc,17β-HSD和AR的mRNA表達(dá)在PND8, PND35和PND90均無變化。 PCB1690.25mg/kg暴露后,StAR mRNA表達(dá)在PND8和PND35被抑制,在PND90無變化；P450scc、3p-HSD、P450c17、AR的mRNA表達(dá)水平在PND8下降,在PND35和PND90均未發(fā)生改變；17β-HSD的mRNA表達(dá)在PND8,PND35和PND90均無變化。 BaP和PCB169聯(lián)合暴露后,在PND8, StAR mRNA表達(dá)在所有劑量組均降低,在PND35和PND90僅在PCB169聯(lián)合BaP10mg/kg及PCB169聯(lián)合BaP25mg/kg劑量組降低；P450scc和P450c17的mRNA在PND8的各劑量組表達(dá)下降,在PND35僅PCB169聯(lián)合BaP25mg/kg劑量組被抑制,在PND90無變化；3p-HSD、17β-HSD及AR的mRNA表達(dá)在PND8所有劑量組下降,在PND35和PND90均未受影響。 BaP和PCB169僅在PND8對StAR和P450c17mRNA表達(dá)的影響具有協(xié)同作用,對其他類固醇合成酶表達(dá)的抑制在各個(gè)發(fā)育階段均無協(xié)同效應(yīng)。 結(jié)果三新生期大鼠BaP和PCB169暴露對StAR, P450c17及AR基因表觀遺傳修飾的影響 BaP暴露后,StAR啟動(dòng)子組蛋白H3K14的乙�；皆赑ND8, PND35和PND90的各劑量組均發(fā)生下降；StAR啟動(dòng)子組蛋白H3的乙�；絻H在PND8的BaP25mg/kg劑量下降,在PND35和PND90無變化；P450c17和AR啟動(dòng)子甲基化狀態(tài)PND8, PND35和PND90未發(fā)生改變。 PCB1690.25mg/kg暴露后,StAR組蛋白H3K14和H3乙�；郊癆R啟動(dòng)子甲基化水平在PND8, PND35和PND90均無影響；P450c17啟動(dòng)子Aval位點(diǎn)的甲基化水平在PND8升高,在PND35和PND90未發(fā)生變化。 BaP和PCB169聯(lián)合暴露后,StAR啟動(dòng)子組蛋白H3K14的乙酰化水平在PND8的各個(gè)劑量被顯著抑制,在PND35的PCB169聯(lián)合BaP1Omg/kg和PCB169聯(lián)合BaP25mg/kg劑量組降低,在PND90的PCB169聯(lián)合BaP25mg/kg劑量組下降；P450c17啟動(dòng)子的甲基化水平在PND8降低,在PND35和PND90無變化；StAR組蛋白H3乙�；胶虯R啟動(dòng)子的甲基化水平在發(fā)育各時(shí)段各劑量組均未發(fā)生改變。 BaP和PCB169的協(xié)同作用僅體現(xiàn)在PND8對StAR啟動(dòng)子組蛋白H3K14乙酰化的抑制方面。 結(jié)論 新生期大鼠BaP暴露可抑制睪丸間質(zhì)細(xì)胞中睪酮合成限速酶系的mRNA表達(dá),在發(fā)育過程中可通過誘導(dǎo)StAR mRNA表達(dá)的持續(xù)下降,來持續(xù)抑制血清中的T水平,進(jìn)而在成年期導(dǎo)致精子生成能力降低。BaP對StAR基因啟動(dòng)子組蛋白H3K14乙酰化水平的持續(xù)抑制是StAR mRNA表達(dá)持續(xù)下降的原因之一。BaP與PCB169聯(lián)合暴露可在比BaP單獨(dú)暴露更低的劑量下產(chǎn)生明顯的毒效應(yīng),但兩種化學(xué)物的協(xié)同效應(yīng)不明顯。
[Abstract]:Benzopyrene (Benzo (a) pyrene; 3,4-Benzypyrene; BaP) is a polycyclic aromatic hydrocarbon, which mainly exists in coal tar, automobile exhaust, smoke produced by various kinds of organic substances, the roasted food and the male reproductive toxicity of.BaP in industrial sewage. It can cause the testicular atrophy, the decrease of sperm quantity and activity, and reduce the essence of semen. The length and weight of the tubules and the reduction of testosterone levels in plasma and testicles. Polychlorinated biphenyls (PCBs) is a synthetic industrial organic compound, which is listed as the male reproductive toxicity and endocrine stem of the 12 most dangerous persistent organic pollutants (Persistent organic pollutants, POPs).PCBs. The disturbing effect has been confirmed by a large number of studies that.BaP and PCBs often coexist in the environment, and in vitro PCB126 can exhibit synergistic effects with BaP by enhancing the oxidative damage toxicity of BaP. The PCB169 toxicity of PCB126 and dioxin polychlorinated biphenyls is slightly lower than PCB126. Our study assumes that PCB169 and BaP are associated with CO exposure. The same effect.
In the toxic mechanism of BaP, BaP enters the cell microsomes to form the epoxidation of benzopyrene and can covalent and combine with DNA to form DNA adducts to cause DNA damage. The target of DNA adducts is often located in the methylation area of DNA. In addition, there are many studies confirmed that BaP storm dew can cause the modification of DNA methylation and histone acetylation. Therefore, epigenetic modification is likely to be an important part of the reproductive toxicity mechanism of BaP.
The new stage is a critical period for the formation and maintenance of methylation. It is also a sensitive period for the organism to be disturbed by chemical substances. Therefore, in this study, the combined exposure of BaP and PCB169 in newborn SD rats was used to evaluate the short-term and long-term combined effects of the two chemicals, and the effect of epigenetic mechanism on the damage of reproductive function caused by BaP was also explored.
On the day of birth (Postnatal Day0, PNDO), all male SD rats were mixed and divided into 10 / nests (within the group, the body weight variation was less than 10%). In PND1, each group was randomly divided into control and treatment groups according to the nests, 30 in each group. For 7 days from PND1, the control and treatment groups were given the same amount of solvent control; or BaP5,10 and 25mg/kg BW; or PC. B1690.25mg/kg BW, or PCB1690.25mg/kg combined with BaP5,10 and 25mg/kg BW. rats were treated with 24h (PND8), puberty (PND35) and adulthood (PND90) after the end of the poisoning. After anesthesia, the rats were sacrificed to collect and preserve the blood, liver, testis and epididymis.
Results the combined exposure of BaP and PCB169 affects the number of adult sperm and serum testosterone.
After BaP exposure, to PND90, the daily sperm production was reduced in BaP10mg/kg, 25mg/kg, and PCB1690.25mg/kg exposure also led to a decrease in the daily sperm production of PND90, and the daily sperm production decreased significantly in each dose group of BaP and PCB169 exposure.
After BaP exposure, the level of serum T decreased in the dose group of PND8 and PND35, and in PND90, the serum T level was inhibited by BaP25mg/kg dose only. PCB1690.25mg/kg exposure in PND8, PND35 and PND90 did not cause changes in serum levels. However, serum T levels were inhibited in PND90, PCB169 combined with BaP10mg/kg and PCB169 combined with BaP25mg/kg dose group.
BaP and PCB169 had no synergistic effect on the number of adult sperm and the serum testosterone levels at various stages of development.
Results two BaP and PCB169 exposure in neonatal rats interfered with the expression of steroid synthase in Leydig cells.
After BaP exposure, in PND8 and PND35, the expression of StARmRNA in BaP10mg/kg, 25mg/kg dose group decreased and the expression of PND90 only decreased in BaP25mg/kg, and the expression level of 17 beta -HSD mRNA decreased only in PND8 BaP25mg/kg dose group. The amount of PND35 and PND90 did not cause P450cl7mRNA change; mRNA expression of P450scc, 17 beta -HSD and AR did not change in PND8, PND35 and PND90.
After PCB1690.25mg/kg exposure, the expression of StAR mRNA was suppressed in PND8 and PND35, and there was no change in PND90; the mRNA expression level of P450scc, 3p-HSD, P450c17, AR was decreased in PND8 and not changed.
After the combined exposure of BaP and PCB169, the expression of StAR mRNA decreased in all dose groups and decreased in PND35 and PND90 only in PCB169 combined BaP10mg/kg and PCB169 combined BaP25mg/kg dose group, and the expression of P450scc and StAR decreased in each dose group. 3 P-HSD, mRNA expression of 17 beta -HSD and AR decreased in all dose groups of PND8, but not in PND35 and PND90.
BaP and PCB169 have synergistic effects only on the effects of PND8 on the expression of StAR and P450c17mRNA, and there is no synergistic effect on the inhibition of the expression of other steroid synthetases at various developmental stages.
Results the effects of BaP and PCB169 exposure on epigenetic modification of StAR, P450c17 and AR genes in three new born rats
After BaP exposure, the acetylation level of the StAR promoter H3K14 was decreased in all the doses of PND8, PND35 and PND90, and the acetylation level of the StAR promoter protein H3 was only decreased in the PND8 BaP25mg/kg dose, and there was no change in PND35 and PND90.
After PCB1690.25mg/kg exposure, the level of StAR histone H3K14 and H3 acetylation and the methylation level of AR promoter had no effect on PND8, PND35 and PND90, and the level of methylation at the Aval loci of P450c17 promoter increased in PND8, and did not change at the PND35 and the Aval.
After the combined exposure of BaP and PCB169, the level of the acetylation of the StAR promoter protein H3K14 was significantly inhibited at every dose of PND8, and decreased in the PND35 PCB169 combined BaP1Omg/kg and PCB169 joint BaP25mg/kg dose group, and decreased in the PND90 PCB169 joint dose group. 0 there was no change; StAR histone H3 acetylation level and AR promoter methylation level did not change in all dose groups at all stages of development.
The synergistic effect of BaP and PCB169 is only reflected in the inhibition of PND8 on StAR promoter histone H3K14 acetylation.
conclusion
BaP exposure in neonatal rats can inhibit the mRNA expression of testosterone synthesis rate limiting enzyme system in Leydig cells. In the process of development, it can continue to inhibit the T level in serum by inducing the continuous decline of StAR mRNA expression, and then in adulthood, spermatogenesis can be reduced by.BaP to the level of H3K14 acetylation of the StAR gene promoter. Persistent inhibition is one of the reasons for the continuous decline in the expression of StAR mRNA. The combined exposure of.BaP and PCB169 can produce significant toxic effects at a lower dose than BaP, but the synergistic effect of the two chemicals is not obvious.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114
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本文編號：1931393