纳米二氧化钛暴露致小鼠生殖毒性及其基因表达的变化
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摘要
纳米氧化钛(TiO_2)是目前国际上生产量最大，在工业、医药卫生、日用化妆品、食品以及环保方面应用最广泛的一种纳米材料。然而随着纳米材料的广泛应用，包括纳米TiO_2在内的纳米材料的潜在环境卫生和职业卫生安全越来越受到人们的高度关注，已成为研究热点。2012年初，美国国家科学院国家研究理事会发布了“工程纳米材料的环境、健康与安全性的研究战略(A Research Strategy forEnvironmental, Health, and Safety Aspects of Engineered Nanomaterials)”报告，提出需要制定整体的研究计划，避免纳米技术快速发展带来的潜在风险(http://www8.nationalacademies.org/onpinews/newsitem.aspx?RecordID=13347)。近年来研究，不仅证实纳米TiO_2暴露可引起动物肝、脾、肺的损伤，尤其是肾和脑的损伤，而且阐明了相应的损伤机制,而肾和脑这两种器官又与生殖系统的功能密切相关。人们长期使用含有纳米TiO_2的化妆品及长期生活或工作在暴露有纳米颗粒的环境下(如生产车间的工人），其纳米颗粒通过皮肤渗透、呼吸和消化系统而进入体内，并可能通过血液循环、血睾屏障在卵巢和睾丸中积累，进而导致生殖系统损伤。但纳米TiO_2暴露对生殖系统的损伤及其机制研究尚不够深入。因此开展纳米TiO_2生殖系统毒性的研究具有非常重要的理论意义和实际意义。鉴于此，本论文以10mg/kg BW的纳米TiO_2（锐钛型5nm）连续灌胃处理雌性和雄性小鼠90天，对其生殖系统的损伤进行了研究；同时结合基因芯片技术深入研究了生殖系统损伤多基因共同作用的分子机制。本研究可为纳米材料长期暴露引起生殖系统的毒性评估提供重要的理论依据。
论文结果如下：
(1)用纳米TiO_2(10mg/kg)对雌性小鼠持续灌胃90天后，研究了雌性小鼠卵巢的损伤和卵巢基因表达特征。结果显示纳米TiO_2颗粒能够进入卵巢中积累并且导致卵巢的损伤如卵巢萎缩，初级卵泡和次级卵泡发育障碍，卵巢细胞的不规则排列和卵泡腔的形状不规则。纳米TiO_2颗粒也可进入卵巢细胞甚至细胞核，进而导致细胞凋亡。纳米TiO_2颗粒暴露造成了卵巢组织钙、钠、钾和锌金属元素的含量增加，而镁、铜和铁金属元素的含量下降。纳米TiO_2颗粒暴露导致了性激素的不平衡，如血清孕酮、促黄体生成素、睾酮和促卵泡激素的水平降低，雌二醇的浓度增加，进而引起生育力下降，如交配率、怀孕率、产仔数都显著下降及子代生长发育减慢。同时纳米TiO_2颗粒暴露引起了卵巢严重的氧化性损伤，如ROS大量积累和DNA氧化水平增加。微阵列分析显示，纳米TiO_2处理的小鼠卵巢组织中有223个已知功能基因上调(diffscore≥13, p<0.05)，65个已知功能基因下调(diffscore-13, p<0.05)。在本研究中，卵巢的差异基因的功能分别涉及细胞分化、免疫炎症反应、离子运输、代谢过程、氧化应激反应、内固醇代谢、凋亡、信号转导、信号通路、转录、运输等。尤其是与内固醇和性激素代谢过程有关的Akr1c18、Cyp17a1和Lgmn显著上调，这些基因的过表达引起雌二醇浓度上升，以及孕酮、促黄体生成素和睾酮浓度下降，进而抑制卵泡发育和降低雌性小鼠的生育力。另外，纳米TiO_2颗粒暴露后导致卵巢Cyp17a1的表达显著增加也可促进雌二醇的生物合成。
(2)分析了10mg/kg纳米TiO_2钛颗粒持续90天灌胃雄性小鼠后生殖毒性和基因表达谱变化。结果表明纳米TiO_2颗粒能够通过血睾屏障在睾丸中沉积，并且导致睾丸的损伤和细胞凋亡，如生精管空泡化、生精层的厚度减小，睾丸细胞的不规则排列和支持细胞的空泡化；附睾尾部的精子发生异常的变化，如总的精子浓度和精子活力下降，精子畸形率显著增加，精子损伤严重如头尾断裂；雄性小鼠的生育力显著下降，如低的交配率、妊娠率和少的产仔数。纳米TiO_2的暴露导致睾丸组织中钙和铁的含量下降，锌含量显著增加。同样，纳米TiO_2暴露也造成性激素的不平衡，如雌二醇和孕酮显著增加，而促卵泡激素、促黄体生成素和睾酮水平显著下降。微阵列分析显示与对照比较，纳米TiO_2处理组的小鼠睾丸组织有155个基因明显上调(diffscore≥13, p<0.05)，100个基因明显下调(diffscore-13,p<0.05)。对已知功能的差异基因分类，表明差异基因的功能涉及的范畴有精子发生、内固醇代谢过程、凋亡、免疫、细胞分化、氧化应激反应、氧化还原活性、激素活性、离子运输等。尤其是Spata19、Tdrd6和Tnp2表达的显著下调直接与精子发生的抑制有关，而Cyp2e1、Mvd、Sc4mol和Srd5a2与内固醇代谢有关,Cyp2e1表达下调，Mvd、Sc4mol和Srd5a2表达上调。
The dynamical development of the nanotechnology industry has led to thewide-scale production and application of nanomaterials. Among the variousnanomaterials, customarily titanium dioxide nanoparticles (TiO_2NPs), owing to theirlarger surface area to volume ratio and high reactivity, have been used as nontoxic,chemical inert and biocompatible pigment products or photocatalysts in industry,medicine and health, cosmetics, paint industries, and environmental protection.However, the potentially human toxicity and environmental impact of TiO_2NPs haveattracted considerable attention with their increased use in industrial applications. Inearly2012, the National Research Council of the National Academy of Sciences of USAreleased " environment, health and safety research strategy of engineered nanomaterials(A Research Strategy for Environmental, Health, and Safety Aspects of EngineeredNanomaterials)" report. The report presented the need for overall research program toavoid potential risks caused by fast development nanotechnology(http://www8.nationalacademies.org/onpinews/newsitem.aspx? RecordID=13347).Recent studies confirmed that the TiO_2NPs exposure can cause liver, kidney, spleen,lung, and brain damages in animals, and clarify their damage mechanisms, particularly,kidney and brain damages are closely related with function of the reproductive system.Long-term use containing TiO_2NPs cosmetics and long-term living or working in theenvironment (such as workers in workshop) exposure to TiO_2NPs, the NPs canpenetrate through the skin, respiratory and digestive system into the body, and possiblythrough blood circulation and blood-testis barrier are accumulated in the ovaries andtestes, which led to damage of the reproductive system. However, the damage by TiO_2NPs exposure and its mechanism needs to be addressed. Thus, the aims of the presentstudy were to test the hypothesis that TiO_2NPs exerts a toxic effect in the reproductivesystem of mice, and elucidate the synergistic molecular mechanisms of multiple genesactivated by TiO_2NPs-induced toxicity of reproductive system in animals and humans.In this study, ovarian and testicular damages and alterations in gene expression profiles in female and male mice induced by intragastric administration of10mg/kg bodyweight of TiO_2NPs for90consecutive days were examined. This study can provideimportant theoretical basis for assessment of reproductive toxicity for long-termexposure to nanomaterials.
Results are listed as follows:
(1) In the current study, ovarian injury and gene-expressed characteristics in femalemice induced by intragastric administration of TiO_2NPs (10mg/kg) for90consecutivedays were investigated. Our findings indicated that TiO_2NPs can accumulate in theovary and result in ovarian damage including ovarian atrophy, disturbance of primaryand second follicle development, irregular arrangement of cells, and a shapelessfollicular antrum. TiO_2NPs can enter ovarian cells and even the nuclei of ovarian cells,thus leading to apoptosis. TiO_2NPs exposure caused increases of calcium, sodium,potassium and zinc contents, and decreases of magnesium, copper, and iron contents, animbalance of sex hormones such as decreased levels of serum progesterone, luteinizinghormone, testosterone and follicle-stimulating, and increased concentration of estradiolin the ovary, which resulted in significant decreases in the mating rate, pregnancy rate,and number of fetuses, and inhibition of growth and development of offspring. TiO_2NPsexposure caused severe ovarian oxidative damage, such as overproduction of ROS andincrease level of DNA oxidation (8-OHdg). Microarray analysis showed that in ovariesfrom mice treated with TiO_2NPs compared to controls,223genes of known functionwere up-regulated(diffscore≥13, p <0.05), while65ovarian genes weredown-regulated(diffscore-13, p <0.05). In the present study, functions of differencegene in the ovaries were involved in cell differentiation, immune and inflammatoryresponse, ion transport, metabolic process, oxidative stress, the steroid metabolism,apoptosis, signal transduction, transcription, transport, etc. Especially Akr1c18,Cyp17a1and Lgmn that related to the steroids and sex hormone metabolism processsignificantly up-regulated, over-expression of these genes caused increased levels ofestradiol, progesterone, luteinizing hormone and decreased level of testosterone, therebyinhibited follicular development and reduced fertility of female mice. In addition, TiO_2NPs exposure resulted in a significant increase in the expression of ovarian Cyp17a1,which also can promote the biosynthesis of estradiol.
(2) In this study, the reproductive toxicity and testicular gene expression profilinginduced by intragastric administration of10mg/kg body weight TiO_2NPs for90 consecutive days were investigated. The results showed that TiO_2NPs can be depositedin the testicles, and cause testicular damage and apoptosis, such as rare sperm, spermbreakages, rarefaction of Sertoli cell and androgone, Sertoli cell apoptosis, androgonenecrosis of the seminiferous tubules, decreased germinative layer thickness, vacuolation,and irregular arrangement of Sertoli cells of the seminiferous tubules, significantdecreases in sperm numbers and sperm motility, and increased abnormal sperm, spermwas severely damaged, such as fracture of the head and tail in the cauda epididymis, thefertility of male mice significantly decreased, such as significant decreases in the matingrate, pregnancy rate, and number of fetuses. TiO_2NPs exposure led to the decline ofcalcium level and increases of iron, zinc contents in testicular tissue. Similarly, TiO_2NPs exposure also caused an imbalance of sex hormones, e.g., estradiol andprogesterone significantly increased, and levels of follicle-stimulating hormone,luteinizing hormone and testosterone were significantly decreased. Microarray analysisshowed that in testes from mice treated with TiO_2NPs compared to controls,155geneswere found to be up-regulated(diffscore≥13, p<0.05), while100genes were found to bedown-regulated (diffscore-13, p<0.05). Functions of difference genes were involved inspermatogenesis, steroid metabolic processes, apoptosis, immune, cell differentiation,oxidative stress, redox activity, hormone activity, ion transport. Of the altered geneexpressions, significantly down-regulated expressions of Spata19, Tdrd6and Tnp2weredirectly related with the suppression of spermatogenesis, and Cyp2e1, Mvd, Sc4mol andSrd5a2were related with steroid metabolic process. Cyp2e1was downregulated, Mvd,Sc4mol and Srd5a2were upregulated. These alterations of gene expressions impairedbalance of sex hormones, and decreased fertility of male mice.
论文结果如下：
(1)用纳米TiO_2(10mg/kg)对雌性小鼠持续灌胃90天后，研究了雌性小鼠卵巢的损伤和卵巢基因表达特征。结果显示纳米TiO_2颗粒能够进入卵巢中积累并且导致卵巢的损伤如卵巢萎缩，初级卵泡和次级卵泡发育障碍，卵巢细胞的不规则排列和卵泡腔的形状不规则。纳米TiO_2颗粒也可进入卵巢细胞甚至细胞核，进而导致细胞凋亡。纳米TiO_2颗粒暴露造成了卵巢组织钙、钠、钾和锌金属元素的含量增加，而镁、铜和铁金属元素的含量下降。纳米TiO_2颗粒暴露导致了性激素的不平衡，如血清孕酮、促黄体生成素、睾酮和促卵泡激素的水平降低，雌二醇的浓度增加，进而引起生育力下降，如交配率、怀孕率、产仔数都显著下降及子代生长发育减慢。同时纳米TiO_2颗粒暴露引起了卵巢严重的氧化性损伤，如ROS大量积累和DNA氧化水平增加。微阵列分析显示，纳米TiO_2处理的小鼠卵巢组织中有223个已知功能基因上调(diffscore≥13, p<0.05)，65个已知功能基因下调(diffscore-13, p<0.05)。在本研究中，卵巢的差异基因的功能分别涉及细胞分化、免疫炎症反应、离子运输、代谢过程、氧化应激反应、内固醇代谢、凋亡、信号转导、信号通路、转录、运输等。尤其是与内固醇和性激素代谢过程有关的Akr1c18、Cyp17a1和Lgmn显著上调，这些基因的过表达引起雌二醇浓度上升，以及孕酮、促黄体生成素和睾酮浓度下降，进而抑制卵泡发育和降低雌性小鼠的生育力。另外，纳米TiO_2颗粒暴露后导致卵巢Cyp17a1的表达显著增加也可促进雌二醇的生物合成。
(2)分析了10mg/kg纳米TiO_2钛颗粒持续90天灌胃雄性小鼠后生殖毒性和基因表达谱变化。结果表明纳米TiO_2颗粒能够通过血睾屏障在睾丸中沉积，并且导致睾丸的损伤和细胞凋亡，如生精管空泡化、生精层的厚度减小，睾丸细胞的不规则排列和支持细胞的空泡化；附睾尾部的精子发生异常的变化，如总的精子浓度和精子活力下降，精子畸形率显著增加，精子损伤严重如头尾断裂；雄性小鼠的生育力显著下降，如低的交配率、妊娠率和少的产仔数。纳米TiO_2的暴露导致睾丸组织中钙和铁的含量下降，锌含量显著增加。同样，纳米TiO_2暴露也造成性激素的不平衡，如雌二醇和孕酮显著增加，而促卵泡激素、促黄体生成素和睾酮水平显著下降。微阵列分析显示与对照比较，纳米TiO_2处理组的小鼠睾丸组织有155个基因明显上调(diffscore≥13, p<0.05)，100个基因明显下调(diffscore-13,p<0.05)。对已知功能的差异基因分类，表明差异基因的功能涉及的范畴有精子发生、内固醇代谢过程、凋亡、免疫、细胞分化、氧化应激反应、氧化还原活性、激素活性、离子运输等。尤其是Spata19、Tdrd6和Tnp2表达的显著下调直接与精子发生的抑制有关，而Cyp2e1、Mvd、Sc4mol和Srd5a2与内固醇代谢有关,Cyp2e1表达下调，Mvd、Sc4mol和Srd5a2表达上调。
The dynamical development of the nanotechnology industry has led to thewide-scale production and application of nanomaterials. Among the variousnanomaterials, customarily titanium dioxide nanoparticles (TiO_2NPs), owing to theirlarger surface area to volume ratio and high reactivity, have been used as nontoxic,chemical inert and biocompatible pigment products or photocatalysts in industry,medicine and health, cosmetics, paint industries, and environmental protection.However, the potentially human toxicity and environmental impact of TiO_2NPs haveattracted considerable attention with their increased use in industrial applications. Inearly2012, the National Research Council of the National Academy of Sciences of USAreleased " environment, health and safety research strategy of engineered nanomaterials(A Research Strategy for Environmental, Health, and Safety Aspects of EngineeredNanomaterials)" report. The report presented the need for overall research program toavoid potential risks caused by fast development nanotechnology(http://www8.nationalacademies.org/onpinews/newsitem.aspx? RecordID=13347).Recent studies confirmed that the TiO_2NPs exposure can cause liver, kidney, spleen,lung, and brain damages in animals, and clarify their damage mechanisms, particularly,kidney and brain damages are closely related with function of the reproductive system.Long-term use containing TiO_2NPs cosmetics and long-term living or working in theenvironment (such as workers in workshop) exposure to TiO_2NPs, the NPs canpenetrate through the skin, respiratory and digestive system into the body, and possiblythrough blood circulation and blood-testis barrier are accumulated in the ovaries andtestes, which led to damage of the reproductive system. However, the damage by TiO_2NPs exposure and its mechanism needs to be addressed. Thus, the aims of the presentstudy were to test the hypothesis that TiO_2NPs exerts a toxic effect in the reproductivesystem of mice, and elucidate the synergistic molecular mechanisms of multiple genesactivated by TiO_2NPs-induced toxicity of reproductive system in animals and humans.In this study, ovarian and testicular damages and alterations in gene expression profiles in female and male mice induced by intragastric administration of10mg/kg bodyweight of TiO_2NPs for90consecutive days were examined. This study can provideimportant theoretical basis for assessment of reproductive toxicity for long-termexposure to nanomaterials.
Results are listed as follows:
(1) In the current study, ovarian injury and gene-expressed characteristics in femalemice induced by intragastric administration of TiO_2NPs (10mg/kg) for90consecutivedays were investigated. Our findings indicated that TiO_2NPs can accumulate in theovary and result in ovarian damage including ovarian atrophy, disturbance of primaryand second follicle development, irregular arrangement of cells, and a shapelessfollicular antrum. TiO_2NPs can enter ovarian cells and even the nuclei of ovarian cells,thus leading to apoptosis. TiO_2NPs exposure caused increases of calcium, sodium,potassium and zinc contents, and decreases of magnesium, copper, and iron contents, animbalance of sex hormones such as decreased levels of serum progesterone, luteinizinghormone, testosterone and follicle-stimulating, and increased concentration of estradiolin the ovary, which resulted in significant decreases in the mating rate, pregnancy rate,and number of fetuses, and inhibition of growth and development of offspring. TiO_2NPsexposure caused severe ovarian oxidative damage, such as overproduction of ROS andincrease level of DNA oxidation (8-OHdg). Microarray analysis showed that in ovariesfrom mice treated with TiO_2NPs compared to controls,223genes of known functionwere up-regulated(diffscore≥13, p <0.05), while65ovarian genes weredown-regulated(diffscore-13, p <0.05). In the present study, functions of differencegene in the ovaries were involved in cell differentiation, immune and inflammatoryresponse, ion transport, metabolic process, oxidative stress, the steroid metabolism,apoptosis, signal transduction, transcription, transport, etc. Especially Akr1c18,Cyp17a1and Lgmn that related to the steroids and sex hormone metabolism processsignificantly up-regulated, over-expression of these genes caused increased levels ofestradiol, progesterone, luteinizing hormone and decreased level of testosterone, therebyinhibited follicular development and reduced fertility of female mice. In addition, TiO_2NPs exposure resulted in a significant increase in the expression of ovarian Cyp17a1,which also can promote the biosynthesis of estradiol.
(2) In this study, the reproductive toxicity and testicular gene expression profilinginduced by intragastric administration of10mg/kg body weight TiO_2NPs for90 consecutive days were investigated. The results showed that TiO_2NPs can be depositedin the testicles, and cause testicular damage and apoptosis, such as rare sperm, spermbreakages, rarefaction of Sertoli cell and androgone, Sertoli cell apoptosis, androgonenecrosis of the seminiferous tubules, decreased germinative layer thickness, vacuolation,and irregular arrangement of Sertoli cells of the seminiferous tubules, significantdecreases in sperm numbers and sperm motility, and increased abnormal sperm, spermwas severely damaged, such as fracture of the head and tail in the cauda epididymis, thefertility of male mice significantly decreased, such as significant decreases in the matingrate, pregnancy rate, and number of fetuses. TiO_2NPs exposure led to the decline ofcalcium level and increases of iron, zinc contents in testicular tissue. Similarly, TiO_2NPs exposure also caused an imbalance of sex hormones, e.g., estradiol andprogesterone significantly increased, and levels of follicle-stimulating hormone,luteinizing hormone and testosterone were significantly decreased. Microarray analysisshowed that in testes from mice treated with TiO_2NPs compared to controls,155geneswere found to be up-regulated(diffscore≥13, p<0.05), while100genes were found to bedown-regulated (diffscore-13, p<0.05). Functions of difference genes were involved inspermatogenesis, steroid metabolic processes, apoptosis, immune, cell differentiation,oxidative stress, redox activity, hormone activity, ion transport. Of the altered geneexpressions, significantly down-regulated expressions of Spata19, Tdrd6and Tnp2weredirectly related with the suppression of spermatogenesis, and Cyp2e1, Mvd, Sc4mol andSrd5a2were related with steroid metabolic process. Cyp2e1was downregulated, Mvd,Sc4mol and Srd5a2were upregulated. These alterations of gene expressions impairedbalance of sex hormones, and decreased fertility of male mice.
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