过氯酸铵对甲状腺毒作用影响及其生物标志物的研究
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摘要
过氯酸铵(Ammonium Perchlorate, AP)是过氯酸盐类的一种,具有强氧化性,其作为固体推进剂已广泛应用于航天及国防工业中,同时也用于烟火制造等民用方面,因而存在着AP职业性接触及危害。有研究报道,AP对动物甲状腺具有明显的毒作用,干扰甲状腺功能的内稳态。研究已表明：AP是甲状腺滤泡上皮细胞上钠碘转运体(NIS)的碘离子的强效抑制剂。目前,已知AP通过竞争性抑制NIS,抑制甲状腺摄取碘,从而扰乱动物的下丘脑-垂体-甲状腺轴的内稳态。但利用人正常甲状腺滤泡上皮细胞(Nthy-ori3-1),在体外探讨AP的氧化损伤机制及对细胞因子合成影响的相关报道较少。AP是如何影响甲状腺滤泡上皮细胞基底膜上NIS表达,AP如何调控碘离子氧化合成有机碘的甲状腺过氧化物酶(TPO)的活性,以及不同剂量AP染毒对合成甲状腺激素的甲状腺球蛋白(Tg)表达水平调控的机制,目前仍不清楚。
环境暴露水平AP是否对人体的甲状腺功能产生健康影响,在国际范围仍认识不一,国外学者认为人体存在健全的甲状腺代偿性保护机制,血清甲状腺功能指标不是指示暴露情况的特异性生物标志物,目前可得到的证据还不能证实甲状腺激素水平的变化和过氯酸盐环境暴露之间的因果关系。已知AP进入体内不经代谢从尿中排出,我们通过染毒动物模型也发现,大鼠尿液中AP随着暴露剂量的增加而呈剂量反应性增加,故通过测定尿中AP水平能很好地评价其暴露情况。以前的研究选取血清甲状腺功能参数作为评价暴露的指标,仅作为效应标志物是非特异性的,无法精确反映暴露剂量与其变化的趋势。如何选择敏感、特异的生物标志物评价作业工人的暴露水平是当务之急。因此,为深入研究AP对甲状腺的毒作用机制,探讨选择合适的内负荷指标,本课题分别从细胞体外实验、染毒动物模型建立及作业人群职业卫生调查三个方面,来研究AP对甲状腺细胞氧化损伤机制及特异性蛋白表达、血清甲状腺功能参数的影响,并评价尿中AP作为职业暴露人群生物标志物的有效性,以期能选择合适的内负荷指标,对职业暴露的作业工人进行早期的健康监护,为保护工人健康和预防控制其可能产生的职业危害提供科学方法和参考依据。
第一部分过氯酸铵对甲状腺细胞毒作用机制的研究
目的：研究AP对人甲状腺细胞(Nthy-ori3-1)氧化应激及诱导凋亡的影响,同时探讨AP对甲状腺细胞因子IL-1β、IL-6、TNF-α含量及对特异性蛋白NIS、Tg、TPO及cAMP蛋白水平表达的的影响。
方法：体外培养Nthy-ori3-1细胞,接触不同浓度AP (0、5、10、20、40mmol/L)及KI(5.25mmol/L)联合染毒后置37℃,5%CO2培养24h,CCK8法检测Nthy-ori3-1细胞的增殖活力,流式细胞仪检测细胞凋亡率及细胞内活性氧(ROS),同时测定氧化损伤指标丙二醛(MDA)及过氧化氢酶(CAT)活力；收集细胞上清液,采用ELISA试剂盒,上酶标仪测定吸光度值,根据标准曲线进行定量；收集细胞裂解后提取蛋白,采用Western blotting测定甲状腺细胞特异蛋白NIS、Tg、TPO及cAMP蛋白水平的变化。
结果：AP抑制Nthy-ori3-1细胞增殖具有明显的剂量依赖性。与对照组相比,各组细胞内ROS生成量随着染毒剂量的增加出现降低趋势,除5mmol/L AP剂量组外,差异均有统计学意义(P<0.05)；较高剂量AP染毒(40mmol/L)甲状腺细胞,MDA生成量比对照组有明显升高,差异有统计学意义(P<0.001)；各染毒剂量组细胞内CAT活力比对照组有升高的趋势,但差异无统计学意义(P>0.05)；高AP剂量组IL-1β水平比低剂量组明显下降(P<0.05)；AP中、高剂量组及AP高碘组(40mmol/L AP+25mmol/L KI)的IL-6水平比对照组明显降低(P<0.01)；AP高剂量组、AP低碘组(40mmol/L AP+5mmol/L KI)及AP高碘组(40mmol/L AP+25mmol/L KI)的TNF-α水平比对照组明显下降(P<0.01)；与对照组(0.758)相比,低、中、高AP染毒剂量组Tg水平分别为0.563、0.484、0.211,呈现明显降低趋势(P<0.05)；低、中、高AP染毒剂量组TPO表达水平分别为0.521、0.489、0.246,随着染毒剂量增加而明显降低(P<0.05)；而NIS、cAMP表达水平随染毒剂量增加无明显变化(P>0.05)。
结论：AP可致Nthy-ori3-1细胞出现氧化应激,诱导细胞凋亡,细胞内ROS量随着染毒剂量的增加而降低,且二者之间存在明显的剂量反应关系。当染毒AP浓度为40mmol/L时,对甲状腺细胞明显产生氧化损伤作用,碘在一定程度上可缓解AP对甲状腺细胞的氧化应激反应；AP染毒Nthy-ori3-1细胞可随着染毒剂量的增加,细胞因子(IL-1β、IL-6、TNF-α)生成量降低的趋势越明显,这可能是细胞自身负反馈调节机制在发挥作用；AP可通过抑制特异性转录因子表达水平从而引起Tg、TPO蛋白表达水平下调,从细胞层面再次验证AP可抑制甲状腺激素合成的关键蛋白的表达水平,从而直接干扰甲状腺激素的合成过程。
第二部分过氯酸铵对作业人群甲状腺功能的影响
目的：了解AP粉尘职业性危害对作业工人健康的影响,为其预防措施提供科学依据。
方法：选择该厂接触AP粉尘的51名作业工人为接触组,不接触AP粉尘的41人为对照组。调查工厂的一般情况,监测作业场所的AP粉尘浓度,对两组人群进行职业体检,检测血常规、肝肾功能、甲状腺激素、尿碘含量,进行肺通气功能检查,同时对职业接触史等基本情况进行健康调查问卷。
结果：粉尘是AP作业场所最主要的职业性有害因素,本次调查各生产车间粉尘浓度均较低,以烘干过筛、称量车间最高,均为1.175mg/m3;复分解车间最低,为0.425mg/m3。体检结果显示,两组人群经比较,接触组血压舒张压(69.67mmHg)明显低于对照组(79.02mmHg)(P<0.05);接触组工人气短症状的检出率(9.80%)明显高于对照组(0%)(P<0.05)；接触组工人尿素氮(BUN)(5.27U/L)明显高于对照组(4.30U/L),差异有统计学意义(P<0.01),但二者均在正常参考值范围内而无临床意义；接触组的TT4(65.61ng/ml)水平明显低于对照组(81.88ng/ml), TSH(3.75mIU/L)明显高于对照组(2.68mIU/L),差异均有统计学意义(P<0.05)；接触组血清FT4(11.67fmol/ml)也低于对照组(13.14fmol/ml),但其差异尚不显著。
结论：接触组工人的血清FT4、TT4水平比对照组降低,而TSH则比对照组升高,可能是接触AP所致,提示长期接触AP粉尘可能会对作业工人甲状腺功能造成不良影响。
第三部分过氯酸铵接触的生物标志物研究
目的：建立敏感、特异、快速测定生物材料中AP含量的方法,为AP职业人群进行早期生物监测提供科学依据。
方法：利用严格控制染毒剂量的大鼠尿液标本进行预实验,对测定方法的各测量参数进行初步探索；采集接触组与对照组工人的生物材料(血、尿样),采用简单稀释的样品前处理方法,同时利用同位素内标技术消除基质效应,运用已初步建立的超高效液相色谱质谱/质谱法(1JHPLC-MS/MS),对两组人群生物材料中AP进行定量分析及评价。
结果：AP经口饮水染毒在0～520mg/kg/day剂量范围内,大鼠尿中AP含量水平与染毒剂量存在明显相关关系(R2=0.8342),回归方程为Y=5.5222X+291.17。方法的准确度及精密度分别为91.7～99.61%、3.37～7.08%；接触组工人血、尿中AP含量的平均值分别为2.463、12.82μg/L,中位数分别为2.59、4.89μg/L;对照组工人血、尿中AP含量平均值分别为1.00、9.10μg/L,中位数分别为0.83、3.56μg/L。接触组工人血、尿中AP含量与对照组相比,差异均有统计学意义(P<0.05)。
结论：尿中AP水平可作为其暴露的有效生物标志物。相比于尿液,血中AP指示环境暴露更为敏感、特异,但样本不易取得,在人群生物监测中应用有其难度。采集特定人群尿样对其过氯酸盐含量进行测定不失为评价环境暴露的优选方法。
Ammonium perchlorate (Ammonium Perchlorate, AP) is a strong oxidizing, which belongs to the perchlorate compounds. It has been widely used as the solid propellant in the aerospace and defense industry, also used in pyrotechnics and other civilian aspects. So AP occupational exposure was prevalent and might do harm to the workers. Researchers reported that AP induced thyroid toxicity by interfering with thyroid function homeostasis of animals. Studies have shown that AP is a potent inhibitor of sodium iodide symporter (NIS), which transports the iodide ion into thyroid follicular cells. Currently, it is well known that AP competitivly inhibit NIS combination of iodine, which is uptaken by thyroid, thereby disrupting the hypothalamic-pituitary-thyroid axis homeostasis of rodent. But there were fewer reports about the mechanism on the oxidative damage and cytokine synthesis being studied on the normal thyroid follicular epithelial cells (Nthy-ori3-1) induced by AP in vitro. How AP affect NIS expression level, which locate at the basal membrane of thyroid follicular epithelial cell, how AP regulate the thyroid peroxidase (TPO) activity, which is responsibl for the catalytically synthesis organic iodine oxidation from iodide ions, as well as how the Tg expression levels are regulated by different AP exposure doses, which is the raw material of the synthetic thyroid hormone molecule, are still unclear.
Whether environmental exposure dose of AP has effects on thyroid function of the human, there is still a different understanding in an international context. Some scholars believed that the human encompasses the perfect thyroid compensatory protective mechanism, and the serum thyroid function indicators are not specific biomarkers indicating exposure intency. The currently available evidences could not prove a causal relationship between thyroid hormone levels variation and perchlorate environmental exposure. AP was known to entered into the body without be metabolized, which was excreted unchanged in prototype. Through the exposed animal models, the dose-response relationship between exposure dose and urinary perchlorate concentration of rats was found. Measuring urinary AP levels might successfully evaluate its exposure. AP dust is the major occupational hazard existed in the workplace air. AP might have impact on the workers' health by the respiratory or dermal routes. Serum thyroid function parameters were selected for evaluation exposure as the indicators in the previous studies, which only used as biomarkers of effect, and were nonspecific and could not accurately reflect the trend of exposure dose change. Choosing a sensitive, specific of biomarkers in assessing the situation of workers exposure is urgent. Therefore, to in-depth study on the toxic mechanism of AP on thyroid, and to explore appropriate biomarkers reflecting the workers actual environmental exposure dose, the study was carried out from the cell experiments in vitro, animals exposure modeling and the occupational exposure workers, respectively, such as the oxidative damage mechanism, thyroid-specific protein expression, and changes of serum thyroid function parameters induced by AP, and evaluation the validity of urine perchlorate as biomarkers of exposure. These studies further improved the understanding of the mechanism of action on thyroid toxicity induced by AP, and contributed to the early health surveillance of occupational exposure workers, as well as provided a scientific reference method for protection workers' health and the prevention and control of occupational hazards.
Part Ⅰ Study on the cytotoxicity mechanism of Ammonium Perchlorate in thyroid cells
Objective:To research the combined effects of AP and KI on the oxidative stress and apoptosis in Nthy-ori3-1thyroid cells, to learn the effects of AP on cytokines IL-1β, IL-6, and TNF-a generated in the supernatant of thyroid cell, and to explore the mechanism of action of AP excerted on thyroid in-depth from the molecular level, by measuring the changes of the protein expression levels in thyroid cell-specific proteins such as NIS, Tg, TPO and c-AMP.
Methods:The Nthy-ori3-1cells cultured at37℃,5%CO2in vitro, were combinedly exposed to different concentrations of AP (10,20,40mmol/L) and KI (5,25mmol/L) for24h, then the cell proliferation activity was detected by the CCK8assay, while the apoptosis and intracellular generated reactive oxygen species (ROS) were mesasured by flow cytometry, simultaneously with the oxidative damage indicators malondialdehyde (MDA) and catalase (CAT) activity being detected. Collecting the supernatants of cells, and the absorbance values were determined on the microplate reader using an ELISA kit, quantitatively analyzing the contents of cytokines based on the establishment of the regression equation after drawing the calibration curve according to the AP standard series. After cells being collected and lysised by lysates, the proteins were totally extracted. The changes of the protein levels of NIS, Tg, TPO and c-AMP in cultured thyroid cell was measured by Western blotting.
Results:The inhibition of Nthy-ori3-1cell proliferation induced by AP expressed as a significant dose-dependent. Compared with the control group, the ROS generation of the cells in each group appeared to be decreased with increasing doses except for the5mmol/LAP dose group, and the statistically significant differences were observed(P<0.05); Comparing with the control group, there was significantly increase in the MDA generation of thyroid cells exposed to higher dose of AP (40mmol/L), and the difference was markedly significant (P<0.001); The CAT activity of thyroid cells exposed to any doses was elevated with the comparison of control group, but the significant difference was not observed(.P>0.05). The levels of IL-1β of the combined effects of high AP and high KI dose group (40mmol/L AP+25mmol/L KI) was significantly lower (P<0.05)when compared to that of the control group and low dose group; IL-6concentration levels of medium, high AP and high AP combined with high iodine(40mmol/L AP+25mmol/L KI) dose groups decreased significantly(P<0.01), when compared with the control group; high AP, high AP combined with low iodine (40mmol/L AP+5mmol/LKJ) and high AP periodate (40mmol/L AP+25mmol/LKI) compared with the control group, the TNF-a concentration levels decreased significantly (P<0.01); Compared with the control group (0.758), Tg expression levels in the low, medium, high exposure dose groups were0.563,0.484and0.211, respectively, and significantly decreased (P<0.05); Meanwhile, TPO expression levels were0.521,0.489and0.246, respectively, and significantly decreased with the increasing exposure dose (P<0.05); However, with increasing the exposure dose, there were no significant change of NIS and c-AMP expression levels were found (P>0.05).
Conclusions:AP might lead to oxidative stress on the Nthy-ori3-1cell, with induction of apoptosis, and intracellular ROS production decreased with the dose exposure increasing, as well as the significant dose-response relationship between the dose and effect When the thyroid cell was exposed up to40mmol/L AP, the apoptosis occurred, and the generation of MDA in thyroid cells markedly increased, which plays a significant role in oxidative damage to the thyroid cells. Iodine might alleviate the oxidative stress to some extent, corresponding to thyroid cells induced by AP. The generation of cytokines IL-1β, IL-6, TNF-a in the supernatant might reduced with the increase of the exposure dose, and the higher dose was given, the more obvious trend of reduction of cytokine occurred. This reflected that the mechanisms of negative feedback might play a key role in the homeostasis regulation of thyroid cell. AP led to reducing Tg and TPO protein expression levels by suppressing the level of expression of specific transcription factors, which confirmed the previous conclusions that AP hindered the key protein expression levels of thyroid hormone synthesis and thus indirectly affected the synthesis of thyroid hormones.
Part Ⅱ Effects of Ammonium Perchlorate on thyroid function in occupational workers
Objective:To understand the occupational hazards of Ammonium Perchlorate dust on operating workers and to provide the basis preventive measures for protecting the workers' health.
Methods:Fifty-one workers exposed to ammonium perchlorate dust and forty-one unexposed workers from the same factory were selected as the exposure and control groups, respectively. Investigations on the general condition, sampling of dust in the workplaces and a special medical examination were conducted on two groups, including clinical manifestations, blood routine test, hepatic and renal functions, thyroid hormone indicators, urinary iodine concentration, and spirometric test, simultaneously filling the questionnaire of history of occupational exposure and other circumstances.
Results:AP dust is the main occupational harmful factors of workplace. And the total dust concentrations in the each workshop were slight low, with the highest concentration for1.175mg/m3in the drying and screening weighing workshop; and the minimum for0.425mg/m3in the metathesis workshop, respectively. The diastolic blood pressure in the exposure group was lower, and the significant difference was observed (69.67VS79.02, P<0.05); The detection rates of breathlessness symptom in the exposure group was markedly higher than that of the control group (9.8%VS0, P<0.05); The BUN in the exposure group notably higher than that of the control group, the difference was statistically significant (5.27VS4.30U/L, P<0.01); TT4level in the exposure group substantially lowered than that of the control group, also the TSH level significantly higher, and both the statistically significant differences were observed(65.61VS81.88ng/ml;3.75VS2.68mIU/L, P<0.05); While serum FT4also lower than that of the control group (11.67VS13.14fmol/ml), but the difference was not significant.
Conclusions:The FT4、TT4levels in the exposure workers were lower, and TSH level higher than those of the control group, which may due to AP exposure, suggesting that long-term, chronic exposure to AP dust might affect thyroid function of workers.
Part Ⅲ Possible biomarkers for Ammonium Perchlorate exposure
Objective:To establish a sensitive, specific and rapid determination method of ammonium perchlorate in biological materials, and to provide a scientific basis for the early biological monitoring of the occupational workers.
Methods:The urine specimens of rats orally administered with strict control of doses of ammonium perchlorate in drinking waters (gavage) were on the initial groping on the measurement parameters of the measurement method by pilot experiment. Collecting biological material (blood, urine) of the exposure group and the control group of occupational workers, using simple dilution method of the sample pre-treatment, as well as the use of isotope internal standard technology to eliminate matrix effects, the concentration of ammonium perchlorate in the biological materials of two groups were quantitatively analyzed, furthermore, the initial established method of the ultra-high performance liquid chromatography mass spectrometry/mass spectrometry (UHPLC-MS/MS) were evaluated as well.
Results:The concentration of urinary perchlorate of rats significantly correlated (R2=0.8342) with orally administrated AP dose in drinking water ranged from0to520mg/kg/day, the regression equation was Y=5.5222X+291.17. The accuracy and precision of the method were91.7～99.61%,3.37～7.08%, repectively; The mean value of perchlorate in urine and blood were12.82,2.463μg/L for the exposure groups, and9.10,1.00μg/L for the control groups, respectively; whereas the the median of perchlorate in the urine and blood were4.89,2.59μg/L for the exposure groups and3.56,0.83μg/L for the control groups, respectively. Furthermore, significant differences of perchlorate concentration in urine and serum samples between the exposure groups and control groups were found (P<0.05).
Conclusions:Urine perchlorate can be used as an effective biomarker of exposure. Comparing to urine perchlorate, blood perchlorate is more sensitive and specific for reflecting the extent of environmental exposure. The application on biomonitoring serum perchlorate of specific population is difficult, since the blood samples are not easily available. Collecting urine samples and detecting urinary perchlorate in the specific populations is certainly to be a gold standard method, which could be chosen for assessment of environmental exposure.
环境暴露水平AP是否对人体的甲状腺功能产生健康影响,在国际范围仍认识不一,国外学者认为人体存在健全的甲状腺代偿性保护机制,血清甲状腺功能指标不是指示暴露情况的特异性生物标志物,目前可得到的证据还不能证实甲状腺激素水平的变化和过氯酸盐环境暴露之间的因果关系。已知AP进入体内不经代谢从尿中排出,我们通过染毒动物模型也发现,大鼠尿液中AP随着暴露剂量的增加而呈剂量反应性增加,故通过测定尿中AP水平能很好地评价其暴露情况。以前的研究选取血清甲状腺功能参数作为评价暴露的指标,仅作为效应标志物是非特异性的,无法精确反映暴露剂量与其变化的趋势。如何选择敏感、特异的生物标志物评价作业工人的暴露水平是当务之急。因此,为深入研究AP对甲状腺的毒作用机制,探讨选择合适的内负荷指标,本课题分别从细胞体外实验、染毒动物模型建立及作业人群职业卫生调查三个方面,来研究AP对甲状腺细胞氧化损伤机制及特异性蛋白表达、血清甲状腺功能参数的影响,并评价尿中AP作为职业暴露人群生物标志物的有效性,以期能选择合适的内负荷指标,对职业暴露的作业工人进行早期的健康监护,为保护工人健康和预防控制其可能产生的职业危害提供科学方法和参考依据。
第一部分过氯酸铵对甲状腺细胞毒作用机制的研究
目的：研究AP对人甲状腺细胞(Nthy-ori3-1)氧化应激及诱导凋亡的影响,同时探讨AP对甲状腺细胞因子IL-1β、IL-6、TNF-α含量及对特异性蛋白NIS、Tg、TPO及cAMP蛋白水平表达的的影响。
方法：体外培养Nthy-ori3-1细胞,接触不同浓度AP (0、5、10、20、40mmol/L)及KI(5.25mmol/L)联合染毒后置37℃,5%CO2培养24h,CCK8法检测Nthy-ori3-1细胞的增殖活力,流式细胞仪检测细胞凋亡率及细胞内活性氧(ROS),同时测定氧化损伤指标丙二醛(MDA)及过氧化氢酶(CAT)活力；收集细胞上清液,采用ELISA试剂盒,上酶标仪测定吸光度值,根据标准曲线进行定量；收集细胞裂解后提取蛋白,采用Western blotting测定甲状腺细胞特异蛋白NIS、Tg、TPO及cAMP蛋白水平的变化。
结果：AP抑制Nthy-ori3-1细胞增殖具有明显的剂量依赖性。与对照组相比,各组细胞内ROS生成量随着染毒剂量的增加出现降低趋势,除5mmol/L AP剂量组外,差异均有统计学意义(P<0.05)；较高剂量AP染毒(40mmol/L)甲状腺细胞,MDA生成量比对照组有明显升高,差异有统计学意义(P<0.001)；各染毒剂量组细胞内CAT活力比对照组有升高的趋势,但差异无统计学意义(P>0.05)；高AP剂量组IL-1β水平比低剂量组明显下降(P<0.05)；AP中、高剂量组及AP高碘组(40mmol/L AP+25mmol/L KI)的IL-6水平比对照组明显降低(P<0.01)；AP高剂量组、AP低碘组(40mmol/L AP+5mmol/L KI)及AP高碘组(40mmol/L AP+25mmol/L KI)的TNF-α水平比对照组明显下降(P<0.01)；与对照组(0.758)相比,低、中、高AP染毒剂量组Tg水平分别为0.563、0.484、0.211,呈现明显降低趋势(P<0.05)；低、中、高AP染毒剂量组TPO表达水平分别为0.521、0.489、0.246,随着染毒剂量增加而明显降低(P<0.05)；而NIS、cAMP表达水平随染毒剂量增加无明显变化(P>0.05)。
结论：AP可致Nthy-ori3-1细胞出现氧化应激,诱导细胞凋亡,细胞内ROS量随着染毒剂量的增加而降低,且二者之间存在明显的剂量反应关系。当染毒AP浓度为40mmol/L时,对甲状腺细胞明显产生氧化损伤作用,碘在一定程度上可缓解AP对甲状腺细胞的氧化应激反应；AP染毒Nthy-ori3-1细胞可随着染毒剂量的增加,细胞因子(IL-1β、IL-6、TNF-α)生成量降低的趋势越明显,这可能是细胞自身负反馈调节机制在发挥作用；AP可通过抑制特异性转录因子表达水平从而引起Tg、TPO蛋白表达水平下调,从细胞层面再次验证AP可抑制甲状腺激素合成的关键蛋白的表达水平,从而直接干扰甲状腺激素的合成过程。
第二部分过氯酸铵对作业人群甲状腺功能的影响
目的：了解AP粉尘职业性危害对作业工人健康的影响,为其预防措施提供科学依据。
方法：选择该厂接触AP粉尘的51名作业工人为接触组,不接触AP粉尘的41人为对照组。调查工厂的一般情况,监测作业场所的AP粉尘浓度,对两组人群进行职业体检,检测血常规、肝肾功能、甲状腺激素、尿碘含量,进行肺通气功能检查,同时对职业接触史等基本情况进行健康调查问卷。
结果：粉尘是AP作业场所最主要的职业性有害因素,本次调查各生产车间粉尘浓度均较低,以烘干过筛、称量车间最高,均为1.175mg/m3;复分解车间最低,为0.425mg/m3。体检结果显示,两组人群经比较,接触组血压舒张压(69.67mmHg)明显低于对照组(79.02mmHg)(P<0.05);接触组工人气短症状的检出率(9.80%)明显高于对照组(0%)(P<0.05)；接触组工人尿素氮(BUN)(5.27U/L)明显高于对照组(4.30U/L),差异有统计学意义(P<0.01),但二者均在正常参考值范围内而无临床意义；接触组的TT4(65.61ng/ml)水平明显低于对照组(81.88ng/ml), TSH(3.75mIU/L)明显高于对照组(2.68mIU/L),差异均有统计学意义(P<0.05)；接触组血清FT4(11.67fmol/ml)也低于对照组(13.14fmol/ml),但其差异尚不显著。
结论：接触组工人的血清FT4、TT4水平比对照组降低,而TSH则比对照组升高,可能是接触AP所致,提示长期接触AP粉尘可能会对作业工人甲状腺功能造成不良影响。
第三部分过氯酸铵接触的生物标志物研究
目的：建立敏感、特异、快速测定生物材料中AP含量的方法,为AP职业人群进行早期生物监测提供科学依据。
方法：利用严格控制染毒剂量的大鼠尿液标本进行预实验,对测定方法的各测量参数进行初步探索；采集接触组与对照组工人的生物材料(血、尿样),采用简单稀释的样品前处理方法,同时利用同位素内标技术消除基质效应,运用已初步建立的超高效液相色谱质谱/质谱法(1JHPLC-MS/MS),对两组人群生物材料中AP进行定量分析及评价。
结果：AP经口饮水染毒在0～520mg/kg/day剂量范围内,大鼠尿中AP含量水平与染毒剂量存在明显相关关系(R2=0.8342),回归方程为Y=5.5222X+291.17。方法的准确度及精密度分别为91.7～99.61%、3.37～7.08%；接触组工人血、尿中AP含量的平均值分别为2.463、12.82μg/L,中位数分别为2.59、4.89μg/L;对照组工人血、尿中AP含量平均值分别为1.00、9.10μg/L,中位数分别为0.83、3.56μg/L。接触组工人血、尿中AP含量与对照组相比,差异均有统计学意义(P<0.05)。
结论：尿中AP水平可作为其暴露的有效生物标志物。相比于尿液,血中AP指示环境暴露更为敏感、特异,但样本不易取得,在人群生物监测中应用有其难度。采集特定人群尿样对其过氯酸盐含量进行测定不失为评价环境暴露的优选方法。
Ammonium perchlorate (Ammonium Perchlorate, AP) is a strong oxidizing, which belongs to the perchlorate compounds. It has been widely used as the solid propellant in the aerospace and defense industry, also used in pyrotechnics and other civilian aspects. So AP occupational exposure was prevalent and might do harm to the workers. Researchers reported that AP induced thyroid toxicity by interfering with thyroid function homeostasis of animals. Studies have shown that AP is a potent inhibitor of sodium iodide symporter (NIS), which transports the iodide ion into thyroid follicular cells. Currently, it is well known that AP competitivly inhibit NIS combination of iodine, which is uptaken by thyroid, thereby disrupting the hypothalamic-pituitary-thyroid axis homeostasis of rodent. But there were fewer reports about the mechanism on the oxidative damage and cytokine synthesis being studied on the normal thyroid follicular epithelial cells (Nthy-ori3-1) induced by AP in vitro. How AP affect NIS expression level, which locate at the basal membrane of thyroid follicular epithelial cell, how AP regulate the thyroid peroxidase (TPO) activity, which is responsibl for the catalytically synthesis organic iodine oxidation from iodide ions, as well as how the Tg expression levels are regulated by different AP exposure doses, which is the raw material of the synthetic thyroid hormone molecule, are still unclear.
Whether environmental exposure dose of AP has effects on thyroid function of the human, there is still a different understanding in an international context. Some scholars believed that the human encompasses the perfect thyroid compensatory protective mechanism, and the serum thyroid function indicators are not specific biomarkers indicating exposure intency. The currently available evidences could not prove a causal relationship between thyroid hormone levels variation and perchlorate environmental exposure. AP was known to entered into the body without be metabolized, which was excreted unchanged in prototype. Through the exposed animal models, the dose-response relationship between exposure dose and urinary perchlorate concentration of rats was found. Measuring urinary AP levels might successfully evaluate its exposure. AP dust is the major occupational hazard existed in the workplace air. AP might have impact on the workers' health by the respiratory or dermal routes. Serum thyroid function parameters were selected for evaluation exposure as the indicators in the previous studies, which only used as biomarkers of effect, and were nonspecific and could not accurately reflect the trend of exposure dose change. Choosing a sensitive, specific of biomarkers in assessing the situation of workers exposure is urgent. Therefore, to in-depth study on the toxic mechanism of AP on thyroid, and to explore appropriate biomarkers reflecting the workers actual environmental exposure dose, the study was carried out from the cell experiments in vitro, animals exposure modeling and the occupational exposure workers, respectively, such as the oxidative damage mechanism, thyroid-specific protein expression, and changes of serum thyroid function parameters induced by AP, and evaluation the validity of urine perchlorate as biomarkers of exposure. These studies further improved the understanding of the mechanism of action on thyroid toxicity induced by AP, and contributed to the early health surveillance of occupational exposure workers, as well as provided a scientific reference method for protection workers' health and the prevention and control of occupational hazards.
Part Ⅰ Study on the cytotoxicity mechanism of Ammonium Perchlorate in thyroid cells
Objective:To research the combined effects of AP and KI on the oxidative stress and apoptosis in Nthy-ori3-1thyroid cells, to learn the effects of AP on cytokines IL-1β, IL-6, and TNF-a generated in the supernatant of thyroid cell, and to explore the mechanism of action of AP excerted on thyroid in-depth from the molecular level, by measuring the changes of the protein expression levels in thyroid cell-specific proteins such as NIS, Tg, TPO and c-AMP.
Methods:The Nthy-ori3-1cells cultured at37℃,5%CO2in vitro, were combinedly exposed to different concentrations of AP (10,20,40mmol/L) and KI (5,25mmol/L) for24h, then the cell proliferation activity was detected by the CCK8assay, while the apoptosis and intracellular generated reactive oxygen species (ROS) were mesasured by flow cytometry, simultaneously with the oxidative damage indicators malondialdehyde (MDA) and catalase (CAT) activity being detected. Collecting the supernatants of cells, and the absorbance values were determined on the microplate reader using an ELISA kit, quantitatively analyzing the contents of cytokines based on the establishment of the regression equation after drawing the calibration curve according to the AP standard series. After cells being collected and lysised by lysates, the proteins were totally extracted. The changes of the protein levels of NIS, Tg, TPO and c-AMP in cultured thyroid cell was measured by Western blotting.
Results:The inhibition of Nthy-ori3-1cell proliferation induced by AP expressed as a significant dose-dependent. Compared with the control group, the ROS generation of the cells in each group appeared to be decreased with increasing doses except for the5mmol/LAP dose group, and the statistically significant differences were observed(P<0.05); Comparing with the control group, there was significantly increase in the MDA generation of thyroid cells exposed to higher dose of AP (40mmol/L), and the difference was markedly significant (P<0.001); The CAT activity of thyroid cells exposed to any doses was elevated with the comparison of control group, but the significant difference was not observed(.P>0.05). The levels of IL-1β of the combined effects of high AP and high KI dose group (40mmol/L AP+25mmol/L KI) was significantly lower (P<0.05)when compared to that of the control group and low dose group; IL-6concentration levels of medium, high AP and high AP combined with high iodine(40mmol/L AP+25mmol/L KI) dose groups decreased significantly(P<0.01), when compared with the control group; high AP, high AP combined with low iodine (40mmol/L AP+5mmol/LKJ) and high AP periodate (40mmol/L AP+25mmol/LKI) compared with the control group, the TNF-a concentration levels decreased significantly (P<0.01); Compared with the control group (0.758), Tg expression levels in the low, medium, high exposure dose groups were0.563,0.484and0.211, respectively, and significantly decreased (P<0.05); Meanwhile, TPO expression levels were0.521,0.489and0.246, respectively, and significantly decreased with the increasing exposure dose (P<0.05); However, with increasing the exposure dose, there were no significant change of NIS and c-AMP expression levels were found (P>0.05).
Conclusions:AP might lead to oxidative stress on the Nthy-ori3-1cell, with induction of apoptosis, and intracellular ROS production decreased with the dose exposure increasing, as well as the significant dose-response relationship between the dose and effect When the thyroid cell was exposed up to40mmol/L AP, the apoptosis occurred, and the generation of MDA in thyroid cells markedly increased, which plays a significant role in oxidative damage to the thyroid cells. Iodine might alleviate the oxidative stress to some extent, corresponding to thyroid cells induced by AP. The generation of cytokines IL-1β, IL-6, TNF-a in the supernatant might reduced with the increase of the exposure dose, and the higher dose was given, the more obvious trend of reduction of cytokine occurred. This reflected that the mechanisms of negative feedback might play a key role in the homeostasis regulation of thyroid cell. AP led to reducing Tg and TPO protein expression levels by suppressing the level of expression of specific transcription factors, which confirmed the previous conclusions that AP hindered the key protein expression levels of thyroid hormone synthesis and thus indirectly affected the synthesis of thyroid hormones.
Part Ⅱ Effects of Ammonium Perchlorate on thyroid function in occupational workers
Objective:To understand the occupational hazards of Ammonium Perchlorate dust on operating workers and to provide the basis preventive measures for protecting the workers' health.
Methods:Fifty-one workers exposed to ammonium perchlorate dust and forty-one unexposed workers from the same factory were selected as the exposure and control groups, respectively. Investigations on the general condition, sampling of dust in the workplaces and a special medical examination were conducted on two groups, including clinical manifestations, blood routine test, hepatic and renal functions, thyroid hormone indicators, urinary iodine concentration, and spirometric test, simultaneously filling the questionnaire of history of occupational exposure and other circumstances.
Results:AP dust is the main occupational harmful factors of workplace. And the total dust concentrations in the each workshop were slight low, with the highest concentration for1.175mg/m3in the drying and screening weighing workshop; and the minimum for0.425mg/m3in the metathesis workshop, respectively. The diastolic blood pressure in the exposure group was lower, and the significant difference was observed (69.67VS79.02, P<0.05); The detection rates of breathlessness symptom in the exposure group was markedly higher than that of the control group (9.8%VS0, P<0.05); The BUN in the exposure group notably higher than that of the control group, the difference was statistically significant (5.27VS4.30U/L, P<0.01); TT4level in the exposure group substantially lowered than that of the control group, also the TSH level significantly higher, and both the statistically significant differences were observed(65.61VS81.88ng/ml;3.75VS2.68mIU/L, P<0.05); While serum FT4also lower than that of the control group (11.67VS13.14fmol/ml), but the difference was not significant.
Conclusions:The FT4、TT4levels in the exposure workers were lower, and TSH level higher than those of the control group, which may due to AP exposure, suggesting that long-term, chronic exposure to AP dust might affect thyroid function of workers.
Part Ⅲ Possible biomarkers for Ammonium Perchlorate exposure
Objective:To establish a sensitive, specific and rapid determination method of ammonium perchlorate in biological materials, and to provide a scientific basis for the early biological monitoring of the occupational workers.
Methods:The urine specimens of rats orally administered with strict control of doses of ammonium perchlorate in drinking waters (gavage) were on the initial groping on the measurement parameters of the measurement method by pilot experiment. Collecting biological material (blood, urine) of the exposure group and the control group of occupational workers, using simple dilution method of the sample pre-treatment, as well as the use of isotope internal standard technology to eliminate matrix effects, the concentration of ammonium perchlorate in the biological materials of two groups were quantitatively analyzed, furthermore, the initial established method of the ultra-high performance liquid chromatography mass spectrometry/mass spectrometry (UHPLC-MS/MS) were evaluated as well.
Results:The concentration of urinary perchlorate of rats significantly correlated (R2=0.8342) with orally administrated AP dose in drinking water ranged from0to520mg/kg/day, the regression equation was Y=5.5222X+291.17. The accuracy and precision of the method were91.7～99.61%,3.37～7.08%, repectively; The mean value of perchlorate in urine and blood were12.82,2.463μg/L for the exposure groups, and9.10,1.00μg/L for the control groups, respectively; whereas the the median of perchlorate in the urine and blood were4.89,2.59μg/L for the exposure groups and3.56,0.83μg/L for the control groups, respectively. Furthermore, significant differences of perchlorate concentration in urine and serum samples between the exposure groups and control groups were found (P<0.05).
Conclusions:Urine perchlorate can be used as an effective biomarker of exposure. Comparing to urine perchlorate, blood perchlorate is more sensitive and specific for reflecting the extent of environmental exposure. The application on biomonitoring serum perchlorate of specific population is difficult, since the blood samples are not easily available. Collecting urine samples and detecting urinary perchlorate in the specific populations is certainly to be a gold standard method, which could be chosen for assessment of environmental exposure.
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