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Studies on the Toxic Effect of Ammonium Perchlorate on Thyroid and It’s Biomarkers of Exposure

Author: ChenHongXia
Tutor: PengKaiLiang
School: Huazhong University of Science and Technology
Course: Occupational and Environmental Health
Keywords: Ammonium perchlorate apotosis oxidative stress cytokine thyroid-specificprotein thyroid hormoneAmmonium Perchlorate dust thyroid function workers health surveillanceAmmonium perchlorate biomarkers biomoitoring isotope internal standard quantitative analysis
CLC: R131
Type: PhD thesis
Year: 2013
Downloads: 2
Quote: 0
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Abstract


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 cellsObjective: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 workersObjective: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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