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Dose-Response Relationship Between Orally Administered Ammonium Perchlorate and Urine Perchlorate Concentrations in Rats: Possible Biomarker to Quantify Environmental Ammonium Perchlorate Exposure on Thyroid Homeostasis ab
b
c
b
b
Hong Xia Chen , Miao Hong Ding , Yong Gan Li , Qin Liu & Kai Liang Peng a
Institute of Biomedicine, Taihe Hospital, Hubei University of Medicine, Shiyan, People's Republic of China b
Click for updates
Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China c
Centers for Disease Control and Prevention of Hubei Provence, Wuhan, People's Republic of China Accepted author version posted online: 27 Jun 2014.
To cite this article: Hong Xia Chen, Miao Hong Ding, Yong Gan Li, Qin Liu & Kai Liang Peng (2015) Dose-Response Relationship Between Orally Administered Ammonium Perchlorate and Urine Perchlorate Concentrations in Rats: Possible Biomarker to Quantify Environmental Ammonium Perchlorate Exposure on Thyroid Homeostasis, Archives of Environmental & Occupational Health, 70:5, 286-290, DOI: 10.1080/19338244.2014.904265 To link to this article: http://dx.doi.org/10.1080/19338244.2014.904265
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Archives of Environmental & Occupational Health (2015) 70, 286–290 C Taylor & Francis Group, LLC Copyright ISSN: 1933-8244 print / 2154-4700 online DOI: 10.1080/19338244.2014.904265
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Dose-Response Relationship Between Orally Administered Ammonium Perchlorate and Urine Perchlorate Concentrations in Rats: Possible Biomarker to Quantify Environmental Ammonium Perchlorate Exposure on Thyroid Homeostasis HONG XIA CHEN1,2, MIAO HONG DING2, YONG GAN LI3, QIN LIU2, and KAI LIANG PENG2 1
Institute of Biomedicine, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China 3 Centers for Disease Control and Prevention of Hubei Provence, Wuhan, People’s Republic of China 2
Received 11 September 2013, Accepted 19 February 2014
To evaluate the feasibility of urine perchlorate as a biomarker of ammonium perchlorate (AP) exposure and to explore the correlation between the thyroid function indicators and the perchlorate concentrations, a sensitive and selective ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) method was developed to detect perchlorate in urine samples. Rats were orally administrated with different doses of perchlorate. Serum free thyroxine (FT4 ), free triiodothyronine (FT3 ), and thyroid-stimulating hormone (TSH) were determined by radioimmunoassays. The results showed that a dose of AP up to 520 mg kg−1 body weight induced a significant increase of TSH, with a decrease of FT4 . Particularly, the levels of urine perchlorate increased dose-dependently on AP exposure from drinking water. The findings highlighted that urine perchlorate may be a useful biomarker for AP environmental exposure. Keywords: ammonium perchlorate, biomarker, thyroid hormone, urine perchlorate
Recently, there has been an increased awareness of the possible detrimental thyroid-related effects of environmental perchlorate, especially in iodine-deficient populations.1 Therefore, there is considerable interest and an urgent need to develop a highly sensitive and selective biomonitoring method to accurately assess perchlorate exposure for specific populations. Human biomonitoring data indicate that exposure to perchlorate occurs worldwide, including highly populated countries such as the United States, China, Canada, Japan, and India.2–5 Accurate assessment of exposure to perchlorate is critical to detecting biochemical end points potentially related to exposure. In the past, serum thyroid-stimulating hormone (TSH) and thyroxine (T4 ) were considered sensitive biomarkers of thyroidal effects induced by AP in light of the compensatory mechanism.6–8 However, the thyroidal function Address correspondence to Hong Xia Chen, Institute of Biomedicine, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, People’s Republic of China. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/vaeh.
indicators are not specific biomarkers due to the complexity of hypothalamic-pituitary-thyroid axis (HPT axis) regulation mechanism. Furthermore, other antithyroid agents such as carbimazole can have similar effects on thyroid homeostasis. The likelihood of widespread human exposure coupled with potential health effects supports the need to develop a biomonitoring method for assessing the actual perchlorate exposure by directly measuring perchlorate in biological matrixes, such as urine. This refers to the biomarkers of exposure, which can accurately reflect the level of internal exposure in the human body burden. Useful human exposure data have been obtained by directly measuring levels of perchlorate in human urine, milk, amniotic fluid, saliva, and blood.9–14 Quantification of perchlorate in biological tissues and fluids is an excellent method for evaluating both the prevalence and magnitude of exposure.15 Furthermore, measurement of perchlorate in biological matrices provides data on the total dose of perchlorate integrated from all sources.16 In the present study, the feasibility of urine perchlorate as a biomarker of ammonium perchlorate (AP) exposure was evaluated, and the potential correlation of the urinary perchlorate concentrations to the thyroid function was investigated.
Archives of Environmental & Occupational Health
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Table 1. Changes of Body Weight Gain in AP-Treated Rat Cumulative net increase in the amount of weekly weight (g)
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Control (0 mg kg−1 bw)
Low (130 mg kg−1 bw)
Medium (260 mg kg−1 bw)
High (520 mg kg−1 bw)
Exposure time (week)
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Initial body weight 1 2 3 4 5 6 7 8 9 10 11 12 13
188.00 73.60 136.60 183.20 221.20 250.40 283.40 312.80 326.40 350.00 373.00 392.20 407.60 417.40
38.68 6.62 9.13 12.58 19.27 21.62 26.37 24.95 28.45 25.48 30.19 33.18 37.71 41.62
182.33 60.17∗∗ 106.67∗∗ 151.83∗ 178.83∗ 224.50 252.83 274.00 283.50 294.33 305.17 311.83∗ 320.50∗ 333.17
27.85 9.75 18.43 28.90 40.20 50.42 58.72 67.86 74.42 81.53 87.37 91.79 99.71 104.61
178.17 55.17∗∗ 100.50∗∗ 135.33∗∗ 159.67∗∗ 191.33∗ 208.33∗∗ 224.00∗∗ 233.17∗∗ 239.67∗∗ 245.50∗∗ 242.00∗∗ 246.00∗∗ 254.33∗∗
18.94 6.05 10.39 14.90 19.24 28.01 33.04 32.03 38.63 51.06 56.65 54.98 57.47 69.74
176.17 59.50∗∗ 115.00∗ 148.17∗∗ 168.00∗∗ 206.83∗ 216.17∗ 214.50∗∗ 210.00∗∗ 209.83∗∗ 208.00∗∗ 209.83∗∗ 213.13∗∗ 216.33∗∗
25.95 4.51 9.96 12.12 15.21 25.94 31.88 38.39 40.46 44.49 45.66 48.73 51.04 102.27
∗p
< .05 and ∗∗ p < .01 compared with control group values.
The study focused on combining urinary perchlorate data and thyroid function data in assessing the population exposed to perchlorate.
Methods Animal and Treatment Twenty-four healthy male Sprague-Dawley rats (28 days old, weighing 80–100 g) were randomly divided into 4 groups of 6 rats. The control was treated without AP, orally administrated by tap water only, with the 3 exposure groups given different doses of AP (130, 260, and 520 mg kg−1 body weight [bw], respectively), in tap water every day by oral administration (gavage) for 13 consecutive weeks. The rats were weighed regularly every week, and the daily administrate doses based on the rats’ body weight. The concentration of AP in tap water was adjusted weekly for these groups, based on measured body weight, in order to achieve the desired dosage levels. The doses used in the present study were based on our preliminary experiment, which determined the oral doses required to alter T4 concentrations. Doses selected which would range from a no significant effect on serum hormone level to a maximum reduction in thyroid hormone. The rats were euthanized after orally administered with AP for 13 consecutive weeks. Urine and serum samples were collected, especially with approximately 1 mL reflex voiding urine sample by extrusion from rats’ bladder. The apparatus used in the urine sampling should be kept clean and dry, avoiding perchlorate contamination in the environmental background; and the feces must be separated from urine, avoiding the urine samples being contaminated. All procedures on animals followed the Guide for the Care and Use of Laboratory Animals published by Ministry of Health of People’s Republic of China.
Instrumental Analysis Urinary perchlorate concentrations were determined by highperformance liquid chromataography (HPLC) based on the method as described by Li and George.17 All solutions were prepared from American Chemical Society (ACS) reagentgrade chemicals in 18 M water obtained from a Water Pro PS purification system (Labconco, Kansas City, MO, USA). The urine perchlorate separation was carried out on Agilent Technologies 1200 ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) system with a ZORBAX SB C18 column (2.1 mm × 50 mm, 1.8 μm) (Agilent, USA). Supernatants of ethanol precipitated rat urine were evaporated to dryness under nitrogen and reconstituted in deionized water. All test solutions were syringe filtered, mixed with an 18O4 -labeled perchlorate internal standard, and directly injected with 5 μL volumes of matric samples into a liquid chromatography–tandem triple-quadrupole mass spectrometry (LC-MS/MS) system. Ionization was accomplished using electrospray ionization in negative mode. The labeled internal standard was corrected for any sample matrix effects on measured signals. Four parent-to-product ion transitions, for loss of oxygen, were monitored for native and 18O4 -labeled perchlorate anion, respectively: 35Clperchlorate, m/z 99 → 83 and 107 → 89; 37Cl-perchlorate, m/z 101 → 85 and 109 → 91. Hormone Analyses Radioimmunoassay (RIA) kits for the hormone measurements were all from the same batch number and with the same expiration date for all the standard and unknown samples. Serum free thyroxine (FT4 ), free triiodothyronine (FT3 ), and thyroid-stimulating hormone (TSH) radioimmunoassays were performed in triplicate, following the manufacturer’s
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Chen et al. Table 3. The Urinary Perchlorate Concentrations of Rats Orally Administrated Different Ammonium Perchlorate (AP) Doses in Each Group Urinary perchlorate (mg/L) Group
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Fig. 1. Body weight gain of each group over the treatment period.
instructions (North Biotechnology Institute, Beijing, China). Tracer (125I) radioactivity was measured using a γ counter (GC-1200 gamma radioimmunoassay counter; Anhui, China).
AP exposure dose (mg kg−1 bw)
Mean
SD
0 130 260 520
0.738 856.99 2535.87∗ 2796.23∗†
0.288 355.22 2095.90 1650.24
Control (n = 6) Low (n = 4)a Medium (n = 6) High (n = 6) aIn
the feeding process, 2 animals died accidentally. with low-dose group, p < .05. ∗ Compared with control group, p < .01. †Compared
Effect of Ammonium Perchlorate on Serum Hormones (FT4 , FT3 , and TSH)
Statistical Method Statistical analysis of the data was performed with SPSS (version 12.0; SPSS, Chicago, IL, USA, 2003). Comparisons between the treatment groups and control group were done with 1-way analysis of variance (ANOVA). The results were expressed as the mean ± SD, and statistical significance was set at p < .05.
As shown in Table 2, FT4 levels were significantly decreased in the low- (130 mg kg−1 bw), medium- (260 mg kg−1 bw), and high- (520 mg kg−1 bw) dose AP groups (p < .05, p < .05, and p < .01, respectively). TSH level of the high-dose AP group (520 mg kg−1 bw) was significantly higher than that of the control group (p < .05). FT3 levels of the low- and medium-dose AP groups have a decreasing trend, but the only significant mean difference was observed in low-dose group (130 mg kg−1 bw) when compared with the control group.
Results Effect of Ammonium Perchlorate on Body Weight in Rats As seen in Table 1, compared with control group, 13-week treatment with 130 mg kg−1 bw AP did not lead to significant change of body weight, but a significant decrease in body weight gain was observed in medium- (260 mg kg−1 bw) and high- (520 mg kg−1 bw) dose AP groups (p < .01, respectively). And the body weight gain of rats decreased in a dose-dependent manner with the AP exposure, which gradually slowed down, with the weight growth curve stabilizing at the 6th week. Body weight gain of each group over the treatment period is shown in Figure 1. Both medium and high AP exposure can obviously impede the body weight gain of rats (Figure 1).
Correlation Between Environmental Perchlorate Exposure and Urinary Perchlorate Concentration Figure 2 shows that the concentration of AP in urine increased proportionally in a dose-related manner, with a linear relationship between exposure dose in the drinking water and urinary perchlorate levels (R2 = .8324). As shown in Table 3, a statically significant increase of urinary perchlorate was observed in the medium- and high-dose AP group (p < .01, respectively) compared with the control group. Furthermore, markedly higher levels of the urinary perchlorate were detected in rats with high dose of AP than those in the low-dose group, demonstrating that urine perchlorate could be used as the biomarkers indicating the actual exposure intensity.
Table 2. Values of Thyroid Hormone Parameters in Each Group
Group Control Low Medium High
FT4 (fmol/mL)
FT3 (fmol/mL)
TSH (mIU/L)
AP exposure dose (mg kg−1 bw)
No. of animal
Mean
SD
Mean
SD
Mean
SD
0 130 260 520
6 4a 6 6
12.76 9.22∗ 8.06∗ 5.71∗∗
2.72 2.76 2.10 2.97
1.55 0.88∗ 1.05 1.65
0.78 0.41 0.51 1.12
0.88 1.00 0.86 1.17∗
0.37 0.30 0.30 0.34
Note. AP = ammonium perchlorate; FT4 = free thyroxine; FT3 = free triiodothyronine; TSH = thyroid-stimulating hormone. aIn the feeding process, 2 animals died accidentally. ∗ p < .05 and ∗∗ p < .01 compared with control group values.
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Fig. 2. The correlation between perchlorate dose in drinking water and the perchlorate concentration in urine of rats.
Comment Perchlorate is highly water soluble and environmentally stable,18 so that the general population is vulnerable to the perchlorate exposure through drinking water and food. Serum thyroid function parameters (e.g., FT3 , FT4 , TSH) were selected 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 intensity. Just as seen in Table 2, only FT3 level in the low-dose group decreased and TSH level in the high-dose group increased with the increased dose. Choosing a sensitive, specific biomarker in assessing the exposure of occupational workers is urgent. In this study, we established a determination method of urine perchlorate in rats and evaluated the dose-response relationship between AP exposure and urine perchlorate. The linear relationship between AP exposure dose in drinking water and urinary perchlorate levels was observed, and the concentration of AP in urine increased proportionally in a dose-related manner (Table 3). Furthermore, the urinary perchlorate level of the high-dose group was markedly higher than that of the low-dose group, which demonstrated that urine perchlorate could be used as the biomarker indicating the actual exposure intensity. Previous studies have indicated that urine is the main excretory pathway for absorbed perchlorate, which could make urine a convenient testing medium for perchlorate levels. Furthermore, urine perchlorate has previously been confirmed as a biomarker that is specific for exposure to perchlorate.19 Urine perchlorate is reasonably indicative of human exposure because 70%–95% of a perchlorate dose is excreted unchanged in the urine, with a half-life of 8–14 hours.19,20 Urine collection is less invasive than blood collection, and urinary perchlorate levels tend to be much higher than serum levels due to efficient renal clearance of perchlorate. Nevertheless, measuring perchlorate in human urine assesses the combined exposure from all sources.21 Determination of perchlorate in urine is the preferred choice for evaluation of environmental exposure. Following the development of sensitive and specific measurement method such as the UHPLC system, urinary perchlorate has the potential to be a novel and effective biomarker for evaluation the low level of occupational exposure.
289 In this study, both medium- (260 mg kg−1 bw) and high(520 mg kg−1 bw) dose AP groups revealed a significant decrease in body weight gain. Furthermore, both FT3 levels in the low-dose group and FT4 levels in the medium- and high-dose groups were significantly lower than those of the control group. In addition, the TSH levels in the high-dose group were significantly increased when compared with the control group. These results suggest that perchlorate could significantly decrease FT4 levels and elevate TSH levels, which are consistent with previous studies.1,22,23 Levels of T3 , T4 , and TSH in serum are considered to be biomarkers of perchlorate exposure on thyroid homeostasis for a long time.19 Since perchlorate anions have an identical charge and similar ionic radius to iodide (I−), perchlorate is known as a strong inhibitor of the sodium-iodide importer (NIS), which is the primary mechanism by which iodide enters thyroid follicle cells from the blood, and is the first step in the uptake of iodide into the thyroid leading to the formation of thyroid hormones.24,25 Thyroid hormones (THs) play a crucial role in differentiation, metabolism, growth, and development. TH is the key factor required for the normal function of nearly all tissues, with major effects on oxygen consumption and metabolic rate. If the thyroid gland is exposed to some specific thyroid toxicants such as perchlorate, thiocyanate, and nitrate over a period of time, the biosynthesis of thyroid hormones may be weakened.26,27 With thyroid hormone levels in the circulation lower, the negative-feedback (HPT axis) regulation mechanism will produce more TSH, which in turn impels the thyroid to produce more thyroid hormones in order to maintain the balance of thyroid homeostasis.28,29 The present study confirmed that perchlorate caused toxicity to the thyroid. The effects of medium- and high-dose perchlorate on body weight gain in rat were presented in this study. These results were generally consistent with our previous findings22 and other findings.30,31 The current study has provided preliminary but important data for further study of perchlorate biomonitoring. The method for measuring the concentration of urinary perchlorate in rats were preliminarily established, and the urinary perchlorate levels combined with the thyroid function data contributed to better understanding of the potential linkage between perchlorate exposure and health effect on rats. But the relationship between urinary perchlorate concentration and perchlorate exposure dose on occupational workers is still unclear. Further studies are required to test the real-world applicability of our method to determine the concentration of urinary perchlorate in AP-exposed occupational workers. Future studies may further investigate the dosage range over which the dose-response relationship can be assessed.
Funding This work was supported by the National Natural Science Foundation of the People’s Republic of China (no. 30972452).
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References 1. Blount BC, Pirkle JL, Osterloh JD, et al. Urinary perchlorate and thyroid hormone levels in adolescent and adult men and women living in the United States. Environ Health Perspect. 2006;114:1865–1871. doi: 10.1289/ehp.9466. 2. Trumbo PR. Perchlorate consumption, iodine status, and thyroid function. Nutr Rev. 2010;68:62–66. 3. Zhang T, Qian W, Hong WS, et al. Perchlorate and iodide in whole blood samples from infants, children, and adults in Nanchang, China. Environ Sci Technol. 2010;44:6947–6953. 4. Krska R, Becalski A, Braekevelt E, et al. Challenges and trends in the determination of selected chemical contaminants and allergens in food. Anal Bioanal Chem. 2012;402:139–162. 5. Dyke JV, Ito K, Obitsu T, et al. Perchlorate in dairy milk. Comparison of Japan versus the United States. Environ Sci Technol. 2007;41:88–92. 6. Hershman JM. Perchlorate and thyroid function: what are the environmental issues? Thyroid. 2005;15:427–31. 7. Stoker TE, Ferrell JM, Laws SC, et al. Evaluation of ammonium perchlorate in the endocrine disruptor screening and testing program’s male pubertal protocol: ability to detect effects on thyroid endpoints. Toxicology. 2006;228:58–65. 8. McLanahan ED, Andersen ME, Campbell JR Jr, et al. Competitive inhibition of thyroidal uptake of dietary iodide by perchlorate does not describe perturbations in rat serum total T4 and TSH. Environ Health Perspect. 2009;117:731–738. 9. Kirk AB, Martinelango PK, Tian K, et al. Perchlorate and iodide in dairy and breast milk. Environ Sci Technol. 2005;39:2011–2017. 10. Valentin-Blasini L, Mauldin JP, Maple D, et al. Analysis of perchlorate in human urine using ion chromatography and electrospray tandem mass spectrometry. Anal Chem. 2005;77:2475–2481. 11. Blount BC, Rich DQ, Valentin-Blasini L, et al. Perinatal exposure to perchlorate. thiocyanate, and nitrate in New Jersey mothers and newborns. Environ Sci Technol. 2009;43:7543–7549. 12. Oldi JF, Kannan K. Analysis of perchlorate in human saliva by liquid chromatography-tandem mass spectrometry. Environ Sci Technol. 2009;43:142–147. 13. Kannan K, Praamsma ML, Oldi JF, et al. Occurrence of perchlorate in drinking water, groundwater, surface water and human saliva from India. Chemosphere. 2009;76:22–26. 14. Otero-Santos SM, Delinsky AD, Valentin-Blasini L, et al. Analysis of perchlorate in dried blood spots using ion chromatography and tandem mass spectrometry. Anal Chem. 2009;81: 1931–1936. 15. Blount BC, Valent´ın-Blasini L. Using Biomonitoring to Assess Human Exposure to Perchlorate. Atlanta, GA: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention; 2006:97–98.
Chen et al. 16. Yu L, Cheng QQ, Canas J, et al. Challenges in determining perchlorate in biological tissues and fluids: implications for characterizing perchlorate exposure. Anal Chim Acta. 2006;567:66–72. 17. Li YT, George EJ. Analysis of perchlorate in water by reversedphase LC/ESI-MS/MS using an internal standard technique. Anal Chem. 2005;77:4453–4458. 18. Srinivasan A, Viraraghavan T. Perchlorate: health effects and technologies for its removal from water resources. Int J Environ Res Public Health. 2009;6:1418–1442. 19. (9) Agency for Toxic Substances and Disease Registry. Toxicological Profile for Perchlorate. Atlanta, GA: US Department of Health and Human Services, Public Health Service; 2008:153–154. Available at: http://www.atsdr.cdc.gov/ toxprofiles/tp162.html. Accessed October 15, 2008. 20. Lamm SH, Braverman LE, Li FX, et al. Thyroid health status of ammonium perchlorate workers: a cross-sectional occupational health study. J Occup Environ Med. 1999;41:248–260. 21. Blount BC, Valent´ın-Blasini L. Biomonitoring as a method for assessing exposure to perchlorate. Thyroid. 2007;17:837–841. 22. Wu FH, Zhou X, Zhang R, et al. The effects of ammonium perchlorate on thyroid homeostasis and thyroid-specific gene expression in rat. Environ Toxicol. 2012;27:445–452. 23. York RG, Funk KA, Girard MF, et al. Oral (drinking water) developmental toxicity study of ammonium perchlorate in SpragueDawley rats. Int J Toxicol. 2003;22:453–464. 24. Carrasco N. Iodide transport in the thyroid gland. Biochim Biophys Acta. 1993;1154:65–82. 25. Kopp P. Thyroid hormone synthesis In: Braverman LE, Cooper DS, eds. Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text. 10th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2012:48–74. 26. Capen CC. Mechanisms of chemical injury of thyroid gland. Prog Clin Biol Res. 1994;387:173–191. 27. Park JW, Rinchard J, Liu F, et al. The thyroid endocrine disruptor perchlorate affects reproduction, growth, and survival of mosquitofish. Ecotoxicol Environ Saf. 2006;63:343–352. 28. Capen CC. Mechanistic data and risk assessment of selected toxic endpoints of the thyroid gland. Toxicol Pathol. 1997;25:39–48. 29. Capen CC. Correlation of mechanistic data and histopathology in the evaluation of selected toxic endpoints of the endocrine system. Toxicol Lett. 1998;102/103:405–409. 30. Mukhian S, Patino R. Effects of prolonged exposure to perchlorate on thyroid and reproductive function in zebrafish. Toxicol Sci. 2007;96:246–254. 31. McNabb FM, Jang DA, Larsen CT, et al. Does thyroid function in developing birds adapt to sustained ammonium perchlorate exposure? Toxicol Sci. 2004;82:106–113. 32. Wolff J. Perchlorate and thyroid gland. Pharmacol Rev. 1998;50:89–106.
Archives of Environmental & Occupational Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vaeh20
Dose-Response Relationship Between Orally Administered Ammonium Perchlorate and Urine Perchlorate Concentrations in Rats: Possible Biomarker to Quantify Environmental Ammonium Perchlorate Exposure on Thyroid Homeostasis ab
b
c
b
b
Hong Xia Chen , Miao Hong Ding , Yong Gan Li , Qin Liu & Kai Liang Peng a
Institute of Biomedicine, Taihe Hospital, Hubei University of Medicine, Shiyan, People's Republic of China b
Click for updates
Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China c
Centers for Disease Control and Prevention of Hubei Provence, Wuhan, People's Republic of China Accepted author version posted online: 27 Jun 2014.
To cite this article: Hong Xia Chen, Miao Hong Ding, Yong Gan Li, Qin Liu & Kai Liang Peng (2015) Dose-Response Relationship Between Orally Administered Ammonium Perchlorate and Urine Perchlorate Concentrations in Rats: Possible Biomarker to Quantify Environmental Ammonium Perchlorate Exposure on Thyroid Homeostasis, Archives of Environmental & Occupational Health, 70:5, 286-290, DOI: 10.1080/19338244.2014.904265 To link to this article: http://dx.doi.org/10.1080/19338244.2014.904265
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Archives of Environmental & Occupational Health (2015) 70, 286–290 C Taylor & Francis Group, LLC Copyright ISSN: 1933-8244 print / 2154-4700 online DOI: 10.1080/19338244.2014.904265
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Dose-Response Relationship Between Orally Administered Ammonium Perchlorate and Urine Perchlorate Concentrations in Rats: Possible Biomarker to Quantify Environmental Ammonium Perchlorate Exposure on Thyroid Homeostasis HONG XIA CHEN1,2, MIAO HONG DING2, YONG GAN LI3, QIN LIU2, and KAI LIANG PENG2 1
Institute of Biomedicine, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China 3 Centers for Disease Control and Prevention of Hubei Provence, Wuhan, People’s Republic of China 2
Received 11 September 2013, Accepted 19 February 2014
To evaluate the feasibility of urine perchlorate as a biomarker of ammonium perchlorate (AP) exposure and to explore the correlation between the thyroid function indicators and the perchlorate concentrations, a sensitive and selective ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) method was developed to detect perchlorate in urine samples. Rats were orally administrated with different doses of perchlorate. Serum free thyroxine (FT4 ), free triiodothyronine (FT3 ), and thyroid-stimulating hormone (TSH) were determined by radioimmunoassays. The results showed that a dose of AP up to 520 mg kg−1 body weight induced a significant increase of TSH, with a decrease of FT4 . Particularly, the levels of urine perchlorate increased dose-dependently on AP exposure from drinking water. The findings highlighted that urine perchlorate may be a useful biomarker for AP environmental exposure. Keywords: ammonium perchlorate, biomarker, thyroid hormone, urine perchlorate
Recently, there has been an increased awareness of the possible detrimental thyroid-related effects of environmental perchlorate, especially in iodine-deficient populations.1 Therefore, there is considerable interest and an urgent need to develop a highly sensitive and selective biomonitoring method to accurately assess perchlorate exposure for specific populations. Human biomonitoring data indicate that exposure to perchlorate occurs worldwide, including highly populated countries such as the United States, China, Canada, Japan, and India.2–5 Accurate assessment of exposure to perchlorate is critical to detecting biochemical end points potentially related to exposure. In the past, serum thyroid-stimulating hormone (TSH) and thyroxine (T4 ) were considered sensitive biomarkers of thyroidal effects induced by AP in light of the compensatory mechanism.6–8 However, the thyroidal function Address correspondence to Hong Xia Chen, Institute of Biomedicine, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, People’s Republic of China. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/vaeh.
indicators are not specific biomarkers due to the complexity of hypothalamic-pituitary-thyroid axis (HPT axis) regulation mechanism. Furthermore, other antithyroid agents such as carbimazole can have similar effects on thyroid homeostasis. The likelihood of widespread human exposure coupled with potential health effects supports the need to develop a biomonitoring method for assessing the actual perchlorate exposure by directly measuring perchlorate in biological matrixes, such as urine. This refers to the biomarkers of exposure, which can accurately reflect the level of internal exposure in the human body burden. Useful human exposure data have been obtained by directly measuring levels of perchlorate in human urine, milk, amniotic fluid, saliva, and blood.9–14 Quantification of perchlorate in biological tissues and fluids is an excellent method for evaluating both the prevalence and magnitude of exposure.15 Furthermore, measurement of perchlorate in biological matrices provides data on the total dose of perchlorate integrated from all sources.16 In the present study, the feasibility of urine perchlorate as a biomarker of ammonium perchlorate (AP) exposure was evaluated, and the potential correlation of the urinary perchlorate concentrations to the thyroid function was investigated.
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Table 1. Changes of Body Weight Gain in AP-Treated Rat Cumulative net increase in the amount of weekly weight (g)
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Control (0 mg kg−1 bw)
Low (130 mg kg−1 bw)
Medium (260 mg kg−1 bw)
High (520 mg kg−1 bw)
Exposure time (week)
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Initial body weight 1 2 3 4 5 6 7 8 9 10 11 12 13
188.00 73.60 136.60 183.20 221.20 250.40 283.40 312.80 326.40 350.00 373.00 392.20 407.60 417.40
38.68 6.62 9.13 12.58 19.27 21.62 26.37 24.95 28.45 25.48 30.19 33.18 37.71 41.62
182.33 60.17∗∗ 106.67∗∗ 151.83∗ 178.83∗ 224.50 252.83 274.00 283.50 294.33 305.17 311.83∗ 320.50∗ 333.17
27.85 9.75 18.43 28.90 40.20 50.42 58.72 67.86 74.42 81.53 87.37 91.79 99.71 104.61
178.17 55.17∗∗ 100.50∗∗ 135.33∗∗ 159.67∗∗ 191.33∗ 208.33∗∗ 224.00∗∗ 233.17∗∗ 239.67∗∗ 245.50∗∗ 242.00∗∗ 246.00∗∗ 254.33∗∗
18.94 6.05 10.39 14.90 19.24 28.01 33.04 32.03 38.63 51.06 56.65 54.98 57.47 69.74
176.17 59.50∗∗ 115.00∗ 148.17∗∗ 168.00∗∗ 206.83∗ 216.17∗ 214.50∗∗ 210.00∗∗ 209.83∗∗ 208.00∗∗ 209.83∗∗ 213.13∗∗ 216.33∗∗
25.95 4.51 9.96 12.12 15.21 25.94 31.88 38.39 40.46 44.49 45.66 48.73 51.04 102.27
∗p
< .05 and ∗∗ p < .01 compared with control group values.
The study focused on combining urinary perchlorate data and thyroid function data in assessing the population exposed to perchlorate.
Methods Animal and Treatment Twenty-four healthy male Sprague-Dawley rats (28 days old, weighing 80–100 g) were randomly divided into 4 groups of 6 rats. The control was treated without AP, orally administrated by tap water only, with the 3 exposure groups given different doses of AP (130, 260, and 520 mg kg−1 body weight [bw], respectively), in tap water every day by oral administration (gavage) for 13 consecutive weeks. The rats were weighed regularly every week, and the daily administrate doses based on the rats’ body weight. The concentration of AP in tap water was adjusted weekly for these groups, based on measured body weight, in order to achieve the desired dosage levels. The doses used in the present study were based on our preliminary experiment, which determined the oral doses required to alter T4 concentrations. Doses selected which would range from a no significant effect on serum hormone level to a maximum reduction in thyroid hormone. The rats were euthanized after orally administered with AP for 13 consecutive weeks. Urine and serum samples were collected, especially with approximately 1 mL reflex voiding urine sample by extrusion from rats’ bladder. The apparatus used in the urine sampling should be kept clean and dry, avoiding perchlorate contamination in the environmental background; and the feces must be separated from urine, avoiding the urine samples being contaminated. All procedures on animals followed the Guide for the Care and Use of Laboratory Animals published by Ministry of Health of People’s Republic of China.
Instrumental Analysis Urinary perchlorate concentrations were determined by highperformance liquid chromataography (HPLC) based on the method as described by Li and George.17 All solutions were prepared from American Chemical Society (ACS) reagentgrade chemicals in 18 M water obtained from a Water Pro PS purification system (Labconco, Kansas City, MO, USA). The urine perchlorate separation was carried out on Agilent Technologies 1200 ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) system with a ZORBAX SB C18 column (2.1 mm × 50 mm, 1.8 μm) (Agilent, USA). Supernatants of ethanol precipitated rat urine were evaporated to dryness under nitrogen and reconstituted in deionized water. All test solutions were syringe filtered, mixed with an 18O4 -labeled perchlorate internal standard, and directly injected with 5 μL volumes of matric samples into a liquid chromatography–tandem triple-quadrupole mass spectrometry (LC-MS/MS) system. Ionization was accomplished using electrospray ionization in negative mode. The labeled internal standard was corrected for any sample matrix effects on measured signals. Four parent-to-product ion transitions, for loss of oxygen, were monitored for native and 18O4 -labeled perchlorate anion, respectively: 35Clperchlorate, m/z 99 → 83 and 107 → 89; 37Cl-perchlorate, m/z 101 → 85 and 109 → 91. Hormone Analyses Radioimmunoassay (RIA) kits for the hormone measurements were all from the same batch number and with the same expiration date for all the standard and unknown samples. Serum free thyroxine (FT4 ), free triiodothyronine (FT3 ), and thyroid-stimulating hormone (TSH) radioimmunoassays were performed in triplicate, following the manufacturer’s
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Chen et al. Table 3. The Urinary Perchlorate Concentrations of Rats Orally Administrated Different Ammonium Perchlorate (AP) Doses in Each Group Urinary perchlorate (mg/L) Group
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Fig. 1. Body weight gain of each group over the treatment period.
instructions (North Biotechnology Institute, Beijing, China). Tracer (125I) radioactivity was measured using a γ counter (GC-1200 gamma radioimmunoassay counter; Anhui, China).
AP exposure dose (mg kg−1 bw)
Mean
SD
0 130 260 520
0.738 856.99 2535.87∗ 2796.23∗†
0.288 355.22 2095.90 1650.24
Control (n = 6) Low (n = 4)a Medium (n = 6) High (n = 6) aIn
the feeding process, 2 animals died accidentally. with low-dose group, p < .05. ∗ Compared with control group, p < .01. †Compared
Effect of Ammonium Perchlorate on Serum Hormones (FT4 , FT3 , and TSH)
Statistical Method Statistical analysis of the data was performed with SPSS (version 12.0; SPSS, Chicago, IL, USA, 2003). Comparisons between the treatment groups and control group were done with 1-way analysis of variance (ANOVA). The results were expressed as the mean ± SD, and statistical significance was set at p < .05.
As shown in Table 2, FT4 levels were significantly decreased in the low- (130 mg kg−1 bw), medium- (260 mg kg−1 bw), and high- (520 mg kg−1 bw) dose AP groups (p < .05, p < .05, and p < .01, respectively). TSH level of the high-dose AP group (520 mg kg−1 bw) was significantly higher than that of the control group (p < .05). FT3 levels of the low- and medium-dose AP groups have a decreasing trend, but the only significant mean difference was observed in low-dose group (130 mg kg−1 bw) when compared with the control group.
Results Effect of Ammonium Perchlorate on Body Weight in Rats As seen in Table 1, compared with control group, 13-week treatment with 130 mg kg−1 bw AP did not lead to significant change of body weight, but a significant decrease in body weight gain was observed in medium- (260 mg kg−1 bw) and high- (520 mg kg−1 bw) dose AP groups (p < .01, respectively). And the body weight gain of rats decreased in a dose-dependent manner with the AP exposure, which gradually slowed down, with the weight growth curve stabilizing at the 6th week. Body weight gain of each group over the treatment period is shown in Figure 1. Both medium and high AP exposure can obviously impede the body weight gain of rats (Figure 1).
Correlation Between Environmental Perchlorate Exposure and Urinary Perchlorate Concentration Figure 2 shows that the concentration of AP in urine increased proportionally in a dose-related manner, with a linear relationship between exposure dose in the drinking water and urinary perchlorate levels (R2 = .8324). As shown in Table 3, a statically significant increase of urinary perchlorate was observed in the medium- and high-dose AP group (p < .01, respectively) compared with the control group. Furthermore, markedly higher levels of the urinary perchlorate were detected in rats with high dose of AP than those in the low-dose group, demonstrating that urine perchlorate could be used as the biomarkers indicating the actual exposure intensity.
Table 2. Values of Thyroid Hormone Parameters in Each Group
Group Control Low Medium High
FT4 (fmol/mL)
FT3 (fmol/mL)
TSH (mIU/L)
AP exposure dose (mg kg−1 bw)
No. of animal
Mean
SD
Mean
SD
Mean
SD
0 130 260 520
6 4a 6 6
12.76 9.22∗ 8.06∗ 5.71∗∗
2.72 2.76 2.10 2.97
1.55 0.88∗ 1.05 1.65
0.78 0.41 0.51 1.12
0.88 1.00 0.86 1.17∗
0.37 0.30 0.30 0.34
Note. AP = ammonium perchlorate; FT4 = free thyroxine; FT3 = free triiodothyronine; TSH = thyroid-stimulating hormone. aIn the feeding process, 2 animals died accidentally. ∗ p < .05 and ∗∗ p < .01 compared with control group values.
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Fig. 2. The correlation between perchlorate dose in drinking water and the perchlorate concentration in urine of rats.
Comment Perchlorate is highly water soluble and environmentally stable,18 so that the general population is vulnerable to the perchlorate exposure through drinking water and food. Serum thyroid function parameters (e.g., FT3 , FT4 , TSH) were selected 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 intensity. Just as seen in Table 2, only FT3 level in the low-dose group decreased and TSH level in the high-dose group increased with the increased dose. Choosing a sensitive, specific biomarker in assessing the exposure of occupational workers is urgent. In this study, we established a determination method of urine perchlorate in rats and evaluated the dose-response relationship between AP exposure and urine perchlorate. The linear relationship between AP exposure dose in drinking water and urinary perchlorate levels was observed, and the concentration of AP in urine increased proportionally in a dose-related manner (Table 3). Furthermore, the urinary perchlorate level of the high-dose group was markedly higher than that of the low-dose group, which demonstrated that urine perchlorate could be used as the biomarker indicating the actual exposure intensity. Previous studies have indicated that urine is the main excretory pathway for absorbed perchlorate, which could make urine a convenient testing medium for perchlorate levels. Furthermore, urine perchlorate has previously been confirmed as a biomarker that is specific for exposure to perchlorate.19 Urine perchlorate is reasonably indicative of human exposure because 70%–95% of a perchlorate dose is excreted unchanged in the urine, with a half-life of 8–14 hours.19,20 Urine collection is less invasive than blood collection, and urinary perchlorate levels tend to be much higher than serum levels due to efficient renal clearance of perchlorate. Nevertheless, measuring perchlorate in human urine assesses the combined exposure from all sources.21 Determination of perchlorate in urine is the preferred choice for evaluation of environmental exposure. Following the development of sensitive and specific measurement method such as the UHPLC system, urinary perchlorate has the potential to be a novel and effective biomarker for evaluation the low level of occupational exposure.
289 In this study, both medium- (260 mg kg−1 bw) and high(520 mg kg−1 bw) dose AP groups revealed a significant decrease in body weight gain. Furthermore, both FT3 levels in the low-dose group and FT4 levels in the medium- and high-dose groups were significantly lower than those of the control group. In addition, the TSH levels in the high-dose group were significantly increased when compared with the control group. These results suggest that perchlorate could significantly decrease FT4 levels and elevate TSH levels, which are consistent with previous studies.1,22,23 Levels of T3 , T4 , and TSH in serum are considered to be biomarkers of perchlorate exposure on thyroid homeostasis for a long time.19 Since perchlorate anions have an identical charge and similar ionic radius to iodide (I−), perchlorate is known as a strong inhibitor of the sodium-iodide importer (NIS), which is the primary mechanism by which iodide enters thyroid follicle cells from the blood, and is the first step in the uptake of iodide into the thyroid leading to the formation of thyroid hormones.24,25 Thyroid hormones (THs) play a crucial role in differentiation, metabolism, growth, and development. TH is the key factor required for the normal function of nearly all tissues, with major effects on oxygen consumption and metabolic rate. If the thyroid gland is exposed to some specific thyroid toxicants such as perchlorate, thiocyanate, and nitrate over a period of time, the biosynthesis of thyroid hormones may be weakened.26,27 With thyroid hormone levels in the circulation lower, the negative-feedback (HPT axis) regulation mechanism will produce more TSH, which in turn impels the thyroid to produce more thyroid hormones in order to maintain the balance of thyroid homeostasis.28,29 The present study confirmed that perchlorate caused toxicity to the thyroid. The effects of medium- and high-dose perchlorate on body weight gain in rat were presented in this study. These results were generally consistent with our previous findings22 and other findings.30,31 The current study has provided preliminary but important data for further study of perchlorate biomonitoring. The method for measuring the concentration of urinary perchlorate in rats were preliminarily established, and the urinary perchlorate levels combined with the thyroid function data contributed to better understanding of the potential linkage between perchlorate exposure and health effect on rats. But the relationship between urinary perchlorate concentration and perchlorate exposure dose on occupational workers is still unclear. Further studies are required to test the real-world applicability of our method to determine the concentration of urinary perchlorate in AP-exposed occupational workers. Future studies may further investigate the dosage range over which the dose-response relationship can be assessed.
Funding This work was supported by the National Natural Science Foundation of the People’s Republic of China (no. 30972452).
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References 1. Blount BC, Pirkle JL, Osterloh JD, et al. Urinary perchlorate and thyroid hormone levels in adolescent and adult men and women living in the United States. Environ Health Perspect. 2006;114:1865–1871. doi: 10.1289/ehp.9466. 2. Trumbo PR. Perchlorate consumption, iodine status, and thyroid function. Nutr Rev. 2010;68:62–66. 3. Zhang T, Qian W, Hong WS, et al. Perchlorate and iodide in whole blood samples from infants, children, and adults in Nanchang, China. Environ Sci Technol. 2010;44:6947–6953. 4. Krska R, Becalski A, Braekevelt E, et al. Challenges and trends in the determination of selected chemical contaminants and allergens in food. Anal Bioanal Chem. 2012;402:139–162. 5. Dyke JV, Ito K, Obitsu T, et al. Perchlorate in dairy milk. Comparison of Japan versus the United States. Environ Sci Technol. 2007;41:88–92. 6. Hershman JM. Perchlorate and thyroid function: what are the environmental issues? Thyroid. 2005;15:427–31. 7. Stoker TE, Ferrell JM, Laws SC, et al. Evaluation of ammonium perchlorate in the endocrine disruptor screening and testing program’s male pubertal protocol: ability to detect effects on thyroid endpoints. Toxicology. 2006;228:58–65. 8. McLanahan ED, Andersen ME, Campbell JR Jr, et al. Competitive inhibition of thyroidal uptake of dietary iodide by perchlorate does not describe perturbations in rat serum total T4 and TSH. Environ Health Perspect. 2009;117:731–738. 9. Kirk AB, Martinelango PK, Tian K, et al. Perchlorate and iodide in dairy and breast milk. Environ Sci Technol. 2005;39:2011–2017. 10. Valentin-Blasini L, Mauldin JP, Maple D, et al. Analysis of perchlorate in human urine using ion chromatography and electrospray tandem mass spectrometry. Anal Chem. 2005;77:2475–2481. 11. Blount BC, Rich DQ, Valentin-Blasini L, et al. Perinatal exposure to perchlorate. thiocyanate, and nitrate in New Jersey mothers and newborns. Environ Sci Technol. 2009;43:7543–7549. 12. Oldi JF, Kannan K. Analysis of perchlorate in human saliva by liquid chromatography-tandem mass spectrometry. Environ Sci Technol. 2009;43:142–147. 13. Kannan K, Praamsma ML, Oldi JF, et al. Occurrence of perchlorate in drinking water, groundwater, surface water and human saliva from India. Chemosphere. 2009;76:22–26. 14. Otero-Santos SM, Delinsky AD, Valentin-Blasini L, et al. Analysis of perchlorate in dried blood spots using ion chromatography and tandem mass spectrometry. Anal Chem. 2009;81: 1931–1936. 15. Blount BC, Valent´ın-Blasini L. Using Biomonitoring to Assess Human Exposure to Perchlorate. Atlanta, GA: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention; 2006:97–98.
Chen et al. 16. Yu L, Cheng QQ, Canas J, et al. Challenges in determining perchlorate in biological tissues and fluids: implications for characterizing perchlorate exposure. Anal Chim Acta. 2006;567:66–72. 17. Li YT, George EJ. Analysis of perchlorate in water by reversedphase LC/ESI-MS/MS using an internal standard technique. Anal Chem. 2005;77:4453–4458. 18. Srinivasan A, Viraraghavan T. Perchlorate: health effects and technologies for its removal from water resources. Int J Environ Res Public Health. 2009;6:1418–1442. 19. (9) Agency for Toxic Substances and Disease Registry. Toxicological Profile for Perchlorate. Atlanta, GA: US Department of Health and Human Services, Public Health Service; 2008:153–154. Available at: http://www.atsdr.cdc.gov/ toxprofiles/tp162.html. Accessed October 15, 2008. 20. Lamm SH, Braverman LE, Li FX, et al. Thyroid health status of ammonium perchlorate workers: a cross-sectional occupational health study. J Occup Environ Med. 1999;41:248–260. 21. Blount BC, Valent´ın-Blasini L. Biomonitoring as a method for assessing exposure to perchlorate. Thyroid. 2007;17:837–841. 22. Wu FH, Zhou X, Zhang R, et al. The effects of ammonium perchlorate on thyroid homeostasis and thyroid-specific gene expression in rat. Environ Toxicol. 2012;27:445–452. 23. York RG, Funk KA, Girard MF, et al. Oral (drinking water) developmental toxicity study of ammonium perchlorate in SpragueDawley rats. Int J Toxicol. 2003;22:453–464. 24. Carrasco N. Iodide transport in the thyroid gland. Biochim Biophys Acta. 1993;1154:65–82. 25. Kopp P. Thyroid hormone synthesis In: Braverman LE, Cooper DS, eds. Werner and Ingbar’s The Thyroid: A Fundamental and Clinical Text. 10th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2012:48–74. 26. Capen CC. Mechanisms of chemical injury of thyroid gland. Prog Clin Biol Res. 1994;387:173–191. 27. Park JW, Rinchard J, Liu F, et al. The thyroid endocrine disruptor perchlorate affects reproduction, growth, and survival of mosquitofish. Ecotoxicol Environ Saf. 2006;63:343–352. 28. Capen CC. Mechanistic data and risk assessment of selected toxic endpoints of the thyroid gland. Toxicol Pathol. 1997;25:39–48. 29. Capen CC. Correlation of mechanistic data and histopathology in the evaluation of selected toxic endpoints of the endocrine system. Toxicol Lett. 1998;102/103:405–409. 30. Mukhian S, Patino R. Effects of prolonged exposure to perchlorate on thyroid and reproductive function in zebrafish. Toxicol Sci. 2007;96:246–254. 31. McNabb FM, Jang DA, Larsen CT, et al. Does thyroid function in developing birds adapt to sustained ammonium perchlorate exposure? Toxicol Sci. 2004;82:106–113. 32. Wolff J. Perchlorate and thyroid gland. Pharmacol Rev. 1998;50:89–106.
