Radioactive Iodide (131I−) Excretion Profiles in Response to Potassium Iodide (KI) and Ammonium Perchlorate (NH4ClO4) Prophylaxis

Radioactive iodide (131I−) protection studies have focused primarily on the thyroid gland and disturbances in the hypothalamic-pituitary-thyroid axis. The objective of the current study was to establish 131I− urinary excretion profiles for saline, and the thyroid protectants, potassium iodide (KI) and ammonium perchlorate over a 75 hour time-course. Rats were administered 131I− and 3 hours later dosed with either saline, 30 mg/kg of NH4ClO4 or 30 mg/kg of KI. Urinalysis of the first 36 hours of the time-course revealed that NH4ClO4 treated animals excreted significantly more 131I− compared with KI and saline treatments. A second study followed the same protocol, but thyroxine (T4) was administered daily over a 3 day period. During the first 6–12 hour after 131I− dosing, rats administered NH4ClO4 excreted significantly more 131I− than the other treatment groups. T4 treatment resulted in increased retention of radioiodide in the thyroid gland 75 hour after 131I− administration. We speculate that the T4 treatment related reduction in serum TSH caused a decrease synthesis and secretion of thyroid hormones resulting in greater residual radioiodide in the thyroid gland. Our findings suggest that ammonium perchlorate treatment accelerates the elimination rate of radioiodide within the first 24 to 36 hours and thus may be more effective at reducing harmful exposure to 131I− compared to KI treatment for repeated dosing situations. Repeated dosing studies are needed to compare the effectiveness of these treatments to reduce the radioactive iodide burden of the thyroid gland.

radioiodide in the thyroid gland. Our findings suggest that ammonium perchlorate treatment accelerates the elimination rate of radioiodide within the first 24 to 36 hours and thus may be more effective at reducing harmful exposure to 131 I − compared to KI treatment for repeated dosing situations. Repeated dosing studies are needed to compare the effectiveness of these treatments to reduce the radioactive iodide burden of the thyroid gland.

Introduction
Stable iodide ( 127 I − ), as potassium iodide (KI) or "dietary iodine", has been recognized for over 30 years as a practical thyroid radioprotectant for people exposed to radioactive isotopes of iodide [1,2]. In a recent National Academies of Science report on the distribution and administration of KI in the event of a nuclear incident [3], sodium perchlorate (Irenat ® ) was recommended for adults if KI treatment was contraindicated, such as in patients with preexisting thyroid disease. Successful thyroid radioprotectant treatments, such as KI or ammonium perchlorate (NH 4 ClO 4 ), are judged by their ability to block or limit thyroidal uptake of trace amounts of radioactive iodide (e.g., 131 I − ), while limited research has focused on treatment doses promoting excretion of the isotopes. Currently, the United States Food and Drug Administration (US FDA) recommends KI tablets as a preventative treatment for 131 I − poisoning of the thyroid gland [4].
Studies utilizing rats in the 1950s and 1960s demonstrated that perchlorate (ClO 4 − ) altered the serum pharmacokinetics of 131 I − [5][6][7]. The kinetic profile of 131 I − in laboratory animals treated with ClO 4 − was characterized by decreased serum 131 I − levels and increased urine 131 I − levels [6]. In the presence of ClO 4 − , 131 I − is excreted in urine more rapidly, presumably because perchlorate blocks basolateral and/or apical uptake of 131 I − into sodium iodide symporter-or pendrin-protein rich tissues, such as the thyroid and small intestine [5,[8][9][10][11][12]. Additionally, perchlorate administration displaces non-organified iodide from the thyroid, and the perchlorate discharge test has been used to diagnose iodide organification defects in humans for decades [10]. Perchlorate has been shown to have side effects ranging from rash and fever to several fatal cases of aplastic anemia [13,14], though these effects typically manifest from large bolus doses administered over weeks or months [15].
Rats administered ClO 4 − intravenously (0.1-3.0 mg/kg) excreted approximately 83% of the administered ClO 4 − dose over 24 hours [16]. In a similar experiment, these authors also intravenously dosed rats with 3.3 mg/kg of isotonically labeled ClO 4 − ( 36 ClO 4 ) and reported that 96% of the dose was excreted in 24 hours and 99.5% by 48 hours [17]. In a recent study, an oral dose of 30 mg/kg of ClO 4 − was administered to rats, and 38% of the dose was recovered in urine within the first 12 hours after dosing [15]. In the present rat study, the effectiveness of KI and NH 4 ClO 4 to increase excretion of 131 I − into urine over a 3 day period was assessed, with and without T 4 administration. In previous rat studies we reported that over a 15 hour post dosing period, KI and NH 4 ClO 4 were equally effective at reducing thyroid gland exposure to 131 I − [15]. However, NH 4 ClO 4 was more effective at increasing urinary clearance of 131 I − than KI. This study extends the timeframe of the experiment from 15 to 75 hours and also evaluates the influence of T 4 administration on 131 I − kinetics. Rats, unlike humans, have short thyroxine half-lives in serum, and are sensitive to up-regulation of the thyroid gland by thyroid stimulating hormone (TSH) [18]. To control the potential for TSH mediated stimulation of the thyroid gland, one subset of rats for each treatment group received replacement doses of T 4 to suppress the potential for TSH release [19,20].

Animals and Experimental Design
Male Sprague-Dawley rats (330 ± 30 g, approximately 11 weeks old) from Harlan Laboratories (Indianapolis, IN, USA) were provided LabDiet™ Laboratory Rodent Diet 5001 rat chow and water ad libitum. The animals used in this study were handled in accordance with the procedures of The University of Georgia Institutional Animal Care and Use Committee (IACUC), AUP# A2005-10110-0. The rats experienced a 12 hour light and dark cycle, with room air temperature at 22 ± 2 °C and relative humidity at 50 ± 20%.
Rats were housed individually in metabolism cages for a 5 day acclimation period prior to the start of the experiments. Twelve hours before the experiment commenced, food was removed from the animals to ensure complete absorption of the radiotracer and treatment doses. A summary of the experiments is shown in Table 1. The general experimental protocol was to dose rats orally with 2.91 µCi (6 ng/kg) 131 I − in saline solution (1 mL) by oral gavage and then return the rats to their metabolism cages. Rats from Group 1 (n = 6 for each treatment group) were removed from their metabolism cages after 3 hours and dosed by oral gavage with 1 mL of either 0.9% saline, 30 mg/kg of KI (calculated as iodide) or 30 mg/kg of NH 4 ClO 4 (calculated as ClO 4 − ) dissolved in 0.9% aqueous saline. Rats from Group 2 (n = 6 for each treatment group) followed the same experimental protocol as Group 1 except each animal received a 0.1 mL intraperitoneal (ip) injection of 15 µg/kg of T 4 (based on euthyroid replacement T 4 doses administered by [21]) dissolved in 0.1 M NaOH and 1 animal from each treatment group received a 0.1 mL ip injection of 0.1 M NaOH (controls). Data from animals in Group 2 that received ip injections of NaOH had not statistically different from animals of a similar treatment in Group 1. As a result, data from animals in Group 2 that received ip injections of NaOH were assimilated with animals of a similar treatment in Group 1. The rats were held in metabolism cages for 75 hours for urine collections. Urine from Groups 1 and 2 were collected via metabolism cage vials at +3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, and 75 hours. Blood was collected from the tail vein of animals in dose Groups 1 and 2 at 15 hours after dosing with 131 I − and at sacrifice. At sacrifice (75 hours after dosing with 131 I − ), animals in Groups 1 and 2 were anesthetized with a ketamine cocktail (50 mg/kg ketamine, 3.3 mg/kg xylazine, and 3.4 mg/kg acepromazine administered at 0.1 mL per 100 g BW), then killed by asphyxiation at +75 hours. Blood was collected via cardiac puncture and serum prepared by centrifugation at 1,500 rpm at 4 °C for 15 min. Thyroid lobes were removed from the trachea and weighed. Urine was removed from the bladder via syringe. Sera, urine, and thyroid glands were stored at −80 °C until analysis.

131 I − Analysis
Serial urine samples were placed on a gamma counter (1470 Wallac Wizard) equipped with one detector and 131 I − counts/minute (cpm) were measured within 2 hours after collection. 131 I − cpm were also assessed in whole thyroids and serum samples within 2 hours of sacrifice. Raw counts were recorded. Urine and sera were then stored at −80 °C for no less than 80 days (10 half-lives for 131 I − ) in order for the radioactivity to decay.
Serum TSH measurements were made using a rat TSH radioimmunoassay kit from A. F. Parlow and the National Hormone & Peptide Program (lot numbers AFP329691Rb, AFP11542B, and AFP5512B).

127 I − and ClO 4 − Analysis
Non-radioactive analytes ( 127 I − and ClO 4 − ) were quantified using ion chromatography coupled with tandem mass spectrometry. Serum samples were spiked with internal standard ( 129 I − and Cl 18 O 4 − ), treated to remove proteins, and analyzed by ion chromatography electrospray ionization tandem mass spectrometry [22]. Urine samples were spiked with internal standard ( 129 I − and Cl 18 O 4 − ) and analyzed by ion chromatography electrospray ionization tandem mass spectrometry [23].

Urinary Excretion Kinetics of Anions
All urine samples for Groups 1 and 2 were analyzed for 131 I − excretion kinetics. Animals in Groups 1 and 2 that received 127 I − or ClO 4 − also had urine samples analyzed for 127 I − or ClO 4 − excretion kinetics. All half-life calculations were prepared using Win Non Lin 5.2 software.

Statistical Analysis
Single factor analysis of variance (ANOVA) was used initially to determine significance across the treatment groups (control saline, KI and NH 4 ClO 4 ) with statistical significance set at p < 0.05. Once statistical significance was determined across the treatment groups by ANOVA, a limited number of comparisons were carried out using a two-sample t-test (assuming equal variance) to compare each treatment group (p < 0.05) to control and to each other. All calculations were performed using Microsoft Excel. It should be noted that animals in Group 2 that received ip injections of NaOH were lumped together with animals in Group 1 of a similar treatment dose, i.e., saline, KI, or ClO 4 − .

131 I − Excretion in Urine
Rats were administered 131 I − , then 3 hours later either saline, 30 mg/kg of KI or 30 mg/kg of NH 4 ClO 4 by oral bolus gavage. Group 2 was distinguished from Group 1 ( Table 1) (Figure 1(a,b)). The 24-hour urinary excretion half-lives for 131 I − (Table 2) in control and KI treated rats ranged from 3.5 to 4 hours; conversely, NH 4 ClO 4 treated rats excreted 131 I − more rapidly (2.6 hour urinary excretion half-life, p < 0.001). 2.6 ± 0.4 * ,# 2.6 ± 0.4 * ,# Rats for each Group were orally dosed with 131 I − and then 3 hours later orally dosed with saline, KI (30 mg/kg), or NH 4 ClO 4 (30 mg/kg). Group 2 rats also received ip injections of thyroxine (0.015 mg/kg) 3 hours following 131 I − administration and then every 24 hours until the conclusion of the experiment. Urine for each Group was collected for 24 hours; * statistically significantly lower than saline treatment (p < 0.001); # statistically significantly lower than KI treatment (p < 0.001). Figure 1. (a) Percent of total 131 I − dose excreted in urine 75 hour of Group 1 male rats collected via metabolism cages dosed with 131 I − followed by saline, KI (30 mg/kg), or perchlorate (30 mg/kg) 3 hours later (n = 12), statistical analysis was conducted on concentration data for each individual time-point (data not shown); (b) Percent of total 131 I − dose excreted in 75 hour urine of Group 2 male rats collected via metabolism cages dosed with 131 I − followed by saline, KI (30 mg/kg), or perchlorate (30 mg/kg) at +3 hours and dosed with replacement T 4 at +3, +27, and +51 hours (n = 6), statistical analysis was conducted on concentration data for each individual time-point (data not shown).

131 I − in Serum and Thyroid
The mean 131 I − concentration in the Group 1 control serum at 15 hours after 131 I − dosing was 1.37 ± 0.41 pg/mL and decreased to 0.50 ± 0.15 pg/mL at 75 hours post dosing (Figure 2a). In Group 1, the mean 131 I − serum levels in the KI and the NH 4 ClO 4 treatment groups at 15 hours were 0.91 ± 0.40 and 0.76 ± 0.29 pg/mL, and decreased to 0.16 ± 0.04 and 0.18 ± 0.09 pg/mL, respectively, at 75 hours post dosing. The 131 I − serum concentrations in the KI and NH 4 ClO 4 treatment groups were significantly less than saline controls for both sampling times (p < 0.05). The addition of T 4 proved to have little effect on mean serum 131 I − concentration for Group 2 (Figure 2b). The mean serum 131 I − concentrations at 15 hours following T 4 and saline, KI and NH 4 ClO 4 treatments was 1.2 ± 0.35, 1.06 ± 0.42, and 0.64 ± 0.33 pg/mL respectively, and decreased to 0.61 ± 0.33, 0.17 ± 0.12, and 0.22 ± 0.15 pg/mL at 75 hours post dosing. At 15 hours post 131 I − dosing, only the NH 4 ClO 4 treatment group 131 I − concentrations were significantly less (p < 0.05) than controls, while both KI and NH 4 ClO 4 treatment group 131 I − concentrations were significantly less than controls at the 75 hour sampling time.
Compared with control saline, KI and NH 4 ClO 4 treatment reduced levels of 131 I − in thyroid gland at 75 hours post exposure (Figure 3a). Also the residual 131 I − levels in the thyroid gland in the KI treatment group were lower than the NH 4 ClO 4 treatment group (p < 0.01). KI and NH 4 ClO 4 treatment reduced the thyroid content of 131 I − by 77 and 61%, respectively, 3 days after administration of 131 I − . Group 2 animals treated with T 4 displayed a different thyroidal 131 I − content (Figure 3b). The mean residual percentage of 131 I − doses were less in both the KI (38% of control) and NH 4 ClO 4 (48% of control) treatment groups, compared with saline controls; however, only the KI treatment was significantly less than controls (p < 0.01). Interestingly, control, KI, and NH 4 ClO 4 treated rats from Group 2 retained more thyroidal 131 I − than rats from Group 1, which did not receive T 4 treatment. Figure 4 compares the thyroidal 131 I − concentrations for Groups 1 and 2. In all cases, T 4 treatment resulted in increased thyroidal 131 I − concentrations (p < 0.05).

127 I and ClO 4 − : Urinary Excretion and Serum Concentrations
In Stable iodide urinary half lives determined using 24 h urine collections in rats that were treated with KI for Groups 1 and 2 were 3.5 ± 0.6 and 3.9 ± 0.3 hours respectively, with no significance between groups. Perchlorate 24 hour urinary half lives in rats that were treated with NH 4 ClO 4 for Groups 1 and 2 were 2.5 ± 0.5 and 2.5 ± 0.4 hours, respectively.  and 72 hours after oral administration of either saline, KI (30 mg/kg), or perchlorate (30 mg/kg) as described in Figure 1a. Data are means ± standard deviation (n = 12); (b) 131 I − concentrations (pg/mL) in serum of Group 2 male rats collected via tail vein bleed at +15 hours and via cardiac puncture at 75 hours after oral administration of 131 I − . Animals were dosed with 131 I − followed by saline, KI (30 mg/kg), or perchlorate (30 mg/kg) and dosed with replacement T 4 as described in Figure 1b. Data are means ± standard deviation (n = 6).
(b) * Significantly less than control (p < 0.05).  Figure 1a. Data are means ± standard deviation (n = 12); (b) 75 hour + T 4 percent of total 131 I − in the thyroid of Group 2 male rats dosed with 131 I − via gavage followed by saline, KI (30 mg/kg), or perchlorate (30 mg/kg) and dosed with T 4 replacement as described in Figure 1b. Data are means ± standard deviation (n = 6).

Figure 4.
Comparison of 131 I − concentration in the thyroid of male rats administered T 4 versus rats not administered hormone replacement therapy (n = 12 for non-T 4 and n = 6 for T 4 ). * Saline treatment without T 4 has a significantly less 131 I − thyroid concentration than saline with T 4 (p < 0.05); ** KI and perchlorate treatment without T 4 has a significantly less 131 I − thyroid concentration than KI and perchlorate with T 4 (p < 0.001).

Serum TSH Levels
The mean serum TSH concentrations for Group 1 was 4.12 ± 1.1, 4.04 ± 1.5, and 3.35 ± 1.8 ng/mL for the saline, KI, and NH 4 ClO 4 treatments, respectively. No statistical significance was determined between treatment groups. Serum TSH concentrations were below the limit of detection of the assay (1.4 ng/mL) in Group 2.

Discussion
The objective of this study was to compare the relative efficacy of stable iodide and perchlorate to purge 131 I − from the body of rats. We evaluated the efficacy of these post-exposure treatments by monitoring 131 I − in the thyroid gland, urine, and serum for up to 3 days after dosing. This study design was based on a previous study in our laboratory [15], which showed that at 15 hours after treatment with KI or NH 4 ClO 4 , the perchlorate treated rats excreted three times the amount of 131 I − in urine compared with control treatments, while stable iodide treated rats excreted only twice the amount of 131 I − in urine compared with control treatments. In the current study, we confirm our earlier results that perchlorate treatment led to faster 131 I − excretion compared with KI treatment (Table 2). Interestingly, by 75 hours after dosing there was no difference in the total amount of 131 I − excreted in urine between treatment groups and the controls. This attenuation of the initial efficacy can be attributed to the short half-lives of perchlorate and iodide, 7.3 and 6 hours respectively [17,24], and suggests that repeated dosing with either treatment may most effectively clear 131 I − from the human body and thus protect the thyroid from radiation poisoning.
One interesting finding in this study is that rats treated with T 4 retained more thyroidal 131 I − than rats without T 4 treatment for all treatment groups at 75 hours after dosing (Figure 4). Other metrics of 131 I − internal exposure for urine and serum were comparable for Groups 1 and 2, i.e., no significant differences were determined between groups in the cumulative amounts of 131 I − , stable iodide, or perchlorate excreted in the urine at 75 hours after dosing or in the concentrations of 131 I − , stable iodide, or perchlorate in serum at 15 and 75 hours after dosing. Although the goal of T 4 administration was to inhibit spikes in serum TSH levels after administration of KI or NH 4 ClO 4 , the outcome was diminished serum TSH levels below euthyroid levels, thus altering the thyroid gland function. The stimulatory effect of the reduced levels of TSH in T 4 -treated rats on thyroid hormone secretion was probably very low compared with the non-T 4 treated rats, resulting in increased residual 131 I − radioactivity. The increase in residual thyroid radioactivity can likely not be attributed to the well known Wolff-Chaikoff effect given its transient nature relative to the duration of the experiment. However, the Wolff-Chaikoff effect must be considered if multiple doses of the treatments are administered over time.
The effectiveness of stable iodide and perchlorate as radioprotectants for 131 I − uptake into the thyroid gland is time-dependent [25,26]. Zanzonico and Becker (2000) [25] determined that when KI is administered to rats 2 hours after 131 I − exposure there is an 80% reduction in uptake of radioactive iodide in the thyroid. However, when KI is administered 8 hours after 131 I − exposure the reduction in uptake of radioactive iodide is reduced by 50%. Sinadinovic and Jovanovic (1971) [26] concluded that when rats were administered perchlorate and KI 30 min or 24 hours prior to radioiodide tracer administration, the perchlorate treated animals accelerated the elimination of radioiodide from the body, reducing its systemic biological half life, compared to KI treated rats. In our experiments, the KI treated rats excreted the same total amount of radioiodide at 3 days post exposure as perchlorate treated animals, but at a slower excretion rate. We plan further experiments to evaluate the efficacy of repeated treatments of perchlorate and KI.
In conclusion, our findings suggest that stable iodide and perchlorate appear equally potent at enhancing urinary elimination of radioiodide over a 3 day period. However, the perchlorate treatment offers an accelerated elimination rate of radioiodide within the first 24 to 36 hours and has a significant reduction in the biological half-life of 131 I − in the first 24 hours. This suggests that repeated doses of perchlorate post radioiodide exposure may increase the effectiveness of blocking uptake in the thyroid and eliminating radiation in the urine. These data support continued consideration of perchlorate as a prophylactic drug to treat radioiodide poisoning. Intentional and unintentional releases of radiation remain a public health concern in many countries and more research is needed to identify the most effective therapies for protecting the thyroid from radioiodide poisoning.

Disclaimer
The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry or the U.S. Food and Drug Administration.