Ammonium perchlorate (99.8%), 100% ethanol, and sodium hydroxide were purchased from Aldrich (Milwaukee, WI, USA). Nonradioactive thyroxine was purchased from Sigma Chemical Corporation (St. Louis, MO, USA). Potassium iodide was obtained from J. T. Baker (Phillipsburg, NJ, USA), carrier-free iodide-131 (¹³¹I⁻) from Amersham Biosciences (29.4 mCi/µg), acepromazine maleate (10 mg/mL) and ketamine HCl (100 mg/mL) from Fort Dodge Animal Health (Fort Dodge, IA, USA) and xylazine (20 mg/mL) from Ben Venue Laboratories (Bedford, OH, USA). Isoflurane (99.9%) was purchased from Abbott Laboratories (Abbott Park, IL, USA).

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) ¹³¹I⁻ 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 NH₄ClO₄ (calculated as ClO₄⁻) 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 T₄ (based on euthyroid replacement T₄ 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 ¹³¹I⁻ and at sacrifice. At sacrifice (75 hours after dosing with ¹³¹I⁻), 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.

Serial urine samples were placed on a gamma counter (1470 Wallac Wizard) equipped with one detector and ¹³¹I⁻ counts/minute (cpm) were measured within 2 hours after collection. ¹³¹I⁻ 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 ¹³¹I⁻) 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).

Non-radioactive analytes (¹²⁷I⁻ and ClO₄⁻) were quantified using ion chromatography coupled with tandem mass spectrometry. Serum samples were spiked with internal standard (¹²⁹I⁻ and Cl¹⁸O₄⁻), treated to remove proteins, and analyzed by ion chromatography electrospray ionization tandem mass spectrometry [22]. Urine samples were spiked with internal standard (¹²⁹I⁻ and Cl¹⁸O₄⁻) and analyzed by ion chromatography electrospray ionization tandem mass spectrometry [23].

All urine samples for Groups 1 and 2 were analyzed for ¹³¹I⁻ excretion kinetics. Animals in Groups 1 and 2 that received ¹²⁷I⁻ or ClO₄⁻ also had urine samples analyzed for ¹²⁷I⁻ or ClO₄⁻ excretion kinetics. All half-life calculations were prepared using Win Non Lin 5.2 software.

Single factor analysis of variance (ANOVA) was used initially to determine significance across the treatment groups (control saline, KI and NH₄ClO₄) 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 ClO₄⁻.

Rats were administered ¹³¹I⁻, then 3 hours later either saline, 30 mg/kg of KI or 30 mg/kg of NH₄ClO₄ by oral bolus gavage. Group 2 was distinguished from Group 1 (Table 1) by repeated T₄ administration, first at the time of dosing with saline, KI or NH₄ClO₄, then at +27 hours and +51 hours during the time-course. The 3-day cumulative volumes of urine produced were 39.7 ± 7.7 mL for Group 1 and 41.3 ± 5.7 mL for Group 2. In Group 1, 71, 63, and 62% of the administered ¹³¹I⁻ doses for NH₄ClO₄, KI, and control saline treatment groups, respectively (Figure 1a), were excreted in urine by 75 hours after dosing with ¹³¹I⁻. In Group 2, 72, 71, and 63% of the administered doses of ¹³¹I⁻ were excreted in urine for the NH₄ClO₄, KI, and saline control treatment groups, respectively (Figure 1b) by 75 hours after dosing with ¹³¹I⁻. Most of the ¹³¹I⁻ collected over the 3-day period (60.5 ± 6%) was excreted in urine by 24 hours after dosing in all treatments for Groups 1 and 2 (Figure 1(a,b)). The 24-hour urinary excretion half-lives for ¹³¹I⁻ (Table 2) in control and KI treated rats ranged from 3.5 to 4 hours; conversely, NH₄ClO₄ treated rats excreted ¹³¹I⁻ more rapidly (2.6 hour urinary excretion half-life, p < 0.001).

The mean ¹³¹I⁻ concentration in the Group 1 control serum at 15 hours after ¹³¹I⁻ 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 ¹³¹I⁻ serum levels in the KI and the NH₄ClO₄ 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 ¹³¹I⁻ serum concentrations in the KI and NH₄ClO₄ treatment groups were significantly less than saline controls for both sampling times (p < 0.05). The addition of T₄ proved to have little effect on mean serum ¹³¹I⁻ concentration for Group 2 (Figure 2b). The mean serum ¹³¹I⁻ concentrations at 15 hours following T₄ and saline, KI and NH₄ClO₄ 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 ¹³¹I⁻ dosing, only the NH₄ClO₄ treatment group ¹³¹I⁻ concentrations were significantly less (p < 0.05) than controls, while both KI and NH₄ClO₄ treatment group ¹³¹I⁻ concentrations were significantly less than controls at the 75 hour sampling time.

Compared with control saline, KI and NH₄ClO₄ treatment reduced levels of ¹³¹I⁻ in thyroid gland at 75 hours post exposure (Figure 3a). Also the residual ¹³¹I⁻ levels in the thyroid gland in the KI treatment group were lower than the NH₄ClO₄ treatment group (p < 0.01). KI and NH₄ClO₄ treatment reduced the thyroid content of ¹³¹I⁻ by 77 and 61%, respectively, 3 days after administration of ¹³¹I⁻. Group 2 animals treated with T₄ displayed a different thyroidal ¹³¹I⁻ content (Figure 3b). The mean residual percentage of ¹³¹I⁻ doses were less in both the KI (38% of control) and NH₄ClO₄ (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 NH₄ClO₄ treated rats from Group 2 retained more thyroidal ¹³¹I⁻ than rats from Group 1, which did not receive T₄ treatment. Figure 4 compares the thyroidal ¹³¹I⁻ concentrations for Groups 1 and 2. In all cases, T₄ treatment resulted in increased thyroidal ¹³¹I⁻ concentrations (p < 0.05).

In Group 1 rats, the cumulative amounts of ¹²⁷I⁻ and ClO₄⁻ excreted in urine over 72 hours were 128 and 92%, respectively, of the administered doses of ¹²⁷I⁻ as KI, and ClO₄⁻, as NH₄ClO₄. Most of the excretion of ¹²⁷I⁻ (93%) and ClO₄⁻ (97%) occurred within 24 hours of dosing. In the T₄ treated group, 106% and 86% of ¹²⁷I⁻ and ClO₄⁻, respectively, was excreted in urine by 72 hours after dosing. Eighty four and 96% of the excreted ¹²⁷I⁻ and ClO₄⁻ anions occurred within 24 hours.

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 NH₄ClO₄ for Groups 1 and 2 were 2.5 ± 0.5 and 2.5 ± 0.4 hours, respectively.

Serum concentrations of ¹²⁷I⁻ and ClO₄⁻ 72 hours after administration were 200.4 ± 199.8 and 81.3 ± 31.6 ng/mL, respectively, in Group 1 and 56.7 ± 15.9 ng/mL and 26.3 ± 14.6 ng/mL, respectively in Group 2.

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 NH₄ClO₄ 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.