Biodistribution of 131I in mice is influenced by circadian variations

Effects of radiation and biodistribution of radionuclides are often studied in animal models. Circadian rhythm affects many biological functions and may influence the biokinetics of radionuclides and observed responses. The aim of this study was to investigate if the time during the day of 131I injection affects the biodistribution and absorbed dose to tissues in mice. Biodistribution studies were conducted on male C57BL/6 N mice for three diurnal time-series: the animals were i.v. injected with 160 kBq 131I at 8 am, 12 pm or 4 pm. The activity concentration in organs and tissues was measured at 1 h to 7 days after administration and absorbed dose at day 7 was determined. Comparison between the three time-series showed statistically significant differences in activity concentration in all investigated tissues and organs. Administration performed at 12 pm resulted in general in higher absorbed dose to the organs than injection performed at 8 am and 4 pm. Time of day of administration affects the biodistribution of 131I in mice and consequently the absorbed dose to individual organs. These findings advocate that subsequent biodistribution studies and dosimetry calculations should consider time-point of administration as a variable that could influence the results.


Materials and methods
Animal model. Animals used were 9-10 weeks old male C57BL/6 N mice (Charles River Laboratories International, Inc., Sulzfeld, Germany) weighing 24 g (SD = 2 g). The mice were kept under standard laboratory day and night cycle, i.e. darkness from 6 pm to 6 am. Water and laboratory food with iodine concentration of 0.87 µg/g were given ad libitum. The study was approved by the Ethics Committee for Animal Research in Gothenburg (no. 146-2015). All experiments were performed in accordance with relevant guidelines and regulations.

I administration and organ sampling.
Altogether 165 animals were divided into three main groups and were i.v. injected with 160 kBq 131 I (GE Healthcare, Braunschweig, Germany) in physiological saline solution (0.1 ml) in the tail vein at 8 am, 12 pm or 4 pm, respectively. The animals were killed by cardiac puncture under anesthesia with sodium pentobarbital (APL, Sweden) after 1,4,8,18,24,72, or 168 h following injection (n = 5 -10 per subgroup). The thyroid, salivary glands, lungs, heart, spleen, liver, kidneys, and stomach were excised and blood and stomach contents were sampled. Several millimeters of the large intestine (starting from the sigmoid colon) and of the small intestine (starting from the duodenum) were excised and contents from the large and small intestine were collected. Sample weight and radioactivity content were measured directly after excision. The average thyroid weight of 46 collected thyroids (weighing ca 3 -5 mg) was 3.9 mg (SEM = 0.1), a value well in accordance with literature 12 . A selection of thyroids was fixed in formalin immediately after weighing; 35, 30 and 30 samples for injection at 8 am, 12 pm and 4 pm, respectively. Radioactivity measurements. The 131 I activity in the stock solutions was measured using a CRC-15 dose calibrator ion chamber (Capintec, IA, USA) and the activity concentration determined. For each animal, the syringe was weighed before and after injection. To compensate for adsorbed 131 I inside the syringes, control syringes were used to determine the actual activity of the injected solution. The 131 I content of the control syringes was measured using a Wallac 1480 Wizard® 3″ NaI(Tl) gamma counter (Wallac Oy, Turku, Finland). 131 I activity in tissue samples was measured in the gamma counter. Corrections were performed for background and dead time loss. Self-attenuation of 131 I in the sample and geometric effects were investigated and found negligible. All data were decay corrected to time of administration.
The activity concentration in organs and tissues at different times after injection, c tissue (t) , was calculated as percent of injected activity per organ mass (%IA/g): where A tissue (t) is the activity in the sample at time t corrected for radioactive decay to time of administration (t = 0), A inj is the injected activity at time t = 0, and m tissue is the mass of the sample. Due to the uncertainties in the thyroid mass measurements, the 131 I activity in the thyroid is presented as percent of injected activity (%IA).
Histological evaluation. Altogether, 37 thyroid samples with low 131 I activity concentration (less than 20% of group max) were analyzed. The samples were fixed in formalin immediately after weighing and then embedded in paraffin, sectioned in 4 μm slices and stained with haematoxylin-eosin, according to standard protocols. Thyroid tissue samples containing less than approximately 25% thyroid tissue were excluded from the study.
Absorbed dose calculation. The absorbed dose from 131 I was calculated according to the MIRD formalism 16 : where Ã (r S , T D ) is the time-integrated activity over the dose-integrated period T D in the source organ r S and M(r T ) is the mass of the target organ r T . Y i is the yield of radiation i with energy E i and ϕ(r S ← r T ) is the fraction of the radiation in the source organ that is absorbed in the target organ 16 . For thyroid, the average weight (3.9 mg, SD = 0.5) was used. The calculations were based on assumption of homogeneous activity distribution in the organs.
Only the electron emission was considered in the absorbed dose calculations, and therefore E i Y i was set to 191 keV/Bqs 17 . The self-absorbed fraction for blood was set to 1. For the salivary glands the self-absorbed fraction was estimated to be 0.880 by interpolation of data for absorbed fraction and tissue weight for organs of similar weight, i.e. lungs and stomach. The cross-absorbed fractions from the salivary glands to the other organs was set to 0. The absorbed fractions for the other investigated organs were taken from a mouse model matching our study design 18 .
The time-integrated activity was estimated with the trapezoidal rule from the time of administration to the dose determination time based on the data from the biodistribution. The activity in the blood at t = 0 was assumed to be equal to the injected activity. For the rest of the organs and tissues the activity at t = 0 were set to zero.
The relative difference between the absorbed dose to the organs in two groups with different time of day of 131 I injection (groups 1 and 2) were calculated: www.nature.com/scientificreports/ Statistical analyses. ANOVA with Tukey HSD test was used to determine the statistical significance of differences in activity concentration between groups. For the thyroid, the statistical significance of differences in 131 I activity was determined by Kruskal-Wallis one-way ANOVA with pairwise comparison, using IBM SPSS Statistics for Windows 25.0. Statistical significance was considered for probabilities higher than 95% (p < 0.05). Uncertainties in the measurements are given as the standard error of the mean (SEM).

Results
Biodistribution of 131 I. The biodistribution of 131 I was determined for 1 h up to 7 days post injection (p.i.) performed at 8 am, 12 pm or 4 pm. The 131 I activity in the thyroid (%IA) is presented in Fig. 1. Highest median value was observed at 18 h p.i. for all administration time-points; 6.3 (SEM = 1.0) for 8 am, 6.1 (SEM = 0.9) for 12 pm and 4.2 (SEM = 0.7) for 4 pm. Maximum for the 4 pm group was statistically significant lower than for the 8 am group (p = 0.046). Statistically significant differences were also observed between the 8 am and 12 pm groups at 4 h p.i., 0.77 (SEM = 0.47) vs. 3.9 (SEM = 0.6), respectively (p = 0.018). A total of 37 thyroid samples had unexpectedly low 131 I activity and 31 of were excluded from the study due to low thyroid tissue content based on histological analysis. The 131 I concentration in the other investigated tissues is presented in Table 1. High 131 I activity concentration was found in stomach content, stomach, and salivary glands. In total, statistically significant differences between the administration time-points were observed in 38 out of 91 time-points and samples. For most investigated organs and tissues, the maximum activity concentration was reached at the first time-point (1 h p.i.) followed by an exponential decrease. For injection performed at 4 pm, the decrease was generally more rapid and the decrease was less rapid for injection performed at 12 pm. The activity concentration in the kidneys did not depict a monotonic decline: a local maximum was observed at 3 days.
(3) When investigating biodistribution of radioiodine, the thyroid is the primary organ of interest. In this study, we demonstrated a difference in uptake of 131 I in the thyroid during the first day. Maximum was reached at the same time-point in all three studies, but there was a 33% difference in maximum activity between the 8 am and 4 pm groups. These findings are in agreement with Walinder's study on mouse resulting in lower thyroid uptake 24 h after injection at 4 pm compared with injection at 8 am or 1 pm 12 . On the contrary, studies on rat showed that the 131 I uptake in thyroid was higher if the injection was performed in the afternoon (7 pm or 6 pm) compared with injection in the morning (7 am or 8 am) 13,14 . The different diurnal patterns could be explained by the species difference and the much lower iodine concentration in the food (0.07 µg/g) given to the rats. Table 1. The activity concentration in mouse tissues at 1 h to 7 days after injection of 160 kBq 131 I at three injection times (8 am, 12 pm and 4 pm). Data are presented as mean (n = 4-10) and SEM (italics). *, † , ‡ indicate statistically significant differences from injection at *8am, † 12 pm and ‡ 4 pm, respectively.  www.nature.com/scientificreports/ The circadian rhythm of the hypothalamus-pituitary-thyroid (HPT) axis may be an underlying cause of the diurnal variations in iodine uptake in the thyroid. The HPT axis regulates the secretion of T3, T4 and TSH 8 , resulting in oscillating levels of the hormones in the blood 19,20 . TSH increases the iodine uptake by stimulating iodide transport into the thyrocytes via the sodium/iodide symporter (NIS) [21][22][23] . It is therefore possible that variations in thyroidal uptake are caused by fluctuating TSH levels in the blood.
Most of the T3 is produced from conversion of T4 to T3 by the type 1 iodothyronine deiodinase (D1) in the liver, kidneys and thyroid 24 , where iodide is released from the hormone. This could explain the increase of 131 I observed in the kidney after 24 h. The local maximum could be an effect of the decrease of 131 I in the thyroid caused by excretion of T3 and T4 containing 131 I. Since the kidneys store more T4, T3 and iodide per organ weight than the liver 25 and due to its important role in excretion of iodide, it is possible that this effect is observable in the kidney and not in the liver.
In most organs the decrease in activity concentration (after maximum) was less steep for injection performed at 12 pm. This difference resulted in higher mean absorbed dose to most organs of the animals injected at 12 pm. The maximal relative difference in mean absorbed dose to the thyroid delivered during the first 7 days was 9%, depending on the time-point of administration, while greater differences were observed in other organs, such as the salivary glands and stomach. Unfortunately, no statistical methods can be applied to determine if these differences are statistically significant. Furthermore, the biological significance of 9% difference in absorbed dose to the thyroid is unknown and needs to be further investigated.
Large inter-individual differences in uptake of radioiodine in thyroid have been shown in previous animal studies 26,27 . Individual metabolic differences and food intake may partly explain the spread within an animal group, since intake of stable iodine via the food may reduce the uptake of radioiodide 12 . In this study the animals were given food ad libitum, in agreement with standard laboratory care.
In chronotherapy, administration of, e.g., anti-cancer drugs is performed at specified time points of day in order to optimize the treatment 11 . Chronotherapy with cytotoxic drugs have shown promising results with variations of over 50% in efficiency dependent on time of day of administration 28 . A few studies have also investigated chronotherapy with external radiotherapy [29][30][31] and it has been proposed that adaptation to the circadian rhythm can result in a more sufficient personalized treatment, especially for pediatric patients 32 . The number of studies on diurnal effects related to radiopharmaceuticals are even fewer. A handful of studies from the 50 s, 60 s and 70 s have observed circadian variations in 32 P (orthophosphate) uptake in mammary tumors 11,[33][34][35] . The intention then was to use 32 P as a marker for mitotic activity in the tumors and thereby be able to choose a time-point for external radiotherapy when tumor is more radiosensitive. However, as far as we know, chronotherapy with radiopharmaceuticals is yet unexplored. www.nature.com/scientificreports/ The findings in the present study suggest that 131 I-based radionuclide therapy may be a potential candidate for chronotherapy. Interestingly, our data show that in mice about 30-50% lower absorbed dose to salivary glands, stomach and small intestines could be obtained if administration was made at 4 pm compared with at 12 pm. Administration at 4 pm also gave lower absorbed dose to thyroid (that might reflect tumor behavior) but to a lower extent, demonstrating that lower side effects could potentially be obtained in salivary glands and gastrointestinal tract if 131 I was administered late during the day. Clinically, the relevance of a difference in absorbed dose to thyroid of 9% might be of minor importance if thyroid is the target and of greater importance if it is a risk organ. If biodistribution of 131 I could be optimized with respect to time of administration also in humans, chronotherapy of patients with differentiated thyroid cancer could work as an additional therapeutic tool or method for improving quality of life.
In the present study animals were i.v. injected with 131 I, while in the clinic, many patients with thyroid diseases receive 131 I orally in pill or in liquid form. The difference in route of administration should not affect the scope of this study, since absorption of orally administered 131 I to the blood is rapid and virtually complete 36,37 . Hence, i.v. injection of 131 I can be seen as a simulation of oral administration. Furthermore, an equivalent patient study would have required several test doses for one and the same patient due to the large inter-individual differences in thyroid uptake. Repeated administrations of 131 I, even with several months between, would pose a great risk of thyroid stunning which not only would affect the results of the study but also the treatment effect. From an ethical point of view, we therefore found it appropriate to investigate potential effects of circadian rhythm in mice before clinical studies are initiated. Another reason for animal studies was the possibility to obtain data also for non-thyroid tissues.

Conclusion
The results of this study demonstrated differences in biodistribution and biokinetics and consequently differences in absorbed dose due to time of 131 I administration. These findings suggest that diurnal variations should be considered in dosimetric evaluations of radiopharmaceuticals. Chronotherapy using 131 I-based radiopharmaceuticals could be favorable for reduction of short-and long-term side effects, and should be further investigated.

Data availability
The data generated and analyzed during this study are available from the corresponding author upon reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.