The influence of circadian rhythm and biological sex on the biodistribution of iodine-131 in mice

Transcription

1 SAHLGRENSKA ACADEMY The influence of circadian rhythm and biological sex on the biodistribution of iodine-131 in mice Mikael Elvborn Essay/Thesis: 30 hp Program and/or course: Medical Physicist Programme Level: Second Cycle Semester/year: Spring 2016 Supervisor: Eva Forssell-Aronsson, Britta Langen and Johan Spetz Examiner: Magnus Båth

2 Abstract Essay/Thesis: 30 hp Program and/or course: Medical Physicist Programme Level: Second Cycle Semester/year: Spring 2016 Supervisor: Eva Forssell-Aronsson, Britta Langen and Johan Spetz Examiner: Magnus Båth Keyword: 131 I, circadian rhythm, biodistribution, biokinetics, C57BL, mice, sex difference Purpose: Theory: Method: Result: The purpose of this study was to validate previous biodistribution data on circadian rhythm (Andersson, 2015) and to investigate a novel early time point. A further aim was to investigate the potential difference between the sexes concerning the biodistribution and biokinetics of 131 I in mice. In addition, the impact of iodine concentration in animal food was studied. The thyroid is both a risk and target organ in radionuclide therapy. The gland takes up iodine to synthesize thyroidal hormones which are important for various cellular mechanisms throughout the body. 131 I, a radioactive isotope, is used in nuclear medicine, but can also be encountered from fallout of nuclear accidents. The circadian rhythm affects many bodily functions, and has recently been shown to also influence the biodistribution of 131 I. Physiological differences between the sexes represent another intrinsic variable that is thought to impact the biodistribution of 131 I. In total, 159 C57BL/6N mice (124 males and 35 females) were used in the experiments and injected intravenously with 131 I prepared in physiological saline. The mice were administered with kbq of 131 I at different time points during the day (8 am, 12 pm and 4 pm) and killed after 1, 4, 8, 18, 24, 72 or 168 h following injection. Various organs, tissue samples, gastrointestinal contents, and the remaining carcass were collected, weighed, and subjected to gamma counter measurement to determine 131 I activity concentration. The results supported previous work showing that 131 I biodistribution is influenced by the time of day of administration. Furthermore, the results demonstrated a difference in 131 I biodistribution data between male and female mice, notably in the kidneys. The results also showed that the extent of diurnal differences in biodistribution data is influenced by the iodine concentration in animal food.

3 Table of content Abbreviations... 1 Introduction... 2 Aims... 4 Materials and Methods... 5 Animal model... 5 Administration of radionuclides... 5 Biodistribution and biokinetics... 7 Radioactivity measurements... 8 Detector calibrations... 8 Measurement corrections... 8 Statistical analysis... 9 Results Detector calibrations Measurement corrections Volume effect Adsorption Biodistribution of 131 I Comparison between males at time point 8 am, 12 pm, and 4 pm Sex difference in 131 I biodistribution Influence of iodine concentrations in food at 12 pm Influence of iodine concentrations in food at 4 pm Effect of circadian rhythm at standard iodine concentration in food Discussion Conclusion and Outlook Acknowledgements Reference list Appendix Biokinetics for comparison with normal iodine concentrations in food for mice Administration at 12 pm Administration at 4 pm... 43

4 Abbreviations β-particle cps ev - Electron or positron - Counts per second - Electron volt 127 I - Iodine I - Iodine 131 i.p. i.v. NIS SEM T3 T4 TSH - Intraperitoneally - Intravenous - Sodium-iodine symporter - Standard Error of Mean - Triiodothyronine - Thyroxine - Thyroid stimulating hormone 131 Xe - Xenon 131 1

5 Introduction All animals, including humans, are subject to basic needs such as eating or sleeping. These are recurring and oscillating needs that are usually satisfied in a periodic manner across the day. Periodicity is within us and all around us, with cycles of varying length: from day lengths of 24 h up to seasonal changes. The cycle of day length is also referred to as the circadian rhythm. The circadian rhythm affects functions in our bodies. Among these are the sleep/wake cycle, secretion of hormones like TSH (Thyroid stimulating hormone), and body temperature (Schulz & Steimer, 2009). These functions are regulated by biological clocks in the body with the SCN (suprachiasmatic nucleus) in the hypothalamus of the brain as the main regulator. This regulator is reset by a range of factors, called zeitgebers (time givers), with daylight being the most important one. Responsible for maintaining circadian rhythm on the tissue level are certain genes expressed in the cells, called molecular clock genes, that are stimulated by molecular signals from the SCN (Reppert & Weave, 2002). Although circadian rhythm is an intrinsic factor in basically all biological activities, it is generally not accounted for in current research or treatment schedules in nuclear medicine. There are a few exceptions to this. One is the study of radiation-induced gene regulation with i.v. administration of 131 I in mice, where the outcome of gene regulation in tissues was dependent on the time of day when 131 I was injected (Langen et al., 2015). Recently, a biodistribution study on 131 I in mice was performed (Andersson, 2015), where mice were injected with 131 I at different times of day. Several decades ago, uptake of 131 I in mice was also studied with consideration of varying time points of administration (Walinder, 1971). That study, however, focused on the thyroid and thus did not consider the impact of circadian variation on radionuclide biodistribution in other tissues. The thyroid is an endocrine gland of approximately 20 grams, in humans, located at the sides of the trachea, just below the larynx (Sand et al., 2007). This gland is subject to circadian variation of follicular lumen size and concordantly weight variation due to change in colloid (water) volume (Wright et al, 1995). The thyroid produces the thyroid hormones T3 (triiodothyronine) and T4 (thyroxine) that regulate important cellular mechanisms such as metabolic activity throughout the body (Sand et al., 2007; Yen, 2001). T3 and T4 hormones are regulated by TSH (released from the pituitary gland) and synthesized in the follicular structure of the thyroid, normally with stable iodine (Sand et al., 2007). This nuclide, 127 I, is the only stable isotope among the 37 known iodine isotopes (The Lund/LBNL Nuclear Data Search, 1999). Thyroid hormones can also be synthesized with other isotopes of iodine, e.g. 131 I, since the molecular mechanisms cannot distinguish between isotopes. Iodine is transported from the blood to the thyroid as iodide (I - ) via the sodium-iodine symporter (NIS) (Sand et al., 2007). Inside the follicular lumen of the thyroid, iodide is covalently bound to a tyrosinebased molecule, creating the T3 and T4 hormones (Yen, 2001). The hormones are then secreted through the blood into various tissues in the body. The thyroid produces mostly the T4 hormone, which is a prohormone and subsequently reduced to T3 in the body, in particular in the kidneys and liver (Larsen et al., 2012; Gereben et al., 2008; Bianco et al., 2002). Because of its high iodine uptake, the thyroid is both a risk organ as well as a target when it comes to dealing with radioactive iodine such as 131 I. This is utilized in e.g. targeted radionuclide therapy treatment for hyperthyroidism, where free 131 I is administered and taken up by the thyroid (Lee, 2012). Patients with some types of cancer, e.g. breast carcinoma, where the tumor cells express sufficiently high levels of NIS, can be treated in a similar fashion (Yao et al., 2015). 131 I has a half-life of 8.03 days (MIRD, 2006) and disintegrates via β - decay into 131 Xe: this energy is mostly absorbed locally due to the short range of respective β-particles (Berger et al., 1984). Because of this, the surrounding tissues around the source organ are mostly spared from absorbed dose. Beside the β-particles, 131 I also has a gamma component with an energy of kev (The Lund/LBNL 2

6 Nuclear Data Search, 1999), which enables detection with e.g. a gamma counter or gamma cameras in clinical applications. Exposure to 131 I can occur from several sources. One example is radioactive iodine being released from 131 I-MIBG (Metaiodo-benzylguanidine) upon autoradiolysis. 131 I-MIBG is used in treatment and diagnostics of e.g. neuroblastomas, paragangliomas and pheochromocytomas, where the aim is to target the cancer with a norepinephrine analogue bound to the radionuclide (Kayano & Kinuya, 2015). Exposure to 131 I can also occur upon nuclear accidents. An example is the nuclear accident that occurred in Chernobyl, where nearly Bq of 131 I were released to the environment and caused an increased incidence of thyroid cancer in Belarus in the following years (McLaughlin et al., 2012; Kazakov et al., 1992). Since iodine is distributed via the blood, it will reach all organs in the body and it is therefore imperative to have a well-based knowledge about the potential risks and dose-responses in normal tissues when dealing with radioactive iodine, regardless of the types of exposure. Biodistribution studies are a good way of collecting these data. Animal models are often used to study biokinetics and various species have been used: mice (Garg et al., 1990), rats (Spetz et al, 2013) and hamsters (Peronace & Houssay, 1970), among others. In clinical research, there is often reluctance towards using females in studies (Holdcroft, 2007). This is of particular concern when considering that women are more prone to certain thyroid-related diseases such as Grave s disease and Hashimoto s thyroiditis, with an incidence ratio of more than 7 10:1 (Beery & Zucker, 2011; Jacobson et al., 1997). There are many benefits to having data for both biological sexes, especially when underlying processes are unknown (Beery & Zucker, 2011; National Institutes of Health, 2001). The purpose of this project was to study the biodistribution of 131 I in various tissues in mice with three different time points of administration, with special consideration of biological sex and iodine diet as experimental variables. 3

7 Aims The purpose of this work was to determine the biokinetics of 131 I in mice in dependence of the time of day of injection. To achieve this, a partial aim was to validate previous data from Charlotte Andersson s master thesis from fall 2015 (Andersson, 2015), i.e. to investigate the effect of circadian rhythm for injections performed at 12 pm and 4 pm. Moreover, this work aimed to characterize 131 I biodistribution for an additional early time point of administration, at 8 am. A further aim was to assess potential differences between the sexes concerning the biodistribution and biokinetics of 131 I in mice. In addition, the impact of iodine concentration in animal food on 131 I biodistribution was studied. To minimize measurement errors of the gamma counters used for this work, the aim was also to investigate and, if needed, correct for self-attenuation in samples and for adsorption in syringes due to varying time spans between preparation of syringes and injection of subjects. 4

8 Materials and Methods Animal model In total, 159 C57BL/6N mice (124 males and 35 females) between 6 and 10 weeks of age, and weighing g for the males and g for the females, were used in the study. The mice were kept in groups of 5 individuals per cage. One group (24 h with administration at 4 pm) was reduced to 4 individuals, since one mouse was unwell and had to be killed before start of the experiments due to ethical guidelines. Drinking water and food were given ad libitum. As standard, the mice were given breeding chow with a reduced iodine concentration of 0.87 µg/g. The reduced iodine nutrition was chosen to limit the individual variation of 127 I amounts in the animals, which may have an effect on the uptake of 131 I during the experiments. This setup also matched the study design in previously performed experiments (Andersson, 2015). An iodine concentration, in food, of 1.20 µg/g was given to 30 males in this work, see Table 1. Standard laboratory day and night cycle was maintained for the mice during the experiments, i.e. dark from 6 pm to 6 am. The studies were approved by the Gothenburg Ethical Committee on Animal Research (no ). Administration of radionuclides 131 I was obtained from GE Healthcare (Braunschweig, Germany) as Na 131 I, and diluted with saline solution to the desired activity concentration for administration. Syringes (0.1-1 ml Braun syringes with mm cannulas) were prepared with an activity of approximately 165 kbq 131 I from stock solutions prepared before each injection time point. All the syringes were prepared according to previous procedure (Andersson, 2015), i.e. were weighed after preparation as well as after injection to determine the administered activity to each subject. At preparation, control syringes were prepared in the same manner and emptied, in a 20 ml scintillation vial, after a similar time span to the waiting period between preparation of animal syringes and injection of animals. The weight of the content of each control syringe was then correlated to measurements in gamma counters, giving an activity/massratio. The time points and administered activity are given in Table 1. 5

9 Table 1: Experimental overview. The table shows mean values, with SEM (italics), for administered activity, weight, and age for each group. It is also indicated if the diet was regular or iodine reduced. Group Number of animals Injected activity [kbq] Weight [g] Age [weeks] Reduced iodine diet (Y/N) Males Injection at 8 am 1 h n= Y 4 h n= Y 8 h n= Y 18 h n= Y 24 h n= Y 72 h n= Y 168 h n= Y Injection at 12 pm 1 h n= Y 4 h n= Y 8 h n= Y 18 h n= Y 24 h n= Y 72 h n= Y 1 h n= N 4 h n= N 72 h n= N Injection at 4 pm 1 h n= Y 4 h n= Y 8 h n= Y 18 h n= Y 24 h n= Y 72 h n= Y 1 h n= N 4 h n= N 72 h n= N Females Injection at 8 am 1 h n= Y 4 h n= Y 8 h n= Y 18 h n= Y 24 h n= Y 72 h n= Y 168 h n= Y 6

10 Table 2: Sampling overview. Organs, part of organs, as well as gastrointestinal contents were excised or sampled and put into 20 ml scintillation vials for measuring in gamma counters. Gastrointestinal contents were excised from the sampled parts of their respective organ. Organ Blood Thyroid Neck Lungs Heart Liver Kidney left Kidney right Salivary glands Spleen Small intestine Large intestine Stomach Small intestine content Large intestine content Gastric content Intestines rest Carcass Method of sampling Sampled Excised Excised Excised Excised Excised Excised Excised Excised Excised Excised Excised Excised Sampled Sampled Sampled Excised As is after excisions Biodistribution and biokinetics The subjects were injected intravenously (in the tail vein) at 8 am, 12 pm, or 4 pm, and then killed after 1, 4, 8, 18, 24, 72 or 168 h following injection, as shown in Table 1. Animals were injected i.p. with sodium pentobarbithal (APL, Sweden) and killed via cardiac puncture, which was the designated time of death. Organs, or part of organs, as well as gastrointestinal contents were then excised or sampled and put into 20 ml scintillation vials (see Table 2). The large intestines were sampled starting from the sigmoid colon, the small intestines were sampled starting from the duodenum, and the contents of each intestine were sampled from the excised parts. The carcass was also committed to a 20 ml scintillation vial after excision of the remaining gastrointestinal tract. The thyroid was, after weighing, embedded in 4% formaldehyde solution to enable validation at a later point. All vials were weighted before and after adding the samples and subsequently measured in the gamma counters. Some of the thyroid samples were of unreasonably high weight (up to ~6.5 mg) in comparison with literature. These weights were corrected according to previous procedures, i.e. the weights above 4.4 mg were reduced to 3.9 mg (Andersson, 2015). 7

11 Radioactivity measurements A CRC-15R dose calibrator ion chamber, produced by Capintec, IA, USA, with known calibration was used as a reference detector to measure 131 I stock solutions of activity above 1 MBq for preparation of syringes and calibration samples. Injected activity was also determined using between 4 and 5 control syringes for each stock solution. A mean value from the control syringes was used to reduce weighing errors from the scale. Activities of 131 I in organs, tissue samples, and control syringes were measured ex-vivo using two Wallac 1480 Wizard 3" NaI(Tl) gamma counters, produced by Wallac Oy, Turku, Finland. Gamma counter with serial number is hereinafter referred to as gamma counter 1 and gamma counter with serial number is referred to as gamma counter 2. For measurements, pre-installed protocols for 131 I were used with the energy window of kev for both detectors. Percentage of administered activity of 131 I per organ mass (%IA/g) was determined based on weight measurements and activity concentration in the samples, decay-corrected to the time of 131 I administration. Detector calibrations To establish the relationship between sample activity and measured counts per second (cps) in the gamma counter, a calibration of the gamma counters was performed. Calibration samples were prepared with a constant volume of 500 µl and decreasing concentrations of 131 I from 800 kbq to 1.6 kbq by performing a two-fold serial dilution. The ion chamber with known calibration was used as a reference detector. By plotting measured cps vs. sample activity and making a linear fit to the curve, a calibration factor was obtained. The calibration factor was then used to determine 131 I activity in the samples from gamma counter measurements. Measurement corrections Due to self-attenuation in large samples and limitations in sensitivity of the gamma counter, an investigation of potential volume effects was performed. An activity of 20 kbq 131 I was pipetted into a 20 ml scintillation vial and then diluted with ultrapure water (Merck Millipore, Darmstadt, Germany) to a total volume of 1, 2, 3, 4, 5, 7.5, 10, 12.5, 15, 17.5 and 20 ml. The activity was chosen with regard to the sensitivity of the gamma counter and expected activity of 131 I in organ samples. Due to varying time spans between preparation of syringes and injection of mice (ranging from 1 h to 8 h after preparation), an investigation of adsorption effects, i.e. the binding of activity on the inside surface in the syringe resulting in a lower activity concentration than originally prepared, was performed to ensure correct estimation of the individual administered activities. The tests were made with the same activity concentration as the one administered to animals. Measurements were corrected for background radiation and dead time. Measurement time was adjusted so that each organ had a minimum of 1000 counts over background and the measured activities were corrected to the time of 131 I administration. 8

12 Statistical analysis Statistical uncertainties in measurement analysis are represented as Standard Error of Mean, SSSSMM xx, calculated with the following equation: SSSSSS xx = SSSS 1 nn = nn 1 nn ii=1 (xx ii xx ) 2, nn where SD is the sample standard deviation, n is the sample number in the group, {xx 1, xx 2,, xx nn } is the value of the observed sample items and xx is the mean value of these observations. Determination of statistically significant differences in radioactivity concentration in organs between administration time points was performed with a Student's t-test where probabilities above 95% (p<0.05) were considered statistically significant. Student's t-test was also used to determine statistical significance of potential volume effects in radioactivity measurements, where probabilities above or equal to 99% (p 0.01) were considered statistically significant in this case. 9

13 Results Detector calibrations The results from the calibrations showed similar relationships between measured cps and 131 I activity in the two gamma counters. Both gamma counters display the same dead time factor for 25 kbq for all observations, i.e. 11%. Thus were measurements with dead time factors under 11% accepted, and tissues with higher dead time factors were left to decay until meeting this criterion. The measurements that met the dead time criterion were corrected by the dead time factor acquired for that measurement. The activity calibration factors were acquired by linear curve fit to data points up to 25 kbq and were found to be k 1= cps/kbq (with an R 2 -value of ) and k 2= (with an R 2 -value of ) cps/kbq for gamma counters 1 and 2, respectively, see Figure 1. The calibration curve can be considered to be linear up to about cps. a b Figure 1: Activity calibration of the gamma counters. The relationship between measured cps and 131 I activity for gamma counter 1 (a), and 2 (b), is shown in the graphs. The activities were prepared by means of linear two-fold serial dilution, with the ionization chamber as a reference detector, creating linear decreasing concentrations with equal volumes of 0.5 ml. The line is a linear curve fit to the data points up to 25 kbq. Data points are mean values of three dilution series and the error bars indicate SEM; error bars not visible are smaller than the data symbol. 10

14 Measurement corrections Volume effect Results from the self-attenuation and sensitivity investigation are shown in Figure 2. The figure shows a decreasing count ratio with increasing sample volume for a constant 131 I activity of 20 kbq. The sample sizes 15, 17.5 and 20 ml show a statistically significant difference compared to the 1 ml sample used as a reference. The count rate of a 20 ml phantom is considered to be underestimated by 20 % and measured count rates for the carcass were therefore corrected with a factor 1.2 to account for this. Figure 2: Count rate ratio between samples of different volumes and a sample of 1 ml at constant 131 I activity. Statistically significant differences compared with measurements of 1 ml samples (p 0.01) were found at 15, 17.5 and 20 ml sample volumes. Error bars display SEM; n=2. Adsorption Results from the adsorption tests are shown in Figure 3. The presented data are mean values normalized to the control syringes (n=5). The investigation showed no statistically significant difference between control syringes and the syringes where ejection was delayed by 4 or 8 hours. Therefore, no adsorption correction was made in the animal experiments. Figure 3: Investigation of adsorption effect in syringes. The mean values of activity concentration in syringes with 4 or 8 h delay between preparation and ejection are shown, normalized to the activity concentration in control syringes. The x-axis shows the time delay (compared to the control syringe) that would have been administered at the nominal time point 8 am. Error bars display with SEM, n=5. 11

15 Biodistribution of 131 I Biodistribution studies were performed for time spans h after administration with administrations performed at 8 am, 12 pm or 4 pm. For injections at 8 am, the highest activity concentration in tissues was found in the thyroid (5800 %IA/g) at 18 h after administration. High activity concentrations were also found in gastric content and stomach at earlier time points. For administration at 12 pm and 4 pm, the highest activity concentration in tissues was found, also in the thyroid, to be 2400 %IA/g and 2100 %IA/g respectively (both at the time point 8 h following injections). For the administration series of female mice, performed at 8 am, the highest activity concentration in tissues was also found in the thyroid (6000 %IA/g) 18 h following administration of 131 I. Please note that two out of the approximately 3000 samples in this work, i.e. two samples of small intestine content in males after 168 h with administration at 8 am, were not able to meet the criterion of obtaining 1000 counts over background, due to very low activity. Activity concentrations in tissues and gastrointestinal contents for males with administration at 8 am can be seen in Figure 4a and in Figure 4b; for administration at 12 pm in Figure 5a and Figure 5b; and for administration at 4 pm in Figure 6a and Figure 6b. For all tissues in male mice, with exception for the thyroid and large intestine content, the highest activity concentration was found 1 h after injections. for large intestine this happened at 4 h after injections. Activity concentrations in tissues and gastrointestinal contents for females with administration at 8 am can be seen in Figure 7a and in Figure 7b. For the female mice, the highest activity concentrations were found at 1 h after injections, with exceptions for thyroid, salivary gland and intestinal contents. The activity concentration levels in the salivary glands, reaching a maximum 8 h following administrations of 131 I, were found maximally at 24 %IA/g. In comparison, the maximum for the male mice was found 1 h following administration for all time of day of injection, and reached a maximum at almost 14 %IA/g (with administration at 12 pm). 12

16 a b Figure 4: Biodistribution of 131 I activity concentration in male mice at 1 h up to 7 days after administration of kbq, performed at 8 am. Error bars display SEM; n=5 for all data points. Please note the change of scale on the vertical axis. 13

17 a b Figure 5: Biodistribution of 131 I activity concentration in male mice at 1 h up to 3 days after administration of kbq, performed at 12 pm. Error bars display SEM; n=5 for all data points. Please note the change of scale on the vertical axis. 14

18 a b Figure 6: Biodistribution of 131 I activity concentration in male mice at 1 h up to 3 days after administration of kbq, performed at 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration (n=4). Please note the change of scale on the vertical axis. 15

19 a b Figure 7: Biodistribution of 131 I activity concentration in female mice at 1 h up to 7 days after administration of kbq, performed at 8 am. Error bars display SEM; n=5 for all data points. Please note the change of scale on the vertical axis. 16

20 Comparison between males at time point 8 am, 12 pm, and 4 pm Figure 8 24 show the biokinetics for each tissue for 131 I administration in males at 8 am, 12 pm, and 4 pm. The time points where statistically significant differences (p<0.05) were found between the three times of administration are noted in the figures. In general there were large deviations observed during time points from 1 h up to h, especially for gastrointestinal content, see Figure In the thyroid, see Figure 9, a statistically significant difference between administration at 8 am and 12 pm was found after 4 and 18 h, with a global maximum after 18 h for administration at 8 am. A maximum of activity concentration was found after 8 h for administration at 12 pm and 4 pm. For the neck and thyroid combined, plotted in %IA, see Figure 10, the maximum was observed more clearly after 8 h for administration at 12 pm; interestingly, for administration at 4 pm, two peaks were observed after 4 and 18 h. At 1 h after administration, there was a statistically significant difference for the small intestine between 8 am and 12 pm injections (Figure 18). Statistical significance was found 4 h after administration between 8 am and 12 pm in all tissues with exception for small intestine content. Between administrations performed at 12 pm and 4 pm, statistical significance was found after 4 h in all tissues except small intestine and thyroid combined with neck. No statistically significant difference was found 4 h after administration between 8 am and 4 pm. Statistical significance was found after 1 h only in small intestine between the 8 am and 12 pm groups (Figure 18). Significant difference was found between all three administration time points at 18 h for salivary gland and small intestine (Figure 16 and Figure 18). At 24 h after administration, the results showed no statistical significance in tissues, with the exception of stomach (between 8 am and 4 pm, Figure 20) and gastric content (between 8 am and 4 pm, and between 12 pm and 4 pm, Figure 23). At 72 h after administration, significance was found only between 8 am and 12 pm (in salivary gland, small intestine, gastric content and carcass, Figure 16, 18, 23 and 24). Between administrations at 8 am and 4 pm, statistically significant differences were found only after 8 and 18 h, with the exception of 24 h for stomach and gastric contents. Gastric contents showed statistical significance between 8 am and 12 pm for the time points 4 and 72 h; between 12 pm and 4 pm for the time points 4, 8 and 18 h; between 12 pm and 4 pm for the time points 4, 8, 18 and 24 h, see Figure 23. All organs, with the exception of thyroid, thyroid combined with neck, and large intestine content, showed the highest %IA/g at 1 h, after which the activity concentration declined monotonically. An exception was found in the kidneys, however, which showed a local maximum at 72 h with a statistically significant rise between 24 h and 72 h for all injection series (and a statistically significant rise between 18 and 24 h in the left kidney for the administration series performed at 12 pm and 4 pm, as well as the right kidney for the administration series performed at 12 pm), see Figure The injection series at 12 pm depicted a slower decline in activity concentration than injections at 8 am and 4 pm, 1 18 h after administration. 17

21 Figure 8: 131 I concentration in blood for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 and 18 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 4 and 8 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Figure 9: 131 I concentration in thyroid for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 and 18 h between 8 am and 12 pm; after 4 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 18

22 Figure 10: 131 I concentration in neck and thyroid for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 and 18 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 8 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Please note that the graph display %IA. Figure 11: 131 I concentration in lungs for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 4 and 8 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 19

23 Figure 12: 131 I concentration in heart for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 4 and 18 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Figure 13: 131 I concentration in liver for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 4 and 18 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 20

24 Figure 14: 131 I concentration in left kidney for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 and 18 h between 8 am and 12 pm; after 8 h between 8 am and 4 pm; after 4 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Figure 15: 131 I concentration in right kidney for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 h between 8 am and 4 pm; after 4 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 21

25 Figure 16: 131 I concentration in salivary glands for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4, 18 and 72 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 4 and 18 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Figure 17: 131 I concentration in spleen for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 4 and 18 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 22

26 Figure 18: 131 I concentration in small intestine for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 1, 4, 18 and 72 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 18 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Figure 19: 131 I concentration in large intestine for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 h between 8 am and 4 pm; after 4 and 8 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 23

27 Figure 20: 131 I concentration in stomach for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8, 18 and 24 h between 8 am and 4 pm; after 4 and 18 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Figure 21: 131 I concentration in small intestine content for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 24

28 Figure 22: 131 I concentration in large intestine content for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 4, 8 and 18 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Figure 23: 131 I concentration in gastric content for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 and 72 h between 8 am and 12 pm; after 8, 18 and 24 h between 8 am and 4 pm; after 4, 8, 18 and 24 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 25

29 Figure 24: 131 I concentration in carcass for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was found after 4 and 72 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 4 and 8 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). 26

30 Sex difference in 131 I biodistribution Biokinetics for both male and female mice, injected with 131 I at 8 am, are presented in Figure The time points where statistically significant differences were found between the sexes are indicated in the figures. In general, statistically significant differences were found at least at three time points in all samples. Only three samples showed fewer or no significant differences: for the thyroid (see Figure 26) and large intestine content (see Figure 39), significance was found at two time points, whereas for thyroid combined with neck (see Figure 27) no statistical significance was observed. The samples for which the highest number of statistically significant differences were found were the carcass (see Figure 41; statistical significance at all time-points), the spleen (see Figure 34) and the kidneys (see Figure 31 32). In total, statistically significant differences between male and female mice were demonstrated in 70 out of 119 observations for the 17 samples studied. In the thyroid, statistically significant differences were found after 24 and 72 h between male and female mice. The activity concentration peaked after 18 h for both sexes, yet presumably with a somewhat higher activity concentration in females (about 200 %IA/g higher mean value), see Figure 26. For male mice, there was a global maximum after 18 h with a statistically significant difference compared with 24 h after administration. For neck and thyroid together, the biokinetics also depicted a statistically significant maximum at 18 h (showing statistically significant difference between 8 and 18 h, and between 18 and 24 h for both sexes) for both males and females, although there was no statistically significant difference between the sexes, see Figure 27. For the kidneys, see Figure 31 32, distinctly different curve behavior was observed between the sexes: In male mice, there was a faster decrease in activity concentration in the time span between 1 and 18 h, followed by an increase with a local maximum after 72 h and subsequent monotone decrease. In contrast, activity concentration in female mice did not display a local maximum but monotone decrease as observed for various other samples. After 72 and 168 h, there was a comparatively large difference in activity concentration for male and female mice. Statistically significant difference was found at all time-points, with the exception of 1 h. A majority of the remaining tissues showed a fast decrease of activity concentration within the first 18 h for both sexes (where female mice showed a slightly less steep decrease in the time span between 1 and 18 h), with some exceptions: For salivary glands (see Figure 33) and small intestines (see Figure 35), the curve depicted an increase of activity concentration for female mice after 1 4 h reaching a maximum after 4 h. In the large intestine content, see Figure 39, the activity concentration in females depicted a distinct maximum after 8 h, which was about twice as high as the observed maximum for males; statistically significant differences for the large intestine content were observed at 8 and 168 h. 27

31 Figure 25: 131 I concentration in blood for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8, 24, 72 and 168 h. Error bars display SEM; n=5. Figure 28: 131 I concentration in lungs for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8, 24 and 72 h. Error bars display SEM; n=5. Figure 26: 131 I concentration in thyroid for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 24 and 72 h. Error bars display SEM; n=5. Figure 29: 131 I concentration in heart for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8, 18, 24 and 72 h. Error bars display SEM; n=5. Figure 27: 131 I concentration in neck and thyroid for administrations at 8 am for male and female mice. No statistical significance was found Error bars display SEM; n=5. Figure 30: 131 I concentration in liver for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8, 18 and 72 h. Error bars display SEM; n=5. 28

32 Figure 31: 131 I concentration in left kidney for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8, 18, 24, 72 and 168 h. Error bars display SEM; n=5. Figure 34: 131 I concentration in spleen for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 1, 4, 8, 18, 24 and 72 h. Error bars display SEM; n=5. Figure 32: 131 I concentration in right kidney for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8, 18, 24, 72 and 168 h. Error bars display SEM; n=5. Figure 35: 131 I concentration in small intestine for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8, 18, 24 and 72 h. Error bars display SEM; n=5. Figure 33: 131 I concentration in salivary glands for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 1, 4, 8, 18 and 72 h. Error bars display SEM; n=5. Figure 36: 131 I concentration in large intestine for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8, 24 and 72 h. Error bars display SEM; n=5. 29

33 Statistical significance (p<0.05) was found after 8 and 168 h. Error bars display SEM; n=5. Figure 37: 131 I concentration in stomach for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 18, 24 and 72h. Error bars display SEM; n=5. Figure 40: 131 I concentration in gastric content for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 1, 4, 24 and 72 h. Error bars display SEM; n=5. Figure 38: 131 I concentration in small intestine content for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found after 4, 8 and 24 h. Error bars display SEM; n=5. Figure 41: 131 I concentration in carcass for administrations at 8 am for male and female mice. Statistical significance (p<0.05) was found at all timeintervals. Error bars display SEM; n=5. Figure 39: 131 I concentration in large intestine content for administrations at 8 am for male and female mice. 30

34 Influence of iodine concentrations in food at 12 pm Biokinetics for 131 I administration at 12 pm with either normal or reduced levels of iodine in food is presented for blood, thyroid, liver and kidneys in Figure Statistical significance was found at 1 h for all five tissues, with the exception of thyroid where no statistical significance was found. The thyroid showed a variation of activity concentrations at 1 and 4 h, but no statistical significance was found. The data for the remaining samples are shown in the Appendix, Figure Figure 44: 131 I concentration in liver for administrations at 12 pm for normal and reduced iodine concentration in food. Statistical significance (p<0.05) was found after 1 h. Error bars display SEM; n=5. Figure 42: 131 I concentration in blood for administrations at 12 pm for normal and reduced iodine concentration in food. Statistical significance (p<0.05) was found after 1 h. Error bars display SEM; n=5. Figure 45: 131 I concentration in left kidney for administrations at 12 pm for normal and reduced iodine concentration in food. Statistical significance (p<0.05) was found after 1 h. Error bars display SEM; n=5. Figure 43: 131 I concentration in thyroid for administrations at 12 pm for normal and reduced iodine concentration in food. No statistical significance was found. Error bars display SEM; n=5. Figure 46: 131 I concentration in right kidney for administrations at 12 pm for normal and reduced iodine concentration in food. Statistical significance (p<0.05) was found after 1 h. Error bars display SEM; n=5. 31

35 Influence of iodine concentrations in food at 4 pm Biokinetics for 131 I administration at 4 pm with either normal or reduced levels of iodine in food is presented for blood, thyroid, liver and kidneys in Figure The results showed no significant differences for these tissues, with the exception of thyroid for which significant difference was found after both 1 and 4 h. The data for the remaining samples are shown in the Appendix, Figure Figure 49: 131 I concentration in liver for administrations at 4 pm for normal and reduced iodine concentration in food. No statistical significance was found. Error bars display SEM; n=5. Figure 47: 131 I concentration in blood for administrations at 4 pm for normal and reduced iodine concentration in food. No statistical significance was found. Error bars display SEM; n=5. Figure 50: 131 I concentration in left kidney for administrations at 4 pm for normal and reduced iodine concentration in food. No statistical significance was found. Error bars display SEM; n=5. Figure 48: 131 I concentration in thyroid for administrations at 4 pm for normal and reduced iodine concentration in food. Statistical significance (p<0.05) was found after 1, 4 and 72 h. Error bars display SEM; n=5. Figure 51: 131 I concentration in right kidney for administrations at 4 pm for normal and reduced iodine concentration in food. No statistical significance was found. Error bars display SEM; n=5. 32

36 Effect of circadian rhythm at standard iodine concentration in food The comparison between 131 I administration at 12 pm or 4 pm with standard levels of iodine in food is shown for blood, thyroid, liver and kidneys in Figure The activity concentration curves in blood, liver and kidneys intersect between 1 and 4 h and show statistically significant differences at time points 1 and 4 h. Figure 54: 131 I concentration in liver for administrations at 4 pm and 12 pm for normal iodine concentration in food. Statistical significance (p<0.05) was found after 1 and 4 h. Error bars display SEM; n=5. Figure 52: 131 I concentration in blood for administrations at 4 pm and 12 pm for normal iodine concentration in food. Statistical significance (p<0.05) was found after 1 and 4 h. Error bars display SEM; n=5. Figure 55: 131 I concentration in left kidney for administrations at 4 pm and 12 pm for normal iodine concentration in food. Statistical significance (p<0.05) was found after 1 and 4 h. Error bars display SEM; n=5. Figure 53: 131 I concentration in thyroid for administrations at 4 pm and 12 pm for normal iodine concentration in food. No statistical significance was found. Error bars display SEM; n=5. Figure 56: 131 I concentration in right kidney for administrations at 4 pm and 12 pm for normal iodine concentration in food. Statistical significance (p<0.05) was found after 1 and 4 h. Error bars display SEM; n=5. 33

37 Discussion In this work, animal experiments were conducted to reproduce and further extend previous investigations on the influence of circadian rhythm on 131 I biodistribution. Furthermore, potential differences between male and female mice and the impact of iodine concentration in animal food were investigated, concerning the biodistribution and biokinetics of 131 I. In addition, several technical investigations were made to minimize measurement errors. These investigations concerned selfattenuation in samples and the sensitivity of the gamma counter when measuring large samples, and the decrease of activity concentration over time by means of adsorption on the inside surface of syringes. The latter is of concern when the experimental schedule necessitates that syringes are prepared early in the morning, while the injections for different groups are distributed across the day, which leads to that certain syringe batches are used approximately four or eight hours after preparation. In addition to this, calibrations of the gamma counters were performed to translate the counted pulses of the samples in the gamma counters to an activity in kbq. Calibration factors of the gamma counters were acquired for 131 I by plotting measured count rates from samples between 1.6 kbq to 25 kbq, measured in the two gamma counters available, and making a linear curve fit to these data points. The results show that a count rate up to approximately is acceptable for both gamma counters. With obtained R 2 -values , the linear curve fitting was a good approximation and the count rate response could be considered linear up to 25 kbq for 131 I, which corresponded to acceptable dead time factors of up to 11%. A volume effect was observed for large sample sizes, which would lead to underestimation of cps if not corrected for. Although only the volume sizes of 15, 17.5 and 20 ml could be proven statistically significant (p 0.01), the trend indicated that the effect may appear at 10 ml or even smaller volumes. In particular, this issue concerned measurement of the mouse carcass, which constitutes a very large sample that fills nearly the entire volume of a 20 ml scintillation vial. The loss of counts due to this volume effect was estimated to be 20 %, and a correction for this was made to more accurately estimate the activity in the carcass. It should be noted that the activity in the carcass is an important quantity, for instance when designing a compartment model for 131 I biodistribution in future work. The correction, however, was only a rough approximation based on homogenous density of the water phantom in the scintillation vial. A more accurate approximation for the loss of pulses would require a tissue-equivalent phantom with non-homogenous density distribution corresponding to a mouse carcass, or a computer simulation of respective scintillation vials with radiation transport codes. The investigation of 131 I adsorption in syringes (brand "Braun") showed that adsorption did not occur to a significant extent (p>0.01) when injection was delayed up to 8 h. This finding is of particular importance for planning these types of animal studies, since the nominal administered activity is valid even if syringes are used several hours later. This means that all syringes can be prepared once from the same stock solution without the necessity to correct for adsorption with additional (increased) stock concentrations. In previous work, various time points following 131 I administration showed statistically significant difference in %IA/g between administration at 12 pm or 4 pm and a number of these instances was also reproduced in the present studies. For instance, in the lungs after 4 and 8 h following injection, statistically significant differences were demonstrated in both previous and present work. Regarding the thyroid, circadian variation in 131 I biodistribution was also demonstrated with statistical significance, although at another time point after administration compared to previous work (Andersson, 2015). Interestingly, the novel time point of administration revealed a distinctly different biodistribution curve behavior for the thyroid: for the earliest administration time point (8 am), the peak of activity concentration occurred at a much later time point after administration compared with later administrations (12 pm or 4 pm). Moreover, the effective day times for respective peaks did not 34