Radiation and Environmental Biophysics

, Volume 50, Issue 4, pp 501–511

Biokinetics of 90Sr after chronic ingestion in a juvenile and adult mouse model

Authors

  • Nicholas Synhaeve
    • Institut de Radioprotection et Sûreté Nucléaire (IRSN), DRPH, SRBE, LRTOX
  • Johanna Stefani
    • Institut de Radioprotection et Sûreté Nucléaire (IRSN), DRPH, SRBE, LRTOX
  • Elie Tourlonias
    • Institut de Radioprotection et Sûreté Nucléaire (IRSN), DSU, SSTC, BELCY
  • Isabelle Dublineau
    • Institut de Radioprotection et Sûreté Nucléaire (IRSN), DRPH, SRBE, LRTOX
    • Institut de Radioprotection et Sûreté Nucléaire (IRSN), DRPH, SRBE, LRTOX
Original Paper

DOI: 10.1007/s00411-011-0374-9

Cite this article as:
Synhaeve, N., Stefani, J., Tourlonias, E. et al. Radiat Environ Biophys (2011) 50: 501. doi:10.1007/s00411-011-0374-9

Abstract

The aim of our study was to define the biokinetics of 90Sr after chronic contamination by ingestion using a juvenile and adult murine model. Animals ingested 90Sr by drinking water containing 20 kBq l−1 of 90Sr. For the juvenile model, parents received 90Sr before mating and their offspring were killed between birth and 20 weeks of ingestion. For the adult model, 90Sr ingestion started at 9 weeks of age and they were killed after different ingestion periods up to 20 weeks. The body weight, food and water consumption of the animals were monitored on a weekly basis. Before killing and sampling of organs, animals were put in metabolic cages. 90Sr in organs and excreta was determined by liquid scintillation β counting. Highest 90Sr contents were found in bones and were generally higher in females than in males, and 90Sr retention varied according to the skeletal sites. An accumulation of 90Sr in the bones was observed over time for both models, with a plateau level at adult age for the juvenile model. The highest rate of 90Sr accumulation in bones was observed in early life of offspring, i.e. before the age of 6 weeks. With the exception of the digestive tract, 90Sr was below the detection limit in all other organs sampled. Overall, our results confirm that 90Sr mainly accumulates in bones. Furthermore, our results indicate that there are gender- and age-dependent differences in the distribution of 90Sr after low-dose chronic ingestion in the mouse model. These results provide the basis for future studies on possible non-cancerous effects during chronic, long-term exposure to 90Sr through ingestion in a mouse model, especially on the immune and hematopoietic systems.

Introduction

Strontium 90 (90Sr) is a radionuclide of exclusively anthropogenic origin, which was released in large amounts in several situations, such as waste releases in the Techa River during the 1950s, nuclear weapons atmospheric tests during the 1960s and 1970s (Sodeman et al. 1991) and Chernobyl reactor #4 explosion in 1986 (UNSCEAR 2011). 90Sr has a half-life of 28.2 years, and due to its solubility and its chemical properties, it is a long-term remnant in the environment. As a result, large populations were chronically exposed to low concentrations of this radionuclide, either through ingestion or through inhalation (Cooper et al. 1992; De Ruig and Van der Struijs 1992; Hoshi et al. 1994). This prompted a large amount of work on both the biokinetics of strontium and the biological effects of 90Sr, in humans as well as in animal models, mainly during the 1960s and 1970s (Sodeman et al. 1991). As a result, the International Commission on Radiological Protection (ICRP) proposed a biokinetic model for humans based upon the similar biological behaviour of strontium when compared with calcium (ICRP 1993). Strontium is absorbed in humans at a ratio of 30%, depending mainly on the form of the diet (solid or liquid) and on the level of calcium in the diet (Apostoaei 2002; Höllriegl et al. 2006). Once absorbed, 90Sr then accumulates mainly in bones (ICRP 1993). Indeed, experimental studies with 90Sr-fed beagles, swines and rats showed that 90Sr migrates to the bones and can be retained there for a considerable time (Book et al. 1982; Gillett et al. 1992; Nilsson and Book 1987).

The biological effects of 90Sr internal contamination are closely linked to the anatomic site of strontium accumulation and fixation in bones. Studies both in miniature swines and in beagles demonstrated that 90Sr contamination led to primary bone tumours (Book et al. 1982; Gillett et al. 1992; Nilsson and Book 1987), but also to soft tissue cancers adjacent to bone (Raabe et al. 1981), squamous cell carcinoma in the jaws (Parks et al. 1984) and hematopoietic neoplasms (Howard and Clarke 1970; Nilsson 1971). The occurrence of non-cancerous effects following a chronic, low-dose ingestion of 90Sr is less well-documented, with the exception of hematopoietic aplasia induced in beagles after an acute high dose of 90Sr inhalation (Gillett et al. 1987) or in rats after single injection of various doses of 90Sr (Stokke et al. 1968). In fact, recent results showed that the chronic ingestion of low doses of 137Cs induced modifications in various physiological systems (Dublineau et al. 2007; Gueguen et al. 2008; Tissandie et al. 2008). The study of populations exposed to chronic contamination due to the Chernobyl accident equally suggested the occurrence of immune and hematopoietic alterations such as immunoglobulin decrease (Vykhovanets et al. 2000) and CD4/CD8 ratio modification (Chernyshov et al. 1997). In these later cases, these alterations were associated with 137Cs contamination, although the possible role of other radionuclides was not ruled out.

Thus, the possible role of 90Sr in the occurrence of non-cancerous effects in populations exposed to long-term, low-dose chronic internal contamination remains to be studied. We thus used two mouse models of chronic contamination through low concentrations of 90Sr in drinking water, namely a juvenile model and an adult model. In a first step study, these models were used in order to compare the biokinetics and the biodistribution of 90Sr in mice with current knowledge. In fact, most previously published studies were conducted either on rats (Gran 1960; Stokke et al. 1968), large animal models (Howard and Clarke 1970; Nilsson and Book 1987) or on humans who had been accidentally contaminated (Shagina et al. 2003). In the juvenile model, parents were contaminated before mating and until weaning of offspring, which were then contaminated through drinking water until the age of killing. This juvenile model was chosen because a placental transfer of strontium to the foetus was demonstrated (MacDonald et al. 1962; Ruhmann et al. 1963). Moreover, the in utero and post-natal periods are known to be particularly sensitive to toxic compounds (Blakley and Blakley 2005). The adult model was used to verify the age dependence of 90Sr biokinetics. The adult model started with a chronic ingestion of 90Sr by animals at the age of 9 weeks and a maximal ingestion period of 20 weeks. The animals of both models were contaminated through drinking water with a 90Sr concentration of 20 kBq l−1. This concentration was also used in previous studies at our laboratory to determine the biokinetics and biological effects of 137Cs chronic contamination (Bertho et al. 2010; Tourlonias et al. 2010) and represents an expected mean daily intake of 100 Bq per animal. Moreover, in both models male and female animals were used in order to verify the gender dependence of 90Sr biokinetics.

Materials and methods

Animals

Nine-week-old Balb/c mice, purchased from Elevage Janvier (Le Genest Saint Isle, France), were used throughout the study. Animals received ad libitum water and standard rodent chow with a mean calcium concentration of 9 g Ca2+/kg (normal calcium diet, R03-type chow, Safe, Epinay-sur-Orge, France). They were housed in standard cages and were kept at a constant room temperature (21°C ± 2°C) with a 12-h daylight cycle. All experimental procedures were approved by the animal care committee of the Institut de Radioprotection et de Sûreté Nucléaire (IRSN) and conformed to the French regulations for animal experimentation (Ministry of agriculture Act No. 8 7-848, 19 October 1987, modified May 29, 2001).

Experimental design

Two groups of mice were constituted throughout the experiments. One control group receiving normal water and one group receiving water containing 20 kBq l−1 of 90Sr (CERCA-LEA, Pierrelatte, France).

For the juvenile mouse model, male and female parents received normal or 90Sr containing drinking water starting 2 weeks before mating and until the date of killing. Control and contaminated breeding groups were then constituted with one male and two females for 1 week. Male parents were then anesthetized and killed by cervical dislocation and their femurs were collected for 90Sr measurement. Births within breeding groups were carefully recorded. Three weeks after birth, i.e. at the time of weaning, female parents were anesthetized and killed by cervical dislocation and femurs were collected for 90Sr measurement. Sexing of the offspring was made, and the mice were separated into groups of three control to six 90Sr ingesting animals and continued to receive normal or 90Sr contaminated drinking water until killing at 3, 6, 12, 16 and 20 weeks old (Fig. 1) for organ sampling. In order to avoid possible litter effects, groups of killing were constituted with six males and six females originating from different litters. As a result, offspring received 90Sr continuously through placenta during foetal life, through lactation before weaning and through drinking water thereafter.
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Fig. 1

Summary of the 90Sr ingestion schedules used for the juvenile (a) and the adult (b) mouse model. Periods of 90Sr ingestion are indicated by hatched areas. The killing time points of the animals are indicated by vertical bars for both models. For each time-point, six control and 12 90Sr ingesting animals were used, with a sex ratio of 1:1

For the adult model, groups of three controls and six 90Sr ingesting nine-week-old mice were formed. They were killed after the same time periods as for the juvenile mouse model, i.e. after 3, 6, 12, 16 and 20 weeks of chronic ingestion (Fig. 1).

Body weight, food and water consumption

A weekly record of individual body weight was made together with a weekly follow-up record per cage for food and water consumption. For the juvenile model, this was done from the time of weaning. The mean food and water consumption per animal and per day was then obtained by calculation on the basis of the number of mice per cage. Based upon an assumed daily water consumption of 5 ml per animal at adult age, a 90Sr contamination of about 100 Bq per animal per day at adult age was expected.

Metabolic cage experiments

Before killing, animals of the adult mouse model were placed individually in metabolic cages for 48 h. Faeces and urine were collected over the whole period and were measured for 90Sr content.

Organ sampling

For the juvenile model at birth, 3, 6, 12, 16 and 20 weeks old or for the adult model at 3, 6, 12, 16 and 20 weeks of 90Sr ingestion, six control and 12 contaminated mice with equal number of males and females were anesthetized by an intraperitoneal injection of a mixture of ketamine (Imalgene, Mérial, Villeurbanne, France) and xylazine (Rompun, Bayer Healthcare, Monheim, Germany). Blood was drawn by intracardiac puncture using heparinised tubes (Choay, Sanofi Aventis, Paris, France), and the mice were killed by cervical dislocation. The following organs were then collected: skin, liver, spleen, kidneys, digestive tract (from stomach to the rectum), heart, thymus, lungs, skeletal muscle, femurs and central nervous system (CNS). Remaining tissues were separated into two parts: legs, tail, skin on the one hand, and the remaining upper part of the body (the carcass) on the other hand. Samples were weighted before 90Sr measurements.

Animals used for the determination of 90Sr content at different skeletal sites were killed and internal organs were removed. Carcasses were then treated entirely for one hour in boiling water and overnight at 56°C in the presence of papain (1 mg/ml final concentration, Sigma–Aldrich, Saint Quentin Fallavier, France). Bones were then isolated and grouped according to the different skeletal sites under an inverted microscope.

90Sr measurements

Organs were calcined at 500°C in an oven for five hours. Ashes were dissolved in nitric acid (HNO3, 67%) and hydrogen peroxide (H2O2, 30%, both from VWR, Fontenay-sous-bois, France) in a 2:1 ratio. These steps were repeated twice for each sample. Samples were redissolved in 1 ml nitric acid (HNO3, 10%) by shaking overnight. Fifteen millilitre of scintillation liquid was added (Ultima Gold AB, Perkin Elmer, Courtaboeuf, France) to each sample and 90Sr measurements were made with a β counter (TRI-CARB 2700TR Liquid Scintillation Analyzer, Perkin Elmer). 90Sr activity was then measured at least 1 month after organ sampling, the time necessary to reach a secular equilibrium at the time of counting between initial 90Sr content in samples and 90Y. The counting time per sample of organs was 2 h. The count rate (in counts per minute—cpm) obtained for each sample was converted to activity per mass unit Am (in Bq g−1) using the following equation:
$$ A_{\text{m}} { = }\left( {{\text{CPM}}_{\text{gross}} - {\text{CPM}}_{\text{control}} } \right)/\left( {(60 \times E \times m)/100} \right) $$
(1)
where CPMgross is the gross measurement of the sample (cpm), CPMcontrol the mean background cpm of 10 control samples, m the mass of the samples (g) and E the efficiency of 90Sr detection.
This efficiency of detection E (%) was determined with the following equation:
$$ E = \left( {{\text{CPM}}_{\text{add}} - {\text{ CPM}}_{\text{gross}} } \right)/\left( {(60 \times A_{\text{add}} )/100} \right) $$
(2)
where CPMadd is the gross measurement of the sample after a defined activity of 2 Bq of 90Sr was added (cpm) and Aadd the defined activity of 90Sr added (2 Bq). Organs from control animals were counted under the same conditions.

Results were then normalised to the wet weight for each organ and expressed thereafter as Bq g−1. The whole-body specific activity was then calculated as the sum of 90Sr activities (Bq) in all organs divided by the total body mass of the animals (g). The detection limit of 90Sr was between 0.45 Bq and 0.75 Bq per sample depending of the organs.

Transfer rate calculations

The rate of 90Sr accumulation in bones BqΔt (in Bq day−1) was calculated using the following equation:
$$ {\text{Bq}}_{\Updelta t} = \, \left( {{\text{Bq}}_{t} - {\text{Bq}}_{t - 1} } \right)/\left( {\Updelta t} \right) $$
(3)
where Bqt and Bqt−1 are the total activity (in Bq) of 90Sr measured in bones at the times t and t – 1, respectively, and Δt the number of days between the two time points t and t − 1.
The daily intestinal absorption ratio (IAR) was calculated using the following equation:
$$ {\text{IAR}} = \left( {I - {\text{FE}}} \right)/I $$
(4)
where I is the mean daily 90Sr ingestion calculated on the basis of weekly measurements (as described above) in Bq day−1 and FE is the measured individual 90Sr excretion through faeces in Bq day−1.

Statistical analysis

Groups of killing were constituted with offspring originating from different litter. For each group of killing, three control and six contaminated males and three control and six contaminated females were used. All results are presented as mean ± standard deviation (SD). All statistical analyses were performed using Sigmaplot software (Systat software Inc., San Jose, Ca). Comparisons between groups were made with either Student t test or 2-way ANOVA (analysis of variance) test, as indicated in the text. Differences were considered statistically significant for p < 0.05.

Results

Distribution of 90Sr in the juvenile model

A regular weight gain was observed for males and females (Fig. 2a). No significant modification of body mass of animals has been observed between control and 90Sr-ingesting animals, regardless of the age or sex of the offspring (2-way ANOVA analysis, F(1,50) = 1.06, non-significant (n.s.) for males and F(1,50) = 0.017, n.s. for females), although an increasing difference was observed between control and contaminated males at ages of 16 weeks and more. The reason for this observation in males remains unclear, but time-specific statistical analysis (using Student t test) did not showed significant differences (Fig. 2a). A body mass of 30.7 ± 5.2 g for control males, 28.7 ± 5.2 g for 90Sr ingesting males, 25.8 ± 4.7 g for control females and 25.6 ± 1.6 g for 90Sr-ingesting females was reached after 20 weeks of ingestion.
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Fig. 2

a Evolution of the body mass (g) of control and 90Sr-ingesting animals of the juvenile model, according to the age and sex of the animals b90Sr intake (Bq animal−1 day−1) through drinking water, according to the age and sex of animals from the juvenile model. Results are presented as mean ± standard deviation (SD), with n = 3–9 for control mice and n = 6–24 for 90Sr ingesting mice, depending on age

A weekly follow-up of food and drinking water consumption per cage was recorded starting from the time of weaning. The mean food and water intake per animal and per day was obtained by calculation, on the basis of the number of animals per cage. Based on these data, the corresponding daily ingestion of 90Sr was calculated (Fig. 2b). The results indicate that 90Sr ingestion increased rapidly the first weeks post-weaning, reaching 68.2 ± 9.7 Bq animal−1 day−1 for males and 71.2 ± 7.7 Bq animal−1 day−1 for females at 6 weeks of chronic ingestion. No significant difference in 90Sr ingestion was observed between males and females, regardless of their age (2-way ANOVA analysis, F(1,165) = 1.042, n.s.). The mean daily intake of 90Sr over the whole period of 20 weeks of chronic ingestion was 74.3 ± 14.6 Bq for males and 69.0 ± 11.6 Bq for females. This was lower than the expected ingestion of 100 Bq per animal per day at adult age and was caused by a lower drinking water intake than expected. In fact, the mean daily water intake per animal over the whole period of 20 weeks was 4.6 ± 0.9 ml day−1 for control males, 3.7 ± 0.7 ml day−1 for 90Sr ingesting males, 3.4 ± 0.5 ml day−1 for control females and 3.5 ± 0.6 ml day−1 for 90Sr ingesting females, respectively.

In order to investigate the distribution of 90Sr after chronic ingestion, the 90Sr content was measured in different organs [skin, liver, spleen, kidneys, digestive tract (from stomach to the rectum), heart, thymus, lungs, skeletal muscle, femurs and central nervous system (CNS)] of the male and female animals from both control and 90Sr ingestion groups. 90Sr was below the detection limit in the organs of the control animals. The detection limit of 90Sr was between 0.45 Bq and 0.75 Bq per sample. For 90Sr-ingesting animals, the highest 90Sr concentrations were found in the bones. It should be noted that 90Sr content in femurs (Fig. 3a) was measured without separating the bone from the bone marrow. According to the age of the animals, a significant increase in 90Sr content with time in femurs was seen (2-way ANOVA analysis, F(5,64) = 266.5, p < 0.001), reaching 79.7 ± 9.4 Bq g−1 and 75.4 ± 9.9 Bq g−1 at 6 weeks for females and males, respectively. At adult age (6–20 weeks of offspring), 90Sr content in the femurs seems to be stabilised at 72.6 ± 7.0 Bq g−1 for males and 75.0 ± 2.1 Bq g−1 for females. No significant difference appeared in the evolution of 90Sr in the femurs between males and females (2-way ANOVA analysis, F(1,64) = 0.521, n.s.) even if a temporary difference was evidenced at 12 weeks old (p = 0.035) (Fig. 3a).
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Fig. 3

90Sr concentration (Bq g−1) a in the femurs and b the digestive tract of 90Sr-ingesting animals from the juvenile model, according to the age and sex of the animals. All the results are presented as mean ± SD, with n = 6 per group. Time-specific differences between males and females are significant for *p < 0.05 (Student t test)

90Sr was below the detection limit in all other organs tested (data not shown) with the exception of the digestive tract (Fig. 3b). A significant variation with age was observed (2-way ANOVA analysis, F(5,64) = 17.3, p < 0.001) with a peak of 90Sr content at 12 weeks (3.0 ± 1.8 Bq g−1 for males and 2.0 ± 1.1 Bq g−1 for females) and a decrease afterwards. Again, no significant difference was found between males and females (2-way ANOVA analysis, F(1,64) = 1.36, n.s.) with the exception of a temporary difference at 12-week old (p = 0.031) (Fig. 3b). In order to delineate more precisely 90Sr location in the digestive tract, 90Sr activity was measured in different segments of the digestive tract, namely the stomach, the small intestine, the caecum and the colon. Results showed that 90Sr was only detectable in the small intestine with a maximum activity of 0.7 ± 0.3 Bq g−1 both at 6 and 20 weeks and not in other segments (data not shown). Since the villi of the digestive tract are highly prominent in the small intestine, this result suggests that the 90Sr retention was mainly due to a mechanical retention in the villi of the small intestine.

The mean 90Sr whole-body activity (Fig. 4) was calculated by dividing the sum of all 90Sr activity detected by the body mass. Results show that between birth and 12 weeks of offspring, a significant increase in the mean whole-body activity is seen (2-way ANOVA analysis, F(5,64) = 72.32, p < 0.001). After 12 weeks of chronic ingestion, the mean whole-body activity showed a slight decrease over time for both males and females, down to 4.9 ± 0.6 Bq g−1 for males and 5.6 ± 0.3 Bq g−1 for females at 20 weeks of age of offspring. With the exception of 6 weeks (3.6 ± 2.2 Bq g−1 for males and 2.7 ± 1.6 Bq g−1 for females), the mean whole-body activity of females was always higher than for males with significant differences between them at 3 weeks (Student t test, p = 0.043) of age of offspring.
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Fig. 4

Mean whole-body 90Sr activity (Bq g−1) in 90Sr-ingesting animals from the juvenile model, according to the age and sex of the animals. All the results are presented as mean ± SD with n = 6 animals per group. A significant evolution of whole-body activity according to the age of the animals was observed (2-way ANOVA test, F(5, 64) = 72.32, p < 0.001) but not according to the sex of the animals. Time-specific differences between males and females are significant for *p < 0.05 (Student t test)

Distribution of 90Sr in the adult model

A continuous mass gain was observed for males and females, reaching a body weight of 29.7 ± 1.5 g for control males, 30.0 ± 1.2 g for 90Sr ingesting males, 24.4 ± 0.7 g for control females and 24.2 ± 1.4 g for 90Sr ingesting females, after 20 weeks of ingestion (Fig. 5a). No significant differences in mass gain between control and 90Sr-ingesting animals were observed (2-way ANOVA analysis, F(1,444) = 3.71 for males and F(1,444) = 3.56 for females, n.s.). The mean calculated daily ingestion of 90Sr per animal was 91.1 ± 16.2 Bq for males and 75.4 ± 14.3 Bq for females over the whole period of 20 weeks of ingestion. There was a significantly higher 90Sr intake among the males compared with the females (2-way ANOVA analysis, F(1,416) = 192.3, p < 0.001) (Fig. 5b). This was due to lower water intake by the females. In fact, the mean daily water intake per animal over the whole period of 20 weeks of ingestion was 5.1 ± 1.0 ml day−1 for control males, 4.6 ± 0.8 ml day−1 for 90Sr-ingesting males, 4.0 ± 0.8 ml day−1 for control females and 3.8 ± 0.7 ml day−1 for 90Sr-ingesting females (data not shown). As for the juvenile model, the mean daily 90Sr ingestion was lower than envisaged; this was caused by a lower overall water intake than expected. One should note that 90Sr intake showed major week-to-week variations, especially in the first 3 weeks of the experiment. The reasons for these variations are unclear. Since these variations are linked to variation in water intake, we may suggest that this is due to variations in the temperature of the animal care that in turn induced variations in water intake by animals. Nevertheless, one also should note that there was no significant difference in water intake between control and 90Sr-ingesting animals and that all animals showed a regular mass gain.
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Fig. 5

a Evolution of the body mass (g) of control (closed symbols) and 90Sr ingesting animals (open symbols) from the adult model, according to the age and sex of the animals. b90Sr intake (Bq animal−1 day−1) through drinking water according to the age and sex of the animals from the adult model. Results are presented as mean ± standard deviation (SD) with n = 3–9 for control mice and n = 6–30 for 90Sr ingesting mice

A significant continuous increase of 90Sr content in femurs was found (2-way ANOVA analysis, F(4,50) = 29.6, p < 0.001) (Fig. 6a). At 20 weeks of chronic ingestion, this resulted in a 90Sr concentration of 33.6 ± 5.2 Bq g−1 for males and 39.5 ± 2.8 Bq g−1 for females. Two-way ANOVA analysis did not show overall significant differences between males and females (F(1,50) = 1.42, n.s.), although a significantly higher accumulation was found in females after 20 weeks of ingestion compared with males (Student t test, p < 0.05). It has to be noted that after 20 weeks of ingestion the 90Sr content in the femurs from adult animals (36.5 ± 5.1 Bq g−1) was lower than those from juvenile animals (73.6 ± 6.9 Bq g−1).
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Fig. 6

a90Sr concentration (Bq g−1) in the femurs and b mean whole-body 90Sr activity (Bq g−1) of contaminated animals from the adult model, according to the age and sex of the animals. All the results are presented as mean ± SD with n = 6 animals per point. Time-specific differences between males and females are significant for *p < 0.05 and **p < 0.001 (Student t test)

90Sr was also found in the digestive tract in low quantities (data not shown). The mean 90Sr content over the whole 20-week period of ingestion was 0.3 ± 0.2 Bq g−1 for males and 0.3 ± 0.2 Bq g−1 for females. Similarly to what was observed in the juvenile model, most of the detected 90Sr activity was detected in the small intestine (data not shown). However, the lower concentrations of 90Sr found in the adult model compared with the juvenile model may be due to better cleaning of the different segments of the digestive tract. In all other organs than the bones and the digestive tract, 90Sr was below the detection limit.

The mean 90Sr whole-body activity (Fig. 6b) showed also a continuous increase over time (2-way ANOVA analysis, F(4,50) = 90.85, p < 0.001), with females having a systematically and significantly higher mean whole-body activity of 90Sr than males (2-way ANOVA analysis, F(1, 50) = 41.33, p < 0.001). After 6 weeks of contamination, this increase was mainly observed for females, while in males the mean 90Sr whole-body activity remained stable. At 20 weeks of contamination, a mean content of 2.3 ± 0.4 Bq g−1 for males and 3.7 ± 0.5 Bq g−1 for females was found in their body (Student t test, p < 0.001).

Rate of 90Sr accumulation in the bones

We calculated the rate of 90Sr accumulation in the bones (Bq day−1) of animals from both the juvenile and the adult model (Fig. 7). For both models, an increase in this rate was observed during the first 12 weeks of ingestion, with significantly higher rates for the juvenile than the adult model. At 12 weeks, the rate of 90Sr accumulation in the bones was for males 2.5 ± 0.3 Bq day−1 for the juvenile model and 0.8 ± 0.2 Bq day−1 for the adult model (Student t test, p < 0.001), while for females, it was 2.6 ± 0.5 Bq day−1 for the juvenile model and 0.5 ± 0.2 Bq day−1 for the adult model (p < 0.001). After 12 weeks of ingestion, the rate of 90Sr accumulation in the bones decreased for both models, even becoming negative at some time points in the juvenile model. Overall, these facts show that the accumulation of 90Sr in the bones is different during bone growth and bone remodelling.
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Fig. 7

Rate of 90Sr accumulation in the bones of 90Sr-ingesting males (left panel) and females (right panel) from the juvenile (open bars) and adult (closed bars) mouse models. Results are presented as mean ± SD of 6 animals per point. Time-specific differences between juvenile and adults animals are significant for *p < 0.05 and **p < 0.001 (Student t test)

Comparison of 90Sr accumulation at different skeletal sites in the juvenile model

As 90Sr preferentially accumulates in bones, it was investigated whether there was a difference in 90Sr content at the different skeletal sites. We isolated the whole skeleton from animals that were 6 and 20 weeks old from the juvenile model by papain treatment. Results showed that the 90Sr uptake in the skeleton depends on both gender (F(1,150) = 24.4, p < 0.001 at 6 weeks and F(1,120) = 41.9, p < 0.001 at 20 weeks) and skeleton site (F(14,150) = 27.3, p < 0.001 at 6 weeks and F(14,120) = 3.4, p < 0.001 at 20 weeks), regardless of the test age. The highest 90Sr accumulation was found in teeth, both in males and females (Fig. 8), which indeed are continuously growing in rodents, thus accumulating 90Sr over the ingestion period. For other bones, the range of 90Sr concentrations were between 110.7 ± 48.9 Bq g−1 and 154.9 ± 16.8 Bq g−1 for females and between 97.5 ± 14.2 Bq g−1and 135.5 ± 15.5 Bq g−1 for males aged 20 weeks (Fig. 8). These variations are in a lower range than the observed variations in humans (Kulp et al. 1960). Overall, higher 90Sr concentrations were consistently found in females compared with males in all skeleton sites, with the exception of ulna aged 20 weeks (Fig. 8). It should be noted that the mean 90Sr concentration is higher in this set of experiments compared with previous experiments in Fig. 3 for the same age. This is due to the difference in the method used to obtain bones. Whereas bones were isolated by simple dissection and manually cleaned in previous experiments, in this experiment, bones were isolated by enzymatic treatment. This completely removes all soft tissue, including bone marrow and cartilage. However, in both cases, females showed a higher 90Sr concentration in bones compared with males.
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Fig. 8

90Sr content (Bq g−1) at the different skeletal sites of 90Sr-ingesting male (closed bars) and female (open bars) animals of 20 weeks old of the juvenile model. All the results are presented as mean ± SD of five animals per group. Differences between males and females are significant for *p < 0.05 and **p < 0.001 (Student t test)

Metabolic cage experiment in the adult model

In order to monitor the elimination of 90Sr during chronic ingestion, animals of the adult model were housed in metabolic cages for 48 h before euthanasia, and their excreta were collected. The level of 90Sr elimination through faeces was dependent upon both the ingestion period (2-way ANOVA analysis, F(4,50) = 41.4, p < 0.001) and the sex of the animals (F(1,50) = 7.1, p < 0.05) (Fig. 9a), with a low level of 90Sr excretion at 3 weeks of ingestion. By contrast, 90Sr excretion through urine appeared similar in both genders and constant over the ingestion period, with mean 90Sr contents of 1.1 ± 0.3 Bq g−1 for males and 1.5 ± 0.3 Bq g−1 for females (Fig. 9b).
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Fig. 9

90Sr excretion during 24 h in a the faeces (Bq g−1) and b the urine (Bq g−1) of 90Sr-ingesting animals from the adult model, according to the age and sex of the animals. c Daily intestinal absorption ratio (IAR) calculated as described in the Materials and methods section. Results are presented as mean ± SD of six animals. Time-specific differences between males and females are significant for *p < 0.05 and **p < 0.001 (Student t test)

Starting from these results and the calculated daily 90Sr ingestion, we calculated the daily intestinal absorption ratio as indicated in the “Materials and methods” section. This ratio takes into account both direct 90Sr intestinal uptake and 90Sr actively reexcreted in the intestine, as previously described both in humans and in rodents (Wiseman 1964). Results indicated that for the adult model, the ratio of intestinal absorption (Fig. 9c) varied according to both the duration of ingestion (2-way ANOVA analysis, F(4,50) = 48.1, p < 0.001) and the sex of the animals (F(1,50) = 5.02, p < 0.05). The highest rate of intestinal absorption was observed at 3 weeks of ingestion and decreased thereafter. This may be explained by a modification in the balance between intestinal absorption and active reexcretion between 3-week-old and 6-week-old. Moreover, gender differences may be explained by increased intestinal absorption of strontium in males, as has been previously suggested in rats and monkeys (Dahl et al. 2001).

Discussion

The experiments in the present study were made by chronic ingestion of drinking water containing 20 kBq l−1 of 90Sr resulting in a daily intake ranging from 40 Bq up to 91 Bq per animal, depending on age and gender. The detection of 90Sr mainly in bones is in agreement with other experimental studies in various animal models (Gillett et al. 1992; Lloyd et al. 1976; Raabe et al. 1981), including rats (Gran 1960; Nilsson 1970), and the biokinetic model of strontium previously proposed (ICRP 1993; Leggett 1992; Lloyd et al. 1976). However, the juvenile and adult models showed very different patterns of 90Sr accumulation in bones. The rate of 90Sr uptake in bones was rapid during the first weeks of life for the offspring, i.e. during bone growth, reaching a plateau level afterwards at adult age. This high rate of 90Sr accumulation is correlated with bone growth, as has already been shown (Book et al. 1982). A plateau level of 90Sr accumulation at adult age was also observed in a study with beagles which ingested chronically 90Sr from in utero to 1.5 years of age (Parks et al. 1984). Moreover, it has been previously demonstrated that the level of strontium uptake is limited by calcium availability (Apostoaei 2002; Höllriegl et al. 2006). In fact, in the present study the animals received a diet with normal levels of calcium, thus limiting strontium absorption. Previous works showed that, due to their chemical similarities, strontium and calcium share similar transport system mechanisms across the intestinal wall and are absorbed competitively (Apostoaei 2002). Moreover, others showed that the ratio of Sr/Ca accumulation in bones was higher during young ages, possibly due to a higher efficiency of Sr absorption by the small intestine early in life (Sugihira et al. 1990). These mechanisms may explain both the rapid increase in 90Sr accumulation at young ages and the plateau level at the adult age observed in the juvenile model. These mechanisms also suggest that 90Sr accumulation in bones may be limited even in immature skeletons that have the high calcium turnover associated with early life (Book et al. 1982). On the other hand, in our adult model, a continuous increase in 90Sr content in the bones was found during the 20 weeks of chronic 90Sr ingestion and no plateau level was reached. This is possibly explained by the low level of daily ingestion, which may not allow equilibrium between ingestion, excretion and bone accumulation to be reached, but may also be due to the limitation to 20 weeks of ingestion period. This reduced rate of 90Sr uptake in the adult model also suggests that 90Sr uptake in bones is mainly linked to bone remodelling (Dahl et al. 2001; Momeni et al. 1976).

In all the other organs tested (blood, liver, spleen, kidneys, thymus, heart, lungs, CNS, muscles and skin), 90Sr was below the detection limit. In fact, since previous studies show that more than 90% of absorbed strontium is accumulated in bones (ICRP 1993), this indicates that, according to the daily rate of 90Sr intestinal absorption of between 10% and 40% that we have observed, less than 3.5 Bq may be found in all other organs than the skeleton. This estimate strongly suggests that if 90Sr is present in the liver, kidney and/or blood, the 90Sr concentration is below the detection limit of 90Sr in our experiments. Nevertheless, small amounts of 90Sr were found in the digestive tract. Measurement of 90Sr content in different segments of the digestive tract (stomach, small intestine, caecum and large intestine) of animals of both models showed that 90Sr was only detectable in the small intestine and not in the other segments. This suggests rather a weak trapping of 90Sr in the villi of the intestinal mucosa than a real accumulation in the intestinal tissue, as previously described for uranium ingestion (Dublineau et al. 2007). However, this local retention may have an influence on the inflammatory status of the intestine due to strontium irradiation, similar to the inflammatory reaction observed in a rat model of uranium ingestion (Dublineau et al. 2007).

Gender differences were also observed here in 90Sr uptake. For both models, higher 90Sr content was found in the bones of females compared with males. This difference between males and females was observed regardless of the skeletal site tested at the ages of 6 and 20 weeks. It is well known that bone remodelling depends upon hormonal cycles in females (Chiu et al. 1999; Hotchkiss and Brommage 2000; Kalyan and Prior 2010). This may explain the observed discrepancy in 90Sr accumulation in bones, despite the lower 90Sr intestinal absorption in females compared with males.

Overall, the present results indicate that there are age- and gender-dependent variations in the uptake of 90Sr throughout long-term, chronic ingestion. This must be taken into account in our future studies on the potential non-cancerous effects of long-term, chronic ingestion of 90Sr. Indeed, important populations are already chronically exposed to small amounts of 90Sr by ingestion due to the Chernobyl accident (Cooper et al. 1992; De Ruig and Van der Struijs 1992; Hoshi et al. 1994). The potential health effects of 90Sr chronic ingestion may be observed on the hematopoietic system, due to the location of the hematopoietic stem cells close to the bone (Calvi et al. 2003; Taichman 2005). Since the bone marrow is the main anatomical site of B lymphocyte differentiation, potential effects may equally be observed on the immune system. However, absorbed radiation doses to the whole body due to chronic 90Sr intake and accumulation in bones remain low in our model. In fact, calculated absorbed whole-body doses, based upon dose conversion factor for the rat model (ICRP 2008), were 9.7 ± 0.1 mGy for males and 10.6 ± 0.1 mGy for females in the juvenile model and 3.8 ± 0.1 mGy for males and 4.7 ± 0.1 mGy for females in the adult model after 20 weeks of ingestion (E. Blanchardon, personal communication). However, much higher localised absorbed radiation doses—up to fivefold more according to calculations made with specific absorbed fractions of energy (Stabin et al. 2006)—may occur in hematopoietic stem cells due to the proximity of bone (Calvi et al. 2003; Taichman 2005). In this respect, the use of a juvenile mouse model of chronic ingestion to study the potential effects of 90Sr may be of special interest, as the hematopoietic system mainly develops during late foetal and post-natal life. Besides, juveniles show an enhanced sensitivity towards many toxic compounds (Blakley and Blakley 2005) including radionuclides (Hoyes et al. 2000; Lindop and Rotblat 1962), and their lifespan makes them the primary targets for potential long-term effects.

Acknowledgments

The authors wish to thank F. Voyer and T. Loiseau for their expert work in animal care. The secretarial assistance of D. Lurmin and V. Joffres is also warmly acknowledged. This project was part of the ENVIRHOM research programme of the Institut de Radioprotection et Sûreté Nucléaire (IRSN) and was supported by grants from the Ile-de-France Region.

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© Springer-Verlag 2011