Biokinetics of 90Sr after chronic ingestion in a juvenile and adult mouse model
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- Synhaeve, N., Stefani, J., Tourlonias, E. et al. Radiat Environ Biophys (2011) 50: 501. doi:10.1007/s00411-011-0374-9
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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.
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
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).
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 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.
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.
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
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.
Distribution of 90Sr in the juvenile model
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.
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.
Distribution of 90Sr in the adult model
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
Comparison of 90Sr accumulation at different skeletal sites in the juvenile model
Metabolic cage experiment in the adult model
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).
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.
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.