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Keywords:

  • strontium 90;
  • chronic exposure;
  • ingestion;
  • bone;
  • hematopoiesis

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The aim of this work was to delineate the effects of chronic ingestion of strontium 90 (90Sr) at low concentrations on the hematopoiesis and the bone physiology. A mouse model was used for that purpose. Parent animals ingested water containing 20 kBq l−1 of 90Sr two weeks before mating. Offspring were then continuously contaminated with 90Sr through placental transfer during fetal life, through lactation after birth and through drinking water after weaning. At various ages between birth and 20 weeks, animals were tested for hematopoietic parameters such as blood cell counts, colony forming cells in spleen and bone marrow and cytokine concentrations in the plasma. However, we did not find any modification in 90Sr ingesting animals as compared with control animals. By contrast, the analysis of bone physiology showed a modification of gene expression towards bone resorption. This was confirmed by an increase in C-telopeptide of collagen in the plasma of 90Sr ingesting animals as compared with control animals. This modification in bone metabolism was not linked to a modification of the phosphocalcic homeostasis, as measured by calcium, phosphorus, vitamin D and parathyroid hormone in the blood. Overall these results suggest that the chronic ingestion of 90Sr at low concentration in the long term may induce modifications in bone metabolism but not in hematopoiesis. Copyright © 2012 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Large amounts of radionuclides were accidentally released into the environment during the nuclear accidents at Chernobyl and Fukushima accidents and during nuclear waste release in the Techa River and in atmospheric nuclear testing. Depending on the nature of the release, two radionuclides are mainly found in the environment in the long term, namely cesium 137 (137Cs) and strontium 90 (90Sr). Twenty-five years after the Chernobyl accident, 137Cs is the main radionuclide remaining (UNSCEAR, 2011), while along the riversides of the Techa River, 90Sr is the main radionuclide detected (Shutov et al., 2002). These releases resulted in a general contamination of food chains, and large populations living in contaminated territories are chronically exposed to small amounts of these radionuclides by ingestion. Previous studies estimated a daily ingestion of 137Cs between 100 and 1200 Bq per day and for 90Sr 1–100 Bq per day of 90Sr for populations around the Chernobyl power plant (Cooper et al., 1992; De Ruig and Van der Struijs, 1992) with a progressive reduction with time (Bernhardsson et al., 2011). This resulted in significant whole body content of 137Cs, up to 760 kBq as measured by whole body counting with large seasonal and individual variations (Handl et al., 2003). This was confirmed by measurement in organs collected at autopsy showing a 137Cs activity up to 2000 Bq g−1 of tissue (Bandazhevsky, 2003; Dam et al., 1988). Similarly, daily ingestion of 5.7 kBq per day was estimated for residents of some villages along the Techa River, resulting in mean body content of up to 50 kBq of 90Sr (Kozheurov, 1994; Kozheurov and Degteva, 1994).

The health effects of such chronic ingestion of low quantities of radionuclides in the long term remain difficult to define. Several studies showed that, during the 10–15 years after the Chernobyl accident, both clean-up workers and children had quantitative changes in cellular and humoral immunity. These changes were mainly expressed as a decrease in T and B lymphocyte numbers (Chernyshov et al., 1997; Yarilin et al., 1993), in a modified ratio of CD4+/CD8+ lymphocytes (Chumak et al., 2001), and impaired production of IgG or IgM (Titov et al., 1995). In addition, Techa riverside residents showed hematological changes such as granulocytopenia and modifications in immune populations with decreased antigen expression (Akleyev et al., 2010a, 2010b). However, in all of these studies, it is difficult to ascribe observed effects to a specific pathway of exposure. In fact the studied populations are exposed to both external irradiation owing to the presence of radionuclides in the environment and internal exposure owing to ingestion of these radionuclides (Bernhardsson et al., 2011). Moreover, in most cases, the populations are exposed to a mixture of radionuclides in various proportions (Handl et al., 2003; Shutov et al., 2002).

We thus developed in our laboratory a rodent model of chronic contamination through ingestion of drinking water containing radionuclides. Animal models showed that chronic exposure to a low concentration of 137Cs in drinking water induced modifications in several physiological systems, such as the central nervous system (Lestaevel et al., 2006, 2008) and several metabolisms (Grignard et al., 2008; Tissandie et al., 2009). However no pathological consequences were observed in association with these modifications (Lestaevel et al., 2010). Moreover, no modifications of both immune and hematopoietic systems were found in a mouse model of 137Cs chronic ingestion (Bertho et al., 2010, 2011).

We thus hypothesize that part of the observed modifications in some physiological systems and especially in the hematopoietic and immune systems in human populations chronically exposed could be due to the ingestion of 90Sr. In fact, strontium is accumulated mainly in bones (Leggett et al., 1982), especially during bone growth (Synhaeve et al., 2011), thus close to bone-forming cells and also close to mesenchymal cells regulating early hematopoiesis (Taichman, 2005). As a result, cells implicated in the regulation of both bone physiology and hematopoietic differentiation may receive significant radiation doses emitted by neighboring tissues having accumulated 90Sr (Bertho et al., 2012). We thus used our mouse model of chronic contamination through drinking water with a 90Sr concentration of 20 kBq l−1. Previous studies showed that this concentration led to a daily ingestion of 80–100 Bq per day and per animal (Bertho et al., 2010; Synhaeve et al., 2011), and give rise to whole body radiation dose of 10 mGy by the end of the 20 week study period (Bertho et al., 2012). In this model, we study various parameters in order to delineate possible effects of 90Sr chronic ingestion on two physiological systems, the hematopoiesis and bone physiology.

Material and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

Balb/c mice were purchased from Elevage Janvier (Le Genest Saint Isle, France). Animals were housed in standard cages and were kept at a constant room temperature (21 ± 2 °C) with a 12 h daylight cycle. Animals received ad libitum water and standard rodent chow containing a mean calcium concentration of 9 g Ca2+ kg−1 (normal calcium diet, R03-type chow, Safe, Epinay-sur-Orge, France). The animal care committee of the Institut de Radioprotection et de Sûreté Nucléaire reviewed and approved all the animal experiments, which were conducted in accordance with French regulations for animal experimentation (Ministry of agriculture Act no. 2001-464, May 2001).

Strontium 90 Ingestion Schedule

Two groups of mice were constituted throughout all the experiments. One control group received normal drinking water and one group received drinking water containing 20 kBq l−1 90Sr. The strontium main solution (CERCA-LEA, Pierrelatte, France) was in the form of SrCl2 at a concentration of 10 µg ml−1 with an initial mass activity of 0.9 × 106 Bq g−1. As a result, the ratio of 90Sr to stable Sr was approximately 1:61 in drinking water. Based upon an assumed daily water consumption of 4 ml per animal per day at adult age (Synhaeve et al., 2011), a 90Sr ingestion of about 80 Bq per animal per day at adult age was expected, corresponding to 0.94 ng of total Sr (stable + radioactive) per animal and per day. 90Sr ingestion was monitored though measurement of water consumption and internal contamination of animals was verified by measurement of 90Sr in femurs of sacrificed animals as previously described (Synhaeve et al., 2011). Male and female parents received normal or 90Sr-containing water starting two weeks before mating and until their date of sacrifice. Control and contaminated breeding groups were constituted with one male and two females for one week. One week after start of mating male parents were anesthetized by intraperiteoneal injection of a mixture of ketamine (Imalgene, Mérial, Villeurbanne, France) and xylazine (Rompun, Bayer Healthcare, Monheim, Germany) and killed by cervical dislocation. Births within breeding groups were carefully recorded. Three weeks after birth, that is, at the time of weaning, female parents were anesthetized and killed by cervical dislocation. Sexing of the offspring was made and the mice were separated into groups of 6–10 control and 6–10 90Sr-ingesting animals, with a sex ratio of 1:1. In order to avoid possible litter effects, groups of sacrifice were constituted with males and females originating from different litters. The animals continued to receive normal or 90Sr contaminated drinking water until their sacrifice at the age of 3, 6, 12, 16 or 20 weeks for organ sampling.

Organ Sampling and Treatment

At the indicated ages, animals were anesthetized and blood was drawn by intracardiac puncture using a heparinized syringe (Choay, Sanofi Aventis, Paris, France) and the animals were killed by cervical dislocation. Femurs, tibia and spleen were then harvested, weighed and used for further analysis. A complete blood cell count was made using a hematologic automated counter (MS9 vet, Melet-Schlossing, Osny, France). Blood was then centrifuged for 5 min at 400 g and plasma was harvested and frozen for later use. Femurs were either frozen in liquid nitrogen for gene expression analysis, or decalcified (RDC, CML, France) and fixed in 4% formaldehyde for histological analysis or flushed to collect hematopoietic cells. A tibia or a femur from each animal was used in order to measure 90Sr concentration in bones, as previously described (Synhaeve et al., 2011).

Spleens were crushed with a Tenbrock′s potter in minimum essential medium alpha (MEM-α, Life technologies, Cergy Pontoise, France) supplemented with 1% glutamine, 1% penicillin–streptomycin and 10% fetal calf serum (FCS; all from Life Technologies). Cell suspension was harvested and washed; cells were numerated and viability was assessed by trypan blue exclusion.

Colony-forming Cell Assay

Spleen cells were plated at 2 × 105 and bone marrow cells were plated at 2.5 × 104 in 1.1 ml of complete methylcellulose medium with cytokines (Stem Cell Technologies, Vancouver, Canada) in 30 mm diameter Petri dishes (Greiner Bio-One, Courtaboeuf, France). Cultures were incubated at 37 °C in a humidified atmosphere with 95% air and 5% CO2. Colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E) and CFU-granulocyte erythrocyte monocyte megakaryocyte (CFU-GEMM) were scored on day 12 of culture.

Phenotypic Analysis of Bone Marrow Cells

Directly coupled specific antibodies used in this study were IgG1-FITC, IgG2b-PE, IgG2a-PE-CY5 and IgG2a-PE-CY7 as isotypic controls, anti-mouse CD4-PE, anti-mouse CD8-PE-CY5, anti-mouse CD49b-PE, anti-mouse CD117-PE-CY5 (all from BD Pharmingen, Le pont de Claix, France), anti-mouse CD3-FITC, anti-mouse CD11b/Mac-1-PE, anti-mouse CD19-PE, anti-mouse CD45-FITC, anti-mouse Ly-6A/E-PE (all from Beckman coulter, Villepinte, France), and anti-mouse lineage mixture (lin) coupled to an Alexa fluor 488 (Caltag, Life Technologies). Suspension of bone marrow cells were adjusted to 1 × 106 cells ml−1 in phosphate buffered saline solution (PBS, Life Technologies), supplemented with 0.5% bovine serum albumin (BSA, Sigma, St Quentin Falavier, France). Cell suspension was then mixed with a pre-defined concentration of a mix of directly coupled specific antibodies. Cells were then incubated for 20 min at 4 °C. After washing in PBS 0.5% BSA, cells were then analyzed onto an FACSort flow cytometer (BD Biosciences) with at least 10 000 events per point and data were analyzed with Cellquest software (BD Biosciences).

General Biochemical Parameters

An automated spectrometric system (Konelab, Thermo Electron Corp., Cergy Pontoise, France) with the manufacturer's biological chemistry reagents was used to measure levels of calcium, phosphorus and alkaline phosphatase in plasma.

Measurement of Plasma Proteins

Plasma concentrations of Fms-like tyrosine kinase-3 ligand [Flt3-l; R&D Systems, Abingdon, UK; detection limit (DL), 15 pg ml−1], parathyroid hormone (PTH; Immunotopics, San Clemente, CA, USA; DL 3 pg.ml−1), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; Immunodiagnostics Systems, Paris, France; DL 2.5 pg ml−1], bone morphogenetic protein-2 (BMP-2;USCN Life Science Inc., Wuhan, China; DL 6.7 pg.ml−1), osteocalcin (OCN; USCN Life Science Inc.; DL 4.2 pg ml−1), bone-specific alkaline phosphatase (bALP; USCN Life Science Inc.; DL 0.143 U l−1), procollagen type 1 N-terminal telopeptide (PINP; Immunodiagnostics Systems; DL 0.7 ng.ml−1), osteoclast-specific tartrate resistant acid phosphatase 5b (TRAP5b; USCN Life Science Inc.; DL 0.29 U ml−1) and collagen type 1 C-telopeptide (CTX; Immunodiagnostics Systems; DL 2 ng ml−1) were measured by commercially available ELISA according to manufacturer's recommendations.

Gene Expression Analysis

Frozen femurs were pulverized using a T25 Ultra-Turrax (IKA, Staufen, Germany) and total RNA was isolated from bone powder using Trizol reagent (Sigma). Obtained RNA was purified by RNeasy Total RNA Isolation Kit (Qiagen, Courtabœuf, France) according to the manufacturer's recommendations. RNA concentration and integrity were checked by OD measurement at 230 nm and the ratio of the OD at 260/280 nm (Thermo Scientific NanoDrop 1000, Labtech, Palaiseau, France). One microgram of total RNA was reverse transcribed with random hexamers using a Staufen, Germany) and total RNA was isolated from bone powder using trizol reagent (Sigma). Obtained RNA was purified by RNeasy Total RNA isolation kit (Quiagen, Courtaboeuf, France) according to manufacturer's recommendations. RNA concentration and integrity were cheked by optical density (OD) measurement at 230 nm and ration of the OD at 260 nm/280 nm (Thermo Scientific NanoDrop 1000, Labtech, Palaiseau, France). One microgram of total RNA was reverse transcribed with random hexamers by use of the high-capacity cDNA Reverse Transcription Kit according to the manufacturer's recommendations (Applied Biosystems, Courtaboeuf, France). Expression of genes was measured by polymerase chain reaction (PCR) in real time. An aliquot of 5 or 10 ng of cDNA was amplified in duplicate for each reaction using SYBR Green PCR Master Mix or Taqman fast universal PCR Master Mix (both from Applied Biosystems). Forward and reverse primers used in this study are listed in Table 1 (either from Life Technologies or Applied Biosytems). Amplification and detection of PCR products were performed with the Abi Prism 7900 Sequence Detection System (Applied Biosystems). The resulting fractional cycle number of the threshold (Ct) was used for transcript quantification. The expression level of each sample was normalized to the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, used as an internal reference gene. Relative expression to the control group was calculated for each gene by the 2−∆∆Ct method previously described (Livak and Schmittgen, 2001).

Table 1. Primers and probes used in polymerase chain reaction experiments. All products were obtained from either Life Technologies or Applied Biosystems
GeneForward primerReverse primerAccession number
  1. RunX2, Run-related transcription factor 2; ALP, alkaline phosphatase; BSP, bone sialoprotein; OPN, osteopontin; OCN, osteocalcin; Coll, collagen; RankL, receptor activator of NFκ-b ligand; OPG, osteoprotegerin; TRAP5b, osteoclast-specific tartrate resistant acid phosphatase 5b; PTHr, parathyroid hormone receptor.

Runx25′-AAATGCCTCCGCTGTTATGAA-3′5′-GCTCCGGCCCACAAATCT-3′NM_001145920.1
ALP5′-CCGATGGCACACCTGCTT-3′5′-GAGGCATACGCCATCACATG-3′NM_007431.2
BSP5′-ACCCCAAGCACAGACTTTTGA-3′5′-CTTTCTGATCTCCAGCCTTCT-3′NM_008318.3
OPN5′-CCCGGTGAAAGTGACTGATTC-3′5′-ATGGCTTTCATTGGAATTGC-3′NM_009263.2
OCN5′-CCGGGAGCAGTGTGAGCTTA-3′5′-AGGCGGTCTTCAAGCCATATC-3′NM_007541.2
Coll 15′-CTTCACCTACAGCACCCTTGTG-3′5′-CTTGGTGGTTTTGTATTCGATCACT-3′NM_007742.3
Coll 35′-TTCCTGAAGATGTCGTTGATGTG-3′5′-TTTTTGCAGTGGTATGTAATGTTCTG-3′NM_009930.2
RankL5′-TCAGCTGATGGTGTATGTCGTTAA-3′5′-TTCGTGCTCCCTCCTTTCAT-3′NM_011613.3
OPG5′-GGGCGTTACCTGGAGATCG-3′5′-GAGAGAACCCATCTGGACATTT-3′NM_008764.3
TRAP 5b5′-GATGACTTTGCCAGTCAGCAGC-3′5′-GCACATAGCCACACCGTTCTC-3′NM_007388.3
PTHr5′-TGGGTCGGTGTCAGAGCAA-3′5′-CCTGGATGATCCACTTCTTGTG-3′NM_001083936.1
GAPDHProduct number 4352932-0804021, Applied biosystems

Histological Staining and Morphometric Analysis

Decalcified femurs were embedded in paraffin and were cut into serial longitudinal slices of 7 µm thickness. After being deparaffinized and dehydrated, six sections per femur were used for modified Trichrome Goldner staining. The distal diaphysis of femurs was examined under a Leica DM4000B light microscope and images were captured using a digital Sony XCD-U100CR camera. Images were analyzed with Histolab 7.6 software (Microvision instruments, Evry, France). Mean thickness of the growth plate was determined from three measurements at set distances along the growth plate. Furthermore a tissue surface (TS) restricted in a 2 mm region from the growth plate (which was included) was determined for analysis of both trabecular and cortical bone. Automated quantification of bone surface (BS) in the tissue surfaceconfined was performed by user-predefined parameters and the BS/TS ratio (%) was calculated. Results of growth plate thickness and BS/TS ratio were obtained for every femur from six sections and averaged for each femur and finally for each group of animals.

Statistical Analyses

All results are presented as mean ± standard deviation (SD) from 6–12 animals per group, unless otherwise indicated. The analysis of gene expression in bones was performed in two steps. First, an unsupervised method of statistical analysis was used. This analysis was based on the expression levels of genes normalized to GAPDH. Hierarchical clustering was performed using ClassDiscovery package version 2.13.0 (Coombes, 2009) with R statistical software version 2.15.0 (R Development Core Team, 2012). Computation of the matrix of Euclidian distances between samples was performed using the distanceMatrix function. Hierarchical cluster analysis was performed with the hclust function using the complete agglomeration method. In a second step, comparisons between groups were made with either Student's t-test, the rank sum test or the two-way ANOVA (analysis of variance) test, with separate analysis of males and females, as indicated in the text. Differences were considered statistically significant for P < 0.05. All statistical analyses were performed using Sigmaplot software (Systat software Inc., San Jose, CA, USA).

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

90Sr Concentration in Bones

A weekly recording of body mass, food and water consumption was made starting at weaning, which allowed an estimation of daily 90Sr ingestion. Results showed that males ingested significantly higher quantities of 90Sr as compared with females (mean 91.4 ± 12.3 and 66.4 ± 9.5 Bq per day per animal, respectively; n = 24, P < 0.001). This difference was due to lower water consumption by females as compared with males, both in the control group and in the 90Sr-ingesting group (data not shown). This ingestion of 90Sr resulted in a similar 90Sr concentration in bone for males and females, up to 76.0 ± 4.2 Bq g−1 in males and 87.1 ± 10.2 Bq g−1 in females at 20 weeks of age (Fig. 1). This was consistent with a previous study showing that the ratio of 90Sr accumulation in bone is overall higher in females as compared with males (Synhaeve et al., 2011).

image

Figure 1. 90Sr concentration in bones of animals from the 90Sr-ingesting groups according to the age. Results are presented as mean ± SD with n = 6 from one representative experiment. A significant evolution of 90Sr concentration was observed according to the age of animals (Two-way ANOVA test, F(5, 48) = 90.0, p < 0.01). However, no significant differences were evidenced between males and females along the experiments.

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Animals showed regular weight gain throughout the experiment, without any significant difference between 90Sr-ingesting animals and control animals (data not shown). At 20 weeks of age, body weights were 28.2 ± 2.4 g for control males and 29.6 ± 1.6 g for 90Sr-ingesting males (n = 20, n.s.) and 24.9 ± 2.3 for control females and 23.8 ± 1.6 g for 90Sr-ingesting females (n = 20, n.s.).

Blood Cell Counts

As a first evaluation of hematopoietic function, we analyzed blood cell counts. However, none of the main cell lineages analyzed, that is, white blood cells, red blood cells and platelets showed modifications in animals ingesting 90Sr as compared with control animals (Fig. 2). In addition, neither hematocrit (Fig. 2D) nor haemoglobin concentration (data not shown) were modified in 90Sr-ingesting animals. We also analyzed number of lymphocytes, granulocytes and monocytes. However, none of these blood cell populations were quantitatively modified by chronic ingestion of 90Sr as compared with control animals (data not shown).

image

Figure 2. Evolution of A: white blood cell (WBC) number, B: Red blood cell (RBC) number, C: hematocrit and D: platelet (Plt) number according to the age of animals. Data are the mean ± SD of two experiments, with n > 10. No significant difference was evidenced between control (closed symbols) and 90Sr-ingesting animals (open symbols) either for males (circles) or for females (squares), using the Two-way ANOVA test.

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Functional Analysis of Hematopoietic Activity

The analysis of blood cell count suggested that 90Sr ingestion does not have a significant effect on hematopoiesis. In order to confirm this, we measured plasma Flt3-ligand concentration, which can be used as a bio-indicator of functional activity of hematopoiesis (Huchet et al., 2003; Prat et al., 2006). Results did not show any modification of Flt3-ligand concentration when comparing 90Sr-ingesting animals and control animals (two way-ANOVA test; Fig. 3A), although age-dependent variations in Flt3-ligand concentration were observed (two-way ANOVA test, F(5, 85) = 31.5, P < 0.001 for males and F(5, 87) = 46.3, P < 0.001 for females). In addition, the frequency of haematopoietic progenitors (CFU-GM, CFU-GEMM and BFU-E), both in the spleen (data not shown) and in the bone marrow (Fig. 3B and C), were not significantly modified by 90Sr-ingestion, as compared with control animals (two-way ANOVA test). Finally, the phenotypic analysis of bone marrow cells and especially the percentage of linSCA1+ckit+ stem cell population (Fig. 3D) was not affected by 90Sr ingestion as compared with control animals (two-way ANOVA test), although significant age-dependent variations were observed (two-way ANOVA test, F(5, 47) = 14.3, P < 0.001 for males and F(5, 47) = 60.4, P < 0.001 for females). Overall, these results indicate that there was no significant modification of hematopoiesis in animals ingesting 90Sr as compared with control animals.

image

Figure 3. Evolution of A: plasma Flt3-ligand concentration used as a bio-indicator of bone marrow function; B: frequency of colony-forming units-granulocyte macrophages (CFU-GM) and C: Burst-forming units-erythroid (BFU-E) among bone marrow mononuclear cells (BM MNC); D: percentage of Lin-ckit+SCA1+ hematopoietic stem cells in bone marrow. Results are the mean ± SD of two experiments with n = 10 for each point in A and n = 5 for B, C and D. No significant difference was observed between control and 90Sr-ingesting animals (Two-way ANOVA test).

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Bone Physiology: Gene Expression in Femur

We first analyzed in the femur of animals the expression of genes involved in either bone formation (namely Runx2, ALP, BSP, OPN, OCN, collagen 1 and 3) or bone resorption (RankL, OPG, TRAP5b and PTHr). Given the large array of data (11 genes per animal and 10–12 animals per group) and parameters (gender, age and 90Sr ingestion), we first analyzed results of PCR experiments using an unsupervised hierarchical clustering. The results (Fig. 4A) showed that samples segregated primarily according to the age of animals, meaning that this latter parameter has the strongest effect on the expression levels of the selected genes. Indeed, no other parameters (sex, 90Sr ingestion, bone resorption or bone formation) showed a clear-cut influence on the results of this global gene expression analysis.

image

Figure 4. Relative gene Expression analysis in femurs from 90Sr ingesting animals. A: Hierarchical clustering based on complete linkage of Euclidean distances of all available samples using the expression levels of studied genes normalized to GAPDH. With the exception of two animals, clear-cut sample segregation was observed according to the age of animals. Grey and black labels correspond to 6 weeks and 20 weeks mice respectively. B: Detailed analysis of gene expression using Two-way ANOVA test for each group. Results are presented as mean ± SEM from 10 to 12 animals per group, either control animals (closed circles) or 90Sr ingesting animals (open circles). Significant differences between control and contaminated animals were observed in genes implicated in bone formation for males at 6 weeks (F(1, 154) = 9.329, p < 0.005) and for females at 20 weeks (F(1, 141) = 18.699, P < 0.001) and genes implicated in bone resorption for males at 6 weeks (F(1, 88) = 4.504, p < 0.05). Gene specific significant differences are indicated for *: p < 0.05 (Rank sum test). See legend of Table 1 for abbreviations used.

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The results of gene expression in femur were then analyzed using a two-way ANOVA test by separating data according to the age and gender of animals and by separating genes encoding proteins involved in bone formation and genes encoding for proteins involved in bone resorption. This allowed us to test for potential differences in gene expression according to the ingestion status of animals, avoiding potential confounding effects of both age and gender. Results (Fig. 4B) indicated that there was a significant decrease in the expression of genes implicated in bone formation and in bone resorption in males at 6 weeks in animals ingesting 90Sr as compared with control animals (F(1, 154) = 9.329, P < 0.005 and F(1, 88) = 4.504, P < 0.05 respectively). These results suggested that the accumulation of 90Sr in bones during the period of bone growth induces a reduction in bone turnover in males. However, such an effect was not observed in females at 6 weeks.

At the age of 20 weeks (Fig. 4B), a decrease in the expression of genes implicated in bone formation was observed in females (F(1, 141) = 18.699, P < 0.001), but not in males (F(1, 139) = 3.249, n.s.), and without any change in the expression of genes implicated in bone resorption, either in males or in females. This indicates that at the adult age the accumulation of 90Sr in bones induces disequilibrium in bone remodeling towards an increase in bone resorption in females and a decrease in bone formation in male.

Overall these results suggest that 90Sr accumulation in bones following ingestion induces a disequilibrium of bone remodeling towards bone resorption, with a dependency of this effect according to age and gender of animals.

Bone Physiology: Protein Concentration in Plasma

In order to confirm this change in bone metabolism, we measured in the blood of animals several molecules involved in either bone formation (bone-specific ALP, PINP, BMP2 and OCN) or bone resorption (CTX and TRAP5b; Fig. 5). A significant increase in CTX concentration in the blood of males ingesting 90Sr was observed at 6 and 20 weeks of age. Since CTX is produced during bone resorption by osteoclast (Brzoska and Moniuszko-Jakoniuk, 2005a, 2005b), this result suggests that 90Sr ingestion may promote bone resorption, in line with results obtained by gene expression analysis in femur.

image

Figure 5. Evolution of plasma concentration of proteins linked to bone formation (two upper panels) or to bone resorption (lower panel) in males (left panel) and females (right panel) from either control group (closed bars) or 90Sr-ingesting group (open bars). Results are presented as mean ± SD of 15 to 17 animals from two independent experiments. Significant difference between control and 90Sr ingesting males at the age of 6 weeks and 20 weeks was observed for CTX (Two-way ANOVA test, F(1, 56) = 9.88, p < 0.005). Age specific differences between control and 90Sr ingesting males are significant for *: p < 0.05.

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Phosphocalcic Homeostasis

Such a modification in bone metabolism may be due to a modification in the regulation of phosphocalcic homeostasis. We thus evaluated possible changes in the blood of 90Sr-ingesting animals by measuring calcium, phosphorus and alkaline phosphatase. Results indicated that there was a significant evolution of both calcium phosphorus and alkaline phosphatase according to the age of animals, both in males and in females (Fig. 6A–C). The chronic ingestion of 90Sr also induced a significant change in alkaline phosphatase concentration in males (two-way ANOVA test, F(1,90) = 15.4, P < 0.001), but not in females (F(1,90) = 1.78, n.s.). Nevertheless, when making a time-specific analysis, a significant increase in phosphatase alcaline in 90Sr-ingesting animals as compared with control animals was observed only at birth (Student's t-test, n = 12, P < 0.001). By contrast, the chronic ingestion of 90Sr did not induce significant changes in both calcium and phosphorus concentration.

image

Figure 6. Evolution of phosphocalcic homeostasis according to the age of animals. A: Calcium concentration; B: Phosphorus concentration; C: Alkaline phosphatase (ALP) concentration; D: Vitamin D concentration; E: Parathyroid hormone (PTH) concentration in plasma. Results are the mean ± SD of two experiments with n = 12 for each point except for vitamin D (n = 8). No significant difference was observed between control and 90Sr-ingesting animals, whatever the sex of animals for all parameter tested (Two-way ANOVA test) except for ALP. A significant different evolution of ALP concentration was observed between control and 90Sr ingesting males (Two way ANOVA test, F(5, 90) = 15.4, p < 0.001), but not between females. Time-specific significant differences between control and 90Sr-ingesting males are indicated for **: p < 0.001. By contrast, a significant evolution of both parameters according to the age of animals was observed, both in males (F(5, 90) = 4.51, p < 0.005, F(5, 90) = 12.8, p < 0.001 and F(5, 90) = 751.7, p < 0.001 for calcium, phosphorus and ALP, respectively) and in females (F(5, 92) = 8.53, p < 0.001, F(5, 92) = 20.5, p < 0.001 and F(5, 92) = 166.7, p < 0.001 for calcium, phosphorus and ALP, respectively).

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When looking at vitamin D [1,25(OH)2D3]and PTH, two key regulators of phosphocalcic homeostasis, we did not observe significant modification in the plasma concentration of these two molecules according to the ingestion status of animals and whatever their age and gender. Overall, these results suggest that the modifications observed in both the gene expression in femur and protein concentration in blood are probably not due to a modification in the hormonal regulation of bone physiology.

Bone Histomorphometric Analysis

The observed modification in bone physiology towards bone resorption may induce in the long term a change in bone morphology. A comparative histomorphometric analysis of bones at 20 weeks of age was performed, using six sections per femur stained using a modified Trichrome Goldner procedure. Six animals per gender and per group of ingestion were analyzed. No significant difference between groups was observed in the total surface of section analyzed (Figure 7). The growth plate thickness and the ratio of bone surface to total surface (BS/TS) were then analyzed and, again, no significant difference between groups was observed (Figure 7). This suggests that the modifications observed in bone physiology towards bone resorption did not induce a major change in bone morphology at the time point studied.

image

Figure 7. A: Representative bone section stained with Hematoxilin-Eosin-Safran (HES) and definition of area of analysis. The horizontal line represents the 2 mm length starting at the center of the growth plate for bone surface analysis. The three short lines are the three growth plate thickness measurements. B, C and D: Results of histomorphometric analysis of male and female femurs from either control (closed bars) or 90Sr ingesting (open bars) groups of 20 weeks of age, presented as mean ± SD of 6 animals per group. B: Mean surface analysed; C: Mean growth plate thickness, and D: bone surface to total surface ratio (BS/TS ratio). No significant difference was observed between groups, whatever the gender and the ingestion status of animals (Student t test).

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Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The experimental schedule used here resulted in a continuous exposure to 90Sr, through placental transfer during fetal life, through lactation after birth and through drinking water after weaning. The follow-up of water consumption showed a mean daily ingestion of 91.4 ± 12.3 Bq per day (1.074 ± 0.145 ng.day−1 of strontium) for males and 66.4 ± 9.5 Bq per day (0.780 ± 0.112 ng per day of strontium) for females. Such an ingestion rate is consistent with estimates of 90Sr ingestion by humans after the Chernobyl accident (Cooper et al., 1992; De Ruig and Van der Struijs, 1992; Handl et al., 2003) or for residents of Techa riverside settlements (Kozheurov and Degteva, 1994). This ingestion rate resulted in 90Sr accumulation in mouse bones up to 76.0 ± 4.2 Bq g−1 in males and 87.1 ± 10.2 Bq g−1 in females at 20 weeks of age. 90Sr accumulated mainly before the age of 6 weeks, that is, during bone growth. These results are consistent with a previous study on the biokinetics of 90Sr with the use of the same mouse model (Synhaeve et al., 2011) and give rise to a whole body absorbed dose between 0.3 mGy at birth and 10 mGy at 20 weeks depending on the age of animals, as previously estimated (Bertho et al., 2012).

We then investigated the effect of 90Sr chronic ingestion on the hematopoietic system by analysis of complete blood cell counts, colony-forming cells, both in the bone marrow and in the spleen, phenotypic analysis of bone marrow cells and Flt3-ligand concentration in plasma. This later parameter was previously described as a bio-indicator of haematopoietic functionality, both in mice (Prat et al., 2006) and in humans (Huchet et al., 2003). However, although age-specific differences were observed for some of these parameters, no significant differences were detected when comparing 90Sr ingesting animals and control animals. These results showed that the chronic ingestion of 90Sr did not induce significant change in the hematopoietic system, despite the proximity between hematopoietic cells and the site of 90Sr accumulation. This result is in accordance with the absorbed radiation dose owing to 90Sr ingestion. In fact, previous work showed that, with this model of chronic ingestion, the absorbed radiation dose to the whole body is about 10 mGy by the end of the study period (Bertho et al., 2012). This absorbed radiation dose is considered to be lower than the threshold dose for the appearance of deterministic effects on the haematopoietic system (Goans, 2002). Moreover, hematopoiesis is strictly controlled to produce the necessary number of mature and functional blood cells, and as such is able to respond to stressful situations. Nevertheless, some late effects of 90Sr ingestion on the hematopoietic system were described in the Techa River population (Akleyev et al., 2010c), with decreased numbers of platelets and neutrophils. Thus we cannot exclude that such effects may appear at later time points in our model. In fact, one has to consider that the β rays emitted by 90Sr may induce higher local radiation doses (as compared with the whole-body radiation dose), especially to bone lining cells such as osteoblasts, osteoclasts and cells constituting the hematopoietic stem cell niche (Moore, 2004; Taichman, 2005). Thus in order to confirm our present results, it should be of interest to look at other parameters, such as the number of hematopoietic stem cell niches or the cell cycle status of hematopoietic stem cells in 90Sr-ingesting animals and to study later time points, 12 and 18 months, for instance, a time at which bone loss may become important (Ferguson et al., 2003).

By contrast, we observed a marked modification in bone metabolism at the gene expression level mainly at the age of 20 weeks. The observed decreased expression of genes encoding proteins involved in bone formation (ALP, BSP, OPN and OCN) and increased expression of gene encoding RankL, a molecule implicated in bone matrix resorption (Boyce and Xing, 2007), suggest that 90Sr ingestion induced a trend to increased bone matrix resorption. This is line with the increased level of CTX concentration in the plasma of males, both at 6 weeks and at 20 weeks. It was previously shown in a rat model of cadmium exposure that an increase in CTX concentration is associated with an enhanced degradation of bone matrix (Brzoska and Moniuszko-Jakoniuk, 2005b). Surprisingly, this effect was observed in males and not in females. The later have in fact a skeleton more susceptible to damage resulting from a modification in phosphocalcic homeostasis linked to changes in the hormone profile (Xing and Boyce, 2005). However, when looking at the phosphocalcic homeostasis, we did not observe any change in calcium and phosphorus in the blood of 90Sr-ingesting animals as compared with control animals, whatever the gender. Moreover, the hormones 1,25-(OH)2 vitamin D3 and PTH, which play a central role in phosphocalcic homeostasis (Baek and Kang, 2009; Morgan, 2001; Quarles, 2008) remained unchanged in 90Sr-ingesting animals as compared with control animals, whatever the gender. Overall, these results suggest that the observed effect of 90Sr ingestion on bone metabolism is the result of a direct effect of irradiation by 90Sr on either osteoblasts or osteoclasts, the two main cell types responsible for bone matrix turnover (Clarke, 2008; Seeman, 2008).

The results showing a trend towards bone matrix resoption may indicate that 90Sr ingestion accelerates the age-related loss of bone mass, by enhancing the rate of bone turnover. In order to verify this point we looked at bone morphology at the age of 20 weeks. Nevertheless, we did not observe differences between 90Sr-ingesting animals and control animals, both in growth plate thickness and in bone surface to tissue surface ratio, regardless of gender. Thus the observed modifications in gene expression and protein concentration in 90Sr-ingesting animals do not induce a major pathological modification in bone morphology. However, it should be of interest to more precisely analyze trabecular bone surface, since the rate of bone turnover is higher in trabecular bone as compared with cortical bone (Dahl et al., 2001). Moreover, we cannot exclude that such modifications to bone morphology may appear at later time point than 20 weeks. In fact, since we observed a trend toward bone resorption, it should be of interest to look at both gene expression and bone morphology at time points at which bone resorption become prominent, that is, in ageing animals after 12 or 24 months of exposure. In fact, it was described by others that major changes in bone physiology appear after 40 weeks in the mouse model (Ferguson et al., 2003; Silbermann et al., 1987).

We used a strontium source containing stable strontium as a carrier. Earlier work showed that chronic ingestion of stable strontium in high quantities is able to induce bone deficiency (Corradino et al., 1971; Omdahl and DeLuca, 1972). Thus, one can hypothesize that the observed effect is due to stable strontium present in drinking water. However, such an effect of stable strontium was observed only for daily ingestion of more than 27 mg per day of stable strontium in rats, and no toxic effect was observed for a daily ingestion of <13.5 mg per day (Morohashi et al., 1994). In our experiments, the measured daily ingestion of strontium was in the range of 0.8–1.1 ng.day−1, that is, about 28 × 106 fold less than the toxic concentration previously defined (Morohashi et al., 1994). So, it is very unlikely that the observed effect is due to stable strontium present in drinking water. This is in accordance with the toxicologic profile of stable strontium (ATSDR, 2004), indicating that, at a concentration relevant to environmental exposure, stable strontium does not have any health effect. Also, it was shown that such bone deficiency induced by high levels of strontium was due to the inhibition of 1,25-(OH)2 vitamin D3 synthesis in the kidney (Omdahl and DeLuca, 1972) that in turn reduced calcium uptake by the intestine (Corradino et al., 1971). By contrast, we did not observe any modification in calcium concentration or in \1,25-(OH)2 vitamin D3 in the plasma of strontium ingesting animals as compared with controls. Thus, we assume that, in our experimental conditions, stable strontium do not have any effect. Rather, stable strontium is able to stimulate bone cells in vitro (Braux et al., 2011; Marie, 1984) and in vivo at low concentration in the diet (<13.5 mg per day; Lymperi et al., 2008; Marie and Hott, 1986). This suggests that the observed effect of 90Sr on bone physiology is mainly due to its radiological nature. As a consequence, and as the β emission of 90Sr and its daughter product 90Y have a short track in living tissue, one can propose that the observed effect of 90Sr ingestion is due to direct irradiation of cells close to the bone matrix. In this respect, mesenchymal stem cells, osteoblasts and osteoclasts have to be considered primary targets of the 90Sr effect.

High doses of 90Sr were previously described to induce osteosarcoma in animal models of either chronic or acute contamination (Nilsson and Book, 1987; White et al., 1993). By contrast, although the appearance of osteosarcoma was described in the Techa River cohort (Kossenko et al., 2005), no excess radiation-related risk of bone cancer was found in these populations exposed to 90Sr ingestion (Krestinina et al., 2005). In the same population, recent results indicated that bone mineral density decreased according to the age of people but not according to either bone surface dose or 90Sr content in bones (Akleyev et al., 2010c). These recent results are not in contradiction with ours, since we observed modifications in expressions of genes encoding for proteins implicated in bone resorption in 90Sr ingesting animals as compared with control animals, and without any major effect on bone histology. Nevertheless, since we observed a tendency to increased bone resorption in 90Sr ingesting animals, we cannot formerly exclude that a decrease in bone density or a modification in bone histology may appears at later time points.

Overall, our results indicate that the chronic ingestion of 90Sr at a daily rate comparable with estimated daily ingestion of populations living on contaminated territories induces an accumulation of this radionuclide in bones, which in turn induces modifications in bone physiology, but not in hematopoiesis. However, this model has some limitations. Among others, a major limitation is the use of a single radionuclide. In fact, populations living in contaminated countries are exposed to both external irradiation and internal contamination, the latter being a mixture of radionuclides. Thus, it is necessary to improve our model by using a mix of 90Sr and 137Cs, the two main radionuclides found in the long term in the environment after a nuclear accident (Cooper et al., 1992; De Ruig and Van der Struijs, 1992). Moreover, in the context of the radioprotection of population, it is necessary to make dose–response experiments, in order to define the nonobservable effect limit, and also to assess the limit for the appearance of pathological effects in the high dose range.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors wish to thank T. Loiseau, J.M. Guischet and F. Voyer for their expert work in animal care. The secretarial assistance of V. Joffres and D. Lurmin is warmly acknowledged. This work is part of the ENVIRHOM research program of the institute de Radioprotection et de Sûreté Nucléaire. N. Synhaeve was supported by a grant from the Ile-de-France region.

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  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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