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Effect of cadmium on bone tissue in growing animals
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Effect of cadmium on bone tissue in growing animals Juliana Rodríguez, Patricia Mónica Mandalunis* University of Buenos Aires, Department of Histology and Embryology, School of Dentistry, Argentina, Marcelo T. de Alvear 2142–1er Piso Sector “A”, C1122AAH Buenos Aires, Argentina
A R T I C L E I N F O
Article history: Received 12 August 2015 Received in revised form 20 May 2016 Accepted 1 June 2016 Keywords: Cadmium Bone volume Bone marrow Osteoclast TRAP
A B S T R A C T
Accumulation of cadmium (Cd), an extremely toxic metal, can cause renal failure, decreased vitamin D synthesis, and consequently osteoporosis. The aim of this work was to evaluate the effect of Cd on two types of bone in growing Wistar rats. Sixteen 21-day-old male Wistar rats were assigned to one of two groups. The Cd group subcutaneously received 0.5 mg/kg of CdCl2 5 times weekly for 3 months. The control group similarly received bidistilled water. Following euthanasia, the mandibles and tibiae were resected, fixed, decalcified and processed histologically to obtain sections for H&E and tartrate-resistant acid phosphatase (TRAP) staining. Photomicrographs were used to determine bone volume (BV/TV%), total growth cartilage width (GPC.Wi) hypertrophic cartilage width (HpZ.Wi), percentage of yellow bone marrow (%YBM), megakaryocyte number (N.Mks/mm2), and TRAP + osteoclast number (N.TRAP + Ocl/ mm2). Results were statistically analyzed using Student’s t test. Cd exposed animals showed a significant decrease in subchondral bone volume and a significant increase in TRAP+ osteoclast number and percentage of yellow bone marrow in the tibia, and an increase in megakaryocyte number in mandibular interradicular bone. No significant differences were observed in the remaining parameters. The results obtained with this experimental design show that Cd would seemingly have a different effect on subchondral and interradicular bone. The decrease in bone volume and increase in tibial yellow bone marrow suggest that cadmium inhibits differentiation of mesenchymal cells to osteoblasts, favoring differentiation into adipocytes. The different effects of Cd on interradicular bone might be due to the protective effect of the mastication forces. ã 2016 Elsevier GmbH. All rights reserved.
1. Introduction In recent years, Cadmium, an extremely toxic metal, has attracted considerable interest as an environmental pollutant. Cadmium is released into the environment mainly from uncontrolled e-waste recycling (Guo et al., 2010; Leung et al., 2008), fertilizers, cigarette smoke, leather industry effluents, and even some metals used in jewelry electroplating (Weidenhamer et al., 2011). It must be pointed out that the half life of cadmium in the body is approximately 10–30 years (Jarup and Akesson, 2009). According to clinical studies, exposure to cadmium causes mainly renal failure and osteoporosis (Engström et al., 2012; Jin et al., 2004; Sughis et al., 2011). Two mechanisms of action have been proposed for cadmium: a direct and an indirect mechanism (Kazantzis, 2004). The indirect mechanism suggests that Cd is
* Corresponding author. E-mail addresses: [email protected] (J. Rodríguez), [email protected] (P.M. Mandalunis).
taken up by cells in the proximal convoluted tubules of the kidney and accumulates in mitochondria, generating disruption of the respiratory chain and causing oxidative stress due to generation of reactive oxygen species (ROS) (Bertin and Averbeck, 2006; Johri et al., 2010). These ROS would seemingly block production of an enzyme responsible for vitamin D activation, thus decreasing calcium absorption in the digestive tract. The ensuing hypercalciuria, caused by the inability of the kidneys to reabsorb calcium in addition to decreased calcium absorption in the duodenum, would deleteriously affect bone formation and mineralization (Alfvén et al., 2000; Horiguchi et al., 2005). The direct mechanism suggests that Cd exerts an effect on bone tissue cells (Chen et al., 2009). Biochemical assays performed in vivo in rats showed that consumption of 1 mg of CdCl2/L of drinking water for 24 months affects the bone remodeling process by decreasing alkaline phosphatase activity and increasing serum levels of C-terminal cross-linking telopeptide of type I collagen (Brzóska and Moniuszko-Jakoniuk, 2004). In vitro studies in human cell cultures have shown that Cd induces activation of caspase-3 and 8, both involved in osteoblast apoptosis (Coonse et al., 2007).
http://dx.doi.org/10.1016/j.etp.2016.06.001 0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: J. Rodríguez, P.M. Mandalunis, Effect of cadmium on bone tissue in growing animals, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.001
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In addition, cell exposure (RAW264.7 mouse monocytes/macrophage cell line (TIB-71; ATCC) to Cd in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) was found to increase levels of tartrate- resistant acid phosphatase (TRAP) activity and formation of TRAP+ cells, suggesting that Cd could increase bone mass loss by inducing osteoclast formation and differentiation (Chen et al., 2011a). Toxic agents affect the entire skeletal system, including the jaw bones. Alveolar bone is a part of the jaw bones that forms the primary supporting structure for teeth. Although alveolar bone and long bones are composed of cortical or compact bone and trabecular bone, their different origin and function determine different responses to the same stimulus (Sodek and McKee, 2000). Studies by Aghaloo et al. (2010) showed that mesenchymal cells derived from mandibular bone marrow have a greater osteogenic potential than those derived from long bone marrow cells. Because of the small size and anatomical complexity of alveolar bone in experimental animals, there are few experimental toxicological studies in the literature using this type of bone. Given the distribution of Cd in the environment and its use in a number of applications, people are exposed to this metal throughout life. According to the literature, mean U-Cd values per mg/g of creatinine range between 0.32 and 0.4 in areas with low contamination (Wang et al., 2014a, 2014b), and can be higher than 10 mg/g of creatinine in highly contaminated zones (Nakagawa et al., 2006). Although studies in the literature have reported several effects of this toxic metal on a number of body tissues and organs, there is not sufficient evidence of the effect of Cd on different types of bone in growing animals. Thus, the aim of the present work was to evaluate the effect of Cd on two types of bone in growing animals, a long bone (tibia) and mandibular bone (interradicular bone at the first lower molar). 2. Materials and methods 2.1. Experimental animals Sixteen healthy male Wistar rats aged 21 days and weighing 65 10 g were assigned to one of two groups: Control and Cd. The rats were housed in galvanized steel cages, 4 animals per cage, at 21–24 C and 52–56% humidity, under 12 h light/dark cycles. The animals had free access to food (Standard diet rat-mouse chow, Cooperación, Argentina) and water; they were fed standard chow containing 23% protein, 1-1.4% calcium and 0.5-0.8% phosphorus (Bernhart and Tomarelli, 1966). The animals in group Cd were subcutaneously administered a 0.5 mg/kg dose of CdCl2 (SigmaAldrich, US) 5 times weekly for 3 months (Chen et al., 2011b). The animals in the control group were similarly treated with vehicle (bidistilled water). Body weight was recorded weekly. Three months after the onset of the experiment, the animals were weighed, anesthetized by intraperitoneal injection of xylazine (5 mg/kg, König Laboratory, Argentina) and ketamine (50 mg/kg, Holliday Laboratory, Argentina), and euthanized by intracardiac injection of 0.2 ml of euthanyl (Brouwer Laboratory, Argentina). The experimental protocol was approved by the Ethics Committee of School of Dentistry (28/11/2012-38), University of Buenos Aires, and is in keeping with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The mandible and tibiae were excised from all the animals. The tibiae were measured using a Vernier type caliper, and were weighed using a precision balance (E. Mettler, Zürich). All the material was fixed in a 4% formaldehyde-buffer solution pH 7.4, decalcified in 10% EDTA pH 7 for 30 days, and processed histologically for embedding in paraffin. Longitudinal sections
were obtained from the tibia, and mesio-distally oriented sections were obtained from the first lower molar in order to evaluate the interradicular bone. Interradicular bone sections and one set of tibia sections, approximately 7-8 mm thick, were stained with hematoxylin-eosin. In addition, one longitudinal section of each tibiae was processed for TRAP staining (Minkin (1982)Minkin C, 1982). Briefly, the method involves incubation in a TRIS buffer solution (pH 5) containing naphtol phosphate AS-BI (Sigma-Aldrich, Switzerland), fast red violet (Sigma, Germany), and sodium tartrate (Sigma, Germany). Bone histomorphometry studies (Demster et al., 2013) were performed using Image Pro Plus 4.5 software. The following parameters were measured in the areas of subchondral bone shown in Fig. 1a and b: BV/TV(%): Bone volume: percentage of bone tissue in the studied area Tb.N (1/mm): Trabecular number Tb.Wi (mm): Trabecular width Tb.Sp (mm): Trabecular spacing GCP.Wi (mm): Total growth cartilage width (Fig. 1b) HpZ.Wi (mm): Hypertrophic cartilage width (Fig. 1b) N.Mk/mm2: Number of megakaryocytes in bone marrow (Fig. 1a*) YBM (%): Percentage of yellow bone marrow (Fig. 1a*) The number of TRAP+ osteoclasts (NTRAP + Oc/mm2) was determined in a region immediately below the growth cartilage, using a light field microscope at 400X magnification. Cells presenting 2 or more nuclei and that were close to trabeculae were considered osteoclastic cells. The following histomorphometric parameters were assessed in the areas of interradicular bone of the first lower molar shown in Fig. 2a and b: - BV/TV(%): Bone volume: percentage of bone tissue in the total studied area. - N.Mk/mm2: Number of megakaryocytes in bone marrow (Fig. 2b*) - YBM (%): Percentage of yellow bone marrow (Fig. 2b*)
Statistical analysis The results are expressed as mean standard deviation. The data were statistically analyzed by Student’s t Test, using “Primer of Biostatistics” software. Values of p below 0.05 were considered statistically significant. 3. Results Animal body weight and length were recorded throughout the experiment. As shown in Fig. 3, body weight and size were similar in all animals throughout the study, and no significant differences were observed between the Cd and the control groups. Likewise, no significant differences were found in tibia weight (g) (control: 0.978 0.068; Cd: 0.908 0.068) or length (cm) (control:4.28 0.118; Cd:4.33 0.131) three months post-exposure. Although no significant morphometric differences were observed, the histomorphometric study showed a decrease in subchondral bone volume and an increase in the percentage of yellow bone marrow in the tibia of Cd exposed animals. Significant differences (p < 0.05) in tibial trabecular bone volume were observed between groups, as a result of lower trabecular number Tb.N (1/mm) (control: 2.07 0.76; Cd: 1.31 0.26) (p<0.05) and greater trabecular spacing
Please cite this article in press as: J. Rodríguez, P.M. Mandalunis, Effect of cadmium on bone tissue in growing animals, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.001
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Fig. 1. Photomicrograph of H&E stained longitudinal tibia sections showing: a) Areas used for determination of histomorphometric parameters of trabecular subchondral bone and yellow bone marrow and b) Area used for determination of growth cartilage width. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. a) Schematic drawing of the rat mandible showing localization of alveolar bone at the first molar. b) Photomicrograph of H&E stained mesio-distal section of interradicular bone showing the areas used for determination of histomorphometric parameters of interradicular bone and yellow bone marrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Tb.Sp (mm) (control:0.47 0.19; Cd:0.71 0.17) (p<0.05), but no differences in trabecular width Tb.Wi (mm) (control:0.071 0.006; Cd:0.077 0.004) (p>0.05) (Fig. 4) were found between groups. In order to determine whether Cd exposure affected the bone
resorption process, TRAP staining was used to analyze osteoclast number. Results showed a greater number of TRAP+ osteoclasts (N. TRAP + Oc/mm2) in Cd exposed animals (26.63 5.06) compared to controls (15.96 4.19) (p<0.05) (Fig. 5). As regards the metaphyseal
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Fig. 3. Graphs showing animal body weight expressed in g (a) and length expressed in cm (b) throughout the experiment.
Fig. 4. Photomicrographs of H&E stained longitudinal sections of the tibia showing the decrease in bone volume and increase in yellow bone marrow (YBM) observed in Cdexposed animals (b) as compared to controls (a). Graphs showing BV/TV% in tibial trabecular bone; p < 0.05 (c), trabecular number; p < 0.05 (d), trabecular width; p > 0.05 (e) and trabecular spacing p < 0.05 (f) in the studied area. (*) Indicates statistically significant difference compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
cartilage, no significant differences in total growth cartilage width (GPC.Wi) (mm) (control: 307 34; Cd: 305 27) (p>0.05) or in hypertrophic cartilage width HpZ.Wi (mm): (control: 135 17; Cd: 133 17) (p > 0.05) were observed between groups (data not
shown). The histomorphometric study of bone marrow showed a significant increase (p < 0.05) in the percentage of yellow bone marrow (%) in Cd-exposed rats (Cd: 27.64 9.16 vs control: 17.81 6.21), but no significant differences in the number of
Please cite this article in press as: J. Rodríguez, P.M. Mandalunis, Effect of cadmium on bone tissue in growing animals, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.001
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Fig. 5. Photomicrographs of longitudinal sections of the tibia showing TRAP+ staining: Control group (a) and Cd group (b). The arrows show TRAP-positive osteoclasts.
megakaryocytes (N.Mks/mm2) (Cd: 5.57 1.39 vs control: 5.70 1.14) (p > 0.05) when comparing the Cd and control groups (data not shown). Unlike tibial subchondral bone volume, interradicular bone volume (BV/TV%) (control: 47.36 2.83; Cd: 48.93 2.75) (p > 0.05) and the percentage of yellow bone marrow (%) (control: 2.42 0.55; Cd: 2.73 0.80) (p > 0.05) were not significantly different between groups. The number of megakaryocytes (N. Mks/mm2), however, was higher in Cd-exposed animals (15.69 3.77) than in controls (10.31 3.53) (p < 0.05) (Fig. 6).
4. Discussion Exposure to toxic metals has become a worldwide problem. Cadmium, in particular, has gained importance on account of the increasing human use of Cd in a number of applications, its long half-life in the body, and its deleterious health effects. Bone is a dynamic structure undergoing constant remodeling throughout life. The bone remodeling/modeling process is accomplished by precise coordination of resorption, synthesis, and mineralization of the bone matrix by osteoclasts and
Fig. 6. Photomicrographs of H&E stained mesio-distal histological sections of the lower first molar showing no significant changes in interradicular bone volume between control (a) and Cd (b) groups. Graphs showing BV/TV% in interradicular bone, p > 0.05 (c); and number of megakaryocytes in the studied area p < 0.05 (d). (*) Indicates statistically significant difference compared to control.
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osteoblasts. Any factor that alters the normal functioning of any of these two cell types will cause an imbalance in these processes. The present work sought to evaluate the effect of a high dose of Cd on two types of bone, the tibia (long bone formed by endochondral ossification) and interradicular bone (mandibular bone, formed by intramembranous ossification) in growing animals. The experimental design used in this study is based on a study by Chen et al. (2011c) who found that administration of 0.5 mg/kg of CdCl2, for 5 days, resulted in urine cadmium and bone mineral density values equivalent to those observed in humans living in areas highly contaminated with cadmium, or exposed to cadmium at their workplace. Our results showed a different effect of Cd on each of these types of bone. It caused a decrease in trabecular bone volume in the tibia, resulting from a decrease in the number of trabeculae, but did not affect growth cartilage width. This finding is partly consistent with reports by Brzóska and Moniuszko-Jakoniuk (2005) showing that exposure to CdCl2 in drinking water caused a dose/time dependent decrease in bone mineral density and in serum and bone alkaline phosphatase enzyme activity in healthy female rats. In vitro studies in the literature (Miyahara et al., 1991; Wang et al., 2014a, 2014b; Wilson et al., 1996) have shown low concentrations of Cd to induce osteoclast differentiation from progenitor cells, and to increase mature osteoclast activity. The present study is the first to demonstrate in vivo that Cd causes an increase in the number of TRAP positive osteoclasts. From these results, it could be inferred that Cd has a deleterious effect on subchondral bone remodeling during growth, stimulating osteoclastogenesis and decreasing bone volume. In addition, our results showed a significant increase in adipocyte number in tibial bone marrow of Cd-exposed animals. There is sufficient evidence of the relation between bone marrow cells and bone tissue homeostasis, as well as of the negative association between the proportion of yellow bone marrow and number of osteoblasts (Gimble et al., 1996; Rosen and Bouxein, 2006). Mesenchymal cells in the bone marrow have the ability to differentiate into osteoblasts or adipocytes, according to the expression of different transcription factors (Gimble and Nuttall, 2004). An imbalance in the expression of these transcription factors, as could be caused by an external factor, can favor differentiation into one or the other cell lineage. The increase in the proportion of yellow bone marrow of subchondral bone observed in Cd-exposed animals suggests that this metal could inhibit differentiation of mesenchymal cells into osteoblasts, favoring their differentiation into adipocytes. Interradicular and subchondral bones have different functions. Over the last years, a number of studies have reported different response of mandibular bone and long bones to systemic diseases and conditions. Mavropoulos et al. (2014, 2007) showed that estrogen deficiency-induced osteoporosis generated a greater impact on subchondral bone than on alveolar bone, and that administration of a soft diet to ovariectomized rats caused a greater loss of alveolar bone volume than a solid diet. In addition, Liu et al. (2014) found that a loss of occlusal stimuli could lead to mandibular bone resorption, and recovery of the stimuli could restore the bone architecture to normal. The present study is the first to report in vivo evidence of the effect of Cd on interradicular bone. Unlike the findings observed in subchondral bone, no significant differences in interradicular bone volume and percentage of yellow bone marrow were found between Cd-exposed animals and controls at the dose and experimental time studied here. During growth, the body is subjected to a wide variety of mechanical stimuli that promote and modulate the normal development of tissues. Mechanical forces can regulate cell proliferation and differentiation via stimulation or inhibition of gene expression (Hao et al., 2015). A study by Liu et al. (2015)
showed that mechanical stimulation of mesenchymal cells cultured in adipogenic differentiation medium increased levels of Runx2, Osx, and collagen type I, and decreased levels of PPARg-2 and C/EBPa (CCAAT enhancer binding protein a), promoting their differentiation into osteoblasts and impeding their differentiation into adipocytes. The results obtained here suggest that the adipogenic effect of Cd observed in the bone marrow of long bones might be mitigated in alveolar bone by the anabolic effect of the mechanical stress exerted by masticatory movements on interradicular bone marrow cells. Megakaryocytes have been found to play a role in both osteoclastogenesis and osteoblastogenesis. The direct effect of megakaryocytes on osteoclasts is complex, since they have the ability to express and or secrete factors that promote osteoclast differentiation, such as RANKL, under certain conditions, but can also secrete factors that impede the process, as is the case of OPG, IL-10, IL-13 y TGF-b (Bord et al., 2005; Kacena and Ciovacco, 2010). Megakaryocytes have also been shown to secrete bone matrix growth factors and proteins, promoting osteoblast differentiation (Kacena et al., 2006; Kelm et al., 1992; Thiede et al., 1994). Our results showed no differences in megakaryocyte number in tibial bone marrow between Cd-exposed and control animals. Interestingly, however, two things must be pointed out with regard to our observations in interradicular bone. First, despite finding no alterations in bone tissue or yellow bone marrow, we observed a significant increase in Mks number in Cdexposed animals. Second, the number of Mks observed in interradicular bone marrow was two-fold that observed in the tibial bone marrow of the same animal. Although both observations need to be further investigated, it could be thought that the differences might be associated with the mechanical stimuli, type of ossification, and embryologic origin of both types of bone: mandibular bone mainly originates from the neural crests and somitomeres, whereas long bones originate from the lateral plate mesoderm. Thus, both bone types develop in different microenvironments, subjected to different signals and stimuli. In conclusion, the present study shows in vivo evidence of the different effect of a toxic metal on different types of bone in healthy growing animals. We found alveolar bone to be less sensitive to the effects of long-term exposure to Cd than subchondral bone. This difference may be associated with the protective effect of masticatory forces. The results of the evaluation of subchondral bone microarchitecture suggest that the effect of Cd on long bone remodeling could be attributed on one hand to the promotion of mesenchymal cell differentiation into adipocytes and inhibition of their differentiation into osteoblasts, and on the other, to the promotion of osteoclastogenesis. Acknowledgments Grant: UBACyT O 406, University of Buenos Aires. The authors wish to express their gratitude to Ht Mariela Lacave, Ht Ivana A. Sánchez Rojas and Vet Marianela Lewicki for their technical assistance. References Aghaloo TL, Chaichanasakul T, Bezouglaia O, Kang B, Franco R, Dry SM, et al. Osteogenic potential of mandibular vs long-bone marrow stromal cells. J. Dent. Res. 2010;89:1293–8. Alfvén T, Elinder CG, Carlsson MD, Grubb A, Hellström L, Persson B, et al. Low-level cadmium exposure and osteoporosis. J. Bone Miner Res 2000;15:1579–86. Bernhart FW, Tomarelli RM. A salt mixture supplying the National Research Council estimates of the mineral requirements of the rat. J. Nutr. 1966;89:495–500. Bertin G, Averbeck D. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review). Biochimie 2006;88:1549–59.
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Effect of cadmium on bone tissue in growing animals Juliana Rodríguez, Patricia Mónica Mandalunis* University of Buenos Aires, Department of Histology and Embryology, School of Dentistry, Argentina, Marcelo T. de Alvear 2142–1er Piso Sector “A”, C1122AAH Buenos Aires, Argentina
A R T I C L E I N F O
Article history: Received 12 August 2015 Received in revised form 20 May 2016 Accepted 1 June 2016 Keywords: Cadmium Bone volume Bone marrow Osteoclast TRAP
A B S T R A C T
Accumulation of cadmium (Cd), an extremely toxic metal, can cause renal failure, decreased vitamin D synthesis, and consequently osteoporosis. The aim of this work was to evaluate the effect of Cd on two types of bone in growing Wistar rats. Sixteen 21-day-old male Wistar rats were assigned to one of two groups. The Cd group subcutaneously received 0.5 mg/kg of CdCl2 5 times weekly for 3 months. The control group similarly received bidistilled water. Following euthanasia, the mandibles and tibiae were resected, fixed, decalcified and processed histologically to obtain sections for H&E and tartrate-resistant acid phosphatase (TRAP) staining. Photomicrographs were used to determine bone volume (BV/TV%), total growth cartilage width (GPC.Wi) hypertrophic cartilage width (HpZ.Wi), percentage of yellow bone marrow (%YBM), megakaryocyte number (N.Mks/mm2), and TRAP + osteoclast number (N.TRAP + Ocl/ mm2). Results were statistically analyzed using Student’s t test. Cd exposed animals showed a significant decrease in subchondral bone volume and a significant increase in TRAP+ osteoclast number and percentage of yellow bone marrow in the tibia, and an increase in megakaryocyte number in mandibular interradicular bone. No significant differences were observed in the remaining parameters. The results obtained with this experimental design show that Cd would seemingly have a different effect on subchondral and interradicular bone. The decrease in bone volume and increase in tibial yellow bone marrow suggest that cadmium inhibits differentiation of mesenchymal cells to osteoblasts, favoring differentiation into adipocytes. The different effects of Cd on interradicular bone might be due to the protective effect of the mastication forces. ã 2016 Elsevier GmbH. All rights reserved.
1. Introduction In recent years, Cadmium, an extremely toxic metal, has attracted considerable interest as an environmental pollutant. Cadmium is released into the environment mainly from uncontrolled e-waste recycling (Guo et al., 2010; Leung et al., 2008), fertilizers, cigarette smoke, leather industry effluents, and even some metals used in jewelry electroplating (Weidenhamer et al., 2011). It must be pointed out that the half life of cadmium in the body is approximately 10–30 years (Jarup and Akesson, 2009). According to clinical studies, exposure to cadmium causes mainly renal failure and osteoporosis (Engström et al., 2012; Jin et al., 2004; Sughis et al., 2011). Two mechanisms of action have been proposed for cadmium: a direct and an indirect mechanism (Kazantzis, 2004). The indirect mechanism suggests that Cd is
* Corresponding author. E-mail addresses: [email protected] (J. Rodríguez), [email protected] (P.M. Mandalunis).
taken up by cells in the proximal convoluted tubules of the kidney and accumulates in mitochondria, generating disruption of the respiratory chain and causing oxidative stress due to generation of reactive oxygen species (ROS) (Bertin and Averbeck, 2006; Johri et al., 2010). These ROS would seemingly block production of an enzyme responsible for vitamin D activation, thus decreasing calcium absorption in the digestive tract. The ensuing hypercalciuria, caused by the inability of the kidneys to reabsorb calcium in addition to decreased calcium absorption in the duodenum, would deleteriously affect bone formation and mineralization (Alfvén et al., 2000; Horiguchi et al., 2005). The direct mechanism suggests that Cd exerts an effect on bone tissue cells (Chen et al., 2009). Biochemical assays performed in vivo in rats showed that consumption of 1 mg of CdCl2/L of drinking water for 24 months affects the bone remodeling process by decreasing alkaline phosphatase activity and increasing serum levels of C-terminal cross-linking telopeptide of type I collagen (Brzóska and Moniuszko-Jakoniuk, 2004). In vitro studies in human cell cultures have shown that Cd induces activation of caspase-3 and 8, both involved in osteoblast apoptosis (Coonse et al., 2007).
http://dx.doi.org/10.1016/j.etp.2016.06.001 0940-2993/ã 2016 Elsevier GmbH. All rights reserved.
Please cite this article in press as: J. Rodríguez, P.M. Mandalunis, Effect of cadmium on bone tissue in growing animals, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.001
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In addition, cell exposure (RAW264.7 mouse monocytes/macrophage cell line (TIB-71; ATCC) to Cd in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) was found to increase levels of tartrate- resistant acid phosphatase (TRAP) activity and formation of TRAP+ cells, suggesting that Cd could increase bone mass loss by inducing osteoclast formation and differentiation (Chen et al., 2011a). Toxic agents affect the entire skeletal system, including the jaw bones. Alveolar bone is a part of the jaw bones that forms the primary supporting structure for teeth. Although alveolar bone and long bones are composed of cortical or compact bone and trabecular bone, their different origin and function determine different responses to the same stimulus (Sodek and McKee, 2000). Studies by Aghaloo et al. (2010) showed that mesenchymal cells derived from mandibular bone marrow have a greater osteogenic potential than those derived from long bone marrow cells. Because of the small size and anatomical complexity of alveolar bone in experimental animals, there are few experimental toxicological studies in the literature using this type of bone. Given the distribution of Cd in the environment and its use in a number of applications, people are exposed to this metal throughout life. According to the literature, mean U-Cd values per mg/g of creatinine range between 0.32 and 0.4 in areas with low contamination (Wang et al., 2014a, 2014b), and can be higher than 10 mg/g of creatinine in highly contaminated zones (Nakagawa et al., 2006). Although studies in the literature have reported several effects of this toxic metal on a number of body tissues and organs, there is not sufficient evidence of the effect of Cd on different types of bone in growing animals. Thus, the aim of the present work was to evaluate the effect of Cd on two types of bone in growing animals, a long bone (tibia) and mandibular bone (interradicular bone at the first lower molar). 2. Materials and methods 2.1. Experimental animals Sixteen healthy male Wistar rats aged 21 days and weighing 65 10 g were assigned to one of two groups: Control and Cd. The rats were housed in galvanized steel cages, 4 animals per cage, at 21–24 C and 52–56% humidity, under 12 h light/dark cycles. The animals had free access to food (Standard diet rat-mouse chow, Cooperación, Argentina) and water; they were fed standard chow containing 23% protein, 1-1.4% calcium and 0.5-0.8% phosphorus (Bernhart and Tomarelli, 1966). The animals in group Cd were subcutaneously administered a 0.5 mg/kg dose of CdCl2 (SigmaAldrich, US) 5 times weekly for 3 months (Chen et al., 2011b). The animals in the control group were similarly treated with vehicle (bidistilled water). Body weight was recorded weekly. Three months after the onset of the experiment, the animals were weighed, anesthetized by intraperitoneal injection of xylazine (5 mg/kg, König Laboratory, Argentina) and ketamine (50 mg/kg, Holliday Laboratory, Argentina), and euthanized by intracardiac injection of 0.2 ml of euthanyl (Brouwer Laboratory, Argentina). The experimental protocol was approved by the Ethics Committee of School of Dentistry (28/11/2012-38), University of Buenos Aires, and is in keeping with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The mandible and tibiae were excised from all the animals. The tibiae were measured using a Vernier type caliper, and were weighed using a precision balance (E. Mettler, Zürich). All the material was fixed in a 4% formaldehyde-buffer solution pH 7.4, decalcified in 10% EDTA pH 7 for 30 days, and processed histologically for embedding in paraffin. Longitudinal sections
were obtained from the tibia, and mesio-distally oriented sections were obtained from the first lower molar in order to evaluate the interradicular bone. Interradicular bone sections and one set of tibia sections, approximately 7-8 mm thick, were stained with hematoxylin-eosin. In addition, one longitudinal section of each tibiae was processed for TRAP staining (Minkin (1982)Minkin C, 1982). Briefly, the method involves incubation in a TRIS buffer solution (pH 5) containing naphtol phosphate AS-BI (Sigma-Aldrich, Switzerland), fast red violet (Sigma, Germany), and sodium tartrate (Sigma, Germany). Bone histomorphometry studies (Demster et al., 2013) were performed using Image Pro Plus 4.5 software. The following parameters were measured in the areas of subchondral bone shown in Fig. 1a and b: BV/TV(%): Bone volume: percentage of bone tissue in the studied area Tb.N (1/mm): Trabecular number Tb.Wi (mm): Trabecular width Tb.Sp (mm): Trabecular spacing GCP.Wi (mm): Total growth cartilage width (Fig. 1b) HpZ.Wi (mm): Hypertrophic cartilage width (Fig. 1b) N.Mk/mm2: Number of megakaryocytes in bone marrow (Fig. 1a*) YBM (%): Percentage of yellow bone marrow (Fig. 1a*) The number of TRAP+ osteoclasts (NTRAP + Oc/mm2) was determined in a region immediately below the growth cartilage, using a light field microscope at 400X magnification. Cells presenting 2 or more nuclei and that were close to trabeculae were considered osteoclastic cells. The following histomorphometric parameters were assessed in the areas of interradicular bone of the first lower molar shown in Fig. 2a and b: - BV/TV(%): Bone volume: percentage of bone tissue in the total studied area. - N.Mk/mm2: Number of megakaryocytes in bone marrow (Fig. 2b*) - YBM (%): Percentage of yellow bone marrow (Fig. 2b*)
Statistical analysis The results are expressed as mean standard deviation. The data were statistically analyzed by Student’s t Test, using “Primer of Biostatistics” software. Values of p below 0.05 were considered statistically significant. 3. Results Animal body weight and length were recorded throughout the experiment. As shown in Fig. 3, body weight and size were similar in all animals throughout the study, and no significant differences were observed between the Cd and the control groups. Likewise, no significant differences were found in tibia weight (g) (control: 0.978 0.068; Cd: 0.908 0.068) or length (cm) (control:4.28 0.118; Cd:4.33 0.131) three months post-exposure. Although no significant morphometric differences were observed, the histomorphometric study showed a decrease in subchondral bone volume and an increase in the percentage of yellow bone marrow in the tibia of Cd exposed animals. Significant differences (p < 0.05) in tibial trabecular bone volume were observed between groups, as a result of lower trabecular number Tb.N (1/mm) (control: 2.07 0.76; Cd: 1.31 0.26) (p<0.05) and greater trabecular spacing
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Fig. 1. Photomicrograph of H&E stained longitudinal tibia sections showing: a) Areas used for determination of histomorphometric parameters of trabecular subchondral bone and yellow bone marrow and b) Area used for determination of growth cartilage width. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. a) Schematic drawing of the rat mandible showing localization of alveolar bone at the first molar. b) Photomicrograph of H&E stained mesio-distal section of interradicular bone showing the areas used for determination of histomorphometric parameters of interradicular bone and yellow bone marrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Tb.Sp (mm) (control:0.47 0.19; Cd:0.71 0.17) (p<0.05), but no differences in trabecular width Tb.Wi (mm) (control:0.071 0.006; Cd:0.077 0.004) (p>0.05) (Fig. 4) were found between groups. In order to determine whether Cd exposure affected the bone
resorption process, TRAP staining was used to analyze osteoclast number. Results showed a greater number of TRAP+ osteoclasts (N. TRAP + Oc/mm2) in Cd exposed animals (26.63 5.06) compared to controls (15.96 4.19) (p<0.05) (Fig. 5). As regards the metaphyseal
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Fig. 3. Graphs showing animal body weight expressed in g (a) and length expressed in cm (b) throughout the experiment.
Fig. 4. Photomicrographs of H&E stained longitudinal sections of the tibia showing the decrease in bone volume and increase in yellow bone marrow (YBM) observed in Cdexposed animals (b) as compared to controls (a). Graphs showing BV/TV% in tibial trabecular bone; p < 0.05 (c), trabecular number; p < 0.05 (d), trabecular width; p > 0.05 (e) and trabecular spacing p < 0.05 (f) in the studied area. (*) Indicates statistically significant difference compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
cartilage, no significant differences in total growth cartilage width (GPC.Wi) (mm) (control: 307 34; Cd: 305 27) (p>0.05) or in hypertrophic cartilage width HpZ.Wi (mm): (control: 135 17; Cd: 133 17) (p > 0.05) were observed between groups (data not
shown). The histomorphometric study of bone marrow showed a significant increase (p < 0.05) in the percentage of yellow bone marrow (%) in Cd-exposed rats (Cd: 27.64 9.16 vs control: 17.81 6.21), but no significant differences in the number of
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Fig. 5. Photomicrographs of longitudinal sections of the tibia showing TRAP+ staining: Control group (a) and Cd group (b). The arrows show TRAP-positive osteoclasts.
megakaryocytes (N.Mks/mm2) (Cd: 5.57 1.39 vs control: 5.70 1.14) (p > 0.05) when comparing the Cd and control groups (data not shown). Unlike tibial subchondral bone volume, interradicular bone volume (BV/TV%) (control: 47.36 2.83; Cd: 48.93 2.75) (p > 0.05) and the percentage of yellow bone marrow (%) (control: 2.42 0.55; Cd: 2.73 0.80) (p > 0.05) were not significantly different between groups. The number of megakaryocytes (N. Mks/mm2), however, was higher in Cd-exposed animals (15.69 3.77) than in controls (10.31 3.53) (p < 0.05) (Fig. 6).
4. Discussion Exposure to toxic metals has become a worldwide problem. Cadmium, in particular, has gained importance on account of the increasing human use of Cd in a number of applications, its long half-life in the body, and its deleterious health effects. Bone is a dynamic structure undergoing constant remodeling throughout life. The bone remodeling/modeling process is accomplished by precise coordination of resorption, synthesis, and mineralization of the bone matrix by osteoclasts and
Fig. 6. Photomicrographs of H&E stained mesio-distal histological sections of the lower first molar showing no significant changes in interradicular bone volume between control (a) and Cd (b) groups. Graphs showing BV/TV% in interradicular bone, p > 0.05 (c); and number of megakaryocytes in the studied area p < 0.05 (d). (*) Indicates statistically significant difference compared to control.
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osteoblasts. Any factor that alters the normal functioning of any of these two cell types will cause an imbalance in these processes. The present work sought to evaluate the effect of a high dose of Cd on two types of bone, the tibia (long bone formed by endochondral ossification) and interradicular bone (mandibular bone, formed by intramembranous ossification) in growing animals. The experimental design used in this study is based on a study by Chen et al. (2011c) who found that administration of 0.5 mg/kg of CdCl2, for 5 days, resulted in urine cadmium and bone mineral density values equivalent to those observed in humans living in areas highly contaminated with cadmium, or exposed to cadmium at their workplace. Our results showed a different effect of Cd on each of these types of bone. It caused a decrease in trabecular bone volume in the tibia, resulting from a decrease in the number of trabeculae, but did not affect growth cartilage width. This finding is partly consistent with reports by Brzóska and Moniuszko-Jakoniuk (2005) showing that exposure to CdCl2 in drinking water caused a dose/time dependent decrease in bone mineral density and in serum and bone alkaline phosphatase enzyme activity in healthy female rats. In vitro studies in the literature (Miyahara et al., 1991; Wang et al., 2014a, 2014b; Wilson et al., 1996) have shown low concentrations of Cd to induce osteoclast differentiation from progenitor cells, and to increase mature osteoclast activity. The present study is the first to demonstrate in vivo that Cd causes an increase in the number of TRAP positive osteoclasts. From these results, it could be inferred that Cd has a deleterious effect on subchondral bone remodeling during growth, stimulating osteoclastogenesis and decreasing bone volume. In addition, our results showed a significant increase in adipocyte number in tibial bone marrow of Cd-exposed animals. There is sufficient evidence of the relation between bone marrow cells and bone tissue homeostasis, as well as of the negative association between the proportion of yellow bone marrow and number of osteoblasts (Gimble et al., 1996; Rosen and Bouxein, 2006). Mesenchymal cells in the bone marrow have the ability to differentiate into osteoblasts or adipocytes, according to the expression of different transcription factors (Gimble and Nuttall, 2004). An imbalance in the expression of these transcription factors, as could be caused by an external factor, can favor differentiation into one or the other cell lineage. The increase in the proportion of yellow bone marrow of subchondral bone observed in Cd-exposed animals suggests that this metal could inhibit differentiation of mesenchymal cells into osteoblasts, favoring their differentiation into adipocytes. Interradicular and subchondral bones have different functions. Over the last years, a number of studies have reported different response of mandibular bone and long bones to systemic diseases and conditions. Mavropoulos et al. (2014, 2007) showed that estrogen deficiency-induced osteoporosis generated a greater impact on subchondral bone than on alveolar bone, and that administration of a soft diet to ovariectomized rats caused a greater loss of alveolar bone volume than a solid diet. In addition, Liu et al. (2014) found that a loss of occlusal stimuli could lead to mandibular bone resorption, and recovery of the stimuli could restore the bone architecture to normal. The present study is the first to report in vivo evidence of the effect of Cd on interradicular bone. Unlike the findings observed in subchondral bone, no significant differences in interradicular bone volume and percentage of yellow bone marrow were found between Cd-exposed animals and controls at the dose and experimental time studied here. During growth, the body is subjected to a wide variety of mechanical stimuli that promote and modulate the normal development of tissues. Mechanical forces can regulate cell proliferation and differentiation via stimulation or inhibition of gene expression (Hao et al., 2015). A study by Liu et al. (2015)
showed that mechanical stimulation of mesenchymal cells cultured in adipogenic differentiation medium increased levels of Runx2, Osx, and collagen type I, and decreased levels of PPARg-2 and C/EBPa (CCAAT enhancer binding protein a), promoting their differentiation into osteoblasts and impeding their differentiation into adipocytes. The results obtained here suggest that the adipogenic effect of Cd observed in the bone marrow of long bones might be mitigated in alveolar bone by the anabolic effect of the mechanical stress exerted by masticatory movements on interradicular bone marrow cells. Megakaryocytes have been found to play a role in both osteoclastogenesis and osteoblastogenesis. The direct effect of megakaryocytes on osteoclasts is complex, since they have the ability to express and or secrete factors that promote osteoclast differentiation, such as RANKL, under certain conditions, but can also secrete factors that impede the process, as is the case of OPG, IL-10, IL-13 y TGF-b (Bord et al., 2005; Kacena and Ciovacco, 2010). Megakaryocytes have also been shown to secrete bone matrix growth factors and proteins, promoting osteoblast differentiation (Kacena et al., 2006; Kelm et al., 1992; Thiede et al., 1994). Our results showed no differences in megakaryocyte number in tibial bone marrow between Cd-exposed and control animals. Interestingly, however, two things must be pointed out with regard to our observations in interradicular bone. First, despite finding no alterations in bone tissue or yellow bone marrow, we observed a significant increase in Mks number in Cdexposed animals. Second, the number of Mks observed in interradicular bone marrow was two-fold that observed in the tibial bone marrow of the same animal. Although both observations need to be further investigated, it could be thought that the differences might be associated with the mechanical stimuli, type of ossification, and embryologic origin of both types of bone: mandibular bone mainly originates from the neural crests and somitomeres, whereas long bones originate from the lateral plate mesoderm. Thus, both bone types develop in different microenvironments, subjected to different signals and stimuli. In conclusion, the present study shows in vivo evidence of the different effect of a toxic metal on different types of bone in healthy growing animals. We found alveolar bone to be less sensitive to the effects of long-term exposure to Cd than subchondral bone. This difference may be associated with the protective effect of masticatory forces. The results of the evaluation of subchondral bone microarchitecture suggest that the effect of Cd on long bone remodeling could be attributed on one hand to the promotion of mesenchymal cell differentiation into adipocytes and inhibition of their differentiation into osteoblasts, and on the other, to the promotion of osteoclastogenesis. Acknowledgments Grant: UBACyT O 406, University of Buenos Aires. The authors wish to express their gratitude to Ht Mariela Lacave, Ht Ivana A. Sánchez Rojas and Vet Marianela Lewicki for their technical assistance. References Aghaloo TL, Chaichanasakul T, Bezouglaia O, Kang B, Franco R, Dry SM, et al. Osteogenic potential of mandibular vs long-bone marrow stromal cells. J. Dent. Res. 2010;89:1293–8. Alfvén T, Elinder CG, Carlsson MD, Grubb A, Hellström L, Persson B, et al. Low-level cadmium exposure and osteoporosis. J. Bone Miner Res 2000;15:1579–86. Bernhart FW, Tomarelli RM. A salt mixture supplying the National Research Council estimates of the mineral requirements of the rat. J. Nutr. 1966;89:495–500. Bertin G, Averbeck D. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review). Biochimie 2006;88:1549–59.
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Please cite this article in press as: J. Rodríguez, P.M. Mandalunis, Effect of cadmium on bone tissue in growing animals, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.06.001