Toxicology 319 (2014) 63–68
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Does methamphetamine affect bone metabolism? Masafumi Tomita a,∗ , Hironobu Katsuyama b , Yoko Watanabe c , Toshiko Okuyama a , Shigeko Fushimi b , Takaki Ishikawa e , Masayuki Nata f , Osamu Miyamoto d a
Department of Medical Toxicology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Japan Department of Public Health, Kawasaki Medical School, 577 Matsushima, Kurashiki, Japan Department of Natural Sciences, Kawasaki Medical School, 577 Matsushima, Kurashiki, Japan d Department of Physiology II, Kawasaki Medical School, 577 Matsushima, Kurashiki, Japan e Division of Legal Medicine, Faculty of Medicine, Tottori University, 86 Nishimachi, Yonago, Japan f Department of Forensic Medicine and Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Japan b c
a r t i c l e
i n f o
Article history: Received 18 November 2013 Received in revised form 26 December 2013 Accepted 26 January 2014 Available online 26 February 2014 Keywords: Methamphetamine Bone metabolism Osteoporosis Mice
a b s t r a c t There is a close relationship between the central nervous system activity and bone metabolism. Therefore, methamphetamine (METH), which stimulates the central nervous system, is expected to affect bone turnover. The aim of this study was to investigate the role of METH in bone metabolism. Mice were divided into 3 groups, the control group receiving saline injections, and the 5 and 10 mg/kg METH groups (n = 6 in each group). All groups received an injection of saline or METH every other day for 8 weeks. Bone mineral density (BMD) was assessed by X-ray computed tomography. We examined biochemical markers and histomorphometric changes in the second cancellous bone of the left femoral distal end. The animals that were administered 5 mg/kg METH showed an increased locomotor activity, whereas those receiving 10 mg/kg displayed an abnormal and stereotyped behavior. Serum calcium and phosphorus concentrations were normal compared to the controls, whereas the serum protein concentration was lower in the METH groups. BMD was unchanged in all groups. Bone formation markers such as alkaline phosphatase and osteocalcin significantly increased in the 5 mg/kg METH group, but not in the 10 mg/kg METH group. In contrast, bone resorption markers such as C-terminal telopeptides of type I collagen and tartrate-resistant acid phosphatase 5b did not change in any of the METH groups. Histomorphometric analyses were consistent with the biochemical markers data. A significant increase in osteoblasts, especially in type III osteoblasts, was observed in the 5 mg/kg METH group, whereas other parameters of bone resorption and mineralization remained unchanged. These results indicate that bone remodeling in this group was unbalanced. In contrast, in the 10 mg/kg METH group, some parameters of bone formation were significantly or slightly decreased, suggesting a low turnover metabolism. Taken together, our results suggest that METH had distinct dose-dependent effects on bone turnover and that METH might induce adverse effects, leading to osteoporosis. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Methamphetamine (METH) possesses a high potential for abuse and addiction, and METH use has become a serious social problem worldwide (Nordahl et al., 2003; Marshall and Werb, 2010). In Japan, more than 10,000 persons annually have been arrested for METH abuse for the last several decades (Wada, 2011). METH abuse often causes an increase in crime, traffic and non-traffic accidents, and physical and psychological injuries. METH not only
∗ Corresponding author. Tel.: +81 86462 1111. E-mail addresses: [email protected], [email protected] (M. Tomita). http://dx.doi.org/10.1016/j.tox.2014.01.014 0300-483X/© 2014 Elsevier Ireland Ltd. All rights reserved.
affects the central nervous system but also causes degeneration in many organs (Wijetunga et al., 2003). An increasing number of clinical and autopsy studies reported that METH use is associated with angina, tachycardia, hypertension, myocarditis, dilated cardiomyopathy, arrhythmia, and sudden death (Pilgrim et al., 2009; Schep et al., 2010; Volkow et al., 2010). Routine dental examinations performed on some convicts showed that METH abusers suffer from tooth decays or periodontal disease more often than other prisoners and also show oral manifestations of rampant caries (“meth mouth”) (Curtis, 2006; Hamamoto and Rhodus, 2009). Although there are a limited number of human studies regarding the potential relationship between molar tooth number and osteoporotic status, it has been suggested that dental condition might represent trabecular bone structure (Darcey et al., 2013). Bone is a dynamic
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organ, continuously remodeling itself. Bone resorption and bone formation are regulated by osteoclasts and osteoblasts, respectively (Hill, 1998; Eimar et al., 2013). It has recently been shown that neurons and neuropeptides in the central nervous system play an important role in bone remodeling (Takeda, 2009; Wang et al., 2010; Crockett et al., 2011). Osteoporosis is a neuroskeletal disease and, as mentioned above, bone remodeling is under the control of the nervous system. Therefore, we hypothesized that METH, which stimulates the central nervous system, may alter bone metabolism. Interestingly, Katsuragawa (1999) reported that the cancellous speed of sound, an indicator of bone strength, was significantly lower in METH abusers than in the control group. Other investigators, using dual energy Xray absorptiometry, observed that the bone mineral density (BMD) in the lumbar spine was lower in METH abusers (n = 46) than in the controls (n = 188) (Kim et al., 2009). However, the effects of METH on bone metabolism remain unknown. The aim of this study was to clarify the effects of METH on bone metabolism, especially on the trabecular bone metabolism in the femurs, in mice exposed to METH every other day for 8 weeks. We performed bone histomorphometry and quantified biochemical markers of bone turnover. Our results indicate that METH might affect bone metabolism in a dose-dependent manner, potentially leading to osteoporosis.
Table 1 Body weight, bone mineral density (BMD), and serum protein, calcium, and phosphorus concentrations. Control Body weight Pre (g) Post (g) BMD (mg/cm2 ) Protein (g/dL) Calcium (mg/dL) Phosphorus (mg/dL)
21.7 28.9 228.8 5.1 8.6 7.8
± ± ± ± ± ±
METH 5 0.8 1.5 19.8 0.2 0.2 1.2
21.9 27.7 223.1 4.9 8.6 6.8
± ± ± ± ± ±
METH 10 0.7 0.7 25.4 0.2 0.3 0.7
22.1 28.6 231.9 4.7 8.7 8.2
± ± ± ± ± ±
0.7 1.2 43.3 0.2a 0.3 1.0
METH (METH 5; 5 mg/kg, METH 10; 10 mg/kg) or vehicle was administered every other day for 8 weeks. Data are expressed as mean ± SD. (n = 6 for each treatment group; a p < 0.01 versus control group using ANOVA, followed by Tukey–Kramer multiple comparison test).
2.4. Trabecular bone mineral density (BMD) The fixed right bones were analyzed using a X-ray computed tomography system for small experimental animals (Model LaTheta, LCT-200; Aloka, Osaka, Japan). The bone was placed horizontally inside a tube and scanned using a 96-m voxel. The scan line was adjusted using the scout view. The trabecular BMD was quantified and calculated in the second cancellous bone proximal to the growth plate of the femoral distal end. The data were quantified using an automated image analysis software supplied with the device. 2.5. Bone histomorphometry
2. Materials and methods 2.1. Animal experiments Eight-week-old male C57BL/6J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). The animals were given a normal rodent chow and tap water and acclimated to the conditions for 1 week. They were then divided into 3 groups: control (saline), and 5 and 10 mg/kg METH groups (n = 6 in each group). METH or saline were administered intraperitoneally every other day for 8 weeks. To assess bone mineralization, mice were double-labeled with subcutaneous injections of 20 mg/kg tetracycline hydrochloride and 10 mg/kg calcein (Sigma–Aldrich Corp., St. Louis, MO), 5 and 2 days before sacrifice, respectively. The body weight of each animal was measured every other day until the final day of administration. Urine samples were collected over a 16-h period, starting immediately after last METH administration and were stored at −80 ◦ C until analysis. The day after the final drug administration and after urine samples collection, the mice were anesthetized under isoflurane, and blood samples were collected from the abdominal aorta into a syringe. Samples were left at room temperature for 30 min and then centrifuged at 3200 × g for 10 min to separate the serum. The serum obtained was stored at −80 ◦ C. After collecting the blood samples, the bilateral femurs were removed, cleaned from soft tissues, and fixed in 70% ethanol. The experimental protocol was approved by the Animal Research Committee of the Kawasaki Medical School.
Bone histomorphometry was performed on the second cancellous bone of the left femoral distal end at the Ito Bone Histomorphometry Institute (Niigata, Japan). The tissue volume (TV, m2 ), bone volume (BV, m2 ), bone surface (BS, m), singlelabeled surface (sLS), double-labeled surface (dLS), interlabeling thickness (L. Th), and the number of osteoclasts (N.Oc, cells) and osteoblasts (N.Ob, cells) were evaluated. The osteoblast cells were further classified into type II to type IV according to morphological classification criteria (Parfitt, 1984). Type I osteoblasts cannot be detected by any microscopic method. The following parameters were then estimated from the primary parameters mentioned above: bone volume ratio (BV/TV, %), osteoid surface ratio (OS/BS, %), osteoid volume ratio (OV/BV, %), eroded surface ratio (ES/BS, %), trabecular thickness (Tb.Th, m), trabecular separation (Tb.Sp, m), trabecular number (Tb.N, N/mm), number of osteoclasts and osteoblasts per bone surface (N.Oc/BS and N.Ob/BS, N/mm), osteoclast and osteoblast surface area ratios (Oc.S/BS, % and Ob.S/BS, %), bone formation rate (BFR/BS, mm3 /mm2 /year), mineral apposition rate (MAR, m/day), and mineralized surface ratio (MS/BS, %). Standard bone histomorphometrical nomenclature, symbols, and units were used as described in the report by the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee (Dempster et al., 2013). 2.6. Statistical analysis All the data were expressed as means ± standard deviation (SD). The data were analyzed using an analysis of variance (ANOVA), followed by a Tukey–Kramer test. P values < 0.05 were considered statistically significant.
2.2. Video tracking of mice behavior Behavior of mice was observed for 60 min after the first injection using a video tracking system. Mice (n = 6 for each group) treated with saline or METH (5 or 10 mg/kg) were placed individually into a standard open field chamber (32 cm × 30 cm × 11 cm), and the behavior of the mice was monitored digitally. Measurements of the distance traveled were also performed using the Top Scan software (CleverSys Inc., Reston, VA).
2.3. Biochemical analysis Serum concentrations of calcium (Ca) and phosphorus (P), as well as alkaline phosphatase (ALP) activity were determined using an autoanalyzer (Hitachi 7180, Hitachi Co., Ltd., Tokyo, Japan). The serum protein concentration was measured using a refractometer (ATAGO, Japan). Serum levels of Gla-osteocalcin (OC) and tartrateresistant acid phosphatase 5b (TRAP5b) were determined using a mouse Gla-OC competitive enzyme immunoassay (EIA) kit (TAKARA Biomedicals Kyoto, Japan) and a mouse TRAP assay kit (Immunodiagnostic Systems, AZ, USA), respectively. The concentration in C-terminal telopeptides of type I collagen (CTX) in the urine was determined using a commercial EIA kit (Immunodiagnostic Systems, AZ, USA) according to the manufacturer’s instructions, and the levels measured were corrected using creatinine (CRE) concentration. The CRE level was measured by SRL Inc. (Tokyo, Japan).
3. Results We did not observe any difference in the body weight of METH-treated animals compared to the controls. The bone mineral densities (BMD) of the second cancellous bone were also similar in all groups. The levels of Ca and P remained unchanged as well (Table 1). However, serum protein concentrations were decreased in animals treated with 10 mg/kg METH compared to the controls. Moreover, we observed that animals that were given METH showed either an increased locomotor activity or an abnormal/stereotyped behavior. The distance traveled in 1 h by mice that received 5 mg/kg METH increased significantly from that of control, while that by the 10 mg/kg METH group was the same as that of control (Fig. 1). The mice that received 10 mg/kg METH exhibited relatively intense and focused oral stereotypes, such as excessive salivation, licking, and walking backward repeatedly. The dose-dependent behavioral effects of METH did not change during our experimental period, although the duration of altered behavior pattern was not quantified.
M. Tomita et al. / Toxicology 319 (2014) 63–68
Fig. 1. Distance traveled (m) in 1 h by mice immediately after METH injection (METH 5: 5 mg/kg, METH 10: 10 mg/kg), measured continuously by overhead video tracking. Data were analyzed using TopScan video tracking software and expressed as mean ± SD (n = 6 for each treatment group; a p < 0.01 versus control group using ANOVA, followed by Tukey–Kramer multiple comparison test). Table 2 Biochemical markers of bone remodeling in experimental mice. Control ALP (IU/L) OC (ng/mL) TRAP 5b (U/L) CTX (ng/mg CRE)
262.5 29.0 11.0 21.7
± ± ± ±
METH 5 22.1 5.7 2.1 3.2
328.4 43.3 9.9 22.7
± ± ± ±
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Fig. 3. Effects of METH on the number of type II–IV osteoblasts on the bone surface. METH (METH 5; 5 mg/kg, METH 10; 10 mg/kg) or vehicle was administered every other day for 8 weeks. Data are expressed as mean ± SD (n = 6 for each treatment group; a p < 0.01 and b p < 0.05 versus control group using ANOVA, followed by Tukey–Kramer multiple comparison test).
METH 10 32.1a 4.2a 1.4 4.8
289.0 36.4 9.3 20.0
± ± ± ±
27.8 8.5 1.7 5.7
METH (METH 5; 5 mg/kg, METH 10; 10 mg/kg) or vehicle was administered every other day for 8 weeks. Data are expressed as mean ± SD. (n = 6 for each treatment group; a p < 0.01 versus control group using ANOVA, followed by Tukey–Kramer multiple comparison test). ALP: alkaline phosphatase, Oc: Gla-osteocalcin, TRAP 5b: tartrate-resistant acid phosphatase 5b, CTX: C-telopeptide degradation products from type I collagen, CRE: creatinine.
The changes in biochemical markers of bone remodeling were studied (Table 2). The levels of markers of bone formation, such as serum ALP activities and OC concentrations, increased significantly in the 5 mg/kg METH group, but were unchanged in the 10 mg/kg METH group. Interestingly, the markers of bone resorption, such as serum TRAP 5b and urine CTX concentrations, did not change among the three groups. Microscopic images of the second cancellous bone stained using the Villanueva bone staining method and fluorescence microscopic images of the calcein and tetracycline layers are shown in Fig. 2. The distance between the calcein and tetracycline layers (L. Th) is correlated with the mineral apposition rate. Histomorphometric parameters involved in
bone metabolism are shown in Table 3. The osteoblast surface ratio (Ob.S/BS) as well as the number of osteoblasts per bone surface (N.Ob/BS) increased significantly in the 5 mg/kg METH group compared to the control group (Ob.S/BS; p < 0.01, N.Ob/BS; p < 0.01), whereas they were similar to control levels in the 10 mg/kg METH group. The distribution of type II to IV osteoblasts is shown in Fig. 3. Type II and III osteoblasts increased significantly after 5 mg/kg METH exposure (type II, p < 0.05; type III, p < 0.01) but not after 10 mg/kg METH injections. Other parameters of bone formation, such as the osteoid volume ratio (OV/BV) and the osteoid surface ratio (OS/BS), were not affected in the 5 mg/kg METH group, whereas OV/BV decreased significantly after 10 mg/kg METH exposure (p < 0.05). The characteristics of bone resorption, such as the eroded surface ratio (ES/BS), the osteoclast surface ratio (Oc.S/BS), and the number of osteoclasts per bone surface (N.Oc/BS) remained unchanged in both METH groups. We did not observe any effect on osteoclast differentiation. The parameters of mineralization such as the mineral apposition rate (MAR), bone formation rate (BFR/BS), mineralized surface ratio (MS/BS), double-labeled surface ratio (dLS/BS), and interlabeling thickness (L. Th) were unchanged in the 5 mg/kg METH group, whereas they tended to decrease in the 10 mg/kg METH group.
Fig. 2. Representative microscopic images of the second cancellous bone of 10 mg/kg METH-treated mice stained with the Villanueva bone staining method (A) and fluorescence microscopic images of the calcein (CL) and tetracycline (TC) layers in the same focus plane (B) (scale bar: 20 m, original magnification: 40×). The distances between CL and TC layers (L.Th, arrowhead) were used to estimate the mineral apposition rate. Green and red arrows indicate an osteoclast and osteoblast, respectively.
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Table 3 Histomorphometric analysis of the second cancellous bone of the left femoral distal end. Control
METH 5
Bone volume
Bone volume Trabecular thickness Trabecular separation Trabecular number
BV/TV Tb.Th Tb.Sp Tb.N
% m m N/mm
10.2 29.4 265.1 3.4
± ± ± ±
2.0 1.6 36.2 0.5
Formation
Osteoid volume Osteoid surface Osteoblast surface Osteoblast number
OV/BV OS/BS Ob.S/BS N.Ob/BS
% % % N/mm
3.8 29.3 17.2 13.7
± ± ± ±
0.7 4.9 3.1 2.1
Resorption
Eroded surface Osteoclast surface Osteoclast number
ES/BS Oc.S/BS N.Oc/BS
% % N/mm
30.1 ± 3.2 12.4 ± 2.6 4.1 ± 1.3
Mineralization
Mineral apposition rate Double labeled surface Label thickness Mineralizing surface Bone formation rate
MAR dLS/BS L.Th MS/BS BFR/BS
m/day % m % mm3 /mm2 /year
1.3 19.4 3.8 24.9 0.12
± ± ± ± ±
0.1 4.6 0.2 5.5 0.02
METH 10
10.6 29.6 255.3 3.6
± ± ± ±
2.2 2.8 44.3 0.5
11.7 31.6 229.2 3.9
± ± ± ±
1.9 2.9 29.3 0.4
4.5 32.0 24.0 19.2
± ± ± ±
0.4 2.4 2.1a 2.2a
2.6 24.9 16.4 13.9
± ± ± ±
0.6b 3.6 2.9 2.1
28.8 ± 5.6 12.9 ± 1.6 3.9 ± 0.4 1.4 22.7 4.3 27.7 0.15
± ± ± ± ±
0.1 7.1 0.6 7.0 0.04
30.3 ± 4.8 13.1 ± 3.5 4.3 ± 0.9 1.2 13.0 3.5 19.2 0.08
± ± ± ± ±
0.2 4.9 0.6 5.4 0.02
METH (METH 5; 5 mg/kg, METH 10; 10 mg/kg) or vehicle was administered every other day for 8 weeks. Data are expressed as mean ± SD. (n = 6 for each treatment group; a p < 0.01 and b p < 0.05 versus Control group using ANOVA, followed by Tukey–Kramer multiple comparison test).
Our results also indicate that METH exposure did not affect the parameters of bone volume such as bone volume ratio (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th).
4. Discussion METH stimulates the central nervous system, suggesting a potential role of METH in bone homeostasis regulation. We examined the effects of METH on femur trabecular bone metabolism. In our mouse model, the animals displayed a significantly different behavior after 5 and 10 mg/kg METH exposure. After 5 mg/kg METH injections, the animals showed an increased locomotor activity, whereas those exposed to 10 mg/kg showed an abnormal and stereotyped behavior, characterized by excessive salivation, straddling, and walking backward patterns. Exercise is generally considered very beneficial for bones and is recommended for the prevention of osteoporosis (Feskanich et al., 2002; Wallace et al., 2009; Scott et al., 2011). Running is positively associated with BMD, and treadmill training is used to study the effects of exercise on the bones in animal models (Yeh et al., 1993; Iwamoto et al., 1998). Our results indicate that the BMD of the trabecular bone were not affected after 5 and 10 mg/kg METH exposure. However, our data confirmed that METH affected the quality of the bones in a dose-dependent manner, which is consistent with the different behaviors observed. Bone formation markers such as serum ALP activities and OC concentrations significantly increased after 5 mg/kg METH administration, but not after 10 mg/kg injections (Table 2). In contrast, concentrations of bone resorption markers such as serum TRAP 5b and urine CTX were not affected after METH injection. These results suggest that bone remodeling was unbalanced after 5 mg/kg METH, whereas it remained steady after 10 mg/kg METH injections. This difference may be related to the differences in behavior observed after METH injection. However, exhaustive running has been shown to induce a sustained increase in bone resorption (Guillemant et al., 2004). In humans, bone resorption assessed using CTX was increased for 4 days after acute exhaustive running (Scott et al., 2010). Other investigators have also reported that bone resorption, but not bone formation, significantly increased after exhaustive running (Kerschan-Schindl et al., 2009). Our data do not support these observations and further investigation will be needed.
Our biomedical markers analyses’ results were consistent with the bone histomorphometric data. Although a previous study indicated that running induced an increase of the trabecular number in a rat injured after tail suspension, restoring the trabecular bone architecture (Ju et al., 2012), the trabecular number was not affected by METH exposure in our study. Moreover, in contrast to the characteristics of bone resorption and mineralization, some parameters of bone formation, in particular the osteoblast surface (Ob.S/BS) and the number of osteoblasts (N.Ob/BS), significantly increased after 5 mg/kg METH administration. Thus, these results suggest that 5 mg/kg METH exposure did not only affect the locomotor activity. Our data indicate that only osteoblasts increased after METH administration and that the process known as uncoupling was unbalanced in the 5 mg/kg METH group. Interestingly, Turner et al. (2013) reported that adipokine leptin increased the osteoblast number and activity probably through a peripheral pathway, whereas other investigators advocated the opposite (Ducy et al., 2000). We, therefore, further analyzed the number of osteoblasts and classified them according to morphological and maturational classification criteria, as follows: classical cuboidal or columnar with adjacent nuclear clear zone (type II), intermediate without adjacent nuclear clear zone (type III), and lining transitional cytoplasm—extremely thin, undulating line (most mature population, type IV). Injections of 5 mg/kg METH significantly increased the number of type III cells, and slightly increased the number of type II cells. Osteoblast differentiation results from the concerted expression of a number of key transcription factors (Crockett et al., 2011; Yu et al., 2013). The reason why the number of type IV osteoblasts remained unchanged needs to be further investigated. The histomorphometric study showed a significant decrease of OV/BV and some other parameters such as OS/BS, MS/BS, dLS/BS, MAR and BFR/BS also showed a tendency to decrease in the 10 mg/kg METH group. These results suggest a low turnover after METH administration. Although the mechanisms responsible for this turnover remain unknown, vitamin D, which has long been considered a fundamental factor for the prevention and treatment of osteoporosis (Chapuy et al., 1997; Rizzoli et al., 2013), could be involved in the turnover observed. Because METH causes damages in many organs such as the heart, liver, and kidney, the transport and/or metabolism of vitamin D into its active form might be altered. A growing body of evidence supports the existence of a crosstalk between brain and bone. Neural signals, primarily originating
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in the hypothalamus, play an important role in regulating bone growth and resorption. METH has been shown to excite the hypothalamic neurons (Tomita et al., 2013), which exhibit high expression of the cocaine and amphetamine-regulated transcript (CART) (Vrang, 2006). CART upregulation decreases the expression of receptor activator of nuclear factor B ligand (RANKL), a transmembrane protein necessary for osteoclast differentiation, resulting in inhibition of bone resorption (Elefteriou et al., 2005). METH administration has also been shown to decrease neuropeptide-Y (NPY) levels, which suppresses osteoblast activity (Kobeissy et al., 2008). Additionally, METH activates central and peripheral adrenergic postsynaptic receptors by releasing catecholamines from the sympathetic nerve terminals (Schep et al., 2010). Takeda et al. (2002) provided evidence of sympathetic regulation of bone growth by reporting a massive decrease in bone mass in mice treated with a beta-adrenergic agonist and increased bone formation following treatment with a beta-adrenergic antagonist. Clinically, beta-blockers were shown to lower the risk of fractures in older adults in a meta-analysis of observational studies (Wiens et al., 2006). Furthermore, effectiveness of beta-blockers in the prevention and treatment of osteoporosis is well-recognized (Song et al., 2012). Taken together, past and current findings demonstrate that the effects of METH on bone remodeling process are extremely complex, with the drug possibly interfering with bone remodeling by affecting neural signals, resulting in increased risk of osteoporosis. Indeed, adverse effects of METH on bone tissue of drug abusers have been reported (Katsuragawa, 1999; Kim et al., 2009). Our results suggest two putative mechanisms by which METH administration may lead to osteoporosis: unbalanced bone remodeling and decreased bone turnover. In conclusion, whereas bone resorption and mineralization remained unchanged after 5 mg/kg METH administration, bone formation biomarkers including serum ALP activity, OC concentration and the number of osteoblasts in the trabecular bone were significantly increased, leading to an unbalanced bone remodeling process. In contrast, 10 mg/kg METH exposure suppressed bone turnover, suggesting the potential development of low turnover osteoporosis. These results suggest that METH induces adverse effects on bone metabolism in a dose-dependent manner. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. References Chapuy, M.-C., Preziosi, P., Maamer, M., Arnaud, S., Galan, P., Hercberg, S., Meunier, P.J., 1997. Prevalence of vitamin D insufficiency in an adult normal population. Osteoporos. Int. 7, 439–443. Crockett, J.C., Rogers, M.J., Coxon, F.P., Hocking, L.J., Helfrich, M.H., 2011. Bone remodelling at a glance. J. Cell Sci. 124 (Pt 7), 991–998. Curtis, E.K., 2006. Meth mouth: a review of methamphetamine abuse and its oral manifestations. Gen. Dent. 54, 125–129. Darcey, J., Horner, K., Walsh, T., Southern, H., Marjanovic, E.J., Devlin, H., 2013. Tooth loss and osteoporosis: to assess the association between osteoporosis status and tooth number. Br. Dent. J. 214, E10. Dempster, D.W., Compston, J.E., Drezner, M.K., Glorieux, F.H., Kanis, J.A., Malluche, H., Meunier, P.J., Ott, S.M., Recker, R.R., Parfitt, A.M., 2013. Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res. 28, 1–16. Ducy, P., Amling, M., Takeda, S., Priemel, M., Schilling, A.F., Beil, F.T., Shen, J., Vinson, C., Rueger, J.M., Karsenty, G., 2000. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 100, 197–207.
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Does methamphetamine affect bone metabolism? Masafumi Tomita a,∗ , Hironobu Katsuyama b , Yoko Watanabe c , Toshiko Okuyama a , Shigeko Fushimi b , Takaki Ishikawa e , Masayuki Nata f , Osamu Miyamoto d a
Department of Medical Toxicology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Japan Department of Public Health, Kawasaki Medical School, 577 Matsushima, Kurashiki, Japan Department of Natural Sciences, Kawasaki Medical School, 577 Matsushima, Kurashiki, Japan d Department of Physiology II, Kawasaki Medical School, 577 Matsushima, Kurashiki, Japan e Division of Legal Medicine, Faculty of Medicine, Tottori University, 86 Nishimachi, Yonago, Japan f Department of Forensic Medicine and Science, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Japan b c
a r t i c l e
i n f o
Article history: Received 18 November 2013 Received in revised form 26 December 2013 Accepted 26 January 2014 Available online 26 February 2014 Keywords: Methamphetamine Bone metabolism Osteoporosis Mice
a b s t r a c t There is a close relationship between the central nervous system activity and bone metabolism. Therefore, methamphetamine (METH), which stimulates the central nervous system, is expected to affect bone turnover. The aim of this study was to investigate the role of METH in bone metabolism. Mice were divided into 3 groups, the control group receiving saline injections, and the 5 and 10 mg/kg METH groups (n = 6 in each group). All groups received an injection of saline or METH every other day for 8 weeks. Bone mineral density (BMD) was assessed by X-ray computed tomography. We examined biochemical markers and histomorphometric changes in the second cancellous bone of the left femoral distal end. The animals that were administered 5 mg/kg METH showed an increased locomotor activity, whereas those receiving 10 mg/kg displayed an abnormal and stereotyped behavior. Serum calcium and phosphorus concentrations were normal compared to the controls, whereas the serum protein concentration was lower in the METH groups. BMD was unchanged in all groups. Bone formation markers such as alkaline phosphatase and osteocalcin significantly increased in the 5 mg/kg METH group, but not in the 10 mg/kg METH group. In contrast, bone resorption markers such as C-terminal telopeptides of type I collagen and tartrate-resistant acid phosphatase 5b did not change in any of the METH groups. Histomorphometric analyses were consistent with the biochemical markers data. A significant increase in osteoblasts, especially in type III osteoblasts, was observed in the 5 mg/kg METH group, whereas other parameters of bone resorption and mineralization remained unchanged. These results indicate that bone remodeling in this group was unbalanced. In contrast, in the 10 mg/kg METH group, some parameters of bone formation were significantly or slightly decreased, suggesting a low turnover metabolism. Taken together, our results suggest that METH had distinct dose-dependent effects on bone turnover and that METH might induce adverse effects, leading to osteoporosis. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Methamphetamine (METH) possesses a high potential for abuse and addiction, and METH use has become a serious social problem worldwide (Nordahl et al., 2003; Marshall and Werb, 2010). In Japan, more than 10,000 persons annually have been arrested for METH abuse for the last several decades (Wada, 2011). METH abuse often causes an increase in crime, traffic and non-traffic accidents, and physical and psychological injuries. METH not only
∗ Corresponding author. Tel.: +81 86462 1111. E-mail addresses: [email protected], [email protected] (M. Tomita). http://dx.doi.org/10.1016/j.tox.2014.01.014 0300-483X/© 2014 Elsevier Ireland Ltd. All rights reserved.
affects the central nervous system but also causes degeneration in many organs (Wijetunga et al., 2003). An increasing number of clinical and autopsy studies reported that METH use is associated with angina, tachycardia, hypertension, myocarditis, dilated cardiomyopathy, arrhythmia, and sudden death (Pilgrim et al., 2009; Schep et al., 2010; Volkow et al., 2010). Routine dental examinations performed on some convicts showed that METH abusers suffer from tooth decays or periodontal disease more often than other prisoners and also show oral manifestations of rampant caries (“meth mouth”) (Curtis, 2006; Hamamoto and Rhodus, 2009). Although there are a limited number of human studies regarding the potential relationship between molar tooth number and osteoporotic status, it has been suggested that dental condition might represent trabecular bone structure (Darcey et al., 2013). Bone is a dynamic
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organ, continuously remodeling itself. Bone resorption and bone formation are regulated by osteoclasts and osteoblasts, respectively (Hill, 1998; Eimar et al., 2013). It has recently been shown that neurons and neuropeptides in the central nervous system play an important role in bone remodeling (Takeda, 2009; Wang et al., 2010; Crockett et al., 2011). Osteoporosis is a neuroskeletal disease and, as mentioned above, bone remodeling is under the control of the nervous system. Therefore, we hypothesized that METH, which stimulates the central nervous system, may alter bone metabolism. Interestingly, Katsuragawa (1999) reported that the cancellous speed of sound, an indicator of bone strength, was significantly lower in METH abusers than in the control group. Other investigators, using dual energy Xray absorptiometry, observed that the bone mineral density (BMD) in the lumbar spine was lower in METH abusers (n = 46) than in the controls (n = 188) (Kim et al., 2009). However, the effects of METH on bone metabolism remain unknown. The aim of this study was to clarify the effects of METH on bone metabolism, especially on the trabecular bone metabolism in the femurs, in mice exposed to METH every other day for 8 weeks. We performed bone histomorphometry and quantified biochemical markers of bone turnover. Our results indicate that METH might affect bone metabolism in a dose-dependent manner, potentially leading to osteoporosis.
Table 1 Body weight, bone mineral density (BMD), and serum protein, calcium, and phosphorus concentrations. Control Body weight Pre (g) Post (g) BMD (mg/cm2 ) Protein (g/dL) Calcium (mg/dL) Phosphorus (mg/dL)
21.7 28.9 228.8 5.1 8.6 7.8
± ± ± ± ± ±
METH 5 0.8 1.5 19.8 0.2 0.2 1.2
21.9 27.7 223.1 4.9 8.6 6.8
± ± ± ± ± ±
METH 10 0.7 0.7 25.4 0.2 0.3 0.7
22.1 28.6 231.9 4.7 8.7 8.2
± ± ± ± ± ±
0.7 1.2 43.3 0.2a 0.3 1.0
METH (METH 5; 5 mg/kg, METH 10; 10 mg/kg) or vehicle was administered every other day for 8 weeks. Data are expressed as mean ± SD. (n = 6 for each treatment group; a p < 0.01 versus control group using ANOVA, followed by Tukey–Kramer multiple comparison test).
2.4. Trabecular bone mineral density (BMD) The fixed right bones were analyzed using a X-ray computed tomography system for small experimental animals (Model LaTheta, LCT-200; Aloka, Osaka, Japan). The bone was placed horizontally inside a tube and scanned using a 96-m voxel. The scan line was adjusted using the scout view. The trabecular BMD was quantified and calculated in the second cancellous bone proximal to the growth plate of the femoral distal end. The data were quantified using an automated image analysis software supplied with the device. 2.5. Bone histomorphometry
2. Materials and methods 2.1. Animal experiments Eight-week-old male C57BL/6J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). The animals were given a normal rodent chow and tap water and acclimated to the conditions for 1 week. They were then divided into 3 groups: control (saline), and 5 and 10 mg/kg METH groups (n = 6 in each group). METH or saline were administered intraperitoneally every other day for 8 weeks. To assess bone mineralization, mice were double-labeled with subcutaneous injections of 20 mg/kg tetracycline hydrochloride and 10 mg/kg calcein (Sigma–Aldrich Corp., St. Louis, MO), 5 and 2 days before sacrifice, respectively. The body weight of each animal was measured every other day until the final day of administration. Urine samples were collected over a 16-h period, starting immediately after last METH administration and were stored at −80 ◦ C until analysis. The day after the final drug administration and after urine samples collection, the mice were anesthetized under isoflurane, and blood samples were collected from the abdominal aorta into a syringe. Samples were left at room temperature for 30 min and then centrifuged at 3200 × g for 10 min to separate the serum. The serum obtained was stored at −80 ◦ C. After collecting the blood samples, the bilateral femurs were removed, cleaned from soft tissues, and fixed in 70% ethanol. The experimental protocol was approved by the Animal Research Committee of the Kawasaki Medical School.
Bone histomorphometry was performed on the second cancellous bone of the left femoral distal end at the Ito Bone Histomorphometry Institute (Niigata, Japan). The tissue volume (TV, m2 ), bone volume (BV, m2 ), bone surface (BS, m), singlelabeled surface (sLS), double-labeled surface (dLS), interlabeling thickness (L. Th), and the number of osteoclasts (N.Oc, cells) and osteoblasts (N.Ob, cells) were evaluated. The osteoblast cells were further classified into type II to type IV according to morphological classification criteria (Parfitt, 1984). Type I osteoblasts cannot be detected by any microscopic method. The following parameters were then estimated from the primary parameters mentioned above: bone volume ratio (BV/TV, %), osteoid surface ratio (OS/BS, %), osteoid volume ratio (OV/BV, %), eroded surface ratio (ES/BS, %), trabecular thickness (Tb.Th, m), trabecular separation (Tb.Sp, m), trabecular number (Tb.N, N/mm), number of osteoclasts and osteoblasts per bone surface (N.Oc/BS and N.Ob/BS, N/mm), osteoclast and osteoblast surface area ratios (Oc.S/BS, % and Ob.S/BS, %), bone formation rate (BFR/BS, mm3 /mm2 /year), mineral apposition rate (MAR, m/day), and mineralized surface ratio (MS/BS, %). Standard bone histomorphometrical nomenclature, symbols, and units were used as described in the report by the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee (Dempster et al., 2013). 2.6. Statistical analysis All the data were expressed as means ± standard deviation (SD). The data were analyzed using an analysis of variance (ANOVA), followed by a Tukey–Kramer test. P values < 0.05 were considered statistically significant.
2.2. Video tracking of mice behavior Behavior of mice was observed for 60 min after the first injection using a video tracking system. Mice (n = 6 for each group) treated with saline or METH (5 or 10 mg/kg) were placed individually into a standard open field chamber (32 cm × 30 cm × 11 cm), and the behavior of the mice was monitored digitally. Measurements of the distance traveled were also performed using the Top Scan software (CleverSys Inc., Reston, VA).
2.3. Biochemical analysis Serum concentrations of calcium (Ca) and phosphorus (P), as well as alkaline phosphatase (ALP) activity were determined using an autoanalyzer (Hitachi 7180, Hitachi Co., Ltd., Tokyo, Japan). The serum protein concentration was measured using a refractometer (ATAGO, Japan). Serum levels of Gla-osteocalcin (OC) and tartrateresistant acid phosphatase 5b (TRAP5b) were determined using a mouse Gla-OC competitive enzyme immunoassay (EIA) kit (TAKARA Biomedicals Kyoto, Japan) and a mouse TRAP assay kit (Immunodiagnostic Systems, AZ, USA), respectively. The concentration in C-terminal telopeptides of type I collagen (CTX) in the urine was determined using a commercial EIA kit (Immunodiagnostic Systems, AZ, USA) according to the manufacturer’s instructions, and the levels measured were corrected using creatinine (CRE) concentration. The CRE level was measured by SRL Inc. (Tokyo, Japan).
3. Results We did not observe any difference in the body weight of METH-treated animals compared to the controls. The bone mineral densities (BMD) of the second cancellous bone were also similar in all groups. The levels of Ca and P remained unchanged as well (Table 1). However, serum protein concentrations were decreased in animals treated with 10 mg/kg METH compared to the controls. Moreover, we observed that animals that were given METH showed either an increased locomotor activity or an abnormal/stereotyped behavior. The distance traveled in 1 h by mice that received 5 mg/kg METH increased significantly from that of control, while that by the 10 mg/kg METH group was the same as that of control (Fig. 1). The mice that received 10 mg/kg METH exhibited relatively intense and focused oral stereotypes, such as excessive salivation, licking, and walking backward repeatedly. The dose-dependent behavioral effects of METH did not change during our experimental period, although the duration of altered behavior pattern was not quantified.
M. Tomita et al. / Toxicology 319 (2014) 63–68
Fig. 1. Distance traveled (m) in 1 h by mice immediately after METH injection (METH 5: 5 mg/kg, METH 10: 10 mg/kg), measured continuously by overhead video tracking. Data were analyzed using TopScan video tracking software and expressed as mean ± SD (n = 6 for each treatment group; a p < 0.01 versus control group using ANOVA, followed by Tukey–Kramer multiple comparison test). Table 2 Biochemical markers of bone remodeling in experimental mice. Control ALP (IU/L) OC (ng/mL) TRAP 5b (U/L) CTX (ng/mg CRE)
262.5 29.0 11.0 21.7
± ± ± ±
METH 5 22.1 5.7 2.1 3.2
328.4 43.3 9.9 22.7
± ± ± ±
65
Fig. 3. Effects of METH on the number of type II–IV osteoblasts on the bone surface. METH (METH 5; 5 mg/kg, METH 10; 10 mg/kg) or vehicle was administered every other day for 8 weeks. Data are expressed as mean ± SD (n = 6 for each treatment group; a p < 0.01 and b p < 0.05 versus control group using ANOVA, followed by Tukey–Kramer multiple comparison test).
METH 10 32.1a 4.2a 1.4 4.8
289.0 36.4 9.3 20.0
± ± ± ±
27.8 8.5 1.7 5.7
METH (METH 5; 5 mg/kg, METH 10; 10 mg/kg) or vehicle was administered every other day for 8 weeks. Data are expressed as mean ± SD. (n = 6 for each treatment group; a p < 0.01 versus control group using ANOVA, followed by Tukey–Kramer multiple comparison test). ALP: alkaline phosphatase, Oc: Gla-osteocalcin, TRAP 5b: tartrate-resistant acid phosphatase 5b, CTX: C-telopeptide degradation products from type I collagen, CRE: creatinine.
The changes in biochemical markers of bone remodeling were studied (Table 2). The levels of markers of bone formation, such as serum ALP activities and OC concentrations, increased significantly in the 5 mg/kg METH group, but were unchanged in the 10 mg/kg METH group. Interestingly, the markers of bone resorption, such as serum TRAP 5b and urine CTX concentrations, did not change among the three groups. Microscopic images of the second cancellous bone stained using the Villanueva bone staining method and fluorescence microscopic images of the calcein and tetracycline layers are shown in Fig. 2. The distance between the calcein and tetracycline layers (L. Th) is correlated with the mineral apposition rate. Histomorphometric parameters involved in
bone metabolism are shown in Table 3. The osteoblast surface ratio (Ob.S/BS) as well as the number of osteoblasts per bone surface (N.Ob/BS) increased significantly in the 5 mg/kg METH group compared to the control group (Ob.S/BS; p < 0.01, N.Ob/BS; p < 0.01), whereas they were similar to control levels in the 10 mg/kg METH group. The distribution of type II to IV osteoblasts is shown in Fig. 3. Type II and III osteoblasts increased significantly after 5 mg/kg METH exposure (type II, p < 0.05; type III, p < 0.01) but not after 10 mg/kg METH injections. Other parameters of bone formation, such as the osteoid volume ratio (OV/BV) and the osteoid surface ratio (OS/BS), were not affected in the 5 mg/kg METH group, whereas OV/BV decreased significantly after 10 mg/kg METH exposure (p < 0.05). The characteristics of bone resorption, such as the eroded surface ratio (ES/BS), the osteoclast surface ratio (Oc.S/BS), and the number of osteoclasts per bone surface (N.Oc/BS) remained unchanged in both METH groups. We did not observe any effect on osteoclast differentiation. The parameters of mineralization such as the mineral apposition rate (MAR), bone formation rate (BFR/BS), mineralized surface ratio (MS/BS), double-labeled surface ratio (dLS/BS), and interlabeling thickness (L. Th) were unchanged in the 5 mg/kg METH group, whereas they tended to decrease in the 10 mg/kg METH group.
Fig. 2. Representative microscopic images of the second cancellous bone of 10 mg/kg METH-treated mice stained with the Villanueva bone staining method (A) and fluorescence microscopic images of the calcein (CL) and tetracycline (TC) layers in the same focus plane (B) (scale bar: 20 m, original magnification: 40×). The distances between CL and TC layers (L.Th, arrowhead) were used to estimate the mineral apposition rate. Green and red arrows indicate an osteoclast and osteoblast, respectively.
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Table 3 Histomorphometric analysis of the second cancellous bone of the left femoral distal end. Control
METH 5
Bone volume
Bone volume Trabecular thickness Trabecular separation Trabecular number
BV/TV Tb.Th Tb.Sp Tb.N
% m m N/mm
10.2 29.4 265.1 3.4
± ± ± ±
2.0 1.6 36.2 0.5
Formation
Osteoid volume Osteoid surface Osteoblast surface Osteoblast number
OV/BV OS/BS Ob.S/BS N.Ob/BS
% % % N/mm
3.8 29.3 17.2 13.7
± ± ± ±
0.7 4.9 3.1 2.1
Resorption
Eroded surface Osteoclast surface Osteoclast number
ES/BS Oc.S/BS N.Oc/BS
% % N/mm
30.1 ± 3.2 12.4 ± 2.6 4.1 ± 1.3
Mineralization
Mineral apposition rate Double labeled surface Label thickness Mineralizing surface Bone formation rate
MAR dLS/BS L.Th MS/BS BFR/BS
m/day % m % mm3 /mm2 /year
1.3 19.4 3.8 24.9 0.12
± ± ± ± ±
0.1 4.6 0.2 5.5 0.02
METH 10
10.6 29.6 255.3 3.6
± ± ± ±
2.2 2.8 44.3 0.5
11.7 31.6 229.2 3.9
± ± ± ±
1.9 2.9 29.3 0.4
4.5 32.0 24.0 19.2
± ± ± ±
0.4 2.4 2.1a 2.2a
2.6 24.9 16.4 13.9
± ± ± ±
0.6b 3.6 2.9 2.1
28.8 ± 5.6 12.9 ± 1.6 3.9 ± 0.4 1.4 22.7 4.3 27.7 0.15
± ± ± ± ±
0.1 7.1 0.6 7.0 0.04
30.3 ± 4.8 13.1 ± 3.5 4.3 ± 0.9 1.2 13.0 3.5 19.2 0.08
± ± ± ± ±
0.2 4.9 0.6 5.4 0.02
METH (METH 5; 5 mg/kg, METH 10; 10 mg/kg) or vehicle was administered every other day for 8 weeks. Data are expressed as mean ± SD. (n = 6 for each treatment group; a p < 0.01 and b p < 0.05 versus Control group using ANOVA, followed by Tukey–Kramer multiple comparison test).
Our results also indicate that METH exposure did not affect the parameters of bone volume such as bone volume ratio (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th).
4. Discussion METH stimulates the central nervous system, suggesting a potential role of METH in bone homeostasis regulation. We examined the effects of METH on femur trabecular bone metabolism. In our mouse model, the animals displayed a significantly different behavior after 5 and 10 mg/kg METH exposure. After 5 mg/kg METH injections, the animals showed an increased locomotor activity, whereas those exposed to 10 mg/kg showed an abnormal and stereotyped behavior, characterized by excessive salivation, straddling, and walking backward patterns. Exercise is generally considered very beneficial for bones and is recommended for the prevention of osteoporosis (Feskanich et al., 2002; Wallace et al., 2009; Scott et al., 2011). Running is positively associated with BMD, and treadmill training is used to study the effects of exercise on the bones in animal models (Yeh et al., 1993; Iwamoto et al., 1998). Our results indicate that the BMD of the trabecular bone were not affected after 5 and 10 mg/kg METH exposure. However, our data confirmed that METH affected the quality of the bones in a dose-dependent manner, which is consistent with the different behaviors observed. Bone formation markers such as serum ALP activities and OC concentrations significantly increased after 5 mg/kg METH administration, but not after 10 mg/kg injections (Table 2). In contrast, concentrations of bone resorption markers such as serum TRAP 5b and urine CTX were not affected after METH injection. These results suggest that bone remodeling was unbalanced after 5 mg/kg METH, whereas it remained steady after 10 mg/kg METH injections. This difference may be related to the differences in behavior observed after METH injection. However, exhaustive running has been shown to induce a sustained increase in bone resorption (Guillemant et al., 2004). In humans, bone resorption assessed using CTX was increased for 4 days after acute exhaustive running (Scott et al., 2010). Other investigators have also reported that bone resorption, but not bone formation, significantly increased after exhaustive running (Kerschan-Schindl et al., 2009). Our data do not support these observations and further investigation will be needed.
Our biomedical markers analyses’ results were consistent with the bone histomorphometric data. Although a previous study indicated that running induced an increase of the trabecular number in a rat injured after tail suspension, restoring the trabecular bone architecture (Ju et al., 2012), the trabecular number was not affected by METH exposure in our study. Moreover, in contrast to the characteristics of bone resorption and mineralization, some parameters of bone formation, in particular the osteoblast surface (Ob.S/BS) and the number of osteoblasts (N.Ob/BS), significantly increased after 5 mg/kg METH administration. Thus, these results suggest that 5 mg/kg METH exposure did not only affect the locomotor activity. Our data indicate that only osteoblasts increased after METH administration and that the process known as uncoupling was unbalanced in the 5 mg/kg METH group. Interestingly, Turner et al. (2013) reported that adipokine leptin increased the osteoblast number and activity probably through a peripheral pathway, whereas other investigators advocated the opposite (Ducy et al., 2000). We, therefore, further analyzed the number of osteoblasts and classified them according to morphological and maturational classification criteria, as follows: classical cuboidal or columnar with adjacent nuclear clear zone (type II), intermediate without adjacent nuclear clear zone (type III), and lining transitional cytoplasm—extremely thin, undulating line (most mature population, type IV). Injections of 5 mg/kg METH significantly increased the number of type III cells, and slightly increased the number of type II cells. Osteoblast differentiation results from the concerted expression of a number of key transcription factors (Crockett et al., 2011; Yu et al., 2013). The reason why the number of type IV osteoblasts remained unchanged needs to be further investigated. The histomorphometric study showed a significant decrease of OV/BV and some other parameters such as OS/BS, MS/BS, dLS/BS, MAR and BFR/BS also showed a tendency to decrease in the 10 mg/kg METH group. These results suggest a low turnover after METH administration. Although the mechanisms responsible for this turnover remain unknown, vitamin D, which has long been considered a fundamental factor for the prevention and treatment of osteoporosis (Chapuy et al., 1997; Rizzoli et al., 2013), could be involved in the turnover observed. Because METH causes damages in many organs such as the heart, liver, and kidney, the transport and/or metabolism of vitamin D into its active form might be altered. A growing body of evidence supports the existence of a crosstalk between brain and bone. Neural signals, primarily originating
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in the hypothalamus, play an important role in regulating bone growth and resorption. METH has been shown to excite the hypothalamic neurons (Tomita et al., 2013), which exhibit high expression of the cocaine and amphetamine-regulated transcript (CART) (Vrang, 2006). CART upregulation decreases the expression of receptor activator of nuclear factor B ligand (RANKL), a transmembrane protein necessary for osteoclast differentiation, resulting in inhibition of bone resorption (Elefteriou et al., 2005). METH administration has also been shown to decrease neuropeptide-Y (NPY) levels, which suppresses osteoblast activity (Kobeissy et al., 2008). Additionally, METH activates central and peripheral adrenergic postsynaptic receptors by releasing catecholamines from the sympathetic nerve terminals (Schep et al., 2010). Takeda et al. (2002) provided evidence of sympathetic regulation of bone growth by reporting a massive decrease in bone mass in mice treated with a beta-adrenergic agonist and increased bone formation following treatment with a beta-adrenergic antagonist. Clinically, beta-blockers were shown to lower the risk of fractures in older adults in a meta-analysis of observational studies (Wiens et al., 2006). Furthermore, effectiveness of beta-blockers in the prevention and treatment of osteoporosis is well-recognized (Song et al., 2012). Taken together, past and current findings demonstrate that the effects of METH on bone remodeling process are extremely complex, with the drug possibly interfering with bone remodeling by affecting neural signals, resulting in increased risk of osteoporosis. Indeed, adverse effects of METH on bone tissue of drug abusers have been reported (Katsuragawa, 1999; Kim et al., 2009). Our results suggest two putative mechanisms by which METH administration may lead to osteoporosis: unbalanced bone remodeling and decreased bone turnover. In conclusion, whereas bone resorption and mineralization remained unchanged after 5 mg/kg METH administration, bone formation biomarkers including serum ALP activity, OC concentration and the number of osteoblasts in the trabecular bone were significantly increased, leading to an unbalanced bone remodeling process. In contrast, 10 mg/kg METH exposure suppressed bone turnover, suggesting the potential development of low turnover osteoporosis. These results suggest that METH induces adverse effects on bone metabolism in a dose-dependent manner. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. References Chapuy, M.-C., Preziosi, P., Maamer, M., Arnaud, S., Galan, P., Hercberg, S., Meunier, P.J., 1997. Prevalence of vitamin D insufficiency in an adult normal population. Osteoporos. Int. 7, 439–443. Crockett, J.C., Rogers, M.J., Coxon, F.P., Hocking, L.J., Helfrich, M.H., 2011. Bone remodelling at a glance. J. Cell Sci. 124 (Pt 7), 991–998. Curtis, E.K., 2006. Meth mouth: a review of methamphetamine abuse and its oral manifestations. Gen. Dent. 54, 125–129. Darcey, J., Horner, K., Walsh, T., Southern, H., Marjanovic, E.J., Devlin, H., 2013. Tooth loss and osteoporosis: to assess the association between osteoporosis status and tooth number. Br. Dent. J. 214, E10. Dempster, D.W., Compston, J.E., Drezner, M.K., Glorieux, F.H., Kanis, J.A., Malluche, H., Meunier, P.J., Ott, S.M., Recker, R.R., Parfitt, A.M., 2013. Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res. 28, 1–16. Ducy, P., Amling, M., Takeda, S., Priemel, M., Schilling, A.F., Beil, F.T., Shen, J., Vinson, C., Rueger, J.M., Karsenty, G., 2000. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 100, 197–207.
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