Methotrexate Chemotherapy Causes Growth Impairments, Vitamin D Deficiency, Bone Loss, and Altered Intestinal Metabolism-Effects of Calcitriol Supplementation.

Vitamin D deficiency or insufficiency is prevalent in childhood cancer patients and survivors after chemotherapy; further studies are needed to investigate the underlying aetiology and effectiveness of vitamin D supplementation in preventing chemotherapy-induced bone loss. This study used a rat model of treatment with antimetabolite methotrexate to investigate whether methotrexate chemotherapy causes vitamin D deficiency and if vitamin D supplementation attenuates the resultant bone loss. Methotrexate treatment (five daily injections) decreased serum vitamin D levels (from 52 to <30 ng/mL), reduced body and bone lengthening and tibial trabecular bone volume, and altered intestinal vitamin D metabolism, which was associated with intestinal mucosal damage known to cause malabsorption of nutrients, including dietary vitamin D and calcium. During the early stage after chemotherapy, mRNA expression increased for vitamin D activation enzyme CYP27B1 and for calcium-binding protein TRPV6 in the intestine. During the intestinal healing stage, expression of vitamin D catabolism enzyme CYP24 increased, and that of TRPV6 was normalised. Furthermore, subcutaneous calcitriol supplementation diminished methotrexate-induced bone loss due to its effect suppressing methotrexate-induced increased bone resorption. Thus, in young rats, methotrexate chemotherapy causes vitamin D deficiency, growth impairments, bone loss, and altered intestinal vitamin D metabolism, which are associated with intestinal damage, and vitamin D supplementation inhibits methotrexate-induced bone loss.

Individual or combination treatments with lapatinib and paclitaxel cause potential bone loss and bone marrow adiposity in rats.

Cancer treatments with cytotoxic drugs have been shown to cause bone loss. However, effects on bone are less clear for ErbB-targeting tyrosine kinase inhibitors or their combination use with cytotoxic drugs. This study examined the effects of individual or combination treatments with breast cancer drugs lapatinib (a dual ErbB1/ErbB2 inhibitor) and paclitaxel (a microtubule-stabilizing cytotoxic agent) on bone and bone marrow of rats. Wistar rats received lapatinib (240 mg/kg) daily, paclitaxel (12 mg/kg) weekly, or their combination for 4 weeks, and effects on bone/bone marrow were examined at the end of week 4. Microcomputed tomographical structural analyses showed a reduction in trabecular bone volume in tibia following the lapatinib, paclitaxel or their combination treatments ( P < 0.05). Histomorphometry analyses revealed marked increases in bone marrow adipocyte contents in all treatment groups. Reverse transcription polymerase chain reaction gene expression studies with bone samples and cell culture studies with isolated bone marrow stromal cells showed that the all treatment groups displayed significantly reduced levels of osterix expression and osteogenic differentiation potential but increased expression levels of adipogenesis transcription factor peroxisome proliferator-activated receptor γ. In addition, these treatments suppressed the expression of Wnt10b and/or increased expression of Wnt antagonists (secreted frizzled-related protein 1, Dickkopf-related protein 1 and/or sclerostin). Furthermore, all treatment groups showed increased numbers of bone-resorbing osteoclasts on trabecular bone surfaces, although only the lapatinib group displayed increased levels of osteoclastogenic signal (receptor activator of nuclear factor κΒ ligand/osteoclastogenesis inhibitor osteoprotegrin expression ratio) in the bones. Thus, inhibiting ErbB1 and ErbB2 by lapatinib or blocking cell division by paclitaxel or their combination causes significant trabecular bone loss and bone marrow adiposity involving a switch in osteogenesis/adipogenesis potential, altered expression of some major molecules of the Wnt/ß-catenin signalling pathway, and increased recruitment of bone-resorbing osteoclasts.

Effects of Resveratrol Supplementation on Methotrexate Chemotherapy-Induced Bone Loss.

Intensive cancer chemotherapy is known to cause bone defects, which currently lack treatments. This study investigated the effects of polyphenol resveratrol (RES) in preventing bone defects in rats caused by methotrexate (MTX), a commonly used antimetabolite in childhood oncology. Young rats received five daily MTX injections at 0.75 mg/kg/day. RES was orally gavaged daily for seven days prior to, and during, five-day MTX administration. MTX reduced growth plate thickness, primary spongiosa height, trabecular bone volume, increased marrow adipocyte density, and increased mRNA expression of the osteogenic, adipogenic, and osteoclastogenic factors in the tibial bone. RES at 10 mg/kg was found not to affect bone health in normal rats, but to aggravate the bone damage in MTX-treated rats. However, RES supplementation at 1 mg/kg preserved the growth plate, primary spongiosa, bone volume, and lowered the adipocyte density. It maintained expression of genes involved in osteogenesis and decreased expression of adipogenic and osteoclastogenic factors. RES suppressed osteoclast formation ex vivo of bone marrow cells from the treated rats. These data suggest that MTX can enhance osteoclast and adipocyte formation and cause bone loss, and that RES supplementation at 1 mg/kg may potentially prevent these bone defects.

Effects of resveratrol supplementation on bone growth in young rats and microarchitecture and remodeling in ageing rats.

Osteoporosis is a highly prevalent skeletal disorder in the elderly that causes serious bone fractures. Peak bone mass achieved at adolescence has been shown to predict bone mass and osteoporosis related risk fracture later in life. Resveratrol, a natural polyphenol compound, may have the potential to promote bone formation and reduce bone resorption. However, it is unclear whether it can aid bone growth and bone mass accumulation during rapid growth and modulate bone metabolism during ageing. Using rat models, the current study investigated the potential effects of resveratrol supplementation during the rapid postnatal growth period and in late adulthood (early ageing) on bone microarchitecture and metabolism. In the growth trial, 4-week-old male hooded Wistar rats on a normal chow diet were given resveratrol (2.5 mg/kg/day) or vehicle control for 5 weeks. In the ageing trial, 6-month-old male hooded Wistar rats were treated with resveratrol (20 mg/kg/day) or vehicle for 3 months. Treatment effects in the tibia were examined by µ-computer tomography (µ-CT) analysis, bone histomorphometric measurements and reverse transcription-polymerase chain reaction (RT-PCR) gene expression analysis. Resveratrol treatment did not affect trabecular bone volume and bone remodeling indices in the youth animal model. Resveratrol supplementation in the early ageing rats tended to decrease trabecular bone volume, Sirt1 gene expression and increased expression of adipogenesis-related genes in bone, all of which were statistically insignificant. However, it decreased osteocalcin expression (p = 0.03). Furthermore, serum levels of bone resorption marker C-terminal telopeptides type I collagen (CTX-1) were significantly elevated in the resveratrol supplementation group (p = 0.02) with no changes observed in serum levels of bone formation marker alkaline phosphatase (ALP). These results in rat models suggest that resveratrol supplementation does not significantly affect bone volume during the rapid growth phase but may potentially have negative effects on male skeleton during early ageing.

Effects of Maternal Hypoxia during Pregnancy on Bone Development in Offspring: A Guinea Pig Model.

Low birth weight is associated with reduced bone mass and density in adult life. However, effects of maternal hypoxia (MH) on offspring bone development are not known. Objective. The current study investigated the effects of fetal growth restriction induced by MH during the last half of gestation on bone structure and volume in the offspring of the fetus near term and the pup in adolescence. Methods. During 35-62-day gestation (term, 69d), guinea pigs were housed in room air (21% O2; control) or 12% O2 (MH). Offspring femur and tibia were collected at 62d gestation and 120d after birth. Results. MH decreased fetal birth weight but did not affect osteogenic potential pools in the fetal bone marrow. Histological analysis showed no effects of MH on tibial growth plate thickness in either fetal or postnatal offspring, although there was increased VEGF mRNA expression in the growth plate of postnatal offspring. MH did not change primary spongiosa height but lowered collagen-1 mRNA expression in postnatal offspring. There was increased mRNA expression of adipogenesis-related gene (FABP4) in bone from the MH postnatal offspring. Conclusion. MH during late gestation did not change the pool of osteogenic cells before birth or growth plate heights before and after birth. However, MH reduced expression of bone formation marker (collagen-1) and increased expression of fat formation marker (FABP4) in postnatal offspring bone.

Widespread differential maternal and paternal genome effects on fetal bone phenotype at mid-gestation.

Parent-of-origin-dependent (epi)genetic factors are important determinants of prenatal development that program adult phenotype. However, data on magnitude and specificity of maternal and paternal genome effects on fetal bone are lacking. We used an outbred bovine model to dissect and quantify effects of parental genomes, fetal sex, and nongenetic maternal effects on the fetal skeleton and analyzed phenotypic and molecular relationships between fetal muscle and bone. Analysis of 51 bone morphometric and weight parameters from 72 fetuses recovered at day 153 gestation (54% term) identified six principal components (PC1-6) that explained 80% of the variation in skeletal parameters. Parental genomes accounted for most of the variation in bone wet weight (PC1, 72.1%), limb ossification (PC2, 99.8%), flat bone size (PC4, 99.7%), and axial skeletal growth (PC5, 96.9%). Limb length showed lesser effects of parental genomes (PC3, 40.8%) and a significant nongenetic maternal effect (gestational weight gain, 29%). Fetal sex affected bone wet weight (PC1, p < 0.0001) and limb length (PC3, p < 0.05). Partitioning of variation explained by parental genomes revealed strong maternal genome effects on bone wet weight (74.1%, p < 0.0001) and axial skeletal growth (93.5%, p < 0.001), whereas paternal genome controlled limb ossification (95.1%, p < 0.0001). Histomorphometric data revealed strong maternal genome effects on growth plate height (98.6%, p < 0.0001) and trabecular thickness (85.5%, p < 0.0001) in distal femur. Parental genome effects on fetal bone were mirrored by maternal genome effects on fetal serum 25-hydroxyvitamin D (96.9%, p < 0.001) and paternal genome effects on alkaline phosphatase (90.0%, p < 0.001) and their correlations with maternally controlled bone wet weight and paternally controlled limb ossification, respectively. Bone wet weight and flat bone size correlated positively with muscle weight (r = 0.84 and 0.77, p < 0.0001) and negatively with muscle H19 expression (r = -0.34 and -0.31, p < 0.01). Because imprinted maternally expressed H19 regulates growth factors by miRNA interference, this suggests muscle-bone interaction via epigenetic factors.

Molecular and structural basis of androgen receptor responses to dihydrotestosterone, medroxyprogesterone acetate and Δ(4)-tibolone.

Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3ß-OH-tibolone, and a delta-4 isomer (Δ(4)-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ(4)-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ(4)-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ(4)-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA.

Adequate dietary vitamin D and calcium are both required to reduce bone turnover and increased bone mineral volume.

Clinical studies indicate that the combination of vitamin D and dietary calcium supplementation is more effective for reducing fracture risk than either supplement alone. Our previous dietary studies demonstrated that an adequate serum 25-hydroxyvitamin D3 (25D) of 80nmol/L or more reduces bone RANKL expression, osteoclastogenesis and maintains the optimal levels of trabecular bone volume (BV/TV%) in young rats. The important clinical question of the interaction between vitamin D status, dietary calcium intake and age remains unclear. Hence, 9 month-old female Sprague-Dawley rats (n=5-6/group) were pair-fed a semi-synthetic diet containing varying levels of vitamin D (0, 2, 12 or 20IU/day) and dietary calcium (0.1% or 1%) for 6 months. At 15 months of age, animals were killed, for biochemical and skeletal analyses. While changes to serum 25D were determined by both dietary vitamin D and calcium levels, changes to serum 1,25-dihydroxyvitamin D3 (1,25D) were consistently raised in animals fed 0.1% Ca regardless of dietary vitamin D or vitamin D status. Importantly, serum cross-laps levels were significantly increased in animals fed 0.1% Ca only when combined with 0 or 2 IUD/day of vitamin D, suggesting a contribution of both dietary calcium and vitamin D in determining bone resorption activity. Serum 25(OH)D3 levels were positively correlated with both femoral mid-diaphyseal cortical bone volume (R(2)=0.24, P<0.01) and metaphyseal BV/TV% (R(2)=0.23, P<0.01, data not shown). In multiple linear regressions, serum 1,25(OH)2D3 levels were a negative determinant of CBV (R(2)=0.24, P<0.01) and were not a determinant of metaphyseal BV/TV% levels. These data support clinical data that reduced bone resorption and increased bone volume can only be achieved with adequate 25D levels in combination with high dietary calcium and low serum 1,25D levels. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Discordant effects of vitamin D deficiency in trabecular and cortical bone architecture and strength in growing rodents.

We have previously shown that vitamin D deficiency in young male rats results in significant reduction in femoral trabecular bone volume (BV/TV). However, the effects of vitamin D deficiency and its impact on other relevant skeletal sites remain unclear. Ten week old male Sprague-Dawley rats were fed various levels of vitamin D3 (2, 4, 8, and 12 IU/day) with standard Ca (0.4%) until 30 weeks of age and achieved stable serum 25-hydroxyvitamin D3 (25D) levels between 16 and 117 nmol/L. At time of death, femora, L2 vertebrae and tibiae were processed for bone histomorphometric analyses and tibial cortical strength by 3-point mechanical testing. A significant association between serum 25D and trabecular bone occurred for both the distal femoral metaphysis (R2=0.34, P<0.05) and L2 vertebrae (R2=0.24, P<0.05). Tibia mid-shaft cortical bone was not, however, changed in terms of total volume, periosteal surface or endosteal surface as a function of vitamin D status. Furthermore, no changes to mechanical and intrinsic properties of the cortices were observed. We conclude that cortical bone is maintained under conditions of vitamin D deficiency in preference to cancellous bone in young growing rats.