Vitamin D Analogues Tacalcitol and Calcipotriol Inhibit Proliferation and Migration of T98G Human Glioblastoma Cells.

The active form of vitamin D (1α,25-dihydroxyvitamin D) acts as a steroid hormone and binds to the vitamin D receptor. This receptor is expressed in most cell types including cells in the central nervous system (CNS). Vitamin D has several functions in the body including effects on brain development, neuroprotection and immunological regulation. It has been shown that vitamin D has antiproliferative activities in different cancer cell lines. Tacalcitol and calcipotriol are synthetic analogues of 1α,25-dihydroxyvitamin D with reduced effect on calcium metabolism. The aim of this study was to analyse the effects of tacalcitol and calcipotriol on cell viability, proliferation and migration in the human glioblastoma cell line T98G. Glioblastoma is the most lethal type of primary tumours in the CNS. Both analogues decreased cell viability and/or growth, dose-dependently, in concentrations between 1 nM and 10 µM. Manual counting indicated suppressive effects by the vitamin D analogues on proliferation. Treatment with tacalcitol strongly suppressed thymidine incorporation, indicating that the vitamin D analogues mainly inhibit proliferation. Also, effects on cell migration were measured with wound-healing assay. Both calcipotriol and tacalcitol reduced the migration rate of T98G cells compared to vehicle-treated cells. However, they had no effect on caspase-3 and -7 activities, suggesting that their mechanism of action does not involve induction of apoptosis. The current results indicate that the vitamin D analogues tacalcitol and calcipotriol strongly reduce proliferation and migration of human glioblastoma T98G cells, suggesting a potential role for this type of compounds in treatment of brain cancer.

Unfavorable effect of calcitriol and its low-calcemic analogs on metastasis of 4T1 mouse mammary gland cancer.

Low vitamin D status is considered as a risk factor for breast cancer and has prognostic significance. Furthermore, vitamin D deficiency increases after adjuvant cancer therapy, which alters bone metabolism increasing the risk of osteoporosis. It is now postulated that vitamin D supplementation in breast cancer treatment delays the recurrence of cancer thereby extending survival. We evaluated the impact of calcitriol and its low-calcemic analogs, PRI­2191 and PRI­2205, on the tumor growth, angiogenesis, and metastasis of 4T1 mouse mammary gland cancer. Gene expression analysis related to cancer invasion/metastasis, real­time PCR, ELISA, western blotting, and histochemical studies were performed. In vitro studies were conducted to compare the effects of calcitriol and its analogs on 4T1 and 67NR cell proliferation and expression of selected proteins. Calcitriol and its analogs increased lung metastasis without influencing the growth of primary tumor. The levels of plasma 17ß-estradiol and transforming growth factor ß (TGFß) were found to be elevated after treatment. Moreover, the results showed that tumor blood perfusion improved and osteopontin (OPN) levels increased, whereas vascular endothelial growth factor (VEGF) and TGFß levels decreased in tumors from treated mice. All the studied treatments resulted in increased collagen content in the tumor tissue in the early step of tumor progression, and calcitriol caused an increase in collagen content in lung tissue. In addition, in vitro proliferation of 4T1 tumor cells was not found to be affected by calcitriol or its analogs in contrast to non-metastatic 67NR cells. Calcitriol and its analogs enhanced the metastatic potential of 4T1 mouse mammary gland cancer by inducing the secretion of OPN probably via host cells. In addition, OPN tumor overexpression prevailed over the decreasing tumor TGFß level and blood vessel normalization via tumor VEGF deprivation induced by calcitriol and its analogs. Moreover, the increased plasma TGFß and 17ß-estradiol levels contributed to the facilitation of metastatic process.

Vitamin D: a novel therapeutic approach for keloid, an in vitro analysis.

BACKGROUND: Vitamin D and its metabolites play an important role in calcium homeostasis, bone remodelling, hormone secretion, cell proliferation and differentiation. Recent studies also suggest a beneficial role of vitamin D in slowing the progression of tissue fibrosis. However, their effects on dermal fibrosis and keloids are unknown. Objectives To investigate the effect of 1,25-dihydroxyvitamin D3 (1,25D) in the pathogenesis of tissue fibrosis by keloid fibroblasts (KFs). METHODS: KFs were cultured and exposed to different concentrations of 1,25D in the presence or absence of transforming growth factor (TGF)-ß1. KF phenotypes and protein production were analysed by real-time reverse transcriptase-polymerase chain reaction, Western blot, immunofluorescence and multiplex enzyme-linked immunosorbent assay techniques. Collagen synthesis was evaluated by measuring (3) H-proline incorporation. The effect of 1,25D on cell proliferation and viability was evaluated by Formazan assay, proliferating cell nuclear antigen expression and the colorimetric conversion of 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. RESULTS: We confirmed the presence of vitamin D receptors (VDRs) in cultured keloid fibroblasts. Fibroblasts transfected with a vitamin D response element reporter construct and exposed to the active vitamin D metabolite 1,25D showed increased promoter activity indicating VDR functionality in these cells. Incubation of KFs with 1,25D suppressed TGF-ß1-induced collagen type I, fibronectin and α-smooth muscle actin expression. 1,25D also modulated plasminogen activator inhibitor-1 and matrix metalloproteinase-9 expression induced by TGF-ß1. Interestingly, 1,25D induced hepatocyte growth factor mRNA expression and protein secretion in keloid fibroblasts. CONCLUSIONS: This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for keloid and related fibrotic disorders.

Influence of vitamin D(3) analogues in combination with budesonid R on proliferation of nasal polyp fibroblasts.

UNLABELLED: Vitamin D (VD) and its different analogues, besides their classic role as regulators of calcium and phosphor homeostasis, have emerged as a large family of antiproliferative agents. Such properties suggested VD potential as a therapy for chronic inflammatory diseases, including nasal polyposis (NP). NP growth involves both an inflammatory process and the proliferation of fibroblast as an important factor inducing aberrations in the phenotype of the epithelium. The aim of this study was to investigate the possible influence of 1alpha,25-dihydroxyvitamin D(3) (calcitriol) and 1alpha,24(R)-dihydroxyvitamin D(3) (tacalcitol) in monotherapy and in combination with budesonid R (BR) on NP fibroblast proliferation. MATERIAL AND METHODS: The study involved 26 samples of NP. NP cells were cultured on 96-well plates beginning with a concentration of 5 x 10(3) cells per well with RPMI 1640 medium supplemented with antibiotics and 10% foetal bovine serum. After the fourth to sixth passage the medium was replaced with a nutrient medium with calcitriol or tacalcitol in a defined concentration (from 10(-9) M to 10(-3) M) alone or in combination with BR in 1:1, 1:3 or 3:1 ratios, each at concentrations from 10(-5) M to 10(-3) M. RESULTS: Growth inhibition of nasal fibroblasts exposed to calcitriol or tacalcitol was noted. Significant antiproliferating activity was observed at calcitriol concentrations of 10(-4) M and 10(-3) M after 48 h, and at a concentration of 10(-3) M after 72 h with the percentage of proliferating cells reduced to 30% compared to the control samples (P < 0.05). In cells treated with tacalcitol the maximal effect was seen at 10(-4) M after 48 h and at 10(-3)M after 72 h with a 60% inhibition with respect to the control (P < 0.05). The inhibition of fibroblast proliferation reached the maximal level when they were exposed to calcitriol: BR (1 : 1) or tacalcitol: BR (1 : 1), each at a concentration of 10(-4) M, after 72 h (82% and 69%, respectively). CONCLUSIONS: The antiproliferative activity of calcitriol and tacalcitol in NP cultures was confirmed. Because of its lower toxicity and higher activity tacalcitol seems to be the more promising agent in NP therapy, both as a single medication and in treatment protocols with BR.

Effect of 2-methylene-19-nor-(20S)-1 alpha-hydroxy-bishomopregnacalciferol (2MbisP), an analog of vitamin D, on secondary hyperparathyroidism.

UNLABELLED: Vitamin D analogs are being developed that retain therapeutic effects but are less calcemic and phosphatemic, a concern in CKD patients who are prone to vascular calcification. We tested a new analog of vitamin D, 2MbisP, and found that it suppresses PTH at doses that do not affect serum Ca or P. INTRODUCTION: Calcitriol is used for the treatment of secondary hyperparathyroidism. However, its use is often limited by the development of hypercalcemia and hyperphosphatemia, an important consideration in patients with chronic kidney disease (CKD) because they are prone to vascular calcification. To minimize this toxicity, structural modifications in the vitamin D molecule have led to the development of calcitriol analogs with selective actions. MATERIALS AND METHODS: In this study, we compared the effects of 1,25(OH)(2)D(3) and a new analog, 2-methylene-19-nor-(20S)-1 alpha-hydroxy-bishomopregnacalciferol (2MbisP), on the development of secondary hyperparathyroidism and established secondary hyperparathyroidism in uremic rats and on mobilization of calcium and phosphorus from bone in parathyroidectomized rats. The clearance from circulation, half-life, and binding affinities to the vitamin D-binding protein and vitamin D receptor of this compound were also evaluated. RESULTS: Uremia produced a marked rise in plasma PTH, but treatment every other day for 2 wk with either 1,25(OH)(2)D(3) (4 ng) or 2MbisP (250, 750, 1500, or 3000 ng) suppressed this increase by >50%. The suppression by 1,25(OH)(2)D(3), however, was accompanied by increases in ionized calcium, phosphorus, and the calcium x phosphorus product, whereas these three parameters were unchanged by 2MbisP. The binding affinity of 2MbisP was 10-20 times less for the vitamin D receptor and 1000 times less for the serum vitamin D-binding protein compared with 1,25(OH)(2)D(3). Also, 2MbisP was cleared more rapidly from the circulation (t1/2 = 10 min) than 1,25-(OH)(2)D(3) (t1/2=7-9 h). In parathyroidectomized rats fed calcium-or phosphorus-deficient diets, daily injections of 2MbisP (1500 or 3000 ng), unlike 1,25(OH)(2)D(3) (50 ng), had no effect on calcium or phosphorus mobilization from bone. CONCLUSIONS: In uremic rats, 2MbisP can suppress PTH at doses that do not affect plasma calcium, phosphorus, and calcium x phosphorus product. This new vitamin D analog may represent an important tool in the treatment of secondary hyperparathyroidism in patients with CKD.

Profile of clinical efficacy and safety of topical tacalcitol.

Several topical treatments such as ointments, keratolytics, dithranol, tar, corticosteroids and Vitamin D3 analogues are commonly used in the treatment of mild and/or moderate psoriasis. These treatments can be associated with a variety of local and systemic side effects, as well as to very often unsatisfactory results. The purpose of this critical review of the literature is to evaluate the efficacy and tolerability of the synthesis of new analogues of the Vitamin D3 Tacalcitol, which is formulated in ointment form at a concentration of 4 microg/g, for the treatment of mild and/or moderate psoriasis (involvement of <20% of the surface of the skin) and to evaluate whether this drug can be used in the treatment of other skin conditions. Based on existing data in the literature, Tacalcitol is an effective drug for the topical treatment of psoriasis and is also able to ensure that the effects last over time, even after treatment has stopped. Tacalcitol is also well tolerated because the onset of side effects, such as local irritation, pruriginous or burning sensations, were reported in only a small percentage of the subjects who were treated. Lastly, the marked regulatory effects it has on the proliferation and differentiation of keratinocytes, as well as on the immunocompetent cells, has led to suggestions that Tacalcitol may be used in other keratinisation disorders and in some hyperproliferative skin diseases. Evaluation of the effective indications to use in these conditions still requires further data confirming its effectiveness, opening the way to wider use of this molecule in dermatology.

Metallothionein induction in calcifying growth plate cartilage chondrocytes.

Metallothionein (MT) induction was studied in mineralizing cultures of chicken growth plate chondrocytes and quantitated using a Cd-saturation assay. In serum free media, MT induction was observed for Cd concentrations of 0.1 microM and greater and at Zn concentrations of 100 microM and greater. Supplementation of culture media with cysteine and/or methionine resulted in higher levels of MT induction and reduced toxicity during Cd exposure. Maximum MT induction appeared to coincide with the earliest culture stages during which important enzymes and matrix components are being synthesized. Of non-metal MT inducers tested, sodium butyrate caused a low level induction of MT while interleukin-1 had no effect on basal MT levels. 1,25-dihydroxyvitamin D increased MT induction. The steroid hormone dexamethasone caused a reduction in basal and induced MT levels. These findings suggest that MT regulation in growth plate chondrocytes differs significantly from what is known in other cell types and that this difference may be related to the mineralization of this tissue.

Expression of sodium-dependent phosphate (NadPi) transport in Xenopus laevis oocytes induced by mRNA from 1 alpha, 25-dihydroxyvitamin D3-treated rat osteoblast-like cells.

Injection of messenger ribonucleic acid (mRNA) isolated from 1 alpha, 25-dihydroxyvitamin D3-treated osteoblast-like (PyMS) cells leads to an enhanced sodium-dependent phosphate (NadPi) transport in Xenopus laevis oocytes, when compared to untreated cells. After mRNA size fractionation, mRNA with an average size of 2.2-3.8 kilobases showed up to a 1.8-fold stimulation of NadPi transport encoding either directly a NadPi transporter(s) or proteins controlling their activity. No hybridization was observed in Northern blots with RNA from rat bone or PyMS cells with the recently cloned rat renal brush border NadPi transporter NaPi-2; hybrid depletion with a NaPi-2 antisense oligonucleotide did not abolish the PyMS mRNA-induced NadPi transport in oocytes. We present the first evidence for functional expression in Xenopus laevis oocytes of a new type of NadPi transport system in bone cells, which is different from the renal type.

Vitamin D deficiency in the chick embryo: effects on prehatching motility and on the growth and differentiation of bones, muscles, and parathyroid glands.

Vitamin D-deficient chicken embryos were obtained by feeding laying hens diets in which 3-7 micrograms calcitriol replaced the vitamin D3 supplement. A large proportion of the D-deficient embryos failed to complete the prehatching positional changes required to start pulmonary respiration. For this reason most of them became cyanotic and had subcutaneous edema and hemorrhages in the head and neck and died without hatching. Total as well as leg-bone and muscle weights were significantly lower in the deficient embryos than in the controls and these changes probably explain the inability of the embryos to complete the movements required to place the beak in contact with the air chamber and start pulmonary respiration. The histological study of the tibiae showed decreased mineralization with narrower trabeculae and enlarged osteoid seams; bone resorption at the inner surface was also significantly decreased. The ultrastructural study of parathyroid glands showed increased functional activity reflected by increased number and size of cisternae of rough endoplasmic reticulum. Injection of 10 ng calcitriol, 1 microgram 24,25-(OH)2D3, or 2 micrograms 25OHD3 to deficient embryos on the 14th day of incubation improved hatchability, bone and muscle weights, and both bone mineralization and resorption.

A direct effect of 24,25-(OH)2D3 and 1,25-(OH)2D3 on the modeling of fetal mice long bones in vitro.

To examine the effects of 24,25-(OH)2D3 and 1,25-(OH)2D3 on fetal long bone modeling the radii and ulnae of 16 day fetal mice were grown in vitro for 2 days. Their growth, mineralization, and resorption were assessed by measuring diaphyseal length, calcium and phosphorus content, hydroxyproline-protein ratios, and the release of incorporated 45Ca. The results showed that 24,25-(OH)2D3 at concentrations of 10(-10)-10(-8) M stimulated the growth of the bones as indicated by their increased diaphyseal length, periosteal bone area, and hydroxyproline content. Calcium and phosphorus content was significantly increased; 45Ca release was unaltered. Bones incubated in media containing 10(-6) M 24,25-(OH)2D3 responded in a similar fashion to bones incubated in media containing 10(-10)-10(-8) M 1,25-(OH)2D3, with inhibition of bone growth as indicated by reduced diaphyseal length, periosteal bone area, hydroxyproline-protein ratios, and calcium and phosphorus content; 45Ca release was significantly increased. Neither metabolite affected total bone length. The results suggest a role for 24,25-(OH)2D3 in the growth of fetal mice bones in vitro and also confirm the findings from previous studies that 1,25-(OH)2D3 and high concentrations of 24,25-(OH)2D2 stimulate bone resorption.

Increased mechanical strength of the vitamin D-replete rat femur by the treatment with a large dose of 24R,25(OH)2D3.

The mechanical properties of the rat femur treated with a large dose of 24R,25(OH)2D3 were examined and their relationship with the mass and mineral contents of the bone were investigated. Male Wistar rats were fed diet containing 0, 0.025, 1.25, 4.0, or 12.5 ppm 24R,25(OH)2D3 for two years starting six weeks after birth. The rats were killed and their right femurs were removed. The adhering soft tissue were stripped off. Radiographs were made of the femur and its bone mineral content was measured by single photon absorptiometry. Then a three-point bending test was done with pressure exerted in the plane of natural extension. After mechanical testing, non-decalcified cross-sections of the femur were prepared at the mid diaphysis as close to the test fracture site as possible. On x-ray images, the cortical thickness was clearly increased in groups treated with larger doses, and the mid-cortical segmental mineral content of the femur increased dose-dependently to about 150%. The mechanical parameters in the treated animals also increased significantly, strength to 120% (p less than 0.01), energy-absorption capacity to 124% (p less than 0.05), and structural stiffness to 183% (p less than 0.01). Segmental bone mineral contents showed the positive correlations with strength and structural stiffness in both the control and the 24R,25(OH)2D3 treated groups as well. We concluded that the increase in the bone density in animals administered high doses of 24R,25(OH)2D3 was accompanied by an increase in mechanical strength of the bone.

Effect of vitamin D status on the activity of carbonic anhydrase in chicken epiphysis and kidney.

Chickens were raised for 6 weeks from the date of hatch under red light on a vitamin D-free diet; controls were given an oral vitamin D supplement. Vitamin D-deficient animals showed decreased total serum calcium concentration and decreased DNA content in epiphysis and kidney homogenates. In calcifying epiphysis, total carbonic anhydrase (CA) activity was decreased, but activity per microgram DNA was slightly increased and specific activity was double that of the controls. Polyacrylamide gel isoelectric focusing after preparation of the enzyme showed a picture similar to that seen after parathyroid hormone (PTH) administration in chicks; therefore, this could be considered a secondary hyperparathyroidism. The CA activation was not seen in the kidney which can be explained by induction of an endogenous inhibitor protein of the cyclic AMP-dependent protein kinase exclusively in the kidney in vitamin D deficiency. In an additional experiment, chickens were raised for 3 weeks from the date of hatch under red light on a vitamin D-free diet. Daily oral substitution by different vitamin D metabolites (1,25(OH)2D3, 25OHD3, 24,25(OH)2D3) over 7 days led to CA activation compared with controls probably by restoring protein kinase activity in the kidney. Our results show that CA activity is inversely correlated with serum calcium concentrations which is in agreement with a regulatory mechanism recently proposed by us.

Rat epiphyseal cells in culture: responsiveness to bone-seeking hormones.

Cell cultures derived from young rat epiphyseal cartilage were grown for approximately 2 wk in BGJb medium supplemented with 10% fetal bovine serum to reach confluence. These cells were identified as chondrocytes as checked by morphology, the presence of alkaline phosphatase, and a positive type II collagen antibody reaction. The cells also responded to different hormonal treatment. Parathyroid hormone (PTH) increased cyclic AMP production by 50% within 15 min of treatment, whereas prostaglandin E2 (PGE2) caused an increase of 160%. Calcitonin (CT) did not affect cAMP production in these cells. DNA synthesis 24 h after hormonal treatment was increased by PTH (2.5-fold) and PGE2 (2-fold), but not by CT. Among the vitamin D metabolites, 24,25(OH)2D3 increased significantly the [3H]thymidine incorporation into DNA, whereas 1,25(OH)2D3 effect was minimal. These results provide evidence for the use of these cell cultures as a model for cartilage in vitro when studying biological and hormonal responsiveness.

Effects of vitamin D deficiency in the chicken embryo.

Vitamin D-deficient chicken embryos were obtained by feeding laying hens a diet in which 5 micrograms 1,25(OH)2D3/kg feed were substituted for the vitamin D3 supplement in the control diet. Hatchability, total Ca and inorganic P concentration in blood, and tibial ash/dry weight ratio were determined in the vitamin D-deficient embryos and in embryos obtained from hens fed the control diet supplemented with 1100 IU vitamin D3/kg feed. After 5 weeks on the substituted diet the hens laid eggs that showed decreased hatchability in spite of excellent shell quality. All determinations in blood and bones were made on embryos of eggs laid after 6-12 weeks on the diets. On the 17th day of incubation the embryos derived from hens fed the substituted diet showed significant hypocalcemia and hyperphosphatemia and a low tibial ash/dry weight ratio. Injection of 1,25(OH)2D3 3 days before killing corrected the hypocalcemia of the deficient embryos. Those chicks that managed to hatch had normal levels of calcium and inorganic phosphate 1 day after hatching. These findings support previous suggestions by us and other authors that vitamin D metabolites are required by the embryo in order to mobilize calcium from the shell, and decreased hatchability in vitamin D-deficient embryos is related to a defect in calcium mobilization from the shell. While in previous studies a decrease in hatchability was the only parameter used to judge D deficiency of the embryos in our present studies, the deficiency is confirmed by demonstrating a deficit in mineral metabolism which is a more specific sign of D deficiency.

The effect of vitamin D on bone in vivo.

The linear rate of bone mineral apposition (BMAR) was measured in vitamin D-deficient and vitamin D-sufficient adult rats before and during treatment with either 25-hydroxyvitamin D3 (25OHD3), 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3]. Dietary vitamin D restriction caused a fall in BMAR which began after 1 week and fell progressively to a value of 35-50% of control values by 4 weeks. The fall in BMAR was related to a fall in the serum concentrations of 25(OH)D3 and 24,25-(OH)2D3, without a fall in the 1,25-(OH)2D3 concentration. Dietary supplementation of the D-deficient animals with either 25OHD3 or 24,25-(OH)2D3 at doses of 200 ng/day restored BMAR. If vitamin D-deficient animals were thyroparathyroid-ectomized before supplementation with vitamin D metabolites, 24,25-(OH)2D3 administration was without effect on BMAR. The combined administration of PTH and 24,25-(OH)2D3 to such animals led to a restoration of the BMAR to normal. In vitamin D-sufficient animals, parathyroidectomy led to a 50% reduction in BMAR, which could be restored by treatment with PTH alone but not with 24,25-(OH)2D3. Simultaneous treatment of these animals with PTH and 24,25-(OH)2D3 led to a greater than normal increase in BMAR (130% of control) in these animals. These data support the concept that 24,25-(OH)2D3 has a role in the regulation of bone formation and/or mineralization, and demonstrate the interrelation between the effects of PTH and 24,25-(OH)2D3 on bone.

The action of various vitamin D3 metabolites on calcium and phosphorus metabolism in chick embryo calvariae.

Chick embryos from vitamin D-deficient hens given physiological doses of 1,25-dihydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 or both become severely hypocalcemic, hyperphosphatemic and fail to hatch as compared to those derived from hens given 25-hydroxyvitamin D3 or 24,25-difluoro-25-hydroxyvitamin D3. Calvariae from the former contain less mineral and on incubation in vitro produce significantly lower calcium and higher phosphate concentration in the medium than do the calvariae derived from the embryos of hens supported on 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3.

Regulatory effect of calcium-regulating hormones on the synthesis and/or secretion of bone gamma-carboxyglutamic acid-containing protein in chick embryonic calvaria in vitro.

Regulatory effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), 24,25-dihydroxyvitamin D3 (24,25-(OH)2-D3), parathyroid hormone (PTH) and calcitonin (CT) on BGP synthesis and/or secretion were studied using chick embryonic calvaria in vitro. BGP contents in calvaria and culture medium were determined by radioimmunoassay using antiserum to purified chick BGP. After 72 h culture, 1,25-(OH)2-D3 (10(-10)-10(-7) M) increased the BGP content in culture medium significantly, and the effect was maximum at 10(-8)M, while the BGP content in calvaria was not changed by 1,25-(OH)2-D3. 24,25-(OH)2-D3 increased the BGP content both in medium at 10(-6) M-10(-5) M after 24 h culture and in calvaria at 5 X 10(-7)-10(-5) M after 48 h culture. PTH increased BGP contents both in calvaria after 72 h culture and medium at 1 U/ml after 24 h culture, while it decreased them at 5-10 U/ml after 24 h culture. CT (0.5-10 U/ml) had no effect on BGP contents in calvaria and medium. 1,25-(OH)2-D3-induced increase of BGP in culture medium was observed from 24 to 72 h of culture and then reached a plateau at 120 h of culture. 1,25-(OH)2-D3-induced increase in the BGP level in medium after 72 h culture was 3-4 times the control value. On the other hand, the BGP content in calvaria was not affected by 1,25-(OH)2-D3 until 72 h of culture and then significantly increased at 120 h. The addition of 1,25-(OH)2-D3 (10(-8) M), 24,25-(OH)2-D3 (10(-6) M), and 1 U PTH (and 1 U CT added to those hormones) into culture medium of chick embryonic calvaria significantly increased BGP contents in calvaria and medium at 72 h after culture. The effect of three hormones was not synergistic, but had a tendency to be greater than that of a single addition of 1,25-(OH)2-D3, 24,25-(OH)2-D3 or PTH. Further addition of 1 U CT into culture medium did not affect the combined effects of 1,25-(OH)2-D3, 24,25-(OH)2-D3 and PTH except for the calcium content in calvaria. The addition of individual calcium-regulating hormones into culture medium of chick embryonic calvaria did not affect the calcium and phosphorus contents in calvaria, but the calcium content in calvaria was significantly increased by the addition of 1,25-(OH)2-D3, 24,25-(OH)2-D3 and PTH to the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of vitamin D3 metabolites on calcium and phosphorus metabolism in chick embryos.

The biochemical nature of the physiological defect found in chick embryos from hens supported on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D is described. Vitamin D-deficient hens (44-wk-old) were divided into six groups of five and dosed daily for 19 wk with either 2.0 micrograms of 25-hydroxyvitamin D3, 2.0 micrograms of 24,24-difluoro-25-hydroxy-vitamin D3, 0.4 micrograms of 1,25-dihydroxyvitamin D3, 2.0 micrograms of 24,25-dihydroxyvitamin D3, 0.4 micrograms of 1,25-dihydroxyvitamin D3 plus 2.0 micrograms of 24,25-dihydroxyvitamin D3, or vehicle only. Normal embryonic development was found in eggs from hens given 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, whereas embryos from hens given 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, or their combination were abnormal and failed to hatch. Embryos from hens fed 1,25-dihydroxyvitamin D3 and/or 24,25-dihydroxyvitamin D3 had vitamin D deficiency: low bone ash, low plasma calcium, low total body calcium, and extremely high plasma phosphorus. Because the shell is the major source of calcium for the developing embryo, calcium transport from the shell to the embryos across the chorioallantoic membrane apparently fails, giving rise to the observed defects in embryonic development.

[Effect of 1,25-dioxycholecalciferol and 24,25-dioxycholecalciferol on calcium homeostasis in rats given hydrocortisone in the diet with various phosphorus contents]. / Vliianie 1,25-dioksikholekal'tsiferola i 24,25-dioksikholekal'tsiferola na gomeostaz kal'tsiia u krys poluchaiushchikh gidrokortizon na fone ratsiona s razlichnym soderzhaniem fosfora.

High content of phosphorus in the diet (1.8% of phosphorus in the diet, Ca:P ratio 1:3) accelerated the development of hypocalcemia and osteoporosis and increased their degree in rats which received hydrocortisone (3.5 mg/10 g bw a day for 4 weeks). Reduction of phosphorus consumption to 0.3% (Ca:P ration 1:0.5) essentially retarded the development of these disturbances and lowered their degree. The use of 1,25-dioxycholecalciferol and 24,25-dioxycholecalciferol in doses of 0.03 and 1.5 mg, respectively promoted the normalization of phosphorus-calcium metabolism and improvement of the status of the osseous tissue in rats given hydrocortisone coupled with both diets. The most beneficial affect on calcium homeostasis in exogenous hypercorticoidism was attained with the use of active metabolites of vitamin D3 coupled with the diet with a low phosphorus content (0.3%). In this case there was a complete normalization of the density of the osseous tissue and of the calcium and phosphorus content. In view of this fact it is advisable to combine active metabolites of vitamin D3 and the diet with a low phosphorus content.