Caffeine at a Moderate Dose Did Not Affect the Skeletal System of Rats with Streptozotocin-Induced Diabetes.

Diabetes may lead to the development of osteoporosis. Coffee drinking, apart from its health benefits, is taken into consideration as an osteoporosis risk factor. Data from human and animal studies on coffee and caffeine bone effects are inconsistent. The aim of the study was to investigate effects of caffeine at a moderate dose on the skeletal system of rats in two models of experimental diabetes induced by streptozotocin. Effects of caffeine administered orally (20 mg/kg aily for four weeks) were investigated in three-month-old female Wistar rats, which, two weeks before the start of caffeine administration, received streptozotocin (60 mg/kg, intraperitoneally) alone or streptozotocin after nicotinamide (230 mg/kg, intraperitoneally). Bone turnover markers, mass, mineral density, histomorphometric parameters, and mechanical properties were examined. Streptozotocin induced diabetes, with profound changes in the skeletal system due to increased bone resorption and decreased bone formation. Although streptozotocin administered after nicotinamide induced slight increases in glucose levels at the beginning of the experiment only, slight, but significant unfavorable changes in the skeletal system were demonstrated. Administration of caffeine did not affect the investigated skeletal parameters of rats with streptozotocin-induced disorders. In conclusion, caffeine at a moderate dose did not exert a damaging effect on the skeletal system of diabetic rats.

Effects of fenoterol on the skeletal system depend on the androgen level.

BACKGROUND: The role of sympathetic nervous system in the osseous tissue remodeling is not clear enough. METHODS: The effects of fenoterol, a selective ß2-adrenomimetic drug, on the skeletal system of normal and androgen deficient (orchidectomized) rats were studied in vivo. Osteoclastogenesis and mRNA expression in osteoblasts were investigated in vitro in mouse cell cultures. RESULTS: Fenoterol administered to animals with physiological androgen level unfavorably affected the skeletal system, damaging the bone microarchitecture. Androgen deficiency induced osteoporotic changes, and fenoterol protected the osseous tissue from consequences of androgen deficiency. The results of in vitro studies correlated with the in vivo observations. A significantly increased number of osteoclasts in bone marrow cell cultures to which testosterone and fenoterol were added simultaneously was demonstrated. In cultures without the addition of testosterone, fenoterol significantly inhibited osteoclastogenesis in comparison with control cultures. CONCLUSIONS: The results indicate the favorable action of fenoterol in conditions of testosterone deficiency, and its destructive influence upon the skeleton in the presence of androgens. The results confirm the key role of sympathetic nervous system in the regulation of bone remodeling.

Cyclic guanosine monophosphate in the regulation of the cell function.

Intracellular concentration of cGMP depends on the activity of guanylate cyclase, responsible for its synthesis, on the activity of cyclic nucleotide degrading enzymes - phosphodiesterases (PDEs). There are two forms of guanylate cyclase: the membrane-bound cyclase and the soluble form. The physiological activators of the membrane guanylate cyclase are natriuretic peptides (NPs), and of the cytosolic guanylate cyclase - nitric oxide (NO) and carbon monoxide (CO). Intracellular cGMP signaling pathways arise from its direct effect on the activity of G protein kinases, phosphodiesterases and cyclic nucleotide dependent cation channels. It has been shown in recent years that cGMP can also affect other signal pathways in cell signaling activity involving Wnt proteins and sex hormones. The increased interest in the research on the role of cGMP, resulted also in the discovery of its role in the regulation of phototransduction in the eye, neurotransmission, calcium homeostasis, platelet aggregation, heartbeat, bone remodeling, lipid metabolism and the activity of the cation channels. Better understanding of the mechanisms of action of cGMP in the regulation of cell function can create new opportunities for the cGMP affecting drugs use in the pharmacotherapy.

Effects of Trigonelline, an Alkaloid Present in Coffee, on Diabetes-Induced Disorders in the Rat Skeletal System.

Diabetes increases bone fracture risk. Trigonelline, an alkaloid with potential antidiabetic activity, is present in considerable amounts in coffee. The aim of the study was to investigate the effects of trigonelline on experimental diabetes-induced disorders in the rat skeletal system. Effects of trigonelline (50 mg/kg p.o. daily for four weeks) were investigated in three-month-old female Wistar rats, which, two weeks before the start of trigonelline administration, received streptozotocin (60 mg/kg i.p.) or streptozotocin after nicotinamide (230 mg/kg i.p.). Serum bone turnover markers, bone mineralization, and mechanical properties were studied. Streptozotocin induced diabetes, with significant worsening of bone mineralization and bone mechanical properties. Streptozotocin after nicotinamide induced slight glycemia increases in first days of experiment only, however worsening of cancellous bone mechanical properties and decreased vertebral bone mineral density (BMD) were demonstrated. Trigonelline decreased bone mineralization and tended to worsen bone mechanical properties in streptozotocin-induced diabetic rats. In nicotinamide/streptozotocin-treated rats, trigonelline significantly increased BMD and tended to improve cancellous bone strength. Trigonelline differentially affected the skeletal system of rats with streptozotocin-induced metabolic disorders, intensifying the osteoporotic changes in streptozotocin-treated rats and favorably affecting bones in the non-hyperglycemic (nicotinamide/streptozotocin-treated) rats. The results indicate that, in certain conditions, trigonelline may damage bone.

Modifications of histamine receptor signaling affect bone mechanical properties in rats.

Histamine receptors are expressed on bone cells and histamine may be involved in regulation of bone metabolism. The aim of the present study was to investigate the effects of loratadine (an H(1) receptor antagonist), ranitidine (an H(2) receptor antagonist) and betahistine (an H(3) receptor antagonist and H(1) receptor agonist) on bone mechanical properties in rats. Loratadine (5 mg/kg/day, po), ranitidine (50 mg/kg/day, po), or betahistine dihydrochloride (5 mg/kg/day, po), were administered for 4 weeks to non-ovariectomized and bilaterally ovariectomized (estrogen-deficient) 3-month-old rats, and their effects were compared with appropriate controls. Serum levels of bone turnover markers, bone mineralization and mechanical properties of the proximal tibial metaphysis, femoral diaphysis and femoral neck were studied. In rats with normal estrogen level, administration of loratadine slightly favorably affected mechanical properties of compact bone, significantly increasing the strength of the femoral neck (p < 0.05), and tending to increase the strength of the femoral diaphysis. Ranitidine did not significantly affect the investigated parameters, and betahistine decreased the strength of the tibial metaphysis (cancellous bone, p < 0.01). There were no significant effects of the drugs on serum bone turnover markers. In estrogen-deficient rats, the drugs did not significantly affect the investigated skeletal parameters. In conclusion, the effects of histamine H(1), H(2) and H(3) receptor antagonists on the skeletal system in rats were differential and dependent on estrogen status.

Favorable effect of moderate dose caffeine on the skeletal system in ovariectomized rats.

SCOPE: Caffeine, a methylxanthine present in coffee, has been postulated to be responsible for an increased risk of osteoporosis in coffee drinkers; however, the data are inconsistent. The aim of the present study was to investigate the effects of a moderate dose of caffeine on the skeletal system of rats with normal and decreased estrogen level (developing osteoporosis due to estrogen deficiency). METHODS AND RESULTS: The experiments were carried out on mature nonovariectomized and ovariectomized Wistar rats, divided into control rats and rats receiving caffeine once daily, 20 mg/kg p.o., for 4 wk. Serum bone turnover markers, bone mass, mass of bone mineral, calcium and phosphorus content, histomorphometric parameters, and bone mechanical properties were examined. Caffeine favorably affected the skeletal system of ovariectomized rats, slightly inhibiting the development of bone changes induced by estrogen deficiency (increasing bone mineralization, and improving the strength and structure of cancellous bone). Moreover, it favorably affected mechanical properties of compact bone. There were no significant effects of caffeine in rats with normal estrogen levels. CONCLUSION: In conclusion, results of the present study indicate that low-to-moderate caffeine intake may exert some beneficial effects on the skeletal system of mature organisms.

Do effects of propranolol on the skeletal system depend on the estrogen status?

BACKGROUND: Propranolol, a nonselective ß-adrenergic receptor antagonist, was reported to favorably affect the skeletal system in different animal models. The aim of the study was to investigate whether the effects of propranolol on the skeletal system depend on the estrogen status. METHODS: The in vivo experiments were carried out on the following groups of mature female Wistar rats: sham-operated control rats, sham-operated rats receiving propranolol, ovariectomized (OVX) control rats, OVX rats receiving propranolol, OVX rats receiving estradiol, OVX rats receiving estradiol and propranolol. Propranolol hydrochloride (10 mg/kg po) and/or estradiol (0.1 mg/kg po) were administered daily for 4 weeks. Bone mass, mineral and calcium content, macrometric and histomorphometric parameters, and mechanical properties were examined. In vitro, effects of estradiol and propranolol on the formation of mouse osteoclasts and on the mRNA expression of genes related to osteoclastogenesis, bone formation and mineralization, as well as adrenergic and estrogen signalling in mouse osteoblasts were investigated. RESULTS AND CONCLUSION: Propranolol exerted some favorable effects on the rat skeletal system in vivo, independently of the estrogen status. However, in vitro studies indicated a possibility of some antagonistic relations between the estradiol and propranolol effects.

Bone remodeling after administration of proton pump (H+/K+-ATPase) inhibitors and alendronate in ovariectomized rats.

During osteoporosis therapy with alendronate, esophagitis and stomach ulceration may occur, requiring the concurrent use of drugs which decrease gastric juice production. The aim of the present study was to investigate the effect of concurrent administration of proton pump (H+/K+ATP-ase) inhibitors: omeprazole or pantoprazole and alendronate on bone remodeling in ovariectomized rats. The experiments were carried out on 3-month-old Wistar rats, divided into following groups (n = 8-10): NOVX - non-ovariectomized control rats; OVX - ovariectomized control rats; OVX + alendronate; OVX + omeprazole; OVX + omeprazole + alendronate; OVX + pantoprazole; OVX + pantoprazole + alendronate. The drugs were administered for 28 days: alendronate (3 mg/kg p.o.), omeprazole or pantoprazole (3 mg/kg i.p.). Bone remodeling was estimated based on histomorphometric evaluation of the tibia (endosteal and periosteal transverse growth and the osteoid width, transverse cross-section surface of the diaphysis and of the marrow cavity) and the femur (width of trabeculae in the distal epiphysis and metaphysis). Bone mass/100 g body mass and mass of bone mineral/100 mg bone mass in the tibia and femur were also determined. Pantoprazole stronger than omeprazole intensified bone remodeling disorders caused by estrogen deficiency in ovariectomized rats. Bone remodeling disorders were the result of intensification of bone resorption with concurrent inhibition of bone formation and mineralization. Pantoprazole, and to lesser extent omeprazole, weakened the preventive effect of alendronate on bone remodeling in ovariectomized rats.

Effects of vigabatrin on the skeletal system of young rats.

Long-term administration of antiepileptic drugs may be connected with the risk of impairment of bone remodeling. Contrary to the reported unfavorable effect of classic antiepileptic drugs on bone metabolism, little is known about the effect of the next generation antiepileptics on bone remodeling. The aim of the present study was to investigate the effect of vigabatrin, as a representative of new antiepileptics, on the skeletal system of young rats, in comparison with conventional drugs--phenytoin and valproic acid. The experiments were carried out on 4-week-old male Wistar rats, divided into the control rats, and rats receiving vigabatrin (250 mg/kg p.o. daily), phenytoin (20 mg/kg p.o. daily) or valproic acid (250 mg/kg p.o. daily). The drugs were administered for 28 days. Histomorphometric parameters of the tibia and femur, mechanical properties of the femur, and bone length, diameter, mass, content of mineral substances and calcium were examined. After administration of phenytoin or valproic acid, the investigated bone parameters did not significantly differ from those observed in the control rats. Administration of vigabatrin caused profound impairment of bone accrual with impairment of bone histomorphometric parameters, along with the significant decrease in the body mass gain.

Effects of propranolol on the development of glucocorticoid-induced osteoporosis in male rats.

Glucocorticoid-induced osteoporosis is the most frequently occurring type of secondary osteoporosis. Antagonists of ß-adrenergic receptors are now considered to be potential drugs under investigation for osteoporosis. The aim of the present study was to investigate the effects of propranolol, a nonselective ß-receptor antagonist, on the skeletal system of mature male rats and on the development of bone changes induced by glucocorticoid (prednisolone) administration. The experiments were performed on 24-week-old male Wistar rats. The effects of prednisolone 21-hemisuccinate sodium salt (7 mg/kg, sc daily) or/and propranolol hydrochloride (10 mg/kg, ip daily) administered for 4 weeks on the skeletal system were studied. Bone and bone mineral mass in the tibia, femur and L-4 vertebra, length and diameter of the long bones, mechanical properties of tibial metaphysis, femoral diaphysis and femoral neck, bone histomorphometric parameters and turnover markers in serum were determined. Prednisolone-induced unfavorable skeletal changes led to disorders in bone mechanical properties. Propranolol not only did not improve bone parameters, but even caused deleterious effects on the skeletal system. Concurrent administration of propranolol with prednisolone did not counteract the changes induced by prednisolone. The results of this study may help to understand the equivocal results of human studies on the effects of ß-blockers on the skeletal system. It is possible that the drugs exert biphasic effects on the skeletal system, both favorable and deleterious, depending on the dose or individual susceptibility.

Fenoterol did not enhance glucocorticoid-induced skeletal changes in male rats.

Glucocorticoids and ß(2)-adrenergic receptor agonists are the most commonly used drugs in the treatment of asthma. Both therapies are potentially dangerous to the skeletal system. The aim of the present study was to investigate the effects of fenoterol, a ß(2)-receptor agonist, on the development of bone changes induced by glucocorticoid (prednisolone) administration in mature male rats. The experiments were carried out on 24-week-old male Wistar rats. The effects of prednisolone 21-hemisuccinate sodium salt (7 mg/kg s.c. daily) or/and fenoterol hydrobromide (1.4 mg/kg i.p. daily), administered for 4 weeks, on the skeletal system were studied. Bone turnover markers, geometric parameters, mass, mass of bone mineral in the tibia, femur and L-4 vertebra, bone histomorphometric parameters and mechanical properties of tibial metaphysis, femoral diaphysis and femoral neck were determined. Both prednisolone and fenoterol had damaging effects on the skeletal system of mature male rats. However, concurrent administration of fenoterol and prednisolone did not result in the intensification of the deleterious skeletal effect of either drug administered separately.

Administration of caffeic acid worsened bone mechanical properties in female rats.

Natural phenolic acids, commonly present in plants that are normally consumed in the diet, have been reported to exert antiresorptive and/or bone formation increasing activity. The aim of the present study was to investigate the effects of ferulic, caffeic, P-coumaric, and chlorogenic acids on the skeletal system of normal, mature female rats. The phenolic acids (10 mg/kg p. o. daily for 4 weeks) were administered to 3-month-old female Wistar Cmd:(WI)WU rats. Bone mass, mineral and calcium content, macrometric and histomorphometric parameters, and mechanical properties were examined. Phenolic acids had differential effects on the rat skeletal system. Although none of them affected bone macrometric parameters, mass and mineralization, all of them increased the width of femoral trabeculae. Administration of caffeic acid worsened bone mechanical properties (decreasing ultimate load sustained by the femur in three-point bending test). In conclusion, high intake of caffeic acid may unfavorably affect the skeletal system.

Effect of low-dose tacrolimus coadministered with raloxifene on the skeletal system in male rats.

Tacrolimus is an immunosuppressive drug, used to prevent organ transplant rejection. Immunosuppresants are known to unfavorably affect the osseous system. However, in our previous study on bone histomorphometric parameters we observed that low-dose tacrolimus intensified bone formation. The aim of the present study was to investigate the effects of low-dose tacrolimus on bone mechanical properties and mineralization in male rats. The effects of concurrent administration of tacrolimus and raloxifene were also studied. Raloxifene is a selective estrogen receptor modulator, used in the prevention and treatment of postmenopausal osteoporosis. In male rats raloxifene induces moderate intensification of bone mineralization. The experiments were carried out on mature male Wistar rats. The animals were divided into four groups (n = 7): control rats, rats treated with tacrolimus (0.3 mg/kg po), rats treated with raloxifene hydrochloride (5 mg/kg po) and rats treated with tacrolimus and raloxifene hydrochloride concurrently at abovementioned doses. The drugs were administered daily for 4 weeks. Body mass, bone mass and bone mineral content in the tibia, femur and L-4 vertebra, as well as mechanical properties of the whole femur (extrinsic stiffness, ultimate and breaking load, deformation caused by the applied load) and the femoral neck (load at fracture) were examined. Administration of tacrolimus at a dose of 0.3 mg/kg po for 4 weeks did not affect bone mechanical properties and mineralization. Concurrent administration of tacrolimus and raloxifene resulted in changes similar to those caused by raloxifene alone (statistically significant increases in the bone mass/body mass ratio, bone mineral content/body mass ratio and bone mineral content/bone mass ratio in comparison with the control rats, and no effect on bone mechanical properties). Results of the present study do not support the hypothesis that tacrolimus may be useful as a drug stimulating bone formation in skeletal diseases.

A comparative study of the effects of genistein, estradiol and raloxifene on the murine skeletal system.

Genistein, a major phytoestrogen of soy, is considered a potential drug for prevention and treatment of postmenopausal osteoporosis. The aim of the present study was to compare the effects of genistein, estradiol and raloxifene on the skeletal system in vivo and in vitro. Genistein (5 mg/kg), estradiol (0.1 mg/kg) or raloxifene hydrochloride (5 mg/kg) were administered daily by a stomach tube to mature ovariectomized Wistar rats for 4 weeks. Bone mass, mineral and calcium content, macrometric parameters and mechanical properties were examined. Also the effects of genistein, estradiol and raloxifene (10(-9)-10(-7) M) on the formation of osteoclasts from neonatal mouse bone marrow cells and the activity of osteoblasts isolated from neonatal mouse calvariae were compared. In vivo, estrogen deficiency resulted in the impairment of bone mineralization and bone mechanical properties. Raloxifene but not estradiol or genistein improved bone mineralization. Estradiol fully normalized the bone mechanical properties, whereas genistein augmented the deleterious effect of estrogen-deficiency on bone strength. In vitro, genistein, estradiol and raloxifene inhibited osteoclast formation from mouse bone marrow cells, decreasing the ratio of RANKL mRNA to osteoprotegerin mRNA expression in osteoblasts. Genistein, but not estradiol or raloxifene, decreased the ratio of alkaline phosphatase mRNA to ectonucleotide pyrophosphatase phosphodiesterase 1 mRNA expression in osteoblasts. This difference may explain the lack of genistein effect on bone mineralization observed in ovariectomized rats in the in vivo study. Concluding, our experiments demonstrated profound differences between the activities of genistein, estradiol and raloxifene towards the osseous tissue in experimental conditions.

Effects of raloxifene on development of the methotrexate-induced changes in bone mechanical properties of male rats.

Methotrexate, a cytostatic and immunosuppressive drug, has been reported to deteriorate the osseous system. Raloxifene, a selective estrogen receptor modulator, is used in the prevention and treatment of postmenopausal osteoporosis. There is a lack of data on possible ways of preventing the unwanted skeletal effects of prolonged immunosuppressive therapy. The aim of the present study was to investigate the effects of raloxifene on mechanical properties of the femur in male rats administered methotrexate. The experiments were carried out on mature male Wistar rats, which were divided into 6 groups: controls, rats administered raloxifene hydrochloride (5 mg/kg p.o.), rats administered methotrexate (0.5 mg/kg p.o. or i.m.), and rats administered raloxifene hydrochloride (5 mg/kg p.o.) plus methotrexate (0.5 mg/kg p.o. or i.m.). Raloxifene was administered for 28 days and methothrexate was administered for the first 10 days of the experiment. After 28 days of drug administration, mechanical parameters of the whole femur were determined: the extrinsic stiffness, the ultimate load and the breaking load, deformation caused by the ultimate and breaking loads, and the load causing fracture of the femoral neck. Additionally, the mass of isolated femurs and their mineral and calcium content were determined. Intragastrically or intramuscularly administered methotrexate impaired the endurance of the tested bones. Administration of raloxifene alone had no significant effects on the mechanical parameters of the femur. Administration of raloxifene resulted in a reduction of the adverse changes in the osseous system induced by methotrexate. Concluding, the experiments demonstrated a protective action of raloxifene against the effects of methotrexate on the osseous system in male rats.

Raloxifene similarly affects the skeletal system of male and ovariectomized female rats.

Raloxifene, a selective estrogen receptor modulator, is used for prevention and treatment of osteoporosis in postmenopausal women. Raloxifene use in male subjects is increasingly considered and a few clinical studies of its effect on bone turnover have already been performed. The aim of the present study was to investigate the effects of raloxifene on the skeletal system of healthy mature male rats. The experiments were performed on mature male Wistar rats, treated daily with raloxifene hydrochloride at a dose of 5 mg/kg po for 4 weeks. Bone mass, mineral content, macrometric and histomorphometric parameters, as well as mechanical properties were examined. For comparison, we also studied the effects of raloxifene on the skeletal system of mature ovariectomized female rats. Raloxifene administration to male rats caused statistically significant increases in the bone mass/body mass ratio, bone mineral content/body mass ratio and bone mineral content/bone mass ratio in comparison with those of the control rats. Bone mechanical properties and most of histomorphometric parameters remained unchanged. Also in ovariectomized female rats, raloxifene administration caused statistically significant increases in the bone mass/body mass ratio, bone mineral content/body mass ratio and bone mineral content/bone mass ratio in comparison with the results obtained in the ovariectomized control rats, to the level of sham-operated control rats. Moreover, raloxifene counteracted the development of changes in histomorphometric parameters caused by ovariectomy in female rats, but did not significantly affect bone mechanical properties. In conclusion, the changes induced by raloxifene in the skeletal system of male rats were similar to those induced by the drug in ovariectomized female rats.

Effects of standard heparin and low-molecular-weight heparins on the formation of murine osteoclasts in vitro.

Long-term administration of heparin can lead to development of osteoporosis. The aim of the present study was to examine the effects of standard heparin and low-molecular-weight heparins (nadroparin, enoxaparin, parnaparin and dalteparin) on osteoclast formation from neonatal murine bone marrow cells stimulated by 1,25-dihydroxyvitamin D3 in vitro. Standard heparin (0.1-10 IU/ml) and low-molecular-weight heparins (1-100 anti-Xa IU/ml) affected the formation of osteoclasts in two directions. In the rat bone marrow cell cultures, at lower concentrations, the heparins tended to increase the formation of osteoclasts, whereas at the highest concentrations (10 IU/ml and 100 anti-Xa IU/ml, respectively) they tended to decrease the number of osteoclasts. In the mouse bone marrow cell culture, the heparins suppressed the formation of osteoclasts, with the exception of standard heparin at 0.1 IU/ml which intensified the process. Results of the present study indicate species differences in the sensitivity of bone marrow cells to standard heparin and low-molecular-weight heparins.

In vivo effects of high-dose methotrexate on bone remodeling in rats.

Methotrexatae (MTX) is a folate antagonist. MTX osteopathy is well recognized to accompany a high-dose therapy with this drug for the treatment of childhood malignancy. Clinical tests also show that low-dose MTX used in the treatment of rheumatoid arthritis may impair bone formation in a population already predisposed to osteoporosis. However, results of clinical tests are hard to interpret, as it is necessary to take into account malignancy-induced changes in the osseous tissue, long-term immobility and concurrent administration of glucocorticosteroids. We conducted in vivo tests to evaluate the effects of oral and intramuscular administration of high dose of MTX on bone remodeling processes in rats. Effects of MTX on the processes of bone remodeling were evaluated by assessing macrometric and histomorphometric parameters as well as mechanical properties of the femur. The tests were carried out on male Wistar rats. Animals were divided into four groups, composed of 7 animals each: Control group (0.9% NaCl solution), MTX-1 po group (MTX at the dose of 1 mg/kg po daily for 10 days: every day for the first five days, and after an 18-day interval, every day for five days), MTX-1 im group (MTX at the dose of 1 mg/kg im daily for 10 days: every day for the first five days, and after an 18-day interval, every day for five days), MTX-5 im group (MTX at the dose 5 mg/kg im daily for 2 days a week for the period of four weeks). Changes in bone remodeling were examined 4 weeks after the first MTX administration. These results show that MTX administered intramuscularly at high doses inhibited the formation and mineralization of new osseous matrix and impaired mechanical properties of the femoral bone, whereas its oral administration had no effect on bone remodeling in rats.

Effects of 6-mercaptopurine (6-MP) on histomorphometric parameters of the rat bones.

6-Mercaptopurine (6-MP) is an antimetabolite. The drug inhibits DNA synthesis of cells by acting as a false analog of purines. 6-MP displays a cytostatic and immunosuppressive activity. The studies carried out so far focus mostly on recognizing the clinical effectiveness of 6-MP, particularly in treating autoimmune diseases, whereas its effect on the osseous tissue still remains unknown. We examined the effect of 6-MP on the osseous system in rats after a 4-week period of its administration by analyzing histomorphometric parameters and bone mass, mineral and calcium content. We also tested the reversal of the above changes after the lapse of 4 week-period during which 6-MP was not administered. Male Wistar rats were divided into 6 groups, each group was composed of 8 animals. Treatment of 6-MP started on day 2 of the experiment, and the drug was administered for 28 days, i.e. between day 2 and 29 in all groups examined. Changes in the histomorphometric parameters were examined in 3 groups 4 weeks after the first 6-MP or 0.9% NaCl administration: I - control group (0.9% NaCl solution), II - 6MP group (6-MP at the dose of 2.5 mg/kg po), III - 6-MP group (6-MP at the dose of 5 mg/kg po). Reversal of the changes was examined 8 weeks after the first 6-MP or 0.9% NaCl administration in 3 groups: IV - control group (0.9% NaCl solution), V - 6-MP group (6-MP at the dose of 2.5 mg/kg po), VI - 6-MP group (6MP at the dose of 5 mg/kg po). We demonstrated that 6-MP administered orally at a dose of 2.5 mg/kg po or at a dose of 5 mg/kg po for the period of 4 weeks inhibited the synthesis and mineralization of the osseous tissue by causing a disorder in histomorphometric parameters, which were not totally reversed after 4 weeks. 6-MP administered at a dose of 5 mg/kg also caused a reduction of mineral and calcium content, mostly in the cancellous bone, which returned to normal after 4 weeks.

Differential effects of genistein, estradiol and raloxifene on rat osteoclasts in vitro.

Genistein, a major phytoestrogen of soy, is considered a potential drug for prevention and treatment of postmenopausal osteoporosis. It is not clear whether mechanism of action of genistein on bone turnover is distinct from that of estradiol or raloxifene. The aim of the present study was to investigate the effects of genistein on the formation of osteoclasts from neonatal rat bone marrow cells in vitro, and compare them with those of estradiol and raloxifene. Formation of osteoclasts was stimulated by 1,25-dihydroxyvitamin D3, added to the culture media on the second day after plating, together with genistein (10(-8)-10(-5) M), estradiol (10(-10)-10(-7) M) or raloxifene (10(-9)-10(-6) M). The bone marrow cell culture lasted 7 or 9 days. Number of osteoclasts and number of osteoclast "ghosts" (necrotic giant cells) were determined. Genistein, estradiol and raloxifene, at some concentrations, decreased the number of osteoclasts after 9-day culture of bone marrow cells. Genistein decreased the number at 10(-8) M because of decreasing the viability of osteoclasts, whereas at 10(-5) M due to attenuation of osteoclast formation. Estradiol decreased the osteoclast number at 10(-9) M due to decreasing their viability, whereas at 10(-8) and 10(-7) M it was the effect of both decreasing the viability and inhibition of the formation. Decreases in the number of osteoclasts caused by raloxifene (10(-9), 10(-8) M were the effect of decreasing the viability of these cells.