Mechanical properties, bone mineral content, and bone composition (collagen, osteocalcin, IGF-I) of the rat femur: influence of ovariectomy and nandrolone decanoate (anabolic steroid) treatment.

Nandrolone decanoate (ND) is an anabolic steroid with a positive effect on bone mass in osteoporotic patients. The mechanism of action, (i.e., reduction of bone resorption and/or stimulation of bone formation), the ultimate effect on mechanical properties, and the most effective dosage are not yet clear. To address these issues, dose-related effects of the long-term effect of ND on serum and bone biochemistry, bone mineral content, and bone mechanical properties in ovariectomized (OVX) rats (12 weeks old at the start of the experiment) were studied for 6 months. The results were compared with those obtained in age-matched, intact, and OVX rats. OVX caused in the femur a significant increase in net periosteal bone formation and net endosteal bone resorption of bone collagen content and torsional strength, and of serum alkaline phosphatase, osteocalcin, and insulin-like growth factor-I (IGF-I) levels, whereas cortical bone density and calcium/creatinine and phosphorus/creatinine in 24-hour urine were significantly reduced. Treatment of OVX rats with 1 mg ND/14 days resulted in a significant increase in periosteal bone formation, femur length, cortical and trabecular bone mineral content and density, torsion stiffness and strength, and bone IGF-I content, and a decrease in serum osteocalcin, urinary calcium/creatinine levels, and bone collagen content compared with OVX controls. The higher ND dosage of 2.5 mg/14 days did not improve the results. ND treatment did not reverse all changes induced by OVS to the level of the intact controls. These results indicate that ND acts as an antiresorptive drug and as a home formation stimulating drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone and mineral metabolism in the androgen-resistant (testicular feminized) male rat.

Androgens have important effects on bone in vivo, possibly by direct activation of the androgen receptors in osteoblasts. To test this hypothesis, calcium homeostasis, bone mass, and bone turnover were evaluated in mature (4-month-old) androgen-resistant (testicular feminized, TFM) male rats. Data were compared with data from both female and male littermates of the same age and strain. Compared to normal males, TFM had similar serum testosterone, twofold higher estradiol and estrone, and sixfold higher androstenedione concentrations. Compared to normal females, TFM rats showed lower estradiol but also elevated concentrations of androstenedione and estrone. Despite similar free 1,25-(OH)2D3 concentrations, both TFM and male rats maintained higher serum calcium and phosphate concentrations than their female littermates. Serum IGF-I concentrations in TFM rats were decreased compared to male rats (-12%) or female rats (-27%). Serum osteocalcin concentrations, however, were twofold higher in TFM rats than in females but not significantly different from males. Femoral length, diameter, and cortical thickness were intermediate between those of males and females. The cancellous bone density of the femur and cancellous bone volume of the proximal metaphysis of the tibia, however, were not significantly different between groups. The ash weight of the tibia was also not significantly different, and the ash weight of the four distal lumbar vertebrae ranged between male and female values. Bone mechanical properties as measured by torsional strength and energy absorption of the femur were lower in TFM than in females but not different from males.(ABSTRACT TRUNCATED AT 250 WORDS)

The action of anabolic steroids on bone in experimental animals.

Anabolic steroids are currently used in the treatment of established osteoporosis. It has been demonstrated that, at least partly, anabolic steroids increase bone density by stimulating bone formation. Very little is known about how anabolic steroids affect bone in experimental animals. Because bone studies in animals have been performed only with the anabolic steroid nandrolone, or its long-acting ester nandrolone decanoate (ND), we present a general overview in this paper of the effect of these anabolic agents in various steroid-affected animal models for osteoporosis, viz. gonadectomized rats, heparin-treated mice and intact or ovariectomized dogs. In rats and mice these agents increase longitudinal and periosteal bone growth and bone mass, thus demonstrating their anabolic action. They also decrease trabecular bone resorption in ovariectomized and orchidectomized rats, which indicates that they have anti-catabolic effects. In ovariectomized rats, ND was found to increase the mechanical strength of cortical bone, which is an important property in a drug that is intended to be used in treating established osteoporosis. In elderly dogs, ND was found to stimulate endosteal bone formation. These findings indicate that nandrolone and ND have beneficial effects on bone in both oestrogen and androgen-deficient animals.

Bone and mineral metabolism in aged male rats: short and long term effects of androgen deficiency.

Both short and long term effects of androgen deficiency and steroid replacement therapy on skeletal homeostasis were investigated in aged (13-month-old) male rats. The animals were either sham operated (n = 28) or orchidectomized (orch; n = 89). The orch animals were divided into 5 groups; 26 rats received an empty sc Silastic implant (orch), all others received an implant containing testosterone (T), 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E2), or nandrolone (Nandro; 15-16 rats in each group). Half of the rats were killed 1 month (short term experiment) after implantation; the others were killed 4 months after implantation (long term experiment). Short term androgen deficiency caused a significant increase in both serum osteocalcin and histomorphometric parameters of bone turnover measured at the proximal tibial metaphysis, but not in a significant decrease in bone mass at this site. This increase in bone turnover was prevented not only by T and DHT, but also by E2 and Nandro. Long term androgen deficiency resulted in a decrease in the calcium content of both tibia and lumbar vertebrae. Cancellous bone volume in the proximal tibial metaphysis was +/- 50% lower in the orch group (P less than 0.001) 4 months after orchidectomy. At the same time, cortical bone was lost in orch rats; femoral cortical thickness was reduced by 12% (P less than 0.01), and cortical density tended to be lower. T, DHT, E2, or Nandro treatment completely prevented this decrease in cortical thickness and density. T and Nandro were also able to prevent the cancellous bone loss. DHT could only partly prevent cancellous bone loss. E2 treatment resulted not only in a sustained decrease in both serum osteocalcin concentrations and histomorphometric indices of bone turnover, but also in a net gain of cancellous bone volume (P less than 0.05 vs. sham). No significant differences in serum concentrations of vitamin D metabolites or nephrogenous cAMP were observed between groups in both short and long term experiments. We conclude that bone mass in aged male rats was significantly decreased 4 months after orchidectomy, preceded by an early increase in bone turnover. Both the early increase in bone turnover and the later decrease in bone mass were prevented by aromatizable and nonaromatizable androgens by estrogen and by nandralone.

Oestrogen and progestogen synergistically stimulate human and rat osteoblast proliferation.

Oestrogens play an important role in bone metabolism; they preserve bone mass after the menopause. Their action in bone has recently been shown to be, partly, a direct one, as oestrogen receptors and their effects have been demonstrated in bone cells. The role of progestogens in bone metabolism is less clear. In this study it has been shown that 17 beta-oestradiol exerts only a small, although not significant, stimulatory action with regard to SaOS-2 human osteosarcoma cell proliferation. A pure progestogen (Org 2058) has no effect when added alone. In combination with 17 beta-oestradiol, however, it has a highly synergistic action on SaOS-2 cell proliferation. The same effect was observed in primary rat osteoblasts, showing that this synergism is a general phenomenon in osteoblastic cells. High numbers of oestrogen and progestogen receptors have been demonstrated in SaOS-2 cells, indicating that the effects of these steroids are mediated via the normal route of steroid receptors. These data provide a cellular basis for the clinically recognized positive effect of oestrogen/progestogen combinations on bone formation.

Osteoclast formation together with interleukin-6 production in mouse long bones is increased by insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) is a potent stimulator of bone formation. Whether this growth factor also induces bone resorption has not been studied in detail. We used two organ culture systems to examine the direct effect of IGF-I on bone resorption. Fetal mouse radii/ulnae, containing mature osteoclasts, showed no response to IGF-I, indicating that osteoclastic activity is not influenced by IGF-I. Fetal mouse metacarpals/metatarsals, containing just osteoclast precursors and progenitors, showed an increase in resorption in response to IGF-I, indicating that IGF-I stimulates the formulation of osteoclast precursors/progenitors and thereby increases the number of osteoclasts. Interleukin-6 (IL-6) has been hypothesized to be a mediator of bone resorptive agents such as parathyroid hormone (PTH). Both radii/ulnae and metacarpals/metatarsals reacted to IGF-I with an increase in IL-6 production. IL-6 production by UMR-106 osteogenic osteosarcoma cells was positively modulated by IGF-I, indicating that osteoblasts are likely to be the cells responsible for increased IL-6 production by the bones, and that IL-6 might be a mediatory of IGF-I-stimulated bone resorption.

Calcium-deficient diet in ovariectomized dogs limits the effects of 17 beta-estradiol and nandrolone decanoate on bone.

Postmortem measurements by dual-photon absorptiometry of the femur and the second lumbar vertebra in adult dogs indicated bone loss after ovariectomy, which was more pronounced when calcium-deficient diet was given in ovariectomized dogs. This bone loss was nonhomogeneous throughout the femur. Ovariectomy resulted in trabecular and cortical bone loss, and additional calcium-deficient diet resulted in a further highly significant trabecular bone loss at the proximal epiphysis of the femur and in the vertebra. This bone loss was presumably the result of increased bone turnover, as reflected by the highly significant increase in serum alkaline phosphatase. Estrogens could only partially prevent the bone loss induced by calcium deficiency after ovariectomy, and nandrolone decanoate was not effective. We conclude that (1) ovariectomy results in bone loss in adult dogs, (2) this bone loss is more pronounced after calcium-deficient diet, (3) calcium deficiency could be a limiting factor for the preventive effect of estrogens and nandrolone decanoate, and (4) dual-photon absorptiometry allows the evaluation of nonhomogeneous bone loss throughout excised bones.

Short-term effects of Org OD 14 and 17 beta-oestradiol on bone and lipid metabolism in early post-menopausal women.

The effects of 8 weeks of daily oral treatment with 1 mg 17 beta-oestradiol (E2), 2.5 mg Org OD 14 [7 alpha, 17 alpha)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one) , a steroid with weak androgenic, weak oestrogenic and weak progestational activity, or placebo on calcium and lipid metabolism were compared in 21 healthy, early post-menopausal women in a randomised double-blind study. The treatment period was followed by a treatment-free period of 8 weeks to study the reversibility of drug-induced effects. The results show that both E2 and Org OD 14 reduce bone resorption, as indicated by the decreases in the urinary hydroxyproline/creatinine and calcium/creatinine ratios in 2-h fasting urine. In contrast to E2, Org OD 14 did not reduce serum calcium levels. As regards lipid parameters, E2 reduced the concentration of serum cholesterol and Org OD 14 decreased serum levels of high-density-lipoprotein cholesterol and triglycerides. All these effects appeared to be reversible after cessation of treatment. It is concluded that both of these steroids reduce bone resorption in early post-menopausal women, but that their mechanisms of action are most likely different.

Non-linear increase in vertebral density induced by a synthetic steroid (Org OD 14) in women with established osteoporosis.

The results of a 2-year placebo-controlled study in 38 female patients with osteoporosis are presented. This study was conducted to evaluate the efficacy of a daily oral dose of 2.5 mg Org OD 14 ((7 alpha, 17 alpha)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one) in the treatment of established osteoporosis. Org OD 14 is a steroid which shows combined weak oestrogenic, androgenic and progestational activity. A total of 31 patients completed a 12-month study period (17 placebo, 14 Org OD 14) and 25 of these went on to complete the full 24-months (15 placebo, 10 Org OD 14). A significant increase in bone mineral density as measured by dual photon absorptiometry was recorded in the lumbar spine in the Org OD 14-treated patients at 8, 16 and 24 months. The gain in bone mass after 8 months averaged 4% (P less than 0.01) and after 24 months 8% (P less than 0.001). In the control group, a bone loss rate of 2% per year was recorded in the lumbar spine. No significant changes in bone density in the forearm as assessed by single photon absorptiometry were found in either group. The increase in spinal bone density in the Org OD 14 group was non-linear and followed an S-shaped upward pattern. Org OD 14, while inducing no appreciable endometrial stimulation, was found to be a bone-active compound with anti-resorbing as well as anabolic activity. Org OD 14 warrants consideration not only for the long-term prevention of bone loss but also for curative treatment of post-menopausal osteoporosis.

Pathophysiology of bone loss in castrated animals.

The pathophysiology of bone loss in castrated animals is reviewed. Both male and female rats rapidly lose metaphyseal trabecular bone from the tibia and the femur due to an imbalance between bone resorption and bone formation. The aetiology of sex hormone deficiency-induced bone loss is not fully understood. It seems unlikely that the bone loss is due to changes in the circulating levels of the calciotropic hormones or to an increase in the spontaneous release from peripheral blood monocytes of the bone resorption stimulating cytokine IL-1. Changes in the sensitivity of bone of castrated rats to calciotropic hormones may play a role as well as the lack of direct stimulatory effects of gonadal oestrogens and androgens on bone cells. In addition several data indicate that prostaglandins may be involved.

Sex steroids and osteoporosis: effects of deficiencies and substitutive treatments.

Adult mammalian bone is continuously renewed by the process of remodelling. In young healthy adults the amount of bone that is resorbed by osteoclasts is replaced by osteoblasts so that no net loss of bone occurs. In a situation of reduced sex hormone levels, such as in females after menopause or ovariectomy, in males after orchidectomy, or in patients of either sex with gonadal dysfunction, there is an imbalance between bone resorption and bone formation resulting in bone loss. The various hypotheses to explain the aetiology of this imbalance are reviewed. Substitution therapy of females with oestrogen results in the prevention of oestrogen deficiency-induced bone loss. It is generally agreed that the effect is due to inhibition of bone resorption. Recent in vitro data, however, indicate that oestrogens also have the capacity to stimulate the proliferation and functioning of bone-forming cells. Prevention of oestrogen deficiency-induced bone loss can also be achieved by treatment with high doses of progestagens. Available data suggest that this too is caused by resorption inhibition. The aim of treatment of females, who have lost so much bone that there is an increased risk of fractures after minimal trauma, is to increase bone mass rather than to prevent further bone loss. This can be accomplished by treatment with anabolic steroids. Both biochemical and histological data indicate that anabolics stimulate the activity of functioning osteoblasts. The increase in bone mass during continuous treatment is temporary because anabolics most probably also inhibit bone resorption. Substitution therapy with anabolics or androgens in males is equally effective and increases trabecular bone mass in the spine.

Therapeutic effects of nandrolone decanoate, tibolone, lynestrenol and ethylestrenol on Sjögren's syndrome-like disorder in NZB/W mice.

Growth of mononuclear cell infiltration in submandibular glands is significantly inhibited by Org OD14 (tibolone), lynestrenol and ethylestrenol given orally to New Zealand Black/White (NZB/W) mice from 26 weeks of age onwards. In addition, the extent of already established mononuclear cell infiltrations is significantly inhibited and reduced by nandrolone decanoate injected from 43 weeks of age onwards. Tibolone and nandrolone decanoate are the most potent of the four drugs. The therapeutic effect of these four steroids on the Sjögren's syndrome-like disorder in NZB/W mice is not related to their endocrine activities.

Effects of sex, gonadectomy and several steroids on the development of insulin-dependent diabetes mellitus in the BB rat.

The spontaneous development of the putative autoimmune insulin-dependent diabetes mellitus (IDDM) in BB rats was not affected by the following factors: (a) sex, (b) gonadectomy, (c) administration of testosterone decanoate, 11 alpha-hydroxynandrolone decanoate, ethinyloestradiol or Org OD14 (tibolone). Treatment with nandrolone decanoate lead to a statistically significant decrease in the incidence of diabetes in male rats in one experiment, but failed to do so in the second. These results and various data from the literature suggest that the influence of sex and steroids on the development of diabetes mellitus in the BB rat is different to that of autoimmune disorders in other animal models (New Zealand Black/White, New Zealand Black mice, Obese Strain chickens) and in man.

Effects of tibolone, lynestrenol, ethylestrenol, and desogestrel on autoimmune disorders in NZB/W mice.

The effects of two progestagens--lynestrenol, desogestrel--an anabolic steroid--ethylestrenol--and a compound with weak progestational, anabolic, androgenic, and estrogenic activities--tibolone--on the development of systemic lupus erythematosus and Sjögren's syndrome-like disorders were studied in the NZB/W mouse. All four compounds inhibited the expression of autoimmune disease. Tibolone was 10-40 times more potent--depending on the parameter used--in preventing symptoms of autoimmunity than the second most effective compound lynestrenol. Ethylestrenol was the third effective compound and desogestrel the least effective compound in this series. Combined with literature data, these results show that steroids with different endocrine profiles can prevent the development of autoimmunity in the NZB/W mice. Since the NZB/W mouse is a good animal model for human systemic lupus erythematosus and Sjögren's syndrome and since tibolone, lynestrenol, and ethylestrenol have endocrinological profiles which are not prohibitive for treatment of male and female patients, investigation whether these compounds have a value in the treatment of human autoimmune diseases seems warranted.

Experimental work with anabolics in autoimmunity models.

19-nortestosterone (nandrolone) or its decanoate was tested in six animal models for autoimmune disease. The compound was found capable of inhibiting the development of disease symptoms or the deterioration of certain immune parameters in three out of five models of spontaneous autoimmune disease: NZB/NZW F1 (NZB/W) mice (lupus glomerulonephritis, Sjögren's disease-like syndrome), NZB (lupus glomerulonephritis, autoimmune haemolytic anaemia) and OS chickens (thyroiditis). No effect of the compound was seen in MRL lpr/lpr mice (lupus glomerulonephritis, lymphoproliferative disease) and in BB rats (insulin-dependent diabetes mellitus; IDDM). Nandrolone decanoate also seems to be active in myasthenia gravis evoked in rats immunised with acetylcholine receptor but further experiments are necessary to confirm this. In a first experiment also ethylestrenol was found to give favourable effects in the model.

Immunocytochemical demonstration of neuropeptides and serotonin in the tapeworm Diphyllobothrium dendriticum.

The present immunocytochemical study concerns the distribution of four neuropeptides, FMRF-amide, vasotocin, leu-enkephalin and neurotensin, and of the bioamine serotonin in the plerocercoid larva of Diphyllobothrium dendriticum. Anti-FMRF-amide and vasotocin-reactivity occurs in perikarya and nerve fibres in the CNS and PNS of this worm. The peptide-containing fibres surround and seem to innervate the musculature and to terminate beneath the basal lamina of the tegument at the inner surface of the bothridia, suggesting a neurotransmitter function. Anti-leu-enkephalin reaction occurs in perikarya and fibres in the main nerve cords and in the PNS. Anti-neurotensin reactive fibres were observed in the neuropile of the nerve cords. Serotonin immunoreactivity was found in neurons in the ganglionic commissure of the brain and along the main nerve cords. This study is the first immunocytochemical identification of neuropeptides and serotonin in a parasitic flatworm and the information gained may be of importance for the development of new antihelminthics.

Effect of nandrolone decanoate on Sjögren's syndrome like disorders in NZB/NZW mice.

The effects of 2.5-40 mg/kg nandrolone decanoate (ND) on the development and growth of mononuclear cell infiltrations in the submandibular glands of NZB/W (B/W) mice have been studied using a quantitative histological method. Injections with ND for 9 months, once every 3 weeks, starting at 4 weeks of age, reduced the number and total area of infiltrations in submandibular glands of female and castrated male B/W mice. Since ND has relatively weak virilizing properties, this substance may be useful for the treatment of Sjögren's syndrome in humans.

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide.

The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.

Characterisation of multiple immunoreactive neurons in the central nervous system of the pond snail Lymnaea stagnalis with different fixatives and antisera adsorbed with the homologous and the heterologous antigens.

In the central nervous system of the pond snail Lymnaea stagnalis a large number of elements (cells and fibers) can be identified with antisera (a-FM) to the molluscan cardioactive tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2). Of these elements some are also reactive to antivasotocin (a-VT) and/or anti-gastrin (a-Gas). These observations suggest that the a-FM positive elements belong to more than one type. Previous results had already indicated that the immunoreactivity of many a-FM positive cells is influenced by the type of fixation. Taking into account the effects of three fixatives on the reactivity of the cells, and their staining characteristics with the two other antisera used, 8 a-FM positive types could be distinguished. Homologous and heterologous adsorptions were carried out to test the specificity of a-FM, a-VT and a-Gas. After homologous adsorptions no staining was obtained. After heterologous adsorptions only part of the multiple staining cells were identified. This indicates that in a-FM, a-VT and a-Gas in addition to (more) selective IgG molecules, less specific IgG molecules occur that can bind to other peptides than those used to raise the antisera (cross-reaction). The (more) selective IgG molecules in a-FM bind to 6 of the a-FM positive types, suggesting that in L. stagnalis a family of FMRFamide-like substances occurs. This conclusion is sustained by results obtained with a-FM adsorbed with fragments of FMRFamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Co-existence of immunoreactivity to anti-dopamine, anti-serotonin and anti-vasotocin in the cerebral giant neuron of the pond snail Lymnaea stagnalis.

Consecutive sections of certain neurons in the central ganglia of the pond snail Lymnaea stagnalis appear to be immunoreactive to anti-dopamine and anti-serotonin. The Cerebral Giant Neurons stain in addition with anti-vasotocin. The observations indicate the presence of two biogenic amines within the same neuron and in addition their co-existence with a biologically active peptide.