Marta Ladizesky

Melatonin effects on bone: experimental facts and clinical perspectives

Abstract: Bone formation proceeds through a remodeling process that runs continuously, involving the resorption of old bone by osteoclasts, and the subsequent formation of new bone by osteoblasts. This is controlled by growth factors and cytokines produced in bone marrow microenvironment and by the action of systemic hormones, like parathyroid hormone, estradiol or growth hormone (GH). One candidate for hormonal modulation of osteoblast and osteoclast formation is melatonin. Because circulating melatonin declines with age, its possible involvement in post‐menopausal and senescence osteoporosis is considered. This review article discusses early studies on melatonin–bone relationships and recent data that suggest a direct effect of melatonin on bone. Melatonin could act as an autacoid in bone cells as it is present in high quantities in bone marrow, where precursors of bone cells are located. Melatonin dose‐dependently augmented proteins that are incorporated into the bone matrix, like procollagen type I c‐peptide. Osteoprotegerin, an osteoblastic protein that inhibits the differentiation of osteoclasts is also augmented by melatonin in vitro. Another possible target cell for melatonin is the osteoclast, which degrades bone partly by generating free radicals. Melatonin through its free radical scavenger and antioxidant properties may impair osteoclast activity and bone resorption. At least in one study melatonin was both inhibitory to osteoclastic and osteoblastic cells. Therefore, the documented bone‐protecting effect of melatonin in ovariectomized rats can depend in part on the free radical scavenging properties of melatonin. Additionally, melatonin may impair development of osteopenia associated with senescence by improving non‐rapid eye movement sleep and restoring GH secretion. Whether melatonin can be used as a novel mode of therapy for augmenting bone mass in diseases deserves to be studied.

Seasonal variations of 25 hydroxyvitamin D and parathyroid hormone in Ushuaia (Argentina), the southernmost city of the world

Serum levels of calcium, phosphorus, alkaline phosphatase, 250HD, 1.25(OH)2D and PTH were studied in a group of 42 children aged 8.5 +/- 1.8 years (X +/- SD) from the city of Ushuaia (latitude 55 degrees S), at both the end of the winter and the end of summer. Calcium, phosphorus, alkaline phosphatase and 1.25(OH)2D serum levels were not different in summer and winter. The levels of serum 25OHD were significantly higher in summer (18.4 +/- 7.3 ng/ml) than in winter (9.8 +/- 3.8 ng/ml P < 0.001). The levels of 25OHD in children with fair or dark skin were similar in winter but were significantly higher in children with fair skin in summer (20.0 +/- 7.2 ng/l vs 15.3 +/- 5.1 ng/ml (P < 0.05). Serum levels of PTH were higher in winter (58.2 +/- 30.5 pg/ml) than in summer (47.9 +/- 28.3 pg/ml) (P < 0.03). The results demonstrate the existence of a population with low serum levels of 25OHD in winter. The higher levels of PTH in winter when serum 25OHD levels are lower could be the cause of the lack of seasonal variation in serum calcium and 1.25(OH)2D levels. Further studies are needed to establish whether these changes besides increasing the incidence of rickets, could also affect the mineral density of the skeleton in the population of this vitamin-D-deficient area.

Melatonin increases oestradiol-induced bone formation in ovariectomized rats

Abstract: To assess the effect of melatonin on bone metabolism in ovariectomized rats, receiving oestradiol therapy or not, melatonin was administered in the drinking water (25 μg/mL water) and oestradiol (10 μg/kg body weight) or vehicle was given subcutaneously 5 days/week for up to 60 days after surgery. Urinary deoxypyridinoline (a marker of bone resorption) and circulating levels of bone alkaline phosphatase activity (a marker of bone formation), as well as serum calcium and phosphorus levels, were measured every 15 days. Bone area (BA), bone mineral content (BMC), bone mineral density (BMD) and total body fat (expressed as 100 g body weight) were measured by dual‐energy X‐ray absorptiometry at the end of the experiment. Body weight and total body fat were augmented after ovariectomy, and decreased after melatonin or oestradiol treatment. The effect of melatonin on body weight was seen in sham‐operated rats only. Ovariectomy augmented, and melatonin or oestradiol lowered, urinary deoxypyridinoline excretion. This effect of melatonin and oestradiol was seen mainly in ovariectomized rats. The efficacy of oestradiol to counteract ovariectomy‐induced bone resorption was increased by melatonin. Melatonin or oestradiol lowered serum bone alkaline phosphatase activity. Melatonin inhibition was seen mainly on the increase of bone alkaline phosphatase activity that followed ovariectomy. Serum phosphorus levels decreased after melatonin administration and were augmented after oestradiol injection; overall, melatonin impaired the increase of serum phosphorus caused by oestradiol. Ovariectomy decreased, and oestradiol increased, serum calcium levels while melatonin augmented serum calcium in sham‐operated rats only. On day 60 after surgery, BMD and content decreased after ovariectomy and were increased after oestradiol injection. Melatonin augmented BA of spine and BMC of whole of the skeleton and tibia. The highest values observed were those of rats treated concurrently with oestradiol and melatonin. The present results indicate that: (i) melatonin treatment restrained bone remodelling after ovariectomy; (ii) the effect of melatonin required adequate concentrations of oestradiol; (iii) melatonin augmented oestradiol effects on bone in ovariectomized rats; (iv) a counter‐regulation by melatonin of the increase in body fat caused by ovariectomy was uncovered. The melatonin doses employed were pharmacological in terms of circulating melatonin levels but not necessarily for some other fluids or tissues.

Effect of melatonin on bone metabolism in ovariectomized rats

To assess the effect of pharmacological dose of melatonin on bone metabolism in ovariectomized rats, urinary deoxypyridinoline (a marker of bone resorption) and calcium excretion, circulating levels of calcium, phosphorus and bone alkaline phosphatase activity (a marker of bone formation), and bone mineral density (BMD), mineral content (BMC) and bone area (BA) of total body, were measured in adult rats for up to 60 days after surgery. Rats received melatonin in the drinking water (25 microg/ml water) or drinking water alone. Urinary deoxypyridinoline increased significantly after ovariectomy by 51% (30 days after surgery) and by 47% (60 days after surgery). The increase in urinary deoxypyridinoline found 30 days after ovariectomy was not observed in melatonin-treated rats. Urinary calcium concentration was similar in the 4 experimental groups studied, as was the circulating calcium concentration at every time interval examined. Fifteen days after surgery, a significant increase in serum phosphorus and bone alkaline phosphatase levels occurred in ovariectomized rats receiving melatonin as compared to their controls. Sixty days after surgery BMD, BMC and BA decreased significantly in ovariectomized rats, an effect not modified by melatonin. Serum estradiol decreased significantly by 30 days after ovariectomy to attain values close to the limit of detection of the assay by 60 days after ovariectomy. The results support the conclusion that a pharmacological amount of melatonin modifies bone remodeling after ovariectomy and that the effect may need adequate concentrations of estradiol.

Changes in Parathyroid Hormone and Calcium Levels after Superior Cervical Ganglionectomy of Rats

To assess the role of peripheral sympathetic nerves in the regulation of parathyroid hormone (PTH) release rats subjected to bilateral superior cervical ganglionectomy (SCGx) 8-24 h earlier were used. SCGx did not result in significant changes of basal serum calcium (Ca) or immunoreactive PTH (iPTH) levels. The i.p. administration of EDTA every 30 min for up to 3 h brought about an impending decrease of serum Ca levels in both sham-operated and SCGx rats when assessed 23 h after surgery. The extent of hypocalcemia was significantly larger in the SCGx group. In sham-operated controls serum iPTH increased by 32-145%, 1-3 h after beginning EDTA treatment whereas such increases were considerably lower or absent in SCGx rats. In 3 out of 7 SCGx animals that survived to a double EDTA dose, iPTH increased to levels indistinguishable from controls. When SCGx rats subjected to surgery 23 h earlier and receiving d-1-propranolol (5 mg/kg) or phentolamine (10 mg/kg) 4 h and 30 min earlier were submitted to iterative EDTA injection serum iPTH increased, whereas no changes were detected in SCGx rats treated with vehicle and subjected to the same EDTA treatment. These data indicate that (1) SCGx does not affect basal release of iPTH or serum Ca concentration; (2) 23-26 h after SCGx there is a significant impairment of homeostatic iPTH responses to low Ca levels which can be overcome by suitable Ca stimulus; (3) circulating catecholamines may affect denervated parathyroid cells, as revealed by the changes in serum iPTH and Ca elicited by alpha- and beta-adrenoceptor-blocker treatment of SCGx rats.

Effect of unilateral superior cervical ganglionectomy on bone mineral content and density of rat's mandible.

To assess the effect of a local sympathectomy on bone metabolism, the effect of a unilateral superior cervical ganglionectomy (Gx) on growth and bone mineral content and density of the ipsi- and contralateral mandibles was examined in female rats. A significant increase in the hemi-mandibular bone ipsilateral to Gx was found as compared to the contralateral, sham-operated side 30 days, but not 15 days, after surgery. Bone mineral content of the hemi-mandibular bones was significantly lower in the side ipsilateral to Gx in the group of rats killed on the 30th day after surgery. Since no difference in areas between innervated and denervated hemi-mandibles was found, bone mineral density was also significantly lower in the hemi-mandible ipsilateral to Gx. The results further support that a regional sympathectomy causes qualitative alterations in bone modeling and remodeling, leading to bone resorption.

Involvement of the Cervical Sympathetic Nervous System in the Changes of Calcium Homeostasis during Turpentine Oil-Induced Stress in Rats

Hypocalcemia is a common finding during stress. The objective of this study was to examine: (a) the changes in circulating calcium, parathyroid hormone (PTH) and calcitonin (CT) concentration in rats stressed by being given a subcutaneous injection of turpentine oil, and (b) the involvement of the sympathetic cervical pathway in stress-induced changes of calcium homeostasis. Four hours after receiving turpentine oil or vehicle, rats were subjected either to hypocalcemia, by being given EDTA intraperitoneally, or to hypercalcemia, by being injected CaCl2 intraperitoneally. Significant changes in serum calcium (10% decrease), serum PTH (28% increase) and CT levels (40% decrease) were observed in stressed rats. EDTA administration brought about a significantly greater hypocalcemia, and a higher PTH secretory response in turpentine oil-stressed rats. During stress, the increase of serum calcium after CaCl2 was significantly smaller, and the rise of CT was greater than in controls. In the case of CT the changes were still observed in rats subjected to superior cervical ganglionectomy (SCGx) 14 days earlier. In the case of PTH, the increase found in stressed rats, but not the augmented response after EDTA, was blunted by SCGx. The potentiation of hypocalcemia brought about by turpentine oil was no longer observed in SCGx rats. In vehicle-treated controls, SCGx delayed PTH response to hypocalcemia, but did not affect the increased response of CT to CaCl2 challenge. The results indicate that a number of changes in calcium homeostasis arise during turpentine oil stress in rats. SCGx was effective to modify the set point for PTH release, but played a minor role in affecting the augmentation of CT release during stress.

Changes in Calcitonin Release during Sympathetic Nerve Degeneration after Superior Cervical Ganglionectomy of Rats

To assess the role of peripheral sympathetic nerves in the regulation of calcitonin release, rats subjected to superior cervical ganglionectomy (SCGx) 16-28 h earlier were used. The time periods selected allowed us to examine C cell response during the supraliminal release of sympathetic transmitter that accompanies anterograde degeneration of nerve varicosities as well as during the neural paralysis that ensues thereafter. At the time intervals examined, SCGx did not result in significant changes of basal serum calcitonin or Ca levels. The intraperitoneal administration of CaCl2 brought about an impending increase of serum Ca to the same extent in SCGx and sham-operated rats. A significant depression of calcitonin release was observed in rats killed around the time of nerve terminal degeneration (16-21 h post SCGx) but not about 10 h later. Additionally a delay to achieve a maximal calcitonin response was apparent during nerve degeneration. Injection of the alpha-adrenoceptor blocker phenoxybenzamine significantly increased basal calcitonin levels and restored the depressed calcitonin response to hypercalcemia seen in SCGx rats. Treatment with the beta-adrenoceptor-blocker propranolol counteracted phenoxybenzamine activity but was unable to modify per se calcitonin release in SCGx or sham-operated rats. Basal Ca levels and their increase after intraperitoneal CaCl2 were similar in all examined groups regardless of the drug injected. In an additional experiment phenoxybenzamine injected into SCGx rats in doses one-fifth those employed earlier still reversed both the depression in maximal calcitonin response as well as the delay to attain maximal release after CaCl2, but was unable to affect basal calcitonin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of unilateral superior cervical ganglionectomy on mandibular incisor eruption rate in rats.

To assess the effect of sympathectomy on rat tooth eruption, the effect of a unilateral superior cervical ganglionectomy (SCGx) on eruption rate of ipsi- and contralateral lower incisors was examined. Two experiments were performed. In a first experiment, the eruption rate of ipsilaterally denervated incisors was similar to that of contralaterally innervated incisors, when assessed for up to 28 days after surgery. In a second experiment, under conditions of unilateral unimpeded eruption of incisors performed ipsilaterally or contralaterally to a unilateral SCGx, a significantly lower eruption rate of denervated incisors at the impeded eruption side, and a significantly higher eruption rate of denervated incisors at the unimpeded side were observed, when computed every 2 days. Significant differences in individual Students t tests at every time interval occurred mainly during the first and the last week of examination. When average daily eruption rate was computed in weekly intervals, a significant interaction between SCGx and the side of impeded or unimpeded eruption was found in a factorial ANOVA, that is, for each of the 4 weeks of examination, sympathetically denervated incisors showed lower eruption rates at the impeded eruption side, and higher eruption rates at the unimpeded side. These results indicate that incisor eruption is not modified by a local sympathetic denervation unless the contralateral lower rat incisor is cut out of occlusion.

Compensatory parathyroid hypertrophy after hemiparathyroidectomy in rats feeding a low calcium diet

SummaryThe functional and anatomic compensatory response of the parathyroid gland was examined in hemiparathyroidectomized (HPTx) rats whose parathyroid hormone (PTH) secretion was stimulated by a low calcium diet. These responses were compared with those observed in the thyroid gland of hemithyroidectomized (HTx) rats. Rats kept on a low calcium diet for 10 days were subjected to HPTx, HTx, or sham operations. Throughout the experiment (up to 28 days after surgery), serum calcium levels of HPTx rats were lower than the basal, with Δ values (mg/dl, mean±SEM) of −0.66±0.17 and −0.84±0.17, (P<0.05) 3 and 28 days after surgery, respectively. Serum PTH decreased significantly from 7 to 21 days after HPTx, reaching normality at day 28 after surgery. In HTx rats, serum thyroxine (T4) levels diminished significantly 7 days after surgery, and attained normality thereafter. The mitotic index (number of metaphases/1,000 cells) in parathyroid glands of colchicine-treated HPTx rats increased significantly in comparison to sham-operated controls, when examined 2 or 40 days after surgery. The mitotic index of thyroid follicular cells was significantly higher than that of their respective controls, 2 but not 40 days after HTx. These results indicate that after HPTx, a delayed compensatory response is found when the animals are kept under a low calcium diet. Parathyroid response is both delayed and of a minor degree compared to that found in the thyroid gland after HTx.