Lack of micronuclei formation in bone marrow of rats after oral exposure to thiocyclam insecticide

In this study, a nereistoxin analogue insecticide, thiocyclam, was administered to adult male albino rats by gavage dose of 135, 270 and 540 mg/kg b.w. repeated for 5 days at 24 h intervals. Control animals received only water. Thiocyclam was tested for its potential to cause genotoxic effects in rat bone marrow cells using an in vivo micronucleus assay. After 24 h of the last treatment, rats from all dose levels were sacrificed. Bone marrow cells were collected and assayed for the presence of micronuclei. Thiocyclam did not cause any increase in the incidence of micronucleated polychromatic erythrocytes in rats bone marrow at any of the dose levels. The polychromatic erythrocytes/normochromatic erythrocytes (PCE:NCE) ratio was found to be in the range from 0.50 ± 0.11 to 0.55 ± 0.02. The results of this study demonstrate that the effect of thiocyclam is not significant in the rat in vivo micronucleus assay.     

Animal housing influences the response of bone to spaceflight in juvenile rats.

The rat has been used extensively as an animal model to study the effects of spaceflight on bone metabolism. The results of these studies have been inconsistent. On some missions, bone formation at the periosteal bone surface of weight-bearing bones is impaired and on others it is not, suggesting that experimental conditions may be an important determinant of bone responsiveness to spaceflight. To determine whether animal housing can affect the response of bone to spaceflight, we studied young growing (juvenile) rats group housed in the animal enclosure module and singly housed in the research animal holding facility under otherwise identical flight conditions (Spacelab Life Science 1). Spaceflight reduced periosteal bone formation by 30% (P < 0.001) and bone mass by 7% in single-housed animals but had little or no effect on formation (-6%) or mass (-3%) in group-housed animals. Group housing reduced the response of bone to spaceflight by as much as 80%. The data suggest that housing can dramatically affect the skeletal response of juvenile rats to spaceflight. These observations explain many of the discrepancies in previous flight studies and emphasize the need to study more closely the effects of housing (physical-social interaction) on the response of bone to the weightlessness of spaceflight.     

Lack of micronuclei formation in bone marrow of rats after repeated oral exposure to nickel sulfate hexahydrate

Workplace exposures to mixtures of nickel compounds have been associated with excess respiratory cancer risk. Animal studies with individual nickel compounds indicate that not all nickel substances have the same potency or potential to induce tumors. The bioavailability of nickel ions at critical cellular sites seems to be important to determine the potential of a substance to induce tumors in animals, but much less is understood about the exact nature (genotoxic or non-genotoxic) of the nickel effects. Within many regulatory frameworks (e.g., European Union), substances are classified for mutagenicity based on the available data and this classification will often influence the mode of action assigned to carcinogenic substances and the way in which risk assessment will be conducted. The objective of this study was to evaluate the ability of nickel sulfate hexahydrate to induce micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow. This study was conducted according to OECD and EU protocol guidelines. In the dose range-finding assays, the maximum tolerated dose was estimated to be 500 mg/kg/day. The doses used in the micronucleus assay were 125, 250, and 500 mg/kg/day. At least 2000 PCEs per animal were analyzed for micronuclei in PCEs. Cytotoxicity was assessed by scoring a minimum of 500 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Nickel sulfate hexahydrate did not induce statistically significant increases in micronucleated PCEs at any dose examined. The negative results in the present study contribute significantly to the weight of evidence evaluation of the mutagenicity (chromosomal level) of nickel substances. These results are consistent with a non-genotoxic mode of action for soluble nickel that could explain the enhancement of cancer risk seen among refinery workers with mixed exposures and its lack of carcinogenicity in animal studies with single exposures.     

Discovery of highly selective EP4 receptor agonists that stimulate new bone formation and restore bone mass in ovariectomized rats

Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening. Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated robust bone anabolic effects when administered in vivo in rat models of osteoporosis.     

Additive effect of vitamin K2 and risedronate on long bone mass in hypophysectomized young rats.

Hypophysectomy (HX) arrests bone growth and induces osteopenia in the long bones of rats. The present study investigated the combined effect of vitamin K(2) and risedronate on long bone mass in HX rats, in order to determine whether treatment with these two agents had an additive effect. Forty female Sprague-Dawley rats were hypophysectomized at 6 weeks of age by the supplier, and were shipped to our laboratory at three days after surgery along with ten intact rats that served as age-matched controls. The study was started on the day when the rats were received. Three HX rats were excluded from the study because of the failure of HX. Forty-seven rats (6 weeks old) were assigned to the following 5 groups by the stratified weight randomization method: intact controls, HX alone, HX + vitamin K(2) (30 mg/kg, p.o., daily), HX + risedronate (2.5 microg/kg, s.c., 5 days a week), and HX + vitamin K(2) + risedronate. The dosing period was 4 weeks. HX resulted in a decrease of the femoral bone area, bone mineral content (BMC) and bone mineral density (BMD), as well as a decrease in the cancellous bone mass of the proximal tibial metaphysis and the total tissue and cortical areas of the tibial diaphysis. These changes were associated with a marked reduction in the serum level of insulin like growth factor (IGF)-I and with elevation of serum alkaline phosphatase (ALP) and pyridinoline. Administration of vitamin K(2) increased the serum ALP level in HX rats, but did not affect any of the other parameters. On the other hand, risedronate ameliorated the decrease of femoral BMD and cancellous bone mass at the proximal tibial metaphysis in HX rats without affecting the serum IGF-I level, as a result of not causing a significant elevation of serum pyridinoline. Vitamin K(2) and risedronate combined had an additive effect on the femoral bone area, BMC and BMD, and the combined treatment group did not show any significant reduction of the total tissue and cortical areas at the tibial diaphysis, as well as a reduced serum pyridinoline level compared with untreated rats and an increased serum ALP level compared with untreated or risedronate-treated rats. These results suggest that risedronate had a positive effect on the BMD and cancellous bone mass of long bones in HX rats. Despite the lack of a significant effect of vitamin K(2) on bone mass parameters, it had an additive effect with risedronate on the BMC, BMD and cortical bone mass of long bones in HX rats.     

Lack of prostaglandin effect on sodium balance and hyperreninemia in adrenalectomized rats

Glucocorticoids are known inhibitors of prostaglandin production. Prostaglandin E2 (PGE2) and prostacyclin (PGI2) are promoters of natriuresis and renin release. Excessive prostaglandin production, therefore, might contribute to the altered sodium balance and renin release observed in primary adrenal insufficiency. To test this hypothesis, sodium balance and prostaglandin production were measured in adrenalectomized rats and in animals receiving prostaglandin inhibitors or replacement dexamethasone. Compared to sham-operated controls, adrenalectomized rats had decreased two-day sodium balance and elevated plasma renin concentration (PRC), renal PGE2 production, and renal 6-ketoprostaglandin F1α (6kPGF1α, the nonezymatic metabolite of PGI2); however, no appreciable change in aortic 6kPGF1α production was observed. Dexamethasone given to adrenalectomized rats normalized PRC but had no effect on sodium balance or prostaglandin production. Likewise, prostaglandin inhibitors did not alter the sodium balance or decrease the PRC post adrenalectomy.These data confirm renal prostaglandin production is increased in adrenalectomized rats, but suggest that the elevation is not due directly to glucocorticoid deficiency. Further, PRC levels in adrenal insufficiency do not appear to be prostaglandin mediated. In conclusion, excessive renal prostaglandin production does not contribute to altered sodium balance or increased PRC in adrenalectomized rats.     

Lack of effect of melatonin on sexual maturation in female rats

Daily subcutaneous injections of 100 micrograms melatonin given to prepubertal female rats housed in 14L:10D or 12L:12D failed to delay puberty as evidenced by the age at which vaginal opening occurred; neither the Sprague-Dawley nor the Wistar strain rats were responsive to melatonin treatment. Reproductive organ weights (ovaries and uteri) at vaginal opening were unaffected by such treatment. Administration of melatonin through the drinking water in doses of 100, 500 or 1000 micrograms/day did not alter the timing of puberty or the reproductive organ weights in rats of the Sprague-Dawley or Long-Evans strains (housed in 12L:12D). Our experimental methods are identical to a previous report and we have no explanation for our failure to reproduce the earlier results.     

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS-degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh-heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh-heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild-type (WT) and hpa-transgenic (hpa-tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa-tg mice. Heparanase expression was absent in osteoclasts. Micro-computed tomographic and histomorphometric skeletal analyses in male and female hpa-tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase.     

Lack of effect of dynorphin on consummatory behaviors in obese and normal rats

The possible role of dynorphin, an endogenous opioid peptide, in the regulation of appetite was studied in male genetically-obese (Zucker) rats and their litter mates of normal weight. Eighteen pairs were divided into 3 treatment groups: control, acutely dynorphin-treated (5 mg/rat), and implanted with Alzet mini-osmotic pumps containing 2 mg dynorphin to be delivered at a rate of 10 micrograms/hr. Body weights and food and water consumption were determined daily for 7 days. Body weights were not significantly changed from initial values for any treatment group. Food and water consumption per 24 hours were generally the same for obese rats and their normal littermates, but in terms of consumption per 100 g body weight, the obese rats generally consumed less food and water. Neither acute nor continuous dynorphin administration affected consummatory levels.     

Lack of prostaglandin effect on sodium balance and hyperreninemia in adrenalectomized rats.

Glucocorticoids are known inhibitors of prostaglandin production. Prostaglandin E2 (PGE2) and prostacyclin (PGI2) are promoters of natriuresis and renin release. Excessive prostaglandin production, therefore, might contribute to the altered sodium balance and renin release observed in primary adrenal insufficiency. To test this hypothesis, sodium balance and prostaglandin production were measured in adrenalectomized rats and in animals receiving prostaglandin inhibitors or replacement dexamethasone. Compared to sham-operated controls, adrenalectomized rats had decreased two-day sodium balance and elevated plasma renin concentration (PRC), renal PGE2 production, and renal 6-ketoprostaglandin F1 alpha (6kPGF1 alpha, the nonenzymatic metabolite of PGI2); however, no appreciable change in aortic 6kPGF1 alpha production was observed. Dexamethasone given to adrenalectomized rats normalized PRC but had no effect on sodium balance or prostaglandin production. Likewise, prostaglandin inhibitors did not alter the sodium balance or decrease the PRC post adrenalectomy. These data confirm renal prostaglandin production is increased in adrenalectomized rats, but suggest that the elevation is not due directly to glucocorticoid deficiency. Further, PRC levels in adrenal insufficiency do not appear to be prostaglandin mediated. In conclusion, excessive renal prostaglandin production does not contribute to altered sodium balance or increased PRC in adrenalectomized rats.     

Effects of spaceflight on structural and material strength of growing bone

Rats in space for 18.5 days did not exhibit the normal gain in femoral bone strength of terrestrial controls. The strength deficit may have been caused by multiple factors including a diminished bone formation and an inhibition of the gain in tissue material strength. Centrifugation at 1g in space substantially enhanced bone strength, possibly by promoting more normal tissue maturation. Full recovery of bone strength was achieved 25 days after reentry.     

Progressive isometric force training and bone mass in rats

In ten growing male Wistar rats, isometric strength training for 69 days (3-6 times vertical gripping position on a wire-netting during 2 x 30 s, with progressive loading of the tail through a 50-200 g indwelling clip), fat mass and plasma leptin concentrations were lower on day 70 than in ten sedentary controls. Muscle mass and femoral trabecular and cortical bone mineral density were simultaneously higher in exercised animals than in controls. Such an effect might result from decreased bone resorption. At the end of the training period no difference concerning plasma osteocalcin concentration was observed between exercised and resting rats while urinary deoxypyridinoline excretion was lower in the former than in the latter.     

Conditional deletion of gremlin causes a transient increase in bone formation and bone mass

Gremlin is a glycoprotein that binds bone morphogenetic proteins (BMPs) 2, 4, and 7, antagonizing their actions. Gremlin opposes BMP effects on osteoblastic differentiation and function in vitro and in vivo, and its overexpression causes osteopenia. To define the function of gremlin in the skeleton, we generated gremlin 1 (grem1) conditional null mice by mating mice where grem1 was flanked by lox(P) sequences with mice expressing the Cre recombinase under the control of the osteocalcin promoter. grem1 null male mice displayed increased trabecular bone volume due to enhanced osteoblastic activity, because mineral apposition and bone formation rates were increased. Osteoblast number and bone resorption were not altered. Marrow stromal cells from grem1 conditional null mice expressed higher levels of alkaline phosphatase activity. Gremlin down-regulation by RNA interference in ST-2 stromal and MC3T3 osteoblastic cells increased the BMP-2 stimulatory effect on alkaline phosphatase activity, on Smad 1/5/8 phosphorylation, and on the transactivation of the BMP/Smad reporter construct 12xSBE-Oc-pGL3. Gremlin down-regulation also enhanced osteocalcin and Runx-2 expression, Wnt 3a signaling, and activity in ST-2 cells. In conclusion, deletion of grem1 in the bone microenvironment results in sensitization of BMP signaling and activity and enhanced bone formation in vivo.     

Effect of treadmill exercise on bone mass in female rats.

Increasing peak bone mass at skeletal maturity, minimizing bone loss during middle age and after menopause, and increasing bone mass and preventing falls in advanced age are important measures for preventing osteoporotic fractures in women. Exercise has generally been considered to have a positive influence on bone health. This paper reviews the effects of treadmill exercise on bone in young, adult, ovariectomized, and osteopenic female rats. Treadmill exercise increases cortical and cancellous bone mass of the tibia as a result of increased bone formation and decreased bone resorption in young and adult rats. The increase in lumbar bone mass seems to be more significant when long-term exercise is applied. Treadmill exercise prevents cancellous bone loss at the tibia as a result of suppressed bone resorption in ovariectomized rats, and increases bone mass of the tibia and mechanical strength of the femur, as a result of suppressed bone resorption and increased bone formation in osteopenic rats after ovariectomy. Treadmill exercise transiently decreases the serum calcium level as a result of accumulation of calcium in bone, resulting in an increase in serum 1,25-dihydroxyvitamin D(3) level and a decrease in serum parathyroid hormone level. We conclude that treadmill exercise may be useful to increase bone mass in young and adult rats, prevent bone loss in ovariectomized rats, and increase bone mass and bone strength in osteopenic rats, especially in the long bones at weight-bearing sites. Treadmill exercise may have a positive effect on the skeleton in young, and adult, ovariectomized, and osteopenic female rats.     

Control of red blood cell mass during spaceflight

Data are reviewed from twenty-two astronauts from seven space missions in a study of red blood cell mass. The data show that decreased red cell mass in all astronauts exposed to space for more than nine days, although the actual dynamics of mass changes varies with flight duration. Possible mechanisms for these changes, including alterations in erythropoietin levels, are discussed.     

Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass

Aging reduces the number of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts in the bone marrow, which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct MSCs to the bone surface by attaching a synthetic high-affinity and specific peptidomimetic ligand (LLP2A) against integrin α4β1 on the MSC surface to a bisphosphonate (alendronate, Ale) that has a high affinity for bone. LLP2A-Ale induced MSC migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation studies and in immunocompetent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or as a result of estrogen deficiency. These results provide proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength.     

Lack of effect of butylparaben and methylparaben on the reproductive system in male rats

BACKGROUND: Parabens are widely used preservatives in cosmetics and pharmaceutical products, and approved as food additives. Parabens have been considered safe for these uses for many years. Recently, adverse effects on male reproductive parameters in rats have been reported when parabens were given orally for 8 weeks starting at three weeks of age. Our studies used two representative parabens, methyl‐ and butylparaben, to try to replicate these studies and thereby evaluate potential reproductive effects in male Wistar rats. METHODS: Diets containing 0, 100, 1000 or 10,000 ppm of either butyl‐ or methylparaben were fed to male rats for eight weeks. Rats were 22 days of age at the start of exposure. Parameters evaluated included organ weights, histopathology of reproductive tissues, sperm production, motility, morphology and reproductive hormone levels (butylparaben only). RESULTS: None of the parameters evaluated for either paraben showed compound‐ or dosage‐dependent adverse effects. Metabolism experiments of butylparaben indicate that it is rapidly metabolized by non‐specific esterases to p‐hydroxybenzoic acid and butanol, neither of which is estrogenic. CONCLUSIONS: Exposure to methyl‐ or butylparaben in the diet for eight weeks did not affect any male reproductive organs or parameters at exposures as high as 10,000 ppm, corresponding to a mean daily dose of 1,141.1±58.9 or 1,087.6±67.8 mg/kg/day for methyl‐ and butylparaben, respectively. The rapid metabolism of parabens by esterases probably explains why these weakly estrogenic substances elicit no in vivo effects when administered by relevant exposure routes (i.e., topical and oral). Birth Defects Research (Part B) 2008. 2008 Wiley‐Liss, Inc.