Acute toxicity of subcutaneously administered vitamin E isomers delta- and gamma-tocotrienol in mice.

The toxicity of parenterally administered vitamin E isomers, delta-tocotrienol (DT3) and gamma-tocotrienol (GT3), was evaluated in male and female CD2F1 mice. In an acute toxicity study, a single dose of DT3 or GT3 was administered subcutaneously in a dose range of 200 to 800 mg/kg. A mild to moderately severe dermatitis was observed clinically and microscopically in animals at the injection site at doses above 200 mg/kg. The severity of the reaction was reduced when the drug concentration was lowered. Neither drug produced detectable toxic effects in any other tissue at the doses tested. Based on histopathological analysis for both DT3 and GT3, and macroscopic observations of inflammation at the injection site, a dose of 300 mg/kg was selected as the lowest toxic dose in a 30-day toxicity study performed in male mice. At this dose, a mild skin irritation occurred at the injection site that recovered completely by the end of the experimental period. At a dose of 300 mg/kg of DT3 or GT3, no adverse effects were observed in any tissues or organs.

Exercise training modifies the bone and endocrine response to graded reductions in energy availability in skeletally mature female rodents.

Introduction: Reductions in energy availability leading to weight loss can induce loss of bone and impact important endocrine regulators of bone integrity. We sought to elucidate whether endurance exercise (EX) can mitigate bone loss observed in sedentary (SED) skeletally mature rodents subjected to graded energy deficits. Methods: Female virgin rats (n=84, 5-mo-old; 12/group) were randomized to baseline controls and either sedentary (SED) or exercise (EX) conditions, and within each exercise status to adlib-fed (ADLIB), or moderate (MOD) or severe (SEV) energy restriction diets for 12 weeks. Rats assigned to EX groups performed treadmill running to increase weekly energy expenditure by 10%. MOD-ER-SED, SEV-ER-SED, MOD-ER-EX and SEV-ER-EX were fed modified AIN93M diets with 20%, 40% 10%, and 30% less energy content, respectively, with 100% of all other nutrients provided. Results: Energy availability (EA) was effectively reduced by ~14% and ~30% in the MOD-ER and SEV-ER groups, respectively. MOD-ER for 12 weeks resulted in few negative impacts on bone and, except for serum leptin in MOD-ER-SED rats, no significant changes in endocrine factors. By contrast, SEV-ER in SED rats resulted in significantly lower total body and femoral neck bone mass, and reduced serum estradiol, IGF-1 and leptin. EX rats experiencing the same reduction in energy availability as SEV-ER-SED exhibited higher total body mass, lean mass, total BMC, and higher serum IGF-1 at the end of 12 weeks. Bone mechanical properties at 3 bone sites (mid-femur, distal femur, femoral neck) were minimally impacted by ER but positively affected by EX. Discussion: These findings indicate that combining increased EX energy expenditure with smaller reductions in energy intake to achieve a targeted reduction in EA provides some protection against loss of bone mass and lean mass in skeletally mature female rats, likely due to better preservation of circulating IGF-1, and that bone mechanical integrity is not significantly degraded with either moderate or severe reduced EA.

Restriction of dietary energy intake has a greater impact on bone integrity than does restriction of calcium in exercising female rats.

We sought to elucidate the effects of restricting calcium, energy, or food on the skeletal integrity of exercising female rats. Female Sprague-Dawley rats (4 mo old) were randomly assigned to 5 groups (n = 10/group): ad libitum intake of an AIN-93M diet (Research Diets D10012M, Research Diets, Inc.) with no exercise (AL-S) or with exercise (AL-EX) or to 1 of 3 exercising restriction groups [40% restriction of calcium only (CAR-EX), energy only (ER-EX), or food (FR-EX)]. All EX rats were treadmill trained 3 d/wk, 45 min/d for 12 wk at ~60% maximal oxygen consumption. After 12 wk, total body bone mineral content (by DXA) and body mass, but not lean mass, were lower in ER-EX (-17%) and FR-EX rats (-13%) compared with the AL-EX group. CAR-EX had few negative effects on bone geometry (by peripheral quantitative computed tomography) or histomorphometry. However, declines in total volumetric bone mineral density at the proximal tibia metaphysic (PTM) were observed in ER-EX (-6%) and FR-EX (-8%) groups; only FR-EX rats exhibited increased osteoclast surface and decreased mineral apposition rate in PTM cancellous bone. Decrements in serum estradiol, uterine weights, or both in these 2 groups implicate altered estrogen status as contributory. Urine pH declined significantly by 12 wk in all restricted groups, but net acid excretion increased only in CAR-EX rats. These findings, when compared with published data on sedentary rats, suggest that treadmill running exercise may mitigate some, but not all, deleterious effects on bone after chronic energy or food restriction but is more protective during calcium restriction.

Fat and Lean Mass Predict Bone Mass During Energy Restriction in Sedentary and Exercising Rodents.

Energy restriction (ER) causes bone loss, but the impact of exercise during ER is less understood. In this study, we examined the impact of metabolic hormones and body composition on both total body bone mineral content (BMC) and local (proximal tibia) volumetric bone mineral density (vBMD) during short- (4 weeks) and long-term (12 weeks) ER with and without exercise in adult female rats. Our first goal was to balance energy between sedentary and exercising groups to determine the impact of exercise during ER. Second, we aimed to determine the strongest predictors of bone outcomes during ER with energy-matched exercising groups. Methods: Female Sprague-Dawley rats were divided into three sedentary groups (ad libitum, -20% ER, and -40% ER) and three exercising groups (ad libitum, -10% ER, and -30% ER). Approximately a 10% increase in energy expenditure was achieved via moderate treadmill running (∼60-100 min 4 days/week) in EX groups. n per group = 25-35. Data were analyzed as a 2 × 3 ANOVA with multiple linear regression to predict bone mass outcomes. Results: At 4 weeks, fat and lean mass and serum insulin-like growth factor-I (IGF-I) predicted total body BMC (R 2 = 0.538). Fat mass decreased with ER at all levels, while lean mass was not altered. Serum IGF-I declined in the most severe ER groups (-40 and -30%). At 12 weeks, only fat and lean mass predicted total body BMC (R 2 = 0.718). Fat mass declined with ER level regardless of exercise status and lean mass increased due to exercise (+5.6-6.7% vs. energy-matched sedentary groups). At the same time point, BMC declined with ER, but increased with exercise (+7.0-12.5% vs. energy-matched sedentary groups). None of our models predicted vBMD at the proximal tibia at either time point. Conclusion: Both fat and lean mass statistically predicted total body BMC during both short- and long-term ER. Fat and lean mass decreased with ER, while lean mass increased with EX at each energy level. Measures that predicted total body skeletal changes did not predict site-specific changes. These data highlight the importance of maintaining lean mass through exercise during periods of ER.

Skin wound trauma, following high-dose radiation exposure, amplifies and prolongs skeletal tissue loss.

The present study investigated the detrimental effects of non-lethal, high-dose (whole body) γ-irradiation on bone, and the impact that radiation combined with skin trauma (i.e. combined injury) has on long-term skeletal tissue health. Recovery of bone after an acute dose of radiation (RI; 8 Gy), skin wounding (15-20% of total body skin surface), or combined injury (RI+Wound; CI) was determined 3, 7, 30, and 120 days post-irradiation in female B6D2F1 mice and compared to non-irradiated mice (SHAM) at each time-point. CI mice demonstrated long-term (day 120) elevations in serum TRAP 5b (osteoclast number) and sclerostin (bone formation inhibitor), and suppression of osteocalcin levels through 30 days as compared to SHAM (p<0.05). Radiation-induced reductions in distal femur trabecular bone volume fraction and trabecular number through 120 days post-exposure were significantly greater than non-irradiated mice (p<0.05) and were exacerbated in CI mice by day 30 (p<0.05). Negative alterations in trabecular bone microarchitecture were coupled with extended reductions in cancellous bone formation rate in both RI and CI mice as compared to Sham (p<0.05). Increased osteoclast surface in CI animals was observed for 3 days after irradiation and remained elevated through 120 days (p<0.01). These results demonstrate a long-term, exacerbated response of bone to radiation when coupled with non-lethal wound trauma. Changes in cancellous bone after combined trauma were derived from extended reductions in osteoblast-driven bone formation and increases in osteoclast activity.

Mechanical loading increases detection of estrogen receptor-alpha in osteocytes and osteoblasts despite chronic energy restriction.

Estrogen receptor-α (ER-α) is an important mediator of the bone response to mechanical loading. We sought to determine whether restricting dietary energy intake by 40% limits the bone formation rate (BFR) response to mechanical loading (LOAD) by downregulating ER-α-expressing osteocytes, or osteoblasts, or both. Female rats (n = 48, 7 mo old) were randomized to ADLIB-SHAM and ADLIB-LOAD groups fed AIN-93M purified diet ad libitum or to ER40-SHAM and ER40-LOAD groups fed modified AIN-93M with 40% less energy (100% of all other nutrients). After 12 wk, LOAD rats were subjected to a muscle contraction protocol three times every third day. ER40 produced lower proximal tibia bone volume (-22%), trabecular thickness (-14%), and higher trabecular separation (+127%) in SHAM but not LOAD rats. ER40 rats exhibited reductions in mineral apposition rate, but not percent mineralizing surface or BFR. LOAD induced similar relative increases in these kinetic measures of osteoblast activity/recruitment in both diet groups., but absolute values for ER40 LOAD rats were lower vs. ADLIB-LOAD. There were fourfold and eightfold increases in proportion of estrogen receptor-α protein-positive osteoblast and osteocytes, respectively, in LOAD vs. SHAM rats, with no effect of ER40. These data suggest that a brief period of mechanical loading significantly affects estrogen receptor-α in cancellous bone osteoblasts and osteocytes. Chronic energy restriction does result in lower absolute values in indices of osteoblast activity after mechanical loading, but not by a smaller increment relative to unloaded bones; this change is not explained by an associated downregulation of ER-α in osteoblasts or osteocytes.

Beta-1 adrenergic agonist treatment mitigates negative changes in cancellous bone microarchitecture and inhibits osteocyte apoptosis during disuse.

The sympathetic nervous system (SNS) plays an important role in mediating bone remodeling. However, the exact role that beta-1 adrenergic receptors (beta1AR) have in this process has not been elucidated. We have previously demonstrated the ability of dobutamine (DOB), primarily a beta1AR agonist, to inhibit reductions in cancellous bone formation and mitigate disuse-induced loss of bone mass. The purpose of this study was to characterize the independent and combined effects of DOB and hindlimb unloading (HU) on cancellous bone microarchitecture, tissue-level bone cell activity, and osteocyte apoptosis. Male Sprague-Dawley rats, aged 6-mos, were assigned to either normal cage activity (CC) or HU (n = 18/group) for 28 days. Animals were administered either daily DOB (4 mg/kg BW/d) or an equal volume of saline (VEH) (n = 9/gp). Unloading resulted in significantly lower distal femur cancellous BV/TV (-33%), Tb.Th (-11%), and Tb.N (-25%) compared to ambulatory controls (CC-VEH). DOB treatment during HU attenuated these changes in cancellous bone microarchitecture, resulting in greater BV/TV (+29%), Tb.Th (+7%), and Tb.N (+21%) vs. HU-VEH. Distal femur cancellous vBMD (+11%) and total BMC (+8%) were significantly greater in DOB- vs. VEH-treated unloaded rats. Administration of DOB during HU resulted in significantly greater osteoid surface (+158%) and osteoblast surface (+110%) vs. HU-VEH group. Furthermore, Oc.S/BS was significantly greater in HU-DOB (+55%) vs. CC-DOB group. DOB treatment during unloading fully restored bone formation, resulting in significantly greater bone formation rate (+200%) than in HU-VEH rats. HU resulted in an increased percentage of apoptotic cancellous osteocytes (+85%), reduced osteocyte number (-16%), lower percentage of occupied osteocytic lacunae (-30%) as compared to CC-VEH, these parameters were all normalized with DOB treatment. Altogether, these data indicate that beta1AR agonist treatment during disuse mitigates negative changes in cancellous bone microarchitecture and inhibits increases in osteocyte apoptosis.

The androgen metabolite, 5α-androstane-3ß,17ß-diol, decreases cytokine-induced cyclooxygenase-2, vascular cell adhesion molecule-1 expression, and P-glycoprotein expression in male human brain microvascular endothelial cells.

P-glycoprotein (Pgp), a multiple drug resistance transporter expressed by vascular endothelial cells, is a key component of the blood-brain barrier and has been shown to increase after inflammation. The nonaromatizable androgen, dihydrotestosterone (DHT), decreases inflammatory markers in vascular smooth muscle cells, independent of androgen receptor (AR) stimulation. The principal metabolite of DHT, 5α-androstane-3ß,17ß-diol (3ß-diol), activates estrogen receptor (ER)ß and similarly decreases inflammatory markers in vascular cells. Therefore, we tested the hypothesis that either DHT or 3ß-diol decrease cytokine-induced proinflammatory mediators, vascular cell adhesion molecule-1 (VCAM-1) and cyclooxygenase-2 (COX-2), to regulate Pgp expression in male primary human brain microvascular endothelial cells (HBMECs). Using RT-qPCR, the mRNAs for AR, ERα, and ERß and steroid metabolizing enzymes necessary for DHT conversion to 3ß-diol were detected in male HBMECs demonstrating that the enzymes and receptors for production of and responsiveness to 3ß-diol are present. Western analysis showed that 3ß-diol reduced COX-2 and Pgp expression; the effect on Pgp was inhibited by the ER antagonist, ICI-182,780. IL-1ß-caused an increase in COX-2 and VCAM-1 that was reduced by either DHT or 3ß-diol. 3ß-diol also decreased cytokine-induced Pgp expression. ICI-182,780 blocked the effect of 3ß-diol on COX-2 and VCAM-1, but not Pgp expression. Therefore, in cytokine-stimulated male HBMECs, the effect of 3ß-diol on proinflammatory mediator expression is ER dependent, whereas its effect on Pgp expression is ER independent. These studies suggest a novel role of 3ß-diol in regulating blood-brain barrier function and support the concept that 3ß-diol can be protective against proinflammatory mediator stimulation.

Cancellous bone formation response to simulated resistance training during disuse is blunted by concurrent alendronate treatment.

The purpose of this study was to assess the effectiveness of simulated resistance training (SRT) exercise combined with alendronate (ALEN) in mitigating or preventing disuse-associated losses in cancellous bone microarchitecture and formation. Sixty male Sprague-Dawley rats (6 months old) were randomly assigned to either cage control (CC), hind limb unloading (HU), HU plus either ALEN (HU + ALEN), SRT (HU + SRT), or a combination of ALEN and SRT (HU + SRT/ALEN) for 28 days. HU + SRT and HU + SRT/ALEN rats were anesthetized and subjected to muscle contractions once every 3 days during HU (four sets of five repetitions, 1000 ms isometric + 1000 ms eccentric). Additionally, HU + ALEN and HU + SRT/ALEN rats received 10 µg/kg of body weight of ALEN three times per week. HU reduced cancellous bone-formation rate (BFR) by 80%, with no effect of ALEN treatment (-85% versus CC). SRT during HU significantly increased cancellous BFR by 123% versus CC, whereas HU + SRT/ALEN inhibited the anabolic effect of SRT (-70% versus HU + SRT). SRT increased bone volume and trabecular thickness by 19% and 9%, respectively, compared with CC. Additionally, osteoid surface (OS/BS) was significantly greater in HU + SRT rats versus CC (+32%). Adding ALEN to SRT during HU reduced Oc.S/BS (-75%), Ob.S/BS (-72%), OS/BS (-61%), and serum TRACP5b (-36%) versus CC. SRT and ALEN each independently suppressed a nearly twofold increase in adipocyte number evidenced with HU and inhibited increases in osteocyte apoptosis. These results demonstrate the anabolic effect of a low volume of high-intensity muscle contractions during disuse and suggest that both bone resorption and bone formation are suppressed when SRT is combined with bisphosphonate treatment.