Dosing Time‐Dependent Effect of Raloxifene on Plasma Fibrinogen Concentration in Ovariectomized Rats

The chronopharmacological effect of raloxifene, a selective estrogen‐receptor modulator, was evaluated by repeated dosing of ovariectomized rats. Bilateral ovariectomy or sham operation was performed at age 12 wks, and animals were kept in rooms with a 12 h light‐12 h dark cycle. Raloxifene (3 mg/kg, once daily for 10 wks) or vehicle was given repeatedly at either 2 h after lights‐on (2 HALO) or 14 h after lights‐on (14 HALO). Plasma fibrinogen concentration at the end of the study was reduced by the drug, and the reduction was significantly prominent in rats in whom the drug was dosed at 2 HALO rather than 14 HALO. Femur bone density decreased, and urinary excretion of deoxypyridinoline, an index of bone resorption capacity of osteoclasts, increased in ovariectomized animals at the end of the study. Treatment with raloxifene ameliorated these changes in a dosing time‐independent manner. Serum calcium, ALT, and total protein concentrations at the end of the study also did not differ acccording to treatment regime, which indicates that protein synthesis and liver function may not contribute to the effects. This is the first study to determine dosing time‐dependent changes in the efficacy of raloxifene in an animal model of osteoporosis. Because fibrinogen concentration is reported to be a marker of cardiovascular events, consideration of dosing time of raloxifene may be important to obtain a better cardioprotective effect of this medication when it is prescribed to postmenopausal women with osteoporosis.     

Chronopharmacology of oxacalcitriol in 5/6 nephrectomized rats

We previously reported on the merits of the chronopharmacological effects of 1,25(OH) 2 vitaminD3 in 5/6 nephrectomized rats (Tsuruoka et al, Life Scineces 2002; 71: 1809-1820). In this study, the chronopharmacological effect of 22-oxacalcitriol (OCT), a newly developed active vitaminD3 analogue with less calcemic activity, was evaluated by a single and repeated dosing of the drug. The 5/6 nephrectomized animals were kept in rooms with a 12-h light/dark cycle. Single (12.5 microg/kg, i.v.) and repeated (5 microg/kg, i.v. three times a week for 12 weeks) dosing of OCT or vehicle was given at either 2 hours after lights on (2HALO) or 14 hours after lights on (14HALO). The severity of hypercalcemia and hyperphosphatemia was significantly milder when the drug was given at 14HALO. Serum concentrations of total OCT and albumin of the 2HALO and 14HALO trials did not differ significantly. The decrease of parathyroid hormone concentration was greater in the 14HALO trial while the increase in urinary ratio of Ca to creatinine was greater in the 2HALO trial. The suppression of urinary deoxypyridinoline excretion, an index of bone resorption capacity of osteoclast, and the increase in bone density of both femurs were greater in the 14HALO trial. These results suggest that the adverse reactions of OCT were ameliorated and its efficacy was enhanced after dosing of the drug at 14HALO. Chronopharmacological differences of OCT were more prominent than those seen with other vitamin D analogues. Dosing-time-dependent variation in the sensitivity of the drug to osteoclast were involved in the mechanisms of these events.     

Cyclosporine A dosing-time-dependent effects on plasma creatinine and body weight in male Wistar rats treated for 3 weeks.

Cyclosporine A (CsA) nephrotoxicity was assessed in 120 male Wistar rats (350 +/- 50 g) entrained to a 12-h cycle (light-dark 12:12); plasma creatinine level and body weight were examined in controls and in rats that had been treated daily with oral CsA or vehicle alone (olive oil-ethanol 90:10) for 21 days; daily dosing (40 mg/kg) was at one of six equally spaced given times during the 24-h cycle. The variations observed in both indexes were shown to be circadian dosing stage dependent. Nephrotoxicity was present as early as the third day of treatment with CsA; plasma creatinine level was enhanced by about 50% in rats dosed around the time of the change from darkness to light: at 22 HALO, 146.7 +/- 4.5 mumol/L, against 92.0 +/- 2.8 mumol/L for controls (p less than 0.05); and at 2 HALO, 148.3 +/- 10.0 mumol/L, against 95.0 +/- 4.3 mumol/L for controls (p less than 0.05). Thereafter, a remission episode was observed between days D5-D9. The more drastic effects were seen on days D16 and D21, in animals dosed in the beginning of the dark span (14 HALO): 185 +/- 10 mumol/L for CsA and 98.0 +/- 5.3 mumol/L for controls (p less than 0.01) and, to a lesser extent, in rats treated at the early resting phase (2 HALO): 152.4 +/- 31 mumol/L for CsA and 95.0 +/- 4 mumol/L for controls (p less than 0.05). The normal increase in body weight during the 21-day period (about 14 +/- 8% in controls) was impeded in CsA-administered rats, especially those dosed at the beginning of the activity span (14 HALO) that even suffered weight reduction. Differences in percentages of survivors were noticed, depending on dosing stage. About 40% of the animals in every time CsA-treatment group died, except for those dosed at the end of the resting period (10 HALO), when all animals died. In surviving rats, the cessation of CsA dosing resulted in a reversible effect on the study variables.     

Contribution of diet to the dosing time-dependent change of vitamin D3-induced hypercalcemia in rats

We have recently reported that the degree of hypercalcemia as an adverse effect induced by a single large-dose of active vitamin D3 varied with its dosing time without alteration in therapeutic effect for secondary hyperparathyroidism in patients with chronic renal failure. The present study was conducted to elucidate an effect of intestinal calcium (Ca) absorption on the chronopharmacological profiles of vitamin D3. 1, 25-dihydroxy-cholecalciferol (D3, 2 microg/kg) or vehicle alone was orally administered at two different times (2 and 14 hours after lights on; HALO) to male Wistar rats (n= 10) kept in rooms with a 12 h light-dark cycle. Blood samples for serum Ca concentration were taken before and 3, 6, 9, and 12 hours after the administration. Urine was collected for 6 hours after dosing. An identical protocol was repeated using the same animals after 16 hours fasting by a cross-over fashion. Under free-fed condition, basal concentration of serum Ca was higher at a resting period (lights on) than during an active period (lights off). Serum Ca reached its peak at 6 hours after dosing in both timings, while the value was significantly higher in the 2 HALO trial than in the 14 HALO trial. Area under the serum Ca concentration-time curve from 0 to 12 hours (AUC0-12h) and urinary excretion of Ca for 6 hours were also significantly higher in the 2 HALO trial than in the 14 HALO trial. When fasted, basal Ca concentration was reduced compared with the free-fed condition, while the daily variation was maintained. Serum Ca concentration profiles from 3 to 12 hours after dosing were not significantly different between the 2 HALO and 14 HALO trials. The AUC0-12h of serum Ca or its urinary excretion was not different between both trials. Serum concentrations of parathyroid hormone and total protein, measured before and 6 hours after the dosing were not affected by the dosing schedule. We have concluded that intestinal Ca absorption is a major factor for the chronopharmacological phenomenon of D3-induced hypercalcemia in intact rats, while intestinal and renal involvement may be relatively small in the mechanism of the intrinsic diurnal variation of serum Ca.     

Dosing-time dependent effect of dexamethasone on bone density in rats

AimsWhile glucocorticoids are widely used to treat patients with various diseases, they often cause adverse effects such as bone fractures. In this study, we investigated whether the decrease in bone density induced by glucocorticoid therapy was ameliorated by optimizing a dosing-time.Main methodsRats were administered with dexamethasone (Dex) orally (1 mg/kg/day) for 6 weeks at a resting or an active period. After the end of the treatment, bone density of femur, biomarkers of bone formation and resorption, and other biomedical variables were measured.Key findingsBone density of femur was significantly decreased by the 6-week treatment with Dex, and the degree of decrease in the 14 HALO (hours after light on) dosing group (an active period) was larger than that in the 2 HALO dosing group (a resting period). Although urinary calcium excretion was accelerated by Dex treatment, secondary hyperparathyroidism was not detected. Histomorphometry analysis showed that Dex suppressed bone resorption, which was larger in the 2 HALO than in the 14 HALO groups. These data indicate that Dex equally suppressed bone formation in the 2 and 14 HALO groups, but inhibited bone resorption more in the 2 HALO than in the 14 HALO groups.SignificanceThis study shows that the decrease in bone density induced by Dex was changed by its dosing-time.     

Time of day improves efficacy and reduces adverse reactions of vitamin D3 in 5/6 nephrectomized rat

Time-dependent differences in adverse reactions and efficacy by a repeated administration of 1,25(OH)2 vitamin D3 (vit D, 0.3 microg/Kg/day for 12 weeks) were examined in 5/6 nephrectomized rats under a condition of 12-hour light-dark cycle. The 5/6 nephrectomy increased serum concentrations of phosphate, osteocalcin and PTH, and urinary excretions of phosphate and deoxypyridinoline (DPD) while the maneuver reduced serum Ca concentration and its urinary excretion. Animals with a dosing of the drug at 2 hours after light on (HALO) had more grade of hypercalcemia and hyperphosphatemia than those at 14 HALO. Reduction of serum intact PTH and increase of serum vit D were observed in both groups with a similar extent. Increase of osteocalcin by the drug was greater in 14 HALO trial. Urinary excretion of DPD was not influenced by the treatment. The increase in bone density of femur was greater in 14 HALO than in 2 HALO trials. These results suggest that adverse reactions of vit D were ameliorated and its efficacy was enhanced after the repeated dosing of the drug at 14 HALO. Time-dependent variation in the sensitivity of the drug to osteoblast was involved in the mechanism of these events, while the roles of pharmacokinetic alteration and renal response were small, if any.     

Circadian time-dependent differences in murine tolerance to the antihistaminic agent loratadine

Loratadine is a second-generation histamine H(1)-receptor antagonist used in the treatment of allergic diseases. The aim of the study was to assess whether lethal toxicity and motor incoordination (neurotoxicity) of loratadine is circadian rhythm-dependent. A total of 210 male Swiss mice, aged 10 wk, were synchronized for 3 wk to 12 h light (rest span)/12 h dark (activity span). The drug was administered per os. The choice of the sublethal (TD(50) = 82 mg/kg body weight) and the lethal (LD(50) = 4 g/kg body weight) dosage was based on preliminary studies. Each of these two doses was administered to comparable groups of animals at six different circadian time points (1, 5, 9, 13, 17, and 21 Hours After Light Onset [HALO]). The survival duration was dosing time-dependent (chi(2) = 16.96; p < 0.001). Drug dosing at 17 HALO resulted in best (67%) survival rate; whereas, dosing at 9 HALO resulted in poorest (21%) survival rate. Cosinor analyses (with a trial period tau = 24 h) validated a statistically significant circadian rhythm in survival rate (p < 0.04) with an acrophase (peak time ? of best tolerance to loratadine) being at 17.5 HALO +/- 4.65 h. Troughs of motor incoordination were located at the administration times of 5 and 17 HALO (60% and 32% of animals affected, respectively), whereas peaks were located at 9 and 21 HALO (87% and 68% of animals affected, respectively). The 24 h mean of the motor incoordination was 61%, the mean proportion of animals affected by the treatment for the six different circadian times studies. The extent of this neurotoxic effect varied as a function of loratadine dosing time (p < 0.001). A statistically significant ultradian component rhythm (p < 0.01) with a trial period tau = 12 h was also validated. The obtained results show that the dosing time of loratadine at the mid-activity (dark) span seems to be optimal, since it corresponds to the longest (21 vs. 12 days) survival span and to least neurotoxicity.     

Gentamicin-induced ototoxicity and nephrotoxicity vary with circadian time of treatment and entail separate mechanisms

The aminoglycoside antibiotic gentamicin can cause both ototoxicity and nephrotoxicity, the severity of which varies with circadian time of daily treatment. However, it is not yet resolved if such drug-induced adverse effects are independent or interdependent phenomena. Two groups of 9 female Sprague-Dawley rats (200–250?g), each housed separately and entrained to a 12?h light (06:00–18:00?h) – 12?h dark cycle, received a daily subcutaneous injection of 100?mg/kg gentamicin. One group was treated at the beginning of the activity span, 2 Hours After Lights On (HALO), and the other at the beginning of the rest span, 14 HALO. Global toxicity was gauged by both body weight loss relative to the pre-treatment baseline and number of deaths. Ototoxicity, i.e., hearing loss, was assessed by changes in auditory brainstem response (ABR) for pure tone stimuli of 8, 16, 24, and 32?kHz before and after 2 and 4 weeks of gentamicin treatment. Renal toxicity was evaluated by changes in urinary N-acetyl-β-glucosaminidase (NAG)/creatinine (CR) concentration ratio before and after each week of treatment. In a complementary substudy of separate but comparable 2 and 14 HALO groups of rats, blood samples were obtained before and 30, 60, 120, and 240?min post-subcutaneous injection of 100?mg/kg gentamicin. Number of animal deaths was greater in the 2 (4 deaths) than 14 HALO (1 death) group, mirroring more severe initial (first two weeks of treatment) body weight losses from baseline, being more than 2-fold greater in animals of the 2 than 14 HALO group. Ototoxicity progressively worsened during the treatment; although, the extent of hearing loss varied according to circadian time of treatment across all frequencies (p?<?0.05), particularly the 24 and 32?kHz ones (both p?<?0.005), both at the 2 and 4 week assessments. At 32?kHz after 4 weeks of gentamicin dosing, the 2 HALO group showed an average 42?dB hearing loss, while the 14 HALO group exhibited only an average 10?dB loss. ABR response latencies were longer for the 2 than 14 HALO rats. The time course of nephrotoxicity differed from that of ototoxicity. The mean urinary NAG/CR ratio peaked after the first week of treatment, averaging 13.64-fold greater than baseline for the 2 HALO-treated animals compared to 7.38-fold greater than baseline for the 14 HALO-treated ones. Ratio values declined thereafter; although, even after the second week of dosing, they remained greater in the 2 than 14 HALO group (averaging 8.15-fold greater and 2.23-fold greater than baseline, respectively). Pharmacokinetic analysis of the blood gentamicin values revealed slower clearance, on average by ～25% (p?<?0.001), in the rats of the 14 than 2 HALO group (x?±?S.E.: 3.22?±?0.49 and 4.53?±?0.63?mL/min/kg, respectively). The study findings indicate robust difference of the time course in rats of both treatment groups of gentamicin-induced ototoxicity and nephrotoxicity, supporting the hypothesis these organ toxicities are independent of one another, and further suggest the observed treatment-time differences in gentamicin adverse effects may be more dependent on local cell, tissue, or organ circadian (chrono) pharmacodynamic than (chrono) pharmacokinetic mechanisms.     

Circadian rhythms in toxic effects of the serotonin antagonist ondansetron in mice

The aim of the study was to learn whether the lethal and the motor incoordination (ataxia) side effect of ondansetron (Zophren) administration is dosing-time dependent. Ondansetron is a serotonin 5-HT3 receptor antagonist used primarily to control nausea and vomiting arising from cytotoxic chemo- and radiotherapy. A total of 210 male Swiss mice 10 to 12 weeks of age were synchronized for 3 weeks by 12 h light (rest span)/12 h dark (activity span). Different doses of ondansetron were injected intraperitoneally (i.p.) at fixed times during the day to determine both the sublethal (TD50) and lethal (LD50) doses, which were, respectively, 3.7 +/- 0.6 mg/kg and 4.6 +/- 0.5 mg/kg. In the chronotoxicologic study a single dose of ondansetron (3.5 mg/kg, i.p.) was administered to different and comparable groups of animals at four different circadian stages [1, 7, 13, and 19 h after light onset (HALO)]. The lethal toxicity was statistically significantly dosing time-dependent (chi2 = 21.51, p < 0.0001). Drug dosing at 1 HALO resulted in 100% survival rate whereas drug dosing at 19 HALO was only one-half that (52%). Similarly, lowest and highest ataxia occurred when ondansetron was injected at 1 and 19 HALO, respectively (chi2 = 22.24, p < 0.0001). Effects on rectal temperature were also dosing-time related (Cosinor analysis, p < 0.0001). The characteristics of the waveform describing the temporal patterns differed between the studied variables, e.g., lethal toxicity and survival rate showing two peaks and rectal temperature showing one peak in the 24 h time series waveform pattern. Cosinor analysis also revealed a statistically significant ultradian (tau = 8 h) rhythmic component in the considered variables. Differences in curve patterns in toxicity elicited by ondansetron on a per end point basis are hypothesized to represent the phase relations between the identified 24 h and 8 h periodicities.     

Effect of hormone replacement therapy on the elemental contents of uterine tissue

For the past years, different therapies based on steroid hormone supplementation or modulators of estrogen receptors have been used after menopause to prevent or manage osteoporosis. Although these treatments seem to be beneficial, they have some negative effects in the uterus and breast. The objective of this study was to assess variations for the concentrations of K, Ca, Mn, Fe, Cu, Zn, and Se in uterine tissue of Wistar rats. Ovriectomized rats were subjected to estrogen, progesterone, raloxifene, and tibolone supplementation and compared with nonovariectomized control animals. Elemental contents determined by the particle-induced X-ray emission (PIXE) technique revealed major alterations in Fe, Ca, Mn, and Se in the uterus of ovariectomized rats relative to control animals. After ovariectomy, a significant increase in Ca and Fe and a significant decrease in Mn and Se contents were determined in the uterus. For the ovariectomized groups in which animals, received raloxifene, tibolone, estrogen, and estrogen combined with progesterone supplementation, an overall recovery in Mn, Fe, and Se contents was verified. Elemental concentration in the progesterone-supplemented group did not significantly differ from ovariectomized animals receiving placebo. The alterations found for ovariectomized animals receiving placebo and progesterone suggest tissue impairment and trace element imbalance, contrasting with the remaining supplemented groups where an enhancement of tissue activity might justify similar concentration levels relative to controls, because most of the elemental contents altered after ovariectomy.     

Circadian Rhythms in Toxic Effects of the Serotonin Antagonist Ondansetron in Mice

The aim of the study was to learn whether the lethal and the motor incoordination (ataxia) side effect of ondansetron (Zophren®) administration is dosing‐time dependent. Ondansetron is a serotonin 5‐HT₃ receptor antagonist used primarily to control nausea and vomiting arising from cytotoxic chemo‐ and radiotherapy. A total of 210 male Swiss mice 10 to 12 weeks of age were synchronized for 3 weeks by 12h light (rest span)/12h dark (activity span). Different doses of ondansetron were injected intraperitoneally (i.p.) at fixed times during the day to determine both the sublethal (TD₅₀) and lethal (LD₅₀) doses, which were, respectively, 3.7 ± 0.6 mg/kg and 4.6 ± 0.5 mg/kg. In the chronotoxicologic study a single dose of ondansetron (3.5 mg/kg, i.p.) was administered to different and comparable groups of animals at four different circadian stages [1, 7, 13, and 19h after light onset (HALO)]. The lethal toxicity was statistically significantly dosing time‐dependent (χ² = 21.51, p < 0.0001). Drug dosing at 1 HALO resulted in 100% survival rate whereas drug dosing at 19 HALO was only one‐half that (52%). Similarly, lowest and highest ataxia occurred when ondansetron was injected at 1 and 19 HALO, respectively (χ² = 22.24, p < 0.0001). Effects on rectal temperature were also dosing‐time related (Cosinor analysis, p < 0.0001). The characteristics of the waveform describing the temporal patterns differed between the studied variables, e.g., lethal toxicity and survival rate showing two peaks and rectal temperature showing one peak in the 24h time series waveform pattern. Cosinor analysis also revealed a statistically significant ultradian (τ ≡ 8h) rhythmic component in the considered variables. Differences in curve patterns in toxicity elicited by ondansetron on a per end point basis are hypothesized to represent the phase relations between the identified 24h and 8h periodicities.     

Dosing time-dependent nephrotoxicity of cyclosporin A during 21-day administration to Wistar rats

The incidence of cyclosporine A (CsA) nephrotoxicity with reference to the temporal stage of administration was studied during a chronic 21-day treatment in male Wistar rats. Oral administration (20 mg/kg/day) was given at four different times: 1, 7, 13, or 19 hours after light onset (HALO). Plasma creatinine and blood urea nitrogen (BUN) levels were determined at regular intervals over the 24 h: before treatment (day 0); 7, 14, and 21 days after the beginning of treatment (days 7, 14, and 21); and 7 and 14 days after CsA withdrawal (days 28 and 35). At the same times, creatinine clearance and g-glutamyl transferase urinary excretion were determined in the groups of animals treated at 7 and 19 HALO. Residual concentrations of CsA in the renal tissue were measured at the end of the treatment period (day 21) in all groups. Nephrotoxicity of CsA was dependent on the temporal stage of administration. The renal vasoconstriction showed by the increase in plasma creatinine and BUN levels and the decrease in creatinine clearance was maximal when the CsA was given at 7 and 19 HALO and was correlated to the tissue concentrations of CsA. Tubular injury seems to occur earlier and the return to normal function less rapidly in animals treated at 19 HALO compared with animals treated at 7 HALO.     

Dosing-time-dependent differences in lipopolysaccharide-induced liver injury in rats

Dosing-time-dependent differences in lipopolysaccharide (LPS)-induced liver injury were examined in rats housed under a 12 h light:dark (LD) cycle. LPS (5 mg/kg) was intravenously injected into different groups of rats at 2, 14, or 20 h after light on (HALO). Elevations in serum liver enzymes after 14 HALO were significantly greater than those after 2 HALO. These parameters were lower in rats given LPS at 20 HALO, compared to 14 HALO. The number of polymorphonuclear cells (PMN) in the liver and the amount of hepatic myeloperoxidase activity, which reflects the number of PMN in liver tissues, was significantly greater in the 14 than in the 2 HALO group. In addition, hepatic interleukin-6 (IL-6) production in the 14 HALO group was enhanced compared to that in the 2 HALO trial. These results suggest that LPS-induced liver injury is greater during the early active than during the early resting period. Dosing-time-dependent variation in the accumulation of PMN in the liver and, potentially, subsequent IL-6 production in liver tissues might be involved in this phenomenon.     

Dosing‐Time–Dependent Differences in Lipopolysaccharide‐Induced Liver Injury in Rats

Dosing‐time–dependent differences in lipopolysaccharide (LPS)‐induced liver injury were examined in rats housed under a 12 h light∶dark (LD) cycle. LPS (5 mg/kg) was intravenously injected into different groups of rats at 2, 14, or 20 h after light on (HALO). Elevations in serum liver enzymes after 14 HALO were significantly greater than those after 2 HALO. These parameters were lower in rats given LPS at 20 HALO, compared to 14 HALO. The number of polymorphonuclear cells (PMN) in the liver and the amount of hepatic myeloperoxidase activity, which reflects the number of PMN in liver tissues, was significantly greater in the 14 than in the 2 HALO group. In addition, hepatic interleukin‐6 (IL‐6) production in the 14 HALO group was enhanced compared to that in the 2 HALO trial. These results suggest that LPS‐induced liver injury is greater during the early active than during the early resting period. Dosing‐time–dependent variation in the accumulation of PMN in the liver and, potentially, subsequent IL‐6 production in liver tissues might be involved in this phenomenon.     

Bone affinity of a bisphosphonate-conjugated protein in vivo

Growth factors capable of stimulating bone formation are potential therapeutic agents for osteoporosis treatment. It is essential, however, that a targeting mechanism is incorporated into the growth factors to deposit them at osseous tissue with minimal distribution to extraskeletal sites. To this end, a strategy has been developed in which a bone-seeking molecule, 1-amino-1,1-diphosphonate methane (aminoBP), was chemically conjugated to a model protein, bovine serum albumin (BSA). This study was carried out to assess the bone affinity of the conjugates in a tibia injection model. Using ovariectomized (OVX) rats, initial (3 h) retention of BSA and aminoBP-BSA were found to be equivalent when injected into the medullary cavity of tibia. After 1 day, an 8- and 12-fold higher tibiae retention of the protein was obtained in normal and OVX rats as a result of aminoBP conjugation. A similar result ( approximately 12-fold difference) was also obtained in OVX rats after 3 days. We concluded that aminoBP conjugation to BSA imparted a high bone affinity and enhanced bone retention of proteins in normal and OVX rats.     

Protective effect of apigenin on ovariectomy-induced bone loss in rats

Recent studies have shown that apigenin not only inhibits bone resorption by osteoclasts but also induces osteoclast apoptosis. However, the influence of apigenin on osteoporosis in animals is relatively unknown. The purpose of this study was to examine the bone-protective effects of apigenin in estrogen-deficient ovariectomized rats. Three-month-old female Sprague-Dawley rats were either sham-operated or ovariectomized and fed AIN-93G diet for 7 weeks to induce bone loss. To confirm bone loss, we used a newly developed non-invasive technique involving zoom-in micro-computed tomography. Apigenin was administered at a dose of 10 mg/kg three times a week for 15 weeks. Our results indicate that apigenin not only increased the mineral content and density of the trabecular bone at the neck of the left femur, but also decreased body weight and dietary consumption. Moreover, our biochemical results indicate that apigenin has a positive effect on bone turnover. The present data suggest that apigenin should be considered for use in the treatment of osteoporosis.     

Differential effects of 17beta-estradiol and raloxifene on VSMC phenotype and expression of osteoblast-associated proteins

Several studies demonstrate an association between osteoporosis and arterial calcific disease, both of which being common in elderly women. Estradiol and raloxifene, a selective estrogen receptor modulator, prevent bone loss in postmenopausal women. Little is known regarding how these agents affect arterial calcification. The aim of this study was to determine whether or not 17beta-estradiol and raloxifene reduced vascular smooth muscle cell (VSMC) differentiation and expression of bone-associated proteins during phosphate-induced calcification in vitro. Aortic VSMC were cultured from adult, gonadally intact, and ovariectomized (OVX) female pigs. Calcifying medium was added, and cells were treated with solvent (control), 17beta-estradiol (E(2)), or raloxifene. Extent of calcification and phenotypic expression of bone-associated proteins [matrix gla protein (MGP), osteoprotegerin (OPG), and bone sialoprotein (BSP)] were examined at 3-day intervals over 2 wk. Calcium content increased in all groups but was greater in VSMC derived from intact compared with OVX animals. E(2) reduced calcification and preserved a contractile phenotype. Expression of OPG significantly decreased with time; this decrease was significantly greater in VSMC derived from OVX compared with gonadally intact pigs. E(2) and raloxifene preserved expression of OPG only in VSMC from intact pigs. Expression of MGP increased significantly with time and was not affected by E(2) or raloxifene treatments. E(2) treatment significantly inhibited synthesis of BSP in cells from both groups. In conclusion, E(2) slows differentiation of VSMC induced by excess phosphate. Effectiveness of raloxifene to preserve expression of bone cell-associated proteins depends on the hormonal status of the tissue donor.     

Circadian variation of cytotoxicity and genotoxicity induced by an immunosuppressive agent “Mycophenolate Mofetil” in rats

Immunosuppressive drugs such as Mycophenolate Mofetil (MMF) are used to suppress the immune system activity in transplant patients and reduce the risk of organ rejection. The present study investigates whether the potential cytotoxicity and genotoxicity varied according to MMF dosing-time in Wistar Rat. A potentially toxic MMF dose (300 mg/kg) was acutely administered by the i.p. route in rats at four different circadian stages (1, 7, 13 and 19 hours after light onset, HALO). Rats were sacrificed 3 days following injection, blood and bone marrow were removed for determination of cytotoxicity and genotoxicity analysis. The genotoxic effect of this pro-drug was investigated using the comet assay and the micronucleus test. Hematological changes were also evaluated according to circadian dosing time. MMF treatment induced a significant decrease at 7 HALO in red blood cells, in the hemoglobin rate and in white blood cells. These parameters followed a circadian rhythm in controls or in treated rats with an acrophase located at the end of the light-rest phase. A significant, thrombocytopenia was observed according to MMF circadian dosing time. Furthermore, abnormally shaped red cells, sometimes containing micronuclei, poikilocytotic in red cells and hypersegmented neutrophil nuclei were observed with MMF treatment. The micronucleus test revealed damage to chromosomes in rat bone marrow; the comet assay showed significant DNA damage. This damage varied according to circadian MMF dosing time. The injection of MMF in the middle of the dark-activity phase produced a very mild hematological toxicity and low genotoxicity. Conversely, it induced maximum hematological toxicity and genotoxicity when the administration occurred in the middle of the light-rest phase, which is physiologically analogous to the end of the activity of the diurnal phase in human patients.     

Combined treatment with dexamethasone and raloxifene totally abrogates osteoporosis and joint destruction in experimental postmenopausal arthritis

Introduction  

Postmenopausal patients with rheumatoid arthritis (RA) are often treated with corticosteroids. Loss of estrogen, the inflammatory disease and exposure to corticosteroids all contribute to the development of osteoporosis. Therefore, our aim was to investigate if addition of the selective estrogen receptor modulator raloxifene, or estradiol, could prevent loss of bone mineral density in ovariectomized and dexamethasone treated mice with collagen-induced arthritis (CIA).     

24-Hour Changes in Circulating Prolactin,Follicle-Stimulating Hormone,Luteinizing Hormone,and Testosterone in Young Male Rats Subjected to Calorie Restriction

This work analyzes the effect of calorie restriction on the 24 h variation of pituitary-testicular function in young male Wistar rats by measuring the circulating levels of prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Control animals were provided an equilibrium calorie diet and the experimental animals a calorie-restriction diet equivalent to 66% of food restriction for four weeks starting on day 35 of life. Different groups of control and experimental rats were killed at 6 h intervals around the clock, beginning 1 h after light on (HALO). Compared to the control animals, the mean secretion of prolactin was augmented and that of LH and testosterone decreased in calorie-restricted rats, whereas FSH release remained unchanged. Significant changes in the 24 h secretory pattern of circulating prolactin, LH, and testosterone occurred in the calorie-restricted rats. These include the appearance of a second maximum of plasma prolactin at 21 HALO, blunting of the LH peak seen at 13 HALO, and phase-shift of the testosterone peak from 13 HALO in controls to 17 HALO in calorie-restricted rats. The significant positive correlation between individual LH and testosterone levels found in controls was no longer observed in calorie-restricted rats. Availability of nutrients presumably affects the mechanisms that modulate the circadian variation of the pituitary-gonadal axis in growing male rats.