Bone DNA Content Research Articles

An Analysis of Micronuclei and DNA Damage Induced by Methotrexate Treatment of Male Albino Mice

Background: Methotrexate is an antineoplastic, antipsoriatic and antirheumatic agent belongs to the group of antimetabolites and inhibits folic acid metabolism. Materials and methods: To investigate its possible effect, sixty male mice were randomly assigned to one of four groups (one control and three treated groups with different doses of methotrexate). Mice of groups 1, 2 and 3 were intraperitoneally injected with 2.5, 5 and 10 mg /kg b.wt. methotrexate respectively. All the control and treated animals were sacrificed at 24, 48 and 72 hour by cervical dislocation post treatment. Results:Micronucleus assay results showed that methotrexate treatment induced genotoxicity in bone marrow cells, the number of micronucleated polychromatic erythrocytes (MNPCEs) and the ratio of polychromatic erythrocytes / normochromatic erythrocytes was gradually increased significantly (P < 0.001) by increasing dose and time of treatment in methotrexate treated groups in comparison with the control group. An analysis of randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR) showed different ranges of DNA modifications in the treated groups after 24, 48 and 72 hour of treatment in comparison with the control group. Results of this study indicate that methotrexate treatment induced cytotoxic and genotoxic effect on bone marrow cells and DNA content of male albino mice even after a low dose and single treatment. Conclusion: Therefore, the therapeutic uses of methotrexate should be restricted to a very narrow range border.

Role of Serum Albumin in fracture healing

Albumin is the most abundant plasma protein with molecular weight of 69 kd and it accounts for 55-60% measured in total serum protein, produced in liver. The multipotent progenitor cells of bone marrow differentiate into functional hepatocyte-like cells which expresses albumin. The 66kDa albumin protein is very prominent, and this protein component is present in the bone tissue as a major bone protein. The increase in IGF-I with fracture healing induce the production of albumin in the femoral-diaphyseal tissues and thus the osteoblastic proliferation takes place. The albumin also enhanced the bone calcium and DNA content that further promotes fracture healing, and its deficiency may be a significant determinant in healing process which may lead to fracture impairment. In this review we focused on the valuable significant role of albumin in fracture healing as well as the nonunion.

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as “mad cow disease”), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application.

Maternal low‐protein diet affects bone mass and mineral metabolism in suckling rats

This study was performed to analyse the effects of low-protein diet (7%) on bone mass and mineral metabolism in rat pups whose mothers were submitted to protein-restricted diet during late pregnancy and early post-natal periods. For this purpose, 12 pregnant Wistar rats were divided into two groups of six animals each: a control group with free access to standard diet (20% protein) and protein-restricted group (PR) fed with low-protein diet (7% protein) from the 14th day of pregnancy until day 14 after delivery. Body weight, femur bone mass, plasma thyroid hormones (FT(4) and FT(3)), biochemical bone marker levels [alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (ACP)] and bone DNA content were recorded. In undernourished pups, a decrease in body weight (-47%, p < 0.001) in skeletal growth (-11%, p < 0.001) and in bone DNA content (-48%, p < 0.001) were observed. Plasma protein and albumin levels and thyroid status were also decreased in undernourished rat pups compared with those of control group. The circulating amino acid levels were decreased in pups. While in their mothers, some of them were increased and the others were decreased. A positive correlation was observed between bone mass and plasma thyroid hormone levels and ALP activity and plasma protein levels, and a negative correlation with ACP activity. Plasma ALP levels were decreased by 19%, whereas plasma tartrate-resistant ACP levels were increased by 33% indicating a hyperactivity of bone remodelling. These results showed that protein-restricted diet given to mothers during late pregnancy and early post-natal periods induced changes in body weight, skeletal growth and bone metabolism in their offspring.

The effect of sheng bai solution on irradiated mice bone marrow cell division index and DNA content.

Before and after general irradiation with 60Co-gamma, mice were orally given Sheng Bai Solution (SBS) for one week. SBS alleviated the irradiation-induced reduction of bone marrow cell chromosome division index. The irradiation-induced decrease of marrow DNA amount, thymic and splenic fractions, and total leukocyte number were restored to some extent. SBS also helped to ameliorate general condition of patients.

Nucleoside-nucleotide mixture increases bone marrow cell number and small intestinal RNA content in protein-deficient mice after an acute bacterial infection.

The aim of this study was to determine if intraperitoneal administration of a nucleoside-nucleotide mixture would affect small intestinal morphology, bone marrow cell number, and DNA content in protein-deficient mice subjected to acute bacterial infection. Mice were randomized into two groups and orally fed protein-free diet or nucleotide-free 20% casein diet for 10 d. The mice in each group were divided into two subgroups and intraperitoneally administered 0.35 mL saline or nucleoside-nucleotide mixture (17.5 mL/kg body weight) for 10 d. On day 10, one subgroup from each major dietary group was either inoculated intravenously with methicillin-resistant Staphylococcus aureus or saline. Three days later, small intestinal morphology, bone marrow cell number, and DNA content were evaluated in infected and noninfected mice. Protein-deficiency in association with infection significantly (P < 0.05) reduced body weight, small intestinal weight, crypt depth, villous height, and wall thickness. All dietary groups exhibited similar small intestinal DNA and protein contents (protein:DNA ratio, RNA:DNA ratio) at 3 d postinfection. However, small intestinal RNA content in the infected protein-free dietary group administered nucleoside-nucleotide mixture was higher (P < 0.05) and tended to be higher relative to the infected nucleotide-free 20% casein group administered nucleoside-nucleotide mixture compared with the rest of the groups. In the infected protein-free dietary group administered nucleoside-nucleotide mixture, bone marrow cell number and bone marrow DNA content were higher (P < 0.05) relative to the infected protein-free dietary group, nucleotide-free 20% casein diet administered saline, or nucleoside-nucleotide mixture, respectively. We conclude that intraperitoneal administration of nucleoside-nucleotide mixture may stimulate bone marrow cell proliferation, DNA content, and small intestinal RNA content during periods of relative deficiency such as protein-deficiency in combination with infection.

Anabolic effect of genistein on bone metabolism in the femoral-metaphyseal tissues of elderly rats is inhibited by the anti-estrogen tamoxifen.

Whether the anabolic effect of genistein on bone metabolism in the femoral-metaphyseal tissues of elderly rats is inhibited by the anti-estrogen tamoxifen was investigated in vitro. Bone tissues were cultured for 24 h in the medium containing either vehicle or genistein. The presence of genistein (10(-6) and 10(-5) M) caused a significant increase in calcium content, alkaline phosphatase activity and DNA content in the bone tissues. The effect of genistein (10(-5) M) in increasing bone alkaline phosphatase activity and DNA content was equal to the stimulatory effect of 17 beta-estradiol (10(-10) and 10(-9) M) on the bone components. The genistein effect was not significantly enhanced by the presence of 17 beta-estradiol (10(-10) and 10(-9) M). The anti-estrogen tamoxifen (10(-7) M) completely blocked the effect of 17 beta-estrogen in increasing bone alkaline phosphatase activity and DNA content. The genistein (10(-5) M)-induced increases in bone alkaline phosphatase activity and DNA content were completely abolished by the presence of tamoxifen (10(-7) and 10(-6) M), although tamoxifen itself had no effect on the bone components. The present study demonstrated that the anabolic effect of genistein on bone components is inhibited by anti-estrogen tamoxifen. The effect of genistein may be mediated through estrogen-like action.

Effect of estrogen on bone metabolism in tissue culture: enhancement of the steroid effect by zinc

The present investigation was undertaken to clarify the effect of estrogen (17 beta-estradiol) on bone metabolism in tissue culture. Calvariae were removed from weanling rats (3-week-old females) and cultured for periods up to 96 h in Dulbecco's Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-10) to 10(-8) M estrogen. All cultures were incubated at 37 degrees C in 5% CO2/95% air. Bone calcium content was significantly increased by the presence of 10(-10) to 10(-8) M estrogen. The steroid (10(-10) to 10(-8) M) also significantly increased alkaline phosphatase activity in the bone, whereas it did not significantly alter acid phosphatase activity. No appreciable effect on bone alkaline phosphatase activity was produced with 17 alpha-estradiol (10(-9) and 10(-8) M). Tamoxifen (10(-6) M), an anti-estrogen, completely blocked the effect of estrogen (10(-9) M) of increasing bone alkaline phosphatase activity. Furthermore, bone DNA content was significantly increased by 10(-10) to 10(-8) M estrogen. Meanwhile, the presence of 10(-4) M zinc, which can stimulate bone protein synthesis, significantly enhanced the effect of 10(-9) M estrogen to increase DNA content in rat calvaria, while the metal did not enhance the steroid effect on bone calcium content and alkaline phosphatase activity. The presence of 10(-7) M cycloheximide completely prevented the stimulatory effect of estrogen (10(-9) M) on calcium content, alkaline phosphatase activity, and DNA content in rat calvaria. The present study demonstrates that estrogen has a direct stimulatory effect on bone metabolism in tissue culture and that zinc can enhance the steroid effect on bone DNA.

Stimulatory Effect of Beta-Alanyl-&lt;i&gt;L&lt;/i&gt;-Histidinato Zinc on Bone Formation in Tissue Culture

The present investigation was undertaken to clarify the in vitro effect of beta-alanyl-L-histidinato zinc (AHZ) on bone metabolism in tissue culture. Calvaria were removed from weanling rats (3-week-old male) and cultured for periods up to 96 h in Dulbecco's modified Eagle medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8) to 10(-4) mol/l AHZ. The bone cellular zinc content was significantly increased in cultures with concentrations of AHZ greater than 10(-6) mol/l. With 10(-5) mol/l zinc sulfate, the bone cellular zinc content was significantly elevated. Bone calcium content was significantly increased by the presence of 10(-7) to 10(-4) mol/l AHZ. This increase was blocked by the presence of 10(-7) mol/l cycloheximide. Bone alkaline phosphatase activity was elevated in the presence of AHZ (10(-7) to 10(-4) mol/l), whereas it did not significantly alter acid phosphatase activity Bone collagen and DNA contents were significantly increased by 10(-7) to 10(-5) mol/l AHZ, while they were not significantly elevated by zinc sulfate (10(-7) and 10(-6) mol/l). The AHZ (10(-5) mol/l)-induced increase in bone alkaline phosphatase activity and DNA content were prevented by 10(-4) mol/l dipicolinate, a chelator of zinc. Furthermore, the AHZ (10(-5) mol/l)-induced increase in bone alkaline phosphatase activity, collagen and DNA contents were blocked by 10(-7) mol/l cycloheximide. These findings indicate that AHZ had a direct stimulatory effect on bone mineralization in vitro, and that bone protein synthesis was a necessary component of this response. The AHZ effect was more intensive than that of zinc sulfate.

A new zinc compound, β-alanyl-l-histidinato zinc, stimulates bone growth in weanling rats

The effect of a new zinc compound. beta-alanyl-L-histidinato zinc (AHZ), on bone metabolism in weanling rats was investigated. Rats were orally administered AHZ (0.5-2.5 mg/100 g body weight) for 3 days, and 24h later they were killed. Administration of AHZ (1.0 and 2.5 mg/100 g) caused a significant increase of zinc content in the femoral diaphysis and a corresponding elevation of calcium content, alkaline phosphatase activity, and DNA content. A dose of 0.5 mg/100g AHZ did not produce an appreciable increase in bone components. When zinc sulfate (0.55 mg Zn/100g) was orally administered in rats for 3 days, the bone zinc content, calcium content, and alkaline phosphatase activity were raised significantly, but bone DNA content was not appreciably affected. Thus, the stimulation of AHZ (2.5 mg/100g), which corresponds to 0.55 mg Zn/100g, on bone metabolism was more intensive than that of zinc sulfate. These results suggest that AHZ can stimulate bone growth in weanling rats, and that the compound has a greater effect in comparison with zinc sulfate.

Effects of intraperitoneal administration of hexasodium N,N,N′,N′-ethylenediaminetetramethylenephosphonate on rat bone metabolism

The influence of hexasodium N,N,N′,N′-ethylenediaminetetramethylenephosphonate (EDITEMP.Na 6) on biochemical parameters in bone has been studied to help explain recently reported histological and pathological changes in bone induced by this agent. A single, intraperitoneal (ip) injection of EDITEMP.Na 6 (350 μmol/kg on Day 0) significantly decreased the activity of serum alkaline phosphatase (Alp) in mature male Wistar rats for up to 10 days following the injection. However, in immature rats the initial EDITEMP.Na 6-induced reduction of serum Alp activity preceded a steady increase in serum Alp activity, which was significantly elevated between Days 6 and 13. This increase coincided with a significant increase in bone Alp activity (an indicator of bone formation) on Day 8. Diaphyseal acid phosphatase (Acp) activity (an indicator of bone resorption) was significantly reduced, on Days 4 and 8 but not on Day 1, following treatment with EDITEMP.Na 6. Repeated exposure of immature male Wistar rats to EDITEMP.Na 6 (70 μmol/kg/day, ip for 7 days) also led to a significant and persistent increase in serum and bone Alp activities and a reduction in diaphyseal Acp activity. The data are in accord with published histomorphological evidence of reduced bone resorption and suggest delayed mineralization of osteoid tissue laid down during the treatment with EDITEMP.Na 6. There were only transient and limited changes in bone DNA content and [ 3H]thymidine incorporation throughout the studies, indicating the absence of marked cytotoxicity or altered bone cell proliferation associated with EDITEMP.Na 6-induced changes in phosphatase enzyme activities.

Beta-Alanyl-&lt;i&gt;L&lt;/i&gt;-Histidinato Zinc Prevents the Toxic Effect of Aluminium on Bone Metabolism in Weanling Rats

The preventive effect of beta-alanyl-L-histidinato zinc (AHZ) on the toxic action of aluminium on bone metabolism was investigated in the femoral diaphysis of weanling rats. Aluminium chloride (5.0, 10.0 and 20.0 mumol A1/100 g body weight) was orally administered for 3 days. The dose of 10.0 and 20.0 mumol A1/100 g caused a significant increase in serum calcium concentration and bone acid phosphatase activity, while bone alkaline phosphatase activity and calcium content were not altered significantly. Moreover, the bone DNA content was significantly decreased by the doses of 10.0 and 20.0 mumol A1/100 g. Meanwhile, the increase in serum calcium concentration caused by the administration of aluminium (20 mumol/100 g) was completely prevented by the simultaneous administration of AHZ (1.0 and 2.5 mg/100 g) for 3 days, although AHZ alone did not have any effect. Also, the effects of aluminium (20.0 mumol/100 g) to increase bone acid phosphatase activity and to decrease the bone DNA content were completely blocked by the simultaneous administration of AHZ (1.0 and 2.5 mg/100 g). AHZ (1.0 and 2.5 mg/100 g) alone had the effect to increase bone DNA content but not bone acid phosphatase activity. The present study indicates that AHZ can prevent the revelation of the toxic effect of aluminium on bone metabolism in rats.

Effects of Intraperitoneal Administration of Hexasodium N,N,N′,N′-Ethylenediaminetetramethylenephosphonate on Rat Bone Metabolism

Effects of Intraperitoneal Administration of Hexasodium N,N,N′,N′-Ethylenediaminetetra-methylenephosphonate on Rat Bone Metabolism. MOORE, N. C, AND CHIPMAN, J. K. (1990). Fundam. Appl. Toxicol. 14, 491–501. The influence of hexasodium N,N,N′,N′-ethylenedi-aminetetramethylenephosphonate (EDITEMP.Na6) on. biochemical parameters in bone has been studied to help explain recently reported histological and pathological changes in bone induced by this agent. A single, intraperitoneal (ip) injection of EDITEMP.Naj (350 μmol/kg on Day 0) significantly decreased the activity of serum alkaline phosphatase (Alp) in mature male Wistar rats for up to 10 days following the injection. However, in immature rats the initial EDITEMP.Na6-induced reduction of serum Alp activity preceded a steady increase in serum Alp activity, which was significantly elevated between Days 6 and 13. This increase coincided with a significant increase in bone Alp activity (an indicator of bone formation) on Day 8. Diaphyseal acid phosphatase (Acp) activity (an indicator of bone resorption) was significantly reduced, on Days 4 and 8 but not on Day 1, following treatment with EDITEMP.Na6. Repeated exposure of immature male Wistar rats to EDITEMP.Na* (70 μmol/kg/day, ip for 7 days) also led to a significant and persistent increase in serum and bone Alp activities and a reduction in diaphyseal Acp activity. The data are in accord with published histomorphological evidence of reduced bone resorption and suggest delayed mineralization of osteoid tissue laid down during the treatment with EDITEMP.Na6 There were only transient and limited changes in bone DNA content and [3H]thymidine incorporation throughout the studies, indicating the absence of marked cytotoxicity or altered bone cell proliferation associated with EDITEMP.Na6-induced changes in phosphatase enzyme activities.

Effect of dipicolinate, a chelator of zinc, on bone protein synthesis in tissue culture: The essential role of zinc

The present investigation was undertaken to clarify the essential role of zinc on bone protein synthesis in tissue culture. Calvariae were removed from 3-week-old male rats and cultured for periods up to 72 hr in Dulbecco's Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The calvariae were incubated for 24 hr at 37° in 5% CO 2/95% air in medium containing 10 −6–10 −3 M diplcolinate, a chelator of zinc, and then the bones were transferred into medium containing either 10 −4 M zinc sulfate or vehicle without diplcolinate. Zinc content in bone tissues was decreased when the culture was treated with 10 −4 and 10 −3 M dipieolinate for 24 hr. When calvariae treated with 10 −4 M dipicolinate for 24 hr were further cultured in medium without dipicolinate for 24 and 48 hr, bone alkaline phosphatase activity was decreased by about 40% (P < 0.01) of untreated bone enzyme activity. The decreased alkaline phosphatase activity was increased markedly by the presence of 10 −4 M zinc (about 2.5-fold of control value). This effect of zinc was blocked completely by the presence of 10 −7 M cycloheximide, but 10 −8 M actinomycin D caused only a partial inhibition. When calvariae treated with 10 −4 M dipicolinate were pulsed with [ 3H]proline, the incorporation of [ 3H]proline into the acid-insoluble residues of bone tissue was decreased by about 40% (P < 0.01) of the value obtained from calvariae not treated with dipieolinate. The presence of 10 −4 M zinc caused an increase of about 2-fold in [ 3H]proline incorporation. Bone DNA content was not altered significantly by treatment with 10 −4 M dipicolinate or 10 −4 M zinc. These results clearly indicate that endogenous zinc induces the stimulation of protein synthesis at the translational process in bone cells. The present study further supports the view that zinc plays an essential role for protein synthesis in bone cells.

Effect of 1,25-dihydroxyvitamin D 3 on bone metabolism in tissue culture : Enhancement of the steroid effect by zinc

The present investigation was undertaken to clarify the interaction of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] and zinc on bone metabolism in tissue culture. Calvaria were removed from weanling rats (3-week-old males) and cultured for periods up to 96 hr in Dulbecco's Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10 −10 to 10 −6M 1,25(OH) 2D 3. All cultures were incubated at 37° in 5% CO 2/95% air. Bone calcium content was increased significantly by the presence of 10 −9 to 10 −7M 1,25(OH) 2D 3. The steroid (10 −9 to 10 −7M) also significantly increased alkaline phosphatase activity in the bone, whereas it did not alter significantly acid phosphatase activity. [ 3H]Leucine incorporation by the bone was raised significantly by 10 −8 to 10 −7 M 1,25(OH) 2D 3. Furthermore, bone DNA content was increased significantly by 10 −9 to 10 −7 M 1,25(OH) 2D 3. Meanwhile, the presence of 10 −4 M zinc, which can stimulate bone formation, significantly enhanced the effect of 10 −7 M 1,25(OH) 2D 3 to increase alkaline phosphatase activity and DNA content in rat calvaria. The present study demonstrates that 1,25(OH) 2D 3 has a direct stimulatory effect on bone metabolism in tissue culture and that zinc can enhance the steroid effect.

Effect of vanadium on bone metabolism in weanling rats: zinc prevents the toxic effect of vanadium.

The effect of vanadium on bone metabolism was investigated in the femoral diaphysis of weanling rats. Vanadium pentoxide (1.0-20.0 mumol V/100 g b.wt.) was administered orally for 3 days. The doses of 15.0 and 20.0 mumol V/100 g caused a significant increase in serum calcium concentration. Bone alkaline phosphatase activity was increased significantly by the doses of 1.0-20.0 mumol V/100 g, while bone acid phosphatase activity was not altered significantly. Bone DNA content was increased significantly by the dose of 1.0-10.0 mumol V/100 g. Bone calcium content was not altered significantly by administration of vanadium. The increase in serum calcium concentration caused by administration of vanadium (20.0 mumol/100 g) was prevented completely by simultaneous injection of zinc sulfate (15.3 mumol Zn/100 g) for 3 days, although zinc alone did not have any effect. Administration of zinc (15.3 mumol/100 g) produced an appreciable increase in bone alkaline phosphatase activity, DNA content, and calcium content. These increases were not enhanced significantly by simultaneous injection of vanadium (2.0 and 20.0 mumol V/100 g). The present study indicates that a comparatively low dose of vanadium may play a nutritional role in bone formation of weanling rats, and that zinc can prevent the relevation of the toxic effect of vanadium with higher doses.

Stimulatory effect of calcitonin on bone formation in tissue culture.

The present investigation was undertaken to clarify the in vitro effect of synthetic [Asu1,7]eel calcitonin (CT) on bone metabolism in tissue culture. Calvaria were removed from weanling rats (3-week-old male) and cultured for periods up to 96 h in Dulbecco's Modified Eagle Medium supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 1.0 to 100 ng/ml CT. All cultures were incubated at 37 degrees C in 5% CO2-95% air. Bone calcium content was increased significantly by the presence of 10 and 100 ng/ml CT. This increase was blocked by the presence of 10(-6) M cycloheximide or 10(-7) M actinomycin D. Bone alkaline phosphatase activity was significantly increased by the presence of 100 ng/ml CT for 48 and 96 h. Bone acid phosphatase activity was not altered significantly by CT (1-100 ng/ml). The incorporation of [3H]proline into the acid-insoluble residues of bone tissue was significantly increased by the presence of CT (1-100 ng/ml) for 96 h. This increase was completely blocked by the presence of 10(-7) M cycloheximide. Bone DNA content was significantly raised by the presence of 10 and 100 ng/ml CT for 96 h. Furthermore, the culture with CT (10 and 100 ng/ml) produced a significant decrease in glucose concentration in the medium. Also, CT (10 and 100 ng/ml) stimulated the production of pyruvic acid from bone tissue. These results suggest that CT had a direct stimulatory effect on bone formation and mineralization in vitro, and that the hormone stimulates energy metabolism in bone cells.

Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate.

We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.

Stimulatory effect of zinc on bone formation in tissue culture

The present investigation was undertaken to clarify the in vitro effect of zinc on bone metabolism in tissue culture. Calvaria were removed from weanling rats (3-week-old males) and cultured for periods up to 96 hr in Dulbecco's Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10 −7 to 10 −3M zinc sulfate. All cultures were incubated at 37° in 5% CO 2/95% air. Zinc uptake by bone was increased significantly in cultures with concentrations of zinc greater than 10 −6 M. Bone calcium content was increased significantly by the presence of 10 −4M zinc. This increase was blocked by the presence of 10 −6 M cycloheximide. Bone alkaline phosphatase activity was elevated in the presence of zinc (10 −6 to 10 −3 M), but the effect was inhibited by 10 −7M cycloheximide or 10 −8M actinomycin D. Zinc (10 −4M) also significantly increased ATPase activity in the bone, whereas it did not alter significantly by pyrophosphatase, acid phosphatase and β- N-acetylglucosaminidase activities. Furthermore, bone collagen content was raised by 10 −6 to 10 −4 M zinc. This elevation was prevented by 10 −7 cycloheximide or 10 −8 M actinomycin D. Bone DNA content and [ 3H]thymidine incorporation by the bone were not altered significantly by 10 −4 M zinc. These findings indicate that the zinc had a direct stimulatory effect on bone mineralization in vitro, and that bone protein synthesis was a necessary component of this response. Zinc may stimulate bone formation in tissue culture.

Interleukin-1 has independent effects on deoxyribonucleic acid and collagen synthesis in cultures of rat calvariae.

Interleukin-1 (IL-1), a monokine known to be important in host defense mechanisms and recently reported to stimulate bone resorption, was studied for its effects on bone formation in cultures of 21-day-old fetal rat calvariae. IL-1 at 0.1-5 U/ml stimulated the incorporation of [3H] thymidine into acid-insoluble residues (DNA) by 29-123% in calvariae treated for 24-96 h. IL-1 also increased the bone DNA content and the number of mitoses after colcemid arrest. IL-1 stimulated total protein synthesis. Treatment with IL-1 at 0.01-1 U/ml for 24 h caused a small increase in the incorporation of [3H]proline into collagenase-digestible protein (CDP) and non-collagen protein (NCP). However, higher doses of IL-1 (5 U/ml) or longer exposure to the agent (1 U/ml for 96 h) inhibited the labeling of CDP but not of NCP. IL-1 affected only type I collagen. The stimulatory effects of IL-1 on DNA, CDP, and NCP labeling were independent, since they were observed at different doses, and hydroxyurea abolished the effect on DNA without changing that on CDP and NCP labeling. Indomethacin blocked the stimulatory effect on CDP and NCP labeling, suggesting a prostaglandin-mediated effect, but did not change the IL-1 effect on DNA synthesis. These studies indicate that IL-1 stimulates calvarial DNA, collagen, and NCP synthesis, but exposure of the calvariae to high IL-1 doses or to IL-1 for prolonged periods of time results in an inhibition of collagen synthesis.