Opposing effects of bisphosphonates and advanced glycation end-products on osteoblastic cells

Patients with long-standing Diabetes mellitus can develop osteopenia and osteoporosis. We have previously shown that advanced glycation endproducts reduce the bone-forming activity of osteoblasts. Bisphosphonates are used for the treatment of various bone disorders, since they reduce osteoclastic function and survival, and stimulate osteoblastic bone-forming capacity. In this work we have investigated whether bisphosphonates are able to revert advanced glycation endproducts-induced deleterious effects in osteoblasts. MC3T3E1 and UMR106 osteoblastic cells were incubated with control or advanced glycation endproducts-modified bovine serum albumin, in the presence or absence of different doses of the bisphosphonates Alendronate, Pamidronate or Zoledronate. After 24–72 h of culture, we evaluated their effects on cell proliferation and apoptosis, type-1 collagen production, alkaline and neutral phosphatase activity, and intracellular reactive oxygen species production. Advanced glycation endproducts significantly decreased osteoblast proliferation, alkaline phosphatase activity and type 1 collagen production, while increasing osteoblastic apoptosis and reactive oxygen species production. These effects were completely reverted by low doses (10^− 8 M) of bisphosphonates. High doses of bisphosphonates (10^− 4–10^− 5 M) were toxic for osteoblasts. Nifedipine (L-type calcium channel blocker) did not affect the advanced glycation endproducts-induced decrease in osteoblastic proliferation, although it blocked the reversion of this effect by 10^− 8 M Alendronate. Both advanced glycation endproducts and Alendronate inhibited the activity of intracellular neutral phosphatases. In conclusion, we show that bisphosphonates revert the deleterious actions of advanced glycation endproducts on osteoblastic cells, and that these effects of bisphosphonates depend on: (a) Ca²⁺ influx through L-type voltage-sensitive channels, and (b) blockage of advanced glycation endproducts-induced reactive oxygen species generation.     

Characterization of cadmium uptake and cytotoxicity in human osteoblast-like MG-63 cells

Since bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation, alterations in osteoblast proliferation and differentiation may disturb the equilibrium of bone remodeling. Exposure to cadmium (Cd) has been associated with the alteration of bone metabolism and the development of osteoporosis. Because little information is available about the direct effects of Cd on osteoblastic cells, we have characterized in vitro the cellular accumulation and cytotoxicity of Cd in human osteoblastic cells. Incubation of osteoblast-like MG-63 cells with increasing concentrations of Cd in serum-free culture medium reduced cell viability in a time- and concentration-dependent manner, suggesting that Cd accumulates in osteoblasts. Consequently, an uptake time-course could be characterized for the cellular accumulation of ¹⁰⁹Cd in serum-free culture medium. In order to characterize the mechanisms of Cd uptake, experiments have been conducted under well-defined metal speciation conditions in chloride and nitrate transport media. The results revealed a preferential uptake of Cd²⁺ species. The cellular accumulation and cytotoxicity of Cd increased in the absence of extracellular calcium (Ca), suggesting that Cd may enter the cells in part through Ca channels. However, neither the cellular accumulation nor the cytotoxicity of Cd was modified by voltage-dependent Ca channel (VDCC) modulators or potassium-induced depolarization. Moreover, exposure conditions activating or inhibiting capacitative Ca entry (CCE) failed to modify the cellular accumulation and cytotoxicity of Cd, which excludes the involvement of canonical transient receptor potential (TRPC) channels. The cellular accumulation and cytotoxicity of Cd were reduced by 2-APB, a known inhibitor of the Mg and Ca channel TRPM7 and were increased in the absence of extracellular magnesium (Mg). The inhibition of Cd uptake by Mg and Ca was not additive, suggesting that each ion inhibits the same uptake mechanism. Our results indicate that Cd uptake in human osteoblastic cells occurs, at least in part, through Ca- and Mg-inhibitable transport mechanisms, which may involve channels of the TRPM family. The effect of Cd on bone metabolism may be enhanced under low Ca and/or Mg levels.     

Effects in vitro of cadmium ions on some membrane and nuclear parameters of normal and irradiated thymic lymphoid cells

Effects of cadmium chloride upon ³H-Con A binding, number of autologous rosette-forming cells (ARFC), cell viability and the degree of DNA supercoiling were studied in normal and irradiated thymic lymphoid cells, isolated from rats and incubated up to 6 h in vitro. Cd (10–100 M) did not significantly alter the patterns of surface markers and viability of normal thymocytes, as measured by supravital staining or nuclear pyknotic criteria. The following effects of Cd were noted for irradiated thymic cells: 1) Cd ions (25 M) caused elimination of radiation-induced increase of Con A binding; 2) the characteristic loss of ARFC receptors, like development of nuclear pyknosis, was prevented in the presence of CdCl₂ (10–100 M); 3) the postradiation relaxation of nuclear supercoiled DNA was distinctly less pronounced with Cd. Possible reasons for these effects of Cd are discussed. Irradiated lymphoid cells are proposed as a suitable experimental model for the studies of different toxic actions of Cd and other heavy metals.     

淫羊藿苷和仙茅苷协同抑制破骨细胞的形成、分化和骨吸收功能

目的 研究淫羊藿苷和仙茅苷配伍抑制破骨细胞形成、分化和骨吸收的相互作用关系.方法 用1,25-(0H)2Vitamin D3和地塞米松;或人巨噬细胞集落刺激因子(MCSF)和核因子κB受体活化因子配体(RANKL)诱导骨髓单核细胞使其分化为破骨细胞抗酒石酸酸性磷酸酶(TRAP)染色进行阳性破骨细胞鉴定;磷酸苯二钠法测定抗酒石酸酸性磷酸酶活性;将破骨细胞和骨片共同培养,以计算机图像分析测定骨吸收陷窝的面积;以Hoechst 33258和罗丹明-鬼笔环肽染色,激光共聚焦显微镜观察破骨细胞肌动蛋白环(F-actin Ring)的形态;用Western-blot分析细胞骨架相关蛋白的表达.结果 淫羊藿苷和仙茅苷在1×10-6mol/L浓度下可协同抑制破骨细胞的形成、降低抗酒石酸酸性磷酸酶的活性,减少破骨细胞在骨片上形成的骨吸收陷窝面积,抑制破骨细胞骨架F-actin环的构建及其调控因子Rho GTPases和灶性黏附激酶(focal adhesion kinase,FAK)的表达.结论 淫羊藿苷和仙茅苷协同抑制破骨细胞性的骨吸收,为药对淫羊藿仙茅的配伍提供了实验依据.     

Effects of phthalazinol on smooth muscle cells of the porcine coronary artery and on neuromuscular junction on the guinea-pig vas deferens

Summary In smooth muscle cells of the porcine coronary artery, phthalazinol (10^–5 M) did not modify the membrane potential and the membrane resistance. At a concentration of 10^–4 M or higher, it hyperpolarized the membrane, reduced the membrane resistance and enhanced the rectifying property of the membrane. At the concentration of 10^–5 M, phthalazinol raised the threshold for the induction of a contraction and suppressed nonselectively the amplitude of the contraction evoked by application of high [K]₀, acetylcholine or electrical depolarization of the membrane. Phthalazinol (10^–5–10^–4 M) did not modify the membrane properties of smooth muscle cells from the guinea-pig vas deferens. However, it suppressed the amplitude of the excitatory junction potentials and the facilitation phenomenon produced by repetitive stimulation at various rates. The action potential recorded from the adrenergic nerves was not affected in the presence of phthalazinol (10^–5 and 10^–4 M). The mean amplitude of the miniature excitatory junction potentials (m.e.j.p.s.) was not affected by treatment with phthalazinol (10^–5–10^–4 M), but the rate of which of m.e.j.p.s. appeared was reduced by phthalazinol (>5×10^–5 M). These results indicate that the vasodilator property of phthalazinol may result from a suppression of Ca-mobilization in both the smooth muscle cells and the adrenergic nerve terminals. The former affects the mechanical response directly and the latter leads to an inhibition of noradrenaline release.     

β-alanyl-l-histidinato zinc and bone resorption

• 1.1.β-Alanyl-l-histidinato zinc (AHZ), in which zinc is chelated to β-alanyl-l-histidine, is a new zinc compound. β-Alanyl-l-histidine can uniquely chelated zinc ion in various essential trace metals. More recently, it has been demonstrated that this compound has more intensive effect than zinc sulfate on bone metabolism, suggesting a role as pharmacological tool in osteoporosis. This review describes mainly the action of AHZ on bone resorption as summarized in the following.
• 2.2. The prolonged oral administration of AHZ (10–100 mg/kg/day) can completely prevent bone loss in the femur of ovariectomized rats, indicating the preventive effect of AHZ on bone resorption in vivo.
• 3.3. The decrease in bone calcium content induced by various bone resorbing factors was completely inhibited by the presence of AHZ (10⁻⁶-10⁻⁴ M) in bone tissue culture system in vitro.
• 4.4. Many bone resorbing agents can stimulate the formation (differentiation) of osteoclasts from marrow cells. AHZ (10⁻⁶-10⁻⁴ M) clearly inhibited osteoclast-like cell formation in mouse marrow culture in vitro.
• 5.5. AHZ may act on the process of parathyroid hormone-induced protein kinase C activation which is involved in Ca²⁺-signaling in osteoclastic cells.

     

The tachykinin NK1 receptor mediates the migration-promoting effect of substance P on human skin fibroblasts in culture

Fibroblast migration is an important component of the tissue response during the repair process, and substance P (SP) has been shown to exert trophic effects. In the present study, cell migration was evaluated as the distance travelled by adherent human skin fibroblasts (HF) at 96 h and by the number of individual cells moving across a filter within 5 h.In control conditions (1% calf serum) adherent fibroblasts moved from the starting line by approximately 700 m. The addition of SP (10^–11–10^–7 M) increased HF mobilisation in a concentration-dependent manner, with maximal activity at 10^–8 M (50% increase in migration over control). Migration of individual HF in suspension was also promoted by SP in a concentration-dependent manner, with an EC₅₀ of 2.2×10^–9 M. The response produced by the maximally effective concentration of SP was equal to 65 and 90% of the effect elicited by 100 ng/ml Platelet-Derived Growth Factor AB (PDGF A/B) on adherent and individual cells respectively. The synthetic NK₁ receptor agonist [Sar⁹]SP-sulphone (10^–11–10^–6 M) reproduced the SP effect. The NK₂ and NK₃ receptor agonists [Ala⁸]NKA(4–10) and [MePhe⁷]NKB were devoid of any effect. The effect of SP was antagonised by two selective antagonists of NK₁ receptors, namely (±) CP 96,345 (10^–10–10^–8 M) and FK 888 (10^–9–10^–7 M), while the NK₂ receptor antagonist MEN 10627 (10^–8–10^–7 M) was not effective.Our data indicate that SP is a potent effector of fibroblast migration and the NK₁ receptor is responsible for this effect. These observations further support the specific role of the NK₁ receptor in mediating the trophic function of SP at the cutaneous level.     

Cadmium uptake and induction of metallothionein synthesis in a renal epithelial cell line (LLC-PK1)

LLC-PK₁ cells, an established cell line from pig kidney with proximal tubule properties, were cultivated in vitro at confluence on plastic dishes. They were then exposed (apical side) to inorganic cadmium (CdCl₂, 5 M) for periods ranging between 1 to 24 h. Analysis of the cell supernatant after homogenisation and ultracentrifugation indicated that Cd taken up in the first 3 h was bound to cytosolic high molecular weight proteins, but was redistributed to low molecular weight proteins at later stages. Induction of Cd-metallothionein (Cd-Mt) synthesis, as judged from Cd-Mt binding to a specific anti-Cd-Mt antibody and from the rate of³⁵S-cys incorporation into a specific protein fraction, was apparent 3–6 h after the addition of Cd to the incubation medium.     

In vitro effects of epoxide-bearing alepotriates on mouse early hematopoietic progenitor cells and human T-lymphocytes

The epoxide-bearing valepotriates valtrate/isovaltrate and dihydrovaltrate, isolated from Valerianaceae roots and used as common sedative drugs, were investigated for their effects on untransformed hematopoietic cells after an alkylating potential and cytotoxicity to tumor cells had been found. The compounds were added to sensitive in vitro assays using granulocyte/macrophage (GM-CFC) and erythrocyte (E-CFC) colony forming cells from murine bone marrow early progenitor cells as well as colony forming PHA-stimulated T-lymphocytes from human peripheral blood.The ID₅₀ for both GM-CFC and T-lymphocytes incubated with valtrate was found to be about 3×10^–6 M, with dihydrovaltrate about 2×10^–6 M. On erythrocyte colonies (E-CFC) both compounds showed an ID₅₀ of about 3×10^–8 M. Valtrate effects were exhibited at lower concentrations than effects caused by the known alkylating agent epichlorohydrin. The non-alkylating epoxide l-scopolamine taken for reference was clearly the least effective. Valtrate and dihydrovaltrate effects on GM-CFC were not reversible by washing the cultures.It is concluded that the valepotriates investigated are likely to be strongly cytotoxic to the untransformed hematopoietic cells studied.

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Zusammenfassung Die epoxidhaltigen Valepotriate Valtrat/Isovaltrat und Dihydrovaltrat, die aus den Wurzeln von Valerianaceae isoliert und als sedative Arzneimittel benutzt werden, wurden hinsichtlich ihrer Wirkung auf untransformierte hämatopoietische Zellen untersucht, nachdem ein Alkylierungspotential und Zytotoxizität auf Tumorzellen gefunden worden waren. Die Stoffe wurden in empfindlichen in vitro-Tests bezüglich ihrer Wirkung auf Vorläuferzellen für Granulo/yten/Makrophagen (GM-CFC) und Erythrozyten (E-CFC) aus dem Knochenmark von Mäusen sowie auf Kolonie-bildende PHA-stimulierte T-Lymphozyten des menschlichen peripheren Blutes geprüft.Bei Valtrat-Zugabe betrug die ID₅₀ für GM-CFC und T-Lymphozyten ca. 3×10^–6 M, bei Dihydrovaltrat ca. 2×10^–5 M. Für E-CFC-Kolonien ergab sich bei beiden Substanzen eine ID₅₀ von ca. 3×10^–8 M. Die Wirkung von Valtrat zeigte sich bei niedrigerer Konzentration als die Wirkung des bekannten Alkylans Epichlorhydrin. Das zum Vergleich untersuchte nichtalkylierende Epoxid l-Scopolamin war eindeutig am wenigsten wirksam. Die Valtrat- und Dihydrovaltrat-Effekte waren nicht reversibel durch Auswaschen der Kulturen.Es kann geschlossen werden, daß die untersuchten Valepotriate wahrscheinlich stark zytotoxisch sind für die in den Tests verwendeten untransformierten hämatopoietischen Zellen.

     

Endothelial cells contain beta adrenoceptors

Summary The direct identification of beta adrenoceptors in endothelial cell cultures has not been possible until the advent of a new beta-adrenergic radioligand, [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP). Using [¹²⁵I]ICYP, we report thes successful identification of a beta adrenoceptor in cultured bovine aortic endothelial cells. At 37°C, specific binding is saturable, stable and reversible. There is a single class of binding sites (21,500±2,900 sites/cell) with an equilibrium dissociation constant (K _d) of 109±26 pM. The rate constant of association, k ₁, is 1.22×10⁹ M^–1 min^–1 and of dissociation, k _–1, is 0.01 min^–1. Binding studies on monolayers of endothelial cells grown in microtiter plates yield similar data (K _d=53±9 pM, B _max=20,000±1,900 sites/cell). Stereoselectivity of binding for the (–)-isomer is demonstrable for both agonists and antagonists. A series of adrenergic agonists competes with [¹²⁵I]ICYP for binding with an order of potency suggesting beta₂ subselectivity; isoproterenol (0.73 M) > epinephrine (15 M) > norepinephrine (71 M). Furthermore, the beta₂ inhibitor butoxamine is more potent than the beta₁ inhibitor practolol (7.7 M vs 22 M respectively). The GTP analogue, Gpp(NH)p, reduces isoproterenol affinity to 1.9 M and increases the Hill coefficient from 0.62–0.90.     

Dihydrolipoamide dehydrogenase and cAMP are associated with cadmium-mediated Leydig cell damage

Cadmium (Cd) directly inhibits testosterone production in Leydig cells, but its mechanism is still unclear. To further explore the signaling pathway of Cd-mediated toxicity to Leydig cells, various concentrations of Cd were cultured with R2C cells for 24 h, and two-dimensional gel electrophoresis (2DE)-based proteomics profiling was used to analyze the change of protein expressions. Cd caused a concentration-dependent inhibition of cell viability with IC₂₅, IC₅₀ and IC₇₅ of 2.42 × 10⁻⁵ M, 4.83 × 10⁻⁵ M and 7.39 × 10⁻⁵ M, respectively. Cd significantly reduced progesterone production and mitochondrial membrane potential (ΔΨ_m) in a concentration-dependent manner. 2DE-based proteomics showed 34 protein spots with altered expression by 2-folds or more, and dihydrolipoamide dehydrogenase (DLD) was the hub in the network of these altered proteins. Real-time polymerase chain reaction (PCR) and Western blotting showed that Cd downregulated the expression of DLD. Cd also decreased intracellular levels of cyclic adenosine monophosphate (cAMP). The results suggest that DLD and cAMP may be key elements related to Cd toxicity to Leydig cells.     

Coenzyme Q10 attenuates cyanide-activation of the ATP-sensitive K+ channel current in single cardiac myocytes of the guinea-pig

Summary The effect of coenzyme Q₁₀ (CoQ₁₀) on the cyanide (CN^–)-induced ATP-sensitive K⁺ channel current (K_ATP) was examined in single atrial myocytes, using the patch clamp technique. Superfusion of the cells with a CN^–/low glucose bathing solution induced an outward current in the whole-cell clamp condition. Glibenclamide (1 M) abolished this current, indicating that the current was carried through the K_ATP channel. After steady-state activation by CN^–, pinacidil (a K_ATP channel opener, 300 M) failed to further increase the current. In cell-attached patches, CN^–, when applied to the bath, induced bursting openings of an 80 pS channel (the K_ATP channel). In cells preincubated for 30 min in a solution containing CoQ₁₀ (100 g/ml), CN^–-activation of the K_ATP channel was markedly attenuated both at the whole cell and at the single channel level. At the steady-state effect of CN^– in CoQ₁₀-treated cells, pinacidil (300 M) activated the current to the maximum level achieved by CN^– in the control cells. These results suggest that CoQ₁₀ reduces in the CN^–-induced KATP current not by affecting the channel itself but by preventing depletion of intracellular ATP caused by CN^–. Send offprint requests to Y. Kurachi at Mayo Foundation     

Field stimulation-induced responses of circular smooth muscle from guinea-pig stomach

Summary Field stimulation of circular smooth muscle of guinea-pig stomach from the regions of the cardia and fundus caused contraction responses at low stimulation frequencies (0.25–1 Hz) with relaxation at higher frequencies (1–10 Hz), whilst tissues of the body and antrum responded with contraction throughout the frequency range. Atropine (10^–9–10^–8 M) antagonised the contraction responses of all tissues, with relaxation developing at higher concentrations (except for antral tissue). In contrast, metoclopramide (10^–8–10^–6 M) caused modest (cardia, fundus) or marked (body, antrum) enhancement of contractions to field stimulation, whilst domperidone (10^–8–10^–7 M), haloperidol (10^–8–10^–6 M), prazosin, propranolol and methysergide (10^–8–10^–6 M) failed to modify the contraction responses. However, whilst yohimbine and guanethidine failed to modify the contractions of the cardia, fundus and body tissues, those of the antral preparations were antagonised by nanomolar concentrations of yohimbine and by guanethidine (10^–6–5×10^–5 M). To optimise the relaxation responses for study, atropine was included in the physiological solution. Relaxation to field stimulation of preparations from the body and cardia, but not the fundus, was antagonised by reserpine pretreatment (5 mg/kg i.p., 24h), addition of guanethidine (10^–5–10^–4 M), phentolamine, prazosin or propranolol (10^–7–10^–6 M) (the effects of prazosin and propranolol being additive). Higher concentrations of haloperidol and domperidone antagonised the relaxation responses of the body preparations only. Metoclopramide, yohimbine and methysergide (10^–8–10^–6 M) were ineffective. Thus, it is concluded that the contractile effects of the 4 stomach areas to field stimulation reflects a major cholinergic involvement, with an additional ₂-adrenoceptor contractile component in antral tissue. Relaxation responses of cardia and body tissue involve ₂- and -adrenoceptors plus a further, unidentified, non-adrenergic component; the latter represents the total relaxation response of the fundic preparation.     

Cytotoxic effects of cadmium in mammary epithelial cells: Protective role of the macrocycle [15]pyN5

Human exposure to cadmium (Cd) occurs via different routes, including diet. The increasing amount of data linking Cd with different cellular effects in the mammary gland justifies additional toxicological assessments using human mammary epithelial cells. This work aimed therefore to assess the cytotoxic effects of Cd in MCF10A cells and to characterize the cytoprotective role of the macrocycle [15]pyN₅ in the form of calcium salt. Cadmium chloride revealed to be cytotoxic to MCF10A cells, decreasing cell viability and proliferation in a concentration-dependent manner. Comparable dose–response curves and IC50 values (57–63 μM, 24 h treatment) were obtained using the MTT reduction, crystal violet and BrdU assays. In terms of reactive oxygen species formation, only a slight increase in superoxide radical anion was observed at very high Cd concentrations (?100 μM). Chelation should thus constitute the primary strategy to mitigate the cytotoxic effects induced by Cd in mammary cells. In this context, [15]pyN₅ which presents appropriate chemical and thermodynamic features was studied as a Cd chelator. This macrocycle (25 and 50 μM) significantly reduced or even abolished Cd-induced cytotoxicity. Protective effects were observed in terms of cell viability, cell proliferation and morphological alterations, being the protection mostly attributed to a chelating-based mechanism.     

Effects of CIS-19, a novel PAF receptor antagonist,on PAF-induced eosinophil recruitment and enhancement of superoxide anion generation in guinea-pigs

We examined the effects of a novel plateletactivating factor (PAF) receptor antagonist, CIS-19 [cis-2-(3,4-dimethoxyphenyl)-6-isopropoxy-7-methoxyl-1-(N-methylformamido)-1,2,3,4-tetrahydronaphthalene], on PAF-induced inflammatory cells recruitment into airways and enhancement of superoxide anion (O _inf2 ^sup– ) generation from cells retrieved by bronchoalveolar lavage (BAL) in urethane-anesthetized guinea-pigs. Administration of PAF (30 ng/kg, Lv.) produced a selective increase of eosinophils into airways, but no significant increase of the number of macrophages, neutrophils or lymphocytes. CIS-19 (2.5 and 5 mg/kg, Lv.) significantly inhibited the eosinophil recruitment induced by PAF. In vitro, PAF, phorbol 12-myristate 13-acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) directly stimulated generation of O _inf2 ^sup– from BAL cells in a concentration-dependent manner. CIS-19 (10^–7 – 10^–4 M) inhibited production of O _inf2 ^sup– induced by PAF (10^–7 M) in a concentration-dependent manner with an EC₅₀ value of 0.84 M, but not induced by PMA (0.5 g/ml) or FMLP (10^–7 M). Administration of PAF (5 ng/kg, i.v.) enhanced markedly PMA (0.5 g/ml) and FMLP (10^–7 M)-induced generation of O _inf2 ^sup– by 80.2% and 51.3%, respectively. The enhancing effect of PAF was maximal in cells harvested 5 min after the addition of PAF and then declined to baseline level at 60 min. These responses were inhibited by administration of CIS-19 (0.5—2.5 mg/kg, i.v.) or BN 52021(5 mg/kg, i.v.). The results indicate that CIS-19 is potent in inhibition of PAF-induced airway inflammatory response and may have therapeutic potential as an anti-inflammatory drugs.     

Role of fos and src in cadmium-induced decreases in bone mineral content in mice

Cadmium decreases bone mineral in mice and stimulates osteoclast formation and activity in cell culture. Bones from fos-deficient mice contain very few osteoclasts; src-/- bones contain osteoclasts that fail to activate. Fos-/- and src-/- mice develop osteopetrotic bones and their teeth do not erupt. These mice were used to determine if cadmium requires c-Fos or c-Src and secondarily functional osteoclasts to decrease bone mineral content. Mice heterozygous for fos deficiency were mated to produce fos-/- and fos+/o (wild-type) offspring. Pups were divided into four groups: fos+/o, Cd-; fos+/o, Cd+; fos-/-, Cd-; and fos-/-, Cd+. Cd+ pups received daily sc injections (50 microg Cd/kg) on days 17-20 and Cd in drinking water thereafter (10 ppm, days 21-27; 20 ppm, days 27-50). An analogous protocol was followed mating mice heterozygous for src deficiency. For acute exposures, 50-day-old mice were placed on a low-calcium diet and given two sequential 100 microg Cd doses by gavage, and feces were monitored for excretion of bone calcium. Continuous exposure results demonstrate that cadmium (1) significantly decreased bone calcium content (14-15%) and concentration (12-13%) in both fos+/o and fos-/- mice, (2) doubled multinucleated osteoclast-like cell number in fos-/- bones, and (3) stimulated tooth eruption in 40% of fos-/- mice. Cd gavage stimulated bone calcium excretion in both fos+/o and fos-/- mice. In contrast, cadmium had no effect on bones or teeth in src-/- mice. Results indicate that cadmium can decrease bone mineral via a c-Fos-independent pathway; however, c-Src is required for cadmium to stimulate bone remodeling and tooth eruption pathways.