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We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 microM and 0.1 microM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 microM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 microM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 mumol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity.

We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 microM and 0.1 microM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 microM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 microM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 mumol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity.

We found that tyrosine kinase pp60(c-src) coisolates with acid-transporting osteoclast membranes and hypothesized that this kinase regulates hydrochloric acid transport. We assayed the membrane acid transport and bone degradation effects of tyrosine kinase inhibitors in avian osteoclasts. Isoflavone, tyrphostin, and benzoquinonoid inhibitors were compared with inactive analogues to determine nonspecific effects. Acid-secreting membranes, isolated by nitrogen cavitation, were assayed as reconstituted vesicles by using acridine orange to indicate ATP-dependent hydrogen ion transport. The soy isoflavone genistein and the benzoquinonoid antibiotic herbimycin inhibited hydrochloric acid transport with 50% inhibition at approximately 10 and approximately 2 micromol/L, respectively; effects appeared in <2 min and were reversible. In membrane incubated with inhibitors, the herbimycin effect also inhibited Cl- transport by variable amounts, suggesting that this compound affects Cl- channel activity. However, genistein and tyrphostins did not produce chloride dependent effects. After 30 min with ATP, tyrphostin A47 irreversibly inhibited hydrochloric acid transport with 50% inhibition at approximately 10 micromol/L. Tyrphostin A25 and controls, tyrphostin A1 and daidzein (a genistein congener), were inactive despite preincubation. Osteoclastic bone resorption was more sensitive to the inhibitors over 3-5-d assays than was membrane acid transport, except for tyrphostins. Herbimycin and genistein inhibited bone resorption with half maximal effects at 0.5 and 10 micromol/L and complete inhibition at 3 d in 1 and 20 micromol/L, respectively. None of the tyrphostins, including A47, nor daidzein inhibited resorption to >20 micromol/L. We conclude that tyrosine kinase inhibition directly inhibits osteoclast membrane hydrochloric acid transport; differences among inhibitors may reflect chemical reactivity and permeability.

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