D-penicillamine: its actions on lead transport in bone organ culture.

Summary: The purpose of this study was to develop a strictly controlled chemical system in vitro to evaluate the efficacy of lead (Pb) chelators. Moreover, these experiments were carried out to understand further the complex interactions between Pb chelating agents, calcium-regulating hormones, and bone cell metabolism. The effects of D-penicillamine (PCA) on previously incorporated 203Pb transport from fetal rat bone explants were examined in bone organ culture. In several experiments, 203Pb and stable bone Pb content were measured, and proportional net losses or gain were found. Experimental medium/control medium (EM/CM) ratios were used to reflect inhibition or stimulation of 203Pb release because these data were expressed as the net cpm released from treated bones into the EM, defined as a change in one variable, divided by the net cpm released from control bones into the appropriate CM. EM/CM ratios increased significantly (1.96 ± 0.04 to 7.28 ± 0.08) in a dose-response relationship as PCA concentrations were raised from 1.0 to 10.0 mM. Addition of parathyroid hormone (PTH) to the medium resulted in increased EM/CM ratios for 203Pb at lower PCA concentrations (EM/CM 2.79 ± 0.14 at 0.10 mM PCA; 6.71 ± 0.08 at PCA 5.0 mM), compared to PCA in the media alone. This addition of PTH effected release of 45calcium (45Ca) (EM/CM, 1.45 ± 0.07 to 3.62 ± 0.10) at PCA concentrations from 0.10 to 10.0 mM. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] in combination with PCA enhanced 203Pb release, evidenced by an EM/CM ratio of 5.69 ± 0.14 at 1.0 mM PCA compared to an EM/CM ratio of 1.96 ± 0.04 when only 1.0 mM PCA was present in the medium. A decrease in medium Ca concentration from 1.40 to 0.70 mM in the presence of 1.0 mM PCA and 20 ng/ml 1,25-dihydroxyvitamin D3 enhanced the net release of 203Pb as well. In contrast, increasing medium concentrations of Ca (2.80 mM) or phosphate from 2.0 to 4.0 mM lowered EM/CM ratios in the presence of PCA, PTH, and 1,25-(OH)2D3. Calcitonin markedly inhibited 1.0 mM PCA-induced 203Pb efflux (EM/CM, 0.62 ± 0.06). Calcium disodium ethylene-diaminetetra-acetic acid at a medium concentration of 0.001 mM produced an equivalent net percentage of release of 203Pb as that measured for 1.0 mM PCA. Duration of calcium disodium ethyl-enediaminetetra-acetic acid activity was sustained for 72 hr compared to 48 hr for PCA. It appears, therefore, that net release of 203Pb produced by PCA was enhanced by PTH, 1,25-(OH)2D3, and decreased medium levels of Ca. On the other hand, the effects of PCA were inhibited by calcitonin and increased medium concentrations of Ca or phosphate. Calcium disodium ethylene diaminetetra-acetic acid, affected by the same hormones and ionic changes as those in PCA cultures, appears to be considerably more potent and longer acting as a chelator of Pb than PCA in this in vitro system. Speculation: In bone organ culture, a rapidly mobile subcompartment of total bone lead (Pb) has been demonstrated to be the source of Pb chelated by D-penicillamine (and CaNa2EDTA or calcium disodium ethylenediaminetetra-acetic acid). This subcompartment of bone Pb in vitro is regulated in large part by the same hormones and ions that normally control bone cell metabolism. Moreover, it is likely that the effects of D-penicillamine's chelation on bone Pb in vitro, like parathyroid hormone 1,25-(OH)2D3, and calcitonin, produce its actions on bone cell metabolism by changes in the permeability of plasma membranes, or by modifications of ionic (calcium, phosphate, and Pb) transport and redistribution from intracellular and/or extracellular sites, particularly in the cytosol and mitochondria. Therefore, the efficacy of Pb chelation by D-penicillamine from bone explants depends, in great measure, on the complexity of coexisting factors that determine the degree of inhibition and stimulation of bone resorption, as well as on translocation of ions between intra- and extracellular stores.