The subcellular distribution of [14C]dichloromethylenebisphosphonate and [14C]1-hydroxyethylidene-1,1-bisphosphonate in cultured calvaria cells.

Rat calvaria cells were cultured for 6 days in the presence or absence of [14C]dichloromethylenebisphosphonate [( 14C]Cl2MBP) or [14C]1-hydroxyethylidene-1, 1-bisphosphonate [( 14C]HEBP), after which cell organelles were separated by differential centrifugation. The distribution of protein, glutamate dehydrogenase, acid phosphatase, and 5'-nucleotidase was similar for cells treated or not treated with Cl2MBP. About 70-80% of the [14C]Cl2MBP and [14C]HEBP was found to be present in the supernatant. This was the only fraction that showed a ratio higher than 1 for the relative specific radioactivity, indicating that the bisphosphonates accumulated mainly in the cytosol. Rapid separation of particulate components and soluble cytoplasm of cells treated with [14C]Cl2MBP confirmed this finding, showing that it is unlikely that the result was due to leakage from the organelles. The uptake of [14C]Cl2MBP into cells was similar in different cell types. The binding of both bisphosphonates to macromolecules in the medium was 0.1-0.2% and 1-4% in the cells. This binding is not due to metabolic activity of the cells. About 15-20% of [14C]HEBP and [14C]Cl2MBP was modified by the living cells.