Relationship between bisphosphonate concentration and osteoclast activity and viability.

Difluoromethylidene bisphosphonate (F2MBP) is one of the many bisphosphonates known to inhibit bone resorption in vitro and in vivo. We have developed an analytical method, employing anion exchange and postcolumn indirect fluorescence detection, by which F2MBP can be quantified in bone samples. The objective of this study was to relate the concentration of F2MBP in embryonic bones treated in organ culture to the physiological effects of the compound, such as bone resorption (i.e., the amount of 45Ca released into the medium from prelabeled bones) and viability of the osteoclast population (i.e., the incidence of abnormal osteoclasts). Osteoclasts in bones treated with F2MBP exhibited morphological features of apoptosis, such as nuclear fragmentation. Both the number and percentage of these abnormal cells increased with dose of F2MBP and duration of incubation. The decrease in normal osteoclasts was correlated with the decreased amount of 45Ca released into the medium. Bones treated with F2MBP for only the first 5 min of the 48-h incubation period had similar numbers of abnormal osteoclasts and amounts of 45Ca released, as had bones incubated with F2MBP continuously for 48 h. The uptake of F2MBP into the bone was rapid. Bones treated with F2MBP for 6 h were similar to bones treated with F2MBP for the entire 48-h incubation period, both in F2MBP concentration and the 45Ca release ratios. These relationships between concentrations of F2MBP within bone and osteoclast activity and viability implicate apoptosis in the mechanism by which this bisphosphonate inhibits bone resorption.

An evaluation of the effectiveness of lead paint hazard reduction when conducted by homeowners and landlords.

This research project was conducted in collaboration with the Iowa Department of Public Health to evaluate whether property owners who follow recommended procedures for lead-based paint removal/repair can do the work safely and effectively. This study included 29 homes where a lead-based paint hazard had been identified and lead-based paint was removed or repaired (hazard reduction). Exposure evaluation included pre-project surface dust wipe sampling, air monitoring during lead-based paint removal, post-project surface dust wipe sampling, and pre- and post-project blood samples from adult study participants. The comparison of surface dust wipe samples taken before and after lead paint hazard reduction was used to evaluate the effectiveness of lead paint hazard reduction. The lead loadings on window sill surfaces in the work area were significantly lower after completion of the project (p = 0.04), and the lead-based paint removal did not contaminate the adjoining living area. The proportion of homes with surface dust lead loading exceeding Department of Housing and Urban Development (HUD) clearance standard was 73 percent pre-project and 38 percent post-project. Personal airborne exposures during lead removal activities (geometric mean = 59.3 micrograms/m3) reinforce the need for respiratory protection and good hygiene. There was no difference in adult pre-/post-blood levels, indicating that participants did remove lead in a safe manner with respect to their own exposures. The results indicate that hazard reduction can be done effectively when recommended procedures for the removal of lead-based paint are followed.

Uptake of a fluorinated bisphosphonate by cultured bones.

The uptake of bisphosphonates into bone was studied using 19-day-old fetal rat bones cultured with a new fluorinated bisphosphonate, difluoromethylidene bisphosphonate (F2MBP). F2MBP uptake was assessed by determining the weight percent of fluoride using electron probe microanalysis. By 30 min the weight percent of fluoride was significantly greater in the F2MBP-treated bones than in controls and continually increased throughout the duration of the experiment to reach a fluoride concentration 6-fold greater than controls after 120 h of incubation. When the peripheral cortical bone was analyzed separately from the interior trabecular bone in the F2MBP-treated bones, the fluoride concentration in the periphery increased until 24 h and then remained somewhat constant, while the interior, which is more actively remodeling, showed a continual increase. The uptake of F2MBP during the 1 to 6 h time intervals demonstrated no differences between vital and devitalized bone and, thus, is not cell-mediated. Because analysis of free fluoride in F2MBP media incubated with bones showed that the concentration of fluoride was less than 1% of the total amount of fluoride, the fluoride detected by the probe was most likely that of the intact molecule and not free fluoride. The rapid uptake of the F2MBP molecule was supported by assessing the effects of short-term F2MBP treatment on subsequent bone resorption, as determined by the release of 45Ca from prelabeled bones. Bones treated with F2MBP for only 5 min exhibited reductions in the percentage of 45Ca released during the remainder of the 120 h incubation period similar to that when F2MBP was continuously in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies of aromatase inhibitors in cultured human breast cancer cells.

The presence of aromatase activity, estrogen receptors, and estrogenic responsiveness in MCF-7 human breast cancer cells has allowed this cell line to be used as a unique in vitro system for investigating the biological activities of potentially therapeutic aromatase inhibitors. We now report the results of studies which have examined the cytotoxicity, antiaromatase, and intrinsic estrogenic activities of aminoglutethimide, 1,2-dehydrotestolactone (testolactone), dihydrotestosterone, 4-hydroxy-4-androstene-3,17-dione, and 10-propargylestr-4-ene-3,17-dione within MCF-7 monolayer cultures. Cell viability was determined by trypan blue exclusion, and aromatase activity was assessed by quantifying the amounts of [3H]estradiol formed from [3H]testosterone. Estrogenic activity was assessed by examining the ability of each inhibitor to increase cytoplasmic progesterone receptor and deplete cytoplasmic estrogen receptor concentrations in these cells during a 5-day incubation period. Cytoplasmic progesterone and estrogen receptors were measured by the single-saturating-dose technique using [17 alpha-methyl-3H]-17 alpha, 21-dimethyl-19-norpregna-4,9-diene-3,20-dione and [3H]estradiol as the labeled ligands for each assay, respectively. The results showed that all of these compounds were noncytotoxic aromatase inhibitors in MCF-7 cells but that these agents demonstrated marked differences in inhibitory potency (10-propargylestr-4-ene-3,17-dione greater than 4-hydroxy-4-androstene-3,17-dione much greater than dihydrotestosterone much greater than testolactone = aminoglutethimide). The incubation of cells with 4-hydroxy-4-androstene-3,17-dione resulted in cytoplasmic progesterone and estrogen receptor responses that were similar in magnitude to those observed in other cultures incubated with equimolar concentrations of estradiol. None of the other four agents demonstrated estrogenic activity in this system. However, we have previously observed that dihydrotestosterone has substantial antiestrogenic action in this system. Taken together, these results indicate that some aromatase inhibitors may influence the hormonal regulation of human breast cancer cells by more than one mechanism.

The specific binding of estradiol and estrone and the subsequent distribution of estrogen-receptor complexes within MCF-7 human breast cancer cells.

We have examined the specific binding of estradiol (E2) and estrone (E1) within MCF-7 cells using both cytosol and whole cell suspension binding assay techniques. The results of these studies have revealed that each of these estrogens binds the high but different affinity to the same single class of cytosol receptor (ER). The incubation of intact cells with E2 at 37 degrees resulted in the rapid formation, nuclear binding, and nuclear processing of E2-ER complexes. Reduction of incubation temperature to 15 degrees C and 4 degrees C resulted in slowed but continued E2-ER formation and nuclear binding, and in a marked inhibition of nuclear receptor processing. The incubation of intact cells with E1 at 37 degrees also was associated with the formation, nuclear binding, and nuclear processing of estrogen-ER complexes. Interestingly, however, E2 rather the E1 represented the major species of specifically bound estrogen under these conditions. This observation suggests that the estrogenic action of E1 in MCF-7 cells may be mediated largely by the intracellular formation of E2. This phenomenon provides a likely explanation of the unexpected potency of E1 in this system previously observed by other workers. Furthermore, our results suggest that E1 may represent a major source of estrogenic stimulation for some hormone-dependent human breast tumors.

An antiestrogenic action of androgens in human breast cancer cells.

We have recently observed that androgens prevent the estrogen-dependent augmentation of cytoplasmic progesterone receptor (PRc) in MCF-7 human breast cancer cells and now report the results of studies that further characterize this new example of sex steroid antagonism. Using a single saturating dose assay to monitor changes in MCF-7 PRc concentration, we have observed that androgens are capable of inhibiting both the estrogenic induction and the ongoing stimulation of PRc synthesis, but have no apparent effect upon basal concentrations of this receptor. Both testosterone and dihydrotestosterone (DHT) demonstrate similar degrees of antiestrogenic activity at concentrations between 10(-10)--10(-8) M. Furthermore, a 10(-8)-M concentration of either androgen completely inhibits the stimulation of PRc synthesis by 10(-11)--10(-8) M 17 beta-estradiol (E2). This inhibitory effect is maintained during the continued presence of either testosterone or DHT, but rapidly disappears after the withdrawal of androgen from the culture medium. The specific nuclear binding of 17 beta-[3H]estradiol over time appears to be similar in cultures incubated in the presence and absence of 10(-8) M DHT. This observation suggests that androgens do not inhibit estrogen action by interfering with the formation, activation, nuclear binding, or nuclear processing of estrogen-receptor complexes. The estrogenic stimulation of PRc is not diminished by the 5 beta-epimer of DHT, and the inhibitory activity of DHT itself is blocked by several different antiandrogens. These findings provide substantial support for the concept that the antiestrogenic effect of androgens is mediated by an androgen receptor mechanism. These results may provide new insights into the clinically apparent antagonistic effects of estrogens and androgens upon both normal and malignant human breast tissues.