Epidemiological data suggest an association between exposures to bromodichloromethane (BDCM), a trihalomethane found in drinking water as a result of drinking water disinfection, and an increased risk of spontaneous abortion. We previously hypothesized that BDCM targets the placenta and showed that the secretion of chorionic gonadotrophin (CG) was reduced in primary cultures of human term syncytiotrophoblasts exposed to BDCM. In the present study we extend this observation by evaluating the effects of BDCM on the morphological differentiation of mononucleated cytotrophoblast cells to multinucleated syncytiotrophoblast-like colonies. Addition of BDCM to cytotrophoblast cultures inhibited the subsequent formation of multinucleated colonies in a dose-dependent manner, as determined by immunocytochemical staining for desmosomes and nuclei. The effect was seen at BDCM concentrations between 0.02 and 2 mM and was confirmed by quantitative image analysis. Secretion of bioactive and immunoreactive chorionic gonadotropin was also significantly inhibited in a dose-dependent manner under these culture conditions, and cellular levels of CG were also reduced. Trophoblast viability was not compromised by exposure to BDCM. We conclude that BDCM disrupts syncytiotrophoblast formation and inhibits CG secretion in vitro. Although other tissue targets are not ruled out, these data substantiate the idea that BDCM targets the placenta and could have implications for understanding the adverse pregnancy outcomes associated with BDCM exposure in humans.

The anionic requirements and the stoichiometric relationships of Na+-taurine cotransport into rat renal brush-border membrane vesicles (BBMV) were evaluated. External Cl- (100 mM) or Br- (100 mM) gradients supported the full overshoot of Na+-taurine symport and yielded similar high-affinity transport systems for taurine uptake. No active uptake of taurine was evident in the presence of external (100 mM) NaF, NaI, Na gluconate, or Na p-aminohippurate (PAH). Na+:taurine stoichiometry was 2.18:1 in the presence of Cl- and 1.60:1 in the presence of Br-. When the external anion gluconate was employed, Na+-dependent taurine uptake was negligible over the whole range of Na+ concentrations examined. Cl-:taurine and Br-:taurine stoichiometries in the presence of external Na+ were 0.97:1 and 0.81:1, respectively. External furosemide (1 mM) or bumetanide (1 mM) did not change taurine accumulation and kinetic parameters. The anionic transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (5 x 10(-4) M), N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (10(-3) M) and p-chloromercuribenzoate (5 x 10(-4) M) significantly decreased initial rate of taurine uptake by 48, 31, and 31%, respectively. These data suggest that Na+-taurine cotransport into rat renal BBMV is Cl- or Br- dependent and probably operates by means of 2 Na+:1 Cl- or Br-:1 taurine carrier complex. Na+-taurine symport across the rat renal brush-border membrane surface is not affected by diuretics that influence NaCl cotransport but is affected by selected anionic transport inhibitors. An intact anionic binding site may be needed for this translocation process.

Accelerator Mass Spectrometry is an established technology whose essentiality extends beyond simply a better detector for radiolabeled molecules. Attomole sensitivity reduces radioisotope exposures in clinical subjects to the point that no population need be excluded from clinical study. Insights in human physiochemistry are enabled by the quantitative recovery of simplified AMS processes that provide biological concentrations of all labeled metabolites and total compound related material at non-saturating levels. In this paper, we review some of the exploratory applications of AMS 14C in toxicological, nutritional, and pharmacological research. This body of research addresses the human physiochemistry of important compounds in their own right, but also serves as examples of the analytical methods and clinical practices that are available for studying low dose physiochemistry of candidate therapeutic compounds, helping to broaden the knowledge base of AMS application in pharmaceutical research.

This was a single dose mass balance and metabolite characterization study of the antimalarial agent pyronaridine. Six healthy male adults were administered a single oral dose of 720 mg pyronaridine tetraphosphate with 800 nCi of radiolabeled (14)C-pyronaridine. Urine and feces were continuously collected through 168 h post-dose, with intermittent 48 h collection periods thereafter through 2064 h post-dose. Drug recovery was computed for analyzed samples and interpolated for intervening time periods in which collection did not occur. Blood samples were obtained to evaluate the pharmacokinetics of total radioactivity and of the parent compound. Total radioactivity in urine, feces, and blood samples was determined by accelerator mass spectrometry (AMS); parent concentrations in blood were determined with LC/MS. Metabolite identification based on blood, urine, and feces samples was conducted using a combination of LC + AMS for identifying radiopeaks, followed by LC/MS/MS for identity confirmation/elucidation. The mean cumulative drug recovery in the urine and feces was 23.7 and 47.8 %, respectively, with an average total recovery of 71.5 %. Total radioactivity was slowly eliminated from blood, with a mean half-life of 33.5 days, substantially longer than the mean parent compound half-life of 5.03 days. Total radioactivity remained detectable in urine and feces collected in the final sampling period, suggesting ongoing elimination. Nine primary and four secondary metabolites of pyronaridine were identified. This study revealed that pyronaridine and its metabolites are eliminated by both the urinary and fecal routes over an extended period of time, and that multiple, varied pathways characterize pyronaridine metabolism.

The in vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid metabolism in human luteinized granulosa cells (hLGC) have been summarized as a decreased estradiol (E(2)) production without altering either E(2) metabolism or cytochrome P450 aromatase activity. In the present study, hLGC were used to analyze the fate of different substrates for cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450(c17)) in the presence or absence of TCDD. Human LGCs were plated directly on plastic culture dishes in medium supplemented with 2 IU/ml of hCG. TCDD (10 nM) or its solvent was added directly to the cells at the time of medium change, every 48 h for 8 days. The objective of the experiment was to test the hypothesis that exogenous steroid, substrate for P450(c17), would reduce the TCDD effects on E(2) synthesis. With dehydroepiandrosterone (DHEA) (a P450(c17) product), a dose-related increase in E(2) production was observed and the effect of TCDD on lowering E(2) production disappeared. In contrast, with increasing doses, up to 10 micro M, of pregnenolone (P(5)), no change in E(2) production was observed. However, 17alpha-hydroxypregnenolone (17P(5)) at 10 micro M produced a modest but significant increase in the E(2) production. Treatments with P(5) and 17P(5) did not alter the effect of TCDD on E(2) production. Radiolabeled substrate utilization by hLGC suggests that the principal metabolic pathway for Delta5 substrates is the conversion to a Delta4 product probably by a very active 3beta-hydroxysteroid dehydrogenase. We conclude that estrogen production by hLGC is limited at the level of lyase activity. Thus, these data suggest that the most likely target for the TCDD-induced inhibition of estrogen synthesis by hLGC is the 17,20-lyase activity of the P450(c17) enzyme complex.