To assess the estrogenic activity potentially stemming from bisphenol A (BPA) in drinking water, APCI/LC/MS and NMR were used to identify the products of its aqueous chlorination under the following conditions: 500 microg/L bisphenol A and 1.46 mg/L sodium hypochlorite (pH 7.5) at 25 degrees C. The 13 products (4-chloro-BPA; 2,6'-dichloro-BPA; 2,6-dichloro-BPA; 2,2',6'-trichloro-BPA; 2,2',6,6'-tetrachloro-BPA; trichlorophenol; 4-isopropyl-2'-hydroxylphenol; and six kinds of polychlorinated phenoxyphenols (PCPPs)) were found in the chlorinated BPA solution. Three main pathways are proposed: (1) chlorine-substitution reactions on the aromatic ring, followed by dehydration to form the chlorine-substituted BPA, (2) chlorine substitution reactions followed by cleavage of the alpha-C on the isopropyl moiety with positive partial charge and beta'-C on the benzene moiety with a negative partial charge to form trichlorophenol and 4-isopropyl-2'-hydroxylphenol, and (3) the formation of PCPPs. Especially for pathway 2, the reaction mechanism was clarified based on semiempirical quantum mechanical calculations. The reaction proceeded by attack of the OH and Cl (from HOCl) on the alpha-C on the isopropyl moiety with a positive partial charge and on the beta'-C with a negative partial charge on the benzene moiety. The activation energies forthe HOCl/4-chloro-BPA and 2,2',6,6'-tetrachloro-BPA reactions were 0.14 and 0.15 kcal/mol, respectively. Finally, the estrogenic activity of the aqueous chlorinated BPA solution was assessed by an estrogen receptor binding assay and a yeast two-hybrid system. It was found that the binding affinity of the chlorinated aqueous BPA at 60 min was 24 times that before chlorination. The transcriptional activation-induced by products were detected by a yeast two-hybrid system based on the ligand-dependent interaction of two proteins, a human ER and a coactivator, suggesting that the chlorinated BPA solution elicits an ability to mimic the effect of the estrogen hormone.

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl(-) ion pump. For efficient Cl(-) transport, the existence of Cl(-)-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl(-) to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl(-) to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.

To assess the estrogenic activity potentially stemming from 17beta-estradiol (E2) in drinking water, ESI-LC-MS was used to identifythe products of its aqueous chlorination under the following conditions: 50 microg/L E2, 1.46 mg/L sodium hypochlorite, pH 7.5, 25 degrees C. Seven products, including 2,4-dichloro-17beta-estradiol, monochloroestrone, 2,4-dichloroestrone, and the four byproducts such as 4-[2-(2,6-dichloro-3-hydroxyphenyl)ethyl]-7alpha-methyloctahydroinden-5-one (product C in the text) were identified in chlorinated E2 solution. The estrogenic activities of the aqueous chlorinated E2 solution at 10, 30, 60, 120, and 180 min contact time were assessed by a yeast two-hybrid system based on the ligand-dependent interaction of two proteins, a human estrogen receptor (ER) and a coactivator. All five solutions elicited transcriptional activation induction. The maximal beta-galactosidase activities induced by the chlorinated solution at 10, 30, and 60 min were similar and slightly lower than those before chlorination, while the activities of the chlorinated solution at 120 and 180 min were about 40% of those before chlorination. Finally, 4-chloro-17beta-estradiol (4-chloro-E2) (we failed to synthesize the 2-chloroestrone (2-chloro-E1)), 2,4-dichloro-17beta-estradiol (2,4-dichloro-E2), and 2,4-dichloroestrone (2,4-dichloro-E1) were synthesized, and product C was fractionated by HPLC. It was found that 4-chloro-E2 elicited strong estrogenic activity, at almost the same level as that of estrone (EC50 = 10(2) nM), while 2,4-dichloro-E2 elicited weaker beta-galactosidase activity compared with that of 4-chloro-E2. The EC50 was ca. 10(3) nM. The maximal beta-galactosidase activity for 2,4-dichloro-E1 was lower than that of 2,4-dichloro-E2, while its EC50 was similar to that of 2,4-dichloro-E2. In addition, product C, 4-[2-(2,6-dichloro-3-hydroxyphenyl)ethyl]-7alpha-methyloctahydroinden-5-one, induced high beta-galactosidase activity at the relatively higher concentration of 3.5 x 10(5) nM. On the basis of the dose-response curve of a single byproduct of chlorinated E2, the estrogenic activity at 120 and 180 min appears to be induced mainly by 2,4-dichloro-E2 and 2,4-dichloro-E1.

Hayase et al. show that R-Spondin1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spondin1 or recombinant α-defensin prevents dysbiosis mediated by graft-versus-host disease, representing a novel approach to restore intestinal ecosystems and homeostasis.