The endocrine-disrupting activities of bisphenol A (BPA) and 19 related compounds were comparatively examined by means of different in vitro and in vivo reporter assays. BPA and some related compounds exhibited estrogenic activity in human breast cancer cell line MCF-7, but there were remarkable differences in activity. Tetrachlorobisphenol A (TCBPA) showed the highest activity, followed by bisphenol B, BPA, and tetramethylbisphenol A (TMBPA); 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,1-bis(4-hydroxyphenyl)propionic acid and 2,2-diphenylpropane showed little or no activity. Anti-estrogenic activity against 17beta-estradiol was observed with TMBPA and tetrabromobisphenol A (TBBPA). TCBPA, TBBPA, and BPA gave positive responses in the in vivo uterotrophic assay using ovariectomized mice. In contrast, BPA and some related compounds showed significant inhibitory effects on the androgenic activity of 5alpha-dihydrotestosterone in mouse fibroblast cell line NIH3T3. TMBPA showed the highest antagonistic activity, followed by bisphenol AF, bisphenol AD, bisphenol B, and BPA. However, TBBPA, TCBPA, and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA, and 3,3'-dimethylbisphenol A exhibited significant thyroid hormonal activity towards rat pituitary cell line GH3, which releases growth hormone in a thyroid hormone-dependent manner. However, BPA and other derivatives did not show such activity. The results suggest that the 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are required for these hormonal activities, and substituents at the 3,5-positions of the phenyl rings and the bridging alkyl moiety markedly influence the activities.

We previously demonstrated that the estrogenicity of either bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane] or bisphenol B [BPB; 2,2-bis(4-hydroxyphenyl)butane] was increased several times after incubation with rat liver S9 fraction (Yoshihara et al., 2001). This metabolic activation, requiring both microsomal and cytosolic fractions, was observed with not only rat liver, but also human, monkey, and mouse liver S9 fractions. To characterize the active metabolites of BPA and BPB, we investigated the structures of the isolated active metabolites by negative mode LC/MS/MS and GC/MS. The active metabolite of BPA gave a negative mass peak at [M-H](-) 267 on LC/MS and a single daughter ion at m/z 133 on MS/MS analysis, suggesting an isopropenylphenol dimer structure. Finally, this active metabolite was confirmed to be identical with authentic 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (MBP) by means of various instrumental analyses. The corresponding peaks of the BPB metabolite were [M-H](-) 295 and m/z 147, respectively, suggesting an isobutenylphenol dimer structure. Further, coincubation of BPA and BPB with rat liver S9 afforded an additional active metabolite(s), which gave a negative mass peak at [M-H](-) 281 and two daughter ion peaks at m/z 133 and m/z 147 on MS/MS analysis. These results strongly suggest that the active metabolite of either BPA or BPB might be formed by recombination of a radical fragment, a one-electron oxidation product of carbon-phenyl bond cleavage. It is noteworthy that the estrogenic activity of MBP, the active metabolite of BPA, is much more potent than that of the parent BPA in several assays, including two reporter assays using a recombinant yeast expressing human estrogen receptor alpha and an MCF-7-transfected firefly luciferase plasmid.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical found in the environment. To assess the metabolic modulation of estrogenic activity of BPA after ingestion, we investigated whether the incubation of BPA with rat liver S9 fraction results in metabolic activation or inactivation of estrogenic activity using a recombinant yeast expressing human estrogen receptor and MCF-7 transfected firefly luciferase plasmid for a reporter assay. When 0.1 mM BPA was incubated with rat liver S9 for 1 h, the estrogenic activity was increased about two to five times compared with that of the control, in which the S9 was inactivated prior to incubation. This metabolic activation was inhibited by SKF 525-A, an inhibitor of cytochrome P450. With increasing incubation time, the estrogenic activity increased time-dependently. Interestingly, however, the metabolic activation did not proceed with either microsomes or cytosol alone and was restored by a recombination of both fractions. The active metabolite was eluted at later retention time than that of BPA on HPLC with a reversed-phase column. Bisphenol B and methoxychlor were also activated by incubation with rat liver S9, whereas 4-tert-octylphenol and 4-nonylphenol, as well as 17beta-estradiol, were metabolically inactivated. The present results clearly indicate that BPA is metabolically activated in terms of estrogenicity under the conditions existing only with combined rat liver microsomes and cytosol.

]m reduction. Cytosolic Na ϩ concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal ⌬⌿ m and 7.6 mM at ⌬⌿m dissipation. We conclude that 1) the mitochondrial Ca 2ϩ uniporter accumulates Ca 2ϩ in a manner that is dependent on ⌬⌿m at the physiological range of [Ca 2ϩ ]c; 2) ⌬⌿m dissipation opens the mPTP and results in Ca 2ϩ influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes Ca 2ϩ from the matrix even after ⌬⌿m dissipation. permeability transition pore; Na ϩ /Ca 2ϩ exchange; depolarization; ischemia-reperfusion injury ACCUMULATING EVIDENCE REVEALS that mitochondria play primary roles in fatal cell damage during ischemia-reperfusion (33). Key events that occur during ischemia include cytosolic Ca 2ϩ elevation, ATP depletion, high P i concentration, depolarized membrane potential, and acidotic pH. On reperfusion and recovery of normal pH, a burst of reactive oxygen species occurs, mitochondrial Ca 2ϩ overload ensues, and these lead to opening of the mitochondrial permeability transition pore (mPTP; Refs. 8,12,34). Opening of the mPTP allows water and solutes Յ1,500 Da in size to enter the matrix and cause mitochondrial swelling, rupture of the outer mitochondrial membrane, and release of cytochrome c or apoptosis-inducing factor, which initiates apoptotic programmed cell death (12,24,34). Because previous studies (8,12,13) (10,11). Furthermore, recent studies (4,8,10,11,18) also suggest a possible contribution by the mPTP to Ca 2ϩ homeostasis in both the cytosol and mitochondria. Despite the considerable attention given to the pathophysiological significance of mitochondrial Ca 2ϩ , the regulation and/or modulation of mitochondrial Ca 2ϩ during pathophysiological conditions such as ischemia-reperfusion injury are unclear. In previous studies, information about mitochondrial Ca 2ϩ was obtained using isolated mitochondria, whereby the structural and functional properties of organelles were seriously affected, and other cellular architectures were separated from the mitochondria. In this study, we measured [Ca 2ϩ ] m in saponin-permeabilized rat ventricular myocytes and investigated how ⌬⌿ m depolarization affects [Ca 2ϩ ] m and mitochondrial Ca 2ϩ transport systems such as the Ca 2ϩ uniporter, the mPTP, and mitoNCX.

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