Repeated exposure to 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) accelerates ligand-independent activation of estrogen receptors in long-term estradiol-deprived MCF-7 cells.

It was previously identified that there may be an active metabolite of bisphenol A (BPA), 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). An in vitro system was developed to detect MBP toxicity to the Michigan Cancer Foundation-7 (MCF-7) cells that had been repeatedly exposed to a low dose of the metabolite. MBP profoundly activated estrogen receptor (ER)-dependent transcription as a ligand, with an EC50 of 2.8 nM. Women are continuously exposed to numerous estrogenic environmental chemicals; but their susceptibility to these chemicals may be significantly altered after menopause. Long-term estrogen-deprived (LTED) cells, which display ligand-independent ER activation, are a postmenopausal breast cancer model derived from MCF-7 cells. In this study, we investigated the estrogenic effects of MBP on LTED cells in a repeated exposure in vitro model. The results suggest that i) nanomolar levels of MBP reciprocally disrupt the balanced expression of ERα and ERß proteins, leading to the dominant expression of ERß, ii) MBP stimulates ERs-mediated transcription without acting as an ERß ligand, and iii) MBP utilizes mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling to evoke its estrogenic action. Moreover, the repeated exposure strategy was effective for detecting low-dose estrogenic-like effects caused by MBP in LTED cells.

4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) Targets Estrogen Receptor ß, to Evoke the Resistance of Human Breast Cancer MCF-7 Cells to G-1, an Agonist for G Protein-Coupled Estrogen Receptor 1.

Bisphenol A (BPA) has been shown to induce the activation of nuclear estrogen receptor α/ß (ERα/ß) in both in vitro and in vivo settings. We originally obtained a 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), a possible active metabolite of BPA, strongly activating the ERs-mediated transcription in MCF-7 cells with an EC50 of 2.8 nM (i.e., BPA's EC50 = 519 nM). Environmental estrogens can also target G protein-coupled estrogen receptor 1 (GPER1), a membrane-type ER. However, the effects of BPA/MBP on GPER1, have not yet been fully resolved. In this study, we used MCF-7, a ERα/ERß/GPER1-positive human breast cancer cell line, as a model to investigate the effects of the exposure to BPA or MBP. Our results revealed that at concentrations below 1 nM MBP, but not BPA, downregulates the expression of GPER1 mRNA via upregulated ERß, and the MCF-7 cells pre-treated with MBP display resistance to GPER1 agonist G-1-mediated anti-proliferative effects. Because GPER1 can act as a tumor suppressor in several types of cancer including breast cancer, the importance of MBP-mediated decrease in GPER1 expression in breast cancer cells is discussed.

Repeated Exposure to 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an Active Metabolite of Bisphenol A, Aggressively Stimulates Breast Cancer Cell Growth in an Estrogen Receptor ß (ERß)-Dependent Manner.

Bisphenol A (BPA), recognized as an endocrine disruptor, is thought to exert its activity through a mechanism involving the activation of estrogen receptors (ERs) α/ß However, a major problem is that very high concentrations of BPA are required (i.e., those in excess of environmental levels) for effective activation of ERα/ß-mediated transcriptional activities in vitro, despite the BPA-induced estrogenic effects observed in vivo. To elucidate the causal reasons, we successfully identified a BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which exhibits highly potent estrogenic activity both in vivo and in vitro. We have focused on the biologic relationship between breast tumor promotion and MBP/BPA, because BPA is considered to be a human carcinogen owing to its breast tumor-promoting properties. In general, humans are exposed to many endocrine disruptors, including BPA. In the present study, we used the ERα/ß-positive human breast cancer cell line MCF-7 as an experimental model to investigate the effects of repeated exposure to BPA/MBP at concentrations found in the environment on the expression of ERα/ß and to determine the particular ER subtype involved. We demonstrated that repeated exposure to MBP, but not to BPA, significantly downregulated ERα protein expression and stimulated the proliferation of MCF-7 cells through the activation of ERß-mediated signaling.

Bisphenol A modulates the metabolic regulator oestrogen-related receptor-α in T-cells.

Bisphenol A (BPA) is a widely used plastics constituent that has been associated with endocrine, immune and metabolic effects. Evidence for how BPA exerts significant biological effects at chronic low levels of exposure has remained elusive. In adult men, exposure to BPA has been associated with higher expression of two nuclear receptors, oestrogen receptor-ß (ERß) and oestrogen-related-receptor-α (ERRα), in peripheral white blood cells in vivo. In this study, we explore the expression of ESR2 (ERß) and ESRRA (ERRα) in human leukaemic T-cell lymphoblasts (Jurkat cells) exposed to BPA in vitro. We show that exposure to BPA led to enhanced expression of ESRRA within 6 h of exposure (mean±s.e.m.: 1.43±0.08-fold increase compared with the control, P<0.05). After 72 h, expression of ESRRA remained significantly enhanced at concentrations of BPA ≥1 nM. Oxidative metabolism of BPA by rat liver S9 fractions yields the potent oestrogenic metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). Exposure of cells to 1-100 nM MBP increased the expression of both ESRRA (significantly induced, P<0.05, at 1, 10, 100 nM) and ESR2 (1.32±0.07-fold increase at 100 nM exposure, P<0.01). ERRα is a major control point for oxidative metabolism in many cell types, including T-cells. Following exposure to both BPA and MBP, we found that cells showed a decrease in cell proliferation rate. Taken together, these results confirm the bioactivity of BPA against putative T-cell targets in vitro at concentrations relevant to general human exposure.

[A memoir of my researches on xenobiotic metabolism for 48 years--researches on Kanemi Yusho and endocrine disrupting chemicals].

The author started a research on xenobiotic metabolism at Graduate School of Pharmaceutical Sciences, Kyushu University in 1965. In 1968, an epidemic of a "strange disease", called Yusho, occurred in western Japan. The epidemic was soon identified to be a food poisoning caused by the ingestion of commercial Kanemi rice bran oil which had been accidentally contaminated with large amounts of polychlorinated biphenyls (PCBs) and their related compounds such as polychlorinated dibenzofurans (PCDFs.) At first, in this review, our toxicological studies on Yusho during the early thirty years were briefly described. Next, the studies on aldehyde oxidase, a molybdenum hydroxylase, which is involved in the lactam formation reaction such as 1-phenyl-2-(2-oxopyrrolidine)pentane(oxoprolintane) from 1-phenyl-2-pyrrolidinopentane(prolintane) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) lactam from 1-methyl-4-phenyl-2,3-dihydropyridinium ion (MPDP⁺) were also presented. Finally, we investigated how the xenobiotic metabolism of endocrine disrupting chemicals such as bisphenol A (BPA) and some isoflavones affects their estrogenic activities. In this study, we demonstrated that BPA is converted to 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an active metabolite as estrogen, by rat liver S9. In the cases of isoflavones, although genistein was inactivated, biochanin A, 4'-methoxy analogue of genistein, was activated to genistein by O-demethylation with rat liver S9.

Novel pathway of metabolic activation of bisphenol A-related compounds for estrogenic activity.

We previously demonstrated that estrogenic activity of bisphenol A (BPA) in the yeast estrogen screening assay was increased severalfold after incubation with rat liver S9 fraction in the presence of a NADPH-generating system. In this study, we investigated whether eight BPA-related compounds are similarly activated metabolically by rat liver S9 fraction. Three of the analogs exhibited an increase of estrogenic activity after incubation with rat liver S9 fraction but not with microsomal or cytosolic fraction alone. The structures of the metabolites formed were examined by liquid chromatography/mass spectrometry. In addition to oxidized metabolites such as catechols, we found novel dimer-type metabolites. Some of the putative metabolites were chemically synthesized to confirm their structures. The structural requirements for formation of the metabolites, some of which showed more potent estrogenic activity than the parent substrates, were examined. We have uncovered a new pathway of metabolic activation of certain phenolic compounds, such as BPA analogs, to estrogenic dimer-type compounds.

In vivo estrogenic potential of 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, an active metabolite of bisphenol A, in uterus of ovariectomized rat.

4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an active metabolite of bisphenol A (BPA), has more potent estrogenic activity than BPA in vitro, but its activity in vivo is not established. Here, we examined in vivo estrogenic activity of MBP by means of uterotrophic assay in ovariectomized (OVX) female rats. MBP exhibited dose-dependent estrogenic activity, as evaluated in terms of effects on uterus weight, uterine luminal epithelial cell height and myometrium thickness. The highest concentration of MBP (10 mg/kg/day) completely reversed the changes caused by OVX, and its activity was equivalent to that of 5 microg/kg/day 17beta-estradiol (E2). We also investigated the effects of MBP on transcription of several estrogen-related genes. The changes of mRNA levels of estrogen receptors alpha and beta, c-fos and insulin-like growth factor 1 in MBP-treated OVX rats were qualitatively similar to those in E2-treated rats. BPA did not show any significant effect on OVX rat in these conditions. This study is the first to demonstrate that MBP, an active metabolite of BPA, has potent in vivo estrogenic activity, being about 500-fold more potent than BPA in OVX rats.

Metal complexes with superoxide dismutase-like activity as candidates for anti-prion drug.

Various compounds were evaluated for ability to inhibit the formation of the abnormal protease-resistant form of prion protein (PrP-res) in two cell lines infected with different prion strains. Examination of the structure-activity relationships indicated that compounds with copper-selective chelating ability and whose copper complexes have high SOD-like activity are candidates for anti-prion drug.

New aspects of cadmium as endocrine disruptor.

Cadmium (Cd) is an industrial and environmental pollutant that exerts adverse effects on a number of organs in humans and animals. Reproductive organs, such as the testis and placenta, are sensitive to the toxic effects of Cd. In animal experiments, high-dose exposure to Cd induced severe testicular interstitial hemorrhage with edema, and increased incidence of fetal death and placental necrosis. Low-dose exposure to Cd affects steroid synthesis in male and female reproductive organs. In 1998, the Ministry of Environment in Japan listed Cd in the strategy plan SPEED98 as one of the chemicals suspected of having possible endocrine disrupting activity. Recently, it has been shown that Cd has potent estrogen- and androgen-like activities in vivo and in vitro, by directly binding to estrogen and androgen receptors. However, the precise mechanisms underlying the effects of Cd as an endocrine disruptor remain to be elucidated. In this review, we will discuss evidence thus far presented concerning the effects of Cd on the endocrine system.

Toxicity to early life stages and an estrogenic effect of a bisphenol A metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on the medaka (Oryzias latipes).

In a recent study, it was reported that 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), a metabolite of bisphenol A (BPA; 2,2-bis(4-hydroxyphenyl)propane), showed estrogenic activity in several in vitro assays, and the estrogenic activity of MBP was higher than that of BPA. In this study, we have investigated the early life stage toxicity and estrogenic effect of MBP on medaka (Oryzias latipes). The 96-h median lethal concentration value of MBP and BPA with 24-h-old larvae was estimated to be 1,640 and 13,900 microg/l, respectively. The hatchability of fertilized eggs exposed to MBP and BPA over 14 days was significantly decreased at doses of 2,500 microg/l and 12,500 microg/l, respectively. Moreover, to compare the potency of estrogenic activity in vivo, male medaka were exposed to various concentrations of MBP and BPA for 21 days. The lowest-observed-effect concentrations of MBP and BPA for hepatic vitellogenin induction in male medaka were estimated to be 4.1 and 1,000 microg/l, respectively. These results suggest that MBP has high toxicity for early life stages of the medaka, and that the estrogenic activity of MBP was about 250-fold higher than that of BPA to male medaka.

Comparative study of the endocrine-disrupting activity of bisphenol A and 19 related compounds.

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.

Potent estrogenic metabolites of bisphenol A and bisphenol B formed by rat liver S9 fraction: their structures and estrogenic potency.

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.

Estrogenic activity of an environmental pollutant, 2-nitrofluorene, after metabolic activation by rat liver microsomes.

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.

Estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes.

In this study we examined estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes. Trans-1,2-diphenylcyclobutane (TCB), cis-1,2-diphenylcyclobutane (CCB), 1,3-diphenylpropane, 2,4-diphenyl-1-butene, 2,4,6-triphenyl-1-hexene, and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin were negative in the yeast estrogen screening assay and estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, TCB exhibited estrogenic activity after incubation with liver microsomes of phenobarbital-treated rats in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Minor activity was observed when liver microsomes of untreated or 3-methylcholanthrene-treated rats were used instead of those from phenobarbital-treated rats. CCB, 1,3-diphenylpropane, and 2,4-diphenyl-1-butene also exhibited estrogenic activity after metabolic activation by liver microsomes, but the activity was lower than that of TCB. 2,4,6-Triphenyl-1-hexene and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin did not show estrogenic activity after such incubation. When TCB was incubated with liver microsomes of phenobarbital-treated rats in the presence of NADPH, three metabolites were detected by high-performance liquid chromatography (HPLC). One metabolite isolated by HPLC exhibited a significant estrogenic activity. The active metabolite was identified as trans-1-(4-hydroxyphenyl)-2-phenylcyclobutane by mass and nuclear magnetic resonance spectral analysis. These results suggest that the estrogenic activity of TCB was caused by the formation of the 4-hydroxylated metabolite.