2-Methoxyestradiol as an Antiproliferative Agent for Long-Term Estrogen-Deprived Breast Cancer Cells.

To identify effective treatment modalities for breast cancer with acquired resistance, we first compared the responsiveness of estrogen receptor-positive breast cancer MCF-7 cells and long-term estrogen-deprived (LTED) cells (a cell model of endocrine therapy-resistant breast cancer) derived from MCF-7 cells to G-1 and 2-methoxyestradiol (2-MeO-E2), which are microtubule-destabilizing agents and agonists of the G protein-coupled estrogen receptor 1 (GPER1). The expression of GPER1 in LTED cells was low (~0.44-fold), and LTED cells displayed approximately 1.5-fold faster proliferation than MCF-7 cells. Although G-1 induced comparable antiproliferative effects on both MCF-7 and LTED cells (IC50 values of >10 µM), 2-MeO-E2 exerted antiproliferative effects selective for LTED cells with an IC50 value of 0.93 µM (vs. 6.79 µM for MCF-7 cells) and induced G2/M cell cycle arrest. Moreover, we detected higher amounts of ß-tubulin proteins in LTED cells than in MCF-7 cells. Among the ß-tubulin (TUBB) isotype genes, the highest expression of TUBB2B (~3.2-fold) was detected in LTED cells compared to that in MCF-7 cells. Additionally, siTUBB2B restores 2-MeO-E2-mediated inhibition of LTED cell proliferation. Other microtubule-targeting agents, i.e., paclitaxel, nocodazole, and colchicine, were not selective for LTED cells. Therefore, 2-MeO-E2 can be an antiproliferative agent to suppress LTED cell proliferation.

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.

Cannabidiolic acid activates the expression of the PPARß/δ target genes in MDA-MB-231 cells.

Cannabidiolic acid (CBDA) can activate peroxisome proliferator-activated receptor-α (PPARα) and PPARγ. Whether CBDA can activate PPARß/δ has not been examined sufficiently to date. Since previous studies showed that triple-negative breast cancer cells respond to activation of PPARß/δ, the present study examined the effect of CBDA in MDA-MB-231 cells and compared the activities of CBDA with known PPARß/δ agonists/antagonists. Expression of the PPARß/δ target genes angiopoietin-like 4 (ANGPTL4) and adipocyte differentiation-related protein (ADRP) was increased by CBDA. Interestingly, ligand activation of PPARß/δ with GW501516 caused an increase in expression of both ANGPTL4 and ADRP, but the magnitude of this effect was markedly increased when co-treated with CBDA. Specificity of these effects were confirmed by showing that CBDA-induced expression of ANGPTL4 and ADRP is mitigated in the presence of either a PPARß/δ antagonist or an inverse agonist. Results from these studies suggest that CBDA can synergize with PPARß/δ and might interact with endogenous agonists that modulate PPARß/δ function.

Perfluorooctanoic acid (PFOA) as a stimulator of estrogen receptor-negative breast cancer MDA-MB-231 cell aggressiveness: Evidence for involvement of fatty acid 2-hydroxylase (FA2H) in the stimulated cell migration.

Detailed in vitro studies on the effects of perfluorooctanoic acid (PFOA) have demonstrated that activation of peroxisome proliferator-activated receptor α (PPARα) is a key process by which PFOA affects the malignancy of estrogen receptor α (ERα)-positive breast cancer cells. However, there is very little information on the PPARα-regulated genes responsible for the effects of PFOA in ERα-negative breast cancer cell malignancy. We recently demonstrated that fatty acid 2-hydroxylase (FA2H) stimulates the migration of ERα-negative human MDA-MB-231 cells, and PPARα is a key factor for the induction of FA2H in these cells. However, evidence for the relationship between PFOA exposure and PPARα-FA2H axis-driven migration has not been obtained. Here we analyzed the effects of PFOA on PPARα transcription and FA2H expression in relation to MDA-MB-231 cell migration. We found that simultaneously with stimulated migration, PFOA upregulated FA2H and activated the transcription of PPARα. FA2H-selective siRNA, but not siRNA control, clearly dampened PFOA-mediated cell migration. There is an inhibitory interaction between PPARα and PPARß/δ (i.e., PPARß/δ can suppress PPARα-mediated transcription) in MDA-MB-231 cells, but even in the presence of PPARß/δ expression, PFOA appeared to free PPARα to upregulate FA2H. Collectively, our findings show that i) PFOA activates PPARα-mediated transcription, ii) PFOA stimulates migration dependent on FA2H expression, and iii) mechanistically, PFOA relieves PPARß/δ suppression of PPARα activity to upregulate FA2H in MDA-MB-231 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.

Cadmium-stimulated invasion of rat liver cells during malignant transformation: Evidence of the involvement of oxidative stress/TET1-sensitive machinery.

Cadmium (Cd) is recognized as a highly toxic heavy metal for humans in part because it is a multi-organ carcinogen. To clarify the mechanism of Cd carcinogenicity, we have established an experimental system using rat liver TRL1215 cells exposed to 2.5 µM Cd for 10 weeks and then cultured in Cd-free medium for an additional 4 weeks (total 14 weeks). Recently, we demonstrated, by using this experimental system, that 1) Cd stimulates cell invasion by suppression of apolipoprotein E (ApoE) expression, and 2) Cd induces DNA hypermethylation of the regulatory region of the ApoE gene. However, the underlying mechanism(s) as well as other potential genetic participants in the Cd-stimulated invasion are undefined. In the present work, we found that concurrent with enhanced invasion, Cd induced oxidative stress, coupled with the production of oxidative stress-sensitive metallothionein 2A (MT2A), which lead to down-modulation of ten-eleven translocation methylcytosine dioxygenase 1 (TET1: DNA demethylation) in addition to ApoE, without impacting DNA methyltransferases (DNMTs: DNA methylation) levels. Furthermore, the expression of tissue inhibitor of metalloproteinase 2 and 3 (TIMP2 and TIMP3) that are positively regulated by TET1, were decreased by Cd. The genes (ApoE/TET1/TIMP2/TIMP3) suppressed by Cd were further suppressed by hydroquinone (HQ; a reactive oxygen species [ROS] producer), whereas N-acetyl-l-cysteine (NAC; a ROS scavenger) prevented the suppression of their expression by HQ. In addition, NAC reversed their expression suppressed by Cd. Cd-stimulated cell invasion was clearly dampened by NAC in a concentration-dependent manner. Overall these findings suggest that 1) altered TET1 expression and activity together with ApoE are likely involved in the enhanced invasiveness due to Cd exposure, and 2) Cd down-regulation of TET1 likely evokes a reduction in ApoE expression (possible by DNA hypermethylation), and 3) anti-oxidants are effective in abrogation of the enhanced invasiveness that occurs concurrently with Cd-induced malignant transformation.

Fatty acid 2-hydroxylase (FA2H) as a stimulatory molecule responsible for breast cancer cell migration.

The functional role of fatty acid 2-hydroxylase (FA2H) is controversial in the field of cancer biology due to the dual role of FA2H, particularly related to its interaction with triple-negative breast cancer (TNBC). A previous biochemical- and clinical-focused study suggested that FA2H could dampen TNBC aggressiveness. However, another epidemiological study demonstrated that FA2H expression is associated with shorter disease-free survival in TNBC cases. We reported that FA2H is a peroxisome proliferator-activated receptor α (PPARα)-regulated gene in human breast cancer MDA-MB-231 cells, in vitro experimental models for TNBC analysis. PPARα activation by its ligand reportedly results in an aggressive MDA-MB-231 cell phenotype, as well as estrogen receptor α (ERα)-positive MCF-7 cells. The results of this study show that i) MDA-MB-231 cells express very low levels of FA2H compared to the MCF-7 cells, reflecting a low basal-level PPARα-driven transcriptional activity compared to the MCF-7 cells, and ii) the increased FA2H expression stimulates the MDA-MB-231 and MCF-7 breast cancer cell migration without affecting proliferation. Taken together, our findings indicate that FA2H might be a breast cancer cell migration stimulator, independently of the ERα expression status.

Cannabidiolic acid dampens the expression of cyclooxygenase-2 in MDA-MB-231 breast cancer cells: Possible implication of the peroxisome proliferator-activated receptor ß/δ abrogation.

A growing body of experimental evidence strongly suggests that cannabidiolic acid (CBDA), a major component of the fiber-type cannabis plant, exerts a variety of biological activities. We have reported that CBDA can abrogate cyclooxygenase-2 (COX-2) expression and its enzymatic activity. It is established that aberrant expression of COX-2 correlates with the degree of malignancy in breast cancer. Although the reduction of COX-2 expression by CBDA offers an attractive medicinal application, the molecular mechanisms underlying these effects have not fully been established. It has been reported that COX-2 expression is positively controlled by peroxisome proliferator-activated receptor ß/δ (PPARß/δ) in some cancerous cells, although there is "no" modulatory element for PPARß/δ on the COX-2 promoter. No previous studies have examined whether an interaction between PPARß/δ-mediated signaling and COX-2 expression exists in MDA-MB-231 cells. We confirmed, for the first time, that COX-2 expression is positively modulated by PPARß/δ-mediated signaling in MDA-MB-231 cells. CBDA inhibits PPARß/δ-mediated transcriptional activation stimulated by the PPARß/δ-specific agonist, GW501516. Furthermore, the disappearance of cellular actin stress fibers, a hallmark of PPARß/δ and COX-2 pathway activation, as evoked by the GW501516, was effectively reversed by CBDA. Activator protein-1 (AP-1)-driven transcriptional activity directly involved in the regulation of COX-2 was abrogated by the PPARß/δ-specific inverse agonists (GSK0660/ST-247). Thus, it is implicated that there is positive interaction between PPARß/δ and AP-1 in regulation of COX-2. These data support the concept that CBDA is a functional down-regulator of COX-2 through the abrogation of PPARß/δ-related signaling, at least in part, in MDA-MB-231 cells.

Metalloestrogenic effects of cadmium are absent in long-term estrogen-deprived MCF-7 cells: Evidence for the involvement of constitutively activated estrogen receptor α and very low expression of G protein-coupled estrogen receptor 1.

Cadmium (Cd) has estrogen-like activities in breast cancer; it acts as a metalloestrogen in humans. Prospective cohort studies of Cd and breast cancer risk suggest a significant relationship between increased Cd intake and cancer incidence, with more pronounced effects for estrogen receptor α (ERα)-positive breast cancers. However, a recent systematic review with the highest level of evidence demonstrated no such relationship in post-menopausal women. Thus, the reported effects of Cd in pre- and post-menopausal ERα-positive breast cancers are inconsistent. MCF-7 human breast cancer cells normally exhibit growth through estradiol-triggered ERα signaling; however, the MCF-7 cells cultured in estrogen-deprived conditions for a long time (∼ 6 months) eventually result in LTED cells that can be used to utilize to study the proliferation of ERα-positive breast cancer cells obtained from post-menopausal women. Our results showed that unlike MCF-7 cells, LTED cells showed estradiol-independent growth because of constitutively activated ERα. Moreover, Cd (∼10 nM) stimulated ERα signaling in MCF-7 cells and ERα-expressing LTED cells, but not in LTED cells; in ERα-expressing LTED cells, this effect was reversed by ICI 182,780 (an ERα antagonist). Furthermore, in comparison with MCF-7 cells, the LTED cells expressed very low levels of G protein-coupled estrogen receptor 1 (GPER1), a membrane ER capable of stimulating the estrogenic activity of Cd. These findings suggest that the estrogenic action of Cd may be suppressed in LTED cells, and potentially in post-menopausal breast cancer.

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.

Δ9-Tetrahydrocannabinol upregulates fatty acid 2-hydroxylase (FA2H) via PPARα induction: A possible evidence for the cancellation of PPARß/δ-mediated inhibition of PPARα in MDA-MB-231 cells.

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear transcription factors, with three characterized subtypes: PPARα, PPARß/δ, and PPARγ. The biological correlation between the two PPAR subtypes PPARα and γ and carcinogenesis is well-characterized; however, substantially less is known about the biological functions of PPARß/δ. PPARß/δ has been reported to repress transcription when PPARß/δ and PPARα or PPARγ are simultaneously expressed in some cells, and MDA-MB-231 cells express functional levels of PPARß/δ. We have previously reported that Δ9-tetrahydrocannabinol (Δ9-THC), a major cannabinoid component of the drug-type cannabis plant, can stimulate the expression of fatty acid 2-hydroxylase (FA2H) via upregulation of PPARα expression in human breast cancer MDA-MB-231 cells. Although the possibility of an inhibitory interaction between PPARα and PPARß/δ has not been demonstrated in MDA-MB-231 cells, we reasoned if this interaction were to exist, Δ9-THC should make PPARα free to achieve FA2H induction. Here, we show that a PPARß/δ-mediated suppression of PPARα function, but not of PPARγ, exists in MDA-MB-231 cells and Δ9-THC causes FA2H induction via mechanisms underlying the cancellation of PPARß/δ-mediated inhibition of PPARα, in addition to the upregulation of PPARα.

Cadmium down-regulates apolipoprotein E (ApoE) expression during malignant transformation of rat liver cells: direct evidence for DNA hypermethylation in the promoter region of ApoE.

There is adequate evidence for the carcinogenicity of cadmium (Cd). However, a significant unaddressed question remains as to how this metal actually causes malignant transformation (tumor initiation). Since it has been shown that Cd only has the weak direct interaction potential with DNA, the metal is recognized as an indirect genotoxicant and mutagen. Currently, Cd-mediated "epigenetic" modifications, such as changes in DNA methylation resulting in alteration in target gene expression, coupled with cancer progression, are the focus of mechanistic research. We have reported that the apolipoprotein E (ApoE) gene, a suppressor of cell invasion, is an early Cd target, and is involved in the malignant transformation of TRL 1215 rodent liver cells. Cd exposure suppresses ApoE expression which can be re-activated with 5-aza-2'-deoxycytidine, a DNA demethylating agent. In the present study, we sought direct evidence of Cd-induced DNA hypermethylation of the ApoE promoter region by performing bisulfite sequencing and real-time quantitative methylation-specific PCR. Our data clearly suggest that Cd can down-regulate the expression of ApoE via introduction of excess DNA methylation in the promoter region of ApoE during malignant transformation of TRL 1215 cells.

Bisphenol AF as an activator of human estrogen receptor ß1 (ERß1) in breast cancer cell lines.

Bisphenol AF (BPAF) is now recognized as one of the replacements for bisphenol A (BPA). Although considerable experimental evidence suggests that BPA is an endocrine-disrupting chemical, the toxicological profile of BPAF has been investigated in less detail than that of BPA, even at the in vitro level. BPAF has been established as an activator of estrogen receptor α (ERα) in many cell lines; however, controversy surrounds its effects on the other isoform, ERß (i.e., whether it functions as a stimulator). Five human ERß isoforms have been cloned and characterized. Of these, we focused on the interactions between BPAF and the two isoforms, ERß1 and ERß2. We demonstrated that i) BPAF functioned as a stimulator of ERß1 (and ERα), which is transiently expressed in the two types of human breast cancer cells (MDA-MB-231 and SK-BR-3 cells) (EC50 values for ERß: 6.87 nM and 2.58 nM, respectively, and EC50 values for ERα: 24.7 nM and 181 nM, respectively), ii) the stimulation of ERß1 by BPAF (1-25 nM) was abrogated by PHTPP (an ERß selective antagonist), and iii) the expression of ERß1 and ERß2 was not modulated by BPAF at nanomolar concentrations up to 25 nM. These results indicate that BPAF activates not only human ERα, but also the ERß1 isoform in breast cancer cells, and exhibits higher activation potency for ERß1.

Cadmium-induced malignant transformation of rat liver cells: Potential key role and regulatory mechanism of altered apolipoprotein E expression in enhanced invasiveness.

Cadmium is a transition metal that is classified as human carcinogen by the International Agency for Research on Cancer (IARC) with multiple target sites. Many studies using various model systems provide evidence of cadmium-induced malignancy formation in vivo or malignant cell transformation in vitro. Nonetheless, further studies are needed to completely understand the mechanisms of cadmium carcinogenicity. Our prior studies have utilized a rat liver epithelial cell line (TRL 1215) as a model for cadmium-induced malignant transformation. In the present study, we focused on the molecular mechanisms of this malignant transformation, especially with regard to hyper-invasiveness stimulated by cadmium transformation. By performing a series of biochemical analyses on cadmium transformed cells, it was determined that cadmium had significantly down-regulated the expression of apolipoprotein E (ApoE). ApoE was recently established as a suppressor of cell invasion. A key factor in the suppression of ApoE by cadmium appeared to be that the metal evoked a 5-aza-2'-deoxycytidine-sensitive hypermethylation of the regulatory region of ApoE, coupled with interference of the action of liver X receptor α (LXRα), a transcriptional regulator for ApoE. Furthermore, the expression of LXRα itself was suppressed by cadmium-mediated epigenetic modification. Re-expression of ApoE clearly abrogated the cell invasion stimulated by cadmium-induced malignant transformation. Together, the current results suggest that the cadmium-mediated enhanced cell invasion is linked to down-regulation of ApoE during malignant transformation these liver cells.

Cannabidiolic Acid-Mediated Interference with AP-1 Transcriptional Activity in MDA-MB-231 Breast Cancer Cells.

We reported that cannabidiolic acid (CBDA), a non-psychotropic constituent of fiber-type cannabis plants, down-regulates the mRNA expression of cyclooxygenase-2 (COX-2) in highly aggressive MDA-MB-231 human breast cancer cells. However, the molecular mechanism(s) underlying the CBDA suppression of COX-2 have not yet been elucidated in detail. In MDA-MB-231 cells, COX-2 expression is known to be tightly regulated by the transcriptional activity of activator protein-I (AP-1), which is composed of a heterodimer of c-Fos and c-Jun. AP-1-mediated transcriptional activity was inhibited by CBDA in a dose-dependent manner. The expression of c-fos was maintained at markedly lower levels (0.035) than basal c-jun expression levels (1.0), implicating c- fos as a limiting factor in the regulation of COX-2. Analyses indicated that CBDA abrogated the expression of c-fos mRNA without affecting c-jun. Collectively, these results suggest that CBDA abolishes the expression of COX-2 by interfering with AP-I activity in MDA-MB3-231 cells.

Preventive effects of metallothionein against DNA and lipid metabolic damages in dyslipidemic mice under repeated mild stress.

The effects of repeated mild stress on DNA and lipid metabolic damages in multiple organs of dyslipidemic mice, and the preventive role of metallothionein (MT) were investigated. Female adult wild-type and MT-null mice fed high-fat diet (HFD) or standard diet (STD) were repeatedly subjected to fasting or restraint for three weeks. The liver, pancreas, spleen, bone marrow and serum samples were taken for evaluating DNA damage, MT, glutathione (GSH), corticosterone, carnitine and adiponectin. Body weights of restraint groups were reduced with the intensity of stress increased, even if the energy intakes were higher than those of STD group. Hepatic GSH levels were reduced in HFD control group and were further reduced in stress groups, especially in restraint groups, while the hepatic MT and serum corticosterone levels were increased in concert with the intensity of stress. Cellular DNA damages were generally increased by the restraint stress, especially in MT-null mice. Hepatic carnitine levels of MT-null mice were markedly lower than those of wild-type mice. The data suggest that MT plays a preventive role by acting as an antioxidant in corporation with GSH decreased by repeated stress and that MT may be an essential factor for inducing carnitine under the stress.

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.

Development of high-fat-diet-induced obesity in female metallothionein-null mice.

Oxidative stress accelerates adipocyte differentiation and lipid accumulation, leading to endoplasmic reticulum (ER) stress, which causes insulin resistance. Because metallothionein (MT) has a role in prevention of oxidative and ER stress, we examined the effects of MT on the development of obesity induced by 27 wk of a high-fat diet (HFD) in female MT-I- and MT-II-null (MT(-/-)) and wild-type (MT(+/+)) mice. Body weight, fat mass, and plasma cholesterol increased at a greater rate in MT(-/-) mice fed an HFD than in MT(-/-) mice fed a control diet (CD) and MT(+/+) mice fed an HFD, indicating that MT(-/-) mice fed an HFD became obese and hypercholesterolemic and that MT could prevent HFD-induced obesity. The observed increases in the levels of plasma leptin and leptin mRNA in the white adipose tissue of MT(-/-) mice fed the HFD suggested a leptin-resistant state. Enhanced expression of a mesoderm-specific transcript, which regulates the enlargement of fat cells, was accompanied by enlarged adipocytes in the white adipose tissue of young MT(-/-) mice before obesity developed after 3 and 8 wk of feeding the HFD. Thus, MT may have a preventive role against HFD-induced obesity by regulating adipocyte enlargement and leptin signaling.

Tissue-dependent preventive effect of metallothionein against DNA damage in dyslipidemic mice under repeated stresses of fasting or restraint.

AIMS: To investigate the effect of repeated stress on DNA damage in seven organs of dyslipidemic mice, and the preventive role of metallothionein (MT). MAIN METHODS: Female adult 129/Sv wild-type and MT-null mice fed high-fat diet (HFD) were repeatedly subjected to mild stress of fasting or restraint in weeks 2 to 4 of 4-week study period. Serum cholesterol level, DNA damage in the liver, pancreas, spleen, bone marrow, kidney, lung and gastric mucosa, and other parameters were determined. KEY FINDINGS: Body weights were increased in both types of mice fed HFD compared to those fed standard diet (STD), and further increased by 12 h-fasting, while they were markedly decreased by 1-3 h-restraint. Fasting accelerated accumulation of fat in the liver, and increase in serum cholesterol of both types of mice fed HFD. Feeding of HFD increased DNA damage in the pancreas, spleen and bone marrow of both types of mice, compared with those fed STD. In the wild-type mice fed HFD, 24 h-fasting increased DNA damage in the liver and spleen, while restraint increased the damage in the liver, pancreas, spleen and bone marrow. DNA damage in the cells of organs was markedly increased in the MT-null mice. Specifically, damage in the liver, pancreas, spleen and bone marrow was greatly increased with the intensity of stress increased, and the damage was much greater in the restraint mice than in the fasting mice. SIGNIFICANCE: MT plays a tissue-dependent preventive role against DNA damage in various murine organs induced by repeated stress.