Robert M. Bigsby

Hydroxylated metabolites of polybrominated diphenyl ethers in human blood samples from the United States.

Background A previous study from our laboratory showed that polybrominated diphenyl ethers (PBDEs) were metabolized to hydroxylated PBDEs (HO-PBDEs) in mice and that para-HO-PBDEs were the most abundant and, potentially, the most toxic metabolites. Objective The goal of this study was to determine the concentrations of HO-PBDEs in blood from pregnant women, who had not been intentionally or occupationally exposed to these flame retardants, and from their newborn babies. Methods Twenty human blood samples were obtained from a hospital in Indianapolis, Indiana, and analyzed for both PBDEs and HO-PBDEs using electron-capture negative-ionization gas chromatographic mass spectrometry. Results The metabolite pattern of HO-PBDEs in human blood was quite different from that found in mice; 5-HO-BDE-47 and 6-HO-BDE-47 were the most abundant metabolites of BDE-47, and 5′-HO-BDE-99 and 6′-HO-BDE-99 were the most abundant metabolites of BDE-99. The relative concentrations between precursor and corresponding metabolites indicated that BDE-99 was more likely to be metabolized than BDE-47 and BDE-100. In addition, three bromophenols were also detected as products of the cleavage of the diphenyl ether bond. The ratio of total hydroxylated metabolites relative to their PBDE precursors ranged from 0.10 to 2.8, indicating that hydroxylated metabolites of PBDEs were accumulated in human blood. Conclusions The quite different PBDE metabolite pattern observed in humans versus mice indicates that different enzymes might be involved in the metabolic process. Although the levels of HO-PBDE metabolites found in human blood were low, these metabolites seemed to be accumulating.

Measurement of polybrominated diphenyl ethers and metabolites in mouse plasma after exposure to a commercial pentabromodiphenyl ether mixture.

Background Previous studies have shown that polybrominated diphenyl ethers (PBDEs) behave as weak estrogens in animal and cell culture bioassays. In vivo metabolites of PBDEs are suspected to cause these effects. Objectives To identify candidate metabolites, mouse plasma samples were collected after continuous oral and subcutaneous exposure to DE-71, a widely used commercial pentabromodiphenyl ether product, for 34 days. Methods Samples were extracted, separated into neutral and phenolic fractions, and analyzed by gas chromatographic mass spectrometry. Results In the plasma samples of orally treated animals, 2,2′,4,4′,5,5′-hexabromodiphenyl ether (BDE-153) represented 52% of total measurable PBDEs, whereas it represented only 4.3% in the DE-71 mixture. This suggested that BDE-153 was more persistent than other congeners in mice. Several metabolites were detected and quantitated: 2,4-dibromophenol, 2,4,5-tribromophenol, and six hydroxylated PBDEs. The presence of the two phenols suggested cleavage of the ether bond of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99), respectively. The hydroxylated (HO)-PBDEs might come from hydroxylation or debromination/hydroxylation. Among the quantitated hydroxylated metabolites, the most abundant was 4-HO-2,2′,3,4′-tetra-BDE, which suggested that there was a bromine shift during the hydroxylation process. para-HO-PBDEs have been proposed to behave as endocrine disruptors. Conclusions There seem to be three metabolic pathways: cleavage of the diphenyl ether bond, hydroxylation, and debromination/hydroxylation. The cleavage of the diphenyl ether bond formed bromophenols, and the other two pathways formed hydroxylated PBDEs, of which para-HO-PBDEs are most likely formed from BDE-47. These metabolites may be the most thyroxine-like and/or estrogen-like congeners among the HO-PBDEs.

Effect of Estradiol on Estrogen Receptor Expression in Rat Uterine Cell Types

Abstract In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERα) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERα. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERα expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17β. Uteri were collected and analyzed for changes in ERα mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERα. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERα. Expression of ERα in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERα mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERα mRNA occurs independent of new protein synthesis. A decrease in ERα mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERα expression. The results indicate that down-regulation of ERα in the epithelia and up-regulation of stromal ERα play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.

Enhanced peritoneal ovarian tumor dissemination by tissue transglutaminase

Tissue transglutaminase (TG2) is involved in Ca(2+)-dependent aggregation and polymerization of proteins. We previously reported that TG2 mRNA is up-regulated in epithelial ovarian cancer (EOC) cells compared with normal ovarian epithelium. Here, we show overexpression of the TG2 protein in ovarian cancer cells and tumors and its secretion in ascites fluid and define its role in EOC. By stable knockdown and overexpression, we show that TG2 enhances EOC cell adhesion to fibronectin and directional cell migration. This phenotype is preserved in vivo, where the pattern of tumor dissemination in the peritoneal space is dependent on TG2 expression levels. TG2 knockdown diminishes dissemination of tumors on the peritoneal surface and mesentery in an i.p. ovarian xenograft model. This phenotype is associated with deficient beta(1) integrin-fibronectin interaction, leading to weaker anchorage of cancer cells to the peritoneal matrix. Highly expressed in ovarian tumors, TG2 facilitates i.p. tumor dissemination by enhancing cell adhesion to the extracellular matrix and modulating beta(1) integrin subunit expression.

Epithelial-to-mesenchymal transition and ovarian tumor progression induced by tissue transglutaminase

Tissue transglutaminase (TG2), an enzyme that catalyzes Ca(2+)-dependent aggregation and polymerization of proteins, is overexpressed in ovarian cancer cells and tumors. We previously reported that TG2 facilitates tumor dissemination using an i.p. xenograft model. Here we show that TG2 modulates epithelial-to-mesenchymal transition (EMT), contributing to increased ovarian cancer cell invasiveness and tumor metastasis. By using stable knockdown and overexpression in epithelial ovarian cancer cells, we show that TG2 induces a mesenchymal phenotype, characterized by cadherin switch and invasive behavior in a Matrigel matrix. This is mediated at the transcriptional level by altering the expression levels and function of several transcriptional repressors, including Zeb1. One mechanism through which TG2 induces Zeb1 is by activating the nuclear factor-kappaB complex. The effects of TG2 on ovarian cancer cell phenotype and invasiveness translate into increased tumor formation and metastasis in vivo, as assessed by an orthotopic ovarian xenograft model. Highly expressed in ovarian tumors, TG2 promotes EMT and enhances ovarian tumor metastasis by activating oncogenic signaling.

Hydroxylated metabolites of the polybrominated diphenyl ether mixture DE-71 Are weak estrogen receptor-α ligands

Background Polybrominated diphenyl ethers (PBDEs) are widely found in the environment and are suspected endocrine disruptors. We previously identified six hydroxylated metabolites of PBDE (OH-PBDEs) in treated mice. Objective We tested the hypothesis that OH-PBDEs would interact with and alter activity of estrogen receptor-α (ER-α). Methods We tested estrogenicity using two assays: 3H-estradiol (3H-E2) displacement from recombinant ER-α and induction of reporter gene (ERE-luciferase) in cultured cells. We incubated the PBDE mixture DE-71 with rat liver microsomes and tested the resultant metabolite mixture for estrogenic activity. We also determined relative estrogenic potential of individual hydroxylated PBDE congeners. Results Reporter gene activity was increased by DE-71 that had been subjected to microsomal metabolism. DE-71 did not displace E2 from ER-α, but all six of the OH-PBDE metabolites did. para-Hydroxylated metabolites displayed a 10- to 30-fold higher affinity for ER-α compared with ortho-hydroxylated PBDEs, and one produced a maximal effect 30% higher than that produced by E2. Coadministration of E2 and DE-71, or certain of its metabolites, yielded reporter activity greater than either chemical alone. Two ortho-OH-PBDEs were antiestrogenic in the reporter assay. Conclusions The observations—that the DE-71 mixture did not displace 3H-E2 from ER-α while the hydroxylated metabolites did—suggest that the weak estrogenic effects of DE-71 are due to metabolic activation of individual congeners. However, the behavior of DE-71 and its metabolites, when co-administered with E2, suggest a secondary, undetermined mechanism from classical ER-α activation.

Estrogen promotes tumor progression in a genetically defined mouse model of lung adenocarcinoma

Numerous epidemiological observations point to sex differences in lung cancer etiology and progression. The present study was aimed at understanding the bases of these sex differences. To test the effect of estradiol on tumor progression, we used a mouse model based on conditional Kras expression and concurrent deletion of Tp53 following inhalation of an adenoviral vector expressing Cre recombinase (AdeCre). Ovariectomized females and males were treated with estradiol via a continuous-release capsule. Tumor multiplicity, tumor volume, and histological grade were determined at 10 weeks after AdeCre administration. Cell proliferation was monitored by Ki67 immunohistochemistry at 4 and 10 weeks after AdeCre administration. At 10 weeks, female mice had more than twice the number of tumors evident on the surface of the lungs than male mice; ovariectomy eliminated this sex difference. The estrogen treatment significantly increased tumor number and volume in ovariectomized females and in males. Histological character of the tumors ranged from adenoma to adenocarcinoma. Ovary-intact females exhibited higher grade tumors than ovariectomized females or males. Progression to higher histological grade was stimulated by estrogen in male mice but not in ovariectomized females. At 10 weeks after AdeCre administration, tumor cell Ki67-labeling varied widely, precluding assessment of an estrogen effect; however, at 4 weeks, Ki67 labeling of lung parenchymal cells was increased 3.5-fold by estrogen. In conclusion, estrogen acts as a promoter for lung adenocarcinoma in a genetically defined lung cancer model; estrogen-induced cell proliferation in the oncogene-initiated cells is likely to play a role in this tumor promoter activity.

Apigenin inhibits antiestrogen-resistant breast cancer cell growth through estrogen receptor-α-dependent and estrogen receptor-α-independent mechanisms

Breast cancer resistance to the antiestrogens tamoxifen (OHT) and fulvestrant is accompanied by alterations in both estrogen-dependent and estrogen-independent signaling pathways. Consequently, effective inhibition of both pathways may be necessary to block proliferation of antiestrogen-resistant breast cancer cells. In this study, we examined the effects of apigenin, a dietary plant flavonoid with potential anticancer properties, on estrogen-responsive, antiestrogen-sensitive MCF7 breast cancer cells and two MCF7 sublines with acquired resistance to either OHT or fulvestrant. We found that apigenin can function as both an estrogen and an antiestrogen in a dose-dependent manner. At low concentrations (1 μmol/L), apigenin stimulated MCF7 cell growth but had no effect on the antiestrogen-resistant MCF7 sublines. In contrast, at high concentrations (>10 μmol/L), the drug inhibited growth of MCF7 cells and the antiestrogen-resistant sublines, and the combination of apigenin with either OHT or fulvestrant showed synergistic, growth-inhibitory effects on both antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. To further elucidate the molecular mechanism of apigenin as either an estrogen or an antiestrogen, effects of the drug on estrogen receptor-α (ERα); transactivation activity, mobility, stability, and ERα-coactivator interactions were investigated. Low-dose apigenin enhanced receptor transcriptional activity by promoting interaction between ERα and its coactivator amplified in breast cancer-1. However, higher doses (>10 μmol/L) of apigenin inhibited ERα mobility (as determined by fluorescence recovery after photobleaching assays), down-regulated ERα and amplified in breast cancer-1 expression levels, and inhibited multiple protein kinases, including p38, protein kinase A, mitogen-activated protein kinase, and AKT. Collectively, these results show that apigenin can function as both an antiestrogen and a protein kinase inhibitor with activity against breast cancer cells with acquired resistance to OHT or fulvestrant. We conclude that apigenin, through its ability to target both ERα-dependent and ERα-independent pathways, holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer. [Mol Cancer Ther 2008;7(7):2096–108]

The polybrominated diphenyl ether mixture DE-71 is mildly estrogenic.

Background Polybrominated diphenyl ethers (PBDEs) are widely found in the environment, and they may act as endocrine disruptors. Objective Our goal in this study was to test the PBDE mixture DE-71 for estrogenic activity. Methods We used proliferation of cultured breast cancer cells (MCF-7) and trophic effects in the reproductive tracts of ovariectomized mice as estrogen bioassays. DE-71 was administered to mice by subcutaneous injection (sc) or oral gavage (po), alone or in combination with estradiol, for 3 or 34 days. Liver weights and cytochrome P450 enzyme activities were also measured. Results DE-71 increased MCF-7 cell proliferation, and this was prevented by antiestrogen. DE-71 cotreatment reduced the effect of estradiol in MCF-7 cells. In the mouse 3-day assay, DE-71 administered alone had no effect on uterine weight, uterine epithelial height (UEH), or vaginal epithelial thickness (VET); however, when DE-71 was administered as a cotreatment, it potentiated estradiol’s effect on uterine weight. DE-71 administered sc to BALB/c mice for 34 days slightly increased UEH and VET, and attenuated the estradiol-induced increase in UEH; these effects were not seen in BALB/c mice treated po or in C57BL/6 mice treated sc. DE-71 increased liver weight in BALB/c, C57BL/6, and estrogen receptor-α knockout mice. We also found an increase in liver cytochrome P450 1A (CYP1A) and CYP2B activities when DE-71 was administered po, but only CYP2B increased after sc treatment. Conclusion DE-71 behaves as a weak estrogen. In mice, the treatment route and duration determined if DE-71 was estrogenic. BALB/c mice are more susceptible to DE-71 effects in estrogen target tissues than C57BL/6 mice. DE-71 increased liver weight independently of estrogen receptor-α.

Interleukin-1α Promotes Tumor Growth and Cachexia in MCF-7 Xenograft Model of Breast Cancer

Progression of breast cancer involves cross-talk between epithelial and stromal cells. This cross-talk is mediated by growth factors and cytokines secreted by both cancer and stromal cells. We previously reported expression of interleukin (IL)-1α in a subset of breast cancers and demonstrated that IL-1α is an autocrine and paracrine inducer of prometastatic genes in in vitro systems. To understand the role of IL-1α in breast cancer progression in vivo, we studied the growth of MCF-7 breast cancer cells overexpressing a secreted form of IL-1α (MCF-7IL-1α) in nude mice. MCF-7IL-1α cells formed rapidly growing estrogen-dependent tumors compared to parental cells. Interestingly, IL-1α expression alone was not sufficient for metastasis in vivo although in vitro studies showed induction of several prometastatic genes and matrix metalloproteinase activity in response to cross-talk between IL-1α-expressing cancer cells and fibroblasts. Animals implanted with MCF-7IL-1α cells were cachetic, which correlated with increased leptin serum levels but not other known cachexia-inducing cytokines such as IL-6, tumor necrosis factor, or interferon gamma. Serum triglycerides, but not blood glucose were lower in animals with MCF-7IL-1α cell-derived tumors compared to animals with control cell-derived tumors. Cachexia was associated with atrophy of epidermal and adnexal structures of skin; a similar phenotype is reported in triglyceride-deficient mice and in ob/ob mice injected with leptin. Mouse leptin-specific transcripts could be detected only in MCF-7IL-1α cell-derived tumors, which suggests that IL-1α increases leptin expression in stromal cells recruited into the tumor microenvironment. Despite increased serum leptin levels, animals with MCF-7IL-1α cell-derived tumors were not anorexic suggesting only peripheral action of tumor-derived leptin, which principally targets lipid metabolism. Taken together, these results suggest that cancer cell-derived cytokines, such as IL-1α, induce cachexia by affecting leptin-dependent metabolic pathways.