Short-term tetrabromobisphenol A exposure promotes fibrosis of human uterine fibroid cells in a 3D culture system through TGF-beta signaling.

Tetrabromobisphenol A (TBBPA), a derivative of BPA, is a ubiquitous environmental contaminant with weak estrogenic properties. In women, uterine fibroids are highly prevalent estrogen-responsive tumors often with excessive accumulation of extracellular matrix (ECM) and may be the target of environmental estrogens. We have found that BPA has profibrotic effects in vitro, in addition to previous reports of the in vivo fibrotic effects of BPA in mouse uterus. However, the role of TBBPA in fibrosis is unclear. To investigate the effects of TBBPA on uterine fibrosis, we developed a 3D human uterine leiomyoma (ht-UtLM) spheroid culture model. Cell proliferation was evaluated in 3D ht-UtLM spheroids following TBBPA (10-6 -200 µM) administration at 48 h. Fibrosis was assessed using a Masson's Trichrome stain and light microscopy at 7 days of TBBPA (10-3  µM) treatment. Differential expression of ECM and fibrosis genes were determined using RT² Profiler™ PCR arrays. Network and pathway analyses were conducted using Ingenuity Pathway Analysis. The activation of pathway proteins was analyzed by a transforming growth factor-beta (TGFB) protein array. We found that TBBPA increased cell proliferation and promoted fibrosis in 3D ht-UtLM spheroids with increased deposition of collagens. TBBPA upregulated the expression of profibrotic genes and corresponding proteins associated with the TGFB pathway. TBBPA activated TGFB signaling through phosphorylation of TGFBR1 and downstream effectors-small mothers against decapentaplegic -2 and -3 proteins (SMAD2 and SMAD3). The 3D ht-UtLM spheroid model is an effective system for studying environmental agents on human uterine fibrosis. TBBPA can promote fibrosis in uterine fibroid through TGFB/SMAD signaling.

Cobalt-induced oxidative stress contributes to alveolar/bronchiolar carcinogenesis in B6C3F1/N mice.

Rodent alveolar/bronchiolar carcinomas (ABC) that arise either spontaneously or due to chemical exposure are similar to a subtype of lung adenocarcinomas in humans. B6C3F1/N mice and F344/NTac rats exposed to cobalt metal dust (CMD) by inhalation developed ABCs in a dose dependent manner. In CMD-exposed mice, the incidence of Kras mutations in ABCs was 67% with 80% of those being G to T transversions on codon 12 suggesting a role of oxidative stress in the pathogenesis. In vitro studies, such as DMPO (5,5-dimethyl-1-pyrroline N-oxide) immune-spin trapping assay, and dihydroethidium (DHE) fluorescence assay on A549 and BEAS-2B cells demonstrated increased oxidative stress due to cobalt exposure. In addition, significantly increased 8-oxo-dG adducts were demonstrated by immunohistochemistry in lungs from mice exposed to CMD for 90 days. Furthermore, transcriptomic analysis on ABCs arising spontaneously or due to chronic CMD-exposure demonstrated significant alterations in canonical pathways related to MAPK signaling (IL-8, ErbB, Integrin, and PAK pathway) and oxidative stress (PI3K/AKT and Melatonin pathway) in ABCs from CMD-exposed mice. Oxidative stress can stimulate PI3K/AKT and MAPK signaling pathways. Nox4 was significantly upregulated only in CMD-exposed ABCs and NOX4 activation of PI3K/AKT can lead to increased ROS levels in human cancer cells. The gene encoding Ereg was markedly up-regulated in CMD-exposed mice. Oncogenic KRAS mutations have been shown to induce EREG overexpression. Collectively, all these data suggest that oxidative stress plays a significant role in CMD-induced pulmonary carcinogenesis in rodents and these findings may also be relevant in the context of human lung cancers.

Immunohistochemical evaluation of immune cell infiltration in canine gliomas.

Evasion of the immune response is an integral part of the pathogenesis of glioma. In humans, important mechanisms of immune evasion include recruitment of regulatory T cells (Tregs) and polarization of macrophages toward an M2 phenotype. Canine glioma has a robust immune cell infiltrate that has not been extensively characterized. The purpose of this study was to determine the distribution of immune cells infiltrating spontaneous intracranial canine gliomas. Seventy-three formalin-fixed, paraffin-embedded tumor samples were evaluated using immunohistochemistry for CD3, forkhead box 3 (FOXP3), CD20, Iba1, calprotectin (Mac387), CD163, and indoleamine 2,3-dioxygenase (IDO). Immune cell infiltration was present in all tumors. Low-grade and high-grade gliomas significantly differed in the numbers of FoxP3+ cells, Mac387+ cells, and CD163+ cells (P = .006, .01, and .01, respectively). Considering all tumors, there was a significant increase in tumor area fraction of CD163 compared to Mac387 (P < .0001), and this ratio was greater in high-grade tumors than in low-grade tumors (P = .005). These data warrant further exploration into the roles of macrophage repolarization or Treg interference therapy in canine glioma.

Differential Receptor Tyrosine Kinase Phosphorylation in the Uterus of Rats Following Developmental Exposure to Tetrabromobisphenol A.

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant that induces endometrial adenocarcinoma and other uterine tumors in Wistar Han rats; however, early molecular events or biomarkers of TBBPA exposure remain unknown. We investigated the effects of TBBPA on growth factor receptor activation (phospho-RTK) in uteri of rats following early-life exposures. Pregnant Wistar Han rats were exposed to TBBPA (0, 0.1, 25, 250 mg/kg/day) via oral gavage on gestation day 6 through weaning of pups (PND 21). Pups were exposed in utero, through lactation, and by daily gavage from PND 22 to PND 90. Uterine horns were collected (at PND 21, PND 33, PND 90) and formalin-fixed or frozen for histologic, immunohistochemical, phospho-RTK arrays, or western blot analysis. At PND 21, the phosphor-RTKs, FGFR2, FGFR3, TRKC and EPHA1 were significantly increased at different treatment concentrations. Several phospho-RTKs were also significantly overexpressed at PND 33 which included epithelial growth factor receptor (EGFR), Fibroblast Growth Factor Receptor 3-4 (FGFR2, FGFR3, FGFR4), insulin-like growth factor receptor 1 (IGF1R), INSR, AXL, MERTK, PDGFRa and b, RET, Tyrosine Kinase with Immunoglobulin Like and EGF Like Domains 1 and 2 (TIE1; TIE2), TRKA, VEGFR2 and 3, and EPHA1 at different dose treatments. EGFR, an RTK overexpressed in endometrial cancer in women, remained significantly increased for all treatment groups at PND 90. Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2) and IGF1R were overexpressed at PND 33 and remained increased through PND 90, although ERBB2 was statistically significant at PND 90. The phospho-RTKs, FGFR3, AXL, DTK, HGFR, TRKC, VEGFR1 and EPHB2 and 4 were also statistically significant at PND 90 at different dose treatments. The downstream effector, phospho-MAPK44/42 was also increased in uteri of treated rats. Our findings show RTKs are dysregulated following early life TBBPA exposures and their sustained activation may contribute to TBBPA-induced uterine tumors observed in rats later in life.

The Life Cycle of the Uterine Fibroid Myocyte.

PURPOSE OF REVIEW: Uterine fibroids are common benign tumors of women in the USA and worldwide, yet the biological nature and pathogenesis of these tumors remain largely unknown. This review presents our view of the stages in the life cycle of a subset of uterine fibroid myocytes, introduces hypothetical concepts and morphological data to explain these changes, and relates these changes in individual myocytes to the phases of fibroid tumor development. RECENT FINDINGS: The observations gained from light and electron microscopic, immunohistochemical, and morphometric studies in our laboratory have led to the hypothesis that fibroid changes over time may relate to the excessive production of collagen by phenotypically transformed myocytes. This accumulation of collagen results in decreased microvessel density, followed by myocyte injury and atrophy, with eventual senescence and involution through ischemic cellular degeneration and inanition. SUMMARY: Uterine leiomyomas, or fibroids, are characterized by two histologic features-proliferation of myocytes and production of an extracellular collagenous matrix. In the larger tumors, the collagenous matrix is often abundant. Within those regions in which the accumulating collagen is excessive, the myocytes are progressively separated from their blood supply, resulting in myocyte atrophy and eventually cell death. It is within these hypocellular, hyalinized areas that the complete lifecycle of the fibroid myocyte is realized. It begins with the phenotypic transformation of a contractile cell to one characterized by proliferation and collagen synthesis, progresses through an intermediate stage of atrophy related to interstitial ischemia, and eventuates in cell death due to inanition. Lastly, resorption of inanotic cells appears to occur by a non-phagocytic, presumably enzymatic process of degradation and recycling that we refer to as reclamation.

Epigenetic regulation of transcription factor promoter regions by low-dose genistein through mitogen-activated protein kinase and mitogen-and-stress activated kinase 1 nongenomic signaling.

BACKGROUND: The phytoestrogen, genistein at low doses nongenomically activates mitogen-activated protein kinase p44/42 (MAPKp44/42) via estrogen receptor alpha (ERα) leading to proliferation of human uterine leiomyoma cells. In this study, we evaluated if MAPKp44/42 could activate downstream effectors such as mitogen- and stress-activated protein kinase 1 (MSK1), which could then epigenetically modify histone H3 by phosphorylation following a low dose (1 µg/ml) of genistein. RESULTS: Using hormone-responsive immortalized human uterine leiomyoma (ht-UtLM) cells, we found that genistein activated MAPKp44/42 and MSK1, and also increased phosphorylation of histone H3 at serine10 (H3S10ph) in ht-UtLM cells. Colocalization of phosphorylated MSK1 and H3S10ph was evident by confocal microscopy in ht-UtLM cells (r = 0.8533). Phosphorylation of both MSK1and H3S10ph was abrogated by PD98059 (PD), a MEK1 kinase inhibitor, thereby supporting genistein's activation of MSK1 and Histone H3 was downstream of MAPKp44/42. In proliferative (estrogenic) phase human uterine fibroid tissues, phosphorylated MSK1 and H3S10ph showed increased immunoexpression compared to normal myometrial tissues, similar to results observed in in vitro studies following low-dose genistein administration. Real-time RT-PCR arrays showed induction of growth-related transcription factor genes, EGR1, Elk1, ID1, and MYB (cMyb) with confirmation by western blot, downstream of MAPK in response to low-dose genistein in ht-UtLM cells. Additionally, genistein induced associations of promoter regions of the above transcription factors with H3S10ph as evidenced by Chromatin Immunoprecipitation (ChIP) assays, which were inhibited by PD. Therefore, genistein epigenetically modified histone H3 by phosphorylation of serine 10, which was regulated by MSK1 and MAPK activation. CONCLUSION: Histone H3 phosphorylation possibly represents a mechanism whereby increased transcriptional activation occurs following low-dose genistein exposure.

Role of the store-operated calcium entry protein, STIM1, in neutrophil chemotaxis and infiltration into a murine model of psoriasis-inflamed skin.

Stromal interaction molecule 1 (STIM1) is a Ca(2+) sensor protein that initiates store-operated calcium entry (SOCE). STIM1 is known to be involved in the chemoattractant signaling pathway for FPR1 in cell lines, but its role in in vivo functioning of neutrophils is unclear. Plaque-type psoriasis is a chronic inflammatory skin disorder associated with chemoattractants driving neutrophils into the epidermis. We investigated the involvement of STIM1 in neutrophil chemotaxis in vitro, as well as during chronic psoriatic inflammation. To this end, we used conditional knockout (KO) mice lacking STIM1 in cells of myeloid lineage (STIM1(fl/fl) LysM-cre). We demonstrate that STIM1 is required for chemotaxis because of multiple chemoattractants in mouse neutrophils in vitro. Using an imiquimod-induced psoriasis-like skin model, we show that KO mice had less neutrophil infiltration in the epidermis than controls, whereas neither chemoattractant production in the epidermis nor macrophage migration was decreased. KO mice displayed a more rapid reversal of the outward signs of psoriasis (plaques). Thus, KO of STIM1 impairs neutrophil contribution to psoriatic inflammation. Our data provide new insights to our understanding of how STIM1 orchestrates the cellular behavior underlying chemotaxis and illustrate the important role of SOCE in a disease-related pathologic model.

Development of phenotypic and transcriptional biomarkers to evaluate relative activity of potentially estrogenic chemicals in ovariectomized mice.

BACKGROUND: Concerns regarding potential endocrine-disrupting chemicals (EDCs) have led to a need for methods to evaluate candidate estrogenic chemicals. Our previous evaluations of two such EDCs revealed a response similar to that of estradiol (E2) at 2 hr, but a less robust response at 24 hr, similar to the short-acting estrogen estriol (E3). OBJECTIVES: Microarray analysis using tools to recognize patterns of response have been utilized in the cancer field to develop biomarker panels of transcripts for diagnosis and selection of treatments most likely to be effective. Biological effects elicited by long- versus short-acting estrogens greatly affect the risks associated with exposures; therefore, we sought to develop tools to predict the ability of chemicals to maintain estrogenic responses. METHODS: We used biological end points in uterine tissue and a signature pattern-recognizing tool that identified coexpressed transcripts to develop and test a panel of transcripts in order to classify potentially estrogenic compounds using an in vivo system. The end points used are relevant to uterine tissue, but the resulting classification of the compounds is important for other sensitive tissues and species. RESULTS: We evaluated biological and transcriptional end points with proven short- and long-acting estrogens and verified the use of our approach using a phytoestrogen. With our model, we were able to classify the diarylheptanoid D3 as a short-acting estrogen. CONCLUSIONS: We have developed a panel of transcripts as biomarkers which, together with biological end points, might be used to screen and evaluate potentially estrogenic chemicals and infer mode of activity.

The natural history of uterine leiomyomas: morphometric concordance with concepts of interstitial ischemia and inanosis.

Based upon our morphologic observations, we hypothesize and also provide morphometric evidence for the occurrence of progressive developmental changes in many uterine fibroids, which can be arbitrarily divided into 4 phases. These developmental phases are related to the ongoing production of extracellular collagenous matrix, which eventually exceeds the degree of angiogenesis, resulting in the progressive separation of myocytes from their blood supply and a condition of interstitial ischemia. The consequence of this process of slow ischemia with nutritional and oxygen deprivation is a progressive myocyte atrophy (or inanition), culminating in cell death, a process that we refer to as inanosis. The studies presented here provide quantitative and semiquantitative evidence to support the concept of the declining proliferative activity as the collagenous matrix increases and the microvascular density decreases.

Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells.

Estrogen and growth factors play a major role in uterine leiomyoma (UtLM) growth possibly through interactions of receptor tyrosine kinases (RTKs) and estrogen receptor-alpha (ERα) signaling. We determined the genomic and nongenomic effects of 17ß-estradiol (E(2)) on IGF-IR/MAPKp44/42 signaling and gene expression in human UtLM cells with intact or silenced IGF-IR. Analysis by RT(2) Profiler PCR-array showed genes involved in IGF-IR/MAPK signaling were upregulated in UtLM cells by E(2) including cyclin D kinases, MAPKs, and MAPK kinases; RTK signaling mediator, GRB2; transcriptional factors ELK1 and E2F1; CCNB2 involved in cell cycle progression, proliferation, and survival; and COL1A1 associated with collagen synthesis. Silencing (si)IGF-IR attenuated the above effects and resulted in upregulation of different genes, such as transcriptional factor ETS2; the tyrosine kinase receptor, EGFR; and DLK1 involved in fibrosis. E(2) rapidly activated IGF-IR/MAPKp44/42 signaling nongenomically and induced phosphorylation of ERα at ser118 in cells with a functional IGF-IR versus those without. E(2) also upregulated IGF-I gene and protein expression through a prolonged genomic event. These results suggest a pivotal role of IGF-IR and possibly other RTKs in mediating genomic and nongenomic hormone receptor interactions and signaling in fibroids and provide novel genes and targets for future intervention and prevention strategies.

Effects of hormonally active agents on steroid hormone receptor expression and cell proliferation in the myometrium of ovariectomized macaques.

Hormone replacement therapy and selective estrogen receptor modulators have been controversial treatment options for postmenopausal women because of their potential health benefits and/or risks. In this study, we determine the effects of the hormonally active compounds, conjugated equine estrogens (CEE), medroxyprogesterone acetate (MPA), CEE + MPA, and tamoxifen (TAM) on the myometrium of ovariectomized macaques. Immunoexpression of estrogen receptor-α (ERα), progesterone receptor (PR), and Ki-67 in the myometrium is assessed. We found no significant difference in ERα myometrial expression in the CEE, MPA, and CEE + MPA treatment groups, but there was a significant decrease in expression in animals administered TAM versus controls. Conjugated equine estrogen-, TAM-, and CEE + MPA-treated animals had significantly increased expression of PR in myometrial cells and there was no difference in PR expression in cells from MPA-treated animals versus control animals. Myometrial cell proliferation did not significantly differ between the controls and any of the treatment groups, although normalized Ki-67 values were somewhat higher in the CEE and TAM groups. These data suggest that ERα and PR expression in the myometrium is influenced by treatment with hormonally active agents.

CD34 expression by hair follicle stem cells is required for skin tumor development in mice.

The cell surface marker CD34 marks mouse hair follicle bulge cells, which have attributes of stem cells, including quiescence and multipotency. Using a CD34 knockout (KO) mouse, we tested the hypothesis that CD34 may participate in tumor development in mice because hair follicle stem cells are thought to be a major target of carcinogens in the two-stage model of mouse skin carcinogenesis. Following initiation with 200 nmol 7,12-dimethylbenz(a)anthracene (DMBA), mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 20 weeks. Under these conditions, CD34KO mice failed to develop papillomas. Increasing the initiating dose of DMBA to 400 nmol resulted in tumor development in the CD34KO mice, albeit with an increased latency and lower tumor yield compared with the wild-type (WT) strain. DNA adduct analysis of keratinocytes from DMBA-initiated CD34KO mice revealed that DMBA was metabolically activated into carcinogenic diol epoxides at both 200 and 400 nmol. Chronic exposure to TPA revealed that CD34KO skin developed and sustained epidermal hyperplasia. However, CD34KO hair follicles typically remained in telogen rather than transitioning into anagen growth, confirmed by retention of bromodeoxyuridine-labeled bulge stem cells within the hair follicle. Unique localization of the hair follicle progenitor cell marker MTS24 was found in interfollicular basal cells in TPA-treated WT mice, whereas staining remained restricted to the hair follicles of CD34KO mice, suggesting that progenitor cells migrate into epidermis differently between strains. These data show that CD34 is required for TPA-induced hair follicle stem cell activation and tumor formation in mice.

Gene expression analysis offers unique advantages to histopathology in liver biopsy evaluations.

Liver diseases that induce nonuniform lesions often give rise to greatly varying histopathology results in needle biopsy samples from the same patient. This study examines whether gene expression analysis of such biopsies could provide a more representative picture of the overall condition of the liver. We utilized acetaminophen (APAP) as a model hepatotoxicant that gives a multifocal pattern of necrosis following toxic doses. Rats were treated with a single toxic or subtoxic dose of APAP and sacrificed 6, 24, or 48 hours after exposure. Left liver lobes were harvested, and both gene expression and histopathological analysis were performed on biopsy-sized samples. While histopathological evaluation of such small samples revealed significant sample to sample differences after toxic doses of APAP, gene expression analysis provided a very homogeneous picture and allowed clear distinction between subtoxic and toxic doses. The main biological function differentiating animals that received sub-toxic from those that had received toxic doses was an acute stress response at 6 hours and signs of energy depletion at later time points. Our results suggest that the use of genomic analysis of biopsy samples together with histopathological analysis could provide a more precise representation of the overall condition of a patient's liver than histopathological evaluation alone.

Early effects of iodine on DNA synthesis in sulfur mustard-induced skin lesions.

Sulfur mustard (SM) is powerful alkylator and highly cytotoxic blisterogen in both humans and animals. This study in male guinea pigs shows that, at an early stage (5 h) after SM exposure, a marked increase occurred in epithelial nuclear vacuolation, epidermal thickening, and dermal acute inflammation. Topical iodine treatment reduced the severity of these parameters. The rate of DNA synthesis expressed by incorporation of bromodeoxyuridine was reduced upon topical treatment with iodine only or SM only by 46 and 72%, respectively. Iodine treatment following SM exposure exerted an effect similar to that of SM only, indicating that DNA synthesis is not directly involved in the mechanism of action of iodine-induced protection.

Chemical-induced atrial thrombosis in NTP rodent studies.

Cardiac thrombosis, one of the causes of sudden death throughout the world, plays a principal role in several cardiovascular diseases, such as myocardial infarction and stroke in humans. Data from studies of induction of chemical thrombosis in rodents help to identify substances in our environment that may contribute to cardiac thrombosis. Results for more than 500 chemicals tested in rodents in 2-year bioassays have been published as Technical Reports of the National Toxicology Program (NTP) http://ntp-server.niehs.nih.gov/index. We evaluated atrial thrombosis induced by these chemical exposures and compared it to similarly induced lesions reported in the literature. Spontaneous rates of cardiac thrombosis were determined for control Fischer 344 rats and B6C3F1 mice: 0% in rats and mice in 90-day studies and, in 2-year studies, 0.7% in both genders of mice, 4% in male rats, and 1% in female rats. Incidences of atrial thrombosis were increased in high-dosed groups involving 13 compounds (incidence rate: 20-100%): 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3'-dimethoxybenzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4'-thiobis(6-t-butyl-m-cresol). The main localization of spontaneously occurring and chemically induced thromboses occurred in the left atrium. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis, hypercoagulability, and impaired atrial mechanical activity, such as atrial fibrillation, which could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis. Supplementary data referenced in this paper are not printed in this issue of Toxicologic Pathology. They are available as downloadable files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 33(5), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.