Norris D. Flagler

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.

Spontaneous and Chemically Induced Proliferative Lesions in Tg.AC Transgenic and p53-Heterozygous Mice

Recently, the use of selected genetically altered mouse models in the detection of carcinogens after short-term chemical exposures has been evaluated. Studies of several chemicals conducted by the National Toxicology Program in Tg.AC transgenic and heterozygous p53-deficient mice have been completed recently and represent a major contribution to this effort, as well as the largest accumulation to date of toxicologic pathology data in these 2 lines of mice. The purpose of this report is to describe the proliferative target organ effects observed in this set of studies, as well as to present the tumor profile in the control groups of this data set. These findings provide a comprehensive toxicologic assessment of these 2 genetically altered mouse strains, which are of emerging importance in toxicologic pathology.

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 17β-estradiol (E2) on IGF-IR/MAPKp44/42 signaling and gene expression in human UtLM cells with intact or silenced IGF-IR. Analysis by RT2 Profiler PCR-array showed genes involved in IGF-IR/MAPK signaling were upregulated in UtLM cells by E2 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. E2 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. E2 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.

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.

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.

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. Citation: Hewitt SC, Winuthayanon W, Pockette B, Kerns RT, Foley JF, Flagler N, Ney E, Suksamrarn A, Piyachaturawat P, Bushel PR, Korach KS. 2015. Development of phenotypic and transcriptional biomarkers to evaluate relative activity of potentially estrogenic chemicals in ovariectomized mice. Environ Health Perspect 123:344–352; http://dx.doi.org/10.1289/ehp.1307935

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.

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.

Chemical-Induced Atrial Thrombosis in NTP Rodent Studies—Supplementary Data:

Figure 1 presents the chemical names and structures of these 13 compounds. We summarize below the information concerning usages and effects in humans of these chemicals. Table 1 shows data from rats indicating the relationship between the severity of myocardial lesions and atrial thrombosis which were induced by bis(2-chloroethoxy)methane. Bis(2-chloroethoxy)methane (CEM): The majority is used for the production of polysulfide polymers, which are used in a variety of sealant applications because of their resistance to high temperatures and solvent degradation. No specific data for human toxicity are available, except those implicating it as an irritant of the skin and eye (NTP, 2003a, 2003b). 2-Butoxyethanol (2-BE): This substance is used extensively as a solvent in surface coatings, such as lacquers, enamels, varnishes, and latex paint; paint thinners and paint stripping formulations; inks; and degreasers and industrial and household cleaners. In humans, the chemical can induce hemolytic anemia and hemoglobinurea (ATSDR, 1998; Burkhart and Donovan, 1998; Gualtieri et al., 2003; NTP, 1998a). Only a few human cases involving hypotension, ventricular tachycardia, and/or arrhythmia exhibited cardiovascular effects (ATSDR, 1998; Gualtieri et al., 2003). C.I. Acid Red 114 (Red 114): This chemical is a benzidine congener-based dye, used to color textiles, paper, plastic, rubber, and leather. No epidemiological studies or reports of human health effects related to exposure to this chemical were found in the literature (NTP, 1991). C.I. Direct Blue 15 (Blue 15): A benzidine congener-based dye, this chemical is used as a dye to color textiles, paper, plastic, rubber, and leather. No epidemiological studies or reports of adverse health effects in humans related to exposure to this chemical were discovered in the literature (NTP, 1992a). C. I. Pigment Red 23 (Red 23): This chemical, a bluish-red dye used as a coloring agent in paints, inks, rubber, plastics, lacquers, and papers, is considered a genotoxic compound (Moller, 2000). No epidemiological studies or reports of human health effects related to exposure were found in the literature (NTP, 1992b). Diazoaminobenzene (DAB): Used as an intermediate, complexing agent and polymer additive, this chemical also occurs as an impurity in certain color additives used in cosmetics, food products, and pharmaceuticals. No information related to the toxicity of this compound in humans was found in a review of the available literature (NTP, 2002b). Diethanolamine (DEL): This compound is widely used in the preparation of diethanolamides and diethanolamine salts that are formulated into soaps and surfactants used in liquid laundry and dishwashing detergents, cosmetics, shampoos, and hair conditioners. No references to human side effects caused by this chemical were found in a review of the literature (NTP, 1992c, 1999). 3,3′-Dimethoxybenzidine dihydrochloride: Used principally as an intermediate in the production of commercial bisazobiphenyl dyes, this compound is used to color textiles, paper, plastic, rubber, and leather. No epidemiological studies or reports of effects, especially hematological, upon human health related to exposure were discovered in the literature (NTP, 1990). Hexachloroethane (HCE): This chemical is used in organic synthesis as a retarding agent in fermentation and camphor substitute in nitrocellulose, in pyrotechnics and smoke devices, in explosives, and as a solvent. No epidemiological studies or reports of human health effects, including cardiovascular, related to exposure were found in the literature (ATSDR, 1997; NTP, 1989, 2002a). Isobutene (IBT): This compound, which has been detected in the urban atmosphere, is used primarily to produce diisobutylene, trimers of butyl rubber, and other polymers. During halothane anesthesia, a 10-fold increase over normal occurs in the concentration of isobutene in the breath of patients; the source was speculated to be terpenes or ubiquinones (Hempel et al., 1980). No epidemiological studies or reports of human health effects related to exposure were discovered in the literature (NTP, 1998b). Methyleugenol (MEG): The chemical acts as a flavoring agent in jellies, baked goods, nonalcoholic beverages, chewing gum, candy, pudding, relish, and ice cream and is also

The Life Cycle of the Uterine Fibroid Myocyte

Purpose of ReviewUterine 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 FindingsThe 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.SummaryUterine 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.