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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.

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-receptor tyrosine kinases (RTKs)] in uteri of rats following early-life exposures. Pregnant Wistar Han rats were exposed to TBBPA (0, 0.1, 25, and 250 mg/kg bw/day) via oral gavage on gestation day 6 through weaning of pups on postnatal day (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, and PND 90) and formalin-fixed or frozen for histologic, immunohistochemical, phospho-RTK arrays, or western blot analysis. At PND 21, the phospho-RTKs, fibroblast growth factor receptor 2 and 3 (FGFR2 and FGFR3), neurotrophic tyrosine kinase receptor type 3 (TRKC), and EPH receptor A1 (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), FGFR2, FGFR3, FGFR4, insulin-like growth factor receptor 1 (IGF1R), insulin receptor (INSR), AXL receptor tyrosine kinase (AXL), MER proto-oncogene, tyrosine kinase (MERTK), platelet derived growth factor receptor alpha and beta (PDGFRA and PDGFRB), ret proto-oncogene (RET), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 and 2 (TIE1 and TIE2), TRKA, kinase insert domain receptor (KDR;VEGFR2), fms related receptor tyrosine kinase (FLT4; VEGFR3), and EPHA1 at different treatment concentrations. EGFR, a 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, TYRO3 protien tyrosine kinase (TYRO3; DTK), HGFR, TRKC, FLT1/VEGFR1, and EPHB2 and 4 were also statistically significant at PND 90 at different treatment doses. 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.

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

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. Electronic supplementary material The online version of this article (doi:10.1186/s12964-016-0141-2) contains supplementary material, which is available to authorized users.

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.

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.

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.

The S-phase hepatocyte labeling index (LI) in rodents has been routinely evaluated by using the labeling agent 5-bromo-2' -deoxyuridine (BrdU) administered by surgically implanted osmotic minipumps or intraperitoneal injections. In this study, BrdU was administered to untreated B6C3F1 mice and F344 rats in drinking water (40, 60, and 80 mg/100 mL) or by osmotic minipumps (30 mg/mL) for 24, 48, or 72 h. In addition, one group of rats, of which half were singly housed, and one group of mice were treated with the hepatotoxic agent furan or corn oil vehicle while exposed for 7 days to BrdU in drinking water (80 mg/100 mL) or by osmotic minipumps. There was an additive increase in the LI of the minipump-dosed mice, but not in the rats, from 24 to 72 h or in either species dosed in drinking water. A significant elevation in the LI in response to furan treatment was detected in mice regardless of the route of BrdU administration. In furan-treated rats, the minipump-dosed group and the singly housed, but not the ...

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

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.

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. [Cancer Res 2007;67(9):4173–81]

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 .

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.

These studies define the site and mechanisms of reproductive toxicity of ethylene glycol monomethyl ether (EGME) in a nongravid female animal model using in vivo and in vitro methods. In vivo studies assessed vaginal cytology and histology, ovarian histology, and serum hormones in 80- to 90-day-old, adult, regularly cycling, female Sprague-Dawley rats treated daily with EGME or vehicle by oral gavage. Dose-response and time-course studies (four to nine rats per group per treatment) determined that 300 mg/kg EGME suppressed cyclicity without systemic toxicity within 3 to 8 days, and doses less than 100 mg/kg had no effect. Pathogenesis studies (six to nine rats per time and treatment) determined that 300 mg/kg EGME elevated serum progesterone within 32 hr after dosing, while serum estradiol, FSH, LH, and prolactin remained at baseline levels. In EGME-treated rats, cyclicity was suppressed, ovulation was inhibited, and corpora lutea were hypertrophied. Thus, EGME appeared to target the ovarian luteal cell. To further examine the toxicity in vitro, luteal cells were recovered from 23-day-old, hCG-primed Sprague-Dawley rats and treated with 0-10 mM methoxy acetic acid (MAA), the proximate toxic metabolite of EGME. MAA (1-10 mM) maintained elevated progesterone levels as production declined in untreated cells at 24 and 48 hr of culture. Progesterone production was maintained independent of LH-stimulated cAMP levels. MAA decreased ATP, but only at 48 hr and at 2.5 mM or greater concentrations. Thus, these studies establish that the ovarian luteal cell is a target of EGME and MAA in vivo and in vitro and that the effect on luteal cell progesterone production is likely independent of LH-stimulated cAMP pathways.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a multisite carcinogen. Although the hepatocarcinogenic actions of TCDD have received the most attention, it has been demonstrated in several rodent carcinogenicity bioassays that TCDD causes a dose-related increase in thyroid follicular cell adenomas and carcinomas. The purpose of the present experiment was to investigate the dose-response relationship for thyroid function alterations in female Sprague-Dawley rats following chronic treatment with TCDD. TCDD was administered via oral gavage biweekly for 30 weeks at average daily equivalent doses of 0.1-125 ng/kg/day, thereby more than encompassing the dose range historically used in previous TCDD rodent bioassays. The endpoints examined include serum levels of thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH). In addition, the induction of the dioxin-responsive genes UDP-glucuronosyltransferase-1 (UGT1) and cytochrome P450 1A1 (CYP1A1) in liver were measured using reverse-transcriptase-polymerase chain reaction (RT-PCR). In agreement with previous hypotheses, TCDD appears to alter thyroid function via a secondary mechanism, namely increased excretion of T4-glucuronide resulting from TCDD induction of UGT1. The observed follicular cell hyperplasia and hypertrophy are consistent with the observed elevated TSH levels and may represent the early stages in the progression of thyroid carcinogenesis. Therefore, TCDD induces alterations in thyroid hormone function, probably as a result of chronic perturbations of liver-pituitary-thyroid axis.