Angela R. Buckalew

Methoxychlor-induced alterations in the histological expression of angiogenic factors in pituitary and uterus ∗

Within the reproductive system, oestrogenic stimulation of uterine and pituitary tissue typically causes a proliferative response accompanied by an angiogenic induction of new blood vessels from existing ones, thereby providing nutrients and oxygen to the growing tissue. The pro-oestrogenic pesticide methoxychlor (MXC), however, has shown a differential effect on proliferative activity. An increase in uterine growth is present, while the pituitary undergoes a decrease in size, even though the effect is accompanied by a characteristic oestrogen-induced elevation in pituitary prolactin concentration. The focus of the current study was whether the observed differences in tissue growth between uterus and pituitary in response to MXC administration were paralleled by a corresponding disparity in the expression of those growth factors (members of the vascular endothelial growth factor (VEGF) and angiopoietin families and their receptors) that are involved in the angiogenic cascade. Ovariectomized adult Sprague–Dawley female rats were administered MXC (0–200 mg/kg, oral) for 1 or 3 weeks. Immunohistochemical staining of uteri and pituitaries was performed under strictly controlled conditions for VEGF and its receptor VEGFR2, Angiopoietin-1 (Ang1) and angiopoietin-2 and their tyrosine kinase receptor Tie2, and platelet endothelial adhesion factor (as an index of vascularity). Image acquisition and densitometric assessments of staining intensity were conducted under blind conditions. The results showed uterine MXC-induced increases in the expression of VEGFR2 and Ang1, changes consistent with a normal proliferative response to oestrogenic stimulation. For VEGF, staining tended to be most pronounced in the stromal region, although there did not appear to be a progressive increase with dose. VEGFR2 expression showed significant dose-related trends in luminal and glandular epithelia by 1 week. Similar effects at 1 week were evident for Ang1 in glandular epithelium. In the anterior pituitary, a dose-related increase in VEGF was present for the 1 and 3 week treatments, and the number of pituitary vessels per unit area was also increased after 3 weeks. The effects indicate that even though the insecticide has not been found to cause an augmentation in pituitary growth, a dose-related rise in the expression of at least one principal angiogenic factor is present that may be associated with an increase in vascular density.

The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

Background, goals, and scopeIn response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.MethodsA previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and “negative” chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.ResultsWith a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.Discussion and conclusionsWith one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).PerspectivesBased on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.

Understanding the effects of atrazine on steroidogenesis in rat granulosa and H295R adrenal cortical carcinoma cells

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) was introduced in the 1950s as a broad spectrum herbicide, and remains one of the most widely used herbicides in the United States. Several studies have suggested that atrazine modifies steroidogenesis and may disrupt reproductive function and development in a variety of species. A primary concern has been whether atrazine increases the synthesis of estrogens, perhaps by enhancing aromatase gene expression and activity. In this study, the effect of atrazine was compared in cultures using primary granulosa cells and H295R adrenal cortical carcinoma cells. Atrazine (10 μM), but not its metabolite, 2-chloro-4,6-diamino-1,2,5-triazine (DACT), significantly increased estradiol production and aromatase activity in granulosa cell cultures only when measured for 1-h following 24h of exposure. In H295R cells, atrazine (10 μM) increased estradiol and estrone production. Importantly, atrazine (10 μM) increased progesterone production from both cell types suggesting a broader effect of atrazine on steroidogenesis.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on fetal mouse urinary tract epithelium in vitro.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the fetal C57BL/6N mouse urinary tract. This study tests the hypothesis that the late fetal urinary tract epithelial cells respond to TCDD with increased proliferation and that the responses do not require contributions from other maternal or fetal tissues. This was achieved by exposing late gestation fetal urinary tract cells to TCDD in an in vitro model. Isolated ureteric cells from gestation day (GD) 18 fetal ureters were plated in medium supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. Both epithelial and mesenchymal cells remain viable under these conditions. The cultures were exposed to 0.1% dimethylsulfoxide (DMSO), 1x10(-8), 1x10(-9) or 1x10(-10) M TCDD. Exposure to 1x10(-10) M TCDD did not affect the cultures, while 1x10(-8) and 1x10(-9) M TCDD supported epithelial, but not mesenchymal, cell survival and stimulated epithelial cell proliferation and differentiation. The TCDD-exposed cells expressed high levels of keratin and little or no vimentin, confirming that the cells, which survive and differentiate are epithelial. However, after continuous exposure to epidermal growth factor (EGF), the TCDD-induced stimulation of ureteric epithelial growth could not be detected. In conclusion, this study demonstrates that late gestational ureteric cells respond to TCDD in vitro with the stimulation of epithelial cell growth and differentiation.

Development of a screening approach to detect thyroid disrupting chemicals that inhibit the human sodium iodide symporter (NIS)

The U.S. EPAs Endocrine Disruptor Screening Program aims to use high-throughput assays and computational toxicology models to screen and prioritize chemicals that may disrupt the thyroid signaling pathway. Thyroid hormone biosynthesis requires active iodide uptake mediated by the sodium/iodide symporter (NIS). Monovalent anions, such as the environmental contaminant perchlorate, are competitive inhibitors of NIS, yet limited information exists for more structurally diverse chemicals. A novel cell line expressing human NIS, hNIS-HEK293T-EPA, was used in a radioactive iodide uptake (RAIU) assay to identify inhibitors of NIS-mediated iodide uptake. The RAIU assay was optimized and performance evaluated with 12 reference chemicals comprising known NIS inhibitors and inactive compounds. An additional 39 chemicals including environmental contaminants were evaluated, with 28 inhibiting RAIU over 20% of that observed for solvent controls. Cell viability assays were performed to assess any confounding effects of cytotoxicity. RAIU and cytotoxic responses were used to calculate selectivity scores to group chemicals based on their potential to affect NIS. RAIU IC50 values were also determined for chemicals that displayed concentration-dependent inhibition of RAIU (≥50%) without cytotoxicity. Strong assay performance and highly reproducible results support the utilization of this approach to screen large chemical libraries for inhibitors of NIS-mediated iodide uptake.

Evaluation of triclosan in the Hershberger and H295R steroidogenesis assays

Triclosan (TCS) is an antibacterial widely used in personal care products that exhibits endocrine disrupting activity in several species, with reports of altered thyroid, estrogen and androgen signaling pathways. To evaluate the androgenic mode of action, TCS was evaluated for androgen receptor mediated effects in the Hershberger assay and for altered androgen synthesis in the H295R steroidogenesis assay. In the Hershberger assay, castrated males were dosed by oral gavage for 10 days with corn oil (vehicle) or TCS (50 or 200 mg/kg/day) in the presence or absence of testosterone proprionate (TP, 0.2 mg/kg/day) prior to assessing accessory sex tissues (ASTs) weights. TCS alone or in combination with TP did not alter androgen dependent AST weights. Assessment of serum thyroxine (T4) demonstrated a significant dose-dependent decrease by TCS (50 or 200 mg/kg/day) co-administered with TP and TCS (200 mg/kg) without TP, but no differences in liver or thyroid weights. In the H295R assay, TCS from 0.01 to 10 μM had no effect on testosterone production but TCS at 3 μM and above did induce a significant increase in estrogen production. At 10 μM, TCS produced significant cytotoxicity which confounded the interpretation of the estrogenic effect at that concentration. Thus, TCS had no effect on androgen synthesis or activity in the models used, but did enhance estrogen production and suppress serum T4.

High-Throughput Screening and Quantitative Chemical Ranking for Sodium-Iodide Symporter Inhibitors in ToxCast Phase I Chemical Library

Thyroid uptake of iodide via the sodium-iodide symporter (NIS) is the first step in the biosynthesis of thyroid hormones that are critical for health and development in humans and wildlife. Despite having long been a known target of endocrine disrupting chemicals such as perchlorate, information regarding NIS inhibition activity is still unavailable for the vast majority of environmental chemicals. This study applied a previously validated high-throughput approach to screen for NIS inhibitors in the ToxCast phase I library, representing 293 important environmental chemicals. Here 310 blinded samples were screened in a tiered-approach using an initial single-concentration (100 μM) radioactive-iodide uptake (RAIU) assay, followed by 169 samples further evaluated in multi-concentration (0.001 μM-100 μM) testing in parallel RAIU and cell viability assays. A novel chemical ranking system that incorporates multi-concentration RAIU and cytotoxicity responses was also developed as a standardized method for chemical prioritization in current and future screenings. Representative chemical responses and thyroid effects of high-ranking chemicals are further discussed. This study significantly expands current knowledge of NIS inhibition potential in environmental chemicals and provides critical support to U.S. EPAs Endocrine Disruptor Screening Program (EDSP) initiative to expand coverage of thyroid molecular targets, as well as the development of thyroid adverse outcome pathways (AOPs).