Ashley S. Murr

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 effects of prenatal exposure to atrazine on pubertal and postnatal reproductive indices in the female rat.

Atrazine (ATR) is an herbicide that exerts negative reproductive effects. We examined the effects of vehicle or ATR (1, 5, 20 and 100mg/kg-d), administered to Sprague-Dawley rats on gestational days 14-21, once daily or divided into two doses per day, on female offspring reproductive indices. Offspring body weights at birth were reduced and mortality increased in the 100mg/kg-d group shortly after birth; by PND 21 there were no significant effects. Vaginal opening was delayed in this group, indicating delayed puberty. No significant differences in mammary gland development were apparent at PND 45, or estrous cyclicity through PND 272. There were no differences between dosing regimens. Lower ATR doses (0-20mg/kg-d) showed few effects in females prenatally exposed to ATR, while the high dose (100mg/kg-d) reduced offspring body weight and delayed vaginal opening. Nonetheless, it is unlikely that environmental exposure comparable to the high dose would be encountered.

Dibromoacetic acid-induced elevations in circulating estradiol: effects in both cycling and ovariectomized/steroid-primed female rats.

Oral exposures to high concentrations of the drinking water disinfection by-product dibromoacetic acid (DBA) over the course of 14 days have been found to disrupt estrous cyclicity in the female rat. In order to investigate possible alterations in the relevant hormonal regulatory mechanisms, female Sprague-Dawley rats were gavaged for 2 weeks with 270 mg/kg DBA, ovariectomized (OVX) and implanted with estradiol capsules. For these females, the induced luteinizing hormone (LH) surge in these animals showed a borderline suppression in peak LH concentrations that was accompanied by a marked increase in circulating estradiol. This elevation in estradiol was DBA dose-related and, for intact, normally cycling females receiving lower doses of DBA (60 and 120 mg/kg, 14 days), was present on the day of estrus, at a time when a dramatic fall from proestrous concentrations is normally evident. Evaluations of liver microsomal cytochrome p450 activity in OVX/estradiol-implanted rats showed a suppression in ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD) activity (indications of the activity of CYP1A and 2B, respectively-two key enzymes in estradiol oxidative metabolism). Phenobarbital (PhB) exposure in these animals did show induction of this activity, but was unable to lower E2 concentrations. This suggests that a DBA-induced suppression in estradiol catabolism is present and may either involve a targeted effect on the estrogen binding site on the CYP2B1/2 and CYP1A genes apart from the PhB-responsive unit, or a second pathway (possibly sulfation) that is not PhB-inducible.

Alterations in ovarian follicular progesterone secretion by elevated exposures to the drinking water disinfection by-product dibromoacetic acid: examination of the potential site(s) of impact along the steroidogenic pathway.

Previous data from our laboratory indicated that the drinking water disinfection by-product, dibromoacetic acid (DBA), when applied in vitro to rat preovulatory follicles at a concentration consistent with blood levels found to disrupt estrous cyclicity, was able to block the stimulated secretion of progesterone. The present experiments focused on establishing a dose-response for such an effect and identifying the point(s)of impact of this compound along the steroidogenic pathway that underlie this suppression. Immature Sprague-Dawley rats were primed with PMSG on day 26 and killed 48 h later. Preovulatory follicles were removed and paired in culture with or without DBA (2-50 microg/ml) to reassess progesterone secretion under hCG-stimulated or baseline conditions. In addition, media supplemented with pregnenolone or 22(R)-hydroxycholesterol (22R-HC) were used to determine the effects of 50 microg/ml DBA on the initial steps leading to progesterone synthesis. Samples taken over the course of 24 h reaffirmed a significant DBA-associated suppression in baseline and stimulated progesterone release, while estradiol secretion was unaffected. This effect was mirrored by a reduction in follicular progesterone content in these DBA groups. The addition of pregnenolone eliminated this decrease, with the DBA-exposed follicles exhibiting a linear increase in progesterone release over the sampling period. The follicular progesterone content at 24 h showed that DBA treatment under pregnenolone supplementation caused marked elevations under both the hCG stimulated and non-stimulated conditions, something not reflected in the release data. Substitution of 22R-HC for pregnenolone eliminated the effect on baseline progesterone release, although the attenuation in stimulated secretion was still present. This suggests both an effect of DBA exposure on mitochondrial cholesterol transport by steroidogenic acute regulatory protein (StAR) and a possible impact on the receptor or postreceptor events triggered by hCG.

Reproductive toxicity of a mixture of regulated drinking-water disinfection by-products in a multigenerational rat bioassay.

Background Trihalomethanes (THMs) and haloacetic acids (HAAs) are regulated disinfection by-products (DBPs); their joint reproductive toxicity in drinking water is unknown. Objective We aimed to evaluate a drinking water mixture of the four regulated THMs and five regulated HAAs in a multigenerational reproductive toxicity bioassay. Methods Sprague-Dawley rats were exposed (parental, F1, and F2 generations) from gestation day 0 of the parental generation to postnatal day (PND) 6 of the F2 generation to a realistically proportioned mixture of THMs and HAAs at 0, 500×, 1,000×, or 2,000× of the U.S. Environmental Protection Agency’s maximum contaminant levels (MCLs). Results Maternal water consumption was reduced at ≥ 1,000×; body weights were reduced at 2,000×. Prenatal and postnatal survival were unaffected. F1 pup weights were unaffected at birth but reduced at 2,000× on PND6 and at ≥ 1,000× on PND21. Postweaning F1 body weights were reduced at 2,000×, and water consumption was reduced at ≥ 500×. Males at 2,000× had a small but significantly increased incidence of retained nipples and compromised sperm motility. Onset of puberty was delayed at 1,000× and 2,000×. F1 estrous cycles and fertility were unaffected, and F2 litters showed no effects on pup weight or survival. Histologically, P0 (parental) dams had nephropathy and adrenal cortical pathology at 2,000×. Conclusions A mixture of regulated DBPs at up to 2,000× the MCLs had no adverse effects on fertility, pregnancy maintenance, prenatal survival, postnatal survival, or birth weights. Delayed puberty at ≥ 1,000× may have been secondary to reduced water consumption. Male nipple retention and compromised sperm motility at 2,000× may have been secondary to reduced body weights. Citation Narotsky MG, Klinefelter GR, Goldman JM, DeAngelo AB, Best DS, McDonald A, Strader LF, Murr AS, Suarez JD, George MH, Hunter ES III, Simmons JE. 2015. Reproductive toxicity of a mixture of regulated drinking-water disinfection by-products in a multigenerational rat bioassay. Environ Health Perspect 123:564–570; http://dx.doi.org/10.1289/ehp.1408579

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.

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).

Regional differences in the pituitary distribution of luteinizing hormone in the gonadectomized and proestrous female rat

Previous data have shown that regional differences in the presence of anterior pituitary luteinizing hormone (LH) generally correlate with the comparable disparities in distribution of gonadotropes throughout the gland. In female rats, the differences are apparent over the estrous cycle, but are more prominent during the hours preceding the proestrus surge of LH. The current experiments examined (1) if such regional disparities are present throughout the surge window, (2) if differences are mirrored by release of LH in vitro and (3) if the appearance of regional differences is altered in ovariectomized females. Results showed that a comparative elevation in the rostral portion of the pituitary during the pre-surge period diminishes and finally disappears concurrent with the rise in circulating LH. This increase in rostral LH concentrations is reflected in this region by a comparable effect in vitro on stimulated LH secretion from pituitary fragments, although the effect is somewhat diminished by referencing release against tissue concentrations of LH present in a contralateral rostral fragment. Ovariectomies conducted at 1500h on proestrus, at a time when a significant regional difference has faded, resulted in a prompt increase in LH across all areas of the pituitary, and the emergence of a marked augmentation in rostral concentrations over the ensuing 72h. The effect was not seen when ovariectomies were performed on estrus. These data show that, while a regional disparity in anterior pituitary LH is present as circulating concentrations of estradiol rise prior to the LH surge, the removal of this steroid feedback at a time when LH synthesis is normally amplified accentuates the difference between the rostral region and other areas of the pituitary.