An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats.

Identifying xenobiotics that interrupt the thyroid axis has significant public health implications, given that thyroid hormones are required for brain development. As such, some developmental and reproductive toxicology (DART) studies now require or recommend serum total thyroxine (T4) measurements in pregnant, lactating, and developing rats. However, serum T4 concentrations are normally low in the fetus and pup which makes quantification difficult. These challenges can be circumvented by technologies like mass spectrometry, but these approaches are expensive and not always widely available. To demonstrate the feasibility of measuring T4 using a commercially available assay, we examine technical replicates of rat serum samples measured both by liquid chromatography mass spectrometry (LC/MS/MS) and radioimmunoassay (RIA). These samples were obtained from rats on gestational day 20 (dams and fetuses) or postnatal day 5 (pups), following maternal exposure to the goitrogen propylthiouracil (0-3 ppm) to incrementally decrease T4. We show that with assay modification, it is possible to measure serum T4 using low sample volumes (25-50 µL) by an RIA, including in the GD20 fetus exposed to propylthiouracil. This proof-of-concept study demonstrates the technical feasibility of measuring serum T4 in DART studies.

High-Throughput Screening of ToxCast PFAS Chemical Library for Potential Inhibitors of the Human Sodium Iodide Symporter.

Over the past decade, there has been increased concern for environmental chemicals that can target various sites within the hypothalamic-pituitary-thyroid axis to potentially disrupt thyroid synthesis, transport, metabolism, and/or function. One well-known thyroid target in both humans and wildlife is the sodium iodide symporter (NIS) that regulates iodide uptake into the thyroid gland, the first step of thyroid hormone synthesis. Our laboratory previously developed and validated a radioactive iodide uptake (RAIU) high-throughput assay in a stably transduced human NIS cell line (hNIS-HEK293T-EPA) to identify chemicals with potential for NIS inhibition. So far, we have tested over 2000 chemicals (US EPA's ToxCast chemical libraries PI_v2, PII, and e1K) and discovered a subset of chemicals that significantly inhibit iodide uptake in the hNIS assay. Here, we utilized this screening assay to test a set of 149 unique per- and polyfluoroalkyl substances (PFAS) (ToxCast PFAS library) for potential NIS inhibition. For this evaluation, the 149 blinded samples were screened in a tiered approach, first in an initial single-concentration (≤100 µM) RAIU assay and subsequent evaluation of the chemicals that produced ≥20% inhibition using multiconcentration (MC) response (0.001-100 µM) testing in parallel RAIU and cell viability assays. Of this set, 38 of the PFAS chemicals inhibited iodide uptake ≥20% in the MC testing with 25 displaying inhibition ≥50%. To prioritize the most potent PFAS NIS inhibitors in this set, chemicals were ranked based on outcomes of both iodide uptake and cytotoxicity and normalized to perchlorate, a known positive control. Consistent with previous findings, PFOS and PFHxS were again found to be potent NIS inhibitors, yet significant inhibition was also observed for several other screened PFAS chemicals. Although further studies are clearly warranted, this initial screening effort identifies NIS as a molecular target for potential thyroid disruption by this persistent and structurally diverse class of chemicals.

Expanded high-throughput screening and chemotype-enrichment analysis of the phase II: e1k ToxCast library for human sodium-iodide symporter (NIS) inhibition.

The sodium-iodide symporter (NIS) mediates the uptake of iodide into the thyroid. Inhibition of NIS function by xenobiotics has been demonstrated to suppress circulating thyroid hormones and perturb related physiological functions. Until recently, few environmental chemicals had been screened for NIS inhibition activity. We previously screened over 1000 chemicals from the ToxCast Phase II (ph1v2 and ph2) libraries using an in vitro radioactive iodide uptake (RAIU) with the hNIS-HEK293T cell line to identify NIS inhibitors. Here, we broaden the chemical space by expanding screening to include the ToxCast e1k library (804 unique chemicals) with initial screening for RAIU at 1 × 10-4 M. Then 209 chemicals demonstrating > 20% RAIU inhibition were further tested in multiple-concentration, parallel RAIU and cell viability assays. This identified 55 chemicals as active, noncytotoxic RAIU inhibitors. Further cytotoxicity-adjusted potency scoring (with NaClO4 having a reference score of 200) revealed five chemicals with moderate to strong RAIU inhibition (scored > 100). These data were combined with our previous PhII screening data to produce binary hit-calls for ~ 1800 unique chemicals (PhII + e1k) with and without cytotoxicity filtering. Results were analyzed with a ToxPrint chemotype-enrichment workflow to identify substructural features significantly enriched in the NIS inhibition hit-call space. We assessed the applicability of enriched PhII chemotypes to prospectively predict NIS inhibition in the e1k dataset. Chemotype enrichments derived for the combined ~ 1800 dataset also identified additional enriched features, as well as chemotypes affiliated with cytotoxicity. These enriched chemotypes provide important new information that can support future data interpretation, structure-activity relationship, chemical use, and regulation.

Evaluation of potential sodium-iodide symporter (NIS) inhibitors using a secondary Fischer rat thyroid follicular cell (FRTL-5) radioactive iodide uptake (RAIU) assay.

The Fischer rat thyroid follicular cell line (FRTL-5) endogenously expresses the sodium-iodide symporter (NIS) and has been used to identify environmental chemicals that perturb thyroid hormone homeostasis by disruption of NIS-mediated iodide uptake. Previously, a high-throughput radioactive iodide uptake (RAIU) screening assay incorporating the hNIS-HEK293T-EPA cell line was used to identify potential human NIS (hNIS) inhibitors in 1028 ToxCast Phase I (ph1_v2) and Phase II chemicals. In this study, the FRTL-5 cell line was evaluated and applied as a secondary RAIU assay coupled with cell viability assays to further prioritize highly active NIS inhibitors from the earlier screening. Assay validation with ten reference chemicals and performance assessment by chemical controls suggest the FRTL-5 based assays are robust and highly reproducible. Top-ranked chemicals from the ToxCast screening were then evaluated in both FRTL-5 and hNIS RAIU assays using newly sourced chemicals to strengthen the testing paradigm and to enable a rat vs. human species comparison. Eighteen of 29 test chemicals showed less than 1 order of magnitude difference in IC50 values between the two assays. Notably, two common perfluorinated compounds, perfluorooctanesulfonic acid (PFOS) and perfluorohexane sulfonate (PFHxS), demonstrated strong NIS inhibitory activity [IC50 - 6.45 (PFOS) and - 5.70 (PFHxS) log M in FRTL-5 RAIU assay]. In addition, several chemicals including etoxazole, methoxyfenozide, oxyfluorfen, triclocarban, mepanipyrim, and niclosamide also exhibited NIS inhibition with minimal cytotoxicity in both assays and are proposed for additional testing using short-term in vivo assays to characterize effects on thyroid hormone synthesis.

High-throughput screening and chemotype-enrichment analysis of ToxCast phase II chemicals evaluated for human sodium-iodide symporter (NIS) inhibition.

In support of the Endocrine Disruptor Screening Program (EDSP), the U.S.EPA's Office of Research and Development (ORD) is developing high-throughput screening (HTS) approaches to identify chemicals that alter target sites in the thyroid hormone (TH) pathway. The sodium iodide symporter (NIS) is a transmembrane glycoprotein that mediates iodide uptake into the thyroid as the initial step of TH biosynthesis. Previously, we screened 293 ToxCast chemicals (ph1v2) using a HEK293T cell line expressing human NIS in parallel radioactive iodide uptake (RAIU) and cell viability assays to identify potential environmental NIS inhibitors. Here, we expanded NIS inhibitor screening for a set of 768 ToxCast Phase II (ph2) chemicals, and applied a novel computational toxicology approach based on the ToxPrint chemotype to identify chemical substructures associated with NIS inhibition. Following single-concentration screening (at 1 × 10-4 M with a 20% inhibition cutoff), 235 samples (228 chemicals) were further tested in multiple-concentration (1 × 10-9 - 1 × 10-4 M) format in both RAIU and cell viability assays. The 167 chemicals that exhibited significant RAIU inhibition were then prioritized using combined RAIU and cell viability responses that were normalized relative to the known NIS inhibitor sodium perchlorate. Some of the highest ranked chemicals, such as PFOS, tributyltin chloride, and triclocarban, have been previously reported to be thyroid disruptors. In addition, several novel chemicals were identified as potent NIS inhibitors. The present results were combined with the previous ph1v2 screening results to produce two sets of binary hit-calls for 1028 unique chemicals, consisting of 273 positives exhibiting significant RAIU inhibition, and 63 positives following application of a cell viability filter. A ToxPrint chemotype-enrichment analysis identified >20 distinct chemical substructural features, represented in >60% of the active chemicals, as significantly enriched in each NIS inhibition hit-call space. A shared set of 9 chemotypes enriched in both hit-call sets indicates stable chemotype signals (insensitive to cytotoxicity filters) that can help guide structure-activity relationship (SAR) investigations and inform future research.

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. EPA's Endocrine Disruptor Screening Program (EDSP) initiative to expand coverage of thyroid molecular targets, as well as the development of thyroid adverse outcome pathways (AOPs).

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

The U.S. EPA's 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.

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.

Comprehensive assessment of a chlorinated drinking water concentrate in a rat multigenerational reproductive toxicity study.

Some epidemiological studies report associations between drinking water disinfection byproducts (DBPs) and adverse reproductive/developmental effects, e.g., low birth weight, spontaneous abortion, stillbirth, and birth defects. Using a multigenerational rat bioassay, we evaluated an environmentally relevant "whole" mixture of DBPs representative of chlorinated drinking water, including unidentified DBPs as well as realistic proportions of known DBPs at low-toxicity concentrations. Source water from a water utility was concentrated 136-fold, chlorinated, and provided as drinking water to Sprague-Dawley rats. Timed-pregnant females (P0 generation) were exposed during gestation and lactation. Weanlings (F1 generation) continued exposures and were bred to produce an F2 generation. Large sample sizes enhanced statistical power, particularly for pup weight and prenatal loss. No adverse effects were observed for pup weight, prenatal loss, pregnancy rate, gestation length, puberty onset in males, growth, estrous cycles, hormone levels, immunological end points, and most neurobehavioral end points. Significant, albeit slight, effects included delayed puberty for F1 females, reduced caput epidydimal sperm counts in F1 adult males, and increased incidences of thyroid follicular cell hypertrophy in adult females. These results highlight areas for future research, while the largely negative findings, particularly for pup weight and prenatal loss, are notable.

Atrazine-induced elevation or attenuation of the LH surge in the ovariectomized, estrogen-primed female rat: role of adrenal progesterone.

Multiple exposures to the herbicide atrazine (ATRZ) were shown to suppress the LH surge in both cycling female rats and those ovariectomized (OVX) and primed with estradiol (E2). A single ATRZ administration was found to induce a prompt and marked increase in progesterone (P4). As exogenous P4 is known to have a differential effect on the LH surge depending on its temporal relationship with the surge, it was hypothesized that a single treatment in an OVX, E2-primed rat would augment the surge, whereas several exposures would cause a decrease. Following four daily treatments with 100 mg/kg, LH surge was suppressed. In contrast, a single ATRZ exposure elevated the surge. Two treatments were without effect. The single administration caused a large increase in P4 at 30 and 60 min that was likely attributable to adrenal secretion. Four exposures also elevated P4 after the final treatment, although the duration of the increase was shortened. A single treatment with 0, 10, 30, and 100 mg/kg ATRZ showed similar elevations at the highest concentration in P4, the LH peak, and area under the curve (AUC), whereas four exposures reduced the AUC. An increase at 1 h in the expression of Kiss1 in the anteroventral periventricular nucleus suggests that this regional kisspeptin neuronal population has a role in the ATRZ augmentation of the surge. These data support the hypothesis that ATRZ-induced changes in adrenal P4 can either augment or attenuate the surge depending on the temporal proximity of exposure to the rise in LH.

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.

Suppression of the steroid-primed luteinizing hormone surge in the female rat by sodium dimethyldithiocarbamate: relationship to hypothalamic catecholamines and GnRH neuronal activation.

In female rodents, hypothalamic norepinephrine (NE) has a role in stimulating the secretion of gonadotropin-releasing hormone (GnRH) that triggers the ovulatory surge of luteinizing hormone (LH). NE synthesis from dopamine (DA) is catalyzed by dopamine-beta-hydroxylase (DbetaH) which contains a copper cofactor. Sodium dimethyldithiocarbamate (DMDC) is a pesticide with metal chelating properties that has been found to reduce DbetaH activity. The resultant decrease in NE causes a suppression of both the LH surge and ovulation. The present study examined the dose-related impact of DMDC on hypothalamic GnRH neuronal activation indicated by the nuclear presence of the early gene product c-fos. It represents an essential link between effects on NE and suppression of the surge. Ovariectomized (OVX), estradiol-, and progesterone-primed Sprague-Dawley rats were given a single ip injection of 0, 3.6, 7.1, 14.2, or 28.4 mg/kg DMDC in separate groups of females to assess tissue GnRH/c-fos immunostaining, hypothalamic catecholamines, and serial blood samplings for LH. A dose-related decline in hypothalamic NE and increase in DA at 2 h after DMDC administration were consistent with a decrease in c-fos-positive GnRH neurons, with an almost complete absence of c-fos at the two highest doses. The effects correlated well with a suppression of the surge, although the percentage decrease in c-fos neurons at 7.1 mg/kg only attenuated the surge peak, not the overall amount of circulating LH. The present data offer further evidence that the impact of DMDC on the LH surge is central in origin and in doing so defines the toxic pathway for this effect on ovulation.

Moderating influence of the drinking water disinfection by-product dibromoacetic acid on a dithiocarbamate-induced suppression of the luteinizing hormone surge in female rats.

The disinfection by-product dibromoacetic acid (DBA) has been found in female rats to increase circulating concentrations of both estradiol (E2) and estrone (E1). This effect is apparently due, at least in part, to a suppression in hepatic catabolism. The present study investigated whether DBA, by increasing sex steroid levels, is able either to augment the hypothalamic up-regulation involved in triggering a luteinizing hormone (LH) surge, or to affect the ability of the neurotoxicant sodium dimethyldithiocarbamate (DMDC) to block the surge. Sprague-Dawley rats were gavaged for 14 days with DBA (0-150mg/kg) and ovariectomized on dosing day 11, and at the same time implanted with an estradiol capsule to generate daily LH surges. An injection of 0.1mM/kg DMDC was administered at 13:00h on day 14 and blood was sampled over the afternoon. DBA induced a dose-related increase in total estrogens. For identified surges, areas under the LH curve partitioned into two groups, comprising the two lower (0 and 37.5mg/kg DBA) and the two higher (75 and 150mg/kg) treatment groups. Consequently, low and high DBA groups were compared and found to be significantly different. At 150mg DBA/0.1mM DMDC, the timing of an identifiable LH peak was comparable to non-DMDC females, unlike the 37.5mg DBA/0.1mM DMDC group in which the appearance of peak concentrations was delayed. A significant effect with DBA treatment alone was not present. Results indicated that this exposure to DBA induced a dose-related increase in total estrogen concentrations that paralleled a diminished DMDC blockade of the LH surge. The effect appeared to be attributable to an augmentation in the estrogen-associated up-regulation in brain mechanisms stimulating the surge.

The rodent estrous cycle: characterization of vaginal cytology and its utility in toxicological studies.

While an evaluation of the estrous cycle in laboratory rodents can be a useful measure of the integrity of the hypothalamic-pituitary-ovarian reproductive axis, it can also serve as a way of insuring that animals exhibiting abnormal cycling patterns are disincluded from a study prior to exposure to a test compound. Assessment of vaginal cytology in regularly cycling animals also provides a means to establish a comparable endocrine milieu for animals at necropsy. The procedure for obtaining a vaginal smear is relatively non-invasive and is one to which animals can become readily accustomed. It requires few supplies, and with some experience the assessments can be easily performed in fresh, unstained smears, or in fixed, stained ones. When incorporated as an adjunct to other endpoint measures, a determination of a female's cycling status can contribute important information about the nature of a toxicant insult to the reproductive system. In doing so, it can help to integrate the data into a more comprehensive mechanistic portrait of the effect, and in terms of risk assessment, may provide some indication of a toxicant's impact on human reproductive physiology.

Reproductive functions and hypothalamic catecholamines in response to the soil fumigant metam sodium: adaptations to extended exposures.

Metam sodium (MS) is a soil fumigant and Category II pesticide with a relatively low toxicity in mammals. Previous data have shown an ability to impair reproductive mechanisms in ovariectomized, estradiol-primed rats. A single i.p. injection blocked the luteinizing hormone (LH) surge that in gonadal-intact females initiates the final stages of follicular and oocytic maturation and serves as the trigger for ovulation. The effect paralleled a fall in hypothalamic norepinephrine (NE) and rise in hypothalamic dopamine (DA) that was likely due to a suppression in dopamine-beta-hydroxylase activity. In addition to determining the influence on catecholamine (CA) concentrations from a single oral exposure to MS, the present study explored effects of longer, 3-week treatments on estrous cyclicity, the LH surge, ovulation and hypothalamic CAs. Normally cycling 90 d S-D rats were administered MS (0-200 mg/kg/d, oral) and cyclicity was monitored daily. At the end of the 3rd week, proestrous blood was sampled over the afternoon from regular 4-day cyclers for a determination of LH. These animals were then killed on the following day of estrus (treatment days 21-26) for oocyte retrieval and assessment of hypothalamic CAs. Results showed that shortly after treatment began there occurred a dose-related period of persistent diestrus that typically lasted 8-16 d before regular cycles were reinstated. After 3 weeks, no effects were seen on the magnitude/timing of the LH surge or ovulated oocyte numbers. Anterior and posterior hypothalamic NE and DA were not significantly different from controls, although DA turnover (reflected by the ratio of DOPAC {3,4-dihydroxy-phenylacetic acid} to DA) in both anterior hypothalamic and caudate regions was decreased at all dosages. The data indicate that a 3 week oral exposure to MS induced an initial period of extended diestrus before the resumption of apparently normal reproductive activity, with previously reported CA alterations (apart from a persistent alteration in the DOPAC/DA ratio) being normalized by the end of dosing.

Twenty-week exposures to the drinking water disinfection by-product dibromoacetic acid: reproductive cyclicity and steroid concentrations in the female Sprague-Dawley rat.

Elevated gavage exposures to the drinking water disinfection by-product dibromoacetic acid (DBA) have been found to disrupt estrous cyclicity in the rat and induce increases in estradiol concentrations in both cycling (day of estrus) and ovariectomized/estradiol-implanted females. The present study was designed to investigate both effects in Sprague-Dawley rats following an extended 20-week treatment with lower dosages of DBA administered in the drinking water (calculated mean intake concentrations of 5, 16, and 33 mg/kg/d). No treatment-related effects on cyclicity were present, although elevations in serum estradiol on the day of vaginal estrus were noted in regularly cycling rats when assessed at the 3rd and 11th weeks of exposure. By the 19th week, this effect was no longer present in cycling animals, but its absence was attributable to a marked increase in control estradiol concentrations, which may be associated with endocrine alterations that precede a disruption in estrous cyclicity in middle-aged females. In the 20th week, diestrous estrone levels were elevated at all dosages without effects on serum androstenedione or progesterone. Uterine and pituitary weights were unchanged at this time, although there were modest increases in liver weights at the two highest dosages. A small number of rats in persistent estrus (PE) did show a general increase in pituitary weight associated with DBA exposure, possibly reflecting an added layering of treatment on the PE-associated rise in estradiol normally seen in these females. The results indicate that increases in circulating estradiol from drinking water exposures to DBA were not linked to a premature disruption of estrous cyclicity in this moderately estrogen-sensitive rat strain.

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.

Continuous exposure to dibromoacetic acid delays pubertal development and compromises sperm quality in the rat.

Previously our work on the haloacid by-products of drinking water disinfection focused on adult exposures. Herein we evaluate the consequence of continuous exposure to dibromoacetic acid (DBA) via drinking water through reproductive development into adulthood. An initial study in which offspring were exposed from gestation day (GD) 15 through adulthood revealed significant delays in preputial separation and vaginal opening, dose-related decreases in the fertility of cauda epididymal sperm, and dose-related diminutions in the sperm membrane protein SP22. Subsequent studies consisted of groups in which exposure ceased on postnatal day 21 (PND 21) versus adulthood. For each exposure, animals were evaluated after puberty (PND 56) as well as at adulthood (PND 120). Exposure to 4, 40, or 400 ppm DBA from GD 15 through PND 21 failed to result in any significant reproductive alterations. By contrast, continuous exposure until adulthood resulted in dose-related alterations consistent with those observed in the dose-finding study. Preputial separation and vaginal opening were delayed 4 and 3 days in males and females exposed to 400 ppm (76.3 mg/kg) DBA. This was associated with increased responsiveness of both the testis and ovary to hCG ex vivo; hCG-stimulated testosterone production by testicular parenchyma on PND 56 was increased at 4 ppm (0.6 mg/kg) DBA and higher. Finally, the quality of proximal cauda epididymal sperm was compromised by continuous exposure to DBA. The sperm membrane proteome was altered in a dose-related manner with SP22, and one of its charged variants, diminished at 40 ppm (3.6 mg/kg) DBA and higher. As more sensitive endpoints are evaluated, lower effect levels can be attributed to haloacid exposure. We are now extending our evaluations to epidemiology studies designed to evaluate sperm quality in men exposed to varying levels of disinfection by-products.

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.