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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 NaClO 4 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.

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 IC 50 values between the two assays. Notably, two common perfluorinated compounds, perfluorooctanesulfonic acid (PFOS) and perfluorohexane sulfonate (PFHxS), demonstrated strong NIS inhibitory activity [IC 50 - 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.

Background: Extensive clinical and experimental research documents the potential for chemical disruption of thyroid hormone (TH) signaling through multiple molecular targets. Perturbation of TH signaling can lead to abnormal brain development, cognitive impairments, and other adverse outcomes in humans and wildlife. To increase chemical safety screening efficiency and reduce vertebrate animal testing, in vitro assays that identify chemical interactions with molecular targets of the thyroid system have been developed and implemented., Objectives: We present an adverse outcome pathway (AOP) network to link data derived from in vitro assays that measure chemical interactions with thyroid molecular targets to downstream events and adverse outcomes traditionally derived from in vivo testing. We examine the role of new in vitro technologies, in the context of the AOP network, in facilitating consideration of several important regulatory and biological challenges in characterizing chemicals that exert effects through a thyroid mechanism., Discussion: There is a substantial body of knowledge describing chemical effects on molecular and physiological regulation of TH signaling and associated adverse outcomes. Until recently, few alternative nonanimal assays were available to interrogate chemical effects on TH signaling. With the development of these new tools, screening large libraries of chemicals for interactions with molecular targets of the thyroid is now possible. Measuring early chemical interactions with targets in the thyroid pathway provides a means of linking adverse outcomes, which may be influenced by many biological processes, to a thyroid mechanism. However, the use of in vitro assays beyond chemical screening is complicated by continuing limits in our knowledge of TH signaling in important life stages and tissues, such as during fetal brain development. Nonetheless, the thyroid AOP network provides an ideal tool for defining causal linkages of a chemical exerting thyroid-dependent effects and identifying research needs to quantify these effects in support of regulatory decision making. https://doi.org/10.1289/EHP5297.

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., (Copyright © 2019. Published by Elsevier Ltd.)

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).

Exposure to endocrine disrupting chemicals has been associated with compromised testosterone production leading to abnormal male reproductive development and altered spermatogenesis. In vitro high-throughput screening (HTS) assays are needed to evaluate risk to testosterone production, yet the main steroidogenesis assay currently utilized is a human adrenocortical carcinoma cell line, H295R, which does not synthesize gonadal steroids at the same level as the gonads, thus limiting assay sensitivity. Here, we propose a complementary assay using a highly purified rat Leydig cell assay to evaluate the potential for chemical-induced alterations in testosterone production by the testis. We evaluated a subset of chemicals that failed to decrease testosterone production in the HTS H295R assay. The chemicals examined fit into one of two categories based on changes in substrates upstream of testosterone in the adrenal steroidogenic pathway (17α-hydroxyprogesterone and 11-deoxycorticosterone) that we predicted should have elicited a decrease in testosterone production. We found that 85% of 20 test chemicals examined inhibited Leydig cell testosterone production in our assay. Importantly, we adopted a 96-well format to increase throughput and efficiency of the Leydig cell assay. We identified a selection criterion based on the AC50 values for 17α-hydroxyprogesterone and 11-deoxycorticosterone generated from the HTS H295R assay that will help prioritize chemicals for further testing in the Leydig cell screen. We hypothesize that the greater dynamic range of testosterone production and sensitivity of the Leydig cell assay permits the detection of small, yet significant, chemical-induced changes not detected by the HTS H295R assay.

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 IC 50 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., (Published by Elsevier Ltd.)

Tamoxifen is an endocrine-active pharmaceutical (EAP) that is used world-wide. Because tamoxifen is a ubiquitous pharmaceutical and interacts with estrogen receptors, a case study was conducted with this compound to (1) determine effects on reproductive endpoints in a nontarget species (i.e., a fish), (2) compare biologically-active metabolites across species, (3) assess whether in vitro assays predict in vivo results, and (4) investigate metabolomic profiles in tamoxifen-treated fish to better understand the biological mechanisms of tamoxifen toxicity. In reproductive assays, tamoxifen exposure caused a significant reduction in egg production and significantly increased ovarian aromatase activity in spawning adult cunner fish (Tautogolabrus adspersus). In plasma from tamoxifen-exposed cunner, the predominant metabolite was 4-hydroxytamoxifen, while in rats it was N-desmethyltamoxifen. Because 4-hydroxytamoxifen is a more biologically active metabolite than N-desmethyltamoxifen, this difference could result in a different level of risk for the two species. The results of in vitro assays with fish hepatic microsomes to assess tamoxifen metabolism did not match in vivo results, indicating probable differences in excretion of tamoxifen metabolites in fish compared with rats. For the first time, a complete in vitro characterization of the metabolism of tamoxifen using fish microsomes is presented. Furthermore, a metabolomic investigation of cunner gonad extracts demonstrates that tamoxifen alters the biochemical profile in this nontarget species. Understanding the consequence of tamoxifen exposure in nontarget species, and assessing the discrepancies between sex- and species-mediated endpoints, is a step toward understanding how to accurately assess the risks posed by EAPs, such as tamoxifen, in the aquatic environment. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1834-1850, 2016., (© 2015 Wiley Periodicals, Inc.)

Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and testis (intact) proteomes in rats after 3 days of exposure. The adrenal accounted for most of the serum progesterone and all of the corticosterone increases in intact and castrated males. Serum luteinizing hormone, androstenedione, and testosterone in intact males shared a non-monotonic response suggesting transition from an acute stimulatory to a latent inhibitory response to exposure. Eight adrenal proteins were significantly altered with dose. There were unique proteomic changes between the adrenals of intact and castrated males. Six testis proteins in intact males had non-monotonic responses that significantly correlated with serum testosterone. Different dose-response curves for steroids and proteins in the adrenal and testis reveal novel adverse outcome pathways in intact and castrated male rats., (Published by Elsevier Inc.)

The steroidogenic enzyme aromatase catalyzes the conversion of androgens to estrogens and therefore plays a central role in reproduction. In contrast to most vertebrates, teleost fish have two distinct forms of aromatase. Because brain aromatase activity in fish is up to 1000 times that in mammals, fish may be especially susceptible to negative effects from environmental endocrine-disrupting chemicals (EDCs) that impact aromatase activity. In this study, the effects of estradiol (E2), ethynylestradiol (EE2), octylphenol (OP), and androstatrienedione (ATD) on reproduction and aromatase activity in brains and gonads from the marine fish cunner (Tautogolabrus adspersus) was investigated. The purpose of the study was to explore the relationship between changes in aromatase activity and reproductive output in a marine fish, as well as compare aromatase activity to two commonly used indicators of EDC exposure, plasma vitellogenin (VTG) and gonadosomatic index (GSI). Results with E2, EE2, and ATD indicate that aromatase activity in cunner brain and ovary are affected differently by exposure to these EDCs. In the case of E2 and EE2, male brain aromatase activity was signficantly increased by these treatments, female brain aromatase activity was unaffected, and ovarian aromatase activity was significantly decreased. Treatment with the aromatase inhibitor ATD resulted in significantly decreased aromatase activity in male and female brain, but had no significant impact on ovarian aromatase activity. Regardless of test chemical, a decrease or an increase in male brain aromatase activity relative to controls was associated with decreased egg production in cunner and was also correlated with significant changes in GSI in both sexes. E2 and EE2 significantly elevated plasma VTG in males and females, while ATD had no significant effect. Treatment of cunner with OP had no significant effect on any measured endpoint. Overall, results with these exposures indicate EDCs that impact aromatase activity also affect reproductive output in spawning cunner., (Published by Elsevier B.V.)

Introduction: Commercially available radioimmunoassays (RIAs) are frequently used to evaluate the effects of endocrine disrupting chemicals (EDCs) on steroidogenesis in rats. Currently there are limited data comparing steroid concentrations in rats as measured by RIAs to those obtained using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This study evaluates the concordance of serum and urine steroid concentrations as quantified by select RIA kits and LC-MS/MS following exposure to an EDC, atrazine (ATR)., Methods: Adult male rats were orally dosed with ATR (200 mg/kg/day) or methylcellulose (1%, vehicle control) for 5 days. Serum was collected and separated into aliquots for analysis. Serum was assayed by RIA for androstenedione (ANDRO), corticosterone (CORT), estradiol (E2), estrone (E1), progesterone (P4), and testosterone (T). Serum was extracted prior to LC-MS/MS analysis with positive electrospray ionization in multiple-reaction monitoring mode for ANDRO, CORT, P4, and T. E1 and E2 concentrations were quantified similarly by LC-MS/MS, following derivatization with dansyl chloride. To compare CORT values from urine, pregnant adult rats were orally dosed with either ATR (100 mg/kg/day) or methylcellulose for 5 days (i.e., gestational days 14-18). Urine samples were collected daily and assayed for CORT by RIA and LC-MS/MS as described above., Results: Data analyses demonstrated significant agreement between the two detection methods as assessed by Pearson product-moment correlation coefficient, Bland-Altman analysis, and the interclass correlation coefficient. No statistically significant differences were observed between RIA and LC-MS/MS means for any of the steroids assayed., Discussion: These findings indicate a significant correlation between the measurement of steroids within rat serum and urine using RIA kits and LC-MS/MS. Differences in the absolute measurements existed, but these were not statistically significant. These findings indicate that steroids may be reliably measured in rat biological media using RIAs or LC-MS/MS., (© 2013.)

Legislation and prospective legislative proposals internationally (may) require that chemicals be tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive in vitro are considered to be endocrine active substances (EAS) and may be putative endocrine disruptors (EDs) in vivo. While there is a growing body of international in vitro test guidelines addressing EAS mechanisms and modes of action, to date there are still few or no standardized methods to incorporate metabolic and toxicokinetic aspects into these in vitro tests for EAS. In vitro assays for EAS should incorporate metabolic enzyme systems to better address the relevance of EAS tests to in vivo adverse outcome pathways, and a previous OECD review paper indicated how this could be done. This paper revisits those recommendations, addressing where research and funding efforts are needed to expedite the development of suitable in vitro metabolism systems to improve the accuracy of in vitro assays for identifying EAS and EDs. Recommendations are made for projects to support short, medium, and long-term goals. The complexity of in vivo metabolism presents major challenges for the development of predictive models suitable for the extrapolation of data from in silico/in vitro approaches to models that can occur in vivo. Therefore, the long-term recommendations are intended to foster an international harmonization of databases, delineation of metabolic pathways, and development of predictive tools that will provide a fundamental understanding of the processes by which metabolism occurs, increasing the predictive accuracy of in silico/in vitro methods.

Previous work has shown that a single oral administration of atrazine (ATR), a chlorotriazine herbicide, causes rapid increases in plasma adrenocorticotropic hormone (ACTH), serum corticosterone (CORT) and progesterone. The mechanism for these effects is unknown. To test whether administration of ATR causes hypothalamic-pituitary-adrenal (HPA) axis activation through the production of a generalized stress response resulting from gastrointestinal distress, we conducted both conditioned taste avoidance (CTA) and pica behavior experiments. Body temperature data were also collected to detect the presence of stress-induced hyperthermia. Adult male Wistar rats were given a single oral dose of ATR (0, 5, 25, 50, 100, or 200 mg/kg) or the primary ATR metabolite diamino-s-chlorotriazine (DACT; 135 mg/kg). Increases were observed in ACTH (LOEL, 12.5 mg/kg), CORT (LOEL, 5 mg/kg) and progesterone (LOEL, 5 mg/kg) 15 min following a single dose of ATR. DACT (135 mg/kg) increased ACTH (1.3-fold), CORT (2.9-fold) and progesterone (1.9-fold) above vehicle control concentrations, but the magnitude of the responses was much lower than that observed for an equal molar dose of ATR (200 mg/kg; 7.0, 9.0 and 11.0-fold above ACTH, CORT, progesterone controls, respectively). CTA results demonstrated conditioned taste avoidance to ATR, with a NOEL of 5 mg/kg. Animals dosed with DACT developed avoidance responses comparable to the highest dose of ATR. In the pica experiment, lower doses (5-50 mg/kg) of ATR had no effect on pica behavior, as measured 6 and 24 h post-dosing, nor did DACT. However, the highest dose of ATR (200 mg/kg) did induce pica behavior at both time points. No differences in body temperature were observed. Overall, results indicate that increases in ACTH and CORT secretion following administration of ATR occur at doses that are without effect on the display of pica behavior, indicating that the HPA-axis activation caused by ATR is not likely the result of gastrointestinal distress., (Published by Elsevier Inc.)

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., (Published by Elsevier Inc.)

Atrazine (ATR) has recently been shown to activate the hypothalamic-pituitary-adrenal (HPA) axis in rodents. The current study investigated the effect of ATR and two of its chlorinated metabolites, desisopropylatrazine (DIA) and diamino-s-chlorotriazine (DACT), on the HPA axis in the Long-Evans female rat. A single oral gavage administration of 75 mg/kg ATR or 60.2 mg/kg DIA (a dose equimolar to the applied ATR dose) during the morning of proestrus resulted in significant, acute increases in circulating adrenocorticotropic hormone (ACTH), corticosterone, and progesterone. Oral doses of ATR or DIA were given daily over the course of the 4-day ovarian cycle starting on the day of vaginal estrus, resulted in a similar, dose-responsive activation of the HPA axis. The increase in ACTH, corticosterone, and progesterone by these compounds was of a similar magnitude to that produced by 5-min restraint stress. Single or multiple oral exposures to DACT, on the other hand, did not significantly alter pituitary-adrenal hormone release. These results were observed despite plasma levels of DACT being higher than any other metabolite at the time of hormone measurement. Overall, circulating metabolite concentrations following equimolar dosing were much higher than those observed after ATR administration. Additional studies indicated that the activation of the HPA axis by oral exposure to ATR and DIA was not due simply to the stimulation of gastrointestinal afferents. Similar responses were observed in rats which received an oral dose of ATR following bilateral subdiaphramatic vagotomy and following intravenous administration of DIA in jugular vein catheterized animals. We conclude that ATR and the metabolite DIA significantly activate the HPA axis following oral exposure in the female rat. Activation of this endocrine axis by these chlorotriazines could contribute to the induced changes of female reproductive function reported previously.

Previously, we reported that atrazine (ATR) alters steroidogenesis in male Wistar rats resulting in elevated serum corticosterone (CORT), progesterone, and estrogens. The increase in CORT indicated that this chlorotriazine herbicide may alter the hypothalamic-pituitary-adrenal axis. This study characterizes the temporal changes in adrenocorticotropic hormone (ACTH), CORT, and P4 in male Wistar rats following a single dose of ATR (0, 5, 50, 100, and 200 mg/kg), simazine (SIM; 188 mg/kg), propazine (PRO; 213 mg/kg), or primary metabolites, deisopropylatrazine (DIA; 4, 10, 40, 80, and 160 mg/kg), deethylatrazine (DEA; 173 mg/kg), and diamino-s-chlorotriazine (DACT; 3.37, 33.7, 67.5, and 135 mg/kg). The maximum dose for each chemical was the molar equivalent of ATR (200 mg/kg). Significant increases in plasma ACTH were observed within 15 min, following exposure to ATR, SIM, PRO, DIA, or DEA. Dose-dependent elevations in CORT and progesterone were also observed at 15 and 30 min post-dosing with these compounds indicating an activation of adrenal steroidogenesis. Measurement of the plasma concentrations of the parent compounds and metabolites confirmed that ATR, SIM, and PRO are rapidly metabolized to DACT. Although DACT had only minimal effects on ACTH and steroid release, dosing with this metabolite resulted in plasma DACT concentrations that were 60-fold greater than that observed following an equimolar dose of ATR and eightfold greater than equimolar doses of DIA or DEA, indicating that DACT is not likely the primary inducer of ACTH release. Thus, the rapid release of ACTH and subsequent activation of adrenal steroidogenesis following a single exposure to ATR, SIM, PRO, DIA, or DEA may reflect chlorotriazine-induced changes at the level of the brain and/or pituitary.

There is a paucity of relevant experimental information available for the evaluation of the potential health and environmental effects of many man made chemicals. Knowledge of the potential pathways for activity provides a rational basis for the extrapolations inherent in the preliminary evaluation of risk and the establishment of priorities for obtaining missing data for environmental chemicals. The differential step in many mechanisms of toxicity may be generalized as the interaction between a small molecule (a potential toxicant) and one or more macromolecular targets. An approach based on computation of the interaction between a potential molecular toxicant and a library of macromolecular targets of toxicity has been proposed for preliminary chemical screening. In the current study, the interaction between a series of environmentally relevant chemicals and models of the rat estrogen receptors (ER) was computed and the results compared to an experimental data set of their relative binding affinities. The experimental data set consists of 281 chemicals, selected from the U.S. EPA's Toxic Substances Control Act (TSCA) inventory, that were initially screened using the rat uterine cytosolic ER-competitive binding assay. Secondary analysis, using Lineweaver-Burk plots and slope replots, was applied to confirm that only 15 of these test chemicals were true competitive inhibitors of ER binding with experimental inhibition constants (K(i)) less than 100 microM. Two different rapid computational docking methods have been applied. Each provides a score that is a surrogate for the strength of the interaction between each ligand-receptor pair. Using the score that indicates the strongest interaction for each pair, without consideration of the geometry of binding between the toxicant and the target, all of the active molecules were discovered in the first 16% of the chemicals. When a filter is applied on the basis of the geometry of a simplified pharmacophore for binding to the ER, the results are improved, and all of the active molecules were discovered in the first 8% of the chemicals. In order to obtain no false negatives in the model that includes the pharmacophore filter, only 8 molecules are false positives. These results indicate that molecular docking algorithms that were designed to find the chemicals that act most strongly at a receptor (and therefore are potential pharmaceuticals) can efficiently separate weakly active chemicals from a library of primarily inactive chemicals. The advantage of using a pharmacophore filter suggests that the development of filters of this type for other receptors will prove valuable.

The U.S. Environmental Protection Agency is currently validating assays that will be used in a Tier I Screening Battery to detect endocrine disrupting chemicals. A primary concern with the Protocols for the Assessment of Pubertal Development and Thyroid Function in Juvenile Male and Female Rats is that a nonspecific reduction in body weight (BWT) during the exposure period may potentially confound the interpretation of effects on the endocrine endpoints. Wistar rats were underfed 10, 20, 30, or 40% less than the ad libitum food consumed by controls from postnatal days (PNDs) 22 to 42 (females) or PNDs 23 to 53 (males). Terminal BWT of females and males were 2, 4, 12, and 19% and 2, 6, 9, and 19% lower than controls, respectively. In the females, neither the age of pubertal onset nor any of the thyroid hormone endpoints were affected by food restriction (FR) that led to a 12% decrease in BWT. Similarly, none of the male reproductive endpoints examined were altered by FR that led to a 9% BWT decrease. However, decreased triiodothyronine and thyroxin was observed in FR males with a 9% reduced BWT. While these data support the use of the maximum tolerated dose for BWT (10%) for the female protocol, effects on the male thyroid endpoints indicate that a slightly lower limit (

Atrazine, a chlorotriazine herbicide, is used to control annual grasses and broadleaf weeds. In this review, we summarize our laboratory's work evaluating the neuroendocrine toxicity of atrazine (and related chlorotriazines) from an historic perspective. We provide the rationale for our work as we have endeavored to determine: 1) the underlying reproductive changes leading to the development of mammary gland tumors in the atrazine-exposed female rat; 2) the cascade of physiological events that are responsible for these changes (i.e., the mode of action for mammary tumors); 3) the potential cellular mechanisms involving adverse effects of atrazine; and 4) the range of reproductive alterations associated with this pesticide.

The U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 male pubertal protocol was designed as a screen to detect endocrine-disrupting chemicals which may alter reproductive development or thyroid function. One purpose of this in vivo screening protocol is to detect thyrotoxicants via a number of different mechanisms of action, such as thyroid hormone synthesis or clearance. Here we evaluate the ability of this EDSP male pubertal protocol to detect the known thyrotoxicant ammonium perchlorate as an endocrine disruptor. Ammonium perchlorate is a primary ingredient in rocket fuel, fertilizers, paints, and lubricants. Over the past 50 years, potassium perchlorate has been used to treat hyperthyroidism in humans. Perchlorate alters thyroid hormone secretion by competitively inhibiting iodide uptake by the thyroid gland. In this study, ammonium perchlorate was administered at 62.5, 125, 250, and 500 mg/kg to male Wistar rats based on a pilot study of oral dosing. Doses of 125-500 mg/kg perchlorate decreased T4 in a dose-dependent manner. TSH was significantly increased in a dose-responsive manner at the same doses, while T3 was unchanged at any dose. Thyroid histology was significantly altered at all doses, even at the 62.5 mg/kg, with a clear dose-dependent decrease in colloid area and increase in follicular cell height. No effects on preputial separation, a marker of pubertal progression, or reproductive tract development were observed at any dose. These results demonstrate that the male pubertal protocol is useful for detecting thyrotoxicants which target the thyroid axis by this mechanism (altered uptake of iodide). This study also found that perchlorate exposure during this period did not alter any of the reproductive developmental endpoints.

This study was conducted to characterize the estrogen receptor (ER)-binding affinities of 50 chemicals selected from among the high production volume chemicals under the U.S. EPA's (U.S. Environmental Protection Agency's) Toxic Substances Control Act inventory. The chemicals were evaluated using the rat uterine cytosolic (RUC) ER-competitive binding assay, with secondary analysis using Lineweaver-Burk plots and slope replots to confirm true competitive inhibition and to determine an experimental K(i). Data from these ER-competitive binding assays represent the types of competitive binding curves that can be obtained when screening chemicals with broad structural diversity. True competitive inhibition was observed in 17 of 50 chemicals. Binding affinities were much lower than that of estradiol (E(2)) with K(i) concentrations ranging from 0.6 to 373 microM as compared with that of E(2) (0.77 nM). Other chemicals that appeared to displace radiolabeled E(2) binding to ER were, in fact, found not to be competitive inhibitors in the secondary K(i) experiments. These seven chemicals likely altered the stability of the assay by changing the buffer pH, denaturing ER, or disrupting the ER-binding kinetics. Thus, several conditions that may confound interpretation of RUC ER-binding assay data are illustrated. For another group of eight chemicals, neither an IC(50) nor K(i) could be determined due to solubility constraints. These chemicals exhibited slight (20-40%) inhibition at concentrations of 10-100 microM, suggesting that they could be competitors at very high concentrations, yet K(i) experiments were not possible as the limit of chemical solubility in the aqueous assay buffer was well above the IC(50). An additional 18 of the 50 chemicals were classified as nonbinders because in concentrations up to 100 microM they produced essentially no displacement of radiolabeled E(2). These results show that although the ER-competitive binding assay is a valuable tool for screening chemicals, secondary tests such as a double reciprocal Lineweaver-Burk experiment with slope replot should be used to confirm true competitive inhibition. This information will be useful for the ongoing validation of the RUC ER-competitive binding assay under the U.S. EPA's Endocrine Disruptor Screening Program, as well as to support research efforts to develop computational models designed to identify chemicals with the ability to bind to ER.

DE-71, a commercial mixture, was used to test the sensitivity of the female and male pubertal protocol to detect thyroid active chemicals. These protocols are being evaluated for the U.S. EPA's Endocrine Disruptor Screening Program as part of a Tier I Screening Battery. To examine the ability of these protocols to screen for chemicals that induce the clearance of thyroid hormone, we examined male and female Wistar rats following DE-71 exposure. Rats were gavaged daily with 0, 3, 30, or 60 mg/kg DE in corn oil from postnatal day (PND) 23-53 in the male or PND 22-41 in the female. The temporal effects of DE-71 on liver enzymes and thyroid hormones were measured in another group of males and females following only 5 days of dosing (PND 21 to 26 in females and PND 23 to 28 in males). Serum T4 was significantly decreased at 30 and 60 mg/kg following the 5-day exposures and in the 21-day exposed females. Doses of 3, 30, and 60 mg/kg decreased T4 in 31-day exposed males. Serum T3 was decreased and TSH elevated by 30 and 60 mg/kg in the 31-day exposed males only. Decreased colloid area and increased follicular cell heights (indicative of the hypothyroid state) were observed in thyroids of the 60 mg/kg groups of 20- and 31-day exposed female and males. Increased liver-to-body weight ratios coincided with a significant induction of uridinediphosphate-glucuronosyltransferase (UDGPT; two to four-fold), and ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD) at the two highest doses in all exposures. Of the androgen dependent tissues in the 31-day exposed males, seminal vesicle (SV) and ventral prostate (VP) weights were reduced at 60 mg/kg, while testes and epididymal weights were not affected. Preputial separation (PPS) was also significantly delayed by doses of 30 and 60 mg/kg. In the female, the 60 mg/kg dose also caused a significant delay in the age of vaginal opening. Based upon the thyroid hormone response data, this study provides evidence that the 31-day alternative Tier 1 male protocol is a more sensitive test protocol than the 5-day or female pubertal protocol for thyrotoxic agents that act via up-regulation of hepatic metabolism. This apparent greater sensitivity may be due a greater body burden attained following the longer dosing regimen as compared with that of the female protocol, or to gender specific differences in thyroid hormone metabolism. Also, the delay in PPS and reduction in SV and VP weights may indicate a modification or inhibition of endogenous androgenic stimulation directly by DE-71 or a secondary effect that occurs in response to a DE-induced change in thyroid hormones.

The screening and testing program the US Environmental Protection Agency (EPA) is currently developing to detect endocrine-disrupting chemicals (EDCs) is described. EDCs have been shown to alter the following activities: hypothalamic-pituitary-gonadal (HPG) function; estrogen, androgen, and thyroid hormone synthesis; and androgen and estrogen receptor-mediated effects in mammals and other animals. The value and limitations of mammalian in vivo assays are described that involve the use of the laboratory rat, the EPA Endocrine Disruptor Screening and Testing Advisory Committee species of choice. The discussion includes the evaluation of high-priority chemicals positive in the Tier 1 Screening (T1S) battery, and of subsequent testing in the Tier 2 (T2) battery, with additional short-term screening assays proposed for use in T1.5 to eliminate any uncertainty about T1S results. Descriptions include the in vivo uterotropic assay, which detects estrogens and antiestrogens; the pubertal female assay, which assesses steroidogenesis, antithyroid activity, antiestrogenicity, and HPG function; and the Hershberger assay, which detects the weight of androgen-dependent tissues in castrate-immature-male rats (antiandrogens). Of the several alternative mammalian in vivo assays proposed, a short-term pubertal male rat assay appears most promising for inclusion in T1 or T1.5. An additional in utero-lactational screening protocol is being evaluated, but appears to be better suited for T1.5 or T2 due to the size, complexity, and duration of the assay. The adult intact male assay, also proposed as an alternative for T1, attempts to identify EDCs in a hormonal battery, but has limited value as a screen due to lack of sensitivity and specificity. For Tier 2 testing, the number of endocrine-sensitive endpoints and offspring (F1) examined in multigenerational tests must be thoughtfully expanded for EDCs on a mode-of-action-specific basis, with consideration given to tailoring T2 based on the results of T1S.

We showed previously that the chlorotriazine herbicide, atrazine (ATR), delays the onset of pubertal development in female rats. ATR and its biotransformation by-products are present in soil and groundwater. Since current maximum contaminant levels are set only for ATR, it is important to determine whether these by-products can also alter pubertal development and possibly pose a cumulative exposure hazard. We evaluated the effects of two ATR by-products, diamino-s-chlorotriazine (DACT) and hydroxyatrazine (OH-ATR), and a structurally similar chlorotriazine, propazine (PRO), on female pubertal development. Rats were gavaged from postnatal days (PNDs) 22 through PND 41 with DACT (16.7, 33.8, 67.5, 135 mg/kg), OH-ATR (22.8, 45.7, 91.5, 183 mg/kg), or PRO (13, 26.7, 53, 106.7, 213 mg/kg). The dose range for each chemical was selected as the molar equivalent of ATR (12.5, 25, 50, 100, 200 mg/kg). The females were monitored daily for vaginal opening (VO) and killed on PND 41. DACT, a by-product of ATR that occurs in the environment and is also the primary chlorinated metabolite of ATR in animal tissue, delayed VO by 3.2, 4.8, and 7.6 days compared to the controls (33.1 +/- 0.4 (SE) days of age) following exposure to 33.8, 67.5, and 135 mg/kg, respectively. The no effect level (NOEL) for DACT (16.7 mg/kg) was identical to the equimolar NOEL for ATR (25 mg/kg). Although the body weight (BW) on PND 41 was reduced by the high dose of DACT (8.4% reduction), this reduction did not exceed the criteria for selecting the maximum tolerated dose (e.g., a dose that causes >10% decrease in BW at necropsy). None of the lower doses of DACT caused a significant difference in BW gain. Additionally, 33.8, 67.5, and 135 mg/kg of DACT significantly increased the BW on the day of VO. PRO (107 or 213 mg/kg) delayed VO by 4 days but did not alter the BW on PND 41. While no significant delays in pubertal development were observed in two separate dose-response studies with doses ranging up to 183 mg/kg (OH-ATR), a minor but statistically significant delay in the onset of puberty in a pilot study using OH-ATR raises the possibility that an effect might occur following exposure to higher doses. However, it is clear from these data that OH-ATR has a much lower potency when compared with equimolar doses of DACT and PRO. Together, these data demonstrate that PRO and DACT can delay the onset of puberty in the female rat at doses equimolar to ATR and provide the scientific basis for the use of additivity in the upcoming risk assessments.

The US Environmental Protection Agency (EPA) is developing a screening and testing program for endocrine disrupting chemicals (EDCs) to detect alterations of hypothalamic-pituitary-gonadal (HPG) function, estrogen (ER), androgen (AR) and thyroid hormone synthesis and AR and ER receptor-mediated effects in mammals and other animals. High priority chemicals would be evaluated in the Tier 1 Screening (T1S) battery and chemicals positive in T1S would then be tested (Tier 2). T1S includes in vitro ER and AR receptor binding and/or gene expression, an assessment of steroidogenesis and mammalian (rat) and nonmammalian in vivo assays (Table 1). In vivo, the uterotropic assay detects estrogens and antiestrogens, while steroidogenesis, antithyroid activity, (anti)estrogenicity and HPG function are assessed in a 'Pubertal Female Assay'. (Anti-) androgens are detected in the Hershberger Assay (weight of AR-dependent tissues in castrate-immature-male rats). Fish and amphibian assays also are being developed. The fathead minnow assay can identify EDCs displaying several mechanisms of concern, including AR and ER receptor agonists and antagonists and inhibitors of steroid hormone synthesis. An amphibian metamorphosis assay is being developed to detect thyroid-active substances. Several alternative mammalian in vivo assays have been proposed. Of these, a short-term pubertal male rat assay appears most promising. An in utero-lactational screening protocol also is being evaluated. For Tier 2, the numbers of endocrine sensitive endpoints and offspring (F1) examined in multigenerational tests need to be expanded for EDCs. Consideration should be given to tailoring T2, based on the results of T1S. Tier 1 and 2 also should examine relevant mixtures of EDCs. Toxicants that induce malformations in AR-dependent tissues produce cumulative effects even when two chemicals act via different mechanisms of action.

Recently we reported that atrazine (ATR), a chlorotriazine herbicide, alters the onset of puberty in male Wistar rats. In this study, we examined the same reproductive parameters in the developing male rat following a similar exposure to the primary, chlorinated metabolites of atrazine. Intact male Wistar rats were gavaged from postnatal day (PND) 23 through PND 53 and several reproductive endpoints were examined. The doses selected were the molar equivalents to atrazine in our previous work. Deethylatrazine (DEA), deisopropyl-atrazine (DIA), and diaminochlorotriazine (DACT) were administered by gavage at doses equivalent to the atrazine equimolar doses (AED) of 6.25, 12.5, 25, 50, 100, or 200 mg/kg. Preputial separation (PPS) was significantly delayed by DEA at 25, 100, and 200 AED, by DIA at 25, 100, and 200 AED, and by DACT at 12.5 through 200 AED. When the males were killed on PND 53, DEA (100 and 200 AED), DIA (50 through 200 AED), and DACT (200 AED) treatments caused a significant reduction in ventral prostate weight, while only the highest doses of DIA and DEA resulted in a significant decrease in lateral prostate weight. Seminal vesicle weight was reduced by DEA (25, 100, and 200 AED), DIA (100 and 200 AED), and 100 and 200 AED of DACT. Epididymal weights were reduced in the DEA (200 AED), DIA (200 AED), and DACT (100 and 200 AED) treatment groups. Serum testosterone was reduced only in the males receiving the 2 highest doses of DIA. Serum estrone was increased in the 2 highest doses of the DACT group, while serum estradiol was not different in any group. No differences were observed in any of the thyroid measures. In summary, the metabolites of ATR delay puberty in a manner similar to that observed in the previous study testing atrazine. These data also suggest that the 3 chlorinated metabolites are similar to ATR, by affecting the CNS control of the pituitary/gonadal axis and subsequent development of the reproductive tract.

The effects of atrazine (ATR), a chlorotriazine herbicide, on the onset of puberty were evaluated in Wistar rats. Female rats were dosed by oral gavage from postnatal day(s) (PND) 22 through PND 41 with 0, 12.5, 25, 50, 100, or 200 mg ATR/kg. Vaginal opening (VO) was significantly delayed 3.4, 4.5, or greater than 6.8 days by 50, 100, and 200 mg/kg, respectively. VO had not occurred in 4 of 15 females in the 200 mg/kg group by the time of necropsies (PND 41). Body weight (bw) at necropsy was reduced in the 200 mg/kg group by 11.6%, but was not different from the control (0) in the 50 and 100 mg/kg groups. To examine the influence of reduced bw on pubertal development, a group of pair-fed controls was included whose daily food intake was dependent upon the amount consumed by their counterpart in the 200 mg/kg group. Although necropsy bw was reduced to the same extent as the ATR females, VO in the pair-fed controls was not significantly delayed. Adrenal, kidney, pituitary, ovary, and uterine weights were reduced by 200 mg/kg ATR. Serum T(3), T(4), and TSH were unaltered by ATR, which was consistent with no histopathologic/morphologic changes in the thyroid. Estrous cyclicity was monitored in a second group of females from VO to PND 149. The number of females displaying regular 4- or 5-day estrous cycles during the first 15-day interval after VO was lower in the 100 and 200 mg/kg ATR and pair-fed controls. Irregular cycles were characterized by extended periods of diestrus. By the end of the second 15-day interval (PND 57-71), no effects on estrous cyclicity were observed. These data show that ATR can delay the onset of puberty and alter estrous cyclicity in the female Wistar rat ( NOAEL of 25 mg/kg). Reduced food consumption and bw did not account for the delay in VO, because this effect was not observed in the pair-fed controls. In addition, the effect on estrous cyclicity was observed in the 100 mg/kg ATR group where no significant reduction in bw was observed.

Since atrazine (ATR), a chlorotriazine herbicide, has been shown previously to alter the secretion of luteinizing hormone (LH) and prolactin (PRL) through a direct effect on the central nervous system (CNS), we hypothesized that exposure to ATR in the EDSTAC male pubertal protocol (juvenile to peripubertal) would alter the development of the male rat reproductive system. We dosed intact male Wistar rats from postnatal day (PND) 23 to 53 and examined several reproductive endpoints. ATR (0, 12.5, 25, 50, 100, 150, or 200 mg/kg) was administered by gavage and an additional pair-fed group was added to compare the effects of any decreased food consumption in the high dose group. Preputial separation (PPS) was significantly delayed in the 12.5, 50, 100, 150, and 200 mg/kg ATR dose groups. PPS was also delayed in the pair-fed group, although significantly less than in the high dose-ATR group. The males were killed on PND 53 or 54, and pituitary, thyroid, testes, epididymides, seminal vesicles, and ventral and lateral prostates were removed. ATR (50 to 200 mg/kg) treatment resulted in a significant reduction in ventral prostate weights, as did the reduced food consumption of the pair-fed group. Testes weights were unaffected by atrazine treatment. Seminal vesicle and epididymal weights were decreased in the high dose-ATR group and the control pair-fed group. However, the difference in epididymal weights was no longer significantly different when body weight was entered as a covariable. Intratesticular testosterone was significantly decreased in the high dose-ATR group on PND 45, but apparent decreases in serum testosterone were not statistically significantly on PND 53. There was a trend for a decrease in luteinizing hormone (LH) as the dose of ATR increased; however, dose group mean LH was not different from controls. Due to the variability of serum prolactin concentrations on PND 53, no significant difference was identified. Although prolactin is involved in the maintenance of LH receptors prior to puberty, we observed no difference in LH receptor number at PND 45 or 53. Serum estrone and estradiol showed dose-related increases that were significant only in the 200 mg/kg-ATR group. No differences were observed in thyroid stimulating hormone (TSH) and thyroxine (T4) between the ATR groups and the control; however triiodothyronine (T3) was elevated in the high dose-ATR group. No differences in hormone levels were observed in the pair-fed animals. These results indicate that ATR delays puberty in the male rat and its mode of action appears to be altering the secretion of steroids and having subsequent effects on the development of the reproductive tract, which appear to be due to ATR's effects on the CNS. Thus, ATR tested positive in the pubertal male screen that the Endocrine-Disrupter Screening and Testing Advisory Committee (EDSTAC) is considering as an optional screen for endocrine disrupters.

Endocrine disrupting chemicals have recently drawn increased interest. The delayed implanting rat model is a method that can identify and quantify the estrogenic activity of a chemical. In rats hypophysectomized after breeding, the administration of progesterone delays embryo implantation, and exposure to one dose of an estrogenic substance initiates implantation. Although methoxychlor was ineffective at dosages below 400 mg/kg when given by injection, the administration of the chemical by gavage resulted in an increase in the percent of fertilized rats exhibiting implantation sites. These results were statistically significant at dosages of 50, 100, 200, and 300 mg methoxychlor/kg. When bisphenol A was administered, by subcutaneous injection, dosages of 50, 100, and 200 mg/kg induced implantation. Only the 400 mg/kg dose of 4-tert-octylphenol was effective. Doses of beta-sitosterol up to 30 mg/kg failed to initiate implantation. These data confirm previous evidence of the availability of this model for evaluating estrogenic activity and provide estimates of the estrogenic potencies of several environmentally important chemicals.

Considerable attention has recently been focused on environmental chemicals that disrupt the reproductive system by altering steroid receptor function. Although numerous in vitro and in vivo methods have been shown to be useful approaches for identifying chemicals that can disrupt reproduction through a direct interaction with the estrogen receptor, it is imperative that the protocols selected be capable of detecting chemicals with a broad range of estrogenic activity. Here we evaluate the reliability of the 3-day uterotrophic assay for detecting chemicals with strong or weak estrogenic activity in both prepubertal and ovariectomized adult Long Evans rats. These data were compared to additional measures of estrogenic activity, which included the age of vaginal opening, the induction of cornified vaginal epithelial cells in ovariectomized adult rats, and estrous cyclicity in intact adult rats. Test chemicals selected for these studies included 17-beta-estradiol, ethynyl estradiol, methoxychlor, 4-tert-octylphenol, 4-nonylphenol and bisphenol A. Data from in vitro receptor binding assays compared the ability of the test chemicals to compete with [3H]-estradiol or [3H]-promegestone for binding to estrogen or progesterone receptors. As expected, the binding affinities for the estrogen receptor ranged from high to low, as reflected by Ki concentrations of 0.4 nM for 17-beta-estradiol and ethynyl estradiol, and 0.05-65 microM for 4-tert-octyphenol, 4-nonylphenol, and methoxychlor. Although none of the test chemicals demonstrated a high affinity for binding to the progesterone receptor, 4-tert-octylphenol and 4-nonylphenol exhibited a weak affinity, with Ki concentrations ranging from 1.2 to 3.8 microM. In vivo studies indicated that the 3-day uterotrophic assay in prepubertal rats was the best method for detecting estrogenic activity when compared with all other end points, based upon the dose-response data for ethynyl estradiol (0.01-0.1 mg/kg), 4-tert-octylphenol (50-200 mg/kg, oral), and 4-nonylphenol (25-100 mg/kg, oral). Although oral doses of ethynyl estradiol (0.01 mg/kg) and 4-nonylphenol (50 mg/kg) induced a significant increase in uterine weight in the prepubertal rats, these doses were ineffective for stimulating a similar response in ovariectomized adult rats. The age of vaginal opening was advanced following oral exposure from postnatal days 21-35 to ethynyl estradiol (0.01 mg/kg), methoxychlor (50 mg/kg), 4-tert-octylphenol (200 mg/kg), and 4-nonylphenol (50 mg/kg). Although bisphenol A (200 mg/kg, oral) induced a significant uterotrophic response within 3 days in prepubertal rats, doses up to 400 mg/kg failed to advance the age of vaginal opening. Monitoring changes in the vaginal epithelium of ovariectomized adult rats was the least effective method for detecting estrogenic activity for 4-tert-octylphenol and bisphenol A. The number of 4-5 day estrous cycles was reduced during a 25-day exposure to ethynyl estradiol (0.01 mg/kg), methoxychlor (50 mg/ kg), 4-tert-octylphenol (200 mg/kg), 4-nonylphenol (100 mg/kg), and bisphenol A (100 mg/kg) by oral gavage. Although long periods of extended diestrus (7-14 days) were generally correlated with exposure to ethynyl estradiol and 4-tert-octylphenol, the cycling patterns following exposure to methoxychlor, 4-nonylphenol and bisphenol A were not as clearly defined, with shorter periods of extended diestrus (4-7 days) and/or estrus (3-5 days) intermittently observed throughout the exposure period. Together these data provide a comparison of the 3-day uterotrophic assay with alternative measures of estrogenic activity for a group of test chemicals with a broad range of affinities for the estrogen receptor. These data can be useful during the assessment and validation of methods for screening environmental chemicals for endocrine disrupting activity.

In 1996, the US Environmental Protection Agency was given a mandate by Congress to develop a screening program that would evaluate whether variously identified compounds could affect human health by mimicking or interfering with normal endocrine regulatory functions. Toward this end, the Agency chartered the Endocrine Disruptor Screening and Testing Advisory Committee in October of that year that would serve to recommend a series of in vitro and in vivo protocols designed to provide a comprehensive assessment of a chemical's potential endocrine-disrupting activity. A number of these protocols have undergone subsequent modification by EPA, and this review focuses specifically on the revised in vivo screening procedure recommended under the title Research Protocol for Assessment of Pubertal Development and Thyroid Function in Juvenile Female Rats. Background literature has been provided that summarizes what is currently known about pubertal development in the female rat and the influence of various forms of pharmaceutical and toxicological insult on this process and on thyroid activity. Finally, a section is included that discusses technical issues that should be considered if the specified pubertal endpoints are to be measured and successfully evaluated.

To test the hypothesis that a transient increase in prolactin (PRL) secretion prior to puberty can result in an alteration of the adult prostate, male rats were exposed from postnatal Days (PND) 22 to 32 to compounds that increase PRL secretion. These compounds included pimozide (a dopamine antagonist), estradiol-17beta, and bisphenol A (a monomer of polycarbonate plastics reported to have weak estrogenic activity). During dosing, pimozide (PIM), bisphenol A (BPA), and estradiol-17beta (E(2)) stimulated an increased secretion of PRL. At 120 days of age, the lateral prostate weight was increased in the PIM and BPA groups as compared to the vehicle-injected controls. Examination of the prostates revealed inflammation in the lateral lobes of all treated groups. Results of a myeloperoxidase assay, a quantitative assay to assess acute inflammation, indicated an increase in the percentage of males with neutrophil infiltrate in the lateral prostates of the PIM and E(2) treatment groups compared to their respective controls. The histological evaluations of these tissues confirmed an increase in luminal polymorphonuclear cells and interstitial mononuclear cells of the lateral prostates in all treatment groups. Administration of the dopamine agonist, bromocriptine, to the estradiol-implanted males from PND 22 to 32 reversed the induction of lateral prostate inflammation by estradiol, suggesting that PRL was necessary for the inflammatory effect. This study demonstrates that prepubertal exposures to compounds that increase PRL secretion, albeit through different mechanisms, can increase the incidence of lateral prostate inflammation in the adult.

The reproductive properties of estrogen are well established, but it is now evident that this steroid hormone has substantial modulatory capabilities in nonreproductive systems. For example, estrogen may be neuroprotective as Alzheimer's disease progresses more slowly in women receiving hormone replacement therapy, and Parkinson's disease affects more men than women. Gender affects both the functional biochemical responses of the nigral-striatal pathway to dopaminergically active compounds. To begin to evaluate the possible neuroprotective effects of estrogen in this pathway, we first determined if gender affected dopaminergic striatal neurotoxicity induced by two different neurotoxicants, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Both agents induced greater neurotoxicity in males than females as evidenced by greater striatal dopamine (DA) depletions. An examination of striatal levels of 1-methyl-4-phenylpyridium ion (MPP+) following MPTP treatment established that the observed gender differences were not due to metabolic/pharmacokinetic variables. The neurotoxicity of MPTP was then examined in ovariectomized (OVX) mice. Estrogen replacement reduced the DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) depletions as well as the glial fibrillary acidic protein (GFAP) elevation induced by MPTP, which indicates that estrogen has neuroprotective properties in this model of striatal dopaminergic neurotoxicity. Surprisingly, estrogen supplementation did not protect against the neurotoxic effects of MPTP in intact 2-yr-old intact female mice, suggesting that low endogenous levels of estrogen may provide neuroprotection.

The reproductive properties of estrogen are well established, but it is now evident that this steroid hormone has substantial modulatory capabilities in nonreproductive systems. For example, estrogen may be neuroprotective as Alzheimer's disease progresses more slowly in women receiving hormone replacement therapy, and Parkinson's disease affects more men than women. Gender affects both the functional and biochemical responses of the nigral-striatal pathway to dopaminergically active compounds. To begin to evaluate the possible neuroprotective effects of estrogen in this pathway, we first determined if gender affected the dopaminergic striatal neurotoxicity induced by two different neurotoxicants, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Both agents induced greater neurotoxicity in males than females as evidenced by greater striatal dopamine (DA) depletions. An examination of striatal levels of 1-methyl-4-phenylpyridium ion (MPP + ) following MPTP treatment established that the observed gender differences were not due to metabolic/pharmacokinetic variables. The neurotoxicity of MPTP was then examined in ovariectomized (OVX) mice. Estrogen replacement reduced the DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) depletions as well as the glial fibrillary acidic protein (GFAP) elevation induced by MPTP, which indicates that estrogen has neuroprotective properties in this model of striatal dopaminergic neurotoxicity. Surprisingly, estrogen supplementation did not protect against the neurotoxic effects of MPTP in intact 2-yr-old intact female mice, suggesting that low endogenous levels of estrogen may provide neuroprotection.

Comparative molecular field analysis (CoMFA), a three-dimensional quantitative structure-activity relationship (3D-QSAR) paradigm, was used to examine the estrogen receptor (ER) binding affinities of a series of structurally diverse natural, synthetic, and environmental chemicals of interest. The CoMFA/3D-QSAR model is statistically robust and internally consistent, and successfully illustrates that the overall steric and electrostatic properties of structurally diverse ligands for the estrogen receptor are both necessary and sufficient to describe the binding affinity. The ability of the model to accurately predict the ER binding affinity of an external test set of molecules suggests that structure-based 3D-QSAR models may be used to supplement the process of endocrine disruptor identification through prioritization of novel compounds for bioassay. The general application of this 3D-QSAR model within a toxicological framework is, at present, limited only by the quantity and quality of biological data for relevant biomarkers of toxicity and hormonal responsiveness.

Vinclozolin, a dicarboximide fungicide, alters morphological sex differentiation in male rats following perinatal exposure. The occurrence of these abnormalities correlates with the in vivo formation of two antiandrogenic metabolites of vinclozolin, (i.e. 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (Mt) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2)), which are potent inhibitors of rat androgen receptor binding. As steroid hormone receptors exhibit promiscuity in their ability to bind different ligands, the present study evaluated the ability of these vinclozolin metabolites to bind to the estrogen (ER) and progesterone (PR) receptors in vitro, and to alter ER and PR function following in vivo exposure. To this end, in vitro ligand binding assays demonstrated that both M1 and M2 can compete with endogenous ligand for binding to the PR (Ki = 400 and 60 microM, respectively). In contrast, neither metabolite exhibited the ability to bind ER. Subsequent in vivo studies to evaluate the potential of vinclozolin to alter ER or PR function demonstrate that, (1) the estrogen-dependent increases in uterine weight and PR induction were not altered by vinclozolin; (2) the distribution of nuclear and cytosolic PR was not altered following short-term vinclozolin exposure; and (3) vinclozolin did not disrupt ovulation in cycling female rats. These studies indicate that although vinclozolin metabolites can compete for binding to the PR in vitro, concentrations of these metabolites do not reach sufficient levels to disrupt female reproductive function following short-term in vivo exposure to vinclozolin. In addition, these studies demonstrate the importance of correlating in vitro receptor binding data with in vivo studies in order to understand the physiological consequences of exposure to environmental toxicants.

Epidermal growth factor (EGF) and its receptor (EGF-R) have been implicated as mediators for estrogen induced cellular growth. This study examines whether the action of the estrogenic pesticide methoxychlor (MXC) parallels the action of 17 beta-estradiol (E2) on uterine EGF-R. Administration of 20 micrograms E2/sexually immature female rat increased 125I-EGF binding to membranes extracted from whole uteri 175% over endogenous levels, while 500 mg MXC/kg led to a 156% increase. E2 in both 20 and 40 micrograms/rat doses and 500 mg MXC/kg led to maximal stimulation over endogenous levels, 12-h posttreatment. Rats were treated with E2, MXC, or vehicle plus 100 micrograms actinomycin-D (ACT-D) or 100 micrograms cycloheximide (CYCLO) per rat to determine if mRNA transcription and translation are involved in the increased EGF-R binding following estrogenic treatment. Only ACT-D inhibited the estrogenic stimulation of EGF-R binding, resulting in a 44% decrease when given concurrently with E2 or MXC, suggesting transcription is required. Additionally, ACT-D decreased endogenous receptor levels by 55%. No other differences were detected. When EGF-R binding data were analyzed by the method of Scatchard, both E2 and MXC, at maximal dosages, elevated uterine EGF-R binding sites by over 200% after 12 h as measured by maximal binding (Bmax) with no significant difference in dissociation constant (Kd) values. These results demonstrate that both E2 and MXC can stimulate the number of EGF-R binding sites without significantly altering the receptor binding affinity (Kd). Further, this stimulation is time dependent and is affected by dose.

The increase in the number of reports of abnormalities in male sex development in wildlife and humans coincided with the introduction of 'oestrogenic' chemicals such as DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) into the environment. Although these phenotypic alterations are thought to be mediated by the oestrogen receptor, they are also consistent with inhibition of androgen receptor-mediated events. Here we report that the major and persistent DDT metabolite, p,p'-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene), has little ability to bind the oestrogen receptor, but inhibits androgen binding to the androgen receptor, androgen-induced transcriptional activity, and androgen action in developing, pubertal and adult male rats. The results suggest that abnormalities in male sex development induced by p,p'-DDE and related environmental chemicals may be mediated at the level of the androgen receptor.

Prolonged exposure of the GnRH receptor to high concentrations of GnRH leads to receptor down-regulation. The role of altered receptor biosynthesis in this agonist-induced receptor down-regulation was investigated in the mouse gonadotrope cell line, alpha T3-1 cells. After exposure to 1 microM GnRH for 24 h, the number of GnRH receptor-binding sites in alpha T3-1 cells decreased to 25 +/- 6% of the control levels. No corresponding changes were observed in GnRH receptor messenger RNA (mRNA) using either quantitative ribonuclease protection/solution hybridization assay or Northern blot analysis. However, when the ability of this RNA to direct the synthesis of functional GnRH receptors was examined by quantitative assessment of the voltage clamp response in Xenopus oocytes, GnRH-induced currents in oocytes injected with RNA isolated from down-regulated cells was reduced to 40 +/- 13% of the response obtained after the injection of RNA from control alpha T3-1 cells. Thus, although GnRH receptor mRNA levels were not altered, the ability of cellular RNA isolated from alpha T3-1 cells to direct the synthesis of functional GnRH receptors was regulated in concert with receptor binding. To investigate the possibility that GnRH receptor mRNA translational efficiency was reduced, the distribution of polyribosome-associated GnRH receptor mRNA was studied. Polyribosome-associated mRNA was separated by linear sucrose gradient, and GnRH receptor mRNA distribution was determined by ribonuclease protection assay. GnRH receptor mRNA distribution shifted from the largest to smaller polyribosome and monosome fractions in cells exposed to GnRH compared to controls. The weighted mean of GnRH receptor mRNA distribution among eight fractions shifted from fraction 5.924 +/- 0.06 in control polysomes to fraction 5.45 +/- 0.219 for polysomes from down-regulated cells (P < 0.05). These results demonstrate that GnRH receptor down-regulation is accompanied by decreased GnRH receptor mRNA translation in the absence of any change in GnRH receptor mRNA levels. These data suggest that decreased efficiency of GnRH receptor mRNA translation contributes to the down-regulation of this receptor in alpha T3-1 cells.

Lindane, gamma-1,2,3,4,5,6-hexachlorocyclohexane (gamma-HCH), has been shown to disrupt reproductive function in mammals. Many of these adverse effects on female reproduction such as alterations in sexual receptivity, disrupted ovarian cyclicity, reduction in uterine weight and termination of pregnancy are thought to be due to altered ovarian hormone secretions and/or an impaired response to circulating estrogen. It has been suggested that gamma-HCH may block the response of estrogen-dependent tissues to estradiol via an interaction with the estrogen receptor. To test this hypothesis, estrogen (ER) and progesterone (PR) receptor affinity and number were evaluated in sexually immature, 17 beta-estradiol-3-benzoate (EB)-primed Long Evans female rats following exposure to vehicle or gamma-HCH (40 mg/kg) for 7 days (Study 1) and in adult, ovariectomized EB-primed Long-Evans rats following gavage with vehicle or gamma-HCH (0, 10, 20, or 40 mg/kg) for 5 days (Study 2). Chlordecone (kepone; 40 mg/kg; i.p.) was used in Study 2 as a positive control for the alteration of the estrogen-induction of PR in the pituitary. Neither gamma-HCH nor chlordecone altered serum estradiol concentrations. gamma-HCH did not change the ER number (1, 24, or 30 h after EB) or the estrogen-dependent induction of PR (24 or 48 h after EB) in the hypothalamus (HYP), pituitary, or uterus. These data indicate that the effects of gamma-HCH on the female reproductive system do not involve an alteration in the ER and that heterogeneity exists between target tissues in their response to xenobiotics.

Recent studies with vinclozolin, a dicarboximide fungicide, demonstrate that perinatal exposure to 100 mg vinclozolin/kg/day from Gestational Day 14 through Postnatal Day 3 inhibits morphological sex differentiation. At 1 year, treated male rats exhibited hypospadias, cleft phallus, suprainguinal ectopic testes, a vaginal pouch, epididymal and testicular granulomas, and atrophic seminal vesicles and ventral prostate glands. This pattern of malformations suggests that this fungicide possesses antiandrogenic activity. To test this hypothesis, we examined the ability of vinclozolin to inhibit the conversion of testosterone to the more potent androgen 5 alpha-dihydrotestosterone via 5 alpha-reductase (EC 1.3.1.22) and to compete with androgen for binding to the androgen receptor. The results indicate that neither vinclozolin nor its degradation products, 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), inhibit 5 alpha-reductase activity. Although the ability of vinclozolin to compete with androgen for binding to the androgen receptor was weak (Ki > 700 microM), the two vinclozolin metabolites, M1 and M2, were effective antagonists of androgen receptor binding (Ki = 92 and 9.7 microM, respectively). As the concentrations of M1 in the serum of pregnant rats and their pups on Postnatal Day 3 meet or exceed the in vitro Ki for androgen receptor inhibition, we suggest that the demasculinizing effects of vinclozolin exposure in vivo also may be mediated via the antiandrogenic metabolites M1 and/or M2.

In primary cultures of rat pituitary, GnRH receptor has been reported to up-regulate when exposed to GnRH. The molecular basis of this regulation could, in theory, involve modulation of gene transcription, RNA processing or stability, translation, and/or receptor degradation. The role of altered biosynthesis was investigated in the mouse gonadotrope cell line, alpha T3-1 cells, by studying the homologous up-regulation of GnRH receptor binding, mRNA levels, and mRNA activity. After GnRH exposure, ligand binding was performed on alpha T3-1 cell membranes. To correlate changes in receptor number and measurements of biosynthetic activity, GnRH receptor mRNA levels were quantitated by solution hybridization/RNase protection assay and Northern blot analysis, and the capacity of RNA from treated cells to direct the synthesis of new receptors (mRNA activity) was evaluated using a Xenopus oocyte-based bioassay. GnRH agonist radioligand binding assay results showed that exposure of alpha T3-1 cells to 10(-10) or 10(-8) M GnRH for 20 min induced an approximately 50% increase in the number of GnRH receptors, similar to previously reported results in rat pituitary primary culture. Despite the increase in receptor number, however, cytosolic and total GnRH receptor mRNA levels assayed by solution hybridization/RNase protection assay with GnRH receptor cRNA probes and by Northern blot analysis were not altered. In contrast to the unchanging mRNA levels, the measurements of mRNA activity paralleled the changes observed at the binding level. alpha T3-1 RNA from treated and control cells were injected into Xenopus oocytes, and the GnRH-induced Cl- current was quantified 48 h later by voltage clamp recording of the response to GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies demonstrate that gonadotroph responsiveness to GnRH, GnRH binding, and the apparent number of GnRH receptors are all increased by 17 beta-estradiol (E) or inhibin (IN) in ovine pituitary cultures. Progesterone (P) attenuates these effects. To explore differences between the effects of IN and E on GnRH binding, a detailed time-course study was performed. The results indicate that after 48 h, IN had a greater effect on binding of a GnRH agonist (5-fold increase) than E (3-fold increase), but was slower to act initially. A combined treatment of IN and E gave a partially additive effect at 48 h (6.5-fold increase). The mechanism of receptor regulation in this system is not known, but could involve synthesis, recycling, or modification of GnRH receptors. To investigate the contribution of altered receptor biosynthesis to the regulation of receptor levels, a functional Xenopus oocyte-based assay for GnRH receptor mRNA activity was employed. After 48 h of treatment, IN or E each led to a 7- to 8-fold increase in GnRH receptor mRNA activity. Treatment with both hormones led to a 19-fold increase. The increase in mRNA activity induced by either hormone was greatly attenuated by P. Modulation of GnRH receptor mRNA levels suggests that IN, E, and P regulate responsiveness to GnRH in the ovine pituitary at least in part by altering de novo synthesis of GnRH receptors. The differing time courses of action, as assayed by GnRH binding, and the additivity of effects at the mRNA level suggest that IN and E alter mRNA levels via different mechanisms.

17 beta-Estradiol (E) rapidly (5-12 h) induced ovine pituitary cultures to increase their binding of des-Gly10-[D-Ala6] GnRH ethylamide by 2.5- to 4.5-fold. The ED50 for E was near 0.1 nM. Scatchard analysis indicated that E increased binding by increasing receptors for GnRH. GnRH-stimulated release of LH increased 2-fold along with the increase in GnRH receptors, but heightened responsiveness to GnRH disappeared after 27 h of E treatment even though GnRH receptors remained elevated. These data are consistent with the concepts that 1) E increased GnRH receptors, which initially enhanced gonadotroph responsiveness to GnRH, and 2) E subsequently nullified the increased response to GnRH by blocking a mechanism of LH secretion not associated with GnRH receptor number. Further support for this hypothesis came from studies with porcine follicular inhibin. Inhibin in ovine pituitary culture concomitantly increased GnRH receptors by 5-fold and GnRH-stimulated LH secretion by 2-fold. Inhibin maintained both effects long term (48 h). When E was added along with inhibin, the increase in GnRH receptor number became greater than with either hormone alone (7-fold increase at 48 h), but the initial increase in GnRH-stimulated LH secretion was fully inhibited by E at 48 h. The increase in GnRH receptors caused by E may be important physiologically to help create the preovulatory LH surge; the delayed inhibitory action of E on GnRH-stimulated LH secretion may limit the LH surge.

Treatments (48 h) with highly purified bovine or porcine inhibins (10 ng/ml) induced ovine pituitary cells to increase their binding for des-Gly10-[D-Ala6]LHRH-ethylamide by 3.6- and 5-fold, respectively. Studies with less pure inhibin from porcine follicles showed that increased binding could reach 7-fold within 48 h and was due to higher numbers of receptors for GnRH. The 48-h increase in GnRH receptors was linear with time and was rapidly reversible, since removal of inhibin at 24 h decreased GnRH binding below control levels at 48 h. Inhibin (bovine or porcine) also increased GnRH-stimulated secretion of LH by 2-fold. The ED50 for both inhibin actions noted above was in the range of 0.5-2.0 ng/ml (in terms of highly purified bovine inhibin). Progesterone (P) totally counteracted inhibin induction of GnRH binding and GnRH-stimulated LH secretion at 48 h. In the absence of inhibin, P decreased GnRH binding below control levels by as much as 80% within 48 h, but did not affect GnRH-stimulated LH secretion at 48 h. The ED50 for P action was near 1 nM, which is within the physiological range for P during the luteal phase of the sheep estrous cycle. The data suggest that P may act during the luteal phase to decrease receptors for GnRH. The rapid decrease in P during the 48 h before the preovulatory LH surge should permit GnRH receptors to rise under the influence of inhibin (and estradiol) to boost gonadotroph responsiveness to GnRH so the LH surge may occur to its fullest.

The acaricide chlordimeform has been reported to have adverse effects in mammals that may be mediated by an interaction with alpha-adrenergic receptors. Since the hormonal signals involved in the regulation of reproductive function are themselves under hypothalamic adrenergic control, the present study was designed to investigate the effects of acute exposure to this compound on the hypothalamic-pituitary-testicular axis. Male rats given two intraperitoneal injections of chlordimeform-HCl (20 or 50 mg/kg) spaced 12 hr apart showed 24-hr declines in serum gonadotropins at 50 mg/kg that were paralleled by a drop in testosterone. These changes returned to control levels by 96 hr. Thyroid-stimulating hormone exhibited a dose-response decline that was accompanied by a similar decrease in serum thyroid hormone levels. The norepinephrine-stimulated secretion in vitro of gonadotropin-releasing hormone from hypothalamic explants was suppressed at the higher dose, while LH release from pituitary fragments in culture was unaffected. Although measurements of the in vitro release of other pituitary hormones suggest that there could be some direct pituitary effects of the compound, it appears likely that chlordimeform is able to influence endocrine regulation adversely within the reproductive system by interfering with hypothalamic alpha-adrenergic activity.

Groups of male Long-Evans rats 30, 50, or 70 d old were injected subcutaneously (sc) with a single dose of 0, 5.5, 11.5, or 24.6 mumol Cd/kg as cadmium chloride. All animals were killed 60 d after treatment. At 2 h prior to sacrifice, the rats were injected sc with 100 IU human chorionic gonadotrophin (hCG) to maximally stimulate serum testosterone concentrations. After sacrifice the testes, epididymides and seminal vesicles were removed and weighed. Cardiac blood was taken, and serum concentrations of testosterone (sT) and follicle-stimulating hormone (sFSH) were determined. Sperm concentration in luminal fluid collected from the vas deferens was determined. Significant (p less than 0.01) dose-dependent effects for all measured reproductive parameters were noted in the 70-d-old animals, while no effects were seen in the 30- or 50-d-old rats in either seminal vesicles weight or hCG-stimulated sT concentration. In the absence of significant (p greater than 0.05) changes in body weight gain, effects were seen in testes and epididymides weight, sperm concentration, and sFSH in the 70-d-old rats at Cd doses that were lower than those necessary to bring about similar changes in the 30- or 50-d-old animals. The sensitive indicators of Cd exposure in all age groups were testicular weight greater than epididymal weight greater than vas deferens sperm concentration greater than sFSH concentration. Seminal vesicle weight and sT concentration were found to be the least sensitive. Regression analyses indicated a significant interaction of age with dose; the 70-d-old rats required 30-61% less Cd/kg to cause a 50% change in a measured parameter than did the 30-d-old animals, while the 50-d-old rats required 15-47% less.

Adult male Sprague-Dawley rats were injected sc with cadmium (Cd, as cadmium chloride) in doses ranging from 1.6 to 152 mumol Cd/kg body weight (body wt). Fourteen days after dosing, animals were evaluated for reproductive damage. Evaluations for each animal included testes, seminal vesicles, and epididymides weights, vas deferens sperm concentration, and human chorionic gonadotropin (hCG)-stimulated serum testosterone concentration. Since 10 to 60% mortality occurred in the two highest dose groups (74 and 152 mumol/kg), no additional evaluations were conducted in these groups. The weights of the testes, seminal vesicles, and epididymides were reduced at least 40 to 50% in groups receiving 16 or 33 mumol Cd/kg while vas deferens sperm concentrations and hCG-stimulated serum testosterone concentrations were essentially zero. Significant depressions in the sperm concentrations and in the hCG-stimulated serum testosterone concentrations were found in animals receiving the two lowest doses (1.6 and 7.4 mumol Cd/kg) although no changes in tissue weights were observed in these animals. Curve-linear regression analyses for the dose responsiveness of these parameters demonstrated that serum testosterone concentration initially decreased at a rate of 19%/mumol Cd/kg, respectively, and was the most sensitive to Cd exposure. The initial rates of decrease for sperm concentrations and for seminal vesicles, testes, and epididymides weight were 6.45, 5.30, 4.19, and 2.45%/mumol Cd/kg, respectively, and were less responsive to Cd exposure than serum testosterone levels.

Long-Evans rat pups were dosed orally from birth to 21 d with particulate Mn3O4 to obtain a daily dose of 0, 71, or 214 micrograms Mn/body weight . d. Assessments of the hypothalamic, pituitary, or testicular functions were determined by measuring the endogenous or stimulated serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and/or testosterone (T) at 21 or 28 d of age. Body, testes, and seminal vesicles weight and tissue concentrations of Mn were also evaluated. Only slight Mn treatment effects were seen in body and testes weights. No effects were seen either on unstimulated or stimulated FSH or LH serum concentrations. Although no Mn treatment effects were seen on endogenous or 2 h human chorionic gonadotropin (hCG) stimulate serum T concentrations, there was a reduction in the serum T following 7 d of hCG stimulation. The hypothalamic Mn concentrations in animals with these reproductive effects were three times those where alterations in the dopaminergic pathway have been reported. However, no indication of hypothalamic or pituitary malfunction was found. These results suggest that the site of Mn damage that causes depression of sustained serum T concentration is in the testicular Leydig cell.