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.

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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).

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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 (E 2 ). A single ATRZ administration was found to induce a prompt and marked increase in progesterone (P 4 ). As exogenous P 4 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, E 2 -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 P 4 at 30 and 60 min that was likely attributable to adrenal secretion. Four exposures also elevated P 4 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 P 4 , 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 P 4 can either augment or attenuate the surge depending on the temporal proximity of exposure to the rise in LH.

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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.

BackgroundTrihalomethanes (THMs) and haloacetic acids (HAAs) are regulated disinfection by-products (DBPs); their joint reproductive toxicity in drinking water is unknown.ObjectiveWe aimed to evaluate a drinking water mixture of the four regulated THMs and five regulated HAAs in a multigenerational reproductive toxicity bioassay.MethodsSprague-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).ResultsMaternal 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×.ConclusionsA 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.CitationNarotsky MG, Klinefelter GR, Goldman JM, DeAngelo AB, Best DS, McDonald A, Strader LF, Murr AS, Suarez JD, George MH, Hunter ES III, Simmons JE. 2015. Reproductive toxicity of a mixture of regulated drinking-water disinfection by-products in a multigenerational rat bioassay. Environ Health Perspect 123:564–570; http://dx.doi.org/10.1289/ehp.1408579