Maternal and Neonatal Effects of Maternal Oral Exposure to Perfluoro-2-methoxyacetic Acid (PFMOAA) during Pregnancy and Early Lactation in the Sprague-Dawley Rat.

Perfluoro-2-methoxyacetic acid (PFMOAA) is a short-chain perfluoroalkyl ether carboxylic acid that has been detected at high concentrations (∼10 µg/L) in drinking water in eastern North Carolina, USA, and in human serum and breastmilk in China. Despite documented human exposure there are almost no toxicity data available to inform risk assessment of PFMOAA. Here we exposed pregnant Sprague-Dawley rats to a range of PFMOAA doses (10-450 mg/kg/d) via oral gavage from gestation day (GD) 8 to postnatal day (PND) 2 and compared results to those we previously reported for perfluorooctanoic acid (PFOA) and hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX). Newborn pups displayed reduced birthweight (≥30 mg/kg), depleted liver glycogen concentrations (all doses), hypoglycemia (≥125 mg/kg), and numerous significantly altered genes in the liver associated with fatty acid and glucose metabolism similar to gene changes produced by HFPO-DA. Pup survival was significantly reduced at ≥125 mg/kg, and at necropsy on PND2 both maternal and neonatal animals displayed increased liver weights, increased serum aspartate aminotransferase (AST), and reduced serum thyroid hormones at all doses (≥10 mg/kg). Pups also displayed highly elevated serum cholesterol at all doses. PFMOAA concentrations in serum and liver increased with maternal oral dose in both maternal and F1 animals and were similar to those we reported for PFOA but considerably higher than HFPO-DA. We calculated 10% effect levels (ED10 or EC10) and relative potency factors (RPF; PFOA = index chemical) among the three compounds based on maternal oral dose and maternal serum concentration (µM). Reduced pup liver glycogen, increased liver weights and reduced thyroid hormone levels (maternal and pup) were the most sensitive end points modeled. PFMOAA was ∼3-7-fold less potent than PFOA for most end points based on maternal serum RPFs, but slightly more potent for increased maternal and pup liver weights. PFMOAA is a maternal and developmental toxicant in the rat producing a constellation of adverse effects similar to PFOA and HFPO-DA.

In vitro activity of a panel of per- and polyfluoroalkyl substances (PFAS), fatty acids, and pharmaceuticals in peroxisome proliferator-activated receptor (PPAR) alpha, PPAR gamma, and estrogen receptor assays.

Data demonstrate numerous per- and polyfluoroalkyl substances (PFAS) activate peroxisome proliferator-activated receptor alpha (PPARα), however, additional work is needed to characterize PFAS activity on PPAR gamma (PPARγ) and other nuclear receptors. We utilized in vitro assays with either human or rat PPARα or PPARγ ligand binding domains to evaluate 16 PFAS (HFPO-DA, HFPO-DA-AS, NBP2, PFMOAA, PFHxA, PFOA, PFNA, PFDA, PFOS, PFBS, PFHxS, PFOSA, EtPFOSA, and 4:2, 6:2 and 8:2 FTOH), 3 endogenous fatty acids (oleic, linoleic, and octanoic), and 3 pharmaceuticals (WY14643, clofibrate, and the metabolite clofibric acid). We also tested chemicals for human estrogen receptor (hER) transcriptional activation. Nearly all compounds activated both PPARα and PPARγ in both human and rat ligand binding domain assays, except for the FTOH compounds and PFOSA. Receptor activation and relative potencies were evaluated based on effect concentration 20% (EC20), top percent of max fold induction (pmaxtop), and area under the curve (AUC). HFPO-DA and HFPO-DA-AS were the most potent (lowest EC20, highest pmaxtop and AUC) of all PFAS in rat and human PPARα assays, being slightly less potent than oleic and linoleic acid, while NBP2 was the most potent in rat and human PPARγ assays. Only PFHxS, 8:2 and 6:2 FTOH exhibited hER agonism >20% pmax. In vitro measures of human and rat PPARα and PPARγ activity did not correlate with oral doses or serum concentrations of PFAS that induced increases in male rat liver weight from the National Toxicology Program 28-d toxicity studies. Data indicate that both PPARα and PPARγ activation may be molecular initiating events that contribute to the in vivo effects observed for many PFAS.

Quantification of tetrabromo benzoic acid and tetrabromo phthalic acid in rats exposed to the flame retardant Uniplex FPR-45.

The first withdrawal of certain polybrominated diphenyl ethers flame retardants from the US market occurred in 2004. Since then, use of brominated non-PBDE compounds such as bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP) and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) in commercial formulations has increased. Assessing human exposure to these chemicals requires identifying metabolites that can potentially serve as their biomarkers of exposure. We administered by gavage a dose of 500 mg/Kg bw of Uniplex FRP-45 (>95 % BEH-TEBP) to nine adult female Sprague-Dawley rats. Using authentic standards and mass spectrometry, we positively identified and quantified 2,3,4,5-tetrabromo benzoic acid (TBBA) and 2,3,4,5-tetrabromo phthalic acid (TBPA) in 24-h urine samples collected 1 day after dosing the rats and in serum at necropsy, 2 days post-exposure. Interestingly, TBBA and TBPA concentrations correlated well (R (2) = 0.92). The levels of TBBA, a known metabolite of EH-TBB, were much higher than the levels of TBPA both in urine and serum. Because Uniplex FRP-45 was technical grade and EH-TBB was present in the formulation, TBBA likely resulted from the metabolism of EH-TBB. Taken together, our data suggest that TBBA and TBPA may serve as biomarkers of exposure to non-PBDE brominated flame retardant mixtures. Additional research can provide useful information to better understand the composition and in vivo toxicokinetics of these commercial mixtures.

Dose Addition Models Accurately Predict the Subacute Effects of a Mixture of Perfluorooctane Sulfonate and Perfluorooctanoic Acid on Japanese Quail (Coturnix japonica) Chick Mortality.

Biomonitoring data have consistently demonstrated that fish, wildlife, and humans are exposed to multiple per- and polyfluoroalkyl substances (PFAS) in drinking water and foods. Despite ubiquitous exposure to mixtures of PFAS, there is a lack of in vivo PFAS mixture research that addresses whether these chemicals act in a cumulative, dose-additive (DA) manner or whether they behave independently. For this reason, there is a critical need for mixtures studies designed to evaluate the cumulative toxicity and potential chemical interactions to support the assessment of human and ecological risks and also to define appropriate regulatory actions. Our primary objective was to evaluate the previously published Japanese quail chick mortality concentration-response data for perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and the mixture of PFOS + PFOA and to use statistical modeling to determine whether the effects of the mixtures were accurately predicted by either DA or response addition modeling. In addition, we wanted to compare different DA models to determine whether one model produced more accurate predictions than the others. Our results support the hypothesis of cumulative effects on shared endpoints from PFOA and PFOS co-exposure and DA approaches for predictive estimates of cumulative effects. Given the limited number of in vivo studies that have been executed with enough individual PFAS and PFAS mixture concentration-response data to test the hypothesis of DA for PFAS mixtures, this re-analysis of the data is an important contribution to our understanding of how PFAS mixtures act. The analysis will provide support for regulatory agencies as they begin to implement PFAS cumulative hazard assessments in higher vertebrates. Environ Toxicol Chem 2024;43:97-104. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Use of genomic data in risk assessment case study: I. Evaluation of the dibutyl phthalate male reproductive development toxicity data set.

A case study was conducted, using dibutyl phthalate (DBP), to explore an approach to using toxicogenomic data in risk assessment. The toxicity and toxicogenomic data sets relative to DBP-related male reproductive developmental outcomes were considered conjointly to derive information about mode and mechanism of action. In this manuscript, we describe the case study evaluation of the toxicological database for DBP, focusing on identifying the full spectrum of male reproductive developmental effects. The data were assessed to 1) evaluate low dose and low incidence findings and 2) identify male reproductive toxicity endpoints without well-established modes of action (MOAs). These efforts led to the characterization of data gaps and research needs for the toxicity and toxicogenomic studies in a risk assessment context. Further, the identification of endpoints with unexplained MOAs in the toxicity data set was useful in the subsequent evaluation of the mechanistic information that the toxicogenomic data set evaluation could provide. The extensive analysis of the toxicology data set within the MOA context provided a resource of information for DBP in attempts to hypothesize MOAs (for endpoints without a well-established MOA) and to phenotypically anchor toxicogenomic and other mechanistic data both to toxicity endpoints and to available toxicogenomic data. This case study serves as an example of the steps that can be taken to develop a toxicological data source for a risk assessment, both in general and especially for risk assessments that include toxicogenomic data.

Dose additive maternal and offspring effects of oral maternal exposure to a mixture of three PFAS (HFPO-DA, NBP2, PFOS) during pregnancy in the Sprague-Dawley rat.

Simultaneous exposure to multiple per- and polyfluoroalkyl substances (PFAS) is common in humans across the globe. Individual PFAS are associated with adverse health effects, yet the nature of mixture effects after exposure to two or more PFAS remains unclear. Previously we reported that oral administration of hexafluoropropylene oxide-dimer acid (HFPO-DA, or GenX), Nafion byproduct 2 (NBP2), or perfluorooctane sulfonate (PFOS) individually during pregnancy produced maternal and F1 effects. Here, we hypothesized that responses to the combined exposure to these three PFAS would be dose additive. Pregnant Sprague-Dawley rats were exposed to a fixed-ratio equipotent mixture where the top dose contained each PFAS at their ED50 for neonatal mortality (100 % dose = PFOS 3 mg/kg; NBP2 10 mg/kg; HFPO-DA 110 mg/kg), followed by a dilution series (33.3, 10, 3.3, and 1 %) and vehicle controls (0 % dose). Consistent with the single chemical studies, dams were exposed from gestation day (GD)14-18 or from GD8-postnatal day (PND2). Fetal and maternal livers on GD18 displayed multiple significantly upregulated genes associated with lipid and carbohydrate metabolism at all dose levels, while dams displayed significantly increased liver weight (≥3.3 % dose) and reduced serum thyroid hormones (≥33.3 % dose). Maternal exposure from GD8-PND2 significantly reduced pup bodyweights at birth (≥33.3 % dose) and PND2 (all doses), increased neonatal liver weights (≥3.3 % dose), increased pup mortality (≥3.3 % dose), and reduced maternal bodyweights and weight gain at the top dose. Echocardiography of adult F1 males and females identified significantly increased left ventricular anterior wall thickness (~10 % increase), whereas other cardiac morphological, functional, and transcriptomic measures were unaffected. Mixture effects in maternal and neonatal animals conformed to dose addition using a relative potency factor (RPF) analysis. Results support dose addition-based cumulative assessment approaches for estimating combined effects of PFAS co-exposure.

Modelling defined mixtures of environmental oestrogens found in domestic animal and sewage treatment effluents using an in vitro oestrogen-mediated transcriptional activation assay (T47D-KBluc).

There is growing concern of exposure of fish, wildlife and humans to water sources contaminated with oestrogens and the potential impact on reproductive health. Environmental oestrogens can come from various sources including concentrated animal feedlot operations (CAFO), municipal waste, agricultural and industrial effluents. US EPA's drinking water contaminant candidate list 3 (CCL3) includes several oestrogenic compounds. Although these contaminants are currently not subject to any proposed or promulgated national primary drinking water regulations, they are known or anticipated to occur in public water systems and may require future regulation under the Safe Drinking Water Act. Using an in vitro transcriptional activation assay, this study evaluated oestrogens from CCL3 both individually and as a seven oestrogen mixture (fixed ray design) over a broad range of concentrations, including environmentally relevant concentrations. Log EC(50) and Hillslope values for individual oestrogens were as follows: estrone, -11.92, 1.283; estradiol-17α, -9.61, 1.486; estradiol-17ß, 11.77, 1.494; estriol, -11.14, 1.074; ethinyl estradiol-17α, -12.63, 1.562; Mestranol, -11.08, 0.809 and Equilin, -11.48, 0.946. In addition, mixtures that mirrored the primary oestrogens found in swine, poultry and dairy CAFO effluent (fixed-ratio ray design), and a ternary mixture (4 × 4 × 4 factorial design) of oestrogens found in hormone replacement therapy and/or oral contraceptives were tested. Mixtures were evaluated for additivity using both the concentration addition (CA) model and oestrogen equivalence (EEQ) model. For each of the mixture studies, a broad range of concentrations were tested, both above and below environmentally relevant concentrations. Results show that the observed data did not vary consistently from either the CA or EEQ predictions for any mixture. Therefore, either the CA or EEQ model should be useful predictors for modelling oestrogen mixtures.

Cumulative maternal and neonatal effects of combined exposure to a mixture of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) during pregnancy in the Sprague-Dawley rat.

Globally, biomonitoring data demonstrate virtually all humans carry residues of multiple per- and polyfluoroalkyl substances (PFAS). Despite pervasive co-exposure, limited mixtures-based in vivo PFAS toxicity research has been conducted. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are commonly detected PFAS in human and environmental samples and both produce adverse effects in laboratory animal studies, including maternal and offspring effects when orally administered during pregnancy and lactation. To evaluate the effects of combined exposure to PFOA and PFOS, we orally exposed pregnant Sprague-Dawley rats from gestation day 8 (GD8) to postnatal day 2 (PND2) to PFOA (10-250 mg/kg/d) or PFOS (0.1-5 mg/kg/d) individually to characterize effects and dose response curve parameters, followed by a variable-ratio mixture experiment with a constant dose of PFOS (2 mg/kg/d) mixed with increasing doses of PFOA (3-80 mg/kg/d). The mixture study design was intended to: 1) shift the PFOA dose response curves for endpoints shared with PFOS, 2) allow comparison of dose addition (DA) and response addition (RA) model predictions, 3) conduct relative potency factor (RPF) analysis for multiple endpoints, and 4) avoid overt maternal toxicity. Maternal serum and liver concentrations of PFOA and PFOS were consistent between the individual chemical and mixture experiments. Combined exposure with PFOS significantly shifted the PFOA dose response curves towards effects at lower doses compared to PFOA-only exposure for multiple endpoints and these effects were well predicted by dose addition. For endpoints amenable to mixture model analyses, DA produced equivalent or better estimates of observed data than RA. All endpoints evaluated were accurately predicted by RPF and DA approaches except for maternal gestational weight gain, which produced less-than-additive results in the mixture. Data support the hypothesis of cumulative effects on shared endpoints from PFOA and PFOS co-exposure and dose additive approaches for predictive estimates of mixture effects.

Developmental toxicity of Nafion byproduct 2 (NBP2) in the Sprague-Dawley rat with comparisons to hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) and perfluorooctane sulfonate (PFOS).

Nafion byproduct 2 (NBP2) is a polyfluoroalkyl ether sulfonic acid that was recently detected in surface water, drinking water, and human serum samples from monitoring studies in North Carolina, USA. We orally exposed pregnant Sprague-Dawley rats to NBP2 from gestation day (GD) 14-18 (0.1-30 mg/kg/d), GD17-21, and GD8 to postnatal day (PND) 2 (0.3-30 mg/kg/d) to characterize maternal, fetal, and postnatal effects. GD14-18 exposures were also conducted with perfluorooctane sulfonate (PFOS) for comparison to NBP2, as well as data previously published for hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX). NBP2 produced stillbirth (30 mg/kg), reduced pup survival shortly after birth (10 mg/kg), and reduced pup body weight (10 mg/kg). Histopathological evaluation identified reduced glycogen stores in newborn pup livers and hepatocyte hypertrophy in maternal livers at ≥ 10 mg/kg. Exposure to NBP2 from GD14-18 reduced maternal serum total T3 and cholesterol concentrations (30 mg/kg). Maternal, fetal, and neonatal liver gene expression was investigated using RT-qPCR pathway arrays, while maternal and fetal livers were also analyzed using TempO-Seq transcriptomic profiling. Overall, there was limited alteration of genes in maternal or F1 livers from NBP2 exposure with significant changes mostly occurring in the top dose group (30 mg/kg) associated with lipid and carbohydrate metabolism. Metabolomic profiling indicated elevated maternal bile acids for NBP2, but not HFPO-DA or PFOS, while all three reduced 3-indolepropionic acid. Maternal and fetal serum and liver NBP2 concentrations were similar to PFOS, but ∼10-30-fold greater than HFPO-DA concentrations at a given maternal oral dose. NBP2 is a developmental toxicant in the rat, producing neonatal mortality, reduced pup body weight, reduced pup liver glycogen, reduced maternal thyroid hormones, and altered maternal and offspring lipid and carbohydrate metabolism similar to other studied PFAS, with oral toxicity for pup loss that is slightly less potent than PFOS but more potent than HFPO-DA.

A mixture of 15 phthalates and pesticides below individual chemical no observed adverse effect levels (NOAELs) produces reproductive tract malformations in the male rat.

Humans carry residues of multiple synthetic chemicals at any given point in time. Research has demonstrated that compounds with varying molecular initiating events (MIE) that disrupt common key events can act in concert to produce cumulative adverse effects. Congenital defects of the male reproductive tract are some of the most frequently diagnosed malformations in humans and chemical exposures in utero can produce these effects in laboratory animals and humans. Here, we hypothesized that in utero exposure to a mixture of pesticides and phthalates, each of which produce male reproductive tract defects individually, would produce cumulative effects even when each chemical is present at a no observed adverse effect level (NOAEL) specific for male reproductive effects. Pregnant Sprague-Dawley rats were exposed via oral gavage to a fixed-ratio dilution mixture of 5 pesticides (vinclozolin, linuron, procymidone, prochloraz, pyrifluquinazon), 1 pesticide metabolite (dichlorodiphenyldichloroethylene (DDE)), and 9 phthalates (dipentyl, dicyclohexyl, di-2-ethylhexyl, dibutyl, benzyl butyl, diisobutyl, diisoheptyl, dihexyl, and diheptyl) during the critical window of rat fetal masculinization (gestation day 14-18). The top dose (100% dose) contained each compound at a concentration 2-fold greater than the individual chemical NOAEL followed by a dilution series that represented each chemical at NOAEL, NOAEL/2, NOAEL/4, NOAEL/8, NOAEL/15, NOAEL/100, NOAEL/1000. Reduced fetal testis gene expression occurred at NOAEL/15, reduced fetal testis testosterone production occurred at NOAEL/8, reduced anogenital distance, increased nipple retention, and delayed puberty occurred at NOAEL/4, and severe effects including genital malformations and weight reductions in numerous reproductive tissues occurred at NOAEL/2. This study demonstrates that these phthalates and pesticides acted cumulatively to produce adverse effects at doses below which any individual chemical had been shown to produce an effect alone and even though they have different MIEs.

Hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) alters maternal and fetal glucose and lipid metabolism and produces neonatal mortality, low birthweight, and hepatomegaly in the Sprague-Dawley rat.

Hexafluoropropylene oxide dimer acid (HFPO-DA or GenX) is an industrial replacement for the straight-chain perfluoroalkyl substance (PFAS), perfluorooctanoic acid (PFOA). Previously we reported maternal, fetal, and postnatal effects from gestation day (GD) 14-18 oral dosing in Sprague-Dawley rats. Here, we further evaluated the perinatal toxicity of HFPO-DA by orally dosing rat dams with 1-125 mg/kg/d (n = 4 litters per dose) from GD16-20 and with 10-250 mg/kg/d (n = 5) from GD8 - postnatal day (PND) 2. Effects of GD16-20 dosing were similar to those previously reported for GD14-18 dosing and included increased maternal liver weight, altered maternal serum lipid and thyroid hormone concentrations, and altered expression of peroxisome proliferator-activated receptor (PPAR) pathway genes in maternal and fetal livers. Dosing from GD8-PND2 produced similar effects as well as dose-responsive decreased pup birth weight (≥30 mg/kg), increased neonatal mortality (≥62.5 mg/kg), and increased pup liver weight (≥10 mg/kg). Histopathological evaluation of newborn pup livers indicated a marked reduction in glycogen stores and pups were hypoglycemic at birth. Quantitative gene expression analyses of F1 livers revealed significant alterations in genes related to glucose metabolism at birth and on GD20. Maternal serum and liver HFPO-DA concentrations were similar between dosing intervals, indicating rapid clearance, however dams dosed GD8 - PND2 had greater liver weight and gestational weight gain effects at lower doses than GD16-20 dosing, indicating the importance of exposure duration. Comparison of neonatal mortality dose-response curves between HFPO-DA and previously published perfluorooctane sulfonate (PFOS) data indicated that, based on serum concentration, the potency of these two PFAS are similar in the rat. Overall, HFPO-DA is a developmental toxicant in the rat and the spectrum of adverse effects is consistent with prior PFAS toxicity evaluations, such as PFOS and PFOA.

In vitro effects-based method and water quality screening model for use in pre- and post-distribution treated waters.

Recent urban public water supply contamination events emphasize the importance of screening treated drinking water quality after distribution. In vitro bioassays, when run concurrently with analytical chemistry methods, are effective tools to evaluating the efficacy of water treatment processes and water quality. We tested 49 water samples representing the Chicago Department of Water Management service areas for estrogen, (anti)androgen, glucocorticoid receptor-activating contaminants and cytotoxicity. We present a tiered screening approach suitable to samples with anticipated low-level activity and initially tested all extracts for statistically identifiable endocrine activity; performing a secondary dilution-response analysis to determine sample EC50 and biological equivalency values (BioEq). Estrogenic activity was detected in untreated Lake Michigan intake water samples using mammalian (5/49; median: 0.21 ng E2Eq/L) and yeast cell (5/49; 1.78 ng E2Eq/L) bioassays. A highly sensitive (anti)androgenic activity bioassay was applied for the first time to water quality screening and androgenic activity was detected in untreated intake and treated pre-distribution samples (4/49; 0.93 ng DHTEq/L). No activity was identified above method detection limits in the yeast androgenic, mammalian anti-androgenic, and both glucocorticoid bioassays. Known estrogen receptor agonists were detected using HPLC/MS-MS (estrone: 0.72-1.4 ng/L; 17α-estradiol: 1.3-1.5 ng/L; 17ß-estradiol: 1.4 ng/L; equol: 8.8 ng/L), however occurrence did not correlate with estrogenic bioassay results. Many studies have applied bioassays to water quality monitoring using only relatively small samples sets often collected from surface and/or wastewater effluent. However, to realistically adapt these tools to treated water quality monitoring, water quality managers must have the capacity to screen potentially hundreds of samples in short timeframes. Therefore, we provided a tiered screening model that increased sample screening speed, without sacrificing statistical stringency, and detected estrogenic and androgenic activity only in pre-distribution Chicago area samples.

Pilot-scale expanded assessment of inorganic and organic tapwater exposures and predicted effects in Puerto Rico, USA.

A pilot-scale expanded target assessment of mixtures of inorganic and organic contaminants in point-of-consumption drinking water (tapwater, TW) was conducted in Puerto Rico (PR) to continue to inform TW exposures and corresponding estimations of cumulative human-health risks across the US. In August 2018, a spatial synoptic pilot assessment of than 524 organic and 37 inorganic chemicals was conducted in 14 locations (7 home; 7 commercial) across PR. A follow-up 3-day temporal assessment of TW variability was conducted in December 2018 at two of the synoptic locations (1 home, 1 commercial) and included daily pre- and post-flush samples. Concentrations of regulated and unregulated TW contaminants were used to calculate cumulative in vitro bioactivity ratios and Hazard Indices (HI) based on existing human-health benchmarks. Synoptic results confirmed that human exposures to inorganic and organic contaminant mixtures, which are rarely monitored together in drinking water at the point of consumption, occurred across PR and consisted of elevated concentrations of inorganic contaminants (e.g., lead, copper), disinfection byproducts (DBP), and to a lesser extent per/polyfluoroalkyl substances (PFAS) and phthalates. Exceedances of human-health benchmarks in every synoptic TW sample support further investigation of the potential cumulative risk to vulnerable populations in PR and emphasize the importance of continued broad characterization of drinking-water exposures at the tap with analytical capabilities that better represent the complexity of both inorganic and organic contaminant mixtures known to occur in ambient source waters. Such health-based monitoring data are essential to support public engagement in source water sustainability and treatment and to inform consumer point-of-use treatment decision making in PR and throughout the US.

De Facto Water Reuse: Bioassay suite approach delivers depth and breadth in endocrine active compound detection.

Although endocrine disrupting compounds have been detected in wastewater and surface waters worldwide using a variety of in vitro effects-based screening tools, e.g. bioassays, few have examined potential attenuation of environmental contaminants by both natural (sorption, degradation, etc.) and anthropogenic (water treatment practices) processes. This study used several bioassays and quantitative chemical analyses to assess residence-time weighted samples at six sites along a river in the northeastern United States beginning upstream from a wastewater treatment plant outfall and proceeding downstream along the stream reach to a drinking water treatment plant. Known steroidal estrogens were quantified and changes in signaling pathway molecular initiating events (activation of estrogen, androgen, glucocorticoid, peroxisome proliferator-activated, pregnane X receptor, and aryl hydrocarbon receptor signaling networks) were identified in water extracts. In initial multi-endpoint assays geographic and receptor-specific endocrine activity patterns in transcription factor signatures and nuclear receptor activation were discovered. In subsequent single endpoint receptor-specific bioassays, estrogen (16 of 18 samples; 0.01 to 28 ng estradiol equivalents [E2Eqs]/L) glucocorticoid (3 of 18 samples; 1.8 to 21 ng dexamethasone equivalents [DexEqs]/L), and androgen (2 of 18 samples; 0.95 to 2.1 ng dihydrotestosterone equivalents [DHTEqs]/L) receptor transcriptional activation occurred above respective assay method detection limits (0.04 ng E2Eqs/L, 1.2 ng DexEqs/L, and 0.77 ng DHTEqs/L) in multiple sampling events. Estrogen activity, the most often detected, correlated well with measured concentrations of known steroidal estrogens (r2 = 0.890). Overall, activity indicative of multiple types of endocrine active compounds was highest in wastewater effluent samples, while activity downstream was progressively lower, and negligible in unfinished treated drinking water. Not only was estrogenic and glucocorticoid activity confirmed in the effluent by utilizing multiple methods concurrently, but other activated signaling networks that historically received less attention (i.e. peroxisome proliferator-activated receptor) were also detected.

Cumulative effects of in utero administration of mixtures of "antiandrogens" on male rat reproductive development.

Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act (FQPA) required the Environmental Protection Agency (EPA) to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures to provide a framework for assessing the cumulative effects of "antiandrogenic" chemicals. Rats were dosed during pregnancy with antiandrogens singly or in pairs at dosage levels equivalent to about one half of the ED50 for hypospadias or epididymal agenesis. The pairs include: AR antagonists (vinclozolin plus procymidone), phthalate esters (DBP plus BBP and DEHP plus DBP), a phthalate ester plus an AR antagonist (DBP plus procymidone), and linuron plus BBP. We predicted that each chemical by itself would induce few malformations; however, by mixing any two chemicals together, about 50% of the males would be malformed. All binary combinations produced cumulative, dose-additive effects on the androgen-dependent tissues. We also conducted a mixture study combining seven "antiandrogens" together. These chemicals elicit antiandrogenic effects at two different sites in the androgen signaling pathway (i.e., AR antagonist or inhibition of androgen synthesis). In this study, the complex mixture behaved in a dose-additive manner. Our results indicate that compounds that act by disparate mechanisms of toxicity display cumulative, dose-additive effects when present in combination.

Generalized Concentration Addition Model Predicts Glucocorticoid Activity Bioassay Responses to Environmentally Detected Receptor-Ligand Mixtures.

Many glucocorticoid receptor (GR) agonists have been detected in waste and surface waters domestically and around the world, but the way a mixture of these environmental compounds may elicit a total glucocorticoid activity response in water samples remains unknown. Therefore, we characterized 19 GR ligands using a CV1 cell line transcriptional activation assay applicable to water quality monitoring. Cells were treated with individual GR ligands, a fixed ratio mixture of full or partial agonists, or a nonequipotent mixture with full and partial agonists. Efficacy varied (48.09%-102.5%) and potency ranged over several orders of magnitude (1.278 × 10-10 to 3.93 × 10-8 M). Concentration addition (CA) and response addition (RA) mixtures models accurately predicted equipotent mixture responses of full agonists (r2 = 0.992 and 0.987, respectively). However, CA and RA models assume mixture compounds produce full agonist-like responses, and therefore they overestimated observed maximal efficacies for mixtures containing partial agonists. The generalized concentration addition (GCA) model mathematically permits < 100% maximal responses, and fell within the 95% confidence interval bands of mixture responses containing partial agonists. The GCA, but not CA and RA, model predictions of nonequipotent mixtures containing both full and partial agonists fell within the same statistical distribution as the observed values, reinforcing the practicality of the GCA model as the best overall model for predicting GR activation. Elucidating the mechanistic basis of GR activation by mixtures of previously detected environmental GR ligands will benefit the interpretation of environmental sample contents in future water quality monitoring studies.

Adverse Maternal, Fetal, and Postnatal Effects of Hexafluoropropylene Oxide Dimer Acid (GenX) from Oral Gestational Exposure in Sprague-Dawley Rats.

BACKGROUND: Hexafluoropropylene oxide dimer acid [(HFPO-DA), GenX] is a member of the per- and polyfluoroalkyl substances (PFAS) chemical class, and elevated levels of HFPO-DA have been detected in surface water, air, and treated drinking water in the United States and Europe. OBJECTIVES: We aimed to characterize the potential maternal and postnatal toxicities of oral HFPO-DA in rats during sexual differentiation. Given that some PFAS activate peroxisome proliferator-activated receptors (PPARs), we sought to assess whether HFPO-DA affects androgen-dependent development or interferes with estrogen, androgen, or glucocorticoid receptor activity. METHODS: Steroid receptor activity was assessed with a suite of in vitro transactivation assays, and Sprague-Dawley rats were used to assess maternal, fetal, and postnatal effects of HFPO-DA exposure. Dams were dosed daily via oral gavage during male reproductive development (gestation days 14-18). We evaluated fetal testes, maternal and fetal livers, maternal serum clinical chemistry, and reproductive development of F1 animals. RESULTS: HFPO-DA exposure resulted in negligible in vitro receptor activity and did not impact testosterone production or expression of genes key to male reproductive development in the fetal testis; however, in vivo exposure during gestation resulted in higher maternal liver weights ([Formula: see text]), lower maternal serum thyroid hormone and lipid profiles ([Formula: see text]), and up-regulated gene expression related to PPAR signaling pathways in maternal and fetal livers ([Formula: see text]). Further, the pilot postnatal study indicated lower female body weight and lower weights of male reproductive tissues in F1 animals. CONCLUSIONS: HFPO-DA exposure produced multiple effects that were similar to prior toxicity evaluations on PFAS, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), but seen as the result of higher oral doses. The mean dam serum concentration from the lowest dose group was 4-fold greater than the maximum serum concentration detected in a worker in an HFPO-DA manufacturing facility. Research is needed to examine the mechanisms and downstream events linked to the adverse effects of PFAS as are mixture-based studies evaluating multiple PFAS. https://doi.org/10.1289/EHP4372.

A mixture of seven antiandrogens induces reproductive malformations in rats.

To date, regulatory agencies have not considered conducting cumulative risk assessments for mixtures of chemicals with diverse mechanisms of toxicity because it is assumed that the chemicals will act independently and the individual chemical doses are not additive. However, this assumption is not supported by new research addressing the joint effects of chemicals that disrupt reproductive tract development in the male rat by disrupting the androgen signalling pathway via diverse mechanisms of toxicity [i.e. androgen receptor (AR) antagonism in the reproductive tract vs. inhibition of androgen synthesis in the foetal testis]. In this study, pregnant rats were exposed to four dilutions of a mixture containing vinclozolin, procymidone, linuron, prochloraz, benzyl butyl phthalate, dibutyl phthalate and diethylhexyl phthalate during the period of sexual differentiation and male offspring were assessed for effects on hormone sensitive endpoints including: anogenital distance, infant areolae retention and reproductive tract tissue weights and malformations. The ratio of the chemicals in the mixture was based upon each chemical's ED(50) for inducing reproductive tract malformations (hypospadias or epididymal agenesis). The observed responses from the mixture were compared with predicted responses generated with a toxic equivalency approach and models of dose addition, response addition or integrated addition. As hypothesized, we found that the mixture of chemicals that alter the androgen signalling pathway via diverse mechanisms disrupted male rat reproductive tract differentiation and induced malformations in a cumulative, dose-additive manner. The toxic equivalency and dose addition models provided the best fit to observed responses even though the chemicals do not act via a common cellular mechanism of action. The current regulatory framework for conducting cumulative risk assessments needs to consider the results, including those presented herein, which indicate that chemicals that disrupt foetal tissues during sexual differentiation act in a cumulative, dose-additive manner irrespective of the specific cellular mechanism of toxicity.

Diverse mechanisms of anti-androgen action: impact on male rat reproductive tract development.

Scientists have identified environmental chemicals that display anti-androgenic activity via multiple mechanisms of action. Early studies focused on pesticides acting as androgen receptor (AR) antagonists but it soon became apparent that was not the only endocrine mode by which compounds affected the androgen signalling pathway. Classes of chemicals currently known to interfere with the androgen signalling pathway include dicarboximide fungicides (e.g. vinclozolin), organochlorine-based insecticides (e.g. p,p'-DDT and -DDE), conazole fungicides (e.g. prochloraz), plasticizers (phthalates) and urea-based herbicides (linuron). Phthalate esters (PEs) and vinclozolin appear to act primarily via a single mechanism of action, while others such as linuron and prochloraz, appear to display dual mechanisms of action. Exposure to PEs decreases mRNA expression of key steroidogenic enzymes and also the peptide hormone insulin-like peptide 3 (insl3) from the foetal Leydig cells. Hence, both androgen- and inls3-dependent tissues are affected. Vinclozolin and procymidone act solely through binding to the AR as antagonists thus blocking the action of androgen at the cellular level but do not affect foetal testosterone synthesis or insl3 gene expression. The compounds linuron and prochloraz are AR antagonists but also inhibit foetal testosterone synthesis, although unlike the PEs, mRNA expression of steroidogenic enzymes and insl3 are not affected. All the above chemicals disrupt androgen signalling in the foetal male rat and produce some malformations in common, but the precise profiles of effects in the offspring are pathognomonic for each mode of action. For example, the 'phthalate syndrome' vs. the 'vinclozolin syndrome' each displays a profile of effects which is clearly different. In summary, as more and more molecular studies with anti-androgenic compounds are conducted, the number of mechanisms by which compounds can affect the androgen signalling pathway is likely to increase. Furthermore, the effects of mixtures of these compounds are just beginning to be explored.

Building a scientific framework for studying hormonal effects on behavior and on the development of the sexually dimorphic nervous system.

There has been increasing concern that low-dose exposure to hormonally active chemicals disrupts sexual differentiation of the brain and peripheral nervous system. There also has been active drug development research on the therapeutic potential of hormone therapy on behaviors. These different research goals have in common the need to develop reliable animal models to study the effect of hormones on brain function and behaviors that are predictive of effects in humans. This paper summarizes presentations given at the June 2007 11th International Neurotoxicology Association (INA-11) meeting, which addressed these issues. Using a few examples from the bisphenol A neurobehavioral literature for illustrative purposes, Dr. Abby Li discussed some of the methodological issues that should be considered in designing developmental neurobehavioral animal studies so they can be useful for human health risk assessment. Dr. Earl Gray provided an overview of research on the role of androgens and estrogens in the development of the brain and peripheral nervous system and behavior. Based on this scientific foundation, Dr. Gray proposed a rational framework for the study of the effects of developmental exposures to chemicals on the organization of the sexually dimorphic nervous system, including specific recommendations for experimental design and statistical analyses that can increase the utility of the research for regulatory decision-making. Dr. Michael Baum and by Dr. Feng Liu presented basic research on the hormonal mechanisms underlying sexual preference and estrogenic effects of cognition, respectively. These behaviors are among those studied in adult animals following in utero exposure to hormonally active chemicals, to evaluate their potential effects on sexual differentiation of the brain. Understanding of the hormonal mechanisms of these behaviors, and of relevance to humans, is needed to develop biologically plausible hypotheses regarding the potential effects of hormonally active chemicals in humans.