Long-chain perfluoroalkylether carboxylic acids PFO5DoA and PFO4DA alter glucose, bile acid, and thyroid hormone homeostasis in fetal rats from 5-day maternal oral exposure.

Chemical monitoring studies in North Carolina, USA and Shandong, China have reported detections of perfluoroalkylether carboxylic acids of increasing chain length with ether bonds between each fluorinated carbon. Despite detection there is limited hazard data available to inform risk assessment. Here, we exposed pregnant Sprague-Dawley rats to two of these compounds, perfluoro-3,5,7,9-butaoxadecanoic acid (PFO4DA) and perfluoro-3,5,7,9,11-pentaoxadodecanoic acid (PFO5DoA), from gestation days 18-22 across a series of doses (0.

Exploring maternal and developmental toxicity of perfluoroalkyl ether acids PFO4DA and PFO5DoA using hepatic transcriptomics and serum metabolomics.

Production of per- and polyfluoroalkyl substances (PFAS) has shifted from long-chain perfluoroalkyl acids to short-chain compounds and those with ether bonds in the carbon chain. Next-generation perfluoroalkylether PFAS include HFPO-DA ("GenX chemicals"), Nafion Byproducts, and the PFOx homologous series that includes perfluoro-3,5,7,9-butaoxadecanoic acid (PFO4DA) and perfluoro-3,5,7,9,11-pentaoxadodecanoic acid (PFO5DoA). PFO4DA and PFO5DoA have been detected in serum and/or tissues from humans and wildlife proximal to contamination point sources.

Using targeted fetal rat testis genomic and endocrine alterations to predict the effects of a phthalate mixture on the male reproductive tract.

Administration of phthalates disrupts gene expression and hormone levels in the fetal rat testis, which are key events in an Adverse Outcome Pathway (AOP) for the Phthalate Syndrome. These measures can be used to predict the postnatal adverse effects of phthalate esters (PEs) on male rat sexual differentiation. Here, pregnant rats were exposed to dibutyl (DBP)- and diisononyl (DINP) phthalate on gestational days 14 to 18 individually and as a mixture (DBP,250 mg/kg/d; DINP, 750 mg/kg/d; and DBP 250 mg/kg/d plus DINP 750 mg/kg/d).

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).

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.

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.

In Utero Exposure to a Mixture of the Perfluoroalkyl-Isopropyl Pesticide Pyrifluquinazon With Dibutyl Phthalate Cumulatively Disrupts Male Rat Reproductive Development via Different Mechanisms of Action.

Administration of individual chemicals and mixtures during sexual differentiation that disrupt the androgen signaling pathway can induce reproductive abnormalities in male rats. In this study, we coadministered the heptafluoroisopropyl pesticide pyrifluquinazon (PFQ), and dibutyl phthalate (DBP) to pregnant rats during sexual differentiation of the reproductive tract. Both chemicals have been shown to disrupt reproductive tract differentiation in a dose-related manner reducing male anogenital distance, permanently reducing androgen-dependent tissue weights and sperm counts, and inducing reproductive malformations in male offspring, albeit by different mechanisms of action that converge downstream in the androgen signaling pathway on a common key event.

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.

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.

Genomic and Hormonal Biomarkers of Phthalate-Induced Male Rat Reproductive Developmental Toxicity Part II: A Targeted RT-qPCR Array Approach That Defines a Unique Adverse Outcome Pathway.

Previously, we demonstrated that exposure to some diortho-phthalate esters during sexual differentiation disrupts male reproductive development by reducing fetal rat testis testosterone production (T Prod) and gene expression in a dose-related manner. The objectives of the current project were to expand the number of test compounds that might reduce fetal T Prod, including phthalates, phthalate alternatives, pesticides, and drugs, and to compare reductions in T Prod with altered testis mRNA expression. We found that PEs that disrupt T Prod also reduced expression of a unique "cluster" of mRNAs for about 35 genes related to sterol transport, testosterone and insulin-like hormone 3 hormone syntheses, and lipoprotein signaling and cholesterol synthesis.

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.

Quantification of the Uncertainties in Extrapolating From In Vitro Androgen Receptor Antagonism to In Vivo Hershberger Assay Endpoints and Adverse Reproductive Development in Male Rats.

Multiple molecular initiating events exist that disrupt male sexual differentiation in utero including androgen receptor (AR) antagonism and inhibition of synthesis, and metabolism of fetal testosterone. Disruption of androgen signaling by AR antagonists in utero reduces anogenital distance (AGD) and induces malformations in F1 male rat offspring. We are developing a quantitative network of adverse outcome pathways that includes multiple molecular initiating events and key events linking anti-AR activities to permanent reproductive abnormalities.

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.

A Conflicted Tale of Two Novel AR Antagonists In Vitro and In Vivo: Pyrifluquinazon Versus Bisphenol C.

Chemicals that disrupt androgen receptor (AR) function in utero induce a cascade of adverse effects in male rats including reduced anogenital distance, retained nipples, and reproductive tract malformations. The objective of this study was to compare the in vitro and in utero activities of two novel AR antagonists, bisphenol C (BPC) and pyrifluquinazon (PFQ). In vitro, BPC was as potent an AR antagonist as hydroxyflutamide.

In utero exposure to simvastatin reduces postnatal survival and permanently alters reproductive tract development in the Crl:CD(SD) male rat.

We showed previously that in utero exposure to the cholesterol-lowering drug simvastatin (SMV) during sex differentiation lowers fetal lipids and testicular testosterone production (T Prod) in Hsd:SD rats. Here, the effects of SMV on fetal lipids and T Prod in Crl:CD(SD) rats were correlated with postnatal alterations in F1 males. The current study was conducted in two parts: 1) a prenatal assessment to confirm and further characterize the dose response relationship among previously reported alterations of SMV on fetal T Prod and the fetal lipid profile and 2) a postnatal assessment to determine the effects of SMV exposure during the periods of major organogenesis and/or sexual differentiation on F1 offspring growth and development.

Mixed "Antiandrogenic" Chemicals at Low Individual Doses Produce Reproductive Tract Malformations in the Male Rat.

Biomonitoring efforts have clearly shown that all humans are exposed to chemical mixtures. Of concern is whether or not exposure to mixtures during pregnancy contributes to congenital abnormalities in children even when each chemical is at an individual dose that does not affect the fetus. Here, we hypothesized that in utero exposure to a mixture of chemicals covering multiple "antiandrogenic" mechanisms of action at doses that individually have no adverse effect would result in permanent reproductive tract alterations in the male rat after birth.

Validation of an automated counting procedure for phthalate-induced testicular multinucleated germ cells.

In utero exposure to certain phthalate esters results in testicular toxicity, characterized at the tissue level by induction of multinucleated germ cells (MNGs) in rat, mouse, and human fetal testis. Phthalate exposures also result in a decrease in testicular testosterone in rats. The anti-androgenic effects of phthalates have been more thoroughly quantified than testicular pathology due to the significant time requirement associated with manual counting of MNGs on histological sections.

Establishing the "Biological Relevance" of Dipentyl Phthalate Reductions in Fetal Rat Testosterone Production and Plasma and Testis Testosterone Levels.

Phthalate esters (PEs) constitute a large class of compounds that are used for many consumer product applications. Many of the C2-C7 di-ortho PEs reduce fetal testicular hormone and gene expression levels in rats resulting in adverse effects seen later in life but it appears that relatively large reductions in fetal testosterone (T) levels and testis gene expression may be required to adversely affect reproductive development (Hannas, B. R.

Dose Addition Models Based on Biologically Relevant Reductions in Fetal Testosterone Accurately Predict Postnatal Reproductive Tract Alterations by a Phthalate Mixture in Rats.

Challenges in cumulative risk assessment of anti-androgenic phthalate mixtures include a lack of data on all the individual phthalates and difficulty determining the biological relevance of reduction in fetal testosterone (T) on postnatal development. The objectives of the current study were 2-fold: (1) to test whether a mixture model of dose addition based on the fetal T production data of individual phthalates would predict the effects of a 5 phthalate mixture on androgen-sensitive postnatal male reproductive tract development, and (2) to determine the biological relevance of the reductions in fetal T to induce abnormal postnatal reproductive tract development using data from the mixture study. We administered a dose range of the mixture (60, 40, 20, 10, and 5% of the top dose used in the previous fetal T production study consisting of 300 mg/kg per chemical of benzyl butyl (BBP), di(n)butyl (DBP), diethyl hexyl phthalate (DEHP), di-isobutyl phthalate (DiBP), and 100 mg dipentyl (DPP) phthalate/kg; the individual phthalates were present in equipotent doses based on their ability to reduce fetal T production) via gavage to Sprague Dawley rat dams on GD8-postnatal day 3.

Simvastatin and dipentyl phthalate lower ex vivo testicular testosterone production and exhibit additive effects on testicular testosterone and gene expression via distinct mechanistic pathways in the fetal rat.

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and consequently, lowering fetal androgen and Insl3 hormone levels. Simvastatin (SMV) is a cholesterol-lowering drug that directly inhibits HMG-CoA reductase.

A short-term in vivo screen using fetal testosterone production, a key event in the phthalate adverse outcome pathway, to predict disruption of sexual differentiation.

This study was designed to develop and validate a short-term in vivo protocol termed the Fetal Phthalate Screen (FPS) to detect phthalate esters (PEs) and other chemicals that disrupt fetal testosterone synthesis and testis gene expression in rats. We propose that the FPS can be used to screen chemicals that produce adverse developmental outcomes via disruption of the androgen synthesis pathway more rapidly and efficiently, and with fewer animals than a postnatal one-generation study. Pregnant rats were dosed from gestational day (GD) 14 to 18 at one dose level with one of 27 chemicals including PEs, PE alternatives, pesticides known to inhibit steroidogenesis, an estrogen and a potent PPARα agonist and ex vivo testis testosterone production (T Prod) was measured on GD 18.

Genomic biomarkers of phthalate-induced male reproductive developmental toxicity: a targeted RT-PCR array approach for defining relative potency.

Male rat fetuses exposed to certain phthalate esters (PEs) during sexual differentiation display reproductive tract malformations due to reductions in testosterone (T) production and the expression of steroidogenesis- and INSL3-related genes. In the current study, we used a 96-well real-time PCR array containing key target genes representing sexual determination and differentiation, steroidogenesis, gubernaculum development, and androgen signaling pathways to rank the relative potency of several PEs. We executed dose-response studies with diisobutyl (DIBP), dipentyl (DPeP), dihexyl (DHP), diheptyl (DHeP), diisononyl (DINP), or diisodecyl phthalate (DIDP) and serial dilutions of a mixture of nine phthalates.

Dose-response assessment of fetal testosterone production and gene expression levels in rat testes following in utero exposure to diethylhexyl phthalate, diisobutyl phthalate, diisoheptyl phthalate, and diisononyl phthalate.

Several phthalate esters have been linked to the Phthalate Syndrome, affecting male reproductive development when administered to pregnant rats during in utero sexual differentiation. The goal of the current study was to enhance understanding of this class of compounds in the Sprague Dawley (SD) fetal rat following exposure on gestational days (GDs) 14-18 by determining the relative potency factors for several phthalates on fetal testes endpoints, the effects of a nine phthalate mixture on fetal testosterone (T) production, and differences in SD and Wistar (W) strain responses of fetal T production and testicular gene expression to di(2-ethylhexyl) phthalate (DEHP). We determined that diisobutyl phthalate (DIBP) and diisoheptyl phthalate (DIHP) reduced fetal testicular T production with similar potency to DEHP, whereas diisononyl phthalate (DINP) was 2.

Dipentyl phthalate dosing during sexual differentiation disrupts fetal testis function and postnatal development of the male Sprague-Dawley rat with greater relative potency than other phthalates.

Phthalate esters (PEs) constitute a large class of plasticizer compounds that are widely used for many consumer product applications. Ten or more members of the PE class of compounds are known to induce male fetal endocrine toxicity and postnatal reproductive malformations by disrupting androgen production during the sexual differentiation period of development. An early study conducted in the rat pubertal model suggested that dipentyl phthalate (DPeP) may be a more potent testicular toxicant than some more extensively studied phthalates.