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 in utero 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). We found that each PE reduced testosterone production (T Prod) and related gene transcripts by about 50 % and that they acted in a dose additive manner, reducing T Prod and gene expression by 75 % as a mixture. Based upon effects on T Prod, DINP was 0.33 times as potent as DBP and thus the DBP + DINP mixture was predicted to be equivalent to 500 mg DBP/kg/d. Logistic regression models of T Prod predicted that the adverse effects of the DBP + DINP mixture group versus the DBP and DINP individual treatments would reduce anogenital distance (AGD) by 27 % versus 10 %, increase hypospadias in 18 % versus < 1 %, induce epididymal agenesis in 46 % versus 10 %, and increase areolae/nipples in 4.8 % versus < 0.1 % of the, respectively. These predictions were highly consistent with effects from previously published dose response studies on the male reproductive effects of DBP. In summary, these results support the use of this New Approach Method to predict the detrimental effects of PEs and PE mixtures, replacing or reducing the need to run long-term, resource and animal use intensive extended one-generation reproduction studies for this class of chemicals.

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.

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.

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.

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. Rats were orally dosed from gestation days 14-18 with dilutions of PFQ and DBP at 0%, 12.5%, 25%, 50%, 75%, and 100% of the top dose (100 mg/kg PFQ and 750 mg/kg DBP). The mixture ratio was selected such that each chemical would contribute equally to multiple effects on the male offspring reproductive tract and the dose range was designed to determine if the mixture produced additive effects predicted by dose addition (DA) or response addition (RA) models, or whether significant interactions occurred. Observed data were compared with DA and RA model predictions. As hypothesized, the mixture reduced F1 male anogenital distance, reproductive organ weights and sperm counts and induced hypospadias with DA consistently providing a better prediction of the observed effects than RA. These results support our hypothesis that chemicals that disrupt the androgen signaling pathway induce dose-additive male reproductive abnormalities regardless of the specific mechanism of action.

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.

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. However, phthalates had little or no effect on mRNA expression of genes in peroxisome proliferator-activated receptor (PPAR) pathways in the fetal liver, whereas the 3 PPAR agonists induced the expression of mRNA for multiple fetal liver PPAR pathway genes without reducing testis T Prod. In summary, phthalates that disrupt T Prod act via a novel adverse outcome pathway including down regulation of mRNA for genes involved in fetal endocrine function and cholesterol synthesis and metabolism. This profile was not displayed by PEs that did not reduce T Prod, PPAR agonists or the other chemicals. Reductions in fetal testis gene expression and T Prod in utero can be used to establish relative potency factors that can be used quantitatively to predict the doses of individual PEs and mixtures of phthalates that produce adverse reproductive tract effects in male offspring.

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.

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. Here, our objective was to determine how accurately the EC50s for AR antagonism in vitro or ED50s for reduced tissue growth in the Hershberger assay (HA) (key events in the adverse outcome pathway) predict the ED50s for reduced AGD in male rats exposed in utero to AR antagonists. This effort included in-house data and published studies from the last 60 years on AR antagonism in vitro and in vivo effects in the HA and on AGD after in utero exposure. In total, more than 250 studies were selected and included in the analysis with data from about 60 potentially antiandrogenic chemicals. The ability to predict ED50s for key events and adverse developmental effects from the in vitro EC50s displays considerable uncertainty with R2 values for HA and AGD of < 6%. In contrast, there is considerably less uncertainty in extrapolating from the ED50s in the HA to the ED50s for AGD (R2 value of about 85%). In summary, the current results suggest that the key events measured in the HA can be extrapolated with reasonable certainty to predict the ED50s for the adverse in utero effects of antiandrogenic chemicals on male rat offspring.

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 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. Furthermore, BPC inhibited fetal testis testosterone production and testis gene expression ex vivo. However, when BPC was administered at 100 and 200 mg/kg/d in utero, the reproductive tract of the male offspring was minimally affected. None of the males displayed reproductive malformations. For comparison, in utero administration of flutamide has been shown to induce malformations in 100% of males at 6 mg/kg/d. In vitro, PFQ was several orders of magnitude less potent than BPC, vinclozolin, or procymidone. However, in utero administration of 12.5, 25, 50, and 100 mg PFQ/kg/d on GD 14-18 induced antiandrogenic effects at all dosage levels and 91% of the males displayed reproductive malformation in the high dose group. Overall, BPC was ∼380-fold more potent than PFQ in vitro, whereas PFQ was far more potent than BPC in utero. Incorporating toxicokinetic and toxicodynamic data into in vitro to in vivo extrapolations would reduce the discordance between the in vitro and in utero effects of PFQ and BPC and combining in vitro results with a short-term Hershberger assay would reduce the uncertainty in predicting the in utero effects of antiandrogenic chemicals.

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. We hypothesized that SMV would have adverse effects on postnatal development and sexual differentiation as a consequence of the disruptions of fetal lipid levels and testicular T Prod since fetal cholesterol is essential for normal intrauterine growth and development and steroid synthesis. In the prenatal assessment, SMV was administered orally at 0, 15.6, 31.25, 62.5, 80, 90, 100, and 110 mg SMV/kg/d from GD 14-18, the period that cover the critical window of sex differentiation in the male rat fetus. T Prod was maximally reduced by ~40% at 62.5 mg/kg/d, and higher doses induced overt maternal and toxicity. In the postnatal assessment, SMV was administered at 0, 15.6, 31.25, and 62.5 mg/kg/d from GD 8-18 to determine if it altered postnatal development. We found that exposure during this time frame to 62.5 mg SMV/kg/d reduced pup viability by 92%, decreased neonatal anogenital distance, and altered testis histology and morphology in 17% of the F1 males. In another group, SMV was administered only during the masculinizing window (GD14-18) at 62.5 mg/kg/d to determine if male rat sexual differentiation and postnatal reproductive development were altered. SMV-exposed F1 males displayed female-like areolae/nipples, delayed puberty, and reduced seminal vesicle and levator ani-bulbocavernosus weights. Together, these results demonstrate that in utero exposure to SMV reduces offspring viability and permanently disrupts reproductive tract development in the male offspring. While the effects of high dose, short term in utero exposure to SMV in the adult male are likely androgen-dependent and consistent with the 40% reduction in T Prod in the fetal testes, long-term, lower dose administration induced some effects that were likely not mediated by decreased T Prod.

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. Pregnant dams were exposed to a range of dilutions (100%, 50%, 25%, 12.5%, 6.25%, or vehicle control) of a mixture containing pesticides, phthalates, and drugs (p, p'-DDE, linuron, prochloraz, procymidone, pyrifluquinazon, vinclozolin, finasteride, flutamide, simvastatin, and 9 phthalates [dipentyl, dicyclohexyl, di-2-ethylhexyl, dibutyl, benzyl butyl, diisobutyl, diisoheptyl, dihexyl, and diheptyl]). The top dose contained each chemical at 20% of its lowest observed adverse effect level (LOAEL) for the most sensitive male reproductive alteration following in utero exposure. We found that male rat offspring displayed a variety of neonatal, pubertal, and permanent adult effects across all dose levels. Even at the lowest dose (each chemical approximately 80-fold below lowest observed adverse effect level) there were permanent reductions in several reproductive tract tissue weights. In the top dose group, 100% of male offspring displayed permanent severe birth defects including genital malformations. Despite acting via 5 different molecular initiating events, a mixture of 18 chemicals can combine to produce additive effects even when each compound is at is at a relatively low dose.

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. SMV may also disrupt steroid biosynthesis, but through a different mode of action (MOA) than the PEs. As cholesterol is a precursor of steroid hormone biosynthesis, we hypothesized that in utero exposure to SMV during the critical period of sex differentiation would lower fetal testicular testosterone (T) production without affecting genes involved in cholesterol and androgen synthesis and transport. Secondly, we hypothesized that a mixture of SMV and a PE, which may have different MOAs, would reduce testosterone levels in an additive manner. Pregnant Sprague Dawley rats were dosed orally with SMV, dipentyl phthalate (DPeP), or SMV plus DPeP from gestational days 14-18, and fetuses were evaluated on GD18. On GD18, SMV lowered fetal T production and serum triglycerides, low density lipoprotein, high density lipoprotein, and total cholesterol levels, and downregulated two genes in the fetal testis that were different from those altered by PEs. When SMV and DPeP were administered as a mixture, fetal T production was significantly reduced in an additive manner, thus demonstrating that a mixture of chemicals can induce additive effects on fetal T production even though they display different MOAs.

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. We also included some chemicals with "unknown" activity including DMEP, DHeP, DHEH, DPHCH, DAP, TOTM, tetrabromo-diethyl hexyl phthalate (BrDEHP), and a relatively potent environmental estrogen BPAF. Dose-response studies also were conducted with this protocol with 11 of the above chemicals to determine their relative potencies. CD-1 mice also were exposed to varying dose levels of DPeP from GD 13 to 17 to determine if DPeP reduced T Prod in this species since there is a discrepancy among the results of in utero studies of PEs in mice. Compared to the known male reproductive effects of the PEs in rats the FPS correctly identified all known "positives" and "negatives" tested. Seven of eight "unknowns" tested were "negatives", they did not reduce T Prod, whereas DAP produced an "equivocal" response. Finally, a dose-response study with DPeP in CD-1 mice revealed that fetal T Prod can be inhibited by exposure to a PE in utero in this species, but at a higher dose level than required in rats.Key words. Phthalate Syndrome, Fetal endocrine biomarkers, Phthalate adverse outcome pathway, testosterone production, fetal rat testis.

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. All phthalates, with the exception of DIDP, reduced fetal testicular T production. Several genes involved in cholesterol transport, androgen synthesis, and Insl3 also were downregulated in a dose-responsive manner by DIBP, DPeP, DHP, DHeP, DINP, and the 9-PE mixture. Despite speculation of peroxisome proliferator activated receptor (PPAR) involvement in the effects of PEs on the fetal testis, no PPAR-related genes were affected in the fetal testes by exposure to any of the tested PEs. Furthermore, the potent PPARα agonist, Wy-14,643, did not reduce fetal testicular T production following gestational day 14-18 exposure, suggesting that the antiandrogenic activity of PEs is not PPARα mediated. The overall sensitivity of the fetal endpoints (gene expression or T production) for the six phthalates from most to least was Cyp11b1 > Star = Scarb1 > Cyp17a1 = T production > Cyp11a1 = Hsd3b = Insl3 > Cyp11b2. The overall potency of the individual phthalates was DPeP > DHP > DIBP ≥ DHeP > DINP. Finally, the observed mixture interaction was adequately modeled by the dose-addition model for most of the affected genes. Together, these data advance our understanding of the collective reproductive toxicity of the PE compounds.

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 2.3-fold less potent. DINP was also less potent at reducing StAR and Cyp11a gene expression levels, whereas DIBP was slightly more potent than DEHP. We observed that administration of dilutions of a mixture of nine phthalates (DEHP, DIHP, DIBP, dibutyl-, benzyl butyl-, dicyclohexyl-, diheptyl-, dihexyl-, and dipentyl phthalate) reduced fetal T production in a dose-dependent manner best predicted by dose addition. Finally, we found that the differential effects of in utero DEHP treatment on epididymal and gubernacular differentiation in male SD and W rats (0, 100, 300, 500, 625, 750, or 875 mg DEHP/kg/day) are likely due to tissue-specific strain differences in the androgen and insl3 signaling pathways rather than differential effects of DEHP on fetal testis T and insl3 production.

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. Regulatory agencies require dose-response and potency data to facilitate risk assessment; however, very little data are currently available for DPeP. The goal of this study was to establish a more comprehensive data set for DPeP, focusing on dose-response and potency information for fetal and postnatal male reproductive endpoints. We dosed pregnant rats on gestational day (GD) 17 or GD 14-18 and subsequently evaluated fetal testicular testosterone (T) production on GD 17.5 and GD 18, respectively. We also dosed pregnant rats on GD 8-18 and evaluated early postnatal endpoints in male offspring. Comparison of these data to data previously obtained under similar conditions for di (2-ethylhexyl) phthalate indicates that DPeP is approximately eightfold more potent in reducing fetal T production and two- to threefold more potent in inducing development of early postnatal male reproductive malformations. Additionally, fetal testicular T production was more sensitive to inhibitory effects of DPeP exposure than was gene expression of target genes involved in male reproductive development, supporting the use of this endpoint as a critical effect in the risk assessment process.

Cumulative and antagonistic effects of a mixture of the antiandrogens vinclozolin and iprodione in the pubertal male rat.

Vinclozolin and iprodione are dicarboximide fungicides that display antiandrogenic effects in the male rat, which suggests that a mixture would lead to cumulative effects on androgen-sensitive end points. Iprodione is a steroid synthesis inhibitor, but androgen receptor antagonist activity, which is displayed by vinclozolin, has not been fully evaluated. Here, we demonstrate that iprodione binds to the human androgen receptor (IC(50) = 86.0 microM), reduces androgen-dependent gene expression, and reduces androgen-sensitive tissue weights in castrated male rats (Hershberger assay). Since vinclozolin and iprodione affect common targets in the pubertal male rat, we tested the hypothesis that a mixture would have cumulative antiandrogenic effects. An iprodione dose, that does not significantly affect androgen-dependent morphological end points, was combined with vinclozolin doses (2 x 5 factorial design). Sprague-Dawley rats were dosed by gavage with vinclozolin at 0, 10, 30, 60, and 100 mg/kg/day with and without 50 mg iprodione/kg/day from postnatal day (PND) 23 to 55-57 (n = 8 per group). The age at puberty (preputial separation [PPS]), organ weights, serum hormones, and ex vivo testis steroid hormone production were measured. Vinclozolin delayed PPS, reduced androgen-sensitive organ weights, and increased serum testosterone. The addition of iprodione enhanced the vinclozolin inhibition of PPS (PND 47.5 vs.49.1; two-way ANOVA: iprodione main effect p = 0.0002). The dose response for several reproductive and nonreproductive organ weights was affected in a cumulative manner. In contrast, iprodione antagonized the vinclozolin-induced increase in serum testosterone. These results demonstrate that these fungicides interact on common targets in a tissue-specific manner when coadministered to the pubertal male rat.