@article{howdeshell_wilson_furr_lambright_rider_blystone_hotchkiss_gray_2008, title={A mixture of five phthalate esters inhibits fetal testicular testosterone production in the sprague-dawley rat in a cumulative, dose-additive manner}, volume={105}, ISSN={["1096-6080"]}, DOI={10.1093/toxsci/kfn077}, abstractNote={Phthalate diesters are chemicals to which humans are ubiquitously exposed. Exposure to certain phthalates during sexual differentiation causes reproductive tract malformations in male rats. In the fetal rat, exposure to the phthalates benzylbutyl phthalate (BBP), di(n)butyl phthalate (DBP), and diethylhexyl phthalate (DEHP) decreases testicular testosterone production and insulin-like 3 hormone mRNA levels. We characterized the dose-response effects of six individual phthalates (BBP, DBP, DEHP, diethyl phthalate [DEP], diisobutyl phthalate [DiBP], and dipentyl phthalate [DPP]) on gestation day (GD) 18 testicular testosterone production following exposure of Sprague-Dawley rats on GD 8-18. BBP, DBP, DEHP, and DiBP were equipotent (ED50 of 440 +/- 16 mg/kg/day), DPP was about threefold more potent (ED50 = 130 mg/kg/day) and DEP had no effect on fetal testosterone production. We hypothesized that coadministration of these five antiandrogenic phthalates would reduce testosterone production in a dose-additive fashion because they act via a common mode of toxicity. In a second study, dams were dosed at 100, 80, 60, 40, 20, 10, 5, or 0% of the mixture. The top dose contained 1300 mg of total phthalates/kg/day including BBP, DBP, DEHP, DiBP (300 mg/kg/day per chemical), and DPP (100 mg DPP/kg/day). This mixture ratio was selected such that each phthalate would contribute equally to the reduction in testosterone. As hypothesized, testosterone production was reduced in a dose-additive manner. Several of the individual phthalates and the mixture also induced fetal mortality, due to pregnancy loss. These data demonstrate that individual phthalates with a similar mechanism of action can elicit cumulative, dose additive effects on fetal testosterone production and pregnancy when administered as a mixture.}, number={1}, journal={TOXICOLOGICAL SCIENCES}, author={Howdeshell, Kembra L. and Wilson, Vickie S. and Furr, Johnathan and Lambright, Christy R. and Rider, Cynthia V. and Blystone, Chad R. and Hotchkiss, Andrew K. and Gray, Leon Earl, Jr.}, year={2008}, month={Sep}, pages={153–165} }
 @article{rider_furr_wilson_gray_2008, title={A mixture of seven antiandrogens induces reproductive malformations in rats}, volume={31}, ISSN={["1365-2605"]}, DOI={10.1111/j.1365-2605.2007.00859.x}, abstractNote={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.}, number={2}, journal={INTERNATIONAL JOURNAL OF ANDROLOGY}, author={Rider, Cynthia V. and Furr, Johnathan and Wilson, Vickie S. and Gray, L. Earl, Jr.}, year={2008}, month={Apr}, pages={249–262} }
 @article{wilson_blystone_hotchkiss_rider_gray_2008, title={Diverse mechanisms of anti-androgen action: impact on male rat reproductive tract development}, volume={31}, ISSN={["1365-2605"]}, DOI={10.1111/j.1365-2605.2007.00861.x}, abstractNote={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.}, number={2}, journal={INTERNATIONAL JOURNAL OF ANDROLOGY}, author={Wilson, Vickie S. and Blystone, Chad R. and Hotchkiss, Andrew K. and Rider, Cynthia V. and Gray, L. Earl, Jr.}, year={2008}, month={Apr}, pages={178–185} }
 @article{howdeshell_furr_lambright_wilson_ryan_gray_2008, title={Gestational and lactational exposure to ethinyl estradiol, but not bisphenol a, decreases androgen-dependent reproductive organ weights and epididymal sperm abundance in the male long evans hooded rat}, volume={102}, ISSN={["1096-0929"]}, DOI={10.1093/toxsci/kfm306}, abstractNote={Many chemicals released into the environment are capable of disrupting normal sex steroid balance, including the oral contraceptive ethinyl estradiol (EE) and the plastic monomer bisphenol A (BPA). EE and BPA are reported to impair reproductive organ development in laboratory animals; however, effects of lower doses of these chemicals have been debated. The goal of the current study was to determine whether relatively low oral doses of EE or BPA would alter male reproductive morphology and associated hormone levels of Long Evans hooded rat. Dams were gavaged with corn oil vehicle, EE (0.05-50 mug/kg/day) or BPA (2, 20, and 200 mug/kg/day) during pregnancy through lactation from gestational day 7 to postnatal day (PND) 18. Anogenital distance was measured at PND2 and nipple retention was measured at PND14 in male pups. Male offspring were euthanized beginning at PND150, and sera and organs were collected for analyses. Adult body weight was significantly decreased in males exposed to 50 mug EE/kg/day. Developmental EE exposure reduced androgen-dependent tissue weights in a dose-dependent fashion; for example, seminal vesicle and paired testes weights were reduced with >/= 5 mug EE/kg/day. Epididymal sperm counts were also significantly decreased with 50 mug EE/kg/day. In contrast, treatment with 2, 20, or 200 mug BPA/kg/day or EE at 0.05-1.5 mug/kg/day did not significantly affect any male endpoint in the current study. These results demonstrate that developmental exposure to oral micromolar doses of EE can permanently disrupt the reproductive tract of the male rat.}, number={2}, journal={TOXICOLOGICAL SCIENCES}, author={Howdeshell, Kembra L. and Furr, Johnathan and Lambright, Christy R. and Wilson, Vickie S. and Ryan, Bryce C. and Gray, L. Earl, Jr.}, year={2008}, month={Apr}, pages={371–382} }
 @article{hartig_cardon_blystone_gray_wilson_2008, title={High throughput adjustable 96-well plate assay for androgen receptor binding: A practical approach for EDC screening using the chimpanzee AR}, volume={181}, ISSN={["0378-4274"]}, DOI={10.1016/j.toxlet.2008.07.008}, abstractNote={The issue as to whether natural and man-made chemicals interfere with endocrine function has raised concerns. This interference could be biologically significant even at very low doses if the chemicals interact deleteriously with hormone receptors at low concentrations. Therefore, the United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that a nonhuman mammalian androgen receptor binding assay be developed for possible use in their Endocrine Disruptor Screening Program (EDSP). Ideally, this assay would be high throughput, not use animals as a source of receptor protein, easily deployed throughout the scientific community, utilize reagents available to both the public and private sector, and have the potential for future automation. We developed a highly modified 96-well plate assay which meets these criteria. It employs a baculovirus expressed recombinant primate androgen receptor which is publically available and exploits the unique ability of some mammalian androgen receptors to remain biologically active after guanidine hydrochloride (GdnHCl) solubilization. This GdnHCl treated receptor remains soluble and requires no additional purification prior to use. We provide a very detailed description of the assay protocol itself, and similarly detailed method for producing and solubilizing the receptor.}, number={2}, journal={TOXICOLOGY LETTERS}, author={Hartig, P. C. and Cardon, M. C. and Blystone, C. R. and Gray, L. E., Jr. and Wilson, V. S.}, year={2008}, month={Sep}, pages={126–131} }
 @inproceedings{howdeshell_rider_wilson_gray_2008, title={Mechanisms of action of phthalate esters, individually and in combination, to induce abnormal reproductive development in male laboratory rats}, volume={108}, number={2}, booktitle={Environmental Research (New York, N.Y.)}, author={Howdeshell, K. L. and Rider, C. V. and Wilson, V. S. and Gray, L. E.}, year={2008}, pages={168–176} }
 @article{blystone_lambright_furr_wilson_gray_2007, title={Iprodione delays male rat pubertal development, reduces serum testosterone levels, and decreases ex vivo testicular testosterone production}, volume={174}, ISSN={["0378-4274"]}, DOI={10.1016/j.toxlet.2007.08.010}, abstractNote={Iprodione (IPRO) is a dichlorophenyl dicarboximide fungicide similar to procymidone and vinclozolin. All three of these fungicides induce Leydig cell tumors in the rat testis in long-term studies and an endocrine mode of action has been hypothesized to mediate this effect. Although both procymidone and vinclozolin antagonize the androgen receptor (AR) in vitro and in vivo, IPRO does not appear to be an AR antagonist. We proposed that pubertal exposure to IPRO would delay male rat pubertal development and reduce testosterone production within the testis. Sprague-Dawley weanling rats were dosed by gavage with 0, 50, 100, or 200mg/kg/day of IPRO from post-natal day (PND) 23 to 51/52. The onset of puberty (progression of preputial separation (PPS)) was measured starting on PND 37. Organ weights, serum hormones, and ex vivo testis steroid hormone production under stimulated (+human chorionic gonadotropin (hCG)) and unstimulated (-hCG) conditions were measured at necropsy. IPRO delayed PPS at 100 and 200mg/kg/day and decreased androgen sensitive seminal vesicle and epididymides weights at 200mg/kg/day. Furthermore, IPRO increased adrenal and liver weights at 200mg/kg/day, presumably by different mechanism(s) of action. Serum testosterone levels were decreased along with serum 17alpha-hydroxyprogesterone and androstenedione whereas serum LH was unaffected. IPRO reduced ex vivo testis production of testosterone and progesterone. Taken together, these results suggest that IPRO affects steroidogenesis within the testis, not through disruption of LH signaling, but possibly through enzyme inhibition of the steroidogenic pathway before CYP17. These data, along with the reported failure of IPRO to elicit an AR antagonism in vitro, provide evidence that IPRO differs from the dicarboximides procymidone and vinclozolin in that the effects on male rat pubertal development result from an inhibition of steroidogenesis and not AR antagonism.}, number={1-3}, journal={TOXICOLOGY LETTERS}, author={Blystone, Chad R. and Lambright, Christy S. and Furr, Jfohnathan and Wilson, Vickie S. and Gray, L. Earl, Jr.}, year={2007}, month={Nov}, pages={74–81} }
 @article{hotchkiss_lambright_ostby_parks-saldutti_vandenbergh_gray_2007, title={Prenatal testosterone exposure permanently masculinizes anogenital distance, nipple development, and reproductive tract morphology in female Sprague-Dawley rats}, volume={96}, ISSN={["1096-6080"]}, DOI={10.1093/toxsci/kfm002}, abstractNote={In mammals, abnormal increases in fetal androgens disrupt normal development of the female phenotype. Due to the recent concern regarding environmental androgen-active chemicals, there is a need to identify sources of fetal androgen variation and sensitive developmental markers for androgenic activity in female rats. Anogenital distances (AGD), nipple retention, reproductive tract, and external genitalia are morphological parameters organized by prenatal androgens and are predictive of altered masculinized/defeminized phenotype in adult female mice and rats. The objectives of this study were to (1) characterize the natural prenatal androgen environment of rats including the magnitude of the intrauterine position (IUP) effect, (2) characterize the permanent effects of prenatal androgen exposure on female rats, and (3) determine the ability of AGD and areolas to predict these permanent androgenic alterations in female rats. Untreated male fetal rats had higher tissue testosterone (T) concentrations than females in the amniotic fluid, reproductive tract, gonad, and fetal body. The intrauterine position (IUP) of male and female fetuses did not affect T concentrations or AGD in male or female rats at gestational day (GD) 22. Female offspring exposed to 0, 1.5, and 2.5 mg/kg/day testosterone propionate (TP) on GDs 14–18 displayed increased AGD at postnatal day (PND) 2 and decreased nipples at PND 13 and as adults. TP-induced changes in neonatal AGD and infant areola number were reliable indicators of permanently altered adult phenotype in female rats. Further, females in the two high-dose groups displayed increased incidences of external genital malformations and the presence of prostatic tissue, not normally found in female rats.}, number={2}, journal={TOXICOLOGICAL SCIENCES}, author={Hotchkiss, Andrew K. and Lambright, Christy S. and Ostby, Joseph S. and Parks-Saldutti, Louise and Vandenbergh, John G. and Gray, Leon E., Jr.}, year={2007}, month={Apr}, pages={335–345} }
 @article{blystone_furr_lambright_howdeshell_ryan_wilson_leblanc_gray_2007, title={Prochloraz inhibits testosterone production at dosages below those that affect androgen-dependent organ weights or the onset of puberty in the male Sprague Dawley rat}, volume={97}, ISSN={["1096-0929"]}, DOI={10.1093/toxsci/kfm004}, abstractNote={Prochloraz (PCZ) is an imidazole fungicide that inhibits gonadal steroidogenesis and antagonizes the androgen receptor (AR). We hypothesized that pubertal exposure to PCZ would reduce testosterone production and delay male rat reproductive development. Sprague Dawley rats were dosed by gavage with 0, 31.3, 62.5, or 125 mg/kg/day of PCZ from postnatal day (PND) 23 to 42 or 51. There was a significant delay in preputial separation (PPS) at 125 mg/kg/day PCZ and several of the androgen-dependent organ weights were decreased significantly, but the significant organ weight effects were not consistent between the 2 necropsies (PND 42 vs. 51). At both ages, serum testosterone levels and ex vivo testosterone release from the testis were significantly decreased whereas serum progesterone and 17α-hydroxyprogesterone levels were significantly increased at dose levels below those that affected PPS or reproductive organ weights. The hormone results suggested that PCZ was inhibiting CYP17 activity. In a second pubertal study (0, 3.9, 7.8, 15.6, 31.3, or 62.5 mg/kg/day PCZ), serum testosterone levels and ex vivo testosterone production were significantly reduced at 15.6 mg/kg/day PCZ. In order to examine the AR antagonism effects of PCZ, independent of its effects on testosterone synthesis, castrated immature male rats were dosed with androgen and 0, 15.6, 31.3, 62.5, or 125 mg/kg/day PCZ for 10–11 days (Hershberger assay). In this assay, androgen-sensitive organ weights were only significantly decreased at 125 mg/kg/day PCZ. These data from the pubertal assays demonstrate that PCZ decreases testosterone levels and delays rat pubertal development, as hypothesized. However, the fact that hormone levels were affected at dosage eightfold below that which delayed the onset of puberty suggests that rather large reductions in serum testosterone may be required to delay puberty and consistently reduce androgen-dependent tissue weights.}, number={1}, journal={TOXICOLOGICAL SCIENCES}, author={Blystone, Chad R. and Furr, Johnathan and Lambright, Christy S. and Howdeshell, Kembra L. and Ryan, Bryce C. and Wilson, Vickie S. and LeBlanc, Gerald A. and Gray, Leon Earl, Jr.}, year={2007}, month={May}, pages={65–74} }
 @article{gray_wilson_stoker_lambright_furr_noriega_howdeshell_ankley_guillette_2006, title={Adverse effects of environmental antiandrogens and androgens on reproductive development in mammals}, volume={29}, ISSN={["1365-2605"]}, DOI={10.1111/j.1365-2605.2005.00636.x}, abstractNote={Within the last decade, several classes of chemicals have been shown in laboratory studies to disrupt reproductive development by acting as androgen receptor (AR) antagonists and/or inhibitors of fetal Leydig cell testosterone production. Some phthalate esters alter gubernacular differentiation by reducing insulin-like 3 (insl3) mRNA levels. We have found that AR antagonists and inhibitors of fetal testis hormone production generally induce cumulative, apparently dose-additive adverse effects when administered in mixtures. New research has also revealed the presence of androgens in the environment. Effluents from pulp and paper mills display androgenic activity of sufficient potency to masculinize and/or sex-reverse female fish. Effluent from beef cattle concentrated animal feedlot operations from the United States also displays androgenic activity in vitro, due, in part, to the presence of a steroid used to promote growth in beef cattle. In summary, we are only beginning to identify the classes of chemicals that have the potential to alter the androgen signalling pathway in utero. This review will (i) present information on the classes of environmental chemicals that display antiandrogenic and androgenic activities in vitro and in vivo, and (ii) provide an insight into how exposure to mixtures these chemicals might behave in utero.}, number={1}, journal={INTERNATIONAL JOURNAL OF ANDROLOGY}, author={Gray, LE and Wilson, VS and Stoker, T and Lambright, C and Furr, J and Noriega, N and Howdeshell, K and Ankley, GT and Guillette, L}, year={2006}, month={Feb}, pages={96–104} }
 @article{gordon_gray_monteiroriviere_miller_1995, title={TEMPERATURE REGULATION AND METABOLISM IN RATS EXPOSED PERINATALLY TO DIOXIN - PERMANENT CHANGE IN REGULATED BODY-TEMPERATURE}, volume={133}, ISSN={["0041-008X"]}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:A1995RH95400019&KeyUID=WOS:A1995RH95400019}, DOI={10.1006/taap.1995.1138}, abstractNote={2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to lower thyroxine levels and cause hypothermia in the adult rat; however, there is little known regarding the perinatal effects of TCDD on metabolism and temperature regulation of the offspring. To address this issue, thermoregulatory responses were assessed in adult male rat offspring exposed perinatally to 1.0 micrograms TCDD/kg body wt by gavage on Gestational Day 15. Individual castrated offspring were placed in a gradient-layer calorimeter for 5 hr during their nocturnal period while ambient temperature (Ta) was maintained at 10, 16, 24, or 28 degrees C. Metabolic rate (M), as measured from the total heat loss in the calorimeter, was determined along with evaporative heat loss (EHL), dry thermal conductance, and body core temperature (Tc). Animals exposed to TCDD had a significantly lower body temperature at TaS of 10, 16, and 24 degrees C and a higher thermal conductance. M was unaffected by TCDD, indicating that TCDD did not impair the effector to regulate Tc during cold exposure. EHL was also unaffected by TCDD. Skin blood flow of the interscapular area was measured in anesthetized rats with laser Doppler velocimetry and found to be the same in control and TCDD groups. The reduction in body temperature over a wide range of TaS concomitant with normal thermoregulatory effector function suggests that perinatal exposure to TCDD results in a reduction in the regulated body temperature (i.e., decrease in set-point).}, number={1}, journal={TOXICOLOGY AND APPLIED PHARMACOLOGY}, author={GORDON, CJ and GRAY, LE and MONTEIRORIVIERE, NA and MILLER, DB}, year={1995}, month={Jul}, pages={172–176} }