@article{blystone_lambright_cardon_furr_rider_hartig_wilson_gray_2009, title={Cumulative and Antagonistic Effects of a Mixture of the Antiandrogens Vinclozolin and Iprodione in the Pubertal Male Rat}, volume={111}, ISSN={["1096-0929"]}, DOI={10.1093/toxsci/kfp137}, abstractNote={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.}, number={1}, journal={TOXICOLOGICAL SCIENCES}, author={Blystone, Chad R. and Lambright, Christy S. and Cardon, Mary C. and Furr, Johnathan and Rider, Cynthia V. and Hartig, Phillip C. and Wilson, Vickie S. and Gray, Leon E., Jr.}, year={2009}, month={Sep}, pages={179–188} }
 @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{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={Summary}, 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{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} }
 @article{goetz_ren_schmid_blystone_thillainadarajah_best_nichols_strader_wolf_narotsky_et al._2007, title={Disruption of testosterone homeostasis as a mode of action for the reproductive toxicity of triazole fungicides in the male rat}, volume={95}, DOI={10.1093/toxsci/kfl124}, abstractNote={Triazole fungicides associated with a range of reported male reproductive effects in experimental animals were selected to assess potential toxic modes of action. Wistar Han rats were fed myclobutanil (M: 100, 500, or 2000 ppm), propiconazole (P: 100, 500, or 2500 ppm), or triadimefon (T: 100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 120. One male per litter was necropsied on PND1, 22, 50, or 92. Measurements included anogenital distance (AGD) at PND0, body and organ weights, serum hormone levels, age at preputial separation (PPS), sperm morphology and motility, and fertility and fecundity. AGD was increased by the high dose of all three triazoles, indicating hypervirilization. Triadimefon delayed PPS, consistent with delayed puberty, at 1800 ppm. Relative liver weights were increased at PND1, 50, and 92 by all three triazoles. Hepatocellular hypertrophy was present at PND50 from propiconazole and triadimefon and at PND92 from all three high-dose triazole treatments. Relative pituitary weights were decreased at PND92 by middle- and high-dose myclobutanil treatment. Absolute testis weights were increased at PND1 by myclobutanil, at PND22 by myclobutanil and triadimefon, and at PND50 by propiconazole and triadimefon treatment. Relative ventral prostate weights were increased at PND92 by myclobutanil and triadimefon treatment. Serum testosterone was increased at PND50 by triadimefon and at PND92/99 by all three triazole treatments. Insemination and fertility were impaired by myclobutanil and triadimefon treatment. In addition to the reproductive system effects, total serum thyroxine levels were decreased at PND92 by high-dose triadimefon. These reproductive effects are consistent with the disruption of testosterone homeostasis as a key event in the mode of action for triazole-induced reproductive toxicity.}, number={1}, journal={Toxicological Sciences}, author={Goetz, A. K. and Ren, H. Z. and Schmid, J. E. and Blystone, C. R. and Thillainadarajah, I. and Best, D. S. and Nichols, H. P. and Strader, L. F. and Wolf, D. C. and Narotsky, M. G. and et al.}, year={2007}, pages={227–239} }
 @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 200 mg/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 200 mg/kg/day and decreased androgen sensitive seminal vesicle and epididymides weights at 200 mg/kg/day. Furthermore, IPRO increased adrenal and liver weights at 200 mg/kg/day, presumably by different mechanism(s) of action. Serum testosterone levels were decreased along with serum 17α-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{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 17alpha-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{blystone_lambright_howdeshell_furr_sternberg_butterworth_durhan_makynen_ankley_wilson_et al._2007, title={Sensitivity of fetal rat testicular steroidogenesis to maternal prochloraz exposure and the underlying mechanism of inhibition}, volume={97}, ISSN={["1096-6080"]}, DOI={10.1093/toxsci/kfm055}, abstractNote={The fungicide prochloraz (PCZ) induces malformations in androgen-dependent tissues in male rats when administered during sex differentiation. The sensitivity of fetal testicular steroidogenesis to PCZ was investigated to test the hypothesis that the reported morphological effects from maternal exposure were associated with reduced testosterone synthesis. Pregnant Sprague-Dawley rats were dosed by gavage with 0, 7.8, 15.6, 31.3, 62.5, and 125 mg PCZ/kg/day (n = 8) from gestational day (GD) 14 to 18. On GD 18, the effects of PCZ on fetal steroidogenesis were assessed by measuring hormone production from ex vivo fetal testes after a 3-h incubation. Lastly, PCZ levels in amniotic fluid and maternal serum were measured using liquid chromatography/mass spectroscopy and correlated to the inhibition of steroidogenesis. Fetal progesterone and 17alpha-hydroxyprogesterone production levels were increased significantly at every PCZ dose, whereas testosterone levels were significantly decreased only at the two high doses. These results suggest that PCZ inhibits the conversion of progesterone to testosterone through the inhibition of CYP17. To test this hypothesis, PCZ effects on CYP17 gene expression and in vitro CYP17 hydroxylase activity were evaluated. PCZ had no effect on testicular CYP17 mRNA levels as measured by quantitative real-time polymersase chain reaction. However, microsomal CYP17 hydroxylase activity was significantly inhibited by the fungicide (K(i) = 865nM). Amniotic fluid PCZ concentrations ranged from 78 to 1512 ppb (207-4014nM) and testosterone production was reduced when PCZ reached approximately 500 ppb, which compares favorably with the determined CYP17 hydroxylase K(i) (326 ppb). These results demonstrate that PCZ lowers testicular testosterone synthesis by inhibiting CYP17 activity which likely contributes to the induced malformations in androgen-dependent tissues of male offspring.}, number={2}, journal={TOXICOLOGICAL SCIENCES}, author={Blystone, Chad R. and Lambright, Christy S. and Howdeshell, Kembra L. and Furr, Johnathan and Sternberg, Robin M. and Butterworth, Brian C. and Durhan, Elizabeth J. and Makynen, Elizabeth A. and Ankley, Gerald T. and Wilson, Vickie S. and et al.}, year={2007}, month={Jun}, pages={512–519} }
 @article{rockett_narotsky_thompson_thillainadarajah_blystone_goetz_ren_best_murrell_nichols_et al._2006, title={Effect of conazole fungicides on reproductive development in the female rat}, volume={22}, number={4}, journal={Reproductive Toxicology (Elmsford, N.Y.)}, author={Rockett, J. C. and Narotsky, M. G. and Thompson, K. E. and Thillainadarajah, I. and Blystone, C. R. and Goetz, A. K. and Ren, H. and Best, D. S. and Murrell, R. N. and Nichols, H. P. and et al.}, year={2006}, pages={647–658} }