Systems Toxicology of Male Reproductive Development: Profiling 774 Chemicals for Molecular Targets and Adverse Outcomes

Background: Trends in male reproductive health have been reported for increased rates of testicular germ cell tumors, low semen quality, cryptorchidism, and hypospadias, which have been associated with prenatal environmental chemical exposure based on human and animal studies. Objective: In the present study we aimed to identify significant correlations between environmental chemicals, molecular targets, and adverse outcomes across a broad chemical landscape with emphasis on developmental toxicity of the male reproductive system. Methods: We used U.S. EPA’s animal study database (ToxRefDB) and a comprehensive literature analysis to identify 774 chemicals that have been evaluated for adverse effects on male reproductive parameters, and then used U.S. EPA’s in vitro high-throughput screening (HTS) database (ToxCastDB) to profile their bioactivity across approximately 800 molecular and cellular features. Results: A phenotypic hierarchy of testicular atrophy, sperm effects, tumors, and malformations, a composite resembling the human testicular dysgenesis syndrome (TDS) hypothesis, was observed in 281 chemicals. A subset of 54 chemicals with male developmental consequences had in vitro bioactivity on molecular targets that could be condensed into 156 gene annotations in a bipartite network. Conclusion: Computational modeling of available in vivo and in vitro data for chemicals that produce adverse effects on male reproductive end points revealed a phenotypic hierarchy across animal studies consistent with the human TDS hypothesis. We confirmed the known role of estrogen and androgen signaling pathways in rodent TDS, and importantly, broadened the list of molecular targets to include retinoic acid signaling, vascular remodeling proteins, G-protein coupled receptors (GPCRs), and cytochrome P450s. Citation: Leung MC, Phuong J, Baker NC, Sipes NS, Klinefelter GR, Martin MT, McLaurin KW, Setzer RW, Darney SP, Judson RS, Knudsen TB. 2016. Systems toxicology of male reproductive development: profiling 774 chemicals for molecular targets and adverse outcomes. Environ Health Perspect 124:1050–1061; http://dx.doi.org/10.1289/ehp.1510385

[1]  Test No. 443: Extended One-Generation Reproductive Toxicity Study , 2018, OECD Guidelines for the Testing of Chemicals, Section 4.

[2]  W. Barfield,et al.  CDC releases a National Public Health Action Plan for the Detection, Prevention, and Management of Infertility. , 2015, Journal of women's health.

[3]  M. Mark,et al.  Role of retinoic acid receptor (RAR) signaling in post-natal male germ cell differentiation. , 2015, Biochimica et biophysica acta.

[4]  Richard A. Anderson,et al.  Comparative Effects of Di(n-Butyl) Phthalate Exposure on Fetal Germ Cell Development in the Rat and in Human Fetal Testis Xenografts , 2014, Environmental health perspectives.

[5]  Changjiang Liu,et al.  BPA-induced apoptosis of rat Sertoli cells through Fas/FasL and JNKs/p38 MAPK pathways. , 2014, Reproductive toxicology.

[6]  Lude Franke,et al.  Genome-wide association analyses identify variants in developmental genes associated with hypospadias , 2014, Nature Genetics.

[7]  M. Pasanen,et al.  The unique characteristics of the placental transcriptome and the hormonal metabolism enzymes in placenta. , 2014, Reproductive toxicology.

[8]  Y. Kurauchi,et al.  Treatment of mid-pregnant mice with KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, induces abnormal retinal vascular patterning in their newborn pups. , 2014, Birth defects research. Part B, Developmental and reproductive toxicology.

[9]  Lee B. Smith,et al.  Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells , 2014, Proceedings of the National Academy of Sciences.

[10]  E. Prossnitz,et al.  Activation of GPER-1 Estradiol Receptor Downregulates Production of Testosterone in Isolated Rat Leydig Cells and Adult Human Testis , 2014, PloS one.

[11]  H. Frederiksen,et al.  Phthalate-induced pathology in the foetal testis involves more than decreased testosterone production. , 2014, Reproduction.

[12]  Xiaodong Han,et al.  Antagonistic Effects of a Mixture of Low-Dose Nonylphenol and Di-N-Butyl Phthalate (Monobutyl Phthalate) on the Sertoli Cells and Serum Reproductive Hormones in Prepubertal Male Rats In Vitro and In Vivo , 2014, PloS one.

[13]  Susan M. Huse,et al.  Differential response to abiraterone acetate and di-n-butyl phthalate in an androgen-sensitive human fetal testis xenograft bioassay. , 2014, Toxicological sciences : an official journal of the Society of Toxicology.

[14]  B. Jégou,et al.  A critical assessment of the endocrine susceptibility of the human testis to phthalates from fetal life to adulthood. , 2014, Human reproduction update.

[15]  Manuel C. Peitsch,et al.  Systems Toxicology: From Basic Research to Risk Assessment , 2014, Chemical research in toxicology.

[16]  Carlo L. Acerini,et al.  Anogenital Distance and Penile Length in Infants with Hypospadias or Cryptorchidism: Comparison with Normative Data , 2013, Environmental health perspectives.

[17]  C. Myers,et al.  Using networks to measure similarity between genes: association index selection , 2013, Nature Methods.

[18]  John S Witte,et al.  Hypospadias and genes related to genital tubercle and early urethral development. , 2013, The Journal of urology.

[19]  K. McGlynn,et al.  Congenital malformations and testicular germ cell tumors , 2013, International journal of cancer.

[20]  Viral G. Jain,et al.  Shorter anogenital distance correlates with undescended testis: a detailed genital anthropometric analysis in human newborns. , 2013, Human reproduction.

[21]  Y. Wada,et al.  KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, induces intrauterine growth restriction in mice. , 2013, Birth defects research. Part B, Developmental and reproductive toxicology.

[22]  B. Katzenellenbogen,et al.  Integrative genomics of gene and metabolic regulation by estrogen receptors α and β, and their coregulators , 2013, Molecular systems biology.

[23]  Lee B. Smith,et al.  Diethylstilboestrol Exposure Does Not Reduce Testosterone Production in Human Fetal Testis Xenografts , 2013, PloS one.

[24]  N. Jørgensen,et al.  Identification of a novel androgen receptor mutation in a family with multiple components compatible with the testicular dysgenesis syndrome. , 2013, The Journal of clinical endocrinology and metabolism.

[25]  Akifumi Shioda,et al.  The effects of interleukin-6 signal blockade on immune system, reproductive and skeletal development in juvenile mice. , 2013, Birth defects research. Part B, Developmental and reproductive toxicology.

[26]  S. Zeng,et al.  Nuclear receptors PXR and CAR: implications for drug metabolism regulation, pharmacogenomics and beyond , 2013, Expert opinion on drug metabolism & toxicology.

[27]  Makoto Ono,et al.  Disorders of sex development: new genes, new concepts , 2013, Nature Reviews Endocrinology.

[28]  T. Mitchard,et al.  Assessment of male rodent fertility in general toxicology 6-month studies. , 2012, Birth defects research. Part B, Developmental and reproductive toxicology.

[29]  J. Laskey,et al.  Novel molecular targets associated with testicular dysgenesis induced by gestational exposure to diethylhexyl phthalate in the rat: a role for estradiol. , 2012, Reproduction.

[30]  K. V. van Erpecum,et al.  Anti-inflammatory and metabolic actions of FXR: insights into molecular mechanisms. , 2012, Biochimica et biophysica acta.

[31]  M. Seifert,et al.  The aryl hydrocarbon receptor ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene regulate distinct genetic networks , 2012, Molecular and Cellular Endocrinology.

[32]  W. Xie,et al.  Targeting xenobiotic receptors PXR and CAR for metabolic diseases. , 2012, Trends in pharmacological sciences.

[33]  Kim Boekelheide,et al.  Of mice and men (and rats): phthalate-induced fetal testis endocrine disruption is species-dependent. , 2012, Toxicological sciences : an official journal of the Society of Toxicology.

[34]  M. Aalabaf-Sabaghi,et al.  Networks, Crowds and Markets: Reasoning about a Highly Connected World , 2012 .

[35]  T. Singer,et al.  The effects of interleukin-6 signal blockade on fertility, embryo-fetal development, and immunization in vivo. , 2012, Birth defects research. Part B, Developmental and reproductive toxicology.

[36]  David M. Reif,et al.  Update on EPA's ToxCast program: providing high throughput decision support tools for chemical risk management. , 2012, Chemical research in toxicology.

[37]  S. Eggener,et al.  Environmental toxicology of testicular cancer. , 2012, Urologic oncology.

[38]  S. Brunak,et al.  A genome-wide association study of men with symptoms of testicular dysgenesis syndrome and its network biology interpretation , 2011, Journal of Medical Genetics.

[39]  D. Bishop-Bailey.  PPARs and angiogenesis. , 2011, Biochemical Society transactions.

[40]  T. Knudsen,et al.  Disruption of embryonic vascular development in predictive toxicology. , 2011, Birth defects research. Part C, Embryo today : reviews.

[41]  H. Hamada,et al.  Retinoic acid signaling regulates sonic hedgehog and bone morphogenetic protein signalings during genital tubercle development. , 2011, Birth defects research. Part B, Developmental and reproductive toxicology.

[42]  Robert J Kavlock,et al.  Predictive models of prenatal developmental toxicity from ToxCast high-throughput screening data. , 2011, Toxicological sciences : an official journal of the Society of Toxicology.

[43]  Miho Suzuki,et al.  Induction of high-dose tolerance to the rat anti-mouse IL-6 receptor antibody in NZB/NZW F1 mice , 2011, Rheumatology International.

[44]  V. Trudeau,et al.  Neuroendocrine Disruption: More than Hormones are Upset , 2011, Journal of toxicology and environmental health. Part B, Critical reviews.

[45]  M. Cohn.  Development of the external genitalia: Conserved and divergent mechanisms of appendage patterning , 2011, Developmental dynamics : an official publication of the American Association of Anatomists.

[46]  T. Woodruff,et al.  Environmental Chemicals in Pregnant Women in the United States: NHANES 2003–2004 , 2011, Environmental health perspectives.

[47]  Suzanne M. McCahan,et al.  Insulin-Like 3 Exposure of the Fetal Rat Gubernaculum Modulates Expression of Genes Involved in Neural Pathways1 , 2010, Biology of reproduction.

[48]  M. Celander,et al.  One-way inhibiting cross-talk between arylhydrocarbon receptor (AhR) and estrogen receptor (ER) signaling in primary cultures of rainbow trout hepatocytes. , 2010, Aquatic toxicology.

[49]  Y. Kojima,et al.  Genetic pathway of external genitalia formation and molecular etiology of hypospadias. , 2010, Journal of pediatric urology.

[50]  J. Bailar,et al.  Toxicity Testing in the 21st Century: A Vision and a Strategy , 2010, Journal of toxicology and environmental health. Part B, Critical reviews.

[51]  Ju Liu,et al.  Differential induction of MAP kinase signalling pathways by cadmium in primary cultures of mouse embryo limb bud cells. , 2010, Reproductive toxicology.

[52]  Daniel L Villeneuve,et al.  Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment , 2010, Environmental toxicology and chemistry.

[53]  R. Sharpe,et al.  Steroidogenesis in the fetal testis and its susceptibility to disruption by exogenous compounds. , 2009, Endocrine reviews.

[54]  L. Richiardi,et al.  Does a testicular dysgenesis syndrome exist? , 2009, Human reproduction.

[55]  Richard S. Judson,et al.  Profiling the activity of environmental chemicals in prenatal developmental toxicity studies using the U.S. EPA's ToxRefDB. , 2009, Reproductive toxicology.

[56]  Robert J Kavlock,et al.  Profiling the reproductive toxicity of chemicals from multigeneration studies in the toxicity reference database. , 2009, Toxicological sciences : an official journal of the Society of Toxicology.

[57]  B. Abbott.  Review of the expression of peroxisome proliferator-activated receptors alpha (PPAR alpha), beta (PPAR beta), and gamma (PPAR gamma) in rodent and human development. , 2009, Reproductive toxicology.

[58]  Mathieu Bastian,et al.  Gephi: An Open Source Software for Exploring and Manipulating Networks , 2009, ICWSM.

[59]  M. Balls.  The Way Forward for Reproductive/Developmental Toxicity Testing , 2008, Alternatives to laboratory animals : ATLA.

[60]  Brad T. Sherman,et al.  Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists , 2008, Nucleic acids research.

[61]  David M. Reif,et al.  Profiling Chemicals Based on Chronic Toxicity Results from the U.S. EPA ToxRef Database , 2008, Environmental health perspectives.

[62]  L Earl Gray,et al.  Diverse mechanisms of anti-androgen action: impact on male rat reproductive tract development. , 2008, International journal of andrology.

[63]  D. Veeramachaneni,et al.  Impact of environmental pollutants on the male: effects on germ cell differentiation. , 2008, Animal reproduction science.

[64]  F. Collins,et al.  Transforming Environmental Health Protection , 2008, Science.

[65]  R. Sharpe,et al.  Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects. , 2008, Fertility and sterility.

[66]  R. Sharpe,et al.  Role of androgens in fetal testis development and dysgenesis. , 2007, Endocrinology.

[67]  R. Sharan,et al.  Network-based prediction of protein function , 2007, Molecular systems biology.

[68]  N. Skakkebaek,et al.  Testicular dysgenesis syndrome: possible role of endocrine disrupters. , 2006, Best practice & research. Clinical endocrinology & metabolism.

[69]  J. Furr,et al.  Adverse effects of environmental antiandrogens and androgens on reproductive development in mammals. , 2006, International journal of andrology.

[70]  P. Chambon,et al.  Function of retinoid nuclear receptors: lessons from genetic and pharmacological dissections of the retinoic acid signaling pathway during mouse embryogenesis. , 2006, Annual review of pharmacology and toxicology.

[71]  J. Toppari,et al.  Testicular dysgenesis syndrome and the development and occurrence of male reproductive disorders. , 2005, Toxicology and applied pharmacology.

[72]  J. Toppari,et al.  Cryptorchidism: an indicator of testicular dysgenesis? , 2005, Cell and Tissue Research.

[73]  T. Ideker,et al.  Systematic interpretation of genetic interactions using protein networks , 2005, Nature Biotechnology.

[74]  C. Handschin,et al.  Regulatory network of lipid-sensing nuclear receptors: roles for CAR, PXR, LXR, and FXR. , 2005, Archives of biochemistry and biophysics.

[75]  A. Giwercman,et al.  Linkage between cryptorchidism, hypospadias, and GGN repeat length in the androgen receptor gene. , 2004, The Journal of clinical endocrinology and metabolism.

[76]  R. Paules,et al.  An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. , 2003, Molecular cell.

[77]  J. Brennan,et al.  Divergent vascular mechanisms downstream of Sry establish the arterial system in the XY gonad. , 2002, Developmental biology.

[78]  T. Willson,et al.  Pxr, car and drug metabolism , 2002, Nature Reviews Drug Discovery.

[79]  Atar Baer,et al.  Maternal, Delivery, and Perinatal Characteristics Associated with Cryptorchidism: A Population-Based Case-Control Study among Births in Washington State , 2002, Epidemiology.

[80]  N. Skakkebaek,et al.  Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects , 2001, Human reproduction.

[81]  L. Gray,et al.  The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[82]  R. Foote,et al.  The rabbit as a model for reproductive and developmental toxicity studies. , 2000, Reproductive toxicology.

[83]  P. Foster,et al.  Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to Di(n-butyl) phthalate during late gestation. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[84]  R. Cooper,et al.  Estrogenic activity of octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[85]  P. Foster,et al.  Male reproductive tract malformations in rats following gestational and lactational exposure to Di(n-butyl) phthalate: an antiandrogenic mechanism? , 1998, Toxicological sciences : an official journal of the Society of Toxicology.

[86]  W. Daniell.  Male reproductive toxicity. , 1990, The Western journal of medicine.

[87]  J. H. Ward.  Hierarchical Grouping to Optimize an Objective Function , 1963 .

[88]  S. Kharb.  Toxicology , 1936 .

[89]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[90]  A. Vinggaard,et al.  A computational approach to mechanistic and predictive toxicology of pesticides. , 2014, ALTEX.

[91]  J. Bowles,et al.  Male sex determination: insights into molecular mechanisms. , 2012, Asian journal of andrology.

[92]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[93]  Matthieu Latapy,et al.  Basic notions for the analysis of large two-mode networks , 2008, Soc. Networks.

[94]  These authors contributed equally to this manuscript. , 2007 .

[95]  Hongbing Wang,et al.  Role of Orphan Nuclear Receptors in the Regulation of Drug-Metabolising Enzymes , 2003, Clinical pharmacokinetics.

[96]  Paul M D Foster,et al.  Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate. , 2002, Reproductive toxicology.

[97]  D. Redmer,et al.  Angiogenesis in the placenta. , 2001, Biology of reproduction.

[98]  G P Daston,et al.  Leydig cell hyperplasia and adenoma formation: mechanisms and relevance to humans. , 1997, Reproductive toxicology.