Abstract
Endocrine disrupting chemicals may disrupt developing neuroendocrine systems, especially when the exposure occurs during a critical period. This study aimed to investigate whether prenatal exposure to di-(2-ethylhexyl) phthalate (DEHP), a major component of plasticizers used worldwide, disrupted the development of a network of genes important for neuroendocrine function in male rats. Pregnant rats were treated with corn oil (vehicle control), 2, 10 or 50 mg/kg DEHP by gavage from gestational day 14 to 19. The neuroendocrine gene expressions were quantified using a 48-gene Taqman qPCR array in the whole hypothalamus of neonatal rats (postnatal day 1) and in the anteroventral periventricular nucleus (AVPV), medial preoptic nucleus (MPN) and arcuate nucleus (ARC) of adult rats (postnatal day 70). Immunofluorescent signals of ERα and CYP19 were detected using the confocal microscopy in adult AVPV, MPN and ARC. The results showed that prenatal DEHP exposure perturbed somatic and reproductive development of offspring. Eleven genes were down-regulated in neonatal hypothalamus and showed non-monotonic dose–response relationships, that the 10 mg/kg DEHP dosage was associated with the greatest number of gene expression changes. Different from this, 14 genes were altered in adult AVPV, MPN and ARC and most of alterations were found in the 50 mg/kg DEHP group. Also, 50 mg/kg DEHP reduced ERα expression in the ARC, but no alterations were observed in CYP19 expression. These results indicated that prenatal DEHP exposure may perturb hypothalamic gene programming and the influences are permanent. The effects showed dependence on developmental stages and nuclei region.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andrade AJM, Grande SW, Talsness CE, Grote K, Chahoud I (2006) A dose–response study following in utero and lactational exposure to di-(2-ethylhexyl)-phthalate (DEHP): non-monotonic dose–response and low dose effects on rat brain aromatase activity. Toxicology 227(3):185–192. doi:10.1016/j.tox.2006.07.022
Araki A, Mitsui T, Miyashita C et al (2014) Association between maternal exposure to di(2-ethylhexyl) phthalate and reproductive hormone levels in fetal blood: the Hokkaido study on environment and children’s health. PLoS One 9(10):2014. doi:10.1371/journal.pone.0109039 (doi:ARTN e109039, eCollection 2014)
Axelsson J, Rylander L, Rignell-Hydbom A, Lindh CH, Jonsson BA, Giwercman A (2015) Prenatal phthalate exposure and reproductive function in young men. Environ Res 138C:264–270. doi:10.1016/j.envres.2015.02.024
Bai YY, Chang F, Zhou R et al (2011) Increase of anteroventral periventricular kisspeptin neurons and generation of E2-induced LH-surge system in male rats exposed perinatally to environmental dose of bisphenol-A. Endocrinology 152(4):1562–1571. doi:10.1210/en.2010-1042
Bakker J, De Mees C, Douhard Q et al (2006) Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens. Nat Neurosci 9(2):220–226. doi:10.1038/nn1624
Bateman HL, Patisaul HB (2008) Disrupted female reproductive physiology following neonatal exposure to phytoestrogens or estrogen specific ligands is associated with decreased GnRH activation and kisspeptin fiber density in the hypothalamus. Neurotoxicology 29(6):988–997. doi:10.1016/j.neuro.2008.06.008
Bosch OJ, Pfortsch J, Beiderbeck DI, Landgraf R, Neumann ID (2010) Maternal behaviour is associated with vasopressin release in the medial preoptic area and bed nucleus of the stria terminalis in the rat. J Neuroendocrinol 22(5):420–429. doi:10.1111/j.1365-2826.2010.01984.x
Cao JY, Patisaul HB (2011) Sexually dimorphic expression of hypothalamic estrogen receptors alpha and beta and Kiss1 in neonatal male and female rats. J Comp Neurol 519(15):2954–2977. doi:10.1002/cne.22648
Carbone S, Samaniego YA, Cutrera R et al (2012) Different effects by sex on hypothalamic–pituitary axis of prepubertal offspring rats produced by in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP). Neurotoxicology 33(1):78–84. doi:10.1016/j.neuro.2011.11.009
Christiansen S, Boberg J, Axelstad M et al (2010) Low-dose perinatal exposure to di(2-ethylhexyl) phthalate induces anti-androgenic effects in male rats. Reprod Toxicol 30(2):313–321. doi:10.1016/j.reprotox.2010.04.005
Clarkson J, Herbison AE (2009) Oestrogen, kisspeptin, GPR54 and the pre-ovulatory luteinising hormone surge. J Neuroendocrinol 21(4):305–311. doi:10.1111/j.1365-2826.2009.01835.x
Davis EC, Shryne JE, Gorski RA (1996) Structural sexual dimorphisms in the anteroventral periventricular nucleus of the rat hypothalamus are sensitive to gonadal steroids perinatally, but develop peripubertally. Neuroendocrinology 63(2):142–148
Dewing P, Shi T, Horvath S, Vilain E (2003) Sexually dimorphic gene expression in mouse brain precedes gonadal differentiation. Brain Res Mol Brain Res 118(1–2):82–90
Dickerson SM, Cunningham SL, Patisaul HB, Woller MJ, Gore AC (2011) Endocrine disruption of brain sexual differentiation by developmental PCB exposure. Endocrinology 152(2):581–594. doi:10.1210/en.2010-1103
Do RP, Stahlhut RW, Ponzi D, Vom Saal FS, Taylor JA (2012) Non-monotonic dose effects of in utero exposure to di(2-ethylhexyl) phthalate (DEHP) on testicular and serum testosterone and anogenital distance in male mouse fetuses. Reprod Toxicol 34(4):614–621. doi:10.1016/j.reprotox.2012.09.006
ECPI (2010) Plasticisers and flexible PVC information centre—plasticisers. http://www.plasticisers.org. Accessed 30 Mar 2015
EFSA (2005) Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to Bis(2-ethylhexyl)phthalate (DEHP) for use in food contact materials. EFSA J 243:1–20
Engel SM, Zhu C, Berkowitz GS et al (2009) Prenatal phthalate exposure and performance on the Neonatal Behavioral Assessment Scale in a multiethnic birth cohort. Neurotoxicology 30(4):522–528. doi:10.1016/j.neuro.2009.04.001
EURAR (2008) European Union risk assessment report of bis(2-ethylhexyl) phthalate (DEHP). https://www.echa.europa.eu/documents/10162/e614617d-58e7-42d9-b7fb-d7bab8f26feb. Accessed 14 Sept 2015
Foster PM (2006) Disruption of reproductive development in male rat offspring following in utero exposure to phthalate esters. Int J Androl 29(1):140–147. doi:10.1111/j.1365-2605.2005.00563.x (discussion 181–185)
Fuchsl AM, Langgartner D, Reber SO (2013) Mechanisms underlying the increased plasma ACTH levels in chronic psychosocially stressed male mice. PLoS One 8(12):e84161. doi:10.1371/journal.pone.0084161
Fudvoye J, Bourguignon JP, Parent AS (2014) Endocrine-disrupting chemicals and human growth and maturation: a focus on early critical windows of exposure. Vitam Horm 94:1–25. doi:10.1016/B978-0-12-800095-3.00001-8
Ge RS, Chen GR, Dong Q et al (2007) Biphasic effects of postnatal exposure to diethylhexylphthalate on the timing of puberty in male rats. J Androl 28(4):513–520. doi:10.2164/jandrol.106.001909
Gore AC (2001) Environmental toxicant effects on neuroendocrine function. Endocrine 14(2):235–246. doi:10.1385/ENDO:14:2:235
Gore AC (2008) Developmental programming and endocrine disruptor effects on reproductive neuroendocrine systems. Front Neuroendocrinol 29(3):358–374. doi:10.1016/j.yfrne.2008.02.002
Gore AC (2010) Neuroendocrine targets of endocrine disruptors. Hormones 9(1):16–27
Gore AC, Heindel JJ, Zoeller RT (2006) Endocrine disruption for endocrinologists (and others). Endocrinology 147(6 Suppl):S1–S3. doi:10.1210/en.2005-1367
Gore AC, Walker DM, Zama AM, Armenti AE, Uzumcu M (2011) Early life exposure to endocrine-disrupting chemicals causes lifelong molecular reprogramming of the hypothalamus and premature reproductive aging. Mol Endocrinol 25(12):2157–2168. doi:10.1210/me.2011-1210
Gore AC, Chappell VA, Fenton SE et al (2015) Executive summary to EDC-2: the endocrine society’s second scientific statement on endocrine-disrupting chemicals. Endocr Rev. doi:10.1210/er.2015-1093
Gorski RA, Harlan RE, Jacobson CD, Shryne JE, Southam AM (1980) Evidence for the existence of a sexually dimorphic nucleus in the preoptic area of the rat. J Comp Neurol 193(2):529–539. doi:10.1002/cne.901930214
Grande SW, Andrade AJ, Talsness CE, Grote K, Chahoud I (2006) A dose–response study following in utero and lactational exposure to di(2-ethylhexyl)phthalate: effects on female rat reproductive development. Toxicol Sci 91(1):247–254. doi:10.1093/toxsci/kfj128
Gray LE Jr, Ostby J, Furr J, Price M, Veeramachaneni DN, Parks L (2000) Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol Sci 58(2):350–365
Guo Y, Kannan K (2011) Comparative assessment of human exposure to phthalate esters from house dust in China and the United States. Environ Sci Technol 45(8):3788–3794. doi:10.1021/es2002106
Hass U, Scholze M, Christiansen S et al (2007) Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environ Health Perspect 115(Suppl 1):122–128. doi:10.1289/ehp.9360
Hotchkiss AK, Ostby JS, Vandenburgh JG, Gray LE Jr (2002) Androgens and environmental antiandrogens affect reproductive development and play behavior in the Sprague-Dawley rat. Environ Health Perspect 110(Suppl 3):435–439
Jakubowski M, Blum M, Roberts JL (1991) Postnatal development of gonadotropin-releasing hormone and cyclophilin gene expression in the female and male rat brain. Endocrinology 128(6):2702–2708. doi:10.1210/endo-128-6-2702
Jarfelt K, Dalgaard M, Hass U, Borch J, Jacobsen H, Ladefoged O (2005) Antiandrogenic effects in male rats perinatally exposed to a mixture of di(2-ethylhexyl) phthalate and di(2-ethylhexyl) adipate. Reprod Toxicol 19(4):505–515. doi:10.1016/j.reprotox.2004.11.005
Jasoni CL, Todman MG, Han S-K, Herbison AE (2006) Expression of mRNAs encoding receptors that mediate stress signals in gonadotropin-releasing hormone neurons of the mouse. Neuroendocrinology 82(5–6):320–328. doi:10.1159/000093155
Jin YX, Lin XJ, Miao WY et al (2014) Chronic exposure of mice to environmental endocrine-disrupting chemicals disturbs their energy metabolism. Toxicol Lett 225(3):392–400. doi:10.1016/j.toxlet.2014.01.006
Kanno J, Kato H, Iwata T, Inoue T (2002) Phytoestrogen-low diet for endocrine disruptor studies. J Agric Food Chem 50(13):3883–3885
Kavlock R, Barr D, Boekelheide K et al (2006) NTP-CERHR expert panel update on the reproductive and developmental toxicity of di(2-ethylhexyl) phthalate. Reprod Toxicol 22(3):291–399
Kay VR, Chambers C, Foster WG (2013) Reproductive and developmental effects of phthalate diesters in females. Crit Rev Toxicol 43(3):200–219. doi:10.3109/10408444.2013.766149
Kermath BA, Riha PD, Woller MJ, Wolfe A, Gore AC (2014) Hypothalamic molecular changes underlying natural reproductive senescence in the female rat. Endocrinology 155(9):3597–3609. doi:10.1210/en.2014-1017
Kim SH, Chun S, Jang JY, Chae HD, Kim CH, Kang BM (2011) Increased plasma levels of phthalate esters in women with advanced-stage endometriosis: a prospective case-control study. Fertil Steril 95(1):357–359. doi:10.1016/j.fertnstert.2010.07.1059
Kobrosly RW, Evans S, Miodovnik A et al (2014) Prenatal phthalate exposures and neurobehavioral development scores in boys and girls at 6–10 years of age. Environ Health Perspect 122(5):521–528. doi:10.1289/Ehp.1307063
Lauber ME, Sarasin A, Lichtensteiger W (1997) Transient sex differences of aromatase (CYP19) mRNA expression in the developing rat brain. Neuroendocrinology 66(3):173–180
Lee HC, Yamanouchi K, Nishihara M (2006) Effects of perinatal exposure to phthalate/adipate esters on hypothalamic gene expression and sexual behavior in rats. J Reprod Dev 52(3):343–352
Lephart ED (1996) A review of brain aromatase cytochrome P450. Brain Res Brain Res Rev 22(1):1–26
Li X, Jiang L, Cheng L, Chen H (2014) Dibutyl phthalate-induced neurotoxicity in the brain of immature and mature rat offspring. Brain Dev 36(8):653–660. doi:10.1016/j.braindev.2013.09.002
Lin Y, Wei J, Li Y et al (2011) Developmental exposure to di(2-ethylhexyl) phthalate impairs endocrine pancreas and leads to long-term adverse effects on glucose homeostasis in the rat. Am J Physiol Endocrinol Metab 301(3):E527–E538. doi:10.1152/ajpendo.00233.2011
Liu X, Herbison AE (2013) Dopamine regulation of gonadotropin-releasing hormone neuron excitability in male and female mice. Endocrinology 154(1):340–350. doi:10.1210/en.2012-1602
Lomniczi A, Wright H, Castellano JM, Sonmez K, Ojeda SR (2013) A system biology approach to identify regulatory pathways underlying the neuroendocrine control of female puberty in rats and nonhuman primates. Horm Behav 64(2):175–186. doi:10.1016/j.yhbeh.2012.09.013
Maggi R, Zasso J, Conti L (2014) Neurodevelopmental origin and adult neurogenesis of the neuroendocrine hypothalamus. Front Cell Neurosci 8:440. doi:10.3389/fncel.2014.00440
Markakis EA, Swanson LW (1997) Spatiotemporal patterns of secretomotor neuron generation in the parvicellular neuroendocrine system. Brain Res Brain Res Rev 24(2–3):255–291
Mayer CM, Fick LJ, Gingerich S, Belsham DD (2009) Hypothalamic cell lines to investigate neuroendocrine control mechanisms. Front Neuroendocrinol 30(3):405–423. doi:10.1016/j.yfrne.2009.03.005
Mittelman-Smith MA, Williams H, Krajewski-Hall SJ et al (2012) Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight. Endocrinology 153(6):2800–2812. doi:10.1210/en.2012-1045
Moore RW, Rudy TA, Lin TM, Ko K, Peterson RE (2001) Abnormalities of sexual development in male rats with in utero and lactational exposure to the antiandrogenic plasticizer di(2-ethylhexyl) phthalate. Environ Health Perspect 109(3):229–237
Navarro VM, Bosch MA, Leon S et al (2015) The integrated hypothalamic tachykinin-kisspeptin system as a central coordinator for reproduction. Endocrinology 156(2):627–637. doi:10.1210/en.2014-1651
Oakley AE, Clifton DK, Steiner RA (2009) Kisspeptin signaling in the brain. Endocr Rev 30(6):713–743. doi:10.1210/er.2009-0005
Paxinos W (2006) The rat brain in stereotaxic coordinates, 6th edn. Elsevier, pp 126–186
Pocar P, Fiandanese N, Secchi C et al (2012) Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring. Endocrinology 153(2):937–948. doi:10.1210/en.2011-1450
Rance NE, Krajewski SJ, Smith MA, Cholanian M, Dacks PA (2010) Neurokinin B and the hypothalamic regulation of reproduction. Brain Res 1364:116–128. doi:10.1016/j.brainres.2010.08.059
Roselli CE, Resko JA (2001) Cytochrome P450 aromatase (CYP19) in the non-human primate brain: distribution, regulation, and functional significance. J Steroid Biochem Mol Biol 79(1–5):247–253
Schecter A, Lorber M, Guo Y et al (2013) Phthalate concentrations and dietary exposure from food purchased in New York State. Environ Health Perspect 121(4):473–494. doi:10.1289/ehp.1206367
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108
Scott MM, Marcus JN, Pettersen A et al (2011) Hcrtr1 and 2 signaling differentially regulates depression-like behaviors. Behav Brain Res 222(2):289–294. doi:10.1016/j.bbr.2011.02.044
Silva MJ, Barr DB, Reidy JA et al (2004) Urinary levels of seven phthalate metabolites in the US population from the National Health and Nutrition Examination Survey (NHANES) 1999–2000. Environ Health Perspect 112(3):331–338
Sioen I, Fierens T, Van Holderbeke M et al (2012) Phthalates dietary exposure and food sources for Belgian preschool children and adults. Environ Int 48:102–108. doi:10.1016/j.envint.2012.07.004
Sousa-Ferreira L, de Almeida LP, Cavadas C (2014) Role of hypothalamic neurogenesis in feeding regulation. Trends Endocrinol Metab TEM 25(2):80–88. doi:10.1016/j.tem.2013.10.005
Specht IO, Toft G, Hougaard KS et al (2014) Associations between serum phthalates and biomarkers of reproductive function in 589 adult men. Environ Int 66:146–156. doi:10.1016/j.envint.2014.02.002
Suzuki Y, Yoshinaga J, Mizumoto Y, Serizawa S, Shiraishi H (2012) Foetal exposure to phthalate esters and anogenital distance in male newborns. Int J Androl 35(3):236–244. doi:10.1111/j.1365-2605.2011.01190.x
Swan SH (2008) Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans. Environ Res 108(2):177–184. doi:10.1016/j.envres.2008.08.007
Swan SH, Main KM, Liu F et al (2005) Decrease in anogenital distance among male infants with prenatal phthalate exposure. Environ Health Perspect 113(8):1056–1061. doi:10.1289/ehp.8100
Tabatadze N, Sato SM, Woolley CS (2014) Quantitative analysis of long-form aromatase mRNA in the male and female rat brain. PLoS One 9(7):e100628. doi:10.1371/journal.pone.0100628
Takagi H, Shibutani M, Lee KY et al (2005) Impact of maternal dietary exposure to endocrine-acting chemicals on progesterone receptor expression in microdissected hypothalamic medial preoptic areas of rat offspring. Toxicol Appl Pharmacol 208(2):127–136. doi:10.1016/j.taap.2005.02.002
Takumi K, Iijima N, Higo S, Ozawa H (2012) Immunohistochemical analysis of the colocalization of corticotropin-releasing hormone receptor and glucocorticoid receptor in kisspeptin neurons in the hypothalamus of female rats. Neurosci Lett 531(1):40–45. doi:10.1016/j.neulet.2012.10.010
USEPA (1996) Guidelines for reproductive toxicity risk assessment. Fed Reg 61(212):56274–56322
van den Driesche S, Scott HM, MacLeod DJ, Fisken M, Walker M, Sharpe RM (2011) Relative importance of prenatal and postnatal androgen action in determining growth of the penis and anogenital distance in the rat before, during and after puberty. Int J Androl 34(6):E578–E586. doi:10.1111/j.1365-2605.2011.01175.x
Vandenberg LN, Colborn T, Hayes TB et al (2012) Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses. Endocr Rev 33(3):378–455. doi:10.1210/er.2011-1050
Walker DM, Juenger TE, Gore AC (2009) Developmental profiles of neuroendocrine gene expression in the preoptic area of male rats. Endocrinology 150(5):2308–2316. doi:10.1210/en.2008-1396
Walker DM, Goetz BM, Gore AC (2014) Dynamic postnatal developmental and sex-specific neuroendocrine effects of prenatal polychlorinated biphenyls in rats. Mol Endocrinol 28(1):99–115. doi:10.1210/me.2013-1270
Wilson CA, Davies DC (2007) The control of sexual differentiation of the reproductive system and brain. Reproduction 133(2):331–359. doi:10.1530/REP-06-0078
Wittassek M, Wiesmuller GA, Koch HM et al (2007) Internal phthalate exposure over the last two decades—a retrospective human biomonitoring study. Int J Hyg Environ Health 210(3–4):319–333. doi:10.1016/j.ijheh.2007.01.037
Wolstenholme JT, Edwards M, Shetty SRJ et al (2012) Gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression. Endocrinology 153(8):3828–3838. doi:10.1210/En.2012-1195
Wu D, Gore AC (2010) Changes in androgen receptor, estrogen receptor alpha, and sexual behavior with aging and testosterone in male rats. Horm Behav 58(2):306–316. doi:10.1016/j.yhbeh.2010.03.001
Yin W, Maguire SM, Pham B et al (2015a) Testing the critical window hypothesis of timing and duration of estradiol treatment on hypothalamic gene networks in reproductively mature and aging female rats. Endocrinology 156(8):2918–2933. doi:10.1210/en.2015-1032
Yin W, Sun Z, Mendenhall JM et al (2015b) Expression of vesicular glutamate transporter 2 (vGluT2) on large dense-core vesicles within GnRH neuroterminals of aging female rats. PLoS One 10(6):e0129633. doi:10.1371/journal.pone.0129633
Acknowledgements
This work was supported by National Natural Science Foundation of China (81273027, to Zengrong Sun). We thank Dr. Wenli Lu and Dr. Changping Li (Department of Health Statistics) for providing statistical analysis help.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Funding
This study was funded by National Natural Science Foundation of China (81273027, to Zengrong Sun).
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Gao, N., Hu, R., Huang, Y. et al. Specific effects of prenatal DEHP exposure on neuroendocrine gene expression in the developing hypothalamus of male rats. Arch Toxicol 92, 501–512 (2018). https://doi.org/10.1007/s00204-017-2049-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-017-2049-z