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)
Highlights
► We investigated the effect of the endocrine disruptors DEHP on the reproductive neuroendocrine regulation in prepubertal male and female rats. ► DEHP decrease GABA and increase aspartate and serum gonadotrophin in male rats. ► DHEP increased aspartate and GABA in female rats, both stimulatory on gonadotrophin. ► DEHP produces qualitative and quantitative changes in the hypothalamic amino acid neurotransmitters that regulate reproduction in prepubertal male and female rat.
Introduction
Reproductive abnormalities in laboratory and wildlife animals were associated with exposure to industrial chemicals. This fact generated public concern that this chemicals, named “environmental oestrogens” or “xenoestrogens may impair human reproductive health (Akingbemi et al., 2001, Sharpe, 2001). Several high weight phthalates, including the widely used plasticizer di-(2-ethylhexyl) phthalate (DEHP), have been early identified as endocrine disruptors capable of altering male sexual differentiation in animals (Sharpe, 1995, Joblin, 1995, Sharpe, 2001, Gray et al., 2000, Akingbemi et al., 2001). DEHP is used in a variety of polyvinyl chloride-based consumer products including infant toys, food wraps, cosmetics and many surgical and medical consumables and devices such us tubing, blood bags and dialysis equipment (Heudorf et al., 2007, Latini et al., 2003, NTP-CERHR, 2006). This substance can leach readily from plastic products into foods, beverages or directly into body fluids (Latini, 2000, Lovekamp-Swan and Davis, 2003, Gartner et al., 2009). Consequently the potential for non-occupational exposure to DEHP is high and humans are daily exposed to DEHP through ingestion, inhalation and by dermal contact (NTP-CERHR, 2006, Schettler, 2006). Moreover, occupational and medical exposure increase body burden levels (Calafat et al., 2004, Calafat and McKee, 2006, Doull et al., 1999, Faouzi et al., 1999, Koch et al., 2006, Latini et al., 2003, Pak et al., 2007) reaching much higher concentrations in this population.
DEHP induces anti-androgenic action and abnormalities of the reproductive system in animals depending of the dose, time and stage of development at exposure (Latini, 2000, Akingbemi and Hardy, 2001). Several mechanisms by which DEHP affects reproductive function in rodents are relevant to humans, e.g., an androgen receptor-independent mechanism of anti-androgenicity (Akingbemi et al., 2004, Bonefeld-Jorgensen et al., 2001, Foster et al., 2001, Gray et al., 2000, Latini et al., 2003, Parks et al., 2000), reduction in the expression of steroidogenesis related factors and activation of peroxisome proliferator-activated receptors (Borch et al., 2006, Howdeshell et al., 2007, Lovekamp-Swan and Davis, 2003, Wilson et al., 2008).
The reproductive toxicity of gestacional and lactational exposure to DEHP is attributable to the action of its primary metabolite, mono-(2-ethylhexyl) phthalate (MEHP), which cross the placental barrier and pass into breast milk (Latini et al., 2003, Main et al., 2006, Stroheker et al., 2005). However, in humans the urine excretion of MEHHP, MEOHP and MECCP, secondary oxidized metabolites of DEHP, reflect better the exposure level than MEHP (Koch et al., 2006). At gonadal level, in uterus and lactational exposure to DEHP produces abnormalities in androgen-dependent processes (Akingbemi et al., 2001, Albro, 1987, Borch et al., 2005, Gray et al., 2000, Parks et al., 2000), alters sexual differentiation (Andrade et al., 2006), and affects the reproductive function and sexual behavior in male rat offspring (Dalsenter et al., 2006). At hypothalamic-pituitary level, prenatal and lactational DEHP exposure at a dose of 30 mg/kg bw/day modifies the content of amino acid neurotransmitters in hypothalamus and FSH secretion in 30-day-old peripubertal male rats (Carbone et al., 2010). The possibility that DEHP exerts adverse effects on androgen synthesis and thereby interferes with reproductive tract development in the human male fetus has been raised in light of evidences indicating that boys born from women exposed to phthalates during pregnancy have an increased incidence of inborn genital malformations, such as shortened anogenital distance (Swan et al., 2005, Swan, 2008), cryptorchidism and hypospadias which are symptoms of one entity named testicular dysgenesis syndrome (TDS) (Skakkebaek et al., 2001). Phthalates induce these adverse effects as a result of abnormal Leydig cells function and the decrease in testosterone synthesis (Hu et al., 2009). Also, prenatal phthalates exposure has been associated with alterations in the neurodevelopment in humans, as neonatal and childhood behaviour, and executive functioning (Engel et al., 2009, Engel et al., 2010).
Little information concerning the reproductive effects of pre and perinatal exposure to DEHP on female rats is presently available, although they have been associated with an increase in the number of ovarian atretic tertiary follicles in adult female offspring rats (Grande et al., 2007). Exposure of adult rats to DEHP results in hypoestrogenic anovulatory cycles and polycystic ovaries (Davis et al., 1994, Lovekamp-Swan and Davis, 2003). MEHP, the active metabolite of DEHP, reduces estradiol production by decreasing aromatase, the rate-limit enzyme that converts testosterone to estradiol, in granulose cell cultures of immature rats (Lovekamp and Davis, 2001). In vitro assays demonstrated that DEHP and the metabolite MEHP may directly inhibit antral follicle growth in adult mice, via a mechanism that includes reduction in levels of estradiol production and a decreased expression of cell cycle regulators (Gupta et al., 2010). On the pituitary–gonadal axis of immature female rats DEHP exerts dual effects stimulating the hormonal function of the pituitary and, at the same time, inhibiting steroidogenesis by the ovarian granulose cells (Svechnikova et al., 2007).
The hypothalamic amino acid system appears to play an important role in the different neuroendocrine processes involved in sexual maturation and in the onset of puberty (Moguilevsky and Wutke, 2001). The excitatory amino acid aspartate (ASP) stimulates GnRH release (Brann and Mahesh, 1994). Previously we have demonstrated that N-methyl-d-aspartate and non-N-methyl-d-aspartate receptors of the excitatory amino acid system stimulate the release of Gn-RH from hypothalamic fragments in vitro and also increase serum gonadotropin levels during maturation in male and female rats (Carbone et al., 1996, Moguilevsky et al., 1995). Gamma amino butyric acid (GABA) exerts the inhibitory control of the hypothalamic–pituitary–gonadal (HPG) axis in peripubertal and adult rats. However, this neurotransmitter stimulates gonadotropin secretion in prepubertal (15 days of age) female rats (Feleder et al., 1996, Moguilevsky et al., 1991). It is also demonstrated that the regulatory effects of amino acid neurotransmitters system on GnRH neurons are modulated by sexual hormones acting on different receptor-subtypes of these neurotransmitters in the anterior preoptic and medial basal areas (APOA-MBH) of the hypothalamus (Moguilevsky et al., 1995, Szwarcfarb et al., 1994).
Despite the potential adverse effects on reproductive system associated with DEHP exposure, there is little information about the neuroendocrine mechanisms by which this endocrine disrupter could alter sexual development. The purpose of this work was to investigate whether DEHP modifies gonadotropin levels and the hypothalamic content of the amino acid neurotransmitters ASP, GLU (glutamate) and GABA in 15-day-old prepubertal male and female rat offspring in DEHP-exposed dams during gestational and nursing periods. An oral route in DEHP administration was chosen for this study to mimic the most likely route.
Section snippets
Animals and drug
Wistar rats used for this work were provided by the Department of Physiology, School of Medicine, University of Buenos Aires. They were allowed at least 7-day acclimatization-period and observed for signs of illness before starting experimental procedures. Animals were raised under light (lights on from 7 am to 7 pm), temperature (22–24 °C) and humidity controlled conditions. Rats were fed with a pellet diet (ACA Animal Nutrition Division, Complete balanced animal aliment, Argentine Industry)
Results
Litter size was not altered by maternal DEHP treatment (8–10 pups per litter in DEHP and control groups). Also, exposure to DEHP did not produced changes on pup weights at birth (control: 6.14 ± 0.26; DEHP 3 mg/kg bw/day: 6.58 ± 0.38 and 30 mg/kg bw/day: 5.94 ± 0.25) and viability (number of pups at sacrifice relative to number of pups at birth).
Discussion
The processes of sexual maturation and adult reproduction function are under the control of numerous hormone-mediated feed back mechanisms that are sensitive to the regulation of many endogenous and exogenous factors, including the environmental endocrine disruptors. Since several of these mechanisms begin to operate during fetal life, the pre and perinatal exposure to chemicals that alter the hormonal balance become very significant in these stages.
Previous studies of phthalates were conducted
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by Grants from Agencia de Promoción Científica y Técnica BID 1728 PIC 2003 and the Universidad Buenos Aires (UBACYT 20020090200080 and UBACYT M006). We thank Angela Ciocca for the manuscript revision.
DISCLAIMER
The findings and conclusions in this report are those of the authors.
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