Effects Of Perinatal Exposure To Bisphenol-A On Development Of The N-methyl-D-aspartate Receptor Of The Male Offspring Rat's Brain

Endocrine disrupting chemicals (EDCs) are chemicals that can disrupt thyroid hormones, Androgens, estrogens and other endocrine progresses. EDCs disrupt development by interfering with the hormonal signals that control normal development of the brain and other organ systems. EDCs can also affect adults by similar mechanisms because these same hormones also play important regulatory roles in adults. EDCs can act at very low levels of exposure to produce profound effects on the course an organism follow from fertilizes oocyte through to maturity, adulthood and death. The effects of EDCs on developing organisms are greatest concern. The developing brain exhibits specific and often narrow windows during which exposure to endocrine disruptors can produce permanent changes in its structure and function.Expoure to BPA is widespread. Bisphenol-A (BPA) is a monomer used in the manufacture of resins that line the inner surface of food cans and beverage containers. BPA leach from these products and disrupt endocrine fuction. BPA profoundly disrupt fetal development induces the brain development.BPA may be ingested by humans as it reportedly leaches from the lining of tin cans into foods, from dental sealants in to saliva, and from polycarbonate bottles into their contents. A large number of articles have been showing that BPA cause harm in laboratory animals at levels to which the average human is exposed. Up to now, there is little information about the effect of BPA on the brain development. N-methyl-D-aspartate(NMDA) receptor (N-methyl-D-aspartate receptor, NR), a subtype of the glutamate receptor, is crucial for normal CNS function and is ubiquitously distributed in high levels throughout the brain and participates in a wide variety of physiological processes, including synaptogenesis and synaptic plasticity, neurogenesis and migration long-term potentiation (LTP) and learning-memory. There are two types of subunits known, NR1 and NR2. The latter consists of 4 subtypes: NR2A～2D. Each NMDA receptor consists of one NR1 subunit and at least one NR2 subunit. Whereas the NMDA receptors play a good important in the developing of brain, estrogen has a relation with the development of CNS. Herein we investigate the effects of perinatal exposure to bisphenol-A on development of the N-methyl-D-aspartate receptor, ERβand P450arom in the rat's brain to lay the foundation for the molecular mechanisms of endocrine disrupting chemicals on the central nervous system (CNS) effects.Methods : Male and female Sprague-Dawley rats were purchased from Experimental Animal Center, Zhejiang Academy of Medical Science and maintained with free access to food and water. After acclimatization for 1 week, female rats were placed with males and vaginal smears were examined daily. A sperm-positive smear determined gestational day (GD) 0. After detection, the pregnant dams were placed individually and assigned to an exposure condition randomly (n=8～9 per condition). Dams were orally exposed to BPA dissolved in sesame oil (200, 50, 5 or 0.5 mg/kg/day) or only sesame oil as a vehicle control at between 8:00 and 9:30 per day from GD 7 through postnatal day (PND) 21. The oral route of BPA administration was chosen to mimic the most likely route of exposure to the compound in humans and wildlife. Dams were weighed daily before dosing. After parturition (PND 0), the pups were counted, weighed, and culled to eight pups, maintaining only male offspring if possible. The pups were identified individually on PND 4 and separated into same-sex littermates and housed at weaning (PND 21). The male pups were sacrificed on PND 4, 7,14, 21 and 56 respectively. The hippocampal regions of the brain were dissected and then stored at -70℃until use.Western-blot was used to check the protein expression of NMDA receptor subunits,ERβand P450arom with rabbit aoti-NR1, NR2A, NR2B and ERβpolyclonal and a mouse anti-P450arom polyclonal antibody. Beta-actin was adopted as intra-reference. Results: 1. In the hippocampus, the expressions of NMDA receptor subunits in the all groups of BPA exposure were remarkable decreased when compared with the control group. At the lower dose of 0.5～50 mg/kg/day, BPA concentration-dependently down-regulated the expression of NMDA receptor subunits; however, at the high dose (200 mg/kg/day), the effects of BPA on these subunits were different, with a larger down-regulation of NR1 expression and a less down-regulation of NR2A, 2B expression when compared with those at the lower dose of BPA. In the premotor cortex, the expression of NMDAR subunits is less sensitive to BPA. Only the higher dose (50, 200 mg/kg/day) of BPA exposure down-regulated the expression of subunits NR2A, 2B. In addition, BPA changed the component of NMDAR subunits in a different pattern in the hippocampus and the premotor cortex. The rates of NR2A and NR1, NR2B and NR1 were increased by 200 mg/kg/day of BPA in the hippocampus, but by 0.5 mg/kg/day of BPA in the premotor cortex; however, the other dose of BPA decreased the rates, when compared with the control.2. In the hippocampus, BPA concentration-dependently down-regulated the protein expression of estrogen beta receptor;especially during the postnatal first week when compared with the control group (P<0.05,P<0.01 or P<0.001). In the premotor cortex, the lower dose (0.5, 5 mg/kg/day) of BPA exposure down-regulated the expression of it with slightly; However, during the whole development period,the higher dose (0.5, 5 mg/kg/day) of BPA exposure down-regulated the expression of it with remarkable (P<0.05, P<0.01 or P<0.001) .3. In the hippocampus, BPA concentration-dependently up-regulated the expression of the protein of P450arom;especially during the postnatal third weekwhen compared with the control group (P<0.05, P<0.01 or P<0.001.). In the premotor cortex, BPA concentration-dependently up-regulated the expression of it during the development period with remarkable (P<0.05, P<0.01 or P<0.001) .Conclution: Perinatal exposure to BPA down-regulated NMDA receptor and changed the component of NMDA receptor subunits; down-regulated the expression of ERβprotein and up-regulated P450arom in a concentration-dependent, which may have relation with the down-regulated NMDA receptor expression.