The Effects Of Low-dose PBDE-209Exposure During Pregnancy And Lactation On Placenta NO/ET-1and Filial Generation Rats’ Hippocampus Cofilin-1/Vimentin

【Background】Brominated flame retardants have faced renewed attention inrecent years. Polychlorinated biphenyls (PCBs) which is the earliestflame retardant were banned in1977when it was discovered that theyare toxic. Industries shifted to using brominated flameretardants instead, but these are now receiving closer scrutiny. TheEU has banned several types of polybrominated diphenylethers (PBDEs) as of2008,10years after Sweden discovered thatthey were accumulating in breast milk. U.S. producers of DecaBDE(a flame retardant that has been used in electronics, wire and cableinsulation, textiles, automobiles and airplanes, and other applications)promise to phase out decaBDE for most uses in the United States byDecember31,2012, and to end all uses by the end of2013. Decabrominated diphenyl ether（PBDE-209）, the major congener inthe high volume industrial flame retardant mixture “DecaBDE”, canbe transferred to the baby via the placenta and umbilical cord.Herbstman showed that children who had higher cord bloodconcentrations of polybrominated diphenyl ethers (PBDEs) scoredlower on tests of mental and motor development at1–4and6years ofage. Roze demonstrated for the first time that transplacental transferof polybrominated flame retardants was associated with the cognitiveabilities (intelligence, visual perception, visuomotor integration,inhibitory control, verbal memory, and attention) of children at schoolage. What’s more, exposure to PBDE-209above the permissiblelevels among pregnants may cause adverse effects on newborns in theform of Low Birth Weight and Preterm Labor, etc.Authors did find exposure to PBDE-209during pregnancy mayhave implications for fetal development. But the mechanisms of itstoxicity were not fully clear. The placenta is an organ that connectsthe developing fetus to the uterine wall to allow nutrient uptake, wasteelimination, and gas exchange via the mother’s blood supply.Endothelin (ET)and prostanoids cause vasoconstriction in placentalarteries, while nitric oxide (NO) vasodilation. The objectives of ourstudy were to find out the effects of low-dose PBDE-209exposureduring pregnancy on the SD rat’s placenta ET-1and eNOSlevel.PBDE-209(decabrominated diphenyl ether) is one of thepersistent organic pollutants, which can accumulate in pregnantwomen when gestational exposure and can through the placenta andumbilical cord to the fetus to affect perinatal outcome. Part IThe effects of low-dose PBDE-209exposure during pregnancy on placenta NO/ET-1[Objective]We established a low-dose PBDE-209pregnancy exposed SD rats model. And we evaluated the effects of low-dose PBDE-209exposure during pregnancy on SD rats’placenta ET-1and eNOS mRNA expression level and on SD rats’placenta tissue ET-1、eNOS and total NO expression level, to investigate the effects of maternal exposure to BDE-209on SD rats’ placenta.[Materials and methods]1. We purchased40healthy female and20male SD rats,10weeks of age. Rats were raised in a specific pathogen free (SPF) laboratory animal facility in Guangzhou Medical University. Two females were caged overnight with one male rat. Mating was confirmed by the presence of sperm in vaginal smears. When a positive pregnancy was identified, this was considered day1of gestation. Unfertilized female rats were still with the male rat cage, until the conception.2. Forty10-week-old pregnant SD rats were randomly divided into five groups from the first gestation day (8rats per group). Group A:control group (normal feeding group); Group B:Gavage5ml/(kg·d) peanut oil; Groups C, D and E:Gavage1,5and10mg/(kg·d) PBDE-209, respectively, which was dissolved in an equal amount of peanut oil. Pregnant rats were normal feeding or gastric irrigation until22days of gestation.3. Placentas and newborn mouse in each group were taken by cesarean section at the22gestation day of each pregnant rats. Counting and weighing the Placentas and newborn mouse to preliminary understand the placenta function.4. Randomly selected four of the placentas from each rat and soaked them in4%paraformaldehyde. These were used to detect the expression of ET-1and eNOS in placenta tissues using immunohistochemical staining. The rest of the placentas in each group were divided into two parts, one part was used to measure total nitric oxide (NO) using a special ELISA kit, the other part was used to extract RNA and then using real-time quantitative reverse transcription-PCR (RT-qPCR) technology to detect the ET-1gene and eNOS gene mRNA expression levels in placenta.[Results]Compared with the control group (6.24±0.384), the mean birth weight of offspring rats in group D (5.49±0.498) andE (5.45±0.455) were reduced(,P<0.05). Compared with the control group(1.000±0.000), the mRNA expression levels of ET-1in group C (1.580±0.039)、D (1.680±0.086)and E(1.912±0.097)were increased(t=-21.234、-11.230and-13.358, P<0.05), and the mRNA expression levels of eNOS in group C (0.867±0.019)、D (0.792±0.058) and E (0.729±0.059) were reduced (t=9.341,4.685and3.537, P<0.05); Compared with the control group (1.00±0.00), the expression level of ET-1in group C(1.53±0.13), D (2.14±0.21) and E (2.57±0.15) were increased (P <0.05). And the expression level of eNOS in group C(0.82±0.11)、D (0.64±0.12) and E (0.49±0.20) were decreased (P<0.05).Compared with the control group (27.179±1.226), expression levels of total NO in group D (79.744±0.577) and E (98.141±1.442) were increased (t=-77.587、-74.984, P<0.05)[Conclusion]PBDE-209exposure during pregnancy affects the placenta expression level of ET-1gene and eNOS gene, which affects the placenta function, thus reduce offspring birth weight. Part ⅡThe effects of low-dose PBDE-209exposure during pregnancy and lactation on filial generation rats’ hippocampus cofilin-1/Vimentin[Objective]To investigate maternal exposure of BDE-209on learning and memory in filial generation rats.[Materials and methods]1. We purchased40healthy female and20male SD rats,10weeks of age. Rats were raised in a specific pathogen free (SPF) laboratory animal facility in Guangzhou Medical University. Two females were caged overnight with one male rat. Mating was confirmed by the presence of sperm in vaginal smears. When a positive pregnancy was identified, this was considered day1of gestation. Unfertilized female rats were still with the male rat cage, until the conception.2. Forty10-week-old pregnant SD rats were randomly divided into five groups from the first gestation day (8rats per group). Group A: control group (normal feeding group); Group B:Gavage5ml/(kg·d) peanut oil; Groups C, D and E:Gavage1,5and10mg/(kg·d) PBDE-209, respectively, which was dissolved in an equal amount of peanut oil. Rats in group B、C、D and E were gavaged from GO to28d after childbirth.3. Then, randomly selected20offspring (10males and10females)in each group to detect their learning and memory abilities using morris water maze;4. When morris water maze test finished, offspring’s hippocampus in each group were taken to detect the mRNA and protein expression levels of the cofilin-1and Vimentin gene.[Results]①Compared with the control group, the mean escape latency of offspring rats in group D and E were prolonged (P<0.05);②Compared with the control group (1.000±0.000), the mRNA expression levels of cofilin-1in group C(0.888±0.045)、D(0.787±0.041)and E(0.610±0.021) were decreased (t=-2.904、-4.555and-3.131, P<0.05), and the mRNA expression levels of Vimentin in group C (0.634±0.031)、D mRNA expression levels of Vimentin in group C（0.634±0.031）、D（0.428±0.022）and E（0.291±0.034）were also decreased（t＝－20.168、－45.852and－36.457，P＜0.01）;③Compared with the control group（1.60±0.07）, protein expression levels of the cofilin-1in group D（0.88±0.14）and E（0.37±0.13）were decreased（t＝3.152、5.432，P＜0.05）; And compared with the control group（1.52±0.20）, proteinexpression levels of the Vimentin in group D （0.51±0.22） and E（0.22±0.18）were also decreased（t＝5.884、8.368，P＜0.05）.【Conclusion】PBDE-209pregnancy and lactating exposure resulting decline inoffspring rats’ learning and memory abilities, this may be related with thedecline of mRNA and protein expression levels of cofilin-1gene andVimentin gene in offspring rat hippocampus.