| Phthalates are mainly used as plasticizers for polyvinyl chloride (PVC) resins,adhesives and cellulose film coating and they are widely exists in Daily necessities.More and more studies have been focus on the precious puberty in Children.Epidemiological studies found that the puberty age of girls was advanced which isassociated with exposing phthalates. Although there is some evidence on femalereproductive toxicity in adult, little is known about the effects of neonatal andjuvenile exposure to low–dose phthalate on female puberty development. Therefore,a better understanding of the effects and mechanism on puberty development effectsof phthalate is needed. Part I Effects of neonatal and juvenile exposure to Di(2-ethylhexyl)/Di-n-butyl phthalate on pubertydevelopment in female ratsObjectFrom the aspects of changes in sex and reproductive development to investigatethe effects of neonatal and juvenile exposure to Di(2-ethylhexyl)/Di-n-butylphthalate/on puberty development in female rats.Methods(1) Animal model: adult male and female SD rats were purchased at3monthsfor breeding. One-day-timed pregnant rats were housed individually. On littering(PND1), the pups were culled to only females and cross-fostered among dams. Thisstudy was divided into two parts: neonatal exposure and juvenile exposure. Rats wererandomly assigned to treating groups and dosed via subcutaneous injection duringPND1-5(neonatal exposure group) or PND26-30(juvenile exposure group) withsesame oil, DBP at0.5,5,50mg/kg/day, DEHP at0.5,5,50mg/kg/day or17-βestradiol at0.1mg/kg/day.(2) Evaluation of risks to female rats development: changes of body weight atdifferent period.(3) Evaluation of risks to female rats sex development: changes of Anogenitaldistance （AGD）at different period.(4) Evaluation of risks to female rats puberty and reproductive devopment:vaginal opening age, estrous cycle length and phase, serum estradiol and luteinizinghormone levels, ovary weight/body weight, numbers of different kinds of follicle. Result1. Effects of exposure to E2on puberty and reproductive development(1) Neonatal exposure to0.1mg/kg E2, rats’ body weight was significantlydecreased at PND15. In a gradual increase, rats body weight were no significantchang compare to control groups at PND25and35. Juvenile exposure to0.1mg/kg E2,rats body weight were no significant change at PND35.(2) Neonatal exposure0.1mg/kg E2, rat normalized AGD had no significantchang but an increasing trend at PND35. Juvenile exposure to0.1mg/kg E2, ratsnormalized AGD had no significant change at PND35.(3) Exposure to0.1mg/kg E2, compared to control group, rats vaginal openingage were significant advance. Exposure timing was no significant effects on ratvaginal opening age.(4) Exposed to0.1mg/kg E2, female rats’ estrous cycle length was increased.The various stages of the estrous cycle were disordered and can not form a regularestrous cycle.(5) Female rats were exposed to0.1mg/kg E2significant increase in serumestrogen levels, neonatal exposure of rat serum estrogen levels higher than theprepubertal exposure group.(6) Neonatal and juvenile exposure to0.1mg/kg E2, serum luteal generationhormone (LH) levels of rats was no significant impact.(7) Exposure to0.1mg/kg E2, rats ovarian/weight were no significant change,but number of different kinds of follicles had no significant change and no obviouspathological changes was observed.2. Effects of exposure to DBP on puberty and reproductive development(1) Neonatal exposure to DBP, compared to control group, female rats bodyweight were significant increased in0.5,5and50DBP mg/kg groups at PND15. AtPND25, female rats body weight were significant increased in0.5and50mg/kg DBP groups. At PND35, female rats body weight were significant increased only in50mg/kg DBP group.Juvenile exposure to DBP had no significant effects on female body weight atPND35compare to the control group.(2) Neonatal exposure to DBP, compared to the control group, female ratsnomalized AGD were decreased in5and50mg/kg DBP groups at PND15. At PND25,female rats nomalized AGD were significant increased in0.5and5mg/kg DBPgroups. At PND35, female rats nomalized AGD were significant increased in0.5and50mg/kg DBP groups. No significant change of PND35normalized AGD inprepubertal exposure to DBP.(3) Exposure to0.5,5,50mg/kg DBP, as compared to the control group, femalerats’ vaginal opening age significant advance in each group, and the two exposuretiming were without a significant impact on rats vaginal opening age. The exposuretiming had an interaction with DBP on the vaginal opening age, especially in PND1-50.5mg/kg DBP and PND26-305mg/kg groups DBP, female rats’ vaginal opening agewere advanced than other groups.(4) Exposure to0.5,5and50mg/kg DBP, rat estrous cycle length wassignificantly increased in0.5and50mg/kg DBP groups, rat estrous cycle length ofjuvenile exposure group were longer than that of neonatal exposure group. Rats’estrous cycle length of PND26-300.5and50mg/kg DBP groups were extended thanother groups. The various stages of the estrous cycle were disordered and can notform a regular estrous cycle.(5) In0.5mg/kg DBP group, serum estrogen level of female rats was significantlyincreased. Exposure timing and DBP had an interaction on serum estrogen level;serum estrogen level of PND26-300.5mg/kg DBP group was significantly higherthan that of other groups. (6) Exposure to DBP, compared to the control group, in0.5,5and50mg/kg DBPgroups, female rats’ serum LH levels was no significant difference, but an upwardtrend.(7) Exposed to DBP, in5mg/kg and50mg/kg groups, female rat ovarian/bodyweight significant increased, but number of different kinds of follicles had nosignificant change and no obvious pathological changes was observed.3. Effects of exposure to DEHP on puberty and reproductive development(1) Neonatal exposure to DEHP, compared to control group, in0.5,5and50mg/kg DEHP groups, rats’ body weight were significantly increased at PND15and25, and rats’ body weight was significant increased in50mg/kg DEHP group atPND35. Juvenile exposure to DEHP, compared to the control group, in5and50mg/kg DEHP groups, rats body weight were significantly reduced at PND35.(2) Neonatal exposure to DEHP, compared to control group, rats normalizedAGD in0.5,5and50mg/kg DEHP groups were no significant changes at PND15, atPND25rats normalized AGD were significantly increased in50mg/kg DEHP groups,at PND35, rats normalized AGD were significantly reduced in5mg/kg DEHP group.Juvenile exposure to DEHP, compared to control group, in0.5mg/kg DEHP grouprats normalized AGD were increased at PND35.(3) After exposure to DEHP, the vaginal opening of the female rats had nosignificant changes compared to control group, the timing of exposure did notsignificantly affect the vaginal opening age of the female rats.(4) After exposure to DEHP, the female rats estrous cycle length were significantincreased and each stage of the estrous cycle. The various stages of the estrous cyclewere disordered and can not form a regular estrous cycle.(5) Exposed to DEHP, female rats serum estrogen levels were not significantlychanged, but0.5and5mg/kg DEHP group of rats serum estrogen levels increased slightly. Exposure timing had no significant effect on serum estrogen levels.(6) Exposure to DEHP, although there is no significant change in female ratsserum LH levels, compared to the control group,0.5and5mg/kg DEHP serum LHlevels were increased.(7) Exposure to DEHP, female rats ovarian/weight was significantly increased in50mg/kg DEHP group. The two exposure timing had no significant effect on rats theovarian/weight. Female rat ovarian/body weight significant increased, but number ofdifferent kinds of follicles had no significant change and no obvious pathologicalchanges were observed.Conclusion1. Neonatal exposure to DBP and DEHP can increase the weight of the femalerats and AGD. The effect of increasing rats’ body weight was weakened gradually.The effects were different from exposure to E2dufing neonatal period, early exposureto E2reduced rats body weight, but this effect is weakened gradually. The mechanismof DBP and DEHP may be different from E2.2. Neonatal and juvenile exposure to DBP, female rats’ onset of puberty wassignificantly advanced and the various stages of the estrous cycle were disordered.Rats’ estrous cycle length was increased. Neonatal and juvenile exposure to DEHPhad no significant effect on female rats’ onset of puberty, but can disordered femalerats estrous cycle and increased the length of estrous cycle. The effect of DBPonfemale rats’ pubertal development was stronger than DEHP. The effects of DBP wassimilar to E2, but was more weak than it.3. Neonatal and juvenile exposure to DBP and DEHP, the ovaries/weight offemale rats in creased in high dose group. Although no effect on the number andphysiological structure of ovarian follicles, the impact of short-term exposure toDBP/DEHP on the reproductive system should be of concern. Part II Exploring mechanism of neonatal and juvenileexposure to Di (2-ethylhexyl)/Di-n-butyl phthalate onpuberty development in female ratsObject1. Using the hypothalamus in vitro culture model, to explore whetherDBP and DEHP can act on the hypothalamus and impact on the expressionof key endocrine genes.2. Exploring mechanism of neonatal and juvenile exposure to Di (2-ethylhexyl)/Di-n-butyl phthalate on puberty development in female ratsMethods1. Hypothalamus Short-term exposure to DEHP/DBP effects on kisspeptin/GPR54system(1) Hypothalamuses of juvenile female rat were dissected and randomly assignedto24-well plate with500μl Medium in every well. They were Pre-incubated for1hour in medium, then taken into different well with medium,10-6M DBP,10-6MDEHP,10-7M E2and incubated for1hour, finally they were transferred into wellwith medium.(2)At the end of incubation, hypothalamuses were collected.(3)Total RNA were isolated, and then for reverse transcription. The RT-PCRwas performed for analyzing expression of GnRH, kiss1, GRP54and ERα mRNA.1. Exploring mechanism of neonatal and juvenile exposure to Di (2-ethylhexyl)/Di-n-butyl phthalate on puberty development in female rats(1) Hypothalamuses were dissected from one part rats in every groups, totalRNA were isolated, and then for reverse transcription. The RT-PCR was performedfor analyzing expression of GnRH, kiss1, GRP54, ERα mRNA. (2) Other rats were primed and dissected into40μm sections. Immunohistochemistry was performed for analyzing density of kisspeptin fibers in AVPV and ARC.Result1. Hypothalamus Short-term exposure to DEHP/DBP effects on kisspeptin/GPR54system.Compared to the control group, Hypothalamus key genes GnRH, kiss1, ERamRNA expression level was significantly reduced exposure to10-6M DBP,10-6MDEHP and10-7M E2, no significant changes in expression of GPR54mRNA.2. Neonatal and juvenile exposure to Di (2-ethylhexyl)/Di-n-butyl phthalateeffects on kisspeptin/GPR54system of female rats hypothalamus.2.1Neonatal and juvenile exposure to E2effects on kisspeptin/GPR54system offemale rats hypothalamus(1) Neonatal and juvenile exposure to0.1mg/kg E2, hypothalamic kisspeptin,GPR54, ERα and ERβ mRNA expression levels were influenced. In AVPV area,neonatal exposure to0.1mg/kg E2, kisspeptin, GPR54, ERα and ERβ mRNA weredown-regulation, juvenile exposure to0.1mg/kg E2, kisspeptin, GPR54and ERαexpression were slightly lower than that in control group. In ARC area, juvenileexposure to0.1mg/kg E2, kisspeptin, GPR54and ERα were up-regulation.(2) Neonatal exposure to0.1mg/kg E2, immunohistochemical kisspeptinexpression density in AVPV was increased. In the ARC, immunohistochemicalkisspeptin expression density decreased slightly. Juvenile exposure to0.1mg/kg E2,immunohistochemical kisspeptin expression density decreased in AVPV and ARCarea.2.2Neonatal and juvenile exposure to Di-n-butyl phthalate effects on kisspeptin/GPR54system of female rats hypothalamus. (1) Neonatal exposure to DBP, in the ARC area, expression of ERα, ERβ,GPR54mRNA were down-regulated, but up-regulated or unchanged in juvenileexposure groups. Neonatal exposure to DBP, Kisspeptin mRNA expression were up-regulated in the ARC area, which were down-regulated or unchanged in juvenileexposure to DBP. In the AVPV area, ER α, ERβ, GPR54mRNA expression increasedor unchanged in PND1-50.5,5and50mg/kg DBP and PND26-200.5mg/kg DBPgroups. Juvenile exposure to DBP, kisspeptin, GPR54, ERβ mRNA wasdown-regulated or unchanged in the high dose group, but ERα mRNA wasupregulation.(2) Neonatal exposure to DBP, kisspeptin expression density was increased inboth ARC and AVPV area. Juvenile exposure to DBP, in the ARC area, kisspeptinexpression density was significantly decreased in5and50mg/kg DBP group, in theAVPV area, kisspeptin expression density were decreased.2.3Neonatal and juvenile exposure to Di (2-ethylhexyl) phthalate effects onkisspeptin/GPR54system of female rats hypothalamus(1) In the AVPV area, neonatal exposure to DEHP, expression of ERα, ERβ,GPR54mRNA were no significant changes, and in0.5,5mg/kg DEHP groups,expression of ERα, ERβ, and GPR54mRNA were declined. In50mg/kg DEHP group,expression of ERα, ERβ, and GPR54mRNA were increased or remain unchanged. Inthe ARC area, neonatal exposure to DEHP, ERα, ERβ and GPR54mRNA expressionswere decreased, there was no significant change in the juvenile exposure groups. InAVPV area, kisspeptin mRNA expression was up-regulation in neonatal exposuregroups, but down-regulation in juvenile groups. In ARC area, kisspeptin mRNAexpression was down-regulation.（2）Neonatal exposure to DEHP, kisspeptin expression density were significantimpacted, in the AVPV area, kisspeptin expression density was significantly higher than the control group, in the ARC area, kisspeptin expression density was nosignificant changes with a increased trend. Juvenile exposure to DEHP, in the AVPVarea, kisspeptin expression density was increased in5and50mg/kg DEHP groups, inthe ARC area, kisspeptin expression density was lower in0.5,5and50mg/kgDEHP group than the control.ConclusionExposure to DBP, DEHP and E2, expressions of GnRH, kisspeptin, GPR54andERα mRNA were significantly affected. Neonatal and juvenile exposure to DBP,DEHP and E2, mainly affected the expression of kisspeptin and GRP54mRNA, andexpressions of ERα and ERβ had a little effect. Trend of kisspeptin protein andmRNA expression in AVPV areas was consistent with that in ARC area in neonataland juvenile DBP treatment groups. All that are different in DEHP and E2teatmentgroups. The possible reason is that non-monotonic dose-response relationship of DBPand DEHP, and different compensatory ways in neonatal and juvenile exposure period.Hypothalamic gene and protein changes may further affect hormone levels, and thefeedback effect of the endocrine system, and ultimately affect pubertal andreproductive development. |
