Neurotoxicity Of Dibutyl Phthalate On Immature Rat Brain Following Perinatal Exposure And The Underlying Mechanism

PART Ⅰ EFFECTS OF DIBUTYL PHTHALATE ONHIPPOCAMPAL NEURONS IN RATS OF DIFFERENTDEVELOPMENTAL STAGES FOLLOWING PERINATAL EXPOSUREObjective: To observe the effects of Dibutyl Phthalate (DBP)at dose of500mg/(kg.d) on hippocampal neurons in rat ofdifferent developmental stages following perinatal exposure,and to investigate whether the effects have age and genderdifferences，finally to find out whether the neurotoxicity ofDBP is still significant for adulthood offspring rats aftercessation of exposure in early developmental stages.Methods: Pregnant Sprague Dawley rats were gavaged dailywith500mg/(kg.d) DBP or with corn oil (control) from gestationday (GD)6to postnatal day (PND)21and the Offspring rats werefed normally after weaning on PND21, then the hippocampus ofoffspring rats were obtained on PND5、PND21and PND60and the following will be carried out:①Counting surviving neurons andobserving morphology changes by Nissl staining;②Observingultrastructural changes of neurons using transmission electronmicroscopy;③Detecting neuronal apoptosis and necrosis byTUNEL and by flow cytometry with Annexin V-PI dual parameters;④Detecting caspase-3activity by Microplate Reader.Results:①Neurons in hippocamps disordered and the nisslbody degraded in DBP-treated group on PND5and PND21, neuronalnumber was also significantly decreased compared with controlgroup，no significant difference was found between male andfemale rats. Moreover, compared with control, neuronal numberwas reduced by13.4%and15.3%in female and male offspringsrespectively on PND5, while was reduced by7.9%and4.7%in femaleand male offsprings on PND21. Neuronal number in DBP-treated ratson PND60also decreased compared with control, but withoutsignificant difference.②Ultrastructure of hippocampalneurons showed mitochondria swelling, even vacuolization andendocytoplasmic reticulum dilatation in male and femaleDBP-treated rats on PND21, while none obvious pathologic changeswere detected in DBP-treated rats on PND60and in control rats.③Compared with control, significant increasing neoronalapoptosis were found in male and female DBP-treated rats on PND5 and PND21both by TUNEL assay and flow cytometry assay; Comparedwith DBP-treated rats on PND21, a more significant increasingneuronal apoptosis was found in DBP-treated rats on PND5.Although neuronal apoptosis in DBP-treated rats on PND60increased compared with control, but there was no significantlydifference.④Both male or female rats in DBP-treated group onPND5and PND21,caspase-3activity were significantly increasedwhen compared to control, and the increasing of caspase-3activity in DBP-treated rats on PND5was more apparent than ratson PND21.Similar with the results above, no significantdifference was observed between DBP-treated rats on PND60andrats in control group on PND60.Conclusion:①Perinatal exposure to500mg/(kg.d) DBP mayincrease neuronal death and apoptosis in hippocampus of bothnewborn and childhood offspring rats without gender differences.And the damage was more severe in newborn rats than in childhoodrats.②DBP failed to cause adulthood hippocampal neurons damagesignificantly after cessation of exposure in early stages ofdevelopment. PART Ⅱ EFFECTS OF DIBUTYL PHTHALATE ONHIPPOCAMPAL SYNAPSES OF IMMATURE OFFSPRING RATSFOLLOWINGPERINATA EXPOSUREOBJECTIVES: To investigate the effects of DBP on hippocampalsynapses of immature offspring rats following perinatal exposure,and to clarify whether the effects have gender differences,whether the effects are still significant for adulthoodoffspring rats after cessation of exposure in early stages ofdevelopment.METHODS: Pregnant Sprague Dawley rats were gavaged dailywith500mg/(kg.d) DBP or with corn oil (control) from gestationday (GD)6to postnatal day (PND)21and the Offspring rats werefed normally after weaning on PND21, then synaptophysin levelin hippocampus were detected by immunohistochemistry and westernblot, ultrastructural changes of hippocampal synapses wereobserved using transmission electron microscopy, and fieldexcitatory postsynaptic potentials (fEPSPs) in CA1area ofhippocampal slices after high-frequency stimulation(HFS) wereanalyzed with patch clamp when offspring rats were on PND21andPND60. RESULTS:①Synaptophysin level decreased markly inhippocampus in both male and female DBP-treated pups on PND21compared with the control (P<0.01), though synaptophysin levelalso decreased in both male and female DBP-treated pups on PND60,but the difference was not statistically significant comparedwith control.②Ultrastructure of hippocampal synapses showedthe number of synapses reduced, synaptic vesicles decreased andpostsynaptic density got thinner in both male and femaleDBP-treated pups on PND21compared with the control, but nonesimilar abnormal changes were detected in DBP-treated rats onPND60.③The changing degrees of fEPSPs slope and amplitudeafter HFS were significantly lower in both male and femaleDBP-treated pups on PND21than the control. However, all the datain DBP-treated rats on PND60decreased too but withoutsignificant difference compared with the ras in control group.CONCLUSIONS:①Perinatal exposure to500mg/(kg.d) DBP cansignificantly damage hippocampal synapse structure andeffectiveness of synaptic transmission in DBP-treated childhoodrat without gender differences.②The impact of DBP perinatalexposure on synapse structure and function in adult offspringrats was minor, and was not statistically significant comparedwith rats in control group, which showed DBP also failed to cause adulthood hippocampal synapse damage significantly aftercessation of exposure in early developmental stages. PART Ⅲ POSSIBLE MECHANISMS OF DBP NEUROTOXICITYON OFFSPRING RATS FOLLOWING PERINATAL EXPOSUREOBJECTIVES:To observe sex hormone levels in serum and AROM、ER-β、p-CREB and BDNF levels in hippocampus of offspring ratsfollowing perinatal DBP exposure, and to probe possiblemechanisms of neurotoxicity of DBP.METHODS: Drug administration and animal grouping in thispart are the same as that in part I and II, then the serum andhippocampus of off-spring rats were obtained on PND21and PND60for testing thefollowings:①Testosterone and estradiol levels in serum bychemiluminescent enzyme-linked immunosorbent assay;②AROM,ER-β, p-CREB and BDNF levels in hippocampus of offspring ratsby immune-histochemistry and Western blot.RESULTS:①Sex hormone levels in Serum: Compared with thecontrol, testosterone levels in serum significantly decreasedin male DBP-treated pups on PND21and PND60, testosterone level also decreased significantly in female DBP-treated pups on PND21;estradiol levels in serum significantly increased for both maleand female DBP-treated pups on PND21and PND60.②AROM, ER-β,p-CREB, BDNF levels: The AROM level increased apparently and thelevels of ER-β, p-CREB and BDNF decreased significantly inhippocampus in both male and female DBP-treated pups on PND21compared with the control, but no significant differences weredetected in DBP-treated rats on PND60compared with rats incontrol group. And there was no significant difference betweenmale and female pups in all groups.CONCLUSIONS:①Perinatal DBP exposure reduced testosteronelevels in serum in both male and female childhood offspring ratsand the reducing of testosterone level continued till adulthoodin male offspring rats. While perinatal DBP exposure increasedestradiol levels in serum in childhood and adulthood offspringrats in both genders.②DBP increased AROM level and inhibitedER-β、p-CREB and BDNF expression in hippocampus of childhoodoffspring rats. The reducing of serum testosterone and ER-βexpression in hippocampus led to the decreasing ofp-CREB-dependent BDNF level, which may be the key reason for theneurotoxic effect caused by DBP.③The changing levels of AROM、ERβ、p-CREB and BDNF in hippocampus after DBP expore were without gender differences, and returned to normal in adult rats,suggesting cessation of exposure in early developmental stagescan avoid persistent neurotoxic damage caused by DBP.