| Nonylphenol(NP) is a typical representative of environmentalestrogen-like chemicals. NP is widely used as nonionic surfactants,emulsifiers, etc., in household, agricultural and industrial products, which isdifficult to biodegrade after use. NP has high liposolubility andbio-accumulation. NP can enter the environment through sewage dischargeinto surface water. NP can enter the body through various channels such asfood and food chain, air, water through oral cavity, skin, etc., to exertadverse effects on population health.The exposure level of NP was reported to arrange from3.6to31.4μg/kg/day for unprofessional exposed population groups in variouscountries through different exposure routes. In the last several decades,many studies unveiled the reproductive and developmental toxicity of NP.These studies demonstrated that NP toxic mechanism stems from theinterference of NP with endocrine system. Evidences indicated that theestrogenic activity of NP is10-4-10-5as strong as estradiol (E2) in vivo and invitro. NP has the potential to mimic the effects of endogenous hormone, which combines estrogen receptor in multiple organs and tissues to play anestrogen-like effect. NP has deleterious effects on multiple systems in fish,amphibians and mammals, including its effects on the function of thereproductive, endocrine, immune, digestive, urinary and other systems.However, toxic effects of NP on neuro development in F1rats have not beenelucidated.Central nervous system(CNS) is very sensitive to the alterations indiverse body hormone levels during CNS development. Children are moresensitive to NP neurotoxicity than adults due to their immaturity ofblood-brain barrier or other physiological factors. During the critical window,expose to NP would be implicated to the impairments of various organs'development and function. CNS experiences the most rapid developmentand is vulnerable to external toxicant's impairment during the period oforganogenesis in embryonic stage and perinatal period. The detriment ofCNS in fetuses or infants exposed to NP in perinatal period could be moreserious than that in other periods. But there is little comprehensive andprofound research on the impairment of CNS in F1rats from dams exposedto NP during gestation and its etiological mechanism. NP has been reportedto combine with other environmental endocrine disrupters to havesignificant toxic effects on organs' development and function in vivo and invitro, however these study results are diverse, and conclusion is stillunidentified. The study on expose to NP causing CNS' damage in children and its mechanism is a hot research topic in the field of public health.In this study, SD rats were exposed to NP during the embryodevelopmental sensitive period of CNS; Toxic effects of environmentalestrogen NP on nerve development in F1rats were examined, alterations inlearning and memory ability in F1rats were detected, and NP's toxicmolecular mechanism were also further explored from various angles suchas apoptosis, neurotrophic factor, glial fibrillary acidic protein and oxidativedamage. Our experimental findings were provided for hazard assessment inNP exposure during CNS development, providing science basis for furtherresearch and protection against the neurotoxic effects of alkylphenolsenvironment hormone,and enacting the related environmental pollutionstandards.Part1Adverse effects of NP on reproductive system in dams andgrowth and development in F1ratsObjective: The study on adverse effects of NP on reproductive systemin dams and growth and development in F1rats.Methods: The28dams were assigned to4groups. Dams weregavaged with NP at a dose level of50mg/kg/day,100mg/kg/day or200mg/kg/day daily from gestational day9to15and groundnut oil alone(vehicle control). Each group had6-7dams. Dams' weight,eating,generalconditions after NP contamination and reproductive health were observed. Physiological developmental landmarks were detected.Result: No obvious toxic symptoms were observed in dams in alltreatment groups. Exposure to100and200mg/kg/day NP caused anincrease in mean litter size, number of live pups per litter and number ofdead pups per litter (p<0.05). Anogenital distance (AGD), body length andtail length decreased in pups exposed to200mg/kg/day NP (p<0.05). Incontrast to the control group, oral exposure to NP treatment groups showedno statistically significant effect on weights of placenta and viscera (brain,liver, kidney, heart, etc.) in pups (p>0.05). Oral exposure to NP at doses200mg/kg/day caused a decrease in body weight at PND1,7,14,21,28compared with controls (p<0.01). The early physiological development (earopening, eye opening, fur development and eruption of incisors) in pupswere significantly retarded in200mg/kg/day NP treatment group comparedwith the negative control group. No difference in contrast to control was seenin the50mg/kg NP groups (p>0.05).Conclusion: exposed to200mg/kg/day NP might have toxic effects onreproductive capacity in dams. NP may cross the placental barrier andinfluence the growth and development of, rats, which exerts adverseeffects on birth landmarks, weight and physiological development. Part2. Toxic effects of NP on neurobehavioral development in F1ratsObjective: To explore the adverse effects of NP on neurobehavioraldevelopment in rats.Methods: Animals and treatments are same as Part1. At specific ageafter birth, the effects of NP on nerve early motor ability and behavioraldevelopment in offspring rats were detected;8-week old pups were selectedto perform Morris Water Maze Task and Step-Down Avoidance Test in orderto examine the effects of NP on learning and memory ability. Brain tissuewas embedded in paraffin and stained with haematoxylin and eosin toobserve pathological changes under an optical microscope; transmissionelectron microscopy was used to observe the ultrastructural changes ofhippocampus.Results: In contrast to the negative control, early neurobehavioraldevelopment (cliff-drop aversion reflex, surface righting reflex, forelimbgrip reflex, air righting, auditory startle and visual placing) in pups weresignificantly retarded in the200mg/kg/day nonylphenol-treated group (p<0.05). In Morris water maze task, pups significantly increased escapelatency to find the platform in the200mg/kg/day nonylphenol-treated group(p<0.05). Pups exposed to NP significantly spent more reaction time andpresented lower latency to first step-down as well as increased number oferror compared with the control pups in Step-down Avoidance Test (p <0.05).Pathological examination showed congestive and edema hippocampalneurons. The morphology of hippocampus of the200mg/kg/day NP-treated group was characterized by the presence of swollen mitochondrial with avacuole-like appearance and chromatin condensation occurred in the borderof the nuclei.Conclusion: Gestational exposure to NP might lead to toxic effects ofF1rats on early neurobehavioral development and spatial learning andmemory.Part3. Deleterious effects of NP on gene expression profile ofhippocampus during the weaning periodObjective: To investigate gene expression in hippocampus tissue inboth NP-treated and control groups and screen part of neurotoxicity-relatedsignificant genes based on microarray data for further verification.Methods: Establishment of the animal model for NP exposure (100mg/kg/day) during gestational and lactational periods. mRNA content in thehippocampus in both NP-treated and control groups were isolate at PND21to examine brain gene expression by DNA microarray. Axon GenePix4000B scanner was used to scan the microarray, and then fluorescent valueswere analyzed using the GenePix Pro software (4.1.1.31Axon).Results: A total of1254differentially expressed genes were screenedout.619up-regulated and635down-regulated genes were identified bycDNA microarray. Part of differentially expressed genes, whoseresponsibilities are already understood, are①neurotrophy-related gene: Nerve Growth Factor (NGF) gene was down-regulated, Galanin (Gal) genewas down-regulated, and growth associated protein43(GAP43) gene wasdown-regulated.②Apoptosis-related genes: Apoptotic protease activatingfactor-1(Apaf-1) gene was up-regulated, Cysteine-aspartic Acid Protease(Caspase7) gene was up-regulated, and defender against apoptotic death-1(DAD1) gene was down-regulated.③Signal transduction-related and ionchannel-related genes: glutamate receptor (AMPA2) gene wasdown-regulated, Calcium/calmodulin-dependent Protein Kinase II Delta(Camk2d) ion transport-related genes was down-regulated. Conclusion:gestational and lactational exposure to NP might involve influencingneurotrophy-related, signal transduction-related and ion channel-related aswell as apoptotic factor-related, etc., gene expression in hippocampusneurons toward having effects on function and development of CNS in pups.Part4. A study on neurodevelopmental toxic action of NP and itsmechanism in pupsObjective: To investigate the effects of NP in pups from dams exposedduring gestational and lactational periods on neuronal apoptosis, signaltransduction, glial cell and nerve growth correlation factors, thereby toexplore the possible mechanism of action of NP on learning and memoryability in pups.Methods:31pregnant dams were randomly assigned to4groups. The treatment groups received gavage with NP at dose levels of25mg/kg/day(low dose),50mg/kg/day (middle dose),100mg/kg/day (high dose) andgroundnut oil alone (vehicle control), respectively. The pregnant dams werehoused individually and fed intragastrically. NP exposure time was limitedfrom gestational day6to postnatal day21. Serum estradiol and testosteronelevels in pups at PND21were analyzed using radioimmunoassay.Cholineacetylase (ChAT) and Acetylcholinesterase (AchE) activities inhippocampus were detected by spectrophotometric colorimetry. Expressionof immediate early gene (c-fos and c-jun) in hippocampus was examined byimmunohistochemistry technique. The occurrences of hippocampal neuronsapoptosis at PND21and60were observed using terminaldeoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nickend labeling (TUNEL) assay. The glial fibrillary acidic protein (GFAP) inastrocytes of cerebral cortex and hippocampus and alterations in theexpression of neural growth-associated protein (GAP-43) were examinedusing immunohistochemistry. Alterations in GFAP and GAP-43mRNAexpressions were detected by Real Time PCR. These indicators associatedwith the impairment of learning and memory ability were statisticallyanalyzed therewith.Results:①The effects of NP on serum hormone levels in pups duringweaning period: radioimmunoassay showed serum testosterone level in pupsat PND21decreased with the increase in the NP concentration in a dose-dependent manner (r=-0.889,p <0.05). In contrast to the control group,serum testosterone level significantly decreased in the50and100mg/kgnonylphenol-treated groups. While serum estradiol level increased with anincrease in the NP concentration, significant differences between control andhigh dosage group (100mg/kg/day) were observed (p <0.05). No differencescompared with control were seen in the25and50mg/kg NP groups (p>0.05).②the effects of NPon neuronal apoptosis in pups during weaning period andmaturity: TUNEL indicated that apoptosis index is higher in pups exposed to100mg/kg/day NP compared with the control group (p<0.01) at PND21and60, there were no significant differences between50,100mg/kg/day NPtreatment and control groups. Comprehensive analysis of the relationshipbetween hippocampal apoptosis and learning and memory abilities showedthat the higher hippocampal apoptosis rate or apoptotic index, the moreserious earning and memory impairment or the worse learning and memoryabilities in pups.③the effects of NP on neural signal transduction in pupsfrom dams exposed during gestational period: A. The effects of NP onCholineacetylase (ChAT) and Acetylcholinesterase (AchE) activities inhippocampus: exposure to50and100mg/kg/day NP induced significantdecrease in ChAT activity in hippocampus of pups. Offspring rats from damstreated with NP at high doses (100mg/kg/day) during gestation produced asignificant decrease in AchE activity compared with the control group (p<0.05). B. Alterations in expression of c-fos and c-jun protein in hippocampus: the expression of c-fos and c-jun protein in normalhippocampus is weak. Decreases in the number of c-los and c-jun-positivecells were observed in25mg/kg NP-treated group, whereas exposure to NPat middle doses (50mg/kg/day) and high doses (100mg/kg/day) exhibitedan increase in the number of c-fos and c-jun-positive cells, especially at highdoses, the color of these cells was dark, and cells concentrated.④theeffects of NP on hippocampal glial cells in pups during weaning period andmaturity: immunohistochemistry unveiled that the protein expression ofGFAP, including the numbers of GFAP immunoreaction positive cells andintegral optical density, in astrocytes of cerebral cortex and hippocampus ofpups at PND21and60at high dose (100mg/kg/day) were significantlyhigher than that in control groups (p <0.05). Real Time PCR showed thatsignificant differences between control and high-dose experimental animalswere observed for GFAP mRNA expression in hippocampus of pups at PND21and60(p<0.05). Analysis of the association between GFAP expression inhippocampus and learning and memory abilities indicated that learning andmemory abilities of pups might decrease after enhanced expression of GFAP.⑤the effects of NP on neural growth-associated protein (GAP-43) in pupsduring weaning period and maturity: immunohistochemistry displayed thatthe protein expression of GAP-43(including the numbers of GAP-43immunoreaction positive cells and integral optical density) were increased athigh dose (100mg/kg/day) compared with the negative control group in the cerebral cortex and hippocampus of pups at PND21and60. Real Time PCRdemonstrated that significant differences between control and high-doseexperimental animals were observed for GAP-43mRNA expression inhippocampus of pups at PND21and60(p<0.05). Compared with thenegative control, GAP-43mRNA expression in hippocampus of pupssignificantly decreased in the100mg/kg nonylphenol-treated groups at PND21and60, differences were statistically significant (p<0.05). Analysis of therelationship between GAP-43mRNA expression in hippocampus andlearning and memory abilities implied that there was a positive correlationbetween GAP-43mRNA expression and learning and memory abilities ofpups.Conclusion: Exposure to NP during embryonic period and lactationmight cause an increase in estrogen levels and decrease in androgen levels inrats. The detecting results of neuronal apoptosis, signal transduction, glialcell and nerve growth correlation factors, etc., indicate that the potentialmechanism of NP-induced neurotoxicity may stem from: NP's weakestrogen-like activity vies for estrogen receptor to cause an imbalance ofendocrine system, to alter the endocrine milieu of brain development, tointerfere GAP-43protein expression in the developing nerve cells, to inhibitnerve cell differentiation and development in offspring rats, meanwhile, toaffect further cholinergic neurotransmitter to deliver Ach and expression ofc-los and c-jun in hippocampus through the adverse effects on morphology, structures and functions of astrocytes and alteration in GFAP proteinexpression, thereby promote apoptosis, and then lead to the retardation ofneural reflex time in nerve developmental stages, and learning and memoryimpediment in maturity of rats.
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