Effect Of Di-(2-ethylhexyl) Phthalate On Neurobehavioral Development Of Rats And Endocrinologic Mechanism Exploration

ObjectivesThis study is designed to evaluate the developmental neurotoxicity of di-2-ethylhexyl phthalate (DEHP) on rats when exposed to DEHP during different growth and development stages, observe the toxic effect change over time of DEHP and the self-repair ability of rats to to adaptation of DEHP toxicity, and explore the endocrine mechanism of developmental neurotoxicity.Methods1. Animals and DEHP administrationHealthy pregnant female Sprague-Dawley (SD) rats were randomly divided into six groups based on pregnancy time and body weight, one group was treated with olive oil by gavage as control, and five groups were orally administered different concentrations of DEHP (2.5,5,50,500,1000 mg/kg BW).The exposure time for different stages was respectively from GD6 to delivery(Embryonic exposure,Indirect exposure through pregnant rats);from PND1 to weaning(Lactation exposure, Indirect exposure through maternal rats);from PND22 to PND30 (Preadolescence exposure, Direct exposure through offspring rats);from GD6 to PND30(Whole developmental phase exposure, indirect exposure and Direct exposure through maternal rats and offspring rats).2. The evaluation of the developmental neurotoxicity of DEHPWhen rats were exposed to DEHP during different growth and development stages, effects of DEHP on the neurotoxicity of central nervous system of rats were observed,and related indexes were as follows:general conditions and reproductive effects; Early physical development indicators of offspring rat including body weight,survival rate, and ages at first appearance of pinna detachment, incisor eruption, eye opening and appearance of hair were observed; early neurological development indexes of offspring:negative geotaxis, righting reflex, cliff avoidance reflex, auditory startle habituation, forelimb hanging and air righting reflex.Neurobehavioral testsduring young adulthood(Open Field Test and Morris water maze test).The hippocampus and cortex morphology observation and the brain weights and brain coefficient.Synaptic plasticity related proteins and genes expression (Western blot, RT-PCR),content change of neuropeptide in hypothalamus (Western blot, RIA Radio Immunoassay).3. Possible endocrine mechanism of developmental neurotoxicity of DEHP. After rats were exposed to DEHP during different growth and development stages,serum and hippocampus tissue of rats were collected to detect the estrogen and thyroid hormones serum levels (ELISA, RIA), the mRNA expression of hormone receptors in hippocampus (TR-PCR).Results1 The effects of DEHP on general conditions of Fo and growth and development of offspring rats1.1 General observations of maternal rats.During embryonic period, infant period,and preadolescence period, there were no symptoms of intoxication were observed. For rats in 1000mg/kg bw DEHP group during whole developmental phase,, the pregnancy time was postponed/the number of baby rats at a birth and survival rates at PND0,PND4 and PND21 were reduced compared with control group (P<0.05)1.2 Growth and development situation of offspring ratsThere was no difference on body weights of offspring rats after exposed to DEHP during embryonic period, infant period,and preadolescence period.Lactation weight of offspring rats was decreased compared with control group when exposed to 50 and 1000 mg/kg DEHP(P<0.05). No difference of Indexes of ages at first appearance of pinna detachment, incisor eruption, eye opening and appearance of hair were observed compared with controls(P>0.05).2. Effects of DEHP on neurobehavioral development2.1 The effects of DEHP on early neurobehavioral developmentDuring embryonic period, there were statistically significant (p<0.05), dose-related delays in successful time of righting reflex, cliff avoidance reflexand air righting reflex in 50,500, 1000mg/kg bw groups (p<0.05), the forelimb hanging time of 50,500,1000 mg/kg bw groups were shorter than other groups(P<0.05). There were no statistically significant changes between groups in negative geotaxisand auditory startle habituationtests. For infant period, air righting reflex of 50,500,1000 mg/kg bw groups were delayed. During whole developmental phase, negative geotaxis and air righting reflex ages of offspring rats exposed to 5 to 1000mg/kg bw DEHP groups were delayed,50,500 and 1000 mg/kg bw DEHP groups rats, righting reflex ages delayed and forelimb hanging time were shorter than controls,500 and 1000 mg/kg bw DEHP groups cliff avoidance reflex delayed(P<0.05).2.2 Open Field TestFor animals at 50、500、1000mg/kg bw DEHPgroups during embryonic period, the time of the rats stayed at center area increased, and distance moved, changes in activity and rearing time decreased(P<0.01,P<0.05). During whole developmental phase, there were statistically significant increase in center time, decrease in distance moved (5 to 1000 mg/kg.bw DEHP groups), changes in activity (500,1000mg/kg bw DEHP groups) and rearing time(50,500,1000mg/kg bw DEHP groups). No abnormal changes were found during infant period and preadolescence period.2.3 The Morris water maze testThe learning and memory ability of offspring rats was improved as the training time went on. Swimming speed in visible platform test for rat offspring was almost same, and it indicated that all animals had a healthy condition about swimming and had no deficits in sensorimotor processes.During embryonic period, rats of 50 mg/kg bw and above groups at the third to fifth day spent more time on looking for the platform compared to the control group(p<0.05). In the space probe trial of Morris Water Maze test, rats of 50,500 and 1000 mg/kg bw groups spent significantly less time in the target quadrant(TA) than those of control group(P<0.05),and time percent during platform quadrant was also relatively reduced. The frequency of crossing platformat 500 and 1000 mg/kg bwwas decreased(P<0.05).During whole developmental phase, at the third to fifth day, male rats of 5 mg/kg bw and above groups and female rats of50 mg/kg bw and above groups spent more time on looking for the platform compared to a control group(p<0.05). In the space probe trial of Morris Water Maze test, rats of 5,50,500 and 1000 mg/kg bw groups swim significantly shorter distance in the target quadrant (TA) and the frequency of crossing platform went down (P<0.05), time percent during platform quadrant was also relatively reduced at 50,500 and 1000 mg/kg bw groups.3. The hippocampus and cortex morphology observationAfter exposed to DEHP, no differences were found about the brain weight and brain coefficient. There were light microscopic morphological changes of brains observedinthe 1000mg/kgbw group offspring during embryonic period. Compared to control group, some changes were found including degeneration of pyramidal neurons in cerebral cortex with dark stained nuclei anddisappearance ofaustenite.The neurons in the hippocampal CA1, CA2, CA3and DG regions appeared in disordered arrangement and neuroglia cellinfiltration were found in hippocampal CA3 and DG regions. When exposed to DEHP during infant period, rats of 5 mg/kg bw group were find The neurons in the hippocampal CA1, CA2, CA3 and DG regions/the neuropil vacuolation, pyramidal neurons in cerebral cortex with dark stained nuclei. In 50mg/kg bw DEHP group, the neuropil vacuolation was found, pyramidal neurons in cerebral cortex with dark stained nuclei. There were no light microscopic morphological changes of brains observed in control and experimental groups offspring during preadolescence period. When exposed to DEHP during whole developmental phase, rats of 1000 mg/kg bw group were find The neurons in the hippocampal DG regions,the neuropil vacuolation, pyramidal neurons in cerebral cortex with dark stained nuclei.4. The effect of DEHP on neuropeptide content in hypothalamusThe content of neuropeptide VP in hypothalamus reduced compared with controls after exposed to DEHP during embryonic period. During infant period, the ACTH increased at 50 and 500 mg/kg bw DEHP groups and the NPY reduced (P<0.05). After whole developmental phase DEHP exposure, the content of neuropeptide VP (5,500 and 1000 mg/kg bw DEHP), ACTH (2,5,5,50 and 500 mg/kg bw DEHP),and NPY(500、1000mg/kg bw DEHP) reduced(P<0.01,P<0.05).5. Effects of DEHP on the expression of synaptic plasticity related proteins and genes in the HippocampusDuring embryonic period, infant period,and the whole developmental phase, the expression of PSD95 was restrained, the expression of PKA was suppressed, the CREB expression level decreased, theproportionof p-CAMKII decreased, and the expression of BDNF decreased. The decrease of proteins mentioned above affected synaptic plasticity and then influenced the learning and memory process, resulting changes in neurobehavior. 6. Effects of DEHP on the estrogen and thyroid hormones serum levels and the mRNA expression of hormone receptors in hippocampus.Thethyroid hormones serum levels were affected by DEHP exposureduring embryonic period and infant period. For rats in 500、1000mg/kg bw groups during the whole developmental phase,,thethyroid hormones (T3, T4) serum levels were decreased, and TSH level increased. Thethyroid hormones serum levels were not affected after preadolescence DEHP exposure. At the same time, the estrogen level was not changed after DEHP exposure at different growth and development period. The expression of ER-β was suppressedin 500、1000mg/kg bw groups,however, the expression of ER-a and THRwere not changed when exposed to DEHP during embryonic period and infant period. The DEHP exposure during the whole developmental period could suppress the expression of THR and ER-β, but had no effect on ER-a.Conclusion1. DEHP exposure during embryonic period, infant period,and the whole developmental phase can cause adverse effects on the early neurological development and the learning and memory ability.2. DEHP has no effect on the rats after exposed to DEHP during preadolescent period under the condition of this experiment. Rats are susceptible to DEHP when exposed during embryonic period, and the toxicity increases with the expose time.3. DEHP can affect thethyroid hormones serum levels and down-regulates the gene expression of THR and ER-β in hippocampus, which may be the most important endocrine mechanism of developmental neurotoxicity.Synaptic plasticity related proteins such as PSD95, PKA, CREB, p-CAMKIIandBDNF may also participate in the regulation of this process.