Study On Mechanism Of Neurodevelopmental Toxicity Induced By Benzo[a]pyrene In Second Trimester Of Rats

Objective:People can be exposed to a large amount of B[a]P in daily life,it’s characteristics of lipophilic and hydrophobic,which makes it easy to combine with the lipid-rich nerve tissues and produce neurotoxic effects.As a key node in the development of the fetal nervous system,the second trimester of pregnancy is susceptible to interference from foreign chemicals.In order to study the short and long term neurodevelopmental toxicity of SD rats caused by B[a]P exposure in the second trimester,this study selected three Stage indicators:embryonic intrauterine neural tube development,postnatal neural and motor reflex functions,and adult learning and memory functions,to explore the development neurotoxicity and molecular mechanism of B[a]P exposure to offspring.Methods:1.Sixty healthy 10 week old SD rats,male and female（1:1）were kept in cages.The next day,it was found that Vaginal plug was recorded as gestational day0（GD0）.The number of 24 pregnant rats were randomly divided into 4 groups,with 6rats in each group:control group,B[a]P low dose group,B[a]P medium dose group and B[a]P high dose group.GD 8 was administered 2.5,25 and 250 mg/kg B[a]P solution by gavage once a day for 7 consecutive days.The control group was given the same volume of vegetable oil,and the health status of the animals was observed and recorded daily.GD15 pregnant rats were anesthetized and laparotomized to take out the uterus and embryo.The total number of embryos,live fetus,dead fetus,absorbed fetus,growth retardation,neural tube malformation and other malformations were recorded.The expression ofβ-catenin in brain tissue of GD15 embryo was detected by immunohistochemistry.The expression of GSK-3βandβ-catenin in brain tissue of GD15 embryo was detected by Western blot.2.120 healthy 10 week old SD rats,male and female（1:1）,were kept in cages.The next day,it was found that Vaginal plug was recorded as gestational day 0（GD0）.54pregnant rats were randomly divided into 9 groups with 6 rats in each group:blank control group,solvent control group,B[a]P group,Li Cl low、meddle、hight groups and Li Cl low、meddle、hight intervention groups.GD8 was administered by gavage once a day for 7 days.The Li Cl intervention group was given 10,20 and 40 mg/kg Li Cl solution by gavage,and 25 mg/kg B[a]P solution was given after two hours.The Li Cl group was given 10,20 and 40 mg/kg Li Cl solution by gavage,and the control group was given normal saline or vegetable oil by gavage.The day of natural childbirth was PND0,and the number of each litter was adjusted to 10（5 females and 5 males）in the early postnatal period.Before weaning（PND21）,the developmental nodes（birth weight,ear opening,tooth sprouting,hair growing and eye opening）of offspring were recorded,and the neural and motor reflex tests（SRR,FGS and SNT）were performed in PND2-PND10.Continue to feed until PND56,male began behavioral test（NOR,MWM）.The expression of GSK-3βandβ-catenin was detected by Western blot.SPSS20.0 and graphpad 7 were used to analyze the data.Measurement data are expressed as mean±standard error.After normality and homogeneity of variance tests are met,two independent sample t-tests are used for comparison between the two groups,and one-way analysis of variance is used for comparison between multiple groups（one way ANOVA）,post-test using Tukey’s multiple comparison;two-factor design data and repeated measures data meet the normality,homogeneity of variance and"spherical symmetry"test after the use of two-way ANOVA and repeated measures analysis of variance（Repeated measurement ANOVA）;counting data is expressed as rate,and the chi-square test of R×C table is used for comparison between multiple groups.The two-sided test levelа=0.05,P<0.05 means the difference is statistically significant.Results:1.The fetal death rate（12.04%）,absorbed fetal rate（14.45%）,growth retardation rate（16.86%）and neural tube malformation rate（13.25%）of B[a]P high-dose group were significantly higher than those of control group（P<0.05）.The results of immunohistochemistry showed that the gray value ofβ-catenin-positive cells in embryonic brain of the B[a]P high-dose group was higher than that of the control group,and the difference was statistically significant（P<0.001）.Western blot results showed that the expression of GSK-3βwas increased and the expression ofβ-catenin was decreased in B[a]P medium and high dose groups（P<0.05）.2.The results of the PND0-PND21 progeny development node showed that compared with the control group,the birth weight of the offspring of the B[a]P group was reduced,the time of ear opening and the time of teeth eruption were delayed.Li Cl high-dose intervention can significantly improve the delays of the above-mentioned developmental nodes of the offspring of the B[a]P group,the were all statistically different（birth weight:control group:6.766±0.235,B[a]P group:5.798±0.135,Li Cl high-dose intervention group:7.013±0.170;ear opening time:control group:3.667±0.142,B[a]P group:4.500±0.151,Li Cl high-dose intervention group:3.333±0.142;tooth eruption time:control group:10.67±0.333,B[a]P group 11.75±0.179,Li Cl high-dose intervention group:10.92±0.193）.There were no statistically significant differences in the prolonged hair growth and the opening time of both eyes among the groups（P>0.05）.The neural and motor reflex test results of the PND3-PND10 offspring showed that the offspring of the B[a]P group were delayed in flat-righting and cliff avoidance time compared with the control group.The high-dose Li Cl intervention can significantly increase the offspring caused by B[a]P.The passing rate of the above-mentioned nerve and motor reflex tests has a statistically significant difference（eligible time for plane correction:control group:5.333±0.284,B[a]P group 6.833±0.112,Li Cl high-dose intervention group:5.33±0.310;cliff avoidance qualified time:control group:6.667±0.284,B[a]P group:8.5±0.151,Li Cl high-dose intervention group:7.33±0.142）.There was no statistically significant difference in the qualified time of the offspring’s negative drive test among the groups（P>0.05）.The canonical correlation analysis results of offspring developmental nodes and neural and motor reflex function showed that two pairs of canonical correlation coefficients were statistically different（CR₁=0.594,P<0.001;CR₂=0.201,P<0.05）.The first pair of typical variables is slightly negatively correlated with birth weight（typical load factor-0.444）and moderately positively correlated with the pass time of plane straightening（typical load factor 0.659）.The second pair of typical variables is moderately positively correlated with ear opening time（typical load factor 0.575）and slightly positively correlated with the cliff avoidance pass time（typical load factor0.308）.3.At PND56,the results of the novelty recognition experiment showed that the number of novelty recognitions in the male offspring of the B[a]P group after adulthood（control group:9.2±1.304,B[a]P group:5.857±0.595）,novelty recognition ratio（control group:40.41±6.555%,B[a]P group:25.12±1.304%）was lower than that of the control group（P<0.05,P<0.01）.The Li Cl high-dose intervention group can significantly improve the above indicators（9.0±0.577,P<0.05;38.56±3.898%,P<0.05）.The novelty recognition ratio in female offspring of B[a]P group（control group:35.14±5.952%,B[a]P group:27.42%±4.60%）was lower than that in the control group（P<0.05）.The above indexes were significantly improved in the high-dose Li Cl intervention group（38.57 4±2.795%,P<0.01）.Morris water maze test results found that the latency of the offspring rats of the B[a]P group to find a platform was longer than that of the control group（solvent control group:8.488±1.115 s,B[a]P group:18.3±3.332 s）on Day 5.After evacuating the platform,the number of male offspring crossing the platform on Day6 was less than that of the control group（solvent control group:4.8±0.374,B[a]P group:2.5±0.525,P<0.05）,and the Li Cl high-dose intervention group could be significantly improved The above indicators（10.26±1.704,P<0.05;4.875±0.515,P<0.05）.There was no statistically significant difference of Morris water maze in female offspring.Western Blot results showed that GSK-3βexpression increased andβ-catenin expression decreased in the hippocampal tissues of male rats in the B[a]P group,the difference was statistically significant（P<0.05）.expression levels of GSK-3βandβ-catenin in the Li Cl medium and high-dose intervention group were better than those in the B[a]P group（P<0.05）.There was no significant difference in the expression of GSK-3βandβ-catenin in female offspring.Conclusion:1.B[a]P exposure in the second trimester of pregnancy can cause the occurrence of rat embryonic brain/meningocele.Both medium and high-dose exposure of B[a]P changed the expression levels of key Wnt pathway proteins in embryonic brain tissue.2.B[a]P exposure in the second trimester can cause the offspring’s birth weight to decrease,the time of ear opening and eruption,and the delay of the offspring’s plane straightening and cliff avoidance.Li Cl intervention can significantly improve B[a]P exposure causes delays in the construction of developmental nodes,nerves,and motor reflex functions.3.B[a]P exposure in the second trimester can cause impairment of work and spatial learning and memory in adult offspring.Li Cl intervention can significantly improve the impairment of learning and memory caused by B[a]P exposure.The underlying mechanism may be related to the down-regulation of Wnt/β-catenin signaling pathway in the hippocampus of offspring by B[a]P exposure in the second trimester.