Effects Of Exposure To Benzo(α)pyrene During Pregnancy On Neurobehavior And BDNF Gene 5hmc In Offspring Rats

Objective:To study the effects of Benzo(α)pyrene(B(α)P)exposure during pregnancy on neurobehavior and hippocampal BDNF gene 5hmc in offspring rats.Methods:120 healthy SPF SD(Sprague Dawley)rats at 3 month-old(1:1 male-female ratio)were selected,After mating,60 pregnant SD rats were randomly divided into blank control group,olive oil group and10,20,40 mg/kg B(α)P exposure groups,12 rats in each group,the female rats were infected by intraperitoneal injection on the 17 th,18th and 19 th day of pregnancy once a day,the dosage volume was 0.2mL/kg,and the blank control group did not give any treatment.The body weights of the rats were recorded in PND1(Postnatal 1 day),PND4,PND7,PND14,PND45 and PND75;The time required for the erecting ears,fluff,teething and opening of the eyes in all rats in each litter were recorded separately;In PND4 and PND7,negative geotaxis test and cliff avoidance experiment were implemented;In the PND12 and PND14,the forelimb suspension experiment was performed separately;In the PND14,the aerial correction experiment was performed;In the PND45 and PND75,the Morris water maze experiment was performed to detect the corresponding spatial learning and memory ability.In PND1,PND4,PND7,PND14,PND45 and PND75,immunohistochemistry was used to detect the total level of 5hmc(5-hydroxymethylcytosine)and the protein level of Tet1(Ten-eleven translocation 1),Tet2 and Tet3 in the hippocampus of SD rats,detection of BDNF(Brain-derived trophic factor)protein level was achieved by immunohistochemistry and Western blot,and hippocampal Tet1,Tet2,Tet3 and BDNF mRNA expression levels were tested by RT-PCR.In PND1,PND4,PND7,PND14,PND45 and PND75,the hMeDIP-PCR method was used to detect the 5hmc level of the hippocampal BDNF coding region in the selected 6 time periods.Results:1.Postnatal physiological development test results: B(α)P exposure during pregnancy causes a decrease in body weight of PND1-PND45 in the offsprings and prolonged teeth appearing in rats.2.Early neurobehavioral test results after birth:(1)In PND7,negative geotaxis test showed that,compared with olive oil group(100%),the compliance rate of 40mg/kg group(80%)decreased significantly(P<0.05);(2)In PND7,cliff avoidance experiment showed the compliance rate of 20mg/kg group(63.33%)and 40mg/kg group(60.00%)decreased significantly(P<0.05)when compared with olive oil group(98.18%);(3)Forelimb suspension experiments showed that,compared with olive oil group and 10 mg/kg group,duration in the 20 mg/kg group and the 40 mg/kg group was significantly reduced in PND12.In PND14,compared with the olive oil,the forelimb suspension time of the three B(α)P treatment groups significantly reduced(P<0.05);(4)In PND14,aerial righting experiment showed the rate of compliance in 10mg/kg group(61.90%),20mg/kg group(30.95%)and the 40mg/kg group(28.57%)significantly decreased when compared with olive oil group(66.67%)(P<0.05).3.Morris water maze experimental results:(1)Repeated measurement analysis of escape latenc shown that,there was no interaction between the training time and the dose in PND45(F = 1.049,P = 0.410),In PND75,there was an interaction between training time and dose(F=195.351,P=0.005).With the increase of training days,the escape latency of PND45 and PND75 mice decreased gradually(F1=85.274,P=0.000;F2=110.667,P=0.000).One-way analysis of variance was used to analyze the difference in escape latency between groups on a single training day.Compared with the olive group,the 40mg/kg group shown significantly prolonged escape latency in the first,third and fourth days of training in PND45(P<0.05);in PND75,compared with the olive group,40mg/kg group shown significantly prolonged escape latency in the first and second days of training(P<0.05).(2)The results of space exploration experiments showed that the target quadrant residence time in three exposure groups in PND45 and 20 mg/kg group in PND75 decreased when compared with the olive oil group(P<0.05);Compared with the olive oil group,the number of crossing platforms in the 10 mg/kg and 40 mg/kg groups was significantly reduced in PND45 and PND75(P<0.05).4.Results of Western blot: Exposure of benzo(α)pyrene in pregnancy caused the decrease of BDNF protein level in hippocampus of PND7-PND75 offspring rats.Compared with olive oil group,the expression of BDNF protein in 20mg/kg and 40mg/kg groups decreased significantly(P<0.05).5.Results of immunohistochemistry:(1)In PND1-PND75,compared with the olive oil group,the global 5hmc in the 20mg/kg and 40mg/kg groups was significantly increased(P<0.05).(2)In PND14,Tet1 protein level was increased in the 20 mg/kg and 40 mg/kg groups when compared with the olive oil and 10 mg/kg groups(P<0.05);in PND45 and PND75,the level of Tet1 protein in the 40 mg/kg group was significantly increased when compared with the olive oil groups(P<0.05).(3)The level of Tet2 protein in the 20 mg/kg and 40 mg/kg groups was significantly elevated when compared with the olive oil group in all the selected time periods(P <0.05).(4)In PND4,Tet3 protein levels were decreased in the 20 mg/kg and 40 mg/kg groups when compared with the olive oil and 10 mg/kg groups.However,in PND7,up-regulating Tet3 protein was observed in the three B(α)P treatment groups.In PND14,the level of Tet3 protein was significantly increased only in the 40 mg/kg group when compared with the olive oil group(P < 0.05).(5)Exposure to B(α)P during pregnancy caused a decrease in the expression of BDNF protein in hippocampus of SD rats,and decreased most in the early postnatal growth(PND1-PND7).6.Results of RT-PCR:(1)Exposure to B(α)P during pregnancy caused a decrease in the expression of Tet1 mRNA in PND1,but increased the expression levels of Tet1 mRNA in PND45 and PND75,all difference was statistically significant(P <0.05).(2)Exposure to B(α)P during pregnancy caused an increase in the expression of Tet2 mRNA in hippocampus in PND1-PND75(P<0.05).(3)Exposure to B(α)P during pregnancy had no effect on the level of Tet3 mRNA in rat hippocampus.(4)Exposure to B(α)P during pregnancy caused rats with decreased expression of BDNF mRNA in PND1,PND4 and PND7 in a dose dependent manner(P<0.05).7.Results of hMeDIP-PCR: In PND1 and PND4,the levels of BDNF 5hmc in the 20mg/kg and 40mg/kg groups were increased when compared with the olive oil group in a dose dependent manner(P<0.05).In PND7,the level of 5hmc in the 20mg/kg and 40mg/kg groups decreased significantly in a dose dependent manner when compared with the olive oil group(P<0.05).Conclusion:1.Exposure to B(α)P during pregnancy caused growth retardation and neurobehavioral changes.2.Exposure of B(α)P during pregnancy caused up-regulation of global 5hmc and protein level of Tet family in hippocampus.3.The expression of BDNF protein and mRNA in the hippocampus decreased in B(α)P treatment groups.4.Exposure to B(α)P during pregnancy caused changes in the level of 5hmc in the BDNF coding region of hippocampus in early developmental rats.