DNA Hydroxymethylation Of Synaptogenesis Genes Mediates Gestational 1-nitropyrene Exposure Induces Anxiety-like Behaviors In Adult Male Offspring

Objective Pollution by nitrated polycyclic aromatic hydrocarbons(NPAHs)is a global environmental problem,which is particularly serious in China.NPAHs contain a variety of toxic and harmful substances,and the study of the toxic mechanism of its key components is the key to clarify the toxic mechanism of NPAHs.1-nitropyrene(1-NP)is one of the typical representative substances of the key components of NPAHs.A previous study by our group found that 1-NP was developmentally toxic.This study aimed to investigate the effects of gestational 1-NP exposure on anxiety-like behavior in offspring and gestational 1-NP exposure causes anxiety-like behavior partially by altering hippocampal epigenetic reprogramming of synaptic plasticity in adult offspring.This study elucidates the neurodevelopmental toxicity of key components of NPAHs such as 1-NP from the perspective of interfering with DNA hydroxymethylation modification,and provides a toxicological basis for the control of NPAHs.MethodsICR mice were used for this study.Mice were subjected in a 12 h alternating light and dark environment at 25℃and 50% humidity.Animal experiments consisted of four parts.In experiment 1,to evaluate the effect of 1-NP exposure during pregnancy on anxiety-like behavior in male adult pups,twenty-nine pregnant mice were used and separated into three groups.Different doses of 1-NP(0,10,100 μg/kg)were given to pregnant mice by gavage daily from GD0 to GD17.All dams were delivered spontaneously.At postnatal week(PNW)12,Elevated-Plus Maze(EPM)and Open-Field Test(OFT)were used to estimate anxiety-like behaviors in male adult offspring.After behavioral tests,sixteen mice were euthanized and hippocampal tissue was collected for Nissl staining,Golgi-Cox staining,immunofluorescence and Western blotting.In experiment 2,to explore the effects of gestational 1-NP exposure on the development of fetal hippocampus,thirty-three pregnant mice were daily exposed to1-NP(0,10,100 μg/kg)by gavage.Eighteen pregnant mice were administrated with1-NP from GD0 to GD17 and euthanized on GD18.Hippocampus was collected from male fetuses at end of pregnancy(GD18)for Western blotting.Fifteen pregnant mice were administrated with 1-NP from GD0 to GD13 and euthanized on GD14.Because the hippocampus could not be dissected out on GD14,we used forebrain instead of hippocampus to detect RT-PCR,TET enzyme activity,α-KG and 5hm C in Cp G fragment.In experiment 3,to investigate the effect of α-KG supplement on1-NP-induced impairment of fetal brain development,twenty pregnant mice were used and separated into four groups: control(Ctrl),α-KG,1-NP,α-KG+1-NP.The intragastric dose of 1-NP was 100 μg/kg.And the intragastric dose of α-KG was 2 g/kg.Exposure duration of all pregnant mice was from GD0 to GD13.Forebrains were collected from fetuses on GD14 for detection of RT-PCR and Western blotting.In Experiment 4,to investigate the protective effect of α-KG supplement on1-NP-induced anxiety-like behavior,30 pregnant mice were separated into four groups:control(Ctrl),α-KG,1-NP,and α-KG+1-NP.The exposure protocol of pregnant mice was same as Experiment 3.Exposure duration of all pregnant mice in Experiment 4was from GD0 to GD17.All dams were delivered spontaneously.At PNW12,anxiety-like behavior was evaluated in male adult offspring.After behavioral tests,hippocampal tissue was collected for immunofluorescence,Golgi-Cox staining and Western blotting.ResultsAnxiety-like behaviors,as determined by Elevated Plus-Maze and Open-Field Test,were showed in male adult pups whose mothers were exposed to 1-NP.Gestational1-NP exposure reduced dendritic complexity in ventral hippocampus of male adult offspring.Additional experiments showed that gephyrin,an inhibitory synaptic marker,was reduced in fetal forebrain and ventral hippocampus in male adult offspring.Nrg1 and Erb B4,two gephyrin-related genes,were reduced in 1-NP-exposed fetuses.Accordingly,5hm C contents in two Cp G sites(32008909 and 32009239)of Nrg1 gene and three Cp G sites(69107743,69107866 and 69107899)of Erb B4 gene were decreased in 1-NP-exposed fetuses.Mechanistically,ten-eleven translocation(TET)activity and alpha-ketoglutarate(α-KG)content,a cofactor for TET dioxygenases,were decreased in 1-NP-exposed fetal forebrain.Supplementation with α-KG alleviated 1-NP-induced downregulation of gephyrin-related genes,prevented hippocampal synaptic damage,and improved anxiety-like behavior in adult pups.These results indicate that early-life exposure to 1-NP causes anxiety-like behavior in adulthood partially by altering hippocampal epigenetic reprogramming of synaptic plasticity.ConclusionIn summary,the current study investigated the mechanism by which gestational 1-NP induced anxiety-like behavior in male adult offspring.Our results showed that gestational 1-NP exposure reduced dendritic complexity in the ventral hippocampus of adult pups.Moreover,gestational 1-NP exposure altered epigenetic reprogramming of hippocampal synaptic-related genes in fetuses and adult offspring.Pretreatment withα-KG alleviated 1-NP-induced downregulation of gephyrin-related genes,hippocampal synaptic damage,and anxiety-like behavior.These results provide novel evidences that early-life exposure to environmental stress leads to affective disorder in later life by altering epigenetic reprogramming in developing brain.