| Objective:Drinking water disinfection is a critical measure to guarantee the safety of drinking water,but in the disinfection process,the disinfectant will produce a series of new compounds with natural organic or inorganic substances in the source water,namely drinking water disinfection byproducts(DBPs).The human body can be exposed to DBPs through ingestion,respiratory tract and skin contact,which could affect human health.Halobenzoquinones(HBQs)are a new class of DBPs discovered in recent years,among which 2,6-dichloro-1,4-benzoquinone(DCBQ)is widely detected in drinking water and swimming pool water,and has potential neurodevelopmental toxic effects.However,studies on the neurodevelopmental toxicity of DCBQ in vivo are lacking.The aim of this study was to examine the effect of DCBQ on hippocampal tissue in offspring rats,and to reveal the neurodevelopmental toxicity and its possible mechanism of DCBQ on offspring by detecting levels of neurotransmitters and brainderived neurotrophic factors,and intestinal flora and related metabolites,as well as the expression of related genes in hippocampal tissue,to provide a scientific basis for drinking water hygiene standards.Methods:SPF grade SD rats,10 males and 40 females,weighting 240-280 g,were used.Males and females were caged together 1:1,and the exposure to DCBQ was started from the discovery of the cathartic bolus for 6 weeks through intragastric administration.The pregnant rats were randomly divided into four groups: solvent control group(equal volume of medical sesame oil),DCBQ low-dose group(0.5mg/kg),DCBQ medium-dose group(5 mg/kg),and DCBQ high-dose group(50mg/kg),with 8 females in each group.The feeding and drinking status of rats were observed daily,and their body weight was recorded every 3 days;after DCBQ exposure was finished,the mother rats were executed,and the serum and organs were collected.Surface right reflex test and cliff avoidance test were performed PND6.After the lactation period,the males and females offsprings were kept separately.The8 week-old offspring rats were used to perform the open field test,and after finishing the open field test,the offspring rats were executed and the serum was collected;the feces and intestinal contents of the offspring were collected in a sterile environment;the neurotransmitters 5-HT,GABA,Glu,DA and brain-derived neurotrophic factor(BDNF)in serum and hippocampus were detected by ELISA.The m RNA expression of 5-HT4 R,Glu R1,NMDAR1,D1 R and BDNF in hippocampal tissues was detected by real-time quantitative fluorescence PCR;the composition of the gut microbiota was detected by 16 S r RNA high-throughput method;and the changes of relevant metabolites were detected by Liquid chromatography-mass spectrometry.Results:1.Effect of DCBQ on the general condition of mother rats: compared with the control group,the body weight of female rats in the DCBQ high-dose group on Day 0,3,6,9,and 12 of lactation was significantly lower(P<0.05);the liver organ index was significantly lower in the DCBQ high-dose group(P<0.05),and the kidney organ index was significantly lower in the DCBQ medium and high dose groups(P<0.001).2.Effect of DCBQ on the general condition of offspring rats: compared with the control group,the offspring rats in the high-dose group of DCBQ had significantly higher kidney organ index(P<0.05)and lower organ index of uterus and ovaries(P<0.05);the offspring rats in the low-dose group of DCBQ had significantly lower liver organ index(P<0.001);the brain organ index were significantly reduced in the medium and high dose groups of DCBQ(P<0.001).3.Effects of DCBQ on neurobehaviors of offspring rats: compared to the control group,the time required for plane flip-flopping was significantly increased in the offspring of the medium and high dose groups of DCBQ(P<0.0001);the time required for the cliff avoidance reflex was significantly increased in the DCBQexposed offspring rats(P<0.05);in the open field test,compared to the control group,the offspring of the DCBQ-exposed group had significantly lower total distance and mean velocity(P<0.05);the proportion of time in the central region was significantly lower and in the peripheral region was significantly higher in the DCBQ high-dose group(P<0.05).4.Effects of DCBQ on neurotransmitters and BDNF in offspring rats:(1)Compared with the control group,the levels of 5-HT,DA and BDNF in the serum of offspring rats in the medium dose and high dose groups of DCBQ were significantly reduced(P<0.01);the levels of Glu in the serum of the offspring rats in the medium dose group of DCBQ were significantly reduced(P<0.05).(2)Compared with the control group,the levels of 5-HT,DA and BDNF in the hippocampus of offspring rat in the medium and high dose groups of DCBQ were significantly reduced(P<0.05).5.Effects of DCBQ on m RNA expression of neurotransmitter receptors and BDNF in offspring rats: m RNA expression levels of 5-HT4 R,Glu R1,NMDAR1,D1 R and BDNF were significantly decreased in the hippocampus of offspring of DCBQ medium and high dose groups compared with the control group(P<0.05).6.Effect of DCBQ on intestinal flora of offspring rats: compared with the control group,there were significant differences in the composition of intestinal flora of DCBQ group(P<0.05).The differential bacteria in male rats mainly included the order Pasteurellales,Pasteurellaceae,Clostridium＿IV and Anaerofustis;the differential bacteria in female rats mainly included Butyrivibrio.7.Effects of DCBQ on intestinal metabolites in offspring rats: compared with the control group,123 differential metabolites appeared in the DCBQ group(P<0.05),with effects on several metabolic pathways including lysine metabolism,m TOR signaling pathway,PPAR signaling pathway,and vitamin digestion and absorption..Conclusions:1.Exposure to DCBQ during pregnancy and lactation has caused alterations in maternal liver and kidney organ index and alterations of organ index in offspring rats.2.Exposure to DCBQ during pregnancy and lactation has caused prolonged neuroreflex behavior in early time of offspring and increased anxiety-like behavior and reduced locomotor activity in offspring rats.3.Exposure to DCBQ during pregnancy and lactation has caused a decrease in the levels of neurotransmitters and BDNF content in the serum and hippocampus of the offspring rats.4.Exposure to DCBQ during pregnancy and lactation has caused a decrease in m RNA expression of neurotransmitter receptor in the hippocampus of offspring rats.5.Exposure to DCBQ during pregnancy and lactation has induced alterations in intestinal flora and intestinal metabolites in offspring rats. |
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