Effects And Mechanisms Of Pregnant Mice Ingested Various Food Additives And Pesticides On Offspring Development

Objective:The aim of this study was to investigate the effects of simultaneous exposure of pregnant mice to a variety of safe dose of food additives and pesticides contained in human daily diets on the inflammatory state and emotion-related behavioral development of offspring mice,and to further explore the possible impact mechanisms.Methods:1.The first part of the experiment: 24 SPF grade C57BL/6 mice were randomly divided into control group and experimental group on Gestational day 0(GD0).The treatment method of the experimental group was that pregnant mice freely ingested a variety of food additive and pesticide(Acceptable/Tolerable daily intake of food additive and pesticide chemical mixtures,AFACM)dissolved in drinking water,the treatment is referred to as AFACM exposure.AFACM exposure started from day 0 of gestation in mice until the end of parturition.At the end of AFACM exposure,the effects of maternal AFACM exposure on pregnancy outcomes,general health status of offspring,levels of interferon(IFN)-γ,interleukin(IL)-6,interleukin(IL)-12p70,tumour necrosis factor(TNF)-αand monocyte chemoattractant protein-1 in blood,prefrontal cortex and hippocampus were assessed on the day of delivery.On postnatal day 56(PND56),the effects of AFACM exposure on emotional behaviors were assessed.According to relevant references,the chemical composition and exposure dose of AFACM used in this study are as follows: carbaryl(0.0075 mg/kg/d),dimethoate(0.001 mg/kg/d),glyphosate(0.5 mg /kg/d),methomyl(0.0025 mg/kg/d),methyl parathion(0.003 mg/kg/d),triadimefon(0.03 mg/kg/d),aspartame(40 mg/kg/d),sodium benzoate(5mg/kg/d),calcium disodium ethylene diamine tetra-acetate(2.5 mg/kg/d),ethylparaben(10 mg/kg/d),butylparaben(0.5 mg/kg/d),bisphenol A(0.004 mg/kg/d)and acacia gum(34 mg/kg/d).2.The second part of the experiment: 24 SPF grade C57BL/6 mice were randomly divided into CON group and AFACM group on GD0.The AFACM exposure protocol is the same as the “Part 1 experiment”.The duration of AFACM exposure was from GD0 to GD12.5 or GD18.The effects of AFACM exposure on the levels of inflammatory factors in fetal mice serum and PFC on embryonic day 12.5(ED12.5)and ED18 were evaluated.Thirty SPF C57BL/6 mice were randomly divided into CON group,AFACM group,AFACM+anti-IFN-γ group,AFACM+Ig G1 group and anti-IFN-γ group on GD0.The pregnant mice in the CON group and the anti-IFN-γ group freely ingested ordinary drinking water,while the pregnant mice in the other experimental groups freely ingested drinking water dissolved in AFACM.The AFACM exposure protocol is the same as the “Part 1 experiment”.The time of injection of anti-IFN-γ antibody was from GD12.5 to delivery.On PND7 and PND14,the effects of AFACM exposure on serum and PFC inflammatory factor levels in offspring mice were assessed.On PND56,the effects of AFACM exposure on emotional behaviors were assessed.To assess whether anti-IFN-γ blocks/attenuates the effects of AFACM exposure on inflammatory status and emotional behavior in offspring mice.3.The third part of the experiment: 30 SPF grade C57BL/6 mice were randomly divided into CON group,AFACM group,AFACM+anti-IFN-γgroup,AFACM+Ig G1 group and anti-IFN-γ group on GD0.The pregnant mice in the CON group and the anti-IFN-γ group freely ingested ordinary drinking water,while the pregnant mice in the other experimental groups freely ingested drinking water dissolved in AFACM.The composition,dose and duration of AFACM exposure are the same as those in the “Part 1Experiment”.The method of injecting anti-IFN-γ antibody is the same as“Part II experiment”.On PND7,the level of ATP phospholipid transporting 8A2(ATPase phospholipid flippase 8A2,ATP8A2)in the PFC of each group was detected by Western Blot and immunofluorescence to assess whether AFACM exposure has an effect on ATP8A2 and the possible mediation of IFN-γ in the process.Results:1.Compared with the control group,the pregnancy outcomes in the AFACM group did not change significantly,and the liver function indicators,body weight in the third trimester of pregnancy and the weight of the uterus/ovary after childbirth did not change significantly in the AFACM group(all P > 0.05).Compared with the control group,the liver function indicators of the mice in the AFACM group did not change significantly(P > 0.05),but the levels of serum IFN-γ,the levels of IFN-γ,IL-6 and TNF-α in the PFC,and the levels of IFN-γ in the hippocampus of the offspring mice in the AFACM group were significantly increased(all P< 0.05).In addition,compared with the control group,the offspring mice in the AFACM group showed abnormal emotional behaviors in adulthood(all P < 0.05).2.Compared with the control group,AFACM exposure during pregnancy increased the levels of IFN-γ in fetal mice serum on ED18(P <0.01),and increased the levels of IFN-γ and IL-6 in PFC(both P < 0.001).AFACM exposure did not alter the levels of inflammatory factors in fetal mice on ED12.5.Compared with the control group,AFACM exposure during pregnancy increased the levels of IFN-γ in serum(P < 0.05),IFN-γ(P <0.01),IL-6(P < 0.01)and TNF-α in PFC of offspring on PND7(P <0.001),under the condition that anti-IFN-γ antibody was administered to neutralize IFN-γ,the effects of AFACM exposure during pregnancy on serum IFN-γ,PFC IFN-γ,PFC IL-6,and PFC TNF-α were completely blocked(AFACM+anti-IFN-γ group vs.AFACM group: all P < 0.05;AFACM+anti-IFN-γ group vs.CON group: all P > 0.05),administration of isotype control antibody Ig G1 had no significant effect on serum IFN-γ,PFC IFN-γ,PFC IL-6,and PFC TNF-α after exposure to AFACM during pregnancy(AFACM+Ig G1 group vs.AFACM group: all P > 0.05;AFACM+Ig G1 group vs.AFACM+anti-IFN-γ group: all P < 0.05).Compared with the control group,AFACM exposure during pregnancy did not alter the levels of inflammatory factors in serum and PFC of offspring on PND14(all P > 0.05).In addition,compared with the control group,the offspring of the AFACM group showed abnormal emotional behaviors in adulthood(all P < 0.05),under the condition that anti-IFN-γ antibody was administered to neutralize IFN-γ,the effects of AFACM exposure during pregnancy on abnormal emotional behavior were completely blocked(AFACM+anti-IFN-γ group vs.AFACM group: all P < 0.05;AFACM+anti-IFN-γ group vs.CON group: all P > 0.05),administration of isotype control antibody Ig G1 had no significant effect on the detection indexes of emotional behaviors after exposure to AFACM during pregnancy(AFACM+Ig G1 group vs.AFACM group: all P > 0.05;AFACM+Ig G1 group vs.AFACM+anti-IFN-γ group: all P < 0.05),which indicated that the blocking effect of anti-IFN-γ antibody on the changes of emotional behaviors after exposure to AFACM during pregnancy was credible,IFN-γ is the key mediator of abnormal emotional behaviors after AFACM exposure during pregnancy factor.In addition,we also observed whether the injection of anti-IFN-γ antibody to normal pregnant mice affected the performance of emotional behaviors,the results showed that anti-IFN-γ antibody administration under physiological conditions had no significant effect(anti-IFN-γ group vs.CON group: all P > 0.05).3.Western Blot result showed that,compared with the control group,AFACM exposure during pregnancy reduced the expression level of ATP8A2 in PFC of offspring mice on PND7(P < 0.001),under the condition that anti-IFN-γ antibody was administered to neutralize IFN-γThe effect of AFACM exposure during pregnancy on the expression level of ATP8A2 was completely blocked(AFACM+anti-IFN-γ group vs.AFACM group: P < 0.01;AFACM+anti-IFN-γ group vs.CON group: P >0.05),administration of isotype control antibody Ig G1 had no significant effect on the expression level of ATP8A2 after exposure to AFACM during pregnancy(AFACM+Ig G1 group vs.AFACM group: P > 0.05;AFACM+Ig G1 group vs.AFACM+anti-IFN-γ group: P < 0.05),which indicated that the blocking effect of anti-IFN-γ antibody on the change of ATP8A2 expression level after exposure to AFACM during pregnancy is credible,IFN-γ is a key mediator of the decreased expression level of ATP8A2 after exposure to AFACM during pregnancy.In addition,we also observed whether the injection of anti-IFN-γ to normal pregnant mice affected the expression of ATP8A2,the results showed that anti-IFN-γunder physiological conditions had no significant effect(anti-IFN-γ group vs.CON group: P > 0.05).The results of Immunofluorescence showed that AFACM exposure during pregnancy reduced the expression level of ATP8A2 in PFC of offspring mice on PND7 compared with the control group(P < 0.01).Under the condition that IFN-γ was neutralized by administration of antiIFN-γ antibody,the effect of AFACM exposure during pregnancy on the expression level of ATP8A2 was completely blocked(AFACM+anti-IFN-γ group vs.AFACM group: P < 0.001;AFACM+anti-IFN-γ group vs.CON group: P > 0.05).Conclusion:1.Exposure to AFACM during pregnancy can induce an inflammatory state in offspring and lead to abnormal emotional behaviors in offspring.2.IFN-γ is the key mediator of inflammatory state in brain and abnormal emotional behaviors of offspring induced by AFACM exposure during pregnancy.3.AFACM exposure during pregnancy down-regulated the expression of ATP8A2 in the prefrontal cortex of offspring mice,and IFN-γ is the key mediator of this effect.