Critical Time Window Of Fenvalerate-induced Fetal Intrauterine Growth Restriction In Mice

ObjectivesAt present,there have been a lot of studies on the population that exposure to pyrethroids during pregnancy can lead to intrauterine growth restriction,and animal studies have confirmed.But there are few studies on the critical time window that exposure to pyrethroids during pregnancy can lead to intrauterine growth restriction.In this study,fenvalerate,a representative of type II pyrethroids,was selected to study the critical time window of fetal intrauterine growth restriction caused by fenvalerate exposure during pregnancy,and the role of oxidative stress was also discussed from the perspective of placenta.Through this study,the study on the effect of pyrethroids exposed during pregnancy on the growth and development of progeny was further deepened,which has great public health significance.MethodsTo establish a model of intrauterine growth restriction induced by fenvalerate exposure in different pregnancy.To investigate the effects of maternal FEN exposure at different gestational stages on fetal development,86 pregnant mice were divided into 6groups.According to our previous study the dose of 20mg/kg,about 1/10 LD50 of FEN,was chosen as the dose.Our preliminary data showed that no signs of maternal toxicity were observed in pregnant mice that were administered with FEN(20mg/kg)during pregnancy.Our previous study found that fetal IUGR were appeared in pregnant mice exposed to FEN(20mg/kg).Pregnant mice were divided into three groups randomly,each group allocated into 2 subgroups(control and FEN).Controls or FEN subgroups were administered corn oil or FEN(20 mg/kg)by gavage daily at early gestational stage(GD0-GD6),middle gestational stage(GD7-GD12)and late gestational stage(GD13-GD17),respectively.The volume for FEN or corn oil was 1 ml/100 g body weight.Food intake(4 mice per cage)and weight of pregnant mice were measured daily.All pregnant mice were sacrificed on GD18.Gravid uterine weights and the number of implantations,live fetuses,dead fetuses,and resorption sites were counted.Male and female live fetuses were weighed respectively.Anal reproductive distance was used to distinguish the sex of fetal mice.If this way could not clearly identify the sex of the fetuses,we identify fetal sex through the uterus and testicles.In this study,the threshold of IUGR was further determined through evaluating the distribution of male and female fetal weights in control group.Fetuses with weights<10th percentile were designated as IUGR.Placentas were weighted respectively.Because only FEN exposure at late gestational stage resulted in IUGR,therefore,we only examined the placental mechanism of mice exposed FEN at late gestational stage.Placental histology was used to analyze the effects of FEN exposure during different pregnancy on the blood sinus area of the placenta,and the content of glutataione(GSH)and malondialdehyde(MDA)was measured after placental tissue homogenation.The expression level of placental3-nitrotyrosine(3-NT)was detected by immunohistochemistry,the placental vascular density was labeled by CD34+,the expression levels of heme oxygenase 1(HO-1)and heme oxygenase 2(HO-2)were detected by Western blotting.The placental mRNA of inducible nitric oxide synthase(iNOS),nuclear factor erythroid-related factor2(Nrf2),Catalase(CAT),superoxide dismutase 3(SOD3),glutathione peroxidase 1(GPx1),and peroxiredoxin-3(PRDX3)was detected by RT-PCR.ResultsThere was no significant difference in the food consumption of pregnant mice exposed to FEN among the different groups.Moreover,maternal FEN exposure did not affect maternal weight.Correspondingly,regardless of the gestational stage the pregnant mice were exposed to FEN,there was no significant difference in maternal weight gain of the pregnant mice.No significant differences in the numbers of resorptions,dead fetuses and live fetuses per litter were observed among the different groups.Although maternal FEN exposure at the early and middle gestational stages did not affect fetal weight,maternal FEN exposure at the late gestational stage significantly reduced fetal weight.Correspondingly,further analysis showed that fetal weight was significantly reduced not only in male fetuses but also in female fetuses from mice exposed to FEN at the late gestational stage.Maternal FEN exposure at different gestational stages did not affect fetal crown-rump length.The rate of IUGR was significantly increased in the pregnant mice exposed to FEN at the late gestational stage.Further analysis found that the rate of IUGR was markedly increased not only in male fetuses but also in female fetuses of mice exposed to FEN at the late gestational stage.The effects of maternal FEN exposure during different gestational stages on placental weight and placental diameter were analyzed.There was no statistically significant difference in placental weight and placental diameter between the two subgroups of mice exposed to FEN at different gestational stages.There was a significant reduction in the blood sinusoid area of the placental labyrinthine region not only in male fetuses but also in female fetuses whose mothers were exposed to FEN at the late gestational stage.The effects of maternal FEN exposure at the late gestational stages on CD34~+microvessel density in the placental labyrinthine region were further analyzed.The CD34~+microvessel density in the placental labyrinthine region was significantly decreased in both male fetuses and female fetuses whose mothers were exposed to FEN at the late gestational stage.The effects of maternal FEN exposure at the late gestational stage on placental GSH and MDA were analyzed.Placental GSH was significantly decreased in both male fetuses and female fetuses whose mothers were exposed to FEN at the late gestational stage.The level of placental MDA,a marker of lipid peroxidation,was markedly increased in female fetuses whose mothers were exposed to FEN at the late gestational stage but not in male fetuses.The effect of maternal FEN exposure at the late gestational stage on placental 3-NT was determined.Representative photomicrographs of placental histological specimens from mice treated with FEN at the late gestational stage are shown.As expected,there was a significant increase in the placental 3-NT immunoreactivity of both male fetuses and female fetuses whose mothers were exposed to FEN at the late gestational stage.The expression of placental iNOS mRNA in male and female fetuses was markedly upregulated in mice exposed to FEN at the late gestational stage.There was no significant difference in the mRNA expression of placental Nrf2,SOD3 and GPx1 in the mice exposed to FEN at the late gestational stage.Interestingly,the mRNA expression levels of placental CAT and PRDX3 of female fetuses were upregulated in mice exposed to FEN at the late gestational stage.The level of placental HO-1 protein was significantly increased in both male and female fetuses whose mothers were exposed to FEN at the late gestational stage.However,no difference in the expression of placental HO-2,a constitutive protein of heme oxygenase,was observed between the two subgroups.ConclusionsThe late gestational stage was a critical time window of FEN-induced fetal IUGR.We provide evidence that placental oxidative stress is,at least partially,involved in the process of FEN-induced placental damage and fetal IUGR.