The Effects Of One Of Environmental Estrogens 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin On Cardiac Development Of Rat Embryos And Suckling Rats

Background and objective:With the development of modem industry and agriculture,there are various chemical compounds discharged into environment,some of which with estrogen-like activity can mimic the physiological and biochemical actions of endogenous estrogen or antagonize effects of androgen are defined as environmental estrogens:The fact that these environmental estrogens that can be very dangerous to the health of both mankind and animals,resulting in reproduction obstacles,developmental anomalies,certain kinds of cancers,and the related ecological effects has attracted extensive attention.The most notorious of the environmental contaminants is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),the byproducts of incompletely burnt wastes,abusage of pesticides containing chlorine,and industrial wastes,that can enter the bodies of human beings and animals through food chain or direct contact.Due to the stable property and unavailable biodegradation,TCDD,with a half life period reaching as long as 7-11 years in vivo,can hardly or can not be discharged,can be distributed in all organs,and can be even detected in the fat tissues,milk,and sera of both human beings and animals as well.TCDD can accumulate in placental tissues by easily breaking through the placental barrier,leading to toxic effect on the placenta and on the neonatals through milk while breastfeeding.It has been reported that embryos and neonatals are more sensitive to TCDD toxicity than adults.Cardiovascular malformation is the congenital defect with the highest incidence rate in mankind,reaching 1%in live-born infants and even as high as 10%in dead-born ones.The effects of TCDD on the circulating system of embryos and neonatals and the related mechanisms are currently poorly known.However,inadequate animal experiments have proved that dioxins can have toxic effect on the circulating systems of multiple living species including fishes,rats,chick embryos,and hamsters such as subcutaneous edema, peficardial edema,and hemorrhage.In an accidental exposure event,the subcutaneous edema was found in a human fetal face.These studies mentioned above indicate that the cardiovascular system may be one of the target organs on which TCDD acts and that identical toxic mechanisms of TCDD may be found in the cardiovascular systems of different vertebrates.Therefore,in our study,we applied in vivo toxicant exposure model to study the effects of TCDD on the growth and development and cardiovascular morphology and functional development of rat embryos and suckling rats for the purpose of providing theoretical and experimental proofs for the prevention of embryonic anomalies as well as laying the foundation for the further studies of mechanisms in TCDD-induced anomalies.Methods:1.Establishment of in vivo toxicant exposure model in rat embryos In vivo toxicant exposure model was established by intraperitoneal administration of TCDD in rat embryos at post-gestational 8 d.2.Observation of the effects of different concentrations of TCDD on the growth and development of rat embryos.â‘ Rat embryos were divided into 4 toxicant exposure groups, i.e.0.05,0.5,5,10μg/kg.DMSO group served as the control group because TCDD had the property of liposolubility.â‘¡Indexes including the body weight,body length,and tail length of the rat embryos(post-gestational 19 d) and the placental weight were observed.3.Observation of the effects of different concentrations of TCDD on the cardiac morphology development of the rat embryo.Indexes including heart weight,ratio of heart weight to body weight of the rat embryo(post-gestational 19 d) were observed.4.Observation of effects of TCDD on cardiac functions.â‘ The effects of different doses of TCDD on the concentrations of intracellular free calcium ions in the cultured myocardial cells of suckling rats were detected by laser scanning confocal microscopy.â‘¡Changes in mRNA expression of atrial natriuretic factor or poly peptide(ANF/ANP) in the myocardial tissues of rat embryos and suckling rats were detected by semiquantitative RT-PCR analysis.Results:1.Statistical analysis showed that there was no significant difference in embryonic development of rat fetuses at the identical developmental stage between the empty control group and the toxicant exposure groups(no occurrence of fetal death,P＞0.05).2.TCDD had remarkable effects on the rat embryonic development at post-gestational 19 d in a dose-dependent manner.The body weight,body length,and tail length of the rat embryos showed a tendency of decline with the increasing doses of TCDD.Compared with those in the toxicant exposure groups,no significant decrease in all indexes was found when TCDD at the dose of 0.05μg/kg was administered(no occurrence of fetal death,P＞0.05). After administration of TCDD at the dose of 0.5μg/kg,further decline was found in all indexes,among which the tail length of the rat embryo was significantly different from that in the toxicant exposure groups(P≤0.001,occurrence of fetal death:4/22,death fetus rate: 18.2%).After administration of TCDD at the dose of 5μg/kg,all indexes continued to decline more significantly as compared with those in the toxicant exposure groups(P＜0.05) and amount of fetal death increased(12/22,death fetus rate:54.5%).However,administration of TCDD at the dose of 10μg/kg revealed the most significant difference in all indexes as compared with those in the toxicant exposure groups(P≤0.001) and occurrence of fetal death in all groups(24/24,fetal death rate:100%),but there was no significant difference in placental weight in all groups (P＞0.05).3.TCDD had significant effects on the cardiovascular system development of the rat embryos at post-gestational 19 d in a dose-dependent manner.With the increasing doses of TCDD, the ratio of embryonic heart weight to body weight decreased in a dose-dependent manner.There was significant difference in the ratio in rat embryos between the control and the toxicant exposure groups when TCDD was administered at the doses of 0.5,5,and 10μg/kg(P＜0.05). Decreased cross section area and heart volume were also observed macroscopically.In addition,different degrees of hemorrhage and edema were found in the toxicant exposure groups.4.TCDD resulted in the occurrence of "calcium overload" in the myocardial cells of suckling rats,i.e.increase in concentration of intracellular free calcium ions.TCDD increased[Ca²⁺]_i concentration in myocardial cells of suckling rats in a dose-dependent manner and there was significant difference as compared with that in the empty control group(P≤0.001),but there was no significant difference in[Ca²⁺]_i concentration in the toxicant exposure group at the dose of 0.05μg/kg as compared with the empty control group(P＞0.05).5.TCDD could up-regulate ANF mRNA expression in the myocardial tissues of rat embryos and suckling rats.Analysis of the ratios of optical density of the corresponding bands with different ANF mRNA expressions at different time points in the control and the toxicant exposure groups showed certain degrees of up-regulation of ANF mRNA in the three groups in a dose-dependent manner,but no significant difference in ANF mRNA expression was found in the toxicant exposure group at the dose of 0.05μg/kg as compared with that in the control group.There was no statistical significance in ANF mRNA expression in the myocardial tissues of the rat embryos at post-gestational 19 d as compared with that of 5 d neonatals(P＞0.05).Conclusions:1.Toxicant exposure model in vivo of rat embryos at the target-window stage of cardiovascular system development had been successfully established.2.TCDD can inhibit the growth and development of rat embryos at the target-window stage of cardiovascular system development.3.Rat heart at the developmental stage is one of the target organs of TCDD action.(1) TCDD can induce developmental anomaly of cardiovascular structures of rat embryos and suckling rats in a dose-dependent manner.(2) TCDD can cause injuries of cardiac functions of rat embryos and suckling rats:â‘ TCDD can increase[Ca²⁺]_i content in a dose-dependent manner,resulting in calcium overload;â‘¡TCDD can up-regulate ANF mRNA expression in myocardial cells.