【作者】 李月；

【导师】 谭凤珠；

【作者基本信息】 河北医科大学 ， 劳动卫生与环境卫生学， 2009， 硕士

【摘要】 目的2 , 3 , 7 , 8 -四氯二苯-对-二恶英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)是毒性最强的化合物之一,国际癌症研究中心将其列为Ⅰ级致癌物。长期接触TCDD可在体内蓄积,对机体造成严重的毒害作用。目前对TCDD的急性毒性作用及妊娠期暴露对子代的影响已深入研究,但关于哺乳期暴露对子代影响的研究甚少。本实验以小鼠为模型,于其分娩后立即腹腔注射低剂量TCDD,通过乳汁使仔鼠染毒,观察哺乳期暴露于TCDD对仔鼠发育,心脏、肾脏和睾丸中细胞色素P450 1A1酶(cytochrome P450 1A1, CYP1A1)、Bcl-2(B-Cell lymphoma/ leukemia-2)和Bax(Bcl-associated x protein)表达的影响,为哺乳期暴露于低剂量TCDD对子代造成的毒性危害提供科学依据。方法1实验动物选取4月龄健康成年昆明小鼠,雌鼠24只,雄鼠8只,按3:1比例同笼受孕。分娩8-10只仔鼠的保留,调整使每窝仔鼠数为8只,雌雄对半,其余淘汰。2实验试剂TCDD购买浓度为10μg/m(lTCDD/甲苯),纯度为99%,注射前按TCDD:花生油=1:4的体积比配制,终浓度为2μg/ml。溶剂对照为1:4的甲苯和花生油混合液。3实验分组将母鼠和仔鼠作为整体(窝)完全随机分组,设2个实验组:40μg和20μg TCDD/kg体重组;2个溶剂对照组:40μg和20μg溶剂/kg体重组;1个空白对照组;共5组,每组3窝(简写为40μg TCDD组、20μg TCDD组、40μg溶剂对照组、20μg溶剂对照组和空白对照组)。雌鼠分娩后,立即称重并腹腔注射。母鼠哺乳仔鼠(即染毒)至出生后第21天,停止哺乳,此后仔鼠自由进食。出生后第35天处死。4仔鼠生长发育指标的测定:仔鼠体重,肾脏、睾丸重量,睾丸下降时间。5仔鼠心脏、肾脏和睾丸中CYP1A1的测定。6仔鼠心脏、肾脏和睾丸细胞凋亡的测定6.1用显微镜观察H.E染色的仔鼠心脏、肾脏和睾丸组织的形态变化。6.2用免疫组织化学法对仔鼠心脏、肾脏和睾丸中Bcl-2和Bax的测定。结果1 TCDD对仔鼠体重的影响出生时各组仔鼠体重无显著差异(P>0.05);出生后第7天,20μg TCDD组仔鼠平均体重低于溶剂对照组和空白对照组(P<0.05);7天后40μg和20μg TCDD组平均体重低于溶剂对照组和空白对照组(P<0.05),40μg TCDD组低于20μg TCDD组(P<0.05);停止染毒后(出生后第28天和35天),平均体重仍低于溶剂对照组和空白对照组(P<0.05)。2 TCDD对仔鼠肾脏重量的影响40μg和20μg TCDD组雌性仔鼠肾脏绝对重量显著低于溶剂对照组和空白对照组(P<0.05),但雄性仅40μg TCDD组显著低于溶剂对照组(P<0.05);雌雄40μg和20μg TCDD组肾脏相对重量与其溶剂对照组均无显著性差异(P>0.05),但雌性实验组显著性低于空白对照组(P<0.05)。40μg TCDD组与20μg TCDD组肾脏绝对重量和相对重量均无差异(P>0.05);雌雄间比较,雌性各指标均显著性低于雄性(P<0.05)。3 TCDD对仔鼠睾丸重量的影响40μg和20μg TCDD组睾丸绝对重量和相对重量低于其溶剂对照组和空白对照组,40μgTCDD组低于20μg TCDD组,但仅40μg TCDD组与其溶剂对照组有显著性差异(P<0.05)。4 TCDD对仔鼠睾丸下降时间的影响40μg和20μg TCDD组睾丸下降时间迟于溶剂对照组和空白对照组(P<0.05),40μg TCDD组明显迟于20μg TCDD组(P<0.05)。5 TCDD对仔鼠心脏、肾脏和睾丸CYP1A1表达的影响免疫组化的结果显示,CYP1A1可表达于心肌血管内皮细胞、肾脏和睾丸中。哺乳期低剂量暴露于TCDD,使仔鼠心肌细胞、肾脏和睾丸中CYP1A1表达量均明显高于其对照组(P<0.05),心肌细胞中无性别差异(P>0.05),但肾脏中雌性显著性高于雄性(P<0.05)。6 TCDD对仔鼠心脏、肾脏和睾丸组织细胞结构的影响以及对组织Bcl-2和Bax表达的影响哺乳期低剂量暴露于TCDD,使仔鼠心血管内皮细胞的完整性破坏、管壁增厚、肌纤维排列混乱;肾脏中肾小球数量增加、体积增大、炎性细胞浸润;睾丸细胞皱缩等。抑制凋亡基因Bcl-2可表达于心肌细胞中,40μg TCDD组与20μg TCDD组无差异(P>0.05);肾脏中Bcl-2主要表达于肾小球细胞中,40μg TCDD组显著性低于20μg TCDD组(P<0.05);睾丸中Bcl-2主要表达于睾丸细胞中,40μg TCDD组与20μg TCDD组无差异(P>0.05)。心脏、肾脏和睾丸的实验组均明显低于其溶剂对照组和空白对照组(P<0.05),心脏和肾脏中Bcl-2的表达无性别差异(P>0.05)。凋亡基因Bax可表达于心肌细胞中,40μg TCDD组与20μg TCDD组无差异(P>0.05),无性别差异(P>0.05);肾脏中Bax主要表达于肾小管和肾小球,40μg TCDD组显著性高于20μg TCDD组(P<0.05),雌性高于雄性(P<0.05);睾丸中Bax主要表达于睾丸细胞中,40μg TCDD组显著性高于20μg TCDD组(P<0.05)。心脏、肾脏和睾丸的实验组均明显高于其溶剂对照组和空白对照组(P<0.05)。雌雄仔鼠心脏、肾脏和睾丸中Bcl-2/Bax的比值显著性低于溶剂对照组和空白对照组(P<0.05),无性别差异(P>0.05),肾脏中40μg TCDD组显著性低于20μg TCDD组(P<0.05)。结论哺乳期低剂量暴露于TCDD,可以:阻碍仔鼠体重的增长;阻碍仔鼠肾脏重量的增加,影响肾脏的生长发育;阻碍仔鼠睾丸重量的增加,使睾丸下降时间延长,抑制生殖器官的发育,影响生殖功能;诱导仔鼠心脏、肾脏和睾丸组织CYP1A1蛋白表达的增加,可能是TCDD导致子代肾脏和睾丸重量下降和睾丸发育迟缓的原因之一;诱导心脏、肾脏和睾丸细胞凋亡,使Bcl-2蛋白表达减低,Bax蛋白表达增高,Bcl-2/Bax比值降低,并且雌性凋亡大于雄性。更多还原

【Abstract】 ObjectiveThe contaminant 2, 3, 7, 8-tetrachlordibenzo-p-dioxin (TCDD) is one of the most toxical compounds and ranked asⅠlevel carcinogen by International Agency for Research on Cancer (IARC). TCDD can accumulate in human body after long-time exposure. As a result, TCDD induce severe toxic effects on the organism. By far now, more attention is devoted to research the mechanism of acute toxicity and the effect of pregnant exposure TCDD on offspring. However, the effect of lactational exposure to low level of TCDD is ignored.In this study, the female mice got TCDD by intraperitoneal injection immediately after parturition and the offspring got by breast feeding. The present study would identify the toxic effect of lactational exposure to low level of TCDD on mice offspring by observing body weight, the histological changes and expression of cytochrome P450 1A1 enzyme (CYP1A1), apoptosis gene B-Cell lymphoma/leukemia 2 (Bcl-2) and Bcl-associated x protein (Bax) in heart, kidney and testes tissue. The results would provide evidence for the toxic effects of lactational exposure to low level of TCDD on mice offspring. Methods1 Animals4-month-old mature Kunming mice with 24 female and 8 male were grouped by 3:1. After parturition, the female mice with 8-10 pups were retained, and 8 pups were adjusted as a unit, including 4 female and 4 male offspring. The others were eliminated.2 ChemicalsTCDD was dissolved in methylbenzene with a concentration of 10μg/ml and a purity of 99% when purchased, and diluted with peanutoil by 1:4 and the final concentration is 2μg. The control vehicle was the mixed liquor with methylbenzene and peanutoil by 1:4.3 GroupsThe female mice and its offspring, regarded as a unit, were divided into 5 groups randomly. In this study, there were 2 doses of treatment: 40μg and 20μg TCDD/kg body weight. Corresponding to 2 doses of TCDD treatment, there were 2 vehicle controls: 40μg and 20μg vehicle/kg body weight, and 1 animal control without any treatment. There were 5 groups totally and each group had 3 units (They noted as 40μg TCDD, 20μg TCDD, 40μg vehicle control, 20μg vehicle control and animal control group respectively). After parturition, the female mice were weighted and intraperitoneally injected. The female mice stop lactation on offspring postnatal days (PND) 21, and then offspring were fed on animal feeds. Mice offspring were killed on PND 35.4 Measurement of mice offspring’s development indexes including body weight, the weight of kidney and testes, descending time of testes.5 CYP1A1 protein expression in heart, kidney and testes tissue of mice offspring.6 Detection of apoptosis in heart, kidney and testes tissue of mice offspring6.1 Observation of structure changes in heart, kidney and testes tissue of mice offspring by H.E staining method.6.2 Localization and semi-quantitative analysis of Bcl-2 and Bax protein expression in heart, kidney and testes tissue of mice offspring by immunohistochemistry.Results1 The effect of TCDD on the body weight of mice offspringThere was no difference in the average body weight of mice offspring between 5 groups when the offspring were born (P>0.05). On PND 7, the average body weight in 20μg TCDD group decreased compared with 20μg vehicle control and animal control groups (P<0.05). After 7 days, the average body weight in 40μg and 20μg TCDD groups decreased compared respectively with vehicle control and animal control groups (P<0.05), and that in 40μg TCDD group also decreased compared with 20μg TCDD group (P<0.05). After mice offspring were weaned (on PND 28 and PND 35), the average body weight in two TCDD groups also decreased compared with vehicle control and animal control groups (P<0.05).2 The effect of TCDD on the kidney’s weight of mice offspringThe absolute weight of kidney in two TCDD groups of the female was decreased significantly compared with those in vehicle control and animal control groups (P<0.05). But only the absolute weight of kidney in 40μg TCDD group of the male were decreased significantly compared with vertical control group (P<0.05). The relative weight of kidney in two TCDD groups of the female and male were no significant difference with the vehicle control (P>0.05). However, the relative weight of kidney in two TCDD groups of the female were also decreased significantly compared respectively with animal control groups (P<0.05). The absolute weight of kidney and relative weight of kidney in 40μg TCDD groups of the female and male were no significant difference with the 20μg TCDD groups (P>0.05). All the indexes of female were lower significantly than the male’s (P<0.05).3 The effect of TCDD on the testes weight of mice offspringThe absolute weight and relative weight of testes in two TCDD groups was decreased compared respectively with vehicle control and animal control groups. Meanwhile, 40μg TCDD groups was lower than those in 20μg TCDD groups, but there was no significant difference (P>0.05). Only the 40μg TCDD groups were decreased significantly compared with the vehicle control groups (P<0.05). 4 The effect of TCDD on descending time of testes of offspring miceThe descending time of testes in two TCDD groups was latter significantly than those in vehicle control and animal control groups and had significant difference compared respectively with vehicle control and animal control groups (P<0.05). The descending time of testes in 40μg TCDD groups was latter significantly than those in 20μg TCDD groups (P<0.05).5 The effect of TCDD on CYP1A1 protein expression in heart, kidney and testes tissue of mice offspringCYP1A1 protein can express mainly in the vascular endothelial cell, kidney and testes of mice offspring. Lactational exposure to low level of TCDD would induce CYP1A1 protein in myocardium increased in TCDD groups (P<0.05), but there was no significant difference between male and female (P>0.05). Lactational exposure to low level of TCDD would induce CYP1A1 protein in kidney increased in TCDD groups (P<0.05), and the female offspring’s CYP1A1 protein expression was higher significantly than the male offspring’s (P<0.05). Meanwhile lactational exposure to low level of TCDD also induced CYP1A1 protein expression in testes increased significantly in TCDD groups (P<0.05).6 The effect of TCDD on morphologic and Bcl-2 or Bax protein expression in heart, kidney and testes tissue of mice offspring Lactational exposure to low level of TCDD led to vascular endothelial cell apoptosis such as disordered structure, cell body crenation in heart of mice offspring. Lactational exposure to low level of TCDD led to the number of glomerular decreased and the dimension of glomerular increased. Meanwhile, inflammatory cells were also found in kidney. Meanwhile, cell body crenation of testes was also increased.Bcl-2 protein mainly expressed in vascular endothelial cell in heart of mice offspring, which can inhibit the cell apoptosis. Lactational exposure to low level of TCDD led to the Bcl-2 protein expression of 40μg TCDD groups in heart of mice offspring were no significant difference with the 20μg TCDD groups (P>0.05). Bcl-2 protein mainly expressed in glomerular of kidney. Lactational exposure to low level of TCDD led to the Bcl-2 protein expression and the Bcl-2 protein expression in 40μg TCDD groups in kidney of mice offspring were lower significantly than those in 20μg TCDD group’s (P<0.05). Bcl-2 protein also expressed in testes and Lactational exposure to low level of TCDD led to the Bcl-2 protein expression of 40μg TCDD groups in testes of mice offspring were no significant difference with the 20μg TCDD groups (P>0.05). Bcl-2 protein expression in two TCDD groups were lower significantly than those in vehicle control and animal control groups (P<0.05). Bcl-2 protein expression in heart and kidney were no significant difference between male and female (P>0.05).Bax protein expressed mainly in cardiac muscle cell of mice offspring. Lactational exposure to low level of TCDD led to the Bax protein expression of 40μg TCDD groups in heart of mice offspring were no significant difference with the 20μg TCDD groups (P>0.05). Meanwhile, there was no significant difference between male and female (P>0.05). Bax protein can express in glhemerular and renal tubules of the kidney. Lactational exposure to low level of TCDD, the Bax protein expression in 40μg TCDD groups in kidney of mice offspring were higher significantly than those in 20μg TCDD group’s (P<0.05) and the female offspring’s Bax protein expression was higher significantly than those of the male offspring (P<0.05). Bax protein also expressed in testes and Bax protein expression in 40μg TCDD groups were higher significantly than those in 20μg TCDD groups (P<0.05).Bax protein expression in two TCDD groups were higher significantly than those in vehicle control and animal control groups (P<0.05).In heart, kidney and testes, the ratio of Bcl-2/Bax decreased significantly in two TCDD groups of mice offspring compared respectively with vehicle control and animal control groups (P<0.05), there was no significant difference between male and female (P>0.05). Lactational exposure to low level of TCDD led to the the ratio of Bcl-2/Bax of 40μg TCDD groups in kidney of mice offspring were higher significantly than 20μg TCDD group’s (P<0.05).ConclusionLactational exposure to low level of TCDD delaied the body weight gain of mice offspring;inhibited the weight of kidney. The toxic effects are responsible for the growth retardation of kidney;hampered the weight of testes. TCDD can display the toxic effect and damage the development of testes;decreased the descending time of testes,disturb the endocrine system and affect the function of testes;increased CYP1A1 protein expression in heart, kidney and testes. TCDD has the relation to the CYP1A1 protein expression;induced the cell apoptosis in heart, kidney and testes. Meanwhile, Lactational exposure to low level of TCDD led to decrease of Bcl-2 protein expression and the increase of Bax protein expression. The change of of Bcl-2 Protein expression is correlated with accelerated apoptosis. In addition, the ratio of Bcl-2/Bax was also decreased and the ratio of Bcl-2/Bax in female offspring was higher than male offspring.更多还原