二噁英染毒模型小鼠相关受体及代谢酶的分布

Distribution of dioxin receptors and metabolizing enzymes of 2,3, 7,8-tetrachlorodibenzo-p-dioxin exposure mice models

目的 探讨引起小鼠早期妊娠毒性敏感与相关受体、代谢酶水平的关系,了解子宫组织的敏感性是否与二噁英（TCDD）代谢活化而导致局部毒性蓄积有关.方法 收集TCDD妊娠早期毒性模型小鼠的子宫、肝脏和肾脏组织,用免疫组织化学法对芳香碳氢化合物受体（AhR）,受体核转位蛋白（ARNT）和细胞色素氧化酶（Cyp1a2）在组织细胞中分布进行检测.结果 正常妊娠小鼠的子宫、肝脏和肾脏组织细胞质有AhR、Cyp1a2弱阳性信号,细胞核内ARNT有强阳性信号.用不同浓度TCDD处理小鼠,各组织细胞质内的AhR阳性信号随浓度的提高而增加.50 ng/L剂量TCDD暴露使肝脏（18.038±3.0916,P＜0.01）,子宫（8.8140±1.4574,P＜0.01）的AhR阳性面积比提高,以及吸光度（A）值显著升高（0.1143±0.0066,0.2399±0.0054,P＜0.01） 而肾脏中2种指标均未见显著性差异.相同处理组Cyp1a2变化则主要表现在吸光度的变化,肝脏（0.2047±0.0150,0.31112±0.0107,P＜0.01）,子宫（0.1892±0.0232,0.3152±0.0432,P＜0.05）和肾脏（0.2545±0.0202,0.3334±0.0168,P＜0.05）A值均显著升高 组织细胞核内ARNT阳性吸光度在肝脏（0.7496±0.0469,0.2458±0.006,P＜0.01）和子宫（0.4766±0.0359,0.2139±0.0253,P＜0.01）显著下降,在肾脏则表现为阳性信号从细胞核进入细胞质.100 ng/L剂量TCDD暴露则引起所有组织AhR和Cyp1a2阳性显著增强 肝脏和子宫ARNT信号消失,肾脏中ARNT信号从核内转入细胞质.结论 肝脏、子宫和肾脏AhR和CYP1A2酶可受TCDD类物质诱导,细胞信号面积随TCDD暴露量的增加而增大 肝脏和子宫细胞核内的ARNT随TCDD暴露量的增加,细胞信号减弱至消失,但肾脏ARNT进入胞质.说明子宫与肝脏具有相同的TCDD诱导特征,具有活化内外源性有毒化合物的功能,这应该是引起小鼠早期生殖毒性的原因之一.

Objective To investigate relationships between high sensitivity to toxicity in NIH mice during early pregnancy and level of receptors and metabolizing enzymes to 2,3,7,8-tetrachlorodibenzo-pdioxin （TCDD）, and to elucidate whether the sensitivity is associated with the TCDD accumulation induced by metabolic activation in the uterus. Methods Liver, kidney and uterus tissues in mice models of toxicity exposure during the early pregnancy were collected and fixed. Distribution of the aryl hydrocarbon receptor （AhR）, aryl hydrocarbon receptor nuclear translocator （ARNT） and cytochrome P4501a2 （Cyp1a2） was then determined by immunohistochemical staining. Results In unexposed mice, the tissues of liver, kidney and uterus, displayed weak-positive signal of AhR and Cyp1a2 in cytoplasma and strong-positive signal of ARNT in the nuclei. The positive signal of cytoplasma AhR in these three types of tissues was enhanced to varying degrees along with increasing dose of TCCD exposure. Exposure to 50 ng/L TCDD increased both the percentage of AhR-positive area in liver （18.038±3.0916,P〈0.01）and uterus （8.8140±1.4574,P〈0.01） and optic density of AhR signals（0.1143±0.0066,0.2399±0.0054, P〈0.01 ）, while no change was found in kidney tissues. With the same treatment, changes in Cyp1a2 were mainly manifested by significant increase in optic density of Cypla2 signals in liver （0.31112±0.0107, P〈0.01 ）, uterus （0.3152±0.0432, P〈0.05） and kidney （0.3334±0.0168, P〈0.05 ）, while the optic density of positive ARNT signals in nuclei decreased significantly in liver （0.2458±0.006, P〈0.01 ）and uterus（0.2139±0.0253, P〈0.01 ）, was largely unchanged but associated with a translocation from nuclei to cytoplasma in kidney. Exposure to 100 ng/L TCDD resulted in enhanced AhR and Cyp1a2 positive signals in all the tissues examined, abolition of ARNT signals in liver and uterus,and traslocation of ARNT signal from nuclei to cytoplasma in the kidney. Conclusions The AhR and Cyp1a2 in liver, uterus and kidney induced by TCDD-like agents may show increasing percentage of signalpositive area along with higher dose of TCDD exposure. In contrast, ARNT signal is weakened and subsequently abolished in nuclei of liver and uterus cells, and displays a traslocation from nuclei to cytoplasma in the kidney. These findings indicate that liver and uterus may share the same TCDD-induced signaling pathway which activates the exogenous toxic agents and should be one of the factors causing sensitivity to TCDD during early pregnancy in mice.