二氧化硫及其衍生物对小鼠几种脏器超微结构和细胞凋亡的影响
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摘要
二氧化硫是一种常见的全球性大气环境污染物，它对人体健康的影响是许多学者关注的焦点。山西省作为煤炭大省，在为全国提供能源的同时也给本省带来了非常严重的环境污染问题。因此迫切需要我们尽快搞清二氧化硫的实际健康危害。我们前期的一些研究提示二氧化硫可能不象通常认为的那样，仅对上呼吸道有损伤和刺激作用，而可能具有比较广泛的毒理学损伤作用，并且也可能导致机体的组织形态发生一定的改变，为此进行了本次实验。
本研究体内实验部分重点探讨了二氧化硫对小鼠肺组织、脾组织的细胞凋亡诱导作用和组织学损伤作用，此外还观察了二氧化硫对小鼠胸腺、肝脏、大脑皮质的组织学结构的影响。体外实验主要研究了二氧化硫代谢衍生物对小鼠分离脾淋巴细胞和人胚肺成纤维细胞的毒性作用。
二氧化硫熏气染毒的体内实验结果表明，在本次实验的浓度范围内(56mg／m~3、112mg／m~3、168mg／m~3低、中、高三个浓度)：(1)通过透射电镜、DNA凝胶电泳分析和流式细胞分析发现二氧化硫吸入染毒一周对小鼠肺脏没有明显的凋亡诱导作用，但通过透射电镜观察发现二氧化硫可引起肺脏明显的超微结构改变，引起Ⅱ型肺泡上皮细胞板层体空泡化，微绒毛减少，线粒体致密化或肿胀变性；肺泡血管内皮细胞和Ⅰ型肺泡上皮细胞之间基膜增厚，使氧气弥散功能出现障碍，从而降低肺功能。(2)二氧化硫吸入可引起小鼠脾脏细胞出现明显的凋亡改变，红髓区和白髓区淋巴细胞出现核固缩，染色质凝聚、边集；DNA凝胶电泳分析发现168mg／m~3二氧化硫染毒组出现典型的DNA梯形条带；流式细胞分析也发现高剂量染毒组的小鼠脾细胞凋亡率增加，并且与对照组相比有显著性差异，P＜0.05。(3)二氧化硫染毒对小鼠胸腺组织学结构影响较小：用透射电镜观察发现胸腺组织中有部分淋巴细胞变形且异染色质增多，胸腺上皮细胞中可见次级溶酶体增多，线粒体变形，这说明二氧化硫可能对中枢免疫器官也有一定的不良影响。(4)二氧化硫染毒对小鼠肝脏的组织学结构有明显影响，可引起肝脏点状坏死、灶状坏死甚至片状坏死，伴随不同程度的炎性细胞浸润；透射电镜观察发现二氧化硫可引起肝细胞脂肪变性、嗜酸性颗粒变和坏死，脂肪变肝细胞中可见大小不等的脂滴存在，嗜酸性颗粒变肝细胞中可见线粒体明显增生，坏死肝细胞可见细胞核结构破坏，细胞器减少，细胞膜不完整。这些均可影响肝脏正常功能的发挥。(5)二氧化硫染毒对小鼠大
山西大学博士学位论文
脑皮层组织学结构没有影响。说明二氧化硫可能对多个脏器具有损伤作用，但不同脏器
的损伤程度明显不同。在本次实验的染毒浓度和时间条件下，肝脏的损伤最为明显，肺
脏、脾脏、胸腺也有一定程度的病理表现，大脑皮层在本次实验过程中未见病理改变。
二氧化硫代谢衍生物体外实验结果表明:(l)二氧化硫代谢衍生物对小鼠脾细胞具
有明显的细胞毒性作用，染毒4小时和24小时细胞的半数存活抑制浓度LCS。分别为
1.12932r以mol几和1.37777~ol几。而且一定浓度范围内可引起小鼠脾淋巴细胞DNA
发生特征性降解，出现DNA LadderS，引起染毒组细胞凋亡率显著性升高，但细胞凋亡
率与剂量不呈直线相关关系。此外二氧化硫代谢衍生物还可引起小鼠脾细胞外乳酸脱氢
酶活性明显降低，细胞内还原性谷肤甘肤含量明显升高，并且均有一定的剂量反应关系。
(2)二氧化硫代谢衍生物对人胚肺成纤维细胞具有明显的细胞毒性作用，4小时和24
小时细胞半数存活抑制浓度LCS。分别为1.1133和2.0070二o1/L。此外二氧化硫代谢衍
生物可引起人胚肺成纤维细胞形态发生明显改变和细胞内外乳酸脱氢酶活性明显降低。
说明二氧化硫代谢衍生物对人胚肺成纤维细胞和小鼠脾细胞都有一定毒性，并且在一定
范围内对小鼠脾细胞具有一定的凋亡诱导作用。
SO2 is a common irritant gas all over the world. Many researchers are devoting themselves on studying its toxicity.
In the present study, we studied the apoptotic induction on mouse lung and spleen and their pathological structural changes of short-term sulfur dioxide inhalation. We also investigated the pathological changes of mouse liver, thymus and cerebrum cortex challenged by SO2 inhalation by in vivo tests. We studied the apoptotic induction on mouse spleen cells and cytotoxicity of human embryo lung fibroblasts of SO2 derivatives by in vitro tests.
In vivo tests of sulfur dioxide inhalation showed:
(1) Effects on mouse lung of SO2 challenge: We found no significant apoptotic changes induced by SO2 inhalation but obvious pathological changes of lung with vacuolating of osmiophilic multilamellar bodies which maybe related with the decrease of surfacant and decrease of microvillus of type II alveolar cells; We also found thickening of part of basement lamina between type I alveolar cells and capillary endothelium cells which may inhibit the dispersion of oxygen and contribute to lung dysfunction.
(2) Effects on mouse spleen of SO2 challenge: We found significant apoptotic changes of mouse spleen through TEM observation and DNA electrophoresis analysis and flow cytometric analysis. We found condensed, marginating, half-moon like apoptotic lymphocytes both in white pulp and red pulp; We found significant DNA degradation with DNA ladders from the DNA electrophoresis analysis in the 168 mg/m3 SO2 treated group; We also found marked increase of apoptotic rate between 168 mg/m3 SO2 treated group and control group from the flow cytometric analysis. These suggested that SO2 may affect body immunity to a certain degree.
(3) Effects on mouse thymus of SO2 challenge : HE staining showed no obvious structure changes of thymus in all treatment groups compaired with the control group; The ultrastructure of thymus can be seen injured in SO2 treated groups from TEM observation. We found nuclear deformation lymphocytes with increased heterochromatin and impaired thymus epithelium cells with increased lysosomes and deformation of mitochondrias.
(4) Effects on mouse liver of SO2 challenge: SO2 can cause significant liver injury. HE
staining showed several kinds of necrosis of liver including spot necrosis, focal necrosis and submassive necrosis infiltrated with lymphocytes, monocytes, few neutrophils and eosinophils;TEM observation showed fatty degeneration with dispersion of fatty droplets and dilation of rough endoplasmic reticulums, acid degeneration with significant hyperplasia of mitochondrias, necrosis of hepatocytes with karyorrhexis and other organelles losing their normal structure.
(5) Effects on mouse cerebrum cortex of SO2 challenge: we found no pathological changes of mouse cerebrum cortex challenged by SO2 by HE staining and TEM observation,
In vitro tests of SO2 derivatives challenge showed that:
(1) SO2 derivatives has significant toxicity on mouse spleen cells, the LC50 of 4h and 24h exposure is 1.1293 mmol/L and 1.3777mmol/L respectively. It has marked apoptotic induction on mouse spleen cells tested by DNA agarose gel electrophoresis analysis and flow cytometric assay in certain degree. It also can decrease the activity of lactate dehydrogenase
(LDH) outside of mouse the spleen cells and increase the quantity of reduced GSH inside the mouse spleen cells.
(2) SO2 derivatives has significant toxicity on human embryo lung fibroblasts, the LC50 of 4h and 24h exposure is 1. 1133 and 2. 0070mmol/L respectively. It also cause signigicant decrease of LDH activity both inside and outside of the human embryo lung fibroblasts.
In conclusion, SO2 can not only affect respiratory system, but also other organs such as spleen, liver, thymus on their histological structures and every organ performs very differently under SO2 exposure. SO2 derivatives has cytotoxicity both on mouse spleen cells and human embryo lung fibroblasts. SO2 may injure other
本研究体内实验部分重点探讨了二氧化硫对小鼠肺组织、脾组织的细胞凋亡诱导作用和组织学损伤作用，此外还观察了二氧化硫对小鼠胸腺、肝脏、大脑皮质的组织学结构的影响。体外实验主要研究了二氧化硫代谢衍生物对小鼠分离脾淋巴细胞和人胚肺成纤维细胞的毒性作用。
二氧化硫熏气染毒的体内实验结果表明，在本次实验的浓度范围内(56mg／m~3、112mg／m~3、168mg／m~3低、中、高三个浓度)：(1)通过透射电镜、DNA凝胶电泳分析和流式细胞分析发现二氧化硫吸入染毒一周对小鼠肺脏没有明显的凋亡诱导作用，但通过透射电镜观察发现二氧化硫可引起肺脏明显的超微结构改变，引起Ⅱ型肺泡上皮细胞板层体空泡化，微绒毛减少，线粒体致密化或肿胀变性；肺泡血管内皮细胞和Ⅰ型肺泡上皮细胞之间基膜增厚，使氧气弥散功能出现障碍，从而降低肺功能。(2)二氧化硫吸入可引起小鼠脾脏细胞出现明显的凋亡改变，红髓区和白髓区淋巴细胞出现核固缩，染色质凝聚、边集；DNA凝胶电泳分析发现168mg／m~3二氧化硫染毒组出现典型的DNA梯形条带；流式细胞分析也发现高剂量染毒组的小鼠脾细胞凋亡率增加，并且与对照组相比有显著性差异，P＜0.05。(3)二氧化硫染毒对小鼠胸腺组织学结构影响较小：用透射电镜观察发现胸腺组织中有部分淋巴细胞变形且异染色质增多，胸腺上皮细胞中可见次级溶酶体增多，线粒体变形，这说明二氧化硫可能对中枢免疫器官也有一定的不良影响。(4)二氧化硫染毒对小鼠肝脏的组织学结构有明显影响，可引起肝脏点状坏死、灶状坏死甚至片状坏死，伴随不同程度的炎性细胞浸润；透射电镜观察发现二氧化硫可引起肝细胞脂肪变性、嗜酸性颗粒变和坏死，脂肪变肝细胞中可见大小不等的脂滴存在，嗜酸性颗粒变肝细胞中可见线粒体明显增生，坏死肝细胞可见细胞核结构破坏，细胞器减少，细胞膜不完整。这些均可影响肝脏正常功能的发挥。(5)二氧化硫染毒对小鼠大
山西大学博士学位论文
脑皮层组织学结构没有影响。说明二氧化硫可能对多个脏器具有损伤作用，但不同脏器
的损伤程度明显不同。在本次实验的染毒浓度和时间条件下，肝脏的损伤最为明显，肺
脏、脾脏、胸腺也有一定程度的病理表现，大脑皮层在本次实验过程中未见病理改变。
二氧化硫代谢衍生物体外实验结果表明:(l)二氧化硫代谢衍生物对小鼠脾细胞具
有明显的细胞毒性作用，染毒4小时和24小时细胞的半数存活抑制浓度LCS。分别为
1.12932r以mol几和1.37777~ol几。而且一定浓度范围内可引起小鼠脾淋巴细胞DNA
发生特征性降解，出现DNA LadderS，引起染毒组细胞凋亡率显著性升高，但细胞凋亡
率与剂量不呈直线相关关系。此外二氧化硫代谢衍生物还可引起小鼠脾细胞外乳酸脱氢
酶活性明显降低，细胞内还原性谷肤甘肤含量明显升高，并且均有一定的剂量反应关系。
(2)二氧化硫代谢衍生物对人胚肺成纤维细胞具有明显的细胞毒性作用，4小时和24
小时细胞半数存活抑制浓度LCS。分别为1.1133和2.0070二o1/L。此外二氧化硫代谢衍
生物可引起人胚肺成纤维细胞形态发生明显改变和细胞内外乳酸脱氢酶活性明显降低。
说明二氧化硫代谢衍生物对人胚肺成纤维细胞和小鼠脾细胞都有一定毒性，并且在一定
范围内对小鼠脾细胞具有一定的凋亡诱导作用。
SO2 is a common irritant gas all over the world. Many researchers are devoting themselves on studying its toxicity.
In the present study, we studied the apoptotic induction on mouse lung and spleen and their pathological structural changes of short-term sulfur dioxide inhalation. We also investigated the pathological changes of mouse liver, thymus and cerebrum cortex challenged by SO2 inhalation by in vivo tests. We studied the apoptotic induction on mouse spleen cells and cytotoxicity of human embryo lung fibroblasts of SO2 derivatives by in vitro tests.
In vivo tests of sulfur dioxide inhalation showed:
(1) Effects on mouse lung of SO2 challenge: We found no significant apoptotic changes induced by SO2 inhalation but obvious pathological changes of lung with vacuolating of osmiophilic multilamellar bodies which maybe related with the decrease of surfacant and decrease of microvillus of type II alveolar cells; We also found thickening of part of basement lamina between type I alveolar cells and capillary endothelium cells which may inhibit the dispersion of oxygen and contribute to lung dysfunction.
(2) Effects on mouse spleen of SO2 challenge: We found significant apoptotic changes of mouse spleen through TEM observation and DNA electrophoresis analysis and flow cytometric analysis. We found condensed, marginating, half-moon like apoptotic lymphocytes both in white pulp and red pulp; We found significant DNA degradation with DNA ladders from the DNA electrophoresis analysis in the 168 mg/m3 SO2 treated group; We also found marked increase of apoptotic rate between 168 mg/m3 SO2 treated group and control group from the flow cytometric analysis. These suggested that SO2 may affect body immunity to a certain degree.
(3) Effects on mouse thymus of SO2 challenge : HE staining showed no obvious structure changes of thymus in all treatment groups compaired with the control group; The ultrastructure of thymus can be seen injured in SO2 treated groups from TEM observation. We found nuclear deformation lymphocytes with increased heterochromatin and impaired thymus epithelium cells with increased lysosomes and deformation of mitochondrias.
(4) Effects on mouse liver of SO2 challenge: SO2 can cause significant liver injury. HE
staining showed several kinds of necrosis of liver including spot necrosis, focal necrosis and submassive necrosis infiltrated with lymphocytes, monocytes, few neutrophils and eosinophils;TEM observation showed fatty degeneration with dispersion of fatty droplets and dilation of rough endoplasmic reticulums, acid degeneration with significant hyperplasia of mitochondrias, necrosis of hepatocytes with karyorrhexis and other organelles losing their normal structure.
(5) Effects on mouse cerebrum cortex of SO2 challenge: we found no pathological changes of mouse cerebrum cortex challenged by SO2 by HE staining and TEM observation,
In vitro tests of SO2 derivatives challenge showed that:
(1) SO2 derivatives has significant toxicity on mouse spleen cells, the LC50 of 4h and 24h exposure is 1.1293 mmol/L and 1.3777mmol/L respectively. It has marked apoptotic induction on mouse spleen cells tested by DNA agarose gel electrophoresis analysis and flow cytometric assay in certain degree. It also can decrease the activity of lactate dehydrogenase
(LDH) outside of mouse the spleen cells and increase the quantity of reduced GSH inside the mouse spleen cells.
(2) SO2 derivatives has significant toxicity on human embryo lung fibroblasts, the LC50 of 4h and 24h exposure is 1. 1133 and 2. 0070mmol/L respectively. It also cause signigicant decrease of LDH activity both inside and outside of the human embryo lung fibroblasts.
In conclusion, SO2 can not only affect respiratory system, but also other organs such as spleen, liver, thymus on their histological structures and every organ performs very differently under SO2 exposure. SO2 derivatives has cytotoxicity both on mouse spleen cells and human embryo lung fibroblasts. SO2 may injure other
引文
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