甲基叔丁基醚诱导细胞增殖的毒物兴奋效应及其毒理学意义
摘要
研究目的：
研究甲基叔丁基醚(Methyl Tertiary Butyl Ether, MTBE)是否能诱导细胞增殖的毒物兴奋效应(Hormesis)及其剂量-反应关系,同时探讨MTBE在环境中的安全剂量范围。
研究方法：
1、采用MTT试验观察MTBE对体外培养的人支气管上皮细胞(Human bronchial epithelial cells,16HBE)的细胞毒性作用,同时观察MTBE作用于16HBE细胞能否产生细胞增殖的毒物兴奋效应,并确定其剂量范围(Hormetic Zone)。
2、采用单细胞凝胶电泳(Single cell gel electrophoresis, SCGE)试验观察MTBE在其细胞增殖的Hormetic Zone的剂量下能否导致16HBE细胞的DNA损伤。
3、通过小鼠经口亚慢性染毒,采用SCGE试验观察MTBE在其细胞增殖的Hormetic Zone的剂量下能否导致小鼠全血细胞和外周血淋巴细胞的遗传物质的损伤效应,同时用微核试验检测其能否导致小鼠骨髓网织红细胞的染色体损伤效应。
研究结果：
1、MTT试验的结果显示,一定剂量的MTBE作用于16HBE细胞能诱导细胞增殖的毒物兴奋效应,其Hormetic Zone为1g／L～10g／L。
2、SCGE试验结果表明,40g／L的MTBE染毒16HBE细胞4小时,细胞已绝大部分凋亡。经IMI图像分析软件分析,将各剂量组的尾长、尾DNA%、尾／头(长)、Olive尾矩、尾头光强比等指标值与正常对照组相比较,结果表明1g／L～30g／L剂量组的各指标值均较正常对照组高,且差异有统计学意义(P<0.05)。说明1g／L～30g／L的MTBE能造成16HBE细胞DNA损伤,MTBE浓度与16HBE细胞DNA损伤的程度呈剂量-反应关系。
3、小鼠经口亚慢性(3个月)染毒的试验结果表明,2.5～5g／L剂量的MTBE能够造成小鼠全血细胞和淋巴细胞的DNA损伤效应,并有一定的剂量效应关系。MTBE对小鼠骨髓网织红细胞微核试验的结果表明,雄性和雌性小鼠骨髓网织红细胞与正染红细胞的比值(PEC/NEC)各染毒剂量组与空白对照组相比,均无显著性差异(P>0.05),表明该试验剂量范围的MTBE长期染毒对小鼠骨髓的造血功能无明显影响；除了剂量为1.25g／L时雌性小鼠的微核率与空白对照组相比存在显著性差异之外,其它各个剂量组中,无论雌性或雄性小鼠的微核率与空白对照组相比均无显著性差异(P>0.05)。该试验结果提示,本试验设计的染毒剂量的MTBE对小鼠网织红细胞染色体尚无法观察到确定的损伤作用。
结论：
1.一定剂量的MTBE作用于16HBE细胞会促进细胞增殖加速,表现出明显的毒物兴奋效应,其毒物兴奋效应作用带(Hormetic Zone)为1 g/L～10 g／L。
2.1 g/L～10 g／L剂量范围的MTBE具有促进16HBE细胞增殖的作用,同时也能导致细胞DNA损伤,却没有观察到染色体水平的严重损伤效应。据此我们认为,能诱导细胞增殖Hormesis的剂量范围的MTBE并非安全,它具有DNA损伤作用,同时促进细胞增殖加速,很可能具有致癌危险性。
Research Aim:
To study whether Methyl Tertiary Butyl Ether (MTBE) has hormesis on cell proliferation and how its dose-effect relationship is. At the same time to investigate the safe dose of MTBE in the environment.
Methods:
1. The MTT test was used to detect the cytotoxic effect of MTBE in Human Bronchial epithelial cells (16HBE cells). At the same time to observe whether MTBE could induce 16HBE cells to occur hormesis and determine its dose range (Hormetic Zone).
2. Us Single cell gel electrophoresis to observe whether the MTBE within the dose of Hormetic Zone can lead to DNA damage in 16HBE cells.
3. Set several dose groups of MTBE within the dose of Hormetic Zone. Let the mice continuously be exposed to these groups of MTBE by way of drinking for three months. Then detecte the DNA damage in their whole blood cells and leucocytes by SCGE while test the DNA damage in their bone marrow reticulocytes using micronucleus test.
Result
1. MTT test revealed that MTBE could induce 16HBE cells to occur hormesis and its dose range of Hormetic Zone is 1 g/L～10 g/L.
2. SCGE experiment showed that apoptosis take place in most 16HBE cells after exposed to 40g/L dose of MTBE for 4h. Each index value in MTBE dose groups of lg/L～30g/L is higher than that in normal control group. It revealed that lg/L～30g/L doses of MTBE can cause DNA damage in 16HBE cells. It also showed clear dose-effect relationship.
3. After continuous exposure to each dose group of MTBE for 90 days, the whole blood cells, leucocytes of blood and bone marrow reticulocyte were collected from the mice. We used these tissues to carry out related experiments. SCGE test showed that MTBE can result in DNA damage in whole blood cells and reticulocyte at the dose of 2.5～5g/L, and it also showed clear dose-effect relationship. Compared with the normal control group, no significant difference has been found in each exposure group (including male and female mouse) about the PCE/NCE value. It demonstrated that long-time exposure to low-dose MTBE is harmless to hematopoiesis. There was no significant difference between the normal control group and each exposure group about the micronucleus rate. Only at the dose of 1.25g/L, the micronucleus rate of female mice was significantly increased compared with the normal control group. Micronucleus test revealed that the dose of MTBE set in this experiment cannot cause chromosome damage.
Conclusion:
1. MTBE could induce 16HBE cells to occur hormesis on proliferation and its dose range of Hormetic Zone is 1 g/L～10 g/L.
2. 1g/L～10g/L doses of MTBE can cause DNA damage in 16HBE cells and shows clear dose-effect relationship.2.5～5g/L doses of MTBE can cause DNA damage in whole blood cells and reticulocytes and has dose-effect relationship.2.5～5g/L doses of MTBE have no significant influence on PCE/NCE value and micronucleus rate. Accordingly, we think that the doses of MTBE in Hormetic Zone is dangerous to organisms, can cause slight DNA damage, but cannot lead to serious damage such as Chromosome breakage.
研究甲基叔丁基醚(Methyl Tertiary Butyl Ether, MTBE)是否能诱导细胞增殖的毒物兴奋效应(Hormesis)及其剂量-反应关系,同时探讨MTBE在环境中的安全剂量范围。
研究方法：
1、采用MTT试验观察MTBE对体外培养的人支气管上皮细胞(Human bronchial epithelial cells,16HBE)的细胞毒性作用,同时观察MTBE作用于16HBE细胞能否产生细胞增殖的毒物兴奋效应,并确定其剂量范围(Hormetic Zone)。
2、采用单细胞凝胶电泳(Single cell gel electrophoresis, SCGE)试验观察MTBE在其细胞增殖的Hormetic Zone的剂量下能否导致16HBE细胞的DNA损伤。
3、通过小鼠经口亚慢性染毒,采用SCGE试验观察MTBE在其细胞增殖的Hormetic Zone的剂量下能否导致小鼠全血细胞和外周血淋巴细胞的遗传物质的损伤效应,同时用微核试验检测其能否导致小鼠骨髓网织红细胞的染色体损伤效应。
研究结果：
1、MTT试验的结果显示,一定剂量的MTBE作用于16HBE细胞能诱导细胞增殖的毒物兴奋效应,其Hormetic Zone为1g／L～10g／L。
2、SCGE试验结果表明,40g／L的MTBE染毒16HBE细胞4小时,细胞已绝大部分凋亡。经IMI图像分析软件分析,将各剂量组的尾长、尾DNA%、尾／头(长)、Olive尾矩、尾头光强比等指标值与正常对照组相比较,结果表明1g／L～30g／L剂量组的各指标值均较正常对照组高,且差异有统计学意义(P<0.05)。说明1g／L～30g／L的MTBE能造成16HBE细胞DNA损伤,MTBE浓度与16HBE细胞DNA损伤的程度呈剂量-反应关系。
3、小鼠经口亚慢性(3个月)染毒的试验结果表明,2.5～5g／L剂量的MTBE能够造成小鼠全血细胞和淋巴细胞的DNA损伤效应,并有一定的剂量效应关系。MTBE对小鼠骨髓网织红细胞微核试验的结果表明,雄性和雌性小鼠骨髓网织红细胞与正染红细胞的比值(PEC/NEC)各染毒剂量组与空白对照组相比,均无显著性差异(P>0.05),表明该试验剂量范围的MTBE长期染毒对小鼠骨髓的造血功能无明显影响；除了剂量为1.25g／L时雌性小鼠的微核率与空白对照组相比存在显著性差异之外,其它各个剂量组中,无论雌性或雄性小鼠的微核率与空白对照组相比均无显著性差异(P>0.05)。该试验结果提示,本试验设计的染毒剂量的MTBE对小鼠网织红细胞染色体尚无法观察到确定的损伤作用。
结论：
1.一定剂量的MTBE作用于16HBE细胞会促进细胞增殖加速,表现出明显的毒物兴奋效应,其毒物兴奋效应作用带(Hormetic Zone)为1 g/L～10 g／L。
2.1 g/L～10 g／L剂量范围的MTBE具有促进16HBE细胞增殖的作用,同时也能导致细胞DNA损伤,却没有观察到染色体水平的严重损伤效应。据此我们认为,能诱导细胞增殖Hormesis的剂量范围的MTBE并非安全,它具有DNA损伤作用,同时促进细胞增殖加速,很可能具有致癌危险性。
Research Aim:
To study whether Methyl Tertiary Butyl Ether (MTBE) has hormesis on cell proliferation and how its dose-effect relationship is. At the same time to investigate the safe dose of MTBE in the environment.
Methods:
1. The MTT test was used to detect the cytotoxic effect of MTBE in Human Bronchial epithelial cells (16HBE cells). At the same time to observe whether MTBE could induce 16HBE cells to occur hormesis and determine its dose range (Hormetic Zone).
2. Us Single cell gel electrophoresis to observe whether the MTBE within the dose of Hormetic Zone can lead to DNA damage in 16HBE cells.
3. Set several dose groups of MTBE within the dose of Hormetic Zone. Let the mice continuously be exposed to these groups of MTBE by way of drinking for three months. Then detecte the DNA damage in their whole blood cells and leucocytes by SCGE while test the DNA damage in their bone marrow reticulocytes using micronucleus test.
Result
1. MTT test revealed that MTBE could induce 16HBE cells to occur hormesis and its dose range of Hormetic Zone is 1 g/L～10 g/L.
2. SCGE experiment showed that apoptosis take place in most 16HBE cells after exposed to 40g/L dose of MTBE for 4h. Each index value in MTBE dose groups of lg/L～30g/L is higher than that in normal control group. It revealed that lg/L～30g/L doses of MTBE can cause DNA damage in 16HBE cells. It also showed clear dose-effect relationship.
3. After continuous exposure to each dose group of MTBE for 90 days, the whole blood cells, leucocytes of blood and bone marrow reticulocyte were collected from the mice. We used these tissues to carry out related experiments. SCGE test showed that MTBE can result in DNA damage in whole blood cells and reticulocyte at the dose of 2.5～5g/L, and it also showed clear dose-effect relationship. Compared with the normal control group, no significant difference has been found in each exposure group (including male and female mouse) about the PCE/NCE value. It demonstrated that long-time exposure to low-dose MTBE is harmless to hematopoiesis. There was no significant difference between the normal control group and each exposure group about the micronucleus rate. Only at the dose of 1.25g/L, the micronucleus rate of female mice was significantly increased compared with the normal control group. Micronucleus test revealed that the dose of MTBE set in this experiment cannot cause chromosome damage.
Conclusion:
1. MTBE could induce 16HBE cells to occur hormesis on proliferation and its dose range of Hormetic Zone is 1 g/L～10 g/L.
2. 1g/L～10g/L doses of MTBE can cause DNA damage in 16HBE cells and shows clear dose-effect relationship.2.5～5g/L doses of MTBE can cause DNA damage in whole blood cells and reticulocytes and has dose-effect relationship.2.5～5g/L doses of MTBE have no significant influence on PCE/NCE value and micronucleus rate. Accordingly, we think that the doses of MTBE in Hormetic Zone is dangerous to organisms, can cause slight DNA damage, but cannot lead to serious damage such as Chromosome breakage.
引文
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