环境污染物的双相剂量—效应与多氯联苯双相剂量—效应的作用机制研究
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摘要
剂量-效应关系是毒理学研究的基础。近年来研究表明,与毒理学传统阈值模型(Threshold model)及线性非阈值模型(Linear non-threshold)的描述不同,环境污染物诱导的生物学响应往往表现出低浓度促进、高浓度抑制的双相剂量-效应关系。双相剂量-效应关系的存在,对传统剂量-效应模型的正确性提出了质疑,对环境风险评估具有潜在而深远的影响。
随着相关报道的逐年增加,双相剂量—效应关系逐步得到科学界认可,但其作用机制并不明确。本论文采用多种生物毒性测试方法对典型污染物诱导的双相剂量-效应关系进行了验证。重金属的发光菌生物毒性检测结果表明,铜、锌、镉、铬(Ⅵ)等4种重金属处理对淡水发光菌——青海弧菌(Vibrio qinghaiensis sp. Q67)和海洋发光菌——明亮发光杆菌(Photobacterium phosphoreum sp. T3)生长的影响均表现出倒“U”形双相剂量-效应关系。总体上讲,处理浓度小于1mg/l时,无毒常量重金属铜、锌对两种发光菌的影响表现为生长促进作用,10mg/l的处理浓度则在各个处理时间点均抑制发光菌生长。非必需有毒重金属镉、铬(Ⅵ)对两种发光菌的毒性抑制作用具有选择性,淡水发光菌Q67对镉处理较为敏感,铬(Ⅵ)对海洋发光菌T3的毒性抑制作用则更为显著。
Vero细胞离体毒性测试结果表明,无论重金属锌还是持久性有机污染物多氯联苯(Polychlorinated biphenyls, PCBs)对Vero细胞生长的影响均符合双相剂量-效应曲线的描述。处理浓度低于25mg/l时,重金属锌的3种化合物形式均能促进Vero细胞生长,但随着处理时间的延长,生长刺激效应会逐渐转变为抑制作用。处理浓度高于200mg/l时,锌盐显著抑制细胞生长,其生长率降至对照的50%以下。不同结构PCBs对Vero细胞生长的影响表明,低浓度PCBs(浓度小于0.01μg/ml)能刺激Vero细胞生长,刺激效应达到对照的120%左右。另外,非共面PCB 52、PCB 153对Vero细胞的毒性抑制作用分别大于共平面结构的PCB 77和PCB 126。
本论文采用Vero细胞离体毒性测试方法,以共平面PCB 126和非共面PCB153作为目标污染物,分低剂量刺激、高剂量抑制两部分对双相剂量-效应关系的作用机制进行了研究。结果表明,无论是共平面结构的PCB 126还是非共面结构的PCB 153在10μg/ml的处理浓度下均表现出显著的细胞毒性。透射电镜及扫描电镜测试发现,PCBs处理会诱导细胞结构的显著变化,PCB 153处理细胞表现出凋亡的形态特征。激光共聚焦显微镜则从细胞核形态变化角度证实PCB153以凋亡形式诱导Vero细胞死亡。流式细胞术定量分析表明,与PCB 126相比,PCB 153能更显著的诱导Vero细胞凋亡。PCBs对细胞膜完整性的破坏是其诱导Vero细胞死亡的一个可能机制。PCBs处理引发的细胞线粒体功能损伤则可能是非共面PCB 153细胞毒性的一个重要途径。
低剂量刺激作用机制研究方面,主要从细胞周期分布变化及细胞信号转导通路激活等方面探讨PCBs诱导Vero细胞生长刺激的可能机制。低浓度PCBs通过诱导S期细胞比例的增加实现对Vero细胞的生长促进作用,高浓度PCBs处理导致的细胞G1期阻滞则是造成PCBs细胞毒性的原因之一。两种不同结构的PCB同系物在低剂量暴露诱导细胞增殖的作用机制上存在差异：共平面结构的PCB 126通过诱导细胞周期正性调控蛋白CDK2、Cyclin D1、Cyclin E表达增加、破坏细胞接触抑制效应实现对细胞增殖的促进。非共面结构的PCB 153则通过上调ERK1/2蛋白表达诱导Vero细胞增殖。
The most fundamental concept used in toxicology to determine risk assessment and regulation is the dose-response relationship, for which two models have traditionally been used. The threshold model is used in the assessment of risks for non-carcinogens, and the linear non-threshold (LNT) model to extrapolate risks to very low doses of carcinogens. But many reports have revealed that the most fundamental shape of the dose response curve is neither threshold nor linear, but U-shaped. The U-shaped biphasic dose-response relationship is commonly represented hormesis. Hormesis is a dose response phenomenon, characterizes the dose-response continuum as stimulatory at low doses and inhibitory at high doses, leading to the biphasic, hormetic dose-response curve. This stimulatory effect in low concentrations of toxic chemicals on organismal metabolism has been exhibited across the spectrum of models, endpoints, and agents. Hormesis rejects the standard toxicological assumption that effects at low doses can be extrapolated from data obtained from high doses, thus challenge the current risk assessment practices.
Bioassay is a useful tool in assessing environmental risk of harmful substances. During the past years, a variety of biological models such as bacteria, microalgae, cultured cells and other organisms were adapted to toxicological studies and made the toxicity testing reliable, sensitive, cost-effective, and rapid. In this study, we took luminescent bioassay and cell culture toxic test as testing models to examine whether the hormetic response is ubiquitous in testing chemicals. In luminescent bioassay, effects of Cu (Ⅱ), Zn (Ⅱ), Cd (Ⅱ) and Cr (Ⅵ) on marine and freshwater luminescent bacteria, named Photobacterium phosphorem and Vibrio qinghaiensis were studied, and biphasic dose-response curve was clearly observed. In cell culture test, the toxicity of zinc salts and PCBs were investigated in Vero cell, an established line of monkey kidney cell which is recommended for screening chemical toxicity in vitro. These chemicals showed a typical hormetic response, which stimulated Vero cell proliferation at low doses, and caused cell death at high concentrations. All our data demonstrated that hormesis response is a common phenomenon accurse in different tests with different chemicals.
Although hormetic effect is a common phenomenon, the cellular mechanisms of the hormetic dose-response are not well clarified yet. In this thesis, we took Vero cell as the testing model to study the possible mechanism of biphasic dose-response relationship. The morphological and biochemical changes in Vero cell cultures manifested clear evidence of PCBs toxicity. The difference between MTT results and cellular configuration alteration indicated that different cytotoxic mechanisms might operate in the induction of cell death between PCB 126 and PCB 153 treated cells. Apoptosis was involved in PCB 153 induced cell death and alteration of membrane integrity, damage of mitochondrial function might associated with the cytotoxicity induced by PCB 153 treatment.
The present data confirm that both coplanar PCB 126 and ortho-substituted PCB 153 presented low doses stimulation on Vero cells proliferation. The modulation of cell cycle with PCBs exposure might be one mechanism involved in low dose proliferation promotion. Low doses PCBs exposure significantly increased the percentage of cells in the S phase, which might associated with cell proliferation caused by low doses PCBs exposure. Meanwhile, the massive accumulation of cell in G1 phase might reflect an arrest of cells in G1 phase, which might response for the cytotoxicity of high concentration PCBs treatment. Our data showed the increase expression of CDK2, Cyclin D1 and Cyclin E caused by PCB 126 exposure, the cell proliferation promotion with coplanar PCB 126 exposure might ascribe to the release from contact inhibition caused by the AhR agonist characteristic of coplanar PCB congener. The capacity of ERK 1/2 signal transduction pathways activation was response for cell proliferation promotion in low dose PCB 153 exposure.
随着相关报道的逐年增加,双相剂量—效应关系逐步得到科学界认可,但其作用机制并不明确。本论文采用多种生物毒性测试方法对典型污染物诱导的双相剂量-效应关系进行了验证。重金属的发光菌生物毒性检测结果表明,铜、锌、镉、铬(Ⅵ)等4种重金属处理对淡水发光菌——青海弧菌(Vibrio qinghaiensis sp. Q67)和海洋发光菌——明亮发光杆菌(Photobacterium phosphoreum sp. T3)生长的影响均表现出倒“U”形双相剂量-效应关系。总体上讲,处理浓度小于1mg/l时,无毒常量重金属铜、锌对两种发光菌的影响表现为生长促进作用,10mg/l的处理浓度则在各个处理时间点均抑制发光菌生长。非必需有毒重金属镉、铬(Ⅵ)对两种发光菌的毒性抑制作用具有选择性,淡水发光菌Q67对镉处理较为敏感,铬(Ⅵ)对海洋发光菌T3的毒性抑制作用则更为显著。
Vero细胞离体毒性测试结果表明,无论重金属锌还是持久性有机污染物多氯联苯(Polychlorinated biphenyls, PCBs)对Vero细胞生长的影响均符合双相剂量-效应曲线的描述。处理浓度低于25mg/l时,重金属锌的3种化合物形式均能促进Vero细胞生长,但随着处理时间的延长,生长刺激效应会逐渐转变为抑制作用。处理浓度高于200mg/l时,锌盐显著抑制细胞生长,其生长率降至对照的50%以下。不同结构PCBs对Vero细胞生长的影响表明,低浓度PCBs(浓度小于0.01μg/ml)能刺激Vero细胞生长,刺激效应达到对照的120%左右。另外,非共面PCB 52、PCB 153对Vero细胞的毒性抑制作用分别大于共平面结构的PCB 77和PCB 126。
本论文采用Vero细胞离体毒性测试方法,以共平面PCB 126和非共面PCB153作为目标污染物,分低剂量刺激、高剂量抑制两部分对双相剂量-效应关系的作用机制进行了研究。结果表明,无论是共平面结构的PCB 126还是非共面结构的PCB 153在10μg/ml的处理浓度下均表现出显著的细胞毒性。透射电镜及扫描电镜测试发现,PCBs处理会诱导细胞结构的显著变化,PCB 153处理细胞表现出凋亡的形态特征。激光共聚焦显微镜则从细胞核形态变化角度证实PCB153以凋亡形式诱导Vero细胞死亡。流式细胞术定量分析表明,与PCB 126相比,PCB 153能更显著的诱导Vero细胞凋亡。PCBs对细胞膜完整性的破坏是其诱导Vero细胞死亡的一个可能机制。PCBs处理引发的细胞线粒体功能损伤则可能是非共面PCB 153细胞毒性的一个重要途径。
低剂量刺激作用机制研究方面,主要从细胞周期分布变化及细胞信号转导通路激活等方面探讨PCBs诱导Vero细胞生长刺激的可能机制。低浓度PCBs通过诱导S期细胞比例的增加实现对Vero细胞的生长促进作用,高浓度PCBs处理导致的细胞G1期阻滞则是造成PCBs细胞毒性的原因之一。两种不同结构的PCB同系物在低剂量暴露诱导细胞增殖的作用机制上存在差异：共平面结构的PCB 126通过诱导细胞周期正性调控蛋白CDK2、Cyclin D1、Cyclin E表达增加、破坏细胞接触抑制效应实现对细胞增殖的促进。非共面结构的PCB 153则通过上调ERK1/2蛋白表达诱导Vero细胞增殖。
The most fundamental concept used in toxicology to determine risk assessment and regulation is the dose-response relationship, for which two models have traditionally been used. The threshold model is used in the assessment of risks for non-carcinogens, and the linear non-threshold (LNT) model to extrapolate risks to very low doses of carcinogens. But many reports have revealed that the most fundamental shape of the dose response curve is neither threshold nor linear, but U-shaped. The U-shaped biphasic dose-response relationship is commonly represented hormesis. Hormesis is a dose response phenomenon, characterizes the dose-response continuum as stimulatory at low doses and inhibitory at high doses, leading to the biphasic, hormetic dose-response curve. This stimulatory effect in low concentrations of toxic chemicals on organismal metabolism has been exhibited across the spectrum of models, endpoints, and agents. Hormesis rejects the standard toxicological assumption that effects at low doses can be extrapolated from data obtained from high doses, thus challenge the current risk assessment practices.
Bioassay is a useful tool in assessing environmental risk of harmful substances. During the past years, a variety of biological models such as bacteria, microalgae, cultured cells and other organisms were adapted to toxicological studies and made the toxicity testing reliable, sensitive, cost-effective, and rapid. In this study, we took luminescent bioassay and cell culture toxic test as testing models to examine whether the hormetic response is ubiquitous in testing chemicals. In luminescent bioassay, effects of Cu (Ⅱ), Zn (Ⅱ), Cd (Ⅱ) and Cr (Ⅵ) on marine and freshwater luminescent bacteria, named Photobacterium phosphorem and Vibrio qinghaiensis were studied, and biphasic dose-response curve was clearly observed. In cell culture test, the toxicity of zinc salts and PCBs were investigated in Vero cell, an established line of monkey kidney cell which is recommended for screening chemical toxicity in vitro. These chemicals showed a typical hormetic response, which stimulated Vero cell proliferation at low doses, and caused cell death at high concentrations. All our data demonstrated that hormesis response is a common phenomenon accurse in different tests with different chemicals.
Although hormetic effect is a common phenomenon, the cellular mechanisms of the hormetic dose-response are not well clarified yet. In this thesis, we took Vero cell as the testing model to study the possible mechanism of biphasic dose-response relationship. The morphological and biochemical changes in Vero cell cultures manifested clear evidence of PCBs toxicity. The difference between MTT results and cellular configuration alteration indicated that different cytotoxic mechanisms might operate in the induction of cell death between PCB 126 and PCB 153 treated cells. Apoptosis was involved in PCB 153 induced cell death and alteration of membrane integrity, damage of mitochondrial function might associated with the cytotoxicity induced by PCB 153 treatment.
The present data confirm that both coplanar PCB 126 and ortho-substituted PCB 153 presented low doses stimulation on Vero cells proliferation. The modulation of cell cycle with PCBs exposure might be one mechanism involved in low dose proliferation promotion. Low doses PCBs exposure significantly increased the percentage of cells in the S phase, which might associated with cell proliferation caused by low doses PCBs exposure. Meanwhile, the massive accumulation of cell in G1 phase might reflect an arrest of cells in G1 phase, which might response for the cytotoxicity of high concentration PCBs treatment. Our data showed the increase expression of CDK2, Cyclin D1 and Cyclin E caused by PCB 126 exposure, the cell proliferation promotion with coplanar PCB 126 exposure might ascribe to the release from contact inhibition caused by the AhR agonist characteristic of coplanar PCB congener. The capacity of ERK 1/2 signal transduction pathways activation was response for cell proliferation promotion in low dose PCB 153 exposure.
引文
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