环境中苯基缩水甘油醚和多溴联苯醚的基因和细胞毒性初步研究
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摘要
本研究报道了两种广泛存在的环境有机污染物所引起的基因和细胞毒性。苯基缩水甘油醚(PGE)是一种常见的工业原料；多溴联苯醚((PBDEs)是一种阻燃剂，它们在环境中广泛存在而且具有生物累积效应，人类持续暴露在含有这些污染物的环境中对健康造成的影响还没有足够的研究。
首先，利用高效液相串联质谱(HPLC-MS／MS)表征了不同序列和不同碱基含量的寡核甘酸。在三种流动相(TEAA，TEAB，和HFIP)中利用HPLC-MS／MS表征了G富集，T富集，和A+T富集的寡核甘酸片段以及单核甘酸多态性(SNPs)片段。在TEAA和TEAB的流动相中，片段中G和T的含量显著影响它们的保留时间，而在HFIP的流动相中，保留时间与片段的长度有关。在TEAA和TEAB的流动相中，离子流的强度比其在HFIP的流动相中的响应值要低很多。具有自互补序列的A+T富集的寡核甘酸片段在TEAA和TEAB的流动相中的保留时间要明显少于没有自互补序列片段的保留时间，而在HFIP的流动相中二者没有明显的差别。在HFIP的流动相中没有自互补序列的片段的电荷分布状态要明显高于具有自互补序列的片段。另外，在本研究选取的寡核甘酸多态性的保留时间顺序为C＜G＜A＜T，多态性表现在3’端的时候更容易从质谱的离子碎片中加以区分。在以上研究的基础上，本研究进一步利用HPLC-MS／MS分别表征了苯基缩水甘油醚和寡核甘酸加合物。具有加合物的片段保留时间要比没有加合物的片段的长，利用于离子碎片信息确定了加合物的结构和其在不同片段中的位点，明确了加合物的形成与片段的序列有很大的关系。
其次，利用两种细胞系对羟基多溴联苯醚诱导的细胞毒性进行了研究。H4IIE是公认的检测环境含氯污染物及其相似物毒性的有效细胞。H295R细胞包含了与肾上腺素合成相关的细胞通路，非常适合研究环境有机污染物的毒性反应。单细胞电泳可以检测基因断裂和修复过程。在本研究中，羟基多溴联苯醚没有明显的基因损伤。为了进一步研究此类化合物对其它细胞毒性的影响，我们研究了羟基多溴联苯醚对细胞存活率，细胞分裂和细胞生长周期分布的影响。结果表明，羟基多溴联苯醚可以导致细胞死亡，抑制细胞分裂，对细胞周期合成有抑制作用。2’OH-BDE85比2’OH-BDE47具有更高的毒性。
根据以上研究，最后本研究利用实时定量PCR检测了25种多溴联苯醚以及衍生物对肾上腺素基因表达的影响。某些化合物可以对的的基因表达有显著影响。利用基因芯片检测了2种羟基多溴联苯醚对整个基因组的基因表达影响。在本研究中，与2’OH-BDE47相比，2’OH-BDE85能引起更多的基因表达变化异常。这与细胞毒性实验结果一致。实时定量PCR验证了基因芯片数据，证明基因芯片关于基因表达变化的数据可信。
因为没有发现明显的基因毒性，本研究指出多溴联苯醚的生物影响是通过后生效应而不是通过基因毒性的路径来发生作用的。如何规避风险需进一步研究化合物的暴露风险。在学科交叉的基础上，本论文对环境污染物与健康的关系进行的探索，为更好的了解环境有机毒害物质的基凶毒理和细胞毒性影响机制具有重要意义。
This research investigates the potential gene and cellular toxicology of two key environmental organic pollutants, phenyl glycidyl ether (PGE) and polybrominated diphenyl ethers (PBDEs). PGE is a material used ubiquitously wild world in the synthesis of plastics; PBDEs are hydrophobic and persistent additive flame retardants that seemingly transfer into environmental compartments where they bioaccumulate i.e. in human biota. Humans are therefore exposed continuously to these agents; the risks of this continual exposure being unknown.
In the initial research, a HPLC-MS/MS method was developed to firstly separate oligonucleotides of differing sequences and the developed method then used to investigate PGE adduct formation in specific oligonucleotides. HPLC was used to indicate PGE adduct formation and to separate the isomeric adducted oligo-products. On-line MS/MS analysis was then used to identify exactly the site of adduct formation at base resolution. The location of the adduct differed for the different oligonucleotides studied, possibly due to secondary structure formation which is known to affect adduct formation.
Next, an investigation into the possible genotoxic effects of PBDEs was undertaken using a pair of cell lines (H4IIE, rat hepatoma cells and H295R, human adrenocortical carcinoma cells), using two versions of the Comet assay to assess single strand breaks and 'total-repairable' DNA damage. However, after exhaustive study, there was no evidence of any of the PBDE's exhibiting significant genotoxicity. Other cellular endpoints were then studied such as cell viability, proliferation and cell cycle arrest. Studies of cell viability and proliferation indicated the PBDEs to possess a range of effects, whilst cell cycle analysis indicated the more potent of these agents have an effect on cell cycle distribution by initiating temporary cell cycle arrest, primarily in the S phase.
These studies were then followed-up by investigations of steroidogenesis and genome-wide gene expression changes by BPDEs and their derivatives. QRT-PCR analysis indicated that parent BPDEs, MeO-BDEs and OH-BDEsare able to dramatically alter steroidogenic gene expression, with the OH-moiety of the hydroxylated derivatives seeming to have a great effect. 'Microarray analysis' of genome-wide gene expression using two of the more potent OH-PBDEs (8A and 13A) indicated 13A to mediate more changes than 8A. 13A prominently altered nuclear acid metabolism genes whilst 8A altered amino acid metabolism genes. 13A changed more genes related to cell cycle and apoptosis than 8A. This is consistent with the results of the proliferation studies. Both chemicals can affect the gene expression of transcription pathways. There is no obvious concentration-dependent changes in neither 8A nor 13A induced expression upon 24 hours treatment in the present study. The Microarray data was validated, and the further PBDE concentrations tested, by real time QRT-PCR.
As there was no evidence of BPDE genotoxicity apparent (Comet assay), we conclude that the biological effects of PBDEs are primarily via epigenetic routes and not via genotoxic routes. These should be further studied to more fully establish the risk of exposure and to seek possible preventative strategies.
首先，利用高效液相串联质谱(HPLC-MS／MS)表征了不同序列和不同碱基含量的寡核甘酸。在三种流动相(TEAA，TEAB，和HFIP)中利用HPLC-MS／MS表征了G富集，T富集，和A+T富集的寡核甘酸片段以及单核甘酸多态性(SNPs)片段。在TEAA和TEAB的流动相中，片段中G和T的含量显著影响它们的保留时间，而在HFIP的流动相中，保留时间与片段的长度有关。在TEAA和TEAB的流动相中，离子流的强度比其在HFIP的流动相中的响应值要低很多。具有自互补序列的A+T富集的寡核甘酸片段在TEAA和TEAB的流动相中的保留时间要明显少于没有自互补序列片段的保留时间，而在HFIP的流动相中二者没有明显的差别。在HFIP的流动相中没有自互补序列的片段的电荷分布状态要明显高于具有自互补序列的片段。另外，在本研究选取的寡核甘酸多态性的保留时间顺序为C＜G＜A＜T，多态性表现在3’端的时候更容易从质谱的离子碎片中加以区分。在以上研究的基础上，本研究进一步利用HPLC-MS／MS分别表征了苯基缩水甘油醚和寡核甘酸加合物。具有加合物的片段保留时间要比没有加合物的片段的长，利用于离子碎片信息确定了加合物的结构和其在不同片段中的位点，明确了加合物的形成与片段的序列有很大的关系。
其次，利用两种细胞系对羟基多溴联苯醚诱导的细胞毒性进行了研究。H4IIE是公认的检测环境含氯污染物及其相似物毒性的有效细胞。H295R细胞包含了与肾上腺素合成相关的细胞通路，非常适合研究环境有机污染物的毒性反应。单细胞电泳可以检测基因断裂和修复过程。在本研究中，羟基多溴联苯醚没有明显的基因损伤。为了进一步研究此类化合物对其它细胞毒性的影响，我们研究了羟基多溴联苯醚对细胞存活率，细胞分裂和细胞生长周期分布的影响。结果表明，羟基多溴联苯醚可以导致细胞死亡，抑制细胞分裂，对细胞周期合成有抑制作用。2’OH-BDE85比2’OH-BDE47具有更高的毒性。
根据以上研究，最后本研究利用实时定量PCR检测了25种多溴联苯醚以及衍生物对肾上腺素基因表达的影响。某些化合物可以对的的基因表达有显著影响。利用基因芯片检测了2种羟基多溴联苯醚对整个基因组的基因表达影响。在本研究中，与2’OH-BDE47相比，2’OH-BDE85能引起更多的基因表达变化异常。这与细胞毒性实验结果一致。实时定量PCR验证了基因芯片数据，证明基因芯片关于基因表达变化的数据可信。
因为没有发现明显的基因毒性，本研究指出多溴联苯醚的生物影响是通过后生效应而不是通过基因毒性的路径来发生作用的。如何规避风险需进一步研究化合物的暴露风险。在学科交叉的基础上，本论文对环境污染物与健康的关系进行的探索，为更好的了解环境有机毒害物质的基凶毒理和细胞毒性影响机制具有重要意义。
This research investigates the potential gene and cellular toxicology of two key environmental organic pollutants, phenyl glycidyl ether (PGE) and polybrominated diphenyl ethers (PBDEs). PGE is a material used ubiquitously wild world in the synthesis of plastics; PBDEs are hydrophobic and persistent additive flame retardants that seemingly transfer into environmental compartments where they bioaccumulate i.e. in human biota. Humans are therefore exposed continuously to these agents; the risks of this continual exposure being unknown.
In the initial research, a HPLC-MS/MS method was developed to firstly separate oligonucleotides of differing sequences and the developed method then used to investigate PGE adduct formation in specific oligonucleotides. HPLC was used to indicate PGE adduct formation and to separate the isomeric adducted oligo-products. On-line MS/MS analysis was then used to identify exactly the site of adduct formation at base resolution. The location of the adduct differed for the different oligonucleotides studied, possibly due to secondary structure formation which is known to affect adduct formation.
Next, an investigation into the possible genotoxic effects of PBDEs was undertaken using a pair of cell lines (H4IIE, rat hepatoma cells and H295R, human adrenocortical carcinoma cells), using two versions of the Comet assay to assess single strand breaks and 'total-repairable' DNA damage. However, after exhaustive study, there was no evidence of any of the PBDE's exhibiting significant genotoxicity. Other cellular endpoints were then studied such as cell viability, proliferation and cell cycle arrest. Studies of cell viability and proliferation indicated the PBDEs to possess a range of effects, whilst cell cycle analysis indicated the more potent of these agents have an effect on cell cycle distribution by initiating temporary cell cycle arrest, primarily in the S phase.
These studies were then followed-up by investigations of steroidogenesis and genome-wide gene expression changes by BPDEs and their derivatives. QRT-PCR analysis indicated that parent BPDEs, MeO-BDEs and OH-BDEsare able to dramatically alter steroidogenic gene expression, with the OH-moiety of the hydroxylated derivatives seeming to have a great effect. 'Microarray analysis' of genome-wide gene expression using two of the more potent OH-PBDEs (8A and 13A) indicated 13A to mediate more changes than 8A. 13A prominently altered nuclear acid metabolism genes whilst 8A altered amino acid metabolism genes. 13A changed more genes related to cell cycle and apoptosis than 8A. This is consistent with the results of the proliferation studies. Both chemicals can affect the gene expression of transcription pathways. There is no obvious concentration-dependent changes in neither 8A nor 13A induced expression upon 24 hours treatment in the present study. The Microarray data was validated, and the further PBDE concentrations tested, by real time QRT-PCR.
As there was no evidence of BPDE genotoxicity apparent (Comet assay), we conclude that the biological effects of PBDEs are primarily via epigenetic routes and not via genotoxic routes. These should be further studied to more fully establish the risk of exposure and to seek possible preventative strategies.
引文
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