栉孔扇贝对苯并芘（Benzo［a］pyrene）生物标志物筛选技术的初步研究
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摘要
多环芳烃化合物(Polycyclic Aromatic Hydrocarbons,简称PAHs)是自然环境中持久性有机污染物的主要代表,具有致癌、致畸和致突变作用,随着煤、石油在工业生产、交通运输以及生活中被广泛应用,海上PAHs的污染日益加重。本论文以栉孔扇贝为研究对象,根据生物标志物筛选和评价原则,全面系统的研究了B[α]P对栉孔扇贝解毒代谢酶、DNA和蛋白质的影响,阐明了B[α]P在栉孔扇贝体内的毒性效应过程。采用统计学方法对所选指标进行了剂量效应和相关性分析,并将在实验室研究中筛选出的标志物应用于青岛近海环境中PAHs的监测。所得结果如下:
1 B[α]P对栉孔扇贝组织解毒代谢指标的影响
本文研究了苯并(α)芘(B[α]P)对栉孔扇贝(Chlamys farreri)鳃丝和消化盲囊毒理学指标(包括AHH、GST、GSH和MDA)的影响,结果显示B[α]P对扇贝毒理学指标影响显著(P＜0.05),鳃丝和消化盲囊中各指标具有相似的变化规律:在为期15d的B[α]P一次染毒实验过程中,AHH活力和MDA含量被诱导升高,被诱导程度与曝污浓度呈现正相关,其中鳃丝被诱导的程度较消化盲囊要大,低浓度处理组GST活力和GSH含量先表现为被诱导升高,之后被抑制并低于对照组水平,高浓度处理组则表现为直接被抑制,被抑制程度与B[α]P浓度相关;当污染消除后,栉孔扇贝表现出良好的自我调节能力,各毒理指标较快地恢复到对照组水平,其中低浓度处理组的恢复速度较高浓度要快,消化盲囊的氧化损伤的修复能力较鳃丝强;经过为期30d的恢复实验后,立即进行二次染毒实验,除高浓度处理组表现为氧化损伤的加剧外,其它标志物的变化趋势与一次染毒实验时基本一致,表现出很好的重现性。本实验表明,栉孔扇贝鳃丝和消化盲囊毒理学指标(AHH、GST、GSH和MDA)能指示B[α]P污染程度,并具有较强的敏感性和良好的重现性,可以作为B[α]P污染的氧化压力生物标志物。
2 B[α]P对栉孔扇贝组织DNA和蛋白质损伤效应的研究
在B[α]P胁迫下研究了栉孔扇贝(Chlamys farreri)鳃丝和消化盲囊DNA单链断裂(分别用碱解旋分析法和单细胞凝胶电泳实验测定)、DNA-蛋白质交联(DPC)和蛋白质羰基化含量的影响。结果显示,B[α]P对栉孔扇贝DNA和蛋白质损伤的诱导显著,且表现出明显的时间效应规律(P＜0.05)。在一次染毒阶段DNA单链断裂水平1d内被显著诱导,之后随着B[α]P在生物体内的不断累积,激活了体内DNA修复系统,是DNA单链断裂水平有所下降。最高浓度组由于浓度过高抑制了体内的修复系统,造成了不可逆的损伤,从而使扇贝具有了一定的本底水平。清除阶段,各处理组均显示了明显的恢复过程。二次染毒阶段处理组DNA单链断裂水平与一次染毒阶段类似,表现出很好的重现性。DPC与蛋白质羰基化含量变化在一次染毒过程中未表现出明显的修复过程,可见蛋白质损伤较之酶和DNA损伤更难恢复。清除阶段各处理组DPC和蛋白质羰基含量恢复较慢,且只有低浓度组恢复至对照组水平。二次染毒阶段与一次染毒阶段变化类似,但稳定后DPC水平和羰基含量均高于一次染毒阶段。总之,与单细胞凝胶电泳实验相比,碱解旋分析法更适于测定DNA单链断裂水平的变化,蛋白质损伤具有作为PAHs污染生物标志物的潜质。
3栉孔扇贝对B[α]P生物标志物筛选技术的研究
本文根据生物标志物的筛选和评价原则,运用统计学方法研究了B[α]P对栉孔扇贝鳃丝和消化盲囊解毒代谢酶和损伤效应指标的剂量效应、一次染毒和二次染毒间指标的相关性以及所有指标间的相关性。结果显示,B[α]P胁迫下1d和15d栉孔扇贝鳃丝和消化盲囊解毒代谢酶和损伤效应指标的变化具有明显的线性剂量效应关系(P＜0.05);除了GSH和MDA其他各指标一次染毒和二次染毒间都变现为极显著性相关(P＜0.05);运用Pearson相关系数对所有指标进行回归分析发现,各指标均表现为极显著性相关,并且酶指标之间、DNA和蛋白质损伤指标之间的相关性要大于二者间的相关性。因此,AHH活力、GST活力、DNA单链断裂、DNA-蛋白质交联和蛋白质羰基含量均符合生物标志物评价的前三条原则,将用于下一章野外实验的进一步验证。
4青岛近海海水中B[α]P含量与栉孔扇贝生物标志物的现场验证与评价
本章在青岛近海选择了三个位点(黄岛红石崖、太平角和8号码头)采取海水和栉孔扇贝样品,测定了海水中B[α]P含量以及栉孔扇贝鳃丝和消化盲囊AHH活力、GST活力、DNA单链断裂、DNA-蛋白质交联和蛋白质羰基含量的变化。结果显示,海水中B[α]P含量大小为:红石崖〈太平角〈8号码头;AHH活力和DPC在红石崖和太平角两个位点无显著差异(P＞0.05)。GST活力、DNA单链断裂和蛋白质羰基含量都很好的反映了海水中B[α]P的含量,且8号码头损伤最为严重。8号码头在两次取样间指标差异显著(P＜0.05),我们最终确定DNA单链断裂水平和蛋白质羰基含量作为基于双壳贝类海洋PAHs污染监测的生物标志物,为海洋生态保护和人类健康提供参考。
Polycyclic Aromatic Hydrocarbons are carcinogenic,teratogenic and mutagenic. They are the typical persisitent organic pollutants in nature.Because of development of maritime petroleum and marine traffic and industry sewerage discharge,polycyclic aromatic hydrocarbon(PAHs) pollution became seriously.Author studied the influence of B[α]P on the process of detoxifcation and metabolism,DNA and protein of Chlamys farreri.Then we adoped the statistical method to analyze the all the index we chosed,and applied them to the offing of Qingdao.The following results were obtained:
1.The toxicological effect of PAHs on the tissue of C.farreri.
We studied the index in gill and digestive gland of C.farreri exposed to B[α]P: inclued AHH,GST,GSH and MDA.The results indicate that index in all treatment groups were induced,and there's a time-course dependent character.In the first exposure period,AHH and MDA were induced significantly and showed a direct correlation with the concentration of B[α]P.The GST and GSH of the lower concentration group were elevated first and restrained to be the control level.While the higher concentration group were restrained at first.In the elimination period,the index in all the group resumed to the control level,and the lower concentration groups resumed quickly than the higher concentration groups.Also the digestive gland had a better resume capacity than gill of C.farreri.The index in the second exposed period showed a similar change with the first exposure period.
2.The toxicological effect of PAHs on DNA and protein in gills and digestive glands of C.farreri.
In this study,the DNA single strand breakage used two methods(Alkaline Unwinding Assay and Comet Assay),DNA-protin crosslink(DPC) and protein carbonyl content in gill and digestive gland of C.farreri exposed to B[α]P were exzamined.We found that all the index showed a significant time- course dependent character.In the firsr exposed period,DNA single strand breakage increased first and becomed restored because of the repair systems initiated,but the repair systems of the highest concentration group were restrained.While the DNA-protin crosslink and protein carbonyl content did not show any repair progress.In the elimination period, DNA-protin crosslink and protein carbonyl content recoverd slower than DNA single strand breaks.Only the lower concentration group recoverd to the control level.In the second exposure period,the index showed similar changes with the first exosure period.And the DNA-protin crosslink and protein carbonyl content were higher than the first exposed period.So contrast with the comet assay,alkaline unwinding assay was more suitable to assess the PAHs pollution.The damage to protein was harder to recover than enzyme and DNA,it could be a potential biomarker of PAHs pollution.
3.Study on the biomarkers identification technology of B[α]P based on C.farreri.
In this study,we used the statistic methods analized the dose-course relationship, correlation of two exposed period and the Pearson correlation coefficient of all the index.Results showed all the index were dose-dependent with the concentration of B[α]P.Except the GSH and MDA,other index showed a linearity relation between two exposed period.And the Pearson coefficient among all the index showed a extremely correlation.The Pearson coefficient among all the enzyme and the Pearson coefficient among DNA and protein damage were higher than it between enzyme and DNA,protein damage.Therefor,we chose AHH activity,GST activity,DNA single strand breaks,DNA-Protein crosslink and protein carbonyl content to be applied in the offing of Qingdao.
4.The wild application and Assessment of B[α]P biomarkers pollution in Chlamys farreri and B[α]P content in seawater from offing of Qingdao
Sub-surface seawater and scallop(Chlamys farreri) samples at 3 sites(include Honsshiya,Taipjiao and NO.8 dock) in the offing of the Qingdao in Apr.30 and Jul. 30 of 2007,Benzo[α]Pyrene(B[α]P) content of seawater and toxicological index of gill and digestive gland of were analyzed.Result indicated the B[α]P in seawater was NO.8 dock＞Taipjiao＞Honsshiya,in indicidual site April＜July.AHH activity and DPC between Taipjiao and Honsshiya both showed no difference(P＞0.05).GST activity, DNA single strand breaks and protein carbonyl content indicated well corellation with the B[α]P in seawater.Well the mussels in NO.8 dock damaged most among the three sample sites.So,we chose DNA single strand breaks and protein carbonyl content as the biomarkers of PAHs pollution in marine environment,based on bivalve mussel.It is very important for security of aquatic food,protection of halobios and people health.
1 B[α]P对栉孔扇贝组织解毒代谢指标的影响
本文研究了苯并(α)芘(B[α]P)对栉孔扇贝(Chlamys farreri)鳃丝和消化盲囊毒理学指标(包括AHH、GST、GSH和MDA)的影响,结果显示B[α]P对扇贝毒理学指标影响显著(P＜0.05),鳃丝和消化盲囊中各指标具有相似的变化规律:在为期15d的B[α]P一次染毒实验过程中,AHH活力和MDA含量被诱导升高,被诱导程度与曝污浓度呈现正相关,其中鳃丝被诱导的程度较消化盲囊要大,低浓度处理组GST活力和GSH含量先表现为被诱导升高,之后被抑制并低于对照组水平,高浓度处理组则表现为直接被抑制,被抑制程度与B[α]P浓度相关;当污染消除后,栉孔扇贝表现出良好的自我调节能力,各毒理指标较快地恢复到对照组水平,其中低浓度处理组的恢复速度较高浓度要快,消化盲囊的氧化损伤的修复能力较鳃丝强;经过为期30d的恢复实验后,立即进行二次染毒实验,除高浓度处理组表现为氧化损伤的加剧外,其它标志物的变化趋势与一次染毒实验时基本一致,表现出很好的重现性。本实验表明,栉孔扇贝鳃丝和消化盲囊毒理学指标(AHH、GST、GSH和MDA)能指示B[α]P污染程度,并具有较强的敏感性和良好的重现性,可以作为B[α]P污染的氧化压力生物标志物。
2 B[α]P对栉孔扇贝组织DNA和蛋白质损伤效应的研究
在B[α]P胁迫下研究了栉孔扇贝(Chlamys farreri)鳃丝和消化盲囊DNA单链断裂(分别用碱解旋分析法和单细胞凝胶电泳实验测定)、DNA-蛋白质交联(DPC)和蛋白质羰基化含量的影响。结果显示,B[α]P对栉孔扇贝DNA和蛋白质损伤的诱导显著,且表现出明显的时间效应规律(P＜0.05)。在一次染毒阶段DNA单链断裂水平1d内被显著诱导,之后随着B[α]P在生物体内的不断累积,激活了体内DNA修复系统,是DNA单链断裂水平有所下降。最高浓度组由于浓度过高抑制了体内的修复系统,造成了不可逆的损伤,从而使扇贝具有了一定的本底水平。清除阶段,各处理组均显示了明显的恢复过程。二次染毒阶段处理组DNA单链断裂水平与一次染毒阶段类似,表现出很好的重现性。DPC与蛋白质羰基化含量变化在一次染毒过程中未表现出明显的修复过程,可见蛋白质损伤较之酶和DNA损伤更难恢复。清除阶段各处理组DPC和蛋白质羰基含量恢复较慢,且只有低浓度组恢复至对照组水平。二次染毒阶段与一次染毒阶段变化类似,但稳定后DPC水平和羰基含量均高于一次染毒阶段。总之,与单细胞凝胶电泳实验相比,碱解旋分析法更适于测定DNA单链断裂水平的变化,蛋白质损伤具有作为PAHs污染生物标志物的潜质。
3栉孔扇贝对B[α]P生物标志物筛选技术的研究
本文根据生物标志物的筛选和评价原则,运用统计学方法研究了B[α]P对栉孔扇贝鳃丝和消化盲囊解毒代谢酶和损伤效应指标的剂量效应、一次染毒和二次染毒间指标的相关性以及所有指标间的相关性。结果显示,B[α]P胁迫下1d和15d栉孔扇贝鳃丝和消化盲囊解毒代谢酶和损伤效应指标的变化具有明显的线性剂量效应关系(P＜0.05);除了GSH和MDA其他各指标一次染毒和二次染毒间都变现为极显著性相关(P＜0.05);运用Pearson相关系数对所有指标进行回归分析发现,各指标均表现为极显著性相关,并且酶指标之间、DNA和蛋白质损伤指标之间的相关性要大于二者间的相关性。因此,AHH活力、GST活力、DNA单链断裂、DNA-蛋白质交联和蛋白质羰基含量均符合生物标志物评价的前三条原则,将用于下一章野外实验的进一步验证。
4青岛近海海水中B[α]P含量与栉孔扇贝生物标志物的现场验证与评价
本章在青岛近海选择了三个位点(黄岛红石崖、太平角和8号码头)采取海水和栉孔扇贝样品,测定了海水中B[α]P含量以及栉孔扇贝鳃丝和消化盲囊AHH活力、GST活力、DNA单链断裂、DNA-蛋白质交联和蛋白质羰基含量的变化。结果显示,海水中B[α]P含量大小为:红石崖〈太平角〈8号码头;AHH活力和DPC在红石崖和太平角两个位点无显著差异(P＞0.05)。GST活力、DNA单链断裂和蛋白质羰基含量都很好的反映了海水中B[α]P的含量,且8号码头损伤最为严重。8号码头在两次取样间指标差异显著(P＜0.05),我们最终确定DNA单链断裂水平和蛋白质羰基含量作为基于双壳贝类海洋PAHs污染监测的生物标志物,为海洋生态保护和人类健康提供参考。
Polycyclic Aromatic Hydrocarbons are carcinogenic,teratogenic and mutagenic. They are the typical persisitent organic pollutants in nature.Because of development of maritime petroleum and marine traffic and industry sewerage discharge,polycyclic aromatic hydrocarbon(PAHs) pollution became seriously.Author studied the influence of B[α]P on the process of detoxifcation and metabolism,DNA and protein of Chlamys farreri.Then we adoped the statistical method to analyze the all the index we chosed,and applied them to the offing of Qingdao.The following results were obtained:
1.The toxicological effect of PAHs on the tissue of C.farreri.
We studied the index in gill and digestive gland of C.farreri exposed to B[α]P: inclued AHH,GST,GSH and MDA.The results indicate that index in all treatment groups were induced,and there's a time-course dependent character.In the first exposure period,AHH and MDA were induced significantly and showed a direct correlation with the concentration of B[α]P.The GST and GSH of the lower concentration group were elevated first and restrained to be the control level.While the higher concentration group were restrained at first.In the elimination period,the index in all the group resumed to the control level,and the lower concentration groups resumed quickly than the higher concentration groups.Also the digestive gland had a better resume capacity than gill of C.farreri.The index in the second exposed period showed a similar change with the first exposure period.
2.The toxicological effect of PAHs on DNA and protein in gills and digestive glands of C.farreri.
In this study,the DNA single strand breakage used two methods(Alkaline Unwinding Assay and Comet Assay),DNA-protin crosslink(DPC) and protein carbonyl content in gill and digestive gland of C.farreri exposed to B[α]P were exzamined.We found that all the index showed a significant time- course dependent character.In the firsr exposed period,DNA single strand breakage increased first and becomed restored because of the repair systems initiated,but the repair systems of the highest concentration group were restrained.While the DNA-protin crosslink and protein carbonyl content did not show any repair progress.In the elimination period, DNA-protin crosslink and protein carbonyl content recoverd slower than DNA single strand breaks.Only the lower concentration group recoverd to the control level.In the second exposure period,the index showed similar changes with the first exosure period.And the DNA-protin crosslink and protein carbonyl content were higher than the first exposed period.So contrast with the comet assay,alkaline unwinding assay was more suitable to assess the PAHs pollution.The damage to protein was harder to recover than enzyme and DNA,it could be a potential biomarker of PAHs pollution.
3.Study on the biomarkers identification technology of B[α]P based on C.farreri.
In this study,we used the statistic methods analized the dose-course relationship, correlation of two exposed period and the Pearson correlation coefficient of all the index.Results showed all the index were dose-dependent with the concentration of B[α]P.Except the GSH and MDA,other index showed a linearity relation between two exposed period.And the Pearson coefficient among all the index showed a extremely correlation.The Pearson coefficient among all the enzyme and the Pearson coefficient among DNA and protein damage were higher than it between enzyme and DNA,protein damage.Therefor,we chose AHH activity,GST activity,DNA single strand breaks,DNA-Protein crosslink and protein carbonyl content to be applied in the offing of Qingdao.
4.The wild application and Assessment of B[α]P biomarkers pollution in Chlamys farreri and B[α]P content in seawater from offing of Qingdao
Sub-surface seawater and scallop(Chlamys farreri) samples at 3 sites(include Honsshiya,Taipjiao and NO.8 dock) in the offing of the Qingdao in Apr.30 and Jul. 30 of 2007,Benzo[α]Pyrene(B[α]P) content of seawater and toxicological index of gill and digestive gland of were analyzed.Result indicated the B[α]P in seawater was NO.8 dock＞Taipjiao＞Honsshiya,in indicidual site April＜July.AHH activity and DPC between Taipjiao and Honsshiya both showed no difference(P＞0.05).GST activity, DNA single strand breaks and protein carbonyl content indicated well corellation with the B[α]P in seawater.Well the mussels in NO.8 dock damaged most among the three sample sites.So,we chose DNA single strand breaks and protein carbonyl content as the biomarkers of PAHs pollution in marine environment,based on bivalve mussel.It is very important for security of aquatic food,protection of halobios and people health.
引文
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