苯并（a）芘、三丁基锡及其混合物对褐菖鲉的毒性效应
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摘要
本文以海洋鱼类褐菖鲉为实验动物,研究海洋环境中两种典型有机污染物多环芳烃和有机锡对其产生的毒性效应。采用的苯并(a)芘(BaP)浓度为10、100、1000 ng/L,三丁基锡(TBT)浓度为1、10、100 ng/L,联合组浓度为5 ng/L BaP+0.5ng/L TBT、50 ng/L BaP+5 ng/L TBT、500 ng/L BaP+50 ng/L TBT,对鱼体进行水体暴露,分别于暴露后0 d、7 d、25 d、50 d和污染解除后7 d、20 d取样。从生理、生化与分子生物学等层次分析了BaP和TBT在鱼体内的代谢,对解毒酶系统的影响,对鱼类造成的氧化损伤与DNA损伤的单独作用和联合作用效应,进而对这些生物标志物在监测海洋环境污染中的有效性进行评价。主要研究结果
如下:
1.胆汁中BaP典型代谢产物浓度随着暴露浓度的增加而增加,能够较好地反映环境中BaP的暴露情况,但是要结合EROD和GST活性情况综合考虑其时间-效应关系。鱼胆汁中的总Sn~(2+)含量作为监测外界TBT污染程度的生物标志物也有可行性,可以指示出高浓度的有机锡污染,而且从联合作用结果看,BaP促进了TBT的代谢排出。
2.环境浓度的BaP和TBT对解毒酶系统造成了显著的影响。Western blot结果表明CYP1A蛋白含量随着BaP暴露浓度的增加而增加,可以作为监测环境中PAHs污染的良好的生物标志物。Ⅰ相解毒酶EROD活性与BaP、TBT及其混合物污染之间存在较为复杂的时间-效应与剂量-效应关系,而且在暴露25 d后,联合作用表现为拮抗作用,仅仅以EROD酶活性指标衡量环境中BaP、TBT或者二者的联合污染水平会出现失真现象。所以,将EROD活性与CYP1A蛋白水平或者BaP典型代谢产物水平,或者胆汁中总锡含量等指标结合起来考虑才能较为客观地认识环境中这两类污染物的污染程度。ECOD活性在BaP、TBT或者二者的联合作用下主要表现出被诱导效应,也能反映出高浓度的BaP和TBT联合污染情况,可以作为高污染环境中的生物标志物参考指标。Ⅱ相解毒酶UDPGT在暴露中期对两种污染物均未表现出显著的效应关系。
3.BaP和TBT对抗氧化体系指标产生了复杂的联合作用结果,主要表现为拮抗作用。SOD、GST活性与GSH含量在BaP暴露50 d后都表现出被诱导效应,将三者结合起来作为监测海洋环境PAHs污染的生物标志物有一定的可行性。TBT暴露50 d后会增加MDA含量,诱导SOD、GPx酶活性,并且降低GSH含量,将四者结合起来能较好地反映有机锡污染。BaP和TBT联合作用50 d后会增加MDA含量,并且诱导GPx、GST酶活性,因此将三者结合起来用于监测海洋环境中PAHs和有机锡的联合污染有一定的可行性。
4.DNA单链断裂损伤随着BaP、TBT及其混合物的暴露浓度增加和暴露时间的延长而加重,可以作为指示水体BaP、TBT或者二者的联合污染的潜在生物标志物。同时,从暴露25 d和50 d后的结果看,联合作用减轻了DNA损伤,这与TBT抑制BaP的代谢活化和BaP促进TBT的代谢排出的结果相一致。
根据以上结果,我们认为环境水平浓度的BaP和TBT所产生的联合毒理学效应有复杂的时间-效应和剂量-效应关系,主要是拮抗作用,表现为TBT对BaP代谢的抑制,BaP对于TBT的代谢排出起到了促进作用。
Marine fish Sebastiscus marmoratus was adopted as experimental animal. Toxicological effects were studied on two kinds of typical organic contaminants in marine environments, Polycyclic aromatic hydrocarbons (PAHs) and organotins. Sebastiscus marmoratus exposed to BaP (10, 100,1000 ng/L), TBT (1, 10, 100 ng/L) and their mixture (5 ng/L BaP+0.5 ng/L TBT, 50 ng/L BaP+5 ng/L TBT, 500 ng/L BaP+50 ng/L TBT) were randomly sampled after exposure for 0, 7, 25, 50 days and transferred to clean water to recover for 7 and 20 days, respectively. The physiological, biochemical and molecular parameters were analyzed to assess the effects of BaP, TBT and their mixture to metabolism, hepatic detoxification enzyme system, antioxidant system and DNA damage. Furthermore, the availability of these biomarkers to monitor marine pollution by BaP, TBT and their mixture were assessed. The main results as follows:
1. Levels of BaP metabolites in the bile were increased in a dose-dependent manner in the fish exposed through water to BaP, and reflected contamination gradients of PAHs. However, EROD and GST activities should be taken into account to assess time-effect relation. Total biliary Sn~(2+) content in fish is feasible to serve as an indicator to monitor severe organotin pollution, and as far as combined effects are concerned, BaP increased the excretion of TBT to bile.
2. Environmentally relevant concentrations of BaP and TBT influenced hepatic detoxification enzyme system significantly. CYPIA level inceased in harmony with BaP concentration with Western blot analysis, so CYPIA level is feasible to serve as a good biomarker to monitor PAHs pollution. EROD and ECOD are both phase I enzymes. There were complicated relations of time-effect and dose-effect between EROD activity and exposure of BaP, TBT or their mixture. Antagonistic interactions occurred after 25 days exposure to the mixure of BaP and TBT. It is fuzzy to merely use EROD activity as biomarker to assess PAHs or TBT pollution. Therefore, it is vital to assemble CYPIA level or BaP-type metabolites to assess PAHs pollution and integrate other biomarkers (e.g. total biliary Sn~(2+)content) to assess TBT pollution in marine environment. ECOD activity was induced by BaP, TBT or their mixture at high concentrations after 50 days exposure, and it is advisable to use ECOD activity as a biomarker to assess severe PAHs or TBT pollution. UDPGT was analyzed as phase II enzyme. There were scarcely significant effects of BaP, TBT or their mixture to UDPGT activity after 25 d exposure.
3. Combined effects of BaP and TBT to antioxidant system were complicated, and antagonistic interactions were dominating. SOD, GST activities were induced and GSH content were increased after 50 days exposure to BaP, and it is feasible to integrate them into PAHs pollution monitoring program. MDA content was increased and SOD, GPx activities were induced, and GSH content was decreased after 50 days exposure to TBT, so it is feasible to integrate them into TBT pollution monitoring program. In the same way, MDA content was increased and GST, GPx activities were induced after 50 days exposure to the mixture of BaP and TBT, so it is feasible to integrate them into PAHs with the addition of TBT pollution monitoring program in marine environment.
4. DNA single strand break damage was aggravated along with the increase of exposure concentration and the elongation of exposure time, so DNA single strand break damage is feasible to serve as a potential biomarker to monitor the pollution of PAHs, TBT or their mixture, because good relations of time-effect and dose-effect existed. As far as the results after 25 days and 50 days exposure are concerned, combination of BaP and TBT alleviated the degree of DNA damage which was consistent with the inhibition of BaP metabolism by TBT and the promotion of TBT excretion by BaP.
Based on the results we obtained, we conclude that combined toxicological effects of BaP and TBT at environmentally relevant concentrations were complicated, and antagonistic interactions were dominating. TBT inhibited BaP metabolism, and BaP facilitated TBT metabolism and excretion.
如下:
1.胆汁中BaP典型代谢产物浓度随着暴露浓度的增加而增加,能够较好地反映环境中BaP的暴露情况,但是要结合EROD和GST活性情况综合考虑其时间-效应关系。鱼胆汁中的总Sn~(2+)含量作为监测外界TBT污染程度的生物标志物也有可行性,可以指示出高浓度的有机锡污染,而且从联合作用结果看,BaP促进了TBT的代谢排出。
2.环境浓度的BaP和TBT对解毒酶系统造成了显著的影响。Western blot结果表明CYP1A蛋白含量随着BaP暴露浓度的增加而增加,可以作为监测环境中PAHs污染的良好的生物标志物。Ⅰ相解毒酶EROD活性与BaP、TBT及其混合物污染之间存在较为复杂的时间-效应与剂量-效应关系,而且在暴露25 d后,联合作用表现为拮抗作用,仅仅以EROD酶活性指标衡量环境中BaP、TBT或者二者的联合污染水平会出现失真现象。所以,将EROD活性与CYP1A蛋白水平或者BaP典型代谢产物水平,或者胆汁中总锡含量等指标结合起来考虑才能较为客观地认识环境中这两类污染物的污染程度。ECOD活性在BaP、TBT或者二者的联合作用下主要表现出被诱导效应,也能反映出高浓度的BaP和TBT联合污染情况,可以作为高污染环境中的生物标志物参考指标。Ⅱ相解毒酶UDPGT在暴露中期对两种污染物均未表现出显著的效应关系。
3.BaP和TBT对抗氧化体系指标产生了复杂的联合作用结果,主要表现为拮抗作用。SOD、GST活性与GSH含量在BaP暴露50 d后都表现出被诱导效应,将三者结合起来作为监测海洋环境PAHs污染的生物标志物有一定的可行性。TBT暴露50 d后会增加MDA含量,诱导SOD、GPx酶活性,并且降低GSH含量,将四者结合起来能较好地反映有机锡污染。BaP和TBT联合作用50 d后会增加MDA含量,并且诱导GPx、GST酶活性,因此将三者结合起来用于监测海洋环境中PAHs和有机锡的联合污染有一定的可行性。
4.DNA单链断裂损伤随着BaP、TBT及其混合物的暴露浓度增加和暴露时间的延长而加重,可以作为指示水体BaP、TBT或者二者的联合污染的潜在生物标志物。同时,从暴露25 d和50 d后的结果看,联合作用减轻了DNA损伤,这与TBT抑制BaP的代谢活化和BaP促进TBT的代谢排出的结果相一致。
根据以上结果,我们认为环境水平浓度的BaP和TBT所产生的联合毒理学效应有复杂的时间-效应和剂量-效应关系,主要是拮抗作用,表现为TBT对BaP代谢的抑制,BaP对于TBT的代谢排出起到了促进作用。
Marine fish Sebastiscus marmoratus was adopted as experimental animal. Toxicological effects were studied on two kinds of typical organic contaminants in marine environments, Polycyclic aromatic hydrocarbons (PAHs) and organotins. Sebastiscus marmoratus exposed to BaP (10, 100,1000 ng/L), TBT (1, 10, 100 ng/L) and their mixture (5 ng/L BaP+0.5 ng/L TBT, 50 ng/L BaP+5 ng/L TBT, 500 ng/L BaP+50 ng/L TBT) were randomly sampled after exposure for 0, 7, 25, 50 days and transferred to clean water to recover for 7 and 20 days, respectively. The physiological, biochemical and molecular parameters were analyzed to assess the effects of BaP, TBT and their mixture to metabolism, hepatic detoxification enzyme system, antioxidant system and DNA damage. Furthermore, the availability of these biomarkers to monitor marine pollution by BaP, TBT and their mixture were assessed. The main results as follows:
1. Levels of BaP metabolites in the bile were increased in a dose-dependent manner in the fish exposed through water to BaP, and reflected contamination gradients of PAHs. However, EROD and GST activities should be taken into account to assess time-effect relation. Total biliary Sn~(2+) content in fish is feasible to serve as an indicator to monitor severe organotin pollution, and as far as combined effects are concerned, BaP increased the excretion of TBT to bile.
2. Environmentally relevant concentrations of BaP and TBT influenced hepatic detoxification enzyme system significantly. CYPIA level inceased in harmony with BaP concentration with Western blot analysis, so CYPIA level is feasible to serve as a good biomarker to monitor PAHs pollution. EROD and ECOD are both phase I enzymes. There were complicated relations of time-effect and dose-effect between EROD activity and exposure of BaP, TBT or their mixture. Antagonistic interactions occurred after 25 days exposure to the mixure of BaP and TBT. It is fuzzy to merely use EROD activity as biomarker to assess PAHs or TBT pollution. Therefore, it is vital to assemble CYPIA level or BaP-type metabolites to assess PAHs pollution and integrate other biomarkers (e.g. total biliary Sn~(2+)content) to assess TBT pollution in marine environment. ECOD activity was induced by BaP, TBT or their mixture at high concentrations after 50 days exposure, and it is advisable to use ECOD activity as a biomarker to assess severe PAHs or TBT pollution. UDPGT was analyzed as phase II enzyme. There were scarcely significant effects of BaP, TBT or their mixture to UDPGT activity after 25 d exposure.
3. Combined effects of BaP and TBT to antioxidant system were complicated, and antagonistic interactions were dominating. SOD, GST activities were induced and GSH content were increased after 50 days exposure to BaP, and it is feasible to integrate them into PAHs pollution monitoring program. MDA content was increased and SOD, GPx activities were induced, and GSH content was decreased after 50 days exposure to TBT, so it is feasible to integrate them into TBT pollution monitoring program. In the same way, MDA content was increased and GST, GPx activities were induced after 50 days exposure to the mixture of BaP and TBT, so it is feasible to integrate them into PAHs with the addition of TBT pollution monitoring program in marine environment.
4. DNA single strand break damage was aggravated along with the increase of exposure concentration and the elongation of exposure time, so DNA single strand break damage is feasible to serve as a potential biomarker to monitor the pollution of PAHs, TBT or their mixture, because good relations of time-effect and dose-effect existed. As far as the results after 25 days and 50 days exposure are concerned, combination of BaP and TBT alleviated the degree of DNA damage which was consistent with the inhibition of BaP metabolism by TBT and the promotion of TBT excretion by BaP.
Based on the results we obtained, we conclude that combined toxicological effects of BaP and TBT at environmentally relevant concentrations were complicated, and antagonistic interactions were dominating. TBT inhibited BaP metabolism, and BaP facilitated TBT metabolism and excretion.
引文
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