几种典型持久性有机污染物对斑马鱼胚胎发育的毒性效应研究
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摘要
本文以斑马鱼(Danio Rerio)作为研究对象,结合国内外环境污染现状,选择2,3,7,8-四氯代二苯并二噁英(TCDD)、五氯酚(PCP)和溴化阻燃剂(BFRs)作为模式化合物,利用鱼类多种生长发育的生物标志物,研究这些化合物在单一、复合暴露下,对鱼类早期生命发育的毒性效应和潜在的致毒机理,为准确评估这些化合物的生态安全性提供科学依据。
通过观察斑马鱼鱼卵发育情况以及不同发育阶段鱼体内细胞色素P4501A(CYP1A)和钙调素(regucalcin,RC)基因的表达,评估了痕量TCDD(50 pg/mL)短期暴露后对斑马鱼早期发育的毒性效应。研究发现:痕量TCDD短期暴露后同样可对斑马鱼胚胎造成显著的毒性效应,如水肿、心跳变缓以及胚胎死亡率升高等;CYP1A基因的表达量随暴露时间延长,表现出先升高(96 hpf)后降低(168 hpf)的变化趋势,而RC基因表达水平在各发育时期均与对照组无显著性差异。
结合我国洞庭湖地区目前严重的五氯酚污染现状以及八氯代二苯并二噁英(OCDD)是五氯酚生产的主要副产物之一,初步研究了PCP和OCDD对斑马鱼胚胎发育的复合毒性效应。实验结果表明:在所测试的浓度范围内(25μg/L～5 mg/L),PCP单独暴露对斑马鱼胚胎发育具有较强的毒性效应,而高浓度OCDD(250μg/L)对斑马鱼胚胎发育没有明显的毒性效应;此外,OCDD与环境浓度下的PCP复合暴露对斑马鱼胚胎的存活与发育等无显著效应,对斑马鱼胚胎内CYP1A基因表达以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的酶活力均没有影响,二者共存没有明显的复合毒性效应。但环境中高浓度(100μg/L)的PCP对斑马鱼胚胎发育仍具有一定的毒性,PCP与其它的二噁英异构体是否存在复合毒性仍需进一步研究。
此外,选择四溴双酚A(TBBPA)和1,2,5,6,9,10-六溴环十二烷(HBCD)作为溴化阻燃剂的模式化合物,初步探讨了这些污染物对斑马鱼胚胎发育的毒性效应。TBBPA单一暴露实验结果表明,高浓度的TBBPA(0.75 mg/L和1.5 mg/L)可引起斑马鱼胚胎发育畸形,并导致胚胎死亡率显著升高,鱼体内SOD酶活力、丙二醛(MDA)含量和热激蛋白Hsp70的表达水平均显著高于对照组;HBCD单一暴露下(0.002 mg/L～10 mg/L)对斑马鱼胚胎发育无明显的毒性效应,但高浓度的HBCD暴露组斑马鱼体内SOD酶活力、MDA含量及Hsp70表达水平与对照组相比有显著性差异,其中SOD酶活力先升高后降低,而MDA含量和Hsp70表达水平较对照组均有显著性的升高;另外,高浓度的TBBPA(0.75 mg/L和1.5 mg/L)可缩短斑马鱼胚胎的出膜时间,但低浓度的HBCD(0.002 mg/L～0.5 mg/L)则造成斑马鱼胚胎发育迟缓;上述实验结果表明不同结构的溴化阻燃剂存在不同的致毒机制。
In this work,zebrafish(Danio Rerio) embryo-larval toxicity tests were performed to assess the toxicities of some typical persistent organic pollutants(POPs).Basing upon the actuality of worldwide environmental pollution,2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD),pentachlorophenol(PCP) and brominated flame retardants(BFRs) were studied. In order to evaluate their ecological safety,kinds of biological markers,related to the development of zebrafish embryos,were chose to investigate the toxicities and potential mechanisms under solo or combined exposure.
Mortality,heart rates,edema severity,CYP1A and regucalcin(RC) gene expressions were investigated to study TCDD-induced toxicity in zebrafish during the early life stage. Results indicated that the 50pg/mL TCDD caused severe and visible developmental toxicity,such as blue sac syndrome,decreased heart rate and increasing mortality. Furthermore,CYP1A mRNA expression was notably induced by TCDD at 96hpf,while fell to the normal at 168hpf.On the other hand,there was no significant difference in RC mRNA expression at each selected time compared to the control.
Due to the existing pollution of pentachlorophenol(PCP) in Dongting Lake region and octachlorodibenzo-p-dioxin(OCDD) as one of the important byproducts in the industrial process of PCP,studies were accomplished to assess the combined toxicities of PCP and OCDD.Embryo and larval survival,hatching,morphological development, CYP1A gene expression and activities of SOD and CAT were measured.The results showed that PCP(25μg/L～5mg/L) caused severe and visible developmental toxicity, while no effects were observed at the 250μg/L OCDD respectively.Moreover,zebrafish embryos did not show any effective toxicity under the combined exposure of 250μg/L OCDD and PCP with lower concentration,and simultaneously there were no significant differences in CYP1A expression and activities of SOD and CAT between the co-exposed embryos and the control.In a word,the co-exposure of PCP and OCDD had no remarkable toxicity,while the higher concentration of PCP solo did cause visible developmental toxicity to zebrafish embryos.Still,more investigations on the combined effect of PCP and other dioxin isomers are indispensable.
In order to explore the developmental toxicity of BFRs,4,4'-isopropylidenebis (TBBPA) and 1,2,5,6,9,10-hexabromocyclododecane(HBCD) were chosen as the typical BFRs.The results revealed that higher doses of TBBPA(0.75 mg/L and 1.5mg/L) caused obvious malformations and increasing mortality,besides,SOD,MDA and Hsp70 level were induced in TBBPA-treated group,compared to the control.With regard to HBCD, no developmental toxicity can be observed in all range of the concentrations from the lowest 0.002mg/L to the highest 10mg/L,but during its high doses there were significant differences in SOD,MDA and Hsp70 level between the HBCD-treated group and the control.Concretely,the trend of MDA and Hsp70 level were the same with TBBPA,but SOD showed a different trend,which was an initial decrease followed by a decrease. Moreover,higher doses of TBBPA(0.75mg/L and 1.5mg/L) accelerated the hatching of embryos,while relative lower doses of HBCD(0.002mg/L～0.5mg/L) delayed the occurrence of hatching.Thus,it can be concluded that BFRs with different chemical constitutions may have different mechanisms causing toxicities to zebrafish embryos.
通过观察斑马鱼鱼卵发育情况以及不同发育阶段鱼体内细胞色素P4501A(CYP1A)和钙调素(regucalcin,RC)基因的表达,评估了痕量TCDD(50 pg/mL)短期暴露后对斑马鱼早期发育的毒性效应。研究发现:痕量TCDD短期暴露后同样可对斑马鱼胚胎造成显著的毒性效应,如水肿、心跳变缓以及胚胎死亡率升高等;CYP1A基因的表达量随暴露时间延长,表现出先升高(96 hpf)后降低(168 hpf)的变化趋势,而RC基因表达水平在各发育时期均与对照组无显著性差异。
结合我国洞庭湖地区目前严重的五氯酚污染现状以及八氯代二苯并二噁英(OCDD)是五氯酚生产的主要副产物之一,初步研究了PCP和OCDD对斑马鱼胚胎发育的复合毒性效应。实验结果表明:在所测试的浓度范围内(25μg/L～5 mg/L),PCP单独暴露对斑马鱼胚胎发育具有较强的毒性效应,而高浓度OCDD(250μg/L)对斑马鱼胚胎发育没有明显的毒性效应;此外,OCDD与环境浓度下的PCP复合暴露对斑马鱼胚胎的存活与发育等无显著效应,对斑马鱼胚胎内CYP1A基因表达以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的酶活力均没有影响,二者共存没有明显的复合毒性效应。但环境中高浓度(100μg/L)的PCP对斑马鱼胚胎发育仍具有一定的毒性,PCP与其它的二噁英异构体是否存在复合毒性仍需进一步研究。
此外,选择四溴双酚A(TBBPA)和1,2,5,6,9,10-六溴环十二烷(HBCD)作为溴化阻燃剂的模式化合物,初步探讨了这些污染物对斑马鱼胚胎发育的毒性效应。TBBPA单一暴露实验结果表明,高浓度的TBBPA(0.75 mg/L和1.5 mg/L)可引起斑马鱼胚胎发育畸形,并导致胚胎死亡率显著升高,鱼体内SOD酶活力、丙二醛(MDA)含量和热激蛋白Hsp70的表达水平均显著高于对照组;HBCD单一暴露下(0.002 mg/L～10 mg/L)对斑马鱼胚胎发育无明显的毒性效应,但高浓度的HBCD暴露组斑马鱼体内SOD酶活力、MDA含量及Hsp70表达水平与对照组相比有显著性差异,其中SOD酶活力先升高后降低,而MDA含量和Hsp70表达水平较对照组均有显著性的升高;另外,高浓度的TBBPA(0.75 mg/L和1.5 mg/L)可缩短斑马鱼胚胎的出膜时间,但低浓度的HBCD(0.002 mg/L～0.5 mg/L)则造成斑马鱼胚胎发育迟缓;上述实验结果表明不同结构的溴化阻燃剂存在不同的致毒机制。
In this work,zebrafish(Danio Rerio) embryo-larval toxicity tests were performed to assess the toxicities of some typical persistent organic pollutants(POPs).Basing upon the actuality of worldwide environmental pollution,2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD),pentachlorophenol(PCP) and brominated flame retardants(BFRs) were studied. In order to evaluate their ecological safety,kinds of biological markers,related to the development of zebrafish embryos,were chose to investigate the toxicities and potential mechanisms under solo or combined exposure.
Mortality,heart rates,edema severity,CYP1A and regucalcin(RC) gene expressions were investigated to study TCDD-induced toxicity in zebrafish during the early life stage. Results indicated that the 50pg/mL TCDD caused severe and visible developmental toxicity,such as blue sac syndrome,decreased heart rate and increasing mortality. Furthermore,CYP1A mRNA expression was notably induced by TCDD at 96hpf,while fell to the normal at 168hpf.On the other hand,there was no significant difference in RC mRNA expression at each selected time compared to the control.
Due to the existing pollution of pentachlorophenol(PCP) in Dongting Lake region and octachlorodibenzo-p-dioxin(OCDD) as one of the important byproducts in the industrial process of PCP,studies were accomplished to assess the combined toxicities of PCP and OCDD.Embryo and larval survival,hatching,morphological development, CYP1A gene expression and activities of SOD and CAT were measured.The results showed that PCP(25μg/L～5mg/L) caused severe and visible developmental toxicity, while no effects were observed at the 250μg/L OCDD respectively.Moreover,zebrafish embryos did not show any effective toxicity under the combined exposure of 250μg/L OCDD and PCP with lower concentration,and simultaneously there were no significant differences in CYP1A expression and activities of SOD and CAT between the co-exposed embryos and the control.In a word,the co-exposure of PCP and OCDD had no remarkable toxicity,while the higher concentration of PCP solo did cause visible developmental toxicity to zebrafish embryos.Still,more investigations on the combined effect of PCP and other dioxin isomers are indispensable.
In order to explore the developmental toxicity of BFRs,4,4'-isopropylidenebis (TBBPA) and 1,2,5,6,9,10-hexabromocyclododecane(HBCD) were chosen as the typical BFRs.The results revealed that higher doses of TBBPA(0.75 mg/L and 1.5mg/L) caused obvious malformations and increasing mortality,besides,SOD,MDA and Hsp70 level were induced in TBBPA-treated group,compared to the control.With regard to HBCD, no developmental toxicity can be observed in all range of the concentrations from the lowest 0.002mg/L to the highest 10mg/L,but during its high doses there were significant differences in SOD,MDA and Hsp70 level between the HBCD-treated group and the control.Concretely,the trend of MDA and Hsp70 level were the same with TBBPA,but SOD showed a different trend,which was an initial decrease followed by a decrease. Moreover,higher doses of TBBPA(0.75mg/L and 1.5mg/L) accelerated the hatching of embryos,while relative lower doses of HBCD(0.002mg/L～0.5mg/L) delayed the occurrence of hatching.Thus,it can be concluded that BFRs with different chemical constitutions may have different mechanisms causing toxicities to zebrafish embryos.
引文
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