石油烃污染对马粪海胆的毒性效应及分子机制研究
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摘要
在近海海洋环境中,石油烃的沉降和积累特性导致海底环境的油污染程度很高,石油烃污染已成为威胁全球海洋环境的最严重的污染问题之一。为探讨石油烃污染对海洋生物可能的损害机制,并寻找适合检测石油烃污染的生物标志物,本文选取海洋底栖环境的模式生物—海胆作为受试生物,分别从个体、组织和分子水平上较系统地研究了石油烃污染对马粪海胆成体及胚胎的毒性效应及其机制,主要研究内容与结果如下:
1.从个体水平方面,为了明确石油污染以及消油剂的生物毒性效应,本文以0号柴油为污染物,研究使用消油剂前后0号柴油分散液和乳化液对马粪海胆(Hemicentrotus pulcherrimus)幼胆生长发育的急性毒性效应。结果表明:0号柴油分散液、加入消油剂的乳化液对马粪海胆96h的LC_(50)值分别为9.6mg/L和5.1mg/L,乳化液的急性毒性大于分散液。通过气相色谱-质谱测定,表明乳化液中芳香族化合物的含量增加近2倍,从而导致石油烃毒性效应显著提高。
2.为了完善海洋石油烃污染生物监测体系,本文选取马粪海胆的配子、胚胎及幼虫为实验材料,采用胚胎静态毒性试验的方法,研究石油烃污染对马粪海胆的配子以及胚胎、幼虫的生长发育的影响。研究结果表明:石油烃污染使配子受精能力下降,影响程度与油浓度的高低成正比;使用消油剂导致石油烃毒性增加,0号柴油对卵子受精率的EC_(50)由0.86mg/L降为0.38mg/L,对精子受精率的EC_(50)由1.57 mg/L减小为0.38mg/L;石油烃暴露使海胆胚胎发育明显滞后,出现死亡、畸形、体长缩短等现象,0号柴油乳化液抑制海胆胚胎发育的48h、72h EC_(50)分别为0.83mg/L和0.44mg/L;0号柴油分散液对海胆胚胎发育抑制的48h、72h EC_(50)分别为3.39mg/L和1.87mg/L,可知0号柴油乳化液的生物毒性明显大于分散液。海胆的精子与胚胎、幼虫对石油污染敏感,可以作为指示生物用于海洋石油污染监测。
3.从组织水平方面,为了研究石油烃污染对马粪海胆抗氧化防御系统的影响,寻找合适的海洋石油污染的生物标志物,采用低剂量长期暴露的方法,选取抗氧化防御系统的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)、谷胱甘肽硫转移酶(GST)以及谷胱甘肽的含量(GSH)作为研究目标,结果表明:马粪海胆各生化指标存在组织差异性,对石油烃污染的反应有一定的剂量-效应特征和显著的时间-效应特征。随着浓度的增加和时间的延长,抗氧化酶活性基本上都是先增加后减小的趋势,GSH被消耗而逐渐减小,脂质过氧化加剧,在抗氧化酶系的各参数中,GST、GPx酶活性及GSH含量反应最灵敏,比较适合作为监测石油烃污染程度的标志物。
4.从分子水平方面,为了检测石油烃暴露对马粪海胆免疫系统及生长发育的遗传损伤机制,本文选择补体C3基因及调控生长发育的Hox1基因,应用PCR-SSCP技术和DNA测序方法,对不同暴露剂量与时间下的马粪海胆的补体C3基因及Hox1基因突变情况进行检测分析,结果表明:在暴露于一定浓度的石油烃后,马粪海胆补体C3基因编码区的1184位发生了单碱基变化,即C→T的转换突变,氨基酸由苏氨酸变成异亮氨酸;对调控生长发育的Hox1基因检测结果显示编码区184位发生了单碱基变化,即T→C的转换突变;氨基酸由丝氨酸变成脯氨酸。随着污染时间的延长和浓度的加大,突变率呈增加趋势。说明石油烃对海胆的毒性效应影响首先是从分子遗传损伤开始的。
5.为了探讨石油烃污染对马粪海胆表观遗传学的改变,本文采用高效液相色谱技术,对石油烃暴露后马粪海胆全基因组DNA甲基化水平进行检测,结果表明:石油烃污染对马粪海胆的全基因组DNA甲基化没有明显的剂量-效应关系。随着暴露时间延长,0号柴油分散液暴露下的马粪海胆全基因组DNA甲基化水平比对照组显著降低,说明长时间石油烃暴露会使马粪海胆体内的DNA甲基转移酶活性受到抑制,表明石油烃污染对海洋生物机体产生遗传毒性同时,还存在着DNA甲基化等非基因序列导致的基因表达的改变。
Nowadays,oil pollution has threaten the marine environment all over the world and become one of the most serious pollution problems.Petroleum hydrocarbons are liable to deposition and accumulation,which leads to a higher level of oil contamination in the seabed.In this paper,model organisms-sea urchin,Hemicentrotus pulcherrimus was selected as test organism.The effects of petroleum hydrocarbon on the adult sea urchins and their embryos were studied from molecular level to explore the toxicity mechanism on aquatic life and to find suitable biomarkers for oil pollution detection. The main conclusions of the study are as follows:
1.From the individual level,the acute toxicity effects of No.0 diesel water-accomodated fractions(WAFs) and dispersed No.0 diesel water-accomodated fractions(dis-WAFs) on sea urchin,Hemicentrotus Pulcherrimus was studied.The results show that:the 96h LC_(50) value of No.0 diesel oil WAF and dis-WAFs are 5.1mg/L and 9.5mg/L respectively.The dis-WAFs is more toxic than WAFs.Gas chromatography-mass spectrometry analysis results show that the content of aromatic compounds in dis-WAFs is nearly 3 times of the latter.
2.By using the method of static embryo toxicity test,sea urchin gametes and embryos were selected as experimental material and the effects of petroleum hydrocarbons on fertilization and embryos development were studied.The results show that petroleum hydrocarbon pollution decreased the fertilization ability of gametes.The inhibition degree is derectly proportional to the oil concentration.The 30minEC_(50) for eggs decreased from 0.86mg/L to 0.36mg/L after adding dispersant into the WAFs,and 30minEC_(50) for sperms decreased from 1.57mg/L to 0.38mg/L.Embryonic growth retardation,shorter body length,deformity and death can be observed in exposure experimental groups.The 48h and 72h EC_(50) for No.0 dis-WAFs were 0.83mg/L and0.44mg/L;while the 48h and 72h EC_(50) for No.0 oil diesel WAFs were 3.39mg/L and 1.87mg/L.The No.0 diesel dis-WAFs is obviously more toxic than WAFs.
3.From an organizational level,the effects of No.0 diesel WAFs on the antioxidant defense system and lipid peroxidation of Hemicentrotus pulcherrimus after low-dose exposure were investigated.The results show different distributions of antioxidant enzyme activity in Hemicentrotus pulcherrimus.The dose-effect and time-effect relations were between the biochemical response and petroleum hydrocarbon pollution. The time-effect pattern of antioxidant defense system follows the law of increasing first and then decreasing.GSH has been gradually reduced because of the consumption and lipid peroxidation intensified by prolonging the exposure time and increasing exposure dose.GST activity is the most sensitive to petroleum hydrocarbon pollution and is more suitable to be the biomarker for monitoring and testing the extent of petroleum hydrocarbon contamination in the seabed.
4.From the molecular level,complement C3 gene mutation was detected through PCR-SSCP technique and sequence determination.The results suggest that single base mutation has taken place in coding region of the 1184,with the conversion of C→T, where amino acid changed from threonine into isoleucine.Hox1 gene mutation detection results show that single base mutation have taken place in coding region of the 184,with the conversion of T→C,where amino acid is changed from serine into proline.It is also observed that with the extension of exposure time and concentration, mutation rate increased gradually.
5.From the molecular level,the globle genomic DNA was detected by high performance liquid chromatography.The results show that there are no significant dose-effect relationship and with the time prolonging,hypomethylation happens under the exposure.It suggests that methyltransferase activity in sea urchin is inhibited after petroleum hydrocarbon exposure.The exposure can not only produces genetic toxicity effects,but also change the DNA methylation level.
1.从个体水平方面,为了明确石油污染以及消油剂的生物毒性效应,本文以0号柴油为污染物,研究使用消油剂前后0号柴油分散液和乳化液对马粪海胆(Hemicentrotus pulcherrimus)幼胆生长发育的急性毒性效应。结果表明:0号柴油分散液、加入消油剂的乳化液对马粪海胆96h的LC_(50)值分别为9.6mg/L和5.1mg/L,乳化液的急性毒性大于分散液。通过气相色谱-质谱测定,表明乳化液中芳香族化合物的含量增加近2倍,从而导致石油烃毒性效应显著提高。
2.为了完善海洋石油烃污染生物监测体系,本文选取马粪海胆的配子、胚胎及幼虫为实验材料,采用胚胎静态毒性试验的方法,研究石油烃污染对马粪海胆的配子以及胚胎、幼虫的生长发育的影响。研究结果表明:石油烃污染使配子受精能力下降,影响程度与油浓度的高低成正比;使用消油剂导致石油烃毒性增加,0号柴油对卵子受精率的EC_(50)由0.86mg/L降为0.38mg/L,对精子受精率的EC_(50)由1.57 mg/L减小为0.38mg/L;石油烃暴露使海胆胚胎发育明显滞后,出现死亡、畸形、体长缩短等现象,0号柴油乳化液抑制海胆胚胎发育的48h、72h EC_(50)分别为0.83mg/L和0.44mg/L;0号柴油分散液对海胆胚胎发育抑制的48h、72h EC_(50)分别为3.39mg/L和1.87mg/L,可知0号柴油乳化液的生物毒性明显大于分散液。海胆的精子与胚胎、幼虫对石油污染敏感,可以作为指示生物用于海洋石油污染监测。
3.从组织水平方面,为了研究石油烃污染对马粪海胆抗氧化防御系统的影响,寻找合适的海洋石油污染的生物标志物,采用低剂量长期暴露的方法,选取抗氧化防御系统的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPx)、谷胱甘肽硫转移酶(GST)以及谷胱甘肽的含量(GSH)作为研究目标,结果表明:马粪海胆各生化指标存在组织差异性,对石油烃污染的反应有一定的剂量-效应特征和显著的时间-效应特征。随着浓度的增加和时间的延长,抗氧化酶活性基本上都是先增加后减小的趋势,GSH被消耗而逐渐减小,脂质过氧化加剧,在抗氧化酶系的各参数中,GST、GPx酶活性及GSH含量反应最灵敏,比较适合作为监测石油烃污染程度的标志物。
4.从分子水平方面,为了检测石油烃暴露对马粪海胆免疫系统及生长发育的遗传损伤机制,本文选择补体C3基因及调控生长发育的Hox1基因,应用PCR-SSCP技术和DNA测序方法,对不同暴露剂量与时间下的马粪海胆的补体C3基因及Hox1基因突变情况进行检测分析,结果表明:在暴露于一定浓度的石油烃后,马粪海胆补体C3基因编码区的1184位发生了单碱基变化,即C→T的转换突变,氨基酸由苏氨酸变成异亮氨酸;对调控生长发育的Hox1基因检测结果显示编码区184位发生了单碱基变化,即T→C的转换突变;氨基酸由丝氨酸变成脯氨酸。随着污染时间的延长和浓度的加大,突变率呈增加趋势。说明石油烃对海胆的毒性效应影响首先是从分子遗传损伤开始的。
5.为了探讨石油烃污染对马粪海胆表观遗传学的改变,本文采用高效液相色谱技术,对石油烃暴露后马粪海胆全基因组DNA甲基化水平进行检测,结果表明:石油烃污染对马粪海胆的全基因组DNA甲基化没有明显的剂量-效应关系。随着暴露时间延长,0号柴油分散液暴露下的马粪海胆全基因组DNA甲基化水平比对照组显著降低,说明长时间石油烃暴露会使马粪海胆体内的DNA甲基转移酶活性受到抑制,表明石油烃污染对海洋生物机体产生遗传毒性同时,还存在着DNA甲基化等非基因序列导致的基因表达的改变。
Nowadays,oil pollution has threaten the marine environment all over the world and become one of the most serious pollution problems.Petroleum hydrocarbons are liable to deposition and accumulation,which leads to a higher level of oil contamination in the seabed.In this paper,model organisms-sea urchin,Hemicentrotus pulcherrimus was selected as test organism.The effects of petroleum hydrocarbon on the adult sea urchins and their embryos were studied from molecular level to explore the toxicity mechanism on aquatic life and to find suitable biomarkers for oil pollution detection. The main conclusions of the study are as follows:
1.From the individual level,the acute toxicity effects of No.0 diesel water-accomodated fractions(WAFs) and dispersed No.0 diesel water-accomodated fractions(dis-WAFs) on sea urchin,Hemicentrotus Pulcherrimus was studied.The results show that:the 96h LC_(50) value of No.0 diesel oil WAF and dis-WAFs are 5.1mg/L and 9.5mg/L respectively.The dis-WAFs is more toxic than WAFs.Gas chromatography-mass spectrometry analysis results show that the content of aromatic compounds in dis-WAFs is nearly 3 times of the latter.
2.By using the method of static embryo toxicity test,sea urchin gametes and embryos were selected as experimental material and the effects of petroleum hydrocarbons on fertilization and embryos development were studied.The results show that petroleum hydrocarbon pollution decreased the fertilization ability of gametes.The inhibition degree is derectly proportional to the oil concentration.The 30minEC_(50) for eggs decreased from 0.86mg/L to 0.36mg/L after adding dispersant into the WAFs,and 30minEC_(50) for sperms decreased from 1.57mg/L to 0.38mg/L.Embryonic growth retardation,shorter body length,deformity and death can be observed in exposure experimental groups.The 48h and 72h EC_(50) for No.0 dis-WAFs were 0.83mg/L and0.44mg/L;while the 48h and 72h EC_(50) for No.0 oil diesel WAFs were 3.39mg/L and 1.87mg/L.The No.0 diesel dis-WAFs is obviously more toxic than WAFs.
3.From an organizational level,the effects of No.0 diesel WAFs on the antioxidant defense system and lipid peroxidation of Hemicentrotus pulcherrimus after low-dose exposure were investigated.The results show different distributions of antioxidant enzyme activity in Hemicentrotus pulcherrimus.The dose-effect and time-effect relations were between the biochemical response and petroleum hydrocarbon pollution. The time-effect pattern of antioxidant defense system follows the law of increasing first and then decreasing.GSH has been gradually reduced because of the consumption and lipid peroxidation intensified by prolonging the exposure time and increasing exposure dose.GST activity is the most sensitive to petroleum hydrocarbon pollution and is more suitable to be the biomarker for monitoring and testing the extent of petroleum hydrocarbon contamination in the seabed.
4.From the molecular level,complement C3 gene mutation was detected through PCR-SSCP technique and sequence determination.The results suggest that single base mutation has taken place in coding region of the 1184,with the conversion of C→T, where amino acid changed from threonine into isoleucine.Hox1 gene mutation detection results show that single base mutation have taken place in coding region of the 184,with the conversion of T→C,where amino acid is changed from serine into proline.It is also observed that with the extension of exposure time and concentration, mutation rate increased gradually.
5.From the molecular level,the globle genomic DNA was detected by high performance liquid chromatography.The results show that there are no significant dose-effect relationship and with the time prolonging,hypomethylation happens under the exposure.It suggests that methyltransferase activity in sea urchin is inhibited after petroleum hydrocarbon exposure.The exposure can not only produces genetic toxicity effects,but also change the DNA methylation level.
引文
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