芳香族污染物基因毒性作用的分子水平研究
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摘要
芳香族污染物,特别是芳香族染料和芳香族表面活性剂,在环境中大量存在,并对生物体造成危害。芳香族污染物及其复合污染物(芳香族污染物之间或芳香族污染物与其他类物质形成的复合物)进入生物体后与DNA直接或间接发生相互作用,改变DNA的结构与构象或引起DNA损伤,从而影响其功能或改变其遗传特性。因此,研究芳香族污染物及其复合污染物与DNA的相互作用和其基因毒性作用的评价方法,将有助于人们从基因分子水平上了解芳香族污染物毒性作用机理,有利于正确认识和评估污染物的毒性。
本论文主要利用光谱学技术对环境中常见的芳香族污染物及其复合污染物的基因毒性作用机理进行了研究,利用光谱学响应信号对这些污染物的基因毒性作用强度进行了评价。论文主要包括以下四个部分:
第一部分:利用吸收光谱法和圆二色谱法从基因分子水平上研究了甲基紫对ctDNA的毒性作用。结果表明,在低甲基紫浓度时,甲基紫与DNA的结合方式为“嵌入作用”方式;而在较高甲基紫浓度时,甲基紫与ctDNA的结合模式为“静电作用”,求得其结合常数为K_(25℃)~θ为1.80×10~3 L·mol~(-1)。本部分还利用紫外可见吸收光谱信号(△A)评价了甲基紫的基因毒性作用强度。当甲基紫与ctDNA的作用模式为静电式结合时,在pH为6.50、甲基紫浓度为4.0×10~(-5) mol L~(-1)的条件下,甲基紫基因分子毒性作用最强;在0～40 min内,随时间增加,甲基紫基因分子毒性作用强度逐渐减弱;在40～120 min内,随时间增加,作用强度减弱程度变小,趋于稳定;当NaCl浓度大于0.008 mol/L时,随离子强度增大,甲基紫与ctDNA的作用强度明显降低,进一步说明甲基紫与ctDNA的结合模式为“静电作用”。圆二色谱的实验结果表明,甲基紫可使DNA构象发生改变,并呈现明显的直线型剂量-效应关系。
第二部分:在体外研究了甲苯胺蓝(TB)对ctDNA的基因毒性作用。紫外-可见吸收光谱、荧光光谱和荧光偏振的实验结果表明ctDNA可以通过两种作用模式与甲苯胺蓝发生作用而表现出一定的基因毒性。在低甲苯胺蓝浓度下,甲苯胺蓝嵌插入ctDNA碱基对中。在高浓度甲苯胺蓝浓度下,甲苯胺蓝与ctDNA外部磷酸根发生静电结合。静电结合和嵌插结合的键合常数分别为1.01×10~6 L mol~(-1)和2.17×10~7 L mol~(-1),键合位点数分别为0.9和0.4。圆二色谱的研究结果表明这两种结合方式对ctDNA构象有不同的影响。以DNA对甲苯胺蓝的荧光猝灭程度评价了甲苯胺蓝的基因毒性强度。在甲苯胺蓝浓度为4.0×10~(-6) mol L~(-1),pH值在6.50～8.50范围内时,甲苯胺蓝的基因毒性作用最强。
第三部分:模拟研究了生物体内偶氮染料碱性橙与CTMAB芳香族复合污染物的基因毒性作用。吸收光谱、共振光散射光谱、圆二色谱和透射电镜的实验结果均表明碱性橙本身基因毒性不强,而碱性橙-CTMAB复合污染物则表现出明显的毒性。污染物在ctDNA表面的聚集产生毒性,聚集程度反映基因毒性大小。基于这一点,利用ctDNA加入前后碱性橙-CTMAB复合污染物共振光散射强度的改变值评价其基因毒性作用强度。当碱性橙浓度为3.0×10.4 mol L~(-1)、CTMAB浓度为1.6×10~(-5) mol L~(-1)、pH大于7.00时,碱性橙-CTMAB复合污染物对ctDNA的基因毒性作用最强。圆二色谱的结果表明碱性橙-CTMAB复合污染物在基因毒性作用过程中能使ctDNA构象发生改变。透射电镜的结果表明碱性橙-CTMAB复合污染物与ctDNA发生作用,生成了离子缔合物。各种光谱学手段的实验结果表明碱性橙-CTMAB复合污染物的基因毒性作用比碱性橙或CTMAB单独存在时毒性作用都强。
第四部分:利用吸收光谱、共振光散射及透射电镜等技术在体外模拟研究了芳香族污染物十六烷基三甲基溴化砒啶(CPB)与纳米银复合污染物所产生的基因毒性。研究发现,粒径20-50 nm范围内的纳米银颗粒单独存在时其基因毒性不强。而nanoAg与芳香族阳离子表面活性剂CPB作用后形成的nanoAg-CPB复合物则有明显的基因毒性。利用ctDNA加入前后nanoAg-CPB复合体系共振光散射强度的改变值评价其基因毒性。在pH为7.6、3.3×10~(-6)g mL~(-1) nano-Ag和6.0×10~(-6) mol L~(-1) CPB协同作用下,nanoAg-CPB复合污染物显示出最强的基因毒性作用。本部分同时考察了纳米银在这一环境行为中的形态变化,当nanoAg与CPB形成复合物时,nanoAg发生聚集并且粒径变大。加入ctDNA后,三者发生相互作用,使nanoAg变得分散并且粒径变小,这也间接说明纳米银-CPB复合污染物具有基因毒性。
A large amount of aromatic pollutants,particularly aromatic dyes and aromatic surfactants,exist in the environment and have toxic effects on organism.Aromatic pollutants or aromatic combined pollutants may interact with DNA directly or indirectly,changing the structure and conformation of DNA or causing DNA damage, thus affect DNA function or change the genetic character of DNA.Therefore,the study of the interaction between aromatic pollutants and DNA and the genotoxicity assessment methods of aromatic pollutants and aromatic combined pollutants will contribute to the understanding of the toxicity mechanism of aromatic pollutants in the molecular level of gene,also to the correct understanding and evaluation of the toxicity of aromatic pollutants.
Utilizing spectroscopy techniques,the dissertation investigate the genotoxicity mechanism of aromatic pollutants and aromatic combined ones.The genotoxicity assessment methods of them are also established utilizing the spectroscopy responding signals.The dissertation consists of the following four parts.
In the first part,utilizing UV-Vis spectrophotometry and circular dichroism(CD) techniques,the toxic effects of methyl violet(MV) to calf thymus DNA(ctDNA) was studied in the molecular level.The results showed that MV interacted with ct DNA by two binding models.At low MV concentrations,MV intercalated into ctDNA base pairs.At higher MV concentrations,MV was attached to the negative charged phosphate groups.The binding constant(K~θ25℃) of the electrostatic binding model was 1.80×10~3 L·mol~(-1).Utilizing the absorbency signal,the genotoxicity of MV was estimated.MV had a strong genotoxicity interaction intensity at the 4.0×10~(-5) mol L~(-1) MV and pH 6.50.The genotoxicity of MV decreased with the time in 0～40 min;the weakening extent the genotoxicity of MV decreased gradually with the time in 40～120 min.When the NaCl concentration was greater than 0.008 mol/L,the interaction intensity of MV with ctDNA decreased significantly with increasing ionic strength,further illuminating that MV was attached to the negative charged phosphate groups.CD results showed that MV can change the conformation of DNA,showing a significant linear dose-response relationship.
In the second part,the genotoxicity mechanism of toluidine blue(TB) was examined in vitro.The absorption spectra,fluorescence spectra and fluorescence polarization showed that TB interacted with ctDNA by two binding models to show gene toxicity.At low dye concentrations,TB intercalated into base pairs of ctDNA.At higher dye concentrations,TB was attached to the negative charged phosphate groups. The binding constant of the intercalation binding model and electrostatic binding model was 2.17×10~7 L mol~(-1) and 1.01×10~6 L mol~(-1) and the number of binding sites was 0.4 and 0.9,respectively.CD results showed that the two binding models had different effects on ctDNA conformation.Utilizing the fluorescence quenching extent of TB by ctDNA,the genotoxicity interaction intensity of TB was estimated and the interaction intensity of TB was strongest at the 4.0×10~(-1) mol L~(-1) TB and pH range 6.50～8.50.
In the third part,the genotoxicity of the azo dye chrysoidine hydrochloride combined with cetyltrimethyl ammonium bromide(CTMAB) in the living tissue was studied in vitro.The absorption spectrum,resonance light scattering(RLS),CD and transmission electron microscopy(TEM) results showed that the genotoxicity of chrysoidine hydrochloride itself was weak,while the chrysoidine hydrochloride-CTMAB combined aromatic pollutant showed obvious genotoxicity. The assembly of the contaminants on ctDNA,which can be shown by RLS,is considered as a sign of genotoxicity.The genotoxicity of chrysoidine hydrochloride-CTMAB combined aromatic pollution was evaluated by the change of the RLS intensity signal of the system due to the addition of ctDNA.The genotoxicity interaction intensity of the combined pollutant to ctDNA showed a strong co-effect at the dose of 3.0×10~(-4) mol L~(-1) chrysoidine hydrochloride,1.6×10~(-5) mol L~(-1) CTMAB and pH higher than 7.00.The CD results showed that the conformation of ctDNA changed in the genotoxic process of chrysoidine hydrochloride-CTMAB combined contamination.The TEM results showed that ctDNA interact with the chrysoidine hydrochloride-CTMAB combined contamination to form an ion associated complex. The experimental results showed that the combination of chrysoidine hydrochloride-CTMAB had higher genotoxicity than either chrysoidine hydrochloride or CTMAB individually.
In the last part,the environmental genotoxic behavior of the aromatic detergent cetylpyridine bromide(CPB) combined with silver nanoparticles(nanoAg) was examined in vitro.The experimental results showed that the genotoxicity of nanoAg itself was very weak,but when combined with CPB,nanoAg showed obvious genotoxicity.By means of the change of the RLS intensity signal of the nanoAg-CPB system caused by the addition of ctDNA to it,the genotoxicity interaction intensity of nanoAg-CPB combined pollution was estimated.The combined materials had a strong co-effect on ctDNA at the concentration of 3.3×10~(-6) g mL~(-1) nanoAg and 6.0×10~(-6) mol L~(-1) CPB and pH 7.6.After the formation of a nanoAg-CPB complex,the nanoAg aggregate.After the addition of ctDNA to the nanoAg-CPB system,the particles were scattered and the diameter decreased,which illuminated that CPB-nanoAg has genotoxicity.
本论文主要利用光谱学技术对环境中常见的芳香族污染物及其复合污染物的基因毒性作用机理进行了研究,利用光谱学响应信号对这些污染物的基因毒性作用强度进行了评价。论文主要包括以下四个部分:
第一部分:利用吸收光谱法和圆二色谱法从基因分子水平上研究了甲基紫对ctDNA的毒性作用。结果表明,在低甲基紫浓度时,甲基紫与DNA的结合方式为“嵌入作用”方式;而在较高甲基紫浓度时,甲基紫与ctDNA的结合模式为“静电作用”,求得其结合常数为K_(25℃)~θ为1.80×10~3 L·mol~(-1)。本部分还利用紫外可见吸收光谱信号(△A)评价了甲基紫的基因毒性作用强度。当甲基紫与ctDNA的作用模式为静电式结合时,在pH为6.50、甲基紫浓度为4.0×10~(-5) mol L~(-1)的条件下,甲基紫基因分子毒性作用最强;在0～40 min内,随时间增加,甲基紫基因分子毒性作用强度逐渐减弱;在40～120 min内,随时间增加,作用强度减弱程度变小,趋于稳定;当NaCl浓度大于0.008 mol/L时,随离子强度增大,甲基紫与ctDNA的作用强度明显降低,进一步说明甲基紫与ctDNA的结合模式为“静电作用”。圆二色谱的实验结果表明,甲基紫可使DNA构象发生改变,并呈现明显的直线型剂量-效应关系。
第二部分:在体外研究了甲苯胺蓝(TB)对ctDNA的基因毒性作用。紫外-可见吸收光谱、荧光光谱和荧光偏振的实验结果表明ctDNA可以通过两种作用模式与甲苯胺蓝发生作用而表现出一定的基因毒性。在低甲苯胺蓝浓度下,甲苯胺蓝嵌插入ctDNA碱基对中。在高浓度甲苯胺蓝浓度下,甲苯胺蓝与ctDNA外部磷酸根发生静电结合。静电结合和嵌插结合的键合常数分别为1.01×10~6 L mol~(-1)和2.17×10~7 L mol~(-1),键合位点数分别为0.9和0.4。圆二色谱的研究结果表明这两种结合方式对ctDNA构象有不同的影响。以DNA对甲苯胺蓝的荧光猝灭程度评价了甲苯胺蓝的基因毒性强度。在甲苯胺蓝浓度为4.0×10~(-6) mol L~(-1),pH值在6.50～8.50范围内时,甲苯胺蓝的基因毒性作用最强。
第三部分:模拟研究了生物体内偶氮染料碱性橙与CTMAB芳香族复合污染物的基因毒性作用。吸收光谱、共振光散射光谱、圆二色谱和透射电镜的实验结果均表明碱性橙本身基因毒性不强,而碱性橙-CTMAB复合污染物则表现出明显的毒性。污染物在ctDNA表面的聚集产生毒性,聚集程度反映基因毒性大小。基于这一点,利用ctDNA加入前后碱性橙-CTMAB复合污染物共振光散射强度的改变值评价其基因毒性作用强度。当碱性橙浓度为3.0×10.4 mol L~(-1)、CTMAB浓度为1.6×10~(-5) mol L~(-1)、pH大于7.00时,碱性橙-CTMAB复合污染物对ctDNA的基因毒性作用最强。圆二色谱的结果表明碱性橙-CTMAB复合污染物在基因毒性作用过程中能使ctDNA构象发生改变。透射电镜的结果表明碱性橙-CTMAB复合污染物与ctDNA发生作用,生成了离子缔合物。各种光谱学手段的实验结果表明碱性橙-CTMAB复合污染物的基因毒性作用比碱性橙或CTMAB单独存在时毒性作用都强。
第四部分:利用吸收光谱、共振光散射及透射电镜等技术在体外模拟研究了芳香族污染物十六烷基三甲基溴化砒啶(CPB)与纳米银复合污染物所产生的基因毒性。研究发现,粒径20-50 nm范围内的纳米银颗粒单独存在时其基因毒性不强。而nanoAg与芳香族阳离子表面活性剂CPB作用后形成的nanoAg-CPB复合物则有明显的基因毒性。利用ctDNA加入前后nanoAg-CPB复合体系共振光散射强度的改变值评价其基因毒性。在pH为7.6、3.3×10~(-6)g mL~(-1) nano-Ag和6.0×10~(-6) mol L~(-1) CPB协同作用下,nanoAg-CPB复合污染物显示出最强的基因毒性作用。本部分同时考察了纳米银在这一环境行为中的形态变化,当nanoAg与CPB形成复合物时,nanoAg发生聚集并且粒径变大。加入ctDNA后,三者发生相互作用,使nanoAg变得分散并且粒径变小,这也间接说明纳米银-CPB复合污染物具有基因毒性。
A large amount of aromatic pollutants,particularly aromatic dyes and aromatic surfactants,exist in the environment and have toxic effects on organism.Aromatic pollutants or aromatic combined pollutants may interact with DNA directly or indirectly,changing the structure and conformation of DNA or causing DNA damage, thus affect DNA function or change the genetic character of DNA.Therefore,the study of the interaction between aromatic pollutants and DNA and the genotoxicity assessment methods of aromatic pollutants and aromatic combined pollutants will contribute to the understanding of the toxicity mechanism of aromatic pollutants in the molecular level of gene,also to the correct understanding and evaluation of the toxicity of aromatic pollutants.
Utilizing spectroscopy techniques,the dissertation investigate the genotoxicity mechanism of aromatic pollutants and aromatic combined ones.The genotoxicity assessment methods of them are also established utilizing the spectroscopy responding signals.The dissertation consists of the following four parts.
In the first part,utilizing UV-Vis spectrophotometry and circular dichroism(CD) techniques,the toxic effects of methyl violet(MV) to calf thymus DNA(ctDNA) was studied in the molecular level.The results showed that MV interacted with ct DNA by two binding models.At low MV concentrations,MV intercalated into ctDNA base pairs.At higher MV concentrations,MV was attached to the negative charged phosphate groups.The binding constant(K~θ25℃) of the electrostatic binding model was 1.80×10~3 L·mol~(-1).Utilizing the absorbency signal,the genotoxicity of MV was estimated.MV had a strong genotoxicity interaction intensity at the 4.0×10~(-5) mol L~(-1) MV and pH 6.50.The genotoxicity of MV decreased with the time in 0～40 min;the weakening extent the genotoxicity of MV decreased gradually with the time in 40～120 min.When the NaCl concentration was greater than 0.008 mol/L,the interaction intensity of MV with ctDNA decreased significantly with increasing ionic strength,further illuminating that MV was attached to the negative charged phosphate groups.CD results showed that MV can change the conformation of DNA,showing a significant linear dose-response relationship.
In the second part,the genotoxicity mechanism of toluidine blue(TB) was examined in vitro.The absorption spectra,fluorescence spectra and fluorescence polarization showed that TB interacted with ctDNA by two binding models to show gene toxicity.At low dye concentrations,TB intercalated into base pairs of ctDNA.At higher dye concentrations,TB was attached to the negative charged phosphate groups. The binding constant of the intercalation binding model and electrostatic binding model was 2.17×10~7 L mol~(-1) and 1.01×10~6 L mol~(-1) and the number of binding sites was 0.4 and 0.9,respectively.CD results showed that the two binding models had different effects on ctDNA conformation.Utilizing the fluorescence quenching extent of TB by ctDNA,the genotoxicity interaction intensity of TB was estimated and the interaction intensity of TB was strongest at the 4.0×10~(-1) mol L~(-1) TB and pH range 6.50～8.50.
In the third part,the genotoxicity of the azo dye chrysoidine hydrochloride combined with cetyltrimethyl ammonium bromide(CTMAB) in the living tissue was studied in vitro.The absorption spectrum,resonance light scattering(RLS),CD and transmission electron microscopy(TEM) results showed that the genotoxicity of chrysoidine hydrochloride itself was weak,while the chrysoidine hydrochloride-CTMAB combined aromatic pollutant showed obvious genotoxicity. The assembly of the contaminants on ctDNA,which can be shown by RLS,is considered as a sign of genotoxicity.The genotoxicity of chrysoidine hydrochloride-CTMAB combined aromatic pollution was evaluated by the change of the RLS intensity signal of the system due to the addition of ctDNA.The genotoxicity interaction intensity of the combined pollutant to ctDNA showed a strong co-effect at the dose of 3.0×10~(-4) mol L~(-1) chrysoidine hydrochloride,1.6×10~(-5) mol L~(-1) CTMAB and pH higher than 7.00.The CD results showed that the conformation of ctDNA changed in the genotoxic process of chrysoidine hydrochloride-CTMAB combined contamination.The TEM results showed that ctDNA interact with the chrysoidine hydrochloride-CTMAB combined contamination to form an ion associated complex. The experimental results showed that the combination of chrysoidine hydrochloride-CTMAB had higher genotoxicity than either chrysoidine hydrochloride or CTMAB individually.
In the last part,the environmental genotoxic behavior of the aromatic detergent cetylpyridine bromide(CPB) combined with silver nanoparticles(nanoAg) was examined in vitro.The experimental results showed that the genotoxicity of nanoAg itself was very weak,but when combined with CPB,nanoAg showed obvious genotoxicity.By means of the change of the RLS intensity signal of the nanoAg-CPB system caused by the addition of ctDNA to it,the genotoxicity interaction intensity of nanoAg-CPB combined pollution was estimated.The combined materials had a strong co-effect on ctDNA at the concentration of 3.3×10~(-6) g mL~(-1) nanoAg and 6.0×10~(-6) mol L~(-1) CPB and pH 7.6.After the formation of a nanoAg-CPB complex,the nanoAg aggregate.After the addition of ctDNA to the nanoAg-CPB system,the particles were scattered and the diameter decreased,which illuminated that CPB-nanoAg has genotoxicity.
引文
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