蚯蚓生物标志物在手性有机磷农药污染评价中的应用研究
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摘要
当今使用的有机磷农药超过30%是手性的,且目前对于手性有机磷农药的对映体选择性的研究多集中在水生毒性上,对于非靶标生物特别是土壤生物的研究比较少。本研究挑选蚯蚓的乙酰胆碱酶、体腔细胞作为生物标志物来评价甲胺磷、马拉氧磷、异马拉硫磷三个手性有机磷农药的对映体选择性,相关研究内容与结果如下:
(1)以乙酰胆碱酶半抑制浓度(IC_(50))为检测指标,研究了甲胺磷、马拉氧磷、异马拉硫磷三个手性有机磷农药对蚯蚓体内外乙酰胆碱酶抑制作用的立体选择性,结果均表现出显著的对映体选择性。体内抑制研究中,外消旋体和(+)-甲胺磷的IC50值分别为0.0281μg/cm2和0.0860μg/cm~2,相差了3.1倍;马拉氧磷中,(+)-、(-)-异构体的IC50值分别为0.0014μg/cm~2和0.0058μg/cm~2,两者相差4.1倍;异马拉硫磷中,1R,3R-异构体与外消旋体的IC_(50)值相差达331倍之多,差异非常显著。体外抑制的动力学常数中我们也发现了明显的对映体差异性:(-)-和(+)-甲胺磷的k_i值(双分子反应常数,表征乙酰胆碱酶的抑制程度)差异是5倍;(+)-和(-)-马拉氧磷的k_i值差异是3.23倍,异马拉硫磷四个对映体异构体的毒性大小关系是1R,3R-异马拉硫磷>1S,3R-异马拉硫磷>1S,3S-异马拉硫磷>1R,3S-异马拉硫磷,k_i值差异在1.4~12.12倍之间。三个有机磷农药对乙酰胆碱酶的抑制均表现出明显的剂量-效应关系,随着抑制浓度的增加,酶活性的抑制随之增强。酶抑制动力学研究表明不同异构体对AChE的空间取向和磷酰化性能均不相同。
(2)利用中性红保留时间(NRRT)来评价体腔细胞溶酶体的膜稳定性,研究结果显示有机磷农药污染会使蚯蚓的溶酶体膜发生损害,从而破坏其稳定性,在(±)-甲胺磷中,当浓度为0.0016μg/cm~2时,NRRT值为76.88min,然而当浓度的升高到0.16μg/cm~2时,NRRT值降为29.78min,随着浓度的增加(±)-马拉氧磷的NRRT值从61.13min降到34.13min,(±)-异马拉硫磷的NRRT值从47.60min缩短到34.32min,中性红保留时间越短,说明溶酶体膜稳定性的越差,溶酶体膜破坏的越严重。实验结果也显示了一定的对映体选择性,但是差异不是特别显著,均在2倍以下。蚯蚓作为环节无脊椎动物,它的体腔是免疫系统的重要组成,说明有机磷农药污染会对蚯蚓造成免疫伤害。
(3)实验还研究了马拉硫磷对蚯蚓乙酰胆碱酶抑制后的恢复状况,表明马拉硫磷对蚯蚓乙酰胆碱酶抑制是一个可逆的过程,蚯蚓经过48h暴露处理后,乙酰胆碱酶的活性被抑制了80%多,转移到干净土壤后的7天内,抑制表现出持续加强的现象,之后酶活并开始逐渐恢复,第30天检测时,酶活只被抑制了27.7%,活性恢复了70%左右的水平。
(4)通过彗星实验研究了马拉氧磷对蚯蚓体腔细胞的DNA损伤,实验结果表明马拉氧磷对蚯蚓体腔细胞的DNA损伤非常的严重,在最低浓度0.0016μg cm~(-2)的暴露水平下就出现了非常明显的拖尾现象,空白对照组也有一点程度的损伤。
More than 30% of the current used organophosphorus (OPs) insecticides are chiral. The studies of enantioselectivity in the aquatic toxicity of chiral OPs have been steadily increasing, however, research concerning enantioselectivity in the toxicity of OPs to nontarget and nonmammalian species, particularly soil organisms, is still inadequate. In this study, the acetylcholinesterase (AChE) activity and coelomocytes lysosomal membrane stability of earthworm (Eisenia fetida) were selected as biomarkers, and the enantioselective effects of three chiral OPs (methamidophos, malaoxon, and isomalathion) were evaluated. Related research content and results are as follows:
(1)The inhibition of acetylcholinesterase (AChE) activity by the chiral OPs all showed significant enantioselectivity. In the vivo research, the IC50 value of (±)-methamidophos on the inhibition of the AChE was 0.0281μg/cm~2, the value of (+)-methamidophos was 0.0860μg/cm~2,with 3.1 fold differences between them. The most toxic of malaoxon to AChE was (+)-malaoxon, the IC~(50) value was 0.0014μg/cm~2, the value of the weakest ((-)-malaoxon)was 0.0058μg/cm~2, with 4.1 fold differences. The most obvious difference in the three chiral OPs was 1R,3R-isomalation with (±)-isomalation, they were 331 fold differences. From the value of inhibition kinetic constants we also could find enantioselective difference, the value of ki expression the degree of AChE inhibition, the value of ki of (-)-methamidophos was 5 times higher than (+)-methamidophos, and (+)-malaoxon was 3.23 times higher than (-)-malaoxon, as to the four stereoisomers of isomalation the order of toxicity was 1R,3R-isomalation> 1S,3R-isomalation>1S,3S-isomalation>1R,3S-isomalation,with 1.4~12.12 fold differences. The relationship between the concentratin of organophosphorus pesticide and AChE inhibtion showed obvious dose effect relationship, with the increase in inhibitory concentration, result in stronger inhibition of enzyme activity. The enzyme inhibition dynamic assays further indicated that both the spatial orientations and phosphorylation properties of different stereoisomers towards AChE were different.
(2)The research of using neutral red retention time (NRRT) evaluation lysosomal membrane stability of coelomocytes showed organophosphorus pesticide contamination could injure lysosomal membrane stability of earthworm. As the concentration of (±)-methamidophos was 0.0016μg /cm~2, the value of NRRT was 76.88min, with the concentration increase to 0.16μg/cm~2, the value of NRRT reduced to 29.78min, the value of NRRT of (±)-malaoxon dropped from 61.13min to 34.13min, (±)-isomalation’s from 47.60min to 34.32min. The shorter neutral red retention time was, the worse the stability of lysosomal membrane, the more serious damage of lysosomal membrane. Also these researches showed enantioselective effect, but no significant difference contrast to acetylcholine activity study, the differences were maintained at 2 times under. Earthworms as part of invertebrates, its coelom is an important component of the immune system, illustrated that organophosphorus pesticide-contaminated will result in immune injury of earthworms.
(3)The recovery of AChE of the earthworm after treated by malathion, was studied that malathion inhibited acetylcholine of earthworms is a reversible process, after 48h exposure treatment, AChE activity of earthworms was inhibited 80%,when transfer to a clean soil within 7 days after,sustained inhibition enhanced,after, AChE activity gradually recovered,when detectioned in the 30th day, AChE activity was only inhibited 27.7%,AChE activity recovered 70%.
(4)The comet assay was used to evaluate the damagement of coelomocytes DNA of earthworm by malaoxon, the result showed that malaoxon cause very serious DNA damage, when at the lowest expose concentration of 0.0016μg/cm~2, Very obvious smearing was observed, and on the control group also have a little degree of injury, We believed that high levels of genetic damage in itself was a universal feature of earthworms.
(1)以乙酰胆碱酶半抑制浓度(IC_(50))为检测指标,研究了甲胺磷、马拉氧磷、异马拉硫磷三个手性有机磷农药对蚯蚓体内外乙酰胆碱酶抑制作用的立体选择性,结果均表现出显著的对映体选择性。体内抑制研究中,外消旋体和(+)-甲胺磷的IC50值分别为0.0281μg/cm2和0.0860μg/cm~2,相差了3.1倍;马拉氧磷中,(+)-、(-)-异构体的IC50值分别为0.0014μg/cm~2和0.0058μg/cm~2,两者相差4.1倍;异马拉硫磷中,1R,3R-异构体与外消旋体的IC_(50)值相差达331倍之多,差异非常显著。体外抑制的动力学常数中我们也发现了明显的对映体差异性:(-)-和(+)-甲胺磷的k_i值(双分子反应常数,表征乙酰胆碱酶的抑制程度)差异是5倍;(+)-和(-)-马拉氧磷的k_i值差异是3.23倍,异马拉硫磷四个对映体异构体的毒性大小关系是1R,3R-异马拉硫磷>1S,3R-异马拉硫磷>1S,3S-异马拉硫磷>1R,3S-异马拉硫磷,k_i值差异在1.4~12.12倍之间。三个有机磷农药对乙酰胆碱酶的抑制均表现出明显的剂量-效应关系,随着抑制浓度的增加,酶活性的抑制随之增强。酶抑制动力学研究表明不同异构体对AChE的空间取向和磷酰化性能均不相同。
(2)利用中性红保留时间(NRRT)来评价体腔细胞溶酶体的膜稳定性,研究结果显示有机磷农药污染会使蚯蚓的溶酶体膜发生损害,从而破坏其稳定性,在(±)-甲胺磷中,当浓度为0.0016μg/cm~2时,NRRT值为76.88min,然而当浓度的升高到0.16μg/cm~2时,NRRT值降为29.78min,随着浓度的增加(±)-马拉氧磷的NRRT值从61.13min降到34.13min,(±)-异马拉硫磷的NRRT值从47.60min缩短到34.32min,中性红保留时间越短,说明溶酶体膜稳定性的越差,溶酶体膜破坏的越严重。实验结果也显示了一定的对映体选择性,但是差异不是特别显著,均在2倍以下。蚯蚓作为环节无脊椎动物,它的体腔是免疫系统的重要组成,说明有机磷农药污染会对蚯蚓造成免疫伤害。
(3)实验还研究了马拉硫磷对蚯蚓乙酰胆碱酶抑制后的恢复状况,表明马拉硫磷对蚯蚓乙酰胆碱酶抑制是一个可逆的过程,蚯蚓经过48h暴露处理后,乙酰胆碱酶的活性被抑制了80%多,转移到干净土壤后的7天内,抑制表现出持续加强的现象,之后酶活并开始逐渐恢复,第30天检测时,酶活只被抑制了27.7%,活性恢复了70%左右的水平。
(4)通过彗星实验研究了马拉氧磷对蚯蚓体腔细胞的DNA损伤,实验结果表明马拉氧磷对蚯蚓体腔细胞的DNA损伤非常的严重,在最低浓度0.0016μg cm~(-2)的暴露水平下就出现了非常明显的拖尾现象,空白对照组也有一点程度的损伤。
More than 30% of the current used organophosphorus (OPs) insecticides are chiral. The studies of enantioselectivity in the aquatic toxicity of chiral OPs have been steadily increasing, however, research concerning enantioselectivity in the toxicity of OPs to nontarget and nonmammalian species, particularly soil organisms, is still inadequate. In this study, the acetylcholinesterase (AChE) activity and coelomocytes lysosomal membrane stability of earthworm (Eisenia fetida) were selected as biomarkers, and the enantioselective effects of three chiral OPs (methamidophos, malaoxon, and isomalathion) were evaluated. Related research content and results are as follows:
(1)The inhibition of acetylcholinesterase (AChE) activity by the chiral OPs all showed significant enantioselectivity. In the vivo research, the IC50 value of (±)-methamidophos on the inhibition of the AChE was 0.0281μg/cm~2, the value of (+)-methamidophos was 0.0860μg/cm~2,with 3.1 fold differences between them. The most toxic of malaoxon to AChE was (+)-malaoxon, the IC~(50) value was 0.0014μg/cm~2, the value of the weakest ((-)-malaoxon)was 0.0058μg/cm~2, with 4.1 fold differences. The most obvious difference in the three chiral OPs was 1R,3R-isomalation with (±)-isomalation, they were 331 fold differences. From the value of inhibition kinetic constants we also could find enantioselective difference, the value of ki expression the degree of AChE inhibition, the value of ki of (-)-methamidophos was 5 times higher than (+)-methamidophos, and (+)-malaoxon was 3.23 times higher than (-)-malaoxon, as to the four stereoisomers of isomalation the order of toxicity was 1R,3R-isomalation> 1S,3R-isomalation>1S,3S-isomalation>1R,3S-isomalation,with 1.4~12.12 fold differences. The relationship between the concentratin of organophosphorus pesticide and AChE inhibtion showed obvious dose effect relationship, with the increase in inhibitory concentration, result in stronger inhibition of enzyme activity. The enzyme inhibition dynamic assays further indicated that both the spatial orientations and phosphorylation properties of different stereoisomers towards AChE were different.
(2)The research of using neutral red retention time (NRRT) evaluation lysosomal membrane stability of coelomocytes showed organophosphorus pesticide contamination could injure lysosomal membrane stability of earthworm. As the concentration of (±)-methamidophos was 0.0016μg /cm~2, the value of NRRT was 76.88min, with the concentration increase to 0.16μg/cm~2, the value of NRRT reduced to 29.78min, the value of NRRT of (±)-malaoxon dropped from 61.13min to 34.13min, (±)-isomalation’s from 47.60min to 34.32min. The shorter neutral red retention time was, the worse the stability of lysosomal membrane, the more serious damage of lysosomal membrane. Also these researches showed enantioselective effect, but no significant difference contrast to acetylcholine activity study, the differences were maintained at 2 times under. Earthworms as part of invertebrates, its coelom is an important component of the immune system, illustrated that organophosphorus pesticide-contaminated will result in immune injury of earthworms.
(3)The recovery of AChE of the earthworm after treated by malathion, was studied that malathion inhibited acetylcholine of earthworms is a reversible process, after 48h exposure treatment, AChE activity of earthworms was inhibited 80%,when transfer to a clean soil within 7 days after,sustained inhibition enhanced,after, AChE activity gradually recovered,when detectioned in the 30th day, AChE activity was only inhibited 27.7%,AChE activity recovered 70%.
(4)The comet assay was used to evaluate the damagement of coelomocytes DNA of earthworm by malaoxon, the result showed that malaoxon cause very serious DNA damage, when at the lowest expose concentration of 0.0016μg/cm~2, Very obvious smearing was observed, and on the control group also have a little degree of injury, We believed that high levels of genetic damage in itself was a universal feature of earthworms.
引文
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