苯胺对黑斑蛙蝌蚪外周血红细胞的遗传毒性效应

Genotoxic Effects of Aniline on Peripheral Blood Erythrocytes in Black-spotted Pond Frog(Rana nigromaculata) Tadpoles

为从不同遗传终点检测苯胺对黑斑蛙(Rana nigromaculata)蝌蚪红细胞的遗传毒性,将黑斑蛙蝌蚪暴露于0、3.45、17.26、34.53、69.06μg/L不同浓度的苯胺96 h后,显微镜下观察红细胞形态和数目的变化,采用微核试验测定红细胞微核率,通过彗星试验测定彗星尾长和尾距的变化。从17.26μg/L浓度组开始出现红细胞变形拉长和细胞膜破裂,且随着苯胺浓度的增加而增多。另外,各浓度组蝌蚪红细胞数目随着苯胺溶液浓度的增加而逐渐减少,且与空白对照相比差异显著(P<0.01)。微核试验结果显示,各浓度处理组微核率均显著高于空白对照组(P<0.05),但由于苯胺所致的红细胞破裂和Heinz小体的影响,微核率和浓度之间并未出现明显的浓度-效应关系。彗星试验结果显示,不同浓度苯胺处理组与空白对照组相比,蝌蚪红细胞尾长和尾距均显著增加(P<0.05或P<0.01),并与处理浓度之间存在显著的浓度-效应关系。上述结果表明,苯胺可诱发黑斑蛙蝌蚪红细胞的染色体、DNA损伤,具有较强的遗传毒性效应;苯胺最高浓度处理组69.06μg/L蝌蚪红细胞DNA损伤水平与5 mg/L环磷酰胺相近,显现明显的DNA损伤,因此建议渔业水质标准对水体中苯胺限量的规定不应高于此值。

To explore the genotoxicity of aniline on the erythrocytes of black-spotted pond frog（Rana nigromaculata） tadpoles, black-spotted pond frog tadpoles were exposed to different concentrations of 0, 3.45, 17.26, 34.53 and 69.06 μg/L aniline respectively for 96 h; in the meantime some tadpoles were exposed to 5 mg/L cyclophosphamide as a positive control. The blood samples were collected from each tadpole by the docking method. The changes of the erythrocytes in morphology and number were observed by optical microscopy, the micronuclei frequency of the erythrocytes was assessed by micronucleus test（MNT）, the tail length and the tail moment of the erythrocytes were measured by comet assay（CA）. The morphological observation showed that the erythrocytes began to be deformed and lengthened, the erythrocyte membrane began to be broken（Fig. 1） in 17.26 μg/L group, with the increase in a aniline-dose-dependent manner. Erythrocyte number analysis showed that in all treatment groups the erythrocyte numbers were decreased gradually with the increase of the concentration of aniline, and there was significant difference compared with the blank control group（P 0.01）（Table 1）. The results of MNT showed that the micronuclear frequency in all treatment groups were significantly higher than that of the blank control group（P 0.05）（Fig. 1, Table 2）, but as a result of the influence of cell rupture and Heinz corpuscle resulted from aniline, there was no obvious concentration-effect relation between the micronuclear frequency and the aniline concentration. The results of CA showed that both the tail length and the tail moment in all treatment groups were significantly increased compared with the blank control group（P 0.05, P 0.01）（Fig. 2, Table 3）; moreover, there was obvious concentration-effect relation among different treatment concentrations. The results aforementioned suggest that aniline may induce chromosome and DNA damage in the erythrocytes of black-spotted pond frog tadpole, indicating a strong genotoxic effect. Furthermore, the level of DNA damage at the highest concentration（69.06 μg/L） of aniline was similar to that of 5 mg/L cyclophosphamide（the positive control）, therefore it is recommended that the upper limit of aniline content in water body set by the water quality standard for fisheries should not be higher than this value.