砷暴露的体外及体内微核试验研究

Micronucleus test of arsenic exposure in vitro and in vivo

目的探讨砷的遗传损伤作用。方法体外试验用胞质分裂阻滞法微核试验(CBMN)进行,观察不同浓度砷(0μg/L、50μg/L、100μg/L、200μg/L、400μg/L)对人淋巴细胞微核率的影响。在RPMI1640培养基中加入正常人淋巴细胞和不同浓度砷在37℃中培养72 h(44 h时加入细胞松弛素B),经低渗固定等收获细胞后染色观察双核淋巴细胞中微核率。体内试验取40只ICR雄性小鼠应用完全随机化的方法分为4个剂量组[阴性对照组、10 mg/(kg·bw)、20 mg/(kg·bw)、30 mg/(kg·bw)],每组10只,将亚砷酸钠按剂量要求经口灌胃染毒3次,染毒后取尾静脉按常规血涂片进行涂片染色,观察外周血嗜多染红细胞微核率。结果体外试验砷接触72 h后,砷浓度200μg/L、400μg/L的双核淋巴细胞微核率分别为10.4‰和20.2‰,与砷浓度0μg/L比较差异均有统计学意义(P<0.05);体内试验ICR小鼠染毒亚砷酸钠后,阴性对照组、10 mg/(kg·bw)、20 mg/(kg·bw)、30 mg/(kg·bw)各剂量组动物外周血嗜多染红细胞微核率分别为0.7‰、0.8‰、1.6‰、2.7‰,呈剂量-反应关系,30 mg/(kg·bw)剂量组外周血嗜多染红细胞微核率明显升高,与阴性对照组比较差异有统计学意义(P<0.05)。结论亚砷酸钠体外试验可导致人双核淋巴细胞微核率增加,体内试验也可导致外周血嗜多染红细胞微核率升高,体内与体外试验结果均提示砷具有遗传损伤作用。

Objective To study on the genetic damage of arsenic by micronucleus test in vitro.Methods The effects of different concentrations of arsenic(0μg/L,50μg/L,100μg/L,200μg/L,and 400μg/L)on the micronucleus rate of human lymphocytes were observed in vitro by cytokinesis blocked micronucleus test(CBMN).In RPMI1640 medium,normal human lymphocytes and different concentrations of arsenic were added and cultured at 37℃ for 72 hours(cytochalasin B was added at 44 h),and the micronucleus rate of binuclear lymphocytes was observed by staining of harvested cells after hypotonic fixation.For in vivo test,40 male ICR mice were randomly divided into four dose groups:negative control group,10 mg/(kg·bw),20 mg/(kg·bw),30 mg/(kg·bw),with 10 mice in each group.Sodium arsenite was administrated by oral gavage for three times according to the dose requirements,then the tail vein was stained by smear staining according to routine blood smear.The micronucleus rate of polychromatic erythrocytes in peripheral blood were observed.Results After 72 h of arsenic exposure in vitro,the themicronucleus rate of binuclear lymphocytes with arsenic concentration of 200μg/L and 400μg/L was 10.4‰ and 20.2‰,respectively,with no significant difference in the arsenic concentration 0μg/L(P<0.05);After ICR mice exposed to sodium arsenite in vivo test,the micronucleus rate of polychromatic erythrocytes in peripheral blood was 0.7‰,0.8‰,1.6‰,2.7‰ in negative control group,10 mg/(kg·bw),20 mg/(kg·bw),30 mg/(kg·bw),and there was dose-response relationship in each group.The micronucleus rate of polychromatic erythrocytes in peripheral blood of 30 mg/(kg·bw)dose group was significantly higher than that in negative control group,and the difference was statistically significant(P<0.05).Conclusion Sodium arsenite can increase the micronucleus rate of human binucleate lymphocytes in vitro tset and peripheral blood polychromatic erythrocytes in vivo test.The results in vivo and in vitro suggest that arsenic has genetic damage.