Moore F R, Urda G A, Krishna G, Theiss J C
Department of Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA.
Mutat Res. 1995 Oct;335(2):191-9. doi: 10.1016/0165-1161(95)90055-1.
The mouse micronucleus assay has long been used as an indicator of in vivo genotoxicity. Recently, it was shown that no single protocol is adequate to detect all clastogens. As a first step in developing a potentially more sensitive assay, micronucleus induction by cyclophosphamide (CP) was assessed in an in vivo/in vitro system using rat bone marrow and spleen cells. In each of two independent experiments, two rats/dose were treated i.p. with 0, 20, or 40 mg CP/kg and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested and slides prepared 24 h after initiation, while spleen cells were harvested at 48 h. One thousand cells/tissue/group were scored for cell cycle kinetics and 1000 binucleate (BN) cells were scored for micronuclei. In addition, spleen cells were concurrently assayed for chromosome aberrations. A dose-related cell cycle delay was observed in both tissues in both experiments. Bone marrow showed a 6% average background frequency of micronucleated BN cells, while the low dose induced an average of 20%, and the high dose 31%. For spleen, the average control frequency of micronucleated BN cells was 3%, the low dose induced a 40% average frequency, and the high dose 65%. Also in splenocytes, a dose-dependent increase in chromosome aberrations was observed, with an almost 40-fold increase observed over the control value at the high dose. Thus, the in vivo/in vitro approach described here shows great potential in detecting drug induced genotoxicity. Also, spleen appears more sensitive than bone marrow to CP.
小鼠微核试验长期以来一直被用作体内遗传毒性的指标。最近有研究表明，没有单一的方案足以检测所有的致断裂剂。作为开发一种可能更敏感的检测方法的第一步，在体内/体外系统中使用大鼠骨髓和脾细胞评估了环磷酰胺（CP）诱导的微核形成。在两个独立实验中，每组两只大鼠分别腹腔注射0、20或40mg CP/kg，并在6小时后处死。然后在生长刺激剂（骨髓用白细胞介素-3和粒细胞-巨噬细胞集落刺激因子；脾用脂多糖和伴刀豆球蛋白A）和细胞松弛素B（一种胞质分裂抑制剂）存在的情况下建立培养体系。启动培养24小时后收获骨髓细胞并制备玻片，而脾细胞在48小时收获。对每组组织的1000个细胞进行细胞周期动力学评分，对1000个双核（BN）细胞进行微核评分。此外，同时对脾细胞进行染色体畸变检测。在两个实验中的两个组织中均观察到剂量相关的细胞周期延迟。骨髓中微核化BN细胞的平均背景频率为6%，低剂量组平均诱导率为20%，高剂量组为31%。对于脾，微核化BN细胞的平均对照频率为3%，低剂量组平均诱导频率为40%，高剂量组为65%。同样在脾细胞中，观察到染色体畸变呈剂量依赖性增加，高剂量组比对照值增加了近40倍。因此，本文所述的体内/体外方法在检测药物诱导的遗传毒性方面显示出巨大潜力。此外，脾对CP的敏感性似乎比骨髓更高。
