Genetic Toxicology and Safety Pharmacology, Preclinical Safety, Novartis Institutes for Biomedical Research, Werk Klybeck, Klybeckstrasse 141, CH-4057 Basel, Switzerland.
Mutagenesis. 2011 Nov;26(6):763-70. doi: 10.1093/mutage/ger044. Epub 2011 Jul 26.
In order to minimise the number of positive in vitro cytogenetic results which are not confirmed in rodent carcinogenicity tests, biological systems that are p53 and DNA repair proficient should be recommended. Moreover, an appropriate cytotoxicity parameter for top dose selection should be considered. Recent International Conference on Harmonisation draft S2 and Organisation for Economic Co-operation and Development (OECD) 487 guideline accepted the in vitro micronucleus test (MNT) as a valid alternative method for in vitro chromosome aberration test within the in vitro cytogenetic test battery. Since mitosis is a prerequisite for expression of the micronuclei, it is compulsory to demonstrate that cell division occurred, and if possible, to identify the cells that completed mitosis. The OECD guideline recommends the use of a cytokinesis block for the assessment of proliferation in primary T-lymphocytes. The work presented in this manuscript was initiated to develop a novel flow cytometry-based primary human lymphocyte MNT method. This new assay is based on a three-step staining procedure: carboxyfluorescein succinimidyl ester as a proliferation marker, ethidium monoazide for chromatin of necrotic and late apoptotic cells discrimination and 4,6-diaminodino-2-phenylindole as a DNA marker. The proof of principle of the method was performed using genotoxic and non-genotoxic compounds: methyl methanesulfonate, mitomycin C, vinblastine sulphate, cyclophosphamide, sodium chloride and dexamethasone. It has been shown that the new flow cytometry-based primary human lymphocyte MNT method is at least equally reliable method as the standard Cytochalasin B MNT. However, further validation of the assay using a wide selection of compounds with a variety of mechanisms of action is required, before it can be used for regulatory purposes. Moreover, a miniaturisation of the technology may provide an additional advantage for early drug development.
为了将在啮齿类动物致癌性试验中未得到确认的体外细胞遗传学阳性结果的数量降至最低，应当推荐使用 p53 和 DNA 修复功能健全的生物系统。此外，还应考虑选择合适的细胞毒性参数来确定最高剂量。最近的国际协调会议（ICH）草案 S2 和经济合作与发展组织（OECD）487 准则接受了体外微核试验（MNT）作为体外细胞遗传学检测组合中体外染色体畸变试验的一种有效替代方法。由于有丝分裂是微核表达的前提，因此必须证明细胞分裂已经发生，如果可能的话，还需要识别完成有丝分裂的细胞。OECD 准则建议在原代 T 淋巴细胞中使用细胞有丝分裂阻断来评估增殖情况。本文介绍的工作旨在开发一种新的基于流式细胞术的原代人淋巴细胞 MNT 方法。该新检测方法基于三步染色程序：羧基荧光素琥珀酰亚胺酯作为增殖标记物，乙锭单叠氮化物用于区分坏死和晚期凋亡细胞的染色质，4,6-二脒基-2-苯基吲哚作为 DNA 标记物。该方法的原理验证使用了遗传毒性和非遗传毒性化合物：甲磺酸甲酯、丝裂霉素 C、长春花碱硫酸盐、环磷酰胺、氯化钠和地塞米松。结果表明，该新的基于流式细胞术的原代人淋巴细胞 MNT 方法与标准细胞松弛素 B MNT 至少同样可靠。然而，在将该检测方法用于监管目的之前，需要使用多种作用机制的化合物对该检测方法进行进一步验证。此外，该技术的小型化可能为早期药物开发提供额外优势。
