Chen Tao, Dusinska Maria, Elespuru Rosalie
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, United States.
Health Effects Laboratory, NILU-Norwegian Institute for Air Research, Kjeller, Norway.
Front Toxicol. 2022 Jun 8;4:864753. doi: 10.3389/ftox.2022.864753. eCollection 2022.
The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM characterization, sample preparation and dose selection, relevant to all genotoxicity assays, are found in an accompanying paper. The present paper describes methods for evaluation of mutagenicity in the mammalian (mouse) () gene occurring in L5178Y mouse lymphoma (ML) cells and in the designated gene in human lymphoblastoid TK6 cells. Mutations change the functional genotype from TK to TK, detectable as cells surviving on media selective for the lack of thymidine kinase (TK) function. Unlike cells with TK enzyme function, the TK cells are unable to integrate the toxic selection agent, allowing these cells to survive as rare mutant colonies. The ML assay has been shown to detect a broad spectrum of genetic damage, including both small scale (point) mutations and chromosomal alterations. This assay is a widely used mammalian cell gene mutation assay for regulatory purposes and is included in the core battery of genotoxicity tests for regulatory decision-making. The TK6 assay is an assay using a human cell line derived similarly via mutagenic manipulations and optimal selection. Details are provided on the materials required, cell culture methods, selection of test chemical concentrations, cytotoxicity, treatment time, mutation expression, cloning, and data calculation and interpretation. The methods describe the microwell plate version of the assays without metabolic activation.
本文概述的方法是专门为纳米材料（NM）遗传毒性评估设计的一系列论文的一部分。在一篇配套论文中可以找到与所有遗传毒性试验相关的常见注意事项，如NM表征、样品制备和剂量选择。本文描述了评估L5178Y小鼠淋巴瘤（ML）细胞中哺乳动物（小鼠）（）基因以及人淋巴母细胞TK6细胞中指定基因致突变性的方法。突变会将功能基因型从TK变为TK，可通过在缺乏胸苷激酶（TK）功能的选择培养基上存活的细胞检测到。与具有TK酶功能的细胞不同，TK细胞无法整合有毒选择剂，从而使这些细胞作为罕见的突变菌落存活下来。ML试验已被证明能检测广泛的遗传损伤，包括小规模（点）突变和染色体改变。该试验是一种广泛用于监管目的的哺乳动物细胞基因突变试验，并且包含在用于监管决策的遗传毒性测试核心组合中。TK6试验是一种使用类似地通过诱变操作和最佳选择获得的人细胞系的试验。文中提供了所需材料、细胞培养方法、测试化学物质浓度选择、细胞毒性、处理时间、突变表达、克隆以及数据计算和解释的详细信息。这些方法描述了无代谢激活的微孔板版本试验。
