Australian Rivers Institute, School of Environment and Science, Griffith University, Southport, Qld, 4222, Australia.
Australian Rivers Institute, School of Environment and Science, Griffith University, Southport, Qld, 4222, Australia.
Chemosphere. 2022 Jul;298:134349. doi: 10.1016/j.chemosphere.2022.134349. Epub 2022 Mar 16.
Anthropogenic contaminants can have a variety of adverse effects on exposed organisms, including genotoxicity in the form of DNA damage. One of the most commonly used methods to evaluate genotoxicity in exposed organisms is the micronucleus (MN) assay. It provides an efficient assessment of chromosomal impairment due to either chromosomal rupture or mis-segregation during mitosis. However, evaluating chromosomal damage in the MN assay through manual microscopy is a highly time-consuming and somewhat subjective process. High-throughput evaluation with automated image analysis could reduce subjectivity and increase accuracy and throughput. In this study, we optimised and streamlined the HiTMiN assay, adapting the MN assay to a miniaturised, 96-well plate format with reduced steps, and applied it to both primary cells from green turtle fibroblasts (GT12s-p) and a freshwater fish hepatoma cell line (PLHC-1). Image analysis using both commercial (Columbus) and freely available (CellProfiler) software automated the scoring of MN, with improved precision and drastically reduced time compared to manual scoring and other available protocols. The assay was validated through exposure to two inorganic (chromium and cobalt) and one organic (the herbicide metolachlor) compounds, which are genotoxicants of concern in the marine environment. All compounds tested induced MN formation below cytotoxic concentrations. The HiTMiN assay presented here greatly increases the suitability of the MN assay as a quick, affordable, sensitive and accurate assay to measure genotoxicity of environmental samples in different cell lines.
人为污染物会对暴露的生物体产生各种不良影响，包括以 DNA 损伤形式出现的遗传毒性。评估暴露生物体遗传毒性的最常用方法之一是微核（MN）检测法。它可以有效地评估由于染色体断裂或有丝分裂过程中的错误分离而导致的染色体损伤。然而，通过手动显微镜评估 MN 检测法中的染色体损伤是一个非常耗时且有些主观的过程。通过自动化图像分析进行高通量评估可以减少主观性并提高准确性和通量。在这项研究中，我们优化并简化了 HiTMiN 检测法，将 MN 检测法适应于微型 96 孔板格式，减少了步骤，并将其应用于绿海龟成纤维细胞（GT12s-p）的原代细胞和淡水鱼肝癌细胞系（PLHC-1）。使用商业软件（Columbus）和免费提供的软件（CellProfiler）进行图像分析，自动化 MN 的评分，与手动评分和其他可用方案相比，提高了精度并大大减少了时间。该检测法通过暴露于两种无机（铬和钴）和一种有机（除草剂甲草氯）化合物进行了验证，这些化合物是海洋环境中遗传毒性物质。所有测试的化合物在细胞毒性浓度以下均诱导 MN 形成。这里提出的 HiTMiN 检测法大大提高了 MN 检测法作为一种快速、经济、敏感和准确的检测法，用于测量不同细胞系中环境样品遗传毒性的适用性。
