Optimized High-Content Imaging Screening Quantifying Micronuclei Formation in Polymer-Treated HaCaT Keratinocytes

Abstract

Research on nano- and micro-plastic particles (NMPPs) suggests their potential threat to human health. Some studies have even suggested genotoxic effects of NMPP exposure, such as micronuclei (MN) formation, while others found the opposite. To clarify the ability of NMPP to induce MN formation, we used non-malignant HaCaT keratinocytes and exposed these to a variety of polystyrene (PS) and poly methyl methacrylate (PMMA) particle types at different concentrations and three different sizes. Investigations were performed following acute (one day) and chronic exposure (five weeks) against cytotoxic (amino-modified NMPPs) and genotoxic (methyl methanesulfonate, MMS) positive controls. An optimized high-content imaging workflow was established strictly according to OECD guidelines for analysis. Algorithm-based object segmentation and MN identification led to computer-driven, unsupervised quantitative image analysis results on MN frequencies among the different conditions and thousands of cells per condition. This could only be realized using accutase, allowing for partial cell detachment for optimal identification of bi-nucleated cells. Cytotoxic amino-modified particles were not genotoxic; MMS was both. During acute and long-term studies, PS and PMMA particles were neither toxic nor increased MN formation, except for 1000 nm PS particles at the highest concentration of unphysiological 100 µg/mL. Interestingly, ROS formation was significantly decreased in this condition. Hence, most non-charged polymer particles were neither toxic nor genotoxic, while aminated particles were toxic but not genotoxic. Altogether, we present an optimized quantitative imaging workflow applied to a timely research question in environmental toxicity.

Cold Physical Plasma Toxicity in Breast and Oral Squamous Carcinoma In Vitro and in Patient-Derived Cancer Tissue Ex Vivo

“…One aspect of assessing safety is studying genotoxic effects [30]. We have previously established a highthroughput, high-content imaging micronucleus assay incorporating the requirements of OECD guidelines on the CBMN assay and utilizing the power of algorithm-driven object segmentation for unsupervised analysis and quantification of micronuclei in binucleated cells [19]. Several studies have demonstrated that cold physical plasma-induced reactive oxygen species (ROS) are responsible for plasma-induced toxicity, have been associated with some hallmarks of cancer and are thought to spur malignancy and DNA damage when applied chronically at higher concentrations [31].…”

“…The cells were then incubated for an additional hour and treated with various treatment times of cold physical plasma (0, 20 s, 40 s, 60 s, 90 s, 120 s, 180 s, 240 s, and 300 s) using the atmospheric pressure argon plasma jet kINPen IND (neoplas, Greifswald, Germany) [18] operated with 2.5 and 1.0 standard liters of argon gas per minute for cytotoxicity and genotoxicity analysis, respectively. The lower feed gas flux was necessary for the latter as the higher fluxes were too toxic for the assay to be performed adequately based on our previously optimized OECD protocol with algorithm-driven image quantification analysis [19]. The distance between the plasma jet nozzle and the cell culture liquid covering the adherent cells was 10 mm.…”

“…After incubation overnight to allow attachment, cells were exposed to cold physical plasma for 10 s (1 slm) and incubated for 6 h. To achieve maximum binucleated cells (BNCs) as per OECD guideline [20], cells were treated with cytochalasin B (final concentration: 2 ug/mL) to block cytokinesis and allow the identification of MN in the cells that have completed one cell division and created BNC. The assay was performed as recently described [19].…”