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.

Keywords

• algorithm
• micro-plastic
• nano-plastic
• cytotoxicity
• Operetta CLS