Proper cell division is a fundamental process for the development and sustainability of healthy living organisms. Defective cell division can have deleterious effects on tissue homeostasis and can represent the first step towards disease development. The overall goal of this work was to develop and validate a new, mitosis-based, single-cell toxicity approach. This contributes to the current need of toxicology research to replace animal testing with predictive in vitro models. Cell division-based assays would be better at predicting risk than other commonly used in vitro measurements, such as persistent cell cycle arrest or cell death. Finally, single-cell microscopic analysis provides far deeper insight into the underlying toxicity mechanism(s) than bulk cell population measurements. To meet our goal, we investigated the toxicity of silver nanoparticles (AgNPs) on immortalized human retinal pigmented epithelial (RPE-1) cells. AgNPs are a major nanomaterial employed in product manufacturing due to desirable antimicrobial properties, yet toxicity reports are still confounding. RPE-1 cells were cultured in the presence of low and high doses of polyvinylpyrrolidone (PVP)-coated AgNPs for a single 24-hour treatment (acute treatment), for six 24-hour treatments administered over a period of 3 weeks (moderate treatment), or for twelve 24-hour treatments administered over a period of 6 weeks (chronic treatment). Time-lapse, phase-contrast microscopy of acutely treated cells showed that 100% of cells engulfed AgNPs, which was further confirmed by electron microscopy. Moreover, we found that higher concentrations of AgNPs resulted in large numbers of acutely treated cells becoming arrested in mitosis, dying, or dividing abnormally. In contrast, untreated cells displayed normal mitotic behavior. High-resolution fluorescence microscopy performed in treated cell populations identified an increased percentage of abnormal nuclear morphologies compared to the untreated cells. Further live-cell analysis indicated that treated cells failed cytokinesis or slipped out of mitosis more often than untreated cells. Overall, our results indicate that AgNPs impair cell division, not only further confirming toxicity to human cells, but also revealing previously unreported toxicity mechanisms and highlighting the propagation of adverse phenotypes within the cell population after exposure. Furthermore, this work illustrates that cell division-based single-cell analysis could provide an alternative to animal experimentation in the future. / Master of Science / Multiple agencies, including the U.S. Environmental Protection Agency and the National Academy of Science, are urging for a radical paradigm shift from standard, whole-animal testing to alternative and novel technologies. To meet this urgent need, we aimed to develop a new, cell division-focused toxicity assay by investigating the mechanism of toxicity from silver nanoparticles (AgNPs) on human retinal pigment epithelial (RPE-1) cells. Cultured RPE-1 cells were treated with varying concentrations of AgNPs and live-cell microscopy was used to analyze the behavior of cells undergoing cell division over a 24 hour time period. Physical interaction between cells and particles was visually observed and 100% of treated cells appeared to engulf particles. We found that higher concentrations of AgNPs resulted in large numbers of cells stalling in mitosis and/or dying. In contrast, untreated cells displayed normal mitotic behavior. High-resolution fluorescence microscopy performed in chronically treated cell populations identified an increased percentage of binucleated cells. Further live-cell analysis indicated that one major cell division defect could explain the binucleated cell phenotype. Indeed, treated cells failed cytokinesis (cytoplasmic division following mitotic chromosome segregation) more often than control cells. Overall, our results indicate that AgNPs specifically impair cell division, not only further confirming toxicity to human cells, but also revealing specific, previously unreported toxicity mechanisms and highlighting the propagation of adverse phenotypes within the cell population after exposure. Furthermore, this work illustrates that cell division-based assays and ingle-cell analysis could greatly benefit chemical safety experimentation in the future.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/102095 |
| Date | 26 January 2021 |
| Creators | Garcia, Ellen Brook |
| Contributors | Biological Sciences, Cimini, Daniela, Hauf, Silke, Marr, Linsey C. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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