Application of Genomics-Based Methodologies in Genetic Toxicology to Advance In Vitro Chemical Assessment
Public Deposited- Resource Type
- Creator
- Abstract
The current standard regulatory tests in genetic toxicology are inadequate for effectively addressing the growing number of chemicals needing assessment. Specifically, the standard in vitro assays do not provide sufficient mechanistic information to inform relevance to in vivo toxicity. The resulting reliance on in vivo models in regulatory toxicology has hindered efficient chemical assessment. Thus, international efforts are underway to reduce animal testing and modernize toxicity assessment by developing and promoting non-animal alternatives, or new approach methodologies (NAMs). The goal of my thesis was to develop and demonstrate the application of genomics NAMs for quantifying genotoxic hazards and obtaining information on mechanism of action to advance in vitro chemical assessment. First, the inter-platform transferability of the TGx-DDI biomarker of DNA damage-induction was investigated. TGx-DDI is a 64-gene biomarker developed using DNA microarrays in human TK6 cells that provides mechanistic support to conventional genotoxicity assays. The biomarker demonstrated a conserved performance when measured by qPCR, demonstrating that transcriptomic biomarkers can be adapted to this widely available platform. Additional chemical testing methods were explored using two next-generation sequencing-based technologies: TempO-Seq, a targeted gene expression platform, and Duplex Sequencing (DuplexSeq), an emerging technology that enables direct and accurate detection of mutations in cells. A transcriptomic biomarker of histone deacetylase inhibition, TGx-HDACi, was developed from TempO-Seq whole transcriptome profiles to address the limited assays available for detecting epigenetic mechanisms of toxicity. The availability of TGx-HDACi contributes to diversifying the transcriptomic biomarkers that can be applied in high-throughput screening of transcriptomic profiles of chemicals. Next, a comparative, inter-laboratory study in TK6 cells identified an optimal experimental design for applying DuplexSeq as a mutagenicity assay. DuplexSeq detected a robust concentration-response in cells exposed to an alkylating agent, with high sensitivity. The results also revealed strong inter-laboratory reproducibility and the power of DuplexSeq in providing a comprehensive view of chemical-induced mutagenesis. Lastly, an Adverse Outcome Pathway (AOP) describing oxidative DNA damage leading to chromosomal aberrations and mutations was developed to provide a mechanistic framework for combining NAMs in integrated testing. Collectively, my thesis lays the foundation for development and advancement of NAMs and AOPs in genetic toxicology.
- Subject
- Language
- Publisher
- Thesis Degree Level
- Thesis Degree Name
- Thesis Degree Discipline
- Identifier
- Rights Notes
Copyright © 2023 the author(s). Theses may be used for non-commercial research, educational, or related academic purposes only. Such uses include personal study, research, scholarship, and teaching. Theses may only be shared by linking to Carleton University Institutional Repository and no part may be used without proper attribution to the author. No part may be used for commercial purposes directly or indirectly via a for-profit platform; no adaptation or derivative works are permitted without consent from the copyright owner.
- Date Created
- 2023
Relations
- In Collection:
Items
Thumbnail | Title | Date Uploaded | Visibility | Actions |
---|---|---|---|---|
cho-applicationofgenomicsbasedmethodologiesin.pdf | 2023-05-05 | Public | Download |