Investigation of the cytotoxicity of thymidine analogues used to monitor DNA replication in trypanosomatids

• Grant number: 23/07193-0
• Support Opportunities: Scholarships in Brazil - Master
• Effective date (Start): December 01, 2023
• Effective date (End): April 30, 2025
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Marcelo Santos da Silva
• Grantee: Marcos Antonio Fernandes de Oliveira
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 19/10753-2 - Investigation on the role of inositol pyrophosphates (PP-IPs) in DNA repair pathways and telomere dynamics using trypanosomatids as a model, AP.JP

Abstract

Thymidine analogs (most common 5-Bromo-22-deoxyuridine - BrdU, and 5-Ethynyl-2'-deoxyuridine - EdU) are widely used to monitor DNA replication in eukaryotic cells. However, recent articles have been reporting certain levels of cytotoxicity in mammalian cells, which can bias data related to the replication capacity of the analyzed cells. In trypanosomatids (unicellular eukaryotic parasites), there are no reports of toxic effects related to the use of thymidine analogs. Thus, there are three possibilities: BrdU and EdU are not toxic to these parasites at standard working concentrations; BrdU and EdU are toxic at standard working concentrations and no research group has evaluated this toxicity; or only one of these analogs is toxic. To find out which of these possibilities is correct, we intend to investigate in this project the possible cytotoxic effects of BrdU and EdU on Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. Preliminary data suggest that EdU (but not BrdU) is toxic at working concentrations to L. major. Considering that the only difference between EdU and BrdU is the presence of the ethynyl group in place of the bromine atom in position 5, this preliminary result justifies the beginning of a further investigation into the cytotoxicity of alkyne groups present in the DNA molecule. We intend to treat the aforementioned trypanosomatids with different concentrations of BrdU and EdU (and dT - thymidine, as a control) and evaluate their growth curves, concomitantly with cell viability assays. Moreover, we will investigate the presence of possible cell cycle arrests by the analysis of DNA content using flow cytometry. We also intend to investigate the presence of DNA damage and DNA damage response (DDR) through immunofluorescence assays (IFA) using ±-³H2A, and by using Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay. Of note, a better understanding of the essential molecular mechanisms during the life cycle of these organisms (the core of this project) can contribute to the development of specific strategies to combat the diseases caused by these parasites.