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Single-mitosis dissection of acute and chronic DNA mutagenesis and repair

ABSTRACT: The mammalian genome is constantly exposed to genotoxins that can lead to mutations and ultimately tumorigenesis. Despite cancer being recognized as a genetic disease, much is still unknown regarding tumor induction and evolution of the cancer genome. To study gradual (reactive oxygen species, ROS) and acute (ultraviolet radiation, UV) mutagenic processes both in vitro and then in vivo, we first use microfluidic separation of cells after a single mitosis following pulse mutagenesis. While UV mutations tend to be fixed after one cell cycle and show mirror image mutation patterns between sister cells from a single mitosis, ROS mutations do not. We next show that while both ROS and UV mutation rates are reduced as a function of transcription, only UV has a strand-specific repair signature. Lastly, we resolve pulse induced lesion segregation to single haplotypes by analysing liver tumour genomes from F1 mice treated with N-nitroso-diethylamine (DEN). In summary, we have established highly controlled systems to study 3 distinct forms of mutagenesis at their inception in mammalian cells, providing insights into how these mutations are fixed at the genome scale.

ORGANISM(S): Mus musculus Mus musculus castaneus

PROVIDER: GSE230579 | GEO | 2024/01/26

REPOSITORIES: GEO

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Project description:N-nitroso compounds (NOC) may be implicated in human colon carcinogenesis, but the toxicological mechanisms involved have not been elucidated. Since it was previously demonstrated that nitrosamines and nitrosamides, representing two classes of NOC, induce distinct gene expression effects in colon cells that are particularly related to oxidative stress, we hypothesized that different radical mechanisms are involved. Using ESR spectroscopy, we investigated radical generating properties of genotoxic NOC concentrations in human colon adenocarcinoma cells (Caco-2). Cells were exposed to nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosurea) or nitrosamines (N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine). Nitrosamines caused formation of reactive oxygen species (ROS) and carbon centered radicals which was further stimulated in presence of Caco-2 cells. N-methyl-N-nitrosurea exposure resulted in a small ROS signal, and formation of nitrogen centered radicals (NCR), also stimulated by Caco-2 cells. N-methyl-N'-nitro-N-nitrosoguanidine did not cause radical formation at genotoxic concentrations, but at increased exposure levels, both ROS and NCR formation was observed. By associating gene expression patterns with ROS formation, several cellular processes responding to nitrosamine exposure were identified, including apoptosis, cell cycle blockage, DNA repair and oxidative stress. These findings suggest that following NOC exposure in Caco-2 cells, ROS formation plays an important role in deregulation of gene expression patterns which may be relevant for the process of chemical carcinogenesis in the human colon, in addition to the role of DNA alkylation. Keywords: Nitrosamines, nitrosamides, N-nitroso compounds, free radicals, toxicogenomics, colon carcinogenesis The study investigated differential gene expression in Caco-2 cell line mRNA following 1, 6 or 24 hours of exposure to six different N-nitroso compounds. Two biological replicates per sample compound. One compound per array, hybridized against vehicle control. Dye-swap between biological replicates.

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Single-mitosis dissection of acute and chronic DNA mutagenesis and repair

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