Novel In Vivo CometChip Reveals NDMA-Induced DNA Damage and Repair in Multiple Mouse Tissues

Norah Owiti, Joshua Corrigan, Lee Pribyl, Jennifer Kay, Bevin Engelward

https://doi.org/10.3390/ijms231911776

The comet assay is a versatile assay for detecting DNA damage in eukaryotic cells. The assay can measure the levels of various types of damage, including DNA strand breaks, abasic sites and alkali-sensitive sites. Furthermore, the assay can also be modified to include purified DNA glycosylases so that alkylated and oxidized bases can be detected. The CometChip is a higher throughput version of the traditional comet assay and has been used to study cultured cells. Here, we have tested its utility for studies of DNA damage present in vivo. We show that the CometChip is effective in detecting DNA damage in multiple tissues of mice exposed to the direct-acting methylating agent methylmethane sulfonate (MMS) and to the metabolically activated methylating agent N-nitrosodimethylamine (NDMA), which has been found to contaminate food, water, and drugs. Specifically, results from MMS-exposed mice demonstrate that DNA damage can be detected in cells from liver, lung, kidney, pancreas, brain and spleen. Results with NDMA show that DNA damage is detectable in metabolically competent tissues (liver, lung, and kidney), and that DNA repair in vivo can be monitored over time. Additionally, it was found that DNA damage persists for many days after exposure. Furthermore, glycosylases were successfully incorporated into the assay to reveal the presence of damaged bases. Overall, this work demonstrates the efficacy of the in vivo CometChip and reveals new insights into the formation and repair of DNA damage caused by MMS and NDMA.

DOI: None

Zenodo URL: None

Created at: 22nd Dec 2023 at 16:41

Contents

Chemical Challenge - Metadata

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CHM-samples-publish2023-09-13_11-34.xlsx

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MUS-samples-publish2023-09-13_11-48.xlsx

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P.ENG-230913-V1_Chemicals-and-Reagents.docx

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  • Chemicals and Reagents.docx

P.ENG-230913-V1_In-Vivo-Comet-Chip---MUS.docx

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  • In Vivo Comet Chip - MUS.docx

Tissue Collection - Metadata

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MUS-samples-publish2023-09-13_11-48.xlsx

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TIS-samples-publish2023-09-13_12-27.xlsx

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  • samples-publish2023-09-13_12-27.xlsx

P.ENG-230913-V1_In-Vivo-Comet-Chip---MUS.docx

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  • In Vivo Comet Chip - MUS.docx

P.ENG-230912-V1_MMS-and-NDMA-TIS.docx

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  • MMS and NDMA-TIS.docx

Comet Chip - Data Linked

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TIS-samples-publish2023-09-13_12-27.xlsx

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  • samples-publish2023-09-13_12-27.xlsx

CEL-samples-publish2023-09-19_13-11.xlsx

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D.IMG-samples-publish2023-09-13_12-43.xlsx

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P.ENG-230912-V1_Cell-Culture.docx

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  • Cell Culture.docx

P.ENG-230912-V1_In-Vivo-Comet-Assay-Protocol.docx

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  • In Vivo Comet Assay Protocol.docx

Comet Chip Analysis - Data Attached

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Figure 2B MMS 211203.pzfx

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  • Figure 2B MMS 211203.pzfx

Figure 3A hAAG 211201_Enzymes.pzfx

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  • Figure 3A hAAG 211201_Enzymes.pzfx

Figure 3B MMS FPG.pzfx

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  • Figure 3B MMS FPG.pzfx

Figure 4B.pzfx

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  • Figure 4B.pzfx

Figure 5A Liver Time Course.pzfx

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  • Figure 5A Liver Time Course.pzfx

Figure 5B Lung time course.pzfx

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  • Figure 5B Lung time course.pzfx

Figure 6A Liver hAAG.pzfx

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  • Figure 6A Liver hAAG.pzfx

Figure 5C Kidney NDMA Time Course.pzfx

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  • Figure 5C Kidney NDMA Time Course.pzfx

Figure 6B Liver Fpg.pzfx

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  • Figure 6B Liver Fpg.pzfx

Figure 6C-D Lung with Enzymes.pzfx

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  • Figure 6C-D Lung with Enzymes.pzfx

Figure 6E-F In_vivo_NDMA_Kidney w Enzymes copy.pzfx

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  • Figure 6E-F In_vivo_NDMA_Kidney w Enzymes copy.pzfx

Figure S2 MEFs and HepG2.pzfx

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  • Figure S2 MEFs and HepG2.pzfx

Figure S5A In_vivo_NDMA_Pancreas.pzfx

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  • Figure S5A In_vivo_NDMA_Pancreas.pzfx

Figure S5B NDMA Spleen.pzfx

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  • Figure S5B NDMA Spleen.pzfx

D.IMG-samples-publish2023-09-13_12-43.xlsx

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P.ENG-230912-V1_In-Vivo-Comet-Assay-Protocol.docx

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  • In Vivo Comet Assay Protocol.docx
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