Abstract
Background
Exposure to ionizing is known to cause persistent cellular oxidative stress and NADPH oxidase (Nox) is a major source of cellular oxidant production. Chronic oxidative stress is associated with a myriad of human diseases including gastrointestinal cancer. However, the roles of NADPH oxidase in relation of long-term oxidative stress in colonic epithelial cells after radiation exposure are yet to be clearly established.
Methods and results
Mice were exposed either to sham or to 0.5 Gy γ radiation, and NADPH oxidase, oxidative stress, and related signaling pathways were assessed in colon samples 60 days after exposure. Radiation exposure led to increased expression of colon-specific NADPH oxidase isoform, Nox1, as well as upregulation of its modifiers such as Noxa1 and Noxo1 at the mRNA and protein level. Co-immunoprecipitation experiments showed enhanced binding of Rac1, an activator of NADPH oxidase, to Nox1. Increased 4-hydroxynonenal, 8-oxo-dG, and γH2AX along with higher protein carbonylation levels suggest increased oxidative stress after radiation exposure. Immunoblot analysis demonstrates upregulation of Ras/p38 pathway, and Gata6 and Hif1α after irradiation. Increased staining of β-catenin, cyclinD1, and Ki67 after radiation was also observed.
Conclusions
In summary, data show that exposure to a low dose of radiation was associated with upregulation of NADPH oxidase and its modifiers along with increased Ras/p38/Gata6 signaling in colon. When considered along with oxidative damage and proliferative markers, our observations suggest that the NADPH oxidase pathway could be playing a critical role in propagating long-term oxidative stress after radiation with implications for colon carcinogenesis.
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Abbreviations
- NADPH:
-
Nicotinamide adenine dinucleotide phosphate-reduced
- Nox:
-
NADPH oxidase
- Noxo1:
-
NADPH oxidase organizer 1
- Noxa1:
-
NADPH oxidase activator 1
- Hif1α:
-
Hypoxia inducible factor 1
- Pak1-PBD:
-
P21 activated kinase-protein binding domain
- Rac1:
-
Ras-related C3 botulinum toxin substrate 1
- Cdc42:
-
Cell division cycle 42
- ROS:
-
Reactive Oxygen Species
- 4HNE:
-
4-Hydroxynonenal
- DSB:
-
Double Strand Break
- Mapk:
-
Mitogen-activated protein kinases
- IACUC:
-
Institutional Animal Care and Use Committee
- DAB:
-
3,3'Diaminobenzidine
- SDS-PAGE:
-
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis
- DNPH:
-
2,4-Dinitrophenylhydrazine
- SEM:
-
Standard Errors of the Mean
- RT-PCR:
-
Reverse transcription-polymerase chain reaction
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Acknowledgements
We are thankful to Ms. Pelagie Ake for animal facility supports.
Funding
This study was supported in part by National Aeronautics and Space Administration (NASA) Grant Numbers NNX13AD58G, NNX09AU95G, and 80NSSC22K1279. The authors acknowledge the Lombardi Comprehensive Cancer Shared Resources (MSR), which are in part supported by award number P30CA051008 (P.I. Louis Weiner) from the National Cancer Institute.
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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. A protocol approved by the Georgetown University Animal Care and Use Committee (GUACUC) was followed for the care and use of animals. This article does not contain any studies with human participants performed by any of the authors.
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Kumar, S., Suman, S., Moon, BH. et al. Low dose radiation upregulates Ras/p38 and NADPH oxidase in mouse colon two months after exposure. Mol Biol Rep 50, 2067–2076 (2023). https://doi.org/10.1007/s11033-022-08186-3
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DOI: https://doi.org/10.1007/s11033-022-08186-3