Free radical stress induces DNA damage response in RAW264.7 macrophages during Mycobacterium smegmatis infection.

Affiliation

Department of Molecular Biology and Bioinformatics, Tripura University, Suryamaninagar, Tripura, 799022, India. [Email]

Abstract

Genomic instability resulting from oxidative stress responses may be traced to chromosomal aberration. Oxidative stress suggests an imbalance between the systemic manifestation of reactive free radicals and biological system's ability to repair resulting DNA damage and chromosomal aberration. Bacterial infection associated insult is considered as one of the major factors leading to such stress conditions. To study free radical responses by host cells, RAW 264.7 macrophages were infected with non-pathogenic M. smegmatis mc2155 at different time points. The infection process was followed up with an assessment of free radical stress, cytokine, toll-like receptors (TLRs) and the resulting DNA damage profiles. Results of CFU count showed that maximum infection in macrophages was achieved after 9 h of infection. Host responses to the infection across different time periods were validated from nitric oxide quantification and expression of iNOS and were plotted at regular intervals. IL-10 and TNF-α expression profile at protein and mRNA level showed a heightened pro-inflammatory response by host macrophages to combat M. smegmatis infection. The expression of TLR4, a receptor for recognition of mycobacteria, in infected macrophages reached the highest level at 9 h of infection. Furthermore, comet tail length, micronuclei and γ-H2AX foci recorded the highest level at 9 h of infection, pointing to the fact that breakage in DNA double strands in macrophage reaches its peak at 9 h of infection. In contrast, treatment with ROS inhibitor N-acetyl-L-cysteine (NAC) prevented host cell death through reduction in oxidative stress and DNA damage response during M. smegmatis infection. Therefore, it can be concluded that enhanced oxidative stress response in M. smegmatis infected macrophages might be correlated with DNA damage response.

Keywords

Cytokines,DNA damage,Free radical stress,Genomic instability,Mycobacterium smegmatis,

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