Original ResearchBasic and Translational—Alimentary Tract4-Hydroxy-2-Nonenal Mediates Genotoxicity and Bystander Effects Caused by Enterococcus faecalis–Infected Macrophages
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Cell Lines and Bacteria
The near-diploid HCT116 human colon cancer cell (American Type Culture Collection, Manassas, VA), YAMC primary mouse colon epithelial cell (Ludwig Institute for Cancer Research, New York, NY), and RAW264.7 murine macrophage cell (American Type Culture Collection) lines were grown as previously described.12, 25 E faecalis strain OG1RFSS was spontaneously derived from OG1RF, a gram-positive human commensal, and expressed high-level resistance to spectinomycin and streptomycin.26 menB was deleted
E faecalis–Infected Macrophages Produce 4-HNE
We initially assayed E faecalis–infected RAW264.7 cells for 4-HNE production and confirmed its structure by 2-dimensional nuclear magnetic resonance. 1H and 13C shifts were consistent with trans-4-hydroxy-2-nonenal (Figure 1A). Compared with uninfected controls, RAW264.7 cells infected with OG1RF at an MOI of 1000 had a 2-fold increase in 4-HNE concentration in supernatants at 24 to 72 hours postinfection (P < .01) (Figure 1B). No significant increase was noted for supernatants from RAW264.7
Discussion
We previously reported that E faecalis–infected macrophages generate bystander effects that cause G2–M cell cycle arrest and CIN in epithelial cells.12, 13 In this study, we show that bystander effects induced by E faecalis are associated with increased production of 4-HNE by infected macrophages. This aldehyde is a breakdown product of lipid peroxidation16 and acts as a diffusible clastogen19 that causes γH2AX deposition due to DNA double-strand breaks. It also induces G2–M cell cycle arrest
Acknowledgments
The authors thank Jim Henthorn in the Flow Cytometry Laboratory and the Rodent Barrier Facility at the University of Oklahoma Health Sciences Center, Robert Whitehead of Vanderbilt University for YAMC cells, and Graça Dores for helpful discussions.
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Conflicts of interest The authors disclose no conflicts.