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Hydrogen peroxide (H2O2) is originally an endogenous small molecule which is reduced into water in cells. In order to know the
H2O2-induced oxidative stress in RAW 264.7 cells, first of all, the optimum concentration of exogenous H2O2 which show reactive cellular
responses was determined as 40 μM by MTT assay, and followed by 40 μM H2O2 application in RAW 264.7 cells for 30 min, 1, or
2 hours. The expressional changes of essential proteins for cellular proliferation, epigenetic modification, inflammation, apoptosis, survival,
and protection were assessed by immunoprecipitation high performance liquid chromatography (IP-HPLC) using 51 antisera. 40 μM H2O2
treatment down-regulated proliferation-related proteins, Ki-67, PCNA, CDK4, cyclin D2, cMyc, and PLK4, induced histone methylation/
deacetylation and DNA methylation by increasing levels of HDAC10 and DMAP1 and by decreasing levels of DNMT1 and KDM4D,
activated inflammatory reaction by increasing levels of MCP-1, COX-2, CD68, LTA4H, CXCR4, and lysozyme, and dramatically up-regulated
cellular apoptosis-, survival-, and protection-related proteins, AIF, PARP-1, caspase 9, c-caspase 9, pAKT1/2/3, SOD-1, HO-1, NF-kB, NRF2,
and GSTO1 in RAW 264.7 cells. These observations suggest exogenous 40 μM H2O2-induced oxidative stresses which resulted global
cellular responses including not only antioxidant, inflammation, and apoptosis but also proliferation and epigenetic modification. Particularly,
40 μM H2O2-induced apoptosis was mainly derived from PARP-1/AIF signaling leading parthanatos, and 40 μM H2O2-induced suppression
of cMyc/MAX/MAD network was relevant to reduction of RAW 264.7 cell proliferation. Accordingly, H2O2 appears to affect RAW 264.7
macrophages in several ways eliciting not only oxidative stresses but also genome-wide DNA damage.
Key words : Hydrogen peroxide, Protein expressions, RAW 264.7 cells, IP-HPLC