DNA Microarray Analysis of Macrophage-like Cells Exposed to Ni Ions

Objective: Ni ions are known to cause cellular damage and allergic reaction. The purpose of this study was to clarify how sublethal dose of Ni ions alters gene expression of macrophages. Methods: Sub-confluent mouse macrophage-like RAW264 cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum with and without 500 µmol/L Ni²⁺ ions for short 5 hours and long 18 hours at 37 ^oC in a humidified 5% CO₂ atmosphere. Total RNAs (at least 30 µg) were collected from the cells with and without Ni ions using the TRIZOL reagent (Invitrogen). Laser Techno Co. (Nagoya, Japan) performed the Cy-3/Cy-5 labeling, microarray hybridizations using 10k oligo DNA chip (Nippon Gene Lab., Miyagi, Japan) and scanning. Genes that showed an alteration in normalized signal expression of more than 5 fold and less than 0.2 fold compared to the control were designated as significantly up-regulated and down-regulated, respectively. Results: When exposed to Ni ions for short 5 hours, 2 genes were down-regulated and 62 genes were up-regulated. Their five most up-regulated genes (expressed by gene symbols) were Csnk2a1, Rpl3, Ldh1, Ero1l and Ly9. When exposed to Ni ions for long 18 hours, 20 genes were down-regulated and 90 genes were up-regulated. Their five most up-regulated genes were Csnk2a1, 2510002 J07Rik, Tsp10-pending, 2410003M15Rik and HIP14l-pending. Csnk2a1 was casein kinase II, alpha 1 polypeptide. Characteristically, after 18 hours culture in Ni-ion-containing medium, antidotal protein and enzyme related genes such as MT2 (metallothionein 2), Hsp8 (heat shock protein 8) and Sod1 (superoxide dismutase 1, soluble) genes were also considerably up-regulated. Conclusion: Many toxic Ni ions absorbed inside the cells were neutralized by producing metal-protein complex. The formed complex appeared to be phagocytized by free radicals (superoxide etc.). Surplus superoxide might damage DNA of RAW264 cells, thereby reducing the viable cell number through necrosis.