Method to Monitor Cellular Function during Contact to Metals

Objective:  We have recently developed the assay system to investigate the interaction between metals and cultured cells.  Using this system, we investigated the possible changes in amino acid utilization (production and consumption) during the contact of various cells with Au, Ag, Cu and Pd, metals used as constituents in dental alloys, to establish the sensitive method for monitoring the effect of metals on cellular function.  Methods: All metals (20 x 20 x 0.5 mm³) were polished, and its surface smoothness was confirmed by SEM.  Three human leukemia cell lines were cultured in RPMI-1640+10%FBS.  Human gingival fibroblasts were cultured in DMEM + 10% FBS as monolayer, and detached by mild trypsinization.  Cell suspension (10⁶/mL, 0.5 mL) were inoculated onto each metal plate and incubated at 37^oC for up to 24 hours in 5%CO₂ incubator.  Viable cell number was determined by trypan blue dye exclusion, apoptosis induction by DNA fragmentation and caspase activation, autophagy induction by acridine staining and TEM, and amino acid concentration by amino acid analyzer.  Results: All cells utilized GLN at the highest rates, followed by SER in the case of leukemic cells.  Contact with Cu plate induced non-apoptotic cell death, accompanied by the dramatic increase in CYS consumption, and cessation of GLN.  Contact with Ag, Pd, but not with Au occationally induced cell death, accompanied by enhanced production of GLU and GLY and the cessation of utilization of GLN.  Conclusion:  Enhanced consumption of CYS is characteristics of Cu-induced cell death, and can be utilized as a marker of oxidative stress. On the other hand, the cessation of production of GLN may be common in metal-induced cell death.  The production of GLN can be utilized as a functional marker of cell viability for the study of metal-cell interaction, without the necessity of recovering the cells.