Effect of saliva, epigallocatechin gallate and hypoxia on Cu-induced oxidation and cytotoxicity

We have previously reported that contact with copper (Cu) induced immediate cell death via an oxidation- involved mechanism in human promyelocytic leukemic HL-60 cells, whereas contact with other metals (Au, Ag, Pd) produced no discernible effect. In the present study, we investigated the conditions under which Cu-induced oxidative stress can be reduced. Contact with a Cu plate in the absence of cells enhanced the rate of consumption of cystine to the greatest extent, followed by that of methionine and histidine. Under hypoxic conditions, the consumption of all these amino acids was significantly reduced. On the other hand, the addition of saliva slightly, but not significantly, reduced the amino acid oxidation. The addition of epigallocatechin gallate (EGCG) slightly, but significantly reduced the consumption of cystine and histidine. The inhibitory effect of EGCG on the methionine consumption was more prominent, especially at higher concentrations. The Cu-induced cell death was significantly inhibited when freshly-prepared human gingival fibroblasts were incubated under hypoxic conditions. The present study demonstrates for the first time that the Cu- induced oxidation and cell death were effectively alleviated under hypoxic conditions. Dental alloys have been reported to induce allergic reactions in the oral cavity, though infrequently (1), which may be related to the stimulated release of metal ions from the alloys under acidic oral environments caused by inflammation, bacterial infection, and intake of soft drinks and coffee (2, 3). Metal ions may be incorporated into cells, possibly via metal transporter-mediated endocytosis (4, 5). Although numerous studies have shown cytotoxic activity and tissue-damaging activity of metal extracts (6-8), no detailed study of cytotoxicity induced by the direct contact with metals has been reported. We have recently constructed an assay system to investigate the interaction between metals and cultured cells (9). Using this system, we found that the direct contact of cells with a copper (Cu) plate induced rapid, non-apoptotic cell death, characterized by a smear pattern of DNA fragmentation, minor caspase activation, loss of the membrane barrier, cytotoplasmic damage prior to nuclear damage, maintenance of cell surface microvilli, and vacuolization in the human promyelocytic leukemic cell line (HL-60), whereas other metals, such as gold, silver and palladium, were essentially inactive (9). It is very important to explore the method by which the cytotoxicity of Cu can be reduced. Here, we investigated the effect of three oral environmental factors: saliva, hypoxic conditions and epigallocatechin gallate (EGCG, a popular green tea polyphenol) on Cu-induced oxidative stress. The changes in the consumption rate of easily oxidizable amino acids in the culture medium was monitored without cells, and the viability of cultured human gingival fibroblast (HGF) at different oxygen concentrations after contact with Cu plate was determined.