Copper toxicity of inflection point in human intestinal cell line Caco-2 dissected: influence of temporal expression patterns

We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 mu M CuSO4 (the early inflection point) was more toxic than 25 mu M CuSO4 in Caco-2 cells. We employed global proteomics to investigate this observation. The altered expression levels of a small number of proteins displaying a temporal response may provide the best indication of the underlying mechanism; more well-known copper response proteins including the metal binding metallothioneins (MT1X, MT1F, MT2A) and antioxidant response proteins including Heme oxygenase were upregulated to a similar level in both copper concentrations and so are less likely to underpin this phenomenon. The temporal response proteins include Granulins, AN1-type zinc finger protein 2A (ZFAND2A), and the heat shock proteins (HSPA6 and HSPA1B). Granulins were decreased after 4 h only in 25 mu M CuSO4 but from 24 h, were decreased in both copper concentrations to a similar level. Induction of ZFAND2A and increases in HSPA6 and HSPA1B were observed at 24 h only in 25 mu M CuSO4 but were present at 48 h in both copper conditions. The early expression of ZFAND2A, HSPs, and higher levels of alpha-crystallin B (CRYAB) correlated with lower levels of misfolded proteins in 25 mu M CuSO4 compared to 3.125 mu M CuSO4 at 48 h. These results suggest that 3.125 mu M CuSO4 at early time points was unable to activate the plethora of stress responses invoked by the higher copper concentration, paradoxically exposing the Caco-2 cells to higher levels of misfolded proteins and greater proteotoxic stress.

Automated summary

At low copper concentrations, 3.125μM CuSO4 was found to be more toxic than 25μM CuSO4, potentially due to its inability to activate sufficient stress responses at early time points, leading to higher levels of protein misfolding and proteotoxic stress.