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

Abstract

We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 μM CuSO₄ (the early inflection point) was more toxic than 25 μM 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 μM 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 μM CuSO₄ but were present at 48 h in both copper conditions. The early expression of ZFAND2A, HSPs, and higher levels of α-crystallin B (CRYAB) correlated with lower levels of misfolded proteins in 25 μM CuSO₄ compared to 3.125 μM CuSO₄ at 48 h. These results suggest that 3.125 μM 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.