Understanding toxicity pathways of gold nanoparticles: RNA expression study on Caco-2 human colon carcinoma cells

A safer design and higher eficacy of nano-gold therapeutic formulations requires examination of cellular responses to gold nanoparticles (AuNPs). In this work we compared cellular uptake, cytotoxicity and RNA expression patterns induced in Caco-2 cells of citrate-stabilized Au NP of two different sizes (5 and 30 nm). The toxicity was measured with Colony Forming Efficiency (CFE) and Trypan Blue assays at 24 and 72 h after exposure to AuNPs in the 10 - 300 mM range. Toxicity was observed only in the CFE assay, at 200 and 300 mM exposure levels, and exclusively for smaller AuNPs size. Cellular internalization was dose and time-dependent for both AuNPs. The most pronounced changes in gene expression, evaluated with Agilent microarrays, were detected at 72 h (300 mM) for 5 nm AuNPs, with 103 up and 708 down regulated transcripts. For 30 nm AuNPs, only 4 gene transcripts were repressed under these conditions. The biological processes affected by 5 nm AuNPs were: RNA/zinc ion/transition metal ion binding (decreased), cadmium/copper ion binding and glutathione metabolism (increased). Some Nrf2 responsive genes (several metallothioneins, HMOX, G6PD, OSGIN1 and GPX2) were highly up regulated. Members of the selenoproteins (SELT, SELK, 15 kDa selenoprotein, SEPP1 and GPX2) were also differentially expressed. Our findings indicate that at high concentrations, smaller AuNPs can induce metal exposure and oxidative stress signaling pathways, and might influence selenium homeostasis. Therefore, some observed effects associated with nano-gold cytotoxicity can be further explored as potential enhancers of anti-cancer properties of already existing AuNPs based nanomedicin. Caco-2 cells were expoxed to spherical gold nanoparticles of two different sizes and concentrations (5 and 30 nm AuNPs, 100 and 300 microM). Cells were collected after 24 or 72 hours