The widespread production and application of carbon nanotubes (CNT) have raising concerns about their release into the environment and, the joint toxicity of CNT with pre-existing contaminants needs to be assessed. This is the first study that investigated the co-exposure of oxidized multiwalled carbon nanotubes (ox-MWCNT) and cadmium (Cd) using a zebrafish liver cell line (ZFL). Two in vitro co-exposure protocols differing by the order of ox-MWCNT interaction with Cd and fetal bovine serum (FBS) proteins were evaluated. Ox-MWCNT was physical and chemical characterized and its adsorption capacity and colloidal stability in cell culture medium was determined in both protocols. Cytotoxicity was investigated by MTT, neutral red, trypan blue, lactate dehydrogenase assays and the necrosis and apoptosis events were determined using flow cytometer. The Cd presence in medium did not interfere in the protein corona composition of MWCNT but the order of interaction of FBS and Cd interfered in its colloidal stability and metal adsorption rate. The ox-MWCNT increased Cd toxicity at low concentration probably by a "Trojan horse" and/or synergistic effect, and induced apoptosis and necrosis in ZFL cells. Although it was not observed differences of toxicity between protocols, the interaction of ox-MWCNT first with Cd led to its precipitation in cell culture medium and, as a consequence, to a possible false viability result by neutral red assay. Taken together, it was evident that the order of compounds interactions disturbs the colloidal stability and affects the in vitro toxicological assays. Considering that Protocol A showed more ox-MWCNT stability after interaction with Cd, this protocol is recommended to be adopted in future studies.
Keywords: Cd adsorption; Cell viability; Colloidal stability; Flow cytometry; Nanoecotoxicology; Protein corona.
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