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.

碳纳米管（CNT）的广泛生产和应用引起了人们对其释放到环境中的担忧，并且需要评估CNT与预先存在的污染物的联合毒性。这是第一项使用斑马鱼肝细胞系（ZFL）研究氧化多壁碳纳米管（ox-MWCNT）和镉（Cd）共同暴露的研究。两次体外评估了ox-MWCNT与Cd和胎牛血清（FBS）蛋白质相互作用的顺序不同的共同暴露方案。Ox-MWCNT具有物理和化学特性，在两种方案中都确定了其在细胞培养基中的吸附能力和胶体稳定性。通过MTT，中性红，锥虫蓝，乳酸脱氢酶测定法研究细胞毒性，并使用流式细胞仪测定其坏死和凋亡事件。介质中Cd的存在不会影响MWCNT的蛋白电晕组成，但FBS和Cd相互作用的顺序会影响其胶体稳定性和金属吸附率。ox-MWCNT可能通过“特洛伊木马”和/或协同作用增加了低浓度下Cd的毒性，并诱导ZFL细胞凋亡和坏死。尽管未观察到方案之间毒性的差异，但是ox-MWCNT首先与Cd相互作用导致其在细胞培养基中沉淀，结果，通过中性红分析可能导致假生存力。综上所述，很明显，化合物相互作用的顺序扰乱了胶体稳定性并影响了胶体的稳定性。体外毒理学测定。考虑到协议A在与Cd相互作用后显示出更多的ox-MWCNT稳定性，因此建议在以后的研究中采用该协议。