Identification and characterization of Gd-binding proteins in NIH-3T3 cells

Gadolinium-based contrast agents (GBCAs), frequently applied in clinical diagnosis, may cause nephrogenic systemic fibrosis (NSF) probably due to the gadolinium ion (Gd³⁺) released from the GBCAs. However, Gd-binding proteins and related mechanism responsible for Gd toxicity remained to be understood. In this study, NIH-3T3 cells were chosen as a model for Gd exposure assays and identification of Gd-binding proteins. A comparative assay showed that gadolinium chloride (GdCl₃) was much more toxic than gadolinium diamide (Gd-DTPA-BMA, a GBCAs). Majority of Gd were absorbed by cells and existed in the fractions of the cell fragment and soluble proteins. High performance liquid chromatography-inductively coupled plasma mass spectrometry(HPLC-ICP-MS), polyacrylamide gel electrophoresis (SDS PAGE) and liquid chromatography-triple time of flight mass spectrometry (LC-Triple TOF) were employed for the identification and characterization of potential Gd-binging proteins. Tubulin was identified as a novel Gd-binding protein in the NIH-3T3 cells. The binding of Gd to tubulin might inhibit assembling of tubulin or depolymerize microtubules in cells. Our results suggested that the formation of microtubules interfered by binding of free Gd³⁺ to tubulin could be an important molecular mechanism of Gd toxicity.