TY - JOUR
T1 - Incompatibility of silver nanoparticles with lactate dehydrogenase leakage assay for cellular viability test is attributed to protein binding and reactive oxygen species generation
AU - Oh, Seok Jeong
AU - Kim, Hwa
AU - Liu, Yingqiu
AU - Han, Hyo Kyung
AU - Kwon, Kyenghee
AU - Chang, Kyung Hwa
AU - Park, Kwangsik
AU - Kim, Younghun
AU - Shim, Kyuhwan
AU - An, Seong Soo A.
AU - Lee, Moo Yeol
PY - 2014/3/21
Y1 - 2014/3/21
N2 - A growing number of studies report that conventional cytotoxicity assays are incompatible with certain nanoparticles (NPs) due to artifacts caused by the distinctive characteristics of NPs. Lactate dehydrogenase (LDH) leakage assays have inadequately detected cytotoxicity of silver nanoparticles (AgNPs), leading to research into the underlying mechanism. When ECV304 endothelial-like umbilical cells were treated with citrate-capped AgNPs (cAgNPs) or bare AgNPs (bAgNPs), the plasma membrane was disrupted, but the LDH leakage assay failed to detect cytotoxicity, indicating interference with the assay by AgNPs. Both cAgNPs and bAgNPs inactivated LDH directly when treated to cell lysate as expected. AgNPs adsorbed LDH and thus LDH, together with AgNPs, was removed from assay reactants during sample preparation, with a resultant underestimation of LDH leakage from cells. cAgNPs, but not bAgNPs, generated reactive oxygen species (ROS), which were successfully scavenged by N-acetylcysteine or ascorbic acid. LDH inhibition by cAgNPs could be restored partially by simultaneous treatment with those antioxidants, suggesting the contribution of ROS to LDH inactivation. Additionally, the composition of the protein corona surrounding AgNPs was identified employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In sum, the LDH leakage assay, a conventional cell viability test method, should be employed with caution when assessing cytotoxicity of AgNPs.
AB - A growing number of studies report that conventional cytotoxicity assays are incompatible with certain nanoparticles (NPs) due to artifacts caused by the distinctive characteristics of NPs. Lactate dehydrogenase (LDH) leakage assays have inadequately detected cytotoxicity of silver nanoparticles (AgNPs), leading to research into the underlying mechanism. When ECV304 endothelial-like umbilical cells were treated with citrate-capped AgNPs (cAgNPs) or bare AgNPs (bAgNPs), the plasma membrane was disrupted, but the LDH leakage assay failed to detect cytotoxicity, indicating interference with the assay by AgNPs. Both cAgNPs and bAgNPs inactivated LDH directly when treated to cell lysate as expected. AgNPs adsorbed LDH and thus LDH, together with AgNPs, was removed from assay reactants during sample preparation, with a resultant underestimation of LDH leakage from cells. cAgNPs, but not bAgNPs, generated reactive oxygen species (ROS), which were successfully scavenged by N-acetylcysteine or ascorbic acid. LDH inhibition by cAgNPs could be restored partially by simultaneous treatment with those antioxidants, suggesting the contribution of ROS to LDH inactivation. Additionally, the composition of the protein corona surrounding AgNPs was identified employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In sum, the LDH leakage assay, a conventional cell viability test method, should be employed with caution when assessing cytotoxicity of AgNPs.
KW - Cytotoxicity
KW - Lactate dehydrogenase leakage assay
KW - Protein adsorption
KW - Reactive oxygen species
KW - Silver nanoparticles
UR - http://www.scopus.com/inward/record.url?scp=84893655513&partnerID=8YFLogxK
U2 - 10.1016/j.toxlet.2014.01.015
DO - 10.1016/j.toxlet.2014.01.015
M3 - Article
C2 - 24463055
AN - SCOPUS:84893655513
SN - 0378-4274
VL - 225
SP - 422
EP - 432
JO - Toxicology Letters
JF - Toxicology Letters
IS - 3
ER -