Assay to detect lipid peroxidation upon exposure to nanoparticles.
This chapter describes a method for the analysis of human hepatocarcinoma cells (HEP G2) for lipid peroxidation products, such as malondialdehyde (MDA), following treatment with nanoparticle formulations. Oxidative stress has been identified as a likely mechanism of nanoparticle toxicity, and cell-based in vitro systems for evaluation of nanoparticle-induced oxidative stress are widely considered to be an important component of biocompatibility screens. The products of lipid peroxidation, lipid hydroperoxides, and aldehydes, such as MDA, can be measured via a thiobarbituric acid reactive substances (TBARS) assay. In this assay, which can be performed in cell culture or in cell lysate, MDA combines with thiobarbituric acid (TBA) to form a fluorescent adduct that can be detected at an excitation wavelength of 530 nm and an emission wavelength of 550 nm. The results are then expressed as MDA equivalents, normalized to total cellular protein (determined by Bradford assay).
Nanotechnology Characterization Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD, USA.
Timothy M
TM
Potter
Barry W
BW
Neun
Stephan T
ST
Stern
D000447:Aldehydes / Q000032:analysis; D001681:Biological Assay; D005456:Fluorescent Dyes / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D015227:Lipid Peroxidation; D008054:Lipid Peroxides / Q000032:analysis; D008315:Malondialdehyde / Q000032:analysis* / Q000378:metabolism; D053758:Nanoparticles / Q000633:toxicity*; D018384:Oxidative Stress; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D012015:Reference Standards; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis* / Q000378:metabolism
D000447:Aldehydes; D005456:Fluorescent Dyes; D008054:Lipid Peroxides; D011506:Proteins; D017392:Thiobarbituric Acid Reactive Substances; D008315:Malondialdehyde