Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay.
The lactate dehydrogenase (LDH) assay accurately quantifies cytotoxicity of chemicals via the measurement of LDH released from damaged cells. In the assay, LDH catalyzes formation of a reporter chromophore that can be quantified spectrophotometrically at its 490 nm peak, a standard assay, and related to the released LDH concentration. However, certain engineered nanomaterials have been reported to produce aberrant values, resulting in inaccurate assessment of toxicity as measured by LDH levels in media. We studied this effect spectroscopically by measuring unexpected changes in the complete visible spectrum of the product chromophore resulting from using either purified LDH or LDH from lysed cells in the presence of varying concentrations of single walled carbon nanotubes (SWCNTs) or carbon nanohorns (SWCNH-oxs). Basically, at constant LDH concentrations, the 490 nm product peak decreased with increasing carbon nanotube concentration, while the 580 nm peak increased to a lesser extent and the maximum absorbing wavelength increased. The product chromophore spectrum was altered in different ways by potential interactions with a number of components in the reaction mixture including: BSA, LDH, SWCNTs, SWCNT-oxs, or various combinations of these species. We propose to improve the accuracy of the LDH assay when evaluated in the presence of varying concentrations of these carbon nanostructures by use of both the 490 and 580 nm peak absorbances combined via regression analysis. Our results indicate that molecular probes of cytotoxicity must be assessed individually for accuracy in the presence of engineered nanomaterials.
Department of Chemistry, University of Massachusetts, Lowell, MA 01854, United States.
Gang
G
Wang
Jianping
J
Zhang
Abiche H
AH
Dewilde
Anoop K
AK
Pal
Dhimiter
D
Bello
Joel M
JM
Therrien
Susan J
SJ
Braunhut
Kenneth A
KA
Marx
D000818:Animals; D002460:Cell Line; D004285:Dogs; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology; D046529:Microscopy, Electron, Transmission; D037742:Nanotubes, Carbon / Q000737:chemistry* / Q000648:ultrastructure; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D018675:Toxicity Tests / Q000379:methods*
D037742:Nanotubes, Carbon; D007770:L-Lactate Dehydrogenase