A SPIN DECAY ASSAY FOR TUMOR-NECROSIS-FACTOR CYTOTOXICITY

Tumor necrosis factor (TNF) is believed to elicit its response primarily by an oxidative process. We present a sensitive biophysical assay system to monitor TNF-induced cell killing by electron paramagnetic resonance (EPR) spectroscopy using the spin label 2,2,6,6-tetramethyl-1-piperidine-n-oxyl (TEMPO). The rate of TEMPO spin decay by the TNF sensitive L929 cells was hypothesized to be an indicator of TNF cytolytic response. The cell-induced rate of loss of TEMPO signal was sensitive to concentration as well as time of incubation with TNF. The TEMPO spin decay first derivative plot correlated well with corresponding TNF cytotoxicity curve as obtained by a standard tetrazolium dye reduction bioassay. While dimethyl sulfoxide (DMSO) (up to 500 mM) and alpha-tocopherol (1 mM) inhibited the TNF induced inhibition of rate of loss of TEMPO decay, superoxide dismutase (SOD) (100 U/ml), catalase (1000 U/ml), and histidine (6 mM) had little effect on the TEMPO decay rate. The loss of TEMPO signal was determined to be at an outer domain of the lipid bilayer as illustrated by its decay and broadening in presence of NiCl2. Thus the reduction of a lipid soluble spin label like TEMPO as monitored by EPR could be a useful reporter to study TNF cytotoxicity in target cells,