Abstract:
: In this study the effect of
diphtheria toxin (DT) on nitric oxide (NO)
production from RAW 264.7 murine
macrophages was investigated. Griess
reagent was used to determine NO
production by measuring nitrite levels in the
culture supernatants. Corynebacterium
diphtheriae G12/6 strain-produced DT (Limes
flocculation activity and immunodiffusion
assays were positive) demonstrated a dosedependent
effect on RAW 264.7
macrophages to induce NO production. LNAME,
an L-arginine analogue, inhibited NO
production from DT-stimulated RAW 264.7
macrophages. At the given concentrations of
DT, lipopolysaccharide (LPS or endotoxin)-
induced NO production from RAW 264.7
macrophages was also inhibited. RAW 264.7
macrophage cells, incubated with various
concentrations of DT for 3 days, were killed
by DT in a dose dependent manner.
Endotoxin contamination of DT was
demonstrated with limulus amoebocyte
lysate assay. Polymyxin B, frequently added
to neutralize the effects of LPS in vitro,
inhibited DT-induced NO production from
RAW 264.7 cells. The misleading data
concerning NO inducing capacity of DT was
seemed to be related to endotoxin
contamination. Thus, DT does not seem to be
capable of inducing NO production from
macrophages.
