Effect of Cyclophosphamide and Sulfur Mustard on Cell Viability and the Role of Vitamin E

Peritoneal macrophages live in tissue culture and in response to LPS and y-IFN produce nitric oxide (NO). After that, the number of viable cells might decrease because of depletion of culture media or substrate, production of inhibitory factors such as TGF-f3, and the toxicity of NO itself for the cells. Alkylating agents like cyclophosphamide and sulfur mustard have inhibitory effect on immune system and macrophage function. The aim of this study was to evaluate the effect of vitamin E as an antioxidant on cell viability and NO release by peritoneal macrophages of Cyc/SM-tread mice.

Peritoneal cells were collected by inter peritoneal injection of 5 ml cold PBS. Cells were washed three times, counted and plated out at 1x105 cells in 96 well plates in 0.2 ml RPMI. Plates were incubated at 37°C with 5% CO2 for 2 hours. The nonadherent cells were removed by washing with warm PBS. Adherent cells were then cultured at above condition in phenol red free medium in the presence or absence of sulfur mustard, cyclophosphamide, and vitamin E. Cell viability was checked with trypan blue and nitrite was measured as an indicator of NO by Griess method.

In the absence of vitamin E, cyclophosphamide enhanced NO release by peritoneal macrophages. Vitamin E reduced cell death was induced by cyclophosphamide and SM. However, by increasing the concentrations of alkylating agent, vitamine E was less effective. Vitamine E also reduced NO release induced by cyclophosphamide and sulfur mustard. Treating the cells with 10. ig/ml cyclophosphamide, resulted in NO release up to 520 nm while in the presence of vitamine E, it decreased to 410 nm (21.15% reduction, P<0.05). Vitamin E inhibited No release by cyclophosphamide or sulfur mustard treated macrophages in all concentrations. However, in the presence of high concentrations of sulfur mustard, vitamin E was less effective. So, in the presence of 60 or 160. im of sulfur mustard, the effect of vitamin E on NO release was not significant (P<0.07).

The enhancement of cell death in the presence of cyclophosphamide or sulfur mustard could be mediated by nitric oxide release. Vitamin E may enhance cell viability through NO reduction.