Induction of oxidative stress and TNF-alpha secretion by dichloroacetonitrile, a water disinfectant by-product, as possible mediators of apoptosis ornecrosis in a murine macrophage cell line (RAW)

The water disinfectant by-product dichloroacetonitrile (DCAN) is a direct-a cting mutagen and induces DNA strand breaks in cultured human lymphoblastic cells. Cellular activation bg environmental agents may exert detrimental e ffects to the cells. Activated macrophages produce reactive oxygen intermed iates such as H2O2, -OH and O-2. Therefore, the effect of various concentra tions of DCAN (100-400 mu M) on the activity macrophage cells (RAW 264.7) w as studied. In these cells, DCAN-induced oxidative stress was characterized by the production of reactive oxygen intermediates (ROI), Also, the ratios of intracellular GSH/GSSG was assessed and used as a biomarker for oxidati ve stress. The secretion of TNF-alpha was assessed since macrophages are kn own to secrete TNF-alpha as a result of cellular oxidative stress. Electrop horetic detection of DNA degradation and Light microscopy was utilized for the characterization of DCAN-induced apoptosis, Lactate dehydrogenase (LDH) leakage and trypan blue exclusion were used as markers of cellular necrosi s, Following exposure to DCAN (200 mu M and 400 mu M), intracellular GSSG w as increased (2.5-fold of control, P < 0.05), DCAN activation of RAW cells was detected by elevated levels of intracellular ROI (1.9-2.5-fold than con trol, P < 0.05) and increased secretion of TNF-alpha (4.5 fold-than control , P < 0.05), Elecrophoresis of genomic DNA of treated cells indicated a dos e-dependent increase in degradation of genomic DNA, Morphological studies a lso indicated that exposure of RAW cells to 100 mu M or 200 mu M DCAN incit es apoptic cell death. At higher concentrations (400 mu M), however, signif icant (P < 0.05) increase in LDH leakage and decrease in cell viability (55 % of control) indicative of cellular necrosis, were observed. These studies indicate that DCAN induces dose-dependent apoptosis or necrosis in RAW cel ls that could be due to the disturbance in intracellular redox: status and initiation of ROI-mediated oxidative mechanisms of cellular damage. (C) 200 0 Elsevier Science Ltd. All rights reserved.