Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxid ase causing: histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induc ed apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was se nsitive to elevated levels of extracellular Ca2+ and was attenuated by Zn2 (modulator of Ca2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fra gmentation induced by 5 mM KCN was preceded by loss of mitochondrial integr ity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen specie s (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragme ntation (Hoechst 33342 staining), a phenomenon that characterises the 'apop totic' type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5-3 h ) of various pharmacological agents viz., Trolox(R) (antioxidant), EGTA (Ca 2+ modulator) and aurintricarboxylic acid (ATA; Ca2+/Mg2+-dependent endonuc lease inhibitor). In addition, Quercetin (antioxidant) was tested as simult aneous treatment alone and was found to be ineffective. On the basis of var ious biochemical indices and DNA fragmentation (quantitative and qualitativ e), simultaneous treatment of Trolox(R) was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an earl y event preceding DNA damage. Both EGTA and ATA could not prevent this dama ge. Trolox(R) also increased the LD50 of KCN in mice 2.5-fold as compared t o 1.8- and 1.6-fold for EGTA and ATA, respectively. (C) 2001 Elsevier Scien ce Ireland Ltd. All rights reserved.