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Cyanide induced DNA fragmentation in mammalian cell cultures
R Bhattacharya1, P V Lakshmana Rao
1Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior, India. root@drdrde.ren.nic.in
Abstract:
Cyanide is a mitochondrial poison and its toxicity is mediated through histotoxic hypoxia. Although cyanide is regarded as a neurotoxin, its other toxic manifestations are also well documented. Cyanide triggers all those events which can lead to DNA damage, but its genotoxic potential has not been established yet. The present investigation addresses the DNA damage induced by cyanide in rat thymocytes in vitro. Cell viability (eosin Y exclusion and LDH leakage) along with DNA strand breaks were measured in thymocytes exposed to 1.25-10 mM KCN for various time intervals. Cleavage into oligonucleosomal fragments of extracted DNA from cyanide treated thymocytes were visualized on gel electrophoresis. Cyanide produced both time and dose dependent DNA fragmentation accompanied by cytotoxicity. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was minimal in Ca2+ free medium. The DNA fragmentation was attenuated by Zn2+ (modulator of Ca2+/Mg2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). Cyanide induced DNA damage was further observed in baby hamster kidney cells (BHK-21), where unlike thymocytes, internucleosomal DNA fragmentation was not observed. Thymocytes were more sensitive to cyanide as compared to BHK-21 cells.
Insights
Cyanide exposure causes DNA damage and cell death in rat thymocytes. This genotoxicity is linked to calcium levels and can be reduced by specific inhibitors.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Cyanide is a known mitochondrial poison and neurotoxin.
- Its potential to cause DNA damage (genotoxicity) has not been fully established.
- Understanding cyanide's effects on DNA is crucial for assessing its overall toxicity.
Purpose of the Study:
- To investigate the genotoxic potential of cyanide in rat thymocytes in vitro.
- To characterize the mechanisms underlying cyanide-induced DNA damage.
- To compare cyanide's effects on different cell types.
Main Methods:
- Rat thymocytes and baby hamster kidney cells (BHK-21) were exposed to potassium cyanide (KCN).
- Cell viability was assessed using eosin Y exclusion and lactate dehydrogenase (LDH) leakage assays.
- DNA strand breaks and fragmentation into oligonucleosomal fragments were analyzed via gel electrophoresis.
- The influence of extracellular calcium (Ca2+), zinc (Zn2+), N-acetylcysteine, and diltiazem on DNA damage was evaluated.
Main Results:
- Cyanide induced dose- and time-dependent DNA fragmentation and cytotoxicity in thymocytes.
- DNA damage was exacerbated by elevated extracellular Ca2+ and reduced in Ca2+-free medium.
- Zinc, N-acetylcysteine, and diltiazem attenuated cyanide-induced DNA fragmentation.
- While cyanide caused DNA damage in BHK-21 cells, internucleosomal fragmentation was not observed, indicating differential sensitivity.
Conclusions:
- Cyanide exhibits genotoxic effects, inducing DNA fragmentation and cytotoxicity in rat thymocytes.
- The observed DNA damage is modulated by extracellular calcium levels and potentially involves endonuclease activity.
- Thymocytes are more susceptible to cyanide-induced DNA damage than BHK-21 cells, highlighting cell-type specific responses to cyanide toxicity.