Oxidative stress induction by T-2 toxin causes DNA damage and triggers apoptosis via caspase pathway in human cervical cancer cells.
T-2 toxin is the most toxic trichothecene and both humans and animals suffer from several pathological conditions after consumption of foodstuffs contaminated with trichothecenes. We investigated the molecular mechanism of T-2 toxin induced cytotoxicity and cell death in HeLa cells. T-2 toxin at LC50 of 10 ng/ml caused time dependent increase in cytotoxicity as assessed by dye uptake, lactatedehydrogenase leakage and MTT assay. The toxin caused generation of reactive oxygen species as early as 30 min followed by significant depletion of glutathione levels and increased lipid peroxidation. The results indicate oxidative stress as underlying mechanism of cytotoxicity. Single stranded DNA damage after T-2 treatment was observed as early as 2 and 4h by DNA diffusion assay. The cells exhibited apoptotic morphology like condensed chromatin and nuclear fragmentation after 4h of treatment. Downstream of T-2 induced oxidative stress and DNA damage a time dependent increase in expression level of p53 protein was observed. The increase in Bax/Bcl2 ratio indicated shift in response, in favour of apoptotic process in T-2 toxin treated cells. Western blot analysis showed increase in levels of mitochondrial apoptogenic factors Bax, Bcl-2, cytochrome-c followed by activation of caspases-9, -3 and -7 leading to DNA fragmentation and apoptosis. In addition to caspase-dependent pathway, our results showed involvement of caspase-independent AIF pathway in T-2 induced apoptosis. Broad spectrum caspase inhibitor z-VAD-fmk could partially protect the cells from DNA damage but could not inhibit AIF induced oligonucleosomal DNA fragmentation beyond 4 h. Results of the study clearly show that oxidative stress is the underlying mechanism by which T-2 toxin causes DNA damage and apoptosis.
Chaudhari
Manjari
M
Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India.
Jayaraj
R
R
Bhaskar
A S B
AS
Lakshmana Rao
P V
PV
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects; D005260:Female; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D008315:Malondialdehyde / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D015398:Signal Transduction; D013605:T-2 Toxin / Q000633:toxicity*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology
D000590:Amino Acid Chloromethyl Ketones; D004273:DNA, Neoplasm; D018696:Neuroprotective Agents; D016159:Tumor Suppressor Protein p53; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D008315:Malondialdehyde; D005978:Glutathione; D013605:T-2 Toxin