Involvement of caspase and reactive oxygen species in cyanobacterial toxin anatoxin-a-induced cytotoxicity and apoptosis in rat thymocytes and Vero cells.
The hepatotoxins and neurotoxins produced by bloom-forming cyanobacteria have been the cause of human and animal health hazards and even death. The most common cyanobacterial neurotoxin is anatoxin-a, and intoxications by these toxins can be fatal through muscular paralysis causing respiratory arrest. We report here anatoxin-a-induced apoptosis in two non-neuronal cells, viz. cultured rat thymocytes and African green monkey kidney cells (Vero). Anatoxin-containing cell-free extracts (ACE) from Anabena flosaquae and purified anatoxin-a were used in the study. The toxin-induced cytotoxicity was characterized by loss of viability, lactate dehydrogenase leakage, loss of mitochondrial function, and DNA fragmentation. The toxin-induced apoptosis was characterized by plasma membrane blebbing, condensed chromatin, nuclear fragmentation and formation of apoptotic bodies. Toxin-treated thymocytes showed typical internucleosomal DNA fragmentation in agarose gel electrophoresis. Ultrastructure studies confirmed the apoptotic morphology in thymocytes. ACE and anatoxin-a showed caspase-3 activation, and pretreatment with the caspase-3-specific tetrapeptide inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) abolished the DNA fragmentation and reduced the incidence of apoptotic cells. The thymocytes also showed dose- and time-dependent toxin-induced generation of reactive oxygen species. The study demonstrates that anatoxin-induced apoptosis is possibly mediated by generation of reactive oxygen species and caspase activation.
Lakshmana Rao
P V
PV
Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India. pvlrao@rediffmail.com
Bhattacharya
R
R
Gupta
Nidhi
N
Parida
M M
MM
Bhaskar
A S B
AS
Dubey
Rupa
R
D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis; D001427:Bacterial Toxins / Q000633:toxicity*; D020169:Caspases / Q000378:metabolism*; D002522:Chlorocebus aethiops; D000087522:Cyanobacteria Toxins; D053938:DNA Fragmentation; D004789:Enzyme Activation; D008297:Male; D008387:Marine Toxins / Q000633:toxicity*; D052998:Microcystins; D008854:Microscopy, Electron; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects* / Q000648:ultrastructure; D013997:Time Factors; D014326:Tropanes; D014709:Vero Cells
D001427:Bacterial Toxins; D000087522:Cyanobacteria Toxins; D008387:Marine Toxins; D052998:Microcystins; D017382:Reactive Oxygen Species; D014326:Tropanes; C509783:anatoxin a; D020169:Caspases