Assessment of phosphamidon-induced apoptosis in human peripheral blood mononuclear cells: protective effects of N-acetylcysteine and curcumin.
The molecular mechanism for noncholinergic toxicity of phosphamidon, an extensively used organophosphate pesticide, is still not clear. The aim of the present study is to find the possible molecular mechanism of this pesticide to induce apoptosis and the role of different drugs for attenuation of such effects. Human peripheral blood mononuclear cells (PBMC) were incubated with increasing concentrations of phosphamidon (0-20 μM) for 6-24 h. The MTT assay reveals that phosphamidon induces cytotoxicity in a dose-dependent manner. Cellular glutathione (GSH) is depleted in a dose-dependent manner from 55% to 70% at concentrations between 10 and 20 μM. The percentage of cells that bind to Annexin-V, which is a representative of cells either undergoing apoptosis or necrosis during 24 h incubation, increases in a dose-dependent manner. Above 5 μM, significant necrosis of cells was observed. DNA fragmentation assay revealed that at low concentration of phosphamidon (1 μM), no appreciable change in DNA fragmentation was seen; however, distinct fragmentation was observed beyond 2.5 μM. Phosphamidon was found to cause significant depletion of GSH, which correlates well with the percentage of cells undergoing apoptosis. An increasing trend in levels of cytochrome c was observed with increasing concentration of phosphamidon, indicating that the apoptotic effect of phosphamidon is mediated through cytochrome c release. Coadministration of the antioxidants N-acetylcysteine and curcumin attenuated phosphamidon-induced apoptosis. This further supports our hypothesis that oxidative stress, as indicated by GSH depletion, results in the induction of apoptosis by release of cytochrome c.
Ahmed
Tanzeel
T
Environmental Biochemistry and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences and G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, India.
Tripathi
Ashok K
AK
Ahmed
Rafat S
RS
Banerjee
Basu Dev
BD
D000111:Acetylcysteine / Q000494:pharmacology*; D017304:Annexin A5 / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents / Q000378:metabolism; D003474:Curcumin / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000172:deficiency; D006801:Humans; D007306:Insecticides / Q000633:toxicity; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects*; D009336:Necrosis; D018384:Oxidative Stress / Q000187:drug effects; D010707:Phosphamidon / Q000633:toxicity*; D020011:Protective Agents / Q000494:pharmacology; D015203:Reproducibility of Results; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors
D017304:Annexin A5; D000975:Antioxidants; D004396:Coloring Agents; D007306:Insecticides; D020011:Protective Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D010707:Phosphamidon; D045304:Cytochromes c; C022616:thiazolyl blue; D005978:Glutathione; D003474:Curcumin; D000111:Acetylcysteine