| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2148192 | Mutation Research/Genetic Toxicology and Environmental Mutagenesis | 2012 | 10 Pages |
The in vitro interaction of the organophosphorous insecticide (OPs) phorate with calf thymus DNA (ctDNA), and its potential to cause changes in cell cycle, membrane damage, and cytotoxicity leading to cell death (necrosis) was investigated in human amnion epithelial (WISH) cells. Fluorescence quenching revealed high binding affinity (Ka = 5.62 × 104 M−1) of phorate to ctDNA. Molecular modeling of the phorate–ctDNA interaction suggested the binding of phorate at AT rich regions on minor groove of DNA. The interaction ensued alkylation of the N-6, N-7 of adenine and C-4 carbonyl oxygen of thymine. Binding of phorate was stronger in the presence of the transition metal ion copper II (Cu2+), and has accentuated the destabilization of the DNA secondary structure. A discernable change in the voltammetric E1/2 (E0′) with lesser cathodic (ipc) and anodic (ipa) peak currents confirmed the formation of phorate–DNA and phorate–DNA–Cu (II) association complexes. Furthermore, the MTT and NRU assays demonstrated substantial phorate cytotoxicity due to loss of mitochondrial and lysosomal membrane integrity, and reduction in mitochondrial membrane potential (ΔΨm) of treated WISH cells. Cell cycle analysis of WISH cells treated with 1000 μM phorate exhibited 13.7-fold (p < 0.01) augmentation in the sub-G1 peak. Annexin V-PE and 7-ADD staining of phorate treated cells reaffirmed the development of late apoptotic or necrotic cell population in a concentration dependent manner. Thus, this study demonstrated the phorate induced DNA structural alterations and cellular damage in cultured human cells.
► This is the first report on the interaction of phorate with DNA and WISH cells. ► Phorate specifically binds to AT rich region of DNA at minor groove. ► WISH cells treated with phorate exhibited late apoptosis and necrosis. ► Toxicity of phorate is also associated with intracellular ROS generation. ► Phorate disrupts membrane potential and cause mitochondrial dysfunction.
