Cytotoxic and necrotic responses in human amniotic epithelial (WISH) cells exposed to organophosphate insecticide phorate.
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 (K(a)=5.62×10(4)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 (Cu(2+)), and has accentuated the destabilization of the DNA secondary structure. A discernable change in the voltammetric E(1/2) (E(0')) with lesser cathodic (i(pc)) and anodic (i(pa)) 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 1000μM phorate exhibited 13.7-fold (p<0.01) augmentation in the sub-G(1) 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.
Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.
Quaiser
Q
Saquib
Javed
J
Musarrat
Maqsood A
MA
Siddiqui
Sansa
S
Dutta
Swagata
S
Dasgupta
John P
JP
Giesy
Abdulaziz A
AA
Al-Khedhairy
D000650:Amnion / Q000166:cytology* / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects*; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects; D006801:Humans; D007306:Insecticides / Q000633:toxicity*; D053078:Membrane Potential, Mitochondrial; D009336:Necrosis / Q000139:chemically induced; D010755:Organophosphates / Q000633:toxicity; D010702:Phorate / Q000633:toxicity*; D017382:Reactive Oxygen Species
D007306:Insecticides; D010755:Organophosphates; D017382:Reactive Oxygen Species; D010702:Phorate; D004247:DNA; C427203:calf thymus DNA