Cytotoxic and necrotic responses in human amniotic epithelial (WISH) cells exposed to organophosphate insecticide phorate

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• 作者：Quaiser Saquib^a ; Javed Musarrat^a ; ^b ; ^{musarratj1@yahoo.com} ; Maqsood A. Siddiqui^a ; Sansa Dutta^c ; Swagata Dasgupta^c ; John P. Giesy^a ; ^d ; ^e ; Abdulaziz A. Al-Khedhairy^a
• 关键词：Phorate ; Cytotoxicity ; Necrosis ; Cell cycle ; Membrane ; ROS
• 刊名：Mutation Research/Genetic Toxicology and Environmental Mutagenesis
• 出版年：2012
• 期刊代码：31_13835718
• 类别：pha
• 出版时间：15 May, 2012
• 卷：744
• 期：2
• 页码：125-134
• 文件大小：1743 K

摘要

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 脳 10⁴ M^鈭?) 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 E_1/2 (E^{0鈥?/sup>) 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 (螖唯m) of treated WISH cells. Cell cycle analysis of WISH cells treated with 1000 渭M 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.}