Date of Award

2005

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Environmental Science

First Advisor

Dr. Hazem A. H. Kataya

Second Advisor

Dr. Tahir Rizvi

Third Advisor

Dr. Evelyn Tiffany-Castiglioni

Abstract

While acute organophosphorous compound poisoning due to inhibition of acetylcholinesterase is a well-established clinical entity, the existence of chronic poisoning due to exposure to low levels of organophosphorous compounds (below the threshold required for cholinergic clinical symptoms) is a hotly debated issue. In this study, we have evaluated the effects of noncholinergic doses of malathion (0.01-20 µM) on apoptosis of murine L929 fibroblasts. Employing flow cytometric and caspase activation analyses, we demonstrate that malathion induces apoptosis in L929 cells in a dose- and time-dependent manner. The initiator caspases (caspase-8 and caspase-9) as well as the effector caspase (caspase-3) were activated by the treatment of L929 cells with malathion. Exposure of L929 cells to malathion in the presence of a general inhibitor of caspase, z-VAD-FMK, abolished the apoptotic effect of the compound. In addition, malathion induced an increase in the expression of the pro-apoptotic protein p53. However, the induction of p53 expression was subsequent to activation of the caspase cascades. The present findings suggest that the cytotoxicity of malathion at noncholinergic doses is mediated through caspase-dependent apoptosis.

By employing caspase specific inhibitors, we extended our observations to elucidate the sequence of events involved in malathion-stimulated apoptosis. Pretreatment of L929 cells with the caspase-9 specific inhibitor zLEHD-fmk attenuated malathion induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor, zIETD-fmk, had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to malathion treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in malathion-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, malathion triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that malathion disrupted mitochondrial transmembrane potential, induced formation of reactive oxygen species and caused loss of mitochondrial cardiolipin independent of the activation of caspase cascades. Finally, malathion treatment also resulted in a time-dependent up-regulation and translocation of the pro-apoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that malathion induces apoptosis in L929 cells through direct effects on mitochondrial functions, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.

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