Toxic Mechanisms of Pyrethroids Studied in Vitro

.................................................................................................8 INTRODUCTION .......................................................................................10 REVIEW OF LITERATURE......................................................................12 1. Chemical structure and biotransformation of pyrethroids .................................12 2. Acute toxicity of pyrethroids .............................................................................15 3. Chronic toxicity of pyrethroids..........................................................................16 4. Neurotoxic mechanisms of pyrethroids .............................................................17 4.1. Neural membrane as a target of pyrethroids..........................................17 4.2. Effects of pyrethroids on sodium channel .............................................19 5. Oxidative stress as a toxic mechanism of pyrethroids .......................................20 6. Endocrine disrupting effects ..............................................................................21 THE AIMS OF THE PRESENT STUDY...................................................23 MATERIALS AND METHODS ................................................................24 1. Pyrethroid compounds (I, II, III, IV) .................................................................24 2. Experimental animals and synaptosome preparation (I, II) ...............................24 3. Protein determinations (I,II)...............................................................................25 4. ATPase measurements in the exposure of synaptosomes to pyrethroids (I, II) .......................................................................................................................25 5. Cell cultures and exposure to pyrethroids (III, IV)...........................................25 5.1. SH-SY5Y cell cultures (III)..................................................................25 5.2. MCF-7 cell cultures (IV) .......................................................................26 6. Cell viability tests ..............................................................................................27 6.1. The bioluminescent assay for ATP determinations (III, IV).................27 4 6.2. Mitochondrial metabolic enzyme activity measurement (WST-1 test)(III, IV) ..................................................................................................27 7. Microscopic photographing of cell cultures ......................................................27 8. Statistical calculations........................................................................................28 9. Ethical considerations ........................................................................................28 RESULTS ................................................................................................... 29 1. Effects of pyrethroids and piperonyl butoxide (PBO) on synaptosomal ATPase activity (I, II) ............................................................................................29 2. Effects of pyrethroids on the viability of cultured neural SH-SY5Y cells, evaluated with ATP and WST-1 tests (III) ............................................................30 3. The cytotoxicity of pyrethroid compounds in SH-SY5Y cell cultures, evaluated with microscopic photographs (III) .......................................................31 4. Comparison of the toxicities of pyrethroid compounds in neural (SHSY5Y) and non-neural (MCF-7) cell lines (III, IV) ..............................................31 5. The hormone-like effects of pyrethroid compounds in MCF-7 cell cultures (IV). ..........................................................................................................33 DISCUSSION ............................................................................................. 35 1. Neurotoxicity of pyrethroid compounds in vitro ...............................................35 2. Hormone-like effects of pyrethroids in vitro .....................................................38 3. In vitro cell culture methods in the evaluation of the toxic effects of pyrethroids .............................................................................................................39 SUMMARY AND CONCLUSIONS ......................................................... 41 ACKNOWLEDGEMENTS........................................................................ 42 REFERENCES............................................................................................ 44 ORIGINAL PUBLICATIONS ................................................................... 55