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Insecticides

Insecticides. 632 Lecture 6 - Application of cellular neuroscience to a practical problem. Cellular Neuroscience - Revision. Resting potential Action potential Channels: voltage gated, ligand gated, ionotropic & metabotropic Chemical synaptic transmission. Aims of lecture.

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Insecticides

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  1. Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem

  2. Cellular Neuroscience - Revision • Resting potential • Action potential • Channels: • voltage gated, • ligand gated, ionotropic & metabotropic • Chemical synaptic transmission

  3. Aims of lecture • to know problems of effective application of insecticides • to know the main types of insecticides • to know their site(s) of action • possible mechanisms of resistance

  4. Reading Matters • Web see http://biolpc22.york.ac.uk/632 • Book: • Tomlin, CD S (1997) The pesticide manual • Papers • Narahashi T (1996) Neuronal ion channels as the target sites of insecticides Pharmacology & Toxicology 79: 1-14 • ffrench-Constant, RH et al (1998) Why are there so few resistance-associated mutations in insecticide target genes? Phil.Trans. R. Soc. B 353 1685-1693 • Matsuda et al (2001) Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors Trends pharm. 11:573-580

  5. Delivering insecticide effectively? • rapidity • specificity • to target species • side effects • stability • light & air (oxygen) • not too persistent • solubility • cheap

  6. Main targets • development • ecdysis [moulting] specific to insects • cuticle specific to insects • respiration • CNS

  7. Why Knockdown • resting insects have low metabolic demand • unlike mammals • general respiratory or muscular poisons not so good? • knockdown insecticides • disable insect quickly • OK to kill slowly • target CNS

  8. Main classes • organochlorine (1940s) • cyclodiene • organophosphorus • pyrethroids (1975-) • Imidacloprid (1990s) • phenyl pyrazoles

  9. Organophosphorus • example: malathion • carbamates have similar action • more toxic to insects • phosphorylate acetylcholinesterase • raises [ACh], so use atropine as antidote if humans are poisoned

  10. Organophosphorus • phosphate group, with two CH3 / C2H5 and one longer side chain • often S replaces O malathion

  11. Carbamates also related • originally derived from calabar beans in W Africa • aldicarb LD50 5mg/kg

  12. More toxic to insects • Insects • OP  oxidase  much more toxic • cytochrome P450 oxidase in mitochondria, etc • Vertebrates • OP  carboxyesterase  non-toxic

  13. maloxon Phosphorylate acetylcholinesterase • active site of enzyme has serine - OH • active site binds P from phosphate • half like very long (80 min) acetylcholine

  14. e.g. Dieldrin, Lindane once widely used like other organochlorines, very lipid soluble Cyclodiene

  15. affects GABAA which carry Cl- currents binds to picrotoxin site not GABA site enhances current faster desensitisation Cyclodiene mode of action dieldrin GABA induced Cl- current

  16. insects are more sensitive to GABAA insecticides because receptor is a pentamer the b-subunit binds the insecticide insect homooligomer b3 receptors mammals have heterooligomer a b g Cyclodiene sensitivity

  17. Phenyl pyrazoles • fipronil • also targets GABAA receptors • same site as Lindane

  18. Organochlorine • DDT • low solubility in water, high in lipids • at main peak of use, Americans ate 0.18mg/day • human mass 80kg • Na Channel effect • more toxic to insects

  19. DDT • symptoms of poisoning are bursty discharges

  20. Na current effect • Na current is slower to end in DDT orange bar marks stimulus

  21. Pyrethroids • very quick knockdown • need an oxidase inhibitor • photostable and effective • 30g/hectare (1% of previous insecticides\)

  22. major current insecticide derived from chrysanthemum Na channel effect more toxic because of differences in Na sequence may also have other effects ? typically esters of chrysanthemic acid Pyrethroids

  23. typical pyrethroids ... • No CN • hyperexcitation • convulsions aromatic rings & Cl or Br contribute to toxicity Deltamethrin most toxic • CN next to ester bond • hypersensitive • paralysis

  24. Na channel effect single voltage • Sodium current lasts longer • Voltage clamp • Note tail current voltage series control tetramethrin

  25. Na channel effect - ii • Unitary sodium current lasts longer • patch clamp • type II open even less often but for even longer

  26. more toxic because • of differences in Na channel sequence • rat mutant isoleucine  methionine in intracellular loop of domain 2 (I874M)

  27. other effects ? • Pyrethroids have been reported to affect • calcium channels • GABA, ACh, glutamate receptors

  28. Imidacloprid • newer nicotinic • binds to ACh receptor

  29. Imidacloprid ii stimulate nerve and record EPSP apply carbamylcholine

  30. Summary so far • Na+ channels targets of DDT, pyrethroids • AChEsterase targets of OPs • ACh receptor target of Imidacloprid • GABAA receptor target of cyclodienes & fipronil

  31. Problem of Resistance • resistance means that the insects survive! • some species never develop, • e.g. tsetse flies - few offspring • most very quick • e.g. mosquitoes - rapid life, many offspring • cross resistance, e.g. to DDT and pyrethroids because same target is used. • [behavioural resistance]

  32. Resistance mechanisms • organophosphates • organochlorine • cyclodiene • pyrethroids • see Ann Rev Entomology 2000

  33. carboxylesterase genes amplified e.g. in mosquito, Culex, up to 250 x copies of gene/cell carboxylesterase gene mutated higher kinetics and affinity (Tribolium) detoxified by glutathione-S-transferases (i.e. addition of glutathione) Organophosphates

  34. Organochlorine • DDT detoxified by glutathione-S-transferases (i.e. addition of glutathione) • Na channel resistance

  35. Cyclodiene • target site change known as Rdl • resistance to dieldrin • GABAA receptor • alanine 302  serine [or glycine] • change affects cyclodiene, picrotoxin binding • and reduces desensitisation

  36. Pyrethroids • non-target resistance P450 oxidase • more transcription giving more expression • leads to cross-resistance to organophosphates & carbamates • target resistance Na+ channel

  37. Na+ channel • kdr : leucine  alanine (L1014F) • 9 Musca strains • super-kdr : methionine  threonine (M918T)

  38. Effect on currents M918T blocks current completely

  39. Comparative mutations

  40. Conclusions • Cellular neuroscience helps understand many insecticide actions • binding to channel proteins • ligand-gated • voltage gated • mutation leads to resistance • target site • enzymatic degradation • Web page • http://biolpc22.york.ac.uk/632/

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