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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 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 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 resistanceassociated 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 Delivering insecticide effectively? rapidity specificity to target species side effects stability light & air (oxygen) not too persistent solubility cheap Main targets development ecdysis [moulting] specific to insects cuticle specific to insects respiration CNS 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 Main classes organochlorine (1940s) cyclodiene organophosphorus pyrethroids (1975-) Imidacloprid (1990s) phenyl pyrazoles Organophosphorus example: malathion carbamates have similar action more toxic to insects phosphorylate acetylcholinesterase raises [ACh], so use atropine as antidote if humans are poisoned Organophosphorus phosphate group, with two CH3 / C2H5 and one longer side chain often S replaces O malathion Carbamates also related originally derived from calabar beans in W Africa aldicarb LD50 5mg/kg More toxic to insects Insects oxidase much more toxic OP cytochrome P450 oxidase in mitochondria, etc Vertebrates carboxyesterase non-toxic OP Phosphorylate acetylcholinesterase active site of enzyme has serine - OH active site binds P from phosphate half acetylcholine like very long (80 min) maloxon Cyclodiene e.g. Dieldrin, Lindane once widely used like other organochlorines, very lipid soluble Cyclodiene mode of dieldrin action affects GABAA which carry Cl- currents binds to picrotoxin site not GABA site enhances current faster desensitisation GABA induced Cl- current Cyclodiene sensitivity 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 Phenyl pyrazoles fipronil also targets GABAA receptors same site as Lindane 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 DDT symptoms of poisoning are bursty discharges Na current effect Na current is slower to end in DDT orange bar marks stimulus Pyrethroids very quick knockdown need an oxidase inhibitor photostable and effective 30g/hectare (1% of previous insecticides\) Pyrethroids 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 typical pyrethroids ... aromatic rings & Cl or Br contribute to toxicity Deltamethrin most toxic No CN CN next to ester bond hyperexcitation hypersensitive convulsions paralysis Na channel effect single voltage Sodium current lasts longer Voltage clamp Note tail current voltage series control tetramethrin Na channel effect - ii Unitary sodium current lasts longer patch clamp type II open even less often but for even longer more toxic because of differences in Na channel sequence rat mutant isoleucine methionine in intracellular loop of domain 2 (I874M) other effects ? Pyrethroids have been reported to affect calcium channels GABA, ACh, glutamate receptors Imidacloprid newer nicotinic binds to ACh receptor Imidacloprid ii stimulate nerve and record EPSP apply carbamylcholine Summary so far Na+ channels targets of DDT, pyrethroids AChEsterase targets of OPs ACh receptor target of Imidacloprid GABAA receptor target of cyclodienes & fipronil 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] Resistance mechanisms organophosphates organochlorine cyclodiene pyrethroids see Ann Rev Entomology 2000 Organophosphates 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-Stransferases (i.e. addition of glutathione) Organochlorine DDT detoxified by glutathione-Stransferases (i.e. addition of glutathione) Na channel resistance Cyclodiene target site change known as Rdl resistance to dieldrin GABAA receptor 302 serine [or glycine] change affects cyclodiene, picrotoxin binding and reduces desensitisation alanine Pyrethroids non-target resistance P450 oxidase more transcription giving more expression leads to cross-resistance to organophosphates & carbamates target resistance Na+ channel + Na channel kdr : leucine alanine (L1014F) 9 Musca strains super-kdr : methionine threonine (M918T) Effect on currents M918T blocks current completely Comparative mutations 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/
