Insecticides

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Insecticides

• 632 Lecture 6 - Application of cellular
  neuroscience to a practical problem

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Cellular Neuroscience - Revision

• Resting potential
• Action potential
• Channels
• voltage gated,
• ligand gated, ionotropic metabotropic
• Chemical synaptic transmission

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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

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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. 11573-580

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Delivering insecticide effectively?

• rapidity
• specificity
• to target species
• side effects
• stability
• light air (oxygen)
• not too persistent
• solubility
• cheap

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Main targets

• development
• ecdysis moulting specific to insects
• cuticle specific to insects
• respiration
• CNS

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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

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Main classes

• organochlorine (1940s)
• cyclodiene
• organophosphorus
• pyrethroids (1975-)
• Imidacloprid (1990s)
• phenyl pyrazoles

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Organophosphorus

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

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Organophosphorus

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

malathion
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Carbamates also related

• originally derived from calabar beans in W Africa
• aldicarb LD50 5mg/kg

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More toxic to insects

• Insects
• OP ? oxidase ? much more toxic
• cytochrome P450 oxidase in mitochondria, etc
• Vertebrates
• OP ? carboxyesterase ? non-toxic

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Phosphorylate acetylcholinesterase

• active site of enzyme has serine - OH
• active site binds P from phosphate
• half like very long (80 min)

acetylcholine
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Cyclodiene

• e.g. Dieldrin, Lindane

• once widely used
• like other organochlorines, very lipid soluble

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Cyclodiene mode of action
dieldrin

• affects GABAA which carry Cl- currents
• binds to picrotoxin site
• not GABA site
• enhances current
• faster desensitisation

GABA induced Cl- current
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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

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Phenyl pyrazoles

• fipronil
• also targets GABAA receptors
• same site as Lindane

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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

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DDT

• symptoms of poisoning are bursty discharges

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Na current effect

• Na current is slower to end in DDT

orange bar marks stimulus
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Pyrethroids

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

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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

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typical pyrethroids ...
aromatic rings Cl or Br contribute to toxicity
Deltamethrin most toxic

• No CN
• hyperexcitation
• convulsions

• CN next to ester bond
• hypersensitive
• paralysis

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Na channel effect
single voltage

• Sodium current lasts longer
• Voltage clamp
• Note tail current

voltage series
control
tetramethrin
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Na channel effect - ii

• Unitary sodium current lasts longer
• patch clamp
• type II open even less often but for even longer

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more toxic because

• of differences in Na channel sequence
• rat mutant isoleucine ? methionine in
  intracellular loop of domain 2 (I874M)

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other effects ?

• Pyrethroids have been reported to affect
• calcium channels
• GABA, ACh, glutamate receptors

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Imidacloprid

• newer nicotinic
• binds to ACh receptor

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Imidacloprid ii
stimulate nerve and record EPSP apply
carbamylcholine
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Summary so far

• Na channels targets of DDT, pyrethroids
• AChEsterase targets of OPs
• ACh receptor target of Imidacloprid
• GABAA receptor target of cyclodienes fipronil

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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

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Resistance mechanisms

• organophosphates
• organochlorine
• cyclodiene
• pyrethroids
• see Ann Rev Entomology 2000

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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-S-transferases (i.e.
  addition of glutathione)

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Organochlorine

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

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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

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Pyrethroids

• non-target resistance P450 oxidase
• more transcription giving more expression
• leads to cross-resistance to organophosphates
  carbamates
• target resistance Na channel

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Na channel

• kdr leucine ? alanine (L1014F)
• 9 Musca strains
• super-kdr methionine ? threonine (M918T)

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Effect on currents
M918T blocks current completely
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Comparative mutations
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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/