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

Dec 22, 2015

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

Insecticides632 Lecture 6 - Application of cellular neuroscience to a practical problem

Chris Elliott
slide 13 should come earlier
Chris Elliott
check grapics in slide 31 (cyclodiene resistance)
Chris Elliott
what is P450 in slide 32
Page 2: 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

Page 3: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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

Page 4: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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

Page 5: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Delivering insecticide effectively?

rapidity specificity

to target species side effects

stability light & air (oxygen) not too persistent

solubility cheap

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

Main targets

development ecdysis [moulting] specific to insects cuticle specific to insects

respiration CNS

Page 7: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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

Page 8: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Main classes

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

phenyl pyrazoles

Page 9: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Organophosphorus

example: malathion carbamates have similar action more toxic to insects phosphorylate acetylcholinesterase raises [ACh], so use atropine as

antidote if humans are poisoned

Page 10: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Organophosphorus

phosphate group, with two CH3 / C2H5

and one longer side chain often S replaces O

malathion

Page 11: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Carbamates also related

originally derived from calabar beans in W Africa

aldicarb LD50 5mg/kg

Page 12: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Insects OP oxidase much

more toxic cytochrome P450

oxidase in mitochondria, etc

Vertebrates OP carboxyesterase

non-toxic

More toxic to insects

Page 13: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Phosphorylate acetylcholinesterase

active site of enzyme has serine - OH

active site binds P from phosphate half like very long (80 min)

acetylcholine maloxon

Page 14: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Cyclodiene

e.g. Dieldrin, Lindane

once widely used like other

organochlorines, very lipid soluble

Page 15: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Cyclodiene mode of action

affects GABAA which carry Cl- currents binds to picrotoxin

site not GABA site enhances current faster

desensitisation

dieldrin

GABA induced Cl- current

Page 16: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Cyclodiene sensitivity

insects are more sensitive to GABAA insecticides because receptor is a

pentamer the -subunit binds

the insecticide insect homooligomer

3 receptors mammals have

heterooligomer

Page 17: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Phenyl pyrazoles

fipronil also targets

GABAA receptors same site as

Lindane

Page 18: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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

Page 19: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

DDT

symptoms of poisoning are bursty discharges

Page 20: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Na current effect

Na current is slower to end in DDT

orange bar marks stimulus

Page 21: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Pyrethroids

very quick knockdown need an oxidase inhibitor photostable and effective

30g/hectare (1% of previous insecticides\)

Page 22: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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

Page 23: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

typical pyrethroids ...

aromatic rings & Cl or Br contribute to toxicity

Deltamethrin most toxic

No CN hyperexcitatio

n convulsions

CN next to ester bond

hypersensitive paralysis

Page 24: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Na channel effect

Sodium current lasts longer Voltage clamp

Note tail current

control tetramethrin

single voltage

voltage series

Page 25: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Na channel effect - ii

Unitary sodium current lasts longer patch clamp type II open even

less often but for even longer

Page 26: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

more toxic because

of differences in Na channel sequence rat mutant isoleucine methionine in

intracellular loop of domain 2 (I874M)

Page 27: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

other effects ?

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

Page 28: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Imidacloprid

newer nicotinic binds to ACh

receptor

Page 29: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Imidacloprid iistimulate nerve and record EPSP apply carbamylcholine

Page 30: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Summary so far

Na+ channels targets of DDT, pyrethroids

AChEsterase targets of OPs ACh receptor target of Imidacloprid GABAA receptor target of cyclodienes

& fipronil

Page 31: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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]

Page 32: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Resistance mechanisms

organophosphates organochlorine cyclodiene pyrethroids

see Ann Rev Entomology 2000

Page 33: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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)

Page 34: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Organochlorine

DDT detoxified by glutathione-S-transferases (i.e. addition of glutathione)

Na channel resistance

Page 35: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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

Page 36: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Pyrethroids

non-target resistance P450 oxidase more transcription giving more

expression leads to cross-resistance to

organophosphates & carbamates target resistance Na+ channel

Page 37: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Na+ channel

kdr : leucine alanine (L1014F) 9 Musca strains

super-kdr : methionine threonine (M918T)

Page 38: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Effect on currents

M918T blocks current completely

Page 39: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

Comparative mutations

Page 40: Insecticides 632 Lecture 6 - Application of cellular neuroscience to a practical problem.

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/