Insecticides632 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 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

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

Insects OP oxidase much

more toxic cytochrome P450

oxidase in mitochondria, etc

Vertebrates OP carboxyesterase

non-toxic

More toxic to insects

Phosphorylate acetylcholinesterase

active site of enzyme has serine - OH

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

acetylcholine maloxon

Cyclodiene

e.g. Dieldrin, Lindane

once widely used like other

organochlorines, very lipid soluble

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

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

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 hyperexcitatio

n convulsions

CN next to ester bond

hypersensitive paralysis

Na channel effect

Sodium current lasts longer Voltage clamp

Note tail current

control tetramethrin

single voltage

voltage series

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

Organochlorine

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

Na channel resistance

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

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/