insecticides

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/