Insect Control: Biological and Synthetic Agents - Index of

et al., 1992). Salehzadeh et al. (2003) prepared tubulin
from pig brains and repolymerized the protein
in vitro in the presence of 0.1 mM azadirachtin,
but this treatment only reduced polymerization by
about 20%. In addition, tubulin and repolymerized
microtubules prepared from sheep brains failed to
specifically bind [ 3 H]dihydroazadirachtin in vitro
(Nisbet et al., unpublished data).
5.7.4. Effects on Protein Synthesis
Azadirachtin directly inhibits protein synthesis in a
variety of tissues where cells are producing enzymes:
midgut cells producing trypsin (Timmins and
Reynolds, 1992), midgut and fat body cells producing
20-monooxygenases for ecdysone catabolism
(Bidmon et al., 1987; Smith and Mitchell, 1988),
and midgut and fat body cells producing detoxification
enzymes in insecticide-resistant insects (Lowery
and Smirle, 2000). These effects in whole insects
are seen also in insect cell lines where both cell
division and protein synthesis are inhibited in a
dose-dependent manner. Using S. gregaria injected
with azadirachtin, Annadurai and Rembold (1993)
were able to study polypeptide profiles of brain,
corpora cardiaca, subesophageal ganglion, and
hemolymph using high-resolution two-dimensional
gel electrophoresis. Using Sf9 cells Robertson (2004)
also carried out polypeptide separation with twodimensional
gels with spot analysis by hierarchical
and K means clustering. Both groups concluded that
differential effects on protein synthesis occurred
although overall protein synthesis was reduced.
Robertson (2004) showed that in Sf9 cells out of
381 protein spots 52.2% were decreased significantly
in intensity, 28.9% were increased in size,
and 18.9% were unchanged.
5.7.5. Studies Using Mammalian Cell Lines
Azadirachtin is not effective against mammalian
cells (measured as effects on proliferation, direct
toxicity, and morphological alterations, e.g., micronucleation)
until concentrations above 10 mM are
reached (Reed et al., 1998; Akudugu et al., 2001;
Salehzadeh et al., 2002; Robertson, 2004). On
cultured rat dorsal root ganglion neurons, neuronal
excitability was altered by modulation of potassium
conductances, but only after azadirachtin treatment
at 10 mM or higher (Scott et al., 1999). In contrast,
the EC50 for the effect of the compound on the
viability of insect cells (Sf9) in vitro is several orders
of magnitude lower than that for mammalian cell
lines, e.g., 150 pM for Sf9 cells, >10 mM for mouse
L929 cells (Salehzadeh et al., 2002). Using mammalian
cell lines to investigate azadirachtin mode
of action it was shown that azadirachtin inhibited
5: Azadirachtin, a Natural Product in Insect Control 197
protein synthesis in mouse mammary cells at concentrations
above 100 mM but had no effects on
protein secretion from these cells (Nisbet et al., unpublished
data). Cytosolic concentrations of Ca 2þ in
mouse mammary cells were unaffected by the presence
of 500 mM azadirachtin. It is therefore apparent
that azadirachtin does not inhibit protein synthesis
in mammalian cells as a secondary effect of instantaneous
mobilization of calcium stores from the
endoplasmic reticulum, as has been demonstrated
for other protein synthesis inhibitors, e.g., vasopressin
(Reilly et al., 1998), arachidonate, carbachol,
or 1,2-bis(o-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic
acid tetra(acetoxy-methyl) ester (BAPTA/
AM) (Wong et al., 1993). It is also clear from the
lack of effect on resting calcium levels or protein
secretion that azadirachtin does not induce reductions
in protein synthesis through general toxicity
or structural cellular damage and that the molecule
may interfere with a specific cellular event involved
in protein synthesis.
5.7.6. Resolving the Mode of Action of
Azadirachtin
It would appear from current knowledge that azadirachtin
may have more than one mode of action.
First, it alters or prevents the formation of new
assemblages of organelles or cytoskeleton resulting
in the disruption of cell division, blocked transport
and release of neurosecretory peptides, and inhibition
of spermatozoa formation. Second, it inhibits
protein synthesis in cells that are metabolically
active and have been switched on to produce large
amounts of protein such as digestive enzymes by
midgut cells, cultured cells with high rates of proliferation,
or cell production of mixed function
oxidases for detoxification of xenobiotics. At the
molecular level azadirachtin may act by altering or
preventing transcription or translation of proteins
expressed at particular stages of the cell cycle. Future
work to elucidate this novel mode of action must now
concentrate on the characterization and identification
of binding sites using proteomic, microarray,
and differential display techniques.
The mode of action of azadirachtin is unlikely
to be resolved through the use of mammalian cell
lines or proteins. The differences between homologous
proteins in mammals and insects may in fact
be the key to the specificity of azadirachtin. The use
of modern protein purification and production
methods should assist in resolving this question.
The rapidly expanding range of proteomic tools
available for synthesizing short peptides or recombinant
polypeptides (e.g., phage display) could provide
a high-throughput method of determining the