Insecticide metabolism

1. DR. BALASAHEB SAWANT KONKON KRISHI VIDYAPEETH,
DAPOLI
College Of Agriculture, Dapoli
Department Of Agricultural
Entomology
Topic Name :- Metabolism of
Insecticides
Presented by :-
MR. RUSHIKESH GORAKH KALE
(ADPM/20/2731)
Submitted to :-
Dr. M. S. Karmarkar
Associate Professor
(Dept. Of Agril. Entomology)

2. METABOLISM OF
INSECTICIDES
Presented by :- Mr. Rushikesh Kale
Reg. No. :-ADPM/20/2731

3. POINTS TO COVER
I. Introduction
II. Phase I Reactions
i. Oxidation
ii. Hydrolysis
iii. Reduction
III. Phase II Reactions
i. Glucose Conjugation
ii. Glucuronic Acid Conjugation
iii. Sulphate Conjugation
iv. Phosphate Conjugation
v. Amino Acid Conjugation
vi. Glutathione Conjugation
IV. Metabolic Pathways Of Selected Insecticides
i. Carbamates
ii. Organo-phosphates
iii. Organo-chlorineates
iv. Necotinates

4.  Insects are faced with numerous toxins (xenobiotics) as they go
through their life cycle, some produced naturally by plants
(allelochemicals) and others produced by humans (insecticides).
 To protect themselves against the natural toxins, insects have
evolved Various detoxification mechanisms. These mechanisms
also sometimes allow insects to survive insecticides, and the level
and type of mechanisms differ greatly. This results in differing
toxicity among different stages, populations, and species of insects.
 Knowledge of detoxification helps us understand mechanisms of
insecticide resistance, and hence develop sound resistance
management. It also allows us to better incorporate chemical
resistance mechanisms in crop plants so that resistant crops can be
developed.
 In this chapter, we will study the major biochemical mechanisms
involved in the transformation of insecticides. We will also consider
some specific examples of insecticide metabolism in major classes.
Normally, lipophilic xenobiotics that enter an insect body are rapidly
detoxified.
I. INTRODUCTION

5.  Defination:-
 When an insecticide is applied on any living organism
(plant or animal), it undergoes some chemical changes
resulting in the formation of new products called
Metabolites and process is known as Metabolism.
 Greek word- Meabolite means Change
 Metabolism of organic insecticides – Two broad Groups –
1. Activation- Metabolic reaction that converts an inactive
compound to an active compound or an active
compound to another active compound.
2. Detoxification- The metabolic reaction converts the
compound into nontoxic compound.

6. Importance of Insecticide Metabolism
studies
(1) Studies in mammals as metabolic predictors for man:
(2) Studies in animals and plants to evaluate the potential for residue
occurrence in human foods:
(3) Studies to elucidate activation and detoxification phenomena and
mode of action:
(4) Studies to evaluate effects on non-target organisms :
(5) Studies to define metabolic basis for pesticide selectivity:
(6) Studies to satisfy regulatory requirements:
(7) Studies to find out the role of various interactions for per sistence of
pesticides:
(8) Studies to help for generating data for fixing up tolerances:

7. • Detoxification can be divided into phase I (primary) and
phase II (secondary) processes.
• Phase I reactions consist of oxidation, hydrolysis, and
reduction. The phase I metabolites are sometimes polar
enough to be excreted, but are usually further converted
by phase II reactions.
• In phase II reactions, the polar products are conjugated
with a variety of endogenous compounds such as sugars,
sulfate, phosphate, amino acids, or gluta thione and
subsequently excreted. Phase I reactions are usually
responsible for decreasing biological activity of a toxicant
and, therefore, the enzymes involved are rate limiting with
respect to toxicity.
• The most important function of biotransformation is to
decrease the lipophilicity of xenobiotics, so that ultimately
they can be excreted.

8. Figure

9. II. PHASE I REACTIONS
It includes
A. Oxidation
B. Hydrolysis
C. Reduction
A . Oxidation:-
Oxidation is taken place through mixed
function oxidases(MFO) in which one of molecule
of oxygen is reduced to water while the other is
used to oxidise the substrate.
R-H+O2+2H R-OH+H2O

10. 1. Oxidation is considered the most important among the
phase I reactions.
2. The oxidative reactions are carried out mainly by a group
of enzymes called cytochrome P450 mono-oxygenase [
also known as mixed function oxidases(MFO)].
3. Insect cytochrome P450 enzymes have been reviewed
recently by Feyereisen (2005). These enzymes, located
mainly in the endoplasmic reticulum of eukaryotic cells,
are commonly found in mammals, birds, reptiles, fish,
crustaceans, mollusks, insects, bacteria, yeast and
higher plants.
4. Microsomal cytochrome P450 monooxygenases are
three components system comprising
• Cytochrome P450,
• NADPH-cytochrome P450 reductase and
• A phospholipid (phosphatidylcholine).

11. B. Hydrolysis:-
Definition:
A chemical reaction in which water is used to
break down a compound; this is achieved by breaking a
covalent bond in the compound by inserting a water
molecule across the bond.
• Insecticides such as organophosphates, carbamates,
pyrethroids, and some juvenoids, which contain ester
linkages, are susceptible to hydrolysis.
• Esterases are hydrolases that split ester compounds by
the addition of water to yield an acid and an alcohol.
R’COOR + H₂O → R’COOH + ROH

12. Esterases that metabolize organophosphates can be
divided into three groups:
1. A-esterases, which are not inhibited by
organophosphates but hydrolyze them;
2. B-esterases, which are susceptible to
organophosphate inhibition; and
3. C-esterases, which are uninhibited by
organophosphates and do not degrade them.
There are two types of esterases that are important in
metabolizing insecticides, namely,
• Carboxylesterases and
• Phosphatases (also called phosphorotriester
hydrolases or phosphotriesterases).

13. C. Reduction
Definition:-
Reduction involves a half-reaction in which a
chemical species decreases its oxidation number, usually
by gaining electrons.Chemical reactions in which the
number of electrons associated with an atom or a group of
atoms is increased.
• In reduction preocess Halogen is replaced by Hydrogen
atom
• Example : Conversion of DDT to DDD
It includes
• Nitrobenzene reductase
• Aldehyde reductases

14. • Certain reductases are important in the detoxification of
allelochemicals.
For example, the milkweed cardinolide is metabolized by
aldehyde reductase to calactin or calotropin (an
enantiomer) in the monarch butterfly (Danacus
plexippus) (Marty and Krieger, 1984).
• Quinones such as juglone and plambagin were reduced
by quinone reductase in fall army worms, corn earworms,
tobacco budworm, and velvetbean caterpillars (Yu,
1987b).
• Among the phase I reactions, oxidation mediated by
cytochrome P450 monooxygenases is the most important
in insects.
• Resistance to insecticides caused by enhanced
microsomal monooxygenase activities has been reported
in numerous insects.

15. III. PHASE II REACTION
 Phase I reactions with xenobiotics result in the addition
of functional groups such as hydroxyl, carboxyl, and
epoxide.
 These phase I products can further undergo conjugation
reactions with endogenous molecules. These
conjugations are called phase II reactions.
 The endogenous molecules include sugars, amino
acids, glutathione, phosphate, and sulfate.
 Conjugation products are usually more polar, less toxic,
and more readily excreted than their parent compounds.
Thus, the process, with only a few exceptions, results in
detoxification.

16. • Phase II reaction involves conjugation of natural or
foreign compounds or their metabolites with readily
available, endogeneous conjugating agents (e.g.,
glucuronic acid, sulfate, acetyl, methyl, glycine) to form
conjugates.
• Conjugation process may be viewed as a normal
biochemical reaction serving as a dual role in
intermediary metabolism which is responsible for
detoxification of pesticides.
• Being a biosynthetic process, conjugation is generally
energy dependent, so directly or indirectly linked with
high energy compounds.

17. Phase 2 reactions are of the following types:
Type I: Pesticide / metabolite + Activated conjugating agent = Conjugated product
Here, pesticide conjugates into endogenous substance which is already activated
by high energy compound and finally forms a conjugated product.
Type I reactions include such conjugations as methylation, acetylation, and the
formation of glucuronides,glucosides, and sulfates.
Type II: Activated Pesticide /metabolite + conjugating agent = Conjugated product
In this case, pesticide is first activated with the high energy compound and then
conjugates with the conjugating agent forming the product.
Type III: Reactive Pesticide / metabolite + reduced glutathione = Conjugated product
Here, the reactive pesticide or their metabolites conjugates with the conjugating
agent forming conjugated product. No activation with the energy compound is
required.
In this type of conjugation, the pesticides or their metabolites possess certain
chemical groups such as halogens, alkenes, NO2, epoxides, aliphatic and
aromatic compounds.

18. • The chemical groups required for type I are OH, NH₂,
COOH, and SH (glucose conjugation, sulphate
conjugation, and phosphate conjugation);
• For type II, COOH (amino acid conjugation); and
• For type III, halogens, alkenes, NO,, epoxides, ethers, and
esters (glutathione conjugation).
• In general, conjugated products are ionic, polar, less lipid
soluble, less toxic and easily excretable frombody. Among
the above three types of conjugating reactions.
• Type I is very common, and occurs in almost all
pesticides.
Types of reaction include are:
• Glucose Conjugation
• Glucuronic Acid Conjugation
• Sulphate Conjugation
• Phosphate Conjugation
• Amino Acid Conjugation
• Glutathione Conjugation

19. i. Glucose conjugation:-
• Glucose conjugation was regarded as most important
reaction in pesticide detoxification, both in animals and
plants.
• In this reaction, glucose is first activated in presence of
enzyme pyrophosphorylase and then conjugated with
pesticide in presence of glycoxyl transferase and form the
product which is highly polar and thus excreted from body.
• In mammals, glycoxyl transferase is located primarily in liver
microsomal fraction while in insect, it is distributed to the
subcellular level.

20. ii. Glucoronic acid conjugation:-
• In glucuronic acid conjugation, the reaction intermediate is
UDPG (Uridine diphosphate Glucose), which further
changes into UDPGA (Uridine diphosphate Glucoronic acid),
and this conjugate with the pesticide in presence of the
enzyme glucoronyltransferase.

21. • Reactions (a) and (b) are catalyzed by enzymes
present in the nuclear and soluble fraction of the liver,
respectively.
• The enzyme responsible for reaction ( c ), UDP
glucoronyltransferase is located in the microsomal
fraction.
• Glucuronide formation occurs mainly in the liver,
although other organs and tissues such as kidney,
intestines, and skin also possess enzyme activity.
• A wide variety of chemicals can be conjugated with
glucuronic acid, the most common functional groups
involved being the hydroxyl, carboxyl, and amino
moieties.

22. iii. Sulphate conjugation:-
• Sulphate ester formation readily occurs in phenolic
hydroxyl, alcoholic hydroxyl and aromatic amino group.
Sulphate ester in biological conjugation is in reality half
ester which is completely ionized and highlysoluble in
H2O.
• Conjugation by sulphate formation requires two stable
activations. The sulphate ion is activated by ATP-
sulfurylase, in the following reactions:

23. • Enzymes responsible for reaction (a) and (b) are located in
the soluble fraction of the cell.
• Reaction (c) occurs with a very broad spectrum of natural
and foreign substrates which include phenols, steroids,
arylamines, chondroitin, choline, tyrosine methyl ester,
luciferin, galactocerebroside, and heparin.
• In general, this enzyme system is located in the soluble
fraction of the cell and the liver, while the presence of
sulfotransferases in the (Prodenia gut tissues of the
southern armyworm eridania) has also been reported.
• This enzyme system is active toward 4-nitrophenol as well
as toward several naturally occurring mammalian, insect,
and plant steroids, including cholesterol, α- ecdysone, and
β-sitosterol.

24. iv. Phosphate Conjugation:-
• Although the biosynthesis of phosphate esters is a
common occurrence in intermediary metabolism, the
conjugation of foreign compounds with phosphate is
rarely encountered in nature.In insects phosphate
conjugation is reported in several members of
coleopterans, Lepidoptera and hymenoptera.
• It is reported that an active phosphotransferase in insects
catalyzed the phosphorylation of 4-nitrophenol in the
presence of ATP and Mg2+.
• It is possible that ATP may serve as the activated
conjugating agent in the enzymatic phosphorylation of
foreign compounds by analogy with other type 1
conjugations.

25. v. Amino Acid Conjugation:-
• Aromatic acids are often conjugated with amino acids în
animals, glycine being the most fre quently used amino
acid.
• Conjugation of aromatic acids with glycine has been
demonstrated in several species of insects.
• Glycine conjugation occurs in two steps. The first step
involves the activation of the substrate (RCOOH) by an
enzyme system requiring ATP and coenzyme A. and the
second step involves the condensation of the activated
substrate with glycine:

26. vi. Glutathion Conjugation (Mercapteric
Acid Formation)
• This is type 3 of reaction, here neither pesticide nor
conjugating agent is get activated, but both are reactive .
The main enzyme involved in this reaction is Glutathion-s-
transferase which are group of enzymes that catalyze
conjugation of electrophilic xenobiotic compounds with
endogenous reduced glutathione. In this reaction, formation
of mercapturic acid involves 4 important steps:

27. • In this type of reaction, the substrate is first conjugate with
reduced glutathione (GSH) in presence of enzyme
Glutathion-s-transferase which further conjugate with
cysteine and glycine to form cys-glyconjugate, and then in
presence of peptidase form premercapturic acid.
Subsequently cys-gly conjugate is acetylated to form
mercapturic acid which becomes highly polar and eliminated
in urine.
• Glutathion-s-transferase are involved in wide variety of
electrophillic insectides conjugation which can be
metabolized by glutathione dependent reaction
• e.g. lindane, DDT and also many organophosphorus
pesticide that are dealkylated or dearylated.
• Glutathion-s-transferase work for binding protein and serve
as a storage place for toxic compound that have lipophillic
nature.In certain strains of insects Glutathion-s-transferase
plays an important role in development to resistenceto
pesticide e.g Housefly.

28. IV. METABOLIC PATHWAYS OF
SELECTED INSECTICIDES
i. Metabolism of Carbamate Insecticides :-
• Carbamate insecticides are susceptible to biochemical
alternations through a multitude of enzyme catalysed
reactions. However, the principal modes of
detoxification are hydrolysis, oxidation and
conjugation.
• Since carbamate insecticides are esters, they are
prone to cleavage by esterases, giving products which
are identical to those formed by chemical hydrolysis
namely parent phenol, oxime or enol, plus methyl or
dimethyl carbamic acid.
Eg. Carbofuran, Carbaryl, Aldicarb, Methomyl

29. 🧪Carbofuran
• House fly metabolizes the carbofuran by oxidation to the
3 hydroxy derivative followed by conjugation this
metabolite as a glucoside.
• Other metabolites are 3 hydroxy-N hydroxymethyl
carbofuran, N hydroxymethyl carbofuran and 3 keto
carboluran.
• The primary metabolites of carbofuran isolated from
mice were 3-hydroxy carbofuran and a small amount of
from carbofuran. N-demethylation was a minor
degradative pathway of carbofuran in mice.
• Conjugated metabolites were mostly derived 3-keto from
hydrolytic products, such as 3 keto phenol and some
carbofuran phenol, but the carbamate 3 hydroxy
carboturan was also present as carboluran a conjugate.
No 3 –hydroxy N hydroxymethyl luran appeared either
free or conjugated in mice.

30. • In bean plant carbofuran
metabolized into 3-hydroxy
carbofuran (60%), 3 keto
carbofuran (6%), conjugated 3-
hydroxy N hydroxymethyl
carbofuran. (Less than 1%),
conjugates of carbofuran
phenal and phenolic derivatives
of 3-hydroxy and 3- keto
carbofuran (6%).
• The remaining were unknown
metabolites.

31. ii. Metabolism of Organophosphate Insecticides :-
• Organophosphatos (Ops) may be phosphate,
Phosphorothionate, Phosphorothiolate, phosphorodithioate,
phosphonate, phosphonothionate, phosphonothiolothionate
and phoroamidate.
• Eg. Malathion, Dichlorvos, Parathion, Dimethoate, Phorate
🧪Malathion
• Malathion belongs to phosphorodithioate group gets
converted to phosphate analogues. Malathion undergoes
following metabolic process.
1. Activation to malaoxon (oxidation).
2. Conversion to desmethyl malathion (dealkylation)
3. Hydrolysis of malathion as such.
4. Conversion of malathion to malathion monoacide and diacid
fol lowed by their hydrolysis.

32. • The conversion of malathion to malaoxon has been
seen in 200 insects, mammals and plants (Fig).
• The malaoxon is further metabolised to dimethyl
thiophosphoric acid which converts into dimethyl
phosphoric acid and then to phosphoric acid.
• Due to dealkylation malathion is converted into
desmethyl malathion and then to phosphoric acid.
• Malathion also gets converted into mono-acid and
diacids by carboxyesterase enzyme. Further
hydrolysis of these acids takes place by
phosphatase, producing phosphorothioate and then
to phosphoric acid.

34. • The toxicity of malathion to insect is due to higher
production of malaoxon and lower activity of
carboxyesterase.
• In malathion resistant flies the carboxyesterase activity
is higher which degrades malathion to its
monocarboxylic acid analogues Dimethyl dithiophate
has been reported in plants when treated with
malathion.
• In insects malathion is oxidised to more toxic ester of
thiophosphoric acid whereas in vertebrates the
hydrolysis in the side chain takes place by the hydrolytic
enzymes carboxyesterase and carboxyamidase
producing non-toxic products. These enzymes are not
present in insects.

35. iii. Metabolism of Organo Chlorine Insecticides
Examples:- DDT, Lindane, Aldrin, Heptachlore, Endrin,
Endosulfan
🧪DDT
• Main routes of DDT metabolism in organisms are following
types.
(i) Oxidation to DDA
(ii) Oxidation to kelthane
(iii) Oxidation to dichlorobenjophenone
(iv) Dehydrochlorination to DDE
(v) Reductive dechlorination to DDD.
• DDT gets converted into DDE (non-toxic compound) in
housefly due to dehydrochlorinase enzyme and this reaction is
known as dehydrochlorination of DDT. Rates convert only
small amounts of DDT to DDE and monkeys not at all.
• DDT in the form of DDE is stored in fatty tissues of man .

37. • In Drosophila the metabolism of DDT to kelthene (Dicofol)
is taken place due to oxidation. The hydroxylation of DDT
to 4.4’ dichloroalpha (trichloromethyl) benzhydrol, in which
the hydrogen on the tertiary carbon is replaced by the
hydroxyl group has been shown to occur in Drosophila.
• The conversion of DDT to PP dichlorobenzophenone
(DBP) has been reported in fruit fly and cockroaches. The
principal metabolite of DDT in vertebrates is DDA (bis (p-
chloro-phenyl) acetic acid) which gets excreted in urine
and faeces.
• The metabolism of DDT to DDD (dichloro-diphenyl
dichloro ethane) is taken place in plants. DDD has also
been detected in insects and mammals.

38. iv. Metabolism of
Nicotine:-
• It is evident from the Fig.
That in plants and
animals nicotine is
metabolized to cotinine
through the
hydroxylation product, 2-
hydroxynicotine.
• Further metabolism of
cotinine to desmothyl
continine has also been
reported.

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