. INTRODUCTION  Insecticides are chemicals specifically designed to kill or control insect populations. They are widely used in agriculture, public health, and other industries to protect crops, livestock, and human health from insect-related damage and diseases. Once applied, insecticides undergo various metabolic processes in insects, which can affect their effectiveness and potential environmental impact. The metabolism of insecticides in insects involves several key mechanisms: 1. Absorption: Insecticides can enter an insect's body through various routes, such as ingestion, contact with the exoskeleton, or inhalation. The mode of entry depends on the formulation and application method of the insecticide. 2. Phase I metabolism: In this initial phase, insecticides are often transformed by enzymes into more polar compounds through processes such as oxidation, reduction, or hydrolysis. These metabolic reactions aim to make the insecticides more water-soluble and facilitate their elimination from the body. 3. Phase II metabolism: Once insecticides undergo phase I metabolism, they may be further conjugated with endogenous compounds such as sugars, amino acids, or glutathione. Conjugation reactions increase the water solubility of the insecticides even more, making them easier to excrete from the insect's body. 4. Detoxification mechanisms: Insects have developed various enzymatic systems to break down insecticides and render them less toxic. For example, insects possess enzymes like cytochrome P450 monooxygenases, esterases, and glutathione-S-transferases, which are involved in the detoxification of many insecticides. These enzymes can modify the chemical structure of insecticides, making them less harmful to the insect. 5. Excretion: Once metabolized, insecticides and their metabolites are eliminated from the insect's body. This process generally occurs through excretory organs such as Malpighian tubules, which function similarly to the kidneys in vertebrates. Insecticides and their metabolites can be excreted in the faeces, urine, or through other excretory pathways.  Microsomal oxidation refers to a type of metabolic reaction that occurs in the microsomes, which are subcellular organelles found in cells. Microsomes contain various enzymes, including cytochrome P450 enzymes, responsible for catalyzing oxidative reactions in the body. A. Cytochrome P450 enzymes are a family of enzymes involved in the metabolism of a wide range of endogenous and exogenous compounds, including pesticides, toxins, and foreign substances. These enzymes play a crucial role in the oxidation, reduction, and hydrolysis of various molecules, making them more water-soluble and easier to eliminate from the body. B. Microsomal oxidation mediated by cytochrome P450 enzymes involves the addition of an oxygen atom to a substrate molecule, resulting in the oxidation of the substrate.

1. Insect Toxicology and Residues
WELCOME
Speaker : Prajwal Gowda M.A
Roll No : 12292
Ph.D. : 1st year
Course-incharge :
Dr. Suresh M. Nebapure
(Senior Scientist)
DIVISION OF ENTOMOLOGY
ICAR-INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI - 110 012
METABOLISM AND MICROSOMAL OXIDATION
OF INSECTICIDES

2.  When insecticides are applied, they undergo various metabolic processes in insects, resulting in
the formation of new products called metabolites. This process is known as metabolism.
The metabolism of insecticides in insects involves:
1. Absorption: Insecticides can enter an insect's body through various routes.
a) Activation : The metabolic reaction that converts an inactive compound to an active
compound or an active compound to another active compound.
b) Detoxification : The metabolic reaction that converts the compound in to non toxic
compounds.
2. Detoxification mechanisms: Insects have various enzymatic systems (cytochrome P450
monooxygenases, esterases, and glutathione-S-transferases) to break down insecticides and
render them less toxic.
3. Phase I metabolism: In this initial phase, insecticides are often transformed by enzymes into more
polar compounds.
4. Phase II metabolism: Once insecticides undergo phase I metabolism, they may be further
conjugated, making them easier to excrete from the insect's body.

3. Sequential or Simultaneous metabolism of pesticides by Phase
I and Phase II reactions
Insecticide

4. Phase I reactions: (Non synthetic)
1. Oxidation : This class includes all those enzymes in which one atom of a
molecule of oxygen is reduced to water while the other is used to oxidize
the substrate. This takes place by mixed function oxidases.
2. Reduction : In these reactions halogen is replaced by hydrogen atom.
3. Hydrolytic processes : These takes place chiefly by esterases. Three
types of esterase are found in insects namely phosphatases,
carboxyesterases and carboxyamidases.
4. Glutathione mediated reaction : In this reactions glutathione is utilized
either in a purely catalytic manner or consumed by the direct binding to
the substrate.

5. Phase 2 reactions are known as "conjugation or synthetic" because the metabolites
are conjugated with glucuronic acid, sulphate, acetyl, methyl, and glycine moieties,
which are large in size and strongly polar in nature.
 Phase 2 reactions are of the following types:
• Type I: Insecticide / metabolite + Activated conjugating agent Conjugated product
Type I is very common, and occurs in almost all pesticides which include such
conjugations with methyl, acetyl, glucuronides, glucosides, and sulfates.
• Type II: Activated Insecticide /metabolite + conjugating agent Conjugated product.
Consist of amino acid conjugations.
• Type III: Reactive Insecticide / metabolite + reduced glutathione Conjugated product.
In this type of conjugation, the insecticides or their metabolites possess certain
chemical groups such as halogens, alkenes, NO2, epoxides, aliphatic and aromatic
compounds.

6. i). Glutathione conjugation : In these reactions the harmful electrophilic compounds are
conjugated with GSH (reduced glutathione). This is type III reaction. It is carried out by
glutathione-S-transferase (GST).
ii). Glucoside conjugation : In these reactions the harmful xenobiotics or their metabolites
combine with glucose to form conjugates. Most important reaction in insecticide
detoxification. Compounds with hydroxyl and carboxylic acid group undergo this.
iii). Amino acid conjugation : It occurs by the activation of the xenobiotic acid through the
enzyme requiring ATP and followed by condensation with endogenous amino acid.
iv). Acetylated conjugation : Compounds having amino or hydrazine are conjugated with
the help of acetyl coenzyme A.
v) Methylated conjugation : Compounds with amines and phenols exhibit this.

7. These are classified into 2 types:
A. Microsomal enzymes  Phase 1 metabolism
• Microsomes are subcellular organelles found in cells. It contains various enzymes,
including cytochrome P450 enzymes (CYPs), responsible for catalyzing oxidative
reactions in the body.
 Chemically, they are made up of lipoproteins and ribonucleic acid (RNA). Earlier,
microsomes were called "cytoplasmic basophilia’.
 Examples are monoxygenases, Cytochrome P450, Epoxidase, Hydrolases etc.
B. Non microsomal enzymes  Phase 2 metabolism
• These are present in the cytoplasm, mitochondria and in other tissues including
plasma. Ex: All conjugations except glucuronidation are carried out by Non-microsomal
enzymes.

8. Phase 1 reactions are mainly catalyzed by the cytochrome P450 group of enzymes.
 Phase 1 reactions include, Microsomal oxidation.
 The NADPH-requiring general oxidation system, commonly referred to as the
“Microsomal oxidase system” (also known as monooxygenase or mixed function
oxidase, MFO).
 The monooxygenase system gets its name from the way the atoms from the oxygen
molecules are separated from one another and end up in different substances.
(i.e, one oxygen molecule gets inserted into an appropriate substrate, R-H. The other
oxygen atom eventually produces a water molecule)
R-H + O₂ + [2H]  R-OH + H₂O
 & the Term ‘Mixed function oxidase’ refers to the enzyme function to oxidize two
separate substrates at the same time.

9.  MFOs are characterized by the oxidization of many different kinds of substrates.
The major components of the MFOs are:
A. NADPH-cytochrome c reductase, Flavoprotein, (as a cofactor)
 It is a mediator of electron flow from NADPH to the oxygen activating enzyme.
 It has 2 moles of FAD (flavin adenine dinucleotide) per mole.
B. Cytochrome P450 (electron transport system)
 It is the common oxygen-activating enzyme for the entire family of microsomal mixed function
oxidases.
 These are actually hemoprotein of b cytochrome type.
 Iron in reduced state can bind with high affinity to carbon monoxide and this CO-bound CYP
complex shows a large absorbance at 450nm.

10. • CYPs contains the main group of enzymes for Phase I metabolism.
• The oxidizing sites in these enzymes is the heme centre and is responsible for the
oxidation of hydrophobic compounds to hydrophilic metabolites for excretion.
• It catalyze the transfer of one atom of oxygen to a substrate producing an oxidized
substrate along with a molecule of water, as given below,

11.  The process of microsomal oxidation integrates the transfer of electrons from NADPH with
the binding of substrate and oxygen at CYP450.
 Two separate one-electron reductions are involved i.e, the first occurs with the substrate-
oxidized CYP450 complex and the second with the reduced cytochrome
P450/substrate/oxygen complex.
 Subsequently to catalysis, oxidized CYP450 is regenerated by dissociation of the hydroxylated
product and water.
The mechanism of microsomal oxidation involves three basic events
i. Substrate binding
ii. Reduction &
iii. Oxygen binding and activation.
Catalytic Events of Microsomal
Oxidase System

12. Interaction of electrons, oxygen, and substrate in the microsomal oxidase system

13. i) Substrate binding :
 Here, the electrons from NADPH integrate the of substrate and oxygen at CYP450.
ii) Reduction :
 It occurs in two separate one-electron steps due to the transfer of electrons from NADPH to CYP450.
a) Reduction of CYP450-substrate complex via NADPH-cytochrome c reductase (in the presence of
carbon monoxide with peak absorbance at 450 nm.
b) The second electron is introduced at the OxyCYP450-substrate complex.
iii) Oxygen binding and Activation:
 Binding of carbon monoxide to cytochrome in competition with oxygen indicates the role of
cytochrome in oxygen activation.
 So, the oxygen molecule is activated and split, one atom being inserted into the substrate and
the other reduced to water.

14.  Most organic insecticides and synergists are subject to
microsomal oxidation
 Many of them possess multiple sites at which oxidation
can occur.
Oxidative transformation of insecticides will be
discussed in five categories:
1. O-, S-, and N- Alkyl Hydroxylation
2. Desulfuration
3. Epoxidation
4. Thio ester oxidation
5. Aromatic hydroxylation Examples of multiple sites for
microsomal oxidation
Microsomal Oxidation of
Insecticides

15.  Here, an alkyl group adjacent to a hetero atom such as
oxygen, sulfur, and or nitrogen is a potential target for
microsomal hydroxylation.
 But because of the electronegativity of the hetero-
atom, the reaction often leads to dealkylation.
O- dealkylation
 Dealkylation of O-alkyl groups of ester or ether
containing insecticides occurs readily.
 First an unstable hydroxyl intermediate is produced
which spontaneously releases an aldehyde in the case
of a primary alkyl group or a ketone in the case of a
secondary alkyl group .
phorate
1. O-, N-, and S-
Dealkylation
O- dealkylation

16. S- dealkylation
 S- demethylation of several ethylthio compounds has been
reported, and its involvement in phorate metabolism leads
to conversion of the methyl thiocarbon to carbondioxide.
N- dealkylation
 Occurs in the metabolism of many organophosphates and
carbamates.
 This reaction often yields a fairly stable N-hydroxy alkyl
derivative, probably because nitrogen is less electronegative
than oxygen.
 The metabolite then may undergo non-oxidative cleavage to
a dealkylated product and an aldehyde.
S- dealkylation

17. N-
dealkylatio
n

18. 2. Desulfuration
 Desulfuration is one of the most common metabolic pathway of organophosphorus insecticides
which contains phosphorothioate and phosphorodithioate esters.
 P S structure of organophosphorus insecticides are desulfurated to their corresponding P
O analogues by microsomal oxidases.
 The detached sulfur is eventually excreted as inorganic sulfate.
 Oxidative desulfuration of P=S P=O is always lead to more toxic products.
(phosphorothioate activation to phosphate).
 The formation of oxons require NADPH and molecular oxygen

19. Desulfuration

20. 3. Thioether or Sulfur Oxidation
 Sulfoxidation of phorate or aldicarb, where one atom
of oxygen is attached with S, forming sulfuroxide and
when two atoms of oxygen are attached with S,
forming sulfone.
 Enzyme responsible in this reaction is called
sulfoxidase.
 Usually, the alkyl sulfur in insecticide is rapidly oxidized
to sulfoxide and more slowly to sulfones.
 Recently, it was observed that these sulfoxides enter
into phase 2 reaction, where they conjugate with
glutathione and finally excreted from body.

21. 4. Aromatic hydroxylation
or NIF shift
 The NIF shift (named after the National
Institute of Health, where it was discovered) is
a characteristic of aromatic hydroxylation by all
mixed function oxidases.
 During such hydroxylation reactions, the
hydrogen atom replaced by the hydroxyl group
is not always expelled from the molecule, but
may migrate to an adjacent position in the ring.
 The degree of hydrogen retention varies with
different substrates.

22. 5.
Epoxidatio
n
 Stable and environmentally persistant epoxides of
dihydrodiols are formed.
 Enzymes epoxidase catalyzes these reactions to
form dihydrodiols.
 Important degradation reaction in case of
cyclodiene compounds e.g., heptachlor, aldrin.
When this reaction occurs, the oxygen gets
detached to the place where chlorine is absent, but
double bond is present.
 These epoxides may further go for hydroxylation
and form trans-diols.

23. • The Toxicology and Biochemistry of Insecticides. 2015. Simon J. Yu.
• Applied Entomology Toxicology of Insecticides. 2007. Dileep K. Singh.
Referen
ces

24. Thank you VERY MUCH…