Normally, lipophilic xenobiotics that enter an animal’s body are rapidly detoxified. Detoxification can be divided into phase I (primary) and phase II (secondary) processes (Figure 8.1). 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 glutathione 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. In insects, the major tissues involved in the metabolism of xenobiotics are the midgut, fat body, and Malpighian tubules.

1. Pesticide
Metabolism

2. • Assessing residual hazards after exposure to insecticides
• Metabolites in food - nature of these products and their toxicity
• Understanding the selective toxicity and mode of action of
insecticides (i.E., Activation/ detoxification) in target and
nontarget organisms
• To develop novel and environmentally friendly insecticides

3. • Detoxification can be divided into phase I (primary) and phase II (secondary)
processes.
• phase I metabolites - polar enough to be excreted but further converted by
phase II reactions
• phase II reactions- polar products conjugated with a variety of endogenous
compounds such as sugars, sulfate, phosphate, amino acids, or glutathione and
subsequently excreted.
• most important function of biotransformation - to decrease the lipophilicity of
xenobiotics so that ultimately they can be excreted.

4. • In insects, the major tissues involved in the metabolism of xenobiotics are
1. midgut,
2. fat body
3. malpighian tubules.

5. Phase 1
Oxidation
Hydrolysis
Reduction

6. Oxidation
• the most important among phase I reactions
• carried out mainly by a group of enzymes called cytochrome P450
monooxygenases (mixed-function oxidases [MFO], polysubstrate
monooxygenases [PSMO], microsomal oxidases, or P450 enzymes)
• located in the endoplasmic reticulum of eukaryotic cells
Cytochrome P450 monoxygenases
Cytochrome P450
NADPH- CYP450 reductase
Phospholipids

8. • The overall reaction occurs according to the equation as follows:
RH + NADPH + H+ + O2 → ROH + NADP+ + H2O
• Phospholipid - essential for electron transfer from NADPH to cytochrome
P450, : coupling of NADPH-cytochrome P450 reductase and cytochrome
P450 and in the binding of the substrate to the cytochrome.
• Addition to oxygenations, CYP450 systems also catalyze various atypical
reactions such as reductions, dehydrations, dehydrogenations, isomerizations,
and ester cleavage.

9. • CYP450 protein is named CYP followed by an Arabic number to designate
the family (with >40% sequence identity), a capital letter to designate the
subfamily (with >55% sequence identity), and an Arabic number to designate
the individual protein, for example, CYP6A1.
• More than 660 insect P450 genes, distributed in CYP4, CYP6, CYP9,
CYP12, CYP15A, CYP18A, CYP28A, CYP29A, CYP48, CYP49, CYP301–
CYP318, CYP319A, CYP321A, CYP324, CYP325, CYP329, and CYP332–
CYP343 families and subfamilies

10. • In addition to microsomal cytochrome P450s, several mitochondrial cytochrome
P450s have been shown to be involved in insecticide metabolism.
Eg: mitochondrial CYP12A4 - lufenuron resistance in D. melanogaster

11. Microsomal Oxidations
Epoxidation
Hydroxylation
N- dealkylation
O- dealkylation
Desulfuration
Sulfoxidation

12. Epoxidation:
• important microsomal reaction.
• aldrin can be oxidized to its epoxide dieldrin and
• no great increase in toxicity, but epoxides are more environmentally persistent than their
precursors.
• some of the epoxides produced in the microsomal oxidation - highly reactive - form
adducts with cellular macromolecules such as proteins, RNA, and DNA, often resulting in
chemical carcinogenesis.

13. Hydroxylation
• DDT and the carbamate insecticide carbaryl are known to be hydroxylated by
cytochrome P450 monooxygenases.
• Microsomal hydroxylation usually results in detoxification.

14. N-Dealkylation
• Common reaction in the metabolism of xenobiotics, - OP and carbamates
• Microsomal N-dealkylation results in detoxification

15. O-Dealkylation
• O-Dealkylation of alkyl groups of the ester or ether structures of insecticides
occurs frequently.
• results in detoxification

16. Desulfuration
• also known as phosphorothioate oxidation.
• OP insecticides with the P=S group are oxidatively desulfurated by CYP450
monooxygenases to their corresponding P=O analogs.
• results in activation - the product, P=O, binds more tightly to AchE - thus a
more potent acetylcholinesterase inhibitor.
• For example, parathion is desulfurated to paraoxon

17. Sulfoxidation
• thioether-containing insecticides - OP and carbamates - oxidized by CYP450
monooxygenases to their corresponding sulfoxides.
• sulfoxide formation represents an oxidative activation process leading to an
increase in anticholinesterase activity.

19. • addition to the CYP450 monooxygenases, the oxidation of certain sulfur- and
nitrogen-containing insecticides also performed by another group of
microsomal enzymes known as the flavin-containing monooxygenases
(FMOs)
• the FMO system requires NADPH and oxygen for activity and exists as
multiple forms in various tissues.

20. Hydrolysis
• OPs, carbamates, pyrethroids, and some juvenoids containing 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:
R1COOR + H2O → R1COOH + ROH
Esters have the general formula R – COO – R ′ , where R may be an alkyl group/an aryl group/
a hydrogen atom, and R′ may be an alkyl group or an aryl group but not a hydrogen atom

21. Esterases
Carboxylesterases
Phosphatases
• Carboxylesterases- play significant roles in degrading OPs, carbamates,
pyrethroids, and some juvenoids.
malathion hydrolysis, which yields both α- and β-monoacids and ethanol

22. • Phosphatases - A-esterases that detoxify many organophosphorus
insecticides, especially phosphates in insects
In houseflies, paraoxon can be hydrolyzed to diethyl phosphoric acid and p-nitrophenol

23. • amide-containing OPs - dimethoate and acephate - hydrolyzed by
carboxylamidases to their corresponding carboxylic acid derivatives

24. Reduction
• reduction less common than oxidation
Reduction
rxns
Nitro reduction
Azo reduction
Aldehyde/ ketone
reduction
• NADPH-dependent nitroreductase - cytosol of adult
female houseflies- reduces parathion to aminoparathion

25. • Aldehyde reductases - catalyze the reduction of aldehydes and ketones -
widely distributed in animal species including insects

26. During azo reduction, the double bond between two nitrogen atoms is reduced and
cleaved to produce two primary amines

27. • Phase I reactions with xenobiotics - addition of functional groups - hydroxyl,
carboxyl, and epoxide.
• phase I products - undergo conjugation reactions with endogenous molecules.
• These conjugations are called phased 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

28. Conjugation
reactions
Type 1
Activated conjugating agent +
substrate = conjugated product
Glucose
sulphate
phosphate conjugation
Type 2
Activated substrate + endogenous
molecule = conjugated product
amino acid conjugation
Type 3
Substrate + conjugating agent
(no activation) = conjugated product
Glutathione conjugation

29. Glucose Conjugation
• Common reaction - insects and plants, rarely in mammals
• Glucoside formation accomplished by a reaction between an activated
intermediate, uridine diphosphate glucose (UDPG), and the xenobiotic, with
the enzyme UDP glucosyl transferase as catalyst.
• O-glucosides have been identified from some insecticide metabolism studies,
including carbaryl, propoxur, carbofuran, DDT, and allethrin

31. Glucuronic Acid Conjugation
• Glucuronide formation is accomplished by a reaction between an activated
intermediate, uridine diphosphate glucuronic acid (UDPGA), and the
xenobiotic, with the enzyme UDP glucuronyl transferase as catalyst.
• It does not occur in insects

32. Sulfate Conjugation
• requires prior activation of inorganic sulfate by ATP to an active intermediate,
3′-phosphoadenosine-5′-phosphosulfate (PAPS), from which the sulfate group
is transferred to a substrate (ROH).
• final step catalyzed by sulfotransferase.

33. Phosphate Conjugation
• rare in animals.
• Insects major groups - phosphate conjugation has been demonstrated.
• An active phosphotransferase requires ATP and Mg+ for phosphorylation of
p-nitrophenol
ROH + ATP ---------------------- ROPO2-
3 + ADP
Mg+2
phosphotransferase

34. Amino Acid Conjugation
• Aromatic acids often conjugated with amino acids
• glycine - most frequently used amino acid.
• Two steps
RCOOH
RCOSCoA
RCONHCH2COOH
Activation by ATP and CoA
Condensation with glycine (H2NCH2COOH)

35. Glutathione Conjugation
• performed by - group of multifunctional enzymes known as
glutathione S-transferases (GSTs)
• These enzymes catalyze the conjugation of reduced glutathione (GSH)
with electrophilic substrates.
• broad substrate specificities, GSTs are responsible for the detoxification of
numerous toxicants including insecticides, herbicides, and drugs.

37. parathion dearylated by GST to produce diethyl phosphorothioic acid and
S-(p-nitrophenyl) glutathion

38. dehydrochlorination

39. • GSTs - metabolism of toxic allelochemicals such as α,β-unsaturated
carbonyl compounds (e.g., trans-cinnamaldehyde, trans-2-hexenal),
isothiocyanates (e.g., allyl isothiocyanate, benzyl isothiocyanate), and
organothiocyanates (e.g., benzyl thiocyanate) in lepidopterous insects.

41. • phase III reactions - reactions affecting the products of phase II reactions.
• After phase II reactions - xenobiotic conjugates further metabolized and
then excreted from cells.
• common example - modification and excretion of glutathione conjugates
• excretion of phase II products performed by a variety of membrane
transporters (P-glycoproteins) also known as ATP-binding cassette
transporters (ABC transporters).

42. • ABC transporter - two transmembrane domains (TMDs) and two
nucleotide-binding domains (NBDs).
• Each TMD consists six membrane-spinning alpha helices, embedded in the
membrane bilayer.
• The NBDs, on the other hand, are located
in the cytoplasm, which are the site of ATP
binding.