*5.1.1. Cytochrome P450 monooxygenases (E.C. 1.14.-.-)*

Oxidation is considered the most important among phase I reactions. The oxidative reactions are carried out mainly by a group of enzymes called cytochrome P450 monooxygenases [also known as mixed function oxidases (MFO) or polysubstrate monooxygenases (PSMO), microsomal oxidase, P450 enzymes]. Cytochrome P450, or *CYP* genes, constitutes one of the largest family of genes, with representatives in virtually all living organisms, from bacteria to protists, plants, fungi and animals [84].

In insects, P450 monooxygenases are involved in many processes including roles in the metabolism of plant allelochemicals by herbivores and in detoxification of insecticides. The human genome carries about 57 CYP genes, and insect genomes can carry from 36 CYP genes in the body louse *Pediculus humanus* [85] to 170 in a mosquito [86]. Each P450 protein is the product of a distinct CYP gene, and P450 diversity is the result of successive gene (or genome) duplications followed by sequence divergence [84].

The typically 45–55-kDa P450 proteins are heme-thiolate enzymes. Their essential common feature is the absorbance peak near 450 nm of their FeII–CO complex for which they are named [87]. P450 enzymes are best known for their monooxygenase role, catalysing the transfer of one atom of molecular oxygen to a substrate and reducing the other to water. The simple stoichiometry commonly describes the monooxygenase or mixed function oxidase reaction of P450:

> RH+O +NADPH+H ROH+H O+NADP 2 2 + + ®

However, oxygen atom transfer is not the only catalytic function of P450 enzymes. They also show activities such as oxidases, reductases, desaturases, isomerases, etc. and collectively are known to catalyse at least 60 chemically distinct reactions (Table 2).

The first insect P450 cloned and sequenced was CYP6A1 from *Musca domestica* in 1989 [88]. The P450 gene complement (CYPome) size of an insect genome is not a definite number [89]. Insects can survive with small CYPomes even in toxic environments. The human body louse *Pediculus humanus*, with 36 CYP genes, is known to become highly resistant to many classes of insecticides [85], and the honeybee, with just 46 CYP genes [90], is not more sensitive than


**Table 2.** Enzymatic reactions catalysed by insect P450 enzymes (adapted from Feyereisen, 2005).

other species in a comparison to the toxicity of 62 insecticides [91]. The main driver of CYPome evolution is of course gene duplication, followed by divergence (by neofunctionalization or subfunctionalization) or death (pseudogenization or deletion) [84].

#### *5.1.2. Carboxylesterases (EC 3.1.1.1)*

Carboxylesterase or esterase is a collective term for the enzymes that hydrolyse carboxylic esters [92]. Classification of these enzymes is difficult because of their overlapping substrate

specificity [93]. However, the esterase classification of Aldridge [94] is generally recognized. According to that classification, esterases inhibited by paraoxon in a progressive and temper‐ ature-dependent manner are called B-esterases and those which are not inhibited are Aesterases [94]. Some A-esterases can hydrolyse OPs, through an acylated cysteine in their active site, and are termed phosphoric triester hydrolases (EC 3.1.8.) [95, 96]. The term carboxyles‐ terase is now mainly attributed to B-esterases [95, 96]. These enzymes have an active site serine residue, hence the terms B-esterase and serine hydrolase are synonymous. 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 alcohol.

Esterases that metabolize organophosphates can be divided into three groups: A-esterases which are not inhibited by organophosphates but hydrolyse them; B-esterases, which are susceptible to organophosphate inhibition; and C-esterases which are uninhibited by organo‐ phosphates and do not degrade them [41].

There are two types of esterases that are important in metabolizing insecticides, namely, carboxylesterases and phosphatases (also called phosphorotriester hydrolases or phosphortriesterases). Carboxyl esterases, which are B-esterases, play a significant role in degrading organophosphates, carbamates, pyrethroids, and some juvenoids in insects. The best example is malathion hydrolysis, which yields both α- and β-monoacids and ethanol [41]. Phosphatases are A-esterases that detoxify many organophosphorous insecticides especially phosphates in insects. In houseflies, paraoxon can be hydrolysed to diethyl phosphoric acid and *p*-nitrophe‐ nol. Phosphatases also hydrolyse the alkyl groups of organophosphates. Paraoxon is hydro‐ lysed by the enzyme in houseflies. Several amides containing organophosphorous insecticides such as dimethoate and acephate have been shown to be hydrolysed by carboxylamidases to their corresponding carboxylic acid derivatives [41].

#### **5.2. Phase II reactions**

other species in a comparison to the toxicity of 62 insecticides [91]. The main driver of CYPome evolution is of course gene duplication, followed by divergence (by neofunctionalization or

CYP405A2 CYP332A3 CYP6M2

CYP4G1

– –

+?(Sterols, ecdysteroid)

+(Defensive steroids)

Carboxylesterase or esterase is a collective term for the enzymes that hydrolyse carboxylic esters [92]. Classification of these enzymes is difficult because of their overlapping substrate

subfunctionalization) or death (pseudogenization or deletion) [84].

**Table 2.** Enzymatic reactions catalysed by insect P450 enzymes (adapted from Feyereisen, 2005).

Reaction catalysed P450

Dehalogenation CYP6G1

Aromatic hydroxylation CYP6D1, CYP6G1, CYP6M2

N-dealkylation CYP6A5, CYP12A1

N-oxidation +(Nicotine) S-oxidation +(Phorate) Aldehyde oxidation CYP18A1

Phosphorothioate ester oxidation CYP6A1, CYP6A2, CYP6D1, CYP12A1

O2 to H2O, H2O2, O2 <sup>−</sup> CYP6A1 (and probably most P450 enzymes)

Epoxidation CYP6A1, CYP6A2, CYP6B8, CYP6B27, CYP6AB3, CYP6

CYP4C7, CYP6A1, CYP6A2, CYP6A8, CYP6G1, CYP6M2, CYP6CM1vQ, CYP9T2, CYP12A1, CYP18A1, CYP302A1

CYP6A1, CYP6D1, CYP6A5, CYP6B4, CYP6B17, CYP6B21, CYP6G1, CYP6Z2, CYP6CM1vQ, CYP9A12, CYP9A14,

CYP6AB11, CYP9E1, CYP12A1, CYP15A1, CYP321A1

CYP306A1, CYP312A1, CYP314A1, CYP315A1

CYP12A1, CYP321A1,

Oxidase activity

358 Insecticides Resistance

O-dealkylation

Aliphatic hydroxylation C–H hydroxylation

Heteroatom oxidation and dealkylation

Complex and atypical reactions Cyanogenic glucoside biosynthesis:

Decarbonylation with C–C cleavage

Endoperoxide isomerisation

Val/Ile to oximes Oximes to cyanohydrins Aryl ether cleavage Carbon–carbon cleavage

Aromatization Reduction

*5.1.2. Carboxylesterases (EC 3.1.1.1)*

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 sulphate. 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 detoxifications.

Three types of conjugation reactions occur in insects. Type I requires an activated conjugating agent that then combines with the substrate to form the conjugated product. Type II involves the activation of the substrate to form an activated donor that then combines with an endog‐ enous molecule to yield a conjugated product. In Type III, conjugation can proceed directly between the substrate and the conjugating agent without involving activation. Thus, Type I and II require information of high-energy intermediates before the conjugation reactions proceed. 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, NO2, epoxides, ethers and esters (glutathione conjugation).

#### *5.2.1. Glutathione S-transferases (EC 2.5.1.18.)*

Glutathione conjugations are performed by a group of multifunctional enzymes known as glutathione S-transferases and are involved in detoxification mechanisms of many molecules. GSTs are involved in the transport of physiologically important lipophilic compounds. These enzymes catalyse reactions in which the sulphur atom of glutathione provides electron for nucleophilic attack on a second electrophilic substrate; the latter can be endogenous natural substrates such as epoxides, organic hydroperoxides, or activated alkenals resulting from oxidative metabolism. These enzymes catalyse the conjugation of reduced glutathione (GSH) with electrophilic substrates. Glutathione S-transferases perform a variety of reactions including:

The S-alkylation of GSH by alkyl halides and related compounds.

The replacement of labile aryl halogen or nitro groups by GSH.

The replacement of labile arakyl halogen and ester groups by GSH.

The addition of GSH to various epoxides.

The addition of GSH to α-, β-unsaturated compounds including aldehydes, ketones, lactones, nitriles and nitro compounds.

The *O*-alkyl and *O*-aryl conjugation of phosphorothiotes and phosphates with GSH.

The glutathione conjugate is subsequently transformed to mercapturic acid through the stepwise loss of glutamic acid and glycine to a cysteine conjugate, which is finally acetylated before excretion. Because of their broad substrate specificities, glutathione S-transferases are responsible for the detoxification of numerous xenobiotics [97]. More than 40 GST genes have been identified in insects [98, 99]. Mammalian GSTs have been classified into eight cytosolic classes (alpha, mu, pi, theta, sigma, zeta, kappa and omega) and a microsomal class on the basis of their amino acid sequence, immunological properties and substrate specificity. Each class shares 40% or higher amino acid in common. The classification of insect GST is not clear. The majority of insect GSTs do not belong to mammalian classes. Insect glutathione Stransferases consist of two subunits (homodimers and heterodimers) of molecular weight between 19 and 35 kD. Two classes of insect GSTs (Class I and Class II) were reported [100], which have been referred to as the Delta and Sigma class, respectively. Recently, a new class of insect GSTs, referred to as Epsilon has been described in several species of insects including *Anopheles gambiae* [101]. Purified cytosolic and microsomal glutathione S-transferase isozymes from fall armyworm larvae possessed cumenehydrperoxide peroxidase [102]. A Delta class GST purified from German cockroaches also showed high peroxidase activity [103]. The name of each GST is composed of the initials of the species scientific name, followed by the acronym GST, a capital letter to designate the class name and an Arabic number for the individual protein, such as AgGSTD2.

Glutathione S-transferases are important in the metabolism of organophosphorous insecti‐ cides resulting in detoxification [99, 104]. For example, methyl parathion is dealkylated by glutathione S-tranferases to form desmethyl parathion and methyl glutathione [41]. On the contrary, parathion can be de-arylated by glutathione S-transferases to produce diethyl phosphorothioic acid and S-(*p*-nitrophenyl) glutathione [41]. Interestingly, a glutathione Stransferase isozyme from the housefly exhibits DDT-dehydrochlorinase activity, showing that DDT-dehydrochlorinase (DDTase) is one of the glutathione S-tranferases [104]. DDT-dehy‐ drochlorinase converts DDT to DDE, resulting in detoxification [41].
