**6. Detoxifying enzymes and insecticide metabolism**

conjugation); and for Type III, halogens, alkenes, NO2, epoxides, ethers and esters (glutathione

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

The addition of GSH to α-, β-unsaturated compounds including aldehydes, ketones, lactones,

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

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

conjugation).

360 Insecticides Resistance

including:

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

The addition of GSH to various epoxides.

nitriles and nitro compounds.

protein, such as AgGSTD2.

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

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

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

The metabolism of insecticides by P450 enzymes is very often a key factor in determining toxicity to insects and to non-target species. The importance of monooxygenases in insecticide resistance became evident in the early 1960s, when it was shown that resistance to carbaryl could be abolished by the P450 inhibitor sesame [106]. Additional evidence of monooxygenasebased resistance quickly amassed [107, 108]. Monooxygenase-mediated detoxification is frequently found as a major mechanism of resistance, and unlike target site resistance, detoxification has the potential to confer cross-resistance to toxins independent of their target sites [109, 110]. Most cases of monooxygenase-mediated resistance result from an increase in detoxification (Table 3). However, in cases where the parent insecticide must undergo monooxygenase-mediated bioactivation, as is the case for many organophosphates, it is also possible that resistance could be achieved through decreased activation [111]. Although this has been reported once, it does not appear to be a common mechanism of resistance. This may explain why esterases are relatively more common than monooxygenases in resistance to some organophosphates [110, 112]. The classical example is probably the metabolism of phosphor‐ othioate insecticides. In many cases, the active ingredients of organophosphorus insecticides are phosphorothioate (P=S) compounds (also known as phosphorothionates), whereas the molecule active at the acetylcholinesterase target site is the corresponding phosphate (P=O) (Figure 2).

P450 enzymes that metabolize OPs can metabolize other insecticides as well, and this sometimes leads to potentially useful interactions. Thus, enhanced detoxification of dicofol in spider mites can lead to enhanced chlorpyriphos activation, and hence negative crossresistance [113]. Similarly, permethrin resistance in horn flies is suppressible by piperonyl butoxide, and negatively related to diazinon toxicity [114]. In *H*. *armigera* populations from West Africa, triazophos shows negative cross-resistance with pyrethroids, and in this case, the synergism shown by the OP towards the pyrethroid appears due to an enhanced activation to the oxon form [115]. Organophosphorus compounds (disulfoton and fenthion) are also activated by thioether oxidation (formation of sulphoxide and sulfone). The metabolism of pyrethroid by P450 enzymes is well studied in insects. Hydroxylations and further metabolism make pyrethroid metabolism and has been noticed for the single enantiomer of deltamethrin (Figure 3).

**Figure 2.** Metabolism of diazinon by cytochrome P450. Following an insertion of oxygen into the substrate, a reactive intermediate collapses (1) by desulphuration or (2) by cleavage of the ester linkage. DEP, diethylphosphate; DEPT, di‐ ethylphosphorothioate; P-ol, 2-isopropoxy-4-methyl-6-hydroxypyrimidine; [S], reactive form of sulphur released dur‐ ing the reaction. Adopted from Feyereisen, 2012.

**Figure 3.** Metabolism of deltamethrin by insect P450 enzymes: (1) deltamethrin; (2) 4′ hydroxydeltamethrin; (3) *trans*hydroxymethyl-deltamethrin; (4) cyano (3-hydroxyphenyl) methyl deltamethrate; (5) deltamethric acid. Adopted from Feyereisen, 2012.

The currently banned cyclodiene insecticides aldrin, heptachlor and isodrin are epoxidized by P450 enzymes to the environmentally stable toxic epoxides dieldrin, heptachlor epoxide and endrin, respectively [116]. Recombinant CYP6A1, -A2, -A8, -B8 and -B27; CYP12A1; and CYP321A1 can catalyse these epoxidations. Examples of pro-insecticide metabolism include the activation of chlorfenapyr by N-dealkylation [117] and diafenthiuron by S-oxidation [118]. In each case, the insect P450-dependent activation is a key in the selective toxicity of these proinsecticides that target mitochondrial respiration. Recombinant housefly CYP6A1 catalyses the activation of chlorfenapyr (Figure 4).

**Figure 4.** Chlorfenapyr and cypermethrin metabolism. The same P450 in *Heliothis virescens* probably activates the pyr‐ role and inactivates the pyrethroid, resulting in negative cross-resistance. Adopted from Feyereisen, 2012.

In *H*. *virescens*, the toxicity of chlorfenapyr is negatively correlated with cypermethrin toxicity [119]. The metabolism of imidacloprid is also of interest, particularly in relation to resistance. Piperonylbutoxide can synergize the toxicity of imidacloprid, and two P450 enzymes, CYP6G1 of *D*. *melanogaster* and CYP-6CM1vQ of *Bemisia tabaci*, have been shown to metabolize this neonicotinoid [120, 121] (Figure 5). Hydroxylations at the 4 and 5 positions can lead to the olefinic metabolite or to the dihydroxylated metabolite. In the whitefly, the 5-hydroxy metabolite is not toxic, but the 4-hydroxy metabolite is as toxic as the parent compound, so region selectivity may be of importance.

Despite the continuous use of insecticides, there are repeated failures in controlling the sucking insect pest species in recent years [11, 21] in different conventional tea plantations of Terai, the Dooars and Darjeeling foothill regions. Such a failure occurs due to changes in the suscepti‐ bility level of the pest species to the applied insecticides. Susceptibility level changes mainly due to metabolic detoxification of the insecticides through higher level of activity of some insecticide detoxifying enzymes under the stress of different management practices [8, 10, 11, 22]. In another mirid pest, *Lygus lineolaris,* metabolic resistance to insecticides due to enhanced level of activity has been reported by many authors [177, 178].

**Figure 3.** Metabolism of deltamethrin by insect P450 enzymes: (1) deltamethrin; (2) 4′ hydroxydeltamethrin; (3) *trans*hydroxymethyl-deltamethrin; (4) cyano (3-hydroxyphenyl) methyl deltamethrate; (5) deltamethric acid. Adopted from

**Figure 2.** Metabolism of diazinon by cytochrome P450. Following an insertion of oxygen into the substrate, a reactive intermediate collapses (1) by desulphuration or (2) by cleavage of the ester linkage. DEP, diethylphosphate; DEPT, di‐ ethylphosphorothioate; P-ol, 2-isopropoxy-4-methyl-6-hydroxypyrimidine; [S], reactive form of sulphur released dur‐

Feyereisen, 2012.

ing the reaction. Adopted from Feyereisen, 2012.

362 Insecticides Resistance

**Figure 5.** Metabolism of imidacloprid by insect P450 enzymes: (1) imidacloprid; (2) 5-hydroxyimidacloprid; (3) 4-hy‐ droxyimidacloprid; (4) dihydroxyimidacloprid; (5) Non-enzymatically derived dehydroimidacloprid. Adopted from Feyereisen, 2005.



**Table 3.** Over-expressed CYP genes in insecticide-resistant strains.

**Figure 5.** Metabolism of imidacloprid by insect P450 enzymes: (1) imidacloprid; (2) 5-hydroxyimidacloprid; (3) 4-hy‐ droxyimidacloprid; (4) dihydroxyimidacloprid; (5) Non-enzymatically derived dehydroimidacloprid. Adopted from

CYP6A1 OP, carbametes [122]

CYP6A5v2, CYP6A36 Pyrethroids [125–127] CYP6D1, CYP6D3 Pyrethroids [128–130] CYP6D1 Pyrethroids [131] CYP6D1, CYP6D3v2 Pyrethroids [132] CYP6A24 Pyrethroids [133] CYP12A1 Pyrethroids [134]

CYP6A2 DDT, malathion [135–137]

IGR [123, 124]

Malathion [137, 138] Malathion [137] DDT [139]

Lufenuron, propoxur [141] Imidacloprid [141] Imidacloprid [142] DDT [143]

DDT [140, 141] DDT [142]

**Species P450 Over-expressed Resistance pattern**

Feyereisen, 2005.

364 Insecticides Resistance

*Musca domestica*

*Drosophila melanogaster* CYP6A8

CYP6G1

In Western Flower Thrips, *Frankliniella occidentalis*, metabolic detoxification of insecticides has been reported by many authors [179]. In *Bemisia tabaci*, metabolic resistance due to enhanced activity of insecticide resistance-related enzymes has also been reported [180].
