**4. GSTs in insects**

to be efficiently conjugated to GSH by GST A4-4 [36,37]. A membrane-bound mouse GSTA4-4 [38]and a rat GSTA8-8 [39,40] were also shown efficiently conjugating 4-hydroxyalkenals. It was reported that transfected mGSTA4 protects HL 60 [41] and K562 human erythroleukemia

Free radicals can also cause DNA peroxidation. The toxicity of thymine propenal, which is generated by oxidative damage to DNA, was shown to be substantially reduced when HeLa cells received GSTP1-1 and GSH [43]. The rat GST6-6 had previously shown active toward

Within the cells, peroxides occur either as hydrogen peroxide (H2O2) or organic hydroperox‐ ides, such as fatty acid and phospholipid hydroperoxides. GSTs protect tissues from endoge‐ nous organic hydroperoxides produced during oxidative stress [35,45]. Some GSTs have shown selenium-independent H2O2 and organic hydroperoxidase activity, which are involved in free radical reactions during oxidative stress [46]. A microsomal GST A1-1 of sheep liver exhibited peroxidase activity toward fatty acid hydroperoxides such as linoleic and arachi‐ donic acid hydroperoxides [47]. Human GSTs, such as hGSTA1-1 and hGSTA2-2 [48,49], also exhibited glutathione peroxidase activity toward phospholipid hydroperoxide [50]. Other workers observed elevated GSTs in *Nilaparvata lugens* when treated with pyrethroids, which

Recent studies of GSTs have demonstrated that a Pi class GST is involved in regulation of c-Jun N-terminal kinase (JNK) signaling in mammals. GSTP interacts with c-Jun N-terminal kinase 1 (JNK1) suppressing the basal kinase activity [52,53]. A model of GST inhibition of JNK signaling was proposed [52]. Under a nonstressed condition, GSTp can exist as free dimeric enzyme or complexed with Jun–JNK thus inhibiting JNK. Upon stress, GSTP forms larger aggregates, which are unable to associate with the Jun–JNK complex, thus enabling the JNK phosphorylation of c-Jun. Phosphorylated Jun can act as a stable and active transcription factor. The accumulation of ROS in response to oxidative stress results in the activation of multiple

Apoptosis signal-regulating kinase 1 (ASK 1) can activate the JNK and the p38 signaling pathways. It plays important role in stress-induced apoptosis. Mouse GSTM1-1 was shown to

It has also been reported that human GST class Omega, GSTO1-1, modulates calcium channel (ryanodine receptors, RyRs) protecting mammalian cells from apoptosis induced by calcium (Ca2+) mobilization [57]. It was suggested that RyRs has two binding sites for GSTO1-1. The mammalian protein Bax (21 kDa) is an inducer of apoptosis. A study [58] has reported a plant GST (Theta class) as one of the Bax-inhibiting plant proteins, which prevent apoptosis in plants. GSTP1-1 was proposed [59] interacting with physiological nitric oxide (NO) carriers such as S-nitrosoglutathione (GSNO) and dinitrosyl-diglutathionyl iron complex (DNDGIC). In the absence of GSH, GSNO interacts with and modifies Cys47 and Cys101 residues of GSTP1-1 by an *S*-nitrosylation reaction. Thus, in the cellular depletion of GSH, GSTP1-1 acts directly as an

cells [42] against 4-hydroxynonenal-induced apoptosis.

induces oxidative stress and lipid peroxidation in insects [51].

stress kinase cascades and an elevated level of GSTp expression [54].

physically interact with ASK1 and repress ASK1-mediated signaling [55,56].

**3.3. Involve in cells regulatory functions**

thymine hydroperoxide [44].

320 Insecticides Resistance

In insects, GSTs are classified into two groups, class I and class II GSTs [61]. According to a recent proposed classification [62], an insect-specific Delta class GST is classified as a class I GST. This includes those from *Drosophila melanogaster*, DmGSTD1 to DmGST10 [62,63]; *Musca domestica*, MdGSTD1 to MdGSTD5 [49,61] and *Anopheles gambie*, AgGSTD1 to AgGSTD6 [64,65]. Class II consists primarily of Sigma class GSTs as identified in *D. melanogaster*, DmGSTS1 [66] and *A. gambie*, AgGSTS1 [67]. A third proposed class of insect's GSTs (class III) [68] that comprises GSTs classified as the Epsilon class in *Drosophila* and the AgGST3-1 and AgGST3-2 of *Anopheles gambiae*.

Studies on insect GSTs were reviewed in detail [7,23,69]. GSTs have been detected in Lepi‐ doptera, Diptera, Coleoptera, Dictyoptera, and Hymenoptera [23]. At present, only few insect GST structures are known. These include that from the Australian sheep blowfly, *L. cuprina* (Theta classs) [66], mosquito, *Anopheles dirus* (isoenzymes 1–3 and 1–4 by[70,71] and fruit fly, *D. melanogaster* (GSTS1) [72,73].

## **4.1. Characterization of GSTs and its challenges**

A problem faced during the extraction of insect GSTs is the possible presence of endogenous inhibitors [74]. Quinones and catecholamines released during homogenization can inhibit the GSTs' activity [75]. The last-named authors suggested the inclusion of GSH in the homoge‐ nizing buffer to protect the GSTs from inhibition. Polyphenol pigments have also been shown to inactivate the GSTs. The inclusion of 5–10 mM cysteine in the homogenate prevents the formation of polyphenol. The endogenous inhibitors can also be removed by initial loading of the crude homogenate through an ion exchange or gel filtration column [7]. There are many different strategies employed to purify the GSTs from insects. One of those is by using affinity chromatography with several different affinity matrices. A ligand, sulfobromophthaleinglutathione conjugate (BSP), has been immobilized to an agarose matrix by using either cyanogen bromide [76] or epichlorohydrin activation [77]. The enzymes bind selectively to the resulting matrices when a crude homogenate is applied directly to the columns. The bound enzymes can be eluted by using 0–5 mM of BSP [77] or 1–5 mM GSH solution [76]. The matrix has been used to purify a GST to homogeneity from *Galleria mellonella* [76,78], *Costelytra zealandica* [79], *Musca domestica* [77,80,81], *Drosophila melanogaster* [82] and *Wiseana cervinata* [83]. Another form of affinity matrix that has been used widely is the immobilized GSH- agarose matrix [84]. The bound enzymes are best eluted from this support with GSH solution. This technique has been used to purify GSTs from a number of insects. These include *Drosophila* [85]. In another instance, a study [86] had used another form of affinity column, namely immobilized *S*-hexylglutathione [87] to purify GSTs from *Drosophila*.

Investigation of multiple forms of GSTs with different isoelectric points could be performed by using isoelectrofocusing [78,88,89] or chromatofocusing [90]. Purification by affinity chromatography followed by isoelectrofocusing revealed the existence of multiple forms of GSTs [91], in house fly strains Rutgers, Cornell R, and Hirokawa. The presence of multiple isoenzymes of GSTs have also been reported in other species, such as *Aedes aegypti* [92], *G. mellonella* [78], *Plutella xylostella* [92], *C. zealandica* [79] and *Tenebrio molitor* [94].

#### **4.2. GSTs and insecticide resistance**

The majority of studies on insect GSTs have focused on their role in conferring insecticide resistance. A review [95] has indicated the importance of genetic and biochemical mechanisms in *Drosophila* in countering toxins and thus developing resistance. There are two types of biochemical mechanism of insecticide resistance outlined [96,97].The first is by selection of an altered target molecule so that no interaction between the molecules and the toxins occurs, resulting in target site resistance. The target sites of insecticides in insects include the GABA receptor, sodium channel, acetylcholine esterase, acetylcholine receptor, and juvenile hormone receptor. Second, through the process of metabolism, these toxins may be converted to less toxic and more easily excretable derivatives before reaching the target sites. This is a metabolic resistance. Several enzymes play important roles in these types of resistance, such as hydro‐ lases, mixed function oxidases, and glutathione S-transferases and a variety of other conju‐ gating (Phase II) enzymes.

High levels of GSTs have consistently been observed in resistant insect strains and play a major role in insecticide resistance [77,98,99]. Increased activity of GSTs in housefly was found to be associated with resistance to azinphosmethyl [100], parathion [98], phyrethroids [101], tetrachlorovinphos [102] and malathion [103]. *D. melanogaster* develops resistance toward several insecticides. These include malathion [104,105] and 1,1,1-trichloro-2,2-bis-(p-chloro‐ phenyl)ethane (DDT) dehydrochlorinase [106]. Resistance to diazinon, chlorpyrifos, prope‐ tamphos, and dichlofenthion and their correlation to increased GST activity has also been reported in larval *L. cuprina* [107].

GSTs have been shown to play an important role in insecticide resistance. The catalysis of conjugation of insecticides, such as organophosphorus compounds, chlorinated hydrocar‐ bons, and carbamate insecticides is shown in Figure 3. It was classified three types of reactions catalyzed by GSTs in metabolism of organophosphorus insecticides [108]. The detoxification of organophosphates (OP) occurs by the conjugation of GSH to OP via an O-dealkylation or O-dearylation conjugation, which later forms O-alkyl, O-aryl, and phosphonate conjugates which are all less toxic derivatives.

For the organochlorine insecticides the process involves dehydrochlorination and the GSH conjugation to the parent molecules [97]. Phyrethroids, however, trigger oxidative damage in cell. Therefore, GSTs role has been detoxifying the lipid peroxidation products resulted by the insecticide [51]. It is well known that some classes of GSTs have shown peroxidases activities. For example, a Delta class GST of *N. lugens* [51] and Epsilon class GST of *A. aegypti* mosquitoes [109,110] and Sigma class GST of *D. melanogaster* [111,112]. There was however report to suggest that protection against phyrethroids can be achieved through a passive sequestration process in which GSTs are capable to bind to various phyrethroids [113,114].

**Figure 3.** Structures of some (A) halogenated hydrocarbons, (B) organophosphorus, (C) carbamates, and (D) pyreth‐ roids.

#### **5. Summary**

agarose matrix [84]. The bound enzymes are best eluted from this support with GSH solution. This technique has been used to purify GSTs from a number of insects. These include *Drosophila* [85]. In another instance, a study [86] had used another form of affinity column, namely

Investigation of multiple forms of GSTs with different isoelectric points could be performed by using isoelectrofocusing [78,88,89] or chromatofocusing [90]. Purification by affinity chromatography followed by isoelectrofocusing revealed the existence of multiple forms of GSTs [91], in house fly strains Rutgers, Cornell R, and Hirokawa. The presence of multiple isoenzymes of GSTs have also been reported in other species, such as *Aedes aegypti* [92], *G.*

The majority of studies on insect GSTs have focused on their role in conferring insecticide resistance. A review [95] has indicated the importance of genetic and biochemical mechanisms in *Drosophila* in countering toxins and thus developing resistance. There are two types of biochemical mechanism of insecticide resistance outlined [96,97].The first is by selection of an altered target molecule so that no interaction between the molecules and the toxins occurs, resulting in target site resistance. The target sites of insecticides in insects include the GABA receptor, sodium channel, acetylcholine esterase, acetylcholine receptor, and juvenile hormone receptor. Second, through the process of metabolism, these toxins may be converted to less toxic and more easily excretable derivatives before reaching the target sites. This is a metabolic resistance. Several enzymes play important roles in these types of resistance, such as hydro‐ lases, mixed function oxidases, and glutathione S-transferases and a variety of other conju‐

High levels of GSTs have consistently been observed in resistant insect strains and play a major role in insecticide resistance [77,98,99]. Increased activity of GSTs in housefly was found to be associated with resistance to azinphosmethyl [100], parathion [98], phyrethroids [101], tetrachlorovinphos [102] and malathion [103]. *D. melanogaster* develops resistance toward several insecticides. These include malathion [104,105] and 1,1,1-trichloro-2,2-bis-(p-chloro‐ phenyl)ethane (DDT) dehydrochlorinase [106]. Resistance to diazinon, chlorpyrifos, prope‐ tamphos, and dichlofenthion and their correlation to increased GST activity has also been

GSTs have been shown to play an important role in insecticide resistance. The catalysis of conjugation of insecticides, such as organophosphorus compounds, chlorinated hydrocar‐ bons, and carbamate insecticides is shown in Figure 3. It was classified three types of reactions catalyzed by GSTs in metabolism of organophosphorus insecticides [108]. The detoxification of organophosphates (OP) occurs by the conjugation of GSH to OP via an O-dealkylation or O-dearylation conjugation, which later forms O-alkyl, O-aryl, and phosphonate conjugates

For the organochlorine insecticides the process involves dehydrochlorination and the GSH conjugation to the parent molecules [97]. Phyrethroids, however, trigger oxidative damage in

immobilized *S*-hexylglutathione [87] to purify GSTs from *Drosophila*.

**4.2. GSTs and insecticide resistance**

322 Insecticides Resistance

gating (Phase II) enzymes.

reported in larval *L. cuprina* [107].

which are all less toxic derivatives.

*mellonella* [78], *Plutella xylostella* [92], *C. zealandica* [79] and *Tenebrio molitor* [94].

GSTs are enzymes of multi-functional roles. Studies in insect have always directed the role of GSTs in conferring resistance toward insecticides resistance. Several classes of GSTs have been shown to counter the insecticides through direct GSH conjugation process and also their ability to react against lipid peroxidation products. This is due to the fact that some insecticides cause oxidative damage. Direct isolation of responsible GST from insect has been of a challenge due to limited ability of available affinity matrix to capture all classes of GSTs. The characterization of recombinant GSTs could have led to a better understanding of the mechanism of action and thus the regulation of the GSTs upon exposure to insecticides. The availability of fully sequence genomes in model insect such as *D. melanogaster* [115] and recently of *M. domestica* [116] could be of advantage for further studies in glutathione transferase-related insecticides resistance.
