Biochemical defence of pro-oxidant plant allelochemicals by herbivorous insects

Biochemical Systematics and Ecology, Vol. 20, No. 4, pp. 269--296, 1992. 0305-1978/92 $5.00+0.00 Printed in Great Britain. © 1992 Pergamon Press Ltd.

Ecology Review Paper

Biochemical Defence of Pro-oxidant Plant Allelochemicals by Herbivorous Insects

SAMI AHMAD Department of Biochemistry/330, Natural Products Laboratory, University of Nevada, Reno, NV 89557, U.S.A.

Key Word Index--Antioxidants; ascorbate; carotenoids; l~-carotene; glutathione; lutein; c~-tocopherol; urate; antioxidant enzymes; catalase; DT-diaphorase; glutathione peroxidase; glutathione reductase; glutathione-S-transferase; superoxide dismutase; free radicals; hydroxyl radical; peroxyl radical; superoxide radical; hydrogen peroxide; lipid peroxidation; oxidative stress; oxygen toxicity; pro-oxidants; quercetin; xanthotoxin; singlet oxygen; Papilio polyxenes ; Spodoptera eridania ; Trichoplusia nL

Abstract--A new aspect of interactions among insect herbivores and defensive chemistry of plants in the regulation of oxygen toxicity exerted by pro-oxidant allelochemicals is described. Endogenous oxygen toxicity results from activation of the ground state of molecular oxygen to the superoxide anion radical (O 2.-), hydrogen peroxide (H202), hydroxyl radical (.OH), lipid hydroperoxides (LOOHs), and peroxyl radicals (LO 2 • or RO 2 . ). The strongly lipid-peroxidizing singlet oxygen (~AgO 2) is also produced during light activation of photosensitizers. Ingestion of pro-oxidants exacerbates oxygen toxicity by increasing the production of these deleterious forms of oxygen. The role of ascorbate, u.-tocopherol, glutathione, carotenoids and urate as antioxidants in insects is apparent, but needs more work for the elucidation of their roles. The major defence mechanism includes a group of antioxidant enzymes represented by superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase's peroxidative activity (GSTPX), glutathione reductase (GR), and DT-diaphorase. SOD converts O2.- radicals to H202 and 02, CAT decomposes H202 to H20 and 02, GSTPX reduces LOOHs to LOHs with GSH as reductant, and GSSG formed from GSH during the GSTPX reaction is reduced to GSH by GR. DT-diaphorase is an important antioxidant in that it reduces quinones by a two-electron reduction to stable products, thereby preventing the one-electron reduction to semiquinone radicals which generate O2.- radicals. Therefore, these enzymes are crucial for insect herbivores for preventing the free-radical cascade of oxygen, and terminating the toxic lipid peroxidation chain reaction, in response to the endogenous and potential exogenous oxidant-induced injury.

Introduction Genera/background Many selection pressures including plant secondary metabolites called allelochemicals exhibit a pervasive influence on the ecology and population biology of herbivorous insects. Plant allelochemicals defend against plant competitors, pathogens, fungi, and herbivorous insects. In turn, plant competitors including insect herbivores have evolved counter-measures to tolerate toxic allelochemicals (Ehrlich and Raven, 1964; Cates, 1980; Feeny, 1980; Berenbaum, 1981; Harborne, 1982; Ahmad, 1983a; 1983b; Bell and Carde, 1984; Ahmad, 1986; Ehrlich and Murphy, 1988).

Most studies on the mechanisms of tolerance of insects to allelochemicals have focused on enzymes such as mixed-function oxidases (Ahmad et al., 1986) and esterases (Lindroth, 1989) which directly detoxify the allelochemicals. On the other hand, for nearly three decades mammalian toxicologists have investigated the importance of deleterious free radicals and activated forms of oxygen, and their more toxic end-products. They have identified the antioxidants and a group of antioxidant enzymes as major biochemcial defences against oxidant-induced cellular and tissue injury. Such studies were lacking for herbivorous insects until research was initiated in our laboratory in 1986. Rapid progress has since been made in several laboratories on antioxidant defences of insect herbivores. In this paper, the patterns of antioxidant defences, together with their evolutionary and ecological significance in alleviating oxidative stress, are described.

(Received 14 October 1991 )

269

276 S. AHMAD

Basic chemistry of oxygen activation Molecules with paired electrons (opposite in spin) are said to be in singlet state, and

most ground state molecules of biological interest are in the singlet ground state. In the triplet state there are two unpaired electrons, and with the exception of dioxygen whose triplet state is the ground state, other triplet molecules usually represent excited states. Ground state molecular oxygen called dioxygen possesses an even number of electrons, with two unpaired electrons in its molecular orbitals (hence, a diradical), and exists in triplet ground state. Dioxygen may be activated to a free radical or singlet species.

A normal chemical bond is formed by a pair of electrons, which are opposite in spin and share a single molecular orbital. A "free radical" is either an atom or a molecule that contains a single electron with an unopposed spin, hence it represents an open bond or a half-bond, and this attribute renders it "chemically reactive" (McCord, 1985). The free electron is often represented as a dot in the chemical formula. Moreover, depending on gain or loss of an electron, a charge sign depicting an anion or cation radical may also appear in the formula (e.g. 02 • -). Self reaction between the two radicals eliminates both radical species, while a reaction between a radical and a nonradical species always generates another free radical. The latter reaction is responsible for free-radical chain reaction, "which may be thousands of events long" (McCord, 1985). Thus, a polyunsaturated fatty acid (PUFA) may be destroyed by a lipid peroxidation chain reaction initiated by a single free radical.

Dioxygen may also be activated to a high-energy singlet species by moving one of the unpaired electrons to another orbital or changing its spin. Overcoming the spin restriction is an unfavourable process, and is often said to be a forbidden process. Considerable energy is needed for the electron movement to another orbital or change in its spin, and typical sources are UV radiation or the high-energy triplet excited state of many chemicals. The dioxygen (3E-gO2; relative energy, 0 kcal mo1-1) is activated to the first excited state of oxygen (1AGO2; relative energy, 22.5 kcal mol-1), followed by a second excited state of higher relative energy (1T~+gO2; relative energy 37.5 kcal mol-1). The lifetime of the second excited state singlet oxygen is shorter (10-11-10 -9 s) than that of the first excited state (10-6-10 _3 s). This is because the second excited state is transformed initially into the first excited state singlet oxygen by a spin-allowed process, whereas the first excited state singlet oxygen undergoes a spin-forbidden transition to reach the ground state (Bellus, 1978). Therefore, most singlet oxygen (has no unpaired electrons) reactions are mediated by the first excited state, which is often denoted simply as 102 . The electronic configuration of oxygen species is illustrated below:

ground state

first excited state

second excited state

Oxidative stress

O23~:g-O2 + "if'-

O/AgO2 -H" - -

O21Zg+O2 -.f-

The term "oxidative stress" is used for the condition when the balance between oxidants and antioxidants is shifted in favour of the former. Oxidant chemicals that induce direct oxidative stress (oxidative damage to cells/tissues) are, for example, ozone (03), nitrogen oxides (NO and NO 2) and their free radicals (NO~) found in smoke and smog, and singlet and free radical species of dioxygen (102, 02 • -, XO~, etc.). On the other hand, pro-oxidants are like pro-drugs, and these compounds require activation to induce oxidative stress. Oxidative stress is a chain event, and a single initiating event caused by a pro-oxidant may cascade into a widespread chain reaction that produces many deleterious products in concentrations many magnitudes greater than the initiator. The biocidal effects of pro-oxidants are a result of exacerbation of

ANTIOXlDANT DEFENCE OF INSECT HERBIVORES 271

oxidative stress which is endogenous to all aerobic life processes (Fridovich, 1983). Therefore, the endogenous sources of toxic forms of oxygen will be reviewed first, followed by the exogenous sources, and then the biocidal effects of oxygen toxicity.

Endogenous sources of toxic forms of oxygen While dioxygen provides enormous advantages for the maintenance of aerobic life

processes, it also imposes universal toxicity. 'qhis toxicity is largely due to the intermediates of oxygen reduction, i.e. 02 • -, H202 and • OH, and any organism that avails itself of the benefits of oxygen does so at the cost of maintaining an elaborate system of defences against these intermediates" (Fridovich, 1983).

One-electron reduction of 302 generates the superoxide anion radical (02 • -). The endogenous sources for the production of 02 • - include autoxidizable molecules such as catecholamines and ubihydroquinone, and oxidoreductases such as hemoproteins and flavin enzymes; O2. - may thus be generated in subcellular compartments including nuclei, mitochondria, endoplasmic reticulum, and cytosol, and chloroplasts in plants (Chance et al., 1979; Fridovich, 1983). Moreover, O2. - is in equilibrium with the hydroperoxyl radical (HO 2. ) and these radicals can be further reduced to hydrogen peroxide (H202). In turn, H202 can be converted to the hydroxyl radical ( • OH) via the metal-catalyzed Haber-Weiss (Fenton) reaction (Halliwell et al., 1980):

02 • - + Fe3+-,Fe 2+ + 02 (1)

H202+ Fe2÷---Fe 3+ + • OH +-OH (2)

overall Fenton reaction: 02 • - + H202--,O 2 + • OH +-OH. (3)

In the above scheme, H202 formation is by one-electron reduction of dioxygen to 02 • -, followed by 02 • - dismutation to H202. A good example of this reaction is the action of xanthine oxidase (XO) on xanthine:

XO reaction: xanthine 4- H20 4- O2-,urate 4- 02 • - (4)

202 • - dismutation: 20~ - + 2H+--,H202 4- 02 (5)

overall reaction: 2(xanthine) + 2H20 + 02 + 2H+-,2(urate) + H202 4- 02. (6)

In addition, H202 may also be produced without the intermediacy of an 02 • - radical by a direct two-electron reduction of 302 by flavin enzymes, i.e. urate oxidase, glucose oxidase, many enzymes of the tricarboxylic acid (TCA) cycle, and D-amino acid oxidases; the latter class of enzymes is used here to illustrate this reaction:

a D-amino acid + H20 + O2--,a 2-0xo acid + NH 3 + H202. (7)

Dioxygen is activated in photosensitization and other reactions to 102 (Singh, 1989). Only recently it was detected in other biological systems such as during recombination of lipid or other unsaturated organic peroxyl radicals (LO.2; RO. 2), peroxidase- catalyzed reactions and in various medical conditions (Cadenas, 1989).

The structures and inter-relationships of these toxic oxygen forms, including singlet and partially reduced species, are shown in Fig. 1.

Exogenous sources of toxic forms of oxygen Approximately 20,000 structurally highly diverse allelochemicals identified to date

account for the richness and diversity of plant allelochemicals (Harborne, 1982), and some of these compounds exert oxidative stress. Examples of pro-oxidant allelochemicals produced by plants include acetophenones (benzofurans and benzopyrans), I]-carboline alkaloids, furanochromes, furanocoumarins, furanoquinoline alkaloids, extended quinones, isoflavonoid phytoalexins, isoquinoline alkaloids, lignans, poly- acetylenes, and thiophenes (Downum, 1986; Downum and Rodriguez, 1986). All of

272 S AHMAD

0 ~ 0 • • - .O--O.

Singlet oxygen (tOz) i spins Paired, nonrodiccL + e -

Ground stnte molecular oxygen (30 z) i spins unpaired, dirodicaL

'O--O:

I

+ H + + HO~, + e " --~J

H202 ,1- e- ~ OH"

Superoxide anion (O2-) i rodicaLi protonated form is hydroperoxy radical (HO~,)

Hydrogen peroxide

• OH Hydroxyl radical

FIG. 1. THE STRUCTURES AND INTER-RELATIONSHIPS OF TOXIC OXYGEN FORMS, INCLUDING StNGLET AND PARTIALLY REDUCED SPECIES. The singlet oxygen shown represents the ~AgO 2 form.

these compounds, such as quinones and flavonoids, appear to be bioactivated through metabolism (Hassan and Fridovich, 1979; Hodnick et aL, 1986).

There are two activation mechanisms for phototoxins, and the effective wave- lengths of light range from UV to visible spectrum of light (Downum and Rodriguez, 1986). For example, the furanocoumarin, 8-methoxypsoralen (8-MOP; xanthotoxin) is activated by long UV light (320-400-nm range), and the conjugated quinone, hypericin, is activated by visible light (540-610 nm) (Thomas et aL, 1992). As depicted below, in the type I mechanism a photosensitizer in its ground state (Sen) is activated by light to a singlet excited state (1Sen), which by rearrangement (intersystem crossing of electrons) is converted to the triplet excited sensitizer (3Sen or Sen*):

Sen + hv-*lSen--.3Sen. (8)

This oxygen-independent mechanism is considered most injurious because 3Sen generally bonds covalently to critical biornolecules such as DNA, tRNA, and proteins. In the type II mechanism, 102 is formed by transfer of excitation energy between 3Sen and 02 with the formation of Sen:

Sen + hv-~ 1Sen--,3Sen (9)

3Sen + O2--,Sen + 102. (10)

The reaction is thus 02 dependent and its deleterious effects are due to the highly reactive product, 102. Moreover, evidence is accumulating for some 02 • - production from most 102-generating photosensitizers such as hematoporphyrin (Grossweiner et al., 1982; Girotti, 1983), furanocoumarins, e.g. 8-MOP (Joshi and Pathak, 1983), 13- carboline alkaloids (Chae and Ham, 1986), photosensitive dyes, e.g. rose bengal (Burch and Martin, 1988), thiophenes, e.g. 0¢-terthienyl (Kagan etaL, 1989), and quinones, e.g. hypericin (Thomas et al., 1992).

In non-biological media the yield of 02 • - relative to 102 from these photosensitizers is very small, and in the range >1.0 to about 3.0%. However, as emphasized for hematoporphyrin (Grossweiner et al., 1982; Girotti, 1983) and recently demonstrated for rose bengal (Burch and Martin, 1988), a greater yield of 02 • - and other radicals, e.g. • OH radical, may result in biological systems depending on the availability of electrons from a variety of physiological reducing agents (RH 2) such as NAD(P)H, GSH, cysteine, tryptophan, tyrosine, and GTP. Essentially the O2.- radical production involves a chain reaction among SSen, an RH 2, oxygen, and a transition metal (M'+I,-,M'). This scheme has been published (Foote, 1979; Ahmad and Pardini, 1990a), and is provided here in a condensed form:

3Sen + RH2~Sen-+ RH • +H ÷ (11)

Sen- + H+~Sen • (H). (12)


302 + RH • --,O 2 • - + R + H ~-, (13)

302 + Sen • (H)-,O 2 • - + Sen + H +. (14)

Superoxide radical generated from 3Sen may undergo a Fenton reaction to produce H202, and ultimately • OH radical. The oxygen-dependent photosensitization reaction thus may generate both 102 and oxygen free radical species. The biological importance of participation of 02 . - and • OH radicals in photosensitization reaction was recently demonstrated for the bacterium, Escherichia coil B, where in its photodynamic killing, DNA was a target of oxygen radicals. This observation was supported when the induction of superoxide dismutase and catalase (which destroy 0 2 . - and H202, respectively) in the E. coil B cells prior to incubation with the photosensitizers markedly lowered mortality. Also, thiourea and dimethylsulfoxide (scavengers of • OH radical) substantially reduced cell deaths.

Phenolic compounds in general and dihydroxy compounds in particular (e.g. 1,4 dihydroxy = a dihydroquinone; 1,2 dihydroxy = a catechol) are widely distributed in nature in both plant and animal systems. These compounds via one-electron reduction or oxidation in alkaline media, or by a number of flavoenzymes, are transformed from the fully reduced state to semiquinone radicals and quinones and vice versa (Kalyanaraman eta/., 1985). Such a pathway is known as "redox cycling" (Powis, 1987), and it is characteristic of redox-active compounds such as phenolics, and oxidoreductases. The redox cycling of dihydroxy compounds is depicted below:

DHP + 302 ~ SQ" + 02 • - (15)

SQ" + le - ~ DHP (16)

SQ' + 302 ,-- Q + 02 • - (17)

Q + 1 e- *-, SQ ", (18)

where DHP may be a catechol or a dihydroquinone, SQ' is the semiquinone anion free radical, and Q is an ortho-quinone (derived from a catechol) or a quinone (derived from a dihydroquinone). The schemes above clearly show how redox-active phenolic compounds generate SQ" and O2.- radicals. Metal ions facilitate the autoxidation, oxido-reductase may facilitate the process or prevent it, and reducing agents such as GSH and ascorbate (AH 2 and as an anion, AH ) always suppress autoxidation:

In the above schemes, GS • GSQ is the glutathione dehydroascorbate.

SQ" + GSH-,DHP + GS. (19)

SQ' + AH---,DH P + A. (20)

Q+ GSH-,GSQ + H20 (21)

Q+ 2AH--,DHP + DHA. (22)

is the thienyl free radical, A . - is the ascorbyl free radical, conjugate of quinone, and DHA (=A) represents

The linear furanocoumarin, 8-MOP, and the ubiquitous flavonoid, quercetin, are examples of pro-oxidant plant chemicals that are activated by photosensitization or redox cycling (metabolic activation), respectively (Fig. 2). As reviewed above, 8-MOP is primarily a 102 generator, but also produces 02 • -, H202 and • OH radical. Consistent with its redox potential, quercetin is activated by a one-electron oxidation to an o-semiquinone free radical, which, in turn, reacts with 302 to generate 02" -, H202, and • OH radical (Hodnick et al., 1988; 1989).

Biocidal effects of oxygen toxicity Both the • OH radical and 102 are the two most reactive forms of activated 302. They

react with macromolecules such as DNA, RNA and proteins, causing alterations in their structure, and they are responsible for deleterious lipid peroxidation. These reactions,

274

OMe fo o S. AHMAD

OH

H O ~ O ~ / O H

"1" "f OH 0

FIG. 2. MODEL PLANT PRO-OXIDANT ALLELOCHEMICALS. Xanthotoxin (8-methyoxypsoralen or 8-MOP) belongs to the photodynamic pro-oxidant class, while quercetin (3,5,7,3',4'-pentahydroxyflavone) belongs to the redox-active pro-oxidant class.

especially the peroxidation of membrane lipids, cause cell dysfunction, and ultimately cell death. The resultant toxicity is responsible for numerous well-known pathologies such as ischemic injury upon reperfusion, cancer among vertebrates, and contributes to aging (Miquel, 1989).

Peroxidation leads to disruption of membranes, oxidation of thiol groups, inhibition of thiol-containing enzymes and DNA strand breaks. Oxidation of the mitochondrial GSH jeopardizes mitochondrial integrity, causes oxidation of pyridine nucleotides, and ultimately impairs energy production (decreases in ATP production). Semiquinone radicals and quinones generated by autoxidation of dihydroquinones or catechols, covalently bind to nucleic acids and proteins and inhibit a large variety of enzymes. Excited photosensitizers (3Sen) form mono- and bifunctional adducts, and DNA strand breaks (photogenotoxicity), impair mitochondrial and nuclear functions and protein synthesis. They also cause lipid peroxidation and GSH oxidation. Cell death and tissue injury are thus a consequence of altered membranes, impaired neurotransmission, impaired nuclear function and protein synthesis, and inactivation of enzymes.

It is increasingly apparent that lipid peroxidation is a very deleterious process which accounts for a majority of pathologies described above. As depicted below, the • OH radical can initiate peroxidation of PUFA (shown below as LH) to form lipid peroxides (cyclic endoperoxide or a hydroperoxide, LOOH); unless peroxide is removed, its breakdown products, e.g. lipid radical (L.), lipid peroxyl radical (LOO • or LO- 2) and • OH will continue to propagate the lipid peroxidation chain reaction:

LH+ • OH-,L • +H20 (23)

L" + O2- -~LO • 2 (24)

LO" 2 + LH-~LOOH + L" (25)

overall: 2LH + 02 + • OH~H20 + L. + LOOH (26) Tissues may be directly oxidatively damaged by peroxides, or from more reactive

and toxic breakdown products of peroxides such as epoxides, ketones, and aldehydes, e.g. malondialdehyde. Furthermore, LOOHs can be decomposed catalytically by metals to regenerate free radicals which will propagate lipid peroxidation chain reaction (Borg and Schaich, 1988), as mentioned previously. Other unsaturated molecules such as steroids (including cholesterol) and DNA may also be similarly peroxidized. Carbon-carbon double bonds of lipids and many unsaturated organic molecules are peroxidized by the insertion of 102 (Singh, 1989).

In insects, lipid peroxidation is potentially very harmful because lipids are not only essential components of cell membranes, but also have unique physiological functions. For example, cuticular hydrocarbons protect insects from desiccation, cholesterol is a precursor of ecdysteroids (molting hormones), isoprenoid juvenile hormones are important in developmental and reproductive physiology, and many lipids act as conspecific and sex-attractants (Downer, 1985).

All aerobic organisms have consequently evolved elaborate defence mechanisms to


remove toxic forms of oxygen and any peroxides formed. In phytophagous insects, such mechanisms are especially important in that they are subject to both endogenous and exoge_~nous sources of oxidative stress.

Biochemical adaptations against oxygen toxicity Many antioxidant compounds are important for the removal of toxic forms of 302,

but all organisms studied to date have a variety of enzymatic mechanisms that are considered crucial for the termination of both the free radical cascade of 302 and lipid peroxidation chain reaction. The review focuses on recent rapid advancements made in our laboratory on understanding the enzymatic defences. Our work in this area has stimulated research in several other laboratories which are also reviewed. Direct detoxification of pro-oxidants is another important area and is relevant to the choice of model insect species used in our research. This aspect is reviewed first, followed by somewhat scant information on the role of putative antioxidant compounds, and a more detailed account of antioxidant enzymatic defences.

Direct detoxification of pro-oxidant allelochemicals Insects have evolved both behavioral and metabolic defences against allelo-

chemicals. For example, many insect species circumvent the toxicity of photodynamic pro-oxidants by rolling up the leaves of plants of Apiaceae rich in coumarins before eating them so that photoactivation by sunlight is avoided (Berenbaum, 1978). Although a generalist species, the larvae of the cabbage looper moth, Trichoplusia nl; have high preference for plants of the cabbage family, Cruciferae (Hoy and Sheldon, 1987). Recently, it has become a minor pest of celery (Apium graveolens) (Jones and Granett, 1982), and has also been observed to occasionally feed on wild parsnip (Pastinaca sativa) (Ahmad etal., 1987). The domesticated celery grown as a crop does not contain the photoactive furanocoumarin, 8-MOP, but its precursor, umbelliferone, which is not photoactivated. Leaf damage during herbivory has the potential of conversion of some umbelliferone to 8-MOP. This potential risk is avoided by T. ni larvae by feeding on the underside of celery leaves, thereby avoiding the risk of direct exposure to sunlight. On the other hand, feeding on R sativa which contains 8-MOP, occurs by snipping the leaf vein and isolating the feeding site from the plant's mobile chemical defences (Ahmad et al., 1987). These feeding modes of T. ni on plants of Apiaceae minimize the threat of fatal exposure to toxic pro-oxidant allelochemicals.

Highly generalist species, because of their indiscriminate mode of feeding, are at high risk of consuming toxic plants, with fatal consequences. The Japanese beetle, Popilia japonica, despite its elaborate olfactory and gustatory discrimination (Ahmad, 1982), dies when it is attracted to and feeds on photosensitizer-containing geranium (Pelargonium domesticum) leaves and flowers (Fleming, 1972). Generalist species such as R japonica (Ahmad, 1982), the fall armyworm, Spodoptera frugiperda, and the southern armyworm, Spodoptera eridania (Ahmad et al., 1986), possess high activities of allelochemical-detoxifying cytochrome P-450-dependent polysubstrate mono- oxygenase (PSMO; EC 1.14.14.1) in their guts. As demonstrated for S. frugiperda, the armyworms are able to metabolize about 59% of ingested 8-MOP (5 Bg 8-MOP g-1 body mass) in 1.5 h (Bull et al., 1984). Because a sufficient amount of 8-MOP remains intact, the risk of incurring deleterious photosensitizing reactions is high in these polyphagous species.

An excellent example of an insect overcoming pro-oxidant allelochemical deterrence is that of larvae of the black swallowtail butterfly, Papilio polyxenes. They preferentially and successfully feed in direct sunlight on 8-MOP-containing plants of Rutaceae (e.g. Thamnosma texana, a livestock photosensitizing weed; Ivie et al., 1983) and Apiaceae (many wild species, e.g. R sativa and parsley, Petroselinum crispurn; Berenbaum, 1981 ). Papilio polyxenes larvae are very efficient in metabolizing 8-MOP to

276 S. AHMAD

non-toxic metabolites. Nearly 99% of an orally administered dose of 5 ~g 8-MOP g-1 body mass is metabolized within 1.5 h to several products; the two major products are a monohydroxy and a dihydroxy acid with the opening of the furan ring (Bull et aL, 1984).

Felton et aL (1989) have shown that phenolic compounds are oxidized to the corresponding quinones in the midgut of insects such as the tobacco bollworm (Helicoverpa = Heliothis zea). In contrast, in the tobacco hornworm, Manduca sexta, the midgut is strongly reducing which would prevent the oxidation of phenolics to the corresponding SQ. radicals and quinones (Appel and Martin, 1990). Additionally, insects may prevent deleterious oxidation of phenolics by means of a quinone reductase, and in an oxidation-reduction system, where plant ascorbic acid is utilized to reduce quinones to phenolics (Felton and Duffy, 1992). Thus depending upon gut pH, insect adaptations and counter-measures by plants, pro-oxidant phenolic compounds may undergo deleterious oxidation reduction cycles. The flavonoid, quercetin, occurs in many conjugated forms ranging from simple j]-glycoside to the complex rutinoside (rutin). The hydrolysis of these conjugates in the gut is a process of activation. This may explain why many lepidopterans such as the tobacco budworm (Heliothis virescens), H. zea, and cotton bollworm (Pectinophora gossypiella) that continually feed on phenolic rich plants are sensitive to dietary quercetin, as well as rutin (>0.2% w/w is fatal) (Harborne, 1979). Research is revealing the presence of J]- glycosidases in midguts of many insect species including lepidopterans (Lindroth, 1988, and references therein).

Trichoplusia ni is notable for its marked preference for plants of Cruciferae and within Cruciferae it feeds voraciously on white cabbage (Brassica oleracea) which has only trace amounts of quercetin compared to other crucifers which contain up to 50 mg quercetin kg -1 fresh mass (Hermann, 1976). Pesticides are used to prevent huge build up of T. n/populations on white cabbage. These observations indicated that T. ni may be a quercetin-sensitive species. On the other hand, the highly indiscriminate polyphagous mode of feeding of S. eridania larvae exposes them to a wide range and varying amounts of flavonoids including quercetin. Quercetin is one of the most abundant flavonoid compounds in the host plant range of R polyxenes, e.g. as much as 1000 mg kg 1 fresh mass of quercetin is present in the carrot (Daucus carota) leaves (Hermann, 1976). Thus, based on the breadth of diet and exposure to two typical pro- oxidants, 8-MOP and quercetin, our insect model for investigating antioxidant enzymatic defences comprised of T. ni(minimal exposure), S. eridania (occasional to moderate exposure), and R polyxenes (high exposure to both pro-oxidants).

Antioxidant compounds Mammalian literature suggests that many compounds such as carotenoids, amines,

furans, sulfhydryls, tocopherols, ascorbate and urate are essential for quenching singlet excited and triplet excited states of 302 and other compounds, and for destroy- ing oxygen-free radicals (Hochstein etal., 1984; Larson and Marley, 1984; Halliwell and Gutteridge, 1985; Larson, 1986; Larson and Berenbaum, 1988; Cadenas, 1989; Singh, 1989). However, little work has been carried out to clarify their defensive roles as antioxidants in insects.

In the interception of the excited state of a molecule with an antioxidant molecule, generally the excited state returns to harmless ground state, but the antioxidant may or may not be destroyed. In the physical or collisional quenching, the electronic energy of the excited state, either singlet or triplet, is removed by an acceptor (an antioxidant called a quencher) through a combination of energy transfer mechanisms, i.e. electronic, vibrational, rotational, or kinetic energy release (Larson, 1986; Cadenas, 1989). It may also involve radiative energy given off by the quencher, and for this to occur the absorption spectrum of the acceptor must be similar to the emission


spectrum of the donor. Carotenoids are ideally suited for quenching 8-MOP (singlet or triplet state), for absorption/emission spectra for these compounds are in the 450-550 nm range. Yet, another quenching mechanism, a nonradiative process, is possible when the electron clouds of the donor and acceptor molecules overlap; electrostatic transfer of energy then occurs between the overlapping orbital of the electrons. In the chemical quenching process, usually both the donor and acceptor are destroyed.

Carotenoids are well-known quenchers of ~O 2, and the most cited example is 13- carotene. As recently reviewed, carotenoids have been studied in insects because they play an important role as pigments for color patterns in insect mimicry (Berenbaum, 1987, and references therein). Carotenoid biochemistry, origin and biotransformations, have also been reviewed without any reference to their role as antioxidants (Kayser, 1982). In recent analysis, we have found that the major carotenoid in larvae of our three model insect species is lutein (I]-E-carotene-3, 3', diol) rather than 13-carotene. The amounts of lutein are negligible in T. ni larvae, moderate in S. eridania larvae, and highest in R polyxenes larvae which are highly adept for feeding on 8-MOP- and quercetin-containing plants (Ahmad et al., unpublished data). This finding was surprising at first because the natural food plants of all three insect species contain high levels of 13-carotene, and several more polar or oxy-carotenoids.

All animals including insects rely on a dietary supply of the carotenoid skeleton (main synthesizers are plants). Moreover, the carotenoid content of an insect species is dependent upon the carotenoid composition of the food, selective resorption and some meager in vivo transformation of lipophilic carotenoids to more polar oxy- carotenoids (Kayser, 1982). Our unpublished data suggest that the insects examined selectively resorb iutein over all other carotenoids, since the levels mirror that of their food plant(s). This is apparently the case with Lepidoptera which differ from other insects in either absorbing equally both I]-carotene and lutein, e.g. the white cabbage fly, Pieris brassicae, or 13-carotene, e.g. Manduca sexta (Kayser, 1982), or lutein as observed by us for T. ni, S. eridania and R polyxenes. It is not clear what advantage exists for accumulating different carotenoids in insect species. An extensive study has shown that by far the most effective scavenger of 102 is lycopene, a most apolar terpenoid carotene, and it is found together with 13-carotene in mammalian plasma (Di Mascio et al., 1989). The quenching constants for lycopene and 13-carotene are 31, and 13-30 /~ (109 M -~ s-l), respectively. Lutein has a quenching constant similar to 13- carotene, i.e. 8-21 kq (Di Mascio etal., 1989). Lutein is the sole carotenoid present in the vertebrate retina which is subject to photo-oxidative damage more than any other tissue. Unlike I]-carotene, lutein may have advantages because it is soluble both in lipid and aqueous media in contrast to the highly lipophilic 13-carotene which is present in lipid core(s) of the cells and in plasma it must be transported as a complex with lipophorin. Carotenoids are crucial for defence against ~O 2 attack on biomembranes, and lutein may serve this function as well as afford protection against ~O 2 insult in the cytosol. This hypothesis remains to be tested. In the only other study of the role of carotenoids in insects, 13-carotene has been shown to protect M. sexta larvae from phytotoxicity of ~-terthienyl (Aucoin etal., 1990). This protection may be via quenching of ~O 2 generated by ~-terthienyl in oxygen-dependent photoactivation, and also from quenching of singlet, and triplet excited states of 0{-terthienyl.

As extensively reviewed by Bellus (1978). quenching of ~O 2 by carotenoids (CAR) such as I}-carotene is via both a non-destructive physical quenching process, and an unusually non-destructive chemical quenching process, as follows:

102+ CAR--,302 +CAR*

CAR*-,CAR + hv

02 + CAR~CAR-O 2

CAR-O2~CAR + [02].

(27)

(28)

(29)

(30)

278 S. AHMAD

In mechanisms (27) and (28), CAR reacts with 102 to form 302 and triplet excited carotenoid (CAR*), which degenerates to the ground state CAR with release of energy. In mechanisms (29) and (30), 102 reacts with the ground state CAR to form an oxidized product, and an enzyme removes oxygen and regenerates CAR.

Several other 102 quenching amines, furans and tetra-substituted olefins have been detected in insects (Larson, 1986), but their role has not been clarified.

Tocopherols, especially 0c-tocopherol (vitamin E; abbreviated as 0c-tocoph-OH), is an excellent quencher of LO • 2 and RO • 2 radicals as shown below (see Cadenas, 1989, for details):

0c-Tocoph-OH + RO • 2-,0c-Tocoph-O • + ROOH. (31)

Quenching of peroxyl radicals converts 0~-tocopherol to the tocopheryl free radical (0~- tocoph-O • ) which is water soluble. Ascorbate reacts with 0c-tocoph-O • to regenerate 0c-tocopherol but, in turn, AH- is destroyed as a free radical:

0c-Tocoph-O • +AH--*0c-Tocoph OH + A • - (32)

0~-Tocopherol also quenches ~O 2 and triplet excited carbonyl compounds, e.g. 3Sen (3[>C=O]*). Whereas the efficiency for triplet excited state compounds is high (/£ =6.7x109 M -1 s-l), it is a poor quencher (50-fold less than for carotenoids) of 102 and other singlet species (Cadenas, 1989; Di Mascio et al., 1989).

In insects, studies on the antioxidant properties of 0~-tocopherol are limited to the effect of exogenously administered 0~-tocopherol in the M. sexta diet which resulted in marked reduction in larval mortality from phototoxic 0c-terthienyl, a notorious 102 generator (Aucoin eta/ . , 1990). Mechanistic information is not available but the protection may be more from quenching of RO • 2 radicals originating from 102 attack on biomembranes than by direct quenching of 102. This aspect requires scrutiny and more studies on other insect species.

The • OH radical may be quenched by protein and cysteine sulfhydryl groups and other functional groups such as amino groups (Singh, 1989), but in this process proteins themselves will be rendered dysfunctional. Suppressed enzyme activities will be highly deleterious because considerable time is required for restoration by de novo synthesis of the protein.

Ascorbate is an outstanding antioxidant for scavenging O • 2- and • OH radicals, H202 and to a lesser extent 102. The reaction with H202 is an enzymatic process but regardless of whether an enzymatic or nonenzymatic reaction, AH- undergoes a univalent oxidation to the A. - radical (Kramer and Sieb, 1982). As discussed above, AH is necessary for the regeneration of 0c-tocopherol from its free radical. Semi- quinone radicals are reduced by AH- to catechols or dihydroquinones with the formation of A . - radical. This is one of several reactions that may minimize 02 • - production (Kalyanaraman eta/., 1985). AH- may also react in insect gut with quinones (both of plant diet origin) forming hydroquinones and, in turn, its resorption as a dietary source is seriously jeopardized. It is not surprising therefore that many phytophagous insects show no dietary requirement for AH-, presumably relying on de novo synthesis of this crucial antioxidant, while some species, e.g.M, sexta and H. zea are able to obtain ascorbate from their diet (Kramer and Sieb, 1982). No information exists on the fate of insects that do not synthesize ascorbate, and when they are under severe oxidative stress from dietary pro-oxidants. One wonders that in the near- complete absence of AH- under these expected conditions, what alternate mechanisms substitute for the antioxidant qualities of AH .

In mammalian species the capacity of purines, especially uric acid, to rapidly quench 102, 0 . 2 and • OH radicals has been recognized (Hochstein eta/., 1984). While mammalian species produce urea far more than uric acid, insects, in particular terrestrial species, generate higher levels of uric acid (80%+) than other forms of nitrogenous waste (Cochrane, 1985). Yet, the antioxidant properties and contributions


of urate in insects have not been studied. Urate has been implicated in stabilizing AH- from oxidation by the chelation of free and protein-bound iron (Hochstein et al., 1985). This additional property should prompt a full investigation of the role of urate in insects, specifically the stability conferred to AH- and its overall contribution as an antioxidant.

Under extremely reactive radiolysis conditions able to generate free radicals ( • OH, RO • 2, etc.) GSH has been suggested to be an extremely good quencher of oxygen- free radicals (Cadenas, 1989). In turn, GSH is converted to the GS. radical which undergoes several reactions, i.e. diradical annihilation, and reaction with thiolate anion (GS), all of which react to form GSSG. However, there is little evidence of this high reactivity in biological systems. GSH does, however, participate in numerous enzymatic reactions, which, as elaborated later, reduce deleterious peroxides to harmless alcohols, and thus break up the lipid peroxidation chain reaction. Another important role of GSH is in the conjugation of quinones in either the 2 or 3 position, which makes them facile for excretion and thus preventing the redox-cycling of quinones which generates O • 2- radicals. This reaction has been reported to occur in H. zea midgut between plant origin quinones and GSH (Felton and Duffy, 1992). Such a conjugation of quinones may occur to some degree in animal tissues, and in view of toxicity of several different quinones fed to our model insect species, this interaction in insect tissues seems minor (Pardini and Ahmad, 1991). Nevertheless, the SQ. radical may be reduced by GSH to form a catechol or dihydroquinone and GS • radical. Self- reaction of GS • radicals produces GSSG.

Antioxidant enzymes Antioxidant enzymes are essential chain breakers of the oxygen radical cascade as

well as the lipid peroxidation chain reaction. Best known from mammalian species, these enzymes are: superoxide dismutase (SOD; EC 1.15.1.1); catalase (CAT; EC 1.11.1.6); glutathione peroxidase (GPOX; EC 1.11.1.9); glutathione transferase (GST; EC 2.5.1.18) and its peroxidase activity (GSTPX); and glutathione (GSSG) reductase (EC 1.6.4.2). Their sequential attack on toxic oxygen products (Ahmad et al., 1990a) is shown in Fig. 3. The stoichiometry of these reactions is as follows:

SOD: 202 • - + 2H +--, H202 + 02 (33)

CAT: H202 + 2H202--,H20 + 02 (34)

GPOX: (i) H202+2GSH--,2H20+GSSG (35)

(ii) LOOH +2GSH--,H20 + LOH + GSSG (36)

GSTPX:LOOH +2GSH~H20 + LOH + GSSG (37)

GR: GSSG + NAD(P)H + H+--,2GSH + NAD+(P). (38)

At pH 7.8, SOD-catalyzed removal of 0 2 . - is 10~°-fold faster than spontaneous dismutation (Fridovich, 1983). SOD-catalyzed dismutation of O2. - generates H202 which is reduced by CAT to H20. GPOX reduces nearly equally H202, and LOOHs. When peroxidized PUFAs are formed they are cleaved off from membranes by phospholipases in order to facilitate their interaction with GPOX or GSTPX (Tan et al., 1984). GSTPX is also efficient in reducing organic peroxides, but not H202. In the reactions catalyzed by GPOX and GSTPX, GSH provides the reducing equivalents and is converted to GSSG. GSSG is reduced to GSH by GR using NAD(P)H as the reductant. NAD(P)H levels are restored in cells by several systems which reduce NAD+(P) to NAD(P)H, e.g. the glucose-6-phosphate and glucose-6-phosphate dehydrogenase system of the hexose-phosphate shunt. In our herbivorous insect model comprised of T. n~ S. eridania and R polyxenes, we have confirmed the presence of SOD, CAT, GSTPX and GR, but not GPOX. These three insect species

280 S. AHMAD

f ,m 02 im ~ i

R (red. H 2 H20

R Cox ) ""~ 2H + 02,~ H + O~

X - CAT ~ •

OH-- , .____/ . . . . .

LH " ~ I I , , ~ I GSSG 2GSH

i r

~ 1 / I . . ~ NAD[P) H NAD(P}

L02 I~ LH ~ , ~ j b 2GSH-9~7-t "GSH, , ,~

] / GSH "~ LOH f

LOOH GST =- (GSOH) D DH2

FIG. 3. THE SEQUENTIAL ATTACK OF ANTIOXIDANT ENZYMES ON OXYGEN RADICALS, HYDROGEN AND LIPID PEROXIDES. R (red.) = electron donor such as a catechol, ubisemiquinone, and metals or metalloproteins; R (ox.) = oxidized form of electron donors; • OH = hydroxyl radical; LH= polyunsaturated fatty acid, or other organic molecules; L- = lipid radical; LO2 = lipid peroxyl radical; LOOH = lipid hydroperoxide; LOH = lipid alcohol; AH 2 = a two-electron donor; A = a fully oxidized form of AH~; DH 2 and D = non-specified NAD(P) redox system; and GST = peroxidase (GSTPX) activity of glutathione transferase.

represent different natural dietary exposures and sensitivities toward our two model pro-oxidants, redox-active quercetin and photodynamic 8-MOP (Pardini et a/., 1989).

Insect sensitivity to pro-oxidants The relative sensitivity is in the order T. n i > S . eridania >> R polyxenes. All toxico-

logical and enzymological work described herein on T. n/was on UC Davis-UNR strain. This strain was highly sensitive to quercetin (LC50=0.0045% W/W) and 8-MOP (LC50 = 0.0004% W/W under UV, 320-380 nm, 4 h) (Ahmad et a/., 1987). In contrast to quercetin, T. ni larvae are refractory to the conjugate flavonoid rutin. Seeds of Rhamnus utilis (Rhamnaceae) contain rutin alongside a specific glycosidase, rhamnodiastase, which hydrolyses rutin to quercetin. Apparently, T. nilarvae lack this enzyme and rutin is excreted without any adverse effect (Ahmad eta/., 1987). However, since quercetin and rutin are equally toxic to larvae of /-/. virescens, H. zea and R gossypiella (Harborne, 1979), potentiation of rutin by hydroylsis must occur, but occurrence of the enzyme rhamnodiastase has not yet been investigated in any insect species.

Fifth instar larvae of S. eridania are not affected by dietary quercetin up to 1.0% (w/w) dietary concentration, nor do they exhibit any casualty in 24 h from dietary 8-MOP in the 0.001-0.7% (w/w) concentration (under exposure to UV, 320-380 nm, 4 h) (Pritsos et a/., 1988a). Under 8-MOP treatment, the larvae exhibit a decrease in relative growth rates in a dose-dependent manner. This onset of toxicity results in delayed mortalities (in the form of failure of larvae to moult to sixth instars, and deaths among pupae that are formed), and by pupal stage an LC50 for 8-MOP is about 0.01% (w/w) (Ahmad, unpublished data). Moreover, like T. n/the quercetin conjugate, rutin, up to 1.0% concentration (w/w), had no effect on S. eridania development and

ANTIOXIDANT DEFENCE OF INSECT HERBIVORES 281

survival. Together, these data suggest that S. eridania larvae are better adapted to flavonoids in their diet as expected from their high polyphagy, but not from photoactive pro-oxidant, 8-MOP, which is presumably rarely encountered. Nonetheless, S. eridania tolerates 8-MOP better than T. ni.

The fifth instar larvae of R polyxenes which are adept in feeding on pro-oxidants tolerate higher than 2.0% (w/w) dietary concentration of quercetin (Pritsos et al., 1988b), and also tolerate well 8-MOP up to 2.0% (w/w) dietary concentration (Ahmad and Pardini, unpublished data).

Unique features of antioxidant enzymes of insects Earlier studies on insects, unrelated to oxidative stresses in insect-plant inter-

actions, had shown SOD and CAT activities in two species of Diptera and 10 species of Coleoptera (Georgi and Deri, 1976; Best-Belpomme and Ropp, 1982; Nickla etal., 1983; Price and Dance, 1983; Allen etal., 1983; 1984; Colepicolo etal., 1986; Sohal and Allen, 1986). Precious little information was available on larval age-related changes in enzyme levels, except for Drosophila melanogaster (Nickla et al., 1983), and no subcellular distribution of insect antioxidant enzymes was carried out. The response of antioxidant enzymes to pro-oxidants such as the pesticide paraquat, had been studied from the standpoint of aging only in M. domesticaadults (Allen etal., 1983; 1984; Sohal and Allen, 1986). SOD was purified from D. melanogaster (Lee et al., 1981) and M. domestica (Bird et al., 1986), and CAT from D. melanogaster (Nahmias and Bewley, 1984). Except for isolation and some inhibition studies, the enzymatic properties were not reported.

Constitutive and ontogenetic profile Enzymes extracted from early-, mid- and late-stage third, fourth and fifth instars of

our three model insect species were assayed for SOD, CAT and GR activities. In general, the enzyme levels were found to be consistent with the low, moderate and high tolerance to pro-oxidants of T. ni, S. eridania, and R polyxenes, respectively (Ahmad etal., 1987; Pritsos etal., 1988a; 1988b). Enzyme activities increased as larvae advanced from third to fifth instars (Table 1), and in each instar the highest activity was associated with the actively feeding larval stage which declined in late instar with the onset of apolysis (data not shown). This pattern of ontogenetic changes in enzyme levels was parallel for both SOD and CAT, suggesting that these two enzymes acted sequentially for the removal of 02 • - and H202, respectively. The GR activity followed an opposite pattern to that observed for SOD and CAT, i.e. the GR activity increased as larvae advanced in age within each instar and, also, as they moulted from third to fourth and fourth to fifth instars. From both a lack of resolution of GPOX activity in these early studies, and also an inexplicable ontogenetic pattern, the role of GR in these insects remained unclear.

A noteworthy feature of these studies is the very high CAT levels of Lepidoptera (200->300 units) in comparison with those observed in many mammalian systems (about 50 units). In contrast to our model lepidopterous species, CAT activity reported from other insects ranged from as low as 1.4 units in male larvae of D. melanogaster (Nickla et al., 1983) to the highest 42 units in a bioluminescent beetle in the genus Pyrophorini (Colepicolo eta/., 1986).

Response to pro-oxidant challenge An increase in SOD activity appeared to be the initial response of the three model

insect species to dietary exposure to both quercetin and 8-MOP (Ahmad and Pardini, 1990b; Pritsos et al., 1988b; 1990). CAT activity is generally high in these insects and other Lepidoptera (Del Vecchio, 1988; Aucoin et al., 1991). It was not induced in our insect model in response to these pro-oxidants. Quercetin suppressed the CAT activity

282 S. AHMAD

TABLE 1. RANGE OF ANTIOXIDANT ENZYME LEVELS IN EARLY, MID-, AND LATE FIFTH INSTAR LARVAE OF MODEL LEPIDOPTEROUS SPECIES*

Constitutive enzyme levels; mean unitst

Insect species SOD CAT GR

T ni 0.9-1.9 193-308 0.8-1.2 S. eridania 1.1-4.3 63-88 (150-200)1: 2.5-4.2 P. polyxenes 1.1-2.8 129-343 1.0-2.8

*Data are from Ahmad et al. (1987) and Pritsos et al. (1988a, 1988b). For brevity, S.D. values are not provided but have been reported in the references above.

tSOD activity was assayed according to Oberly and Spitz (1984), and is expressed as units rain -1 mg protein -1, as originally defined by McCord and Fridovich (1969); CAT activity was assayed according to Aebi (1984) and one unit is defined as the decomposition of 1 i~mol H202 rain ~ mg protein 1, and GR activity was assayed according to Racker (1955) with GSSG and NADPH as co-substrates and expresed as units, where one unit equals change of 0.001 A~0 min ~ mg protein -1.

~S. eridaniaCAT levels reported earlier (Pritsos eta/., 1988a), are one-half to values observed in more recent studies (Ahmad, unpublished data; Weinhold et aL, unpublished data).

of R polyxenes larvae and GR activity of all three insect species. 8-MOP had either no effect or slightly enhanced the GR activity of T. n ior inhibited this activity following 12 h of dietary exposure to this pro-oxidant.

The profile presented above was obtained from assays of enzyme extracts of partially starved (6-h fast prior to placement on control or pro-oxidant incorporated diets) mid-fifth instar larvae. This starvation was essential to prevent feeding aversion due to the repellency from the bitter taste of both quercetin and 8-MOP. However, in these studies antioxidant enzyme levels were also measured in nonstarved larvae to discern whether starvation per se had any effect on the constitutive levels of the enzymes. Moreover, in all three species, enzyme levels were monitored at 1, 4 and 12 h post-ingestion of control and treated diets, and pro-oxidant treatments were at two levels differing by 10-fold (data for lower concentration not shown). Nonetheless, these studies demonstrated that: (i) the levels of all three enzymes in the control groups of partially starved larvae were essentially the same as previously determined enzyme levels for nonstarved larvae of both T. ni and R polyxenes; (ii) the induction of SOD was rapid and levels observed at 1 and 4 h (4-h data not shown in Table 2) were the same, with minimum change by 12 h; (iii) the partial starvation had a dramatic effect on CAT activity of S. eridania larvae, where it increased to a phenomenal 1000 units, which reached close to the constitutive levels after 12 h of feeding (Table 2); thus, CAT of S. eridan/a appeared to be a unique enzyme in that it was induced by an intrinsic factor, i.e. nutritional stress, and not by extrinsic factors such as the exogenous pro- oxidants; and (iv) inhibition of CAT and GR was apparent only with the highest dosage of dietary administration of the pro-oxidants.

Glutathione peroxidase activity The GPOX-like activity toward H202 as substrate was found to be 2.0, 12.8 and 12.3

units in fifth intars of T. ni, S. eridania, and R polyxenes (Ahmad et al., 1989). In mammalian tissues GPOX is a selenoprotein and is generally found in high levels ranging from 100 to 1000 units (Ahmad et al., 1989). Thus, our phytophagous insect species possess trivial levels of this enzyme in comparison to mammalian systems. Moreover, the essentiality of selenium for the insects' GPOX-like activity has not been demonstrated; therefore, it is feasible that this minor GSH-dependent reducing activity towards H202 may be due to another enzyme.

GSTs are a family of enzymes (or isozymes) that are of ubiquitous occurrence in organisms. These multifunctional enzymes are important in the metabolism of


TABLE 2. EFFECTS OF ADLIBITUMADMINISTERED PRO-OXIDANTS ON ANTIOXlDANT ENZYME LEVELS OF MID-FIFTH INSTAR LARVAE OF MODEL LEPIDOPTEROUS SPECIESt

Enzyme levels; mean units:l:

SOD CAT GR Insect species Basal Altered Basal Altered Basal Altered

(1) Pro-oxdiant = quercetin T. ni 2.3-3.5 5.5*-5.0* 283-322 262-238 1.2-1.2 1.3-0.7* S, erldania 1.8-17.5 4.7"-17.7 960-296 193"-195" 21.9-10.9 10.3"-7.6 R polyxenes 1.9-1.5 3.6-2.8* 124-108 54"-41 * 5.2-2.8 3.1-0.0"

(2) Pro-oxidant = 8-MOP T, ni 2.2-2.9 3.8*-4.9* 302-361 306-351 1.7-2.0 2.5-3.0* S. eridania 2.2-10.0 1.9-11.3 165-250 219"-240 11.8-9.1 11.5-3.1" P~ polyxenes ND ND ND ND ND ND

tData are from Ahmad and Pardini (1990b) and Pritsos et aL (1988b, 1990), S.D. values are given in these publications; *significantly different (P<0.05) from respective control values. Assays were according to the procedures listed in Table 1.

SEnzyme levels are ranges at 1 and 12 h following larval feeding of quercetin- and 8-MOP-incorporated diets. Basal levels were recorded with no pro-oxidant [solvent (acetone) only]. The dietary concentration of quercetin was 0.01% for T. ni, 1.0% for S. eridania, and 2.0% for R polyxenes, w/w. The 8-MOP concentration was 0.001% for ;[ niand 0.1% for S. eridania (insects were exposed to 4 h under UV, 320-380 nrn to activate 8-MOP). ND = activity not determined.

xenobiotics which involves conjugation with GSH to facilitate xenobiotic excretion (Jakoby, 1985). This reaction is frequently demonstrated with a model substrate 1-chloro-2, 4-dinitrobenzene (CDNB) (Habig and Jakoby, 1981) as shown below:

CDNB + GSH-~I-GS-2, 4-dinitrobenzene ÷ H + + CI-. (39)

Relevant to this review is the peroxidase (GSTPX) activity of this non-selenium enzyme which is often demonstrated with a model organic hydroperoxide, cumene hydroperoxide (cumOOH) (Prohaska, 1980; Reddy et al., 1981), by mechanisms depicted below in which cumOOH is shown as a typical organic hydroperoxide (ROOH):

ROOH + GSH-~[GSOH] + ROH (40)

[GSOH] + GSH~H20 + GSSG (41)

overall: ROOH + 2GSH-,ROH + H20 ÷ GSSG. (42)

Thus, although the overall reaction is similar to that of the seleno-enzyme GPOX, the reaction catalyzed by GSTPX proceeds in two steps: the first step is enzymatic, but the second one involves a non-enzymatic reaction between a highly reactive intermediate, the sulfenic acid of glutathione [GSOH], and GSH to generate GSSG. Cysteamine and cyanide are used to distinguish the GSH-dependent activity from that of GPOX activity; cysteamine reacts with [GSOH] to form a mixed disulfide which is not as readily attacked as GSSG by the enzyme GR. On the other hand, KCN rapidly reacts with [GSOH] to form GSCN (and H20), and GSCN also is not a substrate for GR (Prohaska, 1980).

Thus, with cumOOH as substrate and inhibitory effects of cyanide and cysteamine, we distinguished and characterized the peroxidase activity of GST from that of GPOX activity in T. ni larvae (Ahmad and Pardini, 1988; 1989). Previously, Cochrane et al. (1987) had reported peroxidase activity associated with the GST of D. melanogaster, but the crucial requirements for characterization of this peroxidase activity, as given by Prohaska (1989), were not performed. An investigation of the ontogenic changes in constitutive activities of the early, mid- and late third, fourth and fifth instar larvae of our model insect species showed that total GST activity (CDNB assay) remained constant in both T. ni(about 0.2 units) and S. eridania (about 0.1 units) larvae, while in R

284 S. AHMAD

polyxenesthe enzyme activity increased from early fourth to fifth instars by about four- fold (2.0-7.5 units) (Ahmad and Pardini, 1990a).

GSTPX activity is particularly high in these insects and is better correlated to their susceptibility to pro-oxidants than the total GST activity (Table 3). This is not surprising since not all multiple forms of GST catalyze the peroxidase reaction, and the peroxidase isozymes also exhibit marked differences in their specific activity (Mannervik, 1985).

When challenged by pro-oxidants, the GSTPX of T. nilarvae responded quickly by increased levels (Ahmad and Pardini, 1990b). Compared to 50 units of control groups, quercetin treatment enhanced GSTPX activity within 1 h to 67 units and by 12 h to 181 units. In contrast to this gradual induction, 8-MOP which generates lipid-peroxidizing 102, the induction was more rapid accounting for 147 units by 1 h and 207 units by 12 h.

Subcellular distribution The SOD activity in our insect model was found primarily confined to cytosol

(CuZnSOD) as in other eukaryotes (Ahmad etal., 1988a; 1988b; 1990b). The CuZnSOD (sensitive to KCN) in eukaryotes is distinct from the mitochondrial MnSOD (insensitive to KCN), yet both SODs catalyze the same reaction (Fridovich, 1983). In mammalian systems such as livers, 80-85% of the total SOD is in the cytosol in the form of CuZnSOD (Geller and Winge, 1984). The distribution of cytosolic vs mitochondrial SOD is 67:33% in S. eridania, 51:49% in Rpolyxenesand 24:76% in T. n/larvae (Table 4). This pattern of subcellular distribution of insects' SOD, especially of T. m; is unlike that found in mammalian species.

In mammalian species, the peroxisomes where H202 is generated by two-electron reduction enzyme systems have been considered the normal site for CAT activity (Chance et al., 1979). There are other reports of CAT activity in mammalian mitochondria (Jones et al., 1981) and cytosol. Nonetheless, in all three insects examined, CAT activity was found to have a broad intracellular distribution, including nuclei, mitochondria, microsomes (with which peroxisomes were associated in our isolations), and cytosol (Table 4). As discussed further, such a very wide intracellular distribution of CAT in insects has both evolutionary and ecological merit.

Subcellular studies provided an additional proof that unlike mammalian species, insects possessed very low levels of a GPOX-like activity toward H202 as substrate (Table 4). On the other hand, the GSTPX activity was very high (Table 4), suggesting that this enzyme and not GPOX plays a prominent role in scavenging deleterious lipid

TABLE 3. TOTAL AND PEROXlDASE ACTIVITY OF GST iN MID-

FIFTH INSTAR LARVAE OF MODEL LEPIDOPTEROUS SPECIES

Mean enzyme units*

Insect species GSTT GSTPX$

T. ni 0.2 50 S. eridania 0.1 106 R polyxenes 3.2 253

*Data are derived from Weinhold et al. (1990), and S.D.

values are not shown here. 1"GST activity was assayed with CDNB and GSH as

co-substrates according to Habig et al. (1974). One unit of GST activity = 1 limol CDNB conjugated rain -1 mg protein -1.

¢GSTPX activity was assayed according to Ahmad and Pardini (1988, 1989) with cumOOH and GSH as co-substrates, and the reaction was coupled to that of GR; thereby the reduction of GSSG formed to GSH by NADPH could be monitored. One unit of GSTPX activity is defined as the oxidation of 1 nmol NADPH to NAD+P min 1 mg protein 1

ANTIOXIDANT DEFENCE OF INSECT HERBIVORES

TABLE 4. SUBCELLULAR DISTRIBUTION AND ACTIVITIES OF ANTIOXIDANT ENZYMES (mean units) OF

MID-FIFTH INSTAR OF MODEL LEPIDOPTEROUS SPECIES*

285

Subcellular compartments

Insect species Nuc leus Mi tochondr ia Microsomes Cytosol

SOD T. ni - - 3.1 -- 1.0

S. eridania - - 2.1 -- 4.3 P. polyxenes -- 4.5 - - 4.3

CAT

T ni 23 283 142 150

S. eridan~ ND 125 119 163

R polyxenes 283 336 106 135

GSTPX

T. ni 74 0 35 13 S. eridania ND ND ND ND R polyxenes 308 64 46 11

GR

7~ ni 3.7 3.7 2.5 0.5

S. eridama ND 5.5 3.3 0 R polyxenes 92 40 25 4.5

*Data are simplified from reports of Ahmad and Pardini (1988, 1989), and Ahmad et al. (1988a, 1988b,

1990b) and S.D. values are not shown. Assays for SOD, CAT and GR were according to the procedures

listed in Table 1. GSTPX activity was measured by the procedure of Ahmad and Pardini (1988, 1989) as

listed in Table 3.

(LOOHs) and other peroxides (ROOHs). The GR activity as well as GSTPX activity displayed broad subcellular distribution (GR was considered confined to cytosol and mitochondria; Chance etal., 1979), and together these data suggested that GSTPX and GR form a team for the reduction of hydroperoxides and GSSG, respectively.

Tissue profile The specific and relative activities of all four antioxidant enzymes in 10 tissues of the

cabbage looper larvae were examined (Ahmad etal., 1991). Generally, the gonads and several tissues that have high metabolic (mitochondrial) activity, e.g. Malpighian tubules, hindgut and muscles, have high specific activities of these antioxidant enzymes (Table 5). The oxidative stress in tissues of high metabolic activity is potentially high because of the greater O2. - production. In the hemolymph, very minor SOD activity (0.1 units) was detected, and upon further fractionation the site for

TABLE 5. SPECIFIC ACTIVITIES OF ANTIOXlDANT ENZYMES OF TISSUES OF FIFTH INSTAR LARVAE OF TRICHOPLUSIA N! RELATIVE TO WHOLE-BODY EXTRACTS*

Mean enzyme units

Antioxidant enzymes WB EP FG MG HG FB SG MT MC HL GD

SOD 2.6 2.8 5.5 4.1 10.8 2.0 1.0 6.4 8.8 0.1 14.9 CAT 303 106 229 275 408 393 0 0 437 0 0

GST 0.27 0.12 0.47 0.21 0.21 0.17 0.16 0.30 0.39 0.04 0.53 GSTPX 47.8 20.2 19.6 41.6 14.6 20.1 42.5 65.9 29.2 0.2 51.3 GPOX 2.0 0.7 1.0 1A 2.8 1,7 0.4 7.1 3.6 <0.1 16.2 GR 2.6 1.8 1.4 1.6 2.8 5.6 1.5 7.1 25.5 0 14.2

*Data are from Ahmad etal. (1991), and for brevity S.D. values are not shown. Assays for SOD, CAT and GR were according to procedures given in Table 1, GPOX assay was according to Ahmad et aL (1989), and for GST and GSTPX the procedures are given in Table 3. Tissue abbreviations are as follows: WB=whole-body; EP=integumental epithelium; FG=foregut; MG = midgut; HG = hindgut; FB = fat body; SG = salivary glands; MT = Malpighian tubules; MC = muscles; HL = hemolymph; GD = gonads.

286 S. AHMAD

this activity was found to be the hemocytes (9.4 units) and the form of enzyme being exclusively CuZnSOD (Ahmad et al., 1991). This unique finding is analogous to the exclusive presence of CuZnSOD in phagocytic leukocytes. All other tissues possessed both CuZnSOD and MnSOD. GST and GSTPX (15% of total GST activity) and GPOX activities were also detected in the hemotymph.

CAT activity was generally high in all tissues showing this activity, and those of GST and GSTPX exhibited a remarkable parallel tissue profile. The GR activity was particularly high in muscles, gonads and Malpighian tubules. Bromosulfopthalein (BSP) a GST inhibitor (Singh et al., 1987), caused up to 90% inhibition of the GSTPX activity, but that of GPOX was negligibly (< 10%) inhibited by ~-mercaptoethanol or mercaptosuccinate, which are good inhibitors of the mammalian selenoprotein GPOX (Chaudiere et al., 1984). Thus, the identity of a GPOX-like enzyme remained obscure despite the evidence of some GSH-dependent capability to reduce H202, and higher specific activity observed in T. ni's Malpighian tubules and gonads have raised the prospect of even higher activities in tissues of S. eridania and R polyxenes which possess five- to six-fold higher GPOX-like activity relative to T. nL

Despite low specific activities, tissues such as the integument and the gut which have higher amounts of protein than other tissues, possess higher overall amounts of these enzymes (Table 6). Thus, a physiological relationship was demonstrated for the antioxidant enzyme levels in T. ni 's tissues commensurate with the tissues high metabolic activity patterns and associated oxidative stress exerted by dietary redox- active pro-oxidants in the gut, and to the potential of photodynamically mediated oxygen toxicity in peripheral organs such as the integument.

Biological effects When the cytosolic CuZnSOD was inhibited in vivo by a copper chelator, diethyl-

dithiocarbamate (DETC; non-toxic to insects and vertebrates), the quercetin toxicity dramatically increased for larvae of R polyxenes and S. eridania (Pritsos et al., 1991). However, DETC had no effect on quercetin toxicity in T. n/larvae which has the lowest SOD activity of all three insect species (especially CuZnSOD, cf. Table 4). These results show the critical role of cytosolic SOD in the pro-oxidant allelochemical defence of insect herbivores.

That CAT also has a crucial role as an antioxidant was evident when 40% in vivo inhibition of CAT by a specific inhibitor, AT (3-mino-1, 2, 4-triazole), resulted in about 50% mortality of mid-fifth instar larvae of T. ni within 24 h. Preliminary results have shown similar effects of CAT inhibition on survival of the other two insect species of our model insects, because in all three species CAT levels are very high (Ahmad and Pardini, unpublished data).

TABLE 6. RELATIVE ACTIVITIES OF ANTIOXIDANT ENZYMES OF TISSUES AND WHOLE-BODY EXTRACTS OF FIFTH INSTAR

LARVAE OF T. NI

Mean enzyme units* Tota I

Antioxidant Tissues tissue enzymes WB EP FG MG HG FB SG MT MC HL GD yield

SOD 100 40.9 16.1 16.0 15.1 4.3 1.5 4,2 4.5 1.0 5.4 109 CAT 100 27.6 12.0 18.0 10.2 11.5 0 0 4.6 0 0 84.7 GST 100 36.4 28.2 16.1 6.1 7.5 4.5 3.9 4.5 9.4 3.9 120.5 GSTPX 100 33.8 6.6 18.7 2.3 5.0 6.9 4.9 1,9 0,2 2.1 82.4 GPOX 100 30,5 7.6 11.6 10.4 10.0 1.6 12.0 5.6 1.6 9.0 98.9 GR 100 11.3 5.4 8.5 5.2 16.3 2.8 6.2 19.6 0 6.7 62.0

*Data are from Ahmad et aL (1991). Based on specific activities and total protein contents of each larval tissue, the enzyme content of each tissue was calculated and expressed as percentage for enzyme content calculated for the whole-body extract. Tissue abbreviations a r e as for Table 5.


More recently, studies were initiated with GSTPX to document its contribution in protection against toxic oxygen-mediated deleterious lipid peroxidation. Results obtained from this ongoing study have clearly demonstrated that the GST inhibitor, BSP, administered orally together with quercetin, profoundly affected growth and metamorphosis of third instars of S. eridania to fifth instars (Meyers et al., unpublished data). Compared to 85-93% larvae in the control, quercetin, and BSP treatments, only 20% were able to advance to fifth instars. Average mass of larvae moulting into fifth instars was only 310 mg larva -~ compared to 367-391 mg larva -~ in other groups. In addition to these fitness-reducing parameters, the BSP 4-quercetin group suffered 23% mortality while in all other groups there was no mortality. These data are congruent to a marked inhibition of GSTPX activity in the BSP4-quercetin group larvae (51%), and lower inhibitions in the BSP alone (32%), and quercetin alone (26%) groups compared with the GSTPX activity of the control group. Thiobarbituric acid (TBA) assay detects aldehydes such as malondialdehyde formed from the breakdown of peroxidized PUFAs and serves as a convenient index for determining the extent of the lipid peroxidation (Esterbauer and Cheeseman, 1990). In the studies described above, TBA reactant aldehydes were found to be 8% higher in the BSP, and 14% higher in the BSP 4- quercetin-fed larval groups than in the control and quercetin (alone)-fed groups. These data clearly demonstrate the biological importance of GSTPX in affording insects protection from 02.--mediated lipid peroxidation. Similar studies are in progress for the enzyme GR, and the antioxidant, GSH.

Biochemical properties of some enzymes Since CAT, GST and GSTPX have unusually high activities in insects, the emphasis

on biochemical characterization has at this time been on these antioxidant enzymes. CAT exhibits dual enzyme activities (Chance eta/., 1979). It has catalase (or catalatic)

activity when the substrates are two molecules of H202, and a peroxidase (or peroxidative) activity when the substrates are one molecule of H202 as an oxidant, and one molecule of a co-substrate as a reductant, usually a hydrogen donor (DH2), such as an alcohol or catechol or an amine. The peroxidative activity of CAT is analogous to that of peroxidase (POD; EC 1.11.1.7), for which the range of DH 2 substrates is wider and includes ascorbate. Both hydroperoxidases are inhibited by KCN and NaN 3, but only the CAT's peroxidase activity and not POD activity is specifically inhibited by AT (Michiels and Ramacle, 1988). We assayed the POD activity of third to fifth instars of T. n/and S. eridania, using H202 and as DH 2 co-substrate, guaiacol. Surprisingly, no POD activity was found in T. n/which has the highest overall CAT level of our model insect species. On the other hand, in S. eridania 8% of the total hydroperoxidase activity was due to a peroxidase activity and based on inhibition by AT, 60% of its peroxidative activity was attributed to POD, and 40% to CAT (Mitchell et al., unpublished data).

Using ethanol-chloroform fractionation, anion exchange chromatography and hydrophobic chromatography, in conjunction with hydroxylapatite chromatography, CAT was recently purified 276-fold to apparent homogeneity from fifth instars of T. ni (Mitchell et al., 1991). The activities shown are for H202; no activity was evident with the organic hydroperoxides, tert-butylhydroperoxide (t-BOOH) or cumOOH, or the DH 2 co-substrate, guaiacol. However, preincubation of the enzyme with ethanol, a DH 2 substrate which competes with AT for binding sites, lowered the inhibition by AT suggesting that T. ni's CAT has some peroxidase activity. The specific activity of the purified enzyme from T. ni(2.2 × 105 units) is higher than that reported for the purified enzyme of D. melanogaster (6.8 × 104 units; Nahmias and Bewley, 1984). The purified enzyme's native M r was found to be in the 247,000-259,000 range and was tetrameric with an apparent M r of 63,000 for each subunit. Moreover, as judged by the Km and Vm, x parameters, T. ni 's CAT has high catalytic power represented by the turnover number,/(ca t, and the enzyme appears ideal for scavenging large amounts of H202 that

288 S. AHMAD

may be generated intracellularly. This idea is supported by apparent high Kc~ t and a well-established feature of CAT, i.e. the enzyme does not obey saturation kinetics implying that the catalytic rate does not drop from substrate saturation (does not follow Michelis-Menton kinetics).

We have also purified CAT from fifth instars of S. eridania using the same protocols as described for CAT of T. nZ The purification for catalatic activity was 233-fold, and the activity of the purified enzyme was 7.1 x 104 units (Weinhold et al., unpublished data). The S. eridania enzyme exhibited peroxidase activity with o-dianisidine as a DH 2 co-substrate, but not with a guaiacol (a better substrate for POD, but not for peroxidase activity of CAT). This peroxidase activity enriched as much as the catalatic activity, i.e. 228-fold representing 45.5 units of the purified enzyme. High Kca t is indicated for this enzyme as for the T. n/enzyme. The enzyme is a tetrameric protein and the presence of four protoheme IX groups, one per monomer, has been confirmed. However, unlike any other CAT purified, this enzyme is a unique polymer of four heterogeneous subunits (approximately in the 42,000-65,000 M r range), and the native enzyme's M r is 270,000. This enzyme is minimally inhibited by AT and reasons for this are not known at this time. However, the enzyme consists of heterogeneous subunits which may affect enzyme affinity or accessibility of AT to the binding site. These ideas require clarification.

Total GST vs GSTPX activity has also been characterized (Weinhold eta/., 1990). A significant finding is that despite high /(ms (low affinity), the Vma x for both CDNB (substrate for total GST) and cumOOH (substrate for GSTPX) were very high, indicating high catalytic power, Kca t.

Our initial studies of SOD which are not complete, suggest similar properties as SODs of other eukaryotes (Ahmad and Pardini, 1991). Insect GR has not yet been characterized but should be purified and properties clarified. After all, its crucial role in reducing GSSG to GSH is evident.

Additional antioxidant enzymes The one-electron transfer flavoprotein enzyme, NADPH-ferrihemoprotein reductase

(EC 1.6.2.4; also called NADPH-cytochrome P-450 reductase) which is a component of the PSMO system, reduces quinones to semiquinone radicals with concomitant production of 02 • radicals. On the other hand, an enzyme commonly known as DT-diaphorase (EC 1.6.99.2, NAD(P)H oxidoreductase [quinone acceptor]), catalyzes a two-electron reduction of quinones or their epoxides (formed by cytochrome P-450 PSMO system) to stable hydroquinones, at a rate competitive with the one-electron reduction reaction by the NADPH-ferrihemoprotein. Therefore, DT-diaphorase is an important "cellular control device against semiquinone and 02" generation" (Brunmark et aL, 1987). This prompted an investigation of effects of several quinoid compounds on the five major antioxidant enzymes as discussed (Ahmad and Pardini, 1991), and DT-diaphorase.

The effects of administration of two naphthoquinones, a benzoquinone and a dipyridinium compound were investigated with T. ni and S. eridania larvae. The compounds were the fungicide, dichlone (2, 3-dichloro-l,4-naphthoquinone; CNQ), menadione (1, 4-naphthoquinone; MEN), tetrachloro-o-benzoquinone (TCBQ), and the pesticide, paraquat (1, 1-dimethyl-4, 4-dipyridinium dichloride; PQT). Trichoplusia ni larvae fed minimally on diets fortified with these compounds because of repellancy from bitter taste. Spodoptera eridania larvae accepted the diets and exhibited a marked effect on development as reflected by suppressed relative growth rates (Pardini and Ahmad, 1991). The effect on antioxidant enzymes was therefore minimal on T. ni larvae but substantial and significant alterations occurred in S. eridania enzymes. SOD activity was induced, CAT activity was not significantly modified, but the GSTPX and GR activities declined. Most importantly, however, the DT-diaphorase


activity was induced, and MEN and TCBQ were stronger inducers than the other two compounds (Pardini and Ahmad, 1991). These results are similar to that reported for mammalian systems of CNQ which induces oxidative stress (Pritsos et aL, 1982; 1986; Pritsos and Pardini, 1984), and free radical-mediated toxicity of PQT to M. domestica (Allen et al., 1984b). More studies are needed to clarify the role of DT-diaphorase in insects.

The inhibition of GPOX and GR in mammalian systems by phenolics (Pritsos and Pardini, 1984; Elliot and Pardini, 1988), and of GST/GSTPX and GR in insects (Ahmad and Pardini, 1990a; Ahmad and Pardini, 1991; Pardini and Ahmad, 1991) deserves comment. As previously suggested, the inhibition may be via hydrogen bonding of catechols or dihydroquinones, or covalent binding with the quinones or semiquinone radicals of these redox-active compounds (van Sumere etal., 1975; Ahmad and Pardini 1990a). This aspect requires clarification.

Peroxidase has been reported from D. melanogaster and several other insects, and in addition to the phenol oxidase, this enzyme has been implicated in the cuticular tanning process (Armstrong et al., 1978; Nickla et al., 1983; Hasson and Sugumaran, 1987). However, no activity of this enzyme was detectable in T. ni(Ahmad et al., 1991). nor in Manduca sexta larvae (S. Ahmad, unpublished data). In the soybean root nodules, Dalton et al. (1986) reported that peroxidase works together with dehydroascorbate reductase (EC 1.8.5.1) and GR for the removal of H202 as show below.

Peroxidase: H202+AH2--~2H20 + A (43)

DHA reductase: A + 2GSH-,AH 2 + GSSG (44)

GR: GSSG + NADPH + H+~2GSH + NAD÷P. (45)

In equations (43) and (44), A represents dehydroascorbate DHA; cf. scheme (22). Recently, Felton and Duffy (1992) have reported the occurrence of this enzyme

complex in H. zea larvae. This finding may explain the observed minor GSH- dependent reducing activity towards H202 which in mammalian systems is due to the seleno-protein, GPOX. Unless this activity is found widespread in insects, the possi- bility exists that the minor GPOX-like activity observed in our insect model is due to the GSTPX (Mannervik, 1985).

The existence of an ascorbate free radical reductase (AFR) in H. zea has also been claimed. There are previous reports of this enzyme in rat colon and renal tissues (Rose et aL, 1988; Rose, 1989; Choi and Rose, 1989). This enzyme is well known to occur in plants from where it has been isolated and characterized (Borraccino et al., 1986). The demonstration of AFR requires great care in that artifactual reaction is often responsible for the reduction of A • to ascorbate. That so far the enzyme has not been purified from any mammalian species requires more work before existence of AFR in animal species is accepted.

Additional contributions Another group has reported a profile of the enzymes SOD, CAT, GSTPX and GR

from T. nilarvae, which is similar to that reported by us, and in agreement with our earlier study; they also found no induction of the high constitutive levels of CAT from exposure to 8-MOP (Lee and Berenbaum, 1989). They reported, however, a slight (1.2-fold) but significant increase in response to dietary harmine, a photodynamic pro-oxidant I]-carboline alkaloid.

Aucoin et al. (1991) examined the SOD, CAT, GSTPX and GR activities in a lepidopterous insect model consisting of Ostrinia nubilis, M. sexta, and Anaitis plagiata. The latter species is a specialist feeder of the photodynamic hypericin-containing plant, Hypericum perforaturn. In A. plagiata the levels of antioxidant enzymes were

290 S. AHMAD

highest and although SOD activity was not induced, CAT and GR levels increased when larvae are switched from a non-hypericin to a hypericin-containing diet.

Until now, there was no report on inhibition of the SOD activity from redox-active or photodynamic pro-oxidants. Nivsarkar et al. (1991) have provided the first evidence that 0~-terthienyl abolishes SOD activity in the respiratory anal gills of larvae of the mosquito, Aedes aegyptl~ As discussed earlier, ~-terthienyl is a predominant 102 generator, with only a 1% yield of 02. radicals (Kagan et al., 1989). Whether the inhibition is via 102, . OH radical derived from the 02 • - cascade, or from singlet or triplet excited photosensitizer remains obscure.

Spin trapping is a reliable method for detection of the dioxygen free radicals as adducts with nitrones which are detected by the electron paramagnetic resonance (EPR) spectrum. Light-activated ~-terthienyl is a notorious 102 generator, but in non- biological solutions it has been reported to generate about 1% 02' of the total activated dioxygen (Kagan et al., 1989). Using O2. - adduct with PBN (phenyl N-tert- butylnitrone), Nivsarkar et al. (in press) have demonstrated that 02 • - is also generated in vivo in the anal gills of A. aegypti larvae when treated with light-activated ~-terthienyl. This kind of demonstration is pioneering for an insect species, and the use of EPR will no doubt increase for monitoring oxygen radical production in vivo.

The parsnip webworm, Depressaria pastinacella, a specialist feeder of plants of Apiaceae rich in furanocoumarins (e.g. 8-MOP), was found to possess 30 times higher microsomal PSMO metabolizing capacity than that reported for R polyxenes (Nitao, 1989). Not only was the metabolism of 8-MOP PSMO dependent, but the substrate also induced the enzyme. These insects also possess high levels of CAT and SOD activities. Thus, the insect's main defence against pro-oxidant insult is an efficient direct detoxification. The role of antioxidant enzymes present in this unpigmented leaf roller, has recently been discussed (Lee and Berenbaum, 1990).

Ecological and evolutionary considerations The constitutive activities of the antioxidant enzymes of the phytophagous insect

species examined reflect adapted (genetically determined) capacities for detoxification of the pro-oxidants. This adaptation is congruent to the degree these herbivores are likely to encounter various kinds and levels of pro-oxidants in their natural range of host plants. An even more important ecologically relevant feature is the feeding strategies of herbivores, which requires that they be able to adapt quickly (and temporarily) to pro-oxidant exposure of varying amounts and structural diversity. In other words, the insect herbivores need antioxidant enzymes that will induce rapidly in the presence of toxic pro-oxidants with enough speed to provide protection from acute poisoning.

The above requirements are fulfilled in part by fast induction of SOD activity which rapidly destroys 02 • - radicals, as a main response to dietary pro-oxidant exposures of these phytophages. The greater amounts of H202 generated by induced SOD activity can be efficiently destroyed by very high levels of CAT activity in insects (4-6-fold higher than in mammalian tissues). In many instances, CAT activity need not be induced due both to its high constitutive activity and non-saturation kinetics. Nonetheless, when oxidative stress may surpass even this unusual capacity, CAT levels will be expected to increase. This has been amply demonstrated in studies employing pro-oxidants other than quercetin and 8-MOP (Lee and Berenbaum, 1989; Aucoin et al., 1991).

The antioxidant enzymes crucial for the removal of toxic hydroperoxides are GSTPX and GR. Although these enzymes are inhibited at high concentrations of the redox- active phenolic pro-oxidants, their induction in response to the lipid-peroxidizing photoactive pro-oxidants has been demonstrated (Ahmad and Pardini, 1990b; Aucoin et al., 1991).


The antioxidant compounds are also crucial for quenching 102, free radicals of dioxygen, peroxides and their radicals. Our studies on carotenoid contents, and the finding that the amounts of lutein, a dihydroxy 13-carotene, are least in T. m; moderate in the polyphagous S. eridania, and highest in the highly pro-oxidant-adapted R polyxenes larvae, are in agreement with the insects' dietary exposure to pro-oxidants. The role of other antioxidants such as 0{-tocopherol, urate, and ascorbic acid have not been critically examined using model species that differ in their pro-oxidant susceptibility. They are obviously important as shown for the protective effect of 0{- tocopherol from phototoxic 0{-terthienyl in M. sexta larvae (Aucoin et al., 1990). More work is needed before their evolutionary, hence, their ecological significance in herbivorous insects is clarified.

The antioxidant enzymes are of ancient evolutionary origin having been evolved in primitive anaerobes to detoxify deleterious by-products of 302 metabolism. SOD and CAT occur in all obligatory aerobic life forms, including prokaryotes and eukaryotes. However, insects, including phytophagous species, lack a true selenium-dependent GPOX activity. GPOX activity is also absent from plants (Ahmad et al., 1989). In vertebrate species the GPOX is considered a crucial enzyme because it reduces H202 and organic peroxides at comparable rates. Since vertebrate CAT was assumed to be confined to peroxisomes, the need for an enzyme which could efficiently reduce both H202 and ROOHs formed in the cytosol and subcellular membranes, was considered crucial. In plants, the lack of GPOX activity has led to the evolution of a peroxidase-ascorbate reductase system that utilizes GSH and ascorbate to reduce H202. Such a system has thus far been found in only one insect species, H. zea (Felton and Duffy, 1992). Whether this capacity is widespread or is of sporadic occurrence in insects awaits additional research work.

The lack of true GPOX activity may have led to two evolutionary modifications. One modification is enhancement of CAT activity while broadening its subcellular distribution, and another one is the elaboration of GSTPX activity. In this manner, insect survival is better assured by adequate counter-measures against excessive accumulations of H202 and ROOHs under oxidative stress. The elaboration of GSTPX activity accompanied by a wide intracellular distribution required yet another evolutionary modification, i.e. broader subcellular distribution of the enzyme, GR. Our studies confirm this scenario. In addition, GSTPX and GR appear to be a team for the reduction of ROOHs at the expense (oxidation) of GSH by GSTPX activity, which is then recycled back to GSH by GR.

The evolution seems to have favored the elaboration of more than one defence mechanism against pro-oxidant toxicity in insect species that are specialized feeders of pro-oxidant-rich plants. Thus, R polyxenes larvae feed without any harmful effect on 8-MOP and flavonoid-rich plants. These larvae efficiently detoxify 8-MOP by a gut cytochrome P-450 PSMO (possibly via a specific isozyme). This capacity is even more elaborate in D. pastinacella larvae which have no opportunity to feed on other plants of Apiaceae which may not contain 8-MOP (Nitao, 1989). In contrast to D. pastinacella, R polyxenes larvae feed on many plants of Apiaceae such as D. carota which does not contain 8-MOR In both insect species, the presence of antioxidant enzymes has been demonstrated (Pritsos et al., 1988b; Ahmad et al., 1990b; Lee and Berenbaum, 1990). The antioxidant enzyme levels are highest in R polyxenes larvae compared to less well-adapted T. ni and S. eridania larvae. Thus, the defence strategy "is quite remarkable and consists of direct detoxification of toxins such as 8-MOP, and a highly elaborate complement of antioxidant enzymes" (Ahmad et al., 1990b).

The metabolism of dietary redox phenolics such as the flavonoid quercetin have not been studied in any detail. Formerly, quinone reductase (Yu, 1987) was considered a distinct enzyme from DT-diaphorase (Pardini and Ahmad, 1991), but both are now classified as DT-diaphorase. DT-diaphorase has the potential of reducing the

292 S, AHMAD

corresponding quinones formed during the redox-cycling of catechol type phenolics. These products are stable and are prevented from semiquinone formation which generates O 2 • - radicals. In this regard, the role of DT-diaphorase in both mammalian and insect systems seems critical. This enzyme has been elaborated in insects as in the mammalian systems to prevent oxidant stress from phenolics.

Conclusion All aerobic organisms are subject to endogenous oxygen toxicity from generation of reactive cytotoxic products such as H202, 0 2 . - and • OH radicals. This endogenous oxidative stress is exacerbated by exposure of organisms to pro-oxidant compounds which also generate these cytotoxic by-products of oxygen metabolism, as well as 102. Insect herbivores cope with this stress either by direct detoxification of the pro- oxidants, or by antioxidant compounds and antioxidant enzymes which prevent free radical cascade of 302 , and quench 102 , oxygen free radicals, and reduce deleterious lipid peroxidation products to innocuous alcohols. A new aspect of interaction between pro-oxidant allelochemicals and insect herbivores has thus emerged, and it is clear that of all antioxidant defence mechanisms, enzymatic defence is crucial to the survival of these herbivores. Even in the most highly pro-oxidant-adapted species the survival is not solely guaranteed by their remarkable ability to directly detoxify the pro- oxidants, but also by a second line of defence represented by their highly elaborate antioxidant system.

Acknowledgements--Much of the research reported in this paper was supported by two USDA competitive research grants 86-CRCR-1-2038 and 88-37153-3457, and a NSF grant BSR-9112892. The author gratefully acknowledges several investigators who co-authored many publications and, in particular, Dr R. S. Pardini. Thanks are also due to Dr Charles R. Heisler and Dr R. S. Pardini for their thorough review of the manuscript. Lastly, I thank Mrs Jeama K. Bowers for skilfully and rapidly typing the entire manuscript.

References Aebi, H. (1984) Catalase In vitro. Meth. Enzym. 105, 121-126. Ahmad S. (1982) Host location by the Japanese beetle: evidence for a key role for olfaction in a highly

polyphagous insect. J. Exp. ZooL 220, 117-120. Ahmad S. (1983a) Mixed-function oxidase activity in a generalist herbivore in relation to its biology, food

plants, and feeding history. Ecology 64, 235-243. Ahmad, S. (ed.) (1983b) Herbivorous Insects. Host-Seeking Behavior and Mechanisms, Academic Press, New

York. Ahmad S. (1986) Enzymatic adaptations of herbivorous insects and mites to phytochemicals. J. Chem. Ecol. 12,

533-56O. Ahmad S., Beilstein, M. A. and Pardini, R. S. (1989) Glutathione peroxidase activity in insects: a reassessment.

Arch. Insect. Biochem. Physiol. 12, 31-49. Ahmad S., Brattsten, L. B., Mullin, C. A. and Yu, S. J. (1986) Enzymes involved in the metabolism of plant allelo-

chemicals. In Molecular Aspects of lnsect-Plant Associations (Brattsten, L. B. and Ahmad, S., eds), pp. 73- 151. Plenum Press, New York.

Ahmad, $., Duval, D. L, Weinhold, L. C. and Pardini, R. S. (1991) Cabbage looper antioxidant enzymes: tissues specificity. Insect Biochem. 21, 563-572.

Ahmad, S., Pardini, R. S., Pritsos, C. A., Sowen, S. M., Heisler, C. R. and Blomquist, G. J. (1988b) Subcellular distribution and activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase in the southern armyworm, Spodoptera eridania. Arch. Insect. B/ochem, Physio/. 7, 173-186.

Ahmad, S. and Pardini, R. S. (1988) Evidence for the presence of glutathione peroxidase activity towards an organic hydroperoxide in larvae of the cabbage looper moth. Trichoplusia nt~ Insect Biochem. 18, 861-866.

Ahmad, S. and Pardini, R. S. (1989) Corrigendum. InsectBiochem. 19, 109. Ahmad, $. and Pardini, R. S. (1990a) Mechanisms for regulating oxygen toxicity in phytophagous insects. Free

Rad. Biol. Med. 8, 401-413. Ahmad, S. and Pardini, R. S. (1990b) Antioxidant defense of the cabbage looper, Trichoplusia ni: enzymatic

responses to the superoxide-generating flavonoid, quercetin, and photodynamic furanocoumarin, xanthotoxin. Photochem. Photobiol. 51, 305-311.

Ahmad, S. and Pardini, R. S. (1991) An insect model for research on detoxication of prooxidants. Th6 Pharmacologist; Proc. ASPET Meeting 33, 344.


Ahmad, S., Pritsos, C. A., Bowen, S. M., Heisler, C. R., Blomquist, G. J. and Pardini, R. S. (1988a) Antioxidant enzymes of larvae of the cabbage looper moth, Trichoplusia ni: subcellular distribution and activities of superoxide dismutese, catalase and glutathione reductase. Free Rad. Res. Commun. 4, 403-408.

Ahmad, S., Pritsos, C. A., Bowen, S. M., Kirkland, K. E., Blomquist, G. J. and Pardini, R. S. (1987) Activities of enzymes that detoxify superoxide anion and related oxyradicals in Trichoplusia ni. Arch. Insect Biochem. Physiol. 7, 85-98.

Ahmad, S., Pritsos, C. A. and Pardini, R. S. (1990a) insect responses to prooxidant plant allelochemicals. Symp. Biol. Hung. 39, 63-69.

Ahmad, S., Pritsos, C. A. and Pardini, R. S. (1990b) Antioxidant enzyme activities in subcellular fractions of larvae of the black swallowtail butterfly, Papilio polyxenes. Arch. Insect Biochem. Physiol. 15, 101-109.

Allen, R. G., Farmer, K. J., Newton, R. K. and Sohal, R, S. (1984b) Effects of paraquat administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutsse, catalase, glutathione reductase, inorganic peroxides and glutathione in the adult housefly. Comp. Biochem. Physiol. 78C, 283-288.

Allen, R. G., Farmer, K. J. and Sohal, R. S. (1983) Effect of catalase inactivation on levels of inorganic peroxides, superoxide dismutase, oxygen consumption and life span in adult houseflies (Musca domestica). Biochem. J. 216, 503-506.

Allen, R. G., Farmer, K. J. and Sohal, R. S. (1984a) Effect of diamide administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutase, catalase inorganic peroxides and glutathione in adult housefly (Musca domestica). Comp. Biochem. Physiol. 78C, 31-33.

Appel, H. M. and Martin, M. M. (1990) Gut redox conditions in herbivorous lepidopterous larvae. J. Chem. Ecol. 16, 3277-3290.

Armstrong, D., Rinehart, R., Dixon, L. and Reigh, D. (1975) Changes of peroxidase with age in Drosophila. Age 1, 8-12.

Aucoin, R, R., Fields, P., Lewis, M. A., Philogene, B. J. R. and Arnason, J. T. (1990) The phototoxic effect of antioxidants to a phototoxin-sensitive insect herbivore. Manduca sexta. J. Chem. Ecol. 16, 2913-2924.

Aucoin, R. R., Philogene, B. J. R. and Arnason, J. T. (1991) Antioxidant enzymes as biochemical defenses against phototoxin-induced oxidative stress in three species of herbivorous Lepidoptera. Arch. Insec~ Biochem. Physiol. 16, 139-152.

Bell, W. J. and Card6, R. T. (eds) (1984) ChemicalEcology of Insects. Sinauer Associates, Sandedand, MA. Bellus, D. (1978) Quenchers of singlet oxygen--a critical review. In Singlet Oxygen Reactions With Organic

Compounds & Polymers (Ranby, B. and Rabek, J. F., eds), pp. 61-110. John Wiley, Chichester. Berenbaum, M. (1978) Toxicity of a furanocoumarin to armyworms: a case of biosynthetic escape from insect

herbivores. Science 201, 532-534. Berenbaum, M. (1981) Effects of linear furanocoumarins on an adapted specialist insect (Papilio polyxenes.)

Ecol. Ent. 6, 345-351. Berenbaum, M. (1987) Charge of the light brigade. ACS. Symp. Ser, 339 (Hietz, J. R. and Downum, K. R., eds),

pp. 206-216. American Chemical Society, Washington, D.C. Best-Belpomme, M. and Ropp, M. (1982) Catalase is induced by ecodysterone and ethanol in Drosophila cells.

Eur. J. Biochem. 121, 349-355. Bird, T. G., Salin, M. L., Boyle, J. A. and Heitz, J. R. (1986) Superoxide dismutase in the housefly, Musca

domestica (L). Arch. Insect Biochem, Physiol. 3, 31-43. Borraccino, G., Dipierro, S. and Arrigoni, O. (1986) Purification and properties of ascorbate free radical

reductase from potato tubers. Planta 167, 521-526. Borg, D. C. and Schaich, K. M. (1988) Iron and hydroxyl radicals in lipid peroxidation: Fenton reactions in lipid

and nucleic acids co-oxidized with lipids. In Oxy-Radicals in Molecuar Biology and Pathology (Cerruti, P. A., Fridovich, I. and McCord, J. M., eds), pp. 427-441. Alan R. Liss, New York.

Brunmark, A., Cadenas, E., Lind, C., Segura-Aguilar, J. and Emster, L. (1987) DT-diaphorase catalyzed two- electron reduction of quinone epoxides. Free. Rado Biol. Med. 3, 181-188.

Bull, D. L,, Ivie, G. W., Beier, R. C., Pryor, N. W. and Oertli, E. H. (1984) Fate of photosensitizin9 furanocoumarins in tolerant and sensitive insects. J. Chem. Ecol. 10, 893-911.

Burch, P. E. and Martin, J. P. Jr (1988) Protection against dye mediated photodynamic effects is conferred by DNA repair enzymes and oxygen radical scavengers. FASEB J. 2, A766.

Cadenas, E. (1989) Biochemistry of oxygen toxicity. A. Rev. Biochem. 58, 79-110. Cates, R. G. (1980) Feeding patterns of monophagous, oligophagous, and polyphagous herbivores: the effect

of resource abundance and plant chemistry. Oecologia 46, 22-31. Chae, K. H. and Ham, H. S. (1986) Production of single oxygen and superoxide anion radicals by beta-

carbolines. Bull. Korean Chem. Soc. 7, 478-479. Chance, B., Sies, H. and Boveris, A. (1979) Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59,

527-605. Chaudiere, J., Wilhelmsen, E. C. and Tappel, A. L. (1984) Mechanism of selenium-glutathione peroxidase and its

inhibition by mercaptocarboxylic acids and other mercaptsns. J. Biol. Chem. 259, 1043--1050. Cochrane, D. G. (1985) Nitrogenous excretion. In Comprehensive Insect Biochemistry, Physiology and

Pharmacology (Kerkut, G. A. and Gilbert, L. I., eds), Vol 10, pp. 467-506. Pergamon Press, Oxford. Cochrane, B. J., Morrissey, J. J. and LeBlanc, G. A. (1987) The genetics of xenobiotic metabolism in Drosophila--

IV. Purification and characterization of the major glutathione-S-transferase. Insect Biochem. 17, 731-738.

294 S. AHMAD

Choi, J. and Rose, R. C. (1989) Regeneration of ascorbic acid by rat colon. Proc. Soc. Exp. Biol. Med. 190, 369- 373.

Colepicolo, N. P., Bechara, E. J. H. and Costa, C. (1986) Oxygen toxicity in luminescent and nonluminescent elaterid larvae. Insect Biochem. 16, 381-385.

Dalton, D. A,, Russell, S. A., Hanus, F. J., Pascoe, G. A. and Evans, H. J. (1986) Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules. Proc. Natn, Acad, ScL U.S.A. 83, 3811-3815.

Del Vecchio, R. J. (1988). Some physiological effects of gamma radiation on larvae of the navel orangeworm (Amyelosis transitella) Ph.D. dissertation, University of California, Davis, CA.

Di Mascio, P., Kaiser, S. and Sies, H. (1989) Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274, 532-538.

Downer, R. G. H. (1985) Lipid metabolism. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology (Kerkut, G. A. and Gilbert, L. I., eds), VoL 10, pp. 77-113. Pergamon Press, Oxford.

Downum, K, R. (1986) Photoactivated biocides from higher plants. In Natural Resistance to Pests. Roles of Allelochemicals. ACS Symp. Set. 296 (Green, M. B. and Hedin, P. A., eds), pp. 197-205. American Chemical Society, Washington, D.C.

Downum, K. R. and Rodriguez, E. (1986) Toxicological action and ecological importance of plant sensitizers. J. Chem. Ecol. 12, 823-834.

Ehrlieh, P. R. and Murphy, D. D. (1988) Plant chemistry and host range in insect herbivores. Ecology69, 908-909. Ehrlich, P. R. and Raven, P. H. (1964) Butterflies and plants: a study in coevolution. Evolution 18, 586-608. Elliott, A. J. and Pardini, R. S. (1988) Flavonoid inhibition of glutathione reductase: a structure activity study. J.

Ceil BioL 107, 417a. Esterbauer, H. and Cheeseman, K. H. (1990) Determination of aldehyde lipid peroxidation products:

malonaldehyde and 4-hydroxynonenal. Meth. Enzym. 186, 407-421. Feeny, P. (1980) Biochemical coevolution between plants and their insect herbivores. In Coevolution of Animals

andP/ants (Gilbert, L. E. and Raven, P. H., eds), revised edition, pp. 3-19. University of Texas, Austin, TX. Felton, G. W., Donato, K., Del Vicchio, R. J. and Duffy, S. S. (1989) Activation of plant foliar oxidases by insect

feeding reduces nutritive quality of foliage for noctuid herbivores. J. Chem. Ecol. 15, 2667-2694. Felton, G. W. and Duffy, S. S. (1992) Ascorbate oxidation reduction in Helicoverpa zea as a scavenging system

against dietary oxidants. Arch. Insect. Biochem. PhysioL 19, 27-37. Fleming, W. E. (1972) Biology of the Japanese beetle. Tech. Bull 1449, U.S. Dept. Agric., Washington D.C. Foote, C. S. (1979) Photosensitized oxidation and singlet oxygen: consequences in biological systems. In Free

Radicals in Biology(Pryor, W. A., ed.), Vol. II, pp. 85-133. Academic Press, New York. Fridovich, I. (1983) An endogenous toxicant. A. Rev. Pharmac. Toxicol. 23, 239-257. Geller, B. L. and Winge, D. R. (1984) Subcellular distribution of superoxide dismutases in rat liver. Meth. Enzym.

105, 105-114. Georgi, F. and Deri, P. (1976) Cytochemistry of late ovarian chambers of Drosophila melanogaster. Histo-

chemistry 48, 325-334. Girotti, A. W. (1983) Mechanisms of photosensitization. Photochem. Photobiol. 38, 745. Grossweiner, L. I., Patel, A. S. and Grossweiner, J. B. (1982) Type I and type II mechanisms in the photo-

sensitized lysis of phosphatidylcholine liposomes by hematoporphyrin. Photochem. Photobiol. 36, 159-167. Habig, W. H. and Jakoby, W. B. (1981) Glutathione-S-transferase in rat and human. Meth. Enzym. 77, 218-231. Habig, W. H., Pabst, M. H. and Jakoby, W, B. (1974) Glutathione-S-transferase. The first enzyme step in

mereapturic acid formation. J. BioL Chem. 249, 7130-7139. Halliwell, B. and Gutteridge, J. M. C. (1985) Free Radicals in Biology and Medicine. Clarendon Press, Oxford. Halliwell, B., Richmond, R., Wong, S. F. and Gutteridge, J. M. C. (1980) The biological significance of the Haber-

Weiss reaction. In Biological and Clinical Aspects of Superoxide and Superoxide Dismutase (Bannister, W. H. and Bannister, J. V., eds), pp. 32-41. Elsevier, New York.

Harborne, J. B. (1979) Flavonoid pigments. In Herbivores. Their Interaction with Secondary Plant Metabolites (Rosenthal, G. A. and Janzen, D. H., eds), pp. 619-655. Academic Press, New York.

Harborne, J. B. (1982) Introduction to Eco/ogicalBiochemistq4, 2nd edn. Academic Press, London. Hassan, H. M. and Fridovich, I. (1979) Intracellular production of superoxide radical and hydrogen peroxide by

redox active compounds. Arch& Biochem. Biophys. 196, 385-395. Hasson, C. and Sugumaran, M. (1987) Protein cross-linking by peroxidase: possible mechanism for sclerotiza-

tion of insect cuticle. Arch. Insect. Biochem. Physiol. 5, 13-28. Hermann, K. (1976) Flavonols and flavones in food plants: a review. J. Fd Technol. 11, 433-438. Hochstein, P., Hatch, L. and Sevanian, A. (1984) Uric acid: functions and determinations. Meth. Enzym. 105,

162-166. Hodnick, W. F., Kalayaranaman, B., Pritsos, C. A. and Pardini, R. S. (1989) The production of hydroxyl and semi-

quinone free radicals during the autoxidation of redox active fiavonoids. In Oxygen Radicals in Biology and Medicine (Simic, M. G., Taylor, K. A., Ward, J. F. and von Sonntag, C., eds), pp. 149-152. Plenum Press, New York.

Hodnick, W. F., Kung, F. S., Roettger, W. J., Bohmont, C. W. and Pardini, R. S. (1986) Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids: a structure activity study. Biochem. Pharmac. 35, 2345-2357.


Hodnick, W. F., Milosavljevie, E. B., Nelson, J. H. and Pardini, R. S. (1988) Electrochemistry of flavonoids-- relationships between redox potentials, inhibition of mitochondrial respiration and production of oxygen radicals by flavonoids. Biochem. Pharmac. 37, 2607.

Hoy, C. W. and Sheldon, A. M. (1987) Feeding response of Artogeia rapae (Lepidoptera:Pieridae) and Tricho- plusia ni(Lepidoptera:Noctuideae) to cabbage leafage. Environ. Ent. 16, 680-682.

Ivie, G. W., Bull, D. L., Beier, R. C., Pryor, N. W. and Oertil, E. H. (1983) Metabolic detoxification: mechanism of insect resistance to plant psoralens. Science 221, 374-376.

Jakoby, W. B. (1985) Glutathione transferases: an overview. Meth. Enzym. 113, 495-499. Jones, D. P., Eklow, L., Thor, H. and Orrenius, S. (1981) Metabolism of hydrogen peroxide in isolated hepato-

cytes: relative contributions of catalase and glutathione peroxidase in decomposition of endogenously released H202. Archs. Biochem. Biophys. 210, 505-516.

Jones, D. and Granett, J. (1982) Feeding site preferences of seven lepidopterous pests of celery. J. Econ. Ent. 75, 449-453.

Joshi, P. C. and Pathak, M. A. (1983) Production of singlet oxygen and superoxide radicals by psoralens and their biological significance. Biochem. Biophys. Res. Commun. 112, 638-648.

Kagan, J., Bazin, M. and Santus, R. (1989) Photosensitization with alpha-terthienyl: production of superoxide in aqueous media. J. Photochem. Photobiol. B3, 165-174.

Kalyanaraman, B., Felix, C. C. and Sealy, R. C. (1985) Semiquinone anion radicals of catechol(amine)s, catechol estrogens, and their metal ion complexes. Environ. Hlth. Perspect. 64, 185-198.

Kayser, H. (1982) Carotenoids in insects. In Chemistry and Biochemistry (Britton, G. and Goodwin, T. W., eds), pp. 195-210. Pergamon Press, Oxford.

Kramer, K. J. and Seib, P. A. (1982) Ascorbic acid and the growth and development of insects. In AscorbicAcid, Chemistq4, Metabolism, and Uses (Seib, P. A. and Tolbert, B. M., eds), pp. 276-291. American Chemical Society, Washington, D.C.

Larson, R. A. (1986) Insect defenses against phototoxic plant chemicals. J. Chem. Ecol. 12, 859-870. Larson, R. A. and Berenbaum, M. R. (1988) Environmental phototoxicity. Environ. Sci. Technol. 22, 354-360. Larson, R. A. and Marley, K. A. (1984) Quenching of singlet oxygen by alkaloids and related nitrogen hetero-

cyclics. Photochemistry 23, 2351-2354. Lee, K. and Berenbaum, M. R. (1989) Action of antioxidant enzymes and cytochrome P-450 monooxygenases in

the cabbage looper in response to plant phototoxins. Arch. Insect. Biochem. Physiol. 10, 151-162. Lee, K. and Berenbaum, M. R. (1990) Defense of parsnip webworm against phototoxic furanocoumarins: role of

antioxidant enzymes. J. Chem. Ecol. 16, 2451-2460. Lee, Y. M., Ayala, F. J. and Misra, H. P. (1981) Purification and properties of superoxide dismutase from

Drosophila melanogaster. J. Biol. Chem. 256, 8506-8509. Lindroth, R. L. (1988) Hydrolysis of phenolic glycosides by midgut 13-glucosidases in Papilio glaucus subspecies.

Insect Biochem. 18, 789-792. Lindroth, R. L. (1989) Biochemical detoxification: mechanism of differential tiger swallowtail tolerance to

phenolic glycosides. Oecologia. 81, 219-224. Mannervik, B. (1985) Glutathione peroxidase. Meth. Enzym. 113, 490-495. McCord, J. M. (1985) Oxygen-derived free radicals in postischemic tissue injury. NewEngl. J. Med. 312, 159-163. McCord, J. M. and Fridovich, I. (1989) Superoxide dismutase. J. Biol. Chem. 244, 6049-6055. Michiels, C. and Ramacle, J. (1988) Use of the inhibition of enzymatic antioxidant systems in order to evaluate

their physiological importance. Eur. J. Biochem. 177, 435-441. Miquel, J. (1989) Historical introduction to free radical and antioxidant biomedical research. In CRC Handbook of

Free Radicals and Antioxidants in Biomedicine (Miquel, J., Quintanilha, A. T. and Weber, H., eds), Vol. 1, pp. 3-11. CRC Press, Boca Raton, FL.

Mitchell, M. J., Ahmad, S. and Pardini, R. S. (1991) Purification of a highly active catalase from cabbage loopers, Trichoplusia ni. Insect Biochem. 21, 641-646.

Nahmias, J. A. and Bewley, G. C. (1984) Characterization of catalase purified from Drosophila melanogaster by hydrophobic interaction chromatography. Comp. Biochem. Physiol. 77B, 355-364.

Nickla, H., Anderson, J. and Palskill, T. (1983) Enzymes involved in oxygen detoxification during development of Drosophila melanogaster. Experientia 39, 610-612.

Nitao, J. K. (1989) Enzymatic adaptation in a specialist herbivore for feeding on furanocoumarin containing plants. Ecology 70, 629-635.

Nivsarkar, M., Kumar, G. P., Laloraya, M. and Laloraya, M. M. (1991) Superoxide dismutase in the anal gills of the mosquito larvae of Aedes aegypti: its inhibition by alpha-terthienyl. Arch. Insect. Biochem. Physiol. 16, 249- 255.

Nivsarkar, M., Kumar, G. P., Laloraya, M. and Laloraya, M. M. (1992) Generation of superoxide radical by c~- terthienyl in the anal gills of mosquito larvae Aedes aegypti: a new aspect in ~-terthienyl phototoxicity. Arch. InsecL Biochem. Physiol. (in press).

Oberly, L. W. and Spitz, D. R. (1984) Assay of superoxide dismutase activity in tumour tissue. Meth. Enzym. 105, 457-464.

Pardini, R. S. and Ahmad, S. (1991) Effects of quinones on antioxidant enzymes of insects. The Pharmacologist." Proc. ASPET Meeting 33, 345.

Pardini, R. S., Pritsos, C. A., Bowen, S. M., Ahmad, S. and Blomquist, G. J. (1989) Adaptations to plant pro-

296 s. AHMAD

oxidants in a phytophagous insect model: enzymatic protection from oxidative stress. In Oxygen Radicals in Bio/ogy and Medicine (Simic, M G., Taylor, K. A., Ward, J. F. and von Sonntag, C., eds), pp. 725-728. Plenum Press, New York.

Powis, G. (1987) Metabolism and toxicity of quinones. Pharmac. Ther, 35, 57-162. Price, N. R. and Dance, S. J. (1983) Some biochemical aspects of phosphine action and resistance in three

species of stored product beetles. Comp. Biochem. Physiol. 78C, 277-281. Pritsos, C. S., Aaronson, L. M. and Pardini, R. S. (1986) Metabolic consequences of dietary 2,3-dichloro-l,4-

naphthoquinone (CNQ) in the rat. Biochem. Pharmac. 35, 1131-1135. Pritsos, C. A., Ahmad, S., Bowen, S. M., Blomquist, G. J. and Pardini, R. S. (1988a) Antioxidant enzymes in the

southern armyworm, Spodoptera eridania. Comp. Biochem. Physiol. 900, 423-427. Pritsos, C. A., Ahmad, S., Bowen, S. M., Elliott, A. J., Blomquist, G. J. and Pardini, R. S. (1988b) Antioxidant

enzymes of the black swallowtail butterfly, Papilio polyxenes, and their response to the prooxidant allelochemical, quercetin. Arch. Insect Biochem. Physiol. 8, 101-112.

Pritsos, C. A., Ahmad, S., Elliott, A. J. and Pardini, R. S. (1990) Antioxidant enzyme level response to prooxidant allelochemicals in larvae of the southern armyworm moth Spodoptera eridania. Free Rad. Res. Commun. 9, 127-133.

Pritsos, C. A., Jensen, D. E., Pisani, D. and Pardini, R. S, (1982) Involvement of superoxide in the interaction of 2,3-dichloro-l,4-naphthoquinone with mitochondrial membranes. Arch. Biochem. Biophys. 217, 98-100.

Pritsos, C. A. and Pardini, R. S. (1984) A redox cycling mechanism of action for 2,3-dichloro-l,4-naphthoquinone with mitochondrial membranes and the role of sulfhydryl groups. Biochem. Pharmac. 33, 3771-3777.

Pritsos, C. A., Pastore, J. and Pardini, R. S. (1991) Role of superoxide dismutase in the protection and tolerance to the prooxidant allelochemical quercetin in Papilio polyxenes, Spodoptera eridania, and Trichoplusia n/: Arch. Insect Biochem. Physiol. 16, 273-282.

Prohaska, J. R. (1980) The glutathione peroxidase activity of glutathione-S-transferase. Biochim. Biophys. Acta 811, 87-98.

Racker, E. (1955) Glutathione reductase (liver and yeast). Meth. Enzym. 2, 722-725. Reddy, C. C., Tu, C.-P. D., Burgess, H. C.-Y., Scholz, R. W. and Masaaro, E. J. (1981) Evidence for the occurrence

of selenium-independent glutathione transferase. Biochem. Biophys. Res. Commun. 101, 970-978. Rose, R, C. (1989) Renal metabolism of the oxidized form of ascorbic acid (dehydro-L-ascorbic acid). Am. J.

Physiol. 256, F52-F56. Rose, R. C., Choi, J. and Koch, M. J. (1988) Intestinal transport and metabolism of oxidized ascorbic acid

(dehydroascorbic acid). Am. J. Physiol. 255, F52-F56. Singh, A. (1989) Chemical and biochemical aspects of activated oxygen: singlet oxygen, superoxide anion, and

related species. In CRC Handbook of Free Radicals and Antioxidants in Biomedicine (Miquel, J., Quintenilha, A. T. and Weber, H., eds), Vol. 1, pp. 17-28. CRC Press, Boca Raton. FL.

Singh, S. V., Leal, T., Ansari, G. A. S. and Awasthi, Y. C. (1987) Purification and characterization of glutathione-S- transferases of human kidney. Biochem. J. 246, 179-186.

Sohal, R. S. and Allen, R. G. (1986) Relationship between oxygen metabolism, aging and the development. Adv. Free Red. Biol. Med. 2, 117-160.

Tan, K. H., Mayer, D. J., Belin, J. and Ketterer, B. (1984) Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Biochem. J, 220, 243-252.

Thomas, C., MacGill, R. S., Miller, G. C. and Pardini, R. S. (1992) Photoactivation of hypericin generates singlet oxygen in mitochondria and inhibits succinoxidase. Photochem. Photobiol. 55, 47-53.

van Sumere, C. F., Albrecht, J., Dedoner, A., de Poorer, H. and Pe, I. (1975) Plant proteins and phenolics. In The Chemistry and Biochemistry of Plant Proteins (Harborne, J. B. and van Sumere, C. G., eds), pp. 211-256. Academic Press, New York.

Weinhold, L. C., Ahmad, S. and Pardini, R. S. (1990) Insect glutathione-S-transferase: a predictor of allelochemical and oxidative stress. Comp. Biochem. Physiol. 95B, 355-363.

Yu, S. J. (1987) Quinone reductase of phytophagous insects and its induction by allelochemicals. Comp. Biochem. Physiol. 87B, 621-624.

Documents

Biochemical defence of pro-oxidant plant allelochemicals by herbivorous insects