Free RadiealBiology & Medicine, Vol. 8, pp. 401-413, 1990
0891-5849/90 $3.00+ .00 © 1990 Pergamon Press plc
Printed in the USA. All rights reserved.
-~" Review Article MECHANISMS FOR REGULATING OXYGEN TOXICITY IN PHYTOPHAGOUS INSECTS S. AHMAD* a n d R. S. PARDINI Department of Biochemistry, University of Nevada-Reno, Reno, NV 89557-0014, USA
(Received 6 September 1989: Revised and Accepted 9 January 1990)
Abstract--The antioxidant enzymatic defense of insects for the regulation of oxygen toxicity was investigated. Insect species examined were lepidopterous larvae of the cabbage looper (Trichoplusia n i l southern armyworm (Spodoptera eridania), and black swallowtail (Papilio polyxenes). These phytophagous species are subject to both endogenous and exogenous sources of oxidative stress from toxic oxygen radicals, hydrogen peroxide (H_,O2) and lipid peroxides (LOOH). In general, the constitutive levels of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GT), and its peroxidase activity (GTp0, and glutathione reductase (GR), correlate well with natural feeding habits of these insects and their relative susceptibility to prooxidant plant allelochemicals, quercetin (a flavonoid), and xanthotoxin (a photoactive furanocoumarin). Induction of SOD activity which rapidly destroys superoxide radicals, appears to be the main response to dietary prooxidant exposure. A unique observation includes high constitutive activity of CAT and a broader subcellular distribution in all three insects than observed in most mammalian species. These attributes of CAT appear to be important in the prevention of excessive accumulation of cytotoxic H202. Unlike mammalian species, insects possess very low levels of a GPOX-like activity toward H20_~. Irrefutable proof that this activity is due to a selenium-dependent GPOX found in mammals, is lacking at this time. However, the activity of selenium-independent GTox is unusually high in insects, suggesting that GTpx and not G P O X plays a prominent role in scavenging deleterious LOOHs. The GSSG generated from the GPOX and GTp~ reactions may be reduced to GSH by GR activity. A key role of SOD in protecting insects from prooxidant toxicity was evident when its inhibition resulted in enhanced toxicity towards prooxidants. The role of antioxidant compounds in protecting these insects from toxic forms of oxygen has not been explored in depth. A major finding, however, is that these insects are lutein accumulators. Lutein is a dihydroxy (diol) derivative of [?,-carotene, and it is a good quencher of activated forms of oxygen and free radicals. Levels of lutein are highest in P. polyxenes which specializes in feeding on prooxidant-containing plants.
Keywords--Antioxidant enzymes, Ascorbate, [?,-carotene, Catalase, Free radical, Glutathione peroxidase, Glutathione reductase, Glutathione transferase, Lutein, Oxygen toxicity, Papilio polyxenes, Quercetin, Spodoptera eridania, Superoxide dismutase, Tocopherols, Trichoplusia ni, Uric acid
INTRODUCTION
While ground-state molecular oxygen or dioxygen (02; or in the triplet state, 302) provides enormous advan-
tages for the sustenance of aerobic life processes, it also imposes universal toxicity. ~ Extensive literature has accumulated on this subject for mammalian spe-
*Author to whom correspondence should be addressed. Dr. Sami Ahmad received B.S. (chemistry; 1960) and M.S. (zoology; 1962) degrees from the University of Karachi, Karachi, Pakistan. After a 6-month training by the International Atomic Energy Agency, he served as a radiation biologist (1962 to 1965) at the Atomic Energy Center, Lahore, Pakistan. In 1965, Ahmad was awarded a Colombo Plan Merit Fellowship for graduate studies in the United Kingdom. He earned a graduate diploma (entomology; 1969) and a Ph.D. degree (insect physiology and biochemistry; 1969) from the University of London, London. After 2 years of postdoctoral training in toxicology at the University of Missouri, Columbia, MO, in 1971 he became a faculty member in the Department of Entomology and Economic Zoology of Rutgers--the State University of New Jersey, New Brunswick. In 1985 he moved to the Biochemistry Department, University of Nevada, Reno, where he established a research program in insect-plant relationships. His work is particularly focused on biochemical adaptations of phytophagous insects against toxic phytochemicals, some of which exert
oxidative stress. Dr. Ahmad has authored 74 research articles, several book chapters and, has edited two books, one book for Academic Press (1983), and another for Plenum Press (1986). Dr. Ronald S. Pardini, a native of San Francisco, CA received a B.S. ( 1961 ) in Animal Husbandry and Agricultural Chemistry from California State Polytechnic University and a Ph.D. (1965l in Food Science at the University of Illinois. After postdoctoral training in biomedical science at SRI International in Menlo Park, CA, in 1968 he joined the Biochemistry faculty at the University of NevadaReno, where he established research programs on the biochemical action of natural products. Recently his research program has focused on the mechanisms of oxidative stress exerted by natural products. Current research programs include the role of flavoholds and quinones in generating oxidative stress, and the role of allelochemical mediated oxidative stress in plant-insect interactions. He has been the author of 65 research publications principally on the biochemical action of naturally occurring xenobiotics. 401
402
S. AHMADand R. S. PARDINI
cies, and for some insect species in relation to development, aging,-' gluconeogenesis,4'5 bioluminescence, 6 and the toxic action of phosphine. 7 Until recently, however, this area was virtually unexplored for phytophagous insects. This paper summarizes the results of our recent investigations, which have elucidated some of the mechanisms for regulating oxygen toxicity in insects. Our insect model is comprised of three lepidopteran species, the cabbage looper (Trichoplusia ni), southern armyworm (Spodoptera eridania), and black swallowtail (Papilio polyxenes), which differ in sensitivity towards two prooxidant phytochemicals, quercetin (3, 5, 7, 3', 4'-pentahydroxyflavone) and xanthotoxin (8-methoxypsoralen). ~
Endogenous sources of oxidative stress One-electron reduction of 02 generates the superoxide anion radical ( O ~ ) . The endogenous sources for the production of O~ include autoxidizable molecules such as catecholamines and ubihydroquinone, and oxidoreductases such as hemoproteins and flavin enzymes; O~ may thus be generated in subcellular compartments including nuclei, mitochondria, endoplasmic reticulum, and cytosol. ~.9 Moreover, O~ - is in equilibrium with the hydroperoxyl radical (HO~) and these radicals can be converted to hydrogen peroxide (H202). In turn, H202 can be converted to the hydroxyl radical (.OH) via the metal-catalyzed Haber-Weiss reaction. Dioxygen is also activated in photosensitization and other reactions to singlet molecular oxygen (IA gO2; or IO2). J0
Exogenous sources of oxidative stress Approximately 20,000 structurally highly diverse allelochemicals identified to date account for the richness and diversity of plant allelochemicals, t~'~2 and some of these compounds exert oxidative stress. We define prooxidants as compounds that upon activation can react with 02 to generate toxic forms of oxygen. As reviewed recently, ~3.t4examples of prooxidants produced by plants include acetophenones (benzofurans and benzopyrans), [3-carboline alkaloids, furanochromes, furanocoumarins, furanoquinoline alkoloids, extended quinones, isoflavonoid phytoalexins, isoquinoline alkaloids, lignans, polyacetylenes, and thiophenes. All of these compounds are photodynamically activated while phenolic compounds such as quinones and flavonoids appear to be bioactivated through metabolism. 15,16 The photodynamic prooxidants are secondary metabolites that are produced in hundreds of plant species for their defense against microbial, fungal, and herbivorous insect attack. These plants belong to the fam-
ilies Apiaceae (formerly Umbelliferae), Asteraceae (Compositae), Cyperaceae, Euphorbiaceae, Fabaceae (Leguminosae), Hypericaceae (Clugiaceae), Liliaceae, Moraceae, Orchidaceae, Polygnonaceae, Rubiaceae, Rutaceae, and Solanaceae. 13.14Prooxidants of the photoactive type occur most frequently and with the greatest structural diversity in Apiaceae, Asteraceae, and Rutaceae. Nonphotoactive quinones occur quite frequently and flavonoids are ubiquitous in plants, ~7 the latter contribute to flower color in angiosperms and are abundant in leaves, tvls The ubiquitous flavonoid, quercetin, and the linear furanocoumarin, xanthotoxin, are examples of prooxidant plant allelochemicals. Quercetin, an autoxidizable redox active molecule, generates O~ , H202 and •OH radicals in vitro, ~9 which is consistent with its redox potential. 2° Upon ingestion, quercetin is apparently activated by one-electron oxidation to a free radical o-semiquinone, which in turn reacts with 02 to generate O~- and, consequently, H202 and .OH. It should be noted that quercetin and other flavonoids with a catecho121-23 and 3-hydroxyl g r o u p 22"24 possess antioxidant properties which were attributed to their ability to chelate heavy metals. 22 The literature suggests that quercetin has a dual role both as a prooxidant and antioxidant; however, under conditions of our studies, the prooxidant role appears to be predominant. Xanthotoxin is a photosensitizer (S) which is activated by long-UV (320-380 nm; max at ca. 365 nm) to the singlet-excited photosensitizer (~S) which rearranges to the triplet-excited photosensitizer (3S). This oxygen-independent (type I) mechanism is the most injurious because 3S can generally bind covalently to key macromolecules like DNA, tRNA, and proteins. ~3 In another deleterious reaction that is oxygen-dependent, IO2 is formed by transfer of excitation energy between 3S and ~O2 with the formation of S. Moreover, O5 is also generated by xanthotoxin, 2s presumably in a manner analogous to other tO2-generating photosensitizers such as hematoporphyrin, 262v [3-carboline alkaloids, 2s and certain photosensitive d y e s . 29 Burch and Martin > have demonstrated that in the photodynamic killing of E. coil B, DNA is a target of oxygen radicals generated via a modified type I mechanism by synthetic dyes. Xanthotoxin (S; activated form, ~S) may likewise be generating O5-, H 2 0 2 and "OH radical as illustrated below. ) RH- + 3S • (H) + H +
(1)
302 -~ RH"
~ 02- + R + H +
(2)
302 + 3 S ' ( H )
~O2- + S + H +
(3)
+ O~.- + 2H +
) H2O 2 + 302
(4)
~ R + M" + H*
(5)
RH2 + 3S
O2
RH. + M "+~
Insects' antioxidantmechanisms O~ + M"+~ 3S' (H) + M"+j H202 + M"
> 30 2 + M"
(6)
> S + M" + H +
(7)
> .OH + OH- + M"+'
(8)
The first reaction (equation l) is dependent on the availability of electrons from a variety of physiological reducing agents (RH2) including NAD(P)H, GSH, cysteine, tryptophan, tyrosine, and GTP. Desferrioxamine was found to inhibit DNA strand scission in E. coil B indicating that the metal-catalyzed reactions depicted above depend on Fe 3+. The premise that the photodynamic demise of E. coil B was dependent on oxygen radicals generated by photosensitizers was supported from two important observations. 29 First, the induction of superoxide dismutase and catalase in the E. coli B cells prior to incubation with the photosensitizers markedly lowered the mortality. Second, the .OH radical scavengers, thiourea and dimethylsulfoxide, also substantially reduced cell deaths. Doskotch et al. 3° have isolated a cytotoxic oxidative compound, peroxyferolide (allyl hydroperoxy sesquiterpene lactone), with antifeedant properties for the gypsy moth (Lymantria dispar) from the leaves of the tulip tree Liriodendron tulipitera (Magnoliaceae). Many plant allelochemicals such as sesquiterpene lactones and cx,[3-unsaturated ketones may arise from allylic hydroperoxides generated by chlorophyllmediated sensitized photo-oxygenation involving 102.30 Extraction and identification of hydroperoxides poses a problem due to their instability; therefore, the likelihood that plant prooxidant hydroperoxides may be of common occurrence awaits discovery. As depicted below, the .OH radical can cause lipid peroxidation of polyunsaturated fatty acids (LH) to form lipid hydroperoxide (LOOH). LH + .OH L. + 02 LH + LO~
~ L- + H20
(5)
~ LO~
(6)
> LOOH + L.
(7)
Other unsaturated organic molecules such as steroids (including cholesterol) and DNA may also be similarly peroxidized. Carbon-carbon double bonds of many unsaturated organic molecules are peroxidized by the insertion of ~O2. Lipids and substituted phenols generally form hydroperoxides, but depending upon the molecule attacked, ~O2 can also produce cyclic endoperoxides and noncyclic dioxetanes.~°
Oxygen toxicity Both the .OH radical and IO 2 a r e the two most reactive forms of activated 02. They react with macromolecules such as DNA, RNA, and proteins causing
403
alterations in the macromolecular structure and, they are responsible for deleterious lipid peroxidation. In insects, lipid peroxidation is potentially very harmful because lipids not only are essential components of cell membranes, but also have unique physiological functions, that is, prevention of dessication by cuticular hydrocarbons; involvement of isoprenoid juvenile hormones in insect development and reproduction, and sex-attractant pheromones. 3~ Tissues may be directly oxidatively damaged by peroxides, or from reactive breakdown products of peroxides such as epoxides, ketones, and aldehydes, for example, malonaldehyde. 32 Furthermore, LOOH can be decomposed catalytically by metals to regenerate the peroxidizing LO~ and -OH radicals. 33 Peroxidation is implicated in aging and numerous pathologies including cancer. 34 All aerobic organisms have consequently evolved elaborate defense mechanisms to remove toxic forms of dioxygen and peroxides.
THE BIOLOGY OF MODEL INSECTS
The biology of species that comprise our insect model is briefly reviewed from the more detailed account of Tietz 35 and, in particular, it clarifies the life cycles, rearing methods, and diets, and the developmental state-related terminologies used throughout this paper. T. ni and S. eridania are species of the moth family, Noctuidae, and P. polyxenes is a species of the butterfly family, Papilionidae. The immature stages of these insects are called larvae (or caterpillars), and represent the actual plant-feeding stages. The adults only require nectar that for the laboratory cultures is satisfied by honey diluted 10-fold with water. The life cycle begins with the hatch from eggs of diminutive first-instar larvae and the initiation of feeding on natural or semisynthetic diets that incorporate plant materials for optimal growth and development. Larval rearing procedures and diets have been previously reported in detail. 36-3~ T. ni and P. polyxenes have five larval instars while S. eridania has six. The larvae within 6-12 h from previous molt (shedding of skin) are termed early instars, mid-instars represent actively feeding stages corresponding to 1 (T. ni) or 2-3 days (S. eridania and P. polyxenes) from the past molt. Lateinstar stage approximated ca. 12 h prior to the next molt and characterized by cessation of feeding and retraction of the head capsules. The ultimate instars of all three insects form cocoons called pupae during which the adult morphogenesis occurs. The entire life cycle, that is, from eggs to adults, is considerably shorter for T. ni, and the larval stage lasts ca. 10 days. In contrast to T. ni, the life cycles of both S. eridania and P. polyxenes are 2-3 times longer, with larval stages lasting 3-4 weeks. The ontogenic studies were
404
S. AHMADand R. S. PARDINI
performed on early, mid, and late, third-, fourth- and fifth-instar larvae, however, all other studies were performed using midfifth-instar larvae of the three insect species. For the sake of brevity, the midfifth instars (except for the text on ontogeny) are referred to as "fifth instars" or "fifth-instar larvae." DEFENSE MECHANISMS OF INSECTS AGAINST PROOXIDANTS
Antioxidant compounds The lipid soluble antioxidant compounds such as carotenoids and tocopherols are primary defense mechanisms against IO2 attack of biomembranes, while sulfhydryl compounds and o~-tocopherol (vitamin E) defend against damage by .OH and O5-, alkoxyl (ROs) or LO~ radicals.~° Reaction with IO2 consumes antioxidants such as [3-carotene. However, in free radical quenching by vitamin E, the tocopherol radical of vitamin E formed is transformed by ascorbate (vitamin C) back to o~-tocopherol. According to Hochstein et al., 39 recently the capacity of uric acid " t o act as a free-radical scavenger and a potentially important biological antioxidant has been recognized." They further stated that many purine bases, especially urate because of its high reactivity with radicals, may be a unique scavenger of LO2, O5 , and .OH radicals. During the radical scavenging reactions, urate is oxidized to allantoin. In addition, urate may be important in stabilizing vitamin C in biological systems. Uric acid forms complexes with iron; thereby, it could prevent the "chelated as well as free iron-dependent oxidation of ascorbic acid," "without the apparent oxidation of urate. ''3~ Insects, especially phytophagous species, can obtain an adequate supply of carotenoids, tocopherols, and ascorbate and, being uricolytic, they generate more copious amounts of uric acid than do mammals,4° yet their tissue and subcellutar distribution and site-specific interaction with oxyradicals and peroxides has not been investigated. Recently we began analysis of the antioxidant composition and their levels in species that comprise our insect model. At this time, results have been analyzed only for the carotenoid compounds. The major carotenoid in third- to fifth-instar larvae of both P. polyxenes and S. eridania is lutein ([3-e-carotene-3,3',diol). The amounts of lutein are higher in P. polyxenes, a species specialized to feed on prooxidants, than in S. eridania (S. Ahmad, K. R. Downum, and R. S. Pardini, unpublished data, 1989). The concentration of lutein and its different levels in the two insect species mirror the lutein contents of their respective diets. No [3-carotene was detected in these insects despite its presence, for example, in the parsley (Petroselinum crispum) leaves
on which P. polyxenes larvae were raised. Several more polar carotenoids were detected in the diets of both insect species, yet their concentration in the larvae was negligible. Neither the larvae of T. ni, nor its pinto bean (not foliage) based diet yielded amounts of carotenoids in quantities adequate for structural resolution or quantitation. Trace levels of a compound that did elute with a retention time (cf. HPLC analysis) anlogous to that of lutein, requires confirmation by analysis of more concentrated extracts. Insects depend on an exogenous supply of the carotenoid skeleton since no de novo synthesis is known to occur in animals. The carotenoid profile of an insect is apparently dependent upon the carotenoid contents of their food, selective absorption, and some limited capability for in vivo transformation to oxygenated metabolites. 4~ The species of Lepidoptera differ from those of other taxonomic orders in that one type absorbs nearly equally lutein and [3-carotene (e.g., the white cabbage butterfly, Pieris braesicae), and another type absorbs lutein with a high preference. 4t Clearly, the species of our lepidopteran insect model are all lutein accumulators, with no evidence of ability to metabolize the carotenoids.
Antioxidant enzymes Enzymatic defenses are crucial in terminating the oxygen-radical cascade and for the removal of LOOH to terminate peroxidation chain reactions. Mammalian cells are well equipped with an enzymatic defense system against toxic oxygen species, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR). 9 Similar enzymes are observed in insects. 23~64°
Prooxidant susceptibility of model species Fifth-instar larvae of T. ni were found to be highly susceptible to xanthotoxin. The 24-h LCs0s were 0.0013 and 0.0004% (w/w) xanthotoxin for full daylight spectrum (14 h) and long-UV (320-380 nm; 4 h) exposures, respectively. T. ni larvae were also susceptible to quercetin with an LCs0 of 0.0045% (w/w) quercetin. 36 Data on relative consumption rates (RCRs) and relative growth rates (RGRs) indicated that the observed deaths from eating xanthotoxin-containing diets were not the result of acute starvation, because feeding did occur, and loopers on all diets produced fecal pellets. The observed reduced consumption over 24 h accompanied by reduced RGRs may in part reflect the debilitating effects of xanthotoxin. S. eridania is a more broadly polyphagous species than T. ni., and its fifth instars exhibited no casualties
Insects ~ antioxidant m e c h a n i s m s
in 24 h from dietary xanthotoxin at 0.001 to 0.7% (w/ w) concentration. 37 RGRs, however, decreased in a dose-dependant manner indicating the onset of toxicity and supporting the premise that xanthotoxin is toxic to S. eridania at higher concentration in the diet. Quercetin up to 1.0% (w/w) dietary concentration did not cause any mortality, and had no effect on RCRs or RGRs. P. polyxenes is specialized to feed with impunity on many species of Apiaceae and a few species of Rutaceae known to contain high levels of xanthotoxin.~314 The host plants of this insect such as parsley and wild carrot (Daucus carota) are known to contain very high levels of flavonoids; in D. carota leaves quercetin amounts to 400-1500 mg/kg, fresh mass basis. ~7 The finding that fifth instar of P. polyxenes fed as much as 164 mg (20% w/w) of quercetin over 12 h showed no signs of acute toxicity or moribundity, is consistent with its feeding specialization. 3~
Basal activities and ontogenetic profile of antioxidant enzymes Enzyme extracts from early-, mid- and late-stage third, fourth, and fifth instars of T. ni, S. eridania, and P. polyxenes were assayed for activities of SOD, CAT, and GR. 36-38 From the data in Fig. 1-3 the following patterns emerge. In general, the enzyme levels are consistent with the high, moderate, and low tolerance to prooxidants ofP. polyxenes, S. eridania, and T. hi, respectively. SOD activities dropped markedly
350 C
~
300
C
o
.Z
I00
50
0891-5849/90 $3.00+ .00 © 1990 Pergamon Press plc
Printed in the USA. All rights reserved.
-~" Review Article MECHANISMS FOR REGULATING OXYGEN TOXICITY IN PHYTOPHAGOUS INSECTS S. AHMAD* a n d R. S. PARDINI Department of Biochemistry, University of Nevada-Reno, Reno, NV 89557-0014, USA
(Received 6 September 1989: Revised and Accepted 9 January 1990)
Abstract--The antioxidant enzymatic defense of insects for the regulation of oxygen toxicity was investigated. Insect species examined were lepidopterous larvae of the cabbage looper (Trichoplusia n i l southern armyworm (Spodoptera eridania), and black swallowtail (Papilio polyxenes). These phytophagous species are subject to both endogenous and exogenous sources of oxidative stress from toxic oxygen radicals, hydrogen peroxide (H_,O2) and lipid peroxides (LOOH). In general, the constitutive levels of the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GT), and its peroxidase activity (GTp0, and glutathione reductase (GR), correlate well with natural feeding habits of these insects and their relative susceptibility to prooxidant plant allelochemicals, quercetin (a flavonoid), and xanthotoxin (a photoactive furanocoumarin). Induction of SOD activity which rapidly destroys superoxide radicals, appears to be the main response to dietary prooxidant exposure. A unique observation includes high constitutive activity of CAT and a broader subcellular distribution in all three insects than observed in most mammalian species. These attributes of CAT appear to be important in the prevention of excessive accumulation of cytotoxic H202. Unlike mammalian species, insects possess very low levels of a GPOX-like activity toward H20_~. Irrefutable proof that this activity is due to a selenium-dependent GPOX found in mammals, is lacking at this time. However, the activity of selenium-independent GTox is unusually high in insects, suggesting that GTpx and not G P O X plays a prominent role in scavenging deleterious LOOHs. The GSSG generated from the GPOX and GTp~ reactions may be reduced to GSH by GR activity. A key role of SOD in protecting insects from prooxidant toxicity was evident when its inhibition resulted in enhanced toxicity towards prooxidants. The role of antioxidant compounds in protecting these insects from toxic forms of oxygen has not been explored in depth. A major finding, however, is that these insects are lutein accumulators. Lutein is a dihydroxy (diol) derivative of [?,-carotene, and it is a good quencher of activated forms of oxygen and free radicals. Levels of lutein are highest in P. polyxenes which specializes in feeding on prooxidant-containing plants.
Keywords--Antioxidant enzymes, Ascorbate, [?,-carotene, Catalase, Free radical, Glutathione peroxidase, Glutathione reductase, Glutathione transferase, Lutein, Oxygen toxicity, Papilio polyxenes, Quercetin, Spodoptera eridania, Superoxide dismutase, Tocopherols, Trichoplusia ni, Uric acid
INTRODUCTION
While ground-state molecular oxygen or dioxygen (02; or in the triplet state, 302) provides enormous advan-
tages for the sustenance of aerobic life processes, it also imposes universal toxicity. ~ Extensive literature has accumulated on this subject for mammalian spe-
*Author to whom correspondence should be addressed. Dr. Sami Ahmad received B.S. (chemistry; 1960) and M.S. (zoology; 1962) degrees from the University of Karachi, Karachi, Pakistan. After a 6-month training by the International Atomic Energy Agency, he served as a radiation biologist (1962 to 1965) at the Atomic Energy Center, Lahore, Pakistan. In 1965, Ahmad was awarded a Colombo Plan Merit Fellowship for graduate studies in the United Kingdom. He earned a graduate diploma (entomology; 1969) and a Ph.D. degree (insect physiology and biochemistry; 1969) from the University of London, London. After 2 years of postdoctoral training in toxicology at the University of Missouri, Columbia, MO, in 1971 he became a faculty member in the Department of Entomology and Economic Zoology of Rutgers--the State University of New Jersey, New Brunswick. In 1985 he moved to the Biochemistry Department, University of Nevada, Reno, where he established a research program in insect-plant relationships. His work is particularly focused on biochemical adaptations of phytophagous insects against toxic phytochemicals, some of which exert
oxidative stress. Dr. Ahmad has authored 74 research articles, several book chapters and, has edited two books, one book for Academic Press (1983), and another for Plenum Press (1986). Dr. Ronald S. Pardini, a native of San Francisco, CA received a B.S. ( 1961 ) in Animal Husbandry and Agricultural Chemistry from California State Polytechnic University and a Ph.D. (1965l in Food Science at the University of Illinois. After postdoctoral training in biomedical science at SRI International in Menlo Park, CA, in 1968 he joined the Biochemistry faculty at the University of NevadaReno, where he established research programs on the biochemical action of natural products. Recently his research program has focused on the mechanisms of oxidative stress exerted by natural products. Current research programs include the role of flavoholds and quinones in generating oxidative stress, and the role of allelochemical mediated oxidative stress in plant-insect interactions. He has been the author of 65 research publications principally on the biochemical action of naturally occurring xenobiotics. 401
402
S. AHMADand R. S. PARDINI
cies, and for some insect species in relation to development, aging,-' gluconeogenesis,4'5 bioluminescence, 6 and the toxic action of phosphine. 7 Until recently, however, this area was virtually unexplored for phytophagous insects. This paper summarizes the results of our recent investigations, which have elucidated some of the mechanisms for regulating oxygen toxicity in insects. Our insect model is comprised of three lepidopteran species, the cabbage looper (Trichoplusia ni), southern armyworm (Spodoptera eridania), and black swallowtail (Papilio polyxenes), which differ in sensitivity towards two prooxidant phytochemicals, quercetin (3, 5, 7, 3', 4'-pentahydroxyflavone) and xanthotoxin (8-methoxypsoralen). ~
Endogenous sources of oxidative stress One-electron reduction of 02 generates the superoxide anion radical ( O ~ ) . The endogenous sources for the production of O~ include autoxidizable molecules such as catecholamines and ubihydroquinone, and oxidoreductases such as hemoproteins and flavin enzymes; O~ may thus be generated in subcellular compartments including nuclei, mitochondria, endoplasmic reticulum, and cytosol. ~.9 Moreover, O~ - is in equilibrium with the hydroperoxyl radical (HO~) and these radicals can be converted to hydrogen peroxide (H202). In turn, H202 can be converted to the hydroxyl radical (.OH) via the metal-catalyzed Haber-Weiss reaction. Dioxygen is also activated in photosensitization and other reactions to singlet molecular oxygen (IA gO2; or IO2). J0
Exogenous sources of oxidative stress Approximately 20,000 structurally highly diverse allelochemicals identified to date account for the richness and diversity of plant allelochemicals, t~'~2 and some of these compounds exert oxidative stress. We define prooxidants as compounds that upon activation can react with 02 to generate toxic forms of oxygen. As reviewed recently, ~3.t4examples of prooxidants produced by plants include acetophenones (benzofurans and benzopyrans), [3-carboline alkaloids, furanochromes, furanocoumarins, furanoquinoline alkoloids, extended quinones, isoflavonoid phytoalexins, isoquinoline alkaloids, lignans, polyacetylenes, and thiophenes. All of these compounds are photodynamically activated while phenolic compounds such as quinones and flavonoids appear to be bioactivated through metabolism. 15,16 The photodynamic prooxidants are secondary metabolites that are produced in hundreds of plant species for their defense against microbial, fungal, and herbivorous insect attack. These plants belong to the fam-
ilies Apiaceae (formerly Umbelliferae), Asteraceae (Compositae), Cyperaceae, Euphorbiaceae, Fabaceae (Leguminosae), Hypericaceae (Clugiaceae), Liliaceae, Moraceae, Orchidaceae, Polygnonaceae, Rubiaceae, Rutaceae, and Solanaceae. 13.14Prooxidants of the photoactive type occur most frequently and with the greatest structural diversity in Apiaceae, Asteraceae, and Rutaceae. Nonphotoactive quinones occur quite frequently and flavonoids are ubiquitous in plants, ~7 the latter contribute to flower color in angiosperms and are abundant in leaves, tvls The ubiquitous flavonoid, quercetin, and the linear furanocoumarin, xanthotoxin, are examples of prooxidant plant allelochemicals. Quercetin, an autoxidizable redox active molecule, generates O~ , H202 and •OH radicals in vitro, ~9 which is consistent with its redox potential. 2° Upon ingestion, quercetin is apparently activated by one-electron oxidation to a free radical o-semiquinone, which in turn reacts with 02 to generate O~- and, consequently, H202 and .OH. It should be noted that quercetin and other flavonoids with a catecho121-23 and 3-hydroxyl g r o u p 22"24 possess antioxidant properties which were attributed to their ability to chelate heavy metals. 22 The literature suggests that quercetin has a dual role both as a prooxidant and antioxidant; however, under conditions of our studies, the prooxidant role appears to be predominant. Xanthotoxin is a photosensitizer (S) which is activated by long-UV (320-380 nm; max at ca. 365 nm) to the singlet-excited photosensitizer (~S) which rearranges to the triplet-excited photosensitizer (3S). This oxygen-independent (type I) mechanism is the most injurious because 3S can generally bind covalently to key macromolecules like DNA, tRNA, and proteins. ~3 In another deleterious reaction that is oxygen-dependent, IO2 is formed by transfer of excitation energy between 3S and ~O2 with the formation of S. Moreover, O5 is also generated by xanthotoxin, 2s presumably in a manner analogous to other tO2-generating photosensitizers such as hematoporphyrin, 262v [3-carboline alkaloids, 2s and certain photosensitive d y e s . 29 Burch and Martin > have demonstrated that in the photodynamic killing of E. coil B, DNA is a target of oxygen radicals generated via a modified type I mechanism by synthetic dyes. Xanthotoxin (S; activated form, ~S) may likewise be generating O5-, H 2 0 2 and "OH radical as illustrated below. ) RH- + 3S • (H) + H +
(1)
302 -~ RH"
~ 02- + R + H +
(2)
302 + 3 S ' ( H )
~O2- + S + H +
(3)
+ O~.- + 2H +
) H2O 2 + 302
(4)
~ R + M" + H*
(5)
RH2 + 3S
O2
RH. + M "+~
Insects' antioxidantmechanisms O~ + M"+~ 3S' (H) + M"+j H202 + M"
> 30 2 + M"
(6)
> S + M" + H +
(7)
> .OH + OH- + M"+'
(8)
The first reaction (equation l) is dependent on the availability of electrons from a variety of physiological reducing agents (RH2) including NAD(P)H, GSH, cysteine, tryptophan, tyrosine, and GTP. Desferrioxamine was found to inhibit DNA strand scission in E. coil B indicating that the metal-catalyzed reactions depicted above depend on Fe 3+. The premise that the photodynamic demise of E. coil B was dependent on oxygen radicals generated by photosensitizers was supported from two important observations. 29 First, the induction of superoxide dismutase and catalase in the E. coli B cells prior to incubation with the photosensitizers markedly lowered the mortality. Second, the .OH radical scavengers, thiourea and dimethylsulfoxide, also substantially reduced cell deaths. Doskotch et al. 3° have isolated a cytotoxic oxidative compound, peroxyferolide (allyl hydroperoxy sesquiterpene lactone), with antifeedant properties for the gypsy moth (Lymantria dispar) from the leaves of the tulip tree Liriodendron tulipitera (Magnoliaceae). Many plant allelochemicals such as sesquiterpene lactones and cx,[3-unsaturated ketones may arise from allylic hydroperoxides generated by chlorophyllmediated sensitized photo-oxygenation involving 102.30 Extraction and identification of hydroperoxides poses a problem due to their instability; therefore, the likelihood that plant prooxidant hydroperoxides may be of common occurrence awaits discovery. As depicted below, the .OH radical can cause lipid peroxidation of polyunsaturated fatty acids (LH) to form lipid hydroperoxide (LOOH). LH + .OH L. + 02 LH + LO~
~ L- + H20
(5)
~ LO~
(6)
> LOOH + L.
(7)
Other unsaturated organic molecules such as steroids (including cholesterol) and DNA may also be similarly peroxidized. Carbon-carbon double bonds of many unsaturated organic molecules are peroxidized by the insertion of ~O2. Lipids and substituted phenols generally form hydroperoxides, but depending upon the molecule attacked, ~O2 can also produce cyclic endoperoxides and noncyclic dioxetanes.~°
Oxygen toxicity Both the .OH radical and IO 2 a r e the two most reactive forms of activated 02. They react with macromolecules such as DNA, RNA, and proteins causing
403
alterations in the macromolecular structure and, they are responsible for deleterious lipid peroxidation. In insects, lipid peroxidation is potentially very harmful because lipids not only are essential components of cell membranes, but also have unique physiological functions, that is, prevention of dessication by cuticular hydrocarbons; involvement of isoprenoid juvenile hormones in insect development and reproduction, and sex-attractant pheromones. 3~ Tissues may be directly oxidatively damaged by peroxides, or from reactive breakdown products of peroxides such as epoxides, ketones, and aldehydes, for example, malonaldehyde. 32 Furthermore, LOOH can be decomposed catalytically by metals to regenerate the peroxidizing LO~ and -OH radicals. 33 Peroxidation is implicated in aging and numerous pathologies including cancer. 34 All aerobic organisms have consequently evolved elaborate defense mechanisms to remove toxic forms of dioxygen and peroxides.
THE BIOLOGY OF MODEL INSECTS
The biology of species that comprise our insect model is briefly reviewed from the more detailed account of Tietz 35 and, in particular, it clarifies the life cycles, rearing methods, and diets, and the developmental state-related terminologies used throughout this paper. T. ni and S. eridania are species of the moth family, Noctuidae, and P. polyxenes is a species of the butterfly family, Papilionidae. The immature stages of these insects are called larvae (or caterpillars), and represent the actual plant-feeding stages. The adults only require nectar that for the laboratory cultures is satisfied by honey diluted 10-fold with water. The life cycle begins with the hatch from eggs of diminutive first-instar larvae and the initiation of feeding on natural or semisynthetic diets that incorporate plant materials for optimal growth and development. Larval rearing procedures and diets have been previously reported in detail. 36-3~ T. ni and P. polyxenes have five larval instars while S. eridania has six. The larvae within 6-12 h from previous molt (shedding of skin) are termed early instars, mid-instars represent actively feeding stages corresponding to 1 (T. ni) or 2-3 days (S. eridania and P. polyxenes) from the past molt. Lateinstar stage approximated ca. 12 h prior to the next molt and characterized by cessation of feeding and retraction of the head capsules. The ultimate instars of all three insects form cocoons called pupae during which the adult morphogenesis occurs. The entire life cycle, that is, from eggs to adults, is considerably shorter for T. ni, and the larval stage lasts ca. 10 days. In contrast to T. ni, the life cycles of both S. eridania and P. polyxenes are 2-3 times longer, with larval stages lasting 3-4 weeks. The ontogenic studies were
404
S. AHMADand R. S. PARDINI
performed on early, mid, and late, third-, fourth- and fifth-instar larvae, however, all other studies were performed using midfifth-instar larvae of the three insect species. For the sake of brevity, the midfifth instars (except for the text on ontogeny) are referred to as "fifth instars" or "fifth-instar larvae." DEFENSE MECHANISMS OF INSECTS AGAINST PROOXIDANTS
Antioxidant compounds The lipid soluble antioxidant compounds such as carotenoids and tocopherols are primary defense mechanisms against IO2 attack of biomembranes, while sulfhydryl compounds and o~-tocopherol (vitamin E) defend against damage by .OH and O5-, alkoxyl (ROs) or LO~ radicals.~° Reaction with IO2 consumes antioxidants such as [3-carotene. However, in free radical quenching by vitamin E, the tocopherol radical of vitamin E formed is transformed by ascorbate (vitamin C) back to o~-tocopherol. According to Hochstein et al., 39 recently the capacity of uric acid " t o act as a free-radical scavenger and a potentially important biological antioxidant has been recognized." They further stated that many purine bases, especially urate because of its high reactivity with radicals, may be a unique scavenger of LO2, O5 , and .OH radicals. During the radical scavenging reactions, urate is oxidized to allantoin. In addition, urate may be important in stabilizing vitamin C in biological systems. Uric acid forms complexes with iron; thereby, it could prevent the "chelated as well as free iron-dependent oxidation of ascorbic acid," "without the apparent oxidation of urate. ''3~ Insects, especially phytophagous species, can obtain an adequate supply of carotenoids, tocopherols, and ascorbate and, being uricolytic, they generate more copious amounts of uric acid than do mammals,4° yet their tissue and subcellutar distribution and site-specific interaction with oxyradicals and peroxides has not been investigated. Recently we began analysis of the antioxidant composition and their levels in species that comprise our insect model. At this time, results have been analyzed only for the carotenoid compounds. The major carotenoid in third- to fifth-instar larvae of both P. polyxenes and S. eridania is lutein ([3-e-carotene-3,3',diol). The amounts of lutein are higher in P. polyxenes, a species specialized to feed on prooxidants, than in S. eridania (S. Ahmad, K. R. Downum, and R. S. Pardini, unpublished data, 1989). The concentration of lutein and its different levels in the two insect species mirror the lutein contents of their respective diets. No [3-carotene was detected in these insects despite its presence, for example, in the parsley (Petroselinum crispum) leaves
on which P. polyxenes larvae were raised. Several more polar carotenoids were detected in the diets of both insect species, yet their concentration in the larvae was negligible. Neither the larvae of T. ni, nor its pinto bean (not foliage) based diet yielded amounts of carotenoids in quantities adequate for structural resolution or quantitation. Trace levels of a compound that did elute with a retention time (cf. HPLC analysis) anlogous to that of lutein, requires confirmation by analysis of more concentrated extracts. Insects depend on an exogenous supply of the carotenoid skeleton since no de novo synthesis is known to occur in animals. The carotenoid profile of an insect is apparently dependent upon the carotenoid contents of their food, selective absorption, and some limited capability for in vivo transformation to oxygenated metabolites. 4~ The species of Lepidoptera differ from those of other taxonomic orders in that one type absorbs nearly equally lutein and [3-carotene (e.g., the white cabbage butterfly, Pieris braesicae), and another type absorbs lutein with a high preference. 4t Clearly, the species of our lepidopteran insect model are all lutein accumulators, with no evidence of ability to metabolize the carotenoids.
Antioxidant enzymes Enzymatic defenses are crucial in terminating the oxygen-radical cascade and for the removal of LOOH to terminate peroxidation chain reactions. Mammalian cells are well equipped with an enzymatic defense system against toxic oxygen species, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR). 9 Similar enzymes are observed in insects. 23~64°
Prooxidant susceptibility of model species Fifth-instar larvae of T. ni were found to be highly susceptible to xanthotoxin. The 24-h LCs0s were 0.0013 and 0.0004% (w/w) xanthotoxin for full daylight spectrum (14 h) and long-UV (320-380 nm; 4 h) exposures, respectively. T. ni larvae were also susceptible to quercetin with an LCs0 of 0.0045% (w/w) quercetin. 36 Data on relative consumption rates (RCRs) and relative growth rates (RGRs) indicated that the observed deaths from eating xanthotoxin-containing diets were not the result of acute starvation, because feeding did occur, and loopers on all diets produced fecal pellets. The observed reduced consumption over 24 h accompanied by reduced RGRs may in part reflect the debilitating effects of xanthotoxin. S. eridania is a more broadly polyphagous species than T. ni., and its fifth instars exhibited no casualties
Insects ~ antioxidant m e c h a n i s m s
in 24 h from dietary xanthotoxin at 0.001 to 0.7% (w/ w) concentration. 37 RGRs, however, decreased in a dose-dependant manner indicating the onset of toxicity and supporting the premise that xanthotoxin is toxic to S. eridania at higher concentration in the diet. Quercetin up to 1.0% (w/w) dietary concentration did not cause any mortality, and had no effect on RCRs or RGRs. P. polyxenes is specialized to feed with impunity on many species of Apiaceae and a few species of Rutaceae known to contain high levels of xanthotoxin.~314 The host plants of this insect such as parsley and wild carrot (Daucus carota) are known to contain very high levels of flavonoids; in D. carota leaves quercetin amounts to 400-1500 mg/kg, fresh mass basis. ~7 The finding that fifth instar of P. polyxenes fed as much as 164 mg (20% w/w) of quercetin over 12 h showed no signs of acute toxicity or moribundity, is consistent with its feeding specialization. 3~
Basal activities and ontogenetic profile of antioxidant enzymes Enzyme extracts from early-, mid- and late-stage third, fourth, and fifth instars of T. ni, S. eridania, and P. polyxenes were assayed for activities of SOD, CAT, and GR. 36-38 From the data in Fig. 1-3 the following patterns emerge. In general, the enzyme levels are consistent with the high, moderate, and low tolerance to prooxidants ofP. polyxenes, S. eridania, and T. hi, respectively. SOD activities dropped markedly
350 C
~
300
C
o
.Z
I00
50
