Summers and Felton 1993 DHAR

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Biochimica et Biophysica Acta, 1156 (1993) 235-238 35 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/ 06.00

BBAGEN 23786 apid eport

n t iox id an t r o l e o f d e h yd r oas c or b ic ac id r e d u c tas e in in s e c t sClinton B Summers and Gary W Felton

Department of Entomology, University of Arkansas, Fayetteville, AR USA)(Received 23 November 1992)

Key words: Ascorbic acid; Dehydroascorbic acid reductase; Selenium-dependent glutathione peroxidase; Glutathione transferase;Catalase; Hydrogen peroxide; (Insect)Dehydroascorbic acid reductase, which catalyses the regeneration of ascorbic acid from dehydroascorbic acid, is reported here tooccur widely among insects. Due to the reported absence of glutathione peroxidase in insects and the generally low affinity ofcatalase for hydrogen peroxide, dehydroascorbic acid reductase may play a pivotal role in the elimination of hydrogen peroxidein insects.

The antioxidant enzymes used by organisms to con-trol the generation of active oxygen and lipid peroxida-tion have been the focus of much recent research [1,2].In organisms studied so far, superoxide dismutase(SOD) and catalase (CAT) may have a primary respon-sibility for removing superoxide and hydrogen perox-ide, respectively. In mammals, a Se-dependent glu-tathione peroxidase (GPOX) may be more importantthan CAT for removal of H202 in vivo [3]. Oxidizedglutathione (GSSG) generated by GPOX is then recov-ered as reduced glutathione (GSH) by glutathione re-ductase (GR). This route for the removal of H202 maybe especially important at low H202 concentrations,due to the fact that the high K m of CAT may make itineffective for eliminating low levels of H202 [2]. Plantslack the Se-dependent GPOX found in mammaliansystems but possess an alternative mechanism for theremoval of H 2 0 2 utilizing ascorbate peroxidase(APOX), dehydroascorbic acid reductase (DHAR), andGR. In this system, H 2 0 2 is reduced by ascorbic acid,generating dehydroascorbic acid. The dehydroascorbicacid is recovered by the GSH dependent enzyme,DHAR, with GR regenerating GSH in essentially thesame manner as mammalian systems studied [4,5]. In-sects also lack the Se-dependent GPOX, but do pos-sess a glutathione S-transferase that exhibits peroxi-dase activity (GSTPX). This Se-independent enzymeshows little affinity for H202, but is capable of remov-ing organic peroxides [6]. The presence of D HAR in

Correspondence to: C.B. Summers, Department of Entomology,University of Arkansas, Fayetteville, AR 72701, USA.

the insect species examined may indicate that theascorbate dependent removal of H2 0 2 is important inthe insect s ability to control the generation of activeoxygen species.To determine the presence of DHAR in insects,whole larval or adult insects were homogenized in anappropriate volume, 2:1 (vol:wt), of ice-cold 0.1 Mpotassium phosphate buffer, pH 7.0, using a Tissuem-izer (Tekmar Co., Cincinnati, OH) tissue homogenizerfor 15 to 20 sec. The homogenate was then centrifugedfor 20 min at 4000 x g. The supernatant was collectedand kept on ice until needed for the DHAR assay [7].Total protein content of the supernatant was deter-mined according to the method of Bradford [8] usingbovine serum albumin as the standard. Each replicatecontained 0.50-1.00 g insect tissue and a minimum ofsix replicates were performed in duplicate for eachinsect species. All spectrophotometric determinationswere performed using a SLM Aminco 3000 Arrayspectrophotometer (SLM Aminco, Rochester, NY).DHAR activity was measured by monitoring theincrease in absorbance at 265 nm due to the formationof ascorbic acid from dehydroascorbic acid [7]. DHARactivity is expressed as nmol ascorbate fo rm ed /m in /m gprotein. All chemicals were purchased from SigmaChemical Co. (St. Louis, MO) except for dehydroascor-bic acid, which was purchased from Aldrich ChemicalCo. (Milwaukee, WI).Each of the insects tested displayed DHAR activity(see Table I). DHAR levels in Malacosoma americanum and Haematobia irritans were among the high-est observed in the insects tested. Levels of DHARwere similar between coleopteran species feeding on


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2 3 6T A B L E ISurt,ey of DHA R acticity in insectsE n z y m e a c ti v it y i s r e p o r t e d a s n m o l a s c o r b a t e f o r m e d / m i n p e r m g p r o t ei n .S p e c ie s C o m m o n n a m e O r d e r E n z y m e S E n

a c t i v i t yCotinis nitida g r e e n J u n e b e e t l e c o l e o p t e r a 3 . 0 6 0 .5 1 1 3Tenebrio molitor y e l lo w m e a l w o r m c o l e o p t e r a 2 . 7 0 0 . 4 2 1 9Alphitobius diapernius l e s s e r m e a l w o r m c o l e o p t e r a 2 . 32 0 . 3 7 1 2Hippodamia com ergens c o n v e r g e n t l a d y b e e t l e c o l e o p t e r a 3 . 48 0 . 3 9 6Cerotoma trifurcata b e a n l e a f b e e t l e c o l e o p t e r a 2 . 90 0 . 8 9 7Haematobia irritans h o r n f ly d i p t e r a 5 . 4 8 0 . 8 7 8Musca domestica c o m m o n h o u s e f ly d i p t e r a 2 . 17 0 . 3 0 7Pseudoplusia includens a s o y b e a n l o o p e r l e p i d o p t e r a 2 . 8 9 0 . 3 6 9Manduca sexta a t o b a c c o h o r n w o r m l e p i d o p t e r a 3 .7 8 0 . 5 8 9Anticarsia gemmatalis ~ v e l v e t b e a n c a t e r p i l l a r l e p i d o p t e r a 2 . 8 5 0 .3 1 2 1Heliothis cirescens a t o b a c c o b u d w o r m l e p i d o p t e r a t .8 3 0 .3 5 1 7Helicot erpa zea ~ c o r n e a r w o r m l e p i d o p t e r a 2 .8 1 0 .3 1 8Galleria mellonella g r e a t e r w a x m o t h l e p i d o p t e r a 1 .5 6 0 . 1 4 8Malacosoma americanum e a s t e r n t e n t c a t e r p i l l a r l e p i d o p t e r a 6 . 9 7 0 . 9 0 1 1~ ' L e p i d o p t e r a n l a r v a e r e a r e d o n a r t i f i c i a l d i e t .

n a t u r a l d i e t s a n d l e p i d o p t e r a n s p e c i e s f e e d i n g o n a r t i-f ic ia l d ie t s . P r e l i m i n a r y d a t a s u g g e s ts t h a t l e p i d o p t e r a ns p e c i e s m a y p o s s e ss h i g h e r D H A R a c t iv i ti e s w h e nf e e d i n g o n h o s t p l a n t s . I t is p o s s i b l e t h a t i n c r e a s e dl ev e ls o f D H A R m a y b e a m e a n s o f p r o te c t i n g t h ef e e d i n g h e r b i v o r e f r o m s u p e r o x i d e a n d H 2 0 2 g e n er -a t e d b y d i e t a r y p r o o x i d a n t s . M a n y p l a n t s p e c i e s u s e da s f o o d s o u r c e s b y l e p i d o p t e r a n l a r v a e c o n t a i n n u m e r -o u s c o m p o u n d s t h a t a r e c a p a b l e o f p r o d u c i n g s u p e r o x -i d e a n d H 2 0 2 in t h e p r e s e n c e o f o x y g e n. Q u i n o n e s ,d i h y d r o x y p h e n o l ic c o m p o u n d s , a n d s e v e ra l p h o t o a c t i -v a t e d c o m p o u n d s s u c h a s x a n t h o t o x i n a n d h a r m i n eh a v e t h e p o t e n t i a l t o p r o d u c e a c t iv e o x y g e n s p e c i e su n d e r a v a r i e ty o f c o n d i t io n s [ 9,1 0] . E x p e r i m e n t s w i t hH zea h a v e d e m o n s t r a t e d u p t o a 1 0 - f o ld in c r e a s e i nD H A R a c t iv i ty i n m i d g u t t is s u e f r o m l a r v a e f e e d i n g o nn a t u r a l h o s t p l a n t s .

A p a r t i a ll y p u r i f i e d e n z y m e p r e p a r a t i o n w a s o b -t a i n e d f r o m a h o m o g e n a t e o f w h o l e H zea l a r v a e .T a b l e I I d e t a i l s t h e p a r t i a l p u r i f i c a t i o n o f D H A R f r o mt h e H zea h o m o g e n a t e . F i n a l p u r i f i c a t i o n b y i s o e l e c -T A B L E I IDetails of the partial purification of DH AR f rom H. zea laruaeE n z y m e a c ti v it y i s r e p o r t e d a s n m o l a s c o r b a t e f o r m e d / m i n p e r m gp r o t e i n .P u r i f i c a t i o n s t e p y i e l d E n z y m e a c t i v it y P u r i f i c a t i o nC r u d e h o m o g e n a t e 1 0 0 1 .1 1 -( N H 4 ) 2 S O 4 p r e c i p i t a t i o n 8 8 . 5 1 8 .3 1 6 .5S e p h a d e x G - 7 5 4 3 . 9 3 4 . 6 3 1 .2R o t o f o r I E F 6 . 0 2 0 4 . 1 1 8 3 . 9

t r ic f o c u s in g , u s i n g a R o t o f o r I E F c e ll B i o R a d , R i c h -m o n d , C A ) w a s c a r r ie d o u t a t a c o n s t a n t p o w e r o f 12W a t t s f o r 4 h a t 4 o C , u s i n g B i o l y t e a m p h o l y t e s , p H 3t o 9 . I n i t i a l c o n d i t i o n s w e r e 8 0 0 V a n d 1 5 m A ; e q u i l i b -r i u m c o n d i t i o n s w e r e 2 0 0 0 V a n d 6 .0 m A . 2 o f t h e 2 0f r a c t io n s c o l l e c t e d w e r e f o u n d t o h a v e s u b s t a n t i a l a c t iv -i ty a p p r o x . 4 .5 m l t o t a l v o l u m e ) . T h e r e s u l t i n g s o l u t i o nw a s u s e d i n e x p e r i m e n t s t o d e t e r m i n e t h e K m a n d maxf o r t h e e n z y m e a s w e l l a s c o f a c t o r s p e c i f i c i t y . P r o t e i nc o n c e n t r a t i o n w a s d e t e r m i n e d f o l lo w i n g B r a d f o r d [ 8] .

T h e k i n e ti c p a r a m e t e r s w e r e e v a l u a t e d b y v ar y in gt h e c o n c e n t r a t i o n o f t h e s u b s t r a t e d e h y d r o a s c o r b i c a ci d .T h e L i n e w e a v e r - B u r k e p l o t o f t h e r e s u l t in g d a t a g a v e aVmax o f 1 .6 0 S E = 0 .2 3 ) m m o l / m i n / m g p r o t e i n a n d aK m o f 0 .2 7 S E = 0 . 0 39 ) m M f o r d e h y d r o a s c o r b i c a ci d .T h e D H A R f ro m H zea d e m o n s t r a t e d a s t r i c t r e q u i r e -m e n t f o r G S H a n d w a s in a c ti v e w i th N A D H , N A D P H ,d i t h i o t h r e i t o l , o r c y s t e in e .

T h i s i s t h e f i r s t d e t a i l e d e x a m i n a t i o n o f th e o c c u r -r e n c e o f D H A R a m o n g v a r i o u s in s e c t s p e c ie s . P re v i -o u s l y i d e n t i f i e d a n d c h a r a c t e r i z e d i n s e v e r a l p l a n t f a m -i li es , D H A R h a s o n l y b e e n o c c a s i o n a l l y n o t e d i n h i g h e ra n i m a l s. T h e o c c u r r e n c e o f D H A R i n al l o f t h e i n s e c tse x a m i n e d m i g h t i n d i c a t e t h a t i t is w i d e s p r e a d i n In -s e c ta . A n i n t e r e s t i n g a s p e c t o f t h e e n z y m e c h a r a c -t e r i z e d i n H zea i s i t s s i m i l a r i t y t o t h e D H A R i s o l a t e df r o m p l a n t s [ 11 ]. T h e s p e c i f i c r e q u i r e m e n t f o r G S Ha n d t h e a p p a r e n t K m fo r d e h y d r o a s c o r b i c a c i d e x h ib -i te d b y th e e n z y m e p a r a l le l t h e p r o p e r t i e s o f th e D H A Ri n p la n t s . A n e n z y m e o f si m i l ar f u n c t i o n i s o l a t e d f r o mr a t ti s su e s b y C h o i a n d R o s e , h o w e v e r , d e m o n s t r a t e d as t r i c t d e p e n d e n c e u p o n N A D P H [ 1 2 ] .


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The recycling of ascorbic acid may play a major rolein control of endogenous formation of active oxygenspecies and in defense against dietary oxidative stresses.Herbivorous insects, in particular, may benefit in viewof the large number of prooxidants found in their diet.Many common plant secondary chemicals such as thecinnamic acid derivatives, quinones, furanocoumarins,flavonoids, epoxides, and organic peroxides, can act aspotent oxidizing agents. Not only are these compoundspotentially toxic to the insect, many of them maydecrease the nutritive value of ingested proteinsthrough oxidative modification. Plants also possess anumber of oxidative enzymes capable of generatingdamaging chemicals, in particular, polyphenol oxidaseand lipoxygenase, which have been implicated in resis-tance to insects in some agricultural crop plants [13].Polyphenol oxidase catalyses the conversion of dihy-droxyphenolic acids to the corresponding quinones,and lipoxygenase generates fatty acid hydroperoxidesfrom free fatty acids. Many plants also contain theenzyme ascorbate oxidase, which catalyses the conver-sion of ascorbic acid to dehydroascorbic acid, and maybe capable of depriving the insect of ascorbic aciddirectly [13]. Ingestion of these oxidative enzymesshould dictate an oxidative environment for the ac-tively feeding herbivore, potentially depleting thebioavailability of ascorbic acid.Ascorbic acid has been implicated in many metabolicprocesses in insects, as well as being an importantgeneral antioxidant in tissues where exposure to en-dogenous sources of oxidative stress is high [14,15]. Itmay be particularly important for the control of H202formation at low levels, because even low levels ofH2 0 2 may be capable of damaging biomolecules viaFen ton type reactions. Although specific enzymaticmechanisms for eliminating these reactive productsexist (e.g., SOD, CAT, and GSTPX), tissue levels ofascorbic acid may be sufficient for significant chemicalscavenging of superoxide and hydrogen peroxide atmetabolically active sites where levels of the necessaryenzymes may be low. Additionally, the inefficiency ofCAT at low H202 concentrations and the lack of aSe-dependent GPOX suggests that DHAR may play acrucial role in protecting insects against H202 toxicity.Preliminary examination of the distribution of DHARin the tissues of H . z e a shows measurable levels of theenzyme to be present in the lumen, the midgut tissue,the fat body, and the Malpighian tubules. The highestactivities were found in Malpighian tubules (101nmol/min/mg protein) and the midgut (45nmol/min/mg protein), respectively (unpublisheddata). It is noteworthy that CAT activity appears to beabsent from the Malpighian tubules in H . z e a whilesubstantial levels of DHAR were found. The absenceof CAT activity in the Malpighian tubules of T r i c h o p l u -s ia n i has been reported by Ahmad et al. (1991) [2]. If

237CAT is found to be generally lacking in the Malpighiantubules of other lepidoteran species as well, it may bepossible that DHAR performs a critical function inmediating the removal of H202 in lepidopterans. Ithas recently been reported that CAT is the sole meansby which insects, D r o s o p h i l a m e l a n o g a s t e r in particu-lar, eliminate H202 [16]. Considering the crucial na-ture of this task and CAT s poor ability to remove lowconcentrations of H202 , the absence of CAT in certaintissues may suggest that other forces must be at workto compensate for these factors.The oxidative product of ascorbic acid, dehydro-ascorbic acid, has been observed to damage cellularmembranes in leukocytes, erythrocytes, and vesicles,leading to increased membrane permeability [17]. Therapid reduction of dehydroascorbic acid by DHAR mayprotect cellular membranes from damage. In conclu-sion, DHAR may be an important component of asystem that not only maintains nutritive levels of ascor-bic acid but also affords considerable protection fromboth endogenous and exogenous sources of oxidativestress. In the case of herbivorous insects, the suppres-sion of active oxygen formation, especially H20 2, maybe greatly aided by DHAR and ascorbic acid.cknowledgments

We are grateful for the support of the ArkansasScience and Technology Authority (ASTA 91-B-15 and92-B-52), and the USDA/CSRS (89-34195-4378 and92-34195-7162). Published with the approval of theDirector of the Agricultural Experiment Station, Uni-versity of Arkansas. We are indebted to Drs. D.C.Steelman, D.T. Johnson, S.Y. Young, and A.J. Muellerfor providing insects for this project.References

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Summers and Felton 1993 DHAR