Biochimica et Biophysica Acta, 1156 (1993) 235-238 235

1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/$06.00

BBAGEN 23786
Rapid Report
Antioxidant role of dehydroascorbic acid reductase in insects
Clinton 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 to
occur widely among insects. Due to the reported absence of glutathione peroxidase in insects and the generally low affinity of
catalase for hydrogen peroxide, dehydroascorbic acid reductase may play a pivotal role in the elimination of hydrogen peroxide
in 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 important
than CAT for removal of H202 in vivo [3]. Oxidized
glutathione (GSSG) generated by GPOX is then recov-
ered as reduced glutathione (GSH) by glutathione re-
ductase (GR). This route for the removal of H202 may
be especially important at low H202 concentrations,
due to the fact that the high K m of CAT may make it
ineffective for eliminating low levels of H202 [2]. Plants
lack the Se-dependent GPOX found in mammalian
systems but possess an alternative mechanism for the
removal of H202 utilizing ascorbate peroxidase
(APOX), dehydroascorbic acid reductase (DHAR), and
GR. In this system, H202 is reduced by ascorbic acid,
generating dehydroascorbic acid. The dehydroascorbic
acid is recovered by the GSH dependent enzyme,
DHAR, with GR regenerating GSH in essentially the
same 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 enzyme
shows little affinity for H202, but is capable of remov-
ing organic peroxides [6]. The presence of DHAR in
Correspondence to: C.B. Summers, Department of Entomology,
University of Arkansas, Fayetteville, AR 72701, USA.
the insect species examined may indicate that the
ascorbate dependent removal of H20 2 is important in
the insect's ability to control the generation of active
oxygen species.
To determine the presence of DHAR in insects,
whole larval or adult insects were homogenized in an
appropriate volume, 2:1 (vol:wt), of ice-cold 0.1 M
potassium phosphate buffer, pH 7.0, using a Tissuem-
izer (Tekmar Co., Cincinnati, OH) tissue homogenizer
for 15 to 20 sec. The homogenate was then centrifuged
for 20 min at 4000 x g. The supernatant was collected
and 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] using
bovine serum albumin as the standard. Each replicate
contained 0.50-1.00 g insect tissue and a minimum of
six replicates were performed in duplicate for each
insect species. All spectrophotometric determinations
were performed using a SLM Aminco 3000 Array
spectrophotometer (SLM Aminco, Rochester, NY).
DHAR activity was measured by monitoring the
increase in absorbance at 265 nm due to the formation
of ascorbic acid from dehydroascorbic acid [7]. DHAR
activity is expressed as nmol ascorbate formed/ mi n/ mg
protein. All chemicals were purchased from Sigma
Chemical Co. (St. Louis, MO) except for dehydroascor-
bic acid, which was purchased from Aldrich Chemical
Co. (Milwaukee, WI).
Each of the insects tested displayed DHAR activity
(see Table I). DHAR levels in Malacosoma ameri-
canum and Haematobia irritans were among the high-
est observed in the insects tested. Levels of DHAR
were similar between coleopteran species feeding on
236
T ABL E I
Surt,ey of DHAR acticity in insects
Enz yme activity is r e por t e d as nmol as cor bat e f o r me d / mi n per mg pr ot ei n.
Speci es Co mmo n n a me Or de r Enz yme SE n
activity
Cotinis nitida gr e e n J u n e beet l e col eopt er a 3.06 0.51 13
Tenebrio molitor yellow meal wor m col eopt er a 2.70 0.42 19
Alphitobius diapernius l esser me a l wor m col eopt er a 2.32 0.37 12
Hippodamia com'ergens c onve r ge nt l ady beet l e col eopt er a 3.48 0.39 6
Cerotoma trifurcata be a n l eaf beet l e col eopt er a 2.90 0.89 7
Haematobia irritans hor n fly di pt er a 5.48 0.87 8
Musca domestica c o mmo n hous e fly di pt er a 2.17 0.30 7
Pseudoplusia includens a s oybean l ooper l epi dopt er a 2.89 0.36 9
Manduca sexta a t obacco hor nwor m l epi dopt er a 3.78 0.58 9
Anticarsia gemmatalis ~' vel vet bean cat er pi l l ar l epi dopt er a 2.85 0.31 21
Heliothis cirescens a t obacco budwor m l epi dopt er a t.83 0.35 17
Helicot'erpa zea ~ cor n ear wor m l epi dopt er a 2.81 0.31 8
Galleria mellonella gr e a t e r wax mo t h l epi dopt er a 1.56 0. 14 8
Malacosoma americanum e a s t e r n t ent cat er pi l l ar l epi dopt er a 6.97 0. 90 11
~' Le pi dopt e r a n l arvae r e a r e d on artificial di et .
nat ural diets and l epi dopt er an species f eedi ng on arti-
ficial diets. Pr el i mi nar y dat a suggests t hat l epi dopt er an
species may possess hi gher DHAR activities when
f eedi ng on host plants. It is possible t hat i ncreased
levels of DHAR may be a means of pr ot ect i ng t he
f eedi ng herbi vore f r om superoxi de and H2 0 2 gener-
at ed by di et ary prooxi dant s. Many pl ant species used
as f ood sources by l epi dopt er an larvae cont ai n numer -
ous compounds t hat are capabl e of pr oduci ng superox-
ide and H2 0 2 in t he pr esence of oxygen. Qui nones,
di hydroxyphenol i c compounds, and several phot oact i -
vat ed compounds such as xant hot oxi n and har mi ne
have t he pot ent i al to pr oduce active oxygen species
under a vari et y of condi t i ons [9,10]. Exper i ment s with
H. zea have demons t r at ed up to a 10-fold i ncrease in
DHAR activity in mi dgut tissue f r om larvae f eedi ng on
nat ural host plants.
A partially puri fi ed enzyme pr epar at i on was ob-
t ai ned f r om a homogenat e of whol e H. zea larvae.
Tabl e II det ai l s t he part i al puri fi cat i on of DHAR from
t he H. zea homogenat e. Final puri fi cat i on by isoelec-
T ABL E II
Details of the partial purification of DHAR from H. zea laruae
Enz yme activity is r e por t e d as nmol as cor bat e f o r me d / mi n per mg
pr ot ei n.
Pur i f i cat i on s t ep % yi el d Enz yme activity Pur i f i cat i on
Cr ude h o mo g e n a t e 100 1.11 -
( NH4) 2SO 4 pr eci pi t at i on 88.5 18.3 16.5
Se pha de x G-75 43.9 34.6 31.2
Rot of or I EF 6.0 204.1 183.9
tric focusing, using a Rot of or I EF cell (Bi oRad, Rich-
mond, CA) was carri ed out at a const ant power of 12
Wat t s for 4 h at 4 o C, using Biolyte amphol yt es, pH 3
t o 9. Initial condi t i ons wer e 800 V and 15 mA; equilib-
ri um condi t i ons were 2000 V and 6.0 mA. 2 of t he 20
fract i ons col l ect ed wer e f ound t o have substantial activ-
ity (approx. 4.5 ml t ot al volume). The resul t i ng solution
was used in exper i ment s to det er mi ne t he K m and Vma x
for t he enzyme as well as cof act or specificity. Pr ot ei n
concent r at i on was det er mi ned following Br adf or d [8].
The ki net i c par amet er s wer e eval uat ed by varying
t he concent r at i on of t he subst rat e dehydr oascor bi c acid.
The Li neweaver - Bur ke plot of t he resul t i ng dat a gave a
Vma x of 1.60 (SE = 0.23) mmo l / mi n / mg pr ot ei n and a
K m of 0.27 (SE = 0.039) mM for dehydr oascor bi c acid.
The DHAR f r om H. z ea demonst r at ed a strict requi re-
ment f or GSH and was inactive with NADH, NADPH,
di t hi ot hrei t ol , or cysteine.
Thi s is t he first det ai l ed exami nat i on of t he occur-
r ence of DHAR among vari ous insect species. Previ-
ously i dent i fi ed and char act er i zed in several pl ant fam-
ilies, DHAR has only been occasionally not ed in hi gher
animals. The occur r ence of DHAR in all of t he insects
exami ned mi ght i ndi cat e t hat it is wi despr ead in In-
secta. An i nt erest i ng aspect of t he enzyme charac-
t er i zed in H. z ea is its similarity t o t he DHAR isolated
f r om plants [11]. The specific r equi r ement for GSH
and t he appar ent K m for dehydr oascor bi c acid exhib-
i t ed by t he enzyme paral l el t he pr oper t i es of t he DHAR
in plants. An enzyme of similar funct i on isolated f r om
rat tissues by Choi and Rose, however, demonst r at ed a
strict dependence upon NADPH [12].
The recycling of ascorbic acid may play a major role
in control of endogenous formation of active oxygen
species and in defense against dietary oxidative stresses.
Herbivorous insects, in particular, may benefit in view
of the large number of prooxidants found in their diet.
Many common plant secondary chemicals such as the
cinnamic acid derivatives, quinones, furanocoumarins,
flavonoids, epoxides, and organic peroxides, can act as
potent oxidizing agents. Not only are these compounds
potentially toxic to the insect, many of them may
decrease the nutritive value of ingested proteins
through oxidative modification. Plants also possess a
number of oxidative enzymes capable of generating
damaging chemicals, in particular, polyphenol oxidase
and 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 hydroperoxides
from free fatty acids. Many plants also contain the
enzyme ascorbate oxidase, which catalyses the conver-
sion of ascorbic acid to dehydroascorbic acid, and may
be capable of depriving the insect of ascorbic acid
directly [13]. Ingestion of these oxidative enzymes
should dictate an oxidative environment for the ac-
tively feeding herbivore, potentially depleting the
bioavailability of ascorbic acid.
Ascorbic acid has been implicated in many metabolic
processes in insects, as well as being an important
general antioxidant in tissues where exposure to en-
dogenous sources of oxidative stress is high [14,15]. It
may be particularly important for the control of H202
formation at low levels, because even low levels of
H20 2 may be capable of damaging biomolecules via
' Fenton type' reactions. Although specific enzymatic
mechanisms for eliminating these reactive products
exist (e.g., SOD, CAT, and GSTPX), tissue levels of
ascorbic acid may be sufficient for significant chemical
scavenging of superoxide and hydrogen peroxide at
metabolically active sites where levels of the necessary
enzymes may be low. Additionally, the inefficiency of
CAT at low H202 concentrations and the lack of a
Se-dependent GPOX suggests that DHAR may play a
crucial role in protecting insects against H202 toxicity.
Preliminary examination of the distribution of DHAR
in the tissues of H. zea shows measurable levels of the
enzyme to be present in the lumen, the midgut tissue,
the fat body, and the Malpighian tubules. The highest
activities were found in Malpighian tubules (101
nmol / mi n/ mg protein) and the midgut (45
nmol / mi n/ mg protein), respectively (unpublished
data). It is noteworthy that CAT activity appears to be
absent from the Malpighian tubules in H. zea, while
substantial levels of DHAR were found. The absence
of CAT activity in the Malpighian tubules of Trichoplu-
sia ni has been reported by Ahmad et al. (1991) [2]. If
237
CAT is found to be generally lacking in the Malpighian
tubules of other lepidoteran species as well, it may be
possible that DHAR performs a critical function in
mediating the removal of H202 in lepidopterans. It
has recently been reported that CAT is the sole means
by which insects, Drosophila melanogaster in particu-
lar, eliminate H202 [16]. Considering the crucial na-
ture of this task and CAT's poor ability to remove low
concentrations of H202, the absence of CAT in certain
tissues may suggest that other forces must be at work
to compensate for these factors.
The oxidative product of ascorbic acid, dehydro-
ascorbic acid, has been observed to damage cellular
membranes in leukocytes, erythrocytes, and vesicles,
leading to increased membrane permeability [17]. The
rapid reduction of dehydroascorbic acid by DHAR may
protect cellular membranes from damage. In conclu-
sion, DHAR may be an important component of a
system that not only maintains nutritive levels of ascor-
bic acid but also affords considerable protection from
both endogenous and exogenous sources of oxidative
stress. In the case of herbivorous insects, the suppres-
sion of active oxygen formation, especially H20 2, may
be greatly aided by DHAR and ascorbic acid.
Acknowledgments
We are grateful for the support of the Arkansas
Science and Technology Authority (ASTA 91-B-15 and
92-B-52), and the USDA/CSRS (89-34195-4378 and
92-34195-7162). Published with the approval of the
Director 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. Mueller
for providing insects for this project.
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