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Insect Biochemistry and Molecular Biology 39 (2009) 180–188

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Insect Biochemistry and Molecular Biology

journal homepage: www.elsevier .com/locate/ ibmb

Glutathione–ascorbic acid redox cycle and thioredoxin reductase activityin the digestive tract of Leptinotarsa decemlineata (Say)

Natraj Krishnan a,*, Dalibor Kodrık a,b, Barbara K1udkiewicz a,c, Frantisek Sehnal a,b

a Institute of Entomology, Biology Centre, Czech Academy of Sciences, Branisovska 31, �Ceske Budejovice 370 05, Czech Republicb Faculty of Science, University of South Bohemia, Branisovska 31, �Ceske Budejovice 370 05, Czech Republicc Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warszawa, Poland

a r t i c l e i n f o

Article history:Received 1 September 2008Received in revised form30 October 2008Accepted 4 November 2008

Keywords:Ascorbate/ascorbic acidAscorbate peroxidaseGlutathione–ascorbic acid redox cycleGlutathione-dependent dehydroascorbatereductaseLeptinotarsa decemlineataOxidative stressThioredoxin reductase

Abbreviations: AA, ascorbate/ascorbic acid; APOXColorado potato beetle; DNPH, 2,4-dinitrophenylhyd(2-nitrobenzoic acid); GDHAR, glutathione-dehydroreduced glutathione; GSSG, oxidised glutathione; MDnicotinamide adenine dinucleotide (reduced form); TBthiobarbituric acid reactive substances; TCA, trichlororeductase.

* Corresponding author. Present address: DepartmHall, Oregon State University, Corvallis 97331, OR, USþ1 541 737 0501.

E-mail address: krishnan@science.oregonstate.edu

0965-1748/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.ibmb.2008.11.001

a b s t r a c t

In view of the antioxidant role of glutathione (GSH) and ascorbic acid (AA), we have examined capacity ofthe GSH–AA redox cycle in relation to oxidative stress effects in the midgut of the Colorado potato beetleLeptinotarsa decemlineata. Adult gut harbors a higher capacity to cope with oxidative stress than thelarval gut. Protein carbonylation was pronounced in the wall of anterior larval midgut and was generallylower in the food digest than in the gut wall. Restriction of oxidative stress effects in anterior gut lumenmanifested by lipid peroxidation and protein carbonylation is interpreted as a mechanism favoringdigestion and absorption in the posterior midgut. Presence of high GSH in the posterior midgut and AAin both posterior and anterior midguts of adults points to higher utility of the GSH–AA redox systemin limiting oxidative stress to manageable levels. The presence, gene expression and activity of thio-redoxin reductase (TrxR) were demonstrated for the first time in L. decemlineata which was markedlyhigher in the anterior than in the posterior midgut in both stages. It is probably central to the mainte-nance of reduced GSH levels in the whole gut, despite a GSSG/2GSH redox potential tending towardsoxidizing ranging from �183.5 to �124.4 mV. Glutathione-dehydroascorbate reductase (GDHAR) activitywas markedly augmented in adult gut compared with larva, pointing to a more efficient conversion ofdehydroascorbate (DHA) to AA. Also, ascorbate peroxidase (APOX) activity was significantly elevated inall gut compartments of adult except the wall of posterior midgut. The results emphasize the potentialimportance and role of the GSH–AA redox cycle as a defense strategy against oxidative stress in the gut ofL. decemlineata.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Reactive oxygen species (ROS), such as singlet oxygen, super-oxide anion, hydroxyl radical and hydrogen peroxide, are elimi-nated by antioxidant enzymes and several redox systems (Cadenas,1989; Davies, 1995, 2000). It is also well accepted that the triadof glutathione, ascorbic acid, and vitamin E is central to theantioxidant defense in both mammals and insects (Meister, 1994;Barbehenn et al., 2003). Each of these compounds is a major ROS

, ascorbate peroxidase; CPB,razine; DTNB, 5,50-dithiobis-ascorbate reductase; GSH,

A, malondialdehyde; NADPH,A, thiobarbituric acid; TBARS,acetic acid; TrxR, thioredoxin

ent of Zoology, 3029 CordleyA. Tel.: þ1 541 737 5513; fax:

(N. Krishnan).

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reductant with actions distinct from ROS scavenging by superoxidedismutase (SOD) and catalase (CAT). The redox systems based onthiols and ascorbic acid are recognized as essential for the removalof ROS generated during oxidative processes within cells but theirparticipation in neutralizing radicals originating in the environ-ment and consumed in the food has been less examined. This lackof knowledge is particularly intriguing in case of some phytopha-gous insects that generate high amounts of ROS by oxidation ofphenols and similar allelochemicals that plants deploy in theirdefense against herbivores and pathogens (Appel and Schultz,1992; Appel, 1993, 1994). ROS contained in plants and generated bythe oxidation of allelochemicals are partly scavenged by certaindietary components such as ascorbate and carotenoids and elimi-nated by ingested plant enzymes. However, most of the radicals areprobably eliminated by mechanisms innate to insect gut.

In our previous study we demonstrated through direct assaysthat both larvae and adults of the Colorado potato beetle (CPB),Leptinotarsa decemlineata, harbor appreciable levels of ROS in thecontent of anterior midgut (Krishnan et al., 2007a). ROS titerdeclines in posterior midgut and is generally low in the gut wall.

N. Krishnan et al. / Insect Biochemistry and Molecular Biology 39 (2009) 180–188 181

These differences among midgut compartments, and also low ROScontent in adults compared to the larvae, are negatively correlatedwith the activities of SOD and CAT. Taken as a whole, our resultsdemonstrated that the maintenance of ROS gradients along the gutis dependent on the physicochemical conditions (pH, redox poten-tial, oxygen tension) and activity of antioxidant enzymes which helpcurb detrimental effects to digestion (Krishnan et al., 2007a).

Oxidative stress inflicts damage to lipids, proteins and nucleicacids when the rate of ROS generation exceeds capacity of theantioxidant systems (Halliwell and Gutteridge, 1989; Stadtman,1992). The peroxidation of lipids and the carbonylation of proteinsare typical products of excessive oxidation. The variety of lipids andthe random nature of ROS reactions can lead to many products,including malondialdehyde (MDA), which is primarily taken as anindicator of cell membrane damage (Janero, 1990). The content ofcarbonyl groups is a marker of protein oxidation (Chevion et al.,2000; Levine, 2002). Carbonyls are formed from the amino groupsin the side chains of lysine, proline, arginine, and histidine that areexposed to H2O2 or O2 in the presence of redox cycling cations suchas Fe2þ or Cu2þ resulting in Fenton reactions generating hydroxyland alkoxyl (or peroxyl) radicals (Halliwell and Gutteridge, 1989).Both the content of MDA and the level of carbonyls are frequentlystudied as indirect measures of oxidative stress as well as resultantcell damage (Halliwell and Whiteman, 2004) and in this way wealso interpret their occurrence in the gut wall of CPB. We demon-strate a high MDA titer in the leaf digest present in larval anteriormidgut, indicating that ROS interference with digestion includesperoxidation of dietary lipids. In the present study we examine thepossible role of the glutathione–ascorbic acid redox systems inmodulation/amelioration of these oxidative stress effects. In addi-tion, we also report on the presence, gene expression and activity ofthioredoxin reductase (TrxR) in place of glutathione reductase (GR),important in maintaining reduced GSH levels.

Fig. 1. Schematic representation of the maintenance of reduced GSH status in insect gut dutogether with the influence of antioxidant enzyme systems. During peroxide eliminationreductase (Trx–TrxR) system, the GSH redox cycle. The ascorbate cycle functions by the forthione-dehydroascorbate reductase (GSH-DHAR); AA in turn reacts with hydrogen peroxide(O2�) is dismutated by superoxide dismutase (SOD) to H2O2 which is converted to water

dependent on the availability of NADPH, of which the pentose phosphate pathway is a majoand the activity of glucose-6-phosphate dehydrogenase (G6PD). All assayed components of

Glutathione (g-glutamylcysteinylglycine, GSH) is the major lowmolecular weight thiol found in the cell cytosol and other aqueousphases of various living systems (Kosower and Kosower, 1978;Lomaestro and Malone, 1995). In the presence of ROS, GSH isoxidized to GSSG that can be recycled to GSH by an NADPHdependent thioredoxin reductase (TrxR) (Fig. 1). In general, TrxRsexhibit rather broad substrate specificity and in contrast to GR, donot participate in GSSG reduction (Gromer et al., 1998). However,Kanzok et al. (2001) found that the thioredoxin system substitutesfor GR in Drosophila and possibly it is the same for all insects. TheNADPH necessary to maintain sufficiently high GSH levels isgenerated primarily in the hexose monophosphate pathway. Weassume that GSH synthesis via g-glutamylcysteine synthetase andthe production of NADPH in the cells of insect gut wall are highenough not to limit the capacity of the GSH/GSSH system.

The GSH/GSSG ratio is a dynamic indicator of oxidative stresswith a high ratio indicating reducing conditions, while a low ratioindicates pro-oxidative conditions (Asensi et al., 1999). The GSH/GSSG shuttle is also linked to ROS scavenging by ascorbic acid (AA).When AA acts as a scavenger, DHA is formed; but if the ascorbate/dehydroascorbate (AA/DHA) ratio is kept high then the redoxpotential is kept reducing until the unstable DHA undergoeshydrolysis or can be reduced back to AA. GSH with a highly negativeredox potential functions as a reducing agent recycling AA from itsoxidised form DHA using the enzyme glutathione-dehy-droascorbate reductase (GDHAR). The interaction between GSH andAA relates directly to their roles in biological oxidations (Meister,1994). By reacting with activated oxygen more readily than anyother aqueous component, AA protects critical macromoleculesfrom oxidative damage. AA can scavenge ROS directly with andwithout enzyme catalysts, and also indirectly by recyclingtocopherol to its reduced form. For example, the spontaneous AAreaction with the hydroxyl radical is limited only by diffusion, while

ring oxidative challenge. Coupling of GSH–AA redox cycle to NADPH supply is shown, the regeneration of GSH from GSSG is maintained by the thioredoxin–thioredoxinmation of ascorbic acid (AA) from dehydroascorbate (DHA) by reduction using gluta-(H2O2) to produce DHA in presence of ascorbate peroxidase (APOX). Superoxide anionand oxygen by catalase (CAT). The continued function of the redox cycle activity isr source. The cellular rate of NADPH supply is regulated by glucose flux in the pathwaythe system are marked in bold abbreviations.

Fig. 2. A typical chromatogram of RP-HPLC of MDA-(TBA)2 adduct from sample. Fordetails see Section 2.

N. Krishnan et al. / Insect Biochemistry and Molecular Biology 39 (2009) 180–188182

hydrogen peroxide scavenging by AA is catalyzed by ascorbateperoxidase (APOX) and depends on this enzyme capacity (Clar-avon-Mathews et al., 1997; Krishnan and Kodrık, 2006).

The main goal of the present work was to evaluate the efficacy ofthe GSH–AA redox cycle in larval and adult guts of CPB by a)measuring the intracellular levels and ratios of GSH:GSSG andAA:DHA as an indirect measure of the ROS turnover and oxidativestress and b) measuring activities of APOX, GDHAR, and TrxR whichare important for the maintenance of such redox cycling. Thehigher the turnover, the more important is the role of this system inthe management of oxidative stress.

2. Materials and methods

2.1. Sample preparation and chemicals

CPB and their food plant, potato cultivar Desiree, were kept ina greenhouse at 25 �C under long day conditions (16 h light: 8 hdarkness). Potato leaves and four compartments of CPB midgut(see below) were analyzed. Potato leaves from healthy, intactplants and from plants exposed to CPB attack for 24 h werechosen. Midguts were dissected from CPB larvae 3 days after thelast larval ecdysis and from the non-diapausing adults 6 daysafter emergence. The insects were immobilized by chilling at�20 �C for 10 min. The midgut was dissected on an ice plate, bycutting across the oesophagus and at the level of the posteriorpylorus, cleaned of fat body and Malpighian tubules, blottedbriefly on a filter paper, and separated into anterior and posteriorhalves by cutting at the first narrowing region after the initialhighly filled anterior region in about one quarter of its length(Krishnan et al., 2007a). Food digest present in the anterior andposterior midgut regions (anterior and posterior contents, AC andPC, respectively), and the anterior and posterior midgut walls(AW and PW, respectively), were collected separately in corre-sponding buffers purged with N2 gas. The samples were sonicatedor homogenized and processed as described in individual assays.

The content of soluble protein was measured in aliquots afterprecipitation with 5% TCA using the Coomassie Blue binding(Bradford, 1976) and reliability of this procedure was verified withturbidimetric protein estimation (Vera, 1988). Bovine serumalbumin was used as standard. All chemicals used in the assayswere purchased from Sigma Chemical Company, St. Louis, MO, USA.Spectrophotometric measurements were taken in a Spectramax340 PC (Molecular Devices, Sunnyvale, CA, USA) microtitre platereader unless indicated otherwise.

2.2. Lipid peroxidation

The amount of malondialdehyde (MDA) was quantified by theadduct formed with thiobarbituric acid (TBA) and used as a markerof lipid peroxidation. Samples were sonicated in phosphoric acid(0.2%), heated in presence of TBA solution (0.6% TBA stabilized with1 mM butylated hydroxytoluene in 50 mM NaOH) to 95 �C for30 min, and after cooling the TBA–MDA complexes were extractedby partitioning by 1-butanol (Uchiyama and Mihara, 1978). Thebutanol phase was collected and the solvent removed in a vacuumevaporator. The residues were reconstituted in elution buffer (35%methanol in 50 mM KH2PO4–KOH, pH 7.0) and applied on the RP-HPLC C18 column (Purospher RP-18, Merck, Darmstadt, Germany)using Waters� HPLC system to detect the MDA-(TBA)2 adduct. Thesample was run under isocratic conditions at a flow rate of 0.5 ml/min and fluorescence detection at lEx 515 nm and lEm 553 nm.Material from the peak (RT¼ 27.93, Fig. 2) was quantified againsta calibration curve prepared with MDA (1,1,3,3 tetramethox-ypropane) standard. The recovery varied between 92 and 96%when some of the samples were spiked with known concentrations

of MDA to assess loss during the extraction process. The resultswere expressed as MDA equivalents in pmol mg�1 protein.

2.3. Protein carbonyls

Carbonyls were quantified after their reaction with 2,4-dinitrophenylhydrazine (DNPH) (Levine et al., 1990) with somemodifications (Krishnan et al., 2007b). Carbonyls were quantifiedspectrophotometrically in a microtitre plate reader at 370 nm.Results were expressed as nmol mg�1 protein using an extinctioncoefficient of 22,000 M�1 cm�1. Bovine serum albumin (BSA)standard curve was used to quantify protein concentrations inguanidine solutions (measured at 280 nm). Protein carbonyl valueswere corrected for interfering substances by subtracting theA370 mg�1 protein measured without DNPH (controls).

2.4. Glutathione (GSH) and glutathione disulfide (GSSG)

GSH and its oxidized product GSSG were measured togetherspectrophotometrically (Griffith, 1980) with some modifications(for details see Krishnan et al., 2007b). The amounts of reducedglutathione (GSH) were calculated by subtracting the content ofderivatized glutathione (GSSG) from that of the total glutathione(GSHþGSSG).

Redox potential (Eh) of the GSH/GSSG couple was calculatedusing the Nernst equation:

Ehðin mVÞ ¼ Eo þ 30 log�½GSSG�=½GSH�2

�

where GSH and GSSG are molar concentrations. Since many redoxreactions are pH dependent Eh (in mV) were calculated using Eo

values from �151 mV to �210 mV, corresponding to the pH valuesof CPB midgut (pH 5.5–6.5; Felton et al., 1992) following themethod of Schafer and Buettner, 2001:

Eo ¼ �240þ ð5:5� 7:0Þð�59:1ÞmV ¼ �151:35 mV (1)

Eo ¼ �240þ ð6:5� 7:0Þð�59:1ÞmV ¼ �210:45 mV (2)

These values are obtained assuming a value of �240 mV for pH7.0 (Rost and Rapoport, 1964; Jones, 2002) with an adjustment of�5.9 mV/0.1 increase in pH.

2.5. Ascorbic acid (AA) and dehydroascorbate (DHA)

A modified spectrophotometric method (Badrakhan et al., 2004)for microtitre plate was employed to quantify AA and DHA. Thesample extracts were processed step by step by adding freshly

N. Krishnan et al. / Insect Biochemistry and Molecular Biology 39 (2009) 180–188 183

prepared desferrioxamine mesylate (15.4 mM), 15% 1,4 dithio-DL-threitol (DTT) (blank), deionised H2O (sample), methanol andphosphate citrate buffer (400 mM NaH2PO4, 125 mM citric acid-1-hydrate, pH 7.7). Each combination was processed in triplicate. Themicrotitre plate incubated at 37 �C for 30 min, and absorbancerecorded at 346 nm. DHA content was quantified against a standardcalibration curve of 0–400 mM DHA and expressed as nmol mg�1

protein.The content of AA was measured after its conversion to DHA. To

this end, ascorbate oxidase from Cucurbita sp. (76.5 U/ml dissolvedin 100 mM N2 purged KH2PO4/K2HPO4 (pH 6.0) buffer:glycerol, 1:1,v/v) was added to the sample wells (incubated 5 min at RT) and anequal volume of 100 mM potassium phosphate buffer:glycerol, 1:1to the blank wells prior to the treatment with desferrioxaminemesylate. The protocol was otherwise identical to that used in theDHA assay. AA content, expressed as nmol mg�1 protein wasquantified against a standard calibration curve of 0–400 mMascorbic acid dissolved in deionised water. In this process the blankwells measured the DHA present in the samples, while the samplewells measured the AA converted to DHA under the influence ofascorbate oxidase as well as the DHA present, hence, readings in thelatter, i.e. sample wells were corrected by appropriately subtractingthe readings in the blanks and then calculating for unknown valuesof AA against standard curve.

2.6. Western blots for TrxR

Western blots were performed to check the presence of TrxR inthe gut tissue extracts of larvae and adults. Briefly, gut tissueextracts were analyzed electrophoretically on a 10% polyacrylamidegel (SDS-PAGE) (Laemmli, 1970). Positive controls were HeLa wholecell lysates (ab29545, Abcam Inc. Cambridge, UK). Proteins wereblotted (Towbin et al., 1979) onto PVDF membrane that was incu-bated overnight with polyclonal TrxR1 or TrxR2 antibodies (1:2000,LabFrontier, Seoul, Korea). Antigen–antibody complexes wereanalyzed with secondary antibody (SwAR/Px anti-rabbit, 1:20,000)and visualized by Supersignal West Pico chemiluminescentsubstrate (Pierce) using a Fuji LAS3000 imaging system.

2.7. qRT-PCR for TrxR mRNA levels

Total RNA was extracted from guts of larvae and adults sepa-rately using Trizol (Invitrogen, Carlsbad, CA, USA). RNA was purifiedusing RNeasy kit (Qiagen, USA) with Qiagen RNase-Free DNasetreatment to remove genomic DNA contamination. First-strandcDNA was synthesized using Superscript III reverse transcriptase(Invitrogen, Carlsbad, CA, USA) as per manufacturer instructions.Briefly, aliquots of w5 mg RNA were added to 20 ml reactionmixtures containing 200 U Superscript III; 1� first-strand buffer;0.01 M DTT; 500 mM dNTPs and 20 pmol adapter – poly (T) primerdesigned in our lab (50-TGA GCA AGT TCA GCC TGG TTA TTT TTT TTTTTT TTT TTT-30) named TRIKANT. Two ml of first-strand solution wasused for amplification of the cDNA in larvae and adults usingspecific primers for TrxR1 and 2 using a BioRad iCycler. Primerswere designed using the Drosophila sequence with some modifi-cations. qPCR was conducted using SYBR green qPCR mastermix(BioRad) with the following primers for TrxR1: Forward: 50-TGCAGT CCG TAC AGA ACC ACA TCA-30 and Reverse: 50-ACT CCA CTTTCT TGT CGC GCA GAT-30, TrxR2: Forward: 50-AAG TTG GCA GTGCCG AGT CCT AAA-30 and Reverse: 50-TGT GTG TGG AGG ATC GCAGAA ACT-30. Gene rp49 was used as an endogenous control. Reac-tions were performed on ABI Prism 7300 and data analyzed usingthe 2-DDCt method for fold changes in mRNA expression levels.

2.8. Enzymes regenerating the glutathione–ascorbate redox system

All major enzymes that restore the ROS scavenging potential ofthe GSH–AA system (Fig. 1) were measured. DTNB (disulfidesubstrate) reduction assays to 5-thio-2, nitrobenzoic acid (TNB)with NADPH, which produces a strong yellow color (Abs 412 nm)was used for assaying the activity of TrxR (EC 1.6.4.5) (Holmgrenand Bjornstedt, 1995). The reduction is directly proportional to theTrxR activity in the sample (TrxRþDTNBþNADPHþHþ/

2TNBþNADPþ). Measurement of TrxR activity in the absence andpresence of aurothioglucose, an inhibitor of TrxR (Smith et al.,1999), allowed for the correction of non-thioredoxin reductase-independent DTNB reduction. The samples were sonicated (1:5 w/v) in ice cold N2 purged buffer (50 mM potassium phosphate with1 mM EDTA, pH 6.4) and supernatants (10,000 g 15 min at 4 �C)immediately transferred to microtiter wells. Since this is a generalassay for reductases/peroxidases we ran positive control wellscontaining assay buffer, DTNB (3 mM) and TrxR (1 U/ml). In somewells GSSG (0.3 mM) was also added as disulfide substrate to testfor TrxR dependent reduction to GSH [NADPHþ TrxS2þHþ/

NADPþþ Trx(SH)2 (a), Trx(SH)2þGSSG / TrxS2þ 2GSH (b)] Thesample wells contained sample supernatant instead of TrxR.NADPH (0.6 mM in assay buffer) was added to all wells and increaseof absorbance at 412 nm was followed for 5 min at 1 min intervals.TrxR activity was expressed as nmol min�1 mg�1 protein.

Activity of GDHAR (EC 1.8.5.1) was measured in sample extractsin 50 mM phosphate buffer (pH 6.4) added to a reaction mixturecomprising 0.12 mM disodium EDTA, 2.5 mM GSH, 0.057 mM DHAin identical buffer (Ishikawa et al., 1998). The change of absorbanceat 265 nm, which reflects formation of AA from DHA, was measuredin Shimadzu 1601 (UV–VIS) spectrophotometer (Shimadzu Corpo-ration, Kyoto, Japan) for 3 min at 30 s intervals. Molar extinctioncoefficient of 14,700 M�1 cm�1 was used to calculate AA amounts.Non-enzymatic DHA reduction determined in boiled samples wassubtracted and the GDHAR activity was expressed as mmol AAformed min�1 mg�1 protein at 25 �C.

To measure APOX (EC 1.11.1.11) activity, sample in 50 mMphosphate buffer (pH 6.4) containing 0.5 mM AA was added toidentical buffer with 0.3% H2O2. Boiled samples served as controls.The rate of change in absorbance (decrease) at 290 nm wasmeasured for 10 min in a Shimadzu 1601 spectrophotometer.Enzyme activity was expressed as nmol ascorbate oxidized min-�1 mg�1 protein using a molar extinction coefficient of2.8 mM�1 cm�1 (Asada, 1984).

2.9. Statistical analysis

Tissues and samples were pooled from 5 to 8 individuals foreach assay and replicated 3–4 times for microtitre plate assays. Dataare expressed as mean� SEM. Student’s t-test (Figs. 3 and 4, andTable 1) and in some cases Welch’s unpaired t-test (Fig. 7 andTable 2) was used for statistical testing of significance of variousparameters between larva and adult. Graphs were generated usingGraphPad Prism 5 (GraphPad software version 5.01, San Diego, CA,USA).

3. Results

3.1. Oxidative changes in ingested food and gut tissues

That CPB suffers from oxidative stress is evidenced by thepresence of lipid peroxidation products and protein carbonyls inthe gut. Extensive lipid peroxidation was detected in larval AC(Fig. 3a) and maximal carbonyl content in larval AW (Fig. 3b). Thelevel of MDA, which was measured as a marker of lipid perox-idation (for details see Sections 2 and 2.2), was nearly 3 times

Fig. 3. Thiobarbituric acid reactive substances (TBARS) content (top a) and proteincarbonyl content (bottom b) in different gut compartments of last instar larva andadult of L. decemlineata. Values have been expressed as mean� SEM (n¼ 5).(AC¼ anterior midgut content, AW¼ anterior midgut wall, PC¼ posterior midgutcontents, PW¼ posterior midgut wall.) Student’s t-test for significance was used tocompare between larva and adult gut compartments: *p< 0.0001, Jp< 0.03.

Fig. 4. Correlation between TBARS and GSSG contents in the gut of L. decemlineata. Asignificantly positive (p< 0.0001, r2¼ 0.886) correlation was found between the TBARScontent and the GSSG content in all the gut compartments of both larvae and adults.The dotted line represents 95% confidence limits; n¼ 60 individuals, 30 each of adultand larvae.

Table 1Total glutathione (GSHþGSSG) and reduced glutathione (GSH) contents, GSH/GSSG raticontents (AC), anterior midgut wall (AW), posterior midgut contents (PC) and posterior mias mean� SEM (n¼ 6). Tests for significance (Student’s t-test) was conducted between l

Parameter Last instar larva

AC AW PC PW

GSHþGSSG (nmol mg�1 protein) 1.80*� 0.02 0.30� 0.01 0.40� 0.01 0.1GSH (nmol mg�1 protein) 0.55y � 0.10 0.20z � 0.01 0.20� 0.02 0.0GSH/GSSG ratio 0.4 2.0 1.0 1.0AAþDHA (nmol mg�1 protein) 15.46� 1.28 14.95� 0.58 194.85� 2.60 194AA (nmol mg�1 protein) 9.38� 0.71 13.82� 0.22 34.92� 1.34 99DHA (nmol mg�1 protein) 6.07� 0.57 1.13� 0.35 159.94*� 1.24 95AA/DHA ratio 1.5 12.5 0.2 1.0

N. Krishnan et al. / Insect Biochemistry and Molecular Biology 39 (2009) 180–188184

higher in larval AC than in larval PC (Fig. 3a). It was generally lowerin adult midgut. Values established in adult AC, AW, and PC ofadults were significantly (p< 0.0001) lower than in the corre-sponding compartments of larvae. In both larvae and adults, higherlevels of MDA were found in the food digest than in the wall ofmidgut. In analysis of pooled data, the titer of MDA exhibiteda highly significant positive correlation (r2¼ 0.886, p< 0.0001)with the GSSG contents (Fig. 4).

In most midgut compartments, carbonyls were also lower in theadult than in the larva (Fig. 3b). The difference was significant atp< 0.03 probability level in AW and at p< 0.0001 in PC and PW. InAC, however, the value was marginally and insignificantly higher inadults than in the larvae. The extreme amount of carbonyls in larvalAW is probably related to the low levels of AA and GSH (Table 1) andlow activities of GDHAR and APOX (Table 2) in this compartment.

3.2. Glutathione–ascorbic acid redox cycle

Total glutathione (GSHþGSSG) was several times higher inlarval AC than in any other examined gut compartment (Table 1).However, about 70% of total glutathione pool comprised of GSSGin AC and 33% in AW of the larva (Table 1). In adult AC and AWabout 56% of total glutathione pool was comprised of GSSG. Thecontent of reduced GSH is generally higher in the gut lumen thanin the gut wall, suggesting that this system may be moreimportant for ROS management in the food digest than in the guttissues. This capacity is significantly (p< 0.0001) higher in larvalthan in adult AC and slightly higher in adult than in larval PC.The level of reduced glutathione (GSH) in posterior midgut wasalso higher in adult compared to larva (p< 0.0001). GSH levels inthe anterior midgut of adults are significantly lower (p< 0.03 forAC and p< 0.02 for AW). The GSH/GSSG ratio was elevated in thegut contents (anterior and posterior) of adult insect compared tolarva whereas the tissue GSH/GSSG ratio was higher in case oflarva (Table 1). The differences in GSH are generally smaller thanin the GSHþGSSG content or ratio, indicating that lowercapacity is partly compensated for by higher turnover betweenthe oxidized and reduced states. The redox potential of the GSH/GSSG redox couple was uniformly oxidizing with an average Eh

value of �177� 9 mV (at pH 6.4) irrespective of gut region orstage of the insect.

In contrast to GSH, which is synthesized by insect cells, ascor-bate must be obtained with the diet. AA and DHA contents inpotato leaves amounted to 1.5� 0.21 and 5.2� 0.69 nmol mg�1

protein, respectively. Much higher titers in midgut compartments(Table 1) reveal high efficiency of ascorbate sequestration. The sumof AA and DHA was consistently higher in the posterior than in theanterior compartments in both stages, while it was considerablyhigher in all compartments of adults than in larvae. The AA andDHA amounts combined made up about 15 nmol mg�1 protein inlarval AC and AW, 195 in larval PC and PW, 80–90 in adult AC andAW, 209 in adult PC and 600 in adult PW. Similar levels found in

o, ascorbic acid (AA), dehydroascorbate (DHA), AA/DHA ratio in the anterior midgutdgut wall (PW) of the last instar larva and adult of L. decemlineata. Data are presentedarva and adult: *p< 0.0001, Jp< 0.005, yp< 0.03, zp< 0.02.

Adult

AC AW PC PW

0� 0.001 0.25� 0.001 0.25� 0.02 0.50*� 0.01 0.22*� 0.0045� 0.002 0.11� 0.01 0.11� 0.01 0.30J� 0.02 0.10*� 0.002

0.7 0.7 1.5 0.8.99� 3.18 91.54� 34.92 81.33� 16.51 208.74� 16.93 675.30� 37.21

.98� 1.27 82.35*� 29.89 60.06*� 11.26 170.14*� 15.90 490.59*� 28.39

.00� 1.91 9.18� 5.03 21.26*� 5.24 38.59� 1.04 184.71*� 8.818.9 2.8 4.4 2.6

Table 2Activity of enzymes (TrxR¼ thioredoxin reductase; GDHAR¼ glutathione-dehydroascorbate reductase; APOX¼ ascorbate peroxidase) associated with the GSH–AA redox cyclein the anterior midgut contents (AC), anterior midgut wall (AW), posterior midgut contents (PC) and posterior midgut wall (PW) of the last instar larva and adult of L.decemlineata. Data have been expressed as mean� SEM (n¼ 6). In all cases Welch’s unpaired t-test was used to test for significance between larva and adult for variousenzymes: *p< 0.0001, Jp< 0.02, zp< 0.03, yp< 0.01.

Enzyme activity Last instar larva Adult

AC AW PC PW AC AW PC PW

TrxR (nmol min�1 mg�1 protein) 20.3*� 0.06 16.0� 0.04 9.8� 0.2 4.8� 0.04 17.3� 0.03 15.5� 0.2 12.7� 0.14 5.9� 0.05GDHAR (mmol min�1 mg�1 protein) 0.8� 0.02 0.1� 0.01 2.4� 0.2 1.5� 0.3 4.6J� 1.2 1.4� 0.3 8.3*� 0.4 3.6J� 0.2APOX (nmol min�1 mg�1 protein) 0.6� 0.01 0.05� 0.01 2.2� 0.2 3.1y � 0.04 2.6*� 0.1 2.1z � 0.7 2.7J� 0.04 2.7� 0.2

N. Krishnan et al. / Insect Biochemistry and Molecular Biology 39 (2009) 180–188 185

the content and wall of most compartments indicate existence ofa balance in AA distribution between gut cells and gut lumen.However, the AA and DHA ratios were markedly different. Inlarvae, the AA/DHA system was 5–8 times more oxidized in thelumen than in the wall; only 9.4 nmol AA mg�1 protein wasavailable for ROS scavenging in AC but increased to 100 nmol mg�1

protein in PW. In adult midgut, the AA/DHA ratio was higher in thelumen than the gut wall but the total amount of available AA washigh in all compartments and particularly in PW.

3.3. Presence and gene expression of TrxR in larval and adultdigestive tract

Western blots revealed presence of TrxR1 (w58 kDa) in guthomogenates of larvae and adults of CPB when compared withHeLa cell lysates used as positive controls with antibody directedagainst cytosolic TrxR1 (Fig. 5). However, the blots for the presenceof mitochondrial TrxR2 were not positive, possibly because of lackof specificity to mammalian TrxR2 antibodies used in this study(data not shown). However, PCR using specific primers did revealboth TrxR1 and TrxR2 in both larvae and adults (Fig. 6).

Relative mRNA expression (qRT-PCR) levels of TrxR1 in larval gutwere 1.4-fold higher (p< 0.05) than in adult gut. However, therewas no significant difference in TrxR2 expression levels in larvaland adult guts (Fig. 7).

3.4. Enzymes associated with the glutathione–ascorbic acidredox cycle

TrxR (cumulative) activity was higher in anterior midgut ofboth larvae and adults and declined in statistically significantfashion in the sequence AC, AW, PC, and PW in both stages(Table 2). The only difference between larva and adult was foundin AC that contained significantly (p< 0.0001) higher activity inlarvae. Values found in other midgut compartments of larvae and

Fig. 5. Western blots of TrxR1. Presence of TrxR1 (w58 kDa) in both larval and adultguts was demonstrated using specific TrxR1 antibody. Positive controls were HeLa celllysates. For details see Section 2.

adults were alike. Generally high TrxR activity suggests that thisenzyme may be crucial for GSH/GSSG cycling, particularly inanterior midgut.

GDHAR activity was generally higher in the adult than in thelarval midgut but showed in both stages a similar distributionpattern among the examined compartment. It was higher in theposterior than in the anterior midgut sections and in both sectionswas elevated in the lumen compared to the wall. Activities inadults were significantly higher than in larvae in AC (p< 0.02), PC(p< 0.0001) and PW (p< 0.02); difference in AW was insignifi-cant. Lower activities in larvae are consistent with lower AAcontent (Table 1), in particular in anterior midgut. The ratio of AAcontent to the GDHAR activity may be taken as an indication to theimportance of this enzyme for DHA reduction. Coupled with lowGDHAR activity (and also minimal APOX activity) in AW, most ofascorbate in this gut compartment occurred in reduced form(Table 1).

Low APOX activity in larval anterior midgut (Table 2) is consis-tent with low AA levels (Table 1). Anterior midgut of adults con-tained significantly higher APOX activities (p< 0.01) and higher AAlevels. There were relatively small differences in APOX activities inlarval PC and PW and adult AC, AW, PC, and PW. This contrastedwith high differences in the AA and DHA concentration.

Fig. 6. cDNA from both larval and adult guts was amplified using specific primers forTrxR1 and TrxR2 and run on 2.4% Agarose gel using SYTO 60 red fluorescent nucleicacid stain (Invitrogen, Carlsbad, CA, USA). BioRad 100 bp PCR EZ ruler was run as DNAstandard. Distinct single amplified bands revealed the presence of both TrxR1 andTrxR2 in larval and adult guts.

Fig. 7. Relative mRNA levels of TrxR1 (a) and TrxR2 (b) established by qRT-PCR. TrxR1level was significantly higher (p< 0.05) in the larval than the adult gut (a); nosignificant difference between larva and adult was found in the contents of TrxR2mRNA.

N. Krishnan et al. / Insect Biochemistry and Molecular Biology 39 (2009) 180–188186

4. Discussion

Our previous study clearly demonstrated presence of super-oxide anion and general peroxides in CPB midgut (Krishnan et al.,2007a). The level of ROS was highest in AC and declined in thesequence AW, PC, and PW, being in all compartments about 4 timeshigher in larva than in the adult. This difference in ROS contentbetween the two stages seemed to be at least in part controlled byphysicochemical conditions existing along the gut as well as anti-oxidant enzymes SOD and CAT that were more active in adult thanin the larval midgut. High ROS content in the AC of larvae (Krishnanet al., 2007a) is apparently responsible for the high level of lipidperoxidation in this gut region.

A strongly positive correlation (r2¼ 0.886) between lipid per-oxidation and oxidized GSH level was found in all midgutcompartments of both larvae and adults in the present work. Thatperoxidation occurs upon leaf ingestion was confirmed bymeasurements of the MDA content in potato leaves. Leaves ofplants protected from CPB contained 13.8� 1.5 pmol and leavesdamaged by CPB feeding 24.1�3.3 pmol MDA mg�1 protein(Krishnan et al., unpublished data). It is reasonable to assume thatthe plants response to herbivory which also involves the generationof oxygen radicals could have contributed significantly to thehigher levels of lipid peroxides in attacked leaf tissues (Bi andFelton, 1995; Maffei et al., 2006). However, this would still notexplain the relatively high levels of lipid peroxidation productsobserved in both larva and adult guts. It is possible that oxidizingconditions in anterior midgut of this insect promote lipid perox-idation. Photosynthesizing plant tissues contain various glycosyl

glycerides (mostly mono- and digalactosylglycerides), phospho-lipids, and waxes (Harwood, 1980). Digestion of these lipids ininsect gut is poorly understood (Turunen and Crailsheim, 1996). It ispossible that their peroxidation and generation of MDA fromglycerol, fatty acids, and possibly also sugars could facilitate/inhibitlipid utilization. We do not understand, however, why this processis so active in larval AC. In the gut wall, ROS can oxidize poly-unsaturated fatty acids in the cell membranes and thereby impairtheir function (Richter, 1987). Another factor which may influenceROS generation is the different feeding strategies adopted by larvaeand adults of CPB. The foliage of host plant (potato) is consumed byadults and larvae with the larva causing the greatest plant injury(Casagrande, 1987) which may result in more ROS from bothexogenous and endogenous sources. Rates of digestion would alsobe an important issue in this process (Krishnan et al., 2007a).

Carbonyl formation caused by protein oxidation was maximal inthe AW of both larvae and adults but distinctly lower in the latterstage. In posterior midgut of larvae and adults it was also higher inthe wall than in the content (Fig. 3b). Possible damage to the gutcells and their secretions can be conjectured from the ROS action inother systems. A number of studies demonstrated that ROS attackmay alter protein conformation and hydrophobicity, therebyaffecting protein function and stability and potentially endangeringthe whole cells (Cheeseman and Slater, 1993; Dean et al., 1991;Winrow et al., 1993). It has also been reported that MDA adductswith proteins, which routine assays detected as protein carbonyls,render the protein less susceptible to proteolysis (Burcham andKuhan, 1997).

The results of the present study indicate that a lower capacity ofthe GSH–AA system may also contribute to higher ROS titer in thelarval gut. GSH content in the AC and AW compartments is higher inlarvae than in adults but the reducing power of combined GSH andAA content is much higher in the adult gut. Enzymes recoveringGSH and AA from their oxidized forms are in most cases also higherin adult than in the larval gut. Only TrxR in AC and APOX in PWexhibited lower activities in adults than in larvae. Differences in theROS husbandry between larva and adult suggest that these stagesdiffer in the initial steps of digestion. In neither stage does ROS titerin the gut lumen copy that in the gut wall. The lumen harbors ROS,antioxidants, and antioxidant enzymes obtained with the plantfood and apparently larger amounts of these components could beliberated by apocrine secretion as has been documented for otherenzymes from the insect gut cells (Cristofoletti et al., 2001).

The reducing functions of GSH and AA are to some extentcomplementary and both components must be present at a certainminimal level for proper functioning of the GSH–AA redox cycle(Meister, 1994). GSH with its potent electron-donating capacity(redox potential of the GSH/GSSG ‘‘redox couple’’, E0o ¼ �0:33 V;Lewin, 1976) can serve as an antioxidant and also a cofactor forenzymatic reactions that require readily available electron pairs(Kehrer and Lund, 1994). The GSH/GSSG redox couple is uniformlyoxidizing in all compartments of both larval and adult midguts withan average Eh value �177� 9 mV (at pH 6.4) which falls within thelimits of redox potentials (�200 to þ240 mV) reported for insectswith midgut pH ranging from 6 to 11.8 (Johnson and Felton, 1996).The GSH/GSSG system in CPB midgut obviously performs its func-tion in the GSH–AA cycling but the prevalence of AA over GSHamounts indicate that the latter could be crucial for ROS scav-enging. Central role of AA in ROS management was also demon-strated for the gut of caterpillars (Timmerman et al., 1999;Barbehenn et al., 2001). The presence and activity of TrxR seems tobe pivotal to GSH restoration from GSSG in CPB midgut. High TrxRactivity, which more or less reflects variations in the GSH/GSSGcontents in different midgut compartments, indicates a high rate ofGSSG/GSH turnover. Apparently lower activities of GDHAR and APOXsuggest that the rate of AA reduction by ROS is relatively low,

N. Krishnan et al. / Insect Biochemistry and Molecular Biology 39 (2009) 180–188 187

especially in the anterior midgut. However, regulation of the redoxbalance is a complex process in which additional factors, forexample the cysteine/cystine pair, are involved.

Relatively higher GSH content in the lumen than the wall ofmidgut and the opposite distribution of AA are surprising. GSH canbe generated by insect cells and its content was therefore expectedto rise in cells endangered by ROS, while AA must be derived fromthe diet and the insects can control only its uptake and turnoverbetween the oxidized and reduced forms. The content of AA inleaf tissue was 1.5� 0.21 mmol mg�1 protein while DHA was5.2� 0.69 mmol mg�1 protein (Krishnan et al., unpublished data).Hence contribution of high dietary levels of AA and their uptake bycells is a plausible explanation. AA can be stabilized in vivo by thiolsand especially by GSH, and in general, GSH deficiency should leadto deficiency in AA levels (Meister, 1994) except in cases wheredietary sources are high when AA recycling becomes less impor-tant. Interpretation of these and other data is complicated by thefact that primary role of GSH in the gut lumen may also be otherthan ROS scavenging.

In cells possessing glutathione-S-transferase, GSH can formconjugates with various electrophilic xenobiotics (Monks and Lau,1994). The process has not been demonstrated in insect gut lumenbut extrusion of the large volumes of cytoplasm, which is typical forthe apocrine secretion of insect midgut (Cristofoletti et al., 2001),does not exclude it. As a major transporter of sulfhydryl groups,midgut GSH may facilitate movement and absorption of thedegradation products of dietary sulfolipids. Finally, it may alsoprovide a mechanism of amino acid absorption by the microvilli ofmidgut cells. In some insects, amino acids are taken up throughsymport channels driven by alkali ions but this mechanism couldnot be detected in the CPB gut (Reuveni et al., 1993). Since GSH isthe major intracellular non-protein thiol defense against freeradicals, its metabolism is of prime importance. The process shouldalso involve a mechanism by which extracellular GSH is able to betransported to intracellular environment to help combat stress incells. However, there is little or no cellular uptake of intact gluta-thione. This opens way to speculation on a possible role of transportassociated with g-glutamyl transpeptidase (GGT, EC 2.3.2.2), as it isknown in mammalian kidney and liver. GSH in the extracellularenvironment is metabolized by GGT, which cleaves off the g-glu-tamyl group, to cysteinylglycine that is hydrolyzed by a membranedipeptidase. The released cysteine can then be transported into thecell and used as a substrate for de novo synthesis of GSH. This g-glutamyl cycle is thought to maintain cellular GSH levels. The GGTenzyme activity was significantly higher in adult midgut comparedto larvae as also was the case with glutathione-S-transferase (GST,EC 2.5.1.18) (Krishnan et al., unpublished data). This indicatesa more dynamic turnover of GSH in adults than in the larvae.

The augmentation of cellular antioxidant systems like GSH andparticularly AA in adult midgut is thus an adaptation for reducingoxidative stress. It would also not be unexpected to find thatsuperoxide and superoxide dismutase are of significance in GSH–AA interrelationships. Although superoxide seems not to bedirectly toxic, it can lead to iron dependent hydroxyl radicalformation. Generation of radicals in gut tissues could also be dueto a unique epithelial oxidative burst as an immune response forefficient microbial control (Ha et al., 2005). In conclusion, thisstudy proves unequivocally the importance of thiols and theirredox cycling in the modulation of oxidative stress induced effectsin insect gut.

Acknowledgements

This work was supported by the Grant No. 522/07/788 (DK) andNo. 522/06/1591 (FS) from the Grant Agency of the Czech Republic

and Project No. Z500 70508 of the Institute of Entomology(Academy of Sciences of the Czech Republic).

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