Current Eye Research, 34, 274–281, 2009Copyright C© Informa Healthcare USA, Inc.ISSN: 0271-3683 print / 1460-2202 onlineDOI: 10.1080/02713680902725954

Elevated Expression of Indoleamine2,3-Dioxygenase (IDO) and Accumulation

of Kynurenic Acid in the Pathogenesisof STZ-Induced Diabetic Cataract

in Wistar RatsVangipurapu Rajani Kanth

Department of Zoology, OsmaniaUniversity, Hyderabad, India

Katikala LavanyaCentre for Cellular and Molecular

Biology (CCMB), Pisci Culture Lab, andDepartment of Zoology, Osmania

University, Hyderabad, India

Jayanthi SrinivasDepartment of Biochemistry, Osmania

University, Hyderabad, India

ABSTRACT

Purpose: Glycated lens proteins are capable of producing reactive oxygen species (ROS), which, inturn, can oxidize tryptophan (Trp) into kynurenines. Indoleamine 2,3-dioxygenase (IDO), which isexpressed in many tissues and which is inducible by interferon-γ (IFN-γ ), is able to oxidize Trpinto kynurenines. These kynurenines can modify lens proteins and, in fact, kynurenine adducts aremarkedly increased in lenses with age-related nuclear cataract. Therefore, it has been suggested thatlenticular IDO is involved in diabetic cataractogenesis. The aim of the present study was to examinethe possible role(s) of IDO in streptozotocin (STZ)-induced diabetic cataract in rats. Methods: Diabeticcataract was induced in male Wistar-NIN rats by IP injection of STZ (34 mg/kg body wt). Slit lampbiomicroscopy was used to monitor progression of the resulting hyperglycemia-induced cataract.Treated and control rats were sacrificed at 30 and 60 days, at which times changes in lenticular levelsof IDO activity, IDO mRNA, IFN-γ mRNA (IDO inducer), Trp, kynurenic acid (KYNA), oxidativestress markers (malondialdehyde and carbonyls), antioxidant (reduced glutathione), antioxidantenzymes (glutathione peroxidase and superoxide dismutase), and polyol enzymes (aldose reductaseand sorbitol dehydrogenase) were determined and compared. Results: Cataract was observed tobegin at 30 days after STZ treatment, and mature cataract was observed 60 days after STZ treatment.Lenticular levels of IDO activity, IDO mRNA, IFN-γ mRNA, Trp, and KYNA increased significantlyat 30 days and remained elevated through 60 days. Significant increases were also observed in levelsof oxidative stress markers, antioxidant enzymes, and polyol enzymes at 30 and 60 days after STZtreatment. However, the level of reduced GSH decreased by ∼50% at both points of determination.Conclusions: Production of IDO was induced in STZ-induced diabetic cataractous lenses, possiblyby locally produced IFN- γ . IDO-mediated oxidation of Trp may partly explain the increase in lensKYNA and may thus be implicated in cataractogenesis in concert with the non-enzymic oxidationof Trp by glycated lens proteins.

Keywords: diabetic cataract; IDO; INF-γ ; kynurenic acid; oxidative stress; tryptophan metabolism

Received 29 August 2007; accepted 6 January 2009.Correspondence to Prof. Turlapati Naga Raju, Ph.D., Division of Physiology, Department of Zoology, Osmania University, Hyderabad 500 007,India. E-mail: rajutn57@yahoo.com

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Role of IDO and KYNA in Cataract Formation

INTRODUCTION

Cataract is characterized by a progressive opacifica-tion and loss of transparency of the lens. Diabetes-induced cataract is a multifactorial metabolic disor-der characterized by hyperglycemia, accumulation ofpolyols, and changes in the levels of other cellularmetabolites within the lens.1 Although the etiology ofcataract is poorly understood, recent studies of diabeticcataract have demonstrated increases in serum trypto-phan (Trp),2 lenticular kynurenines,3,4 and polyols.5

Changes in these metabolites are implicated in lensopacification. In cataractogenesis, glycation is a firmlyestablished mechanism for modification of lens pro-teins. In this process, sugars, ascorbate, and dicar-bonyl compounds react with the amino groups oflysine and arginine residues of lens proteins to formketoamine adducts,6,7 which, in turn, can produce reac-tive oxygen species (ROS) during their modification toadvanced glycation end-products. Glycation-derivedROS induces the oxidation of Trp and contributesto lens protein modification by kynureninefluorescentderivatives of L-Trp generated via the kynureninemetabolic pathway).8−10 Indoleamine 2,3-dioxygenase(IDO) (EC 1.13.11.42) is a cytosolic monomeric heme-protein that catalyzes the first step of Trp oxidationalong the kynurenine pathway (Fig. 1). The dioxyge-nase is induced by (IFN-γ ) and results in the markedincrease in Trp oxidation.11 IDO plays a protective roleagainst a variety of infectious agents (e.g., those caus-ing influenza, tuberculosis, and malaria) and chronicinflammatory disorders by reducing the cellularconcentration of Trp, the depletion or absence of

Figure 1. Trp, kynurenine, and KYNA in the L-Trp-kynurenine pathway.

which restricts parasitic growth.12−14 IDO also actsas an immuno-modulator, preventing rejection ofthe allogeneic fetus from the placenta.15Additionally,IDO plays a contributory role in the pathogenesisof several neurological disorders (e.g., AIDS demen-tia complex),16 cerebral malaria,17 and chronic renalfailure.18

IDO is expressed in human lens and is suggested tobe implicated in the formation of age-related cataractthrough a modification of lens proteins with Trpmetabolites, namely kynurenic acid (KYNA)3 and 3-hydroxykynurenine.19 However, the precise functionof IDO in ocular tissues remains a matter of debate, assome reports suggest that the enzyme acts as an an-tioxidant in lens and cornea by reducing the rate ofapoptosis and lipid peroxidation during UV-induceddamage.20,21 In light of the above information, we hy-pothesize that, besides glycation, there is possible in-volvement of ROS- and IDO-mediated oxidation of Trpin the progression of diabetic cataract. To test this hy-pothesis, we determined levels of IDO, Trp, and KYNA,as well as oxidative stress/antioxidant status in lensesof Wistar rats with STZ-induced diabetic cataract.

MATERIALS AND METHODS

Chemicals

STZ; Tri reagent; DL-glyceraldehyde; sorbitol dehy-drogenase (SDH); NADPH; 1,1,3,3-tetraethoxypropane(TEP); butylated hydroxytoluene (BHT); and cata-lase were obtained from Sigma Chemical (St. Louis,

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MO, USA). Acetonitrile, acetic acid, zinc acetate, andmethanol (HPLC grade) were obtained from Merck(Darmstadt, Germany). DNA ladder was purchasedfrom New England Biolabs (Beverly, MA, USA).Primers were obtained from Bioserve Biotechnologies(Hyderabad, India) and the RT-PCR kit from Fermen-tas (MD, USA). All other chemicals and solvents wereof analytical grade and obtained from local companies.

Animals

All animal protocols were approved by the Depart-mental Animal Ethical Committee. Male Wistar-NINrats were obtained from the National Center for Labo-ratory Animal Sciences, National Institute of Nutrition(NIN), Hyderabad. Rats (average body weight of 218.9± 7.7 g, 2–3 months old) were housed in individualcages in a room with controlled temperature, humid-ity, and a 12-hr light/dark cycle. Animals had free ac-cess to water and were fed an AIN-93 diet ad libitum.After 72-hr fasting, blood glucose levels were deter-mined. Rats showing blood glucose >155 mg/dl wereincluded in the study. Control rats (n = 30) received anIP injection of 0.1 M citrate buffer (pH 4.5); treated rats(n = 30) received a single IP injection of STZ (34 mg/kgbody wt) in the same buffer. Food and water intake wasmonitored daily, and body weights were monitored atregular weekly intervals. Onset and maturation statesof cataracts were assessed by slit lamp biomicroscopy.Stages of cataractogenesis were designated accordingto Suryanarayana et al.22 Briefly, lenses were examinedon alternate days, and observed opacities were gradedinto four stages: Stage 0 = no vacuoles present or clearlens; Stage 1 = vacuoles of less than one-third of thelens radius; Stage 2 = vacuoles located at the peripheryof the lens occupying an area of one- to two-thirds ofthe radius from the periphery; Stage 3 = vacuoles ex-tending up to two-thirds of the radius from the periph-ery (nuclear opacity may be seen); Stage 4 = vacuolescover the entire lens, which appears white to the nakedeye. The incidence of each stage was expressed as thepercentage of total lenses in each group.

Blood and Lens Tissue Collection andProcessing

Blood for glucose estimation was collected weekly fromthe retro-orbital plexus and processed according toBergmayer and Bernt.23 At pre-set timepoints (days0, 30, and 60), rats were sacrificed by CO2 asphyxi-ation. Lenses were removed by a posterior approachand stored at −80◦C until further analysis. A 10%homogenate was prepared from three to five pooledlenses in 50 mM phosphate buffer (pH 7.4). All bio-chemical parameters, except for lens malondialdehyde

(MDA), were analyzed using the soluble fraction of thelens homogenate (15,000 g at 4◦C); MDA was deter-mined in the total homogenate.

IDO Activity Assay

IDO activity was evaluated according to Matin etal.24 Briefly, a 200-µl standard reaction mixture con-sisted of the cytosol preparation of lens homogenates50 µl, 50 mM potassium phosphate buffer (pH 6.5),20 mM sodium ascorbate, 10 µM methylene blue,100 µg/ml catalase, and 200 µM Trp. The reactionmixture was incubated at 37◦C for 1 hr, and the re-action terminated by the addition of 40 µl of 30%(w/v) trichloroacetic acid (TCA). The mixture wasthen incubated at 60◦C for 15 min to hydrolyze N-formylkynurenine to kynurenine and centrifuged at12,000 rpm for 5 min at 4◦C. Aliquots of supernatantswere injected over a 25-min period into the HPLC ap-paratus (Phenomenex C18; 250 × 4.60-mm column,5 µm), which had a binary solvent system containing agradient of 0% B to 50% A. Solvent A was 10 mM am-monium acetate buffer (pH 6.7) containing 10% (v/v)methanol; solvent B was 100% methanol. For measure-ment of KYNA, the flow rate was 0.5 ml/min.

Quantification of Trp and KYNA

Lenses (1.5 g) were homogenized in 1.5 ml of 20% TCAand centrifuged at 15,000 × g for 10 min. Aliquots ofthe supernatants were analyzed for Trp and KYNAby reverse-phase HPLC using a reversed-phase HPLC(Shimadzu C18 Shimpak; 250 × 4.60 mm column, 5µm) column with a mobile phase containing 50 mMacetic acid, 250 mM zinc acetate (pH 4.9), and 1% (v/v)acetonitrile at a flow rate of 1 ml/min and using afluorescence detector as described by Herve et al.25

Biochemical Analyses

MDA was quantified as thiobarbituric acid-reactingsubstances (TBARS) by reversed-phase HPLC us-ing a Phenomenex column (C18; 250 × 4.60 mm,5 µm).26 The protein carbonyl content of solubleprotein was measured spectrophotometrically using2,4-dinitrophenylhydrazine,27 reduced GSH was mea-sured by spectrofluorometry using o-pthalaldehyde(OPT) to yield a fluorescent complex,28 aldose re-ductase (AR)29 and SDH30 activities were assayedspectrophotometrically, superoxide dismutase (SOD)-specific activity was assayed spectrophotometricallyby monitoring the rate of inhibition of pyrogallolreduction,31 glutathione peroxidase (GPx) was assayedspectrofluorometrically using cumene hydroperoxide

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and GSH as substrate,32 and tryptophan fluorescencewas measured spectrofluorometrically in the solubleprotein fraction (0.15 mg/ml in 50 mM sodium phos-phate buffer, pH 7.4) at an excitation level of 295 nm andemission of 310–400 nm33according to the prescribedmethod. Protein was assayed by the Lowry methodusing BSA as a standard.

RNA Preparation

Total cellular RNA was extracted from lenses (100mg) using Tri reagent according to the manufacturer’sinstructions. The purity and concentrations of RNAwere determined spectrophotometrically by measur-ing absorbance at 260 nm (nucleic acids), 280 nm(proteins), and 320 nm (background). Samples witha ratio of OD260/OD280 values > 1.8 were used forexperiments.

Semi-Quantitative Reverse TranscriptasePolymerase Chain Reaction (RT-PCR)

A two-step RT kit was used per manufacturer’s pro-tocol. Total RNA (∼1.2 µg) was reverse transcribedusing avian myeloblastosis virus (AMV) reverse tran-scriptase, and cDNA was amplified in the presenceof the following gene-specific primers: (i) housekeep-ing gene glyceraldehyde 3-phosphatedehydrogenase(GAPDH) (S: -5′-GCCAAGGTCATCCATGACAAC-3′ and AS: -5′- GTCCACCACCCTGTTGCTGTA-3′)—expected to amplify a segment spanning 600bp; (ii)IDO (S: -5′-GACTTCGTGGATCCAGAC-3′ and AS: -5′-TCTAAGGAGGAGAGGAAG-3′)—expected to am-plify a segment spanning 277 bp; and (iii) IFN-γ(S: -5′-AGGAAAGAGCCTCCTCTTGG-3′ and AS: -5′-GATTCTGGTGACAGCTGGTG-3′)—expected to am-plify a segment spanning 300 bp. The primer concentra-tion was 20 pmol. Amplification conditions were as fol-lows: initial denaturation (95◦C for 5 min for all genes);start of cycle with denaturation (95◦C for 30 sec forGAPDH, and 95◦C for 5 min for IDO and IFN-γ ); an-nealing (55◦C for 30 sec for GAPDH, 48.2◦C for 105 secfor IDO, and 52◦C for 90 sec for IFN-γ ); and extension(72◦C for 10 min for all genes). At the end of amplifica-tion cycles, reactions were given a final extension step at72◦C for 10 min. PCR products were electrophoresed ona 2% agarose gel in Tris-acetate EDTA buffer (pH 8.2).Bands were visualized using ethidium bromide andphotographed using a UV-trans illuminator (Syngene,MD, USA). Band intensity was analyzed densitomet-rically using Gene tools (Syngene) software. The rela-tive percentage expressions of candidate genes in STZ-induced diabetic rats were computed by consideringthe expression of GAPDH in control rats as 100%.

Statistical Analyses

Differences between control and treated groups wereanalyzed using one-way analysis of variance (ANOVA)followed by the post hoc test for multiple comparisons.Differences were considered significant if p was < 0.05.

RESULTS

Effect of STZ-Induction on Plasma GlucoseLevels, Body Weight, and Food and WaterIntake

Plasma glucose levels remained constant (∼90 mg/dl)in control rats throughout the experimental regimen(Fig. 2). A significant (p < 0.05) and persistent (∼three-fold) rise in glucose levels, decrease in body weight,and increase in food and water intake (data not shown)were observed in treated rats following induction ofdiabetes by STZ.

Slit Lamp Examination and Degree of LensOpacification

Cataract onset occurred after day 30 in treated rats, asindicated by slit lamp examination. At that time, allcontrol group lenses were clear and normal. In diabeticrats, 32% of lenses were in Stage 1, 55% in Stage 2, and13% in Stage 3 of cataract formation; none remainedclear after 5 weeks. By day 60, most (81%) of the lensesin diabetic rats showed development of mature (Stage4) cataract.

Changes in Marker Molecules

Several biochemical markers of oxidative stress (MDA,carbonyls), antioxidant GSH, antioxidant enzymes

Figure 2. The effect of STZ-induction on plasma glucoselevels during the course of experimentation. Data are anaverage of all the rats in a given group. ∗Denotes that dataare significantly different from control at p < 0.05.

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Figure 3. Effect of STZ induction on biochemical markers in rat lenses. Tabulated values represent changes in markers betweengroups.

(GPx, SOD), and polyol enzymes (AR, SDH), as well asdisruption of enzyme activity, and changes in mRNAIDO, Trp, and KYNA were tested for at days 30 and60 for both groups of rats. The percent change in thevalue of these parameters for treated rats in compari-son to control rats at the same timepoint is indicatedin Figure 3. The amount of change was calculated asthe difference between the average values for treatedand control rats divided by the average value of theparameter in control rats.

Diabetic rats showed a significant (p < 0.05) decreasein the contents of total and soluble protein. The percentsoluble protein was decreased by 56% and 45% in STZ-induced diabetic rats in comparison to control rats atdays 30 and 60, respectively (Fig. 3).

Kynurenine Metabolism (IDO, Trp, andKYNA)

A significant (p < 0.05) increase in the contents of Trp,KYNA, and IDO was recorded in STZ-induced diabeticrats in comparison to control rats at days 30 and 60. Thisincrease was high for Trp, with treated rats showing agreater than six-fold increase in the average concentra-tion of Trp compared to that of controls (Fig. 3).

Oxidative Markers

TBARS and protein carbonyl contents were signifi-cantly (p < 0.05) elevated in STZ-induced rats in com-parison to controls at days 30 and 60 (Fig. 3). This

elevation was over and above the generalized increasein TBARS and carbonyl content observed in controlrats, possibly attributable to aging.

Antioxidant System

Lenticular GSH content was significantly (p < 0.05) re-duced by 59% and 48% in STZ-induced diabetic ratsin comparison to control rats at days 30 and 60, re-spectively (Fig. 3). GPx and SOD activities were signif-icantly (p < 0.05) higher in treated rats compared tocontrols on day 30 but not on day 60.

Polyol Enzymes

A significant (p < 0.05) rise in the specific activities ofAR and SDH was recorded in STZ-induced diabeticrats in comparison to control rats at days 30 and 60(Fig. 3).

Intrinsic Trp Fluorescence

STZ-treated diabetic rats showed decreases in both to-tal and soluble protein, but an increase in Trp (Fig. 4).To determine if protein integrity was affected in dia-betic rats, spectra of intrinsic Trp fluorescence of celllysates were recorded. A reduction in Trp fluorescencewas observed in samples from STZ-induced diabeticrats at days 30 and 60 in comparison to controls. Thiseffect on protein integrity could be due to oxidativestress resulting from hyperglycemia.

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Figure 4. Tryptophan fluorescence of soluble protein in different groups Protein (0.15 mg/ml) in 0.05 M sodium phosphatebuffer pH 7.4 was excited at 295 nm and emission was monitored 300–400 nm. Data represent five observations.

RT-PCR Analysis

Biochemical analyses indicated an increase in the levelof IDO in STZ-treated rats. To determine if this increaseresulted from elevated transcription of the gene, RT-PCR analysis was performed on lenticular tissue fromboth control and treated rats. The IDO transcript wasundetectable in control rats. The relative expression ofIDO mRNA was increased in STZ-induced diabetic ratsat both timepoints (days 30 and 60) (Fig. 5). The relativeexpression of IFN-γ was mildly elevated (p < 0.05) atboth timepoints in diabetic rats compared to controlrats (Fig. 5).

DISCUSSION

STZ-induced diabetic cataract provides an excel-lent model system for investigating the biochemical

Figure 5. Expression (Upper panel) and quantification(Lower table) of IDO and IFN-γ in experimental rats byRT-PCR. Tabulated values are average of three independentexperiments, expressed as% expression. ∗: denotes that dataare significantly different from control at p < 0.05.∗∗:denotes that data are significantly different from STZ day30 day rats at p < 0.05.

changes that accompany lens opacification.22,34,35 Thelens of the eye is a unique tissue that shows little orno turnover of proteins. However, the abundant chro-mophores make lens proteins susceptible to oxidationby the free radicals generated by various metabolicsystems.36

Accumulated kynurenine pathway metabolites in thelens have been implicated in the development ofcataract.2−4,37 It has been postulated that IDO activitydoes not change in an age-dependent fashion in hu-man lenticular tissue.38 However, we observed higherlevels of IDO mRNA, as well as of enzymatic activity,in lenticular tissue of STZ-treated rats. IDO is knownto be inducible by IFN-γ 11; we observed higher levelsof IFN-γ mRNA in the lens tissue of STZ-treated rats.The apparent increase in Trp in the STZ-induced di-abetic cataract rats may be due to partial vitamin B6deficiency.2

STZ-induced hyperglycemia was accompanied by asignificant increase in oxidative stress, as evidenced notonly by an increase in MDA and carbonyls, but also bya concomitant decrease in GSH and alterations in its as-sociated reserve pools. As indicated in previous stud-ies, the lower concentration of GSH in STZ-inducedrats may potentiate formation of adducts betweenlenticular proteins and kynurenines; these adducts,in turn, could lead to cataract formation via cross-linking, insolubilization, and destabilization of proteinintegrity.8,39,40 Variations in the levels of kynurenine-bound adducts have been observed during aging andother pathologies (e.g., senile cataract).4,41 Further-more, kynurenines could aggravate hyperglycemiathrough inactivation of insulin secretion.42

KYNA formation is mediated by non-enzymatic spon-taneous deamination of kynurenine, followed by

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oxidation with H2O2and ROS.43,44 A two-fold, time-dependent increase in KYNA levels was observed incataractous lenses of STZ-induced diabetic rats, thusimplicating a role for KYNA in cataract progression.3,33

Significant amounts of lens kynurenine is bound toproteins, thereby rendering these lens proteins sus-ceptible to photo-oxidation followed by protein oxida-tion. During senile cataract formation, protein-boundkynureniene is decreased and unbound kynurenine isincreased.39,41 These findings may explain the elevatedconcentration of KYNA in diabetic rats.

Oxidative stress in STZ-induced diabetic rats is re-flected by increased polyol enzymes. The build-up offluorescent products in these lenses could be a result ofa Maillard reaction between reducing sugars and lensproteins.39 However, other studies have suggested thatprotein oxidation may be due to UV filters rather thanMaillard reaction products.45

Our observations of enhanced expression of IDO, accu-mulation of Trp and KYNA acid, and imbalances in theoxidative stress/antioxidant status in lenses of STZ-induced diabetic rats suggest that specific inhibitorsof IDO as well as of general antioxidants are of po-tential value in preventing or ameliorating cataractformation.

ACKNOWLEDGMENT

This research was funded in part by DepartmentalResearch Support (U.G.C.-DRS, SAP-II), Departmentof Zoology. VRK acknowledges the Indian Council ofMedical Research (ICMR) for providing a research fel-lowship for this work.

Declaration of interest: The authors report no conflictof interest. The authors alone are responsible for thecontent and writing of the paper.

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