Proc. Nati. Acad. Sci. USAVol. 91, pp. 2616-2620, March 1994Biochemistry

Formation of 4-hydroxy-2-nonenal-modified proteins in the renalproximal tubules of rats treated with a renal carcinogen,ferric nitrilotriacetateSHINYA TOYOKUNI*t, Koji UCHIDAt, KEISEI OKAMOTO*, YUKARI HATTORI-NAKAKUKI*, HIROSHI HIAI*,AND EARL R. STADTMAN§*Department of Pathology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan; tLaboratory of Food and Biodynamics, Nagoya UniversitySchool of Agriculture, Nagoya 464-01, Japan; and tLaboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health,Bethesda, MD 20892

Contributed by Earl R. Stadtman, December 8, 1993

ABSTRACT An iron chelate, ferric nitrilotriacetate (Fe-NTA), Induces prox l tubular necrosis, a consequence oflipid peroxidatlon, that iall leads to a high Incidence of renaladenocarcinoma in rodents. Lipid peroidation as monitoredby formation of thiobarbituric acdd-reactive suaces andbee 4-hydroxy-2-noena (HNE) was observed in the kidneyhomogenates of rats treated with Fe-NTA. Based on the factthat HNE is capable of reating with cellular proteins, weattempted to detect the localization ofHNE-mopieproteinsin rat kidney tissues with an immustochemical procedure.By _uans of an kW technique using poly-cional antibody against the ENE-modified proteins, it washown that NE-modl pra ns are formed in the target

cells of this carcinogenesis model. HINE-modified proteins weredetectedin the renal proima tubules 1 hr after i.p. admin-istraton of Fe-NTA (15 mg of Iron per kg). Intense positivitywas found In the cells with degeneration. After 6 hr, the levelof HNE-protein conjugates decreased due to the subsequentnecrosis. The inte of the immunoical reaction withNE-modified protIns creased In parallel with an increase

in the m ts of b c acid-reactive substances andfee liNE that were found. Furthermore, hisochemcl detec-tion of ddehydes by cold Schfs reagent demonstrated thatlocation ofaldehydes was identical to that of the HNEmodifledproteins determined by immuhistochemical procedures. Itwould thus appear that the production ofHNE, a genotoxic andmutagenic ahlehyde, and its reaction with proteins may play arole in Fe-NTA-induced renal carcinogenesis.

Nitrilotriacetic acid (NTA) is a synthetic aminotricarboxylicacid that efficiently forms water-soluble chelate complexeswith several metal cations at neutral pH and has been used asa substitute for polyphosphates in detergents for householdand hospital use in the United States, Canada, and Europe (1,2). An experimental model ofiron overload was developed bythe use of ferric nitrilotriacetate (Fe-NTA) (3). Later, re-peated i.p. injections of Fe-NTA were reported to induceacute and subacute renal proximal tubular necrosis and asubsequent high incidence (60-92%) of renal adenocarci-noma in male rats and mice (4-6). We have shown in thismodel that lipid peroxidation as seen biochemically bythiobarbituric acid-reactive substances TBARS) and his-tochemically by the cold Schiffs method (detection of alde-hydes) has been closely associated with carcinogenesis (7-12).

Lipid peroxidation has been implicated in causing a widerange ofbiological effects (13) including heart diseases, aging,retinal degeneration, and cancer. Lipid peroxidation leads to

the generation of a variety of products including 4-hydroxy-2-nonenal (HNE, structure 1) (14).

OH

Structure 1

HNE is an a,4-unsaturated aldehyde that can be formed byperoxidation of w6-unsaturated fatty acids such as linoleicand arachidonic acids (15). HNE originates almost exclu-sively from phospholipid-bound arachidonic acid and may bethe most reliable index of free-radical-induced lipid peroxi-dation. HNE exhibits a variety of cytopathological effectssuch as enzyme inhibition, inhibition of DNA and RNAsynthesis, inhibition of protein synthesis, and induction ofheat shock proteins (16). It is highly cytotoxic to many typesof cells such as hepatocytes, mammalian fibroblasts, andEhrlich ascites tumor cells (16). HNE also shows genotoxicand mutagenic effects as well as inhibitory effects on cellproliferation (16). It has been proposed that HNE exertsthese effects because of its facile reactivity with biologicalmolecules, particularly with proteins. Recently, we haveraised a polyclonal antibody against the HNE-modified pro-teins and developed an immunochemical assay for tissueproteins modified in vitro by HNE during lipid peroxidation(17, 18).

In the present work, we studied the effects of Fe-NTAdosage and time on the generation of HNE and HNE-modified proteins in rat kidney and on the attendant mor-phological changes in renal proximal tubules. The resultssuggest that formation of the genotoxic and mutagenic alde-hyde, HNE, may be involved in Fe-NTA-induced renalcarcinogenesis.

MATERIALS AND METHODSAnimals and Experiment Groups. Male SPF slc: Wistar rats

(Shizuoka Laboratory Animal Center, Shizuoka, Japan),weighing 130-150 g (6 weeks of age) were used. They werekept in a stainless steel cage and given commercial rat chow(Funabashi F-2, Chiba) and deionized water (Millipore) adlibitum. A total of 33 animals were divided into time-coursegroups (Fe-NTA at 15 mg of Fe per kg of body weight, i.p.;0, 1, 3, 6, 16, and 24 hr) and dose-dependency study groups(1 hr; Fe-NTA at 0, 7.5, 10, 15, and 30mg ofFe per kg ofbodyweight, i.p.). Each subgroup contained 3 animals.

Abbreviations: HNE, 4-hydroxy-2-nonenal; Fe-NTA, ferric nitrilo-triacetate; TBARS, thiobarbituric acid-reactive substance(s); 2,4-DNPH, 2,4-dinitrophenylhydrazine.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Materials. The stock solution of trans-HNE was preparedby the acid treatment (1 mM HCl) of HNE diethylacetal.Ferric nitrate enneahydrate, sodium carbonate, hydrogenperoxide, acetone, and ethanol were from Wako Biochemi-cals (Osaka); NTA disodium salt and 2,6-di-tert-butyl-p-cresol were from Nakarai Tesque (Kyoto); bovine serumalbumin was from Sigma. All the chemicals used were ofanalytical quality; deionized water was used throughout.

Preparation and Inijection of Fe-NTA Solution and Histo-logical Examination. Fe-NTA solution was prepared imme-diately before use by the method ofAwai et al. (3) with slightmodification. Ferric nitrate enneahydrate and NTA disodiumsalt were each dissolved in deionized water to form 300 mMand 600 mM solutions, respectively. They were mixed at avolume ratio of 1:2 (molar ratio, 1:4) with magnetic stirring atroom temperature. The pH was adjusted with sodium car-bonate to 7.4. Fe-NTA was injected i.p. to animals. Animalswere sacrificed by decapitation and both kidneys of eachanimal were removed immediately. Half of one kidney wassnap-frozen in Tissue-Tek optimum cutting temperaturecompound (Miles) with dry-ice-cooled acetone and stored at-80'C either for immunohistochemistry or histochemicaldetection ofaldehydes. The other halfofthe kidney was fixedwith Bouin's solution (19) overnight, immersed sequentiallyfor 24 hr in 50%o and 70%6 ethanol to remove picric acid,dehydrated, embedded in paraffin, sectioned at 3.5 pm, andmounted on glass slides either for hematoxylin/eosin stainingor immunohistochemistry. The other kidney was homoge-nized with 1.15% KCl, used for determination of TBARS, orstored at -800C for detection of free HNE.Immunohistochemistry. The avidin-biotin complex method

was used (20). Frozen sections were postfixed with Bouin'ssolution for 5 min to suppress endogenous biotin activity.Incubation in 0.09% hydrogen peroxide in 30%6 (vol/vol)methanol for 30 min was used for the inhibition of endoge-nous peroxidase. After these procedures, normal goat serum(Dako; diluted to 1:75) for the inhibition of nonspecificbinding of secondary antibody, partially purified rabbit poly-clonal antibody against HNE-modified proteins (0.5-2.0 pg/ml) (17, 18), biotin-labeled goat anti-rabbit IgG serum (Dako;diluted 1:300), and avidin-biotin complex (Dako; diluted1:100) were sequentially used. Procedures using normalrabbit serum instead of antibody against HNE-modifiedproteins showed no or negligible positivity.

Histochemical Detection of Aldehydes. Detection was per-formed on the day of sacrifice, using cold Schiff's reagent, bythe method of Pompella et al. (21).

Measurement of TBARS. The tissue TBARS content wasdetermined immediately after sacrifice by using the methodof Buege and Aust with slight modification (22). To preventadditional chromophore formation during the assay, 0.1%2,6-di-tert-butyl-p-cresol was added to the reaction mixture.Protein determination was done by the method of Lowry etal. (23), using bovine serum albumin as a standard.

Detection of Free HNE. An aliquot (200 Au) of kidneyhomogenate obtained from in vivo experiments was treatedwith an equal volume of 0.1% 2,4-dinitrophenylhydrazine(2,4-DNPH) in 2 M HCl. After 4 hr at room temperature, thereaction mixtures were extracted twice with 400 A of chlo-roform and then dried under nitrogen. Then 200 Al of meth-anol was added, and the 2,4-DNPH derivative of HNE wasanalyzed by reversed-phase HPLC using a TSK-GEL ODS-8OTM column (0.46 x 25 cm; Tosoh, Tokyo). Separation wasachieved by eluting with a linear gradient of60% methanol inwater (0.1% trifluoroacetic acid) (solvent A)-lOo methanol(solvent B) (time = 0, 100o A; 30 min, 0% A), at a flow rateof 0.8 ml/min; the elution profile was monitored at 360 nm.The identification of the 2,4-DNPH derivative of HNE wasmade on the basis of the retention time of standard 2,4-DNPH-HNE and on the coelution test performed by addingstandard 2,4-DNPH-HNE to biological samples. Quantita-tion of the 2,4-DNPH-HNE derivative in standards andsamples was done by measurement of peak area.

RESULTSHistology. The time-dependent morphological changes that

occur after a single i.p. injection of Fe-NTA at 15 mg of Feper kg of body weight (maximal tolerated dose) were exam-ined. The changes were basically the same as previouslyobserved with ddY mice (10, 11). Briefly, cytoplasmic de-generation with pyknosis was observed at 1 hr in a smallfraction of renal proximal tubules. Degeneration progressedto necrosis after 3 hr, thereby generating many proteinaceouscasts in the renal tubular lumina (Fig. 1B). More than half ofthe proximal tubular cells were removed at 24 hr (Fig. 1C).A dose-dependency study at 1 hr showed no morphologicalchanges with administration of Fe-NTA at 7.5 mg of Fe perkg of body weight and only faint cytoplasmic degeneration inone-third of the animals at 10 mg of Fe per kg. However,administration of Fe-NTA at 30 mg of Fe per kg of bodyweight (a lethal dose) induced massive degeneration andnecrosis even at 1 hr (data not shown).TBARS and Free HNE. Administration ofFe-NTA resulted

in the accumulation of lipid peroxidation products (TBARS

FIG. 1. Histology of renal cortex. (Hematoxylin/eosin; x380.) (A) Untreated control. (B) Three hours after Fe-NTA injection. Patchydegeneration of the proximal tubular epithelium and necrotic debris in the tubules is observed. (C) Twenty-four hours after Fe-NTA injection.Complete removal of the proximal tubular cells in some tubules is observed.

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2618 Biochemistry: Toyokuni et al.

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8 Time, hr -

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FiG. 2. Quantitation of TBARS and free HNE in the kidneyhomogenates of rats treated with Fe-NTA. *, HNE; o, TBARS. (A)Time course (Fe-NTA, 15mg ofFe per kg ofbody weight, i.p.; 0, 1,3, 6, 16, and 24 hr). (B) Dose dependency (1 hr; Fe-NTA, 0, 7.5, 10,15, and 30 mg of Fe per kg of body weight, i.p.).

and free HNE) in rat kidney homogenates (Fig. 2A). Thetime-dependent and Fe-NTA dose-dependent patterns ofTBARS and free HNE production were very similar. Theamount ofboth lipid peroxidation products reached the peakat 1 hr after administration of Fe-NTA (15 mg of Fe per kg)and decreased gradually thereafter, suggesting that TBARSand HNE once generated by the Fe-NTA-induced lipidperoxidation reacted with other molecules. As the amount ofFe-NTA administered was varied from 0 to 15 mg of Fe perkg of body weight, there was a progressive increase in theamount ofTBARS and free HNE formed during a 1-hr periodafter the Fe-NTA administration (Fig. 2B). However, theyield of both lipid peroxidation products was maximal at the

15-mg dose; raising the concentration to 30 mg of Fe per kghad no added effect. These results provided direct evidencethat HNE was generated in this experimental model of renalcarcinogenesis and support the proposition thatHNE may beinvolved in the early stage of carcinogenesis.

anm _ yEdDetsctl of Al-dehydes. Preliminary studies showed that frozen sections orBouin's solution-fixed paraffin-embedded sections were suit-able for immunohistochemistry using polyclonal antibodyagainst HNE-modified proteins. Neutral formalin-fixed par-affin-embedded sections were usable and exhibited lowersensitivity. Untreated control animals gave a weak antibodyreaction in all the proximal tubules (Fig. 3A). One hour afteradministration of Fe-NTA (15 mg of Fe per kg of bodyweight), there was an increase in HNE antibody labeling ofvarious patches in some of the renal proximal tubular cells.More intense antibody binding was observed in the degen-erating cells, indicated by pyknosis. Some of the protein-aceous casts were also stained (Fig. 3B), suggesting thatHNE-modified proteins are removed in part by the urinarysystem. Staining of the nuclei was not evident because oftheintense staining of cytoplasm. Interestingly, even after 16 hr,appreciable antibody labeling was evident at the residualcytoplasm and basement membrane, after the proximal tu-bular cells were scraped off (Fig. 3C). Thus, HNE-modifiedproteins remain at the site of generation for >15 hr after asingle injection of Fe-NTA.To determine whether there are differences in the anatomic

location of HNE-modified proteins as detected by antibodyreaction and of aldehydes as detected by the cold Schiffsmethod, serial frozen sections were prepared and stained.Results obtained 3 hr after the Fe-NTA treatment are illus-trated in Fig. 4. It is evident that the distribution of Schiff-positive patches in the renal tubules (Fig. 4A) is identical tothat of the HNE-modified proteins (Fig. 4B).

DISCUSSIONResults of earlier studies demonstrated that lipid peroxida-tion is closely associated with Fe-NTA-induced carcinogen-esis (7-12). The present study has clearly demonstrated thatHNE is generated in renal proximal tubules of rats exposedto oxidative stress by Fe-NTA and that HNE reacts withcytoplasmic constituents that can be recognized by anti-HNE-protein antibody.Although targets in the renal proximal tubules of Fe-NTA-

treated rats have not been identified, it seems likely that one

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FIG. 3. Immunohistochemistry of renal cortex with antibody against HNE-modified proteins. (x380.) (A) Untreated control. Diffuse faintpositivity in the proximal tubular epithelium is observed. (B) Three hours after Fe-NTA injection. Intense positivity in the proximal tubular cellswith degeneration is observed. Proteinaceous cast (arrowhead) is also stained. (C) Sixteen hours after Fe-NTA injection. Positivity is foundin the remaining cytoplasm and basement membrane (arrowheads).

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FIG. 4. Renal cortex 3 hr after Fe-NTA injection. (Serial frozensections, x 190.) (A) Cold Schiffs method. (B) Immunohistochem-istry with antibody against HNE-modified proteins. Exactly thesame tubules are stained with the two methods.

or more proteins are implicated since results of other studieshave demonstrated that HNE reacts with histidine (24, 25),tyrosine (25), cysteine (16, 24-26), lysine (16, 25-27), andserine (25) residues of proteins. Studies by Esterbauer et al.

(26) demonstrated that the sulfhydryl groups of cysteineresidues undergo Michael type addition to the a,,B-unsaturated bond ofHNE to yield relatively stable thioetherderivatives. In the meantime, it was demonstrated that inaddition to cysteine, the lysine and histidine residues ofproteins also undergo Michael-addition type reactions (24,27, 28), leading in each case to HNE-protein adducts in whichthe aldehyde function is preserved. This validates the use ofhistochemical procedures based on the cold Schiffs methodfor the detection of an HNE-derived aldehyde function infrozen sections. Validation is obtained also from the obser-vation that the anatomic location of aldehydes determined bythe cold Schiffs method is identical to that determined by theanti-HNE-antibody technique (Fig. 4). Moreover, our resultsindicate that fixation with Bouin's solution gives betterretention of epitopes.

In earlier studies analogous to those described here, anti-bodies raised against HNE-lysine (29) and HNE-derivatizedproteins (30, 31) were used for the immunohistochemicaldetection of HNE-protein adducts in oxidized forms oflow-density lipoprotein and in atherosclerotic lesions (29,31). The use of immunohistochemistry ensures greater epi-tope specificity and has the advantage that it can be appliedto neutral formalin-fixed paraffin-embedded sections, a rou-tine procedure for surgical pathology sections.Although free aldehydes are usually not retained in paraf-

fin-embedded samples, our studies suggest that the presenceof HNE-modified proteins is a fair measure of HNE gener-ation since the intensity of the immunochemical labelingincreases concomitantly with an increase in the concentra-tion of free HNE, as determined by direct measurement.

It has been proposed that necrosis is due in part tomembrane lipid peroxidation that is promoted by the reduc-tion of Fe-NTA by cysteine or cysteinylglycine derived fromthe in situ degradation of glutathione (12, 32, 33). Thisconcept is based on the following observations. (i) In rats, thesecond and third segments of renal proximal tubule are mostsusceptible to Fe-NTA treatment. (ii) The y-glutamyl-transpeptidase activity in the second and third segment isgreater than in the first segment (34). (iii) In the brush bordersurface of the renal proximal tubules, -glutamyltranspepti-dase degrades glutathione to cysteinylglycine, which is rap-idly degraded by dipeptidase to cysteine and glycine (34, 35).

(iv) Whereas most of the iron bound to NTA is transferred totransferrin (36) and taken up by hepatocytes, part of the ironappears in the kidney (37) where the Fe-NTA complex issubject to reduction in the renal proximal tubules by glu-tathione degradation products.The observation that glutathione promotes Fe-NTA-

mediated lipid peroxidation of kidney brush border mem-brane vesicles (32) seems contrary to the generally acceptedview that glutathione protects cells from HNE-mediateddamage by virtue of its ability to undergo rapid Michael-typeaddition to HNE (16). Moreover, this spontaneous conjuga-tion of glutathione with HNE is enhanced by at least twoorders of magnitude by cytosolic glutathione S-transferase(38). Indeed, the exposure of cells to HNE leads to rapid lossof endogenous glutathione (39). Furthermore, reduction ofthe aldehyde moiety of HNE to the corresponding hydroxyderivative by the NADH-dependent alcohol dehydrogenaseconverts it to a relatively unreactive form (40). These pro-tective systems and a variety of antioxidant enzymes, in-cluding superoxide dismutase, catalase, glutathione peroxi-dase, and very likely the recently discovered thiol-specificantioxidant enzyme (41, 42), can inhibit the initiation ofmembrane peroxidation. Since the renal proximal tubules area major target of Fe-NTA damage in vivo, histochemicalstudies on the distribution of these protective enzymes in therat kidney may provide insight to the mechanism of Fe-NTA-mediated carcinogenesis.The demonstration here that antibodies directed against

HNE-protein conjugates interact strongly with epitopes indegenerating cells supports the view that the cytotoxicity ofHNE in vivo is associated with its ability to modify proteins.This notwithstanding, a role ofDNA damage is not excludedsince 8-hydroxydeoxyguanosine (8-OHdG) is also generatedin the Fe-NTA-induced renal carcinogenesis model (43-45),-but the mechanism of 8-OHdG production has not beenestablished. The further observation that lipid peroxidationprovokes 8-OHdG formation (46) leaves open the possibilitythat the mechanism of HNE production may provide a linkbetween lipid peroxidation and oxidative DNA damage.

We are grateful to Aya Fukuda (Nagoya University School ofAgriculture) for her technical assistance for the measurement of freeHNE. This work was supported in part by Grants-in-aid for CancerResearch from the Japanese Ministry of Education, Science andCulture and a research grant from the Fujiwara Foundation of KyotoUniversity.

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