[CANCER RESEARCH 47, 6719-6725, December 15, 1987]

Immunocytochemical Detection of Interaction Products ofciVDiamminedichloroplatinum(II) and ciVDiammine-(1,1-cyclobutanedicarboxylato)platinum(II) withDNA in Rodent Tissue Sections1

Philippe M. A. B. Terheggen, Ben G. J. Floot, Ewalt Scherer, Adrian C. Begg, Anne Marie J. Fichtinger-Schepman,and Leo den Engelse2

Divisions of Chemical Carcinogenesis [P. M. A. B. T., B. G. J. F., E. S., L. d. E.] and Experimental Therapy [A. C. B.], The Netherlands Cancer Institute (Antoni vanLeeuwenhoek Huis), Plesmanlaan 121, 1066 CX Amsterdam: and the Medical Biological Laboratory TNO, 2280 AA Rijswijk [A. M. J. F-S.J, The Netherlands

ABSTRACT

Calf thymus DNA was modified in vitro by c/î-diamminedichloro-platinum(II) (cisDDP), complexed with methylated bovine serum albuminand used to immunize rabbits. The anti-cisDDP-DNA antiserum obtainedwas applied in a double peroxidase-antiperoxidase staining procedure tolocalize cisDDP-DNA and cis-diammine(l,l-cyclobutanedicarboxy-lato)platinum(II) (CBDCA)-DNA interaction products in cryostat tissuesections of mice and rats. Rats received cisDDP (0-10 mg/kg) and werekilled after 24 h. Mice received cisDDP (0-15 mg/kg) or CBDCA (200mg/kg), and were killed after 2 h-162 days. For each time-dose combination two mice or one rat were used; agents were given i.p. Specificnuclear staining was observed in all tissues examined from cisDDP- orCBDCA-treated animals. No significant nuclear staining could be observed in tissue sections from control rats and mice. The extent of stainingafter cisDDP was dose and time dependent. The lowest dose of cisDDPafter which specific nuclear staining could be detected varied from tissueto tissue [e.g., 0.1 mg/kg, pancreas (mouse); 0.5 mg/kg, liver, kidney(mouse, rat)|. The longest time interval after a single dose of 6 mg/kgcisDDP in which adducts could be visualized also depended on the tissueand varied between 9 days (spleen, testis) and 162 days (kidney). Thestaining intensity in liver and kidney, measured microdensitometrically,decreased relatively fast in the first days after treatment, but much slowerthereafter. In the kidney, cisDDP-induced DNA modification showedregional variation: inner cortex > outer cortex > medulla (rat) and cortex> medulla (mouse). In the mouse kidney, a small subpopulation of tubularcells in close association with the renal corpuscles showed a remarkablyhigh staining intensity after both cisDDP and CBDCA administration.Tissues that showed clear cisDDP-induced histológica!alterations (kidney, pancreas, testis, and duodenum) also showed moderate to high levelsof cisDDP-DNA interaction products. A correlation between cell damage(measured histologically) and cisDDP-DNA binding within one tissuetype was demonstrated in the rat inner renal cortex, the murine renalcortex, and in duodenal epithelial cells of both mice and rats.

INTRODUCTION

The potent antitumor drug cisDDP3 is routinely used in the

treatment of testicular, ovarian, and bladder cancer, while thedemonstrated activity against a wide range of other malignancies, such as carcinoma of the head, neck, and stomach, or braintumors, needs further research to establish the drug's usefulness

Received 5/20/87; revised 9/3/87; accepted 9/16/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by grant NKI 86-11 from the Koningin Wilhelmina

Fonds, The Netherlands Cancer Foundation.2To whom requests for reprints should be addressed.3The abbreviations used are: cisDDP, rìs-diamminedichloroplatinum(II);

CBDCA, c»-diammine(1,1-cyclobutanedicarboxylato)platinum(II); MBSA,methylated bovine serum albumin; AAF, /V-acetyl-2-aminofluorene; CT-DNA,calf thymus DNA; GAR, goat anti-rabbit immunoglobulin; NGS, normal goatserum; NRS, normal rabbit serum; AAS, atomic absorption spectroscopy; ELISA,enzyme-linked immunosorbent assay; PAP, peroxidase-(rabbit)antiperoxidasecomplex; DAB, 3,3'-diaminobenzidine-HCl; H&E, hematoxylin and eosin; amol,attomole(10~" mol).

(1). Side effects of cisDDP administration include myelo-suppression, neuro-, and gastrointestinal toxicity, while neph-ropathy is regarded as the dose-limiting factor in humans (1,2)and laboratory animals (3, 4). It is believed that both thecytostatic and the cytotoxic effects of cisDDP, in vitro as wellas in vivo, are mediated by its interaction products with DNA(5-9). For the detailed investigation of the relationship betweenDNA interaction and the in situ cytotoxicity, studies at the levelof the individual cell become necessary. Until recently, however,it has not been possible to study the binding of cisDDP to DNAat the level of the individual cell. We therefore prepared a rabbitantiserum against in v/'iro-modified cisDDP-DNA and used this

anti-cisDDP-DNA antiserum to visualize cisDDP-inducedDNA modifications in tissue sections of mice and rats. We alsostudied the cross-reactivity between our antiserum and DNAadducts induced by CBDCA. This second generation platinumantitumor drug was chosen because of the apparent similaritybetween cisDDP-DNA and CBDCA-DNA interaction products(10).

This report shows the remarkable sensitivity and specificityof the immunocytochemical method, originally developed forthe visualization of carcinogen-DNA adducts in tissue sections(11-13). The immunocytochemical technique provides us withthe opportunity to investigate the formation and persistence ofcisDDP-DNA adducts and to correlate the newly obtained datawith existing data on the cytotoxic and antitumor effects ofboth platinum compounds.

MATERIALS AND METHODS

Animals and Treatments. Inbred male Sprague-Dawley rats (250-270g) from the SPF breeding colony of the Netherlands Cancer Institute,received a single cisDDP dose of 0, 0.5, 2, or 10 mg/kg body weight.For each dose one rat was used. Inbred male and female C3H/He/AfOmice (20-40 g) were obtained from the same breeding colony. Mice ingroup 1 received 15 mg/kg cisDDP and were killed after 2 h. Mice ingroup 2 received 0, 0.1, 0.5, 1, 2, 4, 6, or 8 mg/kg cisDDP and werekilled after 6 h. Mice in group 3 received 6 mg/kg cisDDP and werekilled after 6 h, or 1, 3, 9, 27, 81, or 162 days. Mice in group 4 received200 mg/kg CBDCA and were killed after 4 h. For each dose-timecombination two mice were used (one male and one female in group 1;two males in groups 2-4). cisDDP and CBDCA were dissolved in 0.14M NaCl to give an injection volume of 1 ml/animal. Control animalswere injected with 1 ml 0.14 M NaCl and were killed after 24 h (rats),or after 2 h, 6 h, 1, 3, 9, 27, 81, and 162 days (mice). One rat receivedan injection of 10 mg/kg AAF, dissolved in 1,2-propanediol, anotherrat 150 mg/kg benzo(a)pyrene, dissolved in olive oil, and both werekilled after 24 h. Animals were kept under a 14-10 h light-dark cycleand fed standard laboratory chow (Hope Farms, Woerden, The Netherlands) and water ad libitum. All agents were given by i.p. injection.Mice were killed by cervical dislocation, rats by decapitation.

Chemicals and Immunoreagents. cisDDP for injection into animals:a solution of cisDDP in 0.14 M NaCl, adjusted to pH 2-3 (Platino!;

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Bristol-Myers, Weesp, The Netherlands); for the in vitro modificationof DNA cisDDP from Ventrón (Karlsruhe, FRG) was used. CBDCAwas purchased from Bristol-Myers (each vial contained 150 mg CBDCAand 150 mg mannitol). Other reagents used were MBSA, (Serva,Heidelberg, FRG); H2O2 (Merck-Schuchardt, München,FRG); CT-DNA, ovalbumin, imidazole, DAB, and RNase A from bovine pancreas(Sigma Chemical Co., St. Louis, MO); RNase T, from Aspergillusoryzae (Boehringer, Mannheim, FRG); NGS (The Netherlands RedCross Blood Transfusion Service, Amsterdam, The Netherlands); GAR(against whole immunoglobulin G; Campro Benelux, Elst, The Netherlands); PAP (American Qualex, La Miranda, CA). Antisera were usedin the following dilutions: GAR, 1:640; PAP, 1:3200.

Preparation of Antisera. The modification of CT-DNA by cisDDP[resulting in a drug/nucleotide ratio of 6.7 x 10~2, determined by

AAS], the complex formation of cisDDP-DNA with MBSA and theimmunization of rabbits with the cisDDP-DNA-MBSA complex werecarried out as described by Poirier et al. (14), with the followingexception: immunization of the rabbits with the MBSA complex of 330¿tgcisDDP-DNA in Freund's complete adjuvant at weeks 1, 2, 3, and4 was followed by two booster injections in Freund's incomplete adju

vant at weeks 8 and 14. Sera were tested for antibody activity in a directELISA, essentially as described by Van der Laken et al. (15), and usedwithout purification. Preimmune NRS was collected from the rabbit,which was used for immunization with the cisDDP-DNA-MBSA complex. The preparation of antisera against synthetic cisDDP-nucleotideadducts have been described elsewhere (16, 17). Two of these antiserawere applied in the immunocytochemical assay: the W101 antiserum,raised against the synthetic adduci cisPt(NH3)2dGuoGMP and the 3/65 antiserum, raised against the synthetic adduct cisPt(NH3)2d(ApG).These antisera recognize the major cisDDP-DNA interaction products,i.e., the intrastrand cross-links of cisDDP on neighboring nucleobases

(GG for W101 and AG for 3/65). The use of the antisera W101 and3/65 was restricted to liver sections of cisDDP-treated rats.

Preparation of Tissue Sections. Liver, kidney, heart, muscle (fromthe femur quadriceps), duodenum, spleen, testis, pancreas, brain, andthymus (the last three tissues were only collected from mice) werequickly removed, frozen on dry ice, and stored at -80°C. Cryostat

sections (10 /<m)were cut and mounted on ovalbumin-coated slides. Insome experiments fresh tissues were fixed in 4% formalin (in 42 HIMNaH2PO4 and 36.5 mM Na2HPO4, pH 7), embedded in paraffin,sectioned at 4-6 urn and stained with H&E.

Immunocytochemical Assay. The protocol of Heyting et al. (11) wasused with some modifications, which are given below. The generaloutline of the staining procedure was as follows: cryostat sections weretreated with methanol-H2O2 (to inactivate endogeneous peroxidases),RNases A and Ti (to remove possible cisDDP-RNA adducts) andethanol-NaOH (to denature the DNA and/or to increase the accessibility to antibodies). After incubation with NGS (to prevent nonspecificbinding) and the first antiserum (anti-cisDDP-DNA), the presence ofbound antibodies was visualized by "double PAP staining," i.e., by

subsequent incubation of the sections in GAR, PAP, GAR, and PAP.The peroxidase substrate DAB was then added, together with imidazoleand H2O2. The resulting product of the conversion of DAB by theperoxidase was a stable brown precipitate. Parallel cryostat sectionswere stained with H&E. Modifications of the method described byHeyting et al. (l 1) were: treatment during 10 min with 0.07 M NaOHin 30% ethanol instead of 3 min with 0.07 M NaOH in demineralizedwater; incubation with 10% NGS for l h at room temperature prior toincubation with anti-cisDDP-DNA antiserum; CT-DNA (0.2 /ig/Ml)was added to the anti-cisDDP-DNA antiserum just before incubationto diminish nonspecific nuclear staining; after each incubation withGAR and PAP, sections were washed in PBS, wash buffer and PBS (1min each); final staining included incubation in 10 mM imidazole, 50mM Tris-HCl (pH 7.5), 0.5 mg/ml DAB, and 0.01% H2O2 for 10 minin the dark. The anti-cisDDP-DNA antiserum and the NRS wereapplied in the immunocytochemical assay in a dilution of 1:1800. TheW101 and 3/65 antisera were used in a dilution of, respectively, 1:1600and 1:100. In some experiments, 1:1800 diluted anti-cisDDP-DNAantiserum was preincubated in wells coated with either cisDDP-modi-fied or unmodified DNA (0.2 Mg/well) for 3 x 24 h at 4"C.

Microdensitometry. Staining intensity was determined with a Knott(München,FRG) light-measuring device, coupled to a Leitz Orthoplanmicroscope. From 20 randomly selected cells in each investigated tissuesection, the nuclear and cytoplasmic light transmission were measured(beam diameter, ±5 ^mi). Specific nuclear staining was obtained bycorrecting the measured nuclear transmission for the background,measured in the cytoplasm of the same cells.

Histopathological Evaluation. H&E-stained cryostat and formalin-fixed sections were screened for histológica!changes. Nuclear enlargement, defined as the percentage of increase in mean nuclear diameterwhen compared with untreated animals, was measured with an ocularmicrometer (amplification, X1250). Determination of nuclear enlargement was restricted to liver, kidney [6 h (mice) or 24 h (rats) aftercisDDP administration] and pancreas (6 h after cisDDP administration(mice)].

Statistics. Levels of significance were calculated using Student's t

test; P < 0.05 was considered indicative of a significant differencebetween groups.

RESULTS

Controls of the Immunocytochemical Procedure

Nuclear staining in tissue sections from untreated rats andmice (Fig. 1, right) or in liver sections from AAF- orbenzo(a)pyrene-treated rats was very weak and could not bediscerned from the very low cytoplasmic background staining.This nuclear staining did depend on the antiserum dilution.The proper dilution (1:1800) was the lowest dilution that gaveno significant nuclear staining in sections from untreated animals. Significant nuclear staining was also absent from liversections of cisDDP treated rats when the first antiserum(against cisDDP-modified DNA) was replaced by NRS or hadbeen preincubated in wells coated with cisDDP-DNA. Cytoplasmic staining was not influenced by pretreatment withRNases. PAP-stained sections from untreated animals showedslightly more cytoplasmic and nuclear staining after the amicisDDP-DNA antiserum than after NRS. Addition of CT-DNAto the first antiserum was used routinely since it led to asignificant depression or total elimination of nuclear stainingin tissues from solvent-treated animals.

Visualization of Adducts in Rats and Mice after Different cisDDPDoses (Groups 1 and 2)

Liver. Strong, specific nuclear staining and faint cytoplasmicbackground staining was observed in liver sections of cisDDP-

V.?

I

IFig. 1. Immunocytochemical localization of nuclei with cisDDP-DN A adducts

in mouse liver. Staining procedure: see "Materials and Methods." Liver tissue

was obtained from a mouse 2 h after i.p. 15 mg/kg cisDDP (group 1, left) orfrom a control mouse, 2 h after i.p. 1 ml 0.14 M NaCI (right). Brighi field optics;bar, 25 urn.

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treated rats and mice when the anti-cisDDP-DNA antiserumwas applied in the PAP assay (Fig. 1, left). Nuclear stainingintensity, measured microdensitometrically, correlated positively with dose, both in mice (group 2) (Fig. 2) and in rats(data not shown). cisDDP-induced DNA modifications couldbe visualized in nuclei of parenchymal cells but not in nuclei ofblood vessel cells. It is as yet uncertain if nuclei of othernonparenchymal cells show cisDDP-induced DNA modification, because nonparenchymal cells (e.g., Kupffer cells) couldn't

be discriminated unequivocally from parenchymal cells in theimmunocytochemically stained sections. The distribution ofstained nuclei over the different lobular regions was homogeneous. The minimal dose of cisDDP after which modificai ionscould be visualized was 0.5 mg/kg (Table 1). After all doses ofcisDDP, a weak cytoplasmic staining was observed. This cyto-plasmatic background became lower when the dose of cisDDPincreased, probably as a consequence of an increased competition for the anti-cisDDP-DNA antiserum by the nuclearcisDDP-DNA adducts. This type of cisDDP-related, weak cytoplasmic background staining was only seen in liver sections.Dose-dependent, specific nuclear staining was also seen in liversections of cisDDP-treated rats when the 3/65 (after srj.5 mg/kg cisDDP) or the W101 (after ä2mg/kg cisDDP) antiserawere used. The nuclear staining intensity, however, was muchlower when compared to that obtained with the anti-cisDDP-DNA antiserum. In addition, the W101 antiserum gave rise toa considerable (cytoplasmic) background staining after cisDDPdoses above 2 mg/kg.

Kidney. After administration of ISO.5mg/kg cisDDP to ratsor mice, DNA modifications could be visualized in tubules ofboth the cortex and the medulla (Fig. 3). The staining intensityrelated positively to the cisDDP dose (Fig. 2). Nuclear stainingwas observed neither in blood vessel cells nor in the glomeruli,although in some cases nuclear staining of the parietal epithe-

>. 300 -

i

2468cis-DDP( mg/kg)

Fig. 2. Relationship between the microdensitometrically measured nuclearstaining intensity and the cisDDP dose (mice, group 2). Liver and kidney cryostatsections were stained for cisDDP-DN A adducts according to the standard method(see -Materials and Methods"). Points and bars, mean ±SD (20 measurements).

Table 1 cisDDP-DNA adducts in mouse and rat tissues

Minimal dose of cisDDP after which significant nuclear staining, indicativefor cisDDP-DNA lesions, could be observed in tissue sections of mice (M; 6 hafter injection) and rats (R; 24 h after injection).

Dose (mg/kg body weight)

0.1 0.5

Pancreas (M) Liver (M+R) Muscle (M) Brain (M) Testis (M)Kidney (M+R) Testis (R)Heart (M) Duodenum (M+R)

Spleen (M)Heart (R)

Fig. 3. Immunocytochemical localization of nuclei with cisDDP-induced DNAmodifications in murine kidney. Nuclei with highest staining intensity were closelyassociated with renal corpuscles. Kidney tissue was obtained from cisDDP-treatedmice (group I, 2 h after i.p. IS mg/kg cisDDP). Bright-field optics; bar, 25 /mi.

NJINGINTENSITYMo00NUCLEAR

STAIJ_oucor1erexouter

cortex^^~inner

cortex—

•—^^•"inner

cortexouter

innermedullamedullaB^=T-=H-=-ou

er innermedulla medulla

Fig. 4. Microdensitometrically measured nuclear staining intensities in different renal regions after immunocytochemical staining for cisDDP-induced DNAmodifications. Mouse, kidney 6 h after i.p. 8 mg/kg cisDDP; ral, 24 h after i.p. 4mg/kg cisDDP. This distribution was independent of time after injection anddose of cisDDP. Points and bars, mean ±SD (20 measurements per section) fromtwo animals (mice) or two kidney sections from one rat.

Hum of Bowman's capsules was observed. In the rat kidney, the

staining intensity (measured microdensitometrically) was highest in the inner cortex, intermediate in the outer cortex, andlowest in the medulla (Fig. 4). This distribution was the sameregardless of cisDDP dose or length of time after exposure. Inthe mouse, the differences between staining intensities of theinner and the outer cortex were much smaller, but again themedulla showed a relatively low level of DNA modification(Fig. 4). A striking aspect of the staining pattern of the mousekidney was that nuclei from tubules, adjacent to renal corpuscles, showed a very high staining intensity. It is uncertainwhether these stained nuclei belong to the convulated distaltubules or to the most proximal part of proximal tubules,although some observations suggest the latter (Fig. 5). Cytoplasmic and nuclear background staining in the kidney wereinvariably very low.

Pancreas. Mouse pancreatic acinar cells and islet cells showedsignificant nuclear staining after doses as low as 0.1 mg/kgcisDDP (Fig. 6). Nuclei of intra- and interlobular duct cellsremained unstained. Islet cells exhibited a relatively high cytoplasmic background, which tended to be higher after highercisDDP doses.

Muscle. In muscle tissue cisDDP-DNA adducts could bevisualized after a minimal dose of 1 mg/kg (femur quadriceps)

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•V

Fig. 5. Localization of nuclei with CBDCA-induced DNA modifications inthe mouse kidney. Two parallel sections are shown: left, H&E-stained section;i ¡Kin.immunocytochemically stained section. Renal tissue was obtained frommice 4 h after i.p. 200 mg/kg CBDCA (group 4). Cells with highest nuclearstaining intensity seemed to be a part of the cubic epithelium of the Bowman'scapsule and the most proximal part of the proximal tubuli. Bright-field optics;bar, 25 /im.

Fig. 6. Immunocytochemical localization of cisDDP-induced DNA modifications in pancreas, obtained from mice 6 h after i.p. 8 mg/kg cisDDP (group 2).Distribution of stained nuclei is homogeneous; islet cells show relatively highcytoplasmic background. Bright-Held optics; bar, 25 //m.

Fig. 7) or a4 mg/kg (mice). Nuclear staining intensity wasrelatively high in the interstitial cells and almost absent in cellsof the germinative epithelium.

Persistence of Adducts in Mouse Tissues (Group 3)

After a single dose of 6 mg/kg cisDDP, DNA lesions wereseen up to 9 days (testis, spleen), 27 days (pancreas, duodenum,and muscle), 81 days (liver, brain, and heart), or 162 days(kidney). In general, nuclear staining persisted longer in tissueswith a relatively high initial level of nuclear staining (kidney,liver, heart, see Table 1). Two exceptions were observed: thebrain, which had a low initial staining level, and the pancreas.In the latter organ cisDDP-induced DNA modifications couldbe visualized already after 0.1 mg/kg cisDDP 6 h after injection(see Table 1), but no cisDDP-specific staining could be detectedlonger than 9 days after administration of the much higher doseof 6 mg/kg (see above). This suggests that there is no consistentrelationship between adduct persistence and initial nuclearstaining intensity. Fig. 8 shows the time course of nuclearstaining intensities of murine livers and kidneys; measurementsin the kidney were restricted to the subpopulation of heavilystained nuclei from tubules, adjacent to renal corpuscles (seeabove). Adduct loss in both tissues was relatively fast in thefirst days after drug administration, but became much slowerthereafter. Original nuclear staining intensity in the liver waslower than that in the kidney. Nuclear staining intensity washighest 6 h after treatment in the kidney and 1 day aftertreatment in the liver. These observations probably reflect differences in kinetics of adduct formation and loss between theliver and the kidney.

Visualization of Adducts in Mice after CBDCA Administration(Group 4)

In the kidneys of CBDCA-treated mice, DNA modificationscould be visualized both in the cortex and the medulla, usingthe anti-cisDDP-DNA antiserum. The cortex showed higheradduct levels than the medulla, but within the cortex and themedulla staining intensity was distributed rather homogeneously. Tubular cells close to renal corpuscles showed mostpronounced nuclear staining (Fig. 5), as was the case in kidneys

or 0.5 mg/kg cisDDP (heart). Background staining in muscletissue was very low.

Brain. The mouse brain showed nuclear staining in both thecerebellar and cerebral cortex after doses of £2mg/kg cisDDP.Nuclei from the white matter were heavier stained than thosefrom the gray matter.

Spleen. Nuclear staining was visible in the spleen of mice andrats after i?2 mg/kg cisDDP, both in the white and the redpulp. Nuclear staining was faint, except in some scatteredunidentified cells.

Thymus. Staining of the mouse thymus was always weak andregarded nonspecific, since it was impossible to discriminatecytoplasmic from nuclear staining.

Duodenum. In duodenal cells, DNA modifications could bevisualized after cisDDP doses of ^2 mg/kg. Nuclear stainingintensity in epithelial villus cells was much higher than in anyother duodenal (e.g., crypt) cells. Weak cytoplasmic backgroundstaining was observed in both treated and untreated animals.

Testis. cisDDP-induced DNA modifications in testis tissuecould be made visible after cisDDP doses of a2 mg/kg (rats,

it

st

Fig. 7. Immunocytochemical localization of cisDDP-induced DNA modification in testis. obtained from cisDDP-treated rats (24 h after 10 mg/kg i.p.cisDDP). Nuclear staining intensity was relatively high in interstitial cells andalmost absent in cells of the germinative epithelium, st, seminiferous tubules; it.interstitial tissue. Bright-field optics; bar, 10 jun.

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50 100

time alter treatment (days)150

Fig. 8. Nuclear staining intensities in mouse kidney and liver at different time-points after a single i.p. injection of 6 mg/kg cisDDP (group 3). Tissue sectionswere stained for cisDDP-induced DNA modifications. Points and bars, mean ±SD (20 measurements) from two mice.

of cisDDP-treated mice. In the liver, distribution of stainedcells over the liver lobules was rather homogeneous. DNAmodifications could only be visualized in nuclei of parenchyma!cells; unequivocal identification of other stained cell typesproved to be impossible.

Histológica! Changes

cisDDP-induced histológica! changes were observed in the

kidney, the pancreas, the duodenum, and the testis of both miceand rats. Cell injury in the renal cortex of the rat was mostpronounced in the inner cortex; this observation is in agreementwith earlier reports (18-20). Tubular nuclei in the inner cortexof the rat kidney were enlarged by 35%, 24 h after a dose of 2mg/kg cisDDP; this enlargement is statistically significant (/'

< 0.05). cisDDP did not induce enlargement of tubular cells ofthe outer cortex of the rat kidney under these conditions. Themurine kidney showed statistically significant nuclear enlargement of the proximal tubules (17% at 6 h after 8 mg/kg cisDDP,P < 0.05), while nuclei from the distal tubules were not enlarged. Histológica! changes, like nuclear enlargement and nuclear disposition, were observed to the same extent in the innerand outer cortex of the mouse kidney. In the murine pancreas,cytoplasmic basophilia and edema were observed 6 h after eO. 1mg/kg cisDDP; nuclear enlargement was also observed (10%after 0.5 mg/kg, P < 0.05). Histological changes in the duodenum of mice and rats, observed after S0.5 mg/kg cisDDP,predominantly consisted of irregularities of the epithelial villuscells (vacuolation, edema). These changes in the duodenum,observed up to 27 days after 6 mg/kg cisDDP, were in agreement with literature reports (21). Minor changes were observedin crypt cells. In the testis of mice and rats both interstitialdegeneration and a disturbance of the normal hierarchical organization in the seminiferous tubules were observed after elmg/kg (mice) or £2mg/kg cisDDP (rats). Hypocellularity inthe red pulp of the spleen, observed in both mice and ratsalready 24 h after £0.5mg/kg cisDDP, was visible up to 27days after 6 mg/kg cisDDP. Histological changes were absentfrom heart, muscle (femur quadriceps), thymus, brain, and liverof cisDDP-treated mice and rats.

DISCUSSION

This paper shows that interaction products of the antitumordrugs cisDDP and CBDCA with DNA in rodent tissues can bedetected by an immunocytochemical peroxidase technique, thedouble PAP assay. This assay already proved to be useful forthe localization of carcinogen-DNA adducts (O6-alkyldeoxy-guanosine, deoxyguanosin-8-yl-(acetyl)aminofluorene, anddeoxyguanosin-7-yl-aflatoxin B,) in various rat tissues, such asliver, brain, pancreas, and oesophagus (11-13, 22, 23).4 Thedemonstrated recognition of both CBDCA- and cisDDP-in-duced DNA modifications by the anti-cisDDP-DNA antiserumsuggests (at least partial) epitopic similarity of CBDCA andcisDDP interaction products with DNA. This suggestion is inline with recent results from Knox et al. (10), who reported thatcisDDP and CBDCA induce highly similar if not identicallesions in DNA of cultured cells. The anti-cisDDP-DNA anti-serum showed a very low affinity for unmodified DNA. However, nuclear staining was almost absent from tissue sections ofuntreated animals when CT-DNA was added to the first anti-serum during the peroxidase staining. Since the antiserum wasraised in a similar way as that described by Poirier et al. (14,see also Ref. 24), it can be expected to show its highest affinityfor the bidentate adduci of cisDDP in which two chloride ionshave been replaced by two adjacent deoxyguanosines on thesame DNA strand. Further characterization of the anti-cisDDP-DNA antiserum is the subject of present research.

Heterogeneity of DNA Modification. After a given dose ofcisDDP, nuclear staining intensity varied from tissue to tissue,indicating different levels of cisDDP-induced DNA modifications. The apparent differences in cisDDP-induced DNA modification levels between different organs in the present immunocytochemical experiments parallel literature data on the totalplatinum content in tissues of mice (25), rats (3, 26), rabbits(27), and dogfish (28). In all these species relatively high levelsof total platinum were found in kidney and liver; we observedthe highest levels of cisDDP-induced DNA modification alsoin these organs. Heterogeneity of DNA modification within atissue was observed in the kidney, the testis, and the duodenum.The relatively high level of DNA modification in the innercortex of the rat (Fig. 4) is in line with AAS measurements byChoie et al. (19), who reported the highest total platinumconcentration within the kidney in the corticomedullary junction. The correlation between total platinum levels and extentof DNA modification (nuclear staining intensity) is, however,not an absolute one. The spleen proved to be an exception:whereas high levels of total platinum after cisDDP treatmenthave been reported (3, 27), nuclear staining intensity was faint,even after doses of £4mg/kg cisDDP (Table 1). The heterogeneous distribution of cisDDP-induced DNA modificationsmight reflect differences in blood supply or the existence of acertain blood-tissue barrier. The low modification levels in brainand testis might be explained in this way. However, even a closeor continuous contact of cells with the (drug-containing) bloodseems to be no guarantee for cisDDP-DNA adduct formation,in view of the low modification levels in the spleen and theapparent absence of DNA modification in endothelial cells (inall examined tissues) and renal glomerulus cells. So, otherfactors [e.g., drug uptake, pH, protein or Cr content, (29, 30)]may well influence cisDDP-DNA adduct formation.

Stability of DNA Modification. Our results (Fig. 8) indicatea high rate of adduct loss in mouse tissues in the first daysposttreatment and a very slow rate or no loss at all thereafter,

4C. P. Wild, personal communication.

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especially in heart, brain, and liver (nuclear staining for up to81 days after drug exposure), and kidney (nuclear staining forup to 162 days after drug exposure). Differences in adductpersistence between organs could originate from differences incellular turnover rates and/or differences in DNA repair capacity (31). Adduct stability might differ between mice and rats,since Poirier et al. (32) reported little or no decrease in adductlevels (measured by ELISA) in total renal DNA from ratsbetween 6 h and 13 days following a single i.v. injection of 6mg/kg cisDDP. cisDDP-DNA adducts in cancer patients alsoseemed to be relatively stable: DNA modifications in whiteblood cells accumulated when cisDDP was given in 21- or 28-day cycles (7, 32). Fichtinger-Schepman et al. (17) observed afast removal of cisPt(NH3)2d(pGpG) from white blood cells incancer patients during a 24-h period immediately followingtreatment with cisDDP. Taken together, these data suggest thatin human white blood cells an initial phase of rapid loss ofcisDDP-DNA adducts occurs (17), but that complete adductloss takes more than 1 month (7, 32). It is concluded that asubstantial part of cisDDP-DNA interaction products is highlypersistent, both in animals and humans. This persistence mayhave long-term toxicological or carcinogenic implications, e.g.,late toxic effects or an increased risk of second cancer (3).

Sensitivity of the Immunocytochemical Assay. Determinationof the lowest number of cisDDP-DNA adducts per nucleus,which can be visualized by our immunocytochemical technique,was hampered by the widely diverging literature data on theextent of cisDDP-DNA binding in experimental or clinicalsituations. According to Poirier et al. (32) 300 amol cisDDP-DNA adducts/ng DNA, as measured by ELISA, were presentin bulk DNA of rat kidneys 6 h after i.v. injection of 6 mg/kgcisDDP. Poirier et al. (32) and Reed et al. (7) reported adductlevels of 50-200 amol/¿¿gDNA for white blood cells frompatients, 1 day after (cumulative) doses of 100-1500 mg/m2cisDDP. In contrast, Fichtinger-Schepman et al. (9, 17, 33),who used antisera against specific adducts, reported muchhigher modification levels: the level of the cisDDP-DNA adductcisPt(NH3)2d(pGpG) amounted to 0.5-9 fmol/Mg DNA in whiteblood cells of patients 0-24 h after 3 h infusions of 20-100 mgcisDDP/m2 body area. cisPt(NH3)2d(pGpG) adducts in rats 1h after 10 mg/kg cisDDP were as high as 10-400 fmol//¿gDNA, depending on the tissue (34). The latter data were reported to be in good agreement with modification levels measured by AAS analysis in the same tissues. Until this issue issettled it is impossible to make a reliable estimation of thelowest number of adducts per genome which can be detected byour immunocytochemical assay. The detection limit using thedouble peroxidase technique can be as low as 4000 AAF-DNAadducts per diploid genome (12) or 1000-2000 aflatoxin B,-DNA adducts.4

DNA Modification and Histological Alterations. We foundthat tissues, which showed clear, cisDDP-induced histológica!alterations (kidney, pancreas, testis, and duodenum), alsoshowed moderate to high levels of cisDDP-DNA interactionproducts. In rat kidney, the highest total platinum content (asmeasured by AAS, 19), the highest level of cisDDP-inducednuclear staining (Fig. 4) and the most pronounced cisDDP-induced injury of predominantly proximal tubules (18-20, thisreport) are all localized in the inner cortex. The persistence ofcisDDP-induced DNA modification in the murine kidney up toat least 162 days after drug exposure (this paper) correlatesquite well with the persistence of functional kidney damage inC3H/H«mice for up to 15 to 40 weeks after cisDDP exposure,depending on the dose (35). A similar situation is met in

humans, where renal function (as judged from creatinine levelsand "Cr-EDTA clearance) continued to decrease during 12

months after initiation of cisDDP treatment (36). In the duodenum of mice and rats, the most severe cell injury and thehighest levels of DNA modification were confined to epithelialvillus cells. It is more difficult to relate adduct formation inpancreas (mice) or testis (mice, rats) with cytotoxicity, sincehistológica! changes in these organs were not restricted to acertain group of cells. In the liver and the heart (group 2; tissuescollected 6 h after cisDDP administration), moderate or highlevels of DNA modification were not accompanied by histolog-ical changes, although fine-structural studies are lacking. Morphological changes in these tissues were also absent at latertimes after drug exposure (group 3; tissues collected for up to162 days after cisDDP administration). Hypocellularity in thespleen was not accompanied by high levels of DNA lesions. Inthis case, cell loss could also be the result of cisDDP-inducedtoxicity in other tissues, because the red pulp predominantlyconsists of recirculating lymphocytes. We conclude that theinduction of morphological and/or functional tissue damagecan be observed in general only in tissues with a relatively highlevel of initial and/or persistent cisDDP-induced DNA damage.The reverse is not always true: moderate or even high levels ofDNA modification are not necessarily accompanied by histológica!changes.

In summary, we have been able to visualize cisDDP- andCBDCA-induced DNA modifications in rodent tissue sections.The extent of modification varied not only between differenttissues, but also between different cell types within one organ.We showed that a substantial part of cisDDP-DNA modifications are highly persistent. Future research will be concentratedon the role of cisDDP-DNA adducts in the radiosensitizingeffect of cisDDP and the value of adduct measurement intumors as a predictive tool in cisDDP chemotherapy. Preliminary results have already revealed a dose-dependent nuclearstaining in murine solid tumors and cultured tumor cells aftercisDDP exposure.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. W. J. F. van den Vijgh andI. Klein (Free University, Amsterdam) for skillful AAS analysis ofplatinum in DNA samples.

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1987;47:6719-6725. Cancer Res   Philippe M. A. B. Terheggen, Ben G. J. Floot, Ewalt Scherer, et al.   in Rodent Tissue Sections-Diammine-(1,1-cyclobutanedicarboxylato)platinum(II) with DNA

cis-Diamminedichloroplatinum(II) and cisImmunocytochemical Detection of Interaction Products of

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