the presence of a2u-globulin is necessary for d-limonene ......reversibly to the male rat-specific...

(CANCER RESEARCH 51. 3512-3521. July 1. 1991|

The Presence of a2u-Globulin Is Necessary for d-Limonene Promotion of Male Rat

Kidney TumorsDaniel R. Dietrich1 and James A. Swenberg2

Departments of Environmental Sciences and Engineering and Pathology, University of .\orth Carolina at Chapel Hill. Chapel Hill, \orth Carolina 27599-7095

ABSTRACT

In a 2-year carcinogenesis bioassay, d-limonene (di.) induced kidneytumors in male F344 rats, but not in female F344 rats or either sex ofmice. d-Limonene-l,2-oxidc, a metabolite of di.. has been shown to bindreversibly to the male rat-specific urinary protein, a2ll-globulin (a2u-G),resulting in an u .â€¢â€ž-<>-cliemir:il complex that is more resistant to lysosomaldegradation than .>.>â€ž-<â€¢alone. This reduced degradation of a2u-G-chemicalcomplex leads to an accumulation of this protein in the proximal convoluted tubules of the male rat kidney and to the morphological changescharacteristic for .>.â€ž-globulinnephropathy. The only male rat strainknown to be resistant to this renal disease is the a:il-G deficient NCI-Black-Reiter (NBR) rat. The objectives of this study were to determinewhether or not di. causes sustained increases in cell proliferation and haspromoting activity for renal adenomas in male rats and if the male rat-specific urinary protein, "â€¢â€ž-'â€¢-is required. In a 32-week initiation-promotion assay, male F344 and NBR rats were treated with either 0 or500 ppm /Y-ethyl-yV-hydroxyethylnitrosamine (KHEN) in the drinkingwater for 2 weeks. Experimental groups of 31 to 38 rats then received 0or 150 mg d-limonene/kg/day in corn oil for 30 weeks by p.o. gavage 5days/week. Cell proliferation in the proximal tubules was assessed via 5-bromo-2'-deoxyuridine-filled osmotic mini-pumps and immunohisto-

chemistry after 7 weeks (2 weeks EHEN + 5 weeks di.) and at the endof the study (2 weeks EHEN + 30 weeks dL). Preneoplastic andneoplastic lesions were quantified in perfusion-fixed kidneys.

A 5-fold increase in the labeling index of P2-cells was found after 5weeks and 30 weeks of promotion in all dl.-treated E344 rats, whereasno difference between treatment groups was detected in NBR rats. Noincrease in tumors or prencoplastic lesions was detected in dL-treatedNBR rats, whereas a 10-fold increase in renal adenomas and atypicalhyperplasias was found in the EHEN-dL-treated F344 rats comparedwith F344 rats treated with EHEN-corn oil. d-Limonene treatment alonecaused a significant increase in the number of atypical tubules andatypical hyperplasias in F344 rats when compared with the F344 vehiclecontrol. On the other hand, a significantly lower incidence of liver tumorswas found in EHEN-dl.-treated F344 rats compared with F344 ratstreated with EHEN-corn oil, suggesting a chemopreventative effect ofdL on EHEN-induced liver carcinogenesis in F344 rats. It is thusconcluded that dL promotes preneoplastic lesions and renal tumors onlyin the presence of the male rat-specific urinary protein, a2ll-G. Since a2u-G is a species- and sex-specific protein that is causal for both the cytotoxicand carcinogenic response in the male rat, extrapolation of dL carcino-gcnicity data from rat studies to other species, including humans, isprobably not warranted.

INTRODUCTION

There is a growing list of chemicals that induce a male ratspecific renal disease, known as Â«2U-G1nephropathy ( 1). Neither

Received 2/5/91; accepted 4/23/91.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 L'.S.C. Section 1734 solely to indicate this fact.

' Present address: Institute of Toxicology. Swiss Federal Institute of Technology and University of Zuerich. (TI-8603 Schwerzenbach. Switzerland.

: To whom requests for reprints should be addressed.' The abbreviations used are: <>2u-G.<v2u-globulin:NBR. NCI-Black-Rciter: dL.

d-limonene; TMP. 2.2.4-trimethylpentane: I'G, unleaded gasoline: P2. protein-absorbing: EHEN..V-cthyl-.V-hydroxyethylnitrosamine: BrDl . 5-bromo-2'-deo\-

yuridine; PBS. phosphate-buffered saline; AT. atypical tubules; AH. atypicalhyperplasias; RA. renal adenomas; CPN, chronic progressive nephrosis: DMBA.7.l2-dimethylbenz(a)anthracene: AMG. in-microglobulin; H&E, hematoxylinand eosin.

female rats nor either sex of mice, guinea pigs, dogs, or monkeysdevelop this renal disease upon exposure to these chemicals (1-3). The acute stage of Â«iu-Gnephropathy is characterized by anaccumulation of protein droplets in lysosomes of the ?2-seg-ment of the proximal convoluted tubules (4-8). These proteindroplets have been shown to contain accumulations of Â«2u-Gusing immunohistochemistry (9-11).

Â«2u-G,a low molecular weight M, 18,700 protein, is synthesized under androgenic control by the hepatic parenchymal cellsin mature male rats, secreted into the blood, and excreted inlarge amounts (3-19 mg/day) via the urine (12-15). a?u-G isalso found in the urine of female rats, however, at concentrations between 107- and 680-fold lower than those excreted bythe male rat (15). In addition, the female rat does not synthesizethe hepatic form of <v:,,-G,which makes up the bulk of theurinary Â«:U-Gfound in male rats.

In contrast to the commonly used F344 or Sprague-Dawleymale rats, the inbred NBR male rat does not synthesize theandrogen-dependent Â«;u-G(16). These authors proposed thatthe lack of androgenic induction of a2u-G in the liver of NBRrats is the result of a tissue- and gene-specific regulatory defect,since no endocrinological or reproductive abnormalities areapparent in these animals, and the mRNA for the androgen-repressible hepatic protein, SMP-2, is normally regulated. NBRrats acutely exposed to chemicals that cause Â«2,,-Gnephropathysuch as dL, TMP, UG, isophorone, 1,4-dichlorobenzene, lin-dane, and decalin do not develop the disease (11, 17). Thissuggests that the presence of Â«:U-Gis a prerequisite for theinduction of this syndrome.

Indeed, chemicals such as dL, TMP, 1,4-dichlorobenzene,and isophorone and/or their metabolites were shown to bebound reversibly to <Ã•2,,-Gafter in vivo or in vitro exposure (18-21). In in vitro studies, the Â«2u-G-chemicalcomplex proved tobe less degradatale by endopeptidases than <v2u-Galone, suggesting that a decrease in lysosomal degradation is involved inthe accumulation of Â«2u-Gin male rat kidney lysosomes (22).

Continued exposure to chemicals causing Â«2u-Gnephropathyleads to accumulation of Â«2U-G,degeneration, necrosis, andsloughing of individual cells lining the P:-segment of the proximal tubule, and increased rates of cell replication (6, 7, 11, 19,23-25). Short et al. (25) proposed that reduced cell functionand eventual cell death may result from the combination oflysosomal dysfunction, release of digestive enzymes into thecytoplasm, and decreased plasma membrane availability andturnover. The observed increase of cell replication rates wassuggested to be a compensatory reaction of the kidney to thesustained cell loss (3, 6).

Chronic exposure to Â«:U-Gnephropathy inducing agents results in a low but significant incidence of renal tumors in malerats, but not in female rats or either sex of mice (1, 26-32). Aprevious initiation-promotion bioassay revealed that chronicexposure to UG and TMP resulted in accumulation of a2u-G,elevated cell proliferation, and cytotoxicity in the P:-segmentof the male rat proximal tubules, whereas female rats wereunaffected (25). In addition, significantly increased incidencesof preneoplastic lesions and renal tumors were observed inmale, but not in female rats promoted with UG or TMP. These

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d-LIMONENE PROMOTION REQUIRES Â«Â¡â€ž-GLOBULIN

results suggest that the renal tumors diagnosed in male rats,following chronic exposure to Â«:u-Gnephropathy inducingagents, are a sequelae to the induction of Â«2,,-Gnephropathyand the associated sustained increase in cell proliferation. However, conclusive evidence linking <v:â€ž-Gnephropathy and renaltumor formation that demonstrates the requirement of Â«2u-G

for the chemical induction of male rat kidney tumors, has yetto be provided.

An initiation-promotion study using male F344 and Â«2,,-G-deficient NBR rats would elucidate the role of a2tl-G in dLcarcinogenesis. EHEN is a complete carcinogen that selectivelycauses renal epithelial tumors of the proximal tubule, as wellas liver tumors (33-41). d-Limonene was chosen because it isnongenotoxic. causes a2a-G nephropathy. and induces a significantly increased incidence of renal tumors in the male rat in a2-year carcinogenicity bioassay, but is not tumorigenic in anyother organ of either sex of rat or mice (21, 24, 32, 42, 43).

To determine the role of Â«2u-Gin dL carcinogenicity, a 32-week initiation-promotion study in the kidney of male F344and NBR rats was designed that: (a) exposed the rats to thesame dose of dL (150 mg dL/kg/day. 5 days/week in corn oilby p.o. gavage) that was used in the 2-year carcinogenicitybioassay (32); (k) included measurements of cell proliferationafter 5 and 30 weeks of dL promotion; and (c) assessed bothpreneoplastic and neoplastic lesions using the revised nomenclature for renal lesions proposed by Dietrich and Swenberg(44). To achieve a high tumor incidence within the short timeframe of this study, animals were initiated for 2 weeks with ahigh dose (500 ppm) of EHEN in the drinking water.

MATERIALS AND METHODS

Animals

One hundred thirty-eight F344 and 136 male NBR rats approximately 5 to 6 weeks of age were obtained from Charles River BreedingLaboratories (Raleigh. NC) and from the National Cancer Institute(Frederick, MD), respectively. Barrier-sustained animal rooms weremaintained at 22 Â±1Â°C(SE) with a 12-h light-dark cycle. Animals were

allowed free access to food (Regular Lab Chow 3000; Granville MillingCo., Creedmoor, NC) and tap water. Following a 2-week quarantineperiod, the rats were randomized according to body weight into groupsof 31 to 38 animals (see Fig. 1) and housed in pairs in polycarbonate

2Wks

NBR j EHENÃ•NBR [EHEN

NBR

NBR

30 Weeks

Corn Oil (31)

EHEN |X(6)Corn Oil(32)X

F344 | EHEN

F344

F344 Corn Oil (31)

X = BrDU 7-day Mini-Pump Implantation

EHEN: 500 ppmd-Limonene: 150 mg/kg/day 5 days per week

Fig. I. Initiation-promotion experiment with 8-week-old male NBR and F344rats. Experimental design of d-limonene promotion study to determine thepromoting activity of dL on renal tumorigenesis in male F344 and NBR ratsinitiated with EHEN.

shoebox cages on stainless-steel racks fitted with an inline automatedwater system.

Chemicals

EHEN (purity 99.8%; liquid at room temperature) was purchasedfrom Shigematsu & Co., Ltd. (Osaka, Japan) and mixed every 2 daysin distilled water for a final concentration of 0.05%. Samples frommixing containers and drinking dispensers on each animal rack wereanalyzed daily by reverse-phase high-performance liquid chromatogra-phy lo assure accurate and consistent target concentrations. EHEN wasdetected at Am following an injection of a 50-^1 sample, separationonto a Beckman Ultraspherc ODSSu column (4.6 mm x 25 cm), andeluting with a mobile phase consisting of 50% methanol-50% water ata flow rate of l ml/min. EHEN eluted after 2 min and 50 s. A standardcurve of pure EHEN was used as a reference. d-Limonene (99+%; Lot115F0094) was purchased from Sigma Chemical Co. (St. Louis, MO)and was mixed twice weekly in corn oil (MazÃ³la; Best Foods, CPCInternational Inc., Englewood Cliffs, NJ) to a final concentration of 50g dL/liter of corn oil. BrDU (Lot 69F-0049) was used as an exogenousbiomarker for cell proliferation measurements and purchased fromSigma.

Metabolism of d-Limonenc

The in vitro metabolism of dL was evaluated using the procedureoutlined by Watabe et al. (42. 45). d-Limonene and its metabolites wereextracted from the microsomal suspension into CH2Cli, analyzed, andidentified via gas chromatography coupled with a VG 70-250 SEQhybrid mass spectrometer (gas chromatography-mass spectrometry)using electron impact.

Initiation-Promotion Bioassay

The initiation-promotion bioassay protocol is outlined in Fig. 1. Thewater consumption of all groups was monitored during the initiationphase. All surviving animals were sacrificed within 5 days after the endof the study, with the exception of 6 animals/strain of the initiation-control and initiation-promotion group, respectively, which were sacrificed 7 weeks after the beginning of the experiment for cell proliferation studies. To avoid biasing, the sacrifice at the end of the study wentacross exposure groups. The groups were as described below.

Initiation Control. Thirty-eight F344 and 37 NBR male rats wereexposed to 500 ppm EHEN in the drinking water for 2 weeks and thengavaged with corn oil (3 ml/kg/day, 5 days/week) for 30 weeks.

Experimental Initiation-Promotion. Thirty-eight F344 and 37 NBRmale rats were exposed to 500 ppm EHEN in the drinking water for 2weeks and then gavaged with dL in corn oil (3 ml/kg/day, i.e., 150 mgdL/kg/day. 5 days/week) for 30 weeks.

Promotion Control. Thirty-one F344 and 31 NBR male rats weregiven distilled water ad lihitum for 2 weeks and then gavaged with dLin corn oil (3 ml/kg/day, i.e., 150 mg dL/kg/day. 5 days/week) for 30weeks.

Control. Thirty-one F344 and 31 NBR male rats were given distilledwater ad libitum for 2 weeks and then gavaged with corn oil (3 ml/kg/day, 5 days/week) for 30 weeks.

Biological Observations

Animals were observed clinically twice daily for signs of toxicity,behavioral change, and death. Body weights were recorded 3 times/week during the first 8 weeks and biweekly thereafter. Gross andmicroscopic examination of tissues were performed on all animals dyingduring the study, on interim sacrificed animals, and on those animalssacrificed at termination of the study. Grossly observable lesions wererecorded for quantitation and histopathological assessment.

Cell Proliferation Studies

Cell proliferation measurements were carried out at two separatetime points as indicated in the bioassay protocol (Fig. 1). Alzet 7-dayosmotic mini-pumps (Model 2ML1, Lot 52965, flow rate 10.77 Â¿il/h,7-day delivery; Alza Corp., Palo Alto, CA) were filled with 2 ml BrDU

3513



d-LIMONENE PROMOTION RKOIIRES â€ž2u-Gl.OBl'LIN

solution (20 mg BrDU/ml sterile PBS 9240; Irvine Scientific, SantaAna, CA) and implanted s.c. into the animals. Seven days after implantation of the osmotic minipumps, the animals were killed using theprocedure described below. Kidney cells labeled with BrDU were identified via immunohistochemistry (see below).

Histopathology and Immunohistochemistry

All animals sacrificed, whether at the interim time point or at theend of the experiment, were killed using the same method. This methodinvolved anesthetizing the animals with Metofane (Pitman-Moore Inc.,Mundelein, IL) and perfusing the animals via the abdominal aorta withPBS followed by buffered fixative (PBS containing 1% glutaraldehydeand 2'i paraformaldehyde). A detailed account of this procedure is

described by Short et al. (6). The kidneys were removed, weighed, andpostfixed for 24 h in perfusion fixative and then immersed in cold PBS(4Â°C)until further processing. Livers were removed, weighed, and fixedin 10rr phosphate-buffered formalin solution. Both kidneys from each

rat were cut into 6 transverse slices, each 3 mm thick, embedded insurgipath-paraffin (EM-400 Embedding Medium: Surgipath MedicalIndustries Inc.. Greyslake, IL), and cut to 2-^m-thin sections. Sectionsfrom each block were stained with hematoxylin-eosin for histopatho-logical assessment. All sections were coded and read without knowledgeof treatment to quantitate the number of AT. AH. and RA. The revisednomenclature for preneoplastic and neoplastic lesions of the kidneyproposed by Dietrich and Swenberg (44) was used to distinguish AT.AH, and RA.

Unstained kidney sections from animals with BrDU pump-implantsfor measurement of cell proliferation were stained immunohistochem-ically. This procedure involved using a monoclonal primary antibody(anti-BrDU IgG: Becton-Dickinson Co.. RTP. NC) to detect nucleithat had incorporated BrDU. The primary antibody was in turn detectedwith a secondary link antibody, which links the primary antibody to alabel containing an enzyme for a chromogen reaction. The link andlabel are components of a supersensitive kit available from BioGenexLaboratories Inc. (San Ramon, CA). To quantitate the number oflabeled and unlabeled Pi-cells, respectively, a minimum of 500 P;-cells

in at least 12 randomly chosen microscopic fields of the kidney cortexwere counted at a magnification of x400. The labeling index of P:-cells

was calculated as the percent of labeled cells among the total of labeledand unlabeled cells for each animal analyzed. The labeling indexes ofeach exposure group are given as mean Â±SEM.

Unstained kidney sections from animals with BrDU pump-implantswere stained immunohistochemically for the presence of Â«:U-Gusingthe procedure outlined by Dietrich and Swenberg (11).

Statistical Procedures

Increased incidences of liver tumors, AT. AH. and RA were testedfor significance using the 2-sided Fisher's exact test. The mean number

of AT, AH, or RA per rat, as well as the P;-labeling indices, were

analyzed for significant differences between rat strains and treatmentgroups using the 2-sided Student's / test. Significance was tested at 3

different levels. P < 0.05. P < 0.01. and P < 0.001. These statisticaltests were performed using the RS/1 computerized software package(BBN Software. Cambridge. MA).

RESULTS

Metabolism of d-Limonene

To verify that both NBR and F344 rats metabolize dL to d-limonene-l,2-oxide, the metabolite that was shown to bindreversibly to Â«2u-G(21, 22), liver microsome preparations weremade from livers of both strains. The gas chromatography-mass

spectrometry analysis of the hepatic microsome extracts fromNBR and F344 rats demonstrated comparable formation of d-limonene-1,2-oxide.

EHEN Dosing, Water Consumption, and Body Weight

The actual EHEN concentrations in the drinking water analyzed during the initiation phase averaged 509 Â±8 ppm EHEN(average Â±SEM; // = 30). In comparison with the F344 andNBR rats that received distilled water, the water consumptionof both F344 and NBR rats receiving EHEN containing waterwas decreased. This decrease in water consumption was alsoassociated with lower body weight gain in EHEN-initiatedanimals during the initiation period. The total EHEN consumed by F344 and NBR rats was calculated using a time-weighted average of body weight of the strain exposed duringthe initiation phase of the study. F344 and NBR rats wereshown to consume comparable amounts of EHEN, i.e, 58 and55.3 mg EHEN/kg/rat/day, respectively.

General Animal Observations

Occasional clinical observations were noted during the study,including minimal ocular discharge, ruffled fur. and rare casesof alopecia over the back, sides, and/or ventral abdominal areain male NBR and F344 rats. These changes were observed withsimilar frequency in all exposure groups.

No significant differences were observed in spontaneousdeath rate between exposure groups or strains. Of the 3 F344and 2 NBR rats that died during the study, one F344 succumbedduring implantation of the osmotic mini-pump for cell proliferation measurement. One F344 and one NBR rat were killedduring the study due to their unhealthy appearance and abnormal behavior. One F344 and NBR rat died spontaneouslyduring the study.

No significant terminal body weight, liver weight, kidneyweight, and relative liver/body weight of relative kidney/bodyweight differences were observed in any groups. The averagebody weight of NBR groups was significantly higher than thoseof the F344 groups. These differences, however, were strainand not treatment related.

Cell Proliferation Data

The results of the cell proliferation measurements carried out7 weeks and 32 weeks after the beginning of the study aresummarized in Table I. Exposure of NBR and F344 rats toEHEN did not induce significantly different rates of P2-cellproliferation when compared with those measured in the respective control groups. A 4- to 5-fold increase in cell proliferation was observed following treatment with dL in F344 rats,but not in NBR rats. This significant increase in P:-cell turnoverwas maintained throughout the period of dL exposure. Nostrain differences with regard to cell proliferation rates wereobserved in the control groups.

Table I Average labelitiK index in P<-crll\ measured 7 unti 32 weeks after thebeginning of the study

Labeling index (rÃ)

ExposuregroupsEHEN-corn

oilEHEN-d-limoneneH,O-d-limonencHiO-corn

oilEHEN-cornoilEHEN-d-limoneneHiO-d-limoneneHjO-corn

oilSlrainNBRNBRNBRNBRF344F344F344F3447

wk5.46Â±0.51*5.44

Â±0.44NMfN

M5.95Â±0.2726.19Â±

\.lbjN

MNMif666632

wk5.22

Â±0395.604.664.485.1820.5524.150.450.200.320.391.29*l^4.63

0.26n77776766" n. number of animals." Mean Â±SEM.' NM. not measured.''Significantly higher than the F344 EHEN-corn oil. water-corn oil group, or

any of ihe NBR groups. Student's r test (/>< 0.001).

3514



d-LIMONF.NE PROMOTION REQUIRES â€žÂ¡â€ž-GLOBTL.IN

Pathological Findings

Gross Observations

Kidney. Gross examination revealed one renal tumor in a F344 rattreated with EHEN-corn oil and one renal tumor in a F344 rat treatedwith EHEN-dL. In the first case, the tumor exceeded 15 mm indiameter, whereas in the latter case the tumor diameter was 10 mm.No grossly visible renal tumors were observed in the F344 promotioncontrol, the F344 control, or in any of the NBR groups.

Other Organs. With the exception of one large mammaryfibroma found in a NBR rat treated with EHEN-dL and livertumors found in all groups/strains treated with EHEN, notumors were observed in any other organs, regardless of treatment or strain. Liver tumors were found only in groups treatedwith EHEN. d-Limonene treatment of EHEN-initiated F344rats significantly decreased the liver tumor incidence (Table 2).No such effect was found in NBR rats. However, NBR rats hadsignificantly lower numbers of liver tumors than F344 rats.

Necropsy and microscopic analysis of the 2 rats killed duringthe study revealed extensive enlargement of the spleen interpreted as mononuclear leukemia in the F344 rat and renalinfarcts of unknown etiology in the NBR rat. The cause ofdeath was not identified in the case of the F344 and NBR ratthat died spontaneously during the study, but the deaths didnot appear treatment related.

Histopathological Obsen'ations at 7 Weeks

Microscopic observations of the hematoxylin-eosin sectionstaken from EHEN-dL or EHEN-corn oil-treated NBR ratssacrificed 7 weeks after the beginning of the experiment revealed no hyaline droplets or proteinaceous cases within theouter zone of the outer medulla. F344 rats treated with EHEN-dL, on the other hand, demonstrated increased numbers andsize of hyaline droplets containing accumulations of Â«2u-G,leading to cell necrosis and exfoliation when compared withF344 controls (Fig. 2). In addition, proteinaceous casts werepresent within the outer zone of the outer medulla and regenerative tubules were identified within the P;-segment of the

renal cortex.

Morphology of Preneoplastic and Neoplastic Renal Lesions at32 Weeks

Microscopic observations of the kidney are summarized inTables 3, 4, and 5, respectively.

Atypical Tubules. AT were characterized as tubules lined bya single or multiple layer of altered cells (i.e., basophilic, clear,eosinophilic, oncocytic, or chromophobic). The lumen of ATcould be completely obstructed with altered cells, but the sizeof AT did not exceed twice the size of normal, unchangedtubules of the same segment in the surrounding tissue (Figs. 3and 4).

Table 2 Incidence of grossly observable liver tumors in FÃŒ44and .\HR ratstreated with EHE.\ within the scope of a 32-week initiation-promotion study

Tumor

i

ExposuregroupsEHEN-corn

oilEHEN-d-limoneneEHEN-corn

oilEHEN-d-limoncneStrainNBRNBRF344F344No./total12/3111/3126/3017/31%38.7"35.5"86.754.8*

" Significantly lower than the liver tumor incidence in [-'344 rats. Fisher's exact

test (P< 0.001).'Significantly lower than the corresponding F344 EHEN-corn oil group.

Fisher's exact test (/><0.01).

Fig. 2. Accumulation of Â«3â€ž-G(^) and exfoliation of renal tubule epithelialcells (D)in the renal cortex of a male F344 rat initiated with EHEN and promotedfor 5 weeks with dL. n2ll-G immunohistochcmical stain, x 350.

Table 3 Incidence and total number of AT and mean number of AT per rat inNBR and F344 rats

TreatmentEHEN-corn

oilEHEN-d-limoncncH;O-d-linionencHiO-corn

oilEHEN-cornoilEHEN-d-limoncneH2O-d-limoneneH3O-corn

oilStrainNBRNBRNBRNBRF344F344F344F344Incidence27/3127/3026/3127/3030/30'31/3T30/31'21/31%879084901001009768Total

AT2872411431404692015330103AT/rat"9.6Â±1.2*8.04.64.715.665.010.71.0*0.80.8US'3.4'1.3'3.3

0.7" Mean Â±SEM.'Significantly higher than the NBR water-d-limoncne or water-corn oil group.

Student's/test (P< 0.01).'Significantly higher than the F344 water-corn oil group. Fisher's exact test

1Significantly higher than the F344 water-d-limonene. water-corn oil group,and any of the NBR groups. Student's t test (P < 0.01).

'' Significantly higher than the F344 EHEN-corn oil. water-d-limonene. water-corn oil group, and any of the NBR groups. Student's t test (P < 0.001).

^Significantly higher than the F344 water-corn oil group, NBR waler-d-limonene. and NBR water corn oil group. Student's t test (P < 0.001 ).

Atypical Hyperplasia. AH existed in several forms, i.e., tubules with homogeneous or heterogeneous aggregation of proliferating altered cells that are single- or multilayered, with orwithout central necrosis, that may or may not have mitoticfigures, and are cystic, solid, or dilated in appearance. With theexception of the cystic type of AH. these lesions were largerthan AT but did not exceed 10 times the size of the surroundingnormal tubules and generally involved only one tubule.Compression or infiltration of the surrounding tissue as well asa capsule or pseudocapsule were not observed (Figs. 5 and 6).The cystic types of AH were comparable in size to nonpreneo-plastic cysts but, in contrast to the latter, contained a mono- ormultilayer of altered cells with invaginations or papillary protrusions into the lumen.

3515



d-LIMONENE PROMOTION REQL'IRES â€žÂ¡â€ž-(ilOHI I IN

Renal Adenoma. RA were or were not encapsulated by a verythin layer of fibrous connective tissue and were noninvasive,but the surrounding tissue showed signs of compression (Figs.7 and 8). RA were composed of either a homogeneous orheterogeneous altered cell population. Mitotic figures and/ornecrotic foci were observed. All of the RA observed in thisstudy were of the solid RA type (Fig. 8), which appeared asmassive aggregations of altered cells with either solid, tubular,or cordlike structures. No renal carcinomas were detected.

The majority of AT, AH, and RA were observed in the renalcortex. Few preneoplastic lesions were observed in the outermedulla and none in the inner medulla. The relevance of thePi-segment as an important site for preneoplastic and neoplas-

tic lesion development is depicted in Figs. 3, 4, 5, and 6. InFigs. 3 and 4, it is shown that only the P:-segment contains

altered cell types, i.e., form an AT, while the glomerulus andthe P|-segment remain normal. The next stage of progression,

namely AH, is shown in Figs. 5 and 6. The AH depicted in Fig.6 contains the same altered cell type as the AT shown in Fig.4, and some normal cells within the lesions can positively beidentified as P-cells. This suggests that this AH has most likelyprogressed from an AT within the P:-segment.

Of the two renal tumors observed during necropsy, the largerone of a F344 rat treated with EHEN-corn oil was determined

microscopically to be a mesenchymal tumor. The smaller one,of a F344 rat treated with EHEN-dL. was a renal adenoma

(Fig. 8).

Table 4 Incidence unti lutai number of All anil mean number of All per rat inNBR and I-'ÃŽ44rats

TreatmentEHEN-corn

oilEHEN-d-limonencHjO-d-limoncneHÂ¡O-corn

oilEHEN-cornoilEHEN-d-limonencHiO-d-limoneneHjO-corn

oilStrainNBRNBRNBRNBRF344F344F344F344Incidence6/315/302/313/3020/30*31/31''10/31'0/31%191761067100320Total

AH662437482130AH/rat"0.2

Â±0.10.2Â±0.10.1Â±0.0O.I+0.11.2 Â±0.2'15.5

Â±1.5'0.4Â±0.1*0.0

Â±0.0" Mean Â±SEM.* Significantly higher than the F344 water-d-limonene. water-corn oil group,

and any of the NBR groups. Fisher's exact test (P < 0.01).' Significantly higher than the F344 water-d-limonene. water-corn oil group,

and any of the NBR groups. Student's i test (/>< 0.001).''Significantly higher than the F344 EHEN-corn oil, water-d-limonene. water-

corn oil group, and any of the NBR groups. Fisher's exact test (P < 0.001).' Significantly higher than the F344 EHEN-corn oil. water-d-limonene. water-

corn oil group, and any of the NBR groups. Student's i test (P < 0.001).'Significantly higher than the F344 water-corn oil group, and any of the NBR

groups. Fisher's exact test (P < 0.05).* Significantly higher than the F344 water-corn oil group, and any of the NBR

groups. Student's / test (P < 0.05).

Table 5 Incidence and total number of RA and mean number of R.-tper rat in,\BR and FÃŒ44rats

TreatmentEHEN-corn

oilEHEN-d-limoneneH;O-d-limoneneH2O-corn

oilEHEN-cornoilEHEN-d-limoneneHiO-d-limoncneH2O-corn

oilStrainNBRNBRNBRNBRF344K344F344F344Incidence0/310/300/310/301/309/31*0/310/31%

Total RARA/rat"0000329000000111000.000.000.000.000.030.350.000.000.000.000.000.030.

13r0.000.00

0.00" Mean Â±SEM.'Significantly higher than the corresponding F344 EHEN-corn oil group.

Fisher's exact test (P < 0.05).'Significantly higher than the corresponding F344 EHEN-corn oil group.

Student's t test (/>< 0.05).

Fig. 3. C'hromophobic AT observed in the P./Pi-segment

renal proximal tubule of a male F344 rat treated with dl. only.boundary of the

H&E. x 350.

Fig. 4. Clear cell AT observed in the P2-segmenl of the renal proximal tubuleof a F344 male rat initiated with EHEN and promoted with dl.. H&E. x 350.

3516



d-LlMONF.NE PROMOTION REQUIRES ,<Â¡

Fig. 5. Chromophobic AH observed in the renal cortex of a F344 male rattreated with dL only. H&E. x 175.

%&: y.â€¢> .'"Â£**;' .' ^:" â€¢'I ".â€¢'

Â»L;;jt ; * ^ . ^te'Ã•LJfc.^if'li o * 'rÂ¿v " -*v v-Â¿^''r-'o .; â€¢>^'^--â€¢â€¢\ ' â€¢ Ã• â€¢*</ - /-';?Ar -h^

Fig. 6. Clear cell AH observed in the P2-segment of the renal proximal tubuleof a F344 male rat initiated with EHEN and promoted with dL. H&E. x 350.

'vV-v -*â€¢;Â¿Â«C'V-'-TÃ„-.'vSfc- vÃ¶?V'"<'''*:>; " '" ^'* - :*.'â€¢

Fig. 7. Clear cell adenoma observed in the renal cortex of a F344 male ratinitiated with EHEN and promoted with dL. H&E. x 175.

Incidence of A T, AH, and RA and Mean Number of AT, AH,and R.-Ã•per Rat

The comparison of incidences of AT, AH. and RA and ofmean numbers of AT, AH, and RA per rat observed in NBRrats with those seen in F344 rats, clearly demonstrates that dLhas no effect on renal neoplasia in NBR rats (Tables 3-5). InF344 rats, however. dL treatment with or without precedingEHEN initiation, significantly increased the incidence andmean number of AT and AH per rat, when compared with theF344 control group (Tables 3 and 4). EHEN initiation and dLpromotion resulted in a 10-fold increase in incidence of RA incomparison with the F344 initiation control group (Table 5).This increase was clearly dL treatment related and statisticallysignificant. No tumors were found in the F344 promotioncontrol group, however, many of the AH were large (Fig. 5),suggesting that renal adenomas would have formed, had thebioassay lasted longer. No tumors were found in any of theNBR groups. The incidence and mean number of AT and AHper rat were increased by EHEN treatment in F344 rats and toa lesser extent in NBR rats (Tables 3 and 4). Indeed, NBR ratstreated with EHEN-corn oil had significantly lower incidencesof AT and AH than the corresponding F344 treatment group.

The control groups of both strains had a comparable incidence, total number of AT, and mean number of AT per rat.This demonstrates that an appreciable number of AT are present in untreated rats.

Non-Neoplastic Lesions of the Kidney

Of the non-neoplastic lesions described for the kidney byDietrich and Swenberg (44), predominantly the early stages ofCPN were observed at the end of the bioassay. Lesions char-

3517



d I.IMONKNi: PROMOTION RI OÃIKIÃ•Sâ€žÂ¡â€ž-(Â¡lOBIÃŽLIN

Fig. 8. Large basophilic adenoma found in a male F344 ral inilialed withEHEN and promoted with dL. H&E, x 9.

acteristic of these early stages of CPN included tubules withthickened or wrinkled basement membranes containing basophilic staining cells. No treatment-related increases of CPNwere noted in either strain.

DISCUSSION

The initiation-promotion study reported here clearly demonstrates that dL promotes the formation of atypical tubules,atypical hyperplasias, and renal adenomas only in the presenceof <v:,,-G, thus only in male F344 rats but not in the Â«2U-G-deficient male NBR rats. These conclusions were drawn aftercareful evaluation of the data on biological and statisticalgrounds. F344 as well as NBR rats were shown to metabolizedL to the nongenotoxic epoxide. d-limonene-l,2-oxide, thuscorroborating earlier findings by Watabe et al. (42. 45). Despitecomparable metabolism of dL in both strains. NBR male ratsdid not develop renal disease following 5 weeks of treatmentwith dL. These results were not unexpected, as acute exposureof NBR rats to dL did not elicit any response in the kidneys ofthese rats (II). In contrast, male F344 rats demonstrated increased numbers of hyaline droplets and cell necrosis in therenal cortex, and proteinaceous casts within the outer /one ofthe outer medulla. These lesions compare well with thosecharacteristic for a2u-G nephropathy (2, 3). The proteinaceouscasts observed in male F344 rats, however, were not as prominent as reported for other previous studies with dL (24, 32).This is most likely not a treatment-related effect, but the resultof the kidney perfusions, which may have washed away part ofthe proteinaceous casts. The significantly increased rate of P2-cell proliferation demonstrated only in dL-treated F344 malerats suggests that increased rates of cell proliferation representa compensatory response of the kidney to sustained cell loss.

This suggestion agrees well with the dose-response relationshipsbetween hyaline droplet accumulation and proximal tubule cellproliferation found by Short et al. (6, 25) and Charbonneau etal. (19) on other a2u-G nephropathy inducing chemicals, andwith data published by Alden (46), who has shown a correlationbetween the hyaline droplet response, increased mitotic indexin proximal convoluted tubules, and elevation of the number ofcells excreted hourly in the urine (exfoliated necrotic tubulecells), using 2 dose-levels of dL. The absence of renal lesionsand increased cell proliferation rates in dL-treated NBR ratsfound in this study further corroborate the hypothesis thatincreases in P:-cell proliferation following dL treatment are asequel to Â«:,,-Gnephropathy. Similar conclusions were drawnby Short et al. (25), who found that cell proliferation rates infemale F344 rats were not increased following up to 50 weeksof exposure to UG or TMP.

Cell proliferation rates measured after 30 weeks of dL promotion were similar to those after 5 weeks of dL promotion,being increased only in dL-treated F344, but not in the NBRrats or in the F344 control and F344 initiation control groups.In addition, significantly elevated incidences and mean numbersof AT. AH, and RA per rat were found in dL-treated F344 rats,irrespective of whether they were treated previously with wateror EHEN. No such response was demonstrated in dL-treatedNBR rats. The majority of these lesions were located in the P?-segment of the renal cortical tubules (Figs. 3-8). coincidentwith the location of increased cell proliferation. These observations compare well with data published by Short et al. (25,33), who also found increased cell proliferation rates, as well asthe majority of preneoplastic lesions and tumors in the renalcortex of F344 male rats exposed to UG and TMP. In addition,earlier studies conducted with these agents have shown that P;-cell proliferation rates and tumor incidence respond in a dose-dependent fashion (6). Taken together, these data suggest thatsustained increases in cell proliferation are causally related tothe formation of preneoplastic and neoplastic lesions.

d-Limonene was shown to be carcinogenic in male F344 rats,but not in female F344 rats or either sex of mice in a 2-yearbioassay (32), and UG and TMP induced increased rates of cellproliferation, preneoplastic lesions, and tumors in male but notfemale F344 rats exposed for 65 weeks (33), thus demonstratingthat animals that do not synthesize Â«2U-Gor excrete it in lowamounts do not develop renal tumors. Carcinogenesis is knownto be a multistep process that involves 2 or more genetic eventsand requires cell proliferation (47-51). The genetic events canresult from point mutations, chromosomal rearrangements,insertions or deletions of genes, and gene amplification. Allcells have spontaneous DNA damaging events occurring continuously (52). If cell replication takes place prior to repair ofthese lesions, some of them will be converted to mutations.Chronic increases in cell proliferation decrease the time available for DNA repair prior to DNA replication, thus increasingthe probability of mutations and for donai expansion of initiated cells. Such clonal expansion increases the probability ofadditional genetic events occurring in initiated cell populations,leading to progression (53). Thus increased cell proliferationfollowing exposure to dL could contribute to enhanced fixationof spontaneous mutations, leading to increased numbers ofinitiated cells, clonal expansion of such initiated cell populations, and to the formation of tumors. Spontaneous mutationsare assumed to stem from genetic events such as depurinationand deamination of DNA; formation of covalent DNA adductsby normal, active cellular metabolites; DNA damage by oxygenfree radicals produced in cellular metabolism; and errors in

3518



d-UMONKNK PROMOTION RKQt 1RES (>2u-GLOBl'LIN

DNA replication (52). This assumption is corroborated by theappreciable incidence and number of preneoplastic lesions observed in the control groups of both strains in this study, andby the observation that control animals from various strains oflaboratory rodents show a low but significant incidence ofspontaneous tumors (54).

Additional DNA damage is induced by treating animals withDNA alkylating agents, such as EHEN, which leads to anincreased number and incidence of preneoplastic lesions, incomparison with untreated animals. This was demonstrated inboth strains of rats treated with EHEN, although NBR ratsappear to be somewhat less susceptible to EHEN-initiatedcarcinogenesis. Indeed, significantly lower incidences of livertumors and renal preneoplastic lesions were observed in EHEN-corn oil-treated NBR rats when compared with the corresponding F344 group. The evaluation of carcinogenesis studies isclearly enhanced by knowledge of preneoplastic lesions andmechanisms (33. 36. 37. 39,44, 55-66). As AT and AH containaltered cell types similar to those found in adenomas (Figs. 3-

8) and carcinomas (44, 67), it seems reasonable to assume thatthese early lesions represent tumor precursors. Although notumors were found in the F344 promotion control, many of theAH were large, suggesting that renal adenomas, as found in the2-year bioassay (32). would have formed had this study lastedlonger. Indeed. Dietrich and Swenberg (44) reported that theprobability of neoplastic progression of renal lesions increaseswith the size and progressional stage of the lesion. The importance of lesion size in tumorigenesis and malignancy of renallesions in rodents was also noted by Hard (68). Therefore, thepresence of significantly elevated incidences and total numbersof preneoplastic lesions in the F344 promotion control group,in conjunction with the evidence indicating that the presenceof tt2u-G is necessary to induce the observed increase in cellproliferation, suggests that the presence of Â«2,,-Gis necessaryfor the formation of preneoplastic lesions and tumors in thekidneys of male F344 rats following chronic treatment with dL.

When quantitating the number of liver tumors in EHEN-exposed F344 and NBR rats, it became apparent that althoughthere was no observable difference of liver tumor incidence inNBR rats, F344 rats treated with EHEN-dL had a significantlylower tumor incidence than F344 rats treated with EHEN-cornoil (Table 2). This 30% reduction of tumor incidence in F344rats treated with dL is consistent with a postinitiation anticar-cinogenic effect of dL. Anticarcinogenicity of dL has previouslybeen shown in C57BL/6 Jax mice given s.c. injections ofdibenzypyrene and in strain A mice with spontaneous andcarcinogen-induced lung adenomas (69). In both cases, postinitiation treatment of the animals with dL resulted in a 30%decrease in s.c. and lung tumor incidence. d-Limonene was alsoshown to inhibit DMBA-induced mammary cancer in femalerats when given before, during, or after the initiation withDMBA (70, 71). Postinitiation anticarcinogenicity of dL wasalso reported in Wistar Furth rats treated with A'-nitrosometh-

ylurea (72). In the latter case, postinitiation treatment with dLresulted in a 50% reduction in the incidence of mammarytumors. In addition, rat primary mammary carcinomas inducedwith DMBA were demonstrated to regress partially or completely after prolonged treatment with dL (73). thus indicatingthat not only chemopreventive but also antitumorigenic characteristics are inherent to dL. The anticarcinogenic and antitumorigenic effects of dL stand in contrast to the carcinogenicactivity of dL in male F344 rats reported in this study and inthe 2-year bioassay (32).

Having established that the presence of Â«:â€ž-Gis mandatory

for the formation of male rat kidney tumors following treatmentwith dL. the question arises as to whether extrapolation of dLcarcinogenicity data to other species, including humans, iswarranted. Most compounds that are carcinogenic in animalsare generally assumed to pose some risk to humans. In the caseof dL and other Â«:â€ž-Gnephropathy inducing compounds, the

carcinogenic mechanism is clearly associated with the presenceof a specific urinary protein (o:,,-G) not found in humans orany other species. Several proteins sharing amino acid sequencehomology with Â«:â€ž-Ghave been identified in the serum andurine of various species including humans (74-76). The presence of these partially homologous proteins in humans raisedconcern as to the possible interaction of these low molecularweight proteins with n2u-G nephropathy inducing agents, thusquestioning the male rat specificity of <v:â€ž-Gnephropathy. Assuming that a homologous protein reversibly binds the aforementioned chemicals, the protein needs to be excreted into theplasma in large amounts, freely filtered by the glomerulus.readily reabsorbed in the proximal tubules, and catabolized inthe lysosomes of the proximal tubule epithelial cells at a slowerrate than normal upon binding one of the chemicals in order toinduce similar lesions to Â«2,,-Gnephropathy. Based on estimated daily average urine production and body weights of ratsand humans. Olson et al. (77) showed that rats excrete approximately 90 times more total protein than humans. Of the totalprotein excreted, the predominant fraction in rats consisted oflow molecular weight proteins (M, 18,000), whereas a predominance of high molecular weight proteins (M, 66.000) was foundin humans. The small amount of low molecular weight proteinexcreted by male humans was identified as Â«,-acidglycoprotein,AMG, myoglobin, and /ii-microglobulin. Of these 4 proteins,only Â«i-acid glycoprotein and Â«i-microglobulin share aminoacid sequence homology with Â«:U-G(74, 76). Â«,-Acidglycoprotein and AMG are synthesized in the liver of rats and humans(78-81); have been purified from the urine of rats and humans,as well as the urine of rabbits and guinea pigs in the case ofAMG (75. 77); and thus permit a direct comparison with a2u-G.

If Â«i-acidglycoprotein and AMG reversibly bind Â«:,,-Gneph-ropathy-inducing chemicals and/or their metabolites with thesame affinity as a2ll-G, then one would expect that female rats,male NBR rats, rabbits, and guinea pigs would also developrenal disease following treatment with these chemicals. However, male NBR rats, female rats, and guinea pigs do notaccumulate protein in the renal cortex following treatment withÂ«2U-Gnephropathy-inducing agents and thus are refractory tothis disease (2. 3. 11. 17. 82-84). In addition, mice, whichexcrete comparable amounts of the low molecular weight mouseurinary protein with greatest amino acid sequence homology toÂ«2u-G(approximately 90%) (1), do not develop the proteinrelated nephropathy or renal tumors following chronic exposureto dL and other (V:U-Gnephropathy-inducing agents (26-30,32). Therefore the assumption seems reasonable that dL andother <v:,,-Gnephropathy-inducing compounds are carcinogenicin the kidney of male rats, but are unlikely to be carcinogenicfor humans.

In conclusion, an Â«:,,-G-relatedpromoting effect of dL on P:-

cell proliferation and on formation of AT, AH. and renaladenomas has been demonstrated in male F344, but not in theÂ«2U-G-deficientmale NBR rats. Furthermore, the data alsosuggest that the AT and AH demonstrated in F344 rats treatedwith dL only are most likely precursors to renal tumors asobserved following a 2-year administration of dL to male F344rats (32).

3519



d-LlMONENE PROMOTION REQUIRES a2u-GLOBULIN

ACKNOWLEDGMENTS

We wish to acknowledge the excellent efforts of Dr. David Small,who helped procure adequate numbers of male NBR rats for this study.In addition, we would like to express our deepest gratitude to ourlaboratory technician, Janice E. Weaver, whose motivation and extraordinary dedication throughout the study positively influenced the successof this project. In addition, we thank Patricia Upton for her assistanceduring animal sacrifices at the end of this study. Furthermore, we thankCornelia Dietrich for the excellent services rendered in conjunctionwith the photographs used in this publication. Last but not least, theexcellent animal husbandry services provided by the laboratory animalpersonnel of the University of North Carolina are gratefullyacknowledged.

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1991;51:3512-3521. Cancer Res Daniel R. Dietrich and James A. Swenberg Promotion of Male Rat Kidney Tumors

-Globulin Is Necessary for d-Limonene2uαThe Presence of

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the presence of a2u-globulin is necessary for d-limonene ......reversibly to the male rat-specific...

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