[CANCER RESEARCH 49. 4282-4288. August 1, 1989]

Cadmium Carcinogenesis in Male Wistar [Crl:(WI)BR] Rats: Dose-Response

Analysis of Effects of Zinc on Tumor Induction in the Prostate, in the Testes,and at the Injection SiteMichael P. Waalkes,1 Sabine Rehm, Charles W. Riggs, Robert M. Bare, Deborah E. Devor,2 Lionel A. Poirier,1Martin L. Wenk, and John R. Henneman4

Inorganic Carcinogenesis and Tumor Pathology and Pathogenesis Sections, Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National CancerInstitute, Frederick, Maryland [M. P. W., S. R., R. M. B., L. A. P.]; Data Management Services, Frederick Cancer Research Facility, Frederick, Maryland [C. W. R.];and Microbiological Associates, Inc., Bethesda, Maryland [D. E. D., M. L. W., J. R. H.J

ABSTRACT

The ability of zinc acetate to modify the carcinogenic effects of CdCl2in male Wistar [Crl:(WI)BR] rats was studied over a 2-year period.Groups of rats received a single s.c. injection of Cd (30.0 ¿imol/kg)in thedorsal thoracic midline or ¡.HI.in the right thigh at time 0. Zinc was givenin three separate s.c. doses of 0.1, 0.3, or 1.0 mmol/kg (at —¿�6,0, and

+18 h relative to cadmium) in the lumbosacral area or p.o. at 100 ppmin the drinking water (—2to +100 weeks). Cadmium treatments (s.c.)

resulted in the appearance of tumors at the injection site and in the testes.The incidence of s.c. injection site tumors (mostly mixed sarcomas) wasmarkedly reduced by high dose (1.0 mmol/kg) s.c. zinc (50% reduction)and was almost abolished by p.o. zinc (92% reduction). Testicular tumors(mostly Leydig cell adenomas) induced by s.c. cadmium were reduced ina dose-related fashion by zinc and were found to be highly dependent onthe ability of zinc to prevent the chronic degenerative effects of cadmiumin the testes. Oral zinc had no effect on s.c. cadmium-induced testiculartumors, while i.m. cadmium alone did not induce these tumors. In rats inwhich s.c. cadmium-induced testicular tumors and chronic degenerativeeffects were prevented by zinc (1.0 mmol/kg, s.c.), a marked elevation inprostatic tumors (exclusively adenomas) occurred (control, 9.6%; cadmium plus high zinc, 29.6%). Cadmium given i.m., which did not resultin testicular tumors or degeneration, also induced an elevated incidence(42.3%) of prostatic tumors, again indicating a dependence on testicularfunction. Prostatic tumor incidence was also significantly elevated(25.0%) in rats receiving 1.0 mmol/kg zinc, s.c., in combination with i.m.cadmium. These results indicate that zinc inhibition of cadmium carci-nogenesis is a complex phenomenon, depending not only on dose androute but also on the target site in question.

INTRODUCTION

Various studies have indicated that cadmium is a potentheavy metal carcinogen in experimental animals (1-13) and ispossibly carcinogenic in human populations exposed eitheroccupâtionally or environmentally (14-19). Target sites forcadmium Carcinogenesis in rodents have been shown to includethe s.c. or i.m. injection sites (1-7, 11-13), the testes afterparenteral exposure (4-8, 12, 13), the lung after chronic inhalation (9-10), and, recently, the ventral prostate following parenteral exposure (13). The specific tumor types generated bycadmium exposure include sarcomata at the s.c. or i.m. injectionsite (1-7, 11-13), Leydig cell adenomas in the testes (4-8, 12,13), carcinomas in lung (10), and adenomas of the ventral

Received 11/16/88; revised 4/6/89; accepted 5/3/89.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.

1To whom requests for reprints should be addressed, at Laboratory of Comparative Carcinogenesis, NCI-FCRF, Building 538, Room 205E, Frederick, MD21701.

2 Present address: Laboratory of Comparative Carcinogenesis, NCI-FCRF,

Frederick, MD 21701.3Present address: Division of Chemical Toxicology, NCTR, Building 15,

Jefferson, AR 27079.' Present address: Biological Carcinogenesis Development Program, Program

Resources, Inc., NCI-FCRF, Frederick, MD 21701.

prostate (13). The full spectrum of epithelial tumors of theprostate, including adenocarcinomas, can be induced by directinjection into the tissue (20, 21). Some exceedingly rare tumorshave also been seen in the testes of cadmium-treated rats,including seminomas (22), a rete testes adenocarcinoma, and aSertoli cell tumor (23), although a definitive association withcadmium has not been established due to the small numbersinvolved. In humans, evidence exists that cadmium exposure islinked to tumors of the prostate (14-19), lung (18, 19), andkidney (14). The reports of induction of tumors of the lung (10)or prostate (13) in rats exposed to cadmium are, therefore,consistent with the epidemiológica! data available in the literature.

The toxic effects of cadmium in laboratory animals can bereduced or prevented by prior or concomitant treatment withthe essential trace element zinc (4,5,12,24,25). Zinc treatmentcan ameliorate both the acute effects of cadmium (12, 24, 25)and the chronic effects, such as Carcinogenesis (4, 5). Forinstance, the work of Gunn et al. (4, 5) clearly indicates that,in rodents, zinc, even when given s.c. at a location remote tothe s.c. cadmium injection, will antagonize the formation ofboth injection site tumors and testicular tumors induced bycadmium. The exact mechanism by which zinc reduces thecarcinogenic effects of cadmium is unknown. Furthermore, dataconcerning relationships to dose and/or route of zinc in antagonism of cadmium Carcinogenesis, which could prove key indetermining such a mechanism, are unavailable. Therefore, thepresent study was designed to extend previous findings of zincantagonism of cadmium Carcinogenesis by using several dosagelevels of zinc, as well as several routes of both zinc (s.c., p.o.)and cadmium (s.c., i.m.) administration.

MATERIALS AND METHODS

Animals. A total of 354 male Wistar [Crl:(WI)BR] rats were obtainedfrom the Charles River Breeding Laboratories (Kingston, NY) and wereallowed at least 2 weeks of acclimatization prior to initiation of firsttreatment. Animals were housed 3/polycarbonate cage in a standardbarrier facility and were provided food and water ad libitum.

Chemicals and Treatments. Cadmium(II) chloride (CdCI2), anhydrouspowder (Baker Analyzed Reagent), was purchased from VWR Scientific, Inc. (Baltimore, MD). Zinc(II) acetate dihydrate was purchasedfrom Aldrich Chemical Co. (Milwaukee, WI). Injection solutions wereprepared in sterile normal saline (0.9% NaCl). Rats were randomlyplaced in groups and treated as described in Table 1. All s.c. injectionswere given at 4 ml/kg, either in the dorsal thoracic midline for cadmium(0 h) or in the right lumbosacral (—6h), left lumbosacral (0 h), andlumbosacral midline (+18 h) for zinc. The s.c. zinc treatment schedulewas derived from the work of Gunn et al. (4, 5). All i.m. injections weregiven in the right thigh at 0.4 ml/kg, at time 0. Controls received sterilenormal saline at appropriate volumes and through appropriate routes.The two groups dosed orally (p.o.) with zinc in the drinking waterbegan receiving treatment 2 weeks prior to day 0. Water consumption

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EFFECTS OF ZINC ON CADMIUM CARC1NOGENESIS

Table 1 Experimental design

1.2.3.4.5.6.7.8.9.10.11.12.13.Group

(route)0Control

(s.c.)''Cadmium

(s.c.)Highzinc(s.c.)Control

(s.c.)''Cadmium

(s.c.) +lowzinc(s.c.)Cadmium

(s.c.) + medium zinc(s.c.)Cadmium

(s.c.) +highzinc(s.c.)Control(i.m.)rfCadmium

(i.m.)Control(i.m. +S-C./Cadmium

(i.m.) +highzinc(s.c.)Zinc(p.o.)Cadmium

(s.c.) +zinc(P.o.)Initial

no. ofrats21303021303030213021303030Cadmium

dose*

(^mol/kg)0300303030030030030Zincdosec1.0

mmol/kg0.0mmol/kg0.1mmol/kg0.3

mmol/kg1.0

mmol/kg0.0

mmol/kg1.0mmol/kglOOppmlOOppm

800 -

" Compounds were given either s.c., i.m., or in the drinking water (P.O.).* Given in the dorsal thoracic midline (s.c.) or in the right thigh (i.m.) at time

0. Tumors were then assessed over the next 104 weeks. Controls received salineinstead of cadmium.

' Given in the lumbosacral region (s.c.; right, left, midline) in three separatedoses, as specified, at —¿�6,0, and +18 h relative to cadmium or in the drinkingwater (p.o.) from —¿�2to +104 weeks relative to cadmium. Controls received saline(s.c.) or unaltered drinking water.

''Controls (groups 1, 4, 8, and 10) were found to be statistically invariant in

all respects and were thus pooled and are henceforth referred to as pooled control.

data (biweekly; not shown) indicated no differences between these twogroups. Animals were treated when they were 6 weeks old and weighedapproximately 225 g. Rats were then observed for the next 104 weeks.

Pathology. During the course of the experiment, body weights, survival, clinical symptoms, and necropsy findings were recorded. A complete necropsy was performed on each rat whether found dead or killedby CO2 inhalation when moribund or at 104 weeks. The followingtissues were fixed in 10% buffered formalin, processed by standardhistológica! techniques, stained with hematoxylin and eosin, and examined by light microscopy: all grossly altered tissues, s.c. injectionsites (both cadmium and zinc), testes, dorsal and ventral prostate, lungs,kidneys, adrenals, pancreas, pituitary gland, salivary glands, both thighswith femurs (i.m. groups only), and nasal cavities. Selected testicularspecimens were subjected to special stains: phosphotungstic acid-he-matoxylin, Masson's trichrome, and periodic acid-Schiff reaction.

Data Analysis. In all cases, a probability level of P < 0.05 wasconsidered to indicate a significant difference or trend. In pairwisecomparisons of tumor incidence, the Fisher exact test was used (26).Data within the dose-response groups were further tested for trendsusing the Cochran-Armitage test (27). Body weight data were examinedby analysis of variance and Dunnett's t test procedure for comparing

all means with controls (28). Survival in different groups was comparedwith the methods of Kaplan-Meier and Cox (29).

RESULTS

Survival Times and Body Weights. None of the treatmentsused in the present study had any effect on survival (data notshown). Median survival times ranged from 90 to 104 weeks.Likewise, body weight data showed little difference with treatment, although occasional decreases occurred with s.c. cadmium alone (group 2), i.m. cadmium plus s.c. zinc (group 11),and s.c. cadmium plus p.o. zinc (group 13). The differencesbetween these groups and pooled control were at times statistically significant (Fig. 1).

Tumors at the Site of Cadmium Injection. The cumulativeincidence of injection site tumors is shown in Table 2. Tumorswere typically mesenchymal in origin and classified as fibrosar-conias. The histopathology of this type of cadmium-inducedtumor in [Crl:(WI)BR] rats has been previously described (13).Cadmium given s.c. resulted in a 40% incidence of injection

Fig. 1. Mean body weights of pooled control rats (•)and rats in group 2 (D),11 (O), and 13 (9). Rats were treated as specified in Table 1. Significant difference(P s 0.05) from pooled control is indicated below curves.

site sarcomas. Zinc (s.c.) reduced the incidence of injection sitetumors only at the highest dose and only when consideringsarcomas alone (group 7). On the other hand, p.o. zinc almostabolished s.c. cadmium-induced injection site tumors, reducingincidence to <4% (group 13). Cadmium given i.m. resulted ina 10% incidence of tumors at the site (group 9) and, althoughs.c. zinc did reduce this incidence to 3%, the difference was notsignificantly different.

Testicular Tumors. The incidence of cadmium-induced testicular tumors and chronic testicular degeneration and the effectsof the various zinc treatments on this incidence appear in Table3. Tumors were almost exclusively Leydig cell adenomas, withthe exception of a single mixed Sertoli Leydig cell tumor.Because of the rarity of the latter tumor in rats, its histopathology has been described in detail elsewhere (23). With cadmium treatment, and depending on the dose and route of zinctreatment, the testes showed varying degrees of tubular degeneration with or without peritubular fibrosis and intratubularmineralizations (Fig. 2). The testes from animals treated withcadmium were typically atrophie, as indicated by an approximately 30-75% reduction in size. With regard to the testiculartumors induced by s.c. cadmium, the final incidence showed astrong dependence on the dosage level of s.c. zinc. The highestlevel of s.c. zinc (group 7) resulted in a 6.9-fold decrease intesticular tumor incidence. Oral zinc (group 13) had no effecton s.c. cadmium induction of testicular tumors. Overall tumorincidence showed a highly significant positive correlation withthe level of chronic testicular degeneration (Fig. 3). A similarstrong positive correlation with chronic testicular degenerationand tumor formation has been shown in rats given variouslevels of cadmium in a single s.c. injection (13). Cadmium giveni.m. did not result in degenerative lesions or produce an increasein testicular tumors, probably due to the repository nature ofthis route.

Prostatic Tumors. The incidence of prostatic tumors (exclusively adenomas of the ventral lobe) in rats treated with cadmium is shown in Table 4. A histopathological description ofthese cadmium-induced tumors in rats is available elsewhere(13). In the groups treated with cadmium i.m. (groups 9 and11), significant increases in the incidence of prostatic tumorsoccurred, with increases as high as 4-fold over control. Also, inthe group receiving s.c. cadmium and high dose s.c. zinc (group7), a significant increase (3-fold over control) in the incidence

of prostatic tumors occurred. In all groups in which an elevatedprostatic tumor incidence occurred (groups 7, 9, and 11), tes-

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EFFECTS OF ZINC ON CADMIUM CARCINOGENESIS

Table 2 Cadmium injection site tumors

1.2.3.5.6.7.9.11.12.13.Group"PooledcontrolCadmium(s.c.)High

zinc(s.c.)Cadmium(s.c.) + low zinc(s.c.)Cadmium(s.c.) + medium zinc(s.c.)Cadmium(s.c.) + high zinc(s.c.)Cadmium

(i.m.)Cadmium(i.m.) + high zinc(s.c.)Zinc

(P.O.)Cadmium(s.c.) + zinc (p.o.)Effective

no. ofrats84302930292929292930Sarcoma012"08'10"63101Fibroma1100000000Injection

sitetumors'*Hemangioma0000020000Epidermal'2011010000Total313"19"10"y3101

•¿�See Table I for details.* Dorsal thoracic midline (groups 2-7. 12, 13, and pooled control) or right thigh (groups 9 and 11 and pooled control).' Includes 3 keratoacanthomas, 1 squamous cell carcinoma, and 1 trichoepithelioma.d Significant difference from pooled control (P £0.05).

Table 3 Teslicular tumors ana chronic degeneration

Group"1.

Pooledcontrol2.Cadmium(s.c.)3.High zinc(s.c.)5.Cadmium (s.c.)+low

zinc(s.c.)6.Cadmium (s.c.)+medium

zinc(s.c.)7.Cadmium (s.c.)+high

zinc(s.c.)9.Cadmium(i.m.)11.Cadmium (i.m.)+high

zinc(s.c.)12.Zinc(p.o.)13.Cadmium (s.c.)+zinc

(p.o.)Effective

number Tumorof rats(%)*833029302928293029309(10.8)22

(73.3)''4(13.8)23

(76.7)''14

(48.3)''3(10.7)4(13.8)7

(23.3)2

(6.9)25(83.3)''Degeneration'None83129110282930290+0203800000++030101000001+++0240162000029

•¿�See Table 1 for details.* Primarily Leydig cell adenomas with the exception of a seminoma (group

11) and a mixed Sertoli-Leydig cell tumor (group 5).c Defined as mild (+), moderate (++), or severe (+++)•d Significant difference from pooled control (Fisher exact test; P s 0.05).

ticular tumor levels were comparable to control and a conspicuous absence of cadmium-induced chronic degenerative changeswas seen (see Table 3). However, when all cadmium treatmentgroups were considered (i.e., groups 2, 5, 6, 7, 9, 11, and 13),regardless of other treatments, there was a significant increasein prostatic tumor incidence (51 tumor-bearing rats of 199 rats;26%) over pooled control (8 of 83; 10%).

Other Tumors. Table 5 shows the incidence of tumors atvarious sites that were found to be unrelated to treatment. Themost common such tumors were pancreatic acinar cell and isletcell adenomas, pituitary adenomas, and adrenal pheochromo-cytomas. Beyond those tumors listed, compound osteosarcomasat the site of metallic identification tags in the ear, which werealso independent of treatment, occurred. These tumors havebeen described elsewhere (30).

DISCUSSION

The results of the present study show that zinc treatment canmarkedly reduce the carcinogenic effects of cadmium in rats.However, the efficacy of zinc in this regard was found to behighly dependent on the dose and route of zinc exposure aswell as on the target site of cadmium in question. Previousstudies have indeed indicated that zinc is quite effective inreducing the toxicity and/or carcinogenicity of cadmium (4, 5,12, 24, 25), but the present study is the first to show the highdependency on route of zinc exposure and apparent specificityin cadmium-induced tumor sites.

With regard to injection site sarcomas, several studies havedemonstrated that essential trace elements can effectively antagonize the induction of such tumors by carcinogenic metals(4, 5, 7, 31). For instance, magnesium is quite effective inreducing cadmium injection site sarcomas (7), while manganesecan significantly inhibit the formation of muscle sarcomata atthe site of nickel subsulfide injection (31). The inhibition bymagnesium or manganese of sarcoma induction by cadmium ornickel, respectively, appears to require admixture of the carcinogenic and anticarcinogenic metals prior to injection (7, 31).This mode of coinjection makes assessment of the exact modeof antagonism difficult, because physical interactions betweenthe two agents or toxicokinetic alterations resulting in loss ofcarcinogen from the site are still possible. Thus, actual chemicalantagonism at the molecular target of carcinogenesis, whateverthat may be, cannot be unequivocally assumed. The results withzinc antagonism of cadmium-induced sarcoma formation in thepresent and previous studies (4, 5) are somewhat different, inthat zinc is able to inhibit cadmium-induced injection sitesarcomas when separately injected at some distance from cadmium, eliminating at least in these experiments the possibilityof a purely physical mode of interference. In fact, the findingthat zinc p.o. almost abolished cadmium-induced sarcoma formation indicates that antagonism can occur even through entirely different routes and that transference of high concentrations of the metal between sites through the subcutis is probablynot a reasonable explanation for the effectiveness of s.c. zinc.Hence, the present results indicate that the antagonism ofcadmium carcinogenesis at the s.c. injection site by zinc maybe through direct action at molecular targets rather than by a"wash-out" of the carcinogenic metal from the site. The antag

onism of cadmium carcinogenesis by s.c. zinc in the testes seenin the present and previous studies (4, 5) would similarlyindicate a direct antagonism of the two metals.

The testicular Leydig cell tumors induced by cadmium wereclearly shown to be directly dependent on the level of testicularchronic degeneration following the acute insult by cadmiumtreatment and on the extent to which zinc was able to preventsuch effects. Previous studies have shown that zinc will preventdegenerative testicular lesions (4, 5, 12, 24, 25) caused bycadmium in the rat testes, although until the present studydose-response relationships had not been shown. Likewise,dose-response analysis of cadmium alone indicates that tumor-igenic events are apparently initiated only when the cadmiumdose is well above that required to cause acute testicular lesions(13). Hence, it appears that the severity of the testicular lesioninduced by cadmium is a primary factor responsible for eventualtesticular tumor development.

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Fig. 2. Testes from control rat (/I) and rats with Cd-indueed testicular degeneration classified as mild (B), moderate (C). and severe (D). H & E, 100 A, Normaltesticular tubules of age-matched (24 months) control rat. B, Shrunken, degenerate testicular tubules lined by Sertoli cells only, surrounded by proliferating fibroblastsand myoid cells. Occasional tubules display intraluminal flbrosis and mineralizations (arrows). Prominent appearance of small arteries. ( . Beneath thickened tunicavaginalis only a few atrophied tubules lined by Sertoli cells remain. Most tubules contain amorphous mineralized material. Phosphotungstic acid-hematoxylin. D,Diffuse intra- and peritubular fibrosis. Note extremely small diameters of former tubules.

The present results are in accord with a previous study inrats that indicates cadmium induction of prostatic tumorigen-esis after systemic exposure (13), as well as being consistentwith several epidemiológica! studies that indicate cadmium asone possible cause of human prostatic cancer (14-19). In fact,

although not all investigations have shown a link betweencadmium and prostatic carcinogenesis (32, 33), associationswith cadmium exposure both in the work place and through theenvironment have been repeatedly observed (14-19). Further

more, Feustel et al. (34) found that, in human prostatic tissue,the concentrations of cadmium were highest in adenocarcino-mas, intermediate in prostatic hyperplasia, and lowest in normal tissue. Cadmium was further found to be concentrated inthe epithelial portion of the human prostate (35), while thehighest concentrations were found in the nuclear subcellularfraction (36). Other evidence of an association between cadmium and prostatic neoplasia in rats includes the finding oftumors following direct injection into the prostate (20, 21) and

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EFFECTS OF ZINC ON CADMIUM CARCINOGENESIS

100

? 80

¡SO

h-

« 403

_O

1 20

r2 = 0.98

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Testlcular Degeneration (x)

Fig. 3. Relationship between testicular tumor incidence and level of chronictesticular degeneration. Groups used for assessment include pooled control andrats receiving s.c. cadmium (groups 2, 5, 6, 7, and 13). Regression correlationcoefficient (r2) shows a highly significant positive relationship.

Table 4 Incidence ofproslatic tumors

1.•i.3.5.6.7.9.11.12.13.Group"PooledcontrolCadmium(s.c.)High

zinc(s.c.)Cadmium(s.c.) + low zinc(s.c.)Cadmium(s.c.)+(S.C.)Cadmium

(s.c.)(S.C.)Cadmium

(i.m.)Cadmium(i.m.)(S.C.)Zinc

(p.o.)Cadmium(s.c.) +medium

zinc+

highzinc+

highzinczinc

(p.o.)Effective

no. ofrats83302830282726282930Prostatic

tumorsTO*8

(9.6)7(23.3)6(21.4)6

(20.0)5(17.9)8

(29.6)'1

1(42.3)c7(25.0)c4(13.8)7

(23.3)°See Table 1 for details.* All tumors occurred in the ventral lobe of the prostate and were adenomas.' Significant difference from pooled control (P ==0.05).

the observation that oral cadmium exposure causes dose-dependent increases in prostatic weight when assessed over a 6-month exposure period (37). Cadmium can stimulate thegrowth of human prostatic epithelium (38) and induce morphological transformation of Syrian hamster embryo cells (39) invitro. Recently, Terracio and Nachtigal (40) have shown thatcadmium is capable of in vitro transformation of rat ventralprostate epithelial cells and that such cells will produce tumorsat the site of inoculation in newborn rats (41). Hence, thereappears to be increasing evidence in animal models, includingthe present study, in support of a possible role of cadmium inhuman prostatic cancer.

The final incidence of prostatic tumors in cadmium-treated

rats appears to be dependent on the ability of the testes tosupport the prostatic tissue. The growth and maintenance ofthe mammalian prostate is dependent on circulatory levels oftestosterone (15,42), the largest source of which in males is thetestes, and prostatic atrophy will occur with castration or estrogen therapy (15, 43). Cadmium treatment will also result inprostatic atrophy (12), following a marked reduction in testicular androgen secretion (44, 46), due to its effects on the testesincluding acute ischemie necrosis and eventual degenerationand atrophy. Such degenerative effects were frequently seenwith cadmium treatment in the present study. However, thereis a conspicuous absence of such cadmium-induced testiculardegeneration in those groups showing elevated prostatic tumorincidence (groups 7, 9, and 11; compare Tables 3 and 4). Itappears that, in the rats treated with i.m. carcinogen (groups 9and 11), the repository nature of this route did not providesufficient circulatory levels of cadmium to induce necrosis andeventual degeneration of testes, while such degenerative changesof the testes were prevented by high dose s.c. zinc in the other

group in which enhanced prostatic carcinogenesis occurred(group 7). Likewise, dose-response analysis of cadmium effectsindicates that only at doses well below those which inducetesticular necrosis and degeneration does prostatic neoplasiaoccur (13). Hence, the interplay between the testes and prostateclearly has an important role in cadmium-induced prostaticcarcinogenesis.

The exact molecular mechanism of zinc inhibition of cadmium carcinogenesis in the testes or at the injection site cannotbe determined from the present data. However, it would appearthe inhibition is different between these two targets sites, particularly when the different routes of zinc administration areconsidered. The sustained levels of elevated circulating zincbrought about by exposure p.o. abolished cadmium-inducedinjection sarcomas but had no effect on the incidence of testicular tumors. The repository nature of the cadmium injectionmay account for this difference. The s.c. injection of cadmiumwould allow for a slow release of a relatively high concentrationof cadmium to the local tissues (8, 47) and thus cadmium-induced transformations could potentially occur over a relatively long period of time. In contrast, after the initial peakaccumulation of only a very small portion of a given dose, thetestes steadily lose cadmium (8, 47,48), and thus the initiatingevents would have a lesser chance of occurring with time.Hence, sustained elevations of zinc may not be as effective as asingle large bolus in prevention of testicular tumorigenesis. Inthis regard, the present study shows that if the cadmium-induced testicular degeneration, which results from the acutenecrotic lesions seen very rapidly following cadmium (49), isprevented so are the carcinogenic effects. Hence, it appears thatevents occurring soon after exposure to cadmium must beprevented to prevent testicular tumors, while carcinogenicevents within the injection site may be prevented by zinc overa longer period of time.

Reduced distribution of cadmium to the testes by zinc administration would seem a reasonable possible mechanism for theprevention of tumorigenesis within this tissue. However, available data clearly indicate that zinc has no effect on cadmiumuptake by the testes (48, 50), at doses similar to those used forboth metals in the present study. Alternatively, it is thoughtthat zinc often prevents cadmium toxicity, including farcinogenicity, by induction of the synthesis of a low molecular weightmetal-binding protein with a very high affinity for cadmium,which is called metallothionein. Metallothionein is thought tosequester cadmium in a stable form and thus reduce its potentialtoxicity (51, 52). However, recent work from this and otherlaboratories indicates that the rat testes are clearly deficient inmetallothionein (53-56) and in its place a noninducible bindingprotein with a probable lower affinity for cadmium exists (56).Likewise, in mouse strains genetically resistant to the effects ofcadmium on the testes, metallothionein is also deficient (57)and, in fact, the metallothionein gene appears to be highlymethylated (58), a condition frequently associated with quiescence of this gene. Hence, it would appear that two of the mostlikely possibilities for zinc inhibition of cadmium carcinogenesiswould not be highly probable.

Recent work indicates that zinc pretreatment in vivo willprevent the in vitro cytotoxicity of cadmium in subsequentlyisolated interstitial cells of the rat testes (59). Reduced cytotoxicity was reflected in several parameters, including reduced lossof potassium and glutamic-oxaloacetic transaminase (59). Thisreduction in cytotoxicity occurs despite the observed slightincrease in cadmium uptake into these cells with zinc pretreatment (59). Thus a potential link between zinc pretreatment and

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Table 5 Incidence of tumors found unrelated to treatment

SiteAbdominal

cavityAdrenal

glandHematopoietic

systemIntestineKidneyLiverLungMammary

glandNasal/oral

mucosaPancreasParathyroid

glandParotid

glandPeripheral

nervePituitary

glandSkeletal

systemSkinStomachSubcutisThoracic

cavityThyroid

glandThymusVascular

systemZymbal's

glandTumorFibrosarcomaCortical

adenomaCorticaladenocarcinomaPheochromocytomaGanglioneuromaLymphoma/leukemiaAdenocarcinomaTubular

adenomaTubularadenocarcinomaScirrhouscarcinomaHepatocellular

adenomaHepatocellularcarcinomaAdenomaFibroadenomaAdenocarcinomaSquamous

cellcarcinomaPapillomaSarcomaAcinar

celladenomaIsletcelladenomaAdenomaAdenomaSchwannomaPars

distalisadenomaParsintermediaadenocarcinomaOsteosarcomaPapillomaKeratoacanthomaSquamous

cellcarcinomaTrichoepitheliomaSebaceous

glandadenomaPapillomaSquamous

cellcarcinomaSarcomaFibromaSarcomaLipomaMesotheliomaScirrhous

carcinomaFollicular

celladenomaFoliicularcellcarcinomaC-cell

adenomaC-cellcarcinomaThymomaCarcinomaHemangiomaHemangiosarcomaAdenocarcinomaPooled

control(84 rats)"120411213023121210341310217024400200082000431001314Pooled

cadmium(206 rats)"01115033401130527128031004731185022310176111324320552Pooled

zinc(205rats)"011211233412334271089280166820352021011610300214240553

1Pooled control-groups 1, 4, 8, 10; pooled cadmium-groups 2, 5, 6, 7, 9, 11, 13; pooled zinc-groups 3. 5, 6, 7, 11, 12, 13; see Table 1 for details.

reduced cytotoxicity exists, at least in these target cells ofcadmium carcinogenesis. However, further work will be required to determine the exact mechanism of this inhibition andwhat role it may play in reduced carcinogenicity.

In summary, the present study indicates that zinc can be quiteeffective in preventing or reducing the carcinogenic effects of

cadmium in rats. This effectiveness is, however, highly dependent on the dose and/or route of zinc exposure, as well as thetarget site of cadmium. This study also provides importantconfirmatory evidence that cadmium exposure, which has frequently been suspected in association with human prostaticcancer (14-19), will induce tumors in the rat prostate.

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EFFECTS OF ZINC ON CADMIUM CARCINOGENESIS

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1989;49:4282-4288. Cancer Res   Michael P. Waalkes, Sabine Rehm, Charles W. Riggs, et al.   in the Prostate, in the Testes, and at the Injection SiteDose-Response Analysis of Effects of Zinc on Tumor Induction Cadmium Carcinogenesis in Male Wistar [Crl:(WI)BR] Rats:

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