TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
108,448-456
(199
1)
Effect of Chronic Dietary Zinc Deficiency on Cadmium Toxicity and Carcinogenesis in the Male Wistar [Hsd: (WI)BR] Rat’ MICHAEL P. WAALKES, *S ROBERT KOVATCH,P AND SABINE REHM* *Inorganic Carcinogenesis and Tumor Pathology and Pathogenesis Sections, Laboratory of Comparative Careinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland; and tPathology Associates. Inc., Frederick, Maryland
Received
October
12. 1990: accepted January
9. 1991
Effect of Chronic Dietary Zinc Deficiency on Cadmium Toxicity and Carcinogenesis in the Male Wistar [Hsd: (WI)BR] Rat. WAALKES, M. P., KOVATCH. R., AND REHM, S. (1991). Toxicol. Appl. Pharmacol. 108, 448-456. Though it is known that excess zinc will prevent cadmium carcinogenesis, the impact of zinc deficiency on cadmium carcinogenesis has not been defined. This study assessedthe effect of dietary zinc deficiency on the carcinogenic potential of cadmium in rats. Groups (n = 28 each) of male Wistar [Hsd: (WI)BR] rats were fed diets adequate (60 ppm) or deficient (7 ppm) in zinc and received a single SCdose of cadmium (5, 10, or 30 pmol Cd/kg). Lesions were assessedover the next 92 weeks. All cadmium doses increased the incidence of testicular interstitial cell tumors. The incidence of cadmium-induced testicular tumors was unaffected by dietary zinc status. However, when multiplicity of testicular lesions was considered, zinc-deficient diets markedly increased the number of testicular interstitial cell adenomas generated by cadmium exposure while significantly reducing the number of preneoplastic lesions (interstitial cell hyperplasias). The combined total number of neoplastic and preneoplastic lesions ofthe testes was independent of zinc status clearly indicating a shift from hyperplasia to neoplasia within the testes of zinc-deficient rats. The highest cadmium dose (30 wmol/kg) increased injection site sarcomas in zinc-deficient rats (7 tumors/27 rats at risk) but not zinc-adequate rats (3/26) when compared to control (O/49). Chronic progressive renal nephropathy was accelerated by cadmium in zinc-deficient rats. Results indicate that dietary zinc deficiency enhances carcinogenic response at the injection site of cadmium, promotes the neoplastic progression of cadmium-induced testicular lesions, and enhances chronic progressive nephropathy. Thus, dietary zinc deficiency appears to cause a generalized increase in the chronic toxic effects of cadmium. 8 1991 Academic press, IIIC.
The heavy metal cadmium is a potent carcinogen in laboratory animals and possibly a human carcinogen (see Waalkes and Oberdiirster, 1990 for review). The rodent testes are a well-defined target of cadmium and testicular interstitial cell tumors occur with high frequency following cadmium exposure (Roe d al., 1964; Gunn et al., 1963, 1964; Waalkes et ’ Presented in part at the 1990 Annual Meeting of the American Association for Cancer Research, Washington, DC. ’ To whom correspondence should be addressed at the National Cancer Institute-FCRDC, Building 538, Room 205E. Frederick, MD 2 1702- 120 1. 0041-008X/91
$3.00
Copyright Q 1991 by Academic Press, Inc. All rights of reprcduct~on in any form reserved.
448
al., 1988, 1989). The sites of cadmium injection, both subcutaneous and intramuscular, are also well defined as locations for cadmiuminduced tumors (Heath et al., 1962; Gunn et al., 1963, 1964; Kazantis and Handury, 1966; Waalkes et al., 1988, 1989). More recent evidence indicates that the rat lungs (Takenaka et al., 1983) and prostate (Waalkes et al., 1988, 1989) are target sites of cadmium carcinogenesis under certain constraints of route or dosage. Retrospective epidemiological studies provide some evidence that occupational or environmental cadmium exposure is associated with human prostatic and pulmonary
ZINC
DEFICIENCY:
Cd
TOXICITY
carcinogenesis, although this has not been shown in all cases (Bako et al., 1982; Peters et af., 1986; WaaIkes and Oberdorster, 1990). The most prominent nonneoplastic lesion observed with chronic exposure in both humans and animals is nephropathy (Friberg, 1950; Piscator, 1966; Dudley et al., 1985; Maitani et al., 1988; Foulkes, 1990). Numerous reports indicate that the effects of cadmium in laboratory animals can be prevented or markedly reduced by the administration of excess zinc (Parizek, 1957; Gunn et al., 1963, 1964; Goering and Klaassen, 1984; Waalkes et ai., 1989). This includes both acute (Parizek, 1957; Goering and Klaassen, 1984) and chronic effects such as carcinogenesis (Gunn et al., 1963, 1964; Waalkes et al., 1989). In this regard it is thought that many of the toxic effects of cadmium occur through a metabolic disruption of zinc-mediated metabolic processes (Bremner, 1974). On the other hand, very little is known about cadmium toxicity during periods of zinc deficiency. The few studies available indicate that cadmium toxicity, including transplacental toxicity, can be markedly increased in zinc-deficient animals (Parzyck et al., 1978; Petering et al., 1971). Zinc deficiency in rats will also cause an enhanced disposition of cadmium in various tissues, including the testes and kidney (Waalkes, 1986). The effect of zinc deficiency on cadmium carcinogenesis has not been defined, however. The present study was, therefore, designed to assessthe effect of chronic dietary zinc deficiency on cadmium toxicity and carcinogenesis in the rat. In this regard, a level of dietary zinc resulting in what can be termed a marginal deficiency was selected in that it has been shown to cause a 40% reduction in plasma zinc without affecting body weight gain or causing any overt lesions (Waalkes, 1986). MATERIALS
AND
METHODS
Diet. The zinc-defined diets used in this study were obtained from Teklad Company, Inc. (Madison. WI). The basal diet was first analyzed using atomic absorption spec-
AND
CARCINOGENESIS
449
trometry for zinc content (1 .O ppm) and then adjusted to the desired level with zinc acetate dihydrate. Cadmium was not detectable in the basal diet. This level of dietary zinc will produce a 40% reduction in circulating plasma zinc in rats (Waalkes. 1986). Animals and treatment. A total of 224 male Wistar [Hsd: (WI)BR] tats were obtained at 4 weeks of age from HarlanSprague-Dawley (Indianapolis, IN). They were housed 3 per polycarbonate cage in a standard barrier facility, at a temperature of 68-72”F, with a relative humidity of 50 f 5%, and a 12-hr light/dark cycle. The zinc-defined diet and water were given ad libitum. Cadmium (CdC& .2f H,O; J. T. Baker Co.) solutions were prepared in sterile normal saline. Rats were divided into groups as designated in Table 1. At 8 weeks of age rats were placed on the zinc-defined diets for 2 weeks prior to cadmium exposure (Week 0). Rats were then injected (SC; 4.0 ml/kg) in the dorsal thoracic midline area (henceforth referred to as the injection site) with 0 (saline) or 5, 10. or 30 rmol Cd/kg. Body weights, survival, food consumption, and clinical signs were recorded throughout the experiment. Rats were killed when moribund or at 92 experimental weeks. Body weight and testes weights were recorded for each rat at necropsy. Pathology. A complete necropsy was performed on all animals. Injection site, liver, pancreas, spleen, kidneys, prostate, testes, and any abnormal tissues from each animal were studied microscopically. Lung, pituitary, and adrenals were not inspected. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 pm, and stained with hematoxylin and eosin (HE) for histological examination. For testes the number of hyperplastic (preneoplastic) and neoplastic interstitial cell foci (interstitial cell adenomas) was determined for each rat in a single midline section of both testes. The differentiation of interstitial cell hyperplasia and interstitial cell adenoma is to a certain extent subjective as the exact biological behavior of such lesions is unknown. Interstitial cell hyperplasia consisted of clusters of cells in the testicular interstitium that had a central nucleus and moderately abundant amorphous eosinophilic or vacuolated cytoplasm that did not exceed the diameter of a single normal seminiferous tubule. Such lesions only minimally compressed adjacent tissue. Adenoma was diagnosed when the interstitial cell proliferation exceeded the diameter of a normal seminiferous tubule. In such cases, distinct compression of adjacent testicular tissue was evident. Data analysis. In all cases, a probability level ofp < 0.05 was considered to indicate a significant difference. In pairwise comparison of tumor incidence the Fisher’s exact test was used. Body weight data were examined by Dunnett’s test. Survival was examined with the Cox test and the generalized Kruskal/Wallis test and was only considered significantly different if both tests indicated p values < 0.05. In all cases, the number of rats at risk was defined as the number of rats surviving at the time of appearance of the first tumor of any type (28 weeks).
450
WAALKES,
KOVATCH, TABLE
AND
REHM
I
EXPERIMENTAL DESIGN
Group 1 2 3 4 5 6 7 8
Initial No. of rats
Effective No. of rats0
28 28 28 28 28 28 28 28
24 24 26 25 25 26 27 24
Dietary zinc’ bw)
Subcutaneous cadmium ’ (pmol/kg)
7 7 7 1 60 60 60 60
a Defined as those animals surviving to the appearance of the first tumor of any type (28 weeks). ’ Mixed in the diet at the specified level of zinc in the form of zinc acetate, from -2 weeks to the termination study (+92 weeks). ’ Given in the dorsal thoracic midline at Time 0. Tumors were assessed over the next 92 weeks.
RESULTS
0 5 10 30 0 5 10 30
of the
Likewise, no group showeddifferences in food consumption from their respective control Dietary zinc deficiency alone had no effect groups. on survival. Furthermore, survival was the Zinc deficiency did have a marked effect on same in rats fed zinc-deficient diets regardless the generation of tumors at the injection site of the level of cadmium exposure when com- of cadmium (Table 2). Only in the group fed pared to zinc-deficient or zinc-adequate con- zinc-deficient diets and given the highest dose trols (not shown). On the other hand, survival of cadmium was there a significant elevation was significantly depressedin rats fed a zinc- in the incidence of injection site sarcomas adequate diet and given the highest dose (30 (28%). Overall, zinc-deficient rats developed ymol/kg) of cadmium (Fig. 1) when compared to zinc-adequate controls but not in comparison to zinc-deficient rats receiving the level 1.0 of cadmium. Likewise, zinc deficiency by itself 0.9 had no effect on body weight (not shown). In 0.6 fact, only in thoseanimals receiving the highest I 2 : 0.7 doseof cadmium (30 pmol/kg) was any weight 56 gain suppressionobserved and this proved in,E C o.6 . opmO, Cd,kg $ 2 0.5 dependent of dietary zinc status. For approx0 30 ,,mol Cd/kg 1: imately 10 weeks after cadmium injection a .+c 6” 0.4 0.3 12- 17%reduction in body weight wasdetected ::p l #A 0.2 in animals receiving 30 pmol Cd/kg regardless 0.1 of dietary zinc level. This suppressionof body 0.0 weight was resolved by 15 weeks following 0 10 20 30 40 50 60 70 80 9( cadmium injection and all groups showed the Time (weeks) same or similar body weight through the reFIG. 1. Effect of cadmium exposure (30 rmol/kg, SC, mainder of the study. Time 0) on survival of rats fed a diet adequate in zinc (60 Food consumption was also not affected by ppm). Survival was significantly (p < 0.05) depressed in rats in comparison to control rats. dietary zinc status at any point in the study. cadmium-exposed
ZINC
DEFICIENCY:
Cd TOXICITY
AND
TABLE INCIDENCE
451
CARCINOGENESIS
2
OF CADMIUM-INDUCED INJECTION SITE SARCOMAS IN MALE WISTAR [H.sd:(WI)BR] FED DIETS ADEQUATE OR DEF’ICIENT IN ZINC
Group
N”
1 2 3 4 5 6 7 8
24 24 26 25 25 26
a Rats at risk. * An asterisk indicates
Cadmium dose ( rmol/W
Dietary zinc level (ppm)
0 5 10 30 0 5 10 30
I I 7 7 60 60 60 60
21
24
a significant
difference
from appropriate
more than twice as many cadmium-induced injection site tumors (8 sarcomas/79 rats; 10.1%) than zinc-adequate rats (3/78; 3.9%). Cadmium was very effective in inducing interstitial cell tumors, and dose-related increases in overall tumor incidence (rats bearing tumors/total rats at risk) occurred in rats fed either zinc-adequate and zinc-deficient diets (Fig. 2). A maximum incidence of approximately 70% occurred in both zinc-deficient and zinc-adequate animals at cadmium doses >, 10 pmol/kg. No differences in incidence based on dietary zinc level were detected between groups receiving the same level of cadmium. When the multiplicity of adenomatous (neoplastic) and hyperplastic (preneoplastic) interstitial cell lesions was considered, marked differences occurred between animals fed zincadequate or zinc-deficient diets (Table 3; Fig. 3). Although the total number of preneoplastic plus neoplastic lesions was not affected by dietary zinc, a major shift occurred towards adenoma formation in rats consuming diets deficient in zinc (Table 3). In zinc-deficient rats more than 80% of all lesions in the testes were neoplastic while in zinc-adequate rats fewer than half of the total lesions were adenomas. This shift toward adenoma formation occurred in all cadmium dosage groups (Fig. 3)
control.
Tumors
RATS
Injection sarcomas 0 0 I 7* 0 0 0 3
were mostly
site (%)b
(0.0%) (0.0%) (3.6%) (28.0%) (0.0%) (0.0%) (0.0%) (12.5%)
fibrosarcomas.
and was reflected in an overall decreased incidence of hyperplasia in zinc-deficient animals (Fig. 4). The control rats in this study also showed a fair incidence of moderate to severe chronic nephropathy which was not affected by dietary zinc status (Table 4). The kidney lesions were typical of the age-dependent, progressive chronic nephropathy of rats and included glomerular and tubular basement membrane thickening, glomerular sclerosis, chronic interstitial inflammation, proteinaceous tubular
H O-0
0
5
10 Cadmium
15
Zinc Adequate Zmc Deficient
20
Dose bmollkg.
25
30
se)
FIG. 2. Incidence of cadmium-induced testicular interstitial cell tumors in rats fed diets adequate (60 ppm) or deficient (7 ppm) in zinc. Data are expressed as the proportion (%) of tumor-bearing rats in the total number of rats at risk.
WAALKES,
452
KOVATCH.
AND REHM
TABLE 3 EFFECTOF ZINC DEFICIENCYONCADMIUM-INDUCED NEOPLASTICAND PRENEOPLASTICHYPERPLASTIC INTERSTITIAL CELL FOCI IN THE MALE WISTAR [Hsd:(WI)BR] FCAT TESTES Groups
N
Cadmium dose (wol/kg)
Dietary zinc level (ppm)
I 2-4 5 6-8
24 75 25 77
0 S-30 0 5-30
7 7 60 60
Adenoma’ 3 (100%) 194 (83%) 0 (0%) I 15 (47%)
Hyperplasia’
Total lesions
0 (0%) 39(17%) 1 (100%) 131(53%)
3 233 1 246
” Percentages reflect the proportion of total lesions.
casts, and tubular regeneration. However, in zinc-deficient rats given cadmium, a marked increase in the incidence of chronic nephropathy occurred in a dose-related fashion. Various incidental tumors were detected in the 201 total rats at risk that were not associated with any treatment. These included 149 pancreatic acinar cell adenomas; 22 pancreatic acinar cell carcinomas; 8 pancreatic islet cell adenomas; 2 1 thyroid C-cell adenomas; 7 thyroid C-cell carcinomas; 7 noninjection site sarcomas; 6 noninjection site fibromas; 10 keratoacanthomas; 2 lipomas; 2 squamous cell papillomas; 4 prostatic adenomas; and 3
g
mammary gland fibroadenomas. A single one of each of the following types of tumors also occurred: a mesothelioma; a hepatocellular carcinoma; a brain granular cell tumor; a skin squamous cell carcinoma; a forestomach squamous cell carcinoma; a mammary gland adenocarcinoma; a hemangioma; and a skin basal cell adenoma.
DISCUSSION Although in humans deficiency of zinc in its severest forms occurs only very rarely (Prasad, 1982), marginal deficiency of zinc is considered to be sufficiently common to be a po-
loo-
‘ij B
* O... -&--
H I-"
.z” 2 8 t B
----_-
--
_____
BO-
looh
-
60 40--
l -
v5
10
15
20
25
Odium Dose(rmollkg,sc)
Cadmium
3. Proportion (%) of cadmium-induced testicular neoplasia in the total neoplastic plus preneoplastic (interstitial cell hyperplasia) lesions in rats fed diets adequate (60 ppm) or deficient (7 ppm) in zinc. Asterisks indicate a significant (p Q 0.05) difference from rats fed diets adequate in zinc. Pooled cadmium represents all groups given cadmium (5, 10. and 30 pmol/kg) at a specific dietary zinc level. FIG.
g 80. .9 H c t r” x
60.
40-
E 0” E
zo-
-
t
OL Cadmium
Dose bmollkg,
SC)
FIG. 4. incidence of cadmium-induced testicular interstitial cell hyperplasia in rats fed diets adequate (60 ppm) or deficient (7 ppm) in zinc. Data expressed as the proportion (%) of hyperplasia bearing rats in the total number of rats at risk.
ZINC
DEFICIENCY:
TABLE
Cd TOXICITY
4
INCIDENCE OF CHRONIC NEPHROPATHY IN MALE WISTAR [Hsd:(WI)BR] RATS FED DIETS ADEQUATE OR DEFICIENT IN ZINC
Group
Cadmium ( wol/kg)
1 2 3 4 5 6 I 8
0 5 10 30 0 5 10 30
dose
Dietary zinc level (ppm) 7 I 7 7 60 60 60 60
Nephropathyn 9/24 (38%) 14/24 (58%) 18/26* (69%) 20/25 * (80%) I l/25 (44%) 13126 (50%) 14121 (52%) 15/24 (63%)
‘Data expressed as rats bearing lesion/rats at risk (‘3%total). Only animals showing a moderate to severe lesion are included. An asterisk indicates a significant difference from appropriate control.
tential public health concern (Hambidge et al., 1979; Beach et al., 1982; Prasad, 1982). Marginal zinc deficiency can be brought about by various factors including dietary composition (O’Dell, 1969), pharmaceuticals (Prasad, 1982), and disease states (Prasad, 1982) and is further complicated by the fact that such mild deficiencies are unlikely to be detected (Prasad, 1982). Little is known about how marginal deficiencies in zinc modify the toxicity of other metals. The present study evaluated the carcinogenic potential of cadmium in rats fed zinc-adequate and zinc-deficient diets. The zinc-reduced diets used in the present study produced what can be described as a marginal zinc deficiency condition since such diets are known to produce a 40% reduction in plasma zinc levels (Waalkes, 1986) but did not modify body weight, food consumption, or survival over the course of the present study. Despite the absence of such overt effects, the zinc-deficient diets clearly increased the carcinogenic potential of cadmium, both through an increased incidence of sarcomas at the injection site of cadmium and through an enhancement of the progression towards neoplasia within the testes. Renal lesions associated with cadmium were also exacerbated
AND
CARCINOGENESIS
453
by zinc deficiency. Thus, the results of the present study indicate that even a marginal dietary deficiency of zinc will enhance cadmium carcinogenicity and toxicity. Given the prevalence of such subclinical dietary zinc deficiencies in human populations (Hambidge et al., 1979; Beach et al.. 1982; Prasad, 1982) this may place a multitude of individuals at greater risk from cadmium exposure. It would seem that dietary zinc deficiency could be considered to increase susceptibility to experimental cadmium carcinogenesis. Various other dietary deficiencies can clearly enhance carcinogenesis in rodents (Poirier et al., 1986). For instance, dietary methyl deficiency increases the tumor incidence in rat liver resulting from an initiating dose of diethylnitrosamine (Mikol et al., 1983) even when given for relatively brief periods (Hoover et al., 1984). Zinc deficiency too can be an effective enhancer of carcinogenesis induced by various organic carcinogens in rodents (Barth and Iannaccone, 1986). An increased tumor incidence occurs with dietary zinc deficiency in the esophagus of rats given methylbenzylnitrosamine (Fong et al., 1978, 1984; Van Rensburg et al., 1980). Zinc deficiency has also been associated with an increased incidence of human esophageal cancer (Van Rensburg, 198 1). Enhancement of chemically induced forestomach tumors also occurs with dietary zinc deficiency in rats (Ng et al., 1984; Fong et al., 1984). Thus, as is the case with many organic carcinogens, the efficacy of the inorganic carcinogen, cadmium, can be increased by dietary zinc deficiency. In general it appears that tumor incidence or multiplicity is enhanced by dietary zinc deficiency within a given tissue (Fong et al., 1978, 1984; Van Rensburg et al., 1980; Ng et al., 1984). In contrast, shifts of target tissue do not appear to occur with suboptimal zinc intake. In this regard, cadmium is an effective testicular tumorigen (Gunn et al., 1963, 1964; Waalkes et al., 1988, 1989) and produces sarcomas at the site of SCinjection (Gunn et al., 1963, 1964; Waalkes et al., 1988, 1989). These are in fact the same sites at which the carcinogenic effi-
454
WAALKES.
KOVATCH.
cacy of cadmium wasenhanced by dietary zinc deficiency in the present study. The exact mechanism by which zinc deficiency modifies cadmium carcinogenesiscan not be determined from the present results. In the caseof dialkylnitrosamines it appearsthat zinc deficiency increases microsomal P450mediated metabolic activation (Barth et al., 1984) and thus carcinogenic activity (Barth and Iannaccone, 1986). Cadmium, on the other hand, presumably does not require any such metabolic activation. Zinc deficiency does, however, increase the distribution of cadmium to the testes (Waalkes, 1986) and assuming that higher concentrations of the metal would lead to more tumors, this sort of modification in toxicokinetics could have some bearing. It also appears that zinc deficiency can reduce concentrations of the cadmium binding protein, metallothionein, in some tissues(Waalkes, 1986). Since metallothionein is thought to function in part in detoxication of cadmium (Waalkes and Goering, 1990), reduced levels may increasevarious aspects of cadmium toxicity, including carcinogenicity. Zinc-mediated or -dependent metabolic processes,including roles in cellular production of DNA, RNA, and protein (Brady, 1982) are thought to be a prime target of cadmium toxicity (Brady, 1982; Bremner, 1974). However, the exact molecular target or mechanism by which zinc deficiency enhances cadmium carcinogenesisremains to be determined. Metal-metal interactions, specifically interactions with essentialelements, are clearly a key aspectof metallic carcinogenesis.In this regard there are several examples of such interactions that result in reduction of carcinogenic response.This includes the inhibition of cadmium carcinogenesis at the injection site and in the testes by administration of excess zinc (Gunn et al., 1963, 1964; Waalkes et al.. 1989) and the prevention of nickel-induced sarcomas by excess manganese (Sunderman and McCully, 1983) or magnesium (Kasprzak et al., 1985a). Enhanced carcinogenic response can also be observed. Examples of this include
AND
REHM
the increases in cadmium-induced prostatic tumors brought about by excesszinc (Waalkes et al., 1989) and the enhancement of lead-induced renal tumors by calcium (Kasprzak et al.. 1985b) or magnesium (McCreary et al., 1977). The resultsof the present study indicate that the probable derangement in zinc metabolism brought about by even modest dietary zinc deficiency can also have a marked enhancing effect on tumor incidence and progressionfollowing exposure to cadmium. Thus it appears that alterations in essential metal metabolism are of primary mechanistic importance in the process of metallic carcinogenesis,although this is clearly both tissueand metal specific. Chronic renal lesionsare a common finding in cadmium exposed humans or animals (Friberg, 1950; Piscator, 1966; Dudley et al.. 1985; Maitani et al., 1988; Foulkes, 1990). The kidney lesionsof the rats in the present study were comparable to the age-dependent chronic nephropathy of rats for which exact cause and pathogenesisare unknown (Gray, 1986). As such, the lesions do not precisely resemble those typically observed with multiple injections of cadmium. The control rats in this study also showed a moderate incidence of such nephropathy which was not affected by dietary zinc status. However, dietary zinc deficiency caused a marked increase in the incidence of chronic renal lesions seen in cadmium-treated rats. The observation that zinc deficiency will enhance accumulation of renal cadmium (Waalkes, 1986) may have a very important role in this increase in toxicity of cadmium, particularly sincea fairly low critical concentration seemsto be necessary for cadmium induced renal lesionsto occur (Foulkes, 1990). The apparent suppressionof metallothionein levels within the kidneys of zinc deficient rats (Waalkes, 1986) may also allow a greater effective toxicant concentration at the target site, since metallothionein is thought to detoxicate cadmium by sequestration (Waalkes and Goering, 1990). Hence, multiple mechanisms may contribute to enhanced
ZINC
DEFICIENCY:
Cd TOXICITY
cadmium-induced renal toxicity during dietary zinc deficiency. In summary, it appears that marginal zinc deficiency can increase the carcinogenic potential of cadmium. In combination with the observations that excess zinc is very effective in prevention of cadmium carcinogenesis (Gunn et al., 1963, 1964; Waalkes et al., 1988), these results clearly indicate that zinc has a fundamental role in the mechanism of cadmium carcinogenesis. ACKNOWLEDGMENTS
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WAALKES,
KOVATCH,
induction of rat neoplasms. Am. J. Pdzol. 86, 26A34A. MIKOL, Y. B., HOOVER, K. L.. CREASIA,D.. AND POIRIER, L. A. (1983). Hepatocarcinogenesis in rats fed methyldeficient, amino acid-defined diets. Curcinogenesis 4, 1619-1629. NG, W. L., FONG, L. Y. Y., AND NEWBERNE.P. M. (1984). Forestomach squamous papillomas in the rat: Effect of dietary zinc deficiency on induction. Cancer Left. 22, 329-332. O’DELL, B. L. ( 1969). Effect of dietary components upon zinc availability. A review with original data. Am. J. Clin. Nutr. 22, 1315-1322. PARIZEK. J. (1957). The destructive effect ofcadmium ion on testicular tissueand its prevention zinc. J. Enducrkzol. 15,56-63. PARZYCK, D. C., SHAW. S. M., KESSLER,W. V.. VETTER, R. J.. VANSICKLE, D. C., AND MAYES. R. A. (1978). Fetal effects of cadmium in pregnant rats on normal and zinc-deficient diets. B14ll. Environ. Contam. Toxicol. 19,206-2 14. PETERING. H. G., JOHNSON,M. A., AND STEMMER, K. L. ( 197 1). Studies of zinc metabolism in the rat. 1. Doseresponse effectsof cadmium. ‘4rch. Environ. Health 23, 93-101. PETERS,J. M., THOMAS, D., FALK. H., OBER~RSTER, G.. AND SMITH, T. J. (1986). Contribution of metals to respiratory cancer. Environ. Health Perspect. 70, 7183. PISCATOR. M. (1966). Proteinuria in chronic cadmium poisoning. III. Electrophoretic and immunoelectrophoretie studies on urinary proteins from cadmium workers, with special reference to the excretion of low molecular weight proteins. Arch. Environ. Health 12, 335-344. POIRIER. L. A., NEWBERNE, P. M., AND PARIZA. M. W. (1986). Essential Nutrients in Carcinogenesis. Plenum Press. New York. PRASAD.A. S. (1982). Clinical and biochemical spectrum on zinc deficiency in human subjects. In Clinical. Biochemical, und Nutritional .dspects of Trace Elements (A. S. Prasad, Ed.), p. 3. A. R. Liss, New York. ROE, F. J. C.. DUKES. C. E., CAMERON, K. M.. PUGH, R. C. B.. AND MITCHLEY, B. C. V. (1964). Cadmium
AND REHM
neoplasia: Testicular atrophy and Leydig cell hyperplasia and neoplasia in rats and mice following the subcutaneous injection of cadmium salts. Br. J. Cancer 18,674681. SUNDERMAN, F. W.. JR., AND MCCULLY, K. S. (1983). Effectsof magnesium compounds on carcinogenicity of nickel subsulfide in rats, Carcinogenesis 4,46 I-467. TAKENAKA. S., OLDIGES, H., KONIG, H.. HOCHRAINER. D., AND OBERD~RSTER, G. (1983). Carcinogenicity of cadmium chloride aerosols in W rats. .I. Nazi. Cancer Inst. 70. 367-373. VAN RENSBURG,S. J. ( 198 1). Epidemiological and dietary evidence for a specific nutritional predisposition to esophageal cancer. J. Nail. Cancer Inst. 67, 243-25 1. VAN RENSBURG, S. J.. Du BRUYN. D. B.. AND VAN SCHALKWYK. D. J. (1980). Promotion of methylbenzylnitrosamine-induced esophageal cancer in rats by subclinical zinc deficiency. Nutr. Rep. Int. 22,89 l-897. WAALKES, M. P. (1986). Effect of dietary zinc deficiency on the accumulation of cadmium and metallothionein in selected tissuesof the rat. J. To,xicol. Environ. Health 18, 301-313. WAALKES. M. P., AND GOERING, P. L. (1990). Metallothionein and other cadmium-binding proteins: Recent developments. Chem. Res. To.Gcol. 3, 28 l-288. WAALKES. M. P.. AND OBERDORSTER.G. (1990). Cadmium carcinogenesis. In Biological Effects of Heavy Metals: Volwne II, Metal Carcinogenesis (E. C. Foulkes. Ed.), CRC Press. Boca Raton, FL. pp. 129-I 58. WAALKES. M. P.. REHM, S., RIGGS, C. W., BARE, R. M., DEVOR. D. E., POIRIER. L. A., WENK. M. L., AND HENNEMAN, J. R. (1989). Cadmium carcinogenesis in Wistar [Crl: (WI)BR] rats: Dose-response effectsof zinc on tumor induction in the prostate, in the testes, and at the injection site. Cancer Res. 49, 4282-4288. WAALKES, M. P., REHM, S., RIGGS. C. W., BARE, R. M.. DEVOR, D. E., POIRIER, L. A., WENK, M. L., HENNEMAN, J. R., AND BALASCHAK, M. S. (1988). Cadmium carcinogenesis in the male Wistar [Crl: (WI)BR)J rat: Dose-response analysis of tumor induction in the prostate, the testes,and at the injection site. Cancer Res. 48, 4656-4663.
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APPLIED
PHARMACOLOGY
108,448-456
(199
1)
Effect of Chronic Dietary Zinc Deficiency on Cadmium Toxicity and Carcinogenesis in the Male Wistar [Hsd: (WI)BR] Rat’ MICHAEL P. WAALKES, *S ROBERT KOVATCH,P AND SABINE REHM* *Inorganic Carcinogenesis and Tumor Pathology and Pathogenesis Sections, Laboratory of Comparative Careinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland; and tPathology Associates. Inc., Frederick, Maryland
Received
October
12. 1990: accepted January
9. 1991
Effect of Chronic Dietary Zinc Deficiency on Cadmium Toxicity and Carcinogenesis in the Male Wistar [Hsd: (WI)BR] Rat. WAALKES, M. P., KOVATCH. R., AND REHM, S. (1991). Toxicol. Appl. Pharmacol. 108, 448-456. Though it is known that excess zinc will prevent cadmium carcinogenesis, the impact of zinc deficiency on cadmium carcinogenesis has not been defined. This study assessedthe effect of dietary zinc deficiency on the carcinogenic potential of cadmium in rats. Groups (n = 28 each) of male Wistar [Hsd: (WI)BR] rats were fed diets adequate (60 ppm) or deficient (7 ppm) in zinc and received a single SCdose of cadmium (5, 10, or 30 pmol Cd/kg). Lesions were assessedover the next 92 weeks. All cadmium doses increased the incidence of testicular interstitial cell tumors. The incidence of cadmium-induced testicular tumors was unaffected by dietary zinc status. However, when multiplicity of testicular lesions was considered, zinc-deficient diets markedly increased the number of testicular interstitial cell adenomas generated by cadmium exposure while significantly reducing the number of preneoplastic lesions (interstitial cell hyperplasias). The combined total number of neoplastic and preneoplastic lesions ofthe testes was independent of zinc status clearly indicating a shift from hyperplasia to neoplasia within the testes of zinc-deficient rats. The highest cadmium dose (30 wmol/kg) increased injection site sarcomas in zinc-deficient rats (7 tumors/27 rats at risk) but not zinc-adequate rats (3/26) when compared to control (O/49). Chronic progressive renal nephropathy was accelerated by cadmium in zinc-deficient rats. Results indicate that dietary zinc deficiency enhances carcinogenic response at the injection site of cadmium, promotes the neoplastic progression of cadmium-induced testicular lesions, and enhances chronic progressive nephropathy. Thus, dietary zinc deficiency appears to cause a generalized increase in the chronic toxic effects of cadmium. 8 1991 Academic press, IIIC.
The heavy metal cadmium is a potent carcinogen in laboratory animals and possibly a human carcinogen (see Waalkes and Oberdiirster, 1990 for review). The rodent testes are a well-defined target of cadmium and testicular interstitial cell tumors occur with high frequency following cadmium exposure (Roe d al., 1964; Gunn et al., 1963, 1964; Waalkes et ’ Presented in part at the 1990 Annual Meeting of the American Association for Cancer Research, Washington, DC. ’ To whom correspondence should be addressed at the National Cancer Institute-FCRDC, Building 538, Room 205E. Frederick, MD 2 1702- 120 1. 0041-008X/91
$3.00
Copyright Q 1991 by Academic Press, Inc. All rights of reprcduct~on in any form reserved.
448
al., 1988, 1989). The sites of cadmium injection, both subcutaneous and intramuscular, are also well defined as locations for cadmiuminduced tumors (Heath et al., 1962; Gunn et al., 1963, 1964; Kazantis and Handury, 1966; Waalkes et al., 1988, 1989). More recent evidence indicates that the rat lungs (Takenaka et al., 1983) and prostate (Waalkes et al., 1988, 1989) are target sites of cadmium carcinogenesis under certain constraints of route or dosage. Retrospective epidemiological studies provide some evidence that occupational or environmental cadmium exposure is associated with human prostatic and pulmonary
ZINC
DEFICIENCY:
Cd
TOXICITY
carcinogenesis, although this has not been shown in all cases (Bako et al., 1982; Peters et af., 1986; WaaIkes and Oberdorster, 1990). The most prominent nonneoplastic lesion observed with chronic exposure in both humans and animals is nephropathy (Friberg, 1950; Piscator, 1966; Dudley et al., 1985; Maitani et al., 1988; Foulkes, 1990). Numerous reports indicate that the effects of cadmium in laboratory animals can be prevented or markedly reduced by the administration of excess zinc (Parizek, 1957; Gunn et al., 1963, 1964; Goering and Klaassen, 1984; Waalkes et ai., 1989). This includes both acute (Parizek, 1957; Goering and Klaassen, 1984) and chronic effects such as carcinogenesis (Gunn et al., 1963, 1964; Waalkes et al., 1989). In this regard it is thought that many of the toxic effects of cadmium occur through a metabolic disruption of zinc-mediated metabolic processes (Bremner, 1974). On the other hand, very little is known about cadmium toxicity during periods of zinc deficiency. The few studies available indicate that cadmium toxicity, including transplacental toxicity, can be markedly increased in zinc-deficient animals (Parzyck et al., 1978; Petering et al., 1971). Zinc deficiency in rats will also cause an enhanced disposition of cadmium in various tissues, including the testes and kidney (Waalkes, 1986). The effect of zinc deficiency on cadmium carcinogenesis has not been defined, however. The present study was, therefore, designed to assessthe effect of chronic dietary zinc deficiency on cadmium toxicity and carcinogenesis in the rat. In this regard, a level of dietary zinc resulting in what can be termed a marginal deficiency was selected in that it has been shown to cause a 40% reduction in plasma zinc without affecting body weight gain or causing any overt lesions (Waalkes, 1986). MATERIALS
AND
METHODS
Diet. The zinc-defined diets used in this study were obtained from Teklad Company, Inc. (Madison. WI). The basal diet was first analyzed using atomic absorption spec-
AND
CARCINOGENESIS
449
trometry for zinc content (1 .O ppm) and then adjusted to the desired level with zinc acetate dihydrate. Cadmium was not detectable in the basal diet. This level of dietary zinc will produce a 40% reduction in circulating plasma zinc in rats (Waalkes. 1986). Animals and treatment. A total of 224 male Wistar [Hsd: (WI)BR] tats were obtained at 4 weeks of age from HarlanSprague-Dawley (Indianapolis, IN). They were housed 3 per polycarbonate cage in a standard barrier facility, at a temperature of 68-72”F, with a relative humidity of 50 f 5%, and a 12-hr light/dark cycle. The zinc-defined diet and water were given ad libitum. Cadmium (CdC& .2f H,O; J. T. Baker Co.) solutions were prepared in sterile normal saline. Rats were divided into groups as designated in Table 1. At 8 weeks of age rats were placed on the zinc-defined diets for 2 weeks prior to cadmium exposure (Week 0). Rats were then injected (SC; 4.0 ml/kg) in the dorsal thoracic midline area (henceforth referred to as the injection site) with 0 (saline) or 5, 10. or 30 rmol Cd/kg. Body weights, survival, food consumption, and clinical signs were recorded throughout the experiment. Rats were killed when moribund or at 92 experimental weeks. Body weight and testes weights were recorded for each rat at necropsy. Pathology. A complete necropsy was performed on all animals. Injection site, liver, pancreas, spleen, kidneys, prostate, testes, and any abnormal tissues from each animal were studied microscopically. Lung, pituitary, and adrenals were not inspected. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 pm, and stained with hematoxylin and eosin (HE) for histological examination. For testes the number of hyperplastic (preneoplastic) and neoplastic interstitial cell foci (interstitial cell adenomas) was determined for each rat in a single midline section of both testes. The differentiation of interstitial cell hyperplasia and interstitial cell adenoma is to a certain extent subjective as the exact biological behavior of such lesions is unknown. Interstitial cell hyperplasia consisted of clusters of cells in the testicular interstitium that had a central nucleus and moderately abundant amorphous eosinophilic or vacuolated cytoplasm that did not exceed the diameter of a single normal seminiferous tubule. Such lesions only minimally compressed adjacent tissue. Adenoma was diagnosed when the interstitial cell proliferation exceeded the diameter of a normal seminiferous tubule. In such cases, distinct compression of adjacent testicular tissue was evident. Data analysis. In all cases, a probability level ofp < 0.05 was considered to indicate a significant difference. In pairwise comparison of tumor incidence the Fisher’s exact test was used. Body weight data were examined by Dunnett’s test. Survival was examined with the Cox test and the generalized Kruskal/Wallis test and was only considered significantly different if both tests indicated p values < 0.05. In all cases, the number of rats at risk was defined as the number of rats surviving at the time of appearance of the first tumor of any type (28 weeks).
450
WAALKES,
KOVATCH, TABLE
AND
REHM
I
EXPERIMENTAL DESIGN
Group 1 2 3 4 5 6 7 8
Initial No. of rats
Effective No. of rats0
28 28 28 28 28 28 28 28
24 24 26 25 25 26 27 24
Dietary zinc’ bw)
Subcutaneous cadmium ’ (pmol/kg)
7 7 7 1 60 60 60 60
a Defined as those animals surviving to the appearance of the first tumor of any type (28 weeks). ’ Mixed in the diet at the specified level of zinc in the form of zinc acetate, from -2 weeks to the termination study (+92 weeks). ’ Given in the dorsal thoracic midline at Time 0. Tumors were assessed over the next 92 weeks.
RESULTS
0 5 10 30 0 5 10 30
of the
Likewise, no group showeddifferences in food consumption from their respective control Dietary zinc deficiency alone had no effect groups. on survival. Furthermore, survival was the Zinc deficiency did have a marked effect on same in rats fed zinc-deficient diets regardless the generation of tumors at the injection site of the level of cadmium exposure when com- of cadmium (Table 2). Only in the group fed pared to zinc-deficient or zinc-adequate con- zinc-deficient diets and given the highest dose trols (not shown). On the other hand, survival of cadmium was there a significant elevation was significantly depressedin rats fed a zinc- in the incidence of injection site sarcomas adequate diet and given the highest dose (30 (28%). Overall, zinc-deficient rats developed ymol/kg) of cadmium (Fig. 1) when compared to zinc-adequate controls but not in comparison to zinc-deficient rats receiving the level 1.0 of cadmium. Likewise, zinc deficiency by itself 0.9 had no effect on body weight (not shown). In 0.6 fact, only in thoseanimals receiving the highest I 2 : 0.7 doseof cadmium (30 pmol/kg) was any weight 56 gain suppressionobserved and this proved in,E C o.6 . opmO, Cd,kg $ 2 0.5 dependent of dietary zinc status. For approx0 30 ,,mol Cd/kg 1: imately 10 weeks after cadmium injection a .+c 6” 0.4 0.3 12- 17%reduction in body weight wasdetected ::p l #A 0.2 in animals receiving 30 pmol Cd/kg regardless 0.1 of dietary zinc level. This suppressionof body 0.0 weight was resolved by 15 weeks following 0 10 20 30 40 50 60 70 80 9( cadmium injection and all groups showed the Time (weeks) same or similar body weight through the reFIG. 1. Effect of cadmium exposure (30 rmol/kg, SC, mainder of the study. Time 0) on survival of rats fed a diet adequate in zinc (60 Food consumption was also not affected by ppm). Survival was significantly (p < 0.05) depressed in rats in comparison to control rats. dietary zinc status at any point in the study. cadmium-exposed
ZINC
DEFICIENCY:
Cd TOXICITY
AND
TABLE INCIDENCE
451
CARCINOGENESIS
2
OF CADMIUM-INDUCED INJECTION SITE SARCOMAS IN MALE WISTAR [H.sd:(WI)BR] FED DIETS ADEQUATE OR DEF’ICIENT IN ZINC
Group
N”
1 2 3 4 5 6 7 8
24 24 26 25 25 26
a Rats at risk. * An asterisk indicates
Cadmium dose ( rmol/W
Dietary zinc level (ppm)
0 5 10 30 0 5 10 30
I I 7 7 60 60 60 60
21
24
a significant
difference
from appropriate
more than twice as many cadmium-induced injection site tumors (8 sarcomas/79 rats; 10.1%) than zinc-adequate rats (3/78; 3.9%). Cadmium was very effective in inducing interstitial cell tumors, and dose-related increases in overall tumor incidence (rats bearing tumors/total rats at risk) occurred in rats fed either zinc-adequate and zinc-deficient diets (Fig. 2). A maximum incidence of approximately 70% occurred in both zinc-deficient and zinc-adequate animals at cadmium doses >, 10 pmol/kg. No differences in incidence based on dietary zinc level were detected between groups receiving the same level of cadmium. When the multiplicity of adenomatous (neoplastic) and hyperplastic (preneoplastic) interstitial cell lesions was considered, marked differences occurred between animals fed zincadequate or zinc-deficient diets (Table 3; Fig. 3). Although the total number of preneoplastic plus neoplastic lesions was not affected by dietary zinc, a major shift occurred towards adenoma formation in rats consuming diets deficient in zinc (Table 3). In zinc-deficient rats more than 80% of all lesions in the testes were neoplastic while in zinc-adequate rats fewer than half of the total lesions were adenomas. This shift toward adenoma formation occurred in all cadmium dosage groups (Fig. 3)
control.
Tumors
RATS
Injection sarcomas 0 0 I 7* 0 0 0 3
were mostly
site (%)b
(0.0%) (0.0%) (3.6%) (28.0%) (0.0%) (0.0%) (0.0%) (12.5%)
fibrosarcomas.
and was reflected in an overall decreased incidence of hyperplasia in zinc-deficient animals (Fig. 4). The control rats in this study also showed a fair incidence of moderate to severe chronic nephropathy which was not affected by dietary zinc status (Table 4). The kidney lesions were typical of the age-dependent, progressive chronic nephropathy of rats and included glomerular and tubular basement membrane thickening, glomerular sclerosis, chronic interstitial inflammation, proteinaceous tubular
H O-0
0
5
10 Cadmium
15
Zinc Adequate Zmc Deficient
20
Dose bmollkg.
25
30
se)
FIG. 2. Incidence of cadmium-induced testicular interstitial cell tumors in rats fed diets adequate (60 ppm) or deficient (7 ppm) in zinc. Data are expressed as the proportion (%) of tumor-bearing rats in the total number of rats at risk.
WAALKES,
452
KOVATCH.
AND REHM
TABLE 3 EFFECTOF ZINC DEFICIENCYONCADMIUM-INDUCED NEOPLASTICAND PRENEOPLASTICHYPERPLASTIC INTERSTITIAL CELL FOCI IN THE MALE WISTAR [Hsd:(WI)BR] FCAT TESTES Groups
N
Cadmium dose (wol/kg)
Dietary zinc level (ppm)
I 2-4 5 6-8
24 75 25 77
0 S-30 0 5-30
7 7 60 60
Adenoma’ 3 (100%) 194 (83%) 0 (0%) I 15 (47%)
Hyperplasia’
Total lesions
0 (0%) 39(17%) 1 (100%) 131(53%)
3 233 1 246
” Percentages reflect the proportion of total lesions.
casts, and tubular regeneration. However, in zinc-deficient rats given cadmium, a marked increase in the incidence of chronic nephropathy occurred in a dose-related fashion. Various incidental tumors were detected in the 201 total rats at risk that were not associated with any treatment. These included 149 pancreatic acinar cell adenomas; 22 pancreatic acinar cell carcinomas; 8 pancreatic islet cell adenomas; 2 1 thyroid C-cell adenomas; 7 thyroid C-cell carcinomas; 7 noninjection site sarcomas; 6 noninjection site fibromas; 10 keratoacanthomas; 2 lipomas; 2 squamous cell papillomas; 4 prostatic adenomas; and 3
g
mammary gland fibroadenomas. A single one of each of the following types of tumors also occurred: a mesothelioma; a hepatocellular carcinoma; a brain granular cell tumor; a skin squamous cell carcinoma; a forestomach squamous cell carcinoma; a mammary gland adenocarcinoma; a hemangioma; and a skin basal cell adenoma.
DISCUSSION Although in humans deficiency of zinc in its severest forms occurs only very rarely (Prasad, 1982), marginal deficiency of zinc is considered to be sufficiently common to be a po-
loo-
‘ij B
* O... -&--
H I-"
.z” 2 8 t B
----_-
--
_____
BO-
looh
-
60 40--
l -
v5
10
15
20
25
Odium Dose(rmollkg,sc)
Cadmium
3. Proportion (%) of cadmium-induced testicular neoplasia in the total neoplastic plus preneoplastic (interstitial cell hyperplasia) lesions in rats fed diets adequate (60 ppm) or deficient (7 ppm) in zinc. Asterisks indicate a significant (p Q 0.05) difference from rats fed diets adequate in zinc. Pooled cadmium represents all groups given cadmium (5, 10. and 30 pmol/kg) at a specific dietary zinc level. FIG.
g 80. .9 H c t r” x
60.
40-
E 0” E
zo-
-
t
OL Cadmium
Dose bmollkg,
SC)
FIG. 4. incidence of cadmium-induced testicular interstitial cell hyperplasia in rats fed diets adequate (60 ppm) or deficient (7 ppm) in zinc. Data expressed as the proportion (%) of hyperplasia bearing rats in the total number of rats at risk.
ZINC
DEFICIENCY:
TABLE
Cd TOXICITY
4
INCIDENCE OF CHRONIC NEPHROPATHY IN MALE WISTAR [Hsd:(WI)BR] RATS FED DIETS ADEQUATE OR DEFICIENT IN ZINC
Group
Cadmium ( wol/kg)
1 2 3 4 5 6 I 8
0 5 10 30 0 5 10 30
dose
Dietary zinc level (ppm) 7 I 7 7 60 60 60 60
Nephropathyn 9/24 (38%) 14/24 (58%) 18/26* (69%) 20/25 * (80%) I l/25 (44%) 13126 (50%) 14121 (52%) 15/24 (63%)
‘Data expressed as rats bearing lesion/rats at risk (‘3%total). Only animals showing a moderate to severe lesion are included. An asterisk indicates a significant difference from appropriate control.
tential public health concern (Hambidge et al., 1979; Beach et al., 1982; Prasad, 1982). Marginal zinc deficiency can be brought about by various factors including dietary composition (O’Dell, 1969), pharmaceuticals (Prasad, 1982), and disease states (Prasad, 1982) and is further complicated by the fact that such mild deficiencies are unlikely to be detected (Prasad, 1982). Little is known about how marginal deficiencies in zinc modify the toxicity of other metals. The present study evaluated the carcinogenic potential of cadmium in rats fed zinc-adequate and zinc-deficient diets. The zinc-reduced diets used in the present study produced what can be described as a marginal zinc deficiency condition since such diets are known to produce a 40% reduction in plasma zinc levels (Waalkes, 1986) but did not modify body weight, food consumption, or survival over the course of the present study. Despite the absence of such overt effects, the zinc-deficient diets clearly increased the carcinogenic potential of cadmium, both through an increased incidence of sarcomas at the injection site of cadmium and through an enhancement of the progression towards neoplasia within the testes. Renal lesions associated with cadmium were also exacerbated
AND
CARCINOGENESIS
453
by zinc deficiency. Thus, the results of the present study indicate that even a marginal dietary deficiency of zinc will enhance cadmium carcinogenicity and toxicity. Given the prevalence of such subclinical dietary zinc deficiencies in human populations (Hambidge et al., 1979; Beach et al.. 1982; Prasad, 1982) this may place a multitude of individuals at greater risk from cadmium exposure. It would seem that dietary zinc deficiency could be considered to increase susceptibility to experimental cadmium carcinogenesis. Various other dietary deficiencies can clearly enhance carcinogenesis in rodents (Poirier et al., 1986). For instance, dietary methyl deficiency increases the tumor incidence in rat liver resulting from an initiating dose of diethylnitrosamine (Mikol et al., 1983) even when given for relatively brief periods (Hoover et al., 1984). Zinc deficiency too can be an effective enhancer of carcinogenesis induced by various organic carcinogens in rodents (Barth and Iannaccone, 1986). An increased tumor incidence occurs with dietary zinc deficiency in the esophagus of rats given methylbenzylnitrosamine (Fong et al., 1978, 1984; Van Rensburg et al., 1980). Zinc deficiency has also been associated with an increased incidence of human esophageal cancer (Van Rensburg, 198 1). Enhancement of chemically induced forestomach tumors also occurs with dietary zinc deficiency in rats (Ng et al., 1984; Fong et al., 1984). Thus, as is the case with many organic carcinogens, the efficacy of the inorganic carcinogen, cadmium, can be increased by dietary zinc deficiency. In general it appears that tumor incidence or multiplicity is enhanced by dietary zinc deficiency within a given tissue (Fong et al., 1978, 1984; Van Rensburg et al., 1980; Ng et al., 1984). In contrast, shifts of target tissue do not appear to occur with suboptimal zinc intake. In this regard, cadmium is an effective testicular tumorigen (Gunn et al., 1963, 1964; Waalkes et al., 1988, 1989) and produces sarcomas at the site of SCinjection (Gunn et al., 1963, 1964; Waalkes et al., 1988, 1989). These are in fact the same sites at which the carcinogenic effi-
454
WAALKES.
KOVATCH.
cacy of cadmium wasenhanced by dietary zinc deficiency in the present study. The exact mechanism by which zinc deficiency modifies cadmium carcinogenesiscan not be determined from the present results. In the caseof dialkylnitrosamines it appearsthat zinc deficiency increases microsomal P450mediated metabolic activation (Barth et al., 1984) and thus carcinogenic activity (Barth and Iannaccone, 1986). Cadmium, on the other hand, presumably does not require any such metabolic activation. Zinc deficiency does, however, increase the distribution of cadmium to the testes (Waalkes, 1986) and assuming that higher concentrations of the metal would lead to more tumors, this sort of modification in toxicokinetics could have some bearing. It also appears that zinc deficiency can reduce concentrations of the cadmium binding protein, metallothionein, in some tissues(Waalkes, 1986). Since metallothionein is thought to function in part in detoxication of cadmium (Waalkes and Goering, 1990), reduced levels may increasevarious aspects of cadmium toxicity, including carcinogenicity. Zinc-mediated or -dependent metabolic processes,including roles in cellular production of DNA, RNA, and protein (Brady, 1982) are thought to be a prime target of cadmium toxicity (Brady, 1982; Bremner, 1974). However, the exact molecular target or mechanism by which zinc deficiency enhances cadmium carcinogenesisremains to be determined. Metal-metal interactions, specifically interactions with essentialelements, are clearly a key aspectof metallic carcinogenesis.In this regard there are several examples of such interactions that result in reduction of carcinogenic response.This includes the inhibition of cadmium carcinogenesis at the injection site and in the testes by administration of excess zinc (Gunn et al., 1963, 1964; Waalkes et al.. 1989) and the prevention of nickel-induced sarcomas by excess manganese (Sunderman and McCully, 1983) or magnesium (Kasprzak et al., 1985a). Enhanced carcinogenic response can also be observed. Examples of this include
AND
REHM
the increases in cadmium-induced prostatic tumors brought about by excesszinc (Waalkes et al., 1989) and the enhancement of lead-induced renal tumors by calcium (Kasprzak et al.. 1985b) or magnesium (McCreary et al., 1977). The resultsof the present study indicate that the probable derangement in zinc metabolism brought about by even modest dietary zinc deficiency can also have a marked enhancing effect on tumor incidence and progressionfollowing exposure to cadmium. Thus it appears that alterations in essential metal metabolism are of primary mechanistic importance in the process of metallic carcinogenesis,although this is clearly both tissueand metal specific. Chronic renal lesionsare a common finding in cadmium exposed humans or animals (Friberg, 1950; Piscator, 1966; Dudley et al.. 1985; Maitani et al., 1988; Foulkes, 1990). The kidney lesionsof the rats in the present study were comparable to the age-dependent chronic nephropathy of rats for which exact cause and pathogenesisare unknown (Gray, 1986). As such, the lesions do not precisely resemble those typically observed with multiple injections of cadmium. The control rats in this study also showed a moderate incidence of such nephropathy which was not affected by dietary zinc status. However, dietary zinc deficiency caused a marked increase in the incidence of chronic renal lesions seen in cadmium-treated rats. The observation that zinc deficiency will enhance accumulation of renal cadmium (Waalkes, 1986) may have a very important role in this increase in toxicity of cadmium, particularly sincea fairly low critical concentration seemsto be necessary for cadmium induced renal lesionsto occur (Foulkes, 1990). The apparent suppressionof metallothionein levels within the kidneys of zinc deficient rats (Waalkes, 1986) may also allow a greater effective toxicant concentration at the target site, since metallothionein is thought to detoxicate cadmium by sequestration (Waalkes and Goering, 1990). Hence, multiple mechanisms may contribute to enhanced
ZINC
DEFICIENCY:
Cd TOXICITY
cadmium-induced renal toxicity during dietary zinc deficiency. In summary, it appears that marginal zinc deficiency can increase the carcinogenic potential of cadmium. In combination with the observations that excess zinc is very effective in prevention of cadmium carcinogenesis (Gunn et al., 1963, 1964; Waalkes et al., 1988), these results clearly indicate that zinc has a fundamental role in the mechanism of cadmium carcinogenesis. ACKNOWLEDGMENTS
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E. S. O., HANSON,
J., AND
DEWAR,
R.
(1982). The geographical distribution of high cadmium concentrations in the environment and prostate cancer in Alberta. Can. J. Public Health 73, 92-96. BARCH, D. H., AND IANNACCONE, P. M. (1986). Role of zinc deficiency in carcinogenesis. In Essentiul Nutrients in Carcinogenesis. (L. A. Poirier, P. M. Newberne, and M. W. Pariza, Eds.), pp. 5 17-527. Plenum Press, New York. BARCH, D. H., IANNACCONE,
KUEMMERLE. S.. HOLLENBERG, P., AND P. M. (1984). Esophageal microsomal
metabolism of N-nitrosomethylbenzylamine in zincdeficient rats. Cancer Res. 44, 5629-5633. BEACH. R. S., GERSHWIN, M. E., AND HURLEY, L. S. (1982). Zinc, copper, and manganese in immune function and experimental oncogenesis. Nutr. Cancer 3, I72191. BRADY, F. 0. (1982). The physiological function of metallothionein. Trends Biochem. Sci. 7, 143- 145. BREMNER, I. (1974). Heavy metal toxicities. (2. Rev. Bio-
phys. 7, 75-124. DUDLEY, R. E., GAMMAL, L. M.. AND KLAASSEN, C. D. ( 1985). Cadmium-induced hepatic and renal injury in chronically exposed rats: Likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol. .4pp/.
Pharmacol. 77, 414-426. FONG,
L. Y. Y., LEE, J. S. K., CHAN, W. C., AND NEWP. M. (1984). Zinc deficiency and the development of esophageal and forestomach tumors in BERNE,
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