[CANCER RESEARCH 39, 2451 -2456, July 1979]0008-5472/79/0039-0000502.00

Zinc Deficiencyand Growthof EhrlichAscitesTumor1

Daniel T. Minkel, Patricia J. Dolhun, Barbara L. Calhoun,2 Leon A. Saryan,3 and David H. Petering4

Department of Chemistry, University of Wisconsin at Milwaukee, Milwaukee, Wisconsin 53201

ABSTRACT

The growth rate of Ehrlich ascites tumors has been examinedas a function of the zinc content of the diet of the host mice.Imposition of a diet containing a bowamount of zinc (1 @g/g)on the day of tumor transplant leads to a marked retardation ingrowth. Pretreatment of the mice with this diet slows the growthfurther so that the lifetime of the mice can be doubled. Cells insuch animals are still viable and rapidly proliferate after theanimals are placed on a diet containing zinc. Growth rate ofthe tumor is also recorded at levels of zinc (40, 80, 160, and250 tog/mI) in the drinking water. All of these results areexamined in relationship to the zinc in the ascites fluid, whichprovides the zinc nutriture for the tumor. A direct correlationbetween growth rate and fluid zinc content is observed. Theinfluence of diet and the tumor upon zinc content of the liver ofthe host is examined. The results indicate that the tumoressentially sequesters zinc from the animal under zinc-deficientconditions. Over a 10-fold range of fluid zinc values, there areno clear differences in the concentration of zinc within theascites cells. This occurs despite the facile uptake and effluxof zinc ion by the Ehrlich cell.

INTRODUCTION

Dietary zinc is necessary for the proper development andgrowth of mammals including humans (2, 10, 11, 17). Underconditions of nutritional deficiency of the metal, severe abnormalities develop in fetuses of rats, and young animals areunable to grow at a normal rate. In rapidly proliferating tissues,zinc deficiency leads to an inhibition of DNA synthesis (6, 9,20, 22, 26). These observations have been extended totransplanted, malignant tumors which either do not grow or grow ata much reduced rate after implantation into a zinc-deficienthost(i,5,15,18).

Efforts to determine the cellular basis of these effects usingtissue culture methods have affirmed the dependence of DNAsynthesis upon the presence of zinc in the extracellubar medium(3, 14, 21 , 27, 28). However, all of these studies have resortedto the use of metal-chelating agents to establish a zinc-deficientstate. This necessarily leads to ambiguity in the specificity andsites of metal removal and to the possibility of other reactionsof the ligands or the newly formed metal complexes with thecell population (7).

Because of the importance of zinc in the growth of normaland malignant proliferating tissues, the present study was

â€S̃upportedbyUSPHSandtheGraduateSchooloftheUniversityofWisconsinat Milwaukee.

2 Supported by National Science Foundation Undergraduate Summer Re

search Award to the University of Wisconsin at Milwaukee.3 Recipient of Postdoctoral Fellowship CA 05528 from the National Cancer

Institute.4Towhomrequestsforreprintsshouldbeaddressed.RecipientofResearchGrants CA 16156 and ES 01504.

Received December 27, 1977; accepted March 29, 1979.

initiated to examine in detail the cellular roles of zinc in apopulation of Ehrlich ascites tumor cells for which zinc nutritumeis determined by the food intake of the animal host.

MATERIALS AND METHODS

Materials

A pebletedzinc-deficient diet analyzed to contain zinc (0.7 to1 .0 @sg/g) was prepared by Ziegler Brothers, Inc. , Gardners,

Pa., according to a dietary composition published previouslywhich has been used to produce zinc deficiency in rats (16).The normal diet was the standard laboratory chow of RalstonPurina Co. which contains zinc (70 to 90 @tg/g)according toanalyses of several lots. Six- to 8-week-old female Swiss HA!ICR mice were obtained from ARS/Sprague Dawley, Madison,Wis. Reagent grade zinc chloride (Mallinckrodt ChemicalWorks, St. Louis, Mo.) was used to supplement the zinc-deficient diet. All other chemicals were reagent grade.

Methods

Animals and Ehrlich Ascites Tumors. Groupsof 5 mice 14to i 8 weeks old and weighing 25 to 30 g were housed instainless steel wire cages over wood chip bedding and fed andwatered with stainless steel and glass containers. Animalsplaced on a zinc-controlled diet were maintained in cageswhich were scrubbed weekly with 1 mM EDTA and had accessto drinking bottles previously soaked in the same solution andfitted with bow-zincNalgene stoppers. The mice were kept in aconstant temperature room at 25°equipped with a timer tomaintain a 12-hr light-dark cycle. They were fed ad !ibitumeither laboratory chow and tap water containing zinc (less thani @.tg/ml) or the specially prepared zinc-deficient diet and glass

distilled water. In some experiments, this water was supplemented with zinc chloride at zinc concentrations of 40, 80,160, or 250 sg/ml.

Ehrlich ascites tumor (Arthur Little Co., Cambridge, Mass.)was maintained in vivo by i.p. transplantation of 5 x 106 cellsinto recipient mice. Cells in ascites fluid were harvested on theday of experiment by aspiration of the peritoneal fluid intosterile 10-mbdisposable plastic syringes (Pharmaseal Labomatories, Glendale, Calif.) and deposited into 15-mI plastic Dispoculture tubes (Scientific Products, McGaw Park, Ill.) containinga small drop of sodium heparin (1000 units/mI). Control experiments with tubes, distilled water, and heparin showed thatno detectable zinc (less than 0.01 25 @g/ml)was added to thefluid by this procedure. Cells were obtained as a pellet bycentrifugation. Typically, cells and fluid were separated bycareful decantation, and the fluid was reserved for zinc anabysis. Any emythrocytespresent were removed by careful removalof the medpellet with a Pasteur pipet. Cells were resuspendedin ascites fluid or Ringer's solution and counted microscopicallyusing a Spencer Bright-Line hemacytometer (American Optical

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D. 7'. Minke! et a!.

Corp. Buffalo, N. V.) at x 100. Protein concentrations weremeasured with the biuret reagent as standardized with bysozyme or albumin using a scattered transmission accessory ona Beckman Acta V spectrophotometer to correct for sampleturbidity. The error in cell counting for typical samples was±1 5% (S.D.) and in the biuret test, ±10%.

Growth Measurements. The individual weights of 5 weightmatched mice in a group provided with a specified diet wereaveraged daily, and growth curves were generated. The datafor the log phase of the tumor growth were subjected to a linearregression analysis to yield the best slope, which was thenused as the growth rate of the tumor. In all cases, correlationcoefficients of the data exceeded 0.95. It is recognized that ingrowth retardation not only the slope of the line may changebut also the period between injection of cells and day ofdetectable tumor growth may vary.

Metal Analyses. The zinc concentrationsof cells and fluidwere measured only in tumors which were essentially free ofRBC. Ascites fluid was diluted 1:5 with glass-distilled waterand aspirated directly into the flame of a Perkin-Elmer 360atomic absorption spectrophotometer. One ml of cell suspension previously washed in 0.1 5 M NaCI was digested at 90°ina solution of 50% HCIO4,30% concentrated HNO3, and 20%glass-distilled water. Samples were then diluted to 6 ml foranalysis. Whole livers were dissected from mice following memoval of ascites fluid, rinsed in 0.i 5 M NaCI, dried with KimWipes disposable wipers (Kimberly-Clark Corp., Neenah, Wis.),and weighed. Samples were wet ashed in 3.0 ml 70% HNO3Baker and Adamson Reagent grade (Allied Chemical Corp.,Morristown, N. J.) at low temperatures. HNO3was evaporatedovernight. Then samples were dry ashed at about 100°,redissolved in 1.0 ml HNO3, and diluted 1:100 into glass-distilledwater for aspiration. In all cases, atomic absorption standardsof zinc from Fisher Scientific Co. (Pittsburgh, Pa.) were used tocalibrate the instrument. Over the concentration range of zincencountered in these studies, there is a maximum error of0.oi2 ;zgzincperml.

Transport Studies with Zinc. Isolated cells were placed ina 7:18 v/v ratio of ascites fluid and Eagle's medium or inRinger's solution (9 g/litem):NaCl (0.42 g/liter):KCI (0.25 g/liter):CaCI2 (0.12 g/liter:Tris (pH 7.4) to achieve a given concentration of cells. Suspensions were exposed to zinc chlorideat time zero at 23 ±2°and then stirred continuously. Thetransport process was stopped at intervals by centrifugation ofaliquots of cell suspension to yield a pellet and external medium. The medium was analyzed directly for zinc. The pelletwas washed in cold Ringer's buffer with 1O@ M EDTA, digested, and then analyzed for zinc. The EDTA wash served toremove zinc loosely bound to the outside of the cells. Controlvalues for cell zinc content were also obtained. In efflux expemiments, cells containing excess zinc from the uptake experiment were resuspended rapidly in metal-free Ringer's buffer attime zero. The loss of zinc from these cells was followed asdescribed above.

RESULTS

Growth Studies. The effect of zinc nutritureuponthe growthof Ehrlich ascites tumors has been evaluated in terms of levelof dietary zinc and the relative times of imposition of dietarydeficiency and tumor cell injection. Chart i illustrates a typical

experiment containing several groups of animals. Uninoculatedanimals maintained on the zinc-deficient dietary regimen (lessthan i ;.tg zinc per g in the diet) for i 5 days (Chart i , opensquares) or for 88 days (Chart 1, open circ!es) show little or noweight bossover the course of the experiment. In these groups,there was only a modest external evidence of zinc deficiencyrevealed by the occasional loss of body hair on a few animalsand/or scaling about the eyes and the feet. Tumor growth ininoculated animals as evidenced by increase in weight is rapidfor host mice reared on the same feed supplemented with zinc(80 zg/mI) in the drinking water (Chart i , c!osed triang!e). Bycomparison, tumors in animals placed on the zinc-deficientregimen on the same day as tumor inoculation grow much moreslowly throughout the experiment (Chart 1, c!osed square). Agroup of mice maintained on the deficient regimen for 60 daysprior to tumor cell inoculation declines slowly in weight andshows no evidence of tumor growth (Chart 1, c!osed circ!es).That viable tumor cells are present in such animals is evidencedby the fact that reimposition of 80 @gzinc per ml drinking waterto inoculated on the 22nd day leads to immediate rapid cellproliferation and weight increase (Chart 1, open triangle).

Table 1 summarizes food intake for these and additionalgroups of animals. Groups 3 to 8 comprised animals used inChart 1. Other groups were from separate experiments. Whatis clear from the examination of either the means ±S.D. or thep is that the presence of the tumor is correlated with a reductionin food intake relative to controls. One sees this in the comparison of Groups 1 and 3 and 6 and 7. However, betweengroups with or without the tumor under various levels of zinc inthe drinking water (Groups 8 and 11 and 1 and 2), there is nosignificant difference in feeding habits. Thus, there is no evidence in either Chart i or Table 1 that the effect of zincdeficiency is simply one of inanition beading to slower tumorgrowth because of general nutritional deprivation.

In general, as shown in Chart 2, tumors in the 0 ,zgzinc perml group grow very slowly in comparison with other control

0

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Chart 1. Kinetics of average weight change of mice bearing tumor undervarious dietary regimens. All groups contained 5 mice and were fed the zincdeficient diet and various levels of zinc in the drinking water beginning on Day 0unless otherwise stated. Tumor cells were injected on Day 0. A, tumor and zinc(80 @ug/ml)(3); U, tumor and zinc (0 pg/mI) (8); 0, no tumor and zinc (0 @ug/ml)(2); 0, no tumor and zinc (0 pg/mI) where Day 0 is Day 60 on this regimen, andno weight loss has occurred prior to this time (4, 6); •,tumor and zinc (0 tug/mI),tumor injected on Day 60 of this dietary regimen (7); Ls,zinc (80 big/mI) added todrinking water of Group 7 on Day 22. Numbers in parentheses, group numbersin Table 1. Bars, typical S.D.‘sfor Day 10 points.

3020Doys

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GroupZlnc(jig/mI)Iumor@'Foodintake

(g/day/mouse)StatisticalcomparisoncGroupp10.3.56

±026d203.30±0.571-2NS3

480 0+,02.34 ±1.12

4.50±0.85'2-30.00255“

6-0

04.32±0,74g

5.17±0.34'NS70+,603.82 ±o,78g6-7<0.00180+,03.60±0.30980+,

03.67 ±0.518-9NS10160+,04.10±0.448-10NS1

1250+, 04.10 ±0.538-1 1NS

Tumorcell densityanddietaryzincZincsupplement(pg/mI)Cells/mI xi0@@a05.6

±06b(11)C4011.9±2.6(7)<0.0018012.2±4.1

(7)<0.0011601 0.0 ±3.0(5)<0.00252509.8

±1.8 (7)<0.001

40 80 607n in V*tter ug/mi

Chart 2. Growth rate of Ehrlich tumor versus dietary zinc level. Points, average weight change for a group of 5 mIce. Calculated from data such as thatshown In Chart 1 and as described in “Materialsand Methods.“O, zinc-deficientdiet imposed on day of injection; 0, commercial animal chow.

groups. When all groups are removed from the stock chow dieton the day of tumor cell injection and placed on their respectivespecial diets, mice on the chow diet [zinc (70 to 90 @tg/g)]oron the zinc-deficient diet supplemented with 80 and 150 or160 @g/gzinc per ml of drinking water sustain an optimalgrowth of the tumor.

A close correlationbetweenthe diet withzinc suppliedin thedrinking water at 80 @g/mland that in which zinc is part of thepelleted feed (70 to 90 @g/g)is seen here and in other dataand indicates that zinc is equally available in the 2 diets andthat the feeds are dietary equivalents in these studies. Whiledrinking water including zinc (80 and 160 @g/ml)is optimal forgrowth, the zinc supplementation (40 jig/mI) does not sustainthis rate of growth. The depressed growth is reflected in otherparameters discussed below. Once the zinc level reaches 250@g/ml,one group in 3 revealed evidence of inhibition of growth.

Table 2 shows that the final weight increase of the mice ingroups given 40 to 250 @sgzinc per ml can be correlated withan average cell count of 10 to 12 x 10@cells/mb. In contrast,the concentration of cells in the zinc-deficient groups is dis

Inhibition of Ehr!ich Tumor Growth by Zinc Deficiency

Table1Foodconsumptionof mice

tinctly lower. Thus, weight changes overestimate tumor cellgrowth rates for these animals.

In experiments in which the initiation of the zinc-deficient dietis varied with respect to the time of tumor cell injection, thetumor grows slower in inverse relation to the duration of thezinc-deficient conditions as illustrated in Chart 3. In fact, in oneexperiment using a 5-week pretreatment with the low zincregimen, the tumor was not evident for the entire period ofobservation. The animals survived for at least 34 days compared with 17 days for the controls. After this period, thequiescent population of cells was stimulated into rapid celldivision by the readdition of zinc into the drinking water asindicated in the chart. A similar experiment was portrayed inChart 1, showing that tumor cells could be stimulated to proliferate after remaining dormant for 24 days in zinc-deficientmice. There is a similar dependence of fluid zinc concentrationupon the relative times of tumor injection and imposition of thedeficient diet.

When zinc deficiency is imposed after tumor cell injection,very little control of the tumor can be obtained. By Day 4, thetumor grows as well as do controls on zinc (80 @g/ml).Thisbehavior is paralleled by final fluid zinc ion concentrations asa function of day of onset of zinc deficiency. It appears that,

a All groups except Group 1 receIved the zinc-deficient diet beginning on Day

0. Group 1 was maintained on Ralston Purina laboratory animal chow, zinc (70to 90 @Lg/g)

b If injected (+), day of Inoculation listed relative to day of initiation of zincdeficient diet.

C Groups used in t test comparIson. Significant dIfferences are taken for p@

0.01.d Mean ±S.D.S N5. not sIgnIfIcant.

I Period of observation: Days 52 to 59 after imposition of zinc deficiency.g Period of observation: Days 60 to 80 after imposition of zinc deficiency.

20

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Chart 3. Growth rate of Ehrllch tumor versus day of imposition of zlnc-deficient diet. All groups were injected on Day 0. Abscissa, number of days on thezinc-deficient diet betore (—)or beginning after (+) the day after injection. •,growth data; 0, fluid zinc values at day of sacrifice; V. growth rate of —34daygroup after addition of zinc (80 gig/mI) to drinking water; 0, control growth rateof mice maintained on zinc (80 aug/mI).

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LiverzincvaluesZincsup

plement@L9zinc/gFood(aug/mI)wet

tissuepNo

tumorpDa

PDC0 026.0

± 5,2b (7)c23.3 ± 1.9 (5)d30.3± 8.3 (5)CNSeN5TumorPD

PDPDPD0

8016025015.6±

7.6 (11)c46.4 ±18.1 (11)C53.3 ±21.7 (7)C66.6 ±13.9 (6)c<o.oooi

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chow

DatatakenfrmaintainedonAscites

fluidzinc versusdietaryzincomseveralexperimentsin whichgroupsof 5 mithe zinc-deficientdiet starting on the day ofce

aretumorinjection.Zinc

supplement(tug/mI)Statistical

comparlsonaZinc In fluid

(@ug/ml) pP2P3I

00.23 ±009b(23)c2400.54 ±0.22 (7)<0.00023800.95 ±0.41 (11) <0.00001<0.014

1600.83 ±0.30 (12) <0.00001<0.01NSC52501.03 ±0.23 (14) <0.00001 <0.0001NS

D. T. Minke! et a!.

once the tumor takes hold in the host, it acts as a sink for themetal, competing successfully with the animal for its supply ofmobile zinc.

Since the liver is the site of synthesis of circulating zinccontaining proteins such as a2-macrogbobulin and albuminwhich may comprise the macromolecular zinc of ascites fluid,correlations between liver and tumor zincs may be expected(8). According to Table 3, the presence of the tumor does notdeplete the liver zinc except in the severely zinc-deficient diet.In that case, however, liver zinc is depressed severalfold, evenmore than can be accomplished by extended periods of zincdeficiency in normal mice without the tumor. In fact, liver zincis higher in tumored mice fed adequate levels of zinc than innormal animals. The larger S.D.'s seen in all groups of micewith growing tumors are observed in other data below andseem to reflect the individual response of animals to the external agent, the tumor, which is not under the homeostatic controlof the mice.

Table 4 shows that the fluid zinc concentrations of tumorscorrelate directly with the dietary bevelof zinc imposed on theanimals. Together with Chart 2, this implies that growth rateand fluid zinc values correlate with one another. Interestingly,the exposure of the tumor to zinc is controlled by the host inthe diets containing zinc (80 to 250 @zg/ml).The constancy inzinc concentration in the fluids associated with these diets ismirrored by the values for liver zinc listed in Table 3. As in thattable, one can see that fluid zinc levels fluctuate significantlyfor animals on a given diet. In the studies reported below, fluid

zinc concentration has been chosen as an important independent parameter because it reflects dietary zinc exposure andrepresents the nutrient pool of zinc directly bathing the tumorcells and available in some form for incorporation into newcells.

Although the levels of zinc in ascites fluid and liver varymarkedly with the dietary regimen of the mice, the zinc concentration within the cells of tumors which are grown undervarious conditions do not change on either a per mg or per cellbasis (Chart 4). Hence, it is clear that the bulk of the cellularzinc is not sensitive to or in equilibrium with extraceblular zinc,which can change by more than a factor of 10.

In an effort to understand some of the dynamics of zincmovement into and out of Ehrlich cells, transport studies wereinitiated. Cells from normal and zinc-deficient animals givesimilar results. Chart 5 shows that at concentrations of theorder of magnitude found in ascites fluid, zinc ion can be takenup rapidly from the extracelbular medium to achieve a steadystate internal concentration. In experiments not shown, thefinal internal concentration increases as a function of extracel

0

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a PD, purified diet containing zinc (less than 1 .tg/g; C, laboratory

containing zinc (70 to 90 @ug/g).b Mean ± S.D.C Numbers in parentheses, animals sacrificed on about Day 14.

d Numbers in parentheses, animals sacrificed on Day 80.S NS, not significant, p > 0.01.

Table4

•1 .3 .5 .7 .9 I,,i@gZnI ml fluid

Chart 4. Cellular zinc concentration versus fluid zinc concentration. 0, zincper 1o7 cells; •,zinc per mg cell protein.

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2'

a @‘,Group 1 compared with Groups 2 to 5; P2, Group 2 compared with

Groups 3 to 5; p@,Group 3 compared with Groups 4 and 5.b Mean ± S.D.C Numbers In parentheses, number of animals.

d NS. not significant wfth p > 0.01.

TIME minufes

Chart 5. Ehrllch cell transport of zinc. ConditIons: 7 mg/mI cells and 0.049mM zinc. 0, uptake of zinc; •,efflux of zinc from cells in steady state after zincuptake.

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Inhibition of Ehrlich Tumor Growth by Zinc Deficiency

lubar zinc. Because of the rapidity of uptake, initial rate datacould not be determined over a wide enough range of concentrations of external zinc to see if the rate of transport could besaturated. If one uses a value of 3 @sIcell volume per mg Ehrlichcells as calculated by Spencer and Lehninger (25), a concentration of -@‘0.3mM internal zinc is achieved in the experimentsummarized in Chart 4 relative to a final external concentrationof zinc of 0.04 mM. It is evident that zinc ion can be accumubatedagainst its concentration gradient. The fact that steadystate internal concentrations are attained quickly is consistentwith the finding that cells preloaded with zinc release the metalion intoa zinc-freemedium at a ratecommensuratewithuptake. In future experiments, the thermodynamics and mechanism of this transport must be explored. The rapid uptake ofan essential, charged ion suggests a carrier-mediated transportsimilar perhaps to that recently reported for 3T3 cells (12).

Although bow-molecular-weight zinc can move into and outof cells with ease, equilibration of zinc between ascites fluidand cells is not actually observed. A preliminary examinationof the distribution and stability of zinc in the fluid shows thatthere is no detectable zinc in low-molecular-weight forms, suchas zinc-amino acid complexes. Equilibrium dialysis experiments show that no zinc equilibrates across the cellulosemembrane. That is, the zinc in fluid may not be available tocells through a simple set of equilibria and transport processes.

DISCUSSION

Although zinc is integrally involved in the growth of rapidlyproliferating tissues, relatively few studies have been carriedout on the role of zinc ion in the growth of malignant cells.Petering et a!. (18) first showed that the Walker 256 carcinomasarcoma in rats grows slower in zinc-deficient rats than inanimals given a complete diet and that the inhibition of tumorgrowth was greater than the inhibition of the growth of the host(18). DeWys and Pories (4), DeWys et a!. (5), and McQuitty eta!. (15) repeated and extended these observations to demonstrate that a variety of transplantable tumors, including Walker256 carcinosarcoma,Lewis lung sarcoma, and 3 mouse beukemias including Li 210 can be inhibited or prevented fromgrowing in zinc-deficient hosts (4, 5, 15). In other work, Barrand Harris (1) found that P388 leukemia in mice grew slowerin zinc-deficient animals. Different groups have also found thatexcess exposure of tumor cells to zinc slows their growth invivo (19, 29).

The present studies constitute the initial characterization ofthe effect of zinc upon the growth and properties of Ehrlichascites tumor cells. The results demonstrate clearly that dietaryrestriction of zinc under various conditions produces a gradedeffect upon the growth rate of these cells. The 80-j@g/mlzincsupplementation produces optimal growth of the tumor, with40 @g/mlzinc diet somewhat less adequate and the 1-@sg/gbasic diet with no added zinc strikingly inhibitory of tumorgrowth. These effects are achieved in adult mice without undueharm to the host. This contrasts with the results of DeWys andPories (4) in which growing mice and matswere used. In thatstudy, control animals on the zinc-deficient diet survived foronly about 1 month.

Attempts to slow significantly or halt the Ehrlich tumor byimposition of zinc deficiency after tumor cell injection wereunsuccessful (Chart 3). The established colony of tumor cells

competes successfully with the animal for the diminishing supply of zinc. However, simultaneous administration of cells andthe deficient diet does bead to a much diminished rate ofgrowth. Previous studies had used animals already on a meduced zinc re9imen prior to tumor injection to observe adiminuation in growth (4, 5, 15).

Of particular importance is the finding that populations ofinhibited cells can be rapidly stimulated to divide after morethan 30 days of inhibition. Apparently, the cells are constrainedinto a resting state by the limitation of zinc. Given the existenceof a natural resting state, G0, related to the G1 portion of thecell cycle for normal cells and the evidence that P388 cells arehalted in G1 by a zinc deficiency, it is suggested that a similarphenomenon is occurring in the Ehrlich tumor (1).

A primary correlation in this study is between dietary zincand the zinc concentration of the ascites fluid. There is a sharprise in fluid zinc as the zinc is raised from 1 to 40 @tg/mlin thediet. Upon reaching 80 @g/ml,average fluid zinc varies littlewith increasing zinc in the drinking water. These findings arereflections of the facts that zinc turns over rapidly in organisms,and the uptake and distribution are under strong homeostaticcontrol even when excessive amounts of zinc are placed in thediet (24).

The relatively wide S.D. of fluid concentrations of zinc ateach dietary level is noted in Table 4. Despite grouping byweight and age, individual animals interact with injected tumorcells with a surprising variability. Once this is recognized andone assumes that fluid zinc values are the appropriate measures of the actual exposure of tumor cells to zinc, then thezinc concentration in fluid becomes the prime independentvariable in the analysis of the biochemical properties of a givencellpopulation.

Having argued this and anticipating the presence of biochemical lesions in the tumor cells from zinc-deficient mice describedin the next article, it is intriguing to find in Chart 3 that cellularzinc is approximately constant over the range of fluid zinc from0.1 to 1.3 @sg/mlwithin the precision of the measurements. Inthe past, functional aberrations in cells and tissues frequentlyhave been correlated with dietary levels of zinc and not actualinternal levels of zinc in the organism (13). The current resultis a direct contrast to the result for a model unicellular organism, Euglena graci!is, in which a stringent zinc deficiency ofthe growth medium depleted these cells of zinc to 20% of thecontrol bevel(27).

The fact that the zinc concentration in Ehrlich cells is notsubstantially reduced by zinc deficiency has interesting implications. It suggests that if these cells are to divide to produce2 new cells, they mustacquire grosslynormalamountsof zincin the process. Still, many studies in normal tissues and thepresent investigation with tumor cells point to a number offunctional perturbations, presumably due to lack of zinc (13,23). The zinc necessary to satisfy these processes may besmall since zinc analysis has not detected its lack in the cellsfrom zinc-deficient mice.

These and earlier studies demonstrate that a simple nutritionab deficiency can profoundly affect the course of tumordevelopment. The deficiency is successful even in the fastgrowing Ehrlich tumor because zinc turns over rapidly in organisms, and hence, host supplies can be diminished quickly(24). Furthermore,in adultmicethe short-termrequirementforzinc is met sufficiently well by the deficient diet so that only

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0. T. Minke! et a!.

11. Hurley, L. S., and Swenerton, H. Congenital malformations resulting fromzinc deficiency In rats. Proc. Soc. Exp. Blol. Med., 123: 692—697,1966.

12. Huslngh, J., Potter. S., Munoz, R., and Matrone, P. Trace element interactions In mammalian cell culture. Environ. Health Perspect., 17: 288, 1976.

13. Kirchgessner, M., Roth, H. P., and Weigard, E. Biochemical changes in zincdeficiency. In: A. Prasad(ad.), Trace Elements in HumanHealth and Disease,vol.1, pp.189—225.NewYork:AcademicPress,Inc.,1976.

14. Lieberman, I., Abrams. R., Hunt, N., and Ove, P. Levels of enzyme activityand deoxyribonucleic acid synthesis in mammalian cells cultured from theanimal. J. Biol. Chem., 238: 3955-3962, 1963.

15. McQuitty, J. T., DeWys,W. D., Monaco, L, Strain, W. H., Rob, C. G., Apgar,J., and Pories, W. J. Inhibition of tumor growth by dietary zinc deficiency.Cancer Res., 30: 1387-1 390, 1970.

I 6. Murthy, L, Hlghhouse, S., Levin, L., Menden, E. E., and Petering, H. G., Astudy of the combined toxic effects of oral cadmium and lead in rats. In: D.D. Hemphill (ad.), Trace Substances In Environmental Health, vol. 9, pp.395—401. Columbia, Mo.: University of MIssouri Press, 1975.

17. Pallanf, J., and Klrchgessner, M. Zum Zinkbedarf Wachsender Ratten, Int.J. Vitam, Nutr, Res., 41: 543—553,1971.

18. Peterlng, H. G., Buskirk, H. H., and Crim, J. A. The effect of dietary mineralsupplements of the rat on the anti-tumor activity of 3-ethoxy-2-oxobutyraldehyde bls(thiosemicarbazone). Cancer Ass., 2 7: 1115—1121, 1967.

19. Phillips, J. L., and Sheridan, P. J. Effect of zinc administration on the growthof L1210andBW5147tumorsin mice.J. NatI.CancerInst.,57: 361—363,1976.

20. Prasad, A. S., and Oberleas, D. Thymidine kinase activity and Incorporationof thymidine Into DNA in zinc-deficient tissue. J. Lab. Clin. Med., 83: 634—639, 1974.

21. Rubin, H., and Koide, T. Inhibition of DNAsynthesis In chick embryo culturesby deprivation of either serum or zinc. J. Cell Biol., 56: 777—786,1973.

22. Sandstead, H. H., and Rinaldi, R. A. Impairment of deoxyribonucleic acidsynthesis by dietary zinc deficiency in the rat. J. Cell. Physiol., 73: 81—83,1969.

23. Saryan, L. A., Minkel, D. T., Dolhun, P. J., Calhoun, B. L., Wielgus, S.,Schaller, M., and Petering, D. H. Effects of zinc deficiency on cellularprocesses and morphology In Ehrlich ascites tumor cells. Cancer Res., 39:2457—2465.1979.

24. Shaikh, Z. A., and Lucis, 0. J. Biological differences in cadmium and zincturnover. Arch. Environ. Health, 24: 410—418, 1972.

25. Spencer, T. L, and Lehninger, A. L. i.-Lactate transport in Ehrlich ascltesTumor cells. Biochem. J., 154: 405—414, 1976.

26. Swenerton, H., Shrader, A., and Hurley, L S. Zinc-deficient embryos:reduced thymidine incorporation. Science, 166: 1014—1016, 1969.

27. valIse, B. L. Zinc biochemistry in the normal and neoplastic growth processes. In: J. Schultz and F. Ahmad (ads.)., Cancer Enzymology, vol. 12, pp.159—195. New York: Academic Press, Inc., 1976.

28. WIlliams, R. 0., and Loeb, L. A. Zinc requirement for DNA replication instimulated human lymphocytes. J. Cell Blol.. 58: 594—601, 1973.

29. Woster, A. D., FalIla, M. C., Taylor, M. W., and Weinberg, E. D. Zincsuppression of initiation of Sarcoma 180 growth. J. Nafi. Cancer Inst., 54:1001-1003, 1975.

2456 CANCERRESEARCHVOL. 39

modest side effects are observed. However, in the presentwork, the tumor cells are only arrested and do remain viableover an extended period of time.

Finally, the model developed here for examining the effectsof zinc deficiency on tumor growth has several advantages fornutritional studies. In this system, a population of mammaliancells grows singly in a nutrient broth whose composition canbe manipulated through the diet of the host. This is particularlyimportant in the case of zinc because there is currently a backof tissue culture media available in which zinc ion concentrationcan be systematically varied (20). One can also add reagentsto the tumor in vivo, periodically remove cells for study as withtissue culture systems, and have an abundance of cells forstudy. Finally, since the cells must obtain their nutrition fromthe ascites medium which is readily isolated, one can examinein detail the composition of the fluid and the interaction betweenthe fluid and cells.

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2. Burch, A. E., and Sullivan, J. F. Clinical and nutritional aspects of zincdeficiency and excess. Med. Clin. N. Am., 60: 675—685,1975.

3. Chesters, J. K. Biochemical functions of zinc with emphasis on nucleic acidmetabolism and cell division. In: W. G. Hoekstra, J. W. Suftie, H. E. Ganther,and W. Mertz (eds.), Trace elements metabolism in animals—2,pp. 39—50.Baltimore: University Park Press, 1974.

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7. Fujloka, M., and Lieberman, I. A Zn2@requirement for the synthesis ofdeoxyribonucleic acid by rat liver. J. Biol. Chem., 239: 1164—1167, 1964.

8. Glroux, E. L. DeterminatIon of zinc distribution between albumin and a2-macroglobulln In human serum. Biochem. Med., 12: 258—266,1975.

9. Hsu, J. M., and Anthony, W. L. Effect of zinc deficiency and repletion onthymidine metabolism. Clin. Chem., 21: 544—550,1975.

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1979;39:2451-2456. Cancer Res   Daniel T. Minkel, Patricia J. Dolhun, Barbara L. Calhoun, et al.   Zinc Deficiency and Growth of Ehrlich Ascites Tumor

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