Effect of Growth of a Tumor on the Regulation of Water Intake 1

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1 Effect of Growth of a Tumor on the Regulation of Water Intake 1 S. D. MORRISON,2 Laboratory 01 Physiology, National Cancer Institute,3 Bethesela, Marylanel SUMMARY-The water intake of rats during growth of the Walker 256 tumor remains high in the presence of a spontaneously declining food intake. The water intake associated with food intake can be assessed by regarding the spontaneous food intake as ael libitum, and this leaves a large component of the actual water intake unaccounted for; or it can be assessed by regarding the spontaneous food intake as restricted, and this leaves a small but appreciable component unaccounted for. Rats that have recovered from the acute el'fecfs of lateral hypothalamic lesions and that no longer have the normal drinking response to food deprivation show, during tumor growth, a small component of water intake not accountable to food intake and of a size and time of occurrence comparable to the laher estimate for intact tumor bearers. The elevation in water intake induced by the tumor and not accountable to food intake appears not to depend on lateral hypothalamic mechanisms. The decline of food intake in tumor bearers may have the physiological characteristics of a food restriction. J Nat Cancer Inst 41: ,1968. THE FOOD INTAKE of rats with tumors declines in the later stages of tumor growth (1, 2), but the water intake is sustained at or above precancerous levels much longer (3). In normal rats a proportional relationship between ad libitum food and water intakes has frequently been demonstrated (4-6). If the water intake associated with food intake in tumor bearers conforms to this relationship, then a large part of their actual water intake must be attributed to some effect of the tumor. However, the water intake of normal rats, when food is restricted or withheld but water is allowed ad libitum, is sustained at higher levels than water:food proportionality would indicate and may even increase for a day or two (7). If the water intake associated with food intake in tumor bearers follows this pattern, then possibly little or none of the apparently excessive water intake need be attributed to a direct effect of the tumor on water intake; but the decline of food intake during tumor growth would have to be considered as a physiological food restriction occurring in the presence of nominal ad libitum food. 1 Received June 27, 1968; accepted August 7, I I am grateful to Mrs. B. Moore and Mr. E. McDuffy for technical assistance. a National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare

2 1242 MORRISON Although food intake declines during tumor growth, the requirement of the whole organism (host + tumor) for energy and nutrients is not diminished and may, in fact, increase (8, 9). This imbalance might have the effect of a restriction. As in normal rats, food and water intakes of tumor bearers are reduced to zero by destruction of the lateral hypothalamus (3). The milder mechanical damage produced by "sham operation" depresses both food and water intake in tumor bearers, and the water intake remains depressed relative to the intake of intact tumor bearers (3). Detailed interpretation of the results of these procedures is complicated by the associated operative trauma and the variable rate of recovery from the acute effects of the lesions or the "sham lesions" (3). Several components of the water intake of normal rats are blocked, in whole or in part, in rats that have recovered from the acute effects of lateral hypothalamic lesions (10). One component blocked is the maintained or increased water intake normally occurring during food deprivation (7). This chronic residual effect of lateral lesions is free of any operative trauma and, if its intensity alters, it appears to do so very slowly. This report attempts to establish whether the water intake of tumor bearers can be accounted for by considering their decline in food intake as a restriction of food intake and whether there is any part of the apparently excessive water intake that cannot be accounted for in this way. The problem was approached by comparison of the excess water intake estimated for the intact tumor bearers on each of the two hypotheses offood intake (ad libitum or physiologically restricted) with the excess water intake that persists after recovery from lateral hypothalamic lesions. METHODS Male Sprague-Dawley rats were maintained individually in suspended wire cages and allowed a casein-based, semisynthetic diet [C-21 (2,3)] and water ad libitum. Environmental temperature was C and a constant cycle of 11 hours light: 13 hours dark was maintained at all times. Tumor bearers.-from 55 until 104 days of age (the II th day after tumor transplant) food and water intakes and body weight were measured daily to the nearest g, 5 days a week. From then until the animals were killed, all measurements were made daily. At age 66 days bilateral lesions were made in the lateral hypothalamus of 12 rats. The lesions were made, under pentobarbital sodium and ether, with a commercial version of the Stellar-Krause stereotactic instrument (11) with anodal direct current of 1.5 rna for 15 seconds, at points 1.5 mm caudad to the bregma, 2 mm lateral to the midline, and 2 mm dorsal to the sphenoid. Of these rats, 2 died immediately after operation, 6 were aphagic and adipsic for 2 or more days (acute aphagia), and 4 were hypophagic or were aphagic and adipsic for 1 day only (acute hypophagia). All the rats aphagic for more than 2 days were tube-fed with milk and offered milk and chow mash, as well as water and the standard diet, until they recovered spontaneous food and water intake. All rats with lesions were deprived of food but allowed water for 2 days before lesions and for 2 days after apparent recovery from the lesions. Eight rats did not receive lesions but were deprived of food and water for 2 days corresponding to the immediate postoperative period of the rats with lesions. At age 93 days, about 1 mg of viable Walker 256 tumor tissue was transplanted subcutaneously into the right flank of all rats. All tumor fragments were taken from the same 1 g portion of tumor. When the tumors had grown large enough (about the 11 th day after transplant), three orthogonal diameters were measured with a caliper every second day until the end of the experiment (12). Tumor weights were derived from the products of these diameters by equating the tumor product on the last day to the weight of the excised tumor on that day. The rats were killed by asphyxiation with C02 25 days after transplant (age, 118 days) or sooner if they appeared moribund. Tumors were excised and weighed to the nearest 0.1 g and both adrenals were weighed to the nearest 0.1 mg. A further group of 6 rats received Walker 256 transplants in the same way and at the same age and body weight, but from a different portion of tumor. These rats were deprived of food but allowed water on 1 day before tumor transplant and on the 27th day after tumor transplant. Food-restricted normals.-a group of 12 normal male rats, of the same age and body weight as the JOURNAL OF THE NATIONAL CANCER INSTITUTE

3 TUMOR GROWTH AND WATER INTAKE 1243 tumor bearers at transplant, had their food intake progressively restricted according to the pattern and average amount of decline of food intake shown by intact tumor bearers. Tw(.) other groups of 6 rats were also subjected to progressive food restriction but had their food intake during the last 3 or 6 days restricted more severely than the average spontaneous food intake of the tumor bearers. Food and water intakes and body weights of all these rats were measured daily to the nearest g. The rats were killed at the end of the restricted period and their adrenal weights were measured to the nearest 0.1 mg. RESULTS Total body weight increment, host weight increment, tumor size as measured by product of lineal dimensions and final excised tumor weight, final adrenal weight, and food intake did not, in general, differ significantly between intact tumor bearers and tumor-bearing rats that had recovered from lateral hypothalamic lesions (text-fig. 1, table 1). A difference in average tumor size at 15 days after transplant between intact tumor bearers and those recovered from the lateral lesions (text-fig. 1) was not statistically significant (P>0.05). Food intake started to decline, in both groups, about day after transplant (text-fig. 2). A series of 59 tumor bearers were killed at 3, 5, 11, 14,21, and 28 days after tumor transplant. The first significant increase in adrenal weight, from 47.2 ± 1.8 mg to 60.9 ± 4.1 mg (9 rats per group), occurred between 14 and 21 days after transplant. The adrenal weight at 28 days was ± 10.9 mg (cj. values in table 1). Effect of Lateral Lesions on the Water Intake of Tumor Bearers Mean water intake of intact tumor bearers increased significantly (P<O.OOI) about the 13th day after transplant and started to decline about the 17th day (text-fig. 2A). Although these mean changes were well defined, individual variation was great, particularly while the increased intake was sustained. Water intake of rats with tumor transplants, after recovery from lateral lesions that produced 2 or more days of total aphagia and adipsia, did not increase during this period but was sus- TABLE 1.-Carcass growth, adrenal hypertrophy, and total food intake in tumor-bearing rats and rats su bjected to comparable restriction of food intake (values as mean ± BE) Days Final Final Total Carcass 22-Day* Number post tumor adrenal body weight* food Group of rats trans- weight weight weight* increase intake plant (g) (mg) increase (g) (g) (g) Intact tumor bearer 7t ±4.5 ±10.5 ±1O.3 ±8.2 ±7.8 Tumor bearer recovered from lateral lesion-acute hypophagia 3t ± ±21-34 ± ± ±12.5 Tumor bearer recovered from lateral lesion-acute aphagia ± ±21-5 ±1O.9-64 ± ±11.8 Normal rats in progressive A food restriction ±1.1 ±1.5 ±1.5 ±1.8 Bl ±2.6 ±1.7 ±1.7 ±1.0 B ±2.2 ±3.6 ±3.6 ±3.5 All these values expressed in terms of 22 days after transplant or 22 days after equivalent transplant day. tno valid excised tumor weight available from 1 rat from each of these original groups. VOL. 41, NO.5, NOVEMBER 1968

4 1244 MORRISON o CUMULATIVE TOTAL WEIGHT INCREMENT A Inlacl Tumor Bearers CUMULATIVE HOST WEIGHT INCREMENT TUMOR WEIGHT OL---L-----i----- o DAYS AFTER TUMOR TRANSPLANT TEXT-FIGURE I.-Mean total weight increment, weight increment of host, and tumor size during growth of Walker tumor in 8 intact rats (e) and in 10 rats recovered from the acute effects oflateral hypothalamic lesions (0). tained at pretransplant levels until about the 18th day after transplant (text-fig. 2C). The water intake of rats with tumor transplants, after recovery from lesions that produced not more than 1 day of aphagia, was intermediate at each stage of tumor growth (text-fig. 2B). The average water intake of rats that had recovered from lateral hypothalamic lesions, before and after tumor transplants, was always lower than that of intact rats. Effect of Lateral Lesions or of Tumor on the Water Intake During Food Deprivation Prior to transplant of tumor, the water intake during food deprivation was examined before and after recovery from lateral lesions that produced aphagia acutely. Before placement of lesions, water,.., O'--...L..L"'-.I...Ii<"--1--U<JL-L.lLd...L""'--L.t"'-L"-"--J'-lZ21-J 2: 8 "" Recovered Lateral Tumor Beorers -: 20 L- Acule Hypophogio - >.:: I Z ai a O'---ffl Recovered Lalerol Tumor Bearers 20 Acute Aphagia 10 o Woler Inloke ra Food Intake O'-- LllLJJW_-L=- Pre-Irans plant DAYS AFTER TUMOR TRANSPLANT TEXT-FIGURE 2.-Mean daily water intake (open columns) and food intake (hatched columns) during tumor growth in 8 intact rats (A), and in rats after recovery from lateral hypothalamic lesions that produced mild (B, 4 rats) or severe (C, 6 rats) acute effects. Bars show ± SE. intake was not significantly altered by deprivation of food. After recovery from the acute effects of the lesions, the water intake was significantly depressed during food deprivation (P<O.05) and was only about one-quarter of the deprived water intake before lesions (P<O.05) (text-fig. 3A). Rats that had recovered from hypophagia showed no depression of water intake on food deprivation. In intact rats deprivation of food produced no significant change in water intake before tumor transplant or on the 27th day of tumor growth (text-fig. 3B). Effect of Progressive Food Restriction on the Water Intake of Intact Rats Without Tumors Progressive food restriction of normal rats, according to the average pattern and amount of - JOURNAL OF THE NATIONAL CANCER INSTITUTE

5 TUMOR GROWTH AND WATER INTAKE , A Before Lateral Lesions After "Recoverl From 30 La/eral Lesions 30 PROGRESSIVE FOOD RESJRICTION OF NORMAL RATS o Wafer In/ake Group.A.. (f2 Rats) Food In/oke ,... DAYS TEXT-FIGURE 3.-Water intake during deprivation of food (A) before and after lateral hypothalamic lesions that produced severe acute effects and (B) before and 27 days after transplant of tumor into intact rats. Six rats in each group. Periods of food deprivation shown by hatched columns. Bars show ± SE. spontaneous food intake seen in tumor bearers, did not deplete the rats to the same extent as the tumor-bearing host was depleted (table 1). More severe food restriction increased the depletion of body weight but produced no other significant alteration from the pair-fed restriction (table 1, text-fig. 4). In all the restricted groups water intake increased initially and then decreased and was not in proportion to the decrease in food intake (text-fig. 4). The empirical relationship between food and water intakes in this condition was quite complex, but was well defined and clearly distinct from the relationship expected from food intake ad libitum (text-fig. 5). The total adrenal weight of the restricted rats was close to normal (48 ± 2 mg) for rats of this size and on this diet, but was only about one-third of the adrenal weight of tumor bearers (table 1).,.., <> c 20 w "" 10 :z Group B-[ (6 Rats) Group B-2 (6Ra/s) o Ad-Lib DAYS TEXT-FIGURE 4.-Daily water intake (open columns) and food intake (hatched columns) of normal rats with food intake progressively restricted in the pattern of tumor bearers. In group A, food was restricted to the average amount eaten by the tumor bearers. In groups B-1 and B-2, total food allowed was 83 and 80%, respectively, of that eaten by intact tumor bearers over the same period. Time scale represents the equivalent stage of tumor growth. Bars show ± SE. Water Intake Associated With Food Intake in Intact Tumor Bearers and in Tumor Bearers Recovered From Lateral Lesions The water intake in excess of that associated with food intake was first calculated, for all tumorbearing groups, by assuming the water:food relationship of ad libitum feeding, namely, that the intake associated with food is defined by the water: food ratio before transplant. This estimate would indicate that, from about the 13th day after transplant, g/day, i.e., half or more of the total water intake, is in excess (text-fig. 6A). VOL. 41, NO.5, NOVEMBER 1968

6 1246 MORRISON 140r ' '----' 120 IX! CI 100 f- z,,/ ILl u 80 \ c:: ILl \)I)y c... '/ ILl "" 60 't:.7 f- z \\)/,,,'t:./, c:: ILl 40.. '/ i. f- 20 / 0/ FOOD INTAKE PERCENT AD LIB TEXT-FIGURE 5.-Relationship of water intake and food intake in the progressive food restriction of normal rats shown in text-figure 4. Points are the means of 24 (e), 18 (0), 12 (,), or 6 (0) rats. Bars show ± SE for each group. Curve is fitted by eye. When the water:food relationship in restricted feeding (text-fig. 5) was used to define the water intake associated with food intake, the excess water intake of the intact tumor bearers was small (5 gjday) but significant (P<O.OI) from days after transplant (text-fig. 6B). Before tumor transplant, rats that had been acutely aphagic but had recovered from lateral hypothalamic lesions showed a reduction of water intake on food deprivation (text-fig. 3A). It is assumed from this that the water intake associated with food intake in such rats after tumor transplant can be defined by the water:food ratio before transplant. This assumption leads to an estimate of water intake in excess of feeding requirement (text-fig. 6e) comparable in size to that calculated for the intact tumor bearers on the basis of food restriction and occurring at the same time (text-fig. 6B, e). The rats that recovered from lateral hypothalamic lesions and showed only acute hypophagia had a generally depressed level of water intake -. co o 15 &oj ""!z 10 co o... 5 x f- 3i _A_ Intact Tumor Bearers Calculaled Assuming Ad-Lib Water-Food Relationship O--- B <3 10 Inlact Tumor Bearers Calculated Assuming Restricted Water-Food Relationship I- :;c... 5 &oj :> 0 CD &oj 0 ""... :: 10 I U> ::3 5 <..> x... c Tumor Beafers Recovered From Acute Aphagia, Calculated Assuming Ad-Lib Wafer-Food Relationship TEXT-FIGURE 6.-Excess water intake above that associated with food intake during tumor growth: A) of intact rats, assuming that the pre transplant water: food intake ratio defines the water intake associated with food intake; B) of intact rats, assuming that the relationship shown in text-figure 5 for normal restricted rats defines the water intake associated with food intake; and C) of rats recovered from severe hypothalamic damage, assuming that the pretransplant water: food intake ratio defines the water intake associated with food intake. Bars represent ± SE. but no further depression of intake on deprivation of food. Supposedly, the curvilinear relationship of text-figure 5 had been partially eliminated in these rats, but to an unknown extent. No estimate of excess water intake after tumor transplant has been attempted for these rats. DISCUSSION The ad libitum food intake of rats bearing the Walker 256 tumor declines with growth of the tumor (1-3). Water intake, which, in normal rats, is proportional to ad libitum food intake (5, 6), JOURNAL OF THE NATIONAL CANCER INSTITUTE

7 TUMOR GROWTH AND WATER INTAKE 1247 remains at or above normal levels in tumor bearers almost until the terminal phase of tumor growth (3). The cause of this relatively high water intake has been tentatively attributed (3) to retention of sodium by the tumor (13), adrenocortical hypertrophy (14), and increased aldosterone secretion (15) occurring at this time. Normal rats deprived of food, or whose food is restricted, drink more than would be expected, according to the ad libitum water:food relationship (7). This also happens in normal rats that are progressively food restricted in the pattern of the declining spontaneous food intake of tumor bearers, and in tumor-bearing rats totally deprived of food for a short period. (No theoretical interpretation of the curve in ttxt-fig. 5 is suggested here; it is used only as a predictive device.) The relationship between water and food intake in normal rats progressively food restricted could account for most of the apparently high water intake of tumor bearers. Even so, there does appear to be a moiety that is not accounted for in this way. Rats recovered from lateral hypothalamic lesions that produced severe acute effects do not show the anomalously high water intake on deprivation of food. It can be proposed from this that, in such rats, the water intake necessarily associated with food intake conforms to a constant water:food ratio. During growth of a tumor these rats do show a water intake that is high relative to declining food intake, and this high intake is presumably not activated or integrated in the lateral hypothalamus. The excess water intake above that associated with food intake is about the same and occurs at the same time as the excess estimated, on the basis of food restriction, for the intact tumor bearer. There is great adrenal hypertrophy in the tumor bearer which is not affected by lateral lesions, and this hypertrophy first appears during the period of excess water intake. There is no adrenal hypertrophy in normal rats restricted to corresponding food intake. The independent moiety of excess water intake may be related to adrenal changes induced by the tumor (14, 15) and to retention of sodium by the tumor (13). From comparison of the excess water intake of intact tumor bearers with that of tumor bearers recovered from lateral hypothalamic lesions, the water intake associated with food intake in tumor bearers appears to be better assessed by regarding the spontaneous decline in food intake as a food restriction. The decline in food intake is not matched by a decline in need (8, 9), and it seems reasonable, therefore, that the reduced intake should have some characteristics of a restricted food intake. Further, the decrease of food intake in the tumor bearer appears to be independent of hypothalamically controlled mechanisms (3) and this also suggests some kind of physiological restriction rather than a controlled reduction of food intake. REFERENCES (1) MIDER, G. B., TESLUK, H., and MORTON,].].: Effects of Walker carcinoma 256 on food intake, body weight and nitrogen metabolism of growing rats. Acta Un lnt Caner 6: , (2) MILLAR, F. K., WHITE, ]., BROOKS, R. H., and MIDER, G. B.: Walker carcinosarcoma 256 tissue as a dietary constituent. I. Stimulation of appetite and growth in the tumor-bearing rat.] Nat Cancer Inst 19: , (3) BAILLIE, P., MILLAR, F. K., and PRA'IT, A. W.: Food and water intakes and Walker tumor growth in rats with hypothalamic lesions. Amer ] Physiol 209: , (4) ADOLPH, E. F.: Urges to eat and drink in rats. Amer ] Physiol 151: , (5) CIZEK, L. ]., and NocENTI, M. R.: Relationship between water and food ingestion in the rat. Amer ] Physiol 208: , (6) STROMINGER,]. L.: The relation between water intake and food intake in normal rats and in rats with hypothalamic hyperphagia. Yale ] BioI Med 19: , (7) MORRISON, S. D., MACKAY, C., HURLBRINK, E., WIER, ]. K., NICK, M. S., and MILLAR, F. K.: The water exchange and polyuria of rats deprived of food. Quart] Exp Physiol52: 51-67,1967. (8) MIDER, G. B., FENNINGER, L. D., HAVEN, F. L., and MORTON, ]. ].: The energy expenditure of rats bearing Walker carcinoma 256. Cancer Res 11: , (9) PRA'IT, A. W., and PUTNEY, F. K.: Observations on the energy expenditure of rats receiving Walker tumor 256 transplants. ] Nat Cancer Inst 20: , (10) EpSTEIN, A. N., and TEITELBAUM, P.: Severe and persistent deficits in thirst produced by lateral hypothalamic damage. In Thirst (Wayner, M. ]., ed.). New York, Macmillan, 1964, pp (11) STELLAR, E., and KRAUSE, N. P.: New stereotaxic VOL. 41, NO.5, NOVEMBER 1968

8 1248 MORRISON instrument for use with the rat. Science 120: , (12) SCHREK, R.: A quantitative study ofthe growth ofthe Walker rat tumor and Flexner-J obling rat carcinoma. Amer J Cancer 24: , (13) TOAL, J. N., MILLAR, F. K., BROOKS, R. H., and WHITE, J.: Sodium retention by rats bearing the Walker carcinosarcoma 256. Amer J Physiol 200: , (/4) WHITE, J., TOAL, J. N., MILLAR, F. K., and BROOKS, R. H.: Walker carcinosarcoma 256 tissue as a dietary constituent. III. Sodium as a factor in stimulation of water and food intake and growth in tumor-bearing rats. J Nat Cancer Inst 24: , (15) MILLAR, F. K., TOAL, J. N., BROOKS, R. H., DAVIS, J. 0., and WHITE, J.: Increased aldosterone secretion by tumor-bearing rats. Amer J Physiol 205: , 1963.