Effects of Systemic Acidification of Mice with Sarcoma 1801 · 6 Normal acid-base balance in 5-week-old Swiss mice. Means and ranges of 10 different control mice sampled on Day 0

[CANCER RESEARCH 39, 4364-4371, November 1979]0008-5472/79/0039-OOOOS02.00

Effects of Systemic Acidification of Mice with Sarcoma 1801

Salvador Harguindey,2 Edward S. Henderson, and Carl Naeher

Department of Medicine A [S. H., E. S. H., C. N.], and Computer Center [C. NJ, Roswell Park Memorial Institute, Buffalo. New York 14263

ABSTRACT

The effects of dietary-induced acidosis on the growth andrates of complete regression of Sarcoma 180 in mice havebeen studied. The experiments here reported have demonstrated that mineral acidification of laboratory food produces alate decrease in tumor growth and significantly increases therates of complete tumor regression. Blood acid-base studiesalso demonstrate the effects of these diets in altering the acid-

base balance, and seemingly, this is independent of starvationand/or ketosis. The relationships of such in vivo acid-base

metabolic changes to the control of tumor metabolism arebriefly discussed. A therapeutic potential for this preliminaryapproach is considered.

INTRODUCTION

The effects of starvation in retarding tumor growth are wellknown (4, 34). Similarly, the favorable influence of acidosis ontumor control and regression has long been recognized (1,14,26, 30, 41 ). Different acids added to diets have been shown tobe effective in preventing liver cancer in rats (28), decreasingtumor growth in rabbits (41), and inhibiting the growth of solidtumors and leukemias in mice (1). Spontaneous regressions ofthe s.c. form of Sarcoma 180 transplanted to Swiss mice havebeen reported in up to 23%, varying with the different systemsused by separate investigators (18, 40). Mice transplanted withthis nonmetastasizing tumor presumably die of local necrosisand/or infection and secondary toxemia. Caloric restrictionamounting to more than 33% of the food consumed by controlgroups has been shown to significantly retard the growth ofthis particular tumor (2), and this inhibition has been shown tobe related to caloric restriction per se rather than to a deficiency of specific nutrients (3). A marked effect of lowering pHon the control of tumor glycolysis has been shown repeatedlyin different neoplastic cells (10, 20, 35, 47). These facts haveled many investigators to attempt to interfere with tumor growththrough the use of specific inhibitors of tumor glycolysis (6, 21,29). Unfortunately, in most of these studies the direct effectsof environmental H+ concentration were not taken into account. The experiments reported here were planned and undertaken to delineate the interference of in vivo tumor glycolysis and tumor growth through a low systemic pH (32, 47),independent of starvation in the Sarcoma 180 tumor system.

MATERIALS AND METHODS

Three experiments were performed sequentially. Experiment

' This research was supported in part by USPHS Grants CA-5834 and CA-

09108 from the National Cancer Institute.2 To whom requests for reprints should be addressed, at Department of

Thoracic Surgery, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NewYork 14263.

Received June 30, 1978; accepted July 30, 1979.

1 compared the rates of tumor growth and survival of normallyfed mice with those of mice fed with quantitatively differentacidified diets. Experiment 2 was conducted to determine theeffects of different diets on blood acid-base status as well asthe rates of growth of tumor-free mice subjected to different

dietary measures. Experiment 3 was performed to confirm thefindings of Experiment 1 and also to study the effects ofacidification of both water and food on tumor growth.

Experimental Design

Experiment 1

Experiment 1 consisted of 3 different groups.Group 1. The control group was composed of 20 mice.

Nonacidified laboratory food and water were served ad libitumfrom Day 0.

Group 2. This group consisted of 40 mice implanted withtumor on Day 0 and subjected to 4 different periods of 2 N HCIfood (Days 0 to 6, 7 to 9, 12 to 18 or 19, and 21 to 24 or 25).Between the "acidifying" periods the mice received nonacidi-

fied food (see Chart 1). Four of these mice were sampled onDay 5 to study their acid-base status.

Group 3. Forty mice received a 1 N HCI diet beginning theday of tumor transplant continuously throughout the durationof the experiment (72 days).

Concurrent with these studies, acid-base parameters were

determined in 10 normal mice (Table 1, control group). Another4 normally fed mice growing large tumors were studied toassess the effects of tumor growth on the acid-base balance

(Table 1, 32 days after implantation).

Experiment 2

This experiment did not involve tumor transplantation andwas conducted instead to determine the subacute (Day 5) andchronic (Days 14 and 32) effects of the 1 N HCI diet on acid-

base balance, as well as the amount of food eaten by normallyfed versus acid-fed mice. The rates of weight gain and growthof 1 N HCI-fed mice were compared to those of mice fed under

the same conditions in Experiments 1 and 3 which carried atumor transplant. This experiment involved 25 mice.

Experiment 3

This experiment was designed to confirm and extend Experiment 1. Four different groups with 20 mice in each group weretransplanted with Sarcoma 180 on Day 0 and received thefollowing diets.

Group 1. The control group received a normal diet plus tapwater ad libitum.

Group 2. Group 2 received 1 N HCI food plus tap water adlibitum.

Group 3. Group 3 received 1 N HCI food from Day 0. HCI(0.05 N) water was substituted for tap water beginning on Day4 (25 ml of 1 N HCI to 500 ml of tap water).

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Effects of Systemic Acidification with Sarcoma 180

Table 1

Experiment1Control

group (notumor) Day 0

2 N HCI group, Day 5after transplant0

32 days afterimplantation,normal foodTumor

sizepHmice

Range MeanRange10

7.28-7.36

4 6.72-6.94

4 154-735 479 7.18-7.30.-:.

PCO!MeanRangeMean7.314

34.1-48.8 41.8

6.81 16.1-48.9 37.9

7.24 24.1-46.9 30.1BicarbonateRange

Mean17.6-24.5

21.2

2-10.0 6.3

9.7-17.2 12.5Base

excess MetabolicpHaRange-1.2

â€”8.4-23-

-30

-12 â€” 18Mean

Range-5.1

7.28-7.38

-27.9 6.6-6.97

-14.4 7.11-7.22Mean7.325

6.797.17

Metabolic pH is a combined measurement obtained from the original pH and PCO2 which represent the metabolic component of the acid-base status (Blood GasCalculator, Radiometer, Copenhagen, Denmark).

6 Normal acid-base balance in 5-week-old Swiss mice. Means and ranges of 10 different control mice sampled on Day 0 of Experiment 1 (Charts 1. 2, and 3).c Severe acidosis after 5 days of 2 N HCI acid diet in Swiss mice (Chart 1). Only means and ranges are shown.d Acidifying effect of large Sarcoma 180 tumors on blood acid-base balance of normally fed mice in the chronic situation. A respiratory compensation through

hyperventilation (low PCO2) occurred in the 3 mice with the larger tumors. Only means and ranges are shown.

Group 4. Group 4 received 1 N HCI food from Day 0. HCIwater (0.05 N) was substituted for tap water from Day 14.

Animals and Tumors

Five-week-old male Swiss mice were obtained from the Ros-

well Park Memorial Institute colony. The Sarcoma 180 s.c.tumor line, maintained through serial passages at this institutefor more than 10 years, was inoculated by trocar using piecesmeasuring approximately 1 mm in diameter in Experiment 1and 2 mm in diameter in Experiment 3. The transplants wereobtained from peripheral nonnecrotic areas of 11 -day post-

transplant tumors of the donors and were inoculated s.c. intothe backs of the recipient mice. Tumors grew in 97.3% of theimplants (4 of 100 failed to grow in Experiment 1 and 1 of 80failed in Experiment 3). Immediately after transplantation, micewere separated into 3 groups according to weight (less than26 g, 26 to 28 g, and greater than 28 g). They were thenreallocated into the different experimental groups to obtain asaccurately as possible the same initial mean weights amongthe different groups of each experiment. Mice were lodged 5to a cage. All the groups were maintained in the same roomand subjected to the same environmental factors, e.g., temperature, light, and noise. An interval of 4 months elapsedbetween Experiments 1 and 2, and 2 months elapsed betweenExperiments 2 and 3. By the end of 72 days, all tumor-bearing

animals had either died from tumor growth or had survivedfollowing complete regression of the transplanted tumor.

Diets

Normal and acid diets were prepared by thoroughly grindingpelleted laboratory chow and mixing it with water or HCI solutions. Each 100 g of powdered food was evenly mixed witheither 72 ml of tap water (control diet), 72 ml of 1 N hydrochloricacid (1 N HCI diet), or 72 ml of 2 N hydrochloric acid (2 N HCIdiet). From the paste obtained, 4- x 4-cm pieces were sepa

rated and allowed to dehydrate at room temperature at least 6days to recover initial dry weight. In Experiment 3, tap waterwas acidified with hydrochloric acid to a concentration of 0.05N HCI while normal tap water was permitted in Experiments 1and 2. Food and water were always allowed ad libitum. Micestarted on their different diets from Day 0, i.e., immediatelyafter tumor transplant and/or group separation.

Assessment of Acid-Base Balance

Venous blood (0.1 to 0.15 ml) from a superficial razor blade

cut of the tail vein was collected in heparin-pretreated micro-

collection capillary tubes (Biochemical Instruments, Medfield,Mass.) tightly applied to the skin on the bleeding point and wasimmediately mixed using mixing fleas moved within the tubewith a magnet. The tubes were closed with tube sealer andimmediately stored in ice. Blood gas determinations: pH, PCO2,PO2, calculated bicarbonate, and base excess were performedwithin one-half hour of collection using a Corning Model 165

pH/Blood Gas Analyzer. Metabolic pH was calculated using aBlood Gas Calculator Type BGC (Radiometer, Copenhagen,Denmark). This pH value, corrected to a PCO2 of 40 mm Hg,represents the metabolic component of the pH, independent ofrespiration-induced changes. No significant changes in PO2

were observed in any of the treated groups.Animal and Tumor Evaluation. Mice were periodically

weighed, and all tumors were measured by calipers at thetimes indicated in Charts 1,2,4, 6, and 7. The product of the2 largest perpendicular tumor diameters in sq mm measuredby the same observer was used as an index of tumor mass(23).

RESULTS

Experiment 1-Effect of Acid Diets (1 and 2 N HCI) onGrowth of Sarcoma 180 and on Growth and Survival ofSarcoma ISO-bearing mice. Chart 1 shows the mean weightsfor all surviving mice in each of the different groups. Standarderror of the mouse weights was less than 0.6 g in all experiments. The control group steadily gained weight from thebeginning of the experiment. Animals in Group 2 lost an average of 7 g during the first 5 days on the double acidified (2 NHCI) diet. This diet proved intolerable after a few days, withmice of this group able to ingest only 15 to 20% of the amountof food consumed by controls. Mice under these conditionswould rapidly become cachectic and die when their weightdeclined to about 65% of original weight if maintained on thisregimen. Accordingly, the 2 N HCI diet had to be given intermittently during 4 different periods of starvation-acidificationlasting 3 to 9 days and terminating on Day 26 (see Chart 1).Chart 1 shows the rates of weight loss and of regrowth duringthe alternating periods of normal and acid diet. Remarkably,the mean weight of the mice reached the original mean weightof the group within only 48 hr of restarting normal food.

By Day 23 of this experiment, a time when the survival ofGroups 1 and 2 was practically the same (see Chart 3), anddespite the large standard error in tumor mass (S.E. = 85) for

NOVEMBER 1979 4365



S. Harguindey et al.

40-1

Chart 1. Experiment 1. Means of mouse weight in thedifferent groups. Group 1 represents the normally fed controlgroup. Group 2 was subjected to 4 different periods on 2 NMCI diet. Time periods under arrows, acidification periodsunder 2 N MCI diet. Group 3 was maintained on 1 N HCI dietthroughout the duration of the experiment. All tumor transplants were done on Day 0. (For further details, see text.)

GROUPÂ»1 o Control'2---62N HCI foodÂ»3â€”O IN HCI food

0 6

30 DAYS 4Â° 50 60 70

E

EO"(O

400-

Â«/Â»300ut

Chart 2. Experiment 1. Mean tumor mass in the alivemice of the 3 groups of mice shown in Chart 1.

GROUPÂ»1â€”OControlÂ«2-- 42NHCI foodÂ»3..-Q IN HCI food

10 20 30 DAYS

the control group, the mean tumor size of the living mice in the2 N HCI Group 2 (111 sq mm) is clearly below (p < 0.05) themean tumor size (298 sq mm) of the controls (Chart 2). Only 2mice in the 2 N HCI acid group had died with measurable tumorgrowth by that time. Mice of Group 2 were fed with normalnonacidified food from Day 26, and the final rates of survivalbetween Groups 1 and 2 is not different (Chart 3).

Group 3 (1 N HCI) behaved in a different manner. Mice inthis group initially lost an average of 4 g in weight (Chart 1, day4), but by Day 35, with no change in diet, the mean weight ofthe surviving mice had become identical to that of controls.Mice of Group 3 showed tumor growth rates only slightly lessthan that of controls during the time of maximal growth ofSarcoma 180 (Chart 2, Days 5 to 12), but thereafter the rate oftumor growth was considerably slower. In the control group,one tumor failed to grow and another 3 spontaneously regressed (15%), while all the tumors in Group 3 took and grew.However, the proportion of mice exhibiting complete regression

of tumors was considerably higher in the 1 N HCI-fed group

than in controls. Finally, 50% of the transplanted mice in Group3 survived without any evidence of presence of tumor by theend of the experimental period. Tumors of mice in this 1 N HCI-fed group also began to kill their hosts at a later date than inthe controls (Chart 3). Life tables using a Breslow test showeda significant difference in survival between the groups (p <0.001). The final survival rate between the controls (4 of 20)and the 1 N acid group (20 of 40) show a statistically significantdifference at the p < 0.01 level using a x2 test.

Experiment 2-Effect of Acid Diet (1 N HCI) on Growth ofNormal Swiss Mice. Chart 4 shows the growth of 2 groups ofSwiss mice, with almost identical initial mean weights, whichwere not subjected to tumor transplants. One was normallyfed, and the other one was fed with 1 N HCI food from Day 0.Chart 5 shows the amount of food eaten by the mice and thedays in the normally fed and 1 N HCI-fed mice without transplanted tumors of Experiment 2. There was a progressive,





Chart 3. Experiment 1. Life tables and final survival of mice in thecontrol, 2, and 1 N HCI groups shown in Charts 1 and 2.

100.

80.

13

5 60.

ÃœJOOÃ

io..

20

40-1

OÃ030-1

OLU

20-O Control

â€”O IN HCI

10i

20

DAYS30

Chart 4. Experiment 2. Patterns of body weight of nontransplanted Swissmice fed with nonacidified versus 1 N HCI-acidified laboratory food.

linear adjustment to consumption of single-acidified food fromDays 0 until 6. Food consumption after Day 5 showed nodemonstrable differences between the 2 differently fed groups.Chart 5 also shows an occasional but very significant variationin daily food consumption affecting different cages at differentdays, a feature which is as yet unexplained.

Experiment 3-Effect of Acid Diets (1 and 2 N HCI) andAcidified Water (0.05 N HCI) on Growth of Sarcoma 18O andon Growth and Survival of Sarcoma 180-bearing Swiss Mice.This experiment was conducted to confirm the results obtainedin Experiment 1, while attempting to still further increase the

GROUP#1 â€”â€”Control

#2 2NHCI

#3 1NHCI

12. 2t - 36.

SURVIVflLIN DflYSH8- 60- 72-

8-1

7-

6-

2-

,O

2 4 6 8 10DAYS

20 30

Chart 5. Experiment 2. Food consumption per mouse and day of the same 2groups of mice shown in Chart 4 on control and 1 N HCI food This chart alsoshows the linear adjustment of mice to the acidified dietary conditions.

rates of "spontaneous" tumor regression through the addition

of acidified water at different times during the experimentalperiod. Once more, the final survival and complete regressionof tumors in the group receiving 1 N HCI acidified food plusnormal water reached 50%. Despite the fact that 5 of 20 mice(25%) in the control group enjoyed complete spontaneousregressions of tumors (maximal size reached by these 4 tumors, 215 to 260 sq mm) and despite the smaller number ofmice in the 1 N HCI group of this experiment, life table analysesof survival were again significantly in favor of the acid dietanimals (p < 0.05 using the Breslow Test) (Chart 8). Theaddition of HCI acid to drinking water to 0.05 normal concen-

NOVEMBER 1979 4367




tration beginning Day 4 or 14 did not further increase the rateof tumor regression. Acidification of water from Day 4 provedto be intolerable for the mice; this group behaved similarly tothe 2 N HCI diet group of Experiment 1. Chart 6 also showsthat the addition of acidified water on Day 4 increased theweight loss observed during the first 12 days of the experiment.Similarly, the addition of 0.05 HCI water on Day 14 (Group 4)decreased the subsequent growth rate of that cohort; but, incontrast to the Day 4 acidification group, this group of micesurvived the late acidification of water.

The final survival of Groups 2 and 4 in Experiment 3 (1 N HCIfood and 1 N HCI food plus 0.05 HCI water after Day 14) wasvery similar (Chart 8), and the later addition of acidified waterdid not improve the final results. This suggests that the relativeacidification which induces tumor regression must be presentduring the period of maximal tumor growth between Days 5and 12. The growth patterns of the tumors in the control groupsof Experiments 1 and 3 are slightly different. Observed initialtumor growth for all the groups in Experiment 3 appeared tobe faster than in Experiment 1 (Charts 2 and 7), perhaps theresult of the larger mass inoculated in Experiment 3. Whatever

the cause, this initial growth rate seemed to make no differencein the outcome of similarly treated groups. An unusual coursewas observed in 3 mice of Group 4. Two tumors initially grewto a considerable extent (160 and 168 sq mm), slowly regressed to 30 and 80 sq mm, respectively, and then regrew,killing their hosts as they reached 506 and 442 sq mm in size.A third tumor grew to 30 sq mm during the first initial days andthen "spontaneously" regressed completely by Day 14. How

ever, it too recurred killing its host when it reached 420 sq mm.Acid-Base Metabolic Data. Acid-base data obtained at Day

0 for 10 normal Swiss mice in Experiment 1 are summarized inTable 1, control groups. Venous samples for determination ofacid-base status were also obtained from 4 different mice of

Group 2 (2 N HCI) on Day 5. The results obtained after 5 daysof semistarvation on 2 N HCI diet are summarized in Table 1,2 N HCI group. The chronic effects of growing tumors on theacid-base balance of the mice were assessed in 4 mice with

large tumors (Table 1, 32 days after implantation). These largerneoplasms induced a degree of chronic metabolic acidosis forwhich the mice attempted to compensate through hyperventi-lation. A similar pattern was seen in the most acidotic mouse of

Chart 6. Experiment 3. Means of mouse weight in controls and3 other treated groups. Group 1 is a control, normally fed group.Group 2 was maintained on 1 N HCI food plus tap water ad libitum.Groups 3 and 4 also received 1 N HCI food throughout the experiment. Tap water was substituted by 0.05 N HCI water on Day 4 inGroup 3 and on Day 14 in Group 4.

40-

15

GROUPÂ»1 O ControlÂ»2 O IN HCI food#3 1. Food+0.05N HCI wot.r Day 4Â»4â€”o Food+0.05N HCIwotÂ«Day14

K>

â€”Tâ€”

20

1â€”

30

DAYS

â€”iâ€”40

â€”1â€”

50

â€”râ€”

60

GROUP* 1 O ControlÂ»2â€”0 IN HCI foodÂ»3 ----*>. Food+0.05 N HCI water-Day 4Â«4 O Food+ O.OSN H CI water-Day 14

Chart 7. Experiment 3. Mean tumor mass in the alive micein the controls and treated groups shown in Chart 6.




Table 1, 2 N MCI group as well as to 3 of 4 mice after 5 dayson 1 N HCI diet (Table 2, control group). Despite the relativelysmall number of animals sampled, the difference between themeans of metabolic pH of normally fed mice with active tumorgrowth (pH 7.17) and controls (pH 7.325) is marked enough tobe statistically significant (p < 0.05).

Acid-base determinations performed during Experiment 2(Table 2, control group) demonstrate a degree of acidosisappearing within 5 days of 1 N HCI acid feeding. Table 2,control group also shows that these differences between normally fed and acid-fed mice are maintained after 14 days.

However, at the end of 32 days (Table 2, group controls at Day32 and 32 days on 1 N HCI acidified food) these differenceshave largely disappeared. At that time, both groups also showa higher mean pH, possibly related to aging (see Ref. 16; Table2). It is also important to note that after the initial diet-induced

weight loss had been restored (see Chart 4, Day 14), anacidosis of a metabolic origin still persisted (Table 2, controlgroup at Day 0 and 1 HCI group at Days 5 and 14 of acidifiedfood), indicating a continued effect of these diets on acid-base

homeostasis independent of starvation. In this regard, none ofthe mice fed with 1 N HCI diets exhibited acetonuria at any timeusing a sensitive colorimetrie method (Acetest; Ames Co.,Elkart, Ind.), while this procedure could occasionally detect atract of acetonuria in mice of the control groups.

DISCUSSION

The importance of a persistently elevated pH to cancergrowth and progression has been known for many years (26,27, 30, 50). The relationship of these clinical observations tothe characteristics of cancer cells has recently been stressed(16). Further evidence indicates that severe metabolic acidosiscould very well have been responsible for some scatteredcases of spontaneous regression of solid tumors reported inhumans following surgical procedures such as ureterosig-

moidostomy (11,15). The reason for such antitumor effects isunclear.

More than 50 years ago, Warburg ef al. (44) observed thatin vitro tumor glycolysis was faster in a bicarbonate than in aphosphate buffer, and he was aware as early as 1924 of thesignificant pH dependency of glycolysis. Elevation of extracellular pH can induce different functional and biological characteristics of cancer cells such as an increase in cell disaggre-


gation, detachment, and migration (8, 12, 38, 39); stimulationof phosphofructokinase; glycolysis; cell growth; initiation ofDMA synthesis (35); and also a decrease in contact inhibition(12, 39). Relman (31), in a review of the effects of acid-basechanges on metabolic pathways noted that alkalosis, apartfrom enhancing the production of pyruvate, is able to inhibit itsutilization in the oxidative and gluconeogenic pathways andcan also block mitochondrial oxidation and tricarboxylic cycleturnover rates (31). An excessive decrease in hydrogen concentration is capable of disrupting the ordered integrationbetween glycolysis and tricarboxilic acid cyclic mediated respiration through acceleration of the former process and retardation of the latter, a biochemical feature which still remains acornerstone of cancer biochemistry (7, 44).

Severe chronic lactic acidosis is rarely if every compatiblewith life in clinical situations. However, it has been suggestedthat the cancer patient is always a patient with "potential"

lactic acidosis (45). This metabolic derangement seems toarise more frequently in laboratory animals transplanted with avariety of tumors, in which high levels of lactic acid have beenreported (5, 43). The data in Table 1, 32 days after implantationshows a significantly lower pH in mice carrying large Sarcoma180 tumors as compared to normal controls (metabolic pH7.17 versus 7.325). Although direct lactic acid measurementswere not performed, it is very likely that the chronic acidemiawas mediated by this organic acid which was in turn inducedby the transplanted tumor itself. A pathological accumulationof lactic acid could very well have been responsible at least inpart for some of the spontaneous regressions seen in this orother types of animal tumors. However, it is also likely thatmany animals die secondary to lactic acidemia before tumorregression can take place.

Starvation is known to be a significant inhibitor of tumorgrowth (4, 36). Despite the difficulties in separating the effectsof starvation ketosis from those of a directly induced mineralmetabolic acidosis in these animal experiments, we must stressthe complete lack of ketonuria observed in the successfullytreated 1 N HCI acidotic groups, while an occasional trace ofacetone was detected in the normally fed mice. This findingagrees with other publications which have shown that theinduction of mineral acidosis, apart from decreasing the formation of organic acids such as lactic and pyruvic acids, canalso suppress the production of ketone bodies through a directantiketogenic effect (13, 24). Starvation-induced ketosis was

Table 2

Experiment 2 (no tumor)

Control group (notumor) Day 0

1 N HCI group. Day 5of acidifying diet

1 N HCI group. Day 14of acidified food8No.

ofmice4

4

5pHRange7.19-7.31

7.02-7.16

6.96-7.16PCO2Mean7.24

7.12

7.1Range35.4-44.1

21-49

33.9-42Mean38.8

28.8

37.4BicarbonateRange14-19.2

6.2-16.8

9.3-11.9Mean16.3

9.4

11.4Base

excessRange-6.6

â€” 13.8

-13.7--25

-18- -25Mean-11.2

-20.3

-20.3Metabolic

pHRange7.18-7.30

6.94-7.19

6.97-7.11Mean7.23

7.05

7.06

Control group at Day32

1 N HCI group, Day 32of acidified food

5 7.36-7.48 7.40 35.6-65.8 47.2 19.5-37.6 28.3 -4.8-10.5

5 7.26-7.39 7.33 41-57.7 50.5 21.2-31.4 26 -4.2-5

3.3 7.33-7.53 744

-0.2 7.34-7.46 7.39

Means and ranges of the acid-base balance of transplanted mice fed with normal and 1 N HCI food after 5 and 14 days. The acidifying properties of 1 N HCI dietbecome evident after the original weights have been regained by Day 10 (Charts 4 and 5). A respiratory compensation through hyperventilation (low PCO2) occurredin 3 of 5 mice after 5 days on 1 N HCI diet.

Means and ranges for the different acid-base values of mice of Charts 4 and 5 at Day 32. The acidifying effects of 1 N HCI diet seem to have decreasedsignificantly, but some differences still persist.

NOVEMBER 1979 4369




iOC

Chart 8. Experiment 3. Life tables and final survival ofmice in the control and treated groups also shown in Charts6 and 7.

GROUP# l Control# 2 IN HCI food# 3 Food+0.05 HCI water Day 4# 4 Food+ 0.05 HCI water Day 14

'â€”h v

12. 2H. 36.

SuRVIVRL IN DflYS48. 60. 72.

not demonstrable by Acetest during the first 5 days of theexperiments. A complete adjustment to the acidified dietaryconditions occurred between Days 4 and 5 (Chart 5), and afterthis time, we would not detect any differences in eating patternsor food consumption among the normally fed and the 1 N HCI-fed animals. It is an unlikely possibility that the results at theend of 72 days were affected by the initial loss of 4 g bodyweight in the 1 N HCI group during the first 5-day period ofdietary adjustment or by depletion of some specific nutrientduring this time (3). While a caloric restriction of 33% has beenreported to be necessary to retard the growth of Sarcoma 180(2), this degree of caloric deficit could have occurred onlyduring Days 1 and 2 of these experiments (Chart 5). Also, theinitial observable growth of tumors from Days 5 to 10 ispractically of the same magnitude in the controls and the 1 NHCI-fed groups of Experiment 1 (Chart 2).

An apparent loss of effect in the acidifying properties of the1 N HCI diet in the middle stages of the experiment (Table 2,controls at Day 32 and 32 days on 1 N HCI acidified diet) canexplain the fact that no more than 50% of the animals showedcomplete regression of tumor growth. More severe degrees ofacidosis were unsuccessfully attempted through the additionof more acid to the food (Chart 1, 2 N HCI diet) or by acidification of drinking water in addition to the acid diet shortly aftertumor inoculation (Chart 6, Group 3). Both measures exceededthe capacity of mice to adjust to such acidifying changes(Charts 3 and 8). The addition of acidified water after Day 14(Chart 6, Group 4) did not further increase the final rates oftumor regression (Chart 8, Group 4). It is possible that differentmethods of inducing more severe acidosis, which do not createstarvation conditions, will be able to further increase rates ofcomplete regression of this or other transplanted animal tumors.

Another interesting feature was observed by comparing theslopes of mice weight in the control groups of Experiments 1and 2 and the rates of weight "regain" after Day 5 of the 1 N

HCI-fed Groups 3 and 2 of Experiments 1 and 2, respectively

(Charts 1 and 4). After the first 5 days, at a time when thistumor is already visible and palpable s.c., the Sarcoma 180transplant produces a rapid negative effect on the normalgrowth and weight gain of mice, a situation simulating the"anorexia-cachexia" syndrome seen in humans with advanc

ing cancer.These acid-base considerations strongly suggest that a per

sistent low pH could prove to be synergistic with chemothera-peutic drugs such as 5-fluorouracil, prednisolone, methotrex-ate, 6-mercaptopurine, cyclophosphamide, and actinomycin,

all of which have been shown to be inhibitory of tumor glycolysisor to affect glycolysis and/or respiration (22, 33, 46, 48).Conversely, the stimulatory effects of insulin (17, 49), glucose(42), and high pH (35) on malignant cell growth, glycolysis,and DNA synthesis could be utilized alone or in combination tostimulate cancer cell activity for controlled periods of time tofind a higher specificity for anti-DNA chemotherapeutic agents.

Immunological factors could have also affected the results ofthese studies. The relationships of H+ environmental changesto cell-mediated immune defense cannot be integrated at the

present time because of lack of evidence. A few publications,however, have demonstrated the importance of H+ environment on the glycolysis and function of mature granulocytesand macrophages in inflammation (19, 37) and also in malignant disease (9, 19, 26, 37). Furthermore, a low and falling pHseems to create appropriate environmental conditions for macrophage function in the inflammatory process (25).

In conclusion, total body acidification seems to be a promising avenue in antagonizing tumor growth in animals and itseffects seem to be independent of starvation-ketosis. Acidosis

per se is not likely to favor solid tumor growth, and it has thepotential to delay it at least in some cases. Conversely, environmental field alkalosisperse seems likely to promote malignant growth (16). These concepts are applicable without regardto location or histological type of solid tumor and as suchshould be considered in any attempt to control or preventneoplasia.

ACKNOWLEDGMENTS

The authors are grateful to Dr. A. Bhargava from the Laboratory MedicineDepartment for his expert help in providing blood gas determinations, to Dr. M.Katin for reviewing the paper, and to Bradley Bryant for his technical help with

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NOVEMBER 1979 4371



1979;39:4364-4371. Cancer Res Salvador Harguindey, Edward S. Henderson and Carl Naeher Effects of Systemic Acidification of Mice with Sarcoma 180

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Effects of Systemic Acidification of Mice with Sarcoma 1801 · 6 Normal acid-base balance in 5-week-old Swiss mice. Means and ranges of 10 different control mice sampled on Day 0