the liver in relation to normal and malignant...

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THE LIVER IN RELATION TO NORMAL AND MALIGNANT GROWTH: A REVIEW ALEXANDER HADDOW 1 (From tire Department of Bacteriology, Vniversity of Edinburgh) I In his presidential address delivered before the British Associatioii on " Some Chemical Aspects of Life " Sir Frederick Gowland Hopkins has lately drawn attention to the significance of chemical structure in cellular activity and has illustrated his theme by descriptions of the specific functions exerted in metabolism by such substances as acetyl- choline, thyroxine, adrpnalin, insulin and the sex hormones. Of special interest is his mention of the related conception that kindred sub- stances-hormones or vitamins according to their origin-may exert similar specific functions in the promotion of organismal and tissue growth. Authenticated examples of this type of influence are afforded by the action of the vitamin-like substances required for growth pro- motion in the case of certain micro-organisms (Knight and Fildes, 1933); the stimulation of plant growth by the production of specific tiuxines, and the influence on body growth exerted in a general and specialised manner respectively by vitamins A and D. It is obvious that this subject is of the utmost importance to till students of the physiology of growth and that the elucidation of such mechanisms is the rational path to their effective control in the caw of the normal and the malignant cell alike.' It must be pointed out, however, that throughout the literature pertaining to these subjects there exist certain serious misconceptions and ambiguities with regard to nomenclature. These have been the subject of a timely warning in a recent annotation in the columns of the Lancet (2: 1226, 1932), from which source one may with profit summarise certain principles of terminology and definition. The first essential observation is thtit growth in the biological sense presupposes cell division; it follows that a growth-stimulating factor in the true sense must owe its title to its promotive effects on cell multiplication. In addition, these effects must be produced on cell multiplication as a whole, and not be re- stricted merely to a single cell lineage or system, unless this restriction is clearly stated. A growth-promoting principle should therefore aug- ment cell division, both normal and malignant. Conversely-and here ouc may quote the note in question-" an anti-growth principle should inhibit cell division, and if it had this action it might reasonably be expected to inhibit the cell division of a malignant neoplasm, But if this anti-growth factor inhibited cell division, it should have this effect on the active cell divisions going on in normal tissues such as the testis, 11,aura de Sa%eeto Student, University of London. Working with the aid of a grant from the British Empire Caucer Campaign. 308

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THE LIVER I N RELATION TO NORMAL AND MALIGNANT GROWTH: A REVIEW

ALEXANDER HADDOW 1

(From tire Department of Bacteriology, Vniversity of Edinburgh)

I In his presidential address delivered before the British Associatioii

on " Some Chemical Aspects of Life " Sir Frederick Gowland Hopkins has lately drawn attention to the significance of chemical structure in cellular activity and has illustrated his theme by descriptions of the specific functions exerted in metabolism by such substances as acetyl- choline, thyroxine, adrpnalin, insulin and the sex hormones. Of special interest is his mention of the related conception that kindred sub- stances-hormones or vitamins according to their origin-may exert similar specific functions in the promotion of organismal and tissue growth. Authenticated examples of this type of influence are afforded by the action of the vitamin-like substances required for growth pro- motion in the case of certain micro-organisms (Knight and Fildes, 1933); the stimulation of plant growth by the production of specific tiuxines, and the influence on body growth exerted in a general and specialised manner respectively by vitamins A and D.

It is obvious that this subject is of the utmost importance to till students of the physiology of growth and that the elucidation of such mechanisms is the rational path to their effective control in the caw of the normal and the malignant cell alike.' It must be pointed out, however, that throughout the literature pertaining to these subjects there exist certain serious misconceptions and ambiguities with regard to nomenclature. These have been the subject of a timely warning in a recent annotation in the columns of the Lancet (2: 1226, 1932), from which source one may with profit summarise certain principles of terminology and definition. The first essential observation is thtit growth in the biological sense presupposes cell division; it follows that a growth-stimulating factor in the true sense must owe its title to its promotive effects on cell multiplication. In addition, these effects must be produced on cell multiplication as a whole, and not be re- stricted merely to a single cell lineage or system, unless this restriction is clearly stated. A growth-promoting principle should therefore aug- ment cell division, both normal and malignant. Conversely-and here ouc may quote the note in question-" an anti-growth principle should inhibit cell division, and if it had this action it might reasonably be expected to inhibit the cell division of a malignant neoplasm, But if this anti-growth factor inhibited cell division, it should have this effect on the active cell divisions going on in normal tissues such as the testis,

11,aura de Sa%eeto Student, University of London. Working with the aid of a grant from the British Empire Caucer Campaign.

308

LmER I N RELATION TO NORMAL AND MALIGNANT GROWTH 309

the epithelium of the intestinal mucous membranes, the spleen and lymphoid tissues, where new lymphocytes are formed, and the skin, to mention only a few of the tissues in which growthA.e., the formation of new cells-actively proceeds even in an adult organism. A growth- inhibiting factor should therefore produce lymphopenia, sterility, and lesions in the skin and in the intestinal mucous membranes.”

It should be pointed out that these observations were made in discussing certain results obtained by Thompson (1931) and Robinson and Thompson (1932), whose experiments suggested the existence of an anti-growth factor in the parathyroid gland. It was later sug- gested by Thompson that an anti-growth principle which is effective in a normal animal might also be effective against the growth of a malignant neoplasm, but this relation could not be demonstrated con- vincingly in the case of the parathyroid extracts when tested experi- mentally. In this instance it seemed apparent that the inhibiting prin- ciple acted, not on growth generally, but only on the development of bone.

Much of the diiliculty and confusion attached to the subject has arisen from the fact that ‘‘ the existence of growth-promoting hormones and vitamins is generally postulated when it is found that the size or weight of an animal increases more rapidly in the presence of a certain factor than in its absence.’’ In addition it is pointed out that conclu- sions drawn apply as a rule only to skeletal growth and not to the division of cells generally. A supreme example of the dsculties of the problem is presented by the action of the growth hormone of the anterior pituitary. According to Evans (1933), I ‘ if the anterior lobe has been completely removed, growth stasis is immediate and almost, if not quite, absolute.” Although part of the change is a disturbance of calcium metabolism after hypophysectomy, administration of growth hormone to hypophysectomised animals and to certain normal adult mammalian forms results in growth which is not due to adiposity or to development of the skeleton alone, but which, according to Evans, is participated in by all the tissues of the body.

The suggestion that the anterior pituitary may elaborate a growth hormone in the strictest sense recalls experiments by Baker and Carrel (1928), who showed that peptic digests of the anterior pituitary pro- moted proliferation of rat sarcoma cells in tissue culture to the same degree as embryonic extract. Semura also reported (1929) that ex- tracts of the anterior pituitary remarkably increased the growth of chick embryo ventricle in plasma containing embryonic extract or diluted with Ringer’s solution only. Before passing on one may quote another example of the current confusion, this time from the field of botanical growth studies. Much of the interest in the description of auxin as a “ growth-stimulating substance ” is lost when we find that its action depends on the production of increased cell size without effect on cell division (Kogl, 1933; Bonner, 1933).

It is the main purpose of this communication first to state as a hypothesis that cell division as a function may depend completely on

310 ALEXANItER HADDOW

the presence and activity of a single hormone or vitamin-like substance, or alternatively, that the growth of differentiated cell lineages may be governed in a like manner by the activity of specific growth-promoting substances; and secondly to discuss the significance in this respect of certain recent observations which point to the liver as the source of a growth-promoting substance of paramount importance in the division of both normal and malignant cells.

Before proceeding further, an interesting and highly relevant com- parison may be drawn with the conditions governing the growth process in huemopoiesis. Of all the somatic cells, none compares in growth activity with those in the marrow. The unremitting division of those cells which precede the functional units of the blood is of itself, apart from the fact that it is directioned and that the descendants are re- moved after a functional period which exactly balances the rate of production, of an order comparable with the mitotic rates of the most active malignant tumours. The growth function of the erythron-a single tissue composed of the red cells in the peripheral blood and their precursors in the haemopoietic centres-amounts to the replace- ment of every individual erythrocyte at an interval of a few weeks, at an actual rate of production running into hundreds of thousands of millions of red cells every day. Yet the whole process of growth and maturation involved in this output is completely dependent upon the presence in sufficient concentration of a single and relatively simple substance derived, according to the hypothesis of Castle, from the interaction of an enzyme secreted by the stomach (intrinsic factor) and some extrinsic constituent of the proteins of food stuff. It is perhaps unfortunate that the usual description of this haematinic substance as the " anti-anaemic " factor, expressing its significance from the pathological or therapeutic point of view in the treatment of pernicious anaemia, rather tends to obscure its real nature as a physiologically essential growth promoter. In other words, the prac- tical importance of this substance in clinical medicine has drawn at- tention away from the true biological significance of its controlling influence on the growth of a specific tissue. From this aspect the powerful response which followa the administration of a concentrated preparation in a suitable case of pernicious anaemia should be regarded as nothing less than a specific stimulation of megaloblastic growth and maturation. Conversely, this megaloblastic anaemia may best be re- garded as a specific growth inhibition or retardation due to the lack of this essential substance in metabolism, a condition due in turn to lessened production or to inactivation. Perhaps the most striking character of the haematinic principle is the apparently specific and indispensable nature of its function. Enough has been said to indicato the reality of a conception which in this instance has been abundantly proved both in clinic and laboratory, that an important growth function may completely depend on the presence in adequate amount of a single and relatively simple substance.

Before reviewing the evidence which suggests the liver as the seat

LIVER IN RELATION TO NOBlyLAL AND MALIONANT QBOWTH 311

of production of at least one other growth-promoting substance, it m y in the present connection lend some support to this hypothesis to mention two other circumstances of note. In the first place, it will be recalled that the investigation of the liver diet in pernicious anaemia by Minot and Murphy was suggested in part by the discovery of Whipple and his co-workers that experimental haemorrhagic anaemia in dogs could be favourably influenced by the addition of liver to the diet. Secondly, another reason for this choice of diet was, according to Davidson and Gulland (1930), (‘ the demonstration by McCollum that liver feeding could profoundly affect the growth of animals as the result of a greatly enhanced cell division. Minot hoped that increased cell division in the bone marrow might be produced with a correspond- ingly increased output of red blood corpuscles into the circulation.”

11 In the field of general nutrition the first observation on the physio-

logical growth effects of liver in the diet seems to have been that of Osborne and Mendel (1917), who. demonstrated improved growth re- sulting from the addition of small supplements of liver to a lean beef diet. These workers later (1926) obtained excellent growth responses when small additions of certain “ natural ” foods, such as yeast, liver, and lettuce, were made to synthetic food mixtures fed to male albino rats. An early finding of McCollum has been referred to above, and the extensive experiments of this worker and his associates (1921) on the nutritive properties of animal tissues showed that 20 per cent of whole liver in the diet suffices as the sole source of protein for normal growth, reproduction and the rearing of young. A related observation was made by Evans and Burr (1927), who found that a certain pure diet, insdlicient of itself, allowed gestation and the birth of good litters when supplemented by small amounts of lettuce or liver.

Wilkinson and Nelson (1931) studied the effect of various organs used as a supplement to a soy bean ration which had proved inadequate for lactation in the rat. Extraordinarily good results, shown by low mortality rates and high weaning weights, were obtained on diets sup- plemented with 25 per cent of either hog kidney or beef liver and on that containing 30 per cent of hog liver. The detailed results are shown in Table I, modified from the more extensive data in the original

Additional experiments were carried out to test the effect of a pY* ‘et consisting of casein, butter fat, salt mixture, dextrin, and various levels of hog liver. These experiments are represented in Table I by rations 99, 82, 84 and 83, and the results show a definite quantitative response to liver feeding. Their findings were interpreted by Wil- kinson and Nelson as evidence of the existence in liver of a lactating factor, but they drew attention, in addition, to the superior growth rate in the young.

Daggs (1931) carried out rather similar experiments in which female Boston bulldogs were fed balanced diets containing all of the

TAB

- I:

Rep

rodu

dion

and

Lad

atio

n an

the Rat on V

ario

us D

iets

(afte

r W

ilkin

son

and

Nel

son,

193

1)

Die

d 0-

7 D

ays

Num

ber

Wea

ned

X A

vera

ge

Wei

ght i

n G

ram

s

Die

d D

ied

8-14

Day

s 1

53

0 D

ays

Per

cent

M

orta

lity

rfte

r 30

Day

15

14 0 6 0 0 1 6 6 12 4 17

Ave

rage

W

eigh

t (g

ram

s)

--

22

22

31

45

40

26

32

33

42

63

61

67

--

0 29

13

19

0 34

0

20

5 14

6

22

9 14

1

10

15

20

4 2

12

2 8

3

Tum

ber

oi L

itter

s

0 11 0 0

0 36

12

11

7

8 1

4

Tum

ber

oj

You

ng

Num

ber

Wea

ned

-- 4 2 14

22

29

20

30

31

13

36

36

33

--

Rat

ion

No.

O

rgan

Con

tent

I I

68 (c

ontr

ol so

y bean r

atio

n)

0 8

48

88

88

70

71

72

85

w

74

76

87

78

L

t3

30%

hog

lung

30%

hog spleen

25%

hog

panc

reas

%

%be

ef p

ancr

eas

25%

hog

brai

n 25

70 be

ef b

rain

25%

beef h

eart

2570

bee

f th

ymus

48

48

48

48

48

54

48

54

94

71

48

40

58

43

26

73

44

434

990

1160

52

0 96

0 10

23

546

75

77

79

25%

hog

kidn

ey

25%

beef

liv

er

25%

hog

liver

9 9 10

54

54

60

14

28

26

2268

21

96

2144

81

80

73

5% h

og li

ver

15%

hog

liver

30

% ho

g liv

er

8 10 7

48

60

42

50

39 3

loo 53

31

12

--

21

31

29

38

61

68

798

1891

19

72

5% h

og li

ver

15%

hog

liver

25%

hog

liver

40%

hog

liver

6 9 8 7

36

54

48

42

0 20

33

37

0 55

55

66

0 11

00

1815

2442

99

82

84

83

LIVER IN RELATION TO NORMAL AND MALIGNANT GROWTH 313

2 4

3 1

known vitamins and other requirements. The only variable was the source of protein, whether from beef liver, muscle, kidney, or dried hen's egg. These experimental diets were fed from shortly after the time of conception through the fifth week of lactation and were in- creased to meet the demands of the growing pups. The litters were limited to three pups and the mothers given identical care. In the present connection the observation of greatest interest is that the pups of the liver experiments grew 1.34 times as fast as those for which the protein-souroe was egg, as may be seen from Table 11.

TABLE II: (hmdh AnalyeZs (afler Dag~a, 1031) I I I

Liver (2) 60.40 48.98 " (2s) 47.63}

1.341 : 1.000 E p (1) 36.61

(1)

I Liver : Egg Experiment I Diet I m** Average

* The d u e m daignata the dope of the curve or the rate of change of y (might in gnuns) with respect to z (&ye old), cdcuhted from the Eormulrr: y - mx -t b. (b = l/n@zt - m W : n - number of obeervatkm).

Smith (1932) reported that albino rats on certain &eta containing liver protein were unable to nurse their young, The failure of lacta- tion was not caused by a deficiency of vitamins. The daily adminis- tration of 0.5 to 1 gram of dried whole liver (prepared irt uacw at 100" C.) had no beneficial effect on lactation, whereas, 1.5 grams of raw liver daily enabled the animals (9) to nurse their young successfully, and 26 animals out of the 57 born were weaned at twenty-one days of age. The successful action of raw liver was further shown by the fact that. the animals of this succeeding generation were considerably heavier than the parent generation at the same age.

As a result of further work, Seegers and Smith (1932,1933) brought forward evidence indicating that the increased growth-rate resulting from feeding whole liver is due to some influence other than an in- creased food consumption. This is of interest particularly in relation to such work as that of Graham and Griffith (1933). These authors obtained optimum growth-rate of rats on a highly purified Evans and Burr diet supplemented by liver. While confirming the important acoelerating effect, they related the optimum growth obtained to an increased consumption of food resulting from the presence of liver in the daily supplement. Seegers and Smith also pointed out that the factor in liver responsible for the growth-promoting effect is removed by alcohol, and in later work (Smith and Seegers, 1934a) they produced further evidence in support of this view. In particular they demon- strated that dried whole liver possesses a growth-promoting factor whiah is lost on alcohol-extraction. A still later paper (Smith and Seegem, 1934b) described work designed to demonstrate that the su-

314 ALEXANDER HADDOW

periority of raw liver and of dried whole liver over alcohol-extracted liver was real and not fortuitous. For this purpose whole liver and alcohol-extracted liver were used not as small supplements but as the sole source of protein in otherwise identical diets.

An important advance in this subject was made by Mapson (1932 a, b). This worker undertook an investigation to determine if an animal fed on a basal diet containing all the known vitamins and eswri- tial food factors showed any extra growth when fresh liver was fed in addition. Mapson abundantly substantiated the accelerating influ- ence of fresh liver on the growth rate of the young rat and demonstrated t h d this effect is much more notable than was previously thought. Fresh ox liver alone was used and was fed in the dietary supplements at levels of 0.5 to 1 gram per rat per diem. In the case of young animals 40 to 50 grams in weight at the commencement of the experi- ment the growth response to the feeding of ox liver was found to occur rapidly, and a marked acceleration was usually seen for a period of some five to six weeks.

Two interesting observations appear to have emerged at this stage of hfapson’s work. First, the growth response in the female w a ~ found to be much less marked than in the case of the male. Secondly, the growth response appeared to vary inversely with the age of the test animal. ‘( Whatever the process was which had thfis been stimulated in the organism by the feeding of raw liver, it was one which, in the young animal, had a much greater effect on the growth rate than in an animal some weeks older.” Experiments were then started in which fresh liver was fed to male and female rats before mating. In records of weight from birth onwards no,marked difference was found up to twenty-one days of age between the offspring of liver-fed parents and those of the controls. From this point, however, the former grew very

.much faster than the latter and the growth curves for the two groups rapidly diverged. This growth acceleration occurred approximately when the young began eating for themselves, and its extent at nine weeks is indicated in Table 111. The effect was found to be more prolonged in the case of young whose parents had received ‘liver than in the case of direct liver feeding to the young animal. Another result, of possibly greater importance, arises from the figures given by Mapson for the size of the litters born in his various experimental series (Table 111). Since litter size in these experiments was not limited in any way, there exists the possibility that such limitation might increase still further the growth-acceleration effect as measured, for instance, by the average weight of the male offspring at nine weeks.

In the course of this work Mapson adduced evidence to show that the growth augmentation ip such experiments is an effect quite inde- pendent of non-specific improvements in the general maternal condition. He satisfactorily differentiated the growth-promoting factor from known dietary factors such as vitamins A and D and the vitamin B complex, and practically excluded the possibility that the growth-pro- moting effect of the liver supplement might be due to the correction

LIVER IN RELATION TO NORMAL AND MALIQNANT GROWTH 316

Diet of Psrenta Diet of Offupring hotation Number in Litter

Basal + 11- 11 11 + + 13 11 11 + 11 11 9 + +

11 10.3 + + 1 1 12 +

11 10 + 11 12 + 11 11 + 9

11 11, + B8iIlbl + 8'

7 6 - 6

I1 11 - 10 ,Average 11 8 7.4 +

6 + 7 9 - 7,

11

11 11

Bad + liver

11

11

I 1 lo .Average 11

11

11

11

11

11

11

11 11

11 11

11 11

- Bad -

11

11 11 - l l I 1

11

11

11

11

-

of protein- or amino-acid-inadequacy in the basal diet. In addition, aahed ox liver fed in equivalents of 1 to 5 grams of the fresh tissue was found not to exert any growth-stimulating influence. In their paper published after that of Mapson, Graham and Griffith (1933) raised the question whether the vitamin (3 requirement for food consumption and utilisation had been adequately met in experiments of this type. On the other hand, Smith and Seegers (193413) were of opinion that their collected evidence differentiated the growth factor from messory substances present in yeast as well as from any of the hitherto known dietary essentials. In a subsequent paper (1933) Mapson presented data suggestive of the identity of his substance, which he named physin,

T a m I11 (Afler Mopson, 1958b)

Aversge Weight of d Offupring at 9 Weeks, gm.

246 2456 236 280 270 295 230 280 240 270 276 24s 286

166 - - - - 186

179 - - -

316 ALEXANDER HADDOW

Before bleeding 48 hrs. aftter 1st bleeding

" 2nd "

" 3rd "

11 1 I

11 11

1 week after production

2 weeks after production

3 weeks after production

4 weeks after production

of anaemia

of anaemia

of anaemia

of anaemia

vitamin diet. In an experiment lasting nine weeks, weight increase was studied in rats receiving this basal diet alone and in rats receiving supplementary liver feeding. The liver addition had a decidedly bene- ficial effect upon the growth of the animals,.the average gain in weight being 51 grams greater in the liver-fed animals than in the controls, In a later but similar esperiment lasting three months, the rats which received raw liver averaged 60 grams heavier at the end of the period

2.60 2.60 2.65 2.05

2.65

2.70

2.70

2.70

TABLE IV (A jkr Michael, 1932)

I/-

/I- --

Before bleeding 48 hrs. after 1st bleeding

" 2nd "

" 3rd 1 week after production

of anaemia 2 weeks after production

of anaemia 3 weeks after production

of anaemia 4 weeks after production

of anaemia

11 1 6

11 11

-

2.50 2.60 2.65 2.65

2.85

3.00

3.15

3.20

Control Group (Average of 10 adult female cats).

R.B.C.* :millions per cu. mm.)

8.10 5.94 4.92 3.94

4.96

5.84

6.80

7.00

W.B.C.* (per cu.

mm.)

19,000 19,800 18,000 17,000

17,600

18,000

18,400

18,800

Reticule. cytes

%

- 0.2 1.0 2.0

0.5

-

-

-

Hb' %

74 52 40 36

43

48

54

62

-

-

Blood volume (c.c.)

Erlanger's Bleeding method method

161 125 161 125 164 127 164 127

164 127

167 130

167 130

167 130

Liver Group (Average of 15 adult female cats).

8.60 5.85 4.92 3.64

5.88

8.20

8.98

9.40

18,000 18,800 17,700 16,600

17,000

17,800

18,600

18,800

- -

0.08 2.00

5.00

2.00

-

-

70 60 46 30

50

66

74

82

155 120 161 125 164 127 164 127

177 137

186 144

195 151

198 154

* Note standards for normal adult cat: Average erythrocyte count, 8 millions per c.c.; wer- age white-cell count, 18,000 per c.c.; average haemoglobin value ( & I d h e ' s method), 70 per cent.

t,han the animals which received no liver. Bahrs observed, also, a distinct increase in reproductive efficiency under the influence of liver feeding. The number of animals used in certain of these experiments was rather small and the data received only very inadequate statistical treatment, but the results in aggregate appear nevertheless to give definite support to the findings of others already referred to.

Although not dealing with reproduction and somatic growth, as do the investigations referred to above, the experiments of Michael (1932)

LITER IN RELATION TO NORMAL AND MALIGNANT GROWTH 317

are of some interest at this point. They dealt with the effects of liver feeding on blood regeneration in cats rendered anaemic by bleeding, and thus exemplify the growth-stimulating action of liver on a single tissue. This particular effect of liver feeding was demonstrated early by Whipple and his co-workers, but the work of Michael is of additional interest in that she studied the simultaneous effect of this diet on the endocrines. This aspect will be discussed later in the present review. Table IV shows the remarkable influence of liver feeding in accelerating blood regeneration as measured by erythrocyte count and haemoglobin level. While a return to the pre-experimental blood condition was not complete in the control series at the end of four weeks, this condition was reached by the liver-fed animals after two to three weeks of the liver diet, and was improved on in the following one to two weeks. The table also indicates a weight increase which seems of possible signif- icance. According to Michael, the histology of the liver was sig- nificantly different in the controls and liver-fed animals. I n the former the cells around the intra-lobular vein in many of the hepatic lobules had few granules in them, but contained fat globules, while those towards the periphery were filled with granules and had little or no fat. On the other hand, in the liver-fed cats the cells around the intra-lobular veins in many of the hepatic lobules were filled with granules and those at the periphery were loaded with glycogen. There were also markedly fewer intracellular fat globules than in the controls.

I11 Experimental work to test the relation of liver feeding to the ap-

pearance and growth of tumours seems first to have been undertaken by Maisin and Franqois (1928), and it is of interest to note that this investigation was undertaken after the demonstration by Minot and his collaborators of the influence of the liver diet on pernicious anaemia. Maisin and Franqois studied the action of liver feeding in mice sub- jected to tar painting twice weekly in the interscapular region. All the animals received 50 treatments in 120 days, at which period painting ceased. The controls were fed on a mixture of wheat flour, maize with added water, and white bread in small amounts. The experimental series received three times weekly a mixture of wheat flour and liver pulp fed in amounts increasing to 5 grams liver per mouse. Tables V and VI show the result of this experiment with regard to tumour inception in the experimental animals, while Tables VII and VII I show the percentage of cancerous mice in each series bearing metastases and the mean duration of life of cancerous mice, respectively. Maisin and Franqois drew attention to the fact that, in spite of the shortened life span in the liver-fed mice, metastasis formation was much more fre- quent in these animals than in the controls. This indicates that the majority of these mice died as a result of their cancer, and not on account of any nutritional fault in the liver diet. On the contrary, the administration of liver was found to produce a markedly beneficial

318 ALEXANDER HADDOW

Percentage or mice with tumours in those alive a t stated period.

(Fraction in brackets gives actual figures.)

Control series Liver series 4.16(2/48) 21.5(6/19)

16.4 (7/42) 80 (12/15) 34.2 (13/38) 100 (9/0) 62.85(22/35) 100 75 (18/24)

100 (1/1) 100

effect on the condition of the mice. From these findings Maisin and Franqois concluded that the administration of liver hastens the ap- pearance of tar cancer and markedly accelerates the subsequent devel-

Percentage of mice with tumours with reference to the number of animals st commencement of

experiment. (Fraction in brackets gives actual

figures.)

Control series Liver aeries 4.16(2/48) 21.5(6/19)

18.72(9/48) 03.1(12/19) 35.36(17/48) 79 (15/19) 56.16(27/48) 79 64.48(31/48) 79

72.9(36/48)

Stage of experiment (days after commence-

ment of tarring)

Stage of experiment (days after commencement of tarring)

120 150 180 210

120th day 150th day 180th day 210th day 240t,h day end of experiment, 585th

day

Percentage of mice with tar tumours with reference to number of animals a t commencement of experiment

4 10 17 46 39 69 59 76

Control series (132 animals) Liver series (113 animals)

Days after 1st tar application

Control series Liver series 267 198

~

TABU V I (After Maisin, 1991a, b)

Days after cessation of tar painting

Control aeries Liver series 147 78

TABU VII: Percentqe of Cancerow Mice Baring Metastasea (After Maisin and FraWEoia, 1928; Maisin, 1951a, b)

To date 1 Pulmonary metastases 1 Axillary metastases ~~

~~ 1 Control Liver Control Liver series series series series

55 59.4 49.3 I 72.6

opment of the tumour. They drew attention to the work of Ledecq in the same laboratory, who found that the evolution of tar cancer in rabbits on liver diet was unusually rapid and progressive.

LIVER IN RELATION TO NORMAL AND MALIGNANT GROWTH 319

150th day 180th day 9 18

19 35 46 69

The control experiments in Table VI included animals fed on diets differing from the liver regime only in the substitution of liver by another organ. These results are given in detail in Table IX. TABLE IX: Percentage of Mice Bearing Tar Tumours, with Reference to Number of Animalsat

Commencemenl of Experiment (after Maisin, 1931 b)

Diet supplement I Stage of experiment

240th day 44 55 81

Thymus Muscle Liver

Although it was not possible to state the exact nature of the active substance, it was found potent in desiccated preparations after several months, while desiccation of the residue obtained after discarding the ether-soluble fraction of liver gave a product even more effective than fresh liver. I t was found not to be identical with the anti-anaemic substance of Minot ( Maisin, 1931a).

A contemporary report is that of Caylor (1928), who found that liver feeding had a stimulating effect on the rate of growth of the Flexner-Jobling rat carcinoma. These experiments were insufficient to determine whether the effect observed was due to the liver specifically or to the high-protein diet. Caylor, Baldes and Mann (1931) accord- ingly reported experiments in which the effect of the feeding of muscle was also observed. The ratio of the average volume of the tumour at time of death in the liver-fed group to the average volume of the tumour in the control group was 1.35 to 1. The results indicated that the curves for tumour volume in the control animals and in the animals on a high-protein diet, at the time of death, were almost always below the curve for the volume of the tumour in the liver-fed group. Woglom (1932), impressed by the possible practical value of this observation, studied the influence of liver feeding on the Crocker Institute tumours at a time when none of these was growing satisfactorily. In the course of an extensive investigation approximately 10 grams of fresh ox liver was fed to each tumour-bearing rat, but no demonstrable effect was obtained.

A study by Willis (1932) is of great interest in the present connec- tion as a possible direct indication of the action of liver on the division of the malignant cell. Previous observations by this worker had suggested that liver tissue was a highly favourable soil for the multi- plication of the epidermoid cancer cell. The liver was found to be the most frequent site of secondary growths from primary tumours of the portal-drained viscera and next to the lungs was the favoured site of metastases distributed by the systemic blood stream. This condition was the more remarkable in view of the relatively poor arterial blood supply of the organ. Willis accordingly made a quantitative study of the mitotic activity of a variety of malignant tumours and their

TABLE X : Cmparative Frequency of Mitotic Figurea in Primary carcinomaia and in Their Melaetalic Deposits in the Liver (after WiUia, 1932)

- - Case No.

I -

I1

111

IV

V

VI

VII

VIII

IX

-

Summary of Pathology

Anaplastic epidermoid carci- noma of tongue; metastases in liver, kidneys, and ribs; hepatic metastases numerous and mass- ive. (Liver weight 76 ounces).

Epidermoid carcinoma of tonsil; a few small scattered metastases in the liver.

Anaplastic carcinoma of phar- ynx; metastases in lungs, liver, spleen, kidneys, and ribs; he- patic deposits massive and numerous. (Liver weight 100 ounces).

Epidermoid carcinoma of oe- sophagus; metastases in liver, kidneys, cerebral cortex, and one humerue.

Anaplastic medullary carci- noma of stomach; massive hepatic metastases. (Liver weight 168 ounces).

Carcinoma of breast; metastases in lungs, liver, and spleen; hepatic metastases numerous. (Uver weight 68 ounces).

Adenocarcinoma of kidney; metastases in lufgs, liver, spleen, opposite kidney, and bones; liver contained single nodule 1.5 cm. in diameter.

Carcinoma of cervis uteri; scat- tered metastases in liver, up to 1 cm. in diameter.

Carcinoma of ovary; metastases in lungs and liver; hepatic metastases 30-40 in number. (Liver weight 68 ounces).

Site

Primary carcinoma Liver deposit

Primary carcinoma Liver deposit

Primary carcinoma Liver deposit

Primary carcinoma Liver deposit

Primary carcinoma Liver deposit

Primary carcinoma Liver deposit

Primary carcinoma Liver depoeit

Primary carcinoma Liver deposit

Primary carcinoma Liver deposit

Cells counted

3590

3940

3570

3200

3370

3400

3340

3570

3100

3310

3320

3520

8320

3500

3530

3630

3800

3290

- S* -

6

24

6

47

6

13

6

12

5

17

3

12

1

10

26

29

14

29 -

Mitoses - A * -

2

15

3

35

0

5

5

7

4

8

2

12

0

3

8

7

2

10 -

- D + -

0

5

0

10

0

4

0

2

1

5

1

4

0

2

0

2

1

4 -

- Total

8

44

9

92

6

22

11

21

10

30

6

28

1

15

33

38

17

43

Mitotic [ndex (per thousand)

2.2

11.1

2.5

28.7

1.7

6.4

3.2

6.9

3.2

9.0

1.8

7.9

0.3

4.2

9.3

10.4

4.4

13.0

* S spireme; A = aster; D = diaster.

320

LIVER IN RELATION TO NORMAL AND MALIQNANT QROWTH 321

hepatic metastases. Table X gives the comparative frequency of mi- totic figures in these sites in nine cases whose pathology is summarised. In every case the mitotic index in the hepatic metastases exceeded that in the primary growth. I n some the difference was slight, in others striking; the average hepatic mitotic index f o r the nine cases was five times that of the primary tumour. It was thought that these results might express merely a general anaplasia common to all metastatic deposits, and the mitotic index was accordingly estimated in several other situations. It was found that, while metastases in various situa- tions may exhibit mitotic counts greater than their parent primary, the hepatic deposits take general priority in this respect. Willis drew attention to the frequent clinical and pathological evidence of accelera- tion of growth in secondary tumours in the liver, while the primary growth in breast, bowel, or elsewhere remained relatively small.

These facts indicated that hepatic tissue was a highly fertile medium for the growth of the malignant cell, and Willis suggested that this might be related to the high carbohydrate content and the low arterial vascularity of the liver, but in the light of other facts summarised in the present review it seems not unlikely that a more specific mechanism is responsible.

Watson (1933) reported the result of similar studies on the nutri- tional aspects of tar cancer. He found an increased carcinogenic re- sponse in tar-treated mice when the diet on which they were maintained was supplemented with fresh liver. This increased response was in- dicated by the earlier appearance of benign warts and the fact that larger numbers of animals developed warts and epitheliomata. On the other hand, it was found that the average interval of time between the first treatment with tar and the development of malignancy was not reduced in the liver-fed series. Nevertheless, these results in the main confirm and extend those of Maisin and Franqois.

IV The preceding sections of this review constitute a survey of the

present experimental evidence indicating the presence of a growth- promoting substance o r substances in the liver. It is apparent that sufficient positive evidence already exists to justify the reality of the conception, at least in its broader aspect. It remains only to discuss other cognate problems arising from these facts. In the first place, Mapson’s studies led him to regard experimental growth promotion by physin simply as a manifestation of super-activity in a normal physiological process, and he recognized the phenomenon he described as due possibly to a primary stimulation of the anterior pituitary. It is of considerable interest in this connection, therefore, to note that Michael, in the study referred to above, found an obvious difference in the pituitary histology of her anaemic cats according to the nature of the diet. The pars tuberalis and posterior lobe were normal in both series, and mitoses were present in the oxyphil and basophil cells of

322 ALEXANDER HADDOW

the pars wterior. While oxyphils were more numerous than basophils in the control series, however, the anterior lobe in liver-fed animals contained far more basophils than oxyphils. Further, while the capil- lary spaces of the gland were normal in the control series, dilatation was found in the case of the liver group. These changes were inter- preted by Michael as evidence of heightened activity.

Such observations raise the question as to the possible correlation of these appearances with output of growth hormone. Nelson (1933) recently reviewed the evidence which seems to connect the conspicuous- ness of a given cell type in the pituitary with certain coincident phyaio- logical activities. I n the first place, he found no reason to deny the view that the two chromophilic cell types are derived independently from the chromophobic cells and that they maintain their individuality. He followed this observation by the necessary warning that any conclu- sion relating hormone production with a specific cell type must be ‘ tempered by the acknowledgment that morphological structure cannot

always be considered a true criterion of physiological activity.” In the anterior hypophysis of the foetal pig Nelson found that the baso- phils were the first to arise from the chromophobic cells. The develop- mental stage wherein they appeared in numbers was found to coincide with the earliest stage at which Smith and Dortzbach were able to demonstrate the presence of the growth-promoting hormone. Eosino- phils appeared shortly afterwards, but were not numerous until much later. A marked rise in their numbers was found to occur at the stage in which Smith and Dortzbach were first able to obtain a positive test for the presence of gonad-stimulating hormone. These observations pointed to a state of affairs in the pig opposed to the generally accepted theory-also reviewed by Nelson-that the eosinophile cells elaborate the growth hormone while the basophils are responsible for the gonad- stimulating principle, and this circumstance in itself indicates the difficulty surrounding this specific problem.

A further possibly important relation between the liver and the anterior pituitary is suggested by the finding of White (1933) that a highly significant decrease in liver weight occurs after hypophysectomy in the rabbit. For example, in 9 hypophysectomised male rabbits the change after hypophysectomy expressed as a percentage of the net average weights in 5 normal controls was - 21 per cent for body weight, - 30 per cent for thyroid, - 4 per cent for adrenals, - 15 per cent for the kidneys, - 23 per cent for spleen, and - 45 per cent for liver ; while in 8 female rabbits the corresponding figures after hypophysectomy as compared with 10 normal controls were - 5 per cent for body weight, -20 per cent for thyroid, -62 per cent for adrenals, -26 per cent for the kidneys, -77 per cent for spleen, and -558 per cent for liver. This loss in liver weight was not accompanied by any striking histo- logical change.

Further questions may be asked as to the chemical nature of the active substance. I n this regard little evidence is available beyond that summarised above. Assuming that the substance responsible for the

LIVER IN RELATION TO NORMAL AND MALIGNANT GROWTH 323

effects described by different workers is the same, its aqueous extrac- tion is facilitated by a preliminary partial digestion (Mapson), it is removed from fresh liver by 90 per cent alcohol (Mapson, Seegers and Smith), and it is not contained in the ether-soluble fraction of liver (Maisin). Much further work is obviously necessary before the nature of the substance or the mechanism of stimulation can be known, and it is obviously necessary to define the position of the substance in relation to certain others claimed as stimulants of cell division of wide biological activity as, for instance, by Hammett (1929) in the case of the sulphydryl theory of growth by increase in cell number. One must ever consider the possibility that such a substance or group of sub- stances fulfills the same physiological function of cell division in widely varying forms of life. Evans, Meyer and Simpson (1933) have noted the liberation of hydrogen sulphide from the growth hormone in the presence of certain high concentrations of alkali. It is of no less interest in this regard that Huddleson (1929), in studying the differen- tiation of the species of the genus Brucella, utilised for the purpose of estimating the production of hydrogen sulphide by these organisms a liver-infusion medium to serve as a source of readily available sulphur compounds. Apart from its use in this connection, the medium has been found in bacteriological practice to yield luxuriant growths of certain organisms whose growth on other media is less profuse.

There is much hope for the view that the continued study of specific growth-promoting substances will yield a rich harvest in methods of promotion and control. Although extensive speculation is to be de- precated, it is far from improbable that some of the mechanism of the action of x-rays on the dividing cell may prove to be attributable to an inactivating effect on such substances locally. Along these lines such observations as those of Samssonow (1929) seem worthy of attempted verification. This worker grafted rats with sarcoma and with carci- noma and divided each series into two batches after the tumours had begun to grow, One batch served as controls, while of the others some were x-rayed over the liver and others had radon tubes inserted into the substance of the organ. In the animals so treated the tumours showed rapid regression. Moreover, in rats, large tumours not re- sponding to direct irradiation of the tumour itself regressed entirely after irradiation of the liver. The author suggested that such irradia- tion exerted an indirect action upon distant tumours and that it might be of practical value in the treatment of metastases. Further sug- gested lines of work are the study of the influence of various liver extracts on mitosis in vitro (cf. Baker and Carrel, 1928), and the ques- tion of the possible antigenicity of growth-promoting substances. Work along these lines is being carried on by the writer.

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324 ALEXANDER HADDOW

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