CHAPTER I

PREVIOUS INVESTIGATIONS*

EXPE RI M E NTAL C I R R HOSl S

The experimental investigations which have a bearing upon the present work may in this short review be divided into two main groups, i.e., ( I ) dietary cirrhosis of the liver, and ( 2 ) experimental cirrhosis induced by external agents. However, a sharp line cannot always be drawn between these two groups.

( I ) DIETARY CIRRHOSIS OF THE LIVER

The observation that administration of choline was able to restore the fatty liver of pancreatectomized dogs to normal (4) gave rise to an intensive research on the relationships of diet and liver damage. In most of these investigations rats were used as experimen- tal animals. It was observed that a low-protein and cystine-free diet resulted in liver hemorrhages (63), which could be prevented by the administration of methionine or cystine. It was not clear whether the hemorrhages were accompanied by necrosis, but it was soon shown by other experiments that a diet containing casein as the sole source of protein gave rise to liver necrosis ( 2 3 ) . Yeast extract prevented the necrosis. Both necrosis and cirrhosis of the liver could then be produced a t will if a moderately fatty diet with a protein content of less than 10 per cent casein was used. Choline administered together with cystine, or methionine given alone prevented the liver damage (24, 2 5 ) . While it was shown that protein and methionine prevented both the necrosis and the cirrhosis, it was later observed that cystine prevented only the necrosis and

* Previous investigations dealing especially with the morphological picture and mode of development of dietary cirrhosis are dealt with in Chapter VI.

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the hemorrhages, and choline prevented only the fat accumulation and cirrhosis (12) . The nature of the fats in the diet also played a role, in so far as it was established that the amount of fatty infiltra- tion was related to the amount of saturated fatty acids in the food (8). If the diet consisted chiefly of carbohydrates, 8 per cent of casein was sufficient to prevent necrosis, but if more fat was included in the food a larger amount of casein was required (33). However, with a greater amount of casein, although necrosis was prevented, cirrhosis would still develop to a degree proportional to the severity of the fatty infiltration. Choline prevented the cirrhosis (21,34).

The results obtained in different laboratories, however, differed considerably from each other. British investigators reported a practically IOO per cent incidence of massive necrosis in rats kept on a diet in which yeast furnished the only source of protein. On the other hand, American investigators never reached higher figures than 40-50 per cent in their similar series (41). Further investigation then showed that the British yeast was responsible for the difference in the results, in as much as identical results were obtained when this yeast was used in all experiments (41). This first gave rise to the assumption that British yeast may contain some toxic substance affecting the liver, as the content of known protective factors apparently was identical in both yeast brands. The discrepancy in the results between American and British investigators seems, however, to have been solved by the extraction of a water-soluble protective factor from American brewer’s yeast (57). I t was shown by fractionation experiments that this factor was not identical with vitamin E or cystine, and it was designated as“ factor 3”. Meanwhile it was observed independently by different investigators (26, 5 5 , 56) that vitamin E had a protective effect against liver necrosis due to the absence of sulphur-containing amino-acids in the diet. This effect was limited to the necrotic changes, as supplements of tocopherol had no effect on the incidence of dietary cirrhosis (26). The importance of the pre-experimental diet has also been stressed, as this diet, if poor in vitamin El considerably lowered the resistance of the animals to protein deficiency (35). Administration of vitamin E also prevented the liver injury in rats fed a purified type of casein (casein VI) instead of the ordinary casein. However, the dose of vitamin E in this case had to be about 20 times as great as was usually required for the prevention of vitamin E deficiency ( 5 5 , 56). Curiously enough, this lesion could not be prevented by the addition of methionine or choline to the diet ( 5 5 ) . The closer interrelationship of these different protective substances is not yet clear.

The necrotic injury, also, seems to a certain extent to depend on the nature of the dietary fat, as unsaturated fatty acids appeared to increase the frequency of necrosis ( I , 26). Lard and cod liver oil also seem to promote the development of dietary liver cirrhosis, as compared with the effects of certain other fats. Unsaturated fatty acids also appear to be at least partly responsible for the formation of ceroid pigment in dietary cirrhosis of the liver (26).

Aside from proteins, methionine, choline and vitamin E (vide supra and 37), several other factors have later been found to have a certain protective action in various forms of dietary liver injury. Among these may be mentioned vitamin B,, (17, 18, 27) , liver

9

extract ( 2 7 ) , aureomycin, streptomycin, terramycin and sulfaguanidine ( 2 7 , 28). ‘The manner of action of these substances is not yet quite clear*.

Although the more recent experiments have cleared many of the seeming discrepancies between the results of various groups of inves- tigators, there still are some points in the pathogenesis of dietary cirrhosis which are open to debate. One of them is the relation between fat accumulation and cirrhosis. This question is of great importance because it may have a certain bearing upon the inter- pretation of fatty liver and cirrhosis in man. Based on animal experi- ments, most investigators hold that fatty infiltration per se ultimately will lead to cirrhosis. Others point out that they may be separate, not directly related processes, which depend on related causes. This view has been defended by pointing to conditions where the fatty liver in man disappears before gross fibrosis arises (14, 47).

Other controversial subjects have been the localization in the lobule of the fatty infiltration and the incipient fibrosis, and the apparently still unsolved question of the mechanism by which the fibrosis arises. These questions and previous investigations related to them will be dealt with in Chapter VI.

(2) EXPERIMENTAL CIRRHOSIS INDUCED BY EXTERNAL AGENTS

Reference has already been made above to the effects of unsat- urated fatty acids in the diet. It has also been claimed that cystine given in excess causes liver injury ( 1 1 ) . As an explanation for this fact it has been brought in mind that many enzymes are inhibited by a surplus of substrate (41).

Of the numerous chemical agents which cause liver necrosis and cirrhosis, some of the research work on carcinogens has a bearing upon the present investigation because a carcinogen, 9, Io-dimethyl- 1,z-benzanthracene, was used in the test mixture in the present writer’s preliminary series. It is known that painting of the ears of rabbits with tar can sometimes induce necrosis and cirrhosis of the liver (13). It has been shown that 1,2,5,6-dibenzanthracene

* Previous investigations related to special problems will be described, when necessary, in the pertinent chapters.

I 0

dissolved in lard and injected subcutaneously into rabbits gives rise to both necrotic and cirrhotic changes in the liver (9). The individual variations, however, were great and some of the animals survived up to two years of treatment, showing only fatty infiltration but no actual cirrhosis a t autopsy. Frequent application of the carcinogen seemed to cause mainly necrotic changes in the center of the lobules, while application a t longer intervals caused cirrhosis of the liver. It is not necessary to go into details concerning the large body of literature on the cirrhosis-producing power of carci- nogens related to “butter-yellow”, since no carcinogen of this kind was used in the present work. As to the carcinogen used by us, 9,Io-dimethyl-I ,a-benzanthracene, the present writer has not been able to find any report on induction of cirrhosis of the liver by the oral administration of this substance to rabbits.

I t has been demonstrated that a great number of various other chemical substances induce fibrosis of the liver. I t is thus a well known fact that carbon tetrachloride, chlorine, arsenicals, cincophen, phosphorus, and sulphonamides, as well as certain metals, such as lead, copper, and mangan may cause necrosis and fibrosis of the liver.

Among physical effects which have a cirrhogenic property may be mentioned total body X-irradiation (64).

A special position is occupied by the finding of liver fibrosis in diseases related to storage of macromolecules, such as Gaucher’s disease, xanthomatosis and glycogen disease. In this case the size of the molecule and certain physicochemical properties play a signif- icant role in the induction of the cirrhosis. It was thus possible to produce fibrosis of the liver by intravenous injection of silica particles one to three microns in diameter, while particles with a diameter of six to twelve microns caused no liver changes. On the other hand, aluminum oxide particles one to three microns in dia-

. meter caused appearance of giant cells and lymphocytic infiltration of the liver but not progressive fibrosis (19). Results of investigations with different types of methyl cellulose also showed a close relation- ship between molecular size and biological effect. Intravenous in- jection of any kind of methyl cellulose gave rise to foam cell trans- formation of Kupffer cells and periportal histiocytes and atrophy

of liver cells. However, only methyl cellulose of an average molecular weight of 77,700 was stored in the liver cells themselves and caused derangement of the liver functions (39).

TOXIC EFFECTS O F BILE AND BILE CONSTITUENTS

It is generally regarded as a known fact that bile given in suffi- cient quantities by the oral, subcutaneous, intravenous and intrape- ritoneal routes is toxic (22,38). However, practically all investigations dealing with the toxicity of bile have been done as short-term experi- ments, using fairly large quantities of bile only once or a t the most during a few days. The purpose of these earlier investigations was mostly to study the effects upon the cardiovascular or neuromuscular system and the effect on motility and secretion of the stomach in order to obtain an explanation for the clinical symptoms observed in diseases accompanied by jaundice. Because of the complex com- position of whole bile (Table I) many of the investigations were also done with pure bile constituents in order to find out which constituent was responsible for the effect in question.

TABLE I

COMPOSITION OF GALL BLADDER BILE* Base . . .................................. 280-300 Bicarbonate ............................. 8-1 2

Bile acids: conjugated .................... 6.16-8.32 .................... cholic acid 3.33-4.25

desoxycholic acid 4.3 3-5.49 total bile acids . . . . . . . . . . . . . . . . 7.669.75

. . . . . . . . . . . . . .

free bile acids . . . . . . . . . . . . . . . . . 1.5-1.42 Bile sal ts . ............................... 150-210

Bilirubin ................................ 80-1 35 Calcium. ................................ 25-28 Cholesterol .............................. 1 6 e 2 6 0 Chloride. ................................ 16-19 Fatty acids. ............................. 0.08-1.6 Neutral fat and phosphatide .............. up to I

.................................. Mucin '-4

rnEq/L

per cent _ _

7 7

_ - > ?

,, _ _ - _ ,, _ _ rni'q/L mg/Ioc ml mEq/I, mg/Ioo ml

nig/Ioo ml per cent

mEq/L

_ _ > >

The well known bradycardic effect of bile was thus found to be equally strong in cholic acid, while cholesterol and the pigments were ineffective in this respect. For the achievement of this effect no conjugation of the bile salts was required. The deleterious effect of the bile salts consists in part of this depression of nervous activity and in part of

* Cf. also Normal Values in Clinical Medicine. W. B. Saunders Company, Phila- cytolysis (53, 54).

delphia, 1950.

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In acute toxicity experiments it was shown that intraperitoned administration of bile gave rise to peritonitis, vomiting, diarrhea, albuminuria, bradycardia, hypotonia and coma (38). Systematic investigations then showed that sterilization and freezing of the bile did not abolish the effect. An alcohol extract of bile gave rise only to weak toxic symptoms, while a water extract had an equally strong effect as whole bile. .4fter dialysis of bile through celloidin membranes the nondialyzable portion was found to be nontoxic. The above mentioned toxic effects could then be quantitatively repeated with sodium cholate and sodium glycocholate, properly solubilized (38).

Bile salts were also shown to be very general tissue poisons, causing ulceration and hemorrhages in the stomach when injected intraperitoneally or subcutaneously, and necrosis of the salivary glands when injected locally (59). Oral ingestion of gall bladder bile caused a temporary inhibition of gastric motility and decreased gastric secretion (61).

While thus acute toxic manifestations and the effects upon the cardiovascular systex can be duplicated for the greater part by the use of unconjugated simple bile acids, the choleretic effect of bile cannot be completely simulated by simple, synthetic bile salts. The latter seem to cause only an increase of the volume of secreted bile, without parallel1 increase in the amounts of bile constituents. Conjugation of the bile salts or the bile acids with taurine or glycine seems to be necessary for achievement of a true choleretic effect resulting in an increase also in the amounts of secreted bile constit- uents ( 2 2 ) . The effects of simple constituents of bile cannot therefore be directly compared with the effect of whole bile. Only experiments with whole bile can complete the picture obtained from the investi- gations with pure bile constituents. The present writer has not been able to find any reports on the effects of continued oral admin- istration of whole bile preparations on the parenchymatous organs. Because of its solubilizing properties bile has sometimes been u s d for the solubilization of fat-soluble carcinogens. Thus an ox bile preparation was given as a wetting agent in connection with the peroral administration of methylcholanthrene to mice intermittently for 189 to 215 days. The author does not mention any liver changes. However, because the animals were unwilling to eat the diet the bile had to be intermittently discontinued several times for an average of 12 days a t a time (36). No reliable conclusions as to the effect of the used bile preparation can therefore be drawn from this work. It seems as though the effects on the body of a long-term adminis- tration of whole bile is a practically uninvestigated field.

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