Recent advances in molecular pathology: A review of the effects of alcohol on the liver

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

12, 104-132 (1970)

Recent Advances in Molecular Pathology: A Review of the Effects of Alcohol on the Liver E. A. PORTA, 0. R. KOCH AND W. S. HARTROFT The Research

Institute

of the Hospital Received

July

for

Sick

Children,

Toronto,

Canada

18, 1969

Since the recognition of the frequent association of human chronic alcoholism with fatty liver and cirrhosis in the middle of the nineteenth century (Addison, 1836; HUSS, 1849; Rokitansky, 1849), there has been a constant effort by many investigators to clarify the pathogenesis of these hepatic lesions. It was only natural that researchers in this area had tried to reproduce in animals the spectrum of lesions usually encountered in man. A great deal of experimental work has concentrated in the study of the acute effects of a single dose of ethanol on the liver. By this approach, transient fatty livers and certain ultrastructural alterations were readily induced in rats. From these investigations, a fairly complete picture of the metabolic changes occurring during ethanol oxidation has emerged. Despite these advances, the pathogenesis of the acute morphologic hepatic changes is still unresolved. For those investigators more interested in the multifaceted type of lesion associated with the chronic consumption of large amounts of alcohol, the experimental work was plagued with difficulties and failures. When rats were offered alcohol for prolonged periods in the drinking water, separately from the solid food, they consumed relatively low amounts of alcohol and developed only fatty livers and some hepatofibrosis if the final diets (alcohol + food) were grossly inadequate (Best et al., 1949). This situation generated confusion and controversy, particularly in regard as to whether or not alcohol acted as a hepatotoxic independently of dietary factors. Fortunately, in the last decade most of the difficulties in the chronic models of alcoholism in rats have been successfully solved by two newly devised methods of administering the alcohol and by more carefully controlled nutritional approaches (Lieber et al., 1963; Ports and Gomez-Dumm, 1966, 1968). The reproduction of all the hepatic lesions of the human alcoholic, as well as their prevention and regression by simple dietary means, have been recently achieved in rats. As a result the interrelations of alcohol and food have been greatly clarified (Gomez-Dumm and Porta, 1966; Gomez-Dumm et al., 1968; Hartroft and Porta, 1966, 1967, 1968; Hartroft et al., 1966, 1969; Jabbari and Leevy, 1967; Jones and Greene, 1966; Koch et al., 1968; Lieber and De Carli, 1966; Porta, 1969; Porta and Gomez-Dumm, 1966, 1968; Porta and Hartroft, 1965a; Porta et al., 1965a, 1966, 1967, 1968, 1969a, b, c). While controversial views are likely to persist for awhile yet in the field of acute and chronic alcoholism, it is our intention to review here experimental 104

EFFECTS

data that may hopefully problem.

OF

ALCOHOL

ON

THE

help in the understanding ACUTE

LIVER

of this important

105 medical

ALCOHOLISM

There is little doubt that the transient morphologic changes induced in the livers of fasted rats by the oral administration of a single large dose of ethanol result from the metabolic changes occurring during ethanol oxidation in this organ. A change in the redox potentials of the liver to a more reduced state is probably responsible for most of the effects of ethanol on normal hepatic metabolism (Field et al., 1963; Forsander, 1967; Forsander et al., 1958; Isselbather and Krane, 1961; Lieber and Schmid, 1961; Lundquist et al., 1959; Rosenfeld, 1960). Similar events appear to occur when alcohol is dresented to the liver in situ or in vitro by infusions or perfusions of short duration and even when liver slices are incubated in the presence of ethanol (Forsander et al., 1965; Lieber and S&mid, 1961; Lundsgaard, 1938; Majchrowicz and Quastel, 1961). It can be safely concluded that under certain acute experimental conditions ethanol or its metabolites exert an effect on the metabolism of hepatic tissue to induce morphologic changes. However, in evaluating the nosologic implications of results derived from acute experiments and before extrapolating their relevance to the problem of human alcoholism, several factors should be considered. First of all, the rate of absorption of alcohol in the gastrointestinal tract is significantly more rapid and leads to greater elevations in levels of blood-alcohol, than when the same amount is taken when food is present in the stomach., Fasting per se, if moderately prolonged, can markedly decrease the rate of alcohol oxidation by the intact rat as well as the ability of liver homogenates to oxidize alcohol in vitro (Smith and Newman, 1959; Vitale et al., 1953). Furthermore, fasting increases the mobilization of fatty acids from fat depots to the liver and decreases hepatic glycogen depots. This simply: means that the effects of alcohol oxidation on the function and morphology of the liver depends in part at least on the metabolic phase of this biochemical and organ. In this regard, several of the acute alcohol-induced morphologic events of fasted rats have been found to differ in fed rats with normal livers and in rats with fatty or cirrhotic livers (Forsander et al., 1965; Salaspuro and Maenpiiii, 1966). The manner of alcohol administration such as route, dosage,. concentration, as well as the time of sampling, may also influence the results. Congeners present in commercial beverages do not modify the severity ofthe acute ethanol fatty liver of rats (Di Luzio, 1962). At any rate, most of the data presently available on the hepatic changes in acute alcoholism have been derived from experiments in which male rats, relatively young and previously maintained on a complete and balanced diet, received by oral intubation a single large dose of ethanol (usually at the dosage of 6 gm/kg body weight and as a 40-50s solution), after short periods of fasting (8-16 hours). Is this situation frequently encountered in man? And, ‘if so, how serious would any acute hepatic changes be and what relation might they have with the chronic ones ? After all considerations on species’ differences, and particularly those related to rate of alcohol oxidation, this case

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would be the hypothetical one of a fasted nonalcoholic man of 70 kg consuming within only a few minutes about 200 gm of alcohol (i.e., half bottle of whiskey or even more). We know that he will be seriously inebriated if not comatose, and, if his liver reacts like that of rata, it will develop transient and mild fatty changes as well as other reversible ultrastructural ones, But obviously, this is not the usual way in which alcoholic beverages are consumed by individuals who develop chronic liver diseases. Despite these differences, the acute experimental model still provides very valuable information for the understanding of the whole problem of alcoholconditioned hepatic diseases. We will describe here the morphologic events occurring in the liver of rats treated with a high dose of ethanol, and we will discuss the information related to their possible pathogenesis. It should be kept in mind, however, that the amount of alcohol employed by almost all investigators of this acute model is not far from the lethal dose. Fatty changes Male rats fasted for 8 hours promptly accumulate abnormal amounts of visible fat when treated with a single oral dose of ethanol (6 gm/kg body weight). The early changes are not clearly discerned in paraffin-embedded sections stained with hematoxylin-eosin but can be readily visualized in frozen sections stained with Oil Red 0 (Wilson, 1950), or in epon-embedded sections stained with toluidine blue, or with a variety of other dyes (Porta et al., 1966; Minaker and Porta, 1967). Within 1 hour a few small droplets of fat about equal in size to that of nucleoli appear in hepatocytes predominantly located in periportal zones of the liver lobules. These tiny hepatocytic droplets are usually located just beneath the cell border nearest the sinusoids. At this early stage, only a few small droplets of fat accumulate in the cytoplasm of Kupffer cells diffusely distributed throughout the lobule. Although these fatty changes appear to engorge the cytoplasm of some Kupffer cells, they do not alter significantly hepatocytic size. Two and 4 hours after alcohol administration, the hepatocytic fat droplets are slightly more numerous than before and more strikingly periportal. More numerous and larger hepatocytic fat droplets are observed at 8 and 16 hours after treatment than earlier. These progressive changes in hepatocytes are not paralleled by similar ones in the Kupffer cells in which the minimal changes found at the earliest stage do not progress and often even decrease. At any of the above-mentioned periods, the nuclei of hepatocytes are displaced by the fat droplets which on the other hand almost never become as big as the nuclei themselves. The size of these cells and that of sinusoids are not appreciably altered. Although we have observed few histologic sections at later stages, it was evident that at 24 hours the fatty changes had markedly diminished and practically had disappeared by 36 hours. The progression and regression of fatty changes can also be evaluated by biochemical determinations of hepatic triglyceride levels as attested by

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107

several reports (Butler et al., 1959; Di Luzio, 1958; Mallov and Bloch, 1956; Porta et al., 1965a; Reboucas and Isselbacher, 1961; Wolles, 1966). In our experience, 6 gm/kg of ethanol doubles hepatic triglycerides levels at 4 hours, quadruples them at 8. By ‘16 hours, triglyceride content is normal again. Three gm/kg of ethanol also doubles hepatic triglycerides at 4 hours, but levels have reverted to normal at 8 (Porta et al., 196533). At the ultrastructural levels (Figs. l”@rd 21, the fat droplets observed periportally by light microscopy within one ‘hour usually appear in the form of solid homogeneous and moderately electron-dense spherical masses rarely exceeding 4~ in diameter. Some droplets however, may display the form of hollow rims. The solid forms do not differ from the droplets occasionally found in livers of control rate treated with isocaloric amounts of sucrose or glucose or from untreated rats fasted for short periods. Although few droplets of fat are observed in midzonal hepatocytes, they are rarely encountered in those located centrolobularly at early or late stages. The solid or hollow droplets lack definite limiting membranes, although on occasions membranes of adjacent cytoplasmic structures incompletely follow their contour (“borrowed membranes”). Little qualitative variation in these droplets can be appreciated by electron microscopy at 2, 4, and 8 hours after the administration of alcohol. But at later stages the contour of the droplets

FIG. 1. Solid droplets of fat in hepatocyte of a rat 4 hours after the administration of a single dose of ethanol (6 gm/kg). No definite limiting membranes surround these droplets. Lead stain, x25,000.

108

FIG. tration.

PORTA,

KOCH,

2. Hollow droplet of fat encountered Lead stain, ~40,000.

AND

HARTROFT

in hepatocyte

of a rat 4 hours

after

ethanol

adminis-

usually appears more irregular, and sometimes small vacuoles are found at their peripheries which suggest some sort of resorptive process. in similarly treated rats, other investigators found that at early stages (1 and 3 hours) the contour of the droplets was more irregular (Stein and Stein, 1965). In addition, they have described in these and subsequent periods up to 16 hours the presence of very small (30 to 100 rnp) osmiophilic droplets in the Disse space, in the cisternae of the smooth endoplasmic reticulum (SER), and in the Golgi apparatus, that in their opinion represented lipoproteins synthesized and secreted by the liver. Although in our material similar tiny droplets were abserved in these locations, we were unable to detect any appreciable quantitative or qualitative difference with those also found in the livers of control rats.

Mitochondrial

changes

Changes in these organelles are difficult to evaluate if certain precautions are not taken, for they occurred almost exclusively in periportal hepatocytes (Hartroft and Porta, 1964; Porta and Hartroft, 1965b). The simple subjective observation of periportal mitochondria suggests that they progressively increase in size after the administration of alcohol as compared with those of control rats treated with sucrose or glucose. As early as two hours after alcohol, one is given the impression that this enlargement has taken place and becomes even more evident after 4 hours when a tendency to spherulation

EFFECTS

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is also noted. However, it appears that they start reverting to normal after 16 hours. The enlargement, although associated with spherulation is not accompanied by any appreciable diminution in matrix density associated with simple mitochondrial swelling. Some of the enlarged mitochondria and particularly those located in peripheral cytoplasmic regions facing intercellular borders appear closely packed even to the point of coalescence with dissolution of their limiting membranes (Fig. 3). Giant mitochondria of ghostly appearance are found in these areas and appear to be formed by the coalescence of two or more enlarged mitochondria. Most of the enlarged mitochondria have obvious defects in their limiting membranes which are usually markedly attenuated or even absent, apparently presaging the dissolution of some of these organelles. Despite these alterations the number of mitochondria appears unchanged after administration of alcohol. While mitochondrial budding is the only alcohol-induced change reported by Stein and Stein (19651, in these organelles 16 hours after the administration of a single dose of ethanol alone, Ashworth et al. (196513) did not observe any difference between hepatocytic mitochondria of rata treated with a mixture of alcohol and corn oil and those treated with glucose and corn oil or even with those of rats fasted for 22 hours. However, the detection of abnormalities in mitochondria and the possible significance of the variations that may occur in their population, size, and shape between members of differently treated groups cannot be properly ascertained by the simple subjective inspection of no matter how

FIG. 3. Aspects of the mitochondria Lead stain, x25,ooO.

in hepatocytes

of a rat 4 hours

after

ethanol

administration.

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PORTA,

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AND

Average

HARTROFT

Volume

ps

3

0 Glucose Control

4

8

16

Hours

FIG. 4. Average ethanol

(6 gm/kg)

volumes and their

of hepatocytic controls. (From

mitochondria in rata treated Porta et al., 1969c). Average

100

Glucose Control

with

a single

oral

dose of

Number p3 Cytoplaslm

4

g

16

Hours

FIG. 5. Average treated

with

a single

number of hepatocytic mitochondria per standard cytoplasmic volume in rata oral dose of ethanol (6 gm/kg) and in their controls. (From Porta et al., 1969c).

many electron micrographs. Presently, there are methods for the quantitative analyses of the morphologic variations of cellular components at the ultrastructural levels based on the actual measurement and volumetric determinations of the organelles in question that permit accurate evaluations (Porta et al., 1960; Loud. 1968).

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When these quantitative methods were recently applied to the particular problem of acute alcohol-induced mitochondrial variations (Porta et al, 1969c), it was found that at 4 and 8 hours after treatment, the average volume of these organelles had increased when compared with the values obtained in the controls. By the 16th hour, however, a regression was noted (Fig. 4). At any rate the statistical analyses showed that the differences between values of alcoholtreated rats at the different periods or between those of alcohol and isocaloric sucrose groups were not significant. The determinations of the average number of mitochondria per 100~” (standard volume) of cytoplasm indicated that alcohol had induced a relative decrease in their number with time, but again the differences were not statistically significant (Fig. 5). At any rate it was important to note that the total volume occupied by mitochondria per standard volume of cytoplasm (the so-called fractional volume) in the alcohol-treated rats was transiently but significantly enlarged at 4 hours 0,
reticulum

The normal organization of RER in periportal hepatocytes is also altered by the administration of a single dose of alcohol. These changes are already mani100

Gluc.ose

Volume p3 y3 Cytoplasm

4

8

16

Hours

FIG. 6. Fractional volume of hepatocytic cytoplasm occupied by mitochondria of rata treated with a single oral dose of ethanol (6 gm/kg) and their controls. (From Porta et al., 1969c).

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Axial

Glucose Control

HARTROFT

Ratio

4

8

16

Hours

FIG. 7. Axial ratios (6 gm/kg) and in their

of hepatocytic mitochondria of rats treated controls. (From Porta et al., 1969c). Mitochondrial

Envelope

Volume

aCristae

with

a single

oral dose of ethanol

%

ZMatrix

100

a0

60

40

20

0 Glucose Control

,t

8

16

Hours

with

FIG. 8. Volumetric proportions a single oral dose of ethanol

of mitochondria envelopes, cristae, and matrices of rats treated (6 gmikg) and in their controls. (From Porta et al., 1969c).

fested 2 hours after treatment, but at this time they are usually of minimal degree and consist in only slight diminution of the number of parallel lamellae with obvious dilatation of the cisternae, particularly evident at cell peripheries. It can be also observed that the cisternae of RER are fragmented and form mul-

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tiple small vesicules. This vesiculation is frequently accompanied by an apparent detachment of the. ribosomes which appear now free and dispersed in the cytoplasmic ground substance. Four hours after alcohol administration, the vesiculization of RER is more conspicuous. However, organized polysome aggregates are still frequently observed in the micrographs. The changes do not apparently progress much further for at 8 hours and 16 hours less vesiculation and more numerous parallel lamellar arrays are found, although the lamellae show a tendency to surround mitochondria. We have not yet evaluated these complex changes by quantitative analyses but plan to do so. The SER of similarly located hepatocytes does not suffer visible modifications after alcohol administration. Stein and Stein (1965) mentioned only the accumulation of small droplets of fat in the cistema of SER but no other changes. It was always our impression that no proliferation of this reticulum occurs in this acute model. It would be rather surprising for those who consider alcohol a direct hepatotoxic drug, particularly since it is well known that the administration of a variety of hepatotoxic and nonhepatotoxic agents induce, even after a single dose administration, a marked proliferation of SER cistemae (Conney, 1967; Meldolesi, 1967). However, our results on the quantitative analyses of the surface densities of SER per cubic unit of cytoplasm (Fig. 9) showed that in this acute model the alcohol, instead of inducing an increase of this component, had on the contrary resulted in a transient but statistically significant decrease at 8 hours (Porta et al., 1969c). These changes, however, are not apparently associated with any decrease in the amount of cytochrome P-450 as is the case in CCL intoxication (Sasame et al., 1968). SER p2 p3Cytoplasm

*r

0 Glucose Control

4

8

16

Hours

FIG. in their

9. Density of hepatocytic SER in rats treated controls. (From Porta et al., 19691~).

with

a sin@e

dose of ethanol

(6 gm/kg)

and

114 Other ultrastructural

PORTA,

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AND

HARTROFT

changes

Acute alcohol administration greatly affects the content of glycogen granules normally present in hepatocytes of rats which have not been fasted for more than 8 hours. Although we have not quantitated the changes, it was clearly evident that the granules were scanty after only 2 hours and were almost completely absent at 4, 8, and 16 hours. Changes in the Golgi apparatus are not observed after 2 hours of alcohol administration, but at 4 hours its vesicles and vacuoles appear more numerous than in control rats and contain spherical osmiophilic masses and sometimes also have more amorphous but homogeneous material of variable electron-density. These changes persist after 8 and 16 hours, but at this later stage the elements of the Golgi apparatus are frequently intermingled with lysosomes or autophagic vacuoles. The latter organelles, as well as the microbodies and nuclei of hepatocytes, appear not generally affected by the acute administration of alcohol, but more precise quantitative data are necessary here. There is no evidence that the acute administration of alcohol modifies the ultrastructural configuration of the sinusoidal, intercellular, or biliary aspects of the hepatocytic plasma membrane. On the other hand, little is known about the possible changes in other cellular elements of the liver. Although Kupffer cells accumulate some abnormal amounts of fat, the ultrastructure of the cytoplasm appears generally unchanged. Biliary ductal cells and other mesenchyma1 cells are not visibly modified. Comments

These and other studies indicate that the effects of a large single dose of ethanol to fasted rats are manifested in the liver by a transient deposition of abnormal amounts of fat and by other also transient ultrastructural changes. These changes, which occur almost exclusively in the periportal hepatocytes, can be already observed 2 hours after treatment, become increasingly more pronounced during the following few hours, and finally regress. While lower doses mimic to a lesser degree these hepatic changes, higher lethal ones usually killed the rats before the development of morphologic lesions. Because of their transient and zonal characteristics and by all biochemical and histopathological standards considered, the degree of severity of these acutely induced changes is minimal or at the most only moderate. They are certainly not comparable in severity with those induced by the single administration of very much smaller doses of true hepatotoxic agents such as carbon tetrachloride (Recknagel, 1967), phosphorous (Ghoshal et al., 1969), etc. Most of the biochemical changes detected in the liver or blood simply illustrate the metabolic modalities in ethanol oxidation but not significant degrees of cellular injury. Changes in serum activities of enzymes considered to be sensitive indices of liver damage such as glutamic-oxalacetic transaminase (SGOT), glutamic-pyruvic transaminase (SGPT), and lactic dehydrogenase (LDH) are more informative. However, no significant alterations of these enzymatic activities has been found in this acute model (Takeuchi et al., 1968).

EFFECTS

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The changes found in the RER are not apparently associated with any significant biochemical changes indicative of altered protein metabolism. Studies in this area indicated that protein and lipoprotein synthesis were not impaired (Ashworth et al., 1965a; Seakins and Robinson, 1964). Other studies also indicated that the functions of RER as well as that of the SER and possibly the Golgi apparatus, related to the hepatic triglyceride transport or secretory mechanism, were not even transitorily impaired but, on the contrary, were enhanced (Di Luzio, 1966; Zakim et al., 1964). It seems thus probable that at least some of the transient morphologic changes in these membranous components of the ceils can be simple reflections of hyperactivity. Little is known on the functional state of the mitochondria in this acute experimental model. According to Di Luzio (1966), ethanol oxidation induces mitochondrial lipoperoxidation and results in the morphologic changes of these organelles as reported by us (Porta et al., 1966), as well as in the depression of fat oxidation. On the other hand, it is known that ethanol oxidation has a depressing effect on the function of the citric acid cycle (Forsander et al. 1965). In spite of this depression, French (1966) found a transient but significant increase in the concentration of ATP 8 hours after alcohol administration, which may have resulted from the electron transport from cytoplasmic NADH to the mitochondrial cytochrome system. However, an ethanol-induced depression of ATP dephosphorylation could accomplish the same end. At present, we still do not know precisely how mitochondria of acute alcohol-treated rats handle different substrates. The decreased citric acid cycle activity during ethanol oxidation results in the inhibition of gluconeogenesis, and this change may explain the marked reduction of glycogen granules noted in electron micrographs. After the administration of ethanol, liver glycogen is not built up either from glucose (Matunaga, 1942) or from fructose (Field et al., 1963). Although we have not determined biochemically the levels of hepatic glycogen in the acute model in rats, Ammon and Estler (1967) found that 15 minutes after the administration of ethanol to rats, glycogen, glucose, and pyruvate contents of the liver significantly decreased, while glucose-Bphosphatase and lactate did not change. Recent results suggested a depressive action of acute ethanol administration in the reticula endothelial system of the liver (Ali and Nolan, 1967). The fatty changes in the Kupffer cells, as mentioned above, although of minimal degree might offer an explanation for this depression. Although not proved, it appears improbable that the depression resulted from a possible decrease in hepatic blood flow; however, an alteration in the opsonin system necessary for phagocytosis is another possibility. No matter how lenient alI these hepatic changes, they really occur and obviously are the consequence of a metabolic stress upon the liver. However, since alcohol is a highly diffusible substance almost exclusively oxidized in the liver, it is not quite correct to compare its effects with those induced by the administration of isocaloric amounts of carbohydrates, fat, or protein. The latter dietary ingredients cannot create a comparable stress on the liver because they are digested in the gut before absorption, are taken up more slowly by the

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cells, and are to variable extents also metabolized in tissues other than the liver. On the other hand, comparisons with control rats fasted but not treated or receiving just saline are not quite correct either, but, of course, all these controls together help in the interpretation of the results. Taking a closer view now of the transient ethanol-induced fatty changes in the liver, it is intriguing to note that they occur almost exclusively in periportal hepatocytes of the hepatic lobules, or in Zone 1 if Rappaport’s acinar structure is preferred (Rappaport et al., 1954). The hepatic cells situated in this zone are the first to be supplied with blood rich in oxygen, nutrients, and in our case with ethanol. However, the possible different concentrations of ethanol in various parts of the lobules or acinus does not offer a plausible explanation for the preferential hepatocytic changes since at the concentration that ethanol reaches these cells, it is insufficient to induce directly any serious effects before oxidation. Since the fatty changes result from the metabolism of alcohol, the preferential location must depend to a considerable extent on the metabolic peculiarities of the periportal hepatocytes. It is known that a large series of enzymatic activities differs quantitatively in the various parts of the lobules or acini (Novikoff and Essner, 1960; Schumacher, 1957; Shank et al., 1959). Thus it would appear probable that ethanol can be metabolized preferentially in the cells containing more enzymes capable of doing so. Unfortunately, the data reported in this regard are conflictual. While histochemical studies of Greenberger et al. (1965) have indicated a predominance of alcohol dehydrogenase activity in the portal zone of normal rat livers, more recent studies based on quantitative measurements of this enzyme showed a centrolobular predominance (Morrison and Brock, 1967). Although alcohol dehydrogenase may be the rate-limiting enzyme in fed rats consuming ethanol chronically, the DPN/DPNH ratio may be the actual limiting factor in the acute model with fasting (Smith and Newman, 1959). Complicating this situation is the fact that the acinar distribution of hepatocytic substances and enzymes may change under different physiologic conditions and under stress (Eger, 1961). More information on the kinetics of alcohol metabolism in hepatocytes of different lobular locations is obviously needed to understand the preferential location of fatty changes in the acute model of rats. The periportal fatty changes are exclusively due to an increase in triglycerides (Butler et al., 1959, Eger, 1952; Di Luzio, 1958; Lieber et aE., 1960; Mallov and Bloch, 1956; Porta et al., 1965a; Reboucas and Isselbacher, 1961). This accumulation of triglycerides occurs without pronounced alterations in phospholipid concentration or turnover (Di Luzio, 1966), or in the concentration of total or esterified cholesterol (Di Luzio, 1958; Hartroft et al., 1964). Fatty acids in the accumulated triglycerides consist primarily of unsaturated fatty acids (linoleic and oleic) similar to those found in adipose tissue (Brodie et al., 1961; Scheig and Isselbacher, 1965). Several other studies also demonstrated that adipose tissue is the main source of these fatty acids deposited in the liver of fasted rats after acute ethanol administration (Homing et al., 1960; Poggi and Di Luzio, 1964). It was first suggested by Brodie et al. (1961) that ethanol induces an excessive

EFFECTS

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mobilization of peripheral fatty acids to the liver, but the quantitative importance of this possible mechanism in the pathogenesis of fatty liver is still uncertain. While studies of Poggi and Di Luzio (1964) using triglyceride-labeled epididymal fat pads indicated that adipose tissue triglyceride is mobilized at a normal rate during the development of acute ethanol-induced fatty liver, similar studies of Kessler and Yalovsky-Mishkin (1966) suggested an increased rate. The possibility of increased hepatic synthesis of fatty acids in the acute model, as suggested by Lieber and Schmid (1961), is not supported by the above mentioned findings indicating high content of unsaturated fatty acid in livers of rats treated with ethanol. Other results of Kessler and Yalovsky-Mishkin (1966) and more recent ones of Di Luzio (1966) further indicated that increased triglyceride synthesis or fatty acid formation does not contribute to fatty liver development. As already mentioned, the triglyceride secretory mechanism of the liver is not impaired either (Di Luzio, 1966; Seakins and Robinson, 1964). The results of Di Luzio (1966) strongly suggested that an impairment in intrahepatic utilization of triglycerides is the major factor in the development of the acute ethanol-induced fatty liver. Rather similar conclusions were made recently by Fex and Olivecrona (1969) whose results suggested that this type of fatty liver is caused by a decreased hepatic oxidation of plasma free fatty acids taken up at a normal rate. The concept that during ethanol oxidation the breakdown of other substrates is almost totally blocked is not a new one and was elaborated as early as in 1902 by Atwater and Benedict. Years later, Carpenter and Lee (1937) found that the breakdowns of fat and carbohydrates, but not of protein, were the ones most affected by the metabolism of ethanol. The fact that during oxidation the oxygen consumption of the liver was not affected (Fonda1 and Kochakian, 1951; Forsander et al., 1965), along with the observation that as much as three-quarters of the oxygen consumed may be used for the partial oxidation of ethanol (Leloir and Muiioz, 1938; Lundquist et al., 1962), clearly indicated that the oxidation of other substrates must proportionally decrease. It seems now as if we have been walking in circles in the problem of the pathogenesis of the acute ethanol-induced fatty liver. It would also appear possible that all the transient mitochondrial changes found in this acute model may represent simply expressions of physiologic reactions rather than manifestations of any significant injury. However, Di Luzio and collaborators (Di Luzio, 1963, 1964; Di Luzio and Costales, 1965) have advanced the hypothesis that ethanol oxidation induces mitochondrial lipoperoxidation which in turn may be responsible for the functional morphologic changes in these organelles. In favor of this exciting new view is the fact that several natural and synthetic antioxidants used in the acute model by Di Luzio and by ourselves (Porta and Hartroft, 1965b) are capable of preventing the development of fatty liver, as well as the mitochondrial changes, without affecting in any appreciable way the absorption or metabolism of ethanol. Although a recent report of Hashimoto and Rechnagel (1968) appears in conflict with Di Luzio’s hypothesis, the latter has subsequently replied and commented on that objection (Di Luzio, 1968). While at present the possible mechanisms in the pathogenesis of acute eth-

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anol-induced changes are still being pondered and debated, several investigators are studying ways and means to prevent these changes. Antioxidants appear in the first line of defense, but the list of protective agents at the time of this writing includes LYand @ sympathicolytic agents (Brodie et al., 1961), pyridyne carbinol (Ammon and Zeller, 1965), ATP (Hyams and Isselbacher, 1964), L-asparagine (Lansford et al., 1962), 3-5-dymethylpyrazole (Bizzi et al., 1966), ethyl chlorophenoxyisobutyrate (Brown, 1966), antihistamines (Wooles, 1968)) and stimulants of microsomal enzymes such as SKF-525A and phenobarbital (Wooles and Weymouth, 1968; Vincenzi et al., 1967). Choline does not prevent the acute ethanol fatty liver (Di Luzio, 1958; Hartroft et al., 1964). Conversely, it is so far the most effective factor in preventing the fatty changes that may occur in all experimental chronic models of alcoholism. For this and other reasons to be discussed later, we don’t see any relation whatsoever between the acute ethanol-conditioned hepatic lesions and the chronic ones. Even the metabolic characteristics imprinted in the liver of fasted rats by a single dose of ethanol must be perforce different from those occurring at any given time in the livers of animals offered ethanol and food for prolonged periods. CHRONIC

ALCOHOLISM

A century has elapsed since the first reported experiment on chronic alcoholism (Kremiansky, 1868). From then on and up to the present day, the results and interpretations of many investigators have favored at one moment or another the view that alcohol independently of diet exerted a direct toxic effect on the liver or, conversely, that its potentially injurious effect was almost completely mediated through the induction of nutritional deficiencies. For reviewers like us, who fully adhere to the hypothesis of chronic alcoholconditioned nutritional injury of the liver, there could be many ways to recount the history of experimental achievements in this field. But, we will try here to be equanimous and objective in sufficient measure to pay the deserved tribute to all those investigators that have contributed to the understanding of the pathogenesis of this type of hepatic disease and to give the readers a fair interpretation of this situation. Different routes of alcohol administered to a variety of animal species were used by investigators until the third decade of this century as can be appreciated in the quite complete review of Moon (1934) in whose opinion the experimental evidence up to then had not substantiated the belief that alcohol is a direct cause of liver injury. Since that time, the rat became the animal of choice for this type of experiment, and the most common way of giving alcohol has been to incorporate it in the drinking fluid. A few years after Moon’s review, a group of investigators working then in Bethedsa (Daft et al., 1941; Lillie et al., 1941; Lowry et al., 1941) showed that 20% ethanol in the drinking fluid facilitated the development of cirrhosis induced in rats fed a low-protein, low-choline diet. But they also demonstrated that by correcting the dietary deficiencies by the inclusion of choline, methionine, and casein, singly or in combination, in the diets, the livers were pro-

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tected even though the rats still consumed alcohol. These experimental results as well as others from the clinical field on the beneficial effect of diet in alcoholic cirrhosis, reported almost simultaneously by several laboratories (Fleming and Snell, 1942; Patek and Post, 1941), strongly supported the concept, first advanced by Kennedy (1933), that dietary deficiency was the prevailing etiologic factor. However, some years later, Ashworth (1947) based on the results of his experiments, concluded that alcohol could exert a direct toxic effect on the liver of rats irrespective of the adequacy of the diet. But his experimental design was criticized by Best et al. (1949) who felt that the nutritional adequacy of the basal diet used by Ashworth was questionable. Consequently, the Toronto group reinvestigated this problem by experiments in which isocaloric pairfeeding was for the first time used in the study of the effects of alcohol on the liver. The basal diet was designed to provide just those amounts of all essential food factors, vitamins, and protein sufficient to protect the liver when it was consumed without any additional calories in the form of either alcohol or sucrose. It was also designed not to provide any excess of such factors, particularly choline or its dietary precursor methionine, the surplus of which would obviously mask any possible direct effect of alcohol on the liver. The experimental group consumed, in addition to this diet, a 15% solution of ethanol from the drinking fluid. Several control groups received not only the same amounts of food and alcohol but also were placed on regimens in which isocaloric amounts of sucrose replaced alcohol or given other diets with supplements of choline, methionine, or casein. [Although in the original publication it was stated that the group consuming alcohol had derived only 18 % of the total caloric intake from the drinking fluid, recalculations of the data (Hartroft and Porta, 1968) showed that the animals had at least consumed as much as 27% of the total calories in the form of alcohol.] The results of this experiment indicated that the basal diet did not induce any significant hepatic change (biochemically or histochemically) when consumed alone, but it did result in fatty liver and fibrosis when given with alcohol or sucrose supplements. Under those conditions, sucrose was as harmful as alcohol. Furthermore, it was found that neither of these two supplements affected the normal structure and function of the liver when the basal diet was supplemented with additional lipotropes, choline, methionine, or casein sufficient to take care of the extra calories. These results were later confirmed by Forbes and Duncan (1950) and by Klatskin et al. (1954). The latter workers showed that although the lipotropic requirements to overcome the effect of any extra calories provided by alcohol may be somewhat greater than when they are derived from other sources, alcohol did not appear to produce any direct injurious effect on the liver of rats if the lipotropic factors were not limited in any way in the diet. The method of chronic alcohol administration to rats, as it was used in the majority of experiments discussed above, consisted of putting ethanol in the drinking water at concentration between 10 and 20%. At these concentrations the rata consuming separately a nutritionally adequate diet only took approximately 20 to 30% of their total calories from alcohol. Increasing the concentration invariably resulted in significant drops in the levels of total caloric intakes

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and in rates of growth (Best et al., 1949; Forbes and Duncan, 1950; Gillespie and Lucas, 1958; Klatskin et al., 1954; Mallov, 1955; Porta and Gomez-Dumm, 1968; Scheig et al., 1966; Thorpe and Shorey, 1966). At higher strengths, the rats refused to drink as much; consumed actually less alcohol and grew poorly. To overcome these difficulties, Lieber and associates (1963) introduced a new method which consisted of the administration of a whole liquid diet containing 36% of alcohol as part of the mixture. Under this regimen the rats grew well and developed moderately fatty liver changes which were, however, prevented to a considerable degree as the same investigators themselves showed later by the supplementation of choline or methionine (Lieber and De Carli, 1966). In our laboratories in Toronto we have slavishly reproduced the experiments of Lieber and confirmed his results (Porta et al., 1965a). But in addition, we have shown in more prolonged experiments that when in these liquid diets containing even higher amounts of alcohol (40%), the amounts of protein and choline were sufficiently increased, the livers of rats at the end of the fourth month were not fatty. They even contained less fat than those of isocalorically fed control rats whose diets contained sucrose in lieu of alcohol (Porta et al., 1968). More recently we have presented the results of titration experiments designed to provide approximate guides to the protein and lipotropic requirements needed to protect the liver of rats consuming different amounts of alcohol in liquid diets containing various levels of protein and choline (Hartroft et al., 1969). Under these conditions rats can consume up to 30% of their calories in the form of alcohol as long as the rest of the diet provides some 20% of calories in the form of protein and the lipotropic value is somewhere between 75 to 100 (expressed as mg of choline per 100 kcal). At intakes of 40% of calories as alcohol, a level of protein supplying 25% of calories and a correspondingly high level of choline and its precursors prevented hepatic fatty changes although some mitochondrial modifications eventually develop. The protective effect of protein in this model of chronic alcoholism using liquid diets has been also demonstrated by others (Jabbari and Leevy, 1967; Jones and Greene, 1966). In addition, we have shown that the consumption of alcohol (36% of the total caloric intake) in a nutritionally adequate liquid diet permitted the regression of previously produced cirrhosis to the same degree as in comparably cirrhotic rats given the same diets without the alcohol (Takada et al., 1967). All these results indicated to us that Lieber’s original liquid diet was somewhat deficient in a relative sense; a ‘fact that apparently has not yet been accepted by this investigator who in recent publications still considers that alcohol exerts a direct toxic effect on the livers of rats irrespective of the nutritional quality of the diet (Lieber and Rubin, 1968) and despite the evidence cited above. Despite these controversial interpretations, the method using liquid diets permitted considerable advances in the study of chronic alcoholism. The amounts of alcohol ingested by the rats is certainly higher than that achieved when alcohol is merely added to the drinking water. However, it is difficult with the liquid diets to go much above 40 c/Oof calories from alcohol because beyond this level, the total caloric intakes of the animals fall and they fail to maintain their initial body weights, particularly if the amount of protein is not above

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16%. Furthermore, this model contrary to the situation in man, does not permit the rat a choice between alcohol and food. In addition, liquid diets preclude the use of some insoluble food ingredients, are laborious to prepare, and are extremely expensive, quite materially limiting the feasibility of conducting prolonged experiments. Therefore, in our laboratories, we sought other methods that might overcome these disadvantages. It has been known for years that rats have a preference for sweetened water at certain concentrations (Richter and Campbell, 1940). As a result of a series of experiments we found that the simple addition of sucrose to mixtures of alcohol and water to be offered in place of the drinking fluid appealed greatly to the rata (Porta and Gomez-Dumm, 1968). The optimal concentrations of alcohol and sucrose in the drinking water that permitted the highest consumption of alcohol by rata separately fed a stock diet was the combination of 25% sucrose-32% ethanol (weight by volume). This procedure facilitates their natural selection of solid versus fluid consumption, is not expensive, and permits higher alcohol consumption than with previous methods. There is no interference with the growth of the animals if the solid food is not severely unbalanced. This new method of alcohol administration has been employed by us in a large series of experiments in which the solid food offered separately has been modified in different ways to test the importance of various dietary factors in the progression, regression, and prevention of hepatic changes (,Gomez-Dumm et al., 1968; Koch et al., 1968; Porta et al., 1969a, b). The results of all these experiments can be summarized as follows: (1) Rats consuming high amounts of alcohol for periods up to 5-6 months did not develop hepatic fatty changes if the amounts of protein, vitamins, salts, and lipotropes in the final total regimen (alcohol + food) are provided at adequate levels, although mitochondrial changes eventually developed (including the formation of Mallory bodies). (2) All the factors which prevented the fatty changes clearly enhanced the formation of Mallory bodies but without interfering with the normality of liver function. (3) The metabolism of high amounts of alcohol does not require as much of the dietary lipotropic and antinecrogenic factors as does the metabolism of calories derived from isocaloric amounts of carbohydrate or fat. (4) The fatty liver, hepatofibrosis, and even cirrhosis, as seen in alcoholic man, can now be readily duplicated in rats given either higher amounts of alcohol or sucrose if the contents of dietary lipotropes and vitamins are insufficient. But either with alcohol or sucrose all these changes can be completely prevented if the deficiencies are corrected (“super diet”). (5) The recovery of alcohol or sucrose induced-cirrhosis is not interfered in any appreciable way by the persistent consumption of high amounts of alcohol (up to 50% of the total caloric intake) if provided along with such a “super diet.” Our method of high alcohol administration, as any other method in which large amounts of alcohol are offered to the rats such as that using liquid diets with more than 36% of alcohol, presented the investigators with some difhcul-

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ties. A certain percentage of the animals drink only minimal amounts of the sweetened fluid containing the alcohol and also voluntarily reduce their intake of solid food. As a consequence, they may die of inanition shortly after the initiation of the regimen. This situation is obviously not the case for the control animals drinking only water and eating diets with sucrose replacing alcohol. Although this inequality in mortality between these two groups has been used by Lieber et al. (1969) as an example to illustrate the difference between the effects of alcohol and carbohydrate, this is by no means the case and is of no relevance for the evaluation of effects of alcohol on the liver. The animals died not because of the calories consumed in the form of alcohol and food, but because of the lack of them. On the other hand, the animals that may die, succumb without morphologic evidence of liver damage. Anyway, the mortality can be substantially reduced if during a period of adaptation the concentrations of alcohol are gradually increased to their final strengths. This procedure is particularly important if weanling rats are used, and as a matter of fact this period of adaptation was originally proposed by Lieber himself in experiments using liquid diets (Lieber et al., 19631. It is highly improbable that the mortality in these types of experiments can be even due to an early toxic effect on the liver or other organs of the rat of the small amounts of alcohol consumed. However, we have recently explored this possibility (previously unpublished data) in one experiment in which rats were allotted to one of three different groups and offered ad libitum only water, 15p; solution of alcohol, or an isocaloric solution of sucrose. The animals which did not receive any other source of fluid or calories were maintained under these conditions until death. Body weights and fluid consumption were recorded daily. Autopsy was performed in all animals as soon as possible after death. Livers were processed for light microscopy, and their lipids contents were analyzed biochemically. The results showed that rats given water alone lost weight faster than any in the other groups. The life span of rats consuming water alone (7.80 f 1.47 days) was significantly shorter (p
Triglycerides

LIPID

I LEVELS”

Phospholipids

Cholesterol

Groups mg/gm Water Alcohol Sucrose ” Values

expressed

2.43 3.00 3.20

as means

III 0.20 zk 0.28 k 0.18

+ SE.

13.71 14.60 13.60

fresh f i f

liver 0.36 1.04

4.14 2.60

0.46

2.40

f * f

0.14 0.21 0.22

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sucrose alone @
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became elongated but retain at early stages an almost normal appearance. In more advanced stages, however, they display tortuous and branching configurations, frequently encircling portions of cytoplasm, other mitochondria, and sometimes droplets of fat, when because of the inadequacy of the diet these droplets appear in the hepatocytes. More enlarged mitochondria and more bizarre-shaped types eventually develop with time. Some enlarged mitochondria become enormous (larger than nucleoli) and then are readily visible by light microscopy and display all the tinctorial affinities characteristic of human Mallory bodies. The cristae of these bodies are generally short and scanty, but very long ones running parallel to the inner membranes are also encountered. The matrices are usually of normal or slightly increased density, and the number of opaque matrical granules appear sometimes diminished. Some of the bodies contain helical filaments between inner and outer membranes and in dilated aberrant cristae. These filaments appear to play a role in the eventual disintegration of the rat’s Mallory bodies. In Mallory bodies of human alcoholics we have found this type of filament only occasionally, but other types of filaments, the so-called paracrystalline inclusions, are frequently encountered and may play a similar role in the disintegration of these bodies. Although a great number of nutritional factors appear related to the maintenance of the number, size, and shape of mitochondria (Porta, 1969), planimetric determinations of these organelles from rats consuming several diets of different adequacies suggested the following general events: (1) alcohol per se increases the number and size of mitochondria and consequently also the fractional volume of hepatocytic cytoplasm occupied by mitochondria; (2) the provision of vitamins and lipotropes in the amounts usually recommended or higher, particularly in diets also high in protein, appears to prevent partially the increase in their number, but their effect on changes in size is the opposite because many mitochondria become even larger. Our quantitative data on alcohol-induced mitochondrial variations are in general agreement with the results reported by Kiessling and collaborators (Kiessling and Tobe, 1964; Kiessling and Pilstrom, 1966, 1967, 1968). This author has recently found that the oxidative rate of n-glycerophosphate by the mitochondria of rats chronically consuming alcohol was significantly increased (Kiessling, 1968). In view of this result, he concluded that the enlargement could be the result of an adaptive induction of the cu-glycerophosphate oxidase in the mitochondria. This interesting hypothesis would also offer a sensible explanation for the enhancement of the alcohol-induced mitochondrial enlargement by those dietary factors that are well known to protect the liver, such as protein, vitamins, lipotropes, and salts. In this regard, we have also proposed elsewhere the hypothesis that Mallory bodies (megamitochondria) could represent the morphologic expression of a successful adaptive reaction (Porta, 1969). Other investigators think that the chronic ingestion of alcohol may alter the outer mitochondrial membrane causing increased permeability to certain substrates and probably the disintegration of these organelles (French, 1968; French et al., 1969). The significance of this postulated alteration is not clear at present.

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125

LIVER

It has been recently reported that hyperplasia of the hepatocytic SER occurred in experimental chronic alcoholism in dogs (Meldolesi et al., 19651, rats (Iseri et al., 1966), and man (Rubin and Lieber, 1967). These reports are based only on subjective observations and not on objective quantitative analysis of SER variation. We have made such determinations (Porta et al., 1969c) in one model using liquid diets and another using our new method of high alcohol administration. In the experiment with liquid diets (Fig. 10) the SER in hepatocytes of rats from the alcohol group was more abundant than that of controls but only at the second week, because at 4 and 16 weeks the differences between values from both groups were statistically insignificant. The increase in this model was only transient. In the other model in which several control groups were used (Fig. 11) the density of the SER in the alcohol group was not significantly different from that found in the livers of the controls receiving a regimen in which sucrose replaced alcohol or in those in which the replacement was done with sucrose, fat, and protein. In comparison with all the other three groups, the SER was significantly and remarkably increased in the controls given fat. These results indicated to us that alcohol unlike many drugs does not consistently evoke in rats the hyperplasia of SER. The data in addition suggested that dietary factors could be important in the eventual production of hyperplasia of SER. It appears that under certain dietary conditions the chronic consumption of alcohol by rats could be associated with a disruption of the equilibrium between pro-oxidant and antioxidant components of the hepatic cells which results in the development of fatty liver. Although we found that the supplementation of such liquid diets with 15 mg of cu-tocopherol acetate per 100 kcal did not prevent

&&p* ~3 Cytoplosm

I

Su Al 2

Su Al 4 Weeks

SuAl 16

FIG. 10. Density of hepatocytic SER in rata consuming for prolonged ing alcohol or isocaloric amounts of sucrose. (From Porta et al., 1969c).

time

liquid

diets

contain-

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.

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p3 Cytoplasm

Ale

SUC

Fat

FIG. 11. Density of hepatocytic SER in rats consuming diet, and in the controls fed isocalorically solid regimens mixture of sucrose-fat-protein replaced alcohol. (From Porta

Suc-FotProt

for prolonged time alcohol and a solid in which sucrose, or fat, or a balanced et al., 1969c).

the fatty changes (Porta et al., 1965a), more recently Di Luzio and Hartman (1967) have reported that the administration of the antioxidant DPPD did have some effect. Jabbari and Leevy (1967) found that dietary protein as well as synthetic androgenic anabolic steroids in large doses prevented the deposition of hepatic fat in rats consuming certain alcohol liquid diets. These authors have suggested that the effect of anabolic steroids, which in their opinion induced hyperplasia of SER, may be related to their ability to increase synthesis of nuclear RNA and facilitate incorporation of RNA amino acids into RNA protein. In addition, Leevy’s group has reported that rats fed large amounts of alcohol in the presence of a high-protein, vitamin-supplemented diet has increased ethanoloxidizing capacity; while reduced ethanol-oxidizing capacity and progressive hepatic deterioration was noted in protein deficient animals (Cherrick and Leevy, 1965). Final remarks We feel that the general conclusions that can be drawn from all the experimental evidence gathered up to date are as follows: The use of considerable amounts of alcoholic beverages with a proper food intake does not usually cause untoward effects on the liver. Furthermore, the effects of ethanol in higher amounts than can be metabolized on hepatic structure and function depend, in part, upon dietary patterns since a nutritious diet ameliorates and may even largely prevent hepatic damage from gross overconsumption. Unfortunately, the nutritional adequacy of the food usually consumed by

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KIESSLING, K.-H. (1968). Effect of ethanol on rat liver. VI. A possible correlation between aglycerophosphate oxidase activity and mitochondrial size in male and female rats fed ethanol. Acta Pharmacol.

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KIESSLING, K.-H., and PILSTROM, L. (1966). Effect of ethanol, on rat liver. II. Number, size and appearance of mitochondria. Acta Pharmacot. Toxicol. 24,103-111. , KIESSLING, K.-H., and PILSTROM, L. (1967). Effect of ethanol on rat liver. IV. The influence of vitamins, electrolytes and amino acids on the structure and function of mitochondria from rats receiving ethanol. Brit. J. Nutr. 21, 547-556. KIESSLING, K.-H., and PILSTROM, L. (1968). Effect of ethanol on rat liver. V. Morphological and functional changes after prolonged consumption of various alcoholic beverages. Quart. J. Studies Al. 29, 819-827. KIESSLING, K.-H., and TOBE, U. (1964). Degeneration of liver mitochondria in rats after prolonged alcohol consumption. Erptl. Cell Res. 33, 350-354. KLATSKIN, G., KREHL, W. A., and CONN, H. (1954). The effect of alcohol on the choline requirement. I. Changes in the rat’s liver following prolonged ingestion of alcohol. J. Exptl. Med. 100, 695-614. KOCH, 0. R., PORTA, E. A., and HARTROFT, W. S. (1968). A new experimental approach in the study of chronic alcoholism. III. Role of alcohol uersus sucrose or fat derived calories in hepatic damage. Lab. Inuest. 18, 379-386. KREMIANSKY, J. (1868). Ueber bei Pachymeningitis interna Haemorrhagica bei Menschen und Hunden. Arch. Path. Anut. Physiol. 12, 321-328. LANSFORD, E. M. JR., HILL, J. D., and SHIVE, W. (1962). Effects of asparagine and other related nutritional supplements upon alcohol-induced rat liver triglyceride elevation. J. Nutrition 78, 219-222. LELOIR, F., and Mupoz, J. M. (1938). Ethyl alcohol metabolism in animal tissues. Biochem. J. 32, 299-307. LIEBER, C. S., and DE CARLI, L. M. (1966). Study of the agents for the prevention of fatty liver produced by prolonged alcohol intake. Gastroenterology 60, 316-322.

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