- Email: [email protected]
Studies on metabolic tolerance to alcohol, hepatomegaly and alcoholic liver disease
107
METABOLIC
TOLERANCE
TO ETHANOL
Moderator:
Charles S. Lieber
Rapporteurs:
Kai 0. Lindros C. J. Peter Eriksson
Panel Members:
Bjorn Quistorff James M. Khanna
Drug and Alcohol Dependence, 4 (1979) 109 - 118 0 Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
STUDIES MEGALY
aN METAROLIC AND ALCOHOLIC
109
TOLERANCE TO ALCOHOL, LIVER DISEASE
Y. ISRAEL, J. M. KHANNA, H. ORREGO, A. MACDONALD and H. KALANT
G. RACHAMIN,
Departments of Pharmacology und Medicine, University Research Foundation, Toronto, Ontario (Canada)
S. WAHID,
of Toronto
HEPATO-
R. BRITTON,
and Addiction
Summary We previously reported that chronic ethanol administration to Wistar rats results in an increased rate of ethanol metabolism (EMR) and an increased rate of liver oxygen consumption. These increases, expressed per gram of liver, are obtained in conditions in which little or no hepatomegaly occurs. We have confirmed data showing that when hepatomegaly occurs, it is produced by an increased cell volume rather than by an increase in the number of cells. Intracellular water accounts for 60% of the increase in liver weight while a marked reduction in DNA/g of tissue is seen. When the EMR and oxygen consumption following chronic ethanol treatment are expressed per total liver, marked increases are obtained in both parameters, independent of the production of hepatomegaly. In Wistar-derived male SH rats chronic ethanol treatment produces increases in EMR of the order of 100%. In young naive males of this strain, EMR and liver alcohol dehydrogenase (ADH) are high, but both are markedly reduced at the time of sexual maturity (6 - 10 weeks of age). In male SH rats chronic ethanol administration results in higher EMR and ADH which increase in a parallel fashion. Castration prevents the drop in EMR and ADH in naive animals fed chow or sucrose diet and abolishes the increase in EMR and ADH produced by chronic alcohol administration. It is suggested that, in mature male SH rats, ADH under control of testosterone constitutes the primary rate-limiting step in ethanol metabolism, and that chronic ethanol administration acts as a chemical castration. These studies indicate that, at least in laboratory animals, the mechanisms of metabolic tolerance can vary, and are genetically determined. This may explain some discrepancies found in the literature. We have previously shown that propylthiouracil (PTU) administration reduces liver oxygen consumption and protects animals fed alcohol chronically against hypoxic liver damage. A recently concluded double-blind clinical study in three Toronto hospitals indicates that (PTU) administration (300 mg/day) doubles (p < 0.005)
110
the rate of recovery of alcoholics with active liver disease (hepatocellular necrosis), as measured by a battery of tests.
Alcohol
metabolism
and hepatomegaly
We have previously reported that chronic ethanol administration to Wistar rats results in an increased rate of ethanol metabolism (EMR) and in an increased rate of liver oxygen consumption. These increases, expressed per gram of liver, were obtained in conditions in which little or no hepatomegaly occurred [l - 31. Hepatomegaly normally occurs following the administration of 35% of the total caloric intake as alcohol, in diets containing 19% of the calories as proteins, 41% as lipids and the remainder as carbohydrate [4]. Under these circumstances the expression of ethanol metabolism and oxygen consumption per unit tissue weight might not be correct. Baraona et al. [5] have reported that when hepatomegaly occurs during chronic alcohol treatment, the increase in liver weight is due to an increase in hepatocyte volume rather than in hepatocyte number, and that both DNA and mitochondrial mass remain constant. It is clear, therefore, that in order to correlate ethanol metabolism with liver oxygen consumption, the results should most appropriately be expressed per total liver mass or per kg of body weight. Table 1 shows data from experiments in which ethanol (35% calories) was given in liquid diets for four weeks to male Wistar rats. Proteins and fat comprised 19 and 41% of the total calories, respectively, and the remainder was carbohydrate [4] . Control animals were pair-fed with diets in which alcohol was replaced isocalorically by sucrose. In this paradigm, oxygen consumption per unit weight of liver was either not significantly increased or was only marginally increased, and ethanol metabolism expressed in the same manner was not altered. However, both these activities were markedly increased in all experiments, even with animals of different batches obtained from different sources, when expressed per kg of body weight. These data, taken in conjunction with those of Baraona et al. [5], suggest that these parameters are indeed increased in individual hepatocytes. In our hands, hepatomegaly has been shown to vary not only with the diet (alcohol diets with high protein and low fat result in minimal or no hepatomegaly [4] ) but also with different batches of animals (unpublished data). This may be related to the fact that Wistar rats are very heterogeneous and that many substrains exist. Due to this problem we have further investigated the problem of alcohol-induced hepatomegaly in a highly inbred rat strain, namely spontaneously hypertensive (SH) rats. This was studied first with male and female SH rats of different ages. As can be observed (Fig. la and b), alcohol-induced hepatomegaly is more marked in young animals and in females than in males. In young SH rats we further studied, in separate experimental series, the relative contribution of intra- and extracellular water
111 TABLE
1
Ethanol metabolic rate (EMR) and oxygen Wistar rats fed alcohol chronically
Body weight (g) (Liver/body ratio)
consumption
X 100
EMR EMR
&moles (pmoles
per min per g liver) per min per kg body
QO,
(nmoles
per min per g liver)
902
@moles
per min per kg body
weight)
weight)
Ethanol was present at a concentration *N.S., not significant.
of 9.7
(QO,)
in perfused
Controls
Chronic
226 4.64
t_ 12 + 0.12
229 6.00
_t 10 i 0.07
livers of male
alcohol
1.32
r 0.04
62.9
t 2.4
1.43 85.7
+ 0.09 + 4.6
2.78
r 0.06
3.41
* 0.14
132.1
t 4.1
204.0
+ 7.2
(N.S.)* (p < 0.001) (28%) (N.S.) (p < 0.01) (36%) (p < 0.02) (22%) (p < 0.001) (54%)
m&I. n = 6 pairs.
b
Fmal Age (wks)
Fig. 1. Hepatomegaly induced by chronic alcohol treatment rats as a fanction of animal age. Animals were administered liquid diets, as described previously [4]. n = 4 - 5 pairs.
in male (a) and female (b) SH ethanol and control high fat
in alcohol-induced hepatomegaly. For this purpose rats were administered intravenously 0.2 ml of [ 3H] inulin (2pCi/lOO g rat, prepared in a 5% (w/v) unlabeled inulin solution) and were sacrificed 10 - 15 minutes after the injection. Inulin is known to be a marker of extracellular (including vascular) space and can therefore be used to determine both extracellular and (by difference) intracellular water in the tissue. We have also determined Na+ and K’ in the liver, percentage dry weight, DNA, protein and lipids. Data (Table 2) indicate that in both sexes alcohol-induced hepatomegaly is characterized by a marked increase in intracellular water but not in extracellular water. About 60% of the increase in liver weight could be accounted for by intra-
2
n = 6 *N.S.,
not significant.
- 7 pairs.
Total liver weight (g) Total intracellular Hz0 (ml/liver) Total extracellular Hz0 (ml/liver) Total dry weight (g solids per liver) Total protein (g/liver) Total DNA (mg/liver) Total lipids (mg/liver) Total Na+ (pmoles/liver) Total K+ (pmoles/liver) Protein (mg/g liver) DNA (mg/g liver) Lipids (mg/g liver) Na+ (pmoles/g liver) K+ (pmoles/g liver)
0.47 0.16 0.23 0.15 0.063 1.84 61.0 9.8 43.7 5.1 0.24 7.2 0.96 2.6
6.13 2.75 1.53 1.85 1.01 15.24 501.6 172.5 437.0 165.2 2.49 82.2 27.55 71.77 * + i ii+ t * + ? it i *
7.88 3.70 1.69 2.49 1.18 16.25 616.6 174.4 593.2 150.4 2.08 78.2 22.27 75.68
0, < 0.001) (JJ < 0.005) (N.S.)* (p < 0.001) (n < 0.01) (N.S.) (p < 0.00002) (p < 0.05) @ < 0.001) (p < 0.02) (p < 0.001) (p < 0.0001) (p < 0.01) (N.S.)
5.06 2.31 1.19 1.56 0.863 13.51 374.8 131.4 365.0 198.5 2.68 74.1 26.04 72.00
* 0.35 _+0.24 i 0.08 ? 0.11 * 0.054 i 0.89 i 37.4 k 10.1 * 25.8 + 12.7 + 0.12 i 3.3 + 0.87 t 1.89
7.14 3.50 1.24 2.40 1.076 12.07 753.6 157.8 514.0 171.1 1.70 105.8 22.1 72.09
i s.e.m.) Control
Males (mean Ethanol f 0.25 _+0.11 * 0.08 t 0.07 _+0.038 + 0.59 + 20.0 i 7.0 i 23.7 + 12.7 2 0.08 ? 1.9 r 0.99 + 2.2
in SH rats
Control
(mean i s.e.m.)
hepatomegaly
Ethanol
Females
Changes in cellular water and solids in alcohol-induced
TABLE
+ f + f t t i + + t ? t i i
0.2 0.18 0.09 0.07 0.05 0.94 32.4 27.9 20.9 6.2 0.11 5.3 3.23 3.60
Cp < 0.01) (p < 0.005) (N.S.) (p < 0.02) (p < 0.06) (N.S.) (N.S.) (N.S.) (p < 0.002) (N.S.) (p < 0.05) (N.S.) (N.S.) (N.S.)
G
CI
113
cellular water. Also in both male and female rats, a marked increase in total liver K’ (an intracellular cation) was found, without a change in the concentration of K+ per unit liver weight. This indicates that the cells retain their ability to regulate their internal milieu. Assuming a constant membrane permeability factor (ion flux per unit area of membrane), a larger membrane surface, associated with a larger cell volume, would require a more vigorous active ion-pumping activity. Alternatively, a reduction in membrane passive permeability factor is required. In agreement with studies by Baraona and associates [5] we observed that total DNA is not changed in alcohol-induced hepatomegaly. Therefore the observed increased intracellularvolume occurs with a constant hepatocyte number and is not due to an increased number of cells. We have also confirmed an increase in total protein content of the hepatomegalic livers and not an increase per gram of liver. This might conceivably be explained by an initial inability to export proteins which would remain intracellularly, followed by an osmotic water entrance into one cell. However, it can be calculated, assuming a molecular weight of 50 000 daltons, that for those proteins the colloidal osmotic pressure cannot account for more than one to two percent of the increase in intracellular water. The calculation for the female animals is as follows: alcohol-induced increase in total liver proteins = 1.076 - 0.863 = 0.213 g alcohol-induced increase in intracellular water = 3.50 - 2.31 = 1.19 ml With an assumed molecular weight of 50 000:0.213 g protein = 4.26 X lOA mmoles protein. Since an isotonic solution contains 0.32 mmoles solute per ml HsO, it follows that the water necessary for equilibration with 4.26 X lop3 mmoles protein = 13.31 ~1 of water, or only 1.18% of the increase in intracellular water of 1.19 ml. The same type of calculation and general results can be applied to data obtained by Baraona et al. [ 51. It should be noted that, while both in males and females the increase in intracellular water plus that of solids (dry weight) could well account for 90 - 98% of the total increase in liver weight, we cannot account for the increase in total solids on the basis of proteins + lipids + DNA + ions. Although we did not measure glycogen, this is not likely to play an important role since its content is known to be actually reduced in the liver of alcoholtreated animals. Thus, a search for a substance of low molecular weight as a possible explanation for both an unaccounted increase in solids and for an increase in intracellular water might be fruitful. Despite our inability to determine the exact driving force leading to the increases in cellular volume, a representation of the final static changes is presented in Fig. 2. Perhaps one of the most interesting observations is that hepatomegaly was found to occur without concomitant changes in total extracellular volume. This implies that one (or more) of the extracellular compartments (interstitial, Disse or vascular-sinusoidal) must be compressed. This could occur if the liver capsule acts to restrict the total expansion of the organ. An increase in pressure might be responsible for fibrogenesis. This is currently under investigation.
114
CONTROL
ECVIICV
CHRONIC
>
ETHANOL
ECV/ICV
Fig. 2. Schematic representation of intracellular and extracellular changes produced in the liver by chronic ethanol administration. In the upper part of the figure the increase in intracellular water, with a corresponding increase in cell size, is depicted. In the lower part, emphasis is made to the fact that the total number of cells in the liver does not change and that intracellular volume (ICV) increases without a concomitant increase in extracellular volume (ECV). The solid frame represents the rigid liver capsule compressing liver parenchyma.
Metabolic
tolerance
to alcohol
in SH rats
While some studies have shown that an increase in the oxidative capacity of the liver, as measured by increased oxygen consumption, may be adequate to explain an increased rate of ethanol oxidation in isolated liver preparations [6, 71 (see also Table l), there are two reports in the literature indicating that changes in the rate of oxygen consumption could not account for the increased rate of ethanol metabolism [8, 91. We have further investigated this problem in the male SH rat. We have previously shown that in the male SH rat, the EMR is greatly reduced as the animal ages [S] . Since these changes coincided with the onset of sexual maturity, the effect of castration was studied. Some male SH rats were castrated before puberty while other animals were sham operated. Castration was found to prevent the reduction in EMR while testosterone administration reversed the effect (Fig. 3a). A study of liver alcohol dehydrogenase activity (ADH) showed a virtually identical pattern (Fig. 3b). In order to determine whether the testosterone-dependent reduction in ethanol metabolism was reversible, male SH rats were castrated after attaining sexual maturity, at a time when both EMR and ADH activity had been markedly reduced. Castration in these animals led to an increase in both EMR and ADH activity (Fig. 4a and b). The concomitant changes in ADH and EMR suggested that in these animals the EMR may be limited by the activity of ADH rather than by the rate of mitochondrial reoxidation of reducing equivalents. Thus, an increase in ethanol metabolism could conceivably be associated with an increase in ADH activity, rather than with an increase in mitochondrial Oa consumption. Some studies
115 600
a
r
b
Age (wks) Fig. 3. Effect of castration at pre-puberty and of chronic testosterone administration on ethanol metabolic rate (a) and ADH activity (b) in male SH rats. Castration was performed at 4.7 weeks of age, and animals were administered testosterone chronically in silastic tubing capsules which were implanted in subcutaneous dorsal pockets at the age of 9.7 weeks. n = 4 - 6 animals per point.
500 a ;‘ f ," 400-
f
3oo-
0 2 1
5
loo-
10
12
14
16
18
iol, 10
I,,
12
I,,
14
16
3
t
CaStratIOn or Sham
Age (wks) Fig. 4. Effect of castration (b) in male SH rats. n = 4
at post-puberty (10.3 per point.
- 6 animals
weeks)
on EMR
(a) and ADH activity
116
in the literature have reported that in rats chronic ethanol consumption leads to an increase in liver ADH activity, but this has not been supported by other investigators [ 1 - 31. Chronic alcohol administration has been reported to reduce circulating testosterone levels both in animals [lo, 111 and in man [ 12 - 151. Therefore it seemed conceivable that alcohol might simulate a “chemical castration” in these animals. Determinations in intoxicated male SH rats fed chronically with alcohol revealed a 68% reduction in testosterone levels as compared to control animals, in line with reported data (controls: 5.58 k 1.27 ng/ml; ethanol: 1.76 2 0.23 ng/ml;p < 0.03). Data in Fig. 5a and b indicate that chronic ethanol consumption did result in an increase in liver ADH activity associated with an increased rate of ethanol metabolism in the same animals in SH strain. Furthermore, such increases did not occur in castrated animals. We had previously indicated, on the basis of preliminary experiments, that ADH activity was not modified by chronic ethanol feeding in this strain. In that particular study [9] ADH was measured only at one time and in older animals. The results reported in this investigation are based on studies comprising a very large number of animals obtained from two different suppliers. Because of these factors, the present results are more representative of a general biological phenomenon.
Alcoholic
liver disease
We have proposed that the increased rate of oxygen consumption existing in the liver after chronic ethanol ingestion, plays a role in the nroduction of alcoholic liver disease [16] . Due to this fact, a state of a relative
0 Ethanol u sucrose
600 ~ a, i? 0 z
400 -
PC 001 (5)
P< 0 00001 (5)
b
L f
0, ZY
500 -
g g ?
400 -
NS (5)
q
Ethanol
0
Sucrose
PL 0001 (5)
I
B I? 0 ii
200 -
f. w n Castrated
Fig. rats SH n =
Sham
Castrated
Sham
IL
5. Effect of chronic ethanol administration in castrated and sham-operated male SH on EMR (a) and ADH activity (b). Four-week-old castrated or sham-operated male rats were administered high fat, ethanol or control liquid diets [4] for 8 weeks. 5 pairs.
117
hypoxia is induced in areas of the liver (periacinar region, zone 3 of Rapnaport [ 171) that are farthest from the points of entry of oxygenated blood [ 161 . Therefore necrosis of liver cells in the periacinar zone in alcoholics might result from an imbalance between the availability of oxygen and the requirements of the hepatocyte. It has been shown that rats chronically treated with ethanol and exposed to reduced oxygen tensions [16], experimentally induced anemia [ 181 and hepatic arterial ligation [ 191 have hepatocellular necrosis that is confined to the periacinar area. Those animals also have increases both in serum aspartate aminotransferase (SGOT) and in serum omithine carbamoyltransferase (SOCT) activities. None of these changes are observed after the same procedures in animals that are fed isocaloric carbohydrate instead of alcohol. The hypermetabolic state in the liver induced by ethanol is markedly depressed in surgically thyroidectomized rats [20], and is abolished by administration of propylthiouracil (PTU) [ 161. If in the alcoholic rat thyroid activity is required for the production of liver necrosis during hypoxia, treatment with PTU should constitute an effective treatment for this condition. Indeed, treatment of chronically alcohol-fed rats with PTU for ten days before exposure to reduced oxygen tensions completely prevented the histological and biochemical changes in the alcohol-treated animals [ 161. For these reasons PTU was tested in humans as a treatment for alcoholic liver disease [ 211. The study was designed as a double-blind randomized, shortterm (45 days maximum) trial. Several parameters, both clinical and biochemical, were assessed at weekly intervals. A composite clinical and laboratory index (CCLI) was devised to score the severity of the disease in terms of those signs, symptoms, and test results known to correlate best with histological diagnosis and clinical prognosis. For each patient a recovery rate index (RRI) was calculated: CCL1 (admission) - CCL1 (stable final value) x 100 RR1 = -days to reach final CCL1 Sixty-one patients with different stages of alcoholic liver disease were given PTU (75 mg, 4 times a day); 64 patients received placebo in identical capsules. Biopsied patients (85) included 19 with fatty liver, 8 with mild non-specific inflammation, 11 with severe alcoholic hepatitis, 26 with cirrhosis and hepatitis, and 21 with inactive cirrhosis. In 40 patients biopsy was contraindicated because of abnormal coagulation. The preliminary results were encouraging (see Table 3) in terms of both a greater degree of improvement and a shorter time to reach maximum recovery in both clinical and laboratory findings. The effect of PTU treatment was especially significant in patients with alcoholic hepatitis and in patients with both cirrhosis and hepatitis. PTU therefore appears to be an effective form of treatment in patients with active disease, most likely by prevention of necrosis and the secondary inflammatory response. It is not yet possible to say whether PTU therapy alters the long-term outcome of alcoholic hepatitis. This can be determined only by a long-term
118 TABLE Results
3 of treatment
with PTU of patients
with alcoholic
liver disease
Recovery
rate index
Placebo Alcoholic hepatitis Cirrhosis with hepatitis Severely ill* Abnormal prothrombin *Patients **Group
time
with CCL1 scores
19.8 19.6 22.5 2.6
2 + f i
3.3** 4.5 4.2 3.1
*
PTU
P
43.6 i 4.6 43.3 f 5.7 41.4 * 3.8 32.9 +_6.9
in the upper half of the CCL1 range.
means * s.e.m.
study in which PTU is given continuously or repeatedly as required by evidence of active hepatocellular disease, and the evolution of the disease is followed over a period of years. Such a study is now being undertaken.
References 1 L. Videla, J. Bernstein and Y. Israel, Biochem. J., 134 (1973) 507. 2 J. M. Khanna and H. Kalant, in M. M. Gross (ed.), Alcohol Intoxication and Withdrawal, Vol. IIIa, Plenum Press, New York, 1977, p. 281. 3 Y. Israel et al., in M. M. Gross (ed.), Alcohol Intoxication and Withdrawal, Vol. IIIa, Plenum Press, New York, 1977, p. 343. J. M. Khanna, H. Kalant and G. Lin, Biochem. Pharmacol., 21 (1972) 2215. E. Baraona et al., J. Clin. Invest., 60 (1977) 546. Y. Israel, L. Videla and J. Bernstein, Fed. Proc., 34 (1975) 2052. R. G. Thurman ef al., in M. M. Gross (ed.), Alcohol Intoxication and Withdrawal, Vol. IIIa, Plenum Press, New York, 1977, p. 237. Clin. Exp. Res., 1 (1977) 27. 8 A. E. Cederbaum et al., Alcoholism: Clin. Exp. Res., 1 (1977) 39. 9 Y. Israel et al., Alcoholism: 10 D. H. Van Thiel et al., Gastroenterology, 69 (1975) 326. 11 T. J. Cicero and T. M. Badger, J. Pharmacol. Exp. Ther., 201 (1977) 427. 12 S. M. Badr and A. Bartke, Steroids, 23 (1974) 921. 13 D. H. Van Thiel, R. Lester and R. J. Sherins, Gastroenterology, 67 (1974) 1188. 14 G. G. Gordon et al., J. Clin. Endocrinol. Metab., 40 (1975) 1018. N. K. Mello and J. Ellingboe, J. Pharmacol. Exp. Ther., 202 (1977) 15 J. H. Mendelson, 676. 16 Y. Israel et al., Proc Nat. Acad. Sci., 72 (1975) 1137. in C. Rouiller (ed.), The Liver, Academic Press, New York, 1963, 17 A. M. Rappaport, p. 265. 18 Y. Israel et al., in M. M. Fisher and J. G. Rankin (eds.), Alcohol and the Liver, Plenum Press, New York, 1977, p. 323. 19 H. Kalant et al., Fed. Proc., 34 (1975) 719. Exp. Ther., 192 (1975) 583. 20 J. Bernstein, L. Videla and Y. Israel, J. Pharmacol. 76 (1979) 105. 21 H. Orrego et al., Gastroenterology,
METABOLIC
TOLERANCE
TO ETHANOL
Moderator:
Charles S. Lieber
Rapporteurs:
Kai 0. Lindros C. J. Peter Eriksson
Panel Members:
Bjorn Quistorff James M. Khanna
Drug and Alcohol Dependence, 4 (1979) 109 - 118 0 Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
STUDIES MEGALY
aN METAROLIC AND ALCOHOLIC
109
TOLERANCE TO ALCOHOL, LIVER DISEASE
Y. ISRAEL, J. M. KHANNA, H. ORREGO, A. MACDONALD and H. KALANT
G. RACHAMIN,
Departments of Pharmacology und Medicine, University Research Foundation, Toronto, Ontario (Canada)
S. WAHID,
of Toronto
HEPATO-
R. BRITTON,
and Addiction
Summary We previously reported that chronic ethanol administration to Wistar rats results in an increased rate of ethanol metabolism (EMR) and an increased rate of liver oxygen consumption. These increases, expressed per gram of liver, are obtained in conditions in which little or no hepatomegaly occurs. We have confirmed data showing that when hepatomegaly occurs, it is produced by an increased cell volume rather than by an increase in the number of cells. Intracellular water accounts for 60% of the increase in liver weight while a marked reduction in DNA/g of tissue is seen. When the EMR and oxygen consumption following chronic ethanol treatment are expressed per total liver, marked increases are obtained in both parameters, independent of the production of hepatomegaly. In Wistar-derived male SH rats chronic ethanol treatment produces increases in EMR of the order of 100%. In young naive males of this strain, EMR and liver alcohol dehydrogenase (ADH) are high, but both are markedly reduced at the time of sexual maturity (6 - 10 weeks of age). In male SH rats chronic ethanol administration results in higher EMR and ADH which increase in a parallel fashion. Castration prevents the drop in EMR and ADH in naive animals fed chow or sucrose diet and abolishes the increase in EMR and ADH produced by chronic alcohol administration. It is suggested that, in mature male SH rats, ADH under control of testosterone constitutes the primary rate-limiting step in ethanol metabolism, and that chronic ethanol administration acts as a chemical castration. These studies indicate that, at least in laboratory animals, the mechanisms of metabolic tolerance can vary, and are genetically determined. This may explain some discrepancies found in the literature. We have previously shown that propylthiouracil (PTU) administration reduces liver oxygen consumption and protects animals fed alcohol chronically against hypoxic liver damage. A recently concluded double-blind clinical study in three Toronto hospitals indicates that (PTU) administration (300 mg/day) doubles (p < 0.005)
110
the rate of recovery of alcoholics with active liver disease (hepatocellular necrosis), as measured by a battery of tests.
Alcohol
metabolism
and hepatomegaly
We have previously reported that chronic ethanol administration to Wistar rats results in an increased rate of ethanol metabolism (EMR) and in an increased rate of liver oxygen consumption. These increases, expressed per gram of liver, were obtained in conditions in which little or no hepatomegaly occurred [l - 31. Hepatomegaly normally occurs following the administration of 35% of the total caloric intake as alcohol, in diets containing 19% of the calories as proteins, 41% as lipids and the remainder as carbohydrate [4]. Under these circumstances the expression of ethanol metabolism and oxygen consumption per unit tissue weight might not be correct. Baraona et al. [5] have reported that when hepatomegaly occurs during chronic alcohol treatment, the increase in liver weight is due to an increase in hepatocyte volume rather than in hepatocyte number, and that both DNA and mitochondrial mass remain constant. It is clear, therefore, that in order to correlate ethanol metabolism with liver oxygen consumption, the results should most appropriately be expressed per total liver mass or per kg of body weight. Table 1 shows data from experiments in which ethanol (35% calories) was given in liquid diets for four weeks to male Wistar rats. Proteins and fat comprised 19 and 41% of the total calories, respectively, and the remainder was carbohydrate [4] . Control animals were pair-fed with diets in which alcohol was replaced isocalorically by sucrose. In this paradigm, oxygen consumption per unit weight of liver was either not significantly increased or was only marginally increased, and ethanol metabolism expressed in the same manner was not altered. However, both these activities were markedly increased in all experiments, even with animals of different batches obtained from different sources, when expressed per kg of body weight. These data, taken in conjunction with those of Baraona et al. [5], suggest that these parameters are indeed increased in individual hepatocytes. In our hands, hepatomegaly has been shown to vary not only with the diet (alcohol diets with high protein and low fat result in minimal or no hepatomegaly [4] ) but also with different batches of animals (unpublished data). This may be related to the fact that Wistar rats are very heterogeneous and that many substrains exist. Due to this problem we have further investigated the problem of alcohol-induced hepatomegaly in a highly inbred rat strain, namely spontaneously hypertensive (SH) rats. This was studied first with male and female SH rats of different ages. As can be observed (Fig. la and b), alcohol-induced hepatomegaly is more marked in young animals and in females than in males. In young SH rats we further studied, in separate experimental series, the relative contribution of intra- and extracellular water
111 TABLE
1
Ethanol metabolic rate (EMR) and oxygen Wistar rats fed alcohol chronically
Body weight (g) (Liver/body ratio)
consumption
X 100
EMR EMR
&moles (pmoles
per min per g liver) per min per kg body
QO,
(nmoles
per min per g liver)
902
@moles
per min per kg body
weight)
weight)
Ethanol was present at a concentration *N.S., not significant.
of 9.7
(QO,)
in perfused
Controls
Chronic
226 4.64
t_ 12 + 0.12
229 6.00
_t 10 i 0.07
livers of male
alcohol
1.32
r 0.04
62.9
t 2.4
1.43 85.7
+ 0.09 + 4.6
2.78
r 0.06
3.41
* 0.14
132.1
t 4.1
204.0
+ 7.2
(N.S.)* (p < 0.001) (28%) (N.S.) (p < 0.01) (36%) (p < 0.02) (22%) (p < 0.001) (54%)
m&I. n = 6 pairs.
b
Fmal Age (wks)
Fig. 1. Hepatomegaly induced by chronic alcohol treatment rats as a fanction of animal age. Animals were administered liquid diets, as described previously [4]. n = 4 - 5 pairs.
in male (a) and female (b) SH ethanol and control high fat
in alcohol-induced hepatomegaly. For this purpose rats were administered intravenously 0.2 ml of [ 3H] inulin (2pCi/lOO g rat, prepared in a 5% (w/v) unlabeled inulin solution) and were sacrificed 10 - 15 minutes after the injection. Inulin is known to be a marker of extracellular (including vascular) space and can therefore be used to determine both extracellular and (by difference) intracellular water in the tissue. We have also determined Na+ and K’ in the liver, percentage dry weight, DNA, protein and lipids. Data (Table 2) indicate that in both sexes alcohol-induced hepatomegaly is characterized by a marked increase in intracellular water but not in extracellular water. About 60% of the increase in liver weight could be accounted for by intra-
2
n = 6 *N.S.,
not significant.
- 7 pairs.
Total liver weight (g) Total intracellular Hz0 (ml/liver) Total extracellular Hz0 (ml/liver) Total dry weight (g solids per liver) Total protein (g/liver) Total DNA (mg/liver) Total lipids (mg/liver) Total Na+ (pmoles/liver) Total K+ (pmoles/liver) Protein (mg/g liver) DNA (mg/g liver) Lipids (mg/g liver) Na+ (pmoles/g liver) K+ (pmoles/g liver)
0.47 0.16 0.23 0.15 0.063 1.84 61.0 9.8 43.7 5.1 0.24 7.2 0.96 2.6
6.13 2.75 1.53 1.85 1.01 15.24 501.6 172.5 437.0 165.2 2.49 82.2 27.55 71.77 * + i ii+ t * + ? it i *
7.88 3.70 1.69 2.49 1.18 16.25 616.6 174.4 593.2 150.4 2.08 78.2 22.27 75.68
0, < 0.001) (JJ < 0.005) (N.S.)* (p < 0.001) (n < 0.01) (N.S.) (p < 0.00002) (p < 0.05) @ < 0.001) (p < 0.02) (p < 0.001) (p < 0.0001) (p < 0.01) (N.S.)
5.06 2.31 1.19 1.56 0.863 13.51 374.8 131.4 365.0 198.5 2.68 74.1 26.04 72.00
* 0.35 _+0.24 i 0.08 ? 0.11 * 0.054 i 0.89 i 37.4 k 10.1 * 25.8 + 12.7 + 0.12 i 3.3 + 0.87 t 1.89
7.14 3.50 1.24 2.40 1.076 12.07 753.6 157.8 514.0 171.1 1.70 105.8 22.1 72.09
i s.e.m.) Control
Males (mean Ethanol f 0.25 _+0.11 * 0.08 t 0.07 _+0.038 + 0.59 + 20.0 i 7.0 i 23.7 + 12.7 2 0.08 ? 1.9 r 0.99 + 2.2
in SH rats
Control
(mean i s.e.m.)
hepatomegaly
Ethanol
Females
Changes in cellular water and solids in alcohol-induced
TABLE
+ f + f t t i + + t ? t i i
0.2 0.18 0.09 0.07 0.05 0.94 32.4 27.9 20.9 6.2 0.11 5.3 3.23 3.60
Cp < 0.01) (p < 0.005) (N.S.) (p < 0.02) (p < 0.06) (N.S.) (N.S.) (N.S.) (p < 0.002) (N.S.) (p < 0.05) (N.S.) (N.S.) (N.S.)
G
CI
113
cellular water. Also in both male and female rats, a marked increase in total liver K’ (an intracellular cation) was found, without a change in the concentration of K+ per unit liver weight. This indicates that the cells retain their ability to regulate their internal milieu. Assuming a constant membrane permeability factor (ion flux per unit area of membrane), a larger membrane surface, associated with a larger cell volume, would require a more vigorous active ion-pumping activity. Alternatively, a reduction in membrane passive permeability factor is required. In agreement with studies by Baraona and associates [5] we observed that total DNA is not changed in alcohol-induced hepatomegaly. Therefore the observed increased intracellularvolume occurs with a constant hepatocyte number and is not due to an increased number of cells. We have also confirmed an increase in total protein content of the hepatomegalic livers and not an increase per gram of liver. This might conceivably be explained by an initial inability to export proteins which would remain intracellularly, followed by an osmotic water entrance into one cell. However, it can be calculated, assuming a molecular weight of 50 000 daltons, that for those proteins the colloidal osmotic pressure cannot account for more than one to two percent of the increase in intracellular water. The calculation for the female animals is as follows: alcohol-induced increase in total liver proteins = 1.076 - 0.863 = 0.213 g alcohol-induced increase in intracellular water = 3.50 - 2.31 = 1.19 ml With an assumed molecular weight of 50 000:0.213 g protein = 4.26 X lOA mmoles protein. Since an isotonic solution contains 0.32 mmoles solute per ml HsO, it follows that the water necessary for equilibration with 4.26 X lop3 mmoles protein = 13.31 ~1 of water, or only 1.18% of the increase in intracellular water of 1.19 ml. The same type of calculation and general results can be applied to data obtained by Baraona et al. [ 51. It should be noted that, while both in males and females the increase in intracellular water plus that of solids (dry weight) could well account for 90 - 98% of the total increase in liver weight, we cannot account for the increase in total solids on the basis of proteins + lipids + DNA + ions. Although we did not measure glycogen, this is not likely to play an important role since its content is known to be actually reduced in the liver of alcoholtreated animals. Thus, a search for a substance of low molecular weight as a possible explanation for both an unaccounted increase in solids and for an increase in intracellular water might be fruitful. Despite our inability to determine the exact driving force leading to the increases in cellular volume, a representation of the final static changes is presented in Fig. 2. Perhaps one of the most interesting observations is that hepatomegaly was found to occur without concomitant changes in total extracellular volume. This implies that one (or more) of the extracellular compartments (interstitial, Disse or vascular-sinusoidal) must be compressed. This could occur if the liver capsule acts to restrict the total expansion of the organ. An increase in pressure might be responsible for fibrogenesis. This is currently under investigation.
114
CONTROL
ECVIICV
CHRONIC
>
ETHANOL
ECV/ICV
Fig. 2. Schematic representation of intracellular and extracellular changes produced in the liver by chronic ethanol administration. In the upper part of the figure the increase in intracellular water, with a corresponding increase in cell size, is depicted. In the lower part, emphasis is made to the fact that the total number of cells in the liver does not change and that intracellular volume (ICV) increases without a concomitant increase in extracellular volume (ECV). The solid frame represents the rigid liver capsule compressing liver parenchyma.
Metabolic
tolerance
to alcohol
in SH rats
While some studies have shown that an increase in the oxidative capacity of the liver, as measured by increased oxygen consumption, may be adequate to explain an increased rate of ethanol oxidation in isolated liver preparations [6, 71 (see also Table l), there are two reports in the literature indicating that changes in the rate of oxygen consumption could not account for the increased rate of ethanol metabolism [8, 91. We have further investigated this problem in the male SH rat. We have previously shown that in the male SH rat, the EMR is greatly reduced as the animal ages [S] . Since these changes coincided with the onset of sexual maturity, the effect of castration was studied. Some male SH rats were castrated before puberty while other animals were sham operated. Castration was found to prevent the reduction in EMR while testosterone administration reversed the effect (Fig. 3a). A study of liver alcohol dehydrogenase activity (ADH) showed a virtually identical pattern (Fig. 3b). In order to determine whether the testosterone-dependent reduction in ethanol metabolism was reversible, male SH rats were castrated after attaining sexual maturity, at a time when both EMR and ADH activity had been markedly reduced. Castration in these animals led to an increase in both EMR and ADH activity (Fig. 4a and b). The concomitant changes in ADH and EMR suggested that in these animals the EMR may be limited by the activity of ADH rather than by the rate of mitochondrial reoxidation of reducing equivalents. Thus, an increase in ethanol metabolism could conceivably be associated with an increase in ADH activity, rather than with an increase in mitochondrial Oa consumption. Some studies
115 600
a
r
b
Age (wks) Fig. 3. Effect of castration at pre-puberty and of chronic testosterone administration on ethanol metabolic rate (a) and ADH activity (b) in male SH rats. Castration was performed at 4.7 weeks of age, and animals were administered testosterone chronically in silastic tubing capsules which were implanted in subcutaneous dorsal pockets at the age of 9.7 weeks. n = 4 - 6 animals per point.
500 a ;‘ f ," 400-
f
3oo-
0 2 1
5
loo-
10
12
14
16
18
iol, 10
I,,
12
I,,
14
16
3
t
CaStratIOn or Sham
Age (wks) Fig. 4. Effect of castration (b) in male SH rats. n = 4
at post-puberty (10.3 per point.
- 6 animals
weeks)
on EMR
(a) and ADH activity
116
in the literature have reported that in rats chronic ethanol consumption leads to an increase in liver ADH activity, but this has not been supported by other investigators [ 1 - 31. Chronic alcohol administration has been reported to reduce circulating testosterone levels both in animals [lo, 111 and in man [ 12 - 151. Therefore it seemed conceivable that alcohol might simulate a “chemical castration” in these animals. Determinations in intoxicated male SH rats fed chronically with alcohol revealed a 68% reduction in testosterone levels as compared to control animals, in line with reported data (controls: 5.58 k 1.27 ng/ml; ethanol: 1.76 2 0.23 ng/ml;p < 0.03). Data in Fig. 5a and b indicate that chronic ethanol consumption did result in an increase in liver ADH activity associated with an increased rate of ethanol metabolism in the same animals in SH strain. Furthermore, such increases did not occur in castrated animals. We had previously indicated, on the basis of preliminary experiments, that ADH activity was not modified by chronic ethanol feeding in this strain. In that particular study [9] ADH was measured only at one time and in older animals. The results reported in this investigation are based on studies comprising a very large number of animals obtained from two different suppliers. Because of these factors, the present results are more representative of a general biological phenomenon.
Alcoholic
liver disease
We have proposed that the increased rate of oxygen consumption existing in the liver after chronic ethanol ingestion, plays a role in the nroduction of alcoholic liver disease [16] . Due to this fact, a state of a relative
0 Ethanol u sucrose
600 ~ a, i? 0 z
400 -
PC 001 (5)
P< 0 00001 (5)
b
L f
0, ZY
500 -
g g ?
400 -
NS (5)
q
Ethanol
0
Sucrose
PL 0001 (5)
I
B I? 0 ii
200 -
f. w n Castrated
Fig. rats SH n =
Sham
Castrated
Sham
IL
5. Effect of chronic ethanol administration in castrated and sham-operated male SH on EMR (a) and ADH activity (b). Four-week-old castrated or sham-operated male rats were administered high fat, ethanol or control liquid diets [4] for 8 weeks. 5 pairs.
117
hypoxia is induced in areas of the liver (periacinar region, zone 3 of Rapnaport [ 171) that are farthest from the points of entry of oxygenated blood [ 161 . Therefore necrosis of liver cells in the periacinar zone in alcoholics might result from an imbalance between the availability of oxygen and the requirements of the hepatocyte. It has been shown that rats chronically treated with ethanol and exposed to reduced oxygen tensions [16], experimentally induced anemia [ 181 and hepatic arterial ligation [ 191 have hepatocellular necrosis that is confined to the periacinar area. Those animals also have increases both in serum aspartate aminotransferase (SGOT) and in serum omithine carbamoyltransferase (SOCT) activities. None of these changes are observed after the same procedures in animals that are fed isocaloric carbohydrate instead of alcohol. The hypermetabolic state in the liver induced by ethanol is markedly depressed in surgically thyroidectomized rats [20], and is abolished by administration of propylthiouracil (PTU) [ 161. If in the alcoholic rat thyroid activity is required for the production of liver necrosis during hypoxia, treatment with PTU should constitute an effective treatment for this condition. Indeed, treatment of chronically alcohol-fed rats with PTU for ten days before exposure to reduced oxygen tensions completely prevented the histological and biochemical changes in the alcohol-treated animals [ 161. For these reasons PTU was tested in humans as a treatment for alcoholic liver disease [ 211. The study was designed as a double-blind randomized, shortterm (45 days maximum) trial. Several parameters, both clinical and biochemical, were assessed at weekly intervals. A composite clinical and laboratory index (CCLI) was devised to score the severity of the disease in terms of those signs, symptoms, and test results known to correlate best with histological diagnosis and clinical prognosis. For each patient a recovery rate index (RRI) was calculated: CCL1 (admission) - CCL1 (stable final value) x 100 RR1 = -days to reach final CCL1 Sixty-one patients with different stages of alcoholic liver disease were given PTU (75 mg, 4 times a day); 64 patients received placebo in identical capsules. Biopsied patients (85) included 19 with fatty liver, 8 with mild non-specific inflammation, 11 with severe alcoholic hepatitis, 26 with cirrhosis and hepatitis, and 21 with inactive cirrhosis. In 40 patients biopsy was contraindicated because of abnormal coagulation. The preliminary results were encouraging (see Table 3) in terms of both a greater degree of improvement and a shorter time to reach maximum recovery in both clinical and laboratory findings. The effect of PTU treatment was especially significant in patients with alcoholic hepatitis and in patients with both cirrhosis and hepatitis. PTU therefore appears to be an effective form of treatment in patients with active disease, most likely by prevention of necrosis and the secondary inflammatory response. It is not yet possible to say whether PTU therapy alters the long-term outcome of alcoholic hepatitis. This can be determined only by a long-term
118 TABLE Results
3 of treatment
with PTU of patients
with alcoholic
liver disease
Recovery
rate index
Placebo Alcoholic hepatitis Cirrhosis with hepatitis Severely ill* Abnormal prothrombin *Patients **Group
time
with CCL1 scores
19.8 19.6 22.5 2.6
2 + f i
3.3** 4.5 4.2 3.1
*
PTU
P
43.6 i 4.6 43.3 f 5.7 41.4 * 3.8 32.9 +_6.9
in the upper half of the CCL1 range.
means * s.e.m.
study in which PTU is given continuously or repeatedly as required by evidence of active hepatocellular disease, and the evolution of the disease is followed over a period of years. Such a study is now being undertaken.
References 1 L. Videla, J. Bernstein and Y. Israel, Biochem. J., 134 (1973) 507. 2 J. M. Khanna and H. Kalant, in M. M. Gross (ed.), Alcohol Intoxication and Withdrawal, Vol. IIIa, Plenum Press, New York, 1977, p. 281. 3 Y. Israel et al., in M. M. Gross (ed.), Alcohol Intoxication and Withdrawal, Vol. IIIa, Plenum Press, New York, 1977, p. 343. J. M. Khanna, H. Kalant and G. Lin, Biochem. Pharmacol., 21 (1972) 2215. E. Baraona et al., J. Clin. Invest., 60 (1977) 546. Y. Israel, L. Videla and J. Bernstein, Fed. Proc., 34 (1975) 2052. R. G. Thurman ef al., in M. M. Gross (ed.), Alcohol Intoxication and Withdrawal, Vol. IIIa, Plenum Press, New York, 1977, p. 237. Clin. Exp. Res., 1 (1977) 27. 8 A. E. Cederbaum et al., Alcoholism: Clin. Exp. Res., 1 (1977) 39. 9 Y. Israel et al., Alcoholism: 10 D. H. Van Thiel et al., Gastroenterology, 69 (1975) 326. 11 T. J. Cicero and T. M. Badger, J. Pharmacol. Exp. Ther., 201 (1977) 427. 12 S. M. Badr and A. Bartke, Steroids, 23 (1974) 921. 13 D. H. Van Thiel, R. Lester and R. J. Sherins, Gastroenterology, 67 (1974) 1188. 14 G. G. Gordon et al., J. Clin. Endocrinol. Metab., 40 (1975) 1018. N. K. Mello and J. Ellingboe, J. Pharmacol. Exp. Ther., 202 (1977) 15 J. H. Mendelson, 676. 16 Y. Israel et al., Proc Nat. Acad. Sci., 72 (1975) 1137. in C. Rouiller (ed.), The Liver, Academic Press, New York, 1963, 17 A. M. Rappaport, p. 265. 18 Y. Israel et al., in M. M. Fisher and J. G. Rankin (eds.), Alcohol and the Liver, Plenum Press, New York, 1977, p. 323. 19 H. Kalant et al., Fed. Proc., 34 (1975) 719. Exp. Ther., 192 (1975) 583. 20 J. Bernstein, L. Videla and Y. Israel, J. Pharmacol. 76 (1979) 105. 21 H. Orrego et al., Gastroenterology,