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Fragility of Liver Mitochondria in Ethanol-Fed Rats
Vol. 54, No.6 Printed in U.S.A.
GASTROENTEROLOGY
Copyright ©)968 by The Williams & Wilkins Co.
FRAGILITY OF LIVER MITOCHONDRIA IN ETHANOL-FED RATS SAMUEL
W.
FRENCH,
M.D.
Department of Pathology, San Francisco General Hospital , University of California School of Medicine, San Francisco, California
Evidence that ethanol may cause cellular injury by altering the properties of cell membranes has been presented in many studies, both in viv0 1-6 and in vitro. 7-1o The vesiculation of the endoplasmic reticulum ll -12 and degeneration of the hepatocellular ergastoplasm13 -14 caused by chronic ethanol ingestion may be a result of membrane damage induced by ethanol. The mitochondrial membranes may be the ones mainly affected, since electron microscopic examination of liverll, 15-16 has revealed many structural changes in these organelles as a result of chronic ethanol ingestion. Since mitochondria can be isolated from the cell in relatively pure subcellular fractions, they can be conveniently studied for functional or structural alterations in their membranes induced by ethanol ingestion. In the present investigation, mitochondrial membrane function and structure were examined. The function of the mitochondrial membrane was tested by employing the succinic dehydrogenase (SD) assay method based on the work of Singer and Lusty 17 in which the rate of the reaction is dependent on the rate of penetration of an electron acceptor through the membrane permeability barrier. The permeabilReceived October 9, 1967. Accepted January 19,1968. Address requests for reprints to: Dr. Samuel W. French, Department of Pathology, U.C.L.A. School of Medicine, Harbor General Hospital, Torrance, California 90509. This investigation was supported by United States Public Health Service Research Grant AM-05243-06 from the National Institute of Arthritis and Metabolic Diseases. The author is indebted to Dr. Daniel Friend for his advice and encouragement, and to Mrs. Barbara French, Mr. Todor Todoroff, and Miss Hazel March for their technical assistance.
ity to phenazine methosulfate used as an electron acceptor in the SD assay is ratelimiting. This permeability barrier can be eliminated by mechanical fragmentation or chemical damage of mitochondriaP Preservation of this mitochondrial barrier to phenazine was used as a measure of mitochondrial membrane integrity and loss of the barrier as an indication of mitochondrial fragility. As an adjunct to SD assay, mitochondrial pellets were examined by electron microscopy to study structural changes before and after assay. Mitochondrial fragility shown in SD assay was correlated with loss of the outer mitochondrial membrane as determined by electron microscopy. This method was in part based on the work of Bachmann et aI.,18 who showed that phospholipase digestion of mitochondria caused both a loss of the outer membrane and an increase in SD activity. Experimental Procedure Animals. In each of the three experiments, young, male rats of the Wistar strain were used. The same dietary regimens were employed in each experiment, the only difference being in the duration of the experiment and the starting weight of animals (table 1). All animals were weighed twice weekly during the treatment period. They were housed individually in screen bottom cages. Fresh diet was given twice weekly. The composition of the basal diet has been described previously.'· Ethanol and sucrose were given in the drinking water by means of Richter tubes to minimize evaporation of ethanol. The basal diet was made into a thick paste with water and was offered in small jars to avoid loss by spilling. Six rats were used in experiment 1, 6 in experiment 2, and 1 2 in experiment 3. The total number of animals entered into a n experiment was divided into three groups, according to
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FRAGILITY OF LIVER MITOCHONDRIA
diet. Each ethanol-fed animal (group 1) was paired with a control animal fed the basal diet plus sucrose in isocaloric amounts (group 2) and also with a control animal fed the basal diet ad libitum (group 3). Table 1 shows the proportion of ethanol, fat, carbohydrate, and protein consumed by the ethanol-fed rats (group 1) in experiments 1, 2, and 3. The average consumption of food and of ethanol and the percentage of dietary calories were determined from the beginning of each experiment until the time of assay. Thus it was known that adequate dietary ingredients were ingested despite a high ethanol intake, and changes observed could be assumed to be due to etllanol and not to dietary deficiency or imbalance. The pair-fed sucrose control rats (group 2) consumed the same amount of diet and the same volume of liquid calories as ethanol-fed rats. Their weight gain was the same as that of group 1 in experiments 1 and 3 but exceeded that of group 1 in experiment 2. Preparation of tissues. Liver tissue for assay of succinic dehydrogenase activity was obtained by biopsy while the animals were under ether anesthesia. About 1 g of the liver specimen was gently homogenized in a hand-operated, glass-Teflon homogenizer using ice-cold 0.25 M sucrose containing 5 mM ethylene-diaminetetraacetate (EDTA) and 5 mM Tris-HCI buffer, pH 7.4. Mitochondrial fractions were prepared in two different ways. In experiments 1 and 2 a "contaminated" fraction containing a mixture of microsomes and mitochondria was prepared by the method of Christophersen.s Nuclei and cell debris were removed by centrifugation at 700 X g twice in a refrigerated centrifuge at 0 C. The contaminated mitochondrial fraction was sedimented at 20,000 X g for 15 min and washed once by resuspension in homogenizing medium and recentrifuged. The fraction was resuspended in the homogenizing medium in a concentration of 0.75 g of fresh liver per m!. The fractions were assayed immediately. In the thi rd experiment, a p" urified" mitochondrial fraction containing a minimum of contaminating microsomcs was prepared using a modification of the method of Parsons et al: o The purified and contaminated fractions were both prepared at the same time. The supernatant fraction obtained from the cellular debris removal step in the preparation of the contaminated mitochondrial fraction was spun for 10 min at 9,000 X g in a Spinco ultracentrifuge. The supernatant fluid was sucked off from the surface so that the floating layer of lipid was removed with the supernatant fluid. The fluffy
T ABLE
1. Diet and ethanol consumption Calories in diet
Experiment DO.
Duration
Starting weight
--- - - - - 1 2 3
CarboEthanol Protein hydrate .--
mo
g
%
2 14 13
80 60 80
33.3 39.6 35.2
- -
%
19.2 18.3 18.7
- -
%
36.0 31.1 34.9
Fat
-%
11.5 11.0 11.2
layer was resuspended in the last few milliliters of supernatant fluid by agitation with a disposable pipette and then was sucked off. The surface of the pellet was then lightly washed three times witll a small amount of medium. The mitochondrial pellet was resuspended and spun again for 10 min at 500 X g and the supernatant fraction was respun for 10 min at 9,000 X g. The fluffy layer was again resuspended and the mitochondrial pellet surface was again washed. The mitochondria were resuspended in the medium in a concentration of 0.75 g of fresh liver per ml and assayed immediately. The purified and contaminated fractions were both prepared at the same time and assayed at the same time. Enzyme assays. The SD activity of the mitochondrial fractions was assayed by the succinate-phenazine methosulfate method of Bernath and Singer.21 The assay was performed in a Gilson differential respirometer at 38 C using 0.3 M Tris buffer, pH 7.6. Parallel assays were done with and without 0.75 mM calcium added to the media. Phenazine methosulfate was added at a fixed concentration of 0.67 mg per m!.'7 The reaction rate was calculated at 10-, 30-, and 60-min intervals. Mitochondrial protein was determined by the method of Lowry and co-workers:2 The results were calculated as Q02 (microliters of O2 per milligram of protein per minute). In the first and second experiments the percentage of maximum activity was calculated by dividing the activity measured with calcium added to the assay media into the activity determined without calcium in the media. In the third experiment the purified and contaminated fractions were assayed for SD activity with no calcium added to t he media and the results were compared. Purified fractions from control animals (groups 2 na d 3) were also assayed in this way. Acid phosphatase activity of the purified and contaminated mitochondrial fractions was measured in order to estimate the contamination of the fractions by lysosomes. Enzyme ac'" tivity was preserved by adding om ml of 20%
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FRENCH
acetic acid per ml of mitochondrial suspension; freeze-thawing was used to achieve maximum activity. Acid phosphatase activity was assayed by the method of Andersch and Szczypinski.'· Electron microscopy. Mitochondrial pellets prepared before and after SD assay were examined by electron microscopy. For this purpose pellets were fixed in 3% buffered glutaraldehyde, pH 7.4, and postfixed in buffered 0s0., pH 7.6. Thin sections were cut from specimens embedded in Araldite; they were examined with an RCA model EMU-3H electron microscope. All purified mitochondrial pellets in experiment 3 were examined in this way not only to assess contamination by microsomes but also to determine whether the outer membrane of the mitochondria was intact after the 60-min assay. Some of the pellets from experiments 1 and 2 were examined for contamination of the fraction with microsomes and for the effect of the SD assay on the mitochondria.
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1. The results of experiment 1 and 2 were the same and, therefore, they were pooled for statistical analysis. The SD activity of the fractions from the ethanol-fed group differed very little from that of the paired controls for the first 10 min of assay; by 60 min of assay, the activity was significantly greater (P < 0.02) than that of the controls. Adding calcium to the assay media is a convenient way of eliminating the mitochondrial membrane barrier to phenazine so that maximum enzyme activity can be measured.11 By comparing the activity with calcium in the media with the activity without calcium in the media, the percentage of maximum activity can be calculated for each group of animals. The percentage of maximum SD activity for the ethanol-fed animals was double that of the control animals at 60 min of assay Results (P < 0.02). Liver mitochondrial contaminated fracElectron microscopy of the contaminated tion. The SD activity of the contaminated mitochondrial pellet disclosed numerous mitochondrial fractions is plotted in figure microsomal vesicles separating the mitochondria (fig. 2). After assay almost all 125 of the mitochondria from ethanol-fed rats • EffI....1 (group 1) had lost their outer membranes, • Sue.... o ..... LiII whereas most of the mitochondria from pair-fed sucrose controls (group 2) still 100 retained their outer membranes. c: :! Mitochondrial pellets taken from the ...! purified fraction (experiment 3) were all Ci 75 ~c: examined by electron microscopy. In the o unincubated fractions, a marked diminu'5o tion of microsomal vesicles resulted in i.. 50 close packing of mitochondria, although E ........ scattered vesicles and glycogen bodies were o ~ invariably present in small numbers (fig. 25 3). The mitochondria retained their outer and inner membranes. The cristae often were dilated so that they appeared spherical instead of tubular; the matrix ap30 60 Tim. in Minut•• peared uniformly dense. After SD assay FIG. 1. SD activity of the contaminated liver for 60 min the mitochondria from the mi tochondrial fraction during t he 6O-min assay ethanol-fed animals (group 1) were almost period. Note the acceleration of activity in the invariably swollen in appearance (fig. 4). ' ethanol-fed animals after 30 min of assay, when Their outer membranes were absent and no calcium was added to the media. Note also that when calcium was added to the media and the cristae were no longer detectable. The maximum enzyme activity was measured exper- matrix did not stain, or contained a few, imental and control values are not significantly densely stained, amorphous clumps. These different. changes have been described previously
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FRAGILITY OF UVER MITOCHONDRIA
in in vitro, large amplitude, swelling experiments. 2o Numberous intervening vesicles, probably derived from disrupted mitochondria, were also seen. The assayed mitochondria from the control animals of groups 2 and 3 showed numerous intact mitochondria (fig. 5), with a few swollen ones interspersed. The intact mitochondria retained their outer membranes and cristae. The matrix stained less densely. Darkly stained amorphous clumps were characteristically present. N umerous small vesicles were seen between mitochondria. The differences in mitochondrial degeneration between the ethanol-fed and the control animals was a matter of degree and correlated qualitatively with the amount of SD activity: the mitochondria from the ethano l-fed animals showed a more advanced degree of degeneration after assay as well as evidence of greater loss of the membrane barrier to phenazine methosulfate. The same pattern of accelerated activity in the purified and contaminated mitochondrial fractions over the last half of the 60-min assay (fig. 6) indicated that removing microsomes from the fraction did not alter this pattern. The activity of controls (groups 2 and 3) was linear over the 60 min of assay just as it was in the first and second experiment (fig. 1). Therefore, the presence of microsomal contaminants cannot account for the change in permeability to phenazine observed. The acid phosphatase activity of the purified and contaminated fractions was not significantly different in the three groups; however, purification of fractions resulted in somewhat higher values (table 2) .
Pilot studies have shown that the ethanol effect takes a minimum of 5 weeks on the ethanol diet to appear, and more than 2 and less than 5 days of ethanol withdra"wal before the effect disappears. Discussion
Chronic ethanol feeding induced a change in liver mitochondria. This change was expressed as an increase in SD act.ivity during the assay and as degeneration
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in structure during SD assay, observed by electron microscopy. These findings indicate that the outer membrane of mitochondria from the livers of rats fed ethanol are more likely to disintegrate than control mitochondria. The observed loss of the outer membrane would explain the increase in SD activity since SD is found in the inner mitochondrial membrane. 1s If the rate of membrane penetration of phenazine methosulfate is the rate-limiting factor in the SD assay, as suggested by Singer and Lusty,17 then one would expect the disruption of the outer mitochondrial membrane barrier to phenazine to be associated with an increase in SD activity, as observed in this investigation. It would appear, then, that ethanol ingestion causes an in crease in mitochondrial fragility. This idea is supported by the reports of Edmondson et al. 24 who observed dissolution of the mitochondrial membrane in livers of chronic alcoholic patients and I seri et alY who stated that disruption of mitochondrial membranes was common in livers of rats fed ethano 1. The possibility that microsomal or lysosomal contamination could mediate this effect seems unlikely. The partia.l elimination of microsomes from the mitochondrial fraction by a purification procedure did not alter the pattern of activity during SD assay. Lysosomal contamination of the mitochondria was not greater ill the ethanol-fed animals than in the controls as indicated by acid phosphatase a~say of the mitochondrial fractions. The results suggest that the mitochondrial abnonnalities were due to changes induced ill the mitochondria per se. These changes, however, were not manifested unt il after 30 min of assay, which indicates that at tIl(' time of isolation the mitochondria of the ethanol-fed animals were not det ecta hly different from those of control anima Is. The phenomenon of increased permeahility of isolated mitochondria is not new. For example, Christie and Judah 2G observed that liver mitochondria isolated from rats poisoned with carbon tetrachloride leaked pyridine nucleotide a t an abnormally rapid rate, compared with
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FRENCH
FIGs. 2-5
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June 1968
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FRAGILITY OF LIVER MITOCHONDRIA
controls. In fact, Kiessling and Tilander 4 found evidence that mitochondria from ethanol-fed rats were deficient in pyridine nucleotide and cytochrome c; addition of these substances partly restored the respiration of mitochondria derived from ethanol-fed rats. However, they did not explore the possibility that mitochondrial loss of these substances might have occurred after mitochondrial isolation. Palmieri et al.lO showed that 0.3 Methanol increased mitochondrial permeability to substrates such as succinate or p-hydroxybutyrate, and also nicotinamide adenine dinucleotide in vitro, probably because the ethanol accelerated mitochondrial swelling. These experiments seemed to indicate that ethanol altered the mitochondrial membrane but they gave little indication how it did this. There are several possible mechanisms by which mitochondrial fragility could be increased. For example, peroxidation of lipids leads to swelling and lysis of isolated liver mitochondria. 26 Comporti et aU reported that ethanol induced an increase in lipid peroxidation hy rat liver homogenates. Mitochondrial fragility could also result from an increase in endogenous phospholipase activity. Rossi and co-workers27 demonstrated that mitochondrial phospholipase caused an increase in lysolecithin in aged mitochondria. Either free fatty acids 28 or lysolecithin29 , 30 released from the membrane phospholipid by the phospholipase could induce mitochondrial swelling or lysis. Lysolecithin
......
100
~
0
GO: A-
• CONTAMINATED • PURIFIED
80
..... C(
~
Q
z
60
0
:z: v
g
40
2l
CII
E
......
20
N
0
"i.
60
30 TIME IN MINUTES
FIG. 6. SD activity of contaminated and purified mitochondrial fractions during the 60-min assay. The values shown represent the mean for 4 ethanol-fed animals. Removal of microsomes increased the mitochondrial SD activity by concentrating the mitochondria, but this purification did not affect the acceleration of activity observed after 30 min of assay.
2. Effect of diel on isolated rat liver mitochondl'ial fra ction acid phosphatase activity (acid phosphatase units per g of mitochondrial protein)'
TABLE
Diet
Group
--I 2 3
a
Contaminated mitochondrial fraction
Basal + etha- 6.6 ± 2.0 nol 8 .6 ± 1.0 Basal sucrose Basal ad 6.0 ± 1.0 libitum
+
Mean ±
SE
Purified mitochondrial fraction
11.2 ± 2.6 11 .2 ± 1.9 9.8 ± 0.3
(3 animals per group).
FIG. 2. Electron micrograph of a mitochondrial fraction from the liver of an ethanol-fed animal showing heavy contamination by microsomal vesicles. Note the intact outer mitochondrial membranes and the contracted, densely stained matrix, with distortion and vesicular dilation of pseudo cristae (X 20,000). FIG. 3. Electron micrograph of a purified, unincubated mitochondrial fraction. Scattered microsomal vesicles and glycogen bodies are still present but considerably reduced in number (X 20,000). FIG. 4. Electron micrograph of a mitochondrial fraction from the liver of an ethanol-fed animal after 60 min of assay for SD activity. The outer membrane and the cristae are absent. The smaller vesicles between the swollen mitochondria are probably remnants of disrupted mitochondria (X 20,000). FIG. 5. Electron micrograph of a mitochondrial fraction from the liver of a pair-fed control animal (group 2) after 60 min of SD assay. Note that the mitochondrial outer membrane and cristae persist although vesicular remnants of disrupted mitochondria are present (X 34,000).
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was elevated in the livers of rats fed ethanol for many months. 19 However, Kiessling and Lundquist31 reported that ethanol feeding did not alter mitochondrial phospholipid composition. Alterations in fatty acid composition of phospholipid could result in changes in mitochondrial membrane integrity as is the case in essential fatty acid (EFA) deficiency. Hayashida and Portman32 showed in rats fed an EFA-deficient diet that a fall in mitochondrial arachidonic and linoleic acids preceded an unmasking of latent succinic dehydrogenase activity. They used a succinate-phenazine methosulfate method of assay somewhat similar to the method employed in the present paper. As in EFA deficiency, chronic ethanol feeding also induced a change in the fatty acid composition of the liver. Scheig et aP3 reported that rats fed 15% ethanol developed a small and not significant decrease in the percentage of fatty acids 18: 2 and 20: 4 and a significant rise in 18: 1 in whole liver homogenates. They stated that similar changes were observed in individual subcellular fractions including the mitochondrial fraction. This pattern of change in mitochondrial fatty acids was similar to that reported in EF A deficiency,34 although the degree of alteration caused by ethanol feeding was not nearly as marked as in EFA deficiency. In the present study in which alcohol was given to rats in the drinking water it was noted that a minimum of 5 weeks of chronic ethanol ingestion was necessary before the mitochondrial alterations could be detected. In contrast, Iseri et al.,ll who
incorporated the ethanol in liquid diets, observed mitochondiral alterations in the intact liver cell after 16 days of feeding. Similar mitochondrial alterations have also been found in man a fter relatively short periods of alcohol feeding 12 • 35 during which the ethanol was given in graded doses up to 46% of calories. The shortenin g of the time of appearance of mitochondrial alterations observed when the diet was administered in liquid form could result from an increase in intake of ethanol. However, blood alcohol determined on 6 rats treated in a manner
Vol. 54,
}/Q . (j
comparable to that described in this paper averaged 0.126 ± 0.02%. This value is fairly high considering that rats metabolize alcohol more rapidly than man; i.e., the maximum rate of metabolism of ethanol in man is 100 to 200 mg per kg per hr 3s compared with an average rate of 270 ± 50 mg per kg per hr in the rat. 37 Summary
Changes in the liver mitochondria of rats after chronic ethanol feeding were studied by manometric assay of succinic dehydrogenase (SD) activity and by electron microscopy of isolated liver mitochondrial fractions. Abnormalities were not detectable until the assay of SD activity had been allowed to progress for 30 min. By 60 min of assay, an increase in permeability to phenazine methosulfate was demonstrated. This effect was obliterated by eliminating the membrane barrier to the phenazine by adding calcium to the assay media. The electron micrographs of assayed mitochondria showed that the chronic ethanol feeding induced an increase in the number of mitochondria which had lost their outer membranes and cristae, compared with the mitochondria in pair-fed control animals. This loss of outer membranes was correlated with the loss of the permeability barrier to phenazine methosulfate, probably because the SD, located in the inner mitochondrial membrane, was made more accessible to the phenazine. Indirect evidence indicated that neither microsomal contamination nor an increase of lysosomes in the mitochondrial fraction accounted for the ethanol-induced mitochondrial alteration in the ethanol-fed rat liver. In the case of microsomal contamination, purification of the mitochondrial fraction did not alter the ethanol effect. In the case of lysosomal contamination of the mitochondria, no increase in acid phosphatase was observed in the mitochondrial fractions obtained from the ethanol-fed rats. It is concluded that the fragility of mitochondria induced by ethanol feeding is probably due to an abnormality in the mitochondrial membrane.
June 1968
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FRAGILITY OF LIVER MITOCHONDRIA holic
REFERENCES 1. French, S. W. 1964. Succinic dehydrogenase:
histochemical "shift" in hepatic lobular distribution induced by ethanol. Lab. Invest. 13: 1051-1056. 2. Goldberg, D. M ., and C . Watts. 1965. Serum enzyme changes as evidence of liver reaction to oral alcohol. Gastroenterology 49: 256-261. 3. K alant, H., W. Mons, and M. A. Mahon. 1966. Acute effects of ethanol on tissue elec-
trolytes in the rat. Canad. J. Physiol. Pharmacol. 44 : 1-12. 4. Kiessling, K. H., and K. Tilander. 1961. Biochemical changes in rat tissues after prolonged alcohol consumption. Quart. J . Stud. Alcohol 22: 535-543. 5. Nygren, A. 1966. Serum creatine phosphokinase activity in chronic alcoholism, in connection with acute alcohol intoxication. Acta M ed. Scand. 179: 623-630. 6. R egan, T. J., G. Koroxenidis, C. B. Moschos, H. A. Oldewurtel, P. H. Lehan, and H. K. Hellems. 1966. Acute metabolic and hemodynamic responses of the left ventricle to ethanol. J. Clin . Invest. 45: 270'-280. 7. Comporti, M., A. Hart man , and N. R. DiLuzio. 1967. Effect of in vivo and in vitro ethanol administration on liver lipid peroxidation . Lab. Invest. 16: 616--624. 8. Christophersen, B. O. 1964. Effects of ethanol on mitochondrial oxidations. Biochim. Biophys. Acta 86: 14-20. 9. Greiser, E., and K. Soehring. 1967. Der Transport von P entobarbital durch biologische Membranen und seine Beeinflussung durch Athanol. Arzneimittelforschung 17: 207-214. 10. P almieri, F., A. Francavilla, and P. Dinoi. 1965. Effect of ethanol on mitochondrial membrane permeability. Ital. J. Biochem. 14: 316-320. 11. Iseri, O. A., C . S.Lieber, and L. S. Gottlieb. 1966. The ultrastructure of fatty liver induced by prolonged ethanol ingestion. Amer. J. Path . 48: 535-555. 12. Lane, B. P., and C. S. Lieber. 1966. Ultrastructural alterations in human hepatocytes following ingestion of ethanol with adequate diets. Amer. J. Path. 49: 593-603. 13. Biava, C. 1964. Mallory alcoholic hyalin: a heretofore unique lesion of hepatocellular ergastoplasm. Lab. Invest. 13: 301-320. 14. Reppart, J. T., R L. Peters, H. A. Edmondson, and R F. Baker. 1963. Electron and light microscopy of sclerosing hyaline necrosis of the liver. The development of alco-
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and U. Tobe. 1964. Degeneration of liver mitochondria in rats after prolonged alcohol consumption. Exp. Cell Res. 33: 350'-354. Porta, E. A., W. S. Hartroft, and F. A. de la Iglesia. 1965. H epatic changes associated with chronic alcoholism in rats. Lab . Invest. 14: 1437-1455. Singer, T. P., and C. J. Lusty. 1960. Permeability factors in the assay of mitochondrial dehydrogenases. Biochem. Biophys. Res. Commun. 2: 276-281. Bachmann, E., G. Lenaz, J. F. Purdue, N . Orme-Johnson, and D. E . Green . 1967. The membrane systems of the mitochondrion V. The inner membrane of beef heart mitochondria. Arch. Biochem. Biophys. (Chicago ) 121: 73-87. French, S . W. 1967. Effect of chronic ethanol feeding on rat liver phospholipid. J. Nutrition 91: 292-298. Parsons, D . F., G. R Williams, and B. Chance. 1966. Characteristics of isolated and purified preparations of the outer and inner membranes of mitochondria. Ann. N. Y. Acad. Sci. 187 : 643-666. Bernath, P ., and T. P. Singer. 1962. Succinic dehydrogenase, p. 597-614. In S. P. Colowick and N . D. Kaplan [eds.], Methods in enzymology, Vol. 5. Academic Press, Inc ., New York. Lowry , O. H ., J. N. Rosebrough, A. L. F arr, and R J. Randall. 1951. Protein measurement with Folin phenol reagent. J. BioI. Chem.193: 265-275. Andersch, M. A., and A. J. Szczypinski. 1947. Use of p-nitrophenyl phosphate as the substrate in determination of serum acid phosphatase. Amer. J . Clin. P ath. 17: 571-574. Edmondson, H. A., R. L. Peters, H. H. Frankel, and S. Borowsky. 1967. The early stage of liver injury in the alcoholic. Medicine (Bait .) 46: 119-129. Christie, G. S., and J . O. Judah, 1954. Mechanism of action of carbon tetrachloride on liver cells. Proc. Roy. Soc . Bio!. 142: 241-
257. 26. McKnight, R C., F . E. Hunter, Jr., and W. H . Oehlert. 1965. Mitochondrial membrane
ghosts produced by lipid peroxidation induced by ferrous ion. I. Production and general morphology. J. Bio!. Chern. 240: 3439-3446. 27. Rossi, C. R, L. Sartorelli, L. Tato, and N. Siliprandi. 1964. Relationship between oxi-
dative phosphorylation efficiency and phos-
pholipid content in rat liver mitochondria. Arch. Biochem. Biophys. (Chicago) 107: 170~175.
28. Lehninger, A. L., and L. F. Remmert. 1959.
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An endogenous uncoupling and swelling agent in liver mitochondria and its enzymic formation. J. BioI. Chem. 234: 2459-2464. Nygaard, A. P., M. V. Dianzani, and G. F. Bahr. 1954. The effect of lecithinase A on the morphology of isolated mitochondria. Exp. Cell Res. 6: 453--458. Pesch, L. A., and J. Peterson. 1965. Phospholipid-protein interaction as a determinant for the substrate specificity of mitochondrial nicotinamide-adenine-dinucleotide (phosphate) transhydrogenase . Biochim. Biophys. Acta 96: 390-394. Kiessling, K. H., and C. G. Lundquist. 1966. The lipid composition of liver mitochondria from rats after prolonged ethanol treatment. Scand. J. Clin. Lab. Invest. 18: supp!. 92,80-81. Hayashida, T ., and O. W. Portman. 1963.
Changes in SUCCInIC dehydrogenase activity and fatty acid composition of rat liver mitochondria in essential fatty acid deficiency. J. Nutrition 81: 103-109. 33. Scheig, R., N. M. Alexander, and G. Klatskin. 1966. Effects of prolonged ingestion of glucose or ethanol on tissue lipid composition and lipid biosynthesis in rat. J. Lipid R es. 7: 188-196. 34. Bailey, E., C. B. Taylor, and W. Bartley. 1967. Turnover of mitochondrial compo-
nents of normal and essential fatty acid deficient rats. Biochem. J. 104: 1026--1032. 35. Rubin, E., and C. S. Lieber. 1967. Experimental alcoholic hepatic injury in man : ultrastructural changes. Fed. Proc. 26 : 14581467.
36. Westerfeld, W. W., and M. P. Schulman. 1959. Metabolism and caloric value of alcohol. J. A. M. A.170: 197-203. 37. Aull, J. C., Jr., W. J. Roberts, Jr., and F. W. Kinard. 1956. Rate of metabolism of ethanol in the rat. Amer. J . Physiol.186: 380-382.
GASTROENTEROLOGY
Copyright ©)968 by The Williams & Wilkins Co.
FRAGILITY OF LIVER MITOCHONDRIA IN ETHANOL-FED RATS SAMUEL
W.
FRENCH,
M.D.
Department of Pathology, San Francisco General Hospital , University of California School of Medicine, San Francisco, California
Evidence that ethanol may cause cellular injury by altering the properties of cell membranes has been presented in many studies, both in viv0 1-6 and in vitro. 7-1o The vesiculation of the endoplasmic reticulum ll -12 and degeneration of the hepatocellular ergastoplasm13 -14 caused by chronic ethanol ingestion may be a result of membrane damage induced by ethanol. The mitochondrial membranes may be the ones mainly affected, since electron microscopic examination of liverll, 15-16 has revealed many structural changes in these organelles as a result of chronic ethanol ingestion. Since mitochondria can be isolated from the cell in relatively pure subcellular fractions, they can be conveniently studied for functional or structural alterations in their membranes induced by ethanol ingestion. In the present investigation, mitochondrial membrane function and structure were examined. The function of the mitochondrial membrane was tested by employing the succinic dehydrogenase (SD) assay method based on the work of Singer and Lusty 17 in which the rate of the reaction is dependent on the rate of penetration of an electron acceptor through the membrane permeability barrier. The permeabilReceived October 9, 1967. Accepted January 19,1968. Address requests for reprints to: Dr. Samuel W. French, Department of Pathology, U.C.L.A. School of Medicine, Harbor General Hospital, Torrance, California 90509. This investigation was supported by United States Public Health Service Research Grant AM-05243-06 from the National Institute of Arthritis and Metabolic Diseases. The author is indebted to Dr. Daniel Friend for his advice and encouragement, and to Mrs. Barbara French, Mr. Todor Todoroff, and Miss Hazel March for their technical assistance.
ity to phenazine methosulfate used as an electron acceptor in the SD assay is ratelimiting. This permeability barrier can be eliminated by mechanical fragmentation or chemical damage of mitochondriaP Preservation of this mitochondrial barrier to phenazine was used as a measure of mitochondrial membrane integrity and loss of the barrier as an indication of mitochondrial fragility. As an adjunct to SD assay, mitochondrial pellets were examined by electron microscopy to study structural changes before and after assay. Mitochondrial fragility shown in SD assay was correlated with loss of the outer mitochondrial membrane as determined by electron microscopy. This method was in part based on the work of Bachmann et aI.,18 who showed that phospholipase digestion of mitochondria caused both a loss of the outer membrane and an increase in SD activity. Experimental Procedure Animals. In each of the three experiments, young, male rats of the Wistar strain were used. The same dietary regimens were employed in each experiment, the only difference being in the duration of the experiment and the starting weight of animals (table 1). All animals were weighed twice weekly during the treatment period. They were housed individually in screen bottom cages. Fresh diet was given twice weekly. The composition of the basal diet has been described previously.'· Ethanol and sucrose were given in the drinking water by means of Richter tubes to minimize evaporation of ethanol. The basal diet was made into a thick paste with water and was offered in small jars to avoid loss by spilling. Six rats were used in experiment 1, 6 in experiment 2, and 1 2 in experiment 3. The total number of animals entered into a n experiment was divided into three groups, according to
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June 1968
1107
FRAGILITY OF LIVER MITOCHONDRIA
diet. Each ethanol-fed animal (group 1) was paired with a control animal fed the basal diet plus sucrose in isocaloric amounts (group 2) and also with a control animal fed the basal diet ad libitum (group 3). Table 1 shows the proportion of ethanol, fat, carbohydrate, and protein consumed by the ethanol-fed rats (group 1) in experiments 1, 2, and 3. The average consumption of food and of ethanol and the percentage of dietary calories were determined from the beginning of each experiment until the time of assay. Thus it was known that adequate dietary ingredients were ingested despite a high ethanol intake, and changes observed could be assumed to be due to etllanol and not to dietary deficiency or imbalance. The pair-fed sucrose control rats (group 2) consumed the same amount of diet and the same volume of liquid calories as ethanol-fed rats. Their weight gain was the same as that of group 1 in experiments 1 and 3 but exceeded that of group 1 in experiment 2. Preparation of tissues. Liver tissue for assay of succinic dehydrogenase activity was obtained by biopsy while the animals were under ether anesthesia. About 1 g of the liver specimen was gently homogenized in a hand-operated, glass-Teflon homogenizer using ice-cold 0.25 M sucrose containing 5 mM ethylene-diaminetetraacetate (EDTA) and 5 mM Tris-HCI buffer, pH 7.4. Mitochondrial fractions were prepared in two different ways. In experiments 1 and 2 a "contaminated" fraction containing a mixture of microsomes and mitochondria was prepared by the method of Christophersen.s Nuclei and cell debris were removed by centrifugation at 700 X g twice in a refrigerated centrifuge at 0 C. The contaminated mitochondrial fraction was sedimented at 20,000 X g for 15 min and washed once by resuspension in homogenizing medium and recentrifuged. The fraction was resuspended in the homogenizing medium in a concentration of 0.75 g of fresh liver per m!. The fractions were assayed immediately. In the thi rd experiment, a p" urified" mitochondrial fraction containing a minimum of contaminating microsomcs was prepared using a modification of the method of Parsons et al: o The purified and contaminated fractions were both prepared at the same time. The supernatant fraction obtained from the cellular debris removal step in the preparation of the contaminated mitochondrial fraction was spun for 10 min at 9,000 X g in a Spinco ultracentrifuge. The supernatant fluid was sucked off from the surface so that the floating layer of lipid was removed with the supernatant fluid. The fluffy
T ABLE
1. Diet and ethanol consumption Calories in diet
Experiment DO.
Duration
Starting weight
--- - - - - 1 2 3
CarboEthanol Protein hydrate .--
mo
g
%
2 14 13
80 60 80
33.3 39.6 35.2
- -
%
19.2 18.3 18.7
- -
%
36.0 31.1 34.9
Fat
-%
11.5 11.0 11.2
layer was resuspended in the last few milliliters of supernatant fluid by agitation with a disposable pipette and then was sucked off. The surface of the pellet was then lightly washed three times witll a small amount of medium. The mitochondrial pellet was resuspended and spun again for 10 min at 500 X g and the supernatant fraction was respun for 10 min at 9,000 X g. The fluffy layer was again resuspended and the mitochondrial pellet surface was again washed. The mitochondria were resuspended in the medium in a concentration of 0.75 g of fresh liver per ml and assayed immediately. The purified and contaminated fractions were both prepared at the same time and assayed at the same time. Enzyme assays. The SD activity of the mitochondrial fractions was assayed by the succinate-phenazine methosulfate method of Bernath and Singer.21 The assay was performed in a Gilson differential respirometer at 38 C using 0.3 M Tris buffer, pH 7.6. Parallel assays were done with and without 0.75 mM calcium added to the media. Phenazine methosulfate was added at a fixed concentration of 0.67 mg per m!.'7 The reaction rate was calculated at 10-, 30-, and 60-min intervals. Mitochondrial protein was determined by the method of Lowry and co-workers:2 The results were calculated as Q02 (microliters of O2 per milligram of protein per minute). In the first and second experiments the percentage of maximum activity was calculated by dividing the activity measured with calcium added to the assay media into the activity determined without calcium in the media. In the third experiment the purified and contaminated fractions were assayed for SD activity with no calcium added to t he media and the results were compared. Purified fractions from control animals (groups 2 na d 3) were also assayed in this way. Acid phosphatase activity of the purified and contaminated mitochondrial fractions was measured in order to estimate the contamination of the fractions by lysosomes. Enzyme ac'" tivity was preserved by adding om ml of 20%
1108
FRENCH
acetic acid per ml of mitochondrial suspension; freeze-thawing was used to achieve maximum activity. Acid phosphatase activity was assayed by the method of Andersch and Szczypinski.'· Electron microscopy. Mitochondrial pellets prepared before and after SD assay were examined by electron microscopy. For this purpose pellets were fixed in 3% buffered glutaraldehyde, pH 7.4, and postfixed in buffered 0s0., pH 7.6. Thin sections were cut from specimens embedded in Araldite; they were examined with an RCA model EMU-3H electron microscope. All purified mitochondrial pellets in experiment 3 were examined in this way not only to assess contamination by microsomes but also to determine whether the outer membrane of the mitochondria was intact after the 60-min assay. Some of the pellets from experiments 1 and 2 were examined for contamination of the fraction with microsomes and for the effect of the SD assay on the mitochondria.
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1. The results of experiment 1 and 2 were the same and, therefore, they were pooled for statistical analysis. The SD activity of the fractions from the ethanol-fed group differed very little from that of the paired controls for the first 10 min of assay; by 60 min of assay, the activity was significantly greater (P < 0.02) than that of the controls. Adding calcium to the assay media is a convenient way of eliminating the mitochondrial membrane barrier to phenazine so that maximum enzyme activity can be measured.11 By comparing the activity with calcium in the media with the activity without calcium in the media, the percentage of maximum activity can be calculated for each group of animals. The percentage of maximum SD activity for the ethanol-fed animals was double that of the control animals at 60 min of assay Results (P < 0.02). Liver mitochondrial contaminated fracElectron microscopy of the contaminated tion. The SD activity of the contaminated mitochondrial pellet disclosed numerous mitochondrial fractions is plotted in figure microsomal vesicles separating the mitochondria (fig. 2). After assay almost all 125 of the mitochondria from ethanol-fed rats • EffI....1 (group 1) had lost their outer membranes, • Sue.... o ..... LiII whereas most of the mitochondria from pair-fed sucrose controls (group 2) still 100 retained their outer membranes. c: :! Mitochondrial pellets taken from the ...! purified fraction (experiment 3) were all Ci 75 ~c: examined by electron microscopy. In the o unincubated fractions, a marked diminu'5o tion of microsomal vesicles resulted in i.. 50 close packing of mitochondria, although E ........ scattered vesicles and glycogen bodies were o ~ invariably present in small numbers (fig. 25 3). The mitochondria retained their outer and inner membranes. The cristae often were dilated so that they appeared spherical instead of tubular; the matrix ap30 60 Tim. in Minut•• peared uniformly dense. After SD assay FIG. 1. SD activity of the contaminated liver for 60 min the mitochondria from the mi tochondrial fraction during t he 6O-min assay ethanol-fed animals (group 1) were almost period. Note the acceleration of activity in the invariably swollen in appearance (fig. 4). ' ethanol-fed animals after 30 min of assay, when Their outer membranes were absent and no calcium was added to the media. Note also that when calcium was added to the media and the cristae were no longer detectable. The maximum enzyme activity was measured exper- matrix did not stain, or contained a few, imental and control values are not significantly densely stained, amorphous clumps. These different. changes have been described previously
June 1968
FRAGILITY OF UVER MITOCHONDRIA
in in vitro, large amplitude, swelling experiments. 2o Numberous intervening vesicles, probably derived from disrupted mitochondria, were also seen. The assayed mitochondria from the control animals of groups 2 and 3 showed numerous intact mitochondria (fig. 5), with a few swollen ones interspersed. The intact mitochondria retained their outer membranes and cristae. The matrix stained less densely. Darkly stained amorphous clumps were characteristically present. N umerous small vesicles were seen between mitochondria. The differences in mitochondrial degeneration between the ethanol-fed and the control animals was a matter of degree and correlated qualitatively with the amount of SD activity: the mitochondria from the ethano l-fed animals showed a more advanced degree of degeneration after assay as well as evidence of greater loss of the membrane barrier to phenazine methosulfate. The same pattern of accelerated activity in the purified and contaminated mitochondrial fractions over the last half of the 60-min assay (fig. 6) indicated that removing microsomes from the fraction did not alter this pattern. The activity of controls (groups 2 and 3) was linear over the 60 min of assay just as it was in the first and second experiment (fig. 1). Therefore, the presence of microsomal contaminants cannot account for the change in permeability to phenazine observed. The acid phosphatase activity of the purified and contaminated fractions was not significantly different in the three groups; however, purification of fractions resulted in somewhat higher values (table 2) .
Pilot studies have shown that the ethanol effect takes a minimum of 5 weeks on the ethanol diet to appear, and more than 2 and less than 5 days of ethanol withdra"wal before the effect disappears. Discussion
Chronic ethanol feeding induced a change in liver mitochondria. This change was expressed as an increase in SD act.ivity during the assay and as degeneration
1109
in structure during SD assay, observed by electron microscopy. These findings indicate that the outer membrane of mitochondria from the livers of rats fed ethanol are more likely to disintegrate than control mitochondria. The observed loss of the outer membrane would explain the increase in SD activity since SD is found in the inner mitochondrial membrane. 1s If the rate of membrane penetration of phenazine methosulfate is the rate-limiting factor in the SD assay, as suggested by Singer and Lusty,17 then one would expect the disruption of the outer mitochondrial membrane barrier to phenazine to be associated with an increase in SD activity, as observed in this investigation. It would appear, then, that ethanol ingestion causes an in crease in mitochondrial fragility. This idea is supported by the reports of Edmondson et al. 24 who observed dissolution of the mitochondrial membrane in livers of chronic alcoholic patients and I seri et alY who stated that disruption of mitochondrial membranes was common in livers of rats fed ethano 1. The possibility that microsomal or lysosomal contamination could mediate this effect seems unlikely. The partia.l elimination of microsomes from the mitochondrial fraction by a purification procedure did not alter the pattern of activity during SD assay. Lysosomal contamination of the mitochondria was not greater ill the ethanol-fed animals than in the controls as indicated by acid phosphatase a~say of the mitochondrial fractions. The results suggest that the mitochondrial abnonnalities were due to changes induced ill the mitochondria per se. These changes, however, were not manifested unt il after 30 min of assay, which indicates that at tIl(' time of isolation the mitochondria of the ethanol-fed animals were not det ecta hly different from those of control anima Is. The phenomenon of increased permeahility of isolated mitochondria is not new. For example, Christie and Judah 2G observed that liver mitochondria isolated from rats poisoned with carbon tetrachloride leaked pyridine nucleotide a t an abnormally rapid rate, compared with
1110
FRENCH
FIGs. 2-5
Vol. 54, No.6
June 1968
1111
FRAGILITY OF LIVER MITOCHONDRIA
controls. In fact, Kiessling and Tilander 4 found evidence that mitochondria from ethanol-fed rats were deficient in pyridine nucleotide and cytochrome c; addition of these substances partly restored the respiration of mitochondria derived from ethanol-fed rats. However, they did not explore the possibility that mitochondrial loss of these substances might have occurred after mitochondrial isolation. Palmieri et al.lO showed that 0.3 Methanol increased mitochondrial permeability to substrates such as succinate or p-hydroxybutyrate, and also nicotinamide adenine dinucleotide in vitro, probably because the ethanol accelerated mitochondrial swelling. These experiments seemed to indicate that ethanol altered the mitochondrial membrane but they gave little indication how it did this. There are several possible mechanisms by which mitochondrial fragility could be increased. For example, peroxidation of lipids leads to swelling and lysis of isolated liver mitochondria. 26 Comporti et aU reported that ethanol induced an increase in lipid peroxidation hy rat liver homogenates. Mitochondrial fragility could also result from an increase in endogenous phospholipase activity. Rossi and co-workers27 demonstrated that mitochondrial phospholipase caused an increase in lysolecithin in aged mitochondria. Either free fatty acids 28 or lysolecithin29 , 30 released from the membrane phospholipid by the phospholipase could induce mitochondrial swelling or lysis. Lysolecithin
......
100
~
0
GO: A-
• CONTAMINATED • PURIFIED
80
..... C(
~
Q
z
60
0
:z: v
g
40
2l
CII
E
......
20
N
0
"i.
60
30 TIME IN MINUTES
FIG. 6. SD activity of contaminated and purified mitochondrial fractions during the 60-min assay. The values shown represent the mean for 4 ethanol-fed animals. Removal of microsomes increased the mitochondrial SD activity by concentrating the mitochondria, but this purification did not affect the acceleration of activity observed after 30 min of assay.
2. Effect of diel on isolated rat liver mitochondl'ial fra ction acid phosphatase activity (acid phosphatase units per g of mitochondrial protein)'
TABLE
Diet
Group
--I 2 3
a
Contaminated mitochondrial fraction
Basal + etha- 6.6 ± 2.0 nol 8 .6 ± 1.0 Basal sucrose Basal ad 6.0 ± 1.0 libitum
+
Mean ±
SE
Purified mitochondrial fraction
11.2 ± 2.6 11 .2 ± 1.9 9.8 ± 0.3
(3 animals per group).
FIG. 2. Electron micrograph of a mitochondrial fraction from the liver of an ethanol-fed animal showing heavy contamination by microsomal vesicles. Note the intact outer mitochondrial membranes and the contracted, densely stained matrix, with distortion and vesicular dilation of pseudo cristae (X 20,000). FIG. 3. Electron micrograph of a purified, unincubated mitochondrial fraction. Scattered microsomal vesicles and glycogen bodies are still present but considerably reduced in number (X 20,000). FIG. 4. Electron micrograph of a mitochondrial fraction from the liver of an ethanol-fed animal after 60 min of assay for SD activity. The outer membrane and the cristae are absent. The smaller vesicles between the swollen mitochondria are probably remnants of disrupted mitochondria (X 20,000). FIG. 5. Electron micrograph of a mitochondrial fraction from the liver of a pair-fed control animal (group 2) after 60 min of SD assay. Note that the mitochondrial outer membrane and cristae persist although vesicular remnants of disrupted mitochondria are present (X 34,000).
1112
FRENCH
was elevated in the livers of rats fed ethanol for many months. 19 However, Kiessling and Lundquist31 reported that ethanol feeding did not alter mitochondrial phospholipid composition. Alterations in fatty acid composition of phospholipid could result in changes in mitochondrial membrane integrity as is the case in essential fatty acid (EFA) deficiency. Hayashida and Portman32 showed in rats fed an EFA-deficient diet that a fall in mitochondrial arachidonic and linoleic acids preceded an unmasking of latent succinic dehydrogenase activity. They used a succinate-phenazine methosulfate method of assay somewhat similar to the method employed in the present paper. As in EFA deficiency, chronic ethanol feeding also induced a change in the fatty acid composition of the liver. Scheig et aP3 reported that rats fed 15% ethanol developed a small and not significant decrease in the percentage of fatty acids 18: 2 and 20: 4 and a significant rise in 18: 1 in whole liver homogenates. They stated that similar changes were observed in individual subcellular fractions including the mitochondrial fraction. This pattern of change in mitochondrial fatty acids was similar to that reported in EF A deficiency,34 although the degree of alteration caused by ethanol feeding was not nearly as marked as in EFA deficiency. In the present study in which alcohol was given to rats in the drinking water it was noted that a minimum of 5 weeks of chronic ethanol ingestion was necessary before the mitochondrial alterations could be detected. In contrast, Iseri et al.,ll who
incorporated the ethanol in liquid diets, observed mitochondiral alterations in the intact liver cell after 16 days of feeding. Similar mitochondrial alterations have also been found in man a fter relatively short periods of alcohol feeding 12 • 35 during which the ethanol was given in graded doses up to 46% of calories. The shortenin g of the time of appearance of mitochondrial alterations observed when the diet was administered in liquid form could result from an increase in intake of ethanol. However, blood alcohol determined on 6 rats treated in a manner
Vol. 54,
}/Q . (j
comparable to that described in this paper averaged 0.126 ± 0.02%. This value is fairly high considering that rats metabolize alcohol more rapidly than man; i.e., the maximum rate of metabolism of ethanol in man is 100 to 200 mg per kg per hr 3s compared with an average rate of 270 ± 50 mg per kg per hr in the rat. 37 Summary
Changes in the liver mitochondria of rats after chronic ethanol feeding were studied by manometric assay of succinic dehydrogenase (SD) activity and by electron microscopy of isolated liver mitochondrial fractions. Abnormalities were not detectable until the assay of SD activity had been allowed to progress for 30 min. By 60 min of assay, an increase in permeability to phenazine methosulfate was demonstrated. This effect was obliterated by eliminating the membrane barrier to the phenazine by adding calcium to the assay media. The electron micrographs of assayed mitochondria showed that the chronic ethanol feeding induced an increase in the number of mitochondria which had lost their outer membranes and cristae, compared with the mitochondria in pair-fed control animals. This loss of outer membranes was correlated with the loss of the permeability barrier to phenazine methosulfate, probably because the SD, located in the inner mitochondrial membrane, was made more accessible to the phenazine. Indirect evidence indicated that neither microsomal contamination nor an increase of lysosomes in the mitochondrial fraction accounted for the ethanol-induced mitochondrial alteration in the ethanol-fed rat liver. In the case of microsomal contamination, purification of the mitochondrial fraction did not alter the ethanol effect. In the case of lysosomal contamination of the mitochondria, no increase in acid phosphatase was observed in the mitochondrial fractions obtained from the ethanol-fed rats. It is concluded that the fragility of mitochondria induced by ethanol feeding is probably due to an abnormality in the mitochondrial membrane.
June 1968
1113
FRAGILITY OF LIVER MITOCHONDRIA holic
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