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Inhibition of the ethanol and carbon tetrachloride induced fatty liver by antioxidants
EXPERIMENTAL
AND
MOLECULAR
Inhibition
of Induced
PATHOLOGY
the
141-154
4,
Ethanol Fatty
and
Liver
N. R. DI LUZIO Department
(1965)
AND
March
Tetrachloride
by Antioxidants’
of Physiology, University of Tenrzessee Department of Pathology, Baptist Memorial
Received
Carbon
F.
COSTALES~
Medical Units, Memphis, Tertnessee Hospital, Memphis, Tennessee
and
16, 1964
INTRODUCTION Although the development of a fatty liver in normal rats treated with a single intoxicating dose of ethanol or alcoholic beverages has been amply demonstrated (Mallov and Bloch, 1956; Di Luzio, 1958; Lieber and Schmid, 1961; Brodie et al., 1961a; Di Luzio, 1962), considerabledifferencesin opinion exist regarding the etiology of the triglyceride accumulation. Current concepts regarding its genesiscover essentially all possiblevariables, including increasedhepatic synthesis of lipid (Lieber and Schmid, 1961), increasedtriglyceride formation from free fatty acids (FFA) (Nikkila and Ojala, 1963), increased mobilization of free fatty acids from peripheral adipose depots (Brodie et al., 1961a, 1961b), and a decreasedintrahepatic utilization of triglyceride (Di Luzio, 1958; Elko et al., 1961: Poggi and Di Luzio, 1964; Di Luzio, 1964). In contrast to the mechanismof the ethanol-induced fatty liver, the carbon tetrachloride (CCld)-induced fatty liver has been demonstrated to be associated with a decreasedrate of hepatic triglyceride secretion (Rechnagel et al., 1960; Maling et al., 1962), and an enhanced rate of hepatic triglyceride synthesis (Maling et al., 1962). Gallagher (1962) has demonstrated that the intraperitoneal administration of antioxidants afford protection to rats given lethal dosesof carbon tetrachloride. Since multiple factors obviously exist relative to fatty liver induction and in view of the apparent dissimilar mechanismsbetween the ethanol- and carbon tetrachloride-induced fatty liver, studies were undertaken to evaluate the effects of antioxidant administration on the fatty liver induced by these agents. In view of the observation of Calder (1942), that many insoluble compounds injected intraperitoneally would protect against CC&-induced fatty liver, Judah et al., (1963) questioned the concept that antioxidants exert a specific effect on fatty liver development and indicated that the protective effect noted by Gallagher might be due to a non-specific action of the intraperitoneally administered material. In an effort to evaluate this possibility, antioxidants were administered orally simultaneously with ethanol, and the degree of triglyceride accumulation noted. 1 Supported, in part, by a grant from the C. D. Smithers Foundation, the Licensed Industries, Inc., and U. S. Public Health Service (AM-08084). 2 The capable technical assistance of Mrs. Betty Lyons is gratefully acknowledged. 141
Beverage
N. R. DI LUZIO AND F. COSTALES MATERIALS
AND
METHODS
Female rats (Holtzman Co., Madison, W&c.), previously maintained on a Purina Chow diet were employed. The animals were fasted for 8 hours prior to receiving by oral intubation, a single intoxicating dose of ethanol in the amount of 6 gr/Kg, as a 505% solution or isocaloric glucose. Carbon tetrachloride was administered orally to other rats in the amount of 0.4 ml,/100 gr, while an equivalent volume of saline was administered to control rats. The rats were killed with ether 24 hours later. Alphatocopherol acetate was suspended in saline and administered intraperitoneally in the amount of 10 mg/lOO gr 48, 24, and 2 hours prior to the oral administration of ethanol or glucose. Control rats received intraperitoneal injections of saline and the animals killed 16 hours later. N, N’-Diphenyl-p-phenylenediamine (DPPD) was suspended in corn oil and administered intraperitoneally in the amount of 60 mg/lOO gr 48, 24 and 2 hours prior to oral intubation. Control rats in this instance received corn oil intraperitoneally. The rats were killed 24 hours after oral intubation. In an effort to evaluate the oral effectiveness of DPPD, another group of rats received orally 1OOmg of DPPD/lOOgr of body weight simultaneously with ethanol (6 gr/Kg) and killed 16 hours later. A group of 28 rats were employed for histological studies, being distributed as follows : In the CC& study three control rats received corn oil intraperitoneally and saline orally; six received corn oil intraperitoneally and CC& orally and five rats received Ccl, orally after intraperitoneal administration of DPPD suspended in corn oil as described above. In the ethanol study seven rats received ethanol orally and corn oil intraperitoneally and another seven rats received ethanol after the intraperitoneal administration of DPPD and corn oil as described above. Liver sections of all animals were prepared with hematoxylin-eosin stain in paraffin embedded sections and Sudan IV in frozen sections. All preparations were evaluated without previous knowledge of the treatment received by the animals, and the results were graded from lf to 4+ on the basis of the following morphologic changes: (a) fatty metamorphosis, (b) cloudy swelling, (c) hydropic degeneration or ballooning of the hepatic cells, (d) acidophilic bodies, (e) hepatic cell necrosis, (f) polymorphonuclear cell infiltration and (g) mononuclear cell infiltration. After the slides had been evaluated and identified, a representative section from each group was stained with Gomori stain for reticulum. Plasma triglyceride concentrations were determined (Van Handel and Zilversmit, 19.57) liver lipids were extracted with chloroform-methanol and purified (Sperry, 1955) and the chloroform-methanol extract was analyzed for triglycerides (Van Handel and Zilversmit, 1957). All determinations were done in duplicate. The data were examined by means of the “t” test for the difference between means with a 95% confidence limit.
ANTIOXIDANTS
.4ND
LIVER
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INJURY
RESULTS A.
Chemical findings
Plasma triglyceride concentrations were not significantly altered following ethanol administration (Table I). However, liver triglyceride concentration was significantIy of a-tocopherol increased 350% over control values. -4lthough the administration acetate to glucose-treated rats did not alter the concentration of liver and plasma triglycerides, the liver triglyceride concentration in the vitamin E-treated group which received ethanol was 3670 lower than that of the ethanol group. Plasma triglyceride concentrations were not altered from control values in the vitamin E-ethanol group. The administration of DPPD to glucose-treated rats produced a significant decrease in liver triglyceride concentration of 3372~ (Table II). The administration of corn oil to glucose-treated rats produced a significant increase in liver triglyceride concentration when compared to the saline injected glucose group (Table I). The administration of ethanol, as previously observed, resulted in a significant increase in liver TABLE EFFECT
OF U-TOCOPHEROL
I FMTY
ON THE ETHANOL-INDUCED
Triglyceride
Treatment Intraperitoneal Saline Saline Vitamin E
Oral Glucose Ethanol Glucose Ethanol
LIVERY
Vitamin
Liver w/gr
E
Plasma mg/100mI
10.5
k 2.0
47.1
k
5.5
10.4
t
1.3
22.7 25.9 23.0 33.3
30.2 t 3.1
a Values are means -+ standard error. The liver triglyceride values were derived group, while the plasma triglyceride values are from 5 rats per group. The female rates weighed approximately 2OOg and were killed 16 hr after ethanol
TABLE INFLUENCE
Treatment Intraperitoneal
Oral Glucose Glucose
Ethanol Ethanol a corn oral oral
Corn oil DPPD Corn oil DPPD
OF DPPD
No. per group 5 5 8 8
FATTY
Body weight
-c 2 t IL
Final 7 4 5 6
administration.
193 184 187 187
-c k zk 2
LIVERY
Liver triglyceride m/u
!zt.
197 189 197 193
8 rats per
II
ON THE ETHANOL-INDUCED
Initial
from
* 7.0 k 4.0 2 7.0 It 13.0
9 2 7 4
16.4 10.9 36.0 19.9
-r-e & 2
Plasma triglyceride ms% 1.7 2.7 5.7 3.1
33.5 rt 5.1 20.7 2 3.0 46.8 f 5.4
The glucose and ethanol solutions were administered by stomach tube to female rats, while the oil or DPPD solutions were given by intraperitoneal injection 48, 24 and 2 hours prior to the intubation. The liver and plasma triglyceride concentrations were determined 24 hours after intubation. Values are expressed as means -C standard error.
triglyceride (Table II). In contrast, the administration of ethanol to rats previously injected with DPPD resulted in a liver triglyceride concentration that was unaltered from the glucose and corn oil control group.
144
N.
R. DI LUZIO
AND
F. COSTALES
The administration of Ccl4 produced a more severe fatty liver than ethanol administration and resulted in a mean 400% elevation in liver triglyceride concentration when compared to saline-treated rats (Table III). Plasma triglyceride levels were not significantly altered 24 hours following carbon tetrachloride administration. The administration of CC14 to DPPD treated rats was associated with a 60% reduction in TABLE EFFECT OF DPPD Treatment
Saline ccl, ccl,
per group
Corn oil Corn oil DPPD
III
TETRACHLORIDE-INDUCED
NO.
Intraperitoneal
Oral
ON THE CARBON
7 8 7
FATTY
Liver triglyceride mdgr
Plasma triglyceride w%
17.8 -+ 1.3 88.8 k 9.2 35.0 2 2.4
37.5 f 10.0 56.1 k 10.0 71.8 k 32.0
LIVER”
FFA mEq/L 1.33 5 0.53 1.94 -+ 0.26 1.26 5~ 0.11
a Values are expressed as means k standard error. Ccl, or saline was administered orally in the amount of 0.4m1/100gr to female rats weighing approximately 200 gr DPPD was administered intraperitoneally in corn oil 48, 24 and 2 hours prior to oral intubation. The plasma and liver triglyceride and plasma FFA concentration were determined 24 hours after oral intubation. TABLE EFFECT
OF DPPD
IV
ADMINISTRATION SIMULTANEOUSLY WITH HEPATIC TRIGLYCERIDE CONCENTRATIONS
Body weight
Liver weight gr
gr Initial
ETHANOL
Group
DPPD
Final
Ethanol
-
232 -c 15
221 k 17
7.0 k 0.5
Ethanol
+
238k
224 k
7.5 -c 0.6
9
9
a Values, expressed as means k standard error are derived from 5 female hours after the administration of 6gr/Kg of ethanol orally in the absence and in the amount of lgr/Kg. The figures in parenthesis are the sample range.
ON
Liver triglyceride w/a 58.5 2 6.1 (48.5-79.2) 32.5 5 3.0 (27.0-39.6) rats per group 16 presence of DPPD
accumulation and an unaltered plasma triglyceride level. Plasma free fatty (FFA), although increased above normal levels, were not significantly altered within the various groups. The oral administration of DPPD simultaneously with ethanol resulted in a significant reduction in hepatic triglyceride accumulation when compared to ethanol-treated rats (Table IV). The triglyceride decrease in the DPPD group amounted to a 44% reduction. Liver and body weights were not altered in the DPPD group from control values. triglyceride
acids
B.
Histological
studies
The histological appearance of the liver of various rats in the experimental groups, as well as the chemically determined liver triglyceride concentrations, are tabulated in Table V. The saline control group, although fasted for a 24 hour period, showed no visible fat
ANTIOXIDANTS
100
AND
0000000
LIVER
145
INJURY
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300
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+++
++
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too ~“~+o++o++oooo ++++++ ++++++ ++++-t +++++ ++++ + s++ Ooo+++++++ +++++++++++++ ++++t yzez+ ++++-t + ++z++
146
N.
R. DI
LUZIO
AND
F. COSTALES
with the Sudan IV stain nor any other liver alteration. In the animals which received ethanol, the fat content of the liver was significantly higher, five being graded 2+, one 3+, and another l+. The group that received ethanol after being treated with DPPD showed a lesser quantity of fat and was graded from 0 to I+. It is readily apparent that the histological evaluations were well correlated with the chemical findings as denoted by liver triglyceride concentrations. The fat in both ethanol-treated groups was present as fine droplets in the hepatic cells, primarily at the edges of the cytoplasm adjacent to the sinusoid and was evenly distributed throughout the hepatic lobule. Other morphological changes were inconspicuous. Mild cloudy swelling and hydropic degeneration were occasionally found in both groups. .. The rats that received Ccl+ alone showed the typical alterations of acute Ccl4 intoxication, i.e., fatty metamorphosis and central necrosis (Fig. 1). In the center of the hepatic lobule most of the hepatic architecture was markedly distorted, showing disappearance of the continuity of the hepa.tic cords. The sinusoids in the area were dilated, forming highly irregular vascular spaces. Many acidophilic bodies were present in isolated cells. The hepatic cells also exhibited acidophilic cytoplasm and pyknotic or fragmented nuclei. In the intermediate zone the necrotic changes were less extensive and only groups of cells were involved. The remainder, however, showed cloudy swelling. Many hepatic cells in this intermediate zone of the lobule were enlarged I>/2 to 2 times their normal size, compressing the adjacent sinusoids. These enlarged cells exhibited a clear cytoplasm and a centrally located nucleus. The hepatic cells at the periphery of the lobule were less severely involved, with only scattered cells showing cloudy swelling. Similarly, fatty metamorphosis was present in lesser degree in this area than in the intermediate zone of the lobule. Polymorphonuclear cell infiltration paralleled the necrotic changes. In marked contrast to the above findings the rats that had received Ccl, after the administration of DPPD showed less extensive necrotic changes than the preceding group (Fig. 2). The necrotic alterations, when present, were also predominantly centrolobular. The number of cells totally disrupted or with nuclear loss was smaller, and there was no loss of continuity of the hepatic cords with resulting disruption of the architecture and dilation of the sinusoidal spaces. The ballooning or hydropic degeneration of the hepatic cells in the intermediate zone was however, more conspicuous and marked compression of the sinusoid surrounding the greatly enlarged cells was observed. In contrast to the rats that received only CCII, the animals in this group showed the less severe degenerative changes, such as, cloudy swelling and hydropic degeneration which are usually considered reversible. In agreement with the chemical analysis of hepatic triglyceride, the quantity of fat in the Sudan IV-stained sections of the DPPD group which received Ccl4 was much less than in the CCll group (Table V). There was less polymorphonuclear cell infiltration in the antioxidant protected group, but in contrast with the CCll group a marked infiltration of mononuclear cells was present. These cells had kidney-shaped nuclei and were arranged in groups.
ANTIOXIDANTS
AND
LIVER
IN JURY
147
The Gomori stain for reticulum in a representative section of each group shower da m arked distortion of the architecture of the reticulum frame at the center of the lob la 1e inI the rats that received Ccl, (Fig. 3), while it was well preserved in the CC&-trea ted allimals that received DPPD (Fig. 4).
FIG. 1. Liver section from CC14-treated th e central vein and extensive necrosis. In ajority with a pyknotic or fragmented
rat demonstrating disruption of the architecture Only isolated hepatic cells are present in this nucleus. H & E 400 X.
are und area, the
148
N.
R. DI LUZIO
AND
I ?IG. 2. Liver section obtained from Ccl=,-treated Pal :ed to Fig. 1, there is no loss in the continuity
net :rosis
is reduced
and fewer
hepatic
cells show
F. COSTALES
rat which previously received DPPD. Co lrnof the hepatic cords. The degree of cellc dar pyknotic nuclei. H 8: E 400 X.
ANTIOXIDANTS
AND
FIG. 3. Disruption of the reticulum framework nuity of the hepatic cords is seen in CCll-treated al : one edge of the photograph. Gomori reticulum
LIVER
INJURY
in the centrolohular rat liver. A dilated stain. 400 X.
149
zone, with loss of :oncentrolobular vein is v ie;ible
1
N.
4. Liver section from 3, the reticulum framework
I hG.
Fig
R. DI LUZIO
CCld-treated is well
AND
F. COSTALES
rat which previously received DPPD. In preserved. Gomori reticulum stain. 400 X.
contrast
ANTIOXIDANTS
AND
LIVER
IN JURY
151
DISCUSSION Although disagreement exists relative to the pathogenesis of the fatty liver following acute ethanol ingestion, the development of fatty metamorphosis in normal rats following ethanol administration has been amply demonstrated (Mallov and Bloch, 1956; Di Luzio, 1958, 1962; Lieber and Schmid, 1961; Brodie et al., 1961a, 1961b; Elko et al., 1961). The present data amply confirm these findings. Various antioxidants have been demonstrated to be effective in preventing hepatic alterations and increasing survival following carbon tetrachloride poisoning (Gallagher, 1962). Our findings of a significant reduction in triglyceride accumulation in rats pretreated with a-tocopherol or DPPD appear to support the observations of Gallagher (1962) that hepatic alterations following the administration of hepatotoxins can be prevented, in part, by the use of antioxidants. The oral effectiveness of DPPD, as well as other antioxidants (Di Luzio, 1964) implies that the effect of antioxidants is not a nonspecific action as suggested (Judah et al., 1963), but a reflection of a specific effect of antioxidants (Di Luzio, 1964). Although the mechanism of the protective effect of antioxidants on liver triglyceride accumulation following ethanol or Ccl+ administration remains to be ascertained, the protection of a-tocopherol and other antioxidants against the lethal effects of carbon tetrachloride has been credited to maintenance of normal levels of reduced diphosphopyridine nucleotides (DPNH) and a decreased loss of oxidized pyridine nucleotides (DPN) (Gallagher, 1962 ) . The oxidation of ethanol in the liver is catalyzed by alcohol dehydrogenase with coupled reduction of DPN to DPNH. A factor in determining the rate of ethanol metabolism is the rate at which DPNH is reoxidized to DPN (Westerfield, 1961; Smith and Newman, 1959). Ethanol has been demonstrated to increase the DPNH levels in liver without altering appreciably the total amount of DPN plus DPNH, producing therefore an alteration in the DPN/DPNH ratio (Westerfield, 1961; Reboucas and Isselbacher, 1961). The DPN/DPNH ratio has been suggested to be a limiting factor in ethanol metabolism (Westerfield, 1961; Smith and Newman, 1959). Indeed, Smith and Newman (1959) found that agents which reoxidize DPNH to DPN increase the rate of alcohol metabolism. Based upon these observations it is possible that the protective effect of a-tocopherol and DPPD might be due to a maintenance of DPN-DPNH system, which is required for ethanol metabolism and which can inlluence the rate of alcohol metabolism. This postulate would require that the rate of ethanol metabolism is increased in the antioxidant-treated rats with a resulting decrease in hepatic injury. This concept is not supported by the observation that the degree and duration of the state of ethanol-induced intoxication was not significantly altered in the antioxidant treated group. A second and more likely possibility is that a-tocopherol and DPPD, acting as an intracellular antioxidants, prevents the formation of lipid peroxides resulting in maintenance of normal mitochondrial structure and enzyme activity (Tappel and Zalkin, 1959a, 1959b; Desai and Tappel, 1963). This hypothesis would imply that following administration of certain hepatotoxins the development of lipoperoxides or lipohydroperoxides or other complexes (Lea, 1963) is promoted. In support of this concept (Hove, 1953) has previously reported that various hepatotoxic agents such as carbon tetrachloride, benzene, and, tri-o-cresylphosphate
152
N.
R.
DI
LUZIO
AND
F.
COSTALES
function as pro-oxidants in unsaturated lipid systems and advanced the hypothesis relating their toxicity to the oxidative activity in unsaturated lipid systems. The unsaturated acids such as linoleic and oleic acid have been demonstrated to accumulate in livers of rats exposed to a single large dose of ethanol (Brodie et al., 1961a). It is of interest that linoleic acid appears to be one of the most significant oxidizable lipid component (Horwitt, 1960). The oxidation of unsaturated lipid in tissue and mitochondrial preparations has been demonstrated (Ottolenghi, 1959). It has also been reported that isolated mitochondria deteriorate by lipid peroxidation with impairment in certain mitochondrial enzymes (Tappel and Zalkin, 1959a), and that antioxidants were effective in stabilizing enzymes in isolated mitochondria, among which include DPNH cytochrome C reductase (Tappel and Zalkin, 1959b). The observed inhibition of biological activity, from ultraviolet irradiation has been found to be proportional to the amount of lipid peroxides formed. Similarly, the protection of certain enzyme systems in mitochondria from inhibition by ultraviolet irradiation is proportional to the degree of inhibition of peroxide formation (Barber and Ottolenghi, 1957). It is therefore possible that antioxidants by inhibiting the peroxidation of lipids or the formation of other oxidized lipid complexes (Lea, 1963) maintain essentially normal cell activity even in the presence of hepatotoxic agents with a resulting inhibition or prevention of hepatic injury. Ory and Altschul (1962) have demonstrated a lipase system which in the presence of a-tocopherol increases the hydrolysis of triglycerides. This finding would suggest that a third possible mechanism, at least in the a-tocopherol studies, might be an increased rate of hydrolysis of triglycerides with a resultant increased rate of lipid oxidation in the ethanol rats which received a-tocopherol. Smuckler et al., (1961), demonstrated a severe depression in protein formation by liver in CC14-treated animals and suggested that lipid deposition, mitochondrial changes, and necrosis may be dependent upon a defect in protein metabolism. Seakins and Robinson (1963) indicated that inhibition of plasma lipoprotein formation may be the mechanism of fatty liver development following carbon tetrachloride. Since the defect in plasma lipoprotein formation is the result of possible injury to the endoplasmic reticulum of the liver cell (Seakins and Robinson, 1963), the current studies would suggest that the damage to the endoplasmic reticulum would be greatly reduced in carbon tetrachloride-treated rats which received DPPD. Mitochondrial changes would also be anticipated to be greatly reduced in the antioxidant-treated rats exposed to carbon tetrachloride. It is obvious that additional studies will be required to delineate the role of altered cellular or mitochondrial enzyme activities in the development of the acute ethanol and CC& induced fatty liver. While no definitive conclusion can now be reached regarding the mechanism by which antioxidants inhibited the accumulation of hepatic triglycerides following either ethanol or CC14 exposure, it is significant that all postulated mechanisms involve maintenance of oxidative activity of the hepatic cell. The present studies of a preventative action of antioxidants on fatty liver development, in both the ethanol and CC& induced fatty liver, would suggest that, to some degree, a common factor is involved in the pathogenesis of these fatty livers. The inhibition of triglyceride ac-
ANTIOXIDANTS
AND
LIVER
INJURY
153
cumulation by a-tocopherol and DPPD, as presently reported and by other antioxidants (Di Luzio, 1964), in ethanol and CC14-treated rats provides a possibleinsight for the effective prevention and treatment of liver injury. SUMM.4RY The development of the acute ethanol-induced fatty liver was significantly inhibited by the prior administration of a-tocopherol acetate. Complete inhibition of the ethanol-induced fatty liver was induced by the prior intraperitoneal administration of the antioxidant, N,N’-Diphenyl-p-phenylenediamine (DPPD). DPPD reduced, but did not prevent, the ethanol-induced hepatic-triglyceride accumulation when administered orally simultaneously with ethanol. Significant inhibition of hepatic-triglyceride accumulation in carbon tetrachloride-treated rats, pre-treated with DPPD, was also noted. The CC14-treated group manifested fatty metamorphosis, central necrosis, polymorphonuclear cell infiltration, and a disruption of the architecture of the reticulum. These changes were markedly reduced in CC&-treated rats which had received DPPD. Since both the ethanoland Ccl,-induced fatty livers were significantly modified by antioxidant administration, it is suggestive that, to some degree, a common factor is involved in the pathogenesis of fatty livers from these agents. The possible use of antioxidants in the prevention or modification of liver injury is suggested. REFERENCES A. A., and OTTOLENGHI, A. (1957). Effect of ethylenediaminetetraacetate on lipid peroxide formation and succinoxidase inactivation by ultraviolet light. Proc. Sot. Exptl. Biol. G Med. 96, 471-476. BASSI, M. (1960). Electron microscopy of rat liver after carbon tetrachloride poisoning. Exptl. Cell Res. 20, 313-323. BRODIE, B. B., BUTLER, W. M., HORNING, M. G., MAICKEL, R. P., and MALING, H. M. (1961a). Alcohol-induced triglyceride deposition in liver through derangement of fat transport. Am. J. Clin. Nutr. 9, 432-435. BRODIE, B. B., MALING. H. M., HORNING, M. G., and MAICKEL, R. P. (196Ib). In “Drugs Affecting Lipid Metabolism” (S. Garattini and R. Paoletti, eds.) pp. 104-110. Elsevier Publishing Corn. pany, Amsterdam. CALDER, P. M. (1942). The protective action of xanthene and other insoluble substances on the liver. J. Pathol. Bacterial. 54, 369-373. DESAI, I. D. and TAPPEL, A. L. (1963). Damage to proteins by peroxidized lipids. J. Lipid Res.
BARBER,
4, 204-207.
Dr LUZIO, N. R. (1958). Effect of acute ethanol intoxication on liver and plasma lipid fractions of the rat. Am. J. Physiol. 194, 453-456. DI LUZIO, N. R. (1962). Comparative study of the effect of alcoholic beverages on the development of the acute ethanol-induced fatty liver. Quart. J. Stud. Ale. 23, 557-561. DI LUZIO, N. R. (1964). Prevention of the acute ethanol-induced fatty liver by the simultaneous administration of antioxidants. Life Sci. (In Press). ELI;O, E. E., WOOLES, W. R., and DI LUZIO, N. R. (1961). Alterations and mobilization of lipids in acute ethanol-treated rats. Am. J. Physiol. 201, 923-926. GALLAGHER, C. H. (1962). The effect of antioxidants on poisoning by carbon tetrachloride. The Austral. 1. Erptl. Biol. Med. Sci. 40, 241-254. HORWITT, M. K. (1960). Vitamin E and lipid metabolism in man. ilm. J. Clin. Nutr. 8, 451-461. HOVE, E. L. (1953). The toxicity of tri-o-cresyl phosphate for rats as related to dietary casein level, vitamin E and vitamin A. J. Nuts. 51, 609-622. JUDAH, J. D., AI~MED, K., and MCLEAN, A. E. M. (1963). Action of drugs on hepatic organelles. Ann. N. Y. Acad. Sci. 104, 926-938. LEA, C. H. (1963). In “Lipids and their Oxidation” (H. W. Schultz, ed.), pp. 3-28. The Avi Publishing Company, Inc., Westport, Conn. LIEBER, C. S. and SCHMID, R. (1961). The effect of ethanol on fatty acid metabolism stimulation of hepatic fatty acid synthesis. J. Clin. Invest. 40, 394-399.
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LUZIO
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H. M., FRANK, A., and HORNING, M. G. (1962). Effect of carbon tetracbloride on synthesis and release of triglycerides. Biochim. Biophys. Acta. 64, 540-545. MALLOV, S., and BLOCH, J. L. (1956). Role of hypophysis and adrenals in fatty infiltration of liver resulting from acute ethanol intoxication. Am. J. Physiol. 184, 29-34. NIKKILA, E. A., and OJALA, K. (1963). Role of hepatic L-n-glycerophosphate and triglyceride synthesis in production of fatty liver by ethanol. Proc. Sot. Exptl. Biol. Med. 113, 814-817. ORY, R. L., and ALTSCHUL, A. M. (1962). Participation of tocopherol derivatives in lipolysis. Biochem. Biophys. Res. Comm. 7, 370-374. OTTOLENGHI, A. (1959). Interaction of ascorbic acid and mitochondrial lipids. Arch. Biochem. Biophys. 79, 355-363. POCCI, M., and DI LUZIO, N. R. (1964). The role of liver and adipose tissue in the pathogenesis of the ethanol-induced fatty liver. J. Lipid Res. 5, 437-441, 1964. REBOUCAS, G., and ISSELBACHER, K. (1961). Studies of the pathogenesis of the ethanol-induced fatty liver: I. Synthesis and oxidation of fatty acids by the liver. Z. C&n. Invest. 40, 1355-1362. RECHNAGEL, R. O., LOMBARDI, B., and SCHOTZ, M. C. (1960). A new insight into pathogenesis of carbon tetrachloride fat infiltration. Proc. Sot. Exptl. Biol. Med. 104, 608-610. SEAKINS, A., and ROBINSON, D. S. (1963). The effect of the administration of carbon tetrachloride on the formation of plasma lipoproteins in the rat. Biochem. J. 86, 401-407. SMITH, M. E., and NEWMAN, H. W. (1959). The rate of ethanol metabolism in fed and fasting animals. J. Biol. Chem. 234, 1544-1549. SMUCKLER, E. A., ISERI, 0. A., and BENDITT, E. P. (1961). Studies on carbon tetrach!oride intoxication: I. The effect of carbon tetrachloride on incorporation of labelled amino acids into plasma proteins. Biochem. and Biophys. Rex Comm. 6, 270-275. SPERRY, W. M. (1955). In “Methods of Biochemical Analysis” (D. Blick, ed.), Vol. 2, pp. 83111. Interscience Publisher, Inc., New York. TAPPEL, A. L., and ZALKIN, H. (1959a). Lipid peroxidation in iso!ated mitochondria. Arch. Biochem. Biophys. 80, 326-332. TAPPEL, A. L., and ZALKIN, H. (1959b). Inhibition of lipid peroxidation in mitochondria by vitamin E. Ibid. 80, 333-336. VAN HANDEL, E., and ZILWRSMIT, D. B. (1957). Micromethod for the direct determination of serum triglycerides. J. Lab. Clin. Med. 50, 152-157. WESTERFIELD, W. W. (1961). The intermediary metabolism of alcohol. Am. 1. Clin. Nutr. 9, 426431. WILLIS, E. D. (1961). In “The Enzymes of Lipid Metabolism” (P. Desnuelle, ed.) pp. 74-77. Pergamon Press, New York. MALINC.,
hepatic
AND
MOLECULAR
Inhibition
of Induced
PATHOLOGY
the
141-154
4,
Ethanol Fatty
and
Liver
N. R. DI LUZIO Department
(1965)
AND
March
Tetrachloride
by Antioxidants’
of Physiology, University of Tenrzessee Department of Pathology, Baptist Memorial
Received
Carbon
F.
COSTALES~
Medical Units, Memphis, Tertnessee Hospital, Memphis, Tennessee
and
16, 1964
INTRODUCTION Although the development of a fatty liver in normal rats treated with a single intoxicating dose of ethanol or alcoholic beverages has been amply demonstrated (Mallov and Bloch, 1956; Di Luzio, 1958; Lieber and Schmid, 1961; Brodie et al., 1961a; Di Luzio, 1962), considerabledifferencesin opinion exist regarding the etiology of the triglyceride accumulation. Current concepts regarding its genesiscover essentially all possiblevariables, including increasedhepatic synthesis of lipid (Lieber and Schmid, 1961), increasedtriglyceride formation from free fatty acids (FFA) (Nikkila and Ojala, 1963), increased mobilization of free fatty acids from peripheral adipose depots (Brodie et al., 1961a, 1961b), and a decreasedintrahepatic utilization of triglyceride (Di Luzio, 1958; Elko et al., 1961: Poggi and Di Luzio, 1964; Di Luzio, 1964). In contrast to the mechanismof the ethanol-induced fatty liver, the carbon tetrachloride (CCld)-induced fatty liver has been demonstrated to be associated with a decreasedrate of hepatic triglyceride secretion (Rechnagel et al., 1960; Maling et al., 1962), and an enhanced rate of hepatic triglyceride synthesis (Maling et al., 1962). Gallagher (1962) has demonstrated that the intraperitoneal administration of antioxidants afford protection to rats given lethal dosesof carbon tetrachloride. Since multiple factors obviously exist relative to fatty liver induction and in view of the apparent dissimilar mechanismsbetween the ethanol- and carbon tetrachloride-induced fatty liver, studies were undertaken to evaluate the effects of antioxidant administration on the fatty liver induced by these agents. In view of the observation of Calder (1942), that many insoluble compounds injected intraperitoneally would protect against CC&-induced fatty liver, Judah et al., (1963) questioned the concept that antioxidants exert a specific effect on fatty liver development and indicated that the protective effect noted by Gallagher might be due to a non-specific action of the intraperitoneally administered material. In an effort to evaluate this possibility, antioxidants were administered orally simultaneously with ethanol, and the degree of triglyceride accumulation noted. 1 Supported, in part, by a grant from the C. D. Smithers Foundation, the Licensed Industries, Inc., and U. S. Public Health Service (AM-08084). 2 The capable technical assistance of Mrs. Betty Lyons is gratefully acknowledged. 141
Beverage
N. R. DI LUZIO AND F. COSTALES MATERIALS
AND
METHODS
Female rats (Holtzman Co., Madison, W&c.), previously maintained on a Purina Chow diet were employed. The animals were fasted for 8 hours prior to receiving by oral intubation, a single intoxicating dose of ethanol in the amount of 6 gr/Kg, as a 505% solution or isocaloric glucose. Carbon tetrachloride was administered orally to other rats in the amount of 0.4 ml,/100 gr, while an equivalent volume of saline was administered to control rats. The rats were killed with ether 24 hours later. Alphatocopherol acetate was suspended in saline and administered intraperitoneally in the amount of 10 mg/lOO gr 48, 24, and 2 hours prior to the oral administration of ethanol or glucose. Control rats received intraperitoneal injections of saline and the animals killed 16 hours later. N, N’-Diphenyl-p-phenylenediamine (DPPD) was suspended in corn oil and administered intraperitoneally in the amount of 60 mg/lOO gr 48, 24 and 2 hours prior to oral intubation. Control rats in this instance received corn oil intraperitoneally. The rats were killed 24 hours after oral intubation. In an effort to evaluate the oral effectiveness of DPPD, another group of rats received orally 1OOmg of DPPD/lOOgr of body weight simultaneously with ethanol (6 gr/Kg) and killed 16 hours later. A group of 28 rats were employed for histological studies, being distributed as follows : In the CC& study three control rats received corn oil intraperitoneally and saline orally; six received corn oil intraperitoneally and CC& orally and five rats received Ccl, orally after intraperitoneal administration of DPPD suspended in corn oil as described above. In the ethanol study seven rats received ethanol orally and corn oil intraperitoneally and another seven rats received ethanol after the intraperitoneal administration of DPPD and corn oil as described above. Liver sections of all animals were prepared with hematoxylin-eosin stain in paraffin embedded sections and Sudan IV in frozen sections. All preparations were evaluated without previous knowledge of the treatment received by the animals, and the results were graded from lf to 4+ on the basis of the following morphologic changes: (a) fatty metamorphosis, (b) cloudy swelling, (c) hydropic degeneration or ballooning of the hepatic cells, (d) acidophilic bodies, (e) hepatic cell necrosis, (f) polymorphonuclear cell infiltration and (g) mononuclear cell infiltration. After the slides had been evaluated and identified, a representative section from each group was stained with Gomori stain for reticulum. Plasma triglyceride concentrations were determined (Van Handel and Zilversmit, 19.57) liver lipids were extracted with chloroform-methanol and purified (Sperry, 1955) and the chloroform-methanol extract was analyzed for triglycerides (Van Handel and Zilversmit, 1957). All determinations were done in duplicate. The data were examined by means of the “t” test for the difference between means with a 95% confidence limit.
ANTIOXIDANTS
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RESULTS A.
Chemical findings
Plasma triglyceride concentrations were not significantly altered following ethanol administration (Table I). However, liver triglyceride concentration was significantIy of a-tocopherol increased 350% over control values. -4lthough the administration acetate to glucose-treated rats did not alter the concentration of liver and plasma triglycerides, the liver triglyceride concentration in the vitamin E-treated group which received ethanol was 3670 lower than that of the ethanol group. Plasma triglyceride concentrations were not altered from control values in the vitamin E-ethanol group. The administration of DPPD to glucose-treated rats produced a significant decrease in liver triglyceride concentration of 3372~ (Table II). The administration of corn oil to glucose-treated rats produced a significant increase in liver triglyceride concentration when compared to the saline injected glucose group (Table I). The administration of ethanol, as previously observed, resulted in a significant increase in liver TABLE EFFECT
OF U-TOCOPHEROL
I FMTY
ON THE ETHANOL-INDUCED
Triglyceride
Treatment Intraperitoneal Saline Saline Vitamin E
Oral Glucose Ethanol Glucose Ethanol
LIVERY
Vitamin
Liver w/gr
E
Plasma mg/100mI
10.5
k 2.0
47.1
k
5.5
10.4
t
1.3
22.7 25.9 23.0 33.3
30.2 t 3.1
a Values are means -+ standard error. The liver triglyceride values were derived group, while the plasma triglyceride values are from 5 rats per group. The female rates weighed approximately 2OOg and were killed 16 hr after ethanol
TABLE INFLUENCE
Treatment Intraperitoneal
Oral Glucose Glucose
Ethanol Ethanol a corn oral oral
Corn oil DPPD Corn oil DPPD
OF DPPD
No. per group 5 5 8 8
FATTY
Body weight
-c 2 t IL
Final 7 4 5 6
administration.
193 184 187 187
-c k zk 2
LIVERY
Liver triglyceride m/u
!zt.
197 189 197 193
8 rats per
II
ON THE ETHANOL-INDUCED
Initial
from
* 7.0 k 4.0 2 7.0 It 13.0
9 2 7 4
16.4 10.9 36.0 19.9
-r-e & 2
Plasma triglyceride ms% 1.7 2.7 5.7 3.1
33.5 rt 5.1 20.7 2 3.0 46.8 f 5.4
The glucose and ethanol solutions were administered by stomach tube to female rats, while the oil or DPPD solutions were given by intraperitoneal injection 48, 24 and 2 hours prior to the intubation. The liver and plasma triglyceride concentrations were determined 24 hours after intubation. Values are expressed as means -C standard error.
triglyceride (Table II). In contrast, the administration of ethanol to rats previously injected with DPPD resulted in a liver triglyceride concentration that was unaltered from the glucose and corn oil control group.
144
N.
R. DI LUZIO
AND
F. COSTALES
The administration of Ccl4 produced a more severe fatty liver than ethanol administration and resulted in a mean 400% elevation in liver triglyceride concentration when compared to saline-treated rats (Table III). Plasma triglyceride levels were not significantly altered 24 hours following carbon tetrachloride administration. The administration of CC14 to DPPD treated rats was associated with a 60% reduction in TABLE EFFECT OF DPPD Treatment
Saline ccl, ccl,
per group
Corn oil Corn oil DPPD
III
TETRACHLORIDE-INDUCED
NO.
Intraperitoneal
Oral
ON THE CARBON
7 8 7
FATTY
Liver triglyceride mdgr
Plasma triglyceride w%
17.8 -+ 1.3 88.8 k 9.2 35.0 2 2.4
37.5 f 10.0 56.1 k 10.0 71.8 k 32.0
LIVER”
FFA mEq/L 1.33 5 0.53 1.94 -+ 0.26 1.26 5~ 0.11
a Values are expressed as means k standard error. Ccl, or saline was administered orally in the amount of 0.4m1/100gr to female rats weighing approximately 200 gr DPPD was administered intraperitoneally in corn oil 48, 24 and 2 hours prior to oral intubation. The plasma and liver triglyceride and plasma FFA concentration were determined 24 hours after oral intubation. TABLE EFFECT
OF DPPD
IV
ADMINISTRATION SIMULTANEOUSLY WITH HEPATIC TRIGLYCERIDE CONCENTRATIONS
Body weight
Liver weight gr
gr Initial
ETHANOL
Group
DPPD
Final
Ethanol
-
232 -c 15
221 k 17
7.0 k 0.5
Ethanol
+
238k
224 k
7.5 -c 0.6
9
9
a Values, expressed as means k standard error are derived from 5 female hours after the administration of 6gr/Kg of ethanol orally in the absence and in the amount of lgr/Kg. The figures in parenthesis are the sample range.
ON
Liver triglyceride w/a 58.5 2 6.1 (48.5-79.2) 32.5 5 3.0 (27.0-39.6) rats per group 16 presence of DPPD
accumulation and an unaltered plasma triglyceride level. Plasma free fatty (FFA), although increased above normal levels, were not significantly altered within the various groups. The oral administration of DPPD simultaneously with ethanol resulted in a significant reduction in hepatic triglyceride accumulation when compared to ethanol-treated rats (Table IV). The triglyceride decrease in the DPPD group amounted to a 44% reduction. Liver and body weights were not altered in the DPPD group from control values. triglyceride
acids
B.
Histological
studies
The histological appearance of the liver of various rats in the experimental groups, as well as the chemically determined liver triglyceride concentrations, are tabulated in Table V. The saline control group, although fasted for a 24 hour period, showed no visible fat
ANTIOXIDANTS
100
AND
0000000
LIVER
145
INJURY
oooooooooo+ol~~~~~ +++
300
0000000
0000000+++++++++++ +
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0000000
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0+1 ooooo+++++++++++ y;w;+ +
300
+
++
++
+
0000000+++++++++++ +;;;;+
++++ +
‘oooo+o”++oo+ooo+++++++
+++
++
+++++ ;$f$; ++
too ~“~+o++o++oooo ++++++ ++++++ ++++-t +++++ ++++ + s++ Ooo+++++++ +++++++++++++ ++++t yzez+ ++++-t + ++z++
146
N.
R. DI
LUZIO
AND
F. COSTALES
with the Sudan IV stain nor any other liver alteration. In the animals which received ethanol, the fat content of the liver was significantly higher, five being graded 2+, one 3+, and another l+. The group that received ethanol after being treated with DPPD showed a lesser quantity of fat and was graded from 0 to I+. It is readily apparent that the histological evaluations were well correlated with the chemical findings as denoted by liver triglyceride concentrations. The fat in both ethanol-treated groups was present as fine droplets in the hepatic cells, primarily at the edges of the cytoplasm adjacent to the sinusoid and was evenly distributed throughout the hepatic lobule. Other morphological changes were inconspicuous. Mild cloudy swelling and hydropic degeneration were occasionally found in both groups. .. The rats that received Ccl+ alone showed the typical alterations of acute Ccl4 intoxication, i.e., fatty metamorphosis and central necrosis (Fig. 1). In the center of the hepatic lobule most of the hepatic architecture was markedly distorted, showing disappearance of the continuity of the hepa.tic cords. The sinusoids in the area were dilated, forming highly irregular vascular spaces. Many acidophilic bodies were present in isolated cells. The hepatic cells also exhibited acidophilic cytoplasm and pyknotic or fragmented nuclei. In the intermediate zone the necrotic changes were less extensive and only groups of cells were involved. The remainder, however, showed cloudy swelling. Many hepatic cells in this intermediate zone of the lobule were enlarged I>/2 to 2 times their normal size, compressing the adjacent sinusoids. These enlarged cells exhibited a clear cytoplasm and a centrally located nucleus. The hepatic cells at the periphery of the lobule were less severely involved, with only scattered cells showing cloudy swelling. Similarly, fatty metamorphosis was present in lesser degree in this area than in the intermediate zone of the lobule. Polymorphonuclear cell infiltration paralleled the necrotic changes. In marked contrast to the above findings the rats that had received Ccl, after the administration of DPPD showed less extensive necrotic changes than the preceding group (Fig. 2). The necrotic alterations, when present, were also predominantly centrolobular. The number of cells totally disrupted or with nuclear loss was smaller, and there was no loss of continuity of the hepatic cords with resulting disruption of the architecture and dilation of the sinusoidal spaces. The ballooning or hydropic degeneration of the hepatic cells in the intermediate zone was however, more conspicuous and marked compression of the sinusoid surrounding the greatly enlarged cells was observed. In contrast to the rats that received only CCII, the animals in this group showed the less severe degenerative changes, such as, cloudy swelling and hydropic degeneration which are usually considered reversible. In agreement with the chemical analysis of hepatic triglyceride, the quantity of fat in the Sudan IV-stained sections of the DPPD group which received Ccl4 was much less than in the CCll group (Table V). There was less polymorphonuclear cell infiltration in the antioxidant protected group, but in contrast with the CCll group a marked infiltration of mononuclear cells was present. These cells had kidney-shaped nuclei and were arranged in groups.
ANTIOXIDANTS
AND
LIVER
IN JURY
147
The Gomori stain for reticulum in a representative section of each group shower da m arked distortion of the architecture of the reticulum frame at the center of the lob la 1e inI the rats that received Ccl, (Fig. 3), while it was well preserved in the CC&-trea ted allimals that received DPPD (Fig. 4).
FIG. 1. Liver section from CC14-treated th e central vein and extensive necrosis. In ajority with a pyknotic or fragmented
rat demonstrating disruption of the architecture Only isolated hepatic cells are present in this nucleus. H & E 400 X.
are und area, the
148
N.
R. DI LUZIO
AND
I ?IG. 2. Liver section obtained from Ccl=,-treated Pal :ed to Fig. 1, there is no loss in the continuity
net :rosis
is reduced
and fewer
hepatic
cells show
F. COSTALES
rat which previously received DPPD. Co lrnof the hepatic cords. The degree of cellc dar pyknotic nuclei. H 8: E 400 X.
ANTIOXIDANTS
AND
FIG. 3. Disruption of the reticulum framework nuity of the hepatic cords is seen in CCll-treated al : one edge of the photograph. Gomori reticulum
LIVER
INJURY
in the centrolohular rat liver. A dilated stain. 400 X.
149
zone, with loss of :oncentrolobular vein is v ie;ible
1
N.
4. Liver section from 3, the reticulum framework
I hG.
Fig
R. DI LUZIO
CCld-treated is well
AND
F. COSTALES
rat which previously received DPPD. In preserved. Gomori reticulum stain. 400 X.
contrast
ANTIOXIDANTS
AND
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IN JURY
151
DISCUSSION Although disagreement exists relative to the pathogenesis of the fatty liver following acute ethanol ingestion, the development of fatty metamorphosis in normal rats following ethanol administration has been amply demonstrated (Mallov and Bloch, 1956; Di Luzio, 1958, 1962; Lieber and Schmid, 1961; Brodie et al., 1961a, 1961b; Elko et al., 1961). The present data amply confirm these findings. Various antioxidants have been demonstrated to be effective in preventing hepatic alterations and increasing survival following carbon tetrachloride poisoning (Gallagher, 1962). Our findings of a significant reduction in triglyceride accumulation in rats pretreated with a-tocopherol or DPPD appear to support the observations of Gallagher (1962) that hepatic alterations following the administration of hepatotoxins can be prevented, in part, by the use of antioxidants. The oral effectiveness of DPPD, as well as other antioxidants (Di Luzio, 1964) implies that the effect of antioxidants is not a nonspecific action as suggested (Judah et al., 1963), but a reflection of a specific effect of antioxidants (Di Luzio, 1964). Although the mechanism of the protective effect of antioxidants on liver triglyceride accumulation following ethanol or Ccl+ administration remains to be ascertained, the protection of a-tocopherol and other antioxidants against the lethal effects of carbon tetrachloride has been credited to maintenance of normal levels of reduced diphosphopyridine nucleotides (DPNH) and a decreased loss of oxidized pyridine nucleotides (DPN) (Gallagher, 1962 ) . The oxidation of ethanol in the liver is catalyzed by alcohol dehydrogenase with coupled reduction of DPN to DPNH. A factor in determining the rate of ethanol metabolism is the rate at which DPNH is reoxidized to DPN (Westerfield, 1961; Smith and Newman, 1959). Ethanol has been demonstrated to increase the DPNH levels in liver without altering appreciably the total amount of DPN plus DPNH, producing therefore an alteration in the DPN/DPNH ratio (Westerfield, 1961; Reboucas and Isselbacher, 1961). The DPN/DPNH ratio has been suggested to be a limiting factor in ethanol metabolism (Westerfield, 1961; Smith and Newman, 1959). Indeed, Smith and Newman (1959) found that agents which reoxidize DPNH to DPN increase the rate of alcohol metabolism. Based upon these observations it is possible that the protective effect of a-tocopherol and DPPD might be due to a maintenance of DPN-DPNH system, which is required for ethanol metabolism and which can inlluence the rate of alcohol metabolism. This postulate would require that the rate of ethanol metabolism is increased in the antioxidant-treated rats with a resulting decrease in hepatic injury. This concept is not supported by the observation that the degree and duration of the state of ethanol-induced intoxication was not significantly altered in the antioxidant treated group. A second and more likely possibility is that a-tocopherol and DPPD, acting as an intracellular antioxidants, prevents the formation of lipid peroxides resulting in maintenance of normal mitochondrial structure and enzyme activity (Tappel and Zalkin, 1959a, 1959b; Desai and Tappel, 1963). This hypothesis would imply that following administration of certain hepatotoxins the development of lipoperoxides or lipohydroperoxides or other complexes (Lea, 1963) is promoted. In support of this concept (Hove, 1953) has previously reported that various hepatotoxic agents such as carbon tetrachloride, benzene, and, tri-o-cresylphosphate
152
N.
R.
DI
LUZIO
AND
F.
COSTALES
function as pro-oxidants in unsaturated lipid systems and advanced the hypothesis relating their toxicity to the oxidative activity in unsaturated lipid systems. The unsaturated acids such as linoleic and oleic acid have been demonstrated to accumulate in livers of rats exposed to a single large dose of ethanol (Brodie et al., 1961a). It is of interest that linoleic acid appears to be one of the most significant oxidizable lipid component (Horwitt, 1960). The oxidation of unsaturated lipid in tissue and mitochondrial preparations has been demonstrated (Ottolenghi, 1959). It has also been reported that isolated mitochondria deteriorate by lipid peroxidation with impairment in certain mitochondrial enzymes (Tappel and Zalkin, 1959a), and that antioxidants were effective in stabilizing enzymes in isolated mitochondria, among which include DPNH cytochrome C reductase (Tappel and Zalkin, 1959b). The observed inhibition of biological activity, from ultraviolet irradiation has been found to be proportional to the amount of lipid peroxides formed. Similarly, the protection of certain enzyme systems in mitochondria from inhibition by ultraviolet irradiation is proportional to the degree of inhibition of peroxide formation (Barber and Ottolenghi, 1957). It is therefore possible that antioxidants by inhibiting the peroxidation of lipids or the formation of other oxidized lipid complexes (Lea, 1963) maintain essentially normal cell activity even in the presence of hepatotoxic agents with a resulting inhibition or prevention of hepatic injury. Ory and Altschul (1962) have demonstrated a lipase system which in the presence of a-tocopherol increases the hydrolysis of triglycerides. This finding would suggest that a third possible mechanism, at least in the a-tocopherol studies, might be an increased rate of hydrolysis of triglycerides with a resultant increased rate of lipid oxidation in the ethanol rats which received a-tocopherol. Smuckler et al., (1961), demonstrated a severe depression in protein formation by liver in CC14-treated animals and suggested that lipid deposition, mitochondrial changes, and necrosis may be dependent upon a defect in protein metabolism. Seakins and Robinson (1963) indicated that inhibition of plasma lipoprotein formation may be the mechanism of fatty liver development following carbon tetrachloride. Since the defect in plasma lipoprotein formation is the result of possible injury to the endoplasmic reticulum of the liver cell (Seakins and Robinson, 1963), the current studies would suggest that the damage to the endoplasmic reticulum would be greatly reduced in carbon tetrachloride-treated rats which received DPPD. Mitochondrial changes would also be anticipated to be greatly reduced in the antioxidant-treated rats exposed to carbon tetrachloride. It is obvious that additional studies will be required to delineate the role of altered cellular or mitochondrial enzyme activities in the development of the acute ethanol and CC& induced fatty liver. While no definitive conclusion can now be reached regarding the mechanism by which antioxidants inhibited the accumulation of hepatic triglycerides following either ethanol or CC14 exposure, it is significant that all postulated mechanisms involve maintenance of oxidative activity of the hepatic cell. The present studies of a preventative action of antioxidants on fatty liver development, in both the ethanol and CC& induced fatty liver, would suggest that, to some degree, a common factor is involved in the pathogenesis of these fatty livers. The inhibition of triglyceride ac-
ANTIOXIDANTS
AND
LIVER
INJURY
153
cumulation by a-tocopherol and DPPD, as presently reported and by other antioxidants (Di Luzio, 1964), in ethanol and CC14-treated rats provides a possibleinsight for the effective prevention and treatment of liver injury. SUMM.4RY The development of the acute ethanol-induced fatty liver was significantly inhibited by the prior administration of a-tocopherol acetate. Complete inhibition of the ethanol-induced fatty liver was induced by the prior intraperitoneal administration of the antioxidant, N,N’-Diphenyl-p-phenylenediamine (DPPD). DPPD reduced, but did not prevent, the ethanol-induced hepatic-triglyceride accumulation when administered orally simultaneously with ethanol. Significant inhibition of hepatic-triglyceride accumulation in carbon tetrachloride-treated rats, pre-treated with DPPD, was also noted. The CC14-treated group manifested fatty metamorphosis, central necrosis, polymorphonuclear cell infiltration, and a disruption of the architecture of the reticulum. These changes were markedly reduced in CC&-treated rats which had received DPPD. Since both the ethanoland Ccl,-induced fatty livers were significantly modified by antioxidant administration, it is suggestive that, to some degree, a common factor is involved in the pathogenesis of fatty livers from these agents. The possible use of antioxidants in the prevention or modification of liver injury is suggested. REFERENCES A. A., and OTTOLENGHI, A. (1957). Effect of ethylenediaminetetraacetate on lipid peroxide formation and succinoxidase inactivation by ultraviolet light. Proc. Sot. Exptl. Biol. G Med. 96, 471-476. BASSI, M. (1960). Electron microscopy of rat liver after carbon tetrachloride poisoning. Exptl. Cell Res. 20, 313-323. BRODIE, B. B., BUTLER, W. M., HORNING, M. G., MAICKEL, R. P., and MALING, H. M. (1961a). Alcohol-induced triglyceride deposition in liver through derangement of fat transport. Am. J. Clin. Nutr. 9, 432-435. BRODIE, B. B., MALING. H. M., HORNING, M. G., and MAICKEL, R. P. (196Ib). In “Drugs Affecting Lipid Metabolism” (S. Garattini and R. Paoletti, eds.) pp. 104-110. Elsevier Publishing Corn. pany, Amsterdam. CALDER, P. M. (1942). The protective action of xanthene and other insoluble substances on the liver. J. Pathol. Bacterial. 54, 369-373. DESAI, I. D. and TAPPEL, A. L. (1963). Damage to proteins by peroxidized lipids. J. Lipid Res.
BARBER,
4, 204-207.
Dr LUZIO, N. R. (1958). Effect of acute ethanol intoxication on liver and plasma lipid fractions of the rat. Am. J. Physiol. 194, 453-456. DI LUZIO, N. R. (1962). Comparative study of the effect of alcoholic beverages on the development of the acute ethanol-induced fatty liver. Quart. J. Stud. Ale. 23, 557-561. DI LUZIO, N. R. (1964). Prevention of the acute ethanol-induced fatty liver by the simultaneous administration of antioxidants. Life Sci. (In Press). ELI;O, E. E., WOOLES, W. R., and DI LUZIO, N. R. (1961). Alterations and mobilization of lipids in acute ethanol-treated rats. Am. J. Physiol. 201, 923-926. GALLAGHER, C. H. (1962). The effect of antioxidants on poisoning by carbon tetrachloride. The Austral. 1. Erptl. Biol. Med. Sci. 40, 241-254. HORWITT, M. K. (1960). Vitamin E and lipid metabolism in man. ilm. J. Clin. Nutr. 8, 451-461. HOVE, E. L. (1953). The toxicity of tri-o-cresyl phosphate for rats as related to dietary casein level, vitamin E and vitamin A. J. Nuts. 51, 609-622. JUDAH, J. D., AI~MED, K., and MCLEAN, A. E. M. (1963). Action of drugs on hepatic organelles. Ann. N. Y. Acad. Sci. 104, 926-938. LEA, C. H. (1963). In “Lipids and their Oxidation” (H. W. Schultz, ed.), pp. 3-28. The Avi Publishing Company, Inc., Westport, Conn. LIEBER, C. S. and SCHMID, R. (1961). The effect of ethanol on fatty acid metabolism stimulation of hepatic fatty acid synthesis. J. Clin. Invest. 40, 394-399.
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H. M., FRANK, A., and HORNING, M. G. (1962). Effect of carbon tetracbloride on synthesis and release of triglycerides. Biochim. Biophys. Acta. 64, 540-545. MALLOV, S., and BLOCH, J. L. (1956). Role of hypophysis and adrenals in fatty infiltration of liver resulting from acute ethanol intoxication. Am. J. Physiol. 184, 29-34. NIKKILA, E. A., and OJALA, K. (1963). Role of hepatic L-n-glycerophosphate and triglyceride synthesis in production of fatty liver by ethanol. Proc. Sot. Exptl. Biol. Med. 113, 814-817. ORY, R. L., and ALTSCHUL, A. M. (1962). Participation of tocopherol derivatives in lipolysis. Biochem. Biophys. Res. Comm. 7, 370-374. OTTOLENGHI, A. (1959). Interaction of ascorbic acid and mitochondrial lipids. Arch. Biochem. Biophys. 79, 355-363. POCCI, M., and DI LUZIO, N. R. (1964). The role of liver and adipose tissue in the pathogenesis of the ethanol-induced fatty liver. J. Lipid Res. 5, 437-441, 1964. REBOUCAS, G., and ISSELBACHER, K. (1961). Studies of the pathogenesis of the ethanol-induced fatty liver: I. Synthesis and oxidation of fatty acids by the liver. Z. C&n. Invest. 40, 1355-1362. RECHNAGEL, R. O., LOMBARDI, B., and SCHOTZ, M. C. (1960). A new insight into pathogenesis of carbon tetrachloride fat infiltration. Proc. Sot. Exptl. Biol. Med. 104, 608-610. SEAKINS, A., and ROBINSON, D. S. (1963). The effect of the administration of carbon tetrachloride on the formation of plasma lipoproteins in the rat. Biochem. J. 86, 401-407. SMITH, M. E., and NEWMAN, H. W. (1959). The rate of ethanol metabolism in fed and fasting animals. J. Biol. Chem. 234, 1544-1549. SMUCKLER, E. A., ISERI, 0. A., and BENDITT, E. P. (1961). Studies on carbon tetrach!oride intoxication: I. The effect of carbon tetrachloride on incorporation of labelled amino acids into plasma proteins. Biochem. and Biophys. Rex Comm. 6, 270-275. SPERRY, W. M. (1955). In “Methods of Biochemical Analysis” (D. Blick, ed.), Vol. 2, pp. 83111. Interscience Publisher, Inc., New York. TAPPEL, A. L., and ZALKIN, H. (1959a). Lipid peroxidation in iso!ated mitochondria. Arch. Biochem. Biophys. 80, 326-332. TAPPEL, A. L., and ZALKIN, H. (1959b). Inhibition of lipid peroxidation in mitochondria by vitamin E. Ibid. 80, 333-336. VAN HANDEL, E., and ZILWRSMIT, D. B. (1957). Micromethod for the direct determination of serum triglycerides. J. Lab. Clin. Med. 50, 152-157. WESTERFIELD, W. W. (1961). The intermediary metabolism of alcohol. Am. 1. Clin. Nutr. 9, 426431. WILLIS, E. D. (1961). In “The Enzymes of Lipid Metabolism” (P. Desnuelle, ed.) pp. 74-77. Pergamon Press, New York. MALINC.,
hepatic