EXPERIMENTAL
AND
MOLECULAR
Endotoxin
PATHOLOGY
55,
196-202 (191)
Hepatotoxicity
Augmented
YUROSHIBAYAMA,SHOSAKUASAKA,
by Ethanol
ANDKATSUJI
Department of Pathology, Osaka Medical College, Daigaku-Cho,
NAKATA
Takatsuki City, Osaka, Japan
Received February 14, 1991, and in revised form May 21, 1991 To determine whether alcohol increases endotoxin hepatotoxicity, we administered ethanol (4.8 g/kg body wt in 4 ml of water) to rats through a gastric tube, then immediately injected endotoxin (2, 2.5, or 3 m&g body wt). In the rats pretreated with ethanol, the injection of 2 mg/kg body wt of endotoxin induced a slight rise of serum transaminase. However, when 2.5 mg/kg body wt of endotoxin was given, there were no significant histopathological or biochemical differences between the rats pretreated with ethanol and those pretreated with water. Moreover, there was no significant difference in mortality rates between the rats pretreated with ethanol and the controls when 3 mg/kg body wt (LD,,) of endotoxin was injected. These results suggest that acute administration of alcohol enhances endotoxin hepatotoxicity when the dose of endotoxin is small, but that the effect of alcohol is masked when larger doses of endotoxin are given. o ISI Academic press. hc.
INTRODUCTION There is a recent theory that endotoxemia is related to the development of alcoholic hepatic injury. Small amounts of enterically derived endotoxin are regularly absorbed into the portal blood through the mucous membrane and are usually taken up by Kupffer cells in the process of absorptive phagocytosis (Nolan, 1981; Praaning-van Dalen et al., 1981). Alcohol may induce endotoxemia by depressing the endotoxin-detoxifying capacity of Kupffer cells and increasing the permeability of mucous membranes (Ali and Nolan, 1967; Liu, 1979; Lohnberg et al., 1981; Bjarnason et al., 1984; Bode et al., 1987). Accordingly, alcohol may aggravate endotoxin hepatotoxicity, since alcohol enhances the effects of hepatotoxic agents (Nolan and Camara, 1982; Lieber, 1990). However, it is not known whether there is synergism between alcohol and endotoxin. It is known that alcohol-induced fatty livers are more susceptible to hepatotoxic effects of even minimal doses of endotoxin (Bhagwandeen et al., 1987) and that chronic ethanol consumption potentiates endotoxin hepatotoxicity due to hypersensitivity to endotoxin in the coagulation-fibrinolysis system affected by chronic ethanol consumption (Arai et al., 1989). On the other hand, it has been reported that the acute administration of ethanol prevents endotoxin hepatotoxicity by inhibiting endotoxin-induced platelet activation and intravascular coagulation (Arai et al., 1987). In the present study, the effect of alcohol on the hepatotoxicity caused by endotoxin was determined in a rat model with hepatic damage produced by endotoxin. MATERIALS
AND METHODS
Male Wistar rats weighing approximately 230 g were allowed free access to a standard pellet diet and water. Under ether anesthesia, endotoxin (as a solution of 2, 2.5, or 3 mg/kg body wt in 1 ml of physiological saline solution) was injected into the tail vein immediately after the administration of ethanol (as a solution of 4.8 g/kg body wt in 4 ml of water) through a gastric tube. Endotoxin (lipopoly196 0014-4800/91 $3.00 Copy&ht 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENDOTOXIN
HEPATOTOXICITY
AND
saccharide B, Escherichia cofi 026:B6, Difco Laboratories) tuted in sterile pyrogen-free water at the time of injection. The rats were divided into the following groups: Experiment Group A Group B Group C Group D Experiment Group A Group B Group C Group D Experiment Group A Group B Group C Group D
197
ALCOHOL
was freshly reconsti-
I: Challenge with 2 mg/kg body wt of endotoxin. (N = 10): water + physiological saline. (N = 10): ethanol + physiological saline. (N = 10): water + endotoxin. (N = 10): ethanol + endotoxin. II: Challenge with 2.5 mg/kg body wt of endotoxin. (N = 10): water + physiological saline. (N = 10): ethanol + physiological saline. (N = 10): water + endotoxin. (N = 10): ethanol + endotoxin. III: Challenge with 3 mg/kg body wt of endotoxin. (N = 10): water + physiological saline. (N = IO): ethanol + physiological saline. (N = 23): water + endotoxin. (N = 21): ethanol + endotoxin.
Blood samples were taken for liver function tests 24 hr after the administration of physiological saline or endotoxin under ether anesthesia. The animals were killed, and their livers were extirpated and weighed. Each liver was fixed in 10% neutral formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin for histopathological examination. The grade of hepatocellular necrosis was assessed by the number of necrotic foci per 100 mm2 of liver section. Body weight, liver weight, and liver function tests were expressed as means ? SD and analyzed statistically by the analysis of variance. The Mann-Whitney test was used to compare the extent of hepatocellular necrosis, and the x2 test was used for mortality. The level of significance was P < 0.05 in all tests.
RESULTS Experiment
I
Body weight, liver weight, serum transaminase activities, and grade of hepatocellular necrosis are shown in Table I. There were no deaths during the 24 hr after treatment. There were no conspicuous macroscopic or histological changes in the livers of Group A and Group B rats. Small focal and random hepatocellular coagulative necrotic areas with infiltration of neutrophils and mononuclear cells were seen sporadically in the livers of only a few Group C and Group D rats. There was no significant difference in the morphological changes between Group C and Group D livers. There was no statistically significant difference between the serum transaminase levels of Group A and Group B rats. Glutamic pyruvic transaminase activity was elevated in Group C, but glutamic oxaloacetic transaminase activity was not higher than in Group A and Group B rats. Group D serum transaminase activities were significantly higher than those of Group A, Group B, and Group C.
10 10 10 10
II. Experiment II (2.5 mg per kg body wt of endotoxin) A. Water + physiological saline B. Ethanol + physiological saline C. Water + endotoxin D. Ethanol + endotoxin
228 235 225 231
232 241 236 230
+ +2 -c
2 2 f +
11 17 10 7
13 11 12 15
Body wt w
8.2 8.0 8 .8 9.1
8.3 8.4 8.4 8.3
0.5 0.5 0.6 0.6
-t 0.4 2 0.5 -c_0 .6a.b + 0.5”,b
k * ? _’
Liver wt (8)
1.89 1.93 3.35 3.22
1.84 1.88 1.98 2.13
* + ‘2
+ 2 f f
0.04 0.05 0.4Pb 0.57”ab
0.09 0.10 0.15” 0.19“,b,c
SGOT (log U/liter)
1.28 1.35 3.13 3.03
1.25 1.28 1.43 1.85
+ 2 + +
0.06 0.07 0.36“,’ 0.41a.b
I+_0.08 * 0.08 2 O.l2”,b 2 0.16”.b,c
SGPT (log Uiliter)
10 10 0 0
10 10 8 7
0
0 0 2 1
0 0 2 3
l-4
l
0 0 6 6
0 0 0 0
5-24
3a.b
2a.b
0 0
25+
Grade of hepatocellular necrosis Number of necrotic foci*
Necrosis in Rats Given Ethanol,
Note. N, number of animals; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase. Number of necrotic foci per lOO-mm’ liver section. osb,cP < 0.05, significantly different from A, B, and C in the same experimental group. Mean * SD.
10 10 10 10
I. Experiment I (2 mg per kg body wt of endotoxin) A. Water + physiological saline B. Ethanol + physiological saline C. Water + endotoxin D. Ethanol + endotoxin
N
TABLE I Body Weight, Liver Weight, Serum Transaminase Activities, and Grade of Hepatocellular Then Injected with Endotoxin
ENDOTOXIN
HEPATOTOXICITY
AND ALCOHOL
199
Experiment II
Body weight, liver weight, serum transaminase activities, and grade of hepatocellular necrosis are shown in Table I. There were no deaths during the 24 hr after treatment. The liver weights of Group C and Group D rats were significantly greater than those of Group A and Group B rats. There were no conspicuous macroscopic or microscopic changes in the livers of Group A and Group B rats. In all Group C and Group D rats, focal hepatocellular coagulative necrotic areas were seen with infiltration of neutrophils and mononuclear cells (Fig. 1). In severe cases, the focal hepatocellular coagulative necrotic lesions coalesced. There was no significant difference in the morphological changes between Group C and Group D livers. Serum transaminase activities were much higher in Group C and Group D rats than in Group A and Group B rats. There was no statistically significant difference between the activities in Group C and Group D rats. Experiment III
Body weight, mortality, serum transaminase activities, and grade of hepatocellular necrosis are shown in Table II. There was no mortality during the 24 hr after treatment in Group A and Group B, but the mortality rate was high in Group C and Group D, and there was no statistically significant difference between Group C and Group D. There were no conspicuous morphological changes in the livers of Group A and Group B rats. In all surviving Group C and Group D rats, focal hepatocellular
FIG. 1. Liver from a rat injected with 2.5 mgkg body wt of endotoxin immediately after intragastric administration of 4.8 gikg body wt of ethanol. Focal coagulative hepatocellular necrosis with intiltration of neutrophils and mononuclear cells is seen in the lobule. (H&E, X200).
A. B. C. D.
10 10 23 21
241 238 234 244
? +f t
12 15 11 18
o/10 o/10 12/23 9/21
(0%) (0%) (52%)“,b (43%)“,b
Mortality 1.79 1.84 3.19 3.31
” r 2 f
0.06 0.09 0.6Y,b 0.51”sb
SGOT* (log Uiliter) 1.24 1.30 3.03 3.18
f 2 + k
0.07 0.07 0.46”~~ 0.42”,b
XiPT* (log U/liter)
10 10 0 0
0
0 0 2 3
l-4
0 0 7 5
5-24
4a.b
y,b
0 0
25+
Grade of hepatocellular necrosis* Number of necrotic foci?
Necrosis in Rats Given Ethanol, Then Injected with
Note. N, number of animals; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase. * Data from surviving rats. t Number of necrotic foci per 100-mm2 liver section. a,b,cP < 0.05, significantly different from A, B, and C in the same experimental group. Mean 2 SD.
Water + physiological saline Ethanol + physiological saline Water + endotoxin Ethanol + endotoxin
N
Body wt (g)
TABLE II Body Weight, Mortality Rate, Serum Transaminase Activities, and Grade of Hepatocellular Endotoxin (3 mg/kg body wt)
5
F
i ,s > ?
E B %
ENDOTOXIN
HEPATOTOXICITY
AND
ALCOHOL
201
necrotic areas were found to the same extent as in Group C and Group D rats in Experiment II; there was no significant difference in the morphological changes between Group C and Group D rats. The serum transaminase activities in the surviving Group C and Group D rats were significantly greater than in the Group A and Group B rats; there was no statistically significant difference between Group C and Group D activities. DISCUSSION The present study clearly demonstrates that acute administration of alcohol slightly potentiates endotoxin hepatotoxicity when the dose of endotoxin is small. The-exact mechanism for the increase in endotoxin hepatotoxicity by ethanol is not known, but there are some possible explanations. Depression of Kupffer cell function due to alcohol may be associated with the potentiation of endotoxininduced hepatic injury (Ali and Nolan, 1967; Liu, 1979; Lohnberg et al., 1981; Bode et al., 1987). However, we do not support-this hypothesis, because we know that the phagocytic activity of the reticuloendothelial system is not related to the mortality rate or to the degree of hepatic damage following endotoxemia (Shibayama et al., in press). Increased lipid peroxidation and free radical generation by both endotoxin and ethanol might contribute to the augmentation of endotoxin hepatotoxicity by ethanol (Kalish and Di Luzio, 1966; Comporti, 1985; Arthur et al., 1985; Ghezzi et al., 1986; Sugino et al., 1987). We have no direct evidence against this hypothesis, but our preliminary study shows that the administration of free radical scavengers does not provide significant improvement in endotoxininduced hepatotoxicity. Hepatic hypoxia may contribute to the increase in endotoxinhepatotoxicity caused by alcohol; because it is well known that ethanol produces hepatic hypoxia through an increase in oxygen consumption in the liver (Israel et al., 1975;.Thurman et al., 1979)~and that hepatic hypoxia raises sensitivity to endotoxin hepatotoxicity (Shibayama, 1987). If this is true, one would expect the hepatocellular necrosis to be located primarily in the centrilobular areas. In the present study, however, the extent and the location of hepatocellular necrosis were not affected by ethanol administration. These findings suggest that the augmentation of endotoxin hepatotoxicity, the elevation of serum transaminase activities, is due to injury of liver cell membranes. We therefore believe that the augmentation of endotoxin hepatotoxicity by ethanol is due to hepatic hypoxia rather than to a depression of Kupffer cell function or to increased lipid peroxidation and free radical generation. The present study also demonstrates that the increase in endotoxin hepatotoxicity caused by the acute administration of alcohol is not seen when larger doses of endotoxin are given. Moreover, the mortality rate is not affected by alcohol when the LD5, of endotoxin is given. These results suggest that the augmentation of endotoxin hepatotoxicity by alcohol is masked at sublethal doses of endotoxin, and that endotoxin hepatotoxicity is less affected by alcohol than expected. REFERENCES M. V., and NOLAN, J. P. (1967). Alcohol induced depression of reticuloendothelial function in the rat. J. Lab. Clin. Med. 70, 295-301. ARAI, M., OKUNO, F., HIRANO, Y., NAKANO, S., KOBAYASHI, T., ISHII, H., and TSUCHIYA, M. ALI,
202
SHIBAYAMA,
ASAKA,
AND NAKATA
(1987). Protective effect of acute ethanol administration on endotoxin-induced liver injury. Alcohol Alcohol. (Suppl) 1, 487-491. ARAI, M., NAKANO, S., OKUNO, F., HIRANO, Y., SUJITA, K., KOBAYASHI, T., ISHII, H., and TSUCHIYA, M. (1989). Endotoxin-induced hypercoagulability: A possible aggravating factor of alcoholic liver disease. Hepatology 9, 846-851. ARTHUR, M. J. P., BENTLEY, I. S., TANNER, A. R., KOWALSKI SAUNDERS, P., MILLWARD-SADLER, G. H., and WRIGHT, R. (1985). Oxygen-derived free radicals promote hepatic injury in the rat. Gastroenterology 89, 1114-l 122. BHAGWANDEEN, B. S., APTE, M., MANWARRING, L., and DICKESON, J. (1987). Endotoxin induced hepatic necrosis in rats on an alcohol diet. J. Pathol. 151, 47-53. BJARNASON, I., WARD, K., and PETERS, T. J. (1984). The leaky gut of alcoholism: Possible route of entry for toxic compounds. Lancer i, 179-182. BODE, C., KUGLER, V., and BODE, J. C. (1987). Endotoxemia in patients with alcoholic and nonalcoholic cirrhosis and in subjects with no evidence of chronic liver disease following acute alcohol excess. J. Hepatol. 4, 8-14. COMPORTI, M. (1985). Biology of disease. Lipid peroxidation and cellular damage in toxic liver injury. Lab. invest. 53, 599-623. GHEZZI, P., SACCARDO, B., and BIANCHI, M. (1986). Role of reactive oxygen intermediates in the hepatotoxicity of endotoxin. Immunopharmacology 12, 241-244. ISRAEL, Y., KALANT, H., ORREGO, H., KHANNA, J. M., VIDELA, L., and PHILLIPS, J. M. (1975). Experimental alcohol-induced hepatic necrosis: Suppression by propylthiouracil. Proc. Natl. Acad. Sci. USA 12, 1137-l 141. KALISH, G. H., and Dr LUZIO, N. R. (1966). Peroxidation of liver lipids in the pathogenesis of the ethanol-induced fatty liver. Science 152, 139&1392. KUNIMOTO, F., MORITA, T., OGAWA, R., and FUJITA, T. (1987). Inhibition of lipid peroxidation improves survival rate of endotoxemic rats. Circ. Shock 21, 15-22. LIEBER, C. S. (1990). Mechanism of ethanol induced hepatic injury. Pharmacol. Ther. 46, l-41. LIEBER, C. S. (1990). Interaction of ethanol with drugs, hepatotoxic agents, carcinogens and vitamins. Alcohol Alcohol. 25, 157-176. Lru, Y. K. (1979). Phagocytic capacity of reticuloendothelial system in alcoholics. J. Reticuloendothel. Sot. 25, 605613. LOHNBERG, G., FRIMAN, L., and BERGHEM, L. (1981). Reticuloendothelial function in patients with alcoholic cirrhosis. Stand. J. Gastroenterol. 16, 481-489. NOLAN, J. (1981). Endotoxin, reticuloendothelial function and liver injury. Hepatology 1, 458-465. NOLAN, J. P., and CAMARA, D. S. (1982). Endotoxin, sinusoidal cells and liver injury. In “Progress in Liver Disease” (H. Popper and F. Schaffner, Eds.), Vol. 7, pp. 361-376. Grune & Stratton, New York. PRAANING-VAN DALEN, D. P., BROUWER, A., and KNOOK, D. L. (1981). Clearance capacity of rat liver Kupffer, endothelial and parenchymal cells. Gastroenterology 81, 10361044. SHIBAYAMA, Y. (1987). Enhanced hepatotoxicity of endotoxin by hypoxia. Pathol. Res. Pratt. 182, 390-39s. SHIBAYAMA, Y., HASHIMOTO, K., and NAKATA, K. Relation of reticuloendothelial function to endotoxin hepatotoxicity. Exp. Pathof., in press. SUGINO, K., DOHI, K., YAMADA, K., and KAWASAKI, T. (1987). The role of lipid peroxidation in endotoxin-induced hepatic damage and the protective effect of antioxidants. Surgery 101, 746-752. THURMAN, R. G., YUKI, T., BLEYMAN, M. A., and WENDELL, G. (1979). The adaptive increase in ethanol metabolism due to pretreatment with ethanol: A rapid phenomenon. Drug Alcohol Depend. 4, 119-129.
AND
MOLECULAR
Endotoxin
PATHOLOGY
55,
196-202 (191)
Hepatotoxicity
Augmented
YUROSHIBAYAMA,SHOSAKUASAKA,
by Ethanol
ANDKATSUJI
Department of Pathology, Osaka Medical College, Daigaku-Cho,
NAKATA
Takatsuki City, Osaka, Japan
Received February 14, 1991, and in revised form May 21, 1991 To determine whether alcohol increases endotoxin hepatotoxicity, we administered ethanol (4.8 g/kg body wt in 4 ml of water) to rats through a gastric tube, then immediately injected endotoxin (2, 2.5, or 3 m&g body wt). In the rats pretreated with ethanol, the injection of 2 mg/kg body wt of endotoxin induced a slight rise of serum transaminase. However, when 2.5 mg/kg body wt of endotoxin was given, there were no significant histopathological or biochemical differences between the rats pretreated with ethanol and those pretreated with water. Moreover, there was no significant difference in mortality rates between the rats pretreated with ethanol and the controls when 3 mg/kg body wt (LD,,) of endotoxin was injected. These results suggest that acute administration of alcohol enhances endotoxin hepatotoxicity when the dose of endotoxin is small, but that the effect of alcohol is masked when larger doses of endotoxin are given. o ISI Academic press. hc.
INTRODUCTION There is a recent theory that endotoxemia is related to the development of alcoholic hepatic injury. Small amounts of enterically derived endotoxin are regularly absorbed into the portal blood through the mucous membrane and are usually taken up by Kupffer cells in the process of absorptive phagocytosis (Nolan, 1981; Praaning-van Dalen et al., 1981). Alcohol may induce endotoxemia by depressing the endotoxin-detoxifying capacity of Kupffer cells and increasing the permeability of mucous membranes (Ali and Nolan, 1967; Liu, 1979; Lohnberg et al., 1981; Bjarnason et al., 1984; Bode et al., 1987). Accordingly, alcohol may aggravate endotoxin hepatotoxicity, since alcohol enhances the effects of hepatotoxic agents (Nolan and Camara, 1982; Lieber, 1990). However, it is not known whether there is synergism between alcohol and endotoxin. It is known that alcohol-induced fatty livers are more susceptible to hepatotoxic effects of even minimal doses of endotoxin (Bhagwandeen et al., 1987) and that chronic ethanol consumption potentiates endotoxin hepatotoxicity due to hypersensitivity to endotoxin in the coagulation-fibrinolysis system affected by chronic ethanol consumption (Arai et al., 1989). On the other hand, it has been reported that the acute administration of ethanol prevents endotoxin hepatotoxicity by inhibiting endotoxin-induced platelet activation and intravascular coagulation (Arai et al., 1987). In the present study, the effect of alcohol on the hepatotoxicity caused by endotoxin was determined in a rat model with hepatic damage produced by endotoxin. MATERIALS
AND METHODS
Male Wistar rats weighing approximately 230 g were allowed free access to a standard pellet diet and water. Under ether anesthesia, endotoxin (as a solution of 2, 2.5, or 3 mg/kg body wt in 1 ml of physiological saline solution) was injected into the tail vein immediately after the administration of ethanol (as a solution of 4.8 g/kg body wt in 4 ml of water) through a gastric tube. Endotoxin (lipopoly196 0014-4800/91 $3.00 Copy&ht 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
ENDOTOXIN
HEPATOTOXICITY
AND
saccharide B, Escherichia cofi 026:B6, Difco Laboratories) tuted in sterile pyrogen-free water at the time of injection. The rats were divided into the following groups: Experiment Group A Group B Group C Group D Experiment Group A Group B Group C Group D Experiment Group A Group B Group C Group D
197
ALCOHOL
was freshly reconsti-
I: Challenge with 2 mg/kg body wt of endotoxin. (N = 10): water + physiological saline. (N = 10): ethanol + physiological saline. (N = 10): water + endotoxin. (N = 10): ethanol + endotoxin. II: Challenge with 2.5 mg/kg body wt of endotoxin. (N = 10): water + physiological saline. (N = 10): ethanol + physiological saline. (N = 10): water + endotoxin. (N = 10): ethanol + endotoxin. III: Challenge with 3 mg/kg body wt of endotoxin. (N = 10): water + physiological saline. (N = IO): ethanol + physiological saline. (N = 23): water + endotoxin. (N = 21): ethanol + endotoxin.
Blood samples were taken for liver function tests 24 hr after the administration of physiological saline or endotoxin under ether anesthesia. The animals were killed, and their livers were extirpated and weighed. Each liver was fixed in 10% neutral formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin for histopathological examination. The grade of hepatocellular necrosis was assessed by the number of necrotic foci per 100 mm2 of liver section. Body weight, liver weight, and liver function tests were expressed as means ? SD and analyzed statistically by the analysis of variance. The Mann-Whitney test was used to compare the extent of hepatocellular necrosis, and the x2 test was used for mortality. The level of significance was P < 0.05 in all tests.
RESULTS Experiment
I
Body weight, liver weight, serum transaminase activities, and grade of hepatocellular necrosis are shown in Table I. There were no deaths during the 24 hr after treatment. There were no conspicuous macroscopic or histological changes in the livers of Group A and Group B rats. Small focal and random hepatocellular coagulative necrotic areas with infiltration of neutrophils and mononuclear cells were seen sporadically in the livers of only a few Group C and Group D rats. There was no significant difference in the morphological changes between Group C and Group D livers. There was no statistically significant difference between the serum transaminase levels of Group A and Group B rats. Glutamic pyruvic transaminase activity was elevated in Group C, but glutamic oxaloacetic transaminase activity was not higher than in Group A and Group B rats. Group D serum transaminase activities were significantly higher than those of Group A, Group B, and Group C.
10 10 10 10
II. Experiment II (2.5 mg per kg body wt of endotoxin) A. Water + physiological saline B. Ethanol + physiological saline C. Water + endotoxin D. Ethanol + endotoxin
228 235 225 231
232 241 236 230
+ +2 -c
2 2 f +
11 17 10 7
13 11 12 15
Body wt w
8.2 8.0 8 .8 9.1
8.3 8.4 8.4 8.3
0.5 0.5 0.6 0.6
-t 0.4 2 0.5 -c_0 .6a.b + 0.5”,b
k * ? _’
Liver wt (8)
1.89 1.93 3.35 3.22
1.84 1.88 1.98 2.13
* + ‘2
+ 2 f f
0.04 0.05 0.4Pb 0.57”ab
0.09 0.10 0.15” 0.19“,b,c
SGOT (log U/liter)
1.28 1.35 3.13 3.03
1.25 1.28 1.43 1.85
+ 2 + +
0.06 0.07 0.36“,’ 0.41a.b
I+_0.08 * 0.08 2 O.l2”,b 2 0.16”.b,c
SGPT (log Uiliter)
10 10 0 0
10 10 8 7
0
0 0 2 1
0 0 2 3
l-4
l
0 0 6 6
0 0 0 0
5-24
3a.b
2a.b
0 0
25+
Grade of hepatocellular necrosis Number of necrotic foci*
Necrosis in Rats Given Ethanol,
Note. N, number of animals; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase. Number of necrotic foci per lOO-mm’ liver section. osb,cP < 0.05, significantly different from A, B, and C in the same experimental group. Mean * SD.
10 10 10 10
I. Experiment I (2 mg per kg body wt of endotoxin) A. Water + physiological saline B. Ethanol + physiological saline C. Water + endotoxin D. Ethanol + endotoxin
N
TABLE I Body Weight, Liver Weight, Serum Transaminase Activities, and Grade of Hepatocellular Then Injected with Endotoxin
ENDOTOXIN
HEPATOTOXICITY
AND ALCOHOL
199
Experiment II
Body weight, liver weight, serum transaminase activities, and grade of hepatocellular necrosis are shown in Table I. There were no deaths during the 24 hr after treatment. The liver weights of Group C and Group D rats were significantly greater than those of Group A and Group B rats. There were no conspicuous macroscopic or microscopic changes in the livers of Group A and Group B rats. In all Group C and Group D rats, focal hepatocellular coagulative necrotic areas were seen with infiltration of neutrophils and mononuclear cells (Fig. 1). In severe cases, the focal hepatocellular coagulative necrotic lesions coalesced. There was no significant difference in the morphological changes between Group C and Group D livers. Serum transaminase activities were much higher in Group C and Group D rats than in Group A and Group B rats. There was no statistically significant difference between the activities in Group C and Group D rats. Experiment III
Body weight, mortality, serum transaminase activities, and grade of hepatocellular necrosis are shown in Table II. There was no mortality during the 24 hr after treatment in Group A and Group B, but the mortality rate was high in Group C and Group D, and there was no statistically significant difference between Group C and Group D. There were no conspicuous morphological changes in the livers of Group A and Group B rats. In all surviving Group C and Group D rats, focal hepatocellular
FIG. 1. Liver from a rat injected with 2.5 mgkg body wt of endotoxin immediately after intragastric administration of 4.8 gikg body wt of ethanol. Focal coagulative hepatocellular necrosis with intiltration of neutrophils and mononuclear cells is seen in the lobule. (H&E, X200).
A. B. C. D.
10 10 23 21
241 238 234 244
? +f t
12 15 11 18
o/10 o/10 12/23 9/21
(0%) (0%) (52%)“,b (43%)“,b
Mortality 1.79 1.84 3.19 3.31
” r 2 f
0.06 0.09 0.6Y,b 0.51”sb
SGOT* (log Uiliter) 1.24 1.30 3.03 3.18
f 2 + k
0.07 0.07 0.46”~~ 0.42”,b
XiPT* (log U/liter)
10 10 0 0
0
0 0 2 3
l-4
0 0 7 5
5-24
4a.b
y,b
0 0
25+
Grade of hepatocellular necrosis* Number of necrotic foci?
Necrosis in Rats Given Ethanol, Then Injected with
Note. N, number of animals; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase. * Data from surviving rats. t Number of necrotic foci per 100-mm2 liver section. a,b,cP < 0.05, significantly different from A, B, and C in the same experimental group. Mean 2 SD.
Water + physiological saline Ethanol + physiological saline Water + endotoxin Ethanol + endotoxin
N
Body wt (g)
TABLE II Body Weight, Mortality Rate, Serum Transaminase Activities, and Grade of Hepatocellular Endotoxin (3 mg/kg body wt)
5
F
i ,s > ?
E B %
ENDOTOXIN
HEPATOTOXICITY
AND
ALCOHOL
201
necrotic areas were found to the same extent as in Group C and Group D rats in Experiment II; there was no significant difference in the morphological changes between Group C and Group D rats. The serum transaminase activities in the surviving Group C and Group D rats were significantly greater than in the Group A and Group B rats; there was no statistically significant difference between Group C and Group D activities. DISCUSSION The present study clearly demonstrates that acute administration of alcohol slightly potentiates endotoxin hepatotoxicity when the dose of endotoxin is small. The-exact mechanism for the increase in endotoxin hepatotoxicity by ethanol is not known, but there are some possible explanations. Depression of Kupffer cell function due to alcohol may be associated with the potentiation of endotoxininduced hepatic injury (Ali and Nolan, 1967; Liu, 1979; Lohnberg et al., 1981; Bode et al., 1987). However, we do not support-this hypothesis, because we know that the phagocytic activity of the reticuloendothelial system is not related to the mortality rate or to the degree of hepatic damage following endotoxemia (Shibayama et al., in press). Increased lipid peroxidation and free radical generation by both endotoxin and ethanol might contribute to the augmentation of endotoxin hepatotoxicity by ethanol (Kalish and Di Luzio, 1966; Comporti, 1985; Arthur et al., 1985; Ghezzi et al., 1986; Sugino et al., 1987). We have no direct evidence against this hypothesis, but our preliminary study shows that the administration of free radical scavengers does not provide significant improvement in endotoxininduced hepatotoxicity. Hepatic hypoxia may contribute to the increase in endotoxinhepatotoxicity caused by alcohol; because it is well known that ethanol produces hepatic hypoxia through an increase in oxygen consumption in the liver (Israel et al., 1975;.Thurman et al., 1979)~and that hepatic hypoxia raises sensitivity to endotoxin hepatotoxicity (Shibayama, 1987). If this is true, one would expect the hepatocellular necrosis to be located primarily in the centrilobular areas. In the present study, however, the extent and the location of hepatocellular necrosis were not affected by ethanol administration. These findings suggest that the augmentation of endotoxin hepatotoxicity, the elevation of serum transaminase activities, is due to injury of liver cell membranes. We therefore believe that the augmentation of endotoxin hepatotoxicity by ethanol is due to hepatic hypoxia rather than to a depression of Kupffer cell function or to increased lipid peroxidation and free radical generation. The present study also demonstrates that the increase in endotoxin hepatotoxicity caused by the acute administration of alcohol is not seen when larger doses of endotoxin are given. Moreover, the mortality rate is not affected by alcohol when the LD5, of endotoxin is given. These results suggest that the augmentation of endotoxin hepatotoxicity by alcohol is masked at sublethal doses of endotoxin, and that endotoxin hepatotoxicity is less affected by alcohol than expected. REFERENCES M. V., and NOLAN, J. P. (1967). Alcohol induced depression of reticuloendothelial function in the rat. J. Lab. Clin. Med. 70, 295-301. ARAI, M., OKUNO, F., HIRANO, Y., NAKANO, S., KOBAYASHI, T., ISHII, H., and TSUCHIYA, M. ALI,
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