Acute Ethanol Intoxication Stimulates Superoxide Anion Production by In Situ Perfused Rat Liver ABRAHAM P. BAUTISTA AND JOHN J. SPITZER Department of Physiology, Louisiana State University Medical Center, New Orleans, Louisiana 70112

This study examines the generation of superoxide anion by the perfused rat liver after ethanol intoxication and acute endotoxemia to assess the potential importance of oxygen-derived free radicals in the ethanol-induced hepatic pathological condition. Hepatic superoxide anion production of 0.65 f 0.06 nmol/min/gm liver weight was measured 1 hr after ethanol infusion; it reached a peak value of 0.8 f 0.07 at 3 hr and was reduced to 0.11 f 0.01 by 7 hr. In a group of animals, 4-methylpyrazolewas injected 5 min before the administration of ethanol to determine whether the metabolism of ethanol moiety is necessary for the observed effects. However, no significant inhibition of superoxide production was observed after 4-methylpyrazole administration. Introduction of ibuprofen into the perfused liver abolished superoxide anion production, suggesting that arachidonic acid metabolites may play an important role in superoxide generation under these conditions. Endotoxin, a potent activator of macrophages, has also been associated with increased superoxide release by the liver. Therefore the combined impact of ethanol and endotoxin on superoxide production by the liver was also examined. Acute ethanol intoxication inhibited the endotoxin-mediated superoxide anion generation by the perfused liver. These data indicate that the ethanol-mediated superoxide production and the ethanol-induced inhibition of the lipopolysaccharideenhancedfree-radical generation by the liver may have a pathophysiological significance in tissue injury and in resistance to infection. (HEPATOLOGY 1992;15892898.)

Oxygen-derived free radicals have been implicated in the pathogenesis of alcohol-induced tissue injury (1-4). Because the liver is the organ largely responsible for the metabolism of alcohol, it is likely to be directly exposed to the deleterious effects of oxygen-derived toxic radicals. The mechanism of radical formation during acute or chronic ethanol intoxication is not fully defined. However, it has been shown that these metabolites may

Received August 2, 1991; accepted December 12, 1991. This work was supported by National Institute on Alcohol Abuse and Alcoholism grants AA 08846 (A. P. Bautista) and AA 07287 (J. J. Spitzer). Address reprint requests to: Dr. Abraham P, Bautista, Department of PhysioIogy, Louisiana State University Medical Center, New Orleans, LA 70112. 3111135674

be generated through the hypoxanthine and xanthine oxidase reactions (5, 61, and in the course of the metabolism of ethanol to acetaldehyde (7, 8). Although more than 90%of the cellular components of the liver are hepatocytes, our previous studies failed to show measurable release of superoxide anion by hepatocytes isolated from alcoholic or endotoxemic rats (9). However, Kupffer cells, the resident macrophages of the liver, were active in superoxide anion production after either endotoxin (9) or tumor necrosis factor (TNF) administration (10). Sequestered neutrophils in the liver have also been shown to generate superoxide anion on stimulation by phorbol ester (9-11) or acetaldehydealtered membranes of hepatocytes (12). The production of such radicals by phagocytic cells is one of the mechanisms involved in the microbicidal activities of these cells. Superoxide anion is an oxygen-derived radical released as a consequence of ethanol metabolism (7, 8) or activation of phagocytic cells with soluble or particulate materials (9, 11, 13, 14). Dorio et al. (15) have shown that low-dose and short-term ethanol treatment can stimulate phosphatidylinositol turnover and the release of superoxide anion by alveolar macrophages. Paradoxically, ethanol has an attenuating effect on the phorbol ester-induced production of superoxide anion (15) and on the lipopolysaccharide (LPS)-mediated activation of hepatic phagocytes (9). It has been postulated that such attenuation may contribute to immunosuppression in alcoholic individuals, thereby enhancing their susceptibility to having infections develop. Although superoxide anion is the least toxic of the oxygen-derived radicals, this molecule spontaneously dismutates to generate hydrogen peroxide. As a consequence, other more toxic radicals are generated, such as the hydroxyl radical (in the presence of a suitable metal catalyst) and hypochlorite through the participation of haloperoxidase (16, 17). The more toxic radicals are difficult to measure, whereas the superoxide anion can be measured in the intact perfused rat liver, as has been performed after endotoxemia (18)and in viva activation of the retimloendothelial system (19). This study is based on the hypothesis that acute ethanol intoxication attenuates some aspects of the immune response and also enhances the production of oxygen-derived radicals in the liver. Because it was

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ETHANOL-INDUCED SUPEROXIDE PRODUCTION IN THE LIVER

found that ethanol exhibited both of these effects, the two following questions were also raised. (a) Is the metabolism of the ethanoI moiety necessary for these effects? (b) Do metabolites of arachidonic acid participate in mediating these effects? MATERIALS AND METHODS Preparatwn of Experimental Animals. On the day before

the experiments, male Sprague-Dawley rats (250-300 gm, Charles River Breeding Laboratories, Wilmington, MA) were cannulated with venous and arterial catheters using aseptic surgical techniques. Food was removed 18 hr before the experiments, but the animals were allowed free access to water. The next day ethanol was injected intravenously as a bolus at a dose of 1.75 gm/kg body wt. This was followed by a continuous infusion of 250 to 300 mg/kg/hr for 1to 7 hr. Using this dose, the blood ethanol level was maintained at 170 +9 mg/dl. In some experiments, rats were treated intravenously with 80 mg/kg 4-methylpyrazole (Sigma Chemical Co., St. Louis, MO) 5 min before ethanol administration. Under this condition, continuous infusion of ethanol was not carried out because blood ethanol levels were maintained at 178 7 mg/d when alcohol dehydrogenase was inactivated by 4-methylpyrazole, as has been demonstrated by previous experiments from this laboratory (20). In another experimental group, Escherichia coli LPS (LPS, Difco, Detroit, MI) was injected intravenously at a dose of 1 mg/kg/body wt at the same time as ethanol was given. The control group received sterile saline instead of ethanol. All experimental animals in this study received humane care accordingto the guidelines outlined in the “Guide for the Care and Use of Laboratory Animals” by the National Academy of Sciences (National Institutes of Health publication no. 80-23). The experimental protocol for the use of live animals in this study was approved by the LSU Medical Center Institutional Animal Care and Use Committee. Isolation of Liver Cells. Hepatocytes, endothelial cells and Kupffer cells were isolated from the liver by collagenasepronase digestion followed by centrifugal elutriation as described previously (14, 21). The liver was perfused in a circulating system for 5 min with 25 mg collagenase (Sigma Chemical Co.) in 100 ml HBSS supplemented with 0.001% deoxyribose (Sigma Chemical Co.) and 1 mmol CaC1,. The organ was surgically removed, cut into small pieces, resuspended in Gey’s balanced salt solution and filtered through four layers of sterile gauze. The resulting cell suspension was centrifuged at 50 g for 2 min to sediment the hepatocytes. The remaining tissues were further digested with 0.2% pronase E (Sigma Chemical Co.), 0.001% deoxyribonuclease in Gey’s balanced salt solution for 40 min at room temperature. Endothelial and Kupffer cells were collected by centrifugal elutriation at 875 g at 23 ml/min (endothelial cells) and 45 ml/min (Kupffer cells) flow rates using a Beckman elutriator (52-21 centrifuge, JE-6B elutriator rotor; Beckman Inc., Palo Alto, CA). Using these methods, the purity of hepatocytes, endothelial cells and Kupffer cells were above 95% as assessed by morphological method (Giemsa-Wright stain) and peroxidase stain. Cell viability was above 97% as determined by the trypan blue exclusion technique. Peroxidase Staining. Peroxidase positive cells were identified using a modified peroxidase staining technique described previously (22). Twenty microliters of cell suspension (10 x 106 cells/ml) was added to 180 pl of Gey’s balanced salt solution containing 1 mg/ml of diaminobenzidine (Sigma Chemical Co.) and 1%hydrogen peroxide (Fisher Scientific,

*

893

Fair Lawn, NJ). The cells were incubated for 10 min at 37” C. An aliquot (5 pl) was taken and examined under a microscope. Brown-stained cells were considered peroxidase positive. Kupffer cells were peroxidase positive whereas endothelial cells were negative. Measurement of Superoxide Anion Release by In Situ Perfised Liver. The portal vein was cannulated with a 20-gauge

Telfon catheter (Delmed, Canton, MA). The liver was perfused with oxygen-saturated calcium-free HBSS. The superior vena cava cannula served as the outflow tract after ligation of the inferior vena cava. The blood was removed from the liver by continuous perfusion of 200 ml of the buffer at 2 to 3 ml/min/gm wet liver at 37” C. Once the liver was cleared of blood, HBSS containing 32 pmol ferricytochrome C (Sigma Chemical Co.), 5 mmol glucose and 0.8% BSA (Sigma Chemical Co.) was perfused through this organ. Phorbol myristate acetate ( P W , l o p 7 mol/L) was added at 4 to 5 min, whereas superoxide dismutase (SOD; 7,500 IU/liver) was introduced into the perfusion medium 8 to 10 min after the substrate had entered the liver. To determine the effect of cyclooxygenase inhibitors on superoxide anion release, ibuprofen (5 mg/ml/min) was infused into the liver without cytochrome C for 5 min followed by a 10-min perfusion in the presence of cytochrome C. Aliquots (2.5 ml) of the perfusates were immediately placed in an ice bath, and when all the tubes were collected they were centrifuged t o remove contaminating cells and debris. The change in absorbance was measured at 550 nm. The concentration of detectable superoxide anion during liver perfusion was measured based on the slope of the delta absorbance of the reduced ferricytochrome C (minus SOD), which was converted in nanomoles using the molecular extinction coefficient of 21.1 mol/L-l* cm-l (11, 13). The value was then expressed as nanomoles per minute (of perfusion) per gram wet weight. This procedure has been described previously (18). Superoxide Anion Assay on Isolated Cells. Isolated hepatocytes, endothelial cells and Kupffer cells in HBSS were each layered onto a 35-cm sterile petri dish (Costar) at a final cell density of 0.75 to 1.0 x lo6 celldplate. Ferricytochrome C (50 pmol) was added to the reaction mixture. SOD (300 unitdwell) was added to the negative control. PMA was added as a stimulant at a final concentration of 1 pmol. Superoxide anion was measured based on a change in absorbance (difference in absorbance with or without SOD) against a cell-free blank. Delta absorbance was converted into nanomoles per milliliter using the molecular extinction coefficient of 21.1 FmoUL- emp l. Superoxideanion was expressed in nmol/106 cellskr. TNF Assay. TNF activity in serum was measured using the L929 cell line cytotoxicity assay as described previously (23). Statistics. Data presented in this paper represent mean 2 S.E. of 5 to 10 independent experiments. Statistical significancewas assessed by Student’s t test and was applicable by nonparametric statistical analysis.

RESULTS Effect of Ethanol Infusion on Superoxide Anion Production by In Situ Perfused Liver. Figure 1 shows

that ethanol infusion for 3 hr induced the reduction of ferricytochrome C by the in situ perfused liver. The rate of reduction was significantly greater than in the saline-treated control. The addition of SOD toward the end of perfusion demonstrated that the reduction was caused by superoxide anions because the reduction of

BAUTISTA AND SPITZER

894

HEPATOLOGY

TABLE 1. Superoxide anion production by in situ perfused liver after ethanol intoxication

1

OJ

Superoxide anion production" (nmol/min/emliver) ~~~~

~

0 1 3 5

7

J r 0

.

I 2

.

I 4

-

m 6

-

.

-

8

i

control

A ElOH 3 hour

A ~ J h a r + S W

FIG.1.Reduction of ferricytochrome C by the in situ perfused liver after ethanol (ETOH) infusion. Arrows indicate the addition of PMA and SOD. Mean ? S.E. of eight independent experiments.

O'O

g

h

O1

0

I n In

i

PMA

I

-I

-

'

v

0.4

z 6

m

ct

m 4

amounts of superoxide anion detected in the perfused liver at different time intervals after ethanol infusion are shown in Table 1. Control livers generated 0.04 & 0.003 nmol/min of perfusiodgm liver of superoxide anion. In the presence of PMA the amount of superoxide anion was increased in the ethanol-treated groups (particularly at 3 hr) compared with the control group (Table 1 and Fig. 2). The PMA effect was consistent in all ethanol-treated rats. The maximum production of superoxide anion in the presence of PMA was 1.04 k 0.17 nmol/min/gm liver at 3 hr. The PMA effect indicated a reproducible priming of superoxide anion production by ethanol.

-

Effect of 4-Methylpyrazole on Ethanol-induced Production of Superoxide Anion. Because the amount of

.

superoxide anion detected in the perfused liver increased after ethanol treatment at 3 hr, this time point was selected to examine whether inhibition of ethanol metabolism during ethanol intoxication would have an effect on the release of superoxide anion by the liver. Figure 3 shows that although a trend was seen for a modest inhibition of the amount of superoxide anion produced by the liver, these changes did not reach statistical significance. Thus the oxidative metabolism of ethanol is not necessary for most of the observed effects. Administration of 4-methylpyrazole alone did not have any effect on superoxide production by the control livers.

0 v1

0 0.82 5 0.14' 1.04 2 0.17" 0.62 t 0.07' 0.29 t 0.05"

10

Time (minutes) 0

~

Mean 2 S.E. of four to nine experiments. "Time-matched controls have been subtracted from the above values. 'Total superoxide produced (basal + PMA). cp < 0.05 vs. time zero.

u 0.0

0 0.65 rfi 0.06" 0.80 5 0.07" 0.39 2 0.055' 0.11 t 0.01"

0.2

-

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a

I

0

.

I

.

I

2

4

.

I

.

6

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8

.

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l

10

Time (minutes) 0 Control V ETOH 5 hour

0 ETOH 1 hour

A ETOH 3 hour 0 ETOH 7 hour

FIG.2. Reduction of ferricytochrome C by the in situ perfused liver after the infusion of ethanol (ETOH). Arrows indicate the addition of PMA or SOD. Mean & S.E. of four to nine independent experiments.

ferricytochrome was inhibited in the presence of this enzyme (Fig. 1). The effect of SOD on the inhibition of ferricytochrome C reduction in control livers was not significant, indicating that this was not the result of superoxide anion production. Time Course of the Ethanol Effect. Figure 2 shows that 1hr after ethanol infusion a significant reduction of ferricytochrome C occurred. The peak effect was observed at 3 hr, and the effect started to decline thereafter. At 7 hr, the SOD-inhibitable reduction of ferricytochrome C was very small. The estimated

Effect of Ibuprofen on the Superoxide Anion Production. We have previously demonstrated that super-

oxide anion produced by latex-stimulated liver and immunologically sensitized hepatic and blood phagocytes was inhibited by ibuprofen, suggesting the involvement of arachidonic acid metabolites in the generation of this radical by phagocytes (16). Figure 4 shows that in uitro administration of ibuprofen into the perfused liver completely inhibited superoxide anion formation after acute ethanol intoxication. The PMAinduced release of superoxide anion was also inhibited (Fig. 4). Effect of In Vivo Ethanol Infusion on Superoxide Production by Isolated Hepatic Cells In Vitro. To determine which cells were responsible for the ethanol-

Val. 15, No. 5, 1992

ETHANOL-INDUCED SUPEROXIDE PRODUCTION IN THE LIVER

TABLE 2. Superoxide anion production by isolated hepatocytes and nonparenchymal cells after the i n t r a v e n o u s infusion of ethanol Superoxide anion (nmoUlO* cellsh) Control 3 hr of ethanol

Cell type

Hepatocytes Endothelid cells Kupffer cells

0.05 f 0.02 (0.06 f 0.01) 0.10 0.03 (0.10 5 0.05) 0.65 f 0.25 (1.2 0.18)

*

*

~

0.03 f 0.01 (0.05 f 0.03) 0.12 f 0.05 (0.24 t 0.1) 2.01 t 0.18” (5.0 f 0.97’)

7 hr of ethanol

0.05 t 0.02 (0.04 t 0.01) 0.1 lr. 0.01 (0.13 t 0.08) 0.9 f 0.3 (1.13 5 0.2)

~

Figures in parentheses refer to amount of superoxide produced in the presence of 1 kmol PMA. N = 7. ”p < 0.05 vs. control. ‘p < 0.01 vs. control.

induced superoxide production by the perfused liver, hepatic cells were isolated and assayed for superoxide anion production in uitro in the presence or absence of PMA. Table 2 shows that a barely detectable amount of superoxide anion was generated spontaneously by hepatocytes and endothelial cells. Kupffer cells generated more superoxide spontaneously as compared with hepatocytes and endothelial cells. After ethanol infusion at 3 hr, the spontaneous release of superoxide by Kupffer cells was increased 2.6-fold. The addition of PMA further increased superoxide anion production by Kupffer cells after 3 hr of ethanol infusion. At 7 hr after ethanol, superoxide production by Kupffer cells returned to normal levels. Superoxideproduction by either endothelial cells or hepatocytes was not altered after ethanol infusion. Ef’rect of Ethanol on Superoxide Production in Endotoxemic Rats. In an earlier study we showed that acute

ethanol intoxication for 7 hr attenuated the endotoxin (LPS)-inducedpriming of hepatic phagocytes to release superoxide anion (9). Figure 5 shows a significant amount of superoxide anion production of 0.7 nmol/min/gm. This amount was further increased to 2.1 nmol on the addition of phorbol ester, as shown by the arrows in Figure 5. The infusion of ethanol in addition to LPS treatment attenuated the LPS-induced superoxide anion release at 3 hr after treatment, although the amount of superoxide anion was still greater than the parallel control. The PMA effect was also inhibited significantly. At 7 hr after ethanol treatment and 3 hr after LPS, the amount of superoxide anion generation by the perfused liver was almost completely abolished (Fig. 6). Serum TNF Concentration after Ethanol Intoxication and Endotoxemia. TNF has been implicated as one of the key mediators in the enhanced production of

superoxide anion by phagocytic cells (24-26); therefore we monitored serum TNF during these experiments. TNF activity in serum was not detected at different time intervals for 7 hr after ethanol infusion. At 90 min after LPS treatment, the amount of serum TNF was 24,250 k 7,230 IU/ml. In the parallel group treated with

895 Phi4

1

0.8 3

5 5v1: 0.6 5

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0.4

-

0.2

-

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E: 9 0

r a Q

0.0 J I

I

2

0

6

4

8

10

TIME (rnin)

FIG. 3. Effect of 4-methylpyrazole on the reduction of ferricytochrome C by the in situ perfused liver. Control + 4-mmethylpyrazole (v), ethanol + 4-methylpyrazole (u). Shaded areas refer to S.E. for 3 h r of ethanol (0) and control (0)without 4-methylpyrazole. Mean t S.E. of 6 to 10 independent experiments.

Pw

E

o*8

1

1

SOD I

0.0 J 1

I

0

2

4

6

8

10

TIME (min)

FIG. 4. Effect of ibuprofen on the reduction of ferricytochrome C by the perfused rat liver. Control + ibuprofen (D), 3 hr of ethanol + ibuprofen ( 1. Shaded areas refer to S.E. for control ( 0 )and 3 hr of ethanol ( 0 ) without ibuprofen. Mean f S.E. of 5 to 10 independent experiments.

+

ethanol, serum TNF was reduced to 13,650 k 3,250 IU/ml (p < 0.05, by nonparametric statistical analysis) (Table 3). This observation is in accordance with our previous findings (27). DISCUSSION

In this study we demonstrate that intravenous infusion of ethanol initiates a chain of events that results in the release of superoxide anion in the isolated perfused rat liver. These events occur at blood ethanol concentrations that are similar to “social drinking.” The generation of superoxide anion was somewhat enhanced when the alcoholic liver was treated with PMA, a known activator of protein kinase C and superoxide anion

BAUTISTA AND SPITZER

-

1.0

TABLE 3. S e r u m TNF activity after acute ethanol infusion and endotoxin t r e a t m e n t

SOD

-E

0.1

-

0.8

-

Serum TNF (IU/ml)"

Treatment group

LPS

0

g v

HEPATOLOGY

~

0.4

-

0.2

-

~

~

5 to 8. "p < 0.05 (nonparametric statistical analysis). N

W

U 0

24,450 2 7,230 13,678? 3,275

LPS plus ethanol =

v)

4

0.0

0

;

2

;I

0

10

Tima (minutes)

0 Control A ETOH 3 houn

0 LPS

0 EIY)H+LPS3 houn

FIG.5. Reduction of ferricytochrome C by in situ perfused liver after 3 hr of ethanol (ETOH) infusion and endotoxin treatment. Mean t S.E. of 5 to 10 independent experiments.

1.0,

-

0.8

-

0.6

-

0.4

-

0.2

-

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0

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.

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.

2

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4

.

,

.

6

,

.

8

,

10

Time (minutes)

0

Control

A ETOH 7 hour

0 LPS 0 ErOH

+ LPS 7 hour

FIG.6. Reduction of ferricytochrome C by in situ perfused liver after 7 hr of ethanol (ETOH) infusion and endotoxin treatment. Mean t S.E. of 5 to 10 independent experiments.

production by phagocytic cells (11, 13). It appears that the Kupffer cells are the likely primary sources of these free radicals, as shown by studies on isolated hepatic cells. These data also indicate that the ethanol-induced superoxide production peaked at 3 hr. Although some superoxide activity was still detected at 7 hr, isolated cells obtained from the animals failed to release a significant amount of superoxide in uitro. This observation indicates that because the in uiuo effect of ethanol on superoxide release by the liver was already declining

the long isolation procedure required to obtain hepatic cells may have contributed to the loss of this activity. Activated Kupffer cells have been shown to spontaneously produce superoxide anion (9,14,19)and cytokines (e.g., interleukin-1) (28, 29) on adherence to plastic in uitro. Oxygen-derived radical formation is further enhanced when phagocytes are stimulated by soluble or particulate stimuli (11, 13, 14). Although superoxide anion production by endothelial cells or hepatocytes could be derived from hypoxanthine-xanthine oxidase or through the microsomal pathway during metabolism of acetaldehyde from ethanol (7, 8), this was unlikely to have taken place to any appreciable extent because the release of superoxide anion by the parenchymal cells was not detectable after acute ethanol intoxication (91, endotoxemia (141, TNF infusion (10) or in uivo stimulation of the reticuloendothelial system (19). Also, because the inactivation of alcohol dehydrogenase (and thus the prevention of the oxidative degradation of ethanol) did not prevent the production of superoxide anions, we concluded that the metabolism of ethanol, the production of redox change and the formation of acetaldehyde were not involved in a major way in the ethanol-induced production of superoxide by the liver. Although we postulate that the resident Kupffer cells (and possibly the sequestered neutrophils in the liver) are the most likely sources of superoxide anion during ethanol intoxication, in view of the well-documented active "cross talk" between parenchymal and nonparenchymal cells of the liver we cannot exclude the potential secondary importance of hepatocytes in these events. The mechanism of ethanol-enhanced production of superoxide anion under our experimental conditions may be mediated by a complex series of events, including some of the prostanoids. Arachidonic acid and its metabolites have been shown t o stimulate superoxide anion release by phagocytes (30, 31). During Fc receptor-mediated phagocytosis by Kupffer cells, prostaglandin D, and superoxide anion are also produced (32). This study shows that the ethanol-induced release of superoxide anion is likely to be mediated by arachidonic acid metabolites because ibuprofen, the inhibitor of cyclooxygenase pathway, completely abolished superoxide anion production by the perfused liver. We have shown previously that superoxide anion formation by PMA or zymosan-stimulated isolated Kupffer cells and blood and hepatic neutrophils from latex-treated rats were also reduced in the presence of ibuprofen (19). The stimulatory effect of ethanol on phagocytes have

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ETHANOL-INDUCED SUPEROXIDE PRODUCTION IN THE LIVER

also been demonstrated by others (15, 33, 34). Short-term in uitro exposure of macrophages to low-dose ethanol-enhanced superoxide anion production by these cells (15). In uzuo ethanol treatment of mice also increased the phagocytic activity of Kupffer cells and the migration of neutrophils into the liver (33, 34). The mechanism of such stimulation is not known. However, it is possible that ethanol has a direct membrane effect in the macrophages by altering membrane fluidity (35) and inducing an activation of phospholipid turnover (36, 37), an activation of protein kinase C and an increased translocation of NAD(P)H oxidase to the membranes and ultimately superoxide anion release. Ethanol has been shown to enhance the influx of Ca2+ in macrophages that can also contribute to increased superoxide anion production by increasing phosphoinositide turnover resulting in the production of inositol 1,4,5triphosphate and the release of intracellular Ca2 (36). Similar mechanisms may be involved in the ethanolenhanced production of superoxide anion by the perfused liver. Our data also demonstrated that ethanol significantly attenuated the endotoxin-induced superoxide anion production. Although the mechanism of this attenuation is not understood at this time, the previously demonstrated effect of ethanol in diminishing the endotoxininduced TNF release may have significance in this regard. TNF is a potent immunomodulator that has been implicated as one of the key mediators of sepsis or endotoxic shock. TNF has also been shown t o prime alveolar macrophages (24), neutrophils (25) and bone marrow4erived macrophages (26) to generate superoxide anion when these cells are cultured in uitro and further stimulated with particulate or soluble stimuli. Assuming that the diminished superoxide anion production by ethanol is a manifestation of immunosuppression, the ethanol-induced immunosuppression could be mediated through the inhibition of TNF production. This may also result in the lack of priming of phagocytes to respond to secondary challenge or stimuli. Alternately, ethanol may also desensitize the cells to stimulation by other agents through modulation of the signal transduction mechanism, thus rendering the cells transiently refectory or resistant. It is likely that both the Kupffer cells and the sequestered hepatic neutrophils are involved in these events. The possible pathophysiological implications of the two sets of observations made by these studies are as follows: (a) acute intake of ethanol may result in oxygen free radical production that, if it is frequently repeated and if it exceeds the local protective defense mechanisms, may cause or contribute to liver damage; and (b) the coincidental presence of ethanol and small amounts of bacterial endotoxins may impair the natural defense mechanisms of the body that under appropriate conditions could lead to an increased incidence (and also severity) of infection and sepsis. In summary, these studies demonstrate that short-term exposure of the liver to ethanol can stimulate the production of superoxide anion and also attenuate +

897

the LPS-mediated activation of the immune defenses. The release of superoxide anion by ethanol alone may be a contributory factor in hepatic injury and the development of alcoholic liver disease, whereas the interactive insults of ethanol plus endotoxin may lead to the increased frequency and severity of infectious incidence in individuals who drink alcohol. Acknowledgments: We thank Dr. Gregory J. Bagby for the TNF assays. We are also grateful to Jean Carnal for excellent technical assistance and to Howard Blakesly for the illustrations. REFERENCES 1. Comporti M. Lipid peroxidation and cellular damage in toxic liver disease. Lab Invest 1985;53:599-623. 2. Younes M, Strubelt 0. Alcohol induced hepatotoxicity: a role for oxygen-free radicals. Free Radic Res Commun 1987;3:1-5. 3. Cederbaum AI. Role of lipid peroxidation and oxidative stress in alcohol toxicity. Free Radic Biol Med 1989;7:537-539. 4. Rosenblum ER, Gavalier JS, Van Thiel D. Lipid peroxidation: a mechanism for alcohol-induced testicular injury. Free Radic Biol Med 1989;7:569-577. 5. Hearse DJ, Manning AS, Downey JM, Yellon DM. Xanthine oxidase, a critical mediator of myocardial injury during ischemia reperfusion. Acta Physiol Scand Suppl 1986;548:65-78. 6. Fried1HP, Till GO, Ryan US, Ward P. Mediator-induced activation of xanthine oxidase in endothelial cells. FASEB J 1989;3:25122518. 7. Lieber CS. Pathophysiology of alcoholic liver disease. Mol Aspects Med 1988;10:107-146. 8. Fridovich I. Oxygen radicals from acetaldehyde. Free Radic Biol Med 1989;7:557-558. 9. Bautista AP, D'Souza NB, Lang CH, Bagwell J, Spitzer JJ. Alcohol-induced downregulation of superoxide anion production by hepatic phagocytes in endotoxemic rats. Am J Physiol 1991; 260:R969-R976. 10. Bautista AF', Schuler A, Spolarics Z, Spitzer JJ. Tumor necrosis factor stimulates superoxide anion production by the perfused liver and Kupffer cells. Am J Physiol 1991;261:G891-G895. 11. Johnston RB, Kitagawa S. Molecular basis for enhanced respiratory burst in activated macrophages. Fed Proc 1985;44:29272932. 12. Williams AJ, Barry ER. Free radical generation by neutrophils: a potential mechanism of cellular injury in acute alcoholic hepatitis. Gut 1987;28:1157-1161. 13. Johnston RB, Godzik CA, Cohn ZA. Increased superoxide anion production by activated and chemically elicited macrophages. J Exp Med 1978;148:115-127. 14. Bautista AP, Meszaros K, Bojta J , Spitzer JJ. Superoxide anion production by the liver during the early stage of endotoxemia in rats. J Leukoc Biol 1990;48:123-128. 15. Dorio RJ,Hoek JB,Rubin E, Forman HJ. Ethanol modulation of rat alveolar macrophage superoxide production. Biochem Pharmacol 1988;37:3528-3531. 16. Kanofsky JR. Singlet oxygen production in superoxide ionhalocarbon systems. J Am Chem Soc 1986108:2977-2979. 17. Klebanoff SJ. Oxygen metabolism and the toxic properties of phagocytes. Ann Intern Med 1980;93:480-489. 18. Bautista AP, Spitzer JJ. Superoxide anion production by in situ perfused liver: In vivo effects of endotoxin. Am J Physiol 1990;260:G907-G913. 19. Bautista AP, Schuler A, Spolarics Z, Spitzer JJ. In vivo latex phagoeytosis primes hepatic neutrophils and Kupffer cells to generate superoxide anion [in press]. J Leukoc Biol. 20. Molina E, Lang CH, Bagby GJ, Spitzer JJ. Ethanol oxidation is not required to attenuate endotoxin-enhanced glucose metabolism. Am J Physiol 1991;260:R1058-R1065. 21. Figdon CG. Leemans JMM, Bont WS, Vries JE. Theory and -

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practice of centrifugal elutriation (CE): factor influencing the separation of human blood cells. Cell Biophys 1983;5:105-112. 22. Johnson GD, Dorling J. Immunofluorescence and immunoperoxidase techniques. In: Thompson RA, ed. Techniques in clinical immunology. London: Blackwell Publishing Co., 1981:131. 23. Wang AM, Creasy AA, Ladner MB, Lin LS, Strickler J, Van Ardesell JN, Yamamoto R, et al. Molecular cloning of the complementary DNA for human tumor necrosis factor. Science 1985;228:149-154. 24. Warren JS, Kunkel SL, Cunningham TW, Johnson KJ, Ward PA. Macrophage-derived cytokines amplify immune complex triggered superoxide response by rat alveolar macrophages. Am J Physiol 1988;130:489-493. 25. Kapp A, Zeck-Kapp G, Blohm D. Human tumor necrosis factor is a potent activator of oxidative metabolism in human polymorphonuclear neutrophilic granulocytes. J Invest Dermatol 1989;92: 348-354. 26. Phillips WA, Hamilton JA. Phorbol-ester stimulated superoxide production by murine bone-marrow derived macrophages requires preexposure to cytokines. J Immunol 1989;142:2445-2456. 27. D’Souza NB, Bautista AP,Bagby GJ, Lang CH, Spitzer JJ. Acute alcohol intoxication suppresses E. coli LPS-enhanced glucose utilization by hepatic non-parenchymal cells. Alcohol Clin Exp Res 1991;15:249-264. 28. Bautista AP, Fletcher DJ, Volkman A. Regulation of insulin and interleukin-1 release after Propwnibacterium acnes-induced macrophage activation in mice. Lab Invest 1989;60:447-454. 29. Kurt-Jones EA, Beller DI, Mizel SB, Unanue ER. Identification of membrane-associated interleukin-1 in macrouhaees. Proc Natl Acad Sci USA 1985:82:1204-1207.

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HEPATOLOGY

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Acute ethanol intoxication stimulates superoxide anion production by in situ perfused rat liver.

This study examines the generation of superoxide anion by the perfused rat liver after ethanol intoxication and acute endotoxemia to assess the potent...
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