1 Department of Internal Medicine, School of Medicine, Keio University, Tokyo 160-8582; and 2 Second Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan
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ABSTRACT |
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Although the negative effect of excessive alcohol consumption on later stressful events has long been recognized, pathophysiological mechanisms are incompletely understood. We examined possible roles of oxygen radicals and glutathione content in mesenteric venules of chronically ethanol-fed rats exposed to ischemia-reperfusion. Changes in microvascular hemodynamics, such as red blood cell (RBC) velocity, leukocyte adherence, and albumin extravasation, were monitored in postcapillary venules by intravital fluorescence microscopy. Chronic ethanol feeding significantly exaggerated the magnitude of the decrease in RBC velocity, the increased number of adherent leukocytes, and increased albumin leakage elicited by 10 min of ischemia followed by 30 min of reperfusion. Oxidative stress in the endothelium of venules monitored by dihydrorhodamine 123 (DHR) fluorescence was more severe in rats fed ethanol chronically. Both superoxide dismutase and N-acetyl-L-cysteine, which is known to increase glutathione content, reduced the ischemia-reperfusion-induced decrease in RBC velocity, the number of adherent leukocytes, and the increase in albumin leakage, as well as oxidative activation of DHR. This suggests that the increased reperfusion-induced microvascular disturbances in the mesenteric venules of rats fed ethanol chronically are significantly correlated with excessive production of oxygen-derived free radicals and decreased glutathione synthesis.
leukocyte adherence; vascular permeability; mast cell degranulation; oxygen radicals; glutathione
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INTRODUCTION |
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ALCOHOL-INDUCED INJURY INVOLVES a number of organs of the central nervous system and digestive system (8). Although deleterious effects of excessive alcohol consumption on subsequent stressful events have long been recognized, pathophysiological mechanisms are incompletely understood (2). There have been several reports showing morphological and functional changes in the microcirculation in various organs after chronic ethanol consumption (25, 44). It has been reported that chronic alcohol ingestion impairs dilatation of cerebral arterioles in response to activation of adenylate cyclase or ATP-sensitive potassium channels (26), and chronic ethanol consumption has been found to increase hepatic sinusoidal responsiveness to the vasoconstrictor endothelin-1, which may contribute to the increased susceptibility of ethanol-fed rats to secondary stresses such as endotoxemia (3). We have also demonstrated that chronic ethanol feeding enhances endotoxin-induced hepatic sinusoidal leukocyte adhesion (35). In contrast, regular alcohol consumption has some beneficial effects on cardiac function against ischemia-reperfusion (I/R) due to preconditioning (27, 30-32). However, no one has studied the effect of alcohol consumption on ischemia-reperfusion-induced injury to the microcirculation.
It is now well recognized that reperfusion of ischemic tissues leads to an injury response that is frequently accompanied by endothelial cell injury, enhanced protein efflux, and increased adherence and emigration of leukocytes in postcapillary venules (19, 22, 36). A variety of bioactive compounds and effector cells have been invoked as mediators of reperfusion injury, including reactive oxygen metabolites, lipid mediators, nitric oxide, platelets, mast cells, and granulocytes (13, 15, 18, 19, 42, 47). Both xanthine oxidase-derived and granulocyte-derived active oxygens are known to play a role in the pathogenesis of microvascular injury, and in vitro and in vivo studies have demonstrated that oxygen-derived free radicals promote leukocyte adherence and emigration and subsequent endothelial injury (41, 42, 45). Thus leukocyte adhesion and the release of oxygen radicals after reperfusion are thought to be closely related in the process of microvascular injury. Although acute ethanol ingestion potentiates leukocyte migration into small intestine (43), there have been no reports showing the role of oxygen radicals in the process of ischemia-reperfusion-induced microvascular damage in animals after long-term treatment with alcohol.
The tripeptide glutathione (-Glu-Cys-Gly) is the major intracellular
thiol compound in most mammalian tissues and is known to be involved in
a variety of cell functions via participation in metabolic and
detoxification reactions (29). Reduced glutathione (GSH)
plays a protective role in the metabolism of a large number of toxic
agents, reacting either with them directly to form adducts or as a
cosubstrate in conjugation and peroxidation systems. Thus decreases in
GSH levels have been associated with the increased toxicity of many
compounds (12). Chronic ethanol consumption has been
demonstrated to increase the liver toxicity of different compounds, and
this effect may be related to the changes induced in glutathione
metabolism (7, 33). It is unknown, however, whether the
changes in glutathione content induced by chronic ethanol consumption
modify the extent of microvascular disturbances after
ischemia-reperfusion.
The present experiment was therefore undertaken to assess ischemia-reperfusion-induced alteration of microvascular hemodynamics, albumin leakage, and leukocyte-endothelial cell interactions in the postcapillary venules of rats chronically fed ethanol. These issues are addressed in a rat mesentery model, which allows for simultaneous measurement of leukocyte adherence, albumin leakage, and release of oxygen-derived free radicals in discrete segments of postcapillary venules exposed to ischemia, and subsequent reperfusion (29, 45). N-acetyl-L-cysteine (NAC), maleic acid diethyl ester, and superoxide dismutase (SOD) were used to assess the relative contribution of GSH concentration or oxygen radical production to the leukocyte-endothelial cell interactions and albumin leakage observed in postcapillary venules exposed to ischemia-reperfusion.
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MATERIALS AND METHODS |
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Animals and chronic ethanol feeding. All animals were handled according to the guidelines of the Keio University Animal Research Committee. Male Wistar rats (200-250 g; Charles River Breeding Laboratories, Tokyo, Japan) were pair fed for 8 wk with a liquid diet containing ethanol that provided 36% of their total energy or an isocaloric control dextrin diet according to the methods of Lieber and DeCarli (21).
Experimental protocol for I/R injury. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital, 30 mg/kg body wt. The right jugular vein was cannulated with a polyethylene catheter. The abdomen was opened via a midline incision 20-30 mm long. The ileocecal portion of the mesentery 20-cm caudal was gently drawn out, exteriorized, and mounted on a transparent plastic stage specially designed for the rat. The mesentery was kept warm and moist by continuous superfusion with Krebs-Ringer-bicarbonate-buffered solution at 37°C. Microcirculatory hemodynamics in the mesentery were observed by a transillumination method by using an inverted microscope (Diaphot TMD-2S; Nikon, Tokyo, Japan). The mesentery was transilluminated with a 12-V, 100-W, direct current-stabilized light source. A video camera mounted on the microscope projected the image onto a color monitor, and the images were recorded with a videocassette recorder. A video time-date generator projected the time and stopwatch function onto the monitor. Single unbranched venules with diameters ranging between 25 and 35 µm and length >200 µm were selected for study.
After 10 min of basal observation of the hemodynamics in the rat mesenteric microvasculature, the I/R was performed by ligation of the mesenteric artery of the feeding branch of the anterior mesenteric artery and the corresponding vein simultaneously with a snare created from a polyethylene tube for 10 min and subsequent release of the blood flow occlusion (18). Because red blood cell (RBC) velocity of the vessels during the ischemia was not zero, there may have been collateral perfusion to the observed area. Thus both artery and vein were ligated to stop blood supply and to induce venular congestion to enhance ischemia. Earlier study showed that 10 min of ischemia followed by reperfusion was enough to induce mesenteric microcirculatory disturbance but minimize intestinal tissue injury (15). Sham-operated rats without I/R were used as controls.Measurement of microvascular parameters. The images of arterioles and venules were obtained through a charge-coupled devise (CCD) color video camera system (CC-090; Flovel, Tokyo, Japan). The diameters of microvessels were determined time dependently using a video measuring gauge (IV-560; Hoei, Tokyo, Japan). The leukocytes that had adhered to the venule walls were identified by reviewing the dynamic images of leukocytes recorded through this system. Adherent leukocytes were defined as cells that attached to the same site for more than 10 s judging from the replayed video images. The number of adherent leukocytes was counted along venules (25-35 µm in diameter, 200 µm in length) randomly selected from the videotape images recorded (22, 40).
The velocity of RBCs in the venule was simultaneously recorded at a rate of 1,000 frames/s by changing the monitor from CCD to a high speed video camera system (Ektapro 1000; Kodak, San Diego, CA), and the recordings were replayed from the high speed stored images at a rate of 30 frames/s. RBC velocity in the venule was measured with a temporal correlation velocimeter (CapiFlow, Kista, Sweden) (40). Mast cells were identified in the same animals by vital staining with topical application of 0.1% toluidine blue to the mesentery 30 min after the onset of I/R. The numbers of both nondegranulated mast cells and degranulated mast cells were counted from the CCD video images, and the ratio of the number of degranulated mast cells to the total number of mast cells is expressed as the degranulated mast cell ratio (40). To quantify albumin leakage across mesenteric venules, the animals were intravenously injected with 50 mg/kg of FITC-labeled bovine serum albumin (Sigma Chemical) 30 min before each experiment as described previously (17). Fluorescence intensity (excitation 420 to 490 nm; emission 520 nm) was detected using a silicon-intensified target camera (C-2400-08; Hamamatsu Photonics, Hamamatsu, Japan). The fluorescence intensity of FITC-albumin in three segments of the venule under study (Iv) and in three contiguous areas of perivenular interstitium (Ii) area were measured at various times after the administration of FITC-albumin with a computer-assisted digital imaging processor (NIH Image 1.35 on a Macintosh computer), with background fluorescence intensity subtracted from each value. The windows to measure average fluorescence intensity within and along the venule were set at 50 µm long and 25 µm wide (16, 18). An index of vascular albumin leakage was determined by dividing Ii by Iv and expressing this ratio as a percentage (i.e., a value of 50% would indicate that Ii is one-half that of Iv). This tissue-to-plasma index is referred to as "albumin leakage" and is reported during the baseline period as well as during specific times of reperfusion. In another set of experiments, the oxidant-sensitive fluorescent probe dihydrorhodamine 123 (DHR; Molecular Probes) was added to the mesenteric superfusate (10 µmol/l) to monitor oxidant stress in venular walls as described previously (16). The fluorochrome was visualized by using the same microscope and image analysis system described above. DHR fluorescence intensity was monitored in a region of mesentery equivalent to twice the area of the venule being examined. An image processor was used to monitor fluorescence intensity just before (baseline value; Ibase) and after (It=x) I/R. Since Ibase varied depending on DHR loading into the cells, the ratio of It=x to Ibase was defined as an index of oxidant stress in the mesenteric tissue and expressed as DHR fluorescence ratio (16), in which DHR was oxidized to fluorescent rhodamine 123 by intracellular and extracellular secondary H2O2-dependent reactions (39).Agents studied.
In a separate group, SOD, a scavenger of superoxide anion, was
continuously infused (12,000 units · kg1 · h
1)
via the jugular vein from 30 min before ischemia until the end of the experiment. In some experiments, we assessed the microvascular parameters described above after superfusion of rat mesentery with 5 mmol/l NAC, a stimulator of glutathione synthesis (34) or
100 µmol/l maleic acid diethyl ester (DMA), a depletor of cell glutathione (34).
Statistical analysis. Data were analyzed by standard statistical methods, i.e., one-way ANOVA and Fisher's post hoc test. All values are reported as means ± SD of values from 4 to 6 rats, and statistical significance was set at P < 0.05. In the experiments of RBC velocity, leukocyte adhesion, and mast cell degranulation, pair-fed rats (dextrin and alcohol groups) were used in the sham-operated group (n = 6), I/R alone group (n = 6), I/R + SOD group (n = 6), I/R + NAC group (n = 5), and I/R + DMA group (n = 6). Albumin leakage was measured in the pair-fed rats from the sham-operated group (n = 4), I/R alone group (n = 6), I/R + SOD group (n = 4), I/R + NAC group (n = 4), and I/R + DMA group (n = 4). DHR fluorescence was examined in the pair-fed rats from the sham-operated group (n = 4), I/R alone group (n = 5), I/R + SOD group (n = 4), I/R + NAC group (n = 4), and I/R + DMA group (n = 4).
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RESULTS |
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Changes in vascular diameter and RBC velocity. The diameter of arterioles and venules under baseline conditions was 22.5 ± 2.9 and 35.9 ± 3.5 µm, respectively, in the control dextrin-fed group (n = 6) and 22.3 ± 2.5 and 36.1 ± 3.0 µm in the ethanol-fed group (n = 6). The values in the two groups were not significantly different. On the basis of a morphometric analysis, the diameters of the mesenteric arterioles and venules were increased in the I/R group, possibly as a result of congestion, whereas the diameter of the arterioles and venules was almost completely maintained under baseline conditions.
Figure 1A compares the time course of changes in RBC velocity in venules after I/R treatment in the dextrin-fed and ethanol-fed groups. There was no significant difference in baseline RBC velocity between the two groups. Just after the ischemic period, a significant decrease in RBC velocity was observed in both the dextrin-fed and ethanol-fed groups, and during the subsequent reperfusion period, RBC velocity decreased in both the dextrin-fed and ethanol-fed groups at 10 min. In the ethanol-fed group, RBC velocity further decreased to 38.0 ± 2.4% (n = 6) of the baseline at the end of observation period, whereas in the dextrin-fed group RBC velocity maintained its value at 70.7 ± 7.3% (n = 6) after 30 min. There were no significant changes in RBC velocity of the sham-operated rats in either the dextrin-fed or ethanol-fed group.
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Changes in the number of leukocytes adhering to venular walls.
Figure 2A compares the time
course of changes in the number of leukocytes adhering to venular walls
before and after the I/R period in the dextrin-fed and ethanol-fed
groups. Before ischemia, no leukocytes adhered to the venular
walls in either the dextrin-fed or ethanol-fed group. In the
sham-operated groups, a small number of leukocytes was found, and their
number gradually increased to 3.5 ± 2.2 (per 100 µm venules;
n = 6) in the dextrin-fed group and 3.9 ± 2.3 (per 100 µm venules; n = 6) in the ethanol-fed group. In the I/R animals, an initial and dramatic increase in the number of
adherent leukocytes was observed in both the dextrin-fed and ethanol-fed groups immediately after the reperfusion. Although the
number of adherent leukocytes further increased thereafter in the
ethanol-fed group and reached to 21.3 ± 2.6 (per 100 µm venules; n = 6) at 30 min after reperfusion, the number
in the dextrin-fed group showed no significant further increase from 10 to 30 min after the reperfusion.
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Changes in albumin leakage from venular walls.
Figure 3, A and B,
shows representative fluorographic images of albumin leakage in the
mesentery of the ethanol-fed animal at baseline and 30 min after the
reperfusion, respectively. Figure 4
demonstrates significant adherence of leukocytes to venular walls with
degranulation of mast cells in the interstitium by using a CCD camera
and toluidine blue staining in the same microvasculature at 30 min in
the higher magnification. Figure
5A illustrates the time course
of changes in albumin leakage from the venular walls before and after
the I/R period in the dextrin-fed and ethanol-fed groups. Before
ischemia, no detectable albumin leakage was observed in either
the dextrin-fed or ethanol-fed group. In the sham-operated groups, a
small amount of albumin leakage was found during the observation period
[1.8 ± 0.8% in dextrin-fed group (n = 4) and 2.0 ± 0.7% in the ethanol-fed group (n = 4) at
the end of experiments]. In the I/R groups, the extent of albumin
leakage from venules initially increased immediately after reperfusion
in both the dextrin-fed and ethanol-fed groups. A further and
significant increase in albumin leakage was observed from 10 to 30 min
after reperfusion in the ethanol-fed groups, but no such increase was observed in the later period in the dextrin-fed group.
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Changes in intensity of DHR fluorescence in the venular walls.
Figure 6 shows the changes in the
fluorescence intensity of the H2O2-sensitive
probe DHR in the ethanol-fed group during the reperfusion period.
Within 10 min, areas of fluorescence intensity were observed along the
venule, then gradually increased until the end of the observation
period. Although DHR activity was largely confined to the vascular
endothelium, an area of fluorescence intensity corresponding to mast
cells and emigrated leukocytes was also observed in the interstitium.
Figure 7A compares the time
course of changes in DHR fluorescence ratio to the baseline intensity
on the venular walls after the I/R period in the dextrin-fed and
ethanol-fed groups. There were no significant changes in DHR intensity
during the observation period in the sham-operated groups. In the I/R
groups, the intensity of DHR fluorescence was significantly increased
just after reperfusion in the ethanol-fed group and beginning at 10 min
after reperfusion in the dextrin-fed group. In the ethanol-fed group,
these values were dramatically increased at 20 min after reperfusion,
and the values of DHR intensity were significantly greater compared
with those in the dextrin-fed group at 20 and 30 min after reperfusion.
However, the further increase in DHR at 20-30 min was not observed
in the dextrin-fed group.
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Mast cell degranulation along microvessels.
Figure 8 shows the ratio of degranulated
mast cells in the area along the microvessels 30 min after the
reperfusion period and the effects of SOD, NAC, and DMA treatment. In
the sham-operated groups, the mast cell degranulation ratio was
21.0 ± 3.0% in the dextrin-fed group (n = 6) and
23.0 ± 4.0% in the ethanol-fed group (n = 6). It
was significantly increased in I/R animals in both the dextrin-fed and
ethanol-fed groups, and there was no significant difference in these
values between the two groups. Both SOD and NAC treatment significantly
attenuated the I/R-induced increase of mast cell degranulation in both
groups. DMA treatment, however, did not alter the I/R-induced increase
of mast cell degranulation in either the dextrin-fed or ethanol-fed
group.
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DISCUSSION |
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One of the major objectives of this study was to compare the responsiveness of microvessels to I/R between normal dextrin-fed rats and chronic ethanol-fed rats by simultaneous measurement of leukocyte adherence and albumin leakage in postcapillary venules. The results obtained in this model indicate that ischemia significantly promotes leukocyte adherence and elicits an increased albumin efflux just after the start of reperfusion. However, the microvascular responsiveness to I/R., i.e., increase in adherent leukocytes, albumin leakage, and decreased RBC velocity, was not significantly enhanced in ethanol-fed rats compared with the control dextrin-fed rats until 10 min after reperfusion. On the other hand, the magnitude of the microvascular disturbances as indicated by the above parameters was significantly increased in the ethanol-fed rats compared with the dextrin-fed animals at 20-30 min after reperfusion. These observations suggest that there are two different stages in the process of microvascular disturbances by I/R and that only the later stage appears to be aggravated by chronic ethanol treatment.
We used the H2O2-sensitive fluorescent probe DHR, which has been successfully used to measure intracellular H2O2 levels in cultured endothelial cells and in other cell types, in an effort to monitor the oxidative stress elicited by I/R (6, 38). DHR has biological properties that make it an ideal agent for detecting secondary H2O2-dependent reactions in living tissue by fluorescence videomicroscopy. Royall and Ischiropoulos (39) recently showed evidence that oxidation of DHR is not due to H2O2 alone but appears to occur by reactions involving reactive oxygen species. We have shown that oxidative stress in microvasculature subjected to I/R in vivo is more enhanced in ethanol-fed animals at 20-30 min after reperfusion. The remarkable difference in DHR oxidation in postcapillary venules between dextrin-fed and ethanol-fed animals in the later stage indicates that a greater oxidative stress occurred in the ethanol-fed rats.
Another important observation in the present study is that the antioxidant SOD effectively attenuated the microcirculatory disturbances induced by I/R at 30 min, particularly in the ethanol-fed group but partially in the dextrin-fed group. The difference in microvascular injury between the two groups was not observed after SOD treatment except after DHR, suggesting that, at least in the later stage, oxygen-derived free radicals may play a significant role as a causative mediator in ethanol-induced microcirculatory disturbances.
On the other hand, the increase in leukocyte adherence preceded the increase in DHR fluorescence, with the former becoming significant just after reperfusion and the latter at 10 min after I/R (Figs. 2A and 7A). These findings suggest that accumulated and activated leukocytes provoke the oxidative stress in microvascular walls subjected to I/R. Although it was first proposed that xanthine oxidase-derived oxidants mediate the microvascular injury associated with reperfusion of the ischemic intestine in 1981 (11), recent studies have suggested that leukocyte NADPH oxidase-derived oxygen radicals also play a role in the process of microvascular injury (10). In the present study, albumin leakage was significantly attenuated by SOD at 10 min, whereas leukocyte adherence was attenuated by SOD after 20 min. These findings support the involvement of xanthine oxidase-derived active oxygens in the early phase and granulocyte-derived oxygen metabolites in the later phase. However, the leukocyte recruitment in the early phase may at least in part be mediated by oxygen radical-independent mechanisms, because SOD treatment did not significantly inhibit leukocyte recruitment immediately after the reperfusion period in either the dextrin-fed or the ethanol-fed animals.
Another important observation in this study was the significant mast cell degranulation elicited by I/R and the ability of antioxidants to prevent this degranulation response, whereas there was no significant difference between the percentage of mast cells degranulated in the dextrin-fed and ethanol-fed animals. Kubes et al. (14) have proposed that inhibition of nitric oxide synthesis allows accumulation of superoxide, which in turn causes mast cell degranulation. They proposed that mast cell products, including platelet-activating factor, released in response to superoxide exposure promote leukocyte adhesion in postcapillary venules. Thus the early burst of mast cells may significantly contribute to the greater oxidant stress in microvasculature at a later time.
In the present study, microvascular disturbances elicited by I/R were
significantly aggravated in chronic ethanol feeding. Greater
lipoperoxidation or increased malondialdehyde production after
reperfusion has been reported in the liver of chronic ethanol-treated rats (9, 46). We have previously demonstrated that chronic ethanol consumption aggravates endotoxin-induced leukocyte adhesion, which may result in hepatic microcirculatory disturbances and leukocyte
adhesion to the sinusoidal wall and may be associated with increased
tumor necrosis factor- levels (35). Altered microvascular responses induced by chronic ethanol feeding, such as an
increased contractile response to endothelin-1 (3) and decreased hepatic sinusoidal perfusion following hemorrhagic shock (2) or impaired dilation of cerebral resistance arterioles (26), have been observed. The microvascular damage due to
alcohol abuse may include various changes in vascular endothelial cells or smooth muscle cells, such as morphometric changes (25,
44), changes in the number of receptors (24, 26),
changes in signal transduction (1) or energy metabolism,
and alteration of redox states (8, 20). However, regular
alcohol consumption produces cardioprotective effects (27,
30-32), suggesting that different responses and
sensitivities to chronic alcohol feeding in the different organs may exist.
Promotion of generation of oxygen free radical species after chronic
ethanol consumption may be closely associated with impairment of
defense systems against oxidative stress in the microvasculature. Our
results clearly demonstrated that perturbations of glutathione content
by two different agents influence susceptibility to I/R-induced injury
in vivo. First, NAC, a permeable precursor for -glutamylcysteine synthase, is known to increase glutathione content by stimulating glutathione synthesis (28, 37). Albumin leakage and
increased DHR, which are thought to be related to
H2O2 production after reperfusion in
particular, were drastically inhibited by NAC in both the dextrin-fed
and ethanol-fed animals. These findings are consistent with glutathione
peroxidase catalyzing GSH-dependent reduction of
H2O2, which is a major pathway of
H2O2 metabolism in many cells
(34). Therefore, the decrease of GSH levels may largely
contribute to oxygen radical-induced microvascular injury in the later
stage, especially in ethanol-fed animals. Chronic ethanol intake has
been reported to induce a decrease in glutathione content, whereas
malondialdehyde, a lipid peroxidation product, was found to be
increased in rat peripheral nerves (4) and in the
erythrocytes of alcoholic patients (23). Fernandez-Checa et al. (7) demonstrated that the GSH transport system from cytosol into mitochondria is impaired in chronically ethanol-fed rats,
resulting in the development of increased susceptibility to oxidant
stress. Second, DMA is known to interact with glutathione by direct
conjugation and via glutathione-S-transferase, thus reducing
GSH level without an increase in the levels of oxidized glutathione
(GSSG) (5). In our study, DMA treatment did not further
exaggerate the I/R-induced microvascular damage except leukocyte
adherence and albumin leakage in either group, suggesting that I/R
itself had already reduced GSH levels. However, even after DMA
treatment, there was still a significant difference in the extent of
various microcirculatory disturbances between the dextrin-fed and
ethanol-fed animals. This suggests that factors other than glutathione
depletion may also be involved in ethanol-induced microvascular injury,
although the specific factors related to chronic ethanol-induced injury
of the microvasculature remain to be elucidated.
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ACKNOWLEDGEMENTS |
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This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by a grant from Keio University School of Medicine.
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FOOTNOTES |
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Address for reprint requests: H. Ishii, Professor of Internal Medicine, School of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (E-mail: hishii{at}med.keio.ac.jp).
Address for other correspondence: S. Miura, Second Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa City, Saitama 359-8513, Japan (E-mail: miura{at}me.ndmc.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 March 2000; accepted in final form 12 October 2000.
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