Phurnlucology & Toxicology 1992, 71, 278-283.
Effect of Antioxidants on Hypoxia/Reoxygenation-Induced Injury in Isolated Perfused Rat Liver M. Younes**, E. Kayser' and 0. Strubelt' 'Institute of Toxicology, Medical University of Liibeck, Ratzeburger Allee 160, D-2400 Liibeck, Germany, and 'Max-von-Pettenkofer-Institute, BGA, P.O. Box 330013, D-1000 Berlin 33, Germany (Received December 11, 1991; Accepted April 28, 1992) Abstract: Isolated perfused livers from rats fasted overnight were subjected to 30 min. of hypoxia followed by reoxygenation for 60 min., resulting in marked cytotoxicity as evidenced by an enhanced release of cytosolic enzymes (lactate dehydrogenase: 14-fold over controls, glutamate-pyruvate-transaminase: 12-fold over controls) and glutathione (twofold over controls) into the perfusate, by calcium accumulation (by a Factor of 1.4) in the tissue and by an 80% inhibition of bile secretion. Virtually no mitochondria1 injury became apparent and no evidence for lipid peroxidation could be found. In the presence of ascorbate, an augmentation of hepatic injury was observed. This might be due to the pro-oxidant activity of ascorbate in the presence of ionized iron, which is easily released from high molecular weight stores under reductive (e.g. hypoxic) conditions. The water soluble vitamin E analogue trolox C as well as propyl gallate clearly protected the liver against hypoxiaireoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model. Due to their water solubility and their high efficacy as free radical scavengers, these antioxidants might be of therapeutic value.
With the increased application of new therapeutic strategies, like organ transplantation and cardiac reperfusion, the interest in the mechanisms underlying the cytotoxic effects observed following reperfusionlreoxygenation of ischaemicl hypoxic tissue has grown. In several studies reactive oxygen species have been implicated as the ultimate injuries species in reperfusionlreoxygenation damage of various organs (Granger et al. 1981; Younes et al. 1984; Das et al. 1986; Younes & Strubelt 1988; Brass et al. 1991). One of the major sources for reactive oxygen species seems t o be the xanthine oxidase reaction (Granger et al. 1981). Under ischaemicl hypoxic conditions, ATP-catabolism leads t o formation of hypoxanthine and xanthine; concomitantly, low oxygen supply results in a transformation of the NADdependent xanthing dehydrogenase into the 0,-dependent xanthine oxidase (Roy & McCord 1983). As soon as oxygen is reintroduced, purine metabolites are readily oxidized; a reaction which is associated with a n activation of molecular oxygen yielding toxic metabolites. The liver seems t o be particularly resistant to hypoxic as well as reperfusion injury, a s long as anaerobic energy supply (due t o glycolysis) is maintained (Anundi et al. 1987; Younes & Strubelt 1988; Anundi & DeGroot 1989). Depletion of glycogen, e.g. by food deprivation (Younes & Strubelt 1988) or inhibition of glycolysis (Younes & Strubelt 1988; Younes et al. 1989) makes the liver susceptible t o tissue damage due to hypoxia solely or hypoxia and reoxygenation. Evidence for the involvement of oxygen radicals produced mainly by the xanthineixanthine oxidase system in hypoxic liver damage has been provided by inhibitor
* To whom correspondence should be addressed at the following address: WHO-European Centre for Environment and Health, P.O. Box I , NL-3720 BA Bilthoven, The Netherlands.
studies using isolated perfused livers from fasted rats (Younes & Strubelt 1988). T h e aim of this study was to investigate the effect of antioxidants including the water soluble vitamin E analogue trolox C (Davies et al. 1988), the synthetic antioxidant propyl gallate (Kahl 1984) and ascorbate in this model of tissue injury in order t o further evaluate the role of oxidative mechanisms in hypoxic liver injury.
Materials and Methods Animals. Male wistar rats (conventional animals, 320 -380 g; breeder: Winkelmann, Borchen) were used throughout. They had free access to a standard diet (Altromin pellets) and tap water until use. Fasting was achieved by deprivation of feed, but not of drinking water 16 hr before surgery. Liverperfusiorz. Removal of the liver and its connection to a recirculating perfusion system was performed as previously described (Strubelt et al. 1986). The perfusion medium consisted of 250 ml Krebs-Henseleit-buffer, pH 7.4 (118 mmol/l NaCI, 6 mmol/l KCI, 1.1 mmol/l MgSO,, 1.2 mmol/l KH2P04, 25 mmol/l NaHCO,). CaClz (1.25 mmol/l) was added to the prewarmed medium (37') immediately before starting the perfusion experiments. The perfusion medium was continuously gassed with carbogen (95% 0,. 5% CO,) yielding an oxygen partial pressure of about 600 mmHg. To induce hypoxia in the appropriate experiments, carbogen was replaced by a mixture of 95% N, and 5% CO, after a 3 min. equilibration period. After 30 min. of hypoxia, reoxygenation was achieved by regassing with carbogen until the end of the experiment, i.e. for 60 min. Sodium taurocholate (26.7 g/l) was infused into the perfusate at a rate of 12 ml/hr to stimulate bile secretion. Oxygen consumption of the isolated livers was calculated from the difference in the oxygen concentrations of the influent and the effluent perfusate using a Micro pH/Blood Gas Analyzer 413 (Instrumentation Laboratory). In the experiments with antioxidants, trolox C, propyl gallate or ascorbate were added to the perfusate at the start of the equilibration period to yield a final concentration of 0.5 mmolil. At the end of the experiments, the livers were weighed, frozen in liquid nitrogen and kept frozen until analysis.
ANTIOXIDANTS ON LIVER HYPOXIA INJURY Biochemical determinations. The activities of cytosolic lactate dehydrogenase and glutamate-pyruvate-transaminaseas well as mitochondrial glutamate dehydrogenase were determined in the perfusate using commercial kits of Boehringer, Mannheim, FRG. Ca2+concentrations in the liver (following acid extraction) and in the perfusate were measured colorimetrically also with reagents of Boehringer, Mannheim. Malondialdehyde was measured in the liver and the perfusate by coupling to thiobarbituric acid (Buege & Aust 1978).Total glutahione was determined according to Brehe & Burch (1976); oxidized glutathione was estimated by the same procedure after blocking reduced glutathione with 2-vinylpyridine (Griffith 1980).
279
BILE FLOW pg /g min
Statistics. Meansf S.E.M. were calculated in the usual way. The difference between two means was checked with Dunnet’s t-test (Dunnet 1964) in the case of multiple comparisons or with Student’s t-test in the case of simple comparisons. The limit of significance was P < 0.05 in all cases.
Results Under normoxic conditions (control experiments) with oxygen partial pressure of around 600 mmHg, oxygen consumption by the isolated perfused liver was between 2.0 and 2.3 pmol/min. . g over the whole perfusion period (fig. 1). When
OXYGEN CONSUMPTION pmol/min. g
0
30T
I
I
I
30
60
90
1
120 min
Fig. 2. Time-dependent bile flow rate by isolated perfused livers from fasted rats. Values given are means and their standard errors (x+S.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min. equilibration period, hypoxia was induced from 30 to 60 min., followed by reoxygenation from 60 to 120 min. 0-0: controls, no hypoxia; 0-0: hypoxia + reoxygenation; 0-0: +0.5 mmol/l trolox C; M-M: f0.5 mmol/I propyl gallate; A-A: +0.5 mmol/l ascorbate.
2
0 min
Fig. 1. Time-dependent oxygen consumption by isolated perfused livers from fasted rats. Values given are means and their standard errors (xkS.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min. equilibration period, hypoxia was induced from 30 to 60 min., followed by reoxygenation from 60 to 120 min. 0---0: controls, no hypoxia; 0-0: hypoxia+ reoxygenation; 0-0: t 0 . 5 mmol/l trolox C; m-m: f0.5 mmol/l propyl gallate: A-A: f0.5 mmol/l ascorbate.
carbogen was replaced by 95%)N2/5%C 0 2 ,oxygen partial pressure fell to 100 mmHg. As a consequence, oxygen consumption dropped abruptly down to a minimum of 0.5 pmol/min. . g at the end of the hypoxic period (i.e. after 30 min.). Following reoxygenation, oxygen consumption rose again but only reached 76% of the value before hypoxia (fig. 1). Ascorbate, trolox C and propyl gallate had no significant effect on the course of oxygen consumption during the experiment, except for a slight reduction of the prehypoxic value in the presence of propyl gallate (fig. 1). Bile flow showed little variation with perfusion time in control experiments without hypoxia (fig. 2). Hypoxia led to a strong decline in bile flow rate which continued also following reoxygenation, reaching finally 20% of the original rate (fig. 2). Ascorbate had no influence on the inhibition of bile flow. In the presence of trolox C and propyl gallate, on the other hand, bile flow rate declined in the hypoxic phase to the same extent as in the experiments without antioxidants, but did not continue to decrease in the reoxygenation phase (fig. 2). Damage to the isolated perfused livers was estimated by measuring the efflux of the cytosolic enzymes lactate
280
M. YOUNES ET AL
Table I . Hepatic concentrations of glutathione, calcium and TBA-reactive material (TBAR) at the end of the perfusion period. Values given are means and their standard errors (%k S.E.M.; n = 5 each). All animals were fasted for 16 hr prior to the start of the perfusion experiments. Hypoxia + reoxygenation - (Controls)
Group 1
Antioxidant (500 pmol/l) Trolox C Propyl gallate Ascorbate
+ + +
2 3 4 5
+
Total glutathione (GSH+ZGSSG) (pmol/g liver)
Oxidized glutathione (pmol/g liver)
3.67 k0.89 1.7 I f0.40* 2.49 & 0.26 2.46$0.16** 1.32f0.46
0.07kO.01 0.05+0.01
0.07+0.01 0.10+0.01
0.04 k 0.04
Ca2+
TBAR
(pmol/g liver)
(pmol/g liver)
1.42k 0. I4 2.02+0.1 I * 1.47kO.l3** 1.78kO.10 2.61 k0.32
24.6k1.1 22.2 5.4 14.1 k0.6 14.2+ 1.7 35.3k7.3
*
* Statistically significant difference (group 2 vs. group I ) (p< 0.05, Student's t-test)
** Statistically significant difference as compared
to group 2 (hypoxia + reoxygenation) (p < 0.05, Dunnet's t-test)
GSH =glutathione GSSG =oxidized glutathione
dehydrogenase (fig. 3) and glutamate-pyruvate-transaminase (fig. 4).Hypoxia and reoxygenation resulted in a strong sustained increase o f enzyme release into the perfusate which was markedly attenuated in the presence of trolox C or propyl gallate but clearly amplified in the presence of ascorbate (fig. 3 & 4). To assess mitochondria1 damage, the release of glutamate dehydrogenase into the perfusate was
GPT U/I
"ol
i
mm-
m-
too0 0
U
30
60
90
120 rnin
Fig. 3. Time-dependent release of lactate dehydrogenase (LDH) by isolated perfused livers from fasted rats. Values given are means and their standard errors (x+S.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min., equilibration period, hypoxia was induced from 30 to 60 min. followed by reoxygenation from 60 to 120 min. 0-0: controls, no hypoxia; 0-0: hypoxia+ reoxygenation; 0-0: +0.5 mmolil trolox C; B-U: +0.5 mmol/l propyl gallate; A-A: +0.5 mmol/l ascorbate.
I
30 Fig. 4. Time-dependent release of glutamate-pyruvate-transaminase (GPT)by isolated perfused livers from fasted rats. Values given are means and their standard errors (x k S.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min. equilibration period, hypoxia was induced from 30 to 60 min., followed by reoxygenation from 60 to 120 min. 0---0: controls, no hypoxia; 0-0: hypoxia + reoxygenation; 0-0: +0.5 mmol/l trolox C; B-B: +0.5 mmol/l propyl galla+0.5 mmol/l ascorbate. te; A-A:
ANTIOXIDANTS ON LIVER HYPOXIA INJURY
28 1
Table 2. Concentrations of glutathione and TBA-reactive material (TBAR) in the perfusate at the end of the perfusion experiment. Values given are means and their standard errors (IfS.E.M.; n = 5 each). All animals were fasted for 16 hr prior to the start of the perfusion experiment.
Group
Hypoxia + reoxygenation
Total glutathione (GSH + 2GSSG) ( p o l / 1)
Antioxidant (500 pmol/l)
Oxidized glutathione (pmol/l)
Oxidized glutathione/ total glutathione
26.8 k 6.40 14.0k 7.20 0.52 52.1 k 7.13* 21.3 f 4.19* 0.41 Trolox C 23.5*4.82** 14.8f 3.58 0.63 Propyl gallate 18.7+2.48** 7.8* 2.75** 0.42 + Ascorbate 102.0k28.4** 52.9k28.0 0.52 * Statistically significant difference (group 2 versus group 1) (P < 0.05, Student’s t-test) ** Statistically significant difference as compared to group 2 (hypoxiaf reoxygenation) (P< 0.05, Dunnet’s t-test) GSH = glutathione GSSG = oxidized glutathione 1
-
+ + +
2 3 4 5
-
(Controls)
-
measured. No marked mitochondria1 injury occurred following hypoxia and reoxygenation as evidenced by the low rate of glutamate dehydrogenase release which started in the reoxygenation phase (fig. 5). Still, a tendency to attenuate this low value was observed with propyl gallate (fig. 5). Hypoxia and reoxygenation led to an increase in tissue calcium content by a factor of 1.4 over control levels at the end of the experiment (table 1). Trolox C totally and propyl
GCDH U/1
T
II
-i
30
30
60
90
120 rnin
Fig. 5. Time-dependent release of glutamate dehydrogenase (GLDH) by isolated perfused livers from fasted rats. Values given are means and their standard errors (x+S.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min. equilibration period, hypoxia was induced from 30 to 60 min., followed by reoxygenation from 60 to 120 min. 0---0: controls, no hypoxia; 0-0: hypoxia + reoxygenation; 0-0: +0.5 mmolll trolox C; W-W: +0.5 mmol/l propyl galla+0.5 mmol/l ascorbate. te; A-A:
TBAR (pmol/ I) 0.96k0.28 1.25k0.54 0.87 k0.28 1.24k0.23 3.05k1.18
gallate partially antagonized this rise in hepatic Ca2+-concentration while with ascorbate a tendency towards even higher calcium levels was observed (table 1). Following hypoxia and reoxygenation, hepatic glutathione was depleted by 53% (table 1). Again, trolox C and propyl gallate clearly inhibited this effect while ascorbate did not (table 1). Following reoxygenation of hypoxic livers, an enhanced release of glutathione into the perfusate was observed, which was prevented by trolox C and propyl gallate but enhanced by ascorbate (table 2). No increased production (table 1) or release (table 2) of thiobarbituric acidreactive material) was evident following hypoxia and reoxygenation.
Discussion The results of this study support earlier findings which indicated that the liver is resistent to hypoxic damage as long as anaerobic energy conservation reactions take place (Younes & Strubelt 1988; Bradford et al. 1986). Hypoxia and reperfusion injury occurred only in livers from rats which were deprived of food 16 hr prior to surgery. Under these conditions, hepatic glycogen is nearly totally depleted (Younes & Strubelt 1988); hence, no endogenous substrate for glycolysis is present. Besides this direct effect of fasting on energy metabolism, food deprivation was recently shown to accelerate the conversion of xanthine dehydrogenase into xanthine oxidase (Brass et al. 1991), favouring thereby the formation of intracellular pro-oxidants. Trolox C and propyl gallate, two potent water soluble antioxidants, protected isolated perfused rat livers against hypoxic injury. In recent studies with cultured rat hepatocytes (Wu et al. 1991) or with single-path perfused rat liver (Videla 1991), trolox C also proved to be protective against reperfusion injury. These observations provide a strong indication for an involvement of oxidatives stress in hypoxic andlor reperfusion hepatic injury, as both were previously shown to react with hydroxyl radicals with rate constants near diffusion limit (Aruoma et al. 1990). Due to their antioxidant capacity, they were able to save glutathione from being extracted from the cells following oxidation. In fact, direct evidence for the increased formation of
282
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reactive oxygen species in reperfused ischaemic rat livers has been recently provided (Okuda et al. 1991). Membrane phospholipids, however, do not seem to be the major targets for oxidant stress in our model, as no lipid peroxidation products could be detected. Oxidation of cellular thiols and the possible formation of mixed disulfides may have occurred, as can be concluded from the observed loss of cellualr glutathione in our study (which cannot be accounted for by severe release into the perfusate). Similar effects on the hepatic glutathione status have also been reported by Jaeschke et al. (1988). Following depletion of cellular glutathione, oxidation of sulfhydryl groups in functional proteins, including membrane-bound Ca2+-translocasesmay occur (Bellomo & Mirabelli 1987). Such an effect results in a sustained increase in cytosolic free calcium and, consequently, to an activation of a number of calcium-dependent catabolic processes (Orrenius et al. 1988). Ascorbate even enhanced hepatic damage in our study. The most plausible explanation for this effect lies in the fact that, in the presence of transition metal ions, ascorbate may act as a pro-oxidant (Miller & Aust 1989). In fact, iron is readily released from ferritin under reductive conditions (as is the case under hypoxia) (Samokyszyn et ul. 1988) and has been shown to play a major role in promoting tissue damage in our model of reoxygenation liver injury (Younes & Strubelt 1988). Ascorbate most probably potentiates reoxygenation damage by reducing ferric iron to the ferrous form. Ferrous then undergoes a Fenton-type reaction with hydrogen peroxide yielding hydroxyl or hydroxyl-like radicals and ferric, which, in turn, is again reduced by ascorbate. This pro-oxidant activity seems to cover the antioxidant properties of ascorbate under our experimental conditions. In conclusion, the inhibition of hypoxia/reoxygenation injury in isolated perfused rat livers by antioxidants strongly substantiates a role for oxidative stress in this type of tissue injury. Trolox C and propyl gallate may be of therapeutic value especially in emergency situations due to their water solubility and their high efficacy as free radical scavengers. cr-Tocopherol itself has already been successfully used under certain clinical situations, e.g. in the treatment of degenerative blood vessel diseases like intermittent claudication (Pinsky 1980; Haeger 1982). in the therapy of various hematological disorders (Clemens 1990), or in the treatment of shock lung (Wolf & Seeger 1982), to name but a few.
References Anundi, I. & H. DeGroot: Hypoxic liver cell death: critical PO2 and dependence of viability on glycolysis. Amer. J. Physiul. 1989, 257. G58-G64. Anundi, I., J. King, D. A. Owens, H. Schneider, J. J. Lemaster & R. G. Thurman: Total protection from hypoxic liver damage by fructose. Res. Cummun. Chem. Pathul. Pharmacol. 1987, 55, 1 1 1-1 16. Aruoma, 0. I., P. J. Evans, H. Kaur, L. Sutcliffe & B. Halliwell: An evaluation of the antioxidant and potential pro-oxidant properties of food additives and trolox C, vitamin E and probucol. Free Rad. Res. Cummun. 1990, 10, 143-157.
Bellomo, G. & F. Mirabelli: Oxidative stress injury studied in isolated intact cells. Mul. Tuxicol. 1987, 1, 281-293. Bradford, B. U., M. Marotto, J. J. Lemaster & R. G. Thurman: New, simple methods to evaluate zone-specific damage due to hypoxia in the perfused rat liver: time course and effect of nutritional state. J. Pharmacul. Exp. Therap. 1986, 236, 263-268. Brass, C. A,, J. Narciso & J. L. Gollan: Enhanced activity of the free radical producing enzyme xanthine oxidase in hypoxic rat liver. Regulation and pathophysiologic significance. J. Clin. Invest. 1991, 87, 424431. Brehe, J. E. & H. B. Burch: Enzymatic assay for glutathione. Anal Biochem. 1976, 74, 189-197. Buege, J. A. & S . D. Aust: Microsomal lipid peroxidation. Meth. Enxymol. 1978, 52, 302-310. Clemens, M. R.: Antioxidant therapy in hematological disorders. In: Antioxidants in therapy andpreven fivemedicine, Ed.: 1. Emerit. Plenum Press, New York, 1991, pp. 423433. Das, D. K., R. M. Engelman, Otani, H.. J. A. Rousou, R. H. Breyer & S . Lemeshow: Effect of superoxide dismutase and catalase on myocardial energy metabolism during ischemia and reperfusion. Clin. Physiul. Biuchem. 1986, 4, 187-198. Davies, M. J., L. G. Forni & R. L. Willson: Vitamin E analogue trolox C - E.S.R. and pulseradiolysis studies of free-radical reactions. Biuchem. J. 1988, 255, 513-522. Dunnet, C. W.: New tables for multiple comparisons with a control. Biometrics 1964, 20, 482491. Granger, D. N., G. Rutili & J. M. McCord: Superoxide radicals in feline intestinal ischemia. Gustruenterol. 1981, 81, 22-29. Griffith, 0.W.: Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 1980, 106, 207-212. Haeger, K.: Long-term study of alpha-tocopherol in intermittent claudication. Ann. N . E Acad. Sci. 1982, 393, 369-374. Jaeschke, H., C. V. Smith & J. R. Mitchell: Reactive oxygen species during ischemia-reflow injury in isolated perfused rat liver. J. Clin. Invest. 1988, 81, 1240-1246. Kahl, R.: Synthetic antioxidants: biochemical actions and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens. Toxicology 1984, 33, 185-228. Miller, D. M. & S. D. Aust: Studies of ascorbate-dependent, ironcatalyzed lipid peroxidation. Arch. Biuchem. Biophys. 1989, 271, 113-1 19. Okuda, M., I. Ikai, B. Chance & C. Kumar: Oxygen radical production during ischemia-reperfusion in the isolated perfused rat liver as monitored by luminol enhanced chemiluminescence. Biochem. Biophys. Res. Cummun. 199 1, 174, 2 1 7-221, Orrenius, S., D. J. McConkey, D. P. Jones & P. Nicotera: Ca2+activated mechanisms in toxicity and programmed cell death. In: IS1 Atlas of Science Pharmacology. Institute for Scientific Information, Philadelphia, 1988, pp. 3 19-324. Pinsky, M. J.: Treatment of intermittent claudication with alphatocopherol. J. Ameu. Pediat. Assoc. 1980, 70,454458. Roy, R. S . & J. M. McCord: Superoxide and ischemic conversion of xanthine dehydrogenase to xanthine oxidase. In: Oxy radicals and their scavenger systems. Cellular and medical aspects. Vol. 2. Eds.: R. A. Greenwald & G. Cohen. Elsevier, New York, 1983, pp. 145-153. Samokyszyn, V. M., C. E. Thomas, D. W. Reif, M. Sat0 & S . D. Aust: Release of iron from ferritin and its role in oxygen radical toxicities. Drug Metab. Rev. 1988, 19, 283-303. Strubelt, 0.. M. Younes & R. Pentz: Influence of extracellular calcium on ally1 alcohol-induced hepatotoxicity. Acta pharmacol. et toxicul. 1986, 59, 47-52. Videla, L. A.: Respective roles of free radicals and energy supply in hypoxic rat liver injury after reoxygenation. Free Rad. Res. Cummun. I99 I , 14, 209-2 15. Wolf, H. R. & H. W. Seeger: Experimental and clinical results in shock lung treatment with vitamin E. Ann. N . I:Acad. Sci. 1982, 393, 392409.
ANTIOXIDANTS ON LIVER HYPOXIA INJURY Wu, T.-W., N. Hashimoto, J.-X. Au, J. Wu, D. A. G. Mickle & D. Carey: Trolox protects rat hepatocytes against oxyradical damage and the ischemic rat liver from reperfusion injury. Hepatology 1991, 13, 575-580.
Younes, M. & 0. Strubelt: Enhancement of hypoxic liver damage by ethanol. Involvement of xanthine oxidase and the role of glycolysis. Biochem. Pharmacol. 1987, 36, 2973-2971. Younes, M. & 0. Strubelt: The involvement of reactive oxygen species in hypoxic injury to rat liver. Res. Commun. Chem. Pathol. Pharmacol. 1988, 59, 369-381.
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Younes, M., M. H. Schoenberg, H. Jung, B. B. Fredholm, U. Haglund & F. W. Schildberg: Oxidative tissue damage following regional intestinal ischemia and reperfusion in the cat. Res. Exp. Med. 1984, 184, 259-264. Younes, M., H. Wagner & 0. Strubelt: Enhancement of acute ethanol hepatotoxicity under conditions of low oxygen supply and ischemia/reperfusion. The role of oxygen radicals. Biochenz. Pharmacol. 1989, 38, 3573-358 I .
Effect of Antioxidants on Hypoxia/Reoxygenation-Induced Injury in Isolated Perfused Rat Liver M. Younes**, E. Kayser' and 0. Strubelt' 'Institute of Toxicology, Medical University of Liibeck, Ratzeburger Allee 160, D-2400 Liibeck, Germany, and 'Max-von-Pettenkofer-Institute, BGA, P.O. Box 330013, D-1000 Berlin 33, Germany (Received December 11, 1991; Accepted April 28, 1992) Abstract: Isolated perfused livers from rats fasted overnight were subjected to 30 min. of hypoxia followed by reoxygenation for 60 min., resulting in marked cytotoxicity as evidenced by an enhanced release of cytosolic enzymes (lactate dehydrogenase: 14-fold over controls, glutamate-pyruvate-transaminase: 12-fold over controls) and glutathione (twofold over controls) into the perfusate, by calcium accumulation (by a Factor of 1.4) in the tissue and by an 80% inhibition of bile secretion. Virtually no mitochondria1 injury became apparent and no evidence for lipid peroxidation could be found. In the presence of ascorbate, an augmentation of hepatic injury was observed. This might be due to the pro-oxidant activity of ascorbate in the presence of ionized iron, which is easily released from high molecular weight stores under reductive (e.g. hypoxic) conditions. The water soluble vitamin E analogue trolox C as well as propyl gallate clearly protected the liver against hypoxiaireoxygenation injury, yielding further evidence for a causative role of oxidative stress in this model. Due to their water solubility and their high efficacy as free radical scavengers, these antioxidants might be of therapeutic value.
With the increased application of new therapeutic strategies, like organ transplantation and cardiac reperfusion, the interest in the mechanisms underlying the cytotoxic effects observed following reperfusionlreoxygenation of ischaemicl hypoxic tissue has grown. In several studies reactive oxygen species have been implicated as the ultimate injuries species in reperfusionlreoxygenation damage of various organs (Granger et al. 1981; Younes et al. 1984; Das et al. 1986; Younes & Strubelt 1988; Brass et al. 1991). One of the major sources for reactive oxygen species seems t o be the xanthine oxidase reaction (Granger et al. 1981). Under ischaemicl hypoxic conditions, ATP-catabolism leads t o formation of hypoxanthine and xanthine; concomitantly, low oxygen supply results in a transformation of the NADdependent xanthing dehydrogenase into the 0,-dependent xanthine oxidase (Roy & McCord 1983). As soon as oxygen is reintroduced, purine metabolites are readily oxidized; a reaction which is associated with a n activation of molecular oxygen yielding toxic metabolites. The liver seems t o be particularly resistant to hypoxic as well as reperfusion injury, a s long as anaerobic energy supply (due t o glycolysis) is maintained (Anundi et al. 1987; Younes & Strubelt 1988; Anundi & DeGroot 1989). Depletion of glycogen, e.g. by food deprivation (Younes & Strubelt 1988) or inhibition of glycolysis (Younes & Strubelt 1988; Younes et al. 1989) makes the liver susceptible t o tissue damage due to hypoxia solely or hypoxia and reoxygenation. Evidence for the involvement of oxygen radicals produced mainly by the xanthineixanthine oxidase system in hypoxic liver damage has been provided by inhibitor
* To whom correspondence should be addressed at the following address: WHO-European Centre for Environment and Health, P.O. Box I , NL-3720 BA Bilthoven, The Netherlands.
studies using isolated perfused livers from fasted rats (Younes & Strubelt 1988). T h e aim of this study was to investigate the effect of antioxidants including the water soluble vitamin E analogue trolox C (Davies et al. 1988), the synthetic antioxidant propyl gallate (Kahl 1984) and ascorbate in this model of tissue injury in order t o further evaluate the role of oxidative mechanisms in hypoxic liver injury.
Materials and Methods Animals. Male wistar rats (conventional animals, 320 -380 g; breeder: Winkelmann, Borchen) were used throughout. They had free access to a standard diet (Altromin pellets) and tap water until use. Fasting was achieved by deprivation of feed, but not of drinking water 16 hr before surgery. Liverperfusiorz. Removal of the liver and its connection to a recirculating perfusion system was performed as previously described (Strubelt et al. 1986). The perfusion medium consisted of 250 ml Krebs-Henseleit-buffer, pH 7.4 (118 mmol/l NaCI, 6 mmol/l KCI, 1.1 mmol/l MgSO,, 1.2 mmol/l KH2P04, 25 mmol/l NaHCO,). CaClz (1.25 mmol/l) was added to the prewarmed medium (37') immediately before starting the perfusion experiments. The perfusion medium was continuously gassed with carbogen (95% 0,. 5% CO,) yielding an oxygen partial pressure of about 600 mmHg. To induce hypoxia in the appropriate experiments, carbogen was replaced by a mixture of 95% N, and 5% CO, after a 3 min. equilibration period. After 30 min. of hypoxia, reoxygenation was achieved by regassing with carbogen until the end of the experiment, i.e. for 60 min. Sodium taurocholate (26.7 g/l) was infused into the perfusate at a rate of 12 ml/hr to stimulate bile secretion. Oxygen consumption of the isolated livers was calculated from the difference in the oxygen concentrations of the influent and the effluent perfusate using a Micro pH/Blood Gas Analyzer 413 (Instrumentation Laboratory). In the experiments with antioxidants, trolox C, propyl gallate or ascorbate were added to the perfusate at the start of the equilibration period to yield a final concentration of 0.5 mmolil. At the end of the experiments, the livers were weighed, frozen in liquid nitrogen and kept frozen until analysis.
ANTIOXIDANTS ON LIVER HYPOXIA INJURY Biochemical determinations. The activities of cytosolic lactate dehydrogenase and glutamate-pyruvate-transaminaseas well as mitochondrial glutamate dehydrogenase were determined in the perfusate using commercial kits of Boehringer, Mannheim, FRG. Ca2+concentrations in the liver (following acid extraction) and in the perfusate were measured colorimetrically also with reagents of Boehringer, Mannheim. Malondialdehyde was measured in the liver and the perfusate by coupling to thiobarbituric acid (Buege & Aust 1978).Total glutahione was determined according to Brehe & Burch (1976); oxidized glutathione was estimated by the same procedure after blocking reduced glutathione with 2-vinylpyridine (Griffith 1980).
279
BILE FLOW pg /g min
Statistics. Meansf S.E.M. were calculated in the usual way. The difference between two means was checked with Dunnet’s t-test (Dunnet 1964) in the case of multiple comparisons or with Student’s t-test in the case of simple comparisons. The limit of significance was P < 0.05 in all cases.
Results Under normoxic conditions (control experiments) with oxygen partial pressure of around 600 mmHg, oxygen consumption by the isolated perfused liver was between 2.0 and 2.3 pmol/min. . g over the whole perfusion period (fig. 1). When
OXYGEN CONSUMPTION pmol/min. g
0
30T
I
I
I
30
60
90
1
120 min
Fig. 2. Time-dependent bile flow rate by isolated perfused livers from fasted rats. Values given are means and their standard errors (x+S.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min. equilibration period, hypoxia was induced from 30 to 60 min., followed by reoxygenation from 60 to 120 min. 0-0: controls, no hypoxia; 0-0: hypoxia + reoxygenation; 0-0: +0.5 mmol/l trolox C; M-M: f0.5 mmol/I propyl gallate; A-A: +0.5 mmol/l ascorbate.
2
0 min
Fig. 1. Time-dependent oxygen consumption by isolated perfused livers from fasted rats. Values given are means and their standard errors (xkS.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min. equilibration period, hypoxia was induced from 30 to 60 min., followed by reoxygenation from 60 to 120 min. 0---0: controls, no hypoxia; 0-0: hypoxia+ reoxygenation; 0-0: t 0 . 5 mmol/l trolox C; m-m: f0.5 mmol/l propyl gallate: A-A: f0.5 mmol/l ascorbate.
carbogen was replaced by 95%)N2/5%C 0 2 ,oxygen partial pressure fell to 100 mmHg. As a consequence, oxygen consumption dropped abruptly down to a minimum of 0.5 pmol/min. . g at the end of the hypoxic period (i.e. after 30 min.). Following reoxygenation, oxygen consumption rose again but only reached 76% of the value before hypoxia (fig. 1). Ascorbate, trolox C and propyl gallate had no significant effect on the course of oxygen consumption during the experiment, except for a slight reduction of the prehypoxic value in the presence of propyl gallate (fig. 1). Bile flow showed little variation with perfusion time in control experiments without hypoxia (fig. 2). Hypoxia led to a strong decline in bile flow rate which continued also following reoxygenation, reaching finally 20% of the original rate (fig. 2). Ascorbate had no influence on the inhibition of bile flow. In the presence of trolox C and propyl gallate, on the other hand, bile flow rate declined in the hypoxic phase to the same extent as in the experiments without antioxidants, but did not continue to decrease in the reoxygenation phase (fig. 2). Damage to the isolated perfused livers was estimated by measuring the efflux of the cytosolic enzymes lactate
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M. YOUNES ET AL
Table I . Hepatic concentrations of glutathione, calcium and TBA-reactive material (TBAR) at the end of the perfusion period. Values given are means and their standard errors (%k S.E.M.; n = 5 each). All animals were fasted for 16 hr prior to the start of the perfusion experiments. Hypoxia + reoxygenation - (Controls)
Group 1
Antioxidant (500 pmol/l) Trolox C Propyl gallate Ascorbate
+ + +
2 3 4 5
+
Total glutathione (GSH+ZGSSG) (pmol/g liver)
Oxidized glutathione (pmol/g liver)
3.67 k0.89 1.7 I f0.40* 2.49 & 0.26 2.46$0.16** 1.32f0.46
0.07kO.01 0.05+0.01
0.07+0.01 0.10+0.01
0.04 k 0.04
Ca2+
TBAR
(pmol/g liver)
(pmol/g liver)
1.42k 0. I4 2.02+0.1 I * 1.47kO.l3** 1.78kO.10 2.61 k0.32
24.6k1.1 22.2 5.4 14.1 k0.6 14.2+ 1.7 35.3k7.3
*
* Statistically significant difference (group 2 vs. group I ) (p< 0.05, Student's t-test)
** Statistically significant difference as compared
to group 2 (hypoxia + reoxygenation) (p < 0.05, Dunnet's t-test)
GSH =glutathione GSSG =oxidized glutathione
dehydrogenase (fig. 3) and glutamate-pyruvate-transaminase (fig. 4).Hypoxia and reoxygenation resulted in a strong sustained increase o f enzyme release into the perfusate which was markedly attenuated in the presence of trolox C or propyl gallate but clearly amplified in the presence of ascorbate (fig. 3 & 4). To assess mitochondria1 damage, the release of glutamate dehydrogenase into the perfusate was
GPT U/I
"ol
i
mm-
m-
too0 0
U
30
60
90
120 rnin
Fig. 3. Time-dependent release of lactate dehydrogenase (LDH) by isolated perfused livers from fasted rats. Values given are means and their standard errors (x+S.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min., equilibration period, hypoxia was induced from 30 to 60 min. followed by reoxygenation from 60 to 120 min. 0-0: controls, no hypoxia; 0-0: hypoxia+ reoxygenation; 0-0: +0.5 mmolil trolox C; B-U: +0.5 mmol/l propyl gallate; A-A: +0.5 mmol/l ascorbate.
I
30 Fig. 4. Time-dependent release of glutamate-pyruvate-transaminase (GPT)by isolated perfused livers from fasted rats. Values given are means and their standard errors (x k S.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min. equilibration period, hypoxia was induced from 30 to 60 min., followed by reoxygenation from 60 to 120 min. 0---0: controls, no hypoxia; 0-0: hypoxia + reoxygenation; 0-0: +0.5 mmol/l trolox C; B-B: +0.5 mmol/l propyl galla+0.5 mmol/l ascorbate. te; A-A:
ANTIOXIDANTS ON LIVER HYPOXIA INJURY
28 1
Table 2. Concentrations of glutathione and TBA-reactive material (TBAR) in the perfusate at the end of the perfusion experiment. Values given are means and their standard errors (IfS.E.M.; n = 5 each). All animals were fasted for 16 hr prior to the start of the perfusion experiment.
Group
Hypoxia + reoxygenation
Total glutathione (GSH + 2GSSG) ( p o l / 1)
Antioxidant (500 pmol/l)
Oxidized glutathione (pmol/l)
Oxidized glutathione/ total glutathione
26.8 k 6.40 14.0k 7.20 0.52 52.1 k 7.13* 21.3 f 4.19* 0.41 Trolox C 23.5*4.82** 14.8f 3.58 0.63 Propyl gallate 18.7+2.48** 7.8* 2.75** 0.42 + Ascorbate 102.0k28.4** 52.9k28.0 0.52 * Statistically significant difference (group 2 versus group 1) (P < 0.05, Student’s t-test) ** Statistically significant difference as compared to group 2 (hypoxiaf reoxygenation) (P< 0.05, Dunnet’s t-test) GSH = glutathione GSSG = oxidized glutathione 1
-
+ + +
2 3 4 5
-
(Controls)
-
measured. No marked mitochondria1 injury occurred following hypoxia and reoxygenation as evidenced by the low rate of glutamate dehydrogenase release which started in the reoxygenation phase (fig. 5). Still, a tendency to attenuate this low value was observed with propyl gallate (fig. 5). Hypoxia and reoxygenation led to an increase in tissue calcium content by a factor of 1.4 over control levels at the end of the experiment (table 1). Trolox C totally and propyl
GCDH U/1
T
II
-i
30
30
60
90
120 rnin
Fig. 5. Time-dependent release of glutamate dehydrogenase (GLDH) by isolated perfused livers from fasted rats. Values given are means and their standard errors (x+S.E.M., n = 5 each). Time point 0 represents the start of the respective experiments. After a 30 min. equilibration period, hypoxia was induced from 30 to 60 min., followed by reoxygenation from 60 to 120 min. 0---0: controls, no hypoxia; 0-0: hypoxia + reoxygenation; 0-0: +0.5 mmolll trolox C; W-W: +0.5 mmol/l propyl galla+0.5 mmol/l ascorbate. te; A-A:
TBAR (pmol/ I) 0.96k0.28 1.25k0.54 0.87 k0.28 1.24k0.23 3.05k1.18
gallate partially antagonized this rise in hepatic Ca2+-concentration while with ascorbate a tendency towards even higher calcium levels was observed (table 1). Following hypoxia and reoxygenation, hepatic glutathione was depleted by 53% (table 1). Again, trolox C and propyl gallate clearly inhibited this effect while ascorbate did not (table 1). Following reoxygenation of hypoxic livers, an enhanced release of glutathione into the perfusate was observed, which was prevented by trolox C and propyl gallate but enhanced by ascorbate (table 2). No increased production (table 1) or release (table 2) of thiobarbituric acidreactive material) was evident following hypoxia and reoxygenation.
Discussion The results of this study support earlier findings which indicated that the liver is resistent to hypoxic damage as long as anaerobic energy conservation reactions take place (Younes & Strubelt 1988; Bradford et al. 1986). Hypoxia and reperfusion injury occurred only in livers from rats which were deprived of food 16 hr prior to surgery. Under these conditions, hepatic glycogen is nearly totally depleted (Younes & Strubelt 1988); hence, no endogenous substrate for glycolysis is present. Besides this direct effect of fasting on energy metabolism, food deprivation was recently shown to accelerate the conversion of xanthine dehydrogenase into xanthine oxidase (Brass et al. 1991), favouring thereby the formation of intracellular pro-oxidants. Trolox C and propyl gallate, two potent water soluble antioxidants, protected isolated perfused rat livers against hypoxic injury. In recent studies with cultured rat hepatocytes (Wu et al. 1991) or with single-path perfused rat liver (Videla 1991), trolox C also proved to be protective against reperfusion injury. These observations provide a strong indication for an involvement of oxidatives stress in hypoxic andlor reperfusion hepatic injury, as both were previously shown to react with hydroxyl radicals with rate constants near diffusion limit (Aruoma et al. 1990). Due to their antioxidant capacity, they were able to save glutathione from being extracted from the cells following oxidation. In fact, direct evidence for the increased formation of
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M. YOUNES ET AL.
reactive oxygen species in reperfused ischaemic rat livers has been recently provided (Okuda et al. 1991). Membrane phospholipids, however, do not seem to be the major targets for oxidant stress in our model, as no lipid peroxidation products could be detected. Oxidation of cellular thiols and the possible formation of mixed disulfides may have occurred, as can be concluded from the observed loss of cellualr glutathione in our study (which cannot be accounted for by severe release into the perfusate). Similar effects on the hepatic glutathione status have also been reported by Jaeschke et al. (1988). Following depletion of cellular glutathione, oxidation of sulfhydryl groups in functional proteins, including membrane-bound Ca2+-translocasesmay occur (Bellomo & Mirabelli 1987). Such an effect results in a sustained increase in cytosolic free calcium and, consequently, to an activation of a number of calcium-dependent catabolic processes (Orrenius et al. 1988). Ascorbate even enhanced hepatic damage in our study. The most plausible explanation for this effect lies in the fact that, in the presence of transition metal ions, ascorbate may act as a pro-oxidant (Miller & Aust 1989). In fact, iron is readily released from ferritin under reductive conditions (as is the case under hypoxia) (Samokyszyn et ul. 1988) and has been shown to play a major role in promoting tissue damage in our model of reoxygenation liver injury (Younes & Strubelt 1988). Ascorbate most probably potentiates reoxygenation damage by reducing ferric iron to the ferrous form. Ferrous then undergoes a Fenton-type reaction with hydrogen peroxide yielding hydroxyl or hydroxyl-like radicals and ferric, which, in turn, is again reduced by ascorbate. This pro-oxidant activity seems to cover the antioxidant properties of ascorbate under our experimental conditions. In conclusion, the inhibition of hypoxia/reoxygenation injury in isolated perfused rat livers by antioxidants strongly substantiates a role for oxidative stress in this type of tissue injury. Trolox C and propyl gallate may be of therapeutic value especially in emergency situations due to their water solubility and their high efficacy as free radical scavengers. cr-Tocopherol itself has already been successfully used under certain clinical situations, e.g. in the treatment of degenerative blood vessel diseases like intermittent claudication (Pinsky 1980; Haeger 1982). in the therapy of various hematological disorders (Clemens 1990), or in the treatment of shock lung (Wolf & Seeger 1982), to name but a few.
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Bellomo, G. & F. Mirabelli: Oxidative stress injury studied in isolated intact cells. Mul. Tuxicol. 1987, 1, 281-293. Bradford, B. U., M. Marotto, J. J. Lemaster & R. G. Thurman: New, simple methods to evaluate zone-specific damage due to hypoxia in the perfused rat liver: time course and effect of nutritional state. J. Pharmacul. Exp. Therap. 1986, 236, 263-268. Brass, C. A,, J. Narciso & J. L. Gollan: Enhanced activity of the free radical producing enzyme xanthine oxidase in hypoxic rat liver. Regulation and pathophysiologic significance. J. Clin. Invest. 1991, 87, 424431. Brehe, J. E. & H. B. Burch: Enzymatic assay for glutathione. Anal Biochem. 1976, 74, 189-197. Buege, J. A. & S . D. Aust: Microsomal lipid peroxidation. Meth. Enxymol. 1978, 52, 302-310. Clemens, M. R.: Antioxidant therapy in hematological disorders. In: Antioxidants in therapy andpreven fivemedicine, Ed.: 1. Emerit. Plenum Press, New York, 1991, pp. 423433. Das, D. K., R. M. Engelman, Otani, H.. J. A. Rousou, R. H. Breyer & S . Lemeshow: Effect of superoxide dismutase and catalase on myocardial energy metabolism during ischemia and reperfusion. Clin. Physiul. Biuchem. 1986, 4, 187-198. Davies, M. J., L. G. Forni & R. L. Willson: Vitamin E analogue trolox C - E.S.R. and pulseradiolysis studies of free-radical reactions. Biuchem. J. 1988, 255, 513-522. Dunnet, C. W.: New tables for multiple comparisons with a control. Biometrics 1964, 20, 482491. Granger, D. N., G. Rutili & J. M. McCord: Superoxide radicals in feline intestinal ischemia. Gustruenterol. 1981, 81, 22-29. Griffith, 0.W.: Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 1980, 106, 207-212. Haeger, K.: Long-term study of alpha-tocopherol in intermittent claudication. Ann. N . E Acad. Sci. 1982, 393, 369-374. Jaeschke, H., C. V. Smith & J. R. Mitchell: Reactive oxygen species during ischemia-reflow injury in isolated perfused rat liver. J. Clin. Invest. 1988, 81, 1240-1246. Kahl, R.: Synthetic antioxidants: biochemical actions and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens. Toxicology 1984, 33, 185-228. Miller, D. M. & S. D. Aust: Studies of ascorbate-dependent, ironcatalyzed lipid peroxidation. Arch. Biuchem. Biophys. 1989, 271, 113-1 19. Okuda, M., I. Ikai, B. Chance & C. Kumar: Oxygen radical production during ischemia-reperfusion in the isolated perfused rat liver as monitored by luminol enhanced chemiluminescence. Biochem. Biophys. Res. Cummun. 199 1, 174, 2 1 7-221, Orrenius, S., D. J. McConkey, D. P. Jones & P. Nicotera: Ca2+activated mechanisms in toxicity and programmed cell death. In: IS1 Atlas of Science Pharmacology. Institute for Scientific Information, Philadelphia, 1988, pp. 3 19-324. Pinsky, M. J.: Treatment of intermittent claudication with alphatocopherol. J. Ameu. Pediat. Assoc. 1980, 70,454458. Roy, R. S . & J. M. McCord: Superoxide and ischemic conversion of xanthine dehydrogenase to xanthine oxidase. In: Oxy radicals and their scavenger systems. Cellular and medical aspects. Vol. 2. Eds.: R. A. Greenwald & G. Cohen. Elsevier, New York, 1983, pp. 145-153. Samokyszyn, V. M., C. E. Thomas, D. W. Reif, M. Sat0 & S . D. Aust: Release of iron from ferritin and its role in oxygen radical toxicities. Drug Metab. Rev. 1988, 19, 283-303. Strubelt, 0.. M. Younes & R. Pentz: Influence of extracellular calcium on ally1 alcohol-induced hepatotoxicity. Acta pharmacol. et toxicul. 1986, 59, 47-52. Videla, L. A.: Respective roles of free radicals and energy supply in hypoxic rat liver injury after reoxygenation. Free Rad. Res. Cummun. I99 I , 14, 209-2 15. Wolf, H. R. & H. W. Seeger: Experimental and clinical results in shock lung treatment with vitamin E. Ann. N . I:Acad. Sci. 1982, 393, 392409.
ANTIOXIDANTS ON LIVER HYPOXIA INJURY Wu, T.-W., N. Hashimoto, J.-X. Au, J. Wu, D. A. G. Mickle & D. Carey: Trolox protects rat hepatocytes against oxyradical damage and the ischemic rat liver from reperfusion injury. Hepatology 1991, 13, 575-580.
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