Accumulation of aldehydic lipid peroxidation products during postanoxic reoxygenation of isolated rat hepatocytes

FreeRadical Biology & Medicine, VoL 15, pp. 125-132, 1993 Printed in the USA.All rightsreserved.

0891-5849/93 $6.00 + .00 Copyright© 1993PergamonPress Ltd.

Original Contribution ACCUMULATION OF ALDEHYDIC LIPID PEROXIDATION PRODUCTS DURING POSTANOXIC REOXYGENATION OF ISOLATED RAT HEPATOCYTES

TILMAN GRUNE,* WERNER G. SIEMS,t and WOLFRAM SCHNEIDER* *Clinics of Physiotherapy, Medical Faculty (Charitr), Humboldt University Berlin, Schumannstr. 20-21, O-1040 Berlin, Germany; and tHerzog-Julius Hospital, Kurhausstr. 13-17, 0-3388 Bad Harzburg, Germany

(Received 14 October 1992; Revised 15 February 1993; Accepted 16 February 1993) Abstract--The accumulation of the aldehydic lipid peroxidation product 4-hydroxynonenal and thiobarbituric acid-reactive substances was demonstrated during anoxia/reoxygenation of isolated rat hepatocytes. 4-Hydroxynonenal was detected as dinitrophenylhydrazone derivative by means of an isocratic HPLC separation. The highest 4-hydroxynonenal level was found 15 min after the beginning of reoxygenation. The concentration of 4-hydroxynonenal was compared with the thiobarbituric acid-reactive substances formation, the glutathione status, and the cell viability. Addition of the xanthine oxidase inhibitor oxypurinol decreased the aldehyde formation during the reoxygenation phase. The same suppression of oxidative load by 20 vM oxypurinol (inhibition of xanthine oxidase) and by 1 mM oxypurinol (inhibition of xanthine oxidase plus radical scavenging) leads to two conclusions: First, the purine degradation is the primary radical source of postanoxic hepatocytes; second, the inhibitionof radical generation by xanthine oxidase is the main component of cell protecting by oxypurinol. On the other hand, oxypurinol addition did not accelerate the adenosine 5'-triphosphate (ATP) restoration. Keywords--Rat hepatocytes, 4-Hydroxynonenal, Aldehydes, Lipid peroxidation, Oxypurinol, Free radicals

during lipid peroxidation in microsomal membranes after stimulation with adenosine 5'-diphosphate (ADP)/Fe2+ or CC14 or in hepatocytes which were exposed to prooxidant stimuli. 6-s Although there are many results on the role of oxygen-derived free radicals in ischemia/reperfusion injury which were obtained in animal experiments and in clinical observations, including the ischemia/reperfusion of the liver or isolated liver cells, 9,1° there are only a few reports on the postanoxic/postischemic formation of aldehydes. These reports of various research groups describe only the formation of TBARS during the reperfusion, n,~2 But there are no data on the behavior of other aldehydic compounds, especially of 4-hydroxyalkenals, as secondary products of lipid peroxidation like HNE. Until now it was found in our laboratory that 4-hydroxynonenal was accumulated during the reperfusion phase of rat small intestine after 1 h of ischemia in in vivo experiments) 2,~3 This study deals with the measurement of in vitro formation of 4-hydroxynonenal in cell suspensions of isolated hepatocytes during anoxia and the following reoxygenation and the comparison of these changes

INTRODUCTION

Lipid peroxidation in biological systems is always combined with the formation of aldehydes, l 4-Hydroxy-2,3-trans-nonenal (4-hydroxynonenal, HNE) and malondialdehyde (thiobarbituric acid-reactive substances) are major aldehydic products of lipid peroxidation in cells. HNE is formed only from omega-6 polyunsaturated fatty acids and malondialdehyde/ thiobarbituric acid-reactive substances from fatty acids with at least three double bonds) -3 4-Hydroxynonenal is one of the biologically most active lipid peroxidation products. This compound is cytotoxic at high concentration (in the range of 100 ~M), disturbs at low concentration cell proliferation, and exhibits genotoxic effects.4 Furthermore, in the submicromolar range HNE is chemotactic5 and stimulates phospholipase C (for review see Ref. 2). HNE is formed

This study was generously supported by the Deutsche Forschungsgemeinschaft(DFG), Bonn. Address correspondence to: Dr. T. Grune. 125

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T. GRUNE et al.

with the glutathione status, purine degradation, and TBARS formation. MATERIALS AND METHODS

Chemicals Aldehyde standards (dinitrophenylhydrazones) were prepared in the laboratory of Prof. H. Esterbauer, Karl-Franzens University, Graz, Austria. The solvents n-hexane, acetonitrile, dichloromethane, methanol, benzene, and the TLC plates (silica gel 60, 0.2 mm thickness) were from Merck (Darmstadt, Germany). 2,4-Dinitrophenylhydrazine (DNPH) was obtained from Union Chimique (Brussels, Belgium). DNPH was dissolved in 5 ml of 1 M hydrochloric acid, extracted three times with 5 ml of n-hexane, and then adjusted to 1.8 mM solution with 1 M hydrochloric acid by means of absorbance measurement at 378 nm. Collagenase (Clostridium histolyticum) and nucleotide standards were from Boehringer (Mannheim, Germany). All other reagents (e.g., for the incubation buffer and the hepatocyte preparation) were purchased from Sigma Chemic G m b H (Deisenhofen, Germany). Thiobarbituric acid and malondialdehyde diethylacetal were obtained from Aldrich Chemic G m b H (Steinheim, Germany). Tetrabutylammonium phosphate was purchased from Waters (Milford, MA).

Cell preparation

37°C under continuous "shaking" (50 rotations per minute). The anoxia was induced by replacement of the O2/CO2 (95:5) gas phase by N2/CO2 (95:5). In each case the gas mixture was continuously administered to the suspension. The hepatocytes from each animal were used for one sample which was incubated under normoxic conditions (control) and three samples which were incubated for 60 min under anoxic conditions and for the following 30 min under reoxygenation conditions. The anoxia/reoxygenation samples were incubated (1) without any additions, (2) with 1 mM oxypurinol, and (3) with 20 uM oxypurinol. Oxypurinol was added at time 0 of the experiment.

Determination of 4-hydroxynonenal The detection of HNE was performed according to Esterbauer et al. 7 and Poli et al. 8

Derivatisation of liNE with DNPH Four milliliters of the cell suspension reacted with 4 ml of DNPH solution (1.8 mM in 1 N HCI) in the presence of 0.1 ml ethanolic solution of BHT (final concentration 10 mM) for 2 h in the dark. The sample was kept thereafter for 1 h in an ice bath in the dark, and then extracted three times with 8 ml of dichloromethane and centrifuged at 900 g and evaporated on a rotary evaporator to dryness at a temperature of 33°C. The residue was transformed with 1 ml of dichloromethane into small vials for spotting on TLC plates.

Male Wistar H-strain rats with a body weight of about 190 g (186 + 21 g) were used. Hepatocytes were prepared according to Berry and Friend ~4with modifications.~5 Starved rats (24 h) were anaesthetized by an intraperitoneal injection of pentobarbital (30 mg/kg bw). After an initial perfusion of the liver (8 min) as described in Ref. 15, the procedure of cell isolation was performed by a retrograde recirculating perfusion with a collagenase-containing solution (10 min/50 mg collagenase per 100 ml of buffer solution). As perfusion medium, a Krebs-Henseleit bicarbonate buffer gassed with O2:CO2 (19:1) was used.

The TLC plates were developed with dichloromethane in comparison with known standards. In this way according to Esterbauer 7 the dinitrophenylhydrazones of carbonyl compounds were separated into three zones. Zone I contains the 4-hydroxyalkenales. Zone I was scraped off, extracted three times with each 10 ml of methanol, and evaporated to dryness. The residue was dissolved in 1 ml of methanol.

Cell incubation

HPLC equipment and chromatographic conditions

The cell viability was determined by cell staining with trypan blue. Only hepatocyte suspensions with more than 85% of all cells excluding trypan blue were used for anoxia/reoxygenation experiments. The cell concentration was adjusted to a cytocrit of 2% (v/v) (5.2* 106 cells/ml). The incubation medium was a Krebs-Henseleit solution, in which the bicarbonate was replaced by HEPES. The cells were incubated at

An HPLC system from Perkin Elmer was used consisting of an M410 pump system, an LC 95 variable wavelength detector, an LCI-100 integrator, and a Rheodyne injector. For some determinations a Photodiode-Array-Detector (Hewlett-Packard) was used additionally? 6 The eluent for the isocratic separation was methanol-water (4:1, v/v). The flow rate was 1 ml/min. A column Nucleosil 5C 18 (Macherey, Nagel

Thin-layer chromatography

Aldehydesduring postanoxicreoxygenation

& Co., Dtiren, Germany/250 × 4.0 mm i.d.) with a 20 × 4.0 mm i.d. precolumn was used. Peak identification was performed by comparison of the retention times of peaks of biological extracts and of standard extracts, and by the coelution of biological extracts with the reference compound and by comparison of the spectra. Quantification of 4-HNE was achieved by separating the HNE-DNPH standard solutions of different concentrations.

Recovery The recovery was determined using an HNE standard solution (10 #M) and amounted to 32 + 5% (mean + S.D.). As one can see, the value of HNE recovery was highly reproducible. The values of liNE were corrected to 100%.

Detection of thiobarbituric acid-reactive substances The TBARS concentration was determined by means of the method of Uchigama and Mihara.17 One milliliter of cell suspension was added to an equal volume of 0.6% thiobarbituric acid, 10 mM BHT, and 1% H2PO 4. The sample was heated and stirred in a boiling water bath for 45 min. After cooling the optical density was determined at 535 and 520 nm with a Shimadzu UV-2100, and the difference of optical densities at the two wavelengths was taken for the calculation of TBARS level (always the absorbance spectrum from 500 to 600 nm was analyzed). A malondialdehyde standard was prepared by hydrolysis of malondialdehyde diethylacetal.

Determination of the glutathione status The analysis of GSH was described by Beutler et al. is and of GSSG by Hissin and Hilf. 19 The concentrations of GSH and GSSG were measured after reaction with 5.5'-dithiobis-(2-nitrobenzoic acid) (DTNB) photometrically at 405 nm or after addition of Nethyl-maleimide (NEM; serves for the blockade of sulfhydryl groups and prevents therefore the autooxidation of GSH to GSSG) and reaction with o-phtalaldehyde fluorimetrically at 350 nm (excitation maximum) and 420 nm (emission maximum).

Purine analysis The purines were determined by a gradient ionpair reversed-phase HPLC method as described by Werner. 16'2° The preparation of samples for HPLC separation of adenine nucleotides and their degradation products included deproteinization (6% HC104),

127

centrifugation (1200 × g for 5 min), neutralization with triethanolamine-K2CO3, and a filtration. After filtration, 50 #1 of the supernatant were analysed. For the HPLC analysis equipment from Perkin Elmer (Norwalk, CT) was used consisting of an M4 l0 pump system, an LC95 variable wavelength detector (adjusted at 254 nm), and an LCI-100 integrator. The columns were C-18 Sil-X-5, 250 m m , 4 . 6 mm i.d., precolumns 25 m m , 4 . 6 mm i.d. As eluents the buffers A and B were used: Buffer A is l0 mM NH4H2PO4 containing 2 mM tetrabutylammonium phosphate and buffer B is 80% of buffer A + 20% acetonitrile (v/v). The elution profile was 12 min a concave gradient from 100% A to 80% B/20% A; 25 min isocratic 80% B/20% A; in 2 min to 100% A. Then the system was flushed with buffer A for 5 min. The flow rate was 1.3 ml/min. RESULTS

In this study HNE could be detected and quantitatively evaluated in the samples of hepatocyte suspensions. The HNE-DNPH peak in HPLC chromatograms was demonstrated by comparison of the elution time of the biological extracts--shown in Fig. 1--with standard solutions and additionally by coelution of the biological extract and the standard solution in one run. There were no differences between HNE levels of control and anoxia/reoxygenation groups with and without oxypurinol at time zero of the experiments (Fig. 2). The HNE concentration during the first 15 min ofreoxygenation rose up to about threefold of the corresponding control level. This increase was only short term. After 30 min of reoxygenation the HNE level was already in the range of the initial value. There were no significant changes of liNE concentrations in hepatocyte suspensions in the presence ofoxypurinol during the whole incubation time (Fig. 2). In Table 1 the concentrations of TBARS in hepatocyte suspensions are demonstrated. The only significant change in the behavior of TBARS concentration was the 2.5-fold increase at 30 rain of reoxygenation in comparison with the initial value. Table 2 shows the data on viability of hepatocytes during the 90-min experiments. A significant decrease of hepatocytes which are able to exclude the trypan blue dye was measured during reoxygenation after anoxia. This anoxic and postanoxic decrease of cell viability was prevented by oxypurinol pretreatment (Table 2), both at 20 ~M and at 1 mM final concentration of the inhibitor of the xanthine oxidoreductase. The cellular content of reduced glutathione was significantly decreased after a 30-min reoxygenation pc-

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T. GRUNE et al.

A

B

C

I

2 mAU

j\ I

I

,t

L

L

!

0

5

10

0

5

10

timelmin)

Fig. 1. Chromatogram of an extract of hepatocyte suspension at 15 min of reoxygenation following 60 rain of ischemia (B). For comparison, in A the chromatogram of a standard solution mixture of the 2,4-dinitrophenylhydrazones of 4-hydroxyhexenal (1), 4-hydroxyoctenal (2), and 4-hydroxynonenal (3) and in C the addition of DNPH-HNE to the extract A is demonstrated. Column: Nucleosil 5Cts (250 × 4.0 mm i.d.); flow rate: 1 ml/min; eluent: water/methanol (80/20; v/v).

riod (Table 3). This decrease could be partially prevented in the presence of the xanthinoxidase inhibitor oxypurinol. The slight increase of reduced and oxidized glutathione during the incubation under normoxic conditions is the result of a real accumulation ofglutathione in the media as the result of an efflux of both forms of glutathione (data are not shown here) nmol/I suspension 1600 1200 800 400 0

0 min

60 min

75 min

90 min

Fig. 2. Concentration of 4-hydroxynonenal in hepatocyte suspensions during anoxia/reoxygenation experiments (60 min ofanoxia; 75 min = 60 min ofanoxia + 15 min ofreoxygenation; 90 min = 60 min of anoxia + 30 min ofreoxygenation). Symbols are: [] Control group: without any anoxic phase = normoxia (n = 4); [] group with 60 min of anoxia, but without oxypurinol treatment (n = 4); [] group with 60 min ofanoxia and 1 mM oxypurinol (n = 4); [] group with 60 min of anoxia and 20 t~M oxypurinol (n = 4). Values are given as (mean +_ SD). *p < .01 in comparison with values at other time points under the same incubation conditions and with 75-min values in the samples incubated under other conditions: normoxia; anoxia/reoxygenation in the presence of oxypurinol.

combined with the non-interrupted intracellular glutathione synthesis. During normoxic incubations the 2 GSSG/(GSH + 2 GSSG) ratio is without any changes, while the ratio during the reoxygenation phase underlies a twofold increase (Table 3). There is a deficiency of the total glutathione (GSH + 2 GSSG) after 60 min anoxia and 30 min ofreoxygenation in comparison to the 90 min of normoxic incubation and to the initial value of total glutathione. During the anoxic phase the adenine nucleotide content is rapidly decreased to about 20% of the initial value (Table 4). After 30 min ofreoxygenation a slight increase, but not a complete restoration, of the sum of adenine nucleotides was detected. The addition of oxypurinol had no effects on the adenine nucleotide content. The hypoxanthine concentrations at the beginning of the experiment are very low (Table 4). During normoxic incubation the hypoxanthine concentration in the cell suspension increased. The values ofhypoxanthine in Table 4 are given as mmol/l cells, which is valuable for estimation of balances in the purine pools; but taking into account the distribution of hypoxanthine inside and outside the cells the real concentration in cell suspension is much lower. So these values represent accumulation rates obtained by relation of the total hypoxanthine to the hepatocytes in which the hypoxanthine was generated. During anoxia the hypoxanthine level increased. In the case of the inhibition of the xanthinoxidoreductase, its increase was much higher. In the reoxygenation phase

Aldehydes during postanoxic reoxygenation

129

Table 1. The Concentration of Thiobarbituric Acid-Reactive Substances in Rat Hepatocytes during Anoxia and Reoxygenation Anoxia/Reoxygenation Incubation Time 0min 60 min 75 min 90min

Without Additions

+ 20 #M Oxypurinol

+ 1 mM Oxypurinol

7.71 + 1.66 11.03 + 1.09 7.82 + 1.11 22.06___4.80*

8.81 _+ 1 . 0 9 6.62 _ 1.90 5.50 _+ 1.46 12.12+2.91

11.03+2.20 5.50 _+ 1.66 8.81 _+ 2.19 11.02+2.13

Normoxia 9.63+4.97 7.72 + 4.40 7.61 _+ 2.20 7.76___1.18

For anoxia/reoxygenation the time values represent the following conditions: 60 min = 60 min of anoxia; 75 min = 60 min ofanoxia + 15 min of reoxygenation; 90 min = 60 min ofanoxia + 30 min of reoxygenation. All values are given as nmol/ml suspension (mean + SEM; n = 4). * p < .01 in comparison with values at other time points under the same incubation conditions and with 90-min values in the samples incubated under other conditions: normoxia; anoxia/reoxygenation in the presence of oxypurinol.

the hypoxanthine level decreased in the cell suspensions without additions, but the treatment with oxypurinol resulted in a further increase of hypoxanthine.

DISCUSSION

The liver cells are well endowed with the e n z y m a t i c machinery necessary to form large amounts of reactive oxygen species. Sources of reactive oxygen species in liver cells include various oxidases such as xanthine oxidase, amine oxidases, and aldehyde oxidase as well as monaminoxidase and cytochromes. Several authors demonstrated that reperfusion of the liver or reoxygenation of hepatocytes results in substantial cellular injury that is mediated by oxiradicals. The resulting lipid peroxidation processes during ischemia/reperfusion of the solid hepatic tissue or isolated liver parenchymal cells H and of other organs or cell

Table 2. Results of the Trypan Blue Exclusion Test in Rat Hepatocytes during Anoxia and Reoxygenation Anoxia/Reoxygenation Incubation Time

Normoxia

Without Additions

+ 20 #M Oxypurinol

+ 1 mM Oxypurinol

0 min 60 min I 90 rain 2

87.9 _+0.2 88.5 _+0.8 88.1 _+ 0.9

87.5 _+ 0.3 81.7 _+ 3.1 77.6 + 1.7"

88.1 + 0.4 85.3 -+ 2.7 88.6 -+ 0.8

87.9 _+ 0.3 84.4 _+ 2.4 87.0 --_ 1.6

l 60 min of anoxia. 2 60 min of anoxia + 30 min of reoxygenation. Values are given as % of not stained cells (mean + SD; n = 6). * p < .01 in comparison with values at other time points under the same incubation conditions and with 90-min values in the samples incubated under other conditions: normoxia; anoxia/reoxygenation in the presence of oxypurinol.

demonstrated by tissue production of thiobarbituric acid-reactive substances/malondialdehyde and conjugated dienes. The occurrence of 4-hydroxyalkenales as very cytotoxic secondary products of lipid peroxidation in ischemia/reperfusion of liver cells has not been described up to now. Obviously in parallel to the formation of aldehydes there occur very fast chemical and enzymatic processes of aldehyde utilization preventing a marked accumulation of aldehydic compounds, even at high aldehyde formation rates. Therefore, one has to expect low levels of 4-hydroxyalkenals as well under physiological and pathological conditions which are not easy to measure with the methodological approaches known from the literature, even taking into account the methods for detection of free HNE 24 and schiff-base bound and -SH l i n k e d 4 - H N E . 2s-27 The physiological (initial) value of about 450 nmol/1 suspension measured, in this study, is in the range of HNE values given in the literature: 0.48 nmol/g w/w for physiological conditions and 2.82 nmol/g w/w (vitamin E deficiency) in the rat liverJ The threefold increase during the first minutes of anoxia is in good agreement with the increase of HNE in reperfused small intestine. 12 The changes in the glutathione status and the TBARS concentrations are also indicators of an oxygen radical damage of cells during reoxygenation. Other authors measured high values of TBARS level of postischemic tissue, too. 11 But there is no report on the measurement of HNE concentrations during reperfusion. It is suggested that the accumulation of HNE cannot directly represent the extent of HNE formation rate during anoxia and especially during reoxygenation of hepatocytes. There is one reason which reduces very effectively the raise of HNE accumulation in liver cells: the fast consumption of HNE. Also for t y p e s 21-23 w e r e

T. GRUNE et al.

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Table 3. The Glutathione Status of Rat Hepatocytes during Anoxia a n d Reoxygenation Anoxia/Reoxygenation Incubation Time Reduced glutathione (GSH) 0 min 60 m i n 90min Oxidized glutathione (GSSG) 0min 60 m i n 90 m i n 2 GSSG/(GSH + 2GSSG) ratio 0 min 60 m i n 90 m i n

Normoxia

Without Additions

+ 20 #M Oxypurinol

+ 1 mM Oxypurinol

1.86 +_ 0.04 2.16 _+ 0.07 2.48_+0.15

1.89 _+ 0.08 1.94 + 0.11 1.43+0.16"

1.86 _ 0.03 2.09 _+ 0.16 1.71-+0.09

1.80 _+ 0.09 2.02 _+ 0.06 1.76_+0.18

63-+ 1 90 +_ 12 151 _+ 28

63_+ 1 87 -+ 18 102 _+ 13

66+_2 86 +_ 21 109 _+ 19

6.24 _+ 0.03 8.11 _+ 0.22 17.43 +_ 0.35*

6.34 _+ 0.07 7.69 _+ 0.23 10.66 _+ 0.29

6.82 _+ 0.04 7.83 _+ 0.70 11.02 _+ 0.21

64_+5 90 _+ 7 105 + 3 6.42 _+ 0,25 7.72 _+ 0,20 7.81 _+ 0,04

For anoxia/reoxygenation the time values represent the following conditions: 0 m i n = start of the experiment; 60 m i n = 60 m i n of anoxia; 90 m i n = 60 m i n of anoxia + 30 m i n of reoxygenation. Reduced glutathione (GSH) is given as # m o l / g w.w., oxidized glutathione (GSSG) as n m o l / g w.w., a n d 2GSSG/(GSH + 2GSSG) as % ( m e a n _+ SEM; n = 5). * p < .01 in comparison with values at other time points under the same incubation conditions a n d with 90-min values in the samples incubated u n d e r other conditions: normoxia; anoxia/reoxygenation in the presence of oxypurinol.

the control experiments all values of Fig. 2 represent the balance between the HNE formation rate on the one side and the rate of the intracellular consuming reactions on the other side. Preliminary results on the reactions of intracellular HNE consumption in hepatocytes of rats were published by Siems et al. 28 In those studies at 37°C even a maximal rate of HNE consumption of 28 nmol/mg w.w./min was measured. About 30% of the HNE is metabolised via the conjugation with glutathione to an HNE-glutathione adduct. 28 This may be the reason for the deficiency of the total glutathione after 60 min anoxia and 30 min

ofreoxygenation in comparison to the 90 min of normoxic incubation and to the initial value of total glutathione. On the different time dependency of the formation of HNE and TBARS (the HNE accumulation peak 15 min after the onset of reperfusion and the later increase of TBARS level) one can only speculate. It is known that the degradation rate of various aldehydes in liver cells is different. So a 14CO2 production of 2.4 nmol/mg protein/min (about 10 nmol/mg w.w./min) in rat liver homogenates from radioactive labeled malondialdehyde was measured. 29 This consumption rate was significantly lower than that for

Table 4. Concentration o f Adenine Nucleotides and of H y p o x a n t h i n e in Rat Hepatocytes during Anoxia and Reoxygenation Anoxia/Reoxygenation Incubation Time S u m o f adenine nucleotides 0 min 60 m i n ~ 90 m i n 2 Hypoxanthine 0 min 60 m i n i 90 m i n 2

Normoxia

Without Additions

+ 1 mM Oxypurinol

2.86 _+ 0.24 2.94 + 0.31 3.06 _+ 0.27

2.99 _+ 0.21 0.53 _+ 0.19 1.36 _+ 0.25

2.79 _+ 0.15 0.56 -+ 0.29 1.38 _+ 0.30

<0.01 0.11 + 0.05 0.18 +_ 0.08

<0.01 0.60 _+ 0.08 0.30 _+ 0.12

<0.01 1.62 + 0.14 2.31 _+ 0.25

60 m i n o f anoxia. 2 60 m i n o f a n o x i a + 30 m i n of reoxygenation. Values are given as m m o l / l cells ( m e a n _+ SD; n = 6). Taking into account the distribution o f hypoxanthine inside and outside the cells, the real concentration in cell suspension (mmol/1 suspension) is m u c h lower and is obtained by dividing the shown values by about 50.

Aldehydes during postanoxic reoxygenation

4-HNE in hepatocytes. 2s Furthermore, the bulk of TBARS may not be the free MDA, but lipidhydroperoxides. 3° Those compounds, with their slower utilization rate and continuous accumulation, may cover a possible peak of free MDA. The findings of the study confirm the demonstration of the involvement of lipid peroxidation processes in the damage of hepatocytes during anoxia and reperfusion, and in disturbances of the energy/ nucleotide metabolism, 9,2° ion imbalances, 9 etc. The protective role of the xanthine oxidase inhibitor oxypurinol, which has further effects on the pufine and pyrimidine nucleotide metabolism and on radical-mediated processes, 31-33 argues for a key role of this enzyme in the postanoxic oxygen free radical formation, lipid peroxidation, aldehyde formation, and oxidative damage of hepatocytes. The supply of the substrate of the xanthineoxidoreductase was demonstrated by the hypoxanthine accumulation. The differences of hypoxanthine concentration after anoxia in cell suspensions without and with oxypurinol one may explain by the metabolism of hypoxanthine by the xanthine dehydrogenase in the case of cell suspensions without any treatment. The partial transition of xanthine dehydrogenase to the oxidase form was already demonstrated. 9'34 The decrease of hypoxanthine during reoxygenation via the oxidase form of xanthineoxidoreductase forms superoxide anion radicals and hydrogen peroxide. The addition of oxypurinol had no effect on the accumulation of adenine nucleotides during reoxygenation. That means that the energy supply is not the reason for the higher viability of cells prevented by oxypurinol. In general, the effects of oxypurinol do not depend on the concentration of the inhibitor. The different concentrations of oxypurinol were used to evaluate the share of radical scavenging by oxypurinol as a component of cell protection by this agent. One may conclude that the main mechanism of the beneficial action of oxypurinol is the inhibition of xanthine oxidase induced radical formation, not an influence on purine nucleotide regeneration and not radical scavenging effects, which are described for concentrations higher than 0.5 mM. 33

4. 5. 6.

7.

8.

9.

10. 11. 12.

13.

14. 15. 16.

17. 18. 19. 20.

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BHT--butylated hydroxytoluene DNPH--2,4-dinitrophenylhydrazine GSH--reduced glutathione GSSG--oxidized glutathione HEPES--2-[4-(hydroxyethyl)- 1-piperazinyl]-ethansulfonic a c i d HNE--4-hydroxy-2,3-trans-nonenal (4-hydroxynonenal) HPLC--high-pefformance liquid chromatography TBARS--thiobarbituric acid-reactive substances TLC--thin-layer chromatography