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Role of Free Radicals in the Mechanism of The Hydrazine- Induced Formation of Megamitochondria
Free Radical Biology & Medicine, Vol. 23, No. 2, pp. 285–293, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00
PII S0891-5849(96)00616-8
Original Contribution ROLE OF FREE RADICALS IN THE MECHANISM OF THE HYDRAZINEINDUCED FORMATION OF MEGAMITOCHONDRIA
TATSUO MATSUHASHI,* XINRAN LIU,* MARIUSZ K ARBOWSKI,* MICHAL WOZNIAK,† JEDRZEJ ANTOSIEWICZ,** and TAKASHI WAK ABAYASHI * *Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, 65-Tsurumai-cho, Showa-ku, Nagoya 466, Japan, †Department of Biochemistry, University of Gdansk Medical School, 80-210 Debinki 1, Gdansk, Poland, and **Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education, 80-336,Gdansk, Poland (Received 26 August 1996; Revised 5 December 1996; Accepted 6 December 1996)
Abstract—The effect of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl(4-OH-TEMPO), a scavenger for free radicals, and 4-hydroxypyrazolo[3,4-d(pyrimidine)allopurinol], a xanthine oxidase inhibitor, on the hydrazine-induced changes of mitochondrial ultrastructure and those in the antioxidant system of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 0.5% hydrazine for 7 d in the presence and absence of a combined treatment with 4-OH-TEMPO or allopurinol. Results obtained were as follows. 4-OHTEMPO completely prevented the hydrazine-induced formation of megamitochondria in the liver, while it was partly prevented by allopurinol. The following changes observed in hydrazine-treated animals were improved almost completely by 4-OH-TEMPO:decreases in the body weight and liver weight; lowered rates of ADP-stimulated respiration and coupling efficiency of hepatic mitochondria; remarkable elevation of the level of lipid peroxidation. Improving effects of allopurinol were incomplete. The present results suggest that free radicals may play a key role in the mechanism of the hydrazine-induced formation of megamitochondria and that a part of free radicals generated during the hydrazine intoxication is ascribed to the degradation of purine nucleotides via xanthine oxidase. A general mechanism of the megamitochondria formation induced in various pathologic conditions besides the case of hydrazine are discussed. q 1997 Elsevier Science Inc. Keywords—Hydrazine, 4-OH-TEMPO, Megamitochondria, Liver, Free radicals, Allopurinol, Oxidative stress, Lipid peroxidation
partly the hydrazine-induced formation of megamitochondria in the rat liver by a-tocopherol 6 and coenzyme Q10 ( CoQ10 ) .7 At the same time, we have found that the lipid peroxidation in the liver is remarkably enhanced by the hydrazine intoxication, which is partly suppressed by a-tocopherol or CoQ10 . In the present study we demonstrate that 4-hydroxy2,2,6,6-tetramethyl-piperidine-1-oxyl ( 4-OHTEMPO ) , an electron spin resonance label, completely suppresses the hydrazine-induced formation of megamitochondria in the rat liver, and at the same time lowers the hydrazine-induced remarkable increases in lipid peroxidation almost to the same level of the control animals. We have also found changes in the body weight, liver weight, and respiratory rates
INTRODUCTION
We have been studying the mechanism and pathophysiological meaning of the phenomenon of the megamitochondria formation using hydrazine and ethanol as experimental models.1 – 4 We have proposed that the fusion of adjacent mitochondria may play a key role in the mechanism of the phenomenon based on physicochemical, biochemical, and morphological changes of the mitochondrial membranes during the formation process of megamitochondria.2,5,6 Recently, we have succeeded in suppressing Address correspondence to: Prof. T. Wakabayashi, Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, 65-Tsurumai-Cho, Showa-ku, Nagoya 466, Japan. 285
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of mitochondria of hydrazine-treated animals are remarkably improved by 4-OH-TEMPO. We propose based on the present study that free radicals may be a common triggering factor for the formation of megamitochondria induced by apparently different pathologic conditions. MATERIALS AND METHODS
Animals Male Wistar rats aged 4 weeks were divided into six groups. Each group consisted of six animals. Animals of the first three groups were placed on a powdered complete diet containing 0.5% hydrazine for 7 d. The latter three groups of animals were fed a normal complete diet. Animals of the second and fifth group were given 4-OH-TEMPO daily for the first 3 d of the experiment by IV via the tail veins (10 mg/100 g body weight). On the fourth day and thereafter, 4-OHTEMPO was given to the animals by SC injection (10 mg/100 g body weight). Animals of the third and sixth group were given allopurinol, a xanthine oxidase (EC 1.1.3.22) inhibitor, daily by SC injection (5 mg/100 g body weight). 4-OH-TEMPO was dissolved in physiological saline , and allopurinol was suspended in 0.5% carboxymethyl cellulose. Preparation of mitochondria and microsomes from the liver Immediately after the animal was sacrificed by decapitation, the liver was removed from the animal. Small pieces of the tissue was used for light and electron microscopy. The rest of the liver was rinsed in 2 mM N-2-hydroxyethylpiperazine-N *-2-ethansulfonic acid (Hepes), pH7.4, with 70 mM sucrose, 220 mM mannitol and 0.1 mM EDTA. Bovine serum albumin (BSA) was added to the isolation medium at a final concentration of 0.05%. Mitochondria were isolated according to the method previously described.1 In the present study, the routine isolation procedure for liver mitochondria, as previously described, was adopted based on the following previous observations: a heavy (rich in megamitochondria) and light mitochondrial fractions obtained from the liver of animals treated with alkyl alcohols including ethanol showed essentially the same phosphorylating ability 5 ; megamitochondria and normal sized mitochondria in cuprizone-treated mouse liver were often connected to each other by narrow stalks in the hepatocyte when they were revealed on one plane of section for electron microscopy. Furthermore, mitochondria apparently separated from each other on one plane of section were connected to each other far more fre-
quently than expected when they were examined by serial sections 8,9 ; similar results were obtained with hydrazine-treated mouse liver mitochondria, megamitochondria and normal sized mitochondria were connected each other when their three-dimensional structures were reconstructed by serial sectioning technique (unpublished observations). These results may allow us to assume that mitochondria connected to each other in the cell are separated each other during the homogenization and the differential centrifugation procedure. Furthermore, some of megamitochondria may be broken into smaller ones during the isolation process. We must bear in mind that we have not succeeded yet in the isolation of megamitochondria maintaining the same three dimensional structures as they are in vivo. The postmitochondrial fraction was centrifuged for 10 min at 12,000 g to sediment lysosomes, microbodies and swollen, light mitochondria.9 The resultant supernatant was centrifuged for 1 h at 105,000 g. The pellet thus obtained was suspended in 1.15% KCl and was used as the microsomal fraction, and the supernatant was used as the soluble fraction. Measurement of oxygen uptake of mitochondria Respiratory rates of mitochondria were measured using Clark-type electrode as previously described.1 Protein assay Protein was determined by the method of Lowry et al.10 using bovine serum albumin as standard. Determination of purine derivatives Hypoxanthine, xanthine, and uric acid in the soluble fraction were determined using high-performance liquid chromatography (HPLC) according to the method of Kojima et al.11 The chromatographic apparatus consisted of Waters 600E controller, Waters 484 detector and Waters 741 module integrator system (Milford, MA, USA). Aliquots of the soluble fraction were injected on 1018 COSMOSIL column composed of guard column (4.6 1 50 mm, Nakali Tesque, Kyoto, Japan), and the main column (4.6 1 250 mm), (Nakali Tesque). The mobile phase was potassium phosphate buffer (40 mM, pH 2.2) containing 20 ml of methanol per liter. The flow rate was set at 1.0 ml/min and the wavelength of detection was set at 254 nm. Determination of D6- and D9-desaturase activities Activities of D6- and D9-desaturase in microsomes were determined by the method of Christiansen et al.12
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The reaction medium contained in the total volume of 1.0 ml, 0.15 M KCl, 5 mM ATP, 5 mM MgCl2 , 0.25 mM CoA, 1.0 mM NADH, 1.5 mM glutathione, 45 mM NaF, 0.5 mM nicotinamide, 1 mg BSA, 0.1 mM potassium phosphate buffer, pH 7.4, microsomes (1 mg), and 75 nmoles [1- 14C]stearic acid or [1- 14C]linoleic acid as 1 mg BSA emulsion. The incubation was carried out at 377C for 20 min in a metabolic shaker. The reaction was terminated by the addition of 1 ml of 2.7 M KOH in methanol followed by the saponification for 45 min at 657C. The reaction medium was then acidified by the addition of 5 M HCl. Fatty acids in the reaction medium were extracted twice with hexane and methylated by the method of Hoshino et al.13 Fatty acid methyl esters were separated on thin layer chromatography (TLC) plates coated with silica gel G (Wako Chemical Co., Osaka, Japan) impregnated with 10% (w/w) AgNO3 . Plates were developed in hexane/diethyl ether (9:1, v/v) for the separation of monounsaturated fatty acids from saturated fatty acids and of triunsaturated fatty acids from diunsaturated fatty acids. The spots were made visible under ultraviolet light by spraying with 2,7-dichlorofluorescein (0.2%, w/v in ethanol). They were scraped off directly into scintillation vials. A liquid scintillation mixture (5 ml) containing 25 mg 2,5-diphenyloxazole (PPO), 1.5 mg 1,4bis-2-(5-phenyloxazolyl)benzene (POPOP), 0.33 ml ethyl acetate and 4.67 ml toluene was added to each vial.14 The radioactivity was counted using ALOKA LSC-1500 liquid scintillation counter (Aloka, Tokyo, Japan). Determination of oxidative stress Oxidative stress induced by the hydrazine intoxication was monitored by measuring the amounts of thiobarbituric acid reactive substances (TBARS) and lipidsoluble fluorophores (LSF) in microsomes and mitochondria as described before.5 TBARS were assayed with a thiobarbituric acid method.15 The concentration of TBARS in the sample was calculated using extinction coefficient of 1.56 1 10 5 cm-1 . LSF was assayed with a fluorometry method.16 Excitation and emission wave lengths were 366 and 460 nm, respectively. The concentration of LSF in the sample was expressed as a relative fluorescence where the fluorescence of quinine dissolved in sulfuric acid at a concentration of 0.1 mg/ml was taken as 100. Light and electron microscopy A part of the liver of each animal was cut into small pieces and processed for electron microscopy according to the method previously described.1 For light mi-
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croscopic examination, thick sections (0.5–2.0 mm) were obtained from Epon-embedded samples and stained with basic fuchsin and methylene blue.9 Distribution of megamitochondria within a hepatic lobule was examined using thick sections in the light microscope. Chemicals Hydrazine dihydrochloride was obtained from Merck (Darmstadt, Germany). 4-OH-TEMPO and allopurinol were obtained from Wakao Pure Chemical Co. (Osaka, Japan). [1- 14C]stearic acid (2.1 Mbq/ mmol; 97.1 radiochemical purity) and [1- 14C]linoleic acid (2.1 Mbq/ mmol; 98.8 radiochemical purity) were purchased from Amersham International (Amersham, England). All other chemicals were of analytical grade. Statistical analysis All the data were expressed as means { standard error (means { SE). Statistical comparisons of data were made using paired t-test with p õ .05 being considered significant. RESULTS
Effects of 4-OH-TEMPO and allopurinol on the body weight and wet weight of the liver of hydrazinetreated rats Animals placed on a hydrazine diet for 7 d became gradually weak losing body weight remarkably (Table 1). On the other hand, animals treated with hydrazine plus 4-OH-TEMPO were as active as the control animals, and the average body weight of the former animals was almost the same as that of the latter animals. Table 1. Effects of 4-OH-TEMPO and Allopurinol on the Body Mass and Wet Mass of the Liver of Hydrazine-Treated Ratsa
Animals Control Control / 4-OH-TEMPO Control / Allopurinol Hydrazine Hydrazine / 4-OH-TEMPO Hydrazine / Allopurinol
Body Mass (g) 149.0 { 152.9 { 140.0 { 74.7 { 136.5 { 86.7 {
0.8 4.1 5.0 2.0b 4.1 2.8d
Wet Mass of the Liver (g) 6.86 { 6.90 { 6.93 { 2.93 { 5.88 { 4.00 {
0.18 0.17 0.21 0.19b 0.21e 0.29c
a Animals were fed with 0.5% hydrazine-diet for 7 d in the presence or absence of a combined treatment with 4-OH-TEMPO or allopurinol. Details in experimental conditions are described in the Materials and Methods section. Data are the averages and standard error (mean { SE) of five different animals. Values are significantly different from those of the corresponding control at b (p õ .001), c (.001 õ p õ .01), d (.01 õ p õ .02), e (.02 õ p õ .05).
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The average body weight of animals after 7 d of treatment with hydrazine plus allopurinol, however, was much smaller than that of the control animals. The averaged wet weight of the liver of hydrazine-treated animals was distinctly smaller than that of the control animals while it was much improved by the treatment with 4-OH-TEMPO although, statistically, it remained smaller than the control (Table 1). The average wet weight of the liver of animals treated with hydrazine plus allopurinol, on the other hand, remained much smaller than that of the control animals. Effects of 4-OH-TEMPO and allopurinol on hydrazine-induced ultrastructural changes of mitochondria in the liver 4-OH-TEMPO completely abolished the hydrazineinduced formation of megamitochodnria in the liver (Fig. 1A). Namely, mitochondria in the hepatocyte of the liver of animals treated with hydrazine for 7 d became enlarged remarkably with decreases in number per hepatocyte (Fig. 1A). Whereas ultrastructural appearances of those of animals treated with hydrazine plus 4-OH-TEMPO (Fig. 1B) were almost the same as those of the control (Fig. 1D). Mitochondria of the hepatocyte of animals treated with hydrazine plus allopurinol (Fig. 1C) were much smaller than those of animals treated with hydrazine alone and yet they remained larger than those of the control. Effects of 4-OH-TEMPO and allopurinol on the respiratory rate of hydrazine-treated rat liver mitochondria We have demonstrated in the present study that both 4-OH-TEMPO and allopurinol exert remarkable effects on the structural changes of mitochondria of the liver of hydrazine-treated animals. Thus, we have examined the effect of 4-OH-TEMPO and allopurinol on the rate of respiration and phosphorylating ability of mitochondria of hydrazine-treated animals (Table 2). The rate of ADP-stimulated respiration (state 3 respiration) of mitochondria was decreased in hydrazine-treated animals compared to those of the control. The ratio of state 3 to state 4 respiration (respiratory control index [RCI]) became lowered compared to that of the control. Phosphorylating ability of these mitochondria was also decreased compared with that of the control. On the other hand, 4-OH-TEMPO remarkably improved such changes observed in hydrazine-treated animals. Such improving effects of 4-OH-TEMPO on mitochondria of hydrazine-treated animals was less remarkable in the case of allopurinol.
Effects of 4-OH-TEMPO and allopurinol on lipid peroxidation in the liver of hydrazine-treated animals Since the hydrazine-induced formation of megamitochondria was completely suppressed by 4-OHTEMPO, we have examined the effects of 4-OHTEMPO and allopurinol on lipid peroxidation in the liver of hydrazine-treated animals (Fig. 2). It is evident from the figure that the amounts of TBARS in microsomes and mitochondria of the liver of hydrazinetreated animals were remarkably increased compared to those of the control. 4-OH-TEMPO completely suppressed lipid peroxidation enhanced by hydrazine intoxication. On the other hand, inhibitory effects of allopurinol on lipid peroxidation were incomplete; the amounts of TBARS in microsomes and mitochondria of the liver of animals treated with hydrazine plus allopurinol were definitely smaller than those of animals treated with hydrazine alone, and yet larger than those of the control. Similar results were obtained with LSF. However, inhibitory effects of 4-OH-TEMPO using LSF on lipid peroxidation in hydrazine-treated animals were less remarkable compared to those obtained with TBARS. Effects of allopurinol on the rate of the degradation of purine derivatives We have shown in the present study that allopurinol partly prevents the hydrazine-induced formation of megamitochondria and at the same time lowers slightly the lipid peroxidation. Thus, we examined effects of allopurinol on the rate of purine nucleotide degradation in hydrazine-treated animals (Fig. 3). The amounts of hypoxanthine, xanthine, and uric acid in hydrazinetreated animals were remarkably elevated compared to those of the control. The level of hypoxanthine in animals treated with hydrazine plus allopurinol was elevated remarkably whereas that of xanthine became decreased remarkably compared to that of the control. These data indicate that allopurinol actually exerted its inhibitory action on the degradation of hypoxanthine into xanthine in vivo. Changes in some fatty acid desaturase activities in hydrazine-treated animals Previously, we have shown that remarkable changes take place in phospholipid classes and fatty acid compositions of phospholipid domains of the mitochondrial and microsomal membranes during the formation process of hydrazine-induced megamitochondria 17 increases in the relative amount of phosphatidylethanolamine (PtdEtn) and acidic phospholipids such as
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Fig. 1. Effects of 4-OH-TEMPO and allopurinol on hydrazine-induced ultrastructural changes in hepatic mitochondria. (A) hydrazine, (B) hydrazine plus 4-OH-TEMPO, (C) hydrazine plus allopurinol, and (D) control. Magnification of electron micrographs: 19000.
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T. MATSUHASHI et al. Table 2. Effects of 4-OH-TEMPO and Allopurinol on the Coupling Efficiency of Hydrazine-Treated Rat Liver Mitochondriaa Glutamate / Malate
Succinate State 3 Animals
State 4
RCI
ADP/O
State 3
(natoms O/mg/min)
Control Hydrazine Hydrazine / 4-OHTEMPO Hydrazine / Allopurinol
State 4
29.3 { 4.9 6.73 { 0.17 27.8 { 9.8 6.39 { 0.22
ADP/O
(natoms O/mg/min)
203.5 { 8.7 29.4 { 1.7 6.94 { 0.13 1.97 { 0.02 119.3 { 1.6 16.5 { 0.3 150.9 { 7.6e 35.9 { 4.2 4.38 { 0.53d 1.75 { 0.08e 90.3 { 4.1d 15.3 { 0.8 197.0 { 2.2 177.4 { 3.9
RCI
1.92 { 0.03 116.7 { 5.8 1.67 { 0.03c 102.9 { 5.4
7.24 { 0.10 5.90 { 0.20c
2.94 { 0.02 2.56 { 0.04c
18.4 { 0.3d 6.37 { 0.40 2.93 { 0.02 17.1 { 0.8 6.03 { 0.04b 2.69 { 0.01c
a Mitochondria were isolated from the liver of the control rats and animals fed with a diet containing 0.5% hydrazine for 7 d in the presence and absence of 4-OH-TEMPO and allopurinol treatment. Details of the treatment of animals are described in the Materials and Methods section. Data are the averages standard error (mean { SE) of three different animals. Values are significantly different from the control at b ( p õ .001), c (.001 õ p õ .01), d (.01 õ p õ .02), e (.02 õ p õ .05).
phosphatidylserine (PtdSer) in mitochondria; increases in the ratio of unsaturated to saturated fatty acids in phopholipid domain of mitochondrial membranes; decreases in that in microsomal membranes. Thus, we
have measured in the present study some fatty acid desaturase activities in the liver of animals treated with hydrazine (Table 3). It is interesting to note that activity of D9-desaturase was remarkably increased in ani-
Fig. 2. Effects of 4-OH-TEMPO and allopurinol on hydrazine-induced changes in the level of lipid peroxidation in hepatic mind megamitochondria tochondria and microsomes. Data are the averages and standard error (mean { SE) of six different experiments. Data on experimental group are statistically different from those of the control at: a (p õ .001), b (.001 õ p õ .01), c (.01 õ p õ .02), and d (.02 õ p õ .05).
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mation in the hydrazine intoxication is xanthine oxidase, which generates superoxide during the catabolism of hypoxanthine into uric acid.20–22 The present study has demonstrated that the hydrazine intoxication accelerates the degradation of purine nucleotide derivatives indicating an increased production of superoxides.
Role of free radicals in the mechanism of the hydrazine-induced formation of megamitochondria
Fig. 3. Effects of allopurinol on hydrazine-induced changes in the level of hypoxanthine, xanthine and uric acid in the liver. Levels of purine derivatives were measured using the postmicrosomal fraction. Data are the averages and standard error (mean { SE) of six different experiments. Data on experimental group are statistically different from those of the control at: a (p õ .001), b (.001 õ p õ .01), c (.01 õ p õ .02).
mals treated with hydrazine for 3 d, while those of both D6- and D9-desaturase became decreased in animals tretaed with hydrazine for 7 d. The discrepancy seen between the two desaturase activities in animals treated with hydrazine for 3 d will be discussed later.
DISCUSSION
In the present study, we have successfully suppressed the hydrazine-induced formation of megamitochondria in the rat liver by 4-OH-TEMPO which at the same time lowered lipid peroxidation induced by the hydrazine intoxication. We have also shown that allopurinol, a xanthine oxidase inhibitor, partly prevents the hydrazine-induced formation of megamitochondria.
The present study has clearly demonstrated that 4OH-TEMPO is more effective than CoQ10 or a-tocopherol in suppressing both hydrazine-induced lipid peroxidation and megamitochondria formation. The possible explanation for this may be as follows. CoQ10 and a-tocopherol are lipid-soluble and are buried within the mitochondrial membranes while 4-OHTEMPO is water-soluble and may be scattered within the cytoplasm of the cell. And a-tocopherol is able to scavenge effectively only peroxy radicals locating near the surface of the biological membranes, while carbon centered radicals locating deep inside the membrane are free from a-tocopherol.23 On the other hand, nitroxides, such as 4-OH-TEMPO, were reported to be effective in scavenging both peroxy radicals 24 and carbon centered radicals.25 If we accept that free radicals are responsible for the mechanism of the formation of megamitochondria, as shown in the present study, then the superiority of 4-OH-TEMPO to a-tocopherol or CoQ10 as a scavenger for free radicals could be related to the fact that the suppressing effect of the megamitochondria formation by 4-OH-TEMPO was complete while that by a-tocopherol or CoQ10 was incomplete. Finally, we should like to refer briefly to the changes observed in some fatty acid desaturase activities in hyTable 3. Changes in D6-Desaturase and D9-Desaturase Activities of Hydrazine-Fed Rat Liver Microsomesa
Animals
Sources of free radicals in the hydrazine intoxication Previously, we have shown that the hydrazine intoxication causes remarkable increases in the level of lipid peroxidation.6 This was confirmed in the present study. It is generally accepted that the major route of metabolism of hydrazine in vivo is its oxidation yielding nitrogen-centered free radicals.16 The mixed-function oxidase system has been reported to metabolize hydrazine in vitro catalyzing the formation of hydrazine radicals.18,19 Another source of free radical for-
Control Hydrazine-3 d Hydrazine-7 d
D6Desaturase (nmol/min/ mg)
D9Desaturase (nmol/min/ mg)
0.21 { 0.01 0.20 { 0.02 0.12 { 0.03b
1.71 { 0.12 2.79 { 0.02b 0.92 { 0.05b
a Microsome were isolated from the liver of animals fed with a diet containing 0.5% hydrazine for 3 and 7 d. Activity of D6-desaturase using linoleic acid as substrate is expressed as nmol linolenic acid synthesized per min per mg microsomal protein. Activity of D9desaturase using stearic acid as substrate is expressed as nmol oleic acid synthesized per min per mg microsomal protein. Data are the averages and standard error (mean { SE) of six different experiments. Data on experimental group are statistically different from those of the control at b ( p õ .001).
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drazine-treataed animals. Previously, we have proposed that the fusion of adjacent mitochondria may play a key role in the mechanism of the formation of megamitochondria based on physicochemical, biochemical, and morphological changes of the mitochondrial membranes during the hydrazine- and ethanol-induced formation of megamitochondria.2 – 4 Namely, we have shown distinct increases in the ratio of unsaturated fatty acids to saturated fatty acids (U/S ratio) in phospholipids extracted from mitochondria of rats treated with hydrazine for 3 d.17 These data may strongly suggest a possible role of the mebrane fusion in the mechanism of the formation of megamitochondria since the elevation of the degree of unsaturation in fatty acyl chains in phospholipids enhances the transition of phospholipids from bilayer to hexagonal II (HII ) phase, which is essential for the process of the membrane fusion.26 However, we have no data at the present time to explain how D9-desaturase activity was enhanced in animals treated with hydrazine for 3 d. Likewise, we need further investigations to explain changes observed in D6- and D9-desaturase activities in animals treated with hydrazine for 7 d. We are now studying the effect of 4-OH-TEMPO and other scavengers for free radicals on various experimental conditions for megamitochondria formations besides hydrazine, and the results will be reported soon. Acknowledgements — This work was supported in part by Grantsin-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The authors thank Mrs. N.Yoshimi for the preparation of the manuscript.
8. 9. 10. 11.
12. 13. 14.
15. 16. 17.
18.
19. 20.
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nigis, J.; Wakabayashi, T. Suppression of the hydrazine-induced formation of megamitochondria in the rat liver by coenzyme Q10 . Toxicol. Pathol. 86:47–54; 1995. Wakabayashi, T.; Green, D. E. On the mechanism of cuprizoneinduced formation of megamitochondria in mouse liver. Bioenergetics 6:179–192; 1974. Wakabayashi, T.; Asano, M.; Kurono, C.: Mechanism of the formation of megamitochondria induced by copper-chelating agents. Acta Pathol. Jpn. 25:15–37; 1975. Lowry, O. H.; Rosenbrough, A. L.; Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265– 275; 1951. Kojima, T.; Nishina, T., Kitamura, M.; Kamatani, N.; Nishioka, K. Reversed-phase liquid-chromatographic determination of purine compounds in serum applied to studies of hypouricemia. Clin. Chem. 32:287–290; 1986. Christiansen, E. N.; Lund, J. S.; Rortveit, T.; Rustan, A. C. Effect of dietary n-3 and n-6 fatty acid desaturation in rat liver. Biochim. Biophys. Acta 1082:57–62; 1991. Hoshino, M.; Williams, M.; Kishimoto, Y. Esterification of fatty acids at room temperature by chloroform-methanolic Hcl-cupric acetate. J. Lipid Res. 14:599–601; 1973. Ayudarte, M. V.; Nunez, M. C.; Boza, Jr, J.; Jimennez, J. Gil, A.; Squarez, M. D. Changes in liver microsome lipids and plasma fatty acids induced by dietary orotate in the weanling rat. Comp. Biochem. Physiol. 103B:65–69; 1992. Buege, J. A.; Aust, S. D. Microsomal lipid peroxidation. Methods Enzymol. 52:302–310; 1978. Dillard, C. J.; Tappel, A. L. Fluorescent damage products of lipid peroxidation. Methods Enzymol. 105:337–341; 1984. Adachi, K.; Matsuhashi, T.; Nishizawa, Y.; Usukura, J.; Momota, M.; Popinigis, J.; Wakabayashi, T. Further studies on physicochemical properties of mitochondrial membrabes during the formation process of megamitochondria in the rat liver by hydrazine. Exp. Mol. Pathol. 61:134–151; 1994. Nelson, S. D.; Gordon, W. P. Chemical mechanism and biological effects. In: Sydney, R; Parke, D. V.; Kocsis, J. J; Jollow, D. G.; Gibson, G. C.; Witmer, C. M., eds. Biological reactive intermediates. II Part B. New York: Plenum Press; 1982:971– 981. Jenner, A. M.; Timbrell, J. A. Hydrazine metabolism in rat liver microsomes. Human Exp. Toxicol. 68:349–357; 1990. Noda, A.; Noda, H.; Ohno, K.; Mikasa, A.; Kanazawa, Y.; Isobe, R.; Hirata, M. Spin trapping of a free radical intermediate formed during microsomal metabolism of hydrazine. Biochem. Biophys. Res. Commun. 133:1086–1091; 1985. Kellog, E. W.; Fridovich, I. Superoxide, hydrogen peroxide, and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J. Biol. Chem. 250:8812–8817; 1975. Fridovich, I. Superoxide radical: an endogenous toxicant. Annu. Rev. Pharmacol. Toxicol. 23:239–257; 1983. Takahashi, M.; Tsuchiya, J.; Niki, E. Scavenging of radicals by vitamine E in the mebranes as studied by spin labeling. Amer. Chem. Soc. 111:6350–6353; 1989. Antosiewicz, J.; Bertoli, E.; Damiani, E.; Greci, L.; Popinigis, J.; Przybylski, S.; Tanfani, F.; Wozniak, M. Indolinolic and quinolic aminoxyls as protectants against oxidative stress. Free Radic. Biol. Med. 15:203–208; 1993. Chateauneuf, J.; Lusztyk, J.; Ingold, K. V. Absolute rate constants for the reactions of some carbon-centered radicals with 2,2,6,6-tetrame-thylpiperidine-N-oxyl. J. Org. Chem. 53:1629– 1632; 1988. Cullis, P. R.; Hope, M. J. Effects of fusogenic agents on the membrane structure of erythrocyte. Nature 271:672–674; 1978.
ABBREVIATIONS
4-OH-TEMPO—4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl
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Allopurinol—1-hydro-pyrazolo[3,4-d]pyrimidine4-ol LSF—lipid soluble fluorophores DPO—2,5-diphenyloxazole POPOP—1,4-bis-2-(5-phenyloxazoyl)benzene BSA—bovine serum albumin Hepes—N-2-hydroxyethylpiperazine-N*-2ethanesulfonic acid EDTA—ethylenediaminetetraacetate
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RCI—respiratory control index PtdSer—phosphatidylserine PtdEtn—phosphatidylethanolamine HPLC—high-performance liquid chromatography TBARS—thibarbituric acid reactive substances TLC—thin layer chromatography U/S ratio—a ratio of unsaturated to saturated fatty acids Enzyme—Xanthine oxidase(EC 1.2.3.2)
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PII S0891-5849(96)00616-8
Original Contribution ROLE OF FREE RADICALS IN THE MECHANISM OF THE HYDRAZINEINDUCED FORMATION OF MEGAMITOCHONDRIA
TATSUO MATSUHASHI,* XINRAN LIU,* MARIUSZ K ARBOWSKI,* MICHAL WOZNIAK,† JEDRZEJ ANTOSIEWICZ,** and TAKASHI WAK ABAYASHI * *Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, 65-Tsurumai-cho, Showa-ku, Nagoya 466, Japan, †Department of Biochemistry, University of Gdansk Medical School, 80-210 Debinki 1, Gdansk, Poland, and **Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education, 80-336,Gdansk, Poland (Received 26 August 1996; Revised 5 December 1996; Accepted 6 December 1996)
Abstract—The effect of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl(4-OH-TEMPO), a scavenger for free radicals, and 4-hydroxypyrazolo[3,4-d(pyrimidine)allopurinol], a xanthine oxidase inhibitor, on the hydrazine-induced changes of mitochondrial ultrastructure and those in the antioxidant system of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 0.5% hydrazine for 7 d in the presence and absence of a combined treatment with 4-OH-TEMPO or allopurinol. Results obtained were as follows. 4-OHTEMPO completely prevented the hydrazine-induced formation of megamitochondria in the liver, while it was partly prevented by allopurinol. The following changes observed in hydrazine-treated animals were improved almost completely by 4-OH-TEMPO:decreases in the body weight and liver weight; lowered rates of ADP-stimulated respiration and coupling efficiency of hepatic mitochondria; remarkable elevation of the level of lipid peroxidation. Improving effects of allopurinol were incomplete. The present results suggest that free radicals may play a key role in the mechanism of the hydrazine-induced formation of megamitochondria and that a part of free radicals generated during the hydrazine intoxication is ascribed to the degradation of purine nucleotides via xanthine oxidase. A general mechanism of the megamitochondria formation induced in various pathologic conditions besides the case of hydrazine are discussed. q 1997 Elsevier Science Inc. Keywords—Hydrazine, 4-OH-TEMPO, Megamitochondria, Liver, Free radicals, Allopurinol, Oxidative stress, Lipid peroxidation
partly the hydrazine-induced formation of megamitochondria in the rat liver by a-tocopherol 6 and coenzyme Q10 ( CoQ10 ) .7 At the same time, we have found that the lipid peroxidation in the liver is remarkably enhanced by the hydrazine intoxication, which is partly suppressed by a-tocopherol or CoQ10 . In the present study we demonstrate that 4-hydroxy2,2,6,6-tetramethyl-piperidine-1-oxyl ( 4-OHTEMPO ) , an electron spin resonance label, completely suppresses the hydrazine-induced formation of megamitochondria in the rat liver, and at the same time lowers the hydrazine-induced remarkable increases in lipid peroxidation almost to the same level of the control animals. We have also found changes in the body weight, liver weight, and respiratory rates
INTRODUCTION
We have been studying the mechanism and pathophysiological meaning of the phenomenon of the megamitochondria formation using hydrazine and ethanol as experimental models.1 – 4 We have proposed that the fusion of adjacent mitochondria may play a key role in the mechanism of the phenomenon based on physicochemical, biochemical, and morphological changes of the mitochondrial membranes during the formation process of megamitochondria.2,5,6 Recently, we have succeeded in suppressing Address correspondence to: Prof. T. Wakabayashi, Department of Cell Biology and Molecular Pathology, Nagoya University School of Medicine, 65-Tsurumai-Cho, Showa-ku, Nagoya 466, Japan. 285
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of mitochondria of hydrazine-treated animals are remarkably improved by 4-OH-TEMPO. We propose based on the present study that free radicals may be a common triggering factor for the formation of megamitochondria induced by apparently different pathologic conditions. MATERIALS AND METHODS
Animals Male Wistar rats aged 4 weeks were divided into six groups. Each group consisted of six animals. Animals of the first three groups were placed on a powdered complete diet containing 0.5% hydrazine for 7 d. The latter three groups of animals were fed a normal complete diet. Animals of the second and fifth group were given 4-OH-TEMPO daily for the first 3 d of the experiment by IV via the tail veins (10 mg/100 g body weight). On the fourth day and thereafter, 4-OHTEMPO was given to the animals by SC injection (10 mg/100 g body weight). Animals of the third and sixth group were given allopurinol, a xanthine oxidase (EC 1.1.3.22) inhibitor, daily by SC injection (5 mg/100 g body weight). 4-OH-TEMPO was dissolved in physiological saline , and allopurinol was suspended in 0.5% carboxymethyl cellulose. Preparation of mitochondria and microsomes from the liver Immediately after the animal was sacrificed by decapitation, the liver was removed from the animal. Small pieces of the tissue was used for light and electron microscopy. The rest of the liver was rinsed in 2 mM N-2-hydroxyethylpiperazine-N *-2-ethansulfonic acid (Hepes), pH7.4, with 70 mM sucrose, 220 mM mannitol and 0.1 mM EDTA. Bovine serum albumin (BSA) was added to the isolation medium at a final concentration of 0.05%. Mitochondria were isolated according to the method previously described.1 In the present study, the routine isolation procedure for liver mitochondria, as previously described, was adopted based on the following previous observations: a heavy (rich in megamitochondria) and light mitochondrial fractions obtained from the liver of animals treated with alkyl alcohols including ethanol showed essentially the same phosphorylating ability 5 ; megamitochondria and normal sized mitochondria in cuprizone-treated mouse liver were often connected to each other by narrow stalks in the hepatocyte when they were revealed on one plane of section for electron microscopy. Furthermore, mitochondria apparently separated from each other on one plane of section were connected to each other far more fre-
quently than expected when they were examined by serial sections 8,9 ; similar results were obtained with hydrazine-treated mouse liver mitochondria, megamitochondria and normal sized mitochondria were connected each other when their three-dimensional structures were reconstructed by serial sectioning technique (unpublished observations). These results may allow us to assume that mitochondria connected to each other in the cell are separated each other during the homogenization and the differential centrifugation procedure. Furthermore, some of megamitochondria may be broken into smaller ones during the isolation process. We must bear in mind that we have not succeeded yet in the isolation of megamitochondria maintaining the same three dimensional structures as they are in vivo. The postmitochondrial fraction was centrifuged for 10 min at 12,000 g to sediment lysosomes, microbodies and swollen, light mitochondria.9 The resultant supernatant was centrifuged for 1 h at 105,000 g. The pellet thus obtained was suspended in 1.15% KCl and was used as the microsomal fraction, and the supernatant was used as the soluble fraction. Measurement of oxygen uptake of mitochondria Respiratory rates of mitochondria were measured using Clark-type electrode as previously described.1 Protein assay Protein was determined by the method of Lowry et al.10 using bovine serum albumin as standard. Determination of purine derivatives Hypoxanthine, xanthine, and uric acid in the soluble fraction were determined using high-performance liquid chromatography (HPLC) according to the method of Kojima et al.11 The chromatographic apparatus consisted of Waters 600E controller, Waters 484 detector and Waters 741 module integrator system (Milford, MA, USA). Aliquots of the soluble fraction were injected on 1018 COSMOSIL column composed of guard column (4.6 1 50 mm, Nakali Tesque, Kyoto, Japan), and the main column (4.6 1 250 mm), (Nakali Tesque). The mobile phase was potassium phosphate buffer (40 mM, pH 2.2) containing 20 ml of methanol per liter. The flow rate was set at 1.0 ml/min and the wavelength of detection was set at 254 nm. Determination of D6- and D9-desaturase activities Activities of D6- and D9-desaturase in microsomes were determined by the method of Christiansen et al.12
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The reaction medium contained in the total volume of 1.0 ml, 0.15 M KCl, 5 mM ATP, 5 mM MgCl2 , 0.25 mM CoA, 1.0 mM NADH, 1.5 mM glutathione, 45 mM NaF, 0.5 mM nicotinamide, 1 mg BSA, 0.1 mM potassium phosphate buffer, pH 7.4, microsomes (1 mg), and 75 nmoles [1- 14C]stearic acid or [1- 14C]linoleic acid as 1 mg BSA emulsion. The incubation was carried out at 377C for 20 min in a metabolic shaker. The reaction was terminated by the addition of 1 ml of 2.7 M KOH in methanol followed by the saponification for 45 min at 657C. The reaction medium was then acidified by the addition of 5 M HCl. Fatty acids in the reaction medium were extracted twice with hexane and methylated by the method of Hoshino et al.13 Fatty acid methyl esters were separated on thin layer chromatography (TLC) plates coated with silica gel G (Wako Chemical Co., Osaka, Japan) impregnated with 10% (w/w) AgNO3 . Plates were developed in hexane/diethyl ether (9:1, v/v) for the separation of monounsaturated fatty acids from saturated fatty acids and of triunsaturated fatty acids from diunsaturated fatty acids. The spots were made visible under ultraviolet light by spraying with 2,7-dichlorofluorescein (0.2%, w/v in ethanol). They were scraped off directly into scintillation vials. A liquid scintillation mixture (5 ml) containing 25 mg 2,5-diphenyloxazole (PPO), 1.5 mg 1,4bis-2-(5-phenyloxazolyl)benzene (POPOP), 0.33 ml ethyl acetate and 4.67 ml toluene was added to each vial.14 The radioactivity was counted using ALOKA LSC-1500 liquid scintillation counter (Aloka, Tokyo, Japan). Determination of oxidative stress Oxidative stress induced by the hydrazine intoxication was monitored by measuring the amounts of thiobarbituric acid reactive substances (TBARS) and lipidsoluble fluorophores (LSF) in microsomes and mitochondria as described before.5 TBARS were assayed with a thiobarbituric acid method.15 The concentration of TBARS in the sample was calculated using extinction coefficient of 1.56 1 10 5 cm-1 . LSF was assayed with a fluorometry method.16 Excitation and emission wave lengths were 366 and 460 nm, respectively. The concentration of LSF in the sample was expressed as a relative fluorescence where the fluorescence of quinine dissolved in sulfuric acid at a concentration of 0.1 mg/ml was taken as 100. Light and electron microscopy A part of the liver of each animal was cut into small pieces and processed for electron microscopy according to the method previously described.1 For light mi-
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croscopic examination, thick sections (0.5–2.0 mm) were obtained from Epon-embedded samples and stained with basic fuchsin and methylene blue.9 Distribution of megamitochondria within a hepatic lobule was examined using thick sections in the light microscope. Chemicals Hydrazine dihydrochloride was obtained from Merck (Darmstadt, Germany). 4-OH-TEMPO and allopurinol were obtained from Wakao Pure Chemical Co. (Osaka, Japan). [1- 14C]stearic acid (2.1 Mbq/ mmol; 97.1 radiochemical purity) and [1- 14C]linoleic acid (2.1 Mbq/ mmol; 98.8 radiochemical purity) were purchased from Amersham International (Amersham, England). All other chemicals were of analytical grade. Statistical analysis All the data were expressed as means { standard error (means { SE). Statistical comparisons of data were made using paired t-test with p õ .05 being considered significant. RESULTS
Effects of 4-OH-TEMPO and allopurinol on the body weight and wet weight of the liver of hydrazinetreated rats Animals placed on a hydrazine diet for 7 d became gradually weak losing body weight remarkably (Table 1). On the other hand, animals treated with hydrazine plus 4-OH-TEMPO were as active as the control animals, and the average body weight of the former animals was almost the same as that of the latter animals. Table 1. Effects of 4-OH-TEMPO and Allopurinol on the Body Mass and Wet Mass of the Liver of Hydrazine-Treated Ratsa
Animals Control Control / 4-OH-TEMPO Control / Allopurinol Hydrazine Hydrazine / 4-OH-TEMPO Hydrazine / Allopurinol
Body Mass (g) 149.0 { 152.9 { 140.0 { 74.7 { 136.5 { 86.7 {
0.8 4.1 5.0 2.0b 4.1 2.8d
Wet Mass of the Liver (g) 6.86 { 6.90 { 6.93 { 2.93 { 5.88 { 4.00 {
0.18 0.17 0.21 0.19b 0.21e 0.29c
a Animals were fed with 0.5% hydrazine-diet for 7 d in the presence or absence of a combined treatment with 4-OH-TEMPO or allopurinol. Details in experimental conditions are described in the Materials and Methods section. Data are the averages and standard error (mean { SE) of five different animals. Values are significantly different from those of the corresponding control at b (p õ .001), c (.001 õ p õ .01), d (.01 õ p õ .02), e (.02 õ p õ .05).
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The average body weight of animals after 7 d of treatment with hydrazine plus allopurinol, however, was much smaller than that of the control animals. The averaged wet weight of the liver of hydrazine-treated animals was distinctly smaller than that of the control animals while it was much improved by the treatment with 4-OH-TEMPO although, statistically, it remained smaller than the control (Table 1). The average wet weight of the liver of animals treated with hydrazine plus allopurinol, on the other hand, remained much smaller than that of the control animals. Effects of 4-OH-TEMPO and allopurinol on hydrazine-induced ultrastructural changes of mitochondria in the liver 4-OH-TEMPO completely abolished the hydrazineinduced formation of megamitochodnria in the liver (Fig. 1A). Namely, mitochondria in the hepatocyte of the liver of animals treated with hydrazine for 7 d became enlarged remarkably with decreases in number per hepatocyte (Fig. 1A). Whereas ultrastructural appearances of those of animals treated with hydrazine plus 4-OH-TEMPO (Fig. 1B) were almost the same as those of the control (Fig. 1D). Mitochondria of the hepatocyte of animals treated with hydrazine plus allopurinol (Fig. 1C) were much smaller than those of animals treated with hydrazine alone and yet they remained larger than those of the control. Effects of 4-OH-TEMPO and allopurinol on the respiratory rate of hydrazine-treated rat liver mitochondria We have demonstrated in the present study that both 4-OH-TEMPO and allopurinol exert remarkable effects on the structural changes of mitochondria of the liver of hydrazine-treated animals. Thus, we have examined the effect of 4-OH-TEMPO and allopurinol on the rate of respiration and phosphorylating ability of mitochondria of hydrazine-treated animals (Table 2). The rate of ADP-stimulated respiration (state 3 respiration) of mitochondria was decreased in hydrazine-treated animals compared to those of the control. The ratio of state 3 to state 4 respiration (respiratory control index [RCI]) became lowered compared to that of the control. Phosphorylating ability of these mitochondria was also decreased compared with that of the control. On the other hand, 4-OH-TEMPO remarkably improved such changes observed in hydrazine-treated animals. Such improving effects of 4-OH-TEMPO on mitochondria of hydrazine-treated animals was less remarkable in the case of allopurinol.
Effects of 4-OH-TEMPO and allopurinol on lipid peroxidation in the liver of hydrazine-treated animals Since the hydrazine-induced formation of megamitochondria was completely suppressed by 4-OHTEMPO, we have examined the effects of 4-OHTEMPO and allopurinol on lipid peroxidation in the liver of hydrazine-treated animals (Fig. 2). It is evident from the figure that the amounts of TBARS in microsomes and mitochondria of the liver of hydrazinetreated animals were remarkably increased compared to those of the control. 4-OH-TEMPO completely suppressed lipid peroxidation enhanced by hydrazine intoxication. On the other hand, inhibitory effects of allopurinol on lipid peroxidation were incomplete; the amounts of TBARS in microsomes and mitochondria of the liver of animals treated with hydrazine plus allopurinol were definitely smaller than those of animals treated with hydrazine alone, and yet larger than those of the control. Similar results were obtained with LSF. However, inhibitory effects of 4-OH-TEMPO using LSF on lipid peroxidation in hydrazine-treated animals were less remarkable compared to those obtained with TBARS. Effects of allopurinol on the rate of the degradation of purine derivatives We have shown in the present study that allopurinol partly prevents the hydrazine-induced formation of megamitochondria and at the same time lowers slightly the lipid peroxidation. Thus, we examined effects of allopurinol on the rate of purine nucleotide degradation in hydrazine-treated animals (Fig. 3). The amounts of hypoxanthine, xanthine, and uric acid in hydrazinetreated animals were remarkably elevated compared to those of the control. The level of hypoxanthine in animals treated with hydrazine plus allopurinol was elevated remarkably whereas that of xanthine became decreased remarkably compared to that of the control. These data indicate that allopurinol actually exerted its inhibitory action on the degradation of hypoxanthine into xanthine in vivo. Changes in some fatty acid desaturase activities in hydrazine-treated animals Previously, we have shown that remarkable changes take place in phospholipid classes and fatty acid compositions of phospholipid domains of the mitochondrial and microsomal membranes during the formation process of hydrazine-induced megamitochondria 17 increases in the relative amount of phosphatidylethanolamine (PtdEtn) and acidic phospholipids such as
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Fig. 1. Effects of 4-OH-TEMPO and allopurinol on hydrazine-induced ultrastructural changes in hepatic mitochondria. (A) hydrazine, (B) hydrazine plus 4-OH-TEMPO, (C) hydrazine plus allopurinol, and (D) control. Magnification of electron micrographs: 19000.
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Succinate State 3 Animals
State 4
RCI
ADP/O
State 3
(natoms O/mg/min)
Control Hydrazine Hydrazine / 4-OHTEMPO Hydrazine / Allopurinol
State 4
29.3 { 4.9 6.73 { 0.17 27.8 { 9.8 6.39 { 0.22
ADP/O
(natoms O/mg/min)
203.5 { 8.7 29.4 { 1.7 6.94 { 0.13 1.97 { 0.02 119.3 { 1.6 16.5 { 0.3 150.9 { 7.6e 35.9 { 4.2 4.38 { 0.53d 1.75 { 0.08e 90.3 { 4.1d 15.3 { 0.8 197.0 { 2.2 177.4 { 3.9
RCI
1.92 { 0.03 116.7 { 5.8 1.67 { 0.03c 102.9 { 5.4
7.24 { 0.10 5.90 { 0.20c
2.94 { 0.02 2.56 { 0.04c
18.4 { 0.3d 6.37 { 0.40 2.93 { 0.02 17.1 { 0.8 6.03 { 0.04b 2.69 { 0.01c
a Mitochondria were isolated from the liver of the control rats and animals fed with a diet containing 0.5% hydrazine for 7 d in the presence and absence of 4-OH-TEMPO and allopurinol treatment. Details of the treatment of animals are described in the Materials and Methods section. Data are the averages standard error (mean { SE) of three different animals. Values are significantly different from the control at b ( p õ .001), c (.001 õ p õ .01), d (.01 õ p õ .02), e (.02 õ p õ .05).
phosphatidylserine (PtdSer) in mitochondria; increases in the ratio of unsaturated to saturated fatty acids in phopholipid domain of mitochondrial membranes; decreases in that in microsomal membranes. Thus, we
have measured in the present study some fatty acid desaturase activities in the liver of animals treated with hydrazine (Table 3). It is interesting to note that activity of D9-desaturase was remarkably increased in ani-
Fig. 2. Effects of 4-OH-TEMPO and allopurinol on hydrazine-induced changes in the level of lipid peroxidation in hepatic mind megamitochondria tochondria and microsomes. Data are the averages and standard error (mean { SE) of six different experiments. Data on experimental group are statistically different from those of the control at: a (p õ .001), b (.001 õ p õ .01), c (.01 õ p õ .02), and d (.02 õ p õ .05).
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mation in the hydrazine intoxication is xanthine oxidase, which generates superoxide during the catabolism of hypoxanthine into uric acid.20–22 The present study has demonstrated that the hydrazine intoxication accelerates the degradation of purine nucleotide derivatives indicating an increased production of superoxides.
Role of free radicals in the mechanism of the hydrazine-induced formation of megamitochondria
Fig. 3. Effects of allopurinol on hydrazine-induced changes in the level of hypoxanthine, xanthine and uric acid in the liver. Levels of purine derivatives were measured using the postmicrosomal fraction. Data are the averages and standard error (mean { SE) of six different experiments. Data on experimental group are statistically different from those of the control at: a (p õ .001), b (.001 õ p õ .01), c (.01 õ p õ .02).
mals treated with hydrazine for 3 d, while those of both D6- and D9-desaturase became decreased in animals tretaed with hydrazine for 7 d. The discrepancy seen between the two desaturase activities in animals treated with hydrazine for 3 d will be discussed later.
DISCUSSION
In the present study, we have successfully suppressed the hydrazine-induced formation of megamitochondria in the rat liver by 4-OH-TEMPO which at the same time lowered lipid peroxidation induced by the hydrazine intoxication. We have also shown that allopurinol, a xanthine oxidase inhibitor, partly prevents the hydrazine-induced formation of megamitochondria.
The present study has clearly demonstrated that 4OH-TEMPO is more effective than CoQ10 or a-tocopherol in suppressing both hydrazine-induced lipid peroxidation and megamitochondria formation. The possible explanation for this may be as follows. CoQ10 and a-tocopherol are lipid-soluble and are buried within the mitochondrial membranes while 4-OHTEMPO is water-soluble and may be scattered within the cytoplasm of the cell. And a-tocopherol is able to scavenge effectively only peroxy radicals locating near the surface of the biological membranes, while carbon centered radicals locating deep inside the membrane are free from a-tocopherol.23 On the other hand, nitroxides, such as 4-OH-TEMPO, were reported to be effective in scavenging both peroxy radicals 24 and carbon centered radicals.25 If we accept that free radicals are responsible for the mechanism of the formation of megamitochondria, as shown in the present study, then the superiority of 4-OH-TEMPO to a-tocopherol or CoQ10 as a scavenger for free radicals could be related to the fact that the suppressing effect of the megamitochondria formation by 4-OH-TEMPO was complete while that by a-tocopherol or CoQ10 was incomplete. Finally, we should like to refer briefly to the changes observed in some fatty acid desaturase activities in hyTable 3. Changes in D6-Desaturase and D9-Desaturase Activities of Hydrazine-Fed Rat Liver Microsomesa
Animals
Sources of free radicals in the hydrazine intoxication Previously, we have shown that the hydrazine intoxication causes remarkable increases in the level of lipid peroxidation.6 This was confirmed in the present study. It is generally accepted that the major route of metabolism of hydrazine in vivo is its oxidation yielding nitrogen-centered free radicals.16 The mixed-function oxidase system has been reported to metabolize hydrazine in vitro catalyzing the formation of hydrazine radicals.18,19 Another source of free radical for-
Control Hydrazine-3 d Hydrazine-7 d
D6Desaturase (nmol/min/ mg)
D9Desaturase (nmol/min/ mg)
0.21 { 0.01 0.20 { 0.02 0.12 { 0.03b
1.71 { 0.12 2.79 { 0.02b 0.92 { 0.05b
a Microsome were isolated from the liver of animals fed with a diet containing 0.5% hydrazine for 3 and 7 d. Activity of D6-desaturase using linoleic acid as substrate is expressed as nmol linolenic acid synthesized per min per mg microsomal protein. Activity of D9desaturase using stearic acid as substrate is expressed as nmol oleic acid synthesized per min per mg microsomal protein. Data are the averages and standard error (mean { SE) of six different experiments. Data on experimental group are statistically different from those of the control at b ( p õ .001).
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drazine-treataed animals. Previously, we have proposed that the fusion of adjacent mitochondria may play a key role in the mechanism of the formation of megamitochondria based on physicochemical, biochemical, and morphological changes of the mitochondrial membranes during the hydrazine- and ethanol-induced formation of megamitochondria.2 – 4 Namely, we have shown distinct increases in the ratio of unsaturated fatty acids to saturated fatty acids (U/S ratio) in phospholipids extracted from mitochondria of rats treated with hydrazine for 3 d.17 These data may strongly suggest a possible role of the mebrane fusion in the mechanism of the formation of megamitochondria since the elevation of the degree of unsaturation in fatty acyl chains in phospholipids enhances the transition of phospholipids from bilayer to hexagonal II (HII ) phase, which is essential for the process of the membrane fusion.26 However, we have no data at the present time to explain how D9-desaturase activity was enhanced in animals treated with hydrazine for 3 d. Likewise, we need further investigations to explain changes observed in D6- and D9-desaturase activities in animals treated with hydrazine for 7 d. We are now studying the effect of 4-OH-TEMPO and other scavengers for free radicals on various experimental conditions for megamitochondria formations besides hydrazine, and the results will be reported soon. Acknowledgements — This work was supported in part by Grantsin-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The authors thank Mrs. N.Yoshimi for the preparation of the manuscript.
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ABBREVIATIONS
4-OH-TEMPO—4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl
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Hydrazine and megamitochondria
Allopurinol—1-hydro-pyrazolo[3,4-d]pyrimidine4-ol LSF—lipid soluble fluorophores DPO—2,5-diphenyloxazole POPOP—1,4-bis-2-(5-phenyloxazoyl)benzene BSA—bovine serum albumin Hepes—N-2-hydroxyethylpiperazine-N*-2ethanesulfonic acid EDTA—ethylenediaminetetraacetate
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RCI—respiratory control index PtdSer—phosphatidylserine PtdEtn—phosphatidylethanolamine HPLC—high-performance liquid chromatography TBARS—thibarbituric acid reactive substances TLC—thin layer chromatography U/S ratio—a ratio of unsaturated to saturated fatty acids Enzyme—Xanthine oxidase(EC 1.2.3.2)
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