Mitochrondrial damage by active oxygen species in vitro

Free Radical Biology & Medicine, Vol. 10, pp. 277-285, 1991 Printed in the USA. All rights reserved.

0891-5849/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc

Original Contribution MITOCHONDRIAL DAMAGE BY ACTIVE OXYGEN SPECIES IN VITRO

SUDHIR MEHROTRA, POONAM KAKKAR,* a n d P . N . VISWANATHAN Ecotoxicology Section, Industrial Toxicology Research Centre, M.G. Marg, P.O. Box No. 80, Lucknow- 226 001, India (Received 19 June 1990; Revised 12 October 1990; Accepted 27 November 1990)

A b s t r a c t - - U n d e r in vitro conditions involving formation of active oxygen species, rat liver mitochondria were found to undergo swelling, peroxidative decomposition of lipids, and distinct disorganization of ultrastructure. Supplementation with free radical scavengers such as superoxide dismutase (SOD), methionine, histidine, and tryptophan accorded considerable protection to the organelle. A possible correlation between oxygen radicals, membrane integrity, and calcium functions is indicated. Keywords--Free radicals, Mitochondria, Lipid peroxidation, Membrane damage, Calcium dynamics

INTRODUCTION

MATERIALS AND METHODS

Animals

Since the major oxidative metabolic processes of the cell take place in the mitochondria, the presence of several electron carriers and polyunsaturated fatty acid rich membranes make this organelle highly susceptible to the attack of active oxygen species produced during the reduction of molecular oxygen into water taking place in four steps of univalent electron reduction. Dearrangement of intracellular ion concentrations occur as an early response to injurious stimuli in many cells and tissues.l'2 Mitochondria has the highest Ca 2÷ accumulating capacity among the various organeUes. 3 Any toxic stress-mediated increase in the cytosolic Ca 2+ concentration modulates calcium dynamics in the mitochondria also. The rapid accumulation of Ca 2÷ through potential dependent uniport mechanism of Ca 2÷ uptake into mitochondria, removes excess Ca 2+ from the cytosol bringing it down to levels from where it can be taken care of by the endoplasmic reticulum. 4 Thus, the mitochondrial Ca 2+ dynamics as well as generation of free radicals are important events during the course of manifestation of toxicity by a number of toxins resulting in cell injury and cell death. Therefore, the role of active oxygen species formation in the morphological and enzymatic integrity of mitochondria were studied in vitro.

Male albino Wistar strain rats weighing 100-150 gm maintained under standard conditions in the Industrial Toxicology Research Centre animal colony, were used for the study. Lipton pellet diet and water ad libitum were given.

Chemicals Hypoxanthine was obtained from BDH, England, xanthine oxidase (Grade III from butter milk) and superoxide dismutase (from bovine erythrocytes) were procured from Sigma, St. Louis, MO, USA, and calcium chloride was obtained from E. Merck Darmstadt, West Germany. All other chemicals were of analytical grade and obtained from local sources. Deionized HPLC grade water was used throughout.

Isolation of mitochondria Rats were killed by cervical dislocation and livers were taken out immediately, rinsed with 0.25 M sucrose, dried on filter paper, cleared of all adhering tissue debris, weighed, minced, and homogenized in a Potter-Elvehjem glass homogenizer. A 10% (w/v) homogenate was prepared in 0.25 M sucrose solution (or in 0.02 M Tris-0.15 M KCI, pH 7.4 for lipid peroxidation studies only). Mitochondria were isolated according to the method of Mustafa et al. 5 After isolation, the

Address correspondence to Poonam Kakkar, Scientist, Ecotoxicology Section, Industrial Toxicology Research Centre, Post Box 80, M.G.Marg, Lucknow--226 001 India. 277

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mitochondrial pellet was suspended in 0.02 M Tris-0.15 M KC1, pH 7.4. Dilution was also carried out in the same medium. All the operations were carried out under cold conditions (0 to 4°C). Swelling and contraction of isolated rat liver mitochondria were carried out according to the spectrophotometric method of Lehninger et al. 6 using Milton Roy Spectronic 1001 spectrophotometer. Calcium chloride (2 mM) was used as swelling agent whereas ethylene diamine tetraacetic acid disodium salt (EDTA.Na2), magnesium chloride, and adenosine triphosphate disodium salt (ATP. Na ) were employed as contracting agents at 5 mM concentration2 each. Hypoxanthine (5 x 10-SM) and xanthine oxidase (0.025 units) systems was used for the generation of superoxide anion (O~). To study the quenching effect, superoxide dismutase (SOD) (70 units) was used as scavenger of superoxide radical; tryptophan as quencher of active oxygen species; 7 histidine as possible quencher for singlet oxygen ('O2) 8and methionine for hydroxyl radical ('OH).9 The swelling was recorded by the instrument as decrease in the optical density at 1-min intervals for a period of 10 rain. Into the same cuvette, the contracting agents were pipetted and contraction was recorded as increase in the optical density at l-rain intervals for a further period of 10 min.

Lipid peroxidation

Malondialdehyde (MDA) liberated during the course of lipid peroxidation was estimated by the thiobarbituric acid (TBA) method 1° at different time intervals in mitochondria subjected to incubation with specified concentrations of CaC12 alone; CaC12 + hypoxanthine + xanthine oxidase; CaC12 + hypoxanthine + xanthine oxidase + SOD and CaC12 + hypoxanthine + xanthine oxidase + tryptophan, methionine, and histidine. The molecular extinction coefficients of 1.56 × 105M was used for calculating the amount of malondialdehyde.

Transmission electron microscopy

After isolation by centrifugation, the mitochondria were washed with sodium cacodylate buffer and suspended in 2.5% glutaraldehyde (fixative agent) for an hour or two and washed twice with sodium cacodylate buffer. The material was fixed in osmium tetraoxide, washed with the same buffer, and dehydrated with acetone. For embedding araldite 502 (resin), DDSA and DMP (all, Ladd, USA) were used and sections were cut using LKB ultratome. After treating with KOH solution and washing with distilled water, the sections were stained with uranyl acetate and examined on Phillips

400 transmission electron microscope. The entire procedure was carried out according to the methodology of Hayat. 11 Statistical analysis

The statistical evaluation of the effects of exogenous calcium and active oxygen species on structural and functional integrity of freshly isolated rat liver mitochondria was accomplished with student's t test. 12 Significant level of p < 0.05 was used for all comparisons. RESULTS

Swelling pattern of mitochondria and formation of TBA reactive material under active oxygen and calcium stress

In freshly isolated rat liver mitochondria, 2 mM of CaCI 2 caused swelling, as shown in Fig. 1, which in the presence of ATP, Mg, 2÷ and EDTA was abolished almost completely bringing the mitochondria almost back to normal. When, in the same system, O~ was generated by hypoxanthine and xanthine oxidase, mitochondrial swelling of higher magnitude was induced. On contraction mitochondria did not go back to the normal state indicating some morphological changes like membrane damage, and so forth. Superoxide dismutase accorded some protection towards the swelling induced by O~ but was unable to bring back mitochondria to normal levels. Free radical quenchers like tryptophan, histidine, and methionine accorded protection against damage caused by O ~ in a concentration-dependent manner indicating interconversion of free radicals during the treatment of mitochondria. Traces of endogenous iron present in the organelle catalyze Haber-Weiss and Fenton reactions giving rise to hydroxyl radicals and other active states of molecular oxygen. As is apparent from Fig. 2, methionine accords more protection in lower concentration, that is, 10-3M against swelling induced by superoxide as compared to higher concentrations like 10-2M and 5 x 10-2M, but still the swelling caused was much less as compared to that in the presence of O~. Also, the swelling caused by CaC12 alone was less compared to the system where O~ generating system and methionine both were present, indicating the role of O~ in damaging the mitochondrial membrane, thus preventing the quencher from bringing mitochondria back to normal level. Similar results were obtained with tryptophan and histidine (Figs. 3 and 4, respectively). Each experiment was repeated four times and the mean value +_ S.E. is given in Tables 1 and 2. Transmission electron micrographs of isolated rat liver mitochondria subjected to incubation with CaClz

Oxygen radicals and mitochondria

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Fig. 1, Swelling and contraction pattern of fresh rat liver mitochondria. Arrow indicates the time at which EDTA, MG 2+, and ATP were added to the system. ( + - - - + ) Ca 2. induced swelling and EDTA, Mg 2÷, and ATP-induced contraction. Effect of O~ formation (FI---[~) on Ca :÷ induced swelling. Effect of SOD on Ca ~'+ and 0 2 induced swelling (x---x). Assay conditions as described in text.

for 10 min show swelling of mitochondria. Damage to inner and outer mitochondrial membranes leading to disorganization of cristae, separation of individual cristae folds, and loss of matrix material, most of it becoming part of the enclosed membrane structure, were also observed (Fig. 6). Hollow space inside the mitochon-

dria was found as compared to untreated freshly isolated mitochondria (Fig. 5). Fusion of mitochondria-forming aggregates as well as electron opaque calcium precipitates adhering mostly to the mitochondrial membrane were evident (Fig. 6). Effects of EDTA, the calcium chelating agent, Mg 2+, the competitive inhibitor of

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uptake in mitochondria and ATP, the sequestering agent on swollen mitochondria are clear in Fig. 7. All three agents through different mechanisms remove the excess calcium from the mitochondria, thereby causing its contraction. However, irreversible damage to the structural integrity of the mitochondria has already taken place under calcium stress. Fig. 8 shows

that O 2 potentiates the membrane damage induced by Ca 2+ as evident from the larger number of nicks present in the membrane as compared to the mitochondria treated with calcium alone (Fig. 6). Maximum disintegration and fragmentation of mitochondrial membrane has taken place in the system indicating a synergistic effect of calcium and active oxygen species in potentiation of mem-

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Fig. 4. Ca2+ induced swelling (+---+) and EDTA, Mg 2+, and ATP-induced contraction. Effect of 0 2 formation (C]---D) on Ca 2+ induced swelling. 1 mM (x---x) histidine. 2 mM (XZ---V--) histidine. 10 mM (~7-.--~z-) histidine.

281

Oxygen radicals and mitochondria Table 1. Statistical Analysis of Figures 1 and 2 Fig. 1

10m in

2 0 m in

Mitochondria + CaC12

0.624 ± 0.019

0.960 _+ 0.024

Mitochondria + CaCI 2 + H.X. + X.O.

0.515 ± 0.014 (p < 0.001)

0.869 -+ 0.044 (p < 0.01)

Mitochondria + CaC12 + H.X. + X.O. + SOD

0.590 --- 0.022 (p < 0.02)

0.920 ± 0.044 (p < 0.1)

Mitochondria + CaC12 + H.X. + X.O. + Tryptophan (M/1000)

0.570 - 0.015 (p < 0.1)

0.913 ± 0.044 (p < 0.1)

Mitochondria + CaCI2 + H.X. + X.O. + Tryptophan (M/500)

0.546 --- 0.031 (p < 0.1)

0.912 -_+ 0.060 (p < 0.1)

Mitochondria + CaCI2 + H.X. + X.O. + Tryptophan (M/100)

0.530 • 0.013 (p < 0.1)

0.931 --- 0.039 (p < 0.1)

Fig. 2

Values are arithmetic mean -+ S.E. of four determinations in each case.

brane damage caused by each alone. Incorporation of SOD into the system shows that Ca 2+ and O I induced damage was counteracted preserving the structural integrity of mitochondrial membrane to a large extent (Fig. 9). Table 3 shows TBA reactive material formation in mitochondria subjected to incubation with CaCI> CaC12 + O I and CaCI 2 + 0 2 + free radical quenchers. After 2 h of incubation the malondialdehyde formation was

more in the case of mitochondria incubated with CaC12 indicating potentiation of membrane damage by excess Ca 2÷. The results are comparable with TEM and swelling and contraction pattern in presence of an O 2 generating system. TBA reactive substances are greater at 30 min but after 2 h are less than those in the presence of CaC12. SOD lowers the level of malondialdehyde formation, indicating its defensive role. Also, in the presence of CaC12 and 0 1 , active oxygen radical scavengers

Table 2. Statistical Analysis of Figures 3 and 4 Fig. 3

10 min

20 min

Mitochondria + CaCI/ + H.X. + X.O. + Histidine (M/1000)

0.580 -+ 0.034 (p < 0.1)

0.912 + 0.041 (p < 0.1)

Mitochondria + CaC12 + H.X. + X.O. + Histidine (M/500)

0.563 + 0.031 (p < 0.1)

0.818 _+ 0.036 (p < 0.1)

Mitochondria + CaCI 2 + H.X. + X.O. + Histidine (M/100)

0.555 ___ 0.025 (p < 0.1)

0.916 ___ 0.054 (p < 0.1)

Mitochondria + CaC12 + H.X. + X.O. + Methionine (M/1000)

0.572 _-+ 0.030 (p < 0.1)

0.929 _± 0.050 (p < 0.1)

Mitochondria + CaCI z + H.X. + X.O. + Methionine (M/500)

0.559 --+ 0.034 (p < 0.1)

0.921 ± 0.033 (p < 0.1)

Mitochondria + CaC12 + H.X. + X.O. + Methionine (M/100)

0.550 • 0.029 (p < 0.1)

0.936 24- 0.044 (p < 0.1)

Fig. 4

Values are arithmetic mean -+ S.E. of four determinations in each case.

282

S. MErtROrRAet al.

Fig. 5. T.E.M. of freshly isolated mitochondria (control) × 18000. Staining procedure as described in text.

accord protection to varying degrees. Since the interconversion of active oxygen species is very rapid, especially in the presence of iron and ascorbic acid, identification of the specific causative species is difficult. 13 Methionine gives near complete protection in 2 h as is evident from comparison of levels of malondialdehyde formed in mitochondria alone and in the system with methionine. Since hydroxyl radical species causes maximum damage to the cellular macromolecules, its scavenging by methionine accords maximum protection against lipid peroxidation. DISCUSSION

It is well documented that active oxygen species cause peroxidative decomposition of polyunsaturated fatty acid

Fig. 6. T.E.M. of mitochondria + 2 mM CaC12 x 18000.

Fig. 7. T.E.M. of mitochondria in presence of CaC12, EDTA, Mg 2. , and ATP × 18000.

component of lipid bilayer of biological membranes thereby inducing membrane damage as well as permeability changes. 14,15Various enzymatic and nonenzymatic antioxidants provide protection to varying degree depending on the mechanism of damage involved. 16 During the conversion of potentially reversible cell injury status to irreversible cell injury leading to cell death, the mitochondria often pass through a transient condensation phase, lose their matrical granules, and eventually undergo swelling. This is accompanied by intramatrical protein denaturation along with changes in mitochondrial phospholipids including cardiolipin leading to ion deregulation. 1 Intramitochondrial accumulation of an amorphous and then crystalline calcium phosphate and calcium hydroxyapatite occurs only in injuries such as those which mediate activated comple-

Fig. 8. T.E.M. of mitochondria in presence of CaC12, EDTA, Mg 2+ , ATP, and O 2"generating system (hypoxanthine + xanthine oxidase) x 18000.

Oxygen radicals and mitochondria

283

enzymatic free radical scavengers seem to be playing a defensive role against peroxidative membrane damage (Table 3). An age-dependent increase in superoxide anion and hydroxyl radical production by respiring mitochondria has been demonstrated in vivo, with ubiquinone and cytochrome b 566 being the probable sites of electron leakage out of sequence directly to molecular oxygen. 17

Fig. 9. T.E.M. of mitochondriam presence of CaC12, EDTA, Mg2+, ATP, O I generatingsystem (hypoxanthine + xanthine oxidase) and SOD x ~18000.Concentrationsof the reagents as specifiedin the text.

ment, direct membrane damage, inhibition of Na + / C a 2+ exchange, or other types of injuries that do not specifically interfere with mitochondrial Ca 2+ transport. 2 As evident from the transmission electron micrographs of mitochondria subjected to exogenously added calcium stress as well as oxidative stress, the damage caused to the inner and outer membranes is enhanced in the presence of O~, which is counteracted to a large extent by SOD added to the system. Also, other non-

A variety of chemically different prooxidants cause Ca 2+ release from the mitochondria via a route which is physiologically relevant. When the released Ca 2÷ is excessively cycled by mitochondria, they are damaged. This leads to uncoupling, a decreased ATP supply 18 and a decreased ability of mitochondria to retain Ca z+. Along with CaC12 induced swelling of mitochondria the peroxidative decomposition of membrane lipids also is enhanced as evident from the data. Recently, it has been reported that the apparent stimulation of lipid peroxidation by low Ca 2+ concentrations is not a direct effect, but reflects independently initiated processes of lipid peroxidation and Ca 2+ translocation, which interact subsequently in a synergistic manner. 19 At the same time, active oxygen radicals impart their toxic effect on mitochondrial structural and functional integrity by causing enhanced swelling of mitochondria by influencing the permeability barrier. Recently, Ungemach et al. have shown that accumulation of lysophosphatides formed as a result of activation of endogenous phospholipase A 2 - - a process dependent on calcium concentration, in-

Table 3. TBA Reactive ChromogenFormationIn Vitro by Rat Liver Mitochondriain the Presence of Calciumand Free Radical Forming/ScavengingSystems (Data Are Expressed in Terms of n Moles of MalondialdehydeLiberated/mgMitochondrialProtein) Incubation mixture

0 min

30 min

2h

Mitochondria + CaC12 (b)

0.047 _+ 0.004

0.452 -+ 0.032 (p < 0.01)*

1.364 _ 0.112 (p < 0.001)*

Mitochondria + CaC1z + Hypoxanthine+ xanthineoxidase

0.063 -+ 0.003

0.486 -+ 0.019 (p < 0.001)**

1.193 - 0.114 (p < 0.001)**

Mitochondria + CaC1z + Hypoxanthine + Xanthineoxidase + SOD

0.036 ___0.006

0.457 - 0.042 (p < 0.1)**

0.792 __- 0.081 (p < 0.001)**

Mitochondria + CaCI2 + Hypoxanthine + Xanthineoxidase + Tryptophan (c)

0.044 _+ 0.001

0.489 -- 0.036 (p < 0.1)**

1.058 _ 0.112 (p < 0.1)**

Mitochondria + CaCI: + Hypoxanthine + Xanthineoxidase + Methionine (c)

0.058 _+ 0.002

0.594 - 0.046 (p < 0.001)**

0.858 _+ 0.176 (p < 0.01)**

Mitochondria + CaC1z + Hypoxanthine + Xanthineoxidase + Histidine (c)

0.086 _ 0.008

0.550 --- 0.028 (p < 0.001)**

0.947 _+ 0.088 (p < 0.02)**

Values are arithmeticmean _+ S.D. of four determinationsin each case. *p (b) vs. (a); **p (c) vs. (b). The concentrationsof various reagents employedin the study are as follows: CaC12--2mM; Hypoxanthine--5 × 10-s M; Xanthineoxidase-0.025 units; Superoxide dismutase--70 units; Tryptophan, methionineand histidine--1 mM each.

284

S. MEHROXRAet al.

creased membrane permeability, and decreased fluidity -- parallel the loss of cell viability. 2° In isolated rat liver and heart mitochondria, it has been shown that accumulation of small amounts of lysophospholipids and free fatty acids generated by the activation of endogenous phospholipase A 2 correlates with the increase in inner membrane permeability, the release of intramitochondrial cations, deenergization, and large amplitude swelling. 2° Further, in vivo ischemia followed by reperfusion, a condition which is associated both with increase in intracellular calcium concentration as well as oxygen free radicals production has been shown to cause structural alterations in the mitochondrial inner membrane which may account for the functional deficiencies of ischemic mitochondria as well as ischemic liver cell injury. 2~ It has been reported that calcium enhances the in vitro free radical induced damage to brain synaptosomes, mitochondria and cultured spinal cord neurons. 22 Potentiation of oxygen radical injury to renal mitochondria by Ca 2+ with ubiquinone as the probable site in the electron transport chain where the defect is produced, has also been well documented. 23 Stimulation of lipid peroxidation in microsomes and mitochondria occurring as a result of the formation of free radical species in carbon tetrachloride (CC14) poisoning has been observed. Mitochondrial electron transport chain is responsible for the activation of CC14 into trichloromethyl free radical (CCL~) which causes damage resulting in uncoupling of oxidative phosphorylation and impairment of Ca 2+ transport. 24 Haloalkanes, for example, CC14 and bromotrichloromethane (CBrC13), get activated to free radical species by the cellular compartments, which attack the cellular macromolecules including Ca 2÷ pumps of mitochondria and endoplasmic reticulum. This results in impairment of the respective calcium-sequestering capacities of these organelles leading to unphysiological sustained rise in the levels of free cytosolic Ca 2+ concentration with pathological consequences. 25'26 It has been suggested that the presumptive rise in cytosolic free Ca 2"- during the course of action of various hepatotoxins playing the role of a "toxicological second messenger" which could lead to pathological events such as phospholipase A 2 activation in parts of the cell such as the plasmamembrane and organelles like endoplasmic reticulum and mitochondria noted for their ability to cause the generation of free radical species. 25 Disintegration of cristae folds, fragmentation of membrane, loss of cristae material and lysis under free radical stress, and calcium overloading is apparent from the TEM pictures presented in the present study. Similar fragmentation and lysis of mitochondria isolated from livers of mice treated with CC14 have been reported. 27 Thus, from the above study, a correlation

between active oxygen species, membrane functions, and calcium dynamics is apparent. Acknowledgements -- Thanks are due to Prof. P.K. Ray, Director, Industrial Toxicology Research Centre for his keen interest in this work. Technical assistance provided by Dr. L.K.S. Chauhan and P.N. Saxena in carrying out the electron microscopic work is highly appreciated.

REFERENCES 1. Trump, B.F.; Berezesky, I.K. Cell injury and cell death. The role of ion deregulation. Comments on Toxicology 3:47-67;1989. 2. Trump, B.F.; Berezesky, I.K.; Toshihide, S.; Laiho, K.U.; Phelps, P.C.; DeClaris, N. Cell calcium, cell injury and cell death. Env. Health Perspec. 57:281-287; 1984. 3. Fiskum, G. Physiological aspects of mitochondrial calcium transport. In: Sigel, H., ed. Metal ions in biological systems - calcium and its role in biology. Vol. 17. New York: Marcel Dekker Inc.; 1984: 187-213. 4. Carafoli, E. Intracellular calcium homeostasis. Ann. Rev. Biochem. 56:395--433; 1987. 5. Mustafa, M.G. Augmentation of mitochondrial oxidation in lung tissue during recovery of animal from acute ozone exposure. Arch. Biochem. Biophys. 165:531-538; 1974. 6. Lehninger, A.L. Reversal of various types of mitochondrial swelling by ATP. J. Biol. Chem. 234:2465-2471; 1959. 7. Singh, A.; Bell, M.J.; Koroll, G.W.; Kremers, K.; Singh, H. Reactions of tryptophan with singlet oxygen, hydroxyl radical and superoxide anion. In: Oxygen and oxyradicals in chemistry and biology. Rodgers, M.A.J.; Powers, E.L., eds., New York: Academic Press; 1981: 461470. 8. Foote, C.S. Photooxidation of biological model compounds. In: Oxygen and oxyradicals in chemistry and biology. Rodgers, M.A.J.; Powers, E.L., eds., New York: Academic Press; 1981: 425439. 9. Hiller, K.O.; Gobl, M.; Masloch, B.; Asmus, K.D. OH" radical induced oxidation of sulphur containing amino acids. In: Oxygen and oxyradicals in chemistry and biology. Rodgers, M.A.J.; Powers, E.L., eds. New York: Academic Press; 1981: 666. 10. Ohkawa, H.; Onisui, N.; Yagi, K. Assay of lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351-358; 1979. 11. Hayat, M.A., ed. Principle and technique of electron microscopy. Vol. 1. New York:Van Nostrand Reinhold; 1981. 12. Fisher, R.A. Statistical methods for research works, 1lth ed. Edinburgh, U.K.: Oliver & Boyd; 1950. 13. Jamieson, D. Oxygen toxicity and reactive oxygen metabolites in mammals. Free Radic. Biol. Med. 7:87-108; 1989. 14. Kappus, H. Lipid peroxidation: mechanisms, analysis, enzymology and biological relevance. In: Sies, H., ed. Oxidative stress. New York:Academic Press; 1985: 273-290. 15. Comporti, M. Three models of free radical-induced cell injury. Chem. Biol. Interact. 72:1-56; 1989. 16. Cotgreave, I.A.; Moldeus, P.; Orrenius, S. Host biochemical defense mechanisms against prooxidants. Ann. Rev. Pharmacol. Toxicol. 28:189-212: 1988. 17. Nohl, H. Oxygen radical release in mitochondria: Influence of age. In Johnson, J.E., Jr.; Walford, R.; Harman, D.; Miquel, J., eds. Free radicals, aging and degenerative diseases. Vol. 8. New York: Alan. R. Liss Inc.; 1986: 77-97. 18. Richter, C.; Frei, B. Calcium release from mitochondria induced by pro-oxidants. Free Radic. Biol. Med. 4:365-375; 1988. 19. Bors, W.; Buettner, G.R.; Michael, C.; Saran, M. Calcium in lipid peroxidation: Does calcium interact with superoxide? Arch. Biochem. Biophys. 278:269-272; 1990. 20. Ungemach, F.R. Prevention of liver cell damage following lipid peroxidation by depression of lysophosphatide formation. In: Chambers, P.L.; Chambers, C.M.; Greim, H., eds. Biological monitoring of exposure and the response of subcellular levels to

Oxygen radicals and mitochondria

21. 22.

23. 24.

25. 26.

toxic substances. Arch. Toxicol. Suppl. Vol. 13. Berlin Heidelburg:Springer-Verlag; 1989: 275-281. Okayasu, T.; Curtis, M.T.; Farber, J.L. Structural alterations of the inner mitochondrial membrane in ischemic liver cell injury. Arch. Biochem. Biophys. 236:638-645; 1985. Braughler, J.M.; Duncan, L.A.; Goodman, T. Calcium enhances in vitro free radical induced damage to brain synaptosomes, mitochondria and cultured spinal cord neurons. J. Neurochem. 45" 1288-1293; 1985. Malis, C.D.; Bonventre, J.V. Mechanism of calcium potentiation of oxygen free radical injury to renal mitochondria. J. Biol. Chem. 261:14201-14208; 1986. Tomasi,A.; Albano, E.; Banni, S.; Botti, B.; Corongiu, F.; Dessi, M.A.; Iannone, A.; Vannini, V.; Dianzani. M.U. Free radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation. Biochem. J. 246"313-317; 1987. Dolak,A.J.; Waller,L.R.; Glende,E.A.,Jr.; Recknagel,O. Liver cell calcium homeostasis in carbon tetrachloride liver cell injury - - new data with Fura 2. J. Biochem. Toxicol. 3:329-342; 1988. Casini,A.F.; Maelloro, E.; Pompella, A.; Feralli, M.; Comporti, M. Lipid peroxidation, protein thiols and calcium homeostasis in bromobenzene induced liver damage. Biochem. Pharmacol. 36:

285

3689-3695; 1987. 27. Rutkowski, J.V.; Roebuck, B.D.; Smith, R.P. Allyl alcohol partially protects murine hepatic mitochondria against carbontetrachloride. Toxicology 40:25-30; 1986.

ABBREVIATIONS -- Superoxide anion O H ° - - H y d r o x y l radical 'O 2 -- Singlet oxygen SOD -- Superoxide dismutase T B A - - T h i o b a r b i t u r i c acid E D T A - - E t h y l e n e d i a m i n e tetra acetic acid ATP -- Adenosine triphosphate CaC12 - - C a l c i u m c h l o r i d e GEl 4 -- Carbon tetrachloride CBrCI 3 -- Bromo tfichloromethane TEM -- Transmission electron microscopy O~