Molecular and Cellular Biochemistry 154: 39-45, 1996. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

tBHP induced in vitro swelling of rat liver mitochondria

Poonam Kakkar, Sudhir Mehrotra and EN. Viswanathan Ecotoxicology Section, Industrial Toxicology Research Centre, P. Box-80, M.G. Marg, Lucknow-226 001, India

Received 19 January 1995; accepted 11 August 1995

Abstract

Tert-butyl hydroperoxide induced swelling of freshly isolated rat liver mitochondria was inhibited by butylated hydroxytoluene, butylated hydroxyanisole and cL-tocopherol by acting at the initial phase. EDTA was more effective than EGTA in reducing the initial swelling and so were desferal and bipyrridyl. Spermine, an allosteric activator of calcium uptake, enhanced swelling whereas lanthanum and ruthenium red, the Ca 2+ uniport blockers, reduced it. Inhibition ofphospholipase A 2 by dibucaine and Ca 2+ activated proteases by antipain and leupeptin also reduced t-BHP induced swelling. The data indicate that peroxidative mitochondrial swelling involves an iron mediated initial rapid phase and a subsequent calcium dependent propagation phase. (Mol Cell Biochem 154: 39--45, 1996)

Key words: free radicals, oxidative stress, mitochondrial swelling, tert-butyl hydroperoxide, calcium deregulation

Introduction

We reported earlier that in vitro generation of superoxide anions could lead to structural and functional changes in rat liver mitochondria [ 1, 2] including peroxidative decompo- sition of polyunsaturated fatty acids. This process was also found to be influenced by Ca > responsive agents, suggest- ing possible alterations in calcium fluxes and functions in oxidative swelling of mitochondria [3]. Whether organic hydroperoxides such as tert-butyl hydroperoxide (t-BHP) used as convenient models for studying oxidative stress [4, 5] could elicit such a response is of interest. Kennedy et al. [6, 7] have characterized the formation of free radicals by t- BHP in rat liver mitochondria in vitro. Similarly cumene hydroperoxide and benzoyl peroxide cause peroxidative membrane changes [8] including mitochondrial swelling [9]. As in the case of superoxide anion mediated oxidative swell- ing, involvement of transition metal ions, thiol depletion and deregulation of Ca 2+ functions could be involved in organic peroxide mediated swelling. Therefore, the effect of t-BHP on mitochondrial swelling in vitro was tested in the absence and presence of agents influencing the above conditions. The agents tested for effect of oxidative swelling included calcium

channel blocker lanthanum chloride, phospholipaseA 2 inhibi- tor dibucaine, calcium dependent protease inhibitors antipain and leupeptin, chelators EDTA, EGTA, desferal and thiol depletor diethylmaleate as used in our earlier paper [3].

Materials and methods

Chemicals

Butylated hydroxyanisole, butylated hydroxytoluene, ethyl- ene glycol bistetraacetic acid (EGTA) and spermine were pro- cured from Sigma Chemicals Co. St. Louis, MO, USA. Desferal was obtained as a generous gift from Ciba Geigy, Basel, Switzerland. All other chemicals were of AnalaR grade from standard commercial sources. Quartz distilled water was used through out the study.

Isolation of mitochondria

Male albino Wistar strain rats (100--150 g) maintained tra- der standard conditions in the IndustrialToxicology Research

Address for offprints: E Kakkar, Ecotoxicology Section, Industrial Toxicology Research Centre, P. Box-80, M.G. Marg, Lucknow-226 001, India

Lipid peroxidation

Centre animal colony were used. Rats were killed by cervi- cal dislocation and livers were taken out immediately, rinsed with 0.25 M sucrose, dried on filter paper, cleared of all ad- hering tissue debris, weighed, minced and homogenised in a Potter-Elvehj em glass homogeniser. A 10% (w/v) homoge- nate was prepared in 0.25 M sucrose solution (or in 0.02M Tris-0.15 M KC1, pH 7.4 for lipid peroxidation studies only). Mitochondria were isolated according to the method of Mustafa et al. [ 10]. After isolation, the washed 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 these operations were performed at 0-4°C. Electron microscopic observations reported earlier indicated that the preparation was generally free from other organelles.

Study of mitochondrial swelling

Swelling of isolated rat liver mitochondria was carried out according to the spectrophotometric method ofLehninger et al. [11] using Milton Roy Spectronic 1001 spectrophotom- eter. The incubation system consisted of mitochondria sus- pended in Tris-KC1 buffer, and supplements as specified in a total volume of 2 ml. The specified amount oft-BHP was added last. In the unsupplemented system the volume was made up with the vehicle, in the same concentration or otherwise with water. The incubation was done at 30°C for the specified periods, t- BHP and other supplements as specified were incorporated into the assay system and decrease in the optical density at 520 nm at 1 min intervals for a period of 5 rain was recorded. Any di- rect effect of the agents tested on swelling other than through lipid peroxidation is accounted by the optical density changes in the incubation system without hydroperoxide. Each experi- ment was repeated four times and comparative data is given.

Statistical analysis

Malonaldehyde (MDA) liberated by lipid peroxidation was estimated by the thiobarbituric acid (TBA) method [12] at different time intervals in mitochondria incubated with specified concentrations of t-BHP and Fe 2+. The molecular extinction coefficients of 1.56 × 10SM-lcm -1 was used for calculating the amount of MDA.

0 . 3 I I I I I 0 1 2

Time in The statistical significance of the changes observed was evaluated by the student's t-test [ 13] with a significant level of p < 0.05, since the purpose of each in vitro experiment was only to compare the swelling pattern in the presence and absence of agents.

Results

Effect of t-BHP on mitochondrial swelling

The dose dependent effect oft-BHP on the swelling ofmito- chondria (Fig. 1) indicates a biphasic pattern. Swelling was rapid in the initial phase upto 2 min which was followed by a relatively slower phase.

Effect of various agents on t-BHP lnduced swelling

Antioxidants Butylated hydroxynisole (BHA) (Fig. 2) at 25 ~tM completely abolished the swelling caused by 75 ~tM t-BHP, a-Tocopherol at 50 ~tM concentration caused the reversal of swelling which was also observed at 75 and 100 laM. In all these cases the ini- tial rapid phase of swelling was drastically reduced by the radi- cal scavengers and antioxidants. Butylated hydroxy toluene (BHT) also abolished the swelling caused by t-BHP (Table 1).

Externally added GSH as such did not influence the t-BHP induced swelling. GSSG at 2.5 and 5 mM concentrations did not influence the swelling significantly. However, the thiol depleting agent diethyl maleate (DEM) significantly en- hanced t-BHP induced mitochondrial swelling in a dose de- pendent manner, 1 mM enhancing it by over 4 fold (Fig. 3). Another thiol depleting agent N-ethyl maleimide (NEM) also potentiated the t-BHP induced swelling. Exogenously added thiols however had very little effect on swelling pattern prob- ably due to permeability problems.

1.0.

0J I 0.8'

= 0.7

"6 0.6

i 0.S

0.4

40

I I I I 1 3 4 5

mrs.

Fig. 1. Kinetics of t-BHP induced swelling of mitochondria as followed by decrease in O.D. at 520 nm. Different concentrations of t-BHP (O--- Q) 25 gM; (O---O) 50 gM; (x---x) 75 gM and 100 gM (A---A) were used for this study.

41

0.8

0.7

0.73

0 .68

\

0 .63 I , I 0 1

I I I 1 3 ~ i b

T ime in mrs. >

Polyamines Spermine (100 gM) enhanced the rapid swelling in the first 2 rain and more gradually in the subsequent 3 rain as com- pared to control. The overall swelling was enhanced by over 6 fold. Fifty laM spermine was far less active. Spermidine produced similar pattern but was less effective than spermine. Even at 500 gM the potentiation of swelling was about 2.5 fold.

Mitochondrial calcium transport inhibitors Lanthanam (La 3+) ions, the Ca 2+ uniport blocker, increased the lag phase ofmitochondrial swelling leading to reduction in swelling. At 1 mM concentration it caused about 90% re- duction whereas 2.5 mM completely abolished the swelling (Fig. 6). Further, the dose dependent protection accorded by ruthenium red, a specific inhibitor of mitochondrial Ca 2+ uniport, against t-BHP induced mitochondrial swelling also suggested Ca 2+ involvement.

Fig. 2. Effect of Butylated hydroxyanisole (BHA) on t-BHP (75 gM; x-- -x) induced swelling of mitochondria as shown in figure using 50 ~tM BHA (A---A); 100/aM BHA (I - - - I ) and 250 ~tM BHA (0---(9).

Chelating agents The unspecific chelator EDTA at 0.5 and 1 mM completely abolished the swelling ofmitochondria. Unlike EDTA, EGTA which is more specific for calcium could only partially re- duce the swelling (Fig. 4). Upto 1 min EGTA did not have much effect on swelling as compared to EDTA indicating that cations other than Ca 2+, possibly endogenous Fe 2÷ or Cu 2+ may also be involved in the initial stages of permeability changes in membranes. Desferal, the specific chelator of Fe 3+ at 0.5 mM reduced the swelling by about 45% and at 1 mM about 75% (Fig. 5). Another Fe 3÷ chelator 2,2'-bipyridyl also showed a similar response. At the concentrations tested, the chelators by themselves did not cause any significant swelling•

Phospholipase A 2 and protease inhibitors

Dibucaine, a phospholipase A 2 inhibitor at 250 gM caused about 60% reduction at 2.5 min and 53% reduction in swell- ing at 5 rain. On doubling the amount of dibucaine, the inhi- bition of swelling also increased to about 71 and 63% respectively (Table 2). Calcium dependent protease inhibi- tors leupeptin and antipain also reduced t-BHP induced swell- ing. Leupeptin caused about 52 and 58% reduction in swell- ing at 10 gM at 2.5 and 5 min respectively. At 25 gM, about 47 and 54% reduction took place. With antipain also similar results were obtained, 10 gM causing 57.3 and 62.4% inhi- bition. With 25 gM antipain corresponding values were 65 and 69% respectively (Table 2).

Table 1. Effect of BHT and cL-tocopherol on t-BHP induced swelling of mitochondria in vitro

Incubation system (Time in minutes) 0

O.D at 520 nM

1 2 3 4 5

Mitochondria + 75/aM t-BHP 0.621 0.465 0.265 0.258 0.243 0.207 (Basal system) (0.034) (0.018) (0.013) (0.019) (0.020) (0.012) B + 10/aM BHT 0.615 0.561 0.554 0.562 0.553 0.552

(0.03 I) (0.025) (0.021) (0.032) (0.027) (0.023) B + 25 gM BHT 0.618 0.580 0.578 0.576 0.573 0.571

(0.026) (0.032) (0.025) (0.031) (0.029) (0.021) B+ 50 gM a-tocopherol 0.626 0.585 0.574 0.559 0.537 0.530

(0.034) (0.028) (0.022) (0.034) (0.031) (0.025) B + 75/aM o~-tocopherol 0.618 0.587 0.582 0.570 0.552 0.530

(0.029) (0.036) (0.027) (0.032) (0.031) (0.026) B + I00 laM ct-tocopherol 0.619 0.605 0.596 0.592 0.588 0.581

(0.036) (0.022) (0.030) (0.021) (0.029) (0.032)

Values are arithmetic mean of four determinations in each case + S.D. is given in parenthesis. B -Basal system, i.e. mitochondria + 75/aM t-BHE

42

F E 0.~ c

0

5 0.4

0.2

O.C 0

I I I I I I I I I

1 2 3 6 5

Time in mrs.

Fig. 3. Enhancement of 75 ~tM t-BHP (I---O) induced swelling in vitro by thiol depleting agent, diethyl maleate. As shown in figure, swelling increased with the increasing concentration ofDEM (A---A 250 I~M; x---x 500 I~M; 0---(9 250 mM) indirectly supporting the protective role of GSH against peroxidative swelling.

T EO.7 c

o

un

~0.6 c5

I 0.5

0.9 - 0.~

T

0 ,3 L I I I i i i / I ]

0 1 2 3 /* 5

< Time in mrs.

Fig. 4. Protection accorded by calcium chelator EGTA against the sweU- ing of mitochondria caused by 75 gM t-BHP (O---Q). 1 mM EGTA (x--- x) reduced the swelling whereas higher swelling concentration (2.5 mM C)---O) completely abolished the swelling•

Lip id perox ida t ion Incubation o f unsupplemented system led to marginal in- crease in TBA reactive products o f lipid peroxidation (Table 3). Addition o f t-BHP (200 laM) did not significantly influ- ence the pattern, even though the activity in the first 10 rnin tended to decrease and by 2 h it was only slightly lower than the control. Any contribution by endogenously present iron

E o

Lr l

0

I i

1.0

q

0.9

0.8

0.7

0.6

0.5

0.6 I I I I I I I I I I

1 2 3 L, 5

.<--------Time in m?s. >

Fig. 5. Protective role of Fe 3÷ chelator, desferal, on 75 ~tM t-BHP (Q---Q) induced swelling of mitochondria. Higher concentration (1 mM desferal C)---C)) further reducing the swelling as compared to 500 ~tM (x---x).

0.9

T0.8 E c

o (%1 t . n

~0.7 0

IT 0.6

0•5 0

I • I I I I i I i I

I 2 3 ~ 5 q,~---Time in mrs. •

Fig. 6. Effect of Lanthanum on 75 I~M t-BHP (Q-HI) induced swelling ofmitochondria. Both the concentrations tested i.e. 1 mM La 3÷ (x---x) and 2.5 mM La 3÷ (C)---O) caused significant reduction in swelling.

could not be assessed. Supplementation with Fe 2÷ progres- sively enhanced the M D A level at all the stages, (p < 0.01), 40 and 200 IxM causing about 85 and 250% increase at 2 h (,p < 0.001). The enhancement was 157 and 215% at 100 and 150 IxM Fe 2+ supplementation•

Table 2. Protective role of calcium dependent phospholipase and protease inhibitors on the swelling of mitochondria caused by t-BHP

OD at 520 nm observed at different time (rains) intervals

Incubation system 0 I 2 3 4 5

43

Mit +75 gM t-BHP 0.953 0.884 0.733 0.662 0.631 0.611 (Basal system) (0.021) (0.027) (0.041) (0.036) (0.019) (0.017) B + 250 laM dibucaine 0.950 0.931 0.881 0.843 0.821 0.806

(0.017) (0.042) (0.037) (0.041) (0.017) (0.015) B + 500 gM dibucaine 0.965 0.944 0.912 0.875 0.859 0.840

(0.012) (0.039) (0.039) (0.023) (0.023) (0.026) B + 10 gM leupeptin 0.962 0.878 0.828 0.813 0.810 0.805

(0.015) (0.018) (0.029) (0.032) (0.029) (0.013) B + 25 gM leupeptin 0.952 0.884 0.837 0.821 0.812 0.809

(0.018) (0.016) (0.025) (0.028) (0.033) (0.027) B + 10 aM antipain 0.957 0.908 0.859 0.842 0.836 0.833

(0.024) (0.043) (0.042) (0.032) (0.026) (0.018) B + 25 gM antipain 0.965 0.953 0.887 0.869 0.857 0.851

(0.031) (0.048) (0.036) (0.044) (0.041) (0.022)

The values are arithmetic mean of four determinations in each case ± S.D. is given in parenthesis. Mit - mitochondria, B - basal system i.e. mitochondria

+ 75 gM t-BHP.

Table 3. TBA reactive substance formation in vitro by rat liver mitochondria in the presence of t-BHP and FC ÷

n moles of MDA liberated/mg mitochondrial protein

Incubation mixture (Time in minutes) 0 10 30 60 120

Mitochondria suspended in KC l (a) 0.245 (0.026)

Mitochondria + 200 ~tM t-BHP (b) 0.272 (0.012)

Mitochondria+t-BHP + 40 gM Fe 2÷ (c) 0.309 (0.024)

Mitochondria+t-BHP + 200 gM Fe 2÷ (c) 0.328 (0.011)

0.117 0.281 0.384 0.427 (0.016) (0.012) (0.020) (0.014) 0.239 0.257 0.311 0.381"** (0.015) (0.017) (0.012) (0.009) 0.379** 0.441"* 0.600** 0.789* (0.026) (0.014) (0.018) (0.019) 0.706* 0.883* 1.131" 1.289" (0.028) (0.025) (0.050) (0.032)

Values are arithmetic mean + SD &five determinations in each case. p values are b vs a and c vs b, *p < 0.001; **p<0.01; ***p < 0.05 ± S.D. is given in parenthesis. The concentration oft-BHP in all the cases was 200 gM. Mit - mitochondria, t-BHP - tert butyl hydroperoxide.

Discussion

Biological effects oft-BHP have been studied as a model for oxidative cellular injury [14]. Masakie tal . [15] andNieminen et al. [ 16] have reported that t-BHP reduces the inner mito- chondrial membrane potential along with loss of ATP and metabolic acidosis. Recently Castilho et al. [17] have reported oxidative damage of mitochondria induced by t-BHP in the presence of Ca 2+. The present results indicate that incubation of freshly isolated rat liver mitochondria with t-BHP caused marked swelling which was inhibited by antioxidants, and transition metal chelating agents like desferal. Depletion of endogenous thiol reserves by diethylmaleate and N-ethyl- maleimide was found to increase the swelling even though the exogenous thiols were not effective due to permeability problem. The involvement of Fe 2+ in oxidative swelling is

indicated by the influence of both permeable and non per- meable iron chelators. Even though high levels of externally added transition metal ions enhanced peroxidation and swell- ing, the contribution by endogenous iron appeared to be of low order, since exogenous supplementation was needed for malonaldehyde formation. The endogenously present iron may not be in sufficient amounts or may be in a form not accessible to the polyunsaturated fatty acid sites. Since peroxidation is a slower process than swelling and continues even after swelling is complete, exact quantitative correla- tion between the two processes is not possible. However, the parallelism in the two phenomenon under a variety of con- ditions, as reported earlier [ 1-3], indicate a possible correla- tion. Also, the formation of active oxygen species during initiation of swelling could not be quantified in the present study. Whether iron ions have any direct effect on calcium

44

movements in addition to free radical processes, is also not clear from these studies. Similarly the mechanism of action of the individual agents and likely processes other than peroxidation in the swelling phenomenon were beyond the objectives of this study. The main characteristics of t-BHP induced swelling was rapid decrease in optical density in the first 2 rain followed by a more gradual swelling. From the effects of the various agents on the time course of swelling, it seems likely that Fe 2+ is involved in the first stage and Ca 2+ related processes in the second. The various agents exerted their effects by increasing the lag phase or by over all inhi- bition of swelling. Spermine, an allosteric activator of Ca 2÷ uptake [18] caused a marked increase in swelling in the ini- tial phase whereas spermidine which was less active showed lesser effect on mitochondria. The protective effect of La 3+ and ruthenium red also indicated that alterations in Ca 2÷ fluxes may be involved in swelling. Active oxygen species scaven- gers and antioxidants also reduced the t-BHP induced mito- chondrial swelling.

The membrane damage by t-BHP was interpreted by Frei et al. [19] as to involve deregulation of Ca 2+ homeostasis. La 3+ and ruthenium red were found to reduce the membrane po- tential changes and Ca 2+ alteration caused by the hydro- peroxide. Earlier reports from our laboratory [ 1, 2, 3, 20] also indicate a possible relation between Ca 2÷ fluxes and peroxi- dative mitochondrial damage. It is likely that similar mecha- nisms may be involved in the t-BHP induced mitochondrial swelling, a rapid initial phase may be accompanied by per- meability changes and the formation of active oxygen spe- cies, which trigger membrane damage and peroxidation. The results obtained with dibucaine indicate that activation of PLA 2 could be an early event in the oxidative swelling of mitochondria. Lenzen et al. [21 ] have reported that lysophos- pholipids inhibited mitochondrial Ca 2+ uptake and induced active Ca 2÷ effiux. Lysophospholipid accumulation was re- ported in brain by us [22] under oxidative stress. The data on the effects of antipain and leupeptin on mitochondria indi- cate Ca 2÷ activated proteases action during swelling and cy- totoxicity as suggested by Nicotera and his colleagues [23, 24] regarding the activation of non-lysosomal proteases in oxidative cell injury. The present results thus strengthen the evidences for possible interrelation between in vitro mito- chondrial damage, active oxygen radicals formation and al- tered calcium fluxes [l, 2, 3].

Acknowledgements

Thanks are due to Dr. R.C. Srimal, Director, Industrial Toxi- cology Research Centre for his keen interest in this work and permission to publish it. I would also like to thank the ITRC reviewing committee for their suggestions and allotting the communication number 1877 to this manuscript.

References

1. Mehrotra S, Kakkar P, Viswanathan PN: Mitochondrial damage by active oxygen species in vitro. Free Radic Biol Med 10: 227-285, 1991

2. Kakkar P, Mehrotra S, Viswanathan PN: Interrelation of active oxy- gen species, membrane damage and altered calcium functions. Mol Cell Biochem 111: 11-15, 1992

3. Mebrotra S, Viswanathan PN, Kakkar P: Influence of some biologi- cal response modifiers on swelling of rat liver mitochondria in vitro. Mol Cell Biochem 124: 101-106, 1993

4. Trible DL, Jones DP, Edmondson E: Effect of hypoxia on tert-butyl hydroperoxide induced oxidative injury in isolated hepatocytes. Mol Pharmacol 34: 413-420, 1988

5. Massa EM, Giulivi C: Alkoxyl and methyl radical formation during cleavage of ten-butyl hydroperoxide by a mitochondrial membrane bound, redox active copper pool: An EPR study. Free Radic Biol Med 14: 559--565, 1993

6. Kennedy CH, Pryor WA, Winston GW, Bhurch DF: Hydroperoxide induced radical production in liver mitoehondria. Biochem Biophys Res Commun 141: 1123-1129, 1986

7. Kennedy CH, Chureh DF, Winston GW, Pryor WA: Tert-butyl hydroperoxide induced radical production in rat liver mitochondria. Free Radic Biol Med 12: 381-387, 1992

8. Cavallini L, Valente M, Bindoli A: Comparison of cumene hydroperoxide and NADPH/FeWADP induced lipid peroxidation in heart and liver submitochondrial particles. Biochem Biophys Acta 795:466-472, 1984

9. Kennedy GH, Winston GW, Church DF, Pryor WA: Benzoyl peroxide interaction with mitochondria: inhibition of respiration and induc- tion of rapid, large amplitude swelling. Arch Biochem Biophys 271: 456-470, 1989

10. Mustafa MG: Augmentation of mitochondrial oxidation in lung tis- sue during recovery of animal from acute ozone exposure. Arch Bio- chem Biophys 165: 531-538, 1974

11. Lehninger AL: Reversal of various types of mitochondrial swelling by ATP. J Biol Chem 234: 2465-2471, 1959

12. Ohkawa H, Onishi N, Yagi K: Assay of lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351-358, 1979

13. Fisher RA(ed): Statistical methods for research works, l l th ed, 1950. Oliver & Boyd, Edinburgh, UK

14. Younes M, Strubelt D: The role of iron and glutathione in t-butyl hydroperoxide induced damage towards isolated perfused rat livers. J Appl Toxicol 10: 319--324, 1990

15. Masaki N, Kyle ME, Serroni A, Farber JL: Mitochondrial damage as a mechanism of cell injury in the killing of cultured hepatocytes by tert-butyl hydroperoxide. Arch Biochem Biophys 270: 672--680, 1990

16. Nieminen AL, Dawson TL, Gores GL, Kawanishi T, Herman B, Lemasters JJ: Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals. Biochem Biophys Res Commun 167: 600-606, 1990

17. Castilho RF, Kowaltowshi AJ, Meinicke AR, Vercesi AE: Oxidative damage of mitochondria induced by Fe (II) citrate or t-butyl hydroperoxide in the presence of Ca2*: Effect of coenzyme Q redox state. Free Radie Biol Med 18: 55-59, 1995

18. Kroner H: Spermine, another allosteric activator of calcium uptake in rat liver mitochondria. Arch Biochem Biophys 267:205-210, 1988

19. Frei B, Winterhalter KH, Richter C: Quantitative and mechanistic aspects of the hydroperoxide induced Ca 2+ release from rat liver mi- tochondria. Eur J Biochem 149: 633q539, 1985

20. Kakkar P, Mehrotra S, Viswanathan PN: Active oxygen radical me-

diated mitochondrial swelling and calcium fluxes. Free Radic Biol Med 9: (suppl. 1) 31, 1991

21. Lenzen S, Gorlich JK, Rustenbeeh I: Regulation of transmembrane ion transport by reaction products on phospholipase A 2. I. Effects of lysophospholipids on mitochondrial Ca > transport. Biochim Biophys Acta 982: 140-146, 1989

22. Awasthi S, Kakkar P, Viswanathan PN, Bhardwaj R: Increase in the lysolecithin content of brain microsomes in phenobarbitone admin-

45

istered rats and its relation to lipid peroxidation. J Neurochem 57: 277-281, 1991

23. Nicotera P, Bellomo G, Orrenius S: Calcium-mediated mechanisms in chemically induced cell death. Annu Rev Pharmacol Toxicol 32: 449-470, 1992

24. Corcoran GB, Fix L, Jones DP, Moslen MT, Nicotera E Oberhammer FA, Buttyan R: Apoptosis - Molecular control point in toxicity. Toxicol Appl Pharrnacol 128: 169-181, 1994