Molecular and Cellular Biochemistry 111: 11-15, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Interrelation of active oxygen species, membrane damage and altered calcium functions

Poonam Kakkar, Sudhir Mehrotra and P.N. Viswanathan Ecotoxicology Section, Industrial Toxicology Research Centre, P.O. Box 80, Mahatma Gandhi Marg,

Lucknow-226 001, India

Abstract

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine - xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O~ in the presence of CaC12 enhanced the peroxidative decomposi- tion of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhance- ment of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tert- butyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca 2+ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics. (Mol Cell Biochem 111" 11-15, 1992)

Key words: superoxide dismutase, oxidative stress, toxicology, calcium fluxes, free radicals

Introduction

In the realm of modern biochemistry the rapid strides of development in the understanding of the messenger/ regulator functions of calcium in health and diseases have been phenomenal, overshadowed perhaps only by the advancements in molecular genetics and immunol- ogy [1, 2]. The modulation of specific Ca functions in the etiophathogenesis of some cardiovascular [3], and neuro muscular [4] diseases has been well established. Similarly, under xenobiotic mediated stress the involve- ment of altered Ca 2+ fluxes in cytotoxicity is becoming increasingly clearer [5]. Many such xenobiotics mani- fest their effects through oxidative stress caused by

active oxygen species and other free radicals [6]. In many cases this is also accompanied by altered function- al organization of biomembranes [7]. However, any mechanistic interrelation between oxidative damage to membranes by xenobiotics and Ca function is not clear yet. Therefore, studies in this direction are being pur- sued recently by our laboratory, using freshly isolated rat liver mitochondria as the test system. Some of the recent findings are reported herein.

Address for offprints: P. Kakkar, Ecotoxicology Section, Industrial Toxicology Research Centre, P.O. Box 80, M.G. Marg, Lucknow-226001, India

12

Materials and methods

Materials

Dithiothreitol, tert-butyl hydroperoxide, ruthenium red, hypoxanthine, xanthine oxidase and bovine eryth- rocyte superoxide dismutase were procured from Sigma Chemicals Co., USA. Cytochrome c, calcium chloride, reduced glutathione were from BDH Analar. All the other chemicals used were E. Merck extrapure.

Enzyme assay

Monoamine oxidase (EC 1.4.3.4) activity was assayed according to the method of Tabor et al. [9] in freshly isolated as well as in mitochondria under the stress of a superoxide radical generating system in vitro. Activity of glutathione peroxidase (EC 1.11.1.9) was measured according to Splittgerber and Tappel [10] and cyto- chrome c oxidase (EC 1.9.3.1) by the method of Coo- perstein and Lazarrow [11].

Treatment of mitochondria

Swelling of isolated rat liver mitochondria was studied spectrophotometrically according to Lehninger [12] us- ing Milton Roy Spectronic 1001 spectrophotometer. Calcium chloride (2mM) was used as swelling agent and swelling of mitochondria was recorded as decrease in optical density at 520nm at lmin intervals for a period of 10 rain. This concentration of calcium chloride was selected as the reported concentration of in vitro studies [121. Hypoxanthine (5 x 10 -5 M) and xanthine oxidase (0.025 units) system was used for the generation of superoxide anion (O{). To study the quenching ef- fect, superoxide dismutase (SOD; 70 units) was used as scavenger of superoxide radical. The effect of tert-butyl hydroperoxide (t-BHP) on mitochondria was observed

in the presence of varying concentrations of ruthenium red and dithiothreitol (DTT) by following the extent of swelling of mitochondria.

Statistical analysis

The statistical evaluation of the effects of exogenous calcium and active oxygen species on functional integri- ty of freshly isolated rat liver mitochondria was ac- complished with Student's 't' test [13]. Significant level of p < 0.05 was used for all comparisons.

Results

Table 1 shows that 2 mM of CaC12 caused swelling of freshly isolated rat liver mitochondria, which increased to a higher magnitude when 02 was generated by hy- poxanthine (5 x 10-5M) and xanthine oxidase (0.025 units) in the same system. Superoxide dismutase (70 units) accorded some protection towards the mitochon- drial swelling induced by 02. The above data indicates that incubation of the freshly prepared mitochondria with O~generating system causes swelling of mitochon- dria. Changes in mitochondrial functional organisation was also evident (Table 2) from the in vitro enhance- ment (46%) of the activity of monoamine oxidase, a marker enzyme of outer membrane, presumably as a result of better accessibility to external substrate. This effect, caused by free radicals, was partially counter- acted by the presence of superoxide dismutase. The effect of oxidative mechanisms on the cytochrome ox- idase system, intimately associated with inner mem- brane showed 27% inhibition due to membrane dam- age. Presence of SOD in the system accorded protec- tion to a great extent and the degree of inhibition of the enzyme was reduced to 1% (Table 2). Glutathione per- oxidase was also inhibited (73%) by radical stress and this was partially reversed by superoxide dismutase.

In order to study the direct effects of peroxidative

Table 1. Optical density at 520 nm.

System 0 min 5 rain 10 min

Mitochondria + CaC12 0.986 + 0.055 0.688 + 0.046 0.624 + 0.038 Mitochondria + CaCI2 + hypoxanthine + xanthine oxidase 0.988 _+ 0.062 0.610 _+ 0.041 0.515 +_ 0.028 Mitochondria + CaCI 2 + hypoxanthine + xanthine oxidase + SOD 0.970 + 0.088 0.630 +_ 0.022 0.590_+ 0.044

Values are arithmatic mean of four determinations + S.D. in each case.

.7- d (:5.6.

.5-

.4-

.3 0

' I

.9

.8-

"l]me (in mts.)

Fig. 1. Swelling pattern of fleshly isolated rat liver mitochondria in the presence of 25/xM (0---0) , 50/xM (O---O), 75txM (x---x) and 100 tzM (A---A) tert-butyl hydroperoxide.

13

1

. 2 " ' 1 ' I

o ;. s Time (in rots.)

Fig. 2. t-BHP (75/xM) induced swelling (O---O) of rat liver mitochon- dria. Effects of 25/xM (0---0), 50/xM (x---x) and 100/zM (A---A) ruthenium red on t-BHP induced swelling.

intermediates, exogenously added tert-butyl hydrope- roxide (t-BHP) was used. t-BHP was also found to cause swelling of mitochondria in vitro in a concentra- tion dependent manner (Fig. 1). 75/xM t-BHP was found to be the optimum concentration to cause swell- ing. This was abolished in the presence of varying con- centrations of ruthenium red, a calcium uniport block- er, suggesting relationship between oxidative damage and Ca 2+ fluxes (Fig. 2). Further, dithiothreitol could also protect mitochondria in vitro against oxidative damage (Fig. 3), 0.5 mM DTT causing complete pre- vention of swelling due to t-BHP. This indirectly in- dicates the oxidising effect of t-BHP is on -SH groups of proteins responsible for maintaining structural and functional integrity of mitochondria.

Table 2. Effect of superoxide generating and scavenging system in vitro on

Discussion

Calcium chloride (2mM) was used as swelling agent and swelling of mitochrondria was recorded spectro- photometrically as decrease in optical density at 520 nm at 1rain intervals for a period of 10min. Since this concentration used in the in vitro studies is much higher than in vivo, direct correlation of the present data with the situation in intact tissue is not attempted. This is justifiable because the purpose of the study was to correlate the swelling of mitochondria with oxidative stress. Our earlier studies [14] showed that formation of active oxygen species caused swelling and peroxidative decomposition of rat liver mitochondria along with dis-

some mitochondrial enzymes.

Enzyme Control (a) Assay system + 02 generating system (b)

Assay system+ O; generating system+ SOD (c)

Glutatione peroxidase Monoamine oxidase Cytochrome c oxidase

0.189 ± 0.020 0.028 ± 0.008 2.030 _+ 0.140

0.05± 0.002 (73%I)* 0.052± 0.03 (46%E)** 1.489_+ 0.089 (27%)*

0.097_+ 0.003 (49%I)* 0.040_+ 0.001 (30%E)* 2.015_+ 0.011 (1%I)*

Values of enzyme activities are expressed/mg mitochondrial protein. Values are arithmetic mean 4- S.D. of four determinations in each case % inhibition (I) and enhancement (E) of the enzyme activity is given in parenthesis. p is (b) vs (a) and (c) vs (b); *P< 0.01; **p< 0.001.

14

.9-

.8-

c5 .7- C5

.6-

• 5 , I I I I I

0 1 2 3 & 5

Time (in mrs.)

Fig. 3. Effects of 0.5 mM (0---0) and 1 mM (x-- -x) dithiothreitol on t-BHP (75/xM) induced swelling (O---O) of rat liver mitochondria.

tinct disorganisation of ultrastructure [15]. Supplemen- tation with free radical scavengers such as superoxide dismutase, methionine, histidine and tryptophan ac- corded considerable protection to the organelle. The likely relation between active oxygen species and al- tered calcium transport indicated in the present study is supported by the potentiation of the free radical in- duced mitochondrial damage by Ca 2+ [16]. According to Beatrice et al. [17] the non specific Ca 2+ release as a consequence of loss of inner membrane impermeability is paralleled by collapse of the membrane potential, proton uptake and large amplitude swelling of mito- chondria. The main manifestation of toxicity can be regarded as the peroxidative decomposition of polyun- saturated fatty acids and the ensuing structural and functional alterations in plasma membrane and orga- nelles. This, in turn could lead to metabolic changes caused by altered substrate and cofactor accessibility, functional changes in enzymes and undesirable fluxes of cations.

The effect of oxidants on mitochondrial structure and function seems to be due to the hydroperoxide medi- ated Ca 2+ efflux as evident from the present data. Any formation of ADP-ribose from oxidised niacinamide coenzymes, which combine with protein and regulate Ca 2+ fluxes [18], cannot be decided at present. The alterations in calcium will effect diverse biological pro- cesses through regulation of ATP and cations, nucleo-

tides and protein kinases, swelling of mitochondria, and through calmodulin binding and its regulation of metab- olism and signal transduction [19]. Since Ca 2+ gradients between the outside and inside of the cell and between mitochondria and cytoplasm are among the steepest, they will be very vulnerable to toxic stress. Such mecha- nisms may be involved in the mitochondrial damage by oxidative toxicants. Mitochondrial structure and func- tion have been known to be the action of many xeno- biotics [20, 21] and mitochondrial changes in toxicity have been well studied [22, 23]. The loss of mitochon- drial membrane potential has been shown to be one of the critical determinants for the development of irre- versible cell injury. The decrease in mitochondrial GSH caused by Ca ionophore A23187 [24] support this alter- ations in Ca 2+ homeostasis as a cause or effect of mem- brane damage as suggested by the studies of Orrenius [25] and the impaired Ca 2+ sequestration by mitochon- dria in the presence of H202 or t-BHP, also indicate similar correlations. The observations of Reed etal. [26] with blockers of mitochondrial Ca 2+ uniport and of Malis and Bonventre [16] on the oxygen radical medi- ated disturbance of Ca 2+ homeostasis also suggest the role of Ca 2+ in cell injury involving mitochondrial ho- meostasis. The depletion of ATP by oxidants and the behaviour of Ca 2+ fluxes in CCI 4 toxicity and the en- hanced formation of arachidonate cascade [27] condi- tions along with the involvement of phospholipases, protein kinases and messenger system can include a cascade of events leading to toxicity. Thus xenobiotic free radicals---> oxidative s t ress~ membrane chang- es--~ Ca alterations could be a central vicious chain in toxicosis, at least in the unspecific common events. The present data has provided useful information contrib- uting towards the validity of such hypothesis.

Acknowledgement

Thanks are due to Prof. P.K. Ray, Director Industrial Toxicology Research Centre, Lucknow, for his interest in this work.

References

1. Rasmussen H, Barrett P, Smallwood J, Bollag W, Islaes C: Calcium ion as intracellular messenger and cellular toxin. Envi- ron Hlth Perspect 84: 17-25, 1990

2. Pounds JG, Rosen JF: Cellular Ca 2+ homeostasis and Ca 2+ medi-

ated cell processess as critical targets for toxicant action conceptual and methodological pitfalls. Toxicol Appl Pharmacol 94: 331-341, 1988 3. Cheung JY, Leaf A, Bonventre JV: Mitochondrial function and

intracellular calcium in anoxic cardiac myocytes. Am J Physiol 250: C18-C25, 1986

4. Bondy SC, Kumulainen H: Intracellular calcium as an index of neurotoxic damage. Toxicology 49: 35-41, 1988

5. Moore L, Schoenberg DR, Rochelle ML: Impact of halogenated compounds on calcium homeostasis in hepatocytes. Environ Hlth Perspect 84: 149-153, 1990

6. Orrenius S, McConkey D J, Nicotera P: Role of calcium in ox- idative cell injury. In: GL Fiskum (eds.) Cell calcium metabo- lism. Plenum Publishing Corporation, New York, 451-461, 1989

7. Masaki N, Kyle ME, Farber JL: Tert-butyl hydroperoxide kills cultured hepatocytes by peroxidising membrane lipids. Arch Bio- chem Biophys 269: 390-399, 1989

8. Mustafa MF: Augmentation of mitochondrial oxidation in lung tissue during recovery of animal from acute ozone exposure. Arch Biochem Biophys 165: 531-538, 1974

9. Tabor CW, Tabor H, Rosenthal MS: Purification of amine ox- idase from beef plasma. J Biol Chem 208: 645-666, 1954

10. Splittgerber AG, Tappel AI L: Steady state and presteady state kinetics of rat liver selenium glutathione peroxidase. J Biol Chem 254: 980%9813, 1979

1I. Cooperstein SJ, Lazarrow A: A microspectrophotometric meth- od for the determination of cytochrome oxidase. J Biol Chem 189: 665-670, 1951

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

13. Fisher RA: Statistical methods for research works, l l th ed. Edin- burgh, U.K., Oliver & Boyd, 1950

14. Kakkar P, Mehrotra S, Viswanathan PN: Interrelationship of active oxygen species, calcium dynamics and biomembrane func- tions as central mechanism in cytotoxicity. In: Biomembranes in health and disease, vol. 1. A.M. Kidwai, R.K. Upreti & P.K. Ray (eds.) Today & Tomorrow's Printers & Publishers, New Delhi, pp 205-215, 1990

15. Mehrotra S, Kakkar P, Viswanathan PN: Mitochondrial damage

15

by active oxygen species in vitro. Free Rad Biol Med 10: 277-285, 1991

16. Malls CD, Bonventre JV: Mechanism of calcium potentiation of oxygen free radical injury to renal mitochondria. A model post- ischemic and toxic mitochondrial damage. J Biol Chem 261: 4201-4208, 1986

17. Beatrice MC, Palmer JW, Pfeiffer DR: The relationship between mitochondrial membrane permeability membrane potential and the retention of calcium by mitochondria. J Biol Chem 255: 8633-8671, 1980

18. Richter C, Frei B: Calcium release from mitochondria induced by pro-oxidants. Free Radic Biol Med 4: 365-375, 1988

19. Chaudhary A, Rubin RP: Mediators of Ca 2+ dependent secre- tion. Environ Hlth Perspect 84: 35-39, 1990

20. Lemasters JL, Gores GJ, Nieminen AL, Dawson TL, Wray BE, Herman B: Multiparameter digitised video microscopy of toxic and hypoxic injury m single cells. Environ. Hlth Perspect 84: 83-94, 1990

21. Knobloch LM, Bondin GA, Harkin JM: Use of submitochon- drial particles for prediction of chemical toxicity in man. Bull Environ Contam Toxicol 44: 661-668, 1990

22. Masaki N, Kyle ME, Serroni A, Farber JL: Mitochondrial dam- age as a mechanism of cell injury in the killing of cultured hepato- cytes by tert-butylhydroperoxide. Arch Biochem 270: 672-680, 1990

23. Haubensticker ME, Meir AG, Mancy KH, Brabec MJ: Rapid toxicity testing based on yeast respiratory activity. Bull Environ Contain Toxicol 44: 669-674, 1990

24. Olofsdottir K, Pascoe GA, Reed D J: Mitochondrial glutathione status during Ca 2+ induced injury to isolated hepatocytes. Arch Biochem Biophys 226--235, 1988

25. Bellomo G, Orrenius S: Altered thiol and calcium homeostasis in oxidative hepatocellular injury. Hepatology 5: 876-882, 1985

26. Reed D J, Pascoe GA, Thomas CE: Extracellular calcium effects on cell viability and thiol homeostasis. Environ Hlth Pcrspect 84: 113-120, 1990

27. Bonventre JV: Calcium in renal cells. Modulation of calcium dependent activation of phospholipase A2. Environ Hlth Per- spect 84: 155-162, 1990