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Dimethyl sulfoxide elicited increase in cytochrome oxidase activity in rat liver mitochondria in vivo and in vitro
Ckem.-Biol Inten~ctions, 66 (1988) 147-155 Elsevier ScientificPublishers Ireland Ltd.
147
DIMETHYL S U L F O X I D E ELICITED INCREASE IN CYTOCHROME OXIDASE A C T I V I T Y IN R A T L I V E R M I T O C H O N D R I A IN VIVO AND IN VITRO
SHYAMALD. DESAI, K.G. CHETTY and D.S. PRADHAN* Biochemistry Di~q~sio~ Bhabha Atomic Research Centre, Bombay-400 085 ¢IndiaJ
(Received August 7th, 1987) (Revision received January 7th, 19881 (Accepted January 21st, 1988)
SUMMARY A single intraperitoneal injection of dimethyl sulfoxide (275 rag/100 g body wt.) to rats stimulated cytochrome oxidase activity in liver mitochondria 2 5-fold. The enzyme activity remained at this level for as long as 5 days postinjection. There was however only 10.5°/o increase in the content of cytochromes a and a 3 ( a s determined spectrophotometrically) in the same period in response to DMSO injection. The addition of either DMSO or dimethyl sulfate (a metabolite of DMSO) to isolated liver mitochondria also caused 2--3-fold increase in cytochrome oxidase activity. The results indicate that enhancement in cytochrome oxidase activity in liver mitochondria after administration of DMSO to rats is on account of activation of cytochrome oxidase caused by structural alterations in mitochondrial membranes rather than de novo synthesis of cytochrome oxidase. Key
words:
DMSO effects
--
Cytochrome oxidase activity
-
Liver
mitochondria
INTRODUCTION Dimethyl sulfoxide (DMSO) is an important chemical compound used in several biological investigations involving both plants and animals [1]. It is a dipolar aprotic substance endowed with unique physical, chemical and solvent properties [2]. Physiologically DMSO has been demonstrated to be a potent diuretic [3] and respiratory stimulant [4] which is known to increase oxygen availability, and it also stabilizes lysosomal membranes [5,6]. Because of its physiological, pharmacological/clinical properties, DMSO has been used *To whomcorrespondence should be sent. 0009-2797/88/$03.50 © 1988 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
148 as a therapeutic agent [7]. It is also used as a vehicle for several medicinal agents and drugs to penetrate through the skin [3,8]. DMSO is well tolerated in the mammalian body in reasonable doses without reported deleterious toxicological effects [9,10]. DMSO has also been used as a solvent for testing a number of chemicals for their mutagenic/carcinogenic potentials in various test systems [11--13]. Although many reports are available on therapeutic and other applications of DMSO, practically nothing is known about the action of DMSO at the level of the subcellular organelles. In an earlier communication we reported the effect of DMSO in vivo on mitochondrial functions in rat liver [4]. It was shown that administration of a single dose of DMSO (275 mg/100 g body wt.) intraperitoneally to rats results in enhanced rates of respiration and uncoupling of oxidative phosphorylation in liver mitochondria from 2 h to 5 days after the administration [4]. These results have prompted a further study on the mechanism of action of DMSO on enzymes and components of respiratory chain in rat liver mitochondria. This communication presents studies on the in vivo and in vitro effect of DMSO on cytochrome oxidase activity and cytochrome contents of rat liver mitochondria. MATERIALS AND METHODS
Chemicals DMSO was obtained from Cambrian Chemicals, U.K., dimethyl sulfate (DMS) was from E. Merck, F.R.G., cytochrome (from horse heart, type III), digitonin and Triton X-100 were procured from Sigma Chemicals Company, U.S.A. All other chemicals used were of analar grade.
Animal experiments Male albino rats of Wistar strain, each weighing 100--125 g and fed on laboratory stock diet, were used. The animals were administered varying quantities of DMSO intraperitoneally and sacrificed at different time intervals. Diet and water were made available ad libitum to both control and DMSO-injected rats.
Isolation of mitochondria Mitochondria were isolated essentially as described by Katyare et al. [14]. In some experiments, isolated mitochondria were suspended in 0.25 M sucrose containing 0.5% digitonin (10--14 mg/ml) since this plant steroid is believed to activate cytochrome oxidase in the intact mitochondria [15].
Assay of cytochrome oxidase The cytochrome oxidase assay was carried out according to the procedure detailed by Smith [16]. Commercial cytochrome c was reduced according to the procedure of Wharton and Tzagaloff [17]. Thus 100 mg of cytochrome c was dissolved in approx. 3 ml of 0.01 M phosphate buffer (pH 7.0) and a small quantity of ascorbic acid (approx. 5 - 6 mg) was added to reduce cytochrome
149
c. The solution was then dialyzed overnight against 0.01 M phosphate buffer (pH 7.0). The resulting solution was diluted to 10 ml and stored frozen ( - 2 0 ° C ) in a stoppered tube. The extent of reduction of cytochrome c was ascertained by measuring the ratio of optical density of the solution at 550 nm to the optical density at 565 nm. A ratio of greater than 10 was observed. The assay of cytochrome oxidase activity followed by the rate of oxidation of reduced cytochrome c was performed using the reaction mixture containing (in a total volume of 3 ml): 0.3 ml of 0.1 M potassium phosphate buffer (pH 7.0); 0.21 ml of reduced cytochrome c; 2.48 ml of distilled water. The contents were mixed and optical density at 550 nm was measured against a cuvette containing 2.69 ml of water and 0.3 ml of potassium phosphate buffer (pH 7.0) as blank. The reaction was started by adding 10 ~l of mitochondrial suspension (with or without digitonin) and the rate of oxidation of cytochrome c was recorded continuously for a period of 1--1.5 min using Perkin-Elmer double-beam spectrometer model-126 in conjunction with a Hitachi recorder model QPD-54. At the end of the reaction the optical density of the completely oxidised cytochrome c was determined by adding a drop of saturated K3Fe(CN)6. Cytochrome oxidase was calculated according to Smith [16] and expressed as K/rain per mg protein.
Determination of contents of cytochromes The procedure followed was essentially that described by Katyare et al. [18] and Satav and Katyare [19]. To approx. 1 ml of mitochondrial suspension (approx. 10 mg mitochondrial proteins), 1 ml of 1 M potassium phosphate buffer (pH 7.4) was added, and mitochondria was solubilised by the addition of 0 . 3 - 0 . 5 ml of 10% (v/v) Triton X-100. The volume was made up to 6 ml with 0.25 M sucrose and thoroughly mixed. Samples in the reference cuvette was completely oxidised by the addition of a few crystals of KsFe(CN) e and those in the experimental cuvette were reduced by the addition of a few mg of sodium dithionate. Difference spectra of cytochromes were recorded by employing Perkin-Elmer double-beam spectrophotometer model-126 in conjunction with a Hitachi recorder model QPD-54. Samples were scanned from 650--500 nm with a slit of 0.5 or 2.0 ram. Contents of cytochromes were calculated by using wavelength pairs and estimation coefficients as described by Satav and Katyare [19].
Estimation of proteins Protein was estimated by the procedure of Lowry et al. [20]. RESULTS In the initial experiments dose-related effects of DMSO at 2 h post-injection were studied. As seen in Fig. 1 an injection of 275 mg of DMSO per 100 g body weight resulted in maximum stimulation in the liver mitochondrial cytochrome oxidase activity. From Fig. 2, it is seen that cytochrome oxidase
~
100
E
i SO
,ID
Ib
P r. o
i
2" o An,cunt
_, I 550 1100 o| OMSO a d m i n i s l e r e d (rag)
Fig. 1. Dose-dependent effects on mitochondrial cytochrome oxidase in rat liver. Rats each weighing 100--110 g were administered intraperitoneslly with various amounts of DMSO and sacrificed after 2 h for assaying cytochrome oxidase. Results shown are the means and S.E.M. of three independent experiments. For each experiment livers from two rats were pooled.
100 8C
~oso-
~=~0
o~ J=
v
5, ol
I
I,
J
I
~.
J
I_,
1
2
3
I.
5
6
7
Time alter DMSO injection (days)
J
12
Fig. 2. Time-course of DMSO-induced enhancement of mitochondrial cytochrome oxidase activity in rat liver. Rats each weighing 100--110 g were administered intraperitoneal]y with 0.25 ml of DMSO and sacrificed at the times indicated for assaying mitochondrial cytochrome oxidase. Results shown are the means and S.E.M. of three independent experiments. For each experiment livers from two rats were pooled.
*Not significant.
DMSO (275 mg/100 g body wt.)
None
Treatment
2 24
2
Post-injection interval (h)
0.1696 ± 0.008* 0.1792 ± 0.009 °
0.1617 ± 0.012
Cytochrome a + a s
0.2413 ± 0.005* 0.2447 ± 0.005*
0.2382 ± 0.006
Cytochrome b
Cytochromes (nmol/mg mitochondrial protein)
0.4446 ± 0.004* 0.4490 ± 0.005*
0.4380 ± 0.007
Cytochrome c + c 1
The cytochromes were determined in Triton X-100 solubilised mitochondria by difference spectra of reduced and oxidized cytochromes as described by Katyare et al. [19] and Satav and Katyare [20]. The results are represented as mean ± S.E.M. of three independent experiments. For each experiment livers from two rats were pooled.
LEVELS OF CYTOCHROMES IN LIVER MITOCHONDRIA FOLLOWING INTRAPERITONEAL INJECTION OF DMSO
TABLE I
152 activity was elevated from 2 h to 5 days post-injection of DMSO. The patterns of dose-related and time-course experiments of cytochrome oxidase activity, as seen in Figs. 1 and 2, respectively, are smaller to those for DMSO-caused increase in the respiration rates by liver mitochondria reported earlier [4]. In our earlier studies [4], significant changes were noted in state 3 and state 4 respiration rates and uncoupling of oxidative phosphorylation in rat liver mitochondria following DMSO injection. Since cytochrome oxidase activity in DMSO-treated rats showed significant elevation, studies were carried out to investigate DMSO-induced changes in the levels of cytochromes in liver mitochondria. As seen from Table I, at 24 h post-injection of DMSO, there was a marginal increase in the levels of cytochromes when compared with the control. The content of cytochrome a + a 3 showed 10.8% increase, whereas those of cytochromes b and c + c~ registered an increase of 2.70/0 and 2.50/0, respectively. The effect of addition of DMSO to liver mitochondria in vitro on cytochrome oxidase activity was investigated in further studies. When DMSO was added to mitochondria suspended in 0.25 M sucrose ( 1 0 - 1 4 mg protein/ ml 2.75 mg of DMSO was added) there was about 2-fold increase and with DMS approximately 2--5-fold increase in cytochrome oxidase activity (Table II). Digitonin, a plant steroid with surface acting properties, has been suggested to give full expression of cytochrome oxidase activity when added to isolated mitochondria and has therefore been used by some authors in the assay of cytochrome oxidase activity in mitochondria [15]. In the present experiment, digitonin addition did not cause any stimulation of cytochrome oxidase activity. A similar observation regarding the action of digitonin on cytochrome oxidase has also been reported by others [21,22]. Further, when DMSO was added to mitochondria suspended in the buffer containing digitonin, cytochrome oxidase activity did not exhibit any elevation (Table II). TABLE II CYTOCHROME OXIDASE ACTIVITY IN RAT LIVER MITOCHONDRIA: EFFECT OF IN VITRO ADDITION OF DMSO OR DMS The mitochondria were suspended in 0.25 M sucrose with or without digitonin. From this an aliquot of mitochondrial protein was added to reaction medium for assay of cytochrome oxidase activity as described by Smith [17]. The results are represented as a mean _+ S.E.M. of three independent experiments. For each experiment livers from two rats were pooled and mitochondria were isolated. *P ~ 0.005; **P ~ 0.001. Additions
Control DMSO DMS •Not significant.
Cytochrome oxidase activity (K/rain per mg protein) In presence of digitonin in assay mixture
In absence of digitonin in assay mixture
25.00 _+ 2.37 23.33 _+ 1.89" 27.18 _+ 1.35"
27.33 _ 1.42 54.29 _+ 4.69* 69.07 _+ 4.5**
153 DISCUSSION
The present studies reveal that the increase in cytochrome oxidase levels in the liver mitochondria following administration to rats are in consonance with the DMSO-induced changes in oxidative phosphorylation and respiratory rates reported earlier [4]. Cytochrome oxidase activity in isolated mitochondria was also stimulated upon the addition of DMSO or DMS. This would imply that the increase in enzyme activity by in vivo administration of DMSO could be a case of enzyme activation rather than de novo synthesis of enzyme protein. This presumption is borne out from the results showing only marginal increase in individual cytochrome components in DMSO-treated rats and the results of in vitro studies on the effect of DMSO or DMS on mitochondrial cytochrome oxidase (increased activity). It may be noted that the increase in cytochrome oxidase activity in the in vivo studies is similar in magnitude to 3-fold increase in cytochrome oxidase activity in vivo in DMSO-treated rat liver mitochondria. In the present studies the action of DMSO or DMS on mitochondrial cytochrome oxidase seems to be identical to the action of deoxycholate reported by Smith and Camerino [23]. An interesting observation in the present study pertains to the effect of DMSO on isolated mitochondria suspended in digitonin, a plant steroid with surface-activity, which has been suggested to give full expression of cytochrome oxidase activity [15]. DMSO is known to cause structural alterations in biomembranes and, in turn, various types of membrane-associated cellular processes [23--25]. The observed increase in cytochrome oxidase activity in mitochondria elicited by DMSO or its metabolites may indicate alterations in membrane properties. In the present investigation it has been observed that addition of DMSO or DMS to isolated mitochondria suspended in digitonin-containing buffer failed to augment cytochrome oxidase activity. However, the increase in cytochrome oxidase activity brought about by the addition of DMSO or DMS could not be reversed by subsequent addition of digitonin. Digitonin is a known membrane-acting agent [15]. Its inhibitory effect on the DMSO-induced activation of cytochrome oxidase in intact mitochondria suggests interesting differences between the modes of action by digitonin and DMSO on mitochondrial membrane. The role of mitochondrial membrane in the assembling of subunits of cytochrome oxidase is the subject of investigation in recent years [25-31]. Both DMSO and digitonin may hence prove to be useful agents in such investigations. REFERENCES 1 2 3
S.W. Jacob, E.E. Rosenbaum and D.C. Wood, in: Dimethyl Sulfoxide, Vol. 1, Basic Concepts of DMSO, Marcel Dekker, Inc., New York, 1971. N. Kharasch and B.S. Thyagarajan, Structural basis for biological activities of dimethyl sulfoxide, Ann. N.Y. Acad. Sci., 411 (1983) 391-403, S.W. Jacob, Pharmacology of DMSO, in: S.W. Jacob, E. Rosenbaum and D.C. Wood (Eds.),
154
4
5 6
7 8
9 10
11 12 13 14
15 16 17 18
19 20 21 22 23 24 25 26 27
Dimethyl Sulfoxide, Vol. 1, Basic Concepts of DMSO, Marcel Dekker, Inc., New York, 1971, pp. 99-- 112. S.S. Mhatre, K.G. Chetty and D.S. Pradhan, Uncoupling of oxidative phosphorylation in rat liver mitochondria following the administration of dimethyl sulphoxide, Bioehem. Biophys. Res. Commun., 110 (1983) 325-331. M.J. Ashwood-Smith, Radioprotective and cryoprotective properties of dimethyl sulfoxide in cellular systems, Ann. N.Y. Acad. Sci., 141 (1967) 45-62. J.W. Finney, H.C. Urschel, G.A. Balla, G.J. Race, B.E. Jay, H.P. Pingree, A.L. Dorman and J.T. Mallams, Protection of the ischemic heart with DMSO alone and DMSO with hydrogen peroxide, Ann. N.Y. Acad. Sci., 141 (1967) 231-241. S.W. Jacob, M. Biechel and R.J. Herschler, Dimethyl sulfoxide (DMS0): A new concept in pharmacotherapy, Curr. Ther. Res., 6 (1964) 134-135. J.C. de la Torre, H.M. Kawanaga, D.W. Rowed, C.M. Johnson, D.J. Goode, K. Kajihara and S. Mullom, Dimethyl sulfoxide in central nervous system trauma, Ann. N.Y. Acad. Sci., 243 (1975) 362--389. R.I). Brobyn, The human toxicology of dimethyl sulfoxide, Ann. N.Y. Acad. Sci., 243 (1975) 497- 506. M.M. Mason, Toxicology of DMSO in animals, in: S.W. Jacob, E. Rosenbaum and D.C. Wood (Eds.), Dimethyl Sulfoxide, Vol. 1, Basic Concepts of DMS0, Marcel Dekker, Inc., New York, 1971, pp. 113--131. K. Pasupathy and D.S. Pradhan, Exthrome chemical mutagen sensitivity of respiratory adaptation in yeast, Mutat. Res, 80 (1981) 65--74. R.K. Bhattacharya, P.F. Firozi and V.S. Aboobaker, Factors modulating the formation of DNA adduct by aflatoxin B~ in vitro, Carcinogenesis, 5 (1984) 359-1362. • R.A. Coulomke, D.W. Sbelton and J.E. Nixon, Comparative mutagenicity of aflatoxins using a S a l m w n e l l ~ r o n t hepatic enzyme activation system, Carcinogenesis, 3 (1982) 1261-1264. S.S. Katyare, P. Fatterpaker and A. Sreenivasan, Effect of 2,4-dinitrophenol (DNP) on oxidative phosphorylation in rat liver mitochondria, Arch. Biochem. Biophys., 144 (1971) 209215. P. Borst, GJ.C.M. Ruttenburg and A.M. Kroon, Mitoehondrial DNA. 1. Preparation and properties of DNA from chick liver, Biochim. Biophys. Acta, 149 (1967) 140-- 155. L. Smith, Spectrophotometric assay of cytochrome c oxidase, Methods Bioehem. Anal., 2 (1955) 427-- 434. D.C. Wharton and A. Tzagaloff, Cytochrome oxidase from beef heart mitochondria, Methods Enzymol., 10 (1987) 245--250. S.S. Katyare, M.V. Joshi, P. Fatterpaker and A. Sreenivasan, Effect of thyroid deficiency on oxidative phosphorylation in rat liver, kidney and brain mitochondria, Arch. Biechem. Biophys., 182 (1977) 155--163. J.G. Satav and S.S. Katyare, Effect of experimental thyrotoxicosis on oxidative phosphoryiation in rat liver, kidney and brain mitochondria, Mol. Cell. Endocrinol., 23 (1982) 173-179. O.H. Lowry, NJ. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265--275. Z. Kaniuga, A. Gardes and M. Jakubiak, The effect of digitonin on the respiratory chain enzymes o f a heart-muscle preparation, Biochim. Biophys. Acta, 118 (1966) 9 - 1 8 . D.W. Haas and W.B. Elliott, Oxidative phosphorylation and respiratory control in digitonin fragments of beef-heart mitochondria, J. Biol. Chem., 23 (1963) 1132-1136. L. Smith and P.W. Camerino, The reaction of particle-bound cytechrome c oxidase with endogenous and exogenous cytochrome c, Biochemistry, 2 (1983) 1432-1439. W.H. Oliver, S.H. Shaw and J.E. Christian, Effect of dimethyl sulfoxide (DMSO) on the absorption of iron applied to piglets, Radiochem. Radioanal. Lett., 2 (1989) 279--283. H.I. Maibach and R.J. Feldmann, The effect of DMSO on percutaneous penetration of hydroeortisone and testosterone in man, Ann. N.Y. Aesd. Sci., 141 (1987) 423--427. R.A. Capaldi, Arrangement of proteins in the mitechondrial inner membrane, Biochim. Biophys. Acta, 694 (1982) 291--306. F.E.A.M. Verheul, J.C.P. Boonman, J.W. Draizer, A.O. Muijsers, D. Borden, G.E. Tarr and
155
28 29
30 31
E. Margoliash, The isolation of bovine-heart eytochrome c oxidase subunits: Dependence on phospholipid and cholate content, Biochim. Biophys. Acta, 548 (1979) 397--416. A. Azzi, Cytochrome c oxidase: Towards a clarification of its structure, interactions and mechanism, Biochim. Biophys. Aeta, 594 (1980) 231--252. N.N. Downer, N.C. Robinson and R-A. Capaldi, Characterization of a seventh different subunit of beef-heart eytochrome e oxidase. Similarities between the enzyme and that from other species, Biochemistry, 15 (1976) 2930--2936. B. Kadenbach and P. Merle, On the function of multiple subunits of cytoehrome c oxidase from higher eukaryotes, FEB8 Lett., 135 (1981) 1 - 1 1 . M. Klingenberg, Membrane protein oligomeric structure and transport function, Nature, 290 (1981) 449--454.
147
DIMETHYL S U L F O X I D E ELICITED INCREASE IN CYTOCHROME OXIDASE A C T I V I T Y IN R A T L I V E R M I T O C H O N D R I A IN VIVO AND IN VITRO
SHYAMALD. DESAI, K.G. CHETTY and D.S. PRADHAN* Biochemistry Di~q~sio~ Bhabha Atomic Research Centre, Bombay-400 085 ¢IndiaJ
(Received August 7th, 1987) (Revision received January 7th, 19881 (Accepted January 21st, 1988)
SUMMARY A single intraperitoneal injection of dimethyl sulfoxide (275 rag/100 g body wt.) to rats stimulated cytochrome oxidase activity in liver mitochondria 2 5-fold. The enzyme activity remained at this level for as long as 5 days postinjection. There was however only 10.5°/o increase in the content of cytochromes a and a 3 ( a s determined spectrophotometrically) in the same period in response to DMSO injection. The addition of either DMSO or dimethyl sulfate (a metabolite of DMSO) to isolated liver mitochondria also caused 2--3-fold increase in cytochrome oxidase activity. The results indicate that enhancement in cytochrome oxidase activity in liver mitochondria after administration of DMSO to rats is on account of activation of cytochrome oxidase caused by structural alterations in mitochondrial membranes rather than de novo synthesis of cytochrome oxidase. Key
words:
DMSO effects
--
Cytochrome oxidase activity
-
Liver
mitochondria
INTRODUCTION Dimethyl sulfoxide (DMSO) is an important chemical compound used in several biological investigations involving both plants and animals [1]. It is a dipolar aprotic substance endowed with unique physical, chemical and solvent properties [2]. Physiologically DMSO has been demonstrated to be a potent diuretic [3] and respiratory stimulant [4] which is known to increase oxygen availability, and it also stabilizes lysosomal membranes [5,6]. Because of its physiological, pharmacological/clinical properties, DMSO has been used *To whomcorrespondence should be sent. 0009-2797/88/$03.50 © 1988 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
148 as a therapeutic agent [7]. It is also used as a vehicle for several medicinal agents and drugs to penetrate through the skin [3,8]. DMSO is well tolerated in the mammalian body in reasonable doses without reported deleterious toxicological effects [9,10]. DMSO has also been used as a solvent for testing a number of chemicals for their mutagenic/carcinogenic potentials in various test systems [11--13]. Although many reports are available on therapeutic and other applications of DMSO, practically nothing is known about the action of DMSO at the level of the subcellular organelles. In an earlier communication we reported the effect of DMSO in vivo on mitochondrial functions in rat liver [4]. It was shown that administration of a single dose of DMSO (275 mg/100 g body wt.) intraperitoneally to rats results in enhanced rates of respiration and uncoupling of oxidative phosphorylation in liver mitochondria from 2 h to 5 days after the administration [4]. These results have prompted a further study on the mechanism of action of DMSO on enzymes and components of respiratory chain in rat liver mitochondria. This communication presents studies on the in vivo and in vitro effect of DMSO on cytochrome oxidase activity and cytochrome contents of rat liver mitochondria. MATERIALS AND METHODS
Chemicals DMSO was obtained from Cambrian Chemicals, U.K., dimethyl sulfate (DMS) was from E. Merck, F.R.G., cytochrome (from horse heart, type III), digitonin and Triton X-100 were procured from Sigma Chemicals Company, U.S.A. All other chemicals used were of analar grade.
Animal experiments Male albino rats of Wistar strain, each weighing 100--125 g and fed on laboratory stock diet, were used. The animals were administered varying quantities of DMSO intraperitoneally and sacrificed at different time intervals. Diet and water were made available ad libitum to both control and DMSO-injected rats.
Isolation of mitochondria Mitochondria were isolated essentially as described by Katyare et al. [14]. In some experiments, isolated mitochondria were suspended in 0.25 M sucrose containing 0.5% digitonin (10--14 mg/ml) since this plant steroid is believed to activate cytochrome oxidase in the intact mitochondria [15].
Assay of cytochrome oxidase The cytochrome oxidase assay was carried out according to the procedure detailed by Smith [16]. Commercial cytochrome c was reduced according to the procedure of Wharton and Tzagaloff [17]. Thus 100 mg of cytochrome c was dissolved in approx. 3 ml of 0.01 M phosphate buffer (pH 7.0) and a small quantity of ascorbic acid (approx. 5 - 6 mg) was added to reduce cytochrome
149
c. The solution was then dialyzed overnight against 0.01 M phosphate buffer (pH 7.0). The resulting solution was diluted to 10 ml and stored frozen ( - 2 0 ° C ) in a stoppered tube. The extent of reduction of cytochrome c was ascertained by measuring the ratio of optical density of the solution at 550 nm to the optical density at 565 nm. A ratio of greater than 10 was observed. The assay of cytochrome oxidase activity followed by the rate of oxidation of reduced cytochrome c was performed using the reaction mixture containing (in a total volume of 3 ml): 0.3 ml of 0.1 M potassium phosphate buffer (pH 7.0); 0.21 ml of reduced cytochrome c; 2.48 ml of distilled water. The contents were mixed and optical density at 550 nm was measured against a cuvette containing 2.69 ml of water and 0.3 ml of potassium phosphate buffer (pH 7.0) as blank. The reaction was started by adding 10 ~l of mitochondrial suspension (with or without digitonin) and the rate of oxidation of cytochrome c was recorded continuously for a period of 1--1.5 min using Perkin-Elmer double-beam spectrometer model-126 in conjunction with a Hitachi recorder model QPD-54. At the end of the reaction the optical density of the completely oxidised cytochrome c was determined by adding a drop of saturated K3Fe(CN)6. Cytochrome oxidase was calculated according to Smith [16] and expressed as K/rain per mg protein.
Determination of contents of cytochromes The procedure followed was essentially that described by Katyare et al. [18] and Satav and Katyare [19]. To approx. 1 ml of mitochondrial suspension (approx. 10 mg mitochondrial proteins), 1 ml of 1 M potassium phosphate buffer (pH 7.4) was added, and mitochondria was solubilised by the addition of 0 . 3 - 0 . 5 ml of 10% (v/v) Triton X-100. The volume was made up to 6 ml with 0.25 M sucrose and thoroughly mixed. Samples in the reference cuvette was completely oxidised by the addition of a few crystals of KsFe(CN) e and those in the experimental cuvette were reduced by the addition of a few mg of sodium dithionate. Difference spectra of cytochromes were recorded by employing Perkin-Elmer double-beam spectrophotometer model-126 in conjunction with a Hitachi recorder model QPD-54. Samples were scanned from 650--500 nm with a slit of 0.5 or 2.0 ram. Contents of cytochromes were calculated by using wavelength pairs and estimation coefficients as described by Satav and Katyare [19].
Estimation of proteins Protein was estimated by the procedure of Lowry et al. [20]. RESULTS In the initial experiments dose-related effects of DMSO at 2 h post-injection were studied. As seen in Fig. 1 an injection of 275 mg of DMSO per 100 g body weight resulted in maximum stimulation in the liver mitochondrial cytochrome oxidase activity. From Fig. 2, it is seen that cytochrome oxidase
~
100
E
i SO
,ID
Ib
P r. o
i
2" o An,cunt
_, I 550 1100 o| OMSO a d m i n i s l e r e d (rag)
Fig. 1. Dose-dependent effects on mitochondrial cytochrome oxidase in rat liver. Rats each weighing 100--110 g were administered intraperitoneslly with various amounts of DMSO and sacrificed after 2 h for assaying cytochrome oxidase. Results shown are the means and S.E.M. of three independent experiments. For each experiment livers from two rats were pooled.
100 8C
~oso-
~=~0
o~ J=
v
5, ol
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I,
J
I
~.
J
I_,
1
2
3
I.
5
6
7
Time alter DMSO injection (days)
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12
Fig. 2. Time-course of DMSO-induced enhancement of mitochondrial cytochrome oxidase activity in rat liver. Rats each weighing 100--110 g were administered intraperitoneal]y with 0.25 ml of DMSO and sacrificed at the times indicated for assaying mitochondrial cytochrome oxidase. Results shown are the means and S.E.M. of three independent experiments. For each experiment livers from two rats were pooled.
*Not significant.
DMSO (275 mg/100 g body wt.)
None
Treatment
2 24
2
Post-injection interval (h)
0.1696 ± 0.008* 0.1792 ± 0.009 °
0.1617 ± 0.012
Cytochrome a + a s
0.2413 ± 0.005* 0.2447 ± 0.005*
0.2382 ± 0.006
Cytochrome b
Cytochromes (nmol/mg mitochondrial protein)
0.4446 ± 0.004* 0.4490 ± 0.005*
0.4380 ± 0.007
Cytochrome c + c 1
The cytochromes were determined in Triton X-100 solubilised mitochondria by difference spectra of reduced and oxidized cytochromes as described by Katyare et al. [19] and Satav and Katyare [20]. The results are represented as mean ± S.E.M. of three independent experiments. For each experiment livers from two rats were pooled.
LEVELS OF CYTOCHROMES IN LIVER MITOCHONDRIA FOLLOWING INTRAPERITONEAL INJECTION OF DMSO
TABLE I
152 activity was elevated from 2 h to 5 days post-injection of DMSO. The patterns of dose-related and time-course experiments of cytochrome oxidase activity, as seen in Figs. 1 and 2, respectively, are smaller to those for DMSO-caused increase in the respiration rates by liver mitochondria reported earlier [4]. In our earlier studies [4], significant changes were noted in state 3 and state 4 respiration rates and uncoupling of oxidative phosphorylation in rat liver mitochondria following DMSO injection. Since cytochrome oxidase activity in DMSO-treated rats showed significant elevation, studies were carried out to investigate DMSO-induced changes in the levels of cytochromes in liver mitochondria. As seen from Table I, at 24 h post-injection of DMSO, there was a marginal increase in the levels of cytochromes when compared with the control. The content of cytochrome a + a 3 showed 10.8% increase, whereas those of cytochromes b and c + c~ registered an increase of 2.70/0 and 2.50/0, respectively. The effect of addition of DMSO to liver mitochondria in vitro on cytochrome oxidase activity was investigated in further studies. When DMSO was added to mitochondria suspended in 0.25 M sucrose ( 1 0 - 1 4 mg protein/ ml 2.75 mg of DMSO was added) there was about 2-fold increase and with DMS approximately 2--5-fold increase in cytochrome oxidase activity (Table II). Digitonin, a plant steroid with surface acting properties, has been suggested to give full expression of cytochrome oxidase activity when added to isolated mitochondria and has therefore been used by some authors in the assay of cytochrome oxidase activity in mitochondria [15]. In the present experiment, digitonin addition did not cause any stimulation of cytochrome oxidase activity. A similar observation regarding the action of digitonin on cytochrome oxidase has also been reported by others [21,22]. Further, when DMSO was added to mitochondria suspended in the buffer containing digitonin, cytochrome oxidase activity did not exhibit any elevation (Table II). TABLE II CYTOCHROME OXIDASE ACTIVITY IN RAT LIVER MITOCHONDRIA: EFFECT OF IN VITRO ADDITION OF DMSO OR DMS The mitochondria were suspended in 0.25 M sucrose with or without digitonin. From this an aliquot of mitochondrial protein was added to reaction medium for assay of cytochrome oxidase activity as described by Smith [17]. The results are represented as a mean _+ S.E.M. of three independent experiments. For each experiment livers from two rats were pooled and mitochondria were isolated. *P ~ 0.005; **P ~ 0.001. Additions
Control DMSO DMS •Not significant.
Cytochrome oxidase activity (K/rain per mg protein) In presence of digitonin in assay mixture
In absence of digitonin in assay mixture
25.00 _+ 2.37 23.33 _+ 1.89" 27.18 _+ 1.35"
27.33 _ 1.42 54.29 _+ 4.69* 69.07 _+ 4.5**
153 DISCUSSION
The present studies reveal that the increase in cytochrome oxidase levels in the liver mitochondria following administration to rats are in consonance with the DMSO-induced changes in oxidative phosphorylation and respiratory rates reported earlier [4]. Cytochrome oxidase activity in isolated mitochondria was also stimulated upon the addition of DMSO or DMS. This would imply that the increase in enzyme activity by in vivo administration of DMSO could be a case of enzyme activation rather than de novo synthesis of enzyme protein. This presumption is borne out from the results showing only marginal increase in individual cytochrome components in DMSO-treated rats and the results of in vitro studies on the effect of DMSO or DMS on mitochondrial cytochrome oxidase (increased activity). It may be noted that the increase in cytochrome oxidase activity in the in vivo studies is similar in magnitude to 3-fold increase in cytochrome oxidase activity in vivo in DMSO-treated rat liver mitochondria. In the present studies the action of DMSO or DMS on mitochondrial cytochrome oxidase seems to be identical to the action of deoxycholate reported by Smith and Camerino [23]. An interesting observation in the present study pertains to the effect of DMSO on isolated mitochondria suspended in digitonin, a plant steroid with surface-activity, which has been suggested to give full expression of cytochrome oxidase activity [15]. DMSO is known to cause structural alterations in biomembranes and, in turn, various types of membrane-associated cellular processes [23--25]. The observed increase in cytochrome oxidase activity in mitochondria elicited by DMSO or its metabolites may indicate alterations in membrane properties. In the present investigation it has been observed that addition of DMSO or DMS to isolated mitochondria suspended in digitonin-containing buffer failed to augment cytochrome oxidase activity. However, the increase in cytochrome oxidase activity brought about by the addition of DMSO or DMS could not be reversed by subsequent addition of digitonin. Digitonin is a known membrane-acting agent [15]. Its inhibitory effect on the DMSO-induced activation of cytochrome oxidase in intact mitochondria suggests interesting differences between the modes of action by digitonin and DMSO on mitochondrial membrane. The role of mitochondrial membrane in the assembling of subunits of cytochrome oxidase is the subject of investigation in recent years [25-31]. Both DMSO and digitonin may hence prove to be useful agents in such investigations. REFERENCES 1 2 3
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