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Differences in lipid peroxidation of rat liver rough and smooth microsomes
91
Biochimica et Biophysics Acta. 750 ( 1983) 9 l-97 Elsevier Biomedical Press
BBA 51288
DIF~~NCES MICROSOMES THOMAS
IN LIPID PERO~DATION
P.A. DEVASAGAYAM,
Biology and Agriculture (Received February (Revised manuscript
CHOLIPARAMBIL
OF RAT LIVER ROUGH AND SMOOTH
K. PIJSHPENDRAN
and JACOB
EAPEN
Division, Bhabha Atomic Research Centre, Bombay 400 085 (lndia)
22nd, 1982) received
Key words: Lipid peroxidation;
September
29th. 1982)
NA DPH; Ascorbate;
(Rut live‘r microsome)
The rough and smooth microsomes of rat liver show signific~t differences in lipid ~roxidation induced by both NADPH and ascorbate. The parameters studied include kinetics, response towards cofactors and sensitivity to inhibitors. Smooth microsomes are more prone to lipid peroxidation with increasing concentrations of NADPH, Fe3+, ascorbate and Fe’+, and are more susceptible to inhibitors than rough microsomes. Smooth microsomes also contain higher amounts of ascorbic acid, NADPH cytochrome c reductase and total lipids, besides possessing a higher degree of unsa~ration in lipids, a11of which promote lipid ~roxidation. Our results suggest that, although smooth microsomes are more sensitive to lipid peroxidation, they are compensated for by being more sensitive to inhibitors of lipid peroxidation.
of information on the lipid-peroxidizing activities of the two fractions. The present communication reports, for the first time, differences between rough and smooth microsomes in their lipid-peroxidizing systems. The parameters studied include time-course, kinetics, response towards co-factors, content of factors which regulate lipid peroxidation and sensitivity to inhibitors with respect to NADPHand ascorbate-induced lipid-peroxidizing systems.
Peroxidation of membrane lipids is of recent research interest because of its prominent role in biochemical toxicology [l] and the ageing process [Z]. Microsomal lipid peroxidation has been extensively studied [ 1,3,4]. NADPH-induced lipid peroxidation has been shown to require ferric iron chelated by ADP [3] or ferrous iron [4] as co-factors, and to be mediated by the enzyme NADPH cytochrome c reductase [5]. The non-enzymatic ascorbate-induced lipid peroxidation requires ferrous iron for its optimal activity 161. Both these types of lipid peroxidation are affected by the lipid and fatty acid composition of microsomal membranes [7,8]. Though the two fractions of hepatic endoplasmic reticulum, i.e., the rough and smooth, have been reported to differ significantly in their content of major lipid constituents [9], enzymes [ 10,l l] and other parameters [ 1 I] besides their response to xenobiotics [ 1I-141, there is a paucity 0~5-2740/83/0~-0000/~~3.00
0 1983 Elsevier Biomedical
Materials and Methods Preparation and characterizafion of wricrosomai fractions Female Wistar rats (250-280 g), starved overnight, were killed by cervical dislocation and livers were excised. Finely minced livers were homogenized in 5 vol. of 0.25 M sucrose and centrifuged at 10000 x g for 30 min. The supernatant was again centrifuged at the same speed to ensure sedimentation of mitochondria and other contamiPress
92
nants. Fractionation of rough and smooth microsomes from the postmitochondrial supernatant was in principle similar to the procedure followed by DePierre and Dallner [ 111. Postmitochondrial supernatant (5 ml) was carefully layered over two discontinuous gradients, the lower one consisting of 4 ml of 15 mM CsCl in 1.3 M sucrose and the upper one of 2 ml of 15 mM CsCl in 0.6 M sucrose. The gradients were spun at 105 000 x g for 90 min in a Beckman L5-65B ultracentrifuge, using a Ti 50 rotor. Separated rough and smooth microsomes were carefully removed and washed free of sucrose in 0.15 M Tris-HCl buffer, pH 7.4 (spun twice at 120000 X g for 45 min). The purity of the microsomal fractions was ascertained by estimating parameters suggested by earlier workers [10,11,15]. The estimations of NADPH cytochrome c reductase, cytochrome P-450, glucose-6phosphatase [ 131, cholesterol [ 161, RNA [ 171, phospholipids [ 181, 5’-nucleotidase, glucose-6-phosphate dehydrogenase [ 191 and succinate dehydrogenase [20] were carried out using standard procedures. Estimation of factors related to lipid peroxidation Other characteristics of microsomes, related to lipid peroxidation, such as content of total lipids [18], ascorbic acid, reduced glutathione [21], (Ytocopherol[22] and degree of unsaturation in lipids
[23] were also estimated. Microsomal protein was estimated [24] facilitate dilution of samples (5 mg protein/ml).
to
In vitro lipid peroxidation The incubation mixture (0.5 ml) for the estimation of ascorbate-induced lipid peroxidation contained 50 pl microsomes, 50 PM FeSO, and 1 mM KC1 in 0.15 M Tris-HCl buffer, pH 7.4, and ascorbic acid (sodium salt, 0.4 mM) which was added to start the reaction. For the NADPH-induced system, instead of FeSO,, 50 PM FeCl, and 4 mM ADP were included and 0.4 mM NADPH was added to start the reaction, in place of ascorbic acid, For studying the kinetics in the ascorbate-induced system, 10-1000 PM ascorbate and IO-250 PM FeSO, were used whereas in the NADPH-induced system 10-1000 PM NADPH and lo-250 PM FeCl,, which gave linear responses, were used. Estimation of lipid peroxidation After incubation at 37°C the malonaldehyde formed was estimated immediately by thiobarbituric acid (0.5% 2-thiobarbituric acid/lo% trichloroacetic acid/2 mM EDTA/0.63 M hydrochloric acid) reaction [25,26]. Malonaldehyde standard was prepared by the acid hydrolysis of tetraethoxypropane. Endogenous malonaldehyde content was estimated by boiling freshly prepared micro-
TABLE 1 SOME CHARACTERISTICS
OF RAT LIVER
ROUGH
Values are mean+S.E. from five animals. Estimations h P -c0.01, compared to the rough microsomes.
AND SMOOTH were carried
MICROSOMES
out as described
Parameters
Rough
microsomes
Protein content (mg/g liver) NADPH cytochrome c reductase (units/min per mg protein) Cytochrome P-450 (nmol/mg protein) Glucose-6-phosphatase (pg P,/min per mg protein) Total lipids (pg/mg protein) Cholesterol (pg/mg protein) RNA/phospholipid ratio Degree of unsaturation in lipids (pmol equiv./mg protein) Ascorbic acid (pg/mg protein) a-Tocopherol (pg/mg protein) Reduced glutathione (pg/mg protein) 5’-Nucleotidase (pg Pi /min per mg protein) Glucose-6-phosphate dehydrogenase (units/min per mg protein) Succinate dehydrogenase (units/min per mg protein)
6.44+ 0.18 13.61 + 1.25 1.25k 0.17 4.06+_ 0.26 220 *21 48 +6 1.02 0.92+ 0.06 0.65+ 0.15 3.26i 0.18 1.48+ 0.41 0.08+0.015 0.10* 0.02 not detected
in Materials
and
Methods.
Smooth
a P < 0.05and
microsomes
4.93 i 0.26 h 18.05+ 1.30” 2.22+ 0.33 h 6.37 + 0.42 h 382 +41 h 97 fgh 0.16 1.48+ 0.14 h 1.74? 0.38’ 2.70+ 0.19 4.68+ 1.21 A 0.17* 0.016’ 0.13+ 0.02 not detected
93
Differences in factors which regulate lipid peroxida-
somes with thiobarbituric acid reagent without incubation. The effect of inhibitors was studied by including 0.2 mM of the respective inhibitors in the incubation mixture. All values were corrected for endogenous lipid peroxide content and lipid peroxidation without added co-factors. Data were analysed using Student’s r-test.
ti0fl
Rough and smooth microsomes also differ from each other in the content of factors which regulate lipid peroxidation (Table I). Factors which enhance lipid peroxidation, such as NADPH cytochrome c reductase, total lipids, degree of unsaturation in lipids and ascorbic acid, are present in larger proportions in smooth microsomes than in rough. Smooth microsomes also have more reduced glutathione, an inhibitor of lipid peroxidation. The microsomal fractions contain similar amounts of &ocopherol, another inhibitor of lipid peroxidation.
Results Purity of microsomal fractions
Table I presents data on the purity of rough and smooth microsomes, in addition to some characteristics related to lipid peroxidation. Succinate the marker enzyme for dehydrogenase, mitochondria, is not present in measurable quantities in these fractions. However, these fractions contain traces of 5’-nucleotidase (marker enzyme for plasma membranes, under 1%) and glucose-6phosphate dehydrogenase (marker for cytosol, under 3%). Among the microsomal constituents, smooth microsomes contain significantly higher amounts of two components of the mixed function oxidase system, namely NADPH cytochrome c reductase and cytochrome P-450, besides glucose6-phosphatase and cholesterol. Rough microsomes, on the other hand, have more microsomal protein and a higher RNA/phospholipid ratio.
TABLE
differences fractions
in content and response of m~crosomai
Smooth microsomes show significantly more endogenous malonaldehyde content and lipid peroxidation without added co-factors. Smooth microsomes are also more prone to lipid peroxidation with added Fe*+ and NADPH (Table 11). Time-course of iipid peroxidation
With 0.4 mM NADPH, the time-course of NADPH-induced lipid pero~dation is almost similar in rough and smooth microsomes (Fig. I). However, with 1.6 mM NADPH smooth micro-
II
DIFFERENCES IN LIPID SMOOTH MICROSOMES
PEROXIDATION
AND RESPONSES
TO ADDED
CO-FACTORS
IN RAT LIVER
ROUGH
AND
Malonaldehyde was estimated in freshly prepared rough and smooth microsomes by the thiobarbituric acid method, without incubation, and the content is expressed as nmol of malonaIdehyde/mg protein. The complete systems were as described in Materials and Methods. Lipid peroxidation values are expressed as nmol malonaldehyde/mg protein after incubation at 37OC for 1 h. All values are mean i S. E. from five animals. a P < 0.05, compared to the rough microsomes. Parameters Malonaldehyde content Lipid peroxidation Without co-factors With complete NADPH-induced With only NADPH With only Fe3+/ADP With complete ascorbate-induced With only ascorbate With only Fe2+
Rough
microsomes
4J39+0.13
system
system
I .42 f 0.27 12.05 f 0.65 2.13+0.48 6.38k 1.02 20.12+ 1.31 5.11+0.65 1.78+0.50
Smooth
microsomes
5.67kO.23
=
3.97+0.71 a 18.26j; 1.46a 3.98 * 0.30 a 6.52 f 0.68 23.40 + 2.22 6. IO rt 0.43 9.79 f 0.74 a
60
Ascorbate-LP
Minutes
C,,M NADPH I -’
of incubation
Fig. 1. Time course of NADPHand ascorbate-induced malonaldehyde (MD) production by hepatic rough (closed symbols) and smooth (open symbols) microsomes. Values represent meanfS.E. from six animals. Incubations were carried out at 37°C using complete systems, as described in Materials and Methods. In NADPH-induced lipid peroxidation (NADPH-LP), either 0.4 mM NADPH (0, 0.0.4 mM) or 1.6 mM NADPH (A, a, 1.6 mM) was added to start the reaction. In ascorbate-induced lipid peroxidation system (Ascorbate-LP), 0.4 mM ascorbate was used to initiate lipid peroxidation. Fig. 2. Lineweaver-Burk plot of NADPH concentration versus rate of malonaldehyde (MD) formation after 1 h incubation at 37°C in hepatic rough (0) and smooth (0) microsomes (mean of six animals). V,,,. 0.28 nmol malonaldehyde/mg protein per min; apparent K,, 526 nM NADPH for rough microsomes. V,,,.. 0.42 nmol malonaldehyde/mg protein per min; apparent K,,. 14 nM NADPH for smooth microsomes.
somes show a considerable increase in the lipid peroxidation, unlike rough microsomes. With 0.4 mM ascorbate, lipid peroxidation is induced earlier in smooth microsomes but eventually the malonaldehyde formed is similar in both the fractions (Fig. 1). 1.6 mM ascorbate, however, inhibits lipid peroxidation uniformly in both (data not included). Kinetics of NADPH-induced lipid peroxidation Double-reciprocal Lineweaver-Burk plots of NADPH (Fig. 2) or Fe 3+ (Fig. 3) concentrations versus the corresponding rates of malonaldehyde evolution after 1 h incubation were different in the two microsomal fractions. Smooth microsomes are more sensitive to lipid peroxidation as a function of NADPH concentration, with low apparent K, Rough microsomes have lower and high V,,,. apparent K, and V,,, values with increasing concentrations of Fe3+. Kinetics of ascorbate-induced lipid peroxidution Lineweaver-Burk plots of ascorbate (Fig. 4) and Fe*+ (Fig. 5) concentrations versus malonalde-
hyde evolution after 1 h incubation show that smooth microsomes, having higher V,_ values, are more prone to lipid peroxidation. Beyond certain
-0.05
0
+o.os (,uM
.
co.1
Fe3+)-’
Fig. 3. Lineweaver-Burk plot of Fei+ concentration versus rate of malonaldehyde (MD) formation in the NADPH-induced system in hepatic rough (0) and smooth (0) microsomes (mean of six animals). V,_, 0.23 nmol malonaldehyde/mg protein per min; apparent K,. 25 pM Fe’+ for rough microsomes. VmdX, 0.36 nmol malonaldehyde/mg protein per min; apparent K,. 77 pM Fe7+ for smooth microsomes.
I
-0.02
-0.01
0
to 02
+0.01
ht.4 AscorbateI-'
(,uM Fez+)-' Fig. 5. Lineweaver-Burk plot of Fe*+ concentration versus rate of malonaldehyde (MD) formation in the ascorbate-induced system in hepatic rough (0) and smooth (0) microsomes (mean protein of six animals). I&,, 0.25 nmol malonaldehyde/mg
Fig. 4. Lineweaver-Burk plot of ascorbate concentration versus rate of malonaldehyde (MD) formation in hepatic rough (0) and smooth (0) microsomes (mean of six animals). V,,,,,. 0.26 nmol malonaldehyde/mg protein per min; apparent K,, 59 PM ascorbate for rough microsomes. I’,,,x, 0.45 nmol malonaldehyde/mg protein per min; apparent K,, 83 PM ascorbate for smooth microsomes.
per min; apparent K,, 45 fiM Fe*+ for rough microsomes. V,,,, 0.34 nmol malonaldehyde/mg protein per min; apparent K,, 49 pM Fe *+ for smooth microsomes.
Discussion
concentrations, ascorbic acid (1 mM) and FeSO, (0.25 mM) caused inhibition of lipid peroxidation in both the fractions.
Lipid peroxidation involving hepatic microsomal membranes is of great importance due to the many vital roles played by the endoplasmic reticulum. Studies on microsomes have invariably been carried out with total microsomes which contain, besides rough and smooth endoplasmic reticulum, Golgi vesicles, plasma membranes and other contaminants [27]. The rough and smooth microsomes also differ from each other in the level and distribution of enzymes [ lo,1 11, and in lipid
Sensitivity to inhibitors All the five inhibitors studied, i.e., EDTA, CoClz, aniline, ethylmorphine and benz[ alpyrene, significantly inhibit lipid peroxidation in both the microsomal fractions, the higher percent inhibition being always associated with smooth microsomes in both NADPH- and ascorbate-induced systems (Table III).
TABLE EFFECT DUCED
III OF INHIBITORS OF LIPID LIPID PEROXIDATION
PEROXIDATION
ON RAT LIVER
MICROSOMAL
NADPH-
AND ASCORBATE-IN-
Values are averages of five animals and represent percent inhibition of lipid peroxidation after incubation at 37’C for complement of co-factors as described in Materials and Methods, having 0.2 mM of the respective inhibitor. Lipid peroxidation
NADPH-induced Ascorbate-induced
Microsomal fractions
Rough Smooth Rough Smooth
Percent
inhibition
I h with full
with:
EDTA
COCI >
Aniline
Ethylmorphine
Benz[ alpyrene
71.72 85.14 87.75 96.79
46.11 66.67 82.86 96.43
47.12 76.74 67.87 85.36
40.79 52.78 47.15 63.21
46.65 48.40 62.86 71.79
96
fractions [9], besides several physical characteristics [ 1I], all of which can significantly affect the extent of lipid peroxidation f28]. Our data on the characteristics of microsomal fractions show that rough and smooth microsomes have negligible contamination with mitochondria, plasma membranes and cytosol. The isolated rough and smooth microsomes differ from each other with respect to markers [ lo,! 1,151 such as mixedfunction oxidases, glucose-6-phosphatase, cholesterol and RNA/phospholipid ratio. A heat-sensitive lipid peroxidation, dependent upon NADPH, ADP and Fe”+ ]3], mediated by NADPH cytochrome c reductase [5] and with a possible role in the turnover of unsaturated fatty acids [29] has been demonstrated in rat hepatic microsomes. Ascorbate-induced lipid peroxidation also occurs in microsomal membranes [ 1,6] and the amount of ascorbic acid in the liver has been shown to be a major factor which controls the extent of microsomal lipid peroxidation 1301. The present study shows that significant differences exist in the NADPH-induced lipid peroxidation of rough and smooth microsomes. In rat liver, the majority of NADPH-requiring enzymes are more in smooth microsames than in the rough [lo]. Our results also show that NADPH cytochrome c reductase activity, total lipid content and degree of unsaturation in lipids are more in smooth microsomes. This may explain the better response of smooth microsomes to added NADPH as well as to increasing concentrations of NADPH and Fe’+. Although both rough and smooth endoplasmic reticuli of hepatocytes are exposed equally to ascorbic acid present in the cytosol [21], added co-factors in the ascorbate-induced, lipid-peroxidizing systems have different effects on rough and smooth microsomes. It has been shown recently that ascorbic acid in combination with vitamin E and other factors can either enhance or inhibit lipid peroxidation [30]. Although cytocopherol content is similar in the microsomal fractions, ascorbic acid is more in smooth microsomes. Hence, a delicate balance between these two factors is important in regulating lipid peroxidation (301, the presence of excess amount of ascorbic acid in smooth microsomes may be responsible for its higher sensitivity to lipid per-
oxidation. Although smooth microsomes contain more reduced glutathione, an inhibitor of lipid peroxidation [21,28], there is minimal manifestation of inhibition. The difference in ascorbate and NADPH-induced lipid peroxidation and the endogenous malonaldehyde content are likely to be due to differences in the delicate balance between several factors, such as lipid content, degree of unsaturation in lipids, ferric and ferrous iron, CCtocopherol, ascorbic acid and gluthatione peroxidase system, besides other characteristics which can affect the extent of lipid peroxidation [ 1,21,28]. Since smooth microsomes are richer in components of the cytochrome P-450-dependent, microsomal electron-transport chain. their capacity as iron-reducing systems may also be an important factor which makes smooth microsomes more prone to lipid peroxidation [ 10,11,28]. Although kinetic parameters conforming to the classical ~~chaelis-Kenton kinetics pertaining to details of reaction mechanisms may not be important in lipid-peroxidising systems, they can be used to compare the different lipid-peroxidizing activities [31]. The results of our kinetic studies suggest significant differences in the enzymic and nonenzymic processes reponsible for lipid peroxidation in hepatic rough and smooth microsomes. Inhibitors of lipid peroxidation used in the present study are more effective on smooth microsomes than on rough, irrespective of their mechanism of action [6,28,32]. Smooth microsomes, though more susceptible to lipid peroxidation, are perhaps protected by being more sensitive to inhibitors. Xenobiotic metabolism mediated by the monooxygenase system is more in smooth microsomes [IO] and the present results show that drugs which serve as substrates for this system can cause a preferential inhibition of lipid peroxidation, thereby protecting the membranes. References 1 Bus. J.S. and Gibson, J.E. (1979) in Reviews in Biochemical Toxicology (Hodgson, E., Bend, J.R. and Philpot, R.M., eds.). Vol. I pp. 125-149, Elsevier/North-Holland. New York 2 Tappel, A.L., Fletcher, B. and Deamer. D.W. (1973) J. Gerontol. 28, 415-424 3 Ilochstein, P. and Ernster, L. (1963) Biochem. Biophys. Res. ~ommun. 12. 388-394
97
4 Kornbrust, D.J. and Mavis, R.D. (1980) Mol. Pharmacol. 17,408-414 5 Pederson, T.C. and Aust, SD. (1972) Boichem. Biophys. Res. Commun. 48, 789-795 6 Ottolenghi, A. (1958) Arch. B&hem. Biophys. 79, 355-363 7 Rowe, L. and Wills, E.D. (1976) Biochem. Pharmacol. 25, 175-179 8 Hammer, C.T. and Wills, E.D. (1978) Biochem. J. 174. 585-593 9 Glaumann, H. and Dallner, G. (1968) J. Lipid Res. 9, 720-729 10 Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., Fouts, J.R. and Mannering, G.J. (1969) Microsomes and Drug Oxidations, Academic Press, New York 11 DePierre, D.W. and Dallner, G. (1975) Biochim. Biophys. Acta 415, 41 l-472 12 Holtzman, J.L., Gram, T.E., Grigan, P.L. and Gillette, J.R. (1968) Biochem. J. 110, 407-412 13 Devasagayam, T.P.A., Pushpendran, C.K. and Eapen, J. (1979) Biochem. Pharmacol. 28, 1731-1734 14 Devasagayam, T.P.A., Pushpendran, C.K. and Eapen, J. (1980) Ind. J. Exp. Biol. 18, 627-630 15 Lloyd, D. and Poole, R.K. (1979) in Techniques in Metabolic Research, PT-1 (Kornberg, H.L., Metcalfe, J.C., Northcote, D.H., Pogson, C.I. and Tipton, K.F., eds.), pp. 19-46, Elsevier/North-Holland, Limerick 16 Pearson, S., Stern, S. and McGavack, T.H. (1953) Anal. Chem. 25, 813-814 17 Munro, H.N. and Fleck, A. (1967) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 14, pp. 113- 176, Interscience. New York
18 Pushpendran, C.K. and Eapen, J. (1973) Biol. Neonate 23, 303-313 19 Bergmeyer, H.U. (1963) Methods in Enzymatic Analysis, Academic Press, New York 20 Caplan, A.I. and Greenwalt. J.W. (1968) J. Cell Biol. 36, 15-31 21 Wright, J.R., Colby, H.D. and Miles, P.R. (1981) Arch. Biochem. Biophys. 206, 296-304 22 Taylor, S.L., Lamden. M.P. and Tappel, A.L. (1976) Lipids 11. 530-539 23 Barrett, M.C. and Horton, A.A. (1975) Biochem. Sot. Trans. 3, 124-126 24 Lowry, O.H., Rosebrough, N.J.. Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 25 Hunter, F.E., Gebicki, J.M.. Hoffsten, P.E., Weinstein, J. and Scott, A. (1963) J. Biol. Chem. 238, 828-835 26 Mimnaugh, E.G., Trush, M.A., Ginsburg, E., Hirokata, Y. and Gram, T.E. (1981) Toxicol. Appl. Pharmacol. 61, 313-325 27 Gram, T.E. (1974) Methods Enzymol. 31, 225-237 28 Vladimirov, Y.A., Olenov. V.I., Suslova, T.B. and Cheremisina, Z.P. (1980) Adv. Lipid Res. 17, 173-249 29 Pfeifer, P.M. and McCay, P.B. (1972) J. Biol. Chem. 247. 676336769 30 Leung, H.W., Vang, M.J. and Mavis, R.D. (1981) Biochim. Biophys. Acta 664, 266-272 31 Willis, R.J. and Recknagel, R.O. (1979) Toxicol. Appl. Pharmacol. 47, 89-94 32 Miles, P.R., Wright, J.R., (1980) Biochem. Pharmacol.
Bowman. L. and 29, 565-570
Colby,
H.D.
Biochimica et Biophysics Acta. 750 ( 1983) 9 l-97 Elsevier Biomedical Press
BBA 51288
DIF~~NCES MICROSOMES THOMAS
IN LIPID PERO~DATION
P.A. DEVASAGAYAM,
Biology and Agriculture (Received February (Revised manuscript
CHOLIPARAMBIL
OF RAT LIVER ROUGH AND SMOOTH
K. PIJSHPENDRAN
and JACOB
EAPEN
Division, Bhabha Atomic Research Centre, Bombay 400 085 (lndia)
22nd, 1982) received
Key words: Lipid peroxidation;
September
29th. 1982)
NA DPH; Ascorbate;
(Rut live‘r microsome)
The rough and smooth microsomes of rat liver show signific~t differences in lipid ~roxidation induced by both NADPH and ascorbate. The parameters studied include kinetics, response towards cofactors and sensitivity to inhibitors. Smooth microsomes are more prone to lipid peroxidation with increasing concentrations of NADPH, Fe3+, ascorbate and Fe’+, and are more susceptible to inhibitors than rough microsomes. Smooth microsomes also contain higher amounts of ascorbic acid, NADPH cytochrome c reductase and total lipids, besides possessing a higher degree of unsa~ration in lipids, a11of which promote lipid ~roxidation. Our results suggest that, although smooth microsomes are more sensitive to lipid peroxidation, they are compensated for by being more sensitive to inhibitors of lipid peroxidation.
of information on the lipid-peroxidizing activities of the two fractions. The present communication reports, for the first time, differences between rough and smooth microsomes in their lipid-peroxidizing systems. The parameters studied include time-course, kinetics, response towards co-factors, content of factors which regulate lipid peroxidation and sensitivity to inhibitors with respect to NADPHand ascorbate-induced lipid-peroxidizing systems.
Peroxidation of membrane lipids is of recent research interest because of its prominent role in biochemical toxicology [l] and the ageing process [Z]. Microsomal lipid peroxidation has been extensively studied [ 1,3,4]. NADPH-induced lipid peroxidation has been shown to require ferric iron chelated by ADP [3] or ferrous iron [4] as co-factors, and to be mediated by the enzyme NADPH cytochrome c reductase [5]. The non-enzymatic ascorbate-induced lipid peroxidation requires ferrous iron for its optimal activity 161. Both these types of lipid peroxidation are affected by the lipid and fatty acid composition of microsomal membranes [7,8]. Though the two fractions of hepatic endoplasmic reticulum, i.e., the rough and smooth, have been reported to differ significantly in their content of major lipid constituents [9], enzymes [ 10,l l] and other parameters [ 1 I] besides their response to xenobiotics [ 1I-141, there is a paucity 0~5-2740/83/0~-0000/~~3.00
0 1983 Elsevier Biomedical
Materials and Methods Preparation and characterizafion of wricrosomai fractions Female Wistar rats (250-280 g), starved overnight, were killed by cervical dislocation and livers were excised. Finely minced livers were homogenized in 5 vol. of 0.25 M sucrose and centrifuged at 10000 x g for 30 min. The supernatant was again centrifuged at the same speed to ensure sedimentation of mitochondria and other contamiPress
92
nants. Fractionation of rough and smooth microsomes from the postmitochondrial supernatant was in principle similar to the procedure followed by DePierre and Dallner [ 111. Postmitochondrial supernatant (5 ml) was carefully layered over two discontinuous gradients, the lower one consisting of 4 ml of 15 mM CsCl in 1.3 M sucrose and the upper one of 2 ml of 15 mM CsCl in 0.6 M sucrose. The gradients were spun at 105 000 x g for 90 min in a Beckman L5-65B ultracentrifuge, using a Ti 50 rotor. Separated rough and smooth microsomes were carefully removed and washed free of sucrose in 0.15 M Tris-HCl buffer, pH 7.4 (spun twice at 120000 X g for 45 min). The purity of the microsomal fractions was ascertained by estimating parameters suggested by earlier workers [10,11,15]. The estimations of NADPH cytochrome c reductase, cytochrome P-450, glucose-6phosphatase [ 131, cholesterol [ 161, RNA [ 171, phospholipids [ 181, 5’-nucleotidase, glucose-6-phosphate dehydrogenase [ 191 and succinate dehydrogenase [20] were carried out using standard procedures. Estimation of factors related to lipid peroxidation Other characteristics of microsomes, related to lipid peroxidation, such as content of total lipids [18], ascorbic acid, reduced glutathione [21], (Ytocopherol[22] and degree of unsaturation in lipids
[23] were also estimated. Microsomal protein was estimated [24] facilitate dilution of samples (5 mg protein/ml).
to
In vitro lipid peroxidation The incubation mixture (0.5 ml) for the estimation of ascorbate-induced lipid peroxidation contained 50 pl microsomes, 50 PM FeSO, and 1 mM KC1 in 0.15 M Tris-HCl buffer, pH 7.4, and ascorbic acid (sodium salt, 0.4 mM) which was added to start the reaction. For the NADPH-induced system, instead of FeSO,, 50 PM FeCl, and 4 mM ADP were included and 0.4 mM NADPH was added to start the reaction, in place of ascorbic acid, For studying the kinetics in the ascorbate-induced system, 10-1000 PM ascorbate and IO-250 PM FeSO, were used whereas in the NADPH-induced system 10-1000 PM NADPH and lo-250 PM FeCl,, which gave linear responses, were used. Estimation of lipid peroxidation After incubation at 37°C the malonaldehyde formed was estimated immediately by thiobarbituric acid (0.5% 2-thiobarbituric acid/lo% trichloroacetic acid/2 mM EDTA/0.63 M hydrochloric acid) reaction [25,26]. Malonaldehyde standard was prepared by the acid hydrolysis of tetraethoxypropane. Endogenous malonaldehyde content was estimated by boiling freshly prepared micro-
TABLE 1 SOME CHARACTERISTICS
OF RAT LIVER
ROUGH
Values are mean+S.E. from five animals. Estimations h P -c0.01, compared to the rough microsomes.
AND SMOOTH were carried
MICROSOMES
out as described
Parameters
Rough
microsomes
Protein content (mg/g liver) NADPH cytochrome c reductase (units/min per mg protein) Cytochrome P-450 (nmol/mg protein) Glucose-6-phosphatase (pg P,/min per mg protein) Total lipids (pg/mg protein) Cholesterol (pg/mg protein) RNA/phospholipid ratio Degree of unsaturation in lipids (pmol equiv./mg protein) Ascorbic acid (pg/mg protein) a-Tocopherol (pg/mg protein) Reduced glutathione (pg/mg protein) 5’-Nucleotidase (pg Pi /min per mg protein) Glucose-6-phosphate dehydrogenase (units/min per mg protein) Succinate dehydrogenase (units/min per mg protein)
6.44+ 0.18 13.61 + 1.25 1.25k 0.17 4.06+_ 0.26 220 *21 48 +6 1.02 0.92+ 0.06 0.65+ 0.15 3.26i 0.18 1.48+ 0.41 0.08+0.015 0.10* 0.02 not detected
in Materials
and
Methods.
Smooth
a P < 0.05and
microsomes
4.93 i 0.26 h 18.05+ 1.30” 2.22+ 0.33 h 6.37 + 0.42 h 382 +41 h 97 fgh 0.16 1.48+ 0.14 h 1.74? 0.38’ 2.70+ 0.19 4.68+ 1.21 A 0.17* 0.016’ 0.13+ 0.02 not detected
93
Differences in factors which regulate lipid peroxida-
somes with thiobarbituric acid reagent without incubation. The effect of inhibitors was studied by including 0.2 mM of the respective inhibitors in the incubation mixture. All values were corrected for endogenous lipid peroxide content and lipid peroxidation without added co-factors. Data were analysed using Student’s r-test.
ti0fl
Rough and smooth microsomes also differ from each other in the content of factors which regulate lipid peroxidation (Table I). Factors which enhance lipid peroxidation, such as NADPH cytochrome c reductase, total lipids, degree of unsaturation in lipids and ascorbic acid, are present in larger proportions in smooth microsomes than in rough. Smooth microsomes also have more reduced glutathione, an inhibitor of lipid peroxidation. The microsomal fractions contain similar amounts of &ocopherol, another inhibitor of lipid peroxidation.
Results Purity of microsomal fractions
Table I presents data on the purity of rough and smooth microsomes, in addition to some characteristics related to lipid peroxidation. Succinate the marker enzyme for dehydrogenase, mitochondria, is not present in measurable quantities in these fractions. However, these fractions contain traces of 5’-nucleotidase (marker enzyme for plasma membranes, under 1%) and glucose-6phosphate dehydrogenase (marker for cytosol, under 3%). Among the microsomal constituents, smooth microsomes contain significantly higher amounts of two components of the mixed function oxidase system, namely NADPH cytochrome c reductase and cytochrome P-450, besides glucose6-phosphatase and cholesterol. Rough microsomes, on the other hand, have more microsomal protein and a higher RNA/phospholipid ratio.
TABLE
differences fractions
in content and response of m~crosomai
Smooth microsomes show significantly more endogenous malonaldehyde content and lipid peroxidation without added co-factors. Smooth microsomes are also more prone to lipid peroxidation with added Fe*+ and NADPH (Table 11). Time-course of iipid peroxidation
With 0.4 mM NADPH, the time-course of NADPH-induced lipid pero~dation is almost similar in rough and smooth microsomes (Fig. I). However, with 1.6 mM NADPH smooth micro-
II
DIFFERENCES IN LIPID SMOOTH MICROSOMES
PEROXIDATION
AND RESPONSES
TO ADDED
CO-FACTORS
IN RAT LIVER
ROUGH
AND
Malonaldehyde was estimated in freshly prepared rough and smooth microsomes by the thiobarbituric acid method, without incubation, and the content is expressed as nmol of malonaIdehyde/mg protein. The complete systems were as described in Materials and Methods. Lipid peroxidation values are expressed as nmol malonaldehyde/mg protein after incubation at 37OC for 1 h. All values are mean i S. E. from five animals. a P < 0.05, compared to the rough microsomes. Parameters Malonaldehyde content Lipid peroxidation Without co-factors With complete NADPH-induced With only NADPH With only Fe3+/ADP With complete ascorbate-induced With only ascorbate With only Fe2+
Rough
microsomes
4J39+0.13
system
system
I .42 f 0.27 12.05 f 0.65 2.13+0.48 6.38k 1.02 20.12+ 1.31 5.11+0.65 1.78+0.50
Smooth
microsomes
5.67kO.23
=
3.97+0.71 a 18.26j; 1.46a 3.98 * 0.30 a 6.52 f 0.68 23.40 + 2.22 6. IO rt 0.43 9.79 f 0.74 a
60
Ascorbate-LP
Minutes
C,,M NADPH I -’
of incubation
Fig. 1. Time course of NADPHand ascorbate-induced malonaldehyde (MD) production by hepatic rough (closed symbols) and smooth (open symbols) microsomes. Values represent meanfS.E. from six animals. Incubations were carried out at 37°C using complete systems, as described in Materials and Methods. In NADPH-induced lipid peroxidation (NADPH-LP), either 0.4 mM NADPH (0, 0.0.4 mM) or 1.6 mM NADPH (A, a, 1.6 mM) was added to start the reaction. In ascorbate-induced lipid peroxidation system (Ascorbate-LP), 0.4 mM ascorbate was used to initiate lipid peroxidation. Fig. 2. Lineweaver-Burk plot of NADPH concentration versus rate of malonaldehyde (MD) formation after 1 h incubation at 37°C in hepatic rough (0) and smooth (0) microsomes (mean of six animals). V,,,. 0.28 nmol malonaldehyde/mg protein per min; apparent K,, 526 nM NADPH for rough microsomes. V,,,.. 0.42 nmol malonaldehyde/mg protein per min; apparent K,,. 14 nM NADPH for smooth microsomes.
somes show a considerable increase in the lipid peroxidation, unlike rough microsomes. With 0.4 mM ascorbate, lipid peroxidation is induced earlier in smooth microsomes but eventually the malonaldehyde formed is similar in both the fractions (Fig. 1). 1.6 mM ascorbate, however, inhibits lipid peroxidation uniformly in both (data not included). Kinetics of NADPH-induced lipid peroxidation Double-reciprocal Lineweaver-Burk plots of NADPH (Fig. 2) or Fe 3+ (Fig. 3) concentrations versus the corresponding rates of malonaldehyde evolution after 1 h incubation were different in the two microsomal fractions. Smooth microsomes are more sensitive to lipid peroxidation as a function of NADPH concentration, with low apparent K, Rough microsomes have lower and high V,,,. apparent K, and V,,, values with increasing concentrations of Fe3+. Kinetics of ascorbate-induced lipid peroxidution Lineweaver-Burk plots of ascorbate (Fig. 4) and Fe*+ (Fig. 5) concentrations versus malonalde-
hyde evolution after 1 h incubation show that smooth microsomes, having higher V,_ values, are more prone to lipid peroxidation. Beyond certain
-0.05
0
+o.os (,uM
.
co.1
Fe3+)-’
Fig. 3. Lineweaver-Burk plot of Fei+ concentration versus rate of malonaldehyde (MD) formation in the NADPH-induced system in hepatic rough (0) and smooth (0) microsomes (mean of six animals). V,_, 0.23 nmol malonaldehyde/mg protein per min; apparent K,. 25 pM Fe’+ for rough microsomes. VmdX, 0.36 nmol malonaldehyde/mg protein per min; apparent K,. 77 pM Fe7+ for smooth microsomes.
I
-0.02
-0.01
0
to 02
+0.01
ht.4 AscorbateI-'
(,uM Fez+)-' Fig. 5. Lineweaver-Burk plot of Fe*+ concentration versus rate of malonaldehyde (MD) formation in the ascorbate-induced system in hepatic rough (0) and smooth (0) microsomes (mean protein of six animals). I&,, 0.25 nmol malonaldehyde/mg
Fig. 4. Lineweaver-Burk plot of ascorbate concentration versus rate of malonaldehyde (MD) formation in hepatic rough (0) and smooth (0) microsomes (mean of six animals). V,,,,,. 0.26 nmol malonaldehyde/mg protein per min; apparent K,, 59 PM ascorbate for rough microsomes. I’,,,x, 0.45 nmol malonaldehyde/mg protein per min; apparent K,, 83 PM ascorbate for smooth microsomes.
per min; apparent K,, 45 fiM Fe*+ for rough microsomes. V,,,, 0.34 nmol malonaldehyde/mg protein per min; apparent K,, 49 pM Fe *+ for smooth microsomes.
Discussion
concentrations, ascorbic acid (1 mM) and FeSO, (0.25 mM) caused inhibition of lipid peroxidation in both the fractions.
Lipid peroxidation involving hepatic microsomal membranes is of great importance due to the many vital roles played by the endoplasmic reticulum. Studies on microsomes have invariably been carried out with total microsomes which contain, besides rough and smooth endoplasmic reticulum, Golgi vesicles, plasma membranes and other contaminants [27]. The rough and smooth microsomes also differ from each other in the level and distribution of enzymes [ lo,1 11, and in lipid
Sensitivity to inhibitors All the five inhibitors studied, i.e., EDTA, CoClz, aniline, ethylmorphine and benz[ alpyrene, significantly inhibit lipid peroxidation in both the microsomal fractions, the higher percent inhibition being always associated with smooth microsomes in both NADPH- and ascorbate-induced systems (Table III).
TABLE EFFECT DUCED
III OF INHIBITORS OF LIPID LIPID PEROXIDATION
PEROXIDATION
ON RAT LIVER
MICROSOMAL
NADPH-
AND ASCORBATE-IN-
Values are averages of five animals and represent percent inhibition of lipid peroxidation after incubation at 37’C for complement of co-factors as described in Materials and Methods, having 0.2 mM of the respective inhibitor. Lipid peroxidation
NADPH-induced Ascorbate-induced
Microsomal fractions
Rough Smooth Rough Smooth
Percent
inhibition
I h with full
with:
EDTA
COCI >
Aniline
Ethylmorphine
Benz[ alpyrene
71.72 85.14 87.75 96.79
46.11 66.67 82.86 96.43
47.12 76.74 67.87 85.36
40.79 52.78 47.15 63.21
46.65 48.40 62.86 71.79
96
fractions [9], besides several physical characteristics [ 1I], all of which can significantly affect the extent of lipid peroxidation f28]. Our data on the characteristics of microsomal fractions show that rough and smooth microsomes have negligible contamination with mitochondria, plasma membranes and cytosol. The isolated rough and smooth microsomes differ from each other with respect to markers [ lo,! 1,151 such as mixedfunction oxidases, glucose-6-phosphatase, cholesterol and RNA/phospholipid ratio. A heat-sensitive lipid peroxidation, dependent upon NADPH, ADP and Fe”+ ]3], mediated by NADPH cytochrome c reductase [5] and with a possible role in the turnover of unsaturated fatty acids [29] has been demonstrated in rat hepatic microsomes. Ascorbate-induced lipid peroxidation also occurs in microsomal membranes [ 1,6] and the amount of ascorbic acid in the liver has been shown to be a major factor which controls the extent of microsomal lipid peroxidation 1301. The present study shows that significant differences exist in the NADPH-induced lipid peroxidation of rough and smooth microsomes. In rat liver, the majority of NADPH-requiring enzymes are more in smooth microsames than in the rough [lo]. Our results also show that NADPH cytochrome c reductase activity, total lipid content and degree of unsaturation in lipids are more in smooth microsomes. This may explain the better response of smooth microsomes to added NADPH as well as to increasing concentrations of NADPH and Fe’+. Although both rough and smooth endoplasmic reticuli of hepatocytes are exposed equally to ascorbic acid present in the cytosol [21], added co-factors in the ascorbate-induced, lipid-peroxidizing systems have different effects on rough and smooth microsomes. It has been shown recently that ascorbic acid in combination with vitamin E and other factors can either enhance or inhibit lipid peroxidation [30]. Although cytocopherol content is similar in the microsomal fractions, ascorbic acid is more in smooth microsomes. Hence, a delicate balance between these two factors is important in regulating lipid peroxidation (301, the presence of excess amount of ascorbic acid in smooth microsomes may be responsible for its higher sensitivity to lipid per-
oxidation. Although smooth microsomes contain more reduced glutathione, an inhibitor of lipid peroxidation [21,28], there is minimal manifestation of inhibition. The difference in ascorbate and NADPH-induced lipid peroxidation and the endogenous malonaldehyde content are likely to be due to differences in the delicate balance between several factors, such as lipid content, degree of unsaturation in lipids, ferric and ferrous iron, CCtocopherol, ascorbic acid and gluthatione peroxidase system, besides other characteristics which can affect the extent of lipid peroxidation [ 1,21,28]. Since smooth microsomes are richer in components of the cytochrome P-450-dependent, microsomal electron-transport chain. their capacity as iron-reducing systems may also be an important factor which makes smooth microsomes more prone to lipid peroxidation [ 10,11,28]. Although kinetic parameters conforming to the classical ~~chaelis-Kenton kinetics pertaining to details of reaction mechanisms may not be important in lipid-peroxidising systems, they can be used to compare the different lipid-peroxidizing activities [31]. The results of our kinetic studies suggest significant differences in the enzymic and nonenzymic processes reponsible for lipid peroxidation in hepatic rough and smooth microsomes. Inhibitors of lipid peroxidation used in the present study are more effective on smooth microsomes than on rough, irrespective of their mechanism of action [6,28,32]. Smooth microsomes, though more susceptible to lipid peroxidation, are perhaps protected by being more sensitive to inhibitors. Xenobiotic metabolism mediated by the monooxygenase system is more in smooth microsomes [IO] and the present results show that drugs which serve as substrates for this system can cause a preferential inhibition of lipid peroxidation, thereby protecting the membranes. References 1 Bus. J.S. and Gibson, J.E. (1979) in Reviews in Biochemical Toxicology (Hodgson, E., Bend, J.R. and Philpot, R.M., eds.). Vol. I pp. 125-149, Elsevier/North-Holland. New York 2 Tappel, A.L., Fletcher, B. and Deamer. D.W. (1973) J. Gerontol. 28, 415-424 3 Ilochstein, P. and Ernster, L. (1963) Biochem. Biophys. Res. ~ommun. 12. 388-394
97
4 Kornbrust, D.J. and Mavis, R.D. (1980) Mol. Pharmacol. 17,408-414 5 Pederson, T.C. and Aust, SD. (1972) Boichem. Biophys. Res. Commun. 48, 789-795 6 Ottolenghi, A. (1958) Arch. B&hem. Biophys. 79, 355-363 7 Rowe, L. and Wills, E.D. (1976) Biochem. Pharmacol. 25, 175-179 8 Hammer, C.T. and Wills, E.D. (1978) Biochem. J. 174. 585-593 9 Glaumann, H. and Dallner, G. (1968) J. Lipid Res. 9, 720-729 10 Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., Fouts, J.R. and Mannering, G.J. (1969) Microsomes and Drug Oxidations, Academic Press, New York 11 DePierre, D.W. and Dallner, G. (1975) Biochim. Biophys. Acta 415, 41 l-472 12 Holtzman, J.L., Gram, T.E., Grigan, P.L. and Gillette, J.R. (1968) Biochem. J. 110, 407-412 13 Devasagayam, T.P.A., Pushpendran, C.K. and Eapen, J. (1979) Biochem. Pharmacol. 28, 1731-1734 14 Devasagayam, T.P.A., Pushpendran, C.K. and Eapen, J. (1980) Ind. J. Exp. Biol. 18, 627-630 15 Lloyd, D. and Poole, R.K. (1979) in Techniques in Metabolic Research, PT-1 (Kornberg, H.L., Metcalfe, J.C., Northcote, D.H., Pogson, C.I. and Tipton, K.F., eds.), pp. 19-46, Elsevier/North-Holland, Limerick 16 Pearson, S., Stern, S. and McGavack, T.H. (1953) Anal. Chem. 25, 813-814 17 Munro, H.N. and Fleck, A. (1967) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 14, pp. 113- 176, Interscience. New York
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Colby,
H.D.