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Changes in enzymes of hepatic rough and smooth microsomes during postnatal development and ageing of rats
Mechanisms of AgetngandDeveloprdent, 21 (1983) 365-375
365
Elsevier ScientificPublishers Ireland Ltd.
CHANGES IN ENZYMES OF HEPATIC ROUGH AND SMOOTH MICROSOMES DURING POSTNATAL DEVELOPMENT AND AGEING OF RATS THOMAS P.A. DEVASAGAYAM, CHOLIPARAMBIL K. PUSHPENDRAN and JACOB EAPEN Biology and Agriculture Division, Bhabha Atomic Research Centre, Bombay 400 085 (India)
(Received July 25th, 1982) (Revision received November2nd, 1982) SUMMARY Significant changes are observed in wet weight, microsomal protein content and enzymes of purified rough and smooth microsomes of liver during postnatal development and ageing of female Wistar rats. Protein content of total microsomes increases up to 15 days of age and remains steady during subsequent development, unlike that of rough and smooth microsomes which shows changes throughout the same period. Activities of cytochrome P-450, cytochrome bs and NADPH-cytochrome c reductase increase during the period of maturation and decline during senescence. The decrease during senescence is at different rates in the two microsomal fractions. Microsomal glucose-6-phosphatase, but not adenosine triphosphatase, shows a similar increase during development and decrease during senescence. K e y words: Monooxygenases, Rat liver microsome; Ageing
~TRODUCTION Age is an important factor which determines an individual's response to drugs. Children as well as old people are often more susceptible to the adverse effects of drugs [1-3]. This may be explained on the basis of the slow onset of the hepatic drugmetabolizing system during postnatal development and the decline in its activity during ageing [1-3]. Hepatic endoplasmic reticulum (ER), containing the mixed-function oxidase (MFO) system, is the major site of xenobiotic metabolism in mammals [4]. There are several, but conflicting, reports on the changes in rat hepatic ER during postnatal development [3,5,6] and ageing [2,7-12]. Most of these studies deal with total microsomes. This fraction, isolated from liver homogenate using different methods, contains fragments of plasma membranes, Golgi apparatus and other organeUes besides rough and smooth ER, which again differ from each other in several respects [13,14]. By morphometric analysis Schmucker et al. [15] have shown that the volume of rat hepatic total and smooth ER increases during maturity but declines during senescence. 0047-6374/83/$03.00 Printed and Publishedin Ireland
© 1983 Elsevier ScientificPublishersIreland Ltd.
366 Since studies using total microsomes fail to show similar quantitative changes, Schmucker and Wang [11] have concluded that changes in the ER in situ may not be truly reflected by the total microsomes. That the constituents of the total microsomal fraction vary depending on the method of fractionation employed may have a bearing on the contradictory results reported by earlier workers. Isolation of the ER as rough and smooth microsomes gives reasonably pure preparations [14]. In the present study using purified rough and smooth microsomes we have investigated changes in the components of microsomal MFO system, microsomal proteins, glucose-6-phosphatase and adenosine triphosphatase during postnatal development and ageing. MATERIALS AND METHODS Five groups of female Wistar rats ranging in age from 1 day to 2 years (1, 15, 75, 365 and 730 days) were used. The animals were maintained on a nutritionally balanced laboratory diet and water was supplied ad libitum. Rats were sacrificed at the same time of the day (9 a.m.) to avoid diurnal variations. Except sucklings (1 day and 15 day old) other animals were starved for 18 h prior to killing. Rats were killed by cervical dislocation; the liver was excised ~ad immediately chilled in ice. The tissue was finely minced and homogenized in 5 vols. of 0.25 M sucrose. An aliquot of the homogenate was taken for protein estimation [16] and the rest was spun at 10 000 g for 30 min. The supernatant was again centrifuged at the same speed to ensure sedimentation of mitochondria and other contaminants. Fractionation of rough and smooth microsomes from the postmitochondrial supernatant, in a sucrose-caesium chloride density gradient, was in principle similar to the method employed by DePierre and DaUner [14]. Postmitochondrial supernatant (5 ml) was carefully layered over two parts of discontinuous gradients, the lower one consisting of 4 ml of 15 mM CsC1 in 1.3 M sucrose and the upper one of 2 ml of 15 mM CsC1 in 0.6 M sucrose. The gradients were spun at 105 000 g (av.) for 90 min in a Beckman L5-65B ultracentrifuge using a Ti 50 rotor. Angular rotors gave better separation than swinging bucket rotors. Separated rough and smooth microsomes were carefully removed and suspended in 0.05 M TrisHC1 buffer (pH 7.55) containing 20 mM KC1 and 5 mM MgCl2 (TKM buffer) [17] and again centrifuged at 105 000 g (av.) for 45 min. Total microsomes were isolated by spinning the postmitochondrial supernatant at 105 000 g (av.) for 60 min. Pellets of total, rough and smooth microsomes were suspended in a known volume of TKM buffer, frozen in liquid nitrogen and stored at -80°C. Samples were never stored for more than a week. Glucose-6-phosphatase (G-6-Pase) (EC 3.1.3.9) and total adenosine triphosphatase (ATPase) (EC 3.6.1.3) were estimated as described previously [17]. Cytochrones P-450 and bs were estimated as described by Beaufay et al. [18]. NADPH-cytochrome c reductase (EC 1.6.2.4) was assayed by the method of Lu et al. [19]. Besides the enzymes mentioned earlier other parameters were measured, as suggested by DePierre and Dallner [14] and Andersson et al. [20], to check the purity of rough and
367 smooth microsomes. Total lipids, cholesterol, phosphatidylcholine and phosphatidylethanolamine were estimated as described in an earlier study [21 ]. Standard procedures were used for the measurement of RNA [22], 5'-nucleotidase, lactate dehydrogenase and succinic dehydrogenase [23]. Age-related changes in the observed data were quantitated statistically using analysis of variance designed for multiple comparisons and correspondingFratios and least-significant differences (LSD) were calculated as described earlier [21]. Student's t-test was used for some of the comparisons between microsomal fractions. RESULTS Table I presents data on purity of rough and smooth microsomes in addition to other characteristics. Succinic dehydrogenase, the marker enzyme for mitochondria, is not present in measurable quantities in these fractions. However, the fractions contain traces of 5'.nucleotidase (marker enzyme for plasma membranes) and lactate dehydrogenase (marker for cytosol). Smooth microsomes contain significantly higher ( p < 0 . 0 5 ) amounts of cholesterol, phosphatidylcholine, G-6.Pase and ATPase than the rough microsomes. RNA is considerably more abundant in rough microsomes than in the smooth. Phosphatidylethanolamine content is similar in the two microsomal fractions. Data presented in Table II show that the body weight of the animals increases up to 2 years. Although the increase in body weight from 1 day to 2 years is about 80 times, rats gain weight at a rapid rate only up to maturity (75 days). Wet weight of the liver also increases during growth and amounts to an increase of 30% during the first year's growth, when expressed on the basis of body weight. There is a significant decrease in liver weight in 2-year.old rats. The total hepatic protein content also shows a similar trend, increasing up to 1 year and declining significantly in 2-year.old, senescent rats. Total microsomal TABLEI CHARACTERISTICSOF ROUGH AND SMOOTH MICROSOMESOF ADULT RATS Parameters
Rough microsomes
Smooth microsomes
Proteina Totallipid a Cholesterola Phosphatidyloholinea Phosphatidylethanolaminea Glucose-6-phosphataseb Adenosine triphosphatase b Ribonucleic acidc 5~-Nueleotidaseb Lactate dehydrogenased
9.44 ± 0.16 1.62 ± 0.09 0.35 ± 0.02 0.25 ± 0.05 0.25 ± 0.05 2.62 ± 0.17 2.11 ± 0.13 0.25 ± 0.01 0.05 ± 0.01 0.002 ± 0.001
6.83 -+ 0.39* 1.90 ± 0.29 0.48 + 0.03* 0.38 ± 0.05" 0.26 ± 0.02 4.41 ± 0.37* 4.83 ± 0.67* 0.03 ± 0.04* 0.11 ± 0.01" 0.023 ± 0.008*
b~molesPiPer 20 min per nag protein; Crag/rag protein; dAe340 per rain per mg protein. *Sisnificantly different from that of rough nderosomes (p < 0.05). Values represent means + S.E. from 5 rats and are expressed as: amg/g liver,
F value
0.001 25.21
Values represent means -+ S.E. from 5 rats each.
Significance at LSD (p < 0.05)
814.36
6.80 27.85 247.20 415.00 557.00
I 15 75 365 730
Analysis of variance
(g)
(days)
+ 0.25 +_ 0.67 +_ 10.41 + 10.49 _+ 10.91
Body weight
Age
0.001 2.93
11.42
21.26 + 0.77 25.83 _+ 0.79 28.52 ± 1.51 29.81 ± 0.53 25.41± 1.04
(g/kg)
Liver weight
t
23.11 0.001 15.57
126.12 175.15 187.00 186.04 167.63
+ 4.33 + 2.81 + 3.72 + 10.32 _+ 1.04
Homogenate
+ 0.54 +_ 1.44 _+ 1.65 ± 1.99 + 0.94
10.83 0.001 4.65
15.41 26.58 26.75 27.79 25.73
Total microsomes
Protein content (mg/g liver)
.....
+ 0.58 +_ 0.74 + 0.32 + 0.71 + 0.22
9.48 0.001 1.77
6.27 9.76 9.44 10.33 6.93
Rough microsomes
6.63 0.01 1.55
3.32 3.85 6.81 4.83 4.51
_+0.43 ~:0.48 -+0.80 + 0.33 -+0.20
Smooth microsomes
AGE-RELATED CHANGES IN BODY WEIGHT, LIVER WEIGHT AND PROTEIN CONTENT OF HEPATIC HOMOGENATE AND MICROSOMES OF' RATS
TABLE II
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369 protein reaches adult level as early as 15 days and remains virtually unvarying up to 2 years. Unlike total microsomes, protein content of rough and smooth microsomes increases during postnatal development and decreases during ageing. Protein content of rough microsomes in the 15-day-old suckling rats is similar to the adult level and a significant decrease is seen in 2-year-old rats. But smooth microsomes show peak values in 75-day-old rats and lower levels of protein in 1-year-old and 2-year-old rats. Statistical analysis reveals that all the parameters under consideration in Table II are altered significantly as a function of age. Age-related changes in the components of the MFO system, namely cytochrome P.450, cytochrome bs and NADPH-cytochrome c reductase, in rough and smooth microsomes are shown in Figs. 1 and 2. Both specific and total activities show significant changes during postnatal development and ageing. The changes are different in rough and smooth microsomes. Newborn rats have low levels of these three components of the MFO system. The specific activity of cytochrome P-450 increases during postnatal development, reaching peak values by 15 days in rough microsomes (3 times) and by 75 days in smooth microsomes (5 times) in comparison with the newborns (Fig. 1). Cytochrome P-450, computed as nmoles/g liver, shows a similar trend of change (Fig. 2). The amount of cytochrome /'-450, computed in rough and smooth microsomes together, is relatively
1 day
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15 days
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2
E O
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c
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Rough Smooth Cytochrome P-~S0
Rough Smooth Cytochrome b5
Rough Smooth NADPH cyt.c reduct~e
0
Fig. 1. Age-rehted changes in the specific activity of the components of mixed-function oxidase system in rat liver rough and smooth miczosomes.Values represent means + S.E. from 5 rats. 'Rough' represents rough microsomes and 'Smooth' the smooth microsomes. Data from analysis of variance: F values (all significant at 0.001 level): Rough mictosomes: cytochrome P-450, 81.25; cytochrome b,, 33.80; NADPH-cytochrome c reductas¢, 19.89. Smooth miczosomes: cytochrome P-450, 10.56; cytochrome bs, 29.33 ; NADPH-cytochrom¢c reductas¢, 16.98. Least-significant differences (p < 0.05); Rough mic~osomes: cytochrome P-450, 0.27; cytoc~ome b,, 0.30; NADPH-cytoc~ome c reductase 2.93. Smooth miczosomes: cytochrome P-450, 0.57; ¢ytochrome b,, 0.40; NADPHcytochrome c reductas¢, 4.06.
370
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.-C
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Rough Smooth Rough Smooth Cytochrome b5 NADPH cyt.c reductase Fig. 2. Age-related changes in the total activity of the components of mixed-function oxidase system in rat liver rough and smooth microsomes. Values represent means from 5 rats. 'Rough' represents rough microsomes, and 'smooth' smooth microsomes. Cytochrome P-I.S 0
high in 15. and 75-day-old rats (Table Ill). Low values are seen in 1-year-old, 2-year-old and 1.day-old, in that decreasing order. The trend of change in microsomal cytochrome bs is almost limflar to that of cytochrome P-450 (Figs. 1 and 2) but the magnitude of change is different. Peak levels of cytochrome bs in rough microsomes are attained by 15 days and in smooth microsomes by 75 days. Total content of microsomal cytochrome bs, accounted for by rough and smooth microsomes, is the highest in 75-day-old rats and the lowest in 1-day-old animals (Table III). Increase in the specific activity of NADPH-cytochrome c reductase during postnatal development is more pronounced in smooth microsomes (Fig. 1). Both the fractions
TABLE Ill COMPUTED VALUES OF CHANGES IN MICROSOMAL (ROUGH PLUS SMOOTH)ENZYMES IN RAT LIVER AS A FUNCTION OF AGE Age (days)
Cytochrome P-450 a
Cytochrome
NADPH cytochrome c reductase b
G-6-Pasec
A TPasec
bs a
I 15 75 365 730
6.43 29.44 27.24 15.51 9.86
2.37 20.89 27.40 20.17 13.76
56.24 148.67 274.66 207.34 84.37
1.32 22.13 54.76 27.35 5.32
36.25 24.06 52.81 24.98 18.43
V~lues axe means of 5 e x t e n t s
reduced per ~
and axe e x p m ~
as: anmoles/g fiver; bnmoles ~ t ~ o m e
per g fiver, e~mo~s Pi pet 20 rain per g liver.
c
371
contain highest levels of the enzyme at 75 days. Significant decline in the enzyme levels is seen in smooth microsomes of 1-year-old rats whereas rough microsomes show such a change in 2-year-old rats. Changes in the total activity of NADPH-cytochrome c reductase of rough and smooth microsomes computed together show a similar trend (Table III). There are significant changes in the levels of microsomal G-6-Pase and ATPase during postnatal development and ageing (Figs. 3 and 4). G-6-Pase levels, low at birth, increase to reach a maximum at 75 days and decline thereafter. G-6-Pase of smooth microsomes shows significant reduction at 1 year whereas in rough microsomes the decline in the enzyme activity is discernible at 2 years. The combined activity of G-6-Pase in rough and smooth microsomes increases 40.fold between 1 day and 75 days and then declines to a low value in 2-year-old rats (Table III). Unlike the other enzymes studied, ATPase shows high levels of activity at birth (Figs. 3 and 4). There is a second peak of activity at 75 days. A similar trend is apparent in the combined ATPase activity of rough and smooth microsomes (Table III). DISCUSSION
Membranes of the hepatic ER contain approximately 60% protein and 30% phospholipid [14]. The MFO system involved in drug metabolism is one of the major enzyme
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Rough Smooth AT Pass
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Rough Smooth G- 6-Pass
Rough Smooth ATProse
Fig. 3. Age-related changes in the specLqc activity of 81ucose-6-phosphataseand adenosine triphosphatase in rat liver rough and smooth microsomes. Values represent means ± S.E. from 5 rats. 'Rough' represents rough mJcrosomes, and 'smooth' smooth microsomes. Data from analysis of var~nc¢: F values: Rough microsomes: G-6-Pase, 28.86 (p < 0.001); ATPase, 34.94 (p- < 0.001). Smooth microsomes: G~-Pase, 25.9? (p < 0.001); ATPase, 5.18 (p < 0.01). Least significant differences (p < 0.05). Rough microsomes: G-6-Pase, 0.61; ATPas¢, 0.54. Smooth microsomes: G-6-Pase, 0.99; ATPase, 1.27. Fig. 4. Age-related changesin the total activity of glucose-6-phosphataseand adenosine triphosphatas¢ in rat liver rough and smooth mlcrosomes. Values represent means from 5 rats. 'Rough' represents rough microsomcs, and 'smooth' smooth microsomes.
372 systems present in ER and phospholipids have been shown to be essential for its activity [4]. There are many differences in enzyme composition between rough and smooth fractions of ER. The majority of the NADPH-requiring enzymes involved in drug metabolism are more abundant in smooth microsomes [14,24]. During development, smooth ER appears later than rough ER and increases in the former occur mainly after birth [5,25]. Neonates of a number of species have a low capacity to metabolize drugs [3,5,26]. This has been correlated to the low levels of hepatic drug.metabolizing enzymes [3,5,6,26]. Earlier attempts to study postnatal changes in the enzymes of ER have centred mainly around total microsomes [3,6,26]. However, DaUner et al. [5] have shown that the enzyme activities in rough and smooth microsomes follow different patterns of change after birth. But the study has been restricted to 7 days of postnatal life. Our results are in agreement with those of the earlier studies which have shown gradual increase in the enzyme activities of ER during postnatal development. We find that different components of the MFO system in rough and smooth microsomes reach peak levels at different times after birth. It has been shown that NADPH-cytochrome c reductase, G-6-Pase and ATPase in rough and smooth microsomes attain peak levels at different times after birth [5]. In addition, the results ofDallner et al. [5] show that the levels of cytochromes P-450 and b 5 of total microsomes increase gradually after birth, and at 7 days the values are 60-70% of those of the adults. It is of interest to note that cytochromes P-450 and bs in rough microsomes reach peak levels earlier than NADPHcytochrome c reductase, unlike in smooth microsomes. MacLeod et al. [3] have shown that total microsomal cytochrome P-450 of Long-Evans rats reaches a peak level in 3 weeks and NADPH.cytochrome c reductase in 4 weeks after birth. Our study shows that the combined value of microsomal cytochrome P-450 is highest at 15 days and that of NADPH-cytochrome c reductase and cytochrome b5 at 75 days. The difference in the particular age groups selected in the two studies may account for this. It is apparent from our results that the optimum development of components of the MFO system, required for maximal activity, is attained by 75 days after birth rather than at 15 days. Increase in the monooxygenases per unit body weight may also be due to increases in protein content of rough and smooth microsomes. Similar increases in rat liver weight and total microsomal protein have been reported [6]. The other enzymes studied, namely G.6-Pase and total ATPase, also show significant changes during postnatal development. Dallner e t al. [5] and Leskes et al. [27] have shown that microsomal G-6-Pase is low at birth but increases to nearly twice the adult activity by 3 days after birth and gradually declines to reach adult values. We may have missed such a peak because we did not estimate G-6-Pase in 3-day-old rats. Our data on ATPase differ from those of Dallner et al. [5]. We have estimated total ATPase unlike the Mg~+-dependentATPase that Dallner et al. [5] assayed. The incidence of adverse drug reactions increases markedly as a function of the patient's age. One of the main contributory factors for this is the reduced capacity to dispose of drugs [ 1]. This conforms to the reduction in hepatic drug metabolism with age in experimental animals [1,2,7,9,12]. Though it has been generally accepted that
373 this age-related decline is due to decrease in cytochromeP-450-dependent monooxygenases [1,12], the available results are not unequivocal. There are reports on age-related reduction in the basal levels of monooxygenase components [2,9-12,28] or microsomal phospholipids [8,12,28]. Other results indicate that there is no lowering of monooxygenase components such as cytochrome P-450 [9,29], NADPH-cytochrome c reductase [7,8] and cytochrome bs [7,30]. Rikans and Notley [12] attributed these contradictions to differences in sex, strain and age of the animals used. Most of these studies have been on total microsomes prepared by a variety of methods. Since the constituents of the micrc~omal fraction can vary with the method of preparation [13,14,20], such variations may l~ave contributed to the contradictions in the earlier reports. Total microsomal protein content and other biochemical data pertaining to the amount of hepatic ER reported by Schmucker and Wang [10,11] and others [2,7] do not show significant variations during ageing. However, it is difficult to correlate these with electron.microscopic observations which show significant age-related decrease in ER [ 15]. Since stereological values represent the membranes in situ, Schmucker and Wang [10,11] have assumed that they are a true reflection of age-related changes. The contamination of microsomes with extraneous membranes is suggested as a cause for the discrepancy [ 11 ]. Our data on microsomal protein content of female Wistar rats show that, though it remains the same in total microsomes, both rough and smooth microsomal fractions show decreased protein content in senescent rats. Based on electron-microscopic studies, Schmucker et al. [15] have reported age-related decrease only in smooth ER membranes of male Fischer 344 rats. Since our studies on male Wistar rats (unpublished data) also show a similar decrease only in smooth microsomes, the differences noticed in the present study may be due to sex difference. We have observed that drug-induced changes in total microsomes do not correspond to those in rough and smooth microsomes in rats of different ages [21], suggesting the presence of other component(s) besides rough and smooth ER. Our data showing age-related decreases in G-6-Pase also agree with those reported earlier [28,31]. The decline in the enzyme activity is similar in the two microsomal fractions. A comparable decrease is seen in total ATPase of both rough and smooth microsomes. Age-related changes in the monooxygenation of different substrates differ [7,9,12]. Different forms of cytochrome P.450, involved in these monooxygenations, undergo senescent decreases to varying degrees [30]. Since different forms of cytochrome P-450 require different phospholipids for activity [ 12] and phospholipids determine the proper organization of microsomal membranes required for the efficient interaction of the components of the MFO system [4,28], age-related changes in the individual phospholipids [21 ] may also have a profound effect on the changes in hepatic drug metabolism. Lipid peroxidation in microsomal membranes has been shown to increase with age [32]. Lipid peroxidation drastically alters the membrane configuration, through its effect on phospholipids [33], and specifically inactivates the cytochrome P-450 [34]. The agerelated decrease in the MFO components and other enzymes may be a consequence of increased lipid peroxidation.
374 REFERENCES 1 J .R. Gillette, Biotransformation of drugs during ageing. Fed. Proc., 38 (1979) 1900-1909. 2 R. Kato and A. Takanaka, Metabolism of drugs in old rats. I. Activities of NADPH.linked electron transport and drag-metabolizing enzyme systems in liver microsomes of old rats. Jpn. J. Pharmaool., 18 (1968) 381-388. 3 S.M. MacLeod, K.W. Renton and N.R. Eade, Development of hepatic microsomal drug oxidizing enzymes in immature male and female rats. Z Pharmacol. Exp. Ther., 183 (1972) 489-498. 4 C.S. Yang, Organization and interaction of monooxygenase enzymes in microsomal membranes. LifeSci., 21 (1977) 1047-1058. 5 G. Dallner, P. Siekevitz and G.E. Palade, Synthesis of microsomal membranes aM their enzymic constituents in developing rat liver. Biochem. Biophys. Res. Commug, 20 (1965) 135-141. 6 T.K. Basu, LW.T. Dickerson and D.V. Parke, Effect of development on the activity of microsomal drug-metabolizing enzymes in rat liver. Btochem. Z, 124 (1971) 19-24. 7 L.S. Bimbaum and M.B. Baird, Induction of hepatic mixed function oxidases in senescent rodents. Exp. Geeontol., 13 (1978) 299-304. 8 L.S. Bimbaum and M.B. Baird, Induction of hepatic mixed function oxidases in senescent rodents. I1. Effect of polychlorinated biphenyls. Exp. C,erontol., 13 (1978) 469-477. 9 J. Kao and P. Hudson, Induction of the hepatic cytochrome P-450-dependent monooxygenase system in young and geriatric rats. Biochem. Ph~raoo~l., 29 (1980) 1191-1194. 10 D.L. Schmucker and R.K. Wang, Age-related changes in liver drug-metabolizing enzymes. Exp. Geeontol., 15 (1980) 321-329. 11 D.L. Schmucker and R.K. Wang, Effects of aging and phenobarbital on the rat liver microsomal drug-metabolizing system. Mech. Ageing Dev., 15 (1981) 189- 202. 12 L.E. Rikans and B.A. Notiey, Decline in hepatic microsomal monooxygenase components in middle-aged Fischer 344 rats.Exp, Geron~oL, 16 (1981) 253-259. 13 S. Fleischer and M. Kervina, Suboellular fractionation of rat liver. Methods Enzymol., 31 (1974) 6-41. 14 J.W. DePierre and G. Dallner, Structural aspects of the membrane of the endoplasmic reticulum. Biochim. Biophys. Aeta, 415 (1975) 411-472. 15 D.L. Schmucker, J.S. Mooney and A.L. Jones, Age-related changes in the hepatic endoplasmic reticulum : A quantitative analysis.Science, 197 (1977) 1005-1007. 16 O.H. Lowry, NJ. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent. J. Biol. Chem., I93 (1951) 265-275. 17 T.P.A. Devasagayam, C.K. Pushpendran and J. Eapen, Induction of rat liver microsomal enzymes by cycloheximide.Biochem. Pharmacol., 28 (1979) 1731-1734. 18 H. Beanfay, A. Amar-Costesec, E. Feytmans, D. Thines-Sempoux, M. Wibo, M. Rohbi and J. Berthet, Analytical study of microsomes and isolated subcenular membranes from rat liver. I. Biochemical methods. J. CeilBiol,, 61 (1974) 188-200. 19 A.Y.H. Lu, K.W. Junk and M.J. Coon, Resolution of the cytochrome P-450 containing co-hydroxylation system of liver microsomes into three components. J. Biol. Chem., 244 (1969) 3714-3721. 20 G.N. Andersson, U. Torndal and L.C. Eriksson, Sequential preparation of rat liver microsomal and Golgi membranes. Biochtm. Biophys. Aera, 512 (1978) 539-549. 21 C.K. Pushpendran, T.P.A. Devasageyam and J. Eapen, Effect of cycloheximide on hepatic microsomal lipids as a function of age in rats. Dev. Pharmacoi. Thee., 4 (1982) 173-180. 22 H.N. Munro and A. Fleck, The determination of nucleic acids. Methods. B~oehem. Anal., 14 (1967) 113-176. 23 H.U. Bexgmeyer, Methods in Enzymatic Analysis, Academic l~ess, New York, 1963. 24 J.R. Gillette, A.H. Conney, G.J. Cosmides, R.W. Estabrook, J .R. Fouts and G.J. Mannering,Microsomes and Drug Oxidations, Academic Press, New York, 1969. 25 A. Chedid and V. Nair, Ontogenesis of cytoplasmic organelles in rat hepatocytes and the effects of prenatal phenobarbital on endoplasmic reticulum development. Dev. Biol., 39 (1974) 49-62. 26 R. Kato, P. Vassanelli, G. Frontino and E. Chiesara, Variation in the activity of liver microsomal drug-metabolizing enzymes in rats in relation to the age. Biochem. Pharvnacol., 13 (1964) 10371051.
375 27 A. Leskes, P. Siekevitz and G.E. Palade, Differentiation of endoplaunic reticulum in hepatocytes. I. Glucose-6-phosphatase distribution in situ. J. CelIBiol., 49 (1971) 264-287. 28 L.S. Grinna and A.A. Barber, Age-related changes in membrane lipid content and enzyme activities. Biochim. Biophys. Acta, 288 (1972) 347-353. 29 M.B. Baird, R.J. Nicolosi, H.R. Massie and H.V. Samis, Microsomal mixed-function oxidase activity and senescence. I. Hexobarbital sleep time and induction of components of the hepatic microsomal enzyme system in rats of different ages. Exp. Geronrol., 10 (1975) 89-99. 30 D.N. McMartin, J.A. O'Connor, Jr., M.J. Fasco and L.S. Kaminsky, Influence of ageing and induction on rat liver and kidney microsomal mixed-function oxidase system. Toxicol. Appl. Pharma¢ol., 54 (1980) 411-416. 31 D.L. Schmucker and R.K. Wang, Effects of animal age and phenobarbital on rat liver glucose-6phosphatase activity. Exp. Geronrol., 15 (1980) 7-13. 32 T.J. Player, D.J. Mills and A.A. Horton, Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation. Bioehem. Biophys. Res. Commun., 78 (1977) 1397-1402. 33 J.S. Bus and J.E. Gibson, Lipid peroxidation and its role in toxicology. In E. Hodgson, J.R. Bend and R.M. Philpot (eds.), Reviews ~ Biochemical Toxicology, Vol. 1, Elsevier/North-Holland, New York, 1979, pp. 125-149. 34 B.A. Svingen, J.A. Buege, F.O. O'Neal and S.D. Aust, The mechanism of NADPH-dependent lipid peroxidation. The propagation of lipid peroxidation. I. Biol. Chem., 254 (1979) 5892-5899.
365
Elsevier ScientificPublishers Ireland Ltd.
CHANGES IN ENZYMES OF HEPATIC ROUGH AND SMOOTH MICROSOMES DURING POSTNATAL DEVELOPMENT AND AGEING OF RATS THOMAS P.A. DEVASAGAYAM, CHOLIPARAMBIL K. PUSHPENDRAN and JACOB EAPEN Biology and Agriculture Division, Bhabha Atomic Research Centre, Bombay 400 085 (India)
(Received July 25th, 1982) (Revision received November2nd, 1982) SUMMARY Significant changes are observed in wet weight, microsomal protein content and enzymes of purified rough and smooth microsomes of liver during postnatal development and ageing of female Wistar rats. Protein content of total microsomes increases up to 15 days of age and remains steady during subsequent development, unlike that of rough and smooth microsomes which shows changes throughout the same period. Activities of cytochrome P-450, cytochrome bs and NADPH-cytochrome c reductase increase during the period of maturation and decline during senescence. The decrease during senescence is at different rates in the two microsomal fractions. Microsomal glucose-6-phosphatase, but not adenosine triphosphatase, shows a similar increase during development and decrease during senescence. K e y words: Monooxygenases, Rat liver microsome; Ageing
~TRODUCTION Age is an important factor which determines an individual's response to drugs. Children as well as old people are often more susceptible to the adverse effects of drugs [1-3]. This may be explained on the basis of the slow onset of the hepatic drugmetabolizing system during postnatal development and the decline in its activity during ageing [1-3]. Hepatic endoplasmic reticulum (ER), containing the mixed-function oxidase (MFO) system, is the major site of xenobiotic metabolism in mammals [4]. There are several, but conflicting, reports on the changes in rat hepatic ER during postnatal development [3,5,6] and ageing [2,7-12]. Most of these studies deal with total microsomes. This fraction, isolated from liver homogenate using different methods, contains fragments of plasma membranes, Golgi apparatus and other organeUes besides rough and smooth ER, which again differ from each other in several respects [13,14]. By morphometric analysis Schmucker et al. [15] have shown that the volume of rat hepatic total and smooth ER increases during maturity but declines during senescence. 0047-6374/83/$03.00 Printed and Publishedin Ireland
© 1983 Elsevier ScientificPublishersIreland Ltd.
366 Since studies using total microsomes fail to show similar quantitative changes, Schmucker and Wang [11] have concluded that changes in the ER in situ may not be truly reflected by the total microsomes. That the constituents of the total microsomal fraction vary depending on the method of fractionation employed may have a bearing on the contradictory results reported by earlier workers. Isolation of the ER as rough and smooth microsomes gives reasonably pure preparations [14]. In the present study using purified rough and smooth microsomes we have investigated changes in the components of microsomal MFO system, microsomal proteins, glucose-6-phosphatase and adenosine triphosphatase during postnatal development and ageing. MATERIALS AND METHODS Five groups of female Wistar rats ranging in age from 1 day to 2 years (1, 15, 75, 365 and 730 days) were used. The animals were maintained on a nutritionally balanced laboratory diet and water was supplied ad libitum. Rats were sacrificed at the same time of the day (9 a.m.) to avoid diurnal variations. Except sucklings (1 day and 15 day old) other animals were starved for 18 h prior to killing. Rats were killed by cervical dislocation; the liver was excised ~ad immediately chilled in ice. The tissue was finely minced and homogenized in 5 vols. of 0.25 M sucrose. An aliquot of the homogenate was taken for protein estimation [16] and the rest was spun at 10 000 g for 30 min. The supernatant was again centrifuged at the same speed to ensure sedimentation of mitochondria and other contaminants. Fractionation of rough and smooth microsomes from the postmitochondrial supernatant, in a sucrose-caesium chloride density gradient, was in principle similar to the method employed by DePierre and DaUner [14]. Postmitochondrial supernatant (5 ml) was carefully layered over two parts of discontinuous gradients, the lower one consisting of 4 ml of 15 mM CsC1 in 1.3 M sucrose and the upper one of 2 ml of 15 mM CsC1 in 0.6 M sucrose. The gradients were spun at 105 000 g (av.) for 90 min in a Beckman L5-65B ultracentrifuge using a Ti 50 rotor. Angular rotors gave better separation than swinging bucket rotors. Separated rough and smooth microsomes were carefully removed and suspended in 0.05 M TrisHC1 buffer (pH 7.55) containing 20 mM KC1 and 5 mM MgCl2 (TKM buffer) [17] and again centrifuged at 105 000 g (av.) for 45 min. Total microsomes were isolated by spinning the postmitochondrial supernatant at 105 000 g (av.) for 60 min. Pellets of total, rough and smooth microsomes were suspended in a known volume of TKM buffer, frozen in liquid nitrogen and stored at -80°C. Samples were never stored for more than a week. Glucose-6-phosphatase (G-6-Pase) (EC 3.1.3.9) and total adenosine triphosphatase (ATPase) (EC 3.6.1.3) were estimated as described previously [17]. Cytochrones P-450 and bs were estimated as described by Beaufay et al. [18]. NADPH-cytochrome c reductase (EC 1.6.2.4) was assayed by the method of Lu et al. [19]. Besides the enzymes mentioned earlier other parameters were measured, as suggested by DePierre and Dallner [14] and Andersson et al. [20], to check the purity of rough and
367 smooth microsomes. Total lipids, cholesterol, phosphatidylcholine and phosphatidylethanolamine were estimated as described in an earlier study [21 ]. Standard procedures were used for the measurement of RNA [22], 5'-nucleotidase, lactate dehydrogenase and succinic dehydrogenase [23]. Age-related changes in the observed data were quantitated statistically using analysis of variance designed for multiple comparisons and correspondingFratios and least-significant differences (LSD) were calculated as described earlier [21]. Student's t-test was used for some of the comparisons between microsomal fractions. RESULTS Table I presents data on purity of rough and smooth microsomes in addition to other characteristics. Succinic dehydrogenase, the marker enzyme for mitochondria, is not present in measurable quantities in these fractions. However, the fractions contain traces of 5'.nucleotidase (marker enzyme for plasma membranes) and lactate dehydrogenase (marker for cytosol). Smooth microsomes contain significantly higher ( p < 0 . 0 5 ) amounts of cholesterol, phosphatidylcholine, G-6.Pase and ATPase than the rough microsomes. RNA is considerably more abundant in rough microsomes than in the smooth. Phosphatidylethanolamine content is similar in the two microsomal fractions. Data presented in Table II show that the body weight of the animals increases up to 2 years. Although the increase in body weight from 1 day to 2 years is about 80 times, rats gain weight at a rapid rate only up to maturity (75 days). Wet weight of the liver also increases during growth and amounts to an increase of 30% during the first year's growth, when expressed on the basis of body weight. There is a significant decrease in liver weight in 2-year.old rats. The total hepatic protein content also shows a similar trend, increasing up to 1 year and declining significantly in 2-year.old, senescent rats. Total microsomal TABLEI CHARACTERISTICSOF ROUGH AND SMOOTH MICROSOMESOF ADULT RATS Parameters
Rough microsomes
Smooth microsomes
Proteina Totallipid a Cholesterola Phosphatidyloholinea Phosphatidylethanolaminea Glucose-6-phosphataseb Adenosine triphosphatase b Ribonucleic acidc 5~-Nueleotidaseb Lactate dehydrogenased
9.44 ± 0.16 1.62 ± 0.09 0.35 ± 0.02 0.25 ± 0.05 0.25 ± 0.05 2.62 ± 0.17 2.11 ± 0.13 0.25 ± 0.01 0.05 ± 0.01 0.002 ± 0.001
6.83 -+ 0.39* 1.90 ± 0.29 0.48 + 0.03* 0.38 ± 0.05" 0.26 ± 0.02 4.41 ± 0.37* 4.83 ± 0.67* 0.03 ± 0.04* 0.11 ± 0.01" 0.023 ± 0.008*
b~molesPiPer 20 min per nag protein; Crag/rag protein; dAe340 per rain per mg protein. *Sisnificantly different from that of rough nderosomes (p < 0.05). Values represent means + S.E. from 5 rats and are expressed as: amg/g liver,
F value
0.001 25.21
Values represent means -+ S.E. from 5 rats each.
Significance at LSD (p < 0.05)
814.36
6.80 27.85 247.20 415.00 557.00
I 15 75 365 730
Analysis of variance
(g)
(days)
+ 0.25 +_ 0.67 +_ 10.41 + 10.49 _+ 10.91
Body weight
Age
0.001 2.93
11.42
21.26 + 0.77 25.83 _+ 0.79 28.52 ± 1.51 29.81 ± 0.53 25.41± 1.04
(g/kg)
Liver weight
t
23.11 0.001 15.57
126.12 175.15 187.00 186.04 167.63
+ 4.33 + 2.81 + 3.72 + 10.32 _+ 1.04
Homogenate
+ 0.54 +_ 1.44 _+ 1.65 ± 1.99 + 0.94
10.83 0.001 4.65
15.41 26.58 26.75 27.79 25.73
Total microsomes
Protein content (mg/g liver)
.....
+ 0.58 +_ 0.74 + 0.32 + 0.71 + 0.22
9.48 0.001 1.77
6.27 9.76 9.44 10.33 6.93
Rough microsomes
6.63 0.01 1.55
3.32 3.85 6.81 4.83 4.51
_+0.43 ~:0.48 -+0.80 + 0.33 -+0.20
Smooth microsomes
AGE-RELATED CHANGES IN BODY WEIGHT, LIVER WEIGHT AND PROTEIN CONTENT OF HEPATIC HOMOGENATE AND MICROSOMES OF' RATS
TABLE II
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369 protein reaches adult level as early as 15 days and remains virtually unvarying up to 2 years. Unlike total microsomes, protein content of rough and smooth microsomes increases during postnatal development and decreases during ageing. Protein content of rough microsomes in the 15-day-old suckling rats is similar to the adult level and a significant decrease is seen in 2-year-old rats. But smooth microsomes show peak values in 75-day-old rats and lower levels of protein in 1-year-old and 2-year-old rats. Statistical analysis reveals that all the parameters under consideration in Table II are altered significantly as a function of age. Age-related changes in the components of the MFO system, namely cytochrome P.450, cytochrome bs and NADPH-cytochrome c reductase, in rough and smooth microsomes are shown in Figs. 1 and 2. Both specific and total activities show significant changes during postnatal development and ageing. The changes are different in rough and smooth microsomes. Newborn rats have low levels of these three components of the MFO system. The specific activity of cytochrome P-450 increases during postnatal development, reaching peak values by 15 days in rough microsomes (3 times) and by 75 days in smooth microsomes (5 times) in comparison with the newborns (Fig. 1). Cytochrome P-450, computed as nmoles/g liver, shows a similar trend of change (Fig. 2). The amount of cytochrome /'-450, computed in rough and smooth microsomes together, is relatively
1 day
I
75 days
15 days
~
365 days
l
730 days
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2
E O
E
c
0
1
Rough Smooth Cytochrome P-~S0
Rough Smooth Cytochrome b5
Rough Smooth NADPH cyt.c reduct~e
0
Fig. 1. Age-rehted changes in the specific activity of the components of mixed-function oxidase system in rat liver rough and smooth miczosomes.Values represent means + S.E. from 5 rats. 'Rough' represents rough microsomes and 'Smooth' the smooth microsomes. Data from analysis of variance: F values (all significant at 0.001 level): Rough mictosomes: cytochrome P-450, 81.25; cytochrome b,, 33.80; NADPH-cytochrome c reductas¢, 19.89. Smooth miczosomes: cytochrome P-450, 10.56; cytochrome bs, 29.33 ; NADPH-cytochrom¢c reductas¢, 16.98. Least-significant differences (p < 0.05); Rough mic~osomes: cytochrome P-450, 0.27; cytoc~ome b,, 0.30; NADPH-cytoc~ome c reductase 2.93. Smooth miczosomes: cytochrome P-450, 0.57; ¢ytochrome b,, 0.40; NADPHcytochrome c reductas¢, 4.06.
370
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i
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.-C
Smooth
Rough Smooth Rough Smooth Cytochrome b5 NADPH cyt.c reductase Fig. 2. Age-related changes in the total activity of the components of mixed-function oxidase system in rat liver rough and smooth microsomes. Values represent means from 5 rats. 'Rough' represents rough microsomes, and 'smooth' smooth microsomes. Cytochrome P-I.S 0
high in 15. and 75-day-old rats (Table Ill). Low values are seen in 1-year-old, 2-year-old and 1.day-old, in that decreasing order. The trend of change in microsomal cytochrome bs is almost limflar to that of cytochrome P-450 (Figs. 1 and 2) but the magnitude of change is different. Peak levels of cytochrome bs in rough microsomes are attained by 15 days and in smooth microsomes by 75 days. Total content of microsomal cytochrome bs, accounted for by rough and smooth microsomes, is the highest in 75-day-old rats and the lowest in 1-day-old animals (Table III). Increase in the specific activity of NADPH-cytochrome c reductase during postnatal development is more pronounced in smooth microsomes (Fig. 1). Both the fractions
TABLE Ill COMPUTED VALUES OF CHANGES IN MICROSOMAL (ROUGH PLUS SMOOTH)ENZYMES IN RAT LIVER AS A FUNCTION OF AGE Age (days)
Cytochrome P-450 a
Cytochrome
NADPH cytochrome c reductase b
G-6-Pasec
A TPasec
bs a
I 15 75 365 730
6.43 29.44 27.24 15.51 9.86
2.37 20.89 27.40 20.17 13.76
56.24 148.67 274.66 207.34 84.37
1.32 22.13 54.76 27.35 5.32
36.25 24.06 52.81 24.98 18.43
V~lues axe means of 5 e x t e n t s
reduced per ~
and axe e x p m ~
as: anmoles/g fiver; bnmoles ~ t ~ o m e
per g fiver, e~mo~s Pi pet 20 rain per g liver.
c
371
contain highest levels of the enzyme at 75 days. Significant decline in the enzyme levels is seen in smooth microsomes of 1-year-old rats whereas rough microsomes show such a change in 2-year-old rats. Changes in the total activity of NADPH-cytochrome c reductase of rough and smooth microsomes computed together show a similar trend (Table III). There are significant changes in the levels of microsomal G-6-Pase and ATPase during postnatal development and ageing (Figs. 3 and 4). G-6-Pase levels, low at birth, increase to reach a maximum at 75 days and decline thereafter. G-6-Pase of smooth microsomes shows significant reduction at 1 year whereas in rough microsomes the decline in the enzyme activity is discernible at 2 years. The combined activity of G-6-Pase in rough and smooth microsomes increases 40.fold between 1 day and 75 days and then declines to a low value in 2-year-old rats (Table III). Unlike the other enzymes studied, ATPase shows high levels of activity at birth (Figs. 3 and 4). There is a second peak of activity at 75 days. A similar trend is apparent in the combined ATPase activity of rough and smooth microsomes (Table III). DISCUSSION
Membranes of the hepatic ER contain approximately 60% protein and 30% phospholipid [14]. The MFO system involved in drug metabolism is one of the major enzyme
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730 days
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i .,,00.
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Rough Smooth G-6-Pus •
Rough Smooth AT Pass
0
Rough Smooth G- 6-Pass
Rough Smooth ATProse
Fig. 3. Age-related changes in the specLqc activity of 81ucose-6-phosphataseand adenosine triphosphatase in rat liver rough and smooth microsomes. Values represent means ± S.E. from 5 rats. 'Rough' represents rough mJcrosomes, and 'smooth' smooth microsomes. Data from analysis of var~nc¢: F values: Rough microsomes: G-6-Pase, 28.86 (p < 0.001); ATPase, 34.94 (p- < 0.001). Smooth microsomes: G~-Pase, 25.9? (p < 0.001); ATPase, 5.18 (p < 0.01). Least significant differences (p < 0.05). Rough microsomes: G-6-Pase, 0.61; ATPas¢, 0.54. Smooth microsomes: G-6-Pase, 0.99; ATPase, 1.27. Fig. 4. Age-related changesin the total activity of glucose-6-phosphataseand adenosine triphosphatas¢ in rat liver rough and smooth mlcrosomes. Values represent means from 5 rats. 'Rough' represents rough microsomcs, and 'smooth' smooth microsomes.
372 systems present in ER and phospholipids have been shown to be essential for its activity [4]. There are many differences in enzyme composition between rough and smooth fractions of ER. The majority of the NADPH-requiring enzymes involved in drug metabolism are more abundant in smooth microsomes [14,24]. During development, smooth ER appears later than rough ER and increases in the former occur mainly after birth [5,25]. Neonates of a number of species have a low capacity to metabolize drugs [3,5,26]. This has been correlated to the low levels of hepatic drug.metabolizing enzymes [3,5,6,26]. Earlier attempts to study postnatal changes in the enzymes of ER have centred mainly around total microsomes [3,6,26]. However, DaUner et al. [5] have shown that the enzyme activities in rough and smooth microsomes follow different patterns of change after birth. But the study has been restricted to 7 days of postnatal life. Our results are in agreement with those of the earlier studies which have shown gradual increase in the enzyme activities of ER during postnatal development. We find that different components of the MFO system in rough and smooth microsomes reach peak levels at different times after birth. It has been shown that NADPH-cytochrome c reductase, G-6-Pase and ATPase in rough and smooth microsomes attain peak levels at different times after birth [5]. In addition, the results ofDallner et al. [5] show that the levels of cytochromes P-450 and b 5 of total microsomes increase gradually after birth, and at 7 days the values are 60-70% of those of the adults. It is of interest to note that cytochromes P-450 and bs in rough microsomes reach peak levels earlier than NADPHcytochrome c reductase, unlike in smooth microsomes. MacLeod et al. [3] have shown that total microsomal cytochrome P-450 of Long-Evans rats reaches a peak level in 3 weeks and NADPH.cytochrome c reductase in 4 weeks after birth. Our study shows that the combined value of microsomal cytochrome P-450 is highest at 15 days and that of NADPH-cytochrome c reductase and cytochrome b5 at 75 days. The difference in the particular age groups selected in the two studies may account for this. It is apparent from our results that the optimum development of components of the MFO system, required for maximal activity, is attained by 75 days after birth rather than at 15 days. Increase in the monooxygenases per unit body weight may also be due to increases in protein content of rough and smooth microsomes. Similar increases in rat liver weight and total microsomal protein have been reported [6]. The other enzymes studied, namely G.6-Pase and total ATPase, also show significant changes during postnatal development. Dallner e t al. [5] and Leskes et al. [27] have shown that microsomal G-6-Pase is low at birth but increases to nearly twice the adult activity by 3 days after birth and gradually declines to reach adult values. We may have missed such a peak because we did not estimate G-6-Pase in 3-day-old rats. Our data on ATPase differ from those of Dallner et al. [5]. We have estimated total ATPase unlike the Mg~+-dependentATPase that Dallner et al. [5] assayed. The incidence of adverse drug reactions increases markedly as a function of the patient's age. One of the main contributory factors for this is the reduced capacity to dispose of drugs [ 1]. This conforms to the reduction in hepatic drug metabolism with age in experimental animals [1,2,7,9,12]. Though it has been generally accepted that
373 this age-related decline is due to decrease in cytochromeP-450-dependent monooxygenases [1,12], the available results are not unequivocal. There are reports on age-related reduction in the basal levels of monooxygenase components [2,9-12,28] or microsomal phospholipids [8,12,28]. Other results indicate that there is no lowering of monooxygenase components such as cytochrome P-450 [9,29], NADPH-cytochrome c reductase [7,8] and cytochrome bs [7,30]. Rikans and Notley [12] attributed these contradictions to differences in sex, strain and age of the animals used. Most of these studies have been on total microsomes prepared by a variety of methods. Since the constituents of the micrc~omal fraction can vary with the method of preparation [13,14,20], such variations may l~ave contributed to the contradictions in the earlier reports. Total microsomal protein content and other biochemical data pertaining to the amount of hepatic ER reported by Schmucker and Wang [10,11] and others [2,7] do not show significant variations during ageing. However, it is difficult to correlate these with electron.microscopic observations which show significant age-related decrease in ER [ 15]. Since stereological values represent the membranes in situ, Schmucker and Wang [10,11] have assumed that they are a true reflection of age-related changes. The contamination of microsomes with extraneous membranes is suggested as a cause for the discrepancy [ 11 ]. Our data on microsomal protein content of female Wistar rats show that, though it remains the same in total microsomes, both rough and smooth microsomal fractions show decreased protein content in senescent rats. Based on electron-microscopic studies, Schmucker et al. [15] have reported age-related decrease only in smooth ER membranes of male Fischer 344 rats. Since our studies on male Wistar rats (unpublished data) also show a similar decrease only in smooth microsomes, the differences noticed in the present study may be due to sex difference. We have observed that drug-induced changes in total microsomes do not correspond to those in rough and smooth microsomes in rats of different ages [21], suggesting the presence of other component(s) besides rough and smooth ER. Our data showing age-related decreases in G-6-Pase also agree with those reported earlier [28,31]. The decline in the enzyme activity is similar in the two microsomal fractions. A comparable decrease is seen in total ATPase of both rough and smooth microsomes. Age-related changes in the monooxygenation of different substrates differ [7,9,12]. Different forms of cytochrome P.450, involved in these monooxygenations, undergo senescent decreases to varying degrees [30]. Since different forms of cytochrome P-450 require different phospholipids for activity [ 12] and phospholipids determine the proper organization of microsomal membranes required for the efficient interaction of the components of the MFO system [4,28], age-related changes in the individual phospholipids [21 ] may also have a profound effect on the changes in hepatic drug metabolism. Lipid peroxidation in microsomal membranes has been shown to increase with age [32]. Lipid peroxidation drastically alters the membrane configuration, through its effect on phospholipids [33], and specifically inactivates the cytochrome P-450 [34]. The agerelated decrease in the MFO components and other enzymes may be a consequence of increased lipid peroxidation.
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