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Decline in hepatic microsomal monooxygenase components in middle-aged Fischer 344 rats
Exp. Geront. Vol. 16, No. 3, pp. 253-259, 1981. Printed in Great Britain.
0531-5565/81/030253~7$02.00/0 PergamonPressLtd.
DECLINE IN HEPATIC MICROSOMAL MONOOXYGENASE COMPONENTS IN MIDDLE-AGED FISCHER 344 RATS L O R A E . RIKANS a n d B R U C E A . NOTLEY Department of Pharmacology, University o f O k l a h o m a , Health Sciences Center, O k l a h o m a City, OK 73190, U.S.A. (Received 21 December 1980)
INTRODUCTION THE AGINGprocess involves physiological changes which alter an animal's response to drugs. Early studies with old rats demonstrated that the altered response could be attributed in part to a decrease in the metabolism of drugs, as demonstrated by in vivo and in vitro experiments in which the capacity of the liver microsomal drug metabolism system declined with age (Kato and Takanaka, 1968a, b, c). It was accepted that this age-related decline was due to decreases in components of the hepatic cytochrome P-450-dependent monooxygenase system. However, conflicting reports on the effects of aging on the microsomal enzyme system have recently appeared. The microsomal monooxygenase system includes three major components, cytochrome P-450, NADPH-cytochrome P-450 reductase (NADPH-cytochrome c reductase) and phospholipid. Cytochrome b5 may be involved, too. Several investigators have reported that cytochrome P-450 specific content and NADPH-cytochrome c reductase activity are decreased in liver microsomes from old rats (Kato and Takanaka, 1968a, b; Kao and Hudson, 1980; McMartin, O'Connor, Fasco and Kaminsky, 1980; Schmucker and Wang, 1980). An age-related reduction in the quantity of microsomal phospholipid has also been found (Birnbaum and Baird, 1978b; Grinna and Barber, 1972; Grinna, 1977). On the other hand, others have shown that aging does not lower cytochrome P-450 content (Baird, Nicholosi, Massie and Samis, 1975; Birnbaum and Baird, 1978b), cytochrome c reductase activity (Aldeman, 1971; Birnbaum and Baird, 1978a, b) or cytochrome b5 content (Birnbaum and Baird, 1978a; McMartin, 1980) in rat liver microsomes. Furthermore, a study with senescent mice indicated that drug metabolism activities could be markedly altered (increased or decreased) while cytochrome P-450 content, reductase activity and phospholipid content remained unchanged (Birnbaum, 1980). Although some of these apparently contradictory results may be attributed to differences in animal sex, strain and species, or to differences in the ages of the " y o u n g " and " o l d " animals used for comparisons, it is clear that the exact mechanisms for the age-related changes in drug metabolism remain unresolved. The transition between young adulthood and old age is important for understanding the underlying causes of aging processes. The recent studies that examined microsomal drug metabolism in middle-aged rats found no significant differences in monooxygenase activities between middle-aged and young-adult animals (Birnbaum and Baird, 1978b; McMartin et aL, 1980). In these studies the middle-aged rats were 12 months and the young-adult rats were 3 or 4 months of age. In this study we found significant differences between middle-aged (14 months) and young-adult (3-5 months) rats in liver microsomal monooxygenase components and drug 253
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metabolism
LORA E . RIKANS A N D B R U C E A. NOTLEY
activities. Changes
in t h e l i p i d c o m p o s i t i o n
of the hepatic microsomal
m e m b r a n e w e r e also seen. A p r e l i m i n a r y r e p o r t o f t h e s e results h a s b e e n p r e s e n t e d ( R i k a n s a n d N o t l e y , 1980). MATERIALS
AND METHODS
Male Fischer 344 rats were purchased from Charles River Breeding Laboratories at 7 weeks of age and maintained in a separate room within our animal facilities for the duration of the study. The rats were given a semipurified diet (A1N 76 semipurified Diet ®, Nutritional Biochemicals) and water ad lib, They were housed 5/cage until 6 months of age and 3/cage from 6 to 16 months of age, in heat-sterilized hard-wood bedding, under filter caps. The room in which the animals were housed was kept free of cigarette smoke, pesticides, fumigants, disinfectants, etc. Temperature, humidity and lighting remained constant. Rats were killed 3 h after the onset of the 12 h light periods. The livers were removed and homogenized in 4 volumes of a solution containing 1.15% KCI and 0.05 M Tris-HCl, pH 7.5. The homogenates were centrifuged at 10,000 g for 15 min and the resulting supernatant fractions centrifuged at 78,000 g for 90 min. The 78,000 g pellets were washed once with homogenization medium and the final microsomal pellets suspended in 0.1 M Tris-HCl, pH 7.5, to a protein concentration of 5-10 mg/ml. All solutions were kept at 0-4°C. Centrifugations were performed at the g values determined for the average radii of the rotors used. The recovery of microsomal membranes was estimated by determining the total activity of the marker enzyme, cytochrome c reductase, in liver homogenates versus the microsomal fraction (Eriksson, DePierre and Dallner, 1978). Cytochrome P-450 was determined in microsomes from the reduced CO difference spectrum and cytochrome b 5 from the NADH-reduced difference spectrum (Omura and Sato, 1964). Rates of cytochrome c reduction by NADPH were determined at 23°C in a high ionic strength buffer as described by Vermilion and Coon (1978). The reduction of cytochrome P-450 in microsomes was measured at 15°C by following the formation of the reduced cytochrome P-450-CO complex after NADPH addition under anaerobic conditions (Zannoni, Flynn and Lynch, 1972). Microsomal fatty acids were determined using the Folch extraction procedure and gas chromatographic analysis of fatty acid methyl esters (May and McCay, 1968). Phospholipids were determinted as inorganic phosphate in the total extracted lipid (Fiske and Subbarow, 1925). Protein was measured in the presence of sodium dodecyl sulfate by a modification (Markwell, Haas, Bieber and Tolbert, 1978) of the Lowry procedure. The drug metabolism assays were performed using freshly-prepared microsomes. Conditions were optimized for each reaction so that activities were proportional to enzyme concentration and linear with time. Incubations were carried out in a shaking water bath at 37°C. The assay for nitroanisole O-demethylase activitiy was based on the procedure of Netter and Seidel (1964). Reaction mixtures, containing 0.3-0.6 mg microsomal protein, 1 mM p-nitroanisole, 6.8 mM glucose-6-phosphate, 0.27 mM NADP, 100 mM Tris-HCl (pH 7.8) and 1 unit of glucose-6-phosphate dehydrogenase, in a total volume of 2.0 ml, were incubated for 15 min. Reactions were stopped by adding 0.2 ml of 2.2 M NaOH, which served to clarify the mixture (Becker, Meehan and Bartholomew, 1978), and the absorbance of the product, p-nitrophenol, was measured at 415 nm. Aniline hydroxylase activity was determined according to Imai et al. (1966) except that the concentrations of aniline and nicotinamide were 1.25 and 2.5 mM, respectively. Incubation mixtures for the assay of benzphetamine N-demethylase contained: microsomes (0.8 1.6 mg protein), 5 mM benzphetamine, 7.5 mM glucose-6-phosphate, 0.3 mM NADP, 3.0 mM nicotinamide, 100 mM Tris-HCI (pH 7.5) and 5 units of glucose-6-phosphate dehydrogenase, in a total volume of 2.0 ml. After incubating for 15 min the reaction was stopped by adding 0.7 ml of 20°7o trichloroacetic acid. Protein was removed by centrifugation and the supernatant solution was assayed for formaldehyde using Nash reagent (Nash, 1953).
RESULTS A l t h o u g h liver w e i g h t s w e r e g r e a t e r in 1 4 - m o n t h - o l d r a t s c o m p a r e d w i t h 3 - 5 - m o n t h - o l d r a t s , t h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s in r e c o v e r y o f m i c r o s o m a l m e m b r a n e s o r i n t h e p r o t e i n c o n t e n t o f t h e m i c r o s o m e s ( T a b l e 1). T h e r e f o r e , t h e specific c o n t e n t o f m i c r o s o m a l monooxygenase components and the specific activities of monooxygenase reactions were t h e s a m e w h e t h e r e x p r e s s e d p e r m i l l i g r a m o f m i c r o s o m a l p r o t e i n o r p e r g r a m o f liver. T h o s e p a r a m e t e r s o f t h e m i c r o s o m a l m o n o o x y g e n a s e s y s t e m t h a t w e r e m e a s u r e d in b o t h 3-month-old and 5-month-old rats (cytochrome P-450, NADPH-cytochrome P-450 reductase, benzphetamine N-demethylase and aniline hydroxylase) were not significantly d i f f e r e n t a n d v a l u e s f o r 3-, 4-, a n d 5 - m o n t h - o l d r a t s w e r e p o o l e d .
DECLINE IN HEPATICMICROSOMALMONOOXYGENASECOMPONENTS
255
TABLE 1. LIVER WEIGHT, MICROSOMALPROTEIN AND RECOVERY OF MICROSOMAL MEMBRANES
Liver weight (g) Microsomal p r o t e i n t (mg/g wet weight) Recovery of microsomal membranes:l: (°70)
Young-adult
Middle-aged
10.9 _+0.2
16.5 _+0.3*
29.0 _+0.9
30.2 _+0.7
42
43
_+2
_+2
Male Fischer 344 rats were housed under strictly controlled environmental conditions and maintained on a semipurified diet and water ad lib. Young-adult rats were 3-5 months of age and averaged 270-350 g of body weight. Middleaged rats were 14 months of age and weighed an average of 530 g. Values are means _+SE; N 2 = 6. * p < 0.01. "[-Corrected for recovery of microsomal membranes. ~ T h e recovery of microsomal membranes was estimated by determining cytochrome c reductase activity in liver homogenate vs microsomes.
Aging from 5 to 14 months was accompanied by decreases in several components of the hepatic microsomal monooxygenase system, including cytochrome P-450 content (32%), NADPH-cytochrome c reductase activity (42%) and phospholipid content (41%). A less pronounced decrease (22%) in cytochrome b5 was also seen (Table 2). Of the microsomal monooxygenase components"measured, only NADPH-cytochrome P-450 reductase activity was not significantly decreased in middle-aged compared with young-adult rats. Interestingly, liver microsomal monooxygenase activities in middle-aged rats were not uniformly decreased as a consequence of the decline in microsomal components. In fact, nitroanisole O-demethylation increased 44% and benzphetamine N-demethylation was not changed significantly. On the other hand aniline hydroxylation decreased 33% (Table 3). The substrate selectivity of these age-associated changes in microsomal drug metabolism are consistent with published observations from studies with senescent rodents (Birnbaum and Baird, 1978a; Birnbaum, 1980; McMartin et al., 1980). TABLE 2. COMPONENTS OF THE HEPATIC MICROSOMAL MONOOXYGENASESYSTEM
Cytochrome P-450 (nmol/mg protein) Cytochrome c reductase activityt ( n m o l / m i n / m g protein) Phospholipids (nmol lipid P / m g protein) Cytochrome b~ (nmol/mg protein) Cytochrome P-450 reductase activity~ ( n m o l / m i n / m g protein)
Young-adult
Middle-aged
0.79_+ 0.01
0.54+_ 0.03*
240_+ 10
140_+ 10"
750 _+20
440 _+ 10"
0.46_+ 0,01
0.36_+ 0.01"
0.67 _+ 0.04
0.76_+ 0.08
Young-adult rats were 3-5 months and middle-aged rats were 14 months, except those used for phospholipid analyses, which were 5 and 16 months of age. Values are means -+ SE; N , 6.
*p<0.01. t n m o l cytochrome c reduced per rain per mg microsomal protein at 23°C, t n m o l cytochrome P-450 reduced per rain per mg microsomal protein at 15°C.
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LORA E. R I K A N S A N D B R U C E A . N O T L E Y
T A B L E 3. H E P A T I C MICROSOMAL M O N O O X Y G E N A S E A C T I V I T I E S
Activity (nmol/min/mg) Young-adult Middle-aged Nitroanisole O-demethylase* Aniline hydroxylasezl: Benzphetamine N-demethylase§
1.02 -+0.04 0.72 _+0.02 4.89 _+0.11
1.47 -+ 0.11 t 0.48 _+0.03t 5.43 _+0.32
Ages were 3-5 months and 14 months. Values are means --- SE; N < 6. *nmol p-nitrophenol produced per min per mg microsomal protein at 37°C. t p < 0.01. ~nmolp-aminophenol produced per min per mg microsomal protein at 37°C. §nmol HCHO produced per min per mg microsomal protein at 37°C. T h e total fatty acid c o n t e n t was significantly less (33%) in liver m i c r o s o m e s f r o m m i d d l e - a g e d c o m p a r e d with y o u n g - a d u l t rats. F u r t h e r m o r e , gas c h r o m a t o g r a p h i c analysis o f fatty acid m e t h y l esters indicated that the c o m p o s i t i o n o f the m i c r o s o m a l fatty acids was significantly altered. Percentage increases in stearic and arachidonic acids and decreases in p a l m i t o l e i c , oleic a n d linoleic acids were o b s e r v e d in microsomes f r o m 16-month-old rats c o m p a r e d with t h o s e f r o m 5 - m o n t h - o l d rats (Table 4), A l t h o u g h these d i f f er en ces a p p e a r e d small, e.g. 9.2 vs 11.6% o f oleic a n d 28.0 vs 2 6 . 0 % o f a r a c h i d o n i c , they were similar in m a g n i t u d e to the changes in m i c r o s o m a l fatty acid c o m p o s i t i o n that o ccu r with p h e n o b a r b i t a l i n d u c t i o n , e.g. 7.71 vs 10.95% o f oleic an d 16.39 vs 13.65% o f a r a c h i d o n i c (Ilyas, de la Iglesia a n d Feuer, 1978). In spite o f this significantly d i f f e r e n t d i s t r i b u t i o n , aging did not alter the ratio o f u n s a t u r a t e d to s a t u r a t e d or p o l y u n s a t u r a t e d to m o r e s a t u r a t e d fatty acids. TABLE 4.
R E L A T I V E C O M P O S I T I O N OF LIVER M I C R O SOMAL F A T T Y ACIDS
Fatty acid 16 : 0 16:1 18 : 0 18 : 1 18 : 2 20 : 4 22:4 22 : 6
Weight percentage of total fatty acids Young-adult Middle-aged 17.9_ 0.2 3.5_+0.1 20.4 _+0.3 11.6_+0.2 8.3 _+0.4 26.0 _+0.4 4.6_+0.1 2.7 _+0.2
17.0_+ 0.3 2.6_+0.1. 22.0 _+0.4t 9.2_+0.2* 6.0_+0.1" 28.0 _+0.3* 4.9_+0.3 2.3 -+0.3
Ages were 5 months and 16 months. Values are means for 6 rats +- SE. Total fatty acids were 377 _+ 12 and 292 _+ 7 tag/rag microsomal protein in young adult and middle-aged rats, respectively. *p < 0.01. t p < 0.05. DISCUSSION P r e v i o u s studies o f age-related changes in liver d r u g - m e t a b o l i z i n g en zy m es showed no significant d i f f e r e n c e s between y o u n g adult and m i d d l e - a g e d rats ( B i r n b a u m and Baird, 1978b; M c M a r t i n , 1980). H o w e v e r , those studies used y o u n g e r an i m al s for their middleaged g r o u p s , as well as d i f f e r e n t strains o f rats, than those used in the present study. Th e
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marked reduction in microsomal monooxygenase components that occurred at 14 months of age in our study may indicate that age-related changes in the monooxygenase system are not related exclusively to senescence. In fact, Richardson and coworkers have observed that in male Fischer 344 rats the decline in liver microsomal cytochrome P-450 is greater between 12 and 18 months than it is between 18 and 27 months of age (Richardson, A., personal communication). Thus, it appears that in different strains of rats the specific contents of microsomal monooxygenase components decrease at different ages (or remain unchanged), and that in male Fischer 344 rats the reductions occur during the middle years. Of interest was the finding that some drug metabolism activities were unchanged or increased in spite of substantial decreases in monooxygenase components. However, there was no decrease in the enzymatic reduction of cytochrome P-450, the putative ratelimiting step in microsomal monooxygenase reactions (Gillette and Gram, 1969; Imai, Sato and Iyanagi, 1977; Yang, Strickhart and Kicha, 1978). Although microsomes from middle-aged rats exhibited lower rates of cytochrome c reduction, this activity cannot be equated with cytochrome P-450 reduction (Vermilion and Coon, 1978; Guenthner, Kahl and Nebert, 1980). Others have shown that changes in monooxygenase activity correlate well with changes in the rate of cytochrome P-450 reduction and not very well with changes in the rate of cytochrome c reduction (Davies, Gigon and Gillette, 1969; Zannoni et al., 1972; Mellon, Witiak and Feller, 1978). Our results indicated that in liver microsomes from middle-aged rats, the components that declined with age were not rate-limiting for the monooxygenation of at least two substrates. The substrate dependency of the changes in drug metabolism activity in middle-aged rats implies that aging affected microsomal metabolism in a complex fashion that was not related to a change in the total quantity of a certain membrane component or enzyme. Cytochrome P-450, the terminal oxidase and specificity-conferring component of the monooxygenase system, exists in multiple forms. Age-related changes in the relative proportions of functionally different forms could selectively increase or decrease the metabolism of different substrates. Evidence for a selective effect of aging on different forms of cytochrome P-450 has been reported by McMartin et al. (1980). Using regioselective metabolism of R-warfarin to identify the presence of different forms of the cytochrome, they showed that liver microsomes from old Wistar rats had reduced amounts of one form of cytochrome P-450 (PB-C) and approximately equal amounts of other forms (cytochromes P-450, BNF-B and PB-B). On the other hand, Birnbaum and Baird (1978a, b) found no detectable age-related variation in rat liver microsomal cytochrome P-450 profiles, based on electrophoretograms of heme-staining polypeptides. Obviously, further work will be necessary to define clearly the effects of aging on different forms of cytochrome P-450. Phospholipid is an integral part of the cytochrome P-450-dependent system and plays a specific role in monooxygenase reactions. Recent evidence suggests that phospholipid facilitates the formation of a binary complex between cytochrome P-450 and its reductase (Lu, Miwa and West, 1980). Reconstitution experiments using purified components of the system have demonstrated that the fatty acid moieties of the phospholipid molecule are important determinants of monooxygenase activity and that different purified forms of the cytochrome require different types and amounts of phospholipid (Strobel, Lu, Heidema and Coon, 1970; Warner and Neims, 1979). Moreover, compositional changes in the fatty acids of microsomai phospholipids are known to occur with treatments which
258
LORAE. RIKANSAND BRUCEA. NOTLEY
change the activity and the substrate selectivity of the microsomal enzyme system (Becker et aL, 1978; Ilyas et aL, 1978; Hammer and Wills, 1979). Thus, it seems reasonable to propose that the substrate-specific changes that occurred in the present study may have resulted also from changes in the fatty acid composition of the phospholipids. However, other possible explanations for the substrate selectivity of the age-related changes in drug metabolism cannot be excluded. For example, age-associated changes in the composition of the microsomal membrane could differentially affect the accessibility of the monooxygenase system to the various substrates. Finally, age-related changes in the organization of microsomal components within the membrane could specifically affect monooxygenase activities. SUMMARY Monooxygenase components and drug metabolism activities were determined in liver microsomes from young-adult (3-5 months) and middle-aged (14 months) male Fischer 344 rats. Several components of the monooxygenase system were decreased in middle-aged rats including total cytochrome P-450, cytochrome bs, NADPH-cytochrome c reductase activity and phospholipids. However, the reduction of cytochrome P-450 by NADPH, thought to be rate-limiting for monooxygenase activity, was not decreased. Drug metabolism activities in microsomes from middle-aged rats were increased (nitroanisole O-demethylation), decreased (aniline hydroxylation) or unchanged (benzphetamine N-demethylation). Aging decreased the microsomal content of phospholipids and changed the relative percentages of several microsomal fatty acids. The substrate selectivity of the age-related changes in drug metabolism activities may be related to changes in the fatty acid composition of microsomal phospholipids. Acknowledgement--This work was supported by NIH grant AG-01202 from the National Institute on Aging.
REFERENCES ALDEMAN, R. C. (1971)Exp. Gerontol. 6, 75. BAIRD, M. B., NICOLOSl, R. J., MASSIE, H. R. and SArdis, H. V. (1975)Exp. Gerontol. 10.89. BECKER, J. F., MEEHAN, T. and BARTHOLOMEW,J. C. (1978)Biochim. biophys. Acta 512, 136. BIRNBAUM,L. S. (1980)Exp. Gerontol. 15,259. BIRNBAUM,L. S. and BAIRD, M. B. (1978a)Exp. Gerontol. 13,299. BIRNaAUM, L. S. and BAIRD, M. I . (1978b) Exp. Gerontol. 13,469. DAVIES, D. S., GIGON, P. L. and GILLETTE, J. R. 0969) LifeSci. 8, 85. ERIKSSON, L. C., DEPIERRE, J. W. and DALLNER,G. (1978) Pharmac. Ther. 2,281. FISKE, C. H. and SUaaAROW,Y. (1925)./. biol. Chem. 66,375. GILLETTE, J. R. and GRAM, Z. E. (1969). In: Microsomes and Drug Oxidations (Edited by J. R. GILLETTE, A. H. CONNEY, G. J. COSMIDES, R. W. ESTABROOK,J. R. FOUTSand G. J. MANNERING), pp. 133-149. Academic Press, New York. GRINNA, L. S. and BARBER,A. A. (1972) Biochim. biophys. Acta 288,347. GRINNA, L. S. (1977)Mech. Ageing Dev. 6, 197. GUENTHNER, T. M., KAHL,G. F. and NEBERT, D. W. (1980)Biochem. Pharmac. 29, 89. HAMMER, C. T. and WILLS,E. D. (1979) Br. J. Nutr. 41,465. ILYAS, M. S., DE LA IGLESIA, F. A. and FEUER, G. (1978) Toxic. appl. Pharmac. 44,491. IMAI, Y., ITO, A. and SATO, R. (1966) J. Biochem. 60, 417. IMAI, Y., SATO, R. and IYANAGI,"F. (1977)J. Biochem. 82, 1237. KAO, J. and HUDSON, P. (1980)Biochem. Pharmac. 29, 1191. KATO, R. and TAKANAKA,A. (1968a) J. Biochem. 63,406. KATO, R. and TAKANAKA,A. (1968b) Jap. J. Pharmac. 18, 381. KATO, R. and TAKANAKA,A. (1968C) Jap. J. Pharmac. 18,389.
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Lu, A. Y. H., MIWA, G. T. and WEST, S. B. (1980) In: Microsomes, Drug Oxidations, and Chemical Carcinogenesis (Edited by M. J. CooN, A. H. CO~NEY, R. W. ESTABROOK, H. V. GELBO1N, J. R. GILLETTE and P. J. O'BRIEN), pp. 59-66. Academic Press, New York. MARKWELL,M. A. K., HAAS, S. M., BIEBER, L. L. and TOLBERT, N. E. (1978) Analyt. Biochem. 87, 206. MAY, H. E. and McCAY, P. B. (1968) J. biol. Chem. 243, 2288. MCMARTIN, D. N., O'CONNOR, J. A. jr., FASCO,J. J. and KAMINSKY,L. S. (1980) Toxic. appl. Pharmac. 54, 411. MELLON, W. S., WITIAK, D. T. and FELLER,D. R. (1978)Biochem. Pharmac. 27, 1055. NASH, T. (1953) J. biol. Chem. 55,416. NETTER, K. J. and SEIDEL,G. (1964) J. Pharmac. exp. Ther. 146, 61. OMURA, T. and SATO, R. (1964) J. biol, Chem. 239, 2370. RIKANS, L. E. and NOTLEY, B. A. (1980) Pharmacologist 22,276. SCHMUCKER,D. L. and WANG, R. K. (1980)Exp. Gerontol. 15,321. STROBEL, H. W., LU, A. Y. H., HEIDEMA,J. and COON, M. J. (1970) J. bioL Chem. 245, 4851. VERMILION,J. L. and CooN, M. J. (1978) J. biol. Chem. 253, 2694. WARNER, M. and NEIMS, A. H. (1979)Drug Metab. Disp. 7, 188. YANG, C. S., STICKHART,F. S. and KICHA, L. P. (1978)Biochim. biophys. Acta 509,326. ZANNONI, V. G., FtYNN, E. J. and LYNCH, M. (1972)Biochem. Pharmac. 21, 1377.
0531-5565/81/030253~7$02.00/0 PergamonPressLtd.
DECLINE IN HEPATIC MICROSOMAL MONOOXYGENASE COMPONENTS IN MIDDLE-AGED FISCHER 344 RATS L O R A E . RIKANS a n d B R U C E A . NOTLEY Department of Pharmacology, University o f O k l a h o m a , Health Sciences Center, O k l a h o m a City, OK 73190, U.S.A. (Received 21 December 1980)
INTRODUCTION THE AGINGprocess involves physiological changes which alter an animal's response to drugs. Early studies with old rats demonstrated that the altered response could be attributed in part to a decrease in the metabolism of drugs, as demonstrated by in vivo and in vitro experiments in which the capacity of the liver microsomal drug metabolism system declined with age (Kato and Takanaka, 1968a, b, c). It was accepted that this age-related decline was due to decreases in components of the hepatic cytochrome P-450-dependent monooxygenase system. However, conflicting reports on the effects of aging on the microsomal enzyme system have recently appeared. The microsomal monooxygenase system includes three major components, cytochrome P-450, NADPH-cytochrome P-450 reductase (NADPH-cytochrome c reductase) and phospholipid. Cytochrome b5 may be involved, too. Several investigators have reported that cytochrome P-450 specific content and NADPH-cytochrome c reductase activity are decreased in liver microsomes from old rats (Kato and Takanaka, 1968a, b; Kao and Hudson, 1980; McMartin, O'Connor, Fasco and Kaminsky, 1980; Schmucker and Wang, 1980). An age-related reduction in the quantity of microsomal phospholipid has also been found (Birnbaum and Baird, 1978b; Grinna and Barber, 1972; Grinna, 1977). On the other hand, others have shown that aging does not lower cytochrome P-450 content (Baird, Nicholosi, Massie and Samis, 1975; Birnbaum and Baird, 1978b), cytochrome c reductase activity (Aldeman, 1971; Birnbaum and Baird, 1978a, b) or cytochrome b5 content (Birnbaum and Baird, 1978a; McMartin, 1980) in rat liver microsomes. Furthermore, a study with senescent mice indicated that drug metabolism activities could be markedly altered (increased or decreased) while cytochrome P-450 content, reductase activity and phospholipid content remained unchanged (Birnbaum, 1980). Although some of these apparently contradictory results may be attributed to differences in animal sex, strain and species, or to differences in the ages of the " y o u n g " and " o l d " animals used for comparisons, it is clear that the exact mechanisms for the age-related changes in drug metabolism remain unresolved. The transition between young adulthood and old age is important for understanding the underlying causes of aging processes. The recent studies that examined microsomal drug metabolism in middle-aged rats found no significant differences in monooxygenase activities between middle-aged and young-adult animals (Birnbaum and Baird, 1978b; McMartin et aL, 1980). In these studies the middle-aged rats were 12 months and the young-adult rats were 3 or 4 months of age. In this study we found significant differences between middle-aged (14 months) and young-adult (3-5 months) rats in liver microsomal monooxygenase components and drug 253
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metabolism
LORA E . RIKANS A N D B R U C E A. NOTLEY
activities. Changes
in t h e l i p i d c o m p o s i t i o n
of the hepatic microsomal
m e m b r a n e w e r e also seen. A p r e l i m i n a r y r e p o r t o f t h e s e results h a s b e e n p r e s e n t e d ( R i k a n s a n d N o t l e y , 1980). MATERIALS
AND METHODS
Male Fischer 344 rats were purchased from Charles River Breeding Laboratories at 7 weeks of age and maintained in a separate room within our animal facilities for the duration of the study. The rats were given a semipurified diet (A1N 76 semipurified Diet ®, Nutritional Biochemicals) and water ad lib, They were housed 5/cage until 6 months of age and 3/cage from 6 to 16 months of age, in heat-sterilized hard-wood bedding, under filter caps. The room in which the animals were housed was kept free of cigarette smoke, pesticides, fumigants, disinfectants, etc. Temperature, humidity and lighting remained constant. Rats were killed 3 h after the onset of the 12 h light periods. The livers were removed and homogenized in 4 volumes of a solution containing 1.15% KCI and 0.05 M Tris-HCl, pH 7.5. The homogenates were centrifuged at 10,000 g for 15 min and the resulting supernatant fractions centrifuged at 78,000 g for 90 min. The 78,000 g pellets were washed once with homogenization medium and the final microsomal pellets suspended in 0.1 M Tris-HCl, pH 7.5, to a protein concentration of 5-10 mg/ml. All solutions were kept at 0-4°C. Centrifugations were performed at the g values determined for the average radii of the rotors used. The recovery of microsomal membranes was estimated by determining the total activity of the marker enzyme, cytochrome c reductase, in liver homogenates versus the microsomal fraction (Eriksson, DePierre and Dallner, 1978). Cytochrome P-450 was determined in microsomes from the reduced CO difference spectrum and cytochrome b 5 from the NADH-reduced difference spectrum (Omura and Sato, 1964). Rates of cytochrome c reduction by NADPH were determined at 23°C in a high ionic strength buffer as described by Vermilion and Coon (1978). The reduction of cytochrome P-450 in microsomes was measured at 15°C by following the formation of the reduced cytochrome P-450-CO complex after NADPH addition under anaerobic conditions (Zannoni, Flynn and Lynch, 1972). Microsomal fatty acids were determined using the Folch extraction procedure and gas chromatographic analysis of fatty acid methyl esters (May and McCay, 1968). Phospholipids were determinted as inorganic phosphate in the total extracted lipid (Fiske and Subbarow, 1925). Protein was measured in the presence of sodium dodecyl sulfate by a modification (Markwell, Haas, Bieber and Tolbert, 1978) of the Lowry procedure. The drug metabolism assays were performed using freshly-prepared microsomes. Conditions were optimized for each reaction so that activities were proportional to enzyme concentration and linear with time. Incubations were carried out in a shaking water bath at 37°C. The assay for nitroanisole O-demethylase activitiy was based on the procedure of Netter and Seidel (1964). Reaction mixtures, containing 0.3-0.6 mg microsomal protein, 1 mM p-nitroanisole, 6.8 mM glucose-6-phosphate, 0.27 mM NADP, 100 mM Tris-HCl (pH 7.8) and 1 unit of glucose-6-phosphate dehydrogenase, in a total volume of 2.0 ml, were incubated for 15 min. Reactions were stopped by adding 0.2 ml of 2.2 M NaOH, which served to clarify the mixture (Becker, Meehan and Bartholomew, 1978), and the absorbance of the product, p-nitrophenol, was measured at 415 nm. Aniline hydroxylase activity was determined according to Imai et al. (1966) except that the concentrations of aniline and nicotinamide were 1.25 and 2.5 mM, respectively. Incubation mixtures for the assay of benzphetamine N-demethylase contained: microsomes (0.8 1.6 mg protein), 5 mM benzphetamine, 7.5 mM glucose-6-phosphate, 0.3 mM NADP, 3.0 mM nicotinamide, 100 mM Tris-HCI (pH 7.5) and 5 units of glucose-6-phosphate dehydrogenase, in a total volume of 2.0 ml. After incubating for 15 min the reaction was stopped by adding 0.7 ml of 20°7o trichloroacetic acid. Protein was removed by centrifugation and the supernatant solution was assayed for formaldehyde using Nash reagent (Nash, 1953).
RESULTS A l t h o u g h liver w e i g h t s w e r e g r e a t e r in 1 4 - m o n t h - o l d r a t s c o m p a r e d w i t h 3 - 5 - m o n t h - o l d r a t s , t h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s in r e c o v e r y o f m i c r o s o m a l m e m b r a n e s o r i n t h e p r o t e i n c o n t e n t o f t h e m i c r o s o m e s ( T a b l e 1). T h e r e f o r e , t h e specific c o n t e n t o f m i c r o s o m a l monooxygenase components and the specific activities of monooxygenase reactions were t h e s a m e w h e t h e r e x p r e s s e d p e r m i l l i g r a m o f m i c r o s o m a l p r o t e i n o r p e r g r a m o f liver. T h o s e p a r a m e t e r s o f t h e m i c r o s o m a l m o n o o x y g e n a s e s y s t e m t h a t w e r e m e a s u r e d in b o t h 3-month-old and 5-month-old rats (cytochrome P-450, NADPH-cytochrome P-450 reductase, benzphetamine N-demethylase and aniline hydroxylase) were not significantly d i f f e r e n t a n d v a l u e s f o r 3-, 4-, a n d 5 - m o n t h - o l d r a t s w e r e p o o l e d .
DECLINE IN HEPATICMICROSOMALMONOOXYGENASECOMPONENTS
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TABLE 1. LIVER WEIGHT, MICROSOMALPROTEIN AND RECOVERY OF MICROSOMAL MEMBRANES
Liver weight (g) Microsomal p r o t e i n t (mg/g wet weight) Recovery of microsomal membranes:l: (°70)
Young-adult
Middle-aged
10.9 _+0.2
16.5 _+0.3*
29.0 _+0.9
30.2 _+0.7
42
43
_+2
_+2
Male Fischer 344 rats were housed under strictly controlled environmental conditions and maintained on a semipurified diet and water ad lib. Young-adult rats were 3-5 months of age and averaged 270-350 g of body weight. Middleaged rats were 14 months of age and weighed an average of 530 g. Values are means _+SE; N 2 = 6. * p < 0.01. "[-Corrected for recovery of microsomal membranes. ~ T h e recovery of microsomal membranes was estimated by determining cytochrome c reductase activity in liver homogenate vs microsomes.
Aging from 5 to 14 months was accompanied by decreases in several components of the hepatic microsomal monooxygenase system, including cytochrome P-450 content (32%), NADPH-cytochrome c reductase activity (42%) and phospholipid content (41%). A less pronounced decrease (22%) in cytochrome b5 was also seen (Table 2). Of the microsomal monooxygenase components"measured, only NADPH-cytochrome P-450 reductase activity was not significantly decreased in middle-aged compared with young-adult rats. Interestingly, liver microsomal monooxygenase activities in middle-aged rats were not uniformly decreased as a consequence of the decline in microsomal components. In fact, nitroanisole O-demethylation increased 44% and benzphetamine N-demethylation was not changed significantly. On the other hand aniline hydroxylation decreased 33% (Table 3). The substrate selectivity of these age-associated changes in microsomal drug metabolism are consistent with published observations from studies with senescent rodents (Birnbaum and Baird, 1978a; Birnbaum, 1980; McMartin et al., 1980). TABLE 2. COMPONENTS OF THE HEPATIC MICROSOMAL MONOOXYGENASESYSTEM
Cytochrome P-450 (nmol/mg protein) Cytochrome c reductase activityt ( n m o l / m i n / m g protein) Phospholipids (nmol lipid P / m g protein) Cytochrome b~ (nmol/mg protein) Cytochrome P-450 reductase activity~ ( n m o l / m i n / m g protein)
Young-adult
Middle-aged
0.79_+ 0.01
0.54+_ 0.03*
240_+ 10
140_+ 10"
750 _+20
440 _+ 10"
0.46_+ 0,01
0.36_+ 0.01"
0.67 _+ 0.04
0.76_+ 0.08
Young-adult rats were 3-5 months and middle-aged rats were 14 months, except those used for phospholipid analyses, which were 5 and 16 months of age. Values are means -+ SE; N , 6.
*p<0.01. t n m o l cytochrome c reduced per rain per mg microsomal protein at 23°C, t n m o l cytochrome P-450 reduced per rain per mg microsomal protein at 15°C.
256
LORA E. R I K A N S A N D B R U C E A . N O T L E Y
T A B L E 3. H E P A T I C MICROSOMAL M O N O O X Y G E N A S E A C T I V I T I E S
Activity (nmol/min/mg) Young-adult Middle-aged Nitroanisole O-demethylase* Aniline hydroxylasezl: Benzphetamine N-demethylase§
1.02 -+0.04 0.72 _+0.02 4.89 _+0.11
1.47 -+ 0.11 t 0.48 _+0.03t 5.43 _+0.32
Ages were 3-5 months and 14 months. Values are means --- SE; N < 6. *nmol p-nitrophenol produced per min per mg microsomal protein at 37°C. t p < 0.01. ~nmolp-aminophenol produced per min per mg microsomal protein at 37°C. §nmol HCHO produced per min per mg microsomal protein at 37°C. T h e total fatty acid c o n t e n t was significantly less (33%) in liver m i c r o s o m e s f r o m m i d d l e - a g e d c o m p a r e d with y o u n g - a d u l t rats. F u r t h e r m o r e , gas c h r o m a t o g r a p h i c analysis o f fatty acid m e t h y l esters indicated that the c o m p o s i t i o n o f the m i c r o s o m a l fatty acids was significantly altered. Percentage increases in stearic and arachidonic acids and decreases in p a l m i t o l e i c , oleic a n d linoleic acids were o b s e r v e d in microsomes f r o m 16-month-old rats c o m p a r e d with t h o s e f r o m 5 - m o n t h - o l d rats (Table 4), A l t h o u g h these d i f f er en ces a p p e a r e d small, e.g. 9.2 vs 11.6% o f oleic a n d 28.0 vs 2 6 . 0 % o f a r a c h i d o n i c , they were similar in m a g n i t u d e to the changes in m i c r o s o m a l fatty acid c o m p o s i t i o n that o ccu r with p h e n o b a r b i t a l i n d u c t i o n , e.g. 7.71 vs 10.95% o f oleic an d 16.39 vs 13.65% o f a r a c h i d o n i c (Ilyas, de la Iglesia a n d Feuer, 1978). In spite o f this significantly d i f f e r e n t d i s t r i b u t i o n , aging did not alter the ratio o f u n s a t u r a t e d to s a t u r a t e d or p o l y u n s a t u r a t e d to m o r e s a t u r a t e d fatty acids. TABLE 4.
R E L A T I V E C O M P O S I T I O N OF LIVER M I C R O SOMAL F A T T Y ACIDS
Fatty acid 16 : 0 16:1 18 : 0 18 : 1 18 : 2 20 : 4 22:4 22 : 6
Weight percentage of total fatty acids Young-adult Middle-aged 17.9_ 0.2 3.5_+0.1 20.4 _+0.3 11.6_+0.2 8.3 _+0.4 26.0 _+0.4 4.6_+0.1 2.7 _+0.2
17.0_+ 0.3 2.6_+0.1. 22.0 _+0.4t 9.2_+0.2* 6.0_+0.1" 28.0 _+0.3* 4.9_+0.3 2.3 -+0.3
Ages were 5 months and 16 months. Values are means for 6 rats +- SE. Total fatty acids were 377 _+ 12 and 292 _+ 7 tag/rag microsomal protein in young adult and middle-aged rats, respectively. *p < 0.01. t p < 0.05. DISCUSSION P r e v i o u s studies o f age-related changes in liver d r u g - m e t a b o l i z i n g en zy m es showed no significant d i f f e r e n c e s between y o u n g adult and m i d d l e - a g e d rats ( B i r n b a u m and Baird, 1978b; M c M a r t i n , 1980). H o w e v e r , those studies used y o u n g e r an i m al s for their middleaged g r o u p s , as well as d i f f e r e n t strains o f rats, than those used in the present study. Th e
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marked reduction in microsomal monooxygenase components that occurred at 14 months of age in our study may indicate that age-related changes in the monooxygenase system are not related exclusively to senescence. In fact, Richardson and coworkers have observed that in male Fischer 344 rats the decline in liver microsomal cytochrome P-450 is greater between 12 and 18 months than it is between 18 and 27 months of age (Richardson, A., personal communication). Thus, it appears that in different strains of rats the specific contents of microsomal monooxygenase components decrease at different ages (or remain unchanged), and that in male Fischer 344 rats the reductions occur during the middle years. Of interest was the finding that some drug metabolism activities were unchanged or increased in spite of substantial decreases in monooxygenase components. However, there was no decrease in the enzymatic reduction of cytochrome P-450, the putative ratelimiting step in microsomal monooxygenase reactions (Gillette and Gram, 1969; Imai, Sato and Iyanagi, 1977; Yang, Strickhart and Kicha, 1978). Although microsomes from middle-aged rats exhibited lower rates of cytochrome c reduction, this activity cannot be equated with cytochrome P-450 reduction (Vermilion and Coon, 1978; Guenthner, Kahl and Nebert, 1980). Others have shown that changes in monooxygenase activity correlate well with changes in the rate of cytochrome P-450 reduction and not very well with changes in the rate of cytochrome c reduction (Davies, Gigon and Gillette, 1969; Zannoni et al., 1972; Mellon, Witiak and Feller, 1978). Our results indicated that in liver microsomes from middle-aged rats, the components that declined with age were not rate-limiting for the monooxygenation of at least two substrates. The substrate dependency of the changes in drug metabolism activity in middle-aged rats implies that aging affected microsomal metabolism in a complex fashion that was not related to a change in the total quantity of a certain membrane component or enzyme. Cytochrome P-450, the terminal oxidase and specificity-conferring component of the monooxygenase system, exists in multiple forms. Age-related changes in the relative proportions of functionally different forms could selectively increase or decrease the metabolism of different substrates. Evidence for a selective effect of aging on different forms of cytochrome P-450 has been reported by McMartin et al. (1980). Using regioselective metabolism of R-warfarin to identify the presence of different forms of the cytochrome, they showed that liver microsomes from old Wistar rats had reduced amounts of one form of cytochrome P-450 (PB-C) and approximately equal amounts of other forms (cytochromes P-450, BNF-B and PB-B). On the other hand, Birnbaum and Baird (1978a, b) found no detectable age-related variation in rat liver microsomal cytochrome P-450 profiles, based on electrophoretograms of heme-staining polypeptides. Obviously, further work will be necessary to define clearly the effects of aging on different forms of cytochrome P-450. Phospholipid is an integral part of the cytochrome P-450-dependent system and plays a specific role in monooxygenase reactions. Recent evidence suggests that phospholipid facilitates the formation of a binary complex between cytochrome P-450 and its reductase (Lu, Miwa and West, 1980). Reconstitution experiments using purified components of the system have demonstrated that the fatty acid moieties of the phospholipid molecule are important determinants of monooxygenase activity and that different purified forms of the cytochrome require different types and amounts of phospholipid (Strobel, Lu, Heidema and Coon, 1970; Warner and Neims, 1979). Moreover, compositional changes in the fatty acids of microsomai phospholipids are known to occur with treatments which
258
LORAE. RIKANSAND BRUCEA. NOTLEY
change the activity and the substrate selectivity of the microsomal enzyme system (Becker et aL, 1978; Ilyas et aL, 1978; Hammer and Wills, 1979). Thus, it seems reasonable to propose that the substrate-specific changes that occurred in the present study may have resulted also from changes in the fatty acid composition of the phospholipids. However, other possible explanations for the substrate selectivity of the age-related changes in drug metabolism cannot be excluded. For example, age-associated changes in the composition of the microsomal membrane could differentially affect the accessibility of the monooxygenase system to the various substrates. Finally, age-related changes in the organization of microsomal components within the membrane could specifically affect monooxygenase activities. SUMMARY Monooxygenase components and drug metabolism activities were determined in liver microsomes from young-adult (3-5 months) and middle-aged (14 months) male Fischer 344 rats. Several components of the monooxygenase system were decreased in middle-aged rats including total cytochrome P-450, cytochrome bs, NADPH-cytochrome c reductase activity and phospholipids. However, the reduction of cytochrome P-450 by NADPH, thought to be rate-limiting for monooxygenase activity, was not decreased. Drug metabolism activities in microsomes from middle-aged rats were increased (nitroanisole O-demethylation), decreased (aniline hydroxylation) or unchanged (benzphetamine N-demethylation). Aging decreased the microsomal content of phospholipids and changed the relative percentages of several microsomal fatty acids. The substrate selectivity of the age-related changes in drug metabolism activities may be related to changes in the fatty acid composition of microsomal phospholipids. Acknowledgement--This work was supported by NIH grant AG-01202 from the National Institute on Aging.
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