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Effects of age, phenobarbital, β-naphthoflavone and dexamethasone on rat hepatic δ-aminolevulinic acid synthase
ELSEVIER
Archives of Gerontology and Geriatrics 20 (1995) 149-158
ARCHIVES OF GERONTOLOGY AND GERlATRICS
Effects of age, phenobarbital, /3-naphthoflavone and dexamethasone on rat hepatic Gaminolevulinic acid synthase Andrzej
Plewka
Department of Histology and Embryology, Silesian School of Medicine, ul. Medykdw 20. 40-7S2 Katowice-Ligota. Poland
Received 20 June 1994; revision received 16 September 1994; accepted 19 September 1994
Abstract
BAminolevulinic acid synthase is a key enzyme leading to the synthesis of cytochrome Pa compound of the mixed function oxidase system. Because of this, we have studied the effects of both age and three typical mixed function oxidase system inducers on this enzyme activity in male Wistar rats. Our results suggest that &aminolevulinic acid synthase activity in intact rats increases up to maturity and then decreases being, however, higher in the oldest animals than in the youngest. Phenobarbital affects &aminolevulinic acid synthase activity very characteristically. This activity is high in 4-week-old animals and then increases slightly until the twelfth month of life. Phenobarbital enzymic inducibility tends to decrease with age. In the /3-naphthoflavone groups, the profile of b-aminolevulinic acid synthase activity looks like that in the control ones. Generally, /3-naphthoflavone is a weaker S-aminolevulinic acid synthase inducer than phenobarbital, but it seems to induce &aminolevulinic acid synthase like dexamethasone, which, being a physiological inducer of the mixed function oxidasc system, is also an inducer of Gaminolevulinic acid synthase and this inducibility is closely connected with age. 450,
Keywords: Age; Rat; Liver; Inducers; &Arninolevulinic
acid synthase
1. Introduction
acid (ALA) synthase, an enzyme of the mitochondrial matrix, of glycine and succinyl-CoA and the formation of ALA (Ades, 1986). This is the first rate-limiting step in heme synthesis (Granick, 1966). The enzyme synthesis is regulated by induction and repression mechanisms, in&Aminolevulinic
catalyzes the condensation
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chiding quantitative changes in the de novo synthesis of this enzymatic protein (Maines and Kappas, 1978). ALA synthase (ALAS) has a very short half-time (about 60 min), thus results of its activity measurements reflect virtually the quantitative production of the enzyme (Granick and Urata, 1963). In a mature rat, the enzyme is appreciably induced by various compounds (Granick and Urata, 1963; Kappas et al., 1968) but, in the fetus, ALA synthase presents low inducibility (Song et al., 1971; Woods and Dixon, 1970). A low hepatic ALAS activity and the lack of its response to induction in early phases of development seem characteristic. Contrary to this, heme oxygenase (HO) activity is high in the neonatal liver but this enzyme is also weakly stimulated by some factors (Maines et al., 1976; Maines and Kappas, 1975a, 1976, 1977a,b,c). ALA synthase, just like heme oxygenase, is under the influence of both heme and various bivalent metal ions (Kondo et al., 1983; Maines and Kappas, 1974, 1975a, 1976, 1977a). On the basis of the similarity between the impacts of heme and metal ions on the ALA synthase, it is suggested that the central metal atom in heme is a physiological regulator of the enzyme synthesis and a tetrapyrrole may be considered an effective iron carrier to cellular regulatory sites connected with ALA synthase synthesis (Ades, 1986; Maines and Kappas, 1977a,b; Marver et al., 1966). Hepatic ALA synthase activity is decreased in newborn rats (Kappas et al., 1968; Woods and Dixon, 1970), while heme oxygenase (HO) activity is greatly increased (Maines and Kappas, 1975b; Thaler et al., 1972). One thinks that the increase of HO activity is a response to hemolysis in neonates, and the decreased ALA synthase activity is caused by various factors (Amelizad et al., 1987; Friedland and Ades, 1985; Song et al., 1971; Woods and Dixon, 1970). Regarding the heme oxygenase, similar investigations were carried out concerning the influences of inducers and ageing on this enzyme activity (Plewka et al., 1994a). It has been proved that ALA synthase activity sharply decreases in the last phase of the prenatal period. At this time, the influence of allylisopropylacetamide (AIA), a well-known ALA synthase inducer, on ALAS activity is remarkably weak (Song et al., 1971; Woods and Dixon, 1970) but, as proved in the quoted report (Maines and Kappas, 1978), the inhibition of synthase synthesis in rats is parallel to the increase of HO activity, hemolysis and degradation of fetal hemoglobin. This repression of the enzyme and its resistance to induction is considerably more dependent on chemical factors than on developmental ones. Based on the connections between b-aminolevulinic acid synthase, heme oxygenase activities and cytochrome P-450 level, the influence of age on these enzymes was investigated in previous studies (Plewka et al., 1994b). The factors leading, in the neonates of various species (including man), to the cellular heme release and causing a sharp decrease in the fetal hemoglobin level during the postnatal period may be described as follows (Maines and Kappas, 1978): heme, released from destroyed erythrocytes increases its oxidation by inducing heme oxygenase, which then frees iron. The metal ion inhibits ALA synthase synthesis and activates heme oxygenase. This process continues until both haemolysis and heme degradation are stopped. Then HO activity decreases, ALA synthase synthesis becomes possible and, in this way, its chemical induction is also allowed.
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2. Materials and methods 2.1. Animals Wistar inbred male rats from the animal colony of the Silesian Medical School were used. Animals were divided into one control group and three experimental groups, which were treated with inducers. Each series comprised eight age groups: 0.5-, l-, 2-, 4-, 8-, 12-, 20- and 2%month-old. The animals in the first group were with their mothers all the time. The older rats were on a standard diet and before decapitation were fasted for 12 h with free access to water. Control animals were not given any injections. Pilot trials proved that intraperitoneal injections of physiological saline solution didn’t cause statistically significant changes in the measured parameters. The first experimental group was given phenobarbital (50 mg/kg intraperitonally) twice, 3 and 2 days before being killed. This dose was slightly smaller than that usually used in other studies. It was proved that the maximal effective dose is 75 mg/kg (Tavolini et al., 1983). It was also proved that such a dose results in an increase of young animals’ mortality (Rikans and Notley, 1981). All the injections were given at 9.00 a.m. The second experimental group was given P-naphthoflavone, a compound belonging to the II group of inducers. It was injected three times (20 mg/kg) 3,2 and 1 days before sacrificing animals. This is the common dose given in many laboratories (e.g., Cresteil et al., 1986; Lum et al., 1985). This inducer was dissolved in corn oil. Finally, the third experimental group was treated intraperitoneally with dexamethasone, a physiological inducer. It was suspended in corn oil and was given three times (10 mg/kg). This is the most common dose given in other laboratories (Leakey et al., 1986), but not the highest used (Riddick and Marks, 1990; Wrighton and Elswick, 1989). 2.2. Isolation of mitochondrial fraction Rats were always decapitated at 9.00 a.m. The livers were immediately isolated and placed in ice cold physiological salt solution. Then they were washed several times, cut into small fragments and homogenized in a Potter-Elvehjem homogenizer with a teflon piston at 400 rpm and with four full piston’s moves. A homogenization medium was 0.25 M saccharose dissolved in 10 mM Tris-HCl buffer (pH 7.4). Five ml of this solution was given per g of the tissue. This step of processing, and those following, were performed at 2-4°C. The homogenate was centrifuged at 900 x g for 10 min to sediment nuclei and cell membranes. The supematant was centrifuged again at 10 000 x g for 20 min to sediment a mitochondrial fraction. The sediment was subtly suspended in the homogenization medium and centrifuged again at 10 Ooo x g for 20 min. Pure mitochondrial fractions were suspended in 20% (w/v) glycerol buffered as described above to obtain a protein concentration not smaller than 5 mg/cm3. Samples were immediately frozen and stored at -20°C until the next day in order to carry out measurements.
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2.3. ALA synthase activity measurements ALA synthase activity was determined by the modified method from Maines and Kappas laboratory (Granick, 1966; Sassa et al., 1979). The mitochondrial suspension was put in 75 mM phosphatic buffer (pH 7.4) with cofactors (glycine, magnesium chloride, sodium citrate, disodium ethylenediamine tetraacetate, pyridoxal phosphate), to get a final volume of 3 cm3. The mixture was incubated at 37°C for 1 h. Then the reaction was stopped by adding 1 cm3 of ice cold 10% trichloroacetic acid and by putting the tubes into ice for 10 min. The protein precipitate was centrifuged at 6000 r-pm for 10 min and the supematant was mixed with 1.5 cm3 of acetylacetone dissolved in 1 M sodium acetate. The mixture was incubated in a water bath at 80°C for 10 min. After cooling, the pH was adjusted to 7.0-7.5 by adding 0.5 cm3 of 0.5 N Na2HP04 mixed with 1 N NaOH (v/v 1:3). The mixture was shook with 4 cm3 of the so-called equilibrated ether (trichloroacetic acid, sodium octate, NaOH, disodium phosphate). A sample of the water layer (1.5 cm3) was pipetted into tubes containing 1.5 cm3 of a modified Ehrlich’s reagent @-dimethylaminobenzene aldehyde, 70% perchloric acid, ice cold acetic acid). After 15 mm, a spectral difference was measured in the range of 500-700 nm. The difference in absorption between 553 and 650 nm was used to determine ALA synthase activity and was expressed in nM/h per mg of protein. 2.4. Protein determination Protein was determined according to the method of Lowry et al. (1951). Bovine serum albumin was used as a standard. 2.5. Statistical analysis The results of the biochemical measurements are mean values from five separate measures. Statistical significance between age groups and individual series was evaluated by means of the Student’s t-test (P = 0.05). 3. Results ALA synthase activity in the control group (Fig. 1) rapidly increases up to the second month of life (150% of the activity in the 2-week-old animals). At the moment of reaching maturity, the enzyme activity begins to decrease rapidly until the fourth month of life and then more slowly until advanced senescence. Nevertheless, this activity, even in the oldest animals is higher than that in the youngest. Phenobarbital very characteristically affects ALA synthase activity (Fig. 1). In the phenobarbital-treated group, ALA synthase activity sharply rises in the first 4 weeks of life (175% of the activity in the youngest group) and then it falls slightly until the twelfth month of life. From this moment it decreases minimally so that it is clearly higher in the 28-month-old animals than in the youngest ones. Comparing the results in both control and phenobarbital-treated groups we notice a clearly stimulating influence of phenobarbital on ALA synthase activity (Fig. 1). Up to the eighth month of life, the enzyme activity oscillates between 165% in the
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Fig. 1. Age-related changes of mitochondrial Gaminolevulinic acid synthase activity in control group (K) and after phenobarbital (PB), fl-naphthoflavone (BNF) and dexamethasone (DEX) injection.
2-week-old animals and 230% in the l-month-old ones; there is not any characteristic correlation within this age range, though. After the eighth month of life the enzyme inducibility tends to decrease with age, but in the oldest animals it is still high (about 190% of the control value). In the /3-naphthoflavone series, the profile of ALA synthase activity, as a function of age, is identical to that in the control group (Fig. 1). The enzyme activity increases until the second month of life (about 125%, compared with the youngest animals) and then decreases until the end of life. This decrease is, however, greater because ALA synthase activity is clearly lower in the oldest animals than in the 2-week-old ones. fl-Naphthoflavone, a well-known inducer of the MFO system, surprisingly appears to be a much weaker ALA synthase stimulator than phenobarbital. In all the age groups, except the youngest animals in which the enzyme activity is identical to that caused by phenobarbital, ALA synthase activity is considerably lower (about 135%). As in the phenobarbital group, a very fast increase in ALA synthase activity is observed in the dexamethasone group from the second to the fourth week of life (Fig. 1). In the older group, the activity is 165% of the younger group activity. From this moment to the second month of life, the enzyme activity stabilizes and then slightly decreases up to the eighth month of life. Later, the enzyme activity is stabilized again until old age, and only in advanced senescence does the activity decrease more quickly. The physiological inducer of the MFO system used in this study is also an inducer
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of ALA synthase (Fig. 1). This inducibility, however, is closely connected with age. In the immature animals inducibility increases remarkably from the second week (about 120% of the control value) to the fourth week of life (almost 160% of the control value). Reaching maturity is connected with a certain decrease in inducibility (130% of the control value) which, however, increases in the older age groups and reaches 150% in the l-year-old animals, compared with the control group. Further ageing does not result in a decrease of inducibility so there are no great changes in the oldest animals, compared with the 12-month-old ones. 4. Discussion Based on the current data, one would think that heme controls the pathway of its own biosynthesis, mostly by influencing the translocation of inactive ALA presynthase from the cytosol to the mitochondrium in which the synthase is activated. Moreover, heme inhibits transcription of the ALA synthase gene. The final result of these processes is a ‘free heme pool’. Xenobiotics, including porphyrogenic compounds, influence this mechanism by decreasing this regulatory free heme pool in three different ways (Marks et al., 1989): (a) by cytochrome P-450 inactivation causing N-alkylprotoporphyrin formation and inhibition of ferrochelatase (the so-called primary mechanism of cytochrome P-450 inactivation); (b) by cytochrome P-450 inactivation bringing about continuous degradation of heme; (c) by enhanced generation of active oxygenic radicals which form a uroporphyrinogen decarboxylase inhibitor after interacting with endogenous substrates. The de novo heme is incorporated into several various hemoproteins, e.g., over half of this heme is used to synthesize cytochrome P-450. Heme, as shown by Granick (1966), also represses ALA synthase synthesis, but this subject is still being studied (Elliot et al., 1989; Srivastava et al., 1989). An important additional mechanism by which heme regulates ALA synthase activity is inhibition of the synthase precursors’ transport from the cytosol to the mitochondrion (Yamauchi et al., 1980) and modification of m-RNA translation into the synthase (Yamamoto et al., 1983). Therefore, heme may repress ALA synthase by inhibiting: transcription, translation and the synthase translocation from the cytosol to the mitochondrion. Ades et al. (1987) and Hamilton et al. (1988) showed that heme influences ALA synthase mainly by translocating the de novo synthesized synthase to the mitochondrion. Their results suggest that there is not any direct correlation between induced ALA synthase activity and m-RNA for this synthase. The final ALA synthase activity depends on both the level of transcribed m-RNA for this enzyme and cellular free heme content. In our study, we show that ALA synthase activity decreases from the moment of reaching maturity to advanced senescence. Lower ALA synthase activity at the end of life, compared with young animals, is in agreement with other studies (Abraham et al., 1985; Bitar and Shapiro, 1987; Bitar and Weiner, 1983; Paternitti et al., 1978,
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1980; Scotto et al., 1983) and moreover, the decrease in the synthase activity in old animals (70% compared with 2-month-old animals) is analogous with the mentioned studies. Kaliman and Nikitchenko (1989) showed that ALA synthase activity decreases until the third month of life and then remains unchanged till the end of life. We are not able to discuss changes in ALA synthase activity in animals under the age of 2 months because of the lack of related publications. It is also difficult for us to find whether, in 2-month-old animals, ALA synthase activity reverses its upward trend. The only study available to us (Scotto et al., 1983), in which ALA synthase activity in middle-aged animals was measured, showed that the enzyme activity in lZmonth-old animals does not differ from that in young ones. We showed that there is a marked difference in the enzyme activity between these two groups of animals. A 30% decrease in mitochondrial ALA synthase activity in old animals is not a result of greater vulnerability of the old animals’ mitochondria to isolation procedures (Scotto et al., 1983). The decrease in mitochondrial ALAS activity in old animals may result from a disordered distribution of this enzyme. In old rats, compared with young ones, a tendency towards a greater accumulation of ALA synthase in the cytosol appears (Scotto et al., 1983). Investigating ALA synthase inducibility, we showed that this enzyme, in each age group, similarly reacted to phenobarbital which enhanced about twice the enzyme activity both in young and old animals. This is in agreement with other reports (Abraham et al., 1985; Canepa et al., 1989, 1990; Kurata et al., 1989; Rosenberg et al., 1985; Tanaka et al., 1987), although one can find higher ALA synthase activity levels after phenobarbital (Canepa et al., 1985a; Srivastava et al., 1989). Phenobarbital insignificantly influences heme degradation (Abraham et al., 1985) but accelerates heme utilization by binding it to apocytochrome P-450. This decreases free heme pool and affects feedback actions between heme and ALA synthase. The observed increase in ALA synthase activity is not as high as one should expect from the feedback mechanism. This mechanism is probably supplemented, as described by Ibrahim et al. (1981), by the inhibition connected with the inhibiting influence of heme on formation of m-RNA for ALA synthase or with inhibition of ALA synthase translocation from the cytosol to the mitochondrion. Canepa et al. (1985a, 1990) and others (Song et al., 1971) maintain that since phenobarbital causes an increase in c-AMP content, it is reasonable to assume that c-AMP is responsible for enhanced ALA synthase activity after phenobarbital (Canepa et al., 1984, 1985a, 1990; Srivastava et al., 1979), although they cannot be directly connected with ALA synthase induction (Friedland and Ades, 1985). May et al. [39] explain phenobarbital induction as follows: this xenobiotic influencing the cytochrome P-450 gene level stimulates production of m-RNA for cytochrome P-450. This increases apocytochrome P-450 synthesis. Free heme pool is used to synthesize cytochrome P-450. Resulting from this a decrease in free heme content leads to increased production of m-RNA for ALA synthase and to enhanced ALA synthase synthesis. Practically, there are no reports on the influence of both fl-naphthoflavone and
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dexamethasone on ALA synthase activity. In the reports available to us, Srivastava et al. (1989) showed that dexamethasone induces ALA synthase and Tanaka et al. (1987) revealed a 150% increase in ALA synthase activity after 3-methylcholanthrene. On the basis of our results, it is interesting that, in almost all the age groups, dexamethasone and /3-naphthoflavone, two different inducers of the mixed function oxidase system (MFO), induce ALA synthase in a comparable degree. We cannot give any reason for these observations. In the liver, heme is indispensable to the synthesis of cytochrome P-450, a component of the MFO system which metabolizes many xenobiotics (Conney, 1967). This system tends to decrease its own adaptability with age both in man and in animals (Adelman and Freeman, 1972). Moreover, it is suggested that the hepatic drug metabolizing capacity may be variously modified by age, but it depends on such parameters as species, strain and sex (Outslander, 1981). References Abraham, N.G., Levere, R.D. and Freedman, M.L. (1985): Effect of age on rat liver heme and drug metabolism. Exp. Gerontol., 20, 277-284. Adelman, R.C. and Freeman, C. (1972): Age-dependent regulation of glucokinase and tyrosine aminotransferase activities of rat liver in vivo by adrenal, pancreatic and pituitary hormones. Endocrinology, 90, 1551-1560. Ades, I.Z. (1986): Regulation of biogenesis of b-aminolevulinate synthase by heme: perturbation in chemically-induced porphyrias. IRCS Med. Sci., 14, 1I70- I 173. Ades, I.Z., Stevens, T.M. and Drew P.D. (1987): Biogenesis of embryonic chick Gaminolevulinate synthase: regulation of the level of m-RNA by hemin. Arch. B&hem. Biophys., 253, 297-304. Amelizad, Z. Narbonne, J.F. Borin, C. Robertson, L.W. Periquet, A. and Oesch, F. (1987): Effect of unbalanced diets on incorporation of b-aminolevulinic acid into cytochrome P-450. FEBS Lett., 220, 231-235. Bitar, MS. and Shapiro, B.H. (1987): Aberration of heme and hemoprotein in aged female rats. Mech. Ageing Dev., 38, 189-197. Bitar, M.S. and Weiner, M. (1983): Modification of age-induced changes in heme and hemoproteins by testosterone in male rats. Mech. Ageing Dev., 23, 285-296. Canepa, E.T., Llambias, E.B.C. and Grinstein, M. (1984): Effect of glucose on the induction 6aminolevulinic acid synthase and ferrochelatase in isolated rat hepatocytes by allylisopropylacetamide. Biochim. Biophys. Acta, 804, 8- 15. Canepa, E.T., Galvagno, M.A., Llambias, E.B.C., Passeron. S. and Grinstein, M. (1985a): Studies on regulatory mechanism of heme biosynthesis in hepatocytes from experimental-diabetic rats. B&him. Biophys. Acta, 847, 191-197. Canepa, E.T., Llambias, E.B.C. and Grinstein, M. (1985b): Effect of glucose on induction of 6aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead. Biochim. Biophys. Acta, 841, 186-194. Canepa, E.T., Llambias, E.B.C. and Grinstein, M. (1989): Studies on regulatory mechanism of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of c-AMP. B&hem. Cell. Biol., 67, 751-758. Canepa, E.T., Llambias, E.B.C. and Grinstein, M. (1990): Studies on induction of b-aminolevulinic acid synthase, ferrochelatase, cytochrome P-450 and cyclic AMP by phenformin. Biochem. Pharmacol., 40, 365-372. Conney, A.H. (1967): Pharmacological implications of microsomal enzymes induction. Pharm. Rev., 19, 317-366. Cresteil, T., Beaune, P., Celier, C., Leroux, J.P. and Guengerich, F.P. (1986): Cytochrome P-450 isozyme content and monooxygenase activities in rat liver: Effect of ontogenesis and pretreatment by phenobarbital and 3-methylcholanthrene. J. Pharm. Exp. Ther., 236, 269-276.
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Marks, G.S., Mackie, J.E., McCluskey, S.A. and Riddick, D.S. (1989): Regulation of heme biosynthesis in chick embryo liver cells. Adv. Exp. Med. Biol., 271, 123-133. Marver, H.S., Tschudy, D.P., Perlroth, M.G. and Collins, A. (1966): &Aminolevulinic acid synthetase. I. Studies in liver homogenates. J. Biol. Chem., 241, 2803-2809. May, B.K., Borthwick, I.A., Srivastava, G., Pirola, B.A. and Elliott, W.H. (1986): Control of 5aminolevulinate synthase in animals. Curr. Top. Ceil. Regul., 28, 233-262. Ouslander, J.G. (1981): Drug therapy in the elderly. Ann. Int. Med., 95, 71 I-722. Paternitti, J.R., Lin, C.P. and Beattie. D.S. (1978): &Aminolevulinic acid synthase: Regulation of activity in various tissues of the aging rat. Arch. B&hem. Biophys., 191, 792-797. Patemitti, J.R., Lin, C.P. and Beattie, D.S. (1980): Regulation of heme metabolism during senescence: activity of several hemecontaining enzymes and heme oxygenase in the liver and kidney of aging rats. Mech. Ageing Dev., 12, 81-91. Plewka, A., Bienioszek, M. and Plewka, D. (1994a): Effects of age, phenobarbital, @-naphthoflavone and dexamethasone on rat hepatic heme oxygenase. Arch. Toxicol., 68, 32-36. Plewka, A., Bienioszek, M. and Plewka, D. (1994b): Changes in the male rat hepatic cytochrome P-450 level, heme oxygenase and &aminolevulinic acid synthase activities at various stages of life. Mech. Ageing Dev., 74, 79-88. Riddick, D.S. and Marks, G.S. (1990): Irreversible binding of heme to microsomal protein during inactivation of cytochrome P-450 by Calkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6trimethylpyridine. B&hem. Pharmacol., 40, I9 I5- I92 1. Rikans, L.E. and Notley, B.A. (1981): Decline in hepatic microsomal monooxygenase components in middle-aged Fischer-344 rats. Exp. Gerontol.. 16, 253-259. Rosenberg, D.W., Sardana, M.K. and Kappas, A. (1985): Altered induction response of hepatic cytochrome P-450 to phenobarbital, 3-methylcholanthrene, and &naphthollavone in organtin-treated animals. B&hem. Pharmacol.. 34, 997- 1005. Sassa, S., Kappas, A., Berstein, S.E. and Alvarez, A.P. (1979): Heme biosynthesis and drug metabolism in mice with hereditary hemolytic anemia. J. Biol. Chem.. 254, 729-735. Scotto, A.W., Rinehart, R.W. and Beattie, D.S. (1983): Aging-related decreases in hepatic mitochondrial and cytosolic Gaminolevulinic acid synthase during experimental porphyria. Arch. Biochem. Biophys., 222, l50- 157. Song, C.S., Moses, H.L., Rosenthal, A.S., Gelb, N.A. and Kappas, A. (1971): The influence of postnatal development on drug-induced hepatic porphyria and the synthesis of cytochrome P-450. J. Exp. Med., 134, 1349-1371. Srivastava, G., Bawden, M.J., Hansen, A.J. and May, B.K. (1989): Heme may not be a positive regulator of cytochrome P-450 gene expression. Eur. J. B&hem., 178, 689-692. Srivastava, G., May, B.K. and Elliot, W.H. (1979): c-AMP-dependent induction of &aminolevulinate synthase in isolated embryonic chick liver cells. Biochem. Biophys. Res. Commun., 90, 42-49. Tanaka, E., Kurata, N., Kohno, M., Yoshida, T. and Kuroiwa, Y. (1987): Induction ofcytochrome P-450 and related drug-oxiding activities in muscone (3-methylcyclopentadecanone)-treated rats. B&hem. Pharmacol., 36, 4263-4267. Tavolini, N., Jones, M.J. and Berk, P.D. (1983): Dose-related effects of phenobarbital on hepatic microsomal enzymes. Proc. Sot. Exp. Biol. Med., 174, 20-27. Thaler, M.M., Gemes, D.L. and Bakken, A.F. (1972): Enzymatic conversion of heme to bilirubin in normal and starved fetuses and newborn rats. Pediatr. Res., 6, 197-201. Woods, J.S. and Dixon, R.L. (1970): Neonatal differences in the induction of hepatic aminolevulinic acid synthetase. B&hem. Pharmacol., 19, 195I - 1954. Wrighton, S.A. and Elswick, B. (1989): Modulation of the induction of rat hepatic cytochromes P-450 by selenium deficiency. B&hem. Pharmacol., 38, 3767-3771. Yamamoto, M., Hayashi, N. and Kikuchi, G. (1983): Translational inhibition by heme of the synthesis of hepatic Gaminolevulinate synthase in a cell-free system. Biochem. Biophys. Res. Commun., 115, 225-231. Yamauchi, K., Hayashi, K. and Kikuchi, G. (1980): Translocation of &aminolevulinate synthase from the cytosol to the mitochondria and its regulation by hemin in the rat liver. J. Biol. Chem., 255, 1746-1751.
Archives of Gerontology and Geriatrics 20 (1995) 149-158
ARCHIVES OF GERONTOLOGY AND GERlATRICS
Effects of age, phenobarbital, /3-naphthoflavone and dexamethasone on rat hepatic Gaminolevulinic acid synthase Andrzej
Plewka
Department of Histology and Embryology, Silesian School of Medicine, ul. Medykdw 20. 40-7S2 Katowice-Ligota. Poland
Received 20 June 1994; revision received 16 September 1994; accepted 19 September 1994
Abstract
BAminolevulinic acid synthase is a key enzyme leading to the synthesis of cytochrome Pa compound of the mixed function oxidase system. Because of this, we have studied the effects of both age and three typical mixed function oxidase system inducers on this enzyme activity in male Wistar rats. Our results suggest that &aminolevulinic acid synthase activity in intact rats increases up to maturity and then decreases being, however, higher in the oldest animals than in the youngest. Phenobarbital affects &aminolevulinic acid synthase activity very characteristically. This activity is high in 4-week-old animals and then increases slightly until the twelfth month of life. Phenobarbital enzymic inducibility tends to decrease with age. In the /3-naphthoflavone groups, the profile of b-aminolevulinic acid synthase activity looks like that in the control ones. Generally, /3-naphthoflavone is a weaker S-aminolevulinic acid synthase inducer than phenobarbital, but it seems to induce &aminolevulinic acid synthase like dexamethasone, which, being a physiological inducer of the mixed function oxidasc system, is also an inducer of Gaminolevulinic acid synthase and this inducibility is closely connected with age. 450,
Keywords: Age; Rat; Liver; Inducers; &Arninolevulinic
acid synthase
1. Introduction
acid (ALA) synthase, an enzyme of the mitochondrial matrix, of glycine and succinyl-CoA and the formation of ALA (Ades, 1986). This is the first rate-limiting step in heme synthesis (Granick, 1966). The enzyme synthesis is regulated by induction and repression mechanisms, in&Aminolevulinic
catalyzes the condensation
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chiding quantitative changes in the de novo synthesis of this enzymatic protein (Maines and Kappas, 1978). ALA synthase (ALAS) has a very short half-time (about 60 min), thus results of its activity measurements reflect virtually the quantitative production of the enzyme (Granick and Urata, 1963). In a mature rat, the enzyme is appreciably induced by various compounds (Granick and Urata, 1963; Kappas et al., 1968) but, in the fetus, ALA synthase presents low inducibility (Song et al., 1971; Woods and Dixon, 1970). A low hepatic ALAS activity and the lack of its response to induction in early phases of development seem characteristic. Contrary to this, heme oxygenase (HO) activity is high in the neonatal liver but this enzyme is also weakly stimulated by some factors (Maines et al., 1976; Maines and Kappas, 1975a, 1976, 1977a,b,c). ALA synthase, just like heme oxygenase, is under the influence of both heme and various bivalent metal ions (Kondo et al., 1983; Maines and Kappas, 1974, 1975a, 1976, 1977a). On the basis of the similarity between the impacts of heme and metal ions on the ALA synthase, it is suggested that the central metal atom in heme is a physiological regulator of the enzyme synthesis and a tetrapyrrole may be considered an effective iron carrier to cellular regulatory sites connected with ALA synthase synthesis (Ades, 1986; Maines and Kappas, 1977a,b; Marver et al., 1966). Hepatic ALA synthase activity is decreased in newborn rats (Kappas et al., 1968; Woods and Dixon, 1970), while heme oxygenase (HO) activity is greatly increased (Maines and Kappas, 1975b; Thaler et al., 1972). One thinks that the increase of HO activity is a response to hemolysis in neonates, and the decreased ALA synthase activity is caused by various factors (Amelizad et al., 1987; Friedland and Ades, 1985; Song et al., 1971; Woods and Dixon, 1970). Regarding the heme oxygenase, similar investigations were carried out concerning the influences of inducers and ageing on this enzyme activity (Plewka et al., 1994a). It has been proved that ALA synthase activity sharply decreases in the last phase of the prenatal period. At this time, the influence of allylisopropylacetamide (AIA), a well-known ALA synthase inducer, on ALAS activity is remarkably weak (Song et al., 1971; Woods and Dixon, 1970) but, as proved in the quoted report (Maines and Kappas, 1978), the inhibition of synthase synthesis in rats is parallel to the increase of HO activity, hemolysis and degradation of fetal hemoglobin. This repression of the enzyme and its resistance to induction is considerably more dependent on chemical factors than on developmental ones. Based on the connections between b-aminolevulinic acid synthase, heme oxygenase activities and cytochrome P-450 level, the influence of age on these enzymes was investigated in previous studies (Plewka et al., 1994b). The factors leading, in the neonates of various species (including man), to the cellular heme release and causing a sharp decrease in the fetal hemoglobin level during the postnatal period may be described as follows (Maines and Kappas, 1978): heme, released from destroyed erythrocytes increases its oxidation by inducing heme oxygenase, which then frees iron. The metal ion inhibits ALA synthase synthesis and activates heme oxygenase. This process continues until both haemolysis and heme degradation are stopped. Then HO activity decreases, ALA synthase synthesis becomes possible and, in this way, its chemical induction is also allowed.
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2. Materials and methods 2.1. Animals Wistar inbred male rats from the animal colony of the Silesian Medical School were used. Animals were divided into one control group and three experimental groups, which were treated with inducers. Each series comprised eight age groups: 0.5-, l-, 2-, 4-, 8-, 12-, 20- and 2%month-old. The animals in the first group were with their mothers all the time. The older rats were on a standard diet and before decapitation were fasted for 12 h with free access to water. Control animals were not given any injections. Pilot trials proved that intraperitoneal injections of physiological saline solution didn’t cause statistically significant changes in the measured parameters. The first experimental group was given phenobarbital (50 mg/kg intraperitonally) twice, 3 and 2 days before being killed. This dose was slightly smaller than that usually used in other studies. It was proved that the maximal effective dose is 75 mg/kg (Tavolini et al., 1983). It was also proved that such a dose results in an increase of young animals’ mortality (Rikans and Notley, 1981). All the injections were given at 9.00 a.m. The second experimental group was given P-naphthoflavone, a compound belonging to the II group of inducers. It was injected three times (20 mg/kg) 3,2 and 1 days before sacrificing animals. This is the common dose given in many laboratories (e.g., Cresteil et al., 1986; Lum et al., 1985). This inducer was dissolved in corn oil. Finally, the third experimental group was treated intraperitoneally with dexamethasone, a physiological inducer. It was suspended in corn oil and was given three times (10 mg/kg). This is the most common dose given in other laboratories (Leakey et al., 1986), but not the highest used (Riddick and Marks, 1990; Wrighton and Elswick, 1989). 2.2. Isolation of mitochondrial fraction Rats were always decapitated at 9.00 a.m. The livers were immediately isolated and placed in ice cold physiological salt solution. Then they were washed several times, cut into small fragments and homogenized in a Potter-Elvehjem homogenizer with a teflon piston at 400 rpm and with four full piston’s moves. A homogenization medium was 0.25 M saccharose dissolved in 10 mM Tris-HCl buffer (pH 7.4). Five ml of this solution was given per g of the tissue. This step of processing, and those following, were performed at 2-4°C. The homogenate was centrifuged at 900 x g for 10 min to sediment nuclei and cell membranes. The supematant was centrifuged again at 10 000 x g for 20 min to sediment a mitochondrial fraction. The sediment was subtly suspended in the homogenization medium and centrifuged again at 10 Ooo x g for 20 min. Pure mitochondrial fractions were suspended in 20% (w/v) glycerol buffered as described above to obtain a protein concentration not smaller than 5 mg/cm3. Samples were immediately frozen and stored at -20°C until the next day in order to carry out measurements.
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2.3. ALA synthase activity measurements ALA synthase activity was determined by the modified method from Maines and Kappas laboratory (Granick, 1966; Sassa et al., 1979). The mitochondrial suspension was put in 75 mM phosphatic buffer (pH 7.4) with cofactors (glycine, magnesium chloride, sodium citrate, disodium ethylenediamine tetraacetate, pyridoxal phosphate), to get a final volume of 3 cm3. The mixture was incubated at 37°C for 1 h. Then the reaction was stopped by adding 1 cm3 of ice cold 10% trichloroacetic acid and by putting the tubes into ice for 10 min. The protein precipitate was centrifuged at 6000 r-pm for 10 min and the supematant was mixed with 1.5 cm3 of acetylacetone dissolved in 1 M sodium acetate. The mixture was incubated in a water bath at 80°C for 10 min. After cooling, the pH was adjusted to 7.0-7.5 by adding 0.5 cm3 of 0.5 N Na2HP04 mixed with 1 N NaOH (v/v 1:3). The mixture was shook with 4 cm3 of the so-called equilibrated ether (trichloroacetic acid, sodium octate, NaOH, disodium phosphate). A sample of the water layer (1.5 cm3) was pipetted into tubes containing 1.5 cm3 of a modified Ehrlich’s reagent @-dimethylaminobenzene aldehyde, 70% perchloric acid, ice cold acetic acid). After 15 mm, a spectral difference was measured in the range of 500-700 nm. The difference in absorption between 553 and 650 nm was used to determine ALA synthase activity and was expressed in nM/h per mg of protein. 2.4. Protein determination Protein was determined according to the method of Lowry et al. (1951). Bovine serum albumin was used as a standard. 2.5. Statistical analysis The results of the biochemical measurements are mean values from five separate measures. Statistical significance between age groups and individual series was evaluated by means of the Student’s t-test (P = 0.05). 3. Results ALA synthase activity in the control group (Fig. 1) rapidly increases up to the second month of life (150% of the activity in the 2-week-old animals). At the moment of reaching maturity, the enzyme activity begins to decrease rapidly until the fourth month of life and then more slowly until advanced senescence. Nevertheless, this activity, even in the oldest animals is higher than that in the youngest. Phenobarbital very characteristically affects ALA synthase activity (Fig. 1). In the phenobarbital-treated group, ALA synthase activity sharply rises in the first 4 weeks of life (175% of the activity in the youngest group) and then it falls slightly until the twelfth month of life. From this moment it decreases minimally so that it is clearly higher in the 28-month-old animals than in the youngest ones. Comparing the results in both control and phenobarbital-treated groups we notice a clearly stimulating influence of phenobarbital on ALA synthase activity (Fig. 1). Up to the eighth month of life, the enzyme activity oscillates between 165% in the
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CIK
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Fig. 1. Age-related changes of mitochondrial Gaminolevulinic acid synthase activity in control group (K) and after phenobarbital (PB), fl-naphthoflavone (BNF) and dexamethasone (DEX) injection.
2-week-old animals and 230% in the l-month-old ones; there is not any characteristic correlation within this age range, though. After the eighth month of life the enzyme inducibility tends to decrease with age, but in the oldest animals it is still high (about 190% of the control value). In the /3-naphthoflavone series, the profile of ALA synthase activity, as a function of age, is identical to that in the control group (Fig. 1). The enzyme activity increases until the second month of life (about 125%, compared with the youngest animals) and then decreases until the end of life. This decrease is, however, greater because ALA synthase activity is clearly lower in the oldest animals than in the 2-week-old ones. fl-Naphthoflavone, a well-known inducer of the MFO system, surprisingly appears to be a much weaker ALA synthase stimulator than phenobarbital. In all the age groups, except the youngest animals in which the enzyme activity is identical to that caused by phenobarbital, ALA synthase activity is considerably lower (about 135%). As in the phenobarbital group, a very fast increase in ALA synthase activity is observed in the dexamethasone group from the second to the fourth week of life (Fig. 1). In the older group, the activity is 165% of the younger group activity. From this moment to the second month of life, the enzyme activity stabilizes and then slightly decreases up to the eighth month of life. Later, the enzyme activity is stabilized again until old age, and only in advanced senescence does the activity decrease more quickly. The physiological inducer of the MFO system used in this study is also an inducer
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of ALA synthase (Fig. 1). This inducibility, however, is closely connected with age. In the immature animals inducibility increases remarkably from the second week (about 120% of the control value) to the fourth week of life (almost 160% of the control value). Reaching maturity is connected with a certain decrease in inducibility (130% of the control value) which, however, increases in the older age groups and reaches 150% in the l-year-old animals, compared with the control group. Further ageing does not result in a decrease of inducibility so there are no great changes in the oldest animals, compared with the 12-month-old ones. 4. Discussion Based on the current data, one would think that heme controls the pathway of its own biosynthesis, mostly by influencing the translocation of inactive ALA presynthase from the cytosol to the mitochondrium in which the synthase is activated. Moreover, heme inhibits transcription of the ALA synthase gene. The final result of these processes is a ‘free heme pool’. Xenobiotics, including porphyrogenic compounds, influence this mechanism by decreasing this regulatory free heme pool in three different ways (Marks et al., 1989): (a) by cytochrome P-450 inactivation causing N-alkylprotoporphyrin formation and inhibition of ferrochelatase (the so-called primary mechanism of cytochrome P-450 inactivation); (b) by cytochrome P-450 inactivation bringing about continuous degradation of heme; (c) by enhanced generation of active oxygenic radicals which form a uroporphyrinogen decarboxylase inhibitor after interacting with endogenous substrates. The de novo heme is incorporated into several various hemoproteins, e.g., over half of this heme is used to synthesize cytochrome P-450. Heme, as shown by Granick (1966), also represses ALA synthase synthesis, but this subject is still being studied (Elliot et al., 1989; Srivastava et al., 1989). An important additional mechanism by which heme regulates ALA synthase activity is inhibition of the synthase precursors’ transport from the cytosol to the mitochondrion (Yamauchi et al., 1980) and modification of m-RNA translation into the synthase (Yamamoto et al., 1983). Therefore, heme may repress ALA synthase by inhibiting: transcription, translation and the synthase translocation from the cytosol to the mitochondrion. Ades et al. (1987) and Hamilton et al. (1988) showed that heme influences ALA synthase mainly by translocating the de novo synthesized synthase to the mitochondrion. Their results suggest that there is not any direct correlation between induced ALA synthase activity and m-RNA for this synthase. The final ALA synthase activity depends on both the level of transcribed m-RNA for this enzyme and cellular free heme content. In our study, we show that ALA synthase activity decreases from the moment of reaching maturity to advanced senescence. Lower ALA synthase activity at the end of life, compared with young animals, is in agreement with other studies (Abraham et al., 1985; Bitar and Shapiro, 1987; Bitar and Weiner, 1983; Paternitti et al., 1978,
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1980; Scotto et al., 1983) and moreover, the decrease in the synthase activity in old animals (70% compared with 2-month-old animals) is analogous with the mentioned studies. Kaliman and Nikitchenko (1989) showed that ALA synthase activity decreases until the third month of life and then remains unchanged till the end of life. We are not able to discuss changes in ALA synthase activity in animals under the age of 2 months because of the lack of related publications. It is also difficult for us to find whether, in 2-month-old animals, ALA synthase activity reverses its upward trend. The only study available to us (Scotto et al., 1983), in which ALA synthase activity in middle-aged animals was measured, showed that the enzyme activity in lZmonth-old animals does not differ from that in young ones. We showed that there is a marked difference in the enzyme activity between these two groups of animals. A 30% decrease in mitochondrial ALA synthase activity in old animals is not a result of greater vulnerability of the old animals’ mitochondria to isolation procedures (Scotto et al., 1983). The decrease in mitochondrial ALAS activity in old animals may result from a disordered distribution of this enzyme. In old rats, compared with young ones, a tendency towards a greater accumulation of ALA synthase in the cytosol appears (Scotto et al., 1983). Investigating ALA synthase inducibility, we showed that this enzyme, in each age group, similarly reacted to phenobarbital which enhanced about twice the enzyme activity both in young and old animals. This is in agreement with other reports (Abraham et al., 1985; Canepa et al., 1989, 1990; Kurata et al., 1989; Rosenberg et al., 1985; Tanaka et al., 1987), although one can find higher ALA synthase activity levels after phenobarbital (Canepa et al., 1985a; Srivastava et al., 1989). Phenobarbital insignificantly influences heme degradation (Abraham et al., 1985) but accelerates heme utilization by binding it to apocytochrome P-450. This decreases free heme pool and affects feedback actions between heme and ALA synthase. The observed increase in ALA synthase activity is not as high as one should expect from the feedback mechanism. This mechanism is probably supplemented, as described by Ibrahim et al. (1981), by the inhibition connected with the inhibiting influence of heme on formation of m-RNA for ALA synthase or with inhibition of ALA synthase translocation from the cytosol to the mitochondrion. Canepa et al. (1985a, 1990) and others (Song et al., 1971) maintain that since phenobarbital causes an increase in c-AMP content, it is reasonable to assume that c-AMP is responsible for enhanced ALA synthase activity after phenobarbital (Canepa et al., 1984, 1985a, 1990; Srivastava et al., 1979), although they cannot be directly connected with ALA synthase induction (Friedland and Ades, 1985). May et al. [39] explain phenobarbital induction as follows: this xenobiotic influencing the cytochrome P-450 gene level stimulates production of m-RNA for cytochrome P-450. This increases apocytochrome P-450 synthesis. Free heme pool is used to synthesize cytochrome P-450. Resulting from this a decrease in free heme content leads to increased production of m-RNA for ALA synthase and to enhanced ALA synthase synthesis. Practically, there are no reports on the influence of both fl-naphthoflavone and
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dexamethasone on ALA synthase activity. In the reports available to us, Srivastava et al. (1989) showed that dexamethasone induces ALA synthase and Tanaka et al. (1987) revealed a 150% increase in ALA synthase activity after 3-methylcholanthrene. On the basis of our results, it is interesting that, in almost all the age groups, dexamethasone and /3-naphthoflavone, two different inducers of the mixed function oxidase system (MFO), induce ALA synthase in a comparable degree. We cannot give any reason for these observations. In the liver, heme is indispensable to the synthesis of cytochrome P-450, a component of the MFO system which metabolizes many xenobiotics (Conney, 1967). This system tends to decrease its own adaptability with age both in man and in animals (Adelman and Freeman, 1972). Moreover, it is suggested that the hepatic drug metabolizing capacity may be variously modified by age, but it depends on such parameters as species, strain and sex (Outslander, 1981). References Abraham, N.G., Levere, R.D. and Freedman, M.L. (1985): Effect of age on rat liver heme and drug metabolism. Exp. Gerontol., 20, 277-284. Adelman, R.C. and Freeman, C. (1972): Age-dependent regulation of glucokinase and tyrosine aminotransferase activities of rat liver in vivo by adrenal, pancreatic and pituitary hormones. Endocrinology, 90, 1551-1560. Ades, I.Z. (1986): Regulation of biogenesis of b-aminolevulinate synthase by heme: perturbation in chemically-induced porphyrias. IRCS Med. Sci., 14, 1I70- I 173. Ades, I.Z., Stevens, T.M. and Drew P.D. (1987): Biogenesis of embryonic chick Gaminolevulinate synthase: regulation of the level of m-RNA by hemin. Arch. B&hem. Biophys., 253, 297-304. Amelizad, Z. Narbonne, J.F. Borin, C. Robertson, L.W. Periquet, A. and Oesch, F. (1987): Effect of unbalanced diets on incorporation of b-aminolevulinic acid into cytochrome P-450. FEBS Lett., 220, 231-235. Bitar, MS. and Shapiro, B.H. (1987): Aberration of heme and hemoprotein in aged female rats. Mech. Ageing Dev., 38, 189-197. Bitar, M.S. and Weiner, M. (1983): Modification of age-induced changes in heme and hemoproteins by testosterone in male rats. Mech. Ageing Dev., 23, 285-296. Canepa, E.T., Llambias, E.B.C. and Grinstein, M. (1984): Effect of glucose on the induction 6aminolevulinic acid synthase and ferrochelatase in isolated rat hepatocytes by allylisopropylacetamide. Biochim. Biophys. Acta, 804, 8- 15. Canepa, E.T., Galvagno, M.A., Llambias, E.B.C., Passeron. S. and Grinstein, M. (1985a): Studies on regulatory mechanism of heme biosynthesis in hepatocytes from experimental-diabetic rats. B&him. Biophys. Acta, 847, 191-197. Canepa, E.T., Llambias, E.B.C. and Grinstein, M. (1985b): Effect of glucose on induction of 6aminolevulinic acid synthase, ferrochelatase and cytochrome P-450 hemoproteins in isolated rat hepatocytes by phenobarbital and lead. Biochim. Biophys. Acta, 841, 186-194. Canepa, E.T., Llambias, E.B.C. and Grinstein, M. (1989): Studies on regulatory mechanism of heme biosynthesis in hepatocytes from normal and experimental-diabetic rats. Role of c-AMP. B&hem. Cell. Biol., 67, 751-758. Canepa, E.T., Llambias, E.B.C. and Grinstein, M. (1990): Studies on induction of b-aminolevulinic acid synthase, ferrochelatase, cytochrome P-450 and cyclic AMP by phenformin. Biochem. Pharmacol., 40, 365-372. Conney, A.H. (1967): Pharmacological implications of microsomal enzymes induction. Pharm. Rev., 19, 317-366. Cresteil, T., Beaune, P., Celier, C., Leroux, J.P. and Guengerich, F.P. (1986): Cytochrome P-450 isozyme content and monooxygenase activities in rat liver: Effect of ontogenesis and pretreatment by phenobarbital and 3-methylcholanthrene. J. Pharm. Exp. Ther., 236, 269-276.
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