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Different developmental profiles for the hepatic N-demethylation of N-methylamphetamine and N,N-dimethylamphetamine in the rat
Gen. Pharraac., 1978, Vol. 9, pp. 59 to 63. Pergamon Press. Printed in Great Britain
DIFFERENT DEVELOPMENTAL PROFILES FOR THE HEPATIC N-DEMETHYLATION OF N-METHYLAMPHETAMINE AND N,N-DIMETHYLAMPHETAMINE IN THE RAT P. TH. HENDERSON Institute of Pharmacology, Faculty of Medicine, University of Nijmegen, Nijmegen, The Netherlands (Received 14 June 1977) Abstract--1. No parallelism in the rate of development was found between the oxidative N-demethylation of N-methyl and N,N-dimethylamphetamine in rat liver. A gradual increase in apparent Vmax values was observed for the conversion of N,N-dimethylamphetamine by hepatic 9000 g supernatants in the period from 2 until about 60 days after birth, whereas the N-demethylating activity of the same liver preparation using N-methylamphetamine as a substrate remained low during the first 20 days after birth and suddenly increased in the 20-30-day period. 2. The mono-oxygenase system of the developing rat liver appeared to be stereoselective with respect to the enantiomers of N-methylamphetamine. The S(+)-isomer was the preferred substrate. No stereoselectivity, however, was observed in the metabolism of the enantiomers of N,N-dimethylamphetamine by the male rats, unlike the female rats. 3. Another difference refers to a decrease in the apparent K m values of both isomers of N-methylamphetamine, whereas the Km values of the N,N-dimethylamphetamines remained unchanged during the maturation. 4. These findings strongly support the concept that multiple forms of N-demethylating enzymes exist, which develop and maturate independently from each other.
INTRODUCTION
chrome P450 or other mono-oxygenases, each of them with their own substrate specificity. This concept has been studied during the present investigation. The oxidative N-demethylation of the enantiomers of N-methylamphetamine and of N , N dimethylamphetamine has been measured in the 9000 g supernatant liver fractions of rats during the postnatal maturation.
In the many studies during the last few decades it has been well established that in non-primate animals, most drug-metabolizing enzymes are absent or have extremely low activities in the fetus and the newborn (Fours & Hart, 1965; G r a m et al., 1969; Henderson, 1971; Dutton, 1973; Short et al., 1976; Neims et al., 1976). All different pathways appear in about the same period, but at varied rates, so that they reach their maximum activities at different ages. The microsomal mono-oxygenase (mixed function oxidase) system of the liver shows a very small increase in activity near term, followed by a marked increase after birth. Very different profiles have been reported in literature for the postnatal development of the mono-oxygenase activity. For instance, a gradual, nearly linear increase in activity, reaching a maximum at about 40 days after birth, has been described for aromatic and aliphatic hydroxylations, as well as for oxidative dealkylations (Short & Stith, 1973; Basu et al., 1971). Another developmental profile, examplified by the N-demethylation of aminopyrine shows a low level during the first 3 weeks after birth, followed by a sudden rise in the 20-30-day period (Henderson, 1971). For the observation of the different developmental pattems of the mono-oxygenase system, various factors can be held responsible; for instance, the use of different liver preparations, different animal species, or various strains of one species. Another more likely explanation is that the distinct pattems are due to separate developments of multiple types of cyto-
MATERIALS AND METHODS Animals The animals used were Wistar rats (Rattus norvegicus), kept in an environment of constant temperature and humidity. The animals had free access to water and received a constant pellet diet ad libitum. All young animals were weaned at 21 days after birth. Liver preparations The rats were killed by decapitation under light ether anesthesia. The livers were rapidly excised, portions were weighed, finely minced and transferred to 9 vol ice-cold 0.25 M sucrose. Homogenates were made using a Teflonglass Potter-Elvehjem type homogenizer. In the preparation of homogenates from rats under 10 days of age, 3 4 livers were pooled to obtain sufficient amounts for analysis. After sedimentation of nuclei and cell debris by centrifugafion at 500 g, the supernatant was centrifuged at 9000 g for 20 min. The 9000g supernatants were used as the enzyme source for the in vitro estimations of the N-demethylation. Enzyme assay Incubations were carried out under air at 30°C, as described earlier (Henderson et al., 1974). The incubation 59
60
P. TH. HENDERSON
media contained: 5 x 10-2M Tris HCI (pH 7.5); 8 x 10-4M MgC12; 8 x 10 6M MnC12; 5 x 10-3M sodium isocitrate; isocitric dehydrogenase 20 #g/ml (Sigma type IV, capable of generating 6.5/zmole NADPH/min per mg at 37°C); 13 x 10 -s M NADP and varying substrate concentrations ranging from 0.5 to 3.0 x 10 -3 M. The mixture was pre-incubated at 37"C for 10min to ensure reduction of all NADP. The reaction was started by addition of the liver preparation. The total volume of the reaction mixture was 3 ml. After incubation for 15 min the reaction was stopped by adding 0.5 ml 25% ZnSO4 and 0.5 ml of a saturated Ba(OH)2 solution. The precipitated protein was removed by centrifugation and the amount of formaldehyde was determined in the supematant according to the method of Nash (1953) as modified by Cochin & Axelrod (1959). Blanks containing all reaction components including heatdenatured enzyme were similarly treated to detect nonenzymic metabolite formation. In some experiments, after incubation the reaction medium was made alkaline with KOH and extracted with ether. The amounts of metabolites were determined by gas liquid chromatography as described earlier (Henderson et al., 1974). It appeared that in the incubation of either isomer of N-methyl- and N,N-dimethylamphetamine, more than 95% of the total amount of metabolites were the mono-demethylated products, amphetamine and N-methylamphetamine, respectively. Further, it was verified that the incubations give rise to equivalent amounts of formaldehyde and demethylated products.
ing concentrations of the different substrates. Lineweaver-Burk plots were made and apparent Vn,~,, values derived by extrapolation. In order to avoid misinterpretations due to less specific changes in the composition of the developing liver (Henderson, 1971) enzyme activities were calculated on the basis of liver dry weights. In Fig. 1 the oxidative N-demethylation of the two enantiomers of N,N-dimethylamphetamine in male rats is presented as a function of age. No differences in the apparent VmaxValues were found between S(+) and R(-)N,N-dimethylamphetamine. The data presented indicate that the N-demethylating activity gradually, but strongly, increases during the first two months of life. By contrast, the activity of N-methylamphetamine demethylase remained relatively low during the first 3 4 weeks, but rapidly increased to the adult level between 4 and 5 weeks of age (Fig. 2). For the monomethylamphetamine a clear-cut difference between the rate of conversion of the dextro and laevo isomer was observed; S(+)N-methylamphetamine appeared to be preferred substrate. This stereoselectivity already exists during the first postnatal week.
Changes in apparent substrate specificity A comparison of the apparent Km values for the N-demethylations in newborns and adult male rats is shown in Table 1. For the N-demethylation of the two enantiomers of N-methylamphetamine, the apparent substrate affinity significantly increased by about 90%, whereas no change has been observed for the N-demethylation of the dimethylamphetamines during maturation of the rat liver.
Synthesis The N-methyl- and N,N-dimethyl-substituted amphetamines have been prepared according to previously published methods (Vree, 1973) and their purity established by m.p., i.r., u.v, NMR and mass spectral data. RESULTS
Age related changes in enzyme activity
Sex related differences
N-Demethylating activities were measured at vary-
The developmental data presented above refer to
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Fig. 1. Age-related change in the hepatic mono-oxygenase activity (apparent Vmax value) towards the enantiomers of N,N-dimethylamphetamine. Enzyme activities, measured with 9000 g supernatants, are expressed as #mole of formaldehyde formed per hour per gram dry liver. Details of the conditions of incubation are given in the Materials and Methods section.
Different developmental profiles in the rat
61
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age (day~) Fig. 2. Age-related change in the hepatic mono-oxygenase activity (apparent Vm~ value) towards both enantiomers of N-methylampbetamine. Enzyme activities, measured with 9000g supernatants, are expressed as /lmole of formaldehyde produced per hour per gram dry liver. Details of incubation are described in the Materials and Methods section. male rats only. It is however well known that in many animal species, including rat and mouse, sexual maturation can also influence to a substantial degree the development of the mono-oxygenase system, with, as a result, a great difference in the rate of drug oxi-
dation between male and female animals (Davies et al., 1969; K a t o & Onoda, 1970; Henderson, 1971). As is shown in Table 2, this holds also true for the N-demethylation of the N-methyl- and N,N-dimethylamphetamines. For the conversion of both enan-
Table 1. Comparison of Michaelis constantsa (K~,) for the oxidative N-demethylation of N-methylamphetamine and N,N-dimethylamphetamine by hepatic 9000 g supernatants from newborn and adult male rats Age groups Substrate S( + )N-methylamphetamine R(-)N-methylamphetamine S( + )N,N-dimethylamphetamine R( - )N,N-dimethylamphetamine
15 days 1.15 1.07 1.06 1.12
+ 0.21 ___0.24 + 0.15 +_ 0.13
60 days 0.58 _ 0.61 _ 1.20 + 1.07 +
0.14b 0.14b 0.06N's" 0.06N's"
"Apparent K,, values are expressed in mM; values are means ___ S.E. for 6 animals. b Significantly different from the value for the newborn at P < 0.05 (Students t-test). N.S., no significant difference (Students t-test). Table 2. Sex-related differences in the oxidative N-demethlyationa of the enantiomers of N-methylamphetamine and N,N-dimethylamphetamine by 9000 g supernatant liver fractions of adult rats Substrateb
Male
Female
S(+)N-methylamphetamine R(-)N-methylamphetamine
1570 ___98 1026 + 106 1.53 1702 _+ 121 1767 + 117 0.97
933 + 67 596 + 65 1.56 641 +_ 77 390 _ 37 1.64
Ratio ( + / - ) S(+)N,N-dimethylamphetamine R(-)N,N-dimethylamphetamine Ratio ( + / - )
Ratio (male/female) 1.68 1.72 2.65 4.53
a Enzyme activities are expressed as nmoles formaldehyde produced per hour per gram fresh liver; values are means + S.E. for 4 two-month old rats. b Substrate concentration was 5 mM.
62
P. TH. HENDERSON
tiomers of N-methylamphetamine the male/female activity ratios were about the same. However, much higher and unequal ratios were found with S(+) or R(-)N,N-dimethylamphetamine as a substrate. A relatively great sex difference was found for the N-demethylation of R(-)N,N-dimethylamphetamine. This compound was converted 4 4 times faster by the liver enzyme preparation of the male rats than by that of the female animals. In part this is due to the fact that microsomes of the females display a steric preference for the conversion of S(+)N,Ndimethylamphetamine, whereas no stereoselectivity can be observed in the N-demethylation of the N , N dimethylamphetamines by the male rats (Fig. 1).
Interestingly, the reversed situation has been reported by Furner et al. (1969), who found that in the metabolism of hexobarbital, microsomes from male rats preferred the dextro isomer, whereas female rats did not show any stereoselectivity with respect to hexobarbital metabolism. These phenomena are indications that in the rat sexual maturation may induce qualitative as well as quantitative differences in the mono-oxygenase systems. Acknowledoements Thanks are due to Mrs E. W. M. Yih-v.d. Hurk for skilful assistance, to Mr P. J. L. v. Gemert for synthesis of the substrates. This study was supported by grants from the Foundation for Medical Research (FUNGO).
DISCUSSION Although from many studies it has become evident that more than one enzyme is involved in the microsomal oxidation of drugs, the multiplicity of the mono-oxygenase system is still the subject of much speculation. The existence of different types of cytochrome P450 has been adequately demonstrated by spectral studies of microsomal preparations (Werringloer & Estabrook, 1975) and by studies on isolation and purification of cytochrome P450 from 3-methylcholanthrene and phenobarbital treated animals (Lu et al., 1972). Strong evidence for the heterogeneity also emerges from data on differential changes in the route or the rate of oxidation of selected substrates in the presence of inhibitors or after induction (Hildebrandt et al., 1968; Lange, 1967; Ullrich et al, 1973). In 1971 G r a m suggested that the N-demethylation of secondary and tertiary amines proceeds via different N-demethylase systems. Our results presented in Figs 1 and 2, show different developmental profiles for the microsomal N-demethylation of N-methylamphetamine and the N-demethylation of N,N-dimethylamphetamine in the rat. These findings strongly support the concept that the hepatocytes contain several mono-oxygenases, which develop independently after birth. Another difference refers to a change in kinetic properties of the N-demethylase during maturation. For both enantiomers of N-methylamphetamine the apparent K,, values decreased with age. No changes in affinity were found with the N,N-dimethylamphetamines as substrate. It should be remarked here that the development of the drug-metabolizing capacity may be more than just an increase in enzyme concentration, but qualitative changes may also play an important role. Changes in affinity between enzyme and substrate may have far-reaching consequences for the enzyme activity in vivo, where in general substrate concentrations are relatively low. For the conversion of the monomethyl-substituted amphetamine a substantial stereoselectivity became manifest. The developmental profiles however, of the enzyme activity were similar for the two enantiomers. As far as N,N-dimethylamphetamine is concerned, no difference between the dextro and the laevo isomer was found in the rate of conversion by the liver preparation from male rats (Fig. 1, Table 2). On the other hand, adult female rats appeared to metabolize the dextro isomer much faster than the laevo isomer of N,N-dimethylamphetamine.
REFERENCES
BASU T. K., DICKERSONJ. W. T. & PARKED. V. W. (1971) Effect of development on the activity of microsomal drug-metabolizing enzymes in rat liver. Biochem. J. 124, 19 24. COCHIN J. & AXELRODJ. (1959) Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmac. exp. Ther. 125, 105 ll0. DAVIESD. S., GIGON P. L. & GILLETTEJ. R. (1969) Species and sex differences in electron transport systems in liver microsomes and their relationship to ethylmorphine demethylation. Life Sci. 8, 85 91. DUTTON G. J. (1973) Developmental studies on some enzymes associated with detoxication. Enzyme 15, 304-317. FOUTSJ. R. & HART L. G. 0965) Hepatic drug metabolism during the perinatal period. Ann. N.Y Acad. Sci. 123, 245-251. FURNER R. L., MCCARTHY J. S. ST1TZEL R. E. 8z ANDERS M. W. (1969) Stereoselective metabolism of the enantiomers of hexobarbital. J. Pharmac. exp. Ther. 169, 153-158. GRAM T. E. (1971) Enzymatic N-, O- and S-dealkylation of foreign compounds by hepatic microsomes. In Handhook of Experimental Pharmacology. Concepts in Biochemical Pharmacology (Edited by BRODIE 8¢ GILLETTE J. R.), Vol. 28, pp. 334-349. Springer, Berlin. GRAM T. E., GUAR1NOA. M., SCHROEDERD. H. & GILLETTE J. R. (1969) Changes in certain kinetic properties of hepatic microsomal aniline hydroxylase and ethylmorphine demethylase associated with postnatal development and maturation in male rats. Biochem. J. 113, 681 ~85. HENDERSONP. Th. (1971) Metabolism of drugs in rat liver during the perinatal period. Biochem. Pharmac. 20, 1225-1232. HENDERSON P. Th., VREE T. B., VAN GINNEKEN C. A. M. VAN ROSSUMJ. M. (1974) Activation energies of or-Coxidation and N-oxidation of N-alkyl-substituted amphetamines by rat liver microsomes. Stereochemistry and deuterium isotope effects. Xenobiotica 4, 121-130. HILDEBRANDT A. G., REMMER H. & ESTABROOK R. W. (1968) Cytochrome P450 of liver microsomes. One pigment or many. Biochem. biophys. Res. Commun. 30, 607~ 12. KATO R. & ONODA K. (1970) Studies on the regulation of the activity of drug oxidation in rat liver microsomes by androgen and estrogen. Biochem. Pharmac. 19, 1649-1660. LANGE C. (1967) Verschiedene Induktion der mikrosomalen para- und N-Hydroxylierung yon Anilin und N-Aethylanilin bei Kaninchen. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 257, 230.
Different developmental profiles in the rat Lu A. Y. K., KUNTZMAN R., WEST S., JACOBSONM. & CONNEY A. H. (1972) Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. J. biol. Chem. 247, 1727-1734. MASH T. (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55, 416-421. NEIMS A. H., WARNER M., LOUGHNAN P. N. & ARANDA J. V. (1976) Developmental aspects of the hepatic cytochrome P450 mono-oxygenase system. A. Rev. Pharmac. 16, 427-445. SHORT C. R., KINDEN D. A. & Sa1TH R. (1976) Fetal and neonatal development of the mono-oxygenase system. Drug Metab. Rev. 5, 1-42.
63
SHORT C. R. & STIrn R. D. (1973) Perinatal development of hepatic mixed function oxidase activity in swine. Biochem. Pharmac. 22, 1309-1319. ULLRICH V., FROMMERU. & WEBER P. (1973) Differences in the O-dealkylation of 7-ethoxycoumarin after pretreatment with phenobarbital and 3-methylcholanthrene. Hoppe Seyler's Z. physiol. Chem. 354, 514-520. VREE T. B. (1973) Pharmacokinetics and metabolism of amphetamines. Ph.D. Thesis, Drukkerij Brakkenstein, Nijmegen, The Netherlands. WERRINGLOER,J. & ESTABROOKR. W. (1975) Heterogeneity of liver microsomal cytochrome P450: The spectral characterization of reactants with reduced cytochrome P450. Archs Biochem. Biophys. 167, 270-280.
DIFFERENT DEVELOPMENTAL PROFILES FOR THE HEPATIC N-DEMETHYLATION OF N-METHYLAMPHETAMINE AND N,N-DIMETHYLAMPHETAMINE IN THE RAT P. TH. HENDERSON Institute of Pharmacology, Faculty of Medicine, University of Nijmegen, Nijmegen, The Netherlands (Received 14 June 1977) Abstract--1. No parallelism in the rate of development was found between the oxidative N-demethylation of N-methyl and N,N-dimethylamphetamine in rat liver. A gradual increase in apparent Vmax values was observed for the conversion of N,N-dimethylamphetamine by hepatic 9000 g supernatants in the period from 2 until about 60 days after birth, whereas the N-demethylating activity of the same liver preparation using N-methylamphetamine as a substrate remained low during the first 20 days after birth and suddenly increased in the 20-30-day period. 2. The mono-oxygenase system of the developing rat liver appeared to be stereoselective with respect to the enantiomers of N-methylamphetamine. The S(+)-isomer was the preferred substrate. No stereoselectivity, however, was observed in the metabolism of the enantiomers of N,N-dimethylamphetamine by the male rats, unlike the female rats. 3. Another difference refers to a decrease in the apparent K m values of both isomers of N-methylamphetamine, whereas the Km values of the N,N-dimethylamphetamines remained unchanged during the maturation. 4. These findings strongly support the concept that multiple forms of N-demethylating enzymes exist, which develop and maturate independently from each other.
INTRODUCTION
chrome P450 or other mono-oxygenases, each of them with their own substrate specificity. This concept has been studied during the present investigation. The oxidative N-demethylation of the enantiomers of N-methylamphetamine and of N , N dimethylamphetamine has been measured in the 9000 g supernatant liver fractions of rats during the postnatal maturation.
In the many studies during the last few decades it has been well established that in non-primate animals, most drug-metabolizing enzymes are absent or have extremely low activities in the fetus and the newborn (Fours & Hart, 1965; G r a m et al., 1969; Henderson, 1971; Dutton, 1973; Short et al., 1976; Neims et al., 1976). All different pathways appear in about the same period, but at varied rates, so that they reach their maximum activities at different ages. The microsomal mono-oxygenase (mixed function oxidase) system of the liver shows a very small increase in activity near term, followed by a marked increase after birth. Very different profiles have been reported in literature for the postnatal development of the mono-oxygenase activity. For instance, a gradual, nearly linear increase in activity, reaching a maximum at about 40 days after birth, has been described for aromatic and aliphatic hydroxylations, as well as for oxidative dealkylations (Short & Stith, 1973; Basu et al., 1971). Another developmental profile, examplified by the N-demethylation of aminopyrine shows a low level during the first 3 weeks after birth, followed by a sudden rise in the 20-30-day period (Henderson, 1971). For the observation of the different developmental pattems of the mono-oxygenase system, various factors can be held responsible; for instance, the use of different liver preparations, different animal species, or various strains of one species. Another more likely explanation is that the distinct pattems are due to separate developments of multiple types of cyto-
MATERIALS AND METHODS Animals The animals used were Wistar rats (Rattus norvegicus), kept in an environment of constant temperature and humidity. The animals had free access to water and received a constant pellet diet ad libitum. All young animals were weaned at 21 days after birth. Liver preparations The rats were killed by decapitation under light ether anesthesia. The livers were rapidly excised, portions were weighed, finely minced and transferred to 9 vol ice-cold 0.25 M sucrose. Homogenates were made using a Teflonglass Potter-Elvehjem type homogenizer. In the preparation of homogenates from rats under 10 days of age, 3 4 livers were pooled to obtain sufficient amounts for analysis. After sedimentation of nuclei and cell debris by centrifugafion at 500 g, the supernatant was centrifuged at 9000 g for 20 min. The 9000g supernatants were used as the enzyme source for the in vitro estimations of the N-demethylation. Enzyme assay Incubations were carried out under air at 30°C, as described earlier (Henderson et al., 1974). The incubation 59
60
P. TH. HENDERSON
media contained: 5 x 10-2M Tris HCI (pH 7.5); 8 x 10-4M MgC12; 8 x 10 6M MnC12; 5 x 10-3M sodium isocitrate; isocitric dehydrogenase 20 #g/ml (Sigma type IV, capable of generating 6.5/zmole NADPH/min per mg at 37°C); 13 x 10 -s M NADP and varying substrate concentrations ranging from 0.5 to 3.0 x 10 -3 M. The mixture was pre-incubated at 37"C for 10min to ensure reduction of all NADP. The reaction was started by addition of the liver preparation. The total volume of the reaction mixture was 3 ml. After incubation for 15 min the reaction was stopped by adding 0.5 ml 25% ZnSO4 and 0.5 ml of a saturated Ba(OH)2 solution. The precipitated protein was removed by centrifugation and the amount of formaldehyde was determined in the supematant according to the method of Nash (1953) as modified by Cochin & Axelrod (1959). Blanks containing all reaction components including heatdenatured enzyme were similarly treated to detect nonenzymic metabolite formation. In some experiments, after incubation the reaction medium was made alkaline with KOH and extracted with ether. The amounts of metabolites were determined by gas liquid chromatography as described earlier (Henderson et al., 1974). It appeared that in the incubation of either isomer of N-methyl- and N,N-dimethylamphetamine, more than 95% of the total amount of metabolites were the mono-demethylated products, amphetamine and N-methylamphetamine, respectively. Further, it was verified that the incubations give rise to equivalent amounts of formaldehyde and demethylated products.
ing concentrations of the different substrates. Lineweaver-Burk plots were made and apparent Vn,~,, values derived by extrapolation. In order to avoid misinterpretations due to less specific changes in the composition of the developing liver (Henderson, 1971) enzyme activities were calculated on the basis of liver dry weights. In Fig. 1 the oxidative N-demethylation of the two enantiomers of N,N-dimethylamphetamine in male rats is presented as a function of age. No differences in the apparent VmaxValues were found between S(+) and R(-)N,N-dimethylamphetamine. The data presented indicate that the N-demethylating activity gradually, but strongly, increases during the first two months of life. By contrast, the activity of N-methylamphetamine demethylase remained relatively low during the first 3 4 weeks, but rapidly increased to the adult level between 4 and 5 weeks of age (Fig. 2). For the monomethylamphetamine a clear-cut difference between the rate of conversion of the dextro and laevo isomer was observed; S(+)N-methylamphetamine appeared to be preferred substrate. This stereoselectivity already exists during the first postnatal week.
Changes in apparent substrate specificity A comparison of the apparent Km values for the N-demethylations in newborns and adult male rats is shown in Table 1. For the N-demethylation of the two enantiomers of N-methylamphetamine, the apparent substrate affinity significantly increased by about 90%, whereas no change has been observed for the N-demethylation of the dimethylamphetamines during maturation of the rat liver.
Synthesis The N-methyl- and N,N-dimethyl-substituted amphetamines have been prepared according to previously published methods (Vree, 1973) and their purity established by m.p., i.r., u.v, NMR and mass spectral data. RESULTS
Age related changes in enzyme activity
Sex related differences
N-Demethylating activities were measured at vary-
The developmental data presented above refer to
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a 5(+) • R(-)
~_~'~-~'
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Fig. 1. Age-related change in the hepatic mono-oxygenase activity (apparent Vmax value) towards the enantiomers of N,N-dimethylamphetamine. Enzyme activities, measured with 9000 g supernatants, are expressed as #mole of formaldehyde formed per hour per gram dry liver. Details of the conditions of incubation are given in the Materials and Methods section.
Different developmental profiles in the rat
61
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age (day~) Fig. 2. Age-related change in the hepatic mono-oxygenase activity (apparent Vm~ value) towards both enantiomers of N-methylampbetamine. Enzyme activities, measured with 9000g supernatants, are expressed as /lmole of formaldehyde produced per hour per gram dry liver. Details of incubation are described in the Materials and Methods section. male rats only. It is however well known that in many animal species, including rat and mouse, sexual maturation can also influence to a substantial degree the development of the mono-oxygenase system, with, as a result, a great difference in the rate of drug oxi-
dation between male and female animals (Davies et al., 1969; K a t o & Onoda, 1970; Henderson, 1971). As is shown in Table 2, this holds also true for the N-demethylation of the N-methyl- and N,N-dimethylamphetamines. For the conversion of both enan-
Table 1. Comparison of Michaelis constantsa (K~,) for the oxidative N-demethylation of N-methylamphetamine and N,N-dimethylamphetamine by hepatic 9000 g supernatants from newborn and adult male rats Age groups Substrate S( + )N-methylamphetamine R(-)N-methylamphetamine S( + )N,N-dimethylamphetamine R( - )N,N-dimethylamphetamine
15 days 1.15 1.07 1.06 1.12
+ 0.21 ___0.24 + 0.15 +_ 0.13
60 days 0.58 _ 0.61 _ 1.20 + 1.07 +
0.14b 0.14b 0.06N's" 0.06N's"
"Apparent K,, values are expressed in mM; values are means ___ S.E. for 6 animals. b Significantly different from the value for the newborn at P < 0.05 (Students t-test). N.S., no significant difference (Students t-test). Table 2. Sex-related differences in the oxidative N-demethlyationa of the enantiomers of N-methylamphetamine and N,N-dimethylamphetamine by 9000 g supernatant liver fractions of adult rats Substrateb
Male
Female
S(+)N-methylamphetamine R(-)N-methylamphetamine
1570 ___98 1026 + 106 1.53 1702 _+ 121 1767 + 117 0.97
933 + 67 596 + 65 1.56 641 +_ 77 390 _ 37 1.64
Ratio ( + / - ) S(+)N,N-dimethylamphetamine R(-)N,N-dimethylamphetamine Ratio ( + / - )
Ratio (male/female) 1.68 1.72 2.65 4.53
a Enzyme activities are expressed as nmoles formaldehyde produced per hour per gram fresh liver; values are means + S.E. for 4 two-month old rats. b Substrate concentration was 5 mM.
62
P. TH. HENDERSON
tiomers of N-methylamphetamine the male/female activity ratios were about the same. However, much higher and unequal ratios were found with S(+) or R(-)N,N-dimethylamphetamine as a substrate. A relatively great sex difference was found for the N-demethylation of R(-)N,N-dimethylamphetamine. This compound was converted 4 4 times faster by the liver enzyme preparation of the male rats than by that of the female animals. In part this is due to the fact that microsomes of the females display a steric preference for the conversion of S(+)N,Ndimethylamphetamine, whereas no stereoselectivity can be observed in the N-demethylation of the N , N dimethylamphetamines by the male rats (Fig. 1).
Interestingly, the reversed situation has been reported by Furner et al. (1969), who found that in the metabolism of hexobarbital, microsomes from male rats preferred the dextro isomer, whereas female rats did not show any stereoselectivity with respect to hexobarbital metabolism. These phenomena are indications that in the rat sexual maturation may induce qualitative as well as quantitative differences in the mono-oxygenase systems. Acknowledoements Thanks are due to Mrs E. W. M. Yih-v.d. Hurk for skilful assistance, to Mr P. J. L. v. Gemert for synthesis of the substrates. This study was supported by grants from the Foundation for Medical Research (FUNGO).
DISCUSSION Although from many studies it has become evident that more than one enzyme is involved in the microsomal oxidation of drugs, the multiplicity of the mono-oxygenase system is still the subject of much speculation. The existence of different types of cytochrome P450 has been adequately demonstrated by spectral studies of microsomal preparations (Werringloer & Estabrook, 1975) and by studies on isolation and purification of cytochrome P450 from 3-methylcholanthrene and phenobarbital treated animals (Lu et al., 1972). Strong evidence for the heterogeneity also emerges from data on differential changes in the route or the rate of oxidation of selected substrates in the presence of inhibitors or after induction (Hildebrandt et al., 1968; Lange, 1967; Ullrich et al, 1973). In 1971 G r a m suggested that the N-demethylation of secondary and tertiary amines proceeds via different N-demethylase systems. Our results presented in Figs 1 and 2, show different developmental profiles for the microsomal N-demethylation of N-methylamphetamine and the N-demethylation of N,N-dimethylamphetamine in the rat. These findings strongly support the concept that the hepatocytes contain several mono-oxygenases, which develop independently after birth. Another difference refers to a change in kinetic properties of the N-demethylase during maturation. For both enantiomers of N-methylamphetamine the apparent K,, values decreased with age. No changes in affinity were found with the N,N-dimethylamphetamines as substrate. It should be remarked here that the development of the drug-metabolizing capacity may be more than just an increase in enzyme concentration, but qualitative changes may also play an important role. Changes in affinity between enzyme and substrate may have far-reaching consequences for the enzyme activity in vivo, where in general substrate concentrations are relatively low. For the conversion of the monomethyl-substituted amphetamine a substantial stereoselectivity became manifest. The developmental profiles however, of the enzyme activity were similar for the two enantiomers. As far as N,N-dimethylamphetamine is concerned, no difference between the dextro and the laevo isomer was found in the rate of conversion by the liver preparation from male rats (Fig. 1, Table 2). On the other hand, adult female rats appeared to metabolize the dextro isomer much faster than the laevo isomer of N,N-dimethylamphetamine.
REFERENCES
BASU T. K., DICKERSONJ. W. T. & PARKED. V. W. (1971) Effect of development on the activity of microsomal drug-metabolizing enzymes in rat liver. Biochem. J. 124, 19 24. COCHIN J. & AXELRODJ. (1959) Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmac. exp. Ther. 125, 105 ll0. DAVIESD. S., GIGON P. L. & GILLETTEJ. R. (1969) Species and sex differences in electron transport systems in liver microsomes and their relationship to ethylmorphine demethylation. Life Sci. 8, 85 91. DUTTON G. J. (1973) Developmental studies on some enzymes associated with detoxication. Enzyme 15, 304-317. FOUTSJ. R. & HART L. G. 0965) Hepatic drug metabolism during the perinatal period. Ann. N.Y Acad. Sci. 123, 245-251. FURNER R. L., MCCARTHY J. S. ST1TZEL R. E. 8z ANDERS M. W. (1969) Stereoselective metabolism of the enantiomers of hexobarbital. J. Pharmac. exp. Ther. 169, 153-158. GRAM T. E. (1971) Enzymatic N-, O- and S-dealkylation of foreign compounds by hepatic microsomes. In Handhook of Experimental Pharmacology. Concepts in Biochemical Pharmacology (Edited by BRODIE 8¢ GILLETTE J. R.), Vol. 28, pp. 334-349. Springer, Berlin. GRAM T. E., GUAR1NOA. M., SCHROEDERD. H. & GILLETTE J. R. (1969) Changes in certain kinetic properties of hepatic microsomal aniline hydroxylase and ethylmorphine demethylase associated with postnatal development and maturation in male rats. Biochem. J. 113, 681 ~85. HENDERSONP. Th. (1971) Metabolism of drugs in rat liver during the perinatal period. Biochem. Pharmac. 20, 1225-1232. HENDERSON P. Th., VREE T. B., VAN GINNEKEN C. A. M. VAN ROSSUMJ. M. (1974) Activation energies of or-Coxidation and N-oxidation of N-alkyl-substituted amphetamines by rat liver microsomes. Stereochemistry and deuterium isotope effects. Xenobiotica 4, 121-130. HILDEBRANDT A. G., REMMER H. & ESTABROOK R. W. (1968) Cytochrome P450 of liver microsomes. One pigment or many. Biochem. biophys. Res. Commun. 30, 607~ 12. KATO R. & ONODA K. (1970) Studies on the regulation of the activity of drug oxidation in rat liver microsomes by androgen and estrogen. Biochem. Pharmac. 19, 1649-1660. LANGE C. (1967) Verschiedene Induktion der mikrosomalen para- und N-Hydroxylierung yon Anilin und N-Aethylanilin bei Kaninchen. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 257, 230.
Different developmental profiles in the rat Lu A. Y. K., KUNTZMAN R., WEST S., JACOBSONM. & CONNEY A. H. (1972) Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. J. biol. Chem. 247, 1727-1734. MASH T. (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55, 416-421. NEIMS A. H., WARNER M., LOUGHNAN P. N. & ARANDA J. V. (1976) Developmental aspects of the hepatic cytochrome P450 mono-oxygenase system. A. Rev. Pharmac. 16, 427-445. SHORT C. R., KINDEN D. A. & Sa1TH R. (1976) Fetal and neonatal development of the mono-oxygenase system. Drug Metab. Rev. 5, 1-42.
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SHORT C. R. & STIrn R. D. (1973) Perinatal development of hepatic mixed function oxidase activity in swine. Biochem. Pharmac. 22, 1309-1319. ULLRICH V., FROMMERU. & WEBER P. (1973) Differences in the O-dealkylation of 7-ethoxycoumarin after pretreatment with phenobarbital and 3-methylcholanthrene. Hoppe Seyler's Z. physiol. Chem. 354, 514-520. VREE T. B. (1973) Pharmacokinetics and metabolism of amphetamines. Ph.D. Thesis, Drukkerij Brakkenstein, Nijmegen, The Netherlands. WERRINGLOER,J. & ESTABROOKR. W. (1975) Heterogeneity of liver microsomal cytochrome P450: The spectral characterization of reactants with reduced cytochrome P450. Archs Biochem. Biophys. 167, 270-280.