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Developmental Changes of Aldehyde Oxidase in Postnatal Rat Liver
Drug Metab. Pharmacokinet. 22 (2): 119–124 (2007).
Note Developmental Changes of Aldehyde Oxidase in Postnatal Rat Liver Yoshitaka TAYAMA1,2, Aya MORIYASU1, Kazumi SUGIHARA1, Shigeru OHTA1 and Shigeyuki KITAMURA1,3 1Division
of Medicinal Chemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan 2Faculty of Pharmaceutical Science, Hiroshima International University, Hiroshima, Japan 3Nihon Pharmaceutical University, Saitama, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: In this study, the developmental changes and variability of aldehyde oxidase in postnatal rat liver were examined. Postnatal day 1, 7 and 14 rats showed little or no liver aldehyde oxidase activity, as evaluated in terms of the activities for oxidation of benzaldehyde to benzoic acid, N-1-methylnicotinamide (NMN) to N-1-methyl-2-pyridone-5-carboxamide (2-PY) and N-1-methyl-4-pyridone-3carboxamide (4-PY), and methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX). However, these oxidase activities were markedly increased in liver cytosol from the rats after postnatal day 14. The activity was then maintained up to 6 weeks. The amounts of 2-PY and 4-PY formed from NMN were almost the same. The development of aldehyde oxidase activity toward benzaldehyde was closely correlated with that of oxidase activity toward NMN and MTX. The expression of aldehyde oxidase at postnatal day 14 was conˆrmed by Western blotting analysis. The density of bands of aldehyde oxidase was closely correlated with the oxidase activity toward benzaldehyde. The developmental changes of aldehyde oxidase activities during postnatal re‰ected the changes in the amount of the oxidase protein. Thus, aldehyde oxidase activity in rats rapidly increases from birth, reaching a plateau within 4 weeks, and is regulated by expression of the protein.
Key words: aldehyde oxidase; developmental change; N-1-methylnicotinamide; benzaldehyde; methotrexate
However, the developmental changes of aldehyde oxidase in animals and humans have not been reported. Deˆciency of drug-metabolizing enzyme activity is associated with adverse eŠects, such as grey baby syndrome due to impaired metabolism of chloramphenicol in neonates, and fatal hepatotoxicity due to altered metabolism of sodium valproate.18,19) The development of drug metabolizing enzymes plays an important role in the susceptibility of children to adverse drug reactions.20–22) Developmental changes of several drugmetabolizing enzymes have been reported. Variability of CYP3A7 expression in human fetal liver and agedependent clearance of cytochrome P450 3A substrates in humans were described.23,24) Lupp et al. reported changes in the expression of cytochrome P450 isoforms (CYP 1A1, 2B1 and 3A2) and in the associated activities in neonatal rats.25) Developmental changes in expression of aldehyde dehydrogenase in rat liver and lung were
Introduction Aldehyde oxidase (EC 1.2.3.1), a cytosolic enzyme, contains FAD, molybdenum and iron-sulfur centers,1) and is thought to be relevant to the pathophysiology of a number of clinical disorders.2–4) The enzyme in liver of various species catalyzes the oxidation of a number of aldehydes and nitrogenous heterocyclic xenobiotics, including methotrexate (MTX) and cyclophosphamide,1,5–7) and is involved in the metabolism of physiological compounds such as retinaldehyde and monoamine neurotransmitters.8,9) Moreover, the enzyme in the presence of its electron donor can mediate the reduction of a variety of compounds, including sulfoxides, Noxides, epoxides, aromatic nitro compounds and 1,2benzisoxazole derivatives.6,10) Marked inter- and intraspecies variations of the enzyme activity in oxidative and reductive reactions have been reported.5,6,10–17)
Received; November 24, 2006, Accepted; February 16, 2007 To whom correspondence should be addressed : Dr. Shigeyuki KITAMURA, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan. Fax. ＋81-82-257-5329, E-mail: skitamu＠hiroshima-u.ac.jp
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Fig. 1.
Metabolic pathways of benzaldehyde, N-1-methylnicotinamide and methotrexate by aldehyde oxidase.
also reported.26) However, there is little information on the changes of aldehyde oxidase activity. In this study, we estimated the aldehyde oxidase activity in rats by using benzaldehyde, N 1-methylnicotinamide (NMN) and MTX as substrates. Benzaldehyde oxidase activity is usually used as a marker for aldehyde oxidase. NMN, which is formed from nicotinamide by nicotinamide methyltransferase, is widely distributed in animals, like nicotinamide.27) It is neurotoxic, and is detoxiˆed by oxidation to N 1-methyl2-pyridone-5-carboxamide (2-PY) and N 1-methyl-4pyridone-3-carboxamide (4-PY).28) The conversion of NMN to 2-PY and 4-PY has been reported to be catalyzed by aldehyde oxidase,14,28,29) and the ratio of 2-PY and 4-PY formed varies among animals and humans. The oxidation of MTX to 7-hydroxymethotrexate (7-OH-MTX) is also catalyzed by aldehyde oxidase (Fig. 1).6) Materials and Methods Chemicals: NMN, N ?-methylnicotinamide and benzaldehyde were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). MTX and 7-OHMTX were kindly donated by Medical Research Laboratories, Lederle Japan (Tokyo, Japan). 2-PY and 4-PY were prepared by the method of Pullman and Colowick,30) and the method of Shibata et al., respectively.31) Animals: Sea:SD rats were obtained from Seiwa Experimental Animals, Ltd. (Fukuoka, Japan). The animals were housed in cages at 229 C with a 12-h light W
dark cycle, with free access to tap water and a standard pellet diet MM-3 (Funabashi Farm, Funabashi, Japan). Male rats were allowed to mate with female rats, and the newborn pups on postnatal day 1 were used. Other animals were killed after 1–4, and 6 weeks, and the livers were taken. Liver preparations: Livers were excised and homogenized in four volumes of 1.15z KCl. The cytosolic fraction was obtained from the homogenate by successive centrifugation at 9,000×g for 20 min and 105,000×g for 60 min. Protein concentration was determined by the method of Lowry et al. with bovine serum albumin as the standard protein.32) Immunoblot analysis of aldehyde oxidase: The levels of aldehyde oxidase protein were determined by immunoblot analysis of liver cytosol protein from rats. Rat cytosolic proteins (5 mg) were separated by SDSpolyacrylamide gel electrophoresis (10z gel) and transferred to polyvinylidene ‰uoride membranes (Bio-Rad, Hercules, CA, USA) by electroblotting. Membranes were then blocked with 5z skimmed milk in 25 mM tris-buŠered saline (pH 7.6)-0.1z Tween 20 for 1 h and probed with anti-rat aldehyde oxidase antibodies (1:1000) for 3 h. The membranes were washed, and antibody binding was detected with horseradish peroxidase-conjugated goat anti-rat IgG, followed by development with ECL Plus (Amersham Pharmacia Biotech, Buckinghamshire, England). Assay for benzaldehyde oxidase activity: Benzaldehyde oxidase activity was assayed by the method of Johns.33) The decrease in absorption at 249 nm conse-
Developmental Changes of Aldehyde Oxidase in Rats
121
Fig. 2. Developmental changes of benzaldehyde and methotrexate oxidase activities in rats. (A) Benzaldehyde oxidase activity in rat liver cytosol. (B) Western blot analysis of aldehyde oxidase in rat liver cytosol. (C) Correlation between benzaldehyde oxidase activity and density of aldehyde oxidase bands. (D) Methotrexate oxidase activity. (E) Correlation between benzaldehyde and methotrexate oxidase activities. Each value represents the mean of four rats±SD. N.S: not signiˆcance. Oxidase activities toward benzaldehyde and MTX were assayed as described in Materials and Methods.
quent upon the oxidation of benzaldehyde to benzoate was monitored in 165 mM K,Na-phosphate buŠer (pH 7.8) at 259C, using an extinction coe‹cient of 17.54 mM－1cm－1. Assay for MTX-hydroxylating activity: The incubation mixture consisted of 0.2 mmol of MTX and cytosol equivalent to 50–100 mg of liver in a ˆnal volume of 1 mL of 0.1 M K,Na-phosphate buŠer (pH 7.4). The incubation was performed at 379 C for 2 hr. After incubation, 20 mg of benzamide (an internal standard) and 2 volumes of acetonitrile were added and the whole was centrifuged. The supernatant was evaporated to about 0.5 mL, and then 10 mL of the solution was subjected to analysis by high-performance liquid chromatography (HPLC) in a Hitachi L-6000 chromatograph (Tokyo, Japan) ˆtted with an Inertsil ODS-3 column (150 mm×4.6 mm, GL Sciences, Tokyo, Japan). The mobile phase was acetonitrile-0.1 M KH2 PO4 (15:85). The chromatograph was operated at a ‰ow rate of 0.3 mL W min at ambient temperature and with a detection wavelength of 254 nm. The elution
times of MTX, 7-OH-MTX and benzamide were 15.3, 22.2 and 30.7 min, respectively. Assay for NMN oxidase activity: NMN oxidase activity was measured according to the method of Ohkubo et al.34) The amounts of 4-PY and 2-PY formed were measured by HPLC. Brie‰y, the incubation mixture consisted of 0.2 mmol of NMN and liver cytosol equivalent to 50–100 mg of liver wet weight in a ˆnal volume of 1 mL of 0.1 M K,Na-phosphate buŠer (pH 7.4). The incubation was performed at 379 C for 10 min. After incubation, the mixture, after addition of 10 mg of N ?-methylnicotinamide (an internal standard), was extracted with ˆve volumes of ethyl acetate and the extract was evaporated to dryness. The residue was redissolved in 0.1 mL of methanol, and an aliquot was subjected to analysis by HPLC. HPLC was performed with a Capcell pak C18 UG120 column (25 cm× 4.6 mm, Shiseido Co. Ltd., Tokyo, Japan) for the separation of 2-PY and 4-PY. The mobile phase was acetonitrile-water (3:97, v W v). The chromatograph was operated at a ‰ow rate of 0.5 mL W min and with a
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Fig. 3. Developmental changes of N-1-methylnicotinamide oxidase activity in rats. (A) N-1-Methylnicotinamide oxidase activity. (B) Ratio of 2-PY to 4-PY formed from N-1-methylnicotinamide. The same liver cytosol was used as in the experiment shown in Fig. 2. The expression of aldehyde oxidase at postnatal day 14 was conˆrmed by Western blotting analysis (Fig. 2-B). Each value represents the mean of four rats±SD. N.S: not signiˆcance. N-1-Methylnicotinamide oxidase activity was assayed as described in Materials and Methods.
detection wavelength of 254 nm. The elution times of 4-PY, 2-PY and N ?-methylnicotinamide (an internal standard) were 13.5, 14.7 and 30.0 min, respectively. Statistical analysis: Data are presented as mean± standard deviation (SD.). The statistical signiˆcance of diŠerences was evaluated by using analysis of variance (ANOVA) followed by a post hoc test. A value of pº0.05 was considered signiˆcant. Results and Discussion Developmental changes of oxidase activities toward benzaldehyde and MTX in rat liver: Benzaldehyde and MTX oxidase activities were assayed as representatives of aldehyde oxidase activity. Little benzaldehyde oxidase activity was observed at 1 day, 1 week or 2 weeks after birth, but at 4 weeks, the activity was markedly increased in rat liver cytosol (Fig. 2A). Furthermore, in Western blot analysis with anti-aldehyde oxidase antibodies, the band density was well correlated with benzaldehyde oxidase activity, supporting the idea that benzaldehyde oxidase activity in neonatal rat liver is due to aldehyde oxidase (Fig. 2B and 2C). The activity for conversion of MTX to 7-hydroxy-MTX was also assayed as an indicator of aldehyde oxidase activity. Little or no activity toward MTX was observed at 1 week or 2 weeks after birth. Low activity was observed at 3 weeks, and the activity was markedly increased at 4 weeks, as found for benzaldehyde oxidase activity (Fig. 2D). The changes of MTX oxidase activity and benzaldehyde oxidase activity were very similar (Fig. 2E). In short, signiˆcant development of hepatic aldehyde oxidase activities toward benzaldehyde and MTX was observed by 4 weeks after birth. In general,
oxidation of aromatic aldehydes may be catalyzed by aldehyde dehydrogenase and possibly also by xanthine oxidase, in addition to aldehyde oxidase. However, we have reported that benzaldehyde and NMN oxidase activities are not inhibited by oxypurinol, an inhibitor of xanthine oxidase.5) These activities were not enhanced by addition of NAD＋ W NADP＋, the cofactors of aldehyde dehydrogenase. Further, we have established that there are marked strain diŠerences of AO activity in rats, and the AO activity in various strains is well correlated with the MTX hydroxylation activity.7,17) Therefore, oxidase activity towards benzaldehyde, MTX and NMN may be due mainly to AO in rats. Developmental changes of NMN oxidase activity in rat liver: The activity of conversion of NMN to 2-PY and 4-PY was also assayed as an indicator of aldehyde oxidase activity. Little activity was observed at 1 day, 1 or 2 weeks after birth. However, at 4 weeks, the oxidase activity was markedly increased (Fig. 3A). The time course of change closely resembled that of benzaldehyde and MTX oxidase activities. It was reported that the ratio of 2-PY and 4-PY exhibited signiˆcant diŠerences among species, so developmental changes of AO may in‰uence the ratio of 2-PY and 4-PY. However, in rats at 6 weeks after birth, the 2-PY W 4-PY ratio was not signiˆcantly diŠerent from that in newborn rats (1 day and 1 week after birth), though there was some diŠerence at 2 and 3 weeks after birth (Fig. 3B). Therefore, it appears that developmental changes do not markedly aŠect the characteristics of AO, at least in rats. The NMN oxidase activity also appeared to be related to the body weight and liver wet weight of rats of the
Developmental Changes of Aldehyde Oxidase in Rats
same age, and rapidly increased at around 4 weeks after birth ((body weight: pº0.01, r＝0.858), (liver wet weight: pº0.01, r＝0.894)). Developmental changes of aldehyde oxidase in rats: Dramatic developmental changes in the physiological and biochemical processes that govern drug-metabolizing enzymes occur in young children. Cresteil classiˆed the development of hepatic CYP activity into three patterns.36) First, enzymes expressed in fetal liver and active towards endogenous substrates (CYP3A7).37) Second, enzymes expressed in the early neonatal period within hours after birth (CYP2D6 and CYP2E1).38,39) Third, enzymes expressed later in neonatal development (CYP1A2, CYP2C and CYP3A4).40,41) In this study, we observed that aldehyde oxidase activity estimated on the basis of benzaldehyde, NMN and MTX oxidase activities increased during physiological development immediately after birth, like CYP1A2, CYP2C and CYP3A4. These results indicate that aldehyde oxidase activity in rats increases shortly after birth, reaching a plateau level within about 4 weeks. Pediatric dosages are often decided based on body weight and body surface area, although the liver weight is a better parameter in the case of drugs eliminated in the liver. Indeed, Takahashi reported that CYP2C9 activity normalized to liver weight is comparable in children and adults, based on an analysis of warfarin clearance in Japanese children and adults.21) As AO is mainly present in liver, it is reasonable to expect a correlation between the AO activity and liver weight. In addition, there should also be a signiˆcant correlation with body weight. Therefore body weight could be a useful parameter for dose adjustment in young patients. Aldehyde oxidase plays an important role in metabolizing many drugs that may be administered to pediatric patients. Developmental changes are therefore important for the clinical application in infants of MTX and other drugs that are detoxiˆed by aldehyde oxidase. References 1)
2)
3)
4)
Beedham, C.: Molybdenum hydroxylases as drug metabolizing enzyme. Drug Metab. Rev., 16: 119–156 (1985). Berger, R., Mezey, E., Clancy, K. P., Harta, G., Wright, R. M., Repine, J. E., Brown, R. H., Brownstein, M. and Patterson, D.: Analysis of aldehyde oxidase and xanthine dehydrogenase W oxidase as possible candidate genes for autosomal recessive familial amyotrophic lateral sclerosis. Somatic Cell Mol Genetics, 21: 121–131 (1995). Moriwaki, Y., Yamamoto, T. and Higashino, K.: Distribution and pathophysiologic role of molybdenum-containing enzymes. Histol. Histopathol., 12: 513–524 (1997). Wright, R. M., Vaitaitis, G. M., Weigel, L. K., Repine, T. B., McManaman, J. L. and Repine, J. E.: Identiˆca-
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
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tion of the candidate ALS2 gene at chromosome 2q33 as a human aldehyde oxidase gene. Redox Report, 1: 313–321 (1995). Krenitsky, T. A., Tuttle, J. V., Cattau, E. L. and Wang, P.: A comparison of the distribution and electron acceptor speciˆcities of xanthine oxidase and aldehyde oxidase. Comp. Biochem. Physiol., 49B: 687–703 (1974). Kitamura, S., Sugihara, K. and Ohta, S.: Drugmetabolizing ability of molybdenum hydroxylases. Drug Metab. Pharmacokinet., 21: 83–98 (2006). Moriyasu, A., Sugihara, K., Nakatani, K., Ohta, S. and Kitamura, S.: In vivo-in vitro relationship of methotrexate 7-hydroxylation by aldehyde oxidase in four diŠerent strain rats. Drug Metab. Pharmacokinet., 21: 485–491 (2006). Huang, D. Y. and Ichikawa, Y.: Two diŠerent enzymes are primarily responsible for retinoic acid synthesis in rabbit liver cytosol. Biochem. Biophys. Res. Commun., 205: 1278–1283 (1994). McGeer, E. J. and McGeer, P. L. (eds): Molecular Neurobiology of Mammalian Brain, Plenum Publishing Corp., New York (1978). Sugihara, K., Kitamura, S. and Tatsumi, K.: Involvement of mammalian liver cytosols and aldehyde oxidase in reductive metabolism of zonisamide. Drug Metab. Dispos., 24: 199–202 (1996). Rodrigues, A. D.: Comparison of levels of aldehyde oxidase with cytochrome P450 activities in human liver in vitro. Biochem. Pharmacol., 48: 197–200 (1994). Sugihara, K., Kitamura, S. and Tatsumi, K.: Strain diŠerences of liver aldehyde oxidase activity in rats. Biochm. Mol. Biol. Int., 37: 861–869 (1995). Sugihara, K., Kitamura, S., Tatsumi, K., Asahara, T. and Dohi, K.: DiŠerences in aldehyde oxidase activity in cytosolic preparations of human and monkey liver. Biochm. Mol. Biol. Int., 41: 1153–1160 (1997). Sugihara, K., Tayama, Y., Shimoniya, K., Yoshimoto, D., Ohta, S. and Kitamura, S.: Estimation of aldehyde oxidase activity in vivo from conversion ratio of N 1methylnicotinamide to pyridones, and intraspecies variation of the enzyme activity in rats. Drug Metab. Dispos., 34: 208–212 (2006). Schoˆeld, P. C., Robertson, I. G. C. and Paxton, J. W.: Inter-species variation in the metabolism and inhibition of N-[(2?-dimethylamino)ethyl]acridine-4-carboxamide (DACA) by aldehyde oxidase. Biochem. Pharmacol., 59: 161–165 (2000). Kitamura, S., Nakatani, K., Sugihara, K. and Ohta, S.: Strain diŠerences of the ability to hydroxylate methotrexate in rats. Comp. Biochem. Physiol. Part C, 122C: 331–336 (1999a). Kitamura, S., Sugihara, K., Nakatani, K., Ohta, S., Oh-hara, T., Ninomiya, S., Green, C. E. and Tyron, C. A.: Variation of hepatic methotrexate 7-hydroxylase activity in animals and humans. IUBMB Life, 48: 607–611 (1999b). Weiss, C. F., Glasko, A. J. and Weston, J. K.: Chloramphenicol in the newborn infant. New Engl. J. Med., 262: 787–794 (1960).
124
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
Yoshitaka TAYAMA, et al.
Sadeque, A. J. M., Fisher, M. B., Korzekwa, K. R., Gonzalez, F. J. and Rettie, A. E.: Human CYP2C9 and CYP2A6 mediate formation of the hepatotoxin 4-enevalproic acid. J. Pharmacol. Exp. Ther., 283: 689–703 (1997). Johnson, T. N.: The development of drug metabolising enzymes and their in‰uence on the susceptibility to adverse drug reactions in children. Toxicology, 192: 37–48 (2003). Takahashi, H., Ishikawa, S., Nomoto, S., Nishigaki, Y., Ando, F., Kashima, T., Kimura, S., Kanamori, M. and Echizen, H.: Developmental changes in pharmacokinetics and pharmacodynamics of warfarin enantiomers in Japanese children. Clin. Pharmacol. Ther., 68: 541–555 (2000). Kawade, N. and Onishi, S.: The prenatal and postnatal development of UDP-glucuronyltransferase activity towards bilirubin and the eŠect of premature birth on this activity in human liver. Biochem. J., 196: 257–260 (1981). Leeder, J. S., Gaedigk, R., Marcucci, K. A., Gaedigk, A., Vyhlidal, C. A., Schindel, B. P. and Pearce, R. E.: Variability of CYP3A7 expression in human fetal liver. J. Pharmacol. Exp. Ther., 314: 626–635 (2005). Cotreau, M. M., von Moltke, L. L. and Greenblatt, D. J.: The in‰uence of age and sex on the clearance of cytochrome P450 3A Substrates. Clin Pharmacokinet, 44: 33–60 (2005). Lupp, A., Danz, M. and Muller, D.: Histomorphological changes and cytochrome P450 isoforms expression and activities in precision-cut liver slices from neonatal rats. Toxicology, 206: 427–438 (2005). Yoon, M., Madden, M. C. and Barton, H. A.: Developmental expression of aldehyde dehydrogenase in rat: a comparison of liver and lung development. Toxicol. Sci., 89: 386–398 (2006). Yan, L., Otterness, D. M., Craddock, T. L. and Weinshiboum, R. M.: Mouse liver nicotinamide Nmethyltransferase. Biochem. Pharmacol., 54: 1139–1149 (1997). Felsted, R. L., Chu, A. E.-Y. and Chaykin, S.: Puriˆcation and properties of the aldehyde oxidases from hog and rabbit livers. J. Biol. Chem., 248: 2580–2587 (1973). Stanulovic', M. and Chaykin, S.: Aldehyde oxidase: Catalysis of the oxidation of N -1-methylnicotinamide and pyridoxal. Arch. Biochem. Biophys., 145: 27–34 (1971).
30)
31)
32)
33)
34)
35)
36)
37)
38)
39)
40)
41)
Pullman, M. P. and Colowick, S. P.: Preparation of 2and 6-pyridones of N -1-methylnicotinamide. J. Biol. Chem., 206: 121–127 (1954). Shibata, K., Kawada, T. and Iwai, K.: Stimultaneous micro-determination of nicotinamide and its major metabolites, N -1-methyl-2-pyridone-5-carboxamide and N -1-methyl-4-pyridone-3-carboxamide, by high-performance liquid chromatography. J. Chromatogr., 424: 23–28 (1988). Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265–275 (1951). Johns, D. G.: Human liver aldehyde oxidase: DiŠerential inhibition of oxidation charged and uncharged substrates. J. Clin. Invest., 46: 1492–1505 (1967). Ohkubo, M., Sakiyama, S. and Fujimura, S.: Puriˆcation and characterization of N -1-methylnicotinamide oxidase I and II separated from rat liver. Arch. Biochem. Biophys., 221: 534–542 (1983). Ueda, O., Sugihara, K., Ohta, S. and Kitamura, S.: Involvement of molybdenum hydroxylases in reductive metabolism of nitro polycyclic aromatic hydrocarbons in mammalian skin. Drug Metab. Dispos., 33: 1312–1318 (2005). Cresteil, T.: Onset of xenobiotic metabolism in children: toxicological implications. Food Addit. Contam., 15 (suppl): 45–51 (1998). Lacroix, D., Sonnier, M., Moncion, A., Cheron, G. and Cresteil, T.: Expression of CYP3A in the human liver. Evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. Eur. J. Biochem., 247: 625–634 (1997). Treluyer, J. M., Jacqz-Aigrain, E., Alvarez, F. and Cresteil, T.: Expression of CYP2D6 in developing human liver. Eur. J. Biochem., 202: 583–588 (1991). Vieira, I., Sonnier, M. and Cresteil, T.: Developmental expression of CYP2E1 in the human liver. Hypermethylation control of gene expression during the neonatal period. Eur. J. Biochem., 238: 476–483 (1996). Sonnier, M. and Cresteil, T.: Delayed ontogenesis of CYP1A2 in the human liver. Eur. J. Biochem., 251: 893–898 (1998). Treluyer, J. M., Gueret, G., Cheron, G., Sonnier, M. and Cresteil, T.: Developmental expression of CYP2C and 2C dependent activities in the human liver: in vivo W in vitro correlation and inducibility. Pharmacogenetics, 7: 441–452 (1997).
Note Developmental Changes of Aldehyde Oxidase in Postnatal Rat Liver Yoshitaka TAYAMA1,2, Aya MORIYASU1, Kazumi SUGIHARA1, Shigeru OHTA1 and Shigeyuki KITAMURA1,3 1Division
of Medicinal Chemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan 2Faculty of Pharmaceutical Science, Hiroshima International University, Hiroshima, Japan 3Nihon Pharmaceutical University, Saitama, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: In this study, the developmental changes and variability of aldehyde oxidase in postnatal rat liver were examined. Postnatal day 1, 7 and 14 rats showed little or no liver aldehyde oxidase activity, as evaluated in terms of the activities for oxidation of benzaldehyde to benzoic acid, N-1-methylnicotinamide (NMN) to N-1-methyl-2-pyridone-5-carboxamide (2-PY) and N-1-methyl-4-pyridone-3carboxamide (4-PY), and methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX). However, these oxidase activities were markedly increased in liver cytosol from the rats after postnatal day 14. The activity was then maintained up to 6 weeks. The amounts of 2-PY and 4-PY formed from NMN were almost the same. The development of aldehyde oxidase activity toward benzaldehyde was closely correlated with that of oxidase activity toward NMN and MTX. The expression of aldehyde oxidase at postnatal day 14 was conˆrmed by Western blotting analysis. The density of bands of aldehyde oxidase was closely correlated with the oxidase activity toward benzaldehyde. The developmental changes of aldehyde oxidase activities during postnatal re‰ected the changes in the amount of the oxidase protein. Thus, aldehyde oxidase activity in rats rapidly increases from birth, reaching a plateau within 4 weeks, and is regulated by expression of the protein.
Key words: aldehyde oxidase; developmental change; N-1-methylnicotinamide; benzaldehyde; methotrexate
However, the developmental changes of aldehyde oxidase in animals and humans have not been reported. Deˆciency of drug-metabolizing enzyme activity is associated with adverse eŠects, such as grey baby syndrome due to impaired metabolism of chloramphenicol in neonates, and fatal hepatotoxicity due to altered metabolism of sodium valproate.18,19) The development of drug metabolizing enzymes plays an important role in the susceptibility of children to adverse drug reactions.20–22) Developmental changes of several drugmetabolizing enzymes have been reported. Variability of CYP3A7 expression in human fetal liver and agedependent clearance of cytochrome P450 3A substrates in humans were described.23,24) Lupp et al. reported changes in the expression of cytochrome P450 isoforms (CYP 1A1, 2B1 and 3A2) and in the associated activities in neonatal rats.25) Developmental changes in expression of aldehyde dehydrogenase in rat liver and lung were
Introduction Aldehyde oxidase (EC 1.2.3.1), a cytosolic enzyme, contains FAD, molybdenum and iron-sulfur centers,1) and is thought to be relevant to the pathophysiology of a number of clinical disorders.2–4) The enzyme in liver of various species catalyzes the oxidation of a number of aldehydes and nitrogenous heterocyclic xenobiotics, including methotrexate (MTX) and cyclophosphamide,1,5–7) and is involved in the metabolism of physiological compounds such as retinaldehyde and monoamine neurotransmitters.8,9) Moreover, the enzyme in the presence of its electron donor can mediate the reduction of a variety of compounds, including sulfoxides, Noxides, epoxides, aromatic nitro compounds and 1,2benzisoxazole derivatives.6,10) Marked inter- and intraspecies variations of the enzyme activity in oxidative and reductive reactions have been reported.5,6,10–17)
Received; November 24, 2006, Accepted; February 16, 2007 To whom correspondence should be addressed : Dr. Shigeyuki KITAMURA, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan. Fax. ＋81-82-257-5329, E-mail: skitamu＠hiroshima-u.ac.jp
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Fig. 1.
Metabolic pathways of benzaldehyde, N-1-methylnicotinamide and methotrexate by aldehyde oxidase.
also reported.26) However, there is little information on the changes of aldehyde oxidase activity. In this study, we estimated the aldehyde oxidase activity in rats by using benzaldehyde, N 1-methylnicotinamide (NMN) and MTX as substrates. Benzaldehyde oxidase activity is usually used as a marker for aldehyde oxidase. NMN, which is formed from nicotinamide by nicotinamide methyltransferase, is widely distributed in animals, like nicotinamide.27) It is neurotoxic, and is detoxiˆed by oxidation to N 1-methyl2-pyridone-5-carboxamide (2-PY) and N 1-methyl-4pyridone-3-carboxamide (4-PY).28) The conversion of NMN to 2-PY and 4-PY has been reported to be catalyzed by aldehyde oxidase,14,28,29) and the ratio of 2-PY and 4-PY formed varies among animals and humans. The oxidation of MTX to 7-hydroxymethotrexate (7-OH-MTX) is also catalyzed by aldehyde oxidase (Fig. 1).6) Materials and Methods Chemicals: NMN, N ?-methylnicotinamide and benzaldehyde were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). MTX and 7-OHMTX were kindly donated by Medical Research Laboratories, Lederle Japan (Tokyo, Japan). 2-PY and 4-PY were prepared by the method of Pullman and Colowick,30) and the method of Shibata et al., respectively.31) Animals: Sea:SD rats were obtained from Seiwa Experimental Animals, Ltd. (Fukuoka, Japan). The animals were housed in cages at 229 C with a 12-h light W
dark cycle, with free access to tap water and a standard pellet diet MM-3 (Funabashi Farm, Funabashi, Japan). Male rats were allowed to mate with female rats, and the newborn pups on postnatal day 1 were used. Other animals were killed after 1–4, and 6 weeks, and the livers were taken. Liver preparations: Livers were excised and homogenized in four volumes of 1.15z KCl. The cytosolic fraction was obtained from the homogenate by successive centrifugation at 9,000×g for 20 min and 105,000×g for 60 min. Protein concentration was determined by the method of Lowry et al. with bovine serum albumin as the standard protein.32) Immunoblot analysis of aldehyde oxidase: The levels of aldehyde oxidase protein were determined by immunoblot analysis of liver cytosol protein from rats. Rat cytosolic proteins (5 mg) were separated by SDSpolyacrylamide gel electrophoresis (10z gel) and transferred to polyvinylidene ‰uoride membranes (Bio-Rad, Hercules, CA, USA) by electroblotting. Membranes were then blocked with 5z skimmed milk in 25 mM tris-buŠered saline (pH 7.6)-0.1z Tween 20 for 1 h and probed with anti-rat aldehyde oxidase antibodies (1:1000) for 3 h. The membranes were washed, and antibody binding was detected with horseradish peroxidase-conjugated goat anti-rat IgG, followed by development with ECL Plus (Amersham Pharmacia Biotech, Buckinghamshire, England). Assay for benzaldehyde oxidase activity: Benzaldehyde oxidase activity was assayed by the method of Johns.33) The decrease in absorption at 249 nm conse-
Developmental Changes of Aldehyde Oxidase in Rats
121
Fig. 2. Developmental changes of benzaldehyde and methotrexate oxidase activities in rats. (A) Benzaldehyde oxidase activity in rat liver cytosol. (B) Western blot analysis of aldehyde oxidase in rat liver cytosol. (C) Correlation between benzaldehyde oxidase activity and density of aldehyde oxidase bands. (D) Methotrexate oxidase activity. (E) Correlation between benzaldehyde and methotrexate oxidase activities. Each value represents the mean of four rats±SD. N.S: not signiˆcance. Oxidase activities toward benzaldehyde and MTX were assayed as described in Materials and Methods.
quent upon the oxidation of benzaldehyde to benzoate was monitored in 165 mM K,Na-phosphate buŠer (pH 7.8) at 259C, using an extinction coe‹cient of 17.54 mM－1cm－1. Assay for MTX-hydroxylating activity: The incubation mixture consisted of 0.2 mmol of MTX and cytosol equivalent to 50–100 mg of liver in a ˆnal volume of 1 mL of 0.1 M K,Na-phosphate buŠer (pH 7.4). The incubation was performed at 379 C for 2 hr. After incubation, 20 mg of benzamide (an internal standard) and 2 volumes of acetonitrile were added and the whole was centrifuged. The supernatant was evaporated to about 0.5 mL, and then 10 mL of the solution was subjected to analysis by high-performance liquid chromatography (HPLC) in a Hitachi L-6000 chromatograph (Tokyo, Japan) ˆtted with an Inertsil ODS-3 column (150 mm×4.6 mm, GL Sciences, Tokyo, Japan). The mobile phase was acetonitrile-0.1 M KH2 PO4 (15:85). The chromatograph was operated at a ‰ow rate of 0.3 mL W min at ambient temperature and with a detection wavelength of 254 nm. The elution
times of MTX, 7-OH-MTX and benzamide were 15.3, 22.2 and 30.7 min, respectively. Assay for NMN oxidase activity: NMN oxidase activity was measured according to the method of Ohkubo et al.34) The amounts of 4-PY and 2-PY formed were measured by HPLC. Brie‰y, the incubation mixture consisted of 0.2 mmol of NMN and liver cytosol equivalent to 50–100 mg of liver wet weight in a ˆnal volume of 1 mL of 0.1 M K,Na-phosphate buŠer (pH 7.4). The incubation was performed at 379 C for 10 min. After incubation, the mixture, after addition of 10 mg of N ?-methylnicotinamide (an internal standard), was extracted with ˆve volumes of ethyl acetate and the extract was evaporated to dryness. The residue was redissolved in 0.1 mL of methanol, and an aliquot was subjected to analysis by HPLC. HPLC was performed with a Capcell pak C18 UG120 column (25 cm× 4.6 mm, Shiseido Co. Ltd., Tokyo, Japan) for the separation of 2-PY and 4-PY. The mobile phase was acetonitrile-water (3:97, v W v). The chromatograph was operated at a ‰ow rate of 0.5 mL W min and with a
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Fig. 3. Developmental changes of N-1-methylnicotinamide oxidase activity in rats. (A) N-1-Methylnicotinamide oxidase activity. (B) Ratio of 2-PY to 4-PY formed from N-1-methylnicotinamide. The same liver cytosol was used as in the experiment shown in Fig. 2. The expression of aldehyde oxidase at postnatal day 14 was conˆrmed by Western blotting analysis (Fig. 2-B). Each value represents the mean of four rats±SD. N.S: not signiˆcance. N-1-Methylnicotinamide oxidase activity was assayed as described in Materials and Methods.
detection wavelength of 254 nm. The elution times of 4-PY, 2-PY and N ?-methylnicotinamide (an internal standard) were 13.5, 14.7 and 30.0 min, respectively. Statistical analysis: Data are presented as mean± standard deviation (SD.). The statistical signiˆcance of diŠerences was evaluated by using analysis of variance (ANOVA) followed by a post hoc test. A value of pº0.05 was considered signiˆcant. Results and Discussion Developmental changes of oxidase activities toward benzaldehyde and MTX in rat liver: Benzaldehyde and MTX oxidase activities were assayed as representatives of aldehyde oxidase activity. Little benzaldehyde oxidase activity was observed at 1 day, 1 week or 2 weeks after birth, but at 4 weeks, the activity was markedly increased in rat liver cytosol (Fig. 2A). Furthermore, in Western blot analysis with anti-aldehyde oxidase antibodies, the band density was well correlated with benzaldehyde oxidase activity, supporting the idea that benzaldehyde oxidase activity in neonatal rat liver is due to aldehyde oxidase (Fig. 2B and 2C). The activity for conversion of MTX to 7-hydroxy-MTX was also assayed as an indicator of aldehyde oxidase activity. Little or no activity toward MTX was observed at 1 week or 2 weeks after birth. Low activity was observed at 3 weeks, and the activity was markedly increased at 4 weeks, as found for benzaldehyde oxidase activity (Fig. 2D). The changes of MTX oxidase activity and benzaldehyde oxidase activity were very similar (Fig. 2E). In short, signiˆcant development of hepatic aldehyde oxidase activities toward benzaldehyde and MTX was observed by 4 weeks after birth. In general,
oxidation of aromatic aldehydes may be catalyzed by aldehyde dehydrogenase and possibly also by xanthine oxidase, in addition to aldehyde oxidase. However, we have reported that benzaldehyde and NMN oxidase activities are not inhibited by oxypurinol, an inhibitor of xanthine oxidase.5) These activities were not enhanced by addition of NAD＋ W NADP＋, the cofactors of aldehyde dehydrogenase. Further, we have established that there are marked strain diŠerences of AO activity in rats, and the AO activity in various strains is well correlated with the MTX hydroxylation activity.7,17) Therefore, oxidase activity towards benzaldehyde, MTX and NMN may be due mainly to AO in rats. Developmental changes of NMN oxidase activity in rat liver: The activity of conversion of NMN to 2-PY and 4-PY was also assayed as an indicator of aldehyde oxidase activity. Little activity was observed at 1 day, 1 or 2 weeks after birth. However, at 4 weeks, the oxidase activity was markedly increased (Fig. 3A). The time course of change closely resembled that of benzaldehyde and MTX oxidase activities. It was reported that the ratio of 2-PY and 4-PY exhibited signiˆcant diŠerences among species, so developmental changes of AO may in‰uence the ratio of 2-PY and 4-PY. However, in rats at 6 weeks after birth, the 2-PY W 4-PY ratio was not signiˆcantly diŠerent from that in newborn rats (1 day and 1 week after birth), though there was some diŠerence at 2 and 3 weeks after birth (Fig. 3B). Therefore, it appears that developmental changes do not markedly aŠect the characteristics of AO, at least in rats. The NMN oxidase activity also appeared to be related to the body weight and liver wet weight of rats of the
Developmental Changes of Aldehyde Oxidase in Rats
same age, and rapidly increased at around 4 weeks after birth ((body weight: pº0.01, r＝0.858), (liver wet weight: pº0.01, r＝0.894)). Developmental changes of aldehyde oxidase in rats: Dramatic developmental changes in the physiological and biochemical processes that govern drug-metabolizing enzymes occur in young children. Cresteil classiˆed the development of hepatic CYP activity into three patterns.36) First, enzymes expressed in fetal liver and active towards endogenous substrates (CYP3A7).37) Second, enzymes expressed in the early neonatal period within hours after birth (CYP2D6 and CYP2E1).38,39) Third, enzymes expressed later in neonatal development (CYP1A2, CYP2C and CYP3A4).40,41) In this study, we observed that aldehyde oxidase activity estimated on the basis of benzaldehyde, NMN and MTX oxidase activities increased during physiological development immediately after birth, like CYP1A2, CYP2C and CYP3A4. These results indicate that aldehyde oxidase activity in rats increases shortly after birth, reaching a plateau level within about 4 weeks. Pediatric dosages are often decided based on body weight and body surface area, although the liver weight is a better parameter in the case of drugs eliminated in the liver. Indeed, Takahashi reported that CYP2C9 activity normalized to liver weight is comparable in children and adults, based on an analysis of warfarin clearance in Japanese children and adults.21) As AO is mainly present in liver, it is reasonable to expect a correlation between the AO activity and liver weight. In addition, there should also be a signiˆcant correlation with body weight. Therefore body weight could be a useful parameter for dose adjustment in young patients. Aldehyde oxidase plays an important role in metabolizing many drugs that may be administered to pediatric patients. Developmental changes are therefore important for the clinical application in infants of MTX and other drugs that are detoxiˆed by aldehyde oxidase. References 1)
2)
3)
4)
Beedham, C.: Molybdenum hydroxylases as drug metabolizing enzyme. Drug Metab. Rev., 16: 119–156 (1985). Berger, R., Mezey, E., Clancy, K. P., Harta, G., Wright, R. M., Repine, J. E., Brown, R. H., Brownstein, M. and Patterson, D.: Analysis of aldehyde oxidase and xanthine dehydrogenase W oxidase as possible candidate genes for autosomal recessive familial amyotrophic lateral sclerosis. Somatic Cell Mol Genetics, 21: 121–131 (1995). Moriwaki, Y., Yamamoto, T. and Higashino, K.: Distribution and pathophysiologic role of molybdenum-containing enzymes. Histol. Histopathol., 12: 513–524 (1997). Wright, R. M., Vaitaitis, G. M., Weigel, L. K., Repine, T. B., McManaman, J. L. and Repine, J. E.: Identiˆca-
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
123
tion of the candidate ALS2 gene at chromosome 2q33 as a human aldehyde oxidase gene. Redox Report, 1: 313–321 (1995). Krenitsky, T. A., Tuttle, J. V., Cattau, E. L. and Wang, P.: A comparison of the distribution and electron acceptor speciˆcities of xanthine oxidase and aldehyde oxidase. Comp. Biochem. Physiol., 49B: 687–703 (1974). Kitamura, S., Sugihara, K. and Ohta, S.: Drugmetabolizing ability of molybdenum hydroxylases. Drug Metab. Pharmacokinet., 21: 83–98 (2006). Moriyasu, A., Sugihara, K., Nakatani, K., Ohta, S. and Kitamura, S.: In vivo-in vitro relationship of methotrexate 7-hydroxylation by aldehyde oxidase in four diŠerent strain rats. Drug Metab. Pharmacokinet., 21: 485–491 (2006). Huang, D. Y. and Ichikawa, Y.: Two diŠerent enzymes are primarily responsible for retinoic acid synthesis in rabbit liver cytosol. Biochem. Biophys. Res. Commun., 205: 1278–1283 (1994). McGeer, E. J. and McGeer, P. L. (eds): Molecular Neurobiology of Mammalian Brain, Plenum Publishing Corp., New York (1978). Sugihara, K., Kitamura, S. and Tatsumi, K.: Involvement of mammalian liver cytosols and aldehyde oxidase in reductive metabolism of zonisamide. Drug Metab. Dispos., 24: 199–202 (1996). Rodrigues, A. D.: Comparison of levels of aldehyde oxidase with cytochrome P450 activities in human liver in vitro. Biochem. Pharmacol., 48: 197–200 (1994). Sugihara, K., Kitamura, S. and Tatsumi, K.: Strain diŠerences of liver aldehyde oxidase activity in rats. Biochm. Mol. Biol. Int., 37: 861–869 (1995). Sugihara, K., Kitamura, S., Tatsumi, K., Asahara, T. and Dohi, K.: DiŠerences in aldehyde oxidase activity in cytosolic preparations of human and monkey liver. Biochm. Mol. Biol. Int., 41: 1153–1160 (1997). Sugihara, K., Tayama, Y., Shimoniya, K., Yoshimoto, D., Ohta, S. and Kitamura, S.: Estimation of aldehyde oxidase activity in vivo from conversion ratio of N 1methylnicotinamide to pyridones, and intraspecies variation of the enzyme activity in rats. Drug Metab. Dispos., 34: 208–212 (2006). Schoˆeld, P. C., Robertson, I. G. C. and Paxton, J. W.: Inter-species variation in the metabolism and inhibition of N-[(2?-dimethylamino)ethyl]acridine-4-carboxamide (DACA) by aldehyde oxidase. Biochem. Pharmacol., 59: 161–165 (2000). Kitamura, S., Nakatani, K., Sugihara, K. and Ohta, S.: Strain diŠerences of the ability to hydroxylate methotrexate in rats. Comp. Biochem. Physiol. Part C, 122C: 331–336 (1999a). Kitamura, S., Sugihara, K., Nakatani, K., Ohta, S., Oh-hara, T., Ninomiya, S., Green, C. E. and Tyron, C. A.: Variation of hepatic methotrexate 7-hydroxylase activity in animals and humans. IUBMB Life, 48: 607–611 (1999b). Weiss, C. F., Glasko, A. J. and Weston, J. K.: Chloramphenicol in the newborn infant. New Engl. J. Med., 262: 787–794 (1960).
124
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
Yoshitaka TAYAMA, et al.
Sadeque, A. J. M., Fisher, M. B., Korzekwa, K. R., Gonzalez, F. J. and Rettie, A. E.: Human CYP2C9 and CYP2A6 mediate formation of the hepatotoxin 4-enevalproic acid. J. Pharmacol. Exp. Ther., 283: 689–703 (1997). Johnson, T. N.: The development of drug metabolising enzymes and their in‰uence on the susceptibility to adverse drug reactions in children. Toxicology, 192: 37–48 (2003). Takahashi, H., Ishikawa, S., Nomoto, S., Nishigaki, Y., Ando, F., Kashima, T., Kimura, S., Kanamori, M. and Echizen, H.: Developmental changes in pharmacokinetics and pharmacodynamics of warfarin enantiomers in Japanese children. Clin. Pharmacol. Ther., 68: 541–555 (2000). Kawade, N. and Onishi, S.: The prenatal and postnatal development of UDP-glucuronyltransferase activity towards bilirubin and the eŠect of premature birth on this activity in human liver. Biochem. J., 196: 257–260 (1981). Leeder, J. S., Gaedigk, R., Marcucci, K. A., Gaedigk, A., Vyhlidal, C. A., Schindel, B. P. and Pearce, R. E.: Variability of CYP3A7 expression in human fetal liver. J. Pharmacol. Exp. Ther., 314: 626–635 (2005). Cotreau, M. M., von Moltke, L. L. and Greenblatt, D. J.: The in‰uence of age and sex on the clearance of cytochrome P450 3A Substrates. Clin Pharmacokinet, 44: 33–60 (2005). Lupp, A., Danz, M. and Muller, D.: Histomorphological changes and cytochrome P450 isoforms expression and activities in precision-cut liver slices from neonatal rats. Toxicology, 206: 427–438 (2005). Yoon, M., Madden, M. C. and Barton, H. A.: Developmental expression of aldehyde dehydrogenase in rat: a comparison of liver and lung development. Toxicol. Sci., 89: 386–398 (2006). Yan, L., Otterness, D. M., Craddock, T. L. and Weinshiboum, R. M.: Mouse liver nicotinamide Nmethyltransferase. Biochem. Pharmacol., 54: 1139–1149 (1997). Felsted, R. L., Chu, A. E.-Y. and Chaykin, S.: Puriˆcation and properties of the aldehyde oxidases from hog and rabbit livers. J. Biol. Chem., 248: 2580–2587 (1973). Stanulovic', M. and Chaykin, S.: Aldehyde oxidase: Catalysis of the oxidation of N -1-methylnicotinamide and pyridoxal. Arch. Biochem. Biophys., 145: 27–34 (1971).
30)
31)
32)
33)
34)
35)
36)
37)
38)
39)
40)
41)
Pullman, M. P. and Colowick, S. P.: Preparation of 2and 6-pyridones of N -1-methylnicotinamide. J. Biol. Chem., 206: 121–127 (1954). Shibata, K., Kawada, T. and Iwai, K.: Stimultaneous micro-determination of nicotinamide and its major metabolites, N -1-methyl-2-pyridone-5-carboxamide and N -1-methyl-4-pyridone-3-carboxamide, by high-performance liquid chromatography. J. Chromatogr., 424: 23–28 (1988). Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265–275 (1951). Johns, D. G.: Human liver aldehyde oxidase: DiŠerential inhibition of oxidation charged and uncharged substrates. J. Clin. Invest., 46: 1492–1505 (1967). Ohkubo, M., Sakiyama, S. and Fujimura, S.: Puriˆcation and characterization of N -1-methylnicotinamide oxidase I and II separated from rat liver. Arch. Biochem. Biophys., 221: 534–542 (1983). Ueda, O., Sugihara, K., Ohta, S. and Kitamura, S.: Involvement of molybdenum hydroxylases in reductive metabolism of nitro polycyclic aromatic hydrocarbons in mammalian skin. Drug Metab. Dispos., 33: 1312–1318 (2005). Cresteil, T.: Onset of xenobiotic metabolism in children: toxicological implications. Food Addit. Contam., 15 (suppl): 45–51 (1998). Lacroix, D., Sonnier, M., Moncion, A., Cheron, G. and Cresteil, T.: Expression of CYP3A in the human liver. Evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. Eur. J. Biochem., 247: 625–634 (1997). Treluyer, J. M., Jacqz-Aigrain, E., Alvarez, F. and Cresteil, T.: Expression of CYP2D6 in developing human liver. Eur. J. Biochem., 202: 583–588 (1991). Vieira, I., Sonnier, M. and Cresteil, T.: Developmental expression of CYP2E1 in the human liver. Hypermethylation control of gene expression during the neonatal period. Eur. J. Biochem., 238: 476–483 (1996). Sonnier, M. and Cresteil, T.: Delayed ontogenesis of CYP1A2 in the human liver. Eur. J. Biochem., 251: 893–898 (1998). Treluyer, J. M., Gueret, G., Cheron, G., Sonnier, M. and Cresteil, T.: Developmental expression of CYP2C and 2C dependent activities in the human liver: in vivo W in vitro correlation and inducibility. Pharmacogenetics, 7: 441–452 (1997).