Inhibitory effects of neurotransmitters and steroids on human CYP2A6. (49/144)

Human CYP2A6 catalyzes the metabolism of nicotine, cotinine, and coumarin as well as some pharmaceutical drugs. CYP2A6 is highly expressed in liver and, also, in brain and steroid-related tissues. In this study, we investigated the inhibitory effects of neurotransmitters and steroid hormones on CYP2A6 activity. We found that coumarin 7-hydroxylation and cotinine 3'-hydroxylation by recombinant CYP2A6 expressed in baculovirus-infected insect cells were competitively inhibited by tryptamine (both K(i) = 0.2 microM), serotonin (K(i) = 252 microM and 167 microM), dopamine (K(i) = 49 microM and 22 microM), and histamine (K(i) = 428 microM and 359 microM). Cotinine formation from nicotine was inhibited by tryptamine (K(i) = 0.7 microM, competitive), serotonin (K(i) = 272 microM, noncompetitive), dopamine, noradrenaline, and adrenaline (K(i) = 11 microM, 54 microM, and 81 microM, uncompetitive). Estrogens (K(i) = 0.6-3.8 microM), androgens (K(i) = 60-149 microM), and corticosterone (K(i) = 36 microM) also inhibited cotinine formation, but coumarin 7-hydroxylation and cotinine 3'-hydroxylation did not. Nicotine-Delta(5'(1'))-iminium ion formation from nicotine was not affected by these steroid hormones, indicating that the inhibition of cotinine formation was due to the inhibitory effects on aldehyde oxidase. The nicotine-Delta(5'(1'))-iminium ion formation was competitively inhibited by tryptamine (K(i) = 0.3 microM), serotonin (K(i) = 316 microM), dopamine (K(i) = 66 microM), and histamine (K(i) = 209 microM). Thus, we found that some neurotransmitters inhibit CYP2A6 activity, being related with inter- and intraindividual differences in CYP2A6-dependent metabolism. The inhibitory effects of steroid hormones on aldehyde oxidase may also contribute to interindividual differences in nicotine metabolism.  (+info)

Genetic polymorphism of aldehyde oxidase in Donryu rats. (50/144)

One of major metabolic pathways of [(+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine] (RS-8359), a selective and reversible monoamine oxidase type A inhibitor, is the aldehyde oxidase-catalyzed 2-hydroxylation at the pyrimidine ring. Donryu rats showed a dimorphic pattern for the 2-oxidation activity with about 20- to 40-fold variations in the Vmax/Km values between a low and a high activity group. The rats were classified as extensive metabolizers (EM) and poor metabolizers (PM) of RS-8359, of which ratios were approximately 1:1. One rat among the EM rats of each sex showed extremely high activity, and they were referred to as ultrarapid metabolizers. There was no significant difference in the expression levels of mRNA of aldehyde oxidase between the EM and PM rats. Analysis of nucleotide sequences showed four substitutions, of which the substitutions at 377G>A and 2604C>T caused 110Gly-Ser and 852Ala-Val amino acid changes, respectively. Amino acid residue 110 is located very near the second Fe-S center of aldehyde oxidase. Its change from nonchiral Gly to chiral Ser may result in a conformational change of aldehyde oxidase protein with the shift of isoelectric point value from 5.0 in the EM rats to 6.2 in the PM rats. The 110Gly-Ser amino acid substitution (377G>A) may be primarily responsible for the variations of aldehyde oxidase activity observed in Donryu rats, in addition to the difference of expression levels of aldehyde oxidase protein. If a new drug candidate is primarily metabolized by aldehyde oxidase, attention should be given to using a rat strain with high aldehyde oxidase activity and small individual variation.  (+info)

Characterization of superoxide production from aldehyde oxidase: an important source of oxidants in biological tissues. (51/144)

Aldehyde oxidase, a molybdoflavoenzyme that plays an important role in aldehyde biotransformation, requires oxygen as substrate and produces reduced oxygen species. However, little information is available regarding its importance in cellular redox stress. Therefore, studies were undertaken to characterize its superoxide and hydrogen peroxide production. Aldehyde oxidase was purified to >98% purity and exhibited a single band at approximately 290 kDa on native polyacrylamide gradient gel electrophoresis. Superoxide generation was measured and quantitated by cytochrome c reduction and EPR spin trapping with p-dimethyl aminocinnamaldehyde as reducing substrate. Prominent superoxide generation was observed with an initial rate of 295 nmol min(-1) mg(-1). Electrochemical measurements of oxygen consumption and hydrogen peroxide formation yielded values of 650 and 355 nmol min(-1) mg(-1). In view of the ubiquitous distribution of aldehydes in tissues, aldehyde oxidase can be an important basal source of superoxide that would be enhanced in disease settings where cellular aldehyde levels are increased.  (+info)

Genetic studies of a cluster of acute lymphoblastic leukemia cases in Churchill County, Nevada. (52/144)

OBJECTIVE: In a study to identify exposures associated with 15 cases of childhood leukemia, we found levels of tungsten, arsenic, and dichlorodiphenyldichloroethylene in participants to be higher than mean values reported in the National Report on Human Exposure to Environmental Chemicals. Because case and comparison families had similar levels of these contaminants, we conducted genetic studies to identify gene polymorphisms that might have made case children more susceptible than comparison children to effects of the exposures. DESIGN: We compared case with comparison children to determine whether differences existed in the frequency of polymorphic genes, including genes that code for enzymes in the folate and purine pathways. We also included discovery of polymorphic forms of genes that code for enzymes that are inhibited by tungsten: xanthine dehydrogenase, sulfite oxidase (SUOXgene), and aldehyde oxidase. PARTICIPANTS: Eleven case children were age- and sex-matched with 42 community comparison children for genetic analyses. Twenty parents of case children also contributed to the analyses. RESULTS: One bilalleleic gene locus in SUOX was significantly associated with either case or comparison status, depending on which alleles the child carried (without adjusting for multiple comparisons). CONCLUSIONS: Although genetic studies did not provide evidence that a common agent or genetic susceptibility factor caused the leukemias, the association between a SUOXgene locus and disease status in the presence of high tungsten and arsenic levels warrants further investigation. RELEVANCE: Although analyses of community clusters of cancer have rarely identified causes, these findings have generated hypotheses to be tested in subsequent studies.  (+info)

Developmental changes of aldehyde oxidase in postnatal rat liver. (53/144)

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-3-carboxamide (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 confirmed 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 reflected 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.  (+info)

Cloning, expression, and characterization of male cynomolgus monkey liver aldehyde oxidase. (54/144)

In this study, we investigated the properties of monkey liver aldehyde oxidase directed toward the clarification of species differences. The aldehyde oxidase preparation purified from male cynomolgus monkey liver cytosol showed a major 150 kDa Coomassie brilliant blue (CBB)-stained band together with a minor 130 kDa band using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both bands were identified as being aldehyde oxidase by a database search of the MS data obtained with nano-liquid chromatography, quardrupole time of flight, mass spectrometry (nano-LC Q/TOF MS). Based on the sequence coverage, the 130 kDa protein was presumed to be deficient in 20-30 kDa mass from the N-terminus. Full male cynomolgus monkey aldehyde oxidase cDNA was cloned and sequenced with the four degenerate primers designed by considering the peptide sequences containing the amino acids specific for monkey aldehyde oxidase. The deduced amino acid sequences had 96% amino acid identity with those of human enzyme. The aldehyde oxidase expressed in Escherichia coli also exhibited two immunoreactive bands on SDS-PAGE/Western blot analysis. Further, the biphasic pattern was observed for Eadie-Hofstee plots of the (S)-enantiospecific 2-oxidation activity of RS-8359 with the expressed and cytosolic monkey liver aldehyde oxidase. The results suggested that two forms of aldehyde oxidase in monkey were the expression products by a single gene. In contrast, the similarly expressed rat aldehyde oxidase showed only one immunoreactive protein and monophasic pattern. The biphasic phenomenon could be caused by the existence of two aldehyde oxidase isoforms or two active sites in a single enzyme or some other reasons. Further studies on the problems of the biphasic pattern and species differences in aldehyde oxidase are needed.  (+info)

Lack of formation of aldehyde oxidase dimer possibly due to 377G>A nucleotide substitution. (55/144)

In addition to the many articles reporting on the marked differences in species and large differences in rat strains in response to aldehyde oxidase (AO), individual differences in some rat strains have also been reported. However, little has been clarified about any related molecular biological mechanisms. We previously revealed that nucleotide substitutions of 377G>A and 2604C>T in the AO gene might be responsible for individual differences in AO activity in Donryu strain rats. By using native polyacrylamide gel electrophoresis/Western blotting in this study, the lack of formation of the AO dimer protein, which is essential for catalytic activity, was shown in poor metabolizer Donryu rats, and this could be a major reason for the individual differences. Rat strain differences were also verified from the same perspectives of nucleotide substitutions and expression levels of a dimer protein. Rat strains with high AO activity showed nucleotide sequences of (377G, 2604C) and a dimer protein. In the case of those with low AO activity, the nucleotide at position 2604 was fixed at T, but varied at position 377, such as G, G/A, and A. An AO dimer was detected in the liver cytosols of rat strains with (377G, 2604T), whereas a monomer was observed in those with (377A, 2604T). These results suggest that the lack of formation of a dimer protein leading to loss of catalytic activity might be due to 377G>A nucleotide substitution. Individual and strain differences in AO activity in rats could be explained by this 377G>A substitution, at least in the rat strains used in this study.  (+info)

In vitro study of 6-mercaptopurine oxidation catalysed by aldehyde oxidase and xanthine oxidase. (56/144)

In spite of over 40 years of clinical use of 6-mercaptopurine, many aspects of complex pharmacology and metabolism of this drug remain unclear. It is thought that 6-mercaptopurine is oxidized to 6-thiouric acid through 6-thioxanthine or 8-oxo-6-mercaptopurine by one of two molybdenum hydroxylases, xanthine oxidase (XO), however, the role of other molybdenum hydroxylase, aldehyde oxidase (AO), in the oxidation of 6-mercaptopurine and possible interactions of AO substrates and inhibitors has not been investigated in more details. In the present study, the role of AO and XO in the oxidation of 6- mercaptopurine has been investigated. 6-mercaptopurine was incubated with bovine milk xanthine oxidase or partially purified guinea pig liver molybdenum hydroxylase fractions in the absence and presence of XO and AO inhibitor/substrates, and the reactions were monitored by spectrophotometric and HPLC methods. According to the results obtained from the inhibition studies, it is more likely that 6- mercaptopurine is oxidized to 6-thiouric acid via 6-thioxanthine rather than 8-oxo-6-mercaptopurine. The first step which is the rate limiting step is catalyzed solely by XO, whereas both XO and AO are involved in the oxidation of 6-thioxanthine to 6-thiouric acid.  (+info)