Mayuko Adachi

Lack of dimer formation ability in rat strains with low aldehyde oxidase activity

Aldehyde oxidase (AO) is a homodimer with a molecular weight of 300 kDa. To clarify the reasons for the well-known differences in rat strains, we set out to study the relationship between AO activity and the expression levels of its dimer. AO-catalyzed 2-oxidation activity of (S)-RS-8359 was measured in liver cytosols from ten rat strains. The expression levels of AO dimeric protein were evaluated by the native-PAGE/Western blot. Rat strains with low AO activity showed only a monomer, whereas strains with high activity overwhelmingly exhibited a dimer. Exceptionally, one strain in the high AO activity group displayed complex mixed expression patterns of low and high AO activity groups. However, there was a good relationship between AO activity and the expression levels of a dimer, but not of a monomer. The results suggest that rat strains with low AO activity lack the ability to produce a dimer necessary for catalytic activity, and AO differences in rat strains should be discussed in terms of the expression levels of the dimer itself.

Genetic polymorphism of aldehyde oxidase in donryu rats

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.

Lack of Formation of Aldehyde Oxidase Dimer Possibly Due to 377G>A Nucleotide Substitution

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.

A Single Amino Acid Substitution Confers High Cinchonidine Oxidation Activity Comparable with That of Rabbit to Monkey Aldehyde Oxidase 1

Aldehyde oxidase 1 (AOX1) is a major member of the xanthine oxidase family belonging to the class of complex molybdo-flavoenzymes and plays an important role in the nucleophilic oxidation of N-heterocyclic aromatic compounds and various aldehydes. The enzyme has been well known to show remarkable species differences. Comparing the rabbit and monkey enzymes, the former showed extremely high activity toward cinchonidine and methotrexate, but the latter exhibited only marginal activities. In contrast, monkey had several times greater activity than did rabbit toward zonisamide and (+)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine [(S)-RS-8359]. In this report, we tried to confer high cinchonidine oxidation activity comparable with that of rabbit AOX1 to monkey AOX1. The chimera proteins prepared by restriction enzyme digestion and recombination methods between monkey and rabbit AOX1s indicated that the sequences from Asn993 to Ala1088 of rabbit AOX1 are essential for the activity. The kinetic parameters were then measured using monkey AOX1 mutants prepared by site-directed mutagenesis. The monkey V1085A mutant acquired the high cinchonidine oxidation activity. Inversely, the reciprocal rabbit A1081V mutant lost the activity entirely: amino acid 1081 of rabbit AOX1 corresponding to amino acid 1085 of monkey AOX1. Thus, cinchonidine oxidation activity was drastically changed by mutation of a single residue in AOX1. However, this might be true for bulky substrates such as cinchonidine but not for small substrates. The mechanism of substrate-dependent species differences in AOX1 activity toward bulky substrates is discussed.

Leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB4 12-HD/PGR) responsible for the reduction of a double-bond of the α,β-unsaturated ketone of an aryl propionic acid non-steroidal anti-inflammatory agent CS-670

CS-670 is a non-steroidal anti-inflammatory agent with an α,β-unsaturated ketone structure. It exerts its pharmacological activity after being transformed to the active metabolite (2S,1′R,2′S)-trans-alcohol. Two consecutive reductions are needed for the formation of the active metabolite, reduction of the double-bond of the α,β-unsaturated ketone moiety, followed by reduction of the resulting saturated ketone. The objective of the current study was to identify the enzyme responsible for reduction of the double-bond. An enzyme purified from rat liver cytosol as a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was analysed by a Mascot database search of nano-LC tandem mass spectrometry (MS/MS) data and the enzyme was identified as 2-alkenal reductase (EC 1.3.1.74), which is known as an β-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent alkenal/one oxidoreductase and has a role for leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB4 12-HD/PGR). The identification was confirmed by cloning LTB4 12-HD/PGR cDNA from rat liver, expressing it in Escherichia coli, and characterizing the properties of the enzyme. The identity was further supported by the subcellular localization in cytosol, a cofactor requirement for NADPH, substrate specificity, and substantial inhibition by 15-ketoPGF2α, benzylideneacetophenone, indomethacin, and quercitrin. In addition to catalysing the biological reduction of eicosanoids, including prostaglandins, leukotrienes, and lipoxins, LTB4 12-HD/PGR was also determined to function as a xenobiotic-metabolizing enzyme.

Heredity mode of genetic polymorphism in aldehyde oxidase activity in Donryu strain rats

Donryu strain rats show genetic polymorphisms in the aldehyde oxidase gene, resulting in the phenotypic expression of ultrarapid metabolizers with homozygous nucleotide sequences (337G, 2604C), extensive metabolizers with heterozygous nucleotide sequences (377G/A, 2604C/T), and poor metabolizers with homozygous nucleotide sequences (377A, 2604T). In the mating experiments the ratio of the number of ultrarapid metabolizers, extensive metabolizers, and poor metabolizers rats in the F1 generation from the heterozygous F0 extensive metabolizers male and female rats was roughly 0.6 : 1.5 : 1, and the ratio converged to approximately 1 : 2 : 1 in the F2 generation from the heterozygous F1 extensive metabolizers male and female rats. On the contrary, all the F2 generation from homozygous F1 ultrarapid metabolizers male and female rats or from homozygous F1 poor metabolizers male and female rats had the ultrarapid metabolizers or the poor metabolizers genotypes and phenotypes. The genotypes completely agreed with the phenotypes in all individuals of F0, F1, and F2 generations. The results indicate that the genetic polymorphism of aldehyde oxidase in Donryu strain rats obeys Mendelian heredity. The reason for a low ratio of the ultrarapid metabolizers rats in the commercially available Donryu strain rats — not more than several per cent — compared with the ratio expected from the Mendelian rule is unknown.