Aldehyde Oxidaseの機能解析—個体差・系統差を中心として—

Aliphatic aldehydes have a high affinity toward aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. In addition, the oxidation of xenobiotic-derived aromatic aldehydes by the latter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase, and xanthine oxidase activities in the oxidation of substituted benzaldehydes in separate preparations. The incubation of vanillin, isovanillin, and protocatechuic aldehyde with either guinea pig liver aldehyde oxidase, bovine milk xanthine oxidase, or guinea pig liver aldehyde dehydrogenase demonstrated that the three aldehyde oxidizing enzymes had a complementary substrate specificity. Incubations were also performed with specific inhibitors of each enzyme (isovanillin for aldehyde oxidase, allopurinol for xanthine oxidase, and disulfiram for aldehyde dehydrogenase) to determine the relative contribution of each enzyme in the oxidation of these aldehydes. Under these conditions, vanillin was rapidly oxidized by aldehyde oxidase, isovanillin was predominantly metabolized by aldehyde dehydrogenase activity, and protocatechuic aldehyde was slowly oxidized, possibly by all three enzymes. Thus, aldehyde oxidase activity may be a significant factor in the oxidation of aromatic aldehydes generated from amines and alkyl benzenes during drug metabolism. In addition, this enzyme may also have a role in the catabolism of biogenic amines such as dopamine and noradrenaline where 3-methoxyphenylacetic acids are major metabolites.

1. Aldehyde oxidase (AO) is a cytosolic enzyme that contributes to the Phase I metabolism of xenobiotics in human and preclinical species.2. Current studies explored in vitro metabolism of zoniporide in various animal species and humans using S9 fractions. The animal species included commonly used pharmacology and toxicology models and domestic animals such as the cat, cow or bull, pig and horse.3. In addition, gender and strain differences in some species were also explored.4. All animals except the dog and cat converted zoniporide to 2-oxozoniporide (M1).5. Michael-Menten kinetic studies were conducted in species that turned over zoniporide to M1.6. Marked differences in KM, Vmax and Clint were observed in the oxidation of zoniporide.7. Although the KM and Vmax of zoniporide oxidation in male and female human S9 was similar, some gender difference was observed in animals especially, in Vmax.8. The domestic animals also showed marked species differences in the AO activity and affinity toward zoniporide.

Aldehyde oxidase (AO) is a cytosolic enzyme belonging to the xanthine oxidoreductase (XOR) family. A variety of aldehyde substrates are known to be formed in cardiovascular disease secondary to inflammation and oxidative stress that are efficient AO substrates. While XOR has been reported to influence heart function in heart failure and is well known to be an important source of superoxide (O 2 • - ), little is known regarding the role of AO in cardiac oxidant signaling. In order to assess the presence of AO in the heart and its potential role in cardiovascular pathophysiology, we studied the distribution of AO in rat myocardium and isolated cardiac myocytes as well as its levels and activities in heart tissue. Furthermore, in isolated perfused rat hearts we measured the functional effect of AO activation or inhibition. Immunohistochemistry, using an AO specific antibody, showed a myofibrillar / sarcomere distribution for AO. In control rat myocardium, the activity of AO was found to be 1.5-fold higher than that of total XOR (0.21±0.02 vs . 0.14±0.01 nmol min -1 mg -1 of protein), and the levels of protein 2.5-fold higher than that of XOR (30.26±2.42 vs . 11.86±1.07 nM), while O 2 • - production (measured by cytochrome c assay) in the presence of optimal substrate levels was estimated to be >10-fold higher for AO than XOR. In isolated perfused rat hearts, infusion with the AO substrate 4-(dimethylamino)cinnamaldehyde ( p -DMAC) (10 μM) resulted in a significant increase in left ventricular developed pressure (LVDP) of 14±3% (p<0.05), while treatment with the potent inhibitor amadone (10 μM) completely inhibited this increase. Thus, AO is present in cardiac myocytes adjacent to the contractile proteins and in the presence of aldehyde substrates it can function as an important source of O 2 • - modulating contractile function. Further studies will be needed to assess the potential role of AO in modulating cardiac function in ischemic syndromes and heart failure.

We previously demonstrated the existence of a minor 130 kDa subunit in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blot analysis of monkey liver cytosol and expressed monkey aldehyde oxidase (AO) in Escherichia coli. In contrast, the 130 kDa subunit was not observed in rat AO. In the current study, the properties of the 130 kDa subunit were investigated from the viewpoint of species differences in the presence of the subunit and AO activity. Monkey AO with His-tag at the N- and C-terminus were expressed, and were immunoanalyzed with anti-AO and anti-His-tag antisera. The results revealed that the minor 130 kDa subunit was produced by cleavage at the N-terminal side of the 150 kDa subunit. The cleavage point was shown to be located between 188Leu and 189Pro of 150 kDa AO subunit by the Edman degradation method. The two amino acids related to the cleavage are contained in the linkage between the 2Fe-2S and FAD domains in AO of human and monkey, but not in AO of rat and mouse. As a fact, the 130 kDa subunit was observed in AO of human and monkey, but not in AO of rat and mouse, suggesting the two amino acids might be one reason of a species difference in the formation of the 130 kDa subunit. However, the existence of the 130 kDa subunit is not associated with the species differences in AO activity, because the cleavage results in the loss of 2Fe-2S cluster domain essential for exertion of AO activity.

Alcohol-induced breast cancer: a proposed mechanism

All-trans-retinoic acid (atRA) is a critical endogenous signaling molecule. atRA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to atRA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to atRA formation in human liver. Human recombinant AOX formed atRA from retinaldehyde (Km ∼1.5 ± 0.4 µM; kcat ∼3.6 ± 2.0 minute-1). In human liver S9 fractions (HLS9), atRA formation was observed in the absence of NAD+, suggesting AOX contribution to atRA formation. In the presence of NAD+, Eadie-Hofstee plots of atRA formation in HLS9 indicated that two enzymes contributed to atRA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of atRA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg-1 S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg-1 S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of atRA in the presence of NAD+ correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to atRA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter atRA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to atRA and contributes to hepatic atRA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic atRA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic atRA concentrations and signaling and alter vitamin A homeostasis.

Differential influence of molybdenum and tungsten on the growth of barley seedlings and the activity of aldehyde oxidase under salinity

Phylogeny and correlations of aldehyde oxidase, xanthine oxidase, xanthine dehydrogenase and peroxidase in animal tissues

Aldehyde oxidase (AOX) is a cytosolic drug-metabolizing enzyme which has attracted increasing attention in drug development due to its high hepatic expression, broad substrate profile and species differences. In contrast, there is limited information on the presence and activity of AOX in extrahepatic tissues including ocular tissues. Because several ocular drugs are potential substrates for AOX, we performed a comprehensive analysis of the AOX1 expression and activity profile in seven ocular tissues from humans, rabbits, and pigs. AOX activities were determined using optimized assays for the established human AOX1 probe substrates 4-dimethylamino-cinnamaldehyde (DMAC) and phthalazine. Inhibition studies were undertaken in conjunctival and retinal homogenates using well-established human AOX1 inhibitors menadione and chlorpromazine. AOX1 protein contents were quantitated with targeted proteomics and confirmed by immunoblotting. Overall, DMAC oxidation rates varied over 10-fold between species (human ˃˃ rabbit ˃ pig) and showed 2- to 6-fold differences between tissues from the same species. Menadione seemed a more potent inhibitor of DMAC oxidation across species than chlorpromazine. Human AOX1 protein levels were highest in the conjunctiva, followed by most posterior tissues, whereas anterior tissues showed low levels. The rabbit AOX1 expression was high in the conjunctiva, retinal pigment epithelial (RPE), and choroid while lower in the anterior tissues. Quantification of pig AOX1 was not successful but immunoblotting confirmed the presence of AOX1 in all species. DMAC oxidation rates and AOX1 contents correlated quite well in humans and rabbits. This study provides, for the first time, insights into the ocular expression and activity of AOX1 among multiple species.

Nitric oxide (NO) plays numerous essential physiological functions in terrestrial animals. In mammals, NO production from l-arginine is catalysed by the enzyme NO synthase (NOS). In recent years, data have begun to emerge on NOS expression and the physiological significance of NO in ectothermic vertebrates such as fish. However, there are relatively fewer data compared to the mammalian system. Although it is already well known that animal molybdoenzymes can convert nitrate and nitrite into NO, there is almost no information on the content and properties of molybdoenzymes in fish organs in the scientific literature. In this regard, the objectives of the present work were to detect the activity of classical molybdoenzymes xanthine oxidase (XO) and aldehyde oxidase (AO) in the liver and to study their possible activity to reduce nitrate and nitrite to nitrogen monoxide. In this work, the intrinsic activity of XO and AO was examined by using their substrates. At the same time, their nitrate (NR) – and nitrite reductase (NiR) activity were determined. It was determined that XO and AO in the fish liver are mainly represented by the molybdenum-free forms. The presence of an additional source of molybdenum can activate both the intrinsic and the NR and NiR activities. The NiR activity of XO and AO was higher than their NR activity. The data shows that treatment at a concentration of sodium molybdate and glutathione of 1.0 mM increases all activities of XO and AO. The optimal conditions for maximum activation of exogenous molybdate for XO and AO were reached by heating at 70 °C for 5 minutes. The activity of XO increased almost 4.7 times, and the activity of AO 7.7 times compared with its intrinsic activity without heat treatment. NO is formed from nitrite by the enzymes XO and AO much more than from nitrate.

Ziprasidone (Geodon, Zeldox), a recently approved atypical antipsychotic agent for the treatment of schizophrenia, undergoes extensive metabolism in humans with very little (<5%) of the dose excreted as unchanged drug. Two enzyme systems have been implicated in ziprasidone metabolism: the cytosolic enzyme, aldehyde oxidase, catalyzes the predominant reductive pathway, and cytochrome P4503A4 (CYP3A4) is responsible for two alternative oxidation pathways. The involvement of two competing pathways in ziprasidone metabolism greatly reduces the potential for pharmacokinetic interactions between ziprasidone and other drugs. Because CYP3A4 only mediates one third of ziprasidone metabolism, the likelihood of interactions between ziprasidone and CYP3A4 inhibitors/ substrates is low. Furthermore, aldehyde oxidase activity does not appear to be altered when drugs or xenobiotics are coadministered. Aldehyde oxidase, a molybdenum-containing enzyme, catalyzes the oxidation of N-heterocyclic drugs such as famciclovir and zaleplon, in addition to reducing some agents such as zonisamide. Both reactions can occur simultaneously. Although in vitro inhibitors of aldehyde oxidase have been identified, there are no reported clinical interactions with aldehyde oxidase inhibitors or inducers. There is no evidence of genetic polymorphism in aldehyde oxidase, and thus it not surprising that ziprasidone exposure demonstrates unimodality in humans. Aldehyde oxidase is unrelated to the similarly named enzyme aldehyde dehydrogenase, which is predominantly responsible for the oxidation of acetaldehyde during ethanol metabolism. Consequently, it is unlikely that there would be any pharmacokinetic interaction between ethanol and ziprasidone.

Thiopurines, such as 6-mercaptopurine (6-MP) and azathioprine, are converted to the inactive metabolites 6-thioxanthin (6-TX) and 6-thiouric acid (6-TUA). Molybdenum-containing oxidoreductases, aldehyde oxidase (AOX) and xanthine oxidase (XO), are involved in the oxidation of 6-MP to 6-TX; XO inhibitors affect the therapeutic efficacy of thiopurines and the incidence of adverse effects, such as liver and blood disorders. However, the role of AOX in the pharmacokinetics of 6-MP remains unclear. To clarify the clinical importance of AOX-mediated drug-drug interactions, we evaluated whether drugs that inhibit AOX affect 6-MP metabolism. The metabolism of 6-MP to 6-TX was strongly inhibited by AOX inhibitors (amitriptyline, chlorpromazine, clomipramine, clozapine, hydralazine, quetiapine, and raloxifene) in a reaction mixture containing human liver cytosol. The inhibition of 6-TX production rate by each AOX inhibitor was 60-70% at high concentrations, although the XO inhibitor febuxostat showed an inhibition rate of 10-30%. Furthermore, the combination of febuxostat and each AOX inhibitor showed greater inhibition than when each compound was added alone. The AOX inhibitor did not alter 6-MP oxidation by recombinant XO. These results suggest that AOX inhibition may affect the pharmacokinetics of thiopurines. However, because of the lower activity of AOX in rats than that in humans, the contribution of AOX could not be assessed using in vivo experiments. Further studies are needed to evaluate the contribution of AOX to the therapeutic and adverse effects of thiopurines, both in clinical studies and in animal models of liver humanization.

Aldehyde oxidase (AO) is a molybdo-flavo enzyme expressed predominantly in the liver, lung, and kidney. AO plays a major role in oxidation of aldehydes, as well as oxidation of various N-heterocyclic compounds of pharmacological and toxicological importance including antiviral (famciclovir), antimalarial (quinine), antitumour (methotrexate), and nicotine. The aim of this study was to investigate cytosolic aldehyde oxidase activity in human liver. Cytosolic AO was characterised using both the metabolism of N-[(2-dimethylamino)ethyl] acridine-4-carboxamide (DACA) and benzaldehyde to form DACA-9(10H)-acridone (quantified by HPLC with fluorescence detection) and benzoic acid (quantified spectrophotometrically). Thirteen livers (10 female, 3 male) were examined. The intrinsic clearance (Vmax/Km) of DACA varied 18-fold (0.03-0.50 m/min/mg). Vmax ranged from 0.20-3.10 nmol/ min/mg, and Km ranged from 3.5-14.2 microM. In the same specimens, the intrinsic clearance for benzaldehyde varied 5-fold (0.40-1.8 ml/min/mg). Vmax ranged from 3.60-12.6 nmol/min/mg and Km ranged from 3.6-14.6 microM. Furthermore, there were no differences in AO activity between male and female human livers, nor was there any relationship to age of donor (range 29-73 years), smoking status, or disease status. In conclusion, our results showed that there are variations in AO activity in human liver. These variations in aldehyde oxidase activity might reflect individual variations or they might be due to AO stability during processing and storage.

The Iberian Peninsula is home to a large number of endemic species that often show strong genetic subdivisions which indicate possible isolation of populations in the past. Numerous phylogeographic and phylogenetic studies on Iberian flora and fauna have revealed this territory to be the origin of genetic differentiation during the Pleistocene Ice Age. To better understand the influence of Plio-Pleistocene climatic oscillations on the cladogenesis and population structuring of refugial taxa restricted to freshwaters of the Iberian Peninsula, we studied the evolutionary history of the small European genus Baldellia, both from a phylogenetic/phylogeographic perspective and regarding its ecological characteristics. Molecular analyses were based on four plastid regions and nuclear ITS2 ribosomal spacer from individuals belonging to geographically isolated Baldellia populations, while the relationships between climate and Baldellia species distribution was investigated by redundancy analysis applied to the climatic data with the occurrence of Baldellia species as external variables. The absence of a clear phylogenetic signal most likely reflects a common and recent cladogenesis for Baldellia. In contrast, the more rapidly evolving plastid sequences indicate a recent phylogeographic history of refugial repartition and ecological segregation probably occurring during the last glaciation, a scenario well supported by marked differences in species’ ecological preferences and climate analysis.

This study presents data showing individual differences in aldehyde oxidase activity in human and monkey liver cytosols. When assayed with benzaldehyde as a substrate, a significant inter-subject variation in the activity was found in the human liver preparations. When assayed with N1-methylnicotinamide as a substrate, the inter-subject variation of the activity was also observed, but to a lesser extent compared with that of the activity with benzaldehyde. Similarly, variations in aldehyde oxidase activity were found in the monkey liver preparations when assayed with benzaldehyde or N1-methylnicotinamide. The present study suggested that at least two isozymes of aldehyde oxidase exist in the human liver preparations.