Cytosol aldehyde oxidase

Saito et al. (134) found that the cytosolic nitroreductase activity was due to DT-diaphorase, aldehyde oxidase, xanthine oxidase plus other unidentified nitroreductases. As anticipated, the microsomal reduction of 1-nitropyrene was inhibited by 0 and stimulated by FMN which was attributed to this cofactor acting as an electron shuttle between NADPH-cytochrome P-450 reductase and cytochrome P-450. Carbon monoxide and type II cytochrome P-450 inhibitors decreased the rate of nitroreduction which was consistent with the involvement of cytochrome P-450. Induction of cytochromes P-450 increased rates of 1-aminopyrene formation and nitroreduction was demonstrated in a reconstituted cytochrome P-450 system, with isozyme P-448-IId catalyzing the reduction most efficiently. 

Aldehyde dehydrogenase Aldehyde oxidase Mitochondria/cytosol Liver cytosol... 

Several compounds are inhibitors of CYP2A6-mediated nicotme metabolism m vitro, mcludmg methoxsalen (8-methoxypsoralen), tranylcypromine, tryptamine and coumarin (Le Gal et al. 2003 MacDougall et al. 2003 Nakajima et al. 1996 Zhang et al. 2001). Raloxifene is a potent inhibitor of aldehyde oxidase and it has been shown to inhibit the formation of cotinine from nicotme-A -iminium ion in human liver cytosol (Obach 2004). 

Klecker, R.W, Cysyk, R.L. and Collins, J.M. (2006) Zebularine metabolism by aldehyde oxidase in hepatic cytosol from humans, monkeys, dogs, rats, and mice influence of sex and inhibitors. Bioorganic and Medicinal Chemistry, 14, 62-66. 

In addition to these classical aromatic ring hydroxylations, many nitrogen heterocycles are substrates for molybdenum-containing enzymes, such as xanthine oxidase and aldehyde oxidase, which are present in the hepatic cytosolic fractions from various animal species. The molybdenum hydroxylases (B-75MI10902) catalyze the oxidation of electron-deficient carbons in aromatic nitrogen heterocycles. The reactions catalyzed by these enzymes are generally represented by equations (2) and (3). 

Studies with various subcellular fractions are useful to ascertain which enzyme systems are involved in the metabolism of a chug candidate. In the absence of added cofactors, oxidative reactions such as oxidative deamination that are supported by mitochondria or by Ever microsomes contaminated with mitochondria membranes (as is the case with microsomes prepared from frozen liver samples) are likely catalyzed by monoamine oxidase (MAO), whereas oxidative reactions supported by cytosol are likely catalyzed by aldehyde oxidase and/or xanthine oxidase (a possible role for these enzymes in the metabolism of... 

Sugihara K, Kitamura S, Tatsumi K. Involvement of mammalian liver cytosols and aldehyde oxidase in reductive metabolism of zonisamide. Drug Metab Dispos 1996 24 199-202. 

Obach (2004) set up a method for inhibition studies in cytosol using phthalazine as a substrate of aldehyde oxidase. In this system 0.05 mg protein/ml was used in 25 mm potassium phosphate buffer ph7.4 containing 0.1 mm ethylendiaminetetraacetic acid. Incubation is terminated after 2.5 min. 

Oxidation CYP, FMO, aldehyde oxidase SER, SER, cytosol Diazinon, aldicarb, fenthion... 

The cytosolic molybdenum hydroxylases, namely aldehyde oxidase and xanthine oxidoreductase, which exist in a dehydrogenase form (XDH) and an oxidase form (XO). 

Other enzymes may also be involved in the oxidation of aldehydes, particularly aldehyde oxidase and xanthine oxidase, which belong to the molybdenum hydroxylases. These enzymes are primarily cytosolic, although microsomal aldehyde oxidase activity has been detected. They are flavoproteins, containing FAD and also molybdenum, and the oxygen incorporated is derived from water rather than molecular oxygen. Aldehyde oxidase and xanthine oxidase in fact oxidize a wide variety of substrates, both aldehydes and nitrogen containing heterocycles such as caffeine and purines (see below). Aldehyde... 

Other oxidoreductases that can play a major or less important role in drug metabolism are hemoglobin, monoamine oxidases (EC 1.4.3.4 MAO-A and MAO-B), which are essentially mitochondrial enzymes, the cytosolic molybdenum hydroxylases (xanthine oxidase, EC 1.1.3.22 xanthine dehydrogenase, EC 1.1.1.204 and aldehyde oxidase, EC 1.2.3.1), d the broad group of copper-containing amine oxidases (EC 1.4.3.6) (36-39). 

Besides the monooxygenases discussed above, a number of other oxidoreductases can oxidize xenobiotics. These enzymes are mostly but not exclusively nonmicrosomal, being present in the cytosol or mitochondria of the liver and extrahepatic tissues. The list includes alcohol dehydrogenases, aldehyde dehydrogenases, dihydrodiol dehydrogenases, haemoglobin, monoamine oxidases, xanthine oxidase and aldehyde oxidase. Some of these enzyme systems are discussed below. 

Hepatic cytosol is a useful source of sulfotransferases (Pacifici, 2004) and aldehyde oxidase (Lake et al., 2002). Compared with cytochrome P450s, these enzymes are rarely utilized in early drug metaboKsm screening and thus may be limited to specific mechanistic studies or later stages of the drug development... 

Very early reports described the reduction of nitro substituents by liver xanthine-oxidase (11), an enzyme located in the cytosol. In the microsomal fraction of the rat liver, nitro reduction is catalyzed by a system of cytochrome P450 in combination with NADPH-cyto-chrome-P450-reductase, as was described, for example, by Harada and Omura (19). Other enzymes that have been shown to reduce nitroaromatic compounds include aldehyde oxidase (51), dihydrolipoic amide dehydrogenase (47), cytochrome reductase (31), and dia-phorases (24). 

Conditions for cytosolic incubations depend on the aim of the assay e.g. to cover specific enzymatic activity present in the cytosol. For this purpose it is essential to fortify the incubation medium with the specific cofactor for the reaction-if needed (Ekins 1999). (J> -Nicotinamide adenine dinucleotide (NAD) is needed for alcohol and aldehyde dehydrogenases, flavin adenine dinucleotide (FAD) for polyamine oxidase, P-nicotinamide adenine dinucleotide phosphate (NADPH) for Dihydropyrimidine dehydrogenase. Phase II reactions depend on PAPS (sulfotransferases,... 

Other mono-oxygenases are not cytochrome P450 dependent, such as flavoproteins located in the endoplasmic reticulum that are involved in the oxidation of tertiary amines to N-oxides and of various sulfur compounds. Yet other oxidative enzymes, including alcohol and aldehyde dehydrogenases and monoamine oxidases, are located in the mitochondria or cytosol. 

Some oxidations are mediated by hepatic enzymes localized outside the microsomal system. Alcohol dehydrogenase and aldehyde dehydrogenase, which catalyse a variety of alcohols and aldehydes such as ethanol and acetaldehyde, are found in the soluble fraction of the liver. Xanthine oxidase, a cytosolic enzyme mainly found in the liver and in small intestine, but also present in kidneys, spleen and heart, oxidizes mercaptopurine to 6-thiouric acid. Monoamine oxidase, a mitochondrial enzyme found in liver, kidney, intestine and nervous tissue, oxidatively deanoinates several naturally occurring amines (catecholamines, serotonin), as well as a number of drugs. 

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