N-Oxygenation of Primary Amines and Hydroxylamines and Retroreduction of Hydroxylamines by Adult Human Liver Microsomes and Adult Human Flavin-Containing Monooxygenase 3

Lin, Jing; Berkman, Clifford E.; Cashman, John R.

doi:10.1021/tx9600614

Cited by 27 publications

(33 citation statements)

References 37 publications

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“…Based on the observed fragments from the CI mass spectral studies outlined above, it is postulated that spermine can indeed act as a free radical scavenger in the cell. These results suggest that formation and conversion of the adduct formed from spermine and hydroxy radical are similar to those observed following flavin-containing monooxygenase-mediated N-oxygenation of tyramine (27) and other aliphatic primary amines (28,29). In the presence of hydroxyl radical, bis-␣-[ 13 C]spermine could be expected to form the corresponding bis-N-hydroxyspermine, and then possibly the unstable bis-di-N-hydroxyspermine (5) (Fig.…”

Section: Resultssupporting

confidence: 64%

The natural polyamine spermine functions directly as a free radical scavenger

Sirisoma

Kuppusamy

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

608

405

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The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.The cellular polycationic polyamines are ubiquitous in nature and are absolutely required for eukaryotic cell growth (1, 2). However, very few specific molecular functions of polyamines have been described (3, 4). Some of the attributes ascribed to polyamines, particularly spermine, include the regulation of gene expression (5), the stabilization of chromatin (6-8), the prevention of endonuclease-mediated DNA fragmentation (9), and the inhibition of DNA damage (8, 10-13). Lovaas has recently reviewed many of these properties (14). Although protection of DNA damage by polyamines has been demonstrated, the mechanisms by which spermine functions in this protection are not known. Most of the proposals suggest that polyamine action is a result of charge neutralization and conformational changes (8,15). Because spermine is thought to be intimately associated with chromatin (7), we sought to investigate if spermine had a direct role in protecting DNA from free radical attack. Free radical and other reactive oxygen species (ROS) generation through normal cellular metabolism and by exogenous insult is a constant problem for which cells have developed multiple protective mechanisms to survive(16). Here we demonstrate that spermine, which is normally found in millimolar concentrations in the nucleus (17), can function directly as a free radical scavenger. MATERIALS AND METHODSAssays for DNA Strand Breaks. DNA strand breakage was measured by the conversion to open circular and linear forms of supercoiled ⌽X-174 RF1 double-stranded DNA (New England Biolabs). To assess DNA cleavage, 0.2 g of DNA was incubated in the presence of 30 M H 2 O 2 and 10 M CuCl 2 in PBS (pH 7.4) in a total volume of 30 l as described (18,19). Polyamines or antioxidants were coincubated as indicated. Following incubation the samples were separated by electrophoresis in a 1% agarose gel containing 40 mM Tris-acetate and 1 mM EDTA in a horizontal slab gel apparatus using Tris/acetate gel buffer. The gel was stained with ethidium bromide (2 g/ml) for 10 min, followed by destaining in water for 10 min, and was then photographed by UV translumination. The gels were photographed using an Eagle Eye digital camera (Stratagene). A single strand break in supercoiled double-stranded DNA results in the formation of open circular DNA, and double strand breaks result in the formation of linea...

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Section: Resultssupporting

confidence: 64%

The natural polyamine spermine functions directly as a free radical scavenger

Sirisoma

Kuppusamy

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

608

405

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“…This stereoselective N-oxygenation to the trans-oxime is also seen in the FMO-dependent N-oxygenation of amphetamine (Cashman et al, 1999), but differs from that seen with 10-N-(n-octylamino)-2-(trifluoromethyl) phenothiazine (Lin et al, 1996) and of straight-chain aliphatic amines discussed previously (Poulsen et al, 1986), for which the product is the cis-oxime. Interestingly, FMO2, which very efficiently N-oxygenates Ndodecylamine, is a poor catalyst of phenethylamine N-oxygenation.…”

Section: Nitrogen-containing Endogenous Substrates-mentioning

confidence: 49%

Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

Krueger

Williams

2005

Pharmacology & Therapeutics

515

444

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Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a "softnucleophile", usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics.In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences.The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration.

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“…5), a high affinity substrate for the FMOs (Poulsen et al, 1974). Nor was the enzyme stabilized by the presence of cofactor (NADH) when pretreated by moderate heat, as the heat-sensitive FMO would (Ziegler and Pettit, 1966), when assayed after the protocol of Grothusen et al (1996) (results not shown), and no correlation between FMO and the hydroxylamine reductase activity has been seen in human liver microsomes (Lin et al, 1996).…”

Section: Discussionmentioning

confidence: 95%

“…The results are not compatible with data previously presented, in which the participation of a CYP2D enzyme in an electron transport chain also including NADH-cytochrome b 5 reductase and cytochrome b 5 , was suggested (Clement el al., 1997;Clement and Lopian, 2003). Furthermore, Lin et al (1996) failed to obtain a correlation between the rate of hydroxylamine reduction (using 10-N-(8-(hydroxylamino)octyl)-2-(trifluoromethyl)phenothiazine) and the presence of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 in human liver microsomes. In our own experiments, we failed to reconstitute the reductase activity using recombinant CYP1A1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5 (T. B. Andersson, unpublished observations).…”

Section: Discussionmentioning

confidence: 99%

CHARACTERIZATION AND PARTIAL PURIFICATION OF THE RAT AND HUMAN ENZYME SYSTEMS ACTIVE IN THE REDUCTION OFN-HYDROXYMELAGATRAN AND BENZAMIDOXIME

Andersson¹,

Hofmann²,

Nordling³

et al. 2005

Drug Metab Dispos

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ABSTRACT:The enzymic basis for intracellular reduction of N-hydroxylated amidines to their corresponding amidines, and hydroxylamines to their corresponding amines, is unknown. The hydroxylated amidines can be used as prodrug moieties, and an understanding of the enzyme system active in the reduction can contribute to more efficient drug development. In this study, we examined the properties of this enzyme system using benzamidoxime and N-hydroxymelagatran as substrates. In rats and humans, the hepatic enzyme system was localized in mitochondria as well as in microsomes, using preferably NADH as cofactor. Potassium cyanide, N-methylhydroxylamine, p-hydroxymercuribenzoate, and desferrioxamine were efficient inhibitors, whereas typical cytochrome P450 (P450) inhibitors were ineffective. In rats, the highest specific activity was found in liver, adipose tissue, and kidneys, whereas in humans, the specific activity in the preparations of adipose tissue examined was lower. A sex difference was observed in rat liver, where 4-fold higher activity was seen in microsomes from female rats. No gender differences were present in any other tissue investigated. Partial purification of the hepatic system was achieved using polyethylene glycol fractionation followed by Octyl Sepharose chromatography at low detergent concentrations, whereas the enzyme was denatured after complete solubilization. The unique appearance of the enzyme activity in adipose tissue, together with the cyanide sensitivity and the failure of typical P450 inhibitors to impede the reaction, indicates that the enzyme system active in reduction of benzamidoxime and N-hydroxymelagatran formation is not of cytochrome P450 origin, but likely consists of an NADH-dependent electron transfer chain with a cyanide-sensitive protein as the terminal component.

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N-Oxygenation of Primary Amines and Hydroxylamines and Retroreduction of Hydroxylamines by Adult Human Liver Microsomes and Adult Human Flavin-Containing Monooxygenase 3

Cited by 27 publications

References 37 publications

The natural polyamine spermine functions directly as a free radical scavenger

The natural polyamine spermine functions directly as a free radical scavenger

Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

CHARACTERIZATION AND PARTIAL PURIFICATION OF THE RAT AND HUMAN ENZYME SYSTEMS ACTIVE IN THE REDUCTION OFN-HYDROXYMELAGATRAN AND BENZAMIDOXIME

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