Abstract
N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.
Highlights
In vitro the mARC-system catalyzes reductions of N-hydroxylated compounds as for instance N-hydroxylated base analogs
In previous studies it was demonstrated that in vitro the reconstituted recombinant mARC enzyme system has the ability to reduce all until now tested N-hydroxylated nucleobases and nucleosides [27]
MARC proteins are assumed to be involved in metabolism to the corresponding canonical nucleobases or nucleosides
Summary
In vitro the mARC-system catalyzes reductions of N-hydroxylated compounds as for instance N-hydroxylated base analogs. N-Hydroxylated bases and nucleosides such as N6-hydroxylaminopurine (HAP), N6-hydroxyadenosine (HAPR), 2-aminoHAP, or N4-hydroxycytidine have been shown to possess toxic and mutagenic properties in procaryotic and eucaryotic cells [1, 2] As base analogs, their mutagenicity is attributed to their ability to act alongside the natural substrates in cellular nucleic acid metabolism and their eventual incorporation into DNA [3]. Its physiological functions, are mostly unknown yet, mARC proteins are assumed to be involved in detoxication of mutagenic and toxic aromatic hydroxylamines like N-hydroxylated DNA-base analogs This hypothesis is supported by a recent study that describes significant down-regulated expression of human mARC2 in colon tumors of human tissue samples [26]. Flow cytometric analysis of Annexin V-PE and 7-AADstained cells as well as detection of caspase-mediated Poly (ADP-ribose) polymerase (PARP) cleavage was used to determine the influence of mARC knockdown on HAP-induced apoptotic effects on cells
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