Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells

228–236 Nucleic Acids Research, 2008, Vol. 36, No. 1 doi:10.1093/nar/gkm1032 Published online 19 November 2007 Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells Janna M. Mundt1, Sang Soo Hah1, Rhoda A. Sumbad1, Vern Schramm2 and Paul T. Henderson1,* 1 Chemistry, Materials, Earth and Life Sciences Directorate, Lawrence Livermore National Laboratory, 7000 East Avenue, L-452, Livermore, CA 94551 and 2Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA Received September 9, 2007; Revised October 18, 2007; Accepted October 29, 2007 ABSTRACT 7,8-Dihydro-8-oxo-2’-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [14C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 106 nucleotides, 5–6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis. INTRODUCTION Reactive oxygen species (ROS) are important metabolic by-products that cause cellular damage, particularly to proteins, lipids and nucleic acids (1,2). ROS are produced through normal cellular metabolism in most cell types, primarily through oxidative phosphorylation. Incomplete transfer of electrons to O2 during oxidative phosphorylation yields products such as hydrogen peroxide, hydroxyl radicals and singlet oxygen species, which jointly comprise ROS (3). The oxidative modifications caused by ROS lead to cell dysfunction and possibly cell death (4) and are implicated in aging, cancer and other diseases (5,6). Much has been published as to how ROS chemically modify DNA and the nature of these modifications. It has been well characterized that when nucleotides are exposed to ROS, a variety of altered bases are formed (7,8). Amongst the four normal nucleobases, guanine (Gua) is the most susceptible to oxidation due to its low oxidation potential (9–11). The most abundant oxidized nucleobase found in DNA is 7,8-dihydro-8-oxoguanine (8-oxoGua, Figure 1A) (12). When present in DNA, 8-oxoGua can pair with both cytosine and adenine leading to G!T transversion mutations during replication and DNA repair. Likewise, 8-oxodGTP can mispair with adenine nucleotides to cause A!C transversion point mutations. The formation of 8-oxoGua in DNA has been shown to occur via two pathways: (i) through direct oxidation of Gua in DNA or (ii) indirectly via oxidation of dGTP in the nucleotide pool to 8-oxodGTP, followed by incorporation of 8-oxodGTP into the DNA by DNA polymerase(s) (1,13). A third pathway, metabolism of the 20 -deoxynucleoside 7,8-dihydro-8-oxo-20 -deoxyguanosine (8-oxodG) to 8-oxodGTP has only recently been reported (14,15), but with incomplete mechanistic detail. The nucleotide pool containing 8-oxodG may be modulated by extracellular sources. It is known that the repair products from other cells, cell turnover and the diet of the individual contribute to the 8-oxodG load as well. *To whom correspondence should be addressed. Tel: +1 925 423 2822; Fax: +1 925 422 2099; Email: The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. ß 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2008, Vol. 36, No. 1 229 A O O N N N H NH N NH HO NH O N O NH 2 OH Gua N NH2 N O OH 8-oxoGua * H N H N NH HO N O NH2 H dG B HO N H O O O H N N H 8-oxodG NH O NH2 O OH N N NH2 OH 8-oxoG Immucillin H PNP Gua HGPRT GMP GDP GTP RNA Pol RNA (free base) dG dC dCK or dGK Hydroxyurea RR dGMP dGDP dGTP DNA Pol DNA Figure 1. (A) Structures and abbreviations of guanine (Gua), deoxyguanosine (dG), 8-oxoguanine (8-oxoGua), 8-oxodeoxyguanosine (8-oxodG) and 8-oxoguanosine (8-oxoG), respectively. The asterisk on 8-oxodG indicates the 14C label. (B) Nucleotide salvage pathways for dG and inhibitors that can be used to elucidate which are the predominant metabolic pathways. In the cytoplasm, dG undergoes phosphorolysis to the free nucleobase Gua and 20 -deoxyribose-10 -phosphate by human purine nucleoside phosphorylase (PNP, top left). The resulting free base is then phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Two additional phosphorylation steps result in formation of GTP, which serves as a substrate for RNA polymerase-dependent incorporation into RNA. Alternatively, deoxycytidine kinase (dCK, bottom left) in the nucleus or deoxyguanosine kinase (dGK) in mitochondria use dG as a substrate to form dGMP. This in turn is phosphorylated twice to form dGTP, a substrate for DNA polymerase-dependent incorporation into DNA. These two major salvage pathways are connected by ribonucleotide diphosphate reductase (RR), which forms dGDP from GDP. Upon subsequent phosphorylation, the resulting dGTP serves as a nucleotide for DNA synthesis. IH, dC and HU stand for Immucillin H, deoxycytidine and hydroxyurea, respectively. Extracellular 8-oxodG has been identified in human plasma (16,17) and cerebrospinal fluid (18). 8-OxoGua is premutagenic and the cell employs a number of repair pathways to limit the presence of this species in DNA. Cells primarily use base excision repair (BER) to remove oxidized purines (19). In humans, the hOGG1 glycosylase removes 8-oxoGua from DNA when incorporated opposite cytidine (20,21). The Escherichia coli (E. coli) MutY homolog, MYH, removes the misincorporated adenine when opposite 8-oxoGua in DNA (19,22). In the nucleotide pool, the E. coli MutT homolog, hMTH1, hydrolyzes 8-oxodGTP to 8-oxodGMP to prevent 8-oxoGua incorporation into DNA (23,24). More recently, evidence has emerged that mismatch repair is synergistic wi (...truncated)