Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells

Abstract

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

Measurement and Meaning of Oxidatively Modified DNA Lesions in Urine

“…A recent report failed to show 8-oxoGua -containing oligomers in urine (71), implying that further processing occurs, perhaps ultimately yielding 8-oxodG, or that they do not exist. However, under normal circumstances, the role of nucleotide excision repair in the removal of 8-oxoGua and perhaps other small oxidatively generated DNA lesions would seem to be negligible (102)(103)(104)(105)(106), although results from xeroderma pigmentosum cell lines have not entirely excluded the possibility (72,103,107,108). …”

Fourier-transform infrared spectroscopy and gas chromatography-mass spectrometry reveal a remarkable degree of structural damage in the DNA of wild fish exposed to toxic chemicals.

“…These reactions, which give rise to bulky adducts of base structures (2), were recently shown to block DNA replication (3) and mRNA transcription (4). However, the cytochrome P-450 reactions also yield H202 (5) which may be converted to the highly reactive hydroxyl radical (-OH) via the Fe2+-catalyzed Fenton reaction (6).…”

“…For example, the -OH-mediated introduction of a single oxygen into guanine, producing 8-hydroxyguanine (8-OH-Gua), results in a significant, 1-2% base misreading (3,8,9) and profoundly alters the methylation of cytosines that control gene expression (10). In fact, -OH produces a variety of OH adducts and ring-opening products in the DNA of a number of cancerous and noncancerous tissues (7,11).…”

“…At this point, the cellular redox status appears to be critical in determining the degree to which ring-opening products vs. 8-OH adducts are formed (7,11). Given that recent evidence indicates that the reductively formed Fapy derivatives block DNA replication (3) and mRNA transcription (4), whereas at least 8-OH-Gua leads to significant base misreading (3,8,9), the exposed fish would be expected to have a significant (although perhaps temporary) advantage in avoiding cancer by favoring the reductive (Fapy) pathway. In addition, the high concentrations of Fapy derivatives in the DNA of the exposed group (the mean Fapy-G concentration was about 1000-fold greater than that of the control) are likely to slow DNA replication, thereby enhancing the surveillance and repair of OH adducts during cell division (7,11).…”

“…This may be partly due to the fact that the replacement of 8-OH-Gua for guanine in DNA profoundly inhibits the methylation of cytosines controlling gene expression (10). Moreover, while the organism has the ability to channel the radical-induced stress into ring-opening products that potentially inhibit carcinogenesis (3,4), the ultimate outcome is about a 20% incidence of liver cancer (e.g., hepatocellular carcinoma) as a consequence of the continual exposure to high concentrations of xenobiotics (16,18). The highest incidence of hepatic tumors is found in fish older than those used in this study (i.e., >2 years old) (16,18).…”