Immunochemical detection of UV-induced DNA damage and repair

The application of an antiserum to ultraviolet radiation (UVR)-damaged DNA is presented. A novel experimental system was employed to ascertain the limits of detection for this antiserum. Using a DNA standard containing a known amount of dimer, the limits of detection were found to be 0.9 fmol of dimer. This was compared to a limit of 20-50 fmol dimer using gas chromatography-mass spectrometry (GC-MS). Induction of thymine dimers in DNA following UVR exposure, as assessed using this antiserum in an enzyme-linked immunosorbent assay (ELISA), was compared with GC-MS measurements. The ELISA method successfully demonstrated the induction of lesions in DNA irradiated either with UVC or UVB, although despite high sensitivity, no discernible binding was seen to UVA-irradiated DNA. The antiserum was also shown to be applicable to immunocytochemistry, localising damage in the nuclei of UVR exposed keratinocytes in culture. The ability of the antiserum to detect DNA damage in skin biopsies of individuals exposed to sub-erythemal doses of UVR was also demonstrated. Moreover, the subsequent removal of this damage, as evidenced by a reduction in antiserum staining, was noted in sections of biopsies taken in the hours following irradiation. © 2003 Elsevier B.V. All rights reserved.

Urinary 8-oxo-2′-deoxyguanosine:Redox regulation of DNA repair in vivo?

DNA is susceptible to damage by reactive oxygen species (ROS). ROS are produced during normal and pathophysiological processes in addition to ionizing radiation, environmental mutagens, and carcinogens. 8-oxo-2′-deoxyguanosine (8-oxodG) is probably one of the most abundant DNA lesion formed during oxidative stress. This potentially mutagenic lesion causes G → T transversions and is therefore an important candidate lesion for repair, particularly in mammalian cells. Several pathways exist for the removal, or repair, of this lesion from mammalian DNA. The most established is via the base excision repair enzyme, human 8-oxoguanine glycosylase (hOgg1), which acts in combination with the human apurinic endonuclease (hApe). The latter is known to respond to regulation by redox reactions and may act in combination with hOgg1. We discuss evidence in this review article concerning alternative pathways in humans, such as nucleotide excision repair (NER), which could possibly remove the 8-oxodG lesion. We also propose that redox-active components of the diet, such as vitamin C, may promote such repair, affecting NER specifically. © 2002 Elsevier Science Inc.

Novel repair action of vitamin C upon in vivo oxidative DNA damage

There appears to be a paucity of data examining the effect of dietary antioxidants on levels of oxidative DNA damage in vivo, limiting evidence-based assessment of antioxidant efficacy, mechanisms and recommendation for optimal intake. We have examined levels of 8-oxo-2'-deoxyguanosine (8-oxodG) in mononuclear cell DNA, serum and urine from subjects undergoing supplementation with 500 mg/day vitamin C. Significant decreases in DNA levels of 8-oxodG were seen, correlating strongly with increases in plasma vitamin C concentration. Furthermore we established a timecourse for sequential, significant increases in serum and urinary 8-oxodG levels. These results illustrate, for the first time in humans, the kinetics of 8-oxodG removal and processing in vivo, suggesting a role for vitamin C in the regulation of DNA repair enzymes and thereby demonstrating a non-scavenging antioxidant effect.

An age-related increase in resistance to DNA damage-induced apoptotic cell death is associated with development of DNA repair mechanisms

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or gamma-irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2-ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up-regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.

Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line

Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.

8-Hydroxydeoxyguanosine. A marker of oxidative DNA damage in systemic lupus erythematosus

8-Hydroxydeoxyguanosine (80HDG) is a specific marker of oxidative damage to DNA. We have observed that patients with SLE (systemic lupus erythematosus), have undetectable levels of urinary 80HDG by HPLC. Further analysis by GC-MS confirmed that levels of 80HDG in SLE urine were 10(3)-fold lower than in an age- and sex-matched control group. Experiments utilising cultures of SLE and normal lymphocytes exposed to H2O2 confirmed the impaired ability of SLE lymphocytes to repair 80HDG. We subsequently observed in SLE patients that 80HDG had accumulated in low molecular weight DNA associated with circulating immune complexes. We suggest that oxygen radicals may induce pathology in SLE by maintaining the presence of an antigenic form of DNA in the circulation.

Deoxycytidine glyoxal:Lesion induction and evidence of repair following vitamin C supplementation in vivo

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p = .001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p = .0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected. © 2003 Elsevier Science Inc.

Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.