Joseph Lunec

Oxidative DNA damage: mechanisms, mutation and disease.

Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Although more than 20 base lesions have been identified, only a fraction of these have received appreciable study, most notably 8‐oxo‐2′deoxyguanosine. This lesion has been the focus of intense research interest and been ascribed much importance, largely to the detriment of other lesions. The present work reviews the basis for the biological significance of oxidative DNA damage, drawing attention to the multiplicity of proteins with repair activities along with a number of poorly considered effects of damage. Given the plethora of (often contradictory) reports describing pathological conditions in which levels of oxidative DNA damage have been measured, this review critically addresses the extent to which the in vitro significance of such damage has relevance for the pathogenesis of disease. It is suggested that some shortcomings associated with biomarkers, along with gaps in our knowledge, may be responsible for the failure to produce consistent and definitive results when applied to understanding the role of DNA damage in disease, highlighting the need for further studies.—Cooke, M. S., Evans, M. D., Dizdaroglu, M., Lunec, J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 17, 1195–1214 (2003)

Vitamin C exhibits pro-oxidant properties

Vitamin C is marketed as a dietary supplement, partly because of its ‘antioxidant’ properties. However, we report here that vitamin C administered as a dietary supplement to healthy humans exhibits a pro-oxidant, as well as an antioxidant, effect in vivo.

ReviewPart of the Series: From Dietary Antioxidants to Regulators in Cellular Signalling and Gene ExpressionReview: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C

Vitamin C (or ascorbic acid) is regarded as the most important water-soluble antioxidant in human plasma and mammalian cells which have mechanisms to recycle and accumulate it against a concentration gradient, suggesting that the vitamin might also have important intracellular functions. In this review we summarize evidence from human trials that have attempted an association between vitamin C supplementation and an effect on biomarkers of oxidative DNA damage. Most studies reviewed herein showed either a vitamin C-mediated reduction in oxidative DNA damage or a null effect, whereas only a few studies showed an increase in specific base lesions. We also address the possible beneficial effects of vitamin C supplementation for the prevention of cancer and cardiovascular disease. Finally, we discuss the contribution of cell culture studies to our understanding of the mode of action of vitamin C and we review recent evidence that vitamin C is able to modulate gene expression and cellular function, with a particular interest in cell differentiation.

Urinary 8-oxo-2′-deoxyguanosine — Source, significance and supplements

Oxidative damage to cellular biomolecules, in particular DNA, has been proposed to play an important role in a number of patholgical conditions, including carcinogenesis. A much studied consequence of oxygen-centred radical damage to DNA is 8-oxo-2′-deoxyguanosine (8-oxodG). Using numerous techniques, this lesion has been quantified in various biological matrices, most notably DNA and urine. Until recently, it was understood that urinary 8-oxodG derives solely from DNA repair, although the processes which may yield the modified deoxynucleoside have never been thoroughly discussed. This review suggests that nucleotide excision repair and the action of a specific endonuclease may, in addition to the nucleotide pool, contribute significantly to levels of 8-oxodG in the urine. On this basis, urinary 8-oxodG represents an important biomarker of generalised, cellular oxidative stress. Current data from antioxidant supplementation trials are examined and the potential for such compounds to modulate DNA repair is considered. It is stressed that further work is required to link DNA, serum and urinary levels of 8-oxodG such that the kinetics of formation and clearance may be elucidated, facilitating greater understanding of the role played by oxidative stress in disease.

Clinical and molecular stratification of disease risk in medulloblastoma

The accurate assessment of disease risk among children with medulloblastoma remains a major challenge to the field of paediatric neuro-oncology. In the current study we investigated the capacity of molecular abnormalities to increase the accuracy of disease risk stratification above that afforded by clinical staging alone. 41 primary medulloblastoma tumour samples were analysed for ErbB2 receptor expression using immunohistochemistry, and for aberrations of chromosome 17 and amplification of the MYC oncogene using fluorescence in situ hybridisation. The ErbB2 receptor and deletion of 17p were detected in 80% and 49% of tumours, respectively. 17p loss occurred either in isolation (20%), or in association with gain of 17q (29%), compatible with an isochromosome of 17q. Amplification of MYC was detected in only 2 tumours. Significant prognostic factors included, ‘metastatic disease’ (P = 0.0006), ‘sub-total tumour resection’ (P = 0.007), ‘high ErbB2 receptor expression’ (P = 0.003) and ‘isolated 17p loss’ (P = 0.003). Combined analysis of clinical and molecular factors enabled greater resolution of disease risk than clinical factors alone, identifying a sub-population of patients with particularly favourable disease outcome. These data support the hypothesis that a combination of clinical and molecular factors may afford a more reliable means of assigning disease risk in patients with medulloblastoma, thereby providing a more accurate basis for targeting therapy in children with this disease.

Molecular and Cellular Effects of Ultraviolet Light-Induced Genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earths stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.

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.

The Determination of Dehydroascorbic Acid and Ascorbic acid in the Serum and Synovial Fluid of Patients with Rheumatoid Arthritis (RA)

Using a novel high performance liquid chromatography (HPLC) determination of ascorbic acid and dehydroascorbic acid, we have measured the relative amounts of ascorbate and dehydroascorbate in 20 normal controls and in paired sera and synovial fluid from 13 patients with rheumatoid arthritis (RA). In complete contrast to previous published findings we were able to detect dehydroascorbate in normal human sera (12.0 +/- 3.7 mumol/l), while the mean and range of ascorbate measured in normals was 69.6 +/- 20.6 mumol/l. These findings were completely reversed in rheumatoid sera (21.8 +/- 8.6 mumol/l and 5.1 +/- 5.0 mumol/l for dehydroascorbate and ascorbic acid respectively). In several rheumatoid sera no ascorbate could be detected. In paired synovial fluid and serum samples, there was always more dehydroascorbate detected in synovial fluids than in the corresponding sera (p less than 0.01). The data suggests that the reduced level of ascorbate and increased level of dehydroascorbate may be a reflection of the increased antioxidant and free-radical scavenging activity of the vitamin in RA, especially within the inflamed joint.

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 8OHDG by HPLC. Further analysis by GC‐MS confirmed that levels of 8OHDG in SLE urine were 103‐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 8OHDG. We subsequently observed in SLE patients that 8OHDG 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.

Quality Assessment of Genetic Markers Used for Therapy Stratification

PURPOSE Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.