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Aims: Urinary 8-oxo-7,8-dihydro-2¢-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical-and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject-and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs Kronos Science, Phoenix, Arizona. ANTIOXIDANTS & REDOX SIGNALINGVolume 18, Number 18, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/ars.2012.4714 2377showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (r p 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine-or SG-adjusted first morning samples are recommended.

Chronic low-dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low-dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation-induced senescence. Cellular proteins were quantified using isotope-coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence-related biological pathways were influenced by radiation, including cytoskeletal organization, cell-cell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation-induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low-dose irradiation.

Recent epidemiology studies highlighted the detrimental health effects of exposure to low dose and low dose rate ionizing radiation (IR): nuclear industry workers studies have shown increased leukaemia and solid tumour risks following cumulative doses of <100mSv and dose rates of <10mGy per year; paediatric patients studies have reported increased leukaemia and brain tumours risks after doses of 30-60mGy from computed tomography scans. Questions arise, however, about the impact of even lower doses and dose rates where classical epidemiological studies have limited power but where subsets within the large cohorts are expected to have an increased risk. Further progress requires integration of biomarkers or bioassays of individual exposure, effects and susceptibility to IR. The European DoReMi (Low Dose Research towards Multidisciplinary Integration) consortium previously reviewed biomarkers for potential use in IR epidemiological studies. Given the increased mechanistic understanding of responses to low dose radiation the current review provides an update covering technical advances and recent studies. A key issue identified is deciding which biomarkers to progress. A roadmap is provided for biomarker development from discovery to implementation and used to summarise the current status of proposed biomarkers for epidemiological studies. Most potential biomarkers remain at the discovery stage and for some there is sufficient evidence that further development is not warranted. One biomarker identified in the final stages of development and as a priority for further research is radiation specific mRNA transcript profiles.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) is one of the mutagenic base modifications produced in DNA by the reaction of reactive oxygen species. The biological significance of 8-oxo-dG is shown by the existence of repair pathways that are able to recognize and remove this lesion from both DNA and the nucleotide pool. The final outcome of these evolutionarily conserved repair mechanisms in man is excretion of 8-oxo-dG/8-oxo-Gua from the intracellular to extracellular milieu including the blood plasma and urine. The aim of this investigation was to establish dose response relations for radiation-induced appearance of extracellular 8-oxo-dG in cellular model systems. Here we report on excretion of 8-oxo-dG after in vitro irradiation of whole blood and isolated lymphocytes with clinically relevant doses. We find that this excretion is dependent on dose and individual repair capacity, and that it saturates above doses of 0.5-1 Gy of gamma radiation. Our data also suggest that the nucleotide pool is a significant target that contributes to the levels of extracellular 8-oxo-dG; hence the mutagenic target for oxidative stress is not limited to the DNA molecule only. We conclude that extracellular 8-oxo-dG levels after in vitro irradiation have a potential to be used as a sensitive marker for oxidative stress.

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.

Tardigrades are highly tolerant to desiccation and ionizing radiation but the mechanisms of this tolerance are not well understood. In this paper, we report studies on dose responses of adults and eggs of the tardigrade Hypsibius dujardini exposed to gamma radiation. In adults the LD50/48h for survival was estimated at ~ 4200 Gy, and doses higher than 100 Gy reduced both fertility and hatchability of laid eggs drastically. We also evaluated the effect of radiation (doses 50 Gy, 200 Gy, 500 Gy) on eggs in the early and late embryonic stage of development, and observed a reduced hatchability in the early stage, while no effect was found in the late stage of development. Survival of juveniles from irradiated eggs was highly affected by a 500 Gy dose, both in the early and the late stage. Juveniles hatched from eggs irradiated at 50 Gy and 200 Gy developed into adults and produced offspring, but their fertility was reduced compared to the controls. Finally we measured the effect of low temperature during irradiation at 4000 Gy and 4500 Gy on survival in adult tardigrades, and observed a slight delay in the expressed mortality when tardigrades were irradiated on ice. Since H. dujardini is a freshwater tardigrade with lower tolerance to desiccation compared to limno-terrestrial tardigrades, the high radiation tolerance in adults, similar to limno-terrestrial tardigrades, is unexpected and seems to challenge the idea that desiccation and radiation tolerance rely on the same molecular mechanisms. We suggest that the higher radiation tolerance in adults and late stage embryos of H. dujardini (and in other studied tardigrades) compared to early stage embryos may partly be due to limited mitotic activity, since tardigrades have a low degree of somatic cell division (eutely), and dividing cells are known to be more sensitive to radiation.

Our findings motivate further research on the shape of the dose-response and the dose rate effect for radiation-induced vascular senescence.