Inhalation of House Dust and Ozone Alters Systemic Levels of Endothelial Progenitor Cells, Oxidative Stress, and Inflammation in Elderly Subjects.

Ambient air pollution including ozone and especially particulate matter represents important causes of cardiovascular disease. However, there is limited knowledge on indoor air dust with respect to this risk and the potential interactions between dust and ozone. Here, we exposed 23 healthy elderly subjects for 5.5 h, to either clean air, house dust at 275 µg/m3 (diameter < 2.5 µm), ozone at 100 ppb or combined house dust and ozone in a double-blinded randomized cross-over study. The combined house dust and ozone exposure was associated with a 48% (95% CI 24%-65%) decrease as compared with the clean air exposure, in CD34+KDR+ late endothelial progenitor cells (EPCs) per leukocyte in the blood shortly after exposure, whereas none of the single exposures resulted in a significant effect. The combined exposure also increased reactive oxygen species production capacity in granulocytes and monocytes as well as an up-regulation of interleukin-8 mRNA levels in leukocytes. Ozone alone reduced the gene expression of tumor necrosis factor and C-C motif chemokine ligand 2, while dust alone showed no effects. The combined exposure to house dust and ozone also reduced levels of oxidized purines in DNA consistent with concomitant up-regulation of mRNA of the repair enzyme 8-oxoguanine DNA glycosylase. The reduction in late EPCs can be an indicator of cardiovascular risk caused by the combination of pulmonary oxidative stress induced by ozone and the inflammatory potential of the house dust. These data were corroborated with in vitro findings from exposed human macrophages and endothelial cells.

Searching for assay controls for the Fpg- and hOGG1-modified comet assay.

The formamidopyrimidine DNA glycosylase (Fpg) and human 8-oxoguanine DNA glycosylase (hOGG1)-modified comet assays have been widely used in human biomonitoring studies. The purpose of this article is to assess differences in reported levels of Fpg- and hOGG1-sensitive sites in leukocytes and suggest suitable assay controls for the measurement of oxidatively damaged DNA. An assessment of the literature showed a large variation in the reported levels of Fpg-sensitive sites (range 0.05-1.31 lesions/106 bp). The levels of Fpg-sensitive sites are lower in studies where Fpg has been obtained from commercial suppliers or unknown sources as compared to Fpg from one particular non-commercial source (χ2 = 7.14, P = 0.028). The levels of hOGG1-sensitive sites are lower (range: 0.04-0.18 lesions/106 bp in leukocytes) compared to the Fpg-sensitive sites. Surprisingly, few publications have reported the use of oxidising agents as assay controls, with the exception of hydrogen peroxide. This may be due to a lack of consensus about suitable controls for the Fpg- and hOGG1-modified comet assay. A major challenge is to find an oxidising agent that only oxidises nucleobases and does not generate DNA strand breaks because this reduces the dynamic range of Fpg- and hOGG1-sensitive sites in the comet assay. Based on a literature search we selected the photosensitiser Ro19-8022 plus light, KBrO3, 4-nitroquinoline-1-oxide, Na2Cr2O7 and ferric nitrilotriacetate as possible assay controls. A subsequent assessment of these compounds for generating cryopreserved assay controls in mononuclear blood cells showed that Ro19-8022 plus light, KBrO3 and 4-nitroquinoline-1-oxide provided suitable assay controls. We recommend these compounds as comet assay controls for oxidatively damaged DNA.

A Flow Cytometry-based Method for the Screening of Nanomaterial-induced Reactive Oxygen Species Production in Leukocytes Subpopulations in Whole Blood.

To date, the use and translation of nanomedicine from the laboratory to the clinic has been relatively slow. Among other issues, one of the reasons for this tardiness is the lack of the availability of quick and reliable toxicity tools for the screening of nanomaterials (NMs). In this investigation, we apply a flow cytometry-based method for the detection of nanomaterial-induced oxidative stress by measurement of reactive oxygen species production in specific leukocyte subpopulations in human whole blood. The screening of a panel of relevant nanomedical-associated materials (liposomes, silica, iron oxide and functionalized single-walled carbon nanotubes) demonstrated that only the carbon nanotubes induced oxidative stress in human circulating leukocytes. In summary, we apply and corroborate a flow cytometry-based method for the simple and effective measurement of NM-induced oxidative stress in human blood subpopulations after realistic and relevant exposure scenarios which is extremely useful in future toxicological applications.

Nanomaterial-induced cell death in pulmonary and hepatic cells following exposure to three different metallic materials: The role of autophagy and apoptosis.

Autophagy is the catabolic process involving the sequestration of the cytoplasm within double-membrane vesicles, which fuse with lysosomes to form autolysosomes in which autophagic targets are degraded. Since most endocytic routes of nanomaterial uptake converge upon the lysosome and the possibility that autophagy induction by NMs may be an attempt by the cell to self-preserve following the external challenge, this study investigated the role of autophagy following exposure to a panel of widely used metal-based NMs with high toxicity (Ag and ZnO) or low toxicity (TiO2) in a pulmonary (A549) and hepatic (HepG2) cell line. The in vitro exposure to the Ag and ZnO NMs resulted in the induction of both apoptosis and autophagy pathways in both cell types. However, the progression of autophagy was blocked in the formation of the autolysosome, which coincided with morphologic changes in the actin cytoskeleton. This response was not observed following the exposure to low-toxicity TiO2 NMs. Overall, the results show that high toxicity NMs can cause a dysfunction in the autophagy pathway which is associated with apoptotic cell death.

Exposure to ultrafine particles, intracellular production of reactive oxygen species in leukocytes and altered levels of endothelial progenitor cells.

Exposure to particles in the fine and ultrafine size range has been linked to induction of low-grade systemic inflammation, oxidative stress and development of cardiovascular diseases. Declining levels of endothelial progenitor cells within systemic circulation have likewise been linked to progression of cardiovascular diseases. The objective was to determine if exposure to fine and ultrafine particles from indoor and outdoor sources, assessed by personal and residential indoor monitoring, is associated with altered levels of endothelial progenitor cells, and whether such effects are related to leukocyte-mediated oxidative stress. The study utilized a cross sectional design performed in 58 study participants from a larger cohort. Levels of circulating endothelial progenitor cells, defined as either late (CD34(+)KDR(+) cells) or early (CD34(+)CD133(+)KDR(+) cells) subsets were measured using polychromatic flow cytometry. We additionally measured production of reactive oxygen species in leukocyte subsets (lymphocytes, monocytes and granulocytes) by flow cytometry using intracellular 2',7'-dichlorofluoroscein. The measurements encompassed both basal levels of reactive oxygen species production and capacity for reactive oxygen species production for each leukocyte subset. We found that the late endothelial progenitor subset was negatively associated with levels of ultrafine particles measured within the participant residences and with reactive oxygen species production capacity in lymphocytes. Additionally, the early endothelial progenitor cell levels were positively associated with a personalised measure of ultrafine particle exposure and negatively associated with both basal and capacity for reactive oxygen species production in lymphocytes and granulocytes, respectively. Our results indicate that exposure to fine and ultrafine particles derived from indoor sources may have adverse effects on human vascular health.

Atherosclerosis and vasomotor dysfunction in arteries of animals after exposure to combustion-derived particulate matter or nanomaterials.

Exposure to particulate matter (PM) from traffic vehicles is hazardous to the vascular system, leading to clinical manifestations and mortality due to ischemic heart disease. By analogy, nanomaterials may also be associated with the same outcomes. Here, the effects of exposure to PM from ambient air, diesel exhaust and certain nanomaterials on atherosclerosis and vasomotor function in animals have been assessed. The majority of studies have used pulmonary exposure by inhalation or instillation, although there are some studies on non-pulmonary routes such as the gastrointestinal tract. Airway exposure to air pollution particles and nanomaterials is associated with similar effects on atherosclerosis progression, augmented vasoconstriction and blunted vasorelaxation responses in arteries, whereas exposure to diesel exhaust is associated with lower responses. At present, there is no convincing evidence of dose-dependent effects across studies. Oxidative stress and inflammation have been observed in the arterial wall of PM-exposed animals with vasomotor dysfunction or plaque progression. From the data, it is evident that pulmonary and systemic inflammation does not seem to be necessary for these vascular effects to occur. Furthermore, there is inconsistent evidence with regard to altered plasma lipid profile and systemic inflammation as a key step in vasomotor dysfunction and progression of atherosclerosis in PM-exposed animals. In summary, the results show that certain nanomaterials, including TiO2, carbon black and carbon nanotubes, have similar hazards to the vascular system as combustion-derived PM.

Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro.

Exposure to diesel exhaust particles (DEP) has been associated with adverse cardiopulmonary health effects, which may be related to dysregulation of lipid metabolism and formation of macrophage foam cells. In this study, THP-1 derived macrophages were exposed to an automobile generated DEP (A-DEP) for 24h to study lipid droplet formation and possible mechanisms. The results show that A-DEP did not induce cytotoxicity. The production of reactive oxygen species was only significantly increased after exposure for 3h, but not 24h. Intracellular level of reduced glutathione was increased after 24h exposure. These results combined indicate an adaptive response to oxidative stress. Exposure to A-DEP was associated with significantly increased formation of lipid droplets, as well as changes in lysosomal function, assessed as reduced LysoTracker staining. In conclusion, these results indicated that exposure to A-DEP may induce formation of lipid droplets in macrophages in vitro possibly via lysosomal dysfunction.

No oxidative stress or DNA damage in peripheral blood mononuclear cells after exposure to particles from urban street air in overweight elderly.

Exposure to traffic-related particulate matter (PM) has been associated with increased risk of lung disease, cancer and cardiovascular disease especially in elderly and overweight subjects. The proposed mechanisms involve intracellular production of reactive oxygen species (ROS), inflammation and oxidation-induced DNA damage studied mainly in young normal-weight subjects. We performed a controlled cross-over, randomised, single-blinded, repeated-measure study where 60 healthy subjects (25 males and 35 females) with age 55-83 years and body mass index above 25 kg/m(2) were exposed for 5h to either particle-filtered or sham-filtered air from a busy street with number of concentrations and PM2.5 levels of 1800/cm(3) versus 23 000/cm(3) and 3 µg/m(3) versus 24 µg/m(3), respectively. Peripheral blood mononuclear cells (PBMCs) were collected and assayed for production of ROS with and without ex vivo exposure to nanosized carbon black as well as expression of genes related to inflammation (chemokine (C-C motif) ligand 2, interleukin-8 and tumour necrosis factor), oxidative stress response (heme oxygenase (decycling)-1) and DNA repair (oxoguanine DNA glycosylase). DNA strand breaks and oxidised purines were assayed by the alkaline comet assay. No statistically significant differences were found for any biomarker immediately after exposure to PM from urban street air although strand breaks and oxidised purines combined were significantly associated with the particle number concentration during exposure. In conclusion, 5h of controlled exposure to PM from urban traffic did not change the gene expression related to inflammation, oxidative stress or DNA repair, ROS production or oxidatively damaged DNA in PBMCs from elderly overweight human subjects.

Controlled exposure to diesel exhaust and traffic noise--Effects on oxidative stress and activation in mononuclear blood cells.

UNLABELLED: Particulate air pollution increases risk of cancer and cardiopulmonary disease, partly through oxidative stress. Traffic-related noise increases risk of cardiovascular disease and may cause oxidative stress. In this controlled random sequence study, 18 healthy subjects were exposed for 3h to diesel exhaust (DE) at 276 µg/m(3) from a passenger car or filtered air, with co-exposure to traffic noise at 48 or 75 dB(A). Gene expression markers of inflammation, (interleukin-8 and tumor necrosis factor), oxidative stress (heme oxygenase (decycling-1)) and DNA repair (8-oxoguanine DNA glycosylase (OGG1)) were unaltered in peripheral blood mononuclear cells (PBMCs). No significant differences in DNA damage levels, measured by the comet assay, were observed after DE exposure, whereas exposure to high noise levels was associated with significantly increased levels of hOGG1-sensitive sites in PBMCs. Urinary levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine were unaltered. In auxiliary ex vivo experiments whole blood was incubated with particles from the exposure chamber for 3h without effects on DNA damage in PBMCs or intracellular reactive oxygen species production and expression of CD11b and CD62L adhesion molecules in leukocyte subtypes. CONCLUSION: 3-h exposure to DE caused no genotoxicity, oxidative stress or inflammation in PBMCs, whereas exposure to noise might cause oxidatively damaged DNA.

In vivo toxicity of cationic micelles and liposomes.

This study investigated toxicity of nanocarriers comprised of cationic polymer and lipid components often used in gene and drug delivery, formulated as cationic micelles and liposomes. Rats were injected intravenously with 10, 25 or 100 mg/kg and sacrificed after 24 or 48 h, or 24 h after the last of three intravenous injections of 100 mg/kg every other day. Histological evaluation of liver, lung and spleen, clinical chemistry parameters, and hematology indicated little effect of treatment. DNA strand breaks were increased in the lung and spleen. Further, in the dose response study we found unaltered expression levels of genes in the antioxidant response (HMOX1) and repair of oxidized nucleobases (OGG1), whereas expression levels of cytokines (IL6, CXCL2 and CCL2) were elevated in lung, spleen or liver. The results indicate that assessment of genotoxicity and gene expression add information on toxicity of nanocarriers, which is not obtained by histology and hematology. FROM THE CLINICAL EDITOR: This study investigates the toxicity of cationic micelles and liposomes utilized as nanocarriers in gene and drug delivery, demonstrating its effects on the lungs, spleen and liver.

Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals.

Increased levels of oxidatively damaged DNA have been documented in studies of metal, metal oxide, carbon-based and ceramic engineered nanomaterials (ENMs). In particular, 8-oxo-7,8-dihydroguanine-2'-deoxyguanosine (8-oxodG) is widely assessed as a DNA nucleobase oxidation product, measured by chromatographic assays, antibody-based methods or the comet assay with DNA repair enzymes. However, spurious oxidation of DNA has been a problem in certain studies applying chromatographic assays, yielding high baseline levels of 8-oxodG. Antibody-based assays detect high 8-oxodG baseline levels, related to cross-reactivity with other molecules in cells. This review provides an overview of efforts to reliably detect oxidatively damaged DNA and a critical assessment of the published studies on DNA damage levels. Animal studies with high baseline levels of oxidatively damaged DNA are more likely to show positive associations between exposure to ENMs and oxidized DNA in tissue than studies showing acceptable baseline levels (odds ratio = 12.1, 95% confidence interval: 1.2-124). Nevertheless, reliable studies indicate that intratracheal instillation of nanosized carbon black is associated with increased levels of oxidatively damaged DNA in lung tissue. Oral exposure to nanosized carbon black, TiO2 , carbon nanotubes and ZnO is associated with elevated levels of oxidatively damaged DNA in tissues. These observations are supported by cell culture studies showing concentration-dependent associations between ENM exposure and oxidatively damaged DNA measured by the comet assay. Cell culture studies show relatively high variation in the ability of ENMs to oxidatively damage DNA; hence, it is currently impossible to group ENMs according to their DNA damaging potential.

Applications of the comet assay in particle toxicology: air pollution and engineered nanomaterials exposure.

Exposure to ambient air particles is associated with elevated levels of DNA strand breaks (SBs) and endonuclease III, formamidopyrimidine DNA glycosylase (FPG) and oxoguanine DNA glycosylase-sensitive sites in cell cultures, animals and humans. In both animals and cell cultures, increases in SB and in oxidatively damaged DNA are seen after exposure to a range of engineered nanomaterials (ENMs), including carbon black, carbon nanotubes, fullerene C60, ZnO, silver and gold. Exposure to TiO2 has generated mixed data with regard to SB and oxidatively damaged DNA in cell cultures. Nanosilica does not seem to be associated with generation of FPG-sensitive sites in cell cultures, while large differences in SB generation between studies have been noted. Single-dose airway exposure to nanosized carbon black and multi-walled carbon nanotubes in animal models seems to be associated with elevated DNA damage levels in lung tissue in comparison to similar exposure to TiO2 and fullerene C60. Oral exposure has been associated with augmented DNA damage levels in cells of internal organs, although the doses have been typically very high. Intraveneous and intraperitoneal injection of ENMs have shown contradictory results dependent on the type of ENM and dose in each set of experiments. In conclusion, the exposure to both combustion-derived particles and ENMs is associated with increased levels of DNA damage in the comet assay. Particle size, composition and crystal structure of ENM are considered important determinants of toxicity, whereas their combined contributions to genotoxicity in the comet assay are yet to be thoroughly investigated.

Oxidative stress and inflammation generated DNA damage by exposure to air pollution particles.

Generation of oxidatively damaged DNA by particulate matter (PM) is hypothesized to occur via production of reactive oxygen species (ROS) and inflammation. We investigated this hypothesis by comparing ROS production, inflammation and oxidatively damaged DNA in different experimental systems investigating air pollution particles. There is substantial evidence indicating that exposure to air pollution particles was associated with elevated levels of oxidatively damaged nucleobases in circulating blood cells and urine from humans, which is supported by observations of elevated levels of genotoxicity in cultured cells exposed to similar PM. Inflammation is most pronounced in cultured cells and animal models, whereas an elevated level of oxidatively damaged DNA is more pronounced than inflammation in humans. There is non-congruent data showing corresponding variability in effect related to PM sampled at different locations (spatial variability), times (temporal variability) or particle size fraction across different experimental systems of acellular conditions, cultured cells, animals and humans. Nevertheless, there is substantial variation in the genotoxic, inflammation and oxidative stress potential of PM sampled at different locations or times. Small air pollution particles did not appear more hazardous than larger particles, which is consistent with the notion that constituents such as metals and organic compounds also are important determinants for PM-generated oxidative stress and inflammation. In addition, the results indicate that PM-mediated ROS production is involved in the generation of inflammation and activated inflammatory cells can increase their ROS production. The observations indicate that air pollution particles generate oxidatively damaged DNA by promoting a milieu of oxidative stress and inflammation.

Role of oxidative stress in carbon nanotube-generated health effects.

The development of products containing carbon nanotubes (CNTs) is a major achievement of nanotechnology, although concerns regarding risk of toxic effects linger if the hazards associated with these materials are not thoroughly investigated. Exposure to CNTs has been associated with depletion of antioxidants, increased intracellular production of reactive oxygen species and pro-inflammatory signaling in cultured cells with primary function in the immune system as well as epithelial, endothelial and stromal cells. Pre-treatment with antioxidants has been shown to attenuate these effects, indicating a dependency of oxidative stress on cellular responses to CNT exposure. CNT-mediated oxidative stress in cell cultures has been associated with elevated levels of lipid peroxidation products and oxidatively damaged DNA. Investigations of oxidative stress endpoints in animal studies have utilized pulmonary, gastrointestinal, intravenous and intraperitoneal exposure routes, documenting elevated levels of lipid peroxidation products and oxidatively damaged DNA nucleobases especially in the lungs and liver, which to some extent occur concomitantly with altered levels of components in the antioxidant defense system (glutathione, superoxide dismutase or catalase). CNTs are biopersistent high aspect ratio materials, and some are rigid with lengths that lead to frustrated phagocytosis and pleural accumulation. There is accumulating evidence showing that pulmonary exposure to CNTs is associated with fibrosis and neoplastic changes in the lungs, and cardiovascular disease. As oxidative stress and inflammation responses are implicated in the development of these diseases, converging lines of evidence indicate that exposure to CNTs is associated with increased risk of cardiopulmonary diseases through generation of a pro-inflammatory and pro-oxidant milieu in the lungs.

Pulmonary exposure to particles from diesel exhaust, urban dust or single-walled carbon nanotubes and oxidatively damaged DNA and vascular function in apoE(-/-) mice.

This study compared the oxidative stress level and vasomotor dysfunction after exposure to urban dust, diesel exhaust particles (DEP) or single-walled carbon nanotubes (SWCNT). DEP and SWCNT increased the production of reactive oxygen species (ROS) in cultured endothelial cells and acellullarly, whereas the exposure to urban dust did not generate ROS. The apoE(-/-) mice, which were exposed twice to 0.5 mg/kg of the particles by intratracheal (i.t.) instillation, had unaltered acetylcholine-elicited vasorelaxation in aorta segments. There was unaltered pulmonary expression level of Vcam-1, Icam-1, Hmox-1 and Ogg1. The levels of oxidatively damaged DNA were unchanged in lung tissue. The exposure to SWCNT significantly increased the expression of Ccl-2 in the lung tissue of the mice. The exposure to DEP and SWCNT was associated with elevated ROS production in cultured cells, whereas i.t. instillation of the same particles had no effect on biomarkers of pulmonary oxidative stress and dilatory dysfunction in the aorta.

Oxidatively damaged DNA in animals exposed to particles.

Exposure to combustion-derived particles, quartz and asbestos is associated with increased levels of oxidized and mutagenic DNA lesions. The aim of this survey was to critically assess the measurements of oxidatively damaged DNA as marker of particle-induced genotoxicity in animal tissues. Publications based on non-optimal assays of 8-oxo-7,8-dihydroguanine by antibodies and/or unrealistically high levels of 8-oxo-7,8-dihydroguanine (suggesting experimental problems due to spurious oxidation of DNA) reported more induction of DNA damage after exposure to particles than did the publications based on optimal methods. The majority of studies have used single intracavitary administration or inhalation with dose rates exceeding the pulmonary overload threshold, resulting in cytotoxicity and inflammation. It is unclear whether this is relevant for the much lower human exposure levels. Still, there was linear dose-response relationship for 8-oxo-7,8-dihydroguanine in lung tissue without obvious signs of a threshold. The dose-response function was also dependent on chemical composition and other characteristics of the administered particles, whereas dependence on species and strain could not be equivocally determined. Roles of cytotoxicity or inflammation for oxidatively induced DNA damage could not be documented or refuted. Studies on exposure to particles in the gastrointestinal tract showed consistently increased levels of 8-oxo-7,8-dihydroguanine in the liver. Collectively, there is evidence from animal experimental models that both pulmonary and gastrointestinal tract exposure to particles are associated with elevated levels of oxidatively damaged DNA in the lung and internal organs. However, there is a paucity of studies on pulmonary exposure to low doses of particles that are relevant for hazard/risk assessment.

Oxidative damage to DNA by diesel exhaust particle exposure in co-cultures of human lung epithelial cells and macrophages.

Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stress-related damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung epithelium and immune cells. Macrophages have important immune defence functions by engulfing insoluble foreign materials, including particles, although they might also promote or enhance inflammation. We investigated the effect of co-culturing type II lung epithelial A549 cells with macrophages upon treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine-DNA glycosylase or oxoguanine DNA glycosylase (hOGG1) sensitive sites, in mono-cultures of A549 or THP-1a and co-cultures of A549 and THP-1a cells. The strongest genotoxic effects were observed in A549 mono-cultures and SRM2975 was more potent than SRM1650b. The ROS production only increased in cells exposed to SRM2975, with strongest concentration-dependent effect in the THP-1a mono-cultures. The basal respiration level in THP-1a cells increased on exposure to SRM1650b and SRM2975 without indication of mitochondrial dysfunction. This is consistent with activation of the cells and there was no direct relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells.

Urinary excretion of 8-oxo-7,8-dihydroguanine as biomarker of oxidative damage to DNA.

Oxidatively damaged DNA may be important in carcinogenesis. 8-Oxo-7,8-dihydroguanine (8-oxoGua) is an abundant and mutagenic lesion excised by oxoguanine DNA glycosylase 1 (OGG1) and measurable in urine or plasma by chromatographic methods with electrochemical or mass spectrometric detectors, reflecting the rate of damage in steady state. A common genetic OGG1 variant may affect the activity and was associated with increased levels of oxidized purines in leukocytes without apparent effect on 8-oxoGua excretion or major change in cancer risk. 8-OxoGua excretion has been associated with exposure to air pollution, toxic metals, tobacco smoke and low plasma antioxidant levels, whereas fruit and vegetable intake or dietary interventions showed no association. In rodent studies some types of feed may be source of 8-oxoGua in collected urine. Of cancer therapies, cisplatin increased 8-oxoGua excretion, whereas radiotherapy only showed such effects in experimental animals. Case-control studies found high excretion of 8-oxoGua in relation to cancer, dementia and celiac disease but not hemochromatosis, although associations could be a consequence rather than reflecting causality of disease. One prospective study found increased risk of developing lung cancer among non-smokers associated with high excretion of 8-oxoGua. Urinary excretion of 8-oxoGua is a promising biomarker of oxidatively damaged DNA.