Pernille Høgh Danielsen

Role of oxidative damage in toxicity of particulates.

Abstract Particulates are small particles of solid or liquid suspended in liquid or air. In vitro studies show that particles generate reactive oxygen species, deplete endogenous antioxidants, alter mitochondrial function and produce oxidative damage to lipids and DNA. Surface area, reactivity and chemical composition play important roles in the oxidative potential of particulates. Studies in animal models indicate that particles from combustion processes (generated by combustion of wood or diesel oil), silicate, titanium dioxide and nanoparticles (C60 fullerenes and carbon nanotubes) produce elevated levels of lipid peroxidation products and oxidatively damaged DNA. Biomonitoring studies in humans have shown associations between exposure to air pollution and wood smoke particulates and oxidative damage to DNA, deoxynucleotides and lipids measured in leukocytes, plasma, urine and/or exhaled breath. The results indicate that oxidative stress and elevated levels of oxidatively altered biomolecules are important intermediate endpoints that may be useful markers in hazard characterization of particulates.

Air pollution, oxidative damage to DNA, and carcinogenesis

There is growing concern that air pollution exposure increases the risk of lung cancer. The mechanism of action is related to particle-induced oxidative stress and oxidation of DNA. Humans exposed to urban air with vehicle emissions have elevated levels of oxidized guanine bases in blood cells and urine. Animal experimental studies show that pulmonary and gastrointestinal exposure is associated with elevated levels of oxidized guanines in the lung and other organs. Collectively, there is evidence indicating that exposure to traffic-related air pollution particles is associated with oxidative damage to DNA and this might be associated with increased risk of cancer.

Oxidative damage to DNA and repair induced by Norwegian wood smoke particles in human A549 and THP-1 cell lines.

Genotoxic effects of traffic-generated particulate matter (PM) are well described, whereas little data are available on PM from combustion of biomass and wood, which contributes substantially to air pollution world wide. The aim of this study was to compare the genotoxicity of wood smoke particulate matter (WSPM), authentic traffic-generated particles, mineral PM and standard reference material (SRM2975) of diesel exhaust particles in human A549 lung epithelial and THP-1 monocytic cell lines. DNA damage was measured as strand breaks (SB) and formamidopyrimidine DNA glycosylase (FPG) sites by the comet assay, whereas cell cytotoxicity was determined as lactate dehydrogenase release. The exposure to WSPM generated SB and FPG sites in both cell lines at concentrations from 2.5 or 25 microg/ml, which were not cytotoxic. Compared to all other studied particles, WSPM generated greater responses in terms of both SB and FPG sites. Organic extracts of WSPM and SRM2975 elicited higher levels of SB than native and washed PM at 25 and 100 microg/ml, whereas assay saturation precluded reliable assessment of FPG sites. During a 6h post-exposure period, in which the medium with PM had been replaced by fresh medium, 60% of the DNA lesions generated by WSPM were removed. In conclusion, WSPM generated more DNA damage than traffic-generated PM per unit mass in human cell lines, possibly due to the high level of polycyclic aromatic hydrocarbons in WSPM. This suggests that exposure to WSPM might be more hazardous than PM collected from vehicle exhaust with respect to development of lung cancer.

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.

Hazard identification of particulate matter on vasomotor dysfunction and progression of atherosclerosis

The development and use of nanoparticles have alerted toxicologists and regulators to issues of safety testing. By analogy with ambient air particles, it can be expected that small doses are associated with a small increase in risk of cardiovascular diseases, possibly through oxidative stress and inflammatory pathways. We have assessed the effect of exposure to particulate matter on progression of atherosclerosis and vasomotor function in humans, animals, and ex vivo experimental systems. The type of particles that have been tested in these systems encompass TiO2, carbon black, fullerene C60, single-walled carbon nanotubes, ambient air particles, and diesel exhaust particles. Exposure to ambient air particles is associated with accelerated progression of atherosclerosis and vasomotor dysfunction in both healthy and susceptible animal models and humans at risk of developing cardiovascular diseases. The vasomotor dysfunction includes increased vasoconstriction as well as reduced endothelium-dependent vasodilatation; endothelium-independent vasodilatation is often unaffected indicating mainly endothelial dysfunction. Pulmonary exposure to TiO2, carbon black, and engineered nanoparticles generate vasomotor dysfunction; the effect size is similar to that generated by combustion-derived particles, although the effect could depend on the exposure period and the administered dose, route, and mode. The relative risk associated with exposure to nanoparticles may be small compared to some traditional risk factors for cardiovascular diseases, but superimposed on these and possible exposure to large parts of the population it is a significant public health concern. Overall, assessment of vasomotor dysfunction and progression of atherosclerosis are promising tools for understanding the effects of particulate matter.

Vascular Effects of Multiwalled Carbon Nanotubes in Dyslipidemic ApoE−/− Mice and Cultured Endothelial Cells

Accumulating evidences indicate that pulmonary exposure to carbon nanotubes (CNTs) is associated with increased risk of lung diseases, whereas the effect on the vascular system is less studied. We investigated vascular effects of 2 types of multiwalled CNTs (MWCNTs) in apolipoprotein E(-/-) mice, wild-type mice, and cultured cells. The ApoE(-/-) mice had accelerated plaque progression in aorta after 5 intracheal instillations of MWCNT (25.6 μg/mouse weekly for 5 weeks). The exposure was associated with pulmonary inflammation, lipid peroxidation, and increased expression of inflammatory, oxidative stress, DNA repair, and vascular activation response genes. The level of oxidatively damaged DNA in lung tissue was unaltered, probably due to increased DNA repair capacities. Despite upregulation of inflammatory genes in the liver, effects on systemic cytokines and lipid peroxidation were minimal. The exposure to MWCNTs in cultured human endothelial cells increased the expression of cell adhesion molecules (ICAM1 and VCAM1). In cocultures, there was increased adhesion of monocytes to endothelial cells after exposure to MWCNT. The exposure to both types of MWCNT was also associated with increased lipid accumulation in monocytic-derived foam cells, which was dependent on concomitant oxidative stress because the antioxidant N-acetylcysteine inhibited the lipid accumulation. Collectively, our results indicate that exposure to MWCNT is associated with accelerated progression of atherosclerosis, which could be related to both increased adherence of monocytes onto the endothelium and oxidative stress-mediated transformation of monocytes to foam cells.

A Multilaboratory Toxicological Assessment of a Panel of 10 Engineered Nanomaterials to Human Health-ENPRA Project-The Highlights, Limitations, and Current and Future Challenges

ABSTRACT ENPRA was one of the earlier multidisciplinary European Commission FP7-funded projects aiming to evaluate the risks associated with nanomaterial (NM) exposure on human health across pulmonary, cardiovascular, hepatic, renal, and developmental systems. The outputs from this project have formed the basis of this review. A retrospective interpretation of the findings across a wide range of in vitro and in vivo studies was performed to identify the main highlights from the project. In particular, focus was placed on informing what advances were made in the hazard assessment of NM, as well as offering some suggestions on the future of “nanotoxicology research” based on these observations, shortcomings, and lessons learned from the project. A number of issues related to the hazard assessment of NM are discussed in detail and include use of appropriate NM for nanotoxicology investigations; characterization and dispersion of NM; use of appropriate doses for all related investigations; need for the correct choice of experimental models for risk assessment purposes; and full understanding of the test systems and correct interpretation of data generated from in vitro and in vivo systems. It is hoped that this review may assist in providing information in the implementation of guidelines, model systems, validation of assessment methodology, and integrated testing approaches for risk assessment of NM. It is vital to learn from ongoing and/or completed studies to avoid unnecessary duplication and offer suggestions that might improve different aspects of experimental design.

Role of oxidative stress in carbon nanotube-generated health effects

Abstract 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.

Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.

Oxidatively damaged DNA in animals exposed to particles

Abstract 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.