Lone Mikkelsen

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.

Inflammatory and genotoxic effects of nanoparticles designed for inclusion in paints and lacquers

Abstract Manufactured nanomaterials are projected to be used on a large scale in paints and lacquers. We selected seven commercially interesting materials: Three titanium dioxide-based (two coated rutile; one uncoated anatase), one carbon black (Flamrüss 101), one kaolinite clay, and two silica products, whereas carbon black, Printex 90, was used as reference material. DNA damaging activity and inflammogenicity (pulmonary cell composition and mRNAs) were determined 24 h after intratracheal instillation of a single dose of 54 μg in mice. Greatest inflammation was induced by Printex 90 and uncoated titanium dioxide. The inflammatory potency correlated with instilled surface area (R2 = 0.94) but not with material volume (R2 = 0.17). The coated titanium dioxides induced DNA damage in lung lining fluid cells. The uncoated titanium dioxide was not DNA damaging by the comet assay 24 h after exposure despite being highly inflammogenic. This suggests that inflammation is not a prerequisite to DNA damage in titanium dioxide-based products.

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.

Aging and oxidatively damaged nuclear DNA in animal organs

Oxidative stress is considered to contribute to aging and is associated with the generation of oxidatively damaged DNA, including 8-oxo-7,8-dihydroguanine. We have identified 69 studies that have measured the level of oxidatively damaged DNA in organs of animals at various ages. In general, organs with limited cell proliferation, i.e., liver, kidney, brain, heart, pancreas, and muscle, tended to show accumulation of DNA damage with age, whereas organs with highly proliferating cells, such as intestine, spleen, and testis, showed more equivocal or no effect of age. A restricted analysis of studies reporting a baseline level of damaged DNA that was fewer than 5 lesions/10(6) dG showed that 21 of 29 studies reported age-associated accumulation of DNA damage. The standardized mean difference in oxidatively damaged DNA between the oldest and the youngest age groups was 1.49 (95% CI 1.03-1.95). There was no difference between age span, number of tested organs, statistical power, sex, strain, or breeding between the studies showing positive and null effects. Citation and publication bias seems to be a problem in the overall dataset, whereas it is less pronounced in the restricted dataset. There is compelling evidence for aging-associated accumulation of oxidatively damaged DNA in organs with limited cell proliferation.

Inflammatory and genotoxic effects of sanding dust generated from nanoparticle-containing paints and lacquers

Abstract Nanoparticles are increasingly used in paints and lacquers. Little is known of the toxicity of nanoparticles incorporated in complex matrices and released during different phases of the life cycle. DNA damaging activity and inflammogenicity of sanding dust sampled during standardised sanding of boards painted with paints with and without nanoparticles were determined 24 h after intratracheal instillation of a single dose of 54 μg in mice. Dusts from nanoparticle-containing paints and lacquers did not generate pulmonary inflammation or oxidative stress. Sanding dust from both the nanoparticle-containing and the conventional lacquer and the outdoor acrylic-based reference paint increased the level of DNA strand breaks in bronchoalveolar fluid cells. In conclusion, addition of nanoparticles to paint or lacquers did not increase the potential of sanding dust for causing inflammation, oxidative stress or DNA damage, suggesting that the paint/lacquer matrix is more important as determinant of DNA damage than the nanomaterial.

Modest effect on plaque progression and vasodilatory function in atherosclerosis-prone mice exposed to nanosized TiO 2

BackgroundThere is growing evidence that exposure to small size particulate matter increases the risk of developing cardiovascular disease.MethodsWe investigated plaque progression and vasodilatory function in apolipoprotein E knockout (ApoE-/-) mice exposed to TiO2. ApoE-/- mice were intratracheally instilled (0.5 mg/kg bodyweight) with rutile fine TiO2 (fTiO2, 288 nm), photocatalytic 92/8 anatase/rutile TiO2 (pTiO2, 12 nm), or rutile nano TiO2 (nTiO2, 21.6 nm) at 26 and 2 hours before measurement of vasodilatory function in aorta segments mounted in myographs. The progression of atherosclerotic plaques in aorta was assessed in mice exposed to nanosized TiO2 (0.5 mg/kg bodyweight) once a week for 4 weeks. We measured mRNA levels of Mcp-1, Mip-2, Vcam-1, Icam-1 and Vegf in lung tissue to assess pulmonary inflammation and vascular function. TiO2-induced alterations in nitric oxide (NO) production were assessed in human umbilical vein endothelial cells (HUVECs).ResultsThe exposure to nTiO2 was associated with a modest increase in plaque progression in aorta, whereas there were unaltered vasodilatory function and expression levels of Mcp-1, Mip-2, Vcam-1, Icam-1 and Vegf in lung tissue. The ApoE-/- mice exposed to fine and photocatalytic TiO2 had unaltered vasodilatory function and lung tissue inflammatory gene expression. The unaltered NO-dependent vasodilatory function was supported by observations in HUVECs where the NO production was only increased by exposure to nTiO2.ConclusionRepeated exposure to nanosized TiO2 particles was associated with modest plaque progression in ApoE-/- mice. There were no associations between the pulmonary TiO2 exposure and inflammation or vasodilatory dysfunction.

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.

Carbon black nanoparticles and vascular dysfunction in cultured endothelial cells and artery segments

Exposure to small size particulates is regarded as a risk factor for cardiovascular disease. We investigated effects of exposure to nanosized carbon black (CB) in human umbilical vein endothelial cells (HUVECs) and segments of arteries from rodents. The CB exposure was associated with increased surface expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) in HUVECs at 100μg/ml. CB exposure was also associated with increased reactive oxygen species production and damage to the cell membranes in the form of increased lactate dehydrogenase leakage, whereas it did not alter the mitochondrial enzyme activity (WST-1) or the nitric oxide level in HUVECs. Incubation of aorta segments with 10μg/ml of CB increased the endothelial-dependent vasorelaxation, induced by acetylcholine, and shifted the endothelium-independent vasorelaxation, induced by sodium nitroprusside, towards a decreased sensitivity. In mesenteric arteries, the exposure to 10μg/ml was associated with a reduced pressure-diameter relationship. Incubation with 100μg/ml CB significantly decreased both acetylcholine and sodium nitroprusside responses as well as decreased the receptor-dependent vasoconstriction caused by phenylephrine. In conclusion, nanosized CB exposure activates endothelial cells and generates oxidative stress, which is associated with vasomotor dysfunction.

Cytotoxicity, oxidative stress and expression of adhesion molecules in human umbilical vein endothelial cells exposed to dust from paints with or without nanoparticles

Abstract Nanoparticles in primary form and nanoproducts might elicit different toxicological responses. We compared paint-related nanoparticles with respect to effects on endothelial oxidative stress, cytotoxicity and cell adhesion molecule expression. Primary human umbilical vein endothelial cells were exposed to primary nanoparticles (fine, photocatalytic or nanosized TiO2, aluminium silicate, carbon black, nano-silicasol or axilate) and dust from sanding reference- or nanoparticle-containing paints. Most of the samples increased cell surface expressions of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), but paint sanding dust samples generally generated less response than primary particles of TiO2 and carbon black. We found no relationship between the expression of adhesion molecules, cytotoxicity and production of reactive oxygen species. In conclusion, sanding dust from nanoparticle-containing paint did not generate more oxidative stress or expression of cell adhesion molecules than sanding dust from paint without nanoparticles, whereas the primary particles had the largest effect on mass basis.

Hypoxia and oxidation levels of DNA and lipids in humans and animal experimental models

The objective of this review was to evaluate the association between hypoxia and oxidative damage to DNA and lipids. Evaluation criteria encompassed specificity and validation status of the biomarkers, study design, strength of the association, dose‐response relationship, biological plausibility, analogous exposures, and effect modification by intervention. The collective interpretation indicates persuasive evidence from the studies in humans for an association between hypoxia and elevated levels of oxidative damage to DNA and lipids. The levels of oxidatively generated DNA lesions and lipid peroxidation products depend on both the duration and severity of the exposure to hypoxia. Largest effects are observed with exposure to hypoxia at high altitude, but other factors, including ultraviolet light, exercise, exertion, and low intake of antioxidants, might contribute to the effect observed in subjects at high altitude. Most of the animal experimental models should be interpreted with caution because the assays for assessment of lipid peroxidation products have suboptimal validity.