Daniel Vest Christophersen

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

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.

PACAP-38 but not VIP induces release of CGRP from trigeminal nucleus caudalis via a receptor distinct from the PAC1 receptor

OBJECTIVE To investigate if PACAP and VIP have an effect on CGRP release or NOS activity in the trigeminal ganglion and trigeminal nucleus caudalis and if there can be a difference in effect between PACAP and VIP on these two systems. Furthermore, we investigate if PACAP co-localize with CGRP and/or nNOS in the two tissues. BACKGROUND The structurally related neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide-38 (PACAP-38) partially share receptors and are both potent vasodilators. However, PACAP-38 but not VIP is an efficient inducer of migraine attacks in migraineurs. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are two signaling molecules known to be involved in migraine. METHODS Rat tissue was used for all experiments. Release of CGRP induced by VIP and PACAP in dura mater, trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) was quantified by EIA. Regulation of NOS-enzymes caused by VIP and PACAP was investigated in dura mater, TG and TNC by measuring the conversion of L-[3H]arginine to L-[3H]citrulline. Co-expression of PACAP, neuronal nitric oxide synthase (nNOS) and CGRP was explored by immunohistochemistry in TG and TNC. mRNA expression studies of VPAC1, VPAC2 and PAC1-receptors were performed by qRT-PCR. RESULTS PACAP-38 administered in increasing concentrations caused a concentration-dependent CGRP-release in the TNC, but not in TG. VIP was without effect in both tissues examined. The PAC1 receptor agonist maxadilan had no effect on CGRP release and the PAC1 antagonist M65 did not inhibit PACAP-38 induced CGRP release. PACAP-38 or VIP did not affect NOS activity in homogenates of TG and TNC. Quantitative PCR demonstrated the presence of VPAC1, VPAC2 and PAC1 receptors in TG and TNC. Immunohistochemistry of PACAP and CGRP showed co-expression in TG and TNC. PACAP and nNOS were co-localized in TG, but not in TNC. PACAP was found to co-localize with glutamine synthetase in TG satellite glial cells. CONCLUSION PACAP-38 cause release of CGRP from TNC but not from TG. We suggest that the release is not caused via activation of PAC1, VPAC1 or VPAC2 receptors. PACAP has no effect on NOS activity in TG or TNC. In TG PACAP was found in neuronal cells and in satellite glial cells. It co-localized with CGRP and nNOS in the neuronal cells. In TNC PACAP was co-localized with CGRP but not with nNOS.

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

Abstract 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.

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.

Cardiovascular health effects of oral and pulmonary exposure to multi-walled carbon nanotubes in ApoE-deficient mice

Exposure to high aspect ratio nanomaterials, such as multi-walled carbon nanotubes (MWCNTs) may be associated with increased risk of atherosclerosis, pulmonary disease, and cancer. In the present study, we investigated the cardiovascular and pulmonary health effects of 10 weeks of repeated oral or pulmonary exposures to MWCNTs (4 or 40μg each week) in Apolipoprotein E-deficient (ApoE-/-) mice fed a Western-type diet. Intratracheal instillation of MWCNTs was associated with oxidative damage to DNA in lung tissue and elevated levels of lipid peroxidation products in plasma, whereas the exposure only caused a modest pulmonary inflammation in terms of increased numbers of lymphocytes in bronchoalveolar lavage fluid. Ultrasound imaging in live animals revealed an increase in the inner and outer wall thickness of the aortic arch at 10 weeks after pulmonary exposure to MWCNTs, which may suggest artery remodelling. However, we did not find accelerated plaque progression in the aorta or the brachiocephalic artery by histopathology. Furthermore, repeated oral exposure to MWCNTs did not cause changes in the composition of gut microbiota of exposed mice. Collectively, this study indicates that repeated pulmonary exposure to MWCNTs was associated with oxidative stress, whereas cardiovascular effects encompassed remodelling of the aorta wall.

Inflammation and Vascular Effects after Repeated Intratracheal Instillations of Carbon Black and Lipopolysaccharide

Inflammation and oxidative stress are considered the main drivers of vasomotor dysfunction and progression of atherosclerosis after inhalation of particulate matter. In addition, new studies have shown that particle exposure can induce the level of bioactive mediators in serum, driving vascular- and systemic toxicity. We aimed to investigate if pulmonary inflammation would accelerate nanoparticle-induced atherosclerotic plaque progression in Apolipoprotein E knockout (ApoE-/-) mice. ApoE -/- mice were exposed to vehicle, 8.53 or 25.6 μg nanosized carbon black (CB) alone or spiked with LPS (0.2 μg/mouse/exposure; once a week for 10 weeks). Inflammation was determined by counting cells in bronchoalveolar lavage fluid. Serum Amyloid A3 (Saa3) expression and glutathione status were determined in lung tissue. Plaque progression was assessed in the aorta and the brachiocephalic artery. The effect of vasoactive mediators in plasma of exposed ApoE-/- mice was assessed in aorta rings isolated from naïve C57BL/6 mice. Pulmonary exposure to CB and/or LPS resulted in pulmonary inflammation with a robust influx of neutrophils. The CB exposure did not promote plaque progression in aorta or BCA. Incubation with 0.5% plasma extracted from CB-exposed ApoE-/- mice caused vasoconstriction in aorta rings isolated from naïve mice; this effect was abolished by the treatment with the serotonin receptor antagonist Ketanserin. In conclusion, repeated pulmonary exposure to nanosized CB and LPS caused lung inflammation without progression of atherosclerosis in ApoE-/- mice. Nevertheless, plasma extracted from mice exposed to nanosized CB induced vasoconstriction in aortas of naïve wild-type mice, an effect possibly related to increased plasma serotonin.