Search

Inhalation of nanosized metal oxides may occur at the workplace. Thus, information on potential hazardous effects is needed for risk assessment. We report an investigation of the genotoxic potential of different metal oxide nanomaterials. Acellular and intracellular reactive oxygen species (ROS) production were determined for all the studied nanomaterials. Moreover, mice were exposed by intratracheal instillation to copper oxide (CuO) at 2, 6, and 12 μg/mouse, tin oxide (SnO 2 ) at 54 and 162 μg/mouse, aluminum oxide (Al 2 O 3 ) at 18 and 54 μg/mouse, zinc oxide (ZnO) at 0.7 and 2 μg/mouse, titanium dioxide (TiO 2 ) and the benchmark carbon black at 162 μg/mouse. The doses were selected based on pilot studies. Post-exposure time points were 1 or 28 days. Genotoxicity, assessed as DNA strand breaks by the comet assay, was measured in lung and liver tissue. The acellular and intracellular ROS measurements were fairly consistent. The CuO and the carbon black bench mark particle were potent ROS generators in both assays, followed by TiO 2 . Al 2 O 3 , ZnO, and SnO 2 generated low levels of ROS. We detected no increased genotoxicity in this study using occupationally relevant dose levels of metal oxide nanomaterials after pulmonary exposure in mice, except for a slight increase in DNA damage in liver tissue at the highest dose of CuO. The present data add to the body of evidence for risk assessment of these metal oxides., (© 2024 The Author(s). Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.)

Humans ingest particles and fibers on daily basis. Non-digestible carbohydrates are beneficial to health and food additives are considered safe. However, titanium dioxide (E171) has been banned in the European Union because the European Food Safety Authority no longer considers it non-genotoxic. Ingestion of microplastics and nanoplastics are novel exposures; their potential hazardous effects to humans have been under the radar for many years. In this review, we have assessed the association between oral exposure to man-made particles/fibers and genotoxicity in gastrointestinal tract cells and secondary tissues. We identified a total of 137 studies on oral exposure to particles and fibers. This was reduced to 49 papers with sufficient quality and relevance, including exposures to asbestos, diesel exhaust particles, titanium dioxide, silver nanoparticles, zinc oxide, synthetic amorphous silica and certain other nanomaterials. Nineteen studies show positive results, 25 studies show null results, and 5 papers show equivocal results on genotoxicity. Recent studies seem to show null effects, whereas there is a higher proportion of positive genotoxicity results in early studies. Genotoxic effects seem to cluster in studies on diesel exhaust particles and titanium dioxide, whereas studies on silver nanoparticles, zinc oxide and synthetic amorphous silica seem to show mainly null effects. The most widely used genotoxic tests are the alkaline comet assay and micronucleus assay. There are relatively few results on genotoxicity using reliable measurements of oxidatively damaged DNA, DNA double strand breaks (γH2AX assay) and mutations. In general, evidence suggest that oral exposure to particles and fibers is associated with genotoxicity in animals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Many in vitro and in vivo studies have shown that exposure to carbon nanotubes (CNTs) is associated with inflammation, oxidative stress and genotoxicity, although there is a paucity of studies on these effects in the pleural cavity. In the present study, we investigated adverse outcomes of pleural exposure to multi-walled CNTs (MWCNT-7, NM-401 and NM-403) and single-walled CNTs (NM-411). Female C57BL/6 mice were exposed to 0.2 or 5 µg of CNTs by intra-pleural injection and sacrificed one-year post-exposure. Exposure to long and straight types of MWCNTs (i.e. MWCNT-7 and NM-401) was associated with decreased number of macrophages and increased number of neutrophils and eosinophils in pleural lavage fluid. Increased protein content in the pleural lavage fluid was also observed in mice exposed to MWCNT-7 and NM-401. The concentration of mesothelin was increased in mice exposed to MWCNT-7 and NM-411. Levels of DNA strand breaks and DNA oxidation damage, measured by the comet assay, were unaltered in cells from pleural scrape. Extra-pleural effects were seen in CNT exposed mice, including enlarged and pigmented mediastinal lymph nodes (all four types of CNTs), pericardial plaques (MWCNT-7 and NM-401), macroscopic abnormalities on the liver (MWCNT-7) and ovaries/uterus (NM-411). In conclusion, the results demonstrate that intra-pleural exposure to long and straight MWCNTs is associated with adverse outcomes. Certain observations such as increased content of mesothelin in pleural lavage fluid and ovarian/uterine abnormalities in mice exposed to NM-411 suggests that exposure to SWCNTs may also be associated with some adverse outcomes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peter Moller reports financial support was provided by Independent Research Fund Denmark. Ulla Vogel reports financial support was provided by Danish Government., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The ubiquitous pollution of plastic particles in most environmental matrices leads to concern about any potential adverse effects on human health. Most studies on the toxicological effect of nanoplastics has focused on standard particles of polystyrene. In reality humans are exposed to a large variety of different types and sizes of plastic material via oral intake and inhalation. In this study, we investigated the effect of polyethylene terephthalate (PET) nanoplastic particles from ground food containers from a supermarket. The aim was to investigate a possible link between exposure to PET nanoplastics and genotoxic response in a cell model of the human airway epithelial (A549) cells. Further, we investigated the combined effect of PET and chemicals known to alter the cellular redox state, as a model of partially compromised antioxidant defense system. DNA damage was assessed by the alkaline comet assay. The ground PET nanoplastics have a mean hydrodynamic diameter of 136 nm in water. The results showed that PET exposure led to increased reactive oxygen species production (approximately 30 % increase compared to unexposed cells). In addition, exposure to PET nanoplastic increased the level of DNA strand breaks (net increase = 0.10 lesions/10 6 base pair, 95 % confidence interval: 0.01, 0.18 lesions/10 6 base pair). Pre- or post-exposure to hydrogen peroxide or buthionine sulfoximine did not lead to a higher level of DNA damage. Overall, the study shows that exposure to PET nanoplastics increases both intracellular reactive oxygen production and DNA damage in A549 cells., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Inhalation studies are the gold standard for assessing the toxicity of airborne materials. They require considerable time, special equipment, and large amounts of test material. Intratracheal instillation is considered a screening and hazard assessment tool as it is simple, quick, allows control of the applied dose, and requires less test material. The particle-induced pulmonary inflammation and acute phase response in mice caused by intratracheal instillation or inhalation of molybdenum disulphide or tungsten particles were compared. End points included neutrophil numbers in bronchoalveolar lavage fluid, Saa3 mRNA levels in lung tissue and Saa1 mRNA levels in liver tissue, and SAA3 plasma protein. Acute phase response was used as a biomarker for the risk of cardiovascular disease. Intratracheal instillation of molybdenum disulphide or tungsten particles did not produce pulmonary inflammation, while molybdenum disulphide particles induced pulmonary acute phase response with both exposure methods and systemic acute phase response after intratracheal instillation. Inhalation and intratracheal instillation showed similar dose-response relationships for pulmonary and systemic acute phase response when molybdenum disulphide was expressed as dosed surface area. Both exposure methods showed similar responses for molybdenum disulphide and tungsten, suggesting that intratracheal instillation can be used for screening particle-induced acute phase response and thereby particle-induced cardiovascular disease., (© 2023 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Biofuel is an attractive substitute for petrodiesel because of its lower environmental footprint. For instance, the polycyclic aromatic hydrocarbons (PAH) emission per fuel energy content is lower for rapeseed methyl ester (RME) than for petrodiesel. This study assesses genotoxicity by extractable organic matter (EOM) of exhaust particles from the combustion of petrodiesel, RME, and hydrogenated vegetable oil (HVO) in lung epithelial (A549) cells. Genotoxicity was assessed as DNA strand breaks by the alkaline comet assay. EOM from the combustion of petrodiesel and RME generated the same level of DNA strand breaks based on the equal concentration of total PAH (i.e. net increases of 0.13 [95% confidence interval (CI): 0.002, 0.25, and 0.12 [95% CI: 0.01, 0.24] lesions per million base pairs, respectively). In comparison, the positive control (etoposide) generated a much higher level of DNA strand breaks (i.e. 0.84, 95% CI: 0.72, 0.97) lesions per million base pairs. Relatively low concentrations of EOM from RME and HVO combustion particles (<116 ng/ml total PAH) did not cause DNA strand breaks in A549 cells, whereas benzo[a]pyrene and PAH-rich EOM from petrodiesel combusted using low oxygen inlet concentration were genotoxic. The genotoxicity was attributed to high molecular weight PAH isomers with 5-6 rings. In summary, the results show that EOM from the combustion of petrodiesel and RME generate the same level of DNA strand breaks on an equal total PAH basis. However, the genotoxic hazard of engine exhaust from on-road vehicles is lower for RME than petrodiesel because of lower PAH emission per fuel energy content., (© The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There is concern about human exposure to nanoplastics from intentional use or degradation of plastics in the environment. This review assesses genotoxic effects of nanoplastics, defined as particles with a primary size of less than 1000 nm. The majority of results on genotoxicity come from studies on polystyrene (PS) particles in mammalian cell cultures. Most studies have measured DNA strand breaks (standard comet assay), oxidatively damaged DNA (Fpg-modified comet assay) and micronuclei. Twenty-nine out of 60 results have shown statistically significant genotoxic effects by PS exposure in cell cultures. A statistical analysis indicates that especially modified PS particles are genotoxic (odds ratio = 8.6, 95 % CI: 1.6, 46) and immune cells seems to be more sensitive to genotoxicity than other cell types such as epithelial cells (odds ratio = 8.0, 95 % CI: 1.6, 39). On the contrary, there is not a clear association between statistically significant effects in genotoxicity tests and the primary size of PS particles, (i.e. smaller versus larger than 100 nm) or between the type of genotoxic endpoint (i.e. repairable versus permanent DNA lesions). Three studies of PS particle exposure in animals have shown increased level of DNA strand breaks in leukocytes and prefrontal cortex cells. Nanoplastics from polyethylene, propylene, polyvinyl chloride and polyethylene terephthalate have been investigated in very few studies and it is currently not possible to draw conclusion about their genotoxic hazard. In summary, there is some evidence suggesting that PS particles may be genotoxic in mammalian cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Acute phase response (APR) is characterized by a change in concentration of different proteins, including C-reactive protein and serum amyloid A (SAA) that can be linked to both exposure to metal oxide nanomaterials and risk of cardiovascular diseases. In this study, we intratracheally exposed mice to ZnO, CuO, Al 2 O 3 , SnO 2 and TiO 2 and carbon black (Printex 90) nanomaterials with a wide range in phagolysosomal solubility. We subsequently assessed neutrophil numbers, protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, Saa3 and Saa1 mRNA levels in lung and liver tissue, respectively, and SAA3 and SAA1/2 in plasma. Endpoints were analyzed 1 and 28 days after exposure, including histopathology of lung and liver tissues., Results: All nanomaterials induced pulmonary inflammation after 1 day, and exposure to ZnO, CuO, SnO 2 , TiO 2 and Printex 90 increased Saa3 mRNA levels in lungs and Saa1 mRNA levels in liver. Additionally, CuO, SnO 2 , TiO 2 and Printex 90 increased plasma levels of SAA3 and SAA1/2. Acute phase response was predicted by deposited surface area for insoluble metal oxides, 1 and 28 days post-exposure., Conclusion: Soluble and insoluble metal oxides induced dose-dependent APR with different time dependency. Neutrophil influx, Saa3 mRNA levels in lung tissue and plasma SAA3 levels correlated across all studied nanomaterials, suggesting that these endpoints can be used as biomarkers of acute phase response and cardiovascular disease risk following exposure to soluble and insoluble particles., (© 2023. The Author(s).)

Large plastic litters degrade in the environment to micro- and nanoplastics, which may then enter the food chain and lead to human exposure by ingestion. The present study explored ways to obtain nanoplastic particles from real-life food containers. The first set of experiments gave rise to polypropylene nanoplastic suspensions with a hydrodynamic particle size range between 100 and 600 nm, whereas the same grinding process of polyethylene terephthalate (PET) produced suspensions of particles with a primary size between 100 and 300 nm. The exposure did not cause cytotoxicity measured by the lactate dehydrogenase (LDH) and water soluble tetrazolium 1 (WST-1) assays in Caco-2 and HepG2 cells. Nanoplastics of transparent PET food containers produced a modest concentration-dependent increase in DNA strand breaks, measured by the alkaline comet assay [net induction of 0.28 lesions/10 6 bp at the highest concentration (95% CI: 0.04; 0.51 lesions/10 6 base pair)]. The exposure to nanoplastics from transparent polypropylene food containers was also positively associated with DNA strand breaks [i.e., net induction of 0.10 lesions/10 6 base pair (95% CI: -0.04; 0.23 lesions/10 6 base pair)] at the highest concentration. Nanoplastics from grinding of black colored PET food containers demonstrated no effect on HepG2 and Caco-2 cells in terms of cytotoxicity, reactive oxygen species production or changes in cell cycle distribution. The net induction of DNA strand breaks was 0.43 lesions/10 6 bp (95% CI: 0.09; 0.78 lesions/10 6 bp) at the highest concentration of nanoplastics from black PET food containers. Collectively, the results indicate that exposure to nanoplastics from real-life consumer products can cause genotoxicity in cell cultures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Roursgaard, Hezareh Rothmann, Schulte, Karadimou, Marinelli and Møller.)

Lyotropic non-lamellar liquid crystalline (LLC) nanoparticles, with their tunable structural features and capability of loading a wide range of drugs and reporter probes, are emerging as versatile injectable nanopharmaceuticals. Secondary emulsifiers, such as Pluronic block copolymers, are commonly used for colloidal stabilization of LLC nanoparticles, but their inclusion often compromises the biological safety (e.g., poor hemocompatibility and enhanced cytotoxicity) of the formulation. Here, we introduce a library of colloidally stable, structurally tunable, and pH-responsive lamellar and non-lamellar liquid crystalline nanoparticles from binary mixtures of a phospholipid (phosphatidylglycerol) and three types of omega-3 fatty acids (ω-3 PUFAs), prepared in the absence of a secondary emulsifier and organic solvents. We study formulation size distribution, morphological heterogeneity, and the arrangement of their internal self-assembled architectures by nanoparticle tracking analysis, synchrotron small-angle X-ray scattering, and cryo-transmission electron microscopy. The results show the influence of type and concentration of ω-3 PUFAs in nanoparticle structural transitions spanning from a lamellar (L α ) phase to inverse discontinuous (micellar) cubic Fd3m and hexagonal phase (H 2 ) phases, respectively. We further report on cell-culture medium-dependent dynamic fluctuations in nanoparticle size, number and morphology, and simultaneously monitor uptake kinetics in two human cell lines. We discuss the role of these multiparametric biophysical transformations on nanoparticle-cell interaction kinetics and internalization mechanisms. Collectively, our findings contribute to the understanding of fundamental steps that are imperative for improved engineering of LLC nanoparticles with necessary attributes for pharmaceutical development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Environmental exposure can increase the production of reactive oxygen species and deplete cellular antioxidants in humans, resulting in oxidatively generated damage to DNA that is both a useful biomarker of oxidative stress and indicator of carcinogenic hazard. Methods of oxidatively damaged DNA analysis have been developed and used in public health research since the 1990s. Advanced techniques detect specific lesions, but they might not be applicable to complex matrixes (e.g., tissues), small sample volume, and large-scale studies. The most reliable methods are characterized by (1) detecting relevant DNA oxidation products (e.g., premutagenic lesions), (2) not harboring technical problems, (3) being applicable to complex biological mixtures, and (4) having the ability to process a large number of samples in a reasonable period of time. Most effort has been devoted to the measurements of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG), which can be analyzed by chromatographic, enzymic, and antibody-based methods. Results from validation trials have shown that certain chromatographic and enzymic assays (namely the comet assay) are superior techniques. The enzyme-modified comet assay has been popular because it is technically simpler than chromatographic assays. It is widely used in public health studies on environmental exposures such as outdoor air pollution. Validated biomarker assays on oxidatively damaged DNA have been used to fill knowledge gaps between findings in prospective cohort studies and hazards from contemporary sources of air pollution exposures. Results from each of these research fields feed into public health research as approaches to conduct primary prevention of diseases caused by environmental or occupational agents.

Biofuels from vegetable oils or animal fats are considered to be more sustainable than petroleum-derived diesel fuel. In this study, we have assessed the effect of hydrogenated vegetable oil (HVO) exhaust on levels of DNA damage in peripheral blood mononuclear cells (PBMCs) as primary outcome, and oxidative stress and inflammation as mediators of genotoxicity. In a randomized cross-over study, healthy humans were exposed to filtered air, inorganic salt particles, exhausts from combustion of HVO in engines with aftertreatment [i.e. emission with nitrogen oxides and low amounts of particulate matter less than 2.5 µm (approximately 1 µg/m 3 )], or without aftertreatment (i.e. emission with nitrogen oxides and 93 ± 13 µg/m 3 of PM 2.5 ). The subjects were exposed for 3 h and blood samples were collected before, within 1 h after the exposure and 24 h after. None of the exposures caused generation of DNA strand breaks and oxidatively damaged DNA, or affected gene expression of factors related to DNA repair (Ogg1), antioxidant defense (Hmox1) or pro-inflammatory cytokines (Ccl2, Il8 and Tnfa) in PBMCs. The results from this study indicate that short-term HVO exhaust exposure is not associated with genotoxic hazard in humans., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Carbon nanotubes (CNTs) are speculated to cause mesothelioma by persistent inflammation, oxidative stress, tissue injury, and genotoxicity. To investigate the pleural response to CNTs, we exposed C57BL/6 mice by intrapleural injection of 0.2 or 5 µg multiwalled CNTs (MWCNT-7, NM-401, and NM-403) or single-walled CNTs (NM-411). Inflammatory response, cellular reactive oxygen species (ROS) production of pleural lavage cells, and genotoxicity in cells from the mesothelial surface were assessed at days 1 and 90 after the exposure. Long and rigid types of MWCNTs (MWCNT-7 and NM-401) caused acute inflammation, characterized by influx of macrophages, neutrophils, and eosinophils into the pleural cavity. The inflammation was still evident at 90 days after the exposure, although it had reduced dramatically. The cellular ROS production was increased at day 90 after the exposure to MWCNT-7 and NM-401. The short and tangled type of MWCNT (ie NM-403) did not cause pleural inflammation or ROS production in pleural fluid cells. The exposure to NM-411 did not cause consistent inflammation responses or cellular ROS production. Levels of DNA strand breaks and DNA oxidation damage were unaltered, except for NM-411-exposed mice that had increased levels of DNA strand breaks at 90 days after the exposure. In conclusion, the long and rigid CNTs caused prolonged inflammatory response and increased ROS production in pleural lavage cells, yet it was not reflected in higher levels of DNA damage in pleural tissue., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Carbon nanotubes (CNTs) were the first nanomaterials to be evaluated by the International Agency for Research on Cancer (IARC). The categorization as possibly carcinogenic agent to humans was only applicable to multi-walled carbon nanotubes called MWCNT-7. Other types of CNTs were not classifiable because of missing data and it was not possible to pinpoint unique CNT characteristics that cause cancer. Importantly, the European Commission's Joint Research Centre (JRC) has established a repository of industrially manufactured nanomaterials that encompasses at least four well-characterized MWCNTs called NM-400 to NM-403 (original JRC code). This review summarizes the genotoxic effects of these JRC materials and MWCNT-7. The review consists of 36 publications with results on cell culture experiments (22 publications), animal models (9 publications) or both (5 publications). As compared to the publications in the IARC monograph on CNTs, the current database represents a significant increase as there is only an overlap of 8 publications. However, the results come mainly from cell cultures and/or measurements of DNA strand breaks by the comet assay and the micronucleus assay (82 out of 97 outcomes). A meta-analysis of cell culture studies on DNA strand breaks showed a genotoxic response by MWCNT-7, less consistent effect by NM-400 and NM-402, and least consistent effect by NM-401 and NM-403. Results from other in vitro tests indicate strongest evidence of genotoxicity for MWCNT-7. There are too few observations from animal models and humans to make general conclusions about genotoxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Carbon nanotubes (CNTs) are fiber-like nanomaterials, which are used in various applications with possible exposure to humans. The genotoxicity and carcinogenic potential of CNTs remain to be fully understood. This study assessed the genotoxicity of three different multi-walled carbon nanotubes (MWCNTs) (MWCNT-7, NM-401 and NM-403) and one single-walled carbon nanotube (SWCNT) (NM-411) in FE1-Muta™Mouse lung epithelial (MML) cells using the alkaline comet assay. With the 2',7'-dichlorodihydrofluorescein diacetate fluorescent probe, we assessed the effect of CNT-exposure on the intracellular production of reactive oxygen species (ROS). We measured the effect of a 10-week CNT exposure on telomere length using quantitative PCR. Two of the included MWCNTs (NM-401 and MWCNT-7) and the SWCNT (NM-411) caused a significant increase in the level of DNA damage at concentrations up to 40 µg/ml (all concentrations pooled, p  < 0.05), but no concentration-response relationships were found. All of the CNTs caused an increase in intracellular ROS production compared to unexposed cells ( p trend < 0.05). Results from the long-term exposure showed longer telomere length in cells exposed to MWCNTs compared to unexposed cells ( p  < 0.01). In conclusion, our results indicated that the included CNTs cause ROS production and DNA strand breaks in FE1-MML cells. Moreover, the MWCNTs, but not the SWCNT, had an impact on telomere length in a long-term exposure scenario.

A key initiating step in atherosclerosis is the accumulation and retention of apolipoprotein B complexing lipoproteins within the artery walls. In this work, we address this exact initiating mechanism of atherosclerosis, which results from the oxidation of low-density lipoproteins (oxLDL) using therapeutic nanogels. We present the development of biocompatible polyethylene glycol (PEG) cross-linked nanogels formed from a single simultaneous cross-linking and co-polymerization step in water without the requirement for an organic solvent, high temperature, or shear stress. The nanogel synthesis also incorporates in situ noncovalent electrostatically driven template polymerization around an innate anti-inflammatory and anti-oxidizing paraoxonase-1 (PON-1) enzyme payload-the release of which is triggered because of matrix metalloproteinase responsive elements instilled in the PEG cross-linker monomer. The results obtained demonstrate the potential of triggered release of the PON-1 enzyme and its efficacy against the production of ox-LDL, and therefore a reduction in macrophage foam cell and reactive oxygen species formation.

Mining rare earth elements (REEs) can release large amounts of metal(loid)-rich dust, which can pose significant health risks to local residents. However, compared to other types of particulates, toxicity of mining dust has been largely overlooked. To provide experimental evidence on toxicity of REE mine dust, the study assessed the oxidative stress potential and genotoxicity of inhalable particles collected in a REE mining area, and associated toxicological response with source compositions. Both source types (i.e., mine and tailing area) and distances from source (i.e., industrial and residential areas) were considered when selecting the 44 sampling sites. The particle samples contained 2.3-3.5 folds higher concentrations of tested metal(loid)s than background concentrations in soil. Specially, elevated Fe, REEs, Cd, Pb were found. In spite of low cytotoxicity in lung epithelial A549 cells, there was increased cellular ROS production by of particle exposure. Samples with higher mining-originated source contributions (Provenance Index <0.3) had higher cellular ROS production (1.72 fold, 95%CI: 1.66-1.79 fold) than samples with lower mining contributions (1.58 fold, 95%CI: 1.52-1.65 fold). The factors soil (~46%), mine (~22%), and heavy metal (~20%) sources were recognized by source apportionment analysis as the main contributors to cellular ROS production; importantly, mine and heavy metal sources counted more in industrial samples. While samples generated genotoxicity, there were no differences in DNA damage between the location groups of sampling. Collectively, the results indicate that particles in mining areas may cause ROS production and DNA damage in lung cells depending on mine dust. Coupled with the long-range transportation potential of mine dust, safety measures on open pit and dust disposal sites should be adopted., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Biodiesel fuels are alternatives to petrodiesel, especially in the transport sector where they have lower carbon footprint. Notwithstanding the environmental benefit, biodiesel fuels may have other toxicological properties than petrodiesel. Particulate matter (PM) from petrodiesel causes cancer in the lung as a consequence of delivery of genotoxic polycyclic aromatic hydrocarbons, oxidative stress and inflammation. We have reviewed articles from 2002 to 2019 (50% of the articles since 2015) that have described toxicological effects in terms of genotoxicity, oxidative stress and inflammation of biodiesel exhaust exposure in humans, animals and cell cultures. The studies have assessed first generation biodiesel from different feedstock (e.g. rapeseed and soy), certain second generation fuels (e.g. waste oil), and hydrogenated vegetable oil. It is not possible to rank the potency of toxicological effects of specific biodiesel fuels. However, exposure to biodiesel exhaust causes oxidative stress, inflammation and genotoxicity in cell cultures. Three studies in animals have not indicated genotoxicity in lung tissue. The database on oxidative stress and inflammation in animal studies is larger (13 studies); ten studies have reported increased levels of oxidative stress biomarkers or inflammation, although the effects have been modest in most studies. The cell culture and animal studies have not consistently shown a different potency in effect between biodiesel and petrodiesel exhausts. Both increased and decreased potency have been reported, which might be due to differences in feedstock or combustion conditions. In conclusion, combustion products from biodiesel and petrodiesel fuel may evoke similar toxicological effects on genotoxicity, oxidative stress and inflammation.

Particulate matter (PM) from combustion processes has been associated with oxidative stress to DNA, whereas effects related to telomere dysfunction are less investigated. We collected air-borne PM from a passenger cabin of a diesel-propelled train and at a training facility for smoke diving exercises. Effects on oxidative stress biomarkers, genotoxicity measured by the comet assay and telomere length in PM-exposed A549 cells were compared with the genotoxicity and telomere length in peripheral blood mononuclear cells (PBMCs) from human volunteers exposed to the same aerosol source. Although elevated levels of DNA strand breaks and oxidatively damaged DNA in terms of Fpg-sensitive sites were observed in PBMCs from exposed humans, the PM collected at same locations did not cause genotoxicity in the comet assay in A549 cells. Nevertheless, A549 cells displayed telomere length shortening after four weeks exposure to PM. This is in line with slightly shorter telomere length in PBMCs from exposed humans, although it was not statistically significant. In conclusion, the results indicate that genotoxic potency measured by the comet assay of PM in A549 cells may not predict genotoxicity in exposed humans, whereas telomere length measurements may be a novel indicator of genotoxic stress in cell cultures and humans., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Several types of engineered nanoparticles (ENP) have been shown to adversely affect male reproduction in rodent studies, but the airway route of exposure has been little investigated. This precludes adequate risk assessment of ENP exposure in occupational settings. Titanium dioxide nanoparticles (TiO 2 NP) have been shown to affect total sperm count in adult male mice after intravenous and oral administration. This study aimed to investigate whether also airway exposure would affect sperm counts in male mice. Mature C57BL/6J mice were intratracheally instilled with 63 μg of rutile nanosized TiO 2 , once weekly for seven weeks. Respirable α-quartz (SRM1878a) was included at a similar dose level as a positive control for pulmonary inflammation. BALF cell composition showed neutrophil granulocyte influx as indication of pulmonary inflammation in animals exposed to TiO 2 NP and α-quartz, but none of the particle exposures affected weight of testes or the epididymis, sperm counts or plasma testosterone when assessed at termination of the study., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The breast milk plays a crucial role in shaping the initial intestinal microbiota and mucosal immunity of the infant. Interestingly, breastfeeding has proven to be protective against the early onset of immune-mediated diseases including type 1 diabetes. Studies have shown that exosomes from human breast milk are enriched in immune-modulating miRNAs suggesting that exosomal miRNAs (exomiRs) transferred to the infant could play a critical role in the development of the infant's immune system. We extracted exomiRs from breast milk of 52 lactating mothers (26 mothers with type 1 diabetes and 26 healthy mothers), to identify any differences in the exomiR content between the two groups. Small RNA-sequencing was performed to identify known and novel miRNAs in both groups. A total of 631 exomiRs were detected by small RNA sequencing including immune-related miRNAs such as hsa-let-7c, hsa-miR-21, hsa-miR-34a, hsa-miR-146b, and hsa-miR-200b. In addition, ~200 novel miRNAs were identified in both type 1 diabetes and control samples. Among the known miRNAs, nine exomiR's were found differentially expressed in mothers with type 1 diabetes compared to healthy mothers. The highly up-regulated miRNAs, hsa-miR-4497, and hsa-miR-3178, increased lipopolysaccharide-induced expression and secretion of tumor necrosis factor α (TNFα) in human monocytes. The up-regulated miRNA target genes were significantly enriched for longevity-regulating pathways and FoxO signaling. Our findings suggest a role of breast milk-derived exomiRs in modulating the infant's immune system., (Copyright © 2019 Mirza, Kaur, Nielsen, Størling, Yarani, Roursgaard, Mathiesen, Damm, Svare, Mortensen and Pociot.)

Background: Short-term controlled exposure to diesel exhaust (DE) in chamber studies have shown mixed results on lung and systemic effects. There is a paucity of studies on well-characterized real-life DE exposure in humans. In the present study, 29 healthy volunteers were exposed to DE while sitting as passengers in diesel-powered trains. Exposure in electric trains was used as control scenario. Each train scenario consisted of three consecutive days (6 h/day) ending with biomarker samplings., Results: Combustion-derived air pollutants were considerably higher in the passenger carriages of diesel trains compared with electric trains. The concentrations of black carbon and ultrafine particles were 8.5 μg/m 3 and 1.2-1.8 × 10 5 particles/cm 3 higher, respectively, in diesel as compared to electric trains. Net increases of NOx and NO 2 concentrations were 317 μg/m 3 and 36 μg/m 3 . Exposure to DE was associated with reduced lung function and increased levels of DNA strand breaks in peripheral blood mononuclear cells (PBMCs), whereas there were unaltered levels of oxidatively damaged DNA, soluble cell adhesion molecules, acute phase proteins in blood and urinary excretion of metabolites of polycyclic aromatic hydrocarbons. Also the microvascular function was unaltered. An increase in the low frequency of heart rate variability measures was observed, whereas time-domain measures were unaltered., Conclusion: Exposure to DE inside diesel-powered trains for 3 days was associated with reduced lung function and systemic effects in terms of altered heart rate variability and increased levels of DNA strand breaks in PBMCs compared with electric trains., Trial Registration: ClinicalTrials.Gov ( NCT03104387 ). Registered on March 23rd 2017.

Diesel-powered trains are used worldwide for passenger transport. The present study aimed to assess air pollution concentrations in passenger cars from diesel and electric trains. Personal exposure monitoring (6-7 h per day) was carried out for 49 days on diesel and 22 days on electric trains. Diesel trains had higher concentrations of all the assessed air pollution components. Average increases (and fold differences) in passenger cars of diesel trains compared with electric trains were for ultrafine particles 212 000 particles/cm 3 (35-fold), black carbon 8.3 μg/m 3 (6-fold), NO x 316 μg/m 3 (8-fold), NO 2 38 μg/m 3 (3-fold), PM 2.5 34 μg/m 3 (2-fold), and benzo( a)pyrene 0.14 ng/m 3 (6-fold). From time-series data, the pull and push movement modes, the engine in use, and the distance to the locomotive influenced the concentrations inside the diesel trains. In conclusion, concentrations of all air pollutants were significantly elevated in passenger cars in diesel trains compared to electric trains.

There has been a steady output of epidemiological studies linking environmental and occupational exposures to altered telomere length, showing mainly positive associations with persistent organic pollutants, inverse association with cadmium and inconsistent results with arsenic and lead. A bell-shaped dose-response relationship has been observed for ionizing radiation with telomere shortening at a low dose. Long-term air pollution is associated with telomere shortening, whereas the short-term exposure studies have shown mixed results. There are surprisingly few studies on telomere dynamics in animals. Studies on telomere dynamics and senescence in target tissues of animal strains used in toxicology are warranted. Cell culture studies on ionizing radiation have shown mixed results on telomere length, whereas both telomerase activity and cellular senescence are increased. Studies on persistent organic pollutants indicate telomere shortening, decreased telomerase activity and increased cellular senescence. Cell culture studies on heavy metals and air pollution particles are inconsistent. There is no coherent relationship between exposures, oxidative stress, telomere length, telomerase activity and cellular senescence in experimental studies on environmental or occupational exposures. This may be due to differences in exposure levels (including dose rate), exposure time and models (i.e. cell types and animal strains). Guidelines are needed for best practices on assays for telomere dynamics and cellular senescence in toxicology. However, it deserves notice that experimental studies in cells and animals have revealed important information on the effects of environmental and occupational agents on the maintenance of telomeres and cellular senescence.

Particles from burning candles contribute to the overall indoor exposure to particulate matter (PM). However, little is known about the effects of indoor sources of particles on cardiovascular disease endpoints. This study investigated the effect of pulmonary exposure to particles from combustion of candles and progression of atherosclerosis. Telomere shortening was assessed in tissues due to its relationship to risk of cardiovascular diseases. The particles were collected from burning candles and used for toxicological studies in cultured endothelial cells and apolipoprotein E (ApoE) knockout mice. Three hours exposure to particles increased the production of reactive oxygen species in endothelial cells, whereas there was no effect on cytotoxicity. Intratracheal instillation of particles (0.5 or 5 mg/kg) once a week for 5 weeks in ApoE-/- mice was associated with an accelerated progression of atherosclerosis in aorta and telomere shortening in the lung and spleen, whereas there was no effect on inflammation in the lungs (i.e. cell numbers), cell damage (i.e. lactate dehydrogenase) and lung barrier damage (i.e. protein concentration) as measured in bronchoalveolar lavage fluid. The results indicate that particles from burning candles are hazardous and this indoor emission source is an important contribution to the health risk of exposure to PM.

Neurotensin, substance P, and insulin have been demonstrated to improve wound healing in vivo. However, the mechanism behind their effect is still not fully understood. This study investigates the effects leading to enhanced scratch closure by these peptides in vitro. The skin keratinocyte cell line, HaCaT, was used to test scratch closure effects of the peptides and alterations of cytokine levels. HUVEC cells were used to test the angiogenic effect of the peptides. Furthermore, clinical isolates of Staphylococcus lugdunensis were used to examine the potential antimicrobial activity of each peptide. Our results demonstrate that neurotensin, substance P, and insulin had significant migratory effects in scratch assays were neurotensin had the lowest effect. Furthermore, we investigated use of the peptides in combination. When substance P was used in combination with neurotensin, the cell migratory capacity was decreased, and the peptides showed a negative correlation (r = -0.298, P < .001). Neurotensin and insulin significantly increased levels of monocyte chemoattractant protein-1 (P < .001) secreted from white blood cells, whereas substance P showed a tendency. Interestingly, neurotensin increased the level of monocyte chemoattractant protein-1 significantly compared to substance P (P < .01). Additionally, the peptides decreased TNFα mRNA levels (P < .001) in HaCaT cells, whereas only neurotensin and insulin decreased IL-8 mRNA (P < .001) but had no significant effect on IL-6 mRNA levels. Surprisingly, substance P increased IL-6 mRNA 9-fold (P < .001). Furthermore, we demonstrate that the peptides increased angiogenesis in the HUVEC cells (P < .001). Finally, S. lugdunensis isolates were not susceptible to the peptides. We demonstrate that the peptides worked differently on HaCaT cells, but substance P acted differently than neurotensin on cytokine levels expression as well as on migration of HaCaT cells. On the contrary, neurotensin and insulin worked similarly. All of these aspects are crucial for proper wound healing, and the results suggest multiple mechanisms for wound-healing properties of these peptides., (© 2018 European Peptide Society and John Wiley & Sons, Ltd.)

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.

Background: Humans are continuously exposed to particles in the gastrointestinal tract. Exposure may occur directly through ingestion of particles via food or indirectly by removal of inhaled material from the airways by the mucociliary clearance system. We examined the effects of food-grade particle exposure on vasomotor function and systemic oxidative stress in an ex vivo study and intragastrically exposed rats., Methods: In an ex vivo study, aorta rings from naïve Sprague-Dawley rats were exposed for 30 min to food-grade TiO 2 (E171), benchmark TiO 2 (Aeroxide P25), food-grade vegetable carbon (E153) or benchmark carbon black (Printex 90). Subsequently, the vasomotor function was assessed in wire myographs. In an in vivo study, lean Zucker rats were exposed intragastrically once a week for 10 weeks to vehicle, E171 or E153. Doses were comparable to human daily intake. Vasomotor function in the coronary arteries and aorta was assessed using wire myographs. Tetrahydrobiopterin, ascorbate, malondialdehyde and asymmetric dimethylarginine were measured in blood as markers of oxidative stress and vascular function., Results: Direct exposure of E171 to aorta rings ex vivo increased the acetylcholine-induced vasorelaxation and 5-hydroxytryptamine-induced vasocontraction. E153 only increased acetylcholine-induced vasorelaxation, and Printex 90 increased the 5-hydroxytryptamine-induced vasocontraction, whereas Aeroxide P25 did not affect the vasomotor function. In vivo exposure showed similar results as ex vivo exposure; increased acetylcholine-induced vasorelaxation in coronary artery segments of E153 and E171 exposed rats, whereas E171 exposure altered 5-hydroxytryptamine-induced vasocontraction in distal coronary artery segments. Plasma levels of markers of oxidative stress and vascular function showed no differences between groups., Conclusion: Gastrointestinal tract exposure to E171 and E153 was associated with modest albeit statistically significant alterations in the vasocontraction and vasorelaxation responses. Direct particle exposure to aorta rings elicited a similar type of response. The vasomotor responses were not related to biomarkers of systemic oxidative stress.

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.

The utilisation of nanoparticles as the means of targeted delivery of therapeutics and/or imaging agents could greatly enhance the specific transport of biologically active payloads to target tissues while avoiding or reducing undesired side-effects. To allow for this to become a reality, the question of potential toxicological effects needs to be addressed. In the present investigation, a cationic liposome with prospective for medical applications was constructed and thoroughly assessed for any material-induced hepatic adverse effects in vivo - in healthy and alcoholic hepatic disease models and in vitro - (HepG2 cells). The data demonstrated that intravenous injection of liposomes did not cause any significant in vivo hepatic toxicity (inflammation, alterations in blood parameters, anti-oxidant depletion, acute phase response and histopathology) at doses of 200 μg per mouse in either healthy or chronically alcohol fed mice. Additionally, the in vitro material-induced adverse effects (cytotoxicity, inflammation or albumin secretion) were all also minimal. The data from this study demonstrated that the intravenous injection of cationic liposomes does not cause hepatic toxicity. This investigation is important as it investigates the toxicity of a nano-sized material in a model of alcoholic hepatic disease in vitro and in vivo . This is an area of research in the field of nanotoxicology that is currently almost entirely overlooked.

Nanosized titanium dioxide (TiO 2 ) has been investigated in numerous studies on genotoxicity, including comet assay endpoints and oxidatively damaged DNA in cell cultures and animal models. The results have been surprisingly mixed, which might be attributed to physico-chemical differences of the tested TiO 2 . In the present review, we assess the role of certain methodological issues and publication bias. The analysis shows that studies on DNA strand breaks without proper assay controls or very low intra-group variation tend to show statistically significant effects. Levels of oxidatively damaged DNA, measured by the enzyme-modified comet assay, tend to show no effect in studies that have not included proper assay controls or they have uncertainty about the measurement. In addition, there are indications of publication and reporting bias. Nevertheless, the analysis shows that Aeroxide P25 generates DNA strand breaks in a concentration-dependent manner, which is not dependent on the duration of exposure. The standard comet assay seems to be able to discriminate between the genotoxicity of different types of TiO 2 , where anatase TiO 2 seems to be the form with strongest genotoxic potential. Cell culture studies also demonstrate increased levels of oxidatively damaged DNA after exposure to TiO 2 . There are relatively few studies on animal models where DNA strand breaks and oxidatively damaged DNA have been tested with reliable methods. Collectively, this review shows that exposure to nanosized TiO 2 is associated with genotoxicity in cells, whereas there are still too few reliable studies to assess the genotoxic potential in animal models.

Candle burning produces a large amount of particles that contribute substantially to the exposure to indoor particulate matter. The exposures to various types of combustion particles, such as diesel exhaust particles, have been associated with increased risk of lung cancer by mechanisms that involve oxidative stress, inflammation and genotoxicity. The aim of this study was to compare pulmonary effects of candle light combustion particles (CP) with two benchmark diesel exhaust particles (A-DEP and SRM2975). Intratracheal (i.t.) instillation of CP (5mg/kg bodyweight) in C57BL/6n mice produced a significant influx of alveolar macrophages and polymorphonuclear leukocytes and increased concentrations of proteins and lactate dehydrogenase activity in bronchoalveolar fluid. Lower levels of these markers of inflammation and cytotoxicity were observed after i.t. instillation of the same dose of A-DEP or SRM2975. The i.t. instillation of CP did not generate oxidative damage to DNA in lung tissue, measured as DNA strand breaks and human 8-oxoguanine glycosylase-sensitive sites by the comet assay. The lack of genotoxic response was confirmed in lung epithelial (A549) cells, although the exposure to CP increased intracellular levels of reactive oxygen species. In conclusion, pulmonary exposure to particles from burning candles is associated with inflammation and cytotoxicity in the lungs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Silver (Ag) nanoparticles (NPs) are currently among one of the most widely used nanomaterials. This in turn, implies an increased risk of human and environmental exposure. Alcohol abuse is a global issue with millions of people in the general population affected by the associated adverse effects. The excessive consumption of alcohol is a prominent cause of chronic liver disease which manifest in multiple disorders. In this study, the adverse health effects of Ag NP exposure were investigated in models of alcoholic hepatic disease in vitro and in vivo. The data showed that Ag NP induced hepatic health effects were aggravated in the alcohol pretreated mice in comparison to controls with regards to an organ specific inflammatory response, changes in blood biochemistry, acute phase response and hepatic pathology. In addition, alcoholic disease influenced the organ's ability for recovery post-NP challenge. Additionally, it is demonstrated that the in vivo data correlated well with in vitro findings where ethanol pretreatment of hepatocytes resulted in significantly increased inflammatory response post-Ag NP exposure. To the best of our knowledge this is the first study of its kind to investigate nano-sized material-induced hepatic pathology in models representative of susceptible individuals (those with pre-existing alcohol liver disease) within the population. This is an area of research in the field of nanotoxicology, and in particular with regard to NP risk assessment that is almost entirely overlooked., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

One type of carbon nanotubes (CNTs) (MWCNT-7, from Mitsui) has been classified as probably carcinogenic to humans, however insufficient data does not warrant the same classification for other types of CNTs. Experimental data indicate that CNT exposure can result in oxidative stress and DNA damage in cultured cells, whereas these materials appear to induce low or no mutagenicity. Therefore, the present study aimed to investigate whether in vitro exposure of cultured airway epithelial cells (A549) to multi-walled CNTs (MWCNTs) could increase the DNA repair activity of oxidatively damaged DNA and drive the cells toward replicative senescence, assessed by attrition of telomeres. To investigate this, H 2 O 2 and KBrO 3 were used to induce DNA damage in the cells and the effect of pre-exposure to MWCNT tested for a change in repair activity inside the cells or in the extract of treated cells. The effect of MWCNT exposure on telomere length was investigated for concentration and time response. We report a significantly increased repair activity in A549 cells exposed to MWCNTs compared to non-exposed cells, suggesting that DNA repair activity may be influenced by exposure to MWCNTs. The telomere length was decreased at times longer than 24h, but this decrease was not concentration dependent. The results suggest that the seemingly low mutagenicity of CNTs in cultured cells may be associated with an increased DNA repair activity and a replicative senescence, which may counteract the manifestation of DNA lesions to mutations., (© The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Scope: Monocyte adhesion to the vascular endothelium is a crucial step in the early stages of atherogenesis. This study aims to investigate the capacity of an anthocyanin (ACN) and phenolic acid (PA) rich fraction (RF) of a wild blueberry, single ACNs (cyanidin, malvidin, delphinidin) and related metabolites (protocatechuic, syringic, and gallic acid) to counteract monocytes (THP-1) adhesion to human umbilical vein endothelial cells (HUVECs) in a tumor necrosis factor α (TNF-α) mediated proinflammatory environment., Methods and Results: HUVECs were incubated with different concentrations (from 0.01 to 10 μg/mL) of the compounds for 24 h. Labeled monocytic THP-1 cells were added to HUVECs and their adhesion was induced by TNF-α (100 ng/mL). ACN-RF reduced THP-1 adhesion to HUVECs with a maximum effect at 10 μg/mL (-33%). PA-RF counteracted THP-1 adhesion at 0.01, 0.1, and 1 μg/mL (-45, -48.7, and -27.6%, respectively), but not at maximum concentration. Supplementation with gallic acid reduced THP-1 adhesion to HUVECs with a maximum effect at 1 μg/mL (-29.9%), while malvidin-3-glucoside and syringic acid increased the adhesion. No effect was observed for the other compounds., Conclusion: These results suggest that ACNs/PA-RF may prevent atherogenesis while the effects of the single ACNs and metabolites are controversial and merit further exploration., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this study was to compare the effects of cationic micelle and liposome drug delivery systems on liver and lung cells in a toxicological in vitro screening model, with observations on cytotoxicity and genotoxicity. A screening battery was established for assessment of a broad range of parameters related to adverse effects. Clear concentration response effects were observed related to impairment of mitochondrial function, membrane integrity and oxidative stress markers, but no effect was observed on genotoxicity. The adverse effects were highest for the liposomes. The High Content Screening seems optimal for initial screening of adverse effects, and combined with standard cytotoxicity measurements initial screening can be performed for predictive toxicological screening., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Blueberries are a rich source of anthocyanins (ACNs) and phenolic acids (PA), which are hypothesized to protect against development of atherosclerosis. The present study examined the effect of an ACN- and PA-rich fractions, obtained from a wild blueberry powder, on the capacity to counteract lipid accumulation in macrophages derived from monocytic THP-1 cells. In addition, we tested the capacity of pure ACNs and their metabolites to alter lipid accumulation., Methods: THP-1-derived macrophages were incubated with fatty acids (500 μM oleic/palmitic acid, 2:1 ratio) and different concentrations (from 0.05 to 10 μg mL(-1)) of ACN- and PA-rich fractions, pure ACN standards (malvidin, delphinidin and cyanidin 3-glucoside), and metabolites (syringic, gallic and protocatechuic acids). Lipid accumulation was quantified with the fluorescent dye Nile red., Results: Lipid accumulation was reduced at all concentrations of the ACN-rich fraction tested with a maximum reduction at 10 μg mL(-1) (-27.4%; p < 0.0001). The PA-rich fraction significantly reduced the lipid accumulation only at the low concentrations from 0.05 µg mL(-1) to 0.3 µg mL(-1), with respect to the control with fatty acids. Supplementation with pure ACN compounds (malvidin and delphinidin-3-glucoside and its metabolic products (syringic and gallic acid)) reduced lipid accumulation especially at the low concentrations, while no significant effect was observed after cyanidin-3-glucoside and protocatechuic acid supplementation., Conclusions: The results demonstrated a potential role of both the ACN- and PA-rich fractions and single compounds in the lipid accumulation also at concentrations close to that achievable in vivo.

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.

Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle-mediated cardiovascular health effects. We hypothesized that the presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. We investigated the interaction between direct exposure to MWCNTs and PA on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar-endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect measured in the lower chamber on HUVECs and THP-1 cells. The exposure to MWCNTs, including a short (NM400) and long (NM402) type of entangled fibers, was associated with elevated levels of reactive oxygen species as well as a decrease in the intracellular glutathione concentration in HUVEC and A549 monocultures. Both effects were found to be independent of the presence of PA. MWCNT exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did not promote THP-1 adhesion to HUVECs in the lower chamber. When THP-1 macrophages were present on the cell culture inserts, there was a modest increase in the adhesion and an increase in interleukin-6 and interleukin-8 levels in the lower chamber whereas no tumor necrosis factor was detected. Overall, this study showed that direct exposure of HUVECs to MWCNTs was associated with oxidative stress and monocyte adhesion and the presence of PA increased the adhesion when exposed to NM400.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (© 2014 Wiley Periodicals, Inc.)

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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fatty acids exposure may increase sensitivity of intestinal epithelial cells to cytotoxic effects of zinc oxide (ZnO) nanoparticles (NPs). This study evaluated the synergistic effects of ZnO NPs and palmitic acid (PA) or free fatty acids (FFAs) mixture (oleic/PA 2:1) on toxicity to human colon epithelial (Caco-2) cells. The ZnO NPs exposure concentration dependently induced cytotoxicity to Caco-2 cells showing as reduced proliferation and activity measured by 3 different assays. PA exposure induced cytotoxicity, and coexposure to ZnO NPs and PA showed the largest cytotoxic effects. The presence of FFAs mixture did not affect the ZnO NPs-induced cytotoxicity. Filtration of freshly prepared suspension of NPs through a 0.45-µm pore size membrane significantly reduced the cytotoxicity, indicating a role of concentration or size of particles in cytotoxic effects. The ZnO NPs and PA coexposure induced production of mitochondrial reactive oxygen species (mROS) but not intracellular ROS production, whereas FFAs mixture exposure did not induce mROS and inhibited intracellular ROS. Both ZnO NPs and fatty acids (PA and FFAs mixture) promoted lysosomal destabilization, which was not correlated with cytotoxicity. These results indicated that PA can enhance ZnO NPs-induced cytotoxicity probably by the augmentation of mROS production, whereas FFAs mixture did not affect ROS production. Synergistic effects between ZnO NPs and fatty acids may be important when considering NPs toxicity via oral exposure., (© The Author(s) 2014.)

The importance of composition, size, crystal structure, charge and coating of metal-based nanomaterials (NMs) were evaluated in human umbilical vein endothelial cells (HUVECs) and/or THP-1 monocytic cells. Biomarkers of oxidative stress and inflammation were assessed because they are important in the development of cardiovascular diseases. The NMs used were five TiO(2) NMs with different charge, size and crystal structure, coated and uncoated ZnO NMs and Ag which were tested in a wide concentration range. There were major differences between the types of NMs; exposure to ZnO and Ag resulted in cytotoxicity and increased gene expression levels of HMOX1 and IL8. The intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) expression were highest in TiO(2) NM-exposed cells. There was increased adhesion of THP-1 monocytic cells onto HUVECs with Ag exposure. None of the NMs increased the intracellular ROS production. There were no major effects of the coating of ZnO NMs. The TiO(2) NMs data on ICAM-1 and VCAM-1 expression suggested that the anatase form was more potent than the rutile form. In addition, the larger TiO(2) NM was more potent than the smaller for gene expression and ICAM-1 and VCAM-1 expression. The toxicological profile of cardiovascular disease-relevant biomarkers depended on composition, size and crystal structure of TiO(2) NMs, whereas the charge on TiO(2) NMs and the coating of ZnO NMs were not associated with differences in toxicological profile.