Nanotoxicology on the right track : focus on metal and metal oxide nanoparticles

The last decade has seen a rapid increase in the manufacture and use of nanomaterials, a development which should be met with appropriate safety assessment strategies in order to ensure the sustainable development of nanotechnology. With decreasing size, the percentage of atoms found at the surface of a given material increases substantially, leading to an increase in surface phenomena and acquisition of novel properties. These new traits can be appealing for industrial purposes, however, they can also enhance the intrinsic toxicity of the materials as compared to their bulk counterparts. Currently, nanotoxicology faces several challenges related to the multitude of materials that need to be tested, the possible interactions of the nanomaterials with the conventional toxicology assays and the potential emergence of novel nano-specific properties. Despite numerous research efforts being made in the last decade to evaluate the toxicity of nanomaterials, most of these studies fall short of several aspects, such as appropriate particle characterization, cellular uptake, relevant doses and exposure duration. The aim of this thesis was to use in vitro models to address some of the challenges in nanotoxicology in order to improve our understanding of the interactions between nanomaterials and biological systems. In Paper I we demonstrated that we can use the ToxTracker assay, which consists of reporter stem cells, to screen and predict the genotoxicity of metal oxide nanoparticles and at the same time obtain information about their mechanism of toxicity. In Paper II we used a panel of thoroughly characterized silver nanoparticles to address the issue of size-dependent toxicity in human lung cells. Our results showed that small (10 nm) particles were more cytotoxic than larger particles (˃40 nm) after acute exposure (24 hours), and that could be related to a ‘Trojan horse’ effect by which the particulate form facilitates the cellular uptake of metal, with subsequent release of toxic metal ions. In Paper III we selected two of the silver nanoparticles tested in Paper II and evaluated the effects following low-dose, long-term (6 week) exposure to human lung cells. By using both conventional assays and systems toxicology approaches (RNA-sequencing, genome wide DNA-methylation) we identified that chronic exposure to low doses of silver nanoparticles induced a cancer-like phenotype and had immunosuppressive effects in human lung cells. In Paper IV we explored the effects of antioxidant cerium oxide nanoparticles, which allegedly have promising therapeutic potential, in neural stem cells. On one hand, we showed that pretreatment with cerium oxide nanoparticles provided a temporary neuroprotective effect when cells were challenged with an oxidative stress inducer. On the other hand, by using both immunofluorescence and RNA-sequencing we revealed that the same antioxidant properties can have detrimental effects by suppressing neuronal differentiation, in which reactive oxygen species play an important role as signaling molecules. In all, our studies show that by using well-characterized nanomaterials together with appropriate experimental setups, and a combination of traditional toxicological assays with novel tools such as ‘omics’, we can improve our understanding of the toxicity of nanomaterials and by these means contribute to the sustainable development of nanotechnology

Open Access

Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag releas

Mild steel welding is associated with alterations in circulating levels of cancer-related proteins

Welding fumes were recently classified as carcinogenic to humans and worldwide millions work as welders or perform welding operations. The purpose of this study was to identify new biomarkers of welding-induced carcinogenesis. We evaluated a panel of 91 putative cancer-related proteins in serum in a cohort of welders working with mild steel (n = 77) and controls (n = 94) from southern Sweden sampled on two occasions 6-year apart using a longitudinal analysis (linear mixed models). The significant results from the longitudinal analysis were tested for reproducibility in welders (n = 88) and controls (n = 69) sampled once during the same sampling period as timepoint 1 or timepoint 2 (linear regression models), i.e., in a cross-sectional setting. The models were adjusted for age, body-mass index, and use of snus. All study participants were non-smokers at recruitment. Exposure to welding fumes was assessed using questionnaires and respirable dust measurement in the breathing zone that was adjusted for personal respiratory protection equipment. The median respirable dust in welders was 0.7 (0.2–4.2) and 0.5 (0.1–1.9) mg/m3 at the first and second timepoints, respectively. We identified 14 cancer-related proteins that were differentially expressed in welders versus controls in the longitudinal analysis, out of which three were also differentially expressed in the cross-sectional analysis (cross-sectional group). Namely, syndecan 1 (SDC1), folate receptor 1 (FOLR1), and secreted protein acidic and cysteine rich (SPARC) were downregulated, in welders compared with controls. In addition, FOLR1 was negatively associated with years welding. Disease and function analysis indicated that the top proteins are related to lung cancer as well as cell invasion and migration. Our study indicates that moderate exposure to welding fumes is associated with changes in circulating levels of putative cancer-related proteins, out of which FOLR1 showed a clear dose–response relationship. It is, however, unclear to which extent these changes are adaptive or potential early biomarkers of cancer

Prenatal arsenic exposure is associated with increased plasma IGFBP3 concentrations in 9-year-old children partly via changes in DNA methylation

Exposure to inorganic arsenic (As), a carcinogen and epigenetic toxicant, has been associated with lower circulating levels of insulin-like growth factor 1 (IGF1) and impaired growth in children of pre-school age. The aim of this study was to assess the potential impact of exposure to As on IGF1 and insulin-like growth factor-binding protein 3 (IGFBP3) as well as DNA methylation changes in 9-year-old children. To this end, we studied 9-year-old children from a longitudinal mother-child cohort in rural Bangladesh (n = 551). Prenatal and concurrent exposure to As was assessed via concentrations in maternal urine at gestational week 8 and in child urine at 9 years, measured by HPLC-HG-ICPMS. Plasma IGF1 and IGFBP3 concentrations were quantified with immunoassays. DNA methylation was measured in blood mononuclear cells at 9 years in a sub-sample (n = 113) using the Infinium HumanMethylation450K BeadChip. In multivariable-adjusted linear regression models, prenatal As (natural log-transformed), but not children's concurrent urinary As, was positively associated with IGFBP3 concentrations (β = 76, 95% CI 19, 133). As concentrations were not associated with IGF1. DNA methylation analysis revealed CpGs associated with both prenatal As and IGFBP3. Mediation analysis suggested that methylation of 12 CpG sites for all children was mediator of effect for the association between prenatal As and IGFBP3. We also found differentially methylated regions, generally hypermethylated, that were associated with both prenatal As and IGFBP3. In all, our study revealed that prenatal exposure to As was positively associated with IGFBP3 concentrations in children at 9 years, independent of IGF1, and this association may, at least in part, be epigenetically mediated

Exposure to Mild Steel Welding and Changes in Serum Proteins With Putative Neurological Function—A Longitudinal Study

Welders are exposed to high levels of metal particles, consisting mainly of iron and manganese (Mn) oxide. Metal particles, especially those containing Mn can be neurotoxic. In this exploratory study, we evaluated associations between welding and expression of 87 putative neurology-related proteins in serum in a longitudinal approach. The study cohort from southern Sweden included welders working with mild steel (n = 56) and controls (n = 67), all male and non-smoking, which were sampled at two timepoints (T1, T2) 6-year apart. Observed associations in the longitudinal analysis (linear mixed models) were further evaluated (linear regression models) in another cross-sectional sample which included welders (n = 102) and controls (n = 89) who were sampled only once (T1 or T2). The median respirable dust levels for welders after adjusting for respiratory protection was at T1 0.6 (5–95 percentile: 0.2–4.2) and at T2 0.5 (0.1–1.8) mg/m3. The adjusted median respirable Mn concentration was at T2 0.049 mg/m3 (0.003–0.314) with a Spearman correlation between adjusted respirable dust and respirable Mn of rS = 0.88. We identified five neurology-related proteins that were differentially expressed in welders vs. controls in the longitudinal sample, of which one (nicotinamide/nicotinic acid mononucleotide adenylyltransferase 1; NMNAT1) was also differentially expressed in the cross-sectional sample. NMNAT1, an axon-protective protein linked to Alzheimers disease, was upregulated in welders compared with controls but no associations were discerned with degree of exposure (welders only: years welding, respirable dust, cumulative exposure). However, we identified five additional proteins that were associated with years welding (GCSF, EFNA4, CTSS, CLM6, VWC2; welders only) both in the longitudinal and in the cross-sectional samples. We also observed several neurology-related proteins that were associated with age and BMI. Our study indicates that low-to-moderate exposure to welding fumes is associated with changes in circulating levels of neurology-related proteins

Calcium-dependent cyto- and genotoxicity of nickel metal and nickel oxide nanoparticles in human lung cells

Abstract Background Genotoxicity is an important toxicological endpoint due to the link to diseases such as cancer. Therefore, an increased understanding regarding genotoxicity and underlying mechanisms is needed for assessing the risk with exposure to nanoparticles (NPs). The aim of this study was to perform an in-depth investigation regarding the genotoxicity of well-characterized Ni and NiO NPs in human bronchial epithelial BEAS-2B cells and to discern possible mechanisms. Comparisons were made with NiCl2 in order to elucidate effects of ionic Ni. Methods BEAS-2B cells were exposed to Ni and NiO NPs, as well as NiCl2, and uptake and cellular dose were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS). The NPs were characterized in terms of surface composition (X-ray photoelectron spectroscopy), agglomeration (photon cross correlation spectroscopy) and nickel release in cell medium (ICP-MS). Cell death (necrosis/apoptosis) was investigated by Annexin V-FITC/PI staining and genotoxicity by cytokinesis-block micronucleus (cytome) assay (OECD 487), chromosomal aberration (OECD 473) and comet assay. The involvement of intracellular reactive oxygen species (ROS) and calcium was explored using the fluorescent probes, DCFH-DA and Fluo-4. Results NPs were efficiently taken up by the BEAS-2B cells. In contrast, no or minor uptake was observed for ionic Ni from NiCl2. Despite differences in uptake, all exposures (NiO, Ni NPs and NiCl2) caused chromosomal damage. Furthermore, NiO NPs were most potent in causing DNA strand breaks and generating intracellular ROS. An increase in intracellular calcium was observed and modulation of intracellular calcium by using inhibitors and chelators clearly prevented the chromosomal damage. Chelation of iron also protected against induced damage, particularly for NiO and NiCl2. Conclusions This study has revealed chromosomal damage by Ni and NiO NPs as well as Ni ionic species and provides novel evidence for a calcium-dependent mechanism of cyto- and genotoxicity

Effect of welding fumes on the cardiovascular system : a six-year longitudinal study

Objective This study investigated whether low-to-moderate exposure to welding fumes is associated with adverse effects on the cardiovascular system.Methods To test this, we performed a longitudinal analysis of 78 mild steel welders and 96 controls; these subjects were examined twice, six years apart (ie, timepoints 1 and 2). All subjects (male and non-smoking at recruitment) completed questionnaires describing their health, work history, and lifestyle. We measured their blood pressure, endothelial function (by EndoPAT), and risk markers for cardiovascular disease [low-density lioprotein (LDL), homocysteine, C-reactive protein]. Exposure to welding fumes was assessed from the responses to questionnaires and measurements of respirable dust in their breathing zones adjusted for use of respiratory protection equipment. Linear mixed-effect regression models were used for the longitudinal analysis.Results Median respirable dust concentrations, adjusted for respirable protection, of the welders were 0.7 (5–95 percentile range 0.2–4.2) and 0.5 (0.1–1.9) mg/m3 at timepoints 1 and 2, respectively. Over the six-year period, welders showed a statistically significant increase in systolic [5.11 mm Hg, 95% confidence interval (CI) 1.92–8.31] and diastolic (3.12 mm Hg, 95% CI 0.74–5.5) blood pressure compared with controls (multi-variable adjusted mixed effect models). Diastolic blood pressure increased non-significantly by 0.22 mm Hg (95% CI -0.02–0.45) with every additional year of welding work. No consistent significant associations were found between exposure and endothelial function, LDL, homocysteine, or C-reactive protein.Conclusion Exposure to welding fumes at low-to-moderate levels is associated with increased blood pressure, suggesting that reducing the occupational exposure limit (2.5 mg/m3 for inorganic respirable dust in Sweden) is needed to protect cardiovascular health of workers

Maternal exposure to cadmium during pregnancy is associated with changes in DNA methylation that are persistent at 9 years of age

Background: Cadmium (Cd) exposure during gestation has been associated with altered DNA methylation at birth, but it is not known if the changes in methylation persist into childhood. Objectives: To evaluate whether gestational Cd-related changes of DNA methylation persist from birth to 9 years of age. Methods: We studied mother–child dyads in a longitudinal cohort in rural Bangladesh. Cadmium concentrations in maternal blood (erythrocyte fraction; Ery-Cd) at gestational week 14 and in child urine (U-Cd, long-term exposure marker) at 9 years were measured using inductively coupled plasma mass spectrometry. The epigenome-wide DNA methylation was measured in mononuclear cells (PBMCs) prepared from cord blood and peripheral blood at 9 years in 71 children (hereafter referred to as the explorative group) by Infinium HumanMethylation450K BeadChip. Replication of one differentially methylated region (DMR; 9 CpG sites) was performed in PBMCs of 160 9-year-old children (validation group) by EpiTyper MALDI-TOF mass spectrometry. Results: The median maternal Ery-Cd concentration was 1.24 µg/kg (range 0.35, 4.55) in the explorative group and 0.83 µg/kg (0.08, 2.97) in the validation group. The median U-Cd concentration in the 9-year-old children was 0.26 µg/L (0.09, 1.06) in the explorative group and 0.32 µg/L (0.07, 1.33) in the validation group. In the explorative group, we identified ten DMRs, both in cord blood and in PBMCs at 9 years, that were associated with maternal Ery-Cd. Eight out of the ten DMRs were hypomethylated and three of the hypomethylated DMRs were located in the HLA region on chromosome 6. One of the DMRs (hypomethylated) in the HLA region (upstream of the zinc finger protein 57 homolog, ZFP57 gene) was replicated in the validation group, and we found that it was hypomethylated in relation to maternal Ery-Cd, but not child U-Cd. Conclusion: Gestational exposure to Cd appears to be associated with regional changes, especially hypomethylated, in DNA methylation that linger from birth up to prepubertal age

Cerium oxide nanoparticles inhibit differentiation of neural stem cells

Cerium oxide nanoparticles (nanoceria) display antioxidant properties and have shown cytoprotective effects both in vitro and in vivo. Here, we explored the effects of nanoceria on neural progenitor cells using the C17.2 murine cell line as a model. First, we assessed the effects of nanoceria versus samarium (Sm) doped nanoceria on cell viability in the presence of the prooxidant, DMNQ. Both particles were taken up by cells and nanoceria, but not Sm-doped nanoceria, elicited a temporary cytoprotective effect upon exposure to DMNQ. Next, we employed RNA sequencing to explore the transcriptional responses induced by nanoceria or Sm-doped nanoceria during neuronal differentiation. Detailed computational analyses showed that nanoceria altered pathways and networks relevant for neuronal development, leading us to hypothesize that nanoceria inhibits neuronal differentiation, and that nanoceria and Sm-doped nanoceria both interfere with cytoskeletal organization. We confirmed that nanoceria reduced neuron specific beta 3-tubulin expression, a marker of neuronal differentiation, and GFAP, a neuroglial marker. Furthermore, using super-resolution microscopy approaches, we could show that both particles interfered with cytoskeletal organization and altered the structure of neural growth cones. Taken together, these results reveal that nanoceria may impact on neuronal differentiation, suggesting that nanoceria could pose a developmental neurotoxicity hazard