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Abstract The fetal development of organs and functions is vulnerable to perturbation by maternal inflammation which may increase susceptibility to disorders after birth. Because it is not well understood how the placenta and fetus respond to acute lung- inflammation, we characterize the response to maternal pulmonary lipopolysaccharide exposure across 24 h in maternal and fetal organs using multi-omics, imaging and integrative analyses. Unlike maternal organs, which mount strong inflammatory immune responses, the placenta upregulates immuno-modulatory genes, in particular the IL-6 signaling suppressor Socs3. Similarly, we observe no immune response in the fetal liver, which instead displays metabolic changes, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. The maternal liver and plasma display similar metabolic alterations, potentially increasing bioavailability of docosahexaenoic acid for the mother and fetus. Thus, our integrated temporal analysis shows that systemic inflammation in the mother leads to a metabolic perturbation in the fetus.

Background: Hexavalent chromium (Cr(Ⅵ)) is classified as a group 1 human carcinogen and increases the risk of lung cancer. Non-coding RNAs (ncRNAs) have key regulatory roles in lung cancer, but less is known about their relation to Cr(Ⅵ) exposure. Objectives: We aimed to 1) measure the expression of lung cancer-related circulating ncRNAs in exposed workers and controls; 2) assess associations between ncRNAs expression and Cr concentrations in red blood cells (RBC) and urine; and 3) evaluate correlations between the ncRNAs. Methods: The study included 111 Cr(VI) exposed workers and 72 controls recruited from the SafeChrom project. Cr concentrations were measured in RBC (biomarker of long-term exposure) and urine (biomarker of short-term exposure) samples. Long ncRNA (lncRNA) and microRNA (miRNA) were extracted from plasma followed by deoxyribonuclease treatment, complementary DNA synthesis, and quantitative real‐time polymerase chain reaction using target-specific assays for three lncRNAs (H19, MALAT1, NORAD), and four miRNAs (miR-142-3p, miR-15b-5p, miR-3940-5p, miR-451a). Results: Expression levels of lncRNAs MALAT1 and NORAD, and all four miRNAs, were significantly lower in Cr(VI) exposed workers compared with controls, and correlated significantly with RBC-Cr concentrations (rS = −0.16 to −0.38). H19 was non-significantly increased in exposed workers but significantly correlated with miR-142-3p (rS = −0.33) and miR-15b-5p (rS = −0.30), and NORAD was significantly positively correlated with all four miRNAs (rS = 0.17 to 0.46). In multivariate regression models adjusting for confounders, expressions of lncRNAs MALAT1 and NORAD and all miRNAs were still significantly lower in the exposed group compared with controls, and the expression decreased with increasing RBC-Cr concentrations. Conclusions: Cr(VI) exposure was inversely and in a dose–response manner associated with the expression of circulating non-coding RNA, which suggests ncRNAs as potential biomarkers for Cr(VI)-induced toxicity. Correlations between miRNAs and lncRNAs suggest that they participate in the same lncRNA-miRNA-messenger RNA regulatory axes, which may play important roles in Cr(VI) carcinogenesis.

The estrogen-synthesizing enzyme aromatase is expressed in adipose tissue where it controls the local concentration of estrogen. It has been suggested that the organic solvents ethanol and ethylene glycol can induce estrogen synthesis by inhibiting PPARγ activity. Since elevated estrogen synthesis in adipose tissue is a risk factor for breast cancer development, it is of interest to further characterize the mechanisms regulating aromatase expression. Here, we explored the mechanisms by which ethanol and ethylene glycol modulate aromatase mRNA expression and the ultimate conversion of androgens into estrogens.NMR spectroscopy revealed that ethanol and ethylene glycol influence the active state of PPARγ. An inhibitory effect on PPARγ was confirmed by adipogenesis assays and PPARγ target gene expression analysis in adipocytes. However, only ethanol increased aromatase mRNA in differentiated human adipocytes. In contrast, ethylene glycol downregulated aromatase in a PPARγ-independent manner. An animal study using female Wistar rats was conducted to assess the acute effects of ethanol and ethylene glycol on aromatase expression in adipose tissue within a physiological context. No changes in aromatase or PPARγ target gene (Adipoq and Fabp4) levels were observed in adipose tissue or ovary in response to the chemical exposures, suggesting an absence of acute PPARγ-mediated effects in these organs.The results suggest that ethanol and ethylene glycol are weak PPARγ antagonists in mouse and human adipocytes as well as in cell-free NMR spectroscopy. Both compounds seem to affect adipocyte aromatase expression in vitro, where ethanol increased aromatase expression PPARγ-dependently and ethylene glycol decreased aromatase expression independently of PPARγ. No acute effects on aromatase expression or PPARγ activity were observed in adipose tissue or ovary in rats in this study design.

Abstract Background III-V semiconductor nanowires are envisioned as being integrated in optoelectronic devices in the near future. However, the perspective of mass production of these nanowires raises concern for human safety due to their asbestos- and carbon nanotube-like properties, including their high aspect ratio shape. Indeed, III-V nanowires have similar dimensions as Mitsui-7 multi-walled carbon nanotubes, which induce lung cancer by inhalation in rats. It is therefore urgent to investigate the toxicological effects following lung exposure to III-V nanowires prior to their use in industrial production, which entails risk of human exposure. Here, female C57BL/6J mice were exposed to 2, 6, and 18 µg (0.12, 0.35 and 1.1 mg/kg bw) of gallium phosphide (III-V) nanowires (99 nm diameter, 3.7 μm length) by intratracheal instillation and the toxicity was investigated 1, 3, 28 days and 3 months after exposure. Mitsui-7 multi-walled carbon nanotubes and carbon black Printex 90 nanoparticles were used as benchmark nanomaterials. Results Gallium phosphide nanowires induced genotoxicity in bronchoalveolar lavage cells and acute inflammation with eosinophilia observable both in bronchoalveolar lavage and lung tissue (1 and 3 days post-exposure). The inflammatory response was comparable to the response following exposure to Mitsui-7 multi-walled carbon nanotubes at similar dose levels. The nanowires underwent partial dissolution in the lung resulting in thinner nanowires, with an estimated in vivo half-life of 3 months. Despite the partial dissolution, nanowires were detected in lung, liver, spleen, kidney, uterus and brain 3 months after exposure. Conclusion Pulmonary exposure to gallium phosphide nanowires caused similar toxicological effects as the multi-walled carbon nanotube Mitsui-7. Graphical Abstract

Abstract Lung cancer is a complex disease influenced by a variety of genetic and environmental factors. The cytokine interleukin 1 encoded by IL1B is an important mediator of the inflammatory response, and is involved in a variety of cellular activities. The effect of single nucleotide polymorphisms (SNP) at IL1B has been investigated in relation to cancer with inconsistent results. This Northeastern-Chinese case–control study involving 627 cases and 633 controls evaluated the role of three haplotype-tagging single nucleotide polymorphisms (htSNP) (rs1143633, rs3136558 and rs1143630) representing 95% of the common haplotype diversity across the IL1B gene and assessed interactions with IL1B, PPP1R13L, POLR1G and smoking duration in relation to lung cancer risk. The analyses of five genetic models showed associations with lung cancer risk for rs1143633 in the dominant model [adjusted-OR (95% CI) = 0.67 (0.52–0.85), P = 0.0012] and rs3136558 in the recessive model [adjusted-OR (95% CI) = 1.44 (1.05–1.98), P = 0.025]. Haplotype4 was associated with increased lung cancer risk [adjusted-OR (95% CI) = 1.55 (1.07–2.24), P = 0.021]. The variant G-allele of rs1143633 was protective in smoking sub-group of > 20 years. Using multifactor dimensionality reduction (MDR) analyses, we identified the three best candidate models of interactions and smoking-duration or IL1B rs1143633 as main effect. In conclusion, our findings suggest that IL1B SNP rs1143633 may associate with lower risk of lung cancer, confirming previously identified marker; IL1B SNP rs3136558 and haplotype4 consisting of IL1B htSNPs may associate with increasing risk of lung cancer; interactions of IL1B with POLR1G or PPP1R13L or smoking-duration, which is independent or combined, may involve in risk of lung cancer and lung squamous cell carcinoma.

Abstract 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, Al2O3, SnO2 and TiO2 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, SnO2, TiO2 and Printex 90 increased Saa3 mRNA levels in lungs and Saa1 mRNA levels in liver. Additionally, CuO, SnO2, TiO2 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.

Inflammatory Bowel Disease (IBD) affects approximately 0.3% of the global population, with incidence rates rising dramatically worldwide. Emerging evidence points to an interplay between exposome factors such as diet and gut microbiota, host genetics, and the immune system as crucial elements in IBD development. ATP-binding cassette (ABC) transporters, including human p-glycoprotein encoded by the Abcb1 gene, influence intestinal inflammation, and their expression may interact with environmental factors such as diet and gut microbes. Our study aimed to examine the impact of protein sources on a genetic colitis mouse model.MethodsAbcb1a-deficient colitis mice were fed either casein or red meat-supplemented diets to investigate potential colitis-aggravating components in red meat and their effects on host-microbiota interactions. We conducted deep label free quantitative proteomic inflammation profiling of gastrointestinal tissue (colon, ileum) and urine, and determined the overall microbiome in feces using 16S rRNA gene sequencing. Microbiota shifts by diet and protein transporter impairment were addressed by multivariate statistical analysis. Colon and systemic gut inflammation were validated through histology and immune assays, respectively.ResultsA quantitative discovery based proteomic analysis of intestinal tissue and urine revealed associations between ileum and urine proteomes in relation to Abcb1a deficiency. The absence of Abcb1a efflux pump function and diet-induced intestinal inflammation impacted multiple systemic immune processes, including extensive neutrophil extracellular trap (NET) components observed in relation to neutrophil degranulation throughout the gastrointestinal tract. The colitis model’s microbiome differed significantly from that of wild-type mice, indicating the substantial influence of efflux transporter deficiency on microbiota.ConclusionThe proteomic and microbiota analyzes of a well-established murine model enabled the correlation of gastrointestinal interactions not readily identifiable in human cohorts. Insights into dysregulated biological pathways in this disease model might offer translational biomarkers based on NETs and improved understanding of IBD pathogenesis in human patients. Our findings demonstrate that drug transporter deficiency induces substantial changes in the microbiota, leading to increased levels of IBD-associated strains and resulting in intestinal inflammation.GRAPHICAL ABSTRACT

Abstract The incorporation of nanomaterials (NMs) in consumer products has proven to be highly valuable in many sectors. Unfortunately, however, the same nano specific physicochemical properties, which make these material attractive, might also contribute to hazards for people exposed to these materials. The physicochemical properties of NMs will impact their interaction with biological surroundings and influence their fate and their potential adverse effects such as genotoxicity. Due to the large and expanding number of NMs produced, their availability in different nanoforms (NFs) and their utilization in various formats, it is impossible for risk assessment to be conducted on an individual NF basis. Alternative methods, such as grouping are needed for streamlining hazard assessment. The GRACIOUS Framework provides a logical and science evidenced approach to group similar NFs, allowing read-across of hazard information from source NFs (or non-NFs) with adequate hazard data to target NFs that lack such data. Here, we propose a simple three-tiered testing strategy to gather evidence to determine whether different NFs are sufficiently similar with respect to their potential to induce genotoxicity, in order to be grouped. The tiered testing strategy includes simple in vitro models as well as a number of alternative more complex multi-cellular in vitro models to allow for a better understanding of secondary NM-induced DNA damage, something that has been more appropriate in vivo until recently.

Background: TP53 encodes a tumor suppressor protein containing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. The effect of TP53 inactivation is well-known, and genetically determined smaller variations in TP53 activity are related to cancer. Lung cancer causes the highest rates of morbidity and mortality in the world. Epidemiology studies have assessed the association of TP53 single nucleotide polymorphisms with lung cancer. Methods: We systematically examined the association of five htSNPs (haplotype-tagging single nucleotide polymorphism) (rs12951053, rs1042522, rs8079544, rs12602273 and rs8064946) across the entire TP53 locus and interaction between genes TP53 and PPP1R13L and CD3EAP and smoking-duration related to lung cancer risk in this Chinese study including 544 cases and 550 controls. Results: No significant associations were observed in analysis of alleles and genotypes with co-dominant, dominant, recessive, and log-additive models after adjustment for smoking status. Haplotype analysis showed that haplotype9 (rs12951053A-rs1042522C-rs8079544C-rs12602273G-rs8064946C) [OR (95% CI) = 0.13 (0.03–0.59), p = 0.0079] was associated with decreased risk of lung cancer after adjusted for smoking-duration. The analysis of smoking-duration within TP53 haplotypes showed that there were more carriers of haplotype1 (AGCCG), 2 (CCCGC) and 4 (CCCCG) in smoking-subgroup of >20 (years) (all p

Abstract Background Pulmonary exposure to high doses of engineered carbonaceous nanomaterials (NMs) is known to trigger inflammation in the lungs paralleled by an acute phase response. Toll-like receptors (TLRs), particularly TLR2 and TLR4, have recently been discussed as potential NM-sensors, initiating inflammation. Using Tlr2 and Tlr4 knock out (KO) mice, we addressed this hypothesis and compared the pattern of inflammation in lung and acute phase response in lung and liver 24 h after intratracheal instillation of three differently shaped carbonaceous NMs, spherical carbon black (CB), multi-walled carbon nanotubes (CNT), graphene oxide (GO) plates and bacterial lipopolysaccharide (LPS) as positive control. Results The LPS control confirmed a distinct TLR4-dependency as well as a pronounced contribution of TLR2 by reducing the levels of pulmonary inflammation to 30 and 60% of levels in wild type (WT) mice. At the doses chosen, all NM caused comparable neutrophil influxes into the lungs of WT mice, and reduced levels were only detected for GO-exposed Tlr2 KO mice (35%) and for CNT-exposed Tlr4 KO mice (65%). LPS-induced gene expression was strongly TLR4-dependent. CB-induced gene expression was unaffected by TLR status. Both GO and MWCNT-induced Saa1 expression was TLR4-dependent. GO-induced expression of Cxcl2, Cxcl5, Saa1 and Saa3 were TLR2-dependent. NM-mediated hepatic acute phase response in terms of liver gene expression of Saa1 and Lcn2 was shown to depend on TLR2 for all three NMs. TLR4, in contrast, was only relevant for the acute phase response caused by CNTs, and as expected by LPS. Conclusion TLR2 and TLR4 signaling was not involved in the acute inflammatory response caused by CB exposure, but contributed considerably to that of GO and CNTs, respectively. The strong involvement of TLR2 in the hepatic acute phase response caused by pulmonary exposure to all three NMs deserves further investigations.

Abstract Air force ground crew personnel are potentially exposed to fuels and lubricants, as raw materials, vapours and combustion exhaust emissions, during operation and maintenance of aircrafts. This study investigated exposure levels and biomarkers of effects for employees at a Danish air force military base. We enrolled self-reported healthy and non-smoking employees (n = 79) and grouped them by exposure based on job function, considered to be potentially exposed (aircraft engineers, crew chiefs, fuel operators and munition specialists) or as reference group with minimal occupational exposure (avionics and office workers). We measured exposure levels to polycyclic aromatic hydrocarbons (PAHs) and organophosphate esters (OPEs) by silicone bands and skin wipes (PAHs only) as well as urinary excretion of PAH metabolites (OH-PAHs). Additionally, we assessed exposure levels of ultrafine particles (UFPs) in the breathing zone for specific job functions. As biomarkers of effect, we assessed lung function, plasma levels of acute phase inflammatory markers, and genetic damage levels in peripheral blood cells. Exposure levels of total PAHs, OPEs and OH-PAHs did not differ between exposure groups or job functions, with low correlations between PAHs in different matrices. Among the measured job functions, the UFP levels were higher for the crew chiefs. The exposure level of the PAH fluorene was significantly higher for the exposed group than the reference group (15.9 ± 23.7 ng/g per 24 h vs 5.28 ± 7.87 ng/g per 24 h, p = 0.007), as was the OPE triphenyl phosphate (305 ± 606 vs 19.7 ± 33.8 ng/g per 24 h, p = 0.011). The OPE tris(1,3-dichlor-2-propyl)phosphate had a higher mean in the exposed group (60.7 ± 135 ng/g per 24 h) compared to the reference group (8.89 ± 15.7 ng/g per 24 h) but did not reach significance. No evidence of effects for biomarkers of systemic inflammation, genetic damage or lung function was found. Overall, our biomonitoring study show limited evidence of occupational exposure of air force ground crew personnel to UFPs, PAHs and OPEs. Furthermore, the OH-PAHs and the assessed biomarkers of early biological effects did not differ between exposed and reference groups.

Abstract Background An important aspect of nanomaterial (NM) risk assessment is establishing relationships between physicochemical properties and key events governing the toxicological pathway leading to adverse outcomes. The difficulty of NM grouping can be simplified if the most toxicologically relevant dose metric is used to assess the toxicological dose-response. Here, we thoroughly investigated the relationship between acute and chronic inflammation (based on polymorphonuclear neutrophil influx (% PMN) in lung bronchoalveolar lavage) and the retained surface area in the lung. Inhalation studies were performed in rats with three classes of NMs: titanium dioxides (TiO2) and carbon blacks (CB) as poorly soluble particles of low toxicity (PSLT), and multiwall carbon nanotubes (MWCNTs). We compared our results to published data from nearly 30 rigorously selected articles. Results This analysis combined data specially generated for this work on three benchmark materials - TiO2 P25, the CB Printex-90 and the MWCNT MWNT-7 - following subacute (4-week) inhalation with published data relating to acute (1-week) to subchronic (13-week) inhalation exposure to the classes of NMs considered. Short and long post-exposure recovery times (immediately after exposure up to more than 6 months) allowed us to examine both acute and chronic inflammation. A dose-response relationship across short-term and long-term studies was revealed linking pulmonary retained surface area dose (measured or estimated) and % PMN. This relationship takes the form of sigmoid curves, and is independent of the post-exposure time. Curve fitting equations depended on the class of NM considered, and sometimes on the duration of exposure. Based on retained surface area, long and thick MWCNTs (few hundred nm long with an aspect ratio greater than 25) had a higher inflammatory potency with 5 cm2/g lung sufficient to trigger an inflammatory response (at 6% PMN), whereas retained surfaces greater than 150 cm2/g lung were required for PSLT. Conclusions Retained surface area is a useful metric for hazard grouping purposes. This metric would apply to both micrometric and nanometric materials, and could obviate the need for direct measurement in the lung. Indeed, it could alternatively be estimated from dosimetry models using the aerosol parameters (rigorously determined following a well-defined aerosol characterization strategy).

Exposure to nanoparticles by various routes results in size-dependent translocation of nanoparticles to the systemic circulation and subsequent accumulation in the liver. The purpose of this study was to determine possible adverse effects in the liver of long-lasting nanoparticle presence in the organ. Mice exposed to a single dose (162 µg/animal equivalent to 9 mg/kg body weight) of TiO2, CeO2 or carbon black nanoparticles by intratracheal instillation or intravenous injection, resulting in relatively low or high liver burdens, were followed for 1, 28 or 180 days. Clinical appearance, feed intake, body and liver weights, hematological indices, and transaminases and alkaline phosphatase activities were unaffected by exposure. Exposure-related foreign material persisted in the liver up to 180 days after intratracheal and intravenous exposure, mainly in sinusoids, near Kupffer cells, or around blood vessels. Increased incidences of histological findings after intratracheal or intravenous exposure included: initially, prominent nuclei of Kupffer cells, the apparent increase in binucleate hepatocytes (TiO2 and carbon black) and inflammatory infiltrations (CeO2); later, cytoplasmic vacuolation, pyknosis and necrosis, especially for CeO2. Thus, neither low nor high nanoparticle burden in the liver affected enzymatic markers of liver injury, but indications of exposure-related necrotic changes, particularly for CeO2 nanoparticles, were noted.

Multiple myeloma (MM) arises following malignant proliferation of plasma cells in the bone marrow, that secrete high amounts of specific monoclonal immunoglobulins or light chains, resulting in the massive production of unfolded or misfolded proteins. Autophagy can have a dual role in tumorigenesis, by eliminating these abnormal proteins to avoid cancer development, but also ensuring MM cell survival and promoting resistance to treatments. To date no studies have determined the impact of genetic variation in autophagy-related genes on MM risk. We performed meta-analysis of germline genetic data on 234 autophagy-related genes from three independent study populations including 13,387 subjects of European ancestry (6863 MM patients and 6524 controls) and examined correlations of statistically significant single nucleotide polymorphisms (SNPs; p < 1 × 10−9) with immune responses in whole blood, peripheral blood mononuclear cells (PBMCs), and monocyte-derived macrophages (MDM) from a large population of healthy donors from the Human Functional Genomic Project (HFGP). We identified SNPs in six loci, CD46, IKBKE, PARK2, ULK4, ATG5, and CDKN2A associated with MM risk (p = 4.47 × 10−4−5.79 × 10−14). Mechanistically, we found that the ULK4rs6599175 SNP correlated with circulating concentrations of vitamin D3 (p = 4.0 × 10−4), whereas the IKBKErs17433804 SNP correlated with the number of transitional CD24+CD38+ B cells (p = 4.8 × 10−4) and circulating serum concentrations of Monocyte Chemoattractant Protein (MCP)-2 (p = 3.6 × 10−4). We also found that the CD46rs1142469 SNP correlated with numbers of CD19+ B cells, CD19+CD3− B cells, CD5+IgD− cells, IgM− cells, IgD−IgM− cells, and CD4−CD8− PBMCs (p = 4.9 × 10−4−8.6 × 10−4) and circulating concentrations of interleukin (IL)-20 (p = 0.00082). Finally, we observed that the CDKN2Ars2811710 SNP correlated with levels of CD4+EMCD45RO+CD27− cells (p = 9.3 × 10−4). These results suggest that genetic variants within these six loci influence MM risk through the modulation of specific subsets of immune cells, as well as vitamin D3−, MCP-2−, and IL20-dependent pathways.

Abstract Background Multi-walled carbon nanotubes (MWCNT) have received attention due to extraordinary properties, resulting in concerns for occupational health and safety. Costs and ethical concerns of animal testing drive a need for in vitro models with predictive power in respiratory toxicity. The aim of this study was to assess pro-inflammatory response (Interleukin-8 expression, IL-8) and genotoxicity (DNA strand breaks) caused by MWCNT with different physicochemical properties in different pulmonary cell models and correlate these to previously published in vivo data. Seven MWCNT were selected; two long/thick (NRCWE-006/Mitsui-7 and NM-401), two short/thin (NM-400 and NM-403), a pristine (NRCWE-040) and two surface modified; hydroxylated (NRCWE-041) and carboxylated (NRCWE-042). Carbon black Printex90 (CB) was included as benchmark material. Human alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1a) were exposed to nanomaterials (NM) in submerged conditions, and two materials (NM-400 and NM-401) in co-cultures of A549/THP-1a and lung fibroblasts (WI-38) in an air-liquid interface (ALI) system. Effective doses were quantified by thermo-gravimetric-mass spectrometry analysis (TGA-MS). To compare genotoxicity in vitro and in vivo, we developed a scoring system based on a categorization of effects into standard deviation (SD) units (

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed as a result of natural cellular processes, intracellular signaling, or as adverse responses associated with diseases or exposure to oxidizing chemical and non-chemical stressors. The action of ROS and RNS, collectively referred to as reactive oxygen and nitrogen species (RONS), has recently become highly relevant in a number of adverse outcome pathways (AOPs) that capture, organize, evaluate and portray causal relationships pertinent to adversity or disease progression. RONS can potentially act as a key event (KE) in the cascade of responses leading to an adverse outcome (AO) within such AOPs, but are also known to modulate responses of events along the AOP continuum without being an AOP event itself. A substantial discussion has therefore been undertaken in a series of workshops named “Mystery or ROS” to elucidate the role of RONS in disease and adverse effects associated with exposure to stressors such as nanoparticles, chemical, and ionizing and non-ionizing radiation. This review introduces the background for RONS production, reflects on the direct and indirect effects of RONS, addresses the diversity of terminology used in different fields of research, and provides guidance for developing a harmonized approach for defining a common event terminology within the AOP developer community.

Abstract Background Airport personnel are at risk of occupational exposure to jet engine emissions, which similarly to diesel exhaust emissions include volatile organic compounds and particulate matter consisting of an inorganic carbon core with associated polycyclic aromatic hydrocarbons, and metals. Diesel exhaust is classified as carcinogenic and the particulate fraction has in itself been linked to several adverse health effects including cancer. Method In this review, we summarize the available scientific literature covering human health effects of exposure to airport emissions, both in occupational settings and for residents living close to airports. We also report the findings from the limited scientific mechanistic studies of jet engine emissions in animal and cell models. Results Jet engine emissions contain large amounts of nano-sized particles, which are particularly prone to reach the lower airways upon inhalation. Size of particles and emission levels depend on type of aircraft, engine conditions, and fuel type, as well as on operation modes. Exposure to jet engine emissions is reported to be associated with biomarkers of exposure as well as biomarkers of effect among airport personnel, especially in ground-support functions. Proximity to running jet engines or to the airport as such for residential areas is associated with increased exposure and with increased risk of disease, increased hospital admissions and self-reported lung symptoms. Conclusion We conclude that though the literature is scarce and with low consistency in methods and measured biomarkers, there is evidence that jet engine emissions have physicochemical properties similar to diesel exhaust particles, and that exposure to jet engine emissions is associated with similar adverse health effects as exposure to diesel exhaust particles and other traffic emissions.

Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) are nanomaterials with one or multiple layers of carbon sheets. While it is suggested that various properties influence their toxicity, the specific mechanisms are not completely known. This study was aimed to determine if single or multi-walled structures and surface functionalization influence pulmonary toxicity and to identify the underlying mechanisms of toxicity. Female C57BL/6J BomTac mice were exposed to a single dose of 6, 18, or 54 μg/mouse of twelve SWCNTs or MWCNTs of different properties. Neutrophil influx and DNA damage were assessed on days 1 and 28 post-exposure. Genome microarrays and various bioinformatics and statistical methods were used to identify the biological processes, pathways and functions altered post-exposure to CNTs. All CNTs were ranked for their potency to induce transcriptional perturbation using benchmark dose modelling. All CNTs induced tissue inflammation. MWCNTs were more genotoxic than SWCNTs. Transcriptomics analysis showed similar responses across CNTs at the pathway level at the high dose, which included the perturbation of inflammatory, cellular stress, metabolism, and DNA damage responses. Of all CNTs, one pristine SWCNT was found to be the most potent and potentially fibrogenic, so it should be prioritized for further toxicity testing.

Inhaled substances, such as consumer products, chemicals at the workplace, and nanoparticles, can affect the lung function in several ways. In this paper, we explore the adverse outcome pathway (AOP) that starts when inhaled substances that reach the alveoli inhibit the function of the lung surfactant, and leads to decreased lung function. Lung surfactant covers the inner surface of the alveoli, and regulates the surface tension at the air–liquid interface during breathing. The inhibition of the lung surfactant function leads to alveolar collapse because of the resulting high surface tension at the end of expiration. The collapsed alveoli can be re-opened by inspiration, but this re-opening causes shear stress on cells covering the alveoli. This can damage the alveolar-capillary membrane integrity, allowing blood components to enter the alveolar airspace. Blood components, such as albumin, can interact with the lung surfactant and further inhibit its function. The collapse of the alveoli is responsible for a decrease in the surface area available for blood oxygenation, and it reduces the volume of air that can be inhaled and exhaled. These different key events lead to decreased lung function, characterized by clinical signs of respiratory toxicity and reduced blood oxygenation. Here we present the weight of evidence that supports the AOP, and we give an overview of the methods available in vitro and in vivo to measure each key event of the pathway, and how this AOP can potentially be used in screening for inhalation toxicity.

Abstract Toxicity testing and regulation of advanced materials at the nanoscale, i.e. nanosafety, is challenged by the growing number of nanomaterials and their property variants requiring assessment for potential human health impacts. The existing animal-reliant toxicity testing tools are onerous in terms of time and resources and are less and less in line with the international effort to reduce animal experiments. Thus, there is a need for faster, cheaper, sensitive and effective animal alternatives that are supported by mechanistic evidence. More importantly, there is an urgency for developing alternative testing strategies that help justify the strategic prioritization of testing or targeting the most apparent adverse outcomes, selection of specific endpoints and assays and identifying nanomaterials of high concern. The Adverse Outcome Pathway (AOP) framework is a systematic process that uses the available mechanistic information concerning a toxicological response and describes causal or mechanistic linkages between a molecular initiating event, a series of intermediate key events and the adverse outcome. The AOP framework provides pragmatic insights to promote the development of alternative testing strategies. This review will detail a brief overview of the AOP framework and its application to nanotoxicology, tools for developing AOPs and the role of toxicogenomics, and summarize various AOPs of relevance to inhalation toxicity of nanomaterials that are currently under various stages of development. The review also presents a network of AOPs derived from connecting all AOPs, which shows that several adverse outcomes induced by nanomaterials originate from a molecular initiating event that describes the interaction of nanomaterials with lung cells and involve similar intermediate key events. Finally, using the example of an established AOP for lung fibrosis, the review will discuss various in vitro tests available for assessing lung fibrosis and how the information can be used to support a tiered testing strategy for lung fibrosis. The AOPs and AOP network enable deeper understanding of mechanisms involved in inhalation toxicity of nanomaterials and provide a strategy for the development of alternative test methods for hazard and risk assessment of nanomaterials.

Abstract Background Previous studies have shown that inhalation of welding fumes may induce pulmonary and systemic inflammation and organ accumulation of metal, to which spermatogenesis and endocrine function may be sensitive. Also obesity may induce low-grade systemic inflammation. This study aimed to investigate the effects on sperm production of inhaled metal nanoparticles from stainless steel welding, and the potential exacerbation by intake of a high fat diet. Both the inbred Brown Norway and the outbred Sprague Dawley rat strains were included to study the influence of strain on the detection of toxicity. Rats were fed regular or high fat (HF) diet for 24 weeks and were exposed to 20 mg/m3 of gas metal arc-stainless steel (GMA-SS) welding fumes or filtered air for 3 h/day, 4 days/week for 5 weeks, during weeks 7–12. Outcomes were assessed upon termination of exposure (week 12) and after recovery (week 24). Results At week 12, the GMA-SS exposure induced pulmonary inflammation in both strains, without consistent changes in markers of systemic inflammation (CRP, MCP-1, IL-6 and TNFα). GMA-SS exposure lowered daily sperm production compared to air controls in Sprague Dawley rats, but only in GMA-SS Brown Norway rats also fed the HF diet. Overall, HF diet rats had lower serum testosterone levels compared to rats on regular diet. Metal content in the testes was assessed in a limited number of samples in Brown Norway rats, but no increase was obsedrved. At week 24, bronchoalveolar lavage cell counts had returned to background levels for GMA-SS exposed Sprague Dawley rats but remained elevated in Brown Norway rats. GMA-SS did not affect daily sperm production statistically significantly at this time point, but testicular weights were lowered in GMA-SS Sprague Dawley rats. Serum testosterone remained lowered in Sprague Dawley rats fed the HF diet. Conclusion Exposure to GMA-SS welding fumes lowered sperm production in two strains of rats, whereas high fat diet lowered serum testosterone. The effect on sperm counts was likely not mediated by inflammation or lowered testosterone levels. The studied reproductive outcomes seemed more prone to disruption in the Sprague Dawley compared to the Brown Norway strain.

Abstract Recently, Borm and Driscoll published a commentary discussing grouping of Poorly Soluble particles of Low Toxicity (PSLTs) and the use of rats as an animal model for human hazard assessment of PSLTs (Particle and Fibre Toxicology (2019) 16(1):11). The commentary was based on the scientific opinion of several international experts on these topics. The general conclusion from the authors was a cautious approach towards using chronic inhalation studies in rats for human hazard assessment of PSLTs. This was based on evidence of inhibition of particle clearance leading to overload in the rats after high dose exposure, and a suggested over reactivity of rat lung cancer responses compared to human risk. As a response to the commentary, we here discuss evidence from the scientific literature showing that a) diesel exhaust particles, carbon black nanoparticles and TiO2 nanoparticles have similar carcinogenic potential in rats, and induce lung cancer at air concentrations below the air concentrations that inhibit particle clearance in rats, and b) chronic inhalation studies of diesel exhaust particles are less sensitive than epidemiological studies, leading to higher risk estimates for lung cancer. Thus, evidence suggests that the chronic inhalation study in rats can be used for assessing lung cancer risk insoluble nanomaterials.

Abstract Background With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver’s recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. Results The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. Conclusion In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

Polycyclic aromatic hydrocarbons (PAHs) are among the chemicals with proven impact on workers’ health. The use of human biomonitoring (HBM) to assess occupational exposure to PAHs has become more common in recent years, but the data generated need an overall view to make them more usable by regulators and policymakers. This comprehensive review, developed under the Human Biomonitoring for Europe (HBM4EU) Initiative, was based on the literature available from 2008–2022, aiming to present and discuss the information on occupational exposure to PAHs, in order to identify the strengths and limitations of exposure and effect biomarkers and the knowledge needs for regulation in the workplace. The most frequently used exposure biomarker is urinary 1-hydroxypyrene (1-OH-PYR), a metabolite of pyrene. As effect biomarkers, those based on the measurement of oxidative stress (urinary 8-oxo-dG adducts) and genotoxicity (blood DNA strand-breaks) are the most common. Overall, a need to advance new harmonized approaches both in data and sample collection and in the use of appropriate biomarkers in occupational studies to obtain reliable and comparable data on PAH exposure in different industrial sectors, was noted. Moreover, the use of effect biomarkers can assist to identify work environments or activities of high risk, thus enabling preventive risk mitigation and management measures.

Abstract In response to the Letter to the Editor by Kevin Driscoll et al., we certainly agree that particle clearance halftimes are increased with increasing lung burden in rats, hamsters and mice, whereas complete inhibition of particle clearance has only been observed in rats, and only at high particle concentrations (50 mg/m3). Where we disagree with Kevin Driscoll and colleagues, is on the implications of the increased clearance halftimes observed at higher lung burden. We argue that it does not hamper the extrapolations from relatively high dose levels to lower dose levels. Furthermore, we highlight, again, the challenges of detecting particle-induced lung cancer in epidemiological studies where occupational, particle-induced lung cancer has to be detected on top of the background lung cancer incidence. Almost all available epidemiological studies on carbon black and titanium dioxide suffer from a number of limitations, including lack of control for smoking, the use of background population cancer rates as reference in the US studies, lack of information regarding particle size of the exposure, and incomplete follow-up for cause of death of the study population.

Abstract Background Coke oven workers are exposed to both free and particle bound PAH. Through this exposure, the workers may be at increased risk of cardiovascular diseases. Systemic levels of acute phase response proteins have been linked to cardiovascular disease in epidemiological studies, suggesting it as a marker of these conditions. The aim of this study was to assess whether there was association between PAH exposure and the blood level of the acute phase inflammatory response marker serum amyloid A (SAA) in coke oven workers. Methods A total of 87 male Polish coke oven workers from two different plants comprised the study population. Exposure was assessed by means of the individual post-shift urinary excretion of 1-hydroxypyrene, as internal dose of short-term PAH exposure, and by anti-benzo[a]pyrene diolepoxide (anti-B[a]PDE)-DNA), as a biomarker of long-term PAH exposure. Blood levels of acute phase proteins SAA and CRP were measured by immunoassay. C-reactive protein (CRP) levels were included to adjust for baseline levels of SAA. Results Multiple linear regression showed that the major determinants of increased SAA levels were urinary 1-hydroxypyrene (beta = 0.56, p = 0.030) and serum CRP levels (beta = 7.08; p

Abstract Background Little is known about the exposure levels and adverse health effects of occupational exposure to airplane emissions. Diesel exhaust particles are classified as carcinogenic to humans and jet engines produce potentially similar soot particles. Here, we evaluated the potential occupational exposure risk by analyzing particles from a non-commercial airfield and from the apron of a commercial airport. Toxicity of the collected particles was evaluated alongside NIST standard reference diesel exhaust particles (NIST2975) in terms of acute phase response, pulmonary inflammation, and genotoxicity after single intratracheal instillation in mice. Results Particle exposure levels were up to 1 mg/m3 at the non-commercial airfield. Particulate matter from the non-commercial airfield air consisted of primary and aggregated soot particles, whereas commercial airport sampling resulted in a more heterogeneous mixture of organic compounds including salt, pollen and soot, reflecting the complex occupational exposure at an apron. The particle contents of polycyclic aromatic hydrocarbons and metals were similar to the content in NIST2975. Mice were exposed to doses 6, 18 and 54 μg alongside carbon black (Printex 90) and NIST2975 and euthanized after 1, 28 or 90 days. Dose-dependent increases in total number of cells, neutrophils, and eosinophils in bronchoalveolar lavage fluid were observed on day 1 post-exposure for all particles. Lymphocytes were increased for all four particle types on 28 days post-exposure as well as for neutrophil influx for jet engine particles and carbon black nanoparticles. Increased Saa3 mRNA levels in lung tissue and increased SAA3 protein levels in plasma were observed on day 1 post-exposure. Increased levels of DNA strand breaks in bronchoalveolar lavage cells and liver tissue were observed for both particles, at single dose levels across doses and time points. Conclusions Pulmonary exposure of mice to particles collected at two airports induced acute phase response, inflammation, and genotoxicity similar to standard diesel exhaust particles and carbon black nanoparticles, suggesting similar physicochemical properties and toxicity of jet engine particles and diesel exhaust particles. Given this resemblance as well as the dose-response relationship between diesel exhaust exposure and lung cancer, occupational exposure to jet engine emissions at the two airports should be minimized.

Abstract 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/m3 and 1.2–1.8 × 105 particles/cm3 higher, respectively, in diesel as compared to electric trains. Net increases of NOx and NO2 concentrations were 317 μg/m3 and 36 μg/m3. 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

Abstract Background Previous findings indicate that in utero exposure to nanoparticles may affect the reproductive system in male offspring. Effects such as decreased sperm counts and testicular structural changes in F1 males have been reported following maternal airway exposure to carbon black during gestation. In addition, a previous study in our laboratory suggested that the effects of in utero exposure of nanoparticles may span further than the first generation, as sperm content per gram of testis was significantly lowered in F2 males. In the present study we assessed male fertility parameters following in utero inhalation exposure to carbon black in four generations of mice. Results Filter measurements demonstrated that the time-mated females were exposed to a mean total suspended particle mass concentration of 4.79 ± 1.86 or 33.87 ± 14.77 mg/m3 for the low and high exposure, respectively. The control exposure was below the detection limit (LOD 0.08 mg/m3). Exposure did not affect gestation and litter parameters in any generation. No significant changes were observed in body and reproductive organ weights, epididymal sperm parameters, daily sperm production, plasma testosterone or fertility. Conclusion In utero exposure to carbon black nanoparticles, at occupationally relevant exposure levels, via maternal whole body inhalation did not affect male-specific reproductive, fertility and litter parameters in four generations of mice.

The molecular effects of exposures to engineered nanomaterials (ENMs) are still largely unknown. In classical inhalation toxicology, cell composition of bronchoalveolar lavage (BAL) is a toxicity indicator at the lung tissue level that can aid in interpreting pulmonary histological changes. Toxicogenomic approaches help characterize the mechanism of action (MOA) of ENMs by investigating the differentially expressed genes (DEG). However, dissecting which molecular mechanisms and events are directly induced by the exposure is not straightforward. It is now generally accepted that direct effects follow a monotonic dose-dependent pattern. Here, we applied an integrated modeling approach to study the MOA of four ENMs by retrieving the DEGs that also show a dynamic dose-dependent profile (dddtMOA). We further combined the information of the dddtMOA with the dose dependency of four immune cell populations derived from BAL counts. The dddtMOA analysis highlighted the specific adaptation pattern to each ENM. Furthermore, it revealed the distinct effect of the ENM physicochemical properties on the induced immune response. Finally, we report three genes dose-dependent in all the exposures and correlated with immune deregulation in the lung. The characterization of dddtMOA for ENM exposures, both for apical endpoints and molecular responses, can further promote toxicogenomic approaches in a regulatory context.

Lung cancer, one of the most common and deadly forms of cancer, is in some cases associated with exposure to certain types of particles. With the rise of nanotechnology, there is concern that some engineered nanoparticles may be among such particles. In the absence of epidemiological evidence, assessment of nanoparticle carcinogenicity is currently performed on a time-consuming case-by-case basis, relying mainly on animal experiments. Non-animal alternatives exist, including a few validated cell-based methods accepted for regulatory risk assessment of nanoparticles. Furthermore, new approach methodologies (NAMs), focused on carcinogenic mechanisms and capable of handling the increasing numbers of nanoparticles, have been developed. However, such alternative methods are mainly applied as weight-of-evidence linked to generally required animal data, since challenges remain regarding interpretation of the results. These challenges may be more easily overcome by the novel Adverse Outcome Pathway (AOP) framework, which provides a basis for validation and uptake of alternative mechanism-focused methods in risk assessment. Here, we propose an AOP for lung cancer induced by nanosized foreign matter, anchored to a selection of 18 standardized methods and NAMs for in silico- and in vitro-based integrated assessment of lung carcinogenicity. The potential for further refinement of the AOP and its components is discussed in relation to available nanosafety knowledge and data. Overall, this perspective provides a basis for development of AOP-aligned alternative methods-based integrated testing strategies for assessment of nanoparticle-induced lung cancer.

BACKGROUND:The association between vitamin D and incidence of colorectal cancer has been thoroughly investigated, but the results are conflicting. The objectives in this study were to investigate whether two functional polymorphisms in GC and CYP2R1, respectively, previously shown to predict vitamin D concentrations, were associated with risk of colorectal cancer; and further, to assess gene-environment interaction between the polymorphisms and intake of vitamin D through diet and supplementation in relation to risk of colorectal cancer. METHODS:A nested case-cohort study of 920 colorectal cancer cases and 1743 randomly selected participants from the Danish prospective "Diet, Cancer and Health" study was performed. Genotypes CYP2R1/rs10741657 and GC/rs4588 were determined by PCR-based KASP™ genotyping assay. Vitamin D intake from supplements and diet was assessed from a validated food frequency questionnaire. Incidence rate ratios were estimated by the Cox proportional hazards model, and interactions between polymorphisms in GC and CYP2R1 and vitamin D intake in relation to risk of colorectal cancer were assessed. RESULTS:Neither of the two polymorphisms was associated with risk of colorectal cancer per se. Heterozygote carriage of CYP2R1/rs10741657 and GC/rs4588, and carriage of two risk alleles (estimated by a genetic risk score) were weakly associated with 9-12% decreased risk of colorectal cancer per 3 μg intake of vitamin D per day (IRRCYP2R1/rs10741657 = 0.88, 95% CI: 0.79-0.97; IRRGC/rs4588 = 0.91, 95% CI: 0.82-1.01, IRRGRS2 = 0.90, 95% CI: 0.81-0.99). CONCLUSIONS:The results suggest that genetic variation in vitamin D metabolising genes may influence the association between vitamin D intake, through food and supplementation, and risk of colorectal cancer. CLINICAL TRIAL REGISTRY:NCT03370432. Registered 12 December 2017 (retrospectively registered).

Abstract Background Ankylosing spondylitis (AS) results from the combined effects of susceptibility genes and environmental factors. Polymorphisms in genes regulating inflammation may explain part of the heritability of AS. Methods Using a candidate gene approach in this case-control study, 51 mainly functional single nucleotide polymorphisms (SNPs) in genes regulating inflammation were assessed in 709 patients with AS and 795 controls. Data on the patients with AS were obtained from the DANBIO registry where patients from all of Denmark are monitored in routine care during treatment with conventional and biologic disease modifying anti-rheumatic drugs (bDMARDs). The results were analyzed using logistic regression (adjusted for age and sex). Results Nine polymorphisms were associated with risk of AS (p A (rs1800629), and − 238 G > A (rs361525); TNFRSF1A -609 G > T (rs4149570), and PTPN22 1858 G > A (rs2476601)), b: the IL23/IL17 pathway (IL23R G > A (rs11209026), and IL18–137 G > C (rs187238)), or c: the NFkB pathway (TLR1 743 T > C (rs4833095), TLR4 T > C (rs1554973), and LY96–1625 C > G (rs11465996)). After Bonferroni correction the homozygous variant genotype of TLR1 743 T > C (rs4833095) (odds ratios (OR): 2.59, 95% confidence interval (CI): 1.48–4.51, p = 0.04), and TNFRSF1A -609 G > T (rs4149570) (OR: 1.79, 95% CI: 1.31–2.41, p = 0.01) were associated with increased risk of AS and the combined homozygous and heterozygous variant genotypes of TNF -308 G > A (rs1800629) (OR: 0.56, 95% CI: 0.44–0.72, p = 0.0002) were associated with reduced risk of AS. Conclusion We replicated associations between AS and the polymorphisms in TNF (rs1800629), TNFRSF1A (rs4149570), and IL23R (rs11209026). Furthermore, we identified novel risk loci in TNF (rs361525), IL18 (rs187238), TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996) that need validation in independent cohorts. The results suggest that genetically determined high activity of the TNF-α, IL23/IL17, and NFkB pathways increase risk of AS.

Abstract Background Engineered nanoparticles are smaller than 100 nm and designed to improve or creating even new physico-chemical properties. Consequently, toxicological properties of materials may change as size reaches the nm size-range. We examined outcomes related to the central nervous system in the offspring following maternal inhalation exposure to nanosized carbon black particles (Printex 90). Methods Time-mated mice (NMRI) were exposed by inhalation, for 45 min/day to 0, 4.6 or 37 mg/m3 aerosolized carbon black on gestation days 4–18, i.e. for a total of 15 days. Outcomes included maternal lung inflammation (differential cell count in bronchoalveolar lavage fluid and Saa3 mRNA expression in lung tissue), offspring neurohistopathology and behaviour in the open field test. Results Carbon black exposure did not cause lung inflammation in the exposed females, measured 11 or 28–29 days post-exposure. Glial fibrillary acidic protein (GFAP) expression levels were dose-dependently increased in astrocytes around blood vessels in the cerebral cortex and hippocampus in six weeks old offspring, indicative of reactive astrogliosis. Also enlarged lysosomal granules were observed in brain perivascular macrophages (PVMs) in the prenatally exposed offspring. The number of parvalbumin-positive interneurons and the expression levels of parvalbumin were decreased in the motor and prefrontal cortices at weaning and 120 days of age in the prenatally exposed offspring. In the open field test, behaviour was dose-dependently altered following maternal exposure to Printex 90, at 90 days of age. Prenatally exposed female offspring moved a longer total distance, and especially males spent significantly longer time in the central zone of the maze. In the offspring, the described effects were long-lasting as they were present at all time points investigated. Conclusion The present study reports for the first time that maternal inhalation exposure to Printex 90 carbon black induced dose-dependent denaturation of PVM and reactive astrocytes, similarly to the findings observed following maternal exposure to Printex 90 by airway instillation. Of note, some of the observed effects have striking similarities with those observed in mouse models of neurodevelopmental disorders.

Abstract Background Selenium is an essential trace element and is suggested to play a role in the etiology of a number of chronic diseases. Genetic variation in genes encoding selenoproteins, such as selenoprotein P and the glutathione peroxidases, may affect selenium status and, thus, individual susceptibility to some chronic diseases. In the present study, we aimed to (1) investigate the effect of mussel and fish intake on glutathione peroxidase enzyme activity and (2) examine whether single nucleotide polymorphisms in the GPX1, GPX4, and SELENOP genes modify the effect of mussel and fish intake for 26 weeks on whole blood selenium, plasma selenoprotein P concentrations, and erythrocyte GPX enzyme activity in a randomized intervention trial in Denmark. Results CC homozygotes of the SELENOP/rs3877899 polymorphism who consumed 1000 g fish and mussels per week for 26 consecutive weeks had higher levels of both selenoprotein P (difference between means − 4.68 ng/mL (95% CI − 8.49, − 0.871)) and whole blood selenium (difference between means − 5.76 (95% CI − 12.5, 1.01)) compared to fish and mussel consuming T-allele carriers although the effect in whole blood selenium concentration was not statistically significant. Conclusions Our study indicates that genetically determined variation in SELENOP leads to different responses in expression of selenoproteins following consumption of selenium-rich foods. This study also emphasizes the importance of taking individual aspects such as genotypes into consideration when assessing risk in public health recommendations.

Abstract Background Semen quality parameters are potentially affected by nanomaterials in several ways: Inhaled nanosized particles are potent inducers of pulmonary inflammation, leading to the release of inflammatory mediators. Small amounts of particles may translocate from the lungs into the lung capillaries, enter the systemic circulation and ultimately reach the testes. Both the inflammatory response and the particles may induce oxidative stress which can directly affect spermatogenesis. Furthermore, spermatogenesis may be indirectly affected by changes in the hormonal milieu as systemic inflammation is a potential modulator of endocrine function. The aim of this study was to investigate the effects of pulmonary exposure to carbonaceous nanomaterials on sperm quality parameters in an experimental mouse model. Methods Effects on sperm quality after pulmonary inflammation induced by carbonaceous nanomaterials were investigated by intratracheally instilling sexually mature male NMRI mice with four different carbonaceous nanomaterials dispersed in nanopure water: graphene oxide (18 μg/mouse/i.t.), Flammruss 101, Printex 90 and SRM1650b (0.1 mg/mouse/i.t. each) weekly for seven consecutive weeks. Pulmonary inflammation was determined by differential cell count in bronchoalveolar lavage fluid. Epididymal sperm concentration and motility were measured by computer-assisted sperm analysis. Epididymal sperm viability and morphological abnormalities were assessed manually using Hoechst 33,342/PI flourescent and Spermac staining, respectively. Epididymal sperm were assessed with regard to sperm DNA integrity (damage). Daily sperm production was measured in the testis, and testosterone levels were measured in blood plasma by ELISA. Results Neutrophil numbers in the bronchoalveolar fluid showed sustained inflammatory response in the nanoparticle-exposed groups one week after the last instillation. No significant changes in epididymal sperm parameters, daily sperm production or plasma testosterone levels were found. Conclusion Despite the sustained pulmonary inflammatory response, an eight week exposure to graphene oxide, Flammruss 101, Printex 90 and the diesel particle SRM1650b in the present study did not appear to affect semen parameters, daily sperm production or testosterone concentration in male NMRI mice.

Abstract Background Little is known about the mechanism underlying the genotoxicity observed in the liver following pulmonary exposure to carbon black (CB) nanoparticles (NPs). The genotoxicity could be caused by the presence of translocated particles or by circulating inflammatory mediators released during pulmonary inflammation and acute-phase response. To address this, we evaluated induction of pulmonary inflammation, pulmonary and hepatic acute-phase response and genotoxicity following exposure to titanium dioxide (TiO2), cerium oxide (CeO2) or CB NPs. Female C57BL/6 mice were exposed by intratracheal instillation, intravenous injection or oral gavage to a single dose of 162 μg NPs/mouse and terminated 1, 28 or 180 days post-exposure alongside vehicle control. Results Liver DNA damage assessed by the Comet Assay was observed after intravenous injection and intratracheal instillation of CB NPs but not after exposure to TiO2 or CeO2. Intratracheal exposure to NPs resulted in pulmonary inflammation in terms of increased neutrophils influx for all NPs 1 and 28 days post-exposure. Persistent pulmonary acute phase response was detected for all NPs at all three time points while only a transient induction of hepatic acute phase response was observed. All 3 materials were detected in the liver by enhanced darkfield microscopy up to 180 days post-exposure. In contrast to TiO2 and CeO2 NPs, CB NPs generated ROS in an acellular assay. Conclusions Our results suggest that the observed hepatic DNA damage following intravenous and intratracheal dosing with CB NPs was caused by the presence of translocated, ROS-generating, particles detected in the liver rather than by the secondary effects of pulmonary inflammation or hepatic acute phase response.

Abstract Background The accumulation of MWCNTs in the lung environment leads to inflammation and the development of disease similar to pulmonary fibrosis in rodents. Adverse Outcome Pathways (AOPs) are a framework for defining and organizing the key events that comprise the biological changes leading to undesirable events. A putative AOP has been developed describing MWCNT-induced pulmonary fibrosis; inflammation and the subsequent healing response induced by inflammatory mechanisms have been implicated in disease progression. The objective of the present study was to address a key data gap in this AOP: empirical data supporting the essentiality of pulmonary inflammation as a key event prior to fibrosis. Specifically, Interleukin-1 Receptor1 (IL-1R1) and Signal Transducer and Activator of Transcription 6 (STAT6) knock-out (KO) mice were employed to target inflammation and the subsequent healing response using MWCNTs as a model pro-fibrotic stressor to determine whether this altered the development of fibrosis. Results Wild type (WT) C57BL/6, IL-1R1 (KO) or STAT6 KO mice were exposed to a high dose of Mitsui-7 MWCNT by intratracheal administration. Inflammation was assessed 24 h and 28 days post MWCNT administration, and fibrotic lesion development was assessed 28 days post MWCNT administration. MWCNT-induced acute inflammation was suppressed in IL-1R1 KO mice at the 24 h time point relative to WT mice, but this suppression was not observed 28 days post exposure, and IL-1R1 KO did not alter fibrotic disease development. In contrast, STAT6 KO mice exhibited suppressed acute inflammation and attenuated fibrotic disease in response to MWCNT administration compared to STAT6 WT mice. Whole genome analysis of all post-exposure time points identified a subset of differentially expressed genes associated with fibrosis in both KO mice compared to WT mice. Conclusion The findings support the essentiality of STAT6-mediated signaling in the development of MWCNT-induced fibrotic disease. The IL-1R1 KO results also highlight the nature of the inflammatory response associated with MWCNT exposure, and indicate a system with multiple redundancies. These data add to the evidence supporting an existing AOP, and will be useful in designing screening strategies that could be used by regulatory agencies to distinguish between MWCNTs of varying toxicity.

Abstract Background Firefighters have increased risk of cardiovascular disease and of sudden death from coronary heart disease on duty while suppressing fires. This study investigated the effect of firefighting activities, using appropriate personal protective equipment (PPE), on biomarkers of cardiovascular effects in young conscripts training to become firefighters. Methods Healthy conscripts (n = 43) who participated in a rescue educational course for firefighting were enrolled in the study. The exposure period consisted of a three-day training course where the conscripts participated in various firefighting exercises in a constructed firehouse and flashover container. The subjects were instructed to extinguish fires of either wood or wood with electrical cords and mattresses. The exposure to particulate matter (PM) was assessed at various locations and personal exposure was assessed by portable PM samplers and urinary excretion of 1-hydroxypyrene. Cardiovascular measurements included microvascular function and heart rate variability (HRV). Results The subjects were primarily exposed to PM in bystander positions, whereas self-contained breathing apparatus effectively abolished pulmonary exposure. Firefighting training was associated with elevated urinary excretion of 1-hydroxypyrene (105%, 95% CI: 52; 157%), increased body temperature, decreased microvascular function (−18%, 95% CI: -26; −9%) and altered HRV. There was no difference in cardiovascular measurements for the two types of fires. Conclusion Observations from this fire extinction training show that PM exposure mainly occurs in situations where firefighters removed the self-contained breathing apparatus. Altered cardiovascular disease endpoints after the firefighting exercise period were most likely due to complex effects from PM exposure, physical exhaustion and increased core body temperature.

Abstract Epidemiological studies link inhalation of particles to increased risk of cardiovascular disease. Inhaled particles may induce cardiovascular disease by several different mechanisms including translocation of particles to systemic circulation, activation of airway sensory nerves resulting in autonomic imbalance and particle-induced pulmonary inflammation and acute phase response. The acute phase response is the systemic response to acute and chronic inflammatory states caused by for example bacterial infection, virus infection, trauma and infarction. It is characterized by differential expression of ca. 50 different acute phase proteins including C-reactive protein and Serum amyloid A, which are the most differentially up-regulated acute phase response proteins. Blood levels of these two acute phase proteins are closely associated with risk of cardiovascular disease in epidemiological studies and SAA has been causally related to the formation of plaques in the aorta in animal studies. In a recent paper in Particle and Fibre Toxicology, Christian Monsé et al. provide evidence that inhalation of ZnO nanoparticles induces dose-dependent acute phase response in humans at dose levels well below the current mass-based occupational exposure limits in a number of countries including Germany, The Netherlands, UK, Sweden, Denmark and the US. Given the evidence suggesting a causal relationship between increased levels of serum amyloid A and atherosclerosis, the current results call for a re-evaluation of occupational exposure limits for a number of particle exposures including ZnO taking induction of acute phase response into account. Furthermore, it underscores cardiovascular disease as an occupational disease.

An amendment to this paper has been published and can be accessed via the original article.

Previous genome-wide association studies have identified loci associated with the risk of multiple myeloma. Here, the authors present a meta-analysis of six genome wide association studies of the disease and identify eight new loci; functional studies identify genes as candidates for the basis of these associations.

Renewable diesel fuels have the potential to reduce net CO2 emissions, and simultaneously decrease particulate matter (PM) emissions. This study characterized engine-out PM emissions and PM-induced reactive oxygen species (ROS) formation potential. Emissions from a modern heavy-duty diesel engine without external aftertreatment devices, and fueled with petroleum diesel, hydrotreated vegetable oil (HVO) or rapeseed methyl ester (RME) biodiesel were studied. Exhaust gas recirculation (EGR) allowed us to probe the effect of air intake O2 concentration, and thereby combustion temperature, on emissions and ROS formation potential. An increasing level of EGR (decreasing O2 concentration) resulted in a general increase of equivalent black carbon (eBC) emissions and decrease of NOx emissions. At a medium level of EGR (13% intake O2), eBC emissions were reduced for HVO and RME by 30 and 54% respectively compared to petroleum diesel. In general, substantially lower emissions of polycyclic aromatic hydrocarbons (PAHs), including nitro and oxy-PAHs, were observed for RME compared to both HVO and diesel. At low-temperature combustion (LTC, O2 < 10%), CO and hydrocarbon gas emissions increased and an increased fraction of refractory organic carbon and PAHs were found in the particle phase. These altered soot properties have implications for the design of aftertreatment systems and diesel PM measurements with optical techniques. The ROS formation potential per mass of particles increased with increasing engine O2 concentration intake. We hypothesize that this is because soot surface properties evolve with the combustion temperature and become more active as the soot matures into refractory BC, and secondly as the soot surface becomes altered by surface oxidation. At 13% intake O2, the ROS-producing ability was high and of similar magnitude per mass for all fuels. When normalizing by energy output, the lowered emissions for the renewable fuels led to a reduced ROS formation potential.

Much of the current innovation in advanced materials is occurring at the nanoscale, specifically in manufactured nanomaterials (MNs). MNs display unique attributes and behaviors, and may be biologically and physically unique, making them valuable across a wide range of applications. However, as the number, diversity and complexity of MNs coming to market continue to grow, assessing their health and environmental risks with traditional animal testing approaches is too time- and cost-intensive to be practical, and is undesirable for ethical reasons. New approaches are needed that meet current requirements for regulatory risk assessment while reducing reliance on animal testing and enabling safer-by-design product development strategies to be implemented. The adverse outcome pathway (AOP) framework presents a sound model for the advancement of MN decision making. Yet, there are currently gaps in technical and policy aspects of AOPs that hinder the adoption and use for MN risk assessment and regulatory decision making. This review outlines the current status and next steps for the development and use of the AOP framework in decision making regarding the safety of MNs. Opportunities and challenges are identified concerning the advancement and adoption of AOPs as part of an integrated approach to testing and assessing (IATA) MNs, as are specific actions proposed to advance the development, use and acceptance of the AOP framework and associated testing strategies for MN risk assessment and decision making. The intention of this review is to reflect the views of a diversity of stakeholders including experts, researchers, policymakers, regulators, risk assessors and industry representatives on the current status, needs and requirements to facilitate the future use of AOPs in MN risk assessment. It incorporates the views and feedback of experts that participated in two workshops hosted as part of an Organization for Economic Cooperation and Development (OECD) Working Party on Manufactured Nanomaterials (WPMN) project titled, “Advancing AOP Development for Nanomaterial Risk Assessment and Categorization”, as well as input from several EU-funded nanosafety research consortia.

Recent findings show that cerium oxide (CeO2) nanoparticles may undergo in vivo-induced size transformation with the formation of smaller particles that could result in a higher translocation following pulmonary exposure compared to virtually insoluble particles, like titanium dioxide (TiO2). Therefore, we compared liver deposition of CeO2 and TiO2 nanoparticles of similar primary sizes 1, 28 or 180 days after intratracheal instillation of 162 μg of NPs in female C57BL/6 mice. Mice exposed to 162 μg CeO2 or TiO2 nanoparticles by intravenous injection or oral gavage were included as reference groups to assess the amount of NPs that reach the liver bypassing the lungs and the translocation of NPs from the gastrointestinal tract to the liver, respectively. Pulmonary deposited CeO2 nanoparticles were detected in the liver 28 and 180 days post-exposure and TiO2 nanoparticles 180 days post-exposure as determined by darkfield imaging and by the quantification of Ce and Ti mass concentration by inductively coupled plasma-mass spectrometry (ICP-MS). Ce and Ti concentrations increased over time and 180 days post-exposure the translocation to the liver was 2.87 ± 3.37% and 1.24 ± 1.98% of the initial pulmonary dose, respectively. Single particle ICP-MS showed that the size of CeO2 nanoparticles in both lung and liver tissue decreased over time. No nanoparticles were detected in the liver following oral gavage. Our results suggest that pulmonary deposited CeO2 and TiO2 nanoparticles translocate to the liver with similar calculated translocation rates despite their different chemical composition and shape. The observed particle size distributions of CeO2 nanoparticles indicate in vivo processing over time both in lung and liver. The fact that no particles were detected in the liver following oral exposure showed that direct translocation of nanoparticles from lung to the systemic circulation was the most important route of translocation for pulmonary deposited particles.

Psoriasis (PsO) is a chronic inflammatory disease with predominantly cutaneous manifestations. Approximately one third of patients with PsO develop psoriatic arthritis (PsA), whereas the remaining proportion of patients has isolated cutaneous psoriasis (PsC). These two phenotypes share common immunology, but with different heredity that might in part be explained by genetic variables.Using a candidate gene approach, we studied 53 single nucleotide polymorphisms (SNPs) in 37 genes that regulate inflammation. In total, we assessed 480 patients with PsO from DERMBIO, of whom 151 had PsC for 10 years or more (PsC10), 459 patients with PsA from DANBIO, and 795 healthy controls. Using logistic regression analysis, crude and adjusted for age and gender, we assessed associations between genetic variants and PsO, PsC10, and PsA, as well as associations between genetic variants and development of PsA in PsO.Eleven polymorphisms in 10 genes were nominally associated with PsO and/or PsC and/or PsA (P < 0.05). After correction for multiple testing with a false discovery rate of 5%, two SNPs remained significant: TNF (rs361525) was associated with PsO, PsC10, and PsA; and IL12B (rs6887695) was associated with PsO.Among a cohort of Danish patients with moderate-to-severe psoriasis, two SNPs in the IL12B and TNF genes were associated with susceptibility of psoriasis. None of the SNPs were specifically associated with isolated cutaneous psoriasis or psoriatic arthritis.

Red meat is a risk factor for colorectal cancer (CRC). We wanted to evaluate whether a functional polymorphism in the HMOX1 gene encoding heme oxygenase modifies risk of CRC or interacts with diet or lifestyle factors because this would identify heme or heme iron as a risk factor of CRC. The HMOX1 A-413T (rs2071746) was assessed in relation to risk of colorectal cancer (CRC) and interactions with diet (red meat, fish, fiber, cereals, fruit and vegetables) and lifestyle (use of non-steroidal anti-inflammatory drug and smoking status) were assessed in a case-cohort study of 928 CRC cases and a comparison group of 1726 randomly selected participants from a prospective study of 57,053 persons. No association between HMOX1 A-413T and CRC risk was found (TT vs. AA + TA; IRR = 1.15, 95% CI: 0.98–1.36, p = 0.10 for the adjusted estimate). No interactions were found between diet or lifestyle and HMOX1 A-413T. HMOX1 A-413T was not associated with CRC risk and no interactions with diet or lifestyle were identified in this large, prospective cohort with high meat intake. The results reproduced the previous findings from the same cohort and did not support a link between heme or heme iron and colorectal cancer. These results should be sought and replicated in other well-characterized cohorts with high meat intake.

We investigated toxicity of 2-3 layered >1 μm sized graphene oxide (GO) and reduced graphene oxide (rGO) in mice following single intratracheal exposure with respect to pulmonary inflammation, acute phase response (biomarker for risk of cardiovascular disease) and genotoxicity. In addition, we assessed exposure levels of particulate matter emitted during production of graphene in a clean room and in a normal industrial environment using chemical vapour deposition. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 μg/mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated. GO induced a strong acute inflammatory response together with a pulmonary (Serum-Amyloid A, Saa3) and hepatic (Saa1) acute phase response. rGO induced less acute, but a constant and prolonged inflammation up to day 90. Lung histopathology showed particle agglomerates at day 90 without signs of fibrosis. In addition, DNA damage in BAL cells was observed across time points and doses for both GO and rGO. In conclusion, pulmonary exposure to GO and rGO induced inflammation, acute phase response and genotoxicity but no fibrosis.

Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) has been linked to an increased risk of developing cardiovascular disease in addition to the well-documented physicochemical-dependent adverse lung effects. A proposed mechanism is through a strong and sustained pulmonary secretion of acute phase proteins to the blood. We identified physicochemical determinants of MWCNT-induced systemic acute phase response by analyzing effects of pulmonary exposure to 14 commercial, well-characterized MWCNTs in female C57BL/6J mice pulmonary exposed to 0, 6, 18 or 54 μg MWCNT/mouse. Plasma levels of acute phase response proteins serum amyloid A1/2 (SAA1/2) and SAA3 were determined on day 1, 28 or 92. Expression levels of hepatic Saa1 and pulmonary Saa3 mRNA levels were assessed to determine the origin of the acute phase response proteins. Pulmonary Saa3 mRNA expression levels were greater and lasted longer than hepatic Saa1 mRNA expression. Plasma SAA1/2 and SAA3 protein levels were related to time and physicochemical properties using adjusted, multiple regression analyses. SAA3 and SAA1/2 plasma protein levels were increased after exposure to almost all of the MWCNTs on day 1, whereas limited changes were observed on day 28 and 92. SAA1/2 and SAA3 protein levels did not correlate and only SAA3 protein levels correlated with neutrophil influx. The multiple regression analyses revealed a protective effect of MWCNT length on SAA1/2 protein level on day 1, such that a longer length resulted in lowered SAA1/2 plasma levels. Increased SAA3 protein levels were positively related to dose and content of Mn, Mg and Co on day 1, whereas oxidation and diameter of the MWCNTs were protective on day 28 and 92, respectively. The results of this study reveal very differently controlled pulmonary and hepatic acute phase responses after MWCNT exposure. As the responses were influenced by the physicochemical properties of the MWCNTs, this study provides the first step towards designing MWCNT that induce less SAA.

Although vitamin A is essential for gut immune cell trafficking (paramount for the intestinal immune system), epidemiological studies on the role of vitamin A in colorectal cancer (CRC) aetiology are conflicting. By using functional polymorphisms, gene−environment (GxE) interaction analyses may identify the biological effects (or “mechanism of action”) of environmental factors on CRC aetiology. Potential interactions between dietary or supplemental vitamin A intake and genetic variation in the vitamin A metabolic pathway genes related to risk of CRC were studied. We used a nested case-cohort design within the Danish “Diet, Cancer and Health” cohort, with prospectively collected lifestyle information from 57,053 participants, and the Cox proportional hazard models and likelihood ratio test. No statistically significant associations between the selected polymorphisms and CRC, and no statistically significant interactions between vitamin A intake and the polymorphisms were found. In conclusion, no support of an involvement of vitamin A in CRC aetiology was found.