Multiple Approaches to Determine Toxicity of Micro and Nano-sized Titanium Dioxide Materials When Exposed to Human Red Blood Cells

Introduction: The utility of engineered nanomaterials’ is growing, particularly the titanium dioxide (TiO2) polymorphs. TiO2 is very useful for brightening paints, and coloring foods. Nano-sized TiO2 is also useful for sunscreens, cosmetics, and can be utilized as a photocatalyst. However, the nanometer size and the large specific surface area of the TiO2 materials are physicochemical characteristics which may contribute to human red blood cell (RBC) damage. Using RBCs as a cellular model, we have evaluated the effects of TiO2 nanoparticle exposure to RBCs by quantifying oxidized glutathione, oxidized membrane vitamin E, hemolysis, hemoglobin adsorption, and cellular aggregation. Results: Red blood cells are rich in the antioxidant glutathione (GSH). HPLC testing revealed that some TiO2 materials have the ability to cause oxidation of GSH to the oxidized form, glutathione disulfide (GSSG). Due to surface area characteristics, some TiO2 materials have the ability to adsorb protein (visualized as hemoglobin) to their surface. Additionally, some TiO2 materials microscopically form red blood cell aggregates, significantly changing the red cell morphology. The aggregation data was quantified using a hemacytometer. Red blood cell membrane vitamin E was also measured by HPLC, and after exposure to these TiO2 polymorphs, some materials caused vitamin E membrane oxidation. Some TiO2 materials have the ability, through multiple different mechanisms, to cause hemolysis of the red blood cell. Conclusions: Our results indicated that some of the TiO2 polymorphs assayed contributed to red blood cell hemolysis via different mechanisms, whereas some polymorphs did not cause cellular damage. These data indicated that red blood cells can ultimately be hemolyzed by biological oxidative damage (BOD), intracellular oxidation of GSH to GSSG, oxidation of vitamin E in the RBC membrane, material adsorption to the RBC membrane, physical contact, or by a combination of these mechanisms

SCREENING FOR OXIDATIVE STRESS ELICITED BY ENGINEERED NANOMATERIALS: EVALUATION OF ACELLULAR DCFH ASSAY

The DCFH assay is commonly used for measuring free radicals generated by engineered nanomaterials (ENM), a well-established mechanism of ENM toxicity. Concerns exist over susceptibility of the DCFH assay to: assay conditions, adsorption of DCFH onto ENM, fluorescence quenching and light scattering. These effects vary in magnitude depending on ENM physiochemical properties and concentration. A rigorous evaluation of this method is still lacking. The objective was to evaluate performance of the DCFH assay for measuring ENM-induced free radicals. A series of diverse and well-characterized ENM were tested in the acellular DCFH assay. We investigated the effect of sonication conditions, dispersion media, ENM concentration, and the use of horseradish peroxidase (HRP) on the DCFH results. The acellular DCFH assay suffers from high background signals resulting from dye auto-oxidation and lacks sensitivity and robustness. DCFH oxidation is further enhanced by HRP. The number of positive ENM in the assay and their relative ranking changed as a function of experimental conditions. An inverse dose relationship was observed for several Carbon-based ENM. Overall, these findings indicate the importance of having standardized assays for evaluating ENM toxicity and highlights limitations of the DCFH assay for measuring ENM-induced free radicals

Haemolytic activity of soil from areas of varying podoconiosis endemicity in Ethiopia

Background: Podoconiosis, non-filarial elephantiasis, is a non-infectious disease found in tropical regions such as Ethiopia, localized in highland areas with volcanic soils cultivated by barefoot subsistence farmers. It is thought that soil particles can pass through the soles of the feet and taken up by the lymphatic system, leading to the characteristic chronic oedema of the lower legs that becomes disfiguring and disabling over time. Methods: The close association of the disease with volcanic soils led us to investigate the characteristics of soil samples in an endemic area in Ethiopia to identify the potential causal constituents. We used the in vitro haemolysis assay and compared haemolytic activity (HA) with soil samples collected in a non-endemic region of the same area in Ethiopia. We included soil samples that had been previously characterized, in addition we present other data describing the characteristics of the soil and include pure phase mineral standards as comparisons. Results: The bulk chemical composition of the soils were statistically significantly different between the podoconiosis-endemic and non-endemic areas, with the exception of CaO and Cr. Likewise, the soil mineralogy was statistically significant for iron oxide, feldspars, mica and chlorite. Smectite and kaolinite clays were widely present and elicited a strong HA, as did quartz, in comparison to other mineral phases tested, although no strong difference was found in HA between soils from the two areas. The relationship was further investigated with principle component analysis (PCA), which showed that a combination of an increase in Y, Zr and Al2O3, and a concurrent increase Fe2O3, TiO2, MnO and Ba in the soils increased HA. Conclusion: The mineralogy and chemistry of the soils influenced the HA, although the interplay between the components is complex. Further research should consider the variable biopersistance, hygroscopicity and hardness of the minerals and further characterize the nano-scale particles

A living cell quartz crystal microbalance biosensor for continuous monitoring of cytotoxic responses of macrophages to single-walled carbon nanotubes

<p>Abstract</p> <p>Background</p> <p>Numerous engineered nanomaterials (ENMs) exist and new ENMs are being developed. A challenge to nanotoxicology and environmental health and safety is evaluating toxicity of ENMs before they become widely utilized. Cellular assays remain the predominant test platform yet these methods are limited by using discrete time endpoints and reliance on organic dyes, vulnerable to interference from ENMs. Label-free, continuous, rapid response systems with biologically meaningful endpoints are needed. We have developed a device to detect and monitor in real time responses of living cells to ENMs. The device, a living cell quartz crystal microbalance biosensor (QCMB), uses macrophages adherent to a quartz crystal. The communal response of macrophages to treatments is monitored continuously as changes in crystal oscillation frequency (Δf). We report the ability of this QCMB to distinguish benign from toxic exposures and reveal unique kinetic information about cellular responses to varying doses of single-walled carbon nanotubes (SWCNTs).</p> <p>Results</p> <p>We analyzed macrophage responses to additions of Zymosan A, polystyrene beads (PBs) (benign substances) or SWCNT (3-150 μg/ml) in the QCMB over 18 hrs. In parallel, toxicity was monitored over 24/48 hrs using conventional viability assays and histological stains to detect apoptosis. In the QCMB, a stable unchanging oscillation frequency occurred when cells alone, Zymosan A alone, PBs alone or SWCNTs without cells at the highest dose alone were used. With living cells in the QCMB, when Zymosan A, PBs or SWCNTs were added, a significant decrease in frequency occurred from 1-6 hrs. For SWCNTs, this Δf was dose-dependent. From 6-18 hrs, benign substances or low dose SWCNT (3-30 μg/ml) treatments showed a reversal of the decrease of oscillation frequency, returning to or exceeding pre-treatment levels. Cell recovery was confirmed in conventional assays. The lag time to see the Δf reversal in QCMB plots was linearly SWCNT-dose dependent. Lastly, the frequency never reversed at high dose SWCNT (100-150 μg/ml), and apoptosis/necrosis was documented in conventional 24 and 48 hr-assays.</p> <p>Conclusion</p> <p>These data suggest that the new QCMB detects and provides unique information about peak, sub-lethal and toxic exposures of living cells to ENMs before they are detected using conventional cell assays.</p

Evaluation of cytotoxic, genotoxic and inflammatory responses of nanoparticles from photocopiers in three human cell lines

Background: Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines. Methods: Three cell types were used: THP-1, primary human nasal- and small airway epithelial cells. Following collection in a large volume photocopy center, nanoparticles were extracted, dispersed and characterized in the cell culture medium. Cells were doped at 30, 100 and 300 μg/mL administered doses for up to 24 hrs. Estimated dose delivered to cells, was ~10% and 22% of the administered dose at 6 and 24 hrs, respectively. Gene expression analysis of key biomarkers was performed using real time quantitative PCR (RT-qPCR) in THP-1 cells at 5 μg nanoparticles/mL for 6-hr exposure for confirmation purposes. Results: Multiple cytokines, GM-CSF, IL-1β, IL-6, IL-8, IFNγ, MCP-1, TNF-α and VEGF, were significantly elevated in THP-1 cells in a dose-dependent manner. Gene expression analysis confirmed up-regulation of the TNF-α gene in THP-1 cells, consistent with cytokine findings. In both primary epithelial cells, cytokines IL-8, VEGF, EGF, IL-1α, TNF-α, IL-6 and GM-CSF were significantly elevated. Apoptosis was induced in all cell lines in a dose-dependent manner, consistent with the significant up-regulation of key apoptosis-regulating genes P53 and Casp8 in THP-1 cells. No significant DNA damage was found at any concentration with the comet assay. Up-regulation of key DNA damage and repair genes, Ku70 and Rad51, were also observed in THP-1 cells, albeit not statistically significant. Significant up-regulation of the key gene HO1 for oxidative stress, implicates oxidative stress induced by nanoparticles. Conclusions: Copier-emitted nanoparticles induced the release of pro-inflammatory cytokines, apoptosis and modest cytotoxicity but no DNA damage in all three-human cell lines. Taken together with gene expression data in THP-1 cells, we conclude that these nanoparticles are directly responsible for inflammation observed in human volunteers. Further toxicological evaluations of these nanoparticles, including across different toner formulations, are warranted

Biomarkers in long COVID-19: A systematic review

PurposeLong COVID, also known as post-acute sequelae of COVID-19, refers to the constellation of long-term symptoms experienced by people suffering persistent symptoms for one or more months after SARS-CoV-2 infection. Blood biomarkers can be altered in long COVID patients; however, biomarkers associated with long COVID symptoms and their roles in disease progression remain undetermined. This study aims to systematically evaluate blood biomarkers that may act as indicators or therapeutic targets for long COVID.MethodsA systematic literature review in PubMed, Embase, and CINAHL was performed on 18 August 2022. The search keywords long COVID-19 symptoms and biomarkers were used to filter out the eligible studies, which were then carefully evaluated.ResultsIdentified from 28 studies and representing six biological classifications, 113 biomarkers were significantly associated with long COVID: (1) Cytokine/Chemokine (38, 33.6%); (2) Biochemical markers (24, 21.2%); (3) Vascular markers (20, 17.7%); (4) Neurological markers (6, 5.3%); (5) Acute phase protein (5, 4.4%); and (6) Others (20, 17.7%). Compared with healthy control or recovered patients without long COVID symptoms, 79 biomarkers were increased, 29 were decreased, and 5 required further determination in the long COVID patients. Of these, up-regulated Interleukin 6, C-reactive protein, and tumor necrosis factor alpha might serve as the potential diagnostic biomarkers for long COVID. Moreover, long COVID patients with neurological symptoms exhibited higher levels of neurofilament light chain and glial fibrillary acidic protein whereas those with pulmonary symptoms exhibited a higher level of transforming growth factor beta.ConclusionLong COVID patients present elevated inflammatory biomarkers after initial infection. Our study found significant associations between specific biomarkers and long COVID symptoms. Further investigations are warranted to identify a core set of blood biomarkers that can be used to diagnose and manage long COVID patients in clinical practice

Urinary biomonitoring of occupational exposures to Bisphenol A Diglycidyl Ether (BADGE) – based epoxy resins among construction painters in metal structure coating

Epoxy resin systems based on Bisphenol A Diglycidyl Ether (BADGE) monomer and its higher oligomers are important commercial formulations used widely in construction for protective coating of steel structures, such as bridges. The literature on occupational exposures and biomonitoring of BADGE-based epoxies among construction painters is remarkably limited. In this first occupational biomonitoring study of epoxies, 44 construction painters performing mid- and top-coating were recruited from 12 metal structure coating sites in New England. Cross-shift changes in the urinary levels of total BADGE and its three major hydrolysis derivatives - BADGE·2H2O, BADGE·H2O, BADGE·HCl·H2O – were assessed. Results for 81 urine samples collected from coating workers were compared with 28 urine samples of a reference group of 14 spray polyurethane foam (SPF) insulation workers.The highest concentrations of all biomarkers were found in the urine samples of mid-coat applicators. The major urinary biomarker of BADGE in this cohort of workers, BADGE·2H2O, was detected in 100% of urine samples. The post-shift BADGE·2H2O (specific gravity normalized data) in mid-coat applicators had a geometric mean (GM) of 1.46 ng/mL and a geometric standard deviation (GSD) of 3.6 (range, 0.2–18.7 ng/mL). The second most abundant biomarker in urine, BADGE·HCl·H2O, was measured in 84% of samples, and had a post-shift GM(GSD) of 0.17 (2.3) ng/mL (range, <0.025–0.59 ng/mL). BADGE·2H2O was 8.6 times more abundant than BADGE·HCl·H2O. BADGE·H2O was quantified only in 10% of the samples (range, 0.11–0.41 ng/mL). Free BADGE in post-shift urine, corrected for background, had GM (GSD) of 0.04 (2.5) ng/mL (range, <0.025–0.16 ng/mL). Urinary BADGE·2H2O were significantly higher (p = 0.01) in mid-coat applicators compared to top-coat and SPF workers. Post-shift urinary BADGE·2H2O in mid-coat applicators increased by ~2.9× (p = 0.02) and 1.36× in top-coat applicators (p = 0.18) compared to pre-shift values, but not in SPF workers (0.95×; p = 0.40).In conclusion, we demonstrate that (i) significant BADGE uptake occurs via inhalation and skin exposures during application of epoxy-containing paintings (mid-coat), suggesting the need for improvements in hygiene practices and personal protective measures; (ii) BADGE·2H2O is a robust and sensitive biomarker for biomonitoring of exposures to BADGE-based epoxies in occupational settings; and (iii) widespread occurrence of BADGE and BADGE·2H2O in the urine of all workers, including SPF workers, suggest common exposures from non-occupational sources, such as ingestion or do-it-yourself consumer applications of epoxy resins. In light of this observation, establishing a reliable biological monitoring guidance value (BMGV) for BADGE·2H2O will require more background biomonitoring and health effect data. An initial reference value for BADGE·2H2O of 0.5 ng/mL (SG-normalized) or 180 nmol/mol creatinine is being proposed as the threshold to discriminate occupational from non-occupational exposures based on the maximum values observed in the reference SPF group