Exposure to coarse, fine, and ultrafine particles is associated with adverse population health impacts. We investigated whether size-fractionated particles collected repeatedly in the vicinity of industrial (steel mills and associated coking operations, wastewater treatment), high traffic, and residential areas display systematic differences in biological potency. Particulate matter (PM10) samples collected at sites within Windsor, Ontario, were screened for biological potency in human A549 lung epithelial and murine J774A.1 macrophage-like cells using cytotoxicity bioassays (cellular ATP, resazurin reduction, lactate dehydrogenase (LDH) release), cytokine production, and transcript profiles. Potency was determined from the slope of each dose-effect relationship. Cytotoxic potency varied across size fractions and within a fraction across sites and sampling periods, suggesting that particle composition, in addition to size and mass, affected particle toxicity. While ATP and LDH profiles showed some similarity, resazurin reduction (a measure of metabolic activity) exhibited a unique pattern of response, indicating that the cytotoxicity assays were sensitive to distinct particle characteristics. Chemical speciation varied in relation to prevailing winds, consistent with enrichment of source emissions (e.g. higher metal and polycyclic aromatic hydrocarbon content downwind of the industrial site). Notwithstanding this variability, site-dependent differences in particle toxicity were evident, including greater potency of coarse fractions at the industrial site and of ultrafine particles at the traffic site (Site × Size interactions, p < 0.05). Regression of potency against particle constituents revealed correlations between resazurin reduction, induction of metal-responsive genes, and metal content, which were particularly strong for the coarse fraction, and between cytokine release and endotoxin, suggesting that these factors were important drivers of biological effects that explain, at least in part, the contrasting potencies of particles compared on an equivalent mass basis. The data show that 1) particle potency and composition can exhibit significant temporal variation in relation to source contributions; 2) sources may differentially impact the potency of specific size fractions; and 3) particle constituents, notably metals and endotoxin, may elicit distinct biological responses. Together, the data are consistent with the notion that sources and composition, in addition to size and mass concentration, are relevant to particle toxicity.

The likelihood of environmental and health impacts of silicon dioxide nanoparticles (SiNPs) has risen, due to their increased use in products and applications. The biological potency of a set of similarly-sized amorphous SiNPs was investigated in a variety of cells to examine the influence of physico-chemical and biological factors on their toxicity. Cellular LDH and ATP, BrdU incorporation, resazurin reduction and cytokine release were measured in human epithelial A549, human THP-1 and mouse J774A.1 macrophage cells exposed for 24 h to suspensions of 5–15, 10–20 and 12 nm SiNPs and reference particles. The SiNPs were characterized in dry state and in suspension to determine their physico-chemical properties. The dose-response data were simplified into particle potency estimates to facilitate the comparison of multiple endpoints of biological effects in cells. Mouse macrophages were the most sensitive to SiNP exposures. Cytotoxicity of the individual cell lines was correlated while the cytokine responses differed, supported by cell type-specific differences in inflammation-associated pathways. SiNP (12 nm), the most cytotoxic and inflammogenic nanoparticle had the highest surface acidity, dry-state agglomerate size, the lowest trace metal and organics content, the smallest surface area and agglomerate size in suspension. Particle surface acidity appeared to be the most significant determinant of the overall biological activity of this set of nanoparticles. Combined with the nanoparticle characterization, integration of the biological potency estimates enabled a comprehensive determination of the cellular reactivity of the SiNPs. The approach shows promise as a useful tool for first-tier screening of SiNP toxicity.

Nanoforms of mesoporous silica (mSiNPs) are increasingly applied in medicine, imaging, energy storage, catalysis, biosensors and bioremediation. The impact of their physicochemical properties on health and the environment remain to be elucidated. In this work, newly synthesized mesoporous silica (sizes: 25, 70, 100, 170 and 600 nm; surface functionalization: pristine, C3- and C11-COOH moieties) were assessed for cytotoxicity and induction of inflammatory responses in vitro (A549, THP-1, J774A.1 cells). All toxicity endpoints were integrated to obtain simple descriptors of biological potencies of these mSiNPs. The findings indicate that mSiNPs are less bioactive than the nonporous reference SiNP used in this study. The C3-COOH modified mSiNPs were generally less cytotoxic than their pristine and C11-modified counterparts in the nano-range (≤100 nm). Carboxyl-modified mSiNPs affected inflammatory marker release across all sizes with cell type specificity, suggesting a potential for immunomodulatory effects. Surface area, size, extent of agglomeration, zeta potential and surface modification appeared to be important determinants of cytotoxicity of mSiNPs based on association tests. Pathway analysis identified particle and cell type-specific alteration of cellular pathways and functions by mSiNPs. The integration of exposure-related biological responses in multiple cell lines to mSINPs allowed for a comprehensive evaluation of the impact of physicochemical factors on their toxicity characteristics. The integrated multi-level toxicity assessment approach can be valuable as a hazard screening tool for safety evaluations of emerging nanomaterials for regulatory purpose.

Industrial sources contribute a significant proportion of anthropogenic particulate matter (PM) emissions, producing particles of varying composition that may differentially impact health. This study investigated the in vitro toxicity of ambient PM collected near industrial sites in relation to particle size and composition. Size-fractionated particles (ultrafine, PM0.1–2.5, PM2.5–10, PM>10) were collected in the vicinity of steel, copper, aluminium, and petrochemical industrial sites. Human lung epithelial-like A549 and murine macrophage-like J774A.1 cells were exposed for 24 h to particle suspensions (0, 30, 100, 300 μg/cm2). Particle potency was assessed using cytotoxic (resazurin reduction, lactate dehydrogenase (LDH) release) and inflammatory (cytokine release) assays, and regressed against composition (metals, polycyclic aromatic hydrocarbons (PAHs), endotoxin). Coarse (PM2.5–10, PM>10) particle fractions were composed primarily of iron and aluminium; in contrast, ultrafine and fine (PM0.1–2.5) fractions displayed considerable variability in metal composition (especially water-soluble metals) across collection sites consistent with source contributions. Semi-volatile and PM-associated PAHs were enriched in the fine and coarse fractions collected near metal industry. Cell responses to exposure at equivalent mass concentrations displayed striking differences among sites (SITE x SIZE and SITE x DOSE interactions, p < 0.05), suggesting that particle composition, in addition to size, impacted particle toxicity. While both J774A.1 and A549 cells exhibited clear particle size-dependent effects, site-dependent differences were more pronounced in J774A.1 cells, suggesting greater sensitivity to particle composition. Plotting particle potency according to cytotoxic and inflammatory response grouped particles by size and site, and showed that particles of similar composition tended to cluster together. Cytotoxic effects in J774A.1 cells correlated with metal and PAH content, while inflammatory responses were associated primarily with endotoxin content in coarse particles. Industrial sources produce particulate emissions with varying chemical composition that differ in their in vitro potency in relation to particle size and the levels of specific constituents.

In vitro cytotoxicity assays are essential tools in the screening of engineered nanomaterials (NM) for cellular toxicity. The resazurin live cell assay is widely used because it is non-destructive and is well suited for high-throughput platforms. However, NMs, in particular carbon nanotubes (CNT) can interfere in assays through quenching of transmitted light or fluorescence. We show that using the resazurin assay with time-point reading of clarified supernatants resolves this problem. Human lung epithelial (A549) and murine macrophage (J774A.1) cell lines were exposed to NMs in 96-well plates in 200 μL of media/well. After 24 h incubation, 100 μL of supernatant was removed, replaced with resazurin reagent in culture media and aliquots at 10 min and 120 min were transferred to black-wall 96-well plates. The plates were quick-spun to sediment the residual CNTs and fluorescence was top-read (λEx=540 nm, λEm=600 nm). The procedure was validated for CNTs as well as silica nanoparticles (SiNP). There was no indication of reduction of resazurin by the CNTs. Stability of resorufin, the fluorescent product of the resazurin reduction was then assessed. We found that polar CNTs could decrease the fluorescence signal for resorufin, possibly through oxidation to resazurin or hyper-reduction to hydroxyresorufin. This effect can be easily quantified for elimination of the bias. We recommend that careful consideration must be given to fluorimetric/colorimetric in vitro toxicological assessments of optically/chemically active NMs in order to relieve any potential artifacts due to the NMs themselves.

Growing interest in non-animal-based models has led to the development of devices to expose cells to airborne substances. Cells/tissues grown at the air-liquid interface (ALI) are more representati...