Anoop K. Pal

Nanoparticles from photocopiers induce oxidative stress and upper respiratory tract inflammation in healthy volunteers

Abstract Photocopiers emit large quantities of nanoparticles (NPs); however, their toxicological properties have not been studied. Here we investigate for the first time early human responses following a days exposure to NPs from photocopiers. Nine healthy subjects spent 6 h at a busy photocopy centre on 2–3 randomly selected days. Matched nasal lavage and urine samples were collected before and at different time points post-exposure. Nasal lavage samples were analysed for 14 cytokines, inflammatory cells and total protein. Urine samples were analysed for 8-hydroxydeoxyguanosine (8-OH-dG). Exposure assessment was conducted using a suite of instruments. The mean total particle number on exposure days was >5 times higher than background, with size distributions in nanoscale range (peak 30–40 nm). Following exposure, 8-OH-dG and several pro-inflammatory cytokines were elevated 2–10 folds compared with pre-exposure levels and remained elevated for up to 36 h. We conclude that NPs from photocopiers induce upper airway inflammation and oxidative stress.

Advanced computational modeling for in vitro nanomaterial dosimetry.

BackgroundAccurate and meaningful dose metrics are a basic requirement for in vitro screening to assess potential health risks of engineered nanomaterials (ENMs). Correctly and consistently quantifying what cells “see,” during an in vitro exposure requires standardized preparation of stable ENM suspensions, accurate characterizatoin of agglomerate sizes and effective densities, and predictive modeling of mass transport. Earlier transport models provided a marked improvement over administered concentration or total mass, but included assumptions that could produce sizable inaccuracies, most notably that all particles at the bottom of the well are adsorbed or taken up by cells, which would drive transport downward, resulting in overestimation of deposition.MethodsHere we present development, validation and results of two robust computational transport models. Both three-dimensional computational fluid dynamics (CFD) and a newly-developed one-dimensional Distorted Grid (DG) model were used to estimate delivered dose metrics for industry-relevant metal oxide ENMs suspended in culture media. Both models allow simultaneous modeling of full size distributions for polydisperse ENM suspensions, and provide deposition metrics as well as concentration metrics over the extent of the well. The DG model also emulates the biokinetics at the particle-cell interface using a Langmuir isotherm, governed by a user-defined dissociation constant, KD, and allows modeling of ENM dissolution over time.ResultsDose metrics predicted by the two models were in remarkably close agreement. The DG model was also validated by quantitative analysis of flash-frozen, cryosectioned columns of ENM suspensions. Results of simulations based on agglomerate size distributions differed substantially from those obtained using mean sizes. The effect of cellular adsorption on delivered dose was negligible for KD values consistent with non-specific binding (> 1 nM), whereas smaller values (≤ 1 nM) typical of specific high-affinity binding resulted in faster and eventual complete deposition of material.ConclusionsThe advanced models presented provide practical and robust tools for obtaining accurate dose metrics and concentration profiles across the well, for high-throughput screening of ENMs. The DG model allows rapid modeling that accommodates polydispersity, dissolution, and adsorption. Result of adsorption studies suggest that a reflective lower boundary condition is appropriate for modeling most in vitro ENM exposures.

Implications of in vitro dosimetry on toxicological ranking of low aspect ratio engineered nanomaterials

Abstract In vitro high throughput screening platforms based on mechanistic injury pathways are been used for hazard assessment of engineered nanomaterials (ENM). Toxicity screening and other in vitro nanotoxicology assessment efforts in essence compare and rank nanomaterials relative to each other. We hypothesize that this ranking of ENM is susceptible to dispersion and dosimetry protocols, which continue to be poorly standardized. Our objective was to quantitate the impact of dosimetry on toxicity ranking of ENM. A set of eight well-characterized and diverse low aspect ratio ENMs, were utilized. The recently developed in vitro dosimetry platform at Harvard, which includes preparation of fairly monodispersed suspensions, measurement of the effective density of formed agglomerates in culture media and fate and transport modeling was used for calculating the effective dose delivered to cells as a function of time. Changes in the dose–response relationships between the administered and delivered dose were investigated with two representative endpoints, cell viability and IL-8 production, in the human monocytic THP-1 cells. The slopes of administered/delivered dose–response relationships changed 1:4.94 times and were ENM-dependent. The overall relative ranking of ENM intrinsic toxicity also changed considerably, matching notably better the in vivo inflammation data (R2 = 0.97 versus 0.64). This standardized dispersion and dosimetry methodology presented here is generalizable to low aspect ratio ENMs. Our findings further reinforce the need to reanalyze and reinterpret in vitro ENM hazard ranking data published in the nanotoxicology literature in the light of dispersion and dosimetry considerations (or lack thereof) and to adopt these protocols in future in vitro nanotoxicology testing.

High Resolution Characterization of Engineered Nanomaterial Dispersions in Complex Media Using Tunable Resistive Pulse Sensing Technology

In vitro toxicity assessment of engineered nanomaterials (ENM), the most common testing platform for ENM, requires prior ENM dispersion, stabilization, and characterization in cell culture media. Dispersion inefficiencies and active aggregation of particles often result in polydisperse and multimodal particle size distributions. Accurate characterization of important properties of such polydisperse distributions (size distribution, effective density, charge, mobility, aggregation kinetics, etc.) is critical for understanding differences in the effective dose delivered to cells as a function of time and dispersion conditions, as well as for nano–bio interactions. Here we have investigated the utility of tunable nanopore resistive pulse sensing (TRPS) technology for characterization of four industry relevant ENMs (oxidized single-walled carbon nanohorns, carbon black, cerium oxide and nickel nanoparticles) in cell culture media containing serum. Harvard dispersion and dosimetry platform was used for preparing ENM dispersions and estimating delivered dose to cells based on dispersion characterization input from dynamic light scattering (DLS) and TRPS. The slopes of cell death vs administered and delivered ENM dose were then derived and compared. We investigated the impact of serum protein content, ENM concentration, and cell medium on the size distributions. The TRPS technology offers higher resolution and sensitivity compared to DLS and unique insights into ENM size distribution and concentration, as well as particle behavior and morphology in complex media. The in vitro dose–response slopes changed significantly for certain nanomaterials when delivered dose to cells was taken into consideration, highlighting the importance of accurate dispersion and dosimetry in in vitro nanotoxicology.

Biological oxidative damage by carbon nanotubes: Fingerprint or footprint?

Abstract Carbon nanotubes (CNTs) have received much attention for performance and toxicity, but vary substantially in terms of impurity type and content, morphology, and surface activity. This study determined the decrease of antioxidant capacity, defined as biological oxidative damage (BOD), of CNTs-exposed serum. The variability in several physicochemical properties of CNTs and their links to BOD elicited in human serum were explored. Tremendous variation in transition metal type and content (104-fold), specific surface area (SSA, nine-fold), and BOD were observed. Mass specific BOD (mBOD) varied from 0.006–0.187 μmol TEU mg−1, whereas surface area specific BOD (sBOD) varied from 0.068–0.42 μmol TEU m−2. The sBOD increased in a stepwise fashion from ∼0.1–0.32 μmol TEU m−2 for tubes with outer diameter less than 10 nm. The mBOD and sBOD may be useful denominators of surface activity and impurity content and assist in designing safer CNTs.

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.

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

BackgroundNumerous 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).ResultsWe 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.ConclusionThese 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.

Understanding and correcting for carbon nanotube interferences with a commercial LDH cytotoxicity assay.

The lactate dehydrogenase (LDH) assay accurately quantifies cytotoxicity of chemicals via the measurement of LDH released from damaged cells. In the assay, LDH catalyzes formation of a reporter chromophore that can be quantified spectrophotometrically at its 490 nm peak, a standard assay, and related to the released LDH concentration. However, certain engineered nanomaterials have been reported to produce aberrant values, resulting in inaccurate assessment of toxicity as measured by LDH levels in media. We studied this effect spectroscopically by measuring unexpected changes in the complete visible spectrum of the product chromophore resulting from using either purified LDH or LDH from lysed cells in the presence of varying concentrations of single walled carbon nanotubes (SWCNTs) or carbon nanohorns (SWCNH-oxs). Basically, at constant LDH concentrations, the 490 nm product peak decreased with increasing carbon nanotube concentration, while the 580 nm peak increased to a lesser extent and the maximum absorbing wavelength increased. The product chromophore spectrum was altered in different ways by potential interactions with a number of components in the reaction mixture including: BSA, LDH, SWCNTs, SWCNT-oxs, or various combinations of these species. We propose to improve the accuracy of the LDH assay when evaluated in the presence of varying concentrations of these carbon nanostructures by use of both the 490 and 580 nm peak absorbances combined via regression analysis. Our results indicate that molecular probes of cytotoxicity must be assessed individually for accuracy in the presence of engineered nanomaterials.

Buoyant Nanoparticles: Implications for Nano-Biointeractions in Cellular Studies

In the safety and efficacy assessment of novel nanomaterials, the role of nanoparticle (NP) kinetics in in vitro studies is often ignored although it has significant implications in dosimetry, hazard ranking, and nanomedicine efficacy. It is demonstrated here that certain nanoparticles are buoyant due to low effective densities of their formed agglomerates in culture media, which alters particle transport and deposition, dose-response relationships, and underestimates toxicity and bioactivity. To investigate this phenomenon, this study determines the size distribution, effective density, and assesses fate and transport for a test buoyant NP (polypropylene). To enable accurate dose-response assessment, an inverted 96-well cell culture platform is developed in which adherent cells are incubated above the buoyant particle suspension. The effect of buoyancy is assessed by comparing dose-toxicity responses in human macrophages after 24 h incubation in conventional and inverted culture systems. In the conventional culture system, no adverse effects are observed at any NP concentration tested (up to 250 μg mL(-1) ), whereas dose-dependent decreases in viability and increases in reactive oxygen species are observed in the inverted system. This work sheds light on an unknown issue that plays a significant role in vitro hazard screening and proposes a standardized methodology for buoyant NP assessments.

Development of Therapeutic Polymeric Nanoparticles for the Resolution of Inflammation

Liver X receptors (LXRs) attenuate inflammation by modulating the expression of key inflammatory genes, making LXRs and their ligands particularly attractive candidates for therapeutic intervention in cardiovascular, metabolic, and/or inflammatory diseases. Herein, enhanced proresolving activity of polymeric nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (LXR-NPs) is demonstrated, developed from a combinatorial library of more than 70 formulations with variations in critical physicochemical parameters. In vitro studies on peritoneal macrophages confirm that LXR-NPs are significantly more effective than the free agonist at downregulating pro-inflammatory mediators (MCP-1 and TNFα), as well as inducing the expression of LXR target genes (ABCA1 and SREBP1c). Through a zymosan-induced acute peritonitis in vivo model, LXR-NPs are found to be more efficient than free GW3965 at limiting the recruitment of polymononuclear neutrophils (50% vs 17%), suppressing the gene expression and secretion of pro-inflammatory factors MCP-1 and TNFα in peritoneal macrophages, and decreasing the resolution interval up to 4 h. Furthermore, LXR-NPs suppress the secretion of MCP-1 and TNFα by monocytes and macrophages more efficiently than the commercial drug dexamethasone. Overall, these findings demonstrate that LXR-NPs are capable of promoting resolution of inflammation and highlight the prospect of LXR-based nanotherapeutics for inflammatory diseases.