Effects of Laser Printer–Emitted Engineered Nanoparticles on Cytotoxicity, Chemokine Expression, Reactive Oxygen Species, DNA Methylation, and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells, Macrophages, and Lymphoblasts

Background Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing. Objectives We assessed the biological responses of a panel of human cell lines to PEPs. Methods Three physiologically relevant cell lines—small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)—were exposed to PEPs at a wide range of doses (0.5–100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated. Results PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure. Conclusions The in vitro findings obtained in this study suggest that laser printer–emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders. Citation Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, Bello D, Kobzik L, Koturbash I, Demokritou P. 2016. Effects of laser printer–emitted engineered nanoparticles on cytotoxicity, chemokine expression, reactive oxygen species, DNA methylation, and DNA damage: a comprehensive in vitro analysis in human small airway epithelial cells, macrophages, and lymphoblasts. Environ Health Perspect 124:210–219; http://dx.doi.org/10.1289/ehp.1409582


Table of Contents
Supplemental Material, Part A Dosimetric considerations for in vitro testing -Example of calculations Table S1. Summary of parameters used in the in vivo lung Multiple Path Particle Deposition model (MPPD2). Table S2. Assays for determination of LINE-1 and Alu methylation. Table S3. Assays for determination of gene expression. Table S4. In vitro administered and delivered doses of SiO 2 and MS-WF. Figure S1. Hydrodynamic diameter as a function of DSE for PEPs (PM 0.1 ) and MS-WF. DSE cr : critical delivered sonication energy, energy required for minimal agglomeration. Figure S2. Fraction of administered dose deposited, f D , as a function of in vitro exposure time for PEPs (PM 0.1 ), SiO 2 and MS-WF calculated using the agglomeration diameter and estimated effective density. Plots are presented for the tested materials in the two media formulations (RPMI/10% FBS and SAGM). Figure S3. Deposition mass flux (left axis) and deposition fraction (right axis) as a function of airway generation number. Figure S4. Quantitative DNA damage assessment (Comet assay) of human lymphoblasts (TK6 cells) exposed for 4 hours to PEPs at various doses. All values are represented as mean ± SE.
References for Supplemental Material, Part A.

Dosimetric considerations for in vitro testing -Example of calculations
The following example shows the step-by-step calculations performed to arrive at the number of hours of PEPs inhalation that match the delivered-to-cell doses (e.g., 0.5 µg/ml) used for the two cell lines (SAECs, THP-1) in the study.
1. Choose the administered dose of interest used in the experiment to determine the corresponding inhalation exposure to PEPs.
For this example, we chose the administered dose of 0.5 µg/ml.

Converting the administered mass to delivered to cell dose as a function of the in vitro exposure time (t = 24 hrs) using the in vitro dosimetric methodology (Cohen et al. 2014):
The fraction of administered particle mass that is deposited on the cells in a standard 96-well plate as a function of in vitro exposure time (f D ) is calculated. For a 24-hour in vitro exposure, the f D was found to be 1.0 for particles suspended in SAGM (SAECs) and 0.518 for particles suspended in RPMI/10%FBS (THP-1). Therefore, the delivered to cell in vitro mass is as follows:

Calculate the mass delivered-to-cells per well surface area (µg/m 2 ).
Dose delivered-to-cells per area (µg/m 2 ) = Mass delivered-to-cells (µg) / Surface area of one well in a 96-well plate (m 2 )