This study is the first investigation of cytotoxicity of real-world nanoparticles emitted from photocopiers and the fifth in a series of articles on the properties and toxicology of such nanoparticles. In our first study, we described the physicochemical and morphological properties of these nanoparticles and established their complex chemistry as a mixture of metal oxides, and poorly-understood organic fraction . In the second, we showed that the nanoparticles caused a 2–10 fold increase in inflammatory cytokines, inflammatory cell influx, and total protein in nasal lavages, and oxidative stress marker 8-Oxo-2′-deoxyguanosine (8-OH-dG) in the urine of human volunteers after acute exposure to such nanoparticles in a photocopy center at modest exposure levels (average 34,000 particles/cm3) . Furthermore, these effects cleared slowly over the next 24–36 hrs post-exposure .
In another publication by the authors , size selective PM0.1 and PM0.1–2.5 size fractions from the same copy center were instilled intratracheally in Blab/c mice  in order to assess lung injury and inflammation of inhaled PM. Mice instilled with PM0.1 size fraction had significant increases in neutrophil number, lactate dehydrogenase and albumin compared to vehicle control. Likewise, several pro-inflammatory cytokines in the bronchoalveolar lavage fluid, including IL-1α, VEGF, and G-CSF, were also elevated in mice exposed to PM 0.1 compared to other groups. Comparatively, the nanoscale fraction was considerably more potent than the PM0.1–2.5 fraction, and comparable to the reference/comparator welding fumes, in inducing these effects .
Furthermore, in a companion paper of a similar design to the current in-vitro work, we found that several cytokines such as IL-6, IL-8, TNF-α and IL-1β were significantly elevated in THP-1 cells following dosing with the PM0.25–2.0 μm fraction . Only IL-8 was significantly elevated in the primary nasal and small airway cells. THP-1 cells also underwent apoptosis in a dose dependent manner. However, no significant differences were found in the extent of DNA damage at any time point . Similar to Pirella’s study in mice , the PM0.25–2.0 μm fraction in Khatri et al.  was significantly less potent in inducing a response in-vitro (e.g. inflammation and apoptosis) than the nanoscale fraction tested in this study.
Here we aimed at investigating cytotoxic properties of the nanoscale fraction of copier-emitted nanoparticles in three cell lines, relevant to respiratory exposures in humans: THP-1 cell as surrogated for human lung macrophages; primary human nasal epithelial cells, for upper airways; and primary human small airways epithelial cells for deeper airways. We purposefully focused on the production of inflammatory cytokines / chemokines by these cells due to their important mediatory roles in inflammation, insights into possible molecular mechanisms, and to match them with cytokines in nasal lavage of human volunteers. Inflammation, genotoxicity, and oxidative stress are frequently reported endpoints in several human [10–14, 17, 41] and in the limited in-vitro studies on printer-emitted nanoparticles [15, 16]. In addition to these end points, we also investigated apoptosis, DNA damage and supplemented further by gene expression data for key markers of inflammation, oxidative stress, genotoxicity, and apoptosis at sub-cytotoxic concentrations in THP-1 cells. DNA damage and aberrant immune responses have been frequently reported in photocopier operators, together with evidence of cellular apoptosis [11, 14]. Consistent with these previous results, we found that NPs emitted from photocopiers are capable of inducing the release of several pro-inflammatory cytokines, in-vitro and, at least in the case of monocytic cells, induce apoptosis in a time and dose-dependent fashion, but with modest acute cytotoxicity to the cells. In THP-1 cells, gene expression data at 5 μ/mL administered dose and 6 hr exposure duration, confirmed up-regulation of key genes controlling inflammation (TNF-α), apoptosis (p53, CASP8), and oxidative stress (HO1), further supporting the likely involvement of oxidative stress in these responses. These results provide important insights into the toxicological potential and the type of immune responses elicited by these nanoparticles.
Cytokine/Chemokine expression was (as one would expect) cell type dependent, with the monocytic THP-1 cell line being more sensitive to NP exposure than the two primary respiratory epithelial cells. The measured levels of secreted cytokines by the THP-1 cell type were about an order of magnitude higher than in the primary epithelial cells. The higher sensitivity of THP-1 cells may be partly attributed to the fact that we used PMA-differentiated THP-1 macrophages, which have greater ability to engulf particles through phagocytic mechanisms, thereby increasing cellular responses to NPs [42, 43]. The primary nasal and small airway epithelial cells in general secreted low cytokine levels (5 pg/mL-1 ng/mL range). Even for IL-8, which had the highest concentrations of 5 ng/mL were ~3 times lower than in THP-1 cells. IL-8 is a chemokine secreted by epithelial cells and is known to induce chemotaxis of inflammatory cells to sites of action and thus has a prominent role in the development of inflammation. This may due to the lack of simultaneous multiple cell type interactions in response to nanoparticles, typical of the living tissues in whole organisms . In addition, the reduced phagocytotic capacity of primary epithelial cells compared to macrophage-like cells, may result in smaller internalized doses in the epithelial cells, causing less activation of the downstream signaling pathways leading to cytokine release and less cytokine production [44, 45]. At the highest administered NP doses, interaction of selected cytokines (such as GM-CSF, VEGF and TNF-α) with NPs might have lead to significant cytokine losses and, as a consequence, underestimation of their true concentration, due to surface absorption by NP and possibly also due to induced conformational changes. Other researchers have also reported cytokine losses due to surface absorption by NPs in in-vitro conditions [29, 35].
The types of secreted cytokines in-vitro were similar to those found in vivo in the nasal lavage (NL) fluid of healthy subjects after acute (6 hrs) NP exposure in the photocopy center environment . Several cytokines, namely IL-6, IL-8 TNFα, IL-1β, G-CSF, EGF, IL-10, MCP-1, Fractalkine and VEGF, were elevated in vivo in the NL fluid of the participating volunteers. Several of these cytokines, specifically GM-CSF, IL-1β, IL-6, IL-8, MCP-1, TNF-α and VEGF, were also expressed in-vitro in the THP-1 cell line in the current study. Fewer of these, namely IL-8, TNF-α, EGF and IL-1α, were induced in both primary epithelial cell lines (summarized in Table 4). Of note, IFN-γ was overexpressed only in primary nasal epithelial cells. Another cytokine, namely G-CSF is also worth noting because it is overexpressed in human NL but not in-vitro. That might be due the reason that a complex activation machinery requiring multiple interactions in live tissue is required for the expression of these cytokines, which is absent in a single cell type . Overexpression of IL-1α in both primary cell lines in-vitro but not in NL may be a reflection of higher in-vitro doses. Taken together, the in-vitro cytokine/chemokine findings are consistent with our previous human volunteers study  and reinforce our earlier conclusion that copier-emitted nanoparticles are directly responsible for the induction of pro-inflammatory responses.
Most of the cytokines secreted in the present study play a major role in the development of inflammation, which is a gateway to many chronic diseases. Pro-inflammatory cytokines such as TNF-α, IL-1β and GM-CSF play important roles in amplification and maintenance of airway inflammation and are known to be key players in the clinical exacerbations and chronicity of bronchial asthma, an inflammatory disorder of the airways . Additionally, the production of chemokines such as IL-8 from alveolar macrophages and epithelial cells is thought to support neutrophil migration into the airspaces of the lung in idiopathic pulmonary fibrosis (IPF) and subsequent alveolar damage and tissue fibrosis [48–50]. VEGF plays important roles in angiogenesis (growth of new blood vessels) and increased vascular permeability . The chemokine MCP-1 is involved in the recruitment of monocytes to sites of injury and infection and elevated levels play a key role in the clinical course of interstitial lung disease [52, 53]. Therefore, the results from the present study confirm the ability of NPs from photocopiers to trigger inflammatory responses in humans, which upon chronic exposures may, in turn, lead to or intensify any preexisting respiratory condition such as asthma or chronic obstructive pulmonary disease (COPD).
It is important to acknowledge that direct quantitative comparisons between in vivo and in-vitro are difficult to make, because of dosimetry consideration and the simplicity of the single cell in-vitro models. As our human deposition flux models employed here indicate, the estimated in vivo dose to the lungs using the highest measured exposures to workers would correspond to 0.13 μg/mL in the in-vitro system. After correcting for the dose delivered to cells, the in-vitro delivered dose (3–30 μg/mL at 6 hrs and 6–60 μg/mL at 24 hrs) is over an order of magnitude higher than in vivo dose to the deep airway. The nasal cavity may receive much higher doses than the deep airway because its total surface area is disproportionately smaller (~150 cm2 for the nasal cavity vs. 120 m2 for the lungs) than the total NP deposition fractions (6% in nasal cavity vs. ~33% in the deep airways, Figure 3) and the in-vitro delivered NP doses are more comparable to in vivo upper airway doses. However, in vivo, the effective dose of nanoparticles reaching the epithelial cells is smaller than the deposited dose, partly due to the active clearance by macrophages and mucociliary transport, and party due to the barrier provided by the mucus layer, the lung surfactants and its antioxidants.
Copier-emitted nanoparticles also induced cell apoptosis in THP-1 cells, which was shown to occur in a dose- and time-dependent manner. The Annexin V staining results were confirmed by gene expression analysis at a much lower dose (5 μg/mL at 6 hrs), showing up-regulation of CASP8 and p53 but not CASP3, implicating the Fas pathway. At high NP concentrations, especially at 300 ng/mL, interference with Annexin V staining is apparent for both copier-emitted and CuO NPs. This is consistent with previous reports in which NPs have been shown to interact with PI , thereby over-representing the results in detection of necrotic cells, which might be the case with the present study, as we observe higher AnnexinV/PI positive cells at all concentration as compared to cell death results obtained using trypan blue staining. Furthermore, we were unsuccessful at studying apoptosis in primary nasal and small airway epithelial cells after NP treatment due to aberrant scattering properties of these cells in the flow cytometer. Poor internalization of NPs by the primary epithelial cells compared to THP-1, would favor attachment of large amounts of NPs to the outside cell membrane, which could not be removed with repeated cell washing, leading to interferences with the forward and side scatter light of the detection system [33, 55].
There are only few relevant studies on nanoparticles from photocopy equipment to compare our results with. Genotoxicity has been a particular focus on the debate of (nanoparticle and toner) emissions from printing and photocopy equipment and the subject of several past studies [16, 56, 57]. Gminski et al.  used DMSO extracts from toner powders to dope A549 lung cells and observed genotoxic effects . The chemical composition of the DMSO extracts was not characterized. More recently, Tang et al.  used an air-to-liquid system to dope cells with actual nanoparticles emitted from different printers. They reported that the printer emissions were found to cause genotoxicity, however no cytotoxicity was observed. These reports did not provide information about inflammatory properties caused by the printer emissions or the toner. These studies, point to the need to consider variability in the chemical composition of emissions across different toner formulations/manufacturers. In the present study, we did not observe any significant DNA damage based on comet assay in any of the three cell types for test nanoparticles originating from one manufacturer at all concentrations tested. However, high level up-regulation for DNA double strand break genes was observed in THP-1 cells at 6 h with 5 μg/mL, indicating DNA damage potential consistent with previous report [11, 12, 17]. One possible explanations for this discrepancy is that the extracts used in the past studies may differ in chemical composition from airborne fractions in significant ways, i.e. the organic fraction may be over-enriched with certain organic components, and lacks the inorganic component. Large variations in chemical composition of emitted nanoparticles between different manufacturers may exist, further contributing to discrepancies between different studies. As mentioned in the introduction, few human studies also reported genotoxic effects in blood cells and significant chromosomal aberrations in lymphocytes of copy center workers [11, 12]. Previously, we found increased levels of urinary 8-OH-dG, a marker of systemic oxidative DNA damage , in human volunteers following a single exposure. Gene expression in this study also confirmed oxidative damage in THP-1 cells, reflected in the up-regulation of HO1, and down-regulation of SOD1 and GPX1. The discrepancy between high oxidative stress in humans and lack of in-vitro genotoxicity may be due to aging of NP aerosols in the ambient environment or, perhaps 8-OH-dG reflected increased turnover of WBC in humans. Further investigations are needed to better understand such effects and the exact molecular pathway involved.
The strengths and limitations of the study should also be acknowledged. Among strengths, is that we performed extensive chemical characterization of collected NPs, a component completely absent from most of the previous reports. This information might be useful to predict which component of these chemically complex NPs is responsible for the observed in-vitro effects. In addition, the in-vitro toxicity endpoints were chosen to match those measured in humans, enabling direct comparisons between the two systems, as illustrated in Table 4. The use of three cell types, representing different parts of the respiratory system is also informative. Among the limitations, high in-vitro doses (especially above 100 ug/mL) was already discussed. One important limitation is that we did not measure directly the endotoxin content of collected NP (in part due to insufficient NP material), even though we addressed possible endotoxin interferences by treating NPs with Polymyxin B. A more thorough characterization of the free radical generation of copier-emitted NP in acellular and cellular systems, and what components of the NP chemistry (organic fraction, water soluble metals, and nanoparticles, aged vs. fresh nanoaerosols), could not be completed due to insufficient NP mass, and should be studied further. It is also important to extend the in-vitro and in vivo evaluations to nanoparticles from other toner formulations (a.k.a. other photocopier brands) because their physicochemical and toxicological properties may differ.