Exposure to sub-10 nm particles emitted from a biodiesel-fueled diesel engine: In vitro toxicity and inflammatory potential

Accepted Manuscript Title: Exposure to sub-10 nm particles emitted from a biodiesel-fueled diesel engine: In vitro toxicity and inflammatory potential Authors: Malorni Livia, Guida Vincenzo, Mariano Sicignano, Genovese Giuliana, Petrarca Claudia, Pedata Paola PII: DOI: Reference:

S0378-4274(17)30057-7 http://dx.doi.org/doi:10.1016/j.toxlet.2017.02.009 TOXLET 9697

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

11-8-2015 1-2-2017 7-2-2017

Please cite this article as: Livia, Malorni, Vincenzo, Guida, Sicignano, Mariano, Giuliana, Genovese, Claudia, Petrarca, Paola, Pedata, Exposure to sub-10nm particles emitted from a biodiesel-fueled diesel engine: In vitro toxicity and inflammatory potential.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2017.02.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Exposure to sub-10nm particles emitted from a biodiesel-fueled diesel engine: in vitro toxicity and inflammatory potential

Corresponding Author: Pedata Paola, Seconda Università degli Studi di Napoli, Dipartimento di Medicina Sperimentale- Sezione di Igiene, Medicina del Lavoro e Medicina Legale, Via Santa Maria di Costantinopoli ,16- 80138 Napoli, Campania, IT tel: 0039 081 5665902 fax: 0039 081 5665898 e-mail: [email protected]

Authors: Malorni Livia1, Guida Vincenzo2, Mariano Sicignano3, Genovese Giuliana2, Petrarca Claudia4, Pedata Paola2 1

Consiglio

Nazionale

delle

Ricerche,

Istituto

di

Scienze

dell’Alimentazione-

via Roma 64, 83100 Avellino, IT tel: 0039 0825 299208 fax: 0039 0825 781585 e-mail: [email protected] 2

Seconda Università degli Studi di Napoli, Dipartimento di Medicina Sperimentale- Sezione di

Igiene, Medicina del Lavoro e Medicina Legale Via Santa Maria di Costantinopoli ,16- 80138 Napoli, Campania, IT tel: 0039 081 5665902 fax: 0039 081 5665898 e-mail: [email protected]; [email protected]. 3

Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale – DICMAPI.

Università degli Studi di Napoli Federico II 4

Fondazione Università “G. D'Annunzio”- Center of Ageing Science, Unit of Allergy and

Immunotoxicology, via dei Vestini, 31- 66100 Chieti, Abruzzo, IT tel: 0039 0871 541290 fax: 0039 0871 3554061 e-mail: [email protected]

HIGHLIGHTS  Toxicological studies have widely demonstrated that engine-generated ultrafine particles are one of the main insult of air pollution on human health.  Among soot particles, sub-10nm particles, mostly constituted by organic carbon, have the peculiarity to contain a large number of aromatic molecules constituting their surface.  We have separated sub-10nm particles from larger sizes particulate matter and we have analyzed their effects on proliferation, apoptosis and secretion of cytokines, chemokines and growth factors networks production in immortalized non-tumorigenic human dermal keratinocyte cell line (HaCaT) and human alveolar epithelial-like cells (A549).  Sub-10nm particles exert different cytotoxic effects in the two cell lines, suggesting that the dermal way of exposure is more sensitive than the inhalator way.  The data open new perspective to investigate the role of nanometric combustion particles on inflammation up to cell transformation.

Abstract Objectives: The inflammatory effects of organic sub-10nm particles generated and emitted from a diesel engine fueled with a biodiesel and a commercial diesel oil are analyzed in this paper. Diesel combustion is the major sources of ultrafine particles (UFP) in the environment, particularly in urbanized areas. In the last years, there is an increasing use of biomass-derived fuels because they are a renewable source of energy that may mitigate climate change through the reduction of net CO2 with respect to conventional fossil fuels. Although there is a general agreement on biofuels ability to reduce conventional pollutants, new and potentially harmful pollutants can be formed during biofuel combustion. In particular, the emission of sub-10nm particles is strongly increased with respect to that of larger soot particles. Methods: Organic sub-10nm particles are separated from larger sizes particulate matter by collection in water suspension for toxicological and inflammatory tests. After exposure to sub10nm particles, the effects on proliferation, apoptosis and secretion of cytokines, chemokines and growth factors networks production is analyzed in immortalized non-tumorigenic human dermal keratinocyte cell line (HaCaT) and human alveolar epithelial-like cells (A549). Results and Conclusion: Nanoparticles exert different cytotoxic effects in the two cell lines, suggesting that the dermal way of exposure is more sensitive than the inhalant way. These differences are most evident in the secretion of pro-inflammatory, angiogenic and proliferative

cytokines and chemokines whose expression is more finely modulated in HaCaT cells compared to A-549 cells. Considering the size of these particles, it is important to promote the culture of prevention also for the dermal way in particularly exposed workers.

Keywords: biodiesel; diesel; nanoparticles; health effects; cytokines.

Introduction Motor vehicle exhaust emissions are ubiquitous, with exposure occurring through indoor and outdoor air, as well as in many occupational settings. Engine exhausts derive from diesel, gasoline and other fuels combustion. They comprise a complex mixture including gases such as carbon monoxide and nitrogen oxides, volatile organic compounds such as benzene and formaldehyde and polycyclic aromatic hydrocarbons (PAHs) and particulates including elemental and organic carbon, metals, sulfates and ash. The composition and quantity of the different components of engine exhausts are dependent on many factors including the type of engine and fuel used, the condition of the engine, presence of emission controlling devices, and the engine operating conditions. It has been estimated that about 3 million workers (2% of the workforce) were exposed to diesel (D) exhaust in the member states of the European Union in 1990–1993. [1] The International Agency for Research on Cancer (IARC) classified D exhaust as carcinogenic to humans (Group 1), based on studies of lung cancer; [2] however, the evidence for other cancers remains limited. Recently, Kachuri et al. reported that the high-level exposure D emissions may increase the risk of rectal cancer. [3] Another study suggests a positive exposure-response relation between occupational exposure to D exhaust and ovarian cancer but do not support previous findings suggesting an association between engine exhausts and risk of esophageal, testicular, kidney, or bladder cancers, or that of leukemia. [4] During the last years, biodiesel oil (BD) have attracted attention as a renewable and environmentally friendly fuel [5,6] the main reason being the mitigation of climate change through the reduction of net CO2 with respect to conventional fossil fuels. BD is a mixture of fatty acid alkyl esters produced from vegetable oils and animal fats via transesterification with an alcohol. The combinations of fatty acids in fats and oils can vary, substantially depending on the source material and influence the resulting BD properties. Although there is a general agreement on biofuels ability to reduce conventional pollutants, their extensive use is limited by the lack of knowledge about some important aspects regarding their combustion and the formation of new and potentially harmful pollutants. [7] It is well known that the use of BD reduces mass concentration of emitted particulate matter, but this reduction is associated with the formation of sub-10nm particles, a huge number of nanometre-sized particles, eventually containing oxygen atoms. [8] Consequently, it is not yet clear if BD combustion-generated particles produce fewer adverse health effects than those of

particulate matter generated by combustion of fossil fuels. In particular, attention should be focused on the ultrafine fraction of the emitted particulate matter, the sub-10nm particles, which are usually collected in the Soluble Organic Fraction (SOF) at the engine exhaust. [8] Relative to D emissions, BD emissions have been shown to contain less mass concentration of particulate matter (PM), unburned hydrocarbons (HC), CO, and PAHs [9-11]. Chen and Wu have found that BD had no impact on mean particles diameter but caused a 24-42% reduction in total mass of submicron particles. [12] Others authors have demonstrated that pure biofuels reduced particles size, number and volume in the accumulation mode, which includes most of the particles mass. [13] Instead, Tsolakis investigated the impact of pure biofuels on emitted particles size and distribution for rapeseed derived BD using an electrical low-pressure impactor (ELPI). He observed a reduction in total particles mass, but an increase in particles number concentration for diameter ≤0.091µm [14]. Brown et al. observed significant reduction in particles mass, no change in average particles diameter, but an increase in particles number. [15] It is important to note that despite the reduction in the total mass of PM, the soluble fraction of the emitted PM is commonly a greater percentage for BD exhaust emissions. It showed a 30% decrease in PM emissions with use of 100% BD, but the soluble fraction increased by roughly 40%. The soluble fraction present in diesel exhaust particles has been associated with both generation of an oxidative stress and magnitude of the cytokine response in a mouse macrophage cell line (RAW264.7). [16-18] Currently, the major part of scientific studies are aimed to the regulated emissions of pollutants (NOx, CO, PAH, particulate), and show both positive and negative effects compared to conventional D. Few data are available for pollutants which are unregulated, present in trace amounts and of particular concern to health. Knowledge of formation/destruction mechanisms of these compounds and their role in the formation of PM is very limited. This implies that their effect on the toxicity is not yet known, too. The toxicological effects of BD need to be characterized in greater detail, especially when a variety biofuels are already being introduced into wider use in heavy-duty diesel engines such as those installed in buses and trucks in cities. [19, 20] Several recent studies have indicated that exposure to BD produces tissue damage, oxidative stress, inflammatory responses, activation of cellular signalling pathways and cytokine response. [11, 21-23] Bünger et al. have evaluated the cytotoxic effects of BD exhaust emissions proving that the cytotoxic effects of BD from rapeseed methyl ester (RME) emissions were greater than those of a diesel fuel. Exhaust from rapeseed BD was 4-fold more potent than petroleum D exhaust in inducing cytotoxicity (measured as the median effective

dose; ED50). [24] Other studies have compared the acute toxicity exerted by BD and D emissions using mutagenicity assays, while the emissions of BD are less mutagenic than D with high sulphur content fuel. [25] Recent studies demonstrated that BD is more mutagenic when compared to ultralow sulphur diesel fuel (ULSD). [26, 27] BD exhaust have effects similar to those observed for D, as shown in animal exposure study. Finch et al. have exposed F344 rats to emissions from a diesel engine burning 100% soybean oil-derived fuel. Biologically significant, exposure-related effects were limited to the lung and were observed primarily at the highest exposure level. There was a dose-related increase in the numbers of alveolar macrophages and the numbers of particles in the macrophages, as expected from repeated exposure. [28] A recent study was published directly comparing effects of inhalation exposure to D and BD in pure form (B100) from soybean ethyl esters (SEE) in Balb/c mice. Compared to D, B100 caused a significantly increased heart rate variability, but no changes in heart rate or blood pressure. MCV and platelets were elevated possibly indicating an enhanced thrombogenic potency of the B100 exhaust. All other blood parameters remained unchanged or gave inconclusive results. [29] This study is devoted to the characterization of the effects of ultrafine fraction of the emitted PM, the sub-10nm particles collected in the soluble organic fraction, on proliferation, apoptosis and alteration expression of cytokines, chemokines and growth factors networks production in human cell lines derived from keratinocytes (HaCaT) and alveolar epithelial like cells (A549). Lung and skin are potential routes for sub-10nm particles occupational and environmental exposure to nanomaterials and serves as one of the principal portals of entry for nanoparticles. [30-33] Therefore, HaCaT and A-549 cells were chosen as in vitro model cells to compare the effects of dermal and inhalation exposure to sub-10nm particles generated by BD and D oils.

2. Materials and methods

2.1 Engine, nanoparticles collection and characterization Nanoparticles used in this study were collected at the exhaust of a three-cylinder, two valves diesel engine with 1028 cm3 of displacement. The engine is installed at the ‘Istituto Motori’ of Consiglio Nazionale delle Ricerche (CNR), it is compact in size and of high performance: maximum torque and maximum power are available at low rotational speeds. The engine is equipped with an electronically controlled common rail injection system. The engine meets Euro 4 exhaust emission regulation without a Diesel particulate filter. It is equipped with two emission reduction systems: a cooled exhaust gas recirculation for NOx reduction and a

catalyst for CO and HC oxidation. Fatty acid methyl esters (FAME), consisting of straight saturated and unsaturated hydrocarbon chain and glycerin, blended with a commercial diesel oil (20% v/v) is used as fuel. The chemical composition of the BD is different from petroleum fuel, having less carbon and more oxygen. The different chemical structure and higher molecular weight result in higher density and viscosity of the BD with respect to D fuel. The blend has a higher cetane number and consequently, at equal conditions, its ignition delay time is shorter compared to that one of D fuel. A smoke meter was used to determine the mass concentration of particulate matter. PM emission for the BD blend, 40 mg/m3, is about 1.5 times less than diesel fuel, about 60 mg/m3. Additional measurements on PM were performed in order to count particles as a function of their sizes by using an engine exhaust particles sizer. These measurements confirmed the reduction of PM emission and showed the increase of the particles number for the BD with respect to the D fuel and a decrease of the mean particles diameter. [34] Sub-10nm particles were collected for toxicological experiments by cooling and condensation of combustion water and bubbling of the sampled exhaust in bi-distilled water. Sub-10nm particles generated by BD and D oils have been isolated from the larger soot particles by allowing them to interact with water; indeed, soot particles are essentially hydrophobic and are not trapped in water whereas organic carbon sub-10nm particles shows a good affinity with water and remain suspended. [35] In previous experiments performed in laboratory flames, we have shown that organic carbon nanoparticles have a good affinity with water and hence they can be removed from the exhausts with an efficiency of about 50% by this simple sampling procedure. [36] Furthermore, organic carbon nanoparticles are isolated from the larger soot particles enabling the characterization of the health effect of the sub-10nm fraction of the emitted particulate matter. [37] Tests were performed at 2000 rpm and full load. A warm up procedure was performed to stabilize the engine before the measurements. All volatile components of the sampled species collected in water were removed by rotary evaporation under mild pressure at ambient temperature in order to avoid evaporation and degradation of nanoparticles. The water sample containing sub-10nm particles was divided in to two parts: the first one was used for nanoparticles characterization, the other for in vitro experiments. UV-visible absorption and fluorescence measurements were performed to characterize the material sampled in water. Absorption measurement showed that nanoparticles have a strong absorption in the UV at about 210 nm, which decreased to negligible values moving towards

the visible range, at about 350 nm. This spectral behavior is typical of aromatic compounds with few fused rings, although gas-phase aromatics have been removed from the sample by rotary evaporation. UV-induced fluorescence confirmed the presence of small aromatic structures characterized by a fluorescence spectrum that extends from 300 to 450 nm. Atomic Force Microscopy, electro-spray differential mobility and dynamic light scattering measurements performed on the water sample have shown the presence of sub-10nm particles (particles size ranges from 1 to 3nm) in the sample, suggesting that the species responsible for UV absorption and UV-induced fluorescence are clusters of aromatic-aliphatic linked molecules with a loose non-planar structure held together by van der Waals interactions. [38] The mass concentration of nanoparticles collected from the engine was determined by UV absorption measurements considering the absorption cross section of organic nanoparticles formed in laboratory flames. [35] Total mass was confirmed by total organic carbon concentration measurement. Different concentrations of nanoparticles were prepared by simply diluting the sample with sterile bi-distilled water to obtain1.2, 4, 8 and 12 ppm solutions.

2.2 Cell culture HaCaT cell line. The immortalized human keratinocyte cell line (HaCaT) was grown in Dulbecco’s Modified Eagle Medium (DMEM) including 2 mM L-glutamine (Lonza) that was supplemented with 100 U/mL penicillin (Lonza) and 100 mg/mL of streptomycin (Lonza) and 10% heat-inactivated fetal bovine serum (Gibco) in a humidified atmosphere (95% air/ 5% CO2) at 37°C. Cells were seeded at a density of 105 cells in 75 cm2 flasks for cell culture expansion. The medium was changed every 48 hours. Specific culturing conditions were applied for testing, which are described in the next paragraphs. A-549 cell line. A human alveolar epithelial-like cell line representing the alveolar type II phenotype was grown in Dulbecco’s Modified Eagle Medium (DMEM) including 2 mM Lglutamine (Lonza) that was supplemented with 100 U/mL penicillin (Lonza) and 100 mg/mL of streptomycin (Lonza) and 10% heat-inactivated fetal bovine serum (Gibco) in a humidified atmosphere (95% air/ 5% CO2) at 37°C. Cells were seeded at a density of 105 cells in 75 cm2 flasks for cell culture expansion. The medium was changed every 48 hours. Specific culturing conditions were applied for testing, which are described in the next paragraphs.

2.3 Cell viability assay For viability/growth inhibition assay, HaCaT and A-549 cells were seeded in 96 well plate at a density of 2x104 cells/cm2. After 24 h, culture media were replaced with media containing sub-

10nm particles produced by D and BD at concentration of 1.2, 4, 8 and 12 ppm. Cells were exposed to sub-10nm nanoparticles for 24 and 48h. Cell viability was evaluated with crystal violet staining, according to the procedure described by Kueng, [39] based on the binding of the dye to proteins and DNA of viable cells that remain attached to the culture plate (while the dead ones lose their adherence). In detail, cells were washed with PBS 1X and fixed by adding 50 µL of a 10% formalin solution. After 15 min, cells were washed with deionized water and stained with 50 µL of 0.1% crystal violet solution in water for 30 min. Excess dye was removed by washing with deionized water and plates were air-dried and analyzed using optical microscope (Leica). Then, the dye was solubilized in 50 µL of 10% acetic acid. The optical density of the solubilized dye, which correlates with the number of viable cells, was measured at 595 nm using a microplate reader (DAS, Italy). All experiments were performed in triplicate and repeated at least 3 times.

2.4 Annexin V-FITC/propidium iodide (PI) apoptosis assay The quantification of apoptosis induced by particles in HaCaT and A-549 cells was measured by flow cytometry (FCM, Becton Dickinson, USA) with Annexin V-FITC/ PI double staining. In viable cells, phosphatidylserine is found mainly in the inner leaflet of the plasma membrane. FITC-labeled annexin was used to determine cell at apoptosis because its capacity to bind with phosphatidylserine exposed on the surface of apoptotic cells. Additional cell staining with propidium iodide (PI) allows to detect necrotic cells. Briefly, cells were harvested after 24 and 48 h of exposure to sub-10nm particles from D and BD (1.2 ppm, 4 ppm and 8 ppm), washed twice with cold PBS (0.15 M, pH = 7.2) and resuspended to 1 × 106 cells/mL in binding buffer. Then, 100 μL of cells suspension was transferred to a 5 mL culture tube, and 5 μL of FITC-conjugated Annexin V (Annexin V-FITC) and 5 μL PI were added at room temperature in the dark. After incubation for 15 min, stained cells were diluted by the same binding buffer and directly analyzed by fluorescence-activated cell sorting (FACS, FACSCalibur, BD Biosciences, USA). At least 10,000 cells were collected and detected by flow cytometry, and the percentages of apoptotic cells were analyzed by FACS Diva 4.1 software.

2.5

Apoptosis Assay with Bio-plex platform

Two distinct signaling pathways, called extrinsic and intrinsic, induce Apoptosis. The extrinsic pathway is triggered by signaling through death receptors at the cell surface such as Fas, followed by downstream activation of Caspase 8 and Caspase 3. The intrinsic pathway is

triggered by cytotoxic stress which leads to translocation of Bcl2 family proteins, Bax and Bak, to the mitochondrial membrane. Oligomerization of Bax and Bak causes release of Cytochrome c into the cytosol, which promotes apoptosome formation, caspase activation, degradation of nuclear Lamin B and cell death. The Bio-Plex Pro RBM apoptosis multiplex assay (Bio-Rad, Lab Inc., Hercules, CA, USA) (panel 3) can detect, by fluorimetric means, a set of four intracellular proteins (Active caspase 3, Bcl-xL/Bak dimer, Mcl-1/Bak dimer, Survivin) involved in the commitment, onset and induction of apoptosis by the intrinsic pathway. In particular: -Active Caspase 3 - cleaves and activates other caspases and is a primary regulator of apoptotic-associated proteolysis. -Bcl-xL/Bak dimer – Anti apoptotic Bcl-xL heterodimerizes with Bak at the mitochondrial outer membrane and inhibits permeability of the mitochondrial membrane, preventing release of cytochrome C. -Mcl-1/Bak – Anti-apoptotic Mcl-1 heterodimerizes with Bax and Bak at the mitochondrial outer membrane to prevent their activation, thus inhibiting cytochrome C release from mitochondria. -Survivin is a member of the Inhibitor of Apoptosis (IAP) family, which also plays a role in chromosome segregation and cytokinesis. The assay is built on magnetic capture beads and detection antibodies to enable robust quantification of these proteins in cell lysates. Cells were seeded at 2 x 104 cells/cm2 in 10 cm Petri dishes and after 24 h were treated with 1.2, 4 and 8 ppm sub-10nm particles from D and BD, for 24h. Total cell extract were prepared using Lysate Dilution Buffer and immediately stored at -80°C. The assay was run according to the manufacturer's instructions and the fluorescence intensities were measured using a Bio-Plex 200 system (Bio-Rad). The detected fluorescence values in each sample were normalized to the corresponding total protein content (measured by BioRad assay) and data were analyzed with Bio-Plex Manager software v5.0 (BioRad).

2.6

Cytokine/chemokine detection with Bio-plex platform

A multiplex biometric immunoassay performed with the Bio-plex platform (Bio-Rad Lab Inc., Hercules, CA, USA), was used to evaluate the concentrations of various soluble cytokine/chemokine in cell culture supernatants. HaCaT and A-549 cells were seeded in 6 well plate at a density of 2x10 4 cells/cm2 in DMEM 10% FCS. After 24 h, cells were washed twice with PBS and culture media were replaced with

DMEM 0% FCS (in order to avoid bovine serum interference with assay) containing sub-10nm particles produced by D and BD at concentration of 1.2 and 4 ppm. Cells were exposed to sub10nm nanoparticles for 24 h. After that time, 1 mL medium was sampled and immediately frozen and stored at -70 °C until the assay was performed. Detection of pro- and anti-inflammatory cytokines released into the culture medium was carried out with the Bio-Plex Pro Human Cytokine 27-Plex Panel (Bio-Rad) for the detection of: interleukin (IL)-1rα, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, Eotaxin, Granulocyte-colony stimulating factor (G-CSF), Granulocyte macrophage colony stimulating factor (GM-CSF), Interferon (IFN)-γ, IFN-γ inducible protein 10 (IP-10), Monocyte chemo attractant protein 1 (MCP-1), Macrophage inflammatory protein 1-alpha/beta (MIP-1α, MIP-1β), RANTES, Tumor necrosis factor alpha (TNF-α), Platelet-derived growth factor (PDGF-bb), Vascular endothelial growth factor (VEGF) and basic Fibroblast growth factor (bFGF). Data were acquired using a Bio-Plex 200 system equipped with Bio-Plex Manager software v5.0 (BioRad). All washing steps were performed on the Bio-Plex magnetic wash station (BioRad). Measurements were performed on single spent medium sample diluted (1:2) according to the manufacturer’s protocol. A sample of DMEM alone was also run to account for background levels of secreted proteins. The standard curves optimization and the calculation of analyte concentrations were performed by using the Bio-Plex Manager software. Statistical significance was ascribed to the results through use of a Student’s t-test and no thresholds on fold changes. All results with P < 0.05 were considered significant. Raw data, expressed as pg of cytokine/mL of conditioned medium (mean ± SD), with N= 3 for each cell line, particles concentration and time combination, were transformed in percentage quantities relative to control.

2.7

Statistical analysis

The data shown were mean values of at least three independent experiments and expressed as mean±SD. Statistical analysis was performed by Student’s t-test, using statistical software SPSS 10.0.

3. Results 3.1

Effects of sub-10nm particles on cell viability

Results of Cristal Violet assay showed a significant increase of cell mortality in both cells lines treated with sub-10nm particles produced by D and BD at different concentrations (1.2 ppm;

4.0 ppm; 8ppm; 12 ppm) for 24 and 48 hours. In Figure 1 are reported the results of cell viability assay performed on HaCaT and A-549 cells treated with sub-10nm particles from D and BD exhaust which clearly show that treatment at lower concentration (1.2 ppm) does not affect cell viability instead of higher concentrations. In both cell lines results demonstrated that cell number decrease in a concentration-dependent manner, with some differences in the two cell lines and depending on the origin of the sub10nm particles, with a clear evidence that the sub-10nm particles from BD induced less cytotoxicity compared to D ones, on both cell models used. In detail, in the HaCaT cells at concentration of 4 ppm, the sub-10nm particles from D caused a decrease of living cells of about 60% after 24 hours and of about 75% after 48 h of exposure, while the BD sub-10nm particles, at the same times, caused a decrease of 10% and 40%, respectively. These differences are canceled out with the raise of concentration. In the A549 cells, instead, no significant effect on the number of viable cells was observed at the concentration of 4.0 ppm of sub-10nm particles from BD. At the same concentration, sub10nm particles from D induced a decrease of cell number of about 25% after 24h and 35% after 48 h of exposure. Sub-10nm particles from D push on their cytotoxic effect also at concentration of 8 and 12 ppm. The effects on cell viability of sub-10nm particles from BD at 8 and 12 ppm is lower. It is interesting to note that in A-549 cells after 24h of treatment both with 8 ppm sub-10nm particles from D and BD about 50% of cells are still alive. Briefly, at 8 and 12 pmm concentration of sub-10nm particles from D and BD, HaCaT cells are almost totally absent, as clearly reported in Figure 1, where are reported cells colored in plate with Cristal Violet assay. Based on these results, the range concentration 1.2 - 8 ppm was chosen to elucidate possible mechanism(s) of cytotoxicity in experiments subsequently reported.

3.2

Annexin V-FITC/propidium iodide (PI) apoptosis assay

HaCaT and A549 cells were exposed at different concentrations of sub-10nm particles from D and BD for 24 hours and the rate of viable, apoptotic and necrotic cells were analyzed quantitatively by flow cytometry. In Figure 2, results indicate that there is a progressive increase of apoptotic activity as concentration of sub 10nm particles increase and, according to viability cell data, this effect is most evident in HaCaT cells exposed to 8 ppm concentration of sub-10nm particles, both from D and BD.

In fact, about 60% of HaCaT cells treated with 8 ppm of sub-10nm particles both from D and BD are apoptotic. The same effect of the progressive increase of apoptotic activity with concentration of sub 10nm particles is evident in A549 cells at 8 ppm albeit with a minor number of apoptotic cells compared to HaCaT cells (about 30% of apoptotic cells). These results were in accordance with the corresponding data on viability given by the Cristal Violet assay.

3.3

Apoptosis Assay with Bio-plex platform

To further investigate about which signaling pathway (extrinsic or intrinsic) induce apoptosis in HaCaT and A549 cell lines, protein expression of Active Caspase-3, Bcl-xL/Bak dimer, Mcl1/Bak dimer and Survivin were investigated, using the Bio-Plex X-Map technology. Results are reported in Figure 3. HaCaT cells treated with 8 ppm sub-10nm particles from D up regulate both the Active Caspase-3 and the Bcl-xL/Bak dimer, Mcl-1/Bak and Survivin protein levels are not influenced by sub -10 nm particles treatment. In A549 cells there’s no evidence of significative change in protein expression of the four markers investigated. In particular, levels of Active Caspase -3 comparable in control and treated sample are in agreement with data obtained with Annexin V-FITC/propidium iodide apoptosis assay.

3.4

Cytokine/chemokine release profile in sub-10nm particles treated cells

In order to investigate the pro-and anti-inflammatory responses in HaCaT and A549 cells exposed to sub-10nm particles from D and BD a multiplex immunoassay using the Bio-Plex Pro Human Cytokine 27-Plex Panel was performed. The expression profile of the cytokine panel for both cell lines treated with 1.2 and 4.0 ppm sub-10nm particles from D and BD are summarized in Figure 4. Data were normalized and reported as percentage quantities relative to control. Concentrations 1.2 ppm and 4 ppm were chosen to this experiment because at these concentrations, after 24 h of treatment, the percentage of apoptotic cells do not exceed 10%. As reported in Figure 4 in HaCaT cells treatment with 1,2 ppm sub-10nm particles from D caused down regulation of several factors, such as: G-CSF, MIP1a, IL-17A, PDGF-bb, MCP-1, MIP1b, IFNγ, and IL-1ra, while only four factors are up regulated: IL-6, IP-10, VEGF, IL-8. Raising of concentration up to 4 ppm of sub- 10nm particles from D induced a higher increase

of IL-6, VEGF and IL-8. Moreover, at this concentration resulted up regulated also the factors G-CSF, IFNγ, TNFα, β-FGF, IL-1b. On the other side, treatment with 1.2 ppm sub-10nm particles from BD resulted in down regulation of MIP1a, IL-17A, PDGF-bb, MIP1b, IL-1ra, Eotaxin, TNFα, GM-CSF, IL-2 and RANTES. RANTES factor resulted down regulated also at concentration of 4 ppm of sub10nm particles from BD, together with IP-10, IL-15, IL-5 and MCP-1. Only three factors are up regulated after treatment with sub-10nm particles from BD: IL-6 after treatment with 1.2 and 4 ppm; G-CSF and IL-1b after treatment with 4 ppm. The expression panel of the 27 cytokine/chemokine reported in Figure 5 shows that in A549 cells treatment with sub-10nm particles from D and BD inhibit cytokine and chemokine production compared to control. In particular, concentration of 1.2 ppm sub-10nm particles from D caused down regulation of 9 factors (G-CSF, PDGF-bb, IL-1ra, IL-4, Eotaxin, β-FGF, VEGF, IP-10 and IL-6) and concentration of 1.2 ppm sub-10nm particles from BD caused down regulation of 7 factors (G-CSF, IL-17A, PDGF-bb, Eotaxin, β-FGF, IL-2, IL-15). Moreover, at 4 ppm sub-10nm particles from D and BD caused down regulation of respectively of 6 factors (PDGF-bb, MCP-1, Eotaxin, IL-12, VEGF, IP-10) and 3 factors (G-CSF, IL-6, IP10). Results are also summarized in Table 1.

4. Discussion The specific routes by which nanoparticles may enter the human body, and potentially elicit adverse effects are the lung, via inhalation, the gastrointestinal tract, via digestion, the skin and blood vessels via intravenous injection. These biological compartments are “innately designed” to act as barriers to the passage of materials introduced by occupational and environmental exposure into the organism. [40] Since the epithelium is the primary structural barrier, the aim of the present study is to elucidate the biological effects of sub -10nm particles generated from D and BD combustion on two epithelial cells, HaCaT and A549. Our study, for the first time, evaluate the effects of sub-10 nm nanoparticles. Indeed, BD combustion does not emit a relevant mass of particles with sizes much larger than 20 nm but only a significant number of very small, sub-10 nm particles. Moreover, sub-10 nm particles, mostly constituted by organic carbon, have the peculiarity to contain a large number of aromatic molecules constituting their surface, so that their effects on health might be more relevant than those of the “bigger” 20-100 nm particles. Skin, with its large surface area, is a potential route for sub -10nm particles exposure both

occupational and environmental. A major function of skin, especially the stratum corneum, which is the most outer layer, is to provide a protective barrier against the hazardous external environment. Skin is exposed to nanomaterials present in cosmetic products such as moisturisers and sunscreens and is also a potential target for drug delivery via nano-carriers. [41] Most of the studies provide evidence that skin is not the major target of nanoparticle delivery.[42,43] However, controversial discussions are ongoing concerning the benefits of nanoparticles in dermatological therapies and skin care products, as well as the potential disadvantages and possible mechanisms of toxicity. [44] Dermal keratinocytes play an important role in the cell renewal system and in maintaining skin integrity and are used as a model for testing dermal toxicity; therefore, HaCaT cells was chosen as the in vitro model for dermal exposure. Lung is a common target of many toxicants due to its large surface area, when inhaled; nanoparticles reach the alveolar epithelial surface where they can interact with alveolar macrophages and epithelial cells; Primary alveolar epithelial cell cultures provide a tight epithelial barrier resembling the pulmonary barrier in vivo. The cell line A549, which originates from human lung carcinoma, [45] belongs to the most well characterized and most widely used in vitro models. [46] It has been shown that the A549 cells have many important biological properties of alveolar epithelial type II cells (e.g. membrane-bound inclusions), which resemble lamellar bodies of type II cells; [47] consequently, A-549 were chosen to assess the effects of inhalation exposure to sub-10 nm particles. Results show that HaCaT and A-549 cells show different response to exposure to sub-10 nm particles; keratinocytes seems more sensitive than cancer cell line. HaCaT cells react to the 8.0 ppm exposure to sub-10 nm particle both from D and BD with a drastic reduction in cell viability. Both HaCaT and A-549 cells show a dose dependent reduction in cell viability, with a major effect of sub-10nm particles from D than BD (Figure 1). Viability data, obtained by Cristal Violet assay, are confirmed by Annexin V-FITC/PI apoptosis assay. Apoptosis, a physiological process of programmed cell death, is one of the major types of cell death caused by most nanoparticles. Annexin V/PI double-staining results indicate that: a) A549 cells exposed to sub-10nm particle mildly dead with apoptosis mechanism compared to HaCaT cells; b) sub-10nm particle from D are more effective to promote apoptotic death. (Figure 2). In addition, expressions of apoptosis-related proteins such as Active Caspase-3, Bcl-xL/Bak dimer, Mcl-1/Bak dimer and Survivin examined by Bio-Plex X-Map technology revealed that there’s no activation of the apoptosis intrinsic pathway. Only in HaCaT cells there’s a sparing

increase in Active Caspase 3 concentration when cells are treated with 8.0 ppm sub-10nm particle from D (Figure 3). Sub-10 m nanoparticles might act as danger-associated molecular patterns (DAMPs) and trigger inflammation, characterized by the release of pro-inflammatory cytokines. Cytokines can be in principle produced by all nucleated cells, including those examined in the present study, to regulate the immune response and their own survival and function. A highly complex crosstalk between various cytokines and their signaling effectors is established upon cell challenge by external insult. Indeed, a pro inflammatory response appear to characterize HaCaT cells: especially upon exposure to D derived sub-10nm particles, there was a dose dependent increment of proinflammatory cytokines and chemotactic/neovascularization factors. On the contrary, exposure to BD sub-10nm particles produced downmodulation of most of the functional molecules measured in HaCaT cells and all (but two) the cytokines in A-549 cells. Once the exogenous stressors has been cleared, the inflammatory process is antagonized by negative feedback loops that guarantee the regulated shutdown of the response. This fact might explain the reduced level of cytokines observed. Alternatively, interference by sub-10nm particles with the protein synthesis process and/or the secretion may have been occurred. Since depletion of the molecules involved in cell cross talk might also have pathogenic consequences, it will be necessary to verify this hypothesis. In conclusion, there was a differential behavior of HaCaT and A-549 in terms of viability and apoptosis and mediators release, being the HaCaT more responsive than A459 to sub-10nm particles in terms of cell survival and cytokine production. D and BD exhaust sub-10nm particles at 1.2 ppm and 4 ppm result not cytotoxic to both cell lines. However, at these concentrations, the network of cytokine, chemokine and growth factor induced by sub-10 nm particles exposure appear to be more complex for HaCaT cells, showing both down modulation and up regulation.

5. Conclusion Nanoparticulates are rapidly becoming a question of concern regarding health effects in occupational field as well as in the environment. Our study, for the first time, evaluate the effects of sub-10nm nanoparticles generated from BD combustion in a diesel engine on two different human cellular models, representing the two major routes of occupational and environmental exposure, i.e., the inhalator and the dermal ones. Nanoparticles exert different cytotoxic effects in the two cell lines, suggesting that the dermal

way of exposure is more sensitive than the inhalator way. These differences are most evident in the secretion of pro-inflammatory, angiogenic and proliferative cytokines and chemokines whose expression is more finely modulated in HaCaT cells compared to A-549 cells. It is important to affirm that our results are limited to an in-vitro study and that in-vivo studies are necessary to show if the results apply also to real working conditions. Moreover, other in-vitro studies will be undertaken to better define changes in cell proteome and metabolome after exposure to nanoparticles.

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7. Figure Legend

Figure 1: Cell viability assay performed with Cristal Violet staining on HaCaT and A549 cells treated with sub- 10 nm particles from D and BD exhaust. Cells were seeded in 96 plate at density of 2x104 cell/cm2 and exposed to sub 10-nm particle from D and BD (1.2, 4,8, 12 ppm) for 24 and 48h. The optical density of the solubilized dye was measured at 595 nm. Experiments were performed in triplicate and repeated three times.

Figure 2: Annexin V-FITC/propidium iodide (PI) apoptosis assay. Cells exposed to sub -10nm particles from D and BD (1.2 ppm, 4 ppm and 8 ppm) for 24 and 48 h were resuspended and stained with FITC-conjugated Annexin V (Annexin V-FITC) and PI. Stained cells were collected and 10,000 cells were analyzed by flow cytometry with FACS Diva 4.1 software. Figure 3: Apoptosis Assay with Bio-plex platform. Cells were seeded at 2x104 cells/cm2 in 10 cm Petri and treated with 1.2, 4 and 8 ppm sub-10 nm particles from D and BD, for 24h. Total cell extract and assay was run according to the manufacturer's instructions using a Bio-Plex 200 system (Bio-Rad). Data were analyzed with Bio-Plex Manager software v5.0 (BioRad).

Figure 4: Cytokine/chemokine detection with Bio-plex platform on HaCaT cells. Cells were seeded in 6 well plate at a density of 2x104 cells/cm2 in DMEM 10% FCS. After 24 h, cells were washed twice with PBS and culture media were replaced with DMEM 0% FCS containing sub-10nm particles produced by D and BD at concentration of 1.2 and 4 ppm. Cells were exposed to sub-10nm nanoparticles for 24 h. Detection of pro- and anti-inflammatory cytokines released into the culture medium was carried out with the Bio-Plex Pro Human Cytokine 27Plex Panel (Bio-Rad). All washing steps were performed on the Bio-Plex magnetic wash station (BioRad). Data were acquired using a Bio-Plex 200 system equipped with Bio-Plex Manager software v5.0 (BioRad). The standard curves optimization and the calculation of analyte concentrations were performed by using the Bio-Plex Manager software. Raw data, expressed as pg of cytokine/mL of conditioned medium (mean ± SD), with N= 3 for each cell line, particles concentration and time combination, were transformed in percentage quantities relative to control.

Figure 5: Cytokine/chemokine detection with Bio-plex platform on A549 cells. Cells were seeded in 6 well plate at a density of 2x104 cells/cm2 in DMEM 10% FCS. After 24 h, cells were washed twice with PBS and culture media were replaced with DMEM 0% FCS containing

sub-10nm particles produced by D and BD at concentration of 1.2 and 4 ppm. Cells were exposed to sub-10nm nanoparticles for 24 h. Detection of pro- and anti-inflammatory cytokines released into the culture medium was carried out with the Bio-Plex Pro Human Cytokine 27Plex Panel (Bio-Rad). All washing steps were performed on the Bio-Plex magnetic wash station (BioRad). Data were acquired using a Bio-Plex 200 system equipped with Bio-Plex Manager software v5.0 (BioRad). The standard curves optimization and the calculation of analyte concentrations were performed by using the Bio-Plex Manager software. Raw data, expressed as pg of cytokine/mL of conditioned medium (mean ± SD), with N= 3 for each cell line, particles concentration and time combination, were transformed in percentage quantities relative to control.

Table 1. Summary of results of Bio-Plex Pro Human Cytokines 27-plex Panel (↑:up-regulation; ↓: down-regulation) Cell Type

HaCaT

Treatment 1.2ppm D

4ppm D

1.2ppm BD

4ppm BD

G-CSF↓ MIP1a ↓ IL-17A ↓ PDGF-bb↓ MCP-1↓ MIP1b↓ IFNγ ↓ IL-1ra ↓

IL-6↑ VEGF↑ IL-8↑ G-CSF↑ IFNγ↑ TNFα↑ β-FGF↑ IL-1b↑

MIP1a ↓ IL-17a ↓ PDGF-bb↓ MIP1b↓ IL-1ra↓ Eotaxin ↓ TNFα ↓ GM-CSF↓ IL-2 ↓ Rantes↓

RANTES↓ IP-10↓ IL-15↓ IL-5↓ MCP-1↓

IL-6↑ IP-10↑ VEGF↑ IL-8↑ G-CSF↓ PDGF-bb↓ IL-1ra ↓ IL-4 ↓ Eotaxin ↓ β-FGF↓ VEGF↓ IP-10↓ IL-6↓

IL-6↑ G-CSF↑ IL-1b↑

IL-6↑

PDGF-bb↓ MCP-1↓ Eotaxin ↓ IL-12 ↓ VEGF↓ IP-10↓

G-CSF↓ IL-17A ↓ PDGF-bb↓ Eotaxin ↓ β-FGF↓ IL-2↓ IL-15↓

G-CSF↓ IL-6↓ IP-10↓