Serum protein oxidation by diesel exhaust particles: Effects on oxidative stress and inflammatory response in vitro

Chemico-Biological Interactions 206 (2013) 385–393

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Serum protein oxidation by diesel exhaust particles: Effects on oxidative stress and inflammatory response in vitro Ling-Ling Chiang a,b,1, Hao-Cheng Chen a,1, Chun-Nin Lee a,b, Kai-Jen Chuang c,d, Tzu-Tao Chen a,e, Chi-Tai Yeh e,f,g, Liang-Shun Wang e,f,g, Wei-Hua Lee h,i, Lian-Yu Lin j, Hsiu-Er Tseng k, Hsiao-Chi Chuang a,b,⇑, On behalf of Taiwan CardioPulmonary Research (T-CPR) Group a

Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan d School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan e Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan f Cancer Center, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan g Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan h Department of Pathology, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan i Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei, Taiwan j Department of Internal Medicine, Division of Cardiology, National Taiwan University Hospital, Taipei, Taiwan k Division of Consultation and Promotion, Taiwan Drug Relief Foundation, Taipei, Taiwan b c

a r t i c l e

i n f o

Article history: Received 8 July 2013 Received in revised form 6 September 2013 Accepted 14 October 2013 Available online 23 October 2013 Keywords: Amino acid Bovine serum albumin Inflammation Peptide sequence Proteomics Oxidative stress

a b s t r a c t Considerable evidence shows a key role for protein modification in the adverse effects of chemicals; however, the interaction of diesel exhaust particles (DEP) with proteins and the resulting biological activity remains unclear. DEP and carbon black (CB) suspensions with and without bovine serum albumin (BSA) were used to elucidate the biological effects of air pollutants. The DEP and CB samples were then divided into suspensions and supernatants. Two important goals of the interaction of DEP with BSA were as follows: (1) understanding BSA modification by particles and (2) investigating the effects of particles bound with BSA and the corresponding supernatants on cellular oxidative stress and inflammation. We observed significant free amino groups production was caused by DEP. Using liquid chromatography–mass spectrometry (LCMS), we observed that BSA was significantly oxidised by DEP in the supernatants and that the peptides ETYGDMADCCEK, MPCTEDYLSLILNR and TVMENFVAFVDK, derived BSA-DEP conjugates, were also oxidised. In A549 cells, DEP-BSA suspensions and the corresponding supernatants reduced 8-hydroxy-20 -deoxyguanosine (8-OHdG) production and increased interleukin-6 (IL-6) levels when compared to DEP solutions without BSA. Our findings suggest that oxidatively modified forms of BSA caused by DEP could lead to oxidative stress and the activation of inflammation. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: TNBS, 2,4,6-trinitrobenzene sulphonic acid; 8-OHdG, 8-hydroxy20 -deoxyguanosine; CB, carbon black; DEP, diesel exhaust particle; DLS, dynamic light scattering; ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin-6; NF-jB, nuclear factor-kappa beta; PBS, phosphate buffered saline; PAHs, polycyclic aromatic hydrocarbons; RSLC, rapid separation liquid chromatography; ROS, reactive oxygen species; TEM, transmission electron microscopy; TNF-a, tumour necrosis factor-alpha. ⇑ Corresponding author. Address: Taiwan CardioPulmonary Research Group (T-CPR), School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan. Tel.: +886 2 27361661x3515; fax: +886 2 27391143. E-mail addresses: [email protected] (L.-L. Chiang), haochengenator@gmail. com (H.-C. Chen), [email protected] (C.-N. Lee), [email protected] (K.-J. Chuang), [email protected] (T.-T. Chen), [email protected] (C.-T. Yeh), [email protected]. edu.tw (L.-S. Wang), [email protected] (W.-H. Lee), hspenos@yahoo. com.tw (L.-Y. Lin), [email protected] (H.-E. Tseng), [email protected] (H.-C. Chuang). 1 These authors contributed equally to the study. 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.10.013

1. Introduction Epidemiological and clinical studies show that respiratory allergies, deaths due to cardiovascular and pulmonary diseases and lung cancer development are all associated with chronic exposure to particulate air pollution, such as diesel exhaust particles (DEP) [1–4]. The human health effects of DEP have been studied over several years; however, an investigation of the chemical components responsible for the macromolecular effects is lacking, and a detailed understanding of the underlying mechanisms remain unclear [5]. DEP is a complex mixture of hundreds of components, consisting of a central core of elemental carbon and adsorbed organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs as well as small amounts of sulfate, nitrate and

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metals (including trace elements) [6,7]. DEP is used to evaluate the potential effects on vasoactive function and the inflammatory response because the nano-scaled fractions are more capable of being inhaled into the deep lung environments and entering the circulation [8–10]. Traffic-related DEP have been identified as a common air pollutant associated with reactive oxygen species (ROS) formation. Certain chemicals in DEP, such as organic carbon, PAHs and metals, are associated with particle bioreactivity, and organic and metal compounds in particles account for oxidative and inflammatory effects [11]. Protein interactions with particles are associated with some degree of protein unfolding, which affects the biological function of the protein [12]. An increasing number of studies have investigated the interaction of carbon-based nanoparticles with blood carrier protein molecules and the effects on the biological activity of the proteins [13–15]. Particle surface chemistry has shown a considerable influence on the amount of protein binding to distinct ligands, leading to alterations in oxidative stress and inflammation [16]. Serum albumin is a globular protein synthesised by the liver in mammals and is the most abundant protein in serum (approximately 60% of the total globular protein in blood plasma) [17]. Albumin maintains the colloidal osmotic blood pressure and is essential for the transportation of several endogenous and exogenous compounds, including proteins and fatty acids, to specific targets [18]. Bovine serum albumin (BSA), a globular, protein consisting of 583 amino acid residues, is commonly used to investigate protein-particle interactions. In terms of its secondary structure, 67% of the protein is composed of alpha helices, and the protein contains 17 disulfide bridges, which increase its stability [18]. Previous studies have investigated the interactions of nanoparticles with serum protein; however, the chemical effects of DEP on serum protein remain unclear. Increasing evidence suggests that the oxidative damage of proteins is an important mechanism in aging and multiple diseases [19]. The important effects of protein oxidation on cellular homeostasis derive from the fact that proteins play critical roles in regulating cell structure, cell signalling and various enzymatic processes of the cell. There are many different modes of protein oxidation such as amino acid oxidation and metal-catalysed oxidation. However, there is a paucity of literature investigating the interaction of DEP with proteins and the resulting oxidative stress and inflammatory responses in biological systems. To pursue this investigation, we examined BSA modification (in supernatants of suspensions and in BSA-particle conjugates) following interactions with DEP, as a model chemical-rich carbonaceous particle, and with carbon black (CB), as a model chemical-less carbonaceous core. We also compared the effects of exposure of human alveolar epithelial Type II (A549) cells to DEP and CB suspensions and the corresponding supernatants, either with or without BSA. These experiments allowed us to investigate the effects of DEP solutions with and without BSA on oxidative stress [measured by 8-hydroxy20 -deoxyguanosine (8-OHdG) levels] and on the pro-inflammatory cytokine levels [measured by interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a)] in cellular systems.

2. Materials and methods 2.1. Cell culture A549 cells were obtained from the American Type Culture Collection and cultured in RPMI containing 10% fetal bovine serum, penicillin and streptomycin. Cells were incubated in air at 37 °C, 95% humidity and 5% CO2. All chemicals used in this study were reagent grade and were obtained from Sigma Aldrich (UK), unless stated otherwise.

2.2. Sample preparation and characterisation In this study, we investigated protein modification as well as oxidative and inflammatory responses after DEP and CB exposure. The DEP were the Standard Reference Material 2975 obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, USA). DEP are chemical-rich particles, and their chemical characteristics have been described previously [20]. DEP were generated by a heavy-duty forklift diesel engine and collected using a filtering system designed for diesel forklifts [5]. Near-pure, manufactured, CB, with an average diameter of 65 nm (Monarch 120; Cabot Corporation, UK), was selected as a control particle. CB is an industrial carbon produced by the thermal decomposition of hydrocarbons. The chemical characteristics of CB have been described previously [21,22]. A solution of 5% BSA was prepared with sterile phosphate-buffered saline (PBS) and then filter-sterilised. DEP and CB were suspended in 5 ml of PBS with (+) or without 5% BSA () (PBS only) after 15 min of sonication. Aliquots of the DEP and CB suspensions were then combined with the 5% BSA solution or the BSA-free PBS (final volume of 10 ml) to yield final particle concentrations of 0, 50, 150 and 1000 lg/ml. The DEP and CB samples (±5% BSA) were vortexed and incubated at 37 °C for 2 h under constant shaking at 500 rpm to ensure thorough mixing. Five milliliters of the samples (0, 50, 150 and 1000 lg/ml DEP and CB) were then separated into the following two fractions by centrifugation (3500 rpm) and filtration: supernatant and pelleted particles. The pelleted particles were washed thoroughly by repeated resuspension in deionised water and centrifugation [23,24]. The DEP and CB supernatants and the corresponding pelleted particles were used to investigate protein modification, whereas the DEP and CB suspensions and their corresponding supernatants were used for cell-based oxidative stress and inflammation analyses. Particle-free solution ±5% BSA in PBS were used as negative controls and were treated in the same manner as the test samples. Dynamic light scattering (DLS; Malvern Zetasizer NanoZS, UK) was used to determine the hydrodynamic diameters of 50 and 150 lg/ml DEP and CB (±5% BSA) suspensions. Transmission electron microscopy (TEM; Philips CM12) was used to investigate the morphology of DEP and CB suspended in 5% BSA. Samples were loaded onto aurum TEM grids (Agar, UK) and investigated at an accelerating voltage of 80 kV with a spot size 1. The samples were kept at 4 °C and used immediately upon removal from storage.

2.3. 2,4,6-Trinitrobenzene sulphonic acid assay A 2,4,6-trinitrobenzene sulphonic acid (TNBS) assay was used to determine the free amino groups produced in proteins by free radicals, as described previously [25]. Briefly, the concentrations of amino groups in the BSA before and after incubation with CB and DEP was directly quantified with a PowerWave microplate reader (BioTek, USA) monitoring the absorbance at 335 nm. CB and DEP suspensions at 0, 50, 150 and 1000 lg/ml were mixed with 100 lg/ml BSA in 0.1 M NaHCO3 (pH 8.5). Then, 0.5 ml of a 0.01% (w/v) TNBS solution was added to the particle-BSA samples and incubated at 37 °C for 2 h. To relate the absorbance of the samples to the amino acid standard solution (2.5 lmol/ml L-alanine, 2.5 lmol/ml ammonium chloride, 2.5 lmol/ml L-arginine, 2.5 lmol/ml L-aspartic acid, 1.5 lmol/ml L-cystine, 2.5 lmol/ml glutamic acid, 2.5 lmol/ml glycine, 2.5 lmol/ml L-histidine, 2.5 lmol/ml L-isoleucine, 2.5 lmol/ml L-leucine, 2.5 lmol/ml L-lysine, 2.5 lmol/ml L-methionine, 2.5 lmol/ml L-phenylalanine, 2.5 lmol/ml L-proline, 2.5 lmol/ml L-serine, 2.5 lmol/ml L-threonine, 2.5 lmol/ml L-tyrosine and 2.5 lmol/ml L-valine), a calibration curve of amino acid levels ranging from 0.008 to 2.5 lmol/ ml was generated. All TNBS assay data are presented as the free

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amino acid concentrations equivalent to free amino group levels. Each experiment was conducted in quadruplicate. 2.4. Determination of BSA modification in particle supernatants BSA control and DEP and CB supernatants (from 1000 lg/ml suspensions) were diluted with a solution of 5% acetonitrile (J.T. Baker, USA)/0.1% formic acid (Fluka, USA). Liquid chromatography–mass spectrometry (LCMS) analyses were performed using a Q Exactive MS (Thermo Fisher Scientific, Bremen, Germany) coupled to an UltiMate 3000 Rapid Separation LC (RSLC) system (Dionex, Sunnyvale, USA). Peptide were separated using LC with a BioBasic-C4 column (100  2.1 mm, 5 lm particle size; Thermo Fisher Scientific, Germany) under the conditions listed in Supplementary information Table S1. Full MS scans were performed with an m/z range of 800–3500, and the mass spectra obtained were deconvoluted using Protein Deconvolution v2.0 (Thermo, Germany) to determine the molecular weight. 2.5. Determination of BSA modification in pelleted particles BSA control and pelleted DEP and CB (from 1000 lg/ml suspensions) were reduced with 6.5 mM dithiothreitol at 37 °C for 1 h and alkylated using 10 mM iodoacetamide in the dark at room temperature for 30 min. The samples were digested with trypsin in 25 mM ammonium bicarbonate at 37 °C for 18 h and then acidified with 0.1% formic acid. The tryptic peptides were analysed with a QExactive MS (Thermo Fisher Scientific, Bremen, Germany) coupled to an UltiMate 3000 RSLC system. Peptide separation was performed using LC with a C18 column (Acclaim PepMap RSLC, 75 lm  150 mm, 2 lm, Dionex) under the conditions listed in Supplementary information Table S2. Full MS scans were performed with an m/z range of 300–2000, and the ten most intense ions from the MS scan were subjected to fragmentation yielding MS/MS spectra. Raw data were processed into peak lists by Proteome Discoverer v1.4 for Mascot database searches (http:// www.matrixscience.com). Search parameters specified variable modification for deamidation (NQ), oxidation (M), methylation (K) and fixed modification for carbamidomethylation (C). The maximum mass tolerance was set to 10 ppm for precursor ions and 0.05 Da for fragment ions. 2.6. A549 cell treatment For the in vitro experiments, A549 cells were seeded onto surface-treated, 24-well transwells at a density of 1  105 cells/ml and incubated for 24 h (3  104 cells/well; BD Biosciences, UK). The RPMI medium was removed before adding 300 ll of sample (DEP suspension, CB suspensions, DEP supernatant or CB supernatant) prepared ±5% BSA in PBS at particle concentrations of 0, 50 and 150 lg/ml; the cells were incubated at 37 °C for 4 h in a humidified atmosphere with 5% CO2. Each experiment was conducted in quadruplicate. The media was collected and used to monitor the levels of oxidative marker 8-OHdG, IL-6 and TNF-a post-exposure. The concentrations of DEP and CB were chosen to produce oxidative and inflammatory effects in 5% BSA and PBS (>80% cell viability) according to criteria described by Wilson et al. (2002) [26].

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microtiter plate that was coated with 8-OHdG; the plates were incubated at 37 °C for 60 min according to the manufacturer’s instructions. After the wells were washed three times, the antibody that remained bound to the 8-OHdG in the samples was further incubated with horseradish peroxidise-conjugated secondary antibody at 37 °C for 60 min. After washing the wells three times, they were incubated with a substrate containing tetramethylbenzidine at room temperature for 15 min. A PowerWave microplate reader (BioTek, USA) was used to determine the concentration of 8-OHdG by measuring the absorbance at 450 nm. The concentration of 8-OHdG was calculated using 8-OHdG standards supplied by the manufacturer. 2.8. Determination of IL-6 and TNF-a levels An ELISA (BD OptEIA™ set, BD Biosciences, USA) was used to determine the IL-6 and TNF-a levels according to the manufacturer’s instructions. 2.9. Statistical analysis The statistical analyses were performed using GraphPad Version 5 for Windows. The Mann–Whitney U-test was used for comparisons between groups [27]. The significance criterion was set at p < 0.05. 3. Results 3.1. Particle characterisation DEP and CB were used to investigate the interactions between proteins and particles and the consequent protein modification as well as the oxidative stress and inflammatory responses. The hydrodynamic diameters observed in 50 and 150 lg/ml suspensions of DEP and CB with or without 5% BSA were determined (Fig. 1). No significant differences between the particle sizes of 50 lg/ml DEP and CB were observed, with average hydrodynamic diameters of 1162 ± 156 nm and 1170 ± 149 nm, respectively. However, 150 lg/ml CB without BSA showed an approximately 1.5-times higher average hydrodynamic diameter (1603 ± 87 nm; p < 0.05) than DEP without BSA (1098 ± 76 nm). The addition of 5% BSA resulted in particle dispersal, which reduced the hydrodynamic diameters of DEP and CB 4–10 times. The average hydrodynamic diameters of 50 lg/ml DEP and CB with BSA were

2.7. Determination of 8-OHdG level The 8-OHdG levels via a competitive enzyme-linked immunosorbent assay (ELISA) kit (Japan Institute for the Control of Aging, Japan) were used to determine oxidative damage. The detectable range of 8-OHdG was 0.5–200 ng/ml. The monoclonal anti-8-OHdG antibody and sample were loaded at a volume of 50 ll onto a

Fig. 1. Alterations of hydrodynamic diameters in 50 and 150 lg/ml CB and DEP suspensions with (+) and without () BSA. The values are the means ± SD (n = 4); ⁄ p < 0.05.

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191 ± 31 nm and 118 ± 23 nm, respectively. The sizes of 150 lg/ml DEP and CB with BSA were 277 ± 25 nm and 284 ± 24 nm, respectively. TEM images revealed spherical CB chains in solutions of 5% BSA in PBS (Fig. 2) and spherical DEP aggregates and clusters in 5% BSA solutions. 3.2. Free amino groups in BSA To investigate the free amino groups in BSA generated by DEP and CB, four particle mass concentrations were used (0, 50, 150 and 1000 lg/ml; Fig. 3). There was no significant difference between 50 and 150 lg/ml DEP and CB samples when compared to the control (0.07 lmol/ml). However, we observed that DEP were associated with more free amino groups production in BSA than CB at 150 and 1000 lg/ml (p < 0.05). Notably, 1000 lg/ml DEP resulted in 11.5-times free amino groups (0.84 lmol/ml) than the control (p < 0.05), whereas CB resulted in 4.2-times free amino groups (0.31 lmol/ml) than the control; the latter result was not statistically significant.

Fig. 3. Production of free amino groups in BSA by CB and DEP at 0, 50, 150 and 1000 lg/ml. The data are presented as the free amino acid concentrations equivalent to the free amino group levels. The values are the means ± SD (n = 4); ⁄ p < 0.05.

3.3. BSA modification in particle supernatants To investigate BSA modification from DEP and CB exposure, we first collected the corresponding supernatants (obtained from 1000 lg/ml DEP and CB suspensions) for LCMS analysis. The mass spectra of the BSA control and the corresponding CB and DEP supernatants are presented in Fig. 4. The results showed no significant difference between the BSA control and the corresponding CB supernatant; however, the BSA mass spectra showed a significant shift towards a lower m/z after exposure to DEP. We deconvoluted the mass spectra, which reveal that the protein molecular weights in the BSA control and the corresponding CB supernatant were 66,427 Da and 66,428 Da, respectively; this suggested no significant difference in the molecular weight of BSA with and without CB addition. Notably, the molecular weight results associated with the corresponding DEP supernatant could not be deconvoluted by the software. Manual inspection confirmed that no protein signal could be extracted from the corresponding DEP supernatant samples. 3.4. BSA modification in pelleted particles To further investigate BSA modification when bound to particles, the trypsin-digested peptides from the BSA control and the CB and DEP pellets were analysed by LCMS and Mascot database. The following three peptides were oxidised in the DEP and CB pellets: ETYGDMADCCEK, MPCTEDYLSLILNR and TVMENFVAFVDK (Fig. 5). To estimate the oxidation degree, the signal intensities from the extracted ion chromatograms of the three peptides with

and without oxidation were compared. The oxidation degree was calculated based on the peak area [(peak area of oxidised peptide/total peak area of peptides with and without oxidation)  100%]. The estimated oxidation degrees of the three peptides for the BSA control and the CB and DEP pellet samples are shown in Table 1. There was no significant oxidation difference for the three peptides between the BSA control and the CB pellet samples. Notably, significant BSA oxidation of the three peptides was observed in the DEP pellets (89.5–99.2%). 3.5. Effects on 8-OHdG production To determine the effects of BSA modification by DEP on 8-OHdG formation, A549 cells were exposed to CB and DEP suspensions with and without BSA and the corresponding CB and DEP supernatants (Fig. 6a and b). Significant dose-dependence was observed for 8-OHdG production when A549 cells were exposed to CB and DEP suspensions without BSA compared with the controls (p < 0.05) with the exception of the 150 lg/ml CB samples. DEP suspensions without BSA produced significantly higher 8-OHdG concentrations than CB suspensions without BSA at the same mass concentration (p < 0.05). The corresponding CB and DEP supernatants exhibited responses similar to those of the suspensions, but the 8-OHdG levels did not increase significantly after exposure to the CB supernatants. Comparisons of the corresponding CB and DEP supernatants at the same concentration showed there was a significant increase in 8-OHdG production after exposure (p < 0.05) with and without

Fig. 2. TEM photographs of (a) CB with BSA and (b) DEP with BSA. Spherical CB with chain-like morphology was observed with BSA. Spherical DEP with significant aggregation and clusters were observed with BSA. The scale bar represents 1000 nm.

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BSA. Comparisons between DEP with and without BSA at the same particle concentrations, after taking into account the corresponding background control, showed that 8-OHdG production was 0.3-times lower in the corresponding DEP suspensions with BSA than without BSA (at particle concentrations of 50 and 150 lg/ml). The 8-OHdG production was 0.7-times lower in the corresponding DEP supernatant with BSA than without BSA (at a particle concentration of 50 lg/ml). 3.6. Effects on IL-6 and TNF-a production The IL-6 levels were higher in DEP suspensions with and without BSA and the corresponding DEP supernatant compared to most control samples and CB-exposed cells (at the same particle concentrations, p < 0.05; Fig. 6c and d). CB suspensions and the corresponding CB supernatant samples (150 lg/ml) increased IL-6 production when suspended with and without BSA (p < 0.05), but the results were inconsistent. Fig. 6e and f show significantly increased TNF-a concentrations for DEP suspensions with and without BSA and the corresponding DEP supernatants with the exception of DEP supernatant without BSA. When comparing the DEP and CB samples at the same particle concentration, the presence of DEP suspensions with and without BSA showed significantly increased TNF-a production (p < 0.05). However, no significant TNF-a production was caused by the corresponding DEP supernatant. Comparisons between DEP with and without BSA at the same particle concentrations, after taking into account the corresponding background control, showed that the IL-6 production was 1.6-times (for 50 lg/ml) and 1.1-times higher (for 150 lg/ml) in DEP suspensions with BSA than without BSA, respectively. The IL-6 levels were also 1.6-times (for 50 lg/ml) and 1.5times (for 150 lg/ml), respectively, higher for the corresponding DEP supernatant. There was no significant alteration in TNF-a production between the DEP samples with and without BSA. 4. Discussion and conclusions Fig. 4. Mass spectra of (a) BSA control, (b) CB supernatant with BSA and (c) DEP supernatant with BSA. The CB and DEP supernatant samples were obtained from the corresponding 1000 lg/ml particle suspensions with BSA.

In the present study, the following two important issues concerning the interaction of carbon-based particles (i.e.,

Fig. 5. Extracted ion chromatograms of ETYGDMADCCEK, MPCTEDYLSLILNR and TVMENFVAFVDK peptides, with and without oxidation, in BSA control and (a)–(c) BSAcoated CB pellet with BSA and (d)–(f) BSA-coated DEP pellet.

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Table 1 Estimated percentages of BSA oxidation in three peptides (ETYGDMADCCEK, MPCTEDYLSLILNR and TVMENFVAFVDK) for the BSA control samples and CB and DEP pellets (each obtained from 1000 lg/ml particle suspensions with BSA). The estimated oxidation percentage was calculated based on the peak area [(peak area of oxidised peptide/total peak area of peptides with and without oxidation)  100%]. Peptide sequence

ETYGDMADCCEK ETYGDMoxADCCEK MPCTEDYLSLILNR MoxPCTEDYLSLILNR TVMENFVAFVDK MoxENFVAFVDK

Calculated Mw (kDa)

1477.5 1493.5 1723.8 1739.8 1398.6 1414.6

BSA

CB

DEP

Observed m/z (2+)

MASCOT score

Oxidation (%)

Observed m/z (2+)

MASCOT score

Oxidation (%)

Observed m/z (2+)

MASCOT score

Oxidation (%)

739.7 747.7 862.9 870.9 700.3 708.3

51 39 91 130 84 69

21.3

739.7 747.7 862.9 870.9 700.3 708.3

68 54 87 115 63 21

15.8

739.7 747.7 862.9 870.9 700.3 708.3

35 57 37 115 31 80

99.1

1.3 1.2

1.2 3.6

99.2 89.5

Mw: molecule weight.

non-functionalised CB and standard DEP) with protein were investigated: (1) the resulting protein modifications when coated and un-coating with particles, and (2) the effects of protein-coated particles on the oxidative stress and inflammatory responses of cells. The main findings in the present study were that free amino group production and BSA oxidation in the ETYGDMADCCEK, MPCTEDYLSLILNR and TVMENFVAFVDK peptides were caused by DEP. BSA in DEP suspensions and in DEP supernatant reduced cellular 8-OHdG production and increased cellular IL-6 levels when compared to samples without BSA. Our findings suggest that the oxidatively modified forms of BSA in cells could also be associated with oxidative and inflammatory mechanisms. Previous studies have indicated that serum protein, such as BSA, acts as an excellent dispersant for nanoparticles in biological solutions [28,29]. We observed a similar result in that the addition of 5% BSA dispersed CB and DEP well. Additionally, no significant differences in the hydrodynamic diameters and morphologies between CB and DEP were observed. Previous studies showed that carbon nanoparticles strongly interacted with serum albumin, resulting in the secondary structure alteration of the protein in the presence of carbon nanoparticles [13]. However, the chemical compositions of DEP may have different effects on protein-particle interactions. The interaction of nanoparticles with proteins has been increasingly investigated in recent years [13,24]. The structure of proteins has also been suggested to be an important factor in their interaction with the particle surface. A protein folds into the most favourable state of the polypeptide chain; structure is an intrinsic determinant of the protein molecule and stabilises its function. BSA consists of 583 amino acids in a single polypeptide chain [15]. We observed that significant levels of free amino groups in BSA were produced by DEP, implying that protein oxidation occurred during DEP interaction. This study used two types of carbonaceous particles with significantly different chemicals (e.g., metallic and organic components) adsorbed onto their surfaces, leading to a difference in oxidative potential [21]. Some transition metals are capable of redox cycling [30] and generate superoxide and hydroxyl radicals [31]. Inhaled PAHs are also converted to hydroxyl derivatives by cytochrome P450, epoxide hydrolase and dihydrodiol dehydrogenase [32]. Amino acid residues of proteins are susceptible to oxidation by hydroxyl radicals [19]. The most sensitive amino acids are those with aromatic side chain groups and those containing sulphydryl groups. Reactive oxygen species attack of the polypeptide backbone could be initialed by the OHdependent abstraction of the a-hydrogen atom of an amino acid residue to form a hydroxyl protein derivative. Taken together, the increase in free amino groups caused by DEP could be the result of chemical-derived ROS; ROS production could be attributed to protein modification, such as oxidation. Supernatant and pellet fractions from CB and DEP suspensions were used to investigate protein modifications by un-coated BSA with particles and BSA-particle conjugates via LCMS. Our results

showed significant protein modification caused by DEP in both the supernatant and pellet fractions; however, no significant modification by CB was observed. This suggested that the chemical composition of particles played an important role in regulating protein modification. The mass spectra and deconvoluted molecular weight results showed no significant differences between the BSA control and CB supernatant samples, whereas the mass spectra for DEP supernatant showed significant shifting, and no deconvoluted molecular weight could be obtained. This observation could be attributed to protein fragmentation resulting from oxidation of the protein backbone. Oxidation of the polypeptide backbone is caused by hydroxyl radical-associated abstraction of the ahydrogen atom of an amino acid residue to produce a carbon-centered radical, followed by formation of an alkoxyl radical [19]. Many steps of this pathway are mediated by metal-catalysed cleavage of hydrogen peroxide (e.g., the Fenton reaction), which is one of the most common mechanisms for inducing protein oxidation. NADH and NADPH oxidase as well as other oxidation systems catalyse the formation of hydrogen peroxide. Transition metals then bind to a specific metal-binding site within the protein and react with hydrogen peroxide to generate sulfenic acid that can attacks the amino acid residues near the metal-binding site [33]. Additionally, peptide bond cleavage can be generated by ROS attack of glutamyl, aspartyl and prolyl side chains [34]. The interactions of DEP with proteins could result in a structural reorganisation of the protein molecules. This re-structuring of proteins during particle binding could lead to the loss of the native conformation and the formation of a metastable transitional confirmation. Furthermore, the progression of protein re-structuring could lead to the appearance of proteins on the particle surface. The alterations in the functional structure of proteins caused by DEP could stimulate biological responses and/or changes to their activity. The chemical effects on protein oxidation by the organic and inorganic components of DEP require further investigation. The modification of protein structure may be accompanied by additional energy in the protein and transitions of the entropy to reversible or irreversible states [35]. Particles with more free radicals on their surfaces may be able to interact with protein molecules and be stabilised by proteins to form protein-particle conjugates. An increasing number of studies have shown that the interaction of nanoparticles with proteins resulted in protein-particle conjugates [13,16,24]. However, we know little concerning the effects of interactions of chemical-rich particles with protein. Therefore, we characterised the modification of BSA protein when it was coated by DEP via the determination of tryptic peptides using LCMS; the results showed that the ETYGDMADCCEK, MPCTEDYLSLILNR and TVMENFVAFVDK peptides were significantly oxidised by DEP. We then attempted to estimate the degree of oxidation by comparison with the BSA controls. This indicated more than 89.5% oxidation of the three peptides. Notably, the oxidation degree provided here is for reference only and is not an

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Fig. 6. A549 cells were exposed to CB and DEP suspensions and the corresponding supernatants with and without BSA. The endpoints were assessed after exposure for 24 h. (a) and (b) Oxidative stress marker 8-OHdG, (c) and (d) IL-6 and (e) and (f) TNF-a levels were determined using suspensions with 0, 50 and 150 lg/ml particles. The values are the means ± SD (n = 4); ⁄p < 0.05.

absolute value due to the varied ionisation efficiencies of different peptides. Cysteine and methionine residues are particularly sensitive to ROS. These residues are converted to disulfides and methi-

onine sulphoxide residues [19]. For example, the peptide ETYGDMADCCEK is a cysteine-containing peptide that was oxidised by DEP. The thiol-containing side chain of cysteine is the

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most reactive species present in naturally occurring peptides and consequently, cysteine-containing peptides must be handled carefully to prevent side reactions. Peptides containing a single cysteine residue can form dimers by oxidation of the cysteine side chain thiols, linking two chains together by a disulphide bridge. Peptides containing two or more cysteines can form intramolecular disulphide bridges yielding cyclic peptides. Protein oxidation may serve as a maker of exposure to environmental pollutants [36]. For example, the corresponding human serum albumin peptides to the three observed in BSA, ETYGEMADCCAK, MPCAEDYLSVVLNQLCVLHEK and AVMDDFAAFVEK, may be markers for DEP exposure in human. However, further confirmatory studies are required. Protein oxidation may trigger deleterious effects in biological systems, such as A549 cells. Oxidative stress is believed to be a key mechanism in the regulation of particle toxicity. Induction of ROS was observed after exposure to solution of BSA-coated DEP and to the corresponding supernatant. This finding is consistent with previous literature reports [29,37], in which increased ROS production was observed after the interaction of carbon-based nanoparticles with surfactant-containing proteins. Notably, the comparison of DEP (suspension and supernatant) with and without BSA showed 0.3–0.7 times less 8-OHdG production when DEP with BSA. This observation may be due to oxidised cysteine residues (i.e., disulfides) and methionine residues (i.e., methionine sulphoxide) that can be converted back to their unmodified forms in biological systems. Furthermore, it was reported that the cyclic oxidation–reduction of methionine residues served as a ROS scavenger system to protect proteins from more extensive irreversible oxidative modification [38,39]. Taken together, we suggest that pro-oxidants from particulate-associated chemicals may interact with proteins, including their cysteine and methionine residues, thus mitigating the oxidative stress induced by DEP on cells. BSA binding to DEP could have protective effects in response to oxidative stress in vitro. Oxidative stress can result in the activation of signalling pathways via transcription factors, which then initiate the production of major pro-inflammatory and immune mediators, such as IL-6 or TNF-a. Inflammation is defined as a non-specific protective reaction of vascularised tissue to injury; inflammation is a critical step in the innate immune response, which leads to the removal of harmful stimuli and to the initiation of the healing process. We observed that suspensions containing BSA-coated DEP and the corresponding supernatants resulted in lower levels of 8-OHdG production than samples without coated BSA; however, DEP led to increase IL-6 production compared to samples without coated BSA. The particle-bound protein may mask the reactive surface of the particles, preventing interaction with cells and resulting in the reduction of oxidative stress. Previous studies showed that pre-coated surfactant protein A increased the uptake of titanium dioxide particles into primary rat alveolar macrophages [40]. Phosphatidylserine bound to single-walled carbon nanotubes could facilitate the recognition and internalisation of particles by macrophages [41]. To investigate the uptake effects of protein-coated particles in inflammation responses, more studies are required in the future. Interestingly, there was no significant alteration in TNF-a production by DEP, which is similar to what was observed in previous in vitro studies [16,42]. Inconsistently, a previous study showed that DEP-induced significant TNF-a production in the isolated rat brain capillaries [43]. It could be resulted form the cell-type specific responses to DEP. Incubation of human artery endothelial cells with BSA showed significant inhibition of TNF-a-induced mRNA, which could be associated with nuclear factor-kappa Beta (NFjB) activation [14]. This finding may partially explain our TNFa results, but further studies are required to investigate the underlying mechanisms.

A better understanding of the processes underlying the interaction of particles with biomolecules is the basis for describing biological activity in response to particulate air pollution. Previous studies have indicated that protein-particle conjugates influenced oxidative stress and inflammatory potency in vitro [13,16,44], suggesting that coating proteins with carbon-based particles should be considered in future in vitro risk assessment studies. Indeed, our results showed that the interaction of DEP with BSA had an important role in the response to protein oxidation, oxidative stress and inflammatory responses in vitro. Our results further suggested that the chemical composition of DEP may be a critical determinant in the regulation of protein oxidation. However, the slight difference in particle size used in this study could influence the oxidative and inflammatory effects. To determine the effects of oxidative stress caused by DEP on protein oxidation, an antioxidant (e.g. N-acetyl cysteine) could be used in future works. The main potential consequence of the interaction of proteins and peptides with particles is the alteration of the biomolecular structure. The major results of protein oxidation, such as amino acid oxidation and peptide bond cleavage, are associated with the production of protein carbonyl groups. Our results provide additional evidence that may fill the gaps between epidemiological and molecular research. Additionally, our results provide a broader understanding of particle-protein interactions in a biological environment, an understanding which depends on knowledge concerning the fate of particles at the molecular level. Authors’ contributions L.L.C and H.C.C. has contributed substantially to the completion of interpretation of the data and the manuscript. T.T.C., C.N.L. and K.J.C. has contributed to the completion of the study. C.T.Y. and L.S.W. has contributed to the completion of interpretation of the proteomic data. L.Y.L., W.H.L. and H.E.T. has contributed to the completion of interpretation of the data. H.C.C. contributed substantially to the concept, the design and the completion of the study, the statistical analyses and critically revising the manuscript for important intellectual content. All authors have read and approved the final manuscript. Funding This study was founded by the Taipei Medical University (TMU101-AE1-B53). Acknowledgements The authors wish to thank the Mass Solutions Technology for their technical assistance of this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cbi.2013.10.013. References [1] P. Colais, A. Faustini, M. Stafoggia, G. Berti, L. Bisanti, E. Cadum, A. Cernigliaro, S. Mallone, B. Pacelli, M. Serinelli, L. Simonato, M.A. Vigotti, F. Forastiere, E.C. Group, Particulate air pollution and hospital admissions for cardiac diseases in potentially sensitive subgroups, Epidemiology 23 (2012) 473–481. [2] G. Polichetti, S. Cocco, A. Spinali, V. Trimarco, A. Nunziata, Effects of particulate matter (PM10, PM2.5 and PM1) on the cardiovascular system, Toxicology 261 (2009) 1–8. [3] I. Hertz-Picciotto, R.J. Baker, P.S. Yap, M. Dostal, J.P. Joad, M. Lipsett, T. Greenfield, C.E. Herr, I. Benes, R.H. Shumway, K.E. Pinkerton, R. Sram, Early childhood lower respiratory illness and air pollution, Environ. Health Perspect. 115 (2007) 1510–1518.

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