Supplementary materials

Toxicity effects of short term diesel exhaust particles exposure to

human small airway epithelial cells (SAEC) and human lung

carcinoma epithelial cells (A549)

Mingjie Tang1, Qifei Li1, Lifu Xiao1, Yanping Li2, Judy L. Jensen2, Theodore G. Liou2, Anhong

Zhou1,*

1Department of Biological Engineering, Utah State University, Logan, UT, U.S.A

2Division of Respiratory, Critical Care and Occupational Pulmonary Medicine, Department of

Medicine, School of Medicine, University of Utah, Salt Lake City, UT, U.S.A.

*Corresponding author:Anhong Zhou, Ph.D.Department of Biological EngineeringUtah State University4105 Old Main HillLogan, UT 84322U.S.A.Tel: 1(435)797-2863Fax: 1(435)797-1248Email: Anhong.Zhou@usu.edu

Raman and FTIR characterization of DEP

Raman spectra were recorded using a Renishaw inVia Raman microscope (controlled by

WiRE 3.0 software) equipped with a 785 nm near-IR laser. Laser light was focused through a

Leica 50 × 0.75 N PLAN optical glass microscope objective. The instrument was wavelength

calibrated with silicon at a static spectrum centered at 520.5 cm-1 for 1 second. Samples of DEP

were dropped on MgF2 and spectra were collected in static mode of 1 accumulation at 10 seconds

laser exposure over a wavenumber range of 100-3200 cm-1.

The FT-IR spectra were recorded using micro transmittance mode by Varian 660-IR

(Agilent Technologies) with Varian Resolution Pro software. The dry DEP sample was 1%

uniformly mixed with KBr powder and the spectra were collected with Universal Reflectance

Accessory at a single point spectrum for 64 scans with an 8 cm-1 resolution.

The Raman spectrum of DEP displays two distinct peaks, both are from the carbon core

of the DEP: a D peak and a G peak (Figure S3 (a)). The D peak (~1362 cm -1) is related to the

interaction between carbon atoms during the breathing vibrational mode, while the G peak

(~1597 cm-1) corresponds to the interaction between carbon atoms during the in-plane stretching

vibrational mode (Zhu et al., 2005).

Figure S1. Raman spectrum of dry DEP, dried DEP after suspended in methanol and dried DEP after

suspended in CH2Cl2 collected over a wavenumber range of 100-3200 cm-1 at 1% laser power for 10

seconds (a). Bright field image of dry DEP (b). Scale bar = 10 µm. FT-IR spectrum of DEP powder (c).

In FTIR spectrum, a few peaks were observed: 2360 cm-1 is from CO2 asymmetric

stretching (WebBook), 1646 cm-1 is assigned to C=N stretching of semicarbazones and/or

a b c

carbohydrazone (Lin-Vien et al., 1991), 1558 cm-1 could be contributed from carboxylic acid

salts (Zhu et al., 2005), 669 cm-1 would be from deformation vibration of CO2, S-N stretch, or

substituted benzenes (Lin-Vien et al., 1991). There were not strong peaks from the functional

groups of nitro-aromatic compounds, nitrate, ammonium, or sulfate compounds that were usually

presented in DEP (Arai, 1993; Polidori et al., 2008; Reff et al., 2005).

Cytokine and Chemokine analysis of DEP treated SAEC and A549 cells

Compared to A549 cells, SAEC released more cytokines and chemokines. Upon DEP treatment, SAEC released higher levels of IL-10, IL-12p70, IL-13, IL- 17,

Eotaxin, IP-10, MCP-1 and Figure S2. DEP induced cytokines and chemokines release from

SAEC and A549 cells before and after DEP treatment. Histograms of IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-

10, IL-12p70, IL-13, IL-15, IL-17, IL-23, IFNγ, TNFα, TNFβ, Eotaxin, I-309, IP-10, MCP-1, MCP-2 and

RANTES showed mean values of three independent experiments of both types of cells. Cells were

exposed to 10µg/mL DEP for 2h before measurement. Unit of Y-axis: ng/ml. Error bars are standard

deviation of the mean.

MCP-2, while higher levels of Eotaxin, MCP-1, MCP-2 and RANTES were only observed in A549 after exposure. Both Eotaxin and MCP-2 levels were found to be increased for SAEC cells more than A549 cells; however the amount of MCP-1 increased for SAEC is less than that of A549 cells (Fig. S4). The higher levels of cytokines/chemokines released due to pro-inflammatory responses (cytokines/chemokines), more sensitive the cells in response to DEP.

Effect of different donors on biochemical and biophysical properties of SAECs

Figure S3. (a) Averaged confocal Raman spectra of SAEC taken from two different donators at three

locations on cells: nucleus, cytoplasm, and cell membrane. (b) Difference spectra for the averages of

SAECs taken from two different donators at three locations on cells: nucleus, cytoplasm, and cell

membrane. (c) Score cluster plots with two principal components (PCs) for SAEC from different donors.

-4 -2 0 2 4 6x 104

-1500

-1000

-500

0

500

1000

1500

PC1

PC3

SAEC-1-nucSAEC-2-nucSAEC-1-cytSAEC-2-cytSAEC-1-memSAEC-2-mem

c d

a b

Spectra are collected on three locations per cell for four randomly selected cells. (d) Young’s modulus

and adhesion force of SAECs measured from two different donors.

To begin to explore sources of potential bias in our results, we repeated measurements using

SAEC from two distinct human donors (Fig. S3). In the three cellular regions selected for

Raman measurement, the SAEC from the two donors were similar in measurements and response

to DEP-treatment (not shown) but were distinguishable based on donor, and the extent of the

differences was further modified by the passage number of cell culture. However, SAEC from

each of the two donors were also similar in being markedly distinct in all measurements

compared with A549 cells (Fig 2-3).

Cell viability assessment

To test cell viability of SAEC and A549 after DEP exposure, the cell viability was analyzed

using LIVE/DEAD Viability/Cytotoxicity Assay Kit (Invitrogen) according to the

manufacturer’s instruction. Briefly, (1) cells cultured in culture flask were harvested and then

SAEC-Ctrl SAEC-DEP A549-Ctrl A549-DEP

BF

FL

Figure S4. Representative fluorescence images of cell viability evaluation. SAEC and A549 cells were

treated without DEP (Row 1, 3), and SAEC and A549 cells were exposed to 10 µg/ml DEP for 2 hours

(Row 2, 4). Cells are stained with Invitrogen LIVE/DEAD Viability/ Cytotoxicity Assay Kit. Green

fluorescence presents live cells, whereas red fluorescence shows dead or membrane-damaged cells. All

images were obtained with 10× lens. These fluorescence images together revealed that SAEC and A549

cells which were used for Raman and AFM experiments were mostly live.

suspended in fresh growth media, and 2 mL of cell suspension was seeded into poly-D-lysine

coated glass-bottom dishes (MatTek Cop. USA) and further cultured for 1 day, following by

DEP treatment; (2) cells were then washed two times with media prior to addition of fluorescent

reagents; (3) 200 µl of mixed solution of 2 µM Calcein AM and 4 µM ethidium homodimer-1

(EthD-1) (both from Invitrogen) was added directly to cells, and incubated cells for 30 mins at

room temperature; (4) cells were imaged using fluorescence microscope with DP30BW CCD

camera (Olympus IX71) to analyze the relative proportion of live/dead cells. Here, a 10×

objective was used to observe fluorescence. Calcein AM is well retained within live cells

producing green fluorescence; however, EthD-1 enters cells with damaged membrane and binds

to nucleic acids, thereby producing a red fluorescence in dead or membrane-damaged cells.

Therefore, the live/dead cells were differentiated visually.

Table S1. Tentative Raman band assignments of human small airway epithelial cells (SAECs)

and human lung carcinoma epithelial cell (A549)

Raman shift (cm-1)

SAEC A549 Band assignment

624 624 Phenylalanine

643 643 C-C twist Phenylalanine

662 662 C-S stretching mode of cystine (collagen type I)

666 666 G, T-tyrosine-G backbone in RNA

669 669 C-S stretching mode of cytosine

719 719 C-C-N + symmetric stretching in

phosphatidylcholine

720 720 DNA

762 762 Tryptophan

786 785 DNA & phosphdiester bands DNA

813 813 Phosphodiester bands RNA

832 832 PO2−¿¿stretch nucleic acids

854 853 Tyrosine

880 881 Tryptophan

939 939 Skeletal modes (polysaccharides)

961 961 Phosphate of HA; Calcium-phosphate stretch band

1006 1006 Phenylalanine

1034 1034 Phenylalanine

1066 1066 PO2−¿¿stretching; chain stretching; C-O, C-C

stretching

1070-90 1070-90 Symmetric PO2−¿¿stretching of DNA (represents more

DNA in cell)

1095 1095 Phosphodioxy group (PO2−¿¿ in nucleic acids);

Lipid

1129 1129 C-C skeletal stretch transconformation

1158 1158 Lipids and nucleic acids (C, G and A )

1179 1176 Cytosine, guanine

1213 1213 Tyrosine, phenylalanine

1254 1254 Lipid; A,T breathing mode (DNA/RNA); Amide III

(protein)

1304 1304 CH2 deformation (lipid), adenine, cytosine

1306 1306 C-N stretching aromatic amines

1317-9 1317-9 Guanine (B,Z-marker)

1343 1342 G (DNA/RNA); CH deformation (proteins and

carbohydrates)

1400-30 1400-30 γ(C=O) O- (amino acids aspartic & glutamic

acid)

1451 1450 CH2 deformation (nucleic acid, proteins,

lipids)

1579 1581 Pyrimidine ring (nucleic acids)

1608 1608 Phenylalanine, Tryptophan

1660 1661 Amide I

1740 1740 Collagen III

Band assignment is base on (Chan et al., 2006; Cheng et al., 2005; Chiriboga et al., 1998;

Movasaghi et al., 2007; Ruiz-Chica et al., 2004; Shetty et al., 2006; Stone et al., 2002; Stone

et al., 2004; Yu et al., 2006).

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