A Quantitative Toxicogenomics Assay for Highthroughput. and Mechanistic Genotoxicity. Assessment and Screening of Environmental
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1 SUPPLEMENTARY DATA A Quantitative Toxicogenomics Assay for Highthroughput and Mechanistic Genotoxicity Assessment and Screening of Environmental Pollutants Jiaqi Lan, Na Gou, Sheikh Mokhlesur Rahman, Ce Gao, Miao He *, and April Gu *, Department of Civil and Environmental Engineering, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, US. Environmental simulation and pollution control (ESPC) State Key Joint Laboratory, School of Environment, Tsinghua University, Beijing, , China * Corresponding Author: april@coe.neu.edu; hemiao@tsinghua.edu.cn Number of pages (including cover sheet): 16 Number of figures: 5 Number of tables: 1 S1
2 Part 1. Chemicals and concentration range in the study Table S1. Chemicals and concentration range applied in the study. Chemical 4-Nitroquinoline N- oxide (4-NQO) Mitomycin C (MMC) H 2 O 2 ( 35%wt. solution in water) Benzo [a] pyrene Lead (II) nitrate Ibuprofen Atrazine Trichloroacetic acid (TCA) N- nitrosodimethylamine (NDMA) Bromodichloromethane (BDCM) Chlorodibromomethane (CDBM) Formaldehyde Acetylsalicylic acid; Aspirin Source (CAS #; CAT#) Acros Organics ( ; AC ) RPI corp. ( ; M ) Sigma-Aldrich ( ; ml) Sigma-Aldrich ( ; B mg) Acros Organics ( ; AC ) Acros Organics ( ; AC ) AccuStandard ( ; P-005S) Fisher chemical ( ; A ) CHEM SERVICE INC ( ; N MG) ACROS Organics ( ; ) ALFA AESAR ( ; No.:A ) 37% by Weight, Fisher Chemical ( ; F79-500) Fisher Scientific ( ; S79881) Concentration range used in the test (mg/l) a , , , 2 dilution , , , , , , , , , ,000, Concentration for Comet test (mg/l) TD50 (mg/kg/day, mice) b 0.2 NA 0.5 NA NA 0.01 NA NA NA No positive S2
3 Tetracycline hydrochloride Erythromycin Bisphenol A Genlantis Inc ( ; M160025) MP Biomedicals (190197; ) Sigma-Aldrich ( ; G) , , , 10 No positive 100 No positive 10 No positive a The highest concentration for each chemical is up to the maximum noncytotoxic concentration with over 95% cells survives for 24 h exposure (LC5), see details in Figure S1. b data collected from the Carcinogenic Potency Database ( S3
4 Part 2. Cytotoxicity assessment of chemicals in this study. 120% Survival percentage 95% 80% 40% 4-N Q O MMC H 2 O 2 BaP+S9 Pb(N O 3 ) 2 Ibuprofen Atrazine NDMA BDCM formaldehyde Tetracycline A spirin Erythromycin BPA 0% TCA Concentration(mg/L) Figure S1. 24-hr cytotoxicity in yeast cells for chemicals tested in this study. X-axis: chemical concentration (mg/l), Y-axis: percentage of surviving cells indicated by OD600 compared to vehicle control. Mean±SD, n=4. The maximum noncytotoxic concentration is defined as survival percentage <95% (LC5, indicated by dashed line). S4
5 Part 3. Data processing. The alteration in protein expression for a given protein at each time point due to chemical exposure compared to untreated control is referred as induction factor I. LnI data are smoothed by calculating the simple moving average of every five data points over exposure time. For proteins that exhibited up-regulations, I (up-regulated) = I, when I 1; for proteins that showed downregulation, I (up-regulated) =1 when I<1. This is based on the the understanding that up-regulation of the biomarkers selected indicate potential activation of specific DNA damage repair, and the overall protein down regulations have been observed to be related to nonspecific cellular suppression effects. To quantify the chemical-induced protein expression level changes of a treatment, Protein Effect Level Index (PELI) for yeast was proposed and derived as quantitative molecular endpoints. 1-3 The accumulative altered protein expression change over the 2 h exposure period for a given protein (ORF) i was calculated as: PELI ORFi = tt tt=0 II (uuuu rrrrrrrrrrrrrrrrrr)dddd eeeeeeeeeeeeeeee tttttttt (Equation 1) Where, t was exposure time in hour. To identify the pathway activation response, the protein expression changes for all the proteins (ORFs) in a pathway were integrated as PELI pathway = nn ii=1 ww ii PELI OOOOOOOO (Equation 2) nn Where, n was the number of ORFs in one particular pathway, and w i was the weight factor of ORF i. For this study, we assigned value of 1 for all the weight factors. Similar to PELI pathway, the overall protein expression effect level for DNA damage and repair was integrated as PELI geno with all the PELI pathway in the pathway ensemble library as folowing: PPPPPPPP gggggggg = NN jj=1 WW jj PELI pppppphwwwwwwww NN (Equation 3) S5
6 Where, N was the number of pathways in this geno-sensor library. Wj was the weight factor of the pathway j and the value is assigned as 1 for this study. For each chemical, six PELI geno values were evaluated by mean±sd as examples shown below. PELI geno based dose-response pattern was modeled using Four Parameter Logistic (4PL) nonlinear regression model (the fitted curves). PELI max was determined as the top of the corresponding 4PL model (horizonal dashed lines). For chemicals with PELI max 1.5 (e.g., 4- NQO below), PELI1.5 was determined as the corresponding concentration that causes the PELI value to reach 1.5 (the vertical dashed line for 4-NQO). Figure S2. An example of dose-response curve fitting and endpoint derivation. References: 1. Lan, J.; Gou, N.; Gao, C.; He, M.; Gu, A., Comparative and Mechanistic Genotoxicity Assessment of Nanomaterials via A Quantitative Toxicogenomics Approach Across Multiple Species. Environmental Science & Technology 2014, 48, (21), O Connor, S. T. F.; Lan, J.; North, M.; Loguinov, A.; Zhang, L.; Smith, M. T.; Gu, A. Z.; Vulpe, C., Genome-wide functional and stress response profiling reveals toxic mechanism and genes required for tolerance to benzo [a] pyrene in S. cerevisiae. Frontiers in genetics 2012, Lan, J.; Hu, M.; Gao, C.; Alshawabkeh, A.; Gu, A. Z., Toxicity Assessment of 4-Methyl-1- cyclohexanemethanol and Its Metabolites in Response to a Recent Chemical Spill in West Virginia, USA. Environ Sci Technol 2015, 49, (10), S6
7 Part 4. Real-time protein expression profiles of the other fourteen chemicals tested in this study. A B S7
8 C D S8
9 E F S9
10 G H S10
11 I J S11
12 K L S12
13 M N S13
14 Figure S3. Temporal protein expression profiles of 38 biomarkers indicative of different DNA damage repair pathways upon exposure to mitomycin C 1 (A), H 2 O 2 (B), BaP 2 (C), Pb(NO 3 ) 2 (D), ibuprofen (E), atrazine (F), trichloroacetic acid (G), NDMA (H), BDCM (I), CDBM (J), formaldehyde (K), tetracycline hydrochloride (L), aspirin (M) and erythromycin (N) across six concentrations. The mean natural log of induction factor (ln I) indicates the magnitude of altered protein expression (represented by a green-black red color scale at bottom. Red spectrum colors indicate up regulation, green spectrum colors indicate down regulation. Values beyond ±1.5 are shown in the same color as ±1.5). X-axis top: concentrations for each chemical, X-axis bottom: testing time in minutes, the first data point shown is at 20 min after exposure due to data smoothing with moving average of every five data points. Y-axis left: clusters of proteins by DNA damage repair pathways; Y-axis right: list of proteins (ORFs) tested, with details in Table 1. n=3. S14
15 Part 5. Results of Comet assay in human A549 cells exposed to different chemicals and controls ** 100 % Tail DNA ** ** * * * ** ** ** ** ** 0 Figure S4. DNA strand breakage measured by alkaline comet assay (%Tail DNA) for 24-hour exposure in human A549 cells. X-axis: treatments at selected concentrations for different chemicals. N: untreated control. Y-axis: % Tail DNA. * compared with vehicle control, p<0.05, ** compared with vehicle control, p<0.01. Mean±SD, n=3. (For MMC and BPA, see reference 1 ) S15
16 Part 6. Principal component analysis (PCA) of the 16 chemicals tested this study. Figure S5. Principal component analysis (PCA) based on differential protein expression profiles (lni, average of triplicates) of 38 DNA damage repair proteins in GFP-tagged yeast cells exposed to sixteen tested chemicals across six concentrations for each chemical. Samples are color coded according to chemicals and each sphere represents one treatment (a chemical at a given concentration) with sphere size indicating the relative level of concentration. S16
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