This study examined changes in the proteome of lung epithelial cells exposed to low doses of two endocrine disruptive chemicals, TCDD and arsenic trioxide. Immortalized human lung epithelial cells were treated with different doses of each chemical for 24 hours. The proteins were then extracted, labeled, and analyzed using mass spectrometry. TCDD exposure induced changes in several proteins involved in extracellular matrix, cell regulation, and mitochondria. In contrast, arsenic trioxide did not cause any significant changes in protein expression levels. The results provide insight into the molecular pathways affected by these endocrine disruptive chemicals.

1. A Proteomic Analysis of Lung Epithelial Cells Following Exposure to the Endocrine
Disruptive Chemicals 2,3,7,8-tetrachlorodibenzo-p-dioxin and Arsenic Trioxide
Ryan P. Lynch & David M. Smalley
Maine Institute for Human Genetics & Health, 246 Sylvan Road, Bangor ME 04401 (rlynch@emh.org)
Abstract 2,3,7,8-tetrachlorodibenzo-p-dioxin Protein Expression Influenced By
TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and arsenic are environmental endocrine
disruptive chemicals (EDCs) that increase risk of lung cancer. While many studies Treated Human Bronchial Epithelial Cells 2,3,7,8-tetrachlorodibenzo-p-dioxin
have attempted to examine the pathways associated with the transformation of normal
epithelial cells into malignant cells, the mechanisms remain unclear. The goal of the Peptides from 2 altered Peptides from 2 proteins
present study was to examine changes in the proteome of lung epithelial cells following proteins with no change
exposure to low doses of these environmental EDCs. Immortalized human lung
epithelial cells (NuLi-1) were treated with TCDD (2, 10, 50 nM) or As2O3 (0.5, 2, 10
µM), or vehicle for 24 hrs, and then lysed with 6M urea/PBS. The proteins were
reduced, alkylated, and digested, and the peptides were isolated. The peptides were
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then differentially labeled with an isobaric tag (iTRAQ) and the samples were mixed.
Following 2D separation (isoelectric focusing and reverse phase-LC), the peptides
were analyzed by MALDI-TOF/TOF using an Applied Biosystems 4800 Plus mass
spectrometer. The spectra were analyzed using Protein Pilot software with the
Paragon algorithm. Each EDC treatment set generated approximately 1000 proteins.
Relative quantitation was performed on proteins with at least 2 peptides identified.
While most proteins remained unchanged, several were significantly altered following
TCDD exposure, and none after arsenic. These included proteins previously reported
to be altered, as well as others that we believe are novel. Proteins involved with the
extracellular matrix, cell regulation, and a number of mitochondrial associated proteins
are shown to be altered. The results of this study are currently being validated using
alternative strategies.
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Materials & Methods
Downregulated Proteins Upregulated Proteins
(>3 SD from mean; >4 peptides) (>3 SD from mean; >4 peptides)
Protein Biological Function Subcellular Location Protein Biological Function Subcellular Location
Beta-actin-like protein 2 Cell motility Cytoplasm; cytoskeleton Blocks the elongation and
Tropomodulin-3 depolymerization of the actin filaments Cytoplasm; cytoskeleton
Non-histone chromosomal protein HMG-17 Chromatin organization Nucleus; cytoplasm Tenascin Signal transduction Extracellular matrix
40S ribosomal protein S21 Translational elongation Transcriptional regulation; cell cycle
High mobility group protein HMGI-C regulation Nucleus
Mitochondrial ATP synthesis coupled Mitochondria; mitochondria Elongation factor 1-beta Translational elongation
ATP synthase subunit b, mitochondrial proton transport inner membrane L-lactate dehydrogenase B chain Anaerobic glycolysis; oxidation reduction Cytoplasm
Histone H2A type 1-C Nucleosome assembly Nucleus Cell redox homeostasis; oxidation Endoplasmic reticulum
Thioredoxin domain-containing protein 12 reduction lumen
Hydroxymethylglutaryl-CoA synthase, Cholesterol/isoprenoid biosynthetic
60S acidic ribosomal protein P1 Translational elongation
cytoplasmic process Cytoplasm
26S proteasome non-ATPase regulatory
Microtubule-based movement; protein
subunit 2 Regulation of protein catabolic process
As2O3 Treated Human
Tubulin beta-3 chain polymerization
Tyrosine-protein phosphatase non- Negative regulation of insulin receptor Endoplasmic reticulum
Intermediate filament organization; skin receptor type 1 signaling pathway membrane
Keratin, type I cytoskeletal 9 development Rho protein signal transduction; actin
PHF3 Isoform 1 of PHD finger protein 3 Transcription Nucleus Myosin IXB isoform 1 filament-based movement Cytoplasm
Bronchial Epithelial Cells
Isoform Beta of Tripartite motif-containing Transcription from RNA polymerase II Methionine adenosyltransferase 2 subunit Extracellular polysaccharide biosynthetic
protein 29 promoter Cytoplasm beta process; one-carbon metabolic process
DNA replication; double-strand break Putative RNA-binding protein 3 (RBM3) Positive regulation of translation Cytoplasm; nucleus
Flap endonuclease 1 repair Nucleus Nucleus envelope;
Transport; lipid metabolic process; Protein S100-A6 Signal transduction cytoplasm
Fatty acid-binding protein, epidermal epidermis development Cytoplasm Actin, cytoplasmic 1 Cellular component movement Cytoplasm; cytoskeleton
Hemoglobin subunit epsilon Oxygen transport Ras GTPase-activating protein-binding
Neurotransmitter catabolic process; Mitochondria outer protein 2 Transport; Ras protein signal transduction Cytoplasm
Monoamine oxidase A oxidation reduction membrane YWHAH 14-3-3 protein eta Transport; cell signalling
40S ribosomal protein S25 Translational elongation Cytoplasm; mitochondria;
Isoform 2 of Proteasome activator Protein ETHE1, mitochondrial Mitochondrial metabolic homeostasis nucleus
complex Apoptosis regulation Cytoplasm; nucleus Histone H1.4 Nucleosome assembly Nucleus
Mucin-16 Cell adhesion Cell membrane
Hepatoma-derived growth factor Cell proliferation; transcription regulation; Cytoplasm; nucleus
Eukaryotic translation elongation factor 1
epsilon-1 Translation regulation; DNA damage repair Cytoplasm; nucleus
Copyright Maine Institute for Human Genetics & Health March 7, 2010
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Summary/Conclusions
•Low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin induced expression of multiple
proteins in human bronchial epithelial cells, whereas no significant changes in
protein expression were observed after treatment with arsenic trioxide.
•Two-fold changes in relative protein levels from 4 samples can easily be
detected for the 100 most abundant proteins using iTRAQ labeling with a
Database Search Results minimum of 2 peptides per protein.
As2O3 Treated Human Bronchial Epithelial Cells
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Set 1 Set 2 Set 3 Cumulative
Spectra (total) 11316 18442 18560 48318
Spectra (identified) 10058 12689 13861 39274
Peptides 5848 8360 8672 18862
Proteins 957 923 1009 1583