F A d Th h 8/31 2015 orm 'oorove roua1 I OMS N 0925 0001 0 . -

Department of Health and Human Services LEAVE BLANK-FOR PHS USE ONLY.

Public Health Services Tvoe IActivitv Number

Grant Application Review Group Formerly

Do not exceed character length restrictions indicated. Council/Board (Month, Year) Date Received

1. TITLE OF PROJECT (Do not exceed 81 characters, including spaces and punctuation.)

2. RESPONSE TO SPECIFIC REQUEST FOR APPLICATIONS OR PROGRAM ANNOUNCEMENT OR SOLICITATION D NO l:8J YES (If "Yes," state number and title)

Number: N/A ntte HICAHS Pilot/Feasibility Research Projects

3. PROGRAM DIRECTOR/PRINCIPAL INVESTIGATOR

3a. NAME (Last, first, middle) 3b. DEGREE(S) 3h. eRA Commons User Name

Dooley, Greg, Patrick BS MS PhD GREGD1

3c. POSITION TITLE 3d. MAILING ADDRESS (Street, city, state, zip code) Assistant Professor 1680 Campus Delivery

3e. DEPARTMENT, SERVICE, LABORATORY, OR EQUIVALENT Colorado State University Center for Environmental Medicine

Fort Collins, CO 80523 3f. MAJOR SUBDIVISION

CVMBS 3g. TELEPHONE AND FAX (Area code, number and extension) E-MAIL ADDRESS:

TEL: 970-491-5128 FAX: gdooley@colostate.edu

4. HUMAN SUBJECTS RESEARCH 4a. Research Exempt lf "Yes," Exemption No.

l:8J No D Yes l:8J No D Yes

4b. Federal-Wide Assurance No. 4c. Clinical Trial 4d. NIH-defined Phase Ill Clinical Trial

l:8J No D Yes l:8J No D Yes

5. VERTEBRATE ANIMALS ~ No D Yes 5a. Animal Welfare Assurance No.

6. DATES OF PROPOSED PERIOD OF 7. COSTS REQUESTED FOR INITIAL 8. COSTS REQUESTED FOR PROPOSED SUPPORT (month. dav. vear- MMIDD!YY) RI Jnr,FT ll

From Through 7a. Direct Costs ($}

09/01/13 9/01/14 $25,000

9. APPLICANT ORGANIZATION Name Colorado State University

Address Sponsored Programs

601 S. Howes Street

Fort Collins, CO 80523-2002

12. ADMINISTRATIVE OFFICIAL TO BE NOTIFIED IF AWARD IS MADE Name Linda Monum

Title Senior Research Administrator

Address Sponsored Programs 601 S. Howes Street

Fort Collins, CO 80523-2002

Tel: 970-491-097 4 FAX: 970-491-6147

E-Mail: linda.monum@colostate.edu 14. APPLICANT ORGANIZATION CERTIFICATION AND ACCEPTANCE I certify that the statements herein are true, complete anel accurate to the best of my l<nowleelge, anel accept the obligation to comply with Public Health Services terms anel conelitions if a grant is awareled as a result of this application. I am aware that any false, fictrtious, or frauelulent statements or daims may subject me to criminal, civil, or aelministrative penalties.

PHS 398 (Rev. 08/12) Face Page

1

PFRIOn 11~ C:: I ·- ,,_,I

7b. Total Costs($) Sa. Direct Costs ($) Sb. Total Costs {$)

25000 $25,000 25000 10. TYPE OF ORGANIZATION

Public: -+ D Federal l:8J State D Local

Private: -+ D Private Nonprofit

For-profit: -+ D General D Small Business

D Woman-owned D Socially and Economically Disadvantaged

11. ENTITY IDENTIFICATION NUMBER 1-846000545-A 1

DUNS NO. 78-597 -9618 I Cong District C0-002

13. OFFICIAL SIGNING FOR APPLICANT ORGANIZATION Name Tracey Castaneda

Title Research Administrator

Address Sponsored Programs 601 S. Howes Street

Fort Collins, CO 80523-2002

Tel: 970-491-1560 FAX: 970-491-6147

E-Mail: tracey.castaneda@colostate.edu SIGNATURE OF OFFICIAL NAMED IN 13. DATE (In ink. "Per" signature not acceptable.)

Form Page 1

Program Director/Principal Investigator (Last, First, Middle): Dooley, Greg, Patrick PROJECT SUMMARY (See instructions):

Poultry workers have lower overall lung function compared to other agricultural workers (Radon et al. 2001) suggesting a need for a better understanding of the underlying cause of the respiratory impairment. Exposure to organic dust is one component of the poultry production process that increases the risk of acute and chronic respiratory impairment (Viegas et al. 2013, Zuskin et al. 1995.) Little research has been conducted to characterize poultry dust with regard to pro-inflammatory small molecules that may be contributing to respiratory inflammation. We plan to develop a protocol to characterize small molecules in poultry dust that are pro-inflammatory using a combinaiton of mass spectrometry and biological assays. Aqueous poultry dust extracts will be fractionated with liquid chromatography and fractions applied to a human monocytic cell line, THP-1, to determine interleukin-8 induction as a marker of inflammation. Characterization of the most reactive fractions will be done using LC-QTOF mass spectrometry with constituent molecules identified by searching against various accurate mass databases. Identified molecules associated with inflammation will be investigated further with analytical standards to provide positive identification of potentially active molecules. Positively identified molecules with pro-inflammatory potential will be evaluated individually in the IL-8 assay to confirm their inflammatory activity. This proposal will provide a protocol to isolate and target pro-inflammatory small molecules from a very complex dust matrix for identification by mass spectrometry. Resutls will expanded the current chemical characterization of poultry dust and the understanding of specific components contributing its inflammatory activity .

RELEVANCE (See instructions): The significance of this project is to fill in the knowledge gap of pro-inflammatory small organic molecules that are present in poultry dust. The understanding of which molecules maybe be contributing to respiratory impairment may indicate the source and potentially provide a means of intervention to reduce their presence and the inflammatory activity of the dust.

PROJECT/PERFORMANCE SITE(S) (if additional space is needed, use Project/Performance Site Format Page) Project/Performance Site Primary Location

Organizational Name: Colorado State University (CSU) DUNS: 78-597-9618 Street 1: 1680 Campus Delivery Street 2:

City: Fort Collins County: Larimer State: CO Province:

Country: USA Zip/Postal Code: 80523 Project/Performance Site Congressional Districts: CO-4 Additional Project/Performance Site Location

Organizational Name: University of Texas Health Science Center (UTHSC) DUNS:

Street 1: 11937 US Highway 271 Street 2:

City: Tyler County: Smith State: TX Province:

Country: USA Zip/Postal Code: 75708 Project/Performance Site Congressional Districts: TX-01 PHS 398 (Rev. 08/12 Approved Through 8/31/2015) OMB No. 0925-0001 Page 2 Form Page 2

Program Director/Principal Investigator (Last, First, Middle): Dooley, Greg, Patrick

SENIOR/KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Program Director(s)/Principal lnvestigator(s) List all other senior/key personnel in alphabetical order, last name first.

Name eRA Commons User Name

Greg Dooley GREGD1

Stephen Reynolds

Rena Saito

OTHER SIGNIFICANT CONTRIBUTORS

Name

Human Embryonic Stem Cells ~ No

Organization

Oves

Organization Role on Project

CSU Principle Investigator

CSU Faculty Collaborator

UTHSC Co-Investigator

Role on Project

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list:

http://stemcells.nih.gov/research/registry/eligibilityCriteria.asp. Use continuation pages as needed.

If a specific line cannot be referenced at this time, include a statement that one from the Registry w ill be used.

Cell Line

PHS 398 (Rev. 08/12 Approved Through 8/31 /2015) Page~

OMB No. 0925-0001 Form Page 2-continued

Number tile following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b.

Program Director/Principal Investigator (Last, First, Middle): Dooley, Greg, Patrick

The name of the program director/principal investigator must be provided at the top of each printed page and each continuation page

RESEARCH GRANT

TABLE OF CONTENTS

Face Page ................................................................................................................................................ .

Description, ProjecUPerformance Sites, Senior/Key Personnel, Other Significant Contributors, and Human Embryonic Stem Cells ...................................................................................................... ..

Table of Contents .................................................................................................................................... .

Detailed Budget for Initial Budget Period ............................................................................................ ..

Budget for Entire Proposed Period of Support ............................................................................................. .

Budgets Pertaining to Consortium/Contractual Arrangements ......................................................... .

Biographical Sketch - Program Director/Principal Investigator (Not to exceed four pages each) ....... . .

Other Biographical Sketches (Not to exceed four pages each- See instructions) .... .. .... .................. .. .

Resources ................................................................................................................................................ .

Checklist .................................................................................................................................................. .

Research Plan ......................... ..... ........................................................................................................... .

1. Introduction to Resubmission Application , if applicable, or Introduction to Revision Application, if applicable * ....... ........................... ...... ... ...................... .... ....... .... .. ....................... .... ....... ................. .

2. Specific Aims * ...... ......... ....... ...... ..... ......... ....... ......... ...... ... ........... ... .... ......... ......... ........... ... .... ......... . .

3. Research Strategy * ....... ....... ......... ...... ... ......... ......... ..... .... ......... ......... ......... ..... .... .... ..... .... ..... .... ..... . .

4. Inclusion Enrollment Report (Renewal or Revision applications only) ............. ... .... ......... .... ..... ......... . .

5. Bibliography and References Cited/Progress Report Publication List.. ......... ......... ........... .. ..... .... ..... . .

6. Protection of Human Subjects ..... ..... ......... ......... ....... ...... ... ........... ....... ......... ......... ........... ... .... .... ..... . .

7. Inclusion of Women and Minorities ....... ... ......... ......... ..... .... ......... ......... ......... ..... .... ......... ......... ......... . .

8. Targeted/Planned Enrollment Table .................. ... .............. ........................ ........ .... ........ ... ....... ......... . .

9. Inclusion of Children ....... ....... ........... ......... ....... ......... ......... ........... ... .... ......... ......... ........... ... .... ......... . .

10. Vertebrate Animals ......... ......... .... ..... .... ... ......... ......... ...... ... ......... ......... ......... ........... .. ..... ....... ...... ..... . .

11 . Select Agent Research ... ....... ........... ......... ....... ......... ......... ........... ... .... ......... ........... ......... ................. .

12. Multiple PD/Pl Leadership Plan ........ .... ... ......... ......... ..... .... ......... ......... ......... ..... .... ......... ......... .... ..... . .

13. Consortium/Contractual Arrangements ... ......... ......... ......... ......... ......... ......... ..... .... ......... ......... ......... . .

14. Letters of Support (e.g., Consultants) ........ ....... ......... ...... ... ........... ... .... ......... ......... ........... ... .... ......... . .

15. Resource Sharing Plan (s) ..... ...... ..... ......... ....... ......... ...... ... ........... ....... ......... ......... ........... ... .... ......... . .

Page Numbers

1

2 4 5

8 10

12 13-17

Appendix (Five identical CDs.) D Check if Appendix is Included

* Follow the page limits for these sections indicated in the application instructions , unless the Funding Opportunity Announcement specifies otherwise.

PHS 398 (Rev. 08/12 Approved Through 8/31/2015) Page 1

OMB No. 0925-0001 Form Page 3

Program Director/Principal Investigator (Last, Rrst, Middle): Dooley, Greg, Patrick

DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY

List PERSONNEL (Applicant organization only) Use Cal, Acad, or Summer to Enter Months Devoted to Project Enter Dollar Amounts Requested (omit cents) for Salary Requested and Fringe Benefits

ROLE ON Cal. Acad. Summer INST.BASE NAME PROJECT Mnths Mnths Mnths SALARY

Greg Dooley PD/Pl 1 -

SUBTOTALS -. CONSUL TANT COSTS

EQUIPMENT (Itemize)

Agilent 1260 Infinity Fraction Collector

SUPPLIES (Itemize by category)

Liquid Chromatography consumables(- ) Analytical Standards (- )

TRAVEL

INPATIENT CARE COSTS

OUTPATIENT CARE COSTS

AL TERA TIONS AND RENOVATIONS (Itemize by category)

OTHER EXPENSES (Itemize by category)

CONSORTIUM/CONTRACTUAL COSTS I SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item la, Face Page)

FROM

10/1/13

SALARY REQUESTED ..

THROUGH

10/1/14

FRINGE BENEFITS TOTAL .. ..

I

-

-

DIRECT COSTS

$ 25,ooo I CONSORTIUM/CONTRACTUAL COSTS I FACILITIES AND ADMINISTRATIVE COSTS

TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD

PHS 398 (Rev. 08/12 Approved Through 8/31/2015) Page§.

$ 25.ooo I OMB No. 0925-0001

Form Page 4

Program Director/Principal Investigator (Last, First, Middle): Dooley, Greg, Patrick

BUDGET JUSTIFICATION GRANT NUMBER N/A

Provide a detailed budget justification for those line items and amounts that represent a significant change from that previously recommended. Use continuation pages if necessary. Personnel ($ ) Dooley, Greg, Patrick, Ph.D. Assistant Professor, Colorado State University. 1 calender person-months (10%) effort in Year 1. Dr. Dooley is an Assistant Professor in the Department of Environmental and Radiological Health Sciences and Director of Analytical Services for the Center for Environmental Medicince at Colorado State Univeristy. As the Director of the CEM Analytical Laboratory, he supervises all the organic chemical analysis performed in the laboratory and handles acquisition and coordination of revenue bases client projects. His research involves the development of analytical methods utilizing state of the art instrumentation to measure organic molecules of current interest, specifically drugs and pharmaceutials in the environment. He has applied determination of illict drugs in wastewater effluent to evaluate community drug usage pattern in Northern Colorado. Dr. Dooley will be the principal investigator on this project and will be responsible for coordination of sampling and assay logistics with UTHSC. He will devote significant effort into development of the sample fractionation protocol, mass spectral data analysis, and data interpretation aspects of this proposal. The LC-QTOF MS system to be used to generate the mass spectral data is housed in the Center for Environmental Medicine Analytical Laboratory under Dr. Dooley's supervison.

CURRENT BUDGET PERIOD FROM 10/1/13

THROUGH 10/1/14

Explain any estimated unobligated balance (including prior year carryover) that is greater than 25% of the current year’s total budget. N/A

PHS 2590 (Rev. 08/12) Page 6 Form Page 3

Program Director/Principal Investigator (Last, First, Middle): Dooley, Greg, Patrick

Budget Justification (cont'd)

~l A state of the art fraction collector that is compatible with our current Agilent LC-MS system is critcal to the

success of this proposal. The ability to collect reproducable LC fractions of aqueous dust extract is necessary to simplify the dust matrix for evaluation of inflammatory potential with the IL-8 assay and characterization of

small molecules by mass spectrometry. An Agilent 1260 Infinity fraction collector is compatible with our current Agilent 1290 LC with a 6530 QTOF MS. This specific fraction collector has the unique feature of retention time

specific fraction collection which will be useful to maximize reproducibility and limit the variation of the IL-8 assay results.

SuQ lies-We will need funds to buy consumable supplies necessary to conduct sample preparation and the liquid chromatagraphy-mass spectrometry aspects of this proposal. This includes LC columns, solvents, chemical

reagents, molecular weight filters, LC vials and caps.

Travel ($0) Funds will not be necessary for travel to Fort Collins, CO to present research finding at a HICAHS seminar

since Dr. Dooley currenly lives in Fort Collins, CO.

OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015) Page.]__ Continuation Format Page

BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contr butors. Follow this format for each person. DO NOT

EXCEED FOUR PAGES. NAME Dooley, Greg Patrick

POSITION TITLE Assistant Professor Director of Analytical Services Center for Environmental Medicine

eRA COMMONS USER NAME (credential, e.g., agency login) GREGD1 EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION DEGREE (if applicable) MM/YY FIELD OF STUDY

Frostburg State University, Frostburg, MD B.S. 06/00 Fisheries Biology University of Georgia, Athens, GA M.S. 06/02 Toxicology Colorado State University, Fort Collins, CO PhD. 06/07 Toxicology Colorado State University, Fort Collins, CO Postdoctoral 2007-2009 Proteomics

A. Personal Statement My research expertise is in the quantitative analysis of organic chemicals and using proteomics to identify interactions of proteins and xenobiotics During my PhD and Post-Doctoral training, I used several proteomic strategies to identify and characterize novel proteins susceptible to covalent modification by commonly applied herbicide atrazine. Currently as the Director of the Center for Environmental Medicine Analytical Laboratory at CSU, I manage the state of the art analytical instrumentation and oversee the development and implementation analytical methods for a variety of quantitative analysis for both small and macromolecules. The CEM Molecular Analytical Laboratory is a core facility for the Department of Environmental Health Sciences at CSU equipped with the instrumentation (LC-QTOF-MS) and expertise required to support the identification of novel pro-inflammatory small molecules from poultry dust. I am confident the resources available at the CEM Analytical Laboratory will greatly facilitate this research proposal.

B. Positions and Honors

Positions and Employment 2000-2002 Graduate Research Assistant - Department of Environmental Health Sciences, University of

Georgia, Athens, GA. 2001-2002 Graduate Teaching Assistant - Department of Environmental Health Sciences, University of

Georgia, Athens, GA. 2002-2003 Analytical Chemist - Maryland Spectral Services, Baltimore, MD. Chemical analysis of

environmental samples using GC and GC-MS. 2003-2005 Graduate Teaching Assistant - Department of Environmental and Radiological Health Sciences,

Colorado State University, Fort Collins, CO. 2005-2007 Graduate Research Assistant - Department of Environmental and Radiological Health Sciences,

Colorado State University, Fort Collins, CO. 2007-2009 Postdoctoral Fellow – Syngenta Crop Corporation, Colorado State University, Fort Collins, CO.

Analysis of in vivo and in vitro interactions between proteins and triazine herbicides. 2009- Assistant Professor and Director of Analytical Services – Center for Environmental Medicine,

Colorado State University, Fort Collins, CO. Trace level analytical analysis with LC-MS/MS and GC-MS/MS.

Other Experience and Professional Memberships 2000-2002 Member, Society of Environmental Toxicology and Chemistry 2000-2002 Member, American Fisheries Society 2003-2007 Member, Society of Toxicology

8

C. Selected Peer-reviewed Publications

Most relevant to the current application 1. Dooley GP, Reardon KF, Prenni JE, Tjalkens RB, Legare ME, Foradori CD, Tessari JE, Hanneman WH.

2008. Proteomic analysis of diaminochlorotriazine adducts in wistar rat pituitary glands and LBT2 rat pituitary cells. Chem Res Toxicol. 21, 844-51. PMID: 18370413

2. Dooley GP, Ashley AK, Legare ME, Handa RJ, Hanneman WH. 2010. Proteomic Analysis of Diaminochlorotriazine (DACT) Adducts in three brain regions of Wister Rats. Toxicol Lett. [Epub ahead of print] PMID: 20688138

3. Dooley, GP, Prenni, JE, Prentiss, PL, Cranmer, BK, Andersen, ME, Tessari, JD. 2006. Identification of a Novel Hemoglobin Adduct in Sprague Dawley Rats Exposed to Atrazine. Chem. Res. Toxicol. 19, 692-700. PMID: 16696572D.

4. Dooley GP, Hanneman WH, Carbone DL, Legare ME, Andersen ME, Tessari JD. 2007. Development of an immunochemical detection method for atrazine-induced albumin adducts. Chem Res Toxicol. 20,1061-6. PMID: 17567052

5. Poole JA, Dooley GP, Saito R, Burrell AM, Bailey KL, Romberger DJ, Mehaffy J, Reynolds SJ. 2010. Muramic acid, endotoxin, 3-hydroxy fatty acids, and ergosterol content explain monocyte and epithelial cell inflammatory responses to agricultural dusts. J Toxicol Environ Health A. 73684-700. PMID: 20391112

6. Garcia J, Bennett DH, Tancredi D, Schenker MB, Mitchell D, Reynolds S, Dooley GP, Mehaffey J, Mitloehner FM. Characterization of endotoxin collected on California dairies using personal and area based methods. 2012 J. Occup and Environ Health. 10, 580-91 PMID: 22946649

7. Dooley GP, Tjalkens RB, Hanneman WH. The atrazine metabolite diaminochlorotriazine suppresses LH release from murine LßT2 cells by suppressing GnRH-induced intracellular calcium transients. 2013 Toxicol. Res. 3, 180 - 186

D. Research Support

Ongoing Research Support NONE

Completed Research Support EPA STAR GRANT R-8286-10-01-0 Tessari (PI) 08/15/03-05/14/06 Chlorotriazine Protein Binding: Biomarkers of Exposure & Susceptibility The main objective of this research project was to test the hypothesis that binding of chlorotriazines by hemoglobin and hair proteins can be used to evaluate differences in exposure and in individual sensitivity toward chlorotriazines. Role: Graduate Research Assistant Syngenta Crop Research Program Hanneman (PI) 05/15/06- 11/30/08 Analysis of in vivo and in vitro interactions between proteins and triazine herbicides. The main objective of this research project was to investigate the formation of covalent atrazine adducts to proteins in vivo and in vitro following atrazine exposure and its relation the suppresion of luetenizing hormore release. Role: Post Doctoral Fellow

9

BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME Saito, Rena

POSITION TITLE Assistant Professor

eRA COMMONS USER NAME (credential, e.g., agency login) EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION DEGREE (if applicable) MM/YY FIELD OF STUDY

The University of Washington, Seattle, WA B.S. 06/04 Environmental Health Colorado State University, Fort Collins, CO Ph.D. 05/08 Environmental Health

(Industrial Hygiene) Colorado State University, Fort Collins, CO Postdoctoral 12/08 Industrial Hygiene

A. Personal Statement Rena Saito, Ph.D., CIH, is an Assistant Professor of the Department of Occupational Health Sciences at The University of Texas Health Science Center at Tyler (UTHSCT). Prior to joining the University in April 2012, she was working in the Division of Respiratory Disease Studies at the National Institute for Occupational Safety and Health (NIOSH) as a research industrial hygienist. At NIOSH, her duties included health hazard evaluation, sampling and exposure assessment, laboratory analyses of bioaerosols, and statistical analysis. During her years at NIOSH, she was involved in various projects, such as flavoring lung disease, metals (indium tin oxide), chemical use in the healthcare industry, and indoor environment quality. Dr. Saito received her Ph.D. in 2008 from Colorado State University with a major in environmental health and industrial hygiene. Her doctoral research focused on chemical analysis of bacterial endotoxins in dust collected from various agricultural environments and studied the variability in endotoxin measurements in agricultural environments. She is a certified industrial hygienist, and she is the person in charge of UTHSCT Occupational and Safety Laboratory housing various industrial hygiene and chemical analysis equipment, including liquid chromatography/ tandem mass spectroscopy.

B. Positions and Honors Positions 2003 Public Health Intern, Seattle & King County Public Health, Seattle, WA 2005 – 2008 Graduate Research Assistant, Colorado State University, Fort Collins, CO 2008 Post-doctoral Fellow, Colorado State University, Fort Collins, CO 2009 – 2012 Associate Service Fellow, DRDS, NIOSH, CDC, Morgantown, WV 2012 – Present Assistant Professor, Occupational Health Sciences, University of Texas Health Science

Center at Tyler, Tyler, TX Honors 1999 – 2001 Washington Promise Scholarship 2004 – 2005 NIOSH Training Program Grant 2005 – 2006 Colorado State Graduate Fellowship 2006 Jerry Lynch Memorial Scholarship, American Industrial Hygiene Foundation 2006 Ken Dillon Memorial Award for the Best Student Poster (AIHce 2006) 2007 – 2008 Kirke L. Martin Memorial Scholarship, Colorado State University 2009 Best Poster, Sampling and Laboratory Analysis Committee (AIHce 2009) 2012 Rising STARS Award, the University of Texas System

10

C. Selected Peer-reviewed Publications 1. Saito R, Cranmer B, Tessari J, Reynolds S. Modification and application of a GC/MS method for endotoxin

analysis in agricultural dusts. Proceedings of the 4th Annual Regional National Occupational Research Agenda (NORA) Young/New Investigators Symposium. 2006;117-125.

2. Saito R, Cranmer BK, Tessari JD, Larsson L, Mehaffy JM, Keefe TJ, Reynolds SJ. Recombinant Factor C (rFC) assay and gas chromatography / mass spectrometry (GC/MS) analysis of endotoxins in four agricultural dusts. Ann Occup Hyg. 2009;53:713-722.

3. Burch JB, Svendsen E, Siegel PD, Wagner SE, Von Essen S, Keefe T, Mehaffy J, Serrano-Martinez A, Bradford M, Baker L, Cranmer B, Saito R, Tessari J, Koehncke J, Prinz L, Reynolds SJ. Endotoxin exposure and inflammation markers among agricultural workers in Colorado and Nebraska. J Environ Tox. 2010;73:5-22.

4. Poole JA, Dooley GP, Saito R, Burrell AM, Bailey KL, Romberger DJ, Mehaffy J, Reynolds SJ. Muramic acid, endotoxin, 3-hydroxy fatty acid, and ergosterol content explain monocyte and epithelial cell inflammatory responses to agricultural dusts. J Tox Environ Health, Part A. 2010;73:684-700.

5. Thorne PS, Perry SS, Saito R, O’Shaughnessy PT, Mehaffy J, Metwali N, Keefe T, Donham KJ, Reynolds SJ. Evaluation of the limulus amebocyte lysate and recombinant factor C assays for assessment of airborne endotoxin. Appl Environ Microbiol. 2010;76:4988-4995.

6. Cox-Ganser J, Ganser G, Saito R, Hobbs G, Boylstein R, Hendricks W, Simmons M, Eide M, Kullman G, and Piacitelli C. Correcting diacetyl concentrations from air samples collected with NIOSH Method 2557. J Occup Environ Hyg. 2011; 8:59-70.

7. Cummings KJ, Nakano M, Omae K, Takeuchi K, Chonan T, Xiao YL, Harley RA, Roggli VL, Hebisawa A, Tallaksen RJ, Trapnell BC, Day GA, Saito R, Stanton ML, Suarthana E, Kreiss K. Indium lung disease. Chest. 2012; 141:1512-1521

8. Cummings KJ, Suarthana E, Edwards N, Liang X, Stanton ML, Day G, Saito R, and Kreiss K. Serial Evaluations at an Indium-Tin Oxide Production Facility. Am. J. Ind. Med. 2013; 56:300-307.

9. Huff S, Stocks JM, Saito R, Bilhartz P, Levin J, Glazer C, Bailer R, Cummings K, Kreiss K, McCague AB. Obliterative Bronchiolitis amongworkers in a coffee-manufacturing facility – Texas, 2009-2012. MMWR Morb Mortal Wkly Rep. 2013; 62:305-307.

D. Research Support Completed

1. 2008: Rocky Mountain Center for Occupational and Environmental Health/ NIOSH – “Chemical analyses of bacterial composition and potency in agricultural dust.” Role: Principle Investigator (PI transferred to L. Goodridge).

2. 2008: High Plains Intermountain Center for Agricultural Health and Safety/ NIOSH – “Inflammatory relationship of gram positive and gram negative bacteria in occupational and agricultural environments.” Role: Co-Investigator (PI: J. Poole).

3. 2010 – 2012: NIOSH Intramural Project – “Developing exposure assessment methods & survey questionnaire for the international study of asthma in healthcare.” Role: Key Personnel (lead field industrial hygienist) (PI: Virji).

4. 3/2013 – 6/2013: Southwest Center for Occupational and Environmental Health/ NIOSH – “Unintentional exposures to antibiotics in healthcare environments: air sampling and analysis.” Role: Principle Investigator.

11

Specific Aims Poultry workers have lower overall lung function compared to other agricultural workers (Radon et al. 2001) suggesting a need for a better understanding of the underlying cause of the respiratory impairment. Exposure to organic dust is one component of the poultry production process that increases the risk of acute and chronic respiratory impairment (Viegas et al. 2013, Zuskin et al. 1995.) The goal of this project is to identify novel pro-inflammatory molecules in poultry dust. The understanding of which molecules may be contributing to respiratory impairment may indicate the source and potentially provide a means to intervention to reduce their presence and the inflammatory activity of the dust. Aim 1 Justification Preliminary liquid chromatography mass spectrometry (LC-MS) analysis of aqueous poultry dust extracts indicated the presence of at least 2500 small molecules (<2000Da) across a wide range of polarities. This very complex sample will require a procedure to simplify the samples in order to be able to identify novel molecules with inflammatory activity. The combination of chemical chromagraphic fractionation and biological activity testing of the factions will provide a novel procedure for simplifying the complex dust extract. Impact: This aim will provide a protocol to isolate and target pro-inflammatory small molecules from a very complex matrix for identification by mass spectrometry. Aim 1: Evaluate chromagraphic fractions of aqueous poultry dust extracts for inflammatory potential using a cellular based IL-8 ELISA. Rationale: A protocol to fractionate the complex poultry dust extract and to evaluate the fraction’s inflammatory potential is necessary to identify novel pro-inflammatory molecules. Approach: Aqueous poultry dust extracts will be fractionated with liquid chromatography and fractions applied to a human monocytic cell line, THP-1, to determine interleukin-8 induction as a marker of inflammation. The IL-8 analysis with be done by Dr. Rena Saito at the University of Texas Health Sciences Center. This represents collaboration between personnel at the Colorado State University and University of Texas Health Sciences Center Agriculture Centers to take advantage of expertise at each Center to complete this aim. Hypothesis: Several dust extract fractions will have greater relative inflammatory activity compared to other fractions justifying further investigation into the molecule composition of each fraction. Aim 2 Justification The identification of pro-inflammatory compounds from agriculture dust has been limited to allergens (pollens, fungal molds, insect fragments), β-glycans, proteins, and endotoxins. A comprehensive characterization of an agricultural dust to identify novel pro-inflammatory compounds limited to small molecules (<2000Da) has not been accomplished. State of the Art mass spectrometry using LC-quadropule-time-of-flight (LC-QTOF) technology currently provides the ability to rapidly identify small molecules based on accurate mass and isotope ratios. Combined with the cellular based assays to determine inflammatory potential, accurate mass spectrometry provides the workflow necessary to potentially identify novel pro-inflammatory molecules. Impact: This aim will expanded the current chemical characterization of poultry dust and the understanding of specific components contributing its inflammatory activity Aim 2: Characterization of the chemical composition of pro-inflammatory dust extract fractions using accurate mass LC-MS Rationale: The ability to Identify unknown molecules in complex mixtures is critical for discovery of novel molecular targets. Accurate mass LC-MS provides a powerful tool to identify unknown molecules when combined with accurate mass molecular databases such as the METLIN Metabolomic Database. Approach: Characterization of the most reactive fractions will be done using LC-QTOF mass spectrometry with constituent molecules identified by searching against various accurate mass databases. Identified molecules associated with inflammation will be investigated further with analytical standards to provide positive identification of potentially active molecules. Positively identified molecules with pro-inflammatory potential will be evaluated individually in the IL-8 assay to confirm their inflammatory activity. Hypothesis: Novel pro-inflammatory molecules present in poultry dust will be identified leading to an expanded characterization and understanding of poultry dust’s inflammatory potential.

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Significance Agricultural workers display significantly increased rates of clinical symptoms associated with inflammation and impairment of the respiratory system (Kirkhorn and Garry, 2000). Workers in intensive animal production facilities such as swine or poultry are at greater risk for respiratory complications than other farmers (Radon et al. 2001, Rimac et al. 2010, May et al. 2012). Poultry workers specifically have shown to have higher rates of asthma (Viegas et al. 2013), chronic cough and bronchitis (Zuskin et al. 1995), and hypersensitivity pneumonitis (Farmers Lung) (Rylander and Carvalheiro, 2006). Compared with swine operations, poultry workers have lower overall lung function (Radon et al. 2001, Kirychuk et al. 2003) suggesting a need for a better understanding of the underlying cause of the respiratory impairment. Exposure to organic dust is one component of the poultry production process that increases the risk of acute and chronic respiratory impairment (Poole and Romberg 2012). Dust from poultry facilities contains feed, fecal particles, feathers, skin, fungus, bacteria, viruses, and litter particles (Koon et al 1963) all of which may act as allergens or sensitizers leading to respiratory impairment. Endotoxin (lipopolysaccharide component of bacterial cell walls) has been most frequently associated with deleterious respiratory effects from dust exposures (Donham et al. 1995, Reynolds et al. 1996, Burch et al. 2010). Although this association is well documented, it has been clearly shown that endotoxins are not the only component to contribute to inflammation (Romberger et al. 2002, Poole et al. 2008, Sundblad et al. 2009). Little has been published on exposure to other components of the very complex dust that may also be contributing to respiratory inflammation. The significance of this project is to fill in the knowledge gap of other non-endotoxin pro-inflammatory small organic molecules that are present in poultry dust. The understanding of which molecules maybe be contributing to respiratory impairment may indicate the source and potentially provide a means of intervention to reduce their presence and the inflammatory activity of the dust. Innovation - Use of novel instrumentation- LC-TOF-MS or any other accurate mass instrumentation has never been used to characterize an agricultural dust - Use of accurate mass spectra databases to identified unknown compounds -Use of combined biological (immunoassay) and chemical methods (non-targeted mass spectrometry) to isolate pro-inflammatory molecules - There is limited knowledge regarding the composition and diversity of pro-inflammatory small molecules present in agricultural dusts - A systematic approach to discover novel pro-inflammatory small molecules in agricultural dust has never been done. - No established workflow for characterizing pro-inflammatory small molecules from an agricultural dust -Use of molecular weight cutoff membrane to eliminate macromolecules and allergens and focus on small molecules - Collaboration between personnel at the Colorado State University and University of Texas Health Sciences Center Agriculture Centers to take advantage of expertise at each Center to complete this project. Approach (i) Phenomenon Studied Occupational exposures of farm workers to agricultural dusts has been linked to impaired respiratory function from both acute and chronic inhalation exposures. Components of the inhaled dust serve as initiators of the inflammatory response that contributes to various impairments of the respiratory system such as asthma and bronchitis. Dust generated in intensive animal production facilities has been shown to be more potent (May et al. 2012) than dust found in other agricultural setting and yet has not been fully characterized with respect to composition of pro-inflammatory molecules. In a preliminary LC-MS characterization of aqueous poultry dust extracts, > 2500 small molecules (< 2000 Da) were identified in the total raw extract (Figure 1). This clearly shows the complexity of this sample and the potential to have many small molecules with pro-inflammatory activity. From this very complex type of sample, only a few components of the dust such as bacterial endotoxins, β-glycans, and peptides have been previously identified as pro-inflammatory molecules, and little has been published characterizing the other small molecule components of

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various agricultural dusts that may also be contributing to respiratory inflammation. This gap in the knowledge related to the contribution of specific components of agricultural dust exposures to respiratory impairment is critical in understanding phenomenon leading to the clinical onset of the respiratory symptoms. Therefore, the goal of this project is to better characterize poultry dust as a model agricultural dust with highly inflammatory activity (Boggoram 2013, personal communication) to identify novel pro-inflammatory small molecules. A more complete understanding of which molecules in the dust are pro-inflammatory and potentially contributing to respiratory impairment following may indicate the source and potentially provide a means of intervention to reduce their presence and the inflammatory activity of the dust.

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Figure 1. LC-MS chromatogram of an aqueous extract of poultry dust showing the complexity (>2500) of small molecules (<2000 Da) present in the sample.

(ii) Data Collection Settled dust samples will be collected from a poultry production facility in Tyler, TX by personnel at the University of Texas Health Sciences Center at Tyler and sent frozen on dry ice to Colorado State University for extraction and fractionation. Three 1 OOmg aliquots of the dust samples will be extracted with 1 ml of LC-MS grade ultra water by ultrasonication for 20min. Samples will be centrifugation to remove suspended particulate and the supernant passed through a 3000 Da molecular weight cut-off filter to remove macromolecules isolating the small molecule fraction. 100ul aliquots of the filtrate will be injected into a preparative HPLC equipped with a fraction collector. Molecular constituents of the sample will be chromagraphicaly separated by polarity with reversed phase chromatography using a preparative C 18 column and a mobile phase gradient starting with 95% water to 100% acetonitrile over 20 mins. The automated fraction collector will collect eluent fractions at 30sec intervals over the course of the chromagraphic run . Fractionation as described here will greatly simplify sample from several thousand different molecules to several hundred or less molecules per fraction. Multiple injections of the dust extract will be made in order to have sufficient material in each collected fraction to evaluate its pro-inflammatory activity. Pooled fractions will then be snap frozen with liquid nitrogen and lyophilized prior to shipment to the University of Texas Health Sciences Center at Tyler for inflammatory response analysis. Evlauation of the pro-inflammatory potential of each fraction with be done by Dr. Rena Saito at the University of Texas Health Sciences Center. This represents collaboration between personnel at the Colorado State University and University of Texas Health Sciences Center Agriculture Centers to take advantage of expertise at each Center to complete this project. At UTHSC, the lyophilized fractions will be reconstituted with LC-MS grade ultra water for cellular exposures. The human leukemic monocytic cell line, THP-1, will be exposed in the media to each reconstituted fraction and incubated for 3h hours at 37C. Following incubation, the supernant of each exposed cell culture will be collected, and the interleukin-8 levels (inflammatory response) in the supernatants measured by ELISA. lnterleukin-8 (IL-8) is a CXC (a) chemokine produced by the cells following exposure to an inflammatory stimulus._From these results, fractions with the highest inflammatory response will be selected for LC-MS characterization.

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Characterization of the most reactive fractions will be done using LC-quadrupole time-of-flight (QTOF) mass spectrometry. QTOF mass spectrometry provides accurate mass measurements to 2 ppm (mass accuracy to 0.0008 Da), which is more than sufficient for unambiguous determination of a chemical formula from a mass measurement. This ability to generate chemical formula and to search the formula and accurate mass against extensive (> 80,000) small molecule databases is the state-of-the art methodology for identification of unknowns from the samples similar to what we will be generating. Only accurate mass measurements could be used to accomplish unknown identification from very complex samples such as agricultural dust. Each fraction will be initially analyzed by LC-MS with data collected using two ionization modes: positive and negative electrospray ionization. By using both positive and negative ionization modes, we will be able to analyze molecules in all potential ionization states unless the molecules remain neutral. Depending on the polarity of the fraction (early fractions being more polar, later fractions more nonpolar), LC mobile phase gradient conditions will be adjusted to maximally chromagraphicaly separate the molecules within that fraction based on polarity. The MS data will be collected in full scan mode from 50-2000 Da at an acquisition rate of 2.5 Hz. Collected MS spectral data will then be qualitatively analyzed with the workflow described in the data analysis section to identify the individual molecules present in the fraction. Once a molecular feature list is constructed through the data analysis workflow, identified features associated with inflammation or those associated with know antigens (potential biomarkers) will be investigated further with analytical standards to provide positive identification. Molecules of interest with confirmed IDs will then be applied individually to the THP-1 cells in the IL-8 assay to evaluate their inflammatory potential. (iii) Data Analysis Raw MS spectra data from each analyzed fraction will be evaluated using the MassHunter Qualitative Analysis program (Agilent Technologies). Molecular feature extraction will be performed to deconvolute the total ion spectra into individual ion/isotope groupings creating a molecular profile for the sample. The software subtracts the chemical background, finds the true ion signals, groups the chemically related ion signals (isotopes, adducts and dimers), and displays as individual peaks in a compound chromatogram (Figure 1). Each peak in this chromatogram represents a molecular feature as defined as an accurate mass ion with associated retention time and ion abundance. Once molecular features have been identified, MassHunter can generate molecular formulas by taking into account the mass accuracy of the ions, isotope ratios, and isotope mass values to produce a narrow list of possible formulas with associated probabilities. Mass accuracy filters will be set at +/- 2ppm to allow for a more strict molecular formula assignment. Each molecular feature will then be searched against various databases to assist in molecule identification by comparison of molecular formula and mass spectral pattern matching (isotope fidelity) to entries in the database library.

Figure 2. Molecular spectra of compound 1124 identified as the fungal metabolite sphingofungin B demonstrating excellent mass accuracy (-0.23 ppm), a high database match score (99.84/100) and accurate isotopic fidelity, which provided assurance of correct identification.

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Potential databases available to search are the METLIN Metabolomics Database (>80,000 compounds), Human Metabolome Database (>40,000), and the Madison-Qingdao Metabolomics Consortium Database (>20,000). Figure 2 shows a typical database search result and confirmatory data using MassHunter. Molecular feature has a retention time of 12.04 min, an accurate mass of 389.2778 Da, and a formula of C20H39NO6. The mass accuracy was very high with a difference of -0.23 Da for the assigned molecular formula. Mass errors of less than 1 ppm are required for confident molecular formula assignments. When this mass spectra and formula data was searched against the METLIN Metabolomics Database, a significant match based on a database match score of 99.84/100 was assigned to the fungal metabolite sphinogfungin B. Manual validation of the identification was done by inspecting the isotope fidelity as isotope ratios provide additional information that further increases confidence of identification. Figure 2 shows the excellent correspondence between measured isotope pattern versus theoretical (shown in red rectangles) which is based on the correct molecular formula. Through this workflow, presumptive identifications of molecular features can be accomplished, but to confirm the IDs, authentic pure standards will need to be analyzed to confirm correlation of chromagraphic retention times. (iv) Evaluation method The specific aims of this project will be evaluated during the course of the data collection with the IL-8 assay completed at UTHSC. Since IL-8 is an (α) chemokine produced by the cells following exposure to an inflammatory stimulus, detection of increased levels IL-8 will serve as a positive evaluation result. In Specific Aim 1, the assay will be used to determine if pro-inflammatory small molecules in aqueous extracts of poultry dust can be fractionated by reversed phase liquid chromatography. It will be used to evaluate the results of Specific Aim 2 where individual molecules identified in extract fractions will be tested for their inflammatory potential. (v) Quality Assurance Measures All aspects of this project will be completed by trained and skilled personal in suitable laboratory environments to assure quality results. Dr. Greg Dooley will properly maintain the LC-QTOF mass spectrometer with daily mass accuracy calibrations, tuning, and source cleaning. All liquid chromatography mobile phases and columns will be dedicated to this project to avoid contamination. All reagents and standards will be stored under proper conditions. Blank samples will be run to identify contamination and establish the background response that will be subtracted during the molecular feature analysis. Samples spiked with a mixture of 12 know pesticides will be analyzed as a positive control for the molecular feature extraction and database searching aspect of this project. To confirm he database identifications of potential novel pro-inflammatory molecules, authentic analytical standards will be used to match chromagraphic retention times and isotope profiles. This QA procedure will be done prior to these molecules being tested individually in the IL-8 assay. Personnel at UTHSC will be blinded to the fraction number collected during the initial IL-8 characterization analysis and to the identity of novel pro-inflammatory molecules during the confirmation analysis to reduce any bias in the data interpretation. Blank LC-MS grade water samples and LC-MS water samples spiked with lipopolysaccharide will be send as blind negative and positive control samples for the IL-8 analysis at UTHSC. Expected Implications of research findings This would be the first research to use accurate mass based mass spectrometry methods to characterize poultry dust. Characterization of the small molecules present in poultry dust would increase the understanding of the component in this complex matrix and fill in the knowledge gap of other non-endotoxin pro-inflammatory small organic molecules that are present in poultry dust. Very little research has been directed to discovery of dust components that may also be contributing to respiratory inflammation. This project would provide a workflow directly applicable to the analysis of other agriculture dust types to search for novel pro-inflammatory molecules and biomarker discovery. It is expected that novel small molecules with pro-inflammatory activity will be elucidated during this project. Identification of these molecules has great value towards understanding why exposure to agriculture dust increases the risk of respiratory impairment in agricultural workers. This expanding understanding through the characterization of the dust may indicate the source of the pro-inflammatory molecules and potentially provide a means of intervention to reduce their presence and the inflammatory activity of the dust or to determine if certain occupational settings are in need of intervention

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Pitfalls and Limitations Using water as extraction solvent has limitations as to the polarity of molecules it is capable of dissolving. Very nonpolar molecules will likely not be extracted and excluded from this characterization.

Neutral species that will not ionize will not enter the mass spectrometer and therefore will not be analyzed and bias the characterization towards ionizable molecules.

Since the field of metabolomics and compound identification by accurate mass is relatively new, only about 20% of known compounds are currently represented in database libraries. This limitation will likely result in many molecular features not being identified by name, but they will still have an associated molecular formula.

The use of reverse phase chromatography will result in very polar molecules not being sufficiently retained on a C18 LC column to appear in an eluent fraction after the void volume. Un retained compounds in the void volume will be collected and analyzed, but the typical presence of salts in this fraction result in ion suppression, reduced quality of mass spectrum for database searching, and poorer database match results.

Future Directions This proposal will utilize water as the extraction solvent, since it is compatible with the IL-8 assay, but other solvents of differing polarity would need to be investigated since no extraction solvent will be universal. Repeating this experiment with solvents such as acetonitrile (mid-polar) and hexane (nonpolar) would likely yield considerable different molecular features with inflammatory potential. Extraction with three or more different solvents will be necessary to get a complete characterization of the dust.

The novel discovered pro-inflammatory molecules might serve as biomarkers of the inflammatory potential of agricultural dust. Quantitative analytical methods for novel compounds in the dust matrix would need to be developed and would provide a tool to be used for standardized comparison of different dust types. Much like endotoxin levels correspond to higher inflammatory potential of dust, other molecules may be a useful biomarker in environments where bacteria are not a large component of the dust matrix. These new biomarkers could also be used to determine if certain occupational settings are in need of intervention to reduce potential employee health complications.

Methods developed in this proposal would be directly applicable to the analysis of other agriculture dust types to search for novel pro-inflammatory molecules and biomarker discovery. It would be interesting to compare the relative fraction compositions between dust collect from different agricultural environments. Principal components analysis can also be done with the same data sets in this proposal to evaluate the similarity and differences with regard to molecular features between dust from different agriculture operations or seasonal variation. Similar agricultural operations in different parts of the country could also be compared to see if poultry operations in warmer/colder climates have differential inflammatory activity.

Data and results from this pilot study will be used in the submission of a R03 or R01 grant application to agencies such as CDC/NIOSH, USDA, EPA, or NIEHS

Timeline

Task Dust sample collection

Fractionation of aqueous dust extracts

IL-8 assay of dust fractions

LC-MS analysis of dust fractions

MS data reduction and database searching

Evaluation of identified molecules with IL-8 assay

Preparation of Manuscript

Sept Nov Jan Mar May July Aug 2013 2013 2014 2014 2014 2014 2014

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References Boggoram, V. Personal Communication, 2013. Burch JB, Svendsen E, Siegel PD, Wagner SE, Von Essen S, Keefe T, Mehaffy J, Serrano Martinez A,Bradford M, Baker L, Cranmer B, Saito R, Tessari J, Prinz L, Andersen C, Christensen O , Koehncke N, Reynolds SJ. Endotoxin Exposure and Inflammation Markers among Agricultural Workers in Colorado and Nebraska. J Tox Env Health Part A, 73:1,5-22, 2009. Donham KJ, Reynolds SJ, Whitten P, Merchant JA, Burmeister L, Popendorf WJ: Respiratory dysfunction in swine production facility workers: Dose-response relationships of environmental exposures and pulmonary function. American Journal of Industrial Medicine, 27(3):405-418, 1995 Kirkhorn SR, Garry VF. Agricultural lung diseases. Environ Health Perspect. 108 Suppl 4:705-12. 2000

Kirychuk SP, Senthilselvan A, Dosman JA, Juorio V, Feddes JJ, Willson P, Classen H, Reynolds SJ, Guenter W, Hurst TS: Respiratory symptoms and lung function in poultry confinement workers in Western Canada. Canadian Respiratory Journal, 10(7):375-380, 2003 Koon J, Howes JR, Grub W, Rollo CA. Poultry dust: origin and composition. Agricultural Engineering. 44: 608-609,1963. May S, Romberger DJ, Poole JA. Respiratory health effects of large animal farming environments. J Toxicol Environ Health B Crit Rev, 15(8):524-41, 2012

Poole JA, Romberger DJ. Immunological and inflammatory responses to organic dust in agriculture. Curr Opin Allergy Clin Immunol. Apr;12(2):126-32, 2012 Poole, J. A., Alexis, N. E., Parks, C., MacInnes,A. K., Gentry-Nielsen, M. J., Fey, P. D., Larsson, L., Allen-Gipson, D., Von Essen, S.G., and Romberger, D. J. Repetitive organic dust exposure in vitro impairs macrophage differentiation and function. J. Allergy Clin. Immunol. 122: 375–382. 2008 Radon K, Weber C, Iversen M, Danuser B, Pedersen S, Nowak D. Exposure assessment and lung function in pig and poultry farmers. Occup Environ Med. 58(6):405-10, 2001 Reynolds, S. J., Donham, K. J., Whitten, P., Merchant, J. A., Burmeister, F., and Popendorf, W. J. Longitudinal evaluation of dose-response relationships for environmental exposures and pulmonary function in swine production workers. Am. J. Ind. Med. 29: 33-40. 1996 Rimac D, Macan J, Varnai VM, Vucemilo M, Matković K, Prester L, Orct T, Trosić I, Pavicić I. Exposure to poultry dust and health effects in poultry workers: impact of mold and mite allergens. Arch Occup Environ Health. 83(1):9-19. 2010 Romberger, D. J., Bodlak, V., Von Essen, S. G., Mathisen, T., and Wyatt, T. A. Hogbarn dust extract stimulates IL-8 and IL-6 release in human bronchial epithelial cells via PKC activation. J. Appl. Physiol. 93:289–296. 2002 Rylander R, Carvalheiro MF. Airways inflammation among workers in poultry houses. Int Arch Occup Environ Health. 79(6):487-90. 2006 Sundblad, B. M., von Scheele, I., Palmberg, L., Olsson, M., and Larsson, K. Repeated exposure to organic material alters inflammatoryand physiological airway responses. Eur. Respir. J. 34: 80–88. 2009 Viegas S, Faísca VM, Dias H, Clérigo A, Carolino E, Viegas C.Occupational exposure to poultry dust and effects on the respiratory system in workers. J Toxicol Environ Health A. 76(4-5):230-9. 2013

Zuskin E, Mustajbegovic J, Schachter EN, Kern J, Rienzi N, Goswami S, Marom Z, Maayani S. Respiratory function in poultry workers and pharmacologic characterization of poultry dust extract. Environ Res. 70(1):11-9, 1995

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August 15, 2013

HICAHS Pilot Grant Review Committee

Re: Dr. Greg Dooley HICAHS Pilot Grant Application

Dear Review Committee,

Colorado State University Campus Delive1y 1681

Fort Collins, CO 8o523-1681

I am writing to confirm my enthusiastic support for Dr. Dooley's proposed research project "Identification of novel pro-inflammatory small molecules in poultry dust''. This project builds on our current HICAHS research on Bioaerosol Exposures and lvl.odels of Human Response in Dairies taking a significant step fo1ward towards understanding the complex relationship between agricultural aerosols and lung disease among livestock workers. The proposed project importantly involves a close collaboration ·with the Southwest Center Ag Center, leveraging expertise and resources at our two centers. This research will develop methods that will help improve occupational health risk management in the rapidly expanding poultry and dairy industries, and other livestock production sectors - a high priority for HICAHS as the global demand for protein grows.

Dr. Dooley has been an important member of our research team, and demonstrated his innovation and productivity in collaborations with our colleagues at the SW Ag Center. I \1.rill be pleased to assist Dr. Dooley with project management, data analysis/interpretation, and in facilitating collaboration ·with the SW Ag Center. This research will position us to compete for funds from a number of sources, and will be particularly important for competitive renewal of HICAHS in 2016.

Sincerely,

Stephen Reynolds, PhD, CIH, Fellow AIHA Professor Environmental and Radiological Health Sciences Director High Plains Intermountain Center for Agricultural Health and Safety Deputy Director Mountain and Plains ERC Stephen.Reynolds@colostate.edu 970-491-3141

www.hicahs:ioolostate.edu

Occupational Health Sciences 11937 US Highway 271

Tyler, Texas 75708 August 14, 2013 Greg Dooley, Ph.D. Center for Environmental Medicine Department of Environmental and Radiological Health Sciences Colorado State University Fort Collins, CO 80523-1680 Dear Dr. Greg Dooley, I am writing to express my support for your study “Identification of novel pro-inflammatory small molecules in poultry dust.” I am an industrial hygienist and an Assistant Professor in the department of occupational health sciences at the University of Texas Health Science Center at Tyler (UTHSCT). My research has been focused on biological and chemical agents in agricultural and non-agricultural dusts. Along with my project mentor Dr. Vijay Boggaram, who is a biochemist and a Professor in the department of cellular and molecular biology, we will measure inflammatory responses (interleukin-8) on human cell lines in this collaborative study between Colorado State University’s High Plains Intermountain Center for Agricultural Health and Safety and UTHSCT’s Southwest Center for Agricultural Health, Injury Prevention, and Education. Respiratory and lung diseases are one of the major concerns among poultry workers, and the poultry dust contains complex mixture of small molecules. However, the inflammatory responses of such small molecules have not been studied well. This proposed collaborative study will provide important information regarding the inflammatory agents present in agricultural environments. I feel my contribution will provide essential elements for successful completion of the project. I look forward to collaborating with you and extend my support on this project.

Rena Saito, Ph.D., C.I.H. Assistant Professor Occupational Health Sciences The University of Texas Health Science Center at Tyler 11937 US Highway 271 Tyler, Texas 75708

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