1. Determination of the Flavin Containing Monooxygenase (Fmo) Distribution in Mouse Lung and Liver Pachida C. Lo Dr. David Williams’ Laboratory Oregon State University, Environmental & Molecular Toxicology Department Linus Pauling Institute

2.  Gene family that oxygenates a wide range of xenobiotics containing nitrogen and sulfur nucleophilic heteroatoms  In animals Fmo1, Fmo2 and Fmo3 are the main drug metabolizing enzymes  Requires NADPH and O 2  Localized in the Endoplasmic Reticulum (ER)  Broad substrate specificity  Exhibits sex, developmental, and tissue specific expression  FMO metabolism may result in detoxification or bioactivation depending on the substrate/product Flavin Monooxygenases (FMOs)

3. Structures of FMO Substrates Pesticides Drugs S-heteroatom N-heteroatom

4. Background  FMO2 is highly expressed in mammalian lung.  Polymorphisms of FMO2 expression exist in humans.  The human FMO2*1 allele  Full-length, functional FMO2 protein  27% of African Americans  5% of Hispanic populations  The human FMO2*2 allele  Truncated and non-functional FMO2 protein  Caucasians and Asians examined

5. Environmental Injustice  Higher incidence of lung diseases in minority populations expressing the FMO2*1 allele 1  Higher Exposure to xenobiotics Socioeconomic Occupational Settings 1 Gadgeel and Kalemlcerian, 2003; Lenair, 1999, Moss and Mannino, 2002

6. Development of a Null-mouse Model  Study the role of human pulmonary FMO2 in xenobiotic metabolism and toxicity  Wild-type mouse Contains the FMO2*1 allele Models 27% of African-Americans and 5% of Hispanic/Latino  Null mouse Does not contain the FMO2*1 allele Models the majority of the population

7. What is a Null-Mouse Model?  Genetically engineered to carry one or more genes that has been made non-functional.  Used to learn about a gene that has an unknown or incompletely known function.  Used in drug development to assess the potential for a human enzyme as a target for therapy.

8. Hypothesis #1: As in human lung, FMO2 is the predominate isoform expressed in mouse.

9. Other Researchers Publish Contradictory Findings Tissue Type Shephard Laboratory Williams Laboratory LungFmo1> Fmo2>Fmo3Fmo2>Fmo3>Fmo1 LiverFmo3>Fmo1>Fmo2 Table 1. Comparison of FMO Isoform Transcripts in Mouse Lung

10. Possible Causes for Discrepancy VariableShephard Laboratory Williams Laboratory Mouse Strain129/SV and C57BL/6JC57BL/6J/129F1 Age8 weeks12 weeks DietHarlan Teklad TRMAIN93G  Diet is known to modulate FMO levels  Plant alkaloids found in chow diet can act as a substrate or inhibitor

11. DIETS AIN93G is a synthetic diet Williams Laboratory Pure ingredients: casein, soy protein, starch, sucrose Highly reproducible Harklan Tekland is a chow-based diet Shephard Laboratory Crude ingredients: wheat, barley Not highly reproducible Contains plant alkaloids

13. 4 male mice AIN-93 G diet 4 female mice NIH-31 diet 4 male mice NIH-31 diet 4 female mice AIN-93 G diet Harvest Lung and Liver Tissues Isolate and Quantify RNA Make cDNA Quantify FMO1, FMO2, FMO3 and Actin via qPCR Fed 5 weeks 8 weeks Test of Dietary Influence on the Level of Fmo Expression

14. Why Test Fmo1, Fmo2 Fmo3 and Actin?  Overlapping substrate specificities  More than one isoform present in the tissue could complicate interpretation of the results from the knock-out mouse model  Actin is the housekeeping gene.

15. Methods  Isolation and quantification of RNA  Trizol Extraction  Qiagen RNeasy Clean-up  cDNA First Strand Synthesis  Gene specific primers (FMOs), oligo dT (actin)  Superscript III reverse transcriptase  Quantification of FMO1, FMO2, FMO3 and Actin  DYNAMO polymerase SYBR Green qPCR Kit double stranded DNA binding dye Fluorescence enhanced upon binding dsDNA

16. Results

17. Future Work  Isolate microsomes containing FMO from tissues for protein verifcation of RT-PCR results  Western blot using isoform-specific antibodies to detect individual FMOs  Further purification for mass spectrometry determination of FMO profile  Isoform-specific enzyme assays

18.  Williams Laboratory  Tanguay Laboratory  Center for Gene Research Biotechnology  Linus Pauling Institute  Laboratory Animal Research Center  Howard Hughes Medical Institute (HHMI) Dr. Kevin Ahern  National Institute of Environmental Health Sciences T-35 Grant Dr. Rosita Rodriguez Proteau Kay Kent  Undergraduate Research Innovation Scholarship Creativity  NIH-HL038650 Acknowledgements

19. Hmm… back to RNA? Williams’ Lab ROCKS!

20. Graph 2. Fmo2 Standard Curve with Mice Samples Figure 1. Melting Curves for Mice Samples using Fmo2 Specific Primers Graph 1. Fmo2 Standard Curve with Random Primers Comparing Random & Specific Primers

21. Nanodrop  Accurate measure of RNA concentration  1 uL sample is required  Data output 260/280 OD values 230/280 OD values

22. Real-Time Polymerase Chain Reaction Graph 2. Melting Curves for Mice Samples using Fmo2 Primers Figure 2. Mice Samples using Fmo2 Primers on RT-PCR 96-Well Plate Graph 6. Fmo2 Standard Curve with Mice Samples

23. Bioanalyzer  Used to check for RNA integrity and purity  Data output  28s/16s ribosomal RNA ratios  RNA Integrity Number (RIN)  RNA concentrations (ng/uL) Electropherogram Result Tables 18S fragment 28S fragment

24. Extraction Methods 1.Trizol Method (used for lungs) 2.RNeasy Mini Kit Method Steps To Take:  Homogenize Tissue Sample  Phase Separation  RNA Precipitation  RNA wash  Redissolve RNA

25. How it works; cyber green; inter-colating DNA ( Indicates more copies of gene

26. Used to quantify protein Reverse Transcription

27. Test of Dietary Influence on the level of Fmo expression  8 male and 8 female mice (129/SV strain) 3 week weanlings  Fed AIN93-G or Harland Tekland diet for 5 weeks  Collect liver and lung tissue at 8 weeks of age  Isolate and quantify the RNA.  Reverse transcribe a portion of the RNA and make cDNA (complementary DNA).  Use RT-PCR to quantify mouse FMO1, FMO2, FMO3, and a housekeeping gene.

28. Preliminary Results  Males on the AIN93-G or NIH-31 diet show greater increase in mass compared to females  The two diets (per gender) do not appear to uniquely influence body weight

29. The Overall Reaction of FMO FMO(FAD ox ) FMO(FADH 2 )+NADP + FMO(FADOOH) FMO(FADHOH) NADPH O 2 RSH RS-OH H 2 O+NADP + 1 23 4 This ability of FMO to oxidize a variety of xenobiotics is crucial to bioactivation and detoxification processes in mammals.

30. How to Make a Knock-out Mouse Model

31. Environmental Injustice: Low Income & Communities of Color suffer the greatest risks and impacts of pesticide use Address asthma as an environmental justice issue. Asthma disproportionately impacts low-income and ethnic minorities. External factors that may contribute to this disparity include diesel emissions and other outdoor air pollutants near low-income housing. Indoor air issues in low-income housing include excessive moisture, mold, pests, and pesticides. Support efforts to mobilize low-income communities. Address asthma as an environmental justice issue. Asthma disproportionately impacts low-income and ethnic minorities. External factors that may contribute to this disparity include diesel emissions and other outdoor air pollutants near low-income housing. Indoor air issues in low-income housing include excessive moisture, mold, pests, and pesticides. Support efforts to mobilize low-income communities. 1.Migrant and seasonal farmworkers are primarily ethnic minorities who are excluded from federal laws that protect other workers. 2.Farmworkers live and work under substandard conditions that place them at increased risk of pesticide-related illness. 3. Possible biological/genetic factors that increase risk of related illness.