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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 Flavin Monooxygenases (FMOs) 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 O2 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 Structures of FMO Substrates S-heteroatom N-heteroatom Drugs Pesticides 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 Environmental Injustice Higher incidence of lung diseases in minority populations expressing the FMO2*1 allele1 Higher Exposure to xenobiotics • Socioeconomic • Occupational Settings 1 Gadgeel and Kalemlcerian, 2003; Lenair, 1999, Moss and Mannino, 2002 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 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. Hypothesis #1: As in human lung, FMO2 is the predominate isoform expressed in mouse. Other Researchers Publish Contradictory Findings Table 1. Comparison of FMO Isoform Transcripts in Mouse Lung Tissue Shephard Williams Type Laboratory Laboratory Lung Fmo1> Fmo2>Fmo3 Fmo2>Fmo3>Fmo1 Liver Fmo3>Fmo1>Fmo2 Fmo3>Fmo1>Fmo2 Possible Causes for Discrepancy Williams Variable Shephard Laboratory Laboratory Mouse Strain 129/SV and C57BL/6J C57BL/6J/129F1 Age 8 weeks 12 weeks Diet Harlan Teklad TRM AIN93G Diet is known to modulate FMO levels Plant alkaloids found in chow diet can act as a substrate or inhibitor 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 Hypothesis #2: The AIN93-G and Harlan Teklad (NIH-31) diets modulate expression of FMO in lung. Test of Dietary Influence on the Level of Fmo Expression 4 female mice 4 female mice 4 male mice 4 male mice Fed 5 AIN-93 G NIH-31 AIN-93 G NIH-31 weeks diet diet diet diet 8 weeks Harvest Lung and Liver Tissues Isolate and Quantify RNA Make cDNA Quantify FMO1, FMO2, FMO3 and Actin via qPCR 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. 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 Results 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 Acknowledgements 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 Williams’ Lab Hmm… ROCKS! back to RNA? Comparing Random & Specific Primers Graph 1. Graph 2. Fmo2 Standard Curve with Random Primers Fmo2 Standard Curve with Mice Samples Figure 1. Melting Curves for Mice Samples using Fmo2 Specific Primers Nanodrop Accurate measure of RNA concentration 1 uL sample is required Data output • 260/280 OD values • 230/280 OD values Real-Time Polymerase Chain Reaction Figure 2. Mice Samples using Fmo2 Primers on RT-PCR 96-Well Plate Graph 2. Graph 6. Melting Curves for Mice Samples using Fmo2 Primers Fmo2 Standard Curve with Mice Samples Bioanalyzer Used to check for RNA integrity and purity 28S fragment Data output 18S fragment 28s/16s ribosomal RNA ratios RNA Integrity Number (RIN) RNA concentrations (ng/uL) Electropherogram Result Tables 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 Indicates more copies of gene How it works; cyber green; inter-colating DNA ( Reverse Transcription Used to quantify protein 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. 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 The Overall Reaction of FMO This ability of FMO to oxidize a variety of xenobiotics is crucial to bioactivation and detoxification processes in mammals. H 2O+NADP + FMO(FAD ox) NADPH 4 1 FMO(FADHOH) FMO(FADH2)+NADP + RS-OH O2 3 2 FMO(FADOOH) RSH How to Make a Knock-out Mouse Model Environmental Injustice: Low Income & Communities of Color suffer the greatest risks and impacts of pesticide use 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.
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