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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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