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

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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

S-heteroatom
N-heteroatom
Drugs
Pesticides

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

 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

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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

 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.

Table 1.
Comparison of FMO Isoform Transcripts in Mouse Lung
Tissue
Type
Shephard
Laboratory
Williams
Laboratory
Lung Fmo1> Fmo2>Fmo3 Fmo2>Fmo3>Fmo1
Liver Fmo3>Fmo1>Fmo2 Fmo3>Fmo1>Fmo2

Variable Shephard Laboratory
Williams
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

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

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

 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.

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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

 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

 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
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NIH-HL038650

Williams’
Lab
ROCKS!
Hmm… back to
RNA?

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

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Accurate measure of RNA concentration
1 uL sample is required
Data output
• 260/280 OD values
• 230/280 OD values

Figure 2.
Mice Samples using Fmo2 Primers on RT-PCR 96-Well Plate
Graph 2.
Melting Curves for Mice Samples using Fmo2 Primers
Graph 6.
Fmo2 Standard Curve with Mice Samples

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

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 (

Used to quantify protein
Reverse Transcription

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.

 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
2
O+NADP +
FMO(FAD ox
)
NADPH
4 1
FMO(FADHOH)
RS-OH
3
FMO(FADOOH)
FMO(FADH
2
)+NADP +
O
2
2
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.