Download Determination of the flavin mono

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
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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
Type
Shephard
Laboratory
Williams
Laboratory
Lung
Fmo1> Fmo2>Fmo3
Fmo2>Fmo3>Fmo1
Liver
Fmo3>Fmo1>Fmo2
Fmo3>Fmo1>Fmo2
Possible Causes for Discrepancy
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
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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
Fed 5
weeks
8 weeks
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
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
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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
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
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.
Melting Curves for Mice Samples using Fmo2 Primers
Graph 6.
Fmo2 Standard Curve with Mice Samples
Bioanalyzer
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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.
FMO(FAD ox)
H 2O+NADP +
4
NADPH
1
FMO(FADH2)+NADP +
FMO(FADHOH)
RS-OH
3
2
FMO(FADOOH)
RSH
O2
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.