determination of the flavin containing monooxygenase (fmo) distribution in mouse lung and liver...
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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
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
Flavin Monooxygenases (FMOs)
Structures of FMO Substrates
Pesticides
Drugs
S-heteroatom
N-heteroatom
Background
FMO2 is highly expressed in mammalian lung.FMO2 is highly expressed in mammalian lung. Polymorphisms of FMO2 expression exist in humans.Polymorphisms of FMO2 expression exist in humans. The human The human FMO2*1FMO2*1 allele allele
Full-length, functional FMO2 proteinFull-length, functional FMO2 protein 27% of African Americans27% of African Americans 5% of Hispanic populations5% of Hispanic populations
The human The human FMO2*2FMO2*2 allele allele Truncated and non-functional FMO2 proteinTruncated and non-functional FMO2 protein Caucasians and Asians examinedCaucasians 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
Tissue Type
Shephard
Laboratory
Williams
Laboratory
Lung Fmo1> Fmo2>Fmo3 Fmo2>Fmo3>Fmo1
Liver Fmo3>Fmo1>Fmo2 Fmo3>Fmo1>Fmo2
Table 1.Comparison of FMO Isoform Transcripts in Mouse Lung
Possible Causes for Discrepancy
Variable Shephard LaboratoryWilliams
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.
4 male miceAIN-93 G
diet
4 female miceNIH-31
diet
4 male miceNIH-31
diet
4 female miceAIN-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
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
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
Hmm… back to RNA?
Williams’ Lab
ROCKS!
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
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
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
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
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
How it works; cyber green; inter-colating DNA (
Indicates more copies of gene
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.
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
FMO(FADox)
FMO(FADH2)+NADP +
FMO(FADOOH)
FMO(FADHOH)
NADPH
O2
RSH
RS-OH
H2O+NADP+
1
23
4
This ability of FMO to oxidize a variety of xenobiotics is crucial to This ability of FMO to oxidize a variety of xenobiotics is crucial to bioactivation and detoxification processes in mammals.bioactivation and detoxification processes in mammals.
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.