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.