Wet Lab

Radiation Damage Measured by Micronucleus (MN) Assay

• BackgroundBackground• EquipmentEquipment• SuppliesSupplies• ProceduresProcedures• Lab DemonstrationsLab Demonstrations

BackgroundBackground

• Types of micronucleus Types of micronucleus assaysassays

• Applications of Applications of micronucleus assaysmicronucleus assays

Background

MN

Micronucleus Assay (MN)Micronucleus Assay (MN)

• It can be used to detect the It can be used to detect the clastogenicclastogenic and and aneugenicaneugenic effects of test agents effects of test agents both both in vitroin vitro and and in vivoin vivo..– Clastogenic:Clastogenic: any substance or process any substance or process

causing chromosome breaks.causing chromosome breaks.– Aneugenic:Aneugenic: Agents which affect cell Agents which affect cell

division and the mitotic spindle apparatus division and the mitotic spindle apparatus resulting in the loss or gain of whole resulting in the loss or gain of whole chromosomes, thereby inducing an chromosomes, thereby inducing an aneuploidy.aneuploidy.

Background

Micronucleus Assay (MN)Micronucleus Assay (MN)

• It is much more cost effective than It is much more cost effective than the metaphase chromosome the metaphase chromosome aberration assay. aberration assay.

• The The in vitroin vitro micronucleus assay is micronucleus assay is considered as the replacement for considered as the replacement for conventional metaphase analysis as conventional metaphase analysis as the screening test of choice for the screening test of choice for clastogenicity.clastogenicity.

Background

Micronucleus Assay (MN)Micronucleus Assay (MN)

• The The in vivoin vivo assay, usually conducted assay, usually conducted in mice, is especially important since in mice, is especially important since no no in vitroin vitro alternative test has been alternative test has been validated to replace the MN test. validated to replace the MN test.

• The cells evaluated in this assay are The cells evaluated in this assay are typically erythrocyte populations in typically erythrocyte populations in either the peripheral blood or bone either the peripheral blood or bone marrow compartment. marrow compartment.

Background

Micronucleus Assays (MN)Micronucleus Assays (MN)

• Cytokinesis-block micronucleus Cytokinesis-block micronucleus (CBMN) assay(CBMN) assay

• lymph, cell lineslymph, cell lines

• Flow cytometric micronucleus Flow cytometric micronucleus (FCMN) assay (FCMN) assay

• red blood cellsred blood cells

Background

CBMN Assay (lymphocytes)CBMN Assay (lymphocytes)

• Cytochalasin-B (actin inhibitor)Cytochalasin-B (actin inhibitor)

Background

Background

CBMN + FISHCBMN + FISH

Background

Criteria for CBMNCriteria for CBMN

• The diameter of the MN should be The diameter of the MN should be less than one-third of the main less than one-third of the main nucleus.nucleus.

• MN should have similar staining as MN should have similar staining as the main nucleus.the main nucleus.

• MN should be separated from or MN should be separated from or marginally overlap with main marginally overlap with main nucleus.nucleus.

Background

Background

CBMN vs. Chromosome SpreadingCBMN vs. Chromosome Spreading

• AdvantagesAdvantages– RapidityRapidity– CheapCheap– SimplicitySimplicity– Statistical powerStatistical power

• DisadvantagesDisadvantages– Not all chromosome aberrations Not all chromosome aberrations

(acentric fragments)(acentric fragments)– Toxicity of cyto-BToxicity of cyto-B

• Lymph. vs. cell linesLymph. vs. cell lines

Background

Applications of MN AssayApplications of MN Assay

• Biological dosimetryBiological dosimetry

• Risk assessment of cancerRisk assessment of cancer– Lung: smokingLung: smoking– Oral mucosa and bladder cancer: arsenicOral mucosa and bladder cancer: arsenic

• Genotoxic effects of pesticidesGenotoxic effects of pesticides

• Cytotoxic effects of ROSCytotoxic effects of ROS

• Radiosensitivity indicator in head and Radiosensitivity indicator in head and neck tumor patientsneck tumor patients

Background

Dose Response

Dose (Gy)

Dic

e ntr

ics /

Cel

l

1.0

2.0

0

Background

Dose (Gy)

% M

N

2.0

4.0

0

???

chromosome MN

Post-irradiationtime

FCMN AssayFCMN Assay

• Rapid scoringRapid scoring• Large number of cellsLarge number of cells• Small changes in MN frequencySmall changes in MN frequency• Reticulocytes from peripheral Reticulocytes from peripheral

bloodblood• DNA contentsDNA contents

DNA content

Where are micro-nucleated cells?

Whole blood

White blood cellsRed blood cells (size)

Reticulocytes (CD71)

Platelets(anti-platelet)

CD61

Nucleated red cells(DNA contents)

Ret-MN

X X

X

Litron LaboratorySteve Dertinger, Ph.D.

Cell size (red cells)

Whole blood

White blood cellsRed blood cells (size)

Platelets(anti-platelet)

CD61

Nucleated red cells(DNA contents)

Ret-MN

X X

X

Reticulocytes(CD71)

Single cellstotal

mature RBC

Single cellsplatelets

Single cellsReticulocytesfor MN

(anti-CD61-PE)

Whole blood

Red blood cells (size)

Platelets(anti-platelet)

CD61

Nucleated red cells(DNA contents)

Ret-MN

X X

Reticulocytes(CD71)

Single cellsReticulocytesNo platelets

MN

MN: DNA content

EquipmentEquipment

• Tissue culture equipmentTissue culture equipment– Sterile tissue culture hood, Incubator (COSterile tissue culture hood, Incubator (CO22, ,

3737ooC, humidified), Microscopes( upright, C, humidified), Microscopes( upright, inverted, dissecting), Pipettes, inverted, dissecting), Pipettes, Hemacytometers (Coulter counter).Hemacytometers (Coulter counter).

• Radiation sourceRadiation source

• Flow cytometerFlow cytometer

SuppliesSupplies

• Tissue culture flasks (dishes)Tissue culture flasks (dishes)

• Tissue culture mediumTissue culture medium

• Glass slidesGlass slides

• Cytochalasin-BCytochalasin-B

• Giemsa stainGiemsa stain

• MN DNA staining kit (Litron)MN DNA staining kit (Litron)

ProceduresProceduresCalibration and color compensation Calibration and color compensation

for flow cytometerfor flow cytometer

• Rat blood cellsRat blood cells– DNA (PI), CD71- (FITC), anti-platelets+ (PE)DNA (PI), CD71- (FITC), anti-platelets+ (PE)

• Parasite infected mouse blood cellsParasite infected mouse blood cells– DNA (PI), CD71+ (FITC)DNA (PI), CD71+ (FITC)

• Parasite infected mouse blood cellsParasite infected mouse blood cells– DNA (PI), CD71+ (FITC), anti-platelets+ (PE)DNA (PI), CD71+ (FITC), anti-platelets+ (PE)

Rat blood cells

DNA (PI)CD71-anti-platelet

(anti-CD61-PE)

Single cellsmature RBC

Single cellsplatelets

Single cellsReticulocytesfor MN

Rat blood cells

Parasite infectedmouse blood cells

DNA (PI)CD71+

(anti-CD61-PE)

DNA ratio20:6000

Parasite infectedmouse blood cells

Parasite infectedmouse blood cells

DNA (PI)CD71anti-platelet

(anti-CD61-PE)

Parasite infectedmouse blood cells

MNUnirradiated (0 Gy)mouse blood cells

DNA (PI)CD71+CD61+

Parasite infectedmouse blood cells

byFACS Vantage sorter

DNA (PI)CD71+CD61+

Human blood cellsfrom irradiated (therapy)

patient

DNA (PI)CD71+CD61+

Lab DemonstrationsLab Demonstrationsroom 3-4151, 3-4157room 3-4151, 3-4157

• FCMN assayFCMN assay

room B-6624, B-6625room B-6624, B-6625

• CBMN assayCBMN assay