micronucleus assay kanazawa, bems 2007 maria rosaria scarfì cnr – irea institute for...
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MICRONUCLEUS ASSAY
Kanazawa, BEMS 2007
Maria Rosaria Scarfì
CNR – IREA
Institute for Electromagnetic Sensing of Environment Naples, Italy
IREA
MICRONUCLEIObservation of chromosomes and counting of
aberrations in metaphases is the most detailed analysis to measure chromosome damage
•Complexity and time consuming
•Confounding effects of artefactual loss of chromosomes from
metaphases
•Stimulated the development of a simpler method to
measure chromosome damage
FragmentsDicentricRing
Kanazawa, BEMS 2007
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MICRONUCLEI
first described in the cytoplasm of erythocytes
small nuclei separated from the main nucleus
produced during nuclear division
contain chromosomes or chromosome fragments, derived from mitotic spindle
dysfunction or acentric fragments
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MICRONUCLEIMNi expressed in cells that have completed nuclear
division
ideally scored in the binucleated stage of the cell cycle
the assay cannot be applied efficiently or quantitatively in non-dividing cells
Kanazawa, BEMS 2007
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X XX
X X X
X X X
X X X
Kanazawa, BEMS 2007
MICRONUCLEIIREA
Chromosome loss
Chromosome breakage
MICRONUCLEI
a need to develop a method that within a cell population could distinguish between cells that are not dividing and cells that are undergoing mitosis
because of the uncertainty of the fate of MNi following more than one nuclear division it is important to identify cells that have completed one nuclear division only
Kanazawa, BEMS 2007
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CBMN ASSAYSeveral methods have been proposed
CITOKINESIS BLOCK METHOD
successfully introduced in many laboratories all over the world
versatility, simplicity and lack of effects on base-line genetic damage
Provides an index of both chromosome loss and chromosome breakage
monitoring exposed population
identifying mutagen-sensitive individuals
in vitro studiesKanazawa, BEMS 2007
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CBMN ASSAY
mitosis
+Cyt-B
-Cyt-B
CITOKINESIS BLOCK METHOD (1985)
Cytochalasin-B (inhibitor of cytokinesis)once divided cells are binucleates
S.B. Carter, 1967
M.Fenech & A.Morley (1985)
Kanazawa, BEMS 2007
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CBMN ASSAY
S phaseanaphase
Cyt-B
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CBMN ASSAY - method
72 hours
t=0 t=44h
Start culture (PHA)
+ Cyt-B
harvest
in human peripheral blood lymphocytes
cultures with whole blood or separate lymphocytes(culture medium and growth factors i.e. FCS and L-
G)
Kanazawa, BEMS 2007
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CBMN ASSAY
Escaped block or
slow proliferation
more than one
nuclear division
Kanazawa, BEMS 2007
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CBMN ASSAY
Mononucleated cells with MN damage accumulated before cultivation of the cells (spontaneous)
Binucleated cells with MN spontaneous damage + damage expressed
during cultivation (treatment)
MN must be counted only
in binucleated cells
Kanazawa, BEMS 2007
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CBMN ASSAY
Experimental procedure in use in our Lab
Kanazawa, BEMS 2007
IREA
In the following, the experimental
procedures currently in use in our lab to
perform the CBMN assay in human
lymphocytes and in several cell lines are
shown.
The protocol for lymphocyte separation starting from a buffy coat obtained by the transfusion unit of a hospital is shown.The buffy coat is transferred in a sterile tube and diluted (1:2) in culture medium, gently mixed and stratified on a density gradient solution in a ratio of 2 to 1 for lymphocytes isolation.
CBMN assay is applied on both isolated and whole blood lymphocytes.
Experimental procedure in use at IREA Lab
confined at the interface between density gradient solution and the culture medium. A portion of medium is discarded and cells can be collected.After two washing steps in sterile PBS solution, cells are stained with Trypan blue and counted with a haemocytometric chamber to seed the required number of cells for each culture.
Experimental procedure in use at IREA Lab
Following a centrifugation at 2100 rpm for 30 minutes, lymphocytes are
RPMI, FCS, L-Glutamine and PHA, as mitogen, to stimulate lymphocytes to enter cell cycle. In the case of whole blood cultures, heparinized blood, collected by venipuncture, is directly diluted in complete medium, according to standard procedures. In both cases, samples are located for 44 h in a CO2 incubator, then Cytochalasin-B is added.
Experimental procedure in use at IREA Lab
Isolated lymphocytes are seeded in complete culture medium composed by
After 72 h of growth, cultures are harvested and slides prepared.
Procedure to make up slides from whole blood cultures:
a)Collection of 1 ml culture
b)Centrifugation at 3000 rpm for 1 minute
c)Red cells break (lysis buffer for 7 minutes)
d)3 washing steps e)Hypotonic
treatment (15 min).
Experimental procedure in use at IREA Lab
cytospinned for 7 min. Slides are air dried, fixed and stained in a Giemsa solution.Stained cells are confined in a spot on the slides and a coverslip is used to preserve the sample for microscope screening.The procedure described for whole blood cultures is also applied for isolated lymphocyte and cell line cultures, except for the lysis step, due to the absence of erythrocytes.
Experimental procedure in use at IREA Lab
Slides are prepared by using a cytospin. Cells in hypotonic solution are
Solutions requested to harvest cells
- Lysis buffer: EDTA disodium salt 0.1 mM; NH4 Cl 155
mM; KHCO3 10 mM
- Wash medium: RPMI medium +2% FBS
- Hypotonic solution: wash medium + distilled water (1:4)
- Fixation: 80% methanol in distilled water
- Staining: 5% Giemsa in phosphate buffer pH 6.8
Experimental procedure in use at IREA Lab
Advantagesvery sensitive and simple indicator of chromosome damage,
which also provides information on cell cycle progression
less time-consuming respect to chromosomal aberrations
Biomarker of effect: relevant for risk assessment of cancer
Scoring performed relatively easily
Applied on different cell types relevant for human biomonitoring: lymphocytes, fibroblasts and exfoliated
epithelial cells
Increased statistical power
DisadvantagesNo information on the type of chromosomal aberration
CBMN ASSAY
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CBMN ASSAY – HUMN projectInternational Collaborative Project
on MN Frequency in Human Population (HUMN)
34 laboratories in 21 countries participate to the Project
Coordinator: Dr. M. Fenech at CSIROto compare the baseline MN frequency from different labs and among different populations
to compare different techniques to define a standard protocol
to evaluate the suitability of MN as biomarker of risk for diseases such as cancerhttp://www.csiro.au/index.asp?type=activity&id=HUMN
Kanazawa, BEMS 2007
launched in 1997 because of world-wide interest in the MN method to assess environmental effects on chromosome damage in blood and epithelial tissues in
human populations
IREA
Factors influencing MN frequencyAge, sex, life style (smoking, alcohol consumption, diet), drugs, X-rays
Criteria for slides scoringnumber of cells and morphological criteria
Recommendations for performing the MN assay on several cell typeslymphocytes, primary cells and cell lines
Kanazawa, BEMS 2007
Main results of the HUMN project
IREACBMN ASSAY – HUMN project
CBMN ASSAY: scoring criteria
Kanazawa, BEMS 2007
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CBMN ASSAY: scoring criteria
Well stained cellsUndamaged cytoplasmic
membrane
main nuclei of similar dimension
No more than 6 MN per cell
MN with same colour of main nuclei
MN morphologically similar to nuclei and no larger than 1/3 of
the main nucleus
Samples in duplicate
1000 binucleated cells/sample
Kanazawa, BEMS 2007
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Nucleoplasmic bridges(dicentric
chromosomes)
Necrotic cells (vacuolated cytoplasm
and fragmented nucleus)
Apoptotic cells (condensed chromatin)
Kanazawa, BEMS 2007
IREA CBMN ASSAY: scoring criteria
by scoring the number of nuclei in 500 cells
a) cytokinesis-block proliferation index (CBPI)mean number of cell cycles per cell
CBPI = ([M1 + 2M2 + 3(M3 + M4)]) / NIndex of cell kinetics
b) binucleate cell index (BCI)% cells in first mitosis
BCI = Binucleated Cells/NIndex of cytotoxicity
Kanazawa, BEMS 2007
IREA CBMN ASSAY: scoring criteria
CBMN ASSAY – Other cell types
Kanazawa, BEMS 2007
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CB-Method adapted to other primary cell types or cell lines
To assess the cell cycle duration to define the right time for Cyt-B addition (first cell
cycle) and for cell harvest (prior to the second mitosis)
To determine the concentration of Cyt-B to block the maximum number of dividing cells
To use cell types with low and stable background frequency of MN (30/1000 CB
cells)to guarantee the sensitivity of the assay to detect
genotoxic effectsKanazawa, BEMS 2007
IREA CBMN ASSAY – Other cell types
Kanazawa, BEMS 2007
IREA CBMN ASSAY – cancer risk
IREA
Kanazawa, BEMS 2007
6718 subjects from of 10 countries, screened in20 labs for MN frequency between 1980 and 2002
selected from the database of the HUMN project and followed up for cancer incidence or mortality.
To standardize for the inter-laboratory variability subjects were classified according to the percentiles of MN distribution within each laboratory as low, medium or high frequency.
CBMN ASSAY – cancer risk
IREA
Kanazawa, BEMS 2007
A significant increase of all cancers incidence was found for the groups with medium and high MN frequency The same groups also showed a decreased cancer-freesurvival.
This association was present in all national cohorts and for all major cancer sites, especially urogenital and gastro-intestinal cancers
The results provide preliminary evidence that MN frequency in PBL is a predictive biomarker of cancer risk within a population of healthy subjects.
CBMN ASSAY – cancer risk
CBMN ASSAY – centromeric staining
Kanazawa, BEMS 2007
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.
.
The combination of the micronucleus assay with staining techniques of the centromeric region of the chromosomes allows discrimination between micronuclei containing a whole chromosome (centromere positive micronucleus) and an acentric chromosome fragment (centromere negative micronucleus)
It is possible by using
– Abs that bind to kinetochore proteins assembled at the centromeric regions (not specific for single
chromosomes) CREST
– probes that are specific for centromeric DNA FISH
CREST assayImmunofluorescence
staining with antibodies anti-kinetochore
Centromeres of MN can be immunologically visualized with
antikinetochore Ab from scleroderma patients of the CREST subtype (calcinosis,
Raynaud phenomenon, esophageal dismotility,
sclerodactily and telangiectasia)
CREST serum contains antibody to a specific protein of the kinetochore region of
chromosomesKanazawa, BEMS 2007
IREACBMN ASSAY – CREST
staining
Kanazawa, BEMS 2007
IREACBMN ASSAY – FISH
pan-centromeric probes - not specific for single chromosomes
chromosome-specific centromeric probes
FISH techniquefluorescence in situ
hybridisation with a DNA probe specific to the centromeric
regions
Kanazawa, BEMS 2007
FISH using pan-centromeric probe
IREACBMN ASSAY – FISH
Chung et al., Mutation Res. 516, 49–56 (2002)
a) one red and two green signals in each sister nucleus and two red signals in one of four MN b) same nuclei counter-stained with DAPI
FISH using chromosome-specific centromeric probes
CBMN ASSAY – FISH
Kanazawa, BEMS 2007
aneuploidy in CB-lymphocytes with MN
a b
Chromosome 7
Chromosome 8
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Information on:
Genotoxicitychromosome loss (clastogenic events)chromosome breakage (aneugenic events)dicentric chromosomes (nucleoplasmic bridges)
Cytotoxicityapoptotic cellsnecrotic cellscell cycle kinetics (CBPI)% of binucleated cells (BCI)Kanazawa, BEMS 2007
IREA CBMN ASSAY
erythroblast matureerythrocytes
Differentiation process from the erythroblast to the mature erythrocyte
ExpulsionOf the nucleus
Type I Type II Type III Type VI
RETICULOCYTES
Micronucleus assay
Kanazawa, BEMS 2007
IREA MN ASSAY in erythrocytes
In vivo MN test in mammalian erythrocytes
Micronucleus in a type I reticulocyte AO
stained
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The frequency of MN in reticulocytes and erythrocytes increases in response to a
genotoxic treatment
MN ASSAY in erythrocytes
As the average life-span is much longer than immature erythocytes, MN assay is reliable when chronic, but not acute, treatments are performed
Advantage - blood can be obtained by tail venupuncture without sacrifying animals (multiple
sample collection)
Largely employed since 1991
Vijayalaxmi, Obe G Radiat Res. 162: 481-96 (2004)
Vijayalaxmi and G.Obe Bioelectromagnetics 26: 412-430 (2005)
Moulder et al., Int J Radiat Biol 81: 189-203 (2005)
Verschaeve L, Toxicol Appl Pharmacol. 207: 336-41 (2005)
IREA
Kanazawa, BEMS 2007
MN ASSAY in BEMS research