1. Institute for Electromagnetic Sensing of Environment Naples, Italy
IREA
MICRONUCLEUS ASSAY
Maria Rosaria Scarfì
CNR – IREA
Institute for Electromagnetic Sensing of Environment Naples, Italy
Kanazawa, BEMS 2007

2. MICRONUCLEI
IREA
Observation 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
Fragments
Dicentric
Ring
Kanazawa, BEMS 2007

3. MICRONUCLEI small nuclei separated from the main nucleus
IREA
MICRONUCLEI
small nuclei separated from the main nucleus
produced during nuclear division
contain chromosomes or chromosome fragments, derived from mitotic spindle dysfunction or acentric fragments
first described in the cytoplasm
of erythocytes
Kanazawa, BEMS 2007

4. IREA
MICRONUCLEI
MNi 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

5. MICRONUCLEI IREA X X X X X X Chromosome loss Chromosome breakage
Kanazawa, BEMS 2007

6. IREA
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

7. CBMN ASSAY CITOKINESIS BLOCK METHOD Several methods have been proposed
IREA
CBMN ASSAY
Several 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 studies
Kanazawa, BEMS 2007

8. CBMN ASSAY CITOKINESIS BLOCK METHOD (1985)
IREA
CBMN ASSAY
CITOKINESIS BLOCK METHOD (1985)
Cytochalasin-B (inhibitor of cytokinesis)
once divided cells are binucleates
+Cyt-B
-Cyt-B
S.B. Carter, 1967
M.Fenech & A.Morley (1985)
mitosis
CYT-B: Inhibitor of actin polymerisation
Kanazawa, BEMS 2007

9. IREA
CBMN ASSAY
S phase
anaphase
Cyt-B
Kanazawa, BEMS 2007

10. CBMN ASSAY - method in human peripheral blood lymphocytes
IREA
CBMN ASSAY - method
in human peripheral blood lymphocytes
cultures with whole blood or separate lymphocytes
(culture medium and growth factors i.e. FCS and L-G)
72 hours
t=0
t=44h
Start culture (PHA)
+ Cyt-B
harvest
Kanazawa, BEMS 2007

11. CBMN ASSAY IREA Escaped block or slow proliferation
more than one nuclear division
Kanazawa, BEMS 2007

12. CBMN ASSAY MN must be counted only in binucleated cells
IREA
CBMN ASSAY
MN must be counted only
in binucleated cells
Mononucleated cells with MN 
damage accumulated before cultivation of the cells (spontaneous)
Binucleated cells with MN  
spontaneous damage + damage expressed during cultivation (treatment)
Kanazawa, BEMS 2007

13. Experimental procedure in use in our Lab
IREA
CBMN ASSAY
Experimental procedure in use in our Lab
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.
Kanazawa, BEMS 2007

14. Experimental procedure in use at IREA Lab
CBMN assay is applied on both isolated and whole blood lymphocytes.
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.

15. Experimental procedure in use at IREA Lab
Following a centrifugation at 2100 rpm for 30 minutes, lymphocytes are
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.

16. Experimental procedure in use at IREA Lab
Isolated lymphocytes are seeded in complete culture medium composed by
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.

17. Experimental procedure in use at IREA Lab
After 72 h of growth, cultures are harvested and slides prepared.
Procedure to make up slides from whole blood cultures:
Collection of 1 ml culture
Centrifugation at 3000 rpm for 1 minute
Red cells break (lysis buffer for 7 minutes)
3 washing steps
Hypotonic treatment (15 min).

18. Experimental procedure in use at IREA Lab
Slides are prepared by using a cytospin. Cells in hypotonic solution are
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.

19. Experimental procedure in use at IREA Lab
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

20. CBMN ASSAY Advantages Disadvantages
IREA
CBMN ASSAY
Advantages
very 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
Disadvantages
No information on the type of chromosomal aberration
Kanazawa, BEMS 2007

21. CBMN ASSAY – HUMN project
IREA
CBMN ASSAY – HUMN project
International Collaborative Project
on MN Frequency in Human Population (HUMN)
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
34 laboratories in 21 countries participate to the Project
Coordinator: Dr. M. Fenech at CSIRO
to 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 cancer
Kanazawa, BEMS 2007

22. CBMN ASSAY – HUMN project
IREA
CBMN ASSAY – HUMN project
Kanazawa, BEMS 2007

23. CBMN ASSAY – HUMN project
IREA
CBMN ASSAY – HUMN project
Main results of the HUMN project
Factors influencing MN frequency
Age, sex, life style (smoking, alcohol consumption, diet), drugs, X-rays
Criteria for slides scoring
number of cells and morphological criteria
Recommendations for performing the MN assay on several cell types
lymphocytes, primary cells and cell lines
Kanazawa, BEMS 2007

24. CBMN ASSAY: scoring criteria
IREA
CBMN ASSAY: scoring criteria
Kanazawa, BEMS 2007

25. 1000 binucleated cells/sample
IREA
CBMN ASSAY: scoring criteria
Well stained cells
Undamaged 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

26. CBMN ASSAY: scoring criteria
IREA
CBMN ASSAY: scoring criteria
Nucleoplasmic bridges
(dicentric chromosomes)
Necrotic cells
(vacuolated cytoplasm and fragmented nucleus)
Bridges: centromeres pulled to the opposite poles of the cell
Apoptotic cells (condensed chromatin)
Kanazawa, BEMS 2007

27. CBMN ASSAY: scoring criteria
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)]) / N
Index of cell kinetics
b) binucleate cell index (BCI)
% cells in first mitosis
BCI = Binucleated Cells/N
Index of cytotoxicity
Kanazawa, BEMS 2007

28. CBMN ASSAY – Other cell types
IREA
CBMN ASSAY – Other cell types
Kanazawa, BEMS 2007

29. CBMN ASSAY – Other cell types
IREA
CBMN ASSAY – Other cell types
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 effects
Kanazawa, BEMS 2007

30. CBMN ASSAY – cancer risk
IREA
CBMN ASSAY – cancer risk
Kanazawa, BEMS 2007

31. CBMN ASSAY – cancer risk
IREA
CBMN ASSAY – cancer risk
6718 subjects from of 10 countries, screened in
20 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.
Kanazawa, BEMS 2007

32. CBMN ASSAY – cancer risk
IREA
CBMN ASSAY – cancer risk
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-free
survival.
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.
Kanazawa, BEMS 2007

33. CBMN ASSAY – centromeric staining
IREA
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 .
Kanazawa, BEMS 2007

34. CBMN ASSAY – CREST staining
IREA
CREST assay
Immunofluorescence 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 chromosomes
Kanazawa, BEMS 2007

35. CBMN ASSAY – CREST staining
IREA
Kanazawa, BEMS 2007

36. CBMN ASSAY – FISH FISH technique
IREA
FISH technique
fluorescence in situ hybridisation with a DNA probe specific to the centromeric regions
pan-centromeric probes - not specific for single chromosomes
chromosome-specific centromeric probes
Kanazawa, BEMS 2007

37. CBMN ASSAY – FISH IREA FISH using pan-centromeric probe
Kanazawa, BEMS 2007

38. CBMN ASSAY – FISH aneuploidy in CB-lymphocytes with MN IREA
FISH using chromosome-specific centromeric probes
Chung et al., Mutation Res. 516, 49–56 (2002)
Chromosome 7
Chromosome 8 a b
aneuploidy in CB-lymphocytes with MN
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
Kanazawa, BEMS 2007

39. CBMN ASSAY Information on: Genotoxicity Cytotoxicity
IREA
Information on:
Genotoxicity
chromosome loss (clastogenic events)
chromosome breakage (aneugenic events)
dicentric chromosomes (nucleoplasmic bridges)
Cytotoxicity
apoptotic cells
necrotic cells
cell cycle kinetics (CBPI)
% of binucleated cells (BCI)
Kanazawa, BEMS 2007

40. MN ASSAY in erythrocytes In vivo MN test in mammalian erythrocytes
IREA
In vivo MN test in mammalian erythrocytes
Expulsion
Of the nucleus
Type I
Type II
Type III
Type VI
RETICULOCYTES
Micronucleus assay
mature
erythrocytes
erythroblast
Differentiation process from the erythroblast to the mature erythrocyte
Kanazawa, BEMS 2007

41. MN ASSAY in erythrocytes
IREA
The frequency of MN in reticulocytes and erythrocytes increases in response to a genotoxic treatment
As the average life-span is much longer than immature erythocytes, MN assay is reliable when chronic, but not acute, treatments are performed
Micronucleus in a type I reticulocyte AO stained
Advantage - blood can be obtained by tail venupuncture without sacrifying animals (multiple sample collection)
Kanazawa, BEMS 2007

42. MN ASSAY in BEMS research Largely employed since 1991
IREA
Largely employed since 1991
Vijayalaxmi, Obe G Radiat Res. 162: (2004)
Vijayalaxmi and G.Obe Bioelectromagnetics 26: (2005)
Moulder et al., Int J Radiat Biol 81: (2005)
Verschaeve L, Toxicol Appl Pharmacol. 207: (2005)
Kanazawa, BEMS 2007