Presentation on theme: "Institute for Electromagnetic Sensing of Environment Naples, Italy"— Presentation transcript:
1 Institute for Electromagnetic Sensing of Environment Naples, Italy IREAMICRONUCLEUS ASSAYMaria Rosaria ScarfìCNR – IREAInstitute for Electromagnetic Sensing of Environment Naples, ItalyKanazawa, BEMS 2007
2 MICRONUCLEIIREAObservation of chromosomes and counting of aberrations in metaphases is the most detailed analysis to measure chromosome damageComplexity and time consumingConfounding effects of artefactual loss of chromosomes from metaphasesStimulated the development of a simpler method to measure chromosome damageFragmentsDicentricRingKanazawa, BEMS 2007
3 MICRONUCLEI small nuclei separated from the main nucleus IREAMICRONUCLEIsmall nuclei separated from the main nucleusproduced during nuclear divisioncontain chromosomes or chromosome fragments, derived from mitotic spindle dysfunction or acentric fragmentsfirst described in the cytoplasmof erythocytesKanazawa, BEMS 2007
4 IREAMICRONUCLEIMNi expressed in cells that have completed nuclear divisionideally scored in the binucleated stage of the cell cyclethe assay cannot be applied efficiently or quantitativelyin non-dividing cellsKanazawa, BEMS 2007
5 MICRONUCLEI IREA X X X X X X Chromosome loss Chromosome breakage Kanazawa, BEMS 2007
6 IREAMICRONUCLEIa need to develop a method that within a cell population could distinguish between cells that are not dividing and cells that are undergoing mitosisbecause 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 onlyKanazawa, BEMS 2007
7 CBMN ASSAY CITOKINESIS BLOCK METHOD Several methods have been proposed IREACBMN ASSAYSeveral methods have been proposedCITOKINESIS BLOCK METHODsuccessfully introduced in many laboratories all over the worldversatility, simplicity and lack of effects on base-line genetic damageProvides an index of both chromosome loss and chromosome breakagemonitoring exposed populationidentifying mutagen-sensitive individualsin vitro studiesKanazawa, BEMS 2007
8 CBMN ASSAY CITOKINESIS BLOCK METHOD (1985) IREACBMN ASSAYCITOKINESIS BLOCK METHOD (1985)Cytochalasin-B (inhibitor of cytokinesis)once divided cells are binucleates+Cyt-B-Cyt-BS.B. Carter, 1967M.Fenech & A.Morley (1985)mitosisCYT-B: Inhibitor of actin polymerisationKanazawa, BEMS 2007
10 CBMN ASSAY - method in human peripheral blood lymphocytes IREACBMN ASSAY - methodin human peripheral blood lymphocytescultures with whole blood or separate lymphocytes(culture medium and growth factors i.e. FCS and L-G)72 hourst=0t=44hStart culture (PHA)+ Cyt-BharvestKanazawa, BEMS 2007
11 CBMN ASSAY IREA Escaped block or slow proliferation more than one nuclear divisionKanazawa, BEMS 2007
12 CBMN ASSAY MN must be counted only in binucleated cells IREACBMN ASSAYMN must be counted onlyin binucleated cellsMononucleated 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 IREACBMN ASSAYExperimental procedure in use in our LabIn 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 ofa 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 ratioof 2 to 1 for lymphocytes isolation.
15 Experimental procedure in use at IREA Lab Following a centrifugation at 2100 rpm for 30 minutes, lymphocytes areconfined at the interface between density gradient solution and theculture medium. A portion of medium is discarded and cellscan be collected.After two washing steps in sterile PBS solution, cells are stained with Trypan blue and counted witha 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 byRPMI, 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 cultureCentrifugation at 3000 rpm for 1 minuteRed cells break (lysis buffer for 7 minutes)3 washing stepsHypotonic treatment (15 min).
18 Experimental procedure in use at IREA Lab Slides are prepared by using a cytospin. Cells in hypotonic solution arecytospinned 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 cellsLysis buffer: EDTA disodium salt 0.1 mM; NH4 Cl 155mM; KHCO3 10 mMWash medium: RPMI medium +2% FBSHypotonic solution: wash medium + distilled water (1:4)Fixation: 80% methanol in distilled waterStaining: 5% Giemsa in phosphate buffer pH 6.8
20 CBMN ASSAY Advantages Disadvantages IREACBMN ASSAYAdvantagesvery sensitive and simple indicator of chromosome damage, which also provides information on cell cycle progressionless time-consuming respect to chromosomal aberrationsBiomarker of effect: relevant for risk assessment of cancerScoring performed relatively easilyApplied on different cell types relevant for human biomonitoring: lymphocytes, fibroblasts and exfoliated epithelial cellsIncreased statistical powerDisadvantagesNo information on the type of chromosomal aberrationKanazawa, BEMS 2007
21 CBMN ASSAY – HUMN project IREACBMN ASSAY – HUMN projectInternational Collaborative Projecton 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 populations34 laboratories in 21 countries participate to the ProjectCoordinator: Dr. M. Fenech at CSIROto compare the baseline MN frequency from different labs and among different populationsto compare different techniques to define a standard protocolto evaluate the suitability of MN as biomarker of risk for diseases such as cancerKanazawa, BEMS 2007
23 CBMN ASSAY – HUMN project IREACBMN ASSAY – HUMN projectMain results of the HUMN projectFactors influencing MN frequencyAge, sex, life style (smoking, alcohol consumption, diet), drugs, X-raysCriteria for slides scoringnumber of cells and morphological criteriaRecommendations for performing the MN assay on several cell typeslymphocytes, primary cells and cell linesKanazawa, BEMS 2007
25 1000 binucleated cells/sample IREACBMN ASSAY: scoring criteriaWell stained cellsUndamaged cytoplasmic membranemain nuclei of similar dimensionNo more than 6 MN per cellMN with same colour of main nucleiMN morphologically similar to nuclei and no larger than 1/3 of the main nucleusSamples in duplicate1000 binucleated cells/sampleKanazawa, BEMS 2007
26 CBMN ASSAY: scoring criteria IREACBMN ASSAY: scoring criteriaNucleoplasmic bridges(dicentric chromosomes)Necrotic cells(vacuolated cytoplasm and fragmented nucleus)Bridges: centromeres pulled to the opposite poles of the cellApoptotic cells (condensed chromatin)Kanazawa, BEMS 2007
27 CBMN ASSAY: scoring criteria IREACBMN ASSAY: scoring criteriaby scoring the number of nuclei in 500 cellsa) cytokinesis-block proliferation index (CBPI)mean number of cell cycles per cellCBPI = ([M1 + 2M2 + 3(M3 + M4)]) / NIndex of cell kineticsb) binucleate cell index (BCI)% cells in first mitosisBCI = Binucleated Cells/NIndex of cytotoxicityKanazawa, BEMS 2007
28 CBMN ASSAY – Other cell types IREACBMN ASSAY – Other cell typesKanazawa, BEMS 2007
29 CBMN ASSAY – Other cell types IREACBMN ASSAY – Other cell typesCB-Method adaptedto other primary cell types or cell linesTo assess the cell cycle durationto 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-Bto block the maximum number of dividing cellsTo 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
30 CBMN ASSAY – cancer risk IREACBMN ASSAY – cancer riskKanazawa, BEMS 2007
31 CBMN ASSAY – cancer risk IREACBMN ASSAY – cancer risk6718 subjects from of 10 countries, screened in20 labs for MN frequency between 1980 and 2002selected 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 IREACBMN ASSAY – cancer riskA significant increase of all cancers incidence was found for the groups with medium and high MN frequencyThe 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 cancersThe 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 IREAThe 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 IREACREST assayImmunofluorescence staining with antibodies anti-kinetochoreCentromeres 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
36 CBMN ASSAY – FISH FISH technique IREAFISH techniquefluorescence in situ hybridisation with a DNA probe specific to the centromeric regionspan-centromeric probes - not specific for single chromosomeschromosome-specific centromeric probesKanazawa, 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 probesChung et al., Mutation Res. 516, 49–56 (2002)Chromosome 7Chromosome 8abaneuploidy in CB-lymphocytes with MNa) one red and two green signals in each sister nucleus and two red signals in one of four MNb) same nuclei counter-stained with DAPIKanazawa, BEMS 2007
39 CBMN ASSAY Information on: Genotoxicity Cytotoxicity IREAInformation 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
40 MN ASSAY in erythrocytes In vivo MN test in mammalian erythrocytes IREAIn vivo MN test in mammalian erythrocytesExpulsionOf the nucleusType IType IIType IIIType VIRETICULOCYTESMicronucleus assaymatureerythrocyteserythroblastDifferentiation process from the erythroblast to the mature erythrocyteKanazawa, BEMS 2007
41 MN ASSAY in erythrocytes IREAThe frequency of MN in reticulocytes and erythrocytes increases in response to a genotoxic treatmentAs the average life-span is much longer than immature erythocytes, MN assay is reliable when chronic, but not acute, treatments are performedMicronucleus in a type I reticulocyte AO stainedAdvantage - 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 IREALargely employed since 1991Vijayalaxmi, 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