Institute for Electromagnetic Sensing of Environment Naples, Italy IREA MICRONUCLEUS ASSAY Maria Rosaria Scarfì CNR – IREA Institute for Electromagnetic Sensing of Environment Naples, Italy scarfi.mr@irea.cnr.it Kanazawa, BEMS 2007

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

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

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

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

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

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

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

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

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

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

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

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

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.

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.

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.

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).

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.

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

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

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 http://www.csiro.au/index.asp?type=activity&id=HUMN Kanazawa, BEMS 2007

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

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

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

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

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

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

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

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

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

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

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

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

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

CBMN ASSAY – CREST staining IREA Kanazawa, BEMS 2007

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

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

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

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

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

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

MN ASSAY in BEMS research Largely employed since 1991 IREA 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) Kanazawa, BEMS 2007