COMPLEMENTARITY OF STANDARD CYTOGENETIC ASSAYS

Standard cytogenetic assays used in genotoxicology usually include chromosome aberrations analysis and micronucleus cytokinesis-block assay. Both tests originate on standard protocol for lymphocyte culture and can be used as complement or substitute to each other. Aim of this study was to evaluate complementarities between results of chromosome aberration analysis assay and results of micronucleus cytokinesis-block assay in representative sample of inhabitants from Bosnia and Herzegovina. The aim was achieved by calculating Pearson correlation coefficient and simple linear regression.


Introduction
Cytogenetic tests in human lymphocytes culture are the most frequently applied in genetic toxicology.Many of these tests are used for testing of genotoxic potential of chemical agents (), drugs (), as well as genotoxicological monitoring of populations environmentally () or professionally exposed to various mutagens (, ).Chromosome aberrations analysis and micronucleus cytokinesis-block assay, as standard cytogenetic tests, can be used as complement or substitute to each other.These tests are also used in cancer risk assessment studies ().Both tests originate on standard protocol for lymphocyte culture ().Chromosome aberrations analysis assay presents method of detection and scoring of structural and numerical chromosome aberrations in metaphase spreads.As micronuclei derive from chromosomal fragments and whole chromosomes lagging behind in anaphase, this assay can be used to show both clastogenic and aneugenic effects (, ).Micronucleus technique is a simple cytogenetic method proposed for measurement of chromosomal damage in mitogenstimulated human lymphocytes ().Aim of this study was evaluation of the correlation between results of chromosome aberration analysis assay and results of micronucleus cytokinesis-block assay in representative sample of inhabitants from Bosnia and Herzegovina.

Materials and Methods
Study was conducted over peripheral blood samples collected from three local Bosnian populations.Examined group included  persons ( males and  females),  of them were smokers, average age was , years.Preparation of whole blood cultures as well as harvesting of cells was carried out according to standardized procedures.General points of applied cytogenetic tests are presented in Table .Microscopic observation of each sample including analysis of  metaphases for chromosome aberrations analysis assay and  binuclear cells for micronucleus cytokinesis-block assay was performed on x magnification on Olympus BX microscope.Two experienced scorers performed microscopic analysis.Chromosome aberrations were detected and classifi ed according to International System for Human Cytogenetic Nomenclature ().In total structural chromosome aberrations frequencies of metaphases with chromatid and chromosome breaks, acentric fragments and dicentric chromosomes were calculated.Gaps were not scored as chromosome aberrations.In total numerical chromosome aberrations all deviations of normal human chromosome complement (n=) were registered.Detailed description of the scoring criteria for the micronucleus cytokinesis-block assay used in this research defined Fenech et al. ().Micronuclei frequencies were calculated in binucleated lymphocytes.In order to determine linear relationship among results of conducted cytogenetic assays, Pearson correlation coeffi cient and simple linear regression, conducted by Winks . Professional software (TexaSoft, Cedar Hill, TX) were applied.Pearson correlation coeffi cient and simple linear regression are measures of the linear relationship strength between two variables, in case of this research, frequencies of chromosome aberrations and micronuclei.Applied statistical methods are conducted over our previously published results (, , ).

Results and Discussion
Signifi cant positive correlation was determined by Pearson correlation coeffi cient for frequencies of structural chromosome aberrations and binuclear cells with micronuclei (p<,).Th e same was determined for frequencies of numerical chromosome aberrations and binuclear cells with micronuclei (p<,).Comparing frequencies of total (structural + numerical) chromosome aberrations and frequencies of binuclear cells with micronuclei determined correlation coeffi cient was also signifi cant (p<,).Linear relationship between the frequencies of binuclear lymphocytes with micronuclei and frequencies of lymphocytes metaphase spreads with structural and numerical chromosome aberrations are shown in Figures . and .Our results match previously published results of similar studies and reported positive correlations between micronuclei frequency and

Conclusion
Applied statistical methods in study sample confi rm complementarities of standard cytogenetic assays: chromosome aberrations analysis and micronucleus cytokinesis-block assay.Results of this study affi rm validity and usage of both assays either independently or simultaneously in genotoxicological studies.Th is conclusion is additionally supported by the fact that both tests need the same infrastructure and conditions for cell culturing, harvesting, slides preparation and analysis.

TABLE 1 .
General points of applied tests