Methods of Methods of genotoxicologygenotoxicology

No 520 Development

Primary event in DNA ► DNA breaks, alkylation or other alteration of bases, „cross links“

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Reparation, defect in reparation, abnormal

reparation, abnormal joining of broken ends of

chromosomes, unrepaired DNA damage

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Chromosomal aberration

Method of mutagenic studyIn vitro:

Ames test – on bacteria – auxotrophic strains of Salmonella typhimurium

In vivo:

Micronucleus test – micronucleus = chromosomal fragment in cytoplasma of erythrocytes or binuclear cells, lymfocytes

Cytogenetic analysis of bone marrow cells of experimental animals or of human peripheral lymphocytes - chromosomal breaks or rearrangements

Comet assay – single strand DNA breaks

Bacteria : Salmonella typhimurium

- auxotrophic strains (bacterial cells are enable to

synthetize histidine because of mutation)

Mutagens cause reverse mutations and bacterial cells

thereby get the property to grow on the medium without

histidine and form colonies

Auxotrophy is the inability of an organism to synthesize

a particular organic compound required for its growth

Ames test

Scoring: counting of a number of colonies by computer (image analysis).

We detects mutagenic effect by Increased number of colonies (revertants) of sample in comparison with number of colonies in controls

Direct mutagens – original molecule has mutagenic

qualities

Indirect mutagens - need metabolic activation = are active only after enzymatic metabolisation

– S9 fraction of liver homogenate from rats treated by a mixture of mutagens – contains enzymes able to metabolize tested compound

Micronucleus testMicronucleus test

Micronuclei is either the whole chromosome or a chromosomal fragment as a small body outside nucleus

Principle: detection of the frequency of micronuclei: a) in erythrocytes of bone marrow of laboratory animals b) in peripheral lymphocytes of persons

Micronucleus testMicronucleus test

Mutagenes induce changes of chromosomes, breaks.

Chromosomal fragment (or whole chromosome) is not

attached to mitotic spindle, stays in cytoplasma after

formation of daughter nuclei.

A mutagenic effect of tested compound is presented by

increased number of micronuclei in 1000 of cells in

comparison with control.

MiMiccronuronuccleus testleus test

Detection of centromere by FISH

Micronucleus in polychromatophile Micronucleus in polychromatophile erythrocyteserythrocytes

http://faculty.ksu.edu.sa/al-saleh/Pages/LabAlbum.aspx

Micronuclei in peripheral lymphocytesMicronuclei in peripheral lymphocytes

Comet assay - single gel electrophoresis

Method testing mutagenic effects by the DNA strand break analysis

Method based on the electrophoresis

Quantitative analysis of DNA breaks

Advantage: it detects actual state of DNA damage, just before reparation.

Method of comet assayMethod of comet assay

Laboratory animals are exposed by the tested compound

Collection of tissue samples (bone marrow, liver, blood, intestine…)

Isolation of cells (trypsinisation)

Lysis of cells

Releasing of DNA double strands in alkali solution

Electrophoresis of DNA on microscopic slide covered with agarose

Staining (by fluorescent dye - ethidiumbromide)

Detection of DNA fragments as „tail of comet“ in fluorescent microscope

Intensity of fluorescence in tail compared with fluorescence in head of comet is quantitatively analyzed by computer program

Comet assay

Analysis of chromosomal aberrations in

human peripheral lymphocytes :

In vivo: in persons treated by mutagens

(professional exposure, or exposure by accident…) –

cultivation of peripheral lymphocytes – cytogenetic

method, staining and scoring of a number and types of

chromosomal aberrations

Scoring of 100-200 of cells per sample

Normal level (not increased) till 2% of cells with

chromosomal aberrations

post-radiation chromosomal aberrations

difragment

dicentric

Chromatid breaks

Chromatid exchange – after chemicals

Chromosomal changes (exchanges) after chemicals

– Cell cultivation (T lymphocytes) in the presence of

bromdeoxyuridine (5-bromo-2-deoxyuridine, BrdU ) during 2

cell cycles

– In the 1st mitosis - both chromatids are dark

– In the 2nd mitosis - one chromatid is dark, second one pale

pale chromatid = both DNA strands substituted with BrdU → delayed DNA spiralization → pale colour

Sister chromatid exchanges

1.

2.

+ BrdU for 2 cycles of division

BrdU = thymidine analogue

First mitosis:

BrdU is introduced to one DNA strand (new strand) of both chromatids during replication→ dark colour

Second mitosis:

One chromatid is dark, second (with both strands substituted by BrdU) is pale - delayed spiralization => pale staining

Observation of sister chromatid exchanges = method of testing of mutagenic effects

Different staining of chromatids in second mitosis with sister chromatid exchanges-marked by arrows

Sister chromatid exchanges – increased level after mutagene action

Metods of mutagenicity testing serve to prediction of carcinogenic effects (mutagenic test are quick in

comparison with long-term tests of carcinogenicity

Each mutagen = potential carcinogen!

but not all carcinogens are mutagenic (non-genotoxic carcinogens – support proliferation or have other effects, but do not induct mutations)

Experimental organismsExperimental organisms

Models in vivo: Drosophila melanogaster Mus musculus Rattus norvegicus Xenopus laevis Ceanorhabditis elegans

Models in vitro: Tissue cultures Cell lines (He-La cells) Primary cultures

Tobacco mosaic virus

Bacillus subtilis

Saccharomyces cerevisiae

Zea maysArabidopsis thaliana

Mus musculus, Rattus norvegicus

70% homology of human and mice genome

Mutation of same gene

Drosophila melanogasterXenopus laevisCaenorhabditis elegansSchizosaccharomyces pombe

Thanks for attention