1. Methods of genotoxicology
No 520 Development

2. Key words:
Genotoxicity, spontaneous and induced mutations, somatic and gametic mutations, gene, chromosome and genome mutations, substitution, deletion, insertion, frame shift mutation, missense, nonsense, elongation mutations, same-sense mutation, transition, transversion, mutagene, carcinogene, teratogene, ultimative carcinogene, promutagene, procarcinogene, indirect mutagene, adduct, initiation-promotion theory, reverse mutation, Ames test, auxotrofic strain, micronucleus test, cytogenetic analysis, comet assay, sister chromatid exchanges

3. Chromosomal aberration
Primary event in DNA ► DNA breaks, alkylation or other alteration of bases, „cross links“ ▼
Reparation, defect in reparation, abnormal reparation, abnormal joining of broken ends of chromosomes, unrepaired DNA damage
Chromosomal aberration

4. Method of mutagenic study
In 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

5. Ames test 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

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

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

9. 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

10. Micronucleus 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

11. Micronucleus 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.

12. Micronucleus test
Detection of centromere by FISH

13. Micronucleus in polychromatophile erythrocytes

14. Micronucleus in binuclear cells after cytochalasin
Micronucleus in binuclear cells after cytochalasin block of cytokinesis

15. Micronuclei in peripheral lymphocytes

16. 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.

17. Method 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

18. Comet assay

19. 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 of cells per sample
Normal level (not increased) till 2% of cells with chromosomal aberrations

20. post-radiation chromosomal aberrations
difragment
dicentric

21. Chromatid breaks

22. Chromatid exchange – after chemicals

23. Chromosomal changes (exchanges) after chemicals

24. Sister chromatid exchanges
– 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

25. + 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 1.
Second mitosis:
One chromatid is dark, second (with both strands substituted by BrdU) is pale - delayed spiralization => pale staining 2.
Observation of sister chromatid exchanges = method of testing of mutagenic effects

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

27. Sister chromatid exchanges – increased level after mutagene action

28. Each mutagen = potential carcinogen!
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)

29. Experimental 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

30. Zea mays Tobacco mosaic virus Saccharomyces cerevisiae
Arabidopsis thaliana
Zea mays
Bacillus subtilis

31. Mus musculus, Rattus norvegicus
70% homology of human and mice genome
Mutation of same gene

32. Drosophila melanogaster
Xenopus laevis
Caenorhabditis elegans
Schizosaccharomyces pombe

33. Models organisms Phage Lambda, Tobacco mosaic virus
Escherichia coli (E. coli) - This common bacterium is the most widely-used organism in molecular genetics.
Bacillus subtilis
Saccharomyces cerevisiae, baker's yeast or budding yeast (used in brewing and baking) Schizosaccharomyces pombe, fission yeast, subject of genetic studies
Arabidopsis thaliana, currently the most popular model plant.
Many phenotypic and biochemical mutants have been mapped.
Arabidopsis was the first plant to have its genome sequenced.
Maize (Zea mays L.) is a cereal grain. It is a diploid monocot with 10 large
chromosome pairs, easily studied with the microscope. Its genetic
features, including many known and mapped phenotypic mutants and a
large number of progeny per cross (typically ) facilitated the
discovery of transposons ("jumping genes").

34. Models organisms
Caenorhabditis elegans, a nematode, usually called C. elegans[7] - an excellent model for understanding the genetic control of development and physiology. C. elegans was the first multicellular organism whose genome was completely sequenced
Drosophila, usually the species Drosophila melanogaster - a kind of fruit fly, famous as the subject of genetics experiments by Thomas Hunt Morgan and others. Easily raised in lab, rapid generations, mutations easily induced, many observable mutations. Recently, Drosophila has been used for neuropharmacological research
Chicken (Gallus gallus domesticus) - used for developmental studies, as it is an amniote and excellent for micromanipulation (e.g. tissue grafting) and over-expression of gene products
Cat (Felis cattus) - used in neurophysiological research
Dog (Canis lupus familiaris) - an important respiratory and cardiovascular model
Hamster - first used to study kala-azar (leishmaniasis)

35. Models organisms
Mouse (Mus musculus) - (Quantitative genetics, Molecular evolution,
Genomics)
Homo sapiens (humans) - used in various clinical studies
Rat (Rattus norvegicus) (Molecular evolution, Genomics)
Rhesus macaque - used for studies on infectious disease and
cognition
Xenopus laevis, the African clawed frog - used in developmental
biology because of its large embryos and high tolerance for physical
and pharmacological manipulation
Zebrafish (Danio rerio), a freshwater fish, has a nearly transparent
body during early development, which provides unique visual access to
the animal's internal anatomy. Zebrafish are used to study
development, toxicology and toxicopathology[9], specific gene function
and roles of signaling pathways

36. Thanks for attention