Ecology Practicals2

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Ecology Practicals2

  1. 1. Mutagenicity and carcinogenicity of environmental factors
  2. 2. Next week <ul><li>A test in molecular biology </li></ul><ul><ul><li>(will be based on professor Goetz and professor Sedláček lectures) </li></ul></ul><ul><li>Don´t be late, please! </li></ul><ul><li>Possibility of presentations (after negotiation with professor Mareš) </li></ul>
  3. 3. Find Kočárek´s presentations in: <ul><li>http://www.medik.cz/medik/kocarek/ </li></ul><ul><li>or </li></ul><ul><li>http://camelot2.lf2.cuni.cz/turnovec/ublg/vyuka/ </li></ul>
  4. 4. Mutation <ul><li>A change in the DNA molecule </li></ul><ul><li>Process which produces changes in the DNA that may be inherited . </li></ul>
  5. 5. Mutagenicity <ul><li>= mutagenic activity </li></ul><ul><li>= genotoxicity (genotoxic activity) </li></ul><ul><li>Mutagenic (genotoxic) compound = a compound that causes mutation </li></ul><ul><li>Mutations in certain genes could lead to malignant transformation of the cell. </li></ul><ul><li>Many mutagens are supposed to be carcinogenic. </li></ul>
  6. 6. Carcinogens <ul><li>Karkinos (Greek) = crayfish </li></ul><ul><li>Compounds or other factors that induce transformation of a normal cell into a tumor one </li></ul>
  7. 7. Classification of mutagens <ul><li>Chemical (different compounds) </li></ul><ul><ul><li>Alkylating agents </li></ul></ul><ul><ul><li>Aromatic hydrocarbons (e.g. benzo(a)pyrene) </li></ul></ul><ul><ul><li>Intercalating agents ( e.g. fluorescent dyes) </li></ul></ul><ul><ul><li>Artificial derivatives of DNA bases </li></ul></ul><ul><li>Physical (ionizing radiation, UV-radiation) </li></ul><ul><li>Biological (viruses, transposable elements) </li></ul>
  8. 8. According to metabolic conversion we distinguish: <ul><li>Direct acting mutagens (active without metabolic conversion) </li></ul><ul><li>Promutagens (require metabolic activation) </li></ul>
  9. 9. Benzo(a)pyrene <ul><li>Polycyclic aromatic hydrocarbon </li></ul><ul><li>Carcinogenic activity confirmed </li></ul><ul><li>A product of combustion processes (e.g. tobacco smoking) </li></ul><ul><li>A typical promutagenic agent </li></ul><ul><li>Metabolic conversion (addition of epoxide group and two OH-groups). </li></ul><ul><li>A metabolic product binds to DNA -> adduct (a compound that results from addition). </li></ul><ul><li>Presence of adduct can cause mutation. </li></ul>Metabolic conversion binds to DNA BaP – guanin adduct
  10. 10. Metabolic activation of mutagens PROMUTAGEN ACTIVE (ULTIMATE) MUTAGEN DNA ADDUCT (OR OTHER CHEMICAL MODIFICATION OF DNA BASES) MUTATION enzymes metabolic activation TUMOR liver
  11. 11. Genotoxicity screening tests <ul><li>On molecular level </li></ul><ul><li>On gene level </li></ul><ul><li>On chromosomal level </li></ul>
  12. 12. Tests on molecular level <ul><li>Identification of adducts </li></ul><ul><li>Unscheduled DNA synthesis – measure of DNA repair using radioactively labeled nucleotide. </li></ul><ul><li>Comet assay </li></ul>
  13. 13. Comet assay <ul><li>Single Cell Gel Electrophoresis (SCGE) </li></ul><ul><li>Identification of small DNA fragments inside nuclei of affected cells </li></ul><ul><li>The fragments result from mutagenic affect. </li></ul>
  14. 14. Comet assay <ul><li>DNA fragments are released from nuclei using electrophoresis </li></ul><ul><li>Isolated nuclei are mounted into electrophoretic gel – after electrophoresis are stained with fluorescent dye. </li></ul><ul><li>If DNA fragments are present a „comet tail“ is present observed in the vicinity of the nuclei. </li></ul>– +
  15. 15. Comet assay
  16. 16. Possible results of a comet assay Normal nucleus without fragments (DNA is not damaged – mutagenicity excluded) Two nuclei with DNA damage
  17. 17. Evaluation of a comet assay
  18. 18. Comet assay – computer analysis
  19. 19. Tests on gene level <ul><li>Induction of in vitro mammalian cells resistant to certain chemical compound </li></ul><ul><ul><li>(e.g. 8-azaguanine, 6-thioguanin, ouabain) </li></ul></ul><ul><li>SOS/umu test – measurement of DNA repair in bacteria </li></ul><ul><ul><li>The bacteria is transformed by plasmide with mutator (repair) genes umuC and umuD fused with the gene for galactosidase. If the DNA is damaged by mutagen both umu mutator genes and gal gene are transcribed and galactosidase turns a specific substrate into a color product. </li></ul></ul><ul><li>Ames test </li></ul>
  20. 20. Ames test Bruce Ames (born 1928)
  21. 21. Ames test <ul><li>Test system – auxotrophic strain of Salmonella typhimurium – survives only in medium with histidine (dies in normal medium without histidine) </li></ul><ul><li>After treatment with mutagen some auxotrophic cells are turned into normal ones that synthesize histidine and survive in a normal medium. </li></ul><ul><li>These cells are called revertants (due to reverse mutation). </li></ul>
  22. 22. Ames test His – bacteria Dies in a normal medium Medium containing histidine Normal medium His + bacteria Reverse mutation Mutagen
  23. 23. Ames test 0 Negative control Positive control A dish with a compound to be tested GENOTOXICITY CONFIRMED S PONTANEOUS REVERTANTS IS USED FOR CONTROL OF THE TEST COMPOUND CONFIRME D MUTAGENIC COMPOUND
  24. 24. Result of the Ames test
  25. 25. Ames test – automatic evaluation of results
  26. 26. Evaluation of mutagenic activity of 1-nitropyren e by means of the Ames test <ul><li>The graph describes results of the Ames test of various 1-nitropyren e doses. </li></ul><ul><li>Describe the relationship between the dose and number of revertant colonies. Is it possible to confirm the mutagenic activity? </li></ul><ul><li>Why we test the compound in various concentrations? </li></ul>Number of revertant colonies Dose of 1-nitropyrene (micrograms per dish)
  27. 27. Result of the task Number of colonies decreases due to toxic effects of the tested compound Number of revertant colonies Dose of 1-nitropyrene (micrograms per dish) Number of colonies increases with the dose – mutagenicity confirmed.
  28. 28. Tests on chromosomal level <ul><li>Micronucleus test </li></ul><ul><ul><li>Micronucleus – a small body in the vicinity of the nucleus (the body usually consists of an acentric chromosomal fragment that originated from deletion) </li></ul></ul><ul><li>Frequency of chromosomal aberrations, mainly breakages </li></ul><ul><ul><li>Possible in laboratory animals, cultivated mammalian cells and humans (e.g. persons exposed to radiation of other supposed mutagenic factors. </li></ul></ul><ul><li>SCE – sister chromatid exchanges </li></ul><ul><ul><li>A special cultivation method that allows identification of exchanges between sister chromatids </li></ul></ul>
  29. 29. Result of micronucleus test
  30. 30. Tests on chromosomal level <ul><li>Micronucleus test </li></ul><ul><ul><li>Micronucleus – a small body in the vicinity of the nucleus (the body usually consists of an acentric chromosomal fragment that originated from deletion) </li></ul></ul><ul><li>Frequency of chromosomal aberrations, mainly breakages </li></ul><ul><ul><li>Possible in laboratory animals, cultivated mammalian cells and humans (e.g. persons exposed to radiation of other supposed mutagenic factors. </li></ul></ul><ul><li>SCE – sister chromatid exchanges </li></ul><ul><ul><li>A special cultivation method that allows identification of exchanges between sister chromatids </li></ul></ul>
  31. 31. Possible results of chromosomal analysis Mutagenic effect confirmed with high probability More than 4% A border result – mutagenic effect is neither confirmed nor excluded 2 – 4 % Normal finding, spontaneous level of aberrations Less then 2% Result Percentage of cells with chromosomal aberrations
  32. 32. Tests on chromosomal level <ul><li>Micronucleus test </li></ul><ul><ul><li>Micronucleus – a small body in the vicinity of the nucleus (the body usually consists of an acentric chromosomal fragment that originated from deletion) </li></ul></ul><ul><li>Frequency of chromosomal aberrations, mainly breakages </li></ul><ul><ul><li>Possible in laboratory animals, cultivated mammalian cells and humans (e.g. persons exposed to radiation of other supposed mutagenic factors. </li></ul></ul><ul><li>SCE – sister chromatid exchanges </li></ul><ul><ul><li>A special cultivation method that allows identification of exchanges between sister chromatids </li></ul></ul>
  33. 33. Sister Chromatid Exchanges Normal chromosome A chromosome with SCE The higher SCE frequency – the higher mutagenic activity.
  34. 34. About the origin of a tumor cell
  35. 35. Carcinogenesis (simplified scheme) mutagen mutagen apoptosis mutation repaired A cell with the first mutation A cell with the second mutation Dividing tumor cells normal cell normal cell „ Rubicon“
  36. 36. Mutagenic X carcinogenic agent <ul><li>All mutagens are supposed to be carcinogenic but in some of them the carcinogenicity was not detected. </li></ul><ul><li>Some carcinogenic agents are not mutagenic – they belong to the group of epigenetic carcinogens. </li></ul><ul><li>Evaluation of carcinogenicity to humans is difficult and requires combination of various tests. </li></ul>
  37. 37. Carcinogenic agents <ul><li>Chemical (mostly mutagenic compounds) </li></ul><ul><li>Physical (radiation, asbestos) </li></ul><ul><li>Biological (oncogenic viruses – e.g. HTLV = human T-leukemia virus). </li></ul>
  38. 38. Direct evaluation of carcinogenicity <ul><li>Laboratory tests </li></ul><ul><ul><li>In animals </li></ul></ul><ul><ul><li>In vitro transformation of cultivated cells </li></ul></ul><ul><li>Epidemiologic studies of exposed human population – mostly reliable </li></ul>
  39. 39. IARC database International Agency for Research of Cancer
  40. 40. IARC database (International Agency for Research of Cancer) <ul><li>Direct connection to „Monographs“: </li></ul><ul><li>http:// monographs.iarc.fr/index.php </li></ul>
  41. 41. IARC classification <ul><li>Group 1: Compounds (mixtures) carcinogenic to humans </li></ul><ul><li>Group 2A: Probable human carcinogens </li></ul><ul><li>Group 2B: Possible human carcinogens </li></ul><ul><li>Group 3: Carcinogenicity not confirmed </li></ul><ul><li>Group 4: Compounds probably not carcinogenic to humans </li></ul>
  42. 42. See you after 3 weeks!

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