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Wongwiwat Tassaneeyakul Department of Toxicology Khon Kaen University

Genetic Toxicology. Wongwiwat Tassaneeyakul Department of Toxicology Khon Kaen University. Learning Objectives. To know the advancement of genetic and genome sciences, Describe how important of mutation to living organisms,

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Wongwiwat Tassaneeyakul Department of Toxicology Khon Kaen University

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  1. Genetic Toxicology Wongwiwat Tassaneeyakul Department of Toxicology Khon Kaen University W. Tassaneeyakul

  2. Learning Objectives • To know the advancement of genetic and genome sciences, • Describe how important of mutation to living organisms, • Explain consequence of genetic toxicity and common genetic toxicants. W. Tassaneeyakul

  3. Outline • The Human Genome • Genetic concepts • Type of mutations • Mechanism of genetic damage and repair • Consequence of genetic damage • Mutagens W. Tassaneeyakul

  4. Genomics The Big3 Technologies Nano Technology Digital Technology W. Tassaneeyakul

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  6. The Human Genome Project • Start by US-DOE & NIH in 1990: to learn all the base sequences (3 billions bp) in human genome • Expected to finish in 2005 (15 yrs project) • Budget 3.2 billion US dollars • ‘working’ draft (June 2000) • ‘finished’ draft (April 2003) W. Tassaneeyakul

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  8. Research Goals of HGP • Sequence the human genome • Collect and distribute data • Study the ethical, legal and social implications (ELSI) of genetic research • Train researchers • Develop technologies and then transfer technologies to the private sectors W. Tassaneeyakul

  9. HUMAN GENOME PROJECT (HGP) • Publicly funded consortium including • 4 large sequence centers in US • Sanger Center in UK • Labs in Japan, France, Germany & China Dr. Francis Collins CELERA GENOMICS Private funded company Dr. Craig Venter W. Tassaneeyakul

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  12. Today we are learning the language in which God created life. It will revolutionize the diagnosis, prevention and treatment of most, if not all human diseases. President William J. Clinton (26/06/00) W. Tassaneeyakul

  13. Surprise finding of HGP • HG contains only 30,000-35,000 genes , much less than initially expected (100,000). • HG are more complex, with made alternative splicing generating a large number of protein products. • Less than 2% of the genome codes for proteins. • Almost all (99.9%) DNA sequences are exactly the same in all people. • The functions are unknown for over 50% of discovered genes W. Tassaneeyakul

  14. 99.9% of DNA between individual are similar only 0.1 % are different. W. Tassaneeyakul

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  18. Genetic concepts Genetic Toxicity = a branch of toxicology that study the effect of chemical or physical agents on the heredity material (DNA) and on the genetic process of living cells. Genome = a complete set of genetic information of an organism W. Tassaneeyakul

  19. Genetic concepts • DNA is the genetic material. • DNA is a double helix. • DNA consists of 2 purines (A,G) and 2 pyrimidines (C,T/U). • Base pairing always consists of 1 purine and pyrimidine (AT,CG). W. Tassaneeyakul

  20. Genetic concepts • Genes consist of both coding (exon) and noncoding (intron) sequence. • The genetic code is triplet. • Each trinucleotide sequence is called codon. W. Tassaneeyakul

  21. DNA Orientation DNA double Helix Nucleotide Base Structure • Most DNA are in nucleus • 0.1 – 10% in • mitochondria • chloroplasts • plasmids • Amount varies • 5.6 kb virus • 5,000 kb bacteria • 6,000,000 kb humans W. Tassaneeyakul

  22. Function of DNA Sequences Learned to Date W. Tassaneeyakul

  23. Mutation • Unexpected and undirected changes in the component of genetic materials. • Spontaneous or external stimuli. • Macro or micro lesions. • Change the sequence of DNA. • Concentrated at hotspots. W. Tassaneeyakul

  24. Types of mutation 1. Genomic mutation (aneuploidy) = abnormal number of chromosomes. 2. Chromosomal aberrations (clastogenesis) = structural changes of chromosomes. 3. Point mutation (genemutation) 3.1 Transition: pur to pur or pyr to pyr. 3.2 Transversion: pur to pyr or vice versa. W. Tassaneeyakul

  25. Causes of mutations Physical Mechanical tearing Cutting by ionizing radiation, 32P Nondisjunction of chromosomes High temperature Chemical Alteration or removal of DNA bases Incorporation of altered bases Intercalation of oligocyclic aromatic compounds Alteration of DNA backbone Enzymatic Production of chemicals affecting DNA Mistakes in DNA replication Alteration of DNA replication system Mistakes in DNA recombination or repair W. Tassaneeyakul

  26. DNA damage • Ionizing radiation: ss or ds breaks • Nonionizing radiation (UV): pyr dimers • Chemicals: base pair alteration 3.1 Directly damage 3.1.1 adduct (covalent binding) e.g. aflatoxin epoxide, benzo[a]pyrene (bulky) 3.1.2 alkylating e.g. cytotoxic drug 3.2 Indirectly e.g. intercalate between ds • Endogenous agents: oxygen/ROS W. Tassaneeyakul

  27. DNA repair Extensive damage apoptosis Less damage repair Base excision repair Nucleotide excision repair DS break repair: homologous recombination nonhomologous end-joining Mismatch repair W. Tassaneeyakul

  28. Formation of gene mutations. • Radiomimetic mutagens: effect all phase of cell replication e.g. bleomycin, 8-ethoxycaffeine. • Nonradiomimetic mutagens: effect only at S-phase. • Somatic vs. germ cells • In somatic cells may lead to neoplasia or malformation. • In germ cells may transmit to phenotype alteration in the next generation. W. Tassaneeyakul

  29. Consequences of gene mutation 1. Silent 2. Missense 3. Nonsense 4. Frame-shift (addition or deletion). W. Tassaneeyakul

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  32. Mutation – Any change in Genetic Material Substitution T e.g. Sickle Cell G T A T A Missense Mut W. Tassaneeyakul

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  34. Frameshift Mutation “Worse mutation” W. Tassaneeyakul

  35. Formation of chromosomal alterations. • Structural changes of chromosomes. • Abnormal number of chromosomes. e.g. colchicine, griseofulvin, vinblastin • Sister chromatid exchange (SCE) W. Tassaneeyakul

  36. Control Suspect Mutagen Environmental Mutagens Ames Assay His- Salmonella typhimurium W. Tassaneeyakul

  37. Other Genetic Assays • Bacterial E. coli K12 Several genes, forward, reverse • Yeast • Drosophila In vivo screening (sex linked recessive lethal) • Chromosome aberrations – eukaryote • CHO (Chinese hamster ovary) • HeLa • Sister Chromatid Exchange W. Tassaneeyakul

  38. Sister Chromatid exchange 5-Bud + hoechst (fl)dye) Normal exchange rate Very high exchange rate In vivo w lymphocytes monitoring See dose response W. Tassaneeyakul

  39. Mutagens 1) Alkylating agents alkyl halides chloroform HCCl3 sulfur mustards S-(Al-X)2 nitrogen mustards N-(Al-X)3 Unstable 3 member rings Epoxides - Dieldrin Unstable lactones Aflatoxin b1, B-propiolactone diazo compounds N = N = R dyes diazomethane W. Tassaneeyakul

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  41. 2) Nitrosamines: alkylate, chromosome breaks, point mut. diethylnitrosamine O=N-N-(C2H5)2 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) O=N-N-CH3 H C-N-NO2 NH mimics DNA base, potent mutagen (lab safety) 3) Hydrazines: H2N-NH2 Produces free radicals and H2O2 React w pyrimidines, break ring, base removal 4) Base analogs: 5-bromouracil 5) Intercalating agents : acridine dyes W. Tassaneeyakul

  42. 6) Heavy Metals Hg chromosome break Cr6+ Cancer (Cr3+ not) As Cd Ni Inhibit DNA replication and RNA synthesis mispairing of bases 7) Others: Formaldyhyde – formaldehyde exposure associate with cancers of the nasal sinuses, nasopharynx, and brain, and possibly leukemia W. Tassaneeyakul

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