w. tassaneeyakul 1 wongwiwat tassaneeyakul department of toxicology khon kaen university genetic...
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W. TassaneeyakulW. Tassaneeyakul 11
Wongwiwat TassaneeyakulDepartment of ToxicologyKhon Kaen University
Genetic ToxicologyGenetic Toxicology
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Learning ObjectivesLearning Objectives To know the advancement of To know the advancement of
genetic and genome sciences,genetic and genome sciences, Describe how important of Describe how important of
mutation to living organisms,mutation to living organisms, Explain consequence of Explain consequence of
genetic toxicity and common genetic toxicity and common genetic toxicants.genetic toxicants.
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The Human Genome Genetic concepts Type of mutations Mechanism of genetic
damage and repair Consequence of
genetic damage Mutagens
Outline
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The Big 3 Technologies
Nano Technology
Digital Technology
Genomics
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The Human Genome ProjectThe Human Genome Project
• Start - 1990by US DOE & NIH in : to le
arn all the base sequences (3 billions
bp) in human genome
• E xpected to finish in 2005 (15 yrs proje
ct)
• Budget 3.2 billion US dollars•‘ 2000working’ draft (June ) •‘ ’ ( 2 0 0 3 )
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Research Goals of HGPResearch Goals of HGP
Sequence the human genomeSequence the human genome Collect and distribute dataCollect and distribute data Study the ethical, legal and social Study the ethical, legal and social
implications (ELSI) of genetic researchimplications (ELSI) of genetic research Train researchersTrain researchers Develop technologies and then transfer Develop technologies and then transfer
technologies to the private sectorstechnologies to the private sectors
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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
Private funded company
CELERA GENOMICS
Dr. Craig Venter
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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)
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Surprise finding of HGPSurprise 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
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99.9% of DNA between individual are similar only 0.1 % are different.
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Genetic concepts
= 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.
Genetic Toxicity
Genome
= a complete set of genetic information of an organism
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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).
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Genes consist of both coding (exon) and noncoding (intron) sequence.
The genetic code is triplet.
Each trinucleotide sequence is called codon.
Genetic concepts
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DNA double Helix
DNA Orientation
NucleotideBaseStructure
• Most DNA are in nucleus• 0.1 – 10% in
mitochondriachloroplastsplasmids
• Amount varies• 5.6 kb virus• 5,000 kb bacteria• 6,000,000 kb humans
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Function of DNA Sequences Learned to DateFunction of DNA Sequences Learned to Date
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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.
Mutation
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Types of mutation
1. Genomic mutation (aneuploidy)= abnormal number of chromosomes.
2. Chromosomal aberrations (clastogenesis)= structural changes of chromosomes.
3. Point mutation (gene mutation)3.1 Transition: pur to pur or pyr to pyr.3.2 Transversion: pur to pyr or vice versa.
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Physical Mechanical tearingCutting by ionizing radiation, 32PNondisjunction of chromosomesHigh temperature
Chemical Alteration or removal of DNA basesIncorporation of altered basesIntercalation of oligocyclic aromatic compoundsAlteration of DNA backbone
Enzymatic Production of chemicals affecting DNAMistakes in DNA replicationAlteration of DNA replication systemMistakes in DNA recombination or repair
Causes of mutations
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DNA damageDNA damage
1. Ionizing radiation: ss or ds breaks
2. Nonionizing radiation (UV): pyr
dimers
3. Chemicals: base pair alteration3.1 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
4. Endogenous agents: oxygen/ROS
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DNA repairDNA repair
Extensive damage apoptosis
Less damage repair
Base excision repairNucleotide excision repairDS break repair:
homologous recombinationnonhomologous end-joining
Mismatch repair
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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.
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Consequences of gene mutation
1. Silent
2. Missense
3. Nonsense
4. Frame-shift (addition or
deletion).
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Mutation – Any change in Genetic Material
SubstitutionT
GT A
TA
Missense Mut
e.g. Sickle Cell
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Frameshift Mutation
“Worse mutation”
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Formation of chromosomal alterations.
Structural changes of
chromosomes.
Abnormal number of chromosomes.
e.g. colchicine, griseofulvin,
vinblastin
Sister chromatid exchange (SCE)
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Control
SuspectMutagen
Environmental MutagensAmes Assay
His- Salmonella typhimurium
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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
Other Genetic Assays
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Sister Chromatid exchange5-Bud + hoechst (fl)dye)
Normal exchange rate
Very high exchange rate
In vivo w lymphocytesmonitoring
See dose response
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1) Alkylating agents alkyl halideschloroform HCCl3
sulfur mustards S-(Al-X)2
nitrogen mustards N-(Al-X)3
Unstable 3 member ringsEpoxides - Dieldrin
Unstable lactonesAflatoxin b1, B-propiolactone
diazo compoundsN = N = R dyes
diazomethane
Mutagens
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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-bromouracil5) Intercalating agents : acridine dyes
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6) Heavy Metals Hg chromosome breakCr6+ Cancer (Cr3+ not)AsCdNi
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
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