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Genetic Toxicology
Wongwiwat Tassaneeyakul
Department of Toxicology
Khon Kaen University
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Learning Objectives
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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.
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Outline
The Human Genome
Genetic concepts
Type of mutations
Mechanism of genetic
damage and repair
 Consequence of
genetic damage
 Mutagens
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The Big 3 Technologies
Digital
Technology
Nano
Technology
Genomics
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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)
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Research Goals of HGP
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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
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HUMAN GENOME PROJECT
(HGP)
Dr. Francis Collins
Publicly funded consortium including
• 4 large sequence centers in US
• Sanger Center in UK
• Labs in Japan, France, Germany &
China
CELERA GENOMICS
Private funded company
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 HGP
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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
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
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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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Genetic concepts
 Genes consist of both coding
(exon) and noncoding
(intron) sequence.
 The genetic code is triplet.
 Each trinucleotide sequence
is called codon.
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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
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Function of DNA Sequences Learned to Date
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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.
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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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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
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DNA damage
1. Ionizing radiation: ss or ds breaks
2. Nonionizing radiation (UV): pyr dimers
3. 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
4. Endogenous agents: oxygen/ROS
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DNA repair
Extensive damage
Less damage
apoptosis
repair
Base excision repair
Nucleotide excision repair
DS break repair:
homologous recombination
nonhomologous end-joining
Mismatch repair
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Formation of gene mutations.
Radiomimetic mutagens: effect all phase
of cell replication e.g. bleomycin, 8ethoxycaffeine.
Nonradiomimetic mutagens: effect only at
S-phase.
Somatic vs. germ cells
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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
Substitution
T
T
G
e.g. Sickle Cell
T
A
A
Missense Mut
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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
Environmental Mutagens
Ames Assay
His- Salmonella typhimurium
Suspect
Mutagen
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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
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Sister Chromatid exchange
5-Bud + hoechst (fl)dye)
Normal exchange rate
Very high exchange rate
In vivo w lymphocytes
monitoring
See dose response
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Mutagens
1) Alkylating agents alkyl halides chloroform
sulfur mustards
nitrogen mustards
HCCl3
S-(Al-X)2
N-(Al-X)3
Unstable 3 member rings
Epoxides - Dieldrin
Unstable lactones
Aflatoxin b1, B-propiolactone
diazo compounds
N= N= R
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diazomethane
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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) Intercalating agents :
5-bromouracil
acridine dyes
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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
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