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

W. TassaneeyakulW. Tassaneeyakul 11

Wongwiwat TassaneeyakulDepartment of ToxicologyKhon Kaen University

Genetic ToxicologyGenetic Toxicology


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.


The Human Genome Genetic concepts Type of mutations Mechanism of genetic

damage and repair Consequence of

genetic damage Mutagens

Outline


The Big 3 Technologies

Nano Technology

Digital Technology

Genomics


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 )


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


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


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)


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


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


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


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).


Genes consist of both coding (exon) and noncoding (intron) sequence.

The genetic code is triplet.

Each trinucleotide sequence is called codon.

Genetic concepts


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


Function of DNA Sequences Learned to DateFunction of DNA Sequences Learned to Date


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


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.


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


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


DNA repairDNA repair

Extensive damage apoptosis

Less damage repair

Base excision repairNucleotide excision repairDS break repair:

homologous recombinationnonhomologous end-joining

Mismatch repair


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.


Consequences of gene mutation

1. Silent

2. Missense

3. Nonsense

4. Frame-shift (addition or

deletion).


Mutation – Any change in Genetic Material

SubstitutionT

GT A

TA

Missense Mut

e.g. Sickle Cell


Frameshift Mutation

“Worse mutation”


Formation of chromosomal alterations.

Structural changes of

chromosomes.

Abnormal number of chromosomes.

e.g. colchicine, griseofulvin,

vinblastin

Sister chromatid exchange (SCE)


Control

SuspectMutagen

Environmental MutagensAmes Assay

His- Salmonella typhimurium


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


Sister Chromatid exchange5-Bud + hoechst (fl)dye)

Normal exchange rate

Very high exchange rate

In vivo w lymphocytesmonitoring

See dose response


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


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


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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