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Genotoxicity upon exposure to
complex mixtures:
Part I: Basic principles and methods
(Part II: Examples of mechanistic in vitro and in vivo human studies –
May 2, 10.45)
Jan Topinka Institute of Experimental Medicine Academy of Sciences of the
Czech Republic
Outline of the lecture
Introduction – toxicology of complex mixtures, possible approaches, models, new solutions
Genetic toxicology and multistep process of chemical carcinogenesis
Methods to study mechanisms of genotoxicity of complex mixtures (selected): Toxicogenomics
DNA adducts
Comet assay
Oxidative damage of DNA, proteins and lipids
Micronucleus test
Cytogenetic analysis
Summary
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Complex mixtures
Air pollution
◦ Traffic – diesel emissions
◦ Industrial emissions – coke oven, metalurgy
◦ Local heating by fossil fuels
◦ Environmental tobacco smoke (ETS)
Water and water sediments
Food (red meet, fish from poluted water)
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Toxicology of complex
mixtures
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Detailed chemical analysis of composition – assessment of individual toxic potencies of components – laborious, costly, without interactions
Biological approach – analysis of interactions of mixture components with biological systems (bioassays)
Newest development in genetic toxicology of complex mixtures : Combinationn of toxicogenomics with chemical analysis and traditional genotoxicity markers
Genetic toxicology
Genetic toxicology describes the properties of
chemical or physical agents that damages the
genetic information (DNA) causing mutation,
which may lead to cancer...
Genotoxic effect is the first event of
the multistep process of chemical
carcinogenesis.
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What is genotoxicity ?
Genotoxicity is defined as ability of specific factor to damage, mainly chemically, DNA.
Most frequent genotoxic event is covalent binding of the chemical or its metabolite with nucleotides in DNA – DNA adduct…
Genotoxic effect is the first event of the
multistep process of chemical carcinogenesis.
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Multistep process of chemical carcinogenesis
Exposure Effect
External
exposure
dose
Internal
dose
Biologically
effective
dose
Early effects Morfological
structural and
functional
changes
Clinical
symptoms
Stacionary
and
personal
monitoring
Level of
compound
/metabolite
Adducts
DNA, prot.
Somatic
mutations
Preneoplastic
lesions Diagnosis
Genetic toxicology
Jan Topinka TOXI-LATIN 2014 15. 7. 2014 7
Toxicogenomics
Toxicogenomics is a field that deals with the information
about gene and protein activity within particular cell or
tissue of an organism in response to toxic substances.
It includes transcriptomics, proteomics and mebolomics.
Transcriptomics is the most frequent approach in
toxicology…
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Transcriptomic approach to study
genotoxic and carcinogenic potential of
complex mixtures
Which kind of additional information might be received by the analysis of gene expression profiles in cells treated by complex mixtures ?
Further step in understanding of the major mechanisms of toxicity of complex mixture
Involves much more effects than genotoxic effects - the effects of genotoxic and non-genotoxic carcinogens
Two basic approaches:
◦ 1. Global gene expression changes – whole genome microarrays (Illumina, Affymetrix platform)
◦ 2. Expression of specific sets of candidate genes is analyzed in pathway-specific arrays or qRT-PCR
Transcriptional changes detected by microarrays should be verified by qRT-PCR and better on protein level.
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Global gene expression analysis
(Illumina platform)
1. Leukocyte separation 2. RNA extraction and quality control 3. cDNA synthesis
4. IVT and labeling 5. Hybridization to chips and scanning
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qRT-PCR verification of microarray data
1. cDNA synthesis from total RNA
2. Quantitative RT-PCR using the TaqMan chemistry
3. Fold change calculation (deltadelta Ct method)
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Gene expression
data analysis
1. Principal component analysis (PCA) – helps to
identify patterns in the data and highlight
similarities and differences
2. Venn diagrams – identify numbers of common and
differentially expressed transcripts
3. Correlations, T-tests, ANOVA, linear models
4. KEGG pathway analysis (Fisher’s exact test)
5. Correlation of microarray and qRT-PCR data
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DNA adducts One of the most frequent genotoxic event is covalent binding of
the chemical or its metabolite on nucleotides in DNA – DNA
adduct formation
Methods of DNA adduct analysis
32P-postlabelling
Mass spectrometry
Fluorescence methods
Immunoassays
Radiolabelled compounds (3H, 14C)
The methods differ by their sensitivity, specificity, applicability…
For complex mixtures, 32P-postlabellng is the best choice for both in vitro and in vivo studies
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Analysis of DNA adducts
(32P-postlabelling)
Individual compounds
Complex mixtures
(extracts from PM)
B[a]P DB[a,l]
P
Control
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TLC Maps of DNA Adducts
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B[a]P DB[a,l]P Control
Prague Winter
Kosice Winter
Sofia Winter
Control DMSO
Individual compounds
Complex mixtures of the air pollutants
Total genotoxic potential: It gives the information on total genotoxicity of complex mixture.
Sensitivity: very sensitive – 1 adduct per 109 normal nucleotides.
Applicability: Can be used in acellular assay, in vitro and in vivo.
DNA quantity: Requires low DNA quantity
Unspecific unless standards are used – not important for complex mixtures
Radioactive labelling usually 32P
Relatively laborious and costly (~80 samples per experiment)
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32P-postlabelling of DNA adducts –
advantages and disadvantages
Comet assay
Single cell gel electrophoresis (SCGE)
Simple and widely used method to study genotoxicity of chemicals including complex mixtures in vitro and in vivo
+ -
Isolated cells
Slide preparation
Lysis
Denaturation and DNA unwinding
Electrophoresis
Staining
Analysis
COMET ASSAY
(Single Cell Gel Electrophoresis)
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HEAD TAIL HEAD TAIL
Assessment of DNA damage
percentage of DNA in the „tail“ of comet
from the total content of DNA in the nucleus
Alkaline version of CA detects single-SB, double-SB, alkalilabile sites,
transient gaps arising as intermediates during base excision repair,
apoptotic DNA fragmentation
Detection of oxidative damage to DNA by Comet assay
Comet assay + enzymes of excision repair
catalyses the excision of broad spectrum of modified purines
(formamidopyrimidine, 8-oxo-guanine)
(formamidopyrimidine DNA glycosylase) FPG
DNA glycosylase activity with a broad substrate specifity for mutated
pyrimidine derivatives (e.g. thymine glycol, 5-hydroxycytosine,
5,6-dihydrothymine
(endonuclease III) ENDO III
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Oxidative stress Oxidative damage of DNA, proteins and lipids
Oxidative stress
Oxidative stress - the result of an inbalance between levels of
oxidants and antioxidants
Conditions leading to oxidative stress:
increased reactive oxygen species (ROS) generation
decreased antioxidant protection
failure to repair oxidative damage
ROS = radicals and other non-radical reactive oxygen derivatives
ROS include:
Radicals: hydroxyl radical (OH•), superoxide (O2
•¯), nitric oxide (NO•), lipid peroxyl
(LOO•)
Non-radicals: peroxynitrite (ONOO¯), hypochlorous acid (HOCl), hydrogen peroxide
(H2O
2), ozone (O
3), and lipid peroxide (LOOH)
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Oxidative stress
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Oxidative stress in cellular components
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Complex mixtures as sources
of oxidative stress
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Sources:
aerobic respiration – during the conversion of oxygen to water in mitochondria
inflammation
environmental factors (prolonged exposure to sunlight, radiation, exposure to carcinogens, air pollution)
life-style factors (smoking, medication, extreme physical exercise, alcohol consumption)
ROS concentration:
at low concentrations - second messengers (regulation of apoptosis, activation of transcription factors, modulation of
expression of genes coding antioxidant enzymes)
in excessive amount - induce apoptosis in healthy cells, inflammation, cause damage to DNA, proteins, lipids, oxidize
sugars (glucose)
oxidative stress and ROS –role in Alzheimer’s disease, Parkinson’s disease, cancer, and aging
Antioxidant defense
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ROS are cleared from the cell by the action of:
enzymes (Cu-Zn and Mn superoxide dismutases (SOD), catalase, glutathione (GSH) peroxidase,
thioredoxin-thioredoxin reductase, heme oxygenase, heat shock proteins)
Fe- and Cu- transporting proteins (transferin, ferritin)
small antioxidant molecules (glutathione, uric acid, bilirubin, glucose, vitamins A, C, E,
carotenoids, flavonoids)
Cu, Zn, Se and other elements necessary for the activity of antioxidant enzymes
enzymes responsible for repair of oxidized macromolecules
Oxidative damage of DNA
purine and pyrimidine oxidation products
targets of ROS in DNA
abasic sites in DNA
DNA strand breaks
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8-Oxodeoxyguanosine (8-oxodG)
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• the most abundant DNA lesion caused by ROS
• highly mutagenic, causes in GC to TA transversions
• after cleavage from DNA as a result of DNA repair, excreted in
urine
• urinary 8-oxodG levels are considered a general biomarker of
oxidative stress
• methods used for 8-oxodG detection: HPLC, tandem mass
spectroscopy,n (MS-MS), competitive ELISA
Oxidative damage of proteins
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Oxidized proteins
• fragmentation and denaturation of proteins
• formation of hydroperoxide (-OOH) and carbonyl groups
• ROS react: directly with the protein, or with sugars and lipids, generating
products that react with the protein
Carbonyl groups - aldehydes and ketones formed on the protein side chains
during its oxidation (especially Pro, Arg, Lys, Thr)
Formation results in:
• cleavage of protein
• cross-linking of protein
• loss of catalytic and structural function
Protein oxidation – detection methods
• mostly based on derivatization of CO groups with 2,4-dinitrophenylhydrazine
(DNPH) and detection of a stable product (DNP – 2,4-dinitrophenyl) with anti-DNP
antibodies
• methods include spectrophotometric assay, HPLC, western blotting, ELISA
Oxidative damage of lipids – lipid
peroxidation
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Lipid peroxidation – polyunsaturated fatty acid in cell membranes are attacked by
ROS – lipid peroxides (LPO) formation; can be measured in blood or plasma
LPO decomposition – malondialdehyde (MDA), 4-hydroxynonenal, ethane, pentane
MDA – detection with thiobarbituric acid is not specific, MDA represents only about
1% of LPO decomposition products
F2-Isoprostanes
Produced non-enzymatically by free radical-induced peroxidation of arachidonic acid
Formed on phospholipids and then released by phospholipase
Four isomers (5-, 8-, 12-, and 15-series), each comprised of 8 racemic diastereomers
Specific markers of lipid peroxidation, very stable, detected in all biological fluids and
tissues
Detection methods: gas chromatography-mass spectroscopy, liquid chromatography-
mass spectroscopy, ELISA
Genetic damage by complex mixtures
on chromosomal level
Micronucleus test
Conventional cytogenetic analysis
FISH cytogenetics
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From DNA to chromosome - nucleosome
Human DNA in numbers:
pb in DNA : 3 billions
length of DNA in cell : 1.8 m
total lenght of DNA in all cells in body : 180 billions km
- The DNA is wound around the
histone core of eight protein
subunits, forming the
nucleosome.
- The nucleosome is clamped by
histone H1.
- About 147 base pairs of DNA
coil around the histone core.
Methylation of histone or DNA usually turns a gene
off.
Acetylation of histone usually turns a gene on.
Phosphorylation - we're not sure what that does.
2((H2A+H2B)+(H3+H4))
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DNA and histone proteins are
packaged in several levels into
structures called chromosomes
From DNA to chromosome
- Average length of mitotic chromosome
in metaphase: 5 µm
- Human cells have 23 pairs of linear
nuclear chromosomes (22 pairs of
autosomes and one pair of sex
chromosomes), a total of 46 per cell.
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Long arm is called q (q is next in alphabet)
Short arm is called p (French for petit)
Telomere is a region of repetitive DNA sequences at the end of a chromosome
Centromere - holds together the duplicates of chromatids, 1–10 million base
pairs of DNA, most of this is repetitive DNA: short sequences (e.g., 171 bp)
repeated
Human chromosome - description
Kinetochore is a complex of >100 different proteins that forms at each
centromere and serves as the attachment point for the spindle fibres that will
separate the sister chromatids as mitosis proceeds into anaphase.
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Estimated number of genes and base pairs on each human chromosome.
Human chromosomes
Human chromosomes - 7 groups & sex chromosomes
(Denver nomenclature – 1960, ~first nomenclature )
--------------------------------------------------------------- A 1-3 Large metacentric 1,2 or submetacentric
B 4,5 Large submetacentric, all similar
C 6-12, X Medium sized, submetacentric
D 13-15 Medium sized acrocentric with satellites
E 16-18 Short metacentric 16 or submetacentric 17,18
F 19-20 Short metacentrics
G 21,22,Y Short acrocentrics with satellites. Y no satellites.
Position of centromere
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Micronucleus test
Simple method to study damage on the level of chromosomes - clastogenic effect of complex mixtures in vitro and in vivo
Very important – belongs to to the standard battery of tests in genetic toxicology
Analysis of micronuclei - history
1959 – Evans et al. – first attempt to use a micronucleus test – application for
evaluation of chromosomal aberrations in roots of Vicia faba
1976 – Countryman & Heddle – first production of micronuclei (MN) in
cultures of human peripheral lymphocytes
1985 – Fenech & Morley – cytochalasin-B was first used to inhibit cytokinesis
(binucleated cells – BNC)
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Cytokinesis-block micronucleus assay
(CBMN)
Micronucleus technique – a method to measure chromosome loss and
chromosome breakage
CBMN – every cell studied in the system is scored for its viability status,
mitotic status and chromosomal instability or damage status
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Application of micronucleus assay
More than 7 000 studies for the keyword „micronucleus“ in the PubMed database
Application and techniques (lots of combinations)
Genotoxicity testing of new drugs, radiation studies, biomonitoring studies,
effect of exposure to various chemicals and complex mixtures (pesticides,
carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) like benzo[a]pyrene
(B[a]P), benzene, particulate matter, chemical exposure in various plants…)
Various cells (peripheral lymphocytes, bone morrow, exfoliated cells, cell
lines…) from different species (human, rat, fish, plants…)
Different staining (non-fluorescent: Giemsa or May-Grűnwald-Giemsa,
fluorescent: DAPI, Hoechst 33529, ethidium bromide, propidium iodide)
in vitro or in vivo studies (ex vivo)
Scoring mono- or binucleated cells
Analysis (visually, automatic: flow cytometry or image analysis)
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HUMN project
= HUman MicroNucleus Project
* 1997 France, Toulouse
Micronuclei (MN) in human peripheral lymphocytes
- collaboration between 40 laboratories from 16 countries
(pooled data ~7000 subjects)
Main aims:
- detail description of the scoring criteria for lymphocytes
- assessment of the sources of variability in the CBMN assay
- analysis of large data set allowed to achieve greater
statistical power in the results
- analysis of the impact of age, gender and smoking on the
frequency of MN
- frequency of MN and predictive risk of cancer
Founding members:
Michael Fenech Wushou Chang Stefano Bonassi Nina Holland Errol Zeiger
http://ehs.sph.berkeley.edu/holland/humn/
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HUMNXL project
Next project starting 2008 - Minimally invasive method (human buccal mucosal tissue)
- Similar aims as in HUMN project
Automated image analysis – aim of a new project
? 15. 7. 2014 Jan Topinka TOXI-LATIN 2014 43
1982 – Hutter & Stöhr – automation by flow cytometry
1990 – Tates et al. – beginning of automated image analysis
1993 – 1996 – Castelain et al., Bocker et al. – testing of other systems (low
effectivity of detection of BNC and MN)
2004 – Schunck et al., Varga et al.
Metafer (MetaSystems)
- DAPI staining
2009 – Decordier et al.
Pathfinder™ (IMSTAR)
- Giemsa staining
History of automated analysis of MN
Present stage of automated image analysis
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Metafer – applications - software
MSearch AutoCapt
DCScore
MetaCyte
RCDetect
CometScan Metafer P
MicNuc
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Automated image analysis of MN – system
description Various endpoints can be analyzed with the same system
(FL/TL, fluorescence or transmitted light)
The automated scanning system Metafer
with 80-position slide feeder
Hardware architecture:
Computer DELL, 19“ LCD monitor, CCD camera JAI M4 CL, Trackball
Microscope:
Metafer is connected to the motorized microscope AxioImager.Z1 with
automated focusing and special scanning stage (Märzhäuser) for 8
slides per scanning that can be extended to 80 slides
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Staining of slides for automated MN
analysis
Varga et al. (2004), An automated scoring procedure for the micronucleus test by image analysis,
Mutagenesis 19, 391-397.
Fluorescent dyes are more suitable for
automation (DAPI is the most common)
4',6-diamidino-2-phenylindole is a
fluorescent stain that binds strongly to A-T
rich regions in DNA
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Automated image analysis of MN – biomonitoring studies
in populations exposed to complex mixtures of air
pollutants
PRAGUE
CESKE BUDEJOVICE
OSTRAVA
Average year concentrations of benzo[a]pyrene (B[a]P) in the air in 2009 in the Czech Republic
Sources of air pollution
- traffic
- industry
- local heating
Czech Republic
- seasonal variability
of air pollution
Carcinogenic polycyclic aromatic
hydrocarbons (c-PAHs)
example: B[a]P
Particulate matter
example: PM2.5 (<2.5μm)
Volatile organic compounds
example: benzene
PM2.5
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Cytogenetic analysis by FISH
Structural and numerical chromosomal
abberations - conventional and FISH methods
History of human cytogenetics
1951 – first use of hypotonic KCl solution (as a mistake instead of isotonic
solution) → observation of well-spread methaphases
1956 – number of human chromosomes was determined
1959 – Down syndrome was described (Trisomy # 21)
1960 – first described chromosomal aberration in cancer
1968-1970 – banding techniques to identify chromosomes
1988 – Fluorescence in situ hybridization (FISH)
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Cell cycle
M (2%)
G1 (39%) - Cells increase in size in
Gap 1. The G1 checkpoint control
mechanism ensures that everything is
ready for DNA synthesis.
S (40%) - DNA replication
occurs during this phase.
G2 (19%) - During the gap between
DNA synthesis and mitosis, the cell will
continue to grow. The G2 checkpoint
control mechanism ensures that
everything is ready to enter the M
(mitosis) phase and divide.
G0 - A resting phase
where the cell has left
the cycle and has
stopped dividing.
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Mitosis COLCHICINE
-extracted from plants of
the genus Colchicium
(Saffron)
-"mitotic poison" or spindle
poison
-inhibits microtubule
polymerization by binding
to tubulin
Most important phases
for cytogenetics
Intherphase – possible to apply fluorochromes
CYTOCHALASINE B
- a cell-permeable
mycotoxin
- inhibits cytoplasmic
division by blocking the
formation of contractile
microfilaments 15. 7. 2014 Jan Topinka TOXI-LATIN 2014 52
Karyotype
Human karyotype
The 22 autosomes (numbered by size) and two sex
chromosomes, X and Y
46, XX – female or 46, XY - male
Ideogram G-banding
A karyotype is the number and appearance of chromosomes in the
nucleus of an eukaryotic cell. The term is also used for the complete set
of chromosomes in a species, or an individual organism
SKY karyotype
40,XX - karyotype of a
normal, female mouse
Chromosome numbers in some plants
-Arabidopsis thaliana – 10
-Rye – 14
-Maize – 20
Chromosome numbers in some animals
-Drosophila melanogaster – 8
-Gorillas – 48
-Guinea Pig – 64
-Goldfish – 100-104 15. 7. 2014 Jan Topinka TOXI-LATIN 2014 53
An International System for Human
Cytogenetic Nomenclature (ISCN – 1995)
-Symbols and abbreviated terms used in the description of
chromosomes and chromosomal abnormalities in medicine.
-Updates in 2009 based upon higher-resolution analysis
EXAMPLES:
Normal karyotype: 46,XX or 46,XY
Numerical aberrations – Aneuploidy: 47,XX,+21, 47,XY, +18, 45,XY,-21
Aberrations in sex chromosomes: 45,X, 47,XXX……..
Polyploidy: 69,XXX, 69,XXY, 92,XXXX………
Mosaicism: 45,X[50]/46,XX[25], 47,XY,+21[50]/46,XY[30]
Robertsonian translocations – balanced
45,XX,t(14;21) or 45,XX,der(14;21)(q10;q10)
Robertsonian translocations – unbalanced
46,XY,t(13;14),+13 or 46,XY,der(13;14)(q10;q10),+13
Structural aberrations – balanced
46,XX,t(16;18) or 46,XX,t(16;18)(q21;p11.2)
……..del…….add…..der…..i……r……… 15. 7. 2014 Jan Topinka TOXI-LATIN 2014 54
Chromosomal aberrations (anomaly or abnormality)
- Reflect an antypical number of chromosomes (NUMERICAL) or
a structural abnormality in one or more chromosomes (STRUCTURAL)
- Conformation by comparison with a normal karyotype
- Occur e.g. when there is an error in cell division following meiosis or mitosis
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Numerical aberrations
-An abnormal number of chromosomes
POLYPLOIDY - more than two paired (homologous) sets of chromosomes
ANEUPLOIDY - occurs when an individual is missing either a chromosome
from a pair (MONOSOMY) or has more than two chromosomes of a pair
(TRISOMY, TETRASOMY)
Examples –Turner Syndrome – Monosomy in the sex chromosomes 45,X
– Down Syndrome - Trisomy # 21
- Patau Syndrome – Tisomy # 13
- Edwards Syndrome – Trisomy # 18
- Triple X Syndrome – Trisomy in the sex chromosomes 47,XXX
- Klinefelter Syndrome - Trisomy in the sex chromosomes 47,XXY
- XYY Syndrome – Trisomy in the sex the chromosomes 47,XYY
- XXXX Syndrome – Tetrasomy in sex the chromosomes 48,XXXX
- Pentasomy (very rarely) in sex the sex chromosomes 49,XXXXX or
49,XXXXY
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Structural aberrations (1) - a structural abnormality in one or more chromosomes
reasons:
1. Non-Homologous End Joining (NHEJ) - a pathway that repairs double-
strand breaks in DNA (results in translocations)
2. Unbalanced recombination - results in duplications or deletions
2. DELETIONS
- a portion of the chromosome
is missing or deleted
1. DUPLICATIONS
- a portion of the
chromosome is duplicated,
resulting in extra genetic
material
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3. TRANSLOCATIONS
A portion of one chromosome is transferred to another chromosome
RECIPROCAL TRANSLOCATIONS
Segments from two different chromosomes have been exchanged
ROBERTSONIAN TRANSLOCATION
An entire chromosome is attached to another at the centromere (in
humans these only occur with chromosomes 13, 14, 15, 21 and 22)
Structural aberrations (2)
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4. INSERTIONS
A portion of one chromosome is inserted into another chromosome
5. INVERSIONS
A portion of the chromosome is broken off, turned upside down and
reattached
Structural aberrations (3)
Paracentric inversions
- do not include the centromere
and both breaks occur in one arm
of the chromosome
Pericentric inversions
- include the centromere and there
is a break point in each arm.
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6. RINGS
A portion of a chromosome is broken off and forms a circle
Normally, the ends of a chromosome are lost, enabling the arms to fuse
together or in rare cases, the telomeres at the ends of a chromosome fuse
without any disappearing of material.
7. ISOCHROMOSOME
A chromosome that lost one of its arms and duplicated other
This is sometimes seen in some females with Turner syndrome or in tumor
cells.
Structural aberrations (4)
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Structural aberrations (5)
8. DICENTRIC CHROMOSOME
- an aberrant chromosome having two centromeres
- forms when two chromosome segments, each with a
centromere, fuse end to end, with loss of their acentric fragments
- radiation exposure causes DNA strand breaks leading to
abnormal chromosome replication, including dicentric
chromosome formation
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Fluorescence In Situ Hybridization - FISH
Principle: Target DNA and fluorescent labeled
DNA probe are denatured and simultaneously
hybridized
Result: Targed DNA retains fluorescence
Probes:
Length of DNA is 300 – 600
bp (no longer!)
DNA is labeled with
fluorescent molecule –
fluorochromes (Texas Red,
FITC, Cy3…)
Preparation - chemical
addition or PCR or nick-
translation
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FISH - probes
(a) Gene-specific probes
- target specific nucleic acid sequences on a
chromosome.
(b) Centromeric probes
- bind to repetitive sequences that are specific
to the centromeric regions.
(c) Telomeric probes
- recognize the repetitive
sequence TTAGGG, and can be
used to visualize all telomeres
simultaneously. Chromosome-
specific telomeric probes
hybridise to subtelomeric,
chromosome-specific repeats.
(d) Whole chromosome-
painting probes
- consist of pools of
chromosome-specific
probes.
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FISH - variants
M-FISH: Multicolor FISH – 5 fluorochromes for 25-1 = 31 combinations (sufficient 24)
m-BAND: multicolor banding - Set of parcial probes with various fluorochromes
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Summary of the presentation
Selected methods shown here are used in genetic toxicology to study genotoxicity of complex mixtures
Use of toxicogenomics together with traditional genotoxicity markers enables to get more insight into mechanism of the action of genotoxicity of complex mixtures
Concept of toxic equivalency factors based on the detailed chemical analysis failed (interaction of components)
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THANK YOU FOR YOUR
ATTENTION
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