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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

15. 7. 2014 Jan Topinka TOXI-LATIN 2014 2

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)


Toxicology of complex

mixtures


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.


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.

Jan Topinka TOXI-LATIN 2014 15. 7. 2014 6

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


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…


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.


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


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)


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


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


Analysis of DNA adducts

(32P-postlabelling)

Individual compounds

Complex mixtures

(extracts from PM)

B[a]P DB[a,l]

P

Control


TLC Maps of DNA Adducts


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)


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)


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


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)


Oxidative stress


Oxidative stress in cellular components


Complex mixtures as sources

of oxidative stress


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


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


8-Oxodeoxyguanosine (8-oxodG)


• 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


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


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


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))


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.


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.


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


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)


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


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)


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/


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


Metafer – applications - software

MSearch AutoCapt

DCScore

MetaCyte

RCDetect

CometScan Metafer P

MicNuc


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


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


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


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)


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.


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


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


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


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)


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


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.


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.




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


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


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.


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


http://www.metasystems.de/downloads/labelling_scheme.pdf

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)


THANK YOU FOR YOUR

ATTENTION


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