Introduction to biomarkers of exposure – what
can they tell us
Andrzej Wojcik
[email protected]
dicentric
acentric
fragment
dicentric
dicentric
acentric
fragment
What is a biomarker?
Any measurement reflecting an interaction between a
biological system and an environmental agent, which may
be chemical, physical or biological.
Biomarkers can be used for multiple purposes in a
toxicological investigation including:
(1) estimation or validation of received dose, thus improving
the validity of a correlation between exposure and
biological responses;
(2) investigation of individual susceptibility;
(3) early detection of a radiation induced health effect.
An ideal biomarker is both sensitive and specific
to the agent of interest
An ideal biomarker is both sensitive and specific
to the agent of interest
How do we know that the described effect is attributed to radiation?
Some more criteria of a good biomarker:
1. Signal must be detectable after external exposure,
even partial-body
2. Signal must be detectable after internal contamination
3. Signal must not fade with time
4. Signal must be specific to radiation
5. It must be possible to generate a calibration curve
The list sounds like the wish to have peace and prosperity
on Earth with all inhabitants being young, healthy and
happy…
Let us see which radiation effects come close to fulfilling the criteria
•
•
•
•
•
All biochemical effects in cells and organisms are transient
The only effects that are stable with time are mutations (really?)
Mutations can be analysed by studying the genotype or the phenotype
However, mutations are not specific to radiation
But some chromosomal aberrations are
A tribute to chromosomal aberrations
How can we block a cell in mitosis?
Colcemid (colchicine) blocks the development of mitotoc spindles
Some important things to remeber
Agents that induce DNA
double strand breaks
• Ionizing radiation
• Bleomycin
• Carcinostatin
• ROS-generating agents
Agents that induce other
forms of DNA damage
• Alkylating agents
• DNA crosslinking agents
• UV radiation
• most other DNA damaging agents
S-phase independent agents
Induce:
Chromosomal-type aberrations
Chromatid-type aberrations
S-phase dependent agents
Induce:
Only chromatid-type aberrations
But chromatid-type aberrations are
also a marker of genomic instability
Examples of chromosome-, and chromatid type aberrations
Chromosome type
Both chromosome arms are involved
Damage was induced before DNA replication
Chromatid type
One chromosome arm (a chromatid) is involved
Damage was induced after DNA replication
Strange exception: iso-chromatid breaks
Types of radiation-induced chromosomal aberrations
with relation to the cell cycle:
chromosome type vs chromatid type
Chromatid-type
Chromosome-type
M
G1
G2
Chemical mutagens
induce only
chromatid-type
aberrations!
S
Chromosome-type
+
Chromatid-type
Stable vs unstable
aberrations
complete
unstable
incomplete
Why are stable aberrations called stable?
How to score chromosomal aberrations?
• Score blind (code slides)
• Note aberration score per cell (distribution)
• Analyse the mitotic index (MI)
MI =
Example of an aberration score sheet
Number of mitoses
Number of nuclei
x 100
Painting of chromosomes – FISH
Fluorescence in situ hybridisation
14
14
8
8
2
2
2
8
2
8
14
14
mFISH – painting of chromosomes with 24 colors
Johannes et al. Rad. Res. 161:540-548, 2004.
mBand – painting of a chromosome
(# 5) in different color bands
C. Johannes et al.
Rad. Res. 161:540-548, 240.
The micronucleus test
Mn
Mn
The micronucleus assay can be used to
distinguish between clastogens and aneugens
The differentiation between Mn containing acentric fragments and whole
chromosomes is done with the help of fluorescent in situ hybridisation
(FISH) with pan-centromeric probes
?
Dose resonse curve for Mn and MnC+
A. Kryscio 2001, PhD thesis
The Mn assay can be used to detect apoptosis and necrosis
The Mn frequency is related to cytotoxicity
Highly damaged cells may become visible at late fixation time points
(applies both to Mn and aberrations)
Lyphocytes
X-rays
Neutrons
Alphas
72h
96h
120h
MN in binucleated
cells (BNC)
Cytochalasin B
Always use three fixation timepoints when analysing MN or
aberrations in cycling cells
TK6 cells
UVA
UVB
UVC
Gamma
Investigation of micronuclei induction in MTH1 knockdown cells exposed to UVA, UVB or UVC
Asal Fotouhi, Nicola Cornella,
Mehrafarin Ramezani,
Andrzej Wojcik
and Siamak Haghdoost
TK6 wild type
Mutation Research, in press
24h
30h
46h
MN in binucleated cells (BNC)
Cytochalasin B
TK6 MTH1 knock down
Developing methods to assess dose response relationships in the
blue mussel (Mytilus edulis) cells exposed to radiation and PAHs
(Polycyclic aromatic hydrocarbons)
Elinor Eriksson, MSc , SU 2012
Developing methods to assess dose response relationships in the
blue mussel (Mytilus edulis) cells exposed to radiation and PAHs
Elinor Eriksson, MSc , SU 2012
Haemolymph extracted from the foot
DNA damage in haemolymph cells analysed by
• Comet assay
• Mn assay (without Cytochalasin B)
Mn in haemolymph cells
Problem: the cell turn over is not known
Aim: to find the optimal time for scoring Mn
Method: Mn analysed 0, 24, 48, 72, 93, 120h after 2 Gy
90
80
70
MN/1000
60
50
40
30
20
10
0
0
24
48
72
Hours after exposure
93
120
F o ld d if fe r e n c e t o c o n t r o l
DNA damage measured by the alkaline comet assay
with cells from blue mussel heamolymph after in
vitro irradiation
10
8
6
4
2
0
0
1
2
3
D o s e (G y )
4
5
6
Trachemys scripta
Lymphocytes culture possible
Candidates for biomarkers in humans
Ionizing radiation biomarkers for potential use in epidemiological studies
Eileen Pernot et al. Mutation Research 751 (2012) 258–286
New potential biomarkers of exposure – the “omics” approach
EGAN analysis showing all
the differentially expressed
genes enriched pathways.
Each circle represents a
gene.
Dark grayed circles are upregulated genes
Light grayed circles are
down-regulated genes.
The lines represent
connections between
different genes belonging to
different pathways.
H. El-Saghire, PhD thesis
2014, University of Ghent
EGAN: Exploratory Gene
Association Networks
Radiation-induced changes in levels of selected proteins in peripheral blood
serum of breast cancer patients as a potential triage biodosimeter for large-scale
radiological emergencies.
Deperas-Kaminska M, Bajinskis A, Marczyk M, Polanska J, Wersäll P, Lidbrink E, Ainsbury EA, Guipaud O,
Benderitter M, Haghdoost S, Wojcik A.
Health Phys. 107(6):555–563; 2014
Blood was collected from 16 breast cancer patients undergoing
standard external beam radiotherapy (IMRT, 2 Gy/freaction)
8 proteins were analysed in
blood serum
Blood was collected at 5 time points:
•
•
•
•
•
•
•
•
1
Before
RT
2
After 1
fraction
2 Gy
3
After 5
fractions
10 Gy
•
•
4
After 10
fractions
20 Gy
5
1 Month
after 25
fractions
50 Gy
Collections 2-4: always 24h after exposure
Analysis of protein levels by ELISA kits
APOE - Apolipoprotein E
APOH - Apolipoprotein H
C7 – Complement protein 7
FX - Factor X - prothrombinase
PANK4- Pantothenate Kinase 4
A2M - Alpha-2-macroglobulin
FETUB - Fetuin B
A1-AC- Alpha-1-Anti-Chymotrypsin
Individual dose responses
Concentrations of the studied proteins in serum of patients collected at different time points
during therapy. Each line represents one patients. Thick black line: mean. Error bars: 95%
confidence limits.
Individual dose responses
Concentrations of the studied proteins in serum of patients collected at different time points
during therapy. Each line represents one patients. Thick black line: mean. Error bars: 95%
confidence limits.
How precise is the discrimination between
dose groups using
FX, PANK4 and APOE?
Results of multi regression model
A
0-2 Gy
90% accuracy
100% accuracy
B
10-20 Gy
Specificity?
Plausibility?
Reproducibility?
C
>20 Gy (1 month pr)
Are there published data on long-term effects of radiation
on the inflammatory response?
Nakachi K, Hayashi T, Hamatani K, Eguchi H, Kusunoki Y. Sixty years of follow-up of
Hiroshima and Nagasaki survivors: current progress in molecular epidemiology studies. Mutat
Res. 659:109-117, 2008.
Plasma ROS levels in Hiroshima and Nagasaki survivors significantly increased with increased
radiation dose (reference to unpublished data - dose levels unknown).
J.B. Reitan, O.J.Mellbye, T.D. Bergan, P. Strand. Immunological effects of low dose radiation.
Absent or minor effects of Chernobyl fallout in Norway? StrålevernRapport 1998:1.
Analysis of immunological parameters in Norwegian individuals most heavily exposed to the
Chernobyl fallout stayed within normal range. Within this range some correlation with dose was
observed.
Radiation effects at the level of the metabolome
Metabolomics is a "systematic study of the unique chemical fingerprints that specific cellular
processes leave behind“. The metabolome represents the collection of all metabolites in a biological
cell, tissue, organ or organism, which are the end products of cellular processes.
S. Grison et al. The metabolomic approach identifies a biological signature of low-dose chronic
exposure to cesium 137. J Radiat Res 53:33-43, 2012.
Rats were chronically exposed to 137Cs in drinking water. The mean whole-body absorbed dose after 9
months of contamination
was estimated at a maximum value of 4 mGy.
Proteins, carbohydrates, lipids, ions and other
metabolites were analysed in serum and urine.
A metabolomic signature discriminated
137Cs-contaminated from control animals .
We must remember that the results come from an
animal study. Inbred animals show a low degree
of individual variability. But it is very interesting to
test the assay on a human cohort or wild animals.
Widening the radiation protection world…