Introduction to biomarkers of exposure what can they tell us
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1 Introduction to biomarkers of exposure what can they tell us Andrzej Wojcik acentric fragment dicentric dicentric dicentric acentric fragment
2 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
3 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?
4 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
5 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
6 A tribute to chromosomal aberrations
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8 How can we block a cell in mitosis? Colcemid (colchicine) blocks the development of mitotoc spindles
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10 Some important things to remeber Agents that induce DNA double strand breaks Ionizing radiation Bleomycin Carcinostatin ROS-generating agents S-phase independent agents Induce: Chromosomal-type aberrations Chromatid-type aberrations Agents that induce other forms of DNA damage Alkylating agents DNA crosslinking agents UV radiation most other DNA damaging agents S-phase dependent agents Induce: Only chromatid-type aberrations But chromatid-type aberrations are also a marker of genomic instability
11 Examples of chromosome-, and chromatid type aberrations Chromosome type Chromatid type Both chromosome arms are involved Damage was induced before DNA replication One chromosome arm (a chromatid) is involved Damage was induced after DNA replication Strange exception: iso-chromatid breaks
12 Types of radiation-induced chromosomal aberrations with relation to the cell cycle: chromosome type vs chromatid type Chromatid-type M Chromosome-type G 2 G 1 Chemical mutagens induce only chromatid-type aberrations! S Chromosome-type + Chromatid-type
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15 Stable vs unstable aberrations complete unstable incomplete
16 Why are stable aberrations called stable?
17 How to score chromosomal aberrations? Score blind (code slides) Note aberration score per cell (distribution) Analyse the mitotic index (MI) MI = Number of mitoses Number of nuclei x 100 Example of an aberration score sheet
18 Painting of chromosomes FISH Fluorescence in situ hybridisation
19 mfish painting of chromosomes with 24 colors Johannes et al. Rad. Res. 161: , 2004.
20 mband painting of a chromosome (# 5) in different color bands C. Johannes et al. Rad. Res. 161: , 240.
21 The micronucleus test Mn Mn
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24 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?
25 Dose resonse curve for Mn and MnC+ A. Kryscio 2001, PhD thesis
26 The Mn assay can be used to detect apoptosis and necrosis
27 The Mn frequency is related to cytotoxicity
28 MN in binucleated cells (BNC) Highly damaged cells may become visible at late fixation time points (applies both to Mn and aberrations) Lyphocytes X-rays Neutrons Alphas Cytochalasin B 72h 96h 120h
29 MN in binucleated cells (BNC) 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 Mutation Research, in press TK6 wild type Cytochalasin B 24h 30h TK6 MTH1 knock down 46h
30 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
31 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)
32 MN/1000 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 Hours after exposure
33 F o ld d iffe re n c e to c o n tro l DNA damage measured by the alkaline comet assay with cells from blue mussel heamolymph after in vitro irradiation D o s e (G y )
34 Trachemys scripta Lymphocytes culture possible
35 Candidates for biomarkers in humans Ionizing radiation biomarkers for potential use in epidemiological studies Eileen Pernot et al. Mutation Research 751 (2012)
36 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
37 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): ; 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 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 Collections 2-4: always 24h after exposure Analysis of protein levels by ELISA kits
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39 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.
40 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.
41 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 Gy C >20 Gy (1 month pr) Specificity? Plausibility? Reproducibility?
42 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: , 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.
43 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, Rats were chronically exposed to 137 Cs 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 137 Cs-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.
44 Widening the radiation protection world
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