Clastogenic factors in blood plasma obtained from subjects exposed to. Carita Lindholm STUK - Radiation and Nuclear Safety Authority

Transcription

1 Clastogenic factors in blood plasma obtained from subjects exposed to ionizing radiation Carita Lindholm STUK - Radiation and Nuclear Safety Authority

2 Objectives: Identify clastogenic factors in blood plasma from irradiated individuals using detection of γ-h2ax foci and cytogenetic analyses Provide information on factors inducing non-targeted (bystander) effects in vivo in humans

3 Irradiated individual Reporter cells obtained from healthy donor Culture W. Morgan 2003

4 Background Existence of clastogenic factors (CFs) has been demonstrated indirectly through cytogenetic damage in cell cultures with blood plasma obtained from e.g. accidentally irradiated persons radiotherapy patients Chernobyl cleanup workers and population living in the contaminated areas victims of the Hiroshima atomic bombing CFs are not induced in all radiation-exposed individuals Also found in other patient t groups: inflammatory diseases inherited radiosensitivity disorders (Fanconi s anaemia, Ataxia telangiectasia) cancer Other exposures (UV, asbestos) Mechanisms of action relatively unknown Assay requirements relatively stringent

5 Testing the presence of clastogenic factors (CFs) in patient plasma with the help of: Chromosome aberration (CA) analysis Micronuclei (MN) assay Detection of γ-h2ax foci formation

6 Patient groups studied: basal cell carcinoma (I) (); 4 patients treatment volumes 1.0 to 3.5 cm 3 ; Iridium-192 high dose rate plesiobrachytherapy 5.0 Gy per fraction (2x/week); total dose of 40.0 Gy osteoarthritis of the knee (II) ; 13 patients 6 fractions (2x/week) with doses of 1.0 Gy; total dose of 6.0 Gy, high-energy h photons (6-MV) 800 to 1150 cm 3 tendinitis of the elbow ( tennis elbow ) / heel (III) ; 15 patients 6 single doses of 1.0 Gy (2x/week), total dose of 6.0 Gy, orthovoltage therapy ( kv) 80 to 160 cm 3

7 Plasma samples Plasma samples taken immediately before and after the treatment period (3-4 weeks)= one pre- and one post-rt sample from each patient Plasma separated from ml blood, stored at -80 ºC. Filtration with 30 kda filters to remove HMW molecules (e.g. albumin)

8 Cytogenetic analyses CAs 48 h cultures with 200µl plasma in 5 ml culture; Giemsa staining 200 metaphases analysed / sample all aberrations scored (chromatid- and chromosometype) MNi 66 h cultures with 200 µl plasma in5mlculture; Cytochalasin B treatment Akridine Orange staining 1000 binucleated cells analysed / sample for micronuclei

9 Summary of cytogenetic analyses Significant increase of CA in five patient samples (4 osteoarthritis; 1 basal cell carcinoma)and two of the radiation victim samples No effect of radiotherapy No elevation in MN yields in any group

10 Assaying of γ-h2ax foci in cells incubated with patient plasma to identify early response of reporter cells to plasma factors kinetics of foci: 15, 30 and 60 min incubation of filtered plasma with reporter lymphocytes from one donor detection: immunofluorescence (anti-γ-h2ax γ + FITC labelled secondary ab:s) quantification of foci using automated scanning program (MetaCyte software, MetaSystems) 1000 lymphocytes scored / sample

11

12 Identification of cells > 40 µm2 Identification of foci: 1) Integration of 5 focal planes into stack images 2) Acceptance of foci > 0.1 µm 2

13 Summary: γ-h2ax foci Radically increased foci formation in some patient plasma No obvious effect of the RT to the induction of foci Large individual differences (up to 10-fold)

14 Conclusions Presence of CF s not as wide as in other studies, but individual variation was observed Apparently no direct association between γ-h2ax foci formation and CA yields Increased yields in the pre-radiotherapy samples -> effect of inflammatory disease Confounders: treatment volume and location, other treatments, antioxidant status, smoking, age, sex Composition of plasma to be investigated with p p g immunoassays/proteomics

15 University of Leipzig: Prof. Guido Hildebrandt Dr. Jutta Jahns Acknowledgments STUK, Radiation Biology: Anna Acheva (Queen s University Belfast) Armi Koivistoinen Marjo Perälä Sisko Salomaa