A SEMI-AUTOMATED MICRONUCLEUS-CENTROMERE ASSAY TO ASSESS LOW DOSE RADIATION EXPOSURE IN T-

LYMPHOCYTES

Ans Baeyens

Introduction

Chromosomal damage induced by IR Cytogenetic assays

- Radiation protection (eg biological dosimetry)

- Radiobiological research (eg chromosomal radiosensitivity)

Standard method for biological dosimetry of individuals accidently exposed to IR = Analysis of dicentric chromosomes very time consuming and need skilled specialists in cytogenetics

small nuclear fragments in

cytoplasm of interphase cells

Micronuclei

biological dosimetry

in vitro radiosensitivity

G0 Micronucleus assay

Micronucleus assay

0.5 ml blood + 4.5 ml culture medium

Cultures irradiated with Co-60 -rays

Stimulation of lymphocytes with phytohaemagglutinin

24h later: addition of cytochalasin B

70h post irradiation: harvesting of cultures with KCl and Methanol/Acetic Acid

Slide preparation: cells dropped on slide

Scoring of micronuclei in 1000 binucleate cells

Micronuclei: types

A) Whole chromosomes centromere positive

(background –spontaneous)

B) Acentric fragments centromere negative

(induced by ionising radiation) (result of mis- or unrepaired DSB)

MN assay

Advantages: Blood sample is easily collected specimen with

little discomfort for patients simple, easy to use technique, quick Reliable method to assess radiation induced

DNA damage Automated slide scanning system and MN

scoring system Metafer, MetasystemsDrawbacks: Relative high and variable spontaneous MN Low dose estimation is restricted to 0.3 Gy

Low dose radiation

- Majority of over exposure occurs in low dose range

- Adaptive response to radiation?- Sensitivity of biomonitoring tests is restricted to

0.2Gy

Aim

Investigate if the sensitivity of the MN assay could be enhanced by combining the automated MN assay with pan-centromere scoring

Determine a standard dose response curve using a semi-automated micronucleus-centromere assay

Methods

Pancentromeric probe

1. DNA extraction: Using the phenol-chloroform method DNA from a male donor is extracted to obtain X, Y and autosomal centromeric material.

2. Probe design: Centromeric DNA is amplified via polymerase chain reaction (PCR) using specially designed primers.

3. Labelling probe: PCR product is labelled with Spectrum Orange using nick translation method and the probe is applied to cells using FISH methodology.

Fluorescence in situ hybridization (FISH)

Hybridisations of the probe with binucleated lymphocytes or metaphase spreads are done overnight. Washing steps are followed by counterstaining with DAPI that result in blue cell nuclei

Scoring is done with automated Metafer System, Metafer

Pancentromeric probe

The centromeric probe is highly specific for the centromeric region of all chromosomes.

In situ hybridization of metaphase spreads with the pan-centromeric probe:

Pancentromeric probe

CM negative MN

CM positive MN

Dose response curves

Semi-automated scoring of centromeres in MN confirms that a high percentage of spontaneous MN contain centromeres, while radiation induced MN are mainly centromere negative.

Differentiation between CM positive and CM negative MN allows the sensitivity of the MN to be increased and doses as low as 0.05 can be detected

0 0.010.020.030.05 0.1 0.2 0.3 0.5 1 20.00

5.00

10.00

15.00

20.00

25.00

error bars = SEM

Number of CM neg MNnumber of CM pos MNtotal number of MN

Dose (Gy)

Num

ber

of

MN

/1000B

N

Low dose area

Percentage of CM neg MN vs dose (Gy)

By scoring only MN CM-, the sensitivity of the MN assay for low dose detection is increased. When the percentage of MN CM- is taken as indicator for radiation exposure a dose of 0.05 Gy can be detected within estimated 95% confidence limits

Automated microscopic system,Metafer, MetaSystems

The total scoring time per slide, using the Metafer for semi-automated analysis of MN CM-, is 5 times reduced compared to manual scoring

Conclusion

As the sensitivity for low dose detection is

enhanced with the semi-automated MN-

centromere assay compared to the

conventional MN assay, this method

presents a better tool to evaluate in vivo

radiation exposure of individuals following

the absorption of low doses of radiation

Acknowledgement

O Herd, R Swanson, X Muller and A Papadopoulos (Radiation Biophysics, NRF-iThemba Labs, Johannesburg and Radiation Sciences, WITS University)

Prof. J. Slabbert and P. Beukes (Radiation Biophysics, NRF-iThemba Labs, Cape Town)

Prof. D. Van der Merve and T. Mabhengu (Medical physics, CMAHJ, Johannesburg)

Dr. P. Willem (Somatic Cell Genetics Units, Dep Haematology, WITS University, Johannesburg)

Prof. A. Vral (Basic Medical Sciences, Ghent University, Belgium)