Detecting radiation damage to human chromosomes by flow cytometry

Mutation Research, 119 (1983) 161-168

161

Elsevier BiomedicalPress

Detecting radiation damage to human chromosomes

by flow cytometry Judith A. Fantes, D.K. Green, J.K. Elder, Patricia Malloy and H . J . Evans Medical Research Council, Clinical and Population Cytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU (Scotland)

(Accepted 15 September 1982)

Summary The flow karyotype profile of ethidium bromide-stained chromosomes from human peripheral blood lymphocytes has been analysed following exposure of lymphocytes to graded series of X-ray doses in vitro. Flow analysis offers the potential for rapid counting of chromosome abnormalities and it is shown that the level of background fluorescence, the distribution of fluorescence and the area of peaks associated with the larger chromosomes, are altered in a dose-related fashion following previous exposures of cultured lymphocytes to 50-400 rad. Moreover, parallel manual analysis of the incidence of chromosome aberrations in metaphase samples of the irradiated cells show a close correlation between flow karyotype profile distortion and aberration frequency. It is estimated that for any given irradiated blood sample doses above 100 rad could be detected with certainty.

Human exposure to most physical and chemical mutagens results in the formation of chromosome aberrations in exposed cells and these may be readily observed in cultured blood lymphocytes. Indeed 'lymphocyte chromosome dosimetry' can provide a measure of exposure to ionising radiations and is used in cases of suspected overexposure (Evans and Lloyd, 1978). Increased aberration frequencies have also been demonstrated in workers occupationally exposed within the maximum permissible radiation levels, where population background frequencies doubled at exposures of 2-3 rad/annum and increased linearly with dose (Evans et al., 1978; Lloyd et al., 1980). However, to detect such a doubling in a single individual requires the analysis of some 10000 cells and since a trained technician analyses 200-300 cells/day, this is not a feasible prospect for population monitoring at 0165-7992/83/0000-0000/$03.00 © Elsevier BiomedicalPress

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suspected low exposures. In practice it is possible to measure doses of above 15 rad with the analysis of 500 metaphase cells. Chromosome analysis by fluorescence activated chromosome sorter offers a possible alternative approach and we show here that the degree of disturbance of the flow-karyotype profile of chromosomes from X-irradiated human lymphocytes is a function of radiation exposure and is correlated with aberration incidence determined by conventional cytogenetic analysis.

Rationale and methods

Original studies undertaken at Livermore, U.S.A. (Gray et al., 1979) had shown that it was possible to at least partially karyotype the chromosome complement of an individual by flow cytometry analysis of chromosomes from cultures of peripheral blood lymphocytes. Such an approach involves isolating chromosomes in suspension, staining them with a DNA-binding fluorescent dye and forcing them, in single file in a fluid stream, down a fine nozzle. A laser light source which picks out the excitation band of the fl/~orescent dye, is focussed onto the liquid stream emerging from the nozzle, and the resulting fluorescent emission of each passing chromosome is detected by a photomultiplier. Emission of any given object is a function of its DNA content and so the size of a given chromosome passing through the laser beam is measured and recorded. In this way it is possible to produce a profile of the chromosome complement based upon chromosome size (see Fig. 2). An important feature of this flow process is that the rate of passage of chromosomes through the nozzle is rapid, so that the chromosome complement of approx. 1000 cells can be analysed in the time of 1 min. The recent development of reliable and reproducible preparation techniques (Sillars and Young, 1981; Young et al., 1981) for chromosome flow cytometry have made it possible to produce a frequency distribution of ethidium bromide stained human chromosomes with between 12 and 20 peaks, where each peak corresponds to a specific chromosome or group of chromosomes. Every chromosome frequency distribution has an underlying continuous background arising from cell debris and damaged chromosomes, but with careful adjustment of the flow cytometer it is possible to prevent the background level from seriously degrading the chromosome distribution. We have found that the flow-karyotype profile of any given individual is very much like a fingerprint in that flow profiles differ between normal individuals and these differences are consistent on repeated analysis of different samples from any particular individual. However, disturbances in a flow profile following exposure to mutagenic agents are to be expected since the presence of chromosome fragments will reduce the heights of the peaks contributing to the larger chromosomes, increase the incidence of chromosome objects of smaller size and, of course, increase the background. Indeed, such changes have been previously noted (Carrano et al., 1979; Otto and Oldiges, 1980) in flow-karyotypes of Chinese

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hamster cell chromosomes when chromosome suspensions were prepared from Xirradiated as opposed to unirradiated cells. In our studies we have investigated the chromosome distribution profiles of chromosomes from human lymphocytes Xirradiated in vitro. Lymphocytes were separated from human blood on a ficoll hypaque gradient and were irradiated at 20°C by a Siemens Stabilipan X-ray machine (250 kV; Th2 filter; 67 rad/min). Doses ranged from 50 to 400 rad. Irradiated cells and controls were cultured at 5 x l0 s lymphocytes/ml in RPMI 1640 medium supplemented with 15o7o foetal calf serum, 100 U/ml penicillin, 100 ~g/ml streptomycin and loT0 phytohaemagglutinin for 52 h at 37°C. Colcemid was added to the cultures for the final 6 h at a concentration of 0.1 /zg/ml. Chromosomes were isolated from irradiated and control cultures using the method of Sillars and Young (1981). Cells were swollen in hypotonic 75 mM KCI, and resuspended after centrifugation in ice-cold buffer containing 15 mM Tris-HC1, 0.2 mM spermine, 0.5 mM spermidine, 2 mM EDTA, 0.5 mM EGTA, 80 mM KCI, 20 mM NaCl and 14 mM ~-mercaptoethanol, final pH 7.2. After centrifugation out of buffer the cells were disrupted by mild detergent lysis in buffer + 0.1°70 digitonin followed by vortex mixing and this mixture of isolated chromosomes, interphase nuclei and debris was stored overnight on ice. Each sample contained 107 chromosomes/ml and had been prepared from 10 ml of blood. Coded and randomised samples were stained with 0.15 mM ethidium bromide and injected into a flow cytofluorimeter (Green and Fantes, 1982) with a motor driven syringe at 0.3 ml/h. The ethidium bromide was excited with a l-W, 488-nm wavelength laser beam, and the fluorescence emission detected in a 550-650 nm wavelength band. Fluorescence profiles were accumulated and recorded for each sample after a 10-min period of stabilisation. Aliquots were taken of each sample of cells in 75 mM KCI. These were fixed in 3:1 methanol:acetic acid and air-dried slides were prepared and stained in orcein. Slides were coded and randomised before 100 cells from each sample were scored for dicentric chromosomes and acentric fragments.

Results

Fig. 1 shows the ethidium bromide fluorescence distribution of samples prepared from human peripheral blood, where the cells were subjected to X-ray exposure before culture and stimulation for cell division. A visual inspection of the distributions in Fig. 1 show that the level of continuous background increases with radiation dose, particularly at low intensity of fluorescence, and that the overall detail in peaks and troughs decreases. A more satisfactory approach would be to describe the dose-related changed characteristics of the flow profiles numerically. Each profile was therefore cut just below the smallest chromosome peak, normalised to the same

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Fig. 1. A series of fluorescence profiles for chromosomes, derived from short-term cultures of human peripheral blood cells, which, prior to growth stimulation, were given controlled X-radiation exposures. In each profile (a to e) fluorescence intensity divided into 512 channels is shown along the horizontal axis and the frequency of events in each channel is shown along the vertical axis.

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Fig. 2. Normalised flow-profile of ethidium bromide stained c h r o m o s o m e suspension. Measurements B, C, D and E (see text) are shown diagrammatically. A, the area above background is shown as the cross hatched area. C h r o m o s o m e groups corresponding to the peaks of the histogram are numbered. The horizontal axis (fluorescence intensity) was normalised by placing the c h r o m o s o m e (1, 2) peak at channel 400 and the c h r o m o s o m e (9-12) peak at channel 225. The total area was normalised to 100000 units.

30 • :CID o =(Oic+Frag)/Cell

211

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Fig. 3. A comparison o f fluorescence profile distortion, measured with the quotient C/D (unbroken line) and the actual dicentric plus fragment counts (broken line) for a series of radiation experiments. The points for C/D a n d the c h r o m o s o m e d a m a g e were calculated from the combined results shown in Table 1 and in the case of C/D each point is extended by one standard deviation.

166 TABLE 1 RELATIONSHIP BETWEEN FLOW CHROMOSOME PROFILE PARAMETERS AND INCIDENCE OF DICENTRICS AND FRAGMENTS IN X-IRRADIATED BLOOD LYMPHOCYTES FROM SEVERAL INDIVIDUALS

Dose (rad)

Flow parameters

Manual scoring

Cells

A

B

C

D

E trics (/cell)

Dicenments (/cell)

Fragscored

Expt. 1 Control 50 100 200 400

73.1 77.5 74.8 69.7 60.5

34.7 35.9 36.8 36.8 37.9

29.6 21.8 28.0 45.1 76.1

5.92 6.25 5.94 4.93 4.14

3.12 2.74 3.15 3.32 2.89

0.0 0.04 0.18 0.43 1.17

0.02 0.06 0.26 0.74 2.11

100 100 100 100 100

Expt. 2 Control Control 50 100 200 400

72.7 74.3 70.5 66.9 64.0 53.9

32.9 34.3 34.0 35.3 35.1 39.7

21.1 20.8 20.8 31.6 40.8 97.5

6.05 4.54 5.03 5.12 4.60 3.65

3.10 2.89 2.74 3.35 3.02 2.97

0.0 0.0 0.05 0.12 0.44 1.28

0.0 0.0 0.07 0.23 0.80 2.43

100 100 100 100 100 1.00

Expt. 3 Control 300

80.2 65.7

37.3 40.1

30.0 75.5

6.43 4.38

3.00 3.28

0.0 0.72

0.0 1.36

50 100

Expt. 4 Control Control 50 100 200 400

83.4 79.2 78.2 75.0 77.6 62.8

34.1 33.9 34.8 36.2 35.9 38.7

9.3 15.4 25.4 26.2 35.9 76.8

7.51 6.02 6.22 6.19 6.38 4.38

2.78 2.46 2.45 2.69 2.65 2.48

0.0 0.01 0.03 0.17 0.41 1.49

0.0 0.02 0.10 0.22 0.67 2.57

100 100 100 100 100 100

horizontal spread and total area, and with manual assistance a line was drawn through the continuous background. The following characteristics were then ascertained: A = percent area above background; B = percent area (including background) up to the central deep trough which lies between c h r o m o s o m e s (9-12) and (13-15) peaks; C = height o f the central deep trough; D = area o f the least squares fitting Gaussian curve over the c h r o m o s o m e s (1, 2) peak, expressed as a percentage o f the total area; E = coefficient o f variation o f the least squares fitting Gaussian curve over the c h r o m o s o m e s (1, 2) peak. Fig. 2 shows a diagrammatic representation of measurements o f the parameters A - E following the analysis o f a given flow profile and the numerical results for a

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series o f experiments where these parameters have been measured are given in Table 1. Blood from different individuals was used for Expts. 1, 2 and 4. Expt. 3 was performed on a separate occasion but with blood from the Expt. 4 donor. Fig. 1 shows the fluorescence distributions resulting from Expt. 4. Most of the factors listed in Table 1 show a general trend which correlates to the radiation levels in each experiment. In particular the level of the deep central trough (parameter C), which increases with dose, and the area of the chromosome (1, 2) peak (parameter D), which decreases with dose, are most closely correlated with radiation level. The quotient C / D which approximately describes the effect of an increased number of chromosome fragments and a depleted number of homologous chromosomes Nos. 1 and 2 in radiation damaged cells, is plotted as an unbroken line against dose in Fig. 3. The results shown in Table 1 were measured from the fluorescence profiles of ethidium bromide stained chromosome suspensions which were approximately 1 day old. There was no significant change when similar measurements were made 1 week later. The 2 principal kinds of chromosome change that were scored by manual methods to assess the overall aberration incidence were dicentric chromosomes and acentric fragments. These particular chromosome anomalies are relatively unambiguous and easy to detect; their frequencies determined by manual scoring are listed in Table 1 and the total data are plotted as a suitably scaled broken line in Fig. 3. The close correlation between the observed incidence of dicentrics and fragments per cell and the quotient C / D is clearly evident but the increase of C / D from zero to 50 tad is not statistically significant.

Discussion There is scope for better resolution o f chromosome flow-profiles through the use of improved nozzle systems, carefully designed optics, alternative fluorochromes including the combination of two fluorochromes (Gray et al., 1979) - and the more effective removal of signals from non-chromosome objects (Green and Fantes, 1982). Such improvements would give increased discrimination in the detection of chromosome anomalies, so that flow analysis should detect chromosome damage induced by mutagen exposures of considerably less than 50 rad of X-irradiation. Ultimately to be of practical use a flow technique must be capable of detecting radiation doses as low as 15 rad. It is estimated that even when a routine flow technique is perfected, detection of low levels of chromosome damage will depend on the comparison of a normal chromosome sample with a suspected damaged sample from the same individual and that flow profiles from persons open to risk of chromosome damage should be kept on record for a future eventuality. This could be particularly important if the parameter "C" continued to be regarded as an important quantitative indication of radiation dose, since any comparison between individuals

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could be jeopardised by the occurrence of polymorphic chromosomes in the 9-15 groups. Using the flow technique described in this letter it is possible to monitor radiation doses greater than 100 rad given that a control fluorescence profile exists. From an operational point of view, the time taken from the collection of peripheral blood to the production of a chromosome suspension for flow analysis is identical to that for the production of a microscope slide for manual scoring and, in either case, batches of samples may be processed in parallel. Scoring a sample by manual means requires approximately 1 man-hour per 50 metaphase cells and the number of cells scored per day is limited by scorer fatigue. In contrast, scoring a sample by the flow method requires approximately 1 h per equivalent 60000 metaphase cells and scorer fatigue is not a problem.

References Carrano, A.V., M.A. Van Dilla and J.W. Gray (1979) Flow cytogenetics: a new approach to chromosome analysis in: M.R. Melemed, P.F. Mullaney and M.L. Mendelssohn (Eds.), Flow Cytometry and Sorting, Wiley, New York, pp. 421-451. Evans, H.J., and D.C. Lloyd (1978) in: H.J. Evans and D.C. Lloyd (Eds.), Mutagen-induced chromosome damage in man, University Press, Edinburgh. Evans, H.J., K.E. Buckton, G.E. Hamilton and A. Carothers (1979) Radiation-induced chromosome aberrations in nuclear-dockyard workers, Nature (London), 277, 531-534. Gray, J.W., R.G. Langlois, A.V. Carrano, K. Burkhart-Schulte and M.A. Van Dilla (1979) High resolution chromosome analysis: One and two parameter flow cytometry, Chromosoma, 73, 9-27. Green, D.K., and J.A. Fantes (1982) Improved accuracy of in-flow chromosome fluorescence measurements by digital processing of multi-parameter flow data, Signal Proc., submitted. Lloyd, D.C., R.J. Purrott and E.J. Reeder (1980) The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated and occupationally exposed people, Mutation Res., 72, 523-532. Otto, F.I., and H. Oldiges (1980) Flow cytogenetic studies in chromosomes and whole cells for the detection of clastogenic effects, Cytometry, 1, 13-17. Sillars, R., and B.D. Young (1981) A new method for the preparation of metaphase chromosomes for flow analysis, J. Histochem. Cytochem., 29, 74-78. Young, B.D., M.A. Ferguson-Smith, R. Sillars and E. Boyd (1981) High resolution analysis of human peripheral lymphocyte chromosomes by flow cytometry, Proc. Natl. Acad. Sci. (U.S.A.), 78, 7727-7731.