int. j. radiat. biol 1998, vol. 73, no. 5, 549 ± 555

Micronucleus frequencies in cytokinesis-blocked human Blymphocytes after low dose c-irradiation

A. VRAL² *, H . LOUAGIE³ , H . THIERENS§, J. PHILIPPEÂ ³ , M . CORNELISSEN²and L. DE RIDDER²

(R eceived 2 8 N ovemb er 1 9 97 ; accept ed 2 4 F eb ruary 1 9 98 )

Abstract. smaller subsets depending upon maturation, di å er-P urpose: To investigate (1) the radiosensitivity of B versus T entiation and activation. To what extent all theselymphocytes with respect to m icronucleus (MN) induction and di å erent subsets di å er in their response to ionizing(2) the possible application of the B cell MN assay for biological

radiation is not yet completely elucidated. M ostdosimetry of individuals after acute exposure to low doses ofliterature data agree upon the high radiosensitiv ityioniz ing radiation.

M ate rials and m ethods: MN analy sis was performed in T and B of B lymphocytes compared to the other subpopula-lymphocytes of six healthy volunteers exposed in vitro to c-ray tions and this w ith respect to di å erent endpoints,doses ranging from 0´05 Gy to 1 Gy. For the MN assay on B such as immune function (Anderson et al. 1974,cells, peripheral blood mononuclear cells were cultured and

Sieber et al. 1985), clonogenic cell survival (Schwartzstimulated with pokeweed mitogen (PWM). Afterwards the Band Gaulden 1980, Stew art et al. 1988), apoptosislymphocytes (characterized by the CD20 + phenoty pe) were

separated with the FACSort ¯ ow cytom eter and the number of (Seki et al. 1994, Philippe et al. 1997) and prolifera-MN in the sorted binucleate cells was scored. For T lymphocytes tion capacity (Schwartz et al. 1983, HoÈ gsted et al.the standard MN protocol was applied. 1993, Holmen et al. 1994). Concerning the radiationR esul ts: The number of spontaneou s and radiation induced MN

response of B lymphocytes w ith respect to cytogen-were signi® cantly higher in B lymphocytes com pared to Tetic damage, such as the formation of dicentriclymphocytes in the low dose range up to 1 Gy. An analy sis of

the present data showed that when the spontaneou s MN frequen- chromosome aberrations and micronuclei, only acies are not known, doses from 0´08 Gy could be detec ted with few publications are available (HoÈ gsted et al. 1993,the B cell MN assay while the conventional MN assay only WuÈ ttke et al. 1993, Holmen et al. 1994). The in vitroallowed detec tion of doses >0´25 Gy. However, in contrad iction

cytokinesis-block micronucleu s (MN) assay forto the linear-quadratic dose-response for T cells, for B cells thehuman PBL (Fenech and M orley 1985) has beeninitial steep increase of the MN yield with the very low dose was

followed by a ¯ attening of the curve towards higher doses. extensively used for the assessment of chromosomalC onclusion: This study shows that B lymphocytes express a high damage induced by ionizing radiation (Prosser et al.number of MN for doses up to 1 Gy c-rays re¯ ecting the highly 1988, Little® eld et al. 1989, Gantenberg et al. 1991,radiosensitive behaviour of B cells. The results also point to the

Thierens et al. 1991, Vral et al. 1992, Livingstonpossible application of the B-cell MN assay for individual doseet al. 1993, Vral et al. 1994). The simplicity of M Nassessm ent. When blood samples can be taken within 24 h after

acute accidental overexposure, the B-cell MN assay can be scoring makes this test very attractive as a biologicalperformed but only as a supplementary test to the conventional dosimeter for radiation protection applicationsMN assay. (MuÈ ller and Stre å er 1994, Thierens et al. 1996,

WuÈ ttke et al. 1996). Dicentric chromosome analysis,the gold standard method for biological dosimetry1. Introductionof individuals accidentally exposed to ionizing radi-

Hum an peripheral blood lymphocytes (PBL) are ation (IAEA 1986), has a major drawback sincea mixture of mainly three groups of cells : dicentric scoring is very laborious and requiresT lymphocytes, B lymphocytes and natural killer highly skilled technicians. Improvement of the M Ncells . These populations can be further divided into assay by scoring M N for the presence of centromeres

by FISH has increased the sensitiv ity of the M Nassay for low-dose detection. Without pre-irradiation*Author for correspondence.

² Departm ent of Anatom y, Embryology and Histology , Section control information a detection level of 0´1 Gy canHistology , L. Pasteu rlaan 2, B9000 Gent, Belgium. be obtained with the M N-centromere technique

³ Departm ent of Clinical Chem istry, Microbiology and (Vral et al. 1997). The detection lim it w ith dicentricsImmunology , University Hospital 1 B2, De Pintelaan 185, B9000

can be lowered to 0´05 Gy (MuÈ ller and Stre å erGent, Belgium.1991) but only when a few thousand metaphases§ Departm ent of Biomedical Physics, University of Gent,

Proeftu instraat 86, B9000 Gent, Belgium. are scored.

0955 ± 3002/98 $12.00 Ñ 1998 Taylor & Francis Ltd

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Another approach to enhance the sensitiv ity of the T lymphocytes. The culture period for T cells was70 h, cytochalasin B being added after 42 h.M N technique is to look for M N in a radiosensitive

subpopulation of lymphocytes. WuÈ ttke et al. (1993),combined the M N assay with immuno¯ uorescence

2.1.2. P rocessing of the sample s after cul ture w ithstaining using speci® c antibodies for B and T lympho-

PW M . After incubation, the cells were washed twicecytes. They demonstrated that the M N frequency in

with PBS/BSA. CD20 (Leu-16, phycoerythrinB lymphocytes is signi® cantly higher compared to

labelled ) monoclonal antibody (10 m l, obtained fromT lymphocytes for doses of 0´5 Gy and 1 Gy c-rays.

Becton-Dickinson (BD), Erembodegem , Belgium)In spite of the promising results obtained with the B

was added and the cells were incubated for 20 mincell M N assay no further investigation was done in

in the dark at room temperature. A fterw ards, thethe low dose range (<0´5 Gy).cells were washed once and FACS brand lysing

This study set out to investigate the e å ect ofsolution (BD) was added. Incubation time was 15 min

radiation on M N induction in B lymphocytes(room temperature, dark). Cells were washed twice

compared to T lymphocytes in the low dose rangeand resuspended in 0´5 ml PBS/BSA. The B cells

up to 1 Gy. For this purpose, B lymphocytes were(CD20 + ) were sorted (based on their ¯ uorescence

optimally stimulated by pokeweed mitogen (PW M ),characteristics) w ith a purity of more than 95% usingimmunostained with CD20 and subsequently sortedthe FACSort ¯ ow cytometer (BD). A minimum of

by the FACSort module of the ¯ ow cytometer to30 000 mono-, bi- and polynucleate CD20+cells

obtain maximal yield s of binucleate B lymphocytes.were sorted and collected in 50 ml Falcon tubes. The

For T lymphocytes the standard M N protocol wasFalcon tubes were centrifuged (10 min, 200 g) and

followed (Vral et al. 1994). M N were scored lightthe cells were resuspended in 200 m l of RPMI.

microscopically in binucleate B and T lymphocytes. Cytospins were made and Giemsa stained asdescribed in §2.1.4. This whole procedure only took2 ± 3 h.

2. M aterials and m ethods

2.1. M icronucleus expe riments2.1.3. P rocessing of the sample s after cul ture w ith PH A. Atthe end of the culture period, the cells were given a2.1.1. S ample preparation and ce ll cul ture conditions .

Heparinized blood samples were drawn by venepunc- hypotonic shock with 75 mm KCl (4 ß C). Immediatelyafter the addition of KCI the cells were centrifugedture from six healthy volunteers aged between

25 and 50 years. Peripheral blood mononuclear for 6 min at 120 g and the supernatant wasdiscarded. As a ® rst ® xative, a methanol5glacialcells (PBM C) were isolated by density gradient

centrifugation on Ficoll-H ypaque (Lymphoprep, acetic acid (ratio 1051) solution and Ringers solution(0´9% NaCl) were mixed in equal parts and addedNyegaard, Denmark). Peripheral blood mononuclear

cells suspended in RPMI were c-irrad iated at 37 ß C to the cell pellet. A fter centrifugation (10 min, 120g) the ® xative was discarded and the cells werein a 60Co beam at a dose rate of 1´0 Gy/min with

doses ranging from 0´05 to 1 Gy. The c-irrad iated resuspended in pure methanol5glacial acetic acid(1051). The latter ® xation step was repeated twice.cells together w ith the non-irradiated controls were

cultured in RPMI 1640 supplemented with 0´05% l- Each time, the ® xative was added slowly underconstant vortexing to prevent cell clumping. A fterglutam ine, 20 mm HEPES bu å er, 50 IU/ml penicil-

lin, 50 mg/ml streptomycin, 10% (v/v) heat inactiv- ® xation, cells were concentrated and dropped gentlyon slides w ith a Pasteur pipette and air dried (Vralated foetal calf serum (FCS) and 2´5% PWM (all

from Gibco BRL, Paisley, Scotland). Each culture et al. 1994).consisted of 2 Ö 106 PBM C in 2 ml of culturemedium. The cultures were kept for 5 days in a 95% 2.1.4. S taining of the sl ides. The slides were stainedair plus 5% CO 2 fully humidi® ed incubator (Nuair,

w ith Romanowsky-G iemsa (RG) (Azur B Ð Eosin YBrussels, Belgium) since a suæ cient yield of binucleate

stock solution kit, Serva, Heidelberg, Germany) forcells was reached after this culture period. A fter 65 h

20 min at room temperature. For staining, the RGcytochalasin B (3´5 mg/ml, Sigma Chemical Corp.,

stock solution was diluted in a HEPES bu å er solutionSt Louis, M i, USA) was added to block cytokinesis.

(Serva), pH 6´5 in a ratio of 1515 (6´5%).Additional cultures consisting of 2 Ö 106 PBM C in2 ml of culture medium (without PWM) supple-mented with a protein puri® ed phytohaemaglu ttinin 2.1.5. S lide scoring. Three thousand B and T

lymphocytes were scored to evaluate the percentages(PHA P, 30 mg/ml, Difco Laboratories, Detroit, M I,USA) were initiated for scoring M N induction in of mononucleate, binucleate and polynucleate cells

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551M icronucleus frequencies in cy tokinesis - b lo cked human B lymphocy tes

on the slides (500 lymphocytes/donor). To deter- this lymphocyte subpopulation is adequately stimu-lated by PHA in the described culture conditions.mine the M N yield , between 300 and 1000 binucle-

ate cells (B lymphocytes) or 1000 binucleate cells(T lymphocytes) were scored per donor (tab le 1).

3. ResultsThe identi® cation of M N was done accord ing to thecriter ia summarized by Fenech (Fenech 1993). In M N analysis was performed in T lymphocytes and

sorted B lymphocytes of six healthy volunteersthe cultures w ith PHA, although no sortings forT lymphocytes were performed, the binucleate cells exposed in vitro to c-ray doses ranging from 0´05 Gy

(B cells ) or 0´1 Gy (T cells ) to 1 Gy. Optimal prolifera-can be considered as T lymphocytes because only

Table 1. Number of spontaneou s and radiation-induced micronuclei (MN) in T and B lymphocytes expressed as number of MN perbinucleate (BN) cell.

T lymphocytes B lymphocytes

Dose Number of Number of Number of Number ofDonor (Gy) BN MN/BN BN MN/BN

1 0 1000 0´0210 754 0´02650´05 990 0´03640´1 1000 0´0320 1000 0´05100´25 1000 0´0380 977 0´06450´5 1000 0´0465 809 0´06061 1000 0´1040 663 0´1220

2 0 1000 0´0195 727 0´03300´05 487 0´06400´1 1000 0´0245 733 0´05050´25 1000 0´0275 447 0´06500´5 1000 0´0405 553 0´06901 1000 0´1000 417 0´1130

3 0 1000 0´0145 700 0´03570´05 1000 0´04300´1 1000 0´0208 777 0´06200´25 1000 0´0253 855 0´06200´5 1000 0´0530 582 0´07701 1000 0´0990 900 0´0844

4 0 1000 0´0285 523 0´03060´05 403 0´04700´1 1000 0´0355 352 0´07100´25 1000 0´0420 480 0´08300´5 1000 0´0620 533 0´0971 1000 0´1030 494 0´1150

5 0 1000 0´0175 439 0´03400´05 472 0´05700´1 1000 0´0285 572 0´06300´25 1000 0´0300 350 0´08200´5 1000 0´0400 566 0´08501 1000 0´0840 357 0´1010

6 0 1000 0´0240 644 0´03100´05 392 0´04600´1 1000 0´0295 366 0´04600´25 1000 0´0420 454 0´06800´5 1000 0´0465 619 0´09051 1000 0´1080 857 0´1110

means 0 6000 0´0208 (0´0049) 3787 0´0318 (0´0032)(SD) 0´05 3744 0´0489 (0´0100)

0´1 6000 0´0285 (0´0052) 3800 0´0573 (0´0093)0´25 6000 0´0342 (0´0075) 3563 0´0708 (0´0093)0´5 6000 0´0480 (0´0083) 3662 0´0798 (0´0136)1 6000 0´0997 (0´0083) 3688 0´1077 (0´0133)

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552 A . V ral et al.

tion of B lymphocytes was obtained after 5 days ofculture w ith PWM. The ratio of binucleate B cellsto the total of binucleate, mononucleate and polynu-cleate cells was found to be 33%. Pokeweed mediumstimulates B-cell division indirectly as a consequenceof selective stimulation of T-helper cells (Clementet al. 1983, O’Donovan et al. 1995). As B lymphocytesrepresent only a small subset (3 ± 12%) of the totallymphocyte population in the peripheral blood, theB lymphocytes were sorted. Two millilitr es of bloodwas needed to prepare two cytospins containingbetween 300 and 1000 sorted binucleate B cells .When B lymphocytes are not enriched and separated

Figure 1. Mean MN number for T ( D ) and B ( E ) lymphocytes ,from T lymphocytes many slides have to be scoredexpressed as the number of MN per binucleate cell, as a

to obtain suæ cient binucleate B cells , even after function of c-ray dose for cultured lymphocytes of sixoptimal stimulation. For T lymphocytes a maximal donors. The full lines represent the results of best ® ts

through the data. The error bars are SD.yield of M N is reached after 70 h culture with PHA(Vral et al. 1994). The yield of binucleate

m Õ 2SD) allows the calculation of the upper andT lymphocytes was 47%.lower dose lim its on the dose assessment for a givenThe individual M N dose-response data togethernumber of M N. The relativ e uncertain ty on dosewith the spontaneous M N data for B andassessment, obtained by dividing the overall uncer-T lymphocytes, expressed as the number oftainty by the dose value obtained for the consideredM N/binucleate cell, are presented in tab le 1. Thenumber of M N from the mean dose response curve,numbers of spontaneous and radiation-induced M Nis shown in ® gure 2. This ® gure shows that w ithoutare signi® cantly higher at the 95% con® dence lim itsinformation of the spontaneous M N backgroundin B cells compared to T cells applying a Wilcoxonfrequencies, the detection lim it w ith the B cell M Ntest for matched paired observations, except for theassay lies around 0´08 Gy, while the conventionalhighest dose of 1 Gy for which the M N frequenciesM N assay in these conditions only allows detectionare comparable. The tab le further shows that for allof doses from 0´25 Gy. On the other hand, it is cleardonors the 0´05 Gy dose values for B cells are system -that for doses exceeding 0´3 Gy the accuracy of doseatically higher than the control values. The meanassessment w ith the conventional M N assay is higherM N dose-response data for B and T lymphocytes,than with the B cell M N assay.which are obtained by combining the data of the six

donors, are also given in tab le 1. The values inbrackets represent the standard deviations (SD) 4. D iscussionderived from the variability in M N frequencies

This paper demonstrates that low doses of c-raysamong the donors. M ean M N dose-response curves(<1 Gy) induce signi® cantly higher amounts of M Ntogether w ith the SD are given in ® gure 1. While forin B lymphocytes than in T lymphocytes after optimalT lymphocytes a linear-quadratic ® t Y=22´7+31´4

D+44´1 D2 (Y=micronucleus yield , D =dose) couldbe obtained (R =0´999), the mean M N dose-responsecurve obtained for B cells shows clearly a di å erentpattern. This curve, which is the result of an inverselinear-quadratic ® t D =0´10 Õ 8´62 Y +158´72 Y

2

(dose versus M N) (R =0´995), is characterized by aninitial steep increase for the lowest doses of 0´05 and0´1 Gy followed by a ¯ attening of the curve towardsthe higher doses. Close to 1 Gy the curves intersect.

To investigate the possible application of the B cellM N assay for biological dosimetry in radiation acci-dents, the overall uncertain ty on dose assessmentversus dose, due to the interindividual variation, wasstudied. Functional ® ts through the mean dose Figure 2. Relative uncertainty on the dose at the 95% con® d-response curves and through the dose response curves ence limits, obtained with the conventional MN assay

(- - -) and the B cell MN assay ( Ð ± ).representing the 95% con® dence lim its (m+2SD,

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553M icronucleus frequencies in cy tokinesis - b lo cked human B lymphocy tes

stimulation of both subpopulations. The highly radio- Because of this high radiosensitiv ity ofB lymphocytes the possible application of the B cellsensitive behaviour of B cells w ith respect to M N

production is in agreem ent w ith the data of WuÈ ttke M N assay was investigated for biological dosimetryof individuals after acute accidental or medicalet al. (1993). The M N frequencies found in the study

of WuÈ ttke et al. (1993) for the applied doses of 0´5 exposure to low doses of ionizing radiation.Figure 2 demonstrates that when the spontaneousand 1 Gy were higher than the values found in this

study. Their results however, were based on M N M N background frequency is not known, as is thecase in accidents, the B cell M N assay allows dosefrequencies in maximum 200 binucleate B lymph-

ocytes scored on slides of seven donors, while the detection down to 0´08 Gy. For doses higher than0´25 Gy the B cell M N assay is not suitable anymoreresults of this study are based on M N frequencies in

3500 ± 3800 binucleate B lymphocytes scored on because the relativ e uncertain ty becomes very highdue to the ¯ attening of the M N dose response curve.slides of six donors (mean of 600 binucleate

B cells/slide). The radiosensitiv ity of B and T cells As only a very small dose range is covered and thedose is by de® nition unknown in radiation accidents,regarding induction of M N and proliferation capacity

was also evaluated in the study of Holmen et al. the B cell M N assay can only be used as a supplemen-tary test to the conventional M N assay.(1994). B lymphocytes were separated and enriched

by an immunomagnetic method (Dynabeads) A second drawback of the B cell M N assay forbiological dosimetry of radiation accidents is related(HoÈ gsted et al. 1993). They found that B cells had

the lowest M N frequencies but also the lowest mitotic to its in vivo lifespan. B lymphocytes have beengrouped in two, broad populations, one rapidlyindex. However, this study lacks good methodology

since B lymphocytes were not optimally stimulated renew ing in 3 ± 4 days, and the other renew ing moreslowly (Freitas and Rocha 1993, M acLennan andas only PHA was used. From the literature it is

known that optimal stimulation of B cell division is Chan 1993, Osmond 1993). Thus about half of theB cells have a short lifespan and are susceptible toblocked in PHA treated PBL cultures by the concom-

itant increase in T-cytotoxic/supressor cells besides apoptosis induction (Philippe et al. 1997), by whichthe application of the M N assay will be restricted tothe T-helper cells (Clement et al. 1983, Sieber et al.

1985). Furthermore these authors did not use the situations where blood samples can be obtainedwithin 24 h after exposure.`cytokinesis block’ method and hence had no control

on lymphocyte kinetics. In practice, when blood is sampled within 24 h,two lymphocyte cultures, one for the conventionalThe initial steep increase up to 0´1 Gy of the B cell

M N dose-response curve, which is shown in ® gure 1, M N assay and one for the B cell M N assay can beinitiated. This implies no extra work. If after 3 daysis followed by a ¯ attening of the curve pointing to

severe damage at the level of the DNA and spindle an elevated dose is detected with the conventionalM N assay, the other culture is not needed further. Ifapparatus which interferes w ith cell division and thus

also with the formation of M N in these cells con- no increase in M N yield is seen, B cells can be sortedafter 5 days and M N can be scored. At present theretaining the largest radiation damage. A higher mitotic

radiosensitiv ity of B lymphocytes compared to is no alternative cytogenetic method which allowsdetection of doses < 0´1 Gy and which is at the sameT lymphocytes was also shown by Schwartz and

Coworkers (Schwartz and Gaulden 1980, Schwartz time practically feasib le.A lthough there are some important restrictions foret al. 1983) and WuÈ ttke et al. (1993).

The highly radiosensitive behaviour of B lym- the use of the B cell M N assay in radiation accidents,the high radiosensitiv ity of B cells (tab le 1) may bephocytes is not yet fully understood. Accord ing to

a recent communication, the radiosensitiv ity of B cells of special value for dose evaluation in patients treateddiagnostically or therapeutically w ith low dose irradi-is correlated with DNA-dependent protein kinase

(DNA-PK ) activity (Muller et al. 1997). DNA-PK ation. In medical applications, a pre-irradiation bloodsample is available and the problem of sampling timeplays a critical role in mammalian DNA double

strand break (dsb) repair (Weaver 1995, Evans et al. after irradiation does not arise.However, before the B cell M N assay can be used1996). B cells would have a variant form of the Ku

protein, resulting in a decreased ability to recru it the for in vivo dose evaluation, a more profound studywill be needed to investigate: (1) the stability ofcatalytic component of the complex, DNA-PKcs,

which leads to the absence of detectab le DNA-PK spontaneous M N frequencies in B lymphocytes;(2) the reproducibility of the results obtained withactivity in B cells (Muller et al. 1997). These results

would indicate that the high radiosensitiv ity of this assay after low dose irradiation; and (3) the in

vivo behaviour of B lymphocytes w ith respect to M NB lymphocytes w ith respect to cytogenetic damage isdue to de® cient DNA dsb repair. induction. In T lymphocytes the M N yield is compar-

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MuÈller, W.-U. and Streffer, C., 1991, Biological indicatorsable after in vitro and in vivo irradiation (Thierensfor radiation damage. I nternational J ournal of R adiatio net al. 1995, WuÈ ttke et al. 1996). For B lymphocytes noB io logy , 59, 863 ± 873.

literature data are available. MuÈller, W.-U. and Streffer, C., 1994, Micronucleus-assays.In Advance s in M utagenes is R esearch, vol. 4, edited by G. Obe(Berlin: Springer), pp. 1 ± 133.Acknowledgem ents

Muller, C., Dusseau, C., Calsou, P . and Salles, B., 1997,Negativ e regu lation of DNA-dependent protein kinaseThis work was supported by the Programme ofactivity in human normal peripheral blood lymphocytes :Scienti® c Support to the Protection of Workers inExpression of a variant form of the Ku 86 protein.

the Area of Health of the Prime M inisters Oæ ceM utatio n R esearch, 379, (Supplem ent 1), PIX.6, p. S63.

of the Belgian GovernmentÐ Federal Oæ ce for O’ Donovan, M. R., Johns, S. and Wilcox, P ., 1995, The e å ectScienti® c, Technical and Cultural A å aires of PH A stimulation on lymphocyte sub-populations in

whole blood cultures. M utagenes is , 10, 371± 374.(ST/021005).Osmond, D. G., 1993, The turnover of B-cell populations.

Immunology Today , 14, 34 ± 37.P hilippe , J., Louagie, H., Thierens, H., Vral, A.,References

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