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Environmental and Molecular Mutagenesis 13:34-43 (1989) Identification of Aneuploidy-Inducing Ag-ents Using Cytokinesis-Blocked Human Lymphocytes and an Antikinetochore Antibody David A. Eastmond and James D. Tucker Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, California The identification of agents causing aneuploidy in humans, a condition associated with carcinogenesis and birth defects, is currently limited due to the highly skilled and time-consuming nature of cytogenetic analyses. We report the development of a new simple and rapid assay to identify aneuploidy-inducing agents (aneuplaidogens). The assay involves the chemical- or radiotioninduced formation of micronuclei in cytokinesisblocked human lymphocytes and the use of an antikinetcchare antibody to determine whether the micronuclei contain centromeres- condition indicating a high potential for aneuploidy. All agents tested produced dose-related in- creases in the frequency o f micronucleated cells. The micronucleated cells induced by the known oneuploidogens-colchicine, vincristine sulfate, and diethylstilbestrol-contained kinetochare- positive micronuclei 92,87, and 76% of the time, respectively. In contrast, the micronucleated cells induced by the potent clastogens-ionizing radiation and sodium arsenite-contained kineto- chore-positive micronuclei only 3 and 19% of the time, respectively. These results indicate that this relatively simple assay can discriminate between aneuploidogens and clostogens ond may allow a more rapid identification of environmental and theropeutic agents with aneuploidy-inducing patentiol. Key words: micronucleus assay, colchicine, vincristine sulfate, diethylstilbestrol, sodium arsenite, ionizing radiation 23.k INTRODUCTION 1985; Rubin, 198.51, a growing body of molecular and cy- togenetic evidence indicates that the induction of aneu- v Aneuploidy, a condition in which the chromosome num- ploidy may play an important and possibly essential role in ber of a cell or individual differs from a multiple of the the neoplastic development of certain tumors [Yunis. 1983; haploid state, is associated with spontaneous abortions, Cavenee et al., 1983; Oshimura and Barrett, 1986; Conti et congenital malformations, mental retardation, and carcino- al., 1986; Oshimura et al., 19881. genesis [Sandberg, 1980; Hecht and Hecht, 19871. Associ- A significant number of human carcinogens, such as ben- ations between aneuploidy and neoplastic development zene, diethylstilbestrol, ethanol, and asbestos, which have have been observed in studies of humans with congenital been reported as nonmutagenic in standard mutation assays. and familial predispositions for cancer [Cavenee et al.. have been shown to interfere with chromosome segregation 1983; Evans, 198.51, as well as in studies of patients and during mitosis and to result in aneuploidy [Danford, 1985; workers with acute nonlymphocytic leukemia resulting Barrett et al., 1987; Waters et al., 19881. In spite of evi- from chemotherapy and benzene exposure [Erdogan and dence for the involvement of aneuploidy and aneuploidoAksoy, 1973; Rowley, 1983; Pedersen-Bjergaard and Phi- gens (aneuploidy-inducing agents) in the carcinogenic pro- lip. 19871. Similar results are observed in animal and cel- lular systems in which the nonrandom gain or loss of certain chromosomes is associated with chemical, radiation, viral. or spontaneous induction of tumors or neoplastic transfor- mation [Sasaki, 1982; Miller and Miller, 1983; Knuutila. 19871. Although in many tumor cells these changes appear to be Received July 5. 1988: revised and accepted September 13. 1988 Address reprint requests to Dr. David A . Eastmond, Biomedical Sciences Division. Lawrence LivermoreNationalLaboratory. P.O. Box 5507, Lirsr- a secondary effect related to rapid cell proliferation [Evans, more. CA 94550. @ 1989 Alan R. Liss, Inc. Assay for Aneuploidy-InducingAgents 35 cess, rapid assays for identifying these agents using systems relevant to humans are not well developed [Dellarco et al., 1 , ` 5 ; Galloway and Ivett, 1986; Oshimura and Barrett, ; , 51. The standard procedure for determining aneuploido- in mammalian systems involves counting individual &romosomes in metaphase plates and identifying the frequency of cells that differ from the diploid or normal number. This method is time consuming, requires highly skilled personnel, and is prone to technical artifacts resulting in artificially high rates of hypodiploidy [Brown et al., 1983; Oshimura and Barrett, 19861. In vitro assays with nonmamndian systems have been developed but have limited value uman risk estimation in that considerable phylogenetic i'erences exist between species both in the metabolism and in the mechanism and kinetics of cell division [Parry and Parry, 19871. Some investigators have proposed using micronuclei induction as an assay for aneuploidy [Pany and Parry, 1987; Oshimura and Bmett, 19861. A major limitation in this approach has been that micronuclei can arise from entire chromosomes or from chromosomal fragments. and thereface the assay fails to discriminate between aneuploidogens a d clastogens. Numerous studies to distinguish between chromosome- and fragment-containing micronuclei based upon DNA content [Heddle and Carrano, 19771, size [Yamamoto and Kikuchi, 1980; Valadaud-Banieu, 1983; Hogstedt and Karlsson, 19851. hybridization experiments [Viaggi et al., 19871, and centromeric heterochromatin [Banduhn and Obe, 19851have been attempted. Recently, a number of investigators [Brinkley et al., 1985; Frackowiak act al., 1986; Vig and Swearngin, 19861have reported using ~n antikinetochore antibody isolated from the serum of deroderma patients to demonstrate the presence of kinetochores in micronuclei. We have combined this approach with the cytokinesis-blocked lymphocyte method developed by Fenech and Morley [19851 to develop a relatively rapid assay for aneuploidy induction using human cells. The scoring of micronuclei in cytokinesis-blocked (i.e., binucleated) cells has several advantages that are invaluable in developing an assay for aneuploidy. Binucleated cells are the result & a single mitosis and are scored during interphase so ar:ifactual loss of chromosomes is not a problem. In addition, scoring is rapid and simple, requiring minimal training and experience, and staining is fast and reliable. This assay is based upon the assumption that a micronucleus containing a kinetochore (and therefore presumably the centromere and entire chromosome) in a dividing micronucleated cell will segregate with only one of the daughter cells. Cytochalasin B is used to block cytokinesis and facilitate scoring. HowWtr. had the micronucleated cell been allowed to divide, +bresulting micronucleated and nonmicronucleated daughk r cells both have a high probability of being aneuploid. We now report that the modified micronucleus test using an antikinetochore antibody has a potential application as an assay for aneuploidy-inducing agents. The method is capable of distinguishing between the aneuploidogens colchicine, vincristine sulfate, and diethylstilbestrol (DES) [Oshimura and Barrett, 19861, and the clastogens sodium arsenite W a n et al., 19821 and ionizing radiation [Heddle and Carrano, 19771. MATERIALS AND METHODS Chemicals and Media Vincristine sulfate, diethylstilbestrol, and sodium arsenite were purchased from Aldrich (Milwaukee, WI). Colchicine, polyoxyethylenesorbitan monolaurate (Tween 20). 4'-6-diamidino-2-phenylindole(DAPI), p-phenylenediamine dihydrochloride, gentamycin sulfate, and cytochalasin B were obtained from Sigma (St. Louis, MO). The antikinetochore antibody (centromere-positive control serum for the antinuclear antibody test) and the fluorescent rabbit antihuman gamma globulin were purchased from Antibodies Incorporated (Davis, CA). Minimum essential medium alpha, Dulbecco's phosphate-buffered saline (PBS), L-glutamine, and phytohemagglutinin were purchased from Gibco (Grand Island, NY). Fetal bovine serum was purchased from Hyclone (Logan, UT) and sodium heparin was purchased from Invenex (Chagrin Falls, OH). Culture Media and Conditions Blood was obtained from a healthy male volunteer by venipuncture using heparinized vacutainers and was mixed thoroughly. Lymphocytes were isolated using LeucoPREP cell separation tubes (Becton Dickinson, Lincoln Park, NJ) and were cultured in the dark for 72 hr under a 5% COz atmosphere at an initial density of 0.5 x lo6 cells/ml. The culture medium consisted of minimum essential medium alpha. 16% fetal bovine serum, gentamycin sulfate (final concentration 0.048 mg/ml), L-glutamine (final concentration 2 mM), 1% sodium heparin, and 2.36% phytohemagglutinin (M form). Radiation treatment was performed at 24 hr using a I3'Cs source at a dose rate of 33.3 radlmin. Chemical additions were performed at 24 hr and continued until 72 hr except for the colchicine and vincristine sulfate cultures in which the treatment was discontinued at 43 hr by the addition of fresh culture medium following centnfugation and washing of the cells. PBS was used as a carrier for each chemical except for diethylstilbestrol in which dimethyl sulfoxide (DMSO) was employed (final concentration 2.5 pUml). Cytochalasin B (final concentration 3 pg/ml) was prepared as described previously [Fenech and iMorley, 19851 and was added at 44 hr. At 72 hr. the cells were centrifuged directly onto slides (48 g, 5 min) using a Leif bucket technique with one or more layers of filter paper (Whatman #3; Maidstone. England) to absorb the excess culture medium. After drying briefly. the cells were fixed in 100% methanol for 15 min. Slides were either used imme- 'I 36 Eastrnond and Tucker diately for kinetochore labeling or stored desiccated under an Nz atmosphere at -20C until use. Antibody Labeling Procedure Slides were placed in PBS-0. 1% Tween for 5 min after which the excess fluid was drained. The antikinetochore antibody solution was diluted with an equal volume of PBS0.1% Tween, and 50 ~1 of this dilution was placed on each' slide. The slides were coverslipped and placed in a humidified box at 37C. After 1 hr, the coverslips were removed. the slides were rinsed twice in PBS-0. I % Tween for 2 min each, and the excess fluid was drained. A 50-p1 aliquot of fluoresceinated rabbit anti-human IgG previously diluted 1:120 with PBS-O.5% Tween was placed on slides, incubated for 1 hr. rinsed. and drained as before. Two drops of DAPI (2.5 kg/ml), in an antifade solution for fluorescent microscopy [Johnson and Nogueira Araujo, 19811, were added onto the slide using a Pasteur pipet. The light-sensitive nature of DAPI required that counterstaining be performed under subdued or yellow fluorescent lighting. The DAPI solution and remaining fluid were mixed gently with the coverslip, and the excess fluid and air bubbles were removed by blotting. The labeled slides were used immediately or stored in the dark at 4C. Using this procedure, the labeling remained adequate for scoring for several days. Scoring Procedure The fluorescein and DAPI fluorochromes excite maximally at 488 and 355 nm, and emit at 520 and 450 nm, respectively. Through the simultaneous use of phase contrast and DAPI excitation, it was possible to identify both the cell membrane and the nuclei while scoring. Scoring procedures involved visualizing cells under both phase contrast and DAPI excitation until a binucleated cell with a micronucleus was observed. The filter setting was then changed so that the fluorescein-labeled kinetochores could be observed to determine whether the micronucleus contained a kinetochore. Slides from each treatment were randomized and coded prior to scoring. Replicate cultures were established for each experiment except arsenite in which only one culture was available. The number of micronuclei at each dose level was determined by scoring 1,000 binucleated cells (i.e., 1,000 mitotic events) except at the vincristine sulfate 0.065 pM and the sodium arsenite 9 ~ I Mdoses in which 929 and 350 cells were scored, respectively. Scoring was performed using a scoring program on an Apple I1 computer developed at Lawrence Livermore National Laboratory (Livermore, CA). Published criteria for micronuclei determinations [Countryman and Heddle, 19763 were followed with the following modifications: 1) binucleated cells containing any number of micronuclei were scored: 2) fluorescence intensity per unit area of scorable micronuclei was either more or less intense that of the main nuclei; 3) only micro. nuclei that were distinctly separate from the main nuclei and located within binucleate cells with intact cytoplasmic and nuclear membranes were scored. In addition, micronucleated cells were considered scorable for kinetochores only if the kinetochores in the main nuclei were distinctly visible. A measurement of nuclear division for each treatment was determined by scoring the number of nuclei in 400 cells and calculating the nuclear division index: I NDI = {[Ml + (2 X M2) + (3 X M3) + (4 x M4)]/N} where 411-M4 represent the number of cells with one to four nuclei, respectively, and N is the total number of cells scored. Statistical Analyses Statistical analyses were performed using the Fisher exact test to compare the micronucleated cell and the kinetochorepositive micronucleated cell frequencies between control and treatment slides. Critical values were determined using a .05 probability of type I error. RESULTS The use of an antikinetochore antibody to determine the location of kinetochores within the nuclei and micronuclei of human lymphocytes is demonstrated in Figures 1 and 2. Figure la shows a binucleated lymphocyte with two micronuclei using the phase/DAPI filter setting. The same cell is shown in Figure 1b under fluorescein excitation and emission. Numerous kinetochores in the main nuclei and two kinetochores in each of the micronuclei are visible in this plane of focus. Figure 2 shows a binucleated cell with multiple micronuclei in which none of the induced micronuclei contain kinetochores. The number of kinetochores visible within a cell varies depending on the stage of the cell cycle, the microscope plane of focus, the overlap of individual spots, and the efficiency of antibody penetration. Rigorous attempts to identify and enumerate individual kinetochores resulted in the identification of 40-46 fluorescein spots per interphase nucleus in normal binucleated cells and 46 fluorescein spots in virtually all metaphase spreads from colcemid-arrested lymphocytes. Interestingly, in a high percentage of the smaller mononuclear cells (i.e., those no1 responding to PHA stimulation), only 12-15 bright fluorescein spots were observable (Fig. 2b). This may indicate a different centromeric organization in nondividing Gocells or a difference in chromatin structure interfering with epitope accessibility. Three spindle-disrupting chemical agents were tested fol their ability to induce cells with kinetochore-positive micronuclei. Colchicine and vincristine sulfate treatments induced the formarion of micronucleated cells in a dose-re. Assay for Aneuploidy-InducingAgents 37 :roand and leay if >le. lent ells . to 211s act rerol 'ng the .lei 2. roI is 11s- wo his ullei J le le, lal IUS , -es )er 'U- 11- :rlot 0- Ite !Is ii- or 0- ne- Fig. 1. A cytokinesis-blocked binucleated lymphocyte with two micronuclei photographed using (a) simultaneous phase contrast and DAPI excitation and emission wavelengths and (b)fluorescein excitation and emis.;ion wavelengths. Multiple kinetochores arc visible in this plane of focus In each of the main nuclei and in the micronuclei (arrows). Fig. 2. A cytokinesis-blocked binucleated lymphocyte with four micronuclei and n mononucleated lymphocyte photographed using (a) simultaneous phase contrast and DAPI excitation and emission wavelengths and (bt tluorescein excitation and emission wavelengths. Fluoresceinnred kinerochores are observed in the main nuclei of both the bi- and mononuclcnted cells but not in the micronuclei. ' :?d manner as illustrated in Figures 3 and 4, respectively, ited minor increases above control values, an unambiguous Table I. A statistically significant increase in total statistically significant increase both in micronucleated cells .~ronucleatedcells and kinetochore-positive micronuclea- and kinetochore-positive micronucleated cells was seen cells was' seen at doses of 0.050 FM and greater for only at the 30-pM concentration of diethylstilbestrol (Fig. colchicine, and 0.026 plM and greater for vincristine SUI- 5 . Table I). The lines representing total micronucleated cells fate. Although the lower doses of diethylstilbestrol exhib- and the scorable kinetochore-positive cells for these three i 4 ! I 38 Eastmond and Tucker TABLE 1. Distribution of Micronuclei Per Binucleated Cell ~~ ~~ No. of cells Total no. of No. of micronuclei per binuclcared cell Agenudose scored micronuclei 0 I 2 3 4 5+ - F'robabilitp Colchicine Ob 1,041 I 1 I .03 I 9 I 0 0 0 - 0.025 1.019 27 I .Go2 15 1 00 I 0.11 0.05 0.075 0. I 1.101 55 1.071 12 6 01 I C.002 1 .ooo 1.012 95 I95 --943 37 I I 5 I 3 914 49 7 ? 19 5 3 <.oOo1 <.oOoI Vincristine sulfate Ob 1.129 15 1.117 IO I 10 0 - 0.026 0.039 0.052 0.065 1.070 I , I94 1.014 929 35 1,041 25 3 0I 0 c.004 101 1.126 49 1 1 3 4 I <.oOoI 73 954 51 7 10 1 <.ooO1 I93 810 84 19 7 5 4 <.OOO1 Diethylstilbestrol Ob 5 IO 20 30 1,049 1.006 1,001 1.173 1,003 8 1.043 4 -7 0 0 0 -24 991 12 1 00 1 -15 990 8 7 I0 0 - .03 .I4 23 1,156 12 5 00 0 .03 57 957 38 5 30 0 <.ooO1 Sodium arsenite Ob 3 1 ,oOo I.Ooo 8 33 993 6 974 13 I 00 0 0 2I 0 - <.001 6 1.Ooo 81 926 65 6 21 0 c.ooo1 9 350 31 322 25 5 00 0 <.oOo1 Radiation w 1.026 I O 1,017 8 1 0 0 0 - 100 I .Ooo 90 915 80 5 00 0 <.oOoI 200 1.ooo 286 757 209 26 7 1 0 <.oOol 300 1.ooo 543 580 318 82 19 I 0 <.oOol .ooo400 1 917 409 353 182 56 8 2 <.oOOl "Probabilityvalues are based upon a one-tailed Fisher exact test comparing the number of micronucleated cells per treatment with the respective conuol. bMicrornolar. `Rads. agents are nearly parallel, indicating that their effects are primarily aneupioidogenic and are observed at both high and low doses. In addition, the majority (77-93%) of the total micronuclei induced by these chemicals were kinetochore positive. The clastogenic agents sodium arsenite and ionizing radiation resulted in a dose-related increase in micronucleated cells which was statistically significant at all doses when compared to controls (Figs. 6, 7 , Table I). The data shown indicate that these agents are primarily clastogenic in nature as expected. However, at the higher doses for both sodium arsenite and ionizing radiation, small but significant increases in kinetochore-positive micronucleated cells were observed. The distribution of micronuclei per binucleated cell for each agent and dose is shown in Table I. Twenty-twoof the 24 distributions differed significantly from the Poisson, due to an excess of cells with multiple micronuclei. This excess indicates that the number of micronuclei per cell is not random; i.e.. a cell with one micronucleus is more likely to contain additional micronuclei. Table LI shows the nuclear division data for each of these five agents. For each agent, a dose-related decrease in the nuclear division index was observed which was particularly apparent for cells that had received continuous chemical treatment. An indication of the specificity of this assay can be estimated by the percentage of kinetochore-positive micronucleated cells relative to the total number of scorable micronucleated cells. These frequencies for colchicine, vincristine sulfate. and diethylstilbestrol treatments are 92, 87, and 76%, respectively. However, only 3 and 19% of the scorable micronucleated cells induced by ionizing radiation and sodium arsenite. respectively, contained kinetochorepositive micronuclei. Occasionally. a micronucleated cell would be found in Assay for Aneuploidy-Inducing Agents 39 - TABLE II. Percent of Cells With Different Numbers of Nuclei Percent of cells with indicated number of nuclei -:y' sutment Dose I 2 3 cdchlcine O b 72 27 I 0.025 59 37 3 0.050 78 19 I 0.075 85 15 0 0. loo 88 1 1 I b'incnstine sulfate Ob 66 28 3 0.026 70 14 4 0.039 81 17 2 0.052 80 16 3 0.065 87 12 1 sh! lstilbestrol O b 54 38 4 5 68 29 1 I O 75 13 1 20 80 20 0 30 94 6 0 40 99 I 0 Sodium arsenite Ob 57 39 3 3 82 17 I 6 89 I I 0 9 98 2 0 12 99 0 I .diauon OE 57 36 6 LOO 59 37 3 200 66 30 4 300 65 32 2 400 7s 21 3 4 0 I 2 0 0 3 2 0 I 0 4 2 I 0 0 0 1 0 0 0 0 1 I 0 1 1 Nuclear division index" 1.29 I .46 I .27 1.16 1.13 1.43 I .37 1.21 I .25 1.15 1.59 1.37 1.30 1.20 I .07 1.03 1.48 1.18 1.11 I .02 1.01 1.52 1.47 1.39 I .40 I .28 +"Nuclear division index = [MI (2 X M2) + (3 X M3) + ( 4 X M4)]/N where M1-M4 are the number of cells with 1-4 nuclei, and N is the total number of cells scored. bMicrornolar. - 'Rads. /e ..hich the kinetochore labeling was inadequate for scoring. pal advantages are that it is a relatively rapid and simple The frequency of scorable micronucleated cells in the lym- assay that can utilize human cells, and can also be applied phocyte cultures varied by experiment, ranging from 85% to established cell lines. Since it does not utilize metaphase for sodium arsenite-treated cells to 98% for radiation- spreads, but rather relies upon cells with intact cytoplasmic ,t treated cells. membranes, technical artifacts due to the loss of chromo- 0 Of 4,196 binucleated cells scored in control cultures somes that are common with standard cytogenetic tech- (pooled from all experiments except diethylstilbestrol in niques are not a problem. Furthermore, in contrast with e which DMSO was used as a carrier), 38 were micronucle- other methods that determine whether a micronucleus con- .e xed (0.9%). Of these, 12 contained at least one kineto- tains an entire chromosome or a fragment [Heddle and Car- Y lore-positive micronucleus for a 0.0029 frequency of po- rano, 1977; Yamamoto and Kikuchi. 1980; Valadaud-Bar11 tentially aneuploid cells. Ninety-two percent (35138) of the rieu. 1983; Banduhn and Obe, 1985; Hogstedt and control micronucleated cells were considered scorable on Karlsson, 1985; Viaggi et al., 19871, the use of the kine- the basis of kinetochore staining. These limited results from tochore antibody allows the rapid determination of the com- 1- a single donor indicate that 34% (12/35) of the scorable position of micronuclei without additional procedures (e.g., micronucleated cells in the control cultures contained a kine- size measurements. autoradiography, DNA quantification). tochore. The results presented in this paper indicate that the assay exhibits considerable specificity in distinguishing between e PISCUSSION n aneuploidogenic and clastogenic agents. Each of the agents tested here resulted in dose-related increases in the forma- The use of a modified micronucleus assay using an anti- tion of micronucleated cells with the possible exception of tiinetochore antibody appears to have considerable promise diethylstilbestrol in which a major increase in micronucle- 1 as an assay to identify aneuploidogenic agents. The princi- ated cells was observed only at the highest dose. However, ti i , I" .. . 40 Eostmond and Tucker ?... d 60 U n 50 w+w< 40 vi 30 3z 0 X 20 i! I 10 0 0 0.02 0.04 0.06 0.08 0.1 50 45 40 35 30 25 20 i n VI I 0 C S '2 '6 29 24 2 5 32 CONCENTRATION (uM) Fig. 3. Induction of micronucleated lymphocytes by various doses of colchicine: (W) total number of binucleated cells with micronuclei: ( + ) number of binucleared cells containing one or mom kinetochore-positive micronuclei: ( 0 )number of binucleated cells containing no kinerochorepositive micronuclei. The difference between the total number of micronucleated cells and the sum of the kinetochore-positive and kinetochorenegative cells represents the number of unscorable micronucleated cells at each dose. The 0 dose has been slightly offset for clarity. 3c 20 -00 a w (I -v! 80 wd U 70 a wwc-<1 U 1 0au I CCF,CEI.ITRPT'OI'! (UP,!) Fig. 5. Induction of nucronucleated lymphocytes by vanous doses of diethylstilbesml. The key to the symbols is given in the legend to Figure 3. ,90 0 0 80 0, E 70 a 60 v-11 w U 50 0 k 40 3U 30 3z 0a 20 2 H 10 0 0 2 4 6 8 10 0 0.02 0.04 0.06 CONCENTRATION (uM) CONCENTRATION (uM ) Fig. 6. Induction of micronucleated lymphocytes by various doses of sodium arsenite. The key to the symbols is given in the legend to Figure 3. Fig. 4. Induction of micronucleated lymphocytes by various doses of vincristine sulfate. The key to the symbols is given in the legend to Figure 3. the frequencies of cells with one or more kinetochore-positive micronuclei induced by the aneuploidogens were substantially higher (76-92%)than the frequencies induced by the clastogens (3-19%).Based upon these results, the relative aneuploidogenic potential of a particular agent may be identifiable by the induced frequency of kinetochore-positive micronucleated cells. Although the proportion of kinetochore-positive micronucleated cells was much higher for aneuploidogens than clastogens, both ionizing radiation and sodium arsenite Assay for Aneuploidy-Inducing Agents 41 micronucleus segregates with the daughter nucleus from which it was derived, the resulting daughter cells will be ':e /,Y /' diploid, although one daughter cell will contain a micronu- 11 cleus. Whether this type of cell will function normally or whether asynchronous DNA replication will occur as has I been described previously [Obe and Beek, 19821 is un- 1 known. If the micronucleus segregates with the daughter nucleus from which it did not originate, then both daughter cells will be aneuploid. The actual frequency of aneuploidy 1 induction is unknown and will be largely dependent upon ,,'. the nature and frequency of micronucleus segregation. In spite of these limitations, the modified micronucleus assay 1 offers potential for identifying a wide variety of aneuploi- dogens. 0 !00 200 300 300 One characteristic of the chemicals tested in this assay was that the observed dose-related increases in micronuclei RADS frequencies were accompanied by decreases in cell growth Fig. 7. Induction of micronucleated lymphocytes by various doses of and nuclear division. This effect can be readily Seen in ionizing radiation ('37cs)T.he key to the symbols is given in the legend to Tables I and 11. F~~ chemical and, to a lesser extent, Figure 3. radiation, a considerable reduction in the nuclear division index and the percentage of binucleated cells was observed as micronuclei were induced by these agents. The extreme 1. ..:merits resulted in a significant increase in kinetochore- case of this phenomenon was observed in preliminary ex- positive micronuclei at the higher dose levels. These results. periments with colchicine and vincristine sulfate in which suggest that these agents, although principally clastogenic the cells received a continuous treatment with these agents. [Heddle and Carrano, 1977; Wan et al., 19821, may exhibit Numerous cells were observed which apparently had been some weak aneuploidogenic properties. In addition. the re- unable to divide resulting in the formation of mononuclear sults observed with diethylstilbestrol suggest that weak cells with multiple micronuclei. For these two chemicals, clastogenicity may accompany diethylstilbestrol genotoxic- the use of 19-hr pulse treatments (24-43 hr following cul- ity, an observation that agrees with previous observations ture setup) enabled the partial release of cells from this [Howard et al., 19851. The identification of a weak aneu- block and allowed the formation of binucleate cells with ,dogenic or clastogenic potential for these agents may be micronuclei. due to the relatively large sample sizes (1,000 cells per The use of the antikinetochoreantibody to determine aneu- treatment) employed in these studies. This is considerably ploidy induction has a potential utility for a variety of:dis- more than those used in standard cytogenetic analyses (typ- ciplines including genetic toxicology, carcinogenesis, and ically, no more than 200 cells per treatment). teratology. Preliminary results from our laboratory have The major limitation of this assay is that only aneuploi- shown that this approach can be readily adapted for use in dogens that produce lagging chromosomes (and therefore vitro with Chinese hamster ovary cells and mouse lympho- micronuclei) can be identified. It is conceivable that certain cytes or in vivo with mouse erythrocytes. The combination classes of aneuploidogens such as those causing nondisjunc- of this antikinetochore approach with biomonitoring studies hiwould not be detected by the antikinetochore approach. using cytokinesis-blocked lymphocytes offers an opportu- hwever, the results presented here and elsewhere indicate nity to observe aneuploidy induction in the cells of workers that micronuclei induction is a characteristic of many dif- or patients exposed to genotoxicor chemotherapeutic agents. ferent classes of aneuploidogens [Heddle et al., 1983; Parry These initial studies have shown that the use of an anti- and Pany, 19871. In addition, since the induced micronu- kinetochore antibody combined with the chemical- and ra- clei have only a probability of segregating during cytoki- diation-induced formation of micronuclei in cytokinesis- nesis with the daughter cell from which they were derived, blocked human lymphocytes is capable of distinguishing the assay provides only an indirect measure of aneuploidy between the aneuploidy-inducingand clastogenic potentials md aneuploidy induction. This principle is illustrated in of a variety of agents. As such, this assay represents a novel F+re 8. Cells are scored at a cytokinesis-blocked stage, * k 4 n is represented in the middle of the figure. If the cells '.\<re allowed to divide normally, the chromosome-contaming micronucleus would segregate with only one of the approach to identify the aneuploidy-inducing potential of chemicals and other agents. Additional studies employing a wide variety of aneuploidogenic and clasrogenic agenrs will be required to properly validate this method as an assay for daughter cells, resulting in several possible outcomes. If the aneuploidy. 42 Eastmond and Tucker DIPLOID i BLOCKED HYPODIPLOID HYPERDIPLOID CELL DAUGHTER CELLS WITH OR WITHOUT FRAGMENT M ETAPHASE ANAPHASE TELOPHASE OR CYTOKINESIS BLOCKED Fig. 8. Possible series of mitotic events resulting in micronucleated and aneuploid cells. Treatment of a normal diploid cell (a) with an aneuploidogen causes an interference with the mitotic spindle apparatus (b). which in turn results in a lagging chromosome during anaphase (e). During late telophase (d), this lagging chromosome forms a micronucleus. which may segregate with one of the daughter nuclei. The segregation of the micronucleus with the daughter nucleus from which it was derived results in two diploid daughter cells (e, f). The "diploid" cell (e) may not replicate normally (see text). The segregation of the micronucleus with the nucleus ,Ifrom which it did not originate results in two aneuploid daughter cells (9, h). Treatment of a normal diploid cell (a) with a clastopn results in a chromosome fragment (i). which lags during the anaphase ti) and results in , a micronucleated cell during late telophase (k). The segregation of this micronucleus with either daughter nucleus results in daughter cells either containing or lacking the fragment. The presence of a kinetochore in the micronucleus of a cytokinesis-blocked cell (d) indicates a cell with a fuite probability for aneuploidy assuming the cell had been allowed to divide normally. ACKNOWLEDGMENTS This research was conducted in part by an appointment to the Alexander Hollaender Distinguished Postdoctoral Fellowship Program supported by the U.S. Department of Energy, Office of Health and Environmental Research, and administered by Oak Ridge Associated Universities (D.A.E.) 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