Mutation Research Letters

ELSEVIER

Mutation Research 348 (1995) 153-162

Advantages and limitations of using fluorescence in situ hybridization for the detection of aneuploidy in interphase human cells
David A. Eastmond *, Maik Schuler, D.S. Rupa
Enz'ironmental Toxicology Graduate Program Department of Entomology Unicersity of California Rit'erside, CA 92521, USA
Received 15 August 1995; revised 29 September 1995: accepted 27 September 1995

Abstract

Fluorescence in situ hybridization with chromosome-specific DNA probes is being increasingly utilized for the detection of chromosome aberrations induced in vitro and in vivo by chemical and physical agents. Although potentially a powerful technique, FISH studies for aneuploidy can be heavily influenced by cellular phcnomcna and hybridization artifacts which make the performancc and interpretation of the results difficult. As a consequence, frequently hyperdiploid frequencies are reported in the literature which arc substantially higher than one would expect based upon frequencies seen in conventional mctaphase analyses. In this article, a number of the potential pitfalls that we havc encountered while performing FISH analyses for aneuploidy arc discussed and their potential impact on the observed hybridization frcqucncics is described. After considering these factors, the frequencies of lymphocyte nuclei containing 3 and 4 chromosome copies are compared between mctaphase values obtained from published human population studies and interphasc values obtained from similar studies using FISH. It is concluded that by using caution in the evaluation of slides, interphase studies using FISH to detect hyperdiploidy and polyploidy can provide estimates of numerical alterations which closely reflect those seen during mctaphase analysis using either FISH or conventional approaches. However, duc to the inability of interphase analysis to distinguish hyperdiploidy from polyploidy as well as other potential problems, frcqucncics of aneuploid nuclei obtained using single label FISH should only bc considered approximations of absolutc frcquencics. For additional accuracy, multi-color FISH with two or more different probes should bc performed.

Keywords: Fluorescence in situ hybridization; Aneuploidy; Intcrphase cytogenctics

I. Introduction

Fluorescence in situ hybridization (FISH), a promising molecular cytogenetic technique, is be-

Corresponding author. Tel.: (909) 787-4497; Fax (909) 787-3087; email, EASTMOND(q~UCRAC1.U(?R.EDU.

ing increasingly utilized in genotoxicity studies to detect chromosome aberrations induced in vitro and in vivo by chemical and physical agents. Over the past several years, a variety of probes and labeling strategies have been employed to detect or characterize chromosomal alterations. For example, probes labeling the centromeric region of

0165-7992/95/$09.50 1995 Elsevier Science B.V. All rights reserved

SSDI 01 65-7992(95 )0(1059-3

154

D.A. Eastmond et al./Mutation Research 348 (1995) 153-162

all or nearly all human or mouse chromosomes have been used to characterize the origin of micronuclei occurring spontaneously or following exposure to various chemical agents (Becker et al., 1990; Miller et al., 1991; Chen et al, 1994; Titenko-Holland et al., 1994). Other probes which label the length of a specific chromosome, commonly known as whole chromosome probes or painting probes, have been shown to be valuable in detecting and quantifying structural chromosomal aberrations, particularly translocations and dicentrics occurring between nonhomologous chromosomes in metaphase preparations (Natarajan et al., 1992; "Fucker et al., 1993). Additionally, probes which target centromeric or pcricentromeric satellite sequences have been used to detect aneuploidy, i.e. numerical chromosome changcs, and more recently chromosomal breakage and exchanges, induced by chemical and physical agents in metaphase and in interphase ceils (Eastmond and Pinkel, 1990; De Sario et al., 199(I; Rupa et al., 1994). For a number of years, we have used centromeric DNA probes to detect aneuploidy and other chromosome alterations in interphase cells. The objective of this article is to discuss some of the strengths and limitations of interphase FISH analysis to aid researchers as they begin performing FISH studies and to assist others involved in the interpretation of FISH results. Although most of these comments are applicable to many types of FISH studies, the primary focus of this article will be on the use of chromosome-specific centromeric probes to detect aneuploidy in somatic ceils. The use of FISH for cytogenetic analysis is gaining increasing acceptance primarily due to its ease of use and the speed of analysis. Thousands of cells can be evaluated in a relatively short period of time increasing sample sizes and enhancing statistical power. This increases assay sensitivity and allows smaller increases in induced alterations to be detected. An additional and possibly more important reason is that interphase analysis can be performed on rarely dividing or nondividing cells allowing cytogenetic information to be obtained from tissues which previously have not been amenable to cytogenetic analysis (Sandberg et al., 1988; Teyssler, 1988). Already,

interphase analysis has provided valuable information on aneuploidy in human sperm, unstimulated lymphocytes, buccal mucosal cells and urothelial cells (Eastmond and Pinkel, 1990; Moore et al., 1993; Bischoff et al., 1994).

2. Technical limitations and potential pitfalls in FISH analysis

Along with the advantages of being able to analyze interphase cells comes one clear disadvantage - the inability to directly see the chromosome and chromosomal regions targeted by the probe. Consequently, only alterations affecting the labeled region can be detected. In addition, hyperdipioid frequencies for individual chromosomes are frequently reported in the literature which are substantially higher than one would expect based upon the results of conventional metaphase analyses (Kibbelaar et al., 1993; Le Beau, 1993; Herrington et al., 1995). This indicates that certain types of hybridization patterns or technical problems may result in euploid cells being classified as aneuploid and to a lessor extent aneuploid cells being characterized as euploid. Typically interphase FISH studies utilize one or two probes simultaneously to quantify variations in chromosome number, which means only alterations affecting a limited portion of the genome can be detected. Although this results in decreased sensitivity, the greatly increased speed of interphase analysis minimizes this problem. In studies of somatic cells, nuclei exhibiting 3 or more hybridization regions are classified as hyperdiploid whereas those with 0 or 1 hybridization region are called hypodipioid. We use the word 'hyperdiploid' literally, meaning greater than diploid so it includes cells which are trisomic and tetrasomic for the labeled chromosome as well as polypioid cells (4N, 8N). The inclusion of polyploid ceils is important when comparing interphase with metaphase results. Cells classified as hypodiploid include cells which have lost 1 or more chromosomes as well as cells which lack one signal due to hybridization artifacts or nuclear organization in interphase cells. Examples

D.A. Eastmond et al. /Mutation Research 348 (1995) 153-162

155

of problems that may result in an inaccurate interpretation of FISH results are summarized in Table 1. Some common fcatures of thcsc patterns or problems will be described in more detail below. Occasionally following FISH, cells which contain 3 hybridization signals are observed in control or untreated cultures. When two signals appear as a doublet [see Fig. la; having a very similar appearance with only a small space separating the adjacent probes; both being smaller than the third signal which is more normal in size for nuclei of this diameter], the doublet signals are scored as one hybridization region and are thought possibly to be either cells in the G 2 stage of the cell cycle or the result of premature chromatid separation during fixation or drying. [The appearance of only one doublet is not surprising as it would be highly unlikely that both chromosomes would land on the slide in an orientation that would permit separation between the hybridization regions on both pairs of sister chromatids to be seen.] In addition, alpha satellite DNA which spans the primary constriction which may also result in these doublet-type signals (Herrington et al., 1995). Other cells containing 3 hybridization regions are occasionally seen in which a thin thread of DNA connects two of the hybridization regions. These connected hybridiza-

tion regions can be widely separated within the nucleus (see Fig. lb). This pattern may represent cells in which the target region is decondensed during DNA replication or repair resulting in hybridization regions that have migrated away from each other during hypotonic treatment, fixation or drying on the slide. In our laboratory, we score with a conservative bias so that nuclei containing the above-described hybridization patterns are scored as euploid. The appearance of 3 fluorescent spots can also reflect chromosome breakage or exchanges. In scoring cells following treatment with clastogenic agents, we saw a high frequency of nuclei containing 3 hybridization regions which are almost certainly the result of breakage within the chromosome region targeted by the DNA probe rather than the presence of three individual chromosomes. This problem is pronounced when working with classical satellite probes which target the centromeric heterochromatin of human chromosomes 1, 9, and 16, regions which are prone to breakage by chemical and physical agents (Brogger, 1977; Rupa et al., 1995). This can complicate the interpretation of studies on aneuploidy in treated and untreated cells, particularly when testing clastogcnic agents (Eastmond and Pinkel, 1990; Eastmond et al., 1994). An analogous situation is seen when using probes that

Table 1 Cellular and technical factors which may influence the interpretation of FISit results

tlybridization patterns that might be incorrectly interpreted as hyperdiploid Breakage or exchanges affecting the chromosomal region targeted by the probe Cells in the G 2 stage of the cell cycle Premature chromatid separation Satellite DNA spanning the primary constriction (e.g. due to pericentric inversion) Cells in S phase with decondensed DNA connecting condensed satellite regions Cells undergoing DNA repair with decondensed DNA connecting condensed satellite regions Nonspecific binding of probe or cross hybridization with satellite regions on other chromosomes Fluorescent debris, particularly combined with small hybridization signals ttybridization patterns that might be incorrectly interpreted as hypodiploid Strong counterstain that obscures small or weak hybridization signals Inefficient filter for detecting fluorochrome of interest Weakening or loss of signals due to high stringency washes Poor hybridization due to poor quality of probe or inefficient probe penetration into nucleus Overlapping of regions hybridizing to probe Nuclei from donors with highly polymorphic satellite regions Nuclear organization in unstimulated and stimulated interphase cells

156

D.A. Ea.$tmond et al. / Mutation Research 348 (1995) 153-162

target the breakage-prone mouse pcricentric heterochromatin (Chen et al., 1994). To distinguish breakage from numerical changes, we have recently developed a multicolor FISH approach t o detect nuclei with multiple hybridization regions formed from breakage

,,

i~

/'

I,

T
a

'

.ZZ . - _ ~ . ~

- "3

Fig. 2. ttybridization strategy using two adjacent or tandem D N A probes to detect chromosome breakage and hyperdiploidy in metaphase and interphase ceils. [From Rupa et al. (1995) Cancer Res. 55, 640-645, reprinted with permission of the American Association for Cancer Research.]

Fig. 1. lnterphase h u m a n lymphocytes following fluorescence in situ hybridization with a classical satellite probe to chromosome 9 and staining with propidium iodide, a) In the larger nucleus, three hybridization regions are visible: two of these are adjacent and smaller in size illustrating the doublet pattern described in the text. In the smaller nucleus, only one hybridization signal is visible. Based on the nucleus and hybridization sizes, this signal is likely composed of the superimposed signals of two chromosomes, b) A nucleus containing 3 hybridization regions with a thin hybridized bridge connecting two of the hybridization regions. Connected hybridization regions such as this are scored as only one copy of the labeled chromosome.

(Eastmond et al., 1994; Rupa et al., 1995). The basis of this new FISH assay is illustrated in Fig. 2. This approach also allows the cytogeneticist to more confidently and accurately classify ceils as ancuploid. In this application, multicolor FISH is used to label two adjaccnt (or tandem) regions on chromosome 1, 9 or 16. The pericentric heterochromatic region which is large and prone to breakage by chemical and physical agents is labeled by a Texas red "~', Rhodamine or Cy-3 classical satellite probe. An adjacent centromeric region which is somewhat smaller and much less prone to breakage is stained with a green fluorescein-labeled alpha satellite probe. The presence of an interphase nucleus containing three alpha satellite probes adjacent to three classical satellite probes indicates a nucleus which has 3 copies of the chromosome of interest. However if a similar interphase nucleus contains only two alpha satellite probes adjacent to two of the three classical satellite probes, this indicates that breakage has occurred within the chromosomal region targeted by one of the classical satellite

D.A. Eastmond et al. /Mutation Research 348 (1995) 153-162

157

probes. Alternatively, a wide separation between the regions labeled by the alpha and classical satellite probes can also indicate that chromosomal breakage has occurred. Cellular phenomena or hybridization artifacts can also influence the frequency of nuclei which contain 0 or 1 hybridization region and result in elevated frequencies of diploid cells being classified as hypodiploid. For example, if two labeled chromosomes lie immediately adjacent or on top of one another, the hybridization signals will of_ ten be superimposed and appear as one signal (Fig. la). The occurrence of this event appears to be highly influenced by the size of the nucleus and the region targeted by the probe, by nuclear organization which differs for example, between stimulated and unstimulated lymphocytcs and possibly by the use of hypotonic treatments to swell the nuclei (Schmid et al., 1983; Eastmond and Pinkel, 1990; Fcrguson and Ward, 1992; Lcwis et al., 1993). Ideally, one would like to use the

smallest probe possible to minimize this problem. However with probes to small regions, scoring becomes more difficult as the visibility of hybridization signals in interphase cells tends to decrease and the signals bccomc harder to distinguish from fluorescent artifacts. In addition, ceils may bc incorrectly identified as hypodiploid duc to sub-optimal hybridization or microscope detection conditions. The use of a nuclear countcrstain at an excessive concentration can obscure small hybridization signals as can thc use of inefficient microscopic filters and washing slides at excessively high stringency. For example, the cffccts of different slide preparation conditions on untreated and diethylstilbestrol-treated cells is shown in Table 2. In this example, the observed hybridization frequencies have been significantly influenced by the probe used, the stringency of the wash, the strength of the counterstain and the chemical treatment. Furthermore, penetration problems due to residual cell membranes or high

Table 2 Influence of hybridization and staining conditions on the frequencies of hypo- and hyperdiploidy in untrea and diethylstilbestrol (DES)-treated cultured h u m a n lymphocytes determined using fluorescence in situ hybridization with an alpha and a classical satellite probe for chromosome 1 Compound DNA probe Washing conditions PI Conc. " N u m b e r of hybridization (p.g/ml) signals/1000 cells
0 1 2 3 4

Hyperdiploid Description (%)

0.1% D M S O alpha I

60% f o r m a m i d e / 0.2 2XSSC; 45C classical 1 50% f o r m a m i d e / 0.2 2XSSC: 45C 2

60% f o r m a m i d e / 0.2 2XSSC; 45C 2

(I 6 9 21 28

57 92 133 153 155

940 899 855 822 814

2 3 2 3 3

1 0 1 1 0

0.3 0.3 0.3 (1.4 0.3

Optimal hybridization conditions Optimal hybridization conditions Excessive counterstain conccntration Excessively stringent washing conditions Excessive counterstain and and conditions Optimal hybridization conditions Optimal hybridization conditions Excessive counterstain concentration Excessively stringent washing conditions Excessive counterstain and washing conditions

DES 20/.tM

60~: f o r m a m i d e / 0.2 2XSSC; 45C classical 1 5(1% f o r m a m i d e / (}.2 2XSSC; 45C 2 60% f o r m a m i d e / 0.2 2XSSC; 45(? 2

alpha 1

63 76 115 134 143

848 837 799 776 794

15 35 31 31 12

72 51) 43 44 19

8.7 8.5 7.4 7.5 3.1

2 12 15 32

a PI, propidium iodide.

158

D.A. Eastmond et al. / Mutation Research 348 (1995) 153-162

nuclear compaction can also result in weak hybridization signals which can result in elevated frequencies of ceils evaluated as hypodiploid. Sub-optimal hybridization conditions can also result in hyperdiploid cells being scored as diploid. As illustrated for the DES-treated ceils in Table 2 using the classical satellite probe for chromosome 1, the frequency of hyperdiploid nuclei declined by greater than 50% (from 8.5% to 3.1%) under conditions of excessive counterstain and elevated stringency washing. It is interesting to note that a 50% decrease from, for example, 0.4% to 0.2% would most likely not be noticed in scoring 1000 to 20(,ud untreated ceils. Furthermore, overlapping of hybridization regions, probably results in nuclei containing 3 or 4 hybridization regions being scored as containing 2 and 3 hybridization regions, respectively. This can be seen by comparing the hyperdiploid frequencies in Table 2 obtained for the alpha satellite probe and the larger classical satellite probe which exhibits interphase associations (Schmid et al., 1983; Fcrguson and Ward, 1992). In addition following nondisjunction (or endoreduplication), it is likely that the centromeric regions of sister chromatids which have failed to disjoin will be located in close physical proximity in the subsequent interphase cell. As a result, it is possible that tetraploid or endoreduplicated ceils will frequently exhibit 2 or 3 larger hybridization regions rather than an expected 4 signals.

3. Use of FISH for studies of aneuploidy

As a result of these potential limitations, we believe that caution should be used in scoring and in the interpretation of the results of FISH analyses. Based on our experience as well as the data presented in Table 2, particular caution should be exercised in interpreting FISH studies when the combined frequencies of nuclei with 0 and 1 hybridization signals in untreated cells exceed 15%. However, in most situations with well designed experiments, a comparison of the treated cells or exposed humans or animals with appropriate controls provides substantial evidence that the agent induces alterations in chromosome

number. Using this approach, centromeric DNA probes have been shown to be a valuable tool for the identification of aneuploidy in cells exposed to chemical and physical agents in vitro as well as for chemically exposed human populations. In vitro studies have demonstrated that hybridization strategies can effectively detect aneuploidy induced by a variety of agents such as colchicine, vincristinc sulfate, diethylstilbestrol, chloral hydrate, Benomyl, griseofulvin, hydroquinone, 1,2,4-benzenetriol, vanadium pentoxide and sodium arsenite (Raimondi et al., 1989; Eastmond and Pinkel, 1990; De Sario et al., 1990; Vagnarelli et ai., 1990; Zhang et al., 1994; Eastmond et al., 1994; Ramirez et al., 1995). In addition, this approach is starting to be applied to cells obtained from human populations. Although many of the studies to date have only been recently published as abstracts or are not yet published, interphase studies using FISH with centromeric probes have detected elevated frequencies of chromosomal alterations in cultured lymphocytes obtained from pesticide-exposed workers (Rupa et al., 1995), cigarette smokers (Eastmond, Rupa, Hasegawa and Thompson, unpublished results) and benzene-exposed workers (Smith et al., 1995) as well as in the buccal mucosal cells of betel quid chewers (Rupa and Eastmond, unpublished results) and the sperm of patients receiving chemotherapy (Robbins et al., 1994). A second potential advantage of FISH analyses is to provide estimates of aneuploidy occurring in various nondividing cells. This will allow the frequencies of aneuploidy to be determined for cell types which do not divide readily in culture. Additionally, this will provide estimates of aneuploidy that are not influenced by cell culture conditions or mitogenic stimulation, and may therefore be more representative of the frequency of aneuploidy in vivo.

4. Comparison of metaphase analysis

FISH

and

conventional

Estimates of aneuploidy in human peripheral blood lymphocytes vary widely being influenced

D.A. Eastmond et al./Mutation Research 348 (19951 153-162

159

by the age a n d sex of the d o n o r (Fitzgerald a n d M c E w a n , 1977; Galloway a n d Buckton, 1978), the scoring criteria (Verschaeve et al., 1978) a n d possibly the length of cell culture as well as the density of m e t a p h a s e s on the slide. In spite of these variables, a c o m p a r i s o n of the a n e u p l o i d y f r e q u e n c i e s o b t a i n e d using c o n v e n t i o n a l m e t h o d s with those f o u n d using F I S H t e c h n i q u e s can provide an estimate of the accuracy of F I S H for i n t e r p h a s e analysis of c u l t u r e d lymphocytes a n d provide insights of the value of F I S H for o t h e r cell types. Since hypodiploidy is difficult to estimate with accuracy by both c o n v e n t i o n a l a n d F I S H a p p r o a c h e s (due to c h r o m o s o m e loss during m e t a p h a s e p r e p a r a t i o n s or the overlap and fusion of hybridization signals in i n t e r p h a s e cells), this c o m p a r i s o n will focus o n the frequencies of hyperdiploidy a n d polyploidy observed using the two types of analyses. For the m e t a p h a s e analyses, 17 separate studies were located which cont a i n e d a n e u p l o i d y data for 4 3 - 5 6 hr lymphocyte cultures. A s u m m a r y of these e x p e r i m e n t a l results is listed in T a b l e 3. Sixteen studies had

useful values for hyperdiploidy and 7 for polyploidy. F r e q u e n c i e s of hyperdiploidy r a n g e d from 0 to 4.17% with a m e a n of 0.73% ( s t a n d a r d deviation of 1.1%). T h e m e a n for the polyploidy f r e q u e n cies was 0.17% ( s t a n d a r d deviation of 0.19%; range 0 - 0 . 5 6 % ) . In comparison, we have conducted 2 p o p u l a t i o n studies using F I S H with centromeric probes on i n t e r p h a s e lymphocytes stimulated with P H A and c u l t u r e d for 48 hr. Using the t a n d e m probes for c h r o m o s o m e 1, hybridization frequencies were d e t e r m i n e d for 19 individuals serving as controls for a study of pesticide exposure ( R u p a ct al., 1995). The average frequency of nuclei c o n t a i n i n g 3 and 4 copies of chromosome 1 was 0.17% ( s t a n d a r d deviation 0.09%; range 0 - 0 . 3 % ) . [Note: This includes both true hyperdiploid and polyploid nuclei]. In a second u n p u b l i s h e d study, FISH with a single classical satellite probe for c h r o m o s o m e 1 was p e r f o r m e d on 57 n o n s m o k i n g males and females who comprised the controls for a study of cigarette smoking. T h e average frequency of nuclei c o n t a i n i n g 3

Table 3 Summary of aneuploidy and polyploidy data from metaphasc analysis of 43-56 h lymphocyte cultures reported in the literature Gender No Age Cells/person Total (approximate) no. of cells Termination Hypodiploid Hyperdiploid Polyploid Relerence time (%) (%.) (%) 4.8 6.1 5.1 4.2 1.4 15.5 7.1 NR 11.8 2.5 NR NR 39.1 6.6 ~ NR NR 13.0 I).94 0h I).59 I).58 0.1 4.17 1.85 0.611 11.13 11.1 NR 0.11 1.85 0.15 b 0 0.2 0.33 0.12 NR NR NR 0 NR I).56 NR NR 11.2 I).09 0.16 f NR I).113 NR NR NR Hirai, 1970 Linlecki al., 1971 et Fitzgerald and McEwan, 1977 Galloway Buckton, 1978 c and Popescu et al., 1979 Verschaeve al., 1978 et Verschaeve al., 1979 'j et Tonomura et al. 1983 Brown et al., 1983 Ding et al., 1983 Clare et al., 1984 Galloway al., 1986 et Monsalve Chlappe, 1987 and Benderet a[., 1989 Yardley-Jones et al., 1990 Barquinero et al. 1993 Richard et al. 1993

F NR ~ M+ F M+ F M M M M+ F M M+ F M M+ F M+ F M+F M M+ F M+F

17 19-74 100 16 NR 96 ~ 67 18-87 100 166 0-89 50 10 34 100 12 40.5 50 20 20-49 10J0 119 0-70 I(X)0~ 280 18-46 NR 20 NR 49 11 NR 200 3(14 NR 100 14 31.6 100 493 1-84 21~1 29 NR 86 10 21-40 200 8 0-77 120

1700 43-48 1534 51 6595 48 6401 48 1000 52 600 48 1950 48 107792 50 31773 48 976 54 2200 48 182911 48-51 1400 48 108950 48-56 2481 50 2008 48 998 48

a Not reported. ~' Contained 47 chromosomes only. c Data separating the "early" 2-day cultures from the "late' 3-day cultures was provided by Sheila Galloway. d Includes values from smoking controls, e 500 cells were scored for the newborn samples, f Sheila Galloway, personal communication; based on a subset of 2538 cells, g Contained 45 chromosomes only.

160

D.A. Eastmond et al. /Mutation Research 348 (1995) 153-162

and 4 hybridization signals was 0.34% (standard deviation 0.24%; range 0-1.1%). Since this study used only a single classical satellite probe, hyperdiploid nuclei cannot be distinguished from similar appearing nuclei resulting from chromosome brcakage. However based on the proportions of breaks vs. numerical changes seen in the controls of the pesticide study, the proportion of these nuclei representing hyperdiploidy can be estimated as ~ 44%. This results in an estimated hyperdiploid frequency of 0.15% which is similar to that seen in the pesticide study. Based on these two FISH studies, the average frequency of nuclei containing 3 or 4 copies of chromosome 1 in 48 hr cultures would then be approximately 0.16%. If one assumes that only one chromosome at a time is inw)lved in hyperdiploidy and that all chromosomes are equally likely to be involved in hyperdiploidy, then the frequency of nuclei containing 3 or 4 copies of a specific chromosome for the studies listed in Table 3 can be estimated as 0.20% {0.17% (polyploidy frequency) + 0.032% [the total hyperdiploid frequency (0.73%) divided by the 23 chromosome complements in humans]}. If the high polyploid frequency reported by Verschaevc ct al. (1979) is omitted, the expected frequency becomes 0.131%. Given the nature of the estimates - variability in the different experiments, different study populations, questionable assumptions, limited number of studies, etc., the estimates provided from the FISH analyses and conventional metaphases arc remarkably similar. In agreement with this, direct comparisons of hyperdiploid (and breakage) frequencies for specific chromosomes in our laboratory and others (Poddighe ct al., 1991; Hasegawa et al., 1995) have generally yielded similar frequencies when the results of metaphase and interphase cells have been compared. When differences between interphase and metaphase FISH studies have been seen in our laboratory, they typically have been those in which higher numbers of nuclei with 3 and 4 chromosome copies were seen in the metaphase preparations, possibly reflecting the conservatism in our scoring or difficulties in distinguishing 2 adjacent diploid metaphases from a polyploid mctaphasc. One point that should be

emphasized from the above calculations is that nuclei exhibiting 3 or 4 hybridization regions in FISH studies of untreated cells almost certainly represent polyploid cells rather than nuclei hyperdiploid for an individual chromosome. Otherwise, the observed hyperdiploid frequencies of 0.16% for one chromosome multiplied by the 23 chromosome pairs would result in a overall hypcrdiptoid frequency of 3.7%, a frequency very different from those seen in mctaphase studies. The results of these analyses indicate that in spite of various cellular and hybridization artifacts. FISH can be used to provide reasonable estimates of the frequency of numerical alterations in human cells when caution is exercised in the scoring process. In summary, wc believe that interphase analysis using FISH with chromosome-specific DNA probes is a valuable complement to conventional metaphase analysis for detecting aneuploidy in the somatic cells of humans and animals. In this article wc have tried to describe many of the possible cellular and hybridization conditions that can affect the interpretation of FISH analyses for aneuploidy. In spite of these potential problems, a number of studies have demonstrated that FISH can be a valuable tool for in vitro screening of agents with aneuploidy-inducing potential and for detecting aneuploidy in chemically exposed human populations. With efficient hybridizations and cautious scoring, intcrphase studies using FISH to detect aneuploidy (and breakage) can provide estimates of chromosomal alterations which closely reflect those seen during metaphasc analysis using either FISH or conventional approaches. Due to the speed of analysis and the ability to evaluate different cell types, cytogenctic analysis using FISH should contribute significantly to our understanding of the etiology and consequences of aneuploidy affecting human populations.

Acknowledgments
We would like to thank Sheila Galloway for her helpful discussions and for providing us with the 48 h hyperdiploidy and polyploidy data from

D.A. Eastmond et al./Mutation Research 348 (199.5) 153-162

161

her population studies. Financial support from the U.S. Environmental Protection Agency (R 820994-01-1) is gratefully acknowledged.

References
Barquinero, J.F., L. Barrios, M.R. Caballin, R. Miro, M. Ribas, A. Subias and J. Egozcue 11993) Cytogenetic analysis of lymphocytes from hospital workers occupationally exposed to low levels of ionizing radiation, Mutation Rcs., 286, 275-9. Becker, P., H. Scherthan and Ii. Zanki (1990) Use of a centromere-specific DNA probe (p82H) in nonisotopic in situ hybridization for classification of micronuclei, Genes, Chromosomes Cancer, 2, 59-62. Bender, M.A., R.J. Preston, R.C. Leonard, B.E. Pyatt, P.C. Gooch PC (1989). Chromosomal aberration and sisterchromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample. II. Extension of age range, Mutation Res., 212, 149-54. Bischoff, F.Z., D.D. Nguyen, K.J. Burr and L.G. Schaffer (1994) Estimates of aneuploidy using multicolor fluorescence in situ hybridization on human sperm, Cytogenet. Cell Genet., 66, 237-243. Brogger, A. (1977) Non-random localization of chromosome damage in human cells and targets for clastogenic action, Chromosomes Today, 6, 297-305. Brown, T., D.P. Fox, F.W. Robertson and I. Bullock I (1983) Non-random chromosome loss in PHA-stimulated lymphocytes from normal individuals, Mutation Res., 122, 403-6. Chen, H.W., R. Tomar and D.A. Eastmond 11994) Detection of hydroquinone-induced nonrandom breakage in the centromeric heterochromatin of mouse bone marrow cells using multicolor fluorescence in situ hybridization with the mouse major and minor satellite probes, Mutagenesis, 9, 563-569. Clare, M., A. Yardley-Jones, A.C. MacLcan and B.J. Dean (1984) Chromosome analysis from peripheral blood lymphocytes of workers after an acute exposure to benzcnc., Br. J. Ind. Med., 41,249-253. De Sario, A., P. Vagnarelli and L. De Carli 11990) Aneuploidy assay on diethylstilbestrol by means of in situ hibridization of radioactive and biotinylated DNA probes on interphase nuclei, Mutation Res., 243, 127-131. Ding, X., Y. Li, Y. Ding and Yang H. (1983) Chromosome changes in patients with chronic benzene poisoning, Chinese Med. J., 96, 681-685. Eastmond, D.A. and D. Pinkel (1990) Detection of aneuploidy and aneuploidy-inducing agents in human lympht~'ytes using fluorescence in situ hybridization with chromosomespecific DNA probes, Mutation Res, 234, 303-318. Eastmond, D.A., D.S. Rupa and L.S. llasegawa (1994) Detection of hyperdiploidy and chromosome breakage in interphase human lymphocytes following exposure to the benzene metabolite hydr~xluinone using multicolor fluores-

cence in situ hybridization with DNA probes, Mutation Res., 322, 9-20. Ferguson, M. and D.C. Ward 11992) Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei, Chromosoma, 101,557-565. Fitzgerald, P.H. and C.M. McEwan (1977) Total aneuploidy and age-related sex chromosome aneuploidy in cultured lymphocytes of normal men and women, Human Genet., 39, 329-337. Galloway, S.M., P.K. Berry, W.W. Nichols, S.R. Wolman, K.A. Soper, P.D. Stolley and P. Archer (1986) Chromosome aberrations in individuals occupationally exposed to ethylene oxide, and in a large control population. Mutation Res., 170, 55-74. Galloway, S.M. and K.E. Buckton (1978) Aneuploidy and ageing: chromosome studies on a random sample of the population using G-banding, Cytogen. Cell Genctics, 20, 78-95. Hasegawa, L.S., D.S. Rupa and D.A. Eastmond (1995) Rapid generation of alpha- and classical satellite probes for human chromosome 9 by polymerasc chain reaction using gcnomic DNA: application to detect chromosomal alterations in interphase cells, Mutagenesis (in press). ttirai, M 11970) Aneuploidy and aging - a preliminary study, J. Anthrop. Soc. Nippon, 78, 187-199. Herrington, C.S., K. Cooper and J.O. McGee (1995) Interphase cyctogenetics: analysis of numerical chromosome aberrations in isolated cells, J. Pathol., 175, 283-95. KJbbelaar, R.E., F. Kok, E.J. Drcef, J.K. Kleiverda, C.J. Cornelisse, A.K. Raap, Ph.M. Kluin 11993) Statistical methods in interphase cytogenetics: an experimental approach. C)'tometry, 14, 716-724. Le Beau, M 11993) Fluorescence in situ hybridization in cancer diagnosis, in: Important Advances in Oncology 1993, in: V.T. DeVita, S. Hellman, S.A. Rosenberg (Eds), J.B. Lippincott Co., Philadelphia, pp. 29-45. Lewis, J.P., H.J. Tankc, A.K. Raap, G.C. Beverstock and !1.(_. Kluin-Nelemans (1993) Somatic pairing of centromeres and short arms of chromosome 15 in the hcmatopoictic and lymphoid system, Human Gcnct., 92, 577-582. Liniecki, J., A. Bajerska and M. Gluszczowa (1971) Analiza kariologiczna limfoc'ytow krwi obwodowej u osob z przebylym przewleklym zatruclem benzenem, Medycyna Pracy, 22, 187-193. Miller, B.M., II.F. Zitzelsberger, H.-U. Weier and I.-D. Adler 11991) Classification of micronuclci in murine crythrocytes: immunofluorescent staining using CREST antibodies compared to in situ hybridization with biotinylated gamma satellite DNA, Mutagenesis, 6, 297-302. Monsalve, M.V. and C. Chiappe 11987) Genetic effects of methylmercury in human chromosomes: I. A cytogenetic study of people exposed through eating contaminated fish. Environ. Mol. Mutagen., 10, 367-76. Moore, L.E., N. Titenko-Holland and M.T. Smith (1993) Use of fluorescence in situ hybridization to detect chromosome-specific changes in exfoliated human bladder and oral mucosa cells, Environ. Mol. Mutagen., 22, 130-137.

162

D.A. Ea.stmond et aL /Mutation Research 348 11995) 153-162

Natarajan, A.T., R.C. Vyas, F. Darroudi and S. Vermeulcn (1992) Frequencies of X-ray-induced chromosome translocations in human peripheral lymphocytes as detected by in situ hybridization using chromosome-specific DNA libraries, Int. J. Radiat. Biol.. 61, 199-203. Poddighe, P.J., O. Moeskcr, D. Smeets, B.H. Awwad, F.C.S. Ramaekers and A.H.N. Hopman (1991) lnterphase cytogcnetics of hematological cancer: Comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes, Cancer Rcs., 51, 1959-1967. Popescu, H.I., I.. Negru, I. Lancranjan 11979)Chromosome aberrations induced by occupational exposure to mercury, Arch. Environ. Health, 34, 461-3. Raimondi, E., S. Scariolo, A. De Sario and L. Dc Carli 11989) Aneuploidy assays on interphase nuclei by means of in situ hybridization with DNA probes, Mutagenesis, 4, 165-169. Ramirez, P., L. Vega. M.E. Gonsebatt and P. Ostrosky-Wegman 11995) The aneugenic capacity of sodium arsenite and vanadium pentoxide using FISH methodology, Environ. Molecul. Mutagen., 25 Suppl. 25, 44. Richard, F, A. Aurias, J. Couturier, A.M., A.M. Dutrillaux, A. Flury-Herard, M. Gerbault-Seureau, F. Hoffschir, E. Lamoliatte, D. Lefrancois, M. Ia~mbard, M. Mulcris, M. Prieur, M. Ricoul, I.. Sabatier, E. Viegas-Pequignot, V. Vololmmcv and B. Dutrillaux 11993) Aneuploidy in human lymphocytes: an extensive study of eight individuals of various ages, Mutation Rcs., 295, 71-80. Robbins, W.A., M.J. Cassel, D.H. Blakey, M.L. Meistrich and A.J. Wyrobek (1994) Induction of aneuploidy in the sperm of Hodgkin's disease patients treated with NOVP chemotherapy (Detection by multi-chromosome fluorescence in situ hybridization), Enrivon. Molecul. Mutagcn., 23 Suppl. 23. 58. Rupa, D.S., L. Hasegawa and D.A. Eastmond (1995) Detection of chromosomal breakage in the lcen-lql2 region of interphase human lymphoeytes using multicolor fluorescence in situ hybridization with tandem DNA probes. Cancer Res., 55. 640-645. Sandberg, A.A., C. Turec-Carel and R.M. Germill 11988) Chromosomes in solid tumors and beyond, Cancer Res.. 48, 1049-1059. Schmid, M., D. Grunert, T. llaaf and W. Engel (1983) A

direct demonstration of somatically paired heterochromatin of human chromosomes, Cytogenet. Cell Genet., 36, 554-51~1. Smith M.T., I.. Zhang. N. Rothman, Y. Wang, R.B. Hayes, G.-L. Li and S.-N. Yin 11995) interphase cytogenetics of workers exposed to benzene, Toxicologist, 15, 87. Teyssier, J.R. 11989) The chromosomal analysis of human solid tumors: a triple challenge, Cancer Genet. Cytogenet., 37, 103-125. Titenko-Ilolland N., L.E. Moore and M.T. Smith (1994)Measurement and characterization of micronuclci in cxfoliated human cells by fluorescence in situ hybridization with a centromeric probe, Mutation Res., 312: 39-51). Tonomura. A., K. Kishi and F. Saito 11983) Types and frequencies of chromosome aberrations in peripheral lymphocytes of general populations, in: T. Ishihara and M.S. Sasaki (Eds.) Radiation-induced Chromosome Damage in Man, Alan R. Liss, New York, pp. 605-616. Tucker J.D., M.J. Ramsey, D.A. Lee and J.L. Minkler 11993) Validation of chromosome painting in human peripheral lymphocytes following acute exposure to ionizing radiation in vitro, Int. J. Radiat. Biol., 64, 27-37. Vagnarelli, P.. A. De Sario and L. De Carli 11990) Aneuploidy induced by chloral hydrate detected in human lymphocytcs with the Y97 probe, Mutagenesis, 5,591-592. Verschaeve, I.., M. Driesen, M. Kirsch-Volders, L. Hens and C. Susanne 11979) Chromosome distribution studies after inorganic lead exposure, Human Genetics, 49, 147-58. Vcrschacvc, L., M. Kirsch-Volders, L. liens and C. Susanne (1978) Chromosome distribution studies in phenyl mercury acetate exposed subjects and in age-related controls, Mutation Res., 57, 335-7. Yardley-Jones, A., D. Anderson. D. l.ovell and P. Jenkinson (1990) Analysis of chromosomal aberrations in workers exposed to low level benzene., Br. J. Ind. Med., 47, 48-51. Zhang I... P. Venkatesh, M.L.R. Creek and M.T. Smith (1994) Detection of 1,2,4-benzenetriol induced aneuploidy and microtubule disruption by fluorescence in situ hybridizaticm and immunocytochemistry, Mutation Res., 320, 315327.

Communicated by S.M. Galloway